World Journal of Science and Technology 2011, 1(5): 01-09 ISSN: 2231 2587

ISOLATION OF CELLULASE PRODUCING FUNGI FROM SOIL, OPTIMIZATION AND MOLECULAR CHARACTERIZATION OF THE ISOLATE FOR MAXIMIZING THE ENZYME YIELD
B.Lalitha kumari1, M.Hanuma sri2 and P.Sudhakar3
1

Prof. & HOD of BES Dept. NIET, Kantepudi, Guntur (Dt.), A.P. India 2 Pharmist, KLE University, Belgaum, Karnataka. 3 Asst.Prof, Dept. of Biotechnology, ANU, Guntur, A.P. India Corresponding author e-mail: lalithayadlapalli@rediffmail.com

Abstract
Cellulose may be hydrolyzed using enzymes to produce glucose, which can be used for the production of ethanol, organic acids and other chemicals. Cellulase is expensive and contributes only 50% to the overall cost of hydrolysis due to the low specific activity. Cellulases provide a key opportunity for achieving tremendous benefits of biomass utilization. Therefore, there has been much research aimed at obtaining new microorganisms producing cellulase enzymes with higher specific activities and greater efficiency. Presently, work is aimed at screening and isolating cellulolytic fungi from the soil samples collected from different areas of Hyderabad and identification of the isolates based on staining and molecular characterization and efforts were taken to optimize the cultural and environmental conditions for maximizing the yield of the enzyme. Identifiation of the fungal isolates was done based on biochemical and molecular characterization by sequencing the 18S rRNA coding gene. Keywords: Cellulase, A. fumigatus, Optimisation, Physico-chemical parameters, Molecular characterization

Introduction
Cellulose is considered as one of the most important sources of carbon on this planet and its annual biosynthesis by both land plants and marine algae occurs at a rate of 0.8510 11 tonnes per annum (Nowak et al., 2005). Cellulase degradation and its subsequent utilisations are important for global carbon sources. The value of cellulose as a renewable source of energy has made cellulose hydrolysis the subject of intense research and industrial interest (Bhat, 2000). Over the years, a number of organisms, in particular fungi, possessing cellulose-degrading enzymes have been isolated and studied extensively (Bhat and Bhat., 1997; Nowak et al., 2005). Cellulose may be hydrolyzed using enzymes to produce glucose, which can be used for the production of ethanol (Olsson and HahnHagerdahl, 1996), organic acids (Luo et al., 1997) and other chemicals (Cao et al., 1997). Cellulase is expensive and contributes only 50% to the overall

cost of hydrolysis (Mandels, 1985) due to the low specific activity. Enzymatic components act sequentially in a synergistic system to facilitate the breakdown of cellulose and the subsequent biological conversion to an utilizable energy source, glucose (Beguin and Aubert, 1994). Cellulases provide a key opportunity for achieving tremendous benefits of biomass utilization (Wen et al., 2005). Therefore, there has been much research aimed at obtaining new microorganisms producing cellulase enzymes with higher specific activities and greater efficiency (Subramaniyan and Prema, 2000). Cellulases have attracted much interest because of the diversity of their applications, and also for facilitating the understanding of mechanism of enzymic hydrolysis of plant carbohydrate polymers (Bhat and Bhat, 1997). The major industrial applications of cellulases are in textile industry for bio-polishing of fabrics and producing stonewashed look of denims, as well as in household laundry

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detergents for improving fabric softness and brightness. Besides, they are used in animal feeds for improving the nutritional quality and digestibility, in processing of fruit juices, in baking etc. Utilisation in de-inking of paper is yet another emerging application (Tolan and Foody, 1999). The cellulases that are used so far for the above-mentioned industrial applications are those from fungal sources (Tolan and Foody, 1999). Fungal cellulases are produced in large amounts, which include all the components of a multienzyme system with different specicities and mode of action, i.e. endoglucanases, cellobiohydrolases (exoglucanases) and b-glucosidase, acting in synergism for complete hydrolysis of cellulose. The present work is aimed at screening and isolating cellulolytic fungi from the soil samples collected from different areas of Hyderabad and identification of the isolates based on staining and molecular characterization. Further, efforts were also made to optimize the cultural and environmental conditions for maximizing of yield of the enzyme. The present study was undertaken with the following objectives To isolate fungi from soil samples that produce cellulase enzyme using a selective medium after enrichment. . To identify the fungal isolates based on biochemical and molecular characterization by sequencing the 18S rRNA coding gene. To optimize various physico-chemical factors such as temperature and pH that influence growth and production of the cellulases. To study the influence of different concentrations organic and inorganic nitrogen sources on the rate of the enzyme production.

The plate screening medium was used which contains Mandels mineral salts solution along with cellulose, triton X-100 and sorbose as an inhibitor, thus enabling the growth of many cellulase secreting fungi. The growth obtained on Mandels enriched agar medium was isolated and inoculated into petri plates containing CMC (Carboxy methyl cellulose) agar medium. The strains isolated were then inoculated into the production medium to identify the ability of the strain for cellulase production under optimized conditions. The obtained pure cultures of the fungi were maintained at 29C and then transffered to Potato dextrose agar (PDA) slants. The isolated fungi were identified based on the morphological, staining and molecular techniques. Identification of the isolated fungi by sequencing of the amplified 18S rRNA gene The most powerful tool to identify the unknown fungi is to sequence the gene (DNA) coding for 18S rRNA, which is present in the genome of the fungi. The gene coding for the 18S rRNA is amplified using the Polymerase Chain Reaction and the amplified product has been subjected to sequencing and the sequence obtained has been compared with the sequence obtained from the Nucleotide Database of National center for biotechnology information (NCBI). Genomic DNA isolation of the fungal isolates The genomic DNA of the fungal species was isolated by using the fungal genomic DNA isolation kit (Sigma) and the quantity and quality of the DNA was determined by agarose gel electrophoresis. Spectrophotometry Spectrometric studies were also carried out at 260 nm and 280 nm. Calculating the purity and yield One absorbance unit at 260 nm of the double stranded DNA is equal to 50 g/ml of the double stranded DNA. One absorbance unit at 260 nm of the single stranded DNA is equal to 40 g/ml of the single stranded DNA. Total A260 Units = (A260) dilution factor Concentration (g/ml) = total A260 units (50 g/ml) Yield (g) = Volume Concentration

Materials and Methods

Isolation of fungal strains Soil collected from different areas of Hyderabad from a depth of 1-15 inches from the top and sieved through a 2 mm sieve constituted the soil sample. The samples were dispensed into bags and were brought to the laboratory and soil enrichment was done by adding 1g of cellulose. Enrichment broth with cellulose as carbon source and peptone as nitrogen source was used for isolation of cellulolytic fungi. The selective medium i.e., Mandels enriched medium with pH 5 was employed to get desired fungi.

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Pure DNA exhibits an absorbance ratio (A260/A280) of 1.8 to 2.0 If the DNA exhibits an absorbance ratio (A260/A280) of less than 1.7, the sample is contaminated by protein

Results and Discussion

Isolation of the cellulase producing fungi The soil sample contained considerable population of the cellulase producing fungi. The Fungi grown on the selective media supported the growth of the fungi by using cellulose as the carbon source (Khalid et al., 2006). Efficient cellulase producing fungi isolates were finally selected based on the zone of the clearing around the fungi on carboxy methyl cellulase agar (CMC agar) plates (Bakare et al., 2005; Immanuel et al., 2006). The appearance of the clear zone around the colony when the Congo red solution was added (Wood and Bhat, 1998), was a strong evidence that the fungi produced cellulase inorder to degrade cellulose. Morphological identification of the fungal isolates obtained from the soil sample The isolated fungi was purified by repeated sub-culturing on the Potato Dextrose Agar medium at regular intervals and incubating at 29C. The isolate was identified based on the colony morphology, microscopic observation and molecular identification (St-Germain et al., 1996; Collier et al., 1998). Systematic position of Aspergillus Kingdom: Fungi. Phylum: Ascomycota Order: Eurotiales Family: Trichocomaceae Genus: Aspergillus, Species:fumigatus

Amplification of the 18S rRNA gene of the fungal chromosome This was carried out by PCR. The amplified DNA was subjected to agarose gel electrophoresis and along with the marker DNA (DNA Ladder). Based on the size of the amplified 18S rRNA (DNA) fragment, it was confirmed that the 18S rRNA gene was amplified. The amplified product was purified using Helini PCR purification kit and the amplified product was subjected to sequencing. The sequence so obtained was compared with already reported results from the public databases (NCBI) and the assembled sequence of the18S rRNA gene (DNA) of the unknown fungi was determined. The identified and selected fungus was Aspergillus fumigatus. Optimization of physico-chemical parameters: In our present investigation the cellulase enzyme production was optimized at different temperature ranges, pH, substrate concentration (carbon) and various concentrations of nitrogen sources. Cellulase Assay was done by DNS method (3, 5-dinitrosalicylic acid)(Miller, 1959) and the activity of the enzyme was expressed in mol/ml/min. It was calculated by the following formula OD of Unknown OD of Test = to the OD of the Colour intensity of the liberated product. From the graph the concentration of the reducing sugar liberated by the action of the enzyme was determined and the enzyme activity is expressed, Enzyme Activity = moles of the product liberated per mole of enzyme per ml per minute Enzyme Activity = Concentration of Glucose Liberated / Incubation time g Conversion of grams in moles, moles = g / Molecular weight of glucose = moles / ml /min

Aspergillus fumigatus

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Molecular characterization based on 18S rRNA sequence The DNA isolated from the desired cellulase producing fungi tentatively identified as species of Apergillus. When checked for purity exhibited an absorbance ratio of 1.823 and 1.9 respectively (A260/A280 ratio 1.8 to 2.0 to be pure), revealing that the DNA isolated from the two sources was pure. The same DNA samples, when run on an agarose gel also, confirmed to be pure as the bands of DNA are single and distinct. Traces of contaminants were found when observed under the Gel doc and photographed (Figure 1).

(Aspergillus 18S rRNA gene) as per method described by Zunaina et al., 2008. The amplified DNA of the isolate was 401bp in length (because only partial sequence of the 18S rRNA Gene was amplified) were subjected to agrose gel electrophorosis along with the DNA marker.

Fig 2. Lane 1, 2, 3: Amplified partial 18S rRNA sequence Optimization of physico-chemical parameters: Effect of pH: Maximum activity of the cellulase enzyme was observed at pH 5 after 8 days of incubation. These observations were in agreement with those reported by others (Gkhan et al., 2002; Immanuel et al., 2006; Abdelnasser and El-diwany, 2007)(Table 1). The amount of the enzyme activity was expressed as IU/ml-1 (Table 2). Effect of temperature Maximum activity of the cellulase enzyme was seen at 30C after 8th day of incubation at pH 5 (Table 3 ) (Meher et al., 2006; Immanuel et al., 2006; Zhiyou et al., 2007; Abdelnasser and El-diwany, 2007; Arun et al., 2007).

Lane 1 and 3: Genomic DNA Lane 2: 1kb DNA ladder Fig 1. Gel showing the isolated genomic DNA Sequencing of the 18S rRNA gene of Aspergillus fumigatus 18S rRNA gene was enzymatically amplified by Taq DNA polymerase by using a universal fungal primer set, (Forward Primer) 5'GACTCAACACGGGGAAACT-3' and (Reverse primer) 5'-AGAAA GGAGG TGATC CAGCC-3'

Table 1. Effect of pH on cellulase activity of Aspergillus fumigatus at different pH over a time interval of 4 days (g/ml/min)
Time in days 4 8 12 16 20 24 4 1.83 2.05 1.9 1.28 0.78 0.00 5* 4.50 6.83 5.6 2.83 1.36 0.0 6 3.00 5.36 3.51 2.61 1.06 0.00 pH 7 1.45 1.71 1.33 0.76 0.63 0.00 8 0.00 0.80 0.50 0.00 0.00 0.00 9 0.00 0.00 0.00 0.00 0.00 0.00

Values expressed in each column followed by the same letter are not significantly different. P 0.01 significantly different. pH 5 is selected for the study of cellulase activity.

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Table 2. Cellulase activity in IU/ml-1 of Aspergillus fumigatus at pH 5 over a time interval of 4 days.
Time in days Cellulase activity in mol/ml/min at pH 5 4 0.025 8 0.037 12 0.031 16 0.015 20 0.007 24 0.00

Effect of the substrate concentration (saw dust) Different concentrations (0,5 %, 1%, 1.5%, 2%, 2.5%) of the natural substrate like saw dust was used here to see the cellulase activity. Maximum activity of the cellulase enzyme was obtained with 1.5% saw dust after 8th day at pH 5 and at 30oC (Table 4). In the same way maximum enzyme activity was obtained with dry coconut coir powder at the 8th day of incubation (Table 5 & 6). Effect of nitrogen sources According to Spiridonov and Wilson (1998), all the microorganisms which have an important industrial application can utilize inorganic or organic nitrogen sources. Here the medium was supplemented with different concentrations (0.05%, 0.1%, 0.15%, 0.2%, 0.25%) of inorganic (ammonium sulphate) and organic (peptone) nitrogen sources (Narasimha et al., 2006; Arun et al., 2007). Effect of inorganic nitrogen source Maximum activity of the cellulase enzyme of Aspergillus fumigatus was at 0.2% of inorganic

nitrogen source, but the highest activity was observed with 0.2% of ammonium sulphate (Tables 7 & 8). Effect of organic nitrogen source Different concs. Of organic nitrogen source like 0.05, 0.1, 0.15, 0.2, and 0.25% were used here and the activity increased from 0.05% to 0.2%. Further increase in concentration of the organic nitrogen source resulted in considerable decrease in enzyme activity. Maximum activity of the cellulase enzyme was at 0.2% for all the different organic nitrogen sources (Peptone) at pH 5 and temperature of 30C after 8th day of incubation (Tables 9 &10).The catalytic effect of an enzyme is quantitatively expressed in terms of units of activity. One unit (U) of enzyme is defined as that amount which will catalyze the transformation of one micromole of substrate per minute under defined conditions. Statistical analysis of Duncans New Multiple Range (DMR) test of the data revealed that at 0.01% level of significance, there is no significant difference in all the parameters study but there is a significant difference among the time intervals.

Table 3. Effect of temperature on cellulase Activity of Aspergillus fumigatus at pH 5 over a time interval of 4 days.
Time in Days 4 8 12 16 20 24 25C 1.93 3.00 2.56 1.43 0.81 0.00 30C* 4.00 6.71 5.43 3.10 1.38 0.00 35C 3.51 6.00 4.96 2.60 1.06 0.00 Temperature 40C 2.96 4.66 2.25 1.11 0.71 0.00 45C 1.33 3.16 1.48 0.93 0.66 0.00 50C 0.00 1.05 0.93 0.76 0.00 0.00 55C 0.00 0.00 0.00 0.00 0.00 0.00

Values expressed in each column followed by the same letter are not significantly different. P 0.01 significantly different. To study Cellulase activity 30C temperature was selected.

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Table 4. Effect of different concentrations of saw dust on cellulase activity of Aspergillus fumigatus at pH 5 and temperature 30C over a time interval of 4 days (g/ml/min).
Time in Days 4 8 12 16 20 24 Concentrations of saw dust (%) 1% 1.5% * 2% 2.93 3.00 2.08 4.76 5.26 4.50 3.5 4.46 3.25 2.33 2.33 2.61 1.10 1.16 0.91 0.00 0.00 0.00

0.5% 2.00 2.83 1.63 1.06 0.61 0.00

2.5% 2.00 2.66 2.08 1.60 0.80 0.00

Values expressed in each column followed by the same letter are not significantly different. P 0.01 significantly different. 1.5% conc. of Saw dust was selected to study cellulase activity.

Table 5. Effect of the concentration (1.5%) of saw dust cellulase activity of A.fumigatus at pH 5 and temperature at 30C over a time interval of 4 days (g/ml/min).
Time in days 4 8 12 16 20 24

Cellulase activity of A. fumigatus, 1.5% saw dust 3.00 5.26 4.46 2.33 1.16 0.00
Values expressed in each column followed by the same letter are not significantly different. P 0.01 significantly different.

Table 6. Cellulase activity in IU/ml-1 at concentration (1.5%) dry coconut powder of A.fumigatus at temperature 30C over a time interval of 4 days
Time in days 4 8 12 16 20 24

Conc.(1.5%) dry coconut coir powder 0.016 0.029 0.024 0.012 0.006 0.000
Values expressed in each column followed by the same letter are not significantly different. P 0.001 significantly different. 1,5% conc. Of dry coconut powder was selected for the present study.

Table 7. Effect of different concentration of nitrogen source (Inorganic) Ammonium Sulphate on cellulase activity of A. fumigatus at pH 5 and temperature 30C over a time interval of 4 days (g/ml/min)
Time in Days 4 8 12 16 20 24 0.05% 1.91 b 2.08 a 2.00 a 1.05a 0.75a 0.00 Concentration of Ammonium sulphate (%) 0.1% 0.15% 0.2%* 2.26 a 2.61 a 2.50 b a a 2.83 3.03 3.00a a b 2.28 2.11 2.56 b 1.25 a 1.31 b 1.60 b a a 0.81 1.2 1.23 b a 0.00 0.00 0.00 0.25% 2.16 a 2.66 a 2.26 a 1.35 a 1.00 a 0.00a

Values expressed in each column followed by the same letter are not significantly different. P 0.01 significantly different. 0.2% conc. was selected for the present study.

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Table 8. Cellulase activity in IU/ml-1 of Aspergillus fumigatus at pH 5 and temperature 30C in the presence of nitrogen source (Inorganic) over a time interval of 4 days
Time in days 4 8 12 16 20 24 Conc. of Nitrogen source (0.2%) 0.013 0.014 0.016 0.008 0.006 0.000 (Ammonium sulphate)
Values expressed in each column followed by the same letter are not significantly different. P 0.001 significantly different. 0.2% of Ammonium sulphate was selected for the present study.

Table 9. Effect of different concentration of nitrogen source (Organic) peptone on cellulase activity of A. fumigatus at pH 5 and temperature 30C over a time interval of 4 days (g/ml/min)
Time in Days 4 8 12 16 20 24 0.05% 2.45 a 2.93 a 2.26 a 1.98 a 0.63 a 0.00 Concentration of peptone (%) 0.1% 0.15% 0.2% * 2.65 a 2.78a 2.60 a a a 3.80 3.93 4.66 a 2.43 a 2.43a 2.66 b b b 2.13 1.71 1.66 c a a 0.66 0.78 0.91 b 0.00 0.00 0.00 0.25% 2.25 a 3.45 a 2.43 a 1.60b 0.66 a 0.00

Values expressed in each column followed by the same letter are not significantly different. P 0.01 significantly different. 0.2% conc.of peptone was selected for the present study.

Table 10. Cellulase activity IU/ml-1 of Aspergillus fumigatus at pH 5 and temperature 30C in the presence of nitrogen source (Organic) over a time interval of 4 days.
Time in days 4 8 12 16 20 24

Conc. of nitrogen source(0.2%)(Peptone) 0.014 0.025 0.014 0.009 0.005 0.000

Values expressed in each column followed by the same letter are not significantly different. P 0.001 significantly different. 0.2% of nitrogen source was selected for the present study.

Conclusion
Cellulose is the primary product of photosynthesis in terrestrial environments and it is the most abundant renewable bioresource produced in the biosphere (~100 billion dry tons/year). Cellulases produced by microorganisms are either cell associated or free form, metabolize the insoluble cellulose. The fungi was isolated from the soil and was enriched with the cellulase and incubated for 30 days and the soil was diluted and inoculated on the Mendals medium (selective media for cellulase producing fungi) and the isolated was inoculated in to the selective Mendals mineral solution which was

kept on the shaker. The fungi capable to produce cellulase were identified as Aspergillus fumigatus based on colony characters, microscopic observation and identification at molecular level based on DNA coding for 18S rRNA. To maximize the cellulase activity the optimum conditions like pH, temperature, and different nitrogen sources were added and tested. The production of cellulase was assayed and optimized. It was found that the production of cellulase was maximum at pH 5 and at temperature 30C. Further, the production also was influenced by the different organic and inorganic nitrogen sources and also under natural sources. The salient features of the present study are:

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The fungal isolate were identified as Aspergillus fumigatus The fungal isolates yielded maximum enzyme at pH 5 and 30C temperature, The cellulase activity of Aspergillus fumigatus in the presence of saw dust and coconut dry powder was analyzed which indicated that waste can be used as the source of fuel. The fungi exhibited higher cellulase activity in the presence of organic and inorganic nitrogen sources. The unknown isolates were identified by amplifying and sequencing of 18S rRNA Gene.

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