Cellulase and xylanase-producing Trichoderma Asperellum and Rhizopus Oryzae for effective saccharification of oil palm frond leaves by solid-state fermentation

Abstract

Cellulase, xylanase and pectinase contribute almost 20% to the world enzyme market. The growing demand for cellulases and xylanases in lignocellulosic degradation and reutilization has instigated the need for their improved production at a low cost. This study, therefore, evaluated oil palm frond leaves (OPFL) as a cheap and sustainable growth substrate for two novel fungi species to produce cellulase and xylanase under solid-state fermentation (SSF). Morphology, 18S rRNA, phylogeny and BIOLOG® analyses identified the cellulase and xylanase-producing fungal strains as Trichoderma asperellum UC1 and Rhizopus oryzae UC2. While UC2 is robust and fast-growing, its enzyme production rate is slower and sustained; in contrast, strain UC1 showed a higher production rate of the same enzymes. Using the one variable at a time (OVAT) method, optimised fermentation parameters for strain UC1 (30 °C, 60-80 % moisture content, 2.5 × 106 spores/g inoculum size, 6.0-12.0 pH) and strain UC2 (30 °C, 40 % moisture content, 2.0 × 108 spores/g inoculum size, 6.0-12.0 pH) resulted in a corresponding 2.7, 2.6, 1.1, 1.7 (strain UC1) and a 2.3, 3.3, 1.2 and 1.0 (UC2)-fold increase in CMCase, FPase, ß-glucosidase and xylanase maximum activities. Cellulases and xylanase were produced within a broad pH range between pH 4.0-12.0. Proteome analysis using SDS-PAGE, of the enzyme complexes from in situ hydrolysis of raw OPFL under SSF by strain UC1 and UC2 revealed existence of four endo-ß-1,4-xylanases and endoglucanases, as well as one exoglucanase and ß-glucosidase each for strain UC1 and one endo-ß-1,4-xylanase, endoglucanase, exoglucanase as well as three ß-glucosidases for strain UC2. Compositional and structural analysis (FESEM) of OPFL before and after in situ hydrolysis confirmed their degradation, that resulted in 31.16 % and 75.5 % hydrolysis efficiency for strain UC1 and UC2 enzymes. Furthermore, the enzyme complexes from both strains showed thermophilic and acidophilic characteristics at 50-60 °C and pH 3.0-5.0. Glucose (16.87 and 26.74 mg/g) and fructose (18.09 and 50.83 mg/g) were among the dominant fermentable sugar products from the hydrolysis of OPFL, aside from cellobiose (105.92 and 58.31mg/g) and xylose (1.08 and 1.44 mg/g), by strain UC1 and UC2 respectively. Thermal and pH stability tests for their enzyme cocktails revealed half-lives for UC1 CMCase, FPase, ß-glucosidase and xylanase to be 15.18, 4.06, 17.47, 15.16 h at 60 °C, as well as 64.59, 25.14, 68.59 and 19.20 h at pH 4.0; UC2 - 5.13, 1.48, 18.81, 9.23 h when incubated at 60 °C and 27.55, 12.23, 18.26, 4.43 h at pH 4.0. Optimisation using response surface methodology resulted in maximum activities of CMCase (126.87 U/g), FPase (85.53 U/g) and xylanase (215.42 U/g) under optimised SSF conditions (30 °C, 2.0 × 107 spores/g, 75 % moisture content, pH 6.0) and ß-glucosidase (131.76 U/g) at 32 °C, 2.0 × 107 spores/g, 50 % moisture content at pH 12.0. Enzymatic saccharification on ultrasonicated OPFL yielded 1240 mg/g of total reducing sugar as well as 56.21, 72.68 and 43.83 mg/g of glucose, xylose and cellobiose. The enzymes also enhanced the clarification of orange juice and rising of dough by 82-88 % and 1.7-2.0-fold. Based on the findings, it was apparent that T. asperellum UC1 and R. oryzae UC2 are robust producers of cellulolytic and xylanolytic enzymes using OPFL as the main SSF substrate for the production of large quantities of reducing sugars.