Presentation Information
[P04-463]Improving Ethanol Production from Gaseous Substractes : Fermentation Characteristics of a Genetically Engineered Moorella thermoacetica
○Yuichi Kuwada1, Junya Kato1,2, Daichi Morimoto2, Setsu Kato1, Yoshiteru Aoi1, Tatsuya Fujii1,2, Yutaka Nakashimada1 (1. Hiroshima Univ. (Japan), 2. AIST (Japan))
Keywords:
Fermentation Engineering,Gas Fermentation,Anaerobic Fermentation,Material production
Syngas fermentation using CO, CO2, and H2 has attracted increasing attention as a sustainable strategy for converting industrial exhaust gases and renewable resources into value-added chemicals. Acetogenic bacteria play a key role in this process because they assimilate one-carbon gases through the Wood–Ljungdahl pathway. The thermophilic homoacetogen Moorella thermoacetica is a representative acetogen capable of utilizing CO, CO2, and H2 as carbon and energy sources (Drake and Daniel, Research in Microbiology, 2004; Ljungdahl, Annual Review of Microbiology, 1986).
In our research team, an ethanol-producing strain, M. thermoacetica ΔpduL2::adhEAMT ΔpduL1::KanR, was constructed by disrupting the phosphotransacetylase genes pduL1 and pduL2 responsible for acetate formation and introducing the NADPH-dependent alcohol dehydrogenase gene adhEAMT. Although this strain shows high ethanol productivity from sugar substrates, its behavior under gas-substrate conditions has not been fully clarified. This study aimed to characterize its gas fermentation properties and evaluate the effects of substrate composition and electron acceptors on its metabolism.
Hydrogen metabolism was examined using dual-substrate cultivation with fructose and H2. In a previously reported ethanol-producing strain, H2 supplementation caused severe growth inhibition (Kobayashi et al., Frontiers in Microbiology, 2022), whereas no marked inhibition was observed in this strain. During exponential growth, the ethanol carbon yield was 0.26 mol-C/mol-C under H2-supplemented conditions, slightly higher than under fructose-only conditions (0.25 mol-C/mol-C), while acetate production decreased. These results suggest that improved redox balance enabled simultaneous utilization of sugar and H2.
Under H2/CO2 conditions without sugar, little consumption of H2 or CO2 was observed and formate accumulated, indicating insufficient ATP supply. Addition of dimethyl sulfoxide (DMSO) enhanced H2 consumption, cell growth, and ethanol production, suggesting that an external electron acceptor improves energy metabolism and redirects reducing equivalents toward ethanol formation.
Syngas fermentation using a CO/H2 mixed gas was conducted to compensate for reduced ATP supply. The strain entered exponential growth after approximately 24 h and produced ethanol as the major product. Metabolic behavior depended on CO partial pressure. When CO was ≧20 kPa, H2 consumption was approximately 3% of CO consumption, whereas at CO <20 kPa, it increased to about 15%. However, acetate production also increased and ethanol selectivity decreased from 97% to 79% at lower CO. These results indicate that CO influences hydrogen metabolism and metabolic flux distribution.
Overall, this study demonstrates that the engineered M. thermoacetica strain exhibits high ethanol production under CO-containing gas conditions, while CO may suppress H2 metabolism. Furthermore, supplying an external electron acceptor restored metabolic activity under H2/CO2 conditions. These findings provide insights into gas utilization mechanisms in acetogenic bacteria and contribute to the development of efficient gas fermentation processes.
In our research team, an ethanol-producing strain, M. thermoacetica ΔpduL2::adhEAMT ΔpduL1::KanR, was constructed by disrupting the phosphotransacetylase genes pduL1 and pduL2 responsible for acetate formation and introducing the NADPH-dependent alcohol dehydrogenase gene adhEAMT. Although this strain shows high ethanol productivity from sugar substrates, its behavior under gas-substrate conditions has not been fully clarified. This study aimed to characterize its gas fermentation properties and evaluate the effects of substrate composition and electron acceptors on its metabolism.
Hydrogen metabolism was examined using dual-substrate cultivation with fructose and H2. In a previously reported ethanol-producing strain, H2 supplementation caused severe growth inhibition (Kobayashi et al., Frontiers in Microbiology, 2022), whereas no marked inhibition was observed in this strain. During exponential growth, the ethanol carbon yield was 0.26 mol-C/mol-C under H2-supplemented conditions, slightly higher than under fructose-only conditions (0.25 mol-C/mol-C), while acetate production decreased. These results suggest that improved redox balance enabled simultaneous utilization of sugar and H2.
Under H2/CO2 conditions without sugar, little consumption of H2 or CO2 was observed and formate accumulated, indicating insufficient ATP supply. Addition of dimethyl sulfoxide (DMSO) enhanced H2 consumption, cell growth, and ethanol production, suggesting that an external electron acceptor improves energy metabolism and redirects reducing equivalents toward ethanol formation.
Syngas fermentation using a CO/H2 mixed gas was conducted to compensate for reduced ATP supply. The strain entered exponential growth after approximately 24 h and produced ethanol as the major product. Metabolic behavior depended on CO partial pressure. When CO was ≧20 kPa, H2 consumption was approximately 3% of CO consumption, whereas at CO <20 kPa, it increased to about 15%. However, acetate production also increased and ethanol selectivity decreased from 97% to 79% at lower CO. These results indicate that CO influences hydrogen metabolism and metabolic flux distribution.
Overall, this study demonstrates that the engineered M. thermoacetica strain exhibits high ethanol production under CO-containing gas conditions, while CO may suppress H2 metabolism. Furthermore, supplying an external electron acceptor restored metabolic activity under H2/CO2 conditions. These findings provide insights into gas utilization mechanisms in acetogenic bacteria and contribute to the development of efficient gas fermentation processes.
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