PRODUCTION, OPTIMIZATION AND

CHARACTERIZATION OF ASTAXANTHIN USING

SUGARCANE BAGASSE AND ITS APPLICATIONS

A DISSERTATION SUBMITTED TO
THE DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY
UNIVERSITY SCHOOL OF SCIENCES
GUJARAT UNIVERSITY
AHMEDABAD - 380 009

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE

OF

Masters of Science
In
Microbiology

By

PREYA D. TRIVEDI

SEAT NO.
APRIL – 2023
 DEPARTMENT OF MICROBIOLOGY Telephone No.079-26303225
& BIOTECHNOLOGY Telegram: UNISCIENCE
UNIVERSITY SCHOOL OF SCIENCES Fax: (079)26301919 (G.U.)
GUJARAT UNIVERSITY Micro: / /2023
AHMEDABAD - 380009 Date: / / 2023
GUJARAT, INDIA
___________________________________________________________________________
___________________________________________________________________________
________

CERTIFICATE

This is to certify that the work presented in this dissertation entitled

“PRODUCTION, OPTIMIZATION AND CHARACTERIZATION OF ASTAXANTHIN USING
SUGARCANE BAGASSE AND IT´S APPLICATIONS” submitted by Miss PREYA
DIPAKKUMAR TRIVEDI has been carried out in the Department of
Microbiology & Biotechnology, University School of Sciences, Gujarat
University, Ahmedabad-380009, during the academic year 2022-23in
partial fulfilment of the requirement for the degree of Master of
Science in MICROBIOLOGY.

This is further certified that this dissertation or part thereof has not
previously been submitted elsewhere for the degree or diploma or any
other academic award of this University or any other university.

Guided by
1) [Link] [Link]
2) Dr. Rakesh R. Panchal
3) Dr. Devayani Tipre
4) Dr. Kiransinh N. Rajput
5) Dr. Vikram H. Raval
6) Dr. Dweipayan Goswami

Date:
Place: Ahmedabad

Through,
Head,
Department of Microbiology & Biotechnology
 Declaration

I hereby certify that I am the sole author of the thesis entitled

“PRODUCTION, OPTIMIZATION AND CHARACTERIZATION OF
ASTAXANTHIN USING SUGARCANE BAGASSE AND IT´S
APPLICATIONS” and that neither any part of this thesis northe whole of the thesis
has been submitted for a degree to any other university or institution.

I certify that, to the best of my knowledge, my thesis does not infringe upon anyone’s
copyright nor violate any proprietary rights and that the ideas, techniques, quotations,
or any other material from the work of other people included in my thesis, published
or otherwise, are fully acknowledged in accordance with the standard referencing
practices. I declare that this is a true copy of my thesis, including any final revision, as
approved by my thesis review committee.

Date: PREYA D. TRIVEDI

 Acknowledgement
This dissertation work cannot be accomplished without help and views of these noble
figures. I convey my deepest regards to all involved in this dissertation. First and
foremost, praise and thanks to the Almighty God, who has blessed me with
intelligence and bravery, and without whom all my efforts would have been in vain.
Throughout my research, His blessings have given me the strength to finish my thesis.
I would like to express our sincere gratitude to Head, Prof. Meenu Saraf, Department
of Microbiology and biotechnology, Gujarat University, for her supervision, advice
and guidance throughout the work. She spent her valuable time and encouraged us
during our entire work.
I am also thankful to faculty members, Dr. Kiransinh Rajput, Dr. Devyani R Tipre,
Dr. Rakesh Panchal, Dr. Vikram Raval and Dr, Dweipayan Goswami Department
of Microbiology, Gujarat University for their valuable advice and suggestion given
during dissertation work. I offer my sincere gratitude to my guide and mentor
[Link] Raval, Assistant Professor, Gujarat University, who helped me very
much whenever i approached him. He has patiently gone through my work, has given
me timely advice and directions. He has been always helpful to me from the
beginning to the end of my task. He also took time out of his busy schedule to review
my thesis. His valuable contribution will always be treasured.
I am greatly indebted to research scholar Mr Prashant Arya for many helpful
suggestions, readiness to share information and help in concern with the
methodology, for the generous help and guidance during dissertation.
I would like to take this opportunity to extend my heartfelt appreciation and thanks to
Mr. Dharamsinh Solanki, Mr. Tejasbhai, Mr. Dharmeshbhai, and the other non-
teaching staff at the Department of Microbiology and Biotechnology.
Here at this juncture, words fail completely to express gratitude towards my parents.
My parents have been constant source of motivation and inspiration. I owe deepest
thanks to all people in my entire family. Last but not the least I thank my team
members Monika Jain and Tarun Sosa, all my well-wishers, friends and all those
who helped me directly or indirectly in my work.
Place: Ahmedabad Preya Trivedi
Date:
 TABLE OF CONTENTS

Chapter 1 1
1.1 Introduction 2
1.2 Major classes of pigments and microbial sources 3
1.2.1 Fungal and yeast pigments 5
1.2.2 Bacterial pigments 6
1.3 Marine bacterial species as sources of bio-pigments 6
1.4 Production of pigment from agro-food waste as a low cost substrate 9
1.4.1 Submerged culture condition for pigment production 13
1.4.2 Pigment extraction and identification 13
1.5 The Economic viability of pigment production from valorization of 14
agro-food wastes
1.6 Applications of microbial pigments 15
1.6.1 Pigments in pharmaceutical industry 15
[Link] Antioxidant activity of bacterial pigments 16
[Link] Anticancer potential of bacterial pigments 17
[Link] Antibacterial properties of pigments 17
1.6.2 Pigments in food industry 18
1.6.3 Pigments in textile industry 19
1.6.4 Bacterial pigments in the cosmetic industry 20
1.7 Conclusion and future perspective 21
Aim and objectives 23
Chapter 2: Materials and methods 24
2.1 Isolation 25
2.2 Characterization of the pigment producing bacteria 25
2.2.1 Cultural and morphological features of the isolates 25
2.2.2 Biochemical tests of the isolates 25
2.3 Screening of agro-food waste 25
2.4 Preparation of Inoculum 26
2.5 Sub-merged fermentation 26
2.6 Pigment solubility 26
2.7 Extraction of the pigment 26
2.8 Optimization of fermentation parameters 27
2.8.1 Effect of substrate concentration 27
2.8.2 Effect of pH 27
2.8.3 Effect of NaCl 27
2.8.4 Effect of glycerol 28
2.8.5 Effect of inoculum size 28
2.9 Purification, identification and characterization of pigment 29
2.9.1 Thin layer chromatography (TLC) 29
2.9.2 High performance thin layer chromatography (HPTLC) 29
 2.9.3 UV-VISIBLE spectrophotometric analysis 30
2.9.4 Fourier-transformed infrared spectroscopy (FT –IR) 30
2.9.5 Mass spectroscopy (MS) 30
2.10 Applications of pigment 31
2.10.1 Antioxidant activity 31
2.10.2 Antibacterial and antifungal activity 31
2.10.3 Phytotoxicity and PGPR 32
2.10.4 Application of pigments on cloth (Dying of cloth) 32
2.10.5 Application of pigments in candle preparation 32
2.10.6 Application of pigments in lip balm 32
2.10.7 Application in food industry 33
Chapter 3: results and discussion 34
3.1 Characterization of the isolate 35
3.1.1 Cultural and morphological characters 35
3.1.2 Biochemical tests 36
3.2 Screening of Agro-food waste 37
3.3 Solubility of pigment 38
3.4 Extraction of pigment 38
3.5 Optimization by one variable at time (OVAT) 39
3.5.1 Effect of substrate concentration 39
3.5.2 Effect of pH 40
3.5.3 Effect of NaCl 41
3.5.4 Effect of glycerol 42
3.5.5 Effect of inoculum size 44
3.6 Characterization of pigment 45
3.6.1 Thin layer chromatography 45
3.6.2 High performance thin layer chromatography (HPTLC) 45
3.6.3 UV- Visible spectroscopy 46
3.6.4 FT-IR analysis 47
3.6.5 Mass spectrometry (MS) 48
3.7 Applications of Astaxanthin 49
3.7.1 Antioxidant effect of astaxanthin 49
3.7.2 Antibacterial and antifungal activity 49
3.7.3 Phytotoxicity and PGPR 50
3.7.4 Application of pigments on cloth (dying of cloth) 51
3.7.5 Application of pigment in lip balm 52
3.7.6 Food application 53
3.7.7 Application of pigment in candle preparation 53
Chapter 4: Summary 54
Chapter 5: References 57
Table Table Legend Page
No. No.
1 Marine bacterial sources of colored pigmented 7
compounds
2 Microbial pigment production by utilizing various 12
agro-industrial wastes as substrates
3 Optimization of pigment production with OVAT 28
method
4 Cultural and morphological characters of S-5-9-3 35
5 Biochemical tests 36
6 Screening of agro-food waste 37
7 Solubility of pigment 38
8 Antifungal activity of pigment produced by S-5-9-3 50
9 Antibacterial activity of pigment produced by S-5-9-3 50
10 PGPR and phytotoxicity test of pigment 51
Figure Figure Legend Page
No. No.
1 Chemical structures of various bacterial pigments 9
2 Pigment production from agro-industrial waste 10
3 Various applications of microbial pigments 21
4 Pure culture of S-5-9-3 35
5 Gram staining of S-5-9-3 35
6 Submerged fermentation 37
7 Extraction of pigment 39
8 Graph of effect of substrate concentration on pigment 40
production
9 Effect of substrate concentration on pigment production 40
10 Graph of effect of pH on pigment production 41
11 Effect of pH on pigment production 41
12 Graph of effect of NaCl concentration on pigment 42
production
13 Effect of NaCl concentration on pigment production 42
14 Graph of effect of glycerol concentration on pigment 43
production
15 Effect of glycerol concentration on pigment production 43
16 Graph of effect of inoculums size on pigment production 44
17 Effect of inoculum size on pigment production 44
18 Thin layer chromatography (TLC) analysis of pigment 45
produced by S-5-9-3
19 HPTLC analysis of pigment extracted from S-5-9-3 46
20 HPTLC graph of pigment extracted from S-5-9-3 46
21 Maximum absorbance of pigment produced by S-5-9-3 47
22 Fourier transform infrared spectroscopy (FT-IR) analysis 48
of pigment extracted from S-5-9-3.
23 Mass spectrometry (MS) analysis of pigment produced 48
by S-5-9-3
24 Phytotoxicity test of pigment 51
25 Pigment application on cloth material 52
26 Application of pigment in lip balm preparation 52
27 Pigment application in white cream as food colorants 53
28 Pigment application in color candle 53
 List of Abbreviations
etc. = Et cetera
g = Gram
ml = Milliliter
°C = Degree Celsius
nm = Nanometer
UV = Ultraviolet
N = Normal (concentration of solute in solvent)
cm = Centimeter
L = Liter
mm = Millimeter
% w/v = Weight-to-volume percent
% v/v = Volume-to-volume percent
pH = Potential of Hydrogen
SmF = Submerged fermentation
SSF = Solid state fermentation
et al. = et alia (which means “and others”)
 Introduction and review of literature

Chapter 1
Introduction and
review of literature

1
 Introduction and review of literature

1.1 Introduction
Color plays a significant role in human activity. Color has many different cultural
connotations, from traffic signs to artwork and clothes. In addition to their many
cultural meanings, colors have enormous market potential in the food, agriculture,
cosmetic, textile, pharmaceutical and nutrition sectors. Color is advantageous for
evaluating whether edible goods are fit for consumption, such as if something is fresh,
ripe, nutritious, safe, or rotting (for example, red vs. grey meat, green vs. yellow
banana) (Sen et al., 2019). Any member of the family of brightly coloured chemicals
known as pigments is used to give other materials color; they absorb the light at
specific visible wavelength spectrum (380–750 nm). Colorants can be largely divided
into two categories viz. pigments and dyes. According to chemical definitions,
pigments are insoluble colorants, while dyes are soluble colorants (Rana et al.,
2021).The market value for dyes and pigments is predicted to reach $33.2-$44.9
billion in value by 2027 (Chatragadda and Dufossé, 2021). Although synthetic dyes
have dominated industry due to their low cost and high performance, growing
concerns about their non-biodegradability, carcinogenicity, and environmental
toxicity have encouraged both industry and researchers to seek natural and safe
alternatives. In this regard, natural and environmental friendly bio pigments have
attracted great interest due to their non-toxicity, biodegradability, non-carcinogenicity
and non-allergenicity, which increases their acceptance by consumers, prevents
occupational health hazards ignoring environmental concerns (Pailliè-Jiménez et al.,
2020; Aman Mohammadi et al., 2021; Mussagy et al., 2021).
Microbial pigments from natural sources are attractive targets compared to plant or
animal sources due to their rapid growth, year-round availability and easy regulation
of microbial cell factories to achieve high production yields. As a result, the
generation of microbial pigments using fermentative technology offers a versatile
toolset for creating a variety of stable and safe pigments. However, financial and
marketing difficulties remain a major problem in commercial microbial production
(Ramesh et al., 2019; Aman Mohammadi et al., 2021). One of the important
parameter contributing to the fermentative production cost is microbial growth
medium. Hence, the low cost of pigment synthesis may be achieved by using cheap
waste substrates as a growth medium. In this situation, the agro-food wastes have the
potential to become excellent substrates for microbial pigment synthesis. These

2
 Introduction and review of literature

leftover residues contribute to waste generation, which is likely to reach 3.40 billion
metric tonnes by 2050 (Usmani et al., 2020; Sodhi et al., 2021).Waste management
has become enormously difficult as a result of unchecked trash disposal and urgent
action is needed to avoid risks to the environment and public health. The traditional
techniques of waste management: land filling, open dumping, burning, or incineration
has been rejected since they raise carbon footprints. The emphasis has now switched
to waste valorisation, which offers the combined advantages of sustainable value-
added product manufacturing and waste management. Conventionally, the majority of
technological advances have been concentrated on producing bio fuels or bio energy
from these waste feed stocks (Jatoi et al., 2021). Yet recently, they have become
effective building blocks for the creation of a wide range of value-added goods, such
as enzymes, platform chemicals, bioactive compounds, etc. (Grewal and Khare, 2017,
2018; Grewal et al., 2020; Lu et al., 2022). Similarly, these agro-food wastes have
been found to be effective nutrient support for growth of microorganisms and pigment
production by solid-state as well as submerged fermentation (SmF). This strategy not
only addresses environmental issues but also offers a sustainable and affordable
foundation for creating the circular bioeconomy (Banu et al., 2021; Muscat et al.,
2021; Yaashikaa et al., 2022).
Since most agro-food wastes are lignocellulosic in nature, pre-treatment of these
residues is required before microbial conversion due to their recalcitrance. For various
lignocellulosics, pre-treatment methods including physical, chemical, physico-
chemical, or biological have been used. Each has its own benefits and limitations (Ali
et al., 2020; Haldar and Purkait, 2020; Mankar et al., 2021). Both submerged and SSF
techniques have been used effectively for further microbial bio processing of these
untreated or pre-treated residual for pigment production (Aman Mohammadi et al.,
2021; Rana et al., 2021; Sodhi et al., 2021).
1.2Major classes of pigments and microbial sources
Based on their structural properties, natural pigments may be broadly divided into the
following categories: Melanins, quinones (including anthraquinone, naphthoquinone,
and benzoquinone), benzopyran derivatives (including flavonoids and anthocyanins),
isoprenoid derivatives (including iridoids and carotenoids), tetrapyrrole derivatives
(including hem and chlorophylls), and other N-heterocyclic compounds including
betalains, phenazines, flavins, phenoxazines, purines, and pterins (Grewal et al.,2022).

3
 Introduction and review of literature

A wide variety of pigments, including carotenoids, flavins, anthraquinones, violacein,

and prodigiosin, are produced by many types of microorganisms, including bacteria,
fungus, yeast, and algae. Some of them are mentioned below.
One of the most varied classes of pigments, carotenoids, is made up of eight isoprene
units and is a member of the isoprenoids subfamily. They are further categorized into
xanthophylls and carotenes. Carotenes are made of carbon and hydrogen, while
xanthophylls, also known as oxycarotenoids, are made of carbon, hydrogen, and
oxygen (Gmoser et al., 2017). Among these carotenoids, lycopene, b-carotene,
capxanthin, lutein, astaxanthin, canthaxanthin, and astaxanthin have extremely
significant market demand (Gmoser et al., 2017; Ramesh et al., 2019). By 2027, the
global market for carotenoids, which is now increasing at a compound annual growth
rate (CAGR) of 2.6%, is anticipated to reach $2 billion (Mussagy et al., 2021).Yet,
80–90% of the market's supply of carotenoids comes from chemical synthesis; as a
result, microbial sources are in great demand to counteract their adverse effects (Saini
and Keum, 2019; Chatragadda and Dufossé, 2021).Although certain businesses, like
Cyanotech (United States), Algatech (Israel), and Parry Nutraceutical (India), have
started generating carotenoids using biotechnology, their market share is far lower
than that of synthetic producers (Mussagy et al., 2021).The carotenoid biosynthetic
pathway and its regulation has attracted enormous interest among various producers
and is one of the most critical and popular targets for the regulation of pigment
production (Grewal et al., 2022).
Unsaturated lipophilic isoprenoid pigment lycopene, designated as a class A nutrient
by the WHO (World Health Organization) and FAO (United Nations Food and
Agricultural Organization), has several physiological roles and a wide range of uses,
particularly in preventative healthcare (Li et al., 2020). The global market for
lycopene which is expanding at a CAGR of 5.0%is anticipated to reach 161 million
dollars in 2025 whereas, another category of significant glycosylated pigments,
anthocyanins are mostly generated by plants as secondary metabolites and are
responsible for a variety of beautiful colors. From an estimated net worth of 192.5
million dollars in 2020, its market value is anticipated to reach 228.4 million dollars
in 2027 (Mussagy et al., 2021).

4
 Introduction and review of literature

1.2.1 Fungal and yeast pigments

Certain filamentous fungus displays a diverse range of pigments, including orange by
Herpotrichi arhodosticta, deep blood red by Cordycepsunil ateralis, and red pigments
by Talaromyces sp. Fungal families such as Chlorociboriaceae, Monascaceae,
Sordariaceae, Trichocomaceae, Chaetomiaceae, Nectriaceae, Xylariaceae,
Hypocreaceae, Cordycipitaceae, Pleosporaceae, etc. are known to be significant
producers of pigment (Ramesh et al., 2019; Pailliè-Jiménez et al., 2020).
Nevertheless, several pigment-producing funguses, including Penicillium sp.,
Aspergillus sp., and Fusarium sp., also emit secondary hazardous metabolites or
mycotoxin, which poses a safety risk for their use in industry. However, these
bottlenecks are influenced by the type of microbial population, the flow of carbon
towards the correct pigment pathway, and national regulations. For example, even if
Monascus sp. have been commonly used in fermented foods in Asian countries for
over 1,000 years, are not approved for use as food coloring in the United States or the
European Union due to the co-production of citrinin, which has hepatotoxic,
nephrotoxic, or carcinogenic properties (Gmoser et al., 2017; He et al.,
2021).However, the main azaphyllo pigment produced by Monascus sp. is of great
interest for various applications. These include yellow pigment (ankaflavin and
monascin), red pigment (rubropunctamine and monascorubramine) and orange
pigment (monascorubrin and rubropunctatin) (Dufossé, 2018; He et al., 2021).
Another fungus species, Blakeslea trispora generated enormous interest in the
production of lycopene and was intensively studied by both researchers and industry.
This strain has shown excellent potential for producing high titer lycopene in both its
native and modified forms (Li et al., 2020). Specific yeast genera such as Pichia,
Rhodotorula, Xanthophyllomyces, Rhodosporidium, Sporobolomyces and
Sporidiobolus are also efficient producers of various carotenoids and other pigments.
The most common carotenoids produced by yeast are β-carotene, torulene,
astaxanthin and canthaxanthin (Grewal et al., 2022). For optically active astaxanthin,
Xanthophyllomyces dendrorhous produces the 3R, 30R isomeric form. Torularhodin
produced by Sporobolomyces or Rhodotorula genus has strong antimicrobial
properties, making it suitable for many pharmaceutical applications (Rapoport et al.,
2021).

5
 Introduction and review of literature

1.2.2 Bacterial pigments

Due to the color they exhibit many bacteria seems very appealing viz. Serratia
marcescens, Gordonia jacobaea (red); Chryseobacterium sp., Hymenobacter sp.,
Micrococcus (yellow); Chryseobacteriumartocarpi, Kocuria sp. (red-yellow);
Corynebacterium insidiosum, Vogesellaindigofera (blue), Pseudomonas sp. (green)
(Venil et al., 2020). The most commonly used bacterial pigments are Carotenoids,
melanins, violacein, prodigiosin, quinones and tambjamines. They have been reported
to have antioxidant, antimalarial, and anticancer properties, making them suitable for
food or biomedical applications (Pailliè-Jiménez et al., 2020; Sajjad et al., 2020).The
genera Dietzia and Paracoccus are important producers of carotenoids, a widespread
class of biopigments of high commercial value (Gharibzahedi et al., 2014; Maj et al.,
2020). It is known that the crt gene cluster is essential for the production of
carotenoids in Paracoccus strains. Their pigment biosynthetic pathway and
corresponding gene functions have been investigated and characterized by several
studies (Ide et al., 2012; Honda et al., 2020; Maj et al., 2020). Besides these the
popularly reported carotenoids producers are Agrobacterium sp., Streptomyces sp.,
Bradyrhizobium sp., Brevibacterium sp., Micrococcus roseus, Corynebacterium
michiganense and G. Jacobaea (Ram et al., 2020; Venil et al., 2020).
1.3 Marine bacterial species as sources of bio-pigments
Over 300,000 species of marine organisms have been studied, which is a tiny portion
of the total number of bacterial species that may be explored and produce pigment.
Streptomyces sp., Pontibacterkorlensis sp., Pseudomonas sp., Bacillus sp., and Vibrio
sp. are bacterial species isolated from maritime sediments or seawater that generate a
variety of coloured chemicals, including prodigiosin, astaxanthin, pyocyanin,
melanin, and beta carotene, respectively are mentioned in the table 1.

6
 Introduction and review of literature

Table 1: Marine bacterial sources of colored pigmented compound

Marine bacterial species Pigments References
Hahellachejuensis sp. Prodigiosin Morgan et al., 2016
Pseudoalteromonas rubra sp. Setiyono et al., 2020
Streptomyces sp. SCSIO 11594 Song et al., 2015
Vibrio sp. (Strain MI-2)
Serratia marcescens sp. IBRL USM 84
Zooshikellaganghwensis gen. nov., sp. nov.
Pseudomonas aeruginosa sp. Pyocyanin Saha at el., 2008
Brevundimonas scallop sp. Zheng & Liu Astaxanthin Liu at el 2020
Corynebacterium glutamicum sp. Henke at el., 2016
Brevundimonas sp. strain N-5 Asker at el., 2017
Sphingomicrobiumastaxanthinifaciens sp. nov Pachaiyappan at el.,
Rhodovulumsulfidophilum sp. 2020
Pontibacterkorlensis sp. AG6 Shi at el., 2016
Exiguobacterium sp. Zhao at el., 2019
Altererythrobacterishigakiensis sp. NBRC 107699
Rhodotorula sp.
Paracoccushaeundaensis sp.
Cyanobacterium Synechococcus sp. Beta carotene Hamidi at el., 2020
Micrococcus sp. Sibero at el., 2018
Vibrio owensii sp. Teramoto at el., 2015
Flavicella marina gen. nov., sp. nov. Loh at el., 2020
Gordonia terrae sp.TWRH01
Blastochloristepida sp. Lycopene Seto at el., 2020
Salinicoccus roseus sp. Ramanathan at el.,
2017

7
 Introduction and review of literature

Sphingomonasphyllosphaerae sp. KODA19-6 Zeaxanthin Bramhachari at el.,

Mesoflavibacteraestuarii sp. nov. 2016
Aquibacterzeaxanthinifaciens gen. nov., sp. nov. Lee at el., 2014
Zeaxanthinibacterenoshimensis gen. nov., sp. nov Hameed at el., 2014
Gramellaplanctonica sp. nov Shahina at el., 2014
Mesoflavibacterzeaxanthinifaciens gen. nov., sp. nov Sowmya at el., 2015
Formosa sp. KMW Seto at el., 2020
T
Sphingomonasphyllosphaerae sp. FA2 Ramanathan at el.,
Sphingomonas (Blastomonas) natatoria sp. DSM 2017
3183T
Muricaudalutaonensis sp. CC-HSB-11T
Streptomyces sp. Melanin Manirethan at el., 2019
Pseudomonas sp. Kurian at el., 2015
Marinomonasmediterranea sp. MMB-1T Sivaperumal at el.,
Pseudomonas stutzeri sp. 2014
Bacillus sp. BTCZ31 Kurian at el., 2017
Streptomyces sp. MVCS13 Kurian at el., 2019
Providencia rettgeri sp. strain BTKKS1 Kamarudheen at el.,
Marinobacteralkaliphilus sp. 2019
Leclercia sp. Kurian at el., 2018
Halomonas meridian sp.
Nocardiopsisdassonvillei sp. strain JN1
Vibrio alginolyticus sp. strain BTKKS3
Pseudoalteromonastunicata sp. Tambjamines Sakai et al., 2019
Pseudoalteromonascitrea sp. Picott et al.,2019

These pigments come from a variety of chemical classes, such as carotenes, an

unsaturated subclass of carotenoids, prodiginines, which have a
pyrrolyldipyrromethene core structure, tambjamines, which are alkaloid molecules,
and violacein compounds, which are indole derivatives produced by the metabolism
of tryptophan. The structures of bacterial pigments from are mentioned in figure 1
(Ramesh et al., 2019).

8
 Introduction and review of literature

Figure 1: Chemical structures of various bacterial pigments

1.4 Production of pigment from agro-food waste as a low cost

substrate
The high production cost of microbial pigment is major obstacle, especially on a pilot
or industrial scale, to compete with synthetic pigments as safe alternatives. Expensive
synthetic media used to grow microbes to produce pigment is one of the critical
factors affecting the cost of biotechnological synthesis. From that perspective, there is
abundant potential for agro-food wastes to be stored as low-cost substrates to reduce
production costs (Venil et al., 2017; Usmani et al., 2020). Large volumes of residue
are produced during the processing of agricultural commodities, post-harvesting
procedures, and by products of industrial activities, which, if not managed, act as

9
 Introduction and review of literature

pollutants to the surroundings. By using these wastes as substrate for pigment

manufacturing, it is possible to value these wastes and battle environmental and health
risks while also advancing the development of cost-effective bioprocesses.
Nevertheless, the nature of their raw composition will determine how effectively they
are utilised and will have an impact on the processing phases. For instance, before
lignocellulosic wastes like corncob, sugarcane bagasse, rice straw, wheat straw, and
rice husk can be utilised for hydrolyzate production by the action of saccharifying
enzymes, an ideal and affordable pre-treatment strategy must be used (Sodhi et al.,
2021; Zhang H. et al., 2021). Figure 2 shows a schematic illustration of the production
pigment from agro-industrial waste.

Figure 2: pigment production from agro-industrial waste

In the process of pigment production, the availability of waste substrate, its raw
composition, nutritional requirements of fermenting microbes and its effective usage
are of utmost importance. Economically competitive pigment synthesis (16.17 ± 0.37
OD500 nm) from acid hydrolyzate of corncob was made possible by Talaromyces
atroroseus GH2's capacity to co-utilize glucose and xylose without the need for
nutrient supplementation (Morales-Oyervides et al., 2020). Another processing step
that could be necessary to increase the titre of pigment synthesis is the detoxification
of lignocellulosic hydrolyzates utilised as a source of nutrients. In the study, which
used the loquat (Eriobotrya japonica) kernels as a substrate for Rhodotorula glutinis

10
 Introduction and review of literature

MT-5 to produce carotenoids, it was discovered that the fermentation of detoxified

and undetoxified loquat kernel extract produced, respectively, 72.36 and 62.73 mg/L
of carotenoids (Grewal et al., 2022). Another method involved creating a mutant
strain of M. purpureus M523, which showed great tolerance to non-detoxified acid
hydrolyzate of rice husk, using an atmospheric and room temperature plasma (ARTP)
screening device. This mutant strain's spores immobilized in sodium alginate were
used to produce 80.7 U/ml of pigment from rice husk (Zhang S. et al., 2021). In
another intriguing research, utilising lignocellulosic hydrolyzate, Monascusruber
tieghem IOC 2225 produced red pigment at a rate that was 2.5 times greater when
using the hydrolyzate from sugarcane bagasse than when using glucose-based
medium under equivalent circumstances. The metabolic pathway might be affected by
the complexity of the medium, which would then have an impact on the quantity and
variety of secondary metabolites generated by the fermenting bacterium. An
alternative method produced 0.15 g/L of violacein by immobilising Chromobacterium
violaceum cells on sugarcane bagasse to create a continuous column system (Venil et
al., 2017). Recently by using groundnut cake as the only source of carbon/nitrogen,
cheaper scaled-up production of prodigiosin (6886 mg/L) by S. marcescens TUN02
was achieved. Purified prodigiosin showed potential for use in agriculture through its
anti-nematode action (Nguyen et al., 2022).
The different agro-industrial wastes used for pigment generation by various
microorganisms via submerged as well as SSF is listed in table 2.

11
 Introduction and review of literature

Table 2: Microbial pigment production by utilizing various agro-industrial

wastes as substrates
Agro- Microorganisms Type of Microbial Titer References
foodwaste fermentati pigment
on
Groundnut Serratia Submerged Prodigiosi 6,886 mg/L Nguyen et
cake marcescens n (red) al., 2022
Whey Monascus purpureus
Monascus purpureus

Rice husk Monascus purpureus Submerged Monascus 38.4 UA510 Mehri et

M523 pigments nm al., 2021
(red) (absorbance
units)
Wheat bran Serratia marcescens Submerged Violacein 1.47 mg/L Cassarini
UCP 1549 (violet) et al., 2021

Waste orange Monascus purpureus SSF, Monascus 9 AU/gds in Kantifedak

peels ATCC 16365 submerged pigments SSF i et al.,
(yellow, 0.58 2018
red, AU/mL in
orange) submerged
Sugarcane Chromobacterium Submerged Violacein 0.822 g/L Venil et
bagasses Violaceum (violet) al., 2017

Cotton seed Pseudomonas Submerged Pyocyanin 4.0 mg/mL El-Fouly et

meal aeruginosa R1 (blue al., 2015
green)

Corn meal Monascus purpureus SSF Monascus 129.63 Grewal et

CMU001 pigments U/gds corn al., 2020
(red) meal,
63.50 U/gds
coconut
residue,
52.50 U/gds
peanut
meal,
22.50 U/gds
Liquid Chromobacterium Submerged Violacein 16,256 + Aruldass et
pineapple violaceum UTM5 (violet) 440 mg/L al., 2015
waste

12
 Introduction and review of literature

1.4.1 Submerged culture condition for pigment production

In general, coloured bacteria, fungus, and yeast have great physicochemical
sensitivity. Hence, in order to produce additional colours in either solid-state or
submerged fermentation, these microorganisms need a range of in vitro growing
conditions. Studying the ideal cultivation conditions for each species or strain is
required. Consequently, the main physicochemical parameters that cause high
pigment synthesis in microorganisms have been identified by optimisation of
experimental design studies utilising artificial neural networks, Box Behnken design,
central composite design, Plackett-Burman design, and response surface modelling. A
survey of the literature reveals that, regardless of species, the majority of pigmented
microorganisms generate pigments at temperatures between 22 and 28 ºC, pH 5 and 6,
and agitation at 100 to 150 rpm (Ramesh et al., 2019).
Since colour pigment can be extracted and generated so easily, fermentation methods
have advanced. For large-scale fermentation, submerged fermentation is frequently
used. In submerged fermentation, microorganisms are cultured and developed
aerobically in a liquid medium with sufficient agitation to ensure a homogenous
development of cells and media components. For pigment production many factors
are required for formation, including temperature, pH, aeration rate, carbon supply,
and nitrogen source. Due to the high cost of the extraction process and strategy used
to produce microbial pigments, researchers created a low-cost method. There have
been several attempts to synthesise microbial pigments on a big scale using
agricultural and industrial waste (Heer and Sharma, 2017).
1.4.2 Pigment extraction and identification
The conventional technique of organic solvent extraction of pigments from
fermentation broth is a difficult and drawn-out procedure with drawbacks of high
cost, limited yield, and potential solvent residue as impurities in the filtered product. It
was also noted by Sen and his team (2019). Unique method of extraction for recovery
of intracellular pigments using submerged fermentation of Talaromyces spp. in a
surfactant rich environment was also noted by (Morales-Oyervides et al., 2017). The
use of non-ionic resin is particularly ideal for large-scale pigment recovery due to
their high loading ability, and the approach also offers the benefit of direct chemical
absorption from the culture broth, hence lowering overall cost.
Microbial pigments are identified and analysed using a variety of analytical methods
that have been developed over time. For the purpose of identifying and characterising

13
 Introduction and review of literature

microbial pigments, methods in common usage include TLC, UV-VIS

spectrophotometry, FTIR, NMR, and HPLC. Microbial pigments have been
discovered using a portable Raman spectrometer that operates on the excitation laser
principle in a variety of settings (Jehlicka and Oren, 2013; Kumar et al., 2015).
1.5 The economic viability of pigment production from valorisation of agro-food
wastes
In a promising research by (Dursun et al., 2020), the generation of astaxanthin from
wheat bran by X. Dendrorphous utilising SSF was the subject of a techno economic
investigation. The outcomes showed a $3.9 million gross profit for astaxanthin
production, supporting the viability of agro-industrial waste valorisation as a pigment
manufacturing technique. Similar to this, C. violaceum UTM5's cost to produce
violacein was reduced to 235.70 USD from 281.20 USD by utilising liquid pineapple
waste as opposed to nutritional broth (Aruldasset al., 2015). Against two strains of
Staphylococcus aureus, one of which was methicillin-resistant, the produced pigment
also shown high stability and anti-microbial action. The use of molasses-based
medium for carotenoid synthesis by Rhodopseudomonas faecalis PA2 lowered cost by
90.88% as compared to synthetic chemical medium although demonstrating identical
carotenoid output in both media, according to a different study proving the economic
potential of utilising wastes (Saejung and Puensungnern, 2020). Rhodotorula
mucilaginosa CCMA 0156 yeast strain was used to ferment coffee waste, and Moreira
et al. (2018) reported that the cost per gram of β -carotene produced was $5.04,
compared to the market price of about $10.40 for synthetic β carotene. Recent
research (Mehri et al., 2021) utilising demineralized whey, a waste product of the
dairy industry, to produce red pigment in M. purpureus found that it had an identical
operational cost (14.92 dollars/kg) to using glucose as a comparable nutrition source.
These findings indicate that it is essential to assess the viability of waste-derived
fermentation systems from an economic standpoint. To support the viability of
pigment production at an industrial scale, there are presently no thorough research
studies available to critically examine the techno-economic and lifecycle assessment
(LCA) elements of waste-derived bio refineries. The manufacture of natural pigments
will be made possible by evaluating the economic feasibility as well as the social and
political ramifications of modifying the health and environmental risks by sustainable
technology.

14
 Introduction and review of literature

1.6 Applications of microbial pigment

The varieties of pigments produced by the microbial world have numerous uses in
various economic industries (Narsing Rao et al., 2017; Chatragadda and Dufossé,
2021; Orlandi et al., 2021; Rana et al., 2021). Figure 3 shows list of various microbial
pigments (Grewal at el., 2022). These secondary metabolites’ distinctive bioactivities
give them special functional qualities that can be taken advantage of for specific uses
using biotechnological methods. Although pigments have been utilised in a variety of
products, including cosmetics, bio indicators, sensors, diagnostic tools, nano-optics,
paints, and plastics, their essential role in the three primary commercial sectors is
extensively covered.
1.6.1 Pigments in pharmaceutical industry
In the recent SARS-CoV-2 pandemic, the evolution of antibiotic resistant bacteria,
and alarming increase in illnesses with multiple antibiotic resistance have all
increased the urgency of biomedical research to identify potential chemicals with anti-
pathogenic activity (Grewal et al., 2022). Microbial pigments have drawn a lot of
attention in this regard due to their pharmacological properties. Prodigiosin,
carotenoids, violacein, flavins, melanins, and quinones, which make up the majority
of microbial pigments, have been found to have bioactive qualities that include
antibacterial, antiproliferative, antioxidant, antiviral, and many more (Narsing Rao et
al., 2017; Venil et al., 2020a; Orlandi et al., 2021). Due to their potential for
treatment, microbial pigments derived from marine habitats have recently attracted
significant pharmaceutical interest (Torregrosa-Crespo et al., 2018; Chatragadda and
Dufossé, 2021; Nawaz et al., 2021). Recently, it was shown that an astaxanthin
pigment isolated from the marine endophytic bacterium Pontibacter korlensis AG6
had remarkable antibacterial and anti-oxidant properties in addition to having a
cytotoxic effect on the human breast cancer cell line (MCF-7) (Pachaiyappan et al.,
2021).
In a different investigation (Abdelfattah et al., 2019) the prodigiosins produced from
an actinomycete isolated from a marine sponge showed gastroprotective effects
comparable to those of the common anti-gastric ulcer medication omepraze. These
were effective pharmaceutical targets for avoiding stomach injury because of their
anti-inflammatory and antioxidant effects. Similar to this, two marine isolates such as

15
 Introduction and review of literature

Streptomyces sp. And Zooshikella sp., produced prodigiosin that was effective against
S. aureus and other human pathogenic strains (Ramesh et al., 2020)
Although many species of fungi, including Penicillium, Aspergillus, Talaromyces,
and Fusarium, have been shown to have anti-microbial and anti-oxidant properties
(Lagashetti et al., 2019; Morales-Oyervides et al., 2020a), Monascus sp. Pigments
have received the most attention because more than 50 pigments with various colours
have been identified from it. The Monascus sp. Pigments have been identified as
promising biomolecules for pharmaceuticals due to their reported anti-inflammatory,
anti-diabetic, anti-obesity, anticancer, and antimicrobial properties (Vendruscolo et
al., 2016; Kim and Ku, 2018; He et al., 2021).
[Link] Antioxidant activity of bacterial pigments
Investigations are also being done on the antioxidant activity of bio-pigments (bpBPs)
generated by marine-derived bacteria. It was discovered that the pigments 3R
saproxanthin and myxol (from the family Flavobacteriacae) exhibit antioxidant
capabilities against lipid peroxidation as well as neuroprotective qualities against L-
glutamate toxicity. Moreover, melanin and zeaxanthin, both generated from marine
Pseudomonas stutzeri sp. bacteria, have been discovered to possess antioxidant
qualities. The marine bacterium Geitlerinema sp TRV57, which produced the pigment
phycocyanin, also displayed high antioxidant activity. Streptomyces bellus sp., a
marine bacterium, produces MSA1, a crude pigment. It has an 82 % DPPH (2, 2-
diphenyl-1-picryl-hydrazylhydrate) activity and is regarded as a radical scavenger
(Nawaz et al., 2020). Pigments produced by Janibactermelonis and Pseudomonas
stutzeri isolated from marine soil gathered from the shore of India's North-West
coastal area shown significant scavenging activity in a DDPH test (Shivale et al.,
2018). The marine filamentous cyanobacteria Geitlerinema sp. TRV57 produced the
blue pigment phycocyanin, which had a maximum absorbance of 0.49 in the
phosphomolybdenum assay, 0.85 in the ferric ion reducing assay, 78.75 % DPPH
scavenging activity, 95.27 % H2O2 scavenging activity, and 185 g/ml IC. Carotenoid
pigments produced by the extremeophile bacteria Halococcusmorrhuae,
Halobacterium salinarium, and Thermus filiformis provide significant antioxidant and
Pigments produced by Janibactermelonis and Pseudomonas stutzeri isolated from
marine soil gathered from the shore of India's North-West coastal area shown
significant scavenging activity in a DDPH test (Shivale et al., 2018). The marine
filamentous cyanobacteria Geitlerinema sp. TRV57 produced the blue pigment

16
 Introduction and review of literature

phycocyanin, which had a maximum absorbance of 0.49 in the phosphomolybdenum

assay, 0.85 in the ferric ion reducing assay, 78.75 % DPPH scavenging activity, 95.27
% H2O2 scavenging activity, and 185 g/ml IC. Carotenoid pigments produced by the
extremeophile bacteria Halococcusmorrhuae, Halobacterium salinarium, and
Thermus filiformis provide significant antioxidant and H2O2 scavenging action
(Renugadevi et al., 2018).
[Link] Anticancer potential of bacterial pigments
Chemotherapy is still the standard treatment for the most severe kind of cancer, and
because it harms healthy cells, it not only promotes the growth of tumours but also
makes patients' prognoses worse (Felisa et al., 2015; Ravin et al., 2017). As a result,
there is a constant need for companies to provide natural cancer treatments that are
less expensive and safer. Bacterial pigments should be thoroughly investigated since
they appear to have enormous potential as a source of anticancer compounds. With
the purpose of creating anticancer medications, it was discovered that the pigment
from Salinicoccus sp. exhibited inhibitory effect against the proliferation of human
cancer cell lines (Srilekha et al., 2018). The new pigment from Rhodococcusmaris's
yellowish pigment has been demonstrated to lower the risk of breast cancer (Elsayed
et al., 2017). According to Afra et al. (2017), the red pigment from the Caspian Sea-
isolated Athrobacter sp. G20 showed exceptional anticancer activity against
esophageal cancer cell lines, while the carotenoids from Kocuria sp. QWT-12 shown
anticancer activity against breast cancer cell lines MCF-7. Violacein, a different
pigment that was obtained from an Antarctic bacterial isolate, inhibited the
proliferation of HeLa cells. It was determined to be a member of the
Janthinobacterium genus (called Janthinobacterium sp. strain UV13). Studies
comparing violacein to the anticancer chemotherapeutic drug cisplatin in the
treatment of cervix cell carcinoma demonstrated the potential of violacein as an
anticancer agent (Nawaz et al., 2020).
[Link] Antibacterial properties of bacterial pigments
Gram-positive and Gram-negative bacteria have shown to be resistant to the
antibacterial effects of bacterial pigments. A violet pigment produced from
Chromobacterium violaceum has been shown to have broad-spectrum antibacterial
activity against Gram-positive and Gram-negative bacteria, including S. aureus and S.
typhi. Similar conclusions were looked at by Suresh et al. They found that H.
alkaliphilus MSRD1 generates a red pigment that has antibacterial properties against

17
 Introduction and review of literature

Salmonella paratyphi, S. aureus, S. typhi, K. pneumonia, and E. coli. Investigations

on the antibacterial properties of pigments derived from fifteen different
microorganisms were conducted. In this study, they examined the antibacterial
activity of pigments from distinct isolates against nine different human pathogenic
bacterial strains, both Gram-positive and Gram-negative. It was discovered that
pigments derived from different bacteria have antibacterial action that was more
effective against Gram-negative pathogenic strains than Gram-positive pathogenic
strains (Rashid et al., 2014). For instance, the marine Streptomyces sp. pigment
"melanin" showed antibacterial action against the bacteria E. coli, Salmonella typhi,
Salmonella paratyphi, Proteus mirabilis, Vibriocholerae, Staphylococcus aureus, and
Klebsiella oxytoca. In addition to E. coli, Klebsiella pneumoniae, Bacillus subtilis,
Proteus vulgaris, and P. aeruginosa, prodigiosin, cycloprodigiosin, and the yellow
pigment extracted from Micrococcus sp. strain MP76 have all demonstrated
antibacterial potential (Nawaz et al., 2020).
1.6.2 Pigments in the food industry
The food business is constantly looking for foods with vibrant, appetising looks as
well as nutritious and health-promoting qualities for increasing consumer
acceptability. As a result, the food industry highly values natural and secure food
colorants as an alternative to synthetic ones as vat green, tartrazine, reactive blue, and
sunset yellow (Rana et al., 2021). Due to the safe increase of food organoleptic
features and the addition of nutraceutical characteristics like the removal of free
radicals, prevention against ageing, and many other diseases, the market potential for
microbial carotenoids in the food business is growing quickly (Mussagy et al., 2021;
Rana et al., 2021). However, microbial pigments’ usage as colorants presents
additional difficulties because of their greater susceptibility to environmental factors
including light, pH, temperature, oxygen, heat, etc. In order to increase their stability
in food matrices, packaging techniques as micro encapsulations, nano-emulsions, and
nanoformulations are therefore considered effective solutions (Sen et al., 2019; Juri’ c
et al., 2020). It was discovered that a safe and stable natural colorant for jellies and
yoghurt may be made by encapsulating the violet pigment made by the violaceum
bacterium in gum arabic and then spray-drying it (Venil et al., 2015).
A number of microbial pigments such as β-carotene, lycopene from B. trispora,
astaxanthin from various bacteria, algae, and X. dendrorhous, Arpink red from
Penicilliumoxalicum, pigments from Monascus sp., riboflavin from Ashbyagossypii,

18
 Introduction and review of literature

Debaryomycesglobosus, Eremotheciumashbyii, and Candida guilliermondii are

currently utilized industrially in different beverages and foods (Sen et al., 2019; Venil
et al., 2020; Meruvu and Dos Santos, 2021). Although many other microbial strains,
particularly fungal sp., have the potential to be used as food colorants, they must first
pass extensive toxicity and quality tests before receiving the go-ahead from the
relevant food regulatory authorities for commercial use (Dufossé, 2018; Chatragadda
and Dufossé, 2021; Poorniammal et al., 2021). Numerous studies (Dufossé, 2018; He
et al., 2021; Poorniammal et al., 2021) have emphasised the commercial viability of
anthraquinone and azaphilone-producing strains such as Talaromyces sp., Penicillium
sp. Along with toxin-free production of Monascus sp. Pigments for food-grade
colours. The carotenoid pigments are highly sought-after as food additives for animals
or aquatic species in addition to human use (Meléndez-Martnez et al., 2020; Pereira
da Costa and Campos Miranda-Filho, 2020). It was demonstrated that the carotenoid-
producing R. faecalis PA2 displays price competitiveness for use as an animal feed
additive after it was cultivated in domestic wastewater (Saejung and Ampornpat,
2019).
1.6.3 Pigments in textile industry
Each year, more than 200,000 tonnes of dye effluents are produced, which result in
ongoing contamination and present health risks. The use of microbial pigments as safe
colorants has sparked a great interest in the textile industry as a means of overcoming
the dangerous environmental issues, health hazards, and allergenicity posed by
synthetic dyes. Introducing antimicrobial characteristics into protective clothing can
also assist to decrease hospital-acquired infections and boost customer acceptance,
particularly in the post-pandemic era. Prodigiosin, an antimicrobial pigment made
from S. marcescens SB08, has recently been demonstrated to have effective and stable
dyeing properties for both cotton and silk fabrics. The pigment-soaked textile yarn
materials showed the antibacterial effect as well (Venil et al., 2021). Similar to this, a
different study reported the preparation of nano-suspension dyeing based on S.
marcescens prodigiosin production. The project demonstrated the effectiveness and
affordability of a green method for dyeing acrylic cloth, which gave it rich colour and
antimicrobial properties (Ren et al., 2021).
The biopigments made by the fungus genera Monascus, Aspergillus, Talaromyces,
Fusarium, Penicillium, Trichoderma, Scytalidium, Chlorociboria, Curvularia,
Cordyceps, Alternaria and Phymatotrichum have been used to colour a variety of

19
 Introduction and review of literature

materials (Venil et al., 2020; He et al., 2021; Meruvu and Dos Santos, 2021). Yet, it is
still important to assess these fungal metabolites’ toxicity before using them. Once the
cytotoxicity of lixiviates of dyed wool fibres as well as fungal pigments was evaluated
on mammalian cell lines NIH/3T3 and HEK293, Hernández et al., 2019 determined
that the pigments generated by Penicillium murcianum and Talaromyces australis
were safe for dyeing. In addition to naturally occurring microbial producers, genetic
engineering techniques can increase the use of pigments in the textile industry. In the
goal of manufacturing environmentally friendly dyes, a promising work by Ghiffary
et al. (2021) showed how to produce indigoidine by constructing an engineered C.
glutamicum. The metabolically modified C. glutamicum strain generated the highest
titer recorded to date from fed-batch fermentation, or 49.30 g/L indigoidine. Blue
synthetic dyes were compared to the produced pigment, which displayed comparable
colour characteristics and fastness on cotton garments.
1.6.4 Bacterial pigments in the cosmetic industry
The global commercial market for cosmetics is expanding quickly. Cosmetic
producers number roughly 2000 in the United States of America. According to
estimates, American men and women use seven different skincare products daily for
personal maintenance. The cosmetic industry is valued 10, 4, 10, 6, and 13, 01 billion
euros in the United Kingdom, France, and Germany, respectively. Researchers have
started looking at the use of marine-derived bpBPs in skincare products in light of the
global cosmetics market. To enhance UV-B (ultraviolet B) protection and raise SPF
(sun protection factor) values by 10% to 30%, the pigment PCA was added to a
commercial sunscreen solution. Equivalent amounts of the seaweed Gelidium
spinosum and the melanin pigment were utilised to create cream F3 that included
melanin (extracted from marine bacterium Halomonasvenusta sp.). Cream F3 is
highly efficient in promoting wound healing and has a high SPF and photoprotective
action. Moreover, Streptococcus pyogenes sp. (MTCC 442) and Streptococcus aureus
sp., two skin pathogens, were resistant to the produced cream's antibacterial effects
(MTCC 96) (Poulose et al., 2020). According to a different research, melanin, which
comes from the marine bacterium Vibrio natriegens sp., is excellent in protecting
mammalian cells from UV rays. HeLa cells survived 90% of the time in melanized
cell culture. Another study indicated that bio lip balm made from raw pigment
(extracted from S. bellus sp. MSA1) in a combination of coconut oil, lanolin, and

20
 Introduction and review of literature

shredded bee wax may be used as a significant ingredient in a range of beauty care
products (Nawaz et al., 2020).

Figure 3: various applications of microbial pigments

1.7 Conclusion and future perspective

Synthetic colorants have many side effects due to which the use of natural colorants is
increasing. When colorants are made by fermentation, there are various benefits. They
consist of less expensive manufacturing, greater yields, simpler extraction, a lack of
seasonal changes, and an abundance of raw resources. A potential alternative source
of colorants for use in textiles, foods, pharmaceuticals, and other products is the use
of colorants created by bacteria, which is gaining popularity quickly. Thus,
biotechnology plays very crucial role for the large-scale fermentation and production
of colorants.
The use of agro-food waste as a low cost susbtrate for microbial pigment production
is sustainable as well as environmentally friendly way of solving a pollution
[Link] for lowering carbon emissions, microbial pigments meet the rise in
demand for natural [Link] microorganism producing pigments are
incredibly sensitive to light, pH and temperature changes which lead to poor stability
and [Link] inquiry should be focused on this aspect. Investment from

21
 Introduction and review of literature

important business and governmental parties is necessary for the commercialization of

microbial pigments as food colorants. By doing this, the difficulties in producing
microbial pigments may be solved, and methods can be developed for the production,
extraction, and purification of new colors from a variety of microbial sources.

22
 Introduction and review of literature

AIM AND OBJECTIVES

Aim: research work was design to produce bacterial pigment with the use of strain

S-5-9-3 by utilization of agro-food as a low-cost substrate

The research work was carried out with the following objectives:

Objectives

• Study the cultural, morphological and biochemical characteristic of isolates

• Production and optimization of pigment production: One variable at a factor
using agriculture wastes
• Extraction and purification of pigment
• Characterization of pigment
• Potent industrial applications of pigment

23
 Materials and Methods

Chapter 2
Materials and
Methods

24
 Materials and Materials

2.1 Isolation
Salinococcus roseus S-5-9-3 was used which was originally isolated from the coastal
region of Somnath, Gujarat, India. This culture from the marine environment was
cultured in the department of microbiology and biotechnology, Gujarat University,
Ahmedabad. Isolate was given to us for our dissertation work by our guide Dr.
Vikram H Raval from the department the bacterium was maintained on Zobell Marine
Broth (ZMB).
2.2 Characterization of the pigment producing bacteria
2.2.1 Cultural and morphological features of the isolates
Colony characters like colony size, shape, elevation, margin, opacity, pigmentation,
texture, etc. were noted down after 24 hours of incubation at 28°C, by growing isolate
on nutrient agar plate. Several staining techniques were used to study morphological
characteristics, including the Gram stain, capsule stain, endospore stain, and hanging
drop method to assess the motility of the cells.
2.2.2 Biochemical tests of the isolates
Different biochemical tests such as methyl red test, Voges-Proskauer test, citrate
utilization test, indole production test, haemolysis test, nitrate reduction test,
decarboxylation test, H2S production test, enzyme screening (catalase, urease, lipase,
protease, amylase, coagulase, oxidase, gelatinase) and sugars utilization test
(galactose, mannitol, dextrose, sucrose, arabinose, fructose, maltose, lactose) were
performed (Bergeys et al., 1994). These tests were performed for the characterization
of pigment producing isolate.
2.3 Screening of agro-food waste
Eight agriculture food waste were selected such as wheat bran (WB), sugarcane
bagasse (SCB), dry potato peels (DPP), rice bran (RB), dry orange peels (DOP),
groundnut shells (GS), sorghum stalk (SS), castor seed shells (CSS). From which
DOP and PP were collected from kitchen and all other wastes from local shop
vendors. All the wastes were washed properly, dried and grinded in a grinder before
use.
Screening of waste was done on the basis of the maximum pigment production and its
color intensity. Submerged fermentation method was carried out for these wastes in
this process; eight 250ml Erlenmeyer flasks containing 3g of each waste were taken,
2% glycerol were added along with 100ml of basal medium containing, 5%NaCl,
25
 Materials and Materials

0.5% peptone, pH 9 and 0.5% dextrose. All the flasks were kept for an incubation
period of 7 days at 28± 2°C and 150 rpm on a rotary shaker.
2.4 Preparation of inoculum
From pure culture a loopful of bacterial culture was inoculated in 50 ml nutrient broth
under aseptic condition and incubated on a shaker at 150 rpm and 30 ± 2°C for 18-24
h. After incubation, the growth of the culture was measured in terms of absorbance at
600nm where O.D 1.0 is considered as 1%v/v inoculum. The blank was set by
uninoculated sterile nutrient broth.
2.5 Sub-merged fermentation
Submerged fermentation was carried out in 250 ml Erlenmeyer flasks which
containing 3g of sugarcane bagasse with 100ml of basal medium containing, 5%
NaCl, 0.5% peptone, 0.5% dextrose, pH 9 and 1% glycerol. The flasks were
autoclaved at 121°C for 15 min and then, inoculated with 3% inoculum from
activated culture after taking optical density of the culture at 600nm after 24hr. The
production prepared was inoculated with 3% inoculum of S-5-9-3. Flasks were
incubated at 30 ± 2°C and 150 rpm on a rotary shaker for 120h. Following the
incubation time extraction, optimization and characterization of pigment was done.
2.6 Pigment solubility
After being collected, the fermented broth was centrifuged at 10,000 rpm for 10
minutes. The cell pellets were suspended in 20 ml of pure water and centrifuged at
10,000 rpm for 10 minutes before being twice rinsed with distilled water. The
supernatant was then discarded. Cell pellets were then once again resuspended in
different solvents and vortexed for 1 minute. This mixture underwent a 10-minute
centrifugation at 10,000 rpm after being incubated for 30 minutes at 60°C. The
supernatant was then used to measure the colour intensity. Acetone, chloroform, di-
ethyl ether, petroleum ether, ethyl acetate, hexane, and methanol were used to test the
pigment's solubility. Based on visual inspection, a suitable solvent was selected due to
the pigment's enhanced solubility in mentioned solvents (Dawoud et al., 2020).
2.7 Extraction of the pigment
After fermentation period, the suspensions were filtered using cheesecloth and the
liquid excess was squeezed out manually (Nalini and Parthasarathi 2014). After this
process the extracts were centrifuged at 10,000 rpm for 10 min this process was
performed twice. After that the tubes containing pigmented cell biomass was
26
 Materials and Materials

incubated in water bath at 70oC for 45 min. Three times this procedure was carried out
and the extracts were collected and centrifuged for 10 min at 10,000 g. The pellets
obtained were washed with distilled water then after pigment was extracted with
solvent extraction method (hexane: methanol) and pigment was evaporated to dryness
and stored at 4oC.
2.8 Optimization of fermentation parameters
The pigment synthesis method was optimized using one variable at a time (OVAT)
studies. The studies were done to establish the best fermentation conditions and to see
how the fermentation parameters affected the synthesis of orange pigment. Different
parameters mentioned below were optimized. Table 3 shows variations selected for
optimization of pigment production.
2.8.1 Effect of substrate concentration
Optimization of different substrate concentration %w/v (1%, 2%, 3%, 4%, 5%) was
done with the use of selected waste. In 250 ml Erlenmeyer flasks containing 1g, 2g,
3g, 4g and 5g waste were prepared with 100 ml of basal medium containing inducing
ingredients was poured in each flask and mixed properly. All the flasks were
autoclaved at 121 °C and 15 psi for 45 min and then, inoculated with 3% inoculum
from activated culture after taking optical density of the culture at 600nm after 24hr.
Flasks were incubated at 30 ± 2°C and 150 rpm on a rotary shaker for 120h followed
by pigment extraction. From this optimization process substrate concentration optimal
for pigment production was examined.
2.8.2 Effect of pH
For the optimization of pH (7, 8, 9, 10, 11), Five 250 ml Erlenmeyer flasks containing
2g optimized substrate concentration was taken and mixed with 100 ml distilled water
dissolved with other inducing ingredients than the flasks were autoclaved at 121 °C
and 15 psi for 45 min and then, inoculated with 3% inoculum from activated culture
after taking optical density of the culture at 600nm after 24hr. After incubating all the
flasks incubated at 30 ± 2°C and 150 rpm on a rotary shaker for 120h pigment was
extracted and optimal pH for highest pigment production and intensity was examined.
2.8.3 Effect of NaCl
For knowing the effect of salt concentration on pigment production various salt %
(1%, 2%, 3%, 4%, 5%) were selected and added in a flask containing optimized 2g
waste, pH 9 and mixture of basal medium prepared was added. All the flasks were

27
 Materials and Materials

autoclaved at 121 °C and 15 psi for 45 min and then, inoculated with 3% inoculum
from activated culture after taking optical density of the culture at 600nm after 24hr
followed by the submerged at conditions 30 ± 2°C and 150 rpm on a rotary shaker for
120h.
2.8.4 Effect of glycerol
To examine the intensity of pigment affected by glycerol, optimization process with
different glycerol amount was done. Five flasks containing optimized 2g waste, 5%
NaCl, pH 9 and 100 ml of basal medium was taken in which various glycerol
concentration such as 1%, 2%, 3%, 4% and 5% were added respectively after
autoclaving at 121 °C and 15 psi for 45 min 24 h old activated culture was inoculated
with 3% inoculum from activated culture after taking optical density of the culture at
600nm. Pigment was extracted after incubating at 30 ± 2°C and 150 rpm on a rotary
shaker for 120h and examined for glycerol optimization.
2.8.5 Effect of inoculum size
Inoculum size is the amount of culture inoculated in the medium prepared. In a five
250ml Erlenmeyer flasks 2g of waste, pH 9, 5% NaCl and 1% glycerol was added all
the flasks were at 121 °C and 15 psi for 45 min than 24h old activated culture was
inoculated with different inoculums size (1%, 2%, 3%, 4%, 5%) after taking optical
density at 600nm. Flaks were incubated at 30 ± 2°C and 150 rpm on a rotary shaker
for 120h and examined.

Table 3: optimization of pigment production with OVAT method

Sr. No. Factors Variations
1 Substrate concentration %w/v 1g, 2g, 3g, 4g, 5g
2 pH 7, 8, 9, 10,11
3 NaCl %w/v 1%, 2%, 3%, 4%, 5%
4 Glycerol %v/v 1%, 2%, 3%, 4%, 5%
5 Inoculums size 1%, 2%, 3%, 4%, 5%

28
 Materials and Materials

2.9 Purification, identification and characterization of pigment

To characterize partial purified pigment from S-5-9-3, the pigment was analysed by
thin layer chromatography (TLC), high performance thin layer chromatography
(HPTLC), UV-VISIBLE Spectrophotometric analysis (λ max), Fourier-Transformed
Infrared Spectroscopy (FT -IR) and Mass spectroscopy (MS).
2.9.1 Thin layer chromatography (TLC)
Pigment was analyzed for purity and identity by thin-layer chromatography (Zeba,
[Link], 2010). Thin layer chromatography was done using commercially available TLC
sheets (Stationary phase) (TLC Silica gel 60 F from MERCK). In this method 10%
DCM: MeOH solvent system (mobile phase) was used and the chromatography
chamber with the solvent was kept for 15-20 min for the equilibrium. The sample was
spotted on the TLC sheet along with standard using capillary tube and air-dried,
allowing the solvent system run up to 75 % on the sheet. The TLC sheet was carefully
removed, air-dried and the retention factor was calculated according to the following
formula.
Rf = 𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅𝒃𝒚𝒕𝒉𝒆𝒄𝒐𝒎𝒑𝒐𝒖𝒏𝒅
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅𝒃𝒚𝒕𝒉𝒆𝒔𝒐𝒍𝒗𝒆𝒏𝒕
2.9.2 High performance thin layer chromatography (HPTLC)
The HPTLC operates using the same principles as TLC, such as the adsorption
principle of separation. The capillary action allows the solvent or mobile phase to
flow. The analytes migrate towards the stationary phase in accordance with their
affinities (adsorbent). The component with stronger affinity moves more slowly in the
direction of stationary phase. A component with low affinity moves quickly in the
direction of the stationary phase.
The pigment in its pure form was determined using “HPTLC CAMAG TLC system
(CAMG, Muttenz, Switzerland)”. The reversed phase HPTLC analysis of pigment
was done via 30×100mm glass plates pre-coated with RP silica gel 60 F254S plates
(E-Merck, Darmstadt, Germany)” the samples were applied as the 8mm bands using a
CAMAG automatic TLC sampler 4 (ATS4) sample applicator “(CAMAG, Geneva,
Switzerland)”. The CAMG micro litre syringe was connected to the sample
applicator. The application rate for determining the orange pigment was kept constant
at 250 nL/s. The mobile phase DCM: MeOH = 0.5:9.5 was used and the development
chamber was saturated previously for 20 min at room temperature. The detection of

29
 Materials and Materials

pigment was done at wavelength of366nm. Each estimation was done in three or six
replicates and the software applied was “WinCAT´s (Version 1.4.3.6336, CAMAG,
Muttenz, Switzerland).”
2.9.3 UV-VISIBLE spectrophotometric analysis
Jasco- 2000 UV- Visible Spectrophotometer was used to analyze the purified pigment
in the range of 400-700nm. The principle is based on the absorption of visible or
ultraviolet light by the pigment, which results in the production of distinct spectra.
Lambda max of the pigment is the wavelength at which the pigment has its strongest
photon absorption. For identification of maximum absorption, purified pigment was
dried and dissolved in methanol and analysed in UV-VIS spectrophotometer.
2.9.4 Fourier-transformed infrared spectroscopy (FT -IR)
It works on the fact that vibrations of groups and bonds occur at different frequencies
so when infrared radiation passes through a sample, some of the radiation is absorbed
at different frequencies which are characteristic of that molecule. With the use of FT-
IR molecular structure or chemical bonding of the sample can be known. On the
specimen a spot is subjected to a modulated IR beam during FT-IR analysis
(Anderson and M.S, 2000). Transmittance and reflectance are translated into IR
absorption plot consisting of reverse peaks. The resulting spectrum is than matched
and identified with known signatures. The partially purified pigments were placed on
the crystal surface region of the FTIR (Bruker, Alpha) instrument, and analyses were
taken using a range of 400- 4000 cm-1 transmission with a resolution of 4 cm-1.
2.9.5 Mass spectroscopy (MS)
It is an analytical technique used to find the molecular mass of the compound and
helps in identification of compound (Holtin et al., 2009). Using an electro spray
ionization (ESI) probe and a Finnigan LCQ outfitted with an API source, mass
spectrometry was carried out than direct injection of pigment into the MS
spectrometer with 3µl/min flow rate pump. Spectra were obtained in positive ion
mode at a capillary temperature and voltage of 180ºC and 3V respectively. At a
relative energy of collision of 20, the MS spectrum of the pigment´s molecular ion
was obtained upon detection.

30
 Materials and Materials

2.10 Applications of pigment

2.10.1 Antioxidant activity
A molecule which can inhibit oxidation is known as antioxidant. Reactive oxygen
species (ROS) and free radicals which are very highly reactive and produced by
normal aerobic metabolism are responsible for oxidative damages. When this
oxidative molecule become high in number they cause lipid and protein oxidation and
DNA damage associated with various disorders by reacting with lipid, proteins and
DNA through chain reaction. Endogenous and exogenous antioxidants are capable of
inhibiting this type of oxidative molecule (Ambati et al., 2014). Antioxidant activity
of the fractions of methanol extract of microbial pigment was measured by DPPH (1,
1-diphenyl-2-picryl-hydrzil). DPPH solution was prepared in methanol (0.002%w/v).
2ml of DPPH solution was added to the 1ml of pigment solution and for control 2ml
of DPPH was added in 1ml of methanol. The mixture was properly shaken and
allowed to stand at room temperature for 30 minutes in dark condition. After
incubation period the absorbance was measured at 517nm by using a UV-Visible
spectrophotometer and the blank was set by 3ml of methanol. The free radical
scavenging activity was calculated by following equation:
DPPH scavenging effect (%) = [(A0-A1/A0] × 100
Where, A0 = Absorbance of the control reaction
A1 = Absorbance of the test reaction
2.10.2 Antibacterial and antifungal activity
Antibacterial and antifungal property was checked by agar well diffusion assay. For
antifungal activity crude pigment was tested against Fusarium chrysogenum,
Aspergillus niger, Candida albicans and Trichoderma harziumand methanol was used
as control. On nutrient agar plates these cultures were spread and wells were made,
presence of zone around the well containing pigment can be concluded as positive
results.
For antibacterial activity crude pigment was tested against Staphylococcus aureus
(gram-positive), Bacillus subtilis (gram-positive), Bacillus megaterium (gram-
positive), Escherichia coli (gram-negative), Serratia marcesence (gram-negative) and
Enterobacter aerogenes (gram-negative). The nutrient agar medium plate was used
and the appearance of zone of inhibition indicated the positive results.

31
 Materials and Materials

2.10.3 Phytotoxicity and PGPR

Toxicity of pigment produced by Salinococcus roseus S-5-9-3 was examined for
seeds of methi (Trigonella foenum-graecum), variyali (Foeniculum vulgare) and
jowar (sorghum bicolour). At first, the seeds were washed with sterile distilled water
and aluminium foil food container was disinfected with methanol. Wet cotton was
placed in each 12 container with Whatman filter paper grade no 1 on it and 10
different seeds were arranged on the filter paper. One set of seeds was kept for control
on which distilled water was spreaded on each container whereas, pigment was
sprinkled on the other set of seeds. All the containers were covered, kept for
incubation and watered once a day. After the incubation period growth was observed
and compared with the control and examined for phytotoxicity (dos Santos et al.,
2021).
2.10.4 Application of pigments on cloth (dying of cloth)
White textile fabric that was easily accessible was cut into little square-shaped pieces
for the experiment. For 1 hour, textile materials were submerged in a solution of 5 ml
extracted pigment in 2 mg/ml methanol. After incubation, the cloth was dried at 70–
80°C in a hot air oven. The textile samples are properly cleaned after the drying
process (Abou Elmaaty et al., 2020).
2.10.5 Application of pigments in candle preparation
Commercial candle wax (50g) were placed in the bowl and melted in a water bath. 2
ml of a 2 mg/ml concentration of methanolic extract of pigments were added and
properly mixed. This was put in the mould and let to cool for two hours at room
temperature (Mehta & Shah, 2015).
2.10.6 Application of pigments in lip balm
A [Link] (v/w/w) blend of vaseline, paraffin wax, and coconut oil was used to
create the bio lip balm. The three components were placed in a bowl and melted and
blended in a water bath. Dried pigment was added to the combination above, properly
dissolved, and well mixed. The liquid was then put into a lip balm container and let to
cool (Dave and Shetty, 2016).

32
 Materials and Materials

2.10.7 Application in food industry

For food based industrial application, ‘whipped cream/ fresh cream’ available in the
market was selected. For this experiment spoonful of whipped cream was taken, 2
mg/ml pigment solution made in methanol and 2 ml of this solution were dried to test
the effectiveness of the pigment as a food colorant. After that it was added and
blended with a spoonful of whipped cream.

33
 Results and discussion

Chapter 3
Results and
Discussion

34
 Results and discussion

3.1 Characterization of the isolate

3.1.1 Cultural and morphological characters
Results of cultural and morphological characters carried out for isolate S-5-9-3 is
presented in Table 4. S-5-9-3 was medium, round, entire, convex, smooth, opaque,
red, non-motile, Gram positive cocci (figure 5), non-capsulated and non-spore former.
The pure culture plate of S-5-9-3 on Zobell marine agar (ZMA) is shown in the figure
4. Previously morphological characterization of S-5-9-3 was done and they got the
similar results such as gram positive, cocci, non-motile and also the color of pigment
was orange (Meraj Fatima and K. Anuradha, 2022).

Figure 4: Pure culture of S-5-9-3 Figure 5: Gram staining of S-5-9-3

Table 4: Cultural and morphological characters of S-5-9-3

Cultural characters
Size Medium
Shape Round
Margin Entire
Elevation Convex
Texure Smooth
Opacity Opaque
Morphological characters
Gram staining Positive
Endospore Negative
staining
Capsule staining Negative
Motility Non-motile

35
 Results and discussion

3.1.2 Biochemical tests

Different biochemical tests were performed for S-5-9-3 is mentioned in the table 5.
Various tests were performed such as MR test, VP test, indole test, citrate utilization,
H2S production, haemolysis test, decarboxylation test and nitrate reduction test. The
enzyme screening of various enzymes such as protease, lipase, amylase, urease,
catalase, gelatinase, coagulase and oxidase was carried out. Along with this
carbohydrate fermentation tests were also performed for different sugars like
dextrose, maltose, lactose, fructose, sucrose, glucose and mannitol. The isolate is
metabolically diverse and was able to produce several enzymes and ferment different
types of sugars.
Table 5: Biochemical tests
Biochemical test S-5-9-3
MR test -
VP test -
Indole test -
Citrate utilization -
H2S production -
Haemolysis test -
Decarboxylation test -
Nitrate reduction test -
Enzyme screening
Protease +
Lipase +
Amylase +
Urease -
Catalase +
Gelatinase +
Coagulase -
Oxidase -
Sugar utilization
Dextrose +
Maltose -
Lactose -
Fructose -
Sucrose -
Glucose +
Mannitol -

36
 Results and discussion

3.2 Screening of Agro-food waste

Pigment intensity and production was screened by submerged fermentation with the
use of eight different wastes such as: wheat bran (WB), sugarcane bagasses (SCB),
dry potato peels (DPP), rice bran (RB), dry orange peels (DOP), groundnut shells
(GS), sorghum stalk (SS) and castor seed shells (CSS) as shown in the figure 6. From
the results it can be concluded that sugarcane bagasse showed the maximum pigment
production for S-5-9-3 whereas some amount of pigment was also produced from
wheat bran, rice bran, groundnut shells and sorghum stalks. Castor seed shells, dry
orange peels and potato peels showed no pigment production as mentioned in the
table 6. From the results sugarcane bagasses was selected as a low cost substrate for
the pigment production of for [Link] low-cost substrates for X. dendrorhous to
produce astaxanthin, a variety of vegetable by-products from the food sector have
been explored by (Amado and Va´zquez, 2015).
Table 6: Screening of agro-food waste
Types of waste Pigment production
Wheat bran (WB) ++
Sugarcane bagasses (SB) +++
Rice bran (RB) ++
Groundnut shell (GS) +
Castor seed shell (CSS) _
Dry orange peels (DOP) _
Potato peels (DPP) _
Sorghum stalks (SS) +

Figure 6: Submerged fermentation

37
 Results and discussion

3.3 Solubility of pigment

Various solvents were used to check the pigment solubility. Pigment solubility was
more in methanol and hexane out of all other solvent listed. The solubility level of
pigments in all the solvents is as shown in table 7.
Table 7: Solubility of pigment
Types of solvent Solubility of pigment
Ethyl acetate +
Choloroform +
Petroleum ether +
Methanol +++
Distilled water -
Hexane +++
Acetone +
Di ethyl acetate +

3.4 Extraction of pigment

Pigment extraction was done by solvent extraction method and methanol was used as
a solvent as our pigment is highly soluble in methanol. After the incubation period,
the suspensions were filtered using cheese cloth and the excess liquid was squeezed
out manually, similar procedure was carried out by (dos Santos et al., 2021) for the
extraction of prodigiosin from agro-food wastes as substrate. After centrifugation
method 20-30ml of methanol was added and all the tubes were kept in water bath for
45min and 70oC, pigment got solubilised in methanol and cell pellet become
colorless. In a crucible let our pigment containing supernatant air dry overnight and
add hexane to remove lipid impurities again dry the pigment and store by adding
methanol at 4oC figure 7.

38
 Results and discussion

Figure 7: Extraction of pigment

3.5 Optimization by one variable at time (OVAT)
Substrate concentration, pH, NaCl, glycerol and inoculum size are the important
parameters for pigment producing marine bacteria on agro-food waste. Some research
work also suggests that supplementing the media with glycerol also results in high
pigment intensity. So glycerol concentration was also optimized to check its effect on
pigment production. From the experiment it was concluded that the most appropriate
parameter for high pigment production involves 2g, pH 9, 5% NaCl, 3% inoculum
size and 1% glycerol concentration. The results and its graph are individually
described.
3.5.1 Effect of substrate concentration
For optimizing this parameter different substrate concentration were selected such as
1g, 2g, 3g, 4g and 5g respectively. The result from the evaluation of pigment
production is shown in the figure 8 which is 400 µg/ml, 550 µg/ml, 490 µg/ml, 400
µg /ml and 340µg/ml respectively. It can be concluded that 2g of sugarcane bagasse
showed intended and maximal pigment production figure 9. Sugarcane bagasse (SB)
as carbon source has also been used for pigment production by Monascus
purpureus in submerged conditions (Silveira et al., 2013).

39
 Results and discussion

Substrate concentration
700
pigment production µg/ml 600 550

500

400

300

200

100

0
1g 2g 3g 4g 5g
Substrate concentration

Figure 8: Graph of effect of substrate concentration on pigment production

Figure 9: Effect of substrate concentration on pigment production

3.5.2 Effect of pH
Different variations of pH such as 7, 8, 9, 10 and 11 were set to check the pigment
production as our microorganism is marine mostly alkaline pH is preferable, results
are shown in the figure 10 in the form of graph and it can be concluded that pH 9 was
most appropriate for S-5-9-3 to produce maximum amount of pigment which is
580µg/ml figure 11, results of other pH variations are 480µg/ml, 520µg/ml, 500µg/ml
and 450µg/ml respectively for pH 7, 8, 10 and 11. Effect of pH on red pigment

40
 Results and discussion

produced by Monascus species was examined and it was found that color of the
pigment changes with the variations in pH of fermentation medium and it was
concluded that pH of fermentation medium is an important factor (Mehri et al., 2021)

pH variations
700
Pigment production µg/ml

580
600

500

400

300

200

100

0
7 8 9 10 11
pH scale

Figure 10: Graph of effect of pH on pigment production

Figure 11: Effect of pH on pigment production

3.5.3 Effect of NaCl
The pigment producing bacteria was isolated from the marine environments and its
pigment activity is mostly dependent on the salinity of the medium hence the range of
NaCl Concentration at which the pigment intensity is highest was checked. Variations
like 1%, 2%, 3%, 4% and 5% were selected to check effect of salt. After extraction of
pigment it was evaluated that pigment production of 1%, 2%, 3%, 4% and 5% were
350µg/ml, 400 µg/ml 420µg/ml, 510µg/ml and 600µg/ml respectively figure 12. So

41
 Results and discussion

from the results it can be concluded that 5% NaCl is most effective for pigment
production. Results of NaCl concentration are shown in the figure 13. In one of the
studies for carotenoid pigments it was found that the carotenoids production increased
with increasing NaCl concentration also the influence of salinity in microbial pigment
production may vary with organisms and could also depend on their origin and
isolation location (Loh et al., 2020).

Salt concentration
700
600
pigment production µg/ml

600

500

400

300

200

100

0
1% 2% 3% 4% 5%
NaCl concentration

Figure 12: Graph of effect of NaCl concentration on pigment production

Figure 13: Effect of NaCl concentration on pigment production

3.5.4 Effect of glycerol
After the evaluation of pigment production it was found that addition of glycerol
boosted the pigment intensity for this parameter different glycerol concentrations
were taken such as 1%, 2%, 3%, 4% and 5% respectively figure 14 and results are
620µg/ml, 510µg/ml, 440µg/ml, 400µg/ml and 320µg/ml from this it can be

42
 Results and discussion

concluded that 1% glycerol concentration found to boost pigment intensity figure 15.
In one of the researches by (Dikshit and Tallapragada, 2014) it was concluded that
among the factors tested for pigment production by Monascus sanguineus glycerol
had the highest impact on the pigment production. One work done on
ssMonascus pilosus MS-1 to check effect of glycerol, results obtained were pigment
production could be enhanced significantly after adding glycerol 2g/l and the
maximum pigment yield (13.78 U/ml) (Feng et al., 2015).

glycerol concentration
800
Pigment production µg/ml

700
620
600

500

400

300

200

100

0
1% 2% 3% 4% 5%
Glycerol %

Figure 14: Graph of effect of glycerol concentration on pigment production

Figure 15: Effect of glycerol concentration on pigment production

43
 Results and discussion

3.5.5 Effect of inoculum size

Different amount of inoculum size was selected such as 1%, 2%, 3%, 4% and 5%
respectively. The results are 380µg/ml; 520µg/ml, 650µg/ml, 490µg/ml and 410µg/ml
respectively figure 16 from the results it can be concluded that 3% inoculums size
showed maximum pigment production as shown in the figure 17. Study was done on
the pigment production by M. purpureus from jackfruit seed using solid-state
fermentation and it was reported that high inoculum concentrations led to excessive
biomass formation depleting the nutrients in the fermentation medium that are
necessary for the product formation while low inoculum concentration produced
insufficient biomass (Mehri et al., 2021).

Inoculum size
800
Pigment production µg/ml

700 650
600
500
400
300
200
100
0
1% 2% 3% 4% 5%
Inoculum size

Figure 16: Graph of effect of inoculums size on pigment production

Figure 17: Effect of inoculum size on pigment production

44
 Results and discussion

3.6 Characterization of pigment

The extracted pigment was characterized through different methods to find out that
the Unknown pigment belongs to which group or family.
3.6.1 Thin layer chromatography
Characterization of the astaxanthin pigment was performed by TLC by comparing the
Retardation factor (Rf) as indicated in Lorenz Todd standard
chromatogram. Conditions used for silica gel-TLC were applied according to the
method of Tolasa et al., 2005. Solvent system used was 10% DCM: MeOH in the
ratio 2:3. In this method and retardation factor found was 0.36 which was compared
with the standard as shown in the figure 18. Similar results was found by Nagaraj et
al. (2012) who separated carotenoid extraction from H. pluvialis and Rf is 0.37 for
astaxanthin and the another research in which Astaxanthin was extracted from shrimp
waste Rf value confirmed was 0.36 (Senthamil and Kumaresan, 2015).

Figure 18: Thin layer chromatography (TLC) analysis of pigment produced by

S-5-9-3
3.6.2 High performance thin layer chromatography (HPTLC)
In the determination of orange pigment HPTLC approach was applied figure 19. The
chromatographic peak of pigment in all studied sample matrices was recorded at R f =
0.36±0.02 without the presence of additional peak figure 20. Similar results were
obtained in the experiment performed by Pachaiyappan et al., 2021.

45
 Results and discussion

Figure 19: HPTLC analysis of pigment extracted from S-5-9-3

Figure 20: HPTLC graph of pigment extracted from S-5-9-3

3.6.3 UV- Visible spectroscopy
When the extracted pigment was analyzed it was found that the pigment showed
maximum absorbance between the range of 400nm-700nm. Absorbance was noted
with three peaks in the range of 400-700nm, which is the caretenoid characteristic.
From the results, as shown in the graph figure 21 it was concluded that the λmax of
the extracted pigment from S-5-9-3 was 491nm with 1.4424Abs. The extracted

46
 Results and discussion

pigment from the isolates belongs to the derivative of astaxanthin which is orange
colored. This data matches with the data of other researchers (Krishna et al., 2011).

Figure 21: Maximum absorbance of pigment produced by S-5-9-3

3.6.4 FT-IR analysis

FTIR spectra stretches are significant for confirming the functional groups and
chemical bonding present in the molecules. The purified pigment dissolved in
methanol and examined for FTIR spectral analysis and the results are shown in the
figure 22. According to (Coates et al., 2006) the peak descriptions of the FTIR spectra
can be illustrated as follows: 3313.12 cm-1 shows the presence of O-H hydrogen bond
and the peak at 2941.73 cm-1 and 2830.8 cm-1is due to C-H stretching. A peak at
1450.04 cm-1 and 1416.12 cm-1 shows the presence of C=C stretching in the
molecule and the peak at 1021.12 cm-1 is of C-H different hydrocarbons. The spectral
behaviour of the pigment strongly resembles the structure of astaxanthin which was
obtained by X. dendrorhous (Chen et al. 2007).

47
 Results and discussion

Figure 22: Fourier transform infrared spectroscopy (FT-IR) analysis of pigment

extracted from S-5-9-3.

3.6.5 Mass spectrometry (MS)

The pigment obtained was analyzed by MS using direct injection into the
spectrometer. Pigment revealed a [M]+ ion at an m/z 598.65 as shown in the figure,
which fits exactly with the mass of the structure of standard astaxanthin as shown in
the figure 23. Similarly, LC-(APCI) MS was performed in positive ion mode, so free
astaxanthin as well as its isomers were detected as a quasimolecular ion at m/z 597.5
(Holtin et al., 2009).

Figure 23: Mass spectrometry (MS) analysis of pigment produced by S-5-9-3

48
 Results and discussion

3.7 Applications of astaxanthin

Astaxanthin is used preferentially in food, feed, aquaculture, nutraceutical,
pharmaceutical, and poultry products (Dursun et al., 2020).
3.7.1 Antioxidant effect of astaxanthin
After incubating all the tubes for 30 min (dark place) optical density was taken at
517nm with the use of UV-VIS spectrophotometer. Blank was set with 3ml of
methanol than O.D of control was taken which was 0.997 and O.D of sample 0.671.
The results were calculated with the following formula:
DPPH scavenging effect (%) = [(A0-A1/A0] × 100 and it was concluded that the
DDPH scavenging activity of Astaxanthin was 48.58%.
Comparison of several carotenoids such as lutein, lycopene, β and α-carotene and
astaxanthin was done by Ambati et al., 2014 and among all Astaxanthin showed more
antioxidant activity. He also concluded than Astaxanthin's antioxidant activity was
100 times more than that of α-tocopherol and 10 times greater than that of zeaxanthin,
lutein, canthaxanthin, and β-carotene.
Carotenoids have long conjugated double bonds in polyene chains that carry out
antioxidant activities by quenching singlet oxygen and scavenging radicals to stop
chain reactions. The antioxidant capabilities of carotenoids, which are attributable to
their physical and chemical interactions with cell membranes, may be the cause of
their biological advantages. An experiment was carried out in rats and it was found
that in H. Pluvialis the best protection from free radicals was offered by Astaxanthin
followed by β-carotene and lutein (Ambati et al., 2014). Astaxanthin has a unique
molecular structure with hydroxyl and keto moieties attached to each ionone ring,
which gives it strong antioxidant capabilities (Liu et al., 2007).
3.7.2 Antibacterial and antifungal activity
The extracted pigments showed antimicrobial activities against the bacterial and
fungal species as shown in table. Astaxanthin showed highest antifungal activity
against candida albicans and Trichoderma harzium whereas antibacterial activity
against Staphylococcus aureus (gram-positive). AST in the form of a nanoemulsion
has been shown to be effective against a variety of bacteria also MIC values ranged
from 500 to 4000 g/ml for both Gram-positive and Gram-negative bacteria
(Shanmugapriya at el., 2018).

49
 Results and discussion

Antifungal activity
Table 8: Antifungal activity of pigment produced by S-5-9-3
Test fungi mm
Methanol 0.0
Fusarium chrysogenum 0.0
Aspergillus niger 0.0
Candida albicans 13
Trichoderma harzium 10

Antibacterial activity
Table 9: Antibacterial activity of pigment produced by S-5-9-3
Test bacteria mm
Methanol 0.0
Bacillus subtilis (gram-positive) 0.0
Staphylococcusaureus (gram- 7
positive)
Bacillus megaterium (gram- 0.0
positive)
Escherichia coli (gram-negative) 0.0
Serratia marcesence (gram- 0.0
negative)
Enterobacter aerogenes (gram- 0.0
negative)

3.7.3 Phytotoxicity and PGPR

A specific benefit of using plants in toxicity studies is the assessment of possible eco-
toxic chemicals in terrestrial habitats. Other benefits of using phytotoxicity tests
include cheap maintenance costs and quick results (Priac et al. 2017). In this study
astaxanthin produced from S-5-9-3 was examined for phytotoxic effects by using
seeds of methi (Trigonella foenum-graecum), variyali (Foeniculum vulgare) and
jowar (sorghum bicolour).The results obtained after 7 days of incubation period
indicated that the tested concentrations of astaxanthin did not have an inhibitory effect

50
 Results and discussion

on the elongation of the roots or in the germination of seeds in the analyzed

vegetables. Longer tuber was also observed for some of the seeds when compared to
control so, it can also be concluded that astaxanthin may also act as a plant growth
promoter figure 24. Similar experiment was conducted on seeds of cabbage
(Brassica oleracea), lettuce (Lactuca sativa), onion (Allium cepa) and cucumber
(Cucumis sativus) to check the plant toxicity of Prodigiosin produced from S.
marcescens, indicating that is a nontoxic compound table 10 (Santos et al., 2021).

Table 10: PGPR and phytotoxicity test of pigment

Test seeds No. of seeds germinated No. of seeds germinated
(control) (for pigment astaxanthin)

Methi (Trigonella foenum- 9 10

graecum)
Variyali (Foeniculum 7 7
vulgare)
Jowar (Sorghum 7 9
bicolour).

Figure 24: Phytotoxicity test of pigment

3.7.4 Application of pigments on cloth (Dying of cloth)

Application of extracted pigment astaxanthin on textile materials was performed by
applying a little amount of partially purified pigment on the white cloth, which later
was converted in to the colored one as shown in the figure 25. Using various types of
mordants on wool dyeing, researchers discovered that isolated pigment from
Streptomyces thinghirensiscanbe effectively used for dyeing wool fabrics to achieve a

51
 Results and discussion

wide variety of soft and light colours (Abou Elmaaty et al., 2020). Similar
experiment was done by Chakraborty et al., 2015; Mandal et al., 2017; Srinivasan et
al., 2017 and successfully coloured the textile materials using bacterial pigments.

Figure 25: Pigment application on cloth material

3.7.5 Application of pigment in lip balm

When the extracted pigment astaxanthin was combined with the heated (about 40–
60°C) lip balm formulation, it was found that the pigments were stable in the
formulation and, as shown in figure 26, it greatly coloured the formulation. The
colour remained intact for up to one month. For the development of a bio-lip balm, a
similar experiment was conducted using red pigment from Streptomyces sp
(Chakraborty et al., 2015; Srinivasan et al., 2017). The colour of the substance
remained unchanged at room temperature, and the lip balm remained consistent
during the 30-day testing period (Tandale et al., 2016).

Figure 26: Application of pigment in lip balm preparation

52
 Results and discussion

3.7.6 Food application

When the extracted pigment was combined with the whipped cream, the coloration
was unchanged after 48 hours at room temperature figure 27. Due to their
antibacterial properties, pigments can also be utilised as preservatives. The FDA
approved marketed pigments include carotene from Blakeslea trispora and Monascus
pigments, ribofavin from Ashbya gossypii, Arpink Red from Penicillium oxalicumand
astaxanthin from Xanthophyllomyces dendrorhous (Venil et al., 2013).

Figure 27: Pigment application in white cream as food colorants

3.7.7 Application of pigment in candle preparation

As shown in the figure, when extracted pigment astaxanthin from isolate was
combined with hot, melted clear gel wax, it was found that the pigment blended into
the wax uniformly and significantly coloured the candle wax figure 28. The candle
has been successfully coloured by Shaikh, 2016, and Mehta & Shah, 2015, utilising a
prodigiosin red coloured pigment from Serratia marcescens. Yellow pigments
extracted from Micrococcus luteus successfully colored the candle in the experiment
done by Dave & Shetty, 2016.

Figure 28: Pigment application in color candle

53
 Results and discussion

CHAPTER 4
Summary

54
 Results and discussion

SUMMARY
• A study was carried out with production, optimization, characterization and
application pigment produced from marine bacterial strain S-5-9-3 isolated
from the coastal region of Somnath, Gujarat, India with the utilization of agro-
food waste as a low-cost substrate. Cultural, morphological and biochemical
properties of the strain was carried out.
• Selection of waste was done by screening of eight different wastes such as
wheat bran (WB), sugarcane bagasse (SCB), dry potato peels (DPP), rice bran
(RB), dry orange peels (DOP), groundnut shells (GS), sorghum stalk (SS) and
castor seed shells (CSS) followed by pre-treatment of the wastes.
• Submerged fermentation was done and in this process, eight 250ml
Erlenmeyer flasks containing 3g of each waste were taken, 2% glycerol were
added along with 100ml of basal medium containing, 5%NaCl, 0.5% peptone
and 0.5% dextrose. All the flasks were kept for an incubation period of 7 days
at 28± 2°C and 150 rpm on a rotary shaker.
• After incubation period solubility of pigment was checked with the use of
different solvents such as Ethyl acetate, chloroform, petroleum ether, distilled
water, methanol, hexane, acetone and Di ethyl acetate out of which methanol
was selected as a solvent as our pigment was highly soluble in methanol.
Further extraction of pigment was carried out with solvent extraction method
and it was concluded that sugarcane bagasses found to produce more amount
and high intensity of pigment so it was selected as a substrate for pigment
productions by S-5-9-3.
• After selection of waste optimization was done with one variable at time
(OVAT) in which parameters used were Substrate concentration(1g, 2g, 3g,
4g, 5g), pH(7, 8, 9, 10, 11), NaCl (1%, 2%, 3%, 4% 5%), glycerol (1%, 2%,
3%, 4% 5%) and inoculum size (1%, 2%, 3%, 4% 5%). Optimization was
done with submerged fermentation and sugarcane bagasses as a substrate.
Results of optimization were 2g substrate concentration, pH 9, 5% NaCl, 1%
glycerol and 3% inoculum size gave maximum pigment production such as
550µg/ml, 580µg/ml, 600µg/ml, 620µg/ml and 650µg/ml respectively.
• Further characterization and identification of pigment was done with the use
of various analytical techniques such as thin layer chromatography (TLC),

55
 Results and discussion

high performance thin layer chromatography (HPTLC), UV-VISIBLE

Spectrophotometric analysis (λ max), Fourier-Transformed Infrared
Spectroscopy (FT -IR) and Mass spectroscopy (MS).
• Results obtained were, with the use of TLC Rf value of the pigment was found
which 0.36 which was compared with standard is and further pigment was
purified with the use of high-performance thin layer chromatography
(HPLTC). Absorbance maximum of the pigment was 491 nm. From FTIR the
peak descriptions of the FTIR spectra can be illustrated as follows: 3313.12
cm-1 shows the presence of O-H hydrogen bond and the peak at 2941.73 cm-1
and 2830.8 cm-1 is due to C-H stretching. A peak at 1450.04 cm-1 and
1416.12 cm-1 shows the presence of C=C stretching in the molecule and the
peak at 1021.12 cm-1 is of C-H different hydrocarbons. The spectral
behaviour of the pigment strongly resembles the structure of astaxanthin.
• From mass spectrometry molecular mass of obtained was 598.65 which fits
exactly with the mass of the structure of standard astaxanthin. From above
analytical techniques it was concluded that pigment produced from S-5-9-3
can be astaxanthin as it matches with the structure of standard astaxanthin.
• Antioxidant activity of pigment was calculated with DPPH scavenging method
and it was concluded that DDPH scavenging activity of Astaxanthin was
48.58%. Antibacterial and antifungal activity was also checked and it was
concluded that Astaxanthin showed highest antifungal activity against candida
albicans and Trichoderma harziumwhereas antibacterial activity against
Staphylococcus aureus (gram-positive).
• Phyototoxicity testing showed that our pigment astaxanthin was non
phyototxic and might also be used as plant growth promoter. Other
applications such as colored candle, cloth dyeing, lip balm preparation and
food application was also done. Applications of pigments concluded that
pigments show antioxidant activity, antibacterial activity against bacterial
pathogens and pigments successfully applied coloration to textiles and
candles; it can also be used in cosmetics or pharmaceutical products as
antioxidant agent and dye.

56
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CHAPTER 5
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57
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