厌氧氨氧化菌的富集培养与分子鉴定

采用传统培养方法与分子生物学方法相结合,进行了厌氧氨氧化菌的分离培养与分子鉴定.富集培养以2种不同反应器的厌氧氨氧化污泥作为分离源,以亚硝酸盐与铵盐为底物.经过2 a传代培养获得2个培养系,对亚硝酸盐及铵盐具有稳定的去除能力,氨氮去除率为85%左右.通过16S rRNA克隆文库方法对培养系中浮霉菌门微生物群落结构进行了多样性解析,结果表明2个培养系中的厌氧氨氧化菌是同一种微生物,代表序列比对结果与"Kuenenia stuttgartiensis"同源性为99%.采用"K.stuttgartiensis"的特异探针对培养系进行了荧光原位杂交分析(FISH),进一步证实"K.stuttgartiensis"是培养系中的优势厌氧氨氧化菌,占总菌群数的80%～90%.对反应器中"K.stuttgartiensis"的丰度变化进行了FISH跟踪检测,发现原始接种污泥中"K.stuttgartiensis"为主要厌氧氨氧化菌,经过2 a运行该菌在污泥中的丰度由11%提高到24%.

Enrichment and Identification of Anaerobic Ammonium Oxidation Bacteria

Anaerobic ammonium oxidation (ANAMMOX) bacteria were investigated by cultivation and molecular approaches. Primary enrichments were made with NH₄HCO₃ and NaNO₂ as substrates and two types of sludge from ANAMMOX reactors as inoculum. Two stable enrichment cultures were obtained after two years of successive transfers, and ammonium removal rate was 85%. The ANAMMOX bacteria in the enrichment cultures were identified by Planctomycetales-specific 16S rRNA gene-cloning analysis and fluorescence in situ hybridization (FISH). The clone analysis revealed that all the clonal sequences obtained from the two enrichment cultures were similar and closely related to Candidatus"Kuenenia stuttgartiensis" (99% similarities in 16S rRNA gene sequence). FISH with Planctomycetales-specific probe Pla46 and K. stuttgartiensis-specific probe Kst1275 revealed that the predominant ANAMMOX bacteria in the two enrichment cultures showed positive signals with the Pla46 and Kst1275 probes, respectively. To detect the variation in abundance of K. stuttgartiensis in the original inoculum reactor during two years of operation, FISH with Pla46 and Kst1275 probes was applied to the same sludge. The results showed that the abundance of K. stuttgartiensis in the sludge was increased from 11% to 24% within two years.