Research Article
Kinetics of Cellulase and Xylanase of Chaetomium thermophile with Respect to Aeration
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Haq Nawaz
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Farooq Latif
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M. Asghar
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Microbial degradation of the biological wastes is a natural process that has occurred since the on set of life on earth. In fermentation processes micro-organisms utilize the wastes as potential energy source for synthesis of very useful products such as enzymes and biomass/single cell proteins. Cellulases are carbohydrases that hydrolyze the B-1, 4 linkages of cellulose or its chemically modified forms, in addition to degrading cellodextrin or cellobiose. Typically they are multienzyme complexes bearing endo-I, 4-βglucanase, cellobihydrolase and β-glucosidase activity (Goyal et al., 1991).
Fungal cellulases are used alone or in combination with pectinase, β-glucanases and amylases in brewing, cereal processing, fruit juice extractions and wine production and alcoholic, fermentation. Cellulases also have been used to improve the palatability of low-quality vegetables, increase the flavour of mushrooms, promote the extraction of natural products and alter the texture of foods (Coughlan, 1985; Maerk et al., 1990). Xylanases are the key enzymes for breakdown of xylan since they depoymerize the backbone structure. They have potential applications in biopulping, nutritional improvement of lignocellulosic feedstock, production of ethanol and methane, and in the processing of food (Wong et al., 1988). Hemicellulases also are used to improve the properties of doughs used in the production of based goods (Kulp, 1968).
Keeping in view the significance of cellulase and xylanase the project was undertaken to study the kinetics of cellulase and xylanase production by culturing Chaetomium thermophile on wheat straw substrate.
Materials and Methods
Substrate: Wheat straw collected from NIBGE was dried in hot air at 70oC. It was ground in an electric grinder (1 mm mesh) and stored in polythene bags for subsequent use as fermentation medium.
Growth Medium: Growth medium was prepared using ingredients from Sigma company, following the method of Eggins and Pough (1962).
Microorganisms: Pure culture of Chaetomium thermophile was obtained from NIBGE fungal stocks and maintained on growth medium.
Inoculum: Inoculum medium was prepared and its pH was adjusted at 4.5. It was sterilized and spores of fungus were transferred into it. The medium was then placed on orbital shaker (120 rpm) for 24 hours at 45oC. Then it was aseptically transferred to the fermenter containing optimum growth medium (Table 1) of wheat straw for production of cellulase and xylanase. The fermentation conditions of bioreactor were: working volume of 20L, aeration, 0.25-0.75 vvm, pH, 4.5-5.5, stirring 300 rpm and temperature 45oC. After optimum fermentation period the biomass was filtered and filtrate was centrifuged for 15 minutes. The supernatant was then filtered through milipore filter to get a clear filterate. This filterate thus obtained was used for enzyme assays.
Table 1: | Composition of inoculum medium (pH of the medium 4.5-5.5) stirring 300rpm and temperature 45°C |
Table 2: | Maximum cellulase activities at different aeration rates |
Enzyme Assays: Xylanase activity was assayed by spectrophotometer (Miller, 1959) using crystalline oatspelt xylan as substrate and DNS as coupling reagent.
β-glucosidase activity was determined according to the method of Roy et al. (1991).
Filter paperase and endoglucanase (CMCase) activities were determined as described by Gadgil et al. (1995) using filter paper strips and carboxymethyl cellulose (CMC) as substrates, respectively.
Bradford (1976) method was followed for the determination of extracellular protein in biomass.
Potential kinetic parameters such as volumetric rate of substrate utilization (QP) and volumetric rate of enzyme production (Qs) by C. thermophile were also determined (Rajoka and Shahid, 1998).
The results relating to effect of different aeration rates on cellulase and xylanase production have been presented and discussed here under:
Effect of Fermentation time and aeration on Xylanase activity: It was observed that xylanase activity started increasing at 16 hours and became maximum at 72 hours.
Table 3: | Kinetic values of different enzymes of Chaetomium thermophile in 20L fermenter |
Enzyme activity was also determined at three aeration rates but 0.25 vvm gave maximum activity (18.2 u/ml) for the breakdown of its xylan (Fig. 1). The results are supported by Palma et al. (1996) who studied the growth of the P. janthiellum and reported the highest enzymic activity (8.90 ml-1) at a very low oxygen supply and ka of 1.24 h-1.
Fig. 1: | Xylanase activity (U/ml) at different aeration rates |
Fig. 2: | Extracellular protein (mg/ml) at different aeration rates |
Effect of aeration on cellulase activity: Three major components of extracellular (free) cellulase (FPase, endoglucanase and β-glucosidase) showed maximum (1.2, 1.3, 1.6 U/ml) respectively activities at aeration rate of 0.25 vvm, while 0.50 and 0.75 vvm gave somewhat less activity (Table 2). It was observed that enzyme activity was also effected by fermentation period. It was maximum at 72 hours (Fig. 2). The results are in line with those of Umikalsom et al. (1998).
Effect of aeration on extracellular protein: The maximum (0.88 mg/ml) protein production was observed in the fermentation medium which was controlled at 0.25 vvm for 72 hours (Fig. 2). These results are in agreement with those of Illanes et al. (1992) who performed solid state fermentation for cellulase production on leach beet pulp by native and mutant strains of Trichoderma aurociride in column type solid substrate fermenters. Protein enrichment of residual solid was significant in all cases.
The results of all enzyme assays showed maximum activity at 72 hours (Fig. 1).
Kinetic values of different enzymes have been presented in Table 3. These values seemed to be in dependent of aeration rates, for all the enzymes.