The acute toxicity of hexanol
To determine whether the ability to produce hexanol confers increased 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. Furthermore, given that C. ljungdahlii is well researched and genetically accessible, a significantly higher tolerance towards hexanol would turn this organism into a potential biocatalyst for hexanol production compared to the less researched natural hexanol producer C. carboxidivorans. Acutely lethal hexanol concentrations were determined in 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 (0, 1, 2, 3 or 4 g/L) hexanol during the exponential growth phase (Fig. 1A,B).
Interestingly, despite the difference in metabolic capability, we observed no difference in hexanol tolerance between these strains under the conditions tested. There was no effect on the growth of either strain in the presence of 10 mM hexanol, but exposure to ≥ 20 mM caused an increase in turbidity immediately after the hexanol was added and turbidity remained 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 regrow. 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 (Fig. 1C), indicating that hexanol affects the cell membranes. Flocculation was also observed during fed-batch bottle fermentations after 3–4 days, and has been reported in other studies focusing on the production of alcohols [19]. The lack in major differences in hexanol tolerance between both clostridial species led to the decision to focus on the natural hexanol producer C. carboxidivorans for further characterization.
Hexanol titers at the onset of inhibition and calculation of IC 50 values
Having evaluated the acute toxicity of hexanol, we next determined the minimal inhibitory concentration (MIC) and IC50 for C. carboxidivorans P7. The MIC is the highest hexanol concentration, at which growth is still observed, whereas the IC50 is the hexanol concentration at which the initial growth rate is reduced by 50%. Given the reported positive effect of lower incubation temperature on alcohol production [19, 27], we decided to exemplarily investigate both parameters, MIC and IC50, at 37 °C as well as 30 °C. The lower temperature was chosen to achieve an acceptable tradeoff between potentially increased resilience towards hexanol at lower temperatures and faster growth at higher temperatures. By still allowing the cells to grow continuously without a distinct temperature shift, this setup will facilitate easier scale up and continuous process design.
As above, cells were grown in anaerobic glass tubes containing modified minimal medium and syngas as a growth substrate. Since now exact inhibitory titers of hexanol were investigated, we chose to add hexanol before inoculation to avoid the increase in observed turbidity in the experiment shown in Fig. 1. For experiments at 30 °C, the medium was supplemented with 0, 12, 14, 15, 16, 18, 20 or 22 mM hexanol. Exponential growth began immediately after inoculation. On the second day at 30 °C in the presence of hexanol, the growth rates decreased rapidly and a near linear growth profile was observed (Supplementary Fig. 1). Both, the initial growth rates and final biomass yields were lower in cultures with higher hexanol concentrations. Cultures supplemented with 22 mM hexanol did not show significant growth, and cultures supplemented with 20 mM hexanol doubled once on the first day and then stagnated (MIC = 18–20 mM hexanol). After two days of growth, cultures containing 12 mM hexanol achieved only ~ 50% of the biomass yield of the control without added hexanol, confirming that significant growth inhibition occurred even at low hexanol titers. The IC50 for hexanol at 30 °C was determined as 17.5 ± 1.6 mM based on normalized initial growth rates. At 37 °C, the impact of 0, 7, 10, 12, 13, 14 and 15 mM hexanol was investigated and an IC50 value of 11.8 mM ± 0.6 mM was calculated from the growth rates observed. At titers ≥ 15 mM no growth was observed at 37 °C (MIC = 13–14 mM hexanol). In conclusion, a shift from 37 °C to 30 °C leads to a 48% increase in hexanol tolerance. Doubling times increased by 33% from 7.5 ± 0.2 h at 37 °C to 10.1 ± 1.0 h at 30 °C.
Oleyl Alcohol Avoids Product Toxicity
Oleyl alcohol has been widely used as an extraction solvent in ABE fermentation and recently also in syngas fermentation [3], but has not yet been used in syngas fermentations with C. carboxidivorans. 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 with gaseous substrates and is able to detoxify the medium with hexanol titers of at least twice the concentration soluble in water. However, in the presence of cells 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 situ hexanol extraction during fed-batch bottle fermentation
To evaluate the effect of the extraction solvent on product formation, fedbatch bottle fermentations were carried out in the presence or absence of 4% (v/v) oleyl alcohol. We inoculated C. carboxidivorans P7 cells into 250-mL serum flasks containing 25 mL medium at either 30 °C or 37 °C and fed them with syngas (65% CO, 15% CO2, 15% N2 and 5% H2) at 1 bar overpressure, with gas-phase renewal every 24 h for 5 days. Maximum biomass at 30 °C was reached after 5 days of growth with OD600 values of 6.3 ± 0.6 in the control culture (Fig. 2A) and 6.2 ± 0.7 in the culture containing 4% (v/v) oleyl alcohol (Fig. 2B), confirming our earlier observation that oleyl alcohol does not affect growth behavior. At 37 °C, maximum biomass was reached one day earlier with OD600 values of 4.9 ± 0.3 in the control (Fig. 2C) and 5.4 ± 0.6 in the cultures containing oleyl alcohol (Fig. 2D). Although growth was faster at the higher temperature, incubation at 30 °C led to higher biomass formation. Furthermore, maximum OD600 values at 30 °C remained stable for several days in the presence of oleyl alcohol but decreased in the absence of the solvent, confirming its detoxifying effect. At 37 °C however, OD600 values decreased both in the presence and absence of oleyl alcohol.
Under all conditions, cells in the exponential growth phase produced mostly acetate and ethanol, and later butyrate and butanol. As expected, at 37 °C products were initially produced faster and in higher amounts compared to 30 °C. Hexanol production started during the late exponential to early stationary phase, and the acetate and butyrate titers decreased in all cultures. The final hexanol titers in the aqueous phase after 8 days at 30 °C were 10.5 ± 2.2 mM in the control culture and 6.0 ± 1.2 in the culture containing 4% oleyl alcohol, whereas the titers of all other fermentation products measured in the aqueous phase were not influenced by the extraction solvent (Fig. 3A). In the oleyl alcohol phase, the hexanol concentration was 448 ± 130 mM (Fig. 3B), corresponding to 17.9 mM normalized for the culture volume. When added to the 6.0 mM hexanol in the aqueous phase, the overall hexanol titer was 23.9 mM, representing a nearly 2.5-fold increase compared to the control without oleyl alcohol (Fig. 4A). Two thirds of the total hexanol were found in the oleyl alcohol phase, corresponding to a concentration factor of approximately 60 over the aqueous phase. In addition to hexanol, the oleyl alcohol phase contained 102 ± 20 mM butanol, 126 ± 43 mM ethanol and traces of caproate. Neither acetate nor butyrate were present in the extraction phase.
At 37 °C, 7.0 ± 0.5 mM hexanol were produced in the absence of oleyl alcohol. In the presence of the extraction solvent a total of only 5.6 ± 1.3 mM hexanol was observed with 1.6 ± 0.4 mM of the total hexanol located in the aqueous phase and titers of 101 ± 27 mM (4.0 ± 1.0 mM normalized to the culture volume) in the extraction phase (Figs. 3D and 4B). The extraction phase also contained 50.4 ± 5.7 mM butanol and 123 ± 40 mM ethanol as well as traces of caproate. Again, neither acetate nor butyrate were present in the extraction phase.
In conclusion, these findings not only confirm that oleyl alcohol is an efficient hexanol extraction solvent during the fermentation of syngas, but also showed a positive effect on produced hexanol titers by removing the toxic product from the fermentation broth. Intriguingly, this positive effect only occurs at 30 °C but not at 37 °C. In summary, cultivation at 30 °C compared to 37 °C led to a 50% increase in hexanol titers in the absence of oleyl alcohol and more than a 4-fold increase in hexanol production in the presence of oleyl alcohol compared to 37 °C.
Analysis Of Cellular Membrane Fatty Acid Composition
To find a potential explanation for the significant difference on the production and growth behavior at both temperatures investigated, a fatty acid analysis of the cell membrane of C. carboxidivorans was performed. We compared the membrane composition at 30 °C in the presence and absence of hexanol to reveal possible adaptation towards this toxic product. The concentration of 10 mM hexanol was chosen for this experiment. At this concentration we already anticipated adaptation to the alcohol without too severe toxic effects that might lead to differences in growth behavior (i.e. growth phase at cell harvest), which would also lead to potential differences in cell membrane composition.
It has already been shown for E. coli that both, growth at increased temperatures as well as growth in the presence of hexanol led to a shift towards more saturated fatty acids [28]. Most interestingly, we could not find adaptation of C. carboxidivorans cell wall lipid composition when grown in the presence of 10 mM hexanol (Supplementary Table 1). However, comparing cells grown at 30 °C to cells grown at 37 °C, we found a shift in fatty acid composition towards more saturated fatty acids and overall less diversity in the membrane composition at the higher growth temperature. While at 30 °C 75.7 ± 6.6% of the membrane consisted of compounds with a chain length of 16 carbons, this was increased to 91.3 ± 0.4% at 37 °C. Furthermore, at 30 °C the membrane composition was more diverse with percentages of both longer and shorter lipids increased compared to 37 °C (Fig. 5). Within 16 carbon compounds there was a higher percentage of saturated lipids (mostly palmitic acid and 16:0 vinyl ether), detected at 37 °C, while mostly 16:1 cis 9 and 16:1 cis 11 were increased at 30 °C compared to the higher growth temperature. The amount of saturated lipids shifted from 42.6 ± 1.9% at 30 °C to 65.7 ± 0.4% at 37 °C with the correlated decrease in (mono-) unsaturated lipids. The only polyunsaturated fatty acid detected was 15:2 with 2.5 ± 1.6% at 30 °C and 1.1 ± 0.5% at 37 °C. Cyclic or branched chain fatty acids were only found in low concentrations of ≤ 0.8% per molecular species.
Utilization Of Plant Oils As Extraction Solvents
As discussed earlier, oleyl alcohol has already widely been used as an extraction solvent in fermentations with clostridia. Important factors for this application are mainly biocompatibility, selectivity for the targeted product, stability of the solvent and bulk price. Oleyl alcohol can be produced from oleic acid ester isolated from animal and plant oils [29]. Using plant oils directly as extraction solvents would conceptually lead to energy and financial savings and more sustainable production. Corn oil and sunflower seed oil were chosen as exemplary plant based solvents. In comparison, the lab scale costs for the extraction solvents used per liter were 300.89 € for oleyl alcohol, 75.20 € for sunflower seed oil and 70.40 € for corn oil. All extraction solvents were supplied by Sigma Aldrich Chemie (Merck, Darmstadt, Germany). In bulk use for larger scale fermentations the prices, especially of plant oils, would conceivably drop considerably. To investigate the viability of plant oils as alternative extraction solvents, cells were grown in 4.5 mL Medium supplemented with the respective extraction solvent in 250 mL anaerobic flasks with 1 bar overpressure syngas. The small culture volume circumvented the need to renew the gas phase since only final hexanol titers and optical densities were investigated. The experiment was done twice with 10% extraction phase and once with 20% extraction phase, showing no positive influence of increased solvent volume in final hexanol titers. Biomass measured as OD600 reached in this experiment was 4.9 ± 0.8 with oleyl alcohol, 3.7 ± 0.6 with sunflower oil and 4.4 ± 0.8 with corn oil (Fig. 6A). Final hexanol titers were similar with all three extraction solvents but extraction efficiency was less pronounced in the plant oils (Fig. 6B). With 10% solvent, when oleyl alcohol was used, 85 ± 2% of the produced hexanol were found in the extraction phase, while only 41 ± 10% were located in the sunflower oil and 43 ± 6% in the corn oil extraction phase.