Studies On Production Of Cellulase Enzyme by isolated Fungus and Its Application In Pulp And

Paper Industry

Presented by: ASHISH KUMAR

Under the Supervision of: Dr. R. K. Jain

Scientist –F (HOD Biotechnology)Central Pulp And Paper Research Institute

(Saharanpur)

Introduction

An enzyme is a protein molecule that is a biological catalyst with three characteristics. First, the basic function of a enzyme is to increase the rate of reaction. Second, most enzymes act specifically with only one reactant (called a substrate) to produce a product. The third and most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa.

Cellulose Cellulose is an organic compound with the formula (C6H10O5) n, a polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1→4) linked D-glucose units.

Fig. Chemical structure of cellulose molecule

Cellulase Enzyme

Cellulolysis is the process of breaking down cellulose into smaller polysaccharides called cellodextrins or completely into glucose units; this is a hydrolysis reaction. Because cellulose molecules bind strongly to each other.

Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis (i.e. the hydrolysis of cellulose). However, there are also cellulases produced by other types of organisms, such as plants, and animals .

Fig. Model of Cellulase Enzyme

Mode of Action of Cellulase Enzyme

Fig. Mode of Action of Cellulase Enzyme

Application of Cellulase Enzyme

Cellulase Enzyme Used for Various Industrial Applications. Such As

•Animal feed•House hold laundry detergent •Fruit juice and beverage processing •Baking Industry •Alcohol production

Application of Cellulase Enzyme In Pulp And Paper Industry

offers new options for many applications, including stickis control, pitch control, deinking, drainage aids, refining aids, wet and slime preservation and boils outs. Enzymes are neutral, non-corrosive and deliver, so they provides reduced environmental impact.

•Bio-refining •Bio-deinking

Objectives

Objective of the present study is to isolate cellulase producing microorganism, production and application of cellulase enzyme produced by the isolate microorganism. Present study is detailed as below- 1.Isolation of cellulase producing microorganisms.2.Purification and screening of isolated fungi for cellulase production.3. Studies on physical, chemical and nutritional parameters of selected fungia)  Determination of Optimal temperature for growth of selected fungal strainsb) Determination of optimum pH for growth & enzyme productionc)Study of effect of various Substrates on growth, cellulase production of selected fungal strains S2 and S3.4. Application of cellulase enzyme in Pulp and Paper Industry – Biodeinking.5. Conservation of Environment and Natural resource.

Material & Methods Sample Collection:

Table . Details of Samples Collected From Different Sources

S.No Sample Name Date Time Place Weather pH

1 C.P.P.R.I.garden

soil 7/2/11 3:15 Hrs

C.P.P.R.I.

Campus

garden

Sunny, slight

windy 6.5

2 Paper Mill soil 7/2/11 4:15 Hrs Paper Mill,

SRE.

Sunny, slight

windy 6.0

3 Vermicompost 9/2/11 1:45 Hrs C.P.P.R.I.

Campus

Very hot,

sunny 6.5

4 Sugercane

Industry soil 10/2/11 9:10 Hrs

Sugercane

Industry

Sunny, Slight

windy 6.2

Media Used for the Present Study

Berg’s media Ist Berg’s media Ind Norkans media Potato Dextrose Agar (PDA) media

Analytical Methods

1) Growth Measurement: By two methods-

a) Diameter of colony. b) Mycelia Dry Weight.

2) Cellullase Enzyme Assay.

a) Filter Paper Assay (FPase Activity)b) Carboxymethyl Cellulase Assay (CMCase Activity)

Application in Pulp and Paper Industry (Bio-deinking)

Raw Material: Laser Waste Paper. Enzyme used: Two enzyme samples were used in this study:- 1. Commercial enzyme 2. Enzyme produced by selected fungal strain S2.Surfactant:ProcedureThe procedure of deinking involves several steps. These are as follows.1.PulpingConditions during pulping of enzymatic deinking

S.No Pulping

conditions Control

Commercial

Enzyme

Crude extract of

(S2 strain)

1 Consistency, (%) 10 10 10

2 Enzyme dose,

(IU/gm) - 0.08 0.008

3 Surfactant, (%) 0.08 0.08 0.08

4 Temperature, oC 45 45 45

5 pH 7.0 7.0 7.0

6 Pulping time

(minutes) 20 20 20

7 Retention time

(minutes) 10 10 10

Flotation Flotation was carried out in 25 liter capacity laboratory voith flotation cell. The flotation conditions are given in the following table.

Screening Floated pulps were screened in the vibrating screener and passed through 60 mm mesh. Brightness pads were prepared for both enzyme treated and untreated pulp samples and analyzed for brightness, yellowness, whiteness by L&W Elrepho (brightness tester).

S. No Flotation conditions

1 Consistency (%) 0.8

2 Air flow (L/min) 12-13

3 Flotation time (minutes) 10

Result And Discussion

Isolation of Cellulase Producing Fungi With Soil From Different Sources To isolate fungi with cellulase activity, the samples were collected from the sources as detailed in material & methods. Isolation from above cited samples was done by serial dilution method. After serial dilution, the inoculated plates were incubated at 34ºC for 3 to 4 days. After incubation, the plates were observed for the microbial growth. Table: Microbial Flora of Different Sources

Media Sample Dilution Factor No. of

colonies

Morphology of Colonies

BERG’ MEDIA-I

C.P.P.R.I. garden soil

10-3 10-4

4 4(2- white, 1-green and 1- black)

2 No growth No growth

Paper mill soil

10-3 10-4

5 3(white) 2 (green)

3 3 (1-white and 2- green)

Vermicompost

10-3

10-4

5

5(3-white and 2-green)

3(2- green and 1- black) 2 2(2- green)

Sugercane Industry soil 10-3

10-4

5

3

5(2-Green ,1- black and 1- white)

3(1 green, 2 black)

Cont…..

BERG’S MEDIA –II

C.P.P.R.I. garden

soil

10-3

10-4

2 2 ( light yellow)

-- No growth.

Vermicompost

10-3

10-4

6 4 (3- white and 1- yellow)

2 (1- yellow and 1- whitish black)

2 1 (light black)

1 (whitish black)

Paper mill soil

10-3

10-4

2 One colony with green color, One colony with

green color

2 One colony with green color, One colony with

brown color

Sugercane Industry 10-3 10-4

3 3(2-green and 1- yellow)

2 2(1- green and 1- brown)

Screening of Isolated Fungi

Screening of isolated fungi for cellulase activityThe results for screening of isolated fungi, one fungal strain from C.P.P.R.I garden soil, one fungal strain from vermicompost, two from paper mill soil shown colorless zone when flooded with Congo red dye on Berg’ medium used. These four fungal strains were used for further study.

Fig:- Screening of Cellulase Producing Fungi by Congo Red Dye

Control S1

S2

S3 S4

Table : Screening of Isolated Fungi

Sample Screened fungi Cellulase positive Selected positive strain

C.P.P.R.I. garden soil

1

2

3

4

5

6

7

-

-

+

-

-

-

-

S1

Paper mill soil

1

2

3

4

5

6

7

8

-

-

-

+

-

-

-

-

S2

Vermicompost

1

2

3

4

5

6

7

-

+

-

-

-

-

-

S3

Sugercane Industry soil

1

2

3

4

5

-

-

+

-

-

S4

Optimization of Temperature for Growth of Selected Fungal Strains

TemperatureIncubation Period (hour)

Diameter of the fungal colony (cm)

S1 strain S2 strain S3 strain S4 strain

280C24 0.85 3.5 1.8 3.0

48 1.5 7.5 2.4 4.5

72 8.5(Full

growth)

8.5(Full growth) 8.5(Full growth) 8.5(full growth)

340C24 1.0 3.5 1.8 1.0

48 4.1 8.5(Full growth) 3.9 2.5

72 8.5(Full

growth)

8.5(Full growth) 8.5(Full growth) 8.5(full growth)

370C24 0.81 1.5 1.2 1.3

48 0.98 2.6 2.3 2.9

72 1.60 3.1 2.9 3.2

420C24 No growth No growth No growth No growth

48 0.25 0.34 0.28 0.30

72 0.35 0.57 0.49 0.48

Fig. Comparative growth of S1 at different temperatures . Fig. Comparative growth of S2 at different temperatures.

Fig Comparative growth of S3 at different temperatures. Fig. Comparative growth S4 at different temperatures.

Table & and Fig. indicated that all four fungal strains S1, S2, S3 and S4 showed maximum growth at 34˚C temperature.

Optimization of pH for Growth of Selected Fungal Strains

S. No. Initial

pH

Final pH Growth (gm) Cellulase Activity (IU/ml) of fungal strains

S2 S3 S2 S3 S2 Strain S3 Strain

CMCase FPase CMCase FPase

1 4.0 4.6 4.8 0.042 0.030 0.080 0.031 0.043 0.027

2 4.5 5.4 5.6 0.035. 0.040 0.092 0.062 0.045 0.032

3 5.0 5.9 5.7 0.038 0.037 0.062 0.032 0.039 0.028

4 5.5 6.9 6.2 0.030 0.023 0.051 0.029 0.034 0.027

5 6.0 6.5 6.5 0.026 0.028 0.053 0.026 0.023 0.021

Above table indicated that fungal strain S2 and S3 showed maximum Cellulase activity i.e. 0.092 IU/ml and 0.045 IU/ml for CMCase and 0.062 IU/ml and 0.032 IU/ml for FPase respectively at 4.5 pH.

Effect of Various Substrates on Biomass Production And Cellulase Enzyme Production By Selected Fungal Strains S2

The isolated fungal strains S2 were used for further enzyme production. In this study Wheat bran and CMC were used as substrates.

Table: Effect of various substrates (Wheat bran and CMC) on growth and Cellulase production of selected fungal strain S2.

Inducers S. No Incubation

period (Days)

Final pH Biomass Enzyme Activity

(IU/ml)

CMCase FPase

CMC

1. 4 6.5 0.010 0.038 0.020

2. 6 6.5 0.012 0.046 0.040

3. 8 6.5 0.015 0.060 0.042

4. 10 6.6 0.019 0.062 0.044

5. 12 6.5 0.025 0.062 0.049

6. 14 6.3 0.028 0.048 0.042

7. 16 6.1 0.031 0.042 0.032

Wheat bran 1. 4 6.4 0.025 0.035 0.019

2. 6 6.2 0.030 0.048 0.032

3. 8 6.6 0.034 0.054 0.040

4. 10 6.3 0.038` 0.058 0.048

5. 12 6.6 0.041 0.068 0.056

6. 14 6.3 0.043 0.062 0.042

7. 16 6.1 0.048 0.040 0.028

FPase activity of cellulase enzyme at different pH and Temperatures

pH FPase activity, Temperature, IU/ml

40 oC 50 oC 60 oC 70 oC

4 0.045 0.048 0.047 0.041

5 0.051 0.052 0.048 0.042

6 0.048 0.047 0.044 0.041

7 0.045 0.045 0.042 0.041

8 0.041 0.043 0.040 0.000

CMCase activity of cellulase enzyme at different pH and Temperatures

pH CMCase activity, Temperature, IU/ml

40 oC 50 oC 60 oC 70 oC

4 0.047 0.047 0.043 0.033

5 0.049 0.052 0.045 0.035

6 0.043 0.045 0.037 0.032

7 0.033 0.034 0.034 0.031

8 0.031 0.032 0.030 0.000

Application In Pulp and Paper Industry (Bio-deinking)• Cellulase enzyme with maximum enzyme activity produced in Norkan’s media supplemented with Wheat Bran harvested at 12 days of incubation was used for deinking studies. (The FPase activity of extract is 0.056 IU/ml).• In the present study an improvement in pulp yield 68.66% was observed with cellulase enzyme when compared with yields of both control and commercial enzyme treated pulp. i.e 66.80% and 67.48% .Data is shown in Table.

Table: Result of deinking of Laser Waste paperParameters Control Commercial

Enzyme

Cellulase enzyme produced by

Isolated (S2 fungal strain)

Slushed Screened Slushed Screened Slushed Screened

Brightness,% 67.11 82.40 71.45 85.33 71.50 88.05

Yellowness,% -11.31 -4.27 -10.66 -5.30 -11.47 -6.40

Whiteness,% 111.74 117.75 117.48 121.84 117.66 127.65

L 82.30 91.25 80.30 92.40 83.24 92.99

A 2.60 2.24 2.60 2.35 2.69 2.52

B -5.94 -2.97 -5.94 -3.52 -6.08 -4.12

Yield,% 66.80 67.48 68.66

Further, from the results it is also seen that brightness (88.05%), whiteness (127.65%, 92.99%) of produced cellulase enzyme from selected S2 fungal strain treated pulps were improved when compared with commercial enzyme treated pulps (85.33%, 121.84% and 92.40%) and control (82.40%, 117.75% and 91.25%).

Paper pulp after Biodeinking. Ink removel Pad.

Summary of the Project Cellulases enzymes are capable of hydrolyzing cellulose and the most common and important industrial enzyme which is having a large number of industrial applications.The present work comprises the cellulase enzyme production by isolated cellulase producing organism and its industrial application. Observations of the present study may be summarized as follows:1.To isolate producing strain four samples were collected from different sources, i.e. paper mill soil sample, C.P.P.R.I field soil, Sugarcane industry soil and waste cotton sample. All isolated colonies were primarily screened by activity zone techniques with congo red dye. 2.Out of 48 colonies, only four colonies have shown positive results for cellulase production by activity zone method. These four fungal strains (S1, S2, S3 and S4) were selected for further study.3.The maximum growth was observed by the fungal strain S2 and S3 out of four selected fungal strain (S1, S2, S3 and S4) at 34˚C. Out of four different temperatures that is 28˚C, 34˚C, 38˚C and 42˚C.4.The fungal strain S2 and S3 showed maximum Cellulase activity i.e. 0.092 IU/ml and 0.045 IU/ml for CMCase and 0.062 IU/ml and 0.032 IU/ml for FPase respectively at 4.5 pH. 5.Cellulase enzyme produced by isolated Fungal strain is having optimum pH and Temperature at 5.0 pH and 50ºc. 6.Cellulase is having (FPase) used for deinking studies enzyme activity 0.056IU/ml in Norkan’s medium supplemented with wheat bran after 12 days of incubation. Enzyme treated deinked pulp showed better properties when compared to control pulp. Enzyme produced by S2 fungal strain showed better results when compared to commercial treated pulp. Improvement in brightness, whiteness and yield are observed in enzyme (from S2 strain) deinked pulp i.e. 88.05, 127.65 and 68.66% respectively, compared to evaluated commercial enzyme treated pulps 85.75, 122.31 and 66.85%.

Conclusion

•We conclude that the strain S2 has better deinking efficiency when compared with commercial deinking enzyme indication that this enzyme can be efficiently used for deinking of laser waste paper.

• The fungal strain S2 and S3 showed maximum Cellulase activity i.e. 0.092 IU/ml and 0.045 IU/ml for CMCase and 0.062 IU/ml and 0.032 IU/ml for FPase respectively at 4.5 pH.

• Other Commercially used enzyme like BIOFUELZYME, CEL50, NOVAZYME show the activity i.e. 0.085 IU/ml for CMCase and 0.054 IU/ml for FPase at 4.5 pH.

• In Pulp and Paper Industries(Biodeinking) S2 Strain show following results:-

• Improvement in brightness, whiteness and yield are observed in enzyme (from S2 strain) deinked pulp i.e. 88.05, 127.65 and 68.66% compared to evaluated commercial enzyme (NOVAZYME) treated pulps 85.75, 122.31 and 66.85%

•This enzyme is cheaper and Ecofriendly because if we used this enzyme (Biodeinking), we may prevent cutting of trees and we can recycled the waste paper.So, by this we can conserve the nature and natural resources(Environment).

ACKNOWLEDGEMENTThis work supported by Biotechnology Division, Central Pulp and paper research institute, Saharnpur (up).

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