Breakdown of hardly degradable carbohydrates (lignocellulose) in a two-stage anaerobic digestion plant is favored in the main fermenter.

The yield and productivity of biogas plants depend on the degradation performance of their microbiomes. The spatial separation of the anaerobic digestion (AD) process into a separate hydrolysis and a main fermenter should improve cultivation conditions of the microorganisms involved in the degradation of complex substrates like lignocellulosic biomass (LCB) and, thus, the performance of anaerobic digesters. However, relatively little is known about such two-stage processes. Here, we investigated the process performance of a two-stage agricultural AD over one year, focusing on chemical and technical process parameters and metagenome-centric metaproteomics. Technical and chemical parameters indicated stable operation of the main fermenter but varying conditions for the open hydrolysis fermenter. Matching this, the microbiome in the hydrolysis fermenter has a higher dynamic than in the main fermenter. Metaproteomics-based microbiome analysis revealed a partial separation between early and common steps in carbohydrate degradation and primary fermentation in the hydrolysis fermenter but complex carbohydrate degradation, secondary fermentation, and methanogenesis in the main fermenter. Detailed metagenomics and metaproteomics characterization of the single metagenome-assembled genomes showed that the species focus on specific substrate niches and do not utilize their full genetic potential to degrade, for example, LCB. Overall, it seems that a separation of AD in a hydrolysis and a main fermenter does not improve the cleavage of complex substrates but significantly improves the overall process performance. In contrast, the remaining methanogenic activity in the hydrolysis fermenter may cause methane losses.

Uncovering Microbiome Adaptations in a Full-Scale Biogas Plant: Insights from MAG-Centric Metagenomics and Metaproteomics.

The current focus on renewable energy in global policy highlights the importance of methane production from biomass through anaerobic digestion (AD). To improve biomass digestion while ensuring overall process stability, microbiome-based management strategies become more important. In this study, metagenomes and metaproteomes were used for metagenomically assembled genome (MAG)-centric analyses to investigate a full-scale biogas plant consisting of three differentially operated digesters. Microbial communities were analyzed regarding their taxonomic composition, functional potential, as well as functions expressed on the proteome level. Different abundances of genes and enzymes related to the biogas process could be mostly attributed to different process parameters. Individual MAGs exhibiting different abundances in the digesters were studied in detail, and their roles in the hydrolysis, acidogenesis and acetogenesis steps of anaerobic digestion could be assigned. Methanoculleus thermohydrogenotrophicum was an active hydrogenotrophic methanogen in all three digesters, whereas Methanothermobacter wolfeii was more prevalent at higher process temperatures. Further analysis focused on MAGs, which were abundant in all digesters, indicating their potential to ensure biogas process stability. The most prevalent MAG belonged to the class Limnochordia; this MAG was ubiquitous in all three digesters and exhibited activity in numerous pathways related to different steps of AD.

The Role of Petrimonas mucosa ING2-E5AT in Mesophilic Biogas Reactor Systems as Deduced from Multiomics Analyses.

Members of the genera Proteiniphilum and Petrimonas were speculated to represent indicators reflecting process instability within anaerobic digestion (AD) microbiomes. Therefore, Petrimonas mucosa ING2-E5AT was isolated from a biogas reactor sample and sequenced on the PacBio RSII and Illumina MiSeq sequencers. Phylogenetic classification positioned the strain ING2-E5AT in close proximity to Fermentimonas and Proteiniphilum species (family Dysgonomonadaceae). ING2-E5AT encodes a number of genes for glycosyl-hydrolyses (GH) which are organized in Polysaccharide Utilization Loci (PUL) comprising tandem susCD-like genes for a TonB-dependent outer-membrane transporter and a cell surface glycan-binding protein. Different GHs encoded in PUL are involved in pectin degradation, reflecting a pronounced specialization of the ING2-E5AT PUL systems regarding the decomposition of this polysaccharide. Genes encoding enzymes participating in amino acids fermentation were also identified. Fragment recruitments with the ING2-E5AT genome as a template and publicly available metagenomes of AD microbiomes revealed that Petrimonas species are present in 146 out of 257 datasets supporting their importance in AD microbiomes. Metatranscriptome analyses of AD microbiomes uncovered active sugar and amino acid fermentation pathways for Petrimonas species. Likewise, screening of metaproteome datasets demonstrated expression of the Petrimonas PUL-specific component SusC providing further evidence that PUL play a central role for the lifestyle of Petrimonas species.