One pot consolidated bioprocess for conversion of Saccharum spontaneum biomass to ethanol-biofuel

Highlights: • Bioprocessing of Saccharum spontaneum biomass (SSB) for biofuel-ethanol production. • Ca(OH)₂ assisted choline chloride-glycerol ChCl-G for biomass pretreatment. • Hydrolysis of pretreated SSB using ChCl-G-stable cellulase/xylanase (B. subtilis G₂) • Optimized consolidated bioprocess for pretreatment and saccharification of SSB. • Mechanisms of SSB pretreatment by SEM, FT-IR, XRD, NMR, WRA, BET, enzyme adsorption. Lignocellulosic biomass (LB) based ethanol-biofuel represents a promising, sustainable, clean and renewable energy resource to meet the ever-growing global energy demand, and to mitigate the environmental issues related to excessive fuel usage. However, the technology faces multifaceted challenges like high cost/energy intensive and environmentally-unsafe LB pretreatments, lack of appropriate saccharification enzymes, poor process efficacy, and others. Deep-eutectic-solvent (DES) based LB pretreatment has recently emerged as an eco-benign approach which offers multitude of advantages over conventional methods of biomass conversion. Consolidated bioprocessing, CBP provides discreet benefits over traditional multi-reactor biomass processing. Scanty reports are available on application of Saccharum spontaneum biomass (SSB) as a promising biorefinery feedstock despite its huge potential. No studies have yet been undertaken on CBP of SSB by using DESs, and the present study is first such report. In the current study, SSB after pretreatment with DES i.e. choline chloride-glycerol (ChCl-G) and Ca(OH)₂ was subjected to in-situ enzymatic hydrolysis with DES-compatible cellulase-xylanase enzymes (Bacillus subtilis G₂) followed by ethanol fermentation of sugars, in one pot consolidated bioprocess. Optimization of CBP for pretreatment and enzymatic saccharification via design of experiment resulted in substantially increased sugar yield (4.94-fold) i.e. from 75.25 mg/g biomass (unoptimized) to 372.3 mg/g biomass (after optimization). Fermentation of sugars under CBP yielded an ethanol content of 173.61 mg/g of SSB (60.28% of the theoretical yield). Extensive characterization of pretreated SSB based on SEM, FT-IR, XRD, NMR, surface area measurements, cellulase adsorption isotherms, and water retention capacity further provided molecular insights into the pretreatment mechanisms.