成果報告書詳細
管理番号20110000000686
タイトル*平成22年度中間年報 微生物機能を活用した環境調和型製造基盤技術開発/微生物群のデザイン化による高効率型環境バイオ処理技術開発/嫌気性アンモニア酸化(ANAMMOX)プロセスを軸とした高効率窒素除去システムの開発
公開日2011/6/7
報告書年度2010 - 2010
委託先名国立大学法人北海道大学大学院工学研究院
プロジェクト番号P07024
部署名バイオテクノロジー・医療技術部
和文要約和文要約等以下本編抜粋:1 研究開発の内容及び成果等 本研究では、高濃度アンモニア性空素含有廃水(例えば、メタン発酵消化液など)を高効率、低コスト、省エネルギーで処理することが可能である部分硝化~嫌気性アンモニア酸化(anammox)プロセスを開発する。バイオデザイン化技術の1つとして、アンモニア性窒素の亜硝酸までの部分硝化およびanammox細菌の増殖促進を図るために微量のヒドロキシルアミン(NH20H)を添加する。 NH20H添カロ法の部分硝化およびanammox反応に及ぼす有効性は、各種分子生物学的手法および各種マイクロセンサーを用いてバイオフィルム内in situの微生物群集構造と機能を随時解析することで評価する(バイオモニタリング技術)。このプロセスの開発導入により、高効率・省エネルギー、低コスト型の窒素除去システムを構築する。また、anammox細菌は培養が困難であるうえ増殖速度がきわめて遅いので、anammoxリアクターの最適化を行うためには長期間を要する。そこで、メタゲノム解析を実施して、ゲノム情報よリアンモニアの酸化経路や炭素固定(同化反応)経路を解析し、増殖に係わる因子(コファクターやその他の基質など)を検討し、増殖の促進、安定化を検討する。
英文要約Title: Development of a high-performance nitrogen removal process based on anaerobic ammonium oxidation (Anammox) for treatment of wastewaters generated from methanogenic treatment processes (FY2007-FY2011) FY2010 Annual Report Anaerobic ammonium oxidation (Anammox) is a microbiological oxidation of ammonium with nitrite as the electron acceptor and produces dinitrogen gas as the main product. The anammox process is a new and promising alternative to conventional nitrogen removal processes. Since Anammox bacteria require nitrite as the slsctron acceptor, ammonium in wastewater has to be oxidized to nitrite in a preceding reactor. However, nitrite can be easily oxidized to nitrate in an oxic reactor. Therefore, inhibition of nitrite oxidation process(i.e.,partial nitrification (PN) process) is needed. In this study, we operated a granular PN reactor to enhance the pertormance and stability of PN process. This granular PN reactor could be operated at the nitrogen loading rate of 1.25-2.5kgNm3day^-1 under ambient temperature (8-20degrees). In order to examine the better operational condition of anammox processes, the influence of cell density of anammox bacteria was investigated. The production rate of 29N2 gas, specific nitrogen species produced through anammox reaction, was determined at the different cell density (1.3x10^8~10^10 cells/ml^-1). In addition, the presence of acyl-homoserine lactones (AHL) in the culture was examined by a reporter assay system. The production of 29N2 gas was confirmed at the cell density above 10^9 cells/ml^-1. 29N2 gas production rates were 53 and 51 amol/cells^-1/h^-1 at 1.3x10^10 and 10^9 cells/ml^-1, respectively although it was only 0.26 amol/cells^-1/h^-1 at 1.3x10^8 cells/ml^-1. This outcome clearly shows that the occurrence of cell-density dependent anammox activity and its threshold was 10^9 cells/ml^-1. The production of AHL by anammox bacteria was examined by the reporter assay system. Chromobacterium violaceum VIR24 gave a positive signal to the extracts of reactor effluents while Chromobacterium violaceum CV026 didn’t give a positive signal. Based on the outcome, the produced AHL should be long-chain group (C6-14) and the concentration in the effluent was estimated in the range of 1-10 nM. To our knowledge, this is the first report that shows the production of AHL by anammox bacteria. Metagenomic analysis was performed to anammox bacteria, Candidatus ‘Brocadia sinica' and their metabolic versatility were elucidated. An up-flow column reactor (60 mL) was operated with inorganic nutrient medium at 38°C and a hydraulic retention time of 0.2 h. Nucleic acids were extracted by DNA Blood Tissue kit (QIAGEN, Tokyo, Japan) and subjected to pyrosequencing analysis with GS FLX (Roche, BSU, Switzerland). In the metagenomic analysis, the authors obtained 1,204,328 sequences with an average read length of 336 bps and then assembled into fourteen contigs and one scaffold. Total size of fifteen fragments was 4.00 Mbps encoding 5,139 predicted proteins. The completeness of B. sinica genome was estimated to be above 97% based on presence/absence of core gene sets. The obtained genomic information enabled us to estimate the metabolic versatility in the cells of B. sinica and to elucidate genetic differences with another anammox bacterium, Candidatus ‘Kuenenia stuttgartiensis'.
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