CO 2 supply is a powerful tool to control homoacetogenesis, chain elongation and solventogenesis in ethanol and carboxylate fed reactor microbiomes.

Anaerobic fermentation technology enables the production of medium chain carboxylates and alcohols through microbial chain elongation. This involves steering reactor microbiomes to yield desired products, with CO ₂ supply playing a crucial role in controlling ethanol-based chain elongation and facilitating various bioprocesses simultaneously. In the absence of CO ₂ supply (Phase I), chain elongation predominantly led to n-caproate with a high selectivity of 96 Cmol%, albeit leaving approximately 80% of ethanol unconverted. During this phase, C. kluyveri and Proteiniphilum -related species dominated the reactors. In Phase II, with low CO ₂ input (2.0 NmL L ^-1  min ^-1 ), formation of n-butyrate, butanol, and hexanol was stimulated. Increasing CO ₂ doses in Phase III (6 NmL L ^-1  min ^-1 ) led to CO ₂ utilization via homoacetogenesis, coinciding with the enrichment of Clostridium luticellarii , a bacterium that can use CO ₂ as an electron acceptor. Lowering CO ₂ dose to 0.5 NmL L ^-1  min ^-1 led to a shift in microbiome composition, diminishing the dominance of C. luticellarii while increasing C. kluyveri abundance. Additionally, other Clostridia , Proteiniphilum , and Lactobacillus sakei -related species became prevalent. This decrease in CO ₂ load from 6 to 0.5 NmL L ^-1  min ^-1 minimized excessive ethanol oxidation from 30%-50% to 0%-3%, restoring a microbiome favoring net n-butyrate consumption and n-caproate production. The decreased ethanol oxidation coincided with the resurgence of hydrogen formation at partial pressures above 1%. High concentrations of butyrate, caproate, and ethanol in the reactor, along with low acetate concentration, promoted the formation of butanol and hexanol. It is evident that CO ₂ supply is indispensable for controlling chain elongation in an open culture and it can be harnessed to stimulate higher alcohol formation or induce CO ₂ utilization as an electron acceptor.
Competing Interests: Author DL was employed by the ChainCraft B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from ChainCraft B.V. The funder had the following involvement in the study: study design, collection, analysis, interpretation of data and the writing of this article.
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