INTRODUCTION:

Cellulases are inducible enzymes which are synthesized by microorganisms during their growth on cellulosic materials ¹. Enormous amounts of agricultural, industrial and municipal cellulosic wastes have been accumulating or used inefficiently due to the high cost of their utilization processes ². In most cellulolytic organisms, cellulose synthesis is repressed in the presence of easily metabolized, soluble carbon sources, and induced in the presence of cellulose. Several applications of cellulases or hemicellulases are being developed for textile, food and paper, pulp processing ³.

Cellulases are produced by large number of microorganisms. They are either cell-bound or extracellular. Although a large number of microorganisms can degrade cellulose, only a few of them produce significant quantities of free enzyme capable of completely hydrolyzing crystalline cellulose ⁴. Due to the vast usefulness of cellulose, the present study investigate the isolation, production and optimization of cellulase from cow dung in order to apply for degradation of cellulose found in agricultural waste.

MATERIALS AND METHODS:

1. Isolation and screening of microorganisms:

Cellulase producing bacteria were isolated from soil by spread plate technique using CMC agar medium. The plates were incubated at 37°C for 24 to 48 hours. The plates were stained with a solution of 0.1% Congo red for 15 min and destained with 1M NaCl ⁵. To indicate the cellulase activity of the organisms, diameters of clear zone around colonies on CMC agar were measured.

2. Bacterial identification:

The bacterial isolates were presumptively identified by means of morphological examination and some biochemical characterizations. The parameters investigated included morphology, Gram reactions, endospore formation, catalase production, Voges Proskauer reaction, indole production, starch hydrolysis, citrate utilization and gelatin hydrolysis. The results were compared with Bergey’s Manual of determinative Bacteria ⁶.

3. Enzyme activity assay:

Cellulase activity was measured by Filter paper assay (FPA) method using Whatman No:1 filter paper. 2M of crystalline cellulose solution was added with 0.5 ml of culture filtrate, the mixture was incubated for 1 hour and the reaction was terminated by adding 2ml DNS reagent, the contents was heated in a boiling water bath for 5 min, then 1 ml of 40% potassium sodium tartarate was added to the warm tubes. The optical density was read at 540nm against blank by spectrophotometer (Version: VI 5.2)

4. Optimization of cellulase production:

The cellulase activity of the selected bacterial culture was measured by using a FPA method ⁷. A bacterial isolate with the highest cellulase activity was selected for optimization of cellulase production. To evaluate the effect of incubation time on cellulase production, the selected bacterial isolate was grown at 24, 48, 72, 96 hours respectively. After incubation the culture broth were centrifuged at 140 rpm for 20 min to obtain supernatants which were later measured cellulase activity.

The effect of temperature on cellulase production, the selected bacterial isolate was grown in CMC broth and incubated at 25, 35, 45, 55, 65, 75, 85°C for 72 hours. The culture broth was centrifuged at 140 rpm for 20 min to obtain supernatants which were later measured cellulase activity. In order to determine the effect of pH on cellulase production was conducted by adjusting the CMC broth using different pH like 3 to 12 before bacterial inoculation. After 72 hours of incubation the culture broth were centrifuged at 140 rpm for 20 min to obtain supernatants which were later measured cellulase activity.

The effect of different carbon sources on cellulase production was conducted by replacing the carbon source and nitrogen source in CMC broth by adding various sources like glucose, sucrose, lactose, maltose, starch, peptone, ammonium nitrate, alanine and isoleucine before sterilization. After 72 hours of incubation at 25⁰C, culture broths were then centrifuged at 140 rpm for 20 minutes to obtain supernatants which were later measured cellulase activity

RESULT:

Identification of cellulase producing Bacteria:

Three strains showing clear zone after staining with Congo red, designed as S1, S2, S3. Among the three isolates S2 showed maximum zone (1.2 mm). According to the morphological and biochemical studies the isolates were primarily identified as Arthrobacter (S1), Ocrobacterium (S2), Flavobacterium (S3).

Effect of Incubation period:

When withdrawing culture broths for cellulase determination at various times, it was found that the cellulase activity was increased with time until reached peak activity after 72 hours of incubation and activity was 568 IU/L (Fig. 1).

Fig. 1.Enzyme activity at various time intervals

The effect of the pH on the cellulase activity of Ocrobacterium (S2) was examined at various pH ranging from 3.0 to 12.0 as shown in (Fig. 2). The enzyme has a broad range of pH activity with optimal pH at 5, the enzyme activity showed 623 IU/L.

Potential of the bacterial isolate S2 for cellulase production, the bacterium was grown at various temperatures. The optimum temperature was found to be 25°C. The isolate S2 had the highest cellulase activity of 569 IU/L (Fig. 3).

Fig. 2.Enzyme activity at different pH

Fig. 3. Enzyme activity on different Temperature

The effect of different carbon sources on cellulase production was conducted by replacing the carbon source and nitrogen source. After 72 hours of incubation at 25°C, maltose showed maximum cellulase activity of 398 IU/L (Fig. 4) and ammonium nitrate showed maximum activity of 568 IU/L (Fig. 5).

Fig. 4. Enzyme activity on different carbon source

Fig. 5. Enzyme activity on different nitrogen source

DISCUSSION:

Cellulose is the major building block of plants and have major fraction of organic carbon in soil. Microorganisms are responsible for recycling of this organic carbon to the environment. Degredation of cellulosic materials is a complex process and requires participation of microbial cellulolytic enzymes. Researchers studying on cellulolytic activity have isolated various bacteria from different environmental sources.

In the present study three different strains was identified from cow dung, namely Arthrobacter (S1), Ocrobacterium (S2), Flavobacterium (S3). Das et al. ⁸ isolated eight strains from cow dung samples and it was found that Bacillus sp. produce maximum amount of cellulase. In another study, Otajevwo and Auyi ⁹ isolated Pseudomonas sp. and Serratia sp. from soil samples, which have greater capability to produce cellulase enzyme.

According to previous studies, cellulases are active at the pH range of 6.0 to 7.0 from A. Niger ¹⁰, 5.0 to 7.0 from Lysobacter sp. ¹¹ and 5.0 to 6.5 from Bacillus strains ¹². As the temperature increased from 30˚C enzyme activity increased. Present findings were significant from Lysobacter sp. and Bacillus strains (pH 5) and optimum temperature at 25°C. It was found that all the carbon and nitrogen sources, which were used in the present study, supported cellulase enzyme production.

CONCLUSION:

Agricultural waste in the form of cellulose which is the most abundant renewable biomass in the biosphere has been shown to be used in the production of valuable products by microorganisms. Several microorganisms capable of converting cellulose into simple carbohydrates had been discovered for decades. Ocrobacterium showed a potential to convert cellulose into reducing sugars which could be readily used in many applications.

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Received on 30.12.2012 Accepted on 02.02.2013

Research J. Science and Tech 5(2): April- June, 2013 page 255-258