厌氧氨氧化颗粒污泥的未培养和可培养细菌多样性的初步研究及新种的鉴定

【作者】 肖义平；

【导师】 全哲学；

【作者基本信息】 复旦大学 ， 微生物学， 2010， 硕士

【摘要】 随着工业化进程的推进,环境污染成为越来越严重的问题,我们必须采取一边污染一边治理的策略。在污水和固体垃圾废弃物填埋的渗滤液中,氮源是一个非常重要的污染源,而氨氮又是氮源里面最主要的污染物。在氨氮的众多生物处理工艺中,厌氧氨氧化处理工艺是最有前景的一种工艺,以不需要额外提供有机物,不需要动力消耗和产生的污泥量少等优点而吸引了众多科学家加入研究。我们选取了一个UASB反应器中厌氧氨氧化颗粒污泥,采用不依赖培养的分子生物学和分离培养两种方法对其中的细菌多样性进行初步研究。基于16S rRNA基因序列的不依赖培养的分子生物学方法研究结果表明,颗粒污泥中存在着较为丰富的微生物多样性,检测了29个克隆,一共有22个OTU(3% cutoff),分属于8个门中,约占RDP中有分类名称的27个细菌门的1/3。基于hzo基因序列的研究发现其中厌氧氨氧化细菌主要为2个类群：Candidatus Kuenenia和Candidatus Jettenia。颗粒污泥中存在大量与氮循环相关的细菌,包括固氮菌、氨氧化细菌,硝化细菌,厌氧氨氧化细菌和反硝化细菌。他们形成了一个完整的氮循环过程。通过分离培养方法,得到34株表型上有差异的细菌,根据16S rRNA基因序列的系统发育分析,这些菌株分别归属于6个属,3个门中。大部分细菌都属于反硝化细菌。其中芽孢杆菌在不依赖培养的细菌多样性研究中未发现,对不依赖培养的细菌多样性研究是一个有力的补充。在分离纯化培养的细菌中,发现有2株细菌HY-14T和HY-24与已知所有细菌标准菌株的16S rRNA基因序列相似度都在97%以下,对其进行了详细的鉴定。基于neighbour-joining和maximum-parsimony两种方法构建的系统发育树均显示HY-14T和HY-24两菌株在系统发育上被归类到假单胞菌属这一大簇中,与假单胞菌属的7个类群均不能聚类成簇而独立成簇。因为这2个菌株在基因型方面与已知假单胞菌属的细菌有较大的差异,造成了其生理生化特征、G+C含量和脂肪酸谱与已知的假单胞菌属的细菌有着较大的差异。基于上述分析,我们建议命名HY-14T和HY-24两菌株为假单胞菌属的一个新种,Pseudomonas caeni sp. nov. (HY-14T=KCTC 22292T=CCTCC AB 208156T)。更多还原

【Abstract】 Environmental contamination was tending to become worse in terms of the industrial development. Our strategy must be taken to control of the pollution while pollutants are produced. The nitrogen compounds were very important pollutant in the wastewater and landfill leachate, of which ammonia was the major pollutant. Anaerobic ammonia oxidation (Anammox) was a promising biological nitrogen removal process and had a lot of merits such as none of additional organics and aeration, and less sludge production. More and more scientists have joined in the research of anammox.The anammox granules sludge of a UASB (upflow anaerobic sludge blanket) reactor was selected to survey the characterization of bacterial communities using the culture-independent and-dependent methods. Culture-independent method in view of 16S rRNA gene phylogenetic analysis indicated that there were abundant bacterial diversity in the granules sludge. There were 22 OTUs within 29 clones, belonged to 8 phyla, which they accounted for one third of 27 phyla in bacterial taxonomy of RDP (3% cutoff). Culture-independent method in view of hzo gene phylogenetic analysis stated that there were two groups of anammox bacteria such as Candidatus Kuenenia and Candidatus Jettenia. The bacteria of nitrogen cycling such as nitrogen-fixing bacteria, ammonia oxidization bacteria, nitrite oxidization bacteria, anammox bacteria and denitrifying bacteria were distributed in the granules sludge, they formed a completed nitrogen cycling.Thirty-four distinct isolates were obtained from culture-dependent method. They were belonged to 6 genera of 3 phyla based on the phylogenetic analysis of 16S rRNA gene sequences. Most of them were belonged to denitrifying bacteria. The isolates of Bacillus were not detected in the culture-independent method. It illustrated that the culture-dependent method should be a supplement to the culture-independent method.Two strains HY-14T and HY-24, of which the similarity to all known typical bacteria were blow 97%, were selected to classify and identify. Phylogenetic trees based on the neighbour-joining and maximum-parsimony methods indicated that two strains formed a monophyletic clade within the genus Pseudomonas. For the large genic difference, two strains had the large different physiological and biochemical characteristics, G+C contents and fatty acid profiles with the type strains of the genus Pseudomonas. Strains HY-14T and HY-24 were classified as a novel species of the genus Pseudomonas, for which the name Pseudomonas caeni sp. nov. is proposed (HY-14T=KCTC 22292T=CCTCCAB 208156T).更多还原