ABSTRACTNeurotoxic methylmercury (MeHg) is produced by anaerobicBacteriaandArchaeapossessing the geneshgcAB, but it is unknown how organic substrate and electron acceptor availability impacts the distribution and abundance of these organisms. We evaluated the impact of organic substrate amendments on mercury (Hg) methylation rates, microbial community structure, and the distribution ofhgcAB+microbes with sediments. Sediment slurries were amended with short-chain fatty acids, alcohols, or a polysaccharide. Minimal increases in MeHg were observed following lactate, ethanol, and methanol amendments, while a significant decrease (∼70%) was observed with cellobiose incubations. Postincubation, microbial diversity was assessed via 16S rRNA amplicon sequencing. The presence ofhgcAB+organisms was assessed with a broad-range degenerate PCR primer set for both genes, while the presence of microbes in each of the three dominant clades of methylators (Deltaproteobacteria,Firmicutes, and methanogenicArchaea) was measured with clade-specific degeneratehgcAquantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted ofProteobacteria,Firmicutes,Bacteroidetes, andActinobacteria. Clade-specific qPCR identifiedhgcA+DeltaproteobacteriaandArchaeain all sites but failed to detecthgcA+Firmicutes. Cellobiose shifted the communities in all samples to ∼90% non-hgcAB-containingFirmicutes(mainlyBacillusspp. andClostridiumspp.). These results suggest that either expression ofhgcABis downregulated or, more likely given the lack of 16S rRNA gene presence after cellobiose incubation, Hg-methylating organisms are largely outcompeted by cellobiose degraders or degradation products of cellobiose. These results represent a step toward understanding and exploring simple methodologies for controlling MeHg production in the environment.IMPORTANCEMethylmercury (MeHg) is a neurotoxin produced by microorganisms that bioacummulates in the food web and poses a serious health risk to humans. Currently, the impact that organic substrate or electron acceptor availability has on the mercury (Hg)-methylating microorganisms is unclear. To study this, we set up microcosm experiments exposed to different organic substrates and electron acceptors and assayed for Hg methylation rates, for microbial community structure, and for distribution of Hg methylators. The sediment and groundwater was collected from East Fork Poplar Creek in Oak Ridge, TN. Amendment with cellobiose (a lignocellulosic degradation by-product) led to a drastic decrease in the Hg methylation rate compared to that in an unamended control, with an associated shift in the microbial community to mostly nonmethylatingFirmicutes. This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies.