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Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30 °C for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.


INTRODUCTION
The bioconversion of cellulosic materials has been receiving a great attention in recent years and the development of large-scale bioconversion process would alleviate shortages of food and animal feeds, solve modern waste disposal problems and diminish the dependence on fossil fuels by providing an energy source in the form of glucose.
Lignocellulolytic enzymes also have significant potential applications in agriculture and in various industries including chemicals, fuel, food, brewery, wine, animal feed, textile, laundry, pulp and paper (9). On the other hand, lignocelluloses are abundant source of renewable biopolymers in the world, which are continually replenished by photosynthetic reduction of carbon dioxide with the help of sunlight as energy source (1,14). For this purpose research on the enzymatic degradation of experiments, the culture was grown under shaking condition and the fermented medium (broth) was employed as the source of extracellular CMCase.

Effect of inoculum size
Inocula with size ratios ranging from 5% to 25% of the total cultural volume were used. Results in Figure 2 indicated that as the percentage of the introduced inoculum size (1.3 x 10 4 CFU/ml) was raised from 5 to 20%, CMCase progressively increased with rice straw as a carbon source. With regard to sugar cane bagasse, the optimal percent of inoculum size was found to be 10 %. Further increase in inoculum size reduced the level of the formed enzyme.

Influence of pH values
Seven pH values ranging from 3.0 to 8.0 were selected for this study. Results obtained are shown in  shaking condition at an agitation speed of 150rpm was optimal for CMCase formation and used in the following experiments.

Effect of additive carbon sources
Different carbon sources were added at a concentration of 1% with rice straw or sugar cane bagasse (2%) as the main carbon source. As shown in Table 3 CMC was found to be the only inducer for CMCase formation in both agricultural wastes.
Alternatively, D-glucose, D-mannose, maltose, sucrose and cellobiose significantly repressed CMCase formation in case of using rice straw and sugar cane bagasse as carbon sources.

CMCase formation as a function of CMC concentrations
The influences of different concentrations of CMC on CMCase activity were shown in Figure 5.     Table 5, which showed that the enzyme production expressed as relative activity was increased by increasing Tween-80 concentrations to a definite limit till reaching its maximum value at a concentration of 0.4% (v/v Tween-80) for rice straw.
However in case of sugar cane bagasse the addition of Tween-80 did not influence CMCase production. The results obtained also indicate that the CMCase relative activity in case of rice straw increased by about 78% than control. In addition, it was found that increasing Tween-80 concentrations above these values resulted in a gradual decrease in CMCase formation for both agricultural wastes. In the present work, the maximum production of CMCase was inoculum's ratio. In agreement with our results, Fadel (12) reported that maximum CMCase production was obtained with inoculum size of 10 % from A. niger F.119. On the other hand, Chang et al. (4) reported that maximal production of CMCase occurred with the inoculum ratio of 6% of Aspergillus glaucus XC9.
Optimum cultivation temperature depends on the growth kinetics of the microorganism employed rather than on the enzyme produced (22,26). In this study optimum CMCase  (18), Penicillium citrinum (36), and A. niger (34). In this connection Chellapandi and Himanshu reported that the extracellular endoglucanase synthesis is cyclic dependent and therefore subject to catabolite repression by soluble sugar accumulation (5). CMCase formation was increased with increasing CMC concentration up to 0.5 and 1.0% in case of rice straw and sugar cane bagasse, then a gradual decrease take place. This is probably due to the high viscosity of the medium, which decreases the oxygen supply to the cells. High viscosity leads to retard cell division, resulted in low production metabolism and cellulase extraction (13) in agreement with our results.
The obtained data revealed that the best inorganic additive nitrogen compounds stimulated CMCase production was found to be ammonium chloride; whileas ammonium sulphate was recorded in case of Pleurotus dryinus, Aspergillus niger, and Aspergillus glaucus (4,9,12). In case of Bacillus subtilis the rate of CMCase production increased with the addition of sodium nitrate to the medium followed by ammonium sulphate (22).
In this study, CMCase expressed as relative activity was increased by increasing Tween 80 concentrations till reaching its maximum value at 0.4% (v/v) for rice straw, however in case of sugar cane bagasse, the increase of Tween 80 led to a gradual decrease of cellulase production (Table5). The stimulatory effect of surfactants may be a consequence of its action on cell membranes causing increased permeability and/or by promoting the release of cell-bound enzymes and increase cellulase stability and prevent denaturizing of enzyme by desorbing it from cellulose substrate (48). In accordance with our results in case of using rice straw, Tween 80 also induced CMCase production by Aspergillus glaucus, Trichoderma viride (4,27). On the other hand, Micales (28) reported that the presence of Tween-80 did not influence the production or secretion of CMCase of Postia placenta.