Genome-resolved assessment of archaeal diversity in full-scale anaerobic digesters reveals variability in mcrA primer coverage.
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.