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Optimization of prodigiosin production by Serratia marcescens SU-10 and

evaluation of its bioactivity

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International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(5) pp. 128-133, May, 2011
Available online http://www.interesjournals.org/IRJOB
Copyright © 2011 International Research Journals

Full Length Research Paper

Optimization of prodigiosin production by Serratia

marcescens SU-10 and evaluation of its bioactivity
Antony V.Samrot1, Chandana K1, Senthilkumar P2 and Narendra Kumar G1
1
Department of Biotechnology, Sathyabama University, Rajiv Gandhi Salai, Chennai, Tamil Nadu – 600 119, India
2
Department of Chemical Engineering, Sathyabama University, Rajiv Gandhi Salai, Chennai, Tamil Nadu – 600 119,
India
Accepted 9 June, 2011

Prodigiosins, a family of natural red pigments characterized by a common pyrrolylpyrromethane

skeleton produced by Serratia marcescens, Pseudomonas magneslorubra, Vibrio psychroerythrous
etc. It is is a promising drug owing to its reported characteristics of having anti-bacterial, anti-fungal,
anti-neoplastic, anti-proliferative, anti-oxidant and anti-malarial activity. Serratia marcescens is known
to produce more concentration of prodigiosin. In this study, the optimal condition for pigment
production found in nutrient broth at 280C and pH 7 for 72 hrs. The water - insoluble red pigment was
extracted using ethanol and hydrochloric acid (9.5:0.5) as well as with acetone and ethyl acetate (1:1).
Prodigiosin was further purified by organic solvents and thin layer chromatography (TLC). The
pigment was found to have higher inhibitory effect on both gram positive bacteria and gram negative
bacteria.

Keyword: Prodigiosin, S.marcescens SU-10 - Serratia marcescens SU-10, Bioactivity

INTRODUCTION

Serratia marcescens is a Gram negative, bacillus shaped
bacteria that belongs to the family Enterobacteriaceae.
These bacteria grow well on standard media and produce
a red to dark pink pigment that aids in its identification
and the red color pigment is so called Prodigiosin.
Prodigiosin (5( (3-methoxy- 5-pyrrol-2-ylidene-pyrrol-2-
ylidene) -methyl) -2- methyl-3-pentyl-1H- pyrrole) is an
alkaloid secondary metabolite with an unique tripyrrole
chemical structure. It is isolated from few species such as
Serratia, Pseudomonas and Streptomyces (Giri et al.,
2004; Song et al., 2006). It is sensitive to light and
insoluble in water but moderately soluble in alcohol and
ether, and soluble in chloroform, methanol, acetonitrile
and DMSO (Khanafari et al., 2006; Grimont et al., 1977).
Prodigiosin have been shown to be associated with
extracellular vesicles or present in intracellular granules
(Kobayashi and Ichikawa, 1991; Matsuyama et al., 1986).
The pigment has no defined role in the physiology of
producing strains, but have been reported to have
*Corresponding author Email: antonysamrot@gmail.com
antifungal, antibacterial, algicidal, antiprotozoal
antimalarial activities, immunosuppressive and
anticancer activities.
The regular liquid media currently being used for
prodigiosin biosynthesis are nutrient broth (Pryce and
Terry, 2000), peptone glycerol broth(Jonas et al., 1993)
etc. According to the medium patented by Nakamura
(1986), has used only triolein as substrate and
reported a yield of 0.69 mg mL prodigiosin. A new
strain of Serratia marcescens UCP1459 isolated from a
semi-arid soil produced the highest level of prodigiosin
(49.5 g/L) at 48 h of cultivation using 6% “manipueira”
(cassava wastewater) supplemented with mannitol (2%)
at pH 7 and 28 °C. Carbohydrates in “manipueira” and
mannitol were found to play a role in the enhanced cell
growth and prodigiosin production (Araújo et al,. 2010).
Acetone and ethyl acetate extract of S.marcescens
SM-2 which contained prodigiosin showed inhibitory
effect against Gram positive and Gram negative bacteria
(Mekhael and.Yousif, 2009). S.marcescens strain F-1-1
shows activity against plant pathogens Clavibater
michiganensis and Erwinia catotovora (Okamoto et al.,
1998).

MATERIALS AND METHOD
Isolation and identification
Serratia marcescens was isolated from urine sample
collected from out patient, Sathyabama Hospital,
Sholinganallur, Chennai – 600 119. The organism was
cultured which showed highly mucoid colonies and cell
associated red pigment on nutrient agar was isolated,
subcultured and stored at 40C.
Identification of organism was done by 16SrRNA
sequencing. DNA was isolated from the organism and the
large fragment of the 16S rRNA gene was amplified by
PCR using the universal primers BAC-F-(5'-AGA GTT
TGA TC(AC) TGG CTC AG-3') BAC-R (5'AAG GAG GTG
(AT)TC CA(AG) CC-3')
The PCR products were purified using a Wizard PCR
Preps DNA Purification System (Promega, USA)
according to the manufacturer's instructions. The PCR
TM
product after purification is sequenced using a BigDye
Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystems, USA) and a model 3100 automatic
sequencer (Applied Biosystems, USA). The closest
known relatives of the new isolates were determined by
performing a sequence database search.
The sequences of closely related strains were retrieved
from GENBANK and the Ribosomal Database Project
(RDP) libraries.
Optimization of prodigiosin production
Effect of incubation time and carbon source on
pigment production
Equal volume of the bacterial isolate was inoculated in
nutrient broth containing either any one of the following
carbohydrate i.e. Glucose, lactose. This was incubated at
different time of incubation viz., 24, 30, 36, 42, 48, 72, 78,
84 and 90. The prodigiosin production was estimated
after incubation.
Effect of initial pH on pigment production
Equal volume of the bacterial isolate was inoculated in
nutrient broth with various initial pH viz.,4, 5, 6, 7and 8.0.
The flasks were incubated at 30°C for 72 h. The
prodigiosin production was estimated after incubation.
The initial pH at which maximum production of
prodigiosin was observed was chosen and maintained in
the following studies.
Effect of temperature on pigment production
Bacterial isolate was inoculated into nutrient broth and
incubated at different temperature viz., 26, 28, 30, 32, 34
Samrot et al. 129
and 36°C for 72 h. The prodigiosin unit/cell was
estimated after incubation.
Effect of NaCl on pigment production
Organism was inoculated in nutrient broth containing
different concentration of NaCl viz. 4,5,6,7,8,9 and 10
g/1000ml.
Thus the optimized condition for prodigiosin production
was determined and this was followed for prodigiosin
production.
Isolation of pigment
Two types of extraction of pigment from Serratia
marcescens was done by either using
ethylacetate:acetone (1:1) or ethanol : HCl (9.5:0.5). The
pigment thus extracted was then dried and used for
further analysis.
Separation and purification of prodigiosin
The pigment component was separated using Thin layer
Chromatography. The TLC plates of silica gel (20x 20
cm) were prepared. The developing solvent which
contains Chloroform and Methanol was standardized and
poured into the chromatography tank that was saturated
using a filter paper soaked in the mobile phase. The Rf
value of chromatogram was observed in the TLC plates
(Lynch et al., 1967).
Estimation Of Prodigiosin
Isolated prodigiosin was estimated using the following
equation (Mekhael and Yousif, 2008)
Prodigiosin unit/cell = [OD499 – (1.381 x OD
620)] x 1000
OD 620
OD 499 – pigment absorbance
OD620 – bacterial cell absorbance
1.381 – constant
Antimicrobial susceptibility assay
Invitro antimicrobial susceptibility assay was done
following Perez et al (1990) agar well diffusion method.
The ATCC strains like Staphylococcus aureus, E.coli and
Pseudomonas sp was used.
RESULT AND DISCUSSION
Serratia marcescens was isolated from clinical sample
and identification was done based on its cultural
characteristics (Figure 1). S. marcescens was a motile,
Gram negative, rod-shaped facultative anaerobe and
130 Int. Res. J. Biotechnol.

Figure 1. Cultural characteristics of S.marcescens SU-10

2000

1800 1793.3
1689
1600

1400 1423
1333.5 1325
Prodigiosin Unit/cell

1283.6
1248.9
1200
1116.9
1075 1054 Ethylalcohol and
1038.966
1000 980.7 976 HCl
Ethylacetate and
866.9 877.37 acetone
800 800
690 698 714.454
678.8
600 616.9

435
400
366.9
316
200

0
24 30 36 42 48 54 60 66 72 78 84 90
Time in hours

Figure 2. Production of Prodigiosin by Serratia marcescens SU-10 at various incubation time in

nutrient broth

colonies on nutrient agar were red. S. marcescens is unit/cell and 1616 unit/cell of prodigiosin in glucose and
recognized as an opportunistic pathogen and strains of it lactose containing medium respectively. This is
are now resistant to commonly used antibiotics (Hejazi comparatively lower than Nakamura (1986), but it is
and Falkiner, 1997). However, up to 1950 the species greater than Sundaramoorthy et al (2009). Chang et al.,
was thought to be a harmless saphrophytic organism (2000) has reported 3 mg/ml of prodigiosin when
(Anía, 2008). dextrose was used in the medium. Oller (2005) reported
In1986, Grimont and Grimont proposed ribotyping as a that glucose and sorbitol had a repressive effect on
general method for molecular bacterial identification. This prodigiosin synthesis, that was found with our studies too.
method can also discriminate between isolates of the The organism was found to produce more prodigiosin
same species and it has proven to be a useful at 280C at pH 7 (Figure 5 and 7) and the rate was
epidemiological tool in the study of various bacteria. 16S reduced as the temperature increases. Williams and
rDNA was amplified, sequenced and identified as Quadri (1980) reported that no prodigiosin was produced
Serratia marcescens. The 16SrDNA sequence was when cultures were incubated at 38°C; however pigment
submitted in GENBANK and the accession number is JF production was observed when the temperature was
511460. shifted to 27°C. A complete block in prodigiosin was
When the organism was allowed to grow in various observed in most of the basically used media tested at
media, the organism was found to produce more 37° C (Pryce and Terry, 2000). Optimal prodigiosin
prodigiosin in nutrient broth, even glucose and lactose did production was observed at 0.75% NaCl containing
not influence the prodigiosin production (Figure 3, 4 and nutrient broth medium (Figure 7).
5). Prodigiosin production normally done in nutrient broth Among all the extraction procedure, ethyl alcohol and
(Pryce and Terry, 2000) and peptone glycerol broth HCl method was found to extract higher quantity out of
(Jonas et al., 1993). Sundaramoorthy et al (2009) found Serratia marcescens as it was reflected on bioactivity.
Serratia marcescens to produce more prodigiosin in Ethyl acetate: acetone extraction did not show any effect
maltose containing medium. Nakamura (1986) has used on any bacteria. Whereas, ethanol:HCl extraction was
triolein and reported a moderate yield prodigiosin. In found to have antibacterial activity and its zone of
this study, the organism was found to produce 1610 inhibition was measured as 16 mm against E.coli, 19 mm
 Samrot et al. 131

1800

1600 1610
1523

1400
1342
1333
1233
1234
1200

Prodigiosin Unit/cell
1123.38 1138
1119 1123
1100
1000 1000.8
945 968
Ethylalcohol
867 872 and HCl
843
800 800 Ethylacetate
743 and acetone
670
600
516.9
514.4

400
345
296
200

0
24 30 36 42 48 54 60 66 72 78 84 90
Time in hours

Figure 3. Production of Prodigiosin by Serratia marcescens SU-10 at various incubation time in

nutrient broth +glucose

1800

1600 1616.9
1536.9

1400
1299
1289
1259.8
1249
1200
Prodigiosin unt/cell

1145
1123
1043 1038.966
1000 990.7 1000.7
963 Ethylalcohol and
921 928
870 HCl
850 Ethylacetate and
800 788 802
acetone
712
656.23
600
560
459.5
400 416.9

200

0
24 30 36 42 48 54 60 66 72 78 84 90
Time in hours

Figure 4. Production of Prodigiosin by Serratia marcescens SU-10 at various incubation time in

nutrient broth + lactose

2000

1800 1793.3

1634
1600
1512
1567
1400
Prodigiosin unit/cell

1200 1189.62
Nutrient broth
1000
950.23 Nutrient broth
916.9
+Glucose
800 816.9 Nutrient broth
716.69 736.9 +Lactose

600 616.9

400
283.566
200
116.9
106.9
83.5
0
4 5 6 7 8
pH

Figure 5. Effect of pH on prodigiosin production by S. marcesens SU-10

132 Int. Res. J. Biotechnol.

1900

1800 1793.3

1700 1700
1662.4
1620 1634
1600 1603

Prodigiosin unit/cell
1567 1567
1532
1500 1506 1509.4
1505 Nutrient broth
1455
1422 Nutrient broth
1400 1400 +Glucose
Nutrient broth
1344 +Lactose
1300
1278
1233
1200

1100

1000
26 28 30 32 34 36
Temperature (0C)

Figure 6. Effect of temperature in prodigiosin production by S.marcescens SU-10

2000

1800 1793.3

1634
1600
1567

1400
1366.9
Prodigiosin unit/cell

1293
1200 1220
1116.9 Nutrient broth
1045.47 1045
1000 986 1003.8 Nutrient broth
943
+Glucose
Nutrient broth
800 800 +Lactose
700
600 600

400
366.9
350
300
253.26
224
200 200

0
4 5 6 7 8 9 10
Gram/100ml

Figure 7. Effect of NaCl on prodigiosin production by Serratia marcescens SU-10

Figure 8. Thin Layer Chromatography analysis of prodiogiosin isolated

from Serratia marcescen SU-10

for Pseudomonas sp and 20mm for S.aureus. (Table 1) 0.87 (Figure 8). Song et al (2006) has extracted the red
The isolated pigment was subjected for TLC. The pigment directly from the internal adsorbent using
prodigiosin was analyzed and the Rf value of fraction is acidified methanol and phase separation. And he has

Table 1. Antibacterial Activity
S.No ORGANISM USED
1 E.coli
2 Pseudomonas sp
3 Staphylococcus aureus
purified by silica gel chromatography and high
performance liquid chromatograph (HPLC). As a result,
pure prodigiosin was identified by structural studies as a
pigment. The pigment was analyzed by UV
spectrophotometry. The maximum UV absorbance was
observed at 536 nm, corresponding to prodigiosin, in
agreement with results for prodigiosin purified from
Serratia sp KH – 95 (Song et al., 2006).
CONCLUSION
Serratia marcescens SU-10 was isolated from clinical
samples. The organism was found to possess novel
16SrRNA sequence and it is submitted in GENBANK
(Accession number JF511460). Nutrient broth was found
to be the best medium to produce prodigiosin.
Ethanol:HCl was found to be effective extraction of
prodigiosin. Prodigiosin isolated in this study was found
to possess antibacterial activity. Structure of prodigiosin
produced by Serratia marcescens SU-10 was elucidated
by GC.MS analysis.
ACKNOWLEDGEMENT
The authors are thankful to Sathyabama University,
Chennai, Tamil Nadu, India for providing infrastructure
facilities for this study. The authors also grateful to Mr.
Mittapalli Nagesh, Ms.Anupama, Ms.Nithya Mudaliar and
Ms. Jawahar Nisha for their constant support.
REFERENCES
Anía-BJ(2008).Serratia: overview.
http://emedicine.medscape.com/article/228495-overview.
Araújo HWC, Fukushima K, Takaki GMC (2010). Prodigiosin
Production by Serratia marcescens UCP 1549 Using Renewable-
Resources as a Low Cost Substrate. Molecules, 15(10): 6931-6940.
Chang S, Sanada M, Johdo O, Ohta S, Nagamatsu Y, Yoshimoto A
(2000). High production of prodigiosin by Serratia marcescens grown
on ethanol. Biotechnol. Lett. 22: 1761–1765.
Giri AV, Anandkumar N, Muthukumaran G, Pennathur G (2004). A
novel medium for the enhanced cell growth and production of
prodigiosin from Serratia marcescens isolated from soil. BMC
Microbiology. 4: 11.
Grimont PAD, Grimont F, Dulong HLC, De Rosnay, Sneath PHA (1977).
Taxonomy of the genus Serratia. J. Gen. Microbiol. 98: 39.
View publication stats
Samrot et al. 133
DIAMETER OF INHIBITION (mm)
Ethanol : HCl Ethyl acetate: acetone
16 0
19 0
20 0
Hejazi A, Falkiner FR (1997). Serratia marcescens. J Med Microbiol.
46 (11): 903–12.
Jonas D, Schultheis B, Klas C, Krammer, Bhakdi S (1993).
Cytocidal effects of Escherichia coli hemolysin on human T
lymphocytes. Infect. Immun. 61: 1715 - 1721.
Khanafari A, Assadi MM, Fakhr FA (2006). Review of Prodigiosin,
Pigmentation in Serratia marcescens. Online J. Biol. Sci. 1: 1-13.
Kobayashi N and Ichikawa Y (1991). Separation of the prodigiosin
localizing crude vesicles which retain the activity of protease and
nuclease in Serratia marcescens. Microbiol. Immunol. 35: 607-
614.
Lynch DL, Worthy TE, Kresheck GC (1968). Chromatographic
separation of the pigment fractions from a Serratia marcescens
strain. Appl Microbiol. 16: 13-20.
Matsuyama T, Murakami T, Fujita M, Fujita S, Yano T (1986).
Extracellular vesicle formation and biosurfactant production by
Serratia marcescens. J. Gen. Microbiol. 132: 865-875.
Mekhael R, Yousif SY (2009). The role of red pigment produced by
Serratia marcescens as antibacterial and plasmid curing agent. J.
Duhok Univ. 12 (1): 268-274.
Nakamura A, Nagai K, Ando K, Tamura G (1986). Selective
suppression by prodigiosin of the mitogenic response of murine
splenocytes. J Antibiot (Tokyo). 39 (8):1155-9.
Okamoto H, Sato Z, Sato M, Koiso Y, Iwasaki S, Isaka M (1998).
Identification of antibiotic red pigments of Serratia marcescen F-1-1 a
biocontrol agent of damping off of cucumber and antimicrobial activity
against other plant pathogens. Ann. Phytopathol. Soc. Jpn. 64: 294-
298.
Oller AR (2005). Media effects of sugars on pigmentation and antibiotic
susceptibility in Serratia marcescens. Sci. & Technol., Transac. of
Missouri Acad. Sci. 2: 243-246.
Perez C, Pauli M, Bazevque P (1990). An antibiotic assay by the agar
well diffusion method. Acta Biologiae et Medicine Experimentalis.
15:113-115.
Pryce LH, Terry FW (2000). Spectrophotometric assay of gene
expression: Serratia marcescens Pigmentation. Bioscience. 26: 3-13.
Song MJ, Bae J, Lee DS, Kim CH, Kim JS, Kim SW, Hong SI (2006).
Purification and Characterization of Prodigiosin Produced by
Integrated Bioreactor from Serratia sp. KH-95. J. Biosci. Bioeng. 101:
157-161.
Sundaramoorthy N, Yogesh P and Dhandapani R (2009). Production of
prodigiosin from Serratia marcescens isolated from soil. Indian
Journal of Science and Technology. 2(10): 32-34.
Williams RP, Quadri SM (1980). The pigments of Serratia. In The
Genus Serratia, pp. 31–75. Edited by A. Von Graevenitz & S. J.
Rubin. Boca Raton, FL: CRC Press Inc.