MATERIALS AND METHODS

Microorganisms
A. niger was locally isolated from the soil of decaying mangrove debries (Vellar estuary, India) using Martins Rose Bengal Agar medium (Mycological peptone, 5 g L^-1; D- Glucose, 10 g L^-1; KH₂PO₄, 1.0 g L^-1; MgSO₄, 7H₂O, 0.5 g L^-1; Rose Bengal 0.03, g L^-1; Streptomycin sulfate, 0.1 g L^-1; Agar, 15 g L^-1) and identified by lacto phenol cotton blue method.

Chemicals
All analytical reagents and media components were purchased from Hi-Media (Mumbai, India) and Sigma chemicals (St. Louis, USA).

Production Medium
The liquid medium was prepared with the composition of Rice bran, 30.0 g L^-1; Yeast extract, 0.5 g L^-1; NaNO_3, 5 g L^-1; KH₂PO_4, 1 g L^-1; MgSo₄, 0.2 g L^-1; NaCl, 5 g L^-1 and the pH of the medium was adjusted to 7.0 before sterilization by adding 1N NaOH or 1N HCl.

Cellulase Production
A loopful of spore was inoculated into 50 mL of the production medium in 250 mL Erlenmeyer flasks and incubated at 30°C for 72 h . Five milliliter quantities of samples were withdrawn from each flask after 72 h of fermentation and centrifuged at 2000 rpm for 10 min. The clear supernatant broth was used to determine the enzyme yield. All the experiments were carried out in triplicate and the average values was calculated and used further.

Cellulase Assay
Assay for the cellulolytic activities of cellulase was treated against carboxymethyl cellulose (CMC) (Himedia, Mumbai). 0.1 mL of enzyme solution and 0.9 mL of 1.1% (W/V) CMC in 110 mM potassium phosphate buffer (pH 6.0) were mixed and incubated for 20 min at 40°C. The reducing sugar formed was quantified as glucose by the determination methods using dinitrosalicylic acid (Miller et al., 1959). One unit (v) of CMCase activity is defined as the amount of enzymes, which produce 1 μmol reducing sugar as glucose per min. in the reaction mixture under the specified conditions.

Medium Optimization for Cellulase Production
The factors like temperature (20, 25, 30, 35, 37 and 40°C,), pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0,), incubation period (24, 48, 72, 96, 120 and 144 h ), various substrates (corn cobs, rice bran, wheat bran, cellulose and carboxy methyl cellulose) and various carbon (Galactose, Glucose, Xylose, Lactose, Sugar and Maltose) and nitrogen (peptone, yeast extract, beef extract, casein and malt extract) additives affecting the production of cellulolytic enzyme by A. niger were optimized by adopting search technique varying parameters one at a time.

RESULTS AND DISCUSSION

The white colonies were changed to black, as culture matured. Mycelial hyphae were septate and hyaline. Conidiophores were typically 900 to 1600 μm long, smooth-walled and terminate in pale-brown colored globose vesicles 40 to 60 μm in diameter.

Each vesicle was entirely covered with biseriate phialides that emerge from brown metulae. The phialides undergo blastic basipetal. Conidiogenesis to produce black globose mitospores 3 to 5 μm in diameter. Based on colony morphology and slide culture observation the strain used in the experiment was confirmed as A. niger.

In 72 h of incubation at 30°C and pH 7.0, when the culture was taken for assay, the cellulase activity was detected as 21 U mL^-1. In the different temperature ranges, the cellulase activity was very high (32 U mL^-1) at 30°C (Table 1). Among the various pH ranges the enzyme production was very high (32 U mL^-1) at the pH 6.5 (Table 2). Among the different incubation periods the enzyme production was very high (36 U mL^-1) at 96 (Table 3). In the different types of substrate, carbon, nitrogen used for cellulase production, the wheat bran, Lactose, peptone was found to be the most effective one (Table 4-6).

Table 1:	Cellulase activity in various temperatures

Table 2:	Cellulase activity in different pH

Table 3:	Various incubation time for cellulase production

Table 4:	Different substrates tested for cellulase production

Table 5:	Various carbon sources tested for cellulase production

Table 6:	Different nitrogen sources tested for cellulase production

The A. niger is known to produce a variety of cellulolytic enzymes. The characterization of cellulase from commercial and crude enzyme products from A. niger have been reported previously (Clarke and Stone, 1965; Woodward et al., 1986). In the present study, it was tried with estuary isolate of Aspergillus niger because of its high adaptive nature. The effect of initial pH of the medium on the rate of enzyme production was reported irrespective of the type of substrate used. The monitoring and control of pH in the fermentation processes is not usually attempted, probably because of the difficulties in measuring the pH of the moist solids. In fungal fermentation processes, the buffering capacity of some medium constituents is employed to eliminate the need for pH control (Chahal et al., 1996). However, the hydrogen ion concentration has a marked effect on enzyme production. This may be to the stability of extracellular enzymes at a particular pH and the rapid denaturation at lower or higher pH values (Kalra and Sandhu, 1986). In the present study also the low and high pH inhibited the biosynthesis of cellulase production. Great enhancement in enzyme biosynthesis was recorded at pH 6.5.

Asquieri and Park (1992) found that the optimum temperature for producing CMCase from thermostable Aspergillus sp., was 37°C. Whereas the maximum cellulase production was observed at 35 and 45°C, Penicillium chrysogenum was used (Sharma et al., 1996). Temperature for cellulase production from different fungi on various substrates were studied in the literature by many investigations.(Lonsane et al., 1985; Abdullah et al., 1985) but the present study the data showed that biosynthesis of cellulase was more inhibited by low and high temperature ranges. The optimum temperature the maximum production of these enzymes in the present study by A. niger was recorded as 30°C. The usual temperature maintained in fermentation systems, is usually dependent on the growth kinetics of the microorganism employed for the fermentation purposes (Lonsane et al., 1985) and the selection of incubation temperature is guided by the optimum growth temperature of the culture.

Short incubation period for enzyme production offers the potential for inexpensive production of enzymes. Incubation time necessary for optimal production varied between different enzymes produced from one substrate (Smitt et al., 1996). In the present study the enzyme production was increasing steadily right form the beginning and reached the maximum at 96 h of incubation, then the level start decreasing i.e., 39 U mL^-1 in 96 h , 31 U mL^-1 in 120 h and 24 U mL^-1 in 144 h. The low activity for cellulase detected after 24 h might be because the original substrate contained no compounds stimulating enzyme production. The action of endoglucanase (CMCase) during the early stage of growth resulted in cellobiose production that can induce the biosynthesis of these enzymes at later stages (Godden et al., 1989). Further it was also been reported by Deschamps et al. (1985), that the incubation time required for the formation of such enzymes also varies according to the substrate and microorganisms under study. whereas in some of the previous attempts the cellulolytic enzymes was obtained after 66 h by Tricoderma harzianum (Deschamps et al., 1985), but on the contrary Dhillon et al. (2000) obtained the maximum yield only after 7 days of incubation when Tricoderma reesei QM 9414 was cultivated on rice straw.

The choice of an appropriate substrate is of great importance for the successful production of cellulases. The substrate not only serves as carbon and energy source but also provides the necessary inducing compounds for the organism, preferentially for an extended period, since then a prolonged production phase can result in an increased overall productivity of the fermentation process. In the present study, the wheat bran was the most effective substrate for cellulase production among the cellulosic substrates. The use of wheat straw for cellulase production especially in combination with wheat bran has been well-documented (Fujian et al., 2002). Further, the wheat bran is a familiar and a complete medium for producing cellulase, amylase and xylase.

Cellulase production was found to be dependent upon the nature of the carbon source used in culture media. In the present study among the difficult carbon sources tested, lactose promoted maximal enzyme yield when compare to others. This could be attributed to the rapid growth accomplished by the early availability of carbon source along with substrate wheat bran. The earlier results also suggested that the sugars (carbon sources) supplementation with substrate was found to be most effective nutrient for promoting the activity of cellulase (Roche et al., 1994). The enzyme production is affected significant by under different concentration of the organic nitrogen source. The production of cellulases is sensitive to the nitrogen source and nitrogen level in the medium. The result of the present study showed that the different sources have different effects on enzyme activity. Among the different nitrogen sources tested, the enzyme activity was higher with peptone.

From the results of the present study it is quite evident that each and every physical and chemical parameters of the production (culture) medium is influencing the enzyme production. Further standardization of these guidelines in the fermentors could help the small and large-scale production of cellulase in the industries.