ISOLATION OF BACTERIA WITH POTENTIAL FOR PRODUCTION OF THERMOSTABLE EXTRACELLULAR CELLULASE FROM SAWDUST

Abstract

Cellulolytic enzyme is a major renewable source of production of cellulase with high potential for degradation of lignocellulosic waste materials into useful end-products. Industrial processes involved in recycling lignocellulosic wastes generate heat which reduce efficiency or denature cellulase used in degradation of such wastes. There is an urgent need to manage bulk wastes effectively and economically by the use of thermostable cellulase. The aim of this study was to isolate thermophilic bacteria with potential for producing thermostable cellulase useful in degradation of lignocellulosic wastes.

Sawdust samples were purposely collected from sawmills in Ede, Osun State. Bacteria were isolated from the samples, identified and screened for cellulase production using standard microbiological procedures. Isolates with cellulase activity were selected, studied for thermostable cellulase production and their stability were determined. Optimisation of cellulase production was carried out using different carbon and nitrogen sources, temperature, pH and metal ions in pre-treated and untreated sawdust, corncob and sugarcane bagasse as substrates. Isolates with significantly high cellulase production were selected and identified by PCR amplification of 16SrDNA genes. Cellulase was purified using ammonium sulphate precipitation, dialysis and affinity chromatography, and used to hydrolyse lignocellulosic materials. Data were analysed using descriptive statistic and ANOVA at p= 0.05.

Out of 79 bacteria isolated from sawdust samples, 20 were cellulase producers and identified as Actinomyces nueslundii (4), Pseudomonas aeruginosa (12), Thermoactinomyces vulgaris (1), Roseomonas sp. A1 (1), and Anoxybacilus rupiensis (2). Roseomonas sp. (A1), Anoxybacilus rupiensis E1 and Anoxybacilus rupiensis 5H were the best three thermostable cellulase producing bacteria. Their extracellular enzymes were stable at 600C to 650C for 1hour, and 500C to 550C for 2hour. These isolates exhibited significant differences in cellulase production in the presence of carbon and nitrogen sources at 0.5% to 2.5% (w/v) concentration. Anoxybacillus rupiensis (5H) in medium supplemented with 1% carboxymethylcellulose produced 9.22 U/mL of cellulase while Anoxybacillus rupiensis (E1) produced 3.0 U/mL of cellulase in medium supplemented 1% (w/v)tryptone. Optimum production of cellulase was at 50oC and pH of 7, while medium supplemented with magnesium sulphate at 1% (w/v) concentration produced 7.78 U/mL of cellulase. Significant difference was observed in the production of cellulase from corncob, sugarcane bagasse and sawdust at varying concentrations of 1.0-3.0 % (w/v) by Roseomonas sp. (A1), Anoxybacillus rupiensis (E1) and Anoxybacillus rupiensis (5H). Purified cellulase obtained using Sephadex G-75 gave the best activity (26.7 U/mg). Highest percentage lignocellulose hydrolysis by enzymes from Roseomonas sp. A1 and Anoxybacillus rupiensis E1 (21.0 and 29.2 respectively) was obtained in sugarcane bagasse; while 38.9 hydrolysis in the corncob was recorded for Anoxybacillus rupiensis 5H.

Roseomonas species and Anoxybacillus rupiensis demonstrated high hydrolytic action on lignocellulosic materials at high temperature. The cellulolytic enzymes produced by these organisms will be of potential application in lignocellulosic industrial processes that require elevated temperature. Keywords: Thermostable-cellulase, Lignocellulosic waste, Roseomonas and Anoxybacillus Word count: 479