Quantitative Metaproteomics Highlight the Metabolic Contributions of Uncultured Phylotypes in a Thermophilic Anaerobic Digester

Abstract

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH 3 -N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter-affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum "Atribacteria." These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and ␤-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor.IMPORTANCE Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the ␤-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane.

Inhibition of anaerobic digestion processes: Applications of molecular tools

“…In contrast, high VFAs were shown to be non-inhibitory to methanogens in a full-scale reactor treating cow manure and food waste, even when propionate concentrations reached 8.7 g L −1 (Franke-Whittle et al, 2014). Regarding ammonia inhibition, studies have reported that acetoclastic methanogens are inhibited more severely than hydrogenotrophic methanogens (Hagen et al, 2017;Niu et al, 2013;Sun et al, 2016;Werner et al, 2014). For example, in one study methanogenesis was completely inhibited when total ammonia nitrogen (TAN) exceeded 9 g N L −1 with the critical threshold for performance decline reported to be 7 g N L −1 (Sun et al, 2016).…”

“…Combining long read SMRT sequence data with high-throughput and high-accuracy sequences from second generation platforms such as Illumina HiSeq can facilitate the construction of longer and more accurate metagenome assemblies (Frank et al, 2016). These hybrid assemblies have also been used to study low-abundance and difficult to sequence phylotypes in AD systems (Hagen et al, 2017), and this technique could allow researchers to study the role that these phylotypes play during inhibition.…”

“…One metaproteomic study on AD of cellulose identified more than 500 non-redundant protein functions (Lü et al, 2014). The only study to our knowledge that used metaproteomics under potentially inhibitory conditions (free ammonia of 367 mg NH 3 -N L −1 ), found that acetoclastic methanogens were present at low abundance and enzymes associated with Methanosaeta thermophile were detected (Hagen et al, 2017). However, the dominant mechanism of acetate removal was found to be through SAO by groups closely related to Thermacetogenium phaeum.…”

Integration of absolute multi-omics reveals translational and metabolic interplay in mixed-kingdom microbiomes

“…Metaproteomics Proteins were extracted from t1 to t8 in triplicate following a previously described method 44 with a few modifications. Briefly, 30ml of cultures containing cells and substrate were centrifuged at 500x g for 5 minutes to pellet the substrate.…”

Untitled

“…Recently, oftentimes multiple biological or technical samples of the same environment are sequenced to produce distinct genome copy numbers across samples, sometimes using different sequencing protocols and technologies, such as Illumina and PacBio sequencing (Hagen et al, 2016). Genome copies are reflected by corresponding read coverage variation in the assemblies which allows to resolve samples with many genomes.…”