prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1855/1/1378S.pdf · iii The Controller of Examinations, University of Agriculture, Faisalabad. "We the supervisory Committee,

i

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

ii

IN THE NAME OF “ALLAH”, THE MOST

BBEENNEEFFIICCEENNTT AANNDD MMEERRCCIIFFUULL

To

iii

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)

iv

DEDICATIONS

To the

HazratMuhammad(PBUH)

(TheComprehensivepersonalityofthe

Universe)

v

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

vi

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

vii

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

viii

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

ix

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





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







x


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


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








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


models representing brix on various concentrations of DAP 4.24

86 Analysis of variance (ANOVA) for the linear model of brix on various


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






models representing RS. 4.30

xi

94 Analysis of variance (ANOVA) for the linear model of RS 4.31


models representing residual sugars on various concentrations of DAP 4.32

98 Analysis of variance (ANOVA) for the linear model of RS on various


103 Effect of varying Brixº, inoculums size and level rise on the ethanol % (w/v) at

27ºC 4.34


32ºC 4.35


37ºC 4.36


42ºC 4.37


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





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


(gl-1) at 32º 4.47


(gl-1) at 37ºC 4.48


(gl-1) at 42ºC 4.49


models representing residual sugars 4.50

xii

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


32ºC 4.53


37ºC 4.54


42ºC 4.55


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








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




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



xiii


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




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




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



xiv

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



mutant strain

4.7

60 Response surface plots of interaction between pH and nutrient

contrations for Invertase activity of parent strain 4.8

60



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


temperature and DAP concentrations for Invertase activity of

mutant strain

4.13

xv

64 Response surface plots of interaction between different pH and

DAP contrations for Invertase activity of parent strain 4.14


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


temperature and pH for ethanol produced by mutant strain 4.17

72


temperature and nutrients concentrations for ethanol produced by

parent strain

4.18

72


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


Temp for ethanol produced by mutant strain 4.24


temperature and DAP conc. for ethanol produced by mutant strain 4.25


DAP conc. for ethanol produced by parent strain 4.26


DAP conc. for ethanol produced by mutant strain 4.27


different temperature for final brix by parent strain 4.28

xvi


different temperature for final brix by parent strain 4.29

84


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


different concentration of p acid/urea for final brix by parent strain 4.32


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


temperature for final brix by mutant strain 4.35

88


temperature and different concentration of DAP for final brix by

mutant strain

4.36

88


temperature and different concentration of DAP for final brix by

parent strain

4.37


different concentration of DAP for final brix by mutant strain 4.38


different concentration of DAP for final brix by mutant strain 4.39


different temperature for residual sugar by parent strain 4.40


different temperature for residual sugar by mutant strain 4.41

96


concentration of urea/phosphoric acid and different temperature for

residual sugars by parent strain

4.42

xvii

96


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


different concentrations of urea/ phosphoric acid for residual


4.45


different temperature for residual sugars by parent strain 4.46


different temperature for residual sugars by mutant strain 4.47

100


temperature and different concentrations of DAP for residual


4.48

100


temperature and different concentrations of DAP for residual


4.49

101


different concentrations of DAP for residual sugars by mutant

strain

4.50

101


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


different temperature for ethanol % (w/v) by mutant strain 4.53


inoculum rates for ethanol % (w/v) by parent strain 4.54


different inoculums rates for ethanol % (w/v) by mutant strain 4.55

xviii

111 Response surface plots of interaction between different inoculums

rise and different temperature for ethanol % (w/v) by parent strain 4.56


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


temperature for final brix by parent strain 4.59


rate and brix temperature for final brix by parent strain 4.60


rise and brix for final brix by parent strain 4.61


rate and different temperature for final brix by parent strain 4.62


rise and different temperature for final brix by parent strain 4.63


different temperature for residual sugars (g/l) by parent strain 4.64


different temperature for residual sugars (g/l) by mutant strain 4.65


rate and brix for residual sugars (g/l) by parent strain 4.66


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


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

xix


different temperature for cell population by mutant strain 4.71

134


rise and different brix for cell population (million/ml) by parent

strain

4.72


rise and brix for cell population by mutant strain 4.73


rates and brix for cell population (million/ml) by mutant strain 4.74

135


rate and different temperature for cell population (million/ml) by

parent strain

4.75

142


rate and different temperature for fermentation efficiency % by

parent strain

4.76


temperature for fermentation efficiency % by mutant strain 4.77


rise and brix for fermentation efficiency % by mutant strain 4.78


rise and brix for fermentation efficiency % by parent strain 4.79

144


rise and different temperature for fermentation efficiency % by

parent strain

4.80

144


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


(ppm) on bacterial population during the ethanol fermentation of

molasses

4.83

xx

146


(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


(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


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


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

xxi

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

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

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

value

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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.

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

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

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

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

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

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

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

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.

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).

17

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

18

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).

19

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

20

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).

21

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).

22

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

23

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

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