Biosugar production from oil palm mesocarp fiber using crude lignocellulosic degrading enzymes from rhizomucor pusillus AK2

Abstract

The oil palm industry is undergoing global expansion as a result of the demand of oil palm. Consequently, similar expansion is occurring in amounts of lignocellulosic residues generated by the industries. The conversion of these residues to biosugar is limited by the uneconomical hydrolysis process and the high cost of lignocellulolytic enzymes. Thus, this study aimed at the production of enzymes for efficient degradation of oil palm mesocarp fiber (OPMF). The sum of 6 fungi isolates, AK1 to AK6, were isolated from OPMF, and screened for lignocellulolytic enzymes production on selective medium. Further screening of these isolates were carried out on untreated OPMF using solid state fermentation (SSF). Design-Expert software version 7.0, was used to operate the 2 level fractional factorial design and Central Composite Design (CCD) for the screening and optimization of significant factors affecting lignocelluose degrading enzymes production. The activity of crude enzyme, Viscozyme and Celluclast were evaluated based on the generation of biosugar from OPMF by determining the effects of pretreatments (2% (v/v) HNO3, 2% (w/v) NaOH and 2% (w/v) 1-Butyl-3-methylimidazolium chloride), solid loading (1-4 % w/v) and enzyme cocktail (1:1 (v/v), crude enzyme and Viscozyme, crude enzyme and celluclast, viscozyme and celluclast, and 1:1:1 (v/v) crude enzyme, Viscozyme and Celluclast). Isolate AK2 exhibited potential for lignocellulase enzyme production based on the hydrolysis zones >1.5 mm on selective media and producing CMCase (25.4 U/g), FPase, (5.5 U/g), β-glucosidase (9.8 U/g), Xylanase (68.4 U/g) and MnP (4.9 U/g) at exceptional level. The isolate was thus identified by 18S RNA gene sequencing using a universal primer ITS1-F and ITS4-R as Rhizomucor pusillus AK2 (KY583064). From the 2 level fractional factorial design pH, temperature, inoculum size and moisture content are the significant factors affecting the production of lignocellulolytic enzymes. Xylanase was observed to be the highest activity in the lignocellulolytic enzymes cocktail (111.01 U/g). Therefore, CCD was carried out focussing on xylanase production. At the optimum condition, xylanase (128 U/g), was obtained at pH 4.98, temperature 40.27 °C, inoculum size at 108.2 spores/g and moisture at 80.64% using CCD. The regression model of the ANOVA was found to be significant with p<0.0001 and R2 of 0.9831.Biochemical characterization of the crude enzymes indicated that the enzyme was stable at pH of 4 to 6 and temperatures of 30 to 60 °C. Enzymatic saccharification was carried out with the crude enzymes comparative to Viscozyme and Celluclast. Maximum sugar production was obtained from celluclast-saccharified OPMF (1.2 g/L) using 1% (w/v) NaOH pretreated OPMF. Maximum reducing sugar generated from enzyme cocktail was 1.8 g/L obtained from 1% (w/v) NaOH pretreated OPMF which translates to polyoses; glucose (2.59 g/L), xylose (2.1 g/L) and arabinose (0.254 g/L) at 1:1:1 (v/v) crude enzyme, viscozyme and celluclast. The performance of the enzyme cocktail customized in this study is superior to that of the individual and cocktail of commercial enzyme. The study also indicates the potential of OPMF as both a substrate for biosugar and lignocellulase enzyme production from R. pusillus AK2.