两种微生物共存下的厌氧氨氧化及协同脱氮机理研究

Anaerobic ammonium-oxidizing (anammox) is the anaerobic oxidation of ammonium by nitrite or nitrate to yield N2, which is due to the anammox bacteria. These presss anammox bacteria are the last major addition to the nitrogen-cycle (N-cycle). They, however, appear to be present in virtually any anoxic place where fixed nitrogen (ammonium, nitrate, nitrite) is found. According to current estimates, about 50% of all nitrogen gas released into the atmosphere in marine nitrogen loss is made by these bacteria. Besides this, the microorganisms may be very well suited to be applied as an efficient, cost-effective, and environmental-friendly alternative to conventional wastewater treatment for the removal of nitrogen. So far, ten different anammox species which are later identified as a deep-branching planctomycete with a peculiar morphology have been enriched, but none of these are in pure culture. This number is only a modest reflection of a continuum of species that is suggested by 16S rRNA analyses of environmental samples.. In this study, anammox bacteria were enriched in an upflow anaerobic sludge bioreactor (UASB) using sludge samples obtained from several kinds of ecosystems. Many bacteria were isolated and purified from the activated sludge in the conventional method. From these purified bacteria, we found that single bacteria strain had no anammox activity, and also found there was high anammox activity when one strain of denitrifying bacteria and one stain of anammox bacteria were mixed together in the process of anammox reaction for the purified bacteria. We conclude that anaerobic ammonia oxidation reaction is likely to occur in the interaction of the two strains. Study on the morphological, physiological, and ecological characteristics of the two stains will be carried out. The anammox activity and denitrification will be studied by using 15N isotope pairing method, from which we can conclude how much N2 from the reaction contributed to the anammox bacteria. We can also conclude the anammox equation from cultivate the pure bacteria by using 15N isotope pairing method and 15N isotope tracer technique. From the intermediate, end product and enzyme of the anammox process, we can conclude the anammox mechanism. The study will provide theoretical support to the biological nitrogen removal of the waste water, and also to the ecological restoration of the eutrophication water.

厌氧氨氧化是在厌氧条件下以亚硝酸盐为电子受体氧化铵盐产生氮气的过程，是近十几年来发现的新型的废水生物脱氮技术。由于厌氧氨氧化的纯培养物至今未获得，因此其内在机制尚不够清晰。申请人利用UASB反应器，以高基质浓度成功富集了厌氧氨氧化菌。用传统的方法从富集物中分离纯化微生物，对每个纯培养的菌株进行厌氧氨氧化反应试验，发现了单一菌株不具有厌氧氨氧化活性，而一株反硝菌和一株厌氧氨氧化菌混合在一起具有厌氧氨氧化活性。由此推测厌氧氨氧化反应可能是在这两株菌的共同作用下发生。本项目拟在此基础上，研究这两株菌株的形态、生理生化和生态学特性。通过15N同位素配对和15N同位素示踪技术研究这两株菌的协同脱氮过程，推导厌氧氨氧化反应的方程式。对反应过程中间代谢产物、终产物、反应酶系的研究，阐明这两株菌的厌氧氨氧化协同脱氮机理。这些研究结果将为废水生物脱氮和富营养化水体生态修复提供理论基础。

厌氧氨氧化技术是一种新型的生物脱氮技术，具有高效低耗的显著特点。然而针对实际废水，无法由单一的厌氧氨氧化菌完成，必需至少由两种菌共同作用下完成。本项目在此基础上展开了两种微生物的协同脱氮研究。. 主要研究内容包括：（1）一株反硝化菌株苏云金芽孢杆菌（Bacillus thuringiensis）的厌氧氨氧化活性研究；（2）不同接种污泥下启动厌氧氨氧化反应及动力学特征；（3）不同基质浓度下启动厌氧氨氧化及研究微生物群落特征；（4）异养反硝化菌与厌氧氨氧化菌协同脱氮及微生物群落变化规律；（5）硫自养反硝化菌与厌氧氨氧化菌协同脱氮及功能微生物的研究；（6）亚硝化菌与厌氧氨氧化菌协同脱氮及处理垃圾渗滤液的研究。. 不同C/N对厌氧氨氧化与反硝化协同脱氮及微生物群落的变化有重要影响。当葡萄糖浓度为56.4 mg L-1（TOC/N为0.1）时，反应器的脱氮性能最佳。随着进水有机物浓度的增加，反应器中优势菌由Candidatus Kuenenia逐渐转变为Diaphorobacter；随着进水有机物浓度减小，优势菌短期转变为Hydrogenophaga。. N/S对DEAMOX反应器的同步脱氮除硫性能和微生物群落具有重要影响。当反应器进水N/S比为2.38时，NH4+-N去除率最大，为82.8%。反应器内的功能微生物主要有Thiobacillus和Candidatus Kuenenia；当NO3--N限制时，除了这两种菌外，还有Sulfurimonas。. 亚硝化菌与厌氧氨氧化菌协同脱氮处理垃圾渗滤液中试试验，试验前45 d在进水添加少量亚硝氮，可实现CANON反应器的快速启动。进水氮素负荷为1.63 kg N/m3/d，曝气率为8.8 m3/m3/h， 脱氮效率为81%。qPCR分析表明，AOB仅占总细菌的1%-3%，而AnAOB含量更低，还存在其它多种多样的微生物。. 本研究揭示了两种微生物协同脱氮规律和代谢机理，构建了自养/异养反硝化协同厌氧氨氧化工艺以及CANON工艺的启动和运行控制策略，探究了功能微生物变化规律。这些研究结果深化了自然界的碳氮硫的生物地球化学循环，为厌氧氨氧化工艺的选取和稳定运行提供理论和技术参考。