Isolation of Potential Cellulolytic Fungul Isolate from Soil Samples

al., 1999; Han et al., 1995) and hemicellulose with the help of extracellular hydrolytic enzymes, viz., cellulase and hemicellulase respectively. Cellulase is a synergistic enzyme that is used to break up cellulose into glucose or other oligosaccharide compounds (Chellapandi and Himansu, 2008). The cellulase system in fungi is considered to comprise three hydrolytic enzymes: endo-(1,4)-β-D glucanase [endoglucanase, endocellulase, CMCase (EC 3.2.1.4)] which cleaves β- linkage at random, exo-(1,4)-β-D glucanase [cellobiohydrolase, exocellulase (EC 3.2.1.91)] which releases cellobiose from non-reducing or reducing end, generally from the crystalline parts of cellulose and β-glucosidase [cellobiase (EC 3.2.1.21)] which releases glucose from cellobiose and short chain potential fungi with better cellulases production so that the wasteful lignocellulosic biomass (LB) could be converted into useful products.

Cellulases producing fungal strains were isolated from 20 soil samples collected from various sites of district Rohtak (30°1` N and 75°17` E) in Haryana (INDIA). One gram of soil from each sample was suspended in 100mL normal saline solution in a 250mL flask and incubated for 30 minutes at 180rpm. The serially diluted samples (10-3-10-7) were spread on the surface of potato-dextrose agar (PDA) media (prepared by adding potato peels 200g, dextrose 20g, agar agar 20g and streptomycin sulphate 70µg/mL to inhibit bacterial growth) and incubated for 3 days at 30ºC. The colonies were picked up and subcultured to obtain pure cultures. Stock cultures were maintained on PDA media at 4ºC.

Each fungal isolate was spot inoculated on solidified Mandels and Reese (1954) media plates containing 1% carboxymethyl cellulose (CMC) and incubated for three days at 30ºC. After 3 days, the plates were flooded with 0.1% Congo red solution for 15 minutes and then destained with 1M sodium chloride (NaCl) solution for 15 minutes. The diameter of zone of decolorization around each colony was measured.

The fungal isolates with larger zone of decolorization were selected for cellulase production in submerged cultivation. In the test tube, the Mandels and Reese medium (pH 5.0) supplemented with 1% CMC was used for enzyme production. A fungal disc of 4.0mm diameter was inoculated in 250mL Erlenmeyer flask containing 50mL Mandels and Reese medium with 1% CMC as carbon source. The flasks were incubated at 30ºC, 180rpm for 3 days. After 3 days, the culture was centrifuged at 10,000rpm for 10 minutes at 4ºC and supernatant was used as crude enzyme.

The endoglucanase/carboxymethyl cellulase (CMCase) activity was measured according to IUPAC (Ghose, 1987). In the test tube, 0.5mL carboxymethyl cellulose (1%, 4.8pH, and 0.05M citrate buffer) was added with 0.5mL appropriately diluted enzyme and incubated at 50ºC for 30 minutes. The reducing sugar concentration was estimated by DNSA method (Miller, 1959). At the end of incubation period, the reaction was stopped by adding 3.0mL DNSA reagent. The tubes were incubated for 5 minutes in boiling water calculated following the concept that 0.185 units of enzymes will liberate 0.5mg of glucose under the assay conditions and was expressed as U/mL.

The fungal isolates with higher zone of decolorization were identified at genera level by staining the mycelium, spores and conidia with lactophenol cotton blue, observing under the microscope and by colony morphology characteristics.

In the present study, a total of 82 fungal isolates were isolated from 20 soil samples collected from various regions of Rohtak from the sites rich in decomposing organic matter. The fungal isolates with larger zones of decolorization (Figure 1) belonged to genera Trichoderma and Aspergillus which were identified by microscopic examination of conidia, spore, mycelium structure and colony morphology characteristics. On the basis of zone of decolorisation (Table 1), the fungal isolates 2 (8.3cm), 4 (9.0cm), 12 (8.5cm) and 15 (8.6cm) belonging to genera Trichoderma and 30 (5.9cm), 38 (5.7cm), 47 (5.7cm) and 80 (5.6cm) belonging to genera Aspergillus were selected for CMCase production in submerged cultivation. The CMCase activities of all the selected fungal isolates were analysed on third day. Among all the selected Trichoderma and Aspergillus isolates, the CMCase activities of isolates 4 and 30 named Trichoderma sp. R-4 (0.193IU/mL) and Aspergillus sp. R-30 (0.0971IU/mL) respectively were found to be maximum (Figure 2). Table 1: Plate Assay Screening of Fungal Isolates

Aspergillus sp. R- 30 Aspergillus sp. R- 38 Aspergillus sp. R- 47 Aspergillus sp. R- 80 Trichoderma sp. R- 2 Trichoderma sp. R- 4 Trichoderma sp. R- 12 Trichoderma sp. R- 15 Figure 1: Zone of decolorisation of different fungal isolates of Aspergillus and Trichoderma The results obtained during this study indicated that cellulase activity of tested Trichoderma sp. R-4 and Aspergillus sp. R-30 were found relatively higher which was found to be commensurate with the results of Upedgraff (2004), Klueczek-Turpeinen et al. (2005) and Makut and Godiya (2010). This investigation may also lead to the development of strains of soil fungi that would be used locally for the biodegradation of only be harnessed for industrial production of the enzyme but also for the management of solid wastes containing cellulose. The cited results may also be conducive to the industries that use cellulases, viz., textile, detergents, laundry, pulp, paper, solid waste management and biofuel industry. However, further studies are required to be carried out to determine quantitatively the optimum catalytic activity of the cellulases produced by each of the cellulases producing fungal isolates so that the tested highly efficient fungal strains could be used optimally in a H effective manner for the protection of environment through solid waste management by the environmental agencies as the solid waste is mostly in the form of cellulose, the world’s most common organic substance (Ruttloff, 1987), which can be decomposed easily by the investigated potential cellulolytic fungal strains, i.e., Trichoderma sp. R-4 and Aspergillus sp. R-30.

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