ISOLATION, EXTRACTION AND PURIFICATION OF GREEN PIGMENT FROM

ACTINOMYCETES
Komal B Patil, Jincy Joseph, K Anand Solomon Raju, Denise William.
M.Sc. Biomedical genetics, School of Biosciences and Technology, VIT University
Vellore-632014, Tamil Nadu, India.

Abstract:
A strain of Actinomycetes producing green coloured pigment was isolated from the soil sample
collected near vellar estuary Chidambaram district. The study involved in identifying a suitable
production media (kusters broth) for mass multiplication of the organism. The Actinomycete was
isolated from the Vellar estuary of Chidambaram district, Tamil Nadu. Preliminary characterization
of the organism was carried out using certain cultural and biochemical methods. Further the growth
patterns and nutrient requirements of the organism were optimized. In order to enhance the yield of
green pigment production the isolate was subcultured on solid substrate (Rice). The extraction was
carried out using a polar solvent- methanol and the solvent eluent ratio system (4.5:0.5 chloroform:
methanol) for Thin Layer Chromatography was optimised.

Introduction:
Marine microorganisms tend to provide pharmacologically important secondary metabolites which
are unique and novel chemical compounds. Marine microbes especially from the ocean sediments are
continuously explored for drug discovery. Actinomycetes are a group of Gram-positive, high G+C
content, filamentous bacteria. They are excellent elaborators of biotechnological products such as
antibiotics, industrial enzymes and other bioactive compounds (Mordarski et al., 1988). The
representative genera of Actinomycetes include Streptomyces, Actinomyces, Frankia, Micrococcus,
Micromonospora, Thermomonospora and several others. The marine environment is a virtually
untapped source of novel Actinomycete diversity and new metabolites (Kin S Lam., 2006). Secondary
metabolites produced by Actinomycetes possess a wide range of biological activities (Renu solanki et
al., 2008). Hence, soil samples from a relatively unexploited area i.e., the vellar estuary in
Chidambaram district of Tamil Nadu, were taken in an attempt to isolate novel bioactive compounds
that may act against multi-drug resistant pathogens. The isolated Actinomycetes strains showed the
production of an extracellular green pigment. As a part of further evaluation of the biological activity
isolation, purification and chemical screening of the pigment produced, the present work was
undertaken. Synthetic colours have been widely used in the food industry for many years. The use of
synthetic agents have some harmful effects, hence natural pigments are being increasingly

emphasized. Natural pigments have reached commercial potential due to their natural character, safety
and use as additives (Pszczolla, 1998).

Streptomyces sp. strain Wak. A-305 produces ferroverdin in two different culture media (A. Ballio et
al, 1962). Ferroverdin production is known to be dependent upon the presence of iron in the culture
medium (A. Ballio et al, 1962).The reported information on green pigments from Actinomycetes are
very scarce so the objective of our study involved

identifying the pure pigment producing

Actinomycetes and elucidating the function by analysing the structure. Further biological application
of the pigment has to be studied such as antibacterial, antifungal, antiviral, anticancer and
antilarvicidal activity etc as well as on an industrial scale in cosmetics, dyeing industry and in
preparation of colouring agents.

Materials and Methods

Growth conditions
Green pigmenting isolates were identified from soil samples of vellar estuary in Chidambaram
district. These isolates were isolated on Actinomycete Isolation Agar (AIA) [0.4g Sodium caseinate,
0.02g L-asparagine, 0.8g sodium propionate, 0.1g dipotassium phosphate, 0.02g MgSO 4, 0.0002g
FeSO4, 3g Agar, 50% sea water and 50% distilled water-200ml media]. The isolates were then
inoculated in Kusters broth [2g glycerol, 0.06g casein, 0.4g K 2HPO4, 0.4g KNO3, 0.4gNaCl, 0.010g
MgSO4.7H2O, 0.04g FeSO4, 0.002g CaCO3, and Distilled water-200 ml] which showed vigorous
growth upon incubation for a period of 7 days. Then the isolates were sub-cultured on various ISP
media to optimize growth pattern. Media used were ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, and
ISP9.
Biochemical tests
Indole utilization: 1g indole added to 50ml distilled water, Citrate utilization: 1.24g citrate with 0.5g
Agar added to 50 ml distilled water and, Triple sugar ion test: 3.25g TSI with 0.5g Agar added to 50
ml distilled water, Methyl red- vogues proskauer test: 0.75g of MR-VP added to 50 ml distilled water.
All the above were inoculated and incubated. For confirmation of biochemical tests the following
reagents were used: - 0.5ml kovacs reagent (indole utilization), Simmons citrate agar (citrate
utilization), TSI media (triple sugar ion test), MR reagent (methyl red test), and baritts reagent [40%
KOH (1ml) + 5% naphthol in absolute ethanol (3ml) (vogues proskauer test).

Pigment extraction
Solid substrate fermentation
2% of the inoculum from the culture broth was inoculated on 20g of parboiled rice and incubated for
7 days at 28C.
Crude extraction
The fermented substrate (rice-25g) containing the grown actinomycete incubated for a week was
producing a green pigmenting compound was added to 100ml of methanol and incubated overnight
using a magnetic stirrer. The solvent layer was collected and filtered using whatmann paper No 3. The
pigment containing solvent phase (methanol) was concentrated using a Rotavapor.
Thin Layer Chromatography
Silica gel was used as a Stationary phase and solvent system (methanol: chloroform in 4.5:0.5 ratio)
had been used. The sample was loaded on to the TLC plate in the form of spot with the help of a
capillary tube. After the solvent had run, the plate was air dried at room temperature for 20 min; the
spots were detected under UV light. Rf Values are noted down which is calculated by finding sample
front / solvent front.
UV spectroscopy
The UV visible spectroscopy reading for the crude extract sample at scan range wavelength of 200700 nm was taken.

Results:
The isolates were cultured from the soil samples of vellar estuary on AIA media (actinomycetes isolation
agar). The gram positive nature of the colonies was determined using Grams differential staining method.
Further biochemical analysis and cultural characterization was carried out and we identified that the
organism belonged to streptomyces genera. The production media used for the extraction of pigment was
kusters broth. The various cultural growth characteristics of the colonies when plated on ISP media were
noted. Biochemical tests were carried out and results were noted. The TLC chromatogram of the extracted
crude pigment was obtained. . Various solvent systems were used for optimizing solvent system for TLC.
The UV-Vis spectrogram reading of the crude extract had shown various peaks in the Ultraviolet region and
visible region suggesting that there are mixtures of compounds within the sample.

Fig-1

Fig-2

Growth of pigments producing Actinomycetes

Gram Character of green pigment producing

(on AIA media)

Actinomycetes

Fig-3: Growth of actinomycetes in production media

Fig-4 Culture Characteristics of actinomycetes

on different ISP media

Bio Chemical Test

Results

In dole Utilization

Negative

Citrate utilization

Positive

TSI test

Negative

MR Test

Positive

Fig-5: Biochemical character

Fig-6: TLC chromatogram

Solvent system(ratio)

No. of spots

Chloroform : methanol(4:1)

Chloroform : methanol(4.5:0.5)

2.0

ethyl acetate : chloroform (4:1)

1.5

ethyl acetate : chloroform (4.5:0.5)

ethyl acetate : chloroform (3:2)

ethyl acetate : pet-ether(4:1)

-0.5

ethyl acetate : pet-ether(4.5:0.5)

-1.0

ethyl acetate : pet-ether(3:2)

solomon-1

Abs
2.5

1.0
0.5

Fig-7:
TLC solvent system and related data

0.0

200

300

400

500

600

700

Fig-8:
UV-Vis spectroscopy graph

800

nm

Discussion and conclusion:

The marine microorganisms are known to be rich sources of novel compounds. To date, about 1000
natural products have been derived from marine microbes. Interestingly marine Actinomycetes
produce many pharmacologically potential compounds with antibiotic and antitumor properties.
Actinobacteria have been proven as a potential source of bioactive compounds and richest source of
secondary metabolites. They are the most economically and biotechnologically valuable prokaryotes.
However, the research on marine Actinobacteria from Indian peninsula is very scanty. In the present
study we have demonstrated that our isolated Actinomycete produces green pigments .Our sampling
site was the vellar estuary near Chidamabaram district, Tamil nadu. The optimized media, gram
character and preliminary biochemical test results in characterizing the actinobacteria as a strain
belonging to streptomyces group. The crude extract obtained upon the growth of the isolate on solid
substrate was analysed using TLC, this showed distinct spots depicting the different compounds
present in the extract. The UV-Visible spectroscopy of the sample showed different peaks observed in
the wavelength range of 200-700nm. The sample was sent for HPLC and 16s rRNA sequencing,
whose results are awaited. Further studies on purification and characterization of the pure compound
from the strain were analysing the pure fractions of separated compounds for mass spectrometry to
determine the molecular weight structural elucidations of pure compounds by 1H and13C Nuclear
Magnetic Resonance Spectroscopy . Identification of the pigment compound through structure
analysis and to further study the biological applications is ongoing. The results of this study suggested
that the marine Actinomycetes from the unexplored Indian coast could provide compounds of
therapeutic value.

Acknowledgment:
We take the opportunity to thank the management of VIT University for providing us the facilities
and encouragement to carry out this work.

References
A. Ballio, H. Bertholdt, A.Carilli, et al, 1963, Studies on ferroverdin, a green iron containing pigment
produced by a Streptomyces Wak. Species, Royal Society of London, 158, 43-70

Chen-chin yang and John Leong, 1982, Production of Deferriferrioxamines B and E from a
Ferroverdin-Producing Streptomyces Species, Journal of bacteriology, 381-383

Hiroshi Tsujibo, Takashi Sato et. al. (1988) Intracellular Accumulation of Phenazine Antibiotics
Produced by an Alkalophilic Actinomycete. I. Taxonomy, Isolation and Identification of the
Phenazine Antibiotics, Agric. Biol. Chem., 52 (2), 301-306.

J.M. Meyer, M.A. Abdallah, 1978, The Fluorescent Pigment of Pseudomonas fluorescens
:Biosynthesis, Purification and Physicochemical Properties, Journal of General Microbiology, 107,
319-328.

K. Suthindhiran and K. Kannabiran, 2009, Cytotoxic and Antimicrobial Potential of Actinomycete

Species Saccharopolyspora salina VITSDK4 Isolated from the Bay of Bengal Coast of India,
American Journal of Infectious Diseases 5 (2): 90-98

Kelecom, A., 2002. Secondary metabolites from marine microorganisms. An Acad. Bras. Cienc., 74:
151-170. DOI: 10.1590S0001- 37652002000100012

Kin S Lam. (2006) Discovery of novel metabolites from marine actinomycetes. Current Opinion in
Microbiology.

Renu Solanki, Monisha khanna and Rup Lal. 2008, Bioactive compounds from marine actinomycetes.
Indian journal of Microbiology (2008).

V.C. Verma, R.N. Kharwar, A.C. Gange, (2009) Nutr. Nat. Resource.

Zakia Sultana Sathi, Naoki Sugimoto, et.al., 2002, Isolation of a Yellowish Antibiotic Pigment 4hydroxy Nitrobenzene from a Strain of Streptomyces, Pakistan journal of biological sciences 5(2);
201-203.