Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas

Abstract

Background: Synthesis gas, a mixture of CO, H2, and CO2, is a promising renewable feedstock for bio-based production of organic chemicals. Production of medium-chain fatty acids can be performed via chain elongation, utilizing acetate and ethanol as main substrates. Acetate and ethanol are main products of syngas fermentation by acetogens. Therefore, syngas can be indirectly used as a substrate for the chain elongation process.

Results: Here, we report the establishment of a synthetic co-culture consisting of Clostridium autoethanogenum and Clostridium kluyveri. Together, these bacteria are capable of converting CO and syngas to a mixture of C4 and C6 fatty acids and their respective alcohols. The co-culture is able to grow using solely CO or syngas as a substrate, and presence of acetate significantly stimulated production rates. The co-culture produced butyrate and caproate at a rate of 8.5 ± 1.1 and 2.5 ± 0.63 mmol/l/day, respectively. Butanol and hexanol were produced at a rate of 3.5 ± 0.69 and 2.0 ± 0.46 mmol/l/day, respectively. The pH was found to be a major factor during cultivation, influencing the growth performance of the separate strains and caproate toxicity.

Conclusion: This co-culture poses an alternative way to produce medium-chain fatty acids and higher alcohols from carbon monoxide or syngas and the process can be regarded as an integration of syngas fermentation and chain elongation in one growth vessel.

Keywords: Butanol; Butyrate; Caproate; Clostridium autoethanogenum; Clostridium kluyveri; Hexanol; Hydrogen.

Fig. 1

Co-culture establishment. a Production profile of C. autoethanogenum grown with CO and H₂, the headspace was refilled with H₂/CO at t = 4. b Production profile of C. kluyveri, at t = 4, 50 kPa CO was introduced to the culture. c A pure culture of C. autoethanogenum mixed in a 1:1 ratio with a pure culture of C. kluyveri at t = 4. d A pure culture of C. kluyveri mixed in a 1:1 ratio with a pure culture of C. autoethanogenum at t = 4. The legend is representative for all displayed graphs. Solid and open circle symbols represent left and right y-axis, respectively

Fig. 2

Standard production profile of the co-culture in non-shaking conditions. On all data series, a standard deviation is displayed over duplicate experiments. Solid and open circle symbols represent left and right y-axis, respectively

Fig. 3

Effect of initial acetate concentration on the production of MCFAs. Data displayed are representative for 13 days after incubation using 130 kPa CO as a substrate. At the end of cultivation, CO was depleted in all cultures. On all graphs, a standard deviation is displayed over duplicate experiments

Fig. 4

The effect of H₂:CO ratio on the production profile of the co-culture. a Pure CO headspace. b 1:2 ratio of H₂/CO c 2:1 ratio of H₂:CO. d H₂/CO₂ headspace. e Product concentrations at the end of incubation. f Mole of acetate consumed per mole of gas (H₂ + CO) consumed. On all graphs a standard deviation is displayed over duplicate experiments. Solid and open circle symbols represent left and right y-axis, respectively

Fig. 5

Effect of shaking and CO pressure on the co-culture. a Production profile under shaking conditions. b Production profile with maintained CO pressure (>50 kPa), under shaking conditions. c Production profile after initial non-shaking incubation and subsequent transfer to shaking conditions (after t = 4). On all data series, a standard deviation is displayed over duplicate experiments. Solid and open circle symbols represent left and right y-axis, respectively

Fig. 6

Co-cultivation under excess CO conditions. Shaking was applied after 4 days (red vertical line). a Acid concentration profile. b Alcohol concentration profile. c Partial gas pressures of CO, CO₂, and H₂. d Estimated total concentration of products formed, approximated by a Gompertz equation. e Total estimated volumetric production rates displayed as the derivative of the Gompertz equation. f Estimated net volumetric production rates after compensation of product formation and consumption according to Eqs. 3 and 4. Solid and open circle symbols represent left and right y-axis, respectively

Fig. 7

Schematic representation of the co-culture of C. autoethanogenum and C. kluyveri. Reaction stoichiometry and ATP yield for each of the cells are not displayed. Conversion of butyrate to butyraldehyde and caproate to caproaldehyde is assumed to proceed via an aldehyde oxidoreductase, as is observed for acetate to acetaldehyde formation. CODH carbon monoxide dehydrogenase, RnF ferredoxin-NAD:oxidoreductase