Saccharification and Co-fermentation of Lignocellulosic Biomass by a Cockroach-gut Bacterial Symbiont and Yeast Cocktail for Bioethanol Production

Background: The eco-friendly transformation of agro-industrial wastes through microbial bioconversion could address sustainability challenges in line with the United Nations' Sustainable Development Goals. The bulk of agro-industrial waste consists of lignocellulosic materials with fermentable sugars, predominantly cellulose and hemicellulose. A number of pretreatment options have been employed for material saccharification toward successful fermentation into second-generation bioethanol. Biological and/or enzymatic pretreatment of lignocellulosic waste substrates offers eco-friendly and sustainable second-generation bioethanol production opportunities that may also contribute to waste management without affecting food security. In this study, we isolated a promising filamentous bacterium from the guts of cockroaches with commendable cellulolytic activity. The matrices of sequential statistics, from one-factor-at-a-time (OFAT) through significant variable screening by Placket-Burman design (PBD) to Box‒Behnken design of a surface methodology (BBD-RSM), were employed for major medium variable modeling and optimization by solid-state fermentation. The optimized solutions were used to saccharify lignocellulose in real time, and the kinetics of reducing sugar accumulation were subsequently evaluated to determine the maximum concentration of sugars extracted from the lignocellulose. The hydrolysate with the highest reducing sugar concentration was subjected to fermentation by Saccharomyces cerevisiae, Klyuveromyces marxianus and a mixture of both, after which the ethanol yield, concentration and fermentation efficiency were determined.

Results: Sequential statistics revealed that rice husk powder, corn cob powder, peptone and inoculum volume were significant variables for the bioprocess at 59.8% (w/w) rice husk powder, 17.8% (w/w) corn cob powder, 38.8% (v/w; 10 cfu/mL) inoculum volume, and 5.3% (w/w) peptone. These conditions mediated maximum cellulolytic and xylanolytic activities of 219.93 ± 18.64 FPU/mL and 333.44 ± 22.74 U/mL, respectively. The kinetics of saccharification of the lignocellulosic waste under optimized conditions revealed two peaks of reducing sugar accumulation between 16 and 32 h and another between 56 and 64 h.

Conclusions: Although K. marxianus had a significantly greater fermentation efficiency than S. cerevisiae, fermentation with a 50:50 (% v/v) mixture of both yeasts led to 88.32% fermentation efficiency with 55.56 ± 0.19 g/L crude bioethanol, suggesting that inexpensive, eco-friendly and sustainable bioethanol production could be obtained from renewable energy sources.