Fermentative production of extracellular pigment from Streptomyces coelicolor MSIS1

Research Journal of Biotechnology Vol. 8 (4) April (2013) Res. J. Biotech

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Fermentative production of extracellular pigment from Streptomyces coelicolor MSIS1

Mohanasrinivasan V.*, SriramKalyan P., Ipsita Nandi, Subathradevi C., Selvarajan E., Suganthi V. and Jemimah Naine S. School of Bio Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, INDIA

*[email protected]

Abstract In the present research work, the pigment producing

actinomycetes was isolated from a rhizosphere soil of

ornamental plants and identified as Streptomyces

coelicolor MSIS1 (FR856603).The pigment was

produced in shake flask as well as in bioreactor. The

results were evident that there was threefold increase

in the pigment production in bioreactor compared

with shake flask.The extracted pigment was

characterized based on TLC, HPLC and FT-IR. The

HPLC data showed that the compound may be one of

actinorhodinic acid but on the other side FT-IR data

infers that there were no presence of aromatic ring

but prominent aliphatic stretch has been found. Keywords: Soil actinomycetes, antibacterial activity,

pigments, pH indicator, molecular characterization.

Introduction The toxicity problems caused by those of synthetic origin

pigments to the environment have created a mounting

interest towards natural pigments. Among natural

pigments, pigments from microbial sources are potentially

good alternative ones to synthetic pigments. Many artificial

synthetic colorants, which have widely been used in

foodstuff, dyestuff, cosmetic and pharmaceutical

manufacturing processes, comprise various hazardous

effects. To counter the ill effect of synthetic colorants,

there is worldwide interest in process development for the

production of pigments from natural sources1. Natural

pigments can be obtained from two major sources, plants

and microorganisms2,3

. The accessible authorized natural

pigments from plants have numerous drawbacks such as

instability against light, heat or adverse pH, low water

solubility and are often non-availability throughout the

year.

The latter are of great interest owing to the stability of the

pigments produced and the availability of cultivation

technology4-6

. The advantages of pigment production from

microorganisms include easy and fast growth in the cheap

culture medium, independence from weather conditions

and colors of different shades. Hence, microbial pigment

production is now one of the emerging fields of research to

demonstrate its potential for various industrial applications.

The pigment secretion is distinctly observed in actinobacteria group than any other, the biological activity

establishes a consequence, to define the identity between

the pigments formed and the bacteria producing those7.

Streptomyces produces various pigments like yellow,

greyish yellow, bluish grey, whitish grey and many other

pigments. Streptomyces coelicolor was first identified in

1908 by Muller after he found it on a potato and he named

it as Strepthiotrix coelicolor8. Later it was known as

Streptomyces coelicolor. The life cycle has three various

states: vegetative hyphae, aerial hyphae and spores9.In the

present research work an attempt has been made to apply

the pigment extracted from Streptomyces coelicolor to

replace the synthetic dyes used as alkaline pH indicator.

Material and Methods Sample collection and processing: The soil sample was

collected from different gardens of VIT-University using

soil augers and stored to pre-sterilized polythene covers

and sampling containers. Containers were sealed properly

to prevent the unnecessary contamination. Gloves were

used for personal safety and to avoid cross contamination

among the samples. The samples were dried in laminar air

flow for 8-12hrs, then kept in a sterile petri dish and these

air dried samples were treated with 0.1g CaCO3 and

incubated at 28°C for 7 days in atmosphere saturated with

moisture.

Isolation and characterization of pigment producing

actinomycetes: One gram of each soil sample was serially

diluted up to dilutions 10-5

and

spread plated on two

different culture media for Streptomyces on glucose -

asparagine and mannitol soya flour medium, these media

were supplemented with 75µg/ml of amphotericin B to

inhibit the development of filamentous fungi and 10 µg/ml

of polymixin to inhibit gram-negative bacteria. All the

plates were incubated for 7-14 days at 32o

C. The pigment

producing colonies were selected and then transferred to

slant culture at 4°C as well as at 20% (v/v) glycerol stock

at -80°C.The sub culturing was done on GAUSE’S media.

MSIS1 colony was cross streaked and incubated at 28 °C

for 7 days. The colony was further identified based on the

inhibitory potency. The morphological, cultural, physio-

logical and biochemical characterization of the isolate were

carried out as described in ISP10

.

Microscopical characterization: The morphology of

MSIS1 the spore bearing hyphae with entire spore chain

along with substrate and areal mycelium was examined

under light microscope.

Growth pattern studies of MSIS1 on various media:

The strain MSIS1 was inoculated on standard culture

media of ISP 2, ISP 3, ISP 4, ISP6 and ISP7 for the

morphological study and color determination. Each


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medium has different nutrient sources ISP-2 (Yeast extract-

Malt extract, Bennett’s medium), ISP-3 (Oatmeal agar),

ISP-4 (inorganic Starch-Salt agar), ISP-6 (peptone yeast

extract iron agar) and ISP7 (Tyrosine agar).

Biochemical Properties: The strain MSIS1 was studied

for production of soluble pigments in organic and

inorganic media. Carbon source utilization was determined

by growth of MSIS1 on carbon utilization medium

supplemented with 1% carbon source at 28°C and starch

hydrolysis11

.Carbon sources and controls used for test are:

No carbon source (negative control), D-glucose (positive

control), L-arabinose, Sucrose, D-fructose, Xylose, I-

inositol, Raffinose, D-mannitol, Cellulose and Rhamnose12

.

Molecular Characterization: The strain MSIS1 was

subjected for 16s rDNA molecular sequencing. The

genomic DNA of strain MSIS1 was isolated from the pure

culture plate and then ~1.4kb rDNA fragment was

amplified using high –fidelity PCR polymerase. The PCR

product was sequenced bi-directionally using the 16s

forward primer 5'-AGAGTRTGATCMTYGCTWAC-3'

and 16s reverse primer 5’-CGYTAMCTTWTTACGRCT-

3'.

Fermentative production and extraction of pigment in shake flasks and bioreactor Inoculum Preparation: The MSIS1 strain was transferred

to the seed medium in which GAUSE’S Medium is

supplemented with 2g beef extract at pH 7.5. The strain

was inoculated on agar slants and incubated for 2 weeks at

30°C to allow for sporulation. The vegetative inoculum is

prepared by adding two loops of spore into the 30ml seed

medium in 250 ml shake flask. The culture was incubated

at 30°C on a rotary shaker at 200 rpm. The mycelium is

washed and re-suspended into same amount of medium

and stored for inoculation.

Fermentation in Shake Flasks: In shake flask studies

GAUSE’S Medium was supplemented with 20g/l mannitol

and 20g/l soybean flour at pH 7.5-7.8, Cultures were

grown in eight 250ml shake flasks containing 100mL

medium on an orbital shaker at 200 rpm and were

incubated for 7 days at room temperature.

Fermentation in Bioreactor : Production of the pigment

was carried out in an in-house developed bioreactor of 5l

capacity.The medium used was GAUSE’S Medium

supplemented with 20g/l mannitol and 20g/l soybean flour

at pH 7.5-7.8. 3 l of medium was prepared and 10 % (V/V)

mycelium culture was inoculated and incubated for 7 days

at room temperature, with aeration of 3 l/min and agitation

at 300 rpm.

Refinement of pigment from fermented broth: After the

incubation, pH of the fermented broth was adjusted to 12 using 2N NaOH and centrifuged at 4000 rpm for 15

minutes. Supernatant was collected and pH of the

supernatant was again adjusted to 2 using 2N HCl. Re-

centrifugation was done at 4000 rpm for 15 minutes. The

obtained pellet was collected and suspended in methanol.

The pigment was extracted from methanol by using rotary

vacuum evaporator and the amaranth colored pellet

obtained was dried and stored at 5°C.

Characterization of pigment: Pigment was characterized

by analyzing various factors like Solubility, TLC, HPLC,

and FT-IR.

Solubility and coloring of the pigment: The dried

amaranth colored pigment was checked for solubility in

several organic solvents like acetone, dimethyl sulfoxide,

acetonitrile, methanol, chloroform and petroleum ether.

The solubility and color variation of the pigment in water

at various pH values was recorded.

Thin Layer Chromatography (TLC) and High

Performance Liquid Chromatography (HPLC): For

TLC assay silica coated plates and solvent combinations

like benzene-acetic acid (9:1), isopropyl alcohol-water

(9:1) were used. For HPLC the amaranth color pigments

were analyzed on the on a HP 1100 series with auto-

injector (25µl). A kromasil ODS C-18 column, 4.6×250

mm (5µM), was used at 30°C with UWD at 260 and 530

nm. The gradient elution followed was 1ml/min with

solvent A (water containing HAc) and solvent B

(acetonitrile).

Fourier Transform-Infra Red Spectroscopy (FT-IR):

For the FT-IR analysis a pinch of pigment was mixed with

KBR salt and made it to fine pellet form. Using JASCO

Spectrophotometer, the FTIR spectra were recorded by

scanning the samples in the range of frequency 400-4000

cm−1

at the resolution of 4 cm−1

.

Application studies of the extracted pigment Reducing Power Assay: Different concentrations like 10

mg/mL, 50 mg/mL, 100mg/mL of the pigment solution

were prepared in DMSO for reducing power assay. The

colour change was noted further by incubating for 20 mins.

Primary Screening of MSIS1 strain for antimicrobial

activity: The antimicrobial activity test was carried out on

some clinical isolates like Staphylococcus areus, Bacillus subtilis, Klebsiella spp, E. coli collected from the Narayani

Hospital and Research Centre, Vellore. The MSIS1 strain

was lawn cultured on gauses media minimizing number of

colonies to 50-100 colonies per plate. Then 1mL of each

clinical isolates (18-24 h) of nutrient broth cultures was

poured and spread properly. Plates were incubated

overnight and results were recorded.

Replacement of Pigment as a pH indicator: As the

pigment was precipitated in the lower pH conditions, its

activity as indicator was aimed for the alkaline end product in the conventional bio-chemical assays of citrate

utilization test. Different concentrations of 0.25 mg/l,

0.5mg/l and 1.0mg/l of the pigment were substituted for


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bromothymol blue in citrate agar with Klebsiella as test

organism. Normal citrate agar slants (Himedia) were used

as positive control.

Results Isolation and characterization of pigment producing

actinomycetes: The selective isolation process resulted in

isolation of a single blue color pigment producing colony

(Fig.1) which was observed on mannitol soya flour agar

medium after 7 days incubation at 30°C which was

demarcated and named as MSIS1. On GAUSE’S medium

the MSIS1 strain produced white colored powdery

appearance and blue colored halo regions around the

colonies (Fig. 2).

Fig.1: Pigment producing actinomycetes colony on

mannitol soya flour media

Fig. 2: Pure-culture plate of MSIS1 on Day 7

Fig. 3: Gram staining for the MSIS1

Microscopical observation: Gram staining of the strain

MSIS1 was gram positive cylindrical branched filaments

with branched hyphae arranged in clusters. The areal

mycelium and substrate mycelium were observed white on

observation of plate directly under the light microscope

(Fig.3).

Morphological and Cultural Characterization on

Various Media: On various ISP media the strain MSIS1

produced different growth patterns and color

morphologies. The MSIS 1 strain showed good growth on

ISP2 with good sporulation with abundant areal mycelium

in white color and substrate mycelium in off white color.

On the ISP 3 and 6 spore color was white and reverse color

was also white. But on ISP 4, 5 and 7 spore color was

white and reverse color was yellow; this shows there is no

prominent melanin pigments production (Table 1,2, fig.4).

Molecular Characterization: The partial sequencing of

16S rDNA gene of the strain MSIS1 yielded 16S rDNA

nucleotide sequence with 1,369 base pairs. The 16S rDNA

sequence of the strain was deposited in the GenBank

(EMBL, UK) under the accession number FR856603. The

BLAST search of 16S rDNA sequence of the strain showed

highest similarity 95% with Streptomyces spp and 94%

Streptomyces coelicolor MULLER strain. The

phylogenetic tree was constructed with bootstrap values. A

neighbor-joining tree based on 16S rDNA sequences

showed that the isolate occupies a distinct Phylogenetic

position within the radiation including representatives of

the Streptomycetes family. Based on the molecular

taxonomy and phylogeny the strain was identified as novel

Streptomyces spp. and designated as Streptomyces coelicolor MSIS1 strain (Fig.5).

The RNA secondary structure of 16s rRNA gene of

(Streptomyces spp.) showed the free energy of the

predicted structure as -332.7 K cal/mol. The restriction

analysis of the sequence is given in fig.6. The restriction

sites present on the bacterial 16S rDNA showed 50

restriction sites and GC and AT content. The phylogenetic

tree arrangement and the genome sequence of S. coelicolor MSIS1 have revealed much about the many adaptations of

these model actinomycetes in the highly competitive soil

environment. The chromosome has acquired the ability to

replicate in a linear form and appears to have expanded by

lateral acquisition and internal duplication of DNA.

Chromosome expansion has provided a wealth of genes,

allowing the organism a more complex life cycle, adapting

to a wider range of environmental conditions and

exploiting a greater variety of nutrient sources.

The abundance of previously uncharacterized metabolic

enzymes, particularly those likely to be involved in the

production of natural products, is a resource of enormous

potential value. Understanding of such enzymes will

facilitate the genetic engineering of pathways to produce

new compounds with potential therapeutic activity including much essential antimicrobials.

Bioreactor studies and pigment production: In the


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fermentor studies the formation of the blue pigment was

observed from day 3 and the color intensity turned to deep

blue color by day 7 (Fig.7).

Refinement of pigment from the broth: On processing of

the incubated MSIS1 culture broth, the yield of crude

pigment obtained was estimated to be about 5.03 g/l in

shake flasks and around 9g/L in the bioreactor. A threefold

increase in the production of the pigment about 9 g/l was

achieved in present work. The standard bioreactor

conditions like rpm of 300, aeration of 3 l/m, pH range of

7.4-7.8 and 30°C temperature were followed and

supplementation of 20 g/l of mannitol and soya flour were

assumed to be the best physical and nutrient conditions for

threefold increase of production.

Pigment characterization Solubility and coloring of the pigment: The amaranth

pigment was readily soluble in alkaline water solution and

organic solvents such as acetone, dimethyl sulfoxide,

acetonitrile and methanol. In chloroform pigment was

weakly soluble and insoluble in petroleum ether. The pH

value was important factor for the solubility and color

change of the pigment. At pH 1-2 the pigment solubility in

water was poor. It was clear that solubility and pH factors

were directly related for this pigment. In acidic conditions,

pigment was red in color and at pH 7 amaranth colors

whereas at alkaline conditions starting from pH 8 pigment

turned to blue color. Increase in color intensity was

observed with pH raise (Fig.8).

Thin Layer Chromatography (TLC): TLC analysis of

the isolated pigment yielded a single red spot with the

same mobility (Rf 0.28) for both intra and extracellular

pigments, clearly differing from that for authentic

actinorhodin (Rf 0.52).Only traces of actinorhodin were

detected under these growth conditions. The major pigment

may be the actinorhodin derivative compound. In TLC the

mobility of actinorhodinic acid was lower than that of

actinorhodin or g-actinorhodin; it remained almost at the

origin in benzene acetic acid (9:1). The plate 9 with blue

colored demarcation shows band formation (Fig.9).

High Performance Liquid Chromatography (HPLC):

The crude blue pigment was dissolved in acetonitrile and

analyzed by HPLC (HP 1100) at 530 and 260 nm (Fig.10 a

and b). The HPLC data recorded at 530 nm found the

pigment extracted from the Streptomyces coelicolor MSIS1

contained 10 compounds The HPLC data at 260 nm

notified 4 compounds. In common to both the reports

which were analyzed at 260 nm and 530 nm, a common

peak with retention time equivalent to 14.02 when

compared with the standard, it may be actinorhodin related

compound.

Fourier Transformation-Infra Red (FT-IR): The FT-IR

report giving sharp peaks of transmittance at 3111.58cm-1

represents –hydroxyl group (OH), 2978.52 cm-1

represents

–CH (aliphatic) stretch, 2879cm-1

represents aliphatic –CH

stretch, 1680.66 cm

-1 represents C=O. Presence of aromatic

group may be expected around 2978.52 cm-1

(Fig.11).

Applications of the isolated pigment Reducing Power Assay: The reducing power assay for the

extracted pigment gave positive results for all the

concentrations 10mg/ml, 50 mg/ml and 100mg/ml. The

change of color conforms the change of oxidation state of

ferrous, these results explains that the pigment has good

oxidizing nature at minimum concentrations of 10 mg/ml

(Fig.12).

Primary Screening of MSIS1 strain for antimicrobial

activity: In primary screening for the antibiotic activity,

MSIS1 strain showed no activity on the selected strains

Staphylococcus areus, Bacillus subtilis, E.coli, Klebsiella.

The data represents the secondary metabolites produced by

strain MSIS1 has no indefinite antibacterial activity on

selected gram negative and gram positive organisms

(Fig.13).

Replacement of Pigment as a pH indicator: Citrate

utilization capability test using the extracted pigment as a

pH Indicator gave positive result. This is confirmed by

change of color of the pigment from light red to blue

indicating change in pH of the medium as the citrate

utilization results in alkaline end product. The test

organism used is Klebsiella spp. as test organism utilizes

citrate as whole carbon source and produces alkaline

sodium end product (Fig.14).

Discussion

The bacterial strain producing a great amount of

bluepigment was observed during submerged fermentation.

Based on morphological characteristics, cell-wall

chemotype and sequence of 16S rRNA gene, the strain

should belong to the genus Streptomyces; it had 99.4%

homology of 16S rRNA gene sequence with that of

Streptomyces indigocolor. The pigment production by the

strain was affected by carbon and nitrogen sources. The

main components of the pigment mixture (detected by

HPLC and TLC) were tentatively classified as

actinorhodin-related compounds. The pigment was

relatively stable against light and higher temperature but

was sensitive to low pH. The preliminary acute-toxicity

determination showed that the pigment was nontoxic

(LD50 > 15 mg/g).

Similar results were obtained in our research work that the

pigment producing actinomycetes was isolated from a

rhizosphere soil of ornamental plants and identified using

16s rDNA molecular sequencing as Streptomyces coelicolor MSIS1 (FR856603).

The blue pigment from the fermentation of Streptomyces coelicolor MSIS1 with a yield as high as 9g/l is a mixture

of 10 compounds. It is soluble in alkaline water solution


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and a number of organic solvents except for the petroleum

ether. The color of the pigment solution changed with pH

value from red at pH<7 through amaranth at pH7-8 to blue

at pH>8. The results of the antimicrobial activity reveal

that the pigment produced by strain MSIS1 has no

indefinite antibacterial activity on selected gram negative

and gram positive organisms.

On the basis of HPLC data and FT-IR data, it was

identified as novel compound having functional groups

with –CH stretch at 2978.75 cm-1

, 2879.0 cm-1

and C=O at

1680

cm-1

. Based on the above results the extracted

pigment was successfully used as a pH indicator in citrate

utilization test.

Conclusion Over the last few years there has been an increasing

number of works on the pigments from microbial sources.

In future good characteristics of the pigment will open the

doors to work in the area of food processing industry as

additive and can be used to make some colorful beverages

and confectionaries. In a broad sense it will also serve as

ingredient in preparation of bio lipsticks to color blue and

red.

Fig.4: Morphological properties on various ISP media A: ISP2, B:ISP3, C:ISP4, D and E:ISP5,6,F: ISP7


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Fig. 5 : Phylogenetic tree for the strain Streptomyces coelicolor MSIS1

Fig. 6: Predicted secondary structure of 16S rRNA of isolate Streptomyces coelicolor MSIS1 using Genbee software.


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Fig.7: Bioreactor studies of MSIS1 for mass pigment production on A: day 3 and B: day 7

Fig. 8: Dried pigment after methanol extraction, demarcated area shows red color of the pigment in acidic conditions

Fig. 9: TLC of extracted pigment


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Fig. 10a: HPLC report of isolated pigment at 530 nm

Fig.10b: HPLC report of isolated pigment at 260nm


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Fig.11: FT-IR report for the pigment crystals

Fig.12: Reducing power assay of the red pigment isolated


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Fig.13: Antibacterial activity of Strain MSIS1 on various bacteria

A: Bacillus subtilis, B: E.coli, C: Staphylococcus. aureus, D: Klebsiella spp.

Fig.14: Citrate utilization capability test for the extracted pigment as a pH Indicator


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Table 1

Morphological properties of Streptomyces coelicolor MSIS1 on various ISP Media

Table 2

Carbohydrate fermentation by Streptomyces coelicolor MSIS1

++ = rapid and complete fermentation,+ = partial fermentation, - = no fermentation.

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(Received 21st November 2012, accepted 18

th January 2013)

S.N. MEDIUM SPORE COLOR REVERSE COLOR

1 ISP 2 Color less to white Off White

2 ISP 3 White White

3 ISP 4 White Yellow


5 ISP 6 White White


S.N. TEST SUGAR FERMENTATION RESULT for

MSIS1

1 No carbon source (negative control) -

2 D-glucose (positive control) ++

3 L-arabinose ++

4 Sucrose ++

5 D-fructose +

6 Xylose +

7 I-inositol +

8 Raffinose, +

9 D-mannitol +

10 Cellulose +

11 Rhamnose +

http://www.ncbi.nlm.nih.gov/pubmed?term=Capstick%20DS%5BAuthor%5D&cauthor=true&cauthor_uid=22296345

http://www.ncbi.nlm.nih.gov/pubmed?term=Di%20Berardo%20C%5BAuthor%5D&cauthor=true&cauthor_uid=22296345

http://www.ncbi.nlm.nih.gov/pubmed?term=Findlay%20KC%5BAuthor%5D&cauthor=true&cauthor_uid=22296345

http://www.ncbi.nlm.nih.gov/pubmed?term=Hesketh%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22296345

http://www.ncbi.nlm.nih.gov/pubmed?term=Hong%20HJ%5BAuthor%5D&cauthor=true&cauthor_uid=22296345

http://www.ncbi.nlm.nih.gov/pubmed?term=Elliot%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=22296345

http://ijs.sgmjournals.org/

Fermentative production of extracellular pigment from Streptomyces coelicolor MSIS1

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