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Distinguishing the respective contributions of various microbes to methylmercury (MeHg) production is critical for predicting MeHg bioaccumulation and exposure risk. Metabolic inhibitors have been commonly used to block the activity of specific microbial groups and identify primary Hg methylating microbes. By reviewing literatures and our empirical data, we demonstrate how multiple factors, including (1) the addition of inappropriate amounts of inhibitors, (2) a tendency to overlook microbial syntrophy, and (3) the absence of comprehensive proxy systems of Hg methylation, would impact result interpretation of this approach. We thus suggest that the design of inhibition assays should consider the environmental properties, e.g., background levels of electron acceptors, concentrations of metabolic substrates, and abundances of Hg methylating microbes. We also recommend that inhibitors should be added at multiple concentrations and that observed changes in Hg methylation should be assessed with comprehensive indicators. Revealing the key factors responsible for the improper usage of this method and inadequate interpretation of the results would help optimize inhibition assays for robust predictions of MeHg production in nature. [ABSTRACT FROM AUTHOR]