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

experimental conditions as described above. The was with 100 mM hexanol and (v/v) oleyl from sterile, anaerobic The onset of growth inhibition was investigated using the same cultivation setup as described above but with optimized trace element composition [19] and an adjusted syngas composition (65% CO, 15% N 2 , 15% CO 2 and 5% H 2 ). The cells were incubated horizontally at the respective temperature while shaking at 150 rpm. Cell growth was monitored by turbidimetry and OD 600 values were calculated using a calibration curve as above. Malthusian growth during the exponential growth phase was calculated using GraphPad Prism v8 (GraphPad Software, San Diego, CA, USA). Growth rates were normalized against the control without hexanol in the same experiment and were plotted as percentages against the hexanol concentrations added to each culture. Four-factor sigmoidal dose-response curves and IC 50 values were calculated using the same program. Fermentations (independent experiments) were carried out ve times at 30 °C or four times at 37 °C each comprising three cultures per condition.


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The utilization of industrial process gases or waste gases to produce fuels and platform chemicals could help to counter the effects of climate change. The production of organic alcohols such as butanol is particularly attractive based on their higher energy density and lower hygroscopic activity compared to ethanol [1]. Butanol can be used directly as a so-called drop-in fuel because it is compatible with current combustion engine designs [2], in addition it is also suitable as an industrial solvent or a platform chemical for conversion into polymers [1]. The similar structure but longer chain length of hexanol also makes this alcohol suitable for these and other applications. Recent publications by companies such as Evonik Creavis GmbH and Siemens AG concerning butanol and hexanol production from syngas are evidence for increasing interest in these processes from an industrial perspective [3].
Syngas, a mixture of CO, CO 2 and H 2 in differing amounts, can be metabolized by a heterogeneous group of bacteria known as acetogens. Acetogenesis is a physiological rather than a phylogenetic trait and is distributed across 23 genera, with many representatives in the genus Clostridium [4]. Unlike chemical catalysts, these bacteria can metabolize syngas under ambient conditions and can accommodate syngas substrates varying in composition and impurity levels [5,6]. Sustainable sources of syngas include process gases from steel mills or it can be produced by organic waste gasi cation plants. In conclusion, the conversion of such resources to value added chemicals could help to offset industrial greenhouse gas emissions. One promising species for this application is Clostridium carboxidivorans P7. The strain was isolated by screening for species that grow well on CO [7] and is noted for its rare ability to synthesize C6 compounds like hexanol directly from syngas [8][9][10].
Fermentation with solventogenic acetogens usually involves two production phases. The rst is an exponential growth phase that involves acetogenesis and is directly linked to energy conservation [11][12][13]. Once acetate and other primary products accumulate, the bacteria shift from acetogenesis to solventogenesis. During solventogenesis, the primary acidic products are reduced to the corresponding alcohols. With electron-rich substrates such as CO, this allows the cells to reestablish redox homeostasis and to conserve additional energy. For the reduction of acids to the corresponding alcohols via alcohol dehydrogenase, NADH is used as the electron donor. The regeneration of reduced NADH is coupled to energy conservation via the buildup of an ion gradient over the membrane, fueling an ATP synthase [12].
Thus, when the formation of more organic acids is inhibited due to already high product concentrations or low pH, production of solvents allows for additional energy conservation, especially when CO is used as the primary electron donor instead of H 2 or an aldehyde:ferredoxin oxidoreductase is used instead of an alcohol dehydrogenase [13]. Because solventogenesis generally occurs in the later phases of growth, it is often associated with sporulation, although sporulation and solventogenesis tend to be regulated independently in Clostridium species [14][15][16][17][18].
Acetogens metabolize syngas via the Wood-Ljungdahl pathway, also known as the reductive acetyl-CoA pathway. Two molecules of C1 substrate are used to form a coenzymeA-bound acetyl group using electrons derived from H 2 or CO. Acetyl-CoA can then be converted to the C2 products acetate and ethanol for energy conversion, or utilized to produce biomass [12]. Some acetogens, including C. carboxidivorans P7, can extend the carbon chain to synthesize the C4 products butyrate and butanol, and the C6 products caproate and hexanol [7,10]. The ability of C. carboxidivorans P7 to produce caproate and hexanol from syngas can be enhanced by medium and process optimization [10,19,20]. For example, the optimization of trace element composition signi cantly improved bacterial growth, leading to yields of 1.33 g/L (13.0 mM) hexanol in combination with a temperature shift to 25 °C after an initial growth phase at 37 °C [19]. Furthermore, a two-step fermentation process has been developed to overcome the limitations of biphasic growth. Generally, most cell growth is associated with acetogenesis and growth is inhibited as the medium becomes more acidic and a switch towards solventogenesis is triggered. In this two-step fermentation process however, the rst fermenter is held at pH 6 for acetogenesis, allowing the accumulation of biomass, acetate and ethanol. The medium in the rst fermenter is renewed and some of the broth is ushed to a second fermenter with cell retention at pH 5 for solventogenesis. This allows longer fermentation runs because the rst fermenter continuously provides cells for the formation of products with longer carbon chain lengths [20].
Although the effect of acids during fermentation is well understood, and toxicity data are available for both, ethanol and butanol, there is no data concerning the potential toxicity of hexanol towards C. carboxidivorans P7 [9]. As reported for C. carboxidivorans P7 grown at 30 °C with CO as a sole carbon and energy source, a 50% decrease in nal biomass was caused by either 14-14.50 g/L butanol or 35 g/L ethanol. IC 50 values for ethanol and butanol for initial growth rates could be deduced from the growth rates reported to lie between 5-10 g/L and 20-25 g/L, respectively. Furthermore, when a mixture of both alcohols was used, the drop in growth rates was even more pronounced compared to the single alcohols, indicating greater adverse effects due to co-toxicity [21]. In another study, the reported IC 50 value of butanol for C. carboxidivorans P7 grown at 25 °C with a gas mixture of 32% CO, 32% H 2 , 28% N 2 and 8% CO 2 was 4.12 g/L, with complete inhibition at 13.92 g/L. In this study, the IC 50 value for butanol was also calculated using the datasets of the previous study as 6.36 g/L [21,22]. The IC 50 of hexanol is expected to be even lower than the values reported for butanol and ethanol, given that alcohol toxicity increases with chain length and the corresponding nonpolar characteristics of the carbon chain as reported for Escherichia coli [23]. Growth of the model acetogen C. ljungdahlii has been tested in the presence of hexanol. With 10 mM hexanol (1 g/L), growth yields were only slightly impaired, while with concentrations ≥ 50 mM (5 g/L) growth was inhibited completely [24].
Product inhibition and toxic effects during alcohol production can be avoided by the use of oleyl alcohol ((Z)-Octadec-9-en-1-ol) as a biocompatible extraction solvent, as already reported for acetone-butanolethanol (ABE) fermentation [25] as well as other hydrophobic products [26] and syngas fermentation [3].
Hydrophobic extraction solvents are advantageous because the product is transferred to an organic phase, reducing the concentration of toxic products in the aqueous fermentation broth containing the cells. Furthermore, the low polarity and low solubility of hexanol in water can be exploited to concentrate the product in the extraction phase. Phase separation also facilitates continuous product harvesting because, without constant agitation, the lipophilic phase containing the hexanol forms a layer on top of the fermentation broth. The extraction solvent is therefore easy to remove and replace, providing an opportunity for extended fermentation runs with minimal downtime. Finally, the large difference between the boiling points of hexanol (157 °C) and oleyl alcohol (330-360 °C) allows rapid product recovery by distillation and potential recycling of the solvent. We therefore measured the toxicity of hexanol towards C. carboxidivorans P7 and the model acetogen C. ljungdahlii, and compared the use of oleyl alcohol to other extraction solvents for the in-line extraction during the production of hexanol during syngas fermentation in order to increase hexanol titers.

Results
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 signi cantly 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 modi ed 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 signi cant 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 ndings 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, signi cant macroscopic occulation 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 IC 50 for C. carboxidivorans P7. The MIC is the highest hexanol concentration, at which growth is still observed, whereas the IC 50 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 IC 50 , 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 modi ed 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 pro le was observed ( Supplementary Fig. 1). Both, the initial growth rates and nal biomass yields were lower in cultures with higher hexanol concentrations. Cultures supplemented with 22 mM hexanol did not show signi cant growth, and cultures supplemented with 20 mM hexanol doubled once on the rst 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, con rming that signi cant growth inhibition occurred even at low hexanol titers. The IC 50 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 IC 50 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 modi ed 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, con rming 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 OD 600 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 asks containing 25 mL medium at either 30 °C or 37 °C and fed them with syngas (65% CO, 15% CO 2 , 15% N 2 and 5% H 2 ) 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 OD 600 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), con rming our earlier observation that oleyl alcohol does not affect growth behavior. At 37 °C, maximum biomass was reached one day earlier with OD 600 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 OD 600 values at 30 °C remained stable for several days in the presence of oleyl alcohol but decreased in the absence of the solvent, con rming its detoxifying effect. At 37 °C however, OD 600 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 nal 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 in uenced 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 ndings not only con rm that oleyl alcohol is an e cient 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 nd a potential explanation for the signi cant 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 nd 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 nancial savings and more sustainable production. Corn oil and sun ower 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 sun ower 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 asks with 1 bar overpressure syngas. The small culture volume circumvented the need to renew the gas phase since only nal 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 in uence of increased solvent volume in nal hexanol titers. Biomass measured as OD 600 reached in this experiment was 4.9 ± 0.8 with oleyl alcohol, 3.7 ± 0.6 with sun ower oil and 4.4 ± 0.8 with corn oil (Fig. 6A). Final hexanol titers were similar with all three extraction solvents but extraction e ciency 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 sun ower oil and 43 ± 6% in the corn oil extraction phase.

Discussion
Impact of temperature on growth and hexanol toxicity Clostridium carboxidivorans P7 has the unusual ability to produce hexanol directly from syngas, but the toxicity of hexanol has not been determined in this species and current maximum titers may be limited by product toxicity. Accordingly, we tested the acute toxicity of hexanol in C. carboxidivorans P7 and C. ljungdahlii (which does not produce hexanol) and found that almost all cells were killed by exposure to 20 mM (2 g/L) hexanol for 24 h at 37 °C. Intuitively, one might expect greater hexanol tolerance in a natural producer than in an organism without natural exposure to this compound. However, the similar sensitivity we observed for both species probably re ects the natural environment of C. carboxidivorans P7, which was rst isolated from an agricultural settling lagoon [7]. This environment is not an optimized medium for hexanol production, so the titers of hexanol would be much lower than achieved in the laboratory, and in any case the lagoon would allow hexanol to diffuse away from its source, reducing the local concentration. Hexanol production by C. carboxidivorans P7 in its natural environment is therefore unlikely to impose selection pressure for improved tolerance. This may also explain why the fatty acid composition of C. carboxidivorans cell membranes did not adapt in the presence of sub-lethal hexanol titers (10 mM at 30 °C).
The apparent IC 50 of hexanol for C. carboxidivorans was 17.5 ± 1.6 mM (1.8 g/L) at 30 °C and 11.8 ± 0.6 mM (1.2 g/L) at 37 °C. This represents a 48% increase in hexanol tolerance at 30 °C compared to the standard cultivation temperature of 37 °C, although the doubling time only increased by 33% at the lower temperature. Because growth at the lower temperature also increased biomass yields, fermentation at 30 °C appears to offer a favorable tradeoff between growth and product tolerance during hexanol production.
On the second day of growth in the presence of hexanol at 30 °C, exponential growth ceased suggesting the cells were experiencing stress. Signi cant growth impairment was observed in the presence of 12 mM hexanol. This suggests that the toxicity of hexanol increases with longer exposure times or that the synthesis of natural products (mostly acetate and ethanol at this stage) increases stress due to cotoxicity. The increase in turbidity following the addition of hexanol (Fig. 1A,B) may impact the observed effect of hexanol on cell growth in the IC 50 experiments. For example, the apparent doubling observed at 30 °C in the presence 20 mM hexanol may well re ect the hexanol-induced increase in turbidity rather than genuine cell growth. The immediate effect of hexanol on turbidity seems to represent an interaction between cell density and hexanol concentration, so it is not possible to calculate adjusted growth rates from the observed data. This might result in artefactual higher apparent growth rates in the presence of increasing hexanol titers. The genuine IC 50 value is therefore likely to be slightly lower than the reported value in each experiment.
The IC 50 value of hexanol we observed was much lower than the values previously reported for ethanol (20-25 g/L) and butanol (4.12-6.36 g/L) [21,22]. The higher toxicity of longer-chain alcohols is anticipated because such products are less polar and interact more strongly with lipid membranes [23,30]. Hexanol toxicity mainly re ects its effect on membrane uidity, as demonstrated in E. coli [31]. The macroscopic occulation of C. carboxidivorans P7 cells in the presence of higher concentrations of hexanol indicates a similar mode of toxicity in Clostridium spp. A different mode of toxicity has been demonstrated in the yeast Saccharomyces cerevisiae, where hexanol inhibited protein synthesis. Strains with enhanced n-hexanol tolerance were shown to carry mutations in the eIF2 and eIF2B complexes that prevented the negative effects of n-hexanol on the translation initiation complex, allowing signi cantly improved growth in the presence of 0.15% n-hexanol compared to the wild-type strain [32]. Despite the differences between the protein synthesis apparatus in eukaryotes and prokaryotes, investigations using an analogous approach might also help to improve hexanol tolerance in Clostridium spp.
Both C. carboxidivorans P7 and C. ljungdahlii appear less sensitive to hexanol than E. coli, the latter showing 45% inhibition of exponential growth in the presence of 0.625 g/L (6.3 mM) hexanol [23] whereas C. carboxidivorans P7 began to experience growth inhibition at approximately double this concentration (10-12 mM) in our experiments. The IC 50 value of hexanol for an enrichment culture of methanogens was 1.5 g/L [33], which is similar to the values reported here.

Cell Membrane Adaptation To Temperature And Hexanol
To nd a potential explanation for increasing hexanol tolerance and improved hexanol productivity at lower temperatures, we investigated the fatty acid composition of C. carboxidivorans. We found that the abundance of saturated C16 fatty acids in the cell membrane nearly doubled when the temperature was increased from 30 °C to 37 °C, at the expense of mostly mono-unsaturated fatty acids with the same chain length. In E. coli the same temperature increase also triggered a shift towards more saturated fatty acids, and interestingly the same shift was observed when E. coli was exposed to 0.08% hexanol even when the temperature was not changed [28]. Furthermore, longer and more saturated fatty acids accumulate in the membrane of Clostridium pasteurianum during n-butanol fermentation [34] as well as in C. acetobutylicum following a shift to higher temperatures, or in the presence of either 0.5 or 1 g/l hexanol [35]. The latter study reported a MIC for hexanol in C. acetobutylicum of 1.4 g/L [35].
We determined the membrane composition of C. carboxidivorans in the presence of 1 g/L (10 mM) hexanol at 30 °C. Given the effects of this hexanol concentration and the similar MICs reported for C. acetobutylicum and determined for C. carboxidivorans in this study, we anticipated that C. carboxidivorans membranes would adapt when exposed to this concentration of hexanol. Intriguingly, we observed no such adaptation, which could be explained in several ways: (1) C. carboxidivorans may be intrinsically unable to adapt to higher concentrations of hexanol; (2) adaptation may be possible, but the mechanism may not be lipid-dependent and thus was not detected in our experiments (for example adaptation may involve membrane proteins); and (3) even though the cells were adapted to grow in the presence of hexanol before fatty acid analysis, long-term adaptation to higher titers may lead to different ndings in terms of membrane composition.
The inhibitory effects of higher temperatures and hexanol are both likely to re ect, at least in part, an increase in membrane uidity and leakage [36]. This can be addressed by the assembly of a more homogenous membrane with a higher content of saturated fatty acids or by the conversion of cisunsaturated fatty acids to their trans counterparts, the latter allowing tighter packaging [37]. However, trans-unsaturated fatty acids are not produced naturally in E. coli or S. saccharomyces [38] and we did not detect them in C. carboxidivorans. In contrast, Pseudomonsa putida can convert cis to trans unsaturated fatty acids due to the presence of a cis-trans isomerase [39]. When this enzyme was expressed in E. coli, the resulting strain was less sensitive to higher temperatures and various inhibitory compounds, including butanol [38]. A similar approach might allow the development of engineered C. carboxidivorans strains with greater hexanol tolerance.

Impact Of Temperature On Hexanol Production And Extraction
Extraction with oleyl alcohol at 30 °C showed promising detoxi cation results and increased the nal hexanol titers by nearly 2.5-fold to 2.4 g/L (23.9 mM). Our results therefore indicate a strong correlation between the inhibitory hexanol concentration of 10-12 mM and the highest hexanol titers of 13.0 mM produced by C. carboxidivorans P7 thus far [19]. Biocompatible extraction solvents such as oleyl alcohol remove lipophilic toxic products from the fermentation broth and thus achieve higher production titers. However, the nding that in situ extraction neither increased cell viability nor hexanol production at 37 °C was unexpected. This is most likely explained by the initially faster production of acetate and ethanol at higher growth temperatures. These compounds display inhibitory effects which cannot be mediated by the extraction solvent since they are not extracted by oleyl alcohol due to their higher polarity and mostly accumulate in the aqueous phase.
With product toxicity addressed, other factors that limit hexanol production can be investigated. The recently reported two-step fermentation process provides a constant in ux of new substrate by holding the rst fermenter at a higher pH for acetogenesis and the second at a lower pH with cell retention for the production of longer-chain compounds, mostly alcohols [20]. The combination of two-step fermentation with in situ extraction in the second fermenter could therefore increase hexanol titers and running times even further, especially if the extraction solvent were replenished to allow for continuous hexanol extraction.

Plant oils for in situ hexanol extraction
The widely-used extraction solvent oleyl alcohol can be produced by the hydrogenation of oleic acid esters derived from animal or plant oils [29]. The direct use of plant oils could reduce the cost of reagents, therefore we tested corn oil and sun ower oil as potential replacements due to their similar composition and anticipated similar performance. Both plant oils contained approximately similar amounts of oleic acid (18:1), with 29.1% and 30.6%, respectively. Furthermore, corn oil and sun ower oil respectively contain 11.8% and 6.5% palmitic acid (16:0), 1.6% and 3.6% stearic acid (18:0) and 55.4% and 56.8% linoleic acid (18:2).
We found that biomass yields were barely affected in the presence of corn oil but declined by ~ 25% in the presence of sun ower oil. Final hexanol titers were similar with all three extraction solvents but oleyl alcohol achieved the best extraction e ciency with 85 ± 2% of total hexanol in the 10% extraction phase compared to 43 ± 6% for the corn oil and 41 ± 10% for the sun ower oil. This tradeoff between production costs and extraction e ciency will need to be evaluated for each application on a case-by-case basis, and is highly dependent on the reusability of the extraction solvent during industrial processes, which we did not investigate in this study.

Conclusions
We have shown that product toxicity is an important factor limiting hexanol production during the fermentation of syngas by C. carboxidivorans P7. The addition of biocompatible solvents led to a signi cant increase in hexanol production and facilitates e cient and selective product extraction at 30 °C, whereas in situ extraction had no positive effect on growth or production occur at 37 °C.
Hexanol tolerance based on IC 50 values increased by 48% when the temperature was reduced from 37 °C (11.8 mM ± 0.6 mM) to 30 °C (17.5 ± 1.6 mM) whereas the growth rate decreased by only 25%. In line with the previous ndings, hexanol production without extraction is increased by 50% from 7.0 mM to 10.5 mM at the lower incubation temperature. The fatty acid composition of the cell membrane does not adapt to the presence of 10 mM hexanol, but switching from 37 °C to 30 °C led to a signi cant increase in the abundance of (mono-) unsaturated lipids at the expense of saturated 16:0 lipids. Growth inhibition due to product toxicity was eliminated by the addition of a biocompatible solvent at a low growth temperature of 30 °C. Having addressed this challenge, C. carboxidivorans was able to convert syngas into hexanol with titers of 23.9 mM (2.4 g/L), which is 80% higher than the best previous reported results [19]. We now have an opportunity to increase titers far beyond current maximum levels by additional strain development and the further optimization of process parameters.

Methods
Chemicals, strains and bacteria cultivation Trace elements and vitamins were prepared as described by ATCC, and 2 mg/L resazurin was used as a redox indicator to ensure strictly anaerobic conditions in the medium. Batches of medium were autoclaved prior to the addition of vitamins and trace elements from sterile stock solutions and were left overnight in a Whitley A55 Anaerobic Workstation (Don Whitley Scienti c, Herzlake, Germany) in an oxygen-free atmosphere comprising 5% H 2 , 10% CO 2 and 85% N 2 . Lcysteine ( nal concentration: 0.75 g/L) was added as a reducing agent. An optimized trace element composition for alcohol production was used for determination of IC 50 and hexanol production and extraction experiments, and membrane fatty acid analysis [19].

Determination of hexanol toxicity
Pre-cultures of C. carboxidivorans P7 and C. ljungdahlii were adapted for several generations to grow on modi ed minimal medium [19] with syngas (33.3% CO 2 , 33.3% CO and 33.3% H 2 ) as the growth substrate. Adapted cells were used to inoculate 5-mL medium in 25-mL anaerobic glass tubes (Glasgerätebau Ochs, Bovenden/Lenglern, Germany) with rubber stoppers, and the headspace was lled with syngas of the same composition. The cultures were maintained horizontally at 37 °C with constant agitation at 150 rpm. Cell growth was measured using an HI93703 microprocessor turbidity meter (Hanna Instruments Deutschland, Vöhringen, Germany) and the OD 600 was calculated using a calibration curve.
Once growth was observed (turbidity corresponding to OD 600 0.2), hexanol was added from a pure, sterile, anaerobic stock solution via a 10-µL SGE syringe (Trajan Scienti c and Medical, Ringwood, Australia). Hexanol extraction with technical grade 85% oleyl alcohol (Merck) was tested under the same experimental conditions as described above. The medium was supplemented with 100 mM hexanol and 5% (v/v) oleyl alcohol from sterile, anaerobic stock solutions.

Determination of minimal inhibitory concentrations (MIC) and IC 50 values
The onset of growth inhibition was investigated using the same cultivation setup as described above but with optimized trace element composition [19] and an adjusted syngas composition (65% CO, 15% N 2 , 15% CO 2 and 5% H 2 ). The cells were incubated horizontally at the respective temperature while shaking at 150 rpm. Cell growth was monitored by turbidimetry and OD 600 values were calculated using a calibration curve as above. Malthusian growth during the exponential growth phase was calculated using GraphPad Prism v8 (GraphPad Software, San Diego, CA, USA). Growth rates were normalized against the control without hexanol in the same experiment and were plotted as percentages against the hexanol concentrations added to each culture. Four-factor sigmoidal dose-response curves and IC 50 values were calculated using the same program. Fermentations (independent experiments) were carried out ve times at 30 °C or four times at 37 °C each comprising three cultures per condition.

Fed-batch bottle fermentation
Adapted cell cultures were inoculated (OD 600 0.01-0.02) into 25 mL of modi ed minimal medium with optimized trace elements [19] in 250-mL serum bottles (Glasgerätebau Ochs, Bovenden/Lenglern, Germany). For the extraction experiments, we added 4% (v/v) oleyl alcohol as above. Cultures were fed with syngas (65% CO, 15% N 2 , 15% CO 2 and 5% H 2 ) at 1 bar overpressure. The cells were incubated at the respective temperature while shaking at 150 rpm. The growth of cells exposed to the extraction solvent was determined by measuring the OD 600 after washing the cells by centrifugation (13,000 × g, 1 min, room temperature) and resuspending them in water. The gas phase was renewed after 1, 2, 3 and 4 days.
Samples were drawn using a sterile syringe and stored at 20 °C for subsequent analysis. At the end of the experiment (8 days), the oleyl alcohol and fermentation broth were separated by centrifugation and stored in separate tubes at -20 °C for analysis by gas chromatography.

Determination of product concentrations via GC/MS analysis
The concentrations of fermentation products in the aqueous phase was measured by gas chromatography as previously described [40]. Extraction phase samples were diluted 1:100 in pure methanol and then handled in the same manner as the aqueous samples, although the nal hold time during gas chromatography was increased to 12 min.
Determination of membrane fatty acid composition Cells were inoculated from adapted exponential-phase pre-cultures and grown overnight in 200 mL modi ed minimal medium with optimized trace elements in 2 L glass bottles with rubber stoppers, fed with syngas (65% CO, 15% N 2 , 15% CO 2 and 5% H 2 ) at 0.6 bar overpressure. When the OD 600 reached 0.4-0.5, the bottles were opened, and the cells were aerobically cooled on ice and harvested by centrifugation (4000xg, 15 min, 4 °C) before freeze drying overnight in a VaCO5 device (Zirbus Technology GmbH, Bad Grund/Harz, Germany). The membrane fatty acid composition was determined by Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) via GC/MS analysis after the following protocol: Following saponi cation, methylation and extraction [41,42], the fatty acid methyl esters (FAMEs) were analyzed by gas chromatography and detected by ame ionization, with peak identi cation following the respectively. Peaks were identi ed based on retention time and mass spectra. To con rm the position of double bonds, samples were derivatized to corresponding dimethyl disul de adducts [43]. To identify branched-chain fatty acid positions, cyclo-positions and multiple double bonds, samples were derivatized to their 3-pyridylcarbinol ("picolinyl") and/or 4,4-dimethyloxazoline (DMOX) adducts [44][45][46].
Comparison of different extraction solvents Cells were cultivated as described above for the fed-batch experiments. We supplemented a reduced media volume of 4.5 mL with either 10% or 20% oleyl alcohol or plant oils in 250 mL bottles (as indicated in the results section), to avoid the need for gas renewal during experiments without growth curves.

Declarations
Availability of data and materials All data generated or analyzed during this study are included in this published article and supplements or are available from the corresponding author on reasonable request.  The syngas composition was 65% CO, 15% N2, 15% CO2 and 5% H2. Symbols: • OD600, ■ ethanol, ▲ butanol, ▼ hexanol, ♦ acetate, ○ butyrate, □ caproate. Each curve shows a representative experiment from at least three independent experiments, each with three cultures per condition tested.  Final hexanol titers of C. carboxidivorans P7 in the presence of 4% oleyl alcohol as an extraction solvent.
(A) Cultures grown at 30 °C. (B) Cultures grown at 37 °C. Cells were grown in modi ed minimal medium with syngas as a growth substrate (65% CO, 15% N2, 15% CO2 and 5% H2). Hexanol titers measured in oleyl alcohol were normalized to the total culture volume to allow for comparison with titers measured in the aqueous phase. Hexanol titers of cultures grown without oleyl alcohol are shown as the negative control (black bars). All data are means ± standard deviations of at least three independent experiments, each comprising three cultures per condition tested.  Biomass yields (OD600) and hexanol production in the presence of different extraction solvents. Cells were grown for 7 days in modi ed minimal medium with 1 bar overpressure of syngas as a growth substrate (65% CO, 15% N2, 15% CO2 and 5% H2). Hexanol titers measured in the extraction phase were normalized to the whole culture volume and are shown as gray bars. Hexanol titers measured in the aqueous phase are shown as black bars. All data are means ± standard deviations of eight cultures from three independent experiments.

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