A local isolate of Aspergillus niger used in the study was isolated from soil contaminated with effluents of cotton ginning mill by Narasimha et al (1999). This culture was maintained on potato dextrose agar slants.

Different native lignocelluloses like saw dust, wheat straw, sugarcane bagasse and rice bran were collected from the industries located around in Nandyal, Andhra Pradesh, India. They were sorted and then washed under running tap water to remove sand and other dirt particles. All these samples were sun-dried for about two weeks and then milled into a powder form.

The different native lignocelluloses (saw dust, wheat straw, sugarcane bagasse and rice bran) were added at the rate of 0.5 %( W/V) level to each 250 ml Erlenmeyer conical flasks in which 50ml of Czapek-Dox liquid medium was distributed. Flasks containing Czapek-Dox liquid medium amended with 0.5% (W/V) cellulose instead of lignocellulose served as control. All these flasks were incubated at 30°C on a rotary shaker (180rpm) under axenic conditions after inoculating with a spore suspension ( 2 x 10 ⁸ ) of A. niger which was prepared by flooding the 6 day old slant with 2ml of sterile distilled water.

Removal of components of lignin and hemicellulose may improve the production of cellulolytic enzymes. Therefore these components were removed from these lignocelluloses by two selected pretreatment processes. The lignocelluloses (saw dust, wheat straw, sugarcane bagasse and rice bran) used in this study were pretreated by two different pretreatment processes, NaOH method and H₂O₂ methods.

In NaOH method, each lignocelluloses were treated with 50ml NaOH (1%W/Vof distilled water) for 1 hr in an autoclave at 121 ^o C and filtered through Whattman No.1filter paper and washed with water until washings turned neutral. The treated samples were dried at 110 ^o C overnight in oven, where as in H₂O₂ method, 1g of each sample was treated with alkaline peroxide in 50ml of distilled water containing H₂O₂ 1%(W/V). The suspension was adjusted to pH 11.5 with NaOH and stirred gently at room temperature (25 ^o C) for 20 hr. The insoluble residue was collected by filtration, washed with distilled water until neutrality. The treated samples were dried at 110 ^o C overnight (Gould, 1984).

Fifty milliliters of Czapek-Dox liquid medium were distributed into 250 ml Erlenmeyer conical flasks. The treated lignocelluloses were added to these flasks at 0.5%(w/v) level. Flasks containing Czapek-Dox liquid medium amended with untreated lignocelluloses at 0.5%(W/V) served as controls. All flasks were inoculated with spores of A. niger and incubated for growth in the manner as specified above.

Dry weight of the samples was determined by drying them in a hot air oven at 70°C for 24hr.

Soluble protein contents were determined in the filterate by the method of Lowry et al.,(1951) using bovine serum albumin as protein standard.

The estimation of total soluble sugar content was according to the method of Miller (1959) using glucose was as sugar standard.

pH was measured in the filtrate after desired intervals by using pH meter(ELICO).

The filtrate obtained after removal of mycelial mat by filtration through Whatman Filter paper No.1 was used as an enzyme source. Flasks with the growing culture of A. niger were withdrawn at every 7-day interval for processing.

Activities of individual enzyme components of cellulase system secreted into the culture medium of A. niger, were estimated in accordance with methods listed by Wood and Bhat (1988).

Filter paper activity of the culture filtrate of A. niger was determined according to the method of Mandels and Weber (1969). Whatman filter paper strips containing 50mg 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 with/without dilution was 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 (DNS). After cooling colour developed in tubes were read at 540 nm in a Spectrophotometer (ELICO SL 164). Appropriate control without of enzyme were simultaneously run.Activity of cellulase was expressed in filter paper units. One unit of filter paper unit (FPU) was defined as the amount of enzyme releasing µ mole of reducing sugar from filter paper per min.

Activity of endoglucanase in the culture filtrate was quantified by carboxymethyl cellulase method (Ghosh, 1987).

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 water bath for one hr. Appropriate control without of enzyme were simultaneously run. The reducing sugar produced in the reaction mixture was determined by dinitro salicylic acid (DNS) method (Miller, 1959). 3,5-Dinitro-salicylic acid reagent was added to aliquots of the reaction mixture and the color developed was read at wavelength 540 nm in a Spectrophotometer(ELICO-SL164). One unit of endoglucanase activity was defined as the amount of enzyme releasing 1 µmole of reducing sugar per minute.

Activity of β-glucosidase activity in the culture filtrate was based on the method of Herr (1979). For the determination of β-D-glucosidase activity the assay mixture contained 0.2 ml of 5 mM p-nitrophenyl β-D-gluco pyranoside (PNPG, Merck) in 0.05 M citrate buffer pH 4.8 and 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 yellow coloured p-nitrophenol liberated was determined in Spectrophotometer (ELICO-SL164) at 420 nm. One unit of β-glucosidase activity was defined as the amount of enzyme liberating 1 µmole of p-nitrophenol per min under standard assay conditions.

Four different natural lignocelluloses were used for cellulase production from A. niger in order to compare the production of cellulase with that of control in which cellulose is amended instead of lignocellulose(Table 1). Of all , wheat straw served as best source for cellulose.

Figure 1

Table 1. Cellulase production by on natural lignocelluloses

Values represented in the Table are averages of results of two experiments.

a. Filter paperase (FPase) is expressed in terms of filter paper units. One unit is the amount of enzyme in the culture filtrate releasing 1µmole of reducing sugar from filter paper per min.

b. Carboxymethyl cellulase (CMCase) is expressed in terms of units. One unit is the amount of enzyme releasing 1 µmole of reducing sugar from carboxymethyl cellulose per min.

c. One unit of β-glucosidase activity is defined as the amount of enzyme liberating 1µmole of p-nitrophenol per min.

d. Contained cellulose at 0.5% (w/v) instead of lignocellulose.

The production of acidic metabolites changed the pH of the medium from 2.59 – 4.64.

Figure 2

Table 2. pH changes in the medium upon the growth of on natural lignocelluloses

Figure 1 showed that the biomass was reached to maximum in the medium with wheat straw after 14 days of incubation, but after 21 days of incubation biomass was more in medium with rice bran as substrate.

Figure 3

Figure 1: Production of biomass of on natural lignocelluloses

Effect of natural lignocelluloses on the Secretion of extracellular protein by A. niger

High amount of extracellular protein was secreted in wheat straw supported medium after 14 days of incubation(Figure 2)

Figure 4

Figure 2: Secretion of extracellular protein by during cultivation on natural lignocelluloses

Figure 3 showed that high amount of soluble sugar was released in medium with wheat straw after 14 days of incubation.

Figure 5

Figure 3: Total soluble sugar content by on cultivation on natural lignocelluloses

More biomass was achieved in medium with treated sawdust after 21 days of incubation, but less than that of the medium with native rice bran in the same incubation period(Figure 4).

Figure 6

Figure 4: Production of biomass of on pretreated lignocelluloses

Secretion of extracellular protein was high in medium with native wheat straw after 14 days of incubation(Figure 5)

Figure 7

Figure 5: Secretion of extracellular protein by on untreated and pretreared lignocelluloses

Figure 6 showed that release of soluble sugar was more in medium with treated wheat straw after 14 days of incubation.

Figure 8

Figure 6: Total soluble sugar content in the culture broth of on untreated and pretreated lignocelluloses

Of all native and pretreated lignocelluloses, treated sawdust yielded maximum cellulase production(Table 3).

Figure 9

Table 3. Cellulase production on pretreated lignocelluloses by

Values represented in the Table are averages of results of two experiments.

a. Filter paperase (FPase) is expressed in terms of filter paper units. One unit is the amount of enzyme in the culture filtrate releasing 1 µmole of reducing sugar from filter paper per min.

b. Carboxymethyl cellulase (CMCase) is expressed in terms of units. One unit is the amount of enzyme releasing 1 µmole of reducing sugar from carboxymethyl cellulose per min.

c. One unit of β-glucosidase activity is defined as the amount of enzyme liberating 1µmole of p-nitrophenol per min.

The production of acidic metabolites changed the pH of the medium from 2.59 – 4.98(Table 4).

Figure 10

Table 4. pH changes in the medium upon the growth of on pretreated lignocelluloses