Uncovering syntrophic potential from genome-resolved metagenomics of suspended and granular anaerobic digestion sludges

Syntrophic microbial interactions are fundamental to the degradation of organic matter (e.g. fatty acids), playing a central role in natural anoxic ecosystems and engineered systems such as anaerobic digestion (AD). Despite their ecological and biotechnological importance, only a limited number of (obligate) syntrophic fatty-acid oxidizers have been successfully isolated. In this study, microbial communities from suspended and granular sludge samples were characterized using 16S rRNA gene amplicon sequencing and shotgun metagenomics. Network analysis of the 16S rRNA gene amplicon data revealed strong positive associations between methanogens and known syntrophic fatty-acid oxidizers, particularly in granular sludge samples. 743 High-Completion Metagenome Assembled Genomes (HC-MAGs) were recovered. This comprehensive HC-MAGs dataset provides a valuable resource for identifying novel microorganisms with genomic potential for syntrophic oxidation of butyrate, propionate, and acetate. This analysis identified multiple interesting novel targets, including the families DTU052 and CALXsZ01 (class Syntrophomonadia) as potential butyrate oxidizers; the families UBA6807, PHBD01, FEN-1087, and FEN-1099 (class Syntrophia) as potential propionate oxidizers; and genus DTU068 (family Thermacetogeniaceae) together with the family-level lineage 4572-78 (phylum Chloroflexota) as potential acetate oxidizers. These findings highlight granular sludges as a reservoir for previously uncharacterized syntrophic microorganisms. The recovered HC-MAG dataset also provides a framework to further elucidating fatty-acid oxidizing bacterial lineages within complex anaerobic communities.