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This study aims to elucidate the role of sulfide and its precursors in anaerobic digestion (i.e., cysteine, representing sulfur-containing amino acids, and sulfate) on microbial oleate conversion to methane. Serine, with a similar structure to cysteine but with a hydroxyl group instead of a thiol, was included as a control to assess potential effects on methane formation that were not related to sulfur functionalities. The results showed that copresence of sulfide and oleate in anaerobic batch assays accelerated the methane formation compared to assays with only oleate and mitigated negative effect on methane formation caused by increased sulfide level. Nuclear magnetic resonance spectroscopy of sulfide-exposed oleate suggested that sulfide reaction with oleate double bonds likely contributed to negation of the negative effect on the methanogenic activity. Methane formation from oleate was also accelerated in the presence of cysteine or serine, while sulfate decreased the cumulative methane formation from oleate. Neither cysteine nor serine was converted to methane, and their accelerating effects was associated to different mechanisms due to establishment of microbial communities with different structures, as evidenced by high-throughput sequencing of 16S rRNA gene. These outcomes contribute with new knowledge to develop strategies for optimum use of sulfur- and lipid-rich wastes in anaerobic digestion processes.