Hexanol Biosynthesis From Syngas by Clostridium Carboxidivorans P7 - Investigation of Product Toxicity, Temperature Dependence and in Situ Extraction 

Abstract BackgroundClostridium carboxidivorans P7 converts synthesis gas (also called syngas, a mixture of CO, CO2 and H2) directly into industrially relevant alcohols (hexanol, butanol and ethanol) and their corresponding acids (caproate, butyrate and acetate). The product titers and ratios are highly dependent on fermentation parameters and the compositions of syngas as well as the growth medium. 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. ResultsWe tested our hypothesis by analyzing IC50 values for hexanol at 30 °C and 37 °C, which we determined as 17.5 ± 1.6 mM and 11.8 mM ± 0.6 mM, respectively, indicating a major influence of growth temperature on hexanol sensitivity. We found that 20 mM hexanol was acutely toxic to C. carboxidivorans P7 at 30 °C and growth was already completely inhibited in the presence of 15 mM hexanol at 37 °C. Membrane fatty acid analysis showed that the cell membrane composition of C. carboxidivorans adapted strongly to the higher growth temperature but surprisingly did not change significantly when grown in the presence of 10 mM hexanol. To avoid product toxicity during hexanol production we added oleyl alcohol as an extraction solvent. At 30 °C, addition of the solvent increased total hexanol titers nearly 2.5-fold from 10.5 to 23.9 mM. However hexanol titers decreased from 7.0 to 5.6 mM in the presence of oleyl alcohol at 37 °C. At 30 °C, the extraction phase contained large amounts of hexanol (448 ± 130 mM) and butanol (102 ± 20 mM). Values were lower at 37 °C with 101 ± 27 mM hexanol and 50 ± 6 mM butanol. Growth was not inhibited by oleyl alcohol. Biomass remained high in the presence of oleyl alcohol at 30 °C, but rapidly decreased in the absence of the solvent. At 37 °C biomass decreased even in the presence of oleyl alcohol. We tested corn oil and sunflower seed oil as potentially cheaper and more sustainable extraction solvents. While oleyl alcohol displayed the highest extraction efficiency for hexanol, total hexanol titers were similar with all solvents tested. ConclusionsBoth, product toxicity and growth temperature were identified as limiting factors during the conversion of syngas to hexanol by C. carboxidivorans P7. At 30 °C the addition of a biocompatible solvent led to detoxification and a significant increase in hexanol titers, 80% higher than highest previously reported titers. These findings help to mediate the limitation of product toxicity in hexanol production from syngas for the development of more efficient process designs and production strains.