Solid-state fermentation and optimization of cellulase production using local fungal isolate

Abstract

Background Cellulase is the most employed industrial enzyme in biological conversion of many cellulosic wastes. In this work, economic cellulase production by fungi in solid-state fermentation (SSF) by using solid wastes of medicinal plants was studied. Optimization of growth conditions for production of cellulase was the main target of this study. Objective The current study aimed to isolate and screen fungal isolates that have the ability to produce enzymes to degrade solid wastes of medicinal plant process and optimization of growth factors that affect cellulase production. Materials and methods Thirty-five fungal isolates were isolated from different sources by plating and screened for their cellulase activities using Czapek–Dox broth medium amended with 1% cellulose. Cellulase production by tested fungal isolates was carried out through utilization of olive (Olea europaea), black seeds (Nigella sativa), and castor bean (Ricinus communis) cakes in SSF. Optimization of the cellulase productivity was performed by Plackett–Burman design (PBD) and Box–Behnken design. Results and conclusion Out of the isolated 35 fungi, only 12 (34%) produced cellulase in SSF using olive, black seeds (Nigella), and castor bean cakes. Out of these fungal isolates, only 4, that is, no. 1, 7, 10, and 17 were superior in reducing sugar production from olive cakes (13.04, 15.61, 17.03, and 12.85 mg/ml), respectively. While four fungal isolates no. (1, 7, 7, and 10) were active producers of reducing sugars from black seeds (15.45, 18.96, 20, and 18.08 mg/ml), respectively. Only a fungal isolate no. 7 gave high reducing sugars (15.34 mg/ml) in castor cake SSF. The most potent fungal isolate (no. 10) produced 20 mg/ml of reducing sugars using black seed cakes as substrate for SSF. The potential fungal isolate was identified as Aspergillus terreus (OQ085169) based on the extracted fungal DNA that was amplified by PCR using specific internal-transcribed spacer primer (ITS1/ITS4). The PCR products were sequenced and compared with the other related sequences in GenBank (NCBI). The screening of seven factors using PBD showed that only three variables: pH, incubation time, and aeration rate (rpm) affected significantly cellulase production. Box–Behnken design was used to estimate the optimal level of the selected variables based on the results of the PBD. All variables increased significantly cellulase using A. terreus (OQ085169). The P value was very low (0.0207) that indicated the significant, high correlation between the predicted and actual values (R2=0.98), this indicating 98% of the variation in the cellulase activity was owing to the selected independent variables.