Production of Ionic Liquid Tolerant Cellulase from Bacillus subtilis G2 Using Agroindustrial Residues with Application Potential for Saccharification of Biomass Under One Pot Consolidated Bioprocess

Abstract

Ionic liquid (IL) based pretreatment of lignocellulosic biomass for facilitating efficient enzymatic saccharification has emerged as an environmentally benign approach that offers several advantages over conventional strategies. However, residues of ionic liquid left in the pretreated biomass may cause inactivation of saccharifying enzymes thus, necessitating the requirement of ionic liquid-stable enzymes. Cost-effective production of industrial enzymes is always desired to enhance the overall process economy. Current study reports IL-stable cellulase production from a newly isolated bacterium Bacillus subtilis G2. Design of experiment (DoE) based on response surface methodology was used in sequential manner for optimizing cultural and environmental variables to enhance cellulase production by 2.66-fold. IL-stable cellulase was used for saccharification of IL-pretreated pine needle biomass (PNB) with 1-ethyl-3-methylimidazolium methanesulfonate in a consolidated single pot process i.e. one pot consolidated bioprocess (OPCB). The saccharification efficiency of 23.57 % was observed under OPCB. The hydrolsate obtained was fermented by dual culture of yeast i.e. Saccharomyces cereviasie NCIM 3078 and Pichia stipitis NCIM 3497, and a yield of 0.092 g ethanol/g of PNB was obtained with fermentation efficiency of 25.62 %. This study is first ever where-in IL-stable cellulase production is accomplished using agroindustrial residues by employing DoE, and assessed for its application potential under OPCB for saccharification of IL-pretreated PNB. IL-stable cellulases would not only preclude expensive washing step following IL-pretreatment of biomass, but their application in a consolidated single pot process (OPCB) offers numerous technoeconomic advantages over conventional multi pot processes. Production of ionic liquid (IL) tolerant cellulase from Bacillus subtilis G2 was enhanced by 2.66-fold using response surface methodology. Cellulase was assessed for its saccharification potential on IL-pretreated pine needle biomass under one pot consolidated bioprocess, and the hydrolysate was fermented to ethanol using Saccharomyces cereviasie and Pichia stipitis.