Metagenome, metatranscriptome, and metaproteome approaches unraveled compositions and functional relationships of microbial communities residing in biogas plants

Abstract

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.

Keywords: Anaerobic digestion; Biogas microbiome; Biomass conversion; Genome-enabled metatranscriptomics; Integrated omics; Metagenome-assembled genomes; Methanogenesis; Methanogenic Archaea; Sewage digesters; Taxonomic profiling.

Fig. 1

Schematic overview on taxonomic profiling of biogas-producing microbial communities applying 16S rRNA gene amplicon sequencing. After extraction of whole community DNA, 16S rRNA gene amplicon libraries were constructed and subsequently sequenced. Obtained sequences were processed with the program QIIME (Caporaso et al., 2010) to calculate taxonomic community profiles

Fig. 2

Taxonomic profiling of microbial communities residing in exemplary mesophilic and thermophilic biogas plants fed with agricultural (by-) products based on 16S rRNA gene amplicon sequencing (Maus et al. 2016b). For amplicon processing, a pipeline including FLASH (Magoč and Salzberg 2011), UPARSE (Edgar 2013), Usearch 8.0 (Edgar 2010), and RDP classifier (Wang et al. 2007) was used as described recently by Maus et al. (2016b). Relative abundances of the most abundant classes and families of bacterial (left) and archaeal (right) communities were shown

Fig. 3

The predominant archaeal methanogenic genera as summarized by recent literature covering 78 mesophilic and thermophilic BGPs (as referenced in Supplemental Table S1). *Methanosarcina mainly occurred as single cells instead of typical aggregates (single-coccoid form, ≤ 1 μm)

Fig. 4

Workflow for functional profiling of microbial biogas communities exploiting metagenome sequence data. After sampling at biogas reactors, total DNA was extracted for construction of whole metagenome shotgun libraries which were subsequently sequenced on high-throughput sequencing platforms. Resulting sequencing data were quality checked and functionally characterized based on single read sequences in order to deduce functional profiles of the underlying biogas community. Moreover, metagenome assembly followed by a binning approach was applied to compile MAGs, which were then analyzed for their metabolic potential

Fig. 5

Metatranscriptome-based analyses of biogas-producing microbial communities. After sampling, whole community RNA was extracted followed by depletion of ribosomal RNAs. Metatranscriptome cDNA libraries were prepared and sequenced. Resulting metatranscriptome reads were mapped on corresponding metagenome data or MAGs. Finally, Transcripts per million (TPM) values were calculated for each gene to deduce transcriptional profiles of biogas microorganisms

Fig. 6

Metaproteomics workflow comprising sampling of the microbial biogas community, protein extraction, tryptic digestion of proteins, mass spectrometry of resulting peptides, and database searching using mass spectrometry data to identify proteins within the metaproteome analyzed