Hexanol biosynthesis from syngas by Clostridium carboxidivorans P7 is enhanced by in‑line extraction with a biocompatible solvent to avoid product toxicity

Abstract Background Clostridium carboxidivorans P7 has the rare ability to metabolize syngas – a mixture of H2, CO and CO2 – by converting it directly into industrially relevant alcohols (hexanol, butanol and ethanol) and the corresponding acids (caproate, butyrate and acetate). The product titers and ratios are highly dependent on the fermentation parameters and the composition of the syngas and growth medium. The hexanol titers produced by C. carboxidivorans P7 have recently been improved by optimizing these conditions, but little is known about the toxicity of hexanol towards Clostridium species. We hypothesized that the hexanol titers currently produced by C. carboxidivorans P7 are limited by product toxicity. Results We tested our hypothesis by exposing C. carboxidivorans P7 to different concentrations of hexanol and found that growth inhibition started at 10–12 mM, with an IC50 of 17.5 ± 1.6 mM. The presence of 20 mM hexanol was acutely toxic to C. carboxidivorans P7 as well as the model acetogen C. ljungdahlii, which does not produce hexanol. To avoid product toxicity, we added a biocompatible solvent (oleyl alcohol) to fed-batch bottle fermentations of C. carboxidivorans. This increased the total hexanol titers by 2.5-fold from 9.4 to 24.4 mM. Cell growth and product profiles in the aqueous phase after fermentation were similar in cultures with and without oleyl alcohol. The extraction phase contained high levels of hexanol (436 ± 101 mM) and butanol (100.3 ± 17.8 mM) as well as low levels of ethanol and traces of caproate. Conclusions Product toxicity was confirmed as a limiting factor during the conversion of syngas to hexanol by C. carboxidivorans P7. The addition of oleyl alcohol as a biocompatible solvent led to a significant increase in hexanol titers by facilitating the efficient and selective extraction of this product. We have therefore identified an in-line extraction strategy that increases the yield of hexanol and should allow for further improvements by genetic adaptation and/or process optimization.