Bioethanol and xylitol production from delignified wild elephant grass using novel tailor-made recombinant crude enzyme cocktail and Candida tropicalis MTCC 230

Abstract

This study establishes an integrated biorefinery framework for valorizing Elephant grass (EG) into sugar-based bio-products and high-value lignin derivatives. EG was subjected to alkaline hydrogen peroxide (AHP) pretreatment, with parameters optimized via Response Surface Methodology (RSM). The optimal conditions (4.2% AHP, 101.8°C, 147.6 min, 7.5% solids) achieved 71% delignification and 720 mg/g total carbohydrate content, alongside enhanced crystallinity and disrupted surface morphology. Concurrently, lignin recovered from AHPpEG hydrolysate (62.5% yield, 88% purity) exhibited strong antioxidant activity (RSI = 5, EC₅₀ = 0.2 mg/mL), with MALDI-TOF MS and ESI-HRMS revealing guaiacyl-guaiacyl dimers. A tailor-made enzyme cocktail was designed through D-optimal methodology, combined five recombinant crude enzymes and a chimera enzyme in a 9:4:2:2:3 ratio. The cocktail exhibited notable stability and gave 544 mg/g with TritonX-100 at 15% solid loading with 435 mg/g glucose and 106 mg/g xylose. The hydrolysate was fermented using Candida tropicalis in a separate hydrolysis and fermentation (SHF) system. Fermentation parameters, initial inoculum, sugar-to-nitrogen ratio and shaking speed were modeled using RSM and Artificial Neural Network Genetic Algorithm (ANN-GA), with ANN-GA outperforming RSM in predictive accuracy. The optimal conditions (10% inoculum, S:N ratio 37.4, 250 rpm) yielded 27.4 g/L ethanol (0.42 g/g glucose) and 5.1 g/L xylitol (0.44 g/g xylose). Scale-up in a 5 L bioreactor confirmed robustness, producing 28 g/L ethanol and 7.5 g/L xylitol. Overall, this work demonstrates a novel, integrated biorefinery approach combining efficient pretreatment, customized enzymatic hydrolysis and ANN-GA optimized fermentation.