OPTIMIZATION OF MICROWAVE-ALKALINE PRE-TREATMENTS CONDITIONS AND CO-IMMOBILIZATION OF XYLANASE, CELLULASE AND FUNGI SPECIES FOR THE PRODUCTION OF BIOETHANOL FROM SOME AGROWASTES

Abstract

The need for bioethanol production arises from drawbacks of fossil fuel such as non renewable feedstock and emission of dangerous gases during combustion. However, the recalcitrant nature of bioethanol feedstock as well as high cost of hydrolytic enzyme and incomplete fermentation of sugars have been major challenges. The research was aimed at optimizing microwave-alkaline (MA) pre-treatment conditions for the effective delignifica tion of some selected locally available agrowastes and co-immobilization of cellulase, xylanase, and as well co-immobilize yeast and fungi for optimal production of bioethanol. Microwave-alkaline pretreatment conditions, generated using Box-Behnken of design expert software version 11, were optimized for the pretreatment of sugarcane bagasse (SB), plantain pseudostem biomass (PS), and corncob (CC). The pre-treated agrowastes were characterized using Fourier Transform infrared spectroscopy (FTIR), X-Ray crystallography (XRD), and Scanning electron microscopy (SEM). Microorganisms were isolated from different soil sources, palm wine and fermented food to produce enzymes and for fermentation process. Biochar made from sugarcane bagasse was mixed with chitosan to co-immobilize xylanase, cellulase, and fermentation organisms forsimultaneous saccharification and co- fermentation (SScF). The functional properties of co-immobilized enzymes and co-immobilized fermentation organisms were characterized. Results showed the optimal MA pre-treatment conditions for plantain pseudostem biomass (1.97% NaOH, 70W, 5 min), sugarcane bagasse (3% NaOH, 96W, 5 min), and corncob biomass (2.8% NaOH, 86W, 4.4 min). Characteristics of pre-treated agrowastes differed from unpre-treated ones. Aspergillus flavus (OP107821) and Aspergillus niger (OP107822) produced cellulase and xylanase, respectively, while Saccharomyces cerevisiae (OP107824) and Mucor indicus (OP107823) fermented glucose and xylose respectively. Free cellulase, free xylanase, and co-immobilized enzymes had optimal temperatures of 50, 60, and 70 °C respectively and pH values of 5, 6, and 5-7 respectively. Free cellulase, free xylanase, and co-immobilized enzymes had Km values of 0.008 mg/mL, 0.028 mg/mL, and 0.022 mg/mL respectively and Vmax values of 129.87, 119.05, and 114.94 μMmin-1 respectively for plantain pseudostem biomass substrate. Likewise, the Km values for free cellulase, free xylanase, and co-immobilized enzymes were 0.017, 0.006, and 0.018 mg/mL, respectively, and the Vmax values were 333.33, 555.47, and 227.25 μMmin-1 for sugarcane bagasse substrate. Free cellulase, free xylanase, and co immobilized enzymes had activation energies of 15.899, 29.218 and 3.450 kJ/mol, respectively, and deactivation energies of 48.235, 39.596, and 52.145 kJ/mol respectively. After 10 usages, the co-immobilized enzymes retained 55.13% of its activity. Co immobilized enzymes released 32.47 ± 0.89 mg/mL, 36.62 ± 1.90, and 17.42 ± 0.41 mg/mL from PS, SB, and CC, respectively. Co-immobilized enzymes and co-immobilized fermentation organisms yielded the most bioethanol, 68.93±0.33g/L from PS and 76.09±0.15g/L from SB. GC-MS profile of distillate showed other valuable products, but SB and PS had 80.84 and 71.15 percent ethanol, respectively. This study showed that microwave-alkaline pre-treatment reduces recalcitrance of lignin, releasing more cellulose and hemicellulose for hydrolysis. The study also found that locally-sourced enzymes and organisms co-immobilized on locally produced matrix can produce high-yield of bioethanol, which may make relatively cheap and eco-friendly fuel for domestic and industrial uses. Considering the seasonal availability of different agrowastes based on harvesting times, it is recommended that in a future study, a mixture of different agrowastes that are available at the same time be optimized for bioethanol production