Metabolic changes of the acetogen Clostridium sp. AWRP through adaptation to acetate challenge

In this study, we report the phenotypic changes that occurred in the acetogenic bacterium Clostridium sp. AWRP as a result of an adaptive laboratory evolution (ALE) under the acetate challenge. Acetate-adapted strain 46 T-a displayed acetate tolerance to acetate up to 10 g L^-1 and increased ethanol production in small-scale cultures. The adapted strain showed a higher cell density than AWRP even without exogenous acetate supplementation. 46 T-a was shown to have reduced gas consumption rate and metabolite production. It was intriguing to note that 46 T-a, unlike AWRP, continued to consume H₂ at low CO₂ levels. Genome sequencing revealed that the adapted strain harbored three point mutations in the genes encoding an electron-bifurcating hydrogenase (Hyt) crucial for autotrophic growth in CO₂ + H₂, in addition to one in the dnaK gene. Transcriptome analysis revealed that most genes involved in the CO₂-fixation Wood-Ljungdahl pathway and auxiliary pathways for energy conservation (e.g., Rnf complex, Nfn, etc.) were significantly down-regulated in 46 T-a. Several metabolic pathways involved in dissimilation of nucleosides and carbohydrates were significantly up-regulated in 46 T-a, indicating that 46 T-a evolved to utilize organic substrates rather than CO₂ + H₂. Further investigation into degeneration in carbon fixation of the acetate-adapted strain will provide practical implications for CO₂ + H₂ fermentation using acetogenic bacteria for long-term continuous fermentation.