Open Culture Ethanol-Based Chain Elongation to Form Medium Chain Branched Carboxylates and Alcohols

Chain elongation fermentation allows for the synthesis of biobased chemicals from complex organic residue streams. To expand the product spectrum of chain elongation technology and its application range we investigated 1) how to increase selectivity towards branched chain elongation and 2) whether alternative branched carboxylates such as branched valerates can be used as electron acceptors. Elongation of isobutyrate elongation towards 4-methyl-pentanoate was achieved with a selectivity of 27% (of total products, based on carbon atoms) in a continuous system that operated under CO2 and acetate limited conditions. Increasing the CO2 load led to more in situ acetate formation that increased overall chain elongation rate but decreased the selectivity of branched chain elongation. A part of this acetate formation was related to direct ethanol oxidation that seemed to be thermodynamically coupled to hydrogenotrophic carboxylate reduction to corresponding alcohols. Several alcohols including isobutanol and n-hexanol were formed. The microbiome from the continuous reactor was also able to form small amounts of 5-methyl-hexanoate likely from 3-methyl-butanoate and ethanol as substrate in batch experiments. The highest achieved concentration of isoheptanoate was 6.4 +/- 0.9 mM Carbon, or 118 +/- 17 mg/L, which contributed for 7% to the total amount of products (based on carbon atoms). The formation of isoheptanoate was dependent on the isoform of branched valerate. With 3-methyl-butanoate as substrate 5-methylhexanoate was formed, whereas a racemic mixture of L/D 2-methyl-butanoate did not lead to an elongated product. When isobutyrate and isovalerate were added simultaneously as substrates there was a large preference for elongation of isobutyrate over isovalerate. Overall, this work showed that chain elongation microbiomes can be further adapted with supplement of branched-electron acceptors towards the formation of iso-caproate and iso-heptanoate as well as that longer chain alcohol formation can be stimulated.

Automated summary

Chain elongation fermentation allows for the synthesis of biobased chemicals from complex organic residue streams. Investigation into increasing selectivity towards branched chain elongation showed that increasing CO2 load led to more in situ acetate formation, which increased overall chain elongation rate but decreased the selectivity of branched chain elongation. This work demonstrates the potential for adapting chain elongation microbiomes with branched-electron acceptors to form longer chain products like iso-caproate and iso-heptanoate.