Metagenomic analysis and functional characterization of an industrial-scale biogas plant by high throughput sequencing

Energy crisis and environmental pollution have led to an increasing interest in renewable energies. Biogas production from plant material, agricultural residual products and food wastes represents one of the most economically attractive alternative technology for biofuel production. Complex consortia of microorganisms are responsible for biomass degradation and biogas production involving several stages. Next-generation high-throughput sequencing provides a powerful tool for dissecting microbial community structure and methane-producing pathways in anaerobic digestion. A taxonomic and functional metagenomic analysis of microbial community residing in an industrial-scale biogas fermenter has been carried out at different steps of biogas production. Sample were collected from an industrial-scale mesophilic plant consisting of a three steps production taking place in a bioreactor, post-reactor and a storage tank. Total DNA was extracted from samples belonging to each steps of biogas production. Metagenomic analysis was carried out by using 16S and shotgun sequencing approach. The 16S datasets were generated by sequencing the bacterial and archaeal V4 hypervariable region. Reads from 16S sequencing were aligned against SILVA ribosomal RNA sequence database by using MALT(1), while shotgun reads were aligned against NCBI-nr protein database by using DIAMOND(2). Taxonomic binning and functional annotation were performed with MEGAN 6 software(3). About 2.9 and 11.5 million high quality reads were generated on the Ion Torrent S5 Sequencing System for 16S and shotgun approach, respectively. Metagenomic analysis revealed that the overall taxa distribution resulting from both sequencing strategies was conserved. In details, the superkingdom of Bacteria was dominant (~93%) along the production steps, whereas Archaea were less represented (~4%). Within the superkingdom of Archaea, only microorganisms belonging to the phylum of Euryarchaeota were detected. Within the key microorganisms involved in methanogenesis, data showed that during biogas production steps the abundance of Methanosarcina genus decreased from bioreactor to storage tank, with a simultaneous increase of Methanoculleus genus. Considering the key methanogenesis pathways, functional analysis supported a shift from acetotrophic methanogens to hydrogenotrophic methanogens. Results showed that the combination of both 16S and shotgun sequencing approach successfully addressed the taxonomical and functional analysis of microbial community, revealing new insights in microbial and functional dynamics during biogas production steps.