Int J Med Res.

2011; 1(3):145-150
IJMBR

ISSN: 0976-8971

International Journal of Medicobiological

Research
(An International peer review journal)

Journal homepage: ijmedres.com

Research article

Optimized Production of Prodigiosin from Serratia Marcescens SU-10 Grown as

Batch Culture and Evaluation of Bioactivity of Produced Prodigiosin
Antony V. Samrot,1

Chandana. K1, Senthilkumar. P2, and Narendrakumar. G1

Department of Biotechnology, Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu - 600 119. India.
Department of Chemical Engineering, Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu - 600 119.
India.

Corresponding author: antonysamrot@gmail.com

Article information

ABSTRACT

Keywords:
Prodigiosin,
Serratia marcescens,
SU-10,
anticancer activity

Serratia marcescens belongs to Gram-negative Enterobacteriaceae family and produce a red to dark
pink pigment prodigiosin. Prodigiosin (5((3-methoxy-5-pyrrol-2-ylidene-pyrrol-2-ylidene)-methyl)-2methyl-3-pentyl-1H-pyrrole) is a secondary metabolite alkaloid with a unique tripyrrole chemical
structure. The pigment has no defined role in the physiology of producing strains, but have
been reported to have antifungal, antibacterial, algicidal, antiprotozoal/antimalarial,
immunosuppressive and anticancer activities. In this study, Serratia marcescens SU-10 was isolated
from clinical sample. The organism was found to produce notable quantity of prodigiosin in nutrient
broth, but it could not produce more in either glucose or lactose containing nutrient broth medium. The
pigment isolated from the organism was found to possess anti-cancerous activity against Hep2 cell
lines.

Received on: 21.04.2011

Revised on: 01.05.2011
Accepted on: 09.06.2011

and he has patented the medium and he has also used oleic acid
and triolein instead of sodium oleate and reported a yield of
Prodigiosin (5((3-methoxy-5-pyrrol-2-ylidene-pyrrol-2-ylidene)- 0.69 mg/mL prodigiosin.
methyl)-2-methyl-3-pentyl-1H-pyrrole)
is
a
secondary
metabolite alkaloid with a unique tripyrrole chemical structure.
It is a red pigment isolated from a few species such as Serratia,
Pseudomonas and Streptomycin [1,2]. It has three rings forming a
pyrrolylpyrromethane skeleton with a C-4 methoxy group, a
molecular formula C20H25N3O and a molecular weight of 323.44
Da (Figure. 1).[2,3,4] It is sensitive to light and insoluble in water.
It is moderately soluble in alcohol and ether, and soluble in
chloroform, methanol, acetonitrile and DMSO.[5,6] Prodigiosin
have been shown to be associated in extracellular vesicles,
Figure. 1. Chemical Structure of Prodigiosin [2,3]
cell associated or present in intracellular granules [7,8].

1.

INTRODUCTION

The regular liquid media currently being used for

prodigiosin biosynthesis are nutrient broth[9] and peptone
glycerol broth.[10] According to the medium patented by
Nakamura[11] used sodium oleate 2% for prodigiosin production

Acetone and ethyl acetate extract of S.marcescens SM-2, which

contained prodigiosin, showed inhibitory effect against Gram
positive and Gram-negative bacteria. The Prodigiosin pigment
was found to be a successful curing agent on plasmids of E.coli

www.ijmedres.com Copyright 2010 Medicobiological Research Publications. All rights reserved.

Antony V. Samrot, et al.,/Int J Med Res. 2011; 1(3):145-150

146

HB101 and S. aureus.[12] Prodigiosin also found to induce the optimized condition for prodigiosin production was
apoptosis in cells derived from human tumors[13] and acute determined and this was followed for prodigiosin production.
human T-cell leukemia.[14] The National Cancer Institute,
Bethesda has also shown that prodigiosin has an average IC50 of 2.3. Estimation of Prodigiosin
116725 nM[15] against cancer cell lines.
Isolated prodigiosin was estimated using the following equation.
[12]
2. MATERIALS AND METHOD
2.1. Isolation and Identification
Prodigiosin unit/cell = [OD499 (1.381 x OD 620)] x 1000
OD 620
S. marcesens was isolated from urine sample collected from
outpatient, Sathyabama Hospital, Sholinganallur, Chennai 600 OD499 pigment absorbance
119. The organism was cultured in the nutrient agar where it OD620 bacterial cell absorbance
grows as red colored pigmented colonies. Identification was 1.381 constant
done by performing routine biochemical tests and 16SrRNA
sequencing method. DNA was isolated from organism. A large 2.4. Isolation of Pigment
fragment of the 16S rRNA gene was amplified by PCR using the
universal primers BAC-F-(5'-AGA GTT TGA TC(AC) TGG The pigment, Prodigiosin of S. marcesens is produced at 28C
CTC AG-3') BAC-R (5'AAG GAG GTG (AT)TC CA(AG) CC- and inhibited at 37C. Two types of extraction of pigment from
3'). The PCR product after purification is sequenced using a S. marcesens was done by ethyl alcohol:HCl (1:1). The pigment
BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit thus extracted was then dried and used for further analysis.
(Applied Biosystems, USA) and a model 3100 automatic
sequencer (Applied Biosystems, USA). The closest known 2.5. Separation and Purification of Prodigiosin
relatives of the new isolates were determined by performing a
sequence database search. The sequences of closely related The pigment component was separated using Thin layer
strains were retrieved from GENBANK and the Ribosomal Chromatography. The TLC plates of silica gel (20x 20 cm) were
prepared. The developing solvent, which contains Chloroform
Database Project (RDP) libraries.
and Methanol, was standardized and poured into the
chromatography tank that was saturated using a filter paper
2.2. Optimization of Prodigiosin Production
2.2.1. Effect of incubation time and carbon source on pigment soaked in the mobile phase.16The Rf value of chromatogram was
observed in the TLC plates .
production
Equal volume of the bacterial isolate was inoculated in nutrient 2.6. Cytotoxicity Activity
broth containing either any one of the following carbohydrate
i.e. Glucose, lactose. This was incubated at different time of In vitro cytotoxicity was analyzed by performing MTT assay
incubation viz., 24, 30, 36, 42, 48, 72, 78, 84 and 90. The against the cancerous cell line Hep2 and normal cell line Vero.
prodigiosin production was estimated after incubation.
3. RESULT AND DISCUSSION
2.2.2. Effect of initial pH on pigment production
S. marcesens was isolated from clinical sample and found to
Equal volume of the bacterial isolate was inoculated in nutrient grow as red-pigmented colony on nutrient agar (Figure.1), the
broth with various initial pH viz.,4, 5, 6, 7and 8.0. The flasks organism was preserved for further study. The 16SrDNA was
were incubated at 30C for 72 h. The prodigiosin production was sequenced and the sequence was submitted in GENBANK
estimated after incubation. The initial pH at which maximum (accession number is JF 511460).
production of prodigiosin was observed was chosen and
When the organism was found to produce more prodigiosin
maintained in the following studies.
in nutrient broth (Figure.2). Prodigiosin production normally
done in nutrient broth [9] and peptone glycerol broth[10]. Chang et
2.2.3. Effect of temperature on pigment production
al., [17] has reported 3 mg/ml of prodigiosin production in
[18]
was
Bacterial isolate was inoculated into nutrient broth and incubated dextrose containing medium. Sundaramoorthy et al.,
at different temperature viz., 26, 28, 30, 32, 34 and 36C for 72 found S. marcesens to produce more prodigiosin in maltose
containing medium. S. marcescens SU-10 was found to produce
h. The prodigiosin unit/cell was estimated after incubation.
least prodigiosin in carbohydrate containing medium. This may
be due to repressive effect of glucose or lactose on prodigiosin
production, which was already observed by Oller.[19] The
2.2.4. Effect of NaCl on pigment production
organism was found to produce more prodigiosin at 280C at pH
Organism was inoculated in nutrient broth containing different 7 with incubation time of 72h and NaCl concentration of
concentration of NaCl viz. 4,5,6,7,8,9 and 10 g/1000ml. Thus, 7gm/1000ml (Figure.3,4 and 5) and the rate was reduced as the
www.ijmedres.com Copyright 2010 Medicobiological Research Publications. All rights reserved.

Antony V. Samrot, et al.,/Int J Med Res. 2011; 1(3):145-150

147

temperature increases. Williams and Quadri[20] also noted no

prodigiosin production when cultures were incubated at 38C;
however pigment production was observed when the
temperature was shifted to 27C. A similar result was observed
by Pryce and Terry.[9]
3.1. Isolation of Prodigiosin Pigment
Using ethyl alcohol and HCl (1:1) prodigiosin was extracted out
of Serratia marcescens SU-10. The isolated pigment was
subjected for TLC and the Rf value of fraction was 0.87
(Figure.6). Song et al.,[2] has extracted the red pigment directly
from the internal adsorbent using acidified methanol and phase
separation. The maximum UV absorbance of the isolated
prodigiosin was observed at 536 nm, it was in correspondence
with Serratia sp KH 95.[2]
3.2. Cytotoxicity
In vitro cytotoxicity was analysed by performing MTT assay, IC
50 of isolated prodigiosin against Vero cells was found to be 2.5
mg/ml and against cancerous cell line, Hep2 was 0.625 mg/ml. It
was found that least concentration of prodigiosin was found to
possess anticancer activity (Figure. 7.). The National Cancer
Figure.1. Cultural characteristics of S. marcescens SU-10
Institute, Bethesda has found to have an average IC50 of 116725
[15]
nM .
2000

1800

1600

Prodigiosin Unit/cell

1400

1200
Nutrient broth
Nutrient broth + Glucose
Nutrient broth + Lactose

1000

800

600

400

200

0
24

30

36

42

48

54

60

66

72

78

84

90

Time in hours

Figure. 2. Production of Prodigiosin by S. marcesens SU-10 at various incubation time in nutrient broth, nutrient broth+glucose and
nutrient broth + lactose

www.ijmedres.com Copyright 2010 Medicobiological Research Publications. All rights reserved.

Antony V. Samrot, et al.,/Int J Med Res. 2011; 1(3):145-150

2000
1800

1793.3

1600
1512

Prodigiosin unit/cell

1400
1200

1189.62

1000

950.23

800
600
400
200
83.5
0
4

pH

Figure.3. Effect of pH on prodigiosin production by S. marcesens SU-10

1900

1800

1793.3
1700

1700

Prodigiosin unit/cell

1662.4
1600
1509.4

1500

1455
1400
1344
1300

1200

1100

1000
26

28

30

32

34

Temperature ( C)

Figure. 4. Effect of temperature in prodigiosin production by S.marcescens SU-10

www.ijmedres.com Copyright 2010 Medicobiological Research Publications. All rights reserved.

36

148

Antony V. Samrot, et al.,/Int J Med Res. 2011; 1(3):145-150

149

2000
1800

1793.3

1600

Prodigiosin unit/cell

1400

1366.9

1200
1116.9
1045.47

1000
800

800

600
400

366.9
253.26

200
0
4

10

Gram/1000ml

Figure. 5. Effect of NaCl on prodigiosin production by S. marcescens SU-10

Figure.6. Thin layer chromatography of isolated pigment

125

Percentage cytotoxicity

100

75

Vero
Hep2
IC50

50

IC50

25

0
5

2.5

1.25

0.625

0.3125

0.156

0.078

0.039

0.0195

0.009

Concentration (mg/ml)

Figure. 7. Bioactivity of prodigiosin extract (IC50 of the extract was determined in Vero cell line and Hep2 cell line)
www.ijmedres.com Copyright 2010 Medicobiological Research Publications. All rights reserved.

Antony V. Samrot, et al.,/Int J Med Res. 2011; 1(3):145-150

4. CONCLUSION
The organism was found to produce more prodigiosin at 280C at [9]
pH 7 with incubation time of 72h and NaCl concentration of
7gm/1000ml. The isolated prodigiosin was found to possess
anticancer activity against Hep2 cell lines.
[10]
ACKNOWLEDGEMENTS
The authors are thankful to Sathyabama University, Chennai, [11]
Tamil Nadu, India for providing infrastructure facilities for this
study. The authors are thankful faculty members, Department of
biotechnology for their help. The authors also grateful to Mr. [12]
Mittapalli Nagesh, Ms. Anupama, Ms. Nithya Mudaliar and Ms.
Jawahar Nisha for their constant support.
5. REFERENCES
[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

Giri A.V., N. Anandkumar, G. Muthukumaran and G.

Pennathur. 2004. A novel medium for the enhanced
cell growth and production of prodigiosin from
Serratia
marcescens
isolated from soil. BMC
Microbiology., 4: 11.
Song M.J., J. Bae,; D.S.Lee, C.H.Kim, J.S.Kim, S.W.;Kim,
and S.I. Hong. 2006. Purification and Characterization of
Prodigiosin Produced by Integrated Bioreactor from
Serratia sp. KH-95. J. Biosci. Bioeng. 101:157-161.
Williamson N.R.; P.C.Fineram, F.J. Leeper and G.P.C.
Salmond. 2006. The biosynthesis and regulation of
bacterial prodiginines. Nat. Rev. Microbiol. 4: 887-899.
Harris A.K.P., N.R.Williamson, H. Slater, A. Cox,
S. Abbasi, I. Foulds, H.T. Simonsen, F.J.Leeper, G.P.C.
Salmond. 2004. The Serratia gene cluster encoding
biosynthesis of the red antibiotic, prodigiosin, shows
speciesand strain-dependent genome context variation.
Microbiol. 150: 3547-3560.
Khanafari A., M.M. Assadi, and F.A. Fakhr. 2006. Review
of Prodigiosin, Pigmentation in Serratia marcescens.
Online J. Biol. Sci. 1: 1-13.
Grimont P.A.D., F. Grimont, H.L.C. Dulong, De Rosnay
and P.H.A. Sneath. 1977. Taxonomy of the genus Serratia.
J. Gen. Microbiol. 98: 39.
Kobayashi, N. and Y. Ichikawa. 1991. Separation of the
prodigiosin localizing crude vesicles which retain the
activity
of
protease
and nuclease
in Serratia
marcescens. Microbiol. Immunol. 35: 607-614.
Matsuyama, T., T.Murakami, M.Fujita, S. Fujita and I.
Yano. 1986. Extracellular vesicle formation and

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

150

biosurfactant production by Serratia marcescens. J. Gen.

Microbiol. 132: 865-875.
Pryce, L.H. and F.W.Terry. 2000. Spectrophotometric
assay of gene expression: Serratia marcescens
Pigmentation. Bioscience. 26: 3-13.
Jonas, D., B.Schultheis, C. Klas, P.H. Krammer and S.
Bhakdi. 1993. Cytocidal effects of Escherichia coli
hemolysin on human T lymphocytes. Infect. Immun. 61:
1715-1721.
Nakamura A., K. Nagai, K. Ando and G. Tamura. 1986.
Selective suppression by prodigiosin of the mitogenic
response of murine splenocytes. J Antibiot. 39 (8):1155-9.
Mekhael R. and S.Y. Yousif. 2009. The role of red
pigment produced by Serratia marcescens as antibacterial
and plasmid curing agent. J. Duhok Univ. 12 (1): 268274.
Montaner, B. and R. Prez-Toms 1998. Prodigiosininduced apoptosis in human colon cancer cells. Life Sci.
68: 2025-2036.
Mangan,D.F., N.S. Taichman, E.T. Lally and S.M. Wahl.
1991.
Lethal
effects
of
Actinobacillus
actinomycetemcomitans leukotoxin on human T
lymphocytes. Infect.Immunol. 56: 3267-3272.
Campas C., M. Dalmau, B. Montaner, M.Barragan, B.
Bellosillo, D. Colomer, G. Pons, R. Perez-Tomas and J.
Gil. 2003. Prodigiosin induces apoptosis of B and T cells
from B-cell chronic lymphocytic leukemia. Leukemia. 17:
746-750.
Lynch D.L., T.E. Worthy and G.C. Kresheck. 1968.
Chromatographic separation of the pigment fractions from
a Serratia marcescens strain. Appl. Microbiol. 16: 13-20.
Chang S, M. Sanada, O. Johdo, S. Ohta, Y.Nagamatsu and
A. Yoshimoto. 2000. High production of prodigiosin by
Serratia marcescens grown on ethanol. Biotechnol. Lett.
22, 17611765.
Sundaramoorthy N, P. Yogesh and R. Dhandapani. 2009.
Production of prodigiosin from Serratia marcescens
isolated from soil. Indian Journal of Science and
Technology. 2 (10): 35-37.
Oller A. R. 2005. Media effects of sugars on pigmentation
and antibiotic susceptibility in Serratia marcescens. Sci.
& Technol., Transac. of Missouri Acad. Sci. 243-246.
Williams, R. P. and S. M. Quadri. 1980. The pigments of
Serratia. In The Genus Serratia, Edited by A. Von
Graevenitz & S. J. Rubin. Boca Raton, FL: CRC Press Inc.
pp. 3175.

Disclaimer: Statements, information, inferences, observations and findings etc., stated in the International Journal of Medicobiological
Research are attributed to the authors and do in no way imply to Journal. Queries related to articles should be directed to authors.
www.ijmedres.com Copyright 2010 Medicobiological Research Publications. All rights reserved.