Massive Cultivation of Methanococcus maripaludis Enabled by Optimized Ni, Se, and Nitrogen Supply

Methanococcus maripaludis is a well-studied archaeon with a physiology characterized by hydrogen and CO₂ consumption linked to methane production. In this study, we systematically optimized the culture medium of M. maripaludis by adjusting the concentrations of essential trace elements (Ni and Se) and ammonium. Under batch culture conditions in small bottles, the optimized medium enhanced cell growth and methanogenesis, showing a 2.13-fold increase in optical density and an approximately 25-fold increase in methane production rate compared to the conventional medium. We further scaled up the process using a 2 L bioreactor to investigate the impact of physical parameters on methanogenesis. Cell optical densities approaching nearly 49 and a maximum dry cell weight of 15.2 g/L were observed, with a methane production rate (MPR) up to 5.58 L_CH4 /L_culture/h under a 0.5 L/min gas flow rate and 1.0 barg (gauge pressure). Pressure and gas flow rate increased both MPR and specific methane productivity (q_CH4 ) in the reactor, consistent with the hypothesis of gas substrate limitation. However, higher gas flow rates reduced methane concentration (CH₄%) in the outlet stream due to the shortened gas residence time and dilution by unconverted H₂/CO₂. These performance data offer practical insights for optimizing bioreactor performance and scale-up design with this genetically tractable model methanogen and highlight the critical role of medium compositions and operational parameters in promoting high-density cultivation and efficient methane generation.