Development of multiple inhibitor tolerant yeast via adaptive laboratory evolution for sustainable bioethanol production.

The present research work aimed at developing robust yeast cell factory via adaptive laboratory evolution (ALE) for improved cellulosic bioethanol production. Kluyveromyces marxianus JKH5, a newly isolated thermotolerant ethanologenic yeast, was engineered by serial passaging for 60 generations in medium supplemented with gradually higher concentration of inhibitors (acetic acid, furfural, and vanillin) that are generated during dilute acid pretreatment. The improved strain K. marxianus JKH5 C60, showed 3.3-fold higher specific growth rate, 56% reduced lag phase and 80% enhanced fermentation efficiency at 42 °C in comparison to parent strain in inhibitor cocktail comprising medium. Bioethanol production by simultaneous saccharification and fermentation of sequential dilute acid-alkali pretreated sugarcane bagasse in presence of inhibitors, resulted in ethanol titre and yield, respectively, 54.8 ± 0.9 g/L and 0.40 g/g. The adapted yeast can be used to ferment unwashed pretreated biomass, thereby, reducing overall cost, time, and wastewater generation, hence making bioethanol production sustainable.