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Genetic heterogeneity is a common trait in microbial populations, caused by de novo mutations and changes in variant frequencies over time. Microbes can thus differ genetically within the same species and acquire different phenotypes. For instance, performance and stability of anaerobic reactors are linked to the composition of the microbiome involved in the digestion process and to the environmental parameters imposing selective pressure on the metagenome, shaping its evolution. Changes at the strain level have the potential to determine variations in microbial functions, and their characterization could provide new insight into ecological and evolutionary processes driving anaerobic digestion. In this work, single nucleotide variant dynamics were studied in two time-course biogas upgrading experiments, testing alternative carbon sources and the response to exogenous hydrogen addition. A cumulative total of 76,229 and 64,289 high-confidence single nucleotide variants were discerned in the experiments related to carbon substrate availability and hydrogen addition, respectively. By combining complementary bioinformatic approaches, the study reconstructed the precise strain count—two for both hydrogenotrophic archaea—and tracked their abundance over time, while also characterizing tens of genes under strong selection. Results in the dominant archaea revealed the presence of nearly 100 variants within genes encoding enzymes involved in hydrogenotrophic methanogenesis. In the bacterial counterparts, 119 mutations were identified across 23 genes associated with the Wood-Ljungdahl pathway, suggesting a possible impact on the syntrophic acetate-oxidation process. Strain replacement events took place in both experiments, confirming the trends suggested by the variants trajectories and providing a comprehensive understanding of the biogas upgrading microbiome at the strain level. Overall, this resolution level allowed us to reveal fine-scale evolutionary mechanisms, functional dynamics, and strain-level metabolic variation that could contribute to the selection of key species actively involved in the carbon dioxide fixation process. [Display omitted] • This is the first use of strain-resolved metagenomics in in-situ biogas upgrading. • A total of 97 high-quality metagenome assembled genomes were reconstructed. • The variants calling approach retrieved 140,518 variants in high abundance genomes. • Two strains were found in M. wolfeii and M. thermophilus at distinct time points. • Eight hydrogenotrophic methanogenesis genes were under selective pressure. [ABSTRACT FROM AUTHOR]

Background: The anaerobic digestion process degrades organic matter into simpler compounds and occurs in strictly anaerobic and microaerophilic environments. The process is carried out by a diverse community of microorganisms where each species has a unique role and it has relevant biotechnological applications since it is used for biogas production. Some aspects of the microbiome, including its interaction with phages, remains still unclear: a better comprehension of the community composition and role of each species is crucial for a cured understanding of the carbon cycle in anaerobic systems and improving biogas production. Results: The primary objective of this study was to expand our understanding on the anaerobic digestion microbiome by jointly analyzing its prokaryotic and viral components. By integrating 192 additional datasets into a previous metagenomic database, the binning process generated 11,831 metagenome-assembled genomes from 314 metagenome samples published between 2014 and 2022, belonging to 4,568 non-redundant species based on ANI calculation and quality verification. CRISPR analysis on these genomes identified 76 archaeal genomes with active phage interactions. Moreover, single-nucleotide variants further pointed to archaea as the most critical members of the community. Among the MAGs, two methanogenic archaea, Methanothrix sp. 43zhSC_152 and Methanoculleus sp. 52maCN_3230, had the highest number of SNVs, with the latter having almost double the density of most other MAGs. Conclusions: This study offers a more comprehensive understanding of microbial community structures that thrive at different temperatures. The findings revealed that the fraction of archaeal species characterized at the genome level and reported in public databases is higher than that of bacteria, although still quite limited. The identification of shared spacers between phages and microbes implies a history of phage-bacterial interactions, and specifically lysogenic infections. A significant number of SNVs were identified, primarily comprising synonymous and nonsynonymous variants. Together, the findings indicate that methanogenic archaea are subject to intense selective pressure and suggest that genomic variants play a critical role in the anaerobic digestion process. Overall, this study provides a more balanced and diverse representation of the anaerobic digestion microbiota in terms of geographic location, temperature range and feedstock utilization. [ABSTRACT FROM AUTHOR]