整合克隆文库16S-rDNA序列和通过454焦磷酸测序获得的宏基因组序列数据对沼气厂微生物群落进行系统发育特征分析。

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

作者信息

Kröber Magdalena, Bekel Thomas, Diaz Naryttza N, Goesmann Alexander, Jaenicke Sebastian, Krause Lutz, Miller Dimitri, Runte Kai J, Viehöver Prisca, Pühler Alfred, Schlüter Andreas

机构信息

Institut für Genomforschung und Systembiologie, Centrum für Biotechnology (CeBiTec), Universität Bielefeld, D-33615 Bielefeld, Germany.

出版信息

J Biotechnol. 2009 Jun 1;142(1):38-49. doi: 10.1016/j.jbiotec.2009.02.010. Epub 2009 Feb 27.

Abstract

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

摘要

采用综合方法,利用克隆文库序列以及通过454焦磷酸测序获得的宏基因组序列数据,分析了以玉米青贮饲料、绿黑麦和液体粪肥为原料的生产规模沼气厂发酵样品中微生物群落的系统发育结构。对细菌和古菌16S - rDNA扩增子文库中的109个克隆进行测序,结果显示,所获得的核苷酸序列与来自不同厌氧环境(包括消化器和生物反应器)的16S - rDNA数据库序列相似但并不相同。大多数细菌16S - rDNA序列可归属于厚壁菌门,其中梭菌纲最为丰富,也可归属于拟杆菌纲,而大多数古菌16S - rDNA序列则聚集在产甲烷菌布氏甲烷袋状菌附近。古菌文库的其他序列很可能代表了甲烷袋状菌属内迄今未鉴定的物种。mcrA克隆序列的系统发育分析也得出了类似结果。mcrA基因产物编码参与甲烷生成最后一步的甲基辅酶M还原酶的α亚基。应用严格设置的BLASTn分析将16S - rDNA宏基因组序列读数与62个16S - rDNA扩增子序列进行了比对,从而能够对16S - rDNA克隆文库序列的丰度频率进行估计。对宏基因组16S - rDNA读数进行核糖体数据库项目(RDP)分类分析,结果显示厚壁菌门、拟杆菌门和广古菌门以及梭菌目、拟杆菌目和甲烷微菌目丰度较高。此外,很大一部分16S - rDNA宏基因组读数无法归属于更低的分类等级,这表明在分析的沼气厂发酵样品中,许多微生物仍未分类或未知。

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