The acute toxicity of hexanol
The acute toxicity of hexanol was established by conducting growth inhibition assays. Cells growing in anaerobic glass roll tubes containing modified minimal medium with syngas as a growth substrate were exposed to 0, 10, 20, 30 or 40 mM hexanol during the exponential growth phase (Figure 1A,B). Growth was measured by turbidimetry, and corresponding OD600 values were calculated from standard curves so that growth behavior could be compared in roll tubes and serum bottles. To determine whether the ability to produce hexanol confers resistance to this product, we compared the growth of the natural hexanol producer C. carboxidivorans P7 with that of the model acetogen C. ljungdahlii, which does not produce hexanol. Interestingly, despite the difference in metabolic capability, we observed no difference in hexanol tolerance between these strains. There was no effect on the growth of either strain in the presence of 10 mM hexanol, but exposure to ≥ 20 mM caused the turbidity to increase immediately after the hexanol was added and to remain stable thereafter. Furthermore, gas consumption ceased in cultures with hexanol concentrations ≥ 20 mM. This indicated the significant inhibition of growth and syngas utilization. To determine whether the cells were killed or merely dormant, 500-µL aliquots of cells were removed after 24 h and transferred to fresh medium without hexanol to see whether recovery was possible. The cultures originally exposed to 0 and 10 mM hexanol recovered fully, whereas only 50% of the cultures originally exposed to 20 mM hexanol were able to continue growing. There was no regrowth in the cultures exposed to higher concentrations of hexanol. These findings indicate that exposure to 20 mM hexanol for 24 h killed nearly all of the cells, and a 500-µL aliquot contained on average less than one viable cell. At higher hexanol concentrations, significant macroscopic flocculation of the cells was observed (Figure 1C), indicating that hexanol has a negative effect on the cell membranes. Flocculation was also observed during fed-batch bottle fermentations after 3–4 days, and has been reported in other studies for the production of alcohols .
Hexanol titers at the onset of inhibition and calculation of the IC50
Having evaluated the acute toxicity of hexanol, we next determined the minimal inhibitory concentration and IC50 for C. carboxidivorans P7. As above, cells were grown in anaerobic glass tubes containing modified minimal medium and syngas as a growth substrate, but this time the medium was supplemented with 0, 12, 14, 15, 16, 18, 20 or 22 mM hexanol before inoculation (Figure 2). Exponential growth began immediately after inoculation. On the second day of growth in the presence of hexanol, the growth rates decreased rapidly and a near linear growth profile was observed instead of exponential growth. Both the initial growth rates and final biomass yields were lower in cultures with higher hexanol concentrations. Cultures containing 22 mM hexanol did not show significant growth, and cultures containing 20 mM hexanol doubled once on the first day and then stagnated. After two days of growth, cultures containing 12 mM hexanol achieved only ~50% of the biomass yield of the control, confirming that significant growth inhibition occurred even at low hexanol titers.
The IC50 of hexanol was 17.5 ± 1.6 mM based on normalized initial growth rates. Given the immediate effect of hexanol on turbidity (Figure 1), the apparent growth rates of cultures containing higher titers of hexanol are likely to be greater than the real growth rates. The immediate effect of hexanol on turbidity appeared to be dependent on both cell density and hexanol concentration, so it was not possible to calculate adjusted growth rates from the empirical data. The real IC50 is therefore likely to be slightly lower than the calculated value (approximately 15–17 mM).
Oleyl alcohol avoids product toxicity
Oleyl alcohol has been widely used as an extraction agent in ABE fermentation but has not been tested with gaseous substrates. We therefore cultivated cells in modified minimal medium with syngas as above, but also added 5% (v/v) oleyl alcohol and 100 mM hexanol. Both C. carboxidivorans P7 and C. ljungdahlii were able to grow robustly, confirming that oleyl alcohol does not impair growth even with gaseous substrates and is able to detoxify the medium with hexanol titers of at least twice the concentration soluble in water. However, the oleyl alcohol formed microscopic bubbles or vesicles that increased the optical density of the medium over time (data not shown), so it was not possible to collect accurate values directly. For subsequent experiments, cells were therefore harvested and washed by centrifugation before OD600 values were determined.
In-line hexanol extraction during fed-batch bottle fermentation
To evaluate the effect of the extraction solvent on product formation, fed‑batch bottle fermentations were carried out in the presence or absence of 4% (v/v) oleyl alcohol. We inoculated C. carboxidivorans P7 cultures into 25 mL medium in 250-mL serum flasks and fed them with syngas (65% CO, 15% CO2, 15% N2, 5% H2) at 1 bar overpressure, with gas-phase renewal every 24 h for 4 days. After 3 further days, the final OD600 values were 5.7 ± 0.3 in the control culture and 5.9 ± 0.6 in the culture containing 4% (v/v) oleyl alcohol, confirming our earlier observation that oleyl alcohol does not affect growth. The experiments were carried out at 30 °C because this supported stable growth and approximately doubled the hexanol titers compared to growth at 37 °C (data not shown). Cells in the exponential growth phase produced mostly acetate and ethanol, and later butyrate and butanol. Hexanol production started during the late exponential to early stationary phase, and the acetate and ethanol titers decreased (Figure 3). The final hexanol titers in the aqueous phase after 7 days were 9.4 ± 2.5 mM in the control culture and 7.0 ± 0.6 in the culture containing 4% oleyl alcohol, whereas the titers of all other fermentation products were similar in both cultures (Figure 4A). In the oleyl alcohol phase, the hexanol concentration was 436 ± 101 mM (Figure 4B), corresponding to 17.4 mM for the entire culture volume. When added to the 7.0 mM hexanol in the aqueous phase, the overall hexanol titer was 24.4 mM, representing a 2.5-fold increase compared to the control without oleyl alcohol. Two thirds of the total hexanol was found in the oleyl alcohol phase, corresponding to a concentration factor of 60 over the aqueous phase. In addition to hexanol, the oleyl alcohol phase contained 100.3 ± 17.8 mM butanol (concentration factor = 5.4) and traces of caproate (Figure 5). These findings not only confirm that oleyl alcohol is an efficient hexanol extraction solvent during the fermentation of syngas, but also shows its positive effect on hexanol titers by removing the toxic product from the fermentation broth.