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

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 165-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 165-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 165-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 165-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 a-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 165-rDNA metagenome sequence reads to 62 165-rDNA amplicon sequences thus enabling frequency of abundance estimations for 165-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 165-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 165-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. (C) 2009 Elsevier B.V. All rights reserved.