ENHANCED PRODUCTION OF ETHANOL FROM

SUGAR CANE MOLASSES THROUGH THERMOTOLERANT

SACCHAROMOMYCESCEREVISIAE CELL

A thesis submitted by

SYED FARMAN ALI SHAH

In fulfillment of the requirement for the degree of

Doctor of Philosophy

in

Chemical Engineering

Department of Chemical Engineering

Faculty of Engineering

Mehran University of Engineering and Technology

Jamshoro

June 2010

ii

DEDICATION

To Holy Prophet

Hazrat Muhammad Mustafa

Salallahu Alaihi Wa Alihi Wa Sallam

iii

iv

ACKNOWLEDGEMENTS

Allah, The omnipotent, The beneficent and The merciful, Who created the universe

and bestowed mankind with the knowledge and ability to think into His secrets.

Peace and blessings of Allah be upon Holy Prophet Muhammad (salallahu alaihi wa

aalihi wasallam, the unique comprehensive personality, the everlasting source of

guidance and knowledge for humanity, I thank to all.

I would like to thank my supervisor, Late Professor Dr. Muhammad Ibrahim Pathan,

Former Dean Faculty of Engineering, Mehran University of engineering and Technology

(MUET), Jamshoro. He rendered his valued services to the noble cause of education. I

pray for his departed soul.

Gratitudes for Professor Dr. Hafeez-ur-Rahman Memon,Director,Institute of Petroleum

and Natural Gas Engineering, MUET, Jamshoro, for his kind supervision after the

death of Dr.Pathan.

Thanks to Professor Dr.Abdul Qadeer Khan Rajput,The Vice Chancellor, Professor Dr.

Abdul Ghani Pathan, Dean Faculty of Engineering, Professor Dr. Ghous Bakhsh

Khaskheli, Director and Mr. Mahboob Ali Abbasi, Assistant Registrar, Postgraduate

Studies.

I am deeply indebted to my Honorable co-supervisor Dr Muhammad Ibrahim Rajoka,

Deputy Chief Scientist, National Institute for Biotechnology and Genetic

v

Engineering (NIBGE) Jhang Road Faisalabad for the continuous help, support and

stimulating suggestions and encouragement during my Ph.D. research work in NIBGE

Faisalabad. Thanks to Mr. Muhammad Ferhan, Junior Scientist, NIBGE, Faisalabad am

also thankful to Mr.Ali Ahmed and Ms. Munazza Afzal, who imparted the heights of

their assistance in my laboratory work, which helped me a lot.

I feel a deep sense of gratitude for my family, who felt difficulties in my absence from

home during my research work but always prayed for my success.

Lastly, I am grateful to my family and friends for the inspiration and moral support they

provided during my Ph.D. work.

vi

TABLE OF CONTENTS

Description Page#

List of Notations xii

List of Abbreviations xiv

List of Tables xvii

List of Figures xxi

Abstract xxviii

CHAPTER 1 INTRODUCTION 1

1.1 General 1

1.2 Ethanol and its scope 2

1.3 Ethanol production 5

1.4 Raw materials for ethanol production 5

1.5 Fermentation by yeast, S. cerevisiae 6

1.6 Aeration in fermentation 7

1.7 Oxygen transfer 8

1.8 Determination of KLa value 9

vii

1.9 Why thermotolerant yeast is used? 9

1.10 General modification of strain 9

1.11 Objectives of present research 10

CHAPTER 2 LITERATURE REVIEW 12

2.1 Ethanol and its by-products 12

2.2 Yeast and invertases 18

2.3 Other parameters 20

2.3.1 Substrate concentration 20

2.3.2 Nitrogen and carbon sources 20

2.3.3 Airflow rate 20

2.3.4 Additives 20

2.3.5. Thermodynamics of ethanol and Ffase

formation

21

CHAPTER 3 MATERIALS AND METHODS 26

3.1 Research centers 26

3.2 Sample centers 26

3.3 Microbial strain 26

3.4 Maintenance of culture 27

viii

3.5 Growth medium composition 28

3.6 Preparation of plates 28

3.7 Preparation of slants 29

3.8 Preparation of the culture of native S. cerevisiae 30

3.8.1. Preparation of yeast growth medium 30

3.9 Effect of gamma rays irradiation on viability of

cells

32

3.10 Selection of mutant of S. cerevisiae 32

3.11 Purification of mutants of S. cerevisiae 33

3.11.1. Slants of purified culture 33

3.12 Propagation of yeast 34

3.12.1 First stage propagation 34

3.12.2 Second stage propagation 38

3.12.3 Fermentation 38

3.13 Effect of carbon and nitrogen sources on ethanol

production

39

3.14 Effect of different additives on ethanol yield by

both wild and mutant culture in 23 liter fermenter

39

3.15 Effectiveness of air in ethanol production 39

3.16 Analytical methods 40

ix

3.16.1 Preparation of standard curve for

biomass estimation

40

3.16.2 Biomass estimation 41

3.16.3 Extraction of ethanol 41

3.16.4 Ethanol estimation through

HPLC

42

3.16.5 Harvesting of intracellular

invertases

43

3.16.5.1 Invertase assay 43

3.16.5.2 Determination of units for

invertase activity

44

3.16.6 Glucose concentration

determination

45

3.16.6.1 Preparation of DNS

(Dinitrosalicylic Acid) solution

45

3.16.6.2 Standard curve of glucose 46

3.16.7 Substrate utilization 46

3.17 Chemical composition of hydrol (starch molasses). 47

3.18 Determination of growth kinetic parameters 49

3.19 Effect of temperature 50

3.20 Determination of thermodynamic parameters 51

x

3.20.1 Thermodynamics of cell mass and

product formation

51

3.20.2 Thermodynamics of ethanol formation 51

3.21. Effect of pH 52

CHAPTER 4 RESULTS AND DISCUSSION 53

4.1 Mutagenesis of S. cerevisiae using -rays 53

4.2 Substrate regulation of invertase and ethanol

production

54

4.3 Initial observations 64

4.4 Substrate concentration dependent formation of

ethanol

66

4.5 Effect of substrate sources 75

4.6 Regulation of ethanol production by nitrogen

sources

79

4.7 Effectiveness of air on productivity of ethanol 85

4.8 Effect of Agitation 90

4.9 Effect of additives 95

4.10 Production from hydrol 99

4.10.1. Effect of temperature on ethanol

production from molasses.

108

xi

4.11 Thermodynamics of ethanol production process 114

4.11.1 Thermodynamics of ethanol production

from hydrol and molasses

114

4.11.2. Thermodynamic parameters of extra-

cellular Ffase production

119

4.12 Effectiveness of pH for alcohol & Ffase

production

122

4.13 Ethanol and Ffase in 150 liter fermenter for the

productivity

123

CHAPTER 5 CONCLUSIONS & RECOMMENDATIONS 129

5.1 CONCLUSIONS 129

5.2 RECOMMENDATIONS 132

REFERENCES 134

xii

LIST OF NOTATIONS

K = Liquid phase mass transfer coefficient

a = Total surface area available for mass transfer

C = Concentration driving force

K = Feed rate for substance

kDa = Kilo Dalton

td = doubling time

nm = nanometer

= Specific growth rate (slope of ln x v/s time)

Qx = g cells per litre per hour

Qs = g substrate consumed per litre per hour

Qp = g product per hour per litre

Yx / s = g cells per gram substrate consumed

Yp/x = g ethanol per gram cell

Yp/s = g ethanol per gram substrate consumed

Qp = g ethanol per gram cells per hour

Qs = Specific ethanol yield in g ethanol per g substrate per hour

x = Cells (g per litre)

P = Product (ethanol g/l)

xiii

S = Substrate

TY = Yield % (Based on maximum yield of ethanol per substrate (0.51 g

Ethanol per g glucose)

xiv

LIST OF ABBREVIATIONS

CFU = Colony Formation Unit

CSL = Corn Steep Liquor

D = Dilution rate

DAP = Di Ammonium Phosphate

DNA = Deoxy Nitrocelicellic Acid

DNS = Di Nitro Sulphate

DO = Dissolved Oxygen

E85 = Blend of 85% ethanol and 15 % gasoline

EtOH = Ethanol

FPL = Fast Product Laboratory

GRAS = Generally Regarded as Safe

h = hour

HCl = Hydrochloric Acid

HPLC = High Performance Liquid Chromatography

Hsp = Heat Shock Proteins

HSuc = High Concentration Sucrose

ICR = Immobilized Cell Reactor

IU = International Units

xv

l = Liter

M = Mass

MSW = Municipal Solid Waste

NaCl = Sodium Chloride

NaF = Sodium Fluoride

NaOH = Sodium Hydroxide

NIBGE = National Institute for Biotechnology and Genetic Engineering

OD = Optical Density

OTR = Oxygen Transfer Rate

OUR = Oxygen Uptake Rate

PAGE = Poly Acryl amide Gel Electrophoresis

PAEC = Pakistan Atomic Energy Commission

ppm = Parts per million

PSI = Proteomic Standard Initiative

RPM = Revolutions per Minute

RQ = Respiratory Quotient

S = Substrate or Glucose content

SOUR = Specific Oxygen Uptake Rate

T = Time

TRS = Total Reducing Sugars

xvi

UV = Ultra Violet

V = Volume

VF = Final Volume

Vi = Initial Volume

xvii

LIST OF TABLES

DESCRIPTION PAGE #

Table 1.1 Important physical properties of ethanol 3

Table 1.2 Major substrates for fermentative production of ethanol 6

Table 3.1 Chemical composition of growth medium: 28

Table 3.2 Chemical composition of inoculum medium 30

Table 3.3 Concentration of glucose for standard curve 46

Table 3.4 Physico-chemical characteristics of hydrol (starch molasses) 48

Table 4.1 Comparative fermentation kinetic parameters of S. cerevisiae

and its mutant derivative M9 for extra-cellular and

intracellular Ffase production and specific growth rate

on different concentrations of sucrose in 23l fermentor

(working volume 15l) at 30 C

62

Table 4.2 Comparative fermentation parameters of S. cerevisiae and its

mutant derivative M9 for production of extracellular and

intracellular Ffase production on different substrates in 23 l

fermenter at 30 C

63

Table 4.3 Kinetics parameters for cell mass production substrate

consumption and ethanol formation by the native (N) and

mutant (M) cells of S. Cerevisiae at different concentrations

of sugars.

70

xviii

Table 4.4 Time dependent molasses concentration on ethanol and cell

mass formation

72

Table 4.5 Substrate concentration dependent kinetics parameters for

ethanol and cell mass formation and substrate utilization by

the mutant organism calculated using data in Table 4.4.

72

Table 4.6 Effect of different substrate sources on ethanol and cell

mass formation with time dependent substrate

consumption from both sources

77

Table 4.7 Kinetics for mutant strain for substrate consumption and

product formation parameters from molasses and hydrol

78

Table 4.8 Time dependent effect of different nitrogen sources on

ethanol, cell mass and substrate present in 15 liter

working volume fermenter.

81

Table 4.9

Kinetics of product formation of ethanol and substrate

consumption parameters for mutant strain of S. cerevisiae

using N-sources.

82

Table 4.10 Effectiveness of air flow on ethanol and cell mass production

during time course uptake of sugars from molasses

85

Table 4.11 Airflow rate dependent kinetic parameters for ethanol

formation and substrate consumption by the mutant strain by

maintaining all other process variables constant except

airflow rate, which had different values.

86

xix

Table 4.12 Effectiveness of agitation for ethanol productivity and for the

growth of cells during the time dependent substrate uptake

standard working conditions

91

Table 4.13 Agitational dependent kinetics parameters of mutant strain

for substrate consumption, and product formation in 15 liter

(working volume) fermenter under standard working

conditions.

92

Table 4.14 Effect of different additives on ethanol and cell mass

formation with time dependent consumption of 15 %

total sugars in molasses (pH =5.5) under optimized

working conditions

96

Table 4.15

Kinetic parameters of S. cerevisiae M -9 for ethanol

production following growth on 15 % sugars in Dextrozyme-

pretreated hydrol in a fully controlled 23-l fermenter

103

Table 4.16

Temperature effect on biomass formation and substrate

Consumption by S. cerevisiae M-9 during ethanol

production following growth on 15 % sugars in Dextrozyme-

pretreated hydrol in a fully controlled 23-l fermenter

104

Table 4.17 Effect of different representative temperatures on ethanol

and cell mass formation during time-dependent sugar

consumption of sugars from molasses medium by both wild

and mutant strains of S. cerevisiae

110

xx

Table 4.18 Temperature-dependent kinetic parameters for ethanol and

cell mass formation during substrate consumption (15%

total sugars in molasses, pH=5.5) by the mutant strain of

S. cerevisiae

111

Table 4.19 Enthalpy and entropy values of ethanol production and

inactivation pathways following growth on hydrol and

molasses

118

Table 4.20 Enthalpy and entropy values of extracellular Ffase

production and inactivation pathway following growth

on molasses at different temperatures

122

Table 4.21 Dependence of ethanol production on culturing condition

namely 23 liter, 150 liter fermenter and shake flask (s. flask)

cultures: Kinetic parameters for substrate consumption

(molasses) and ethanol formation by S. cerevisiae mutant

derivative M- 9.

127

xxi

LIST OF FIGURES

DESCRIPTION PAGE#

Fig. 3.1 Plate culture of fresh thermotolerant S.cerevisiea 27

Fig.3.2 A view of the 23 Liter Fermenter 36

Fig3.3 A view of a 150 Liter Fermenter 37

Fig.3.4 HPLC chromatograms of fermented molasses and untreated

hydrol containing 15 % (TS)

42

Fig.3.4 HPLC chromatogram of properly diluted and filtered

hydrol using RI detector.

48

Fig.4.1 Protein expression profile with and without sucrose in the

growth medium for both wild and derepressed mutant stains

of Sachharomyces cerevisiae.

58

Fig. 4.2(a) Extracellular -fructo-furanosidase (Ffase) by parental cells

(), and mutant cells () and intracellular FFase by parental

() and mutant cells () following growth on 8 % sucrose in

yeast medium which carries substrate.

59

Fig.4.2(b) Extracellular -fructo-furanosidase (Ffase) by parental cells

(), and mutant cells () and intracellular FFase by parental

() and mutant cells () following growth on 10 % sucrose

60

xxii

in yeast medium which carries substrate.

Fig.4.3 Kinetics of production of ethanol (), cell mass () and

substrate in the medium (), extracellular (inverted open

triangle) and intracellular () following growth of mutant

cells body.

61

Fig.4.4(a) Effect of sugar concentrations [ 5 %( , ), 8 %( , ) 10

%(,), 12 %(,) and 15 %( , )] in molasses on

ethanol production by native (empty symbols), and mutant

(filled thick symbols) cells of S. cerevisiae

67

Fig.4.4(b) Effect of sugar concentrations [ 5 %(, ), 8 %(, ) 10

%(,), 12 %(,) and 15 %( ,)] in molasses on

cell mass production by native (empty symbols), and

mutant (filled thick symbols) cells of S. cerevisiae

68

Fig.4.4(c ) Effect of sugar concentrations [ 5 %(,), 8 %( , ) 10

%(,), 12 %(,) and 15 %( ,)] in molasses on

substrate consumption by native empty symbols), and

mutant (filled thick symbols)) cells of S. cerevisiae

69

Fig.4.5(a) Effect of sugar concentration on the production of

ethanol by the wild (open symbols) and mutant

derivative of S. cerevisiae in 15 liter working volume

73

xxiii

fermenter. Each value is a mean of two observations

Fig.4.5(b) Effect of sugar concentration on cell mass formation by the

native (open symbols and mutant derivative of the test

organisms

74

Fig.4.5(c ) Effect of sugar concentration on substrate consumption by

both native (open symbols) and mutant derivative (closed

symbols) done as described in materials and methods.

75

Fig.4.6(a) Effect of substrate sources on cell mass formation by the

native

78

Fig.4.6(b) Effect of substrate sources on substrate consumption by both

organisms, wild and mutated.

79

Fig.4.7(a) Ethanol production from 30 C by both wild (open symbols)

and mutant (closed symbols) strains in 23 liter working

volume fermenter. All conditions were kept constant except

nitrogen sources were altered and maintained at a

concentration of 0.246 % nitrogen.

82

Fig.4.7(b) Ethanol production from 30 C by both wild (open symbols)

and mutant (closed symbols) strains in 15 liter working

volume fermenter. All conditions were kept constant except

nitrogen sources were altered and maintained at a

concentration of 0.246 % nitrogen

83

Fig.4.7(c ) Ethanol production from 30 C by both wild (open symbols)

and mutant (closed symbols) strains in 15 liter working

84

xxiv

volume fermenter. All conditions were kept constant except

nitrogen sources were altered and maintained at a

concentration of 0.246 % nitrogen

Fig.4.8(a) Ethanol by both Native and mutated strains of S. cerevisiae at

30 C All other process variables were kept constant except

air flow rate, which was changed from 0.1 to 0.4 vvm.

87

Fig.4.8(b) Cell growth by both Native and mutated strains of S.

cerevisiae at 30 C all other process variables were kept

constant except air flow rate, which was changed from 0.1 to

0.4 vvm.

88

Fig.4.8( c) Substrate Consumption by both Native and mutated strains

of S. cerevisiae at 30 C All other process variables were

kept constant except air flow rate, which was changed from

0.1 to 0.4 vvm.

89

Fig.4.9(a) Effectiveness of agitation for ethanol productivity from 15 %

total sugars from both of the organism. The data given in the

table is an average of the two readings

93

Fig.4.9 (b) Effect of agitation (rpm) on cell mass formation from 15%

total sugars The data given in the table is an average of the

tow readings .The errors between values were small there

fore it is not shown in the data.

94

Fig.4.9(c ) Effect of agitation on substrate consumption from 15% total

sugars in molasses from the organism. The data given in the

table is an average of the tow readings .The errors between

95

xxv

values were small there fore it is not shown in the da

Fig.4.10(a) Effectiveness of additives shown for ethanol productivity

from molasses (total sugars 15 %, pH =5.5) under optimized

working conditions.

97

Figure 4.10b Effect of additives on cell mass synthesis from molasses

(total sugars 15 %, pH =5.5) under optimized working

conditions. Additives were Tween 80 for parental () and

mutated () strain respectively.

98

Fig.4.10(c ) Effect of additives on substrate consumption by the native

(open symbols) and mutant strain (closed symbols) of S.

cerevisiae.

99

Fig.4.11 Effect of substrate concentration on specific growth rate

(), specific substrate consumption (qS), volumetric rate of

product formation (QP), product yield (YP/S) and

specific productivity (qP) in 23-l fermenter using

Dextrozyme-pretreated hydrol as substrate, and corn steep

liquor (25 g/l) as nitrogen source. Initial flow rate was

1 vvm for 8 h followed by 0.25 vvm in agitated vessel

(250 rpm) at 30 C.

100

Fig.4.12 Determination of activation energy for growth (a) and

formation of Ethanol with the help of native and the mutated

strain. using hydrol based medium (dextrozyme treated 15%

sugars containing hydrol) using Arrhenius relationship.

105

Fig.4.13 Intracellular protein expression profile of derepressed and

thermotolerant mutant M-9 on 15% TS with 3% corn steep

liquor (lanes 1-2), and native culture on this medium (lanes

3-4). M= protein marker and invert = standard invertase

from Sigma-Aldrich

107

Fig.4.14(a) Effect of temperature on the production of ethanol from 112

xxvi

under optimized conditions

Fig.4.14(b) Effect of temperature on cell mass formation from 15%

sugars

113

Fig.4.14(c) Effect of temperature on substrate consumption from 15%

sugars

114

Fig.4.15(a) Enthalpy and entropy requirements for alcohol production

from hydrol in the shown temperature ranges. An average is

shown in the data, = parental and = mutant culture as per

Arrhenius equation.

116

Fig. 4.15(b) Enthalpy and entropy requirements for alcohol production

from hydrol in the shown temperature ranges. An average is

shown in the data, = parental and = mutant culture as per

Arrhenius equation.

117

Fig. 4.16 Arrhenius relationship to calculate enthalpy and entropy of

activation for invertase production and inactivation

pathway

121

Fig.4.17 Effect of controlled pH on Ffase formation by parental ()

and mutant () culture and ethanol production by the

parental () and mutant () cultures respectively from

15% sugars in molasses at 30 C under optimized

conditions of aeration and agitation.

124

Fig.4.18 Representative time course production of ethanol by native

() and mutant (), cell mass by native () and

125

xxvii

mutant () with consumption of sugars by native

() and mutant ()in 150 liter fermenter using

hydrol as a carbon source at 40 C.

xxviii

ABSTRACT

In the present study, Sachharomyces cerevisiae produced invertase and ethanol from

different C-sources and TS. It was catabolite repression sensitive but could grow up to 40

C, though maximum growth and product formation occurred at 30-35 C. The -rays

mutagenesis of Sachharomyces cerevisiae was carried out at 1.2 kGy to select catabolite

repression resistant mutant derivative with retention of its ability to hyperproduce ethanol

and invertase at 43 C. Production of ethanol and invertase by Sachharomyces cerevisiae

wild and its 2-deoxy-D-glucose (DG) resistant mutant (M9) was optimized involving one-

at-a-time approach. The mutant M9 also hyper-produced both ethanol and invertase from

sucrose and molasses-based media. A concentration of 15 % total sugars in molasses was

optimized as the best sugar concentration which produced 74 g/l ethanol in 23 litre

fermenter (working volume 15 litre). Lower concentrations resulted in lower values and

higher sugar concentrations needed more time for complete fermentation. Because of

better results on molasses medium with 15 % total sugars (TS), it was adopted regarding

these studies

CSL was used as N-source and as the only supplement and produced 75 g/l ethanol; 9.4

g/l cell mass and consumed 148 g/l of the sugars. The addition of NaF and Tween 80 as

additives did not show any encouraging results, however, Tween 80 proved better if

utilized for more time up to 72 h.

xxix

Studies have a firmed observation that more than 96% TS are utilized at a rpm of 250-

300 and an optimized rate of oxygen for the maximizing ethanol production.

This mutant of Saccharomyces cerevisiae was employed for ethanol production from

starch-based concentrate (locally called hydrol), in 23-l fermenter for optimization of

process variables by optimizing one variable at a time approach. Maximum ethanol was

attained at 36 h of cultivation of dextrozyme-treated hydrol under optimized fermentation

conditions (sugars 150 g/l; Dextrozyme 1.0 unit/g maltose, maltotriose and

polysaccharides; pH 5.5; ammonium sulphate 10 g/l and temperature 40 C). The

maximum rates i.e., (YP/S) were 2.82 g/g cells h and 0.49 g/g respectively. Determination

of activation energy for cell growth (Eag= 20.8 kJ/mol) and death (Egd= 9.1 kJ/mol) and

product formation and inactivation (EP=35.8 kJ/mol and Edp=33.5 kJ/mol) revealed the

thermo-stability of the organism up to 47 C and can be exploited in a wide temperature

range (in summer) for ethanol production.

Thermodynamic studies revealed that mutation had thermostablization influence on the

growth, ethanol and enzymes production equilibria. The mutant M9 required lower

activation energy (Ea(P)) Gibbs free energy (G*P), enthalpy (H*

P) and entropy (S*P)

magnitudes for ethanol and invertase formation. The activation enthalpy of ethanol and

Ffase formation equilibria by the mutant was lesser in values for ethanol and Ffase

production. In activation pathway were quite comparable and are the criteria of

thermostable metabolic network of thermophilic organisms. The mutant strain is better in

xxx

the inactivation equilibria. Mutation made the organism significantly better with respect

to genetic make up in the glycolytic pathway of the organism.

When molasses and Enzoz hydrol were compared, molasses proved better (74 g/l of

ethanol) than Enzoz hydrol (68 g/l of ethanol). Potential S. cerevisiae during growth in

optimized media in 150 liter fermenter studies indicated that molasses supplemented with

ammonium sulfate supported 1.5-fold higher specific productivity than that by

unoptimized medium and that in 150 liter fermenter aeration and stirring enhanced

enzyme titre by 1.55-fold over optimized media in 23 litre fermenter. Furthermore, the

cell mass productivity (0.34 g/l h) was 1.33- fold and substrate consumption rate (6.3

g/l/h) was 1.66-fold higher than those in the shake flask. The influence of treatments on

all fermentation attributes of ethanol production was highly significant except for q/S,

which was quite non-significant. The values of the kinetic parameters obtained for

ethanol are higher than the values reported by other workers on the same strain.

CHAPTER 1

INTRODUCTION

1.1 GENERAL

This collaborative work of Mehran University of Engineering and Technology

Jamshoro and Pakistan Atomic Energy Commission was carried out. A commercial

yeast strain S. cerevisiae was mutated by Gamma irradiation by employing two

approaches obtaining the desired phenotypes of the desired genotypes and the mutant

derivative and then finally selected strain was designated as M-9.Enhanced

production of ethanol, i.e.; 7.5 % (w/v), 95.4 % (w/w) of the theoretical yield and 9.4

(w/v) cell mass and consumed almost all sugars i.e.; 98.6% (w/v) at elevated

temperatures at optimized parameters in fermenters at laboratory and semi

commercial scale bioreactors, digitally controlled through microprocessors.

The research work is presented in four chapters in this thesis. The first chapter

commences with an introduction to the production of ethanol using different types of

raw materials. Detailed description of the fermentation process by yeast, S. cerevisiae,

is given and the effects of different variables on the production of ethanol are also

described in this introductory chapter. Chapter two provides a detailed literature

review and background to the present work. Chapter three describes the material and

methods used to produce ethanol under different conditions. Effects of various

parameters on the fermentation process are highlighted in this chapter. Chapter four

describes the results obtained in this study and discusses the importance of the data in

a wider context. The Final chapter provides the overall conclusion of this work.

1

2

1.2 ETHANOL AND ITS SCOPE

According to Jeremy (2001), biofuels have the potential to meet the future energy

demands because they are truly renewable energy sources and can be produced

anywhere plants can grow. They are not intermittent and can potentially supply liquid

fuels to the transport sector without major modifications to the existing infrastructure.

Von Sivers et al. (1994) and Wheals et al. (1999) said that ethanol is an important

industrial chemical with emerging potential to be used as biofuel and replace

vanishing fossil fuels. Ethanol (or ethyl alcohol) has been described as one of the

most exotic synthetic oxygen-containing organic chemicals because of its unique

combination of properties as a solvent, a germicide, a beverage, an antifreeze, a fuel, a

depressant, and especially because of its versatility as a chemical intermediate for

other organic compounds.

Ethanol proves itself as a volatile, flammable, clear and color less liquid in normal

conditions. It has pleasant order and suitable taste when diluted with water. The

hydroxyl group is the basis for physical and chemical properties of ethanol (Table

1.1). The group imparts polarity to the molecule and raises the intermolecular

hydrogen bonding. In the liquid state, hydrogen bonds are formed by the attraction of

the hydroxyl hydrogen of one molecule and the hydroxyl oxygen of a second

molecule. This bonding liquefies ethyl alcohol, otherwise it was not possible. The

behavior is similar to that of water in which intermolecular hydrogen bonding is very

strong that water appears to exist in liquid clusters of more than two molecules.

The reactions of dehydration, dehydrogenation, oxidation, and esterification occur

because of the hydroxyl group in ethanol. The hydrogen atom of the hydroxyl group

3

can be replaced by an active metal, such as sodium, potassium and calcium to form a

metal ethoxide (ethylate) with the evolution of hydrogen gas.

Table 1.1: Important physical properties of ethanol

Property Value

Normal boiling point, C 78.32

Density, d420

, g/ml 243.1

Heat of combustion at 25C, J/g 0.7893

Critical temperature, C 793.0

Lower, vol% 4.3

Upper, vol% 19.0

Gong (1999) is of the view that most of ethanol produced in the world today is starch

or sucrose derived. Van Hoek et al. (1998) said that carbohydrates are readily

hydrolyzed by enzymes, and Saccharomyces cerevisiae easily ferments the resulting

sugars (glucose and fructose) to high concentrations of ethanol.

Costello and Chum (1998) proved that ethanol is a clean burning fuel. Its oxygen

contents decrease emissions of pollutant gasses when combusted with gasoline, and

because ethanol is derived originally from plant matrix, therefore its use does not

contribute to the net accumulation of carbon dioxide in the atmosphere, when used as

fuel. Therefore, ethanol blends have been available for over 20 years at about 30% in

gasoline. It was offered by Wheals et al (1999) in a thorough appraisal of literature

and reported ethanol is environmental friendly, as it reduces pollution and green

house gas emission. It has a positive effect on subsurface soils and ground water and

its falls into sustainable bio products. Ethanol can be formulated from C6 sugars as

under:

4

(1.1)

The maximum weight % ethanol from the process would be 92/180 = 51.11% about

50% glucose [88/180 (49%)] is converted to carbon dioxide. Hemicellulose is made

up of the C5 sugar (xylose) arranged in chains with other minor C5 sugars interspersed

as side chains. Just as with cellulose, the hemicellulose can be extracted from the

plant material and treated to release xylose which would be converted into ethanol.

1.3 ETHANOL PRODUCTION

Jones (1989) viewed that ethanol can be synthesized, by direct fermentation of sugars,

or from other carbohydrates that can be converted in to sugars, such as starch and

cellulose.

The ethanol can be prepared be ethylene. In the first step, the hydrocarbon feedstock

containing 35-95% ethylene is exposed to 95-98% sulfuric acid in a column reactor to

form mono- and diethyl sulfate:

CH2CH2 + H2SO4 = CH3CH2OSO3H (1.2)

2(CH2CH2) + H2SO4 = (CH3CH2O)2SO2 (1.3)

Then hydrolyzed with water to give 50-60% aqueous sulfuric acid solution:

CH3CH2OSO3H + H2O = 2 CH3CH2OH + H2SO4 (1.4)

(CH3CH2O)2SO2 + 2 H2O =2 CH3CH2OH + H2SO4 (1.5)

5

Then ethanol and dilute H2SO4 are separated and in last concentrated sulfuric acid is

formed and recycled. Other processes to make ethanol synthetically are not

commercially important.

1.4 RAW MATERIALS FOR ETHANOL PRODUCTION

Zaldivar et al. (2001) reported that there are three major categories of agricultural raw

materials: simple sugars, starch and cellulose

(Table 1.2)

Table 1.2: Major substrates for fermentative production of ethanol

Sugars Starch Cellulose and hemi cellulose

Sugarcane Grains Wood

Sugar beet Potatoes Agricultural residues

Molasses Root crops Municipal solid wastes

Fruit Waste papers, Crop residue

Heinisch and Hollenberg (1993) have summarized the characteristics and documented

the various aspects related to the growth behavior of S. cerevisiae used in the brewing

and baking industry.

This yeast has been extensively studied and applied widely both in the laboratory and

industry.

1.5 FERMENTATION BY YEAST S. CEREVISIAE

It is believed that yeast S. cerevisiae is very commonly used for ethanol production in

the world (Zaldivar et al. 2001 and Kaisa et al 2006).Some researchers (Nevoigt and

Stahl 1996) have used this strain as rich model strain and its shear stress for have

chosen the yeast strain S. cerevisiae for use as the model aerobic organism in the

6

experiments mentioning some reason as it is intensive to shear stress, best for food

and beverages, having simple metabolism.

Reed and Nagodawithana (1991) proved that the engineered yeast strains of S.

cerevisiae exhibited a higher fermentation rate than the wild strains. In the absence of

aeration, yeast has the ability to instantaneously change its respiratory metabolism

from oxidative to fermentative one. This catabolic shift is referred to as the Pasteur

effect. It is manufactured by large scale aerobic fermentation of selected strain of S.

cerevisiae. Aerobic growth of S. cerevisiae on fermentable sugars has been studied

mainly in batch culture experiments. The growth characteristics of S. cerevisiae are

variable depending on the condition to which yeast cells are subjected.

Many researchers have studied the factors affecting the growth patterns of S.

cerevisiae under aerobic conditions (Reed and Nagodawithana 1991). Subsequently,

studies in applications of genetic engineering techniques have become very popular

due to the increasing demands of the industry to improve the strains of yeasts. Control

strategies in industrial aerobic fermentation have been developed to maximize the

growth of yeast and minimize the detrimental factors affecting the yeasts growth

patterns.

1.6 AERATION IN FERMENTATION

Pim et al. (1998) revealed that an amount of Oxygen is supplied to the

microorganisms and uniformity could be maintained by agitation. Both parameters are

important in promoting effective mass transfer to liquid medium in the fermenter. The

main function of a properly designed bioreactor is to provide a controlled

environment in order to achieve the optimal growth and product formation in the

7

particular cell system employed. In laboratory shake flasks, aeration and agitation are

accomplished by the rotary or reciprocating action of the shaker apparatus. Pim et al

(1998) utilized the air stream with the flow rate of 0.5 liter min1

.

1.7 OXYGEN TRANSFER

Oxygen must be supplied as per demand of the microorganism for satisfactory growth

rate. That required oxygen will be transfer through the air intake in the bioreactors, by

bubbles present in the reactor. That must be supplied in any mode of the reactor,

batch, semi-continuous or continuous (Doran 1995).The Charles and Wilson (1994)

revealed that separate calculating of coefficient of mass transfer, KL and a is difficult,

but some times impossible.KLa is coefficient of mass transfer instead of KL,which is

directly proportional to the driving force and the area for the air treanfer.That may me

presented mathematically as:

Oxygen Transfer Rate (OTR) = KLa C (1.6)

and

OTR = KL a (C*L- CL ) (1.7)

Further research was made by other workers too such as Ahmad et al. (1994) and

found an enhanced traditional oxygen transfer rate as the speed of agitator raised

(from 300-600 rpm). Greater agitation produces more dispersion hence the greater

mass transfer rate.Kaster et al. (1990) found that more dispersion could be created in

low agitation if bubble dispersion is utilized for the purpose. If smaller sized bubbles

incorporated then it permits more oxygen and consumes more time to dissolve.

8

1.8 DETERMINATION OF KLa VALUE

Finding KLa in bioreactors is an important aspect with respect to aeration efficiency by

using many techniques to find the rate of oxygen transfer (Klekner 1988) while

keeping that a system of aeration and homogenization be used, construction of the

fermentation and physiological impact of microorganisms and fermentation medium

composition.

1.9 WHY THERMOTOLERANT YEAST IS USED?

Heat is generated in alcohol fermentation at around 140 cal/g of glucose and would

not be possible for the microorganisms to tolerate it and would result poor alcoholic

yiled.That is to be kept under control through cooling systems, an extra load on the

industry. This proves an advantageous, if heat tolerant yeast is utilized for the same.

That leads to economic production of ethanol. As the industry uses non-amylolytic

and non-cellulytic strain there for starchy and cellulosic substrates need to be

converted into simple sugars. The starchy produce maltose glucose fructose the

cellulosic substrates give xylose, arabinose, glucose, mannose and galactose.

1.10 GENERAL MODIFICATION OF STRAIN

Bailey (1991) and Stephanopoulos and Vallino (1991) reported that general

modification and improvement yeast strain is found important and it is relied on

random mutagenesis or traditional breeding and crossing of strains by screening it out

these techniques provides more properties in the strains. Recombinant technology

enhanced more characterized microorganisms by manipulation was done and

achieved more directed approach. An advancement was recorded when Goffeau et al.

9

(1996) improved the cellular properties and engineered it by analysis of the cells was

made to identify the most promising targets for the genetic manipulation.

1.11 OBJECTIVES OF PRESENT RESEARCH

Following are the main objectives of the current research:

i. Development of a mutated S. cerevisae strain tolerant of deoxy-D-

glucose .

ii. Comparative study Native and mutated strain in a fermenter of 23 liter

capacity (15 liter working capacity) at standard conditions.

iii. Effectiveness of air flow rate on production of alcohol by S. cerevisae

mutant culture.

iv. Effectiveness of agitation, using mutant cells.

v. Optimizing the sugar % in substrate concentration in molasses and

hydrol to support maximum product formation.

vi. Effectiveness of temperature on ethanol manufacturing.

vii. Study of influence of nitrogen source on ethanol production in a

fermenter of 15 liter working capacity.

viii. Effect of different additives on ethanol yield by both wild and mutant

culture in a fermenter of 15 liter working capacity.

ix. Comparative study of wild and mutated cultures for cell mass and

product formation under optimized conditions in laboratory, semi

commercial (150 liters) scale reactors.

10

CHAPTER 2

LITERATURE REVIEW

2.1 ETHANOL AND ITS BY-PRODUCTS

Sheikh and Berry (1980) isolated thermotolerant yeast in multiple stages, which grows

on molasses and urea medium. This yields a biomass at 30-41 % at 40 oC. Four of

these strains were tested and found resistant on 55 oC when incubated for 15 minutes

time.

Neelam and Amarjit (1991) utilized over ripped grapes and isolated 6 thermal

resistant strains. These heat resistant mutant strains were isolated at 37 oC, by dilution

techniques in yeast extract medium, when irradiated by UV treatment and ethanol

enhanced quantity of ethanol was yileded.Batch reactor was employed and using 20

% total sugars. Iconomou et al. (1991) also enhanced ethanol production form the the

molasses fermentation medium using gamma-rays

Argiriou et al. (1992) revealed that 17.6 % and 16.5 % alcohol may be obtained from

the two strains of S.cereviae, named as AXAZ-1 and AXAZ-2,respectivel.Grapes

must was used as a source for the sugar substrate.

Laplace et al. (1992) presented the kinetic behavior of six mutated strains of yeast

species on the medium containing D-galactose.

Christer (1993) grew yeast strains of S.cerevisiae through the technique of metabolic

uncoupling. Carbon and energy sources were used for that chemostat culture.

Gardner (1993) presented a study of 14 strains of S. cerevisiae and determined the

growth pattern of these microorganisms by using a variance of 100 to 300 ppm for

glycerol production through a fermentation process.

11

Roukas (1994) revealed the results of the kinetics of ethanol in shake flask

fermentation experiments through S. cerevisiae and presented ethanol yield for 3.5-

6.5 % .on the temperature range of 30-35 oC.

Sonia and Miguel (1994) analyzed the glycolytic flux of yeast S.cerevisiea and

calculated the production rate in chemostat culture and found out coefficients of

metabolic concentration.

Win et al. (1996) presented a study regarding an experiments base upon the yeast S.

cerevisiae by cassava starch syrup and molasses medium in batch fermentation.

Yadev et al. (1996) has isolated yeast, named, HAU-1 on molasses medium in a

reactor, containing columns. That yeast was utilized and found the ratio between

length and diameter o f the reactor. It was asserted that it showed lower efficiency of

that reactor but could be enhanced through using supplements of nutrients.

Banat et al. (1998) reported that the heat resistant yeast of sugarcane molasses is able

to work at a temperature more than 40 C.

De et al. (1998) presented a model fermentation in which biomass, sugar, ethanol,

diacetyl and ethyl acetate are taken into account and all other parameters were also

monitored

Pim et al. (1998) presented a study stae growth rate of at industrial fermentation to

find out the dilution rate, respiration in the system was kept under the study for

ethanol production.

Sheoran et al. (1998) reported an active cell and optimized the rate fof production

through the yeast strain UUA-I in vertical column reactor. It was revealed that the

yeast cells are 30 % active in that reactor and are capable for ethanol fermentation, if

yeast beads were employed in the reactor at 40 oC.

12

Domingues et al. (1999) presented a study for the alcohol fermentation through

K.marxianus and S. cerevisiae species of yeast. An expression was done for LAC12

and LAC4 (lactose permease and (-galactosidase).Ethanol production rate was

recorded as an increased one at seven times and found that the system is stable for

long six month period time.

Newman et al. (1999) worked for the data of a Parental Stress Index [PSI] for the

yeast stress release factor in heat shocked proteins (Hsp104) and it was pointed out

that defense mechanism of yeast release factor Sup35.

A study was carried out for two genes, MIG1 and GAL80, for the utilization of

galactose by Ronnow et al. (1999) for an industrial strain for ethanol distillery.

Physiological characteristics were investigated on mixtures of glucose and galactose

and on molasses for the same.

Abdel-fattah et al. (2000) has reported an enhanced temperature for synthesizing the

Hsp from various microorganisms, during fermentation process

Atiyeh and Dvnjak (2000) used S. cerevisiae ATCC 36858 and beat sugar molasses

medium in batch fermentation utilizing total sugars at 94.9 to 312 g l-1

for ethanol

production at 93 % of the theoretical yield,which was lowered with the lower % age

of total sugars

Sreenath and Jeffries (2000) experienced 43 forest products laboratory (FPL) strains

of Pichia stipitis and Candida Shehatae for their ability to ferment a 1:1 mixture of

glucose and xylose to ethanol prior to fermentation of partially deacidified wood

hydrolyzates. The starting sugar composition, pH, and concentrations of inhibitors

such as acetic acid, furfural, and hydroxyl methyl furfural varied from one batch to

13

another. The delay observed in growth and fermentation depended on the amounts of

inhibitors present and on the capacity of the strain to resist them.

Gimenes et al. (2002) determined xylose concentrations in a shake flask experiment

and significant growth was recorded at increase values of oxygen intake.

Carvalho et al. (2003) has worked on ethanol production through the yeast S.

cerevisiae grown on molasses medium in a fed batch culture system. All fermenter

parameters and the kinetics may also be presented for yields, inoculum, substrate

consumption and inhibition rates.

Alfenore et al. (2004) presented an optimized strategy for aeration rate in the bio

ethanol production. Aeration conditions were also quantified and showed a high

performance for the S.cerevisiae cell

De Neto et al. (2004) screened out some non-flocculating type of yeasts growth

factors for the yeasts, other than the yeasts during grapes juice fermentation medium.

It was also studied that what does it effects if the concentration is at very low one. The

morphologivcl study was the another parameters for this study.

Najafpour et al. (2004) carried out a successful fermentation study of total sugars

concentration consumptions by S. cerevisiae for ethanol productivity through an

immobilized reactor. For its long 24 h operation time.

Rajoka et al. (2004) has investigated the outcomes of carbon resources and its

attentiveness, and different fermentation parameters and their effects on the

production of beta-glucosidase through a high temperature resistant K.marxianus at

shake flasks level.

Marchetti et al. (2005) presented advantages and disadvantages of alternative

technologies for the use of biofuels production. Methanol, Ethanol and Butanol were

14

presented. Sodium hydroxide, potassium hydroxide, sulfuric acid and supercritical

fluids and heterogeneous ones such as lipases were used as catalyst.

Rajoka et al. (2005) mutated and thermotolerant S. cerevisiae ATCC 26602 ,through

multiple screening techniques by the use of UV radioactivity, which was made

possible to work at 40 oC and produce enhanced production of ethanol at 1.6 folds.

Shang et al. (2006) developed a laboratory scale bioreactor of 5 l volume for the yeast

culture at high cell density and other keeping other reactor parameters under control.

He revealed that the feed rate of glucose was adjusted with the ethanol concentration.

Other reactor components were maintained at these values: Temperature, 30 oC, pH

5.5, agitation, 300rpm and fermentation retention time 60 h, while respiration was

kept at 1.0 and ethanol concentration at 1 %.

Jurascik et al. (2006) offered a metabolic pathway model and used a modified

equation of Monod. Found all kinetic parameters keeping growth rate proportional to

enzyme concentration. Three routes for the yeast S.cerevisiae 424A (LNH-ST) were

experienced for glucose and xylose fermentation as: lactose and ethanol oxidation

reduction of lactose, with sugars concentration at 20 g l-1

Muenduen et al. (2006) used flocculating yeast, S. cerevisiae M30 and cane molasses

as a substrate. 12 kinetic parameters for ethanol production, cell mass growth and

sugar consumption were found and temperature effects were recorded in this research.

Activation energy, death rate and ethanol production rate were correlated with

Arrhenius plot.

15

2.2 YEAST AND INVERTASES

A conversion takes place through -Fructo furanosidase (EC 3.2.1.26) in which

sucrose is converted into fructose and glucose. Most of food and pharmaceutical

industries utilize this enzyme. The enzyme also possesses fructosyl- transferase

activity and can lead to formation of fructo-oligosaccharides which have achieved

great attention because of several favourable properties for health foods (Hayashi et

al. 1992; Roberfoid 1993; Tomamatau 1994; Yun 1998). A number of cultures make

this type of enzyme.(Hayashi et al. 1992; Euzenat et al. 1997; Muramatsu &

Nakakuki 1995; Roberfoid 1993; Yun 1998).

It is very important to screen organisms with the help of sucrose as an inducer for the

enhanced production of enzyme at commercial scale. (Hayashi et al. 1992). In our

country, sucrose is needed as sweetener for human consumption and there is no

surplus sucrose to be utilized for production of invertase. Its production from

molasses could improve economics of -fructo-furanosidase (Ffase) production.

Sugarcane molasses contains may contain up to 25-40% glucose and fructose which

exert catabolic repression on Ffase production (Rincon et al. 2001)Enzymatic

manufacture of enzymes is prejudiced with the help of insertion and synthesize

through catabolite (de Groot et al. 2003). Carbon catabolite repression alters with the

help of protein, named as CreA (de Vries et al. 1999). Sucrose is Ffase inducer and

liberates sugars and not feasible for Cre A structure (Hrmova et al. 1991and deVries

et al. 1999).Fungi is also regulated in the same pattern (deGroot et al. 2003; deVries

et al. 1999 )

16

Saccharomyces cerevisiae produces both extracellular and intracellular -fructo-

furanosidase in submerged fermentation (Rincon et al.2001). Enhancement in the

enzymatic expression of Ffase increases substrate consumption allows for permease

(Rincon et al. 2001). Isolation of glucoses is regulated through mutants. (Rajoka et al.

1998; Haq et al. 2001). The separation of this strain is improved and beneficial.

Kaiser et al. (1986) constructed a series of indicators of the enzyme invertase. Agudo

and Zimmermann (1994) observed a low level invertase activity. Vitolo et al. (1995)

permitted this strain to grow through molasses by variation of parameters like DO,pH

and sugar consumption rate.

Sturm (1996) presented observations for the invertases hydrolyzation from sucrose

into glucose and fructose. Zhu et al. (1997) created relationship among activity and

concentration. Niuris et al. (2000) articulated this product and presented its properties.

Tanaka et al. (2000) experimental shows that the product is higher in quality form the

native cells. Ghosh et al. (2001) has purified the invertases and produced high quality

of it. Niuris et al. (2000) has produced a wide range of microorganisms by utilizing

nutrients. Maria et al. (2002) purified production of invertases by using a variety of

nutrients through SDS-PAGE. Rossi et al. (2003) worked on the entrapped cells

grown and shown their growth patterns and also found that they are consuming more

sugars.

17

2.3 OTHER PARAMETERS

2.3.1 Substrate concentration

Sivaraman et al. (1994) reported a high yield at high consumption. Myers et al. (1997)

considered action this thermotolerant yeast and found a high consumption of substrate

and have a direct relationship with HSuc for example bread particles.

2.3.2 Nitrogen and carbon sources

Najafpour et al. (2004) is of the opinion that reported CSL is the best source for this

thermal resistant yeast. It was also found that a reasonable % of ethanol was also

yielded up to 12.5 %.However Sues et al. (2004) found (NH4)2SO4 preferable sources

forth Nitrogen.

2.3.3 Airflow rate

An airflow rate of 0.5 v/v was utilized by Pim et al. (1998) and reported a 60 % of DO

from 800 rpm using smaller bioreactor of 2 liter size.

2.3.4 Additives

Tween 80 was utilized by Castro et al. (1995) and Dragone et al. (2003) as additives

for the fermentation and uphold the ethanol production rates,putting notes that there

was a more time required in this experimental work. Gasch et al. (2000) have used

tween 80 and found that it could possibly be transferred at larger scale production and

concluded that the experiments are that of laboratory scale and need further study at

fermenter and above scales. Vitamins are also experienced by Alfenore et al. (2002)

as additives in high yield ethanol with a disadvantage that glycerol is produced as an

18

additional production which reduces the ethanol production rate. Reddy and Reddy

(2005) experience more time in high yield of ethanol when additives are utilized

during the fermentation process.

2.3.5 Thermodynamics of ethanol and Ffase formation

Many activities are recoded when a cell goes under a metabolic pathway by which an

unordered molecule will under go some changes through a catalyst. An experimental

data was presented by (Agarwal 2006) for the movement and reaction rate. (Hammes-

Schiffer 2002). Very enhanced production of enzymes could be possibly achieved

with the conditions if they are bound the transition state energy. (Eisenmesser et al.

2005). Garcia-Viloca et al. (2004) have worked for the new developed theory for

transition energy for all types of energies that will confirm fluctuated, active effects

for improved enzymatic catalysis. Garcia-Viloca et al. (2004) and Eisenmesser et al.

(2005) have presented almost similar findings for the electrostatic that leads for

entropy. Agarwal (2006) suggested a functionality of enzyme catalysts which is

effective for enhancement in proteins for getting promoters and cross behavior with

the help of current researchers.

Wolfenden and Snider (2001) observed that enzymatic catalysts can expedite the

reactivity of the chemicals at a range of 100-1000 s l

. Siddiqui et al (2002) have

recognized proteins, which are capable in a flexible structure and increase the

coefficients of activity, kcat .

Brisol et al. 1999; Heijen (1999) reveled that in the processing of microorganisms

there are three basic interactive variables; biomass, substrate and product which may

keep the coefficients of maintainability stable ones and helps in finding the rate of

19

productivity. Stephanopoulos et al. (1998) and Maskow and Stockar (2005) have

provided a useful information regarding biological and thermodynamic processes and

their metabolic reactivities and silico predictions. (Goldberg et al. (2004) have applied

the thermodynamic data to industrialized systems for direct calculations of

stiochiometry in nature and are providing assistance to engineers for the energy needs

of the plants.

Garcia-Ochoa et al. (2000) presented a study for an optimized control of the

bioprocess plant .They proved that it requires a model for the various kinetic

parameters too the reason behind is to calculate the stability of the culture and control

of the bioprocess. Liu et al. (2003) have presented an empirical formula, the Monod

equation for the thermodynamic properties the fluid That was used for the study for

the various mediums in their viscosity would possible effect the rheological

characteristics of a fluid issues in materials shifting and lowers the activity of

metabolic conditions and used for the optimizations of the transfer of oxygen and

their rates of aeration and agitation for getting the fluid mixed. Thermal motions

create an enhancement in the reactions of enzymes and rate of transition (Fisher

2005). When the temperature effects on the production it enhances the state of

transition and increase the rate of formation in that. (Benkovic and Hammes-Schiffer

2003). The Monod equation for the kinetic changes and modifications is used by

Kelly (2004) and the research is made for the consuming the substrate .in it. And mass

formation for these cells under the study.

Roels (1983) has explained the way among the three, which are now introduced for

the finding out the thermodynamic values in any enzymatic system. This was also

found useful for the time dependant variables in the reversible equations there. Tow

20

different models of transitional state and Arrhenius theory was joint together by Aiba

(1973) and that was found successful in the systems where thermodynamic and

transitional state is used. However previous workers (Arni et al. 1997) have used the

thermodynamic and transitional parameters in the fermentation systems for the

bioproducts. Arni et al. (1999); Converti and Dominguez (2001) have applied values

for the production and the enzymes ocncentratiuon rate in kinetic thermodynamic

parameters of the reactions .This has been followed by other workers too(Converti

and Del Borghi 1997; Converti and Dominguez 2001; Rajoka et al. 2003).

Rate of change in the consumption depends on the value of the coefficient of transfer

that is equal to the identity (Day et al. 2002)This was proved by the current research

and is mostly applied to the media which is less visoucs (Garcia-Viloca et al.

2004)applicable to non-viscous media. (Agarwal 2006). Ln (A) is also having same

value for the determination (Winzor et al. 2005).

Enzymatic reaction is reversible when folding and unfolding of the enthalpy proteins

are considered. (Beadle et al. 1999; Shiraki et al. 2001).A week interaction creates a

unstable or stable due to entropy which is conformational one. It shows that the

difference e is very small in the entropies. The stability is dependent of the product

formation (Eisenmesser et al. 2005). A reasonable number of the proteins is formed

when the cells are shocked by the heats or the therms and these proteins (Borges and

Ramos 2005).

High rate of enzymatic production could be achieved, if and when the enthalpy

changes with respect to the substrate changes. This is also useful for the contributing

enzymes there. This study was made to make some results on the basis of temperature

profile for the reactions and the systems (Winzor et al. 2005).

21

Agarwal (2006) has freshly recommended that protein ambiance is very important for

finding out the way to bimolecular process to get a rid from the local energy barriers

for the bioprocesses. That will solve the problems of energy at all. That has a very

strong effect on the forming a production and to modify the proteins very efficiently

and rapidly in a fermentation process.

22

CHAPTER 3

MATERIALS AND METHODS

3.1 RESEARCH CENTERS

This thesis was conducted in a close collaboration with the National Institute for

Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. This research

institute is under the administration and management of Pakistan Atomic Energy

Commission (PAEC), Islamabad. The added benefit of this research collaboration has

led to the establishment of an advanced Biochemical Engineering Laboratory in the

Chemical Engineering Department, Mehran University, Jamshoro. It is anticipated

that this laboratory will be ready for project students and advanced research work in

near future.

3.2 SAMPLE CENTERS

The substrates, black strap sugar cane were taken from sugar industries of various

parts of Pakistan.

3.3 MICROBIAL STRAIN

Yeast strain of S. cerevisiae SAF (France) was purchased from a local market and

grown at the most popular yeast medium at minimum composition of the constituents

as shown in Tables 3.1 and 3.2 as used by Rajoka et al. (2005). Then the culture was

further stabilized (through mutagenesis) for higher working temperature and

catabolite repression resistant at the same time to make it thermotolerant with

retention of hyper-production of ethanol power and used for enhanced ethanol

production.

23

3.4 MAINTENANCE OF CULTURE

The strain was maintained according to Sirianuntapiboon et al. (2004) on yeast

growth media (Fig. 3.1). Different chemicals of analytical grade were used for

preparation of the yeast growth medium. The chemicals were added into distilled

water one by one with shaking and volume was made upto 100 ml in an Erlenmeyer

flask of 500 ml capacity. Through the solutions of Hydrochloric acid (1 N) and

Sodium Hydroxide (1 N) the media pH was kept upto 5.5.

Figure 3.1: Plate culture of fresh thermotolerant S.cerevisiea

14

MUTANT CULTURE OF

KLUYVEROMYCES MARXIANUS

24

3.5 GROWTH MEDIUM COMPOSITION

Table 3.1: Chemical composition of growth medium

Chemicals/ Biochemicals w/v (%)

Ammonium sulfate 0.003%

Potassium di hydrogen phosphate 0.001%

MgSO4 0.005%

KCl 0.005%

Yeast extract 0.5%

Malt extract 1.0%

Glucose 1.0%

Peptone 1.0%

Agar 2.5%

All of the chemicals and biochemicals were that of quality and purchase d from well

reputed companies.All chemicals were of analytical grade and were purchased from

Sigma/Aldrich Chemical Company, Oxoid Chemicals (USA), Sharlau, (Spain) and

Merck Co. Germany.

3.6 PREPARATION OF PLATES

Distilled water was dispensed through 500 ml Erlenmeyer flask and the chemicals

required, as described earlier for preparation of the yeast growth media, were added

one by one with shaking. Then the volume was maintained upto 100 mls in a conical

flask for optimization studies or pH was varied for pH optimization studies. This well

plugged and covered with aluminum foil was sterilized in an autoclave machine at a

prescribed machine parameters i.e.,121C, 15 psi for 10 min. After autoclaving, the

yeast growth medium was poured into the Petri plates. Upon solidification of media,

25

the plates were kept at room temperature for one day to confirm the purity. Later on,

Petri plates were inoculated with S.cerevisiae and then by streaked and incubated at a

temperature of 37 C for 24 h. When the colonies were formed, the plates were

properly sealed by parafilm then preserved for the long time at lower temperature of 4

C.

3.7 PREPARATION OF SLANTS

The yeast growth media, prepared by the procedure already discussed in section 3.6,

were equally distributed in 10 test tubes (Pyrex) properly cotton-plugged and covered

with aluminum foil. The tubes were autoclaved at 121 C, 15 psi for 10 min. The

autoclaved tubes were laid down in slanting position for some time for solidification

of media. The slants were inoculated with S. cerevisiae by streaking, and then

incubating at the temperature of 37 C for 24 h.

3.8 PREPARATION OF THE CULTURE OF NATIVE S. CEREVISIAE

3.8.1 Preparation of yeast growth medium

Distilled water (50 ml) was dispensed in an Erlenmeyer flask of 250 ml capacity, and

following chemicals were added and the pH was kept as in section 3.8.

Table 3.2: Chemical composition of inoculum medium

Chemicals/Biochemicals w/v (%)

Ammonium sulfate 0.05

Potassium di hydrogen phosphate 0.05

Magnesium sulfate 0.05

Yeast extract 0.5

Glucose 2.0

pH 5.5

The pH of the inoculum was kept 5.5 as described in the section 3.4

26

I. A well grown single colony of S. cerevisiae was picked up by a loop and was

inoculated in 100 ml of yeast inoculum medium in a conical flask was

incubated in a shaking incubator (Toshiba, Japan) at 37 C at 120 rpm for a

period of 24 h.

II. Properly diluted culture (100 l) was taken after 24 h of growth and spread on

the yeast media plate for incubation as described in section 3.8 part I and

stored as described previously for further utilization.

Yeast growth medium was prepared with the help of biochemical and salts as

mentioned in the table 3.2 and as described before (section 3.8) for the required

volumes and autoclaved as per described method in section 3.4 for 15 minutes. These

sterilized and cooled cells were grown after inoculation and then centrifuged as

mentioned in the previous section 3.4 and 3.5 and analyzed through

Spectrophotometer (LaboMed USA).

A known volume (50 ml) dispensed in a 250 ml Erlenmeyer flask; the chemicals,

with the composition already mentioned in Table 3.2, were added to prepare the yeast

growth media. The pH of the medium maintained as described in section 3.8.

I. Yeast inoculum medium was inoculated with a loopful of purified culture of S.

cerevisiae aseptically in a laminar hood and then the flask was incubated at 30

C at 120 rpm in orbital shaker for 24 h.

II. After growth. 10 ml of culture of S. cerevisiae was taken aseptically in the

autoclaved McCartney vials of 30 ml capacity.

III. McCartney vials were labeled for exposure of different doses of gamma

radiation i.e. (0.20 to 1.00 kGy in the span of .20 kGy ).

27

IV. They were properly sealed with parafilm and packed in polyethylene packets

to avoid the leakage and contamination of culture of S. cerevisiae with water

in the tank during gamma radiation exposure.

V. After exposure of culture of S. cerevisiae to different doses of Gamma

radiations. McCartney vials were stored at 4 C for further usage.

3.9 EFFECT OF GAMMA RAYS IRRADIATION ON VIABILITY OF

CELLS

The untreated culture of S. cerevisiae was taken as control. The 100 l of native and

gamma rays irradiated cultures (0.2, 0.4, 0.6, 0.8 and 1.00 kGy) of S. cerevisiae were

diluted in 900 l of biological saline to make 10 times dilution. The cultures were

further diluted upto 107 and were spread on growth plates separately by spreader

aseptically. The plates were labeled and incubated at 37 C. After growth of 24 h, the

viable colonies were counted and colony forming units/ml (CFU/ml) were determined

as follows:

Example:

Viable Counts = 1324

Colony Forming Unit (CFU)/ml =Viable countsx1

Sample volume x dilution factor

= 1324 x 1

0.1 x 107

=1.324x1011

Cells /ml

3.10 SELECTION OF MUTANT OF S. CEREVISIAE

The survival curve was prepared to select an exposure dosage for mutation, giving 3-

log Kill (Rajoka et al. 1988) and exposure dose of 1.0 kGray (kGy) giving 3-log kill

(0.1 % survival).Two strategies used for mutant selection simultaneously. In first

strategy the mutant was selected by directly spreading the irradiated cells on plates

28

containing DG 1.5 % (w/v), sugars 17% (w/v) in yeast medium and agar 2.5 % (w/v).

In second strategy, the mutants were selected by permitting irradiated cells to express

in broth containing 17% sugars plus 1.5 % deoxyglucose at 45 oC until the OD

reached 0.1 at 10 dilution and plated on PDA plates containing 1.5 % deoxyglucose

and incubated at 45 C .The mutants were grown at different temperature (37, 40, 43,

45 and 47 C) on yeast agar plates. The best mutant of S. cerevisiae was selected on

the basis of its thermotolerance.

Similarly the mutants were grown in yeast fermentation medium at different

temperatures (20, 22, 24, 26, 28, 30, 35, 40, 43, 45 and 47 C) and best mutant of

S.cerevisiae was selected on the basis of thermotolerance.

3.11 PURIFICATION OF MUTANTS OF S. CEREVISIAE:

The mutant cells of S. cerevisiae were purified in the same way as mentioned for

native strain with the exception that mutants were grown at 45 C. Later on plates

were inoculated by S. cerevisiae by streaking, and incubation was done as in section

3.4 to 3.6 and preserved for next experimentation after sealed properly by parafilm

and stored at 4 C.

3.11.1 Slants of purified culture

The yeast growth medium, prepared by the procedure already discussed, was

equally distributed in 10 test tubes (Pyrex) which were properly cotton-plugged and

covered with aluminum foil. The tubes were autoclaved as before and the autoclaved

tubes were laid down in slanting position for some time for the solidification of

media. The slants were inoculated as earlier.

29

3.12 PROPAGATION OF YEAST

The yeast was propagated for 23 liter bioreactors. Then the yeast was propagated for

semi-commercial production in two stages to increase the volume. The naming was

used as the first stage propagation and the second stage propagation.

3.12.1 First stage propagation

At the first stage propagation cell culture was inoculated in a one liter volume flask to

make a volume of 250 mls carrying Di ammonium phosphate, yeast extract along

with glucose ,2.5,2.5 10,2, gl-1

was incubated as mentioned earlier..Fermenter of 23

liter volume (Made in Germany) was used to carry out this research studies. The

operating volume of the reactor was 15 l. All important accessories were attached

with the reactor for pH, DO, aeration, agitation and mixing. The molasses medium

containing 15% or sugar concentrations others in g/l-1

) of the nutrients used in flask

experiment such as ( NH4) 2 SO4, 2.5 and yeast extract 2.0 g and pH was adjusted at

5.5 with sulfuric acid. After steam-sterilization for 45 minutes at 121 C for 30 min at

1 bar. Air and circulation water were opened till temperature came down to 50 C.

The reactor media was inoculated with that of ten percent at 30 C. 15 liters per hour

was utilized an air flow rate for initial eight hours that will make a rich production of

cell mass .Then the rate was reduced upto three liters per hour for next fermentation

operation of the reactor. For the process optimization the reactor operation was made

continuous upto 28 hours and the foam was made under control by the use of

antifoam ,Silicon oil .At this stage, the fermentation broth had a viable cell count at

300(106

cell/ml and total sugars measured as brix was 15-19. That was ready to

transfer to stage II when pilot plant studies were performed.

30

Fig. 3.2: A view of the 23 Liter Fermenter

31

Fig. 3.3: A view of a 150 Liter Fermenter

32

3.12.2 Second stage propagation

At this stage the working capacity of the closed vessel made up of stainless steel of

100 l was used. First of all vessel was sterilized as mentioned in section 3.13.1. Then

the yeast inoculum was transferred in these tanks as stated above and the fermentation

started.

3.12.3 Fermentation

Fermentation tanks were made up of stainless steel, having capacity of 23 and 150 l.

All experiments were performed in batch fermentation (Fig.3.2 and Fig. 3.3). The

temperature was maintained at 30 C for optimization studies of process variables.

The mutant was grown at 43-45 C or as mentioned other wise. The temperature was

controlled by cooling water passing around the mash through the jacketed vessel.

Substrates (molasses and Enzoz hydrol) of the optimized brix/concentrations were

used. Level of the fermenter was raised during agitation so one third volume of vessel

was kept void in each study. This circulation ended after 48 h, by continuous adding

of silicon oil as an antifoaming agent. Fermented mash from the fermenters was

sampled at every four h. A Brix hydrometer utilized for checking the specific and was

confirmed on HPLC. Ethanol (already separated through extractor a t laboratory

scale) was known through HPLC. When molasses was used as a carbon source,

almost 100 % total sugars (TS) were consumed after 24-28 h fermentation. Peaks of

ethanol were observed after a retention time of 19.30 to 19.36 min as mentioned in the

Figure 3.4.

33

3.13 EFFECT OF CARBON AND NITROGEN SOURCES ON ETHANOL

PRODUCTION

Black strap sugar cane molasses was used as a carbon source and compared with corn

molasses (known as Enzoz Hydrol) in the production medium. The effect of different

substrate concentrations i.e. 5, 8, 10, 12, 15, 17.5 and 20 % (w/v) were tested to

determine the optimum substrate concentration for maximum ethanol production by

mutant strain of S. cerevisiae. 0.23 % of nitrogen was utilized from CSL, di

ammonium phosphate (DAP) and Urea for the growth medium as utilized previously

by Favela-Torres et al. (1998) and Gutierrez-Rojas et al. (1995).

3.14 EFFECT OF DIFFERENT ADDITIVES ON ETHANOL YIELD BY

BOTH WILD AND MUTANT CULTURE IN 23 LITER FERMENTER

Tween 80 and NaF were tested as additives for the enhancement of production of

ethanol through S.cerevisiae mutant in the fermenters of 23 liters and then in 150

liters.

3.15 EFFECTIVENESS OF AIR IN ETHANOL PRODUCTION

The air is essential for supporting a basic quantity of cell mass in fermenters. Earlier

studies (Rajoka et al. 2005) suggested that at initial 8 hours air rate may be at 1 v/v

followed by supplying air at slower aeration rate was sufficient for ethanol production

process. Thus the ethanol fermentation was carried out for the producing ethanol

through a thermotolerant yeast S. cerevisiae. As mentioned earlier that the agitation

intensity helps in improving mass transfer of air, production of biomass air-

optimization of air transfer rate and agitation rate was performed. During

fermentation, data on cell mass, ethanol, concentration of sugars in mash were

collected for calculation of different process kinetic parameters.

34

3.16 ANALYTICAL METHODS

3.16.1 Preparation of standard curve for biomass estimation

Small volume (50 ml) of cell culture of S. cerevisiae was harvested after incubation of

24 h at 37 C that was separated in a centrifuge, as mentioned in section 3.5 and the

pellets were washed with saline. Then the material was recentrifuged and was dried

on filter paper in hot air oven. The dry cells were grinded to a fine power. Stock

solution-I was prepared by dissolving 100 mg of grinded dry cells in 4 ml distilled

water and stock solution-II was prepared by adding 400 1of stock solution-I to 9.6

ml distilled water. Various dilutions of stock solution-II (upto 10 fold) were made to

make calibration curve. The optical density of each dilution was noted at 610 nm on a

digital Spectrophotometer (Spectro-UV-VISRS, Labo Med. Inc USA). The

absorbance was adjusted to zero with blank, which was distilled water.

3.16.2 Biomass estimation

Effect of different process variables like nitrogen and carbon sources and their

concentrations, additives, fermentation temperatures, media pH, dissolved oxygen, air

flow rate, agitation intensity, was determined during biomass formation. Different

time course samples of native and mutant strains of S. cerevisiae were subjected to

determine optical density at 610 nm on the spectrophotometer after making proper

dilutions.

The absorbance was adjusted to zero with distilled water as blank. The samples were

diluted and centrifuged as mentioned in the section 3.16.2.Optical density (OD) of

diluted cell free solution noted down and subtracted from total OD of the culture

35

broth with cells. The OD was multiplied by slope of the plot of standard curve to get

biomass in mg cells/ml.

3.16.3 Extraction of ethanol

After fermentation, cells were separated through centrifugation operation as

mentioned earlier. Ethanol was distilled with Soxhlett apparatus (Japan) by setting its

temperature at 80 oC. After getting the distilled sample, either volume was measured

or its volume was made up to its original volume with deionized water, filtered (in

filter paper 0.22 microns) and microfuged (7,000 rpm, 3 min). Ethanol concentration

was confirmed on HPLC as mentioned earlier (Rajoka et al. 2005).

3.16.4 Ethanol estimation through HPLC

Distilled and filtered samples of ethanol were run in High Performance Liquid

Chromatograph (HPLC) (Perkin Elmer, United States of America) using column

HPX-87H (300 x 78 mm) (Bio, Richmond, California) maintained at 45C in a

column oven. Sulphuric acid (0.001 N) and HPLC grade water was utilized as a

mobile phase at 0.6 ml/min. The samples were detected by refractive index detector

and quantified using Turbochron 4 software of Perkin Elmer, USA.

Fig. 3.4:HPLC chromatogram of fermented molasses and untreated hydrol

containing 15 % TS

36

3.16.5 Harvesting of intracellular invertases

The intracellular invertases were extracted by sonication from the culture of native

and mutant strains of S. cerevisiae. After growth of the yeast, the culture was

centrifuged and the cell pellets were washed as earlier .The cell mass pellet was

suspended in normal biological saline (0.89 % w/v of Na Cl). The cells of exact mass

were taken for the sonication. They were vortexed to get homogeneous mixing of

cells and were disintegrated by ultrasonic waves (10 sec impulses 5 seconds rest for

20 cycles) in ice (To avoid the denaturation of intra-cellar proteins at high

temperature attained during sonication, ice was used during this operation). After

sonication, the samples were recentrifuged to settle down the disintegrated cell debris.

The supernatant having intracellular invertases was taken and preserved at -20C.

3.16.5.1 Invertase assay

The activity of invertases checked through was determined (by using of 100 l

enzyme 10 m1 McllVain buffer of 0.15 Molality and maintained a pH of 5.5) or 50

ml sodium acetate (pH 5.0) and 1.5 (w/v) sucrose solution was used as substrate. That

mixture was sent for incubation 50 C for 15 minutes a shaking bath. Then quenching

performed through the placement of that reaction mixture in running water for 5 min.

The amount of glucose was determined by adding 100 l of reaction mixture to 1 ml

of glucose oxidase based glucose measuring kit (Biocons, Germany) and was then

incubation was done then Optical Density (O.D.) was taken through

Spectrophotometer taking a previous method of Hayashi et al. (1992).

3.16.5.2 Determination of units for invertase activity

Glucose concentration in assay was determined using the following equation:

Glucose concentration = A of samp1e conc. of standard (100 mg/dl

37

= A of standard

For example:

A of sample = 0.12 A or standard = 0.039 Glucose concentration = 0.12 100 ml

0.039

= 307.69 mg /100 ml = 3.0769 mg /ml

Total volume of reaction mixture = 2.1 ml

Concentration of glucose in assay = 2 3.0769 = 6.46149 mg/ml

1 mol of glucose = 0.1802 mg Total moles of glucose released by invertase = 6.46149 = 35.86

0.1802

Incubation time {-or invertase activity = 15 min.

moles of glucose released in 15 min = 35.86 moles of glucose released / min. = 35.86 15

= 2.391 mol/min 1000 l of invertase will liberate = 2.391 1000

100

= 23.91 units

Invertase activity = 23.91 U/ml/min. under the assay

conditions

3.16.6 Glucose concentration determination

DNS method was used for the purpose of sugars referring previous method used by

Miller (1959)

3.16.6.1 Preparation of DNS (Dinitrosalicylic Acid) solution

Different ingredients were used for the preparation of DNS. These are as follows:

(i) Distilled water 1416 ml

(ii) 3-5, Dinitrosalicylic acid 10.6 g

(iii) NaOH 19.5g

The above ingredients were dissolved and gently heated in water bath at about 80C

until a clear solution was obtained. Then the following chemicals were added:

(iv) Rochelle salt 19.5 g

(Sodium Potassium tartarate)

38

(v) Phenol (melted at 60C) 7.5 ml

(vi) Sodium metabisulfate 8.3 g

After dissolving all the above ingredients, the solution was filtered through a large

coarse sintered glass filter and stored at room temperature in an amber bottle to avoid

photo-oxidation. It was stable for 6 months.

A zero point absorbance was adjusted by blank containing 3 ml of distilled water and

3 ml of DNS reagent.

3.16.6.2 Standard curve of glucose

Different known concentrations of 0.1 % glucose was taken and diluted to a final

volume of 3.0 ml with citrate phosphate buffer as shown in Table 3.3

Table 3.3: Concentration of glucose for standard curve

S.No Concentration of

glucose solution (l) Distilled

water (l) Buffer

(ml)

Total

volume

(ml)

DNS

reagent

(ml)

Absorbance at

550 nm

1 200 800 2.0 3.0 3 0.235

2 400 600 2.0 3.0 3 0.47

3 600 400 2.0 3.0 3 0.520

4 800 200 2.0 3.0 3 0.690

5 1000 0.00 2.0 3.0 3 0.860

Samples were boiled in boiling water bath for 15 min. The reaction was quenched on

ice for 15 min before taking reading on Spectrophotometer as described earlier. Then

it was plotted against different glucose concentration (g/ml) to draw standard curve

using Slidewrite 3 software.

39

3.16.7 Substrate utilization

The substrate utilization by native and mutant strains of S. cerevisiae, during the time

course study was determined by DNS method and glucose measuring kit to analyze

total and reducing sugars in the samples.

Standard

The 10 l of standard solution and 90 1 of distilled water were added to 1 ml glucose

kit incubation was done for 5-10 minutes and then 500 nm on spectrophotometer as

mentioned earlier.

Blank

Known volume (100 1) of distilled water was added to 1 ml glucose kit and the

absorbance adjusted at zero.

Sample

Known volume (100 l) of diluted sample was added to 1 ml glucose kit and OD

recorded after incubation of 5 to 10 minutes at 500 mn of spectrophotometer.

For Example:

A of sample = 0.61 A of standard = 0.29 Glucose concentration =0.6l/0.29x100=210.35mg/100 ml

= 210.35 mg/100 ml

= 210.35 (dilution factor

= 210.35 ( 20

= 4207 mg/100 ml

= 4.21 g/100 ml

= 42.1 g/1

3.17 CHEMICAL COMPOSITION OF HYDROL (STARCH MOLASSES)

Properly diluted and filtered hydrol was analyzed by HPLC using RI detector. This

substrate contained

40

Fig. 3.5: HPLC chromatogram of properly diluted and filtered hydrol using RI

detector.

Table 3. 4: Physico-chemical characteristics of hydrol (starch molasses)

S.No Description % Ingredients %

i. Dry Substance 70

ii. Total Sugars 82 Glucose 56

Maltose 13

Maltotriose 2

Oligo Sac