OPTIMIZATION OF β-MANNANASE PRODUCTION FROM Bacillus

OPTIMIZATION OF β-MANNANASE PRODUCTION

FROM Bacillus subtilis H AND ITS APPLICATION FOR

THE FORMULATION OF FISH FEED CONTAINING

PALM KERNEL CAKE

SITI NORITA BINTI MOHAMAD

DOCTOR OF PHILOSOPHY

2016

Optimization of β-Mannanase production from Bacillus

subtilis H and its Application for the Formulation of Fish

Feed Containing Palm Kernel Cake

by

Siti Norita Binti Mohamad

A thesis submitted in fulfillment of the requirements for the degree of

Doctor of Philosophy

Faculty of Agro Based Industry

UNIVERSITI MALAYSIA KELANTAN

2016

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THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original research

and has not been submitted for a higher degree to any other University or Institution.

OPEN ACCESS I agree that my thesis is to be made immediately available

As hardcopy or on-line open access (full text).

EMBARGOES I agree that my thesis is to be made available as hardcopy

Or on-line (full text) for a period approved by the Post

Graduate Committee.

Dated from ____________ until _____________

CONFIDENTIAL (Contains confidential information under the Official Secret

Act 1972)*

RESTRICTED (Contains restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of Universiti Malaysia Kelantan.

2. The library of Universiti Malaysia Kelantan has the right to make copies for the

purpose of research only.

3. The library has the right to make copies of the thesis for academic exchange.

_______________________ ___________________________

SIGNATURE SIGNATURE OF SUPERVISOR

_______________________ ___________________________

IC/PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

ACKNOWLEDGEMENTS

All praise to Allah S.W.T. who has guide me safely, through every mile, grant me wealth,

give me health and most of all give me care and love well. I also thank Allah S.W.T. for

giving me the strength to finish my study.

I would like to express my sincere appreciation and deepest gratitude to my supervisor,

Prof. Dato’ Dr. Hj. Ibrahim Che Omar for his invaluable guidance, kind and suggestions

during the course of this study. My deep appreciation is also extent to the members of my

supervisory committee, Prof. Dr. Arbakariya Ariff and Dr. Noor Azlina Ibrahim for their

inputs and guidance.

I would also like to express my gratitude to the Director General and Director of

Research, of Fisheries Department to for their permission to pursue the study. Thanks

also extended to the Director of FRI Glami Lemi for supporting me to continue the study.

I would also like to thank to all the people I have met along the way and which have

helped me in a number of ways during my work at Universiti Malaysia Kelantan

especially to all the staff and post-graduate students.

I also wish to express my thanks to all my friends in Freshwater Fisheries Research

Division, FRI Glami Lemi especially to Reha, Zie, Chew, Kamarul and Wardi for helping

me during the period of my study.

Finally, a great and heartfelt thank you I’ll give to my family, Hjh Rokiah Md. Diah, Hj

Mohamad Hj Abu, all my brothers, sisters in-law, niece and nephew; thank you for your

understanding, caring and moral support given during the period of my study.

TABLE OF CONTENTS

PAGE

THESIS DECLARATION

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

ABSTRAK

ABSTRACT

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ii

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xiii

xviii

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xxxiv

CHAPTER 1 INTRODUCTION

1.1 Status of Animal Feed Industry in Malaysia

1.2 Problem Statements

1.3 Research Objectives

1.4 Organization of Thesis

1

6

7

8

CHAPTER 2 LITERATURE REVIEW

2.1 Utilization of Agro Waste: A Concept of Waste to Wealth

2.2 Type of Agro Waste

2.3 Palm Kernel Cake (PKC)

2.4 Uses of PKC

13

16

19

22

2.5 Problems Associated to the Use of PKC

2.6 Enzymatic Degradation of PKC

2.7 β-Mannanase the Mannan Degrading Enzyme

2.7.1 β-Mannanase Producing Microorganisms

2.7.2 Fermentation Process for β-Mannanase Production

2.8 Enzymatic Saccharification of PKC

2.9 Application of β-Mannanase in Feed Formulation

26

31

32

36

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43

CHAPTER 3 GENERAL MATERIALS AND METHODS

3.1 Chemicals and Reagents

3.2 Equipments

3.3 Overall Experimental Flowchart

3.4 General Methodologies

3.4.1 Palm Kernel Cake (PKC)

3.4.2 Microorganism and Enzyme Production

3.4.3 Analytical Procedures

3.4.3.1 Determination of Total Reducing Sugars

3.4.3.2 Determination of β-Mannanase and

Endoglucanase Activity

3.4.3.3 Determination of α-Galactosidase (pNP-αGal)

and β-Mannosidase (pNP-βMan) Activity

3.4.3.4 Protein Assay by Lowry's Method

3.4.3.5 Proximate Analysis

48

50

52

56

57

58

59

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60

61

62

63

3.4.3.6 High Performance Liquid Chromatography

(HPLC)

3.4.4 Experimental Design

3.4.5 Statistical Analysis

65

66

68

CHAPTER 4 SELECTION AND IDENTIFICATION OF β-

MANNANASE PRODUCER AND THE PURIFICATION AND

CHARACTERIZATION OF THE ENZYME

4.1 Background

4.2 Materials and Methods

4.2.1 Selection and Identification of β-Mannanase Producing

Bacteria

4.2.1.1 Primary Selection Procedure

4.2.1.2 Secondary Selection (Isolation of

Mannanolytic Strain)

4.2.2 Bacterial Identification

4.2.2.1 Gram’s Staining

4.2.2.2 Endospore Staining

4.2.2.3 Biochemical Test

4.2.2.4 Scanning Electron Microscopy (SEM)

4.2.2.5 16S rRNA

4.2.3 Purification of β-Mannanase

4.2.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis (SDS-PAGE)

4.2.5 Zymograms

69

77

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78

79

79

80

81

87

88

90

91

93

4.2.6 Characterization β-Mannanase

4.2.6.1 Effect of pH on Enzyme Activity and Stability

4.2.6.2 Effect of Temperature on Enzyme Activity and

Stability

4.2.6.3 Effect of Metals and Inorganic Compounds on

β-Mannanase Activity

4.2.6.4 Substrate Specificity and Kinetic Parameters

Km and Vmax

4.3 Results

4.3.1 Selection of β-Mannanase Producing Bacteria

4.3.2 Identification of Isolate H

4.3.3 Purification of β-Mannanase

4.3.4 Enzyme Physical Characteristics

4.3.4.1 Substrate Specificity and Kinetic Parameters

Km and Vmax

4.3.4.2 Effect of pH on Activity and Stability

4.3.4.3 Effect of Temperature on Activity and Stability

4.3.4.4 Metal and Inorganic Compounds Effect

4.4 Discussion

4.5 Conclusion

94

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107

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137

CHAPTER 5 FERMENTATION STUDIES – OPTIMIZATION OF

β-MANNANASE PRODUCTION IN SHAKE FLASK AND

FERMENTER SYSTEM

5.1 Background


5.2.1 Optimization of Medium Compositions in Shake Flask

5.2.1.1 Microorganism

5.2.1.2 Partial Factorial Design for Selection of

Medium Component

5.2.1.3 Optimization PKC and Different Nitrogen

Sources Concentrations in Shake Flask by

RSM

5.2.2 Optimization of β-Mannanase Production in 2 L

Fermenter

5.2.2.1 Inoculum Preparation

5.2.2.2 Fermentation Experiments

5.2.2.3 Cell Growth Determination

5.2.2.4 Response Surface Methodology for Culture

Condition in 2 L Fermenter

5.3 Results

5.3.1 Optimization of Medium Compositions in Shake Flask

5.3.1.1 Screening of the Critical Variables

5.3.1.2 Response Surface Methodology

5.3.1.3 Attaining Optimum Conditions

5.3.2 Optimization of β-Mannanase Production in 2 L

Fermenter

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163

5.3.2.1 Regression Models of Response

5.3.2.2 Attaining Optimum Conditions

5.4 Discussion

5.5 Conclusion

163

174

180

202

CHAPTER 6 STABILIZATION OF THE FREEZE DRIED β-

MANNANASE

6.1 Background


6.2.1 Freeze Drying Process

6.2.2 Partial Factorial Design for Selection of Suitable

Stabilizer

6.2.3 Response Surface Methodology for Stabilization of

Freeze Dried β-Mannanase

6.2.4 Enzyme Stability Test

6.3 Results

6.3.1 Partial Factorial Design for Screening of Stabilizers for

Freeze Drying Process

6.3.2 Response Surface Methodology (RSM)

6.3.3 Attaining Optimum Conditions

6.3.4 Validation of the Model

6.4 Discussion

6.5 Conclusion

204

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251

CHAPTER 7 SACCHARIFICATION OF PKC USING CRUDE β-

MANNANASE PREPARATION FROM Bacillus subtilis H

7.1 Background


7.2.1 Pretreatment of PKC

7.2.2 Proximate Analysis

7.2.3 Saccharification Experiment

7.2.3.1 Substrate and Enzyme Concentration

7.2.3.2 Adsorption Experiments

7.2.3.3 Effect of Different pH and Temperature

7.2.3.4 Effect of Heat Pretreatment

7.2.3.5 Effect of Different Particle Size

7.2.3.6 Effect of Surfactant Addition

7.2.4 Comparison with Commercial Enzyme

7.2.5 Scanning Electron Microscopy (SEM) Studies

7.3 Results

7.3.1 Chemical Composition of PKC

7.3.2 Effect of PKC and Enzyme Concentration

7.3.3 Effect of pH and Temperature

7.3.4 Effect of Heat Pretreatment

7.3.5 Effect of Particle Sizes

7.3.6 Effect of Surfactant Addition

253

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7.3.7 Comparison of PKC Saccharification with Commercial

Enzyme

7.3.8 Microscopic Examination of Hydrolyzed PKC

7.4 Discussion

7.5 Conclusion

278

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300

CHAPTER 8 THE GROWTH AND APPARENT DIGESTIBILITY

OF RED TILAPIA HYBRID (Oreochromis sp.) FED

FORMULATED DIETS CONTAINING β-MANNANASE

SUPPLEMENTED PALM KERNEL CAKE

8.1 Background


8.2.1 Enzyme Source

8.2.2 Experimental Dietary Preparation

8.2.3 Experimental Procedures

8.2.3.1 Growth Study

8.2.3.2 Apparent Digestibility Test

8.2.4 Analytical Procedures

8.2.4.1 Biochemical Analysis

8.2.4.2 Determination of Total Protease Activity

8.2.4.3 Determination of Trypsin Activity

8.2.4.4 Determination of Chymotrypsin Activity

8.2.4.5 Determination of Amylase Specific Activity

8.2.4.6 Chemical Analyses

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317

8.2.4.7 Water Quality Analysis

8.2.4.8 High Performance Liquid Chromatography

(HPLC)

8.2.5 Data Calculation and Statistical Analysis

8.3 Results

8.3.1 Proximate and Amino Acid Composition of

Experimental Diets

8.3.2 Growth Performance

8.3.3 Apparent Digestibility Coefficients

8.3.4 Whole Body and Muscle Composition

8.3.5 Digestive Organ Index and Enzyme Activity

8.4 Discussion

8.5 Conclusion

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CHAPTER 9 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS

9.1 Summary

9.2 General Conclusion

9.3 Recommendations

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365

REFERENCES 368

Appendix A - Medium and Substrate Preparation

Appendix B

Appendix C - Sephadex Column Preparation

420

422

423

Appendix D - Sodium Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis (SDS-PAGE) Preparation

426

LIST OF PUBLICATIONS 379

LIST OF TABLES

NO. PAGE

2.1 Chemical composition (on dry matter) and gross energy

of the PKC.

24

2.2 Amino acid composition of palm kernel cake (PKC).

28

2.3 Mineral composition of palm kernel cake (PKC) and palm

kernel shell (PKS).

30

2.4 Nutrient composition of dietary ingredients (% dry

matter).

45

3.1 List of suppliers of the chemicals used.

48

3.2 List of the instruments used.

50

4.1 Summary of purification and characterization of β-

mannanase enzyme by different microorganisms.

72

4.2 Colony characteristics and cell morphologies of isolate H.

102

4.3 The biochemical tests.

104

4.4 Top five hits of similarity search with BLAST.

107

4.5 Summary of β-mannanase purification from the isolated

Bacillus subtilis H.

108

4.6 Substrate specificity of enzymes towards different

substrates.

111

4.7 Effect of metal and inorganic compounds on the activity

of crude and purified enzyme preparations of Bacillus

subtilis H.

119

5.1 Different concentration of medium components in sixteen

experimental runs based on the actual values.

142

5.2 Actual factor levels corresponding to coded factor levels

for different concentration of ingredients.

144

5.3 Full factorial central composite design matrix of four

medium components in actual values.

145


for β-mannanase production by Bacillus subtilis H in 2 l

fermenter.

150

5.5 Full factorial central composite design matrix of three

variables in actual values.

151

5.6 Specification of experiments based on 28-4IV fractional

factorial design and summarized results for each

experimental run

152

5.7 Regression analysis of the 28−4IV fractional factorial

design.

154

5.8 Full factorial central composite design matrix of four

variables in coded units along with the observed

responses for β-mannanase and pH during harvest of

Bacillus subtilis H in batch fermentation.

156

5.9 The least-square fit and parameters (significant of

regression coefficient) for β-mannanase production.

158


regression coefficient) for pH at harvest.

159



responses for β-mannanase, fermentation time, medium

pH and growth (Log10 CFU/ml) at maximum production

of enzyme by Bacillu subtilis H.

164


regression coefficient) for β-mannanase production in 2 l

fermenter.

170


regression coefficient) for fermentation time at maximum

β-mannanase production.

170


regression coefficient) for medium pH at maximum β-

mannanase production.

171


regression coefficient) for growth (CFU per ml) at

maximum β-mannanase production.

171

6.1 Examples of excipients providing an efficient stabilizing

effect during freeze and spray drying of protein (80% of

activity recovery after freeze drying).

208

6.2 Specification of experiment based on 26−3III fractional

factorial design for each experimental run in actual value

[% weight (w) / volume, (v)].

212


for stabilizer used.

213


variables in actual value [% weight (w) / volume, (v)].

214

6.5 Specification of experiment based on 26−3III fractional

factorial design and summarized results for each

experimental run.

218

6.6 Regression analysis of the 26−3III fractional factorial

design for stability of freeze dried enzyme.

218



responses for freeze dried β-mannanase activity retention

after exposure to different conditions.

222


variables in coded units along with the predicted

responses for freeze dried β-mannanase activity retention

after exposure to different conditions.

223

6.9 Estimated regression coefficient and corresponding F-

and P-value for fresh β-mannanase stability just after

freeze dried (FD0).

225


and P-value for β-mannanase stability after reconstituted

and kept in fridge for a month (RC1).

225


and P-value for β-mannanase stability just after freeze

dried and were heat at 70oC for 6 h prior to dissolution

(T706).

226


and P-value for β-mannanase stability after stored in

environment that have 44% relative humidity (K2CO3) for

one month and were heat at 70oC for 6 h prior to

dissolution (RH44-T706).

226


and P-value for β-mannanase stability after stored in a

fridge (4oC) for 12 months, and was heated at 70oC for 6

h prior to dissolution (FG12-T706).

227

6.14 Estimated regression coefficient and corresponding F -

and P-value for β-mannanase stability after stored in

desiccators for 12 months at room temperature, and was

heated at 70oC for 6 h (RT12-T706) prior to dissolution.

227

6.15 Comparison between experimental data using optimized

conditions and calculated data from model equations for

stability of β-mannanase in tested conditions and freeze

dried enzyme without the addition of stabilizer.

237

7.1 Chemical composition of PKC on dried basis.

264

7.2 Effect of different particle sizes on the performance of

20% (weight/volume) PKC saccharification on dry basis.

276

8.1 Formulation of the experimental diets (g/kg of diet) for

red tilapia.

308

8.2 Nutrient composition of experimental diets (% dry

matter).

325

8.3 Initial mean body weight, final mean weight, weight gain

(WG), feed conversion ratio (FCR), specific growth rate

(SGR), survival rate, protein efficiency ratio (PER),

protein productive value (PPV), thermal growth

coefficient (TGC), total ammonia-N (NH3-N) and

phosphate (PO4-3) of red tilapia fed 3 different diets for 12

weeks.

327

8.4 Apparent digestibility coefficients of crude protein, crude

lipid, gross energy, hemicellulose, lignin, cellulose, and

amino acid for tilapia fed 3 experimental diets.

328

8.5 The proximate composition of whole body, and muscle of

tilapia fed experimental diets (% on dry basis) for 12

weeks.

331

8.6 Morphological measurements of tilapia fed diets

containing enzymes for 12 weeks of growth.

333

8.7 Trypsin, chymotrypsin and amylase from stomach and

intestine tissues of red tilapia fed 3 different diets. 334

LIST OF FIGURES

NO. PAGE

2.1 Schematic representation of plant cell walls and their

main constituents (Source: Gidenne, 2003).

18

2.2 Illustrative structures of different forms of mannans and

the enzymes required for their hydrolysis. Typical

structures of (A) linear mannan, (C) branched

galactomannan, (D) linear glucomannan, and (F)

branched galactoglucomannan are shown. The mannan

backbone is hydrolyzed by β-mannanase, whilst α-

galactosidase and acetyl mannan esterase release

galactose and acetyl groups respectively. The products

generated by β-mannanase, (B) mannose and (E)

glucomannose oligosaccharides, are further hydrolysed

by β-mannosidase and β-glucosidase to finally yield the

monosaccharides mannose, glucose and galactose

(Source: van Zyl et al., 2010).

21

2.3 Typical composition chart of the palm fruit production

processes (Source: Dalimin, 1995).

23

2.4 Fresh oil palm fruit photo with its longitudinal section

(Source: Guo & Lua, 2001).

23

2.5 Global classification of dietary fiber (Source: Gidenne,

2003).

27

2.6 Generalized process stages in lignocellulosic waste

bioconversion (Source: Sánchez, 2009).

42

2.7 A framework representing nutrient partitioning in

growing fish (Source: Hua et al., 2010).

44

3.1 Flow chart of the experimental process. 53

3.2 Palm kernel cake. 57

4.1 After 30 days of fermentation. 78

4.2 Purification steps in Sephadex G-75 column, C1) initial

set-up, C2) loading of enzyme and C3) elution of enzyme

fraction.

92

4.3 β-Mannanase activity from 10 isolates after 96 h of

culture. Data are presented as the mean of three replicates

and the error bars shows the standard deviation. Same

alphabet above the columns indicates not statistically

different at P>0.05.

99

4.4 Isolate H on nutrient agar plate after 24 h of incubation at

30oC.

100

4.5 Light microscope images of A) Gram stained bacterial

cells from 24 h old culture and B) Endospore staining

from 72 h old culture (100x magnificent).

100

4.6 Bacterial structure under scanning electron microscope

(SEM), A) 2,000x, 6,000x and B) 15,000x magnificent.

101

4.7 PCR product, A) PCR amplified 16S rRNA gene

fragment of approximately 1.5 kbp from samples. M, 1

kbp DNA Ladder (Fermentas); 1, H; 2, Escherichia coli

(positive control); 3, negative control; B) Recombinant

plasmids containing the approximately 1.5 kbp 16S

rRNA gene fragment from sample and Escherichia coli

and C) PCR product of approximately 1.6 kbp amplified

from the recombinant plasmids, indicating the presence

of insert with expected size. M, 1 kbp DNA Ladder

(Fermentas); 1, H; 2, negative control.

105

4.8 The sequences of the 16S rRNA gene fragment of

unknown isolate H.

106

4.9 Interaction chromatography of major β-mannanase active

fractions, obtained after Sephadex G-75 gel column.

Fractions were tested for protein (—Δ—), β-mannanase

activity (—□—).

108

4.10 SDS-gel electrophoresis on a 12.0% polyacrylamide gel.

A) Purification of β-mannanase from Bacillus subtilis H.

Lane M, broad range molecular weight calibration kit;

lane 1 - 2, empty; lane 3, fraction of 20 - 80%

110

ammonium sulfate; lane 4, empty; and lane 5, fraction of

Sephadex G-75 (57 kDa and 43 kDa) and B)

Zymograms.

4.11 Lineweaver-Burk plot of β-mannanase in different

substrates, A) locust bean gum (LBG) and B) guar gum

(GG). The data points represent individual

measurements. Symbols represent: (●) Crude enzyme (□)

Purified enzyme.

113

4.12 pH optimum of crude and purified β-mannanase

preparation by Bacillus subtilis H assayed for 30 min at

different substrate pHs. The error bars are standard

deviations of triplicate values. Symbols represent: (●)

Crude enzyme (□) Purified enzyme.

114

4.13 pH stability of crude and purified β-mannanase

preparation by Bacillus subtilis H incubated for 24 h

without substrate at different buffered pHs. The error

bars are standard deviations of triplicate values. Symbols

represent: (●) Crude enzyme (□) Purified enzyme.

115

4.14 Temperature optimum of crude and purified β-

mannanase by Bacillus subtilis H. The error bars are

standard deviations of triplicate values. Symbols

represent: (●) Crude enzyme (□) and Purified enzyme.

116

4.15 Temperature stability of A) crude and B) purified

enzyme of Bacillus subtilis H. The error bars are

standard deviations of triplicate values. Symbols

represent: (■) 40oC; (□) 50oC; (▲) 60oC; (Δ) 70oC; (●)

80oC; (○) 90oC.

118

5.1 Stainless steel top plate with inoculums tubing port (red

arrow).

146

5.2 A 2 l stirred tank fermenter with 1 l working volume

(Biostat B Plus).

147

5.3 The half normal plot for 28−4IV fractional factorial design

(initial screening for significant values). (A) PKC, (B)

yeast extract, (C) NaH2PO4.H2O, (D) (NH4)2SO4, (E)

NH4NO3, (F) K2HPO4, (G) ZnCl2 and (H) MgSO4.7H2O.

153

5.4 Contour and surface plot of the model equation and fitted

the experimental data of the central composite design

based on the influence of variation in concentration of A)

yeast extract & PKC, B) (NH4)2SO4 & PKC, C) NH4NO3

& PKC, D) (NH4)2SO4 & yeast extract, E) NH4NO3 &

yeast extract; and F) NH4NO3 & (NH4)2SO4 to the

production of β-mannanase by Bacillus subtilis H when

PKC, yeast extract, (NH4)2SO4 and/or NH4NO3,

concentration was maintained at 55.0 g/l, 8.0 g/l, 4.0 g/l

and 4.0 g/l; respectively.

161




yeast extract & PKC, B) (NH4)2SO4 & PKC, C) NH4NO3

& PKC, D) (NH4)2SO4 & yeast extract, E) NH4NO3 &

yeast extract; and F) NH4NO3 & (NH4)2SO4 to the final

pH of the medium by B. subtilis H when PKC, yeast

extract, (NH4)2SO4 and/or NH4NO3, concentration was

maintained at 55.0 g/l, 8.0 g/l, 4.0 g/l and 4.0 g/l;

respectively.

162

5.6

Time course of A) β-mannanase production (nkat/ml), ■;

cell growth, Log10 (CFU/ml), □; B) dissolved oxygen

(%),○; medium pH,●; and C) reducing sugar, ▲; of

Bacillus subtilis H in 2 l fermenter at agitation speed of

650 rpm, temperature of 35oC, initial pH of 6.5 and

aeration rate of 1.5 vvm. The error bars are standard

deviations of six center point values.

167




agitation speed and temperature, B) initial pH and

temperature and C) initial pH and agitation rate to the β-

mannanase production by Bacillus subtilis H when

agitation speed, temperature, and initial pH, was

maintained at 6.5, 600 rpm and 35oC; respectively.

175

6.2 Freeze dried samples in 20 ml vials. 217





temperature and C) initial pH and agitation rate to the

fermentation time at maximum β-mannanase production

by Bacillus subtilis H when agitation speed, temperature,

and initial pH, was maintained at 6.5, 600 rpm and 35oC;

respectively.

177





temperature, and C) initial pH and agitation rate to the

pH value at maximum β-mannanase production by

Bacillus subtilis H when agitation speed, temperature,

and initial pH, was maintained at 6.5, 600 rpm and 35oC;

respectively.

178





temperature and C) initial pH and agitation rate to the

growth of Bacillus subtilis H at highest β-mannanase

production when agitation speed, temperature, and initial

pH, was maintained at 6.5, 600 rpm and 35oC;

respectively.

179

6.1 State diagram for a water (w) /solute (s) system. Tm (w)

and Tm (s): melting temperatures of water and solute, Teu:

eutectic temperature, Tg (w) and Tg (s): glass transition

temperature of water and solute and Tg0: glass transition

temperature of the maximally freeze-concentrated

solution. Crystallization (black drawings) of a solute

occurs below Teu. In the case of vitrification (gray

drawings), the solute does not crystallize at Teu, freeze-

concentration proceeds and transits into a glass state at Tg

(Source: Kasper & Friess, 2011).

206

Documents

OPTIMIZATION OF β-MANNANASE PRODUCTION FROM Bacillus