Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery

Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A(600 nm)) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg⁻¹, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg⁻¹ in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L⁻¹ of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L⁻¹ ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency.