Biological pretreatment of oil palm empty fruit bunch using crude laccase from Pycnoporus sanguineus for sugar production

• Main Author: Masran, Ruqayyah
• Format: Thesis
• Language: English
• Published: 2020
• Subjects: Crop residues - Utilization - Malaysia; Laccase - Research; Lignocellulose - Biodegradation
• Online Access: http://psasir.upm.edu.my/id/eprint/104602/1/FBSB%202020%2035%20IR.pdf; http://psasir.upm.edu.my/id/eprint/104602/

To date, Malaysia rank in the second place as a global palm oil producer. Despite the rapid growth of oil palm plantation in Malaysia, a non-systematic biomass management contributes to biomass accumulation in huge amount. Oil palm empty fruit bunch (OPEFB) can be categorized as one of the toughest lignocellulosic biomass to be degraded naturally due to its complexity in structure. Conventional industrial practice used chemical and physical pretreatments to treat the OPEFB as it performs faster in hydrolyzing the biomass than biological pretreatment does. However, as the world is moving towards green technology concept, chemical pretreatment is no longer suitable to be practiced because it produces harmful by-products which requires proper management prior to its disposal. One of the methods currently sparking interest is biological pretreatment using laccase. In this study, OPEFB was pretreated biologically using crude laccase and subjected to enzymatic hydrolysis using cellulase to produce sugars. Application of laccase has become a current trend in biological pretreatment of lignocellulosic biomass where laccase aids in degrading and modifying the lignin barrier. This condition subsequently loosens up the biomass structure and helps to improve the cellulase accessibility towards cellulose. Naturally, wild type fungi produce ligninolytic enzymes in low concentration and inducers have shown promising result in enhancing ligninolytic enzymes production. Pycnoporus sanguineus UPM4 was utilized in this study and was found to be a dominant laccase producer. Therefore, enhancement using selected laccase inducers which are veratryl alcohol, ferulic acid, Kraft lignin, copper sulfate and 2,5-xylidine were carried out at different concentration, respectively. Veratryl alcohol, ferulic acid and Kraft lignin were found to be able to enhance the laccase production, meanwhile copper sulfate and 2,5-xylidine inhibited the laccase production. Veratryl alcohol with a concentration of 16 mM has shown to be to best inducer with resulting laccase production of 6.35 U/mL (2 folds increment). Response surface methodology (RSM) was employed to optimize the biological pretreatment of OPEFB using enhanced crude laccase synthesized by P. sanguineus UPM4. Investigation and screening of significant variables was performed using one factor at time (OFAT) and two-level factorial design. From the analysis of variance (ANOVA), temperature, initial pH and laccase loading have resulted in major effect on the pretreatment process, whereas substrate concentration and incubation time were found to be insignificant. These three variables were further analyzed using Central Composite Design (CCD). The optimum pretreatment condition attained from the model were temperature at 50°C, initial pH of 4.5 and 65 U/g of laccase loading. Hence, biological pretreatment performed at this optimum condition resulted in 13.08% of lignin removal and yielded 20.70 g/L of sugars from pretreated OPEFB. Additionally, characterization of pretreated OPEFB has revealed a remarkable change occurred on the substrate which was evident through scanning electron microscope micrograph, surface functional groups and surface oxide groups. Scanning electron microscope micrograph displayed formation of craters on substrate surface due to removal of silica bodies on the pretreated OPEFB. Next, an alteration of surface functional groups on the pretreated OPEFB was demonstrated by FTIR spectrum and it was further explained by surface oxide groups analysis showing an increment of carboxyl groups and decrement of lactone and phenolic groups in the pretreated OPEFB. A study on the feasibility of simultaneous pretreatment and saccharification of OPEFB was carried out. It was observed that cocktail of crude laccase at 45 U/g and Acremonium cellulase at 25 FPU/g has resulted in 8.81% of lignin removal and 8.16 g/L of sugars. As a conclusion, veratryl alcohol has increased the laccase production with 2 folds increment. Optimization of biological pretreatment of OPEFB using crude laccase has increased the lignin removal by 1.2 folds and sugars production by 1.8 folds. Besides that, simultaneous pretreatment and saccharification of OPEFB using cocktail of crude laccase and Acremonium cellulase were a good combination as compared to Trichoderma reesei crude cellulase and Celluclast 1.5L, respectively. Overall, in comparison to previous research, this study demonstrated two novel approaches of biological pretreatment of OPEFB. Optimum operating conditions in separate pretreatment and saccharification of OPEFB has resulted in 56.63% of hydrolysis yield. Meanwhile, the simultaneous pretreatment and saccharification of OPEFB at laboratory scale proved that lignocellulolytic enzymes cocktail was feasible and able to attain hydrolysis yield at 32.62%, in conjunction with reduction of time consumption, number of vessels as well as elimination of substrate washing step.