Chapter-1

Introduction
 1. INTRODUCTION

Color is the most delighting characteristic of any substance. Pigments are light-absorbing
compounds that are responsible for the colors that organisms display. Pigments are
molecules that provide attractive colors and play important role in the development of the
organism and metabolism. Colors are vital to the sensing of the environment and have
evolved in higher living organisms to guide their interactions with others. A variety of
natural and synthetic pigments are available. Pigment producing bacteria are known as
chromobacteria.

1.1. History of pigments

Nature is rich in colors obtained from fruits, vegetables, roots, minerals, plants and
microbes. Due to their origin from biological material they are often called bio-colors.
Addition of colors to the processed food is a very old practice. Saffron, turmeric and
vegetable dyes had been used to color food (Joshi et al., 2003). Since people have been
wearing clothes, they have sought ways to make their garments more attractive, more
aesthetically pleasing, and more colorful. Most dyes derived from plants or animals,
which ranged from the ordinary to the exotic. The first recorded mention of fabric dyeing
dates back to 2600 BC. Originally, dyes were made with natural pigments mixed with
water and oil used to decorate skin, jewelry and clothing. Man learnt to use different
kinds of natural coloring matters to dye cloths. Biochemists have identified that the vital
activity of plants is also dependent on colorants in a way that the bright color of flowers
are important in attracting insects and birds to act as pollinators. When these are used for
dyeing fabrics, they not only impart color to the fabric, but also act as antifungal agent
whereby they impart protection to fabric against bacterial or fungal infections or as moth
reluctant. Some dyes have a cooling sensation also. Natural pigments obtained from
insects, plants and animals were the colorants used since prehistoric period (Venil et al.,
2013). Synthetic dyes which took the place of natural pigments in the middle of 19th
century still rule the field to the maximum extent in spite of its hazardous effect to
humans, animals and environment. Bacterial pigments as an alternative to synthetic
pigments, due to their better bio-degradability and higher compatibility with the
environment, offer promising avenues for various applications. The industry is now able
to produce some bacterial pigments for applications in food, pharmaceuticals, cosmetics
and textiles.
1.2. Role of pigments in living organism
Biological pigments produced by living organisms are basically coloring agents.
Pigments are secondary metabolites which play an important role in the survival of the
microbes (bacteria, fungi, yeast and algae) plants and animals.

a) Role of pigments in microbes: In micro-organisms, pigment formation is associated

with morphological characteristics, cellular activities, pathogenesis, protection and
survival. Autotrophic cyanobacteria contain a green colored pigment, known as
chlorophyll. They also contain accessory pigments phycobilin and chlorophyll b
which are required in photosynthesis. Pigments of photosynthetic bacteria carry out
photosynthesis similarly like plant chlorophyll. Pigments produced by microbes
absorb UV radiation or quench oxygen free radicals. In both ways, pigments play
important role of the cell protection. Pigments help in survival in stress condition.
Bright pigmentation in extremophiles offers protection from oxidative stress.
Pigments confer antibacterial and heavy metal resistance (Ahmed et al., 2013).
b) Role of pigments in animals: Melanin is the main pigment found in the mammals.
Melanin is responsible for the color of the hair and fur of an animal. In animals,
melanin is present in two different forms, pheomelanin and eumelanin (Brown and
Salvo, 1994). These different forms of melanin produce a wide range of colors
varying from black to red to sandy. The presence of biological pigments in animals is
useful in the sense that they may be helpful in protecting their tissues from the
harmful effects of the ultra-violet rays (Frases et al., 2007). Melanin in the skin
protects the mammals from several skin disorders caused by the UV rays. In addition,
biochromes in animals are also said to be helpful in sexual reproduction, indicating
the preparedness to breed and also to recognize the species and gender of animals to
prospective mates. It is also well known that numerous animals use their biological
pigments to protect themselves from predators. In fact, such animals utilize the
pigments as a mean of disguise. The chameleons utilize pigments and display the
 phenomenon of mimicry. They use the biological pigments present in their body to
match their body color with their surroundings by means of regulating the absorption
levels of the electromagnetic spectrum. Hemoglobin is a pigment present in blood and
help in oxygen transport.
c) Role of pigments in plants: In plants, biological pigments are found in a range of
forms and some of them may be intensely compound as well as large structures. Till
date, scientists have been able to categorize in excess of 600 naturally
occurring carotenoid structures in plants. In addition to this over 7,000 flavonoids,
counting 500 anthocyanins, have also been identified. For instance, biological
pigments like chlorophyll are pigmented organic molecules which are caused by the
presence of unsaturated bonds in plants. Chlorophyll is the major pigment in plants
that produce their own food. The fact remains that porphyrin found in the plants help
them to absorb the yellow and blue wavelengths of light, while the green wavelength
of light is reflected by the porphyrin molecules present in most plants. The presence
of chlorophyll in a plant makes the plant look green to the viewer. All green plants
growing on the land and the green algae actually have two different types of green
pigments - chlorophyll a and chlorophyll b. Unlike the green algae, the red algae only
possess chlorophyll a. These diverse varieties of chlorophyll provide the plants with
the fundamental means to intercept light to enable them to undertake process called
photosynthesis. Pigments like anthocyanin, flavanoids and carotenoids display
attractive colors which attract pollinators and help in seed dispersal (Delgado-Vargas
et al., 2000).

1.3. Classification of pigments:

Pigments are classified as either organic/inorganic or natural/synthetic. Plants, animals
and microbes are the major sources of natural pigments. Naturally derived pigments are
represented by carotenoids, flavonoids (anthocyanins), and some tetrapyrroles
(chlorophylls and phycobiliproteins). Synthetic pigments are synthesized in the
laboratories through chemical manufacturing. Pigments from natural sources have been
obtained since long time ago, and their interest has increased due to the toxicity problems
caused by those of synthetic origin. In this way the pigments from microbial sources are a
good alternative. Some of more important natural pigment are the carotenoids, flavonoids
(anthocyanins) and some tetrapirroles (chloropyls, phycobilliproteins). Other less
important groups are the betalains and quinones. The specific color of the pigment is
characteristic for each microbe. Some microbes produce pigments as part of their normal
metabolism. A large number of microorganisms (bacteria, molds and yeasts) produce
pigments.

1.4. Advantages of microbial pigments over synthetic pigments

Microbial pigments offer following advantages:
 Easy propagation and wide strain selection.
 Highly versatile and productive over plants and synthetic sources.
 Fermentation is inherently faster and more productive compared to any other
chemical process.
 Simple and fast culturing techniques allowing continuous bioreactor operation.
 Structural complexity suits for industrial needs.
 Bacterial pigments extracted using simple liquid–liquid extraction technique
minimizing operation cost. Cheap substrates (maltose, glucose, galactose, peanut oil,
sesame oil etc.) used for bulk production (Shahitha and Poornima, 2012).
 Microorganisms produce a large variety of stable pigments such as carotenoids,
flavonoids, quinones, and rubramines, and the fermentation has higher yields in
pigments and lower residues compared to the use of plants and animals (Hobson and
Wales, 1998).
Major advantages of pigment production from micro-organisms include easy and fast
growth in the cheap cultures medium, independence from weather conditions and colors
of different shades. Hence, microbial pigment production is now one of the emerging
fields of research to demonstrate its potential for various industrial applications (Babitha
et al., 2009).
Development of bacterial strains that can utilize cheap and renewable substrates will
make the price of pigments competitive with synthetic pigments. Therefore, discovering
cheap substrates for pigment production is believed to reduce the production cost.
Although, the price of bacterial pigment will be relatively higher compared to the
synthetic dyes, the production cost can be reduced via (Venil et al., 2013):
 Use of agricultural wastes such as pineapple wastes, sugarcane bagasse and molasses
as growth medium for cultivation of bacteria.
 Use of locally isolated wild type bacterial strains eliminates the cost for any genetic
alterations etc.
 Use of simple extraction techniques.
Microbial production has the advantage of being much more environmentally friendly as
compared to chemical synthesis, which is non-degradable.

1.5. Industrial importance of microbial pigments

Pigments are compounds with characteristics of importance to many industries. In the
food industry they are used as additives, color intensifiers, antioxidants etc (Tibor, 2007).
The industrial production of natural food colorants is already well-established and
expanding. However, the range of natural color shades is still limited compared to
synthetic dyes. Besides, the use of plant extracts is known to be expensive and
uncompetitive to synthetic dyes due to their high production costs. Consequently, micro-
organisms are becoming a more popular alternative source for natural food grade
pigments. Development of microbial food grade pigments are likely to cut down the high
production cost of natural colors, thus leading to a cheaper source of natural food
colorants among the modern consumers. Pigments like indigoids, anthraquinones and
naphthoquinones will hold potential applications in food industry in near future
(Jacobson et al., 1997). These pigments are looked upon for their safe use as a natural
food colorants and will not only benefit human health but also preserve the biodiversity,
as harmful chemicals released into the environment while producing synthetic colorants
could be stopped (Neeraj et al., 2011).

1.6. Factors affecting microbial pigment production

Very little is known about the exact chemical nature of pigments, because the formation
of pigment is influenced by the pH of the medium, aeration, temperature of the growth
and carbon and nitrogen sources. Following factors affects pigment production:
a) Temperature:
The production of microbial pigments is greatly affected by the temperature of
incubation, depending upon the type of micro-organism. Dikshit and Tallapragada (2011)
reported that Monascus purpureus has optimum growth and pigment production at 28°C -
30°C.
b) pH:
The growth and type of pigment produced is affected by pH of the medium in which
microorganisms are grown. It differs from one microorganism to another. Slight change
in pH can alter the shade of color (Joshi et al., 2003).
c) Carbon source:
The mycelial growth of pigment producing microorganism is affected by the type of
carbon source like glucose, galactose, maltose, fructose etc. Glucose and its
oligosaccharides are better source for growth and pigment production. Oller (2005)
reported that glucose and sorbitol had a repressive effect on prodigiosin synthesis.
d) Nitrogen source:
The production of microbial pigments is affected by the type of nitrogen source. Organic
nitrogen sources give rise to higher mycelial growth compared with inorganic nitrogen
sources. Meat peptone, casein peptone, peptone–yeast extract mixture and corn steep
powder had a positive effect on pigment production, whereas soy peptone and malt
extract strongly inhibited red pigment synthesis in Monascus purpureus (Kim et al.,
2002).
e) Type of fermentation:
Fermentation (solid or submerged) affects microbial pigment production. The solid state
fermentation yields 3 fold more pigment than the submerged fermentation (Joshi et al,
2003). Nowadays fermentative food grade pigments are in the market like riboflavin, β-
carotene and phycocyanin (Jacobson et al., 1997). To develop a process for the
maximum pigment production, standardization of medium and fermentation condition is
crucial.
1.7. Aim and objectives of the research: Keeping in view the industrial applications
of microbial pigments the present study was undertaken with the following aim
and objectives:

Aim: Characterization of microbial pigments

Objectives: This aim will be achieved by following objectives:

1. Isolation and production of microbial pigments

2. Bio-physical characterization of microbial pigments
3. Antimicrobial activity of crude microbial pigments