循环水养殖系统生物滤池细菌群落的PCR-DGGE分析

PCR-DGGE analysis of bacterial communities in bio-filtors of re-circulating mariculture system

通过模拟实验对循环水养殖系统中不同初始NH4+-N浓度的生物滤池中生物膜上和水中的细菌数量及群落种类组成进行了研究。对成熟生物膜及水体样品中的异养菌、氨氧化菌、亚硝酸盐氧化菌的培养计数结果表明,随着生物滤池初始氨氮浓度增大,除异养细菌数量逐渐下降外,生物膜上的氨氧化菌和亚硝酸盐氧化菌数量呈逐渐增加趋势,且均高出水样3～4个数量级;同时对上述样品的16S rRNA基因片段的PCR扩增产物进行变性梯度凝胶电泳(DGGE)分析及其序列同源性分析的结果表明,生物膜和水中都有较高的细菌多样性,同一初始氨氮浓度的滤池中生物膜上的细菌多样性高于水中的。生物滤池中的细菌主要由拟杆菌门的黄杆菌纲和变形菌门的α-、β-、γ-变形菌纲的15种细菌组成。生物膜上的优势菌包括奥雷氏菌属、湖饲养者菌属、泥滩杆菌属、沉积杆菌属、雷辛格氏菌属、冷蛇形菌属和亚硝化单胞菌属等;水体中的优势菌则有明显差异,主要有蛋黄色杆菌属、Nautella,玫瑰杆菌属和一种硫氧化菌等。初始氨氮越高的滤池中,亚硝化单胞菌属的细菌在生物膜上所占比例越高,逐渐成为优势菌之一。实验证实,挂膜初期,提高水体中初始氨氮浓度,有利于硝化细菌的富集和固着,提高生物滤池的除氮效率。

The numbers and composition of bacterial communities in bio-filters of re-circulating mariculture system were studied.The numbers of total heterotrophic bacteria,ammonium-oxidizing bacteria and nitrate-oxidizing bacteria on the matured bio-film samples from the 4 bio-filters with different initial ammonia-nitrogen concentrations（1#,2#,3#,4#）and from the water sample（5#）of 3# bio-filter were counted.The results showed that,the numbers of ammonia-oxidizing bacteria and nitrate-oxidizing bacteria on the bio-films increased and the number of heterotrophic bacteria declined gradually with the increase of initial ammonia-nitrogen concentrations in bio-filters.The numbers of bacteria on bio-film were higher by 3-4 orders of magnitude than those of the water sample in the same bio-filter.The amplifying products of the fragments of 16S rRNA gene from above samples were analyzed with denaturing gradient electrophoresis（DGGE）,and the sequences of DGGE bands were phylogenetic analyzed.The abundant bacterial diversity was found in bio-filtors of re-circulating systems,and higher on biofilm than in water in the same bio-filtor.The bacteria communities in bio-film and water samples were mainly made up with two bacterial groups：Flavobacteria,and α-,β-,γ-Proteobacteria,including 15 genera.The predominant bacteria on bio-films were Olleya,Lacinutrix,Gaetbulibacter,Sediminibacter,Leisingera,Psychroserpens and Nitrosomonas.While those of water were significantly different,including Vitellibacter,Nautella,Roseobacter,and a sulfur-oxidizing bacterium.Nitrosomonas became predominant bacteria on bio-films gradually with the increase of initial ammonia-nitrogen concentrations.The results suggested that,during the maturation of bio-film,increasing the initial ammonia-nitrogen concentrations is beneficial to the gathering of nitrifying bacteria and the improvement of nitrogen removing efficiency.