MATHEMATICAL MODEL ON THE CELLULASE ENZYMES...

I

MATHEMATICAL MODEL ON THE CELLULASE

AS A FUNCTION OF PRODUCTION SENZYME

VISCOSITY

FAHIMEH ABDEYAZDAN

DISSERTATION SUBMITTED IN FULFILLMENT OF

THE REQUIREMENT FOR THE DEGREE OF MASTER

OF SCIENCE

INSTITUTE OF BIOLOGICAL SCIENCE

FACULTY OF SCIENCE

UNIVERSITY OF MALAYA

KUALA LUMPUR

2011

II

This thesis is dedicated to my parents

Mostafa and Narjes

III

Declaration

Name of Candidate: FAHIMEH ABDEYAZDAN (I.C/Passport No: L13473857)

Registration/Matric No: SGR080115 Name of Degree: MASTER OF SCIENCE

Title of Project Paper/Research Report/Dissertation/Thesis (―this Work‖):

MATHEMATICAL MODEL ON THE CELLULASE ENZYME PRODUCTION AS A

FUNCTION OF VISCOSITY

Field of Study: Biotechnology

I do solemnly and sincerely declare that:

I am the sole author/writer of this Work;

(1) This Work is original;

(2) Any use of any work in which copyright exists was done by way of fair dealing and

for permitted purposes and any excerpt or extract from, or reference to or

reproduction of any copyright work has been disclosed expressly and sufficiently

and the title of the Work and its authorship have been acknowledged in this Work;

(3) I do not have any actual knowledge nor do I ought reasonably to know that the

making of this work constitutes an infringement of any copyright work;

(4) I hereby assign all and every rights in the copyright to this Work to the University of

Malaya (―UM‖), who henceforth shall be owner of the copyright in this Work and

that any reproduction or use in any form or by any means whatsoever is prohibited

without the written consent of UM having been first had and obtained;

(5) I am fully aware that if in the course of making this Work I have infringed any

copyright whether intentionally or otherwise, I may be subject to legal action or any

other action as may be determined by UM.

Candidate’s Signature Date

Subscribed and solemnly declared before,

Witness’s Signature Date

Name:

Designation:

IV

Abstract

As cellulosic material is the most abundant and renewable source of organic

matter, ethanol can be produced cheaply from cellulosic waste. The cellulose must be

hydrolyzed to glucose before it can be fermented to ethanol. The enzymatic hydrolysis

of cellulose using cellulase enzymes is considered an environmental- friendly and less

energy intensive alternative.

White-rot fungi belong to the wood-destroying basidiomycetes and are well

known as the only micro-organisms responsible for the enzyme hydrolysis of all major

wood polymers, including lignin, cellulose and hemicelluloses.

On the other hand, the production cost of cellulase enzymes is a limitation in

ethanol fermentation. Therefore, the goal is to produce the enzymes economically by

means of high-yield cellulase fermentation.

Different type of bacteria and fungi can be used to achieve to this goal. In the

present study, the ability of Pycnoporus sanguineus, a white rot fungus to produce

cellulase was investigated since there are no reports of its cellulase production

available. Different types of basal media were tested and enzyme activity was measured

in the presence of cellulose powder, carboxymethylcellulose (CMC), Tween 80 and

yeast extract. The cost of each medium is an important consideration. The highest

amount of reducing sugar production was observed when CMC and Tween 80 were

V

used as the components of the medium. This formulation is the most cost effective

among the medium formulation.

The study also presents the development of a mathematical model for cellulase

production in submerged fermentation as a function of medium’s viscosity. To achieve

this, the fungus was grown in two different media on agar plates. Then, the inoculation

was carried out from these plates to a medium formulation in the shake flasks using

variable concentration of CMC for determination of CMCase activity. Enzyme activity

and viscosities of media were measured simultaneously. Mathematical function was

explored for each media and then a unique model was reported for each media.

VI

Abstrak

Memandangkan sebatian selulos merupakan bahan organik yang banyak dan

sumber yang boleh diperbaharui secara semula jadi, etanol dapat dihasilkan dengan

kos yang rendah daripada sisa-sisa selulos. Selulos harus di hydrolisiskan kepada

glukos sebelum proses penapaian etanol dapat dilakukan. Pencernaan (hidrolisis)

selulose dengan menggunakan enzim selulase merupakan satu kaedah yang tidak

mencemarkan alam sekitar dan tidak membebankan.

Sebaliknya, kulat putih reput kepunyaan basidiomycetes memusnahkan kayu

dan dikenali sebagai mikroorganisma sahaja bertanggungiawab untuk pemineralan

semnua polimer kayu yang utama, termasuk lignin, selulosa dan hemicelluloses.

Penggunaan enzim selulose dalam proses penapaian etanol memerlukan kos

penghasilan yang tinggi. Oleh itu, tujuan utama kajian ini adalah untuk

menghasilkan enzim ini dalam kuantiti yang banyak tanpa mengeluarkan kos yang

tinggi.

Berbagai jenis bacteria dan kulat boleh digunakan untuk mencapai matlamat

ini. Dalam kajian ini, kemampuan Pycnoporus sanguineus iaitu cendawan pelapuk

putih yang dapat menghasilkan selulase telah dikaji dan diselidik kerana tiada

laporan kajian mengenai pengahasilan enzim ini daripada spesies tersebut. Berbagai

jenis media dasar diuji dan aktiviti enzim diukur dengan adanya serbuk selulosa

carboxymethyl cellulose (CMC), Tween 80 and ekstrak yis.

VII

Kemudian, kos setiap media akan menjadi pelaburan. Jumlah tertinggi

mengurangkan pengeluaran gula apabila CMC dan Tween 80 telah digunakan

sebagai media. Mujurlah media ini adalah media termurah antara yang lain yang

telah disiasat.

Disertasi ini juga menerangkan pembangunan model matematik untuk

penghasilan selulase dalam fermentasi terendam sebagai fungsi dari viskositi

medium. Untuk mencapai matlamat ini, kulat ditumbuhkan dalam dua media yang

berbeza pada plat agar . kemudian, inokulasi dilakukan daripada plat agar ke dalam

flask yang mengandungi medium yang telah diformulasikan dimana kepekatan

CMC telah dimanipulasikan untuk penentuan aktiviti CMCase. Aktiviti enzim dan

viskositi media diukur serentak. Fungsi matematik yang menyeluruh telah dijalankan

dan model yang unik telah dipersembahkan untuk setiap media.

VIII

Acknowledgements

I am indebted to my helpful supervisor, Assoc. Prof. M Suffian M Annuar,

whose encouragement, guidance and support from the initial to the final level

enabled me to develop an understanding of the subject. The supervision and support

that he gave truly help not only my thesis even my life.

I’m heartily thankful to Dr. Ahmad Neyamadpour, for his help during

accomplish my project and his guide to understanding of mathematical modelling

aspect of thesis step by step. Also, Great deals appreciated go to Dr. Ahmad Faris

Mohd Adnan, my co- supervisor for his advices.

To Professors Amru Nasrulhaq and Rosli Hashim, heads of Institute of

Biological Science, I am grateful for the trust deposited in my work and for the

motivation demonstrated.

I offer my regards and blessings to my knowledgeable friend Masoud

Razavian and my lab mate Muhamad Alimin; they supported me in any respect

during the completion of the project.

Lastly, I would also to express my appreciation to my liberal parents and my

brother and sister, Mohammad Ali and Farideh; their love gave me forces to make

this work.

Fahimeh Abdeyazdan

December, 2011

IX

Table of contents

MATHEMATICAL MODEL ON THE CELLULASE ENZYMES

PRODUCTION AS A FUNCTION OF VISCOSITY ............................................. I

Declaration ............................................................................................................... III

Abstract .................................................................................................................... IV

Abstrak ..................................................................................................................... VI

Acknowledgements ............................................................................................... VIII

Table of contents ..................................................................................................... IX

List of Tables ........................................................................................................ XIII

List of Figures ....................................................................................................... XIX

Publication ........................................................................................................... XXII

Abbreviations .................................................................................................... XXIII

Chapter 1 .................................................................................................................... 1

Introduction ................................................................................................................ 1

1.1. Background and scope of research .................................................................... 1

1.1. Objectives of thesis ........................................................................................... 2

1.2. Outline of the thesis .......................................................................................... 3

X

Chapter 2 .................................................................................................................... 4

Literature review ........................................................................................................ 4

2.1. Biological part ................................................................................................... 4

2.1.1. White-rot Fungi .............................................................................................. 4

2.1.2. Cellulose structure ........................................................................................ 10

2.1.3. Cellulase enzyme system ............................................................................. 13

2.1.4. Endogluconases activity using viscometric method .................................... 21

2.2. Mathematical modeling ................................................................................... 23

2.2.1. General Regression Theory .......................................................................... 24

2.2.2. Derivatives and Nonlinear Regression ......................................................... 29

2.2.3. Understanding and Interpreting Regression Results .................................... 30

Residuals ................................................................................................................ 30

Sum of Residuals ................................................................................................... 31

Average Residual ................................................................................................... 31

Residual Sum of Squares ....................................................................................... 32

Error Variance ........................................................................................................ 33

Standard Error of the Estimate ............................................................................... 33

R2, or Coefficient of Multiple Determination ........................................................ 33

2.2.4. Regression Variable (parameter) Results ..................................................... 34

XI

2.2.5. Confidence Intervals .................................................................................... 35

2.2.6. Variance Inflation Factors (VIF) .................................................................. 36

2.2.7. Determining the goodness of fit ................................................................... 37

Chapter 3 .................................................................................................................. 41

Materials and Methods ............................................................................................ 41

3.1. Microorganism ................................................................................................ 41

3.2. Media preparation ........................................................................................... 41

3.3. Inoculation and incubation .............................................................................. 43

3.4. Enzyme production ......................................................................................... 43

3.4.1. Glucose standard calibration ........................................................................ 45

3.4.2. CMCase activity using spectrometer ........................................................... 47

3.4.3. FPase activity using spectrometer ................................................................ 48

3.5. Enzyme activity using commercial cellulase from Aspergilleus niger ........... 48

3.6. Viscometric method for cellulase assay .......................................................... 49

3.6.1. Media composition ....................................................................................... 51

3.6.2. CMCase activity measurement .................................................................... 52

3.6.3. Viscosity measurement ................................................................................ 52

3.6.4. Vibro viscometer system .............................................................................. 53

Chapter 4 .................................................................................................................. 56

XII

Results and discussion ............................................................................................. 56

4.1. Fungal cellulase production in modified media .............................................. 56

4.2. Commercial enzyme production ..................................................................... 66

4.3. Comparison of costs of selected media ........................................................... 67

4.4. Mathematical modelling part .......................................................................... 68

Chapter 5 .................................................................................................................. 83

Conclusions ............................................................................................................... 83

References ................................................................................................................. 86

Appendix A ............................................................................................................... 99

XIII

List of Tables

Table 2.1. Distribution of cellulase in fungi. ............................................................... 7

Table 3.1. Composition of potato dextrose broth. ...................................................... 41

Table 3.2. Basal medium composition. ...................................................................... 43

Table 3.3. Media compositions for fungal cellulase production in shake flasks. ...... 44

Table 3.4. Composition of GYMP Agar. ................................................................... 51

Table 4.1. The viscosity and enzyme activities when inocula were prepared in

GYMP (pH= 5.5 at 25oC). ......................................................................... 69

Table 4.2. The viscosity and enzyme activities when inocula were prepared in PDA

media (pH= 5.5 at 25oC). ........................................................................... 70

Table 4.3. Summary of regression results for the best fit modelling study for media

with GYMP inocula. .................................................................................. 72

Table 4.4. 99% Confidence Intervals, GYMP inocula, 5 g L-1

CMC concentration. 74


CMC concentration.

.................................................................................................................................... 74


CMC concentration.

.................................................................................................................................... 75


CMC concentration.

.................................................................................................................................... 75


CMC concentration.

.................................................................................................................................... 76

XIV

Table 4.9. Summary of regression results for the best fit modelling study for media

with PDA inocula. ..................................................................................... 77

Table 4.10. 99% Confidence Intervals, PDA inocula, 5 g L-1

CMC concentration. .. 79









Table A.1. Regression Variable Results, media= GYMP, 5 g L

-1 CMC concentration.

.................................................................................................................................... 99

Table A.2. 68% Confidence Intervals, media= GYMP, 5 g L-1

CMC concentration.

.................................................................................................................................... 99


CMC concentration.

.................................................................................................................................. 100


CMC concentration.

.................................................................................................................................. 100


CMC concentration.

.................................................................................................................................. 100

Table A.6. Variance Analysis, media= GYMP, 5 g L-1

CMC concentration. ......... 101

Table A.7. Regression Variable Results, media= GYMP, 10 g L-1

CMC

concentration. ........................................................................................ 101


CMC concentration.

.................................................................................................................................. 101


CMC concentration.

.................................................................................................................................. 102

XV

Table A. 10. 95% Confidence Intervals, media= GYMP, 10 g L-1

CMC

concentration. ............................................................................................. 102


CMC

concentration. ............................................................................................. 103


CMC concentration. ....... 103


CMC

concentration. ........................................................................................ 103


CMC

concentration. ........................................................................................ 104


CMC

concentration. ........................................................................................ 104

Table A.16. 95 % Confidence Intervals, media= GYMP, 15 g L-1

CMC

concentration. ........................................................................................ 104

Table A.17. 99 % Confidence Intervals, media= GYMP, 15 g L-1

CMC

concentration. ........................................................................................ 105


CMC concentration. ..... 105


CMC

concentration. ........................................................................................ 105


CMC

concentration. ........................................................................................ 106


CMC

concentration. ........................................................................................ 106


CMC

concentration. ........................................................................................ 107

XVI


CMC

concentration. ........................................................................................ 107



Table A. 25. Regression Variable Results, media= GYMP, 25 g L-1

CMC

concentration. ........................................................................................ 108


CMC

concentration. ........................................................................................ 108


CMC

concentration. ........................................................................................ 108


CMC

concentration. ........................................................................................ 109


CMC

concentration. ........................................................................................ 109



Table A.31. Regression Variable Results, media= PDA, 5 g L-1

CMC concentration.

.................................................................................................................................. 110

Table A. 32. 68% Confidence Intervals, media= PDA, 5 g L-1

CMC concentration.

.................................................................................................................................. 110


CMC concentration.

.................................................................................................................................. 111


CMC concentration.

.................................................................................................................................. 111

Table A.35. 99% Confidence Intervals, media= PDA, 5 g L-1

CMC concentration.

.................................................................................................................................. 111

XVII

Table A.36. Variance Analysis, media= PDA, 5 g L-1

CMC concentration. ........... 112


CMC

concentration. ........................................................................................ 112


CMC concentration.

.................................................................................................................................. 112


CMC concentration.

.................................................................................................................................. 113


CMC concentration.

.................................................................................................................................. 113


CMC concentration.

.................................................................................................................................. 113




CMC

concentration. ...................................................................................... 114


CMC concentration.

.................................................................................................................................. 114


CMC concentration.

.................................................................................................................................. 115


CMC concentration.

.................................................................................................................................. 115


CMC concentration.

.................................................................................................................................. 116



XVIII


CMC

concentration. ...................................................................................... 116


CMC concentration.

.................................................................................................................................. 116


CMC concentration.

.................................................................................................................................. 117


CMC concentration.

.................................................................................................................................. 117


CMC concentration.

.................................................................................................................................. 118




CMC

concentration. ...................................................................................... 118


CMC concentration.

.................................................................................................................................. 119


CMC concentration.

.................................................................................................................................. 119


CMC concentration.

.................................................................................................................................. 119


CMC concentration.

.................................................................................................................................. 120



XIX

List of Figures

Figure 2.1. Diagram of the structural formula for the β-1,4-glucan polymer chain

(cellulose). The repeating unit, cellobiose is indicated in brackets (Brown

et al., 1996).............................................................................................. 11

Figure 2.2. A simplified model to illustrate the cross-linking of cellulose microfibrils

and hemicellulose in the lignocellulosic biomass. .................................. 12

Figure 2.3. Enzymatic hydrolysis of cellulose to glucose. ......................................... 13

Figure 2.4. Typical cellulase structure. (a): Exogluconase, (b): Endogluconase. ...... 15

Figure 2.5. Hypothesis for the role of oligomers during microbially and

enzymatically mediated cellulose hydrolysis .......................................... 16

Figure 2.6. Schematic representation of the hydrolysis of amorphous and

microcrystalline cellulose by non-complexed (a) and complexed (b)

cellulase systems. not shown to scale...................................................... 17

Figure 3.1. Schematic diagram for enzyme activity measurement……………….....43

Figure 3.2. Glucose standard calibration.................................................................... 46

Figure 3.3. Spectrophotometric system (V-630 Jusco). ............................................. 47

Figure 3.4. Schematic diagram for mathematical modeling methodology………....51

Figure 3.5. Glass fibre for sample filtration. .............................................................. 53

Figure 3.6. Vibro viscometer system. ........................................................................ 53

Figure 3.7. Vibro viscometer (detection system). ...................................................... 54

Figure 4.1. Reducing sugars production and enzyme activities from CMCase and

FPase when 20 g L-1

cellulose powder was used as the major source of carbon. ...... 57

XX


FPase when 20 g L-1

cellulose was as the major source of carbon and Tween

80 was included in the basal media. ............................................................... 58


FPase when 10 g L-1

cellulose was as the major source of carbon and 10

g L-1

yeast extract supplementation. ..................................................... 59

Figure 4.4. Reducing sugar production and enzyme activities from CMCase and

FPase when 10 g L-1

cellulose, 10 g L-1

yeast extract and Tween 80 was

included in the medium. .......................................................................... 59

Figure 4.5. The reducing sugars production in different media composition with

cellulose as a major carbon source. T: Tween 80, Y: yeast extract. ....... 60


FPase when 20 g L-1

CMC was the major source of carbon in the media.

................................................................................................................. 61


Fpase when 20 g L-1

CMC was the major source of carbon and Tween

80 was included in the basal media. ........................................................ 62

Figure 4.8. Reducing sugar production and enzyme activities from CMCase and

FPase when 10 g L-1

CMC, 10 g L-1

yeast extract was included in the

medium. ................................................................................................... 63


Fpase when 10 g L-1

CMC, 10 g L-1

yeast extract and Tween 80 were

included in the medium. .......................................................................... 64

XXI

Figure 4.10. The reducing sugars production in different media composition in the

presence of CMC as a major carbon source. ........................................... 65

Figure 4.11. Reducing sugars production using commercial cellulase. ..................... 67

Figure 4.12. Costs in Malaysian Ringgit to produce 1 L of production media and

reducing sugars production after 144 hours. ........................................... 68

Figure 4.13. Agreement between the proposed model outputs and experimental data

(GYMP inocula, a) 5 g L-1

, b) 10 g L-1

, c) 15 g L-1

, d) 20 g L-1

, d)25 gL-1

CMC concentration). ............................................................................... 73

Figure 4.14. Agreement between the proposed model outputs and experimental data

PDA inocula, a) 5 g L-1

, b) 10 g L-1

, c) 15 g L-1

, d) 20 g L-1

, d) 25 g L-1

CMC concentration. ................................................................................ 78

Figure 5.1. Enzyme assay using viscometric and spectrophotometric methods in (a)

GYMP media and (b) PDA media........................................................85

XXII

Publication

The following paper has been published from this thesis:

Conference:

Media strategies to increase cellulase production from Pycnoporus sanguineus.

15th

Biological Graduate Congress, University of Malaya, Kuala Lumpur, Malaysia.

XXIII

Abbreviations

AR Average Residual

Avicelase Avicel hydrolysis activity

BG β-glycosidase

CBH Cellobiohydrolase

CMC Carboxymethylcellulose

CMCase Carboxymcthyl cellulase

Ea Enzyme activity

EG Endoglucanase

Fp Filter paper

FPase Filter paper hydrolyse

mol. wt. molecular weight

Glu Glucose

R2 Coefficient of Multiple Determination

Ra2 Adjusted Coefficient of Multiple Determinations

RSS Residual Sum of Squares

SEE Standard Error of the Estimate

SR Sum of Residuals

T Time

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