Careful CO Addition Enhances Autotrophic <scp>d</scp> ‐Lactate Formation With Engineered <i>Acetobacterium woodii</i>

The acetogen <i>A. woodii</i> efficiently converts CO₂ and H₂ to acetate. Metabolic engineering enabled the autotrophic production of non-native products, for example, d-lactate from CO₂ by overexpression of d-lactate dehydrogenase from <i>Leuconostoc mesenteroides</i> and knockout of the native lactate dehydrogenase. During gas fermentation with acetogens, the addition of CO leads to increased provision of reducing equivalents, and thus increased biomass formation. However, literature data reveal that already small CO partial pressures in the gas phase inhibit the autotrophic growth of <i>A. woodii</i>. This study aims to investigate adding 0.6%-6.0% CO to batch-operated stirred tank bioreactors with continuous gassing to study autotrophic growth and product formation with the d-lactate producing <i>A. woodii</i> mutant. No growth and product formation were observed with 6% CO. Surprisingly, cell growth and metabolic product concentrations are non-linearly dependent on lower CO concentrations in the inlet gas phase. Highest biomass concentrations were observed with 3% CO (3.24 g L^-1, 89% improvement compared to the reference process without CO addition), and the highest d-lactate accumulation was achieved with 0.8% CO (6.2 g L^-1 d-lactate, 189% improvement compared to the reference without CO) after a prolonged lag phase. In conclusion, CO-sensitive <i>A. woodii</i> cells need tight control of CO in syngas to affect autotrophic product selectivities.