A unified compendium of prokaryotic and viral genomes from over 300 anaerobic digestion microbiomes.

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.

Decoding Microbial Responses to Ammonia Shock Loads in Biogas Reactors through Metagenomics and Metatranscriptomics.

The presence of elevated ammonia levels is widely recognized as a significant contributor to process inhibition in biogas production, posing a common challenge for biogas plant operators. The present study employed a combination of biochemical, genome-centric metagenomic and metatranscriptomic data to investigate the response of the biogas microbiome to two shock loads induced by single pulses of elevated ammonia concentrations (i.e., 1.5 g NH4+/LR and 5 g NH4+/LR). The analysis revealed a microbial community of high complexity consisting of 364 Metagenome Assembled Genomes (MAGs). The hydrogenotrophic pathway was the primary route for methane production during the entire experiment, confirming its efficiency even at high ammonia concentrations. Additionally, metatranscriptomic analysis uncovered a metabolic shift in the methanogens Methanothrix sp. MA6 and Methanosarcina flavescens MX5, which switched their metabolism from the acetoclastic to the CO2 reduction route during the second shock. Furthermore, multiple genes associated with mechanisms for maintaining osmotic balance in the cell were upregulated, emphasizing the critical role of osmoprotection in the rapid response to the presence of ammonia. Finally, this study offers insights into the transcriptional response of an anaerobic digestion community, specifically focusing on the mechanisms involved in recovering from ammonia-induced stress.

Adaptation of Anaerobic Digestion Microbial Communities to High Ammonium Levels: Insights from Strain-Resolved Metagenomics.

Ammonia release from proteinaceous feedstocks represents the main inhibitor of the anaerobic digestion (AD) process, which can result in a decreased biomethane yield or even complete failure of the process. The present study focused on the adaptation of mesophilic AD communities to a stepwise increase in the concentration of ammonium chloride in synthetic medium with casein used as the carbon source. An adaptation process occurring over more than 20 months allowed batch reactors to reach up to 20 g of NH4+ N/L without collapsing in acidification nor ceasing methane production. To decipher the microbial dynamics occurring during the adaptation and determine the genes mostly exposed to selective pressure, a combination of biochemical and metagenomics analyses was performed, reconstructing the strains of key species and tracking them over time. Subsequently, the adaptive metabolic mechanisms were delineated by following the single nucleotide variants (SNVs) characterizing the strains and prioritizing the associated genes according to their function. An in-depth exploration of the archaeon Methanoculleus bourgensis vb3066 and the putative syntrophic acetate-oxidizing bacteria Acetomicrobium sp. ma133 identified positively selected SNVs on genes involved in stress adaptation. The intraspecies diversity with multiple coexisting strains in a temporal succession pattern allows us to detect the presence of an additional level of diversity within the microbial community beyond the species level.