Conversion of gaseous substrates (CO, CO2, H2) by acetogenic bacteria is a promising technology to establish sustainable bioproduction scenarios. The thermophilic acetogen Thermoanaerobacter kivui (Topt = 66 °C) grows on H2/CO2 in chemically defined mineral medium with high growth rates (doubling time: 2 h). However, 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 with the ability 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). Genomic analysis of the clonal strain CO-1 using short and long read sequencing indicated the presence of a large transposon which might be required for growth of T. kivui on CO. To characterize the physiology 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. Transcriptomic analysis revealed that the presence of the transposon increased gene expression levels of the energy converting hydrogenase 2 complex (Ech2) involved in energy conservation. Next, the use of syngas obtained via steam gasification of softwood pellets as a feedstock for gas fermentation with T. kivui was evaluated. To that end, we evaluated (i) the impact of potential inhibitors generated during biomass gasification (aromatic compounds BTEX, H2S and cyanide) and (ii) recovery of nitrogen from the syngas stream and utilization as nitrogen source for T. kivui to reduce the resource footprint of the fermentation process. Overall, synthesis gas generated from biomass gasification was successfully utilized for gas fermentation with T. kivui in continuous mode in 200 mL stirred tank bioreactors as well as a 20 L bubble column bioreactor. Collectively, the results of this study represent a first step toward establishing high-temperature gas fermentation processes.