Mercury methylation in methanogenic archaea: A protocol for stabilized cultivation and accurate assessment

Although methanogenic archaea are among the oldest microorganisms capable of mercury methylation, their contribution to methylmercury (MeHg) production has only recently gained attention. Studies with laboratory-cultivated methanogens elucidate the transformation of inorganic mercury (Hg) into MeHg, thereby uncovering underlying microbial methylation mechanisms. However, this field faces challenges such as significant Hg loss and unstable culture systems, which impede accurate assessment of these processes. This study aims to develop a reliable low-Hg-loss cultivation protocol for Hg methylation by methanogens, enabling a more accurate evaluation of their contribution to MeHg production. Our findings demonstrate that redox potential is a critical factor for Hg methylation, affecting Hg speciation and microbial growth. Notably, titanium nitrilotriacetate (Ti(III)-NTA), a reducing agent used in prior studies, was identified as a primary cause of Hg loss, reducing 83.2 % of Hg(II) to elemental Hg(0) at 500 μM. Adding cysteine satisfied both the redox and sulfur requirements of methanogens. Under these optimized conditions, Methanospirillum hungatei JF-1 achieved the highest MeHg production of all methanogens, converting 75.7 % of Hg(II) to MeHg and 630.4 pmol MeHg/mg protein. Overall, this study establishes a stable culture system for investigating Hg methylation by methanogens and indicates that the role of methanogens in mercury methylation is more substantial than previously acknowledged.