Biogas produced from the anaerobic digestion of animal slurry consists mainly of methane (CH) and carbon dioxide (CO), but also includes other minor gases, such as hydrogen sulfide (HS). Since it can act as a potent corrosive agent and presents a health hazard even at low concentrations, HS is considered an undesirable by-product of anaerobic digestion. Sulfate-reducing bacteria (SRBs) have been identified as the main biological source of HS in a number of natural, biological, and human-made habitats, and thus represent likely candidate microorganisms responsible for the production of HS in anaerobic manure digesters. Phylogenetically, SRBs form a divergent group of bacteria that share a common anaerobic respiration pathway that allows them to use sulfate as a terminal electron acceptor. While the composition and activity of SRBs have been well documented in other environments, their metabolic potential remains largely uncharacterized and their populations poorly defined in anaerobic manure digesters. In this context, a combination of in vitro culture-based studies and DNA-based approaches, respectively, were used to gain further insight. Unexpectedly, only low to nondetectable levels of HS were produced by digestate collected from a manure biogas plant documented to have persistently high concentrations of HS in its biogas (2000-3000 ppm). In contrast, combining digestate with untreated manure (a substrate with comparatively lower sulfate and SRB cell densities than digestate) was found to produce elevated HS levels in culture. While a 16S rRNA gene-based community composition approach did not reveal likely candidate SRBs in digestate or untreated manure, the use of the dsrAB gene as a phylogenetic marker provided more insight. In digestate, the predominant SRBs were found to be uncharacterized species likely belonging to the genus Desulfosporosinus (Peptococcaceae, Clostridiales, Firmicutes), while Desulfovibrio-related SRBs (Desulfovibrionaceae, Desulfovibrionales, Proteobacteria) were the most highly represented in untreated manure. Intriguingly, the same species-level OTUs with a similar pattern of opposite relative abundance were also found in two other digesters with lower HS levels in their biogas. Together, our results suggest that elevated HS production in anaerobic digesters requires the combination of biological and nutritional factors from both untreated manure and digestate.