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
Department of Chemical Engineering, Sathyabama University, Rajiv Gandhi Salai, Chennai, Tamil Nadu – 600 119,
India
2
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-2ylidene) -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).
Samrot et al. 129
MATERIALS AND METHOD
and 36°C for 72 h. The prodigiosin unit/cell was
estimated after incubation.
Isolation and identification
Effect of NaCl on pigment production
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.
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).
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.
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
Effect of initial pH on pigment production
Antimicrobial susceptibility assay
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.
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
Effect of temperature on pigment production
Bacterial isolate was inoculated into nutrient broth and
incubated at different temperature viz., 26, 28, 30, 32, 34
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
S.marcescens SU-10
characteristics
of
2000
1800
1793.3
1689
1600
1423
Prodigiosin Unit/cell
1400
1283.6
1333.5
1325
1248.9
1200
1038.966
1000
1116.9
1075
980.7
976
877.37
866.9
800
Ethylalcohol and
HCl
Ethylacetate and
acetone
1054
800
714.454
698
690
678.8
616.9
600
435
366.9
400
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
recognized as an opportunistic pathogen and strains of it
are now resistant to commonly used antibiotics (Hejazi
and Falkiner, 1997). However, up to 1950 the species
was thought to be a harmless saphrophytic organism
(Anía, 2008).
In1986, Grimont and Grimont proposed ribotyping as a
general method for molecular bacterial identification. This
method can also discriminate between isolates of the
same species and it has proven to be a useful
epidemiological tool in the study of various bacteria. 16S
rDNA was amplified, sequenced and identified as
Serratia marcescens. The 16SrDNA sequence was
submitted in GENBANK and the accession number is JF
511460.
When the organism was allowed to grow in various
media, the organism was found to produce more
prodigiosin in nutrient broth, even glucose and lactose did
not influence the prodigiosin production (Figure 3, 4 and
5). Prodigiosin production normally done in nutrient broth
(Pryce and Terry, 2000) and peptone glycerol broth
(Jonas et al., 1993). Sundaramoorthy et al (2009) found
Serratia marcescens to produce more prodigiosin in
maltose containing medium. Nakamura (1986) has used
triolein and reported a moderate yield prodigiosin. In
this study, the organism was found to produce 1610
unit/cell and 1616 unit/cell of prodigiosin in glucose and
lactose containing medium respectively. This is
comparatively lower than Nakamura (1986), but it is
greater than Sundaramoorthy et al (2009). Chang et al.,
(2000) has reported 3 mg/ml of prodigiosin when
dextrose was used in the medium. Oller (2005) reported
that glucose and sorbitol had a repressive effect on
prodigiosin synthesis, that was found with our studies too.
The organism was found to produce more prodigiosin
0
at 28 C at pH 7 (Figure 5 and 7) and the rate was
reduced as the temperature increases. Williams and
Quadri (1980) reported that no prodigiosin was produced
when cultures were incubated at 38°C; however pigment
production was observed when the temperature was
shifted to 27°C. A complete block in prodigiosin was
observed in most of the basically used media tested at
37° C (Pryce and Terry, 2000). Optimal prodigiosin
production was observed at 0.75% NaCl containing
nutrient broth medium (Figure 7).
Among all the extraction procedure, ethyl alcohol and
HCl method was found to extract higher quantity out of
Serratia marcescens as it was reflected on bioactivity.
Ethyl acetate: acetone extraction did not show any effect
on any bacteria. Whereas, ethanol:HCl extraction was
found to have antibacterial activity and its zone of
inhibition was measured as 16 mm against E.coli, 19 mm
Samrot et al. 131
1800
1610
1600
1523
1400
1342
1333
1233
1234
Prodigiosin Unit/cell
1200
1138
1119
1123.38
1100
1000
Ethylalcohol
and HCl
872
867
843
800
1123
1000.8
968
945
800
Ethylacetate
and acetone
743
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
1616.9
1536.9
1600
Prodigiosin unt/cell
1400
1259.8
1249
1200
1299
1289
1145
1123
1000
800
1043
990.7
963
921
1038.966
1000.7
Ethylalcohol and
HCl
Ethylacetate and
acetone
928
870
850
802
788
712
656.23
600
560
459.5
416.9
400
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 innutrient broth + lactose
2000
1800
1793.3
1634
1600
1512
1567
Prodigiosin unit/cell
1400
1200
1189.62
Nutrient broth
1000
950.23
916.9
816.9
800
736.9
716.69
616.9
600
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
Nutrient broth
+Glucose
Nutrient broth
+Lactose
132 Int. Res. J. Biotechnol.
1900
1800
1793.3
1700
1700
Prodigiosin unit/cell
1662.4
1634
1620
1600
1603
1567
1567
1532
1509.4
1505
1506
1500
Nutrient broth
1455
1422
1400
1400
1344
1300
Nutrient broth
+Glucose
Nutrient broth
+Lactose
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
1600
1634
1567
Prodigiosin unit/cell
1400
1366.9
1293
1220
1200
1116.9
1045
1003.8
1045.47
986
943
1000
Nutrient broth
Nutrient broth
+Glucose
Nutrient broth
+Lactose
800
800
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)
The isolated pigment was subjected for TLC. The
prodigiosin was analyzed and the Rf value of fraction is
0.87 (Figure 8). Song et al (2006) has extracted the red
pigment directly from the internal adsorbent using
acidified methanol and phase separation. And he has
Samrot et al. 133
Table 1. Antibacterial Activity
S.No
ORGANISM USED
1
2
3
E.coli
Pseudomonas sp
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.
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