Mandel’s medium (Patel et al., 2007) was prepared by adding the following reagents (g 1000 ml^-1).Urea (0.3 g), (NH)₂SO₄ (1.4 g), KH₂PO₄ (2.0 g), CaCl₂.2H₂O (0.3 g), MgSO₄.7H₂O (0.3 g), Bactopeptone (1.0 g), Tween 80 ( 0.1 g), Carboxymethyl cellulose (10g). Trace elements: FeSO₄.6H₂O (5 mg), MnSO₄.H₂O (16 mg), ZnCl₂.2H₂O (17mg), CoCl_2.6H₂O (2 mg).

The Mandel’s medium was prepared and its pH was adjusted to 5.0. The conical flasks were plugged with cotton and sterilized at 15 lbs per sq.inch for 20 minutes. Each flask was inoculated with various soil isolate. These flasks were incubated at room temperature for 5 days on a shaker at 125 rpm. After five days, the mycelium was separated by filtration through Whatman filter paper No. 1. The filtrate was used for further analyses of the study.

The total amount of reducing sugars in the sample was determined by Dinitrosalicylic acid (DNS) method (Miller, 1959). Cellulase activity (CMCase) of the fungal sample was determined as followed by filter paper activity (FPA), a method proposed by Ghose, (1987).

The culture filtrate was further inoculated with Saccharomyces cerevisiae strain and allowed to ferment for 7 days. After fermentation it was filtered and ethanol content of the cultures was estimated. Ethanol content of the fermented culture filtrate was assessed quantitatively using spectrophotometric method (Cemeg and Cymru, 2006).

The CMCase activities of the two cultures were observed on the 1^st, 3^rd, 5^th, 7^th and 9^th day of the growth.

Figure 1

Figure 1

It is clearly indicated from the figure 1 that the enzyme activity of both the isolates were gradually increasing when CMC was used as a substrate and also the activity of isolate 6 was found to be slightly higher when compared with isolate 5.

Among various soluble organic carbon sources and lignocelluloses tested in a study by Narasimha et al. (2005), carboxymethylcellulose and sawdust at 1% supported maximum production of cellulase by A. niger.

Figure 2 represents the filter paper activity of the selected isolates.

Figure 2

Figure 2

From the graph, it is observed that as the number of days increase, the cellulolytic activity of both the fungal isolates increased. Though similar trend was observed for both the methods, cellulase activity was found to be slightly lesser in FPA activity when compared to CMCase activity.

Our results are in accordance with Rajoka (2004), who has reported that the medium containing soluble carbon sources like CMC, the organism synthesizes low enzyme for filter paper activity, while it synthesizes a high level of enzymes when grown on other cellulosic substrates.

In a study by Ojumu et al., (2003), the filter paper activity of A. flavus on saw dust gave the highest cellulase activity of 0.0743 U/ml.

Estimation of alcohol in the selected sample

Since the enzyme activity was found to be more on 7^th day and 9^th day cultures, alcohol estimation was done only for these two cultures. These two cultures were allowed to undergo fermentation using Saccharomyces cerevisiae at a temperature of 37°C for 7-9 days. The sample was centrifuged and the supernatant was used for alcohol estimation.

Figure 3

Figure 3

The graph clearly shows that alcohol production was found to be more on 9^th day.

In a study by Karimi et al., (2006), the ethanol yield by Saccharomyces cerevisiae on complex substrate like straw yielded only 10.20 g/L of ethanol.

Production of alcohol using the fungal isolates and Saccharomyces cells could be achieved as more sugar molecules could be produced from the substrate like CMC.