Boosting 2G bioethanol production by overexpressing dehydrogenase genes in Klebsiella sp. SWET4adh1+adhE: ANN optimization, scale-up, and economic sustainability

The most significant obstacle in the production of 2G bioethanol lies in transforming the complex carbohydrates found in plant biomass into simpler, fermentable sugars. However, this challenge can be effectively addressed by developing highly efficient lignocellulolytic ethanologenic strains. The current study intended to improve the ethanol productivity of efficient cellulose and hemicellulose metabolizing Klebsiella sp. SWET4 by overexpressing its native ethanologenic genes (adh1, adhE, and nifJ). Among the recombinants, SWET4adh1+adhE exhibited the highest ethanol productivity from banana peel (BP) in facultative anaerobic conditions with a 7.76-fold increase compared to the native strain. Transcript level expression study using qPCR indicated 106.15- and 22.78-fold higher expression of adh1 and adhE gene, respectively at 56 h from BP, whereas, 4.80- and 2.08-fold higher concentrations of respective proteins were obtained after 24 h. ANN and GA-mediated optimization further enhanced biomass and ethanol production to 2.33 and 24.47 g/L, respectively. Hence the ethanol productivity by SWET4adh1+adhE was enhanced by 19.89-fold. The bioreactor kinetic study exhibited diauxic growth of SWET4adh1+adhE and achieved an impressive ethanol yield of 0.44 g/g of carbohydrates, surpassing many 2G bioethanol processes. Despite of single-product scheme, a minimum selling price of $2/kg of distillate was found to make the process economically feasible. The breakeven point of the process was found to be 30 % of its total capacity. Techno-economic analysis underscores the feasibility and economic advantages of the current study eliminating the pretreatment steps, and highlighting the process's innovation and viability in 2G bioethanol production. Elimination of the pretreatment process not only achieved economic sustainability but also significantly enhanced the renewability of bioethanol production endorsing direct biomass conversion to bioethanol.