Method for automated high performance closed batch cultivation of gas-utilizing methanogens

To advance the utilization of microbial cell factories in gas fermentation processes, their physiological and biotechnological characteristics must be understood. Here, we report on the construction and operation of a novel device, the Gas and Pressure Controller (GPC), which is specifically designed for the automated control of the headspace gas pressure of closed cultivation bottles and facilitates automated gassing, sparging, monitoring and regulation of the headspace volume operated in closed batch cultivation mode in real time.As proof of concept, the physiological and biotechnological characteristics of four autotrophic, hydrogenotrophic methanogenic archaea were examined to quantify novel physiological limits through the elimination of gas limitation during growth and methane formation. We determined unprecedented high maximum specific methane productivity (qCH₄) values for: Methanothermobacter marburgensis of 169.59 ± 12.52 mmol g^{- 1} h^{- 1}, Methanotorris igneus of 420.21 ± 89.46 mmol g^{- 1} h^{- 1}, Methanocaldococcus jannaschii of 364.52 ± 25.50 mmol g^{- 1} h^{- 1} and Methanocaldococcus villosus of 356.38 ± 20.79 mmol g^{- 1} h^{- 1}. Obtained qCH₄ of M. marburgensis is more than 10-fold higher compared to conventional closed batch cultivation set-ups and as high as the highest reported qCH₄ value of M. marburgensis from fed-batch gas fermentation in stirred tank bioreactors. Furthermore, the GPC demonstrated reliable functionality with Methanococcus maripaludis, operating safely and autonomous during long term cultivation. This novel device enables optimal headspace pressure control, providing flexibility in application for various gas-fermenting biotechnological processes. It facilitates near optimal cultivation conditions in semi-continuous closed batch cultivation mode, the analysis of limiting factors in high-throughput experimental design and allows for automated biomass production of autotrophic, hydrogenotrophic methanogens.