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The production of biogas by anaerobic digestion (AD) of agricultural residues, organic wastes, animal excrements, municipal sludge, and energy crops has a firm place in sustainable energy production and bio-economy strategies. Focusing on the microbial community involved in biomass conversion offers the opportunity to control and engineer the biogas process with the objective to optimize its efficiency. Taxonomic profiling of biogas producing communities by means of high-throughput 16S rRNA gene amplicon sequencing provided high-resolution insights into bacterial and archaeal structures of AD assemblages and their linkages to fed substrates and process parameters. Commonly, the bacterial phyla Firmicutes and Bacteroidetes appeared to dominate biogas communities in varying abundances depending on the apparent process conditions. Regarding the community of methanogenic Archaea, their diversity was mainly affected by the nature and composition of the substrates, availability of nutrients and ammonium/ammonia contents, but not by the temperature. It also appeared that a high proportion of 16S rRNA sequences can only be classified on higher taxonomic ranks indicating that many community members and their participation in AD within functional networks are still unknown. Although cultivation-based approaches to isolate microorganisms from biogas fermentation samples yielded hundreds of novel species and strains, this approach intrinsically is limited to the cultivable fraction of the community. To obtain genome sequence information of non-cultivable biogas community members, metagenome sequencing including assembly and binning strategies was highly valuable. Corresponding research has led to the compilation of hundreds of metagenome-assembled genomes (MAGs) frequently representing novel taxa whose metabolism and lifestyle could be reconstructed based on nucleotide sequence information. In contrast to metagenome analyses revealing the genetic potential of microbial communities, metatranscriptome sequencing provided insights into the metabolically active community. Taking advantage of genome sequence information, transcriptional activities were evaluated considering the microorganism's genetic background. Metaproteome studies uncovered enzyme profiles expressed by biogas community members. Enzymes involved in cellulose and hemicellulose decomposition and utilization of other complex biopolymers were identified. Future studies on biogas functional microbial networks will increasingly involve integrated multi-omics analyses evaluating metagenome, transcriptome, proteome, and metabolome datasets.

Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus , were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

Background: The production of biogas takes place under anaerobic conditions and involves microbial decomposition of organic matter. Most of the participating microbes are still unknown and non-cultivable. Accordingly, shotgun metagenome sequencing currently is the method of choice to obtain insights into community composition and the genetic repertoire., Findings: Here, we report on the deeply sequenced metagenome and metatranscriptome of a complex biogas-producing microbial community from an agricultural production-scale biogas plant. We assembled the metagenome and, as an example application, show that we reconstructed most genes involved in the methane metabolism, a key pathway involving methanogenesis performed by methanogenic Archaea. This result indicates that there is sufficient sequencing coverage for most downstream analyses., Conclusions: Sequenced at least one order of magnitude deeper than previous studies, our metagenome data will enable new insights into community composition and the genetic potential of important community members. Moreover, mapping of transcripts to reconstructed genome sequences will enable the identification of active metabolic pathways in target organisms.