KEMET - A python tool for KEGG Module evaluation and microbial genome annotation expansion.

Background: The rapid accumulation of sequencing data from metagenomic studies is enabling the generation of huge collections of microbial genomes, with new challenges for mapping their functional potential. In particular, metagenome-assembled genomes are typically incomplete and harbor partial gene sequences that can limit their annotation from traditional tools. New scalable solutions are thus needed to facilitate the evaluation of functional potential in microbial genomes. Methods: To resolve annotation gaps in microbial genomes, we developed KEMET, an open-source Python library devised for the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) functional units. KEMET focuses on the in-depth analysis of metabolic reaction networks to identify missing orthologs through hidden Markov model profiles. Results: We evaluate the potential of KEMET for expanding functional annotations by simulating the effect of assembly issues on real gene sequences and showing that our approach can identify missing KEGG orthologs. Additionally, we show that recovered gene annotations can sensibly increase the quality of draft genome-scale metabolic models obtained from metagenome-assembled genomes, in some cases reaching the accuracy of models generated from complete genomes. Conclusions: KEMET therefore allows expanding genome annotations by targeted searches for orthologous sequences, enabling a better qualitative and quantitative assessment of metabolic capabilities in novel microbial organisms.

In-situ biogas upgrading assisted by bioaugmentation with hydrogenotrophic methanogens during mesophilic and thermophilic co-digestion.

In this study, the effects of bioaugmentation of typically dominant hydrogenotrophic methanogens to CSTR co-digesting cheese whey and manure, under in-situ biomethanation operations were investigated. Reactors working at mesophilic (37 °C) and thermophilic (55 °C) conditions were independently treated and examined in terms of microbial composition and process dynamics. Addition of Methanoculleus bourgensis in the mesophilic reactor led to a stable biomethanation, and an improved microbial metabolism, resulting in 11% increase in CH4 production rate. 16S rRNA and biochemical analyses revealed an enrichment in syntrophic and acidogenic species abundance. Moreover, nearly total volatile fatty acids conversion was observed. Differently, Methanothermobacter thermautotrophicus addition in the thermophilic reactor did not promote biogas upgrading performance due to incomplete H2 conversion and inefficient community adaptation to H2 excess, ultimately favoring acetoclastic methanogenesis. Bioaugmentation constitutes a viable tool to strengthen in-situ upgrading processes and paves the way to the development of more sophisticated and robust microbial inoculants.

Microbial dynamics in biogas digesters treating lipid-rich substrates via genome-centric metagenomics.

Co-digestion with lipid-rich substrates is a likely strategy in biogas plants, due to their high energy content. However, the process stability is vulnerable to inhibition due to the sudden increase of fatty-acid concentration. Therefore, techniques that promote the adaptation of the microorganisms to the presence of lipids have been proposed. In this frame, the initial hypothesis of the work was that a gradual change in feedstock composition would enable us to elucidate the microbial organisation as a result of deterministic (i.e. chemical composition of influent) and stochastic (e.g. interspecies interactions) factors. This study investigates the response of the biogas microbiome to gradual increment of the Organic Loading Rate by supplementing the influent feedstock with Na-Oleate. The results showed that as a response to the feedstock shifts three clusters describing microbes behaviours were formed. The dynamics and the functional role of the formed microbial clusters were unveiled, providing explanations for their abundance and behavior. Process monitoring indicated that the reactors responded immediately to lipid supplementation and they managed to stabilize their performance in a short period of time. The dominance of Candidatus Methanoculleus thermohydrogenotrophicum in the biogas reactors fed exclusively with cattle manure indicated that the predominant methanogenic pathway was hydrogenotrophic. Additionally, the abundance of this methanogen was further enhanced upon lipid supplementation and its growth was supported by syntrophic bacteria capable to metabolize fatty acids. However, with the shift back to the original feedstock (i.e. solely cattle manure), the microbial dynamicity significantly altered with a remarkable increment in the abundance of a propionate degrader affiliated to the order of Bacteroidales, which became the predominant microorganism of the consortium.