Production of Cellulase and Xylanase from Oil Palm Trunk and Frond and Their Applications

Abstract

Oil palm trunk (OPT) and oil palm frond (OPF) are abundant biomass in Southern Thailand. The major chemical composition of OPT varied along its height and found to accumulate starch along its height with the highest quantity at the top end part (1 m long) of 3.79% (w/w of the top end part). After extraction of sugars, the sap from the bottom part and middle part of OPT had the highest glucose (20.13 and 12.74 g/L, respectively) and fructose (3.04 and 6.02 g/L, respectively), respectively. The OPF sap contained the highest glucose concentration (25.42 g/L) followed by fructose (3.66 g/L). The ground OPT and OPF fibers were fermented at various temperatures for 15 days. The maximum carboxymethyl cellulase (CMCase) (0.48 Unit/gds) was obtained from fermentation of OPT at room temperature for 15 days while the maximum xylanase activity (0.44 Unit/gds) was achieved from fermentation of OPF at room temperature for 3 days (0.44 Unit/gds). There was no enzyme activity after fermentation of OPT and OPF at 50 °C. The microbial community analysis of the natural fermentation of OPT and OPF revealed high diversity of bacteria with low diversity of yeasts and fungi. The total of 20 fungal strains were isolated and only eight of them could grow on agar plate containing OPT and OPF residues (after sugar extraction). They were encoded as the isolate TT, TT2, TT3, TT4, TTS, TM1, TM2 and TM3. They were compared for their ability to produce cellulase and xylanase under SSF and SmF. The isolates TTI, TM3 and TT2 produced the highest enzyme activity and identified as Ceratocystis paradoxa, Trichoderma koningiopsis and Hypocrea nigricans, respectively. Time-courses of enzymes production from these strains were conducted. Oil palm trunk residues (OPTr) was a better substrate for enzymes production than oil palm frond residues (OPFr). C. paradoxa TTI gave the highest CMCase (18.16 Unit/gds) in SmF while T. koningiopsis TM3 exhibited the highest xylanase (56.46 Unit/gds) and FPase (2.13 Unit/gds) production in SSF. The inoculums of the two newly isolated strains C. paradoxa TTI and T. koningiopsis TM3 were prepared in packed dried form with the quantity. The inoculums contained of 1.2 x 10° and 1.6 x 10* CFU per g dry weight, respectively. After storage at room temperature and 4 °C for 6 months, only the inoculum of T. koningiopsis TM3 remained at the same level (approximately 10% CFU per g dry weight). In contrast, the survival of the formulated TTI decreased sharply to 30-45% of their original values. Lignocellulolytic enzymes production from the formulated inoculums and spore suspension inoculum of C. paradoxa TTI and T. koningiopsis TM3 (individual and mixed inoculum) were compared with the mixed cultures from the Land Development Department (Super LDD1). Results in SSF and SmF revealed that the formulated inoculums TM3 and TTI produced the lignocellulolytic enzymes higher than the Super LDDI and similar to that of the spore suspension form. The formulated inoculum TM3 produced the lignocellulolytic enzymes higher than the formulated inoculum TTI. Oil palm biomass (OPTr, OPFr, EFB, decanter cake and plam pressed fiber (PPF)) were used as substrates for production of enzymes under SSF and POME under SmF by the formulated inoculum T. koningiopsis TM3. The maximal CMCase and xylanase activities (4.44 and 63.17 Unit/gds, respectively) were obtained when OPTr was used as a carbon source. Characterization of the enzymes revealed the optimum temperature for CMCase and xylanase at 50 °C while the optimum pH of CMCase and xylanase were in the pH range of 4.4-4.8 and 4.8-5.6, respectively. Thermal stability study revealed that CMCase retained more than 75% of their activities after 5 h incubation at 40 °C and lower than 50% of its activity at room temperature (30÷2 °C), 50 and 60 °C. The xylanase exhibited lower thermal stability as it only preserved 75% of its activities at temperature below 40 °C and lost 73% and 79% of its activity within 1 h of incubation at 50 °C and 60 °C, respectively, while CMCase lost only 21% and 31% of its activities under the same condition. The crude enzyme obtained above from the formulated T, koningiopsis TM3 was precipitated by using acetone. The activity of CMCase and xylanase increased 6 and 6.8 fold, respectively (3.22 and 54.14 Unit/ml, respectively) with the recovery yields about 60 and 68%, respectively. The concentrated enzymes were used to hydrolyze OPTr by using the enzymes concentrations in the range of 0-40 Unit/g OPT and incubated at 50 °C for 24 h. Enzymatic hydrolysis of OPTr revealed that the maximum reducing sugars of 11.92 g/L with the yield of 0.48 g/g were obtained by hydrolyzing with 25 Unit/g of the enzymes at 50 °C for 15 h. For ethanol production from the OPTr hydrolysate (without nutrients added), Saccharomyces cerevisiae TISTR5055 was more efficient than Candida shehatae TISTR5843 and the co-cultures. Two-stage process and simultaneous fermentation using the co-cultures of S. cerevisiae and Acetobacter aceti were compared. Supplementation of YM nutrients to the OPTr hydrolysate exhibited strong influence on ethanol production (4.10 g/L at 12 h in two-stage process and 4.01 g/L at 18 h in simultaneous fermentation) but not acetic acid production. Without nutrients addition, the maximum acetic acid concentration and productivity (2.12 g/L at 24 h) were achieved from the simultaneous fermentation of the co-cultures which were 1.7 folds and 4 folds higher than those from the two-stage process (1.23 g/L at 54 h). The efficacy of concentrated enzymes in hydrolyzing sterilized POME and OPTr at 40 and 50 °C was evaluated prior to methane fermentation and its co-digestion. The maximum sugars concentrations of POME hydrolysate were obtained from enzymatic hydrolysis using 15 Unit/g TVS at 50 °C for 18 h incubation (glucose 16.69 g/l, xylose 4.39 g/l, cellobiose 3.49 g/l and arabinose 2.35 g/l) with the yield of 0.3521 g/g TVS. The OPTr hydrolysate had the total sugar concentration of 23.28 g/L (19.41 g/l glucose, 1.09 g/l xylose, 2.18 g/l cellobiose and 0.60 g/l arabinose) under the same condition. Methane potential of the POME hydrolysate (1,243 ml CH,/g VS-added) increased by 15.3% compared to the raw POME (1,078 ml CHug VS-added). Meanwhile, the methane potential of raw OPTr was slightly (3.7%) higher than that of OPTr hydrolysate (1,402 and 1,350 ml CH./g VS-added, respectively). Co-digestion of POME hydrolysate with OPTr gave the best result of methane yield (1,340 ml CHag VS-added). The dominant archaea that played an important role in methane production were Methanoculleus sp. and Methanosarcina sp.. These results indicated that enzymatic pretreatment and co-digestion of POME hydrolysate with OPTr could improve biogas yield from anaerobic fermentation.