EMS-Induced Mutagenesis of Clostridium carboxidivorans for Increased Atmospheric CO 2 Reduction Efficiency and Solvent Production.

Clostridium carboxidivorans (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO ₂ , and H ₂ ) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that were capable of growing in the presence of CO ₂ as a unique source of carbon with increased solvent formation and atmospheric CO ₂ reduction to limit global warming. Phenotypic analysis including growth and end product characterization of the P7 wild type (WT) demonstrated that this strain grew better at 25 °C than 37 °C when CO ₂ served as the only source of carbon. In the current study, 55 mutagenized P7-EMS mutants were developed by using 100 mM and 120 mM EMS. Interestingly, using a forward genetic approach, three out of the 55 P7-EMS mutants showed a significant increase in ethanol, butyrate, and butanol production. The three P7-EMS mutants presented on average a 4.68-fold increase in concentrations of ethanol when compared to the P7-WT. Butyric acid production from 3 P7-EMS mutants contained an average of a 3.85 fold increase over the levels observed in the P7-WT cultures under the same conditions (CO ₂ only). In addition, one P7-EMS mutant presented butanol production (0.23 ± 0.02 g/L), which was absent from the P7-WT under CO ₂ conditions. Most of the P7-EMS mutants showed stability of the obtained end product traits after three transfers. Most importantly, the amount of reduced atmospheric CO ₂ increased up to 8.72 times (0.21 g/Abs) for ethanol production and up to 8.73 times higher (0.16 g/Abs) for butyrate than the levels contained in the P7-WT. Additionally, to produce butanol, the P7-EMS _III-J mutant presented 0.082 g/Abs of CO ₂ reduction. This study demonstrated the feasibility and effectiveness of employing EMS mutagenesis in generating solvent-producing anaerobic bacteria mutants with improved and novel product formation and increased atmospheric CO ₂ reduction efficiency.