CHAPTER -2

Chapter – 2

REVIEW OF LITERATURE

Natural colours are generally defined as materials extracted, isolated or otherwise

derived from plants, animals or microorganisms that are capable of imparting a

distinguishing colour when added.

2.1. Pigments Classification

2.1.1. Based on Origin

Pigments can be classified based on their origin as natural, synthetic or inorganic.

Natural pigments are produced by living organisms such as plants, animals and

microorganisms. Natural and synthetic pigments are organic compounds. Inorganic

pigments are found in nature or reproduced by synthesis (Bauernfeind, 1981).

2.1.2. Based on chemical structure of the chromophore

Pigments can be classified according to the chemical structure of the

chromophore as

Chromophores with conjugated systems (carotenoid, anthocyanin,

betalain,caramel and synthetic pigment) and

Metal-coordinated porphyrins (myoglobin, chlorophyll and their derivatives)

(Wong, 1989)

2.1.3. Based on application as food additives

Based on the application of the pigments as food additives, the Food and Drug

Administration (FDA) has classified pigments into two types

Certifiable (synthetic pigments and lakes) and

Exempt from certification (pigments derived from natural sources such as

vegetables, minerals or animals and synthetic counterparts of natural derivatives)

(Food and Nutrition Board, 1971; Frick and Meggos, 1988 and Wong, 1989)

2.1.4. Based on the structural characteristics of the natural pigments

Natural pigments are also classified based on their structural characteristics as

Tetrapyrrole derivatives (chlorophylls and heme colours),

Isoprenoid derivatives (carotenoids and iridoids),

N-heterocyclic compounds different from tetrapyrroles (purines, pterins,

flavins, phenazines, phenoxazines and betalains),

Benzopyran derivatives (anthocyanins and other flavonoid pigments) and

Quinones (benzoquinone, naphthoquinone, anthraquinone and melanins)

(Bauernfeind, 1981 and Hari, et al., 1994)

2.2. Characteristics of Major Pigments

2.2.1. Quinone

Quinones include large number of structural variants and have a great number of

colouring compounds. Their basic structure consists of a desaturated cyclic ketone that is

derived from an aromatic moncyclic or polycyclic compound. Coenzyme Q, represented

by the ubiquinone has the quinine structure. This group is widely present in animals,

plants and microorganisms and plays an important role in electron transport system in

their cells.

Quinones can be classified based on their structure as benzoquinones,

naphthoquinones, anthraquinones, dibenzoquinones, dianthraquinones and

dinaphthaquinones. Large number of quinines is found due to their variability in the kind

and structure of substituents. Quinones are found in plants: plastoquinones are found in

chloroplasts of higher plants and algae; ubiquinones are ubiquitous in living organisms;

menaquinones are found in bacteria; naphthoquinones in animals and anthroquinones are

found in fungi, lichens, flowering plants and insects. Many types of quinine are

byproducts of the metabolic pathways and a few organisms (fungi) produce large

quantities of quinines. In general, quinines produce yellow, red or brown colourations,

while quinine salts show purple, blue or green colours (Hari, et al., 1994 and Thomson,

1962b).

Structure of Quinone

2.3. Microbial Pigments

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 pigments are the carotenoids, flavonoids (anthocyanins) and some

tetrapirroles (chloropyls, phycobilliproteins). Another group less important is the

betalains and quinones. The carotenoids are molecules formed by isoprenoids units and

the most important used as colorant are the alpha and beta carotene which are precursors

of vitamin A, and some xantophylls as astaxanthin.

Beta-carotene is a pigment used in the industry, which is obtained from some

microalgae and cyanobacteria. Astaxanthin, an important carotenoid (red pigment) of

great commercial value, and it is used in the pharmaceutical feed and aquaculture

industries. This pigment is mainly obtained from Phaffia rhodozyma, Haematococcus

pluvialis and other organisms. The phycobilliproteins obtained from cyanobacteria and

some group of algae, have recently been increased on the food industries. Recently these

pigments are used as fluorescent marker in biochemical assays. Research group has

carried out studies about the factors that improve the production of these pigments

obtained from different microbial species as well as the methods for their extraction and

application (Canizares, et al., 1998).

Among the natural sources of colourants, microorganisms offer great scope and

hope. The ease of cultivation, extraction, the genetic diversity in microbes and

sophistication of technology has made their choice more feasible (Juailova, et al., 1997).

Among the different living organisms, bacteria, yeast, algae, fungi and actinomycetes

appear more efficient and attractive sources of biocolourants.

Nature is rich in colours (minerals, plants etc.) and pigment producing

microorganisms (fungi, yeasts and bacteria). Microbial pigments such as carotenoids,

melanins, flavins and quinines are used in many food industries (Dufosse, 2006). The

ability to synthesis pigments varies with the organism and its environment. Fluorescent

Pseudomonads are ubiquitous bacteria that are common inhabitants of the water, soil,

plants and animals and they are the most studied group (Jayalakshmi, 2008).

There are a number of microorganisms, which have the ability to produce

pigments in high yields, including species of Monascus (Hajjaj, et al., 2000) and Serratia

(Williams, et al., 1971a). The red pigments, produced in solid-state culture by several

species of the genus Monascus, have been traditionally used in many Asian countries for

colouring and securing a number of fermented foods (Francis, 1987).

Due to the global increase in the manufacture of processed foods and the

continued consumer demands for natural food ingredients, the market for natural

colorants for food use is estimated to grow (Downham and Collins, 2000). Currently, the

vast majority of the natural food colorants permitted in the European Union and the

United States are derived by extraction of the pigments from raw materials obtained from

the flowering-plants of the kingdom Plantae (Mortensen, 2006).

The production of many existing natural colorants of plant origin has a

disadvantage of dependence on the supply of raw materials, which are influenced by

agro-climatic conditions – in addition, their chemical profile may vary from batch-to-

batch. Moreover, many of the pigments derived from the contemporary sources are

sensitive to heat, light, and oxygen and some may even change their colour in response to

pH changes as in case of anthocyanins (Mapari, et al., 2005). Many ascomycetous fungi

naturally synthesize and secrete pigments and may thus provide a more reliable source

for natural, "organic" food colorants with improved functionalities (Duran, et al., 2002).

The diversity of fungal pigments is not only found in their chemical structures but also in

the colour range of these pigments that may add new or additional hues to the colour

palette of the existing colorants derived from contemporary sources (Mapari, et al.,

2006).

The use of additives in food plays a significant role to enhance its quality, hence,

acceptability (Corsetti and Settanni, 2007). In this context colors contribute a lot. Of 29

approved food colors, 16 are synthesized from petrochemicals and their long-term intake

is a matter of great concern (Evans and Wang, 1984). Thus, the urge for alternative

combines with the exploration of microorganisms. Several classes of fungi are known to

produce a wide range of water soluble pigments, but low productivity becomes a

significant bottleneck for commercialization (Hejaji and Wijffels, 2004). Since,

basidiomycetous fungi are usually difficult to grow in industrial scale, scientists

emphasized on ascomycetous fungi. The red mold, Monascus purpureus, traditionally

used for preparation of red rice, is a promising source of pigment (Mapari, et al., 2005

and Sandipan Chatterjee, et al., 2009).

The microorganisms such as Monascus, Rhodotorula, Bacillus, Achromobacter,

Yarrowia and Phaffia produce a large number of pigments. Commonly found microbial

pigments are carotenoids and astaxanthin. Carotenoids are yellow, orange and red

pigments, which are widely distributed in nature. They are utilized as food or feed

supplements and as antioxidants in pharmaceutical formulations (Miura, et al., 1998).

Several microorganisms have been shown to produce astaxanthin. They include

Agrobacterium aurantiacum (Misawa, et al., 1995), Phaffia rhodozyma and

Haematococcus pluvalis (Johnson and An, 1991). Among them Phaffia rhodozyma is a

potential candidate for commercial production due to its high astaxanthin content

(Andrews, et al., 1972). Astaxanthin added to poultry feed can improve the colour of

both egg yolks and flesh (Johnson, et al., 1980). Astaxanthin possesses an unusual

antioxidant activity, which has caused a surge in the nutraceutical market for the

encapsulated product.

Astaxanthin has potent antioxidant activity and may have a role in decaying or

preventing degenerative diseases in humans and animals (Schroeder and Johnson, 1993).

Research on the health benefits of astaxanthin is very recent and has mostly been

performed in vitro or at the pre-clinical level with humans (Higuera-ciapara, et al., 2006).

The medical literature suggests that β-carotene may exhibit anticancer activities and aid

in reducing the incidence of cardiovascular diseases (Sies and Krinsi, 1995). Further,

health benefits such as cardiovascular disease prevention, immune system boosting,

bioactivity against Helicobacter pylori, and cataract prevention, have been associated

with astaxanthin consumption (Ciapara, et al., 2006).

Wong and Koehler, 1981 reported that the species of Monascus produces several

natural pigments, including primary colour, red and the secondary colour, orange during

the solid state fermentation. These colours are widely used in Asia as food colourants.

Monascus are traditionally used in oriental countries, originally in China and Thailand, to

prepare fermented rice with strong red colour, which finds several applications ranging

from conferring colour to products such as wine, cheese and meat, to medicinal uses and

as a meat preservative.

List of some of the pigment producing microbes

Organisms Pigment Colour Reference

Janthinobacterium

lividum

Violacein Purple Matz, et al., 2004

Xanthomonas oryzae pv.

Oryzae

Xanthomonadin Yellow Rajagopal, et al., 1997

Staphylococcus aureus Zeaxanthin Yellow Hammond and White,

1970

Pseudomonas aeruginosa Pyocyanin Blue-

green

Baron and Rowe, 1981

Phaffia rhodozyma Astaxanthin Red Florencio, et al., 1998

Haematococcus pluvialis Astaxanthin Red Kobayashi, et al.,2001

Serratia rubidaea Prodigiosin like

pigment

Red Moss, 2002

Vibrio gazogenes Prodigiosin like

pigment

Red Moss, 2002

Alteromonas rubra Prodigiosin like

pigment

Red Moss, 2002

Rugamonas rubra Prodigiosin like

pigment

Red Gerber, 1975

Streptoverticillium

rubrireticuli

Prodigiosin like

pigment

Red Gerber, 1975

Bradyrhizobium Canthaxanthin Orange Lorquin, et al., 1997

Corynebacterium

insidiosum

Indigoidine Blue Starr, et al., 1966

Micrococcus roseus Canthaxanthin Orange-

pink

Cooney, et al., 1966

Dunaliella salina β-carotene Orange Jacobson and Wasileski,

1994

A maroon dye was extracted from the rhizome of Arnebia nobilis (Indrayan, et

al., 2004). Phycoerythrin pigment was isolated from the cyanobacterium, Nostoc

muscorum (Ranjitha and Kaushik, 2005). The nutritional requirements for pyoverdine

pigments from Pseudomonas aeruginosa was reported by Barbhaiya and Rao (1985b).

The pigment Xanthomonadin produced by the genus Xanthomonas has a role against

photo damage (Rajagopal, et al., 1997).

2.4. Pseudomonads

Pseudomonads are ubiquitous bacteria in nature. They possess variable metabolic

abilities that enable them to utilize a wide range of organic compounds, and occupy an

important ecological position in the carbon cycle. An essential pre - requisite for a

detailed investigation of the roles and evolution of Pseudomonads is an accurate system

of classification and identification. However, the classification of Pseudomonas strains is

not fully established due to the lack of an accurate taxonomic system (Satoshi

Yamamoto, et al., 2000).

Bacteria of the genus Pseudomonas are characterized as straight or slightly bent

Gram-negative rods with one or more polar flagellae, not forming spores. Their

metabolism is strictly aerobic and chemo-organotrophic (Palleroni, 1984; Staley, 1984;

Woese, 1992 and Palleroni, 1992). On the basis of rRNA / DNA-homology studies the

genus Pseudomonas has been classified and divided into five groups. However, recent

polyphasic and genomic taxonomic studies indicated that Pseudomonas is a multigeneric

entity and that the five rRNA groups are distantly related to each other. The most of the

rRNA groups are now assigned to new genera such as Comamonas, Hydrogenophaga,

Burkholderia, Ralstonia (Kersters, et al., 1996).

Therefore the genus Pseudomonas sensu stricto is now reserved to the RNA

homology group I of Palleroni, 1984 which presently contains more than 100 fluorescent

and non-fluorescent species, some of them was described only during the last four years

(Doudoroff, 1973; Pecknold and Grogan, 1973 and Palleroni, 1993). The nomenclature

“fluorescent” and “non-fluorescent” which is followed in Table 3.2 is based on the

observation that some of these bacteria, the fluorescent Pseudomonads, produce yellow-

green fluorescent water-soluble pigments (Palleroni, 1984), called pyoverdines

(occasionally also named pseudobactins) (Elliot, 1958).

Some Main Species and Newly Assigned Species Relevant for Pseudomonas Sensu

Stricto (RNA Homology Group I) (Palleroni, 1993 and Meyer, et al., 1997)

Pseudomonas Species

Fluorescent P. aeruginosa

P. putida

P. syringae

P. cichorii

P. agarici

P. monteilii

P. jessenii

P. mandelii

P. libanensis

P. orientalis

P. migulae

P. brassicacearum

P. fluorescens

P. chlororaphis

P. tolaasii

P. viridiflava

P. asplenii

P. rhodesiae

P. veronii

P. mosselii

P. cedrella

P. gessardii

P. thivervalensis

Non-fluorescent P. fragi

P. mendocina

P. pseudoalcaligenes

P. corrugate

P. stutzeri

P. alcaligenes

P. graminis

P. plecoglossicida

Pseudomonas fluorescens has multiple flagella. It has an extremely versatile

metabolism, and can be found in the soil and in water. It is an obligate aerobe but certain

strains are capable of using nitrate instead of oxygen as a final electron acceptor during

cellular respiration. Optimal temperatures for growth of P. fluorescens are 25-30C. It

tests positive for the oxidase test. The word Pseudomonas means 'false unit', being

derived from the Greek words pseudo (Greek: ψευδο 'false') and monas (Latin: monas, fr.

Greek: μονάς/μονάδα 'a single unit'). The word was used early in the history of

microbiology to refer to germs. The name 'fluorescens' is because secretes a soluble

fluorescent pigment called pyoverdine or pyoverdin (formerly called fluorescein), which

is a type of siderophore.

P. fluorescens is also a nonsaccharolytic bacterium. Heat stable lipases and

proteases are produced by Pseudomonas fluorescens and other similar Pseudomonads.

The lipases produced by Pseudomonas sp have become an integral part of the modern

food industry and are used in the preparation of a variety of products including fruit

juices, baked food, vegetable fermentation and dairy enrichment. Lipases have got wide

application in pesticides, detergents, cosmetics and perfume industry. They are also used

in leather industry for processing hides and skins (bating) and for treatment of activated

sludge and other aerobic waste products where they remove the thin layer of the fats and

facilitate oxygen transport. Lipases are of wide spread occurrence throughout the earth's

flora and fauna. They are found abundantly in microbial flora comprising bacteria, fungi

and yeast (Rekha and Ahmed John, 2010).

2.4.1. Importance of Pseudomonads

The strong interest in Pseudomonads is based on their different properties.

Included in this genus are human or phyto-pathogenic and plant growth promoting

bacteria. Other Pseudomonads influence the growth of plants. Rhizobacteria belonging to

the P. fluorescens and P. putida species are able to promote plant growth (O'Sullivan and

O' Gara, 1992; Jacques, et al., 1993 and Yoshikawa, et al., 1993). It has been shown that

these bacteria rapidly colonize plant roots and cause at the plant level statistically

significant yield increases. The presence of Pseudomonas leads to the suppression of

plant deleterious microorganisms both by the production of antibiotically active

substances (Budzikiewicz, 1993) and by the excretion of effective siderophores, the

pyoverdines, with high iron binding constants that cannot be utilized by pathogenic fungi

or bacteria (Kloepper, et al., 1980). Another mechanism is the production of organic

acids like succinic and lactic acid that increase plant growth directly (Yoshikawa, et al.,

1993).

Kloepper and Schroth, 1981 reported that the genus Pseudomonas is one of the

most diverse Gram negative bacterial genera (isolated from sources ranging from plants

to soils and water) which are straight or slightly curved rods, motile by means of polar

flagella. Pseudomonas is characterized by their ability to grow in simple media at the

expense of a great variety of simple organic compounds, without needing organic growth

factors. King’s B media is an optimal for most species of Pseudomonas isolation,

developed the first selective media (King’s A and King’s B) for the isolation of

fluorescent Pseudomonads (King, et al., 1954). In recent years, there has been much

success in obtaining biological control of plant pathogens using bacterization techniques.

Bacteria used as inoculants are mostly Pseudomonas fluorescens and

Pseudomonas putida types obtained from soils and plant surfaces. Some of these bacteria

produce metabolites, which chelate the environmental iron thus making it unavailable to

pathogens. To date a number of these diseases suppressive antibiotic compounds have

been characterized chemically and include N-containing heterocycles such as phenazines,

pyrrole type antibiotics, pyo-compounds and indole derivatives. A small number of

antibiotics like compounds that do not contain nitrogen have also been isolate from

fluorescent Pseudomonads, one of these metabolites 2, 4- diacetylphloroglucinol

(DAPG), is a major factor control of a range of plant pathogens. The antibiotic DAPG is

produced by Pseudomonads of worldwide origin, and its biosynthetic locus is conserved

in Pseudomonads obtained from diverse geographic locations. Bacteria that produce

DAPG play a key role in agricultural environments, and their potential for use in

sustainable agriculture is promising (Prasanna Reddy and Rao, 2009).

2.5. Pyoverdine

2.5.1. Occurrence and Structure

Pyoverdines are chromopeptides that consist of three structural parts, a quinoline

chromophore, a peptide chain and a side chain. Pyoverdines are yellow - green, water

soluble, chromopeptides, fluorescent pigment (Meyer, 2000) and the typical siderophore

of the fluorescent members of the bacterial genus Pseudomonas sp in the rRNA

homology group I of the family pseudomonadaceae (Insa Barelmann,et al., 2002). They

consist of three distinct structural parts, viz. a dihydroxyquinoline chromophore

responsible for their fluorescence, a peptide chain comprising 6 to 12 amino acids bound

to its carboxyl group, and a small dicarboxylic acid (or its monoamide) connected

amidically to the NH2- group (Diana Uria Fernandez, et al., 2003).

The chromophore 5 – amino -2, 3- dihydro- 8, 9- dihydroxy- 1 H- pyrimido - [1,2-

a] quinoline -1- carboxylic acid is identical for all pyoverdines and its catechol unit is one

of the binding sites for iron (III) (Regine Fuchs, et al.,2001). Several pyoverdine

isoforms, which structurally differ one from another by the nature of the dicarboxylic

acid side chains attached to the chromophore (Suc, Mal or their amides, Glu or Kgl),

varies from one strain to another.

Usually several pyoverdins co-occur having the same peptide chain, but differing

in the nature of the dicarboxylic acid. The peptide chains have a twofold function. They

provide two of the ligand sites for Fe3+

, and they are responsible for the recognition of

their Fe3+

complexes at the surface of the producing cell (Budzikiewicz, 1997a). The

members of the fluorescent Pseudomonas group are the best-studied plant growth-

promoting rhizobacteria. Some Pseudomonas species have been used as seed inoculants

on crops to promote plant growth (Kloepper, et al., 1980; Schroch and Hancock, 1982;

Leong, 1986 and Cook and Leong, 1987). Enhanced plant growth caused by these strains

is often accompanied by reductions in populations of deleterious microorganisms, the so

called minor pathogens (Burr and Caesor, 1984; Leong, 1986; Schippers, et al., 1987;

Kloepper, et al., 1980; Loper and Buyer, 1991 and Soeng, et al., 1991). It has been

suggested that Pseudomonads exert their beneficial effects in part by producing yellow-

green fluorescent siderophores, called pyoverdines (or pyoverdins or pseudobactins),

under iron-deficient conditions (Kloepper, et al., 1980; Schippers, et al.,1987 and Seong,

et al.,1991). Pyoverdines are thought to chelate iron efficiently, making it less available

to phytopathogens and thus promoting plant growth.

According to the structural features of the peptide chain, pyoverdines can be

divided into four groups (Regine Fuchs, et al., 2001)

Group 1: The most common structural form of a pyoverdine has a linear peptide

chain and cyclo-Nhydroxy-Orn as the C-terminal amino acid. A tetrahydro-

pyrimidine ring can be observed in some pyoverdines. It results from

condensation of Dab with the preceding amino acid of the chain.

Group 2: The second largest group is characterized by a C-terminal cyclic part

consisting of three to four amino acids, formed by an amide bond between the

carboxyl group of the C-terminal amino acid and the e-amino group of an in-chain

Lys.

Group 3: A small number of pyoverdines has peptide chains with a C-terminal

cyclodepsipeptidic substructure formed by an ester bond between the carboxyl

group of the C-terminal amino acid and an in-chain Ser or Thr.

Group 4: Several pyoverdines have a C-terminal free carboxyl group. Actually,

some or all of them may be hydrolysis products of the rather labile cyclic esters

(Group 3) which are readily hydrolysed at pH values of nine or higher.

2.5.2. Extraction of Pyoverdine

For pigment analysis, cells were grown on King’s B agar and Nutrient agar, and

testing for pigment production and spectral characteristics was performed by extraction

with methanol, according to Hildebrand, et al., 1994, using a visible – UV Kontron

Uvikon 860 spectrophotometer (Alvaro Peix, et al., 2005).

The culture supernatant was concentrated under vacuum. Then, FeC13.6H20 (1

g/liter of original medium) was added, and it was extracted twice with chloroform-phenol

(ml/g); to the pooled organic phase was added about three volumes of ether, and that

solution was extracted several times with small volumes of water. After repeated rinsing

with chloroform to remove the phenol, the pooled aqueous phase was evaporated to

dryness under vacuum; this yielded the crude extract. In some cases, the concentrated

supernatant was first saturated with sodium chloride or ammonium sulfate; the resulting

crude extract was then dissolved in water and the whole procedure was repeated before

further purification. Crude extract, 2-10 g, was dissolved in 0.05 M pyridine-acetate, pH

5, applied to a Carboxymethylethyl (CM)-Sephadex column (1.5 x 30 cm) and eluted

with the same buffer. Three fractions were collected: the first and second are brown and

are labeled A and B, respectively, A being dominant. The third fraction appeared as a

red-orange band that remained at the top of the column; it was eluted with a gradient of

increasing pyridine-acetate concentration (0.05 to 2 M, pH 5) which separates ferribactin

from the rest of the crude extract.

Fraction A, 0.1-0.2 g, was applied to a 50-cm CM-Sephadex column and eluted

with 0.05 M pyridine-acetate buffer. It was resolved into five fractions, numbered I

through V. All are brown except III, which is purple. Fraction IV, the main component,

was further fractionated by chromatography on Sulfopropyl (SP)-Sephadex with 5% (v/v)

acetic acid brought to pH 3.1 with pyridine (0.2%, v/v). This yielded brown ferric

pyoverdine and a minor yellow compound we call ferric pyoverdine A whose presence is

erratic and is likely to represent a modified pyoverdine. Fraction V was not

chromatographically distinguishable from fraction B.

The ferribactin fraction was purified by passage through a CM- cellulose column

in 0.05 M pyridine-acetate, pH 5; this was repeated as necessary. The criterion for purity

was that the optical spectrum showed a single peak in the visible region (460 nm); for the

other compounds, the criterion was that they move as a single spot on thin layer sheets

(Stephen B. Philson and Miguel Llinas, 1982).

2.5.3. Purification and Characterization of Pyoverdine

Bacteria were grown for 40 h at 25C in standard Succinate medium, the pH

being maintained in the range of 7.0 to 7.3 by periodic addition of HCl. At the time of

harvesting, batches were pooled to a volume of 20 1itre; FeCl, (200 mg Fe3+

1-l) was then

added to the culture, which was centrifuged, the pelleted cells being discarded. The

supernatant was concentrated to approximately 1 litre under reduced pressure, saturated

with NaCl, and extracted with 0.5 vol. CHCl3 / phenol (1:1, v/w). The aqueous phase was

discarded, and the organic phase was treated with 2 vol. diethyl ether and 100 ml distilled

water (Meyer and Abdallah, 1978).

The aqueous phase, containing the pigment-iron complex, was re-extracted three

times with ether in order to remove phenol completely, and then evaporated under

reduced pressure. This solution (20 ml) was applied to a column of CM - Sephadex C-25

(2.5 x 90 cm) eluted with 0.1 M pyridine/acetic acid buffer (pH 6.5). A front-running

minor fraction, generally representing less than 10 % of the total, was shown by

electrophoresis to contain a mixture of pigmented degradation products. The major

fraction consisted of the native Fe (III)-pigment complex. Repetition of the CM-

Sephadex C-25 step showed this fraction to be 99% pure, in agreement with the results of

electrophoretic analysis. The yield (after lyophilization) was about 130 mg per litre of the

initial culture medium (Meyer and Abdallah, 1978).

All purification procedures were carried out at room temperature. The cell-free

supernatant was buffered with 1 M HEPES (N-2-hydroxyethylpiperazine – N 9- 2 -

ethanesulfonic acid) buffer (pH 7.0) and then applied to a Chelating Sepharose Fast Flow

column (1.5 by 25 cm) that had been presaturated with CuSO4. The column was

equilibrated with 20 mM HEPES buffer (pH 7.0) containing 100 mM NaCl at a flow rate

of 100 ml/h. The column was then washed with 200 ml of 20 mM HEPES buffer and

eluted with 20 mM acetate buffer (pH 5.0) containing 100 mM NaCl. The A400 of each 10

ml fraction collected was determined.

The pyoverdine-containing fractions (peaks FI, FII, and FIII) were pooled

separately and lyophilized. The dried material from each of the fractions was dissolved in

1 ml of distilled water containing 10 mM EDTA and then applied to a Sephadex G15

column (1.5 by 100 cm) that had been pre-equilibrated with deionized water. Elution was

carried out with distilled water at a flow rate of 20 ml/h. Fractions (3 ml) were collected,

and the A400 of each fraction was determined. In addition, the fluorescence of each

fraction was determined at emission and excitation wavelengths of 460 and 400 nm,

respectively.

The pyoverdine-containing fractions were pooled, lyophilized, and stored at 48C

until they were needed. On the basis of the fraction fluorescence profiles after gel

filtration, peaks FI, FII, and FIII each yielded one major fluorescent compound; since

pyoverdines are the only fluorescent siderophores produced by P. fluorescens 2-79, the

three compounds were designated Pf-A, Pf-B, and Pf-C, respectively. The concentrations

of pyoverdines Pf-A, Pf-B, and Pf-C were estimated by using an extinction coefficient of

20,000 M-1

cm-1

(Rong Xioa and Kisaalita, 1995).

Pyoverdine absorption spectra were determined with a Beckman model DU 650

spectrophotometer. The effects of different pH values on the spectra of iron-free

pyoverdines Pf-A, Pf-B, and Pf-C were determined as follows: pyoverdines were

dissolved in 0.1 M citric acid phosphate buffers (pH 3.0, 5.0, 7.0, and 8.0) and 0.1 M

Tris-HCl buffer (pH 9.0), and then the absorption spectra from 300 to 500 nm were

determined and compared. To study the interaction of Cu2+

with pyoverdine Pf-B, 10.0

mM CuSO4 or 10.0 mM FeCl3 was added to 100 mM acetate buffer (pH 5.0) containing

3.0 mM pyoverdine Pf-B. The reaction mixture was kept at room temperature for 30 min,

and then the absorption spectra between 340 and 500 nm for free pyoverdine Pf-B, Cu2+

pyoverdine Pf-B complexes, and Fe3+ pyoverdine. Pf-B complexes were determined and

compared (Rong Xioa and Kisaalita, 1995).

Pyoverdin was purified by a modification of the methods of Meyer and Abdallah,

1978 and Stephen B. Philson and Llinas, 1982 for pyoverdin siderophores. Strain B301D

was grown from an initial concentration of 5 x 106 CFU/ml in 1-liter flasks containing

250 ml of deferrated NM liquid medium. Cells were grown at 25°C with rotary shaking

(250 rpm); after 24 h, 4 ml of a FeCl3 stock (1 M) was added per liter of medium. After

the mixture was stirred for 20 min, cells and precipitate were removed by centrifugation

at 10,000 X g for 20 min. The culture supernatant was concentrated 50-fold by flash

evaporation at 55°C, and then contaminating proteins were precipitated by saturation

with NaCl. After removal of the precipitate by centrifugation, the concentrated culture

supernatant was extracted with an equal volume of chloroform- phenol (1:1[v/w]) (Cody

and Gross, 1987).

The organic phase was mixed with 3 volumes of diethyl ether, and the pigment

then was partitioned into deionized water (20 ml) until color could no longer be

extracted from the organic phase. The resulting aqueous extract was washed three times

with diethyl ether to remove phenol and reduced to dryness by lyophilization. The crude

siderophore extract, 50 to 150 mg, was further purified through a carboxymethyl (CM)-

Sephadex (Pharmacia, Piscataway, N.J) cation-exchange column (1.5 by 90 cm)

equilibrated with 50 mM pyridine-acetate buffer, pH 5.0. The column was eluted with the

same buffer. Absorbance was monitored at 405 nm and all peaks were collected. The

predominant reddish-brown peak was non-fluorescent and consisted of ferric

pyoverdinpss. The ferric pyoverdinpss was lyophilized, suspended in pyridine-acetate

buffer (1 ml), and again passed through the CM- Sephadex column to increase its

homogeneity.

The purity of the pyoverdine was determined by thin-layer chromatography, using

Silica Gel G (Merck and Co., Inc., Rahway, N.J.) Uniplates (Analtech, Newark, Del.)

developed with a 70:30 (v/v) ethanol-water solvent system. A homogeneous preparation

of pyoverdin was noted by the presence of only one sharp reddish-brown spot; no

fluorescent spots were observed when plates were illuminated with UV light (366 nm)

(Cody and Gross, 1987).

The siderochromes were monitored during various procedures by thin layer

chromatography, using silica gel-impregnated glass fiber sheets (ITLC SA, Gelman) and

70:30 (v/v) ethanol-water as solvent. The colored ferric compounds were observed

directly. Since all the iron-free pigments but deferriferribactin are fluorescent, very small

quantities could be seen under long wavelength UV irradiation, after spraying with an

aqueous EDTA solution (Stephen B. Philson and Miguel Llinas, 1982).

2.5.4. Microbial Production of Pyoverdine

The synthesis under certain growth conditions of yellow-green, fluorescent,

water-soluble pigments is a characteristic property of some Pseudomonas sp. (Stanier, et

al., 1966). These species are all members of the same intra-generic genetic homology

group (Palleroni, et al., 1973). They include P. aeruginosa, P. putida, P. fluorescens and

phytopathogens of the P. syringae type (Palleroni and Doudoroff, 1974). Many different

environmental factors affect the synthesis of these pigments, notably the chemical nature

of the organic carbon and energy source (Lepierre, 1895; Sullivan, 1905; Blancheti Rae,

1920; Giral, 1936; Gouda and Chodat, 1963 and Gouda and Greppin, 1965), the degree of

aeration of the culture medium (Elliot, 1958 and Lenhoff, 1963), pH and light (Greppin

and Gouda, 1965) and the cations Mg2+

(Georgia and Poe, 1931), Zn2+

(Baghdiantz, 1952

and Chakrabarty and Roy, 1964a) and Fe3+

(King, et al., 1948; Totter and Moseley, 1953;

Lenhoff, 1963; Palumbo, 1972 and Lluch, et al., 1973). No clear-cut physiological role

has so far been assigned to this class of pigments.

Various structures have been proposed for the pigments including pteridines

(Giral, 1936 and Chakrabarty and Roy, 1964b), flavines (Birkhoffer and Birkhoffer,

1948) and pyrroles (Lenhoff, 1963 and Greppin and Gouda, 1965), but none is supported

by unequivocal chemical evidence. The factors that affect pigment synthesis, the

purification of the pigment and its physicochemical properties were described by Meyer

and Abdallah (1978). Turfreijer (1942) proposed the term ' pyoverdine ' for the yellow-

green, fluorescent, water-soluble pigment of P. fluorescens. He chose this name by

analogy with that of the phenazine pigment, pyocyanine, produced by P. aeruginosa.

This designation is preferable to those of ' bacterial fluorescein' or ' fluorescin' which

have also sometimes been used in the literature (King, et al., 1948 and Lenhoff, 1963).

Other authors (Elliot, 1958 and Hulcher, 1968) have extended the name of

pyoverdine to include all pigments produced by fluorescent Pseudomonads. However,

since preliminary unpublished studies have shown that there are minor structural

differences between the pigments of the different species of fluorescent Pseudomonads, it

seems desirable to designate the pigments of this class by a suffix indicating the species

responsible for their production, e.g., pyoverdine, for the pyoverdine produced by P.

fluorescens (Meyer and Abdallah, 1978).

P. fluorescens 2-79 was grown on a synthetic succinate medium and the pH was

adjusted to 7.0 by adding a 1 N NaOH solution prior to sterilization. The medium was

dispensed into 500 ml Erlenmeyer flasks, each of which contained 100 ml of medium.

The flasks were then inoculated, at a seed rate of 1.0%, with exponential-phase cells

grown in the same medium. The cultures were incubated at 25C and 200 rpm in a New

Brunswick model Innova 4000 shaker-incubator for 48 h. Batches were pooled and

centrifuged at 10,000 X g for 10 min at 4C. The resulting supernatant was membrane

filtered (pore size, 0.25 mm; Amicon) to yield a cell-free solution of crude pyoverdines.

The amount of pyoverdine produced was estimated by determining the A400 of the

supernatant (Rong Xioa and Kisaalita, 1995).

P. fluorescens SS101 was grown on Pseudomonas agar F (Difco) plates or in

liquid King’s medium B (KB) at 25°C (De Bruijn, et al., 2008). P. fluorescens were

grown in a succinate minimal medium (Budzikiewicz, 1997). For the work-up of the

culture and isolation of the ferri-pyoverdins by chromatography on XAD-4 and Biogel P-

2 (Georgias, et al., 1999). From both strains two fractions were obtained which were

further purified by chromatography on CM - Sephadex C-25 with a pyridinium acetate

buffer (pH 5.0, gradient 0.02 to 0.2 m); final purification by RP-HPLC on Nucleosil-100

with 50 mm acetic acid/methanol (gradient 3 to 30% acetic acid). Decomplexation was

achieved by adsorption of the ferri-pyoverdins on a Sep-Pak cartridge and washing with a

6.5% Sodium oxalate solution (pH 4.3). After removing all salt residues with water the

free pyoverdins were eluted with methanol/ water 1:1 (v/v). The solutions were brought

to dryness and the samples were stored at -25C (Insa Barelmann, et al., 2002).

For qualitative and quantitative analysis of the amino acids by total hydrolysis,

determination of their configuration by GC/MS of their TAP derivatives on a chiral

column and dansyl derivatization of the free amino groups (Briskot, et al., 1986 and

Mohn, et al.,1990). Partial hydrolysis was affected with 6 N HCl at 110C for 15 min.

The peptide fragments were separated by chromatography on Bio-Gel P-2 with 0.1 M

acetic acid. Subsequently they were subjected to total hydrolysis, TAP derivatization and

GC-analysis as above. The 5-hydroxy chromophore from 1 and 2 for CD analysis was

obtained as described by Michels, et al., 1991.

Pseudomonas aeruginosa has the ability to produce a variety of water-soluble

pigments, including pyocyanin, pyoverdin, fluorescein, pyomelanin, and pyorubin.

Although the incidence of strains producing the latter two pigments is low, these

pigments can mask the production of pyocyanin. Two pigment-enhancing media, based

on research using different peptone ingredients in the agar was developed by King, et al.,

1954. Pseudomonas P (Tech) Agar or Pigment Production Agar - A incorporates

pancreatic digest of gelatin, which enhances the production of pyocyanin and inhibits the

pigment fluorescein. Pyocyanin production is also stimulated by the incorporation of low

concentrations of magnesium chloride and potassium sulfate. Once the pigment is

produced, it diffuses into the media giving a characteristic green color.

Pseudomonas F (Flo) Agar or Pigment Production Agar - B incorporates a

combination of casein and animal tissues, along with K2HPO4, which promotes the

production of fluorescein. Pyocyanin production is inhibited. Detection of this pigment,

which produces a yellow-green color, may be aided by the use of ultraviolet light (Gildo

Almeida da silva and Erik, 2006). When these media are used in combination with one

another, they are useful in the identification of P. aeruginosa and P. fluorescens

(Lennette, 1985). A bright yellow-green pigment diffused into the agar is indicative of

fluorescein production. The use of an ultraviolet light may aid in the detection of this

pigment. If fluorescein is present, it will fluoresce with a yellow-green colour. No

pigment production of fluorescence is indicative of a negative test (Washington, 1981).

King Agar B enhances the elaboration of fluorescein and inhibits the pyocyanin

formation. Mixed peptone provides the essential nitrogenous nutrients, carbon, sulphur

and trace elements. Glycerol serves as a C-source and Dipotassium hydrogen phosphate

buffers the medium. Magnesium sulfate is necessary for the activation of fluorescein

production (Mac Faddin, 1985). P. aeruginosa build colonies surrounded by a yellow to

greenish-yellow zone due to fluorescein production which fluoresces under UV light. If

pyocyanin is also synthesized, a bright green colour is produced. For the confirmation of

pyocyanin the coloured pigments can be extracted with chloroform. Most pyocyanin-

producing Pseudomonas strain synthesizes also fluorescein and others produce just one

pigment. The temperature can be a determining factor as most fluorescent strains will not

grow at 35°C. Rather, they grow 25-35°C. Atypical pyocyanin-negative, fluorescein-

positive, P. aeruginosa strains can also be differentiated from P. fluorescens and P.

putida (Blazevic, et al., 1973).

2.5.5. Molecular biology and Genetics

Sequencing a cloned PCR fragment spanning the junction of PA2400 and PA2401

in the P. aeruginosa genome showed that a GC base-pair was missing from the genome

sequence at position 2669175 and when this was included PA2400 and PA2401 form a

single reading frame PA2400/1 (pvdJ). Southern blotting was carried out by standard

methods (Sambrook, et al., 2000), using as probes radiolabelled PCR fragments or cloned

restriction fragments corresponding to individual genes, except that pvdN and pvdO were

part of the same PCR fragment. Membranes were washed at 65°C in 0.1% SDS / 0.1 %

SSC prior to autoradiography.

DNA sequences were obtained from the P. aeruginosa genome project

(http://www.pseudomonas.com) and the P. aeruginosa genome database

(http://pseudomonas.bit.uq.edu.au). Sequences were manipulated using Seqed (Devereux,

et al., 1984) and analysed using NLDNA and Codon use as described previously

(Merriman, et al., 1995). Database searches and analysis of the genomes of other

fluorescent Pseudomonads were carried out at the National Center for Biotechnology

Information (http: // www. ncbi.nlm.nih.gov) with BLAST algorithms (Lamont and

Martin, 2003).

For 16S rRNA gene sequencing and comparative analysis, DNA extraction,

amplification and sequencing of 16S rRNA genes were performed as already reported

(Rivas, et al., 2003). Nearly complete 16S rRNA gene sequences for isolates CH01T and

PA01 were obtained (1532 and 1530 nucleotides, respectively). They were compared

with sequences retrieved from GenBank by using the BLAST program (Altschul, et al.,

1990), and aligned by using CLUSTAL X software (Thompson, et al., 1997 and Alvaro

Peix, et al., 2005).

P. fluorescens is a heterogeneous species that can be subdivided by various

taxonomic criteria into several biotypes.The main aim was: (i) to develop a rapid

polymerase chain reaction (PCR) - based species-specific protocol to identify

P. fluorescens and (ii) to distinguish the different biotypes of this species by combining

molecular techniques with traditional biochemical methods (Mauro Scarpellini, et al.,

2004). The genotyping was performed by PCR-RAPD analysis. The purpose of the study

was to apply the RAPD technique for characterization of P. fluorescens and evaluate the

ability of this technique to differentiate between them (Prasanna Reddy and Rao, 2009).

To confirm strains as fluorescent Pseudomonas, 16S–23SrRNA intervening sequence

specific primers ITS1F (AAGTCGTAACAAGGTAG) and ITS2R

(GACCATATATAACCCCAAG) were used to get an amplicon size of 560 bp

(Gunasekaran, et al., 2002).

Chen and Kuo, 1993 developed a simple and rapid method for the preparation of

Gram negative bacterial genomic DNA. For DNA extraction, the selected strains were

cultivated in LB medium (5 g of yeast extract, 5 g of sodium chloride and 10 g of

peptone/L) during 24 h at 28°C and 40°C. Subsequently, the culture was centrifuged for

10 min at 10000 rpm, and DNA was extracted following the methodology described by

Bramwel, et al., 1995. Finally, 100 µl of buffer was hydrated and frozen at -20°C to be

used. The amplification of subunits 16S rRNA of the B. cepacia and P. fluorescens

strains was performed by PCR. For B. cepacia, the following primers were used: 5'TGG

TAG TCC ACG CCC TAAACG3' (forward) and 5'AGG GCC ATGA GGA CTT GAC

G3' (reverse). For P. fluorescens, the following primers were used: 5'CGT GTG

TGAAGAAGG TCT CGG3' (forward) and 5'TTA CGG CGT GGA CTA CCA GG3'

(reverse).

Amplification was performed in sterile PCR tubes (500 µL) at a volume of 50 µL

of buffer 10X, 1U of polymerase Taq, 100 µM of each d NTP (dATP, dCTP, dTTP and

dGTP), 2 µM of each primer, and 1 µL of purified DNA extract. The first PCR cycle

lasted 5 min, at 94°C for DNA denaturation, 6 min for hybridation (53°C), and extension

(72°C). The other 29 cycles were programmed for 1 min of denaturation at 95°C, and 3

min for hybridation and extension. Final extension was done for 10 min at 72°C.

Amplifications were performed in automatic equipment 2400 (Perkin Elmer) (Rychlik,

1995). As negative control, PCR was done using DNA of A. brasilense Sp7 with the

previous primers. The PCR products were analysed by electrophoresis 1/10 of final

volume of agarose gel reaction at 1% in TBE 1X buffer supplemented with 1 µg/mL of

ethyl bromide, according to the procedure described by Sambrook, et al., 1989.

2.5.6. Application

2.5.6.1. Antagonistic activity of Pseudomonas fluorescens

Nature has been a source of medicinal agents for thousands of years. An

impressive number of modern drugs have been isolated from microorganisms, mainly

based on their use in traditional medicine. In the past century, however, an increasing role

has been played by microorganisms in the production of antibiotics and other drugs

(Fenical, 1993 and Ahmed, et al., 2008).

Microorganisms have been the study of importance in recent years because of

production of novel metabolites, which exhibits antimicrobial, antiviral, antitumor as well

as anticoagulant properties. These secondary metabolites serve as model systems in

discovery of new drugs (Marderosian, 1969; Austin, 1989; Mlinski, 1993; Fenical, 1993

and Bernan, et al., 1997). Most of the current antimicrobial drugs are the derivatives of

the earlier generation and microbial resistance against them further intensify the need for

new drug discovery. Acceptable options available are the metabolites of plants or animal

origin, which are biocompatible, biodegradable and non-toxic in nature. These

metabolites are widely studied and are produced by various groups of microorganisms.

Pseudomonas (Elander, et al., 1968; Flood, et al., 1972; Castric, 1975; Chain and

Mellows, 1977 and Gross and De Vay, 1977) and Streptomyces (Shanshoury, et al., 1966;

Tahvonen, 1982; Yuan and Crawford, 1995 and Trejo Estrada, et al., 1998) are the

groups of microorganisms, studied for their secondary metabolites (Madhava Charyulu,

et al., 2009).

Fungal diseases in agriculture crops pose a great challenge. The currently used

fungicides are either less effective or of great environmental concern. Hence, it is

necessary to develop environmentally safer and potent fungicides of natural origin.

Microbial metabolites have been expected to minimize the deleterious side effects caused

by synthetic fungicides. In this regard, actinomycetes deserve to be studied, as they are

known to produce potent antibiotics. Compared to terrestrial species, marine

actinomycetes are important sources of novel antibiotics (Okami, 1986 and Newman, et

al., 1989). The actinomycetes found in the marine and coastal ecosystems may be viewed

as a rich gene pool possibly containing isolates capable of producing useful metabolites.

However, only few attempts have been made out to isolate marine actinomycetes

especially for their antifungal activity". In the present study, the isolated actinomycetes

from marine samples have been tested against four fungal pathogens that cause

economically important diseases in crops, like rice and sugarcane, to identify potent

antagonistic strains (Kathiresan, et al., 2005).

Strains of Pseudomonas fluorescence showed known biological control activity

against certain soil-borne phytopathogenic fungi and has the potential to produce known

secondary metabolites such as siderophore, HCN and protease that showed antagonistic

activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthoranicotianae

var. parasitica, Pythium sp. and Fusarium sp. Siderophore production in Pseudomonas

strains inhibited the growth of Staphylococcus sp, Escherichia coli and Aeromonas

hydrophila to 96.7% (Ahmadzadeh, et al.,2006).

The genus Pseudomonas has been heterogenous since Migula first named it in

1984 (Migula, 1984). He designated and described the species associated with the genus

in 1985 (Migula, 1985). Pseudomonas is gram-negative, strictly aerobic, polarly

flagellated rods. They are aggressive colonizers of the rhizosphere of various crop plants,

and have a broad spectrum antagonistic activity plant pathogens, such as antibiosis (the

production of inhibitory compounds) (Cartwright, et al., 1995 and Rosales, et al., 1995),

siderophores production (iron-sequestering compounds) and nutrition or site competition

(Winkelmann and Drechsel, 1997).

Some species of Pseudomonas can also produce levels of HCN that are toxic to

certain pathogenic fungi (David and O’Gara, 1994). These characteristics make

Pseudomonas species good candidates for used as seed inoculant and root dips for

biological control of soil-borne plant pathogen against different plant pathogenic fungi

viz. Alternaria cajani, Curvularia lunata, Fusarium sp., Bipolaris sp. and

Helminthosporium sp. in vitro (Srivastava and Shalini, 2008).

Plant protection is an important area which needs attention since most of the

hazardous inputs added into the agricultural system are in the form of plant protection

chemicals. Pseudomonas fluorescens possess a variety of promising properties which

make it a better biocontrol agent. The objectives of the present study were to isolate

P. fluorescens from soil, to check its antagonistic activity and effect of its secondary

metabolites on three fungal plant pathogens by in vitro techniques. P. fluorescens was

isolated from rhizosphere soil on King’s B medium and its antagonistic effect on three

fungal plant pathogens was studied in vitro. Its antagonistic activity was checked by co-

inoculation with the fungal isolates.

In pour plate method, P. fluorescens on coinoculation with fugal pathogens

decreased their growth rate. Maximum inhibition was observed in Pythium ultimim (80%)

followed by Macrophomina phaseolina (70%) and Pyricularia oryzae (50%). Effect of

the separated secondary metabolites on the fungal growth by broth dilution technique and

antifungal activity by agar well diffusion technique was studied. P. fluorescens produces

a broad-spectrum antifungal compound, which inhibits a variety of plant pathogenic fungi

and inhibits Pythium ultimum more when compared to other plant pathogens in the

present study. Further investigations on the type of antifungal components and in vivo

experiments will make P. fluorescens as one of the most suitable biocontrol agent in

suppressing the phytopathogenic fungi and replace chemical fungicides (Goud and

Muralikrishnan, 2009).

Bacteria - bacterial pathogen interactions are having an increased interest in

antibacterial interactions involved Pseudomonas sp. The genus Pseudomonas actively

solubilizes phosphate in vitro and produces several antibiotics with high specificity

against several microorganisms like Salmonella typhi, Streptococcus mutans, Bacillus

subtilis, Shigella sonnei. The Pseudomonas fluorescens strain seems to be highly

potential in controlling bacterial pathogens of health significance and result in

development of a potential biocide (Rekha and Ahmed John, 2010).

The plant growth promoting rhizobacteria (PGPR) improve plant growth either

directly via production of plant growth regulators such as auxine and cytokinines and by

increasing the plant uptake of some micro and macro elements in the rhizosphere

(Lugtenberg, et al., 1991 and Glick, 1995) or indirectly, through biological control of

pathogens or induction of host defense mechanisms (O’Sullivan and O’Gara, 1992;

Thomashow and Weller, 1996; Van Loon, et al., 1998 and Pieterse, et al., 2000).

Production of antifungal secondary metabolites, such as 2, 4 - diacetylphloroglucinol (2,

4-DAPG), pyoluteorin (PLT), pyrrolnitrin (PRN), phenazines, hydrogen cyanide (HCN),

siderophore and lytic enzymes (protease), is a prominent feature of many biocontrol

fluorescent Pseudomonads (Fenton, et al., 1992; Lemanceau, et al., 1992; Dowling and

O’Gara, 1994 and Thomashow and Weller, 1996).

Fluorescent Pseudomonads that produce the polyketide antibiotic 2, 4-DAPG are

an important group of PGPR that are effective against a broad spectrum of soil-borne

plant pathogenic fungi (Shanahan, et al., 1992; Harrison, et al., 1993; Nowak Thompson,

et al., 1994; Duffy and Defago, 1997 and Sharifi Tehrani, et al., 1998).

A proteobacterium isolated from coastal region produced appreciable secondary

metabolite and partial purification of the obtained secondary metabolite demonstrated

antimicrobial activity against both Gram positive and negative organisms such as

Klebsiella pneumonia, Staphylococcus aureus, Shigella flexneri, Pseudomonas

aureginosa, and Bacillus subtilis including MRSA (methicillin resistant Staphylococcus

aureus). Identification of the isolate using biochemical tests, 16S rDNA sequence

analysis, G+C content and electron microscopy studies revealed that the isolate belonged

to Pseudomonas genera. Extraction, purification, characterization and antimicrobial

activity of secondary metabolite carried out using various standard instrumental

techniques suggested that the active fraction was of 272 m/z with a stable fragment of

244 m/z and also displayed stable free radical activity assessed using EPR analysis. This

stable free radical activity of secondary metabolite mediated its antimicrobial activity

(Madhava Charyulu, et al., 2009).

Antibacterial activity of isolated bacterial strains was checked by cross streak and

agar well diffusion method. Crude extract were prepared from antibacterial metabolite

producing strains using organic solvents. A total of six strains showed antibacterial

activity against Aeromonas punctata, Kokuria marina, Rothia marina, Vibrio cholerae,

Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Staphylococcus

aureus, methicillin resistant Staphylococcus aureus and Proteus vulgaris. Production of

antibacterial metabolites varied within 24 to 72 h, in marine broth 2216. A total of 6

crude ethyl acetate extracts of antibacterial substance were prepared from the

antibacterial metabolite producing strains grown on marine agar 2216 and screened for

their antibacterial activity against test bacterial strains. Out of 6 extracts, 3 extracts

showed activity against marine and clinical isolates (Ahmed, et al., 2008).

Antibacterial metabolite producing strains were identified by 16S rRNA gene

sequence analysis. Active bacterial strains belonged mainly to genera of Pseudomonas,

Bacillus and Bravebacterium. The recovery of strains with antimicrobial activity suggests

that marine environment may represent an ecological niche which harbors a largely

uncharacterized microbial diversity and a yet unexploited potential in the search for new

secondary metabolites (Ahmed, et al., 2008).

The antimicrobial activities of these strains were studied against Salmonella

typhimurium, Staphylococcus aureus, Fusarium culmorum, F. oxysporum and Aspergillus

niger. Despite equal siderophore activities with various Pseudomonas sp. as measured by

the chrome azurol S assay, the study shows how siderophore activity does not correlate

with the antibacterial activity against food pathogens or with the antimould activity

against pathogenic moulds. Furthermore, the results illustrate how siderophores are able

to act both as growth inhibitors and stimulators (Laine, et al., 1996).

2.5.6.2. Pigment in Textile and Paper industry

At present, fabrics are dyed mainly with synthetic pigments. However, natural

pigments are still valuable because of their natural colour tones. Janthinobacterium

lividum was isolated from wet silk thread whose color became bluish-purple. This

bacterium produced large amounts of bluish-purple pigment on some media containing

amino acids, such as Wakimoto medium. The pigment was extracted with methanol and

was identified as a mixture of violacein and deoxyviolacein. This pigment could be used

to dye not only natural fibers like silk, cotton and wool, but also synthetic synthetic fibers

like nylon and vinylon, and generally gave a good color tone. The shade depended on the

material. Silk, cotton and wool showed a bluish-purple color, nylon a dark blue color, and

acetate a purple color (Shirata, et al., 2000).

Dyeing could be performed by a simple procedure consisting of either dipping in

the pigment extract or boiling with the bacterial cells. By changing the dipping time and

the temperature of the dye bath, shades ranging from light purple to deep bluish-purple

could be selected. The color fastness of the dyed material was about the same as that

materials dyed with vegetable dyes, but the color faded easily when the material was

exposed to sunlight. However, since the pigment can be mass-produced by culturing, if

these shortcomings could be overcome, the dye may become promising. The pigment

displayed an antimicrobial activity against phytopathogenic fungi like Rosellinia necatrix

which causes white root rot of mulberry. It could also be used as a bio-fungicide (Shirata,

et al., 2000).

2.5.6.3. Pigment in food industry

Use of microbial pigments in processed food is an area of promise with large

economic potential. However, microbial pigments offer challenges due to high cost,

lower stability and variation in shades due to changes in pH. At present, none of the

microbial pigments can replace synthetic pigment. The microorganisms such as

Monascus, Rhodotorula, Bacillus, Achromobacter, Yarrowia and Phaffia produce a large

number of pigments. An ideal pigment-producing microorganism should be capable of

using a wide range of C and N sources, have tolerance to pH, temperature and minerals,

and give reasonable colour yield.

Non-toxic and nonpathogenic nature of pigment-producing microorganisms

coupled with easy separation from the cell biomass is stressed. The various advantages of

producing pigments from microorganisms include independence from weather

conditions, easy and fast growth, colours of different shades and growth on cheap

substances. Studies revealed unstable, largely degradable and sensitive to heat, light,

acidity and water activity as characteristics of microbial colour. Improvement in stability,

safety and solubility can certainly make widespread use of microbial pigments in the food

industry (Joshi, et al., 2003).

In the recent years, colouring of food with pigments produced from natural

sources is of worldwide interest and is gaining importance. These pigments are looked

upon for their safe use as a natural food dye in replacement of synthetic ones because of

undesirable toxic effects including mutagenicity and potential carcinogenicity. Though

many natural colors are available, microbial colorants play a significant role as food

coloring agent, because of its flexibility in production and easy down streaming process.

Among the various pigment-producing microorganisms, Monascus is reported to produce

non-toxic pigments, agent it enhances the flavour of the food and acts a food preservative

(Vidyalakshmi, 2009).

Natural raw materials and by products of industry have wide use as culture media

in fermentation processes because of their low cost since the medium components can

represent from 38 to 73% of the total production cost. Rice is one of the major

agricultural products of economic importance. This primary product could serve as the

sustainable raw material for secondary value-added products through fermentation of

Monascus molds. Rice broken can be utilized for the production of these useful

microbial metabolites at an inexpensive manner and can be applied to varying food

products. In case of red pigment production by Monascus sp., the cost of the culture

medium cost is certainly a significant fraction of the total cost, because of the low cost of

the product and the lack of purification. The raw fermentation medium of semisolid

cultures of Monascus sp. in rice, for example, is used directly as the red pigment in foods

(Schmidell, 2001).

Nature is rich in colours (minerals, plants etc.) and pigment producing

microorganisms (fungi, yeasts and bacteria). Microbial pigments such as carotenoids,

melanins, flavins and quinines are used in many food industries (Dufosse, 2006). The

ability to synthesis pigments varies with the organism and its environment. Fluorescent

Pseudomonads are ubiquitous bacteria that are common inhabitants of the water, soil,

plants and animals and they are the most studied group.

Isolation and identification of Pseudomonas aeruginosa from marine environment

for phenazine pigment production. Pigment production results revealed that, the strain

could able to produce two different pigments namely, pyocyanin and pyorubrin.

Maximum biomass was observed at 66 h of incubation, but the pigment production seems

to be continuous throughout the culture period (72 h). Antibacterial activity of the

pigments was evaluated against pathogenic bacteria, maximum growth inhibitory activity

was observed with pyocyanin and pyorubrin at 20 µl concentration (1.7 and 1.3 cm,

respectively) against Citrobacter sp. Hemolytic activity of the pigments inferred that,

hemolysis was observed with both pigments at 15, 20 and 25 µl, concentration and no

hemolysis was found at 5, 10 and 15 µl. The pigments were evaluated for their potential

as food colourants with agar. Pleasant colouration was observed with pyocyanin and

pyorubrin at 25 mg mL -1

concentration. Pigments were used as food colourants and this

test was made to ensure the reliability of the pigments and their colouring ability at

boiling temperature in food materials (Jayalakshmi, 2008).

The controversial topic of synthetic dyes in food has been discussed for many

years. The scrutiny and negative assessment of synthetic food dyes by the modern

consumer have raised a strong interest in natural colouring alternatives. Nature is rich in

colours (minerals, plants, microalgae, etc.), and pigment-producing microorganisms

(fungi, yeasts, bacteria) are quite common. Among the molecules produced by

microorganisms are carotenoids, melanins, flavins, quinones, and more specifically

monascins, violacein or indigo. The success of any pigment produced by fermentation

depends upon its acceptability on the market, regulatory approval, and the size of the

capital investment required bringing the product to market (Dufosse, 2006).

A few years ago, some expressed doubts about the successful commercialization

of fermentation-derived food grade pigments because of the high capital investment

requirements for fermentation facilities and the extensive and lengthy toxicity studies

required by regulatory agencies. Public perception of biotechnology-derived products

also had to be taken into account. Nowadays some fermentative food grade pigments are

on the market: Monascus pigments, astaxanthin from Xanthophyllomyces dendrorhous,

Arpink Red from Penicillium oxalicum, riboflavin from Ashbya gossypii, β-carotene from

Blakeslea trispora. The successful marketing of pigments derived from algae or extracted

from plants, both as a food colour and a nutritional supplement, reflects the presence and

importance of niche markets in which consumers are willing to pay a premium for all

natural ingredients (Dufosse, 2006).

Recent increasing concern regarding the use of edible coloring agents has banned

various synthetic coloring agents, which have a potential of carcinogenicity and

terratogenicity. This circumstance has inevitably increased the demands for safe and

naturally occurring natural (edible) coloring agents, one of which is pigment from the

fungus Monascus purpureus. It has long been known that the microorganisms of the

genus Monascus produce red pigments, which can be used for coloring the foods.

Monascus pigments are a group of fungal secondary metabolites, called azaphilones,

which have similar molecular structures as well as similar chemical properties. The

pigments can easily react with the amino group containing the compounds in the medium

to form water-soluble pigments. Due to the high cost of the currently used technology for

the microbial pigment production on an industrial scale, there is a need for developing

low cost process for the production of the pigments that could replace the synthetic ones.

Utilizing cheaply available agro-industrial residues as substrate through solid-state

fermentation can attain such an objective (Sumathy Babitha, 2009).

2.5.7. Indian Scenario

In general, the scenario on research on microbial pigments at national level is

limited to very few reports over the years. The role of pigment Xanthomonasdin

produced by the genus Xanthomonas against photo damage was reported (Rajagopal, et

al., 1997). Barbhaiya and Rao, (1985a and 1985b) optimized the nutritional requirements

for pyoverdine production by Pseudomonas aeruginosa. Studies on a new generation

biocolours from Monascus sp. and related fungi for use as a colourant to the foods was

reported (Tamilselvan, 2001). Cellulose acetate membrane was used for the purification

and concentration of natural pigments (Chaudhuri, et al., 2004). A maroon dye was

extracted from the rhizome of Arnebia nobilis (Indrayan, et al., 2004). Effect of hexoses

and di-hexoses on the growth, morphology and pigment synthesis in the transformed root

cultures of red beet was studied (Ravishankar, 2004). A novel medium for enhanced cell

growth and production of prodigiosin from Serratia marcescens, isolated from soil was

optimized (Giri, et al., 2004).