Aug 2026· Journal of Applied Microbiology· 0 citations
Medicine
TL;DR
Commonly used mcrA primer sets differ substantially in their ability to capture methanogenic diversity, with some showing broad representation of reactor-associated methanogens and others exhibiting strong lineage-specific biases.
Abstract
Aims
Methanogenic archaea are key players in anaerobic digestion, driving methane production in biogas reactors. This study aimed to assess the diversity of methanogenic archaea in full-scale anaerobic digesters using genome-resolved metagenomics and to systematically evaluate the taxonomic coverage of commonly used mcrA-targeted qPCR primer sets against this genomic framework.
Methods
AND
Results
Methanogenic diversity was assessed using 113 dereplicated archaeal metagenome-assembled genomes (MAGs) recovered from 109 full-scale anaerobic digesters treating diverse substrates. Genome-resolved analyses revealed a diverse archaeal community spanning multiple phyla, dominated by Halobacteriota and Methanobacteriota, with additional representatives from Methanobacteriota_B, Thermoplasmatota, and Thermoproteota. The presence of the mcrA gene was identified in a subset 55 MAGs, which were subsequently used as the genomic framework to evaluate six commonly used mcrA qPCR primer sets in silico. This subset clustered into nine phylogenetic groups and formed the basis for the primer coverage analysis. The evaluation revealed marked differences in taxonomic coverage among primer sets. Most primers preferentially detected Methanobacteriales and Methanosarcinales, while underrepresenting or excluding other methanogenic lineages, including H₂-dependent methylotrophic Methanomassiliicoccaceae.
Conclusions
Commonly used mcrA primer sets differ substantially in their ability to capture methanogenic diversity, with some showing broad representation of reactor-associated methanogens and others exhibiting strong lineage-specific biases. Genome-resolved metagenomics provides an effective framework for benchmarking primer performance and supports the selection and improvement of molecular tools for more accurate monitoring of anaerobic digestion systems.
The El Tatio geothermal field is identified as a rich source of putative hydrolase-encoding genes for future biochemical and biotechnological exploration and expands current knowledge of microbial diversity and functional potential in high-altitude geothermal ecosystems.
Bernardita Valenzuela, Ignacio Navarrete-Diaz, Mayra Cayo et al.· International Journal of Mol...· 0 citations
The methyl-coenzyme M reductase subunit alpha gene (mcrA) is an important phylogenetic marker for high throughput ecological profiling of methanogenic archaea, central to industrial biological methane production and greenhouse gas emissions. Yet, dedicated reference databases predate current relevant NCBI sequence accumulation and archaeal taxonomic revision. We present three updated mcrA reference databases: (i) one derived from NCBI-catalogued methanogen genomes (1572 sequences); (ii) a database built by expansion of a previously published reference dataset, leveraging the NCBI nucleotide collection (27,942 sequences); (iii) a curated-taxonomy version of the latter. The updated amplicon databases provide a ∼ 3.5-fold sequence richness expansion, extend genus-level richness from 31 to 83 taxa, more than 4-fold species-level richness, and incorporate novel lineages compared with the previous reference dataset (e.g. Thermoplasmatota-encompassed). All databases were formatted to support analysis with relevant contemporary software pipelines and packages. Overall, the generated databases facilitate a highly improved characterization of methanogen diversity and ecology.
S. Vasileiadis, Marios I Valmas, Dimitra S. Pitsikoglou et al.· Systematic and Applied Micro...· 0 citations
Viruses play a major role in regulating microbial community composition and activity, yet their diversity and host associations in anaerobic digestion (AD)—a complex biotechnological process converting organic waste into biogas—remain poorly characterized. Linking viral sequences to metabolically active hosts in such complex communities is particularly challenging. Here, we applied an integrative approach combining
13
C-stable isotope probing (SIP) with shotgun sequencing of viromes and microbiomes to identify and characterize viruses infecting the active formatotrophic guilds in batch AD microcosms.
Microcosms fed with
13
C-labeled formate as sole carbon source selectively enriched two primary formate-consuming guilds: hydrogenotrophic methanogens (Methanobacteriales, predominantly
Methanobacterium subterraneum
) and acetogenic bacteria (family Natronincolaceae, genus
Andreesenella
). From cross-assembly of six viromes on the one hand, and six
13
C-SIP microbiome fractions on the other, we assembled a catalogue of 2,368 vOTUs, of which 261 were selected for detailed analysis. The striking enrichment of vOTUs associated with methanogenic archaeal hosts, specifically in the heavy (
13
C-enriched) SIP fractions, contrasted with their near-absence in total viromes—demonstrates that DNA-SIP successfully resolved viruses of active formatotrophs at the viral community level. Novel viruses were identified for both primary guilds, including archaeal viruses of Methanobacteriales (family Anaerodiviridae and a possible novel family) and two
Andreesenella
viruses carrying diversity-generating retroelements. A provirus predicted to infect
Methanothrix
—a strictly acetoclastic methanogen—further illustrates the capacity of SIP-viromics to capture viral associations across trophic levels. Analysis of auxiliary viral genes (AVGs) revealed numerous defense-associated genes (
dcm
,
metK
,
queC
) and genuine metabolic AVG candidates, such as
cysH
(assimilatory sulfate reduction) and, a contiguous cluster including
uxe, galU
and
serB
(surface polysaccharide biosynthesis), which may influence on host metabolism.
This study demonstrates the power of SIP-viromics for resolving virus-host associations in complex anaerobic communities, linking viral diversity directly to metabolically active formatotrophic and methanogenic guilds. The identification of novel viral lineages infecting key AD microorganisms, combined with AVGs with potential consequences for carbon and sulfur cycling, provides a foundation for understanding the functional role of viruses in AD process performance.
Vuong Quoc Hoang Ngo, Caroline Talleu, François Enault et al.· Microbiome· 0 citations
Antarctic soils harbor highly specialized bacterial communities that perform essential functions in the functioning of polar ecosystems; however, the composition and structure of these communities remain poorly understood in many areas of Antarctica. The objective of this study was to characterize the diversity and structure of bacterial communities present in soils from four islands in the South Shetland Archipelago (Torre, Barrientos, Greenwich, and Dee) using 16S rRNA gene amplicon sequencing. Total genomic DNA was extracted from composite soil samples, followed by amplification of the 16S rRNA gene and sequencing on the Illumina MiSeq platform. Taxonomic classification allowed the sequences to be assigned from the domain level down to the species level, achieving classification rates between 98.47% and 99.39% at the domain level and between 57.39% and 65.88% at the species level. The bacterial communities were dominated by the phyla Bacillota, Pseudomonadota, Actinomycetota, and Bacteroidota, although variations in relative abundance patterns were observed among the composite samples analyzed. Alpha diversity indices revealed high taxonomic complexity, with greater species richness on Dee Island and greater evenness in Greenwich, highlighting differences in the diversity patterns observed among the composite samples analyzed. Taken together, these results expand our understanding of bacterial biodiversity in Antarctic soils and provide a baseline for future research in microbial ecology, biogeography, and the exploration of the biotechnological potential of microorganisms adapted to extreme environments.
María Fernanda Pincay Cantos, Juan J. Garrido, Gabriela María Vergara-Grandes et al.· Bacteria· 0 citations
Ancient DNA and metagenomic studies provide valuable insights into microbial evolution, host–microbe interactions, and the ecology of ancient microbial communities; however, data from urban archaeological contexts remain limited. This study investigated microbial DNA preserved in paired petrous bone and adjacent sediment samples recovered from the Bath–Gymnasium complex of Stratonikeia, a Hellenistic–Roman city in western Anatolia, to characterize microbial diversity and functional gene profiles in an archaeological context. DNA was extracted under contamination-controlled conditions and analysed using high-throughput shotgun metagenomic sequencing. Bioinformatic analyses included taxonomic profiling, microbial diversity assessment, differential abundance analysis, and characterization of virulence-associated and antimicrobial resistance-related genes. The RH1MK1 (Tepidarium) samples exhibited greater microbial diversity and a higher relative abundance of bacterial taxa with recognized pathogenic potential, including Escherichia coli, Shigella flexneri, Vibrio cholerae, and Clostridioides difficile, compared with the SRH1PPG (Palaestra) samples. Virulence-associated genes, including Shiga toxin-, cholera toxin-, and type III secretion system-related sequences, together with antimicrobial resistance-associated genes, were more abundant in RH1MK1, consistent with differences in depositional and microenvironmental conditions between the two archaeological contexts. Comparative analyses revealed both shared and site-specific microbial profiles relative to other Anatolian archaeological sites. These findings demonstrate the potential of shotgun metagenomics for investigating preserved microbial DNA in archaeological materials while emphasizing that the detected microbial taxa and functional genes should be interpreted within their archaeological and depositional context rather than as direct evidence of ancient infectious diseases or epidemic events.