Studies on Pigment Production by Microorganisms Using Raw

Materials of Agro-industrial Origin

Thesis Submitted by
TARANGINI KORUMILLI (509CH107)

In partial fulfillment for the award of the Degree of

DOCTOR OF PHILOSOPHY

IN

CHEMICAL ENGINEERING

Under the guidance of

Dr. Mrs. SUSMITA MISHRA

Department of Chemical Engineering

NATIONAL INSTITUTE OF TECHNOLOGY
Rourkela 769 008, Orissa, India
June-2014
Dr. (Mrs.) Susmita Mishra
Associate Professor
Department of Chemical Engineering
E-mail: smishra1234@gmail.com, smishra@nitrkl.ac.in

CERTIFICATE

This is to certify that the thesis entitled “Studies on Pigment Production by Microorganisms
Using Raw Materials of Agro-industrial Origin” submitted by Tarangini Korumilli (Roll No-
509CH107) to National Institute of Technology, Rourkela in partial fulfillment of the
requirements for the completion of the Ph. D. degree in Chemical Engineering, is an authentic
work carried out by her under my supervision and guidance.

Dr. (Mrs.) Susmita Mishra

i
 Acknowledgements
I express my heartfelt gratitude to Prof. (Mrs.) Susmita Mishra, my supervisor, Department of
Chemical Engineering, NIT, Rourkela for her constant encouragement, invaluable advice and
guidance throughout the course of my research work. I shall remain ever grateful for her care,
concern and sincere interest in my prosperity. I must mention that without her timely help in
writing and correction, this thesis could not have been submitted in time.

I am thankful to Prof. R. K. Singh, HOD, Department of Chemical Engineering NIT, Rourkela

for his help and support during the course of my tenure. I owe a depth of gratitude to Prof. G. K.
Roy, Prof. K. C. Biswal, Prof. S. K. Agarwal and Prof. P. Rath, Chemical Engineering
Department, NIT Rourkela, for their support and encouragement.

A special thanks to Prof. (Mrs.) Madhushree Kundu, Prof. Santanu Paria, Prof. Basudeb munshi
for their for their constant support throughout my programme. I am also thankful to Prof. (Mrs.)
Abanti Sahoo, Prof. Hara Mohan Jena, Prof. Sujit Sen and Prof. Arvind kumar, Department of
Chemical Engineering for their valuable advices and moral support.

I would like to thank all the faculty members and non-teaching staff of Chemical Engineering
department for their constant support during my course work. I am thankful to Prof.(Mrs). A.
Mandal and Prof.(Ms.) Usharani Subuddhi, Department of Chemistry for their analytical support.

My heartfelt thanks to my labmates Ramkrishna, Balaji Patro, Adya das,Vijiya Jha, Shilpi for
their joyous company and for helping me in several ways. Special thank to my friend Sateesh
Sagiri and thanks to all my friends of the department for their company and support.

And it goes without saying, that I am indebted to my parents, my husband and brothers, whose
patience, support and endurance made completion of my course a reality.

ii
Abstract
The recent awareness in human safety and environmental conservation has created fresh
enthusiasm for natural sources of pigments. Compared to synthetic pigments, microbial pigments
shows better biodegradability and higher compatibility with the environment, and have numerous
applications from food to cosmetics. Identification of new microbial sources, utilization of low
cost substrates and optimization of process parameters are the areas under focus towards
economical pigment production. The present study aimed at screening and identification of
microbial isolates from soil and water, which are having pigment producing ability. Efforts have
been made to cultivate them on numerous cheaper and inexpensive substrates with no special
conditions and supplements for effective pigment production. Furthermore, two carotenoid
producing strains were also exploited on numerous inexpensive substrates at ambient conditions
for pigment metabolites. While analyzing pigment metabolites in all cases, key parameters
influencing pigment production by respective strains were optimized by utilizing statistical
techniques like Taguchi method and response surface approaches wherever needed. The
conditions for enhanced pigment production were established employing microbial isolates and
purchase strains individually. The melanin producing Pseudomonas guinea, (bacterial strain) was
isolated from marine water sample and was employed on vegetable waste for effective pigment
production. Another strain of Bacillus safensis was isolated from garden soil and showed its
ability to produce melanin on fruit waste extract (FWE). It is noteworthy that both melanins
produced from marine and soil isolates showed antioxidant, photoprotective and metal ion
chelation activities. Addressing garden soil, a new carotenoid producing bacterial strain Bacillus
clausii was screened and cultivated on FWE for high yield pigment production. The pigment
produced by this strain was observed to be a β-carotenoid type and its stability towards thermal
treatment was also evaluated. Eying on the significance of carotenoids, microorganisms
(Rhodotorula rubra, Xanthophyllomyces dendrorhous) in their developmental stage were
purchased and studied for pigment production on various residues as sole substrates. The
obtained yeasts showed improved carotenoids yield i.e. torularhodin and astaxanthin respectively
on FWE. In a nut shell we could conclude that there is a huge scope for industrial scale
production of Melanin and Carotinoid using easily available agro-industrial raw materials such
as rice powder and fruit waste extract (FWE).

iii
 CONTENTS

Particulars Page no.

Certificate i
Acknowledgements ii
Abstract iii
List of Figures x
List of Tables xxiii

Chapter 1 Introduction 1
1.1 Motivation and scope 5
1.2 Organization of thesis 7
References 9

Chapter 2 Literature Review 13

2.1 Outline 13
2.2 History of colorants 14
2.3 Dyes and pigments – their implications 15
2.4 Biological pigments- different classes 17
2.4.1 Biological pigments 17
2.4.2 Plant pigments 18
2.4.3 Animal pigments 19
2.4.4 Microbial pigments 20
2.5 Significance of microbial pigments as natural colorants 23
2.6 Classification of microbial pigments 25
2.7 Microbial pigments of commercial importance 29
2.8 Benefits and Applications of Microbial pigments 33
2.9 Novel practices of microbial pigment production 35
2.9.1 Strain improvement 35
2.9.2 Fermentation 36
2.9.3 Low-cost substrates 38

iv
 2.10 Objectives of the work 39
References 40

Chapter 3 Screening of inexpensive substrates for pigments production by 55

isolated and known microbial strains - Bridge to Part I and II
of Chapter 4
3.1 Melanins and carotenoids - pigments of marketable importance 55
3.2 Sampling 58
3.3 Screening of pigment producing microorganisms 59
3.4 Different substrates and Cultivation of isolates/purchased strains 59
3.5 Strain/substrate selection and further experimentation 60
3.6 Summary 63
References 63

Chapter 4 Approach and investigations

Part I - Isolation of Pigment Producing Bacteria and the Production of 66

Pigments on Cheaper Substrates

4.1 Melanin from Isolated Marine Pseudomonas sp. using Vegetable 66

waste
4.1.1 Introduction 67
4.1.2 Materials and methods 69
4.1.2.1 Chemicals used 69
4.1.2.2 Screening and isolation of the melanin producing strain 69
4.1.2.3 Pigment production, extraction and purification 69
4.1.2.4 DPPH/SPF/metal chelating assay 70
4.1.2.5 Analytical methods 74
4.1.3 Results and Discussion 74
4.1.3.1 Strain selection and characterization of the microorganism 74
4.1.3.2 Pigment production and characteristics of the produced 78
melanin

v
 4.1.3.3 Spectroscopy, SEM/EDX and IR analysis of melanin 81
4.1.3.4 DPPH assay 85
4.1.3.5 XRD analysis 86
4.1.3.6 SPF values determination 88
4.1.3.7 The metal binding capacity of melanin 89
4.1.4 Conclusions 90
References 90

4.2 Melanin by Soil Microbial Isolate on Fruit Waste Extract: - 93

Optimization of Key Production Parameters
4.2.1 Introduction 93
4.2.2 Materials and methods 95
4.2.2.1 Chemicals and microorganism 95
4.2.2.2 Substrate preparation 96
4.2.2.3 Production and purification of melanin 97
4.2.2.4 Parameters optimization using Taguchi and CCD design 97
4.2.2.5 SPF/DPPH/metal chelating assay 98
4.2.2.6 Analytical methods 101
4.2.3 Results and discussion 101
4.2.3.1 Strain selection and Pigment production 101
4.2.3.2 Taguchi design for screening critical factors 102
4.2.3.3 Optimization by response surface methodology 105
(CCD design)
4.2.3.4 Interaction effects of variables and validation of the 106
model
4.2.3.5 Spectroscopy, IR and XRD analysis of melanin 108
4.2.3.6 SPF values determination 111
4.2.3.7 DPPH assay 112
4.2.3.8 The metal binding capacity of melanin 113
4.2.3 Conclusions 114
References 114

vi
 4.3 Carotenoid by Bacillus clausi Using Rice Powder as the Sole 117
Substrate: Pigment Analyses and Optimization of Key -
Production Parameters
4.3.1 Introduction 117
4.3.2 Materials and methods 119
4.3.2.1 Sampling, microscopy and strain characterization 119
4.3.2.2 Pigment production 119
4.3.2.3 Purification and stability of the pigment 120
4.3.2.4 Optimization of key parameters 121
4.3.3 Results and discussion 121
4.3.3.1 Colony Screening, pigment production 121
4.3.3.2 Microscopy and microbial characterization 123
4.3.3.3 UV-Visible/fluorescence spectrophotometry and stability 125
of the pigment
4.3.3.4 FTIR analysis 127
4.3.3.5 Optimization of key production parameters 129
4.3.4 Conclusions 132
References 132

Part II - Facile Synthesis of Commercial Pigments by Purchased Strains on 138

Cheaper Substrates

4.4 Carotenoid by Rhodotorula sp. on Fruit Waste Extract as a Sole 138

Carbon Source and Optimization of key production parameters
4.4.1 Introduction 138
4.4.2 Experimental section 141
4.4.2.1 Reagents and chemicals 141
4.4.2.2 Microorganism and its maintenance 141
4.4.2.3 Substrate preparation 141
4.4.2.4 Pigment production and extraction 142

vii
 4.4.2.5 DPPH assay 143
4.4.2.6 Analysis 143
4.4.2.7 Experimental design 143
4.4.3 Results and discussion 145
4.4.3.1 Effect of pH on biomass and carotenoid yield 145
4.4.3.2 Biomass growth, pigment yield and glucose utilization 146
4.4.3.3 Box–Behnken design 149
4.4.3.4 DPPH radical scavenging activity 155
4.4.4 Conclusions 156
References 157

4.5 Astaxanthin by Xanthophyllomyces dendrorhous Using Fruit Waste 161

Extract as Sole Source of Energy: Optimization of Culture -
Conditions by Taguchi method for Improved Pigment Production
4.5.1 Introduction 161
4.5.2 Materials and methods 163
4.5.2.1 Chemicals and microorganism 163
4.5.2.2 Substrate preparation 163
4.5.2.3 Culture conditions 163
4.5.2.4 Pigment production and extraction/ Sugar determination 164
4.5.2.5 Taguchi design and statistical analysis 164
4.5.2.6 Determination of free radical scavenging activity 165
4.5.2.7 Analytical methods 166
4.5.3 Results and discussion 167
4.5.3.1 Experimental design by Taguchi method 168
4.5.3.2 Validation of the model 168
4.5.3.3 Pigment yield, biomass trend and glucose utilization 170
4.5.3.4 UV analysis 172
4.5.3.5 FTIR analysis 172
4.5.3.6 DPPH radical scavenging activity 172
4.5.4 Conclusions 173

viii
 References 174

Chapter 5 Conclusions and future prospectives 178

Publications from this work 183
Curriculum vitae 184

ix
List of Figures
Figure No. Title Page No.
Fig. 2.1. Some food grade pigments and their structures from microorganisms. 32
Fig. 4.1.1. Screening of microbial strains obtained from various parts of a 75
seashore. A) near stones, B) near shore and C) 10 m away from sea.
Fig. 4.1.2. Low (a) and high (b) magnification SEM images of the isolated 76
microorganism with black colonies on agar plates.
Fig. 4.1.3. Melanin production on various media: marine broth, blend (marine 80
broth and vegetable waste - (10:90; 20:80 and 30:70 in v/v %), and
diluted marine broth 30 % (a). M indicates melanin in (a). Dark
brownish black colonies due to melanin production in sole marine
broth (b) and in marine broth – vegetable waste blend medium (c).
Fig. 4.1.4. Acid treated (a) and purified melanin (b) after centrifugation. 81
Fig. 4.1.5. UV-visible spectral properties of melanin pigment obtained from 83
marine broth (a) and marine broth –vegetable waste medium (b).
Fig. 4.1.6. SEM images of purified bacterial melanin from a) marine broth and b) 83
marine broth – vegetable waste medium.
Fig. 4.1.7. EDX analysis of elemental composition of melanin from a) marine 84
broth and b) marine broth – vegetable waste medium.
Fig. 4.1.8. The FTIR spectrum of the melanin pigment obtained from a) marine 84
broth and b) marine broth – vegetable waste medium.
Fig. 4.1.9. DPPH radical scavenging activity of synthesized melanin pigment with 86
various doses (a) and UV-vis absorption spectrum of melanin (44.7
µg/mL) - DPPH at different days along with control containing no
melanin (b). Dose dependent scavenging activity of the synthesized
melanin (c). Insert of (a) shows the melanin doses 0, 14.9, 29.8 and
44.7 µg/mL to 0.1 mM DPPH from left to right.

x
Fig. 4.1.10. (a) X-ray diffractograms of the produced melanin (M-marine broth, 87
M-blend) and purchased melanin (M-synthetic). (b) Interlayer spacing
(d-value) and crystallite sizes and (c) % crystallinity of the different
mealnins.
Fig. 4.1.11. Metal ion chelation effect of the melanin (produced from vegetable 89
waste) (a) monitored via complete spectrum and (b). at a fixed
absorption maximum.
Fig. 4.2.1. Steps involved in substrate preparation along with the prepared FWE 96
as figure insert.
Fig. 4.2.2. (a) FWE before (left) and after melanin production (right) by the 102
garden soil microbial isolate, (b) colonies with diffusible melanin on
NA plates, and (c) SEM image of the microorganism. (d) Phylogenetic
tree showing the position of the isolate ZJHD1-43 with reference to
related strains.
Fig. 4.2.3. Main effects of factors or average of obtained results (mg/mL) in 104
which each factor is at a given level. For description of ‘levels’ refer to
Table 4.2.1.
Fig. 4.2.4. Three dimensional response surface curves with surface plot (a) and 108
contour plot (b) showing the effect of interactions of pH and
temperature on melanin yield. (c) melanin yield at optimum conditions
with respect to time.
Fig. 4.2.5. (a) UV-visible spectrum of melanin pigment obtained from FWE. (b) 111
FTIR spectra of standard melanin (upper) and bacterial melanin
(lower). (c) X-ray diffractograms of the obtained melanin (upper) and
purchased melanin (lower) and % crystallinity of both are also shown
as indicated by arrow.
Fig. 4.2.6. (a) Dose dependent scavenging activity of the synthesized melanin 113
from FWE and ascorbic acid as a control. (b) Metal ion chelation effect
of the produced melanin in different doses (monitored spectrally) and
(c) At a fixed absorption maximum.
Fig. 4.3.1. Microbial isolate with orange – red spotted colonies on nutrient agar 122

xi
 slant (a), and orange pigment by the isolated soil microorganism on
rice powder (b). Phase separation of the obtained pigment was shown
in (c). Resultant separated pigment concentrate in liquid (d insert) and
in solid form after vacuum drying was also given in (d).
Fig. 4.3.2. Orange pigments by the isolated soil microorganism on rice powder 123
plates at pH - 6 (a) and 8 (b) respectively.

Fig 4.3.3. Pigment producing bacterial species with an orange pigment (insert) 124
magnified by an optical microscope at 400 X magnification (a). SEM
images of the identified pigment producing bacteria with (b) 5000 X,
(c) 9000 X magnifications and (d) Phylogenetic tree showing the
position of the isolate XJU-3 with reference to related strains.
Fig. 4.3.4. (a) UV- visible spectrum of the extracted orange pigment in ethanol, 126
(b) UV absorption of the oxidized pigment with narrow and wide
(insert of (b)) spectral regions, and (c) Fluorescence emission spectra
of the oxidized pigment at 382 nm.
Fig. 4.3.5. ATR-FTIR analysis of the extracted and purified orange pigment 128
before (a) and after (b) oxidation process.
Fig. 4.3.6. Main effects of factors or average of obtained results (pigment per 3 130
gm rice powder) in which each factor is at a given level. For detail
about ‘levels’ refer to Table 4.3.1.

Fig. 4.4.1. (a) Growth of biomass and carotenoid yield at various pHs and at 146
0
room temperature (30 C). Prepared fruit waste extract (FWE) – left,
and FWE with R. rubra growth with intracellular carotenoid (right)
upon incubation (b). CHNS analysis of FWE before and after
fermentation; upon separation of R. rubra by centrifugation (c). SEM
image of the used yeast.
Fig. 4.4.2. Carotenoid production (mg/l) and biomass yield (mg/ml) along with 148
glucose utilization (mg/ml) given in [A]. The picture insert shows the
colored biomass from 1 to 6 days. UV-visible spectrum of the purified
carotenoid in methanol medium [B]. FTIR spectra of the synthesized

xii
 carotenoid (a) and the purchased β-carotene (b) pigment.
Fig. 4.4.3. (a) Cell mass concentration calculated from regression model equation 154
versus the corresponding experimentally obtained values. (b)
Carotenoid production calculated from the regression model equation
versus the corresponding experimentally obtained values.
Fig. 4.4.4. Response surface and contour plots obtained from Equation (2) and 155
(3) showing the effect of the temperature, agitation and their mutual
interaction on biomass concentration (a), (b); while it is effect of pH
and temperature and their mutual interaction on caroteniod pigment
concentration (c), (d). The displayed units of all the graphs are in
natural units.

Fig. 4.4.5. Free radical scavenging property of the carotenoid by R. rubra. Dose 156
dependent scavenging activity of the pigment compared with ascorbic
acid (control).

Fig. 4.5.1. Pigmentation of FWE (a) by before X. dendrorhous yeast with lower 167
(b) and higher (c) magnification SEM images.
Fig. 4.5.2. Main effects of factors or average of obtained results as mg/g biomass 169
in which each factor is at a given level. For description of ‘levels’ refer
to Table 4.5.1.
Fig. 4.5.3. (a) Time course of the growth and production of astaxanathin by X. 171
dendrorhous in FWE. Experiment was carried out at optimum
conditions i.e. pH (5), temperature (20 oC) and agitation (300 rpm). (b)
UV-Visible and FTIR spectrum (c) of the produced pigment in
methanol. (d) Antioxidant activity (DPPH radical scavenging) of
astaxanthin and ascorbic acid.

xiii
List of Tables
Table No. Title Page No
Table 2.1. Pigment producing microorganisms based on color and 27
appearance.
Table 2.2. Pigments from various microorganisms which are already in use as 30
natural food colorants.
Table 2.3. Different microorganisms and various inexpensive substrates used 39
for pigments production.
Table 4.1.1. Colony characteristics of the isolated melanin producing 76
bacterium.
Table 4.1.2. Normalized product function used in the calculation of SPF by 88
Mansur equation.
Table 4.1.3. SPF values of different melanins. 88
Table 4.2.1. Factors and their levels which were studied by Taguchi approach. 100
Table 4.2.2. Levels of three different factors, applied in each of 18 trials with 100
the obtained results.
Table 4.2.3. Analysis of variance of main effects of factors. 103
Table 4.2.4. Optimum conditions suggested by statistical calculations after 105
performing the tests
Table 4.2.5. Experimental design matrix for the central composite design. 106
Table 4.2.6. Estimated regression coefficients from the model equation. 107
Table 4.3.1. Factors and their levels studied by Taguchi method. 130
Table 4.3.2. Levels of three different factors in each of sixteen trails and 131
obtained results.
Table 4.3.3. Analysis of variance of main effects of factors. SS/MS indicate 131
sum/mean of squares in the table.
Table 4.3.4. Optimum conditions suggested by statistical analysis after 132
performing the tests.
Table 4.4.1. Coded and actual levels of the three variables. 144
Table 4.4.2. Experimental design matrix for the Box-Behnken design. 144

xiv
Table 4.4.3. Statistical significance obtained for the regression coefficients in 151
Eq’s .(1), (2).
Table 4.4.4. ANOVA results for biomass and pigment yields. 151
Table 4.5.1. Factors and their levels studied by Taguchi method. 166
Table 4.5.2. Levels of three different factors applied in each of nine trials, with 166
observed results.
Table 4.5.3. Analysis of variance of main effects of factors. 169
Table 4.5.4. Optimum conditions suggested by statistical calculations after 170
performing the experiments.

xv
Chapter 1 Introduction
Pigments are the chemical substances that absorb the light of visible region. The produced

color is because of the chromophore, a molecule specific structure which captures the sun

energy and causes an excitation of electron from external orbital to higher orbital, where the

non-absorbed energy is refracted or reflected to be captured by eye [1]. The modern meaning

related to the word pigment has its origin in the twentieth century, meaning a substance

constituted of small particles which is practically insoluble in the applied medium and is used

due to its colorant, protective or other properties. Pigments are compounds with uniqueness

of importance to many industries. In the food industry they are used as additives,

antioxidants, color intensifiers, etc. Pigments come in a wide selection of colors, some of

which are water-soluble. The terms pigment and color are generally applied for the food

coloring matters, sometimes indistinctly [2-3]. Until the mid-19th century all colorants were

attained from plant or animal extracts. The textile industry used natural pigments, such as

cochineal, wood madder, turmeric, or henna. In 1856, H. Perkin established the first factory

of organic synthetic colors to produce mauve. A few years later the discovery of diazotization

and a coupling reaction by Peter Griess was the next major step forward for development of

the color industry. In the 19th century, synthetic organic dyes were developed, creating a more

1
economical and broader range of colorants. Since then their quality has been enhanced due to

extensive research and development [4-5]. The economic consequence of the color industry is

clearly reflected in the large number of synthesized compounds; as many as 700 colorants are

currently available. They have widely been used in foodstuff, dyestuff, cosmetic and

pharmaceutical manufacturing processes; encompass various hazardous effects [6]. All

synthetic food components suffered severe criticism, including synthetic additives and

predominantly food pigments. Today, all food color additives are cautiously regulated by

federal authorities to ensure that foods are safe to eat and accurately labeled [3-5]. Pigments

produced from natural sources are of worldwide interest and is gaining significance. These

are looked upon for their safe use as a natural food dye in substitute of synthetic ones in spite

of having of undesirable market [4]. It is therefore, essential to explore various natural

sources of food grade pigments and their potentials [5]. The utilization of natural pigments in

foodstuff, dyestuff, cosmetic and pharmaceutical manufacturing processes has been mounting

in recent years [7-8-9]. Natural colorants or dyes derived from flora and fauna are believed to

be secure because of non-toxic, non-carcinogenic and biodegradable in nature. Natural

pigments are attained from ores, insects, plants and microbes [10-11].

Among all, microbial pigments are dominant sources. The microbial production of

carotenoids, pigments from vegetables or chemical synthesis, have problems of seasonal and

geographic variability in the production and marketing [21].The microbial production of

carotenoids, They are of great interest owing to the stability of the pigments produced and the

accessibility of cultivation technology [2-3, 7-8, 12-13]. The advantages of pigment

production from microorganisms comprise easy and fast growth in the cheap culture medium,

independence from weather conditions and colors of different shades. The economic

2
advantages of microbial pigments include growth on natural substrates such as red rice wine,

red bean curd as carbohydrate source [21-22]. Microbial colorants are in use in the fish

industry already, for example to improve the pink color of farmed salmon [12-15] In nature,

color rich and pigment producing microorganisms (fungi, yeasts, and bacteria) are fairly

common. Microorganisms produce various pigments like carotenoids, melanins, quinones,

flavins, prodigiosins and more specifically monascins, violacein or indigo [7-8, 16-17].

Carotenoids such as β-carotene and xanthophylls like astaxanthin play central roles in

the metabolism of the eye's macula and retina and in retaining healthy vision. β-carotene play

a constructive role in the prevention of cancer and as chemo-protectives [7] [18-19]. In

addition, it, also act as neutraceutical that avert carcinogenesis through anti-oxidative, anti-

free radical or other mechanisms [20].

The production of microbial pigments is very much affected by the temperature of

incubation, depending upon the type of microorganism [23-24]. The growth of Monascus sp.

entails 25-28 °C for the production of pigment, whereas Pseudomonas requires 35-36 °C for

its growth and pigment production [25]. pH of the medium is another parameter that affect

the growth and kind of pigment produced by the in which microorganisms are grown [25].

The yield of astaxanthin from Phaffia rhodozyma was 325 to 212 µg/g astaxanhin at a pH of

6.5 to 3.5 [26]. Pigment production is also affected by carbon source like glucose, fructose,

lactose, maltose, galactose, etc [27-26] and nitrogen source depending upon the

microorganism [28]. Minerals also has significant role in pigment production [29]. Zn (2x10-3

M and 3x10-3 M) inhibited the growth in liquid medium whereas in solid medium vigorous

growth and pigmentation was observed.

3
 The optimization of growth conditions of microorganisms, particularly physical and

nutritional parameters are of prime importance in the development of any pigment production

process owing to their impact on the economy and practicability of the process. Medium

optimization and physical conditions have been customarily performed using one-factor-at-a-

time method. The disadvantages of such a classical method are that it is time consuming,

laborious and expensive; in addition, it fails to resolve the combined effect of different

factors [30-31]. Eyeing on maximizing the pigment yield, productivity and minimizing the

production costs, most of the recent optimization efforts have relied on statistical

experimental design and response surface analysis [32] and, to a smaller extent, artificial

intelligence techniques such as genetic algorithms [33]. Statistical design is a potent tool that

can be used to account for the main as well as interactive influences of fermentation

parameters on process performance. It is an efficient way to generate useful information with

limited experimentation, thereby limiting the process development time and cost [34].

Therefore, researchers are encouraged to apply statistical experimental approaches such as

Taguchi method [35] and response surface methodology (RSM), which provide a great

amount of information based on only a small number of experiments [31].

On summing up, the growing apprehension over the eventual harmful effects of

synthetic colorants on both the consumer and the environment has raised preferential interest

in natural coloring alternatives [5]. Among all, microbial colorants popularly known as

pigments have some advantages over plant and animal based colorants Extensive studies

proved that microbes are known to produce a large amount of stable pigments. [8, 36]. Large

amounts of agro-industrial and domestic residues are generated from diverse economic

activities; utilization of these residues as inexpensive substrates to support the growth of

4
microorganisms to generate value-added products like pigments are of biotechnological

interest in recent years [8, 37]. Several processes and methodologies have been developed

and developing that utilizes a variety of cheaper substrates and wastes as alternative

substrates for the production of microbial pigments [38]. The utilization of several wastes as

or raw materials notably helps in solving pollution problems, while their disposal may

otherwise cause. In addition to the above, screening of different resources, establishing

simple methodologies, and exploring the microbial synthesis of pigments on inexpensive

substrates is an attractive option to develop commercial scale production [2, 37-38]. Aiming

at natural pigments on readily available agro-industrial materials, this study mainly focus on

1) Isolation of pigment producing microorganisms and the production of pigments (especially

carotenoids and melanins), 2) Simple production of commercial pigments in higher amounts

by purchased strains (which are at developmental stage) and 3) Optimization of key

parameters influencing pigment production where ever necessary

1.1 Motivation and scope

The use of synthetic organic colors has been acknowledged for many years as the most

reliable and economical method of restoring some of the food’s original shade to the

processed product. Synthetic colors are superior to natural pigments in tinctorial power, ease

of application, stability, and cost effectiveness. However, from the health safety viewpoint

they are not accepted by consumers, so over the past years growing interest in natural food

colorants has been observed [5].

The utilization of natural pigments in foodstuff, dyestuff, cosmetic and

pharmaceutical manufacturing practices has been increasing in recent years [24]. Natural

5
pigments can be obtained from three major sources i.e. animals, plants [25] and

microorganisms [13]. The accessible authorized natural pigments from animals and plants

have numerous drawbacks such as limited range, volatility against light, heat or adverse pH,

low water solubility and are often non-availability throughout the year. Moreover microbial

pigments are of great interest owing to the stability of the pigments produced and the

availability of cultivation technology [17], [26]. The benefits of pigment production from

microorganisms include easy and fast growth in the cheap culture medium, independence

from weather conditions and colors of different shades. Hence, microbial pigment production

is now one of the promising and emerging fields of research to reveal its potential for various

industrial applications [13], [27], [28].

From an industrial point of view, there is a necessity to develop a high throughput and

cost-effective approaches for large scale production of various microbial pigments.

Conventional media used for the biosynthesis of microbial pigments are rich in a variety of

nutrients. Microorganisms vary in their needs to carbon sources according to their nutrient

nature; the use of pure carbon sources e.g. (glucose, sucrose, and fructose) is expensive from

cost-effective casing, so the industrial processes try to use contemptible carbon sources

especially industrial wastes, variety of plant seed oils etc. have also been used as carbon

substances for obtaining different pigments. From an industrial point of view it is essential to

obtain a suitable medium to simultaneously improve the growth of organism and the pigment

production [17], [29], [30].

Thus, there is an urgent need for alternative colorants that are natural, cost effective

and easily degradable and without production of recalcitrant intermediates when they enter

the environment. There is an increasing interest involving microorganisms as a possible

6
alternate source of colorants used in food, cosmetic and pharma industries. In this direction,

the exploration of several wastes as substrates for the production of microbial pigments could

make huge cut-off in the production costs of these natural biocolorants and makes the

approach promising and worthwhile.

1.2 Organization of thesis

The prime objective of the work presented in this thesis was to select potent pigment

producing microorganisms from natural sources like marine water and soil. Selections of

suitable cheaper substrates for economical productivity of commercial pigments,

development of process by optimization of key parameters are subsequent accomplishments.

Along with above utilization of cheaper substrates for improved pigment production by

carotenoid producing yeast strains is also investigated.

The thesis has been organized into five chapters. Chapters 1 and 2 represent introduction to

the topic and relevant literature review regarding microbial colorants. History of colorants,

significance of dyes and pigments, various classes of pigments, scope of microbial pigments,

available technologies and practices etc., are provided in these chapters. The extensive

summary and descriptions of Chapters 1 and 2 provided ample motivation and facilitated in

framing the research objectives of the work.

Chapter 3 addresses the key role of melanins and carotenoids which are at research project

and development stage pigments and connects the coming chapters as a bridge by justifying

the objectives of the work. This chapter deals with the key issues like sampling, screening,

isolation of pigment producing microbes, synthesis of commercial pigments by using

purchased strains, microbial cultivation strategies, selection of suitable substrates etc.

7
Chapter 4 deal with approach and investigations of the work and is mainly divided into two

parts part I and part II. Studies pertaining to the isolation of pigment producing bacteria and

the production of pigments on cheaper substrates were encompassed in Part I with sub-

chapters of 4.1, 4.2 and 4.3. Sub-chapters 4.1 and 4.2 deals with the isolation of melanin

producing strains from natural resources (like marine water and a soil sample), pigment

production on cheaper substrates like vegetable waste and fruit waste extract, optimization of

key production parameters, analysis of the obtained melanins, evaluating their efficiency as

photoprotective, radical scavenging and metal chelating agents. Additionally, carotenoid

producing activity on rice powder as a sole substrate was described in sub-chapter 4.3 by a

novel garden soil isolate. The pigment was subjected to various analysis and the influential

production parameters were optimized using a simple Taguchi approach.

Part II of Chapter 4 detail facile synthesis of commercial pigments by purchased strains on

cheaper substrates with sub-chapters 4.4 and 4.5. Sub-chapter of 4.4 illustrates the carotenoid

production by the obtained yeast strain Rhodotorula rubra and its ability to utilize the FWE

as sole substrate for pigment production. This study employs a simple two step optimization

of key parameters involved in production. Furthermore, the sub-chapter 4.5 presents

production of the carotenoid astaxanthin by yeast Xanthophyllomyces dendrorhous and the

process of optimization of key parameters using FWE. Antioxidant assay of the obtained

carotenoids by both the yeasts were also evaluated in respective studies.

Finally, Chapter 5 summarises, major findings of all the chapters and suggestions for further

work in the arena of commercial scale pigments production on cheaper substrates using

microorganisms in particular.

8
References

[1] F. Delgado-Vargas, et al., "Natural pigments: carotenoids, anthocyanins, and

betalains—characteristics, biosynthesis, processing, and stability," Critical reviews in

food science and nutrition, vol. 40, pp. 173-289, 2000.

[2] S. Babitha, "Microbial pigments," in Biotechnology for agro-industrial residues

utilisation, ed: Springer, 2009, pp. 147-162.

[3] K. Malik, et al., "Microbial pigments: A review," Int. J. Microbial. Resour. Technol,

vol. 1, pp. 361-365, 2012.

[4] Z. E. Sikorski, Chemical and functional properties of food components: CRC Press,

2006.

[5] C. Socaciu, Food colorants: chemical and functional properties: CRC Press, 2007.

[6] N. Durán, et al., "Ecological-friendly pigments from fungi," Critical reviews in food

science and nutrition, vol. 42, pp. 53-66, 2002.

[7] A. Mortensen, "Carotenoids and other pigments as natural colorants," Pure and

Applied Chemistry, vol. 78, pp. 1477-1491, 2006.

[8] C. K. Venil, et al., "Bacterial pigments and their applications," Process Biochemistry,

vol. 48, pp. 1065-1079, 2013.

[9] D. Cristea and G. Vilarem, "Improving light fastness of natural dyes on cotton yarn,"

Dyes and Pigments, vol. 70, pp. 238-245, 2006.

[10] T. Bechtold and R. Mussak, Handbook of natural colorants: John Wiley & Sons,

2009.

[11] F. Delgado-Vargas and O. Paredes-López, Natural colorants for food and

nutraceutical uses: CRC Press, 2002.

9
[12] N. Nagpal, et al., "Microbial pigments with health benefits-A mini review," Trends in

Biosciences, vol. 4, pp. 157-160, 2011.

[13] L. Dufossé, "Microbial Production of Food Grade Pigments," Food Technology &

Biotechnology, vol. 44, 2006.

[14] L. Dufossé, et al., "Microorganisms and microalgae as sources of pigments for food

use: a scientific oddity or an industrial reality?," Trends in Food Science &

Technology, vol. 16, pp. 389-406, 2005.

[15] T. Storebakken, et al., "Carotenoids in diets for salmonids: IV. Pigmentation of

Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin,"

Aquaculture, vol. 65, pp. 279-292, 1987.

[16] V. Vasanthabharathi, et al., "Melanin production from marine Streptomyces," African

Journal of Biotechnology, vol. 10, pp. 11224-11234, 2013.

[17] F. Pantanella, et al., "Violacein and biofilm production in Janthinobacterium

lividum," Journal of Applied Microbiology, vol. 102, pp. 992-999, 2007.

[18] A. Domínguez-Bocanegra and J. Torres-Muñoz, "Astaxanthin hyperproduction by

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as

sole source of energy," Applied Microbiology and Biotechnology, vol. 66, pp. 249-

252, 2004.

[19] I. Schmidt, et al., "Biotechnological production of astaxanthin with Phaffia

rhodozyma/Xanthophyllomyces dendrorhous," Applied Microbiology and

Biotechnology, vol. 89, pp. 555-571, 2011.

[20] C. K. Venil and P. Lakshmanaperumalsamy, "An insightful overview on microbial

pigment, prodigiosin," Electronic Journal of Biology, vol. 5, pp. 49-61, 2009.

10
[21] L. C. Mata-Gómez, et al., "Biotechnological production of carotenoids by yeasts: an

overview," Microbial Cell Factories, vol. 13, p. 12, 2014.

[22] H. Mohan Kumari, et al., "Safety evaluation of Monascus purpureus red mould rice in

albino rats," Food and Chemical Toxicology, vol. 47, pp. 1739-1746, 2009.

[23] J. Tinoi, et al., "Simplex optimization of carotenoid production by Rhodotorula

glutinis using hydrolyzed mung bean waste flour as substrate," Process Biochemistry,

vol. 40, pp. 2551-2557, 2005.

[24] K. Sanjay, et al., "Optimization of carotenoid production by Aspergillus carbonarius

in submerged fermentation using a response surface methodology," International

Journal of Food Engineering, vol. 3, 2007.

[25] V. Joshi, et al., "Microbial pigments," Indian Journal of Biotechnology, vol. 2, pp.

362-369, 2003.

[26] E. A. Johnson and M. J. Lewis, "Astaxanthin formation by the yeast Phaffia

rhodozyma," Journal of General Microbiology, vol. 115, pp. 173-183, 1979.

[27] K. Boonyapranai, et al., "Optimization of submerged culture for the production of

naphthoquinones pigment by Fusarium verticillioides," Chiang Mai Journal of

Science, vol. 35, pp. 457-466, 2008.

[28] L. K. Chintapenta, et al., "Culture conditions for growth and pigment production of a

mangrove Penicillium species." MalaysianJournal of Scientifc Researchvol. 1, pp. 29-

35, 2014.

[29] H. Manonmani and K. Sreektaniah, "Pigment production by a strain of Aspergillus

sp," Journal of Food Science and Technology, vol. 21, pp. 195-197, 1984.

11
[30] S. N. Surwase, et al., "Optimization of melanin production by Brevundimonas sp. SGJ

using response surface methodology," 3 Biotech, pp. 1-8, 2012.

[31] M. Aghaie-Khouzani, et al., "Decolorization of some synthetic dyes using optimized

culture broth of laccase producing ascomycete Paraconiothyrium variabile,"

Biochemical Engineering Journal, vol. 60, pp. 9-15, 2012.

[32] P. D. Haaland, Experimental design in biotechnology vol. 105: CRC press, 1989.

[33] D. Weuster-Botz, "Experimental design for fermentation media development:

statistical design or global random search?," Journal of bioscience and

bioengineering, vol. 90, pp. 473-483, 2000.

[34] R. H. Myers and C. M. Anderson-Cook, Response surface methodology: process and

product optimization using designed experiments vol. 705: John Wiley & Sons, 2009.

[35] M. Azin, et al., "Production of xylanase by Trichoderma longibrachiatum on a

mixture of wheat bran and wheat straw: Optimization of culture condition by Taguchi

method," Enzyme and microbial technology, vol. 40, pp. 801-805, 2007.

[36] W. A. Ahmad, et al., Application of Bacterial Pigments as Colorant: Springer, 2012.

[37] S. I. Mussatto, et al., "Use of Agro-industrial wastes in solid-state fermentation

processes," Industrial waste. Croatia: InTech, pp. 121-40, 2012.

[38] P. Buzzini and A. Martini, "Production of carotenoids by strains of Rhodotorula

glutinis cultured in raw materials of agro-industrial origin," Bioresource Technology,

vol. 71, pp. 41-44, 2000.

12
Chapter 2 Literature Review

2.1 Outline

Pigments are the colors that we observe at each step of our lives, because pigments are

present in all the organisms in the world, where plants are the principal producers. Pigments

are present in leaves, fruits, vegetables, and flowers; also, they are also found in skin, eyes,

and other animal structures; and in bacteria and fungi. Natural and synthetic pigments are

used in medicines, foods, clothes, furniture, cosmetics, and in other products. However,

natural pigments have important functions besides imparting beauty, such as photosynthesis

by chlorophylls and carotenoids. Respiration in animals by hemoglobin under stress

conditions plan synthesizes flavonoids; the quinones play very important role in the

conversion of light into chemical energy. The melanins act as a protective screen in humans

and other vertebrates, and in some fungi melanins are essential for vital cycles. Pigments

have a well-known pharmacological activity such as anticancer and effective against

cardiovascular diseases [1-2].

In the recent years, pigments produced from natural sources are of worldwide interest

and is gaining importance. The demand for natural source of pigments is increasing day by

13
day because of the consciousness of positive health benefits out of natural compounds [1]. It

is therefore, necessary to explore various natural sources of colorants and their potentials [2].

Though many natural colors are available from ores, insects, plants and microbes; microbial

colorants play a significant role as food coloring agent, because of its production and easy

down streaming process [3-4].

2.2 History of colorants

Until the mid-19th century all dyes were obtained from animal or plant extracts. The textile

industry used natural pigments, such as turmeric, cochineal, wood madder, or henna. In 1856,

H. Perkin established the first industrial unit of organic synthetic dyes to produce mauve. A

few years later the discovery of diazotization and a coupling reaction by Peter Griess was the

next major advance for development of the color industry [5]. In the 19th century, synthetic

organic dyes were developed, creating a more inexpensive and wider range of colorants.

Since then their quality has been enhanced due to extensive research and developments. The

economic significance of the color industry is clearly reflected in the large number of

synthesized compounds; as many as 700 colorants are currently available [4].

Toward the end of the 19th century, when synthetic colors were first adopted for use

on a large scale, they were hailed as a considerable technological breakthrough. The term

'synthetic' was associated with the idea of progress and synthetic colorants were actually

considered safer in food than the naturals, as they were tinctorially much stronger and

consequently a smaller quantity was needed to achieve a specific colored effect [5].

Synthetic colorants were used in foods, medicines, and cosmetics, but through the

years their importance reduced. This cutback of synthetic colorants started about five decades

14
ago. All synthetic food components suffered severe criticism, including synthetic additives

and mostly food pigments. Color additives were one of the first man-made (synthetic)

products regulated by law. Today, all food color additives are cautiously regulated by federal

authorities to ensure that foods are safe to eat and accurately labeled [6].

2.3 Dyes and pigments – their implications

Color is an important marker of food quality. The consumer links food color with good

processing and safety. However, color cannot be studied without taking into account the

human sensory system. Perception of color is associated to three factors: spectral composition

of the light source, physical object characteristics, and eye sensitivity [7]

Coloring agents can be divided primarily into two classes i.e. pigments and dyes.

Dyes are water soluble substances and have at least one salt-forming group. The most

common is the sulfonic acid group; however carboxylic acid residues can also be used. These

dyes are generally isolated as sodium salts. They have colored anions and are well-known as

anionic dyes. The other dyes containing basic groups, like –NH2, -NH-CH3, or –N(CH3)2,

from water –soluble salts with acids. These are the cationic dyes and have positively charged

colored ion. If both acidic and basic groups are present, an internal salt is usually formed [8].

Pigments are the particulate solids disperse into a medium without significant solution or

their interactions. They are oil-soluble or solvent-soluble colorants lack with salt-forming

groups. They occupy a major place in our daily life. Pigments are used in food, cosmetics,

paints, pharmaceuticals, glass, textiles etc. The most primitive known pigments were natural

minerals. Natural iron oxides, anhydrous Fe2O3, charcoal and so on are several well-known

pigments since prehistoric times [9].

15
The color of material such as food is the outcome of the presence of natural pigments or of

added synthetic organic dyes. The definition of a natural colorant is variable from one

country to another, but generally, natural colorants comprise the pigments occurring in

unprocessed food, and those that can be formed upon heating, processing, or storage [5].

These may be isoprenoid derivatives like carotenoids, porphyrins like chlorophylls and

hemes, phenolics like anthocyanins, betalains, quinones, curcuminoids and some

miscellaneous naturally occurring colorants like riboflavins, caramels, melanoidins, melanins

and so on [2, 5]. Based upon major sources, natural pigments may be divided into three major

classes which include plant, animal and microbial groups. All natural pigments are unstable

and participate in different reactions and their color is robustly dependent on conditions of

storage and processing.

Organization of pigments can be done in numerous w ays [2] and can be stated as follows:

By Their Origin

Pigments can be classified by their origin as natural, synthetic, or inorganic. Natural pigments

are produced by living organisms such as plants, animals, fungi, and microorganisms.

Synthetic pigments are obtained from laboratories.

By the Chemical Structure of the Chromophore

Also, pigments can be classified by taking into account the chromophore chemical structure

as Chromophores with conjugated systems: carotenoids,anthocyanins, betalains, caramel,

synthetic pigments, and lakes. Metal-coordinated porphyrins: myoglobin, chlorophyll, and

their derivatives.

By the Structural Characteristics of the Natural Pigments

 Moreover, natural pigments can be classified by their structural characteristics as:

16
  Tetrapyrrole derivatives: chlorophylls and heme colors.

 Isoprenoid derivatives: carotenoids and iridoids.

 N-heterocyclic compounds different from tetrapyrroles: purines, pterins, flavins,

phenazines,

 Phenoxazines, and betalains.

 Benzopyran derivatives (oxygenated heterocyclic compounds): anthocyanins and

other flavonoid pigments.

 Quinones: benzoquinone, naphthoquinone, anthraquinone.

 Melanins.

As Food Additives

By considering the pigments as food additives, their classification by the FDA is

Certifiable: These are manmade and subdivided as synthetic pigments and lakes.

Exempt from certification: This group includes pigments derived from natural sources such

as vegetables, minerals, or animals, and manmade counterparts of natural derivatives.

2.4 Biological pigments- different classes

2.4.1 Biological pigments

In recent times growing concern on the use of edible coloring agents has banned various

synthetic coloring agents which have a potential of carcinogenicity and teratogenicity [10].

Biological pigments are also well-known as pigments or biochromes. These are the

constituents produced by living organisms that have a color resulting from selective color

absorption. The topic of synthetic dyes in food is in conversation for many years. The study

and negative valuation of synthetic food dyes by the modern consumer have raised a strong

17
interest in natural coloring substitute. Nature is rich in colors (minerals, plants, microalgae,

etc.), and pigment-producing microorganisms (fungi, yeast, bacteria) are reasonably common

[11-12]. All biological pigments selectively absorb particular wavelengths of light while

reflecting others. The light that is absorbed may be used by the organism or plant to power

chemical reactions, while the reflected wavelengths of light govern the color of the pigment

that will appear to the eye. Biological pigments based on the source were categorized as

mentioned below.

2.4.2 Plant pigments

Several companies decided to color the food with plant extracts or pigments from plants.

Plant pigments include a variety of diverse kinds of molecules including porphyins,

carotinoids, anthocyanins and batalains. In plants these pigments will also assist in pollination

by attracting the pollinators [13]. Some major plant pigments are as follows [2-3]:

a) Chlorophylls

These are the principal pigments in plants. These porphyin compounds absorb yellow and

blue wavelengths of the light and reflect the green. All land plants, green plants and green

algae possess two forms of this pigment chlorophyll a and chlorophyll b, whereas red

algae possess only chlorophyll a.

b) Carotenoids

These are the orange, red or yellow tetraterpinoids. They gather the wave lengths that are

not generously absorbed by the chlorophylls, most familiar carotenoids are carotene,

lutein, and lycopene. Carotenoids have been shown to act as natural antioxidants

18
 c) Anthocyanins

These are water soluble flavonoid pigments that look like red to blue according to the pH.

The anthocyanin catches the light that has passes the leaf and reflects it back to the

regions having chlorophyll in order to maximize the use of available light. Color range of

pink/red to mauve/blue were obtained from elderberries, black grape skin, black carrots,

red cabbage etc.

d) Betalains

There are water soluble pigments like anthocyanins but unlike anthocyanins they are

indole-derived substances synthesized by tyrosine. These classes are only found in

caryophyllales (including cactus and amaranth) and never co-occur in plants with

anthocyanins. These are responsible for the deep red color of beets and are used as food-

coloring agents commercially.

Although plant pigments are emerging alternatives, they suffer from several bottlenecks i.e.

for example; abundant orange-yellow pigment like curcumin (from plant rhizome of

Curcuma longa) has to be debittered to avoid its odour and sharp taste. And pigments like

anthocyanins, chlorophyll, betanin are pH-dependent, oxygen sensitive, heat sensitive, and

subject to photo-oxidation some limitation of must be [14].

2.4.3 Animal pigments

Our ancestors managed to obtain wide range of pigments from animal sources before

chemical equivalents are manufactured the most rare and difficult to obtain became symbol

of wealth and status for example the color purple is associated with wealth and royalty. The

purple dye of the ancients is one of the old pigments known in 13th century. Murexes types of

drilling snails have a mucus secreting organ called hypobranchial gland. Tyrian purple is

19
eked out in small amount from the mucus of certain mollusks. Similarly caramine made from

cochnieal insects is much more concentrated than the traditional red dye obtained from

madder root. Carminic acid and carmine of orange to red and pink to red comes from a

female cochineal insect from Peru and Equador. The pigment is price-sensitive here. Dye

from kermes and cochneal was of high demand throughout Europe as it is used to color the

fabrics of royalty, nobility and church leaders. For several centuries it was the dye used in

British red coats, hand woven rugs, as paint. Carmine derived from cochneal is used to color

drinks, foods, meat, sausages, processed poultry products, bakery products, pie fillings,

icings, jams, desserts, yogurt, cheese, ice creams and other dairy products. Cosmetic industry

is the major consumer of insoluble carmine pigment, particularly for hair and skin products,

lipsticks, face powders, rouge and blushes. Another major application is in pharmaceutical

industry to color ointments and pills. Lac insect produces red dye similar to that of the

caramine dye, the water soluble red dye comes from the body of the insect is obtained by

aqueous extraction and is processed into seedlac and shellac. They are used in a multitude of

applications including varnishes, paints, printing inks, sealing wax, coat pills, sweets and

chocolates. Shellac is used in making vinyl records and to color Indian military uniforms and

found in oriental carpets [14]. Other pigments like Canthaxanthin of orangish pink color are

obtained from salmon, shrimp and flamingos. While several animal pigments are price

sensitive, many area having the main disadvantages such as limited color range and

availability [14].

2.4.4 Microbial pigments

There are a number of natural pigments but only a few are available in adequate quantities for

industrial production. Production of pigments from microorganisms is beneficial over other

20
sources because microorganisms can grow rapidly which may lead to a high productivity of

the product [15-16].

a) Fungal pigments

Many fungi produce pigments during their growth which are substantive as specified by

the permanent staining that is often associated with mildew growth on textiles and plastics.

Some fungal pigments have been shown to be anthroquinone derivatives, resembling the

important class of vat dyes. Fungal compounds therefore have potential for the direct

production of textile dyes or dye intermediates replacing chemical synthesis. The

production and evaluation of microbial pigments as textile colorants is currently being

explored [17].

b) Bacterial pigments

Some bacteria produce pigments which can be observed after they grow into colonies.

Pigments can aid to identify bacteria. For example certain bacteria produce water soluble

pigments which spread through the medium in which they grow. Others give pigments that

are soluble in fat [18]. The yellow pigment from zeaxanthin from Flavobacterium species

can be used as an additive in poultry feed to fortify the yellow color of the skin of birds or

to accentuate the color of the yolk of the egg. A yellow pigment zeaxanthin from

Flavobacterium sp. can also be used in cosmetic and in food industry. Canthaxanthin from

the photosynthetic bacterium Bradyrhizobium sp. has been used in fish feed for numerous

years. Halobacterium is also one more source of canthaxanthin. astaxanthin from

Agrobacterium aurantiacum [19].

21
 c) Yeast pigments

Some yeast can produce valuable carotenoids in pure culture on low-cost substrates,

thereby providing an alternative to chemical synthesis. Asthaxanthin from

Xanthophyllomyces dendrohous formerly known as Phaffia rhodozyma is a carotenoid

pigment which is used as a food colorant and widely used in the animal feed to impart

color to the animal skin as animals have no capacity to synthesis the carotenoid pigment.

Hundreds of scientific papers and patents deal with asthaxanthin production using this

yeast and pigment production process has not been economically efficient till now. Most

of the research in recent years is also focused on Rhodotorula glutinis which gives

carotenoid pigment. However some papers reported reasonable pigment production with

other species such as R. gracilis, R. rubra (now R. mucilaginosa) and R. graminis too. The

main compounds obtained by these red yeasts are torulene and torularhodin with minute

quantity of beta carotene [16, 19].

Among all the stated pigment sources, microbes have vast potential to produce diverse

bioproducts and one such bioproduct is pigments. Interest in microbial pigments has

increased considerably, mainly due to the benefits to human health and also to the growth of

certain areas such as agriculture, especially aquaculture and poultry industry [20], Britton,

nutritional supplements, food industry where they are used as coloring agents for cooked

sausages, soft drinks, baked goods and pharmaceutical as additive to cosmetics. For example,

carotenoids market has resulted interesting in 2010 estimated at nearly $1.2 billion, but the

expectations for 2018 are increasing considerably supposing to reach $1.4 billion with a

compound annual growth rate of 2.3 [21].

22
 The production and application of microbial pigments as natural colorants has been

studied by various researchers and is one of the emerging fields of research [14-15, 18-19,

21-22]. Most of the microbial pigments productions are still at the R&D phase. And there are

many studies in the literature on various microbial pigments which focus on production and

application of specified pigment in each case.

Efforts have been made in order to ease the production costs of microbial derived

pigments compared to those of synthetic pigments or pigments extracted from natural

sources. Innovations will progress the economy of pigment production by isolating new or

creating better microorganisms, by improving the processes. Hence, work on the microbial

bacterial pigments should be excelled especially in finding cheap and suitable growth

mediums which can reduce the cost and increase its applicability for industrial production

[15].

2.5 Significance of microbial pigments as natural colorants

Microorganisms are the most prevailing creatures in existence and determine the life and

death on this planet. Microorganisms are associated with all the foods that we eat and are

accountable for the formation of certain food products by the process of fermentation and can

also be used as a source of food in the form of single cell proteins and food supplements in

the form of pigments, amino acids, vitamins, organic acids, and enzymes.

In this way the pigments from microbial origin are a good alternative.

Microorganisms are known to produce a range of pigments; therefore they are promising

source of food colorants [23-24]. Some of the most significant natural pigments are

carotenoids, flavonoids, tetrapyrroles and some xanthophylls as astaxanthin. The pigment

23
most frequently used in industries is beta-carotene which is obtained from some microalgae

and cyanobacteria. Astaxanthin produced from Phaffia rohodozoa and Haematococcus

pluviais, is a red pigment of great commercial value and is used in feed, pharmaceutical and

aquaculture industries. Microorganisms which have the capacity to produce pigments in high

yields include species of Monascus, Paecilomyces, Serratia, Cordyceps, Streptomyces and

yellow-red and blue compounds produced by Penicillium herquei and Penicillium

atrovenetum, Rhodotorula, Sarcina, Cryptococcus, Monascus purpureus, Phaffia rhodozyma,

Bacillus sp., Achromobacter, Yarrowia and Phaffia also produce a large number of pigments

[16, 21].

Most of the bacteria and fungi are extensively studied for their potential as a source of

food colorants. Natural pigments possess anticancer activity, contain pro-vitamin A and have

some important properties like stability to light, heat and pH [25]. Thus, the food industry has

become increasingly interested in the use of microbial technology to produce colors for usage

in foods. It can also help to overcome the growing public apprehension over the adverse

health effects of addition of synthetic colors in food products.

Furthermore, natural colorants will not only be valuable to the health of human beings, but it

will be a benefit for the preservation of biodiversity as harmful chemicals released into the

environment while producing synthetic colorants could be stopped. These natural colorants

are used in baby foods, breakfast cereals, sauces, pastas, processed cheese, fruit drinks,

vitamin-enriched milk products, and some energy drinks. Thus, natural colors in addition to

being eco-friendly, can also serve the dual need for visually appealing colors and probiotic

health benefits in food products [26].

24
2.6 Classification of microbial pigments

Pigments produced by organisms as reminiscence of its secondary metabolism are commonly

mentioned as biopigments. These biopigments have extensive synthetic and commercial

application [27]. Biological pigments can be categorized based on structural affinities and

natural occurrence. Some examples of naturally occurring microbial pigments are:

Riboflavin: It is a yellow water-soluble vitamin produced by various microorganisms.

Traditional chemical synthesis of riboflavin is now being exchanged by commercially

competitive biotechnological processes using ascomycetes Ashbya gossypii, filamentous

fungi Candida famata, or bacterium Bacillus subtilis [28]. It is used in baby foods, breakfast

cereals, sauces, pastas, processed cheese, fruit drinks, vitamin-enriched milk products, and

some energy drinks.

Beta-carotene: Phycomyces and Mucor circinelloides (wild type) are a prospective source of

beta-carotene. The European Union Committee considers that beta-carotene obtained by

fermentation of Blakeslea trispora is equivalent to the chemically synthesized material used

as food colorant and is therefore acceptable for use as a coloring agent in foodstuffs [19].

Canthaxanthin: It is produced as the major carotenoid pigment by orange and dark pink-

pigmented bacteriochlorophyll containing Bradyrhizobium (photosynthetic) strains isolated

from stem nodules of Aeschynomene species and Halobacterium sp [29]. Canthaxanthins are

effective antioxidants and inhibit the oxidation of lipids in liposomes [30].

Carotenoids: These are yellow to orange-red pigments that are ubiquitous in nature. Several

numbers of microorganisms produce this pigment such as Serratia and Streptomyces.

Carotenoids are potent antioxidants and are widely used as food colorants. Majority of

25
microbes investigated produce carotenoids belonging to Myxococcus [31], Streptomyces [32],

Mycobacterium, Agrobacterium and Sulfolobus [33].

Prodigiosin: It is a multipurpose red pigment, produced by a number of microorganisms such

as Serratia marcescens, Vibrio psychoerythrus, Rugamonas rubra, actinomycetes, such as

Streptoverticillium rubrireticuli and other eubacteria [26]. It is known to have anti-malarial,

antibacterial, antineoplastic and antibiotic activity.

Phycocyanin: It is a blue pigment, produced by cyanobacteria which contain chlorophyll a.

The blue colorant is known by the name spirulina (blue green alga), and is used as a dietary

supplement which is rich in proteins. Here the supplement consists of dried cyanobacteria

[34].

Violacein: It is a versatile pigment from a bacterium Chromobacterium violaceum that

exhibits numerous biological activities. It has gained immense importance in industrial

markets, such as in medicine, cosmetics, food and textiles [35].

Astaxanthin: Chemically it is 3, 3’-dihydroxy-b, b-carotene-4, 4’- dione and is an orange -

red pigment and produced by microorganisms such as red Basidiomycetous yeast

Xanthophyllomyces dendrorhous [36], green algae Heamatococcus pluvialis and

Agrobacterium aurantiacum [33].

Pigment compounds form microorganisms based on color and appearance can be

classified as shown in Table 2.1.

26
Table 2.1.Pigment producing microorganisms based on color and appearance.

Microorganism(s) Pigments/Molecule Color/appearance Reference

Bacteria
Agrobacterium aurantiacum Astaxanthin Pink-red [19]
Paracoccus carotinifaciens Astaxanthin Pink-red [15]
Bradyrhizobium sp. Canthaxanthin Dark- red [15]
Flavobacterium sp., Paracoccus
Zeaxanthin yellow [37-38]
zeaxanthinifaciens
Achromobacter sp. Red [39]
Brevibacterium sp. Carotenoid like Orange yellow [40]
Greyish to
Corynebacterium michigannise
creamish
Corynebacterium insidiosum Indigoidine Blue [41]
Rugamonas rubra,
Streptoverticillium rubrireticuli,
Prodigiosin Red [22]
Vibrio gaogenes, Alteromonas
rubra
Xanthophyllomyces dendrorhous Astaxanthin Pink [42]
Haloferax alexandrinus Canthaxanthin Dark Red [43]
Staphyloxanthin
Staphylococcus aureus Golden Yellow [44]
Zeaxanthin
Chromobacterium violaceum Violacein Purple [45]
Serratia marcescens, Serratia
Prodigiosin Red [46]
rubidaea,
Pseudomonas aeruginosa Pyocyanin Blue-green [47]
Xanthomonas oryzae Xanthomonadin Yellow [48]
Janthinobacterium lividum Violacein Purple [49]
Algae
Dunaliella salina β-carotene Red [50]

27
Chlorococcum sp. Lutein Orange [51]
Hematococcus sp. Canthaxanthin Yellow-orange-red [52]
Fungi
Aspergillus sp. β -carotene Orange-red [53]
Blakeslea trispora β -carotene Cream [54]
Fusarium sporotrichioides Lycopene Red [55]
Haematococcus Pluvialis Astaxanthin Red [56]
Monascorubramin
Monascus sp. Red - orange [57]
Rubropunctatin
Monascus purpureus Monascin Ankaflavin Red-yellow [22]
Monascus roseus Canthaxanthin Orange-pink [22]
Monascus sp. Ankaflavin Yellow [22]
Penicillium oxalicum Anthraquinone Red [58]
Blakeslea trispora Lycopene Red [59]
Cordyceps unilateralis Naphtoquinone Deep blood-red [60]
Mucor circinelloides,
Neurospora crassa and β-carotene Yellow-orange [61]
Phycomyces blakesleeanus
Pacilomyces farinosus Anthraquinone Red [62]
Yeast
Cryptococus sp. Melanin type compounds Reddish brown [63]
Saccharomyces neoformans var.
Melanin Black [19]
nigricans
Phaffia rhodozyma Astaxanthin Pink-red [64]
Rhodotorula sp. Rhodotorula
Torularhodin Orange-red [65]
glutinis
Yarrowia lipolytica Melanin Brown [66]
Actinomycetes
Streptoverticillium rubrireticuli Prodigiosin Red [67]
Streptomyces echinoruber Rubrolone Red [68]

28
2.7 Microbial pigments of commercial importance

The success of any microbial pigment produced by biotechnological means (for example

fermentation) depends upon its acceptability in the market, regulatory approval, and the size

of the capital investment required in bringing the product to the market. A few years ago,

some expressed doubts about the positive commercialization of fermentation derived food

grade pigments because of the high capital investments requirements for fermentation

facilities and the expensive and time-span toxicity studies required by regulatory agencies

[15, 19].

In addition to the above public perception of biotechnology derived products should

also be taken into account for the absolute production benefit. Table 2.2 states the successful

microbial pigments already in use as food grade and/or nutritional supplements in the market

and are derived from various bacteria, yeast and fungi. Based on the extensive research

reports, these pigments broadly can be categorized into pigments of industrial production

(IP), developmental stage (DS) and research project (RP) phase (Table 2.2) [19, 22].

Microbial colorants are in use in the fish industry already, for instance, to enhance the

pink color of farmed salmon. Further, some natural food colorants have commercial

prospective for use as antioxidants [69]. Nowadays several fermentative food grade pigments

are in the market: Monascus pigments, astaxanthin from Xanthophyllomyces dendrorhous,

Arpink Red from Penicillium oxalicum, riboflavin from Ashbyagossypii, and carotene from

Blakeslea trisporatrispora [15, 19, 24] which are considered harmless and approved by FDA.

The successful marketing of pigments derived from microbes, both as a food color and a

nutritional supplement, reflects the presence and significance of niche markets in which

consumers are willing to pay a premium for ‘all natural ingredients’.

29
Table 2.2. Pigments from various microorganisms which are already in use as natural food

colorants [19, 22].

Pigment Color Microorganism Status

Ankaflavin Yellow Monascus sp. IP

Anthroquinone Red Pencilllium candidum IP

Monascorubramine Red Monascus sp. IP

Riboflavin Yellow Ashbya gossypi IP

Rubropanctatin Orange Monascus sp. IP

β Carotene Yellow-orange Blakeslea trisporia IP

Astaxanthin Pink-red Agrobacterium aurantiacum RP

Astaxanthin Pink-red Paracoccus carotinifaciens RP

Cathaxanthin Dark red Bradirhizobium sp. RP

Lycopene Red Fusarium sporotrichioides RP

Melanin Black Saccharomyces neoformis RP

Napthoquinone Deep blood red Cardyceps unilateralis RP

Zeaxanthin Yellow Paracoccus zeaxanthinifaciens RP

β Carotene Yellow-orange Fusarium sporotrichioides RP

β Carotene Yellow-orange Neurospora crassa RP

β Carotene Yellow-orange Phycomyces blaksleeanus RP

Unknown Red Paecilomyces sinclairii RP

Xanthophyllomyces
Astaxanthin Pink-red DS
dendrohous

30
Lycopene Red Blakeslea trisporia DS

Rubrolone Red Streptomyces echinoruber DS

Torularhodin Orange-red Rhodotorula sp. DS

Zeaxanthin Yellow Flavobacterium sp. DS

β Carotene Yellow-orange Mucor circinelloides DS

Unknown Red Penicillium purpurogenum DS

The number of approved colorants for food industry is restricted. Some approved food

colorants are recognized by their chemical name (for eg. canthaxanthin) while others are

known by source (eg. fruit juice or vegetable juice). The biocolorants identified by their

chemical name can be synthesized easily by cheaper biotechnological sources particularly by

various microorganisms. And technological limitations are the major hold-up for the

commercial exploitation of the source materials [15, 61]. The success of any pigment

produced by fermentation rely up on its acceptability in the market, regulatory approval, and

the size of the capital investment required in bringing the product to market [61]. Some food

grade pigments by microorganisms with commercial value are given in Fig. 2.1.

31
Fig. 2.1. Some food grade pigments and their structures from microorganisms [15].

32
2.8 Benefits and Applications of Microbial pigments

Pigments produced by microorganisms are of traditional use in oriental countries and have

been a subject of intense research in the present decades because of its potential for

applications. Use of microorganisms and biotechnology would offer solutions to the problems

of various industries especially food colorant industry. Fermentative production of colorants

has a number of benefits that include: cheaper production, readily available raw materials,

high yields and no seasonal variations [70].

In contrast to higher plants, single cell algae and fungi are more appropriate for

biotechnological production because they can be grown using existing culture techniques.

Many fungi produce pigments which have application in both textile and food industries [71].

Fungal pigments are routinely utilized as colorants for both foodstuffs and materials. Some of

the more importantly utilized fungal pigments come from the water soluble orange/red

pigments produced by Monascus sp., frequently used in rice wines in eastern countries [72].

Pigments produced from Monascus purpureus Piedallu are used in wool dying [73], and an

anthraquinone pigment obtained from Penicillium oxalicum Currie & Thom is currently

being developed for use as a ‘natural’ food additive that may have some anticancer effects

[14, 16]. Mycelial extracts of some promising mushrooms are Chroogomplus vinicolor

(which gives red tints), Bankera violascens (which gives greens) and Collybia iocephala

(which gives blues), they have a remarkable potential for dyeing wool and silk fabrics [74].

Carotenoids such as β-carotene and lycopene have been known to be produced by fungal cell

factories. For example, β-carotene by Blakeslea trispora, Mucor circinelloides, and

Phycomyces blakesleeanus and lycopene from Fusarium sporotrichioides, Blakeslea trispora

are already in use as for food colorants [33]. Vitamin, riboflavin (vitamin B2) is a yellow

33
food colorant that is fermentatively produced by the fungi Eremothecium ashbyii and Ashbya

gossypi and has huge commercial potential [75].

Microalgae such as Haematococcus (astaxanthin), Spirulina (phycocyanin), and

Dunaliella (ß-carotene and other carotenoids) are already explored as sources of natural food

colorants with some limitations of low productivity and contamination in the open culture

system, where they are grown [76]. Newly, a blue-green polyphenolic antioxidant pigment

termed marennine from the Diatom Haslea ostrearia has been reported [77]. This may aid as

a promising food coloring additive.

Among yeasts, the marketable success has been with the case of astaxanthin

production by Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) and is widely

in use as food additive. Additional example of production of carotenoids is by the yeast

Rhodotorula spp., R. glutinis, R. gracilis, R. rubra, and R. graminis. The chief carotenoids

produced by these yeasts are torulene and torularhodin, with minute quantity of β-carotene

[78-81].

The pigments obtained from bacteria offer the following benefits and advantages [15,

24]; they are progressively attractive to science because of broad ranging activities, easy

propagation and wide-ranging strain selection, high versatile and productive compared to

other sources, fermentation is integrally faster and more productive production when

compared to any other chemical process, simple and fast culturing methods allowing

continuous bioreactor operation, quite easy to manipulate genes, structural complexity suits

for industrial requirements, Cheap substrates used for bulk production and their pigments

extracted using simple liquid-liquid extraction technique minimizing operation cost. Some

important functions of bacterial pigments of commercial potential are 1) Riboflavin

34
synthesized by Bacillus subtilis and used in foods, vitamin enriched milk products and energy

drinks, 2) β-carotene usually produced by Flavobacterium, Agrobacterium aurantiacum and

widely used as food supplement for humans and as food additives for animals and fish, 3)

Prodigiosin by Serratia marcescens, Pseudomonas, Pseudoalteromonas, Alteromonas

denitrificans, Hahella, Vibrio species used as antibacterial, antifungal, antimalarial,

antibiotic, immunosuppressive, anticancer etc., 4) Phycocyanin by Cyanobacteria used as

dietary supplement rich in proteins, and 5) Violacein by Chromobacterium

violaceum,Alteromonas luteoviolacea was used as Antiviral, antibacterial, antiulcerogenic,

antileishmanial, and anticancer properties, potent cytotoxic effects against U937 and HL60

Leukemia cell lines [15, 24].

2.9 Novel practices of microbial pigment production

2.9.1 Strain improvement

Traditionally, strain development was achieved mainly by multiple rounds of random

mutagenesis and selection, which are still very helpful nowadays. In the latest decade, the

development of gene deletion techniques enabled efficient genome DNA inactivation and

greatly improved metabolic engineering of bacteria [15, 82-83]. A more systematic and

integrated approach for biotechnological process for strain improvement became prevalent.

The motivation for industrial strain development is economic, since pigment amounts

produced by wild strains are too low for cost-effective process. It is very essential to isolate

strains which produce pigments with shorter fermentation times. Improvement of microbial

strains for the over production of industrial products has been the characteristic of all

commercial fermentation processes. The strain improvement by common mutagens such as

35
ultraviolet (UV), ethyl methane sulfonate (EMS) and 1-methyl-3-nitro-1-nitrosoguanidine

(NTG) is convenient and can improve pigments yield as proved in several cases [15, 84].

New methods like X-ray and fast neutron of irradiations are also being tried to achieve

microbial strain improvements. For instance, M.purpureus was subjected to the above

irradiations to bring about change in pigmentation and morphology [25].

2.9.2 Fermentation

Fermentation is a metabolic process that converts sugar to acids, gases and/or alcohol and

usually occurs in yeast and bacteria. The production of microbial pigments by fermentation is

an interesting area and a lot of attention is now paid to this biotechnological approach. This

technique can be of solid or submerged fermentation mainly and each approach is having its

own merits and demerits in pigment production.

Submerged fermentation

Submerged fermentation utilizes free flowing liquid substrates, such as molasses and broths.

The bioactive molecules are secreted into the fermentation broth. The substrates are utilized

rapidly; hence need to be constantly replaced or supplemented with nutrients. This

fermentation technique is best suited for microorganisms such as bacteria that require high

moisture content. An added advantage of this technique is that purification of products is

easier [85]. For example Monascus has been successfully cultured submerged condition for

pigment production and versatile substrates like breads, rice and other amylaceous (starch,

dextrins, glucose, maltose and fructose) materials for high productivity of red pigments which

occurs due to glucose and maltose utilization [85]. Carotenoid production by Aspergillus

sp.[86] melanin type pigment by Aspergillus niger [87], water soluble red pigment by

Penicillium purpurogenum [88]are some reported studies from fungi. Furthermore many

36
bacterial pigments too successfully produced using this technique [15]. Various commercial

scale microbial pigments such as prodigiosin, monascorubramie, astaxanthin, canthaxanthin,

beta-carotene and so on are produced using this method. Under submerged conditions, factors

like nitrogen, pH temperature majorly affect the pigment production [15, 25, 33].

Solid- state fermentation

Solid state fermentation (SSF) is defined as any fermentation process performed on a non-

soluble substance that acts both as physical support and source of nutrients in absence of free

flowing liquid [89]. SSF utilizes solid numerous versatile substrates, like bran, bagasse, and

paper pulp etc. The main advantage of using these substrates is that nutrient-rich waste

materials can be easily recycled as substrates [89-90]. In this fermentation technique, the

substrates are utilized very slowly and steadily, so the same substrate can be used for long

fermentation periods. Hence, this technique supports controlled release of nutrients. SSF is

best suited for fermentation techniques involving fungi and microorganisms that require less

moisture content. However, it cannot be used in fermentation processes involving organisms

that require high aw (water activity), such as bacteria [91]. Many pigments were reported

using this technique [92-93] and among all Monascus pigments lead with many reports [94-

96]. For example red rice, a traditional Asian food, has for thousands of years been prepared

by fermentation of steamed rice by Monascus purpureus in SSF. The fungus produces six

different polyketide pigments colored from bright yellow to deep red, which have found

applications both as food additives and pharmaceuticals [25, 97]. For significant amounts of

pigments by molds, substrates like rice, cassava or corn, wheat, oat and barley were used as

substrates [93]. Aspergillus sp. has been grown successfully on starch medium using different

carbon sources wherein dextrin and maltose produced the highest color intensity [25]. Some

37
significant studies include carotenoid by Penicillium sp. [98], riboflavin, a yellow pigment

from Eremothecium ashbyii and Ashbya gossypi, beta-carotene by Dunaliella and D.

bardawil, astaxanthin by Haematococcus pluvialis, phycocianin by Spirulina sp. [92, 99] etc.

2.9.3 Low-cost substrates

Agro-industrial by-products and surpluses that frequently create serious environmental

problems may be possibly used as inexpensive carbohydrate sources for microbial

fermentations, thus decreasing their initial high biological oxygen demand (BOD) while

obtaining biochemical compounds like pigments suitable for pharmaceutical, chemical and

food industries [100]. Low cost by products and residues of agro-industrial origin have shown

their potential in production of different pigments by diverse group microorganisms. Along

this line, variety of substrates and microorganisms has been tested. beta-carotene synthesis by

citrus products [101], carotenoids production using whey ultrafiltrate [102], sauerkraut brine

[81] and peat extract [103], riboflavin in concentrated rectified grape must [104], astaxanthin

on grape juice [105] are some promising studies.

These by-products from industrial processes and other agro or domestic sources are pollutants

to the environmental and their treatment involves high costs. The conversion of these wastes

to value added materials like pigments by microorganisms would provide economic benefits

and reduce waste materials impact on environment as pollutants. Many investigations have

been performed and are under investigation to diminish the costs and optimize the pigments

production [78, 86, 106-111]. Factors such as carbon and nitrogen source are very important

to consider on the selection of wastes as substrates. And pigment production also depends on

minerals and other components in some cases. Pigments produced on several wastes were

shown in Table 2.3.

38
Table 2.3. Different microorganisms and various inexpensive substrates used for pigments

production.

Substrate Microorganism Pigment type References

Whey R. glutinis β -carotene [112]

Potato medium R. mucilaginosa β -carotene [112]

Crude glycerol R. glutinis carotenoids [113]

Chicken feathers R. glutinis carotenoids [114]

Fermented radish brine R. glutinis β -carotene [115]

Mung bean waste flour and sweet

R. glutinis carotenoids [79]
potato extract

Mustard waste X. dendrorhous Astaxanthin [100]

Plant extracts X. dendrorhous Astaxanthin [116]

Coconut milk X. dendrorhous Astaxanthin [117]

Enzymatic hydrolysates of
X. dendrorhous Astaxanthin [118]
prehydrolysed wood

Sugarcane waste marine Streptomyces sp. melanin [119]

2.10 Objectives of the work

The overall objectives of this study are to explore the possibility of pigment production by

different microbial isolates from numerous sources on various substrates. Furthermore, the

enhanced pigment production capability of carotenoid producing microorganisms was also

investigated using fruit waste material as sole substrates. The influential parameters of

increased pigment production by several optimization techniques were also investigated.

39
The specific objectives of this study are:

 Isolation, identification and characterization of the microorganisms from different

sources responsible for various pigment production

 Investigate and determine effects of substrate (cost-effective), pH, temperature,

moisture, agitation, etc., on the pigment production process by respective

microorganism

 Optimize the key parameters for improved pigment production and validation of the

established procedure

 Exploring the potential of the obtained pigments as antioxidants, metal chelating

agents, photoprotective agents etc.

 Investigating known microbial strains towards enhanced pigment production on

various agro-industrial substrates

References

[1] D. Cristea and G. Vilarem, "Improving light fastness of natural dyes on cotton yarn,"

Dyes and Pigments, vol. 70, pp. 238-245, 2006.

[2] F. Delgado-Vargas, et al., "Natural pigments: carotenoids, anthocyanins, and

betalains—characteristics, biosynthesis, processing, and stability," Critical reviews in

food science and nutrition, vol. 40, pp. 173-289, 2000.

[3] T. Bechtold and R. Mussak, Handbook of natural colorants: John Wiley & Sons,

2009.

[4] F. Delgado-Vargas and O. Paredes-López, Natural colorants for food and

nutraceutical uses: CRC Press, 2002.

40
[5] Z. E. Sikorski, Chemical and functional properties of food components: CRC Press,

2006.

[6] D. M. Freedman, "Reasonable Certainty of No Harm: Reviving the Safety Standard

for Food Additives, Color Additives, and Animal Drugs," Ecology LQ, vol. 7, p. 245,

1978.

[7] J. Neitz, et al., "Color perception is mediated by a plastic neural mechanism that is

adjustable in adults," Neuron, vol. 35, pp. 783-792, 2002.

[8] M. Satake and Y. Mido, Chemistry of colour: Discovery Publishing House, 1995.

[9] M. H. Stafsnes, et al., "Isolation and characterization of marine pigmented bacteria

from Norwegian coastal waters and screening for carotenoids with UVA-blue light

absorbing properties," The Journal of Microbiology, vol. 48, pp. 16-23, 2010.

[10] J. Eyal and M. G. Spencer, "Morchella rotunda sp. useful for producing natural blue

pigment," ed: Google Patents, 1992.

[11] S. Babitha, "Microbial pigments," in Biotechnology for agro-industrial residues

utilisation, ed: Springer, 2009, pp. 147-162.

[12] S. Babitha, et al., "Jackfruit Seed--A novel substrate for the production of Monascus

pigments through solid-state fermentation," Food Technology & Biotechnology, vol.

44, 2006.

[13] H. Sheehan, et al., "Color and scent: how single genes influence pollinator attraction,"

in Cold Spring Harbor symposia on quantitative biology, 2012, pp. 117-133.

[14] S. A. Mapari, et al., "Exploring fungal biodiversity for the production of water-

soluble pigments as potential natural food colorants," Current Opinion in

Biotechnology, vol. 16, pp. 231-238, 2005.

41
[15] C. K. Venil, et al., "Bacterial pigments and their applications," Process Biochemistry,

vol. 48, pp. 1065-1079, 2013.

[16] L. Dufossé, et al., "Microorganisms and microalgae as sources of pigments for food

use: a scientific oddity or an industrial reality?," Trends in Food Science &

Technology, vol. 16, pp. 389-406, 2005.

[17] D. K. Hobson and D. S. Wales, "‘Green’dyes," Journal of the Society of Dyers and

Colourists, vol. 114, pp. 42-44, 1998.

[18] A. B. Soliev, et al., "Bioactive pigments from marine bacteria: applications and

physiological roles," Evidence-Based Complementary and Alternative Medicine, vol.

2011, 2011.

[19] L. Dufossé, "Microbial Production of Food Grade Pigments," Food Technology &

Biotechnology, vol. 44, 2006.

[20] H. J. Nelis and A. De Leenheer, "Microbial sources of carotenoid pigments used in

foods and feeds," Journal of Applied Microbiology, vol. 70, pp. 181-191, 1991.

[21] L. C. Mata-Gómez, et al., "Biotechnological production of carotenoids by yeasts: an

overview," Microbial Cell Factories, vol. 13, p. 12, 2014.

[22] C. K. Venil and P. Lakshmanaperumalsamy, "An insightful overview on microbial

pigment, prodigiosin," Electronic Journal of Biology, vol. 5, pp. 49-61, 2009.

[23] A. Aberoumand, "A review article on edible pigments properties and sources as

natural biocolorants in foodstuff and food industry," World J Dairy Food Sci, vol. 6,

pp. 71-78, 2011.

[24] W. A. Ahmad, et al., Application of Bacterial Pigments as Colorant: Springer, 2012.

42
[25] V. Joshi, et al., "Microbial pigments," Indian journal of biotechnology, vol. 2, pp.

362-369, 2003.

[26] N. Nagpal, et al., "Microbial pigments with health benefits-A mini review," Trends in

Biosciences, vol. 4, pp. 157-160, 2011.

[27] A. Shirata, et al., "Isolation of bacteria producing bluish-purple pigment and use for

dyeing," Japan Agricultural Research Quarterly, vol. 34, pp. 131-140, 2000.

[28] K.-P. Stahmann, et al., "Three biotechnical processes using Ashbya gossypii, Candida

famata, or Bacillus subtilis compete with chemical riboflavin production," Applied

Microbiology and Biotechnology, vol. 53, pp. 509-516, 2000.

[29] D. Asker and Y. Ohta, "Production of canthaxanthin by extremely halophilic

bacteria," Journal of bioscience and bioengineering, vol. 88, pp. 617-621, 1999.

[30] A. A. Woodall, et al., "Carotenoids and protection of phospholipids in solution or in

liposomes against oxidation by peroxyl radicals: relationship between carotenoid

structure and protective ability," Biochimica et Biophysica Acta (BBA)-General

Subjects, vol. 1336, pp. 575-586, 1997.

[31] D. F. Browning, et al., "Light‐induced carotenogenesis in Myxococcus xanthus:

functional characterization of the ECF sigma factor CarQ and antisigma factor CarR,"

Molecular microbiology, vol. 48, pp. 237-251, 2003.

[32] H. Takano, et al., "Light-induced carotenogenesis in Streptomyces coelicolor A3 (2):

identification of an extracytoplasmic function sigma factor that directs

photodependent transcription of the carotenoid biosynthesis gene cluster," Journal of

Bacteriology, vol. 187, pp. 1825-1832, 2005.

43
[33] K. Malik, et al., "Microbial pigments: A review," Int. J. Microbial. Resour. Technol,

vol. 1, pp. 361-365, 2012.

[34] O. Ciferri, "Spirulina, the edible microorganism," Microbiological reviews, vol. 47, p.

551, 1983.

[35] K. H. McClean, et al., "Quorum sensing and Chromobacterium violaceum:

exploitation of violacein production and inhibition for the detection of N-

acylhomoserine lactones," Microbiology, vol. 143, pp. 3703-3711, 1997.

[36] M. Rodríguez-Sáiz, et al., "Xanthophyllomyces dendrorhous for the industrial

production of astaxanthin," Applied Microbiology and Biotechnology, vol. 88, pp.

645-658, 2010.

[37] A. Berry, et al., "Paracoccus zeaxanthinifaciens sp. nov., a zeaxanthin-producing

bacterium," International journal of systematic and evolutionary microbiology, vol.

53, pp. 231-238, 2003.

[38] B. Holmes, et al., "Flavobacterium odoratum: a species resistant to a wide range of

antimicrobial agents," Journal of clinical pathology, vol. 32, pp. 73-77, 1979.

[39] J. A. Duerre and P. J. Buckley, "Pigment production from tryptophan by an

Achromobacter species," Journal of bacteriology, vol. 90, pp. 1686-1691, 1965.

[40] D. Jones, et al., "Taxonomically significant colour changes in Brevibacterium linens

probably associated with a carotenoid-like pigment," Journal of general

microbiology, vol. 77, pp. 145-150, 1973.

[41] M. P. Starr, "The blue pigment of Corynebacterium insidiosum," Archiv für

Mikrobiologie, vol. 30, pp. 325-334, 1958.

44
[42] M. Vázquez, "Effect of the light on carotenoid profiles of Xanthophyllomyces

dendrorhous strains (formerly Phaffia rhodozyma)," Food technology and

biotechnology, vol. 39, pp. 123-128, 2001.

[43] D. Asker and Y. Ohta, "Haloferax alexandrinus sp. nov., an extremely halophilic

canthaxanthin-producing archaeon from a solar saltern in Alexandria (Egypt),"

International journal of systematic and evolutionary microbiology, vol. 52, pp. 729-

738, 2002.

[44] G. Y. Liu, et al., "Staphylococcus aureus golden pigment impairs neutrophil killing

and promotes virulence through its antioxidant activity," The Journal of experimental

medicine, vol. 202, pp. 209-215, 2005.

[45] R. S. Blosser and K. M. Gray, "Extraction of violacein from Chromobacterium

violaceum provides a new quantitative bioassay for N-acyl homoserine lactone

autoinducers," Journal of microbiological methods, vol. 40, pp. 47-55, 2000.

[46] A. A. Deorukhkar, et al., "Identification of a red-pigmented bacterium producing a

potent anti-tumor N-alkylated prodigiosin as Serratia marcescens," Research in

microbiology, vol. 158, pp. 399-404, 2007.

[47] B. A. Kenner and H. P. Clark, "Detection and enumeration of Salmonella and

Pseudomonas aeruginosa," Journal (Water Pollution Control Federation), pp. 2163-

2171, 1974.

[48] A. K. Goel, et al., "Genetic locus encoding functions involved in biosynthesis and

outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae,"

Journal of Bacteriology, vol. 184, pp. 3539-3548, 2002.

45
[49] F. Pantanella, et al., "Violacein and biofilm production in Janthinobacterium

lividum," Journal of Applied Microbiology, vol. 102, pp. 992-999, 2007.

[50] A. Ben-Amotz, et al., "Mode of action of the massively accumulated β-carotene of

Dunaliella bardawil in protecting the alga against damage by excess irradiation,"

Plant Physiology, vol. 91, pp. 1040-1043, 1989.

[51] R. J. McLean, "New taxonomic criteria in the classification of Chlorococcum species.

I. Pigmentation," Journal of Phycology, vol. 4, pp. 328-332, 1968.

[52] E. Christaki, et al., "Functional properties of carotenoids originating from algae,"

Journal of the Science of Food and Agriculture, vol. 93, pp. 5-11, 2013.

[53] V. Álvarez, et al., "The crtS gene of Xanthophyllomyces dendrorhous encodes a novel

cytochrome-P450 hydroxylase involved in the conversion of β-carotene into

astaxanthin and other xanthophylls," Fungal Genetics and Biology, vol. 43, pp. 261-

272, 2006.

[54] M. Rodríguez-Sáiz, et al., "Blakeslea trispora genes for carotene biosynthesis,"

Applied and environmental microbiology, vol. 70, pp. 5589-5594, 2004.

[55] L. Dufossé, "Mikrobna proizvodnja pigmenata hrane," Food technology and

biotechnology, vol. 44, pp. 313-321, 2006.

[56] T. Lotan and J. Hirschberg, "Cloning and expression in Escherichia coli of the gene

encoding β-C-4-oxygenase, that converts β-carotene to the ketocarotenoid

canthaxanthin in Haematococcus pluvialis," FEBS letters, vol. 364, pp. 125-128,

1995.

[57] D. Wild, et al., "New Monascus metabolite isolated from red yeast rice (angkak, red

koji)," Journal of agricultural and food chemistry, vol. 50, pp. 3999-4002, 2002.

46
[58] S. Zhang, et al., "Biosorption of reactive dyes by the mycelium pellets of a new

isolate of Penicillium oxalicum," Biotechnology letters, vol. 25, pp. 1479-1482, 2003.

[59] W. Hsu, et al., "Carotenoid biosynthesis in Blakeslea trispora," Phytochemistry, vol.

11, pp. 2985-2990, 1972.

[60] P. Unagul, et al., "Production of red pigments by the insect pathogenic fungus

Cordyceps unilateralis BCC 1869," Journal of Industrial Microbiology and

Biotechnology, vol. 32, pp. 135-140, 2005.

[61] C. Socaciu, Food colorants: chemical and functional properties: CRC Press, 2007.

[62] P. Velmurugan, et al., "Water‐soluble red pigments from Isaria farinosa and structural

characterization of the main colored component," Journal of basic microbiology, vol.

50, pp. 581-590, 2010.

[63] S. Chaskes and R. L. Tyndall, "Pigment production by Cryptococcus neoformans

from para-and ortho-Diphenols: effect of the nitrogen source," Journal of clinical

microbiology, vol. 1, pp. 509-514, 1975.

[64] B. Bjerkeng, et al., "Digestibility and muscle retention of astaxanthin in Atlantic

salmon, Salmo salar, fed diets with the red yeast Phaffia rhodozyma in comparison

with synthetic formulated astaxanthin," Aquaculture, vol. 269, pp. 476-489, 2007.

[65] P. Buzzini, "Batch and fed‐batch carotenoid production by Rhodotorula glutinis–

Debaryomyces castellii co‐cultures in corn syrup," Journal of Applied Microbiology,

vol. 90, pp. 843-847, 2001.

[66] A. Carreira, et al., "Brown pigments produced by Yarrowia lipolytica result from

extracellular accumulation of homogentisic acid," Applied and environmental

microbiology, vol. 67, pp. 3463-3468, 2001.

47
[67] N. N. Gerber and D. P. Stahly, "Prodiginine (prodigiosin-like) pigments from

Streptoverticillium rubrireticuli, an organism that causes pink staining of polyvinyl

chloride," Applied microbiology, vol. 30, p. 807, 1975.

[68] N. Palleroni, et al., "Production of a novel red pigment, rubrolone, by Streptomyces

echinoruber sp. nov. I. Taxonomy, fermentation and partial purification," The Journal

of antibiotics, vol. 31, pp. 1218-1225, 1978.

[69] W. A. Schroeder and E. Johnson, "Antioxidant role of carotenoids in Phaffia

rhodozyma," Journal of general microbiology, vol. 139, pp. 907-912, 1993.

[70] A. Mortensen, "Carotenoids and other pigments as natural colorants," Pure and

Applied Chemistry, vol. 78, pp. 1477-1491, 2006.

[71] N. Durán, et al., "Ecological-friendly pigments from fungi," Critical reviews in food

science and nutrition, vol. 42, pp. 53-66, 2002.

[72] J. C. d. Carvalho, et al., "Biopigments from Monascus: strains selection, citrinin

production and color stability," Brazilian Archives of Biology and technology, vol. 48,

pp. 885-894, 2005.

[73] S. C. Robinson, et al., "Utilizing pigment-producing fungi to add commercial value to

American beech (Fagus grandifolia)," Applied Microbiology and Biotechnology, vol.

93, pp. 1041-1048, 2012.

[74] A. Bessette, Mushrooms of North America in color: a field guide companion to

seldom-illustrated fungi: Syracuse University Press, 1995.

[75] Z. Lin, et al., "Polyketides from Penicillium sp. JP-1, an endophytic fungus associated

with the mangrove plant Aegiceras corniculatum," Phytochemistry, vol. 69, pp. 1273-

1278, 2008.

48
[76] W.-L. Chu, "Potential applications of antioxidant compounds derived from algae,"

Current Topics in Nutraceutical Research, vol. 9, 2011.

[77] J.-B. Pouvreau, et al., "Antioxidant and free radical scavenging properties of

marennine, a blue-green polyphenolic pigment from the diatom Haslea ostrearia

(Gaillon/Bory) Simonsen responsible for the natural greening of cultured oysters,"

Journal of agricultural and food chemistry, vol. 56, pp. 6278-6286, 2008.

[78] M. Vazquez and A. M. Martin, "Optimization of Phaffia rhodozyma continuous

culture through response surface methodology," Biotechnology and bioengineering,

vol. 57, pp. 314-320, 1998.

[79] J. Tinoi, et al., "Simplex optimization of carotenoid production by Rhodotorula

glutinis using hydrolyzed mung bean waste flour as substrate," Process Biochemistry,

vol. 40, pp. 2551-2557, 2005.

[80] E. D. Simova, et al., "Effect of aeration on the production of carotenoid pigments by

Rhodotorula rubra-lactobacillus casei subsp. casei co-cultures in whey ultrafiltrate,"

Zeitschrift fur Naturforschung C-Journal of Biosciences, vol. 58, pp. 225-229, 2003.

[81] C. Shih and Y. Hang, "Production of Carotenoids by Rhodotorula rubra from

Sauerkraut Brine," LWT-Food Science and Technology, vol. 29, pp. 570-572, 1996.

[82] V. A. Portnoy, et al., "Adaptive laboratory evolution—harnessing the power of

biology for metabolic engineering," Current Opinion in Biotechnology, vol. 22, pp.

590-594, 2011.

[83] M. Sonderegger and U. Sauer, "Evolutionary engineering of Saccharomyces

cerevisiae for anaerobic growth on xylose," Applied and environmental microbiology,

vol. 69, pp. 1990-1998, 2003.

49
[84] Y. Chen, et al., "Screening and characterization of astaxanthin-hyperproducing

mutants of Haematococcus pluvialis," Biotechnology letters, vol. 25, pp. 527-529,

2003.

[85] M. A. Dhale, "Physiology of Monascus purpureus in relation to metabolite production

and application as functional food," India: Doctor thesis of University of Mysore,

2007.

[86] K. Sanjay, et al., "Optimization of carotenoid production by Aspergillus carbonarius

in submerged fermentation using a response surface methodology," International

Journal of Food Engineering, vol. 3, 2007.

[87] T. R. Jørgensen, et al., "Submerged conidiation and product formation by Aspergillus

niger at low specific growth rates are affected in aerial developmental mutants,"

Applied and environmental microbiology, vol. 77, pp. 5270-5277, 2011.

[88] A. Méndez, et al., "Red pigment production by Penicillium purpurogenum GH2 is

influenced by pH and temperature," Journal of Zhejiang University SCIENCE B, vol.

12, pp. 961-968, 2011.

[89] A. Pandey, et al., "Types of fermentation and factors affecting it," Biotechnology:

Food Fermentation, Educational Publishers, New Delhi, pp. 383-426, 1999.

[90] S. R. Couto and M. A. Sanromán, "Application of solid-state fermentation to food

industry—a review," Journal of Food Engineering, vol. 76, pp. 291-302, 2006.

[91] K. Babu and T. Satyanarayana, "α-Amylase production by thermophilic Bacillus

coagulans in solid state fermentation," Process Biochemistry, vol. 30, pp. 305-309,

1995.

50
[92] L. Thomas, et al., "Current developments in solid-state fermentation," Biochemical

Engineering Journal, vol. 81, pp. 146-161, 2013.

[93] U. Hölker, et al., "Biotechnological advantages of laboratory-scale solid-state

fermentation with fungi," Applied Microbiology and Biotechnology, vol. 64, pp. 175-

186, 2004.

[94] A. Rosenblitt, et al., "Solid substrate fermentation of Monascus purpureus: growth,

carbon balance, and consistency analysis," Biotechnology progress, vol. 16, pp. 152-

162, 2000.

[95] S. Babitha, et al., "Solid-state fermentation for the production of Monascus> pigments

from jackfruit seed," Bioresource Technology, vol. 98, pp. 1554-1560, 2007.

[96] O. Han and R. E. Mudgett, "Effects of Oxygen and Carbon Dioxide Partial Pressures

on Monascus Growth and Pigment Production in Solid‐State Fermentations,"

Biotechnology progress, vol. 8, pp. 5-10, 1992.

[97] M. R. Johns and D. M. Stuart, "Production of pigments by Monascus purpureus in

solid culture," Journal of industrial microbiology, vol. 8, pp. 23-28, 1991.

[98] J.-R. Han, "Sclerotia growth and carotenoid production of Penicillium sp PT95 during

solid state fermentation of corn meal," Biotechnology letters, vol. 20, pp. 1063-1065,

1998.

[99] A. Pandey, "Recent process developments in solid-state fermentation," Process

Biochemistry, vol. 27, pp. 109-117, 1992.

[100] J. Tinoi, et al., "Utilization of mustard waste isolates for improved production of

astaxanthin by Xanthophyllomyces dendrorhous," Journal of Industrial Microbiology

and Biotechnology, vol. 33, pp. 309-314, 2006.

51
[101] A. Ciegler, et al., "Enhancement of β-carotene synthesis by citrus products," Applied

microbiology, vol. 11, pp. 128-131, 1963.

[102] G. Frengova, et al., "Formation of carotenoids by Rhodotorula glutinis in whey

ultrafiltrate," Biotechnology and bioengineering, vol. 44, pp. 888-894, 1994.

[103] A. M. Martin, et al., "Growth parameters for the yeast Rhodotorula rubra grown in

peat extracts," Journal of fermentation and bioengineering, vol. 76, pp. 321-325,

1993.

[104] P. Buzzini and J. Rossi, "Semi-continuous and continuous riboflavin production by

calcium-alginate-immobilized Candida saitoana 4253 in concentrated rectified grape

must.[Erratum: Feb 1999, v. 15 (1), p. 153-159.]," World journal of microbiology &

biotechnology, 1998.

[105] P. Meyer and J. Du Preez, "Astaxanthin production by a Phaffia rhodozyma mutant

on grape juice," World journal of microbiology and biotechnology, vol. 10, pp. 178-

183, 1994.

[106] S. N. Surwase, et al., "Optimization of melanin production by Brevundimonas sp. SGJ

using response surface methodology," 3 Biotech, vol. 3, pp. 187-194, 2013.

[107] P. Park, et al., "Optimization of carotenoid production by Rhodotorula glutinis using

statistical experimental design," World journal of microbiology and biotechnology,

vol. 21, pp. 429-434, 2005.

[108] F. Mantzouridou, et al., "Optimization of β-carotene production from synthetic

medium by Blakeslea trispora in a stirred tank reactor and relationship between

morphological changes and pigment formation," Food Biotechnology, vol. 16, pp.

167-187, 2002.

52
[109] C. Malisorn and W. Suntornsuk, "Optimization of β-carotene production by

Rhodotorula glutinis DM28 in fermented radish brine," Bioresource Technology, vol.

99, pp. 2281-2287, 2008.

[110] B. D. H. Dung, et al., "Optimization carotenoids production by Rhodotorula glutinis

GBDO Uusing design of Plackett-Burman matrix and central composite design-

response surface methodology," in Conference proceedings of Biotechnology for

Green Solutions and Sustainable Environment, 2010, p. 25.

[111] P. Bhosale and R. Gadre, "Optimization of carotenoid production from

hyper‐producing Rhodotorula glutinis mutant 32 by a factorial approach," Letters in

applied microbiology, vol. 33, pp. 12-16, 2001.

[112] I. Marova, et al., "Use of several waste substrates for carotenoid-rich yeast biomass

production," Journal of Environmental Management, vol. 95, pp. S338-S342, 2012.

[113] C. Saenge, et al., "Potential use of oleaginous red yeast Rhodotorula glutinis for the

bioconversion of crude glycerol from biodiesel plant to lipids and carotenoids,"

Process Biochemistry, vol. 46, pp. 210-218, 2011.

[114] M. Taskin, et al., "Use of waste chicken feathers as peptone for production of

carotenoids in submerged culture of Rhodotorula glutinis MT-5," European Food

Research and Technology, vol. 233, pp. 657-665, 2011.

[115] C. Malisorn and W. Suntornsuk, "Improved β-carotene production of Rhodotorula

glutinis in fermented radish brine by continuous cultivation," Biochemical

Engineering Journal, vol. 43, pp. 27-32, 2009.

[116] S. Kim, et al., "Increased carotenoid production in Xanthophyllomyces dendrorhous

G276 using plant extracts," Journal of Mmicrobiology-Seoul-, vol. 45, p. 128, 2007.

53
[117] A. Domínguez-Bocanegra and J. Torres-Muñoz, "Astaxanthin hyperproduction by

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as

sole source of energy," Applied Microbiology and Biotechnology, vol. 66, pp. 249-

252, 2004.

[118] J. Parajó, et al., "Production of carotenoids by Xanthophyllomyces dendrorhous>

growing on enzymatic hydrolysates of prehydrolysed wood," Food Chemistry, vol.

60, pp. 347-355, 1997.

[119] V. Vasanthabharathi, et al., "Melanin production from marine Streptomyces," African

Journal of Biotechnology, vol. 10, pp. 11224-11234, 2013.

54
Chapter 3

Screening of inexpensive substrates for pigments

production by isolated and known microbial strains -

Bridge to Chapter 3 to 8

3.1 Melanins and Carotenoids - pigments of marketable importance

Owing to a growing worldwide market for pigment compounds by microbial production,

carotenoids and melanin compounds has great potential in terms of applications and strategies

of production. Among both, carotenoids occur as natural colorants with a range of yellow to

red colors, so they have great influence on the acceptability of many foods [1-2]. Moreover,

some carotenoids are precursors of vitamin A; in terms of human health, they are amongst the

bioactive phytochemicals credited that reduce risks for degenerative diseases such as cancer,

cardiovascular diseases, macular degeneration and cataract [3-4]. Carotenoids are naturally

occurring lipid-soluble pigments, the majority being C40 terpenoids, which act as membrane

protective antioxidants scavenging O2 and peroxyl radicals; their antioxidant ability is

55
apparently credited to their structure. Carotenoids pigments occur commonly in

photosynthetic systems of higher plants, algae and phototrophic bacteria. On the other hand,

in non-photosynthetic organisms, carotenoids are significant in protecting against photo-

oxidative damage. Thus, many non-phototrophic bacteria and fungi rely on carotenoids for

protection when growing on conditions where light and air are copious [3-7]. The carotenoids

production by the organisms follow two distinct pathways, the well-known mevalonate

(MVA) pathway and the relatively new 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

This pathway produce isopentenyl diphosphate (IPP) and its isomer of dimethylallyl

diphosphate (DMAPP), the universal building blocks for the synthesis of all carotenoids.

Eukaryotes generally use the MVA pathway to convert acetyl–CoA to IPP, which is

subsequently isomerized to DMAPP. Prokaryotes, with some exceptions, use the MEP

pathway to produce IPP and DMAPP via an initial condensation reaction between pyruvate

and glyceraldehyde-3-phosphate (G3P). Plants and Streptomycetes follow both pathways for

the synthesis of these commercially significant pigments [8-9].

Further coming to melanin compounds, these are interesting materials that are

currently being used in diverse fields such as medicine, pharmacology, cosmetics etc. In

addition, many different technological applications including photo-voltaics, sensors,

electronic devices and may prove to be a novel class of semiconducting polymers, bio-

compatibles and with a readily available natural source as natural materials extracted from

pigmented [10-11]. However, despite momentous scientific effort over the past 30 years, the

basic functions of melanins are still a matter of controversy and speculation. This uncertainty

imparts from the few and poorly defined structural and physical–chemical properties that are

known in these molecules that continue to mystify researchers even though their adaptive

56
importance has already been proven [10, 12]. These materials are predominantly intractable

from an analytical perspective because they are chemically and photo-chemically very stable,

and they are virtually insoluble in most common solvents. In microorganisms, melanins may

be found in both extracellular or intracellular environments, and they pretence very

interesting targets for molecular structure determination and organic synthesis [13-16].

Melanins constitute a general class of complex, polyphenolic heteropolymers and are usually

divided into brown–black melanins (called as eumelanins) and brown, red or yellow melanins

(called as pheomelanins). A number of bacterial species have been testified to produce

melanins [11, 17]. These compounds can be produced in the Actinomiceto or Streptomicetos

groups, Bacillus and Azotobacter genuses [11, 18]; however, very few of these pigments have

been molecularly defined. The well-known melanisation pathway is the classic Mason–Raper

pathway. In this pathway, tyrosinases yield the melanin intermediate dihydroxyphenyl-

alanine (DOPA), dihydroxyindole (DHI) and dihydroxyindole carboxylic acid (DHICA) [10-

11, 19]. They are some other melanin types and other synthesis pathways are also reported

that produce melanins; for example, pyomelanin is derived from the catabolism of tyrosine

via p -hydroxyphenylpyruvate and homogentisic acid (HGA) [20].

First and foremost, production of melanin is one of the most universal adaptations of

living organisms to the variable conditions of the earth and these pigments. As these

pigments are widespread among microorganisms, extensive research focused on exploration

around this ubiquitous pigment which of commercial interest and have great application

potentials in the agriculture, cosmetics and pharmaceutical industries. Additionally, the

widespread synthesis calibre of catotenoids by heterotrophic and non-phototropic bacteria,

fungi and yeast suggests that these pigments are crucial for the viability of these organisms in

57
their natural environment. Carotenoids are the largest and most diverse class of natural

pigments known to mankind and they have enormous commercial significance with

promising applications from food to textiles. The first part of our investigation engaged in

isolation of individual melanin and carotenoid producing microorganisms which are majorly

at RP and DS of research. Here microbial cultures were collected from various natural

resources such as marine and soil samples The second part of our work involves the

employment of the selective pigment producing microbial isolates on various low-cost by-

products and solids as substrates for effective pigment (caroteniod or melanin) production.

This chapter ends by extensive screening of the collected inexpensive wastes as sole

substrates for pigment production by the purchased microbial strains.

3.2 Sampling

Water

Marine water samples were collected from different sites along the coast of Vishakhapatnam

beach, Andhra Pradesh, India. Sampling locations were shown in Table 3.1.

Soil

Soil sample was collected from the garden of NIT campus Rourkela. The obtained soil was

thoroughly washed with sterile distilled water to separate the floating dust and debris.

The soil suspension and the collected water samples were then aseptically diluted

serially (10-7 and 10-10 times where ever necessary) and 1ml of it was inoculated on various

mediums and observed for various diffusible pigments and/or pigmented colonies. All the

isolates were cultivated at ambient conditions i.e. at pH 7 and temperature 30 0C. For liquid

media an agitation of 150 rpm was maintained throughout the incubation process.

58
3.3 Screening of pigment producing microorganisms

Isolates from different sources were cultured on their respective growth media and at

specified growth conditions; these were awaited for 2-10 days for visible colored colonies.

Primarily, after incubation for two days, marine isolate from deep sea water gave creamy and

purple colonies; while isolates from shore (near rocks) gave yellow and black colonies. The

observed colonies were picked and cultured separately for individual pure cultures. Coming

to soil isolate; nutrient agar was used as culture medium. Here various colonies were seen

after 2 days, whereas orange pigmented colonies were observed after 10 days of incubation.

These colored colonies were picked and sub-cultured for further experimentation.

3.4 Different substrates and Cultivation of isolates/purchased strains

In this work, cabbage (Brassica oleracea) waste was prepared from the outer leaves of

cabbage that are peeled off before cabbages are distributed in the market. And potato peels

and jack fruit seeds were collected from domestic disposed wastes. The obtained raw wastes

were dried in oven at 60 0C for 12 hrs, powdered and used as substrates for screening

microbial isolates for pigment production. A 5 % solution was prepared from the respective

powders and used as liquid medium for the cultivation of isolates.

Free water or liquid medium does not appear to be natural habitat for the majority of

the microorganisms. Solid substrates in this case enable favorable conditions (close to the

natural environment to which microorganisms are adapted) of growth especially for wild type

microorganisms. Therefore solid substrates such as rice, corn and wheat flour powders

(obtained from local market) were explored by the isolated microorganisms for pigment

59
production. Substrate medium was prepared by autoclaving (at 15 lb pressure for 10 min) 3 g

of individual powders in a petridish (10 cm) with 8 mL distilled water.

Another waste used in the work was from fruit wastes. Fruit waste was obtained from

a fruit juice shop of a local market. The material used is from single batch i.e. used in all the

experiments to minimise the disturbances in the results due to variations in composition. The

waste contains major portion of pine apple and orange waste and minor portions of

pomegranate waste. Fruit waste includes extracted carpels of oranges, core of pineapples, and

crushed seeds along with arils of pomegranate. The soluble sugars are extracted from 1

kilogram of fruit waste by adding 2 litres of distilled water and boiled at 100 0C for 30

minutes. The resultant straw colour fruit waste extract (FWE) was filtered and stored at 4 0C

for further experimentation.

The prepared solid and liquid media are used for pigment production studies by

marine and soil microbial isolates. These were incubated for 6-7 days at 25 °C and REMI

shaking incubator (operated at 150 rpm) was used for liquid medium substrates. Carotenooid

producing strains Rhodotorula rubra. (MTCC no: 1446) Xanthophyllomyces dendrorhous

(MTCC No: 7536) were obtained from imtech Chandigarh and were cultivated on above

specified wastes for pigment production.

3.5 Strain/substrate selection and further experimentation

Selection of the appropriate substrates - for microbial isolates

The pigment compounds presences in various wastes along with selective media by microbial

isolates are shown in Table 3.1. Upon exploitation, marine isolate taken from near shore area

60
resulted in black color which is moderate in vegetable waste medium as substrate when

compared to significant color change in marine agar.

Furthermore two microbial strains isolated from garden soil; independently resulted in black

color when cultivated on fruit waste extract medium, and an orange color in solid rice powder

medium when used as solitary substrates. Rice powder medium was significantly colored in

orange than the defined nutrient agar medium by garden soil isolate.

The mediums devoid of color change were discarded and omitted from the study. Vegetable

waste from cabbage, fruit waste extract and rice powder are therefore selected for additional

investigations.

Selection of the appropriate substrates - for carotenoid producing microorganisms

Like microbial isolates, various growth mediums were tested by the purchased

microorganisms (yeast) which are potent carotenoid producers on defined and undefined

mediums. Color change of the media was observed for the selection of the best substrate for

pigment production by the obtained microbes (Table 3.2).

Among all the mediums, fruit waste extract medium significantly changed its color when

cultivated by respective yeast. R. rubra produced vibrant pink-orange color, while X.

dendrorhous gave intense orange-red to the medium comparable to the standard nutrient

medium. This FWE medium was subjected to further investigation using these yeasts.

61
 Table 3.1. Screening of various substrates by marine water and soil isolates for pigments

production. ++, + - indicate qualitative significance of high and moderate pigment production

Sampling Color/pigmented Marine Nutrient FWE Vegetable waste Jack Rice Corn Wheat
location colonies agar agar Cabbage Potato fruit powder flour flour
observed waste peels seed
waste
Marine Deep Creamy, purple ++ - - - - - - - -
sea
water
Shore Black ++ - - + - - - - -
Yellow ++ - - - - - - - -
Soil Garden Black ++ ++ - - - - - -

Orange + - - - - ++ - -

Table 3.2. Screening of various substrates for pigments production by purchased strains. ++,

+ - indicate qualitative significance of high and moderate pigment production

Species Nutrient FWE Vegetable Potato Jack fruit Rice Corn Wheat flour
medium waste peels seed powder flour
powder
Rhodotorula rubra. ++ ++ - - - - - -
(MTCC no: 1446)

Xanthophyllomyces ++ ++ - - - - - -
dendrorhous
(MTCC No: 7536)

62
3.6 Summary

Use of microbial pigments in processed foods, cosmetics and in other applications is

promising with large economic potential. However, microbial pigments present several

challenges due to high cost, lower stability and disparity in shades due to changes in pH.

At present, none of the microbial pigment can replace synthetic pigments. But recent

advances and efforts in synthetic biology, metabolic engineering etc. will greatly expand the

pigments that could be produced economically in sufficient amounts for industrial

application.

Extensive efforts on fermentation process, utilization of low-cost by-products and

residues of agro-industrial origin as alternative substrates have shown the possibility of

attaining non-toxic microbial pigments in significant amounts. Research in this area is

escalating as the outputs achieved here are fast, have economic potential with less efforts and

these technologies can be implemented immediately than the time taking genetic engineering

of microorganisms. Utilization of wastes, screening of novel microorganisms for non-toxic

pigments, optimization of production conditions are our major objectives of focus in the

coming chapters of the work.

References

[1] L. Ruan, et al., "Melanin Pigment Formation and Increased UV Resistance in Bacillus

thuringiensis Following High Temperature induction," Systematic and applied

microbiology, vol. 27, pp. 286-289, 2004.

[2] S. Kim, et al., "Increased carotenoid production in Xanthophyllomyces dendrorhous

G276 using plant extracts," Journal of Microbiology-Seoul-, vol. 45, p. 128, 2007.

63
[3] M. B. Sporn, The retinoids vol. 1: Academic Press, 1984.

[4] C. N'Soukpoe-Kossi, et al., "Retinol and retinoic acid bind human serum albumin:

stability and structural features," International journal of biological macromolecules,

vol. 40, pp. 484-490, 2007.

[5] T. Lóránd, et al., "FT-IR study of some seco-and apocarotenoids," Journal of

biochemical and biophysical methods, vol. 53, pp. 251-258, 2002.

[6] K. Jacob, et al., "Stability of carotenoids, phenolic compounds, ascorbic acid and

antioxidant capacity of tomatoes during thermal processing," Archivos

Latinoamericanos de Nutricion (ALAN), vol. 60, p. 192, 2010.

[7] G. T. Hayman, et al., "Production of carotenoids by Phaffia rhodozyma grown on

media composed of corn wet-milling co-products," Journal of industrial

microbiology, vol. 14, pp. 389-395, 1995.

[8] A. Das, et al., "An update on microbial carotenoid production: application of recent

metabolic engineering tools," Applied Microbiology and Biotechnology, vol. 77, pp.

505-512, 2007.

[9] L. C. Mata-Gómez, et al., "Biotechnological production of carotenoids by yeasts: an

overview," Microbial Cell Factories, vol. 13, p. 12, 2014.

[10] S. Sajjan, et al., "Purification and physiochemical characterization of melanin

pigment from Klebsiella sp. GSK," Journal of microbiology and biotechnology, vol.

20, pp. 1513-1520, 2010.

[11] A. M. Gómez-Marín and C. I. Sánchez, "Thermal and mass spectroscopic

characterization of a sulphur-containing bacterial melanin from Bacillus subtilis,"

Journal of Non-Crystalline Solids, vol. 356, pp. 1576-1580, 2010.

64
[12] V. Coyne and L. al-Harthi, "Induction of melanin biosynthesis in Vibrio cholerae,"

Applied and environmental microbiology, vol. 58, pp. 2861-2865, 1992.

[13] J. D. Laskin, et al., "Control of melanin synthesis and secretion by B16/C3 melanoma

cells," Journal of cellular physiology, vol. 113, pp. 481-486, 1982.

[14] S. Youngchim, et al., "Production of melanin by Aspergillus fumigatus," Journal of

medical microbiology, vol. 53, pp. 175-181, 2004.

[15] W. W. van de Sande, et al., "Melanin biosynthesis in Madurella mycetomatis and its

effect on susceptibility to itraconazole and ketoconazole," Microbes and Infection,

vol. 9, pp. 1114-1123, 2007.

[16] E. Harki, et al., "Purification, characterisation and analysis of melanin extracted from

Tuber melanosporum Vitt," Food Chemistry, vol. 58, pp. 69-73, 1997.

[17] J. Ravishankar, et al., "Isolation and characterization of melanin from a marine

fungus," Botanica Marina, vol. 38, pp. 413-416, 1995.

[18] J. M. Wang, et al., "The Research Progress of Melanin," Advanced Materials

Research, vol. 204, pp. 2057-2060, 2011.

[19] F. G. Cánovas, et al., "The role of pH in the melanin biosynthesis pathway," Journal

of Biological Chemistry, vol. 257, pp. 8738-8744, 1982.

[20] S. I. Kotob, et al., "Homogentisic acid is the primary precursor of melanin synthesis

in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana," Applied and

environmental microbiology, vol. 61, pp. 1620-1622, 1995.

65
Chapter 4

Approach and investigations

Part I - Isolation of Pigment Producing Bacteria and the

Production of Pigments on Cheaper Substrates

4.1 Melanin from Isolated Marine Pseudomonas sp. using

Vegetable waste

66
4.1.1 Introduction

Melanins are diverse group of macromolecules, synthesized ubiquitously in living organisms

by oxidative polymerization of various phenolic substances in the process of adaption [1]. In

nature, melanins act as photoprotectants (against UV and visible light), charge transport

mediators, free-radical scavengers, antioxidants, metal ion balancers and etc. [2]. Melanins

also have applications in agriculture, medicine, cosmetic and pharmaceutical industries. In

general, melanins are negatively charged, hydrophobic, high molecular weight compounds

with amorphous nature. These are insoluble in common organic solvents, aqueous acids and

water [1],[3].

Based on color and structural classes primarily there are three types of melanins i.e.

eumelanins, pheomelanins and allomelanins. Eumelanins are black to brown color pigments

produced by melanisation by classic Mason-Raper pathway, which produce tyrosine

intermediates or metabolites by the action of tyrosinases. Pheomelanins are brown, red or

yellow color pigments which are produced in course of oxidation of tyrosine and/or

phenylalanine to dihydroxyphenylalanine (DOPA) and dopaquinone. Pheomelanin results

from cysteinylation of DOPA and these are sulphur containing compounds. Allomelanins

include nitrogen free heterogeneous group of polymers formed from catechol precursors

[3],[4]. The eumelanins and pheomelanins commonly occur in animal species, while

allomelanins can be seen in microorganisms and plants [5]. Some of the funguses known to

produce melanins are Cryptococcus neoformans, Sporothrix schenckii, Sepia officinalis,

Aspergillus niger, Penicillium marneffei, Paracoccidioides brasiliensis,

Histoplasmacapsulatum, C. neoforman [6]. some bacterial species include Aeromonas

salmonicida, Azotobacter, Mycobacterium, Micrococcus, Bacillus, Legionella, Streptomyces,

67
Rhizobium, Vibrio, Proteus, Azospirillum, Pseudomonas aeruginosa, Hypomonas sp,

Burkholderia cepacia, E. coli, Bordetella pertusis, Campylobacter jejuni, Yersinia pestis etc.

[2],[5].

Apart from terrestrial microorganisms, explorations of melanin production by marine

microorganisms appear to be inadequate due to limited literature. For instance, Kotob et al [7]

synthesized marine melanin from Vibrio cholerae, a Hyphomonas strain, and Shewanella

colwelliana. They reported that the formed melanin was pyomelanin that resulted due to

catabolism of tyrosine via Tyrosine degradation pathway. Another study by marine bacterium

genus Alteromonas produced melanin in-vivo with the aid of tyrosine precursors [8].

Similarly, other marine bacteria capable of producing melanin are Marinomonas

mediterranea MMB-1T which belong to the phylum Proteobacteria [9] and thermo-

alkaliphilic Streptomyces (from limestone quarries of the Deccan traps) [10]. Apart from

bacteria, a study by obligate marine fungus Cirrenalia pygmea showed melanin production

ability in its mycelium and conidia [11].

Overall, melanin pigment from various microbial species especially from marine

species is an attractive option of research still in its infancy. In this study, we describe a

procedure for the isolation of melanin producing marine microorganism (Pseudomonas sp.)

and characterized biochemically. Melanin producing ability of the isolate was further tested

on vegetable waste (pure and blended with marine broth) at ambient temperature and pH. The

structure of the bacterium and pigment nature was identified by scanning electron microscopy

and the synthesized melanin was analyzed spectrophotometrically. The extracted and purified

pigment was characterized using energy dispersive spectroscopy and Fourier infrared

spectroscopy.

68
4.1.2 Materials and methods

4.1.2.1 Chemicals used

Marine agar, marine broth, DPPH (2,2-diphenyl-1-picrylhydrazyl) were procured from

HiMedia chemicals, Mumbai, India. Ethanol, NaCl, NaOH, HCL and all other chemicals

used were of analytical reagent grade throughout the study. Ultrapure water used for the

experiments and aseptic conditions were maintained wherever necessary.

4.1.2.2 Screening and isolation of the melanin producing strain

Microorganisms capable of producing melanin were isolated from the sea water samples

collected from three different locations i.e. nearby rocks (sample 1), shore (sample 2) and

from deep sea water [10 m away from shore] (sample 3) of Vishakapatnam beach, Andhra

Pradesh, India. 0.1 mL of diluted water samples of 10-7 dilution was individually spreaded on

marine agar plates at pH 7.0. The media and the glassware were autoclaved at 15 psi (121 0C)

for 20 min prior to the experiment. These agar plates with media and inoculum were

incubated at 25 0C for 48 h. Melanin producing microorganisms were identified by the

presence of microbial colonies with dominant thick black color (diffusible) in agar plates.

Selective colonies were separated out for sub-culturing and characterization.

4.1.2.3 Pigment production, extraction and purification

Marine broth medium was used for inoculum preparation and pigment production. About 10

µL (108 CFU/mL) culture suspension was added to 50 mL marine broth in 250 mL flasks.

This medium was then incubated at 25 0C on a rotary shaker moving at 200 rpm for 48 to 72

h until the liquid medium was darkly pigmented and nearly opaque. All media used for the

study were sterilized by autoclaving unless elsewhere stated. After a specific incubation time,

the medium was centrifuged by a centrifuge (REMI-RM12C, India) at 8000 rpm for 15 min

69
to separate the broth (supernatant) and the cells. The solid pellet of cells was separated and

suspended in distilled water. These cells were again centrifuged to collect the supernatant.

Melanin was extracted from the overall supernatant by acidification with 3 N HCl at pH-2

and allowed to stand for 48 h initially at room temperature. This process was repeated for 3

more days until no further precipitation occurred. Then the suspension thus obtained was

boiled for 5 min to prevent the formation of melanoidins [1]. As a final point, crude pigment

pellet was collected after centrifugation at 4000 rpm for 15 min. In addition to marine broth

medium, the pigment production ability of the isolate was tested by culturing them upon

vegetable waste (from cabbage), blend of marine broth and vegetable waste (10:90, 20:80,

30:70) as a nutrient source. Culture conditions and the rest of the protocol maintained same as

described above.

4.1.2.4 DPPH/SPF/metal chelating assay

DPPH assay

DPPH (1, 1diphenyl2picrylhydrazyl) accepts an electron to form a stable diamagnetic

molecule. The methanolic solution of DPPH of violet color has got a strong UV absorbance

at 517nm. The presence of a reducing agent in this methanolic solution pairs the odd

electrons of DPPH radical and further the solution fade color stiochometrically and also the

absorbance of the solution decreases at 517nm. The schematic representation of the process

was shown below

70
The radical scavenging activity by melanin pigment was investigated by modified method of

Ju et al. [12]. Primarily 1 mL of (15‐100 μg/mL) microbial melanin or standard (Ascorbic

acid) was added to 2 mL of DPPH in ethanol. After 30 minutes incubation at 37 oC the

absorbance was measured at 516 nm using UV‐spectrophotometer. Corresponding blanks

were also taken respectively. The experiment was performed in duplicate. The absorbance of

DPPH as control was measured at 516 nm. Lower absorbance of the reaction mixture

indicated higher radical scavenging activity. The scavenging effect (%) was measured using

the following formula:

DPPH inhibition (%) =[(Control absorbance -test absorbance)/Control absorbance] X 100

In vitro sun protection factor

Sunburn, premature skin aging, skin cancers and suppression of the immune system are all

linked to exposure of skin to UV light. The ultraviolet (UV) spectrum of between 200 nm and

400 nm is commonly divided into three regions: UV-A: 320-400 nm; UV-B: 280-320 nm;

UV-C: 200-280 nm.

71
 The highest energy region, UV-C, is absorbed wholly by ozone in the stratosphere.

Among the total solar UV radiation reaching the earth’s surface, 6% is in the UV-B region

and 94% in the UV-A. The potential of UV radiation to cause skin damage increases

exponentially with decreasing wavelength. UV light at 280 nm is 1000 times more damaging

than light at 340 nm, thus, a sunscreen’s ability to block UV-B is more important to prevent

the negative effects of sun exposure.

The severe effects of UV-A and UV-B exposure are both short-lived and reversible.

These effects include mostly sunburn (or erythema) and tanning (or pigment darkening).

Largely sunscreen products contain ingredients that provide adequate protection only against

UV-B rays. Even those labelled as broad-spectrum sunscreens may offer only partial

protection against UV-A radiation. Sunscreens are products applied to the skin to protect

against the harmful effects of the sun's UV rays. Sunscreens are usually grouped into two key

categories, namely chemical absorbers and physical blockers. Chemical absorbers absorb

high-intensity UV rays while physical blockers reflect or scatter them. Chemical absorber

compounds include avobenzone, padimate O, octyl methoxycinnamate, octisalate, and

octocrylene. Physical blocker compounds include titanium dioxide and zinc oxide. The

international standard for quantifying the damaging effects of UV radiation on skin is the

erythemal action spectrum and the Calculation of Sun Protection Factor (SPF) was performed

according to Mansur equation [13] stated below.

As per the adapted method, spectra of melanin samples were collected over the spectral range

400-280 nm with 1 nm data point resolution on a UV-visible UV-3200 double beam

spectrophotometer (LABINDIA analytical Instruments Pvt Ltd, India). The SPF values of

72
melanin from microbial isolate and purchased melanin were determined by Mansur

mathematical equation as follows:

320
SPF  CF   EE   I  Abs 
290

Where, SPF=Sunscreen protection factor; EE(λ)=Erythremal effect spectrum, I(λ)=Solar

intensity spectrum; Abs(λ)=Absorbance of sunscreen product; CF=Correction factor (=36.2

for SNOW LOTUS® SPF 15 and 30) were taken from Huang et al [13].

Determination of metal chelating activity

The ferrozine-based colorimetric assay permits the quantitation of iron in the solution phases

effectively. Ferrous and ferric iron were detected equally well by the assay and the accuracy

was unaffected by other divalent metal cations. Usually Fe (II) ions form a magenta colored

solution with ferrozine that yields a maximum absorbance at 562 nm. This reaction is used to

quantitatively analyse Fe+2 (aq) in solution using a spectrophotometer. The complex is stable

between pH 4 and 10 approximately. This method is applicable in ranges of concentrations

between ~0.010 mg/L and ~ 3mg/L or between 2 and 10 ppm.

The chelation of ferrous ions by the melanin pigment was estimated by the method of Huang

et al. [13]. Different concentrations of melanin were mixed with a solution of 2 mM FeCl 2

(0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the

mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance

of the solution was then measured spectrophotometrically at 562 nm. All the tests and

73
analyses were done in duplicate and averaged. The inhibition of the melanin pigment, metal

chelating activity in percentage (%) was calculated by the following equation:

Metal chelating effect (%)=[(A0-A1)/A0] X 100 %

where A0 is the absorbance of control reaction and A1 is the absorbance in the presence of the

sample of the melanin pigment or standards. The control used contains FeCl2 and ferrozine.

4.1.2.5 Analytical methods

The morphology of the microorganism and the purified pigment was examined by scanning

electron microscope (SEM) [JEOL JSM-6480LV]. The compositional pattern was determined

by energy dispersive X-ray (EDX) spectroscopy which is coupled to SEM. UV-visible

spectrum of the melanin was observed using UV-vis. spectrophotometer (UV-3600

Shimadzu). The FTIR analysis of pigment was carried out after mixing with KBr using FTIR

spectrophotometer (Perkin Elmer, Model No.S2000, USA).

4.1.3 Results and Discussion

4.1.3.1 Strain selection and characterization of the microorganism

Inoculated sea water samples on marine agar plates were observed each day for melanin

producing microorganism. At ambient conditions, during the end of second day (~ 48 h)

visible black color colonies with diffused black color was evident in the agar plates

inoculated with sea water sample 1 (Fig. 4.1.1). The other isolates devoid of black color were

ignored for further study. Colonies form black color marine agar plates were transferred to

new agar plates and allowed to grow for 2 days.

74
 The visible tiny colonies and morphology of the isolated microorganisms observed by

SEM images (Fig. 4.1.2a and b) states that the microbe might be of bacterial origin. The

microbe size range was 1.2 to 1.7 µm and was rod shaped in appearance. For detail evidence

regarding black pigment producing organism, isolated strain was sent for microbial

phenotypic characterization to Institute of microbial technology (IMTECH), Chandigarh,

India. The microorganism was identified as Pseudomonas sp. and is closely related to

Pseudomonas guinea. Table 4.1.1 list out the key morphological and biochemical

characteristics of the isolated bacterial strain.

Fig. 4.1.1. Screening of microbial strains obtained from various parts of a sea shore. A) near

stones, B) near shore and C) 10 m away from sea.

75
Fig. 4.1.2. Low (a) and high (b) magnification SEM images of the isolated microorganism

with black colonies on agar plates.

Table 4.1.1. Colony characteristics of the isolated melanin producing bacterium.

Characteristics Result

Colony morphology

Configuration Circular

Margin Entire

Elevation Raised

Texture Slimy

Opacity Opaque

Gram’s reaction

Cell shape Rods

Spore (s) -

Motility +

76
Growth temperatures

4, 10, 25, 30, 37, 42, 55 0C -, -, +, +, +, +, -

Growth pH’s

5, 6, 7, 8, 9, 10, 11, 12 -, +, +, +, +, +, +, -

Growth on NaCl (%)

0.5, 2.0, 4.0, 6.0, 8.0, 10.0, 11.0, 12.0 +, +, +, +, -, -, -, -

Tests

Catalase test +

Oxidase test +

Voges Proskauer test -

Casein hydrolysis -

Citrate +

Nitrate -

Arginine dihydrolase -

Gelatin hydrolysis -

Starch hydrolysis -

Esculin hydrolysis -

Tween 20 +

Tween 40 +

Tween 60 +

Tween 80 +

DNase -

77
Acid Production from

Cellobiose -

Trehalose -

Fructose +

Maltose +

Dulcitol -

Sucrose +

Dextrose +

Raffinose +

4.1.3.2 Pigment production and characteristics of the produced melanin

The isolated bacterium was incubated in marine broth and vegetable waste up to 48 h for

melanin production. Here, vegetable waste from cabbage leftovers is selected as a growth

medium as it satisfies the major micronutrient append as that of marine broth. Prior to the

addition of inoculum, vegetable waste was supplemented with 1.9 % NaCl to maintain

salinity as that of marine broth.

Pigment production was tested on three different media i.e., marine broth, blend (marine

broth and vegetable waste - (10:90; 20:80 and 30:70 in v/v %), and diluted marine broth 30 %

v/v. Fig. 4.1.3a shows the visible pigment production at significant amount was observed in

30:70 blend medium. This was selected for further study for economical pigment production

Additionally marine broth – vegetable waste blend in 30:70 ratio also used as melanin

production medium for comparison. As melanin pigment started to produce at proportion

78
(30:70) of blended medium; this was selected for further study as it is having maxmium

proportion of vegetable waste and minimum marine broth.

The dark brown to black color (Fig. 4.1.3b) of the marine broth medium indicates

more melanin production by the bacterial isolate than the marine broth - vegetable waste

blended medium (Fig. 4.1.3c). The sole vegetable waste was not given any color but has

cream slimy growth appearance after incubation of 2 days. The observed behaviors indicate

melanin production was strictly medium dependent (i.e. marine broth here). The influence of

medium was clearly evident from Fig. 4.1.3b and 4.1.3c where a 30:70 ratio of marine broth -

vegetable waste blend gave less intense melanin when compared to marine broth alone. The

melanin produced from pure marine broth and marine broth - vegetable waste blend was

found to be 5.35 ± 0.4 and 2.79 ± 0.2 mg/mL after 72 h of incubation. The melanin from

blended medium was found to be ~ 0.52 times lesser than pure marine broth.

However melanin from both sources after purification (by acid treatment) looked

alike in appearance. The physical appearance of the purified melanin was also shown in Fig.

4.1.4 with a true black color typical of melanins in general [14]. The produced melanin was

insoluble in water, ethanol, chloroform, acetone, benzene and slightly soluble in phenol and

1N NaOH. The melanin was precipitated with 6 N HCl and decolorized with the addition of

H2O2. The observed features when compared with previous reports indicate the synthesized

melanin was similar to that of bacterial melanin in properties [1].

79
Fig. 4.1.3. Melanin production on various media: marine broth, blend (marine broth and

vegetable waste - (10:90; 20:80 and 30:70 in v/v %), and diluted marine broth 30 % (a). M

indicates melanin in (a). Dark brownish black colonies due to melanin production in sole

marine broth (b) and in marine broth – vegetable waste blend medium (c).

80
Fig. 4.1.4. Acid treated (a) and purified melanin (b) after centrifugation.

4.1.3.3 Spectroscopy, SEM/EDX and IR analysis of melanin

For a detail, inference and structural elucidation UV-visible spectroscopy, SEM/EDX and

FTIR analysis were performed for the purified melanin pigments from two different media.

The UV-visible wavelength scan showed the absorption was highest at the UV region of 200

to 300 nm, but diminished towards the visible region (Fig. 4.1.5) for both the melanins

obtained. This phenomenon is characteristic to melanin and was due to actual complex

structure of melanin [1].

Fig. 4.1.6 shows the SEM image of the purified melanin. The appearance from the

figure suggests that the material was an amorphous deposit with no differentiable structures,

similar to past reports of purified bacterial melanin [3]. Furthermore, reported studies show

the ability of Pseudomonas sp (P. aeruginosa and P. stutzeri) to produce pyomelanin type

melanin, where the pathway of synthesis includes catabolism of tyrosine via p -

hydroxyphenylpyruvate and homogentisic acid (HGA) [2],[5]. To know the compositional

examination of the obtained melanin, EDX analysis was performed and shown in Fig. 4.1.7.

81
The analyses revealed that the majority composition of the purified melanin from marine

broth alone is of C, O with ~ 66 and 30 weight % and minor S content with ~3.85 %. While,

melanin from blended medium showed C, O with ~ 35.62, 50.29 weight % and minor Ca

content with ~14.09 %. Some peaks of Fig. 4.1.7 are undetectable as EDX may not be a

reliable method to quantify elements in low weight % [3]. This result serve as an additional

support which reflects the purity of the melanin produced. The compositional variation of

melanin pigments might be due to the change in medium compositions.

FTIR spectroscopic analysis was performed on the acid treated purified melanin

pigments to know the information about functional groups and structure. Fig. 4.1.8 shows the

IR spectrum of melanins pressed into KBr disks. Similar spectral pattern from Fig. 4.1.8a and

4.1.8b indicate both melanin pigments obtained are having similar peaks corresponding to

equivalent functional groups. The details of both spectra are as follows:

A broad absorption at 3373 cm-1 indicate the presence of – OH and NH2 groups and

small band at 2918 cm-1 can be assigned to stretching vibration of aliphatic C-H group [15].

The characteristic strong band at 1625 cm-1 (between 1650 - 1620 cm-1) attributed to

vibrations of aromatic ring C=C of amide I C=O and/or of COO- groups. Bands at ~1400 to

1500 cm-1 can be due to aliphatic C-H groups and weak bands below 700 cm-1 ascribed to

alkene C-H substitution in the melanin pigment [1]. The observed IR patterns for the purified

melanins were similar to the earlier reported DOPA-melanin study [4].

82
Fig. 4.1.5. UV-visible spectral properties of melanin pigment obtained from marine broth (a)

and marine broth –vegetable waste medium (b).

Fig. 4.1.6. SEM images of purified bacterial melanin from a) marine broth and b) marine

broth – vegetable waste medium.

83
Fig. 4.1.7. EDX analysis of elemental composition of melanin from a) marine broth and b)

marine broth – vegetable waste medium.

Fig. 4.1.8. FTIR spectrum of the melanin pigment obtained from a) marine broth and b)

marine broth – vegetable waste medium.

84
4.1.3.4 DPPH assay

Melanin particle were found to possess antioxidant property in biological systems. It can

scavenge free radicals and has the ability to sequester redox active metal ions [12]. Free

radical scavenging activity was evaluated by performing in-vitro DPPH assay. Reduction of

absorbance at 516 nm supplied with different melanin doses was shown in Fig. 4.1.9a. The

colored DPPH solution faded and turned dull during the course of incubation of 3 days (Fig.

4.1.9 insert). This may be due to the reduction of the DPPH molecules and electron transfer

from melanin suspension.

Fig. 4.1.9a also indicates a nonlinear pattern of DPPH reduction for various melanin

doses used. A sharp change in absorbance up to 48 h for the used melanin concentrations

indicates that the rate of reduction is rapid at initial stages. The diminished behavior (from

Fig. 4.1.9a) beyond 48 h indicates the maximum threshold reduction by a particular melanin

dose used. The reduction in spectral behavior at various time intervals (in days) was shown in

Fig. 4.1.9b for a melanin dose of 44.7 µg/mL. Moreover from Fig. 4.1.9a, we can plot %

scavenging activity with respect to melanin dosage after a residual period of 72 h (Fig.

4.1.9c). Henceforth, the minimum time period required by the melanin molecules for the

maximum DPPH reduction and dose dependent scavenging activity were successfully valued

in the present study.

85
Fig. 4.1.9. DPPH radical scavenging activity of synthesized melanin pigment with various

doses (a) and UV-vis absorption spectrum of melanin (44.7 µg/mL) - DPPH at different days

along with control containing no melanin (b). Dose dependent scavenging activity of the

synthesized melanin (c). Insert of (a) shows the melanin doses 0, 14.9, 29.8 and 44.7 µg/mL

to 0.1 mM DPPH from left to right.

4.1.3.5 XRD analysis

The XRD spectrum of bacterial melanins and purchased melanin are shown in Fig. 4.1.10a.

The spectra of melanins are characterized by a broad peak which is commonly seen in

amorphous and disordered materials centered at about 24. The observed 2θvalues and

crystallite sizes of the produced bacterial melanins are very close to synthetic (Fig. 4.1.10b).

The inter layer spacing d, is calculated according to the Bragg equation.

where θ is diffraction angle, m is diffraction order and λ is X- ray wave length by considering

first order diffraction (m = 1). The values of d are in good agreement with reported values of

86
the inter layer spacing in the stacked sheets model of the melanin [16]. An estimate of

average grain size of melanins was calculated from the Dedye - schrerrer equation [16].

where FWHM is full width at half maximum of diffraction peak. The closeness of the grain

size values indicates the quality of the purified bacterial melanins. Furthermore %

crystallinity was also calculated for the stated melanins by considering glass substrate as

background. The calculation is as follows:

( total area - background profile area)

% crystallinity  100
(total area)

% crystallinity values from Fig. 4.1.10c indicated that the bacterial melanin from blend and

marine broth were less crystalline than the synthetic melanin studied. Lack of crystallinity is

significant sign of consistent physical property of melanin [17]. And the above results state

that the obtained bacterial melanins are of high pure and amorphous.

Fig. 4.1.10. (a) X-ray diffractograms of the produced melanin (M-marine broth, M-blend) and

purchased melanin (M-synthetic). (b) Interlayer spacing (d-value) and crystallite sizes and (c)

% crystallinity of the different melanins.

87
4.1.3.6 SPF values determination

The determination of SPF values for samples (bacterial and purchased melanin) was made

through the UV spectrophotometric method and the Mansur equation was applied [13]. The

results of different melanins were given in Table 4.1.2. As melanins are known for their

photoprotective role [18], the comparable SPF values of the bacterial melanins to that of

synthetic melanin (Table 4.1.3) indicate the produced melanin has significant photoprotection

activity.

Table 4.1.2. Normalized product function used in the calculation of SPF by Mansur equation.

Wavelength EE x I Abs(λ)
(nm) (normalized) M-MB M-MB+VW M-control
290 0.015 1.776 1.844 1.980
295 0.082 1.656 1.694 1.852
300 0.287 1.536 1.570 1.733
305 0.328 1.411 1.461 1.623
310 0.186 1.297 1.374 1.522
315 0.084 1.207 1.306 1.430
320 0.018 1.149 1.260 1.345
M-melanin; MB-marine broth;VW-vegetable waste

Table 4.1.3. SPF values of different melanins.

S.No. sample SPF value

1 Purchased melanin 59.336 ± 0.006

2 Melanin-marine broth 51.756 ± 0.008

3 Melanin-vegetable waste 53.738 ± 0.004

88
4.1.3.7 The metal binding capacity of melanin

The metal binding capacities of melanin from vegetable waste was determined by assessing

its ability to compete with ferrozine for the ferrous ions. The concentration dependent metal

chelating activity was shown in Fig. 4.1.11. The reduction in spectrum with increase in

melanin dose (Fig. 4.1.11a) indicates that melanin compound was interfering with the

formation of ferrous and ferrozine complex. The dose dependent activity was also clearly

evident upon plotting % metal chelation effect against different melanin doses. Results show

the better chelating effect of melanin than ferrozine towards ferrous ions and maximum effect

(~ 55 % chelation) was observed in a dose of 0.146 mg/mL (Fig. 4.1.11b).

0 mg/ml
0.123 mg/ml
0.24 0.036 mg/ml
0.061 mg/ml
(a) 0.086 mg/ml
0.20 0.110 mg/ml
0.146mg/ml
0.16 0.183mg/ml 60
Metal chelating effect (%)
Absorbance

0.220 mg/ml (b)

50
0.12
40
0.08 30
20
0.04 at 562 nm
10
0.00 0
0.00 0.05 0.10 0.15 0.20 0.25
450 500 550 600 650 700 Melanin dose (mg/ml)
Wavelength (nm)

Fig. 4.1.11. Metal ion chelation effect of the melanin (produced from vegetable waste) (a)

monitored via complete spectrum and (b). at a fixed absorption maximum

89
4.1.4 Conclusions

Though different marine isolates were used, it was found that the sample 1 contains the

melanin producing bacterium and was found to be Pseudomonas guinea. Upon investigation

for the melanin production in different media, 5.35 ± 0.4 and 2.79 ± 0.2 mg/mL pigment was

produced when cultured in marine broth alone and blended medium (vegetable waste and

marine broth), respectively. Pigments produced from the different nutrient sources have

shown different elemental compositions with varying weight %. Moreover, the FTIR analysis

revealed that functional groups were conserved in both the melanins and were appeared to be

same. The compositional variation in the pigments might be due to the change in the media

composition although P.guinea, only was used in both the cases. Furthermore, the produced

melanin noticed to have efficient free radical scavenging activity (of a model DPPH radical)

and also exhibited photoprotective and metal ion chelation activity. This study confirms that

the antioxidant melanin pigment can be produced from the cheaper substrates without any

functional variation.

References

[1] S. Sajjan, et al., "Purification and physiochemical characterization of melanin

pigment from Klebsiella sp. GSK," Journal of microbiology and biotechnology, vol.

20, pp. 1513-1520, 2010.

[2] J. Geng, et al., "Bacterial melanin interacts with double-stranded DNA with high

affinity and may inhibit cell metabolism in vivo," Archives of microbiology, vol. 192,

pp. 321-329, 2010.

90
[3] A. M. Gómez-Marín and C. I. Sánchez, "Thermal and mass spectroscopic

characterization of a sulphur-containing bacterial melanin from Bacillus subtilis,"

Journal of Non-Crystalline Solids, vol. 356, pp. 1576-1580, 2010.

[4] E. Harki, et al., "Purification, characterisation and analysis of melanin extracted from

Tuber melanosporum Vitt," Food Chemistry, vol. 58, pp. 69-73, 1997.

[5] V. Coyne and L. al-Harthi, "Induction of melanin biosynthesis in Vibrio cholerae,"

Applied and environmental microbiology, vol. 58, pp. 2861-2865, 1992.

[6] S. Youngchim, et al., "Production of melanin by Aspergillus fumigatus," Journal of

medical microbiology, vol. 53, pp. 175-181, 2004.

[7] S. I. Kotob, et al., "Homogentisic acid is the primary precursor of melanin synthesis

in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana," Applied and

environmental microbiology, vol. 61, pp. 1620-1622, 1995.

[8] F. Solano, et al., "Isolation and characterization of strain MMB-1 (CECT 4803), a

novel melanogenic marine bacterium," Applied and environmental microbiology, vol.

63, pp. 3499-3506, 1997.

[9] P. Lucas-Elío, et al., "Complete genome sequence of the melanogenic marine

bacterium Marinomonas mediterranea type strain (MMB-1T)," Standards in genomic

sciences, vol. 6, p. 63, 2012.

[10] S. R. Quadri and D. Agsar, "Detection of melanin producing thermo-alkaliphilic

Streptomyces from limestone quarries of the Deccan traps," World Journal of Science

and Technology, vol. 2, 2012.

[11] J. Ravishankar, et al., "Isolation and characterization of melanin from a marine

fungus," Botanica Marina, vol. 38, pp. 413-416, 1995.

91
[12] K.-Y. Ju, et al., "Bioinspired polymerization of dopamine to generate melanin-like

nanoparticles having an excellent free-radical-scavenging property,"

Biomacromolecules, vol. 12, pp. 625-632, 2011.

[13] S. Huang, et al., "Antioxidant activities and UV-protective properties of melanin from

the berry of Cinnamomum burmannii and Osmanthus fragrans," Medicinal Chemistry

Research, vol. 20, pp. 475-481, 2011.

[14] W. W. van de Sande, et al., "Melanin biosynthesis in Madurella mycetomatis> and its

effect on susceptibility to itraconazole and ketoconazole," Microbes and Infection,

vol. 9, pp. 1114-1123, 2007.

[15] J. Coates, "Interpretation of infrared spectra, a practical approach," Encyclopedia of

analytical chemistry, 2000.

[16] V. Capozzi, et al., "Optical and photoelectronic properties of melanin," Thin solid

films, vol. 511, pp. 362-366, 2006.

[17] Y. Thathachari and M. Blois, "Physical studies on melanins: II. X-ray diffraction,"

Biophysical journal, vol. 9, pp. 77-89, 1969.

[18] M. Brenner and V. J. Hearing, "The protective role of melanin against UV Damage in

human Skin†," Photochemistry and photobiology, vol. 84, pp. 539-549, 2008.

92
4.2 Melanin by Soil Microbial Isolate on Fruit Waste

Extract: Optimization of Key Production Parameters

4.2.1 Introduction

Melanins are the natural pigments which have their presence in animals, plants and in most of

the microorganisms [1]. They are the dark colored negatively charged high molecular weight

pigments which are formed due to polymerized phenolic and/or indolic compounds. These

complex polymers are amorphous in nature and shows solubility in neither aqueous nor

organic solvents. They showed resistance to concentrated acids and are susceptible to

bleaching by oxidizing agents [2]. They play a vital role in defense and protection

mechanisms that improve the survival and competitiveness of the organisms [3]. Melanin is

known for its absorption capacity of radiation of all wavelengths with an optimum

absorbance at UV range [4] which prevents photo induced damage. Hence it is used in the

preparation of photo absorbing optical lenses and in bioplastics. Besides photo protection it

has versatile biological activities such as radical scavenging, antioxidant, antitumor, anti-

inflammatory [5] and as immune stimulating agent [6].

93
 Melanin obtained from microbes has great advantages over melanin from animals and

plants. Microorganisms don’t cause the problems of seasonal variations and are selected

arsenals as they modify them according to the medium and conditions provided to them [7].

Targeting melanogenisis in microbes may help to discover antimicrobial drugs. For example,

melanins produced by Cryptococcus neoformans and Burkholderia cepacia offer virulence

and contribute to the growing resistance of these pathogenic bacteria towards antibiotics [2,

8]. The melanin synthesized by microbes shows metal chelating ability too (sorb the

radioactive wastes uranium) [9]. There are reports that showed the anti HIV properties of

melanin and their role in photo voltage generation and fluorescence studies [10-11].

Therefore all these properties of melanin make them unique and are widely used in cosmetic,

sunscreen protection creams, eye glasses, pharmaceuticals, and food industries

Eying on the potential uses and increasing demand for the melanin pigment there is a

need to conduct studies on the production of melanin from microbes. There are reports on

melanin production from various microorganisms including Bacillus species which are well

known for their pigment production ability in various stress environments [4, 12]. Selection

of substrate for melanin production has economic importance. For instance till date expensive

substrates like NCM media [4], LB (Luria-Bertani) media [12], minimal media supplemented

with L-tyrosine [13], amino acids enriched tryptone broth agar [14] and so on [15-16] were

used for high yield of melanin. Owing to the economy and practicability of the melanin

production process; the need to use economically feasible substrates along with optimization

of key parameters is needed. Taguchi method [17] is a systematic technique of design and

analysis of experiments that has been employed successfully in recent years to design and

94
improve product quality economically [18], CCD design approach has been used to fit a

polynomial model.

In this study, a bacterium capable of producing melanin was isolated from garden soil

and subsequently characterized. The strain was cultivated in the fruit waste extract (FWE) [as

sole source of energy] to produce significant amounts of melanin. The key parameters in

melanin production were identified and optimized using simple two step Taguchi and CCD

(central composite design) approach. Upon purification and characterization, the obtained

melanin was tested for In vitro sun protection effect, free radical scavenging and metal

chelating activities.

4.2.2 Materials and methods

4.2.2.1 Chemicals and microorganism

DPPH (2,2-diphenyl-1-picrylhydrazyl), purchased from HiMedia chemicals, Mumbai, India.

Ascorbic acid was purchased from Merck, India. Ferrozine and melanin (synthetic) was

purchased from Sigma-Aldrich, India. Ethanol, NaCl, NaOH, HCL are from Merck, India and

all other chemicals used were of analytical reagent grade throughout the study. Ultrapure

water was used for the experiments and aseptic conditions were maintained wherever

necessary.

The microorganism used in this study was isolated from garden soil in front of

Department of Chemical Engineering, National Institute of Technology, Rourkela, India

using serial dilution technique on a nutrient agar (NA) [19]. Using 107 dilution soil samples

on NA plates. Melanin producing organism was identified. It was separated by observing a

95
diffusible black pigment on NA plates after 24 h. The isolated culture was preserved on NA

slants at 4 0C and sub-cultured at monthly intervals.

4.2.2.2 Substrate preparation

Fruit waste was obtained from a fruit juice shop of a local market. The material used is from

single batch i.e. used in all the experiments to minimize the disturbances in the results due to

variations in composition. The waste contains major portion of pine apple and orange waste

and minor portions of pomegranate waste. Fruit waste includes extracted carpels of oranges,

core of pineapples, and crushed seeds along with arils of pomegranate. The soluble sugars

were extracted from 1 kilogram of fruit waste by adding 2 liters of distilled water and boiled

at 100 0C for 30 minutes. The resultant straw color fruit waste extract (FWE) was filtered and

stored at 4 0C for further experimentation. The prepared FWE with a flow sheet of

preparation was given in Fig. 4.2.1.

Fig. 4.2.1. Steps involved in substrate preparation along with the prepared FWE as figure

insert.

96
4.2.2.3 Production and purification of melanin

Nutrient broth (peptone-5g/L, beef extract-3g/L, NaCl-5g/L) was used for inoculum

preparation and FWE was used as production medium for melanin. About 10 µL (108

CFU/mL) culture suspension was added to FWE medium in 250 mL flasks with a working

volume 50 ml. The medium was then incubated at 30 0C on a rotary shaker moving at 200

rpm for 24 h. A dark pigmented and nearly opaque FWE medium was observed (Fig. 4.2.2a).

After the incubation time, the medium was centrifuged using REMI-RM12C, India centrifuge

at 8000 rpm for 15 min to separate the broth (supernatant) and the cells. The solid pellet of

cells were separated and suspended in distilled water. The cells were further centrifuged to

collect the supernatant. Melanin was extracted from the overall supernatant by acidification

with 3 N HCl to pH-2 and allowed to stand for 48 h initially at room temperature. This

process was repeated for 7 more days until no precipitate was obtained. The obtained

suspension was boiled for 5 min to prevent the formation of melanoidins. As a final point,

crude pigment pellet was collected after centrifugation at 4000 rpm for 15 min.

4.2.2.4 Parameters optimization using Taguchi and CCD design

Preliminary idea on growth conditions, suggests Taguchi method to be employed for the

optimization of culture conditions for high yield melanin pigment production. Optimization

of three vital factors like pH, temperature and agitation in 6-3-3 levels respectively was done

as a starting point of the study (Table 4.2.1). Then Taguchi method was performed by 18

different experiments by using L18 orthogonal array as shown in Table 4.2.2. Shown values

of the obtained melanin (mg/mL) are the average of results of two replicates. Based on the

obtained results, the optimum conditions of the used parameters were identified and an

analysis of variance (ANOVA) for the obtained results was investigated.

97
 Once the critical factors were identified, in addition to the above, a central composite

design (CCD) for independent variables was used for further optimization. Two variables at

two levels were used to fit a polynomial model. A two level full factorial is performed with a

model equation designed such that the variance of Y is constant for all points equidistant

from the center of the design. Minitab (14.0) statistical software package was used in the

experimental design and data analysis. Response surface graphs were obtained to know the

effect of the variables, individually and in combination, and to determine their optimum

levels for maximum melanin production. All trials were performed in duplicate, and the

average melanin yield was used as response Y.

4.2.2.5 SPF/DPPH/metal chelating assay

In vitro sun protection factor

As per the adapted method, spectra of melanin samples were collected over the spectral range

400-280 nm with 1 nm data point resolution on a UV-visible UV-3200 double beam

spectrophotometer (LABINDIA analytical Instruments Pvt Ltd, India). The SPF values of

melanin from microbial isolate and purchased melanin were determined using Mansur

mathematical equation (1).

320
SPF  CF   EE   I  Abs  (1)
290

Where, SPF=Sunscreen protection factor; EE(λ)=Erythremal effect spectrum, I(λ)=Solar

intensity spectrum; Abs(λ)=Absorbance of sunscreen product; CF=Correction factor (=36.2

for SNOW LOTUS® SPF 15 and 30) Huang et al.[20]

98
DPPH assay

The radical scavenging activity by melanin pigment was investigated by modified method of

Ju et al. [21]. Primarily 1 mL of (15‐100 μg/mL) microbial melanin/standard (Ascorbic acid)

was added to 2 mL of DPPH in ethanol. After keeping for 30 minutes at 37oC the absorbance

at 516 nm was measured using UV‐spectrophotometer with reference blank samples. The

experiment was performed in duplicate. The absorbance of DPPH as control was measured at

516 nm. Lower absorbance of the reaction mixture indicated higher radical scavenging

activity. The scavenging effect was measured using equation (2).

DPPH inhibition (%) = [(Control absorbance -test absorbance)/Control absorbance] X 100

(2)

Determination of metal chelating activity

The chelation of ferrous ions by the melanin pigment was estimated by the method of Huang

et al.[20]. Different concentrations of melanin were mixed with a solution of 2 mM FeCl2

(0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the

mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance

of the solution was then measured spectrophotometrically at 562 nm. All the tests and

analysis were performed in duplicate and averaged. The inhibition of the melanin pigment

metal chelating activity in percentage (%) was calculated using equation (3).

Metal chelating effect (%) = [(A0-A1)/A0] X 100 % (3)

where A0 is the absorbance of control reaction and A1 is the absorbance in the presence of the

sample of the melanin pigment or standards. The control contains FeCl2 and ferrozine.

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Table 4.2.1. Factors and their levels which were studied by Taguchi approach.

Levels
Factor
1 2 3 4 5 6
pH 4.3 5 6 7 8 9
Temperature (oC) 15 30 45
Agitation (rpm) 90 140 180

Table 4.2.2. Levels of three different factors, applied in each of 18 trials with the obtained

results.

pH Temperature Agitation Melanin (mg/mL)

4.3 15 90 0.001
4.3 30 140 0.01
4.3 45 180 0.011
5 15 90 0.08
5 30 140 0.124
5 45 180 0.11
6 15 140 0.309
6 30 180 0.331
6 45 90 0.328
7 15 180 0.522
7 30 90 0.655
7 45 140 0.577
8 15 140 0.356
8 30 180 0.446
8 45 90 0.428
9 15 180 0.219
9 30 90 0.231
9 45 140 0.218

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4.2.2.6 Analytical methods

XRD analysis was performed using the CuKα radiation (λ=1.5406 Ao) X-ray diffractometer

[Philips (PW1830 HT)], in the range of 20–900 (2θ) at 0.05o/s with an accelerating voltage 35

kV and applied current (30 mA). The absorption spectra of the purified melanin solutions at

room temperature were obtained by UV-visible spectrophotometer. Structural functional

groups were identified by FTIR, [Bruker, Germany] equipped with attenuated total

reflectance (ATR) mode with zinc selenide (ZnSe) crystal.

4.2.3 Results and discussion

4.2.3.1 Strain selection and pigment production

The melanin producing soil microbial isolate from NA plates was carefully separated and

cultivated on fresh agar plates (Fig. 4.2.2b) for 24 h. These colonies were examined

microscopically for their morphology as shown in Fig. 4.2.2c. The isolated strain upon 16S

rDNA sequencing identified a novel bacterial species Bacillus safensis strain ZJHD1-43

(GenBank Accession Number:KF585035.1). The phylogenic tree was constructed showing

the position of isolate with reference to related strains in Fig. 4.2.2d. At usual conditions,

FWE appeared to be most suitable medium for melanin production. An intense coloration of

the medium from straw colour to brownish black was observed within 24 h at a pH of 7and

with agitation of 100 rpm.

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Fig. 4.2.2. (a) FWE before (left) and after melanin production (right) by the garden soil

microbial isolate, (b) colonies with diffusible melanin on NA plates, and (c) SEM image of

the microorganism. (d) Phylogenetic tree showing the position of the isolate ZJHD1-43 with

reference to related strains.

4.2.3.2 Taguchi design for screening critical factors

Effect of pH, temperature and agitation were studied employing Taguchi method, which is a

fractional factorial experimental design tool. Experiments performed at experimental

conditions (pH 7; temperature 30 ºC; agitation 90 rpm) produced maximum melanin of 0.655

102
mg/mL on an average as shown in Table 4.2.2. Each of these factors such as pH, temperature

and agitation influenced significantly on melanin production represented as "main effect" and

illustrated in Fig. 4.2.3. Using ANOVA software tool, significance of two important factors

pH and temperature was reflected as per Table 4.2.4. F value represents the relative

contribution of estimated variance to residual variance. Large F value is desirable as small

value indicates significance of pH in this optimization method. Again further confirmation of

the significant effect is understood from P value. Using P-value prob > F test that indicates

probability of F value that will be observed when P < 0.05. Thus we found that pH and

temperature has significant influence in optimization of process conditions towards melanin

production, whereas agitation has negligible effect.

Table 4.2.3. Analysis of variance of main effects of factors

Analysis of Variance for Means

Source DF Seq SS Adj SS Adj MS F P

pH 5 0.659 0.659 0.131873 195.66 0

Temperature 2 0.008 0.008 0.004054 6.02 0.025

Agitation 2 0.001 0.0014 0.000714 1.06 0.39

Residual Error 8 0.0053 0.0053 0.000674

Total 17 0.6742

103
Fig. 4.2.3. Main effects of factors or average of obtained results (mg/mL) in which each

factor is at a given level. For description of ‘levels’ refer to Table 4.2.1.

Furthermore Table 4.2.4 shows the suggested condition as predicted from the optimization

tool. Statistical calculations predicted that at these conditions (Table 4.2.4) the melanin yield

should reach 0.620 mg/mL. However this value is slightly less than and almost equals the

value by trail no: 7 (from the array of experiments given in Table 4.2.2). Hence further

investigation at the suggested conditions (Table 4.2.4) was discontinued.

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Table 4.2.4. Optimum conditions suggested by statistical calculations after performing the

tests

Factor Level description Level Contribution (mg/mL)

pH 7 4 0.584–0.275=0.309

Temperature (oC) 30 2 0.299 –0.275=0.024

Agitation (rpm) 90 1 0.287 –0.275=0.012

4.2.3.3 Optimization by response surface methodology (CCD design)

Optimization of process parameters was carried out using the CCD design with the

parameters found to be significant from the Taguchi approach, including pH (X1) and

temperature (X2). Table 4.2.5 represents the design matrix and the results of the 13

experiments carried out using the CCD design. The data obtained provided the regression

model using ANOVA software.

Y = 6.7014 - 0.3367(X1) + 0.2083 (X2) - 0.6048 (X1*X1) - 0.8598 (X2*X2) - 0.1175 (X1*X2)

---------(4)

Where X1 and X2 represents pH and temperature respectively. The estimated regression

coefficients from response surface analysis of the quadratic regression model (Table 4.2.6)

demonstrate that equation. 4 is a highly significant model with goodness of fit R2 - 0.982 and

adjusted R2 - 0.969. These values indicate that the model equation was adequate for

predicting the melanin production under any combination of values of the variables.

105
Table 4.2.5. Experimental design matrix for the central composite design.

pH Temperature pH values Temperature values Melanin (mg/ml)

0 1 7 35 6.12

0 0 7 30 6.63

0 -1 7 25 5.54

0 0 7 30 6.78

0 0 7 30 6.83

1 0 7.5 30 5.62

-1 1 6.5 35 5.8

-1 -1 6.5 25 5.23

0 0 7 30 6.68

0 0 7 30 6.61

-1 0 6.5 30 6.55

1 1 7.5 35 5.02

1 -1 7.5 25 4.92

4.2.3.4 Interaction effects of variables and validation of the model

The graphical representation provides a method to visualize the relationship between the

response and experimental levels of each variable and the type of interactions between the

test variables in order to identify the optimum conditions. The interaction effects and optimal

levels of the variables were determined by plotting the three dimensional (3D) response

surface curves. The response surface curve in Fig. 4.2.4a,b represents the interaction between

106
pH and temperature, which showed that the maximum melanin yield was obtained toward

neutral pH while melanin yield was significantly affected with alkaline pH.

Validation was carried out under conditions predicted by the model. The optimum

conditions predicted by the model are pH - 6.84, Temp - 30.7 ºC with yield of ~ 6.8 mg/mL

and the actual yield obtained was 6.96 ± 0.6 mg/mL. The close correlation between the

experimental and predicted values signifies the reliability of the response methodology (CCD

design) over traditional optimization approach. In addition, the increased melanin production

was observed with the optimized conditions than the initially used conditions. Additional to

the above, yield of melanin under optimum conditions was observed at different time

intervals and are shown in Fig. 4.2.4c.

Table 4.2.6. Estimated regression coefficients from the model equation.

Response Surface Regression: melanin (mg/ml) versus pH, Temperature

Term Coef SE Coef T P

Constant 6.7014 0.05127 130.714 0

pH -0.3367 0.05041 -6.679 0

Temperature 0.2083 0.05041 4.133 0.004

pH*pH -0.6048 0.07429 -8.141 0

Temperature*Temperature -0.8598 0.07429 -11.573 0

pH*Temperature -0.1175 0.06173 -1.903 0.099

S = 0.1235; R-Sq = 98.20%; R-Sq(adj) = 96.90%

107
Fig. 4.2.4. Three dimensional response surface curves with surface plot (a) and contour plot

(b) showing the effect of interactions of pH and temperature on melanin yield. (c) melanin

yield at optimum conditions with respect to time.

4.2.3.5 Spectroscopy, IR and XRD analysis of melanin

UV- Vis analysis

The absorption spectrum of natural melanin is shown in Fig. 4.2.5a. The UV-visible

wavelength scan showed that absorption was highest in the UV region (200 to 300 nm), but

108
diminished towards the visible region. This phenomenon is characteristic to melanin and was

due to actual complex structure of melanin [1, 13].

FTIR analysis

IR spectroscopy is important for the interpretation of the structure binding capacity, affinity

and sites of metal ions in melanin. Fig. 4.2.5b,c shows strong absorptions at 3500 cm-1, 1700

cm-1, 1300 cm-1 for standard melanin and for bacterial melanin obtained. The signals from

3600-2800 cm-1 attribute to the stretching vibrations of (O-H and N-H) the carboxylic,

phenolic and aromatic amino functional groups of indolic and pyrrolic systems [22-23]. The

spectral area between 1750 - 1550 cm-1represents the bending vibrations of C=O. the OH

bending of phenolic and carboxylic groups are present in 1400 - 1300 cm-1 [22].

XRD analysis

The XRD spectrum of bacterial melanin and purchased melanin are shown in Fig. 4.2.5c. The

spectra of melanin are characterized by a broad peak which is commonly seen in amorphous

and disordered materials centered at about 24°. The observed 2θvalues are 24.83º and 24.32º

for bacterial and purchased melanin respectively (Fig. 4.2.5c). This peak is due to X- ray

diffraction from parallel planer layers. The inter layer spacing d, is calculated according to

the Bragg equation.

. (5)

where θ is diffraction angle, m is diffraction order and λ is X- ray wave length by considering

first order diffraction (m = 1) we obtained d values of 3.582 and 3.656 Aº for bacterial and

purchased melanins respectively. The value of d is in good agreement with reported value of

the inter layer spacing in the stacked sheets model of the melanin [1]. An estimate of average

grain size of melanins can be calculated from the Dedye - schrerrer equation [1].

109
 (6)

where FWHM is full width at half maximum of diffraction peak. The obtained D values are

0.668 and 0.568 nm for the bacterial and purchased melanins. The closeness of the grain size

values indicates the quality of the purified bacterial melanin. Furthermore % crystallinity was

also calculated for the stated melanins by considering glass substrate as background. The

calculation is as follows:

( total area - background profile area) (7)

% crystallinity  100
(total area)

Although both melanin samples exhibited the lack of structure in the diffraction pattern

corresponding to any significant crystallinity, the % crystallinity values (Fig. 4.2.5c, picture

indicated with arrow) further indicate bacterial melanin from FWE was far less crystalline

when compared to the purchased melanin. Lack of crystallinity is significant sign of

consistent physical property of melanin [24].

110
Fig. 4.2.5. (a) UV-visible spectrum of melanin pigment obtained from FWE. (b) FTIR spectra

of standard melanin (upper) and bacterial melanin (lower). (c) X-ray diffractograms of the

obtained melain (upper) and purchased melanin (lower) and % crystallinity of both are also

shown as indicated by arrow.

4.2.3.6 SPF values determination

The determination of SPF values for samples (bacterial and purchased melanin) was made

through the UV spectrophotometer using the Mansur equation [20]. The SPF value for

melanin in FWE was 53.36 ± 0.009, while it was 59.34 ± 0.006 for purchased melanin.

111
As melanins are known for their photoprotective role [25], the obtained SPF values state that

melanin from FWE might have profound protection effect against dermal damage related to

photoaging as that of purchased melanin.

4.2.3.7 DPPH assay

DPPH accepts an electron to become a stable diamagnetic molecule. The ethanolic solution

of DPPH (violet color) has got a strong UV absorbance at 516 nm. The presence of a

reducing agent in this ethanolic solution pairs the odd electrons of DPPH radical and further

the solution losses color stiochometrically and also the absorbance of the solution decreases

at 516 nm. Reduction of absorbance at 516 nm and color of DPPH associated with different

melanin doses was verified. The % increase in radical scavenging activity from Fig. 4.2.6a

indicates the diminished behavior of the radical. The data obtained from Fig. 4.2.6a states that

scavenging activity of the melanin was higher than the control ascorbic acid at each and

every dose studied. This behavior shows 30 % enhanced reductive capability of the obtained

bacterial melanin than ascorbic acid for a constant dose of melanin dose of ~100 µg/mL.

4.2.3.8 The metal binding capacity of melanin

The metal binding capacities of melanin from FWE was determined by assessing its ability to

compete with ferrozine for the ferrous ions. The concentration dependent metal chelating

activity was shown in Fig. 4.2.6b and its insert. The reduction in spectrum with increase in

melanin dose indicates that melanin compound was interfering with the formation of ferrous

and ferrozine complex. This suggests the better chelating effect of melanin and good ability

to capture ferrous ions than ferrozine. Maximum effect (~ 64 % chelation) was observed for a

dose of 0.2 mg/mL (Fig. 4.2.6c). The results suggest that the action of melanins as oxidation

protection factors may be predominantly due to their iron binding capacity.

112
 0 mg/ml
0.012 mg/ml
0.24 0.036 mg/ml
0.061 mg/ml
100 (a) (b) 0.086 mg/ml
0.20 0.110 mg/ml
% Radical scavenging

80 0.123 mg/ml
0.16 0.184 mg/ml

Metal chelating effect (%)

Absorbance
0.246 mg/ml 70
60
60
(c)
0.12
50
40
Melanin-FWE 0.08 40
20 Ascorbic acid 30
0.04 20
at 562 nm
0 10
0.00 0
0 20 40 60 80 100 450 500 550 600 650 700 0.00 0.05 0.10 0.15 0.20 0.25
Pigment dose (µg/mL) Melanin dose (mg/ml)
Wavelength (nm)

Fig. 4.2.6. (a) Dose dependent scavenging activity of the synthesized melanin from FWE and

ascorbic acid as a control. (b) Metal ion chelation effect of the produced melanin in different

doses (monitored spectrally) and (c) At a fixed absorption maximum.

4.2.3 Conclusions

From the results of this study, it is concluded that the use of two step statistical approach not

only helped in locating the optimum levels of the most significant factors considered with

minimum resources and time but also proved to be an useful and satisfactory method in

melanin production-optimizing exercise. Thus, the optimization of vital nutritional

parameters using response surface methodology significantly enhanced the yield of melanin

on fruit waste extract has proved its feasibility for large-scale production by a garden soil

isolate (Bacillus safensis). The melanin obtained in this study has photoprotective, radical

scavenging and metal binding capacity which is of economic importance. So the B. safensis

and fruit waste extract can be potential sources for melanin production.

113
References

[1] V. Capozzi, et al., "Optical and photoelectronic properties of melanin," Thin solid

films, vol. 511, pp. 362-366, 2006.

[2] J. D. Nosanchuk and A. Casadevall, "Impact of melanin on microbial virulence and

clinical resistance to antimicrobial compounds," Antimicrobial agents and

chemotherapy, vol. 50, pp. 3519-3528, 2006.

[3] J. Zhang, et al., "Characterization of melanin produced by a wild-type strain of

Bacillus cereus," Frontiers of Biology in China, vol. 2, pp. 26-29, 2007.

[4] L. Ruan, et al., "Melanin pigment formation and increased UV resistance in Bacillus

thuringiensis following high temperature induction," Systematic and applied

microbiology, vol. 27, pp. 286-289, 2004.

[5] A. El-Obeid, et al., "Herbal melanin modulates tumor necrosis factor alpha (TNF- α),

interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) production,"

Phytomedicine, vol. 13, pp. 324-333, 2006.

[6] V. M. Sava, et al., "A novel melanin-like pigment derived from black tea leaves with

immuno-stimulating activity," Food Research International, vol. 34, pp. 337-343,

2001.

[7] Q. Gao and F. Garcia-Pichel, "Microbial ultraviolet sunscreens," Nature Reviews

Microbiology, vol. 9, pp. 791-802, 2011.

[8] J. D. Nosanchuk and A. Casadevall, "The contribution of melanin to microbial

pathogenesis," Cellular microbiology, vol. 5, pp. 203-223, 2003.

[9] J. McLean, et al., "Role for lichen melanins in uranium remediation," Nature, vol.

391, pp. 649-650, 1998.

114
[10] S. N. Surwase, et al., "Optimization of melanin production by Brevundimonas sp. SGJ

using response surface methodology," 3 Biotech, vol. 3, pp. 187-194, 2013.

[11] I. Hanyz and D. Wróbel, "Photovoltage generation and fluorescence of charged

tetraphenylporphyrins with dopa melanin," Crystal Research and Technology, vol. 38,

pp. 325-330, 2003.

[12] A. M. Gómez-Marín and C. I. Sánchez, "Thermal and mass spectroscopic

characterization of a sulphur-containing bacterial melanin from Bacillus subtilis,"

Journal of Non-Crystalline Solids, vol. 356, pp. 1576-1580, 2010.

[13] S. Sajjan, et al., "Purification and physiochemical characterization of melanin

pigment from Klebsiella sp. GSK," Journal of microbiology and biotechnology, vol.

20, pp. 1513-1520, 2010.

[14] V. Coyne and L. al-Harthi, "Induction of melanin biosynthesis in Vibrio cholerae,"

Applied and environmental microbiology, vol. 58, pp. 2861-2865, 1992.

[15] M. T. Cubo, et al., "Melanin production by Rhizobium strains," Applied and

environmental microbiology, vol. 54, pp. 1812-1817, 1988.

[16] V. Lagunas‐Muñoz, et al., "Optimum melanin production using recombinant

Escherichia coli," Journal of Applied Microbiology, vol. 101, pp. 1002-1008, 2006.

[17] G. Taguchi, Introduction to quality engineering: designing quality into products and

processes, 1986.

[18] B. Harlizius, et al., "Genomics for food safety and sustainable animal production,"

Journal of Biotechnology, vol. 113, pp. 33-42, 2004.

[19] K. Aneja, Experiments in microbiology, plant pathology and biotechnology: New Age

International, 2003.

115
[20] S. Huang, et al., "Antioxidant activities and UV-protective properties of melanin from

the berry of Cinnamomum burmannii and Osmanthus fragrans," Medicinal Chemistry

Research, vol. 20, pp. 475-481, 2011.

[21] K.-Y. Ju, et al., "Bioinspired polymerization of dopamine to generate melanin-like

nanoparticles having an excellent free-radical-scavenging property,"

Biomacromolecules, vol. 12, pp. 625-632, 2011.

[22] M. Magarelli, et al., "Purification, characterization and analysis of sepia melanin from

commercial sepia ink (Sepia officinalis)," Revista CES Medicina Veterinaria y

Zootecnia, vol. 5, pp. 18-28, 2010.

[23] J. Coates, "Interpretation of infrared spectra, a practical approach," Encyclopedia of

analytical chemistry, 2000.

[24] Y. Thathachari and M. Blois, "Physical studies on melanins: II. X-Ray Diffraction,"

Biophysical journal, vol. 9, pp. 77-89, 1969.

[25] M. Brenner and V. J. Hearing, "The protective role of melanin against UV damage in

human skin†," Photochemistry and photobiology, vol. 84, pp. 539-549, 2008.

116
4.3 Carotenoid by Bacillus clausi Using Rice Powder as

the Sole Substrate: Pigment Analyses and Optimization of

Key Production Parameters

4.3.1 Introduction

Pigments are particulate solids that disperse into a medium without significant interaction in

solutions [1]. Their wide applications in fields of food, cosmetics, paints, pharmaceuticals,

textiles, glass etc. has contributed to its increasing demand [2]. Currently there are two

methods employed so far to produce β-carotene; they are based on Wittig reaction developed

by Badische Anilin and Soda Fabrik (BASF) and the second is based on Grinard reaction

elaborated by Hoffman-La Roche [3-4]. However the microbial method has gained maximum

interest as they are naturally produced and are environmentally friendly. Carotenoids are the

secondary metabolites which are usually accumulate in the organisms during biotic and

abiotic stress. Among all natural pigments, carotenoids represent the largest and most diverse

class compounds known to mankind [5].

117
 Carotenoids of microbial origin have found maximum application in the food and

cosmetic industries which has exhibited increase in demand in recent years. Considerable

research has been focused on these pigments as nutritional supplements with some $ 935

million to billion dollars market in 2005 [6-7]. Besides, carotenoids play a significant role in

human health as precursors of vitamin A, scavengers of active oxygen, enhancers of in vitro

antibody production, anticancer agents and so on [6, 8].

In view of high cost of current technology there is a need to develop low cost

processes for the production of pigments which could replace the synthetic pigments in

industrial scale. Production of pigments essentially depends on the type of substrate on which

the microbe grows [9]. A variety of substrates are tested for carotenoids production using

different microbes with special emphasis on cheaper substrates like maple sap [10], coconut

milk [11], sugar manufacture processing waste [12], fermented radish brain [13], corn meal,

pea nut meal, soybean meal, coconut residue [14], whey waste [7], corn syrup [15], grape

must, beet molasses, soybean flour extract, maize flour extract [16], jack fruit seed [2], plant

extract [17], prehydrolysed wood [18] etc. Efforts have been made in order to reduce the

production cost of microbial pigments as compared to synthetic, plant and animal derived

pigments [16, 19].

This paper outlines the following objectives. 1) Isolation of the pigment producing

bacteria from garden soil, 2) Examine the potential of cheap natural substrate like rice

powder for the production of pigment, 3) Extraction, purification and analyzing the chemical

nature of the produced pigment, 4) Investigating the stability of the obtained pigment, and 5)

Optimization of key production parameters, using Taguchi experimental design tool.

118
4.3.2 Materials and methods

4.3.2.1 Sampling, microscopy and strain characterization

Carotenoid producing microbial cultures was isolated from soil sample collected from the

premises of NIT campus Rourkela. Screening procedure adopted was plating of 1 mL (10 -10)

diluted soil samples on nutrient agar (Himedia Pvt Ltd, India) with inoculation at 25 °C. The

orange colored colonies developed on the plates were observed. The isolate showing

maximum diameter was propagated on rice plates at 25 °C for 24 h. Characterization and

identification of the isolate with pigment production ability was carried out morphologically

with optical microscope (Hund-H600). In order to study the morphological characters such as

size and shape of the microorganism, various images were captured at different

magnifications using SEM (Scanning Electron Microscope) analysis [JEOL JSM-6480LV].

16s-rDNA characterization was performed on this isolated strain at Xcelris labs Ltd,

Ahmedabad, India.

4.3.2.2 Pigment production

Finely ground rice powder was used as a substrate for pigment production. Experiments were

conducted in petriplates of 10 cm diameter containing 3 g of rice powder. The substrate was

moistened with double distilled water (8 mL) at neutral pH - 7 and autoclaved to maintain

sterile conditions throughout the study. On cooling to room temperature; moisture content of

the substrate was analyzed using moisture analyzer (Sartorious MA-150) and was estimated

as 52.04 %. The substrate in plates was inoculated with 1 mL inoculum culture and incubated

at ambient room temperature ~ 30 °C for 10 days. After observing intense coloration in rice

plates, batch experiments at pH 6 and 8 were also studied for pigment production.

119
4.3.2.3 Purification and stability of the pigment

Pigment was extracted using a modified method adapted from Babitha et al. [20]. To evaluate

the solubility of the pigment a definite quantity of substrate (rice powder) 1g was extracted

with fixed volume 5 mL of different solvents such as acetone, methanol, ethanol, chloroform,

water and 10 % NaOH [21]. The pigment-solvent mixture was allowed to stand after shaking

for 15 minutes. Based on high extraction of pigment and fair dispersion with 10 % NaOH,

ethanol was selected as extracting solvent. About 1:5 rice powder (grams) to solvent (mL)

ratio was used for pigment extraction by thorough dispersion as per the reported method [2].

The mixture was centrifuged at 9000 rpm and the supernatant was collected. The above

process was repeated until the substrate was colorless. Phase separation of the extracted

pigment in the supernatant was achieved on adding equal volumes of diethyl ether and NaCl

(0.1 M) to obtain pure pigment devoid of cellular disturbances [11]. The obtained pigment

solution was further purified using thin layer chromatography using stationary phase - TLC

silica gel 60 F 254 and hexane: methanol (7:3) as stationary phase and solvent phase

respectively. To obtain orange colored powdered pigment the TLC extract was subjected to

vacuum evaporation.

The extracted pigment was assessed on 0.50 g of dry pigment and adjusted to 50 ml

constant volume of ethanol before measuring its absorbance at (λmax) using a UV-3600

Shimadzu UV-Visible spectrophotometer. The structural analysis of the extracted pigment

was examined by FTIR (Bruker ATR-FTIR, USA) at room temperature. Stability of the

purified pigment was also tested after thermal treatment at 45 0C for 8 h at ambient conditions

in an oven. It was subjected to UV-visible, fluorescence and Attenuated total reflectance

120
Fourier transform infrared (FTIR) spectroscopy analyses to observe the structural

confirmations.

4.3.2.4 Optimization of key parameters

Taguchi experimental design method was employed to evaluate the optimum conditions for

pigment production. The key parameters such as pH, temperature and moisture content were

studied. A standard L16 (43) with 15 degree of freedom was used to examine three factors in

four levels. The levels of the factors studied and the layout of the L16 orthogonal array are

represented in Table 4.3.1 and 2. ANOVA technique was adopted to analyze statistically and

determine the factors that significantly affect the pigment production. The controlling factors

were identified with the magnitude of effect quantified. Consequently, the optimal conditions

were determined by combining the levels of factors that had the highest main effect value. All

calculations were performed using MINITAB® Release 14.1 software.

4.3.3 Results and discussion

4.3.3.1 Colony Screening, pigment production

Distinct microbial colonies were observed on the agar plate after three weeks of incubation.

An orange colored peculiar spot surrounded with cream white color colony was screened and

cultured for the pigment production. After two days of incubation at 35 °C, visible cream

white color colonies were identified on the agar slants. Incubation for another three weeks

resulted in the production of evenly spaced orange-red spots (Fig. 4.3.1a). These colonies

were preserved using paraffin method as per the protocol [22]. Previous investigations have

revealed the utility of rice as a nutrient supplement in fungal pigment production [9, 23]. In

121
our study rice powder alone was used as a sole nutrient medium for orange colored pigment

production through bacterial species. Using rice powder as nutrient medium an intense

orange colored pigment was observed on the plate after 10 days of incubation at ambient

temperature of 30 °C (Fig. 4.3.1b). During the incubation, it was noted that the pigment

intensity was more on the surface of the rice plate than the deeper layers of the plate. The

pigmented rice material was suspended in ethanol for pigment extraction and subjected to

further purification steps as ascribed in the above section via thin layer chromatography and

was shown in Fig. 4.3.1c,d. At constant temperature (30 °C) and varying the pH (6 and 8) did

not show notable color change on rice plates (Fig. 4.3.2).

Fig. 4.3.1. Microbial isolate with orange – red spotted colonies on nutrient agar slant (a), and

orange pigment by the isolated soil microorganism on rice powder (b). Phase separation of

the obtained pigment was shown in (c). Resultant separated pigment concentrate in liquid (d

insert) and in solid form after vacuum drying was also given in (d).

122
Fig. 4.3.2. Orange pigments by the isolated soil microorganism on rice powder plates at pH -

6 (a) and 8 (b) respectively.

4.3.3.2 Microscopy and microbial characterization

Fig. 4.3.3a shows the optical microscopic image of the microbial isolate and the produced

pigment. The nature of the obtained pigment was agglomerative and extracellular. It was also

evident from the small size of the microbe involved that the bulky pigment clumps might be

their possible metabolites. SEM analysis confined the morphology of the microbe (Fig.

4.3.3b,c). It clearly suggests a rod shaped bacterial species of the size range 1.1 to 1.5

micron. These colored colonies based on morphology and phylogenic analysis through 16s

rDNA sequencing were identified as Bacillus clausii XJU-3, This bacterium strain showed

100 % homology with Bacillus clausii (AY960116.1) and the phylogenic relationship is

shown in Fig. 4.3.3d. Reports on production of yellow, orange and pink carotenoids on a

variety of substrates suggest that the identified species has potential for carotenoid pigment

production [24-25].

123
 (d)

Fig 4.3.3. Pigment producing bacterial species with an orange pigment (insert) magnified by

an optical microscope at 400 X magnification (a). SEM images of the identified pigment

producing bacteria with (b) 5000 X, (c) 9000 X magnifications and (d) Phylogenetic tree

showing the position of the isolate XJU-3 with reference to related strains

124
4.3.3.3 UV-Visible/fluorescence spectrophotometry and stability of the pigment

Fig. 4.3.4a shows the UV-Visible spectrum of pigment solution (extracted in ethanol)

produced from the isolated B. clausii on the rice medium. The absorption maxima were

observed at 447 and 472 nm and the spectral pattern were in close resemblance to the

characteristic of β-carotene [26]. Literature shows that β-carotene type carotenoids are

sensitive to oxidation, light and heat [27]. To confirm the pigment kind, pigment stability

under different storage conditions was tested. In general, β-carotene is stored in aluminum

foil bags under inert gas atmosphere at low temperatures < 15 0C to retain > 96% of the

activity [28]. To test the degradability and stability of the observed pigment it was subjected

to thermal drying under aerobic conditions in a closed oven for 8 h at 45 0C. After incubation,

the color of the pigment residue became slightly shaded from orange-red to pale yellow,

which may be due to oxidation of the pigment. It was subjected to further analyses

(UV/fluorescence).

Fig. 4.3.4b shows the spectrum of the shaded pigment and the discrete difference in

spectra of (a) and (b) might be due to oxidation of the produced pigment. The band maximum

of spectrum in (b) was found to be at 330 nm with a shoulder peak at 345 nm, while for the

diluted solution of the same, an additional absorption at 262 nm was observed (Fig. 4.3.4b

insert). The absorption of spectrum (b) at 330 nm of the electromagnetic spectrum indicates

close spectral resemblance of vitamin A class retinol pigment [29]. The additional peak split

at ~ 340 nm and band at 262 nm specify that the extracted pigment was in its oxidized form

[30]. An interesting feature of these retinol class compounds absorption at shorter wavelength

region is due to their highly twisted double-bond in their systems [31]. The above evidence

shows that the nature of the pigment obtained from B. clausii is of wholesome β-carotene and

125
produced products like precursors of vitamin A as a result of aerobic thermal oxidation,

which usually occur via chemical transformation [32]. The evaluation of fluorescence

property of the oxidized pigment also confirms the property of retinol type products [32]. The

maximum excitation was found to be at 315 nm and the λemi is observed at 382 nm as shown

in Fig. 4.3.4c. The fluorescence yield of pigment before thermal treatment has produced no or

negligible spectral signal and was not reported.

6 1.0
1.4 0.8

Absorbance (a.u)
1.2 (a) 5 (b) 0.6

0.4
Absorbance (a.u)

Absorbance (a.u)
0.2

1.0 0.0
4 200 250 300 350 400 450
Wavelength (nm)
500

0.8
3
0.6

0.4 2

0.2
1
400 450 500 550 600 650 700 750 300 320 340 360 380 400 420
Wavelength (nm) Wavelength (nm)

1.0
(c)
0.8
Emission (n.u)

0.6

0.4

0.2

0.0
300 350 400 450 500 550 600
Wavelength (nm)

Fig. 4.3.4. (a) UV- visible spectrum of the extracted orange pigment in ethanol, (b) UV

absorption of the oxidized pigment with narrow and wide (insert of (b)) spectral regions, and

(c) Fluorescence emission spectra of the oxidized pigment at 382 nm.

126
4.3.3.4 FTIR analysis

In order to understand the pigment structure FTIR analysis was performed. The purified

pigment was subjected to FTIR spectroscopy before and after oxidation steps (Fig. 4.3.5a,b).

Fig. 4.3.5a shows the IR spectrum of the pigment with major peaks at 3320, 2943, 2832,

1652, 1448, 1405, 1100 and 1020 cm-1. The majority of the peaks from the obtained pigment

show a close resemblance to a β-carotene structure [33-34]. Peaks at 2943 and 2832 cm-1 are

due to asymmetric and symmetric stretching vibrations of CH2 and CH3 groups [34]. And

peaks at 1020 cm-1 is for wagging vibration of (RH).C=C.(RH) groups of the synthesized

pigment. Peaks at 1405 and 1448 cm-1 are due to symmetric deformation of δ CH3 and

deformation vibration of δ CH2 groups. Moreover the peaks at 3320, 1652 and 1020 cm-1 are

due to vibrational modes of water interference in the analyzed pigment [33, 35].

Fig. 4.3.5b shows FTIR peaks after the oxidation of the pigment. The major peaks

observed here are at 3433, 3229, 3000, 2883, 1672, 1388, 1343, 1089, 1082, 884, 708 and

631 cm-1. The spectral pattern highly resembles retinol compound of carotenoids family [36].

The absorptions in the region of 3200 - 2700 cm-1 are normally characteristic of carbon and

hydrogen containing species, and can be assigned to various forms of C-H stretching. The

absorbance at and above 3000 cm-1 suggest that the compound is likely to have unsaturated

C=C. The absorption at 3433 cm-1 which is in the region of 3650-3250 cm-1 mainly occurs for

hydroxy or amino groups.

The additional intense bands in 1600-1300, 1200-1000 and 800-600 cm-1 in Fig.

4.3.5b confirms that the oxidized compound consists a simple hydroxy compound with O-H

absorption and is likely to be an alcohol. The absorption at the low end and below 1700 cm-1

was due to carboxylate (carboxylic acid salt). In our case the peak at 1672 cm-1 may be due to

127
 conjugation at C=O. (may be our oxidized compound is a mixture of retinol and retinoic

acid). Peaks at 3000 cm-1 and 2986 cm-1 which are just above and below 3000 cm-1 may be

due to unsaturated and saturated C-H absorptions in the compound. Bands at ~ 1000 cm-1 and

880 cm-1 indicate C-H out of plane bending which is usually seen for all trans retinol

compounds. Further the olefinic double bond type unsaturation can be confirmed by a single

peak around ~ 890 cm-1along with the absorptions at ~1650 and ~3000 cm-1 [35]. The

structural confirmations by IR analysis in concurrence with UV-Visible analysis infer that the

produced pigment is exclusively close to β-carotene in all aspects. And the retinol derivatives

of the obtained pigment on thermal treatment further confirm the instability of these β-

carotene type pigments.

1652
% Transmittance (n.u)

2943 2832 1448 1405

1551
1100 1299
3320
2883
1584

(a) 3433
3000
(b) 1343

3229 1388
1672

1089
1082 708

884 631
1020
3500 3000 2500 2000 1500 1000 4000 3600 3200 2800 2400 2000 1600 1200 800
Wavenumber (cm-1) Wavenumber (cm-1)

Fig. 4.3.5. ATR-FTIR analysis of the extracted and purified orange pigment before (a) and

after (b) oxidation process.

128
4.3.3.5 Optimization of key production parameters

Extensive research focused on the optimization of the key process parameters for high yield

carotenoid production by employing simple statistical procedures [37-38]. The above analysis

inferred that the pigment produced by the bacterial isolate B. clausii needs proper attention

while purification and preservation as it is thermally sensitive at normal atmospheric

conditions. By following cautious steps and to know the influential factors for the pigment

production in higher amounts; effect of key parameters like pH, temperature and moisture are

studied by using a simplistic Taguchi method. This method of optimization offers ease in

identifying the key process parameters and is a fractional factorial experimental design. This

method can be effectively used in optimization of biotechnological processes, since it has the

ability to include categorical factors also [39].

The results of the experiment showed that the maximum yield of pigment was 95.8

mg/3 g rice powder, at optimum experimental condition (pH 7, temperature 30 °C and

moisture 55 %) (Table 4.3.2). Fig.4.3.6 depicts the main effect of each factor (Table 4.3.2),

he term ‘main effects’ considered as average of the obtained results in which each factor is at

a given level. Results obtained using ANOVA software (Table 4.3.3) suggests the

significance of the factor in the following order pH > temp with p - values of 0.011 and

0.065. Thus these p-values (on comparison with α – level values) indicate that the parameter

pH and temperature are significant at the levels 0.05 and 0.010 α respectively and are

significantly related to the response. The average yield of the pigment was estimated to be

69.4 mg/1g rice powder (Table 4.3.2). Table 4.3.4 shows the suggested conditions as per

calculations. The statistical calculations as per the conditions suggested in Table 4.3.4

predicted that if the conditions were chosen as shown in Table 4.3.4, should produce 103.3

129
mg pigment per 1 g rice powder which is 48.9 % more yield. However, the experimental

result showed 107 ± 1.2 mg pigment production using 1 g rice powder since the deviation in

actual and predicted value was only about 3.58 %, which is acceptable. Therefore on

comparing with 95.8 mg pigment produced before, a further increase of about 11.6 % was

achieved.

90 pH
Temperature
85 Moisture
Pigment yield (mg)

80

75

70

65

60

55
1 2 3 4
Levels of factors

Fig. 4.3.6. Main effects of factors or average of obtained results (pigment per 3 gm rice

powder) in which each factor is at a given level. For detail about ‘levels’ refer to Table 4.3.1.

Table 4.3.1. Factors and their levels studied by Taguchi method.

Factor Level 1 Level 2 Level 3 Level 4

pH 6 6.5 7 7.5

Temperature (oC) 25 30 35 40

Moisture % (w/w) 40 45 50 55

130
Table 4.3.2. Levels of three different factors in each of sixteen trails and obtained results.

pH Temperature Moisture Pigment yield - mg/3 g substrate

1 1 1 46.1
1 2 2 52.4
1 3 3 68.3
1 4 4 54.0
2 1 2 50.2
2 2 1 49.3
2 3 4 86.6
2 4 3 88.7
3 1 3 78.0
3 2 4 95.8
3 3 1 87.6
3 4 2 92.8
4 1 4 56.4
4 2 3 68.4
4 3 2 70.5
4 4 1 65.3

Table 4.3.3. Analysis of variance of main effects of factors. SS/MS indicate sum/mean of

squares in the table.

Source DF Seq SS Adj SS Adj MS F P

pH 3 2347.7 2347.7 782.55 9.46 0.011
Temperature 3 1032.0 1032.0 343.99 4.16 0.065
Moisture 3 473.0 473.0 157.67 1.91 0.230
Residual
6 496.3 496.3 82.72
error
Total 15 4349.0

131
Table 4.3.4. Optimum conditions suggested by statistical analysis after performing the tests

Contribution greater than avg. yield

Factor Level description Level
in ‘mg’ - in ‘%’.

pH 7 3 19.10 – 27.0

Temperature (oC) 35 3 8.85 – 12.7

Moisture % (w/w) 50 3 6.45 – 9.2

4.3.4 Conclusions

The production of orange pigment showed promising results using a soil microorganism with

rice powder as a sole substrate. Microscopic studies exposed the morphological

characteristics and 16S rRNA sequence analysis confirmed the bacterial isolate as B. clausii.

Observations by UV– visible absorption spectrum and FTIR analysis of the purified pigment

showed it’s resemble to the carotenoid class compound β-carotene. The thermal sensitivity of

the obtained orange pigment was well explored by UV, fluorescence, FTIR analyses and the

residual products were structurally close to β-carotene derivatives like retinol and retinoic

acid. Maximum pigment production was achieved (107 ± 1.2 mg/1 g rice powder) by Taguchi

method of optimization at pH - 7 and 35 oC temperature. The high sensitivity in yield with

respect to close variation in key parameters (like temperature and pH) was studied.

References

[1] H. Zollinger, Color chemistry: syntheses, properties, and applications of organic dyes

and pigments: Wiley. com, 2003.

132
[2] S. Babitha, et al., "Jackfruit Seed--A novel substrate for the production of Monascus

pigments through solid-state fermentation," Food Technology & Biotechnology, vol.

44, 2006.

[3] G. Wittig and H. Pommer, "Ger. Patent 954,247, 1959," in Chem. Abstr, 1959, p.

2279.

[4] T. Storebakken, et al., "Carotenoids in diets for salmonids: IV. Pigmentation of

Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin,"

Aquaculture, vol. 65, pp. 279-292, 1987.

[5] G. Britton, et al., Carotenoids: handbook: Springer, 2004.

[6] P. Park, et al., "Optimization of carotenoid production by Rhodotorula glutinis using

statistical experimental design," World journal of microbiology and biotechnology,

vol. 21, pp. 429-434, 2005.

[7] I. Marova, et al., "Use of several waste substrates for carotenoid-rich yeast biomass

production," Journal of Environmental Management, vol. 95, Supplement, pp. S338-

S342, 2012.

[8] M. M. S. Cabral, et al., "Carotenoids production from a newly isolated Sporidiobolus

pararoseus strain by submerged fermentation," European Food Research and

Technology, vol. 233, pp. 159-166, 2011.

[9] L. Kagliwal, et al., "A novel medium for the production of cephamycin C by

Nocardia lactamdurans using solid-state fermentation," Bioresource Technology, vol.

100, pp. 2600-2606, 2009.

[10] A. E. Wasserman, "Absorption and fluorescence of water-soluble pigments produced

by four species of Pseudomonas," Applied microbiology, vol. 13, pp. 175-180, 1965.

133
[11] A. Domínguez-Bocanegra and J. Torres-Muñoz, "Astaxanthin hyperproduction by

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as

sole source of energy," Applied Microbiology and Biotechnology, vol. 66, pp. 249-

252, 2004.

[12] G. T. Hayman, et al., "Production of carotenoids by Phaffia rhodozyma grown on

media composed of corn wet-milling co-products," Journal of industrial

microbiology, vol. 14, pp. 389-395, 1995.

[13] C. Malisorn and W. Suntornsuk, "Optimization of β-carotene production by

Rhodotorula glutinis DM28 in fermented radish brine," Bioresource Technology, vol.

99, pp. 2281-2287, 2008.

[14] S. I. Mussatto, et al., "Use of Agro-industrial wastes in solid-state fermentation

processes," Industrial waste. Croatia: InTech, pp. 121-40, 2012.

[15] P. Buzzini, "Batch and fed‐batch carotenoid production by Rhodotorula glutinis–

Debaryomyces castellii co‐cultures in corn syrup," Journal of Applied Microbiology,

vol. 90, pp. 843-847, 2001.

[16] P. Buzzini, "An optimization study of carotenoid production by Rhodotorula glutinis

DBVPG 3853 from substrates containing concentrated rectified grape must as the sole

carbohydrate source," Journal of Industrial Microbiology and Biotechnology, vol. 24,

pp. 41-45, 2000.

[17] S. Kim, et al., "Increased carotenoid production in Xanthophyllomyces dendrorhous

G276 using plant extracts," Journal of Microbiology-Seoul-, vol. 45, p. 128, 2007.

134
[18] J. Parajó, et al., "Production of carotenoids by Xanthophyllomyces dendrorhous

growing on enzymatic hydrolysates of prehydrolysed wood," Food Chemistry, vol.

60, pp. 347-355, 1997.

[19] L. Dufossé, et al., "Microorganisms and microalgae as sources of pigments for food

use: a scientific oddity or an industrial reality?," Trends in Food Science &

Technology, vol. 16, pp. 389-406, 2005.

[20] S. Babitha, et al., "Solid-state fermentation for the production of Monascus pigments

from jackfruit seed," Bioresource Technology, vol. 98, pp. 1554-1560, 2007.

[21] W. Decleir and A. Richard, "A study of the orange-red pigment from the accessory

nidamental glands of the cephalopod Sepia officinalis L," Biol. Jb. Dodonaea, vol. 40,

pp. 188-197, 1972.

[22] K. Aneja, Experiments in microbiology, plant pathology and biotechnology: New Age

International, 2003.

[23] X. Lian, et al., "Identification of new red pigments produced by Monascus ruber,"

Dyes and Pigments, vol. 73, pp. 121-125, 2007.

[24] L. H. Duc, et al., "Carotenoids present in halotolerant Bacillus spore formers," FEMS

microbiology letters, vol. 255, pp. 215-224, 2006.

[25] R. Khaneja, et al., "Carotenoids found in Bacillus," Journal of Applied Microbiology,

vol. 108, pp. 1889-1902, 2010.

[26] P. Park, et al., "Chemical disruption of yeast cells for the isolation of carotenoid

pigments," Separation and purification technology, vol. 53, pp. 148-152, 2007.

135
[27] K. Jacob, et al., "Stability of carotenoids, phenolic compounds, ascorbic acid and

antioxidant capacity of tomatoes during thermal processing," Archivos

Latinoamericanos de Nutricion (ALAN), vol. 60, p. 192, 2010.

[28] E. F. S. Authority, "Scientific Opinion on the safety and efficacy of beta-carotene as a

feed additive for all animal species and categories.," 2012.

[29] C. N'Soukpoe-Kossi, et al., "Retinol and retinoic acid bind human serum albumin:

stability and structural features," International journal of biological macromolecules,

vol. 40, pp. 484-490, 2007.

[30] S. Martras, et al., "Kinetics of human alcohol dehydrogenase with ring-oxidized

retinoids: effect of Tween 80," Archives of biochemistry and biophysics, vol. 430, pp.

210-217, 2004.

[31] M. B. Sporn, The retinoids vol. 1: Academic Press, 1984.

[32] S. Adhikari, et al., "Pulse radiolytic oxidation of β-carotene with halogenated

alkylperoxyl radicals in a quaternary microemulsion: formation of retinol,"

Biophysical chemistry, vol. 88, pp. 111-117, 2000.

[33] M. Baranska, et al., "Determination of lycopene and β-carotene content in tomato

fruits and related products: comparison of FT-Raman, ATR-IR, and NIR

spectroscopy," Analytical Chemistry, vol. 78, pp. 8456-8461, 2006.

[34] W. Ammawath and Y. Bin Che Man, "a rapid method for determination commercial

β-carotene in rbd palm olein by fourier transform infrared spectroscopy," Asian

Journal of Food and Agro-Industry, 3 (4), 443-452., 2010.

[35] J. Coates, "Interpretation of infrared spectra, a practical approach," Encyclopedia of

analytical chemistry, 2000.

136
[36] T. Lóránd, et al., "FT-IR study of some seco-and apocarotenoids," Journal of

biochemical and biophysical methods, vol. 53, pp. 251-258, 2002.

[37] P. Bhosale and R. Gadre, "Optimization of carotenoid production from

hyper‐producing Rhodotorula glutinis mutant 32 by a factorial approach," Letters in

applied microbiology, vol. 33, pp. 12-16, 2001.

[38] R. Sarada, et al., "A response surface approach for the production of natural pigment

astaxanthin from green alga, Haematococcus pluvialis: effect of sodium acetate,

culture age, and sodium chloride," Food Biotechnology, vol. 16, pp. 107-120, 2002.

[39] M. Azin, et al., "Production of xylanase by Trichoderma longibrachiatum on a

mixture of wheat bran and wheat straw: Optimization of culture condition by Taguchi

method," Enzyme and microbial technology, vol. 40, pp. 801-805, 2007.

137
Chapter 4 Approach and investigations

Part II - Facile Synthesis of Commercial Pigments by

Purchased Strains on Cheaper Substrates

4.4 Carotenoid by Rhodotorula sp. on Fruit Waste

Extract as a Sole Carbon Source and Optimization of key

production parameters

4.4.1 Introduction

Pigments are of great commercial interest and have received considerable attention because

of their potential beneficial effects on human health besides pigmenting properties [1]. In

recent years there is an increasing demand for microbial pigments as promising alternatives

138
for synthetic pigments that are widely used in food industries [2]. The pigments from

microorganisms are nature selected and have advantages over plant and animal derived

pigments with no seasonal variations, no geographic inconsistency in the production, high

productivity and are easily manipulated in the processing schemes [3-4].

Among microbial derived pigments, carotenoids are fat soluble diverse class of

yellow, orange, red and purple natural pigments which are unanimously produced by a wide

range of microorganisms and plants. Due to the recent discovery of anticancer and

antioxidant properties of carotenoid pigments, their use in pharmaceuticals and

neutraceuticals is expected along with their applications in the fields of food, cosmetics,

chemicals and so on [5-7]. Globally carotenoids are estimated to supersede USD S 280

million in 2015 [8]. In order to improve the yield of carotenoid pigments and to decrease the

cost of production, various studies have been performed using several microorganisms using

numerous different substrates [9-10]. Amongst different microorganisms Rhodotorula sp.

stood as a potential and scientifically favorable candidate for high yield carotenoids

production [11].The yeast Rhodotorula sp. is one of very few types of yeast which is able to

produce a large number of carotenoid pigments which are mainly β - carotene, torulene,

torularhodin [12]. Moreover, many investigations have been done using this genus to make

the process economical [3, 6, 13-14]. Key process parameters like type of substrate, nutrients

availability, pH, temperature and so on are optimized in various processes [15]. However,

there are still extensive investigations focused on this versatile genus Rhodotorula to produce

carotenoids in more economical ways.

The cost of the substrate has an important contribution to the overall pigment

production cost, and it can be minimized by using cheaper organic waste. Substrate

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composition, typically carbon source has a significant effect on carotenoid production and its

cost. Many substrates have been considered as potential substrates for carotenoid production

[16]. Using a low cost or cheap substrate for the production of high value products may help

to make the process economical. Till now, several cheaper substrates have been used (e.g.

sugar cane juice, peat extract, whey, grape must, beet molasses, hydrolyzed mug bean waste

flour, soyabean and corn flour extract and sugar cane molasses etc.) for the production of

carotenoids [17]. In this study, we have used fruit waste extract (FWE) as the substrate for the

production of carotenoids. In general, fruit processing units dispose the wastes which are rich

in soluble sugars and micronutrients that support the microbial growth. Nowadays, fruit waste

disposal is one of the problems that the fruit processing industries are facing. Eyeing on this,

we have chosen fruit waste extract as potential substrate for pigment production which can

reduce the production cost and make the product economical. Using FWE as a suitable sole

substrate; we examined the ability of Rhodotorula rubra for carotenoids production.

In recent years statistical design has been successfully employed to identify the

optimum level of various parameters involved in the process. Statistical design is a powerful

tool that accounts for the main as well as interactive influences of the parameters on the

process performance. The disadvantages of other classical methods are that they are time

consuming, laborious and expensive. In contrast, the use of statistical tools such as RSM

methodology provides a great amount of information based on only a small number of

experiments [18]. In this study a combination of traditional non statistical and statistical

method based experimental design has been employed to optimize the biomass and

carotenoid production. Primarily, one factor at a time, a classical approach was practiced that

involves various levels of one factor when the other factors are constant. Using this method,

140
the key parameters which influenced the pigment production significantly, were identified.

Box–Behnken experimental design method is useful for rapidly optimizing the process with

limited number of experiments. Hence the objective of this study is to explore the

effectiveness of the sole substrate (mixed fruit extract) on pigment production using the Box-

Behnken statistical tool. Optimization of process parameters such as pH, temperature, and

agitation for high yield of biomass and pigment production was experimented.

4.4.2 Experimental section

4.4.2.1 Reagents and chemicals

Methanol, acetone, HCL, NaCl, and diethyl ether were procured from Merck, Mumbai,

India., malt yeast extract medium, malt yeast extract agar were purchased from HiMedia,

India. All other chemicals used were of analytical reagent grade throughout the study. Double

distilled water was used through the study and aseptic conditions were maintained wherever

necessary.

4.4.2.2 Microorganism and its maintenance

The microorganism Rhodotorula rubra (MTCC no: 1446) used in this study was obtained

from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. The

microorganism was grown and maintained on malt yeast extract medium and stock cultures

were preserved on malt yeast extract agar slants at 4 0C and sub-cultured at monthly intervals.

4.4.2.3 Substrate preparation

Fruit waste typically containing pineapple, pomegranate and orange is obtained from a fruit

juice shop of a local market. It includes extracted carpels of oranges, core of pineapples, and

crushed seeds along with arils of pomegranate. The soluble sugars are extracted from 1

141
kilogram of fruit waste by adding 2 liters of distilled water at 100 0C for 30 minutes. The

resultant straw colored fruit waste extract (FWE) is filtered and stored at 4 0C for further

experimentation.

4.4.2.4 Pigment production and extraction

The biomass (5 mg) was activated in malt yeast extract broth (100 ml) at 30 0C for 24 hrs.

1ml of the activated culture was inoculated in 250 ml conical flasks containing 50 mL

autoclaved FWE medium maintained at different pH’s (4.3, 5, 6, 7, 8). Until significant color

appearance the incubation at 30 OC was continued. The pigmented biomass collected after

centrifugation (9000 rpm, 10 min) was washed repeatedly (thrice) with distilled water and

subsequently treated with 1 N HCl (60 0C, 10 min).

The treated cells were further washed in sterile distilled water and subjected to

repeated solvent washes to extract the intracellular pigment. Methanol: acetone (1:1) was

used as solvent for the microbial pellet wash and washing step continued till colorless pellet

was achieved. The washed colored solvent was collected separately and extracted with equal

parts of diethyl ether and NaCl (10 %) as a part of further purification step of the carotenoid

pigment. The obtained pigment here was subjected to thin layer chromatographic purification

step as stated in Chapter 4.3. The total carotenoid concentration (measured as β - carotene) in

diethyl ether extract was determined using standard method [13]. It was estimated according

to the absorbance at 448 nm using spectrophotometer (UV-3600 Shimadzu). The pigment

quantity is estimated using absorption coefficient E1%1cm = 2659 via spectral analysis. To

obtain purified pigment in solid form, the collected ether layer subjected to vacuum drying.

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4.4.2.5 DPPH assay

The effect of carotenoid form R. rubra on DPPH free radical was studied following the

method of [19] with some modifications. 0.1 mM DPPH solution in ethanol (99.5 %) was

prepared. Pigment in ethanol (1.65 mg/ml) in different volumes (10, 20, 30, 50 and 70 µl)

was made up to 100 µl with ethanol and was added to 1 ml DPPH solution. The mixture was

thoroughly mixed and allowed to stand for 40 min at room temperature in the dark. The

decrease in the absorbance at 517 nm was then measured. Ascorbic acid is used as positive

control and the pigment solution without DPPH as negative control. The radical scavenging

activity was measured as a decrease in the absorbance of DPPH and was calculated using the

following formula:

(Ac - As)
% Scavenging acticity   100
(Ac)

Where Ac,As are absorbance’s from control and sample.

4.4.2.6 Analysis

Cell dry weight was measured by harvesting the cells after centrifugation of the growth

medium at 5000 rpm for 10 minutes and subsequent washing thrice with distilled water. The

cells were dried at 105 0C till constant weight was attained. Reducing sugars were estimated

during experimentation by DNS assay method [20]. Elemental analyses were determined on a

Vario EL Cube CHNS Analyzer for FWE, before and after carotenoid production at pH -7.

4.4.2.7 Experimental design

One factor at a time and Box-Behnken design were used for optimization of carotenoid

production. The significant effect of pH towards carotenoid and biomass yield was

determined by one factor at a time method. Knowing the safe operating zones of other

143
production parameters like temperature and agitation, a Box-Behnken design (BBD) was

selected for optimization of the mentioned key process parameters (Table 4.4.1) for

carotenoids production by R. rubra.

Table 4.4.1. Coded and actual levels of the three variables

Variable Symbol Coded and actual levels

-1 0 1
pH x1 6 6.5 7
Temperature x2 25 27.5 30
agitation x3 90 120 250

Table 4.4.2. Experimental design matrix for the Box-Behnken design.

Run pH (x1) Temperature (x2) Agitation (x3) Biomass-mg/ml Pigment-mg/l

1 0 - - 3.33 1.33
2 0 - + 4.01 1.74
3 + 0 - 5.3 2.12
4 + - 0 6.31 2.51
5 + + 0 7.21 2.77
6 - - 0 2.74 1.09
7 0 0 0 6.44 2.47
8 0 0 0 6.76 2.71
9 - 0 + 4.99 1.98
10 0 0 0 6.54 2.62
11 - + 0 5.2 2.09
12 + 0 + 7.75 3.1
13 - 0 - 3.87 1.55
14 0 + + 7.08 2.72
15 0 + - 4.8 1.92

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The experimental design and statistical analysis of the data were carried out using the Minitab

(14.0) statistical software package. BBD with three factors and 15 runs was chosen; a model

was developed and also validated. Table 4.4.1 shows the variables and experimental design

levels for response surface and Table 4.4.2 shows the design matrix of BBD.

4.4.3 Results and discussion

4.4.3.1 Effect of pH on biomass and carotenoid yield

Using first approach the significant parameters that affected carotenoid production were

identified as pH, temperature and agitation. Effect of pH on biomass growth and carotenoid

production is illustrated in Fig. 4.4.1(a). The figure shows native FWE’s pH (4.31) including

different values studied. For evaluating the influence of pH on biomass and pigment

production, R. rubra was cultivated in FWE medium at 30 0C for 7 days. The microbial

culture is inoculated in the medium with adjusted pH's 5, 6, 7, 8, and 9 along with initial pH

4.31. Among various pHs, biomass growth was identified at values of 5 to 9 and pigment

production was observed only at 6 and 7. So, the results suggest that pigment production was

highly dependent on pH irrespective of biomass growth with high yield caused at pH 7. The

prepared FWE and fermented FWE with the yeast R. rubra are shown in Fig. 4.4.1(b).

The reducing sugar content in FWE was identified to be 19.90 mg/ml before

experimentation. CHNS analysis was performed for the prepared FWE so as to estimate the

presence of major and minor nutrients. CHNS analysis was done before and after carotenoid

production (Fig. 4.4.1(c)). The values stated suggests that significant utilization of macro and

micro nutrients by the microorganism followed the following order of H > N > C > S. In

145
addition to the above, near and far view SEM image of the used yeast was also shown in Fig.

4.4.1(d) and its insert.

Fig. 4.4.1. (a) Growth of biomass and carotenoid yield at various pHs and at room

temperature (30 0C). Prepared fruit waste extract (FWE) – left, and FWE with R. rubra

growth with intracellular carotenoid (right) upon incubation (b). CHNS analysis of FWE

before and after fermentation; upon separation of R. rubra by centrifugation (c). SEM image

of the used yeast.

4.4.3.2 Biomass growth, pigment yield and glucose utilization

Fig. 4.4.2A presents the individual values of cell dry weight (mg/ml), total carotenoid content

(mg/l) and glucose concentration (mg/ml) as a function of time in days. Pattern from the

figure shows that carotenoid content paralleled the cell dry weight and substrate

concentration decreased linearly till 3 days (72 h). Fig. 4.4.2A represents the growth of

biomass and production of carotenoid with time. Initially the rate of growth of biomass

superseded the carotenoid production. However after 3 days the biomass yield parallel to

146
carotenoid production rate. On the contrary the substrate utilization rate also diminished after

3 days. This graphical representation suggests that maximum carotenoid production was

achieved at the end of log phase at the expense of substrate utilization. Thereafter with the

approaching stationary phase, the carotenoid production capacity was constant which is

typical of any production process in a batch reactor. The observed condition shows the

biomass’s pigment production ability irrespective of quantitative substrate utilization.

However maximum pigment can be produced within 4 days of incubation using FWE as sole

substrate.

The UV-visible spectrum of the pigment produced was illustrated in Fig. 4.4.2B. The

observed absorption maximum at 507 nm towards the higher wavelength states that the

produced compound resembles Torularhodin type carotenoid, which is usually produced by

Rhodotorula in significant quantities [5]. The conformation study showing structural details

using FTIR analysis was carried out on the produced pigment along with the reference β -

carotene (Fig. 4.4.2C-a, b).

The FTIR spectrum of extracted carotenoid is shown in the Fig. 4.4.2C-a. The peak

descriptions can be described [21] as follows: the broad peak at 3351 cm-1 (which is in

between 2900 to 3500 cm-1) is due to hydrogen bonded O-H; peak at 2873 cm-1 is due to

methyne C-H stretch; peaks at 1692, 1636 cm-1 is for alkenyl C=C and aryl substituted C=C

in the compound; peak at 1429 cm-1 attribute to methyl C-H asymmetric band; peak at 1231

cm-1 is for C-O stretch and 1081 cm-1 is for C-C skeletal vibrations. The peak bands of 3351

along with peak at 1231 cm-1 confirms the presence of carboxylic group in the purified

carotenoid.

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Fig. 4.4.2. Carotenoid production (mg/l) and biomass yield (mg/ml) along with glucose

utilization (mg/ml) given in [A]. The picture insert shows the colored biomass from 1 to 6

days. UV-visible spectrum of the purified carotenoid in methanol medium [B]. FTIR spectra

of the synthesized carotenoid (a) and the purchased β-carotene (b) pigment.

Moreover on comparison with the purchased β - carotene’s spectrum (Fig. 4.4.2C-b), the

obtained carotenoid spectrum has some similarities and some variations. The spectrum of β-

carotene displayed major peaks at 2912 and 2860 cm-1 (for asymmetric and symmetric

vibrations of the CH2 and CH3); peak at 1446 cm-1 (due to CH2 group); peak at 1365 cm-1 (for

splitting due to dimethyl; group); peaks at 1021 cm-1 (is for in plane –CH-) and 965 cm-1 (is

148
for trans conjugated alkene -CH=CH- out-of-plane deformation). Further peaks at 700 to 900

cm-1 are for skeletal vibration of C-C stretch. The IR analysis along with UV spectrum of the

obtained carotenoid closely resembles the structures of torularhodin [14] foremost, and has

some similarities with β-carotene (as shown in Fig. 4.4.2C-b). Also this spectral pattern is

obvious as the mentioned pigments are predominantly obtained by Rhodotoula sp [5].

4.4.3.3 Box–Behnken design

The influence of pH on pigment productivity was identified as described in the above section,

this indicates a slight deviation of pH values from 6 to 7 resulted in negative effects with

low/negligible pigment yield. The above synthesis conditions were at static temperature 30
0
C and agitation (100 rpm). The Box–Behnken design was employed to study the interactions

among the significant factors and also determine their optimal levels. This methodology is

essentially an assortment of statistical and regression techniques. The first step involves

framing a statistically significant empirical model capable of describing the effect of multiple

factors on a response. A commonly used empirical model of the response surface analysis is a

quadratic polynomial of the type:

where y is the predicted response, xixj are input variables which influence the response

variable y; b0 is the offset term; bi is the ith linear coefficient; bii the ith quadratic coefficient

and bij is the ijth interaction coefficient.

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Once a suitable model is obtained, it can be used for optimization which involves finding an

optimum combination of factors that will maximize or minimize a response. The average

yields of biomass and pigment are found to be 5.488 mg/ml and 2.181 mg/l from Table 4.4.2.

By using multiple regression analysis, the experimental responses shown in Table 4.4.2 were

correlated with two significant factors according to Equation (1).

– (2)

Carotenoid production

– (3)

The factors x1 and x2 are indicated in their coded units (shown in Table 4.4.1). The goodness

of fit of the quadratic polynomials is expressed by the coefficient of determination (R2- which

is a measure of how well the model can be made to fit the raw data). The closer the value of

R2 is to 1, the better is the correlation among the observed and predicted values. The R2

values for Equations (2) and (3) are 0.956 and 0.960 respectively; indicating that about ~ 95

% of the variations in biomass and carotenoid yield can be explained by the quadratic

polynomials. This means that Equations (2) and (3) are adequate for correlating the

experimental results. Moreover regression equations (2 and 3) were evaluated by the F-test

for analysis of variance (ANOVA). Statistical significance and responses for biomass and

pigment production are shown in Table 4.4.3 and 4.4.4. Prob > F value for the model is less

than 0.05 infers that the model terms are statistically significant.

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Table 4.4.3. Statistical significance obtained for the regression coefficients in Eq’s .(1), (2).

For Biomass For Pigment

Model term
Coef p Coef p
Intercept 6.5800 0.000 2.6000 0.000
x1 1.2213 0.000 0.4737 0.000
x2 0.9875 0.000 0.3537 0.000
x3 0.8162 0.001 0.3275 0.000
x1*x1 -0.2713 0.166 -0.1125 0.122
x2*x2 -0.9438 0.002 -0.3725 0.002
x3*x3 -0.8313 0.004 -0.3000 0.004
x1*x2 -0.3900 0.059 -0.1850 0.024
x1*x3 0.3325 0.093 0.1375 0.064
x2*x3 0.4000 0.055 0.0975 0.154
R-Sq = 98.4% R-Sq(adj) R-Sq = 98.6% R-
= 95.6% Sq(adj) = 96.0%

Table 4.4.4. ANOVA results for biomass and pigment yields.

ANOVA

For Biomass-mg/ml

Source DF Sum of squares Mean square F P

Regression 9 32.2579 3.58421 34.75 0.001

Linear 3 25.063 8.35433 81 0

Square 3 5.5043 1.83477 17.79 0.004

Interaction 3 1.6906 0.56354 5.46 0.049

Residual Error 5 0.5157 0.10314

Lack-of-Fit 3 0.4621 0.15403 5.75 0.152

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Pure Error 2 0.0536 0.0268

Total 14 32.7736

For Pigment-mg/l

Source DF Sum of squares Mean square F P

Regression 9 4.70655 0.52295 38.78 0

Linear 3 3.65468 1.21823 90.34 0

Square 3 0.80132 0.26711 19.81 0.003

Interaction 3 0.25055 0.08352 6.19 0.039

Residual Error 5 0.06742 0.01348

Lack-of-Fit 3 0.03802 0.01267 0.86 0.576

Pure Error 2 0.0294 0.0147

Total 14 4.77397

The actual and predicted values of responses for biomass and carotenoid

concentrations versus the corresponding values calculated by regression models are shown in

Fig. 4.4.3a,b respectively. Actual values are the measured values for a particular experiment,

whereas predicted values are generated by using the approximating functions. The line of

perfect fit is also shown in these figures and visualization of the two regression models

provides an accurate description of the experimental data. In addition the values of R2 and

adjusted R2 (Table 4.4.3) have advocated a high correlation between actual and predicted

values. The response surface and contour plots were constructed using the regression models

and represented in Fig. 4.4.4a-d. Fig. 4.4.4a depicts the interactive effect of the agitation and

152
temperature on the biomass concentration. At low to moderate agitation values, increases in

temperature led to increased production of biomass initially up to center point and then

decreased. At low to moderate temperature values, increases in agitation values showed

increase in biomass yield. Similar trends were not observed for pigment yield as a function of

pH and temperature (Fig. 4.4.4c). At high pH values, increases in temperature showed

significant increase in pigment yield up to the center point and decreased thereafter. While

with increase in temperature values, increase in pH led to increased pigment production.

The contour plots in Fig. 4.4.4b,c indicate that a local optimum exists in the area

experimentally studied; a set of values on the two factors that leads to maximum biomass or

pigment production. The location of these optimal points can be obtained by differentiating

Equations (2) and (3). Equation (3) was used to derive the most efficient combination of x1 to

x3 to produce carotenoids from Rhodotura sp. According to Equation (3), a maximum

carotenoid recovery of 3.17 mg/l could be attained with pH (7.0), temperature (28.2 0C), and

agitation (150 rpm). Experimental validation of the optimum x1-x3 combination gave a

carotenoid yield of 3.298 ± 0.28 mg/l (with 7.83 mg/ml biomass concentration). Equation (2)

depicted that at pH (7.0), temperature (30 0C), and agitation (150 rpm) maximum yield of

7.97 mg/ml biomass could be achieved. The experimental validation resulted in biomass yield

of 8.27 ± 0.33 mg/ml with 2.43 mg/l carotenoid production.

153
Fig. 4.4.3. (a) Cell mass concentration calculated from regression model equation versus the

corresponding experimentally obtained values. (b) Carotenoid production calculated from the

regression model equation versus the corresponding experimentally obtained values.

The obtained results are in good agreement with the predicted values of pigment and

biomass production with ~ 4 % and ~ 3.6 % deviations. Therefore the results are in

agreement with the effectiveness of the response surface approach described here.

Furthermore, the obtained pigment yield was found to be in comparison with the earlier

reports which employed sugar cane molasses [6] and mug bean wastes [13]. In addition to the

higher yield, the economic aspects (simple methodology, cheap substrate) suggested that the

pigment production can be scaled up to the industrial level without any special conditions.

Till date, most of the pigment production was associated with the special conditions like acid

hydrolysis of their substrate which may hinder the scale up to industrial level.

154
Fig. 4.4.4. Response surface and contour plots obtained from Equation (2) and (3) showing

the effect of the temperature, agitation and their mutual interaction on biomass concentration

(a), (b); while it is effect of pH and temperature and their mutual interaction on caroteniod

pigment concentration (c), (d). The displayed units of all the graphs are in natural units.

4.4.3.4 DPPH radical scavenging activity

DPPH is a stable free radical that displays maximum absorbance at 517 nm. When DPPH

radicals come across a proton-donating substrate such as an antioxidant, the radicals would be

scavenged and the absorbance would be reduced [22]. The decrease in absorbance is taken as

a measure for radical-scavenging action.

155
 After 40 min of incubation, free radical scavenging property with different doses (0 to

100 µg/mL) of R. rubra craotenoid was compared with ascorbic acid as a control (Fig. 4.4.5).

A ~70 % scavenging activity of DPPH was achieved by ~100 µg/mL ascorbic acid, while it is

only 60 % with the produced R. rubra craotenoid. Thus the carotenoid obtained was not a

dominant radical scavenger with ~6 % lesser ability than the commercial ascorbic acid.

Fig. 4.4.5. Free radical scavenging property of the carotenoid by R. rubra. Dose dependent

scavenging activity of the pigment compared with ascorbic acid (control).

4.4.4 Conclusions

In summary, the results obtained from the R. rubra in this study will be useful for efficient

carotenoids production by utilizing the FWE as a sole substrate. The study also screened out

key vital parameter like pH and optimized other imperative parameters such as temperature

and agitation along with the pH using a response surface methodology. The simultaneous

optimization of parameters yielded maximum carotenoid production conditions in an

156
economical way, for its large scale production using a cheaper substrate (FWE). Our results

demonstrated that FWE could be profitably used as a suitable substrate without any

additional growth supplement for noteworthy carotenoid production. The response

optimization of parameters pH (7), temperature (28.2 0C) and agitation (150 rpm) by Box–

Behnken design resulted in 51.2 % more enhancement of the mean carotenoid production.

The produced carotenoid pigment is having potential antioxidant applications in food

industries. This can be valued for industrial scale utilization of FWE to generate high value

carotenoids using R. rubra and opens up scope for exploring other high value microbial

pigments.

References

[1] A. Das, et al., "An update on microbial carotenoid production: application of recent

metabolic engineering tools," Applied Microbiology and Biotechnology, vol. 77, pp.

505-512, 2007.

[2] L. Dufossé, et al., "Microorganisms and microalgae as sources of pigments for food

use: a scientific oddity or an industrial reality?," Trends in Food Science &

Technology, vol. 16, pp. 389-406, 2005.

[3] E. D. Simova, et al., "Effect of aeration on the production of carotenoid pigments by

Rhodotorula rubra-lactobacillus casei subsp. casei co-cultures in whey ultrafiltrate,"

Zeitschrift fur Naturforschung C-Journal of Biosciences, vol. 58, pp. 225-229, 2003.

[4] I. Marova, et al., "Use of several waste substrates for carotenoid-rich yeast biomass

production," Journal of Environmental Management, vol. 95, Supplement, pp. S338-

S342, 2012.

157
[5] P. Park, et al., "Chemical disruption of yeast cells for the isolation of carotenoid

pigments," Separation and purification technology, vol. 53, pp. 148-152, 2007.

[6] P. Park, et al., "Optimization of carotenoid production by Rhodotorula glutinis using

statistical experimental design," World journal of microbiology and biotechnology,

vol. 21, pp. 429-434, 2005.

[7] M. M. S. Cabral, et al., "Carotenoids production from a newly isolated Sporidiobolus

pararoseus strain by submerged fermentation," European Food Research and

Technology, vol. 233, pp. 159-166, 2011.

[8] B. Ribeiro, et al., "Technological Aspects of β-Carotene Production," Food and

Bioprocess Technology, vol. 4, pp. 693-701, 2011/07/01 2011.

[9] H. J. Nelis and A. De Leenheer, "Microbial sources of carotenoid pigments used in

foods and feeds," Journal of Applied Microbiology, vol. 70, pp. 181-191, 1991.

[10] S. Liaaen-Jensen and A. Andrewes, "Microbial carotenoids," Annual Reviews in

Microbiology, vol. 26, pp. 225-248, 1972.

[11] G. I. Frengova and D. M. Beshkova, "Carotenoids from Rhodotorula and Phaffia:

yeasts of biotechnological importance," Journal of industrial microbiology &

biotechnology, vol. 36, pp. 163-180, 2009.

[12] B. D. H. Dung, et al., "Optimization carotenoids production by Rhodotorula glutinis

GBDO Uusing design of Plackett-Burman matrix and central composite design-

response surface methodology," in Conference proceedings of Biotechnology for

Green Solutions and Sustainable Environment, 2010, p. 25.

158
[13] J. Tinoi, et al., "Simplex optimization of carotenoid production by Rhodotorula

glutinis using hydrolyzed mung bean waste flour as substrate," Process Biochemistry,

vol. 40, pp. 2551-2557, 2005.

[14] B. Latha and K. Jeevaratnam, "Purification and characterization of the pigments from

Rhodotorula glutinis DFR-PDY isolated from natural source," Global J Biotech

Biochem, vol. 5, pp. 166-174, 2010.

[15] P. Bhosale and R. Gadre, "Optimization of carotenoid production from

hyper‐producing Rhodotorula glutinis mutant 32 by a factorial approach," Letters in

applied microbiology, vol. 33, pp. 12-16, 2001.

[16] C. Malisorn and W. Suntornsuk, "Optimization of β-carotene production by

Rhodotorula glutinis DM28 in fermented radish brine," Bioresource Technology, vol.

99, pp. 2281-2287, 2008.

[17] P. Buzzini, "An optimization study of carotenoid production by Rhodotorula glutinis

DBVPG 3853 from substrates containing concentrated rectified grape must as the sole

carbohydrate source," Journal of Industrial Microbiology and Biotechnology, vol. 24,

pp. 41-45, 2000.

[18] S. N. Surwase, et al., "Optimization of melanin production by Brevundimonas sp. SGJ

using response surface methodology," 3 Biotech, pp. 1-8, 2012.

[19] A. Gramza-Michałowska and B. Stachowiak, "The antioxidant potential of carotenoid

extract from Phaffia rhodozyma," Acta Scientiarum Polonorum. Technologia

Alimentaria, vol. 9, 2010.

[20] S. K. Thimmaiah, Standard methods of biochemical analysis: Kalyani publishers,

2006.

159
[21] J. Coates, "Interpretation of infrared spectra, a practical approach," Encyclopedia of

analytical chemistry, 2000.

[22] R. Lo Scalzo, "Organic acids influence on DPPH scavenging by ascorbic acid," Food

Chemistry, vol. 107, pp. 40-43, 2008.

160
4.5 Astaxanthin by Xanthophyllomyces dendrorhous

Using Fruit Waste Extract as Sole Source of Energy:

Optimization of Culture Conditions by Taguchi method

for Improved Pigment Production

4.5.1 Introduction

Carotenoid class astaxanthin belongs to the group of lipophilic tetraterpenes, which are

combination of eight isoprene units (C5) that has important application in the neutraceutical,

cosmetics, food and feed industries due to its high antioxidant activity [1-3]. Astaxanthin

synthesized chemically contains only 8% astaxanthin and costs $25,000-30,000 Kg-1[4].

Synthetic astaxanthin is the major source of carotenoid currently being used in fish feeds [5].

However, food and feed additives from biotechnological processes are preferred by

consumers to those produced by chemical technologies [6]. Among the microorganisms

Brevibacterium [7], Mycobacterium lacticola [8], Agrobacterium auratium [9],

Haematococcus pluvalis [10] and X. dendrorhous [4, 11] can be used for commercial

161
production of astaxanthin as 80-90% of the total carotenoids produced by these yeasts

accounts for astaxanthin.

Among all red pigmented heterobasidiomycetous yeast, X. dendrrorhous has the

potential to produce carotenoids on cheaper substrates like different agro-industrial raw

materials with high yields [12]. This yeast strain has ability for the assimilation and

metabolizing mono, di and polysaccharides, organic acid and alcohols. Moreover till date

research is focused on this versatile strain on various cheaper substrates for significant

astaxanthin production. Aiming towards cost cutting strategies, substrates such as waste

streams like those from sugar manufacturing processes or the corn wet milling industry, white

grape juice, enzymatic eucalyptus wood hydrolyzates [13], hemicellulosic hydrolyzates of

eucalyptus globulus [14], yucca medium (based on date juice) [15], peat hydrolysate [16],

corn steep liquor [17] etc. were successfully employed for the production of significant

carotenoid class astaxanthin.

Very limited research has been carried out so far on fruit wastes and their extracts as

sole source of energy for astaxanthin production using X. dendrrorhous. India contributes to

10 % of the world’s fruit production [18]. Over last few years research focus is towards

reprocessing and reuse of various fruit wastes for the conversion of nutritive and valuable

products [19-20]. Previous studies on fruit wastes signify that it is a rich source of

carbohydrates and other minor nutrients to support the microbial growth [19-20]. The surplus

fruit wastes those are frequently disposed in the environment after exhaustive extraction of

their juice may be possibly used as low cost substrates for production of microbial bioactive

compounds like pigments. The availability of readymade simple sugars along with macro and

micro nutrients make fruit waste as a suitable compound for the production of numerous

162
pigments by a variety of microorganisms. The above reported studies used special conditions

and/or enriched the substrate media for high yield pigment production. In contrast, eying on

cheaper, economical and quick synthesis process devoid of providing special conditions, we

explored the capacity of readily prepared fruit waste extract (FWE) as sole substrate for

astaxanthin production by acquired X. dendrrorhous. Thus this paper outlined the optimum

production conditions for carotenoid pigment by X. dendrrorhous on FWE using Taguchi

method.

4.5.2 Materials and methods

4.5.2.1 Chemicals and microorganism

Acetone, ascorbic acid, methanol, NaCl, HCL and diethyl ether were procured from Merck,

India. The strain Xanthophyllomyces dendrorhous (7536) was obtained from MTCC

Chandigarh. The yeast was maintained on Yeast extract/ malt extract (YM) media agar plates

at 4 0C and sub cultured monthly in YM media broth. YM media and agar were purchased

from HiMedia chemicals, India.

4.5.2.2 Substrate preparation

Fruit waste is obtained from local juice shop from local market, Rourkela, Odisha, India,

which includes pineapple waste, orange waste and minor portion of pomegranate waste. Fruit

waste extract (FWE) media is prepared from mixed waste by boiling and extracting the

soluble sugars for 30 min using distilled water.

4.5.2.3 Culture conditions

The inoculum was prepared in YM broth at 22 0C for 48h. The yeast cells were harvested by

centrifugation and washed twice with distilled water. 1 mL of microbial solution (1 % w/v)

163
was inoculated in a 250 ml Erlenmeyer flask containing 50 ml FWE medium. The inoculated

cultures were kept at 22 0C in incubator shaker at a shaking speed of 150 rpm for pigment

production. Yeast extract/malt extract (YM) medium is used as the reference medium for

comparison with FWE medium. Each experiment was performed thrice to minimize the error.

4.5.2.4 Pigment production and extraction/ Sugar determination

Yeast cells obtained after centrifugation was washed twice with distilled water. Pigmented

cells were treated with methanol: acetone (1:1) solvents and centrifuged. This process was

repeated until colorless biomass was obtained. The extracted pigment was subjected to phase

separation using equal volumes of petroleum ether and 10 % NaCl. The carotenoid was

collected from the diethyl ether phase and analyzed at 474 nm by UV-visible

spectrophotometer using an extension coefficient of = 2,100 [21]. Obtained carotenoids

were further purified using thin layer chromatography using stationary phase - TLC silica gel

60 F 254 and hexane: methanol (7:3) as stationary phase and solvent phase respectively. The

purified pigment was further analyzed in FTIR to determine the functional groups.

The 3,5- dinitrosalicyclic acid (DNS) method of miller [22] was used to determine the

reducing sugars in FWE medium. About 1ml sample was centrifuged at 3,500 X g for 5 min,

and 1 mL DNS reagent was added to the supernatant and boiled for 5 min. The sample was

placed in ice –bath for rapid cooling. Distilled water was added to the samples and vortexed

for 5 min. The optical density was calculated at 575 nm. In addition, CHNS analysis was

performed before and after the pigment production

4.5.2.5 Taguchi design and statistical analysis

Simple Taguchi orthogonal array design was selected where there are three factors and three

levels. From the reported studies, pH (4-6), temperature (15-25 0C) and agitation (100-300

164
rpm) were found to be significant in obtaining higher amounts of astaxanthin production by

X. dendrorhous [23-25]. In this study Taguchi method was used for optimization of pigment

production conditions. Optimization of stated factors in three different levels is illustrated in

Table 4.5.1. To perform the Taguchi method, 9 different experiments using L9 orthogonal

array was run as shown in Table 4.5.2. Based on the primary results a verification test was

employed to check the optimum condition. An analysis of variance (ANOVA) for the

obtained results was examined. Design of experiments, ANOVA and optimization of process

were accomplished using MINITAB- 14 software.

4.5.2.6 Determination of free radical scavenging activity

The effect of astaxanthin from X. dendrorhous on DPPH free radical was studied as per the

method of Gramza-Michałowska and Stachowiak[26] with some modifications. A 2 mg/ml

pigment suspension was prepared in varying volumes of ethanol (10 to 60 µl). To the

suspension 1 ml DPPH solution containing 0.1 mM DPPH and 99.5 % ethanol (99.5 %) was

added. The mixture was thoroughly mixed and allowed to stand for 40 min at room

temperature in the dark. Ascorbic acid is used as positive control and the pigment solution

without DPPH as negative control. The radical scavenging activity was measured as a

decrease in the absorbance of DPPH at 517 nm wavelength and was calculated using the

following formula:

(Ac - As)
% Scavenging activity   100
(Ac)

Where Ac, As are absorbance’s from control and sample.

165
4.5.2.7 Analytical methods

UV-visible spectra and radical scavenging activity were measured using an UV-visible-NIR

spectrophotometer (Shimadzu, UV-3600). Morphological characteristics of the used

microorganism was studied by using scanning electron microscopy (SEM, JEOL JSM 6480

LV), active functional groups of the produced pigment was identified through Fourier

infrared spectroscopy (FTIR, Bruker, USA) equipped with a horizontal attenuated total

reflectance (ATR) device with zinc selenide (ZnSe) crystal.

Table 4.5.1. Factors and their levels studied by Taguchi method.

Factor Level 1 Level 2 Level 3
pH 4 5 6
Temperature (oC) 15 20 25
Agitation (rpm) 100 200 300

Table 4.5.2. Levels of three different factors applied in each of nine trials, with observed

results.

pH Temperature(OC) Agitation (rpm) Astaxanthin (mg/g) Biomass (g/L)

4 15 100 0.402 0.3216
4 20 200 0.556 0.548
4 25 300 0.525 0.62
5 15 200 1.24 1.133
5 20 300 1.252 1.34
5 25 100 0.91 0.728
6 15 300 0.978 1.28
6 20 100 0.88 0.704
6 25 200 1.211 1.103

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4.5.3 Results and discussion

Among various yeasts, X. dendrorhous has been exploited as major source of astaxanthin

with potential commercial applications in the fields of cosmetics, pharmaceuticals and

agriculture [5, 7]. However, astaxanthin production from cheaper substrates has limited

reports. So optimization of its process parameters for astaxanthin production has been

extensively studied to emphasize its production in commercial scale. Substrate

utilization/growth and pigment production by yeast was significantly influenced by key

factors like pH, temperature and agitation; hence these factors play a vital role in the cost

effectiveness of pigment production. The pigmentation of the FWE and the SEM image of

the studied yeast were given in Fig. 4.5.1.

Fig. 4.5.1. Pigmentation of FWE (a) by before X. dendrorhous yeast with lower (b) and

higher (c) magnification SEM images.

167
4.5.3.1 Experimental design by Taguchi method

Effects of pH, temperature and agitation were studied using Taguchi method, which is a

fractional factorial experimental design. The results of experiments performed in this section

(Table 4.5.2) showed that the maximum average yield of pigment 1.252 mg/g, at

experimental conditions pH 5, temperature 20 oC and agitation 300 rpm. Fig. 4.5.2 depicts the

main effect of each of these factors that implies the average results obtained for each factor in

order to screen out significant factors the statistical technique of analysis of variance

(ANOVA) with F test was employed to the simulation reflecting the data shown in Table

4.5.3. The ANOVA results implicates from F value of 23.71 that pH has significant effect on

pigment production. Similarly temperature showed negligible effect. Thus the optimum

experimental condition was selected from the obtained result as pH 5, temp 20 oC and

agitation 300 rpm. When the results were analyzed, an optimum condition was proposed by

calculations.

4.5.3.2 Validation of the model

Table 4.5.4 shows the suggested conditions for maximum pigment production. Statistical

calculations predicted that if the conditions were chosen as shown in Table 4.5.4, the pigment

production should reach 1.264 mg/g biomass. However after performing the experiment at

said condition, the produced astaxanthin was 1.40 ± 0.14 mg/g. The difference between

predicted and actual result was about 10 %, and is regarded as acceptable and promising.

168
 1.2
1.1
Astaxanthin (mg/g)

1.0
0.9
0.8
0.7
pH
0.6 o
Temperature ( C)
0.5 Agitation (rpm)
0.4
1.0 1.5 2.0 2.5 3.0
Levels of factors

Fig. 4.5.2. Main effects of factors or average of obtained results as mg/g biomass in which

each factor is at a given level. For description of ‘levels’ refer to Table 4.5.1.

Table 4.5.3. Analysis of variance of main effects of factors

Analysis of Variance for Means

Source DF Seq SS Adj SS Adj MS F P

pH 2 0.7009 0.7009 0.3504 23.71 0.04

Temperature 2 0.0007 0.0007 0.0003 0.03 0.974

Rpm 2 0.1160 0.1160 0.0580 3.93 0.203

Residual Error 2 0.0295 0.0295 0.0147

Total 8 0.8474

169
Table 4.5.4. Optimum conditions suggested by statistical calculations after performing the

experiments.

Factor Level description Level Contribution in ‘mg/g’

pH 5 2 1.134–0.883=0.251

Temperature (oC) 20 2 0.896 –0.883=0.013

Agitation (rpm) 200 2 1.00 –0.883=0.117

4.5.3.3 Pigment yield, biomass trend and glucose utilization

Table 4.5.4, illustrates the optimum condition for astaxanthin pigment production. Growth

kinetics of the pigment production, cell mass and substrate utilization has been studies at

optimum experimental condition as represented in Fig. 4.5.3a. The figure shows that both

biomass and astaxanthin production increased over time as total soluble sugars content

decreased. The maximum levels of biomass (~22.4 g/L) and astaxanthin (~31.5 mg/L) were

reached at 84 and 96 h, while the carbon source (soluble sugars) had been depleted and

reaching a constant level from 96 to 144 h (Fig. 4.5.3a).

From the figure, the time course of the biomass growth showed the characteristic

exponential and stationary phases and reached a maximum level after about 84 h. Moreover it

was also noted that the pigment production was not constant at stationary phase of biomass

growth and went on increased up to 108 h. The observed behavior infers the ability of the

used strain to give significant amounts of astaxanthin even in stationary phase, which is

beneficial effect of the used substrate (under the used conditions).

From this result it is clear that the astaxanthin production was partially dependent on

biomass growth and still occurred even after growth and sugars depletion. Such behavior is

170
certainly related to the fact that X. dendrorhous excretes and stocks some extracellular carbon

intermediates, which stimulate late carotenogenesis [7].

Astaxanthin (mg/L); Biomass (g/L)

35 35
0.9
30 (a) 30
0.8
(b)
Glucose depletion (g/L)

Absorbance (a.u)
25 25 0.7

20 20 0.6

0.5
15 15
Glucose depletion (g/L) 0.4
10 Astaxanthin (mg/L) 10
Biomass (g/L) 0.3
5 5 0.2

0 0 0.1
0 20 40 60 80 100 120 140 300 375 450 525 600 675
Time (hours) Wavelength (nm)
100
2.02

2.00
(c) 80
(d)
% Radical scavenging
Intensity (a.u)

1.98
60
1.96 1736
2970
1380
1.94 40
3362
1.92 Astaxanthin
20 Ascorbic acid
1.90
950
1163
0
1.88
3600 3150 2700 2250 1800 1350 900 0 20 40 60 80 100 120
-1
Wavenumber (cm ) Pigment dose (µg/mL)

Fig. 4.5.3. (a) Time course of the growth and production of astaxanathin by X. dendrorhous

in FWE. Experiment was carried out at optimum conditions i.e. pH (5), temperature (20 oC)

and agitation (300 rpm). (b) UV-Visible and FTIR spectrum (c) of the produced pigment in

methanol. (d) Antioxidant activity (DPPH radical scavenging) of astaxanthin and ascorbic

acid.

171
4.5.3.4 UV analysis

The spectral property of the produced astaxanthin in FWE was shown in Fig. 4.5.3b. The

absorption maximum was identified to be at 474 nm in methanol and was in good agreement

with the reported value [27].

4.5.3.5 FTIR analysis

The purified pigment from the TLC plate was dissolved in ethanol and analyzed for FTIR

spectral analysis and the results are shown in (Fig. 4.5.3c). According to Coates et al. [28] the

peak descriptions of the FTIR spectra can be described as follows: The peak at 3362 shows

the presence of O-H hydrogen bond. The peak at 2970 is due to methyne C-H stretch; a peak

at 1736 shows the presence of C=O bond in the molecule. The peak at 1380 is due to methyl

C-H asymmetric band. The peak at 1163 is for C-O stretch and the peak at 950 represents the

skeletal vibrations. The spectral behavior of the pigment closely resembles the structure of

astaxanthin which is predominantly obtained by X. dendrorhous [29].

4.5.3.6 DPPH radical scavenging activity

DPPH is a stable free radical that displays maximum absorbance at 517 nm. When DPPH

radicals come across a proton-donating substrate such as an antioxidant, the radicals would be

scavenged and the absorbance would be reduced [30]. The decrease in absorbance is taken as

a measure for radical-scavenging action. The DPPH radical-scavenging activity was

investigated at different concentrations (0 to 109 µg/mL) of the produced astaxanthin. The

results are shown in Fig. 4.5.3d.

172
 After 40 min of incubation, it could be observed that the radical scavenging activity

increased promptly with an increase in astaxanthin dose when compared to ascorbic acid

(control) and reached up to ~85 % at the dose of 109 µg/ml. However, it is also noted that

ascorbic acid helped in achieving only ~70 % under the same conditions. Although

astaxanhin and ascorbic acid both showing an increase in % radical scavenging, it is

noteworthy that the former was higher than the latter at any moment within the studied

pigment dose (Fig. 4.5.3d).

In summary, the key factors that independently influence biomass growth and

pigment production were identified by Taguchi method. At identified key parameters, FWE

appears to be an excellent substrate since a remarkable astaxanthin production (1.4 ± 0.14

mg/g biomass) was obtained when compared to YM medium i.e. 1.66 ± 0.21 mg/g biomass

at same culture conditions. YM is an expensive source for astaxanthin production with C to N

proportion of 1.0 and 0.3 %, while FWE is an inexpensive alternative with C to N proportion

found to be 1.0 and 0.066 % (observed by CHNS analysis). This work shows that FWE

medium plays a key role in astaxanthin production by X. dendrorhous and it is worth noting

that, in our case we achieved enhanced astaxanthin production with free radical scavenging

activity without any substrate supplements and special conditions.

4.5.4 Conclusions

This study shows the suitability of the FWE medium for the production of astaxanthin by X.

dendrorhous yeast strain. Critical parameters screened by simple Taguchi approach and

optimum conditions for the enhanced production of pigment were established. Increase yield

was observed at pH - 5, Temp - 20 °C and Agitation-200 rpm with optimum production of

173
1.4 ± 0.14 mg/g biomass. UV-visible and IR analysis of the purified pigment illustrated the

structural confirmations of the pigment. In addition, free radical scavenging activity of the

obtained pigment gave 15 % better activity than ascorbic acid and encourages its applicability

as a constitutional ingredient in cosmetics.

References

[1] H. T. H. AN, et al., "Determination of astaxanthin and other carotenoids in

vietnamese crustaceans by HPLC." TAp Chí Khoa Hoc Và Công Nghe, vol. 46, pp.

47-58, 2008.

[2] R. N. Rao, et al., "Preparative isolation and characterization of some minor impurities

of astaxanthin by high-performance liquid chromatography," Journal of

chromatography A, vol. 1076, pp. 189-192, 2005.

[3] I. Schmidt, et al., "Biotechnological production of astaxanthin with Phaffia

rhodozyma/Xanthophyllomyces dendrorhous," Applied Microbiology and

Biotechnology, vol. 89, pp. 555-571, 2011.

[4] J. Tinoi, et al., "Utilization of mustard waste isolates for improved production of

astaxanthin by Xanthophyllomyces dendrorhous," Journal of Industrial Microbiology

and Biotechnology, vol. 33, pp. 309-314, 2006.

[5] I. Higuera-Ciapara, et al., "Astaxanthin: a review of its chemistry and applications,"

Critical reviews in food science and nutrition, vol. 46, pp. 185-196, 2006.

[6] D. Pleissner and C. S. K. Lin, "Valorisation of food waste in biotechnological

processes," Sustainable Chemical Processes, vol. 1, p. 21, 2013.

174
[7] E. A. Johnson and G.-H. An, "Astaxanthin from microbial sources," Critical Reviews

in Biotechnology, vol. 11, pp. 297-326, 1991.

[8] H. J. Nelis and A. De Leenheer, "Microbial sources of carotenoid pigments used in

foods and feeds," Journal of Applied Microbiology, vol. 70, pp. 181-191, 1991.

[9] J. A. Bon, et al., "Isolation of astaxanthin-overproducing mutants of Phaffia

rhodozyma," Biotechnology letters, vol. 19, pp. 109-112, 1997.

[10] L. Waldenstedt, et al., "Effects of astaxanthin-rich algal meal (Haematococcus

pluvalis) on growth performance, Caecal Campylobacter and Clostridial counts and

tissue astaxanthin concentration of broiler chickens," Animal feed science and

technology, vol. 108, pp. 119-132, 2003.

[11] L. M. Ducrey Sanpietro and M. R. Kula, "Studies of astaxanthin biosynthesis in

Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Effect of inhibitors and low

temperature," Yeast, vol. 14, pp. 1007-1016, 1998.

[12] G. I. Frengova and D. M. Beshkova, "Carotenoids from Rhodotorula and Phaffia:

yeasts of biotechnological importance," Journal of industrial microbiology &

biotechnology, vol. 36, pp. 163-180, 2009.

[13] J. Cruz and J. Parajo, "Improved astaxanthin production by Xanthophyllomyces

dendrorhous growing on enzymatic wood hydrolysates containing glucose and

cellobiose," Food Chemistry, vol. 63, pp. 479-484, 1998.

[14] J. C. Parajo, et al., "Production of carotenoids by Phaffia rhodozyma growing on

media made from hemicellulosic hydrolysates of Eucalyptus globulus wood,"

Biotechnology and bioengineering, vol. 59, pp. 501-506, 1998.

175
[15] . Ram rez, et al., "Optimization of astaxanthin production by Phaffia rhodozyma

through factorial design and response surface methodology," Journal of

Biotechnology, vol. 88, pp. 259-268, 2001.

[16] M. Vazquez and A. M. Martin, "Optimization of Phaffia rhodozyma continuous

culture through response surface methodology," Biotechnology and bioengineering,

vol. 57, pp. 314-320, 1998.

[17] S. S. Kesava, et al., "An industrial medium for improved production of carotenoids

from a mutant strain of Phaffia rhodozyma," Bioprocess Engineering, vol. 19, pp.

165-170, 1998.

[18] FAOSTAT, "These are food and agriculture classification groups. For definition with

list of botanical species covered under each classification, consult FAOSTAT of the

United Nations," 2011.

[19] P. Viswanath, et al., "Anaerobic digestion of fruit and vegetable processing wastes for

biogas production," Bioresource Technology, vol. 40, pp. 43-48, 1992.

[20] M. Mahadevaswamy and L. Venkataraman, "Integrated utilization of fruit-processing

wastes for biogas and fish production," Biological wastes, vol. 32, pp. 243-251, 1990.

[21] W. A. Schroeder and E. Johnson, "Antioxidant role of carotenoids in Phaffia

rhodozyma," Journal of general microbiology, vol. 139, pp. 907-912, 1993.

[22] G. L. Miller, "Use of dinitrosalicylic acid reagent for determination of reducing

sugar," Analytical Chemistry, vol. 31, pp. 426-428, 1959.

[23] S. Kim, et al., "Increased carotenoid production in Xanthophyllomyces dendrorhous

G276 using plant extracts," Journal of Microbiology-Seoul-, vol. 45, p. 128, 2007.

176
[24] Y.-G. Zheng, et al., "Large-Scale Production of Astaxanthin by Xanthophyllomyces

Dendrorhous," Food and bioproducts processing, vol. 84, pp. 164-166, 2006.

[25] A. Domínguez-Bocanegra and J. Torres-Muñoz, "Astaxanthin hyperproduction by

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as

sole source of energy," Applied Microbiology and Biotechnology, vol. 66, pp. 249-

252, 2004.

[26] A. Gramza-Michałowska and B. Stachowiak, "The antioxidant potential of carotenoid

extract from Phaffia rhodozyma," Acta Scientiarum Polonorum. Technologia

Alimentaria, vol. 9, 2010.

[27] M. Buchwald and W. P. Jencks, "Optical properties of astaxanthin solutions and

aggregates," Biochemistry, vol. 7, pp. 834-843, 1968.

[28] J. Coates, "Interpretation of infrared spectra, a practical approach," Encyclopedia of

analytical chemistry, 2000.

[29] X. Chen, et al., "The preparation and stability of the inclusion complex of astaxanthin

with β-cyclodextrin," Food Chemistry, vol. 101, pp. 1580-1584, 2007.

[30] R. Lo Scalzo, "Organic acids influence on DPPH scavenging by ascorbic acid," Food

Chemistry, vol. 107, pp. 40-43, 2008.

177
Chapter 5 Conclusions and future perspectives

Conclusions

The main outcome of this work is that we have screened some novel potent pigment

producing microorganisms, bacteria in particular, which can produce melanins and

carotenoids on cheaper and renewable substrates in high amounts. This work also explores

the pigment producing ability of the obtained microbial strains (yeasts) on various cheaper

substrates and accomplished noticeable high yields on a fruit waste extract medium. The

beauty of the work lies in the identification of the key parameters of pigment production (for

isolated as well as obtained strains) and optimization of the influential parameters for

improved pigments production.

 The present exertion starts with the production of melanin by a marine isolate in part I-

4.1 of Chapter 4. Here marine bacterium capable of melanin production on marine

broth/agar was isolated and identified as Pseudomonas sp. (closely related to guinea) on

phenotypic characterization. Melanin production activity of the isolate was studied in

liquid media such as pure marine broth and vegetable cabbage waste. In pure marine

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 broth, melanin yield was ~5.35 mg/mL and pigment production was negligible in pure

vegetable waste medium. However in the presence of marine broth (as starter culture)

melanin yield increased to ~2.79 mg/mL. This indicates melanin production may be

initiated austerely by marine broth. The characterized melanin was very near to synthetic

dihydroxyphenylalanine (DOPA)-melanin in all aspects. Furthermore, the purified

melanin upon analyzing for sun protection activity showed promising result. SPF value

of the melanin here was found to be 53.74 where, it was 59.34 for synthetic melanin.

Additionally radical scavenging activity of the obtained melanin was higher than the

ascorbic acid (control) at all doses which showed 40% of enhanced reductive capability.

~55% ferrous ion chelation activity was noticed for the dose of 0.146 mg/mL which was

greater than synthetic melanin.

 The pigment outcome from a soil microbial isolate addressed in part I–4.2 of Chapter 4

was a melanin and abundantly produced on a fruit waste extract medium. The garden

soil isolate here was identified to be Bacillus safensis. The optimization of process

parameters influencing melanin production was attempted using a simple two step

methodology. Taguchi approach was adopted for screening of critical parameters for

pigment production, and optimization study was performed using the response surface

methodology (RSM) with a central composite design. At optimum conditions of pH -

6.84 and Temp - 30.7 ºC, a significant yield of 6.96 ± 0.6 mg/mL was observed.

Statistical analysis revealed that the experimental results fitted well to this statistical

model with model R2 value 0.982. The optimization of process parameters using RSM

reported a 15 % increase in the yield of melanin than average yield by the trails of the

studied model. The melanin pigment obtained here too exhibited significant

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 photoprotective capability with sunscreen protection factor 53.36, while it was 59.34 for

synthetic melanin. It also showed ~ 30% greater radical scavenging activity than

ascorbic acid for a constant melanin dose of 100 µg/mL. and metal chelating activity

studies showed that obtained melanin has maximum chelation ~ 64 % for a dose of 0.2

mg/mL.

 Microbial method of carotenoid pigment (β-carotene type) production was attained by

Bacillus clausii (a garden soil isolate) using rice powder as the sole substrate in part I-4.3

of Chapter 4. Influence of substrate (rice powder) on β-carotenoid production has been

extensively studied at optimum process conditions such as pH, temperature etc. The

maximum yield of β-carotenoid 48.9 % using B.clausii was achieved at pH 7 and 35 oC

utilizing rice powder as a sole substrate. A statistical design technique, Taguchi method

was applied to evaluate the optimal process conditions such as pH, temperature etc. for

maximum pigment production.

 The investigations of part II-4.4 and 4.5 of Chapter 4 shows the effectiveness of

abundantly available cheaper fruit waste extract as a sole substrate in obtaining

carotenoids in significant amounts by R. rubra and X. dendrorhous yeast strains. In 4.4,

a two-step simple sequential strategy was employed for the optimization of carotenoid

production by R. rubra. In the first step, one factor at a time was employed to evaluate

the impact of pH on carotenoid production. The outcome revealed that pH has a

noteworthy influence on pigment production at ambient conditions with constant

temperature and agitation. A Box–Behnken design was then applied in the second step to

optimize the pH, temperature and agitation to obtain high pigment yield. The statistical

experimental design predicted the high yield conditions of different responses. The

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 interaction between pH and temperature stood vital for improved carotenoid production

(2.98 ± 0.28 mg/l) with biomass yield of 7.83 mg/ml by the optimization of significant

parameters. The optimum conditions followed for high yield carotenoids are pH (7.0),

temperature (28.2 0C), and agitation (150 rpm). The purified pigment upon testing for

free radical scavenging activity displayed moderately comparable and ~6 % lesser ability

than the tested ascorbic acid as a control.

 While addressing the later study in 4.5, Taguchi method was employed to find the

optimum pigment production conditions of the yeast in FWE medium. Effect of key

parameters (pH, temperature and agitation) was investigated using L9 orthogonal array

by three factor-three level approach and significant parameters influencing pigment yield

was checked by analysis of variance. Key parameter’s contribution was suggested by

statistical calculations and assessed by a validation test. Conducting the experiments at

optimum conditions (pH-5, Temp-20 0C and Agitation-200 rpm), an increase in 10 %

astaxanthin was achieved. This study shows a simple and cost effective synthesis of

astaxanthin by X. dendrorhous using FWE. Additionally free radical scavenging activity

of the purified pigment gave 15 % better activity than the ascorbic acid with a constant

dose of 100 µg/mL.

The above evidences suggest that, FWE has huge potential to be an economical substrate for

various microorganisms for novel pigments production with no medium enrichments and

special conditions. The established optimized conditions in FWE have huge scope to be

scaled-up for the large scale production of melanins and carotenoids by selective

microorganisms which are having significant applications in cosmetics, agriculture and

medicine sectors.

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Future Perspectives

 A systematic screening of other microbial habitants for pigment producing microbes and

their exploitation of a wide range of different cheaper substrates is highly recommended.

 Evaluation of potent pigment producing organisms like fungi and bacteria has to be

exploited on different inexpensive materials as a sole source of substrates will warrant

pigments production economical.

 Robust Taguchi and response surface approaches should be implemented during the

cultivation procedures to identify the key process parameters and to develop a model

based production strategy. Statistical methods not only save time, but also eliminate the

wastage of media and materials.

 Toxicity issues to be thoroughly studied for the novel identified pigments by

developmental stage for marketability and consumer acceptance.

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 Publications from the work

 Tarangini Korumilli and Susmita Mishra, Production of Melanin by Soil Microbial

Isolate on Fruit Waste Extract: Two Step Optimization of Key Parameters.

Biotechnology Reports, 4, 39-146 (2014).

 Tarangini Korumilli and Susmita Mishra, Carotenoid Production by Bacillus clausii

Using Rice Powder as the Sole Substrate: Pigment Analyses and Optimization of

Key Production Parameters, Journal of Biochemical Technology, 5, 788-794 (2014).

 Tarangini Korumilli and Susmita Mishra, Carotenoid production by Rhodotorula sp.

on fruit waste extract as a sole source and optimization of key parameters. Iranian

journal of Chemistry and Chemical Engineering, 33, 97-106 (2014).

 Tarangini Korumilli and Susmita Mishra, Production, Characterization and

Analysis of Melanin from Isolated Marine Pseudomonas sp. using Vegetable waste,

Research Journal of Engineering Science, 2, 40-46 (2013).

 Tarangini Korumilli and Susmita Mishra, Astaxanthin by Xanthophyllomyces

dendrorhous Using Fruit Waste Extract as Sole Source of Energy: Optimization of

Culture Conditions by Taguchi method for Improved Pigment Production.

Biotechnology Reports - Submitted (In review) 2014.

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 CURRICULUM VITAE

TARANGINI KORUMILLI
C/O Annapurna Pharmacy, DoNo:12-21-16
Aryapuram. Rajahmundry-533014,
East Godavari District, Andhra Pradesh, India
Contact: 09439783304
E-mail: k.tarangini@gmail.com

Career Goal
To work in a challenging academic position that gives me an opportunity to prove myself
and to be an outstanding asset to the organization in which I work.

Current status
Doctorial Research scholar engaged in “Microbial pigments” research, especially on
“Exploring simple and economical methods of production of microbial pigments on cheaper
substrates and investigating their biological applications”.

Academics

Class / % of
Course College/University Year of Passing
Marks
M.Tech (Res) National Institute of Technology, First Class/
2009
(Chemical Engg) Rourkela, Orissa, India. (86.80 %)
Godavari Institute of Eng &
B.Tech. First Class/
Technology, JNTU, 2006
(Bio-Technology) (76.40 %)
(Rajahmundry, A.P, India)
Intermediate Aditya Junior College, (Board Of First Class/
2001
(Bi.P.C) Intermediate Education, A.P.) (80.50 %)

Areas of Interest
Biochemical engineering, Food science and technology, Biosorption, Molecular Biology,
Genetics, Bioprocess Engineering, Immunology, and associated areas of Biotechnology.

Skill Profile
 Expertise with instruments like TGA, AAS, UV-visible spectroscopy, Sonicator,
Spray drier, HPLC etc. with all basic analytical techniques.
Other skills: System languages C; Operating System-Windows XP, Windows 7

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Assets
 Good communication, practical & positive thinking, interpersonal and leadership
skills. Thrive in both independent and collaborative work environments.
 Quick learner with an ability to rapidly achieve organizational integration, assimilate
job requirements and employ new ideas, concepts, methods and technologies
 Efficient in handling projects and a team.

Major Achievements
International Publications
 Tarangini Korumilli and Susmita Mishra, Production of Melanin by Soil Microbial
Isolate on Fruit Waste Extract: Two Step Optimization of Key Parameters.
Biotechnology Reports, 4, 39-146 (2014).
 Tarangini Korumilli and Susmita Mishra, Carotenoid Production by Bacillus clausii
Using Rice Powder as the Sole Substrate: Pigment Analyses and Optimization of
Key Production Parameters, Journal of Biochemical Technology, 5, 788-794 (2014).
 Tarangini Korumilli and Susmita Mishra, Carotenoid production by Rhodotorula
sp. on fruit waste extract as a sole source and optimization of key parameters.
Iranian journal of Chemistry and Chemical Engineering, 33, 97-106 (2014).
 Tarangini Korumilli and Susmita Mishra, Production, Characterization and
Analysis of Melanin from Isolated Marine Pseudomonas sp. using Vegetable waste,
Research Journal of Engineering Science, 2, 40-46 (2013).
 Tarangini Korumillii, Arvind Kumar, G. R. Satpathy, Vikas Kumar, SangalStatistical
optimization of process parameters for Cr (VI) biosorption onto mixed cultures of
Pseudomonas aeruginosa and Bacillus subtilis, CLEAN - Soil, Air, Water, Wiley Inter
Science. 37, 319-327 (2009).
 Tarangini Korumilli and G. R. Satpathy, Optimization of heavy metal biosorption using
attenuated cells of Bacillus subtilis and Pseudomonas aeruginosa. Journal of Environmental
Research and Development, 3, 677-684 (2009).
 Tarangini Korumilli and Susmita Mishra, Astaxanthin by Xanthophyllomyces dendrorhous
Using Fruit Waste Extract as Sole Source of Energy: Optimization of Culture Conditions by
Taguchi method for Improved Pigment Production. Biotechnology Reports - Submitted
(In review) 2014.

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National and International Conferences
 Presented a paper in International Congress on Environmental Research and
Development (ICER-08) on Optimization of heavy metal biosorption using attenuated cells
of Bacillus subtilis and Pseudomonas aeruginosa. 2008.
 Secured second place in National Level Paper Presentation on Solar Photo catalyzed
Oxidation Reactions using TiO2 for killing bacteria at S.N.E.T – 2003.
 Secured second place in State Level Paper Presentation on Bio Hydrogen Production
from Rice Bra Oil Mill waste at Srujana – 2005.
 Participated in National Level Symposium, held in Vellore Institute of Technology on
Bio Hydrogen Production from Paper Mill waste – 2005.
 Participated in National Level Symposium, held in Ananthapur J.N.T.U on A Non –
Conventional approach for Bio Hydrogen Production -2005.
 Presented a Paper at National Seminar on Advances in the frontiers of environment
research at Andhra University – Nov 2005.
 Working Model on Microbial analysis of soil in and around Rajahmundry at Srujana –
2004.
 Working Model on Mushroom cultivation at Srujana – 2005.

Academic Projects
Title “Optimization of Heavy metal Biosorption using attenuated cultures of
Bacillus subtilis and Pseudomonas aeruginosa.”
Project theme Eco-friendly and cost effective removal of heavy metals from the aqueous
solutions using Biosorption technique
Application Today’s world is facing serious problem of environmental pollution where
of work water pollution occupies a major place. So, to reduce this water pollution
we are removing dissolved hazardous and carcinogenic metals by
Biosorption technique by using individual and mixed cultures of Bacillus
and Pseudomonas species.

Personnel Details
Date of Birth : 25th Nov 1983
Alternate address : D/o Chittibabu, D.No.2-7-2, Ramaraopet, Peddapuram-533437
East Godavari District, Andhra Pradesh, Phone: 08852 – 244355.

References : Available on request

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