CO₂:H₂-based gas fermentation with acetogenic Clostridium species are at an early stage of development. This work exploited the Adaptive Laboratory Evolution technique to improve the growth of C. carboxidivorans P7 on CO₂ and H₂. An adapted strain with decreased growth lag phase and improved biomass production was obtained. Genomic analysis revealed a conserved frameshift mutation in the catalytic subunit of the hexameric hydrogenase gene. The resulted truncated protein variant, most likely lacking its functionality, suggests that other hydrogenases might be more efficient for H₂-based growth of this strain. Furthermore, the adapted strain generated hexanol as primary fermentation product. For the first time, hexanol was produced directly from CO₂:H₂ blend, achieving the highest maximum productivity reported so far via gas fermentation. Traces of valerate, pentanol, eptanol and octanol were observed in the fermentation broth. The adapted strain shows promising to enrich the product spectrum targetable by future gas fermentation processes.

Gas fermentation by acetogens of the genus Clostridium is an attractive technology since it affords the production of biochemicals and biofuels from industrial waste gases while contributing to mitigate the carbon cycle alterations. The acetogenic model organisms C. ljungdahlii and C. autoethanogenum have already been used in large scale industrial fermentations. Among the natural products, ethanol production has already attained industrial scale. However, some acetogens are also natural producers of 2,3-butanediol (2,3-BDO), a platform chemical of relevant industrial interest. Here, we have developed a lab-scale screening campaign with the aim of enhancing 2,3-BDO production. Our study generated comparable data on growth and 2,3-BDO production of several batch gas fermentations using C. ljungdahlii and C. autoethanogenum grown on different gas substrates of primary applicative interest (CO₂ · H₂, CO · CO₂, syngas) and on different media featuring different compositions as regards trace metals, mineral elements and vitamins. CO · CO₂ fermentation was found to be preferable for the production of 2,3-BDO, and a fair comparison of the strains cultivated in comparable conditions revealed that C. ljungdahlii produced 3.43-fold higher titer of 2,3-BDO compared to C. autoethanogenum. Screening of different medium compositions revealed that mineral elements, Zinc and Iron exert a major positive influence on 2,3-BDO titer and productivity. Moreover, the CO₂ influence on CO fermentation was explored by characterizing C. ljungdahlii response with respect to different gas ratios in the CO · CO₂ gas mixtures. The screening strategies undertaken in this study led to the production of 2.03 ± 0.05 g/L of 2,3-BDO, which is unprecedented in serum bottle experiments.

To successfully scale-up the production of bio-based building blocks through CO₂ and H₂-based gas fermentation, it is crucial to deeply understand and control the microbial catalyst response to the bioreactor environment. This study investigates the effects of key process parameters, such as CO₂ and H₂ partial pressures, gas feeding strategies, and mixture composition, on the production pathways of an evolved Clostridium carboxidivorans strain. The ultimate goal is to optimize 1-hexanol production in elevated-pressure stirred-tank reactors. Continuous gas feeding enhanced acetogenic and solventogenic metabolisms, while gas-limited conditions promoted chain elongation to caproic acid. An optimized process, combining an initial gas-limited step followed by a continuous gas phase, increased 1-hexanol production, achieving a maximum biomass-specific productivity of 0.9 g g_CDW^-1 day^-1. In-situ product extraction improved 1-hexanol carbon selectivity to an unprecedented 60 %. These findings demonstrate the potential of CO₂ and H₂-fed fermentation to produce high-value chemicals other than ethanol and acetate.