dr v v phd thesis - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/45315/7/front.pdf · i...
TRANSCRIPT
�
OPTIMIZATION OF EXOPOLYSACCHARIDE FROM MICROBES AND ITS
KINETICS
A THESIS SUBMITTED TO THE ANNAMALAI UNIVERSITY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY IN CHEMICAL ENGINEERING
Submitted by
A.SIRAJUNNISA
(Reg. Number: 1037050004)
Under the Guidance of
Dr. V. VIJAYAGOPAL M.E.(Chem.), Ph.D.,
P.G. Dip. In Yoga, M.Sc. Yoga
Professor of Chemical Engineering
Department of Chemical Engineering
DEPARTMENT OF CHEMICAL ENGINEERING
FACULTY OF ENGINEERING AND TECHNOLOGY
ANNAMALAI UNIVERSITY
ANNAMALAI NAGAR � 608002
INDIA
November � 2013
�
DECLARATION
Investigations reported in this thesis have been carried out in the Bioprocess
Laboratory, Department of Chemical Engineering, FEAT, Annamalai University and are
original in approach to the best of my knowledge and have not formed part of any thesis
previously submitted for the award of any Degree or Diploma.
Department of Chemical Engineering A.SIRAJUNNISA
Annamalai University
Annamalai Nagar � 608002
Tamilnadu, India
�
ANNAMALAI UNIVERSITY
DEPARTMENT OF CHEMICAL ENGINEERING
FACULTY OF ENGINEERING AND TECHNOLOGY
ANNAMALAI NAGAR 608002. TAMILNADU, INDIA
Dr. V.VIJAYAGOPAL M.E.(Chem.), Ph.D., P.G. Dip. In Yoga, M.Sc. Yoga Professor of Chemical Engineering
Annamalai University
Annamalai Nagar
Date:
This is to certify that the thesis entitled � OPTIMIZATION OF
EXOPOLYSACCHARIDE FROM MICROBES AND ITS KINETICS
submitted to Annamalai University by Ms. A. SIRAJUNNISA for the award of
the degree of DOCTOR OF PHILOSOPHY is a bonafide record of research
work done by her under my supervision. The contents of this thesis have not been
submitted to any other University or Institute for the award of any Degree or
Diploma.
Dr. V. VIJAYAGOPAL
Thesis Guide
Professor
Dept. of Chemical Engineering
Annamalai University
Annamalai Nagar 608002
�
ACKNOWLEDGEMENT
I express my deepest sense of gratitude to my mentor, Dr. V. Vijayagopal,
Professor of Chemical Engineering for his constant support, sustained guidance and
encouragement. I am privileged and honored to work under his supervision.
I sincerely acknowledge Dr. P. Kantha Bhabha, Professor and Head, for the
interest shown upon research and aiding us with essential facilities in the department.
I owe much to Dr. T. Viruthagiri, Professor and Former Head, Department of
Chemical Engineering for being a moral support and assisting me with valuable advices
throughout my course of study.
I heartly thank Dr. S. Velusami, Dean, Faculty of Engineering and Technology
and the University authorities for filliping and letting me flourish proficiently in
academics, access laboratory and providing an august environment to work.
I thank Dr. B. Sivaprakash, Assistant Professor, Department of Chemical
Engineering and Dr. R. Balamurugan, Assistant Professor, Department of Pharmacy, for
their timely and kindest help during the research.
I am indebted to all the Teaching Staff members of this department, who had
often triggered my senses for cerebrating to strive more, thus raising my research to the
pinnacle of success.
My hearty thanks to all the Laboratory Staff for their unceasing cooperation
during my research and catering all requirements whenever needed.
I am grateful to Union Grants Commision, New Delhi for providing the financial
backing for the research.
I highly appreciate and am obliged to all my cheerful labmates and adorable
friends who directly or indirectly had been a constellation of incessant company and bore
my tantrums with all patience during the research.
�
I evince my gratefulness to my dearest parents and siblings for their unbound
love, affection and prayers that steered me to prosperous completion of the study.
Above all, I acknowledge the kindest blessings bestowed upon me by the
Almighty, without which my academics and this Thesis would have not been a possibility
and reality.
A.SIRAJUNNISA
�
TABLE OF CONTENTS
No. TOPIC PAGE
No.
LIST OF TABLES vii
LIST OF FIGURES ix
NOMENCLATURES xiv
ABBREVIATIONS xvi
ABSTRACT xvii
1 INTRODUCTION 1
1.1 Microbial Exopolysaccharides 1
1.2 Biosynthesis of EPS 3
1.3 Production of EPS 4
1.4 Applications 4
1.5 Aim and Scope 5
1.6 Organisation of the thesis 6
2 REVIEW OF LITERATURE 6
2.1 Polymers and Microbial polymers 7
2.2 Microbial polysaccharides 8
2.3 Structure of Cell Wall 9
2.4 Exopolysaccharides 11
2.5 Properties 14
2.5.1 Biofilm formation 14
2.5.2 Adhesion to substratum 14
2.5.3 Rheological properties 15
2.5.4 Composition and Linkages 15
2.6 Biosynthesis of EPS 17
2.7 Isolation/ Extraction Techniques 20
2.8 Quantification and Characterisation of
EPS
22
2.9 Fermentation of EPS 24
�
2.9.1 Batch fermentation 24
2.9.2 Fed batch fermentation 27
2.9.3 Immobilisation 29
2.10 Factors influencing EPS production 33
2.10.1 Effect of carbon sources 33
2.10.2 Effect of nitrogen sources 34
2.10.3 Effect of carbon / nitrogen ratio 35
2.10.4 Effect of industrial agricultural wastes as
carbon sources
36
2.10.5 Effect of vitamins and trace elements 36
2.10.6 Effect of pH, temperature and time 37
2.11 Applications 38
3 MATERIALS AND METHODS 41
3.1 Sample Collection 41
3.2 Isolation of cultures 42
3.3 Morphological identification 43
3.3.1 Gram staining 43
3.3.2 Endospore staining (Schaffer Fulton
method)
43
3.3.3 Negative staining 44
3.3.4 Motility test (hanging drop technique) 45
3.4 Biochemical characterisation (IMViC
tests)
45
3.4.1 Indole production test 45
3.4.2 Methyl red test 46
3.4.3 Voges Proskauer test 46
3.4.4 Citrate Utilisation test 47
3.4.5 Carbohydrate fermentation test 47
3.4.6 Urease test 48
3.4.7 Triple sugar iron test 48
3.4.8 Starch hydrolysis 49
3.4.9 Casein hydrolysis 49
3.4.10 Gelatin hydrolysis 50
3.4.11 Oxidase test 50
3.4.12 Catalase test 51
3.4.13 Nitrate reduction test 51
3.5 Effect of UV radiation on EPS yield 52
3.6 Isolation of bacterial DNA 52
3.7 Molecular identification of the strain 54
3.8 Optimisation studies 54
3.8.1 Pretreatment of agro industrial waste 54
3.8.2 One factor at a time approach 55
3.8.3 Plackett Burman Design 55
3.8.4 Central Composite Design 56
3.8.5 Optimisation of environmental
parameters
57
3.9 Fed batch fermentation of EPS 59
3.10 Immobilisation technique 59
3.11 Isolation of EPS 59
3.12 Solubility of EPS 60
3.13 Determination of total carbohydrate
content
60
3.14 Estimation of total proteins 61
3.15 Fourier Transform Infra Red
Spectroscopic analysis
62
3.16 Particle size distribution 62
3.17 Nanoparticle synthesis 63
3.17.1 UV Visible spectral analysis 63
3.17.2 Fourier Transform Infra Red
Spectroscopic analysis
63
3.17.3 Scanning Electron Microscopy and EDS
studies
63
3.17.4 Evaluation of antioxidant activity- DPPH
scavenging activity
64
3.17.5 Antibacterial activity 64
3.18 Kinetic studies 65
3.18.1 Determination of viscosity and specific
viscosity
65
4 RESULTS AND DISCUSSIONS 67
4.1 Cultures for study 67
4.1.1 Morphological identification 67
4.1.2 Biochemical characterisation 68
4.1.3 Effect of UV radiation on the bacterial
cells for study
70
4.1.4 Molecular identification of the strains 71
4.2 Medium optimisation studies 74
4.2.1 One factor at a time approach 74
4.2.1.1 Effect of synthetic carbon sources on EPS 74
4.2.1.2 Effect of synthetic nitrogen sources on
EPS
76
4.2.1.3 Effect of sodium chloride of EPS 78
4.2.1.4 Effect of minerals on EPS production 80
4.2.1.5 Effect of vitamins and aminoacids on EPS
production
81
4.2.1.6 Effect of cane molasses and rice bran as
carbon substrates on EPS yield
83
4.2.2 Plackett Burman Design 85
4.2.2.1 Variables influencing EPS production
from B.subtilis
85
4.2.2.2 Variables influencing EPS production
from P.fluorescens
89
4.2.3 Central Composite Design 93
� �
4.2.3.1 Optimisation of nutrient concentration for
EPS production from B.subtilis
93
4.2.3.2 Optimisation of nutrient concentration for
EPS production from P.fluorescens
98
4.3 Optimisation of environmental
parameters
102
4.3.1 Preliminary studies 102
4.3.1.1 Influence of temperature and pH on yield
EPS from B.subtilis and P.fluorescens
102
4.3.1.2 Influence of incubation time and
inoculum concentration on yield EPS
from B.subtilis and P.fluorescens
104
4.3.2 Central Composite Design 106
4.3.2.1 Optimisation of parameters for EPS
production from B.subtilis
106
4.3.2.2 Optimisation of parameters for EPS
production from P.fluorescens
110
4.4 Fed batch fermentation 115
4.5 Immobilisation 116
4.6 Characterisation studies 118
4.6.1 Total carbohydrate and protein contents 118
4.6.2 Fourier Transform Infrared Spectroscopy 118
4.6.3 Solubility check 120
4.6.4 Particle size distribution 121
4.7 Applications 121
4.7.1 Antioxidant activity 121
4.7.2 Silver nanoparticle synthesis 122
4.7.2.1 Visual observation 123
4.7.2.2 UV spectroscopy 124
4.7.2.3 Scanning Electron Microscopy 125
4.7.2.4 Energy Dispersive Spectral analysis 126
� �
4.7.2.5 Fourier Transform Infrared Spectroscopy 127
4.7.2.6 Applications 129
4.7.2.6.1 Antibacterial activity 129
4.7.2.6.2 Antioxidant activity 131
5 KINETICS AND MODELING 132
5.1 Substrate Utilisation Kinetics 134
5.2 Growth kinetics 135
5.2.1 Malthus law 135
5.2.2 Contois model 136
5.2.3 Moser Equation 136
5.2.4 Tessier model 136
5.2.5 Verlhurst model 137
5.2.6 Andrew model 137
5.2.7 Monod model 137
5.2.8 Logistic model 138
5.3 Product formation kinetics 138
5.4 Data analysis and modeling 140
5.5 Viscosity check 146
6 CONCLUSIONS 148
Future scope of the study 152
REFERENCES 153
APPENDICES
PUBLICATIONS
� �
LIST OF TABLES
No. TOPIC PAGE
No.
2.1 Various kinds of EPS produced by
microorganisms
12
4.1 Inferences for biochemical tests of the two
isolates used in this study
69
4.2 Plackett Burman design for two levels of 20
variables along with the observed EPS yield by
B.subtilis
86
4.3 Statistical analysis of Plackett Burman Design
for B.subtilis showing coefficient, T and P
values, for each variable
88
4.4 Plackett Burman Design table with coded levels
and response for EPS yield by P.fluorescens
90
4.5 Statistical analysis of Plackett Burman Design
for P.fluorescens showing coefficient, T and P
92
� �
values, for each variable
4.6 Central Composite Design with experimental
and predicted responses of EPS from B.subtilis
93
4.7 Analysis of variance of optimisation of EPS
production by B.subtilis
95
4.8 Central Composite Design with experimental
and predicted responses of EPS from
P.fluorescens
98
4.9 Analysis of variance of optimisation of EPS
production by P.fluorescens
100
4.10 Central composite design matrix with
experimental data showing coded, actual and
response values of EPS from B.subtilis
106
4.11 ANOVA and regression analysis of the
experiment on EPS production from B.subtilis
108
4.12 Central composite design matrix with
experimental data showing coded, actual and
response values of EPS from P.fluorescens
111
4.13 ANOVA and regression analysis of the
experiment on EPS production from
P.fluorescens
112
4.14 Antibacterial and anticandidal effects of EPS
stabilised SNP
130
5.1 Model parameters for EPS production 142
5.2 Experimental and predicted values of cell mass
concentration, substrate utilisation and product
formation of B.subtilis
143
5.3 Experimental and predicted values of cell mass
concentration, substrate utilisation and product
formation of P.fluorescens
145
� �
LIST OF FIGURES
No. TOPIC PAGE
No.
2.1 � � � � � � � � � � � � � � � � � � � � � � � � � � � � 7
2.2 Cell wall structure of Gram positive and
Gram negative bacteria locating
exopolysaccharides (EPS)
9
2.3 Biosynthetic pathways involved in Gram
negative bacterial EPS
18
2.4 Synthesis of EPS in Gram positive bacterium,
Lactococcus lactis
20
2.5 Batch growth curve of microorganisms 25
� �
3.1 Overall process of optimizing EPS production
From Bacillus subtilis
57
3.2 Overall process of optimizing EPS
production from Pseudomonas fluorescens
58
3.3 Calibration plot for total carbohydrate
estimation
61
3.4 Calibration plot for total protein
determination
62
3.5 Simple Ostwald's viscometer 65
4.1 Morphological examination of B.subtilis-
colonies on nutrient agar medium (A), Gram's
staining (B), Capsular staining (C) and
endospore staining (D)
67
4.2 P.fluorescens isolation source (A), colonies
on Pseudomonas F agar (B), culture under
UV lamp (C), Gram's staining (D) and Slimy
EPS (E)
68
4.3 A plot on influence of UV on EPS yield from
B.subtilis
70
4.4 A plot on influence of UV on EPS yield from
P.fluorescens
70
4.5 16S rRNA sequence (A) and phylogenetic
tree for B.subtilis
72
4.6 16S rRNA sequence (A) and phylogenetic
tree for P.fluorescens
73
4.7 Effect of synthetic carbon sources on EPS by
B.subtilis
74
4.8 Effect of synthetic carbon sources on EPS by
P.fluorescens
75
4.9 Effect of synthetic nitrogen sources on EPS
by B.subtilis
77
� �
4.10 Effect of synthetic nitrogen sources on EPS
by P.fluorescens
78
4.11 Influence of NaCl on EPS production 79
4.12 Effect of minerals on EPS production by
B.subtilis
80
4.13 Effect of minerals on EPS production by
P.fluorescens
80
4.14 Effect of vitamins and aminoacids on EPS
production by B.subtilis
82
4.15 Effect of vitamins and aminoacids on EPS
production by P.fluorescens
82
4.16 EPS in different media- nutrient broth (NB),
medium containing cane molasses by
B.subtilis (CM) and medium with rice bran
by P.fluorescens (RB)
83
4.17 Effect of cane molasses and rice bran as
carbon substrates on EPS yield
84
4.18 Pareto chart of the effects of various nutrients
on EPS production by B.subtilis
87
4.19 Pareto chart of the effects of various nutrients
on EPS production by P.fluorescens
91
4.20 Response surface plots of the interactive
effects of variables, cane molasses (X1), yeast
extract (X2), NaCl (X3) and CaCl2 (X4) on
EPS production
97
4.21 Response surface plots of interactive effects
of variables, Ricebran (X1), Peptone (X2),
NaCl (X3) and MnCl2 (X4)
102
4.22 Effect of temperature on EPS production by
B.subtilis
103
4.23 Effect of temperature on EPS production by 103
� �
P.fluorescens
4.24 Effect of incubation time on EPS production 105
4.25 Effect of inoculum concentration on EPS
production
105
4.26 Response and contour plots of the interactive
effects of four significant variables for EPS
production from B.subtilis
110
4.27 Response and contour plots of the interactive
effects of four significant variables for EPS
production from P.fluorescens
114
4.28 Biomass and EPS yield from cultures used in
study (biomass of B.subtilis, biomass of
P.fluorescens, EPS from B.subtilis and EPS
from P.fluorescens)
115
4.29 Alginate beads entrapping microbial cells 116
4.30 Dry cell mass and EPS yield by encapsulated
B.subtilis
117
4.31 Dry cell mass and EPS yield by encapsulated
P.fluorescens
117
4.32 FTIR spectrum of extracted EPS from
B.subtilis
119
4.33 FTIR spectrum of extracted EPS from
P.fluorescens
120
4.34 DPPH scavenging efficiency of EPS 122
4.35 SNP synthesis observed by change in color
from pale yellow to dark brown coloration
(A) P.fluorescens EPS at 120h and (B)
B.subtilis EPS at 72h
123
4.36 Absorbances of P.fluorescens EPS reduced
SNP at various time intervals
124
4.37 Absorbances of P.fluorescens EPS reduced 125
�
SNP at various time intervals
4.38 SEM images of (A) B.subtilis EPS reduced
SNP and (B) P.fluorescens EPS reduced SNP
126
4.39 EDS plots of (A) B.subtilis EPS reduced SNP
and (B) P.fluorescens EPS reduced SNP
127
4.40 FTIR spectrum of P.fluorescens EPS reduced
SNP
128
4.41 FTIR spectrum of B.subtilis EPS reduced
SNP
129
4.42 DPPH scavenging efficiency of EPS reduced
SNP
130
5.1 Certain important parameters, phenomena
and interactions which determine cell
population kinetics
133
5.2 Different perspectives for cell population
kinetic representation
134
5.3 Comparative chart showing experimental and
predicted values of cell concentration of
B.subtilis
142
5.4 Comparative chart showing experimental and
predicted values of substrate consumption of
B.subtilis
142
5.5 Comparative chart showing experimental and
predicted values of product concentration of
B.subtilis
143
5.6 Comparative chart showing experimental and
predicted values of cell concentration of
P.fluorescens
144
5.7 Comparative chart showing experimental and
predicted values of cell substrate
consumption of P.fluorescens
144
�
NOMENCLATURE
5.8 Comparative chart showing experimental and
predicted values of product concentration of
P.fluorescens
145
5.9 Overall experimental and predicted values of
Logistic and Luedeking Piret models for
Bacillus subtilis and Pseudomonas
fluorescens
146
5.10 Relative viscosity of the two culture filtrate
with EPS
147
� �
µm Micrometer
kDa Kilo Dalton
M Molar
ml Millilitre
p.a. Per annum
nm Nanometer
U/ml Unit per millilitre
h Hour
gl-1
Gram per liter
mg l-1
h-1
Milligram per liter per hour
ºC Degree Celsuis
min Minute
sec Second
w/v Weight by volume
lbs/in2
Pounds per inch square
µg/ml Microgram per millilitre
mM Milli Molar
rpm Rotation per minute
µl Microliter
L Liter
N Normality
cm-1
Per centimetre
kV Kilo volt
g gram
keV Kilo electro volt
h-1
Per hour � Viscocity
x0 Initial cell concentration
x � � � � � � � � � � � � � � � � � � � � � � � � � � � � ! � " -1)
� �
xs Cell concentration at stationary phase (gL-1
)
s0 Initial substrate concentration (gL-1
)
P(t) Product � � � � � � � � � � � � � � � � � � � � � � � � ! � " -1)
P(0) Initial Product concentration (gL-1
)
s � � # � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ! � " -1)
Yp/s Yield coefficient for product with respect to substrate
consumed
Yp/x Yield coefficient for product with respect to cell mass
formed
Yx/s Yield coefficient for cell mass with respect to substrate
consumed
k Logistic constant $ % & Luedeking- Piret model constants
µ specific growth rate (time-1
)
µmax maximum specific growth rate (time-1
)
Ks saturation constant
Kis substrate inhibitory constant
� �
ABBREVIATIONS
EPS Exopolysaccharides
CCD Central Composite Design
FTIR Fourier Transformer Infrared
NMR Nuclear Magnetic Resonance
PHA Poly Hydroxy Alkonates
CPS Capsular Polysaccharides
LPS Lipopolysaccharides
HPLC High Performance Liquid Chromatography
TLC Thin Layer Chromatography
GCMS Gas Chromatography-Mass Spectroscopy
HPAEC-PAD High Pressure Anion Exchange Chromatography with
Pulse Amperometric Detection
SSF Solid State Fermentation
SmF Submerged Fermentation
PGA Poly-Glutamic Acid
EDTA Ethylene Diamine Tetra Acetic Acid
SDS Sodium Dodecyl Sulfate
SNP Silver Nano Particles
KBR Potassium Bromate
BLAST Basic Local Alignment Sequence Tool
rRNA ribosomal Ribonucleic Acid
ANOVA Analysis of Variance
� �
DPPH Diphenyl Picryl Hydrazyl
EDS Energy Dispersive Spectroscopy
MATLAB Matrix Laboratory
ABSTRACT
The global polymer research has plunged into the exploration of replacements for
chemically synthesized polymers. Synthetic polymers pose a malicious impact on the
ecosystem, as they are non-degradable, toxic to humans and difficult to dispose, causing a
serious environmental pollution. Therefore, in the recent scenario, the polymer researches
are oriented much towards the discovery and tailoring of new biological polymers.
Biopolymers are generally long chained / cross-linked polysaccharides, naturally
existing as metabolites and structural elements in plants, animals, crustaceans and in
microbial sources like bacteria, fungi and algae. These are considered to be a non-
pollutant, possess efficient biodegrading ability and harmless to humankind.
Exopolysaccharide (EPS) is a biopolymer of differing molecular weight with unique
characteristics and properties, found as hardbound capsular / loosely attached slimy
exopolysaccharide on cell walls of microbes, mainly composed of polysaccharides along
with proteins, uronic acid and nucleic acids. EPS is being used in various industrial and
pharmaceutical purposes, but there is yet a difficulty in commercializing the biopolymers,
due to the slight quantity being produced and downstreaming processes.
The present thesis emphasised on the optimisation of media for higher production
of exopolysaccharide (EPS) from the cultures of study, Bacillus subtilis and
Pseudomonas fluorescens and on the kinetics based on their mechanisms of EPS
production in a batch mode of fermentation.
The bacterial cultures used in the study were isolated from different soil samples
by routine microbiological techniques like morphological identification by staining,
biochemical characterization and through 16S rRNA gene sequencing. The mucoid
cultures were confirmed to be B.subtilis and P.fluorescens. B.subtilis is a Gram positive
� �
bacterium producing capsular EPS whilst P.fluorescens is a Gram negative bacterium
producing slimy EPS. The fermentation factors affecting the EPS production were
studied to ascertain the nutrient requirements and environmental conditions for
maximizing the yield of EPS.
Medium optimization was carried out hierarchically for both the organisms. The
preliminary screening was one factor at a time approach, which dealt with the effects of
various nutrient sources like carbon, nitrogen, NaCl, minerals, vitamins and aminoacids.
For a cost effective and higher production of EPS, agricultural wastes had been used.
Cane molasses for B.subtilis and rice bran for P.fluorescens, as carbon substrates, had
resulted in maximum yield of EPS.
After the primary study, Response Surface Methodology (RSM) was employed to
statistically optimize the medium components. Firstly, an RSM tool, Plackett- Burman
design was implemented for selecting specifically from the screened nutrients through
one factor at a time method. Secondly, to analyse the concentration of the selected
components through Plackett Burman design, Central Composite design of RSM had
been utilized. Response curves indicated the interactions between the four nutritive
components on the EPS yield. The optimized medium components for an efficient
generation of EPS from B.subtilis were, cane molasses ' 2.36 %, yeast extract ' 0.56 %,
NaCl ' 0.71 % and CaCl2 ' 0.05 %, generating a maximum of 4.98 gL-1
. For producing
EPS from P.fluorescens, the optimized medium was composed of rice bran ' 5.02 %,
peptone ' 0.35 %, NaCl ' 0.51 % and MnCl2 ' 0.074 %, producing a yield of 4.65 gL-1
.
To optimize the environmental parameters, the preliminary screening method of
one factor at a time procedure was performed following which Central Composite design
was experimented. Environmental conditions for fermentations namely pH, temperature,
inoculum concentration and incubation time were considered. Through one factor at a
time, the conditions were defined and statistically optimized. The optimised
environmental parameters for EPS production by B.subtilis were, temperature 35 0C, pH
7, incubation time ' 72 h and inoculum concentration ' 2 %, producing a maximum of
5.59 gL-1
EPS. The fermentation conditions optimized for production of EPS from
� �
P.fluorescens were, temperature 28 0C, pH 7, incubation time- 48h and inoculum
concentration ' 2 %, yielding 5.29 gL-1
.
An attempt was also made for generating EPS from the cultures used in the study
by fed batch mode of fermentation in shake flasks in still conditions. The study resulted
in yields lesser than the quantity obtained from batch fermentation. Maxima of 2.769 gL-1
of EPS by B.subtilis and 1.167 gL-1
by P.fluorescens were obtained by fed batch
fermentation. Immobilisation technique was also performed to learn its effect on the EPS
production from both cultures. It was observed that this procedure deteriorated the
growth and subsequently the production of EPS. The maximum production of EPS by
B.subtilis was found to be 1.521 gL-1
. In the case of immobilized P.fluorescens, highest
amount of EPS produced was 2.498 gL-1
. From these experiments, it was inferred that
batch mode of cell cultivation and production of EPS apparently be the best fermentation
technique to enhance the yield of biopolymer using the agrowastes under still conditions.
After isolation EPS from the cultures by ethanol precipitation method, the
samples were lyophilized and characterized. The total carbohydrate and protein content
were analysed and the results revealed that the biopolymers obtained from the
microorganisms were majorly polysaccharides with 81.81 % and 84.21 % of
carbohydrates in EPS from B.subtilis and P.fluorescens. Fourier Transform Spectral
analysis of EPS revealed the presence of essential functional groups like hydroxyl,
carboxylate, methoxyl groups. Viscosity of culture broth containing EPS was analyzed,
which increased with the increase in time, after 72 h and 48 h for B.subtilis and
P.fluorescens, the viscosity remained constant. The relative viscosity of the culture broth
indicated that the EPS solutions exhibited a Non Newtonian behavior. Particle size of
EPS from B. subtilis and P.fluorescens ranged between 10-100 µm, which indicated that
the particles could be biodegradable and used in various biological purposes. EPS
samples were checked for their solubility in various solvents and acids. It was observed
that EPS from B.subtilis dissolve only in hot concentrated sulfuric acid when
continuously stirred while EPS from P.fluorescens readily dissolved in hot concentrated
sulfuric acid.
� �
EPS from the bacterial sources was employed in different activities to learn their
applications. Antioxidant activity was performed for EPS from both cultures. As the
concentration increased the reducing capacity also elevated. The maximum antioxidant
activity of EPS, was at the concentration of 1mg/ml for both systems. EPS from
P.fluorescens exhibited antioxidant activity with a maximum percentage inhibition of
39.98 %, which was comparable with that of reference (27.81 %). EPS from B.subtilis
showed an inhibition activity of 61.19 % which was the highest. The study showed that
the isolated could be an effective antioxidant better than the standard antioxidant. Silver
nanoparticles were synthesized using EPS as the stabilizer and reducing agent. The
nanosilver produced was visually observed and analysed by various techniques like UV
Visible spectroscopy, FTIR spectroscopy, SEM and EDS. The generated nanoparticles
exhibited themselves as potent antibacterial agents and antioxidants.
The kinetic study was performed out to learn the phenomena of substrate
utilization, cellular growth and product formation. EPS production was directly
proportional to the growth of organisms and was found maximum in the exponential
phase. The growth experiments were simulated using MATLAB software and substrate
consumption and product formation were simulated graphically using MS Excel. The
predicted values were in consistent with experimental data. The study confirmed that the
logistic equation for growth kinetics and Luedeking Piret model for substrate utilisation
and product formation model beseemed the experiments.
In conclusion, the capsular EPS from B.subtilis and slimy EPS from
P.fluorescens, though showed disparity in their medium composition and environmental
necessities, and their features exhibited their differences, they produced comparatively
high amount of EPS utilizing respective agrowastes, one of the main perspectives of the
research. The extracted EPS from B.subtilis and P.fluorescens could be potent
alternatives for synthetic polymers and effective pharmaceutical products.