Bacterial diversity and detection of potentially pathogenic species in an agricultural biogas plant - new insights gained by high-throughput DNA sequencing of rRNA gene amplicons

Most environmental bacteria cannot be cultivated in the laboratory, but they can be detected and differentiated, independent of cultivation, by analyses of their 16S rRNA genes. DNA-sequences of 97 identity are grouped together as OTUs (operational taxonomic units), which correspond roughly the species-level. In this study, an agricultural biogas plant was analysed for the diversity of bacterial communities and the potential occurrence of bacterial pathogens during fermentation. The plant consisted of two subsequent fermentation chambers, and two independent lines, both receiving silage, and one, in addition, cattle manure. A total of 3.5 million 16S rRNA gene fragment sequences were analysed. Bioinformatic and phylogenetic analyses indicated that each chamber contained about 2150 OTUs, which could be assigned to 53 different bacterial classes. Clostridia (49-56% of all sequences), Bacilli (8-12%), and Bacteroidia (7-10%) were the most dominant. Only 5.1% of the OTUs responded to the presence of cattle manure. One dominant OTU (6.5-10.9%) belonged to the Streptococcus bovis/Streptococcus equinus complex. The 16S rRNA genes of other potential pathogens occurred only in very low abundances (< 0.01%), including Clostridium tetani, Clostridium perfringens, Clostridium botulinum, or Mycobacterium tuberculosis. None of these OTUs increased between the first and second fermentation chambers. The addition of cattle manure had no significant effect. Clearly 16S rRNA gene sequencing, as applied here, cannot conclusively demonstrate the presence of pathogens, but this approach allows a highly sensitive identification of potentially risky environmental samples which would be relevant for further diagnostic analyses.