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Industrial Production of Bioethanol by Mutant
Strain of Saccharomyces cerevisiae
By
Muhammad Arshad (M.Phil. Biochemistry, UAF)
Thesis submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy in
Biochemistry
Department of Chemistry and Biochemistry
University of Agriculture Faisalabad
Pakistan 2011
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IN THE NAME OF “ALLAH”, THE MOST
BBEENNEEFFIICCEENNTT AANNDD MMEERRCCIIFFUULL
To
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The Controller of Examinations,
University of Agriculture,
Faisalabad.
"We the supervisory Committee, certify that the contents and form of
thesis submitted by Mr. Muhammad Arshad Regd. No. 2000-ag-877,
have been found satisfactory and recommended that it be processed for
evaluation of, by the external examiner (s) for the award of degree".
Supervisory committee:
Chairman ______________________________
(Dr. Muhammad Anjum Zia)
Member ______________________________
(Prof. Dr. Muhammad Asghar)
Member _____________________________
(Prof. Dr. Haq Nawaz Bhatti)
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DEDICATIONS
To the
HazratMuhammad(PBUH)
(TheComprehensivepersonalityofthe
Universe)
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ACKNOWLEDGEMENT
Words are bound and knowledge is limited to praise Allah, the omnipotent,
the beneficent, the merciful, who created this universe. Peace and blessings
of Allah be upon Holy prophet Muhammad (peace be upon him), the unique
comprehensive personality, the everlasting source of guidance and
knowledge for humanity.
I would like to express my thanks to all those who gave me the possibility to
complete this effort. First and foremost I show gratitude for Dr Muhammad
Anjum Zia, Assistant Professor Department of Chemistry & Biochemistry,
University of Agriculture Faisalabad for his keen interest and master advice
throughout the course of my studies. I am deeply inedited to the honorable
Prof. Dr. Muhammad Asghar Bajwa, Department of Chemistry &
Biochemistry, University of Agriculture Faisalabad for his detailed and
constructive comments on this thesis. His extensive discussion on this work
and interesting explorations has been very helpful for this study. My Sincere
thanks are due to Professor Dr Haq Nawaz Bhatti Associate Professor,
Department of Chemistry & Biochemistry, University of Agriculture
Faisalabad for his cooperation.
My sincere thanks to the Prof. Dr. Muhammad Ibrahim Rajoka,
Department of Bioinformatics, GC University Faisalabad for his guidance
through this study.
I am thankful to Shakarganj Mills Ltd Jhang and National Institute for
Biotechnology & Genetic Engineering Jhang road Faisalabad for providing
me the research facilities at their prestigious institutions.
I feel a deep sense of gratitude for my parents and I am also grateful to my
wife and my son Muhammad Talha Arshad and my daughters Tayyaba
Arshad and Tooba Arshad.
In the last I would like to thank Higher Education Commission of Pakistan
for providing the funds under Indigenous PhD Fellowship Scheme.
Muhammad Arshad
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CONTENTS
No. Title Page 1. Introduction 1
2. Review of literature 7
2.1 Microorganism 7
2.2 Substrate 7
2.3 Catabolite Repression 8
2.4 Random Amplified Polymorphic DNA 10
2.5 Invertase 12
2.6 Molasses 15
2.7 Nitrogen source 17
2.8 Phosphorous source 18
2.9 Inoculum 19
2.10 Ethanol Tolerance 19
2.11 Aeration 22
2.12 Thermotolerance 23
2.13 Very High Gravity Technology 27
2.14 By Products 29
2.15 Antibiotic 30
2.16 Response Surface Methodology 32
3. Materials and Methods 34
3.1 Research Stations 34
3.2 Chemicals/Biochemicals 34
3.3 Substrate 34
3.4 Microorganism 34 3.5 Maintenance of the culture 34
3.5.1 Growth Medium Composition 35
3.5.2 Preparation of Plates 35
3.5.3 Preparation of Slants 35
3.6 Preparation of purified parent strain 36
3.6.1 Medium for inoculums 36
3.7 Strain Improvement 37
3.7.1 Survival Curve 37
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3.7.2 Purification of Mutant culture 37
3.7.3 Selection of Mutant of S. cerevisiae 37
3.8 Genetic variability 38
3.8.1 Total Genomic DNA Isolation
38
3.8.2 RAPD Assay 38
3.8.2.1 Data analysis 39
3.9 Invertase and ethanol production at lab. scale 39
3.9.1 Effect of Nitrogen and Phosphate source 39
3.9.2 Effect of Temperature 40
3.9.3 Effect of pH 40
3.10 Industrial Scale Studies 40
3.10.1 Propagation of Yeast Culture 40
3.10.1.1 First Stage 40
3.10.1.2 2nd stage 41
3.10.1.3 3rd Stage 41
3.10.2 Fermentation 41
3.10.2.1Effect of different brix (sugar level) 41
3.10.2.2 Effect of different inoculums size 41
3.10.2.3 Effect of different level rise 42
3.10.2.4 Effect of temperature 42
3.10.3 Effect of Antibiotic 42
3.10.3.1Effect of Sodium Flouride 42
3.10.3.2 Effect of virginiamycin 42
3.10.3 Very High Gravity Technology 42
3.10.3.1Effect of Very High Brix 42
3.10.3.2 Effect of Aeration Rate 42
3.11 Analysis 42
3.11.1 Invertase assay 42
3.11.1.1Calculation of Invertase Activity 43
3.11.2 Brix 43
3.11.3 pH 43
3.11.4 Sugars Analysis 43
3.11.5 Cell Population 43
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3.11.6 Ethanol % 43
3.11.7 Yied 43
3.11.8 Fermentation efficiency 44
3.11.9 Gas Chromatography 44
3.11.10Potassium Permanganate Time Test (PTT)
44
3.11.11Acidity of Alcohol 44
3.12 Statistical Analysis 44
4. Results and Discussion 45
4.1 Genetic Variability between the mutant and parent strains 46
4.1.1 Genomic DNA Isolation 46
4.1.2 RAPD Assay 47
4.2 Laboratory scale study 49
4.3 Industrial Scale studies 102
4.4 Use of antibiotic for ethanol fermentation from
contaminated/deteriorated molasses
145
4.4.1 Use of Virginiamycin 145
4.4.2 Use of Sodium Flouride 148
4.5 Very High Gravity Fermentation 151
4.6 Conclusion 168
5.0 Summary 169
Literature cited 171
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LIST OF TABLES Page No. Title Table
35 Recipe of chemicals/biochemical for growth medium 3.1
36 Chemicals used in inoculums preparation 3.2
48 The total number of amplified fragments and polymorphic fragments with the
primers used in the study 4.1
50 Invertase activity of parent and mutant strain at different nutrients concentration, pH and temperature 27ºC
4.2
51 Invertase activity of parent and mutant strain at different nutrients
concentration, pH and temperature 32ºC 4.3
52 Invertase activity of parent and mutant strain at different nutrients
concentration, pH and temperature 37ºC 4.4
53 Invertase activity of parent and mutant strain at different nutrients
concentration, pH and temperature 42ºC 4.5
55
Regression coefficients, standard errors, t test and significance level for the
models representing invertase activity on various concentrations of
urea/phosphoric acid
4.6
56 Analysis of variance (ANOVA) for the linear model of invertase activity on
various concentrations of urea/phosphoric acid 4.7
60 Regression coefficients, standard errors, t test and significance level for the
models representing invertase activity on various concentration of DAP 4.8
61 Analysis of variance (ANOVA) for the linear model of invertase activity on
various concentration of DAP 4.9
66 Ethanol % (w/v) of parent and mutant strain at different nutrients
concentration, pH and temperature at 27ºC 4.10
67 Ethanol % (w/v) of parent and mutant strain at different nutrients
concentration, pH and temperature at 32ºC 4.11
68 Ethanol % (w/v) of parent and mutant strain at different nutrients
concentration, pH and temperature at 37ºC 4.12
69 Ethanol % (w/v) of parent and mutant strain at different nutrients
concentration, pH and temperature at 42ºC 4.13
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70 Regression coefficients, standard errors, t test and significance level for the
models representing ethanol on various concentrations of urea/phosphoric acid 4.14
70 Analysis of variance (ANOVA) for the linear model of ethanol on various
concentrations of urea/phosphoric acid 4.15
74 Regression coefficients, standard errors, t test and significance level for the
models representing ethanol on various concentrations of DAP 4.16
74 Analysis of variance (ANOVA) for the linear model of ethanol on various
concentrations of DAP 4.17
78 Final brixº of parent and mutant strain at different nutrients concentration, pH
and temperature at 27ºC 4.18
79 Final brixº of parent and mutant strain at different nutrients concentration, pH
and temperature at 32ºC 4.19
80 Final brixº of parent and mutant strain at different nutrients concentration, pH
and temperature at 37ºC 4.20
81 Final brixº of parent and mutant strain at different nutrients concentration, pH
and temperature at 42ºC 4.21
82 Regression coefficients, standard errors, t test and significance level for the
models representing brix on various concentrations of urea/phosphoric acid 4.22
82 Analysis of variance (ANOVA) for the linear model of brix on various
concentrations of urea/phosphoric acid 4.23
86 Regression coefficients, standard errors, t test and significance level for the
models representing brix on various concentrations of DAP 4.24
86 Analysis of variance (ANOVA) for the linear model of brix on various
concentrations of DAP 4.25
90 Residual Sugars (g/l) of parent and mutant strain at different nutrients concentration and pH at 27ºC
4.26
91 Residual Sugars (g/l) of parent and mutant strain at different nutrients
concentration and pH at 32ºC 4.27
92 Residual Sugars (g/l) of parent and mutant strain at different nutrients
concentration and pH at 37ºC 4.28
93 Residual Sugars (g/l) of parent and mutant strain at different nutrients
concentration and pH at 42ºC 4.29
94 Regression coefficients, standard errors, t test and significance level for the
models representing RS. 4.30
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94 Analysis of variance (ANOVA) for the linear model of RS 4.31
98 Regression coefficients, standard errors, t test and significance level for the
models representing residual sugars on various concentrations of DAP 4.32
98 Analysis of variance (ANOVA) for the linear model of RS on various
concentrations of DAP 4.33
103 Effect of varying Brixº, inoculums size and level rise on the ethanol % (w/v) at
27ºC 4.34
104 Effect of varying Brixº, inoculums size and level rise on the ethanol % (w/v) at
32ºC 4.35
105 Effect of varying Brixº, inoculums size and level rise on the ethanol % (w/v) at
37ºC 4.36
106 Effect of varying Brixº, inoculums size and level rise on the ethanol % (w/v) at
42ºC 4.37
107 Regression coefficients, standard errors, t test and significance level for the
models representing ethanol 4.38
108 Analysis of variance (ANOVA) for the linear model of Ethanol 4.39
112 Effect of varying brixº, inoculums size and level rise on the final brix at 27ºC 4.40
113 Effect of varying brixº, inoculums size and level rise on the final brix at 32ºC 4.41
114 Effect of varying brixº, inoculums size and level rise on the final brix at 37ºC 4.42
115 Effect of varying brixº, inoculums size and level rise on the final brix at 42ºC 4.43
116 Regression coefficients, standard errors, t test and significance level for the
models representing brix 4.44
116 Analysis of variance (ANOVA) for the linear model of brix 4.45
120 Effect of varying brixº, inoculums size and level rise on the Residual sugars
(gl-1) at 27ºC 4.46
121 Effect of varying brixº, inoculums size and level rise on the Residual sugars
(gl-1) at 32º 4.47
122 Effect of varying brixº, inoculums size and level rise on the Residual sugars
(gl-1) at 37ºC 4.48
123 Effect of varying brixº, inoculums size and level rise on the Residual sugars
(gl-1) at 42ºC 4.49
124 Regression coefficients, standard errors, t test and significance level for the
models representing residual sugars 4.50
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124 Analysis of variance (ANOVA) for the linear model of residual sugars 4.51
128 Effect of varying brixº, inoculums size and level rise on the cell population at
27ºC 4.52
129 Effect of varying brixº, inoculums size and level rise on the cell population at
32ºC 4.53
130 Effect of varying brixº, inoculums size and level rise on the cell population at
37ºC 4.54
131 Effect of varying brixº, inoculums size and level rise on the cell population at
42ºC 4.55
132 Regression coefficients, standard errors, t test and significance level for the
models representing cell count 4.56
132 Analysis of variance (ANOVA) for the linear model of cell count 4.57
137 Effect of varying brixº, inoculums size and level rise on the fermentation
efficiency at 27ºC 4.58
138 Effect of varying brixº, inoculums size and level rise on the fermentation
efficiency at 32ºC 4.59
139 Effect of varying brixº, inoculums size and level rise on the fermentation
efficiency at 37ºC 4.60
140 Effect of varying brixº, inoculums size and level rise on the fermentation
efficiency at 42ºC 4.61
141 Regression coefficients, standard errors, t test and significance level for the
models representing fermentation efficiency 4.62
141 Analysis of variance (ANOVA) for the linear model of fermentation efficiency 4.63
152 Effect of aeration condition on ethanol % (w/v) at initial brixº 32 4.64
152 Effect of aeration condition on ethanol % (w/v) at initial brixº 36 4.65
153 Effect of aeration condition on ethanol % (w/v) at initial brixº 40 4.66
153 Regression coefficients, standard errors, t test and significance level for the
models representing ethanol 4.67
154 Analysis of variance (ANOVA) for the linear model of Ethanol 4.68
155 Effect of aeration rate (vvm) on final brixº at brixº 32 4.69
156 Effect of aeration rate (vvm) on final brixº at brixº 36 4.70
157 Effect of aeration rate (vvm) on final brixº at brixº 40 4.71
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157 Regression coefficients, standard errors, t test and significance level for the
models representing final brix 4.72
158 Analysis of variance (ANOVA) for the linear model of final brix 4.73
159 Effect of aeration rate (vvm) on cell population at brixº 32 4.74
159 Effect of aeration rate (vvm) on cell population at brixº 36 4.75
160 Effect of aeration rate (vvm) on cell population at brixº 40 4.76
160 Regression coefficients, standard errors, t test and significance level for the
models representing count 4.77
161 Analysis of variance (ANOVA) for the linear model of Count 4.78
163 Effect of aeration rate (vvm) on RS (g/l) at brixº 32 4.79
163 Effect of aeration rate (vvm) on RS (g/l) at brixº 36 4.80
164 Effect of aeration rate (vvm) on RS (g/l) at brixº 40 4.81
164 Regression coefficients, standard errors, t test and significance level for the
models are representing residual sugars 4.82
165 Analysis of variance (ANOVA) for the linear model of residual sugars 4.83
166 Effect of aeration rate (vvm) on by products formation at brixº 32 4.84
166 Effect of aeration rate (vvm) on by products formation at brixº 36 4.85
167 Effect of aeration rate (vvm) on by products formation at brixº 40 4.86
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LIST OF FIGURES
Page No. Title Figure
3 Ethanol fermentation from glucose 1.1
45 Influence of gamma irradiation on Survival of Saccharomyces
cerevisiae 4.1
46 Extracted DNA of the both strains on 0.8% agarose gel 4.2
47 RAPD amplification with primers OPA-1, OPA-2, OPA-3 and
OPA-4 of both parent and mutant strain 4.3
57 Response surface plots of interaction between pH and different
temperatures for Invertase activity of parent strain 4.4
58 Response surface plots of interaction between pH and different
temperatures for Invertase activity of mutant strain 4.5
59
Response surface plots of interaction between nutrients
concentration and different temperatures for Invertase activity of
parent strain
4.6
59
Response surface plots of interaction between nutrients
concentration and different temperatures for Invertase activity of
mutant strain
4.7
60 Response surface plots of interaction between pH and nutrient
contrations for Invertase activity of parent strain 4.8
60
Response surface plots of interaction between nutrients
concentration and different temperatures for Invertase activity of
mutant strain
4.9
62 Response surface plots of interaction between pH and temperature
for Invertase activity of parent strain 4.10
62 Response surface plots of interaction between pH and tempertaure
for Invertase activity of mutant strain 4.11
63
Response surface plots of interaction between different
temperature and DAP concentrations for Invertase activity of
parent strain
4.12
63
Response surface plots of interaction between different
temperature and DAP concentrations for Invertase activity of
mutant strain
4.13
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64 Response surface plots of interaction between different pH and
DAP contrations for Invertase activity of parent strain 4.14
64 Response surface plots of interaction between different pH and
DAP contrations for Invertase activity of mutant strain 4.15
71 Response surface plots of interaction between different
temperature and pH for ethanol produced by parent strain 4.16
71 Response surface plots of interaction between different
temperature and pH for ethanol produced by mutant strain 4.17
72
Response surface plots of interaction between different
temperature and nutrients concentrations for ethanol produced by
parent strain
4.18
72
Response surface plots of interaction between different
temperature and nutrients concentrations for ethanol produced by
mutant strain
4.19
73 Response surface plots of interaction between different nutrient
concentration and pH for ethanol produced by parent strain 4.20
73 Response surface plots of interaction between different nutrient
concentration and pH for ethanol produced by mutant strain 4.21
75 Response surface plots of interaction between different temperature and DAP concentration for ethanol produced by parent strain
4.22
75 Response surface plots of interaction between different DAP
concentration and Temp for ethanol produced by parent strain 4.23
76 Response surface plots of interaction between different pH and
Temp for ethanol produced by mutant strain 4.24
76 Response surface plots of interaction between different
temperature and DAP conc. for ethanol produced by mutant strain 4.25
77 Response surface plots of interaction between different pH and
DAP conc. for ethanol produced by parent strain 4.26
77 Response surface plots of interaction between different pH and
DAP conc. for ethanol produced by mutant strain 4.27
83 Response surface plots of interaction between different pH and
different temperature for final brix by parent strain 4.28
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83 Response surface plots of interaction between different pH and
different temperature for final brix by parent strain 4.29
84
Response surface plots of interaction between different
temperature and different concentration of p acid/urea for final brix
by mutant strain
4.30
84 Response surface plots of interaction between different pHand
different concentration of p acid/urea for final brix by mutant strain 4.31
85 Response surface plots of interaction between different pH and
different concentration of p acid/urea for final brix by parent strain 4.32
85 Response surface plots of interaction between different pH and
different concentration of p acid/urea for final brix by mutant strain 4.33
87 Response surface plots of interaction between different pH and temperature for final brix by parent strain
4.34
87 Response surface plots of interaction between different pH and
temperature for final brix by mutant strain 4.35
88
Response surface plots of interaction between different
temperature and different concentration of DAP for final brix by
mutant strain
4.36
88
Response surface plots of interaction between different
temperature and different concentration of DAP for final brix by
parent strain
4.37
89 Response surface plots of interaction between different pH and
different concentration of DAP for final brix by mutant strain 4.38
89 Response surface plots of interaction between different pH and
different concentration of DAP for final brix by mutant strain 4.39
95 Response surface plots of interaction between different pH and
different temperature for residual sugar by parent strain 4.40
95 Response surface plots of interaction between different pH and
different temperature for residual sugar by mutant strain 4.41
96
Response surface plots of interaction between different
concentration of urea/phosphoric acid and different temperature for
residual sugars by parent strain
4.42
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96
Response surface plots of interaction between different
concentration of urea/phosphoric acid and different temperature for
residual sugars by mutant strain
4.43
97
Response surface plots of interaction between different pH and
different concentrations of urea/ phosphoric acid for residual
sugars by mutant strain
4.44
97
Response surface plots of interaction between different pH and
different concentrations of urea/ phosphoric acid for residual
sugars by mutant strain
4.45
99 Response surface plots of interaction between different pH and
different temperature for residual sugars by parent strain 4.46
99 Response surface plots of interaction between different pH and
different temperature for residual sugars by mutant strain 4.47
100
Response surface plots of interaction between different
temperature and different concentrations of DAP for residual
sugars by mutant strain
4.48
100
Response surface plots of interaction between different
temperature and different concentrations of DAP for residual
sugars by mutant strain
4.49
101
Response surface plots of interaction between different pH and
different concentrations of DAP for residual sugars by mutant
strain
4.50
101
Response surface plots of interaction between different pH and
different concentrations of DAP for residual sugars by mutant
strain
4.51
109 Response surface plots of interaction between different brixº and
different temperature for ethanol % (w/v) by parent strain 4.52
109 Response surface plots of interaction between different brixº and
different temperature for ethanol % (w/v) by mutant strain 4.53
110 Response surface plots of interaction between different brixº and
inoculum rates for ethanol % (w/v) by parent strain 4.54
110 Response surface plots of interaction between different brixº and
different inoculums rates for ethanol % (w/v) by mutant strain 4.55
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111 Response surface plots of interaction between different inoculums
rise and different temperature for ethanol % (w/v) by parent strain 4.56
111 Response surface plots of interaction between different inoculums
rise and different temperature for ethanol % (w/v) by parent strain 4.57
117 Response surface plots of interaction between different brix and
temperature for final brix by parent strain 4.58
117 Response surface plots of interaction between different brix and
temperature for final brix by parent strain 4.59
118 Response surface plots of interaction between different inoculums
rate and brix temperature for final brix by parent strain 4.60
118 Response surface plots of interaction between different inoculums
rise and brix for final brix by parent strain 4.61
119 Response surface plots of interaction between different inoculums
rate and different temperature for final brix by parent strain 4.62
119 Response surface plots of interaction between different inoculums
rise and different temperature for final brix by parent strain 4.63
125 Response surface plots of interaction between different brix and
different temperature for residual sugars (g/l) by parent strain 4.64
125 Response surface plots of interaction between different brix and
different temperature for residual sugars (g/l) by mutant strain 4.65
126 Response surface plots of interaction between different inoculums
rate and brix for residual sugars (g/l) by parent strain 4.66
126 Response surface plots of interaction between different inoculums
rate and brix for residual sugars (g/l) by mutant strain 4.67
127
Response surface plots of interaction between different inoculums
rate and different temperature for residual sugars (g/l)) by parent
strain
4.68
127
Response surface plots of interaction between different inoculums
rate and different temperature for residual sugars (g/l) by parent
strain
4.69
133
Response surface plots of interaction between different brix and
different temperature for cell population (million/ml) by parent
strain
4.70
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133 Response surface plots of interaction between different brix and
different temperature for cell population by mutant strain 4.71
134
Response surface plots of interaction between different inoculums
rise and different brix for cell population (million/ml) by parent
strain
4.72
134 Response surface plots of interaction between different inoculums
rise and brix for cell population by mutant strain 4.73
135 Response surface plots of interaction between different inoculums
rates and brix for cell population (million/ml) by mutant strain 4.74
135
Response surface plots of interaction between different inoculums
rate and different temperature for cell population (million/ml) by
parent strain
4.75
142
Response surface plots of interaction between different inoculums
rate and different temperature for fermentation efficiency % by
parent strain
4.76
142 Response surface plots of interaction between different brix and
temperature for fermentation efficiency % by mutant strain 4.77
143 Response surface plots of interaction between different inoculums
rise and brix for fermentation efficiency % by mutant strain 4.78
143 Response surface plots of interaction between different inoculums
rise and brix for fermentation efficiency % by parent strain 4.79
144
Response surface plots of interaction between different inoculums
rise and different temperature for fermentation efficiency % by
parent strain
4.80
144
Response surface plots of interaction between different inoculums
rate and different temperature for fermentation efficiency % by
mutant strain
4.81
145
The graph showing the effect of virginiamycin concentration
(ppm) on yeast population during the ethanol fermentation of
molasses
4.82
146
The graph showing the effect of virginiamycin concentration
(ppm) on bacterial population during the ethanol fermentation of
molasses
4.83
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146
The graph showing the effect of virginiamycin concentration
(ppm) on ethanol % (w/v) during the ethanol fermentation of
molasses
4.84
147 The graph showing the effect of virginiamycin concentration
(ppm) on final brix during the ethanol fermentation of molasses 4.85
147
The graph showing the effect of virginiamycin concentration
(ppm) on residual sugars (g/l) during the ethanol fermentation of
molasses
4.86
148 The graph showing the effect of NaF concentration (ppm) on yeast
population during the ethanol fermentation of molasses 4.87
148 The graph showing the effect of NaF concentration (ppm) on
bacterial population during the ethanol fermentation of molasses 4.88
149 The graph showing the effect of NaF concentration (ppm) on
ethanol % (w/v) during the ethanol fermentation of molasses 4.89
149 The graph showing the effect of NaF concentration (ppm) on final
brix during the ethanol fermentation of molasses 4.90
150 The graph showing the effect of NaF concentration (ppm) on
residual sugars (g/l) during the ethanol fermentation of molasses 4.91
155 Response surface plots of interaction between time (hrs) and
aeration rates for ethanol % (w/v) 4.92
158 Response surface plots of interaction between time (hrs) and
different aeration rates for ethanol % (w/v) by parent strain 4.93
161
Response surface plots of interaction between time (hrs) and
different aeration rates for cell population during VHG ethanol
fermentation
4.94
165
Response surface plots of interaction between time (hrs) and
different aeration rates for residual sugars during VHG
fermentation
4.95
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List of Abbreviations
RAPD Random Amplified Polymorphic DNA
AFLP Amplified Fragment Length Polymorphism
SSR Specific Sequence Repeats
ALP Aerobic Low Peptone
RPM Revolutions per Minute
HPLC High Performance Liquid Chromatography
VHG Very High Gravity
SML Shakarganj Mills Ltd Jhang
COD Chemical Oxygen Demand
BOD Biological Oxygen Demand
PHE Plate Heat Exchanger
DNA De-oxy Ribose Nucleic Acid
EIA Energy
PCR Polymerase Chain Reaction
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1
Chapter 1
INTRODUCTION The basic driving force behind the socio-economic progress is the frequently
accessibility of energy. The present human progression is honestly relying on
accessible energy means enhancing the productivity through technological
applications. Transportation is one of the three basic major areas (other two are
electricity generation, heating and cooling systems) which utilize most of the
energy. Human’s energy requirements has been met by the fossil fuels (coal; oil;
gas) since many decades. Now these are full filling approximately above 80%,
nuclear sources share only 6% and the balance maintained by the renewable
means (Bose, 2000).
The discharge of green house gases through the burning of fossil fuels alters the
natural equilibrium of environment. Transportation sector is the rapidly growing
consumer and utilize twenty seven percent of primary energy presently. It is
predicted that transportation sector will see 80% rises in consumption of fuels
between 2006 and 2030 (EIA, 2009). Therefore for the reduction of greenhouse
gas emissions, it is the hot area.
Gasoline like products consumption is the 40 percent of the current energy
utilization of Pakistan. Its consumption has been rising much faster during the
last decade and transportation sector is the major consumer (Business Recorder,
2010). It is a general concept that the period of economical energy has come to
end, as accessibility of the fossil fuels is limited in certain areas (IPCC, 2007).
The word has now started to realize the problem and syndromes created by them.
To minimize the fossil fuels role, the exploration of renewable substitutes like
photovoltaics, wind and nuclear power are on rise. In contrast, the bioethanol has
undisputedly emerged as alternative of conventional fuels in transportation
replacing gasoline (3%).
The first fuel used in an automobile engine was the ethanol (Antoni, 2007). The
designer of model T Ford also used ethanol and foreseen it, "the fuel of the
future". However, after many decades during the oil crises of 1970, it was
realized as potential fuel.
It is more oxygenated compound that leads to reduced emission of hazardous
gases like carbon monoxide (CO) and unburnt hydrocarbons. The emission of
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2
these gases were decreased ten times (50 g/km in 1980 to 5.8 g/km in 1995) in
brazil by the use of this fuel (Goldemberg, 2007).
Besides the environmental benefits, the bio-ethanol sector has the extensive
employment capability and may generate lot of opportunities. Such programs are
always attractive for Pakistan like states. The sugar industry, on which the
distilleries are dependent, is the 2nd largest industry of the country. More than
70% peoples living in rural areas are dependent for their bread and butter.
Moreover, sugarcane cultivation also employs some others people with the
ethanol production plants as the additional source of employment.
Pakistan's economy has been overburden due to oil imports especially the
increase in oil prices in last decade. The instability and upward trend of oil
prices had shaken the world’s economic and political situation especially on
developing countries. Renewable biofuels, like ethanol can solve the problem by
diversifying the energy sources with increased energy security and favorable
trade balance.
The low flame temperature, elevated octane number and high heat of
vaporization, make it as outstanding transportation fuel. Furthermore, its mixing
with gasoline increases the octane number without supplementation of any
unwanted substances. Bioethanol is eco-friendly and easily recyclable; therefore
it is appropriate fuel according to the environment (Demirbas, 2009).
Traditionally ethanol had been made by the fermentation of sugars and
responsible for more than 90 % ethanol production, remains is produced
synthetically. For the production of ethanol from biomass, fermentation or
hydrolysis is must. Following are the main categories of biomass used for the
bioethanol production :
(i) The bio-renewables with readily available sugars include sugar-beet
juice, sugarcane juice and molasses,
(ii) The materials with sugars in the form of starches like cassava, cereals,
potatoes and maize
(iii) The materials with the sugars present in complex form, the cellulosic
materials such as wood, rice straw, sugarcane bagasse and even waste materials,
in general
The bioethanol production is the major one of industrial level fermentations
(Antoni, 2007). It is the most aged and renowned process with highest industrial
-
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-
4
highest ethanol level and maximum sugar tolerance are highly demanded in
ethanol production industry.
The optimal fermentation temperature for highest productivity and maintenance
of cell viability is approximately 32 ºC (Aldiguier et al., 2004; Phisalaphong et
al., 2006). The process is exothermic in nature and need cooling to keep the
optimum temperature. The temperature above optimum exerts stress on the
microorganism, consequently the growth is halted and fermentation capability is
lost. The ethanol tolerance and fermentation temperature are interdependent
(Aldiguier et al., 2004).
The ethanol production at high temperature has certain advantages as compared
to in conventional mode, such like the reduced operating costs by the elimination
of cooling of fermentation tanks, contamination chances are minimized,
enhanced productivity and the distillation is eased with low consumption of
energy for the recovery of final product (Banat et al., 1998). The active
performance of microorganism at very high temperatures and ethanol level is
critical to achieve the above mentioned goals (Alfenore et al., 2004).
The other significant factor influencing the ethanol fermentation is catabolite
repression caused by the hexoses. In presence of glucose or fructose, the yeast
Saccharomyces cerevisiae does not utilize other sugars (Rincon et al., 2001)
especially sucrose present in the sugarcane molasses. For the consumption of
the disaccharide, microorganism keeps an extracellular enzyme called invertase
that converts sucrose into glucose and fructose, which are brought into the cell
by hexose transporters (Badotti et al., 2008). In presence of glucose and fructose,
the invertase synthesis and release is extremely inhibited or completely blocked
(Klein et al., 1998).
A glucose de-repressed mutant strain with the characteristics to co-utilize all the
sugars has capability to reduce the production time increasing the fermentation
rate. Highest ethanol formation in a minimum fermentation time is economically
very much considerable at industrial scale ethanol production. (Zhang and
Greasham 1999).
Maximum 8% (v/v) ethanol is achieved in existing industrial scale fermentation
(Arshad et al., 2008) from 15-16% diluted molasses having 80–85% efficiency.
The process creates large amount of waste matter (stillage) (roughly 12
liters/liter of ethanol produced) having biological oxygen demand (BOD) above
-
5
50,000 mgl-1 and chemical oxygen demand (COD) over 90,000 mgl-1 (Selladurai
et al., 2010). No technology is available for treatment of stillage satisfactory
(Piggot et al., 2003). Hence, the process can potentially be make better as an
environment affable and producer of inexpensive ethanol.
Disposal of the stillage is very hard and different environmental problems
created. Very high gravity (VHG) defined as “the preparation and fermentation
of mashes containing 27 g or more dissolved solids per 100 g mash” (Thomas et
al., 1993) has the capability to solve the matter. It raises the ethanol level (12-15
%) in fermentation, and reduces the contamination risks with decreases in the
cost of distillation (Bafrncova et al., 1999).
This technology is quiet attractive for alcohol industry worldwide and especially
for Pakistani producers, as it rescues significant quantity of water. Due to high
yield of ethanol energy requirements reduced, with less chances of
contaminating bacteria.
The yeast produces some unwanted products with the ethanol during
fermentation of molasses. Acetates, aldehydes, higher alcohols and methanol are
the major one. The quality of final products reduced with the presence of such
unlike products.
Keeping in view the above stated problems and requirements of ethanol
industry; a mutant Saccharomyces cerevisiae strain was developed trough
gamma rays irradiation and subsequently selection on 2-Deoxy-D-glucose
(DOG). The glucose-derepressed mutant with high invertase activity selected.
The competence of a strain is directly proportional to the invertase activity
(hydrolysis of sucrose) especially under the stress conditions of molasses
medium (Takeshige and Ouchi, 1995). Process variables optimized at laboratory
scale on complex industrial media (molasses) instead of synthetic media
composed of pure chemicals in precisely known proportions. The synthetic
media offers favorable conditions for the microorganism as compared to
molasses like complex media. The alcohol fermentation performed in industrial
fermenters (300m3 working volume). Moreover, the genetic variability between
mutant and parent tested by the well-reputed molecular methods. The study
seems to be comprehensive that the strain characterized at laboratory scale first
and then optimized at industrial scale. Before this, no study was performed to
quantify the effect of invertase activity on industrial scale ethanol production.
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6
Particular goals of the Project
To develop derepressed mutant strain of Saccharomyces cerevisiae,
(insensitive to glucose catabolite repression in molasses medium).
Optimizing of conditions for hyperproduction of invertase by the
derepressed mutant strain
To explore the optimal process variables for maximum ethanol
production
To evaluate the very high gravity ethanol fermentation technology
To obtain maximum production from contaminated molasses with the
use of antibacterials
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7
Chapter 2
REVIEW OF LITERATURE Humans remained familiar to ethanol, ever since the history begun. Firstly, it
produced by the spontaneous fermentation; however, man got the control and
struggled all the times to advance the process. Although excellent research
performed on many issues of ethanol fermentation, yet the process is open to
explore it more and more (Lopes and S-Penna, 2001).
The above studies were oriented on maximum ethanol production at higher
temperatures reducing the byproducts. The present project focused on strain
improvement that may be well adapted for industrial ethanol production.
2.1 Microorganism
Yeasts are highly demanded unicellular microbes, member of kingdom
ascomycotina, employed by humans for economic objectives. Their fast growth
rate and capability in efficiently conversion of sugars to ethanol make them
economically feasible (Reeves, 2001).
The Saccharomyces yeasts are extensively used and well-liked microorganisms
for wine and fuel ethanol fermentations due to their various unique
characteristics like high growth rates (anaerobically or aerobically), proficient
ethanol fermentation, and capability to tolerate various stresses (Piskur et al.,
2006). Many yeast strains belonging to Saccharomyces genera
(Schizosaccharomyces pombe, Saccharomyces uvarum, Saccharomyces
diastaticus) and from Kluyveromyces genera (Kluyveromyces lactis,
Kluyveromyces maxiranus) are presently in use for ethanol fermentation but the
Saccharomyces cerevisiae is the chief.
Universally Saccharomyces cerevisiae is most frequently employed
microorganism for ethanol production through fermentation. Now
Saccharomyces cerevisiae and ethanol are likely to remain, respectively, the
world’s premier commercial microorganism and biotechnological product for
many coming years (Pretorius et al., 2003).
2.2 Substrate
Each and every substance having sugars can be fermented to produce ethanol.
But inexpensive materials that can be efficiently converted to desired product are
economically significant for the industrial scale process. A number of potential
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8
substrates utilized for industrial scale ethanol fermentation have been reported
(Lee et al., 1995). Most of the ethanol is produce from sugar cane and corn-
derived materials. However, Jerusalem artichoke juice, cellulose, barley and
cassava are also in use.
The sugar cane molasses, the remaining of the sugar juice, having some high
value hexoses and disaccharides can be used for industrial scale ethanol
fermentation (Gopal and Kammen, 2009). About 50% (w/w) sugars are present
in molasses; sucrose 32–34 %, 14 – 16 % reducing sugars (glucose and fructose)
(Arshad, 2005).
2.3 Catabolite Repression
The Saccharomyces cerevisiae has been cultured from thousands of years and lot
of strains have been developed and used for specific purposes. Even though
excellent strains with amazingly high ethanol production efficiency have been
obtained but some responses in certain conditions take it away from industrial
point of view. One of them is the glucose repression being encountered in
presence of mixed sugars (Olsson and Nielsen, 2000).
Molasses having combination of sucrose, glucose and fructose is the frequently
used carbon source for industrial ethanol fermentation. These sugars are utilized
sequentially due to glucose repression posed by the presence of glucose. The
literature on this theme is review under.
Naturally, occurring mutants of Saccharomyces cerevisiae capable to grow in
presence of 2-deoxy-D-glucose with better fermentation characteristics were
selected. Three mutants and wild culture was grown at the same rate but
invertase and maltase production was higher in case of mutants under repression
conditions. The medium utilized was Difco yeast nitrogen 0.17%, ammonium
sulfate 0.5% and glucose 2%. Release of carbon dioxide from dough
fermentation was much higher by the two mutants as compared to wild culture in
laboratory and industrial setting, with the addition of glucose or sucrose. Other
three mutants fermented plain doughs very slowly. The quality of products
obtained from mutant culture was much improved as compared to wild
organism (Rincon et al., 2001).
The fermentation of ethanol can be efficiently performed by the yeast strains that
are insensitive to catabolite repression. A strain Kluyveromyces marxianus KD-
15 insensitive to catabolite repression was employed for the ethanol fermentation
-
9
of sugar beet juice (sugar concentration 200 mg ml-1) supplemented with crude
whey in 50 ml medium. Ethanol concentration remained above 99 mg ml-1 in
every experiment, and its production reduced directly with crude whey
concentration used. At lower temperature 30ºC, the fermentation process
remained bit slower but ethanol produced was higher than 33ºC - 37ºC. Using
1.5 liter medium in 2 liter fermenter, the aeration up to 15-50 ml min-1 increased
the ethanol level but it decreased at 100 ml min-1. After optimization, ethanol
achieved was 102 mg ml-1 with complete utilization of sugars in 72 h by the
strains KD-15 (Oda et al., 2010).
A synthetic media similar to sweet sorghum juice containing hexoses (glucose
and fructose) and combination of sucrose with glucose and fructose were
employed for alcoholic fermentation. The kinetic response and growth behavior
of Saccharomyces cerevisiae on several sugars was studied. The difficulties
faced during natural materials fermentation were explored (Phowchinda and
Strehaiano, 1999).
Mostly industrial ethanol fermentation is performed on a combination of several
sugars. The sugars are consumed successively due to repression caused by the
glucose presence; resulting in extended fermentation time. The metabolic
engineering used for the development of derepressed mutant strains has been
studied (Olsson and Nielsen, 2000).
The shifting of yeast metabolism from respiration to fermentation is regulated by
the oxygen level as well as cells outside glucose concentration. A
Saccharomyces cerevisiae strain relying on the chimeric hexose transporter has
been developed. The switching from respiration to fermentation was independent
of the glucose concentration and shifting occurs only in case of oxygen
deficiency (Otterstedt et al., 2004).
The continuous ethanol fermentation of the media containing sucrose is
regulated by the conversion of the disaccharide to its simple sugars. The effects
of glucose repression was studied in a laboratory scale fermenter, with cell
recycling, The invertase production remained very low due to the repression.
The maximum ethanol concentration achieved was 68 g l-1 h-1 (Fontana et al.,
1992).
The metabolism of sugars in yeasts is mediated by the glucose level and control
is lying at the genome level. The exact mechanism is still unknown but generally
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10
it is accepted that glucose 6-phosphate is control point and intracellular or
extracellular glucose level is highly conserved. The importance of the glucose
level inside and outside the cell during growth phase in continuous culture mode
with nitrogen deficiency was explored (Meijer et al., 1998).
2.4 Random Amplified Polymorphic DNA
In spite of the huge research work performed on alcoholic fermentation, the
subject still needs attention to improve the process through development of high
performance yeast strains. The improvement of different strains for several
processes by mutagenesis (irradiation or chemical) had been performed but the
new strain should be confirmed though trustworthy techniques. Polymerase
chain reaction based molecular techniques (RAPD and SSR) are applicable
successfully in this regards.
The molecular techniques, RAPD, mitochondrial DNA restriction study and
electrophoretic karyotyping were employed to assess genetic variability among
wine producing yeast. RAPD gave much better results as compared to other two.
The geographical base of the strains was confirmed through the examination of
genetic polymorphism (Martinez et al., 2007).
RAPD was used to examine the molecular polymorphism among fifty strains
belonging to two different wineries of Poland. The technique was utilized with
different primers GTG5, GAC5, GACA4 to amplify microsatellite to analyze the
similarity. After each run of RAPD, dendrograms were presented showing
genetic resemblance (Walczak et al., 2007).
Relying on polymerase chain reaction, three different molecular methods RAPD,
AFLP and SSR were utilized to examine the genetic variability among twenty-
seven strains of Saccharomyces cerevisiae. The techniques clearly made
discrimination among the strains as compared to conventional methods (Gallego
et al., 2005).
Different stresses are exerted on the yeast cells during industrial scale ethanol
production and the yeast population may vary accordingly. Therefore a strain
isolated from an industrial ethanol production facility may work well in these
conditions and it may be employed instead of yeasts available in the market.
Polymerase chain reaction with GTG5, a microsatellite amplifying primer was
used to differentiate among the yeast inhabitants in the fermenters of six ethanol
plants. It was concluded that native strains present in the raw material were
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11
potentially much better as compared to commercially available strains (Silva-
Filho et al., 2005).
The extensively used technique RAPD was performed to distinguish among the
yeast strains commercially available for use in Brazilian ethanol production
facilities. Genetic similarity pattern was recognized among sixteen strains. By
using the eight primers, ninety three scorable bands were obtained and 41.9%
bands were polymorphics. The strains were divided into three groups based on
cluster analysis. Eleven strains were lying in group one. Redstar strain was at the
28%, maximum distance (Echeverrigaray et al., 2000).
Two molecular methods, amplified ribosomal DNA restriction analysis and
RAPD were performed for discrimination among 6 strains that were identified as
Saccharomyces cerevisiae by conventional methods. Twenty one primers were
used for characterization and only four primers were able to discriminate among
the studied strains. The six strains tested shown forty to eighty percent similarity
(Xufre et al., 2000).
RAPD analysis was performed to make differentiation among 19 strains from
two genera Saccharomyces and ZygoSaccharomyces. Only 5 primers were used
and differentiation among the strains was satisfactory. The results were reliable
and equivalent as by the restriction fragment length polymorphism (Paffetti et
al., 1995).
The yeast cells from different strain emerging sporadically at the end of
continuous ethanol fermentation process were identified by classical taxonomic
methods, and molecular technique RAPD-PCR. There were present non-
Saccharomyces yeasts 29.6% with Saccharomyces cerevisiae (the inoculated
strain) in two months. RAPD-PCR analysis demonstrated that the non-
Saccharomyces yeast were from Issatchenkia orientalis and Pichia
membranifaciens. One isolate 195B of I. orientalis was able to produce ethanol
at 42°C temperature much faster (Gallardo et al., 2010).
Genetic variability between mutant and parental strains of A. niger was tested
through the amplication of their DNA with twenty eight deca primers. Certain
dissimilar patterns were exhibited between two strains through RAPD analysis.
Homogeneous patterns were shown in mutant strain though parental culture had
heterogeneous amplification patterns. Seven primers identified 42.9% similarity
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12
in the amplification products, showing some genetic variability between the two
strains (Awan et al., 2011).
Several doses; zero, 5, 10, 15, 20 and 25 Kr of gamma irradiation were applied
on Jatropha curcas and similarity among the mutants was indentified through
RAPD. From 23 random primers applied only six primers non-polymorphic.
Different number of band were produced by the primers ranging from (1-8) and
with mean of 3.9 bands per primer of which 2.3 were polymorphic. The
similarity index was ranged from 0 to 100 with an average of 55.16%. The
Jaccard's coefficients of dissimilarity was in the range of (0.324 - 0.397)
(Dhakshanamoorthy et al., 2011).
2.5 Invertase
Saccharomyces cerevisiae keeps the enzyme called sucrase/invertase (extra/intra
cellular), that hydrolyze sucrose (disaccharide) into its constituents hexoses.
Hexose transporters then shift the hexose into the cell for further processing
(Badotti et al., 2008). Invertase activity unit can be defined as micro-mol of
glucose released ml-1min-1 under definite settings
The SUC genes, its expression results in the production of sucrase (Mortimer
and Hawthorne, 1966), regulate sucrose consumption in Saccharomyces
cerevisiae.
The maximum amount of invertase production 16.10 U/ml
was achieved at
temperature 30°C. The analysis of the kinetics data of various parameters like
Yp/x
(enzyme formation/mg
of cells), Yp/s
(enzyme production/mg
of substrate
utilized), Yx/s
(mg biomass produced/mg of sugar utilized), Y
s/x (mg of substrate
used/mg
of biomass formed), qp (enzyme formed/mg substrate/h), qs (mg of
sugar utilized/h), qx (mg of cells/mg sugar used/h), μ (mg biomass formed/h)
were performed. Results indicated that substrate consumption and enzyme
synthesis both were directly influenced by the temperature (Shafiq et al., 2004).
A yeast strain, Saccharomyces cerevisiae303-67 was irradiated through ultraviolet
rays, having glucose repressible gene for the production of invertase. Glucose
repression resistant mutants were developed by irradiating twice. CAMP
concentration was elevated in cells that were cultivated in minimal glucose
media as compared to maximal glucose media. Moreover, the invertase was only
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13
1 to 2 % of the whole cell protein and its synthesis was not was not associated
with the CAMP level (Montenecourt et al., 1973).
The invertase production in Aspergillus niger is initiated by its substrates
raffinose, sucrose and turanose. The hexoses, resulted through hydrolysis by the
enzyme act as regulator at genetic level. The initiation mechanism of the enzyme
synthesis in filamentous fungi is suggested with the involvement of cAMP
different from yeast (Rubio and Navarro, 2006).
Ethanol fermentation was performed by Saccharomyces uvarum on sucrose
containing materials to quantify the invertase synthesis. The invertase activity in
fermentation medium was between 1.4 to 4.8 units/ml and greatly influenced by
the dilution factor, the level of corn steep liquor and the nature of sugar (Chan et
al., 1992).
Conditions were optimized for invertase production from fruit peel waste by
Aspergillus flavus. Maximum invertase production was obtained after 96 hrs at
temperature 30 ºC. The optimum pH was 5.0 and inoculums size 3 %.
Supplementation of sucrose and yeast extract increased the enzyme productivity
(Uma et al., 2010).
Invertase formation from sugar cane bagasse by Aspergillus ochraceus was
optimized and high amount of enzyme was achieved at 40ºC after 96 h of
fermentation. Through DEAE-cellulose and Sephacryl S-200, invertase was
purified by 7.1-times; enzyme recovery remained 24% only. Electrophoresis
showed that the enzyme was homogenized in nature. The enzyme was a dimeric
glycoprotein with carbohydrates 41%. The Optimum pH was 4.5 and
temperature 60ºC (Guimaraes et al., 2007).
The parent and mutant strains (NA6) of Saccharomyces cerevisiae were
compared for inveratse production in a time course study using batch mode.
Addition of urea raised the kinetic values (Yx/s, Yp/s and Yp/x) considerably
higher (p≤0.05). Biomass formation (Qx) was highest at 48 h of fermentation,
and some higher than control. Both Michaelis-Menten constant and Vmax of the
invertase by mutant organism were considerably enhanced (Haq and Ali, 2007).
Saccharomyces cerevisiae was cultured on sucrose containing media having
various levels of glucose. Sucrose was utilized by two ways; extracellular
breakdown into hexoses and transfer of disaccharide into the cells. Initial
glucose level and adaptation condition of yeast cells are the two main factors in
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14
determining the mechanism of sugar metabolism. Preferably, glucose was
utilized in both ways then invertase was produced. At glucose level higher two g
l-1 the invertase secretion was repressed. The yeast cells incubated on sucrose for
a long time were able to consume sucrose in the presence of a repressive glucose
level. In this case sucrose was transferred, into the cell without breaking into
ingredient hexoses. Moreover it was also find that direct transfer of sucrose into
the cell gave major portion of the sucrose utilized (Mwesigye and Barford,
1996).
The production of extracellular invertase on the medium containing mixture of
corn steep liquor and sugars by Saccharomyces uvarum in a chemostat reactor
was quantified. Increase in level of corn steep liquor in the medium directly
amplified the invertase formation. Enzyme secretion was also influenced by the
sugar concentration and temperature. DEAE chromatography raised the enzyme
activity 9 times (Chan et al., 1991).
The fermentation rate of yeast strain, YOY655 on molasses was much slower as
compared to synthetic medium supplemented with different nutrients and
containing same sugar concentration. Low fermentation rate was attributed to
osmotic pressure exerted on the microorganism in molasses medium. Osmolality
in molasses medium was mediated by invertase and it was a significant aspect in
fermentation rate (Takeshige and Ouchi, 1995).
Invertase activity by the yeast strain Saccharomyces cerevisiae was accessed on
different sucrose feeding rates with pH (4.0 to 6.5), dissolved oxygen (0 to 5.0
mg O2 l-1) in batch and fed-batch molasses fermentation. Enzyme activity was
reduced when glucose level was above 0.5 g l-l. The decrease in invertase
formation was due to the glucose repression. Followings were the parameters
optimized for maximal invertase secretion: temperature (30ºC), pH (5.0),
dissolved oxygen (3.3 mg O2 l-1) glucose level (0.5 gl-1) and addition of sucrose
according to the equations: (V-Vo) = t2/16 or (V-Vo) = (Vf- Vo). (e 0.6t)/10
(Vitolo et al., 1995).
Kinetic analysis of invertase formation by 5 wild strains of Saccharomyces
cerevisiae screened from dates was examined. The strain Saccharomyces
cerevisiae GCA-II was better performer with improved Qp and Yp/s than all the
other strains. The influence of sucrose level, invertase formation, pH of the
substrate and varying nitrogen sources in submerged culture mode was explored.
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15
The enzyme yield was increased 47.7% on following optimum parameters,
sucrose level (3%), urea (0.2 % w/v) and pH (6) (Ikram-ul-Haq et al., 2005).
(Haq and Ali, 2005)
Elevated ethanol and sodium chloride level in complex molasses media disable
the invertase reversibly. A considerable difference in activity of the invertase
from Baker’s yeast and an osmotolerant strain of Saccharomyces cerevisiae was
noted. The enzyme inactivation resulted in dissemblance of glycosylated and
protein subunits (Zech and Gorisch, 1994).
The genetic control and metabolic regulation are involved in sugar consumption
by the yeasts. The hereditary difference, catabolite repression and influence of
sucrose, galactose, melibiose, and maltose on sugar utilization has been explored
(Carlson, 1987).
Ethanol tolerance was tested among 13 yeasts strains. The three strains were able
to sustain on ethanol concentration 10 % (v/v) and glucose (25 % w/v). Invertase
activity was much higher in one strain YC3, and possibly, it has a significant role
in alcoholic fermentation of molasses (Ekunsanmi and Odunfa, 1990).
Five mutant of Saccharomyces cerevisia were developed through ultra violet
irradiation. Ethanol fermentation of banana peel in batch mode was performed
by the mutant strains. Maximum ethanol level (9 gl-1) was produced by the strain
four produced the on following optimized conditions; temperature (33 ºC), pH
(4.5) and initial substrate concentration 10 % (w/v) (Manikandan et al., 2008).
Influence of invertase activity on alcohol fermentation of molasses by the
thermotolerant mutant yeast strain was analyzed. The mutant was created
through ultraviolet irradiation and ethanol fermentation was performed in a very
automatic bioreactor. Ethanol production was 1.45 times more in mutant strain.
The high ethanol production by mutant strain was due to maximum intracellular
and extracellular invertase production. The extracellular and intracellular
invertase production by mutant organism was 1.8 and 2.6 times more as
compared to parent strain at 40ºC (Rajoka et al., 2005).
2.6 Molasses
Sugar cane molasses, is the most important byproduct of the sugar refinery, is
commonly utilized for ethanol production. Still, the process requires achieving
of three vital goals including enhancement of ethanol concentration during
-
16
fermentation, reduction in energy utilization during ethanol recovery and
decrease in ecological contamination.
From 13 isolated osmotolerant strains of Saccharomyces cerevisiae, the strain
1912 fermented molasses at very high concentration and gave 13.6% (V/V)
ethanol. The fermentation time was only 72 h in a 5 L fermentor. Fifteen percent
(v/v) ethanol was achieved in shaking flasks experiments after forty-eight hours
with 30% (w/v) preliminary sugar concentration. The efficiency of the strain
compared with the presently employed strain for the ethanol production
(Yansong et al., 2001).
Optimization of various process variables of ethanol fermentation from sugar
molasses by the Saccharomyces cerevisiae was performed. The temperature
35°C, pH 4.0, substrate concentration 300 gm/l, enzyme rate 2 gm/l and
fermentation period 72 h were the optimum process variables with 53% rise in
ethanol yield (Periyasamy et al., (2009).
Alcoholic fermentation of henequen leaf juice supplemented with sugar cane
molasses was done by the co-inoculation of Kluyveromyces marxianus and
Saccharomyces cerevisiae collectively. K. marxianus produced reduced ethanol;
5.22 ± 1.087 % (v/v). S. cerevisiae alone or combination with 25% K.
marxianus at an initial population of 3 x 107 cells ml-1 was recognized optimum
and only 2– 4 gl-1 residual sugars were present. The co-inoculation by varying
yeast strains can be beneficial for ethanolic drinks formation. (C-Farfan et al.,
2008).
Correlation between fermentation period and inoculums size was recognized by
the third degree polynomial equation. Using molasses as substrate, ethanol
fermentation was performed in semi continuous mode. The inoculums size was
kept in the range of 40% to 92%. Inoculums size 58% gave maximum ethanol
level (Teresinha et al., 1992).
The presence of difference impurities in molasses lowers the ethanol production
efficiency. To improve the fermentation, molasses clarification was done by a
ceramic MF membrane having pores diameter 0.05. Ash contents and coloring
compounds were decreased after pretreatment of molasses. The residual sugars
were decreased about 42% with 18.1% rise in ethanol level in batch mode
fermentations after 78 h (Kaseno and Koku, 1997).
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Shake flask experiments were conducted for alcohol fermentation of molasses at
varying brix 30 40 and 50º was conducted. Twenty yeast strains were screend
and utilized at different temperatures varying from 32 to 45 ºC. Only two strains;
Schizosaccharomyces pombe and Saccharomyces cerevisiae performed better at
all molasses brix (Haraldson and Bjorling, 1981).
To maximize the ethanol productivity in fed batch mode, two strategies of
substrate addition, constant feeding rate and decreasing feeding rate were
analyzed. Ethanol productivity was raised in case of exponentially decreasing
feeding rate while the ethanol yield remained unchanged. Monod equation
showed the effects of the initial substrate addition (Carvalho et al., 1990).
Decreasing substrate addition rates were employed in place of consistent
substrate addition ratios in fed-batch ethanol production. The influence on
ethanol yield and productivity was estimated (Krauter et al., 1987).
Ethanol fermentation efficiency in fed-batch mode was enhanced up to 17% of
the theoretical yield as the reactor feeding time was approached with
Saccharomyces cerevisiae. Provisional increase of the product in the cells may
provide the answer of higher efficiency (Borzani et al., 1996).
Different five strains of Saccharomyces cerevisiae available in the market were
employed for ethanol fermentation of sugar beet and cane molasses. Production
of ethanol and byproducts was quantified. All the strains yielded ethanol in the
range of 7–9% (v/v). Safdistil C-70 emerged as the topmost suitable strain
(Patrascu, 2009).
The effects of Ca2+ on ethanol fermentation efficiency of yeast were evaluated
at sugar concentration 20% (w/v). A concentration dependent injurious
influence of Ca2+ on yeast activity was noted. At 0.18% (w/v) Ca2+ in all sugar
levels tested decreased the fermentation rates and ethanol yields a little. Above
this level the influence was more prominent and at 0.72% (w/v), the rates of
fermentation and ethanol yields decreased by 14-25% relative to the control
sample. The concentration of Ca2+, 2.16% (w/v) almost stopped the
fermentation of sucrose (Chotineeranat et al., 2010).
2.7 Nitrogen source
Hyper production of invertase was achieved using Saccharomyces cerevisiae
strain. The three different nitrogen sources including urea was applied in
submerged fermentation. β-fructofuranosidase formation was increased from
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121.35 to 158.26 U/ml with the supplementation of urea in the fermentation
medium (Baig et al., 2003).
The influence of varying nitrogen sources ammonium sulfate, casamino acids
and peptone on alcohol fermentation by 4 Brazilian industrial strains were
examined. The experiments were performed under shaken and static mode, with
sucrose concentration twenty two % (w/v). Structural intricacy of the nitrogen
source and the oxygen supply greatly effected the fermentation capacity of the
strains. The trehalose accumulation was drictly propotional to the fermentation
efficiency (Júnior et al., 2009).
The influence of different initial nitrogen levels on metabolism was analyzed.
Through the estimation of cell physiology, important metabolic activities, cell
population and biomass effects on fermentation rate were acessed. The trehalose
accumulation may be responsible for keeping cells viable in nitrogen deprived
fermentation irrespective to initial assimilable nitrogen (Varela et al., 2004).
The influence of cauliflower waste (CW) addition in ethanol fermentation of
molasses by Saccharomyces cerevisiae was evaluated. Water was added to
molasses to achieve sugar (total) concentration 9.60 and reducing sugars level
3.80% (w/v). The addition of 15 % CW and 0.2 % yeast extract elevated the
ethanol level by 36% and 49 % respectively. Co-addition of cauliflower waste
15 % with yeast extract 0.2 % raised the ethanol level up to 29 %. The addition
of cauliflower waste at 15% yielded optimum fermentation parameters; cells
2.65 mg ml-1, ethanol level 41.2 gl-1, final ethanol value 0.358 gg-1 and
fermentation efficiency 70.11 % (Dhillon et al., 2007).
2.8 Phosphorous source
Saccharomyces cerevisiae was employed for the batch ethanol fermentation of
grapes at temperature 32°C, sugar concentration 100 gl-1 and pH 4.5 to attain
optimum ethanol yield. KH2PO4 was emerged as superior phosphorous source as
compared to K2HPO4. The best nitrogen source was (NH4)2SO4 (Asli, 2010).
A cheap synthetic medium for the invertase production by the strain
Saccharomyces cerevisiae GCB-K5 was formulated. The effects of phosphate
ions level in the fermentation medium on the enzyme formation were studied.
Kinetic values for Yp/x, Yp/s Yx/s and μ was estimated. Di-potassium hydrogen
phosphate was the better source and its optimum concentration was 0.020 % for
maximum invertase production (Ikram-Ul-Haq et al., 2004).
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2.9 Inoculum
Better sugar consumption and product formation during ethanol fermentation
can be achieved through the improved initial cell population. Lot of information
is available on the responses of Saccharomyces cerevisiae at laboratory scale but
inadequate data is available about stresses faced and responses shown by the
organism at industrial scale.
Physiological state of Saccharomyces cerevisiae strains is vital in industrial scale
ethanol fermentations. Through the fermentation process and propagation stage
yeast cells face different stresses.
Different preliminary cell population of Pichia stipitis was employed for the
ethanol fermentation of xylose. Substrate consumption rate, product formation,
and the product yield were raised with the high preliminary cell population. The
maximum ethanol level 41.0gl-1 and yield 0.38gg-1 were achieved at preliminary
cell population of 6.5 gl-1. The byproduct xylitol level was enhanced with higher
preliminary cell population. To calculate the steady state of cell population at the
varying initial cell volumes, a two-parameter mathematical model was used
(Agbogbo et al., 2007).
The influence of inoculums level on metabolism and reaction to stresses was
examined in Saccharomyces cerevisiae in fermentation conditions using 5
inoculum sizes. The gas chromatography equipped with time-of-flight mass
spectrometry was used and definite marks on the metabolic activity of S.
cerevisiae were seen. Glycerol formation, amino acid production and depressed
citric acid cycle intermediates were enhanced as the stress increased. But
reduction in varying metabolites was found with the increase in inoculums size.
Maximum concentration of glycerol and proline in yeast population of higher
inoculum size fermentations showed the protecting role of these compound in
microorganism (Ding et al., 2009).
2.10 Ethanol Tolerance
Saccharomyces cerevisiae strain was mutanted by ethyl methane sulfonate. The
mutants showed elevated ethanol level. The mutant cells were cultured on ALP
medium having ethanol in the range of 2-12% (v/v). Mutant colonies were
appeared at 30ºC after 2-6 days. The potential mutants which were grown in
maximum ethanol level were used for bioethanol production in microfuge tubes.
The Bioethanol concentration was analyzed by the distillation-colorimetric
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technique. All mutant were also tested for invertase formation. Ethanol
production was 17.3% higher in mutant than the parent (M-Dehkordi et al.,
2008).
The ethanol fermentation capability and sugar tolerance of some yeast strains
was evaluated. With the increase in ethanol and glucose level, growth and
fermentation rates were declined respectively, "flor" yeasts was the slightest
influenced. Slow addition of glucose increased the ethanol fermentation rate.
Molasses was better fermented by the strains that had done well at laboratory
medium. Addition of ammonia, biotin and biomass recycling increased the
fermentation rate (Jimnez and Benitez, 1986).
Free and immobilized culture of Saccharomyces cerevisiae (GC-IIB31) were
employed for ethanol production under stationary mode of fermentation.
Different sugar levels (12-21%), pH (4.0-5.5), temperatures (25-30°C), volume
of fermentation medium (200-350 ml) and recycling of immobilized culture was
optimized. Immobilized yeast produced significant results up to four successive
batches. Free cells produced ethanol at maximal rate. Under optimized values,
the highest ethanol formation from both free and immobilized culture was yeast
biomass (2g), sugar level in molasses (15%), pH (4.5), temperature (30°C) and
three hundred ml fermentation media in 500 ml fermentation flasks. The highest
ethanol was obtained in the 4th batch and it declined significantly in further
repetition (Mariam et al., 2009).
From 6 yeast strains screened from orchard soil, Orc 2 and Orc 11 had tolerated
ethanol up to 15% whilst Orc 6 tolerated maximum ethanol 20%.
Saccharomyces yeast, Orc 6 the maximl ethanol tolerant, was tried in ethanol
production. The isolated strain Orc 6 was survived in osmotic stress of 12%
(w/v) sorbitol and 15% (w/v) sucrose showing superiority over the reference
strain. The isolated yeast Orc 6 also expressed better fermentation performance
as invertase level was also elevated (Moneke et al., 2008).
Growth of two Saccharomyces cerevisiae strains, one of commercial base strain
S5 and other isolated from soil strain S6 were cultured on four different agar
based media were analyzed. Malt extract agar was used as starting medium to
optimize the growth requirements with pH 5.0, temperature 37°C growth period
72 hours, rpm 110, inocula volume 1.0 ml. The experimental strains growth was
also influenced by different chemicals (Noor and Dahot, 2008).
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With the disconnection of production phase from growth phase, a fed-batch fully
aerated process was devised to achieve twenty percent (v/v) of ethanol in forty-
five hr of fermentation by Saccharomyces cerevisiae. To expose the cause of
variation in ethanol level complete physiology of cells was studied. High escape
of intracellular metabolites into the fermentation medium declined the cell
viability. Moreover, cell viability loss was proportional to the decrease in
phospholipids of plasma membrane. To overcome the ethanol toxicity, yeast
cells undergo metabolic remodelling to produce maximal level of bioethanol in
aerated fed-batch processes instead of cells entering a quiescent GO/G1 condition
(Cot et al., 2006).
The affects of reactor's shape on the kinetics of ethanol fermentation at
laboratory-scale in batch mode was accessed. Two kinds of reactors were
employed: one was the 1-liter cylinder (glass) and other 2-liter fermentation
flask. The fermentation was performed with 1,000 ml inoculated media each.
The reactor's shape influence was controlled by the relation between the initial
yeast population (X0: ~7 g l-1, ~ 14 g l-1, and ~ 21 g/l-1, dry matter) and the initial
glucose level (S0: ~ 100 g l-1, ~ 150 g l-1 and ~ 200 g l-1). At higher values of
X0/S0 0.038 to 0.219 the affects of the reactor shape minimized, and was zero at
0.22 to 0.24 (Borzani et al., 2006).
Ethanol is accumulated inside the yeast cells at the beginning of fermentation
(3h), but there were almost equal ethanol level inside and outside the cells at 12
h. With the change of osmotic stress, the inside to outside ethanol level was
varied at start of fermentation (3h) but it remained same at 12 h. Rise in osmotic
pressure declined the yeast population and fermentation performance. The
addition of nutrient elevated the growth and fermentation rate with the complete
utilization of glucose. But intracellular ethanol level was unaltered (D'Amore et
al., 1988).
Potato tubers were finely ground and ethanol was produced after cooking and
drying at 70°C. Slurry was made in water 1:4 ratio and application of α-amylase
produced 15.2% total reducing sugars. The ethanol concentration 56.8 g l-1 was
achieved by Saccharomyces cerevisiae HAU-1 at temperature 30°C and
fermentation time 48 h. Ethanol yield was not considerably affected by the
nitrogen supplementation (Rani et al., 2010).
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Different stresses faced by the yeasts at industrial scale are concentrated
substrate, high temperature, high ethanol level, low pH and various organic
acids. The strains that can survive among these stresses are capable to give high
ethanol concentration and would be highly demanded for industrial use. A
“stress model” fermentation system relying on the level of ethanol produced by a
certain strain was made to isolate yeast strains that were relatively stress
resistance. Based on the model, 8 different strains of Saccharomyces cerevisiae
were screened. The total of the stress factors in the model was exceeded above
the tolerance level of most of the strains screened (approximately 40%). The
final ethanol level significantly (P < 0.01) higher, was achieved in only two
strains, J006 and A007, with better fermentative efficiency (Graves et al., 2007).
Potential of four pearl millet genotypes was tested for fuel ethanol production.
The fermentation was done in shake flasks and in a five liter bioreactor using
Saccharomyces cerevisiae strain (ATCC 24860).
In shake flasks fermentation, the final ethanol level was 8.7-16.8% (v/v) at dry
biomass level of 20-35%, and fermentation efficiencies were 90.0-95.6%.
Ethanol fermentation efficiency at 30% biomass in a five liter fermenter
approached 94.2%, which was higher than shake flask experiments 92.9%. The
fermentation efficiencies of pearl millets, on a starch basis, were equivalent to
those of corn and grain sorghum (Wu et al., 2006).
2.11 Aeration
The affect of aeration on yeast propagation and fermentative capability in
continuous alcohol cultures had been very much explored (Hoppe and Hansford
1984; Furukawa et al., 1983; Sweere et al., 1988; Ryu et al., 1984). At a
particular dilution rate, cell mass formation, cell mass/glucose consumption and
cell viability were increased through aeration except ethanol concentration. In
bath and fed batch fermentation limited aeration enhanced biomass and ethanol
formation ( Rosenfeld et al., 2003; Alfenore et al., 2004; Cot et al., 2006; Seo et
al., 2009a; Seo et al., 2009b; Seo et al., 2010).
Varying aeration levels none, 0.13, 0.33, and 0.8 vvm, were used to evaluate the
affects of aeration on ethanol inhibition and glycerol formation in fed-batch
mode of ethanol fermentation. Ethanol concentration, specific ethanol formation
pace, and ethanol yield were improved in aerated conditions as compared to non
aeration. It was shown by the model equation of ethanol inhibition kinetics that
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aeration relieved ethanol inhibition by improving the specific rate of biomass
formation and ethanol production. Moreover the glycerol yield and specific
glycerol formation pace were declined about 50 and