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World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(5):01-09

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 sri

2and P.Sudhakar

3

1Prof. & HOD of BES Dept. NIET, Kantepudi, Guntur (Dt.), A.P. India

2Pharmist, KLE University, Belgaum, Karnataka.

3Asst.Prof, Dept. of Biotechnology, ANU, Guntur, A.P. India

Corresponding author e-mail: [email protected]

AbstractCellulose 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 mostimportant sources of carbon on this planet and itsannual biosynthesis by both land plants and marinealgae occurs at a rate of 0.85×10 11 tonnes per annum(Nowak et al ., 2005). Cellulase degradation and itssubsequent utilisations are important for globalcarbon sources. The value of cellulose as arenewable source of energy has made cellulosehydrolysis the subject of intense research andindustrial interest (Bhat, 2000). Over the years, a

number of organisms, in particular fungi, possessingcellulose-degrading enzymes have been isolated andstudied extensively (Bhat and Bhat., 1997; Nowak et al ., 2005). Cellulose may be hydrolyzed usingenzymes to produce glucose, which can be used for the production of ethanol (Olsson and Hahn-Hagerdahl, 1996), organic acids (Luo et al ., 1997)and other chemicals (Cao et al ., 1997). Cellulase isexpensive and contributes only 50% to the overall

cost of hydrolysis (Mandels, 1985) due to the lowspecific activity.

Enzymatic components act sequentially in asynergistic system to facilitate the breakdown of cellulose and the subsequent biological conversion toan utilizable energy source, glucose (Beguin andAubert, 1994). Cellulases provide a key opportunityfor achieving tremendous benefits of biomassutilization (Wen et al ., 2005). Therefore, there hasbeen much research aimed at obtaining newmicroorganisms producing cellulase enzymes withhigher specific activities and greater efficiency

(Subramaniyan and Prema, 2000).Cellulases have attracted much interest

because of the diversity of their applications, and alsofor facilitating the understanding of mechanism of enzymic hydrolysis of plant carbohydrate polymers(Bhat and Bhat, 1997). The major industrialapplications of cellulases are in textile industry for ‘bio-polishing’ of fabrics and producing stonewashedlook of denims, as well as in household laundry

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detergents for improving fabric softness andbrightness. Besides, they are used in animal feeds for improving the nutritional quality and digestibility, inprocessing of fruit juices, in baking etc. Utilisation inde-inking of paper is yet another emerging application(Tolan and Foody, 1999). The cellulases that are

used so far for the above-mentioned industrialapplications are those from fungal sources (Tolan andFoody, 1999).

Fungal cellulases are produced in largeamounts, which include all the components of a multi-enzyme system with different specificities and modeof action, i.e. endoglucanases, cellobiohydrolases(exoglucanases) and b-glucosidase, acting insynergism for complete hydrolysis of cellulose.

The present work is aimed at screening andisolating cellulolytic fungi from the soil samplescollected from different areas of Hyderabad and

identification of the isolates based on staining andmolecular characterization. Further, efforts were alsomade to optimize the cultural and environmentalconditions for maximizing of yield of the enzyme.The present study was undertaken with the followingobjectives

• To isolate fungi from soil samples that producecellulase enzyme using a selective mediumafter enrichment. .

• To identify the fungal isolates based onbiochemical and molecular characterization by

sequencing the 18S rRNA coding gene.• To optimize various physico-chemical factors

such as temperature and pH that influencegrowth and production of the cellulases.

• To study the influence of differentconcentrations organic and inorganic nitrogensources on the rate of the enzyme production.

Materials and Methods

Isolation of fungal strains Soil collected from different areas of

Hyderabad from a depth of 1-15 inches from the topand sieved through a 2 mm sieve constituted the soilsample. The samples were dispensed into bags andwere brought to the laboratory and soil enrichmentwas done by adding 1g of cellulose. Enrichment brothwith cellulose as carbon source and peptone asnitrogen source was used for isolation of cellulolyticfungi. The selective medium i.e., Mandel’s enrichedmedium with pH 5 was employed to get desired fungi.

The plate screening medium was used whichcontains Mandels mineral salts solution along withcellulose, triton X-100 and sorbose as an inhibitor,thus enabling the growth of many cellulase secretingfungi. The growth obtained on Mandel’s enriched agar medium was isolated and inoculated into petri plates

containing CMC (Carboxy methyl cellulose) agar medium. The strains isolated were then inoculatedinto the production medium to identify the ability of thestrain for cellulase production under optimizedconditions. The obtained pure cultures of the fungiwere maintained at 29°C and then transffered toPotato dextrose agar (PDA) slants. The isolated fungiwere identified based on the morphological, stainingand molecular techniques.

Identification of the isolated fungi by sequencingof the amplified 18S rRNA gene

The most powerful tool to identify theunknown fungi is to sequence the gene (DNA) codingfor 18S rRNA, which is present in the genome of thefungi. The gene coding for the 18S rRNA is amplifiedusing the Polymerase Chain Reaction and theamplified product has been subjected to sequencingand the sequence obtained has been compared withthe sequence obtained from the Nucleotide Databaseof National center for biotechnology information(NCBI).

Genomic DNA isolation of the fungal isolates

The genomic DNA of the fungal species wasisolated by using the fungal genomic DNA isolation kit(Sigma) and the quantity and quality of the DNA wasdetermined by agarose gel electrophoresis.

Spectrophotometry Spectrometric studies were also carried out

at 260 nm and 280 nm.

Calculating the purity and yieldOne absorbance unit at 260 nm of the double

stranded DNA is equal to 50 µg/ml of the double

stranded DNA. One absorbance unit at 260 nm of thesingle stranded DNA is equal to 40 µg/ml of the singlestranded DNA.

• Total A260 Units = (A260) х dilution factor

• Concentration (µg/ml) = total A260 units х (50µg/ml)

• Yield (µg) = Volume х Concentration

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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 iscontaminated by protein

Amplification of the 18S rRNA gene of the fungalchromosome

This was carried out by PCR. The amplifiedDNA was subjected to agarose gel electrophoresisand 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 genewas amplified. The amplified product was purifiedusing Helini PCR purification kit and the amplifiedproduct was subjected to sequencing. The sequenceso obtained was compared with already reported

results from the public databases (NCBI) and theassembled sequence of the18S rRNA gene (DNA) of the unknown fungi was determined. The identifiedand selected fungus was Aspergillus fumigatus.

Optimization of physico-chemical parameters:In our present investigation the cellulase

enzyme production was optimized at differenttemperature ranges, pH, substrate concentration(carbon) and various concentrations of nitrogensources.

Cellulase Assay was done by DNS method

(3, 5-dinitrosalicylic acid)(Miller, 1959) and the activityof the enzyme was expressed in µmol/ml/min. It wascalculated by the following formula

OD of Unknown – OD of Test = to the OD of theColour intensity of the liberated product.

From the graph the concentration of thereducing sugar liberated by the action of theenzyme was determined and the enzyme activity isexpressed,

Enzyme Activity = µ moles of the product liberated per

mole of enzyme per ml per minute

Enzyme Activity = Concentration of Glucose Liberated/ Incubation time µg

Conversion of µ grams in µ moles,

µ moles = µg / Molecular weight of glucose = µ moles/ ml /min

Results and Discussion

Isolation of the cellulase producing fungi

The soil sample contained considerablepopulation 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 producingfungi isolates were finally selected based on the zoneof the clearing around the fungi on carboxy methylcellulase 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 solutionwas added (Wood and Bhat, 1998), was a strongevidence that the fungi produced cellulase inorder todegrade cellulose.

Morphological identification of the fungal isolatesobtained from the soil sample

The isolated fungi was purified by repeatedsub-culturing on the Potato Dextrose Agar medium atregular intervals and incubating at 29°C. The isolatewas identified based on the colony morphology,microscopic observation and molecular identification(St-Germain et al ., 1996; Collier et al ., 1998).

Systematic position of AspergillusKingdom: Fungi.Phylum: AscomycotaOrder: EurotialesFamily: TrichocomaceaeGenus: Aspergillus,Species:fumigatus

Aspergillus fumigatus

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Molecular characterization based on 18S rRNAsequence

The DNA isolated from the desired cellulaseproducing fungi tentatively identified as species of

Apergillus. When checked for purity exhibited anabsorbance ratio of 1.823 and 1.9 respectively

(A260/A280 ratio 1.8 to 2.0 to be pure), revealing thatthe DNA isolated from the two sources was pure. Thesame DNA samples, when run on an agarose gelalso, confirmed to be pure as the bands of DNA aresingle and distinct. Traces of contaminants werefound when observed under the Gel doc andphotographed (Figure 1).

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 Aspergillusfumigatus

18S rRNA gene was enzymatically amplifiedby Taq DNA polymerase by using a universal fungalprimer set, (Forward Primer) 5'-GACTCAACACGGGGAAACT-3' and (Reverseprimer) 5'-AGAAA GGAGG TGATC CAGCC-3'

( Aspergillus 18S rRNA gene) as per methoddescribed by Zunaina et al ., 2008. The amplifiedDNA of the isolate was 401bp in length (because onlypartial sequence of the 18S rRNA Gene wasamplified) were subjected to agrose gelelectrophorosis 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 enzymewas observed at pH 5 after 8 days of incubation.These observations were in agreement with thosereported by others (Gökhan et al ., 2002; Immanuel et al ., 2006; Abdelnasser and El-diwany, 2007)(Table 1).The amount of the enzyme activity was expressed asIU/ml-1 (Table 2).

Effect of temperatureMaximum activity of the cellulase enzyme

was seen at 30°C 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).

Table 1. Effect of pH on cellulase activity of Aspergillus fumigatus at different pH over a

time interval of 4 days (µg/ml/min)

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.

Time in

days

pH

4 5* 6 7 8 94 1.83 4.50 3.00 1.45 0.00 0.00

8 2.05 6.83 5.36 1.71 0.80 0.00

12 1.9 5.6 3.51 1.33 0.50 0.00

16 1.28 2.83 2.61 0.76 0.00 0.00

20 0.78 1.36 1.06 0.63 0.00 0.00

24 0.00 0.0 0.00 0.00 0.00 0.00

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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.

Effect of the substrate concentration (sawdust)

Different concentrations (0,5 %, 1%, 1.5%,2%, 2.5%) of the natural substrate like saw dust wasused here to see the cellulase activity. Maximumactivity of the cellulase enzyme was obtained with1.5% saw dust after 8th day at pH 5 and at 30oC(Table 4). In the same way maximum enzyme activitywas obtained with dry coconut coir powder at the 8 th

day of incubation (Table 5 & 6).

Effect of nitrogen sourcesAccording to Spiridonov and Wilson (1998),

all the microorganisms which have an importantindustrial application can utilize inorganic or organicnitrogen sources. Here the medium wassupplemented with different concentrations (0.05%,0.1%, 0.15%, 0.2%, 0.25%) of inorganic (ammoniumsulphate) and organic (peptone) nitrogen sources(Narasimha et al ., 2006; Arun et al., 2007).

Effect of inorganic nitrogen sourceMaximum activity of the cellulase enzyme of

Aspergillus fumigatus was at 0.2% of inorganic

nitrogen source, but the highest activity was observedwith 0.2% of ammonium sulphate (Tables 7 & 8).

Effect of organic nitrogen sourceDifferent concs. Of organic nitrogen source

like 0.05, 0.1, 0.15, 0.2, and 0.25% were used hereand the activity increased from 0.05% to 0.2%.Further increase in concentration of the organicnitrogen source resulted in considerable decrease in

enzyme activity. Maximum activity of the cellulaseenzyme was at 0.2% for all the different organicnitrogen sources (Peptone) at pH 5 and temperatureof 30°C after 8th day of incubation (Tables 9 &10).Thecatalytic effect of an enzyme is quantitativelyexpressed in terms of units of activity. One unit (U) of enzyme is defined as that amount which will catalyzethe transformation of one micromole of substrate per minute under defined conditions. Statistical analysisof Duncan’s New Multiple Range (DMR) test of thedata 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 thetime intervals.

Table 3. Effect of temperature on cellulase Activity of Aspergillus fumigatus at pH 5 over a

time interval of 4 days.


P ≤ 0.01 significantly different. To study Cellulase activity 30°C temperature was selected.

Time in days 4 8 12 16 20 24

Cellulase activity in

µmol/ml/min at pH 5

0.025 0.037 0.031 0.015 0.007 0.00

Time in

Days

Temperature

25°C 30°C* 35°C 40°C 45°C 50°C 55°C

4 1.93 4.00 3.51 2.96 1.33 0.00 0.00

8 3.00 6.71 6.00 4.66 3.16 1.05 0.00

12 2.56 5.43 4.96 2.25 1.48 0.93 0.0016 1.43 3.10 2.60 1.11 0.93 0.76 0.00

20 0.81 1.38 1.06 0.71 0.66 0.00 0.00

24 0.00 0.00 0.00 0.00 0.00 0.00 0.00

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Table 4. Effect of different concentrations of saw dust on cellulase activity of Aspergillus

fumigatus at pH 5 and temperature 30°C over a time interval of 4 days (µg/ml/min).


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 30°C over a time interval of 4 days (µg/ml/min).


P ≤ 0.01 significantly different.


at concentration (1.5%) dry coconut powder of

A.fumigatus at temperature 30°C 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


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 30°C over a time

interval of 4 days (µg/ml/min)


P ≤ 0.01 significantly different. 0.2% conc. was selected for the present study.

Time in

Days

Concentrations of saw dust (%)

0.5% 1% 1.5% * 2% 2.5%

4 2.00 2.93 3.00 2.08 2.008 2.83 4.76 5.26 4.50 2.66

12 1.63 3.5 4.46 3.25 2.08

16 1.06 2.33 2.33 2.61 1.60

20 0.61 1.10 1.16 0.91 0.80

24 0.00 0.00 0.00 0.00 0.00

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

Time in

Days

Concentration of Ammonium sulphate (%)

0.05% 0.1% 0.15% 0.2%* 0.25%4 1.91

2.26a

2.61a

2.50

2.16a

8 2.08 a 2.83 a 3.03 a 3.00a 2.66 a

12 2.00 a 2.28 a 2.11 b 2.56 b

2.26 a

16 1.05a

1.25a

1.31

1.60

1.35a

20 0.75a 0.81 a 1.2 a 1.23 1.00 a

24 0.00 0.00 0.00a

0.00 0.00a

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of Aspergillus fumigatus at pH 5 and temperature

30°C 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%)(Ammonium sulphate)

0.013 0.014 0.016 0.008 0.006 0.000


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 30°C over a time interval of 4

days (µg/ml/min)

Time in

Days

Concentration of peptone (%)

0.05% 0.1% 0.15% 0.2% * 0.25%

4 2.45a

2.65a

2.78a

2.60a

2.25a

8 2.93a

3.80a

3.93a

4.66a

3.45a

12 2.26 a 2.43 a 2.43a 2.66 2.43 a

16 1.98 a 2.13 b 1.71b 1.66 c 1.60b 20 0.63

a0.66

a0.78

a0.91

0.66

a

24 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. 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 30°C

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


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 themost abundant renewable bioresource produced inthe biosphere (~100 billion dry tons/year). Cellulasesproduced by microorganisms are either cellassociated or free form, metabolize the insolublecellulose.

The fungi was isolated from the soil and wasenriched with the cellulase and incubated for 30 daysand the soil was diluted and inoculated on theMendals medium (selective media for cellulaseproducing fungi) and the isolated was inoculated in tothe selective Mendals mineral solution which was

kept on the shaker. The fungi capable to producecellulase were identified as Aspergillus fumigatus based on colony characters, microscopic observationand identification at molecular level based on DNAcoding for 18S rRNA. To maximize the cellulaseactivity the optimum conditions like pH, temperature,and different nitrogen sources were added and

tested. The production of cellulase was assayed andoptimized. It was found that the production of cellulase was maximum at pH 5 and at temperature30°C. Further, the production also was influenced bythe different organic and inorganic nitrogen sourcesand also under natural sources.The salient features of the present study are:

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• The fungal isolate were identified as Aspergillusfumigatus

• The fungal isolates yielded maximum enzyme atpH 5 and 30°C temperature,

• The cellulase activity of Aspergillus fumigatus inthe presence of saw dust and coconut drypowder was analyzed which indicated that wastecan be used as the source of fuel.

• The fungi exhibited higher cellulase activity inthe presence of organic and inorganic nitrogensources.

• The unknown isolates were identified byamplifying and sequencing of 18S rRNA Gene.

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