OPTIMIZATION OF VARIOUS PARAMETERS FOR THE PRODUCTION OF CELLULASE BY ASPERGILLUS NIGER

By:

Richa Singh

Shachi

Smriti Khandelwal

Tanvi Jain

DEPARTMENT OF BIOTECHNOLOGY

FACULTY OF ENGINEERING & TECHNOLOGY

R.B.S COLLEGE, BICHPURI CAMPUS, AGRA.

May 2009

OPTIMIZATION OF VARIOUS PARAMETERS FOR THE PRODUCTION OF CELLULASE BY ASPERGILLUS NIGER

By:

Richa Singh

Shachi

Smriti Khandelwal

Tanvi Jain

Submitted to the Department of Biotechnology

in partial fulfillment of the requirement for the degree of

Bachelor of Technology

in

BiotechnologyS

Faculty of Engineering and Technology

R.B.S College, Bichpuri Campus, Agra.

U.P. Technical University, Lucknow (U.P.)

May 2009

CONTENTSTitles Page no.

DECLARATION…………………………………………………………………………….....i

CERTIFICATE………………………………………………………………………………...j

ACKNOWLEDGEMENT…….……………………………………………………………....k

ABSTARCT…………………………………………………………………………………….l

LIST OF ABBREVIATIONS…………………………………………………………………m

LIST OF TABLES……………………………………………………………………………..n

LIST OF FIGURES…………………………………………………………………………….o

CHAPTER 1: INTRODUCTION…………………………………………………………......1

CHAPTER 2: REVIEW OF LITRATURE…………………………………………………..5

2.1 General Aspects……………………………………………………………………………....6

2.2 Types of Cellulase…………………………………………………………………………6

2.3 Mechanism of Cellulolysis………………………………………………………………...7

2.4 Cellulase Producing Microorganism……………………………………………………....8

2.4.1 Fungi………………………………………………………………………………...8

2.4.2 Bacteria./……………………………………………………………………………..9

2.5 Substrate Used for the production of Cellulase………………………………………….10

2.6 Method of Production……………………………………………………………………11

2.6.1 Solid State Fermentation……………………………………………………….…..12

2.6.2Different Substrates for Solid State Fermentation………………………………….12

2.6.3 Submerged Fermentation…………………………………………………………...13

2.6.4 Different Substrate for Submerged Fermentation………………………………….14

2.7Enzyme Assay Method……………………………………………………………………15

2.7.1DinitrosalicyclicAcid Method……………………………………………………….15

2.7.2 Filter Paper Assay Method…………………………………………………………16

2.8 Industrial Application of Cellulase ………………………………………………………17

2.8.1 Application of Cellulase to Control Industrial Slime…………………………...….17

2.8.2 Application of Cellulase in the Sterility Test for Fortified Procaine Penecilline .17

2.8.3 Cellulase Usedfor Starch Production……………………………………………….18

2.8.4Cellulase Used for Fuel Production…………………………………………………18

CHAPTER3:MATERIALS AND METHODS……………………………………………….19

3.1Materials…………………………………………………………………………………..20

3.1.1 Chemical Used………………………………………………………………………20

3.1.2 Microorganisms……………………………………………………………………..21

3.1.3 Equipments………………………………………………………………………….21

3.1.4 Media Used…………………………………………………………………………21

3.2 Methods…………………………………………………………………………………..23

3.2.1 Maintenance of Stock Culture………………………………………………………23

3.2.2 Substrate Used for Production of Cellulase…………………………………………23

3.2.3 Pretreatment of Substrate…………….........................................................................24

3.2.4 Cellulase Production by Submerged Fermentation…………………………………..26

3.2.5 Cellulase Production by Solid State Fermentation…………………………………..27

3.2.6 Enzyme Assay………………………………………………………………………..27

3.2.6.1 Preparation of Glucose Standard Curve……………………………………..28

CHAPTER 4: RESULT AND DISCUSSION……………………………………………….31

4.1 Selection of Substrate for Production of Cellulase……………………………………….32

4.2 Selection of Fermentation Conditions for Production of Cellulase………………………35

4.3 Optimization of Temperature for Production of Cellulase……………………………….40

4.4 Optimization of Fermentation Conditions for Production of Cellulase…………………43

CHAPTER 5: CONCLUSION……………………………………………………..………….46

CHAPTER 6:BIBLIOGRAPHY ………………………………………………….…………..48

DECLARATION

We hereby declare that this submission is our own work and that, to the best of our

knowledge and belief, it contains no material previously published or written by another person

nor material which to a substantial extent has been accepted for the award of any other degree or

diploma of the university or other institute of higher learning, except where due acknowledgment

has been made in the text.

Name Roll No. Signature

Richa Singh 0500454020

Shachi 0500454022

Smriti Khandelwal 0500454024

Tanvi Jain 0500454028

Date:

(i)

CERTIFICATE

This is to certify that Project Report entitled “Optimization of various parameters for the

production of cellulase by Aspergillus niger”, which is submitted by Richa Singh,Shachi,Smriti

Khandelwal,Tanvi Jain in partial fulfillment of the requirement for the award of degree B. Tech.

in Biotechnology from U. P. Technical University, is a record of the candidate own work carried

out by him under my supervision. The matter embodied in this thesis is original and has not been

submitted for the award of any other degree.

 

 

Date: Er. V.K.Srivastava

Lecturer

Deptt. Of Biotechnology

F.E.T. R.B.S., College,

Bichpuri Campus, Agra

(j)

ACKNOWLEDGEMENT

It gives us a great sense of pleasure to present the report of the B. Tech Project undertaken

during Final Year. We owe special debt of gratitude to lecturer V.K. Srivastava, Department of

Bio-technology, for his constant support and guidance throughout the course of our work. His

sincerity, thoroughness and perseverance have been a constant source of inspiration for us. It is

only his cognizant efforts that our endeavors have seen light of the day.

We also take the opportunity to acknowledge the contribution of Er. Ashish

Shukla,Lecturer of Biotechnology department for his full support and assistance during the

development of the project.

We would also like to appreciate the contribution of Er. Manoj Kumar, and Er. Shakti

Katiyar Lecturer of Biotechnology department for their kind assistance and cooperation during

the development of our project. We are also grateful to the technical staff without who se

cooperation we would have not been able to complete our project. We would also thank our

friends for their contribution in the completion of the project.

Name Roll No.

1. Richa Singh 0500454020

2. Shachi 0500454022

3. Smriti Khandelwal 0500454024

4. Tanvi Jain 0500454028

Date:

(k)

ABSTRACT

Cellulases are a group of hydrolytic enzyme capable of degrading cellulose to the smaller

glucose units. The goal of the present study was to investigate the cellulase enzyme production

ability of A.niger against cellulosic materials such as Wheat bran, sugarcane, Bagasse, Paper,

cotton and select the best Cellulase enzyme producing substrate . maximum enzyme activity

obtained was Bagasse 19.72µmol/min/ml.

It was used for optimization of temperature which was obtained at 35°C with a maximum

activity of 19.58µmol/min/ml.

Time was also optimized with a maximum enzyme activity of 19.72 µmol/min/ml at 10 days

interval time.

(l)

(Sm)

List of Tables

TABLE 1: Different Fungi worked upon by different authors for Cellulase production

TABLE 2: Different Bacteria worked upon by different authors for Cellulase production

TABLE 3: Different Substrate worked upon by different authors for Cellulase production

TABLE 4: Absorbance of Glucose Standard Curve

TABLE 5: Enzyme Activity found for different Substrates

TABLE 6(a): Enzyme Activity found for different fermentation condition at 30°C

TABLE 6(b): Enzyme Activity found for different fermentation condition at 35°C

TABLE 7: Enzyme Activity found for different Temperatures

TABLE 8: Enzyme Activity found for different days interval

(n)

List of Figures

Figure 1: Prepared slant of Sub cultured A.niger

Figure 2(a): Pretreated substrates Bagasse (left), Wheat Bran (right)

Figure 2(b): Pretreated substrates Cotton (left) , Paper (right)

Figure 3: Glucose Standard Curve

Figure 4: Diagram showing Absorbance for Different Substrates

Figure 5(a): Diagram showing Absorbance for Different Fermentation condition at 30°C

Figure 5(b): Diagram showing Absorbance for Different Fermentation condition at 35°C

Figure 6: Diagram showing Absorbance at Different Temperature

Figure 7: Diagram showing Absorbance at Different Fermentation time (days)

(o)

INTRODUCTION

REVIEW OF LITERATURE

MATERIALS AND

METHODS

CONCLUSION

BIBLIOGRAPHY