optimization of β-mannanase production from bacillus
TRANSCRIPT
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
SIT
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A B
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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
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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
i
ii
iii
xiii
xviii
xxvi
xxxiii
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
38
41
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
59
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
77
77
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
94
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107
110
111
112
116
117
120
137
CHAPTER 5 FERMENTATION STUDIES – OPTIMIZATION OF
β-MANNANASE PRODUCTION IN SHAKE FLASK AND
FERMENTER SYSTEM
5.1 Background
5.2 Materials and Methods
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
138
140
140
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141
143
144
144
146
148
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150
150
150
154
160
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 Materials and Methods
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
210
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211
212
215
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221
229
236
238
251
CHAPTER 7 SACCHARIFICATION OF PKC USING CRUDE β-
MANNANASE PREPARATION FROM Bacillus subtilis H
7.1 Background
7.2 Materials and Methods
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
255
255
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256
258
258
259
260
260
261
262
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263
263
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277
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
280
280
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 Materials and Methods
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
302
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305
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307
307
310
312
312
314
315
316
316
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
319
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356
CHAPTER 9 SUMMARY, GENERAL CONCLUSION AND
RECOMMENDATIONS
9.1 Summary
9.2 General Conclusion
9.3 Recommendations
357
363
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
5.4 Actual factor levels corresponding to coded factor levels
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
5.10 The least-square fit and parameters (significant of
regression coefficient) for pH at harvest.
159
5.11 Full factorial central composite design matrix of three
variables in coded units along with the observed
responses for β-mannanase, fermentation time, medium
pH and growth (Log10 CFU/ml) at maximum production
of enzyme by Bacillu subtilis H.
164
5.12 The least-square fit and parameters (significant of
regression coefficient) for β-mannanase production in 2 l
fermenter.
170
5.13 The least-square fit and parameters (significant of
regression coefficient) for fermentation time at maximum
β-mannanase production.
170
5.14 The least-square fit and parameters (significant of
regression coefficient) for medium pH at maximum β-
mannanase production.
171
5.15 The least-square fit and parameters (significant of
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
6.3 Actual factor levels corresponding to coded factor levels
for stabilizer used.
213
6.4 Full factorial central composite design matrix of three
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
6.7 Full factorial central composite design matrix of three
variables in coded units along with the observed
responses for freeze dried β-mannanase activity retention
after exposure to different conditions.
222
6.8 Full factorial central composite design matrix of three
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
6.10 Estimated regression coefficient and corresponding F-
and P-value for β-mannanase stability after reconstituted
and kept in fridge for a month (RC1).
225
6.11 Estimated regression coefficient and corresponding F-
and P-value for β-mannanase stability just after freeze
dried and were heat at 70oC for 6 h prior to dissolution
(T706).
226
6.12 Estimated regression coefficient and corresponding F-
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
6.13 Estimated regression coefficient and corresponding F-
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
5.5 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 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
5.7 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)
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
5.8 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)
agitation speed and temperature, B) initial pH and
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
5.9 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)
agitation speed and temperature, B) initial pH and
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
5.10 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)
agitation speed and temperature, B) initial pH and
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.
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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).
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