2023年09月, 国際水協会, アーダーン・ロケット賞2023　　　　　　　　　　　　　　　
岡部 聡

2023年06月, 公益社団法人 日本水環境学会, 学術賞　　　　　　　　　　　　　　　
窒素除去にかかわる複合微生物生態系の構造と機能の解析
岡部 聡

2018年03月, 北海道大学, 北海道大学教育研究総長表彰（研究部門）　　　　　　　　　　　　　　　
岡部 聡

2017年03月, 北海道大学, 北海道大学教育研究総長表彰（教育部門）　　　　　　　　　　　　　　　
岡部 聡

2016年, International Water Association, IWA Fellow　　　　　　　　　　　　　　　
岡部 聡

2014年04月, Nagase Science and Technology Foundation, 長瀬研究振興賞　　　　　　　　　　　　　　　
岡部 聡

2009年, 日本水環境学会論文賞　　　　　　　　　　　　　　　
日本国

2008年, 日本学術振興会賞　　　　　　　　　　　　　　　
日本国

2008年, JSPS prize　　　　　　　　　　　　　　　

2006年, The best poster award at The 6th International Conference on Biofilm Systems　　　　　　　　　　　　　　　

2005年, 土木学会環境工学フォーラム優秀ポスター発表賞　　　　　　　　　　　　　　　
日本国

2003年, 環境工学フォーラム論文賞　　　　　　　　　　　　　　　
日本国

2001年, 月刊「水」論文賞　　　　　　　　　　　　　　　

1999年, 日本水道協会論文有効賞　　　　　　　　　　　　　　　
日本国

1999年, 日本水環境学会論文奨励賞（廣瀬賞）　　　　　　　　　　　　　　　
日本国

1999年, 平成10年度土木学会年次学術講演会優秀講演者賞　　　　　　　　　　　　　　　
日本国

1998年, 平成9年度土木学会年次学術講演会優秀講演者賞　　　　　　　　　　　　　　　
日本国

Microbial photoelectrochemical cell using hybrid CuO/ZnO/CuO and Shewanella oneidensis MR-1 anode for hydrogen production
Ryosuke Matsuo, Seiichi Watanabe, Satoshi Okabe
Chemical Engineering Journal, 505, 159093, 159093, Elsevier BV, 2025年02月
研究論文（学術雑誌）

Quantitative association of SARS-CoV-2 in wastewater and clinically confirmed cases in different areas of the Tokyo 2020 Olympic and Paralympic Village
Masaaki Kitajima, Michio Murakami, Hiroki Ando, Syun-suke Kadoya, Ryo Iwamoto, Tomohiro Kuroita, Kiyoshi Yamaguchi, Hiroyuki Kobayashi, Satoshi Okabe, Hiroyuki Katayama, Seiya Imoto
Science of The Total Environment, 960, 178209, 178209, Elsevier BV, 2025年01月
研究論文（学術雑誌）

Salinity Tolerance and Osmoadaptation Strategies in Four Genera of Anammox Bacteria: Brocadia, Jettenia, Kuenenia, and Scalindua
Satoshi Okabe, Akimichi Kamizono, Lei Zhang, Seiya Kawasaki, Kanae Kobayashi, Mamoru Oshiki
Environmental Science & Technology, 58, 12, 5357, 5371, American Chemical Society (ACS), 2024年03月16日, ［査読有り］
英語, 研究論文（学術雑誌）

Quantitative analysis of SARS-CoV-2 RNA in wastewater and evaluation of sampling frequency during the downward period of a COVID-19 wave in Japan.
Tomohiro Kuroita, Akimasa Yoshimura, Ryo Iwamoto, Hiroki Ando, Satoshi Okabe, Masaaki Kitajima
The Science of the total environment, 906, 166526, 166526, 2024年01月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Wastewater-based epidemiology (WBE) is a practical approach for detecting the presence of SARS-CoV-2 infections and assessing the epidemic trend of the coronavirus disease 2019 (COVID-19). The purpose of this study was to evaluate the minimum sampling frequency required to properly identify the COVID-19 trend during the downward epidemic period when using a highly sensitive RNA detection method. WBE was conducted using the Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S), a highly sensitive SARS-CoV-2 RNA detection method, at nine neighboring wastewater treatment plants (WWTPs). These WWTPs were in the same prefecture in Japan, and they had different sewer types, sampling methods, and sampling frequencies. The overall detection rate of SARS-CoV-2 RNA was 97.8 % during the entire study period when the geometric means of new COVID-19 cases per 100,000 inhabitants were between 3.3 and 7.7 in each WWTP. The maximum SARS-CoV-2 RNA concentration in wastewater was 2.14 × 104 copies/L, which corresponded to pepper mild mottle virus (PMMoV)-normalized concentrations of 6.54 × 10-3. We evaluated the effect of sampling frequencies on the probability of a significant correlation with the number of newly reported COVID-19 cases by hypothetically reducing the sampling frequency in the same dataset. When the wastewater sampling frequency occurred 5, 3, 2, and 1 times per week, these results exhibited significant correlations of 100 % (5/5), 89 % (8/9), 85 % (23/27), and 48 % (13/27), respectively. To achieve significant correlation with a high probability of over 85 %, a minimum sampling frequency of twice per week is required, even if sampling methods and sewer types are different. WBE using the EPISENS-S method and a sampling frequency of more than twice a week can be used to properly monitor COVID-19 wave epidemic trends, even during downward periods.

Collaborative metabolisms of urea and cyanate degradation in marine anammox bacterial culture
Mamoru Oshiki, Emi Morimoto, Kanae Kobayashi, Hisashi Satoh, Satoshi Okabe
ISME Communications, 4, 1, Oxford University Press (OUP), 2024年01月01日
研究論文（学術雑誌）, Abstract

Anammox process greatly contributes to nitrogen loss occurring in oceanic oxygen minimum zones (OMZs), where the availability of NH4+ is scarce as compared with NO2−. Remineralization of organic nitrogen compounds including urea and cyanate (OCN−) into NH4+ has been believed as an NH4+ source of the anammox process in oxygen minimum zones. However, urea- or OCN−- dependent anammox has not been well examined due to the lack of marine anammox bacterial culture. In the present study, urea and OCN− degradation in a marine anammox bacterial consortium were investigated based on 15N-tracer experiments and metagenomic analysis. Although a marine anammox bacterium, Candidatus Scalindua sp., itself was incapable of urea and OCN− degradation, urea was anoxically decomposed to NH4+ by the coexisting ureolytic bacteria (Rhizobiaceae, Nitrosomonadaceae, and/or Thalassopiraceae bacteria), whereas OCN− was abiotically degraded to NH4+. The produced NH4+ was subsequently utilized in the anammox process. The activity of the urea degradation increased under microaerobic condition (ca. 32–42 μM dissolved O2, DO), and the contribution of the anammox process to the total nitrogen loss also increased up to 33.3% at 32 μM DO. Urea-dependent anammox activities were further examined in a fluid thioglycolate media with a vertical gradient of O2 concentration, and the active collaborative metabolism of the urea degradation and anammox was detected at the lower oxycline (21 μM DO).

Tracking the effects of the COVID-19 pandemic on viral gastroenteritis through wastewater-based retrospective analyses.
Hiroki Ando, Warish Ahmed, Satoshi Okabe, Masaaki Kitajima
The Science of the total environment, 905, 166557, 166557, 2023年12月20日, ［国際誌］
英語, 研究論文（学術雑誌）, The COVID-19 pandemic possibly disrupted the circulation and seasonality of gastroenteritis viruses (e.g., Norovirus (NoV), Sapovirus (SaV), group A rotavirus (ARoV), and Aichivirus (AiV)). Despite the growing application of wastewater-based epidemiology (WBE), there remains a lack of sufficient investigations into the actual impact of the COVID-19 pandemic on the prevalence of gastroenteritis viruses. In this study, we measured NoV GI and GII, SaV, ARoV, and AiV RNA concentrations in 296 influent wastewater samples collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan between October 28, 2018 and January 12, 2023 using the highly sensitive EPISENS™ method. The detection ratios of SaV and ARoV after May 2020 (SaV: 49.8 % (134/269), ARoV: 57.4 % (151/263)) were significantly lower than those before April 2020 (SaV: 93.9 % (31/33), ARoV: 97.0 % (32/33); SaV: p < 3.5×10-7, ARoV: p < 1.5×10-6). Furthermore, despite comparable detection ratios before (88.5 %, 23/26) and during (66.7 %, 80/120) the COVID-19 pandemic (p = 0.032), the concentrations of NoV GII revealed a significant decrease after the onset of the pandemic (p < 1.5×10-7, Cliff's delta = 0.72). NoV GI RNA were sporadically detected (24.7 %, 8/33) before April 2020 and after May 2020 (6.5 %, 17/263), whereas AiV was consistently (100 %, 33/33) detected from wastewater throughout the study period (95.8 %, 252/263). The WBE results demonstrated the significant influence of COVID-19 countermeasures on the circulation of gastroenteritis viruses, with variations observed in the magnitude of their impact across different types of viruses. These epidemiological findings highlight that the hygiene practices implemented to prevent COVID-19 infections may also be effective for controlling the prevalence of gastroenteritis viruses, providing invaluable insights for public health units and the development of effective disease management guidelines.

Fabrication of ZnO/CuO nanoforests and their applicability to microbial photoelectrochemical cells
Ryosuke Matsuo, Yuki Takahashi, Seiichi Watanabe, Satoshi Okabe
Applied Catalysis B: Environmental, 339, 123097, 123097, Elsevier BV, 2023年12月
研究論文（学術雑誌）

Growth of the Nitrosomonas europaea cells in the biofilm and planktonic growth mode: Responses of extracellular polymeric substances production and transcriptome
Mamoru Oshiki, Takahiro Saito, Yuki Nakaya, Hisashi Satoh, Satoshi Okabe
Journal of Bioscience and Bioengineering, Elsevier BV, 2023年11月
研究論文（学術雑誌）

Identification of SARS-CoV-2 variants in wastewater using targeted amplicon sequencing during a low COVID-19 prevalence period in Japan.
Ryo Iwamoto, Kiyoshi Yamaguchi, Kotoe Katayama, Hiroki Ando, Ken-Ichi Setsukinai, Hiroyuki Kobayashi, Satoshi Okabe, Seiya Imoto, Masaaki Kitajima
The Science of the total environment, 887, 163706, 163706, 2023年08月20日, ［国際誌］
英語, 研究論文（学術雑誌）, Wastewater-based epidemiology is expected to be able to identify SARS-CoV-2 variants at an early stage via next-generation sequencing. In the present study, we developed a highly sensitive amplicon sequencing method targeting the spike gene of SARS-CoV-2, which allows for sequencing viral genomes from wastewater containing a low amount of virus. Primers were designed to amplify a relatively long region (599 bp) around the receptor-binding domain in the SARS-CoV-2 spike gene, which could distinguish initial major variants of concern. To validate the methodology, we retrospectively analyzed wastewater samples collected from a septic tank installed in a COVID-19 quarantine facility between October and December 2020. The relative abundance of D614G mutant in SARS-CoV-2 genomes in the facility wastewater increased from 47.5 % to 83.1 % during the study period. The N501Y mutant, which is the characteristic mutation of the Alpha-like strain, was detected from wastewater collected on December 24, 2020, which agreed with the fact that a patient infected with the Alpha-like strain was quarantined in the facility on this date. We then analyzed archived municipal wastewater samples collected between November 2020 and January 2021 that contained low SARS-CoV-2 concentrations ranging from 0.23 to 0.43 copies/qPCR reaction (corresponding to 3.30 to 4.15 log10 copies/L). The targeted amplicon sequencing revealed that the Alpha-like variant with D614G and N501Y mutations was present in municipal wastewater collected on December 4, 2020 and later, suggesting that the variant had already spread in the community before its first clinical confirmation in Japan on December 25, 2020. These results demonstrate that targeted amplicon sequencing of wastewater samples is a powerful surveillance tool applicable to low COVID-19 prevalence periods and may contribute to the early detection of emerging variants.

Impact of the COVID-19 pandemic on the prevalence of influenza A and respiratory syncytial viruses elucidated by wastewater-based epidemiology.
Hiroki Ando, Warish Ahmed, Ryo Iwamoto, Yoshinori Ando, Satoshi Okabe, Masaaki Kitajima
The Science of the total environment, 880, 162694, 162694, 2023年07月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Since the COVID-19 pandemic, a decrease in the prevalence of Influenza A virus (IAV) and respiratory syncytial virus (RSV) has been suggested by clinical surveillance. However, there may be potential biases in obtaining an accurate overview of infectious diseases in a community. To elucidate the impact of the COVID-19 on the prevalence of IAV and RSV, we quantified IAV and RSV RNA in wastewater collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan, between October 2018 and January 2023, using highly sensitive EPISENS™ method. From October 2018 to April 2020, the IAV M gene concentrations were positively correlated with the confirmed cases in the corresponding area (Spearman's r = 0.61). Subtype-specific HA genes of IAV were also detected, and their concentrations showed trends that were consistent with clinically reported cases. RSV A and B serotypes were also detected in wastewater, and their concentrations were positively correlated with the confirmed clinical cases (Spearman's r = 0.36-0.52). The detection ratios of IAV and RSV in wastewater decreased from 66.7 % (22/33) and 42.4 % (14/33) to 4.56 % (12/263) and 32.7 % (86/263), respectively in the city after the COVID-19 prevalence. The present study demonstrates the potential usefulness of wastewater-based epidemiology combined with the preservation of wastewater (wastewater banking) as a tool for better management of respiratory viral diseases.

Oxygen tolerance and detoxification mechanisms of highly enriched planktonic anaerobic ammonium-oxidizing (anammox) bacteria
Satoshi Okabe, Shaoyu Ye, Xi Lan, Keishi Nukada, Haozhe Zhang, Kanae Kobayashi, Mamoru Oshiki
ISME Communications, 3, 1, Springer Science and Business Media LLC, 2023年05月03日
研究論文（学術雑誌）, Abstract

Oxygen is a key regulatory factor of anaerobic ammonium oxidation (anammox). Although the inhibitory effect of oxygen is evident, a wide range of oxygen sensitivities of anammox bacteria have been reported so far, which makes it difficult to model the marine nitrogen loss and design anammox-based technologies. Here, oxygen tolerance and detoxification mechanisms of four genera of anammox bacteria; one marine species (“Ca. Scalindua sp.”) and four freshwater anammox species (“Ca. Brocadia sinica”, “Ca. Brocadia sapporoensis”, “Ca. Jettenia caeni”, and “Ca. Kuenenia stuttgartiensis”) were determined and then related to the activities of anti-oxidative enzymes. Highly enriched planktonic anammox cells were exposed to various levels of oxygen, and oxygen inhibition kinetics (50% inhibitory concentration (IC₅₀) and upper O₂ limits (DO_max) of anammox activity) were quantitatively determined. A marine anammox species, “Ca. Scalindua sp.”, exhibited much higher oxygen tolerance capability (IC₅₀ = 18.0 µM and DO_max = 51.6 µM) than freshwater species (IC₅₀ = 2.7–4.2 µM and DO_max = 10.9–26.6 µM). The upper DO limit of “Ca. Scalindua sp.” was much higher than the values reported so far (~20 µM). Furthermore, the oxygen inhibition was reversible even after exposed to ambient air for 12–24 h. The comparative genome analysis confirmed that all anammox species commonly possess the genes considered to function for reduction of O₂, superoxide anion (O₂^•-), and H₂O₂. However, the superoxide reductase (Sor)-peroxidase dependent detoxification system alone may not be sufficient for cell survival under microaerobic conditions. Despite the fact that anaerobes normally possess no or little superoxide dismutase (Sod) or catalase (Cat), only Scalindua exhibited high Sod activity of 22.6 ± 1.9 U/mg-protein with moderate Cat activity of 1.6 ± 0.7 U/mg-protein, which was consistent with the genome sequence analysis. This Sod-Cat dependent detoxification system could be responsible for the higher O₂ tolerance of Scalindua than other freshwater anammox species lacking the Sod activity.

Wastewater-based prediction of COVID-19 cases using a highly sensitive SARS-CoV-2 RNA detection method combined with mathematical modeling.
Hiroki Ando, Michio Murakami, Warish Ahmed, Ryo Iwamoto, Satoshi Okabe, Masaaki Kitajima
Environment international, 173, 107743, 107743, 2023年03月, ［国際誌］
英語, 研究論文（学術雑誌）, Wastewater-based epidemiology (WBE) has the potential to predict COVID-19 cases; however, reliable methods for tracking SARS-CoV-2 RNA concentrations (CRNA) in wastewater are lacking. In the present study, we developed a highly sensitive method (EPISENS-M) employing adsorption-extraction, followed by one-step RT-Preamp and qPCR. The EPISENS-M allowed SARS-CoV-2 RNA detection from wastewater at 50 % detection rate when newly reported COVID-19 cases exceed 0.69/100,000 inhabitants in a sewer catchment. Using the EPISENS-M, a longitudinal WBE study was conducted between 28 May 2020 and 16 June 2022 in Sapporo City, Japan, revealing a strong correlation (Pearson's r = 0.94) between CRNA and the newly COVID-19 cases reported by intensive clinical surveillance. Based on this dataset, a mathematical model was developed based on viral shedding dynamics to estimate the newly reported cases using CRNA data and recent clinical data prior to sampling day. This developed model succeeded in predicting the cumulative number of newly reported cases after 5 days of sampling day within a factor of √2 and 2 with a precision of 36 % (16/44) and 64 % (28/44), respectively. By applying this model framework, another estimation mode was developed without the recent clinical data, which successfully predicted the number of COVID-19 cases for the succeeding 5 days within a factor of √2 and 2 with a precision of 39 % (17/44) and 66 % (29/44), respectively. These results demonstrated that the EPISENS-M method combined with the mathematical model can be a powerful tool for predicting COVID-19 cases, especially in the absence of intensive clinical surveillance.

The detectability and removal efficiency of SARS-CoV-2 in a large-scale septic tank of a COVID-19 quarantine facility in Japan.
Ryo Iwamoto, Kiyoshi Yamaguchi, Chisato Arakawa, Hiroki Ando, Eiji Haramoto, Ken-Ichi Setsukinai, Kotoe Katayama, Takuya Yamagishi, Sumire Sorano, Michio Murakami, Shigeru Kyuwa, Hiroyuki Kobayashi, Satoshi Okabe, Seiya Imoto, Masaaki Kitajima
The Science of the total environment, 849, 157869, 157869, 2022年11月25日, ［国際誌］
英語, 研究論文（学術雑誌）, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to be present in sewage, and wastewater-based epidemiology has attracted much attention. However, the physical partitioning of SARS-CoV-2 in wastewater and the removal efficiency of treatment systems require further investigation. This study aimed to investigate the detectability and physical partitioning of SARS-CoV-2 in wastewater and assess its removal in a large-scale septic tank employing anaerobic, anoxic, and oxic processes in a sequential batch reactor, which was installed in a coronavirus disease 2019 (COVID-19) quarantine facility. The amount of SARS-CoV-2 RNA in wastewater was determined with polyethylene glycol (PEG) precipitation followed by quantitative polymerase chain reaction (qPCR), and the association of SARS-CoV-2 with wastewater solids was evaluated by the effect of filtration prior to PEG precipitation (pre-filtration). The amount of SARS-CoV-2 RNA detected from pre-filtered samples was substantially lower than that of samples without pre-filtration. These results suggest that most SARS-CoV-2 particles in wastewater are associated with the suspended solids excluded by pre-filtration. The removal efficiency of SARS-CoV-2 in the septic tank was evaluated based on the SARS-CoV-2 RNA concentrations in untreated and treated wastewater, which was determined by the detection method optimized in this study. Escherichia coli and pepper mild mottle virus (PMMoV) were also quantified to validate the wastewater treatment system's performance. The mean log10 reduction values of SARS-CoV-2, E. coli, and PMMoV were 2.47 (range, 2.25-2.68), 2.81 (range, 2.45-3.18), and 0.66 (range, 0.61-0.70), respectively, demonstrating that SARS-CoV-2 removal by the wastewater treatment system was comparable to or better than the removal of fecal indicators. These results suggest that SARS-CoV-2 can be readily removed by the septic tank. This is the first study to determine the removal efficiency of SARS-CoV-2 in a facility-level sequencing batch activated sludge system.

The Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S): A rapid and cost-effective SARS-CoV-2 RNA detection method for routine wastewater surveillance.
Hiroki Ando, Ryo Iwamoto, Hiroyuki Kobayashi, Satoshi Okabe, Masaaki Kitajima
The Science of the total environment, 843, 157101, 157101, 2022年10月15日, ［国際誌］
英語, 研究論文（学術雑誌）, Wastewater-based epidemiology has attracted attention as a COVID-19 surveillance tool. Here, we developed a practical method for detecting SARS-CoV-2 RNA in wastewater (the EPISENS-S method), which employs direct RNA extraction from wastewater pellets formed via low-speed centrifugation. The subsequent multiplex one-step RT-preamplification reaction with forward and reverse primers for SARS-CoV-2 and a reverse primer only for pepper mild mottle virus (PMMoV) allowed for qPCR quantification of the targets with different abundances in wastewater from the RT-preamplification product. The detection sensitivity of the method was evaluated using wastewater samples seeded with heat-inactivated SARS-CoV-2 in concentrations of 2.11 × 103 to 2.11 × 106 copies/L. The results demonstrated that the sensitivity of the EPISENS-S method was two orders of magnitude higher than that of the conventional method (PEG precipitation, followed by regular RT-qPCR; PEG-QVR-qPCR). A total of 37 untreated wastewater samples collected from two wastewater treatment plants in Sapporo, Japan when 1.6 to 18 new daily reported cases per 100,000 people were reported in the city (March 4 to July 8, 2021), were examined using the EPISENS-S method to confirm its applicability to municipal wastewater. SARS-CoV-2 RNA was quantified in 92 % (34/37) of the samples via the EPISENS-S method, whereas none of the samples (0/37) was quantifiable via the PEG-QVR-qPCR method. The PMMoV concentrations measured by the EPISENS-S method ranged from 2.60 × 106 to 1.90 × 108 copies/L, and the SARS-CoV-2 RNA concentrations normalized by PMMoV ranged from 5.71 × 10-6 to 9.51 × 10-4 . The long-term trend of normalized SARS-CoV-2 RNA concentration in wastewater was consistent with that of confirmed COVID-19 cases in the city. These results demonstrate that the EPISENS-S method is highly sensitive and suitable for routine COVID-19 wastewater surveillance.

Growth of nitrite-oxidizing Nitrospira and ammonia-oxidizing Nitrosomonas in marine recirculating trickling biofilter reactors.
Mamoru Oshiki, Hirotoshi Netsu, Kyohei Kuroda, Takashi Narihiro, Naoki Fujii, Tomonori Kindaichi, Yoshiyuki Suzuki, Takahiro Watari, Masashi Hatamoto, Takashi Yamaguchi, Nobuo Araki, Satoshi Okabe
Environmental microbiology, 24, 8, 3735, 3750, 2022年06月07日, ［国際誌］
英語, 研究論文（学術雑誌）, Aerobic ammonia and nitrite oxidation reactions are fundamental biogeochemical reactions contributing to the global nitrogen cycle. Although aerobic nitrite oxidation yields 4.8-folds less Gibbs free energy (∆Gr ) than aerobic ammonia oxidation in the NH4 + -feeding marine recirculating trickling biofilter reactors operated in the present study, nitrite-oxidizing and not ammonia-oxidizing Nitrospira (sublineage IV) outnumbered ammonia-oxidizing Nitrosomonas (relative abundance; 53.8% and 7.59% respectively). CO2 assimilation efficiencies during ammonia or nitrite oxidation were 0.077 μmol-14 CO2 /μmol-NH3 and 0.053-0.054 μmol-14 CO2 /μmol-NO2 - respectively, and the difference between ammonia and nitrite oxidation was much smaller than the difference of ∆Gr . Free-energy efficiency of nitrite oxidation was higher than ammonia oxidation (31%-32% and 13% respectively), and high CO2 assimilation and free-energy efficiencies were a determinant for the dominance of Nitrospira over Nitrosomonas. Washout of Nitrospira and Nitrosomonas from the trickling biofilter reactors was also examined by quantitative PCR assay. Normalized copy numbers of Nitrosomonas amoA were 1.5- to 1.7-folds greater than Nitrospira nxrB and 16S rRNA gene in the reactor effluents. Nitrosomonas was more susceptible for washout than Nitrospira in the trickling biofilter reactors, which was another determinant for the dominance of Nitrospira in the trickling biofilter reactors.

Correction for Oshiki et al., "Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS)".
Mamoru Oshiki, Komei Nagai, Satoshi Ishii, Yoshiyuki Suzuki, Nobuo Saito, Takashi Yamaguchi, Nobuo Araki, Satoshi Okabe
Applied and environmental microbiology, 88, 9, e0052522, 2022年04月20日, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）

Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).
Mamoru Oshiki, Komei Nagai, Satoshi Ishii, Yoshiyuki Suzuki, Nobuo Saito, Takashi Yamaguchi, Nobuo Araki, Satoshi Okabe
Applied and environmental microbiology, 88, 7, e0241621, 2022年04月12日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE 15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.

Temporal dynamics of Campylobacter and Arcobacter in a freshwater lake that receives fecal inputs from migratory geese.
Mayumi Kobayashi, Qian Zhang, Takahiro Segawa, Mitsuto Maeda, Reiko Hirano, Satoshi Okabe, Satoshi Ishii
Water research, 217, 118397, 118397, 2022年04月04日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Migratory geese could influence the microbiological water quality; however, their impacts on pathogen dynamics remain largely unknown. In this study, we analyzed the population dynamics of Campylobacter and Arcobacter group bacteria (AGB) in a freshwater lake in Japan over two years. The bacteria were quantified by using both culture-dependent and -independent methods. The potential sources of these bacteria were examined by a high-throughput flaA sequencing approach. Campylobacter was abundantly detected both by culture-dependent and -independent methods in the lake, especially when migratory geese were present in the lake. High-throughput flaA sequencing suggests that geese were the likely source of Campylobacter in the lake. The viable population of Campylobacter exceeds the concentrations that can potentially cause 10-4 infections per person per year when water is used to grow fresh vegetables. The occurrence of AGB, on the other hand, was not directly related to the population of migratory geese. AGB were not detected in geese fecal samples. Diverse AGB flaA genotypes occurred in the lake over multiple seasons. Our results suggest that AGB likely comprise a part of the indigenous microbial population of the lake and grow in response to high nutrient, warm temperature, and low dissolved oxygen concentrations in the lake. Geese therefore can indirectly impact the AGB population by providing nutrients to cause eutrophication and lower the dissolved oxygen concentration. Since geese travel long-distance and disperse their fecal microbiota and nutrients to wide areas, they may have significant impacts on water quality and public health.

Effect of poised cathodic potential on anodic ammonium nitrogen removal from domestic wastewater by air-cathode microbial fuel cells.
N'dah Joel Koffi, Satoshi Okabe
Bioresource technology, 348, 126807, 126807, 2022年03月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Performances of anodic ammonia oxidation have been investigated for various bioelectrochemical systems at a wide range of poised anodic potentials in the literature. The effect of poised cathodic potential on ammonium nitrogen (NH4+-N) and total nitrogen (TN, sum of NH4+-N, NO2--N, and NO3--N) removal from domestic wastewater by single chamber air-cathode microbial fuel cells (MFCs) was investigated. Poising the air-cathode potential at +0.7 V vs. SHE significantly increased current generation (from 11 ± 1 mA to 22.8 ± 5 mA) and oxygen permeation into the MFC through the air-cathode (from 75.4 ± 1.2 g-O2/m3/d to 151 ± 3.7 g-O2/m3/d), which consequently resulted in a high NH4+-N removal rate of 150 ± 13 g-NH4+-N/m3/d and TN removal rate of 63 ± 16 g-TN/m3/d. These high NH4+-N and TN removal rates could be attributed to the enhancement of dual respiratory pathways: the electrode-assisted anodic and aerobic NH4+ oxidation.

The Intrapopulation Genetic Diversity of RNA Virus May Influence the Sensitivity of Chlorine Disinfection.
Syun-Suke Kadoya, Syun-Ichi Urayama, Takuro Nunoura, Miho Hirai, Yoshihiro Takaki, Masaaki Kitajima, Toyoko Nakagomi, Osamu Nakagomi, Satoshi Okabe, Osamu Nishimura, Daisuke Sano
Frontiers in microbiology, 13, 839513, 839513, 2022年, ［国際誌］
英語, 研究論文（学術雑誌）, RNA virus populations are not clonal; rather, they comprise a mutant swarm in which sequences are slightly different from the master sequence. Genetic diversity within a population (intrapopulation genetic diversity) is critical for RNA viruses to survive under environmental stresses. Disinfection has become an important practice in the control of pathogenic viruses; however, the impact of intrapopulation genetic diversity on the sensitivity of disinfection, defined as -log10 (postdisinfected infectious titer/predisinfected titer), has not been elucidated. In this study, we serially passaged populations of rhesus rotavirus. We demonstrated that populations with reduced chlorine sensitivity emerged at random and independently of chlorine exposure. Sequencing analysis revealed that compared with sensitive populations, less-sensitive ones had higher non-synonymous genetic diversity of the outer capsid protein gene, suggesting that changes in the amino acid sequences of the outer capsid protein were the main factors influencing chlorine sensitivity. No common mutations were found among less-sensitive populations, indicating that rather than specific mutations, the diversity of the outer capsid protein itself was associated with the disinfection sensitivity and that the disinfection sensitivity changed stochastically. Simulation results suggest that the disinfection sensitivity of a genetically diverse population is destabilized if cooperative viral clusters including multiple sequences are formed. These results advocate that any prevention measures leading to low intrapopulation genetic diversity are important to prevent the spread and evolution of pathogenic RNA viruses in society.

Metagenomic Analysis of Five Phylogenetically Distant Anammox Bacterial Enrichment Cultures
Mamoru Oshiki, Yoshihiro Takaki, Miho Hirai, Takuro Nunoura, Atsushi Kamigaito, Satoshi Okabe
Microbes and Environments, 37, 3, n/a, n/a, Japanese Society of Microbial Ecology, 2022年, ［査読有り］, ［責任著者］
研究論文（学術雑誌）

NH2OH Disproportionation Mediated by Anaerobic Ammonium-oxidizing (Anammox) Bacteria.
Mamoru Oshiki, Lin Gao, Lei Zhang, Satoshi Okabe
Microbes and environments, 37, 2, 2022年, ［査読有り］, ［最終著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, Anammox bacteria produce N2 gas by oxidizing NH4+ with NO2-, and hydroxylamine (NH2OH) is a potential intermediate of the anammox process. N2 gas production occurs when anammox bacteria are incubated with NH2OH only, indicating their capacity for NH2OH disproportionation with NH2OH serving as both the electron donor and acceptor. Limited information is currently available on NH2OH disproportionation by anammox bacteria; therefore, the stoichiometry of anammox bacterial NH2OH disproportionation was examined in the present study using 15N-tracing techniques. The anammox bacteria, Brocadia sinica, Jettenia caeni, and Scalindua sp. were incubated with the addition of 15NH2OH, and the production of 15N-labeled nitrogenous compounds was assessed. The anammox bacteria tested performed NH2OH disproportionation and produced 15-15N2 gas and NH4+ as reaction products. The addition of acetylene, an inhibitor of the anammox process, reduced the activity of NH2OH disproportionation, but not completely. The growth of B. sinica by NH2OH disproportionation (-240.3‍ ‍kJ mol NH2OH-1 under standard conditions) was also tested in 3 up-flow column anammox reactors fed with 1) 0.7‍ ‍mM NH2OH only, 2) 0.7‍ ‍mM NH2OH and 0.5‍ ‍mM NH4+, and 3) 0.7‍ ‍mM NH2OH and 0.5‍ ‍mM NO2-. NH2OH consumption activities were markedly reduced after 7‍ ‍d of operation, indicating that B. sinica was unable to maintain its activity or biomass by NH2OH disproportionation.

Transcriptomic response of HepG2 cells exposed to three common anti-inflammatory drugs: Ketoprofen, mefenamic acid, and diclofenac in domestic wastewater effluents.
Hiroe Hara-Yamamura, Koji Nakashima, Toshikazu Fukushima, Satoshi Okabe
Chemosphere, 286, Pt 2, 131715, 131715, 2021年08月05日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The biological impacts of residual pharmaceuticals in the complex wastewater effluents have not been fully understood. Here, we investigated changes in the transcriptomic responses of hepatobrastoma (HepG2) cells exposed to a single or partially combined three common non-steroidal anti-inflammatory drugs (NSAIDs); ketoprofen (KPF), mefenamic acid (MFA) and diclofenac (DCF), in domestic wastewater effluents. After 48 h sub-lethal exposure to single compounds, the DNA microarray analysis identified 57-184 differently expressed genes (DEGs). The hierarchical clustering analysis and GO enrichment of the DEGs showed that gene expression profiles of the NSAIDs were distinct from each other although they are classified into the same therapeutic category. Four maker genes (i.e., EGR1, AQP3, SQSTM1, and NAG1) were further selected from the common DEGs, and their expressions were quantified by qPCR assay in a dose-dependent manner (ranging from μg/L to mg/L). The results revealed the insignificant induction of the marker genes at 1 μg/L of KPF, MFA, and DCF, suggesting negligible biological impacts of the NSAIDs on gene expression (early cellular responses) of HepG2 at typical concentration levels found in the actual wastewater effluents. Based on the quantitative expression analysis of the selected marker genes, the present study indicated that the presence of wastewater effluent matrix may mitigate the potentially adverse cellular impacts of the NSAIDs.

Bioelectrochemical anoxic ammonium nitrogen removal by an MFC driven single chamber microbial electrolysis cell.
N'Dah Joel Koffi, Satoshi Okabe
Chemosphere, 274, 129715, 129715, 2021年07月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Nitrogen removal from wastewater is an indispensable but highly energy-demanding process, and thus more energy-saving treatment processes are required. Here, we investigated the performance of bioelectrochemical ammonium nitrogen (NH4+-N) removal from real domestic wastewater without energy-intensive aeration by a single chamber microbial electrolysis cell (MEC) that was electrically powered by a double chamber microbial fuel cell (MFC). Anoxic NH4+-N oxidation and total nitrogen (TN) removal rates were determined at various applied voltages (0-1.2 V), provided by the MFC. The MEC achieved a NH4+-N oxidation rate of 151 ± 42 g NH4+-N m-3 d-1 and TN removal rate of 95 ± 42 g-TN m-3 d-1 without aeration at the applied voltage of 0.8 V (the anode potential Eanode = +0.633 ± 0.218 V vs. SHE). These removal rates were much higher than the previously reported values and conventional biological nitrogen removal processes. Open and closed-circuit MEC batch experiments confirmed that anoxic NH4+-N oxidation was an electrochemically mediated biological process (that is, an anode acted as an electron acceptor) and denitrification occurred simultaneously without NO2- and NO3- accumulation. Moreover, ex-situ15N tracer experiment and microbial community analysis revealed that anammox and heterotrophic denitrification mainly contributed to the TN removal. Thus, the bioelectrochemical anodic NH4+-N oxidation was coupled with anammox and denitrification in this MFC-assisted MEC system. Taken together, our MFC-driven single chamber MEC could be a high rate energy-saving nitrogen removal process without external carbon and energy input and high energy-demanding aeration.

Maintenance power requirements of anammox bacteria "Candidatus Brocadia sinica" and "Candidatus Scalindua sp."
Satoshi Okabe, Atsushi Kamigaito, Kanae Kobayashi
The ISME journal, 15, 12, 3566, 3575, 2021年06月18日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Little is known about the cell physiology of anammox bacteria growing at extremely low growth rates. Here, "Candidatus Brocadia sinica" and "Candidatus Scalindua sp." were grown in continuous anaerobic membrane bioreactors (MBRs) with complete biomass retention to determine maintenance energy (i.e., power) requirements at near-zero growth rates. After prolonged retentostat cultivations, the specific growth rates (μ) of "Ca. B. sinica" and "Ca. Scalindua sp." decreased to 0.000023 h-1 (doubling time of 1255 days) and 0.000157 h-1 (184 days), respectively. Under these near-zero growth conditions, substrate was continuously utilized to meet maintenance energy demands (me) of 6.7 ± 0.7 and 4.3 ± 0.7 kJ mole of biomass-C-1 h-1 for "Ca. B. sinica" and "Ca. Scalindua sp.", which accorded with the theoretically predicted values of all anaerobic microorganisms (9.7 and 4.4 kJ mole of biomass-C-1 h-1at 37 °C and 28 °C, respectively). These me values correspond to 13.4 × 10-15 and 8.6 × 10-15 watts cell-1 for "Ca. B. sinica" and "Ca. Scalindua sp.", which were five orders of magnitude higher than the basal power limit for natural settings (1.9 × 10-19 watts cells-1). Furthermore, the minimum substrate concentrations required for growth (Smin) were calculated to be 3.69 ± 0.21 and 0.09 ± 0.05 μM NO2- for "Ca. B. sinica" and "Ca. Scalindua sp.", respectively. These results match the evidence that "Ca. Scalindua sp." with lower maintenance power requirement and Smin are better adapted to energy-limited natural environments than "Ca. B. sinica", suggesting the importance of these parameters on ecological niche differentiation in natural environments.

Glycogen metabolism of the anammox bacterium "Candidatus Brocadia sinica".
Satoshi Okabe, Amrini Amalia Shafdar, Kanae Kobayashi, Lei Zhang, Mamoru Oshiki
The ISME journal, 15, 5, 1287, 1301, 2021年05月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in "Ca. Brocadia sinica" growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)-1 while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that "Ca. B. sinica" synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.

Viral Interference as a Factor of False-Negative in the Infectious Adenovirus Detection Using Integrated Cell Culture-PCR with a BGM Cell Line.
Daisuke Sano, Ryosuke Watanabe, Wakana Oishi, Mohan Amarasiri, Masaaki Kitajima, Satoshi Okabe
Food and environmental virology, 13, 1, 84, 92, 2021年03月, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, This study investigated the influence of viral interference on the detection of enteric viruses using the integrated cell culture (ICC)-PCR with a BGM cell line. It was possible to detect 102 plaque-forming units (PFU)/flask of enterovirus 71 (EV71) in spite of the presence of 104 PFU/flask of adenovirus 40 (AdV40). Meanwhile, 104 PFU/flask of AdV40 was not detected in the presence of 102 PFU/flask of EV71. This inhibition of AdV40 detection using ICC-PCR was attributable to the growth of EV71, because the addition of a growth inhibitor of EV71 (rupintrivir) neutralized the detection inhibition of AdV40. The growth inhibition of AdV40 under co-infection with EV71 is probably caused by the immune responses of EV71-infected cells. AdV is frequently used as a fecal contamination indicator of environmental water, but this study demonstrated that false-negative detection of infectious AdV using ICC-PCR could be caused by the co-existence of infectious EV in a water sample. The addition of rupintrivir could prevent false-negative detection of AdV using ICC-PCR. This study, therefore, emphasizes the importance of confirming the presence of multiple enteric viruses in a sample derived from environmental water prior to the application of ICC-PCR because the viral interference phenomenon may lead to the false-negative detection of target viruses.

Simple assay for colorimetric quantification of unamplified bacterial 16S rRNA in activated sludge using gold nanoprobes.
Meri Nakajima, Reiko Hirano, Satoshi Okabe, Hisashi Satoh
Chemosphere, 263, 128331, 128331, 2021年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Domestic and industrial wastewater treatment systems are vital in the protection of natural ecosystems and human health. Identification of microbial communities in the systems is essential to stable treatment performance. However, the current tools of microbial community analysis are labor intensive and time consuming, and require expensive equipment. Therefore, we developed a simple assay for colorimetric quantification of bacterial 16S rRNA extracted from environmental samples. The assay is based on RNA extraction with commercial kits, mixing the unamplified RNA sample with Au-nanoprobes and NaCl, and analyzing the absorbance spectra. Our experimental results confirmed that the assay format was valid. By analyzing the synthesized DNA, we optimized the operational parameters affecting the assay. We achieved adequate capture DNA density by setting the capture DNA probe concentration at 10 μM during the functionalization step. The required incubation time after NaCl addition was 30 min. The binding site of the target had negligible effect on DNA detection. Under the optimized condition, a calibration curve was created using 16S rRNA extracted from activated sludge. The curve was linear above 5.0 × 107 copies/μL of bacterial 16S rRNA concentration, and the limit of detection was 1.17 × 108 copies/μL. Using the calibration curve, the bacterial 16S rRNA concentration in activated sludge samples could be quantified with deviations between 48% and 208% against those determined by RT-qPCR. The findings of our study introduce an innovative tool for the quantification of 16S rRNA concentration as the activity of key bacteria in wastewater treatment processes, achieving stable treatment performance.

High voltage generation from wastewater by microbial fuel cells equipped with a newly designed low voltage booster multiplier (LVBM).
N'Dah Joel Koffi, Satoshi Okabe
Scientific reports, 10, 1, 18985, 18985, 2020年11月04日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Although microbial fuel cells (MFCs) can produce renewable energy from wastewater, the generated power is practically unusable. To extract usable power from an MFC fed with wastewater, we newly developed a low voltage booster multiplier (LVBM), which is composed of a self-oscillating LVB and multistage voltage multiplier circuits (VMCs). The low output MFC voltage (ca. 0.4 V) was successfully boosted up to 99 ± 2 V, which was the highest voltage that has been ever reported, without voltage reversal by connecting an LVB with 20-stage VMCs. Moreover, the boosted voltage (81 ± 1 V) was stably maintained for > 40 h even after disconnecting the LVBM from the MFC. The energy harvesting efficiency of LVBM was > 80% when an LVB with 4-stage VMCs was charged to 9.3 V. These results clearly suggest that the proposed LVBM system is an efficient and self-starting energy harvester and storage for low-power generating MFCs.

Influence of δ18 O of water on measurements of δ18 O of nitrite and nitrate.
Kanae Kobayashi, Keitaro Fukushima, Yuji Onishi, Kazuya Nishina, Akiko Makabe, Midori Yano, Scott D Wankel, Keisuke Koba, Satoshi Okabe
Rapid communications in mass spectrometry : RCM, 35, 2, e8979, 2020年10月14日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, RATIONALE: Oxygen isotope ratio measurements of NO2- and NO3- by the azide method and denitrifier method are sensitive to the δ18 O of sample water. However, the influence of δ18 OH2O on those measurements has not been quantitatively evaluated and documented so far. Therefore, we investigated the influence of δ18 OH2O of sample on the δ18 O analysis of NO2- and NO3- . METHODS: We prepared NO2- and NO3- standards (with known δ18 ONO2- and δ18 ONO3- values) dissolved in waters having different δ18 OH2O values (δ18 OH2O = -12.6, 25.9, 56.7, and 110.1‰). Nitrite and nitrate were converted to N2 O using the azide method and the denitrifier method, respectively. The isotope ratios of the generated N2 O were measured with a Sercon PT-GC/IRMS system. The measured δ18 O values of the produced N2 O were plotted against known δ18 ONO2- and δ18 ONO3- values to evaluate the influence of exchange of an oxygen atom with H2 O during the conversion of NO2- to N2 O and NO3- to N2 O, respectively. RESULTS: The degree of O isotope exchange was 10.8 ± 0.3% in the azide method and 5.5 ± 1.0 % in the denitrifier method, indicating that the azide method is more susceptible to artifacts arising from differences in the δ18 OH2O value of water than the denitrifier method. Thus, the intercept of the standard calibration curve must be corrected to account for differences in δ18 OH2O . Abiotic NO2 - H2 O equilibrium isotope effect experiments yielded a rate constant of (1.13 ± 007) × 10-2 (h-1 ) and an equilibrium isotope effect of 11.9 ± 0.1‰ under the condition of pH=7.5, 30°C, and 2.5% salinity. CONCLUSIONS: Oxygen isotope ratio measurements of NO2- by the azide method are highly sensitive to δ18 OH2O as a result of significant oxygen isotope exchange between NO2- and H2 O. Therefore, to obtain the most accurate measurements the same δ18 OH2O value as that of the sample must be used to make the NO2- and NO3- standards.

The Effect of GD1a Ganglioside-Expressing Bacterial Strains on Murine Norovirus Infectivity.
Yifan Zhu, Hiroki Kawai, Satoshi Hashiba, Mohan Amarasiri, Masaaki Kitajima, Satoshi Okabe, Daisuke Sano
Molecules (Basel, Switzerland), 25, 18, 2020年09月07日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, we investigated the impact of GD1a-expressing bacterial strains on the infectivity of murine norovirus (MNV). Eligible bacterial strains were screened from a sewage sample using flow cytometry, and their genetic sequences of 16S rRNA were determined. The enzyme-linked immunosorbent assay (ELISA) was employed to analyze the binding between bacteria and MNV particles, and the plaque assay was used to assess the effects of GD1a-positive and negative strains on MNV infectivity. The result from ELISA shows that MNV particles are able to bind to both GD1a-positive and negative bacterial strains, but the binding to the GD1a-positive strain is more significant. The infectivity assay result further shows that the MNV infectious titer declined with an increasing concentration of GD1a-positive bacteria. The addition of anti-GD1a antibody in the infectivity assay led to the recovery of the MNV infectious titer, further confirming that the binding between MNV particles and bacterial GD1a ganglioside compromises MNV infectivity. Our findings highlight the role indigenous bacteria may play in the lifecycle of waterborne enteric viruses as well as the potential of exploiting them for virus transmission intervention and water safety improvement.

N2O production using native nos-deficient denitrifying bacterial strains screened by a genome mining approach
Mamoru Oshiki, Miho Ishimaru, Masashi Hatamoto, Takashi Yamaguchi, Nobuo Araki, Satoshi Okabe
Bioresource Technology Reports, 11, 2020年09月
研究論文（学術雑誌）, Although N2O is a notorious greenhouse and ozone-depleting gas, N2O is a strong oxidant and can be an energy source by combusting with methane gas. The present study screened N2O-producing denitrifying bacteria by genome mining approach, and the activity and efficiency of NO2− reduction to N2O were examined. Presence and absence of the genes encoding nitrite reductase (nir), nitric oxide reductase (nor), and nitrous oxide reductase (nos) on the 2750 prokaryotic genomes were examined to screen nos-deficient denitrifying bacteria capable of NO2− reduction to N2O. The 131 nos-deficient denitrifying bacterial genomes were screened, and Cupriavidus necator H16 and Propionibacterium freudenreichii showed high activity and efficiency of 15NO2− reduction to 15N2O. Nitrogen removal performance and N2O conversion efficiencies were further investigated in a membrane bioreactor, and C. necator H16 showed 0.18 kg-N m−3 d−1 of NO2− removal rate with 75–95% of N2O conversion efficiencies.

Simultaneous removal of nitrate and heavy metals in a continuous flow nitrate-dependent ferrous iron oxidation (NDFO) bioreactor.
Kazuki Jokai, Tomomi Nakamura, Satoshi Okabe, Satoshi Ishii
Chemosphere, 262, 127838, 127838, 2020年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Nitrogen and heavy metals can co-occur in various industrial wastewaters such as coke-oven wastewater. Removal of these contaminants is important, but cost-efficient removal technology is limited. In this study, we examined the usefulness of nitrate-dependent ferrous iron oxidation (NDFO) for the simultaneous removal of nitrate and heavy metals (iron and zinc), by using an NDFO strain Pseudogulbenkiania sp. NH8B. Based on the batch culture assays, nitrate, Fe, and Zn were successfully removed from a basal medium as well as coke-oven wastewater containing 5 mM nitrate, 10 mM Fe(II), and 10 mg/L Zn. Zinc in the water was most likely co-precipitated with Fe(III) oxides produced during the NDFO reaction. Simultaneous removal of nitrate, Fe, and Zn was also achieved in a continuous-flow reactor fed with a basal medium containing 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L Zn. However, when the reactor is fed with coke-oven wastewater supplemented with 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L ZnCl2, the reactor performance significantly decreased, most likely due to the inhibition of bacterial growth by thiocyanate or organic contaminants present in the coke-oven wastewater. Use of mixed culture of NDFO bacteria and thiocyanate/organic-degrading denitrifiers should help improve the reactor performance.

Improvement of Electrochemical Conditions for Detecting Redox Reaction of K₃[Fe(CN)₆] toward the Application in Norovirus Aptasensor
Seiya HIRANO, Junki SAITO, Tomoki YUKAWA, Daisuke SANO, Akihiro OKAMOTO, Satoshi OKABE, Masaaki KITAJIMA
Electrochemistry, 88, 3, 205, 209, The Electrochemical Society of Japan, 2020年05月05日, ［査読有り］
研究論文（学術雑誌）

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain.
Syun-Suke Kadoya, Syun-Ichi Urayama, Takuro Nunoura, Miho Hirai, Yoshihiro Takaki, Masaaki Kitajima, Toyoko Nakagomi, Osamu Nakagomi, Satoshi Okabe, Osamu Nishimura, Daisuke Sano
Journal of virology, 94, 10, 2020年05月04日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, RNA viruses form a dynamic distribution of mutant swarms (termed "quasispecies") due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect, in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability, and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and nonsynonymous substitutions on rotavirus genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate a stronger bottleneck effect can create more sequence spaces for minor sequences.IMPORTANCE In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated.

Simple and reliable enumeration of Escherichia coli concentrations in wastewater samples by measuring β-d-glucuronidase (GUS) activities via a microplate reader.
Hisashi Satoh, Kai Kikuchi, Yutaka Katayose, Shu Tsuda, Reiko Hirano, Yuga Hirakata, Masaaki Kitajima, Satoshi Ishii, Mamoru Oshiki, Masashi Hatamoto, Masahiro Takahashi, Satoshi Okabe
The Science of the total environment, 715, 136928, 136928, 2020年05月01日, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Monitoring of Escherichia coli concentrations at wastewater treatment plants (WWTPs) is important to ensure process performance and protect public health. However, conventional E. coli enumeration methods are complicated and time- and labor-consuming. Here, we report a novel simple and reliable method based on β-d-glucuronidase (GUS) activity assay to enumerate E. coli concentrations in wastewater (WW) samples. An aliquot (20 μL) of the medium with fluorogenic enzyme substrate for E. coli and 180 μL of a WW sample were added to one well of a 96-well microplate. The microplate was placed in a microplate reader at 37 °C. To this end, the fluorescence intensity of a fluorogenic enzyme substrate for E. coli was measured every 10 min over 3 h to determine GUS activity. The linear increase in the fluorescence intensity representing the GUS activities showed a positive correlation with E. coli concentrations in wastewater samples. However, the correlation equations were specific to WWTPs, which could be due to the difference in the E. coli population structures among WWTPs. We observed that the wastewater matrix is not a limitation to measure the GUS activity, and a WWTP-specific correlation equation can be used as a calibration curve to estimate the E. coli concentrations in the samples collected from that site. A comparison of the results with those of culture-dependent Colilert method proved that the current method is simple and useful for the enumeration of E. coli concentrations in wastewater samples reliably.

Ecological niche differentiation among anammox bacteria.
Lei Zhang, Satoshi Okabe
Water research, 171, 115468, 115468, 2020年03月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Anaerobic ammonium oxidizing (anammox) bacteria can directly convert ammonium and nitrite to nitrogen gas anaerobically and were responsible for a substantial part of the fixed nitrogen loss and re-oxidation of nitrite to nitrate in freshwater and marine ecosystems. Although a wide variety of studies have been undertaken to investigate the abundance and biodiversity of anammox bacteria so far, ecological niche differentiation of anammox bacteria is still not fully understood. To assess their growth behavior and consequent population dynamics at a given environment, the Monod model is often used. Here, we summarize the Monod kinetic parameters such as the maximum specific growth rate (μmax) and the half-saturation constant for nitrite (KNO2-) and ammonium (KNH4+) of five known candidatus genera of anammox bacteria. We also discuss potential pivotal environmental factors and metabolic flexibility that influence the community compositions of anammox bacteria. Particularly biodiversity of the genus "Scalindua" might have been largely underestimated. Several anammox bacteria have been successfully enriched from various source of biomass. We reevaluate their enrichment methods and culture medium compositions to gain a clue of niche differentiation of anammox bacteria. Furthermore, we formulate the current issues that must be addressed. Overall this review re-emphasizes the importance of enrichment cultures (preferably pure cultures), physiological characterization and direct microbial competition studies using enrichment cultures in laboratories.

Required Chlorination Doses to Fulfill the Credit Value for Disinfection of Enteric Viruses in Water: A Critical Review.
Andri Taruna Rachmadi, Masaaki Kitajima, Tsuyoshi Kato, Hiroyuki Kato, Satoshi Okabe, Daisuke Sano
Environmental science & technology, 54, 4, 2068, 2077, 2020年02月18日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A credit value of virus inactivation has been assigned to the disinfection step in international and domestic guidelines for wastewater reclamation and reuse. To fulfill the credit value for water disinfection, water engineers need to apply an appropriate disinfection strength, expressed as a CT value (mg × min/L), which is a product of disinfectant concentration and contact time, against enteric viruses in wastewater. In the present study, we extracted published experimental data on enteric virus inactivation using free chlorine and monochloramine and applied the Tobit analysis and simple linear regression analysis to calculate the range of CT values (mg × min/L) needed for 4-log10 inactivation. Data were selected from peer-reviewed papers containing kinetics data of virus infectivity and chlorine residual in water. Coxsackie B virus and echovirus require higher CT values (lower susceptibility) for 4-log10 inactivation than adenovirus and a human norovirus surrogate (murine norovirus) with free chlorine. On the other hand, adenovirus has lower susceptibility to monochloramine compared to murine norovirus, coxsackievirus, and echovirus. The factors that influence the required CT value are virus type, pH, water temperature, and water matrix. This systematic review demonstrates that enteroviruses and adenovirus are appropriate representative enteric viruses to evaluate water disinfection using free chlorine and monochloramine, respectively.

Transcriptomic analysis of HepG2 cells exposed to fractionated wastewater effluents suggested humic substances as potential inducer of whole effluent toxicity.
Hiroe Hara-Yamamura, Toshikazu Fukushima, Lea Chua Tan, Satoshi Okabe
Chemosphere, 240, 124894, 124894, 2020年02月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, We performed a transcriptome-based bioassay (TSB assay) using human hepatoma HepG2 cells to evaluate the potential toxicity of whole wastewater effluents from two membrane bioreactors (MBRs) and a conventional activated sludge process (AS). The biologically active agent(s) in the wastewater effluents were characterized based on expression of the marker genes (i.e., CYP1A1, AKR1B10, GCLM and GPX2) selected by DNA microarray analysis, after the wastewater effluent samples were concentrated by a reverse osmosis (RO) membrane and further fractionated by various manipulations. The qPCR assay of marker genes demonstrated that the induction of CYP1A1 and GPX2 was mitigated after passing through C18 and chelate columns. In addition, clear induction of CYP1A1 was observed in the smallest size fraction with 1 k Da or smaller organic molecules in all the tested effluents. These results together with the water quality data of the fractionated samples suggested that responsible constituents for potentially adverse and abnormal transcriptomic responses in HepG2 could have hydrophobic nature and act with metal-dissolved organic matter (DOM) complexes in 1 k Da or smaller size fraction. Although DOM is known to play two contradictory roles as a protector and an inducer of toxicants, our present study indicated the DOM in wastewater effluent, particularly humic substances with acidic nature, functioned as a toxicity inducer of residual chemicals in the effluents. This study provided a new insight into the nature of "toxic unknowns" in the wastewater effluents, which should be monitored whole through the reclamation process and prioritized for removal.

Mixture toxicity of the combinations of silver nanoparticles and environmental pollutants.
Toshikazu Fukushima, Wongta Jintana, Satoshi Okabe
Environmental science and pollution research international, 27, 6, 6326, 6337, 2020年02月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Although toxicity of silver nanoparticles (AgNPs) has been well studied, the mixture toxicity of the combination of AgNPs and other environmental pollutants is still largely unknown. Here, we investigated the mixture toxicity of the combinations of AgNPs and common environmental pollutants such as arsenic (As), cadmium (Cd), and chromium (Cr) on human hepatoma cell line (HepG2) at noncytotoxic concentrations based on analyses of cytotoxicity, genotoxicity, reactive oxygen species (ROS) generation, and modes of cell death. In addition, DNA microarray analysis was performed to understand the cellular responses at a molecular level. AgNPs-As and AgNPs-Cd combinations exhibited synergistic effect on cytotoxicity while AgNPs-Cr showed additive effect. The AgNPs-Cd combination caused much stronger synergism than AgNPs-As combination. Based on cellular and molecular level analyses, the synergistic effect could be explained by overproduction of reactive oxygen species (ROS), which induced DNA damage and consequently apoptotic cell death. On the other hand, the additive effect caused by AgNPs-Cr could be attributed to reduction of the mixture toxicity by precipitation of Cr ions. Taken together, our results clearly demonstrated that the mixture toxicity of AgNPs with As, Cd, or Cr at noncytotoxic concentrations had different toxicity effects. Particularly, toxicogenomic approach using DNA microarray was useful to assess the mechanisms of the mixture toxicity.

Candidal Prosthetic Hip Infection in a Patient with Previous Candidal Sepsis - A Case Report.
Yoichi Murata, Satoshi Okabe, Shogo Tamagawa, Hideo Ohnishi, Akinori Sakai
Journal of UOEH, 42, 4, 327, 330, 2020年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A 60 year-old woman with hip dysplasia battled with duodenal cancer that was complicated with Candida tropicalis sepsis. Two years later, the patient underwent a total hip arthroplasty (THA). She complained of a persisting low-grade fever and local heat on the THA scar. Arthrocentesis of the hip was performed and the Candida tropicalis was detected. Debridement and polyethylene liner/modular head exchange were performed 28 days after the primary THA. Fluconazole was administrated for one year. The patient reported no symptoms five years later. It was found that periprosthetic infection could be prevented by implant preservation surgery.

Cell Density-dependent Anammox Activity of Candidatus Brocadia sinica Regulated by N-acyl Homoserine Lactone-mediated Quorum Sensing.
Mamoru Oshiki, Haruna Hiraizumi, Hisashi Satoh, Satoshi Okabe
Microbes and environments, 35, 4, 2020年, ［査読有り］, ［最終著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, The activity of anaerobic ammonia-oxidizing (anammox) bacteria is considered to depend on cell density; however, this has not yet been confirmed due to the fastidious nature of anammox bacteria (e.g., slow growth, oxygen sensitivity, and rigid aggregate formation). In the present study, the cell density-dependent occurrence of anammox activity (14-15N2 gas production rate) was investigated using planktonic enrichment cultures of Candidatus Brocadia sinica. This activity was detectable when the density of cells was higher than 107‍ ‍cells‍ ‍mL-1 and became stronger with increases in cell density. At the cell densities, the transcription of the BROSI_A1042 and BROSI_A3652 genes, which are potentially involved in the biosynthesis and reception of N-acyl homoserine lactone (AHL), was detectable in Brocadia sinica cells. The presence of AHL molecules in the MBR culture of B. sinica was confirmed by an AHL reporter assay and gas chromatography mass spectrometry analysis. The exogenous addition of the MBR culture extract and AHL molecules (a cocktail of C6, C8, C10, and C12-homoserine lactones) increased the specific 14-15N2 production rate of B. sinica. These results suggest that the specific anammox activity of B. sinica is regulated by AHL-mediated quorum sensing.

Dual nitrogen and oxygen isotope effects of anaerobic ammonium oxidation (anammox) - Nitrogen and oxygen isotope effects of anammox
Kobayashi Kanae, Makabe Akiko, Yano Midori, Oshiki Mamoru, Kindaichi Tomonori, Casciotti Karen L, Okabe Satoshi
The ISME Journal, 13, 10, 2426, 2436, Springer Science and Business Media LLC, 2019年10月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects (15ε and 18ε) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO3- producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including "Ca. Scalindua japonica", a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH4+ oxidation to N2 (15εNH4→N2) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO2- oxidation to NO3- (15εNO2→NO3 = -45.3‰ to -30.1‰). In contrast, 15εNO2→N2 (NO2- reduction to N2) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO2- oxidation (18ENO2→NO3) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO2- reoxidation (N recycling) in various natural environments.

Fecal Source Tracking in A Wastewater Treatment and Reclamation System Using Multiple Waterborne Gastroenteritis Viruses.
Zheng Ji, Xiaochang C Wang, Limei Xu, Chongmiao Zhang, Cheng Rong, Andri Taruna Rachmadi, Mohan Amarasiri, Satoshi Okabe, Naoyuki Funamizu, Daisuke Sano
Pathogens (Basel, Switzerland), 8, 4, 170, 170, {MDPI} {AG}, 2019年09月30日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Gastroenteritis viruses in wastewater reclamation systems can pose a major threat to public health. In this study, multiple gastroenteritis viruses were detected from wastewater to estimate the viral contamination sources in a wastewater treatment and reclamation system installed in a suburb of Xi'an city, China. Reverse transcription plus nested or semi-nested PCR, followed by sequencing and phylogenetic analysis, were used for detection and genotyping of noroviruses and rotaviruses. As a result, 91.7% (22/24) of raw sewage samples, 70.8% (17/24) of the wastewater samples treated by anaerobic/anoxic/oxic (A2O) process and 62.5% (15/24) of lake water samples were positive for at least one of target gastroenteritis viruses while all samples collected from membrane bioreactor effluent after free chlorine disinfection were negative. Sequence analyses of the PCR products revealed that epidemiologically minor strains of norovirus GI (GI/14) and GII (GII/13) were frequently detected in the system. Considering virus concentration in the disinfected MBR effluent which is used as the source of lake water is below the detection limit, these results indicate that artificial lake may be contaminated from sources other than the wastewater reclamation system, which may include aerosols, and there is a possible norovirus infection risk by exposure through reclaimed water usage and by onshore winds transporting aerosols containing norovirus.

Membrane fouling potentials of an exoelectrogenic fouling-causing bacterium cultured with different external electron acceptors
So Ishizaki, Rimana Islam Papry, Hiroshi Miyake, Yoko Narita, Satoshi Okabe
Frontiers in Microbiology, 9, 3284, 3284, 2018年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Integrated microbial fuel cell (MFC) and membrane bioreactor (MBR) systems are a promising cost-effective and energy-saving technology for wastewater treatment. Membrane fouling is still an important issue of such integrated systems in which aeration (oxygen) is replaced with anode electrodes (anodic respiration). Here, we investigated the effect of culture conditions on the membrane fouling potential of fouling-causing bacteria (FCB). In the present study, Klebsiella quasipneumoniae strain S05, which is an exoelectrogenic FCB isolated from a MBR treating municipal wastewater, was cultured with different external electron acceptors (oxygen, nitrate, and solid-state anode electrode). As results, the fouling potential of S05 was lowest when cultured with anode electrode and highest without any external electron acceptor (p < 0.05, respectively). The composition of soluble microbial products (SMP) and extracellular polymeric substances (EPS) was also dependent on the type of electron acceptor. Protein and biopolymer contents in SMP were highly correlated with the fouling potential (R2 = 0.73 and 0.81, respectively). Both the fouling potential and yield of protein and biopolymer production were significantly mitigated by supplying electron acceptors sufficiently regardless of its types. Taken together, the aeration of MBR could be replaced with solid-state anode electrodes without enhancement of membrane fouling, and the anode electrodes must be placed sufficiently to prevent the dead spaces in the integrated reactor.

Aggregation ability of three phylogenetically distant anammox bacterial species.
Muhammad Ali, Dario Rangel Shaw, Lei Zhang, Mohamed Fauzi Haroon, Yuko Narita, Abdul-Hamid Emwas, Pascal E Saikaly, Satoshi Okabe
Water research, 143, 10, 18, 2018年10月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Anaerobic ammonium-oxidizing (anammox) bacteria are well known for their aggregation ability. However, very little is known about cell surface physicochemical properties of anammox bacteria and thus their aggregation abilities have not been quantitatively evaluated yet. Here, we investigated the aggregation abilities of three different anammox bacterial species: "Candidatus Brocadia sinica", "Ca. Jettenia caeni" and "Ca. Brocadia sapporoensis". Planktonic free-living enrichment cultures of these three anammox species were harvested from the membrane bioreactors (MBRs). The physicochemical properties (e.g., contact angle, zeta potential, and surface thermodynamics) were analyzed for these anammox bacterial species and used in the extended DLVO theory to understand the force-distance relationship. In addition, their extracellular polymeric substances (EPSs) were characterized by X-ray photoelectron spectroscopy and nuclear magnetic resonance. The results revealed that the "Ca. B. sinica" cells have the most hydrophobic surface and less hydrophilic functional groups in EPS than other anammox strains, suggesting better aggregation capability. Furthermore, aggregate formation and anammox bacterial populations were monitored when planktonic free-living cells were cultured in up-flow column reactors under the same conditions. Rapid development of microbial aggregates was observed with the anammox bacterial population shifts to a dominance of "Ca. B. sinica" in all three reactors. The dominance of "Ca. B. sinica" could be explained by its better aggregation ability and the superior growth kinetic properties (higher growth rate and affinity to nitrite). The superior aggregation ability of "Ca. B. sinica" indicates significant advantages (efficient and rapid start-up of anammox reactors due to better biomass retention as granules and consequently stable performance) in wastewater treatment application.

Anammox biomass carrying efficiency of polyethylene non-woven sheets as a carrier material.
Sunja Cho, Minki Jung, Dongjin Ju, Young-Hee Lee, Kuk Cho, Satoshi Okabe
Environmental technology, 39, 19, 2503, 2510, 2018年10月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, To access the effects of the surface modification and fabric structure of polyethylene (PE) non-woven fabric sheets on retaining the attachment efficiency of anammox biomass, three different non-woven sheets were prepared and inserted in an anammox reactor. The hydrophobic surface modification with 10% KMnO4 and gelatin did not improve the attachment efficiency of the anammox biomass on the surface of the PE non-woven fibers. Densely packed PE-755 having the highest specific surface area to volume ratio (SA/V) (755) retained 221.4 mg biomass per unit sheet, whereas PE-181 having the lowest SA/V (181) retained only 66.4 mg biomass per unit. Accordingly, the volumetric anammox activity of non-woven sheet PE-755 was the highest among the three PE non-woven sheets because of the strong positive relationship between the specific anammox activity and biomass amount (R = 0.835, P < .01). The specific surface area to volume ratio (cm2 cm-3) as well as the bulk density should be considered as important parameters for the selection of non-woven biocarriers for anammox biomass.

Free-Chlorine Disinfection as a Selection Pressure on Norovirus.
Andri Taruna Rachmadi, Masaaki Kitajima, Kozo Watanabe, Sakiko Yaegashi, Joeselle Serrana, Arata Nakamura, Toyoko Nakagomi, Osamu Nakagomi, Kazuhiko Katayama, Satoshi Okabe, Daisuke Sano
Applied and environmental microbiology, 84, 13, 2018年07月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure.IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.

Complete Genome Sequence of Klebsiella quasipneumoniae Strain S05, a Fouling-Causing Bacterium Isolated from a Membrane Bioreactor.
Masaaki Kitajima, So Ishizaki, Jeonghwan Jang, Satoshi Ishii, Satoshi Okabe
Genome announcements, 6, 23, e00471-18, American Society for Microbiology, 2018年06月07日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, We report here the complete genome sequence of Klebsiella quasipneumoniae strain S05, a bacterium capable of producing membrane fouling-causing soluble substances and capable of respiring on oxygen, nitrate, and an anodic electrode. The genomic information of strain S05 should help predict metabolic pathways associated with these unique biological properties of this bacterium.

3-[Bis(pyridin-2-ylmethyl)amino]-5-(4-carboxyphenyl)-BODIPY as Ratiometric Fluorescent Sensor for Cu2.
Akira Hafuka, Hisashi Satoh, Koji Yamada, Masahiro Takahashi, Satoshi Okabe
Materials (Basel, Switzerland), 11, 5, MDPI, 2018年05月16日, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, We developed an asymmetric fluorescent sensor 1 for Cu2+, based on 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), by introducing 4-carboxyphenyl and bis(pyridin-2-ylmethyl)amine groups at the 5- and 3-positions, respectively, of the BODIPY core. We then investigated the photophysical and cation-sensing properties of the sensor. BODIPY 1 showed large absorption and fluorescence spectral shifts on binding to Cu2+. The fluorescence peak at 580 nm red-shifted to 620 nm. The binding stoichiometry of BODIPY 1 and Cu2+ was 1:3. The ratio of the fluorescence intensity at 620 nm to that at 580 nm (F620/F580) increased with increasing concentration of Cu2+ (3⁻10 equiv); this enabled ratiometric determination of Cu2+. Although BODIPY 1 showed good selectivity for Cu2+, there was an interfering effect of Fe3+. BODIPY 1 could be used for the naked-eye detection of Cu2+ in a water-containing sample.

Experimental Evidence for in Situ Nitric Oxide Production in Anaerobic Ammonia-Oxidizing Bacterial Granules.
Rathnayake M L D Rathnayake, Mamoru Oshiki, Satoshi Ishii, Takahiro Segawa, Hisashi Satoh, Satoshi Okabe
Environmental science & technology, 52, 10, 5744, 5752, AMER CHEMICAL SOC, 2018年05月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Although nitric oxide (NO) emissions from anaerobic ammonium oxidation (anammox)-based processes were reported previously, the NO production pathways are poorly understood. Here, we investigated the NO production pathways in anammox granules in detail by combining 15N-stable isotope tracer experiments with various inhibitors, microsensor measurements, and transcriptome analysis for key genes of NO2- reduction. NO was emitted from the anammox granules, which account for 0.07% of the N2 emission. 15N-stable isotope-tracer experiments indicated that most of the N2 was produced by anammox bacteria, whereas NO was produced from NO2- reduction by anammox and denitrifying bacteria. The NO emission rate was highest at pH 8.0 and accelerated by increasing NH4+ and NO2- concentrations in the culture media. The microsensor analyses showed the in situ NO production rate was highest in the outer layer of the anammox granule where anammox activity was also highest. The detected in situ NO concentrations of up to 2.7 μM were significantly above physiological thresholds known to affect a wide range of microorganisms present in wastewater. Hence, NO likely plays pivotal roles in the microbial interactions in anammox granules, which needs to be further investigated.

Selection of cellular genetic markers for the detection of infectious poliovirus
D. Sano, M. Tazawa, M. Inaba, S. Kadoya, R. Watanabe, T. Miura, M. Kitajima, S. Okabe
Journal of Applied Microbiology, 124, 4, 1001, 1007, Blackwell Publishing Ltd, 2018年04月01日, ［査読有り］, ［最終著者］
英語, 研究論文（学術雑誌）, Aims: Cellular responses of an established cell line from human intestinal epithelial cells (INT-407 cells) against poliovirus (PV) infections were investigated in order to find cellular genetic markers for infectious PV detection. Methods and Results: Gene expression profile of INT-407 cells was analysed by DNA microarray technique when cells were infected with poliovirus 1 (PV1) (sabin) at multiplicity of infection of 10−3 and incubated for 12 h. Poliovirus infection significantly altered the gene expressions of two ion channels, KCNJ4 and SCN7A. The expression profile of KCNJ4 gene was further investigated by real-time RT-qPCR, and it was found that KCNJ4 gene was significantly regulated at 24 h postinfection of PV1. Conclusions: KCNJ4 gene, coding a potassium channel protein, is proposed as a cellular genetic marker for infectious PV detection. Significance and Impact of the Study: This is the first study to show the availability of cellular responses to detect infectious PV. The selection of cellular genetic markers for infectious viruses using DNA microarray and RT-qPCR can be applicable for the other enteric viruses.

Reverse transcription-quantitative PCR assays for genotype-specific detection of human noroviruses in clinical and environmental samples.
Mohan Amarasiri, Masaaki Kitajima, Akiho Miyamura, Ricardo Santos, Silvia Monteiro, Takayuki Miura, Shinobu Kazama, Satoshi Okabe, Daisuke Sano
International journal of hygiene and environmental health, 221, 3, 578, 585, Elsevier {BV}, 2018年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Circulation of human noroviruses in water environments is suspected to be genotype-dependent, but the established primer and probe sets for noroviruses are usually genogroup-specific, which do not allow to compare the genotype-specific properties, such as persistence in water environments and resistance to disinfectants. In this study, quantitative PCR assays were designed for genotype-specific quantification of four epidemiologically important genotypes, GII.3, GII.4, GII.6, and GII.17. Developed assays were tested using norovirus positive stool samples which were previously confirmed to present target genotypes of this study. The results were 100% in accordance with the previous results. Effect of the co-existence of multiple genotypes in a sample on the target genotype quantification was evaluated using composite stool samples and wastewater samples containing multiple genotypes and the presence of non-target genotypes didn't affect the quantification of target genotype. Sensitivity and specificity was 100% for all four assays developed in this study with no cross-reactions between genotypes demonstrating the validity of our assays and their applicability to clinical and environmental samples.

Disinfection as a Selection Pressure on RNA Virus Evolution.
Andri Taruna Rachmadi, Masaaki Kitajima, Kozo Watanabe, Satoshi Okabe, Daisuke Sano
Environmental science & technology, 52, 5, 2434, 2435, 2018年03月06日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Electricity generation potential of poultry droppings wastewater in microbial fuel cell using rice husk charcoal electrodes
Godwin E. Oyiwona, James Chukwuma Ogbonna, Chukwudi Anyanwu, Satoshi Okabe
Bioresources and Bioprocessing, 5, 13, 1, 6, 2018年03月, ［査読有り］, ［最終著者, 責任著者］
© 2018, The Author(s). Background: Poultry droppings from poultry farms and rice husks obtained from rice milling process are generally considered as wastes and discarded in Nigeria. Although many studies have shown that microbial fuel cells (MFCs) can generate electricity from organic wastes, little or no study have examined MFCs for generating electricity from poultry droppings and rice husk as electrode material. Findings: Laboratory-scale double-chamber MFCs were inoculated with concentrations of poultry droppings wastewater and supplied with rice husk charcoal as anode and cathode electrodes for electricity generation. Power outputs and dissolved organic carbon (DOC) removal efficiencies were compared between MFCs using rice husk charcoal (RHCE) as electrode and those using carbon cloth (CCE) as electrodes. The RHCE-MFC 2 containing 477 mg L−1dissolved organic carbon produced a volumetric power density of 6.9 ± 3.1 W m−3which was higher than the control and the CCE-MFCs by a factor of 2 and achieved at DOC removal efficiencies of 40 ± 1.2%. Conclusions: The results suggest that poultry droppings wastewater is a feasible feedstock for generating electricity in MFCs. The findings also suggest that rice husk charcoal is a potentially useful electrode material in MFCs. [Figure not available: see fulltext.].

Insights into the roles of anammox bacteria in post-treatment of anaerobically-treated sewage　　　　　　　　　　　　　　　
岡部 聡
Critical Reviews in Environmental Science and Technology, 48, 6, 655, 684, 2018年03月, ［査読有り］
英語

菌叢解析再訪 : 変遷と展望 (特集 環境分野に於ける菌叢解析 : 分子生物学が汚泥に光を当てる)
伊藤 司, 金田一 智則, 押木 守, 岡部 聡
EICA : journal of EICA : 環境システム計測制御学会誌, 22, 4, 21, 26, 環境システム計測制御学会, 2018年, ［査読有り］, ［最終著者, 責任著者］
日本語

High-Throughput flaA Short Variable Region Sequencing to Assess Campylobacter Diversity in Fecal Samples From Birds.
Qian Zhang, Gabriel A Al-Ghalith, Mayumi Kobayashi, Takahiro Segawa, Mitsuto Maeda, Satoshi Okabe, Dan Knights, Satoshi Ishii
Frontiers in microbiology, 9, 2201, 2201, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Current approach to identify sources of human pathogens is largely dependent on the cultivation and isolation of target bacteria. For rapid pathogen source identification, culture-independent strain typing method is necessary. In this study, we designed new primer set that broadly covers flaA short variable region (SVR) of various Campylobacter species, and applied the flaA SVR sequencing method to examine the diversity of Campylobacter spp. in geese fecal samples (n = 16) with and without bacteria cultivation. Twenty-three Campylobacter strains isolated from the 16 geese fecal samples were grouped similarly by conventional flaA restriction fragment length polymorphism (RFLP) method and by the flaA SVR sequencing method, but higher discriminant power was observed in the flaA SVR sequencing approach. For culture-independent flaA SVR sequencing analysis, we developed and optimized the sequence data analysis pipeline to identify as many genotypes as possible, while minimizing the detection of genotypes generated by sequencing errors. By using this pipeline, 51,629 high-quality flaA sequence reads were clustered into 16 operational taxonomic units (=genotypes) by using 98% sequence similarity and >50 sequence duplicates. Almost all flaA genotypes obtained by culture-dependent method were also identified by culture-independent flaA SVR MiSeq sequencing method. In addition, more flaA genotypes were identified probably due to high throughput nature of the MiSeq sequencing. These results suggest that the flaA SVR sequencing could be used to analyze the diversity of Campylobacter spp. without bacteria isolation. This method is promising to rapidly identify potential sources of Campylobacter pathogens.

Rapid cultivation of free-living planktonic anammox cells.
Lei Zhang, Satoshi Okabe
Water research, 127, 204, 210, PERGAMON-ELSEVIER SCIENCE LTD, 2017年12月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Despite that anaerobic ammonium oxidizing (anammox) bacteria are key players in the global nitrogen cycle, no pure cultures are still available. Planktonic cell culture with high purity is, therefore, essential for physiological and biochemical studies of anammox bacteria. However, development of such planktonic cell cultures requires an enormous amount of time and effort. Here we developed a novel rapid method for cultivating free-living planktonic anammox cells. First, anammox granules were physically dispersed, immobilized in 6% polyvinyl alcohol-4% sodium alginate (PVA-SA) gel beads, and then pre-cultured in an up-flow column reactor. Anammox bacteria grew rapidly as loosely flocculated micro-clusters in the gel beads. After 18 days of pre-cultivation, mature gel beads were harvested, physically dispersed by vortex and inoculated into a membrane bioreactor (MBR). The MBR was then continuously operated at a low nitrogen loading rate (<0.9 kg-TN m-3 d-1). After 17 days of operation, active free-living planktonic anammox cells with purity >95% were successfully developed in the MBR. Total culture time (gel beads and MBR) to accomplish free-living planktonic anammox cells was only 35 days, which was significantly shorter than the previous reports. This new cultivation technique could greatly facilitate various microbial, physiological and biochemical studies of anammox bacteria.

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents.
Toshikazu Fukushima, Hiroe Hara-Yamamura, Koji Nakashima, Lea Chua Tan, Satoshi Okabe
Chemosphere, 188, 312, 319, PERGAMON-ELSEVIER SCIENCE LTD, 2017年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management.

Microbial competition among anammox bacteria in nitrite-limited bioreactors.
Lei Zhang, Yuko Narita, Lin Gao, Muhammad Ali, Mamoru Oshiki, Satoshi Ishii, Satoshi Okabe
Water research, 125, 249, 258, PERGAMON-ELSEVIER SCIENCE LTD, 2017年11月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Phylogenetically diverse anammox bacteria have been detected in most of anoxic natural and engineered ecosystems and thus regarded as key players in the global nitrogen cycle. However, ecological niche differentiation of anammox bacteria remains unresolved despite its ecological and practical importance. In this study, the microbial competitions for a common substrate (nitrite) among three anammox species (i.e. "Candidatus Brocadia sinica", "Candidatus Jettenia caeni" and "Candidatus Kuenenia stuttgartiensis") were systematically investigated in nitrite-limited gel-immobilized column reactors (GICR) and membrane bioreactors (MBRs) under different nitrogen loading rates (NLRs). 16 S rRNA gene-based population dynamics revealed that "Ca. J. caeni" could proliferate only at low NLRs, whereas "Ca. B. sinica" outcompeted other two species at higher NLRs in both types of reactors. Furthermore, FISH analysis revealed that "Ca. J. caeni" was mainly present as spherical microclusters at the inner part (low NO2- environment), whereas "Ca. B. sinica" was present throughout the gel beads and granules. This spatial distribution supports the outcomes of the competition experiments. However, the successful competition of "Ca. J. caeni" at low NLR could not be explained with the Monod model probably due to inaccuracy of kinetic parameters such as half saturation constant (Ks) for nitrite and a difference in the maintenance rate (m). In addition, the growth of "Ca. K. stuttgartiensis" could not be observed in any experimental conditions, suggesting possible unknown factor(s) is missing. Taken together, NLR was one of factors determining ecological niche differentiation of "Ca. B. sinica" and "Ca. J. caeni".

Target virus log10 reduction values determined for two reclaimed wastewater irrigation scenarios in Japan based on tolerable annual disease burden.
Toshihiro Ito, Masaaki Kitajima, Tsuyoshi Kato, Satoshi Ishii, Takahiro Segawa, Satoshi Okabe, Daisuke Sano
Water research, 125, 438, 448, PERGAMON-ELSEVIER SCIENCE LTD, 2017年11月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Multiple-barriers are widely employed for managing microbial risks in water reuse, in which different types of wastewater treatment units (biological treatment, disinfection, etc.) and health protection measures (use of personal protective gear, vegetable washing, etc.) are combined to achieve a performance target value of log10 reduction (LR) of viruses. The LR virus target value needs to be calculated based on the data obtained from monitoring the viruses of concern and the water reuse scheme in the context of the countries/regions where water reuse is implemented. In this study, we calculated the virus LR target values under two exposure scenarios for reclaimed wastewater irrigation in Japan, using the concentrations of indigenous viruses in untreated wastewater and a defined tolerable annual disease burden (10-4 or 10-6 disability-adjusted life years per person per year (DALYpppy)). Three genogroups of norovirus (norovirus genogroup I (NoV GI), geogroup II (NoV GII), and genogroup IV (NoV GIV)) in untreated wastewater were quantified as model viruses using reverse transcription-microfluidic quantitative PCR, and only NoV GII was present in quantifiable concentration. The probabilistic distribution of NoV GII concentration in untreated wastewater was then estimated from its concentration dataset, and used to calculate the LR target values of NoV GII for wastewater treatment. When an accidental ingestion of reclaimed wastewater by Japanese farmers was assumed, the NoV GII LR target values corresponding to the tolerable annual disease burden of 10-6 DALYpppy were 3.2, 4.4, and 5.7 at 95, 99, and 99.9%tile, respectively. These percentile values, defined as "reliability," represent the cumulative probability of NoV GII concentration distribution in untreated wastewater below the corresponding tolerable annual disease burden after wastewater reclamation. An approximate 1-log10 difference of LR target values was observed between 10-4 and 10-6 DALYpppy. The LR target values were influenced mostly by the change in the logarithmic standard deviation (SD) values of NoV GII concentration in untreated wastewater and the reliability values, which highlights the importance of accurately determining the probabilistic distribution of reference virus concentrations in source water for water reuse.

Enhancement of organic matter degradation and methane gas production of anaerobic granular sludge by degasification of dissolved hydrogen gas.
Hisashi Satoh, Wasala M K R T W Bandara, Manabu Sasakawa, Yoshihito Nakahara, Masahiro Takahashi, Satoshi Okabe
Bioresource technology, 244, Pt 1, 768, 775, ELSEVIER SCI LTD, 2017年11月, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, A hollow fiber degassing membrane (DM) was applied to enhance organic matter degradation and methane gas production of anaerobic granular sludge process by reducing the dissolved hydrogen gas (D-H2) concentration in the liquid phase. DM was installed in the bench-scale anaerobic granular sludge reactors and D-H2 was removed through DM using a vacuum pump. Degasification improved the organic matter degradation efficiency to 79% while the efficiency was 62% without degasification at 12,000mgL-1 of the influent T-COD concentration. Measurement of D-H2 concentrations in the liquid phase confirmed that D-H2 was removed by degasification. Furthermore, the effect of acetate concentrations on the organic matter degradation efficiency was investigated. At acetate concentrations above 3gL-1, organic matter degradation deteriorated. Degasification enhanced the propionate and acetate degradation. These results suggest that degasification reduced D-H2 concentration and volatile fatty acids concentrations, prevented pH drop, and subsequent enhanced organic matter degradation.

Determination of Cadmium in Brown Rice Samples by Fluorescence Spectroscopy Using a Fluoroionophore after Purification of Cadmium by Anion Exchange Resin.
Akira Hafuka, Akiyoshi Takitani, Hiroko Suzuki, Takuya Iwabuchi, Masahiro Takahashi, Satoshi Okabe, Hisashi Satoh
Sensors (Basel, Switzerland), 17, 10, MDPI AG, 2017年10月09日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Simple analytical methods are needed for determining the cadmium (Cd) content of brown rice samples. In the present study, we developed a new analytical procedure consisting of the digestion of rice using HCl, Cd purification using anion exchange resin, and then determining the Cd content using fluorescence spectroscopy. Digestion with 0.1 M HCl for 10 min at room temperature was sufficient to extract Cd from the ground rice samples. The Cd in the extract was successfully purified in preference to other metals using Dowex 1X8 chloride form resin. Low concentrations of Cd in the eluate could be determined using fluorescence spectroscopy with a fluoroionophore. Overall, the actual limit of quantification value for the Cd content in rice was about 0.1 mg-Cd/kg-rice, which was sufficiently low compared with the regulatory value (0.4 mg-Cd/kg-rice) given by the Codex Alimentarius Commission. We analyzed authentic brown rice samples using our new analytical procedure and the results agreed well with those determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Since the fluoroionophore recognized Zn2+ and Hg2+ as well as Cd2+, a sample containing high concentration of Zn2+ or Hg2+ might cause a false positive result.

Genetic diversity of marine anaerobic ammonium-oxidizing bacteria as revealed by genomic and proteomic analyses of 'Candidatus Scalindua japonica'.
Mamoru Oshiki, Keisuke Mizuto, Zen-Ichiro Kimura, Tomonori Kindaichi, Hisashi Satoh, Satoshi Okabe
Environmental microbiology reports, 9, 5, 550, 561, WILEY, 2017年10月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Anaerobic ammonium-oxidizing (anammox) bacteria affiliated with the genus 'Candidatus Scalindua' are responsible for significant nitrogen loss in oceans, and thus their ecophysiology is of great interest. Here, we enriched a marine anammox bacterium, 'Ca. S. japonica' from a Hiroshima bay sediment in Japan, and comparative genomic and proteomic analyses of 'Ca. S. japonica' were conducted. Sequence of the 4.81-Mb genome containing 4019 coding regions of genes (CDSs) composed of 47 contigs was determined. In the proteome, 1762 out of 4019 CDSs in the 'Ca. S. japonica' genome were detected. Based on the genomic and proteomic data, the core anammox process and carbon fixation of 'Ca. S. japonica' were further investigated. Additionally, the present study provides the first detailed insights into the genetic background responsible for iron acquisition and menaquinone biosynthesis in anammox bacterial cells. Comparative analysis of the 'Ca. Scalindua' genomes revealed that the 1502 genes found in the 'Ca. S. japonica' genome were not present in the 'Ca. S. profunda' and 'Ca. S. rubra' genomes, showing a high genomic diversity. This result may reflect a high phylogenetic diversity of the genus 'Ca. Scalindua'.

Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium 'Candidatus Brocadia sapporoensis'.
Yuko Narita, Lei Zhang, Zen-Ichiro Kimura, Muhammad Ali, Takao Fujii, Satoshi Okabe
Systematic and applied microbiology, 40, 7, 448, 457, ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2017年10月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, We successfully enriched a novel anaerobic ammonium-oxidizing (anammox) bacterium affiliated with the genus 'Candidatus Brocadia' with high purity (>90%) in a membrane bioreactor (MBR). The enriched bacterium was distantly related to the hitherto characterized 'Ca. Brocadia fulgida' and 'Ca. Brocadia sinica' with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The bacterium exhibited the common structural features of anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of anammox activity was 20-45°C with a maximum activity at 37°C. The maximum specific growth rate (μmax) was 0.0082h-1 at 37°C, corresponding to a doubling time of 3.5 days. The half-saturation constant (KS) for nitrite was 5±2.5μM. The anammox activity was inhibited by nitrite (IC50=11.6mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched anammox bacterium shared nearly half of genes with 'Ca. Brocadia sinica' and 'Ca. Brocadia fulgida'. The enriched bacterium showed all known physiological characteristics of anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name 'Ca. Brocadia sapporoensis' sp. nov.

Bacteriophage removal efficiency as a validation and operational monitoring tool for virus reduction in wastewater reclamation: Review.
Mohan Amarasiri, Masaaki Kitajima, Thanh H Nguyen, Satoshi Okabe, Daisuke Sano
Water research, 121, 258, 269, Elsevier {BV}, 2017年09月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The multiple-barrier concept is widely employed in international and domestic guidelines for wastewater reclamation and reuse for microbiological risk management, in which a wastewater reclamation system is designed to achieve guideline values of the performance target of microbe reduction. Enteric viruses are one of the pathogens for which the target reduction values are stipulated in guidelines, but frequent monitoring to validate human virus removal efficacy is challenging in a daily operation due to the cumbersome procedures for virus quantification in wastewater. Bacteriophages have been the first choice surrogate for this task, because of the well-characterized nature of strains and the presence of established protocols for quantification. Here, we performed a meta-analysis to calculate the average log10 reduction values (LRVs) of somatic coliphages, F-specific phages, MS2 coliphage and T4 phage by membrane bioreactor, activated sludge, constructed wetlands, pond systems, microfiltration and ultrafiltration. The calculated LRVs of bacteriophages were then compared with reported human enteric virus LRVs. MS2 coliphage LRVs in MBR processes were shown to be lower than those of norovirus GII and enterovirus, suggesting it as a possible validation and operational monitoring tool. The other bacteriophages provided higher LRVs compared to human viruses. The data sets on LRVs of human viruses and bacteriophages are scarce except for MBR and conventional activated sludge processes, which highlights the necessity of investigating LRVs of human viruses and bacteriophages in multiple treatment unit processes.

Membrane fouling induced by AHL-mediated soluble microbial product (SMP) formation by fouling-causing bacteria co-cultured with fouling-enhancing bacteria.
So Ishizaki, Ryoichi Sugiyama, Satoshi Okabe
Scientific reports, 7, 1, 8482, 8482, NATURE PUBLISHING GROUP, 2017年08月16日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Membrane fouling still remains a major obstacle for wider applications of membrane bioreactor (MBR), which is mainly caused by soluble microbial products (SMP). Identification of key bacteria responsible for SMP production is essential for mitigation of membrane fouling. Here, we investigated the effect of microbial interaction on membrane fouling. We measured the membrane fouling potentials of 13 bacterial strains isolated from a pilot-scale MBR treating domestic wastewater when they were cultivated as single-culture and co-culture. We found that fouling-causing bacteria (FCB) displayed much higher fouling potential when co-cultured even with non-FCB and mixed population (activated sludge). In particular, the fouling potential of strain S26, one of FCB, increased 26.8 times when cultivated with strain S22 (fouling-enhancing bacteria, FEB). The secretion of N-octanoyl-L-homoserine lactone (C8-HSL) was increased by co-cultivating S22 and S26 as compared with cultivating as single culture, which stimulated the production of fouling-causing SMP by S26 and consequently resulted in severe membrane fouling. This result suggests that AHL-mediated quorum-sensing (QS) regulatory system was involved in secretion of fouling-causing SMP.

Maximum specific growth rate of anammox bacteria revisited.
Lei Zhang, Yuko Narita, Lin Gao, Muhammad Ali, Mamoru Oshiki, Satoshi Okabe
Water research, 116, 296, 303, PERGAMON-ELSEVIER SCIENCE LTD, 2017年06月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Anammox bacteria have long been considered to be slow-growing bacteria. However, it has recently been reported that they could grow much faster than previously thought when they were cultivated in a membrane bioreactor (MBR) with a step-wise decrease in the solid retention time (SRT). Here, we reevaluated the maximum specific growth rates (μmax) of three phylogenetically distant anammox bacterial species (i.e. "Ca. Brocadia sinica", "Ca. Jettenia caeni" and "Ca. Scalindua sp.") by directly measuring 16S rRNA gene copy numbers using newly developed quantitative polymerase chain reaction (qPCR) assays. When free-living planktonic "Ca. B. sinica" and "Ca. J. caeni" cells were immobilized in polyvinyl alcohol (PVA) and sodium alginate (SA) gel beads and cultivated in an up-flow column reactor with high substrate loading rates at 37 °C, the μmax were determined to be 0.33 ± 0.02 d-1 and 0.18 d-1 (corresponding doubling time of 2.1 day and 3.9 day) from the exponential increases in 16S rRNA genes copy numbers, respectively. These values were faster than the fastest growth rates reported for these species so far. The cultivation of anammox bacteria in gel beads was achieved less than one month without special cultivation method and selection pressure, and the exponential increase in 16S rRNA gene numbers was directly measured by qPCR with high reproducibility; therefore, the resulting μmax values were considered accurate. Taken together, the fast growth is, therefore, considered to be an intrinsic kinetic property of anammox bacteria.

Formation of CuO nano-flowered surfaces via submerged photo-synthesis of crystallites and their antimicrobial activity.
Fumika Nishino, Melbert Jeem, Lihua Zhang, Kazumasa Okamoto, Satoshi Okabe, Seiichi Watanabe
Scientific reports, 7, 1, 1063, 1063, NATURE PUBLISHING GROUP, 2017年04月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We report the fabrication of flower-like CuO nanostructured surfaces via submerged photo-synthesis of crystallites (SPSC), which requires only UV illumination in neutral water. In this paper, we discuss the reaction mechanism of the photochemical formation of the SPSC-fabricated CuO nanostructures in detail based on surface microstructural analyses and a radiation-chemical consideration with additional gamma-ray irradiation. Since the SPSC method for surface nanostructural fabrication can work at low temperatures at atmospheric pressure without using harmful substances, it is a potential fabrication method for green nanotechnology applications. In this vein, the antibacterial activity of the nano-flowered CuO surfaces was tested against Gram-positive (Staphylococcus aureus) bacteria and Gram-negative (Escherichia coli K12) bacteria, and the results demonstrate that the nano-flowered CuO nanostructures act as an effective antimicrobial agent.

Generation of Small Colony Variants in Biofilms by Escherichia coli Harboring a Conjugative F Plasmid.
Yosuke Tashiro, Hiroaki Eida, Satoshi Ishii, Hiroyuki Futamata, Satoshi Okabe
Microbes and environments, 32, 1, 40, 46, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2017年03月31日, ［査読有り］, ［最終著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, A conjugative F plasmid induces mature biofilm formation by Escherichia coli by promoting F-pili-mediated cell-cell interactions and increasing the expression of biofilm-related genes. We herein demonstrated another function for the F plasmid in E. coli biofilms; it contributes to the emergence of genetic and phenotypic variations by spontaneous mutations. Small colony variants (SCVs) were more frequently generated in a continuous flow-cell biofilm than in the planktonic state of E. coli harboring the F plasmid. E. coli SCVs represented typical phenotypic changes such as slower growth, less biofilm formation, and greater resistance to aminoglycoside antibiotics than the parent strain. Genomic and complementation analyses indicated that the small colony phenotype was caused by the insertion of Tn1000, which was originally localized in the F plasmid, into the hemB gene. Furthermore, the Tn1000 insertion was removed from hemB in the revertant, which showed a normal colony phenotype. This study revealed that the F plasmid has the potential to increase genetic variations not only by horizontal gene transfer via F pili, but also by site-specific recombination within a single cell.

Oxidation of glucose by syntrophic association between Geobacter and hydrogenotrophic methanogens in microbial fuel cell.
Godwin E Oyiwona, James Ogbonna, Chukwudi Uzoma Anyanwu, So Ishizaki, Zen-Ichiro Kimura, Satoshi Okabe
Biotechnology letters, 39, 2, 253, 259, SPRINGER, 2017年02月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVE: To investigate a syntrophic interaction between Geobacter sulfurreducens and hydrogenotrophic methanogens in sludge-inoculated microbial fuel cell (MFC) systems running on glucose with an improved electron recovery at the anode. RESULTS: The presence of archaea in MFC reduces Coulombic efficiency (CE) due to their electron scavenging capability but, here, we demonstrate that a syntrophic interaction can occur between G. sulfurreducens and hydrogenotrophic methanogens via interspecies H2 transfer with improvement in CE and power density. The addition of the methanogenesis inhibitor, 2-bromoethanesulfonate (BES), resulted in the reduction in power density from 5.29 to 2 W/m3, and then gradually increased to the peak value of 5.5 W/m3 when BES addition was stopped. CONCLUSION: Reduction of H2 partial pressure by archaea is an efficient approach in improving power output in a glucose-fed MFC system using Geobacter sp. as an inoculum.

Improvement of a Phosphate Ion-selective Microsensor Using Bis(dibromophenylstannyl)methane as a Carrier.
Hisashi Satoh, Yuji Miyazaki, Shou Taniuchi, Mamoru Oshiki, Rathnayake M L D Rathnayake, Masahiro Takahashi, Satoshi Okabe
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 33, 7, 825, 830, JAPAN SOC ANALYTICAL CHEMISTRY, 2017年, ［査読有り］, ［最終著者］, ［国内誌］
英語, 研究論文（学術雑誌）, An ionophore-doped sensing membrane phosphate (PO4) microsensor based on bis(dibromophenylstannyl)methane (Bis microsensor) is described. The Bis microsensor showed a Nernstian response. The response of the Bis microsensor was log-linear down to a monohydrogen phosphate ion (HPO42-) concentration of 0.5 μM (corresponding to 1.0 μM of orthophosphate at pH 7.2), whereas the detection limit of PO4-microsensors based on trialkyl/aryltin chloride was 50 μM of HPO42-. The Bis microsensor showed excellent selectivity for HPO42- against nitrite, nitrate, chloride, bicarbonate and sulfate, as compared with PO4 microsensors based on trialkyl/aryltin chloride. Dissolved oxygen, which is known to interfere with the response of a previously developed cobalt-based potentiometric solid-state PO4 microsensor, had no effect on the response of the ionophore-doped sensing membrane-type microsensors described herein. Only OH- (i.e., pH) interfered with the ionophore-doped sensing membrane-type microsensors.

Draft Genome Sequence of the Anaerobic Ammonium-Oxidizing Bacterium "Candidatus Brocadia sp. 40".
Muhammad Ali, Mohamed Fauzi Haroon, Yuko Narita, Lei Zhang, Dario Rangel Shaw, Satoshi Okabe, Pascal E Saikaly
Genome announcements, 4, 6, e01377-16, American Society for Microbiology, 2016年12月08日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The anaerobic ammonium-oxidizing (anammox) bacterium "Candidatus Brocadia sp. 40" demonstrated the fastest growth rate compared to others in this taxon. Here, we report the 2.93-Mb draft genome sequence of this bacterium, which has 2,565 gene-coding regions, 41 tRNAs, and a single rrn operon.

Evaluation of virus reduction efficiency in wastewater treatment unit processes as a credit value in the multiple-barrier system for wastewater reclamation and reuse.
Toshihiro Ito, Tsuyoshi Kato, Makoto Hasegawa, Hiroyuki Katayama, Satoshi Ishii, Satoshi Okabe, Daisuke Sano
Journal of water and health, 14, 6, 879, 889, IWA PUBLISHING, 2016年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The virus reduction efficiency of each unit process is commonly determined based on the ratio of virus concentration in influent to that in effluent of a unit, but the virus concentration in wastewater has often fallen below the analytical quantification limit, which does not allow us to calculate the concentration ratio at each sampling event. In this study, left-censored datasets of norovirus (genogroup I and II), and adenovirus were used to calculate the virus reduction efficiency in unit processes of secondary biological treatment and chlorine disinfection. Virus concentration in influent, effluent from the secondary treatment, and chlorine-disinfected effluent of four municipal wastewater treatment plants were analyzed by a quantitative polymerase chain reaction (PCR) approach, and the probabilistic distributions of log reduction (LR) were estimated by a Bayesian estimation algorithm. The mean values of LR in the secondary treatment units ranged from 0.9 and 2.2, whereas those in the free chlorine disinfection units were from -0.1 and 0.5. The LR value in the secondary treatment was virus type and unit process dependent, which raised the importance for accumulating the data of virus LR values applicable to the multiple-barrier system, which is a global concept of microbial risk management in wastewater reclamation and reuse.

Source identification of nitrous oxide emission pathways from a single-stage nitritation-anammox granular reactor.
Muhammad Ali, Rathnayake M L D Rathnayake, Lei Zhang, Satoshi Ishii, Tomonori Kindaichi, Hisashi Satoh, Sakae Toyoda, Naohiro Yoshida, Satoshi Okabe
Water research, 102, 147, 157, PERGAMON-ELSEVIER SCIENCE LTD, 2016年10月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Nitrous oxide (N2O) production pathway in a signal-stage nitritation-anammox sequencing batch reactor (SBR) was investigated based on a multilateral approach including real-time N2O monitoring, N2O isotopic composition analysis, and in-situ analyses of spatial distribution of N2O production rate and microbial populations in granular biomass. N2O emission rate was high in the initial phase of the operation cycle and gradually decreased with decreasing NH4(+) concentration. The average emission of N2O was 0.98 ± 0.42% and 1.35 ± 0.72% of the incoming nitrogen load and removed nitrogen, respectively. The N2O isotopic composition analysis revealed that N2O was produced via NH2OH oxidation and NO2(-) reduction pathways equally, although there is an unknown influence from N2O reduction and/or anammox N2O production. However, the N2O isotopomer analysis could not discriminate the relative contribution of nitrifier denitrification and heterotrophic denitrification in the NO2(-) reduction pathway. Various in-situ techniques (e.g. microsensor measurements and FISH (fluorescent in-situ hybridization) analysis) were therefore applied to further identify N2O producers. Microsensor measurements revealed that approximately 70% of N2O was produced in the oxic surface zone, where nitrifiers were predominantly localized. Thus, NH2OH oxidation and NO2 reduction by nitrifiers (nitrifier-denitrification) could be responsible for the N2O production in the oxic zone. The rest of N2O (ca. 30%) was produced in the anammox bacteria-dominated anoxic zone, probably suggesting that NO2(-) reduction by coexisting putative heterotrophic denitrifiers and some other unknown pathway(s) including the possibility of anammox process account for the anaerobic N2O production. Further study is required to identify the anaerobic N2O production pathways. Our multilateral approach can be useful to quantitatively examine the relative contributions of N2O production pathways. Good understanding of the key N2O production pathways is essential to establish a strategy to mitigate N2O emission from biological nitrogen removal processes.

Bactericidal and virucidal mechanisms in the alkaline disinfection of compost using calcium lime and ash.
Nowaki Hijikata, Rui Tezuka, Shinobu Kazama, Masahiro Otaki, Ken Ushijima, Ryusei Ito, Satoshi Okabe, Daisuke Sano, Naoyuki Funamizu
Journal of environmental management, 181, 721, 727, ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2016年10月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In the present study, the bactericidal and virucidal mechanisms in the alkaline disinfection of compost with calcium lime and ash were investigated. Two indicator microorganisms, Escherichia coli and MS2 coliphage, were used as surrogates for enteric pathogens. The alkaline-treated compost with calcium oxide (CaO) or ash resulted primarily in damage to the outer membrane and enzyme activities of E. coli. The alkaline treatment of compost also led to the infectivity loss of the coliphage because of the partial capsid damage and RNA exteriorization due to a raised pH, which is proportional to the amount of alkaline agents added. These results indicate that the alkaline treatment of compost using calcium oxide and ash is effective and can contribute to the safe usage of compost from a mixing type dry toilet.

Impact of Anodic Respiration on Biopolymer Production and Consequent Membrane Fouling.
So Ishizaki, Kotaro Terada, Hiroshi Miyake, Satoshi Okabe
Environmental science & technology, 50, 17, 9515, 23, AMER CHEMICAL SOC, 2016年09月06日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Microbial fuel cells (MFCs) have recently been integrated with membrane bioreactors (MBRs) for wastewater treatment and energy recovery. However, the impact of integration of the two reactors on membrane fouling of MBR has not been reported yet. In this study, MFCs equipped with different external resistances (1-10 000 ohm) were operated, and membrane-fouling potentials of the MFC anode effluents were directly measured to study the impact of anodic respiration by exoelectrogens on membrane fouling. It was found that although the COD removal efficiency was comparable, the fouling potential was significantly reduced due to less production of biopolymer (a major foulant) in MFCs equipped with lower external resistance (i.e., with higher current generation) as compared with aerobic respiration. Furthermore, it was confirmed that Geobacter sulfurreducens strain PCA, a dominant exoelectrogen in anode biofilms of MFCs in this study, produced less biopolymer under anodic respiration condition than fumarate (anaerobic) respiration condition, resulting in lower membrane-fouling potential. Taken together, anodic respiration can mitigate membrane fouling of MBR due to lower biopolymer production, suggesting that development of an electrode-assisted MBR (e-MBR) without aeration is feasible.

Membrane fouling potentials and cellular properties of bacteria isolated from fouled membranes in a MBR treating municipal wastewater.
So Ishizaki, Toshikazu Fukushima, Satoshi Ishii, Satoshi Okabe
Water research, 100, 448, 457, PERGAMON-ELSEVIER SCIENCE LTD, 2016年09月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Membrane fouling remains a major challenge for wider application of membrane bioreactors (MBRs) to wastewater treatment. Membrane fouling is mainly caused by microorganisms and their excreted microbial products. For development of more effective control strategies, it is important to identify and characterize the microorganisms that are responsible for membrane fouling. In this study, 41 bacterial strains were isolated from fouled microfiltration membranes in a pilot-scale MBR treating real municipal wastewater, and their membrane fouling potentials were directly measured using bench-scale cross-flow membrane filtration systems (CFMFSs) and related to their cellular properties. It was found that the fouling potential was highly strain dependent, suggesting that bacterial identification at the strain level is essential to identify key fouling-causing bacteria (FCB). The FCB showed some common cellular properties. The most prominent feature of FCB was that they formed convex colonies having swollen podgy shape and smooth lustrous surfaces with high water, hydrophilic organic matter and carbohydrate content. However, general and rigid biofilm formation potential as determined by microtiter plates and cell surface properties (i.e., hydrophobicity and surface charge) did not correlate with the fouling potential in this study. These results suggest that the fouling potential should be directly evaluated under filtration conditions, and the colony water content could be a useful indicator to identify the FCB.

Ecology and physiology of anaerobic ammonium oxidizing bacteria.
Mamoru Oshiki, Hisashi Satoh, Satoshi Okabe
Environmental microbiology, 18, 9, 2784, 96, WILEY-BLACKWELL, 2016年09月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Anaerobic ammonium oxidation (anammox) is a microbial process in which NH4 (+) is oxidized to N2 gas with NO2 (-) as an electron acceptor. The anammox process is mediated by bacterial members affiliated with the phylum Planctomycetes, which are ubiquitously detected from anoxic natural and man-made ecosystems and a key player in the global nitrogen cycle. In the past two decades, phylogenetically different anammox bacteria have been recognized in natural and synthetic ecosystems (i.e. 'Candidatus Kuenenia', 'Candidatus Brocadia', 'Candidatus Jettenia', 'Candidatus Anammoxoglobus' and 'Candidatus Scalindua' genera), and the geographic distributions of these anammox bacteria indicate that they have genus-specific or species-specific habitats. Recently, we revealed the physiological characteristics of 'Ca. Jettenia' in addition to 'Ca. Kuenenia', 'Ca. Brocadia' and 'Ca. Scalindua', and, as a result, it is possible to compare the physiological characteristics of the anammox bacteria and discuss their niche partitioning. Therefore, we summarize the current knowledge of anammox bacterial ecology and physiology in this review to assess the potential ecological niche partitioning of anammox bacteria in natural and synthetic ecosystems.

Hydroxylamine-dependent anaerobic ammonium oxidation (anammox) by "Candidatus Brocadia sinica".
Mamoru Oshiki, Muhammad Ali, Kaori Shinyako-Hata, Hisashi Satoh, Satoshi Okabe
Environmental microbiology, 18, 9, 3133, 43, WILEY-BLACKWELL, 2016年09月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Although metabolic pathways and associated enzymes of anaerobic ammonium oxidation (anammox) of 'Ca. Kuenenia stuttgartiensis' have been studied, those of other anammox bacteria are still poorly understood. NO2- reduction to NO is considered to be the first step in the anammox metabolism of 'Ca. K. stuttgartiensis', however, 'Ca. Brocadia' lacks the genes that encode canonical NO-forming nitrite reductases (NirS or NirK) in its genome, which is different from 'Ca. K. stuttgartiensis'. Here, we studied the anammox metabolism of 'Ca. Brocadia sinica'. (15) N-tracer experiments demonstrated that 'Ca. B. sinica' cells could reduce NO2- to NH2 OH, instead of NO, with as yet unidentified nitrite reductase(s). Furthermore, N2 H4 synthesis, downstream reaction of NO2- reduction, was investigated using a purified 'Ca. B. sinica' hydrazine synthase (Hzs) and intact cells. Both the 'Ca. B. sinica' Hzs and cells utilized NH2 OH and NH4+, but not NO and NH4+, for N2 H4 synthesis and further oxidized N2 H4 to N2 gas. Taken together, the metabolic pathway of 'Ca. B. sinica' is NH2 OH-dependent and different from the one of 'Ca. K. stuttgartiensis', indicating metabolic diversity of anammox bacteria.

Denitrification and Nitrate-Dependent Fe(II) Oxidation in Various Pseudogulbenkiania Strains
Satoshi Ishii, Kazuki Joikai, Shigeto Otsuka, Keishi Senoo, Satoshi Okabe
MICROBES AND ENVIRONMENTS, 31, 3, 293, 298, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2016年09月, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）, Pseudogulbenkiania is a relatively recently characterized genus within the order Neisseriales, class Betaproteobacteria. This genus contains several strains that are capable of anaerobic, nitrate-dependent Fe(II) oxidation (NDFO), a geochemically important reaction for nitrogen and iron cycles. In the present study, we examined denitrification functional gene diversities within this genus, and clarified whether other Pseudogulbenkiania sp. strains perform denitrification and NDFO. Seventy strains were analyzed, including two type strains, a well-characterized NDFO strain, and 67 denitrifying strains isolated from various rice paddy fields and rice-soybean rotation fields in Japan. We also attempted to identify the genes responsible for NDFO by mutagenesis. Our comprehensive analysis showed that all Pseudogulbenkiania strains tested performed denitrification and NDFO; however, we were unable to obtain NDFO-deficient denitrifying mutants in our mutagenesis experiment. This result suggests that Fe(II) oxidation in these strains is not enzymatic, but is caused by reactive N-species that are formed during nitrate reduction. Based on the results of the comparative genome analysis among Pseudogulbenkiania sp. strains, we identified low sequence similarity within the nos gene as well as different gene arrangements within the nos gene cluster, suggesting that nos genes were horizontally transferred. Since Pseudogulbenkiania sp. strains have been isolated from various locations around the world, their denitrification and NDFO abilities may contribute significantly to nitrogen and iron biogeochemical cycles.

Genome Sequence of Enterobacter cloacae Strain SENG-6, a Bacterium Producing Histo-Blood Group Antigen-Like Substances That Can Bind with Human Noroviruses.
Satoshi Ishii, Mohan Amarasiri, Satoshi Hashiba, Peiyi Yang, Satoshi Okabe, Daisuke Sano
Genome announcements, 4, 4, e00893, 16, American Society for Microbiology, 2016年08月25日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Enterobacter sp. strain SENG-6, isolated from healthy human feces, produces histo-blood group antigen (HBGA)-like substances that can bind with human noroviruses. Based on the genome sequence analysis, strain SENG-6 belongs to the species Enterobacter cloacae The genome sequence of this strain should help identify genes associated with the production of HBGA-like substances.

Specific Single-Cell Isolation of Escherichia coli O157 from Environmental Water Samples by Using Flow Cytometry and Fluorescence-Activated Cell Sorting.
Shuji Ozawa, Satoshi Okabe, Satoshi Ishii
Foodborne pathogens and disease, 13, 8, 456, 61, MARY ANN LIEBERT, INC, 2016年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Contamination of food and water with pathogenic bacteria is of concern. Although culture-independent detection and quantification of pathogens is useful, isolation of pathogenic bacteria is still important when identifying the sources of pathogens. Here, we report the use of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) to specifically detect and isolate individual Escherichia coli O157:H7 cells from water samples. When present at >10 cells/mL water, target pathogen was specifically detected and isolated. The FACS-sorted E. coli O157:H7 population reflected the original population diversity, in contrast to the populations obtained by immunomagnetic separation. Relative abundance of multiple pathogenic strains is important when performing source-tracking studies; therefore, single-cell isolation with FCM-FACS can be a useful tool to obtain pathogenic bacteria for source tracking purpose.

下水再生処理におけるヒト腸管系ウイルスの目標除去効率の算定法
伊藤寿宏, 押木守, 小林直央, 加藤毅, 瀬川高弘, 幡本将史, 山口隆司, 原田秀樹, 北島正章, 岡部聡, 佐野大輔
土木学会論文集, 72, 7, 305, 313, 2016年07月, ［査読有り］

Bacterial histo-blood group antigens contributing to genotype-dependent removal of human noroviruses with a microfiltration membrane.
Mohan Amarasiri, Satoshi Hashiba, Takayuki Miura, Toyoko Nakagomi, Osamu Nakagomi, Satoshi Ishii, Satoshi Okabe, Daisuke Sano
Water research, 95, 383, 91, Elsevier {BV}, 2016年05月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We demonstrated the genotype-dependent removal of human norovirus particles with a microfiltration (MF) membrane in the presence of bacteria bearing histo-blood group antigens (HBGAs). Three genotypes (GII.3, GII.4, and GII.6) of norovirus-like particles (NoVLPs) were mixed with three bacterial strains (Enterobacter sp. SENG-6, Escherichia coli O86:K61:B7, and Staphylococcus epidermidis), respectively, and the mixture was filtered with an MF membrane having a nominal pore size of 0.45 μm. All NoVLP genotypes were rejected by the MF membrane in the presence of Enterobacter sp. SENG-6, which excreted HBGAs as extracellular polymeric substances (EPS). This MF membrane removal of NoVLPs was not significant when EPS was removed from cells of Enterobacter sp. SENG-6. GII.6 NoVLP was not rejected with the MF membrane in the presence of E. coli O86:K61:B7, but the removal of EPS of E. coli O86:K61:B7 increased the removal efficiency due to the interaction of NoVLPs with the exposed B-antigen in lipopolysaccharide (LPS) of E. coli O86:K61:B7. No MF membrane removal of all three genotypes was observed when S. epidermidis, an HBGA-negative strain, was mixed with NoVLPs. These results demonstrate that the location of HBGAs on bacterial cells is an important factor in determining the genotype-dependent removal efficiency of norovirus particles with the MF membrane. The presence of HBGAs in mixed liquor suspended solids from a membrane bioreactor (MBR) pilot plant was confirmed by immune-transmission electron microscopy, which implies that bacterial HBGAs can contribute to the genotype-dependent removal of human noroviruses with MBR using MF membrane.

水中病原体の許容感染リスクに基づいた水質衛生基準候補値の算出方法に関する提案
小林彩乃, 佐野大輔, 加藤毅, 伊藤寿宏, 宮村明帆, 三浦尚之, 石井聡, 岡部聡
土木学会論文集, 72, 3, 40, 49, 公益社団法人 土木学会, 2016年03月, ［査読有り］, ［最終著者］
日本語, 本研究では環境水の水質衛生基準候補値を算出する方法を提案する．まずモニタリング調査結果をもとに指標微生物に対する病原体の濃度比分布を推定し，さらに定量的微生物リスク評価の手法を用いることで仮定された水利用形態における病原体の河川水中許容濃度を算出した．この病原体の水中許容濃度は，指標微生物の病原体に対する濃度比分布と照合され，安全率を勘案した上で水質衛生基準値としての指標微生物濃度を算出した．個体差に関わる安全率を10とした場合，生活環境の保全に関する環境基準（河川）B類型における大腸菌の水質衛生基準値の候補として150 MPN/ 100mLを得た．本研究で提案した手法により，モニタリングデータの活用と仮定の検証を進めることで，「科学的根拠」に基づいた水質衛生基準値の設定が可能となる．

Estimation of concentration ratio of indicator to pathogen-related gene in environmental water based on left-censored data.
Tsuyoshi Kato, Ayano Kobayashi, Toshihiro Ito, Takayuki Miura, Satoshi Ishii, Satoshi Okabe, Daisuke Sano
Journal of water and health, 14, 1, 14, 25, IWA PUBLISHING, 2016年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A stochastic model for estimating the ratio between a fecal indicator and a pathogen based on left-censored data, which includes a substantially high number of non-detects, was constructed. River water samples were taken for 16 months at six points in a river watershed, and conventional fecal indicators (total coliforms and general Escherichia coli), genetic markers (Bacteroides spp.), and virulence genes (eaeA of enteropathogenic E. coli and ciaB of Campylobacter jejuni) were quantified. The quantification of general E. coli failed to predict the presence of the virulence gene from enteropathogenic E. coli, different from what happened with genetic markers (Total Bac and Human Bac). A Bayesian model that was adapted to left-censored data with a varying analytical quantification limit was applied to the quantitative data, and the posterior predictive distributions of the concentration ratio were predicted. When the sample size was 144, simulations conducted in this study suggested that 39 detects were enough to accurately estimate the distribution of the concentration ratio, when combined with a dataset with a positive rate higher than 99%. To evaluate the level of accuracy in the estimation, it is desirable to perform a simulation using an artificially generated left-censored dataset that has the identical number of non-detects as the actual data.

Effects of dissolved oxygen and pH on nitrous oxide production rates in autotrophic partial nitrification granules.
Rathnayake M L D Rathnayake, Mamoru Oshiki, Satoshi Ishii, Takahiro Segawa, Hisashi Satoh, Satoshi Okabe
Bioresource technology, 197, 15, 22, ELSEVIER SCI LTD, 2015年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The effects of dissolved oxygen (DO) and pH on nitrous oxide (N2O) production rates and pathways in autotrophic partial nitrification (PN) granules were investigated at the granular level. N2O was primarily produced by betaproteobacterial ammonia-oxidizing bacteria, mainly Nitrosomonas europaea, in the oxic surface layer (<200μm) of the autotrophic PN granules. N2O production increased with increasing bulk DO concentration owing to activation of the ammonia (i.e., hydroxylamine) oxidation in this layer. The highest N2O emissions were observed at pH 7.5, although the ammonia oxidation rate was unchanged between pH 6.5 and 8.5. Overall, the results of this study suggest that in situ analyses of PN granules are essential to gaining insight into N2O emission mechanisms in a granule.

Anammox-based technologies for nitrogen removal: Advances in process start-up and remaining issues.
Muhammad Ali, Satoshi Okabe
Chemosphere, 141, 144, 53, PERGAMON-ELSEVIER SCIENCE LTD, 2015年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Nitrogen removal from wastewater via anaerobic ammonium oxidation (anammox)-based process has been recognized as efficient, cost-effective and low energy alternative to the conventional nitrification and denitrification processes. To date, more than one hundred full-scale anammox plants have been installed and operated for treatment of NH4(+)-rich wastewater streams around the world, and the number is increasing rapidly. Since the discovery of anammox process, extensive researches have been done to develop various anammox-based technologies. However, there are still some challenges in practical application of anammox-based treatment process at full-scale, e.g., longer start-up period, limited application to mainstream municipal wastewater and poor effluent water quality. This paper aimed to summarize recent status of application of anammox process and researches on technological development for solving these remaining problems. In addition, an integrated system of anammox-based process and microbial fuel cell is proposed for sustainable and energy-positive wastewater treatment.

Bayesian modeling of virus removal efficiency in wastewater treatment processes
T. Ito, T. Kato, K. Takagishi, S. Okabe, D. Sano
WATER SCIENCE AND TECHNOLOGY, 72, 10, 1789, 1795, IWA PUBLISHING, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Left-censored datasets of virus density in wastewater samples make it difficult to evaluate the virus removal efficiency in wastewater treatment processes. In the present study, we modeled the probabilistic distribution of virus removal efficiency in a wastewater treatment process with a Bayesian approach, and investigated how many detect samples in influent and effluent are necessary for accurate estimation. One hundred left-censored data of virus density in wastewater (influent and effluent) were artificially generated based on assumed log-normal distributions and the posterior predictive distribution of virus density, and the log-ratio distribution were estimated. The estimation accuracy of distributions was quantified by Bhattacharyya coefficient. When it is assumed that the accurate estimation of posterior predictive distributions is possible when a 100% positive rate is obtained for 12 pairs of influent and effluent, 11 out of 144, 60 out of 324, and 201 out of 576 combinations of detect samples gave an accurate estimation at the significant level of 0.01 in a Kruskal-Wallis test when the total sample number was 12, 18, and 24, respectively. The combinations with the minimum number of detect samples were (12, 9), (16, 10), and (21, 8) when the total sample number was 12, 18, and 24, respectively.

Rapid and successful start-up of anammox process by immobilizing the minimal quantity of biomass in PVA-SA gel beads.
Muhammad Ali, Mamoru Oshiki, Lashitha Rathnayake, Satoshi Ishii, Hisashi Satoh, Satoshi Okabe
Water research, 79, 147, 57, PERGAMON-ELSEVIER SCIENCE LTD, 2015年08月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Rapid start-up of anaerobic ammonium oxidation (anammox) process in up-flow column reactors was successfully achieved by immobilizing minimal quantity of biomass in polyvinyl alcohol (PVA)-sodium alginate (SA) gel beads. The changes in the reactor performance (i.e., nitrogen removal rate; NRR) were monitored with time. The results demonstrate that the reactor containing the immobilized biomass concentration of 0.33 g-VSS L(-1) achieved NRR of 10.8 kg-N m(-3) d(-1) after 35-day operation, whereas the reactor containing the granular biomass of 2.5 g-VSS L(-1) could achieve only NRR of 3.5 kg-N m(-3) d(-1). This indicates that the gel immobilization method requires much lower seeding biomass for start-up of anammox reactor. To explain the better performance of the immobilized biomass, the biological and physicochemical properties of the immobilized biomass were characterized and compared with the naturally aggregated granular biomass. Effective diffusion coefficient (De) in the immobilized biomass was directly determined by microelectrodes and found to be three times higher than one in the granular biomass. High anammox activity (i.e., NH4(+) and NO2(-) consumption rates) was evenly detected throughout the gel beads by microelectrodes due to faster and deeper substrate transport. In contrast, anammox activity was localized in the outer layers of the granular biomass, indicating that the inner biomass could not contribute to the nitrogen removal. This difference was in good agreement with the spatial distribution of microbes analysed by fluorescence in situ hybridization (FISH). Based on these results, PVA-SA gel immobilization is an efficient strategy to initiate anammox reactors with minimal quantity of anammox biomass.

Impacts of hydrophilic colanic acid on bacterial attachment to microfiltration membranes and subsequent membrane biofouling.
Keitaro Yoshida, Yosuke Tashiro, Thithiwat May, Satoshi Okabe
Water research, 76, 33, 42, PERGAMON-ELSEVIER SCIENCE LTD, 2015年06月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, In order to examine the interactions between physicochemical properties of specific extracellular polymeric substances (EPS) and membrane biofouling, we investigated the impacts of hydrophilic colanic acid, as a model extracellular polysaccharide component, on initial bacterial attachment to different microfiltration (MF) membranes and membrane biofouling by using Escherichia coli strains producing different amounts of colanic acid. In a newly designed microtiter plate assay, the bacterial attachment by an E. coli strain RcsF(+), which produces massive amounts of colanic acid, decreased only to a hydrophobic membrane because the colanic acid made cell surfaces more hydrophilic, resulting in low cell attachment to hydrophobic membranes. The bench-scale cross-flow filtration tests followed by filtration resistance measurement revealed that RcsF(+) caused severe irreversible membrane fouling (i.e., pore-clogging), whereas less extracellular polysaccharide-producing strains caused moderate but reversible fouling to all membranes used in this study. Further cross-flow filtration tests indicated that colanic acid liberated in the bulk phase could rapidly penetrate pre-accumulated biomass layers (i.e., biofilms) and then directly clogged membrane pores. These results indicate that colanic acid, a hydrophilic extracellular polysaccharide, and possible polysaccharides with similar characteristics with colanic acid are considered as a major cause of severe irreversible membrane fouling (i.e., pore-clogging) regardless of biofilm formation (dynamic membrane).

Physiological characterization of anaerobic ammonium oxidizing bacterium 'Candidatus Jettenia caeni'.
Muhammad Ali, Mamoru Oshiki, Takanori Awata, Kazuo Isobe, Zenichiro Kimura, Hiroaki Yoshikawa, Daisuke Hira, Tomonori Kindaichi, Hisashi Satoh, Takao Fujii, Satoshi Okabe
Environmental microbiology, 17, 6, 2172, 89, WILEY-BLACKWELL, 2015年06月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, To date, six candidate genera of anaerobic ammonium-oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus 'Candidatus Jettenia'. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) bacterium with a maximum growth rate of 0.0020 h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name 'Ca. Jettenia caeni' sp. nov. for the bacterial clade of the planctomycete KSU-1.

Removal properties of human enteric viruses in a pilot-scale membrane bioreactor (MBR) process.
Takayuki Miura, Satoshi Okabe, Yoshihito Nakahara, Daisuke Sano
Water research, 75, 282, 91, PERGAMON-ELSEVIER SCIENCE LTD, 2015年05月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In order to evaluate removal properties of human enteric viruses from wastewater by a membrane bioreactor (MBR), influent, anoxic and oxic mixed liquor, and membrane effluent samples were collected in a pilot-scale anoxic-oxic MBR process for 16 months, and concentrations of enteroviruses, norovirus GII, and sapoviruses were determined by real-time PCR using murine norovirus as a process control. Mixed liquor samples were separated into liquid and solid phases by centrifugation, and viruses in the bulk solution and those associated with mixed liquor suspended solids (MLSS) were quantified. Enteroviruses, norovirus GII, and sapoviruses were detected in the influent throughout the sampling period (geometrical mean, 4.0, 3.1, and 4.4 log copies/mL, respectively). Enterovirus concentrations in the solid phase of mixed liquor were generally lower than those in the liquid phase, and the mean log reduction value between influent and anoxic mixed liquor was 0.40 log units. In contrast, norovirus GII and sapovirus concentrations in the solid phase were equal to or higher than those in the liquid phase, and higher log reduction values (1.3 and 1.1 log units, respectively) were observed between influent and anoxic mixed liquor. This suggested that enteroviruses were less associated with MLSS than norovirus GII and sapoviruses, resulting in lower enterovirus removal in the activated sludge process. Enteroviruses and norovirus GII were detected in the MBR effluent but sapoviruses were not in any effluent samples. When MLSS concentration was reduced to 50-60% of a normal operation level, passages of enteroviruses and norovirus GII through a PVDF microfiltration membrane were observed. Since rejection of viruses by the membrane was not related to trans-membrane pressure which was monitored as a parameter of membrane fouling, the results indicated that adsorption to MLSS plays an important role in virus removal by an MBR, and removal properties vary by viruses reflecting different adsorptive behavior to MLSS. Our observations suggested that sapoviruses are more associated with MLSS and removed more efficiently than enteroviruses and norovirus GII.

Draft Genome Sequence of an Anaerobic Ammonium-Oxidizing Bacterium, "Candidatus Brocadia sinica".
Mamoru Oshiki, Kaori Shinyako-Hata, Hisashi Satoh, Satoshi Okabe
Genome announcements, 3, 2, AMER SOC MICROBIOLOGY, 2015年04月16日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, A draft genome sequence of an anaerobic ammonium-oxidizing (anammox) bacterium, "Candidatus Brocadia sinica," was determined by pyrosequencing and by screening a fosmid library. A 4.07-Mb genome sequence comprising 3 contigs was assembled, in which 3,912 gene-coding regions, 47 tRNAs, and a single rrn operon were annotated.

Culture-independent evaluation of nonenveloped-virus infectivity reduced by free-chlorine disinfection.
Daisuke Sano, Takatomo Ohta, Arata Nakamura, Toyoko Nakagomi, Osamu Nakagomi, Satoshi Okabe
Applied and environmental microbiology, 81, 8, 2819, 26, AMER SOC MICROBIOLOGY, 2015年04月, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The inability of molecular detection methods to distinguish disinfected virions from infectious ones has hampered the assessment of infectivity for enteric viruses caused by disinfection practices. In the present study, the reduction of infectivity of murine norovirus S7-PP3 and mengovirus vMC0, surrogates of human noroviruses and enteroviruses, respectively, caused by free-chlorine treatment was characterized culture independently by detecting carbonyl groups on viral capsid protein. The amount of carbonyls on viral capsid protein was evaluated by the proportion of biotinylated virions trapped by avidin-immobilized gel (percent adsorbed). This culture-independent approach demonstrated that the percent adsorbed was significantly correlated with the logarithm of the infectious titer of tested viruses. Taken together with the results of previous reports, the result obtained in this study indicates that the amount of carbonyls on viral capsid protein of four important families of waterborne pathogenic viruses, Astroviridae, Reoviridae, Caliciviridae, and Picornaviridae, is increased in proportion to the received oxidative stress of free chlorine. There was also a significant correlation between the percent adsorbed and the logarithm of the ratio of genome copy number to PFU, which enables estimation of the infectious titer of a subject virus by measuring values of the total genome copy number and the percent adsorbed. The proposed method is applicable when the validation of a 4-log reduction of viruses, a requirement in U.S. EPA guidelines for virus removal from water, is needed along with clear evidence of the oxidation of virus particles with chlorine-based disinfectants.

Substituent Effects at the 5-Position of 3-[Bis(pyridine-2-ylmethyl)amino]-BODIPY Cation Sensor Used for Ratiometric Quantification of Cu2+
Akira Hafuka, Ryosuke Kando, Kohei Ohya, Koji Yamada, Satoshi Okabe, Hisashi Satoh
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 88, 3, 447, 454, CHEMICAL SOC JAPAN, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, In this paper, we investigated the effects of substitution at the 5-position of an asymmetric BODIPY cation sensor to tune its spectroscopic, photophysical, and cation-sensing properties. We introduced substituent groups with differing electron density at the 5-position of 3-[bis(pyridine-2-ylmethyl)amino]-BODIPY, which contains a cation recognition moiety at the 3-position of the BODIPY core, to develop four sensors which all exhibited distinctive ratiometric spectral changes in the presence of Cu2+. Aromatic substitution increased the Stokes shift. Substitution with the electron-withdrawing sulfonylphenyl group resulted in the highest fluorescence quantum yield, largest absorption coefficient, and largest spectral shift in the presence of Cu2+. The sulfonylphenyl-substituted sensor also exhibited excellent selectivity for Cu2+.

Microfluidic quantitative PCR for simultaneous quantification of multiple viruses in environmental water samples.
Satoshi Ishii, Gaku Kitamura, Takahiro Segawa, Ayano Kobayashi, Takayuki Miura, Daisuke Sano, Satoshi Okabe
Applied and environmental microbiology, 80, 24, 7505, 11, AMER SOC MICROBIOLOGY, 2014年12月, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/μl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.

External CO2 and water supplies for enhancing electrical power generation of air-cathode microbial fuel cells.
So Ishizaki, Itto Fujiki, Daisuke Sano, Satoshi Okabe
Environmental science & technology, 48, 19, 11204, 10, AMER CHEMICAL SOC, 2014年10月07日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Alkalization on the cathode electrode limits the electrical power generation of air-cathode microbial fuel cells (MFCs), and thus external proton supply to the cathode electrode is essential to enhance the electrical power generation. In this study, the effects of external CO2 and water supplies to the cathode electrode on the electrical power generation were investigated, and then the relative contributions of CO2 and water supplies to the total proton consumption were experimentally evaluated. The CO2 supply decreased the cathode pH and consequently increased the power generation. Carbonate dissolution was the main proton source under ambient air conditions, which provides about 67% of total protons consumed for the cathode reaction. It is also critical to adequately control the water content on the cathode electrode of air-cathode MFCs because the carbonate dissolution was highly dependent on water content. On the basis of these experimental results, the power density was increased by 400% (143.0 ± 3.5 mW/m(2) to 575.0 ± 36.0 mW/m(2)) by supplying a humid gas containing 50% CO2 to the cathode chamber. This study demonstrates that the simultaneous CO2 and water supplies to the cathode electrode were effective to increase the electrical power generation of air-cathode MFCs for the first time.

嫌気性アンモニア酸化(anammox)細菌の廃水処理への適用 (特集総説 極限生物たちが切り拓く未来の環境バイオテクノロジー)
押木 守, 佐藤 久, 岡部 聡
Journal of environmental biotechnology, 14, 1, 21, 29, 環境バイオテクノロジー学会, 2014年10月, ［査読有り］, ［最終著者, 責任著者］
日本語

Identification of key nitrous oxide production pathways in aerobic partial nitrifying granules.
Satoshi Ishii, Yanjun Song, Lashitha Rathnayake, Azzaya Tumendelger, Hisashi Satoh, Sakae Toyoda, Naohiro Yoshida, Satoshi Okabe
Environmental microbiology, 16, 10, 3168, 80, WILEY-BLACKWELL, 2014年10月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The identification of the key nitrous oxide (N2O) production pathways is important to establish a strategy to mitigate N2O emission. In this study, we combined real-time gas-monitoring analysis, (15)N stable isotope analysis, denitrification functional gene transcriptome analysis and microscale N2O concentration measurements to identify the main N2O producers in a partial nitrification (PN) aerobic granule reactor, which was fed with ammonium and acetate. Our results suggest that heterotrophic denitrification was the main contributor to N2O production in our PN aerobic granule reactor. The heterotrophic denitrifiers were probably related to Rhodocyclales bacteria, although different types of bacteria were active in the initial and latter stages of the PN reaction cycles, most likely in response to the presence of acetate. Hydroxylamine oxidation and nitrifier denitrification occurred, but their contribution to N2O emission was relatively small (20-30%) compared with heterotrophic denitrification. Our approach can be useful to quantitatively examine the relative contributions of the three pathways (hydroxylamine oxidation, nitrifier denitrification and heterotrophic denitrification) to N2O emission in mixed microbial populations.

Simple, rapid and effective preservation and reactivation of anaerobic ammonium oxidizing bacterium "Candidatus Brocadia sinica".
Muhammad Ali, Mamoru Oshiki, Satoshi Okabe
Water research, 57, 215, 22, PERGAMON-ELSEVIER SCIENCE LTD, 2014年06月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, It is still the biggest challenge to secure enough seeding biomass for rapid start-up of full-scale (anaerobic ammonium oxidation) anammox processes due to slow growth. Preservation of active anammox biomass could be one of the solutions. In this study, biomass of anammox bacterium, "Candidatus Brocadia sinica", immersed in various nutrient media were preserved at -80 °C, 4 °C and room temperature. After 45, 90 and 150 days of preservation, specific anammox activity (SAA) of the preserved anammox biomass was determined by measuring (29)N2 production rate and transcription levels of hzsA gene encoding hydrazine synthase alpha subunit. Storage in nutrient medium containing 3 mM of molybdate at room temperature with periodical (every 45 days) supply of NH4(+) and NO2(-) was proved to be the most effective storage technique for "Ca. Brocadia sinica" biomass. Using this preservation condition, 96, 92 and 65% of the initial SAA was sustained after 45, 90 and 150 days of storage, respectively. Transcription levels of hzsA gene in biomass correlated with the SAA (R(2) = 0.83), indicating it can be used as a genetic marker to evaluate the anammox activity of preserved biomass. Furthermore, the 90-day-stored biomass was successfully reactivated by immobilizing in polyvinyl alcohol (6%, w/v) and sodium alginate (2%, w/v) gel and then inoculated to up-flow column reactors. Total nitrogen removal rates rapidly increased to 7 kg-N m(-3) d(-1) within 35 days of operation. Based on these results, the room temperature preservation with molybdate addition is simple, cost-effective and feasible at a practical scale, which will accelerate the practical use of anammox process for wastewater treatment.

Estimation of Contamination Sources of Human Enteroviruses in a Wastewater Treatment and Reclamation System by PCR-DGGE
Zheng Ji, Xiaochang C. Wang, Limei Xu, Chongmiao Zhang, Naoyuki Funamizu, Satoshi Okabe, Daisuke Sano
FOOD AND ENVIRONMENTAL VIROLOGY, 6, 2, 99, 109, SPRINGER, 2014年06月, ［査読有り］
英語, 研究論文（学術雑誌）, A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was employed to estimate the contamination sources of human enteroviruses and understand how their dominant strains vary in a wastewater treatment and reclamation system consisting of sewage collection, wastewater treatment with membrane bioreactor and open lakes for reclaimed water storage and reuse. After PCR-DGGE using a selected primer set targeting enteroviruses, phylogenetic analysis of acquired enterovirus gene sequences was performed. Enteroviruses identified from the septic tank were much more diverse than those from grey water and kitchen wastewater. Several unique types of enterovirus different from those in wastewater samples were dominant in a biological wastewater treatment unit. Membrane filtration followed by chlorination was proved effective for physically eliminating enteroviruses; however, secondary contamination likely occurred as the reclaimed water was stored in artificial lakes. Enterovirus 71 (EV71), a hand-foot-and-mouth disease (HFMD) viral pathogen, was detected mainly from the artificial lakes, implying that wastewater effluent was not the contamination source of EV71 and that there were unidentified non-point sources of the contamination with the HFMD viral pathogen in the reclaimed water stored in the artificial lakes. The PCR-DGGE targeting enteroviruses provided robust evidence about viral contamination sources in the wastewater treatment and reclamation system.

Water quality monitoring and risk assessment by simultaneous multipathogen quantification.
Satoshi Ishii, Takamitsu Nakamura, Shuji Ozawa, Ayano Kobayashi, Daisuke Sano, Satoshi Okabe
Environmental science & technology, 48, 9, 4744, 9, AMER CHEMICAL SOC, 2014年05月06日, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Water quality monitoring and microbial risk assessment are important to ensure safe water for drinking, recreational, and agricultural purposes. In this study, we applied a microfluidic quantitative PCR (MFQPCR) approach to simultaneously quantify multiple waterborne pathogens in a natural freshwater lake in Hokkaido, Japan, from April to November, 2012. Tens of thousands of geese stopped over at this lake during their migration in spring and fall. Because lake water is used for irrigation of the surrounding agricultural area, we assessed infection risks through irrigation water usage based on pathogen concentrations directly measured by MFQPCR. We detected various pathogens in the lake water, particularly during the bird migration seasons, suggesting that migratory birds were the main source of the pathogens. However, neither counts of geese nor fecal indicator bacteria were good predictors of pathogen concentrations. On the basis of quantitative microbial risk assessment, concentrations of Campylobacter jejuni and Shigella spp. in water samples were above the concentrations that can potentially cause 10(-4) infections per person per year when water is used to grow fresh vegetables. These results suggest that direct and simultaneous multipathogen quantification can provide more reliable and comprehensive information for risk assessment than the current fecal indicator-based approach.

Application of fluorescence spectroscopy using a novel fluoroionophore for quantification of zinc in urban runoff.
Akira Hafuka, Hiroaki Yoshikawa, Koji Yamada, Tsuyoshi Kato, Masahiro Takahashi, Satoshi Okabe, Hisashi Satoh
Water research, 54, 12, 20, PERGAMON-ELSEVIER SCIENCE LTD, 2014年05月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Fluorescence spectroscopy has great potential for on-site and real-time monitoring of pollutants in aquatic environments; however, its application to environmental aquatic samples has been extremely limited. In this study, a novel fluoroionophore based on a BODIPY-terpyridine conjugate was developed and applied to determine Zn concentrations in urban runoff. The fluoroionophore selectively bound to Zn(2+) in water, which led to an instant red-shift of the fluorescence peak of the fluoroionophore from 539 nm to 567 nm that could be seen by the naked eye. Zn concentrations could be quantified using the ratio of fluorescence intensities, and the detection limit was 9 μg/L, which is sufficiently low for environmental aquatic samples. To demonstrate applicability of the method to environmental samples, we measured Zn concentrations in urban runoff samples with a complex matrix (∼60 mg/L dissolved organic carbon and ∼20 mS/cm electrical conductivity). The total and dissolved fractions of Zn in the samples could be determined by fluorescence spectroscopy and its relative error was estimated to be less than 30% by inductively coupled plasma-atomic emission spectroscopy analysis. The proposed method is rapid and easy-to-use with simple pretreatment for Zn determination in environmental aquatic samples with complex matrices.

Raoultella electrica sp. nov., isolated from anodic biofilms of a glucose-fed microbial fuel cell.
Zen-Ichiro Kimura, Kyung Mi Chung, Hiroaki Itoh, Akira Hiraishi, Satoshi Okabe
International journal of systematic and evolutionary microbiology, 64, Pt 4, 1384, 1388, SOC GENERAL MICROBIOLOGY, 2014年04月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, A Gram-stain-negative, non-spore-forming, rod-shaped bacterium, designated strain 1GB(T), was isolated from anodic biofilms of a glucose-fed microbial fuel cell. Strain 1GB(T) was facultatively anaerobic and chemo-organotrophic, having both a respiratory and a fermentative type of metabolism, and utilized a wide variety of sugars as carbon and energy sources. Cells grown aerobically contained Q-8 as the major quinone, but excreted Q-9 and a small amount of Q-10 when cultured with an electrode serving as the sole electron acceptor. The G+C content of the genomic DNA of 1GB(T) was 54.5 mol%. Multilocus sequence typing (MLST) analysis showed that strain 1GB(T) represented a distinct lineage within the genus Raoultella (98.5-99.4 % 16S rRNA gene sequence similarity and 94.0-96.5 % sequence similarity based on the three concatenated housekeeping genes gyrA, rpoB and parC. Strain 1GB(T) exhibited DNA-DNA hybridization relatedness of 7-43 % with type strains of all established species of the genus Raoultella. On the basis of these phenotypic, phylogenetic and genotypic data, the name Raoultella electrica sp. nov. is proposed for strain 1GB(T). The type strain is 1GB(T) ( = NBRC 109676(T) = KCTC 32430(T)).

Toxicity assessment of chlorinated wastewater effluents by using transcriptome-based bioassays and Fourier transform mass spectrometry (FT-MS) analysis.
Toshikazu Fukushima, Hiroe Hara-Yamamura, Makoto Urai, Ikuro Kasuga, Futoshi Kurisu, Taro Miyoshi, Katsuki Kimura, Yoshimasa Watanabe, Satoshi Okabe
Water research, 52, 73, 82, PERGAMON-ELSEVIER SCIENCE LTD, 2014年04月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Effects of chlorination on the toxicity of wastewater effluents treated by activated sludge (AS) and submerged membrane bioreactor (S-MBRB) systems to HepG2 human hepatoblastoma cells were investigated. In addition to the cytotoxicity and genotoxicity assays, the DNA microarray-based transcriptome analysis was performed to evaluate the change in types of biological impacts on HepG2 cells of the effluents by chlorination. Effluent organic matter (EfOM) and disinfection by-products (DBPs) were also characterized by using Fourier transform mass spectrometry (FT-MS). Although no significant induction of genotoxicity was observed by chlorination for both effluents, the chlorination elevated the cytotoxicity of AS effluent but reduced that of S-MBRB effluent. The FT-MS analyses revealed that more DBPs including nitrogenated DBPs (N-DBPs) were formed in the AS effluent than in the S-MBRB effluent by chlorination, supporting the increased cytotoxicity of AS effluent. The lower O/C ratio of S-MBRB EfOM suggests that a large number of organic molecules were detoxified by chlorination, which consequently decreased the cytotoxicity of S-MBRB effluent. Integration of all the results highlights that both cytotoxicity and biological impacts of chlorinated wastewater effluents were clearly dependent on the EfOM characteristics such as DBPs and O/C ratio, namely, on types of treatment systems.

培養できないウイルスの感染性の評価法　　　　　　　　　　　　　　　
佐野大輔, 岡部聡
感染と消毒, 21, 2, 24, 27, 2014年, ［招待有り］, ［最終著者］
日本語, 研究論文（その他学術会議資料等）

Bayesian Modeling of Enteric Virus Density in Wastewater Using Left-Censored Data
Tsuyoshi Kato, Takayuki Miura, Satoshi Okabe, Daisuke Sano
FOOD AND ENVIRONMENTAL VIROLOGY, 5, 4, 185, 193, SPRINGER, 2013年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Stochastic models are used to express pathogen density in environmental samples for performing microbial risk assessment with quantitative uncertainty. However, enteric virus density in water often falls below the quantification limit (non-detect) of the analytical methods employed, and it is always difficult to apply stochastic models to a dataset with a substantially high number of non-detects, i.e., left-censored data. We applied a Bayesian model that is able to model both the detected data (detects) and non-detects to simulated left-censored datasets of enteric virus density in wastewater. One hundred paired datasets were generated for each of the 39 combinations of a sample size and the number of detects, in which three sample sizes (12, 24, and 48) and the number of detects from 1 to 12, 24 and 48 were employed. The simulated observation data were assigned to one of two groups, i.e., detects and non-detects, by setting values on the limit of quantification to obtain the assumed number of detects for creating censored datasets. Then, the Bayesian model was applied to the censored datasets, and the estimated mean and standard deviation were compared to the true values by root mean square deviation. The difference between the true distribution and posterior predictive distribution was evaluated by Kullback-Leibler (KL) divergence, and it was found that the estimation accuracy was strongly affected by the number of detects. It is difficult to describe universal criteria to decide which level of accuracy is enough, but eight or more detects are required to accurately estimate the posterior predictive distributions when the sample size is 12, 24, or 48. The posterior predictive distribution of virus removal efficiency with a wastewater treatment unit process was obtained as the log ratio posterior distributions between the posterior predictive distributions of enteric viruses in untreated wastewater and treated wastewater. The KL divergence between the true distribution and posterior predictive distribution of virus removal efficiency also depends on the number of detects, and eight or more detects in a dataset of treated wastewater are required for its accurate estimation.

Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay
Takayuki Miura, Sylvain Parnaudeau, Marco Grodzki, Satoshi Okabe, Robert L. Atmar, Francoise S. Le Guyader
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 79, 21, 6585, 6592, AMER SOC MICROBIOLOGY, 2013年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

フルオロイオノフォアを用いた蛍光分光法による工場廃水中Zn2+の定量
羽深昭, 吉川弘晃, 大屋光平, 山田幸司, 高橋正宏, 岡部聡, 佐藤久
土木学会論文集, 50, 3, 275, 280, Japan Society of Civil Engineers, 2013年11月, ［査読有り］
日本語, 本研究では, 新規に開発した特異的イオン認識蛍光色素(フルオロイオノフォア)を用い, 蛍光分光法により工場廃水中のZn²⁺を定量することを試みた. フルオロイオノフォアはZn²⁺と結合すると蛍光スペクトルが長波長シフトし, Zn²⁺濃度に依存したレシオメトリック型の蛍光スペクトル変化を示した. フルオロイオノフォアを実廃水試料に適用したところ, CrやNiが高濃度に含まれている廃水においても誤差20%以内でZn²⁺を定量できた.

Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers.
Ayano Kobayashi, Daisuke Sano, Asami Taniuchi, Satoshi Ishii, Satoshi Okabe
Applied microbiology and biotechnology, 97, 20, 9165, 73, SPRINGER, 2013年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.

Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.
Takayuki Miura, Daisuke Sano, Atsushi Suenaga, Takeshi Yoshimura, Miyu Fuzawa, Toyoko Nakagomi, Osamu Nakagomi, Satoshi Okabe
Journal of virology, 87, 17, 9441, 51, AMER SOC MICROBIOLOGY, 2013年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.

Chicken- and duck-associated Bacteroides-Prevotella genetic markers for detecting fecal contamination in environmental water.
Ayano Kobayashi, Daisuke Sano, Jun Hatori, Satoshi Ishii, Satoshi Okabe
Applied microbiology and biotechnology, 97, 16, 7427, 37, SPRINGER, 2013年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bacteroides-Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides-Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61 %, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70 % of chicken feces, while detected from 39 % of cow feces, 8.3 % of pig feces, and 12 % of swan feces. Duck-Bac marker was detected from 85 % of wild duck feces and cross-reacted with 31 % of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides-Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.

Acetate oxidation by syntrophic association between Geobacter sulfurreducens and a hydrogen-utilizing exoelectrogen.
Zen-ichiro Kimura, Satoshi Okabe
The ISME journal, 7, 8, 1472, 82, NATURE PUBLISHING GROUP, 2013年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Anodic microbial communities in acetate-fed microbial fuel cells (MFCs) were analyzed using stable-isotope probing of 16S rRNA genes followed by denaturing gradient gel electrophoresis. The results revealed that Geobacter sulfurreducens and Hydrogenophaga sp. predominated in the anodic biofilm. Although the predominance of Geobacter sp. as acetoclastic exoelectrogens in acetate-fed MFC systems has been often reported, the ecophysiological role of Hydrogenophaga sp. is unknown. Therefore, we isolated and characterized a bacterium closely related to Hydrogenophaga sp. (designated strain AR20). The newly isolated strain AR20 could use molecular hydrogen (H2), but not acetate, with carbon electrode as the electron acceptor, indicating that the strain AR20 was a hydrogenotrophic exoelectrogen. This evidence raises a hypothesis that acetate was oxidized by G. sulfurreducens in syntrophic cooperation with the strain AR20 as a hydrogen-consuming partner in the acetate-fed MFC. To prove this hypothesis, G. sulfurreducens strain PCA was cocultivated with the strain AR20 in the acetate-fed MFC without any dissolved electron acceptors. In the coculture MFC of G. sulfurreducens and strain AR20, current generation and acetate degradation were the highest, and the growth of strain AR20 was observed. No current generation, acetate degradation and cell growth occurred in the strain AR20 pure culture MFC. These results show for the first time that G. sulfurreducens can oxidize acetate in syntrophic cooperation with the isolated Hydrogenophaga sp. strain AR20, with electrode as the electron acceptor.

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the "Candidatus Scalindua" Group
Takanori Awata, Mamoru Oshiki, Tomonori Kindaichi, Noriatsu Ozaki, Akiyoshi Ohashi, Satoshi Okabe
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 79, 13, 4145, 4148, AMER SOC MICROBIOLOGY, 2013年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The phylogenetic affiliation and physiological characteristics (e.g., K-s and maximum specific growth rate [mu(max)]) of an anaerobic ammonium oxidation (anammox) bacterium, "Candidatus Scalindua sp.," enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. "Candidatus Scalindua sp." exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.

Development and characterization of the partial nitrification aerobic granules in a sequencing batch airlift reactor.
Yanjun Song, Satoshi Ishii, Lashitha Rathnayake, Tsukasa Ito, Hisashi Satoh, Satoshi Okabe
Bioresource technology, 139, 285, 91, ELSEVIER SCI LTD, 2013年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, partial nitrifying (PN) aerobic granules were developed in a sequencing batch airlift reactor by controlling the airflow rate and NH4(+) loading rate. The PN reactor produced an effluent with a NO2(-)/NH4(+) ratio of approximately one and with an NH4(+) conversion rate of 1.22 kg N m(-3)day(-1). More than 95% of the total organic carbon was removed during the process. On the basis of clone library analysis and fluorescence in situ hybridization, ammonia-oxidizing bacteria (AOB) closely related to Nitrosomonas eutropha and putative heterotrophic denitrifiers were mainly present near the surface of the PN aerobic granules. Microelectrode measurements revealed that both NH4(+) and NO2(-) were consumed near the surface (<200 μm), whereas no nitrate (NO3(-)) accumulation was observed throughout the granules. These results indicate that PN by AOB and nitrite denitrification by heterotrophs, but not nitrite oxidation, simultaneously occurred near the surface of the PN aerobic granules.

Nitrate-dependent ferrous iron oxidation by anaerobic ammonium oxidation (anammox) bacteria
M. Oshiki, S. Ishii, K. Yoshida, N. Fujii, M. Ishiguro, Hisashi Satoh, S. Okabe
Applied and Environmental Microbiology, 79, 13, 4087, 4093, 2013年07月, ［査読有り］
英語, 研究論文（学術雑誌）, We examined nitrate-dependent Fe2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of "Candidatus Brocadia sinica" anaerobically oxidized Fe2+ and reduced NO3- to nitrogen gasat rates of 3.7±0.2 and 1.3±0.1 (mean±standard deviation [SD]) nmol mg protein-1 min-1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of "Ca. Brocadia sinica" (10 to 75 nmol NH4+ mg protein-1 min-1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3- by "Ca. Brocadia sinica." The activities of nitrate-dependent Fe2+ oxidation were dependent on temperature and pH, and thehighest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO3 -±SD of "Ca. Brocadia sinica" was determined to be 51±21 μM. Nitrate-dependent Fe2+ oxidation was further demonstrated by another anammox bacterium, "Candidatus Scalindua sp.," whose rates of Fe2+ oxidation and NO3- reduction were 4.7±0.59 and 1.45±0.05 nmol mg protein-1 min-1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO2 - to consumed NH4 + (δNO2-/δNH4 +) and produced NO3- to consumed NH4 + (δNO3 -/δNH4 +). These reactions are preferable to the application of anammox processes for wastewater treatment. © 2013, American Society for Microbiology.

Physiological characterization of an anaerobic ammonium-oxidizing bacterium enriched from coastal sediments,Hiroshima,Japan　　　　　　　　　　　　　　　
Takanori Awata, Mamoru Oshiki, Tomonori Kindaichi, Noriatsu Ozaki, Akiyoshi Ohashi, Satoshi Okabe
The 2nd International Anammox Symposium, pp.67, The 2nd International Anammox Symposium, 2013年06月, ［査読有り］
研究論文（学術雑誌）, The effect of salinity on organic removal and ammonium oxidation in a down-flow hanging sponge reactor was investigated by conducting a long-term continuous experiment over a period of 800 days. The DHS reactor, constructed by connecting three identical units, was fed with artificial wastewater containing 500 mg-N/L of ammonium nitrogen and 1400 mg-COD/L of phenol. Salinity of the influent was controlled by the addition of 8.0 to 25 g-Cl-/L of NaCl. The DHS reactor was operated at a hydraulic retention time of 12 h in a temperature controlled room at 25 degrees C. No significant inhibition of organic removal and ammonium oxidation was observed at salinities of up to 20 g-Cl-/L, at which levels ammonium oxidation and COD removal both exceeded 90%, respectively. However, at a salinity of 25 g-Cl-/L, organic removal and ammonium oxidation were both severely inhibited. In addition, the ratio of effluent nitrite nitrogen to influent ammonium nitrogen increased from 3.4% at salinities of 8.0 g-Cl-/L to 33% at salinities of 20 g-Cl-/L.

Evaluation of whole wastewater effluent impacts on HepG2 using DNA microarray-based transcriptome analysis.
Hiroe Hara-Yamamura, Koji Nakashima, Asiful Hoque, Taro Miyoshi, Katsuki Kimura, Yoshimasa Watanabe, Satoshi Okabe
Environmental science & technology, 47, 10, 5425, 32, 10, 2013年05月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, DNA microarray-based transcriptome analysis with human hepatoma HepG2 cells was applied to evaluate the impacts of whole wastewater effluents from the membrane bioreactors (MBRs) and the activated sludge process (AS). In addition, the conventional bioassays (i.e., cytotoxicity tests and bioluminescence inhibition test), which were well-established for the evaluation of the overall effluent toxicity, were also performed for the same samples. Transcriptome analysis revealed that 2 to 926 genes, which were categorized to 0 to 225 biological processes, were differentially expressed after exposure to the effluents and the raw wastewater. Among the tested effluents, the effluent from a MBR operated at a relatively long solid retention time (i.e., 40 days) and small membrane pore size (i.e., 0.03 μm) showed the least impacts on the HepG2 even at the level comparable to tap water. The observed gene expression responses were in good agreement with the results of cytotoxicity tests, and provided additional molecular mechanistic information on adverse effects occurred in the sublethal region. Furthermore, the genes related to "lipid metabolism", "response to endogenous stimulus", and "response to inorganic substance" were selected as potential genetic markers, and their expression levels were quantified to evaluate the cellular impacts and treatability of wastewater effluents. Although the harmful impacts and innocuous impacts could not be distinguished at present, the results demonstrated that the DNA microarray-based transcriptome analysis with human HepG2 cells was a powerful tool to rapidly and comprehensively evaluate impacts of whole wastewater effluents.

Simultaneous quantification of multiple food- and waterborne pathogens by use of microfluidic quantitative PCR.
Satoshi Ishii, Takahiro Segawa, Satoshi Okabe
Applied and environmental microbiology, 79, 9, 2891, 8, AMER SOC MICROBIOLOGY, 2013年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.

Introduction of a Degassing Membrane Technology into Anaerobic Wastewater Treatment
W. M. K. R. T. W. Bandara, M. Ikeda, H. Satoh, M. Sasakawa, Y. Nakahara, M. Takahashi, S. Okabe
WATER ENVIRONMENT RESEARCH, 85, 5, 387, 390, WATER ENVIRONMENT FEDERATION, 2013年05月, ［査読有り］
英語, The effectiveness of degasification using a degassing membrane to improve chemical oxygen demand (COD) removal efficiency was investigated using a bench-scale upflow anaerobic sludge blanket (UASB) reactor. Vacuum degasification was able to transfer dissolved gas in the bulk liquid of the UASB reactor inside the membrane. Such a process might provide thermodynamically favorable conditions for the degradation of organic compounds. The COD-removal efficiency improved from 83% during normal operation to 90% during the degassing operation.

Effects of temperature and predator on the persistence of host-specific Bacteroides-Prevotella genetic markers in water.
Ayano Kobayashi, Daisuke Sano, Satoshi Okabe
Water science and technology : a journal of the International Association on Water Pollution Research, 67, 4, 838, 45, 4, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Genetic markers derived from Bacteroidales spp. have been proposed as promising indicators for fecal contamination in the water environment. However, little is known about the persistency of Bacteroidales spp. 16S rRNA genetic markers in the natural environment, which hampers the precise identification of fecal contamination sources. In this study, the persistency of human-specific Bacteroidales spp. genetic markers in river water was investigated during a 3-week agitation. The copy number of Bacteroidales spp. genetic marker was decreased with agitation time, and was very sensitive to water temperature. After the 3-week agitation, three clones of 18S rRNA gene related to Glaucoma scintillans, Spumella-like flagellate, and Colpidium campylum were acquired. The presence of predators that can prey on target bacteria could also be a critical factor affecting the quantified value of genetic markers. It is very important to take these factors, water temperature and the presence of predator, into account for predicting the fate of genetic markers to accurately identify fecal pollution sources.

Spatial and temporal oxygen dynamics in macrofaunal burrows in sediments: a review of analytical tools and observational evidence.
Hisashi Satoh, Satoshi Okabe
Microbes and environments, 28, 2, 166, 79, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2013年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The availability of benthic O2 plays a crucial role in benthic microbial communities and regulates many important biogeochemical processes. Burrowing activities of macrobenthos in the sediment significantly affect O2 distribution and its spatial and temporal dynamics in burrows, followed by alterations of sediment microbiology. Consequently, numerous research groups have investigated O2 dynamics in macrofaunal burrows. The introduction of powerful tools, such as microsensors and planar optodes, to sediment analysis has greatly enhanced our ability to measure O2 dynamics in burrows at high spatial and temporal resolution with minimal disturbance of the physical structure of the sediment. In this review, we summarize recent studies of O2-concentration measurements in burrows with O2 microsensors and O2 planar optodes. This manuscript mainly focuses on the fundamentals of O2 microsensors and O2 planar optodes, and their application in the direct measurement of the spatial and temporal dynamics of O2 concentrations in burrows, which have not previously been reviewed, and will be a useful supplement to recent literature reviews on O2 dynamics in macrofaunal burrows.

A new approach for evaluating the infectivity of noncultivatable enteric viruses without cell culture.
Kazuki Tojo, Daisuke Sano, Takayuki Miura, Toyoko Nakagomi, Osamu Nakagomi, Satoshi Okabe
Water science and technology : a journal of the International Association on Water Pollution Research, 67, 10, 2236, 40, IWA PUBLISHING, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, This study developed a novel approach for evaluating the infectivity of enteric viruses without cell culture. Cumulative carbonyl groups on the viral capsid protein were labeled using biotin hydrazide, and the biotinylated virions were separated using a spin column filled with avidin-immobilized gel. Rotavirus was treated with free chlorine at an initial concentration of 0.3 mg/L for 3 min, and the log reduction in the infectious titer was 0.19 log (standard deviation, SD = 0.05). The log reduction of rotavirus treated with free chlorine at an initial concentration of 0.6 mg/L for 3 min was 2.6 log (SD = 0.37). No significant reductions in the amplicon copy numbers were observed in these free chlorine-treated samples. The recovery levels of intact virions in the first three fractions after biotin-avidin affinity chromatography were 76, 21, and 2.8%, while those of virions treated with free chlorine at an initial concentration of 0.3 mg/L for 3 min were 70, 23, and 5.6%. These results showed that the proposed approach could discriminate a 0.19 log infectivity-reduced population from an intact population, although no reduction in the amplicon copy number was observed. This novel method could be applied to noncultivatable enteric viruses such as human norovirus and sapovirus, and it could be very helpful for evaluating the viral inactivation efficiencies of intervention measures.

BODIPY-Based Ratiometric Fluoroionophores with Bidirectional Spectral Shifts for the Selective Recognition of Heavy Metal Ions
Akira Hafuka, Hiroki Taniyama, Sang-Hyun Son, Koji Yamada, Masahiro Takahashi, Satoshi Okabe, Hisashi Satoh
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 86, 1, 37, 44, CHEMICAL SOC JAPAN, 2013年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Two novel asymmetric BODIPY fluoroionophores with dipicolylamine (BDP-DPA, dipicolylamine: bis(pyridyl-methyl)) and terpyridine (BDP-TPY) are described. These fluoroionophores display opposite wavelength responses on complexation with heavy metal ions. Furthermore, the fluorescence spectra vary depending on the ionic species. In particular, BDP-DPA shows a high affinity toward Cr3+ and upon complexation, the fluorescence spectrum blue-shifts from 591 to 566 nm. In contrast, BDP-TPY preferentially binds to Zn2+ and the fluorescence spectra red-shifts from 539 to 567 nm. BDP-TPY is the first example of asymmetric BODIPY with a pyridyl receptor at the 3 position showing red-shifted fluorescence by complexation with metal ions. The concentration of each metal ion was successfully determined by ratiometric measurement. The wavelength-responses characteristics of these fluoroionophores could be very useful in the development of novel ratiometric fluoroionophores for metal ions.

Hydrogenophaga electricum sp. nov., isolated from anodic biofilms of an acetate-fed microbial fuel cell.
Zen-ichiro Kimura, Satoshi Okabe
The Journal of general and applied microbiology, 59, 4, 261, 6, MICROBIOL RES FOUNDATION, 2013年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A Gram-negative, non-spore-forming, rod-shaped bacterial strain, AR20(T), was isolated from anodic biofilms of an acetate-fed microbial fuel cell in Japan and subjected to a polyphasic taxonomic study. Strain AR20(T) grew optimally at pH 7.0-8.0 and 25°C. It contained Q-8 as the predominant ubiquinone and C16:0, summed feature 3 (C16:1ω7c and/or iso-C15:02OH), and C18:1ω7c as the major fatty acids. The DNA G+C content was 67.1 mol%. A neighbor-joining phylogenetic tree revealed that strain AR20(T) clustered with three type strains of the genus Hydrogenophaga (H. flava, H. bisanensis and H. pseudoflava). Strain AR20(T) exhibited 16S rRNA gene sequence similarity values of 95.8-97.7% to the type strains of the genus Hydrogenophaga. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain AR20(T) is considered a novel species of the genus Hydrogenophaga, for which the name Hydrogenophaga electricum sp. nov. is proposed. The type strain is AR20(T) (= KCTC 32195(T) = NBRC 109341(T)).

Cultivation of planktonic anaerobic ammonium oxidation (anammox) bacteria using membrane bioreactor.
Mamoru Oshiki, Takanori Awata, Tomonori Kindaichi, Hisashi Satoh, Satoshi Okabe
Microbes and environments, 28, 4, 436, 43, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2013年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Enrichment cultures of anaerobic ammonium oxidation (anammox) bacteria as planktonic cell suspensions are essential for studying their ecophysiology and biochemistry, while their cultivation is still laborious. The present study aimed to cultivate two phylogenetically distinct anammox bacteria, "Candidatus Brocadia sinica" and "Ca. Scalindua sp." in the form of planktonic cells using membrane bioreactors (MBRs). The MBRs were continuously operated for more than 250 d with nitrogen loading rates of 0.48-1.02 and 0.004-0.09 kgN m(-3) d(-1) for "Ca. Brocadia sinica" and "Ca. Scalindua sp.", respectively. Planktonic anammox bacterial cells were successfully enriched (>90%) in the MBRs, which was confirmed by fluorescence in-situ hybridization and 16S rRNA gene sequencing analysis. The decay rate and half-saturation constant for NO2(-) of "Ca. Brocadia sinica" were determined to be 0.0029-0.0081 d(-1) and 0.47 mgN L(-1), respectively, using enriched planktonic cells. The present study demonstrated that MBR enables the culture of planktonic anammox bacterial cells, which are suitable for studying their ecophysiology and biochemistry.

Anaerobic treatment of municipal wastewater at ambient temperature: Analysis of archaeal community structure and recovery of dissolved methane.
Wasala M K R T W Bandara, Tomonori Kindaichi, Hisashi Satoh, Manabu Sasakawa, Yoshihito Nakahara, Masahiro Takahashi, Satoshi Okabe
Water research, 46, 17, 5756, 5764, PERGAMON-ELSEVIER SCIENCE LTD, 2012年11月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Anaerobic treatment is an attractive option for the biological treatment of municipal wastewater. In this study, municipal wastewater was anaerobically treated with a bench-scale upflow anaerobic sludge blanket (UASB) reactor at temperatures from 6 to 31 °C for 18 months to investigate total chemical oxygen demand (COD) removal efficiency, archaeal community structure, and dissolved methane (D-CH(4)) recovery efficiency. The COD removal efficiency was more than 50% in summer and below 40% in winter with no evolution of biogas. Analysis of the archaeal community structures of the granular sludge from the UASB using 16S rRNA gene-cloning indicated that after microorganisms had adapted to low temperatures, the archaeal community had a lower diversity and the relative abundance of acetoclastic methanogens decreased together with an increase in hydrogenotrophic methanogens. D-CH(4), which was detected in the UASB effluent throughout the operation, could be collected with a degassing membrane. The ratio of the collection to recovery rates was 60% in summer and 100% in winter. For anaerobic treatment of municipal wastewater at lower temperatures, hydrogenotrophic methanogens play an important role in COD removal and D-CH(4) can be collected to reduce greenhouse gas emissions and avoid wastage of energy resources.

Identification and quantification of key microbial trophic groups of methanogenic glucose degradation in an anaerobic digester sludge
Tsukasa Ito, Kazumi Yoshiguchi, Herto Dwi Ariesyady, Satoshi Okabe
BIORESOURCE TECHNOLOGY, 123, 599, 607, ELSEVIER SCI LTD, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated the major phylogenetic groups and population size of glucose-, propionate-, and acetate-degrading bacteria in the glucose-degrading anaerobic digester sludge by stable-isotope probing analysis of 16S rRNA (RNA-SIP) with [C-13(6)]glucose followed by time course analysis of microautoradiography combined with fluorescent in situ hybridization (MAR-FISH) with [U-C-14]glucose. The results indicated that glucose was predominately degraded to CH4 and CO2 by glucose-degrading Propionibacterium and Olsenella that are belonging to the phylum Actinobacteria, propionate-degrading Smithella and Syntrophobacter, and acetate-degrading Methanosaeta and Synergistes group 4 in this anaerobic sludge. The population size of propionate degraders was the smallest among three trophic groups and the specific degradation rate of propionate was also low. The specific degradation rate of acetate was low even though their population size was comparable to the glucose degraders. These results could explain why the degradation of propionate and acetate was the rate-limiting step in methanogenic glucose degradation. (C) 2012 Elsevier Ltd. All rights reserved.

Occurrence of Hand-Foot-and-Mouth Disease Pathogens in Domestic Sewage and Secondary Effluent in Xi'an, China
Zheng Ji, Xiaochang Wang, Chongmiao Zhang, Takayuki Miura, Daisuke Sano, Naoyuki Funamizu, Satoshi Okabe
MICROBES AND ENVIRONMENTS, 27, 3, 288, 292, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2012年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Hand, foot and mouth disease (HFMD), caused by a group of enteric viruses such as Enterovirus 71 (EV71), Coxsackievirus A16 (CVA16) and Coxsackievirus A10 (CVA10), is heavily epidemic in East Asia. This research focused on investigating the occurrence of HFMD pathogens in domestic sewage and secondary effluent before disinfection in a wastewater treatment plant (WWTP) in Xi'an, the largest megacity in northwest China. In order to simultaneously detect all three HFMD pathogens, a semi-nested RT-PCR assay was constructed with a newly designed primer set targeting conservative gene regions from the 5' untranslated region (UTR) to VP2. As a result, 86% of raw sewage samples and 29% of the secondary effluent samples were positive for the HFMD viral gene, indicating that HFMD pathogens were highly prevalent in domestic wastewater and that they could also persist, even with lower probability, in the secondary effluent before disinfection. Of the three HFMD pathogens, CVA10 was positive in 48% of the total samples, while the occurrences of CVA16 and EV71 were 12% and 2%, respectively. It could thus be stated that CVA10 is the main HFMD pathogen prevailing in the study area, at least during the investigation period. High genetic diversity in the conservative gene region among the same serotype of the HFMD pathogen was identified by phylogenetic analysis, implying that this HFMD pathogen replicates frequently among the population excreting the domestic sewage.

Physiological characteristics of marine anammox bacteria enriched from sea sediments,Hiroshima,Japan.　　　　　　　　　　　　　　　
Takanori Awata, Tomonori Kindaichi, Noriatsu Ozaki, Akiyoshi Ohashi, Mamoru Oshiki, Satoshi Okabe
14th International Symposium on Microbial Ecology(isme14), PS09.457A, PS09.457A, 14th International Symposium on Microbial Ecology(isme14), 2012年08月19日, ［査読有り］
研究論文（学術雑誌）

バイオフィルムの理解・制御から共存へ
岡部 聡
日本海水学会誌 = Bulletin of the Society of Sea Water Science, Japan, 66, 4, 191, 197, The Society of Sea Water Science, Japan, 2012年08月01日
日本語

RelE-mediated dormancy is enhanced at high cell density in Escherichia coli.
Yosuke Tashiro, Koji Kawata, Asami Taniuchi, Kenji Kakinuma, Thithiwat May, Satoshi Okabe
Journal of bacteriology, 194, 5, 1169, 76, AMER SOC MICROBIOLOGY, 2012年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chromosomal relBE and rhaBAD. With this strain, we demonstrated that RelE-mediated dormancy is enhanced at high cell densities compared to that at low cell densities. The initiation of expression of the antitoxin RelB from a plasmid, pCA24N, reversed RelE-mediated dormancy in bacterial cultures. The activation of RelE increased the appearance of persister cells against β-lactams, quinolones, and aminoglycosides, and more persister cells appeared at high cell densities than at low cell densities. Further analysis indicated that amino acid starvation and an uncharacterized extracellular heat-labile substance promote RelE-mediated dormancy. This is a first report on the induction of RelE-mediated dormancy by high cell density. This work establishes a population-based dormancy mechanism to help explain E. coli survival in stressful environments.

Community structure and in situ activity of nitrifying bacteria in Phragmites root-associated biofilms.
Satoshi Okabe, Yoshiyuki Nakamura, Hisashi Satoh
Microbes and environments, 27, 3, 242, 9, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2012年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The amount of oxygen released by Phragmites roots and the community structure and in situ activity of nitrifying bacteria in the root biofilms were analyzed by the combined use of 16S rRNA gene-cloning analysis, quantitative PCR (qPCR) assay and microelectrodes. Axial and radial O₂ microprofiles were obtained for individual roots of Phragmites in a horizontal flow reactor fed with artificial medium continuously. Axial O₂ profiles revealed that O₂ was released at a rate of 0.21 μmol O₂ cm⁻² (root surface area) h⁻¹ only in the apical region (up to ca. 40 mm from the root apex), where there was a high abundance (10⁷ to 10⁸ copies g⁻¹ biomass) of Nitrosomonas-like AOB and Nitrospira-like NOB. This abundance, however, sharply declined to the detection limit at positions more basal than 80 mm. Phylogenetic analysis based on 16S rRNA gene identified strains related to Nitrosomonas oligotropha and Nitrosomonas cryotolerans as the predominant AOB and strains related to Nitrospira marina and Nitrospira moscoviensis as the predominant NOB in the root biofilms. Based on radial O₂ microprofiles, the oxic region only extended about 0.5 mm into the surrounding sediment due to a high rate of O₂ consumption in the rhizosphere. The net NH₄⁺ and O₂ consumption rates in the apical region were higher than those determined at the oxic sediment surface in which the abundance of AOB and NOB was one order of magnitude lower than in the rhizosphere. These results clearly indicated that Phragmites root biofilms played an important role in nitrification in the waterlogged anoxic sediment.

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge
Tsukasa Ito, Kazumi Yoshiguchi, Herto Dwi Ariesyady, Satoshi Okabe
ISME JOURNAL, 5, 12, 1844, 1856, NATURE PUBLISHING GROUP, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope-and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with (14)C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with (13)C(6)-glucose and (13)C(3)-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with (13)C-glucose and (13)C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with (14)C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K(m) for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5-10mM). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.The ISME Journal (2011) 5, 1844-1856; doi:10.1038/ismej.2011.59; published online 12 May 2011

Enterobactin is required for biofilm development in reduced-genome Escherichia coli.
Thithiwat May, Satoshi Okabe
Environmental microbiology, 13, 12, 3149, 62, 12, 2011年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A variety of bacterial cell surface structures and quorum signalling molecules play a role in biofilm development in Escherichia coli. However, here we show that an engineered reduced-genome E. coli mutant that lacks 17.6% of the parental E. coli genome, including the genes involved in the synthesis of various cell surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers and the autoinducer-2 signalling molecule, is able to develop mature biofilms. Using temporal gene expression profiling, we investigated phenotypic changes in reduced-genome biofilms in relation with the genes encoding the synthesis of different amino acids that were differentially expressed during biofilm formation. We identified and characterized entB, marR, dosC, mcbR and yahK genes, as involved in biofilm formation by the reduced-genome E. coli. Of these, for a first time, we demonstrated that overproduction of entB and yahK, which encode an enterobactin for iron transport and a hypothetical oxidoreductase protein, respectively, promoted biofilm development and maturation. Our results indicate that specific types of genes contribute to phenotypic changes in reduced-genome E. coli biofilms. In addition, this work demonstrates that the functions of biofilm-specific genes could be analysed through experiments using the reduced-genome E. coli.

N2O emission from a partial nitrification-anammox process and identification of a key biological process of N2O emission from anammox granules.
Satoshi Okabe, Mamoru Oshiki, Yoshitaka Takahashi, Hisashi Satoh
Water research, 45, 19, 6461, 70, PERGAMON-ELSEVIER SCIENCE LTD, 2011年12月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Emission of nitrous oxide (N(2)O) during biological wastewater treatment is of growing concern. The emission of N(2)O from a lab-scale two-reactor partial nitrification (PN)-anammox reactor was therefore determined in this study. The average emission of N(2)O from the PN and anammox process was 4.0±1.5% (9.6±3.2% of the removed nitrogen) and 0.1±0.07% (0.14±0.09% of the removed nitrogen) of the incoming nitrogen load, respectively. Thus, a larger part (97.5%) of N(2)O was emitted from the PN reactor. The total amount of N(2)O emission from the PN reactor was correlated to nitrite (NO(2)(-)) concentration in the PN effluent rather than DO concentration. In addition, further studies were performed to indentify a key biological process that is responsible for N(2)O emission from the anammox process (i.e., granules). In order to characterize N(2)O emission from the anammox granules, the in situ N(2)O production rate was determined by using microelectrodes for the first time, which was related to the spatial organization of microbial community of the granule as determined by fluorescence in situ hybridization (FISH). Microelectrode measurement revealed that the active N(2)O production zone was located in the inner part of the anammox granule, whereas the active ammonium consumption zone was located above the N(2)O production zone. Anammox bacteria were present throughout the granule, whereas ammonium-oxidizing bacteria (AOB) were restricted to only the granule surface. In addition, addition of penicillin G that inhibits most of the heterotrophic denitrifiers and AOB completely inhibited N(2)O production in batch experiments. Based on these results obtained, denitrification by putative heterotrophic denitrifiers present in the inner part of the granule was considered the most probable cause of N(2)O emission from the anammox reactor (i.e., granules).

Adsorption characteristics of an enteric virus-binding protein to norovirus, rotavirus and poliovirus
Takahiro Imai, Daisuke Sano, Takayuki Miura, Satoshi Okabe, Keishi Wada, Yoshifumi Masago, Tatsuo Omura
BMC BIOTECHNOLOGY, 11, 123, BIOMED CENTRAL LTD, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Water contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies.
Results: A gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure.
Conclusions: EVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples.

Development of long-term stable partial nitrification and subsequent anammox process.
Satoshi Okabe, Mamoru Oshiki, Yoshitaka Takahashi, Hisashi Satoh
Bioresource technology, 102, 13, 6801, 7, ELSEVIER SCI LTD, 2011年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The partial nitrification reactor was successfully started up and operated stably for more than 250 days with a maximum nitrite production rate of 1.12 kg-Nm(-3)day(-1). The important factors for successful partial nitrification were high ammonium loading rate (>1.0 kg-Nm(-3)day(-1)) and relatively high pH (ca. 8.0), giving high free ammonia concentrations (>10mg NH(3)-NL(-1)). In addition, the air flow rate must be controlled at the ratio of air flow rate to ammonium loading rate below 0.1 (m(air)(3)day(-1))/(kg-Nm(-3)day(-1)). After the establishment of stable partial nitrification, the effluent NO(2)(-)-N/NH(4)(+)-N ratio and effluent NO(3)(-)-N concentration were 1.20 ± 0.33 and 1.2 ± 1.0mg-NL(-1), respectively, which was then fed into an granular-sludge anammox reactor. Consistent nitrogen removal was achieved for more than 250 days with a maximum nitrogen removal rate of 15.0 kg-TNm(-3)day(-1).

Physiological characteristics of the anaerobic ammonium-oxidizing bacterium 'Candidatus Brocadia sinica'.
Mamoru Oshiki, Masaki Shimokawa, Naoki Fujii, Hisashi Satoh, Satoshi Okabe
Microbiology (Reading, England), 157, Pt 6, 1706, 1713, SOC GENERAL MICROBIOLOGY, 2011年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m(-3) day(-1). The anammox bacteria were identified as representing 'Candidatus Brocadia sinica' on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (μ(max)) was 0.0041 h(-1), corresponding to a doubling time of 7 days. The half-saturation constants (K(s)) for ammonium and nitrite of 'Ca. B. sinica' were 28±4 and 86±4 µM, respectively, higher than those of 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis'. The temperature and pH ranges of anammox activity were 25-45 °C and pH 6.5-8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50 % inhibition at 16 mM), ethanol (91 % at 1 mM) and methanol (86 % at 1 mM). Anammox activities were 80 and 70 % of baseline in the presence of 20 mM phosphorus and 3 % salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (mol NH (+)(-4))(-1), respectively. This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems.

Removal of residual dissolved methane gas in an upflow anaerobic sludge blanket reactor treating low-strength wastewater at low temperature with degassing membrane.
Wasala M K R T W Bandara, Hisashi Satoh, Manabu Sasakawa, Yoshihito Nakahara, Masahiro Takahashi, Satoshi Okabe
Water research, 45, 11, 3533, 40, PERGAMON-ELSEVIER SCIENCE LTD, 2011年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, we investigated the efficiency of dissolved methane (D-CH(4)) collection by degasification from the effluent of a bench-scale upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater. A hollow-fiber degassing membrane module was used for degasification. This module was connected to the liquid outlet of the UASB reactor. After chemical oxygen demand (COD) removal efficiency of the UASB reactor became stable, D-CH(4) discharged from the UASB reactor was collected. Under 35 °C and a hydraulic retention time (HRT) of 10 h, average D-CH(4) concentration could be reduced from 63 mg COD L(-1) to 15 mg COD L(-1); this, in turn, resulted in an increase in total methane (CH(4)) recovery efficiency from 89% to 97%. Furthermore, we investigated the effects of temperature and HRT of the UASB reactor on degasification efficiency. Average D-CH(4) concentration was as high as 104 mg COD L(-1) at 15 °C because of the higher solubility of CH(4) gas in liquid; the average D-CH(4) concentration was reduced to 14 mg COD L(-1) by degasification. Accordingly, total CH(4) recovery efficiency increased from 71% to 97% at 15 °C as a result of degasification. Moreover, degasification tended to cause an increase in particulate COD removal efficiency. The UASB reactor was operated at the same COD loading rate, but different wastewater feed rates and HRTs. Although average D-CH(4) concentration in the UASB reactor was almost unchanged (ca. 70 mg COD L(-1)) regardless of the HRT value, the CH(4) discharge rate from the UASB reactor increased because of an increase in the wastewater feed rate. Because the D-CH(4) concentration could be reduced down to 12 ± 1 mg COD L(-1) by degasification at an HRT of 6.7 h, the CH(4) recovery rate was 1.5 times higher under degasification than under normal operation.

微生物の多様性―「遺伝子の多様性」/「細胞の多様性」から見た生物多様性 バイオフィルム
押木守, 岡部聡
生物の科学 遺伝, 65, 3, 48, 54, エヌ・ティー・エス, 2011年05月01日
日本語

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages.
Thithiwat May, Kenji Tsuruta, Satoshi Okabe
The ISME journal, 5, 4, 771, 5, NATURE PUBLISHING GROUP, 2011年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.

Ecophysiology of Uncultured Filamentous Anaerobes Belonging to the Phylum KSB3 That Cause Bulking in Methanogenic Granular Sludge
Takeshi Yamada, Kae Kikuchi, Toshihiro Yamauchi, Koji Shiraishi, Tsukasa Ito, Satoshi Okabe, Akira Hiraishi, Akiyoshi Ohashi, Hideki Harada, Yoichi Kamagata, Kazunori Nakamura, Yuji Sekiguchi
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77, 6, 2081, 2087, AMER SOC MICROBIOLOGY, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A filamentous bulking of a methanogenic granular sludge caused by uncultured filamentous bacteria of the candidate phylum KSB3 in an upflow anaerobic sludge blanket (UASB) system has been reported. To characterize the physiological traits of the filaments, a polyphasic approach consisting of rRNA-based activity monitoring of the KSB3 filaments using the RNase H method and substrate uptake profiling using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was conducted. On the basis of rRNA-based activity, the monitoring of a full-scale UASB reactor operated continuously revealed that KSB3 cells became active and predominant (up to 54% of the total 16S rRNA) in the sludge when the carbohydrate loading to the system increased. Batch experiments with a short incubation of the sludge with maltose, glucose, fructose, and maltotriose at relatively low concentrations (approximately 0.1 mM) in the presence of yeast extract also showed an increase in KSB3 rRNA levels under anaerobic conditions. MAR-FISH confirmed that the KSB3 cells took up radioisotopic carbons from [ (14)C] maltose and [ (14)C] glucose under the same incubation conditions in the batch experiments. These results suggest that one of the important ecophysiological characteristics of KSB3 cells in the sludge is carbohydrate degradation in wastewater and that high carbohydrate loadings may trigger an outbreak of KSB3 bacteria, causing sludge bulking in UASB systems.

Effect of feeding regimens on polyhydroxybutyrate production from food wastes by Cupriavidus necator
Akira Hafuka, Kenji Sakaida, Hisashi Satoh, Masahiro Takahashi, Yoshimasa Watanabe, Satoshi Okabe
Bioresource Technology, 102, 3, 3551, 3553, 3, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated the effects of different feeding regimens (1-pulse, stepwise, and continuous) of fermented food-waste liquid on polyhydroxybutyrate (PHB) production. The fermentation liquid was filtered with a membrane filter (pore size, 0.45. μm) to remove anaerobic microorganisms and solids and used as a carbon source for Cupriavidus necator. One-pulse feeding yielded the highest cell concentration of C. necator. However, the PHB concentration was higher in the stepwise- and continuous-feeding regimens. Therefore, the continuous-feeding regimen was used for continuous PHB production. PHB could be produced over 259 h (8 draw-fill cycles) with a maximal PHB content of 87%, but the PHB concentration and content decreased with an increase in the operation time. © 2010 Elsevier Ltd.

Quantification and Genotyping of Human Sapoviruses in the Llobregat River Catchment, Spain
Daisuke Sano, Unai Perez-Sautu, Susana Guix, Rosa Maria Pinto, Takayuki Miura, Satoshi Okabe, Albert Bosch
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77, 3, 1111, 1114, AMER SOC MICROBIOLOGY, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Human sapoviruses (SaVs) were quantified and characterized in an 18-month survey conducted along the Llobregat river catchment area in Spain. Sample types included freshwater, untreated and treated wastewater, and drinking water. All genogroups were recovered, and a seasonal distribution was observed. This is the first report of SaV quantification and genotyping in the environment outside Japan.

Effect of feeding regimens on polyhydroxybutyrate production from food wastes by Cupriavidus necator.
Akira Hafuka, Kenji Sakaida, Hisashi Satoh, Masahiro Takahashi, Yoshimasa Watanabe, Satoshi Okabe
Bioresource technology, 102, 3, 3551, 3, ELSEVIER SCI LTD, 2011年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We investigated the effects of different feeding regimens (1-pulse, stepwise, and continuous) of fermented food-waste liquid on polyhydroxybutyrate (PHB) production. The fermentation liquid was filtered with a membrane filter (pore size, 0.45 μm) to remove anaerobic microorganisms and solids and used as a carbon source for Cupriavidus necator. One-pulse feeding yielded the highest cell concentration of C. necator. However, the PHB concentration was higher in the stepwise- and continuous-feeding regimens. Therefore, the continuous-feeding regimen was used for continuous PHB production. PHB could be produced over 259 h (8 draw-fill cycles) with a maximal PHB content of 87%, but the PHB concentration and content decreased with an increase in the operation time.

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor
Sunja Cho, Naoki Fujii, Taeho Lee, Satoshi Okabe
BIORESOURCE TECHNOLOGY, 102, 2, 652, 659, ELSEVIER SCI LTD, 2011年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (+/- 0.19) kg-N m(-3) d(-1) than the reactor initiated as the partial nitrifying reactor (0.23 (+/- 0.16) kg-N m(-3) d(-1)). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor. (C) 2010 Elsevier Ltd. All rights reserved.

Effect of formation of biofilms and chemical scale on the cathode electrode on the performance of a continuous two-chamber microbial fuel cell.
Kyungmi Chung, Itto Fujiki, Satoshi Okabe
Bioresource technology, 102, 1, 355, 60, ELSEVIER SCI LTD, 2011年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A two-chamber MFC system was operated continuously for more than 500 days to evaluate effects of biofilm and chemical scale formation on the cathode electrode on power generation. A stable power density of 0.57 W/m(2) was attained after 200 days operation. However, the power density decreased drastically to 0.2 W/m(2) after the cathodic biofilm and chemical scale were removed. As the cathodic biofilm and chemical scale partially accumulated on the cathode, the power density gradually recovered with time. Microbial community structure of the cathodic biofilm was analyzed based on 16S rRNA clone libraries. The clones closely related to Xanthomonadaceae bacterium and Xanthomonas sp. in the Gammaproteobacteria subdivision were most frequently retrieved from the cathodic biofilm. Results of the SEM-EDX analysis revealed that the cation species (Na(+) and Ca(2+)) were main constituents of chemical scale, indicating that these cations diffused from the anode chamber through the Nafion membrane. However, an excess accumulation of the biofilm and chemical scale on the cathode exhibited adverse effects on the power generation due to a decrease in the active cathode surface area and an increase in diffusion resistance for oxygen. Thus, it is important to properly control the formation of chemical scale and biofilm on the cathode during long-term operation.

A polyphasic approach to study ecophysiology of complex multispecies nitrifying biofilms.
Satoshi Okabe, Hisashi Satoh, Tomonori Kindaichi
Methods in enzymology, 496, 163, 84, 2011年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, This chapter aims to highlight the great potential of the combined use of microautoradiography (MAR) combined with fluorescent in situ hybridization (FISH) and microsensor technology in studies of complex multispecies nitrifying biofilms. The combination of FISH and microsensor technology is a powerful and reliable tool to link the spatial organization of microbial communities and their in situ function at community levels. MAR-FISH can be used to simultaneously examine the 16S rRNA-based phylogenetic identity and specific metabolic activity of cultivable or uncultivable microorganisms within complex microbial communities at a single-cell level. Information obtained at both resolution levels must be combined to draw a clear picture of a complex multispecies biofilm ecosystem. In addition, ecophysiological interactions among community members in complex multispecies biofilms can be investigated by tracing the fate of radiolabeled [ (14)C] atom incorporated in nitrifying bacteria with MAR-FISH. The structure, function, and ecophysiological interactions among community members in complex multispecies nitrifying biofilms will be illustrated as an example of the combined use of MAR-FISH and microsensor technology.

A Great Leap forward in Microbial Ecology
Satoshi Okabe, Mamoru Oshiki, Yoichi Kamagata, Nobuyasu Yamaguchi, Masanori Toyofuku, Yutaka Yawata, Yosuke Tashiro, Nobuhiko Nomura, Hiroyuki Ohta, Moriya Ohkuma, Akira Hiraishi, Kiwamu Minamisawa
MICROBES AND ENVIRONMENTS, 25, 4, 230, 240, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2010年12月, ［査読有り］
英語, Ribosomal RNA (rRNA) sequence-based molecular techniques emerged in the late 1980s, which completely changed our general view of microbial life. Coincidentally, the Japanese Society of Microbial Ecology (JSME) was founded, and its official journal "Microbes and Environments (M&E)" was launched, in 1985. Thus, the past 25 years have been an exciting and fruitful period for M&E readers and microbiologists as demonstrated by the numerous excellent papers published in M&E. In this minireview, recent progress made in microbial ecology and related fields is summarized, with a special emphasis on 8 landmark areas; the cultivation of uncultured microbes, in situ methods for the assessment of microorganisms and their activities, biofilms, plant microbiology, chemolithotrophic bacteria in early volcanic environments, symbionts of animals and their ecology, wastewater treatment microbiology, and the biodegradation of hazardous organic compounds.

Characterization and global gene expression of F- phenocopies during Escherichia coli biofilm formation.
Thithiwat May, Akinobu Ito, Satoshi Okabe
Molecular genetics and genomics : MGG, 284, 5, 333, 42, SPRINGER HEIDELBERG, 2010年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The ecological role of horizontal gene transfer within biofilms has been recently investigated, and it has been reported that conjugation directly induces bacteria to form biofilms via expression of conjugative pili. In this report, we described the contribution of bacterial conjugation during biofilm formation by Escherichia coli harboring a natural IncF conjugative F plasmid (F(+)). We showed that cell-to-cell pili interactions through the homosexual mating-pair formation among F(+) × F(+) cells (namely, F(-) phenocopy phenomenon) promote E. coli biofilm formation at the early development stage. The presence of F(+) × F(+) population is the result from heterogeneity within biofilms leading to sessile bacteria that grow at different rates, in which the late-stationary phase cells acted as F(-) phenocopy cells. According to global transcriptional analysis, the biofilm lifestyle shared similar gene expression pattern with F(-) phenocopies. F(-) phenocopy cells expressed specific sets of chromosomal genes (e.g., genes for general stress response and two-component systems) that control the regulation regions of F transfer operon by blocking surface exclusion proteins and DNA transfer machineries. However, mating-pair proteins were stabilized and consequently promoted F(+) × F(+) pili assembly. Thus, F(-) phenocopy phenomenon is an effective adaptive behavior of bacterial cells during biofilm formation.

Thermodynamic Properties of N-Isopropylacrylamide in Water: Solubility Transition, Phase Separation of Supersaturated Solution, and Glass Formation
Shigeo Sasaki, Satoshi Okabe, Yuji Miyahara
JOURNAL OF PHYSICAL CHEMISTRY B, 114, 46, 14995, 15002, AMER CHEMICAL SOC, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, The solubility of N-isopropylacrylamide (NIPA) in water was found to discretely change at 25 degrees C. The highly concentrated NIPA solution separated into two solutions, the concentrations of which were higher than the solubility below 25 degrees C and lower than the solubility above 25 degrees C. The X-ray crystallographic analysis indicated that a NIPA crystal formed in the aqueous solution was H(2)O free. When the aqueous NIPA phase-separated solutions were cooled down to -90 degrees C at a rate faster than 35 degrees C/min, the glassy structure formed. On the other hand, the crystalline solid formation of NIPA and H(2)O were observed when the solutions were cooled to 50 degrees C at a slower rate than 3 degrees C/min. The DSC measurements of the phase-separated solutions revealed that the energy levels of NIPA were +15.2, +11.5, and -0.08 kJ/mol (regarding the crystalline solid state at 25 degrees C as the ground state) for the liquid state, the H(2)O-poor solution and the H(2)O-rich solution in the phase-separated state, respectively. The experimental results are explained in terms of the molecular assemblies of NIPA and H(2)O molecules in the solutions.

Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor
Sunja Cho, Yoshitaka Takahashi, Naoki Fujii, Yohei Yamada, Hisashi Satoh, Satoshi Okabe
CHEMOSPHERE, 78, 9, 1129, 1135, PERGAMON-ELSEVIER SCIENCE LTD, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated nitrogen removal performance and responsible microbial community in an anaerobic up-flow granular bed anammox reactor. The anammox reactor was operated more than 1 year. Biomass in the reactor formed granules after about 2 months of operation, and a sufficient amount of the granules was retained in the reactor with a metallic net to avoid biomass washout during the entire operation. The average diameter of the granules was 3.6 mm at day 310. After 8 months of operation, stable nitrogen removal (60%) was achieved at an average total inorganic nitrogen removal rate of 14 kg-N m(-3) d(-1). The phylogenetic analysis and fluorescence in situ hybridization results revealed that the anammox granules consisted of mono species of anammox bacteria, "Candidatus Brocadia-like species", affiliated with "Candidatus Brocadia anammoxidans" with 16S rRNA gene sequence similarity of 95.7%. The relative abundance of the anammox bacteria in the granules was more than 80% of the total bacteria stained with 4',6-diamidino-2-phenylindole. The anammox bacteria were present throughout the granules whereas the other bacterial groups. Chloroflexi-like filamentous bacteria and betaproteobacterial ammonia-oxidizing bacteria, were mainly present on the surface of the anammox granules and around the anammox bacterial clusters. The in situ anammox activity was detected mainly from near the surface of granules to the upper 800 pm of the granules with microsensors. The granular anammox biomass tolerated higher concentrations of nitrite (400 mg-N L(-1)) than did the homogenized biomass (200 mg-N L(-1)) probably due to substrate diffusion limitation. (C) 2009 Elsevier Ltd. All rights reserved.

Microbial community structures and in situ sulfate-reducing and sulfur-oxidizing activities in biofilms developed on mortar specimens in a corroded sewer system.
Hisashi Satoh, Mitsunori Odagiri, Tsukasa Ito, Satoshi Okabe
Water research, 43, 18, 4729, 39, PERGAMON-ELSEVIER SCIENCE LTD, 2009年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microbially induced concrete corrosion (MICC) caused by sulfuric acid attack in sewer systems has been a serious problem for a long time. A better understanding of microbial community structures of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) and their in situ activities is essential for the efficient control of MICC. In this study, the microbial community structures and the in situ hydrogen sulfide production and consumption rates within biofilms and corroded materials developed on mortar specimens placed in a corroded manhole was investigated by culture-independent 16S rRNA gene-based molecular techniques and microsensors for hydrogen sulfide, oxygen, pH and the oxidation-reduction potential. The dark-gray gel-like biofilm was developed in the bottom (from the bottom to 4 cm) and the middle (4-20 cm from the bottom of the manhole) parts of the mortar specimens. White filamentous biofilms covered the gel-like biofilm in the middle part. The mortar specimens placed in the upper part (30 cm above the bottom of the manhole) were corroded. The 16S rRNA gene-cloning analysis revealed that one clone retrieved from the bottom biofilm sample was related to an SRB, 12 clones and 6 clones retrieved from the middle biofilm and the corroded material samples, respectively, were related to SOB. In situ hybridization results showed that the SRB were detected throughout the bottom biofilm and filamentous SOB cells were mainly detected in the upper oxic layer of the middle biofilm. Microsensor measurements demonstrated that hydrogen sulfide was produced in and diffused out of the bottom biofilms. In contrast, in the middle biofilm the hydrogen sulfide produced in the deeper parts of the biofilm was oxidized in the upper filamentous biofilm. pH was around 3 in the corroded materials developed in the upper part of the mortar specimens. Therefore, it can be concluded that hydrogen sulfide provided from the bottom biofilms and the sludge settling tank was emitted to the sewer atmosphere, then oxidized to corrosive compounds in the upper and middle parts of the manhole, and only the upper part of the mortar specimens were corroded, because in the middle part of the manhole the generated corrosive compounds (e.g., sulfuric acid) was reduced in the deeper parts of the biofilm.

Localized expression profiles of rpoS in Escherichia coli biofilms.
Akinobu Ito, Thithiwat May, Asami Taniuchi, Koji Kawata, Satoshi Okabe
Biotechnology and bioengineering, 103, 5, 975, 83, WILEY-BLACKWELL, 2009年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Although importance of the rpoS gene on biofilm formation by Escherichia coli has been suggested, there has not been any report showing where the rpoS is expressed during biofilm formation process. Since physiological state of the cells in the biofilms is considerably heterogeneous, the expression of the rpoS gene must be heterogeneous. In this study, in situ spatial expression of the rpoS gene during biofilm formation was investigated with an rpoS-gfp transcriptional fusion mutant strain. A ribosomal binding site and a gene encoding a green fluorescent protein were introduced into the downstream of the rpoS gene, which enabled us to observe the in situ spatial expression of the rpoS gene during biofilm formation processes without any disturbance of the rpoS expression. In the early stages of the biofilm formation process, the rpoS gene was expressed in the most of the cells. On the other hand, the rpoS expression was observed only at the outside of the biofilms during the late stages of the biofilm formation process. The in situ spatial expression of the rpoS gene in the biofilm was verified by quantifying the expression levels of the rpoS at the outside and the inside of the biofilms with the real time RT-PCR. In addition, global gene expression analysis was performed with DNA microarray to investigate physiological difference between the outside and the inside of the biofilms. This heterogeneous rpoS expression profile suggested that the cells at the outside of the biofilm need to express the rpoS to shift the physiological state to the stationary growth mode such as induction of various stress responses and suppression of the motility.

In vitro toxicity of silver nanoparticles at noncytotoxic doses to HepG2 human hepatoma cells.
Koji Kawata, Masato Osawa, Satoshi Okabe
Environmental science & technology, 43, 15, 6046, 51, AMER CHEMICAL SOC, 2009年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Although it has been reported that silver nanoparticles (Ag-NPs) have strong acute toxic effects to various cultured cells, the toxic effects at noncytotoxic doses are still unknown. We, therefore, evaluated in vitro toxicity of Ag-NPs at noncytotoxic doses in human hepatoma cell line, HepG2, based on cell viability assay, micronucleus test, and DNA microarray analysis. We also used polystyrene nanoparticles (PS-NPs) and silver carbonate (Ag2CO3) as test materials to compare the toxic effects with respect to different raw chemical composition and form of silver. The cell viability assay demonstrated that Ag-NPs accelerated cell proliferation at low doses (< 0.5 mg/L), which was supported by the DNA microarray analysis showing significant induction of genes associated with cell cycle progression. However, only Ag-NPs exposure exhibited a significant cytotoxicity at higher doses (> 1.0 mg/L) and induced abnormal cellular morphology, displaying cellular shrinkage and acquisition of an irregular shape. In addition, only Ag-NPs exposure increased the frequency of micronucleus formation up to 47.9 +/- 3.2% of binucleated cells, suggesting that Ag-NPs appear to cause much stronger damages to chromosome than PS-NPs and ionic Ag+. Cysteine, a strong ionic Ag+ ligand, only partially abolished the formation of micronuclei mediated by Ag-NPs and changed the gene expression, indicating that ionic Ag+ derived from Ag-NPs could not fully explain these biological actions. Based on these discussions, it is concluded that both "nanosized particle of Ag" as well as "ionic Ag+" contribute to the toxic effects of Ag-NPs.

Continuous power generation and microbial community structure of the anode biofilms in a three-stage microbial fuel cell system.
Kyungmi Chung, Satoshi Okabe
Applied microbiology and biotechnology, 83, 5, 965, 77, SPRINGER, 2009年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A mediator-less three-stage two-chamber microbial fuel cell (MFC) system was developed and operated continuously for more than 1.5 years to evaluate continuous power generation while treating artificial wastewater containing glucose (10 mM) concurrently. A stable power density of 28 W/m(3) was attained with an anode hydraulic retention time of 4.5 h and phosphate buffer as the cathode electrolyte. An overall dissolved organic carbon removal ratio was about 85%, and coulombic efficiency was about 46% in this MFC system. We also analyzed the microbial community structure of anode biofilms in each MFC. Since the environment in each MFC was different due to passing on the products to the next MFC in series, the microbial community structure was different accordingly. The anode biofilm in the first MFC consisted mainly of bacteria belonging to the Gammaproteobacteria, identified as Aeromonas sp., while the Firmicutes dominated the anode biofilms in the second and third MFCs that were mainly fed with acetate. Cyclic voltammetric results supported the presence of a redox compound(s) associated with the anode biofilm matrix, rather than mobile (dissolved) forms, which could be responsible for the electron transfer to the anode. Scanning electron microscopy revealed that the anode biofilms were comprised of morphologically different cells that were firmly attached on the anode surface and interconnected each other with anchor-like filamentous appendages, which might support the results of cyclic voltammetry.

A biofilm model for prediction of pollutant transformation in sewers
Feng Jiang, Derek Hoi-wai Leung, Shiyu Li, Guang-Hao Chen, Satoshi Okabe, Mark C. M. van Loosdrecht
WATER RESEARCH, 43, 13, 3187, 3198, PERGAMON-ELSEVIER SCIENCE LTD, 2009年07月, ［査読有り］
英語, 研究論文（学術雑誌）, This study developed a new sewer biofilm model to simulate the pollutant transformation and biofilm variation in sewers under aerobic, anoxic and anaerobic conditions. The biofilm model can describe the activities of heterotrophic, autotrophic, and sulfate-reducing bacteria (SRB) in the biofilm as well as the variations in biofilm thickness, the spatial profiles of SRB population and biofilm density. The model can describe dynamic biofilm growth, multiple biomass evolution and competitions among organic oxidation, denitrification, nitrification, sulfate reduction and sulfide oxidation in a heterogeneous biofilm growing in a sewer. The model has been extensively verified by three different approaches, including direct verification by measurement of the spatial concentration profiles of dissolved oxygen, nitrate, ammonia, and hydrogen sulfide in sewer biofilm. The spatial distribution profile of SRB in sewer biofilm was determined from the fluorescent in situ hybridization (FISH) images taken by a confocal laser scanning microscope (CLSM) and were predicted well by the model. (C) 2009 Elsevier Ltd. All rights reserved.

Increased antibiotic resistance of Escherichia coli in mature biofilms.
Akinobu Ito, Asami Taniuchi, Thithiwat May, Koji Kawata, Satoshi Okabe
Applied and environmental microbiology, 75, 12, 4093, 100, AMER SOC MICROBIOLOGY, 2009年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 microg/ml), kanamycin (25 microg/ml), and ofloxacin (10 microg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.

Comparison of gene expression profiles in HepG2 cells exposed to arsenic, cadmium, nickel, and three model carcinogens for investigating the mechanisms of metal carcinogenesis.
Koji Kawata, Ryuhei Shimazaki, Satoshi Okabe
Environmental and molecular mutagenesis, 50, 1, 46, 59, WILEY-LISS, 2009年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Carcinogenesis is an important chronic toxicity of metals and metalloids, although their mechanisms of action are still unclear. Comparison of gene expression patterns induced by carcinogenic metals, metalloids, and model carcinogens would give an insight into understanding of their carcinogenic mechanisms. In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2, after exposing to two metals (cadmium and nickel), a metalloid (arsenic), and three model carcinogenic chemicals N-dimethylnitrosoamine (DMN), 12-O-tetradecanoylphorbol-13-acetate (TPA), and tetrachloroethylene (TCE) using DNA microarrays with 8,795 human genes. Of the genes altered by As, Cd, and Ni exposures, 31-55% were overlapped with those altered by three model carcinogenic chemical exposures in our experiments. In particular, the metals and metalloid shared certain characteristics with TPA and TCE in remarkable upregulations of the genes associated with progression of cell cycle, which might play a central role in As, Cd, and Ni carcinogenesis. This characteristic of gene expression alteration was partially counteracted by intracellular accumulation of vitamin C in As-exposed cells, whereas the number of cell-cycle associated genes was increased in Cd- and Ni-exposed cells. In our experimental conditions, ROS might have an accelerative effect on the cell proliferation mechanisms of As, but have an inhibitory effect on those of other two heavy metals. Furthermore, based on the results of Q-PCR, the oncogene PTTG1, which was upregulated by all carcinogenic chemical exposures in the array experiments, might be a useful biomarker for evaluation of the carcinogenesis of inorganic carcinogens.

The Response of Pseudomonas aeruginosa PAO1 Efflux Pump-Defective Mutants to N-Octanoyl-L-Homoserine Lactone
Yutaka Yawata, Hideaki Maseda, Satoshi Okabe, Akinobu Ito, Isao Sawada, Hiroaki Kurashima, Hiroo Uchiyama, Nobuhiko Nomura
MICROBES AND ENVIRONMENTS, 24, 4, 338, 342, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2009年, ［査読有り］
英語, 研究論文（学術雑誌）, N-Octanoyl-L-homoserine lactone (C8-HSL) is an acyl-homoserine-lactone signal utilized in the quorum-sensing (QS) systems of Burkholderia cenocepacia and other bacterial species. Although also produced by Pseudomonas aeruginosa, its role in this species has not been elucidated. Here, we report that C8-HSL modulated antibiotic resistance and pyocyanin production in a P. aeruginosa efflux pump-deficient mutant. The rhl/las quorum-sensing system and qscR gene were both shown to be nonessential in the C8-HSL-induced changes in ofloxacin resistance, suggesting that P. aeruginosa possesses a distinct pathway to respond to C8-HSL.

Colorectal carcinoma: Local tumor staging and assessment of lymph node metastasis by high-resolution MR imaging in surgical specimens
Ichiro Yamada, Norio Yoshino, Akemi Tetsumura, Satoshi Okabe, Masayuki Enomoto, Kenichi Sugihara, Jiro Kumagai, Hitoshi Shibuya
International Journal of Biomedical Imaging, 2009, 659836, 2009年, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose. To assess the accuracy of high-resolution MR imaging as a means of evaluating mural invasion and lymph node metastasis by colorectal carcinoma in surgical specimens. Materials and Methods. High-resolution T1-weighted and T2-weighted MR images were obtained in 92 surgical specimens containing 96 colorectal carcinomas. Results. T2-weighted MR images clearly depicted the normal colorectal wall as consisting of seven layers. In 90 (94%) of the 96 carcinomas the depth of mural invasion depicted by MR imaging correlated well with the histopathologic stage. Nodal signal intensity on T2-weighted images (93%) and nodal border contour (93%) were more accurate than nodal size (89%) as indicators of lymph node metastasis, and MR imaging provided the highest accuracy (94%-96%) when they were combined. Conclusion. High-resolution MR imaging is a very accurate method for evaluating both mural invasion and lymph node metastasis by colorectal carcinoma in surgical specimens. Copyright © 2009 Ichiro Yamada et al.

Escherichia coli harboring a natural IncF conjugative F plasmid develops complex mature biofilms by stimulating synthesis of colanic acid and Curli.
Thithiwat May, Satoshi Okabe
Journal of bacteriology, 190, 22, 7479, 90, AMER SOC MICROBIOLOGY, 2008年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F(+) cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.

Quantification of cell specific uptake activity of microbial products by uncultured Chloroflexi by microautoradiography combined with fluorescence in situ hybridization.
Yuki Miura, Satoshi Okabe
Environmental science & technology, 42, 19, 7380, 6, AMER CHEMICAL SOC, 2008年10月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We, for the first time, quantitatively determined cell specific uptake activities of microbial products (bacterial cell detritus and extracellular polymeric substances, EPS) by the member of uncultured Chloroflexiby using a microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) technique. For this MAR-FISH analysis, we prepared [14C]-labeled microbial products from biomass sludge obtained and bacterial strains (Pseudomonas sp. and Acinetobacter sp.) isolated from our pilot-scale membrane bioreactor (MBR) as tracer substrates, which probably represent the more realistic food source in the MBR. The quantitative MAR-FISH analyses clearly showed that most of the uncultured Chloroflexi could indeed uptake the bacterial detritus of the two isolated strains with rates of 1.7-3.5 x 10(-7) g-C microm-2-surface area h(-1) (corresponding to 1.2-1.7 mg-C-bacterial detritus L(-1) h(-1)) in the cultures, which were, however, about 2 orders of magnitude lower than the uptake rates of simple monosaccharides (mannose, arabinose, fucose, and galactose). Based on these results and their high abundance (more than 20% of total bacteria detected with EUB338-mixed probes), it could be estimated that the uncultured Chloroflexi contributes 38-51% of the total degradation of microbial products occurred in the MAR-FISH cultures.

Community structure and function of candidate division TM7 in a wastewater treatment plant.　　　　　　　　　　　　　　　
Tomonori Kindaichi, Hiroshi Kajihara, Takashi Yamamoto, Noriatsu Ozaki, Akiyoshi Ohashi, Satoshi Okabe
12th International Symposium on Microbial Ecology (isme12), 2008年08月, ［査読有り］
研究論文（学術雑誌）

Significance of rpoS during maturation of Escherichia coli biofilms.
Akinobu Ito, Thithiwat May, Koji Kawata, Satoshi Okabe
Biotechnology and bioengineering, 99, 6, 1462, 71, JOHN WILEY & SONS INC, 2008年04月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.

Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering
Lucille Abad, Satoshi Okabe, Nlitsuhiro Shibayama, Hisaaki Kudo, Seiichi Saiki, Charito Aranilla, Lorna Relleve, Alumanda de la Rosa
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 42, 1, 55, 61, ELSEVIER SCIENCE BV, 2008年01月, ［査読有り］
英語, 研究論文（学術雑誌）, The conformational associative properties Of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100kGy. The con Fort national change in X-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and X-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, L-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan > kappa-carrageenan > iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation. (c) 2007 Elsevier B.V. All rights reserved.

Significance of Chloroflexi in performance of submerged membrane bioreactors (MBR) treating municipal wastewater.
Yuki Miura, Yoshimasa Watanabe, Satoshi Okabe
Environmental science & technology, 41, 22, 7787, 94, AMER CHEMICAL SOC, 2007年11月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We operated pilot-scale submerged membrane bioreactors (MBR) treating real municipal wastewater for over 3 months and observed an interesting phenomenon that carbohydrate concentrations in the MBRs rapidly increased, which consequently resulted in membrane fouling, when relative abundance of the member of uncultured Chloroflexi decreased from over 30% of total Bacteria to less than 10%. We, therefore, hypothesized that the uncultured Chloroflexi present in the MBRs could preferentially degrade carbohydrates and consequently prevent membrane fouling. To test this hypothesis, we investigated the phylogenetic identity, diversity, and in situ physiology (substrate utilization characteristics) of Chloroflexi residing in the MBR by using 16S rRNA gene sequencing analysis and microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) technique. Most of the clones related to the phylum Chloroflexiwere affiliated with the Chloroflexi subphylum 1 containing only a few cultured representatives. The MAR-FISH revealed that the members of Chloroflexi were metabolically versatile and could preferentially utilize glucose and N-acetyl glucosamine (a main substantial constituent of the cell wall peptidoglycan) under oxic and anoxic conditions. The utilization of these compounds was low at low pH. These findings suggest that the members of Chloroflexi are ecologically significant in the MBR treating municipal wastewater and are responsible for degradation of SMP including carbohydrates and cellular materials, which consequently reduces membrane fouling potential.

Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules.
Hisashi Satoh, Yuki Miura, Ikuo Tsushima, Satoshi Okabe
Applied and environmental microbiology, 73, 22, 7300, 7, AMER SOC MICROBIOLOGY, 2007年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH(4), H(2), pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH(4), H(2), pH, and ORP revealed that acid and H(2) production occurred in the upper part of the granule, below which H(2) consumption and CH(4) production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H(2) was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH(4) in the inner layer. We determined the effective diffusion coefficient for H(2) in the anaerobic granules to be 2.66 x 10(-5) cm(2) s(-1), which was 57% in water.

Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules
Hisashi Satoh, Yuki Miura, Ikuo Tsushima, Satoshi Okabe
Applied and Environmental Microbiology, 73, 22, 7300, 7307, 2007年11月
英語, 研究論文（学術雑誌）, The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH4, H2, pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH4, H2, pH, and ORP revealed that acid and H2 production occurred in the upper part of the granule, below which H2 consumption and CH4 production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H2 was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH4 in the inner layer. We determined the effective diffusion coefficient for H2 in the anaerobic granules to be 2.66 × 10-5 cm2 s-1, which was 57% in water. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Electrostatic self-assembly of neutral and polyelectrolyte block copolymers and oppositely charged surfactant
Masahiko Annaka, Kanae Morishita, Satoshi Okabe
JOURNAL OF PHYSICAL CHEMISTRY B, 111, 40, 11700, 11707, AMER CHEMICAL SOC, 2007年10月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated the phase behavior and the microscopic structure of the colloidal complexes constituted from neutral/polyelectrolyte diblock copolymers and oppositely charged surfactant by dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The neutral block is poly(N-isopropylacrylamide) (PNIPAM), and the polyelectrolyte block is negatively charged poly(acrylic acid) (PAA). In aqueous solution with neutral pH, PAA behaves as a weak polyelectrolyte, whereas PNIPAM is neutral and in good-solvent condition at ambient temperature, but in poor-solvent condition above similar to 32 degrees C. This block copolymer, PNIPAM-b-PAA with a narrow polydispersity, is studied in aqueous solution with an anionic surfactant, dodecyltrimethylammonium bromide (DTAB). For a low surfactant-to-polymer charge ratio Z lower than the critical value Z(C), the colloidal complexes are single DTAB micelles dressed by a few PNIPAM-b-PAA. Above Z(C), the colloidal complexes form a core-shell microstructure. The core of the complex consists of densely packed DTA(+) micelles, most likely connected between them by PAA blocks. The intermicellar distance of the DTA(+) micelles is similar to 39 angstrom, which is independent of the charge ratio Z as well as the temperature. The corona of the complex is constituted from the thermosensitive PNIPAM. At lower temperature the macroscopic phase separation is hindered by the swollen PNIPAM chains. Above the critical temperature T-C, the PNIPAM corona collapses leading to hydrophobic aggregates of the colloidal complexes.

Persistence of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental waters: effects of temperature and salinity.
Satoshi Okabe, Yoko Shimazu
Applied microbiology and biotechnology, 76, 4, 935, 44, SPRINGER, 2007年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Host-specific Bacteroides-Prevotella 16S rRNA genetic markers are promising alternative indicators for identifying the sources of fecal pollution because of their high abundance in the feces of warm-blooded animals and high host specificity. However, little is known about the persistence of these genetic markers in environments after being released into environmental waters. The persistence of feces-derived four different host-specific Bacteroides-Prevotella 16S rRNA genetic makers (total, human-, cow-, and pig-specific) in environmental waters was therefore investigated at different incubation temperatures (4, 10, 20, and 30 degrees C) and salinities (0, 10, 20, and 30 ppt) and then compared with the survival of conventional fecal-indicator organisms. The host-specific genetic markers were monitored by using real-time polymerase chain reaction (PCR) assays with specific primer sets. Each host-specific genetic marker showed similar responses in non-filtered river water and seawater: They persisted longer at lower temperatures and higher salinities. In addition, these markers did not increase in all conditions tested. Decay rates for indicator organisms were lower than those for host-specific genetic markers at temperature above 10 degrees C. Furthermore, we investigated whether the PCR-detectable 16S rRNA genetic markers reflect the presence of live target cells or dead target cells in environmental waters. The result revealed that the detection of the Bacteroides-Prevotella 16S rRNA genetic markers in environmental waters mainly reflected the presence of 'viable but non-culturable' Bacteroides-Prevotella cells. These findings indicate that seasonal and geographical variations in persistence of these host-specific Bacteroides-Prevotella 16S rRNA genetic markers must be considered when we use them as alternative fecal indicators in environmental waters.

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms
Tomonori Kindaichi, Ikuo Tsushima, Yuji Ogasawara, Masaki Shimokawa, Noriatsu Ozaki, Hisashi Satoh, Satoshi Okabe
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73, 15, 4931, 4939, AMER SOC MICROBIOLOGY, 2007年08月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phyllogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (< 1,000 mu m) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the How direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH4+, and NO2- consumption rates decreased from 0.68 and 0.64 mu mol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 mu mol cm(-2) h(-1) at P3 (the third port, 205 rum from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH4+ and NO2- and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O-2, or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators.
Olga Savichtcheva, Noriko Okayama, Satoshi Okabe
Water research, 41, 16, 3615, 28, PERGAMON-ELSEVIER SCIENCE LTD, 2007年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Occurrence and prevalence of different bacterial enteric pathogens as well as their relationships with conventional (total and fecal coliforms) and alternative fecal indicators (host-specific Bacteroides 16S rRNA genetic markers) were investigated for various water samples taken from different sites with different degrees of fecal contamination. The results showed that a wide range of bacterial pathogens could be detected in both municipal wastewater treatment plant samples and in surface water samples. Logistic regression analysis revealed that total and human-specific Bacteroides 16S rRNA genetic markers showed significant predictive values for the presence of Escheriachia coli O-157, Salmonella, heat-labile enterotoxin (LT) of enterotoxigenic E. coli (ETEC), and heat-stable enterotoxin for human (STh) of ETEC. The probability of occurrence of these pathogenic bacteria became significantly high when the concentrations of human-specific and total Bacteroides 16S rRNA genetic markers exceeded 10(3) and 10(4) copies/100 mL. In contrast, Clostridium perfringens was detected at high frequency regardless of sampling sites and levels of Bacteroides 16S rRNA genetic markers. No genes related to Shigella spp., Staphylococcus aureus and Vibrio cholerae were detected in any samples analyzed in this study. Conventional indicator microorganisms had low levels of correlation with the presence of pathogens as compared with the alternative fecal indicators. These results suggested that real-time PCR-based measurement of alternative Bacteroides 16S rRNA genetic markers was a rapid and sensitive tool to identify host-specific fecal pollution and probably associated bacterial pathogens. However, since one fecal indicator might not represent the relative abundance of all pathogenic bacteria, viruses and protozoa, combined application of alternative indicators with conventional ones could provide more comprehensive pictures of fecal contamination, its source and association with pathogenic microorganisms.

Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge.
Herto Dwi Ariesyady, Tsukasa Ito, Kazumi Yoshiguchi, Satoshi Okabe
Applied microbiology and biotechnology, 75, 3, 673, 83, SPRINGER, 2007年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The phylogenetic and functional diversity of syntrophic propionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR-FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52-62% of the total active POB under all the propionate concentrations tested (0.5-15 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobacter accounted for 16-31% of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration.

Classification of heavy-metal toxicity by human DNA microarray analysis.
Koji Kawata, Hiroyuki Yokoo, Ryuhei Shimazaki, Satoshi Okabe
Environmental science & technology, 41, 10, 3769, 74, AMER CHEMICAL SOC, 2007年05月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microarray technology is proving to be a useful tool to classify undefined environmental toxicants, to investigate underlying mechanisms of toxicity, and to identify candidate toxicant-specific genetic markers by examining global effects of putative toxicants on gene expression profiles. The aim of this study was to evaluate the toxicities of six heavy metals through the comparison with gene expression patterns induced by well-known chemicals. For this purpose, we first identified the genes altered specifically in HepG2 under the exposure of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), phenol, and N-nitrosodimethylamine (DMN), which were selected as the model chemicals, using DNA microarray. On the basis of the expression profiles of these genes, toxicities of six heavy metals, arsenic, cadmium, nickel, antimony, mercury, and chromium, were evaluated. The specific gene alteration and hierarchical clustering revealed that biological action of six heavy metals was clearly related to that of DMNQ which has been reported to be a reactive oxygen species (ROS) generating chemical and which induced the genes associated with cell proliferative responses. These results suggest that cell proliferative responses which are probably caused by ROS are a major apparent biological action of high-dose heavy metals, supporting the previous reports. Overall, a mechanism-based classification by DNA microarray would be an efficient method for evaluation of toxicities of environmental samples.

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors.
Ikuo Tsushima, Yuji Ogasawara, Tomonori Kindaichi, Hisashi Satoh, Satoshi Okabe
Water research, 41, 8, 1623, 34, PERGAMON-ELSEVIER SCIENCE LTD, 2007年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-Nm(-3)day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH(4)(+) to NO(2)(-) molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH(4)(+)/NO(2)(-) molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH(4)(+) and NO(2)(-)) of 4.45 g-N of (NH(4)(+)+NO(2)(-))m(-2)day(-1) was detected in the upper 800 microm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.

Quantification of host-specific Bacteroides-Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater.
Satoshi Okabe, Noriko Okayama, Olga Savichtcheva, Tsukasa Ito
Applied microbiology and biotechnology, 74, 4, 890, 901, SPRINGER, 2007年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9-11 log(10) copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides-Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r (2) = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50-800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems.
Satoshi Okabe, Mitsunori Odagiri, Tsukasa Ito, Hisashi Satoh
Applied and environmental microbiology, 73, 3, 971, 80, AMER SOC MICROBIOLOGY, 2007年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.

Bacterial community structures in MBRs treating municipal wastewater: relationship between community stability and reactor performance.
Yuki Miura, Mirian Noriko Hiraiwa, Tsukasa Ito, Takanori Itonaga, Yoshimasa Watanabe, Satoshi Okabe
Water research, 41, 3, 627, 37, PERGAMON-ELSEVIER SCIENCE LTD, 2007年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bacterial community structures in pilot-scale conventional membrane bioreactors (CMBRs) and hybrid MBRs (HMBRs) which were combined with pre-coagulation/sedimentation were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescence in situ hybridization (FISH) techniques. The results were compared with the community structure in a full-scale activated sludge (AS) process treating the same municipal wastewater. The Dice index (Cs) of similarity analysis of DGGE banding patterns demonstrated that the microbial community in AS was more similar to those in CMBR1 and CMBR2 than HMBR1 and HMBR2. This suggested that influent wastewater composition had a larger impact on bacterial community structures. Long-term community structure changes in the HMBRs and CMBRs were monitored and analyzed over 240 days by Non-metric multidimensional scaling (NMDS) analysis of DGGE banding patterns. The NMDS analysis revealed that both HMBRs and CMBRs had marked changes in community structures during the first about 100 days. Thereafter the perpetual fluctuations of bacterial community structures were observed in both HMBRs and CMBRs, even though the stable MBR performances (the performance was measured as membrane permeability and removal of dissolved organic carbon, DOC) were achieved. These results suggest that not only the stability, but also the adequate dynamics ("flexibility") of the bacterial community structure are important for the stable performance of the MBRs treating complex municipal wastewater.

Influences of infaunal burrows on the community structure and activity of ammonia-oxidizing bacteria in intertidal sediments.
Hisashi Satoh, Yoshiyuki Nakamura, Satoshi Okabe
Applied and environmental microbiology, 73, 4, 1341, 8, AMER SOC MICROBIOLOGY, 2007年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O(2) concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O(2) transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH(4)(+) consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH(4)(+) and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O(2) transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH(4)(+) consumption capacity. Consequently, the infaunal burrow became an important site for NH(4)(+) consumption in the intertidal sediment.

Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR
Ikuo Tsushima, Tomonori Kindaichi, Satoshi Okabe
Water Research, 41, 4, 785, 794, 4, 2007年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9×101 to 8.9×106 copies per PCR, corresponding to the detection limit of 3.6×103 target copies mL-1. A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples. © 2006 Elsevier Ltd. All rights reserved.

Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR.
Ikuo Tsushima, Tomonori Kindaichi, Satoshi Okabe
Water research, 41, 4, 785, 94, 2007年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9x10(1) to 8.9x10(6) copies per PCR, corresponding to the detection limit of 3.6x10(3) target copies mL(-1). A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples.

Influences of infaunal burrows on the community structure and activity of ammonia-oxidizing bacteria in intertidal sediments
Hisashi Satoh, Yoshiyuki Nakamura, Satoshi Okabe
Applied and Environmental Microbiology, 73, 4, 1341, 1348, 2007年02月
英語, 研究論文（学術雑誌）, Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O2 concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O2 transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH4+ consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH 4+ and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O2 transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH4+ consumption capacity. Consequently, the infaunal burrow became an important site for NH4+ consumption in the intertidal sediment. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Membrane biofouling in pilot-scale membrane bioreactors (MBRs) treating municipal wastewater: impact of biofilm formation.
Yuki Miura, Yoshimasa Watanabe, Satoshi Okabe
Environmental science & technology, 41, 2, 632, 8, AMER CHEMICAL SOC, 2007年01月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, For more efficient control and prediction of membrane biofouling in membrane bioreactors (MBRs), a fundamental understanding of mechanisms of membrane biofouling is essential. In this study, we operated full-scale submerged MBRs using real municipal wastewater delivered from the primary sedimentation basin of a municipal wastewater treatment facility over 3 months, and the adhesion and formation of biofilms on 0.4-microm pore size polyethylene hollow-fiber microfiltration (MF) membrane surfaces, separated from simple deposition of sludge cake, were monitored using scanning electron microscopy (SEM). In addition, the compositions of planktonic and biofilm microbial communities in the MBR were analyzed using culture independent molecular-based methods (i.e., fluorescent in situ hybridization (FISH) and 16S rRNA gene sequence analysis). The SEM and LIVE/DEAD staining analyses clearly showed that the biofilms gradually developed on the membrane surfaces with time, which had a strong positive correlation with the increase in trans-membrane pressure (TMP). This indicated that the biofilm formation induced the membrane fouling. The FISH results revealed that the microbial communities on membrane surfaces were quite different from those in the planktonic biomass in the mixed liquor. Moreover, FISH and 16S rRNA gene sequence analyses revealed that a specific phylogenetic group of bacteria, the Betaproteobacteria, probably played a major role in development of the mature biofilms, which led to the severe irreversible membrane biofouling.

Development of a super high-rate Anammox reactor and in situ analysis of biofilm structure and function.
Ikuo Tsushima, Yuji Ogasawara, Masaki Shimokawa, Tomonori Kindaichi, Satoshi Okabe
Water science and technology : a journal of the International Association on Water Pollution Research, 55, 8-9, 9, 17, IWA PUBLISHING, 2007年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The anaerobic ammonium oxidation (Anammox) process is a new efficient and cost effective method of ammonium removal from wastewater. Under strictly anoxic condition, ammonium is directly oxidised with nitrite as electron acceptor to dinitrogen gas. However, it is extremely difficult to cultivate Anammox bacteria due to their low growth rate. This suggests that a rapid and efficient start-up of Anammox process is the key to practical applications. To screen appropriate seeding sludge with high Anammox potential, a real-time quantitative PCR assay with newly designed primers has been developed. Thereafter, the seeding sludge with high abundance of Anammox bacteria (1.7 x 10(8) copies/mg-dry weight) was selected and inoculated into an upflow anaerobic biofilters (UABs). The UABs were operated for more than 1 year and the highest nitrogen removal rate of 24.0 kg-N m-3 day(-1) was attained. In addition, the ecophysiology of Anammox bacteria (spatial distribution and in situ activity) in biofilms was analysed by combining a full-cycle 16S rRNA approach and microelectrodes. The microelectrode measurement clearly revealed that a successive vertical zonation of the partial nitrification (NH4+ to NO2-), Anammox reaction and denitrification was developed in the biofilm in the UAB. This result agreed with the spatial distribution of corresponding bacterial populations in the biofilm. We linked the micro-scale information (i.e. single cell and/or biofilm levels) with the macro-scale information (i.e. the reactor level) to understand the details of Anammox reaction occurring in the UABs.

Views on youth job satisfaction of non-regular employment: Focusing on single male non-regular employees
Satoshi Okabe
SOCIOLOGICAL THEORY AND METHODS, 22, 2, 169, 187, JAPANESE ASSOC MATHEMATICAL SOCIOLOGY, 2007年, ［査読有り］
日本語, 研究論文（学術雑誌）, Since 1990's in Japan, the increase of non-regular employment in youth has had a great impact on the way of getting their achievement of their social status. Ever since then, a lot of studies focusing on the differences among youth, such as income gap, have been provided, whereas studies about consciousness or sense of values of youth are relatively few. In this paper, I pay an attention to job satisfaction of Japanese youth and try to find determinant factors of their job satisfaction. As a result, job satisfaction of single male non-regular employees remains at the lowest level among young people. The youth belonging to this category has an affinity with a jobless youth on life awareness side, and would not have enough living or educational background. Focusing on one's past experience, I found that the experience to come in contact with adults is an essential factor of job satisfaction. To solve the issues of employment in youth, it is important not only to improve the work condition of youth, but also to promote the experience to come in contact with adults at a compulsory education stage.

Structural transition in block and gradient copolymer aqueous solutions
Satoshi Okabe, Chieko Fuse, Shinji Sugihara, Sadahito Aoshima, Mitsuhiro Shibayama
PHYSICA B-CONDENSED MATTER, 385, 756, 758, ELSEVIER SCIENCE BV, 2006年11月, ［査読有り］
英語, 研究論文（学術雑誌）, The structural transitions in block (Block) and gradient copolymer (Grad) aqueous solutions were investigated with small-angle neutron scattering and were compared to each other. The micelle formation was observed both in Grad and in Block systems. The micelle formation in Grad system was rather continuous, while that in Block underwent a stepwise transition. The size and its variation with temperature of the resulting micelles in Grad copolymer were also different from those in Block system. Possible transition mechanisms for these copolymer systems are described by taking account of the continuous variation in the composition along the polymer chain of Grad copolymer. (c) 2006 Elsevier B.V. All rights reserved.

PB-07 異種微生物間コミュニケーションによるPseudomonas aeruginosaの抗生物質耐性、病原性、遺伝子発現制御の発見(共生/相互作用,ポスターセッションB,(1)ポスター発表会,研究発表会)
八幡, 穣, 岡部, 聡, 伊藤, 暁信, 間世田, 英明, 内山, 裕夫, 野村, 暢彦, NAKAJIMA, Toshiaki
日本微生物生態学会講演要旨集, 0, 22, 125, 125, 日本微生物生態学会, 2006年10月
日本語, 研究論文（学術雑誌）

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR
Tomonori Kindaichi, Yoshiko Kawano, Tsukasa Ito, Hisashi Satoh, Satoshi Okabe
BIOTECHNOLOGY AND BIOENGINEERING, 94, 6, 1111, 1121, JOHN WILEY & SONS INC, 2006年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O-2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities. (c) 2006 Wiley Periodicals, Inc.

Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms
H Satoh, T Yamakawa, T Kindaichi, T Ito, S Okabe
BIOTECHNOLOGY AND BIOENGINEERING, 94, 4, 762, 772, JOHN WILEY & SONS INC, 2006年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial waste-water were investigated by combination of the 16S rRNA gene clone analysis, fluorescence in situ hybridization (FISH) and microelectrodes. These results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (IWTP). Both nitrification and denitrification occurred in the primary denitrification (PD) tank and denitrification occurred in the secondary denitrification (SD) tank. In contrast, nitrification and denitrification rates were very low in the nitrification (N) tank. 16S rRNA gene clone sequence analysis revealed that the bacteria affiliated with Alphaproteobacteria, followed by Betaproteobacteria, were numerically important microbial groups in three tanks. The many clones affiliated with Alphaproteobacteria were closely related to the denitrifying bacteria (e.g., Hyphomicrobium spp., Rhodopseudomonas palustris, and Rhodobacter spp.). In addition, Methylophilus leisingeri affiliated with Betaproteobacteria, which favorably utilized methanol, was detected only in the SD-tank to which methanol was added. Nitrosomonas europaea and Nitrosomonas marina were detected as the ammonia-oxidizing bacteria affiliated with Betaproteobacteria throughout this plant, although the dominant species of them was different among three tanks. Nitrifying bacteria were mainly detected in the upper parts of the PD-biofilm whereas their populations were low in the upper parts of the N-biofilm. The presence of denitrifying bacteria affiliated with Hyphomicrobium spp. in SD- and N-biofilms was verified by FISH analysis. Microelectrode measurements showed that the nitrifying bacteria present in the N- and PD-biofilms were active and the bacteria present in the SD-biofilm could denitrify. (c) 2006 Wiley Periodicals, Inc.

Alternative indicators of fecal pollution: relations with pathogens and conventional indicators, current methodologies for direct pathogen monitoring and future application perspectives.
Olga Savichtcheva, Satoshi Okabe
Water research, 40, 13, 2463, 76, PERGAMON-ELSEVIER SCIENCE LTD, 2006年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The ecological and survival characteristics of bacterial, viral and parasitic pathogens vary under environmental conditions, indicating that probably no single indicator organism can predict the presence of all enteric pathogens for all types of waters and different host-associated fecal pollution. If there are true correlations between indicator organisms and pathogens, it is necessary to find out to what extent and under which circumstances these organisms can be used as reliable indicators of fecal pollution. Application of conventional and alternative fecal indicators has greatly enhanced our abilities to predict and reduce health risk associated with the use of surface waters. New molecular-based techniques have shown that combined use of conventional and alternative indicators for fecal pollution increases both the detection sensitivity and specificity of fecal pollution and associated pathogens. In this review, we, therefore, summarize the advantages and limitations of conventional and alternative fecal indicators in terms of predicting pathogen presence as well as current and future methodologies for direct pathogen monitoring in environmental waters. This manuscript is mainly focused on the relationships between microbial fecal indicators and the presence of pathogens, which have not previously been summarized yet and could nicely supplement with recent literature reviews on microbial source tracking.

Community structure, abundance, and in situ activity of nitrifying bacteria in river sediments as determined by the combined use of molecular techniques and microelectrodes.
Yoshiyuki Nakamura, Hisashi Satoh, Tomonori Kindaichi, Satoshi Okabe
Environmental science & technology, 40, 5, 1532, 9, AMER CHEMICAL SOC, 2006年03月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The community structure, spatial distributions, and in situ activity of ammonia-oxidizing bacteria (AOB) representing the Betaproteobacteria and nitrite-oxidizing bacteria (NOB) representing the genus Nitrospira in three different river sediments with different pollution sources and levels along the Niida River, Hachinohe, Japan, were investigated by the combined use of 16S rRNA gene-cloning analysis, real-time quantitative polymerase chain reaction (RTQ-PCR) assays, and microelectrodes. The goal of this research was to evaluate the contribution of nitrifying activity in the sediment to the overall nitrogen elimination rate in this river. The 16S rRNA gene-cloning analysis revealed that the community structures of AOB and Nitrospira-like NOB are present in three sediments. On the basis of the results of 16S rRNA gene-cloning analysis, the RTQ-PCR assay using a TaqMan probe was developed and optimized for the quantification of the Nitrospira-like NOB. In the sediments, AOB specific 16S rRNA genes were detected in the range of 10(6) to 10(7) copies/cm3 and evenly distributed over the sampled sediment depth (0-5 mm), whereas the Nitrospira-like NOB 16S rRNA gene copy numbers per cm3 were 1-2 orders of magnitude higher than the AOB copy numbers. Under light conditions, intensive oxygenic photosynthesis occurred in the surface and increased the maximal O2 concentration and O2 penetration depth in all sediments. This concomitantly stimulated nitrifying bacteria present in diurnally anoxic deeper zones and expanded nitrification zones, which consequently increased the total NH4+ consumption rate in the sediment (i.e., total NH4+ flux into the sediment). The results suggested that the in situ nitrifying activity was restricted mainly to the surface 2 mm of the sediment and linked with photosynthetic activity, which obviously plays an important role in nitrogen elimination in this river.

A vista for microbial ecology and environmental biotechnology
BE Rittmann, M Hausner, F Loffler, NG Love, G Muyzer, S Okabe, DB Oerther, J Peccia, L Raskin, M Wagner
ENVIRONMENTAL SCIENCE & TECHNOLOGY, 40, 4, 1096, 1103, AMER CHEMICAL SOC, 2006年02月, ［査読有り］
英語

Distribution analyses of multi-modal dynamic light scattering data　　　　　　　　　　　　　　　
Satoshi Okabe, Takeshi Karino, Mitsuhiro Shibayama
Polymer Preprints, Japan, 55, 2, 3218, 3219, 2006年
日本語, 研究論文（国際会議プロシーディングス）, A method of data analysis for dynamic light scattering is proposed to evaluate the weight fraction, w(Rh), of a small amount of large aggregates in a dilute solution, where Rh is the hydrodynamic radius. We examined the time-correlation function of scattering intensity for model multi-modal systems, i.e., mixtures of latex solutions having different particle sizes and of polystyrene standard solutions having different molecular weights, by properly taking into account the unknown fractions, w(Rh), and scattering intensities of individual components. We derived an equation to evaluate the weight fractions of the components. The validity of this method was verified by successfully reconstructing the observed correlation functions having fast and slow modes. Examples of application not only to particle systems but also to polymer solutions will be shown.

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water.
Olga Savichtcheva, Noriko Okayama, Tsukasa Ito, Satoshi Okabe
Biotechnology and bioengineering, 92, 3, 356, 63, JOHN WILEY & SONS INC, 2005年11月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.

PB-37 異種微生物間コミュニケーションによるPseudomonas aeruginosaの抗生物質耐性、病原性、遺伝子発現制御の発見(共生/相互作用,ポスターセッションB,ポスター発表)
八幡, 穣, 岡部, 聡, 伊藤, 暁信, 間世田, 英明, 内山, 裕夫, 野村, 暢彦
日本微生物生態学会講演要旨集, 0, 21, 157, 157, 日本微生物生態学会, 2005年10月
日本語, 研究論文（学術雑誌）

Involvement of cyclin D3 in liver metastasis of colorectal cancer, revealed by genome-wide copy-number analysis
H Tanami, H Tsuda, S Okabe, T Iwai, K Sugihara, Imoto, I, J Inazawa
LABORATORY INVESTIGATION, 85, 9, 1118, 1129, NATURE PUBLISHING GROUP, 2005年09月, ［査読有り］
英語, 研究論文（学術雑誌）, The question of whether any genetic differences exist between primary and colorectal cancers (CRCs) and their metastatic foci is controversial. To look for genetic aberrations involved in metastasis of CRCs to the liver, we performed subtractive comparative genomic hybridization (CGH) experiments using paired samples from 20 CRC patients with primary tumors and synchronous or metachronous liver metastases. Relatively frequent gains in DNA copy number were detected at 6p, suggesting the presence of one or more metastasis-related genes in the region. Analysis of 11 CRC cell lines using array-based CGH (CGH-array) revealed one 6p candidate gene, CCND3. Quantitative reverse transcriptase-polymerase chain reaction experiments showed that CCND3 was significantly upregulated in liver-metastatic lesions compared with primary lesions (P<0.0152). In addition, immunohistochemical analysis of 120 primary CRC tumors demonstrated that cyclin D3 expression in the region of rolled edge was significantly associated with total recurrence, especially hematogenous recurrence ( P = 0.0307). The results implied involvement of cyclin D3 in liver metastasis of CRC, and the data may contribute to the development of a novel therapy or diagnostic agent for this currently intractable disease. Our experiments also confirmed the power of subtractive CGH and CGH-array analysis for identifying cancer-related genes.

Use of microelectrodes to investigate the effects of 2-chlorophenol on microbial activities in biofilms
H Satoh, Y Sasaki, Y Nakamura, S Okabe, T Suzuki
BIOTECHNOLOGY AND BIOENGINEERING, 91, 2, 133, 138, JOHN WILEY & SONS INC, 2005年07月, ［査読有り］
英語, 研究論文（学術雑誌）, In order to assess the applicability of using microelectrodes as a tool for inhibition tests, temporal and spatial inhibitory effects of 2-chlorophenol (2-CP) on O-2 respiration and nitrification activities in municipal wastewater biofilms were investigated using microelectrodes for O-2 and NH4+. The time-course microelectrode measurements demonstrated that 2-CP inhibited O-2 respiration and nitrification activities within 6-18 min. The microbial activities were inhibited only in the upper 400 pm of the biofilms by 2-CP, and the bacteria present in the deeper parts of the biofilms were still active, probably due to limited penetration of 2-CP. These results could reasonably explain the difference in inhibitory ratios of the O-2 respiration and nitrification activities in the biofilms. O-2 respiration activity was incompletely inhibited, which was attributed to the presence Of O-2 respiration activities in the deeper parts of the biofilm. In contrast, nitrification activity was significantly inhibited because ammonia-oxidizing bacteria were present in the upper parts of the biofilm. These results indicate that the microelectrodes with a very quick response time and a high spatial resolution are useful tools to study temporal and spatial inhibitory effects of inhibitors on in situ microbial activities in biofilms. (c) 2005 Wiley Periodicals, Inc.

Fate of C-14-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms
S Okabe, T Kindaichi, T Ito
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 71, 7, 3987, 3994, AMER SOC MICROBIOLOGY, 2005年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The cross-feeding of microbial products derived from C-14-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [C-14] bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of C-14 originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of C-14-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized C-14-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of C-14-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.

Succession of internal sulfur cycles and sulfur-oxidizing bacterial communities in microaerophilic wastewater biofilms.
Satoshi Okabe, Tsukasa Ito, Kenichi Sugita, Hisashi Satoh
Applied and environmental microbiology, 71, 5, 2520, 9, AMER SOC MICROBIOLOGY, 2005年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S(0) accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H(2)S was only partially oxidized to S(0) by mainly Thiomicrospira denitirificans with NO(3)(-) as an electron acceptor, leading to significant accumulation of S(0) in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S(0) accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S(0) to SO(4)(2-) under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 10(9) cells cm(-3)) and strain SO07 (ca. 10(8) cells cm(-3)) were found at the sulfur-rich surface (100 microm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 10(7) to 10(8) cells cm(-3). Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H(2)S produced by SRB to SO(4)(2-) in the second phase, indicating establishment of the complete sulfur cycle in the biofilms.

Thiovirga sulfuroxydans gen. nov., sp. nov., a chemolithoautotrophic sulfur-oxidizing bacterium isolated from a microaerobic waste-water biofilm.
Tsukasa Ito, Kenichi Sugita, Isao Yumoto, Yoshinobu Nodasaka, Satoshi Okabe
International journal of systematic and evolutionary microbiology, 55, Pt 3, 1059, 1064, SOC GENERAL MICROBIOLOGY, 2005年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A novel mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain SO07(T), was isolated from a microaerobic waste-water biofilm. Chemolithoautotrophic growth was observed with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor. Anaerobic and heterotrophic growth were not observed. Nitrate was not used as a terminal electron acceptor. The optimum pH and temperature for growth were pH 7.5 and 30 degrees C, respectively. The major isoprenoid quinone was Q-8. The DNA G + C content of strain SO07(T) was 47.1 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that strain SO07(T) formed a monophyletic group in the gamma-Proteobacteria with only 89 % similarity to members of the genus Halothiobacillus, its nearest phylogenetic neighbours. In addition, the isolate differed from members of the genus Halothiobacillus in its requirement for and tolerance of NaCl; strain SO07(T) was unable to grow in NaCl concentrations of more than 180 mM. On the basis of phylogenetic, chemotaxonomic and physiological data, it is proposed that isolate SO07(T) (=JCM 12417(T) = ATCC BAA-1033(T)) represents the type strain of a novel species in a new genus, Thiovirga sulfuroxydans gen. nov., sp. nov.

Isolation, characterization, and in situ detection of a novel chemolithoautotrophic sulfur-oxidizing bacterium in wastewater biofilms growing under microaerophilic conditions.
Tsukasa Ito, Kenichi Sugita, Satoshi Okabe
Applied and environmental microbiology, 70, 5, 3122, 9, AMER SOC MICROBIOLOGY, 2004年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S(0)), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 micro m) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H(2)S and S(0) to SO(4)(2-) under oxic conditions.

Photosynthesis in sediments determined at high spatial resolution by the use of microelectrodes.
Yoshiyuki Nakamura, Hisashi Satoh, Satoshi Okabe, Yoshimasa Watanabe
Water research, 38, 9, 2439, 47, PERGAMON-ELSEVIER SCIENCE LTD, 2004年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The present study investigated photosynthetic rates and their regulation by light within the upper 5mm of sediment in a tidal area of Niida River in Hachinohe, Japan. Steady-state concentration profiles of O(2), NH(4)(+), NO(2)(-), H(2)S, and pH in the sediment were measured with microelectrodes. Microzonation of O(2) respiration, denitrification and SO(4)(2-) reduction was found in the sediment. When light intensities exceeded 1050 micromol photons/m(2)/s, net photosynthetic activity was detected in the upper 0.5mm of the microbial mat colonizing on the sediment surface in the tidal area. In contrast, gross photosynthetic activity was detected in the upper 1.0mm of the microbial mat at 1900 micromol photons/m(2)/s. As light intensity increased, the net photosynthetic rate and O(2) penetration depth increased. The maximal net photosynthetic rate and O(2) penetration depth were 6.1 micromol O(2)/cm(3)/h and 2.2mm, respectively, at 1900 micromol photons/m(2)/s. Net photosynthetic rates in the microbial mat in the tidal area were lower than in the upstream sediment. The analysis of continuous O(2) concentration measurements in different layers of the microbial mat during artificial light-dark cycles demonstrated that the photosynthetic activity response to changes in light intensity was extremely fast (a few seconds) and the O(2) concentration in the microbial mat became stable within 200s. The measurement of physical and chemical parameters in river water revealed that the study site was relatively polluted and sunlight intensity significantly fluctuated temporally. These results suggested that the in situ microbial processes occurring in the sediment fluctuated in accordance with periodic fluctuations in sunlight intensity.

Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization
Tomonori Kindaichi, Tsukasa Ito, Satoshi Okabe
Applied and Environmental Microbiology, 70, 3, 1641, 1650, 3, 2004年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4+ as an energy source were investigated by using a full-cycle 16S RRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (α-Proteobacteria), 23%
γ-Proteobacteria, 13%
green nonsulfur bacteria (GNSB), 9%
Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%
and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. α- and γ -Proteobacteria dominated the utilization of [14C]acetic acid and 14C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1- 14C]glucosamine (NAG). The GNSB accounted for 9% of the 14C-amino acid-consuming bacteria and 27% of the [ 14C]NAG-consuming bacteria but did not utilize [14C] acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.

Macroscale and microscale analyses of nitrification and denitrification in biofilms attached on membrane aerated biofilm reactors
H Satoh, H Ono, B Rulin, J Kamo, S Okabe, KI Fukushi
WATER RESEARCH, 38, 6, 1633, 1641, PERGAMON-ELSEVIER SCIENCE LTD, 2004年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A membrane aerated biofilm reactor (MABR), in which O-2 was supplied from the bottom of the biofilm and NH4+ and organic carbon were supplied from the biofilm surface, was operated at different organic carbon loading rates and intra-membrane air pressures to investigate the occurrence of simultaneous chemical oxygen demand (COD) removal, nitrification and denitrification. The spatial distribution of nitrification and denitrification zones in the biofilms was measured with microelectrodes for O-2, NH4+, NO2-, NO3- and pH. When the MABR was operated at approximately 1.0 g-COD/m(2)/day of COD loading rate, simultaneous COD removal, nitrification and denitrification could be achieved. The COD loading rates and the intra-membrane air pressures applied in this study had no effect on the startup and the maximum rates of NH4+ oxidation in the MABRs. Microelectrode measurements showed that O-2 was supplied from the bottom of the MABR biofilm and penetrated the whole biofilm. Because the biofilm thickness increased during the operations, an anoxic layer developed in the upper parts of the mature biofilms while an oxic layer was restricted to the deeper parts of the biofilms. The development of the anoxic zones in the biofilms coincided with increase in the denitrification rates. Nitrification occurred in the zones from membrane surface to a point of ca. 60 mum. Denitrification mainly occurred just above the nitrification zones. The COD loading rates and the intra-membrane air pressures applied in this study had no effect on location of the nitrification and denitrification zones. (C) 2004 Elsevier Ltd. All rights reserved.

Macroscale and microscale analyses of nitrification and denitrification in biofilms attached on membrane aerated biofilm reactors
H Satoh, H Ono, B Rulin, J Kamo, S Okabe, KI Fukushi
WATER RESEARCH, 38, 6, 1633, 1641, PERGAMON-ELSEVIER SCIENCE LTD, 2004年03月
英語, 研究論文（学術雑誌）, A membrane aerated biofilm reactor (MABR), in which O-2 was supplied from the bottom of the biofilm and NH4+ and organic carbon were supplied from the biofilm surface, was operated at different organic carbon loading rates and intra-membrane air pressures to investigate the occurrence of simultaneous chemical oxygen demand (COD) removal, nitrification and denitrification. The spatial distribution of nitrification and denitrification zones in the biofilms was measured with microelectrodes for O-2, NH4+, NO2-, NO3- and pH. When the MABR was operated at approximately 1.0 g-COD/m(2)/day of COD loading rate, simultaneous COD removal, nitrification and denitrification could be achieved. The COD loading rates and the intra-membrane air pressures applied in this study had no effect on the startup and the maximum rates of NH4+ oxidation in the MABRs. Microelectrode measurements showed that O-2 was supplied from the bottom of the MABR biofilm and penetrated the whole biofilm. Because the biofilm thickness increased during the operations, an anoxic layer developed in the upper parts of the mature biofilms while an oxic layer was restricted to the deeper parts of the biofilms. The development of the anoxic zones in the biofilms coincided with increase in the denitrification rates. Nitrification occurred in the zones from membrane surface to a point of ca. 60 mum. Denitrification mainly occurred just above the nitrification zones. The COD loading rates and the intra-membrane air pressures applied in this study had no effect on location of the nitrification and denitrification zones. (C) 2004 Elsevier Ltd. All rights reserved.

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization.
Tomonori Kindaichi, Tsukasa Ito, Satoshi Okabe
Applied and environmental microbiology, 70, 3, 1641, 50, AMER SOC MICROBIOLOGY, 2004年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH(4)(+) as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (alpha-Proteobacteria), 23%; gamma-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. alpha- and gamma-Proteobacteria dominated the utilization of [ (14)C]acetic acid and (14)C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1-(14)C]glucosamine (NAG). The GNSB accounted for 9% of the (14)C-amino acid-consuming bacteria and 27% of the [ (14)C]NAG-consuming bacteria but did not utilize [ (14)C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms.
Satoshi Okabe, Tomonori Kindaichi, Tsukasa Ito, Hisashi Satoh
Biotechnology and bioengineering, 85, 1, 86, 95, JOHN WILEY & SONS INC, 2004年01月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.

MAR-FISH-An Ecophysiological Approach to Link Phylogenetic Affiliation and in Situ Metabolic Activity of Microorganisms at a Single-Cell Resolution
Satoshi Okabe, Tomonori Kindaichi, Tsukasa Ito
Microbes and Environments, 19, 2, 83, 98, 2004年
英語, 研究論文（学術雑誌）, A major goal of microbial ecology is to study the abundance, localization, and activities of microorganisms in situ in order to understand ecophysiological roles that the microorganisms play in complex natural ecosystems. In fact, in typical microbial habitats such as biofilms, sediments, and microbial aggregates, resources and physicochemical conditions are dynamically changing with time and across even a very tiny distance because of metabolic activities and substrate transport limitation. To directly correlate microbial identity (16S rRNA-based phylogeny) to the specific metabolic function of individual cells within such complex and heterogeneous microbial habitats, several new molecular-based techniques have been developed in the last decade. These techniques exploit in situ simultaneous phylogenetic identification and metabolic capabilities of even uncultured microorganisms without the need to isolate them in culture. Microautoradiography is a powerful but “rather old” tool, with which the in situ uptake of specific radiolabeled substrates by individual cells can be determined. Fluorescence in situ hybridization (FISH) is a new molecular-based technique that allows the in situ phylogenetic identification of individual cells. However, FISH cannot provide sufficient information on metabolic capabilities, because phylogeny and phenotype are rarely congruent. Recently, microautoradiography and FISH have been successfully combined to further improve the complementary strengths of the two methods. Microautoradiography combined with FISH (MAR-FISH) can be used to simultaneously examine the phylogenetic identity and the relative or actual specific activity of microorganisms within a complex microbial community at a single-cell level. This article overviews the principle, experimental protocol and application of the MAR-FISH technique, as well as current developments of other analytical techniques for in situ microbial functions (metabolic activities) from a single-cell level to community levels. © 2004, Japanese Society of Microbial Ecology &
The Japanese Society of Soil Microbiology. All rights reserved.

Analysis of microbial community structure and in situ activity of nitrifying biofilms
Okabe S, Satoh H, Ito T, Watanabe Y
Journal of Water and Environment Technology, 2, 2, 65, 74, Japan Society on Water Environment, 2004年
英語, Wastewater biofilms are very complex multispecies biofilms, displaying considerable heterogeneity with respect to both the microorganisms present and their physicochemical microenvironments. To understand the eco-physiology of individual microorganisms in the biofilm, techniques and tools with a high spatial and temporal resolution are required for direct detection of the spatial distributions of microbial species and their activities in minimally disturbed their natural habitats (e.g., biofilms). In this paper, we will, therefore, address the great potential of the combined use of the current FISH technique and microelectrodes to study the microbial ecology of complex microbial communities such as biofilms. The combination of these two techniques will provide reliable and direct information about relationships between in situ microbial activity and the occurrence of specific microorganisms in biofilms. As an example of the combined study, we will illustrate the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria on fine scale in autotrophic nitrifying biofilms by applying the full-cycle of 16S rRNA approach followed by fluorescence in situ hybridization (FISH), which is linked to their in situ activity distributions at a similar resolution determined by use of microelectrodes. The combination of these techniques allows relating in situ microbial activity directly to occurrence of nitrifying bacteria population.

Cyclooxygenase-2 expression and its relationship with proliferation of colorectal adenomas
T Sato, K Yoshinaga, S Okabe, T Okawa, T Higuchi, M Enomoto, T Takizawa, K Sugihara
JAPANESE JOURNAL OF CLINICAL ONCOLOGY, 33, 12, 631, 635, OXFORD UNIV PRESS, 2003年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Cyclooxygenase (COX)-2 may be linked to carcinogenesis. In the previous study, we examined COX-2 expression immunohistochemically in 95 adenomas and reported a significant correlation between its expression and the grade of dysplasia. To clarify the correlation between COX-2 expression and cell proliferation, we investigated Ki-67 labeling index using immunohistochemistry and its correlation with COX-2 expression.
Methods: Immunohistological staining for Ki-67 antigen was performed on 95 colorectal adenomas previously reported.
Results: The Ki-67 labeling index was significantly higher in the high-COX-2 group than in the low-COX-2 and negative groups in adenomas with moderate (44.5 +/- 6.4% vs 33.0 +/- 2.6%, 39.0 +/- 6.2%; P = 0.01, P < 0.001, respectively) or severe dysplasia (47.2 +/- 7.6% vs 40.3 +/- 7.2%, 35.0 +/- 5.4%; P = 0.02, P = 0.005, respectively). There was no correlation between Ki-67 labeling index and COX-2 expression in mild dysplasia.
Conclusions: These results suggest that COX-2 may play a causal role in cell proliferation in carcinogenesis.

Effect of oxygen concentration on nitrification and denitrification in single activated sludge flocs
H Satoh, Y Nakamura, H Ono, S Okabe
BIOTECHNOLOGY AND BIOENGINEERING, 83, 5, 604, 607, JOHN WILEY & SONS INC, 2003年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Simultaneous nitrification and denitrification (SND) was investigated in the single aeration tank of a municipal wastewater treatment plant. Microelectrode measurements and batch experiments were performed to test for the presence of SND. Microelectrodes recorded the presence of O-2 concentration gradients in individual activated sludge flocs. When the O-2 concentration in the bulk liquid was <45 μM, anoxic zones were detected within flocs with a larger diameter (approximately 3000 pm). The O-2 penetration depth in the floc was found to be dependent on the O-2 concentration in the bulk liquid. Nitrification was restricted to the oxic zones, whereas denitrification occurred mainly in the anoxic zones. The nitrification rate of the activated sludge increased with increasing O-2 concentration in the bulk liquid, up to 40 μM, and remained constant thereafter. SND was observed in the aerated activated sludge when O-2 concentration was in the range of 10 to 35 μM. (C) 2003 Wiley Periodicals, Inc.

Dynamic response of nitrifying activated sludge batch culture to increased chloride concentration
GH Chen, MT Wong, S Okabe, Y Watanabe
WATER RESEARCH, 37, 13, 3125, 3135, PERGAMON-ELSEVIER SCIENCE LTD, 2003年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Dynamic response of nitrifying activated sludge batch cultures to increased chloride concentration was studied in this paper, which focused upon the changes in the specific nitrification rate (SNR) and nitrifier population when the chloride level was gradually or stepwise increased to 30,000 mg Cl L-1. The dominant species of ammonia-oxidizers and nitrite-oxidizers in the population were examined by Fluorescent in situ hybridization technique with 16S rRNA-targeted oligonucleotide probes. It was found that neither chloride increasing approaches affected the SNR of the batch cultures before the chloride concentration exceeded 10,000 mg Cl L-1, after which the stepwise increase approach reduced the SNR more significantly than the gradual increase approach. From 10,000 to 18,000 mg Cl L-1 a down-and-up pattern of the SNR variation appeared in both approaches, which was associated with the change in the dominant species of ammonia-oxidizers from non-saline-resistant species such as Nitrosomonas europaea-lineage and Nitrosomonas eutropha to saline-resistant species, such as the Nitrosococcus mobilis-lineage. Nitrobacter was the only dominant species when the chloride concentration was below 10,000 mg Cl L-1, where no nitrite-oxidizers survived. Therefore, the 10,000 mg Cl L-1 chloride level is a critical level for the shift of the nitrifier population in the nitrifying activated sludge batch cultures. (C) 2003 Elsevier Science Ltd. All rights reserved.

Evaluation of the impact of bioaugmentation and biostimulation by in situ hybridization and microelectrode
H Satoh, S Okabe, Y Yamaguchi, Y Watanabe
WATER RESEARCH, 37, 9, 2206, 2216, PERGAMON-ELSEVIER SCIENCE LTD, 2003年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Three rotating disk biofilm reactors were operated to evaluate whether bioaugmentation and biostimulation can be used to improve the start-up of microbial nitrification. The first reactor was bioaugmented during start-up period with an enrichment culture of nitrifying bacteria, the second reactor received a synthetic medium containing NH4+ and NO2- to facilitate concomitant proliferation of ammonia- and nitrite-oxidizing bacteria, and the third reactor was used as a control. To evaluate the effectiveness of bioaugmentation and biostimulation approaches, time-dependent developments of nitrifying bacterial community and in situ nitrifying activity in biofilms were monitored by fluorescence in situ hybridization (FISH) technique and microelectrode measurements of NH4+, NO2-, NO3-, and O-2. In situ hybridization results revealed that addition of the enrichment culture of nitrifying bacteria significantly facilitated development of dense nitrifying bacterial populations in the biofilm shortly after, which led to a rapid start-up and enhancement of in situ nitrification activity. The inoculated bacteria could proliferate and/or survive in the biofilm. In addition, the addition of nitrifying bacteria increased the abundance of nitrifying bacteria in the surface of the biofilm, resulting in the higher nitrification rate. On the other hand, the addition of 2.1 mM NO2- did not stimulate the growth of nitrite-oxidizing bacteria and did inhibit the proliferation of ammonia-oxidizing bacteria instead. Thus, the start-up of NO2- oxidation was unchanged, and the start-up of NH4+ oxidation was delayed. In all the three biofilm reactors, data sets of time series analyses on population dynamics of nitrifying bacteria determined by FISH, in situ nitrifying activities determined by microelectrode measurements, and the reactor performances revealed an approximate agreement between the appearance of nitrifying bacteria and the initiation of nitrification activity, suggesting that the combination of these techniques was a very powerful monitoring tool to evaluate the effectiveness of bioaugmentation and biostimulation strategies. (C) 2002 Elsevier Science Ltd. All rights reserved.

Effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film as determined by use of microelectrodes.
Satoshi Okabe, Cecilia M Santegoeds, Dirk De Beer
Biotechnology and bioengineering, 81, 5, 570, 7, JOHN WILEY & SONS INC, 2003年03月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microelectrode, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate the effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film. Microelectrode measurements of O(2), H(2)S, NO(3)(-), NO(2)(-), and pH revealed that the addition of NO(2)(-) and NO(3)(-) forced sulfate reduction zones deeper in the agar gel and significantly reduced the in situ sulfide production levels. The sulfate reduction zone was consequently separated from O(2) and NO(2)(-) or NO(3)(-) respiration zones with increasing the concentrations of NO(2)(-) and NO(3)(-). These NO(2)(-) and NO(3)(-) treatments had only a transient effect on sulfide production. The in situ sulfide production quickly recovered to the previous levels when NO(2)(-) and NO(3)(-) were removed. The PCR-DGGE and FISH analyses revealed that 2-day-continuous addition of 500 microM NO(3)(-) did not change the metabolically active sulfate-reducing bacterial (SRB) community. On the basis of these data, it could be concluded that the addition of NO(2)(-) and NO(3)(-) did not kill SRB, but induced the interspecies competition for common carbon source (i.e., acetate) between nitrate-reducing heterotrophic bacteria and SRB and enhanced the oxidation of the produced sulfide, which were main possible causes of the suppression of in situ sulfide production in the agar gel.

Influence of different cultivars on populations of ammonia-oxidizing bacteria in the root environment of rice.
Aurelio M Briones, Satoshi Okabe, Yoshiaki Umemiya, Niels-Birger Ramsing, Wolfgang Reichardt, Hidetoshi Okuyama
Applied and environmental microbiology, 68, 6, 3067, 75, AMER SOC MICROBIOLOGY, 2002年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Comparisons of the activities and diversities of ammonia-oxidizing bacteria (AOB) in the root environment of different cultivars of rice (Oryza sativa L.) indicated marked differences despite identical environmental conditions during growth. Gross nitrification rates obtained by the 15N dilution technique were significantly higher in a modern variety, IR63087-1-17, than in two traditional varieties. Phylogenetic analysis based on the ammonium monooxygenase gene (amoA) identified strains related to Nitrosospira multiformis and Nitrosomonas europaea as the predominant AOB in our experimental rice system. A method was developed to determine the abundance of AOB on root biofilm samples using fluorescently tagged oligonucleotide probes targeting 16S rRNA. The levels of abundance detected suggested an enrichment of AOB on rice roots. We identified 40 to 69% of AOB on roots of IR63087-1-17 as Nitrosomonas spp., while this subpopulation constituted 7 to 23% of AOB on roots of the other cultivars. These results were generally supported by denaturing gradient gel electrophoresis of the amoA gene and analysis of libraries of cloned amoA. In hydroponic culture, oxygen concentration profiles around secondary roots differed significantly among the tested rice varieties, of which IR63087-1-17 showed maximum leakage of oxygen. The results suggest that varietal differences in the composition and activity of root-associated AOB populations may result from microscale differences in O2 availability.

Successional development of sulfate-reducing bacterial populations and their activities in an activated sludge immobilized agar gel film.
Satoshi Okabe, Cecilia M Santegoeds, Yoshimasa Watanabe, Dirk de Beer
Biotechnology and bioengineering, 78, 2, 119, 30, JOHN WILEY & SONS INC, 2002年04月20日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S m(-2) x s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) x s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 10(8) cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel.

Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions.
Tsukasa Ito, Satoshi Okabe, Hisashi Satoh, Yoshimasa Watanabe
Applied and environmental microbiology, 68, 3, 1392, 402, AMER SOC MICROBIOLOGY, 2002年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 microM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 microm from the surface during all stages of biofilm development. The first sulfide production of 0.32 micromol of H(2)S m(-2) s(-1) was detected below ca. 500 microm in the 3rd week and then gradually increased to 0.70 micromol H(2)S m(-2) s(-1) in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 x 10(9) cells cm(-3) and 3.6 x 10(9) cells cm(-3), respectively, in the surface 400 microm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.

Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization.
Tsukasa Ito, Jeppe L Nielsen, Satoshi Okabe, Yoshimasa Watanabe, Per H Nielsen
Applied and environmental microbiology, 68, 1, 356, 64, AMER SOC MICROBIOLOGY, 2002年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [ (14)C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H(2)-utilizing and (14)CO(2)-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H(2)-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H(2) utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.

ANAEROBIC SRB BIOFILMS IN INDUSTRIAL WATER-SYSTEMS - A PROCESS ANALYSIS
S OKABE, WL JONES, W LEE, WG CHARACKLIS
BIOFOULING AND BIOCORROSION IN INDUSTRIAL WATER SYSTEMS, 189, 204, LEWIS PUBLISHERS INC, 1994年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）

NGS解析による環境由来薬剤耐性プラスミドの配列決定および活性汚泥における接合伝達頻度・宿主域の解明
樋口宏介, 新谷政己, 徳田真穂, 北島正章, 岡部聡, 押木守, 日本水環境学会年会講演集, 57th, 2023年

フローサイトメトリーとアンプリコンシーケンスを組み合わせた活性汚泥における多剤耐性プラスミドの伝播頻度・宿主域の実態解明
樋口宏介, 本田樹, 北島正章, 岡部聡, 押木守, 日本水環境学会年会講演集, 56th, 2022年

活性汚泥における多剤耐性IncP-1群プラスミドpB10の伝播頻度・宿主域の解明
樋口宏介, 北島正章, 岡部聡, 押木守, 日本農芸化学会東北支部大会プログラム・講演要旨集, 2022, 2022年

下水疫学調査による新型コロナウイルスの流行状況の把握と変異株の早期検知
安藤宏紀, 岡部聡, 北島正章, Journal of Environmental Biotechnology (Web), 22, 1, 2022年

ファウリング進行細菌の代謝産物に作用する新規ファージの膜抵抗抑制メカニズム
渡部慶彦, 北島正章, 大坪千穂, 石崎創, 荒川千智, 石井聡, 押木守, 岡部聡, 日本水環境学会年会講演集, 55th, 2021年

パイロットスケールMBRを設置した下水処理場における病原・指標ウイルスの定量解析
石崎知依, 木村克輝, 岡部聡, 北島正章, 日本水環境学会年会講演集, 55th, 2021年

Domestic wastewater treatment and energy harvesting by serpentine up-flow MFCs equipped with PVDF-based activated carbon air-cathodes and a low voltage booster
N'Dah Joel Koffi, Satoshi Okabe, Chemical Engineering Journal, 380, 2020年01月15日
© 2019 Elsevier B.V. Microbial fuel cells (MFCs) exhibit high capital cost and low energy output, which are major issues of the expected practical application of MFCs in wastewater treatment. Here, we have developed serpentine up-flow MFCs equipped with polyvinylidene fluoride (PVDF)-based activated carbon (AC) air-cathode (MFC-PVDF/AC) and continuously operated for more than 6 months with real domestic wastewater as a substrate. The MFC-PVDF/ACs achieved average total COD removal rates (5.11 ± 0.94 kg tCOD/m3/d) and power densities (3.96 ± 3.01 W/m3) without major water leakage, which were even higher than those of MFCs equipped with Pt-based air-cathode (MFC-Pts). The MFC-PVDF/ACs also achieved high and stable suspended solid (SS) removal efficiency (>90%) at 1.5-h HRT without any clogging event during the entire operation period. Since the PVDF-based AC air-cathode is less expensive, more durable, and easy to manufacture, expensive Pt cathodes are not necessary for MFCs treating low strength domestic wastewater. Furthermore, we have developed a low voltage booster (LVB) to increase low output voltage of MFC-PVDF/ACs (i.e., <0.4 V). Connecting a single LVB with a single MFC-PVDF/AC increased the voltage from <0.4 V to 4.35–5.2 V without the voltage reversal, which was enough to turn on three LED bulbs for >12 days. Taken together, the MFC-PVDF/AC with a LVB circuit exhibits excellent and stable performance of domestic wastewater treatment and usable power generation, suggesting that it could be used as a cost- and energy-saving primary wastewater treatment system.

鉄還元細菌Shewanellaへのバクテリオファージ感染が細胞外電子伝達に及ぼす影響
石原令梧, 岡本章玄, 岡部聡, 北島正章, 日本水環境学会年会講演集, 54th, 2020年

集団内の遺伝的多様性がもたらすロタウイルス株の遊離塩素感受性変化
門屋俊祐, 西村修, 佐野大輔, 浦山俊一, 布浦拓郎, 北島正章, 岡部聡, 中込とよ子, 中込治, 日本水環境学会年会講演集, 54th, 2020年

水系感染症ウイルスの集団内遺伝的多様性がもたらす塩素感受性変化
門屋俊祐, 浦山俊一, 布浦拓郎, 中込治, 中込とよ子, 北島正章, 岡部聡, 西村修, 佐野大輔, 日本水環境学会シンポジウム講演集, 23rd, 2020年

電子受容体が膜ファウリング進行細菌(FCB)の代謝および膜ファウリングポテンシャルに及ぼす影響
藤平卓也, 石崎創, 木村善一郎, 岡部聡, 日本水環境学会年会講演集, 54th, 2020年

海洋性アナモックス細菌“Ca.Scalindua sp.”の窒素および酸素同位体分別の解析
小林香苗, 福島慶太郎, 大西雄二, 仁科一哉, 眞壁明子, 押木守, 金田一智規, 木庭啓介, 岡部聡, 日本水環境学会年会講演集, 54th, 2020年

無機-生物ハイブリッド人工光合成システムによる酢酸合成
松尾稜介, 高橋優樹, 百瀬皓太, 渡辺精一, 岡部聡, 日本水環境学会年会講演集, 54th, 2020年

Complete Genome Sequence of a Novel Myoviridae Phage, SfΦ01, Infecting Shigella spp.
Masaaki Kitajima, Satoshi Ishii, Tatsuma Takagi, Satoshi Okabe, Microbiology resource announcements, 8, 23, 2019年06月06日, ［査読有り］, ［国際誌］
The Shigella bacterium is one of the most significant causes of waterborne and foodborne bacterial dysentery. A lytic bacteriophage infecting Shigella flexneri was isolated from wastewater in Japan. We report here the complete genome sequence of this bacteriophage, revealing that it belongs to the Myoviridae family and possesses linear genomic DNA., 英語

Sign-constrained linear regression for prediction of microbe concentration based on water quality datasets.
Tsuyoshi Kato, Ayano Kobayashi, Wakana Oishi, Syun-Suke Kadoya, Satoshi Okabe, Naoya Ohta, Mohan Amarasiri, Daisuke Sano, Journal of water and health, 17, 3, 404, 415, 2019年06月, ［査読有り］, ［国際誌］
This study presents a novel methodology for estimating the concentration of environmental pollutants in water, such as pathogens, based on environmental parameters. The scientific uniqueness of this study is the prevention of excess conformity in the model fitting by applying domain knowledge, which is the accumulated scientific knowledge regarding the correlations between response and explanatory variables. Sign constraints were used to express domain knowledge, and the effect of the sign constraints on the prediction performance using censored datasets was investigated. As a result, we confirmed that sign constraints made prediction more accurate compared to conventional sign-free approaches. The most remarkable technical contribution of this study is the finding that the sign constraints can be incorporated in the estimation of the correlation coefficient in Tobit analysis. We developed effective and numerically stable algorithms for fitting a model to datasets under the sign constraints. This novel algorithm is applicable to a wide variety of the prediction of pollutant contamination level, including the pathogen concentrations in water., 英語

Development of a simple analytical method to determine arsenite using a DNA aptamer and gold nanoparticles.
Koji Matsunaga, Yu Okuyama, Reiko Hirano, Satoshi Okabe, Masahiro Takahashi, Hisashi Satoh, Chemosphere, 224, 538, 543, 2019年06月, ［査読有り］, ［国際誌］
A simple analytical method was developed to determine the arsenite (As(III)) concentration using a DNA aptamer and gold nanoparticles (AuNPs). Prior to sample measurements, the method sensing mechanism was confirmed by analyzing the particle size of the AuNPs at each step of the analysis procedure, and the key operational parameters that affect the method performance were optimized. The optimal final NaCl concentration, incubation time with NaCl and pH of a 3-(N-morpholino) propanesulfonic acid buffer were 60 mM, 10 min and 7.3, respectively. A calibration curve was created under optimized operational conditions. The calibration curve was linear from a 1.0- to 10-μM As(III) concentration. The detection limit was 2.1 μM (161 μg/L). Using the calibration curve, we evaluated groundwater samples spiked with As(III). As(III) concentrations in groundwater pretreated with a 0.2-μm-pore-size membrane filter and cation-exchange resin were determined by using the method, which suggests that the proposed method can be used to determine the As(III) concentration in groundwater., PERGAMON-ELSEVIER SCIENCE LTD, 英語

嫌気性アンモニア酸化細菌“Ca.Scalindua sp.”の酸素同位体分別の解析
小林香苗, 福島慶太郎, 大西雄二, 眞壁明子, 矢野翠, 押木守, 金田一智規, 木庭啓介, 岡部聡, 日本微生物生態学会大会(Web), 33rd, 2019年

Specific interactions of rotavirus HAL1166 with Enterobacter cloacae SENG-6 and their contribution on rotavirus HAL1166 removal.
Mohan Amarasiri, Hiroki Kawai, Masaaki Kitajima, Satoshi Okabe, Daisuke Sano, Water science and technology : a journal of the International Association on Water Pollution Research, 79, 2, 342, 348, 2019年01月, ［査読有り］, ［国際誌］
Contribution of specific interactions between human enteric viruses and wastewater suspended solids on human enteric virus removal by microfiltration was studied. A cross-flow microfiltration system was used with rotavirus HAL1166 and Enterobacter cloacae SENG-6 as the model virus and wastewater suspended solid. Cleavage of rotavirus HAL1166 protein VP4 by trypsin produces the VP8* subunit, which specifically interacts with histo-blood group antigen (HBGA). In the presence of Enterobacter cloacae SENG-6, the trypsin-treated rotavirus concentration reduced with time (R2 > 0.6) compared to the reduction of non-trypsin treated rotavirus. Calculation of the gel/cake layer deposited on the membrane, consisting of Enterobacter cloacae SENG-6 and either trypsin-treated or non-trypsin treated rotavirus HAL1166, revealed that the microflocs consisting of trypsin-treated rotavirus and Enterobacter cloacae SENG-6 have lower porosity and permeability, displaying higher resistance to virus passage through the membrane. The results provide evidence that specific wastewater suspended solids-human enteric virus interaction can contribute to increasing the removal of human enteric viruses by microfiltration., {IWA} Publishing, 英語

nos欠損型脱窒菌による窒素含有廃水の処理及びN₂O回収による新エネルギー源の創出
石丸美穂, 押木守, 荒木信夫, 岡部聡, 幡本将史, 山口隆司, 日本水環境学会年会講演集, 52nd, 656, 2018年03月09日
日本語

アナモックス細菌の窒素・酸素同位体効果の解析
小林香苗, 眞壁明子, 押木守, 金田一智規, 岡部聡, 日本水環境学会年会講演集, 52nd, 379, 2018年03月09日
日本語

共存細菌との結合が水系感染症ウイルスの感染効率に与える影響
河合大樹, 北島正章, 岡部聡, 佐野大輔, 日本水環境学会年会講演集, 52nd, 2018年

金属腐食抑制への応用に向けた鉄還元細菌Shewanellaの溶菌性バクテリオファージの単離と特性解析
石原令梧, 岡本章玄, 高木達馬, 岡部聡, 北島正章, 日本水環境学会年会講演集, 52nd, 2018年

下水処理水中のβ-グルクロニダーゼ活性を利用した簡易迅速大腸菌定量法の開発
菊地凱, 片寄由貴, 石井聡, 北島正章, 岡部聡, 高橋正宏, 佐藤久, 日本水環境学会年会講演集, 52nd, 2018年

DNAアプタマーを用いた簡易ノロウイルス検出法の開発
吉原光, 北島正章, 岡部聡, 高橋正宏, 佐藤久, 佐野大輔, 日本水環境学会年会講演集, 52nd, 2018年

環境水から単離した溶菌性バクテリオファージによる水中病原細菌の低減効果
高木達馬, 佐野大輔, 岡部聡, 北島正章, 日本水環境学会年会講演集, 52nd, 2018年

ウイルス干渉現象がIntegrated cell culture-PCRによる感染性ウイルス検出に与える影響
渡邊亮介, 北島正章, 岡部聡, 佐野大輔, 土木学会北海道支部論文報告集(CD-ROM), 74, 2018年

β-グルクロニダーゼを用いた新規簡易大腸菌数測定法の開発
菊地凱, 片寄由貴, 北島正章, 高橋正宏, 岡部聡, 佐藤久, 平野麗子, 下水道研究発表会講演集, 55th, 2018年

ウイルス干渉現象がIntegrated cell culture-PCRによる感染性アデノウイルス検出に与える影響
佐野大輔, 渡邊亮介, 北島正章, 岡部聡, 日本水環境学会シンポジウム講演集, 21st, 2018年

遊離塩素耐性ロタウイルスの変異遺伝子同定に関する研究
門屋俊祐, 浦山俊一, 布浦拓郎, 北島正章, 岡部聡, 佐野大輔, 日本水環境学会年会講演集, 51st, 2017年

DNAアプタセンサー:水中ノロウイルスの即時・高感度検出に適した新技術
平野誠也, 岡本章玄, 佐野大輔, 岡部聡, 北島正章, 日本水環境学会年会講演集, 51st, 2017年

病原性レジオネラの水系感染制御に資する溶菌性バクテリオファージの探索
高木達馬, 佐野大輔, 岡部聡, 北島正章, 日本水環境学会年会講演集, 51st, 2017年

水環境におけるノロウイルスの遺伝子型別存在実態の把握
宮村明帆, 植木洋, 三浦尚之, 風間しのぶ, 北島正章, 岡部聡, 佐野大輔, 日本水環境学会年会講演集, 51st, 2017年

ウイルス吸着性細菌との結合が水系感染症ウイルスの感染能力に与える影響
河合大樹, 中込とよ子, 中込治, 北島正章, 岡部聡, 佐野大輔, 日本水環境学会年会講演集, 51st, 2017年

環境水中からのノロウイルス吸着性大腸菌の単離と吸着因子同定に関する研究
羽柴聡, AMARASIRI Mohan, 原本英司, 北島正章, 岡部聡, 佐野大輔, 日本水環境学会年会講演集, 51st, 2017年

遊離塩素耐性ロタウイルスの変異遺伝子および耐性メカニズムの解明
門屋俊祐, 浦山俊一, 布浦拓郎, 北島正章, 岡部聡, 佐野大輔, 環境工学研究フォーラム講演集, 54th, 2017年

水系感染性病原細菌に対するバクテリオファージの単離および溶菌特性解析
高木達馬, 佐野大輔, 岡部聡, 北島正章, 環境工学研究フォーラム講演集, 54th, 2017年

大腸菌のβ-グルクロニダーゼ生産能の解析
菊地凱, 北島正章, 岡部聡, 高橋正宏, 佐藤久, 環境工学研究フォーラム講演集, 54th, 2017年

DNAアプタマーを用いた簡易ノロウイルス検出法の開発
吉原光, 北島正章, 佐野大輔, 岡部聡, 高橋正宏, 佐藤久, 環境工学研究フォーラム講演集, 54th, 2017年

下水再生利用のためのノロウイルス目標除去効率算定に関する研究
伊藤寿宏, 北島正章, 加藤毅, 石井聡, 瀬川高弘, 岡部聡, 佐野大輔, 日本微生物生態学会大会(Web), 2017, 2017年

組織細胞におけるイオンチャネル遺伝子発現プロファイルに着目した水中感染性エンテロウイルス迅速検出手法の開発
渡邊亮介, 稲葉愛美, 岡部聡, 佐野大輔, 北島正章, 日本水環境学会年会講演集, 51st, 2017年

DNAアプタマーを用いた簡易ノロウイルス検出法の開発
吉原光, 北島正章, 佐野大輔, 岡部聡, 高橋正宏, 佐藤久, 全国会議(水道研究発表会)講演集, 2017, 2017年

Aptamer: The potential application to norovirus research, diagnosis, and therapeutics
Seiya Hirano, Daisuke Sano, Satoshi Okabe, Masaaki Kitajima, Noroviruses: Outbreaks, Control and Prevention Strategies, 223, 242, 2017年01月01日
© 2017 Nova Science Publishers, Inc. Aptamer has been attracting much attention as a new biomolecular tool for viral diagnostic and therapeutic applications. Aptamer is a functional nucleic acid that folds into a unique three-dimensional conformation that binds to a specific target, which can be an alternative for antibodies. Rather, aptamer offers various advantages over antibodies as a target-specific recognition molecule: high affinity, specificity, and thermostability, low production cost, etc. Several studies have reported DNA aptamers for certain human norovirus strains as well as murine norovirus, which has enabled development and evaluation of aptasensor (aptamer-based biosensor) technology for rapid and sensitive norovirus detection. Therapeutic application of aptamer to inhibiting norovirus infections has not been clearly demonstrated to date, but there have been a few reports suggesting the potential of aptamer-based therapeutics. Taken together, it has been implied that aptamer is a new target-specific biomolecular tool that offers various potential applications to norovirus research, diagnosis, and therapeutics, which should contribute to the infection and disease control of norovirus by rapidly identifying potential infection routes and reducing the disease burden through therapeutic approach. Herein we summarize recent development and application of norovirus-specific aptamers and discuss future research directions towards better control of norovirus transmission/infections using this aptamer technology.

ヒドラジンを合成するオルガネラ:嫌気性アンモニウム酸化細菌のアナモキソソーム
押木守, 岡部聡, 日本微生物生態学会大会(Web), 2017, ROMBUNNO.S‐070 (WEB ONLY), 2017年
日本語

Genome Sequence ofEnterobacter cloacaeStrain SENG-6, a Bacterium Producing Histo-Blood Group Antigen-Like Substances That Can Bind with Human Noroviruses
Satoshi Ishii, Mohan Amarasiri, Satoshi Hashiba, Peiyi Yang, Satoshi Okabe, Daisuke Sano, Genome Announcements, 4, 4, e00893, 16, 2016年08月
© 2016 Ishii et al. Enterobacter sp. strain SENG-6, isolated from healthy human feces, produces histo-blood group antigen (HBGA)-like substances that can bind with human noroviruses. Based on the genome sequence analysis, strain SENG-6 belongs to the species Enterobacter cloacae. The genome sequence of this strain should help identify genes associated with the production of HBGA-like substances., American Society for Microbiology

Anammox細菌の窒素同位体分別に関する研究
小林香苗, 押木守, 金田一智規, 眞壁明子, 岡部聡, 日本水環境学会年会講演集, 50th, 664, 2016年03月10日
日本語

遊離塩素処理がノロウイルスの遺伝的多様性に与える影響
中村新, 渡辺幸三, 八重樫咲子, 岡部聡, 中込とよ子, 中込治, 佐野大輔, 日本水環境学会年会講演集, 50th, 398, 2016年03月10日
日本語

ヒト細胞由来遺伝子マーカーを用いた感染性ウイルスの迅速検出
稲葉愛美, 伊藤寿宏, 大村達夫, 岡部聡, 佐野大輔, 日本水環境学会年会講演集, 50th, 2016年

Bayesian modeling of virus removal efficiency in wastewater treatment processes
T. Ito, T. Kato, K. Takagishi, S. Okabe, D. Sano, WATER SCIENCE AND TECHNOLOGY, 72, 10, 1789, 1795, 2015年11月
Left-censored datasets of virus density in wastewater samples make it difficult to evaluate the virus removal efficiency in wastewater treatment processes. In the present study, we modeled the probabilistic distribution of virus removal efficiency in a wastewater treatment process with a Bayesian approach, and investigated how many detect samples in influent and effluent are necessary for accurate estimation. One hundred left-censored data of virus density in wastewater (influent and effluent) were artificially generated based on assumed log-normal distributions and the posterior predictive distribution of virus density, and the log-ratio distribution were estimated. The estimation accuracy of distributions was quantified by Bhattacharyya coefficient. When it is assumed that the accurate estimation of posterior predictive distributions is possible when a 100% positive rate is obtained for 12 pairs of influent and effluent, 11 out of 144, 60 out of 324, and 201 out of 576 combinations of detect samples gave an accurate estimation at the significant level of 0.01 in a Kruskal-Wallis test when the total sample number was 12, 18, and 24, respectively. The combinations with the minimum number of detect samples were (12, 9), (16, 10), and (21, 8) when the total sample number was 12, 18, and 24, respectively., IWA PUBLISHING, 英語

嫌気性アンモニウム酸化細菌由来ヒドラジン合成酵素の精製
押木守, 荒木信夫, 岡部聡, 日本微生物生態学会大会(Web), 30th, PD‐051 (WEB ONLY), 2015年
日本語

Anammox研究開発の動向 (特集 Anammoxプロセスの普及をめざして)
岡部 聡, 水環境学会誌, 37, 9, 316, 320, 2014年09月
日本水環境学会 ; 1992-, 日本語

ノロウイルスの塩素消毒耐性獲得メカニズム解明に関する基礎的研究
富岡哲史, 渡辺幸三, 岡部聡, 佐野大輔, 土木学会北海道支部論文報告集(CD-ROM), 70, ROMBUNNO.G-01, 2014年02月
日本語

O21-02 Identification of key N_2O production pathways in aerobic partial nitrifying granules :
Ishii Satoshi, Song Yanjun, Rathnayake Lashitha, Tumendelger Azzaya, Satoh Hisashi, Sakae Toyoda, Yoshida Naohiro, Okabe Satoshi, 日本微生物生態学会講演要旨集, 2014, 94, 94, 2014年
日本微生物生態学会, 英語

ゲノミクス,プロテオミクス解析から読み解くアンモニア酸化(anammox)細菌“Ca.Scalindua japonica”の生活環
押木守, 水戸佳祐, 木村善一郎, 金田一智規, 佐藤久, 岡部聡, 日本ゲノム微生物学会年会要旨集, 8th, 69, 2014年
日本語

Source identification of nitrous oxide on autotrophic partial nitrification in a granular sludge reactor
R. M. L. D. Rathnayake, Y. Song, A. Tumendelger, M. Oshiki, S. Ishii, H. Satoh, S. Toyoda, N. Yoshida, S. Okabe, WATER RESEARCH, 47, 19, 7078, 7086, 2013年12月, ［最終著者, 責任著者］
Emission of nitrous oxide (N2O) during biological wastewater treatment is of growing concern since N2O is a major stratospheric ozone-depleting substance and an important greenhouse gas. The emission of N2O from a lab-scale granular sequencing batch reactor (SBR) for partial nitrification (PN) treating synthetic wastewater without organic carbon was therefore determined in this study, because PN process is known to produce more N2O than conventional nitrification processes. The average N2O emission rate from the SBR was 0.32 +/- 0.17 mgN L-1 h(-1), corresponding to the average emission of N2O of 0.8 +/- 0.4% of the incoming nitrogen load (1.5 +/- 0.8% of the converted NI-14"). Analysis of dynamic concentration profiles during one cycle of the SBR operation demonstrated that N2O concentration in off-gas was the highest just after starting aeration whereas N2O concentration in effluent was gradually increased in the initial 40 min of the aeration period and was decreased thereafter. Isotopomer analysis was conducted to identify the main N2O production pathway in the reactor during one cycle. The hydroxylamine (NH2OH) oxidation pathway accounted for 65% of the total N2O production in the initial phase during one cycle, whereas contribution of the NO reduction pathway to N2O production was comparable with that of the NH2OH oxidation pathway in the latter phase. In addition, spatial distributions of bacteria and their activities in single microbial granules taken from the reactor were determined with microsensors and by in situ hybridization. Partial nitrification occurred mainly in the oxic surface layer of the granules and ammonia-oxidizing bacteria were abundant in this layer. N2O production was also found mainly in the oxic surface layer. Based on these results, although N2O was produced mainly via NH2OH oxidation pathway in the autotrophic partial nitrification reactor, N2O production mechanisms were complex and could involve multiple N2O production pathways. (C) 2013 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

2S-Cp04 嫌気性アンモニア酸化(anammox) 細菌の代謝多様性と廃水処理への応用(極限生物たちが切り拓く未来の環境バイオテクノロジー,シンポジウム)
押木 守, 佐藤 久, 岡部 聡, 日本生物工学会大会講演要旨集, 65, 89, 89, 2013年08月25日
公益社団法人日本生物工学会, 日本語

変色型ボロンジピロメテン蛍光色素を用いた路面排水中Zn²⁺濃度の定量
羽深昭, 吉川弘晃, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 分析化学討論会講演要旨集, 73rd, 149, 2013年05月04日
日本語

Introduction of a Degassing Membrane Technology into Anaerobic Wastewater Treatment
W. M. K. R. T. W. Bandara, M. Ikeda, H. Satoh, M. Sasakawa, Y. Nakahara, M. Takahashi, S. Okabe, WATER ENVIRONMENT RESEARCH, 85, 5, 387, 390, 2013年05月
The effectiveness of degasification using a degassing membrane to improve chemical oxygen demand (COD) removal efficiency was investigated using a bench-scale upflow anaerobic sludge blanket (UASB) reactor. Vacuum degasification was able to transfer dissolved gas in the bulk liquid of the UASB reactor inside the membrane. Such a process might provide thermodynamically favorable conditions for the degradation of organic compounds. The COD-removal efficiency improved from 83% during normal operation to 90% during the degassing operation., WATER ENVIRONMENT FEDERATION, 英語, その他

MBR膜ファウリングを支配する細菌の探索
井川裕介, 吉田圭太郎, 福島寿和, 岡部聡, 三好太郎, 渡辺義公, 日本水環境学会年会講演集, 47th, 255, 2013年03月11日
日本語

網羅的重金属イオン分析に向けた新規変色型蛍光色素の開発
吉川弘晃, 羽深昭, 菅藤亮輔, 大屋光平, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 47th, 431, 2013年03月11日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 谷内翔, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 47th, 321, 2013年03月11日
日本語

新規変色型蛍光色素を用いた路面排水中亜鉛濃度の定量
菅藤亮輔, 羽深昭, 吉川弘晃, 大屋光平, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 47th, 432, 2013年03月11日
日本語

重金属測定用蛍光色素の理論的分子設計による高感度化
大屋光平, 羽深昭, 菅藤亮輔, 吉川弘晃, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 47th, 692, 2013年03月11日
日本語

リン酸マイクロセンサーのイオノフォアの検討
谷内翔, 宮崎悠爾, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 47th, 322, 2013年03月11日
日本語

表面プラズモン共鳴を利用した病原微生物バイオセンサの開発
坂槙有紀恵, 山田健太, 佐野大輔, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 47th, 436, 2013年03月11日
日本語

遺伝子発現解析に基づく再生水中残留医薬品の毒性評価
中島弘司, 原(山村)宏江, 木村克輝, 岡部聡, 三好太郎, 渡辺義公, 日本水環境学会年会講演集, 47th, 412, 2013年03月11日
日本語

ヒト由来培養細胞を用いた塩素消毒下水処理水の毒性評価
福島寿和, 木村克輝, 岡部聡, 三好太郎, 渡辺義公, 日本水環境学会年会講演集, 47th, 516, 2013年03月11日
日本語

細胞応答に基づいた膜処理再生水中の毒性画分の探索―粒径に着目して
原(山村)宏江, HOQUE Asiful, 木村克輝, 岡部聡, 三好太郎, 渡辺義公, 日本水環境学会年会講演集, 47th, 517, 2013年03月11日
日本語

広島湾により集積されたScalindua属アナモックス細菌の生理学的特性
粟田貴宣, 金田一智規, 尾崎則篤, 大橋晶良, 押木守, 岡部聡, 日本水環境学会年会講演集, 47th, 160, 2013年03月11日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 谷内翔, 押木守, 佐藤久, 高橋正宏, 岡部聡, 土木学会北海道支部論文報告集(CD-ROM), 69, ROMBUNNO.G-02, 2013年02月
日本語

水の再生と再利用の鍵 : 「膜分離技術」と「バイオテクノロジー」の融合
岡部 聡, 水環境学会誌 = Journal of Japan Society on Water Environment, 36, 1, 15, 18, 2013年01月10日
日本水環境学会 ; 1992-, 日本語

Hydrogenophaga sp.AR20のGeobacter sulfurreducensとの水素を介した共生的電気生産
寺田浩太朗, 木村善一郎, 石崎創, 岡部聡, 日本水環境学会年会講演集, 47th, 2013年

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 谷内翔, 押木守, 高橋正宏, 岡部聡, 佐藤久, 衛生工学シンポジウム論文集, 20th, 24, 2012年12月04日
日本語

表面プラズモン共鳴を利用した病原微生物バイオセンサの開発
坂槙有紀恵, 山田健太, ピタックティーラタム ニティ, 石井聡, 佐野大輔, 高橋正宏, 岡部聡, 佐藤久, 環境工学研究フォーラム講演集, 49th, 28, 30, 2012年11月28日
日本語

新規蛍光分子プローブを用いた環境水中重金属イオン分析
菅藤亮輔, 羽深昭, 吉川弘晃, 大屋光平, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 環境工学研究フォーラム講演集, 49th, 31, 33, 2012年11月28日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 谷内翔, 押木守, 佐藤久, 高橋正宏, 岡部聡, 環境工学研究フォーラム講演集, 49th, 25, 27, 2012年11月28日
日本語

PK-24 微生物燃料電池内に成立する水素資化性電気生産細菌・嫌気的酢酸酸化細菌からなる酢酸酸化共生系(PK 相互作用・共生,ポスター発表)
木村 善一郎, 寺田 浩太朗, 岡部 聡, 日本微生物生態学会講演要旨集, 28, 176, 176, 2012年09月19日
日本微生物生態学会, 英語

PGa-04 嫌気性アンモニア酸化細菌Candidatus 'Brocadia sinica'メタゲノム解析による新規アナモックス代謝経路の同定(PGa 増殖・生理・代謝,ポスター発表)
押木 守, 新家子 香織, 佐藤 久, 岡部 聡, 日本微生物生態学会講演要旨集, 28, 160, 160, 2012年09月19日
日本微生物生態学会, 英語

次世代シーケンサーが導く環境研究の革新
岡部 聡, 水環境学会誌 = Journal of Japan Society on Water Environment, 35, 9, 289, 289, 2012年09月10日
日本語

表面プラズモン共鳴を利用した水中病原微生物検出バイオセンサの開発
佐藤久, 坂槙有紀恵, 山田健太, ピタックティーラタムニティ, 石井聡, 佐野大輔, 高橋正宏, 岡部聡, 日本水環境学会シンポジウム講演集, 15th, 233, 234, 2012年09月10日
日本語

嫌気性アンモニア酸化(アナモックス)細菌のゲノム解析が明らかにする生理学的特性
押木守, 佐藤久, 岡部聡, 新家子香織, 日本水環境学会シンポジウム講演集, 15th, 157, 2012年09月10日
日本語

トリベンジルチンクロライドを用いたリン酸マイクロセンサーの開発
谷内翔, 宮崎悠爾, 高橋正宏, 岡部聡, 佐藤久, 日本分析化学会年会講演要旨集, 61st, 324, 2012年09月05日
日本語

新規変色型蛍光色素を用いた路面排水中亜鉛濃度の定量
吉川弘晃, 菅藤亮輔, 羽深昭, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本分析化学会年会講演要旨集, 61st, 315, 2012年09月05日
日本語

表面プラズモン共鳴を用いた水中病原性微生物バイオセンサ
坂槙有紀恵, 山田健太, 高橋正宏, 岡部聡, 佐藤久, 日本分析化学会年会講演要旨集, 61st, 397, 2012年09月05日
日本語

バイオフィルム感染症の新治療戦略 大腸菌RelEが誘導する菌体密度依存的休眠化
岡部 聡, 田代 陽介, 谷内 亜沙美, Thithiwat May, 柿沼 建至, 川田 耕司, 日本生物工学会大会講演要旨集, 平成24年度, 100, 100, 2012年09月
(公社)日本生物工学会, 日本語

重金属センサアレイに向けた変色型ボロンジピロメテン蛍光色素群の開発
菅藤亮輔, 吉川弘晃, 谷山拓生, 羽深昭, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 分析化学討論会講演要旨集, 72nd, 130, 2012年05月05日
日本語

微小電極を用いた堆積型堆肥表層中の一酸化二窒素および一酸化窒素の測定
日向寺崇文, 前田高輝, 岡部聡, 高橋正宏, 佐藤久, 日本水環境学会年会講演集, 46th, 510, 2012年03月14日
日本語

新規変色型蛍光色素を用いた環境サンプル中の重金属イオン分析
谷山拓生, 羽深昭, 菅藤亮輔, 吉川弘晃, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 46th, 506, 2012年03月14日
日本語

新規変色型蛍光色素を用いたクロムイオン分析
吉川弘晃, 羽深昭, 谷山拓生, 菅藤亮輔, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 46th, 676, 2012年03月14日
日本語

新規変色型蛍光色素を用いた亜鉛イオン分析
羽深昭, 谷山拓生, 菅藤亮輔, 吉川弘晃, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 46th, 622, 2012年03月14日
日本語

トキシコゲノミクス的アプローチを用いた再生水の水質評価~残留医薬品の影響~
中島弘司, 原宏江, HOQUE Asiful, 木村克輝, 岡部聡, 三好太郎, 渡辺義公, 日本水環境学会年会講演集, 46th, 93, 2012年03月14日
日本語

トキシコゲノミクス的アプローチを用いた膜処理再生水の水質評価の試み
原(山村)宏江, HOQUE Asiful, 木村克輝, 岡部聡, 中島弘司, 三好太郎, 渡辺義公, 日本水環境学会年会講演集, 46th, 74, 2012年03月14日
日本語

Anammox菌による鉄酸化硝酸還元能の解析
石黒真規, 押木守, 石井聡, 岡部聡, 日本水環境学会年会講演集, 46th, 151, 2012年03月14日
日本語

Anammox活性の菌体密度依存的制御機構の解明
平泉晴菜, 押木守, 岡部聡, 日本水環境学会年会講演集, 46th, 152, 2012年03月14日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 谷内翔, 高橋正宏, 岡部聡, 佐藤久, 化学系学協会北海道支部冬季研究発表会講演要旨集, 2012, 148, 2012年01月31日
日本語

変色型ボロンジピロメテン蛍光色素を用いた重金属イオン分析
菅藤亮輔, 吉川弘晃, 谷山拓生, 羽深昭, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 化学系学協会北海道支部冬季研究発表会講演要旨集, 2012, 101, 2012年01月31日
日本語

PM-09 大腸菌バイオフィルム内で発生する小コロニー形成株の特性解析(PM バイオフィルム・界面,ポスター発表)
榮田 弘明, 田代 陽介, 岡部 聡, 日本微生物生態学会講演要旨集, 28, 182, 182, 2012年
日本微生物生態学会, 英語

Characterization of microbial community structures and their activities in single anaerobic granules by beta imaging, microsensors and fluorescence in situ hybridization
H. Satoh, I. Tsushima, Y. Miura, T. Ito, S. Okabe, WATER SCIENCE AND TECHNOLOGY, 65, 12, 2125, 2131, 2012年
The spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by fluorescence in situ hybridization (FISH), beta imaging and microsensors. FISH results revealed a layered structure of microorganisms in the granule, where Chloroflexi was present in the outermost layer, Smithella spp. and Syntrophobacter spp. were found in a depth of ca. 100 mu m, and Archaea was restricted to the inner layer (below ca. 300 mu m from the surface). Substrate uptake patterns elucidated by beta imaging demonstrated that glucose uptake was highest at 50 mu m depth, whereas propionate uptake had a peak at 200 mu m depth. In addition, microsensor measurements revealed that acid was produced mainly at 100 mu m depth and H-2 production was detected at a depth from 100 to 200 mu m. H-2 consumption and corresponding CH4 production were found below 200 mu m from the surface. Direct comparison of these results implied sequential degradation of complex organic compounds in anaerobic granules; Chloroflexi contributed to fermentation of organic compounds and acid production in the outermost layer, volatile fatty acids were oxidized and H-2 was produced mainly by Smithella spp. and Syntrophobacter spp. at a depth from 100 to 200 mu m, and Archaea produced CH4 below ca. 300 mu m from the surface., IWA PUBLISHING, 英語

N2O emission from a partial nitrification–anammox process and identification of a key biological process of N2O emission from anammox granules
OKABE Satoshi, OKABE Satoshi, OSHIKI Mamoru, OSHIKI Mamoru, TAKAHASHI Yoshitaka, SATOH Hisashi, Water Research, 45, 19, 6461, 6470, 2011年12月
Emission of nitrous oxide (N2O) during biological wastewater treatment is of growing concern. The emission of N2O from a lab-scale two-reactor partial nitrification (PN)-anammox reactor was therefore determined in this study. The average emission of N2O from the PN and anammox process was 4.0 +/- 1.5% (9.6 +/- 3.2% of the removed nitrogen) and 0.1 +/- 0.07% (0.14 +/- 0.09% of the removed nitrogen) of the incoming nitrogen load, respectively. Thus, a larger part (97.5%) of N2O was emitted from the PN reactor. The total amount of N2O emission from the PN reactor was correlated to nitrite (NO2-) concentration in the PN effluent rather than DO concentration. In addition, further studies were performed to indentify a key biological process that is responsible for N2O emission from the anammox process (i.e., granules). In order to characterize N2O emission from the anammox granules, the in situ N2O production rate was determined by using microelectrodes for the first time, which was related to the spatial organization of microbial community of the granule as determined by fluorescence in situ hybridization (FISH). Microelectrode measurement revealed that the active N2O production zone was located in the inner part of the anammox granule, whereas the active ammonium consumption zone was located above the N2O production zone. Anammox bacteria were present throughout the granule, whereas ammonium-oxidizing bacteria (AOB) were restricted to only the granule surface. In addition, addition of penicillin G that inhibits most of the heterotrophic denitrifiers and AOB completely inhibited N2O production in batch experiments. Based on these results obtained, denitrification by putative heterotrophic denitrifiers present in the inner part of the granule was considered the most probable cause of N2O emission from the anammox reactor (i.e., granules). (C) 2011 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Adsorption characteristics of an enteric virus-binding protein to norovirus, rotavirus and poliovirus
Takahiro Imai, Daisuke Sano, Takayuki Miura, Satoshi Okabe, Keishi Wada, Yoshifumi Masago, Tatsuo Omura, BMC BIOTECHNOLOGY, 11, 123, 1, 11, 2011年12月
Background: Water contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies.
Results: A gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure.
Conclusions: EVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples., BIOMED CENTRAL LTD, 英語

Enterobactin is required for biofilm development in reduced-genome Escherichia coli
Thithiwat May, Satoshi Okabe, ENVIRONMENTAL MICROBIOLOGY, 13, 12, 3149, 3162, 2011年12月
A variety of bacterial cell surface structures and quorum signalling molecules play a role in biofilm development in Escherichia coli. However, here we show that an engineered reduced-genome E. coli mutant that lacks 17.6% of the parental E. coli genome, including the genes involved in the synthesis of various cell surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers and the autoinducer-2 signalling molecule, is able to develop mature biofilms. Using temporal gene expression profiling, we investigated phenotypic changes in reduced-genome biofilms in relation with the genes encoding the synthesis of different amino acids that were differentially expressed during biofilm formation. We identified and characterized entB, marR, dosC, mcbR and yahK genes, as involved in biofilm formation by the reduced-genome E. coli. Of these, for a first time, we demonstrated that overproduction of entB and yahK, which encode an enterobactin for iron transport and a hypothetical oxidoreductase protein, respectively, promoted biofilm development and maturation. Our results indicate that specific types of genes contribute to phenotypic changes in reduced-genome E. coli biofilms. In addition, this work demonstrates that the functions of biofilm-specific genes could be analysed through experiments using the reduced-genome E. coli., WILEY-BLACKWELL, 英語

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge
Tsukasa Ito, Kazumi Yoshiguchi, Herto Dwi Ariesyady, Satoshi Okabe, ISME JOURNAL, 5, 12, 1844, 1856, 2011年12月
Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope-and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with (14)C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with (13)C(6)-glucose and (13)C(3)-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with (13)C-glucose and (13)C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with (14)C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K(m) for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5-10mM). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.The ISME Journal (2011) 5, 1844-1856; doi:10.1038/ismej.2011.59; published online 12 May 2011, NATURE PUBLISHING GROUP, 英語

新規蛍光色素を用いた重金属イオン分析
羽深昭, 谷山拓生, 菅藤亮輔, 吉川弘晃, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 環境工学研究フォーラム講演集, 48th, 214, 216, 2011年11月25日
日本語

重金属イオン分析に向けた新規蛍光色素の開発
菅藤亮輔, 羽深昭, 谷山拓生, 吉川弘晃, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 環境工学研究フォーラム講演集, 48th, 220, 222, 2011年11月25日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
谷内翔, 宮崎悠爾, 押木守, 佐藤久, 高橋正宏, 岡部聡, 環境工学研究フォーラム講演集, 48th, 217, 219, 2011年11月25日
日本語

微生物燃料電池内に存在する細胞外キノンの生態学的意義に関する研究
木村善一郎, 伊藤皓亮, 岡部聡, 日本微生物生態学会講演要旨集, 27th, 27, 74, 74, 2011年10月08日
日本微生物生態学会, 日本語

PA-034 Anammox細菌Candidatus "Brocadia sinica"の嫌気性アンモニア酸化活性に及ぼす菌体密度の影響(PA 水処理生態系,ポスター発表)
平泉 晴菜, 新家子 香織, 押木 守, 佐藤 久, 岡部 聡, 日本微生物生態学会講演要旨集, 27, 73, 73, 2011年10月08日
日本微生物生態学会, 日本語

PA-030 メタゲノム・プロテオームアプローチによる嫌気性アンモニア酸化(anammox)細菌Candidatus 'Brocadia sinica'の生理生態学的特性の解明(PA 水処理生態系,ポスター発表)
押木 守, 新家子 香織, 佐藤 久, 岡部 聡, 日本微生物生態学会講演要旨集, 27, 72, 72, 2011年10月08日
日本微生物生態学会, 日本語

部分硝化‐AnammoxプロセスからのN₂O生成
岡部聡, 押木守, 高橋慶考, 佐藤久, 日本水環境学会シンポジウム講演集, 14th, 43, 44, 2011年09月10日
日本語

オンサイト重金属イオン分析に向けた新規変色型蛍光色素の開発
谷山拓生, 羽深昭, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 土木学会年次学術講演会講演概要集(CD-ROM), 66th, ROMBUNNO.VII-192, 2011年08月05日
日本語

新規変色型蛍光色素を用いた水サンプル中重金属イオンの簡易分析
羽深昭, 谷山拓生, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 土木学会年次学術講演会講演概要集(CD-ROM), 66th, ROMBUNNO.VII-191, 2011年08月05日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 押木守, 高橋正宏, 岡部聡, 佐藤久, 土木学会年次学術講演会講演概要集(CD-ROM), 66th, ROMBUNNO.VII-029, 2011年08月05日
日本語

重金属イオンの識別が可能な変色型ボロンジピロメテン蛍光色素の開発
谷山拓生, 菅藤亮輔, 羽深昭, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本化学会北海道支部夏季研究発表会講演要旨集, 2011, 150, 2011年07月01日
日本語

変色型ボロンジピロメテン蛍光色素を用いた含水サンプル中での重金属イオンの簡易分析
菅藤亮輔, 谷山拓生, 羽深昭, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本化学会北海道支部夏季研究発表会講演要旨集, 2011, 149, 2011年07月01日
日本語

Development of long-term stable partial nitrification and subsequent anammox process
Satoshi Okabe, Mamoru Oshiki, Yoshitaka Takahashi, Flisashi Satoh, BIORESOURCE TECHNOLOGY, 102, 13, 6801, 6807, 2011年07月
The partial nitrification reactor was successfully started up and operated stably for more than 250 days with a maximum nitrite production rate of 1.12 kg-N m(-3) day(-1). The important factors for successful partial nitrification were high ammonium loading rate (>1.0 kg-N m(-3) day(-1)) and relatively high pH (ca. 8.0), giving high free ammonia concentrations (>10 mg NH(3)-N L(-1)). In addition, the air flow rate must be controlled at the ratio of air flow rate to ammonium loading rate below 0.1 (m(air)(3) day(-1))/(kg-N m(-3) day(-1)). After the establishment of stable partial nitrification, the effluent NO(2)(-)-N/NH(4)(+)-N ratio and effluent NO(3)(-)-N concentration were 1.20 +/- 0.33 and 1.2 +/- 1.0 mg-N L(-1), respectively, which was then fed into an granular-sludge anammox reactor. Consistent nitrogen removal was achieved for more than 250 days with a maximum nitrogen removal rate of 15.0 kg-TN m(-3) day(-1). (C) 2011 Elsevier Ltd. All rights reserved., ELSEVIER SCI LTD, 英語

二員培養系微生物燃料電池の電力増大因子の解明
木村善一郎, 伊藤皓亮, 岡部聡, 環境バイオテクノロジー学会大会プログラム講演要旨集, 44th, 25, 2011年06月20日
日本語

Physiological characteristics of the anaerobic ammonium-oxidizing bacterium 'Candidatus Brocadia sinica'
Mamoru Oshiki, Masaki Shimokawa, Naoki Fujii, Hisashi Satohl, Satoshi Okabe, MICROBIOLOGY-SGM, 157, 6, 1706, 1713, 2011年06月
The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m(-3) day(-1). The anammox bacteria were identified as representing 'Candidatus Brocadia sinica' on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (mu(max).) was 0.0041 h(-1), corresponding to a doubling time of 7 days. The half-saturation constants (K(s)) for ammonium and nitrite of 'Ca. B. sinica' were 28 +/- 4 and 86 +/- 4 mu M, respectively, higher than those of 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis'. The temperature and pH ranges of anammox activity were 25-45 degrees C and pH 6.5-8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50% inhibition at 16 mM), ethanol (91% at 1 mM) and methanol (86% at 1 mM). Anammox activities were 80 and 70% of baseline in the presence of 20 mM phosphorus and 3% salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (mol NH(4)(+))(-1), respectively. This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems., SOC GENERAL MICROBIOLOGY, 英語

Removal of residual dissolved methane gas in an upflow anaerobic sludge blanket reactor treating low-strength wastewater at low temperature with degassing membrane
Wasala M. K. R. T. W. Bandara, Hisashi Satoh, Manabu Sasakawa, Yoshihito Nakahara, Masahiro Takahashi, Satoshi Okabe, WATER RESEARCH, 45, 11, 3533, 3540, 2011年05月
In this study, we investigated the efficiency of dissolved methane (D-CH(4)) collection by degasification from the effluent of a bench-scale upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater. A hollow-fiber degassing membrane module was used for degasification. This module was connected to the liquid outlet of the UASB reactor. After chemical oxygen demand (COD) removal efficiency of the UASB reactor became stable, D-CH(4) discharged from the UASB reactor was collected. Under 35 degrees C and a hydraulic retention time (HRT) of 10 h, average D-CH(4) concentration could be reduced from 63 mg COD L(-1) to 15 mg COD L(-1); this, in turn, resulted in an increase in total methane (CH(4)) recovery efficiency from 89% to 97%. Furthermore, we investigated the effects of temperature and HRT of the UASB reactor on degasification efficiency. Average D-CH(4) concentration was as high as 104 mg COD L(-1) at 15 degrees C because of the higher solubility of CH(4) gas in liquid; the average D-CH(4) concentration was reduced to 14 mg COD L(-1) by degasification. Accordingly, total CH(4) recovery efficiency increased from 71% to 97% at 15 degrees C as a result of degasification. Moreover, degasification tended to cause an increase in particulate COD removal efficiency. The UASB reactor was operated at the same COD loading rate, but different wastewater feed rates and HRTs. Although average D-CH(4) concentration in the UASB reactor was almost unchanged (ca. 70 mg COD L(-1)) regardless of the HRT value, the CH(4) discharge rate from the UASB reactor increased because of an increase in the wastewater feed rate. Because the D-CH(4) concentration could be reduced down to 12 +/- 1 mg COD L(-1) by degasification at an HRT of 6.7 h, the CH(4) recovery rate was 1.5 times higher under degasification than under normal operation. (C) 2011 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages
Thithiwat May, Kenji Tsuruta, Satoshi Okabe, ISME JOURNAL, 5, 4, 771, 775, 2011年04月
Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation. The ISME Journal (2011) 5, 771-775; doi: 10.1038/ismej.2010.158; published online 21 October 2010, NATURE PUBLISHING GROUP, 英語

一槽型バイオ燃料電池における正極反応の促進による電力上昇
藤木一到, 石崎創, 木村善一郎, 岡部聡, 日本水環境学会年会講演集, 45th, 168, 2011年03月18日
日本語

酢酸を基質とする微生物燃料電池内の電子フローに関する研究
木村善一郎, 岡部聡, 日本水環境学会年会講演集, 45th, 166, 2011年03月18日
日本語

オンサイト重金属イオン分析に向けた新規変色型蛍光色素の開発
谷山拓生, 羽深昭, 菅藤亮輔, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 45th, 293, 2011年03月18日
日本語

新規変色型蛍光色素を用いた水サンプル中の重金属検出
菅藤亮輔, 羽深昭, 谷山拓生, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 45th, 295, 2011年03月18日
日本語

新規変色型蛍光色素を用いたレシオメトリー測定による重金属イオン分析
羽深昭, 谷山拓生, 菅藤亮輔, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 45th, 294, 2011年03月18日
日本語

Anammoxと組み合わせた部分硝化のための好気性グラニュール反応器の開発
SONG Yanjun, 押木守, 岡部聡, 日本水環境学会年会講演集, 45th, 161, 2011年03月18日
日本語

Anammox細菌の代謝活性に及ぼす菌体密度の影響
平泉晴菜, 新家子香織, 押木守, 岡部聡, 日本水環境学会年会講演集, 45th, 47, 2011年03月18日
日本語

新規anammox細菌Candidatus ‘Brocadia sinica’における網羅的プロテオーム解析
新家子香織, 押木守, 佐藤久, 岡部聡, 日本水環境学会年会講演集, 45th, 46, 2011年03月18日
日本語

Ecophysiology of Uncultured Filamentous Anaerobes Belonging to the Phylum KSB3 That Cause Bulking in Methanogenic Granular Sludge
Takeshi Yamada, Kae Kikuchi, Toshihiro Yamauchi, Koji Shiraishi, Tsukasa Ito, Satoshi Okabe, Akira Hiraishi, Akiyoshi Ohashi, Hideki Harada, Yoichi Kamagata, Kazunori Nakamura, Yuji Sekiguchi, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77, 6, 2081, 2087, 2011年03月
A filamentous bulking of a methanogenic granular sludge caused by uncultured filamentous bacteria of the candidate phylum KSB3 in an upflow anaerobic sludge blanket (UASB) system has been reported. To characterize the physiological traits of the filaments, a polyphasic approach consisting of rRNA-based activity monitoring of the KSB3 filaments using the RNase H method and substrate uptake profiling using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was conducted. On the basis of rRNA-based activity, the monitoring of a full-scale UASB reactor operated continuously revealed that KSB3 cells became active and predominant (up to 54% of the total 16S rRNA) in the sludge when the carbohydrate loading to the system increased. Batch experiments with a short incubation of the sludge with maltose, glucose, fructose, and maltotriose at relatively low concentrations (approximately 0.1 mM) in the presence of yeast extract also showed an increase in KSB3 rRNA levels under anaerobic conditions. MAR-FISH confirmed that the KSB3 cells took up radioisotopic carbons from [ (14)C] maltose and [ (14)C] glucose under the same incubation conditions in the batch experiments. These results suggest that one of the important ecophysiological characteristics of KSB3 cells in the sludge is carbohydrate degradation in wastewater and that high carbohydrate loadings may trigger an outbreak of KSB3 bacteria, causing sludge bulking in UASB systems., AMER SOC MICROBIOLOGY, 英語

脱気膜を用いた有機性廃水の水素発酵プロセスの高効率化
佐藤久, 池田真之, 高橋正宏, 岡部聡, 笹川学, 中原禎仁, 環境浄化技術, 10, 1, 85, 90, 2011年02月01日
日本語

Quantification and Genotyping of Human Sapoviruses in the Llobregat River Catchment, Spain
Daisuke Sano, Unai Perez-Sautu, Susana Guix, Rosa Maria Pinto, Takayuki Miura, Satoshi Okabe, Albert Bosch, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77, 3, 1111, 1114, 2011年02月
Human sapoviruses (SaVs) were quantified and characterized in an 18-month survey conducted along the Llobregat river catchment area in Spain. Sample types included freshwater, untreated and treated wastewater, and drinking water. All genogroups were recovered, and a seasonal distribution was observed. This is the first report of SaV quantification and genotyping in the environment outside Japan., AMER SOC MICROBIOLOGY, 英語

Effect of feeding regimens on polyhydroxybutyrate production from food wastes by Cupriavidus necator
Akira Hafuka, Kenji Sakaida, Hisashi Satoh, Masahiro Takahashi, Yoshimasa Watanabe, Satoshi Okabe, BIORESOURCE TECHNOLOGY, 102, 3, 3551, 3553, 2011年02月
We investigated the effects of different feeding regimens (1-pulse, stepwise, and continuous) of fermented food-waste liquid on polyhydroxybutyrate (PHB) production. The fermentation liquid was filtered with a membrane filter (pore size, 0.45 mu m) to remove anaerobic microorganisms and solids and used as a carbon source for Cupriavidus necator. One-pulse feeding yielded the highest cell concentration of C. necator. However, the PHB concentration was higher in the stepwise- and continuous-feeding regimens. Therefore, the continuous-feeding regimen was used for continuous PHB production. PHB could be produced over 259 h (8 draw-fill cycles) with a maximal PHB content of 87%, but the PHB concentration and content decreased with an increase in the operation time. (C) 2010 Elsevier Ltd. All rights reserved., ELSEVIER SCI LTD, 英語

PB-056 アミロイド様細胞外タンパクcurliが引き起こす水処理膜上のバイオフィルム形成(PB 界面/バイオフィルム,ポスター発表)
石川 寛恭, 田代 陽介, 吉田 圭太朗, May Thithiwat, 岡部 聡, 日本微生物生態学会講演要旨集, 27, 98, 98, 2011年
日本微生物生態学会, 日本語

PB-060 バイオフィルム内における形態変異株の発生とその性質の解析(PB 界面/バイオフィルム,ポスター発表)
榮田 弘明, 田代 陽介, 岡部 聡, 日本微生物生態学会講演要旨集, 27, 99, 99, 2011年
日本微生物生態学会, 日本語

A POLYPHASIC APPROACH TO STUDY ECOPHYSIOLOGY OF COMPLEX MULTISPECIES NITRIFYING BIOFILMS
Satoshi Okabe, Hisashi Satoh, Tomonori Kindaichi, METHODS IN ENZYMOLOGY, VOL 46: RESEARCH ON NITRIFICATION AND RELATED PROCESSES, PT B, 496, 163, 184, 2011年
This chapter aims to highlight the great potential of the combined use of microautoradiography (MAR) combined with fluorescent in situ hybridization (FISH) and microsensor technology in studies of complex multispecies nitrifying biofilms. The combination of FISH and microsensor technology is a powerful and reliable tool to link the spatial organization of microbial communities and their in situ function at community levels. MAR-FISH can be used to simultaneously examine the 16S rRNA-based phylogenetic identity and specific metabolic activity of cultivable or uncultivable microorganisms within complex microbial communities at a single-cell level. Information obtained at both resolution levels must be combined to draw a clear picture of a complex multispecies biofilm ecosystem. In addition, ecophysiological interactions among community members in complex multispecies biofilms can be investigated by tracing the fate of radiolabeled [ (14)C] atom incorporated in nitrifying bacteria with MAR-FISH. The structure, function, and ecophysiological interactions among community members in complex multispecies nitrifying biofilms will be illustrated as an example of the combined use of MAR-FISH and microsensor technology., ELSEVIER ACADEMIC PRESS INC, 英語

Analyses of three dominant membrane proteins from anammox planctomycete Candidatus 'Brocadia sinica'
TOJO FUYUMI, ITOH YOSHIKANE, OKABE SATOSHI, Journal of Environmental Biotechnology, 11, 1-2, 1, 81, 2011年
環境バイオテクノロジー学会, 英語

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor
Sunja Cho, Naoki Fujii, Taeho Lee, Satoshi Okabe, BIORESOURCE TECHNOLOGY, 102, 2, 652, 659, 2011年01月
Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (+/- 0.19) kg-N m(-3) d(-1) than the reactor initiated as the partial nitrifying reactor (0.23 (+/- 0.16) kg-N m(-3) d(-1)). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor. (C) 2010 Elsevier Ltd. All rights reserved., ELSEVIER SCI LTD, 英語

Effect of formation of biofilms and chemical scale on the cathode electrode on the performance of a continuous two-chamber microbial fuel cell
Kyungmi Chung, Itto Fujiki, Satoshi Okabe, BIORESOURCE TECHNOLOGY, 102, 1, 355, 360, 2011年01月
A two-chamber MFC system was operated continuously for more than 500 days to evaluate effects of biofilm and chemical scale formation on the cathode electrode on power generation. A stable power density of 0.57 W/m(2) was attained after 200 days operation. However, the power density decreased drastically to 0.2 W/m(2) after the cathodic biofilm and chemical scale were removed. As the cathodic biofilm and chemical scale partially accumulated on the cathode, the power density gradually recovered with time. Microbial community structure of the cathodic biofilm was analyzed based on 16S rRNA clone libraries. The clones closely related to Xanthomonadaceae bacterium and Xanthomonas sp. in the Gammaproteobacteria subdivision were most frequently retrieved from the cathodic biofilm. Results of the SEM-EDX analysis revealed that the cation species (Na(+) and Ca(2+)) were main constituents of chemical scale, indicating that these cations diffused from the anode chamber through the Nation membrane. However, an excess accumulation of the biofilm and chemical scale on the cathode exhibited adverse effects on the power generation due to a decrease in the active cathode surface area and an increase in diffusion resistance for oxygen. Thus, it is important to properly control the formation of chemical scale and biofilm on the cathode during long-term operation. (C) 2010 Elsevier Ltd. All rights reserved., ELSEVIER SCI LTD, 英語

A polyphasic approach to study ecophysiology of complex multispecies nitrifying biofilms
Satoshi Okabe, Hisashi Satoh, Tomonori Kindaichi, Methods in Enzymology, 496, 163, 184, 2011年
This chapter aims to highlight the great potential of the combined use of microautoradiography (MAR) combined with fluorescent in situ hybridization (FISH) and microsensor technology in studies of complex multispecies nitrifying biofilms. The combination of FISH and microsensor technology is a powerful and reliable tool to link the spatial organization of microbial communities and their in situ function at community levels. MAR-FISH can be used to simultaneously examine the 16S rRNA-based phylogenetic identity and specific metabolic activity of cultivable or uncultivable microorganisms within complex microbial communities at a single-cell level. Information obtained at both resolution levels must be combined to draw a clear picture of a complex multispecies biofilm ecosystem. In addition, ecophysiological interactions among community members in complex multispecies biofilms can be investigated by tracing the fate of radiolabeled [14C] atom incorporated in nitrifying bacteria with MAR-FISH. The structure, function, and ecophysiological interactions among community members in complex multispecies nitrifying biofilms will be illustrated as an example of the combined use of MAR-FISH and microsensor technology. © 2011 Elsevier Inc. All rights reserved., 英語

Analyses of Three Dominant Membrane Proteins from Anammox Planctomycete Candidatus 'Brocadia sinica'
TOJO FUYUMI, ITOH YOSHIKANE, OKABE SATOSHI, Journal of environmental biotechnology, 11, 1-2, 1, 81, 2011年
環境バイオテクノロジー学会, 英語

A Great Leap forward in Microbial Ecology
Satoshi Okabe, Mamoru Oshiki, Yoichi Kamagata, Nobuyasu Yamaguchi, Masanori Toyofuku, Yutaka Yawata, Yosuke Tashiro, Nobuhiko Nomura, Hiroyuki Ohta, Moriya Ohkuma, Akira Hiraishi, Kiwamu Minamisawa, MICROBES AND ENVIRONMENTS, 25, 4, 230, 240, 2010年12月
Ribosomal RNA (rRNA) sequence-based molecular techniques emerged in the late 1980s, which completely changed our general view of microbial life. Coincidentally, the Japanese Society of Microbial Ecology (JSME) was founded, and its official journal "Microbes and Environments (M&E)" was launched, in 1985. Thus, the past 25 years have been an exciting and fruitful period for M&E readers and microbiologists as demonstrated by the numerous excellent papers published in M&E. In this minireview, recent progress made in microbial ecology and related fields is summarized, with a special emphasis on 8 landmark areas; the cultivation of uncultured microbes, in situ methods for the assessment of microorganisms and their activities, biofilms, plant microbiology, chemolithotrophic bacteria in early volcanic environments, symbionts of animals and their ecology, wastewater treatment microbiology, and the biodegradation of hazardous organic compounds., JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 英語, 書評論文,書評,文献紹介等

P-46 超高速窒素除去に関与する新種anammox細菌Candidatus "Brocadia gluttonica"のメタゲノム解析(C.水処理生態系,ポスター発表)
新家子 香織, 押木 守, 佐藤 久, 岡部 聡, 日本微生物生態学会講演要旨集, 26, 117, 117, 2010年11月23日
日本微生物生態学会, 英語

蛍光色素を用いたレシオメトリー測定による重金属イオン分析
羽深昭, 谷山拓生, 山田幸司, 高橋正宏, 岡部聡, 佐藤久, 環境工学研究フォーラム講演集, 47th, 184, 186, 2010年11月12日
日本語

環境サンプル測定のためのリン酸マイクロセンサーの開発
宮崎悠爾, 押木守, 佐藤久, 高橋正宏, 岡部聡, 環境工学研究フォーラム講演集, 47th, 4, 6, 2010年11月12日
日本語

微生物燃料電池 : 一石三鳥の次世代型排水処理技術
岡部 聡, 水環境学会誌 = Journal of Japan Society on Water Environment, 33, 11, 347, 347, 2010年11月10日
日本語

Characterization and global gene expression of F- phenocopies during Escherichia coli biofilm formation
Thithiwat May, Akinobu Ito, Satoshi Okabe, MOLECULAR GENETICS AND GENOMICS, 284, 5, 333, 342, 2010年11月
The ecological role of horizontal gene transfer within biofilms has been recently investigated, and it has been reported that conjugation directly induces bacteria to form biofilms via expression of conjugative pili. In this report, we described the contribution of bacterial conjugation during biofilm formation by Escherichia coli harboring a natural IncF conjugative F plasmid (F+). We showed that cell-to-cell pili interactions through the homosexual mating-pair formation among F+ x F+ cells (namely, F- phenocopy phenomenon) promote E. coli biofilm formation at the early development stage. The presence of F+ x F+ population is the result from heterogeneity within biofilms leading to sessile bacteria that grow at different rates, in which the late-stationary phase cells acted as F- phenocopy cells. According to global transcriptional analysis, the biofilm lifestyle shared similar gene expression pattern with F- phenocopies. F- phenocopy cells expressed specific sets of chromosomal genes (e.g., genes for general stress response and two-component systems) that control the regulation regions of F transfer operon by blocking surface exclusion proteins and DNA transfer machineries. However, mating-pair proteins were stabilized and consequently promoted F+ x F+ pili assembly. Thus, F- phenocopy phenomenon is an effective adaptive behavior of bacterial cells during biofilm formation., SPRINGER HEIDELBERG, 英語

微小電極を用いた放牧地からのN₂O生成速度の測定
佐藤久, 日向寺崇文, 高橋正宏, 岡部聡, 土木学会年次学術講演会講演概要集(CD-ROM), 65th, ROMBUNNO.VII-176, 2010年08月05日
日本語

DNA‐SIP法を用いた微生物燃料電池内の炭素フローの解析
木村善一郎, 岡部聡, 日本水環境学会年会講演集, 44th, 507, 2010年03月15日
日本語

純粋培養系と複合系を用いた微生物燃料電池における電力生産の比較
伊藤皓亮, 木村善一郎, CHUNG Kyungmi, 岡部聡, 日本水環境学会年会講演集, 44th, 512, 2010年03月15日
日本語

微小電極を用いたANAMMOXグラニュール内の微生物活性の解析
佐藤久, 高橋慶考, 山田陽平, 高橋正宏, 岡部聡, 日本水環境学会年会講演集, 44th, 396, 2010年03月15日
日本語

Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor
Sunja Cho, Yoshitaka Takahashi, Naoki Fujii, Yohei Yamada, Hisashi Satoh, Satoshi Okabe, CHEMOSPHERE, 78, 9, 1129, 1135, 2010年02月
We investigated nitrogen removal performance and responsible microbial community in an anaerobic up-flow granular bed anammox reactor. The anammox reactor was operated more than 1 year. Biomass in the reactor formed granules after about 2 months of operation, and a sufficient amount of the granules was retained in the reactor with a metallic net to avoid biomass washout during the entire operation. The average diameter of the granules was 3.6 mm at day 310. After 8 months of operation, stable nitrogen removal (60%) was achieved at an average total inorganic nitrogen removal rate of 14 kg-N m(-3) d(-1). The phylogenetic analysis and fluorescence in situ hybridization results revealed that the anammox granules consisted of mono species of anammox bacteria, "Candidatus Brocadia-like species", affiliated with "Candidatus Brocadia anammoxidans" with 16S rRNA gene sequence similarity of 95.7%. The relative abundance of the anammox bacteria in the granules was more than 80% of the total bacteria stained with 4',6-diamidino-2-phenylindole. The anammox bacteria were present throughout the granules whereas the other bacterial groups. Chloroflexi-like filamentous bacteria and betaproteobacterial ammonia-oxidizing bacteria, were mainly present on the surface of the anammox granules and around the anammox bacterial clusters. The in situ anammox activity was detected mainly from near the surface of granules to the upper 800 pm of the granules with microsensors. The granular anammox biomass tolerated higher concentrations of nitrite (400 mg-N L(-1)) than did the homogenized biomass (200 mg-N L(-1)) probably due to substrate diffusion limitation. (C) 2009 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

P-184 水処理膜における微生物付着の新規評価手法の開発(M.界面/バイオフィルム,ポスター発表)
吉田 圭太朗, 田代 陽介, MAY THITHIWAT, 岡部 聡, 日本微生物生態学会講演要旨集, 26, 186, 186, 2010年
日本微生物生態学会, 英語

P-181 アミノグリコシド系抗生物質による大腸菌のバイオフィルム形成誘導(M.界面/バイオフィルム,ポスター発表)
榮田 弘明, 田代 陽介, MAY THITHIWAT, 岡部 聡, 日本微生物生態学会講演要旨集, 26, 185, 185, 2010年
日本微生物生態学会, 英語

1B-2 大腸菌トキシン-アンチトキシンシステム蛋白RelEに対する感受性制御因子の同定(G.生理/代謝/増殖,口頭発表)
柿沼 建至, 田代 陽介, MAY THITHIWAT, 岡部 聡, 日本微生物生態学会講演要旨集, 26, 69, 69, 2010年
日本微生物生態学会, 英語

A great leap forward in microbial ecology.
Satoshi Okabe, Mamoru Oshiki, Yoichi Kamagata, Nobuyasu Yamaguchi, Masanori Toyofuku, Yutaka Yawata, Yosuke Tashiro, Nobuhiko Nomura, Hiroyuki Ohta, Moriya Ohkuma, Akira Hiraishi, Kiwamu Minamisawa, Microbes and environments, 25, 4, 230, 40, 2010年, ［査読有り］, ［国内誌］
Ribosomal RNA (rRNA) sequence-based molecular techniques emerged in the late 1980s, which completely changed our general view of microbial life. Coincidentally, the Japanese Society of Microbial Ecology (JSME) was founded, and its official journal "Microbes and Environments (M&E)" was launched, in 1985. Thus, the past 25 years have been an exciting and fruitful period for M&E readers and microbiologists as demonstrated by the numerous excellent papers published in M&E. In this minireview, recent progress made in microbial ecology and related fields is summarized, with a special emphasis on 8 landmark areas; the cultivation of uncultured microbes, in situ methods for the assessment of microorganisms and their activities, biofilms, plant microbiology, chemolithotrophic bacteria in early volcanic environments, symbionts of animals and their ecology, wastewater treatment microbiology, and the biodegradation of hazardous organic compounds., JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 英語

Characterization of electrochemical activity of a strain ISO2-3 phylogenetically related to Aeromonas sp. isolated from a glucose-fed microbial fuel cell.
Kyungmi Chung, Satoshi Okabe, Biotechnology and bioengineering, 104, 5, 901, 10, 2009年12月01日, ［査読有り］, ［国際誌］
The microbial communities associated with electrodes in closed and open circuit microbial fuel cells (MFCs) fed with glucose were analyzed by 16S rRNA approach and compared. The comparison revealed that bacteria affiliated with the Aeromonas sp. within the Gammaproteobacteria constituted the major population in the closed circuit MFC (harvesting electricity) and considered to play important roles in current generation. We, therefore, attempted to isolate the dominant bacteria from the anode biofilm, successfully isolated a Fe (III)-reducing bacterium phylogenetically related to Aeromonas sp. and designated as strain ISO2-3. The isolated strain ISO2-3 could grow and concomitantly produce current (max. 0.24 A/m(2)) via oxidation of glucose or hydrogen with an electrode serving as the sole electron acceptor. The strain could ferment glucose, but generate less electrical current. Cyclic voltammetry supported the strain ISO2-3 was electrically active and likely to transfer electrons to the electrode though membrane-associated compounds (most likely c-type cytochrome). This mechanism requires intimate contact with the anode surface. Scanning electron microscopy revealed that the strain ISO2-3 developed multiplayer biofilms on the anode surface and also produced anchor-like filamentous appendages (most likely pili) that may promote long-range electron transport across the thick biofilm., JOHN WILEY & SONS INC, 英語

脱気膜を用いた有機性廃水の水素発酵プロセスの高効率化
池田真之, 佐藤久, 高橋正宏, 岡部聡, 中原禎仁, 笹川学, 環境工学研究フォーラム講演集, 46th, 103, 104, 2009年11月27日
日本語

嫌気性消化プロセスへの脱気膜の適用
佐藤久, 池田真之, 高橋正宏, 岡部聡, 中原禎仁, 笹川学, 用水と廃水, 51, 11, 925, 932, 2009年11月01日
産業用水調査会, 日本語

Induction of multidrug resistance mechanism in Escherichia coli biofilms by interplay between tetracycline and ampicillin resistance genes.
Thithiwat May, Akinobu Ito, Satoshi Okabe, Antimicrobial agents and chemotherapy, 53, 11, 4628, 39, 2009年11月, ［査読有り］, ［国際誌］
Biofilms gain resistance to various antimicrobial agents, and the presence of antibiotic resistance genes is thought to contribute to a biofilm-mediated antibiotic resistance. Here we showed the interplay between the tetracycline resistance efflux pump TetA(C) and the ampicillin resistance gene (bla(TEM-1)) in biofilms of Escherichia coli harboring pBR322 in the presence of the mixture of ampicillin and tetracycline. E. coli in the biofilms could obtain the high-level resistance to ampicillin, tetracycline, penicillin, erythromycin, and chloramphenicol during biofilm development and maturation as a result of the interplay between the marker genes on the plasmids, the increase of plasmid copy number, and consequently the induction of the efflux systems on the bacterial chromosome, especially the EmrY/K and EvgA/S pumps. In addition, we characterized the overexpression of the TetA(C) pump that contributed to osmotic stress response and was involved in the induction of capsular colanic acid production, promoting formation of mature biofilms. However, this investigated phenomenon was highly dependent on the addition of the subinhibitory concentrations of antibiotic mixture, and the biofilm resistance behavior was limited to aminoglycoside antibiotics. Thus, marker genes on plasmids played an important role in both resistance of biofilm cells to antibiotics and in formation of mature biofilms, as they could trigger specific chromosomal resistance mechanisms to confer a high-level resistance during biofilm formation., AMER SOC MICROBIOLOGY, 英語

Microbial community structures and in situ sulfate-reducing and sulfur-oxidizing activities in biofilms developed on mortar specimens in a corroded sewer system
Hisashi Satoh, Mitsunori Odagiri, Tsukasa Ito, Satoshi Okabe, WATER RESEARCH, 43, 18, 4729, 4739, 2009年10月
Microbially induced concrete corrosion (MICC) caused by sulfuric acid attack in sewer systems has been a serious problem for a long time. A better understanding of microbial community structures of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) and their in situ activities is essential for the efficient control of MICC. in this study, the microbial community structures and the in situ hydrogen sulfide production and consumption rates within biofilms and corroded materials developed on mortar specimens placed in a corroded manhole was investigated by culture-independent 16S rRNA gene-based molecular techniques and microsensors for hydrogen sulfide, oxygen, pH and the oxidation-reduction potential. The dark-gray gel-like biofilm was developed in the bottom (from the bottom to 4 cm) and the middle (4-20 cm from the bottom of the manhole) parts of the mortar specimens. White filamentous biofilms covered the gel-like biofilm in the middle part. The mortar specimens placed in the upper part (30 cm above the bottom of the manhole) were corroded. The 16S rRNA gene-cloning analysis revealed that one clone retrieved from the bottom biofilm sample was related to an SRB, 12 clones and 6 clones retrieved from the middle biofilm and the corroded material samples, respectively, were related to SOB. In situ hybridization results showed that the SRB were detected throughout the bottom biofilm and filamentous SOB cells were mainly detected in the upper oxic layer of the middle biofilm. Microsensor measurements demonstrated that hydrogen sulfide was produced in and diffused out of the bottom biofilms. In contrast, in the middle biofilm the hydrogen sulfide produced in the deeper parts of the biofilm was oxidized in the upper filamentous biofilm. pH was around 3 in the corroded materials developed in the upper part of the mortar specimens. Therefore, it can be concluded that hydrogen sulfide provided from the bottom biofilms and the sludge settling tank was emitted to the sewer atmosphere, then oxidized to corrosive compounds in the upper and middle parts of the manhole, and only the upper part of the mortar specimens were corroded, because in the middle part of the manhole the generated corrosive compounds (e.g., sulfuric acid) was reduced in the deeper parts of the biofilm. (C) 2009 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

有機性廃棄物からのポリヒドロキシアルカン酸の生産
羽深昭, 佐藤久, 高橋正宏, 岡部聡, 日本水環境学会シンポジウム講演集, 12th, 168, 169, 2009年09月14日
日本語

In Vitro Toxicity of Silver Nanoparticles at Noncytotoxic Doses to HepG2 Human Hepatoma Cells
Koji Kawata, Masato Osawa, Satoshi Okabe, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 43, 15, 6046, 6051, 2009年08月
Although it has been reported that silver nanoparticles (Ag-NPs) have strong acute toxic effects to various cultured the toxic effects at noncytotoxic doses are still unknown. We, therefore, evaluated in vitro toxicity of doses in human hepatoma cell line, HepG2, based on cell viability assay, micronucleus test, and DNA microarray We also used polystyrene nanoparticles (PS-NPs) and silver carbonate (Ag(2)CO(3)) as test materials to compare the toxic with respect to different raw chemical composition and form of silver. The cell viability assay demonstrated that Ag-NPs accelerated cell proliferation at low doses (<0.5 mg/L), was supported by the DNA microarray analysis showing significant induction of genes associated with cell cycle progression. However, only Ag-NPs exposure exhibited a significant cytotoxicity at higher doses (> 1.0 mg/L) and abnormal cellular morphology, displaying cellular shrinkage and acquisition of an irregular shape. In addition, only exposure increased the frequency of micronucleus formation up to 47.9 +/- 3.2% of binucleated cells, suggesting that appear to cause much stronger damages to chromosome than PS-NPs and ionic Ag(+). Cysteine, a strong ionic Ag(+) only partially abolished the formation of micronuclei mediated by Ag-NPs and changed the gene expression, indicating that ionic Ag(+) derived from Ag-NPs could not fully explain these biological actions, Based on these discussions, it is concluded that both "nanosized particle of Ag" as well as "ionic Ag(+)" contribute to the toxic effects of Ag-NPs., AMER CHEMICAL SOC, 英語

Localized Expression Profiles of rpoS in Escherichia coli Biofilms
Akinobu Ito, Thithiwat May, Asami Taninchi, Koji Kawata, Satoshi Okabe, BIOTECHNOLOGY AND BIOENGINEERING, 103, 5, 975, 983, 2009年08月
Although importance of the rpoS gene on biofilm formation by Escherichia coli has been suggested, there his not been any report showing where the rpoS is expressed during biofilm formation process. Since physiological state of the cells in the biofilms is considerably heterogeneous, the expression of the rpoS gene must be heterogeneous. In this study, in situ spatial expression of the rpoS gene during biofilm formation was investigated with an rpoS-gfp transcriptional fusion mutant strain. A ribosomal binding site and a gene encoding a green fluorescent protein were introduced into the downstream of the rpoS gene, which enabled us to observe the in situ spatial expression of the rpoS gene during biofilm formation processes without any disturbance of the rpoS expression. In the early stages of the biofilm formation process, the rpoS gene was expressed in the most of the cells. On the other hand, the rpoS expression was observed only at the outside of the biofilms during the late stages of the biofilm formation process. The in situ spatial expression of the rpoS gene in the biofilm was verified by quantifying the expression levels of the rpoS at the outside and the inside of the biofilms with the real time RTPCR. In addition, global gene expression analysis was performed with DNA microarray to investigate physiological difference between the outside and the inside of the biofilms. This heterogeneous rpoS expression profile suggested that the cells at the outside of the biofilm need to express the rpoS to shift the physiological state to the stationary growth mode such as induction of various stress responses and suppression of the motility. Biotechnol. Bioeng. 2009;103: 975-983. (C) 2009 Wiley Periodicals, Inc., WILEY-BLACKWELL, 英語

A biofilm model for prediction of pollutant transformation in sewers
Feng Jiang, Derek Hoi-wai Leung, Shiyu Li, Guang-Hao Chen, Satoshi Okabe, Mark C. M. van Loosdrecht, WATER RESEARCH, 43, 13, 3187, 3198, 2009年07月
This study developed a new sewer biofilm model to simulate the pollutant transformation and biofilm variation in sewers under aerobic, anoxic and anaerobic conditions. The biofilm model can describe the activities of heterotrophic, autotrophic, and sulfate-reducing bacteria (SRB) in the biofilm as well as the variations in biofilm thickness, the spatial profiles of SRB population and biofilm density. The model can describe dynamic biofilm growth, multiple biomass evolution and competitions among organic oxidation, denitrification, nitrification, sulfate reduction and sulfide oxidation in a heterogeneous biofilm growing in a sewer. The model has been extensively verified by three different approaches, including direct verification by measurement of the spatial concentration profiles of dissolved oxygen, nitrate, ammonia, and hydrogen sulfide in sewer biofilm. The spatial distribution profile of SRB in sewer biofilm was determined from the fluorescent in situ hybridization (FISH) images taken by a confocal laser scanning microscope (CLSM) and were predicted well by the model. (C) 2009 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Continuous power generation and microbial community structure of the anode biofilms in a three-stage microbial fuel cell system
Kyungmi Chung, Satoshi Okabe, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 83, 5, 965, 977, 2009年07月
A mediator-less three-stage two-chamber microbial fuel cell (MFC) system was developed and operated continuously for more than 1.5 years to evaluate continuous power generation while treating artificial wastewater containing glucose (10 mM) concurrently. A stable power density of 28 W/m(3) was attained with an anode hydraulic retention time of 4.5 h and phosphate buffer as the cathode electrolyte. An overall dissolved organic carbon removal ratio was about 85%, and coulombic efficiency was about 46% in this MFC system. We also analyzed the microbial community structure of anode biofilms in each MFC. Since the environment in each MFC was different due to passing on the products to the next MFC in series, the microbial community structure was different accordingly. The anode biofilm in the first MFC consisted mainly of bacteria belonging to the Gammaproteobacteria, identified as Aeromonas sp., while the Firmicutes dominated the anode biofilms in the second and third MFCs that were mainly fed with acetate. Cyclic voltammetric results supported the presence of a redox compound(s) associated with the anode biofilm matrix, rather than mobile (dissolved) forms, which could be responsible for the electron transfer to the anode. Scanning electron microscopy revealed that the anode biofilms were comprised of morphologically different cells that were firmly attached on the anode surface and interconnected each other with anchor-like filamentous appendages, which might support the results of cyclic voltammetry., SPRINGER, 英語

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms
Akinobu Ito, Asami Taniuchi, Thithiwat May, Koji Kawata, Satoshi Okabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75, 12, 4093, 4100, 2009年06月
Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 mu g/ml), kanamycin (25 mu g/ml), and ofloxacin (10 mu g/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses., AMER SOC MICROBIOLOGY, 英語

溶存バイオガスに着目した新規嫌気性廃水処理リアクターの開発
佐藤久, 池田真之, 高橋正宏, 岡部聡, 中原禎仁, 笹川学, 日本水環境学会年会講演集, 43rd, 253, 2009年03月16日
日本語

有機性廃棄物からのポリヒドロキシアルカン酸の生産
羽深昭, 佐藤久, 高橋正宏, 岡部聡, 日本水環境学会年会講演集, 43rd, 623, 2009年03月16日
日本語

P-168 The fate of male-specific filamentous bacteriophage in Escherichia coli biofilm formation :
鶴田 研二, Thithiwat May, 岡部 聡, 日本微生物生態学会講演要旨集, 25, 125, 125, 2009年
日本微生物生態学会, 英語

微生物燃料電池内の嫌気的酢酸分解に寄与する微生物群集
木村善一郎, 藤木一到, 岡部聡, 日本微生物生態学会講演要旨集, 25th, 25, YOSHI74, 74, 2009年
日本微生物生態学会, 日本語

二双培養を用いた微生物燃料電池における電力生産の評価と細菌間相互作用の評価
伊藤皓亮, 木村善一郎, 鄭景美, 岡部聡, 日本微生物生態学会講演要旨集, 25th, 25, YOSHI73, 73, 2009年
日本微生物生態学会, 日本語

The Response of Pseudomonas aeruginosa PAO1 Efflux Pump-Defective Mutants to N-Octanoyl-L-Homoserine Lactone
Yutaka Yawata, Hideaki Maseda, Satoshi Okabe, Akinobu Ito, Isao Sawada, Hiroaki Kurashima, Hiroo Uchiyama, Nobuhiko Nomura, MICROBES AND ENVIRONMENTS, 24, 4, 338, 342, 2009年
N-Octanoyl-L-homoserine lactone (C8-HSL) is an acyl-homoserine-lactone signal utilized in the quorum-sensing (QS) systems of Burkholderia cenocepacia and other bacterial species. Although also produced by Pseudomonas aeruginosa, its role in this species has not been elucidated. Here, we report that C8-HSL modulated antibiotic resistance and pyocyanin production in a P. aeruginosa efflux pump-deficient mutant. The rhl/las quorum-sensing system and qscR gene were both shown to be nonessential in the C8-HSL-induced changes in ofloxacin resistance, suggesting that P. aeruginosa possesses a distinct pathway to respond to C8-HSL., JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 英語

Qualitative and quantitative estimation of fecal pollution using Bacteroides–Prevotella 16S rRNA host-specific genetic markers by T-RFLP and real-time PCR analyses
SAVICHTCHEVA O, OKABE S, Water Science and Technology, 59, 9, 1831, 1840, 2009年
Rapid and reliable determination of the non-point sources of fecal pollution is a critical issue for the environmental microbiologists all over the world. In this work we evaluated the use of anaerobic bacterial group Bacteroides-Prevotella as an alternative fecal pollution indicator. Terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (RT-PCR) analyses were used to monitor and quantify human-, cow- and pig-specific fecal contamination in natural river waters. We also included DNA sequence analysis of the identified fecal markers revealed by T-RFLP in order to clarify the specificity of each marker. It was suggested that the most influent peaks for each fecal source could be used to identify the source of fecal pollution. Development of specific probes based on these markers permit to quantify source of contamination by quantitative RT-PCR. Therefore, we combined the T-RFLP results and RT-PCR assay to quantify fecal contamination by certain host. We can conclude that T-RFLP and RT-PCR analyses showed high reproducibility and sensitivity during analyzing real water samples and can be used to identify, track and quantify host-specific bacterial genetic markers in complex natural water environments. © IWA Publishing 2009., I W A PUBLISHING, 英語

Comparison of Gene Expression Profiles in HepG2 Cells Exposed to Arsenic, Cadmium, Nickel, and Three Model Carcinogens for Investigating the Mechanisms of Metal Carcinogenesis
Koji Kawata, Ryuhei Shimazaki, Satoshi Okabe, ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, 50, 1, 46, 59, 2009年01月
Carcinogenesis is an important chronic toxicity of metals and metalloids, although their mechanisms of action are still unclear. Comparison of gene expression patterns induced by carcinogenic metals, metalloids, and model carcinogens would give an insight into understanding of their carcinogenic mechanisms. In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2, after exposing to two metals (cadmium and nickel), a metalloid (arsenic), and three model carcinogenic chemicals N-dimethylnitrosoamine (DMN), 12-O-tetradeconoylphorbol-3-acetate (TPA), and tetrachloroethylene (TCE) using DNA microarrays with 8,795 human genes. Of the genes altered by As, Cd, and Ni exposures, 31-55% were overlapped with those altered by three model carcinogenic chemical exposures in our experiments. In particular, the metals and metalloid shared certain characteristics with TPA and TCE in remarkable upregulations of the genes associated with progression of cell cycle, which might play a central role in As, Cd, and Ni carcinogenesis. This characteristic of gene expression alteration was partially counteracted by intracellular accumulation of vitamin C in As-exposed cells, whereas the number of cell-cycle associated genes was increased in Cd- and Ni-exposed cells. In our experimental conditions, ROS might have an accelerative effect on the cell proliferation mechanisms of As, but have an inhibitory effect on those of other two heavy metals. Furthermore, based on the results of Q-PCR, the oncogene PTTG I, which was upregulated by all carcinogenic chemical exposures in the array experiments, might be a useful biomarker for evaluation of the carcinogenesis of inorganic carcinogens. Environ. Mol. Mutagen. 50:46-59, 2009. (C) 2008 Wiley-Liss, Inc., WILEY-LISS, 英語

有機性廃棄物からのポリヒドロキシアルカン酸の生産
羽深昭, 坂井田健司, 佐藤久, 深澤達矢, 高橋正宏, 岡部聡, 環境工学研究フォーラム講演集, 45th, 61, 63, 2008年11月28日
日本語

溶存バイオガスに着目した嫌気性廃水処理リアクターの開発
佐藤久, 池田真之, 高橋慶多, 深澤達矢, 高橋正宏, 岡部聡, 中原禎仁, 笹川学, 環境工学研究フォーラム講演集, 45th, 58, 60, 2008年11月28日
日本語

Escherichia coli Harboring a Natural IncF Conjugative F Plasmid Develops Complex Mature Biofilms by Stimulating Synthesis of Colanic Acid and Curli
Thithiwat May, Satoshi Okabe, JOURNAL OF BACTERIOLOGY, 190, 22, 7479, 7490, 2008年11月
It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F(+) cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms., AMER SOC MICROBIOLOGY, 英語

Quantification of cell specific uptake activity of microbial products by uncultured Chloroflexi by microautoradiography combined with fluorescence in situ hybridization
Yuki Miura, Satoshi Okabe, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 42, 19, 7380, 7386, 2008年10月
We, for the first time, quantitatively determined cell specific uptake activities of microbial products (bacterial cell detritus and extracellular polymeric substances, EPS) by the member of uncultured Chloroflexi by using a microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) technique. For this MAR-FISH analysis, we prepared [ (14)C]-labeled microbial products from biomass sludge obtained and bacterial strains (Pseudomonas sp. and Acinetobacter sp.) isolated from our pilot-scale membrane bioreactor (MBR) as tracer substrates, which probably represent the more realistic food source in the MBR. The quantitative MAR-FISH analyses clearly showed that most of the uncultured Chloroflexi could indeed uptake the bacterial detritus of the two isolated strains with rates of 1.7-3.5 x 10(-17) g-C mu m(-2)-surface area h(-1) (corresponding to 1.2-1.7 mg-C-bacterial detritus L(-1) h(-1)) in the cultures, which were, however, about 2 orders of magnitude lower than the uptake rates of simple monosaccharides (mannose, arabinose, fucose, and galactose). Based on these results and their high abundance (more than 20% of total bacteria detected with EUB338-mixed probes), it could be estimated that the uncultured Chloroflexi contributes 38-51% of the total degradation of microbial products occurred in the MAR-FISH cultures., AMER CHEMICAL SOC, 英語

脱気膜を用いたメタン発酵プロセスにおける高効率ガス回収
佐藤久, 高橋慶多, 深澤達矢, 高橋正宏, 岡部聡, 中原禎仁, 笹川学, 土木学会年次学術講演会講演概要集(CD-ROM), 63rd, Disk 2, ROMBUNNO.7-043, 2008年08月13日
日本語

水系生物膜内の微生物活性解析への微小電極の適用
佐藤 久, 岡部 聡, 水, 50, 10, 20, 28, 2008年08月
月刊「水」発行所, 日本語

Bacterial community dynamics and conversion of inorganic nitrogen under aerobic and micro-aerobic conditions
S. -J. Cho, S. Okabe, S. -J. Lee, ENVIRONMENTAL TECHNOLOGY, 29, 4, 463, 471, 2008年04月
To investigate the inorganic nitrogen conversion in reactors that were operated under aerobic and micro-aerobic conditions and to identify populations that became acclimated in the reactors under those oxygen conditions, we operated two reactors with 72 h of hydraulic retention time and an artificial medium containing ammonium- and nitrite-nitrogen without any organic compound. We determined the concentration of inorganic nitrogen in samples from the reactors and the microbial community structure in the reactors by using PCR-DGGE of the partial 16S rRNA gene. The results showed that nitrification to nitrate fully progressed in the aerobic reactor within 24 h, but we could not find any obvious reaction in the micro-aerobic reactor. In a view of microbial community structure, the total number of microorganisms composing the communities in the reactors was dramatically decreased compared to those of the initial inoculated sludge that originated from the Jang-lim, sewage treatment plant in Busan, Republic of Korea because of the limited nutrients present. One Nitrospira sp. was clearly detected under both aerobic (as DO > 2 mg l(-1)) and micro-aerobic (as DO < 0.7 mg l(-1)) conditions while no AOB-Iike bacterium was exactly matched among main bands. By the results, nitrite-oxidizing bacteria may be more tolerant to variations in oxygen, or the occupation of ammonia-oxidizing bacteria as dominant groups may have been inhibited by substrate starvation and high concentrations of nitrite-N in influent., TAYLOR & FRANCIS LTD, 英語

Significance of rpoS during maturation of Escherichia coli biofilms
Akinobu Ito, Thithiwat May, Koji Kawata, Satoshi Okabe, BIOTECHNOLOGY AND BIOENGINEERING, 99, 6, 1462, 1471, 2008年04月
Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG 1655 wild type (WT) and rpoS mutant (Delta rpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the Delta rpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas Delta rpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (arpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcf) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses., JOHN WILEY & SONS INC, 英語

MAR‐FISH法を用いた膜分離活性汚泥槽内の糸状性細菌Chloroflexiの生物由来有機物資化特性評価
三浦佑己, 渡辺義公, 岡部聡, 日本水環境学会年会講演集, 42nd, 170, 2008年03月19日
日本語

04-074 MFC負極中バイオフィルム内の微生物群集構造解析(水処理生態系,研究発表)
熊野 圭一, 鄭 景美, 岡部 聡, 日本微生物生態学会講演要旨集, 24, 95, 95, 2008年
日本微生物生態学会, 日本語

01-010 大腸菌トキシン-アンチトキシンシステム蛋白RelEに対する菌密度依存的感受性変化(生理/代謝/増殖,研究発表)
谷内 亜沙美, 川田 耕司, 岡部 聡, 日本微生物生態学会講演要旨集, 24, 63, 63, 2008年
日本微生物生態学会, 日本語

01-006 大腸菌バイオフォイルム形成課程における薬剤感受性(界面/バイオフィルム,研究発表)
伊藤 暁信, 谷内 亜沙美, May Thithiwat, 岡部 聡, 日本微生物生態学会講演要旨集, 24, 61, 61, 2008年
日本微生物生態学会, 日本語

S-C3 複合系バイオフィルム内の微生物群集構造と機能の解明(シンポジウムC「バイオフィルム研究の様々な切り口-分子、細胞、生態-」,第24回大会シンポジウム)
岡部 聡, 日本微生物生態学会講演要旨集, 24, 191, 191, 2008年
日本微生物生態学会, 日本語

有機性廃棄物からのポリヒドロキシアルカン酸の生産
佐藤久, 坂井田健司, 岡部聡, 渡辺義公, 環境バイオテクノロジー学会大会プログラム講演要旨集, 34th, 27, 2008年
日本語

トキシコゲノミクスとDNAマイクロアレイ
岡部 聡, 環境技術 = ENVIRONMENTAL CONSERVATION ENGINEERING, 36, 11, 778, 782, 2007年11月20日
環境技術学会, 日本語

有機性廃棄物からのポリヒドロキシアルカン酸の生産
佐藤久, 坂井田健司, 岡部聡, 渡辺義公, 環境工学研究フォーラム講演集, 44th, 224, 226, 2007年11月16日
日本語

Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules
Hisashi Satoh, Yuki Miura, Ikuo Tsushima, Satoshi Okabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73, 22, 7300, 7307, 2007年11月
The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH4, H-2, pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeall clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH4, H-2, pH, and ORP revealed that acid and H-2, production occurred in the upper part of the granule, below which H-2, consumption and CH4 production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H-2 was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH4 in the inner layer. We determined the effective diffusion coefficient for H-2 in the anaerobic granules to be 2.66 X 10(-5) cm(2) s(-1), which was 57% in water., AMER SOC MICROBIOLOGY, 英語

Significance of Chloroflexi in performance of submerged membrane Bioreactors (MBR) treating municipal wastewater
Yuki Miura, Yoshimasa Watanabe, Satoshi Okabe, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 41, 22, 7787, 7794, 2007年11月
We operated pilot-scale submerged membrane bioreactors (MBR) treating real municipal wastewater for over 3 months and observed an interesting phenomenon that carbohydrate concentrations in the MBRs rapidly increased, which consequently resulted in membrane fouling, when relative abundance of the member of uncultured Chloroflexi decreased from over 30% of total Bacteria to less than 10%. We, therefore, hypothesized that the uncultured Chloroflexi present in the MBRs could preferentially degrade carbohydrates and consequently prevent membrane fouling. To test this hypothesis, we investigated the phylogenetic identity, diversity, and in situ physiology (substrate utilization characteristics) of Chloroflexi residing in the MBR by using 16S rRNA gene sequencing analysis and microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) technique. Most of the clones related to the phylum Chloroflexi were affiliated with the Chloroflexi subphylum 1 containing only a few cultured representatives. The MAR-FISH revealed that the members of Chloroflexi were metabolically versatile and could preferentially utilize glucose and N-acetyl glucosamine (a main substantial constituent of the cell wall peptidoglycan) under oxic and anoxic conditions. The utilization of these compounds was low at low pH. These findings suggest that the members of Chloroflexi are ecologically significant in the MBR treating municipal wastewater and are responsible for degradation of SMP including carbohydrates and cellular materials, which consequently reduces membrane fouling potential., AMER CHEMICAL SOC, 英語

Persistence of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental waters: effects of temperature and salinity
Satoshi Okabe, Yoko Shimazu, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 76, 4, 935, 944, 2007年09月
Host-specific Bacteroides-Prevotella 16S rRNA genetic markers are promising alternative indicators for identifying the sources of fecal pollution because of their high abundance in the feces of warm-blooded animals and high host specificity. However, little is known about the persistence of these genetic markers in environments after being released into environmental waters. The persistence of feces-derived four different host-specific Bacteroides-Prevotella 16S rRNA genetic makers (total, human-, cow-, and pig-specific) in environmental waters was therefore investigated at different incubation temperatures (4, 10, 20, and 30 degrees C) and salinities (0, 10, 20, and 30 ppt) and then compared with the survival of conventional fecal-indicator organisms. The host-specific genetic markers were monitored by using real-time polymerase chain reaction (PCR) assays with specific primer sets. Each host-specific genetic marker showed similar responses in non-filtered river water and seawater: They persisted longer at lower temperatures and higher salinities. In addition, these markers did not increase in all conditions tested. Decay rates for indicator organisms were lower than those for host-specific genetic markers at temperature above 10 degrees C. Furthermore, we investigated whether the PCR-detectable 16S rRNA genetic markers reflect the presence of live target cells or dead target cells in environmental waters. The result revealed that the detection of the Bacteroides-Prevotella 16S rRNA genetic markers in environmental waters mainly reflected the presence of 'viable but non-culturable' Bacteroides-Prevotella cells. These findings indicate that seasonal and geographical variations in persistence of these host-specific Bacteroides-Prevotella 16S rRNA genetic markers must be considered when we use them as alternative fecal indicators in environmental waters., SPRINGER, 英語

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms
Tomonori Kindaichi, Ikuo Tsushima, Yuji Ogasawara, Masaki Shimokawa, Noriatsu Ozaki, Hisashi Satoh, Satoshi Okabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73, 15, 4931, 4939, 2007年08月
We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phyllogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (< 1,000 mu m) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the How direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH4+, and NO2- consumption rates decreased from 0.68 and 0.64 mu mol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 mu mol cm(-2) h(-1) at P3 (the third port, 205 rum from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH4+ and NO2- and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O-2, or organic compounds, which consequently established suitable microenvironments for anammox bacteria., AMER SOC MICROBIOLOGY, 英語

Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators
Olga Savichtcheva, Noriko Okayama, Satoshi Okabe, WATER RESEARCH, 41, 16, 3615, 3628, 2007年08月
Occurrence and prevalence of different bacterial enteric pathogens as well as their relationships with conventional (total and fecal coliforms) and alternative fecal indicators (host-specific Bacteroides 16S rRNA genetic markers) were investigated for various water samples taken from different sites with different degrees of fecal contamination. The results showed that a wide range of bacterial pathogens could be detected in both municipal wastewater treatment plant samples and in surface water samples. Logistic regression analysis revealed that total and human-specific Bacteroides 16S rRNA genetic markers showed significant predictive values for the presence of Escheriachia coli O-157, Salmonella, heat-labile enterotoxin (IT) of enterotoxigenic E. coli (ETEC), and heat-stable enterotoxin for human (STh) of ETEC. The probability of occurrence of these pathogenic bacteria became significantly high when the concentrations of human-specific and total Bacteroides 16S rRNA genetic markers exceeded 10(3) and 104 copies/100mL. In contrast, Clostridium perfringens was detected at high frequency regardless of sampling sites and levels of Bacteroides 16S rRNA genetic markers. No genes related to Shigella spp., Staphylococcus aureus and Vibrio cholerae were detected in any samples analyzed in this study. Conventional indicator microorganisms had low levels of correlation with the presence of pathogens as compared with the alternative fecal indicators. These results suggested that real-time PCR-based measurement of alternative Bacteroides 16S rRNA genetic markers was a rapid and sensitive tool to identify host-specific fecal pollution and probably associated bacterial pathogens. However, since one fecal indicator might not represent the relative abundance of all pathogenic bacteria, viruses and protozoa, combined application of alternative indicators with conventional ones could provide more comprehensive pictures of fecal contamination, its source and association with pathogenic microorganisms. (c) 2007 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge
Herto Dwi Ariesyady, Tsukasa Ito, Kazurni Yoshiguchi, Satoshi Okabe, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75, 3, 673, 683, 2007年06月
The phylogenetic and functional diversity of syntrophic prop ionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR-FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52-62% of the total active POB under all the propionate concentrations tested (0.515 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobactet- accounted for 16-3 1 % of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration., SPRINGER, 英語

Classification of heavy-metal toxicity by human DNA microarray analysis
Koji Kawata, Hiroyuki Yokoo, Ryuhes Shimazaki, Satoshi Okabe, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 41, 10, 3769, 3774, 2007年05月
Microarray technology is proving to be a useful tool to classify undefined environmental toxicants, to investigate underlying mechanisms of toxicity, and to identify candidate toxicant-specific genetic markers by examining global effects of putative toxicants on gene expression profiles. The aim of this study was to evaluate the toxicities of six heavy metals through the comparison with gene expression patterns induced by well-known chemicals. For this purpose, we first identified the genes altered specifically in HepG2 under the exposure of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), phenol, and N-nitrosodimethylamine (DMN), which were selected as the model chemicals, using DNA microarray. On the basis of the expression profiles of these genes, toxicities of six heavy metals, arsenic, cadmium, nickel, antimony, mercury, and chromium, were evaluated. The specific gene alteration and hierarchical clustering revealed that biological action of six heavy metals was clearly related to that of DMNQ which has been reported to be a reactive oxygen species (ROS) generating chemical and which induced the genes associated with cell proliferative responses. These results suggest that cell proliferative responses which are probably caused by ROS are a major apparent biological action of high-dose heavy metals, supporting the previous reports. Overall, a mechanism-based classification by DNA microarray would be an efficient method for evaluation of toxicities of environmental samples., AMER CHEMICAL SOC, 英語

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors
Ikuo Tsushima, Yuji Ogasawara, Tomonori Kindaichi, Hisashi Satoh, Satoshi Okabe, WATER RESEARCH, 41, 8, 1623, 1634, 2007年04月
To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-N m(-3) day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH4+ to NO2- molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH4+/NO2- molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria), They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH4+ and NO2-) of 4.45 g-N of (NH4++NO2-) m(-2) day(-1) was detected in the upper 800 mu m of the biofilm, which was consistent with the spatial distribution of anammox bacteria. (c) 2007 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester.
Herto Dwi Ariesyady, Tsukasa Ito, Satoshi Okabe, Water research, 41, 7, 1554, 68, 2007年04月, ［査読有り］, ［国際誌］
Functional Bacteria and Archaea community structures of a full-scale anaerobic sludge digester were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescent in situ hybridization (FISH) technique and micromanipulation. FISH analysis with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes based on 16S rRNA clone libraries revealed that the Gram-positive bacteria represented by probe HGC69A-hybridized Actinobacteria (8.5+/-1.4% of total 4', 6-diamidino-2-phenylindole (DAPI)-stained cells) and probe LGC354-hybridized Firmicutes (3.8+/-0.8%) were the major phylogenetic bacterial phyla, followed by Bacteroidetes (4.0+/-1.2%) and Chloroflexi (3.7+/-0.8%). The probe MX825-hybridized Methanosaeta (7.6+/-0.8%) was the most abundant archaeal group, followed by Methanomicrobiales (2.8+/-0.6%) and Methanobacteriaceae (2.7+/-0.4%). The functional community structures (diversity and relative abundance) of major trophic groups were quantitatively analyzed by MAR-FISH. The results revealed that glucose-degrading microbial community had higher abundance (ca. 10.6+/-4.9% of total DAPI-stained cells) and diversity (at least seven phylogenetic groups) as compared with fatty acid-utilizing microbial communities, which were more specialized to a few phylogenetic groups. Despite the dominance of Betaproteobacteria, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups dominated the [ (14)C]glucose-, [ (14)C]propionate-, [ (14)C]butyrate- and [ (14)C]acetate-utilizing microorganism community, and accounted for 27.7+/-4.3%, 29.6+/-7.0%, 34.5+/-7.6% and 18.2+/-9.5%, respectively. In spite of low abundance (ca. 1%), the hitherto unknown metabolic functions of Spirochaeta and candidate phylum of TM7 as well as Synergistes were found to be glucose and acetate utilization, respectively., PERGAMON-ELSEVIER SCIENCE LTD, 英語

有機性廃棄物からのポリヒドロキシアルカン酸の生産
坂井田健司, 佐藤久, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 41st, 335, 2007年03月15日
日本語

膜分離活性汚泥法における糸状性細菌Chloroflexiの生物代謝由来有機物資化特性評価
三浦佑己, 渡辺義公, 岡部聡, 日本水環境学会年会講演集, 41st, 227, 2007年03月15日
日本語

Quantification of host-specific Bacteroides-Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater
Satoshi Okabe, Noriko Okayama, Olga Savichtcheva, Tsukasa Ito, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 74, 4, 890, 901, 2007年03月
Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9-11 log(10) copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides-Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r(2) = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50-800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments., SPRINGER, 英語

Bacterial community structures in MBRs treating municipal wastewater: Relationship between community stability and reactor performance
Yuki Miura, Mirian Noriko Hiraiwa, Tsukasa Ito, Takanori Itonaga, Yoshimasa Watanabe, Satoshi Okabe, WATER RESEARCH, 41, 3, 627, 637, 2007年02月
Bacterial community structures in pilot-scale conventional membrane bioreactors (CMBRs) and hybrid MBRs (HMBRs) which were combined with pre-coagulation/sedimentation were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescence in situ hybridization (FISH) techniques. The results were compared with the community structure in a full-scale activated sludge (AS) process treating the same municipal wastewater. The Dice index (Cs) of similarity analysis of DGGE banding patterns demonstrated that the microbial community in AS was more similar to those in CMBR1 and CMBR2 than HMBR1 and HMBR2. This suggested that influent wastewater composition had a larger impact on bacterial community structures. Long-term community structure changes in the HMBRs and CMBRs were monitored and analyzed over 240 days by Non-metric multidimensional scaling (NMDS) analysis of DGGE banding patterns. The NMDS analysis revealed that both HMBRs and CMBRs had marked changes in community structures during the first about 100 days. Thereafter the perpetual fluctuations of bacterial community structures were observed in both HMBRs and CMBRs, even though the stable MBR performances (the performance was measured as membrane permeability and removal of dissolved organic carbon, DOC) were achieved. These results suggest that not only the stability, but also the adequate dynamics ("flexibility") of the bacterial community structure are important for the stable performance of the MBRs treating complex municipal wastewater. (c) 2006 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR
Ikuo Tsushima, Tomonori Kindaichi, Satoshi Okabe, WATER RESEARCH, 41, 4, 785, 794, 2007年02月
The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9 x 10(1) to 8.9 x 10(6) copies per PCR, corresponding to the detection limit of 3.6 x 10(3) target copies mL(-1). A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples. (c) 2006 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Influences of infaunal burrows on the community structure and activity of ammonia-oxidizing bacteria in intertidal sediments
Hisashi Satoh, Yoshiyuki Nakamura, Satoshi Okabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73, 4, 1341, 1348, 2007年02月
Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O-2 concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O-2 transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 rum from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH4+ consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH4+ and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O-2 transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH4+ consumption capacity. Consequently, the infaunal burrow became an important site for NH4+ consumption in the intertidal sediment., AMER SOC MICROBIOLOGY, 英語

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems
Satoshi Okabe, Mitsunori Odagiri, Tsukasa Ito, Hisashi Satoh, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73, 3, 971, 980, 2007年02月
Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over I year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after I year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S-0, and S2O32-). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and thatA. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below., AMER SOC MICROBIOLOGY, 英語

PB-14 Microbial community structure of biofims in microbial fuel cells(Waste water treatment ecosystem,Poster presentation B) :
Chung Kyungmi, Okabe Satoshi, 日本微生物生態学会講演要旨集, 23, 121, 121, 2007年
日本微生物生態学会, 英語

PB-13 Key physiology of ANAMMOX bacteria(Waste water treatment ecosystem,Poster presentation B) :
Shimokawa Masaki, Tsushima Ikuo, Takahashi Yoshitaka, Cho Sun-Ja, Kindaichi Tomonori, Okabe Satoshi, 日本微生物生態学会講演要旨集, 23, 121, 121, 2007年
日本微生物生態学会, 英語

PB-17 In situ physiology of candidate division TM7 in a wastewater treatment plant(Waste water treatment ecosystem,Poster presentation B) :
Kindaichi Tomonori, Ozaki Noriatsu, Ohashi Akiyoshi, Okabe Satoshi, 日本微生物生態学会講演要旨集, 23, 123, 123, 2007年
日本微生物生態学会, 英語

Ecophysiology of sulphate-reducing bacteria in environmental biofilms
Satoshi Okabe, Sulphate-Reducing Bacteria: Environmental and Engineered Systems, 359, 382, 2007年01月01日
INTRODUCTION Sulphate-reducing bacteria (SRB) are a phylogenetically and physiologically diverse group of bacteria, characterized by their versatile metabolic capability to use various electron acceptors and donors (Widdel, 1988). SRB are therefore universally distributed in diverse environments and play significant ecophysiological roles in anaerobic biomineralization pathways. The degradation of organic matter by a complex microbial community is governed to a large extent by available electron acceptors. The terminal stages of the anaerobic mineralization of organic matter is catalyzed by SRB and methanogens, and their competitive and cooperative interactions have been described previously (Oude Elferink et al., 1994). Typical domestic wastewaters contain sulphate concentrations of 100-1000 µM and relatively low dissolved oxygen due to the lower solubility and rapid depletion of this gas by biological activity. Thus, sulphate reduction can be the dominant terminal electron accepting process and account for up to 50% of mineralization of organic matter in wastewater biofilms (Kühl and Jorgensen, 1992
Okabe et al., 2003a). Multiple electron donors and electron acceptors are present in the wastewaters. As a result, wastewater biofilms are very complex multispecies biofilms, displaying considerable heterogeneity, with regard to both the microorganisms present and their physicochemical microenvironments. Sulphate reduction is anticipated to take place in the deeper anoxic biofilm strata even though the bulk liquid is oxygenated. It is, therefore, thought that a successive vertical zonation of respiratory processes can be found in aerobic wastewater biofilms with a typical thickness of only a few millimeters (Ito et al., 2002b
Kühl and Jorgensen, 1992
Okabe et al., 1999a
2003a
Ramsing et al., 1993)., Cambridge University Press, 英語

二段ステップ流入式上向流型ANAMMOXリアクターにおける処理性の評価
高橋 慶考, 封馬 育夫, 下川 正貴, 岡部 聡, 環境工学論文集, 44, 201, 206, 2007年
本研究は、不織布を生物膜担体として用いた上向流型anammoxリアクターのさらなる窒素除去速度の向上を目的として行った。上向流型anammoxリアクターの中間部付近に基質流入管を挿入し、リアクター下部の流入部と同濃度及び同流量の基質を供給する二段ステップ流入方式運転を行った。また、ステップ流入運転期間中、HRTを段階的に短縮して処理性の評価を行った。二段ステップ流入方式でHRTを0.2hにまで短縮することにより、リアクター後半のpH上昇が抑制され、リアクター後半部の窒素除去速度が上昇し、リアクター全体として最大31.2kg-N m^-3 day^-1の窒素除去速度を達成した。この速度は、これまでに報告されたanammoxリアクターによる窒素除去速度として最高である。, Japan Society of Civil Engineers, 日本語

Development of a super high-rate Anammox reactor and in situ analysis of biofilm structure and function
Ikuo Tsushima, Yuji Ogasawara, Masaki Shimokawa, Tomonori Kindaichi, Satoshi Okabe, WATER SCIENCE AND TECHNOLOGY, 55, 8-9, 9, 17, 2007年
The anaerobic ammonium oxidation (Anammox) process is a new efficient and cost effective method of ammonium removal from wastewater. Under strictly anoxic condition, ammonium is directly oxidised with nitrite as electron acceptor to dinitrogen gas. However, it is extremely difficult to cultivate Anammox bacteria due to their low growth rate. This suggests that a rapid and efficient start-up of Anammox process is the key to practical applications. To screen appropriate seeding sludge with high Anammox potential, a real-time quantitative PCR assay with newly designed primers has been developed. Thereafter, the seeding sludge with high abundance of Anammox bacteria (1.7 X 10(8) copies/mg-dry weight) was selected and inoculated into an upflow anaerobic biofilters (UABs). The UABs were operated for more than 1 year and the highest nitrogen removal rate of 24.0 kg(-N) m(-3) day(-1) was attained. In addition, the ecophysiology of Anammox bacteria (spatial distribution and in situ activity) in biofilms was analysed by combining a full-cycle 16S rRNA approach and microelectrodes. The microelectrode measurement clearly revealed that a successive vertical zonation of the partial nitrification (NH4+ to NO2-), Anammox reaction and denitrification was developed in the biofilm in the UAB. This result agreed with the spatial distribution of corresponding bacterial populations in the biofilm. We linked the micro-scale information (i.e. single cell and/or biofilm levels) with the macro-scale information (i.e. the reactor level) to understand the details of Anammox reaction occurring in the UABs., IWA PUBLISHING, 英語

Membrane biofouling in pilot-scale membrane bioreactors (MBRs) treating municipal wastewater: Impact of biofilm formation
Yuki Miura, Yoshimasa Watanbe, Satoshi Okabe, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 41, 2, 632, 638, 2007年01月
For more efficient control and prediction of membrane biofouling in membrane bioreactors (MBRs), a fundamental understanding of mechanisms of membrane biofouling is essential. In this study, we operated full-scale submerged MBRs using real municipal wastewater delivered from the primary sedimentation basin of a municipal wastewater treatment facility over 3 months, and the adhesion and formation of biofilms on 0.4-mu m pore size polyethylene hollow-fiber microfiltration (MF) membrane surfaces, separated from simple deposition of sludge cake, were monitored using scanning electron microscopy (SEM). In addition, the compositions of planktonic and biofilm microbial communities in the MBR were analyzed using culture independent molecular-based methods (i.e., fluorescent in situ hybridization (FISH) and 16S rRNA gene sequence analysis). The SEM and LIVE/DEAD staining analyses clearly showed that the biofilms gradually developed on the membrane surfaces with time, which had a strong positive correlation with the increase in trans-membrane pressure (TMP). This indicated that the biofilm formation induced the membrane fouling. The FISH results revealed that the microbial communities on membrane surfaces were quite different from those in the planktonic biomass in the mixed liquor. Moreover, FISH and 16S rRNA gene sequence analyses revealed that a specific phylogenetic group of bacteria, the Betaproteobacteria, probably played a major role in development of the mature biofilms, which led to the severe irreversible membrane biofouling., AMER CHEMICAL SOC, 英語

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR
Tomonori Kindaichi, Yoshiko Kawano, Tsukasa Ito, Hisashi Satoh, Satoshi Okabe, BIOTECHNOLOGY AND BIOENGINEERING, 94, 6, 1111, 1121, 2006年08月
Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O-2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities. (c) 2006 Wiley Periodicals, Inc., JOHN WILEY & SONS INC, 英語

Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms
H Satoh, T Yamakawa, T Kindaichi, T Ito, S Okabe, BIOTECHNOLOGY AND BIOENGINEERING, 94, 4, 762, 772, 2006年07月
The bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial waste-water were investigated by combination of the 16S rRNA gene clone analysis, fluorescence in situ hybridization (FISH) and microelectrodes. These results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (IWTP). Both nitrification and denitrification occurred in the primary denitrification (PD) tank and denitrification occurred in the secondary denitrification (SD) tank. In contrast, nitrification and denitrification rates were very low in the nitrification (N) tank. 16S rRNA gene clone sequence analysis revealed that the bacteria affiliated with Alphaproteobacteria, followed by Betaproteobacteria, were numerically important microbial groups in three tanks. The many clones affiliated with Alphaproteobacteria were closely related to the denitrifying bacteria (e.g., Hyphomicrobium spp., Rhodopseudomonas palustris, and Rhodobacter spp.). In addition, Methylophilus leisingeri affiliated with Betaproteobacteria, which favorably utilized methanol, was detected only in the SD-tank to which methanol was added. Nitrosomonas europaea and Nitrosomonas marina were detected as the ammonia-oxidizing bacteria affiliated with Betaproteobacteria throughout this plant, although the dominant species of them was different among three tanks. Nitrifying bacteria were mainly detected in the upper parts of the PD-biofilm whereas their populations were low in the upper parts of the N-biofilm. The presence of denitrifying bacteria affiliated with Hyphomicrobium spp. in SD- and N-biofilms was verified by FISH analysis. Microelectrode measurements showed that the nitrifying bacteria present in the N- and PD-biofilms were active and the bacteria present in the SD-biofilm could denitrify. (c) 2006 Wiley Periodicals, Inc., JOHN WILEY & SONS INC, 英語

Alternative indicators of fecal pollution: Relations with pathogens and conventional indicators, current methodologies for direct pathogen monitoring and future application perspectives
Olga Savichtcheva, Satoshi Okabe, WATER RESEARCH, 40, 13, 2463, 2476, 2006年07月
The ecological and survival characteristics of bacterial, viral and parasitic pathogens vary under environmental conditions, indicating that probably no single indicator organism can predict the presence of all enteric pathogens for all types of waters and different host-associated fecal pollution. if there are true correlations between indicator organisms and pathogens, it is necessary to find out to what extent and under which circumstances these organisms can be used as reliable indicators of fecal pollution.
Application of conventional and alternative fecal indicators has greatly enhanced our abilities to predict and reduce health risk associated with the use of surface waters. New molecular-based techniques have shown that combined use of conventional and alternative indicators for fecal pollution increases both the detection sensitivity and specificity of fecal pollution and associated pathogens. in this review, we, therefore, summarize the advantages and limitations of conventional and alternative fecal indicators in terms of predicting pathogen presence as well as current and future methodologies for direct pathogen monitoring in environmental waters. This manuscript is mainly focused on the relationships between microbial fecal indicators and the presence of pathogens, which have not previously been summarized yet and could nicely supplement with recent literature reviews on microbial source tracking. (c) 2006 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語, 書評論文,書評,文献紹介等

Community structure, abundance, and in situ activity of nitrifying bacteria in river sediments as determined by the combined use of molecular techniques and microelectrodes
Y Nakamura, H Satoh, T Kindaichi, S Okabe, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 40, 5, 1532, 1539, 2006年03月
The community structure, spatial distributions, and in situ activity of ammonia-oxidizing bacteria (AOB) representing the Betaproteobacteria and nitrite-oxidizing bacteria (NOB) representing the genus Nitrospira in three different river sediments with different pollution sources and levels along the Niida River, Hachinohe, Japan, were investigated by the combined use of 16S rRNA gene-cloning analysis, real-time quantitative polymerase chain reaction (RTQ-PCR) assays, and microelectrodes. The goal of this research was to evaluate the contribution of nitrifying activity in the sediment to the overall nitrogen elimination rate in this river. The 16S rRNA gene-cloning analysis revealed that the community structures of AOB and Nitrospira-like NOB are present in three sediments. On the basis of the results of 16S rRNA gene-cloning analysis, the RTQ-PCR assay using a TaqMan probe was developed and optimized for the quantification of the Nitrospira-like NOB. In the sediments, AOB specific 16S rRNA genes were detected in the range of 10(6) to 10(7) copies/cm(3) and evenly distributed over the sampled sediment depth (0-5 mm), whereas the Nitrospira-like NOB 16S rRNA gene copy numbers per cm(3) were 1-2 orders of magnitude higher than the AOB copy numbers. Under light conditions, intensive oxygenic photosynthesis occurred in the surface and increased the maximal O-2 concentration and O-2 penetration depth in all sediments. This concomitantly stimulated nitrifying bacteria present in diurnally anoxic deeper zones and expanded nitrification zones, which consequently increased the total NH4+ consumption rate in the sediment (i.e., total NH4+ flux into the sediment). The results suggested that the in situ nitrifying activity was restricted mainly to the surface 2 mm of the sediment and linked with photosynthetic activity, which obviously plays an important role in nitrogen elimination in this river., AMER CHEMICAL SOC, 英語

A vista for microbial ecology and environmental biotechnology
BE Rittmann, M Hausner, F Loffler, NG Love, G Muyzer, S Okabe, DB Oerther, J Peccia, L Raskin, M Wagner, ENVIRONMENTAL SCIENCE & TECHNOLOGY, 40, 4, 1096, 1103, 2006年02月
AMER CHEMICAL SOC, 英語, その他

C-13 新たな糞便汚染指標としてのBacteroides-Prevotella属遺伝子マーカーの環境水中での挙動に関する研究(環境衛生,(2)口頭発表会,研究発表会)
嶋津 陽子, Olga Savichtcheva, 川田 耕司, 岡部 聡, 日本微生物生態学会講演要旨集, 22, 277, 277, 2006年
日本微生物生態学会, 日本語

B-29 廃水処理システム内に普遍的に存在する新規Betaproteobacteriaの機能解析(水処理生態系,(2)口頭発表会,研究発表会)
小野 香保里, 伊藤 司, 岡部 聡, 日本微生物生態学会講演要旨集, 22, 261, 261, 2006年
日本微生物生態学会, 日本語

B-03 Escheirhica coliバイオフィルム形成におけるrpoS遺伝子の関与(界面/バイオフィルム,(2)口頭発表会,研究発表会)
伊藤 暁信, May Thithiwat, 岡部 聡, 日本微生物生態学会講演要旨集, 22, 235, 235, 2006年
日本微生物生態学会, 日本語

B-30 嫌気性汚泥のバルキング化に関与する未培養糸状性細菌群の機能解析と培養(水処理生態系,(2)口頭発表会,研究発表会)
関口 勇地, 山田 剛史, 白石 皓二, 伊藤 司, 岡部 聡, 大橋 晶良, 原田 秀樹, 鎌形 洋一, 中村 和憲, 日本微生物生態学会講演要旨集, 22, 262, 262, 2006年
日本微生物生態学会, 日本語

UASBグラニュール内の水素濃度分布の解析
佐藤 久, 岡部 聡, 衛生工学シンポジウム論文集, 13, 199, 202, 2005年11月16日
北海道大学衛生工学会, 日本語

ANAMMOX反応を利用した窒素除去リアクターの開発と群集構造解析
金田一 智規, 尾崎 則篤, 對馬 育夫, 小笠原 雄二, 岡部 聡, 衛生工学シンポジウム論文集, 13, 13, 195, 198, 2005年11月16日
第13回衛生工学シンポジウム（平成17年11月17日（木）-18日（金） 北海道大学クラーク会館） . 一般セッション . 6 水処理 . 6-4, 北海道大学衛生工学会, 日本語

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water
O Savichtcheva, N Okayama, T Ito, S Okabe, BIOTECHNOLOGY AND BIOENGINEERING, 92, 3, 356, 363, 2005年11月
To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram (TM) Red(+) Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as ILDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters. (c) 2005 Wiley Periodicals, Inc., JOHN WILEY & SONS INC, 英語

Measurement of growth rate of ammonia oxidizing bacteria in partially submerged rotating biological contactor by fluorescent in situ hybridization (FISH)
A Jang, S Okabe, Y Watanabe, IS Kim, PL Bishop, JOURNAL OF ENVIRONMENTAL ENGINEERING AND SCIENCE, 4, 5, 413, 420, 2005年09月
Optimization of the nitrification processes in biofilms is important for effective nitrogen removal because nitrification in an aerobic biofilm is considered to be a less than reliable process. Thus, one of the main factors to improve biological nitrogen removal processes is a better understanding of the microbiology and population dynamics of ammonia oxidizing bacteria (AOB) in wastewater treatment biofilms. Although the AOB in wastewater treatment have been qualitatively and quantitatively studied, information on their actual populations and activities is still limited. Therefore, the areal cell density of AOB in domestic wastewater biofilms on a partially submerged rotating biological contactor (RBC) was determined by fluorescent in situ hybridization (FISH) with a set of 16S rRNA-targeted oligonucleotide probes. The growth kinetics of the in situ AOB was also studied. Although low numbers of AOB were found at the deeper layers where oxygen was depleted, they were primarily detected in the upper and middle layers of the biofilm. The maximum specific growth rate (mu(b,max)) and half saturation constant (K-s) of AOB in the biofilm were 0.32 d(-1) and 1.7 mM/L of NH4+, respectively., NATL RESEARCH COUNCIL CANADA, 英語

Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms.
Satoshi Okabe, Tomonori Kindaichi, Tsukasa Ito, Applied and environmental microbiology, 71, 7, 3987, 94, 2005年07月, ［査読有り］, ［国際誌］
The cross-feeding of microbial products derived from 14C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [14C]bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of 14C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of 14C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized 14C-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of 14C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm., 英語

Thiovirga sulfuroxydans gen. nov., sp nov., a chemolithoautotrophic sulfur-oxidizing bacterium isolated from a microaerobic waste-water biofilm
T Ito, K Sugita, Yumoto, I, Y Nodasaka, S Okabe, INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 55, 3, 1059, 1064, 2005年05月
A novel mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain SO07(T) was isolated from a microaerobic waste-water biofilm. Chemolithoautotrophic growth was observed with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor. Anaerobic and heterotrophic growth were not observed. Nitrate was not used as a terminal electron acceptor. The optimum pH and temperature for growth were pH 7(.)5 and 30 degrees C, respectively. The major isoprenoid quinone was Q-8. The DNA G + C content of strain SO07(T) was 47(.)1 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that strain SO07(T) formed a monophyletic group in the gamma-Proteobacteria with only 89% similarity to members of the genus Halothiobacillus, its nearest phylogenetic neighbours. In addition, the isolate differed from members of the genus Halothiobacillus in its requirement for and tolerance of NaCl; strain SO07(T) was unable to grow in NaCl concentrations of more than 180 mM. On the basis of phylogenetic, chemotaxonomic and physiological data, it is proposed that isolate SO07(T) (= JCM 12417(T) = ATCC BAA- 1033(T)) represents the type strain of a novel species in a new genus, Thiovirga sulfuroxydans gen. nov., sp. nov., SOC GENERAL MICROBIOLOGY, 英語

Succession of internal sulfur cycles and sulfur-oxidizing bacterial communities in microaerophilic wastewater biofilms
Satoshi Okabe, Tsukasa Ito, Kenichi Sugita, Hisashi Satoh, Applied and Environmental Microbiology, 71, 5, 2520, 2529, 2005年05月
The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S0 accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H2S was only partially oxidized to S0 by mainly Thiomicrospira denitirificans with NO3- as an electron acceptor, leading to significant accumulation of S0 in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S 0 accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S0t to SO 42- under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 109 cells cm-3) and strain SO07 (ca. 108 cells cm-3) were found at the sulfur-rich surface (100 μm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 107 to 108 cells cm -3. Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H 2S produced by SRB to SO42- in the second phase, indicating establishment of the complete sulfur cycle in the biofilms. Copyright © 2005, American Society for Microbiology. All Rights Reserved., 英語

Membrane Bioreactorの運転性に伴う微生物群集構造の解析
三浦佑己, 伊藤司, 渡辺義公, 岡部聡, 日本水環境学会年会講演集, 39th, 41, 2005年03月17日
日本語

ヨシ根圏生物膜内の物質濃度分布および微生物群集構造の解析
中村吉志, 渡辺義公, 岡部聡, 佐藤久, 日本水環境学会年会講演集, 39th, 231, 2005年03月17日
日本語

B-20 MAR-FISH法とRNA-SIP法を用いた嫌気性消化汚泥内における糖分解経路に関与する微生物群集構造解析(水処理生態系,口頭発表)
吉口 和美, 伊藤 司, アリエスヤディ ヘルト ドゥイ, 岡部 聡, 日本微生物生態学会講演要旨集, 21, 240, 240, 2005年
日本微生物生態学会, 日本語

B-19 メンブレンバイオリアクターの膜表面に形成するバイオフィルムの微生物群集構造解析(水処理生態系,口頭発表)
三浦 佑己, 伊藤 司, 渡辺 義公, 岡部 聡, 日本微生物生態学会講演要旨集, 21, 239, 239, 2005年
日本微生物生態学会, 日本語

PA-38 MAR-FISH法とRNA-SIP法を用いた嫌気性消化汚泥内におけるプロピオン酸酸化細菌の多様性解析(水処理生態系,ポスターセッションA,ポスター発表)
伊藤 司, アリエスヤディ ヘルト ドゥイ, 吉口 和美, 岡部 聡, 日本微生物生態学会講演要旨集, 21, 84, 84, 2005年
日本微生物生態学会, 日本語

3S4-AM5 硝化細菌バイオフィルム内における微生物間の生理・生態学的相互作用の解析(微生物コミュニケーション研究の工学的応用への展開,シンポジウム)
岡部 聡, 日本生物工学会大会講演要旨集, 17, 47, 47, 2005年
日本生物工学会, 日本語

A-10 ANAMMOXリアクター内の微生物群集構造および機能解析(群集構造解析,口頭発表)
對馬 育夫, 小笠原 雄二, 金田一 智規, 岡部 聡, 日本微生物生態学会講演要旨集, 21, 206, 206, 2005年
日本微生物生態学会, 日本語

Eco-physiology of autotrophic nitrifying biofilms
S Okabe, T Kindaichi, Y Nakamura, T Ito, WATER SCIENCE AND TECHNOLOGY, 52, 7, 225, 232, 2005年
Microautoradiography combined with fluorescent in situ hybridization (MAR-FISH), a powerful tool for linking physiology with identification of individual cells, was applied to investigate microbial interactions between nitrifying bacteria and coexisting heterotrophic bacteria in an autotrophic nitrifying biofilm community fed with only ammonia as the sole energy source and bicarbonate as the sole carbon source. First, nitrifying bacteria were radiolabeled by culturing the biofilm samples with [C-14]bicarbonate for 6 h, and then the transfer of radioactivity from nitrifying bacteria to heterotrophic bacteria was monitored by using MAR-FISH. MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabofically diverse. We could obtain direct evidence that organic matter derived from nitrifiers was subsequently utilized by mainly filamentous bacteria belonging to the Chloroflexi (green non-sulfur bacteria) group or CFB group in the biofilm, which was clearly visualized by MAR-FISH at single cell resolution for the first time. On the other hand, the members of the alpha- and gamma-Proteobacteria were specialized to utilize low-molecular-weight organic matter. This community represents functionally integrated units that assure maximum access to and utilization of metabolites of nitrifiers., I W A PUBLISHING, 英語

微生物群集構造解析技術の現状と今後の展開・課題　　　　　　　　　　　　　　　
水環境学会誌, 28, 8, 460, 465, 2005年

バイオフィルムの概論・理論 : バイオフィルム : 細菌の生き延びるためのストラテジー (小特集 バイオフィルム)
岡部 聡, 伊藤 暁信, 配管技術, 46, 8, 1, 7, 2004年07月
日本工業出版, 日本語

Isolation, characterization, and in situ detection of a novel chemolithoautotrophic sulfur-oxidizing bacterium in wastewater biofilms growing under microaerophilic conditions
T Ito, K Sugita, S Okabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70, 5, 3122, 3129, 2004年05月
We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S-0), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 mum) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H2S and S-0 to SO42- under oxic conditions., AMER SOC MICROBIOLOGY, 英語

ジルコニウムメゾ構造体による下水汚泥からのリン回収
巽善彦, 渡辺義公, 岡部聡, 日本水環境学会年会講演集, 38th, 196, 2004年03月17日
日本語

MBR(Membrane Bioreactor)を用いた都市下水処理システムにおける女性ホルモン様物質の挙動に関する研究
久保広明, 渡辺義公, 岡部聡, 木村克輝, 日本水環境学会年会講演集, 38th, 363, 2004年03月17日
日本語

Real‐Time PCR法によるBacteroidalesを指標とした糞便汚染度評価
岡山紀子, SAVICHTCHEVA O, 金田一智規, 伊藤司, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 38th, 313, 2004年03月17日
日本語

MAR‐FISH法を用いた硝化細菌生物膜内における硝化細菌由来有機物の追跡
金田一智規, 伊藤司, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 38th, 380, 2004年03月17日
日本語

ANAMMOX集積汚泥の微生物構造および機能解析
対馬育夫, 金田一智規, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 38th, 370, 2004年03月17日
日本語

根に形成される生物膜内の物質濃度および微生物群集構造解析
中村吉志, 岡部聡, 渡辺義公, 佐藤久, 日本水環境学会年会講演集, 38th, 508, 2004年03月17日
日本語

Macroscale and microscale analyses of nitrification and denitrification in biofilms attached on membrane aerated biofilm reactors
H Satoh, H Ono, B Rulin, J Kamo, S Okabe, KI Fukushi, WATER RESEARCH, 38, 6, 1633, 1641, 2004年03月
A membrane aerated biofilm reactor (MABR), in which O-2 was supplied from the bottom of the biofilm and NH4+ and organic carbon were supplied from the biofilm surface, was operated at different organic carbon loading rates and intra-membrane air pressures to investigate the occurrence of simultaneous chemical oxygen demand (COD) removal, nitrification and denitrification. The spatial distribution of nitrification and denitrification zones in the biofilms was measured with microelectrodes for O-2, NH4+, NO2-, NO3- and pH. When the MABR was operated at approximately 1.0 g-COD/m(2)/day of COD loading rate, simultaneous COD removal, nitrification and denitrification could be achieved. The COD loading rates and the intra-membrane air pressures applied in this study had no effect on the startup and the maximum rates of NH4+ oxidation in the MABRs. Microelectrode measurements showed that O-2 was supplied from the bottom of the MABR biofilm and penetrated the whole biofilm. Because the biofilm thickness increased during the operations, an anoxic layer developed in the upper parts of the mature biofilms while an oxic layer was restricted to the deeper parts of the biofilms. The development of the anoxic zones in the biofilms coincided with increase in the denitrification rates. Nitrification occurred in the zones from membrane surface to a point of ca. 60 mum. Denitrification mainly occurred just above the nitrification zones. The COD loading rates and the intra-membrane air pressures applied in this study had no effect on location of the nitrification and denitrification zones. (C) 2004 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Analysis of Size Distribution and Areal Cell Density of Ammonia-Oxidizing Bacterial Microcolonies in Relation to Substrate Microprofiles in Biofilms
Satoshi Okabe, Tomonori Kindaichi, Tsukasa Ito, Hisashi Satoh, Biotechnology and Bioengineering, 85, 1, 86, 95, 2004年01月05日
A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells μm-2, which was half of the cell density of Nitrosomonas (1.12 cells μm-2). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O2 and NH4+ conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by ∼ 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term (∼ 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O2 between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH4 +-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O 2 and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm. © 2004 Wiley Periodicals, Inc., 英語

A-30 コンクリート腐食の進行に関与する微生物群集構造解析(水処理生態系,口頭発表)
小田切 光典, 伊藤 司, 佐藤 久, 岡部 聡, 日本微生物生態学会講演要旨集, 20, 152, 152, 2004年
日本微生物生態学会, 日本語

C-06 ヨシ根圏内の窒素循環と関連細菌群集構造の解析(水圏生態系,口頭発表)
中村 吉志, 岡部 聡, 佐藤 久, 渡辺 義公, 日本微生物生態学会講演要旨集, 20, 208, 208, 2004年
日本微生物生態学会, 日本語

P-020 16S rRNAアプローチとSIP法を用いた嫌気性消化汚泥内における酸生成および酸分解に関与する微生物群の構造解析(水処理生態系,ポスター発表)
吉口 和美, 伊藤 司, Ariesyady Herto Dwi, 岡部 聡, 日本微生物生態学会講演要旨集, 20, 42, 42, 2004年
日本微生物生態学会, 日本語

Effects of hydroxylamine on microbial community structure and function of autotrophic nitrifying biofilms determined by in situ hybridization and the use of microelectrodes
T Kindaichi, S Okabe, H Satoh, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 49, 11-12, 61, 68, 2004年
Effects of hydroxylamine (NH2OH), an intermediate of NH4+ oxidation, on microbial community structure and function of two autotrophic nitrifying biofilms fed with and without NH2OH were analyzed by a 16S rRNA approach and the use of microelectrodes. In the NH2OH-added biofilm, partial oxidation of NH4+ to NO2- was observed, whereas complete oxidation of NH4+ to NO3- was achieved in the control biofilm. In situ hybridization results revealed that no nitrite-oxidizing bacteria (NOB) hybridized with any specific probes were detected in the NH2OH-added biofilm. Thus, the addition of low concentrations of NH2OH (250 muM) completely inhibited the growth of NOR Phylogenetic analysis of 16S rDNA indicated that the ammonia-oxidizing bacteria (AOB) detected in both biofilms were closely related to Nitrosomonas europaea, and that the clone sequences from both biofilm libraries have more than 99% similarity to each other. However, in situ hybridization results revealed that the addition of NH2OH changed the form of growth pattern of the dominant Nitrosomonas spp. from dense clusters mode to single scattered cells mode. Microelectrode measurements revealed that the average NH4+ consumption rate calculated in the NH2OH-added biofilm was two times higher than that in the control biofilm. This clearly demonstrated that the oxidation of NH4+ was stimulated by NH2OH addition., I W A PUBLISHING, 英語

嫌気性アンモニア酸化型メンブレンバイオリアクターを核とした新規浸出水処理システムの開発とDNAチップを用いた処理水の安全性評価手法の確立
渡辺義公, 岡部聡, 木村克輝, 第4回廃棄物対策研究発表会成果発表抄録集 平成16年度廃棄物対策研究推進事業, I.35-I.37, 2004年
日本語

Effects of hydroxylamine on microbial community structure and function of autotrophic nitrifying biofilms determined by in situ hybridization and the use of microelectrodes
T. Kindaichi, S. Okabe, H. Satoh, Y. Watanabe, Water Science and Technology, 49, 11-12, 61, 68, 2004年
Effects of hydroxylamine (NH2OH), an intermediate of NH4+ oxidation, on microbial community structure and function of two autotrophic nitrifying biofilms fed with and without NH2OH were analyzed by a 16S rRNA approach and the use of microelectrodes. In the NH2OH-added biofilm, partial oxidation of NH4+ to NO2- was observed, whereas complete oxidation of NH4+ to NO3- was achieved in the control biofilm. In situ hybridization results revealed that no nitrite-oxidizing bacteria (NOB) hybridized with any specific probes were detected in the NH2OH-added biofilm. Thus, the addition of low concentrations of NH2OH (250 μM) completely inhibited the growth of NOB. Phylogenetic analysis of 16S rDNA indicated that the ammonia-oxidizing bacteria (AOB) detected in both biofilms were closely related to Nitrosomonas europaea, and that the clone sequences from both biofilm libraries have more than 99% similarity to each other. However, in situ hybridization results revealed that the addition of NH2OH changed the form of growth pattern of the dominant Nitrosomonas spp. from dense clusters mode to single scattered cells mode. Microelectrode measurements revealed that the average NH4+ consumption rate calculated in the NH2OH-added biofilm was two times higher than that in the control biofilm. This clearly demonstrated that the oxidation of NH4+ was stimulated by NH2OH addition. © IWA Publishing 2004., 英語

Microbial communities and their interactions in biofilm systems: an overview
S Wuertz, S Okabe, M Hausner, WATER SCIENCE AND TECHNOLOGY, 49, 11-12, 327, 336, 2004年
Several important advances have been made in the study of biofilm microbial populations relating to their spatial structure (or architecture), their community structure, and their dependence on physicochemical parameters. With the knowledge that hydrodynamic forces influence biofilm architecture came the realization that metabolic processes may be enhanced if certain spatial structures can be forced. An example is the extent of plasmid-mediated horizontal gene transfer in biofilms. Recent in situ work in defined model systems has shown that the biofilm architecture plays a role for genetic transfer by bacterial conjugation in determining how far the donor cells can penetrate the biofilm. Open channels and pores allow for more efficient donor transport and hence more frequent cell collisions leading to rapid spread of the genes by horizontal gene transfer. Such insight into the physical environment of biofilms can be utilized for bioenhancement of catabolic processes by introduction of mobile genetic elements into an existing microbial community. If the donor organisms themselves persist, bioaugmentation can lead to successful establishment of newly introduced species and may be a more successful strategy than biostimulation (the addition of nutrients or specific carbon sources to stimulate the authochthonous population) as shown for an enrichment culture of nitrifying bacteria added to rotating disk biofilm reactors using fluorescent in situ hybridization (FISH) and microelectrode measurements of NH4+, NO2-, NO3-, and O-2. However, few studies have been carried out on full-scale systems. Bioaugmentation and bioenhancement are most successful if a constant selective pressure can be maintained favoring the promulgation of the added enrichment culture. Overall, knowledge gain about microbial community interactions in biofilms continues to be driven by the availability of methods for the rapid analysis of microbial communities and their activities. Molecular tools can be grouped into those suitable for ex situ and in situ community analysis. Non-spatial community analysis, in the sense of assessing changes in microbial populations as a function of time or environmental conditions, relies on general fingerprinting methods, like DGGE and T-RFLP, performed on nucleic acids extracted from biofilm. These approaches have been most useful when combined with gene amplification, cloning and sequencing to assemble a phylogenetic inventory of microbial species. It is expected that the use of oligonucleotide microarrays will greatly facilitate the analysis of microbial communities and their activities in biofilms. Structure-activity relationships can be explored using incorporation of C-13-labeled substrates into microbial DNA and RNA to identify metabolically active community members. Finally, based on the DNA sequences in a biofilm, FISH probes can be designed to verify the abundance and spatial location of microbial community members. This in turn allows for in situ structure/function analysis when FISH is combined with microsensors, microautoradiography, and confocal laser scanning microscopy with advanced image analysis., I W A PUBLISHING, 英語

Photosynthesis in sediment at a tidal area of Niida River, Hachinohe, Japan　　　　　　　　　　　　　　　
Water Research, 38, 9, 2439, 2447, 2004年

MAR-FISH: An ecophysiological approach to link phylogenetic identity and in situ metabolic activity of microorganisms at single-cell resolution
Microb. Environ, 19, 2, 83, 98, 2004年

Eco-physiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms
KINDAICHI T, ITO T, OKABE S, Applied and Environmental Microbiology, 70, 3, 1641, 1650, 2004年

Ｒｅａｌ‐Ｔｉｍｅ ＰＣＲを用いた生物膜内における硝化細菌のポピュレーションダイナミックス
金田一 智規, 河野 快子, 伊藤 司, 岡部 聡, 環境工学研究論文集, 41, 41, 321, 330, 2004年
We investigated the population dynamics of nitrifying bacteria in autotrophic nitrifying biofilms by using Real-Time PCR and fluorescent in situ hybridization (FISH). Primers and TaqMan probe specific to genus Nitrospira including uncultured Nitrospira sp. in the biofilms were newly designed and applied to the biofilms. The populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) determined by Real-Time PCR during steady-state condition were 1.2×10⁸cells/cm² and 3.5×10⁸cells/cm², respectively. The population dynamics of nitrifying bacteria corresponded with the reactor performance and the development of nitrifying bacteria in the biofilms determined by FISH. Real-Time PCR revealed that specific growth rates of uncultured Nitrospira sp. were 0.021h^-1 in the suspended enrichment culture and 0.014h^-1 in the biofilms. Average specific growth rates of both nitrifying bacteria during the steady-state condition in the biofilms were significantly lower than that during the log phase, might suggest that metabolic pathway of nitrifying bacteria in the biofilms was different in the log and stationary phase. We speculated that Real-Time PCR was powerful and suitable technique for understanding of microbial population dynamics in the complex microbial communities., 公益社団法人 土木学会, 日本語

バイオフィルム内の細菌の特異的検出とその分布の測定　　　　　　　　　　　　　　　
Bacterial Adherence & Biofilm, 17, 102, 107, 2004年

Sulfate-reducing bacterial community structure and their contribution to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions
S Okabe, T Ito, H Satoh, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 63, 3, 322, 334, 2003年12月
The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga-Flexibacter-Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H-2 as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2-4x10(9) cells cm(-3) and 5x10(7) filaments cm(-3), respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB., SPRINGER-VERLAG, 英語

Eco-physiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by MAR-FISH
Kindaichi Tomonori, Ito Tsukasa, Okabe Satoshi, Watanabe Yoshimasa, 衛生工学シンポジウム論文集, 11, 131, 134, 2003年10月31日
北海道大学衛生工学会, 英語

微小電極を用いた下水道構造物の腐食機構の検討
佐藤 久, 佐々木 裕一, 小山田 浩之, 岡部 聡, 衛生工学シンポジウム論文集, 11, 69, 72, 2003年10月31日
北海道大学衛生工学会, 日本語

MBR(Membrane Bioreactor)を用いた下水処理水の安全性評価およびMicropollutantsの挙動に関する研究
久保広明, 岡部聡, 木村克輝, 渡辺義公, 土木学会年次学術講演会講演概要集(CD-ROM), 58th, Disk 2, VII-085, 2003年09月01日
日本語

コンクリート腐食に関与する微生物群集の構造解析
岡部聡, 小田切光典, 伊藤司, 佐藤久, 小山田浩之, 渡辺義公, 土木学会年次学術講演会講演概要集(CD-ROM), 58th, Disk 2, VII-189, 2003年09月01日
日本語

Effect of oxygen concentration on nitrification and denitrification in single activated sludge flocs
H Satoh, Y Nakamura, H Ono, S Okabe, BIOTECHNOLOGY AND BIOENGINEERING, 83, 5, 604, 607, 2003年09月
Simultaneous nitrification and denitrification (SND) was investigated in the single aeration tank of a municipal wastewater treatment plant. Microelectrode measurements and batch experiments were performed to test for the presence of SND. Microelectrodes recorded the presence of O-2 concentration gradients in individual activated sludge flocs. When the O-2 concentration in the bulk liquid was <45 μM, anoxic zones were detected within flocs with a larger diameter (approximately 3000 pm). The O-2 penetration depth in the floc was found to be dependent on the O-2 concentration in the bulk liquid. Nitrification was restricted to the oxic zones, whereas denitrification occurred mainly in the anoxic zones. The nitrification rate of the activated sludge increased with increasing O-2 concentration in the bulk liquid, up to 40 μM, and remained constant thereafter. SND was observed in the aerated activated sludge when O-2 concentration was in the range of 10 to 35 μM. (C) 2003 Wiley Periodicals, Inc., JOHN WILEY & SONS INC, 英語

Dynamic response of nitrifying activated sludge batch culture to increased chloride concentration
GH Chen, MT Wong, S Okabe, Y Watanabe, WATER RESEARCH, 37, 13, 3125, 3135, 2003年07月
Dynamic response of nitrifying activated sludge batch cultures to increased chloride concentration was studied in this paper, which focused upon the changes in the specific nitrification rate (SNR) and nitrifier population when the chloride level was gradually or stepwise increased to 30,000 mg Cl L-1. The dominant species of ammonia-oxidizers and nitrite-oxidizers in the population were examined by Fluorescent in situ hybridization technique with 16S rRNA-targeted oligonucleotide probes. It was found that neither chloride increasing approaches affected the SNR of the batch cultures before the chloride concentration exceeded 10,000 mg Cl L-1, after which the stepwise increase approach reduced the SNR more significantly than the gradual increase approach. From 10,000 to 18,000 mg Cl L-1 a down-and-up pattern of the SNR variation appeared in both approaches, which was associated with the change in the dominant species of ammonia-oxidizers from non-saline-resistant species such as Nitrosomonas europaea-lineage and Nitrosomonas eutropha to saline-resistant species, such as the Nitrosococcus mobilis-lineage. Nitrobacter was the only dominant species when the chloride concentration was below 10,000 mg Cl L-1, where no nitrite-oxidizers survived. Therefore, the 10,000 mg Cl L-1 chloride level is a critical level for the shift of the nitrifier population in the nitrifying activated sludge batch cultures. (C) 2003 Elsevier Science Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Evaluation of the impact of bioaugmentation and biostimulation by in situ hybridization and microelectrode
H Satoh, S Okabe, Y Yamaguchi, Y Watanabe, WATER RESEARCH, 37, 9, 2206, 2216, 2003年05月
Three rotating disk biofilm reactors were operated to evaluate whether bioaugmentation and biostimulation can be used to improve the start-up of microbial nitrification. The first reactor was bioaugmented during start-up period with an enrichment culture of nitrifying bacteria, the second reactor received a synthetic medium containing NH4+ and NO2- to facilitate concomitant proliferation of ammonia- and nitrite-oxidizing bacteria, and the third reactor was used as a control. To evaluate the effectiveness of bioaugmentation and biostimulation approaches, time-dependent developments of nitrifying bacterial community and in situ nitrifying activity in biofilms were monitored by fluorescence in situ hybridization (FISH) technique and microelectrode measurements of NH4+, NO2-, NO3-, and O-2. In situ hybridization results revealed that addition of the enrichment culture of nitrifying bacteria significantly facilitated development of dense nitrifying bacterial populations in the biofilm shortly after, which led to a rapid start-up and enhancement of in situ nitrification activity. The inoculated bacteria could proliferate and/or survive in the biofilm. In addition, the addition of nitrifying bacteria increased the abundance of nitrifying bacteria in the surface of the biofilm, resulting in the higher nitrification rate. On the other hand, the addition of 2.1 mM NO2- did not stimulate the growth of nitrite-oxidizing bacteria and did inhibit the proliferation of ammonia-oxidizing bacteria instead. Thus, the start-up of NO2- oxidation was unchanged, and the start-up of NH4+ oxidation was delayed. In all the three biofilm reactors, data sets of time series analyses on population dynamics of nitrifying bacteria determined by FISH, in situ nitrifying activities determined by microelectrode measurements, and the reactor performances revealed an approximate agreement between the appearance of nitrifying bacteria and the initiation of nitrification activity, suggesting that the combination of these techniques was a very powerful monitoring tool to evaluate the effectiveness of bioaugmentation and biostimulation strategies. (C) 2002 Elsevier Science Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

MAR‐FISH法を用いた硝化生物膜の生態学的構造と機能の解析
金田一智規, 伊藤司, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 37th, 62, 2003年03月04日
日本語

省エネルギー型の新規窒素除去プロセスの検討
対島育夫, 金田一智規, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 37th, 507, 2003年03月04日
日本語

下水生物膜内における新規硫黄酸化細菌の機能と構造の解析
杉田謙一, 伊藤司, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 37th, 67, 2003年03月04日
日本語

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars
AM Briones, S Okabe, Y Umemiya, NB Ramsing, W Reichardt, H Okuyama, PLANT AND SOIL, 250, 2, 335, 348, 2003年03月
Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO3--N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH4+ and NO3-. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH4+ and NO3- with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar., KLUWER ACADEMIC PUBL, 英語

Effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film as determined by use of microelectrodes
S Okabe, CM Santegoeds, D De Beer, BIOTECHNOLOGY AND BIOENGINEERING, 81, 5, 570, 577, 2003年03月
Microelectrode, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate the effect of nitrite and nitrate on in situ sulfide production in an ac activated sludge immobilized agar gel film. Microelectrode measurements of O-2, H2S, NO3-, NO2-, and pH revealed that the addition of NO2- and NO3- forced sulfate reduction zones deeper in the agar gel and significantly reduced the in situ sulfide production levels. The sulfate reduction zone was consequently separated from 02 and NO2- or NO3- respiration zones with increasing the concentrations of NO2- and NO3-. These NO2- and NO3- treatments had only a transient effect on sulfide production. The in situ sulfide production quickly recovered to the previous levels when NO2- and NO3- were removed. The PCR-DGGE and FISH analyses revealed that 2-day-continuous addition of 500 muM NO3- did not change the metabolically active sulfate-reducing bacterial (SRB) community. On the basis of these data, it could be concluded that the addition of NO2- and NO3- did not kill SRB, but induced the interspecies competition for common carbon source (i.e., acetate) between nitrate-reducing heterotrophic bacteria and SRB and enhanced the oxidation of the produced sulfide, which were main possible causes of the suppression of in situ sulfide production in the agar gel. (C) 2003 Wiley Periodicals, Inc., JOHN WILEY & SONS INC, 英語

28-B-10 MAR-FISH法を用いた下水管渠内の付着生物膜を構成するbeta-, gamma-Proteobacteriaの群集構造と機能解析(水処理生態系,一般講演)
伊藤 司, 岡部 聡, 日本微生物生態学会講演要旨集, 19, 105, 105, 2003年
日本微生物生態学会, 日本語

28-B-11 下水生物膜から単離した新規硫黄酸化細菌の生理機能と生態(水処理生態系,一般講演)
杉田 謙一, 伊藤 司, 岡部 聡, 渡辺 義公, 日本微生物生態学会講演要旨集, 19, 106, 106, 2003年
日本微生物生態学会, 日本語

Effect of dissolved oxygen concentration on the biofilm and in situ analysis by fluorescence in situ hybridization (FISH) and microelectrodes
A Jang, PL Bishop, S Okabe, SG Lee, IS Kim, WATER SCIENCE AND TECHNOLOGY, 47, 1, 49, 57, 2003年
A better understanding of microbiology and ecology of nitrifying bacteria in inner biofilms is an important part of improving process performance and control. Microelectrodes and fluorescent in situ hybridization (FISH) in biofilm research have been used to investigate the spatial distributions of various microbial activities in biofilms and have led to new experimental findings as well as modifications of the homogeneous assumptions in the biofilm kinetic models. The objective of this study is to try the combination of two methods, both FISH and microelectrode measurements, and to provide reliable and in situ information on nitrifying bacterial activity in biofilms. The characteristics of biofilm developed on tygon slides were different according to the change of dissolved oxygen (DO). When the DO increased from 2 to 10 mg DO/L, the rate of the biofilm thickness increased and its dry density changed from 50-70 to 25-90 mg/cm(3). Ammonia oxidizing bacteria were not uniformly distributed in biofilm, and were found at the deeper layer where oxygen is depleted, they were detected primarily in the upper and middle layers of the biofilm., I W A PUBLISHING, 英語

Effect of nitrite and nitrate on biogenic sulfide production in sewer biofilms determined by the use of microelectrodes
S Okabe, T Ito, H Satoh, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 47, 11, 281, 288, 2003年
The effects of O-2 and NO3- concentrations on in situ sulfate reduction and sulfide reoxidation in microaerophilic wastewater biofilms grown on rotating disk reactors were investigated by the use of microelectrodes for O-2, S2-, NO3-, NO2-, and pH. Microelectrocle measurements showed the vertical microzonation of O-2 respiration, NO3- respiration, H2S oxidation and SO42- reduction in the biofilms. The microelectrode measurements indicate that sulfate reducing activity was largely restricted to a narrow anaerobic zone located about 500 mum below the biofilm surface. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from the O-2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium. Measurements of the reduced inorganic sulfur compounds (FeS, FeS2 and SO), total-Mn and total-Fe in the biofilm indicated that the produced H2S became immediately oxidized with O-2, NO3- and other oxidants, mainly ferric/ferrous hydrates. On the basis of the present results, it was estimated that of all sulfide produced, 13% of the sulfide was precipitated by metal ions as FeS and SO just above the sulfate reduction zone, 65% was anaerobically oxidized to SO42- with NO3- as an electron acceptor and 22% was aerobically oxidized within the biofilm incubated in 70 mumol l(-1) of DO and 280 mumol l(-1) of NO3-., I W A PUBLISHING, 英語

Effect of nitrite and nitrate on biogenic sulfide production in sewer biofilms determined by the use of microelectrodes
S. Okabe, T. Ito, H. Satoh, Y. Watanabe, Water Science and Technology, 47, 11, 281, 288, 2003年
The effects of O2 and NO3- concentrations on in situ sulfate reduction and sulfide reoxidation in microaerophilic wastewater biofilms grown on rotating disk reactors were investigated by the use of microelectrodes for O2, S2-, NO3-, NO2-, and pH. Microelectrode measurements showed the vertical microzonation of O2 respiration, NO3- respiration, H2S oxidation and SO42- reduction in the biofilms. The microelectrode measurements indicate that sulfate reducing activity was largely restricted to a narrow anaerobic zone located about 500 μm below the biofilm surface. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from the O2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium. Measurements of the reduced inorganic sulfur compounds (FeS, FeS2 and S0), total-Mn and total-Fe in the biofilm indicated that the produced H2S became immediately oxidized with O2, NO3- and other oxidants, mainly ferric/ferrous hydrates. On the basis of the present results, it was estimated that of all sulfide produced, 13% of the sulfide was precipitated by metal ions as FeS and S0 just above the sulfate reduction zone, 65% was anaerobically oxidized to SO42- with NO3- as an electron acceptor and 22% was aerobically oxidized within the biofilm incubated in 70 μmol l1- of DO and 280 μmol l1- of NO3-., 英語

Effect of dissolved oxygen concentration on the biofilm and in situ analysis by fluorescence in situ hybridization (FISH) and microelectrodes
A Jang, PL Bishop, S Okabe, SG Lee, IS Kim, WATER SCIENCE AND TECHNOLOGY, 47, 1, 49, 57, 2003年
A better understanding of microbiology and ecology of nitrifying bacteria in inner biofilms is an important part of improving process performance and control. Microelectrodes and fluorescent in situ hybridization (FISH) in biofilm research have been used to investigate the spatial distributions of various microbial activities in biofilms and have led to new experimental findings as well as modifications of the homogeneous assumptions in the biofilm kinetic models. The objective of this study is to try the combination of two methods, both FISH and microelectrode measurements, and to provide reliable and in situ information on nitrifying bacterial activity in biofilms. The characteristics of biofilm developed on tygon slides were different according to the change of dissolved oxygen (DO). When the DO increased from 2 to 10 mg DO/L, the rate of the biofilm thickness increased and its dry density changed from 50-70 to 25-90 mg/cm(3). Ammonia oxidizing bacteria were not uniformly distributed in biofilm, and were found at the deeper layer where oxygen is depleted, they were detected primarily in the upper and middle layers of the biofilm., I W A PUBLISHING, 英語

１６Ｓ ｒＤＮＡ解析によるアンモニア酸化細菌の多様性評価
金田一 智規, 伊藤 司, 岡部 聡, 渡辺 義公, 環境工学研究論文集, 40, 40, 71, 79, 2003年
We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in different environments based on 16S ribosomal DNA (rDNA) sequence analysis with the specific primer set. The sequence analysis revealed that the Nitrosomonas oligotropha-like AOB were the dominant in low-ammonium environments such as river and drinking water treatment systems. In contrast, the Nitrosomonas europaea-like AOB were the dominant in high-ammonium environments in which the concentration of ammonium is more than 1000 mg-N/L. In medium-ammonium environments, the coexistence of the distinct AOB related to the Nitrosomonas europaea-, Nitrosomonas oligotropha-, and Nitrosospira sp.-like clusters were found, indicating a high phylogenetic diversity. These observations support the hypothesis that Nitmsomonas europaea is r-strategist, whereas Nitrosospira sp. is K-strategist, indicating that Nitrosomonas europaea can outcompete the Nitrosospira sp. at high ammonium concentration. These results provide insight into the type of AOB responsible for nitrification in different engineered systems, which should help direct future studies aimed at characterizing relevant AOB growth., 公益社団法人 土木学会, 日本語

バイオフィルム内における硫黄循環と関連微生物群集構造の解析　　　　　　　　　　　　　　　
月刊海洋号外「海洋微生物Ⅱ－基礎，応用研究とその利用」, 35, 54, 63, 2003年

微好気性生物膜内における硫酸塩還元細菌の空間分布と硫黄の挙動解析
岡部聡, 伊藤司, 佐藤久, 渡辺義公, 用水と廃水, 44, 11, 961, 970, 2002年11月01日
産業用水調査会, 日本語

t-RFLP法によるBacteroidesを指標とした河川の糞便性汚染の評価
伊藤 司, 岸田 秀, 岡山 紀子, 岡部 聡, 渡辺 義公, 衛生工学シンポジウム論文集, 10, 117, 120, 2002年10月31日
北海道大学衛生工学会, 日本語

微小電極を用いたコンクリート表面に形成された生物膜内の硫酸塩還元反応の解析
小山田 浩之, 佐藤 久, 岡部 聡, 伊藤 司, 衛生工学シンポジウム論文集, 10, 109, 112, 2002年10月31日
北海道大学衛生工学会, 日本語

河川底泥の窒素循環に及ぼす底生動物の影響
中村 吉志, 佐藤 久, 岡部 聡, 衛生工学シンポジウム論文集, 10, 105, 108, 2002年10月31日
北海道大学衛生工学会, 日本語

下水生物膜内における硫黄酸化細菌群集の解析
杉田謙一, 岡部聡, 伊藤司, 渡辺義公, 土木学会年次学術講演会講演概要集(CD-ROM), 57th, VII-284, 2002年09月01日
日本語

Influence of different cultivars on populations of ammonia-oxidizing bacteria in the root environment of rice
AM Briones, S Okabe, Y Umemiya, NB Ramsing, W Reichardt, H Okuyama, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68, 6, 3067, 3075, 2002年06月
Comparisons of the activities and diversities of ammonia-oxidizing bacteria (AOB) in the root environment of different cultivars of rice (Oryza sativa L.) indicated marked differences despite identical environmental conditions during growth. Gross nitrification rates obtained by the N-15 dilution technique were significantly higher in a modern variety, IR63087-1-17, than in two traditional varieties. Phylogenetic analysis based on the ammonium monooxygenase gene (amoA) identified strains related to Nitrosospira multiformis and Nitrosomonas europaea as the predominant AOB in our experimental rice system. A method was developed to determine the abundance of AOB on root biofilm samples using fluorescently tagged oligonucleotide probes targeting 16S rRNA. The levels of abundance detected suggested an enrichment of AOB on rice roots. We identified 40 to 69% of AOB on roots of IR63087-1-17 as Nitrosomonas spp., while this subpopulation constituted 7 to 23% of AOB on roots of the other cultivars. These results were generally supported by denaturing gradient gel electrophoresis of the amoA gene and analysis of libraries of cloned amoA. In hydroponic culture, oxygen concentration profiles around secondary roots differed significantly among the tested rice varieties, of which IR63087-1-17 showed maximum leakage of oxygen. The results suggest that varietal differences in the composition and activity of root-associated AOB populations may result from microscale differences in O-2 availability., AMER SOC MICROBIOLOGY, 英語

Successional development of sulfate-reducing bacterial populations and their activities in an activated sludge immobilized agar gel film
S Okabe, CM Santegoeds, Y Watanabe, D de Beer, BIOTECHNOLOGY AND BIOENGINEERING, 78, 2, 119, 130, 2002年04月
A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S (m-)2 s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 108 cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x 10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel. (C) 2002 Wiley Periodicals, Inc., JOHN WILEY & SONS INC, 英語

生物膜内における硫黄酸化反応機構および関連微生物群集の解析
伊藤司, 岡部聡, 渡辺義公, 杉田謙一, 佐藤久, 日本水環境学会年会講演集, 36th, 40, 2002年03月14日
日本語

ヒドロキシルアミンの添加が硝化反応に及ぼす影響
金田一智規, 岡部聡, 渡辺義公, 対馬郁夫, 日本水環境学会年会講演集, 36th, 375, 2002年03月14日
日本語

t‐RFLP法よるBacteroidesを指標とした河川の糞便性汚染の評価
岸田秀, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 36th, 345, 2002年03月14日
日本語

16S rDNA解析によるアンモニア酸化細菌の多様性評価
河野快子, 岡部聡, 渡辺義公, 高倉恭子, 日本水環境学会年会講演集, 36th, 374, 2002年03月14日
日本語

Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions
T Ito, S Okabe, H Satoh, Y Watanabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68, 3, 1392, 1402, 2002年03月
A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 muM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 mum from the surface during all stages of biofilm development. The first sulfide production of 0.32 mumol of H2S m(-2) s(-1) was detected below ca. 500 mum in the 3rd week and then gradually increased to 0.70 mumol H2S m(-2) s(-1) in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 x 10(9) cells cm(-3) and 3.6 x 10(9) cells cm(-3), respectively, in the surface 400 mum during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm., AMER SOC MICROBIOLOGY, 英語

B-16 下廃水処理系生物膜内の新規硫黄酸化細菌の特異的検出と機能評価(水処理生態系1,口頭発表)
伊藤 司, 杉田 謙一, 岡部 聡, 渡辺 義公, 日本微生物生態学会講演要旨集, 18, 84, 84, 2002年
日本微生物生態学会, 日本語

Structure and function of nitrifying biofilms as determined by molecular techniques and the use of microelectrodes
S Okabe, H Naitoh, H Satoh, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 46, 1-2, 233, 241, 2002年
The phylogenetic diversity of a nitrifying bacterial community of two types of nitrifying biofilms, a domestic wastewater biofilm and an autotrophic nitrifying biofilm grown on rotating disk reactors (RDR), was characterized by 16S ribosomal DNA (rDNA)-cloning analysis. Thereafter, successional development of nitrifying the bacterial community within both biofilms was visualized in situ by fluorescent in situ hybridization (FISH) with a set of fluorescently labeled 16S rRNA-targeted DNA probes. In situ hybridization revealed that Nitrosomonas ureae was the numerically dominant species of the ammonia-oxidizing population in the domestic wastewater biofilm and that a population shift from N. urea to N. europaea and N. eutropha occurred when the culture medium was switched to the synthetic media from the domestic wastewater. After reaching the steady-state condition, microprofiles of NH4+, NO2-, NO3-, and O-2 in the biofilms were measured by use of microsensors, and the spatial distributions of in situ nitrifying activities were determined. The relationship between the spatial organization of nitrifying bacterial populations and the in situ activity of these populations within the biofilms was discussed. Microelectrode measurements revealed that the active ammonia-oxidizing zone was vertically separated from the active nitrite-oxidizing zone. This vertical separation became more evident with increase of the substrate C/N ratio, leading to deterioration of nitrification efficiency. The combined use of these techniques made it possible to relate in situ nitrifying activity directly to the occurrence of nitrifying bacterial populations., I W A PUBLISHING, 英語

Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization
T Ito, JL Nielsen, S Okabe, Y Watanabe, PH Nielsen, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68, 1, 356, 364, 2002年01月
We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [C-14] propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H-2-utilizing and (CO2)-C-14-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H-2-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans NIB lineages, were found to be important 11, utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms., AMER SOC MICROBIOLOGY, 英語

Biofilm formation potentials in drinking waters treated by different advanced treatment processes
S Okabe, T Kokazi, Y Watanabe, 2ND WORLD WATER CONGRESS: WATER DISTRIBUTION AND WATER SERVICES MANAGEMENT, 2, 4, 97, 104, 2002年
When biodegradable organic matter and other nutrients, such as ammonia and phosphorus, are not sufficiently removed during water treatment, bacteria may proliferate in the water distribution system. Bacterial regrowth deteriorates water quality (taste and odor), accelerates corrosion, and potentially increases the risk of microbial diseases. Therefore, this research was conducted to evaluate the impact of four different advanced water treatment processes, including biological treatments such as a rotating biofilm membrane reactor (RBMR) and a biological activated carbon (BAC) filter and ultrafiltration (UF), on reduction of nutrient levels and biofilm formation potentials of the treated water entering model distribution systems (annular reactors). Our results revealed that biological treatments significantly improved the "biostability" of water leaving from the treatment plant. On average, The RBMR and BAC filter reduced easily assimilable organic carbon (AOC) concentration by half when compared with conventional treatment (multi-media filtration; MF) and ultrafiltration (from 35-49 to 18-23 gg C L-1). Consequently, biofilm formation potential was reduced by a factor of 5 to 10 (from 3,200-5, 100 to 490-710 pg ATP cm(-2)). With respect to "biostability" of water, ultrafiltration was less effective in reducing AOC concentrations. In addition, the impact of chlorine disinfection on biofilm accumulation and AOC levels in the distribution system were studied., I W A PUBLISHING, 英語

Structure and function of nitrifying biofilms as determined by molecular techniques and the use of microelectrodes
S Okabe, H Naitoh, H Satoh, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 46, 1-2, 233, 241, 2002年
The phylogenetic diversity of a nitrifying bacterial community of two types of nitrifying biofilms, a domestic wastewater biofilm and an autotrophic nitrifying biofilm grown on rotating disk reactors (RDR), was characterized by 16S ribosomal DNA (rDNA)-cloning analysis. Thereafter, successional development of nitrifying the bacterial community within both biofilms was visualized in situ by fluorescent in situ hybridization (FISH) with a set of fluorescently labeled 16S rRNA-targeted DNA probes. In situ hybridization revealed that Nitrosomonas ureae was the numerically dominant species of the ammonia-oxidizing population in the domestic wastewater biofilm and that a population shift from N. urea to N. europaea and N. eutropha occurred when the culture medium was switched to the synthetic media from the domestic wastewater. After reaching the steady-state condition, microprofiles of NH4+, NO2-, NO3-, and O-2 in the biofilms were measured by use of microsensors, and the spatial distributions of in situ nitrifying activities were determined. The relationship between the spatial organization of nitrifying bacterial populations and the in situ activity of these populations within the biofilms was discussed. Microelectrode measurements revealed that the active ammonia-oxidizing zone was vertically separated from the active nitrite-oxidizing zone. This vertical separation became more evident with increase of the substrate C/N ratio, leading to deterioration of nitrification efficiency. The combined use of these techniques made it possible to relate in situ nitrifying activity directly to the occurrence of nitrifying bacterial populations., I W A PUBLISHING, 英語

生物膜内における硫黄酸化反応および関連微生物の解析
伊藤司, 岡部聡, 渡辺義公, 日本微生物生態学会講演要旨集, 17th, 17, 27, 27, 2001年11月09日
日本微生物生態学会, 日本語

貧栄養条件下にある浄水処理プロセス内の微生物群集解析
志水豊晴, 山川岳志, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 56th, 468, 469, 2001年09月01日
日本語

配水過程における生物膜生成能に及ぼす異なる浄水処理プロセスの影響
岡部聡, 渡辺義公, 小鍛冶利彦, 小沢源三, 土木学会年次学術講演会講演概要集 第7部, 56th, 470, 471, 2001年09月01日
日本語

バイオフィルム内硝化細菌のFISH法と微小電極を併用した動態解析
岡部 聡, バイオサイエンスとインダストリー = Bioscience & industry, 59, 6, 385, 388, 2001年06月01日
バイオインダストリ-協会, 日本語

配水過程における生物膜生成能に及ぼす異なる浄水処理プロセスの影響
小鍛冶利彦, 岡部聡, 渡辺義公, 木村克輝, 全国水道研究発表会講演集, 52nd, 278, 279, 2001年04月27日
日本語

亜硝酸酸化細菌の優占種を決定する環境因子の検討
山川岳志, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 35th, 533, 2001年03月14日
日本語

貧栄養条件下にある浄水処理プロセス内の微生物群集解析
志水豊晴, 山川岳志, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 35th, 318, 2001年03月14日
日本語

Analysis of microbial structure and function of nitrifying biofilms
S Okabe, H Satoh, Y Watanabe, MICROBIAL GROWTH IN BIOFILMS, PT B, 337, 213, 224, 2001年
ACADEMIC PRESS INC, 英語, 書評論文,書評,文献紹介等

Kinetic analysis of nitrifying biofilm growing on the rotating membrane disk
K Kimura, Y Watanabe, S Okabe, H Satoh, WATER SCIENCE & TECHNOLOGY: WATER SUPPLY, VOL 1, NO 4, 1, 4, 111, 118, 2001年
The authors have proposed a novel water treatment process in which nitrifying bacteria are fixed on the surface of rotating membrane disks. This biofilm-membrane process can perform strict solid-liquid separation and oxidation of ammonia nitrogen simultaneously. In this research, applicability of the conventional biofilm model (assuming the biofilm structure to be flat, homogeneous and continuous) to analysis of the biofilm developing in the proposed process was examined. A long-term operation for culturing the active nitrifying biofilm was carried out prior to kinetic investigation. By cryosectioning of the biofilm and image analysis, the thickness of the biofilm was determined to be 87 pm. From this biofilm thickness and the result of the batch ammonia consumption test, the intrinsic zero-order ammonia consumption rate of the biofilm was estimated precisely to be 930 g/m(3)/h. Using these parameters, the ammonia concentration profile in the biofilm was calculated by the conventional model, and the applicability of the model was examined by comparing the calculated profile with the ones measured with a microelectrode. The calculated profile was very close to the measured ones, which indicated feasibility of the conventional model to the analysis of the biofilm grown in the proposed process. The studied biofilm actually had a simple, i.e. flat, homogeneous and continuous, structure due to membrane filtration. This was the reason why the conventional model could still be employed. In the analysis of the data dealing with low concentrations of ammonia, however, first-order kinetics should be used. The first-order ammonia consumption rate constant of the studied biofilm was estimated to be 808 h(-1)., I W A PUBLISHING, 英語

廃水処理プラント内担体付着生物膜のin situ活性及び微生物相の解析
山川岳志, 岡部聡, 渡辺義公, 日本微生物生態学会講演要旨集, 16th, 16, 71, 71, 2000年11月12日
日本微生物生態学会, 日本語

MAR‐FISH法を用いた生物膜内in situでの硫酸塩還元細菌の特異的検出と機能解析
伊藤司, 岡部聡, NIELSEN P H, NIELSEN J L, 渡辺義公, 日本微生物生態学会講演要旨集, 16th, 16, 39, 39, 2000年11月12日
日本微生物生態学会, 日本語

16S rRNAアプローチによる硝化菌生物膜の生態学的構造解析
岡部聡, 渡辺義公, 内藤初夏, 土木学会年次学術講演会講演概要集 第7部, 55th, 122, 123, 2000年08月31日
日本語

微小電極を用いた生物膜内の溶存酸素輸送機構の解析
佐藤久, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 55th, 114, 115, 2000年08月31日
日本語

微小電極と分子生物学的手法を併用した廃水処理プラントの評価
山川岳志, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 55th, 112, 113, 2000年08月31日
日本語

微好気性生物膜内における硫酸塩還元細菌の多様性評価
山川岳志, 伊藤司, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 34th, 372, 2000年03月15日
日本語

混合培養系生物膜内の硝化機構
佐藤久, 山口有希, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 34th, 158, 2000年03月15日
日本語

モデル生物膜内における硫酸塩還元細菌の動態解析
岡部聡, 渡辺義公, 日本水環境学会年会講演集, 34th, 373, 2000年03月15日
日本語

Significance of substrate C/N ratio on structure and activity of nitrifying biofilms determined by in situ hybridization and the use of microelectrodes
H Satoh, S Okabe, N Norimatsu, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 41, 4-5, 317, 321, 2000年
The effect of substrate C/N ratio on the spatial distributions of ammonia-oxidizing bacteria and their activity was investigated by using microelectrodes with high spatial resolution and fluorescent in situ hybridization (FISH) technique. In this study, an interspecies competition for O-2 between ammonia-oxidizing bacteria and heterotrophic bacteria was experimentally evaluated. An autotrophic nitrifying biofilm originally cultured at C/N=0 was used as a model biofilm to study changes in specific NH; oxidation rate profiles in the biofilm when the substrate C/N ratio was varied. As C/N ratio increased, specific NH; oxidation rates decreased in the outer part of the biofilm due to interspecies competition, while they were unchanged in the inner part. The increase in substrate C/N ratio (i.e., addition of acetate) immediately induced the interspecies competition for O-2 between ammonia-oxidizing bacteria and heterotrophic bacteria at the outer part of the biofilm. As a result of the interspecies competition, NH4+ oxidation was restrained, resulting in a decrease in the ammonia-oxidizing bacterial populations. This experimental result clearly explains the stratified spatial distributions of ammonia-oxidizing bacteria within the biofilms at higher substrate C/N ratios. The combined application of microelectrodes and FISH techniques provides new insights into microbial ecology and population dynamics of nitrifying bacteria within multi-species biofilms., I W A PUBLISHING, 英語

Structure and function of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes
S Okabe, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 42, 12, 21, 32, 2000年
Time dependent development of the spatial organization of NH4+- and NO2--oxidizing bacterial populations in a domestic wastewater biofilm and in an autotrophic nitrifying biofilm were investigated by fluorescent in situ hybridization (FISH) with a set of 16S rRNA-targeted oligonucleotide probes. Population dynamics of nitrifying bacteria in the biofilms were correlated with the biofilm performance. In situ hybridization indicated that Nitrosomonas spp. (excluding probe NEU stained NH4+-oxidizing bacteria: i.e., N. marina-lineage, N, europaea-lineage, N. eutropha, and N. halophila) and Nitrospira-like bacteria were the numerically dominant nitrifying species in the domestic wastewater biofilm. However, probe NEU stained NH4+-oxidizing bacteria became dominant populations in the autotrophic nitrifying biofilm (which were initially cultured with the primary settling tank effluent) after switching to the synthetic media. This population shift might be attributed to the effect of NO2--N accumulation and higher growth Fates of N. europaea-lineage and N. eutropha, outcompeting of her Nitrosomonas spp. in the synthetic medium. This evidence indirectly supports that N. europhaea has been most commonly isolated and studied in most of the previous researches.
For the spatial organization of NH4+- and NO2--oxidizing bacterial populations, bacteria of the genus Nitrobacter could not be detected, instead Nitrospira-like bacteria were found as the main nitrite-oxidizing bacteria in both biofilms. Whereas most of the ammonia-oxidizing bacteria were found throughout the biofilms, the location of nitrite-oxidizing bacteria was restricted to the active nitrite-oxidizing zone, which was detected in the inner part of the biofilms.
Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of NO2--oxidizing bacteria, as determined with FISH. These observations have considerable significance to our understanding of microbial nitrification occurring in wastewater treatment processes and in the natural environment., I W A PUBLISHING, 英語

微小電極とFISH法を用いた硝化細菌生物膜の生態学的構造と機能の解析
岡部聡, 佐藤久, 渡辺義公, 日本微生物生態学会講演要旨集, 15th, 36, 1999年11月05日
日本語

Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms
S Okabe, T Itoh, H Satoh, Y Watanabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 65, 11, 5107, 5116, 1999年11月
The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating dish reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O-2, H2S, NO2-, NO3-, NH4+, and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 10(9) to 10(10) cells per cm(3) of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained Desulfobulbus spp. were found to be numerically important members of SRB populations (approximately 10(8) to 10(9) cells per cm(3)). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 mu m below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S-0) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 mu m), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate., AMER SOC MICROBIOLOGY, 英語

Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms
Satoshi Okabe, Tsukasa Itoh, Hisashi Satoh, Yoshimasa Watanabe, Applied and Environmental Microbiology, 65, 11, 5107, 5116, 1999年11月
The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating disk reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O2, H2S, NO2-, NO3-, NH4+, and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 109 to 1010 cells per cm3 of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained Desulfobulbus spp. were found to be numerically important members of SRB populations (approximately 108 to 109 cells per cm3). The result of microelectrode measurements showed that a high sulfate- reducing activity was found in a narrow anaerobic zone located about 150 to 300 μm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S0) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 μm), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate., 英語

FISH法を用いた都市下水生物膜内における硝化細菌の動態解析
岡部聡, 乗松直生子, 内藤初夏, 渡辺義公, 水環境学会誌, 22, 8, 683, 691, 1999年08月10日
Time dependent developments of in situ spatial distributions of NH4+- and NO2-- oxidizing bacterial populations in domestic wastewater biofilms and in synthetic nitrifying biofilms were investigated by fluorescent in situ hybridization (FISH) with a set of 16S rRNA-targeted oligonucleotide probes. NH4+-oxidizing bacterial populations were classified into three groups according to the probe specificity, and population dynamics of these groups was correlated with the biofilm performance. In situ hybridization indicated that Nitrosomonas spp. (excluding probe NEU stained NH4+-oxidizing bacteria : i.e., N. marina-lineage, N. europaea-lineage, N. eutropha, and N. halophila) and Nitrospira-like bacteria were the numerically dominant species in the domestic wastewater biofilms. However, probe NEU stained NH4+-oxidizing bacteria became dominant populations in the synthetic nitrifying biofilms (which were initially cultured with the primary settling tank effluent) after switching to the synthetic media. This population shift might be attributed to the accumulation of NO2--N and relatively higher loads of NH4+-N which accelerate the growth of N. europaea-lineage and N. eutropha. This evidence indirectly supported that N. europaea has been most commonly isolated and studied in most of the previous researches. These observations have considerable significance to our understanding of microbial nitrification process in wastewater treatment processes and in natural environment., 公益社団法人 日本水環境学会, 日本語

テトラゾリウム塩還元法を応用した硝化細菌の検出方法の検討
岡部 聡, 水環境学会誌 = Journal of Japan Society on Water Environment, 22, 8, 645, 645, 1999年08月
日本語

生物膜内への懸濁微粒子の輸送に与える生物膜構造の影響
岡部聡, 渡辺義公, 前嶋恵, 黒田浩史, 土木学会年次学術講演会講演概要集 第7部, 54th, 432, 433, 1999年08月01日
日本語

混合培養系生物膜内の硝化機構
佐藤久, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 54th, 428, 429, 1999年08月01日
日本語

分離膜表面に付着した生物膜の動力学的解析
木村克輝, 渡辺義公, 岡部聡, 佐藤久, 土木学会年次学術講演会講演概要集 第7部, 54th, 430, 431, 1999年08月01日
日本語

In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes
S Okabe, H Satoh, Y Watanabe, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 65, 7, 3182, 3191, 1999年07月
We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH., AMER SOC MICROBIOLOGY, 英語

都市下水生物膜内のアンモニア酸化反応
佐藤久, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 33rd, 189, 1999年03月15日
日本語

都市下水生物膜内における硝化・脱窒同時反応の検討
山川岳志, 佐藤久, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 33rd, 191, 1999年03月15日
日本語

微好気性生物膜内におけるH₂S酸化機構
伊藤司, 佐藤久, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 33rd, 190, 1999年03月15日
日本語

FISH法を用いた硝化細菌生物膜内の生態学的構造解析　　　　　　　　　　　　　　　
水環境学会誌, 22, 8, 683, 691, 1999年

Biofilm modeling: Present status and future directions
DR Noguera, S Okabe, C Picioreanu, WATER SCIENCE AND TECHNOLOGY, 39, 7, 273, 278, 1999年
Biofilm models are commonly used as simulation tools in engineering applications and as research tools to identify and fill gaps in our knowledge of biofilm processes. While models used in engineering applications rely on simplifying assumptions to make them practical, recent experimental evidence of biofilm heterogeneity questions the validity of these assumptions. On the other band, research models are becoming more complex and use advanced computational tools to mathematically investigate which factors determine the structural heterogeneity and the population dynamics of biofilms. One of the goals of advanced models is to evaluate the relevance of three-dimensional heterogeneities to the predictive capability of traditional biofilm models. In addition, biofilm models are used to evaluate experimental observations when studying a diversity of biofilm-related phenomena. Given the variety of applications of biofilm models and the different approaches that modelers have taken in recent years, a specialist group was convened to evaluate the present status and determine future directions of biofilm modeling research. The education of scientists and engineers on the fundamentals of biofilm models, the development of mathematical models for real-time control of biofilm processes, and the ability to "engineer" the biofilm structure and function (or performance) were identified as the most important objectives for the practical application of biofilm models. As mathematical research tools, biofilm models are directed towards gaining a better understanding of biofilm structure and population dynamics. Specific topics identified as priorities on biofilm research include the behavior of specialist microorganisms, the elucidation of attachment and detachment mechanisms, the determination of mechanical properties of exopolymeric substances, and the study of ecological interactions among different microorganisms. The need to evaluate parameter sensitivity in the different models was identified as an essential component of modeling research. A group decision from this meeting was to initiate a collaborative effort to identify similarities and differences among current modeling approaches. Such comparative analysis will enhance our understanding of biofilm processes and mathematical approaches, and will facilitate the future use of biofilm models by scientists and engineers involved in biofilm research. (C) 1999 IAWQ Published by Elsevier Science Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Microbial ecology of sulfate-reducing bacteria in wastewater biofilms analyzed by microelectrodes and fish (fluorescent in situ hybridization) technique
S Okabe, H Satoh, T Itoh, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 39, 7, 41, 47, 1999年
The vertical distribution of sulfate-reducing bacteria (SRB) in microaerophilic wastewater biofilms grown on fully submerged rotating disk reactors (RDR) was determined by the conventional culture-dependent MPN method and in situ hybridization of fluorescently-labelled 16S rRNA-targeted oligonucleotide probes for SRB in parallel. Chemical concentration profiles within the biofilm were also measured using microelectrodes for O-2, S2-, NO3- and pH. In situ hybridization revealed that the SRB probe-stained cells were distributed throughout the biofilm even in the oxic surface zone in ail states from single scattered cells to clustered cells. The higher fluorescence intensity and abundance of SRB probe-stained cells were found in the middle part of the biofilm. This result corresponded well with O-2 and H2S concentration profiles measured by microelectrodes, showing sulfate reduction was restricted to a narrow anaerobic zone located about 500 mu m below the biofilm surface. Results of the MPN and potential sulfate reducing activity (culture-dependent approaches) indicated a similar distribution of cultivable SRB in the biofilm, The majority of the general SRB probe-stained cells were hybridized with SRB 660 probe, suggesting that one important member of the SRB in the wastewater biofilm could be the genus Desulfobulbus. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from O-2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium., PERGAMON-ELSEVIER SCIENCE LTD, 英語

微小電極及びFISH法を用いた都市下水生物膜内のアンモニア酸化機構の検討
佐藤久, 岡部聡, 渡辺義公, 水環境学会誌, 22, 3, 206, 214, 1999年
Ammonium oxidation in wastewater biofilms grown on rotating biological contactors (RBCs) was investigated by using microelectrodes and fluorescent in situ hybridization (FISH) technique. The density of ammonia-oxidizers which formed dense cell cluster was about 5 times higher in the innermost biofilm than in the middle and surface of the biofilm. Due to the presence of ammonia-oxidizers throughout the biofilm, ammonium oxidation zone was expanded with increasing in oxygen penetration depth, which consequently resulted in an increase of the overall ammonium flux into the biofilm. Specific ammonium oxidation rate was dependent upon the density of ammonia-oxidizers. Although more ammonia-oxidizers were detected in the innermost layer, they might be in the dormant state and not contribute ammonium oxidation during the RBC operation because of substrate transport limitation. Furthermore, the fraction of O2 consumption for nitrification to total O2 consumption was in the range of 35% -53% in a medium with ammonium as the sole electron donor, indicating the biofilm consumed a significant amount of oxygen for other than nitrification. It implied that heterotrophs would grow and outcompete nitrifiers in biofilms even though there was no organic carbon source in the medium. In conclusion, the combination of microelectrode measurements and FISH technique made it possible to relate the spatial organization of ammonia-oxidizers with their in situ ammonium oxidation rates in biofilms, which obviously providing more insight into the nitrification process in wastewater biofilm systems., 公益社団法人 日本水環境学会, 日本語

FISH法を用いた都市下水生物膜内におけるアンモニア酸化細菌の動態解析
岡部聡, 内藤初夏, 渡辺義公, 水環境学会誌, 22, 3, 191, 198, 1999年
In situ spatial organization of ammonia-oxidizing bacteria in wastewater biofilms was investigated using fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM) and digital image analysis. In situ hybridization of steady-state biofilm samples indicated more than 65% of ammonia-oxidizing bacteria present in the biofilm formed dense spherical clusters with the diameter of less than 10 μm and 10∼20% formed clusters with the diameter of more than 10 μm. Most of ammonia-oxidizing bacteria were present in states of small clusters (d<5 μm) in the surface biofilm, because the bigger clusters were imposed on the higher shear stress and consequently sloughed off.
On the other hand, about 4∼10 times higher bacterial population were found as the bigger clusters (d=5∼20 μm) in the bottom biofilm than in the surface, creating a spatial gradient of ammonia-oxidizing bacteria throughout the biofilm. Furthermore, evaluation of series of optical sections through the clusters taken by the CSLM showed that a cluster with the diameter of approximately 25 μm consisted of up to 9,000 rod-shaped cells. Areal cell density within the clusters was found to be in the range of 0.9∼1.1 cells·μm^-2 with areal fraction of cells of 51 ± 6%, indicating that these clusters have relatively large openings. These experimental results suggested that the interior biofilm structure was porous and heterogeneous, the spatial distribution of ammonia-oxidizing bacteria was strongly influenced by the hydraulic shear stress and substrate concentration profiles within the biofilm., 公益社団法人 日本水環境学会, 日本語

微小電極及びFISH法を用いた生物膜内アンモニア酸化細菌菌体密度分布及び活性分布に及ぼす流入基質C/N比の影響評価
佐藤久, 岡部聡, 渡辺義公, 水環境学会誌, 22, 9, 763, 769, 1999年
Effects of substrate C/N ratio on the spatial distributions of ammonia-oxidizing bacterial population and their activities in biofilms were investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes and the use of microelectrodes. The increase in substrate C/N ratio (i.e., addition of acetate) immediately intensified the interspecies competition for oxygen between ammonia-oxidizing bacteria and heterotrophic bacteria, and consequently resulted in deterioration of ammonium oxidation rate especially in the surface of the biofilm. This decrease in the ammonium oxidation rate in the surface could explain a steep spatial gradient of ammonia-oxidizing bacterial population within the biofilms cultured at high substrate C/N ratios.Furthermore, development of the spatial distributions of ammonia-oxidizing bacteria and their activities within the biofilm was followed during biofilm development. The active ammonium oxidation zone gradually shifted from the upper layer to the inner layer of the biofilm, which corresponded with the development of the spatial distributions of ammonia-oxidizing bacteria within the biofilm. Based on in situ analyses of microbial structure and activity, we discussed on mechanisms of population dynamics of ammonia-oxidizing bacteria in biofilms., 公益社団法人 日本水環境学会, 日本語

微好気性生物膜内における硫酸塩還元細菌の空間的分布と硫黄の挙動
伊藤司, 佐藤久, 岡部聡, 渡辺義公, 環境工学研究フォーラム講演集, 35th, 147, 149, 1998年11月
日本語

FISHを用いた硝化菌生物膜の生態学的構造解析
岡部聡, 乗松直生子, 渡辺義公, 環境工学研究フォーラム講演集, 35th, 162, 164, 1998年11月
日本語

FISH法を用いた都市下水生物膜内のアンモニア酸化細菌の存在形態
内藤初夏, 佐藤久, 岡部聡, 渡辺義公, 乗松直生子, 土木学会年次学術講演会講演概要集 第7部, 53rd, 152, 153, 1998年10月
日本語

混合培養系生物膜内のアンモニア酸化機構の遷移
佐藤久, 岡部聡, 渡辺義公, 乗松直生子, 土木学会年次学術講演会講演概要集 第7部, 53rd, 154, 155, 1998年10月
日本語

共焦点レーザー顕微鏡を用いた生物膜の構造解析と膜内流速分布の測定
岡部聡, 渡辺義公, 川村美穂, 土木学会年次学術講演会講演概要集 第7部, 53rd, 158, 159, 1998年10月
日本語

FISH法による好気性生物膜内の硫酸塩還元細菌の存在形態
大西徳子, 伊藤司, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 53rd, 156, 157, 1998年10月
日本語

循環汚泥接触型MF膜処理装置に関する研究 (文部省S)
渡辺義公, 岡部聡, 鈴木辰彦, マンガン自触媒反応と嫌気性膜洗浄を利用した膜処理の効率化 平成9年度 No.08555135, 6,7-23, 1998年
日本語

活性炭循環型MF膜処理装置に関する研究 (文部省S)
渡辺義公, 岡部聡, 鈴木辰彦, マンガン自触媒反応と嫌気性膜洗浄を利用した膜処理の効率化 平成9年度 No.08555135, 24,25-49, 1998年
日本語

AOCを指標とした高度浄水処理システムの性能評価
笠原 伸介, 相澤 拓, 渡辺 義公, 小澤 源三, 岡部 聡, 水道協会雑誌, 67, 11, 12, 21, 1998年
日本水道協会, 日本語

テトラゾリウム塩還元法を応用した硝化細菌の検出方法の検討
岡部聡, 水環境学会誌, 21, 2, 88, 97, 1998年

微小電極を用いた微好気性生物膜内の硫酸塩還元反応の測定
佐藤久, 岡部聡, 伊藤司, 渡辺義公, 水環境学会誌, 21, 6, 367, 375, 1998年
The vertical zonation of 0₂ respiration, NO₃^- respiration, H₂S oxidation and SO₄^2- reduction in microaerobic biofilms grown on rotating biological contactors (RBCs) of treating domestic wastewater was studied by measuring concentration profiles in the biofilms with microelectodes for 0₂, NO₃^-, H₂S and pH. From those profiles, in situ specific reaction rates and substrate fluxes were calculated by using a simple one-dimensional deffusion reaction model and were related to population densities of sulfate-reducing bacteria (SRB) and potential sulfate reduction rates determined by standard batch experiments. The maximum population densities and the highest potential sulfate reduction rates were found in the outermost biofilm. On the contrary, the in situ sulfate reduction rates were restricted in a narrow zone located in the middle biofilm incubated DO=100 μmol·l^-1. An increase in the O₂ and/or NO₃^- penetration depth in the biofilm induced a shift of sulfate reduction zone to the deeper biofilm and a decrease in the specific sulfate reduction rate. This is probably because that the potential sulfate reduction rate decreased toward the depth and addition of nitrate enhanced oxidation of the produced sulfide or increased competition for electron donor with denitrifiers, or both. The microelectrode measurements with the high spatial resolution enable us to quantitatively measure in situ SO₄^2- reduction rate in microaerobic biofilms, which could not be detected measureing sulfide or sulfate flux across the water-biofilm interface., 公益社団法人 日本水環境学会, 日本語

Sulfate reduction and sulfide oxidation in aerobic mixed population biofilms
S Okabe, T Matsuda, H Satoh, T Itoh, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 37, 4-5, 131, 138, 1998年
The microzonation of O-2 respiration, H2S oxidation and SO42- reduction in aerobic biofilms grown on rotating disk reactors of a sewage treatment plant was studied by measuring concentration profiles with microelectrodes for O-2, S2- and pH. The vertical distribution of sulfate-reducing bacteria (SRB) in the biofilms was also determined by the conventional culture-dependent MPN method and fluorescently labeled 16S rRNA-targeted oligonucleotide probes for SRB. The SRB probe stained cells were distributed throughout the biofilm with a distinct higher fluorescence intensity in the middle part of the biofilm. This result corresponded well with O-2 and S2- concentration gradients measured by microelectrodes, showing sulfate reducing activity was largely restricted to a narrow anaerobic zone located in the middle of the biofilm. Measurements of accumulation of reduced sulfur compounds (FeS, FeS2 and S-O) in the biofilm indicated that the H2S produced by SRB became oxidized by O-2 and other oxidants, probably ferric/ferrous hydrates, and precipitated as FeS and S-O just above the sulfate reduction zone. (C) 1998 IAWQ. Published by Elsevier Science Ltd., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Significance of biofilm structure on transport of inert particulates into biofilms
S Okabe, H Kuroda, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 38, 8-9, 163, 170, 1998年
Evolutional changes in interior structures of mixed population biofilms grown on domestic wastewater were quantitatively analyzed using a cryosectioning technique and an image analysis. Meanwhile, transport of particulates into the biofilms was also experimentally investigated using fluorescent microbeads as tracers to relate the biofilm structure and particulate transport into the biofilm. Microscopic observation of the cryomicrotomy biofilm sections indicated the biofilms were very porous and consisted of interwinded filamentous biomass acting as a framework of the biofilm. A honeycomb structure was often found, which would make the biofilm more resistant to water flow. There were micropores with the diameter of about 10 mu m microcolony aggregates attached to filamentous biomass and macropores with the diameter of 20-200 mu m in the biomass matrix. These pores did not clog during two months of cultivation. Areal porosity was about 30% in the bottom biofilm and more than 90 % in the surface. Significant difference in transport efficiency was not observed for various sizes of microbeads due to the presence of macropores. Therefore, even 10 mu m tracer beads could quickly traverse throughout a biofilm 640 mu m thick via water channels or macropores and then penetrated into the micropores. Convective transport from the bulk to the bottom biofilm, rather than molecular diffusion, was responsible for this rapid transport. Based on experimental results, it can be concluded that the biofilm structure seems to be well designed to maximize the transport efficiency of substrates and products and the strength of biofilm structure. (C) 1998 IAWQ, Published by Elsevier Science Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, 英語

微小電極を用いた微好気性生物膜内の硫酸塩還元の測定
佐藤 久, 伊藤 司, 岡部 聡, 渡辺 義公, 衛生工学シンポジウム論文集, 5, 222, 227, 1997年11月01日
北海道大学衛生工学会, 日本語

硝化菌生物膜の生態学的構造解析
伊藤司, 乗松直生子, 佐藤久, 岡部聡, 渡辺義公, 境一澄, 環境工学研究フォーラム講演集, 34th, 91, 93, 1997年11月
日本語

混合培養系好気性生物膜における硫酸塩還元及び硫化物酸化
佐藤久, 岡部聡, 伊藤司, 渡辺義公, 松田尚之, 環境工学研究フォーラム講演集, 34th, 94, 96, 1997年11月
日本語

硝化菌生物膜の生態学的構造と機能
佐藤久, 岡部聡, 渡辺義公, 境一澄, 土木学会年次学術講演会講演概要集 第7部, 52nd, 294, 295, 1997年09月
日本語

テトラゾリウム塩還元法を用いた硝化細菌の検出方法の検討
岡部聡, 渡辺義公, 境一澄, 土木学会年次学術講演会講演概要集 第7部, 52nd, 296, 297, 1997年09月
日本語

初期生物膜形成時における流速の生物膜構造に及ぼす影響
岡部聡, 佐藤久, 渡辺義公, 尾原裕昌, 土木学会年次学術講演会講演概要集 第7部, 52nd, 330, 331, 1997年09月
日本語

蛍光遺伝子プローブを用いた硝化菌生物膜の構造解析
岡部聡, 乗松直生子, 境一澄, 渡辺義公, 船田良, 日本水環境学会年会講演集, 31st, 135, 1997年03月
日本語

生物膜の物理学的構造と懸濁微粒子の輸送に関する研究
黒田浩史, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 31st, 134, 1997年03月
日本語

微小電極を用いた微好気条件下での硫化水素の挙動に関する研究
佐藤久, 伊藤司, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 31st, 136, 1997年03月
日本語

INT還元法を用いた生物膜内の硝化細菌の検出方法に関する研究
境一澄, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 31st, 133, 1997年03月
日本語

Uptake and release of inert fluorescence particles by mixed population biofilms
S Okabe, T Yasuda, Y Watanabe, BIOTECHNOLOGY AND BIOENGINEERING, 53, 5, 459, 469, 1997年03月
Inert fluorescent microparticles were used as tracers to investigate the dynamics of spatial distribution of particulate components in mixed population biofilms. The tracer bead spatial distributions in the biofilm were experimentally measured by sectioning the biofilms with a microslicer. The experimental results were compared with model simulations using the biofilm model (BIOSIM) to evaluate the assumption that advective transport (displacement) of particulates balances with cell growth in the model. The tracer beads could traverse throughout a biofilm 360 mu m thick with in less than 23 minutes, which cannot be explained solely by their attachment to the surface followed by molecular diffusion. Advective transport of the tracer beads via ''voids and pores'' could be responsible for such rapid bead penetration. Observation by confocal scanning laser microscopy (CSLM) clearly showed that the biofilm consisted of a thick loose surface layer, varying in thickness, and a semicontiguous base layer separated by water channels. About 80% of attached tracer beads remained in the biofilm for over 20 days. The trapped tracer beads were gradually transferred from the depth of the biofilm to the surface. The observed bead release rate was much slower than the model predictions. This is probably because the cell density increased predominantly near the substratum, resulting in an unbalance of advective transport of the tracer beads and cell growth. The pores, voids, and cell-free spaces in the biofilm were first filled with growing biomass, thereafter, displacement of the beads took place once the cell density reached certain levels. The model assumptions of the temporal and spatial constant cell density and the continuum concept (flat biomass) are clearly oversimplified and should be revised. It was concluded that the dynamics of the inert microbeads in the biofilm was strongly influenced by not only microbial growth, but also by the biofilm structure and growth pattern. Therefore, one dimensional modeling is not adequate for the accurate description of the transport of particulates in a biofilm. (C) 1997 John Wiley & Sons, Inc., JOHN WILEY & SONS INC, 英語

A novel biofilm-membrane reactor for ammonia oxidation at low concentrations
Y Watanabe, K Kimura, S Okabe, G Ozawa, N Ohkuma, WATER SCIENCE AND TECHNOLOGY, 36, 1, 51, 60, 1997年
For the oxidation of low concentrations of NH4+-N, conventional biofilm reactors such as a rotating biological contactor encounter difficulty due to mass transport limitation of NH4+-N. Therefore, the authors have developed a novel biofilm-membrane reactor, in which biomass is fixed on the surface of rotating membrane disks to enhance NH4+-N transport into the biofilm. Three long-term bench-scale experiments were carried out and sufficient nitrification efficiency was obtained even at low levels of NH4+-N. The experimental results were evaluated in comparison with model simulation. (C) 1997 IAWQ. Published by Elsevier Science Ltd., PERGAMON-ELSEVIER SCIENCE LTD, 英語, 書評論文,書評,文献紹介等

木質系および石炭系粒状活性炭を用いたオゾン・生物活性炭処理　　　　　　　　　　　　　　　
水道協会雑誌, 66, 12, 20, 29, 1997年

Significance of the spatial distribution of microbial species in mixed-population biofilms
S Okabe, K Hirata, Y Watanabe, BIOFOULING, 11, 2, 119, 136, 1997年
Dynamic changes in spatial microbial distribution in mixed-population biofilms resulting from interspecies competition between heterotrophs and nitrifiers were experimentally investigated using a microslicer technique. Biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. The results showed that variation in the carbon loading rate (CLR) and the composition of the feed substrate resulted in rapid and substantial changes in the spatial distribution of nitrifiers in mixed population biofilms within a week, which significantly influenced the NH4-N removal rate. Heterotrophs were more successful than nitrifiers in acquiring dissolved oxygen and space and dominated throughout the biofilms, especially in the surface biofilm. Nitrifiers were therefore diluted in the surface biofilm and mainly found in the inner biofilm. The fraction differences of NH4-oxidizers and NO2-oxidizers over the biofilm depth were about 2 orders and 3-4 orders of magnitude at CLR=0.8 and 1.6 g-C m(-2) d(-1) respectively, indicating a steep spatial gradient of nitrifiers. Although total areal density of NH4-oxidizers in the biofilms were in the same order, the NH4-N flux decreased by about 9% and 24% after one month at CLR=0.8 and 1.6 g-C m(-2) d(-1) respectively. This decrease of NH4-N flux was attributed to the extent of the stratified spatial distribution of NH4-oxidizers. Heterotrophs lie near the surface and act as a diffusion barrier, resulting in an increase in internal dissolved oxygen and NH4-N diffusion resistance for NH4-oxidizers. These results suggest that simplifying or neglecting the spatial distribution of microbial species can lead to substantial errors in biofilm kinetic parameters determined from measured substrate removal rates. For implication in reactor design, the extent of the spatial distribution of microbial species in a biofilm is especially important for interpretation of nitrification efficiency in the presence of organic matter., HARWOOD ACAD PUBL GMBH, 英語

生物活性炭におけるアンモニア性窒素と生物同化性有機炭素の挙動
笠原伸介, 相沢拓, 渡辺義公, 小沢源三, 岡部聡, 丹保憲仁, 土木学会年次学術講演会講演概要集 第7部, 51st, 212, 213, 1996年08月
日本語

茨戸湖の底泥における硫酸塩還元菌とメタン生成菌の競合
乗松直生子, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 51st, 340, 341, 1996年08月
日本語

好気性処理における生物膜構造の,微生物活性度分布に及ぼす影響
佐藤久, 黒田浩史, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 51st, 102, 103, 1996年08月
日本語

嫌気化した底泥におけるイオウ化合物茨戸湖下部湖盆を例として
井野場誠治, 岡部聡, 渡辺義公, 土木学会年次学術講演会講演概要集 第7部, 51st, 342, 343, 1996年08月
日本語

生物膜内の微粒子の挙動に関する基礎的研究
岡部聡, 渡辺義公, 安田岳雄, 土木学会年次学術講演会講演概要集 第7部, 51st, 112, 113, 1996年08月
日本語

Spatial microbial distributions of nitrifiers and heterotrophs in mixed-population biofilms
S Okabe, K Hiratia, Y Ozawa, Y Watanabe, BIOTECHNOLOGY AND BIOENGINEERING, 50, 1, 24, 35, 1996年04月
Spatial microbial distributions of nitrifiers and heterotrophs in undefined mixed-population biofilms were experimentally investigated using a microslicer technique and correlated with nitrification efficiency of the biofilm system. The general stratification of different bacterial groups in the biofilm was simulated using a one-dimensional (1-D) mathematical biofilm accumulation model (BAM) and compared with the experimental results. Biofilms were cultured at three C:N ratios of feed solutions In a partially submerged rotating biological contactor (RBC). It was shown that the biofilms were vertically stratified (from biofilm surface to substratum). At C:N = 0, heterotrophs and nitrifiers coexisted in the outermost biofilm and heterotrophs dominated in the innermost biofilm. At C:N = 1.5, heterotrophs outcompeted nitrifiers for dissolved oxygen and space; thus, heterotrophs dominated in the outermost biofilm and nitrifiers were present only in the deeper biofilm. Nitrifiers and heterotrophs coexisted in the innermost biofilm. An increase in the influent C:N ratio resulted in stronger stratification of microbial species, as well as inhibition of nitrification. In batch experiments, NH4-N utilization rate (R(NH4-N)) was almost the same at each substrate C: N ratio even though NH, oxidizers were predominantly present in the deeper biofilm. The biofilm performance could not be sufficiently explained by the obtained microbial spatial distribution, suggesting that one-dimensional description of microbial distribution was not good enough and three-dimensional measurements of microbial spatial distribution is necessary. Total bacterial densities increased by a factor of 3-17 with biofilm depth. The metabolically active cell fraction decreased from 35 +/- 13% in the outermost biofilm to 15 +/- 4% in the innermost biofilm, presumably due to substrate limitation. The model predicted more pronounced stratification of nitrifiers and heterotrophs than the observed results. This discrepancy could be attributed to the real biofilms that were structurally heterogeneous (e.g., water channels), which could not be described by the one-dimensional model. The results of this study clearly indicate the limitation of 1-D biofilm models to describe the extent of stratification of nitrifiers and heterotrophs and suggest a 3-D model is necessary. (C) 1996 John Wiley & Sons, Inc., JOHN WILEY & SONS INC, 英語

好気性生物膜中の硫黄の循環に関する研究
松田尚之, 岡部聡, 渡辺義公, 日本水環境学会年会講演集, 30th, 131, 1996年03月
日本語

Relationship between population dynamics of nitrifiers in biofilms and reactor performance at various C:N ratios
S. Okabe, Y. Oozawa, K. Hirata, Y. Watanabe, Water Research, 30, 7, 1563, 1572, 1996年
The relationship between time dependent population dynamics of nitrifiers and heterotrophs in undefined mixed-population biofilms and their nitrification efficiency was experimentally investigated at various C:N ratios of feed solutions. Five types of biofilms were cultured in partially submerged rotating biological contactors (RBC's) at different C:N ratios and were used as test materials. The results indicated that initial microbial composition in the biofilms and substrate composition (e.g. C:N ratio) strongly influenced the later population dynamics and the nitrification efficiency. Higher influent C:N ratio retarded accumulation of nitrifying bacteria, especially NO2-oxidizers, resulting in a considerably long start-up period for complete and stable nitrification due to competition for dissolved oxygen and space in the biofilm. Furthermore, a start-up inoculum was very important to keep start-up time of nitrification to a minimum. Time-dependent population dynamics in the biofilms reflected well the bulk water quality and microbial community structure in the bulk liquid. These results suggest that the structure of microbial community in the biofilm can be predicted from monitoring the water quality and microbiology of the bulk liquid. Physiologically inactive cells in the biofilm were determined by an INT dehydrogenease assay. These cells gradually accumulated up to about 30% of the total bacterial population within the biofilms. The results of this study will provide a rational basis for developing and controlling desired biofilm population dynamics to maximize nitrification efficiency of wastewater biofilms., Pergamon Press Inc, 英語

混合培養系生物膜内の懸濁微粒子の挙動に関する基礎的研究
岡部 聡, 安田 岳雄, 佐藤 久, 渡辺 義公, 環境工学研究論文集, 33, 103, 114, 1996年
The dynamics of spatial distribution of particulate components in mixed population biofilms was investigated using lμm fluorescent microparticles as tracer. Shifts of the tracer bead spatial distribution in the biofilm were measured by sectioning the biofilm with a microslicer and were compared with model simulations to evaluate the growthadvection concept in the existing biofilm models. The tracer beads could traverse throughout a 360 μm thick biofilm within 23 minutes, could be attributed to advective transport via water channels and pores. The release of the entrapped beads were much slower than predicted by a one dimensional model due to spatial and temporal changes in cell density in the biofilm. This evidence implied that cell growth and advection was not balanced due to the presence of biofilm heterogeneity (e. g., pores and voids). Three dimensional observation by a confocal scanning laser microscope clearly indicated that the biofilm consisted of semicountiguous base film and relatively high thickness variation of loose surface film. The substratum was not uniformly covered by the biofilm and cell-free spaces and voids were observed near the substratum. This suggested that the pores, voids and cell-free spaces in the biofilm were firstly filled with growing biomass, thereafter displacement of the tracer beads took place once the cell density reached certain levels. Model assumptions of constant cell density and a continuum concept (flat biomass) are clearly over simplified and should be revised. It was concluded that microbial population dynamics in the biofilm can be determined by not only microbial growth kinetics and physiology, but also by the biofilm structure and growth pattern. One dimensional approach (modeling) is, therefore, adequate to predict biofilm accumulation and its performance, but inadequate to accurately describe the microbial population dynamics in the biofilm., Japan Society of Civil Engineers

嫌気化した茨戸湖底泥の硫黄循環を中心とした生物化学的現状把握
岡部 聡, 井野場 誠治, 乗松 直生子, 渡辺 義公, 環境工学研究論文集, 33, 331, 340, 1996年
Chemical and biological characterization of anaerobic sediments of a eutrophic brackish lake Barato, Sapporo were performed with emphases on sulfur cycle in the sediment. Vertical distributions of sulfide compounds (FeS, FeS₂ and S⁰) were measured and related to sediment oxygen demand (SOD). The SOD was divided into biological and chemical components by using specific inhibitors. The chemical SOD was further separated by ferrous, sulfide and manganous demands. The experimental results indicated that SOD ranged 0.3-1.4 mgO₂/cm³ and the major component was ferrous demand accounting for 29-75%(avg.=55.4%). The content of sulfide compounds accounted for 1-2% of total dry weight of the sediment, which was responsible for about 40-50% of total SOD. Sulfate reduction and methane production in the sediment were limited by fatty acid (i. e., acetate) production. The addition of molybdate and chloroform to inhibit sulfate reduction and methanogenesis, respectively, gave a quantitative measure of fatty acid utilization rates by sulfate reduction and methane production. The experimental results revealed that acetate, propionate and n-butyrate accounted for approximately 60%, 25%, and 10%, respectively, of the electron donors of the sulfate reducing bacteria in this sediment. Acetate accounted for 40-60% of the electron donor of methane producing bacteria. These experimental results imply that the oxidation of sulfide, generated by microbial sulfate reduction is a key process in the biogeochemistry of brackish sediments. The detail information on the sediment of lake Barato will be useful for development of a new means to improve the overlaying water quality., Japan Society of Civil Engineers

テトラゾリウム塩還元法を用いた硝化細菌の計測方法
岡部聡, 渡辺義公, 境一澄, 土木学会年次学術講演会講演概要集 第2部, 50th, B, 1142, 1143, 1995年09月
日本語

Sulfide product inhibition of Desulfovibrio desulfuricans in batch and continuous cultures
OKABE S, NIELSEN P H, JONES W L, CHARACKLIS W G, Water Research, 29, 2, 571, 578, 1995年01月01日
Sulfide product inhibition kinetics for growth and activity of Desulfovibrio desulfuricans was investigated in batch and continuous cultures at pH = 7.0. A non-competitive inhibition model adequately described sulfide product inhibition kinetics. Inhibition coefficient (Ki) for maximum specific growth rate (μinhmax) was 251 mg l-1S in a batch experiment. Cell yield determined in a chemostat was reduced in half by a sulfide concentration of about 250 mg l-1S, which was very close to the Kivalue for the batch growth. Maximum specific growth rate (μinhmax) and cell yield (Yc Lac) were strongly inhibited by high levels of sulfide concentrations, whereas specific lactate utilization rate increased with increasing sulfide concentrations. The results indicated an increase in the relative energy needed for maintenance to overcome sulfide inhibition and uncoupling growth from energy production. However, D. desulfuricans to some extent could recover from the shock of high sulfide concentrations. Stoichiometry for catabolic reactions (energy producing) did not change at high sulfide concentrations, while anabolic reactions (cellular synthesis) were strongly inhibited by high sulfide concentrations. These results suggested that separation of sulfide product inhibition into growth (cell yield) and activity (substrate utilization rate) was important to incorporate the sulfide product inhibition kinetics in a variety of applications. © 1995.

SIMULTANEOUS REMOVAL OF ORGANIC MATERIALS AND NITROGEN BY MICRO-AEROBIC BIOFILMS
Y WATANABE, S OKABE, K HIRATA, S MASUDA, WATER SCIENCE AND TECHNOLOGY, 31, 1, 195, 203, 1995年
Simultaneous nitrification with denitrification was investigated using a single rotating biological contactor (RBC). The authors proposed two means to achieve simultaneous nitrification with denitrification in the single reactor: (1) to control oxygen transfer rate by reducing oxygen partial pressure (P-O2) in the air phase and (2) to develop a combined partially (aerobic) and fully (anaerobic) submerged RBC (CPFSR) reactor. For the former experiment, the maximum denitrification efficiency of 90 % was obtained at C:N ratio=6 and P-O2=0.10 atm. Moreover, heterotrophs, NH4- and NO2- oxidizers, and denitrifiers were distributed throughout the biofilm, suggesting that nitrification and denitrification can occur wheresoever the local environment meets their growth conditions. For the latter experiment, effects of type of organic matter and influent carbon:nitrogen ratio (C:N ratio) on the efficiency of simultaneous nitrification with denitrification were investigated using the CPFSR reactor. Acetate, ethylene-glycol, phenol, and poly-vinyl-alcohol (PVA) were used as carbon sources for denitrification. An excellent nitrification efficiency was obtained for all experimental runs and all organic substrates could be degraded and used for denitrification, indicating a great potential for simultaneous removals of nitrogen and xenobiotic compounds by the CPFSR reactor., PERGAMON-ELSEVIER SCIENCE LTD, 英語

RATE AND STOICHIOMETRY OF MICROBIAL SULFATE REDUCTION BY DESULFOVIBRIO-DESULFURICANS IN BIOFILMS
S OKABE, PH NIELSEN, WL JONES, WG CHARACKLIS, BIOFOULING, 9, 1, 63, 83, 1995年
The kinetics and stoichiometry of Desulfovibrio desulfuricans attached to a polycarbonate surface were determined in RotoTorque(TM) reactors and compared with those of suspended cells. Biofilm specific cellular growth rate (mu(b)) and detachment rate (q(dx)) were determined from unsteady state biofilm experiments. In the initial biofilm accumulation phase, the specific cellular growth rate was the same as the maximum specific growth rate for D. desulfuricans in suspension (mu(max) = 0.37 . h(-1)); thereafter mu(b) decreased and approached a steady state value of about 0.1 . h(-1). The decrease in average cellular specific growth rate could be attributed to substrate (lactate) limitation in some experiments, but in others there was no evidence of this. Biofilm-specific cellular detachment rate decreased similarly to biofilm-specific cellular growth rate. In biofilms, cellular yield at mu(b) = 0.1 . h(-1) was approximately 18% of planktonic cellular yield partly due to the production of extensive extracellular polymeric substance. A linear relationship between mu(b) and specific lactate utilization rate (q(s)) in the biofilm did not exist. During the steady state biofilm accumulation phase (mu(b) = .0.1 . h(-1)), specific lactate utilization by biofilm cells was about 2-3 times greater than by planktonic cells, whereas it was essentially the same during the initial biofilm accumulation phase. These results suggest that kinetic and stoichiometric data derived from suspended cells must be cautiously incorporated into biofilm accumulation models., HARWOOD ACAD PUBL GMBH, 英語

Dynamic changes in spatial microbial distribution in mixed-population biofilms: Experimental results and model simulation
S Okabe, K Hirata, Y Watanabe, WATER SCIENCE AND TECHNOLOGY, 32, 8, 67, 74, 1995年
Dynamic changes in spatial microbial distribution in mixed-population biofilms were experimentally determined using a microslicer technique and simulated by a biofilm accumulation model (BAM). Experimental results were compared with the model simulation. The biofilms cultured in partially submerged rotating biological contactors (RBC) with synthetic wastewater were used as test materials. Experimental results showed that an increase of substrate loading rate (i.e., organic carbon and NH4-N) resulted In the microbial stratification in the biofilms. Heterotrophs defeated nitrifiers and dominated in the outer biofilm, whereas nitrifiers were diluted out in the outer biofilm and forced into the inner biofilm. At higher organic loading rates, a stronger stratified microbial spatial distribution was observed, which imposed a severe internal oxygen diffusion limitation cn nitrifiers and resulted in the deterioration of nitrification efficiency. Model simulations described a general trend of the stratified biofilm structure. However, the actual stratification was stronger than the simulated results. For implication in the reactor design, when the specific carbon loading rate exceeds a certain limit, nitrification will be deteriorated or require a long start-up period due to the interspecies competition resulting in oxygen diffusion limitation. The extend of microbial stratification in the biofilm is especially important for determination of feasibility of nitrification in the presence of organic matters., PERGAMON-ELSEVIER SCIENCE LTD, 英語

上向流バイオフィルターを用いた都市下水処理
岡部聡, 渡辺義公, 環境工学研究フォーラム講演集, 31st, 124, 126, 1994年11月
日本語

流入基質C:N比の生物膜内の硝化菌の挙動に与える影響
岡部聡, 渡辺義公, 平田貴紅子, 土木学会年次学術講演会講演概要集 第2部, 49th, B, 1074, 1075, 1994年09月
日本語

ハイブリッド高度下水処理システム
渡辺義公, 岩崎義彦, 岡部聡, 水文・水資源学会研究発表会要旨集, 1994, 368, 369, 1994年08月
日本語

生物膜形成時における細菌分布特性に関する研究
岡部聡, 渡辺義公, 大沢祐子, 土木学会西部支部研究発表会講演概要集, 1993, 312, 313, 1994年03月
日本語

上向流バイオフィルターによる都市下水処理
三木幸浩, 岡部聡, 渡辺義公, 土木学会西部支部研究発表会講演概要集, 1993, 310, 311, 1994年03月
日本語

Effects of limiting nutrients and sulfide concentration on kinetics and stoichiometry of desulfovibrio desulfuricants
岡部 聡, 宮崎大學工學部紀要, 23, 49, 68, 1994年02月
宮崎大学, 英語

STUDY ON THE PERFORMANCE OF AN UP-FLOW AERATED BIOFILTER (UAB) IN MUNICIPAL WASTE-WATER TREATMENTS
Y WATANABE, S OKABE, T ARATA, Y HARUTA, WATER SCIENCE AND TECHNOLOGY, 30, 11, 25, 33, 1994年
A comprehensive wastewater treatment system that accomplishes oxidation of organic matter, nitrification, and denitrification was developed, and its characteristics and performance were investigated. A municipal wastewater was treated by an up-flow aerated biofilter (UAB), in which biofilms were developed on stainless meshes installed horizontally. This UAB exhibited a great potential ability of oxidation of organic matter, SS stabilization, and nitrification due to a unique aeration mechanism giving high DO concentrations with relatively low aeration rates. Another unique feature of the UAB was that attached biofilms on stainless meshes physically filtered out and/or adsorbed suspended solids in the wastewater in addition to the biological oxidation of organic matter. A stable nitrification could be achieved at HRT = 10 hours corresponding to a hydraulic loading of 86 L m(-2) d(-1) and at a ratio of aeration rate to wastewater flow rate (A/W) of 2, which is considerably low as compared to aeration rates of typical activated sludge systems. This UAB system also could handle relatively high hydraulic loading rates. The UAB used in this study still have enough space to install more stainless meshes so as to reduce hydraulic loading rates resulting in the reduction of HRT and aeration rate, which leads to improvement of the system performance as well as reduction of the running cost., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Estimation of cellular and extracellular carbon contents in sulfate-reducing bacteria biofilms by lipopolysaccharide assay and epifluorescence microscopic technique
OKABE S, NIELSEN P H, JONES W L, CHARACKLIS W G, . Water Research, 28, 11, 2263, 2266, 1994年01月01日
Measurement of cellular and extracellular carbon contents of sulfate-reducing bacteria (SRB) is essential and important in studies of the role of SRB in corrosion and biofouling. An epifluorescence (EPI) microscopic technique and a lipopolysaccharide (LPS) assay were used to quantify cellular and extracellular carbon contents in Desulfovibrio desulfuricans biofilms. The average contents of lipopolysaccharide (LPS) and cellular carbon were 7.3 ± 2.8 (fg LPS) cell-1 and 39.9 ± 9.9 (fg cellular-C) cell-1, respectively, in a D. desulfuricans chemostat culture. A ratio of cellular carbon content to LPS content was 6.5 ± 2.8, and was used to estimate cellular carbon contents in a D. desulfuricans biofilm. The LPS and EPI methods gave comparable results for suspended samples, but not for biofilm samples. © 1994.

STUDY ON THE PERFORMANCE OF AN UP-FLOW AERATED BIOFILTER (UAB) IN MUNICIPAL WASTE-WATER TREATMENTS
Y WATANABE, S OKABE, T ARATA, Y HARUTA, WATER SCIENCE AND TECHNOLOGY, 30, 11, 25, 33, 1994年
A comprehensive wastewater treatment system that accomplishes oxidation of organic matter, nitrification, and denitrification was developed, and its characteristics and performance were investigated. A municipal wastewater was treated by an up-flow aerated biofilter (UAB), in which biofilms were developed on stainless meshes installed horizontally. This UAB exhibited a great potential ability of oxidation of organic matter, SS stabilization, and nitrification due to a unique aeration mechanism giving high DO concentrations with relatively low aeration rates. Another unique feature of the UAB was that attached biofilms on stainless meshes physically filtered out and/or adsorbed suspended solids in the wastewater in addition to the biological oxidation of organic matter. A stable nitrification could be achieved at HRT = 10 hours corresponding to a hydraulic loading of 86 L m(-2) d(-1) and at a ratio of aeration rate to wastewater flow rate (A/W) of 2, which is considerably low as compared to aeration rates of typical activated sludge systems. This UAB system also could handle relatively high hydraulic loading rates. The UAB used in this study still have enough space to install more stainless meshes so as to reduce hydraulic loading rates resulting in the reduction of HRT and aeration rate, which leads to improvement of the system performance as well as reduction of the running cost., PERGAMON-ELSEVIER SCIENCE LTD, 英語

Corrosion of mild steel underneath aerobic biofilms containing sulfate-reducing bacteria, part I: at low dissolved oxygen concentration
Whonchee Lee, Zbigniew Lewandowski, Sathoshi Okabe, William G. Characklis, Recep Avci, Biofouling, 7, 1, 15, 1993年11月01日
The sulfate-reducing bacteria (SRB)-enhanced corrosion of mild steel in the presence of 1.5 mg.l-1 dissolved oxygen (DO) in bulk liquid was investigated. The biofilm process analysis was combined with microelectrode measurements, electrochemical measurements, and surface analysis. In the early stages of biofilm accumulation, the cathodic polarization and the decreasing corrosion rate were attributed to DO consumption by aerobic bacteria. During that time, limited SRB activity was observed. The DO concentration near the steel surface was between 0.6 and 1 mg.l-1. After total depletion of dissolved oxygen near the steel surface, the cathodic depolarization and the increased corrosion rate were associated with the proliferation of SRB near the steel surface. Auger electron spectroscopy analysis indicated localized sulfide attack. High pit density appeared where the coincidence of oxygen and sulfur occurred. The bottom of the pit was enriched with sulfur. © 1993, Taylor & Francis Group, LLC. All rights reserved.

上向流バイオフィルターに関する研究
岡部聡, 渡辺義公, 春田勇二, 荒田朋睦, 土木学会年次学術講演会講演概要集 第2部, 48th, 1312, 1313, 1993年09月
日本語

Anaerobic biofilms in industrial water-systems　　　　　　　　　　　　　　　
Abstracts of Papers of the American Chemical Society, 204, 9999, 1993年

Effects of temperature and phosphorous concentration on microbial sulfate reduction by Desulfovibrio desulfuricans
OKABE S, CHARACKLIS W G, Biotechnology and Bioengineering, 39, 10, 1031, 1042, 1992年01月01日
The effects of temperature and phosphorous concentration on the rate and the extent of microbial sulfate reduction with lactate as carbon and energy source were investigated for Desulfovibrio desulfuricans. The continuous culture experiments (chemostat) were conducted at pH 7.0 from 12 to 48°C. The maximum specific growth rate (μmax) was relatively constant in the range 25°C–43°C and dramatically decreased outside this temperature range. The half‐saturation coefficient was minimum at 25°C. Cell yield was highest in the optimum temperature range (35°C–43°C) for growth. Maintenance energy requirements for D. desulfuricans were not significant. Two moles of lactate is consumed for every mole of sulfate reduced, and this stoichiometric ratio is not temperature dependent. Steady state rate and stoichiometric coefficients accurately predicted transient behavior during temperature shifts. The extent of extracellular polymeric substance (EPS) is related to the concentration of phosphorous in the medium. EPS production rate increased with decreased phosphorous loading rate. Failure to discriminate between cell and EPS formation by D. desulfuricans leads to significant overestimates of the cell yield. The limiting C:P ratio for D. desulfuricans was in the range of 400:1 to 800:1. Copyright © 1992 John Wiley & Sons, Inc.

Corrosion of mild steel underneath aerobic biofilms containing sulfate-reducing bacteria　　　　　　　　　　　　　　　
Corrosion, 47, 1, 15, 1992年

Factors affecting microbial sulfate reduction by Desulfovibrio desulfuricans in continuous cultures: Limiting nutrients and sulfide concentration
OKABE S, NIELSEN P H, CHARACKLIS W G, Biotechnology and Bioengineering, 40, 6, 725, 734, 1992年01月01日
The effects of sulfate and nitrogen concentrations of the rate and stoichiometry of microbial sulfate reduction were investigated for Desulfovibrio desulfuricans grown on lactate and sulfate in a chemostat at pH 7.0. Maximum specific growth rates (μmax), half‐saturation coefficients (Ksul), and cell yield (Yc/Lac) of 0.344 ± 0.007 and 0.352 ± 0.003 h−1, 1.8 ± 0.3 and 1.0 ± 0.2 mg/L, and 0.020 ± 0.003 and 0.017 ± 0.003 g cell/g lactate, respectively, were obtained under sulfate‐limiting conditions at 35°C and 43°C. Maintenance energy requirements for D. desulfuricans were significant under sulfate‐limiting conditions. The extent of extracellular polymeric substance (EPS) produced was related to the carbon: nitrogen ratio in the medium. EPS production rate increased with decreased nitrogen loading rate. Nitrogen starvation also resulted in decreased cell size of D. desulfuricans. The limiting C : N ratio (w/w) for D. desulfuricans was in the range of 45 : 1 to 120 : 1. Effects of sulfide on microbial sulfate reduction, cell size, and biomass production were also ivestigated at pH 7.0. Fifty percent inhibition of lactate utilization occurred at a total sulfide concentration of approximately 500 mg/L. The cell size of D. desulfuricans decreased with increasing total sulfide concentration. Sulfide inhibition of D. desulfuricans was observed to be a reversible process. © 1992 John Wiley & Sons, Inc. Copyright © 1992 John Wiley & Sons, Inc.

回転円板法(RBC)における物質移動
渡辺義公, 石黒政儀, 岡部聡, 田代雄児, 土木学会年次学術講演会講演概要集 第2部, 41st, 807, 808, 1986年11月
日本語

Nitrification　　　　　　　　　　　　　　　
ASM Press, 2011年

バイオ燃料電池の最新動向　　　　　　　　　　　　　　　
シーエムシー出版, 2011年

Research on Nitrification and Related Processes, Part B　　　　　　　　　　　　　　　
Elsevier, 2011年

Nitrification　　　　　　　　　　　　　　　
ASM Press, 2011年

Recent treands in microbial fuel cells　　　　　　　　　　　　　　　
CMC, 2011年

Research on Nitrification and Related Processes, Part B　　　　　　　　　　　　　　　
Elsevier, 2011年

水再利用学　　　　　　　　　　　　　　　
技報堂出版, 2010年

難培養微生物研究の最新技術II　　　　　　　　　　　　　　　
シーエムシー出版, 2010年

Wastewater Treatment　　　　　　　　　　　　　　　
Caister Academic Press, 2010年

Water Reuse　　　　　　　　　　　　　　　
2010年

Wastewater Treatment　　　　　　　　　　　　　　　
Caister Academic Press, 2010年

非持続的窒素循環を革新する新規生物電気化学的窒素変換技術の創出
科学研究費助成事業
2023年04月01日 - 2027年03月31日
岡部 聡, 押木 守
日本学術振興会, 基盤研究(A), 北海道大学, 23H00192

エネルギー自給式ハイブリッド電気化学的膜分離バイオリアクターの開発
科学研究費助成事業
2022年06月30日 - 2025年03月31日
岡部 聡, 山村 寛
本研究では、酸素供給(曝気)を必要とせず固体電極を最終電子受容体として用いるバイオ燃料電池(MFC)を膜分離バイオリアクター(MBR)に導入することにより、下水処理と電気エネルギーの直接回収を行い、得られた電力を利用し電導性膜分離の電位を人為的に制御し、膜ファウリングを抑制しながら膜分離による高度下水処理を同時に達成する、新規MFC搭載型エネルギー自給式ハイブリッド電気化学的膜分離バイオリアクター (He-MBR)を開発する。
本年度は、既存のMBR槽内にカセット式空気カソードMFCを複数ユニット挿入したHe-MBRを構築した。アノード電極は、高密度で充填可能なグラファイトカーボンブラシを使用した。カソード電極はステンレス膜(膜口径2 ㎜)を基盤とし、微粉末活性炭(PAC)を添加したポリフッ化ビニリデン(PVDF)を塗布することで作製した。鍵となる導電性分離膜は、ステンレススチールメッシュ (孔径: 0.11 mm, 厚さ:0.06 mm) を基盤とし、ポリエチレングリコール(PEG-600)とDimethyl Sulfoxide (DMSO)を添加したPVDF混合高分子溶液を塗布することにより作製することに成功した。作成した電導性分離膜の物理電気化学的特性（孔径、表面凹凸、など）を評価した。さらに、デッドエンド式膜ろ過実験を行い膜の透過性、ファウりング特性、COD除去率、微生物（大腸菌）の除去率を評価した。
日本学術振興会, 挑戦的研究(開拓), 北海道大学, 22K18296

バイオ燃料電池駆動型エネルギー自立式Anammox MECシステムの開発
科学研究費助成事業 基盤研究(A)
2019年04月01日 - 2023年03月31日
岡部 聡, 北島 正章, 佐藤 久, 押木 守
嫌気性アンモニア酸化(Anammox)プロセスは、省エネ型窒素除去プロセスとして工場排水や嫌気性消化脱水ろ液処理などに適用が始まっているが、メインストリームの都市下水処理への適用は一向に進んでいない。メインストリームの都市下水処理への適用の鍵は、前段の部分硝化(NH4+をNO2-まで酸化する)プロセスの安定化・高効率化である。
本研究では、前段に有機物除去を担うバイオ燃料電池(MFC)を設け、MFCで発生する電圧を用いてMECアノード電極電位を制御することで、アンモニア酸化細菌(AOB)がアノード電極を電子受容体としてNH4+をNO2-まで酸化し、Anammox細菌が生成されたNO2-とNH4+を窒素ガス(N2)へ変換するMFC駆動型部分硝化(PN)-Anammox 生物電解セル(MEC)システムを開発するものである。本年度は以下の2点について検討した。① MFCを補助電源とし、アンモニア酸化細菌（AOB）は、アノード電極を電子受容体としてNH4+をNO2-へ酸化できるか？さらに、Anammox細菌は、アノード電極を電子受容体としてNH4+を直接N2へ酸化できるか？
② 亜硝酸酸化細菌(NOB; NO2-をNO3-に酸化する細菌)に特異的に感染し溶菌させるバクテリオファージは存在するのか？そして、それをNOB増殖抑制剤として利用し部分硝化反応(NH4+ → NO2-)の安定化を効率的に達成できるか？その結果、①関してはMFCを補助電源とするMECを構築し、NH4+の除去性能を確認できた。②に関しては、活性汚泥などからファージを濃縮しNitrospiraのバイオマスと混合し、ファージ感染の有無を確認する実験を行ったが、NOBに特異的に感染するバクテリオファージを獲得するには至らなかった。
日本学術振興会, 基盤研究(A), 北海道大学, 19H00776

非生物・生物ハイブリッド人工光合成システムの構築：持続可能な酢酸生成拠点の創出
科学研究費助成事業 挑戦的研究(開拓)
2019年06月28日 - 2022年03月31日
岡部 聡, 渡辺 精一, 佐藤 久
本研究の目的は、CO2と水を原料とし太陽光エネルギー（可視光）と廃水をエネルギー源として、常温常圧、中性条件下で酢酸（2炭素化合物）を生産可能なバイオ‐光電気化学太陽電池（Bio-PEC)ハイブリッドシステムを構築することである。そのために必要なCuOナノワイヤー表面にZnOナノロッドを付加した3次元ナノ構造を持つZnO/CuO複合体を作成することに成功した。また、その電気化学的特性（水素生成速度や電流密度）は、既往の類似する三次元ナノ構造を有するZnO/CuOの値の約3倍であった。これにより、効率の良いBio-PECシステムの構築の可能性が示された。
日本学術振興会, 挑戦的研究(開拓), 北海道大学, 20K20486

水環境におけるヒトノロウイルス未知動態の解明
科学研究費助成事業 基盤研究(A)
2012年04月01日 - 2015年03月31日
大村 達夫, 原田 秀樹, 佐野 大輔, 渡部 徹, 岡部 聡, 真砂 佳史
本研究では、水環境中におけるノロウイルスの未知動態を解明することを目的とし、１）ウイルス外殻タンパク質損傷評価法の適用条件、２）環境由来濃縮サンプルから目的ウイルスゲノムのみを特異的に回収する手法の開発、および３）ノロウイルスによる養殖カキの汚染度に関する調査・研究を行った。その結果、カプシドタンパク質損傷検出手法が水環境中のヒトノロウイルスに適用可能であることが確認され、環境由来濃縮サンプルから損傷を受けていないウイルスゲノムのみを特異的に回収する手法の確立に成功した。さらに養殖カキ中の優占している遺伝子型のみならずマイナーな感染流行株が次世代シーケンスにより検出可能であることが示された。
日本学術振興会, 基盤研究(A), 東北大学, 24246089

糞便汚染マーカー定量検出を基盤とした微生物学的水質管理手法の確立
科学研究費助成事業 基盤研究(A)
2011年04月01日 - 2014年03月31日
岡部 聡, 石井 聡, 佐藤 久, 佐野 大輔
現行の糞便汚染指標である大腸菌群数による微生物学的水質管理は、水環境中での大腸菌群の増殖、糞便に由来しない大腸菌群の存在など、数多くの問題点が指摘されている。このため、合理的な糞便性汚染指標の確立および水域の微生物学的水質管理手法の確立が急務となっている。このような背景のもと、本研究では、宿主特異的遺伝子マーカー（腸内蛋白質分解細菌の最優占種であるBacteroides-Prevotella 属由来遺伝子）をもとに水域の糞便汚染レベルを定量的に評価し、さらに糞便汚染源（ヒト、家畜及び野生動物等）を迅速かつ正確に特定する新規方法論を確立し、具体的な汚染防止対策の構築を含む合理的な微生物学的水質管理を実現することを目的としている。
本年度は、昨年度開発した各宿主特異的糞便汚染マーカーを実際の水環境に適用し、糞便汚染の実態を明らかにした。さらに、遺伝子マーカーを糞便汚染指標として活用するためには、生存細胞と死細胞を区別して定量することが求められる。そこで、Propidium monoazide (PMA)を併用した定量PCR法を確立し、糞便汚染源の特定を行うために重要となる生菌由来の糞便性汚染マーカーの定量が可能となった。
次に、環境水中における糞便汚染マーカー（ヒト、ブタ、ウシ、ニワトリ、カモの各宿主特異的遺伝子マーカー）の挙動を解析した。さらに、既存の糞便汚染指標である大腸菌群数、糞便生大腸菌群及び大腸菌の定量も行い、減衰速度の違いを評価した。
最後に、水系感染する腸管系感染症起因細菌及びノロウイルス等の腸管系ウイルスの特異的検出を行い、各宿主特異的遺伝子マーカーと病原微生物の環境水中における存在比の相関関係を調査した。
日本学術振興会, 基盤研究(A), 北海道大学, 23246094

水循環の基盤となる革新的水処理システムの創出　　　　　　　　　　　　　　　
ＪＳＴ戦略的創造研究推進制度（研究チーム型） （戦略的基礎研究推進事業：ＣＲＥＳＴ）
2009年 - 2014年
競争的資金

Development of innovative water and wastewater treatment systems for sustainable urban water metabolism　　　　　　　　　　　　　　　
JST Basic Research Programs (Core Research for Evolutional Science and Technology :CREST)
2009年 - 2014年
競争的資金

廃水処理、発電、発生汚泥量削減を同時に可能にするバイオ燃料電池の開発
科学研究費助成事業 挑戦的萌芽研究
2011年 - 2012年
岡部 聡
本研究は、廃水処理と電気エネルギー回収が同時に可能となることが期待されているバイオ燃料電池のさらなる発電能力の向上を図ることを目的とした。そこで本年度は、カソード反応を促進するため、すなわちプロトン供給量を促進するために、カソードへ供給する空気中の水分およびCO2濃度の発電量に及ぼす影響について検討した。供給する空気中のCO2濃度が高くなるほど、カソード電極表面上に存在する高pH水に溶解するCO2が増大しプロトンの供給を促し、電気伝導率の向上が確認された。さらに、供給するガス組成(CO2とO2の濃度比)および供給速度を変化させて、最大の電力密度が得られる条件を求めた。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 23656324

トキシコゲノミクスによる新規機能性ナノ高分子(デンドリマー)の毒性評価
科学研究費助成事業 挑戦的萌芽研究
2009年 - 2010年
岡部 聡
PAMAM(polyamidoamine)デンドリマーの生物学的活性は世代が大きくなる程高くなるため、毒性は世代に強く依存すると考えられる。世代依存毒性を含むPAMAMデンドリマーの毒性のより詳細な解明が必須であり、本研究ではPAMAMデンドリマーの毒性及びその発現メカニズムの解明を目的とした。ヒト肝癌由来細胞株HepG2細胞を用いて、PAMAMデンドリマーG4-G7の毒性評価を行った。全ての試験通して、PAMAMデンドリマーとBSAの相互作用による影響を除く為、PAMAMデンドリマーの溶媒にHBSS(Hank's balanced salt solution)にHEPESを10mMとなるように加えたものを用いた。また、暴露濃度にはNeutral red(NR)assayで細胞生存率が80%となる濃度をそれぞれ用いた。その結果、PAMAMデンドリマーの世代依存的な細胞毒性を確認した。同じNR assayで細胞生存率が80%となる濃度でも、世代の大きいPAMAMデンドリマーほどROSの産生能が高いことが明らかとなった。ROS産生は120minで最高値をとった。さらに、DNA損傷性を評価するために、H_2O_2をPositive Controlとしてコメットアッセイを行った。実験結果の解析は、尾の長さ、面積、濃さにより、尾の無いもの(I)から,核内のDNA全てが断片化した頭部の無いものあるいは頭部の極微少なもの(V)までI～Vの5段階に分けて評価した。その結果、AMAMデンドリマーで処理していないHBSS(negative control)の細胞群では、クラスIが大部分であった(75.2%)のに対し、PAMAMデンドリマー暴露系ではクラスIが40%～55%と低く、G7で最低値の40.4%%となった。また、G6,G7では損傷の重篤であるIV,Vの割合が高く、コメット値はG5を除いて世代が増加するに従い、増加する傾向が認められた。以上の結果より、PAMAMデンドリマーがDNA損傷性を有しており、DNA損傷の程度は世代が大きい程より重篤であることが明らかとなった。また、1mM H_2O_2とPAMAMデンドリマーG6はROS産生量がほぼ同程度であったにも関わらず、DNA損傷レベルはH_2O_2の方がPAMAMデンドリマーに比べかなり高い値となった。このことから、酸化ストレスがPAMAMデンドリマーの毒性の直接的な原因ではない可能性が示唆された。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 21656129

トキシコゲノミクス的アプローチによる重金属-農薬複合毒性の解析
科学研究費助成事業 基盤研究(B)
2008年 - 2010年
岡部 聡
本研究では、ヒトDNAマイクロアレイを用いて、代表的な汚染物質である重金属、農薬の混合暴露下におけるヒト由来細胞(HepG2)の遺伝子発現解析を行い、重金属、農薬それぞれの暴露で得られた遺伝子発現パターンと比較し複合毒性作用(相乗、拮抗、もしくは新たな毒性の出現)の解明および有用な毒性マーカー遺伝子の選定を行った。さらに、遺伝子発現解析により選定された遺伝子マーカーを用いて、より詳細な遺伝子発現解析(定量的RT-PCR)を実施し、各重金属-農薬の組合せにおいて、遺伝子発現レベルで作用濃度閾値を明らかにした。
日本学術振興会, 基盤研究(B), 北海道大学, 20360235

ヒトDNAマイクロアレイを用いたナノ粒子の細胞毒性評価
科学研究費助成事業 萌芽研究
2007年 - 2008年
岡部 聡
現在生産されているナノ粒子の毒性に関する情報は限られており、人体への影響は解明されてはいない。本研究では、この技術を銀ナノ粒子存在下ヒト由来細胞に応用し、銀ナノ粒子の毒性の有無およびその毒性作用について考察を行った。ニュートラルレッド法と形態観察の結果より、遺伝子発現解析を実施する暴露濃度を低濃度でありながら形態変化が明らかであった40μg/Lと、より高濃度の1000μg/Lに決定した。対照系で紡錘形であった細胞が、銀ナノ粒子暴露群では仮足が伸びたような形態へと変化し、細胞内に多数の空隙が認められた。データベースを基に有意変動遺伝子の機能分類を行った結果、銀、ポリスチレン両ナノ粒子暴露においてM-phaseに分類され、細胞分裂を促進する作用を持つ遺伝子群の上昇変動が顕著であった。また、DNA-repairに分類される遺伝子群の上昇変動も顕著であった。これは、DNAの修復と対応していると考えられ、遺伝子傷害の可能性が示唆された。また、銀ナノ粒子暴露に特異的な遺伝子発現パターンとして、活性酸素種の解毒に重要な役割を担う遺伝子をはじめとする酸化ストレス応答遺伝子の下降変動が挙げられた。小核試験の結果、銀ナノ粒子暴露系にのみ多くの小核が確認され(50.2%)、銀ナノ粒子暴露による遺伝子障害の可能性を強く支持していると考えられる。また、アスコルビン酸を蓄積させた細胞で同様の実験を行った結果、小核形成率が低下したことから(50.2%→26.3%)、銀ナノ粒子暴露による遺伝子障害性に、活性酸素種の関与が示唆された。以上の結果をまとめると、銀、ポリスチレンナノ粒子において発癌作用に関連すると推測される細胞増殖刺激、遺伝子障害を示唆する遺伝子発現パターンが確認された。さらに、これらの結果は、小核試験によって支持され、また、遺伝子傷害性がアスコルビン酸によって抑制されることから、銀ナノ粒子暴露による遺伝子障害性に対する活性酸素種の関与が示唆された。
日本学術振興会, 萌芽研究, 北海道大学, 19656129

DNAマイクロアレイを用いた環境汚染化学物質の多指標型毒性評価システムの開発
科学研究費助成事業 基盤研究(B)
2005年 - 2007年
岡部 聡
本年度は、発癌作用のモデル化学物質として、ニトロソアミン(DNA障害性)、TPA(DNA非障害性、プロモーター)、TCE(DNA非障害性)を選択し、これら化学物質により特異的に誘導される遺伝子ライブラリーを構築した。また、3種の重金属(As、 Cd、 Ni)暴露における遺伝子発現解析を行い、モデル化学物質暴露における遺伝子発現パターンと比較した。その結果、これら重金属の主要な発癌作用は細胞増殖の促進、DNA傷害であることが明らかとなった。
上記の特徴は活性酸素(ROS)産生物質であるDMNQでも認められたため、ROS産生が重金属の主要な発癌メカニズムと仮定し、抗酸化物質であるアスコルビン酸処理後の細胞についても同様の検討を行った。その結果、ヒ素では細胞増殖、DNA傷害に関わる変動遺伝子数が顕著に減少したのに対し、カドミウムとニッケルではむしろ増加する結果となった。すなわち、ヒ素の発癌性においては活性酸素産生が主要な発癌メカニズムであり、カドミウム、ニッケルにおいては他のメカニズムが関与していることが明らかとなった。
さらに本研究では、発癌性マーカーとして有用であると考えられる遺伝子PTTG1が見出され、供試したすべての発癌物質、重金属において有意に発現することが定量的RT-PCR法によって確認された。重金属の発癌性はエイムズテスト等の既存の発癌評価本手法によって評価することが困難であるため、簡便かつ迅速な評価手法の開発が求められる。本研究によって得られた結果より、DNAマイクロアレイによって重金属を含む広範囲の発癌物質を評価可能であることが明らかとなり、また、見いだされた発癌性マーカーPTTG1遺伝子は機構の異なる発癌物質の包括的なスクリーニングに有用であると考えられる。
日本学術振興会, 基盤研究(B), 北海道大学, 17360250

遺伝子および化学マーカーによる河川糞便性汚染源の特定
科学研究費助成事業 基盤研究(B)
2003年 - 2004年
岡部 聡, 木村 克輝
本研究では糞便性指標微生物の遺伝子マーカーと化学マーカーを組み合わせた迅速(1-2日程度)かつ簡便な、糞便性汚染源の特定と汚染度の定量的評価手法の開発を行うことを目的とした。腸内蛋白質分解細菌の最優占種であるBacteroidesに着目し、培養を必要としない分子生物学手法(16S rDNAクローンライブラリー法やTerminal Restriction Fragment Length Polymorphism : T-RFLP法、定量PCR法)を用い、宿主特有のBacteroidesの16S rRNA遺伝子配列(遺伝子マーカー)を特定・探索することにより、水環境中の糞便汚染源(人間、牛、豚等)を特定する。研究成果は以下のように要約される。
宿主動物(人間、牛、豚)毎に特異的なBacteroides種が存在し、宿主動物を認識することが可能な16SrRNA遺伝子配列(遺伝子マーカー)を見つけ出すことに成功した。これによって、これら特異的なBacteroides属を指標微生物とすることにより、糞便汚染源の特定が可能であることを示唆している。次に、これら宿主特異的遺伝子マーカーを迅速かつ簡便に検出・定量するために、これらの遺伝子マーカーに特異的なPCRプラーマーのセットを7つ設計することができた。これら設計したPCRプラーマーセットを用いたT-RFLP法を確立した。この結果、宿主動物毎に特異的なDNA断片長を有するクローンが河川環境中に多く存在することが確認された。したがって、これらのDNA断片は宿主動物毎の遺伝子マーカーとなることが確認できた。また、遺伝子マーカーの迅速な定量を行うために、それぞれのプライマーセットに対応するReal-timePCR法の反応条件を確立した。T-RFLP法およびReal-timePCR法による解析は、DNA抽出から定量まで約8時間で終了し、かつ培養法によるバイアスを排除できるため、より正確に複合微生物系内に存在する各種糞便性大腸菌および病原細菌の特定・定量および汚染源の特定・モニタリングが可能となると思われる。また、検出限界は極めて低く、高感度かつ特異的な糞便性大腸菌汚染の指標となることが明らかとなった。
日本学術振興会, 基盤研究(B), 北海道大学, 15360283

再生水造水とリン資源回収のためのハイブリッド下水処理システムの構築
科学研究費助成事業 基盤研究(A)
2002年 - 2004年
渡辺 義公, 岡部 聡, 木村 克輝
1)膜ファウリング機構の解明および抑制
実都市下水を用いた多系列の膜分離活性汚泥処理実験を行い、運転条件、前処理の有無が反応槽内汚泥の性状に及ぼす影響について検討した。反応槽内汚泥濃度の上昇に伴って槽内汚泥の粘度が上昇し、膜ろ過運転を困難にするが、特に汚泥濃度が10,000mg/Lを超過する際に粘度の上昇が顕著になる傾向が観察された。前凝集沈殿処理を行うことでこれらの粘度上昇は良好に抑制されうることを見いだした。
2)ハイブリッドMBRでは窒素除去率が低いことを改善するために、PVDF製のMF平膜を用いたMBR内に仕切り板を入れて、MBRの水位を変化させて槽内に好気部と無酸素部を同時に形成する新たなMBRを開発した。このMBRでは処理水の全窒素濃度が5mg/l程度となった。また、MBR内の生物相の解析によって、MBR内には糸状細菌であるChloroflexiが活性汚泥ばっき槽内と比較して極めて高い存在割合を占めていることを明らかにした。Chloroflexiは死滅細菌由来の有機物のスカベンジャーとして機能し、溶解性有機物の蓄積による膜ファウリングの抑制に寄与していることが示唆された。
3)処理水の再利用の観点から、Bacteroides属とPrevotella属を指標として、迅速かつ正確に人獣の糞便由来の病原性微生物の存在をチェックできる新たな分子生物学的手法を開発し、実河川の水質測定に適用した。その結果開発された手法によって、人、牛、豚の糞便汚染を区別できることが明らかになり、処理水の安全性の確認のみではなく、河川の糞便汚染の汚染源を特定できるようになった。
4)新規吸着剤であるジルコニアメゾ構造体(ZS)をリン酸吸着に適用した結果、ZSに含まれるSO_4^<2->とOH^-がリン酸イオンと陰イオン交換することで、リン酸を選択的に吸着する機構を明らかにした。その最大吸着能約3500μM/gZSであり、既存の吸着剤の1.7倍に達した。ZSに吸着されたリン酸はクエン酸溶液(pH=4.5)やNaOH溶液に浸すことで容易に溶出した。今後はZSの再生方法と高濃度・高純度に濃縮されたリン酸溶液からの硝析脱リンによるリン回収技術の確立を計画している。
日本学術振興会, 基盤研究(A), 北海道大学, 14205077

バイオ燃料電池の開発　　　　　　　　　　　　　　　
2004年
競争的資金

Development of microbial fuel cells　　　　　　　　　　　　　　　
2004年
競争的資金

世界の環境改善という視点から見た環境工学の未来とその発展のための政策提案
科学研究費助成事業 基盤研究(C)
2003年 - 2003年
福士 謙介, 飯田 俊彰, 岡部 聡, 大瀧 雅寛, 滝沢 智, 徳永 光晴
我が国の環境工学はこれまで一定の成果をあげてきた。そのおかげで、国民は衛生的かつ快適な生活を送り、世界の最長寿国として知られるようになった。また、生物が住めないような極度に汚染された河川・湖沼・海洋は日本中に存在しない。その一方で、目を東南アジア、南アジア、アフリカ、南アメリカなどに向けると、環境の崩壊は確実に進みつつある。
本研究は世界に目を向けた場合、開発が必要である環境工学の諸分野とそのタスクを明らかにすることを第一の目的とした。次に、それらの研究開発(技術移転を含む)を効率的に行い、内外の研究者が開発に専一に取り組むための政府や助成団体ならびの大学の研究助成体制のあり方や総合的援助システムを提言することを第二の目的とした。最終的には本研究理念を具現化するための研究グループを立ち上げる為の活動を具体的に行う提案をする事を活動の最終的な目的とした。
本研究活動として平成15年8月に北海道イトムカにおいて第1回ワークショップを合宿形式で開催した。約20名の若手研究者が集まり、次世代の環境工学について協議を行った。次に、タイ・バンコク市において外国の研究者を交えて第2回ワークショップを開催した。その結果平成16年度、文部科学省・科学技術振興調整費・我が国の国際的リーダーシップの確保のプログラムへ「アジアの持続的発展のための国際協議会企画」というプロジェクトの提案を行った。この提案課題は中堅〜若手の柔軟性のある研究者や実務者が参集し長時間にわたり、持続的な環境について協議する場を提案するもので、最終的にはアジアにおける、環境を中心テーマとした、いわゆるダボス会議(世界経済フォーラム年次総会)を目指す物である。
日本学術振興会, 基盤研究(C), 東京大学, 15636011

MAR-FISH法による硫酸塩還元細菌の特異的検出と有機物利用特性の同時解析
科学研究費助成事業
2001年 - 2002年
岡部 聡
下水中の全有機物酸化量の約50%は硫酸塩還元反応によって達成されているという報告もあり、硫酸塩還元反応は極めて重要な反応であるが、反応槽及び生物膜内部での硫黄循環の存在や硫酸塩還元細菌の多様な有機物代謝能のため、下水処理生物膜内の有機物分解経路に関与する硫酸塩還元細菌の役割およびその多様性に関する知見は皆無である。本研究では、放射性標識した有機物を用い、微生物細胞内における放射性物質(トレーサー)の取込みを観察するマイクロオートラジオグラフ法(MAR)と蛍光in situハイブリダイゼーション法(FISH)を組み合わせたMAR-FISH法を提案することにより、生物膜内における硫酸塩還元細菌の有機物利用特性(機能)および特異的検出・同定の評価を同時に行った。マイクロオートラジオグラフにおいて細胞レベルで有機物の取込みを検出できる感度を得るためには、前培養時間および本培養時間、基質濃度、トレーサーの濃度、バイオマス濃度、X線写真フィルムヘの感光時間等、全てのパラメーターが適切に設定されなければならない。そのため、予備実験としてホモジナイズした生物膜試料を対象とし、小型バイアル瓶を用いた系を設定し、これらパラメーターを最適化した。放射性物質(トレーサー)には[^3H]Acetate、[^<14>C]CO_2+H_2、[^<14>C]Lactate、[^<14>C]Formate、[^<14>C]Propionateを用いた。これらの培養実験を好気、無酸素(NO_<3^->のみ存在)、嫌気の三つの条件で行った。
その結果、本研究ではマイクロオートラジオグラフィー(MAR)と蛍光in situハイブリダイゼーション法(FISH)を組み合わせることにより、貧酸素環境にある都市下水生物膜内に存在する硫酸塩還元細菌の分子系統学的同定(生態学的構造)および有機物利用特性(機能)をシングルセルレベルの解像度で解析することに成功した。その結果、本生物膜内ではDesulfobulbusの約27%は硝酸塩を電子受容体としてプロピオン酸を酸化することが明らかとなった。更に、Desulfobulbusの約10%は酸素を電子受容体として利用することも可能であった。さらに、微小電極により生物膜内in situにおける正味の硫酸塩還元活性度分布を測定し、MAR-FISH法の結果と比較検討した。これらの実験結果を総合的に解析・評価することにより、実際の下水処理生物膜内においてどのグループの硫酸塩還元細菌が、どの有機物代謝経路に、どの程度関与しているかについて明らかにすることが可能であった。
日本学術振興会, 基盤研究(C), 北海道大学, 13650593

PCR-DGGE法による都市下水生物膜内の硝化細菌群の多様性評価
科学研究費助成事業
1999年 - 2000年
岡部 聡
本研究は、生物学的窒素除去プロセスを安定的に運転管理し、処理効率をより向上させることを目的として、16S rRNA遺伝子のクローニング解析とFISH法により都市下水生物膜内に存在する硝化細菌群の多様性を評価するとともに、in stiuでの優占種の動態解析を行うことを目的としたものである。16S rRNA遺伝子解析により都市下水中で培養された生物膜内ではNitrosomonas ureaeに近縁なクローン(相同性約96%)が、人工基質により培養された生物膜内では増殖速度の大きいNitrosomonas eutrophaに近縁な硝化細菌のクローン(約相同性96%)が高頻度で検出され、これら生物膜内のアンモニア酸化細菌の優占種と推定された。その他にも両生物膜より多種多様のアンモニア酸化細菌に近縁なクローンを検出することができた。さらにこの結果は人工基質によって培養されたことにより、優占種が変化したことを示唆しており、FISH法により生物膜形成過程におけるアンモニア酸化細菌の優占種の遷移について検討した。その結果、生物膜の形成とともにNso190とNsm156の両プローブではハイブリするがNEUではハイブリしないアンモニア酸化細菌(すなわちN.ureae)から、N.eutropha及びN.europaeaへのアンモニア酸化細菌の優占種が変化することを視覚的に示すことができた。この結果は16S rRNA遺伝子のクローニング解析の結果と一致するものであった。一方、亜硝酸酸化細菌に関しては生物膜内における存在比率が非常に小さく、本研究で用いた真正細菌に特異的なプライマーセットではその存在が確認できなかった。
日本学術振興会, 奨励研究(A), 北海道大学, 11750483

バイオフィルムの形成機構に関する研究　　　　　　　　　　　　　　　
2000年
競争的資金

トキシコゲノミクスを用いた化学物質の毒性評価手法の開発　　　　　　　　　　　　　　　
2000年
競争的資金

Study on biofilm development　　　　　　　　　　　　　　　
2000年
競争的資金

Evaluation of chemical toxicity by using toxicogenomics　　　　　　　　　　　　　　　
2000年
競争的資金

16SrRNA蛍光遺伝子プローグと微小電極を用いた生物膜の構造と機能の解析
科学研究費助成事業
1997年 - 1998年
岡部 聡
本研究の目的は、安定した生物学的窒素除去を行うために、(1)混合系生物膜内in situでの硝化細菌の空間的分布及びその動態を微生物種レベルで明らかにすること、(2)硝化細菌分布と硝化活性との関係を明らかにすることである。更にこれら生物膜内のマイクロスケールの情報と生物膜全体の処理能力(メソスケール)との関係を明らかにすることである。本研究の結果は以下のようである。
(1) アンモニア酸化細菌と亜硝酸酸化細菌に特異的な16S rRNA標的蛍光DNAプローブを用いたFluorescent in situ hybridization(FISH)法により、都市下水生物膜内における両硝化細菌の時間的・空間的分布の解析を行った。その結果、アンモニア酸化細菌は主に直径5-10μmの球状の密な集塊(クラスター)を形成して、生物膜中層及び底層部に多く存在することが明らかとなった。また亜硝酸酸化細菌に関しては、Nitrobacter属ではなくNitrospira属が優占種であり、これらの細菌はアンモニア酸化細菌クラスターの周辺に、比較的小さな集塊を形成して存在していた。このことは、アンモニア酸化細菌と亜硝酸酸化細菌の間で、効率の良いNO_2^-の授受が行われていることを示唆するものである。
(2) 生物膜内in situにおける硝化活性を測定するために、先端径が5-10μmのDO,NH_4^+,NO_3^-,NO_2^-及びpH測定用の微小電極を開発した。これら微小電極を用いて生物膜内のアンモニア及び亜硝酸酸化活性分布を測定することが可能となった。生物膜内の硝化細菌分布と硝化活性分布を対応させることにより、生物学的硝化反応のより詳細なメカニズムが明らかとなった。
日本学術振興会, 奨励研究(A), 北海道大学, 09750627

処理水再利用と汚泥中リンの有効利用のためのハイブリッド下水処理システム
科学研究費助成事業
1996年 - 1998年
渡辺 義公, 木村 克輝, 岡部 聡, 但野 利秋, 中埜渡 丈嘉
(1) 凝集・高速固液分離・生物酸化を組み合わせた高度下水処理システム
固液分離プロセスの更なる効率化のために、JMSの多孔板の孔の形状が固液分離性に及ぼす影響について解析して、円孔よりも縦スリット孔が有効であることを明らかにした。凝集剤としても、凝集沈殿汚泥からのリンの回収も考慮して、多用されているアルミニウム系に代わる新しい鉄系のPoly-Silicato-Iron(PSI)の有効性を検討し、凝集pHを6.5とすればAlと同等の添加濃度で同等の凝集効果を持つことを実証した。
(2) 生物膜の構造・機能解析による硝化機能の向上
下・廃水からの窒素除去を担う生物膜内の硝化細菌を対象として、16rRNAを標的とする蛍光遺伝子プローブを用いたFISH(Fluoresent In Situ Hybridization)法と共焦点レーザー走査型顕微鏡による画像解析を併用して、原水の有機物とアンモニア性窒素の濃度比(C/N)と生物膜形成時間が硝化細菌の種類と菌密度について研究した。アンモニア酸化細菌の優占種についての検討し、生物膜内には異なる生理特性を持つ様々なアンモニア酸化細菌が混在し、水質の変化に対応して菌相が入れ替わり、結果として生物膜のアンモニア酸化は安定して維持されることが明らかとなった。
(3) フミン質による膜ファウリング機構
フミン質と濁質が共存する高色度河川水で高度下水処理水に近い水質の千歳川をサンプルとして、回分膜ろ過実験によってUF膜のファウリング特性を明らかにした。高色度原水のUF膜ろ過では、フミン質が膜ファウリングの主原因物質である。膜ろ過で発現するファウリングに由来する抵抗を、(1)ケーキ層抵抗、(2)濃度分極層抵抗、(3)吸着抵抗、に分けてそれぞれを個別に定量化する方法を提案した。フミン質と濁質が共存すると高分子フミン質が大きな濁質粒子の間隙に入り込み、大きなケーキ層抵抗を発現する。膜細孔より大きい高分子フミン質によるケーキ層抵抗を、フミン質濃度の異なる濁質を含まない原水をろ過した場合の実験データを、Ruthのケーキろ過式を変形した式によって解析し、提案した膜ファウリング機構を検証した。
(4) 植物根分泌酸性ファスファターゼの遺伝子解析、機能評価
P処理を施したルーピンの根のRNAから、APaseをコードする2種類のcDNA,LASAP1およびLASAP2を単離した。LASAP1は細胞膜・壁に存在して根の表面に到達する有機態リン酸化合物を分解し、LASAP2は根から分泌されて有機態リン酸化合物を分解する機能をもつことが示された。
日本学術振興会, 基盤研究(B), 北海道大学, 08458160

マンガン自触媒反応と嫌気性膜洗浄を利用した膜処理の効率化
科学研究費助成事業
1996年 - 1997年
渡辺 義公, 鈴木 辰彦, 岡部 聡, 中埜渡 丈嘉
除濁と溶解性マンガン除去を主目的とした、マンガン自触媒汚泥循環式MF膜処理装置に関する研究を、パイロットプラント実験により行った。得られた結果は下記のとおりである。
(1) 生成した二酸化マンガンを含む自触媒汚泥は、粒径約3.3μmの粒子であった。本返送汚泥を塩素で活性化させた時の、溶解性マンガンの吸着等温式はフロントリッヒ型で近似された。
(2) 原水にアンモニアが含まれると、クロラミン生成の方が早く進行し、その後溶解性マンガンの酸化が起こると考えられる。よって、溶解性マンガンの酸化除去には、クロラミン生成に要する塩素量以上を添加する必要がある。モノクロラミン生成に必要な塩素要求量を、添加塩素濃度から差引いた塩素濃度が0.5mg/l以上では、90%以上の溶解性マンガンの除去が可能であった。
(3) 膜透過係数K_<20>はマンガン除去効率に影響された。マンガン除去効率が高い程、K_<20>は緩やかに減少した。
(4) 膜モジュールに付着した金属類を調べると、適切な塩素添加率で運転され、マンガン除去性が良好な時は、膜に直接付着しているマンガン量は少なかった。塩素添加率が不十分でマンガン除去性が低下した時は、膜付着のマンガン量は1オーダー高い値となった。よって、原水に対し適切な運転条件が保持できれば、自触媒汚泥の循環により、膜面でのマンガンの析出は防止できる。
日本学術振興会, 基盤研究(B), 北海道大学, 08555135

好気性生物膜内における硫黄(S)のサイクルに関する研究
科学研究費助成事業
1996年 - 1996年
岡部 聡
本研究では、反応槽におけるSの物質収支によってのみでは解明できない生物膜内の硫黄(S)の循環を明らかにするために、生物膜内の硫酸塩還元活性、硫黄酸化活性及び代謝産物である硫化化合物の濃度分布を測定した。研究の結果、以下のような知見を得た。
(1)硫酸塩還元細菌及び硫黄酸化細菌の細菌密度分布と存在的な活性度分布
硫酸塩還元細菌(SRB)は好気性生物膜表面部に最も多く存在し(5×10^5 MPN/cm^3程度)、膜深さ方向に減少した。硫黄酸化細菌も生物膜全体に一様に分布していた。硫酸塩還元活性は表層部ではほとんど検出されなかったが、表層部から徐々に増加し中層部(膜表面から300μm程度)で最大値(30μm/cm^3/h)を示した。膜表面でSRB菌体密度が高いにもかかわらず活性が低い原因は、表層部では溶存酸素(DO)濃度が高くSRB活性が抑制されているためと考えられる。膜深部でSRB活性は有機物律速のため再び低い値を示した。一方、硫黄脱窒細菌(SDB)活性は膜全体で一様に認められたが深部で最も高い値を示した。膜深部ではDO濃度が低くNO_3,H_2S濃度が比較的高く、SDBの増殖に有利であったと考えられる。
(2)生物膜内の硫化化合物(S^0,FeS及びFeS_2)の濃度分布の測定
生物膜厚が一定となった実験開始から30日目の生物膜内の硫化化合物(S^0,FeS及びFeS_2)の濃度分布を測定した。S^0はFeS及びFeS_2に比較して、膜全体に30μmol-S/cm^3程度蓄積していた。これは硫酸塩還元活性により生成されたH_2Sの生物化学的酸化に起因するものと思われる。FeSはSRB活性の高かった膜表面から250μm付近で著しいピークを示し、表層部及び深層部では検出されなかった。
以上の結果をまとめると、好気性生物膜内にも絶対嫌気性細菌であるSRBが存在すること、また測定された硫化化合物の濃度分布とSRB及びSDBの活性度分布との間には良い相関関係が見られたことから500μm程度の好気性生物膜内にの硫黄循環が存在することが明らかとなった。
日本学術振興会, 奨励研究(A), 北海道大学, 08750661

脱燐・脱窒を行う実下水処理プラントのモデル化と最適運転操作条件の検討
科学研究費助成事業
1995年 - 1996年
高桑 哲男, 岡部 聡, 船水 尚行
有機物除去を主目的として標準活性汚泥法のプロセスとして設計された処理場を大きな建設工事を行うことなく処理機能を向上させる一つの方法として、運転操作のみの変更で栄養塩除去を実現するステップ流入式嫌気好気活性汚泥法がある.本研究では,この方法を採用している実処理場の機能をシミュレートできる数学モデルを開発し,シミュレーションによって栄養塩除去と運転操作の関係を検討した.
処理場のモデルをActivated Sludge Model No. 2を利用して作成し,モデルの有用性を処理場の実測結果ならびに処理場から採取した汚泥,下水を用いた回分実験結果により確認した.流入下水中の有機物の分類結果を用いて,処理場の運転操作条件と処理特性の関係をシミュレーションにより検討した.シミュレーション結果は,ステップ流入量配分によって処理場の窒素除去率を向上させることは難しいことを示した.また,この原因が流入下水中有機物のうち容易に分解する割合が少なく,脱窒速度が遅いことが原因であることも示された.
既存処理場を対象に,運転操作のみの変更による処理の改善を目的としている本研究では,反応槽の増設以外の方法として,窒素除去率の改善の方策として脱窒槽に有機酸を添加し脱窒速度を早くする方法を検討した.回分実験結果とシミュレーションにより有機酸の添加が反応速度を上げ,窒素除去率が向上することを確認した.また,最初沈殿池汚泥の酸発酵による脱窒槽への有機物供給可能性を室内実験により検討した.
日本学術振興会, 基盤研究(C), 北海道大学, 07650632

混合系生物膜内の硝化菌と他栄養性細菌の競合に関する研究
科学研究費助成事業
1995年 - 1995年
岡部 聡
本年度は前処理なしで生物膜を膜深さ方向に10-50μm間隔でスライスできるマイクロスライサ-^を用いて次の3点、(1)細菌密度分布と生物膜の処理特性の関係、(2)流入基質C:N比の硝化菌密度分布に与える影響、(3)In Situでの細菌活性密度分布の測定、について検討した。その結果以下のような知見が得られた。
(1)細菌密度分布と生物膜の処理特性の関係 生物膜内の全硝化細菌数は同じであっても、膜深さ方向の密度分布が異なれば生物膜へのアンモニアfluxが異なる結果を得た。つまり硝化細菌が膜深部に優占的に存在する場合(例えば有機物負荷が高い場合、C:N=1.5)のアンモニアfluxは生物膜全体に一様に分布する場合(例えばC:N=0,0.25)に比べて約25%低い値を示した。この結果より生物膜の基質消費速度などの動力学的係数を正確に求めるためには、生物膜内の菌体量のみでなくその膜深さ方向の密度分布も測定しなければならないと考えられる。
(2)流入基質C:N比の硝化菌密度分布に与える影響 流入基質C:N=1.5の場合、硝化細菌は膜深部に優占的に存在し膜表面では深部の1/100-1/1000程度しか存在しておらず膜深さ方向に険しい密度勾配を形成した。一方、C:N=0,0.25の場合においては、C:N=1.5で見られたような膜深さ方向の密度勾配は存在せず、生物膜全体に一様に分布していた。この結果からC:N=1.5の場合には、硝化細菌と他栄養性細菌が溶存酸素と空間をめぐって競合し、増殖速度の遅い硝化細菌は生物膜表面では存在することができず膜深部のみに存在可能であることが明らかとなった。
(3)In Situでの細菌活性密度分布の測定 テトラゾリウム塩還元法を用いて生物膜内の細菌活性密度分布を測定した。生物膜表面では活性菌は全細菌数の35±13%であり、膜深さ方向に徐々に減少し膜深部では15±4%となった。故に生物膜内には多くの不活性細菌が存在することが明らかとなった。
日本学術振興会, 奨励研究(A), 北海道大学, 07750632

上向流のバイオフィルターによる都市下水処理に関する研究
科学研究費助成事業
1994年 - 1994年
岡部 聡
生物膜による濾過作用と吸着作用により下水中の懸濁性汚濁物を除去し,生物学的酸化作用により溶解性有機物とアンモニア性窒素の酸化を目的とする上向流バイオフィルターを用いて都市下水処理に関する研究を行い以下のような結果を得た。上向流バイオフィルターは装置下部より導入された空気が形成された生物膜によって抑留され空気層を形成するという効率的な曝気機構を持ったため、比較的少ない曝気量(曝気量:下水流量比,A/W=2)で高い槽内DO濃度を得ることができた.そのためBOD,SS,濁度の除去及び硝化率はHRT=5hr(水量負荷=86L/m^2・d)でそれぞれ80%以上と良好な処理水が得られた.フィルター枚数を増加させれば,抑留させる空気層が増加するとともに水量負荷が軽減され,有機物酸化が促進される.そのため,装置後段で高いDO濃度が維持され硝化は促進された.生物膜に抑留された懸濁性有機物は可溶化され生物学的に酸化されるため.余剰汚泥の発生量は少なく,フィルターの目詰まりもほとんど生じなかった.本装置はまだフィルター枚数を増加させることが可能であり,更に処理効率の向上が期待できる.槽内全体に各種細菌(硝化菌及び他栄養性細菌)が棲息しており,運転条件を適切に操作することにより脱窒も可能となることが示唆された.また本法は流入負荷変動に対して十分に対応可能であった.本研究で行った実験条件では,単一槽での最大窒素除去率は曝気位置を最下部に置き,処理水の返送比=1:3,曝気量=360mL/minで50%程度が得られた.上向流バイオフィルターを多段に連結するまたは他の下水処理プロセスと併用する事により,更に安定した処理効率が期待できると思われる.上向流バイオフィルターは構造上の利点より建設・運転費が安価であり維持管理も容易であるため,小規模下水処理システムや合併浄化槽への適用が今後期待できる.
日本学術振興会, 奨励研究(A), 北海道大学, 06750592

噴流撹拌固液分離槽と高効率回転生物膜接触・沈殿装置を組み合わせた下水処理システム
科学研究費助成事業
1992年 - 1993年
増田 純雄, 渡辺 義公, 岡部 聡, 渡辺 義公, 増田 純雄
昨年に引き続き宮崎市木花下水処理場に設置した処理能力100m^3/日のパイロットプラントによる実験的研究を行った。本年度は最初沈殿池を省いて,沈砂池流出水を直接噴流撹拌固液分離槽に流入させるフローにおけるシステムの処理効率を調べた。回転生物膜接触・沈殿装置(RBC)のカバーに太陽光発電パネル(表面積8m^2)を取付けそこでの発電電力によってRBCを稼働させる実験を行った。冬期間ではあったが晴天時には1日に8時間程度の稼働が可能であった。以下に,本年度に得られた成果を記す。
1)処理流量100m^3/日,接触体回転速度2rpm,Al添加量5mg/L,固液分離槽滞留時間45分の条件において処理水のSS濃度は5mg/L,BODは8mg/L程度であった。
2)固液分離槽に多孔板を挿入した場合としない場合の実験により,多孔板の通過噴流速度が10cm/s,間隔が20cmにおいて最大のSS除去率が得られることを確認した。
3)Al添加濃度が高くなる程固液分離槽でのリン除去率は高いが,Al添加濃度が2.5mg/Lと低い場合でも,微細な不溶解化リンがRBC生物膜に付着除去され65%程度のリン除去率が得られた。
4)固液分離槽でSSの90%近くが除去された。分離汚泥の濃縮性は良好で,脱水比抵抗も通常の最初沈殿池汚泥と同等の10^9S^2/gのオーダであった。
日本学術振興会, 試験研究(B), 宮崎大学, 04555136

生物膜の機能と生態学的構造に関する研究　　　　　　　　　　　　　　　
競争的資金

Biofilm structure and function　　　　　　　　　　　　　　　
競争的資金