of 162
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