Continuous gas fermentation with Thermoanaerobacter kivui adapted to carbon monoxide

Abstract

Conversion of gaseous one carbon substrates (CO, CO₂) and H₂ by acetogenic bacteria is a promising technology to establish sustainable bioproduction scenarios for value-added products in the context of a circular carbon economy. The thermophilic acetogen Thermoanaerobacter kivui (Topt = 66 °C) grows on H₂/CO₂ in chemically defined mineral medium with growth rates (doubling time: 2 h), exceeding those of mesophilic acetogens. Nevertheless, there is currently no bioprocessing system available for quantitative characterization of T. kivui as a model thermophilic acetogen under well-defined bioreactor conditions. In this study, we established a continuous high-temperature gas fermentation system and characterized T. kivui wildtype as well as a strain adapted to grow on CO (referred to as CO-1). T. kivui CO-1 grew in a 100 % CO gas phase in chemically defined mineral medium with a growth rate of up to 0.25 h-1 (doubling time: 2.8 h) and adaptation occurred in as little as 30 generations, similar to previous work by Weghoff and Müller (2016) using rich medium [1]. Genomic analysis of the clonal strain CO-1 using short and long read sequencing technologies indicated that a handful of SNPs and large-scale genomic rearrangements are responsible for the successful adaptation of T. kivui to CO. To characterize the physiological behavior of strain CO-1 in more detail, steady state chemostat cultures operated at specific growth rates of 0.10-0.20 h-1 were used to quantify growth, gas consumption, and acetate production on H₂/CO₂, syngas and pure CO and to obtain samples for transcriptomic and proteomic analyses. A stoichiometric core model of T. kivui metabolism was established and used to analyze intracellular flux distributions. Collectively, the knowledge gained in this study represents a first step toward establishing high-temperature gas fermentation processes.