Czapek-Dox medium (HIMEDIA) used in this method contained (g/l); sucrose – 30, NaNO₃ – 2, K₂HPO_{4 -} 1, MgSO₄ – 0.05, KCl – 0.5, FeSO₄ – 0.01. Czapek-Dox liquid medium amended with 1% cellulose was adjusted to different pH ranges (3, 4, 5, 6, 7 & 8) and distributed in 250 ml Erlenmeyer conical flasks. The flasks were sterilized, cooled and inoculated with the fungal spore suspension. The flasks were incubated at 32°C on a rotary shaker at 120 rpm for 7 days. After incubation, dry weight of fungal mass, protein content, reducing sugars and individual enzyme components of cellulase activity was determined.

To check the influence of temperature on cellulase production, the conical flasks containing fermentation medium supplemented with 1% cellulose (pH 5.0) were incubated at different temperatures (25,32 and 40°C). The flasks were inoculated with fungal spores. After incubation, dry mass of fungal mat, the extracellular protein, total soluble sugar, and activity of total cellulase was determined.

To determine the effect of various carbon sources on cellulase production in the Czapek-Dox medium, various carbon sources at 1% (W/V) level were used. Glucose, fructose, lactose, galactose, maltose, and CMC were separately added to different Erlenmeyer flasks containing 100 mL of Czapek-Dox medium, the pH was adjusted to 5.0. The flasks with cellulose amended medium without a carbon source served as a control. The flasks were autoclaved at 121°C, 15 lb pressure for 15 minutes. After sterilization, the flasks were cooled and inoculated with the spore suspension of Aspergillus niger. The flasks were incubated at 32°C on the rotary shaker at 120 rpm for 7 days. Then the contents of flasks were filtered through Whatman No. 1 filter paper. Biomass, the extracellular protein content, total soluble sugar, and the total cellulase activity were determined in the collected culture filtrate.

To know the impact of nitrogen sources on cellulase production, Erlenmeyer conical flasks (250ml) containing 100 ml of Czapek-Dox medium with 1% (W/V) cellulose were separately added with various nitrogen sources like peptone, yeast extract, KNO₃, (NH₄)₂SO₄ and urea at a concentration of 0.03%. The flasks with cellulose amended medium without nitrogen source served as control. All the flasks were aseptically inoculated with spores of Aspergillus niger and the flasks were incubated at 32°C on a rotary shaker at 120 rpm for 7 days. Biomass, extracellular protein content, glucose content and total cellulase activity were determined in the collected culture filtrate.

To detect the supportive native lignocellulosic substrates for the production of cellulase complex, different natural lignocellulosic substrates which include sawdust, rice bran, rice husk, wheat bran, bagasse, litter, groundnut shells, and coir and corn cob were used in the present study. 100 ml of Czapek-Dox broth medium was distributed into separate Erlenmeyer conical flasks. Each flask was provided with one gram of finely powdered untreated lignocelluloses. The pH of the medium was adjusted to 5.0. After sterilization, the flasks were cooled and inoculated with the spore suspension of Aspergillus niger. The flasks were incubated at 32°C for 7 days on a rotary shaker at 120 rpm. After 7 days of incubation, the contents of the flasks were filtered through Whatman filter paper No. 1. The culture filtrates were used for the estimation of biomass, the extracellular protein content, total soluble sugar and total cellulase activities.

The extracellular protein content in the fungal filtrate was determined [20]. Suitable aliquots of filtrate were mixed with 5 ml of alkaline solution. After 30 min, 0.5 ml Folin-Ciocalteu’s - reagent was added. The color developed was read at 550 nm by using the spectrophotometer (Spectronic- 20D). Bovine serum albumin was used as a protein standard.

The total soluble sugar content in the culture filtrates was determined [21]. Glucose was used as a standard. Suitable aliquots of culture filtrates were mixed with 3 ml of DNS reagent. The contents were boiled vigorously in a boiling water bath for exactly five minutes and the colour developed was read at 540 nm by using the spectrophotometer (Spectronic-20D).

The fungal biomass was estimated by incubating the fungal culture for 7 days. After incubation, the contents of the flasks were aseptically passed through a pre-weighed filter paper (Whatman No.1) to separate mycelial mat from culture filtrate. The filter paper along with mycelial mat was dried at 70°C in an oven until constant weight and the weight was recorded. Difference between the weights of the filter paper bearing mycelial mat and weight of pre-weighed filter paper represented fungal biomass, which was expressed in terms of dry weight of mycelial mat (mg/100 ml of Czapek-Dox medium).

The filtrates obtained after removal of mycelia mat by filtration through filter paper was used as an enzyme source. Flasks containing the growing culture of Aspergillus niger were withdrawn at every 7- day interval for processing.

Filter paper activity of the culture filtrates was determined according to the method of Mandels and Weber (1969) [22]. Whatman filter paper strips containing 50 mg weight was suspended in one ml of 0.05 M sodium citrate buffer (pH 4.8) at 50°C in a water bath. Suitable aliquots of enzyme source were added to the above mixture and incubated for 60 minutes at 50°C. After incubation, the liberated reducing sugars were estimated by the addition of 3, 5-Dinitrosalicylic acid [21]. After cooling, colour developed in tubes was read at 540 nm in a spectrophotometer (Spectronic-20D). Appropriate control without enzyme was simultaneously made to run. The activity of cellulase was expressed in filter paper units. One unit of Filter Paper Unit (FPU) was defined as the amount of enzyme releasing one micromole of reducing sugar from filter paper /ml /h.

The activity of endoglucanase in the culture filtrate was quantified by carboxymethylcellulose method [23]. The reaction mixture with 1.0 ml of 1% carboxymethyl cellulose in 0.2 M acetate buffer (pH 5.0) was pre-incubated at 50°C in a water bath for 20 minutes. An aliquot of 0.5 ml of culture filtrate with appropriate dilution was added to the reaction mixture and incubated at 50°C in a water bath for an hour. Appropriate control without enzyme was simultaneously made to run. The reducing sugar produced in the reaction mixture was determined by Dinitrosalicylic acid (DNS) method [21]. 3, 5-Dinitro-salicylic acid reagent was added to aliquots of the reaction mixture and the colour developed was read at a wavelength of 540 nm by using the spectrophotometer (Spectronic-20D). One unit of endoglucanase activity was defined as the amount of enzyme releasing one micromole of reducing sugar /ml /h.

The activity of β-glucosidase in the culture filtrates was determined based on the method of Herr (1979) [24]. 200 microlitres of 5mM p-nitro phenyl β-D-glucopyranoside (PNPG,) in 0.05 M citrate buffer pH 4.8 was added to 0.2 ml of diluted enzyme solution with appropriate controls. After incubation for 30 min at 50°C, the reaction was stopped by adding 4 ml of 0.05 M NaOH-Glycine buffer (pH 10.6) and the liberated yellow colored p-nitrophenol was determined at 450 nm by using the spectrophotometer (Spectronic-20D). One unit of β-glucosidase activity was defined as the amount of enzyme liberating one micromole of þ-nitro phenol /ml /h under standard assay conditions.

The effect of different pH ranges (3-8) on cellulase production by Aspergillus niger was tested and listed (Figs. 1, 2 and Table 1). Though maximum growth (1800mg/100ml of Czapek-Dox medium) of tested organism was obtained at pH 4.0, maximum activities of FPase (14.16 U/ml), CMCase (64.00 U/ml), extracellular protein (1.65 mg/ml), total soluble sugar (7.11 mg/ml) were recorded at pH 5.0. Relatively lower activities of FPase (0.38-1.6 U/ml), CMCase (8.11-10.2 U/ml) and β-glucosidase (0.006 U/ml), lower amounts of total soluble sugar (0.07-0.42 mg/ml), the extracellular protein (0.22-0.41 mg/ml) was found when the culture was grown at pH 3.0, 4.0 and 8.0. The less vegetative growth of 1460 mg/100ml was found at pH value of 8.0. β-glucosidase activity was not detected when pH of the medium was set above 5.0. Thus, it was clear from the above results that pH 5.0 was found to optimum pH value for cellulase production in case of Aspergillus niger.

Fig. (1). Effect of different medium pH on A. niger biomass production. The results are the mean of three different experiments.

Fig. (2). Effect of medium pH on soluble sugar and protein production. The results are the mean of three different experiments.

Temperature highly influences growth and enzymatic activities of microorganisms. The effect of different temperatures on cellulase production was studied and results were depicted in Figs. (3, 4 and Table 2). The culture filtrates obtained from the flasks incubated at 32ºC exhibited higher of FPase activity (14.66 U/ml), CMCase (64.00 U/ml) and β-glucosidase (0.014 U/ml), fungal biomass of 1670 mg/100ml of Czapek-Dox medium, the extracellular protein of 1.65 mg/ml and total soluble sugar of 7.11 mg/ml (Figs. 10). Comparatively lower activities of FPase (2.66 U/ml), CMCase (9.32 U/ml) and β-glucosidase (0.006 U/ml) as well as lower contents of extracellular protein (0.71 mg/ml), total soluble sugar (0.55 mg/ml) and biomass of (1480 mg/100ml) were recorded at 40ºC. The culture filtrate obtained from the flask incubated at 25ºC shows FPase of 7.62 U/ml, CMCase of 30.88 U/ml and β-glucosidase of 0.006 U/ml. The dry weight of fungal biomass was 1580 mg/100ml, the extracellular protein was 1.58 mg/ml and total soluble sugar was 6.88 mg/ml. These values lie in between the activity ranges of the above-discussed temperatures (32ºC, 40ºC).

Fig. (3). Effects of temperature on biomass production.

Fig. (4). Effects of temperature on total sugar and protein content. The results are the mean of three different experiments.

Different carbon sources used in this study exhibited variable effects on the selected parameters (Figs. 5, 6 and Table 3). Of the carbon sources applied, lactose supported the maximal activities of FPase (38.33 U/ml) CMCase (85.54 U/ml), higher extracellular protein (9.6 mg/ml) and total soluble sugar (9.6 mg/ml). β-glucosidase activity was higher (0.02 U/ml) when fructose was used as carbon source. The inclusion of maltose and galactose exhibited higher titres of FPase and CMCase next to lactose. The relatively higher vegetative growth of Aspergillus niger of 3330mg/100ml was obtained in the medium supplemented with carboxymethyl cellulose (CMCellulose) as substrate. The three enzyme components FPase (4.33 U/ml), CMCase (16.11 U/ml) and β-glucosidase (0.007 U/ml) were exhibited at low levels. The extracellular protein content (0.77 mg/ml) and total soluble sugar (0.9 mg/ml) were also recorded at lower levels in the Carboxymethylcellulose(CMCellulose) amended medium. Compared to remaining carbon sources used in the present study, FPase and CMCase activities in glucose and fructose amended medium were found to be intermediate to the above-described ones. β-glucosidase activity was not detected in the medium amended with glucose, lactose, and maltose.

Fig. (5). Effect of carbon source on soluble sugar and protein content.

Fig. (6). Effect of carbon sources on biomass production. The results are the mean of three different experiments.

The results are the mean of three different experiments.

The effects of various nitrogen sources on cellulase production were studied and compared with control (Figs. 7, 8 and Table 4). Among the list of nitrogen sources, urea appeared to be the best as reflected by the highest production of extracellular protein (210mg/ml). 38.88 U/ml of FPase, 68.88 U/ml of CMCase, and total soluble sugar of 8.70 mg/ml. β-glucosidase was undetected not only in urea but also in peptone and KNO₃ amended medium. Though higher β-glucosidase activity (0.230 U/ml) was observed in yeast extract supplemented medium lower activities of FPase (0.77 U/ml), CMCase (7.32 U/ml) was recorded. Maximum (2080 mg/100ml) and minimum (1490 mg/100ml) fungal growth were observed in (NH₄)₂, SO₄ and KNO₃ amended medium, respectively. Considerably lower activities of FPase and CMCase along with lower amounts of extracellular protein and total soluble sugar yeast extract and (NH₄)_2, SO₄supplemented medium.

Fig. (7). Effect of nitrogen sources on soluble sugar and protein content.

Fig. (8). Effect of nitrogen source on biomass production. The results are the mean of three different experiments.

Various natural lignocelluloses were employed at 1% level in Czapek-Dox medium to induce cellulase production by Aspergillus niger. The results were compared to the yields of cellulase synthesized by the same microorganism on cellulose (Figs. 9, 10, Table 5). Among the lignocelluloses supplemented in the present study, the sawdust induced maximum biomass of 1650 mg/100ml, the total soluble sugar of 7.90 mg/ml and the highest titres of FPase (31.11 U/ml) CMCase (67.54 U/ml) and lower yields of extracellular protein (1.71 mg/ml) were noticed. Higher quantities of β-glucosidase (0.43 U/ml) were obtained with bagasse. Supplementation of forest litter induced the yields of FPase (29 U/ml) CMCase (60.44 U/ml), total soluble sugar (5.0 mg/ml) which proved it as a good inducer of cellulase production next to sawdust. The secretion of extracellular protein was maximum (1710 mg/ml) when groundnut shells were added as substrate. The decreased activities of FPase and CMCase were noticed with rice bran, groundnut shells, wheat bran, and bagasse. The lowest β-glucosidase activity (0.02 U/ml) was recorded in wheat bran and groundnut shells. Least values of total soluble sugar (0.87 mg/ml), extracellular protein (0.75 mg/ml) and dry weight (690 mg/100ml) were noticed in the experiments which used bagasse as a substrate.

Fig. (9). Effect of lignocelluloses on biomass production.

Fig. (10). Effect of lignocelluloses on soluble sugar and protein content. The results are the mean of three different experiments.