Immobilization of cellulase and yeast for the hydrolysis and fermentation of pre-treated bagasse for ethanol production

Abstract

Lignocellulose ethanol promises to be the cheapest form of fuel, however, the drawback in the production is in the pretreatment process to remove lignin and the efficient hydrolysis of free cellulose. This research work is designed to delignify sugarcane bagasse, hydrolyze and ferment it with immobilized cellulase from the snail gut isolates and yeast respectively. The biomass were pretreated with Ca(OH)₂ and then placed in the water-bath with temperature of 20⁰C, 40⁰C, 60⁰C, 80⁰C,100⁰C and 120⁰C. The pretreated biomass was hydrolysed with free and immobilized cellulase at 50⁰C for 5-48hrs. The activity, optimum pH, optimum temperature, substrate concentration profile and kinetic parameters, V_max and K_m of cellulase were also determined. The optimum pH for free and immobilized cellulase ranged from 4.0-5.5 and optimum temperature was recorded at 45⁰C and 55⁰Cfor free and immobilized cellulase respectively. The effect of temperature on both free and immobilized cellulases showed that immobilized cellulase has higher resistance to temperature than the free cellulase. Also the yield of glucose (40mg/ml) was higher with immobilized enzyme after 24hrs. The results obtained has also shown that immobilized cellulase has a higher Km when compared with free cellulase The maximum reaction rate (V_max) obtained from Michaelis Menten plots was lower for immobilized cellulase than for the free enzyme. Higher value of V_max for free enzyme indicated that the enzyme converted more substrate to product per unit time upon saturation with substrate. The biomass was fermented for 48hrs with immobilized Saccharomyces cerevisiae and the results showed the ethanol yield of 31.75% at 24hrs and 70.84% at 48hrs. The initial glucose concentration was 40mg/ml and this significantly reduced to 6.21mg/ml after 24hrs and 1.25mg/ml after 48hrs of the fermentation process. These results showed a proportional increase in ethanol yield against a depleting concentration of glucose which is being used up in the fermentation reaction revealing the maximum efficiency of the immobilized yeast cells. In this study, it has shown that the entrapped cellulase cells produced high levels of reducing sugars in hydrolysis compared with their native counterparts and immobilized yeast cells also gave a high yield of ethanol. The immobilization process therefore obtained more thermostable biocatalysts with increased productivity which is more economical for biofuel production.

Keywords: Immobilization, Cellulase, Saccharomyces cerevisiae, biocatalysts, delignification