In the present study it has been investigated that Serratia marcescens MTCC 4822 has good potential for Prodigiosin production. Among the screened media components, maltose was the best carbon source for the production for this strain. The fermentation media supplemented with maltose (2%) and NaCl (0.5%) at pH 6.8-7.0 incubated at 28°C gave maximum prodigiosin production (1390 unit/cell) with the biomass content of 3.45 g L-1 after 96 hrs of incubation period. Prodigiosin, a red pigment, produced by bacterial species Serratia marcescens, belongs to the family of tripyrrole was found to exhibit antibacterial, antimycotic, immunomodulating, anti-tumor and anti-malarial properties. A lot of attention is now paid to the biotechnological synthesis of the colours through the microorganisms. Plant cell and tissue culture, microbial fermentation and gene manipulation have been investigated with respect to the production of biopigments. However, extensive safety testing of such products is required before they are given clearance as safe food additives or other applications.

1. Int.J.Adv.Microbiol.Health.Res.2018; 2(1):17-23
17
International Journal of Advanced Microbiology and Health Research
ISSN: 2457-077X. Volume 2, Issue 1 (January to March 2018), PP. 17-23
Available online at www.ijamhr.com
Original Research Article
Effect of Various Substrate and Process Parameters on the
Production of Prodigiosin by Serratia marcescens MTCC 4822
Bhairav Prasad
College of Health Science, Vidya Jyoti Institution, Derabssi, SAS Nagar Mohali-140508, Punjab, India
ABSTRACT
INTRODUCTION
Colour is one of the most important attributes of
food considered as a quality indicator. Synthetic
colourants have many disadvantages when
compared to natural ones, including higher cost and
lower stability. However, people have increasingly
avoided synthetic colourants, preferring natural
pigments, which are considered to be harmless or
even healthy. These requirements compelled
numerous regulation changes worldwide. For
instance, the USA permitted list of synthetic
colourants was reduced from 700 to only seven
until the beginning of the 21st Century [1]. Nature
produces a variety of compounds adequate for food
colouring, such as the water-soluble anthocyanins,
betalains, and carminic acid, as well as the oil
soluble carotenoids and chlorophylls. The health-
benefit properties of natural pigments have been
focused by many works, especially those of
carotenoids and anthocyanins whose antioxidant
properties have been extensively studied. Betalains,
because of their relative scarceness in nature, have
not been much explored as bioactive compounds,
but some studies have indicated their potential as
antioxidant pigments. These findings have helped
to motivate utilization of betalains as food
colourants. A pigment found in an organism is
called biopigment. The most common biopigments
in Caridina shrimp are carotenoids, which appear to
be obtained through diet. These pigments can form
complexes with proteins, altering the colour or tone
produced, and these complexes are known as
carotenoproteins. When these complexes are
broken, the colour and tone of the original
carotenoid is expressed. Now commonly used in
food coloring, including two categories of pigment:
natural pigments and synthetic pigments. Natural
pigments from natural products, mainly extracted
from the plant tissues, including some from the
animal and microbial pigments. Synthetic artificial
pigment is obtained by chemical synthesis of
organic pigments, mainly isolated from coal tar
aniline dyes made from raw materials. Microalgae
and several classes of fungi are well known to
produce wide range of water-soluble pigments, but
the low productivity of algal cultures is a
Received 19th
February, 2018
Revised 22nd
February, 2018
Accepted 24th
February, 2018
Published online 13th
March, 2018
 Biopigment
 Serratia marcescens
 Prodigiosin
Article Info
In the present study it has been investigated that Serratia marcescens MTCC 4822 has
good potential for Prodigiosin production. Among the screened media components,
maltose was the best carbon source for the production for this strain. The fermentation
media supplemented with maltose (2%) and NaCl (0.5%) at pH 6.8-7.0 incubated at 28°C
gave maximum prodigiosin production (1390 unit/cell) with the biomass content of 3.45 g
L-1
after 96 hrs of incubation period. Prodigiosin, a red pigment, produced by bacterial
species Serratia marcescens, belongs to the family of tripyrrole was found to exhibit
antibacterial, antimycotic, immunomodulating, anti-tumor and anti-malarial properties. A
lot of attention is now paid to the biotechnological synthesis of the colours through the
microorganisms. Plant cell and tissue culture, microbial fermentation and gene
manipulation have been investigated with respect to the production of biopigments.
However, extensive safety testing of such products is required before they are given
clearance as safe food additives or other applications.
Keywords

2. Int.J.Adv.Microbiol.Health.Res.2018; 2(1):17-23
18
significant bottleneck for their commercialization
[2]. The use of such fungi to color foodstuffs is not
a novel practice; the use of Monascus pigments in
the food has been carried out traditionally [3,4].
Serratia marcescens a bacteria is the major
producers of prodigiosin. The production of
prodigiosin in Serratia marcescens is susceptible to
temperature and is substantially inhibited at
temperatures higher than 37°C. Addition of certain
nutrients is particularly suitable for prodigiosin
production. Serratia is a genus of Gram-negative,
facultative anaerobic, rod-shaped bacteria of the
Enterobacteriaceae family [5]. Prodigiosin is a red
pigment produced by many strains of the bacterium
like Serratia marcescens and some other unrelated
microbial strains, such as Vibrio psychroerythrus,
Streptomycin griseoviridis and Hahella chejuensis
was found to exhibit antibacterial, antimycotic,
immunomodulating, anti-tumor and anti-malarial
properties [6]. It has been reported that prodigiosin
revealed a broad range of inhibitory activities
against many bacterial, fungal and protozoan
species [7]. These biopigments have wide synthetic
and commercial application [8].
MATERIALS AND METHODS
Chemicals and Glassware
All the chemicals used for preparing reagents and
solutions were procured from HI -media, sd-fine
chemicals and were of AR grade. For the
production of prodigiosin dehydrated media used
were procured from HI- media and were used such
as per the manufacturer’s directions. All the glass
wares like test tubes, beakers, conical flasks etc.
were of borosilicate grade
Procurement and maintenance of bacterial culture
Serratia marcescens MTCC 4822 used in the
present investigation was procured from Microbial
Type Culture Collection (MTCC), Institute of
Microbial Technology, Chandigarh, India. The
bacterial culture was maintained on growth media
containing: Beef extract (1 g L-1), Yeast extract (2
g L-1), Peptone (5 g L-1), Sodium chloride (5 g L-
1). The culture was maintained by sub culturing,
aseptically at fortnight intervals and stored at 4oC,
until further use.
Preparation of starter culture
The bacterial culture was grown in 50ml of media
in 250ml capacity Erlenmeyer flask, having the
same composition as described above. After
sterilization, the flask were inoculated with loopful
of culture from capsule and incubated at 37o c for
24 h in rotary shaker at 120 rpm.
Preparation of production medium for
prodigiosin
The composition of fermentation medium was
(g/L): NaNO3 (1.28), K2HPO4 (0.87),
MgSO4.7H2O (0.1), NaCl (0.1), KCl (0.2), Tris
(hydroxymethyl) aminomethane (6.5), glucose (20);
mineral salt solution (5mL). The mineral salt
solution contained the following ingredients (g/L):
FeSO4 (NH4)2SO4.6H2O, H3BO3, CoCl2.6H2O,
CuSO4.5H2O,MnSO4.H2O, (NH4)6Mo7O24.4H2
O, ZnSO4.7H2O. The pH of medium was initially
adjusted to 6.8±0.2 by 1.0 M HCl. Different carbon
sources were added to the fermentation media. The
media was inoculated with the Serratia marcescens
MTCC 4822 strain and incubated at shaker at 120
rpm for 72h at 30°C.
Effects of different sugar substrate [9]
The effect of different carbon substrates (Maltose,
Glucose, Lactose, and fructose) on prodigiosin
production was investigated by supplementing
individually in fermentation medium at the
concentration of 2% (w/v).
Effect of Process Parameters [9]
The effect of different process parameters such as
pH (5, 6, 7, 8, 9), temperature (15, 20, 25, 30, 35,
37, 40), salt concentration (0.5, 1.0, 1.5. 2.0, 2.5)
w/v and incubation time (24, 48, 72, 96, 120h) was
monitored by varying the respective parameters.
Recovery and estimation of prodigiosin
[10]
The organisms were harvested by centrifuging at
10,000 rpm for 10 minutes. The supernatant was
discarded and the pellet was resuspended in
acidified ethanol (4% of 1M HCl in 96 mL
ethanol). The mixture was vortexed and the
suspension was centrifuged at 10,000 rpm for 10
minutes. The supernatant was transferred to a fresh
vial and observed under UV-visible
spectrophotometer at 534 nm. The prodigiosin
produced was quantified. Isolated biopigment was
estimated using the following equation [11].

3. Int.J.Adv.Microbiol.Health.Res.2018; 2(1):17-23
19
Prodigiosin unit/cell =
[OD499 – (1.381 x OD620)] x 1000
OD620
Where,
OD 499 = pigment absorbance
OD620 =bacterial cell absorbance
1.381 =constant
RESULTS AND DISCUSSION
The experimentation undertaken to evaluate the
Serratia marcescens MTCC 4822 for production of
biopigment has been presented and discussed
below:
Effects of different sugar substrate
Screening experiments were conducted on four
carbon sources, viz. Maltose, Glucose, Lactose,
fructose for their effectiveness on prodigiosin
production. The results of various carbon sources
for prodigiosin activity under above mentioned
conditions are as shown in (Fig.1). In the present
study the sugar substrate maltose gave the
maximum yield of 1381.38 prodigiosin unit/cell,
which is followed by sucrose, fructose and glucose.
Similarly, 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. Prodigiosin production
normally done in nutrient broth [12] and peptone
glycerol broth [13]. It has been reported that
Serratia marcescens, produce more prodigiosin in
maltose containing medium [9]. Nakamura has
used triolein and reported a moderate yield
prodigiosin of 1610 unit/cell and 1616 unit/cell of
prodigiosin in glucose and lactose containing
medium respectively[14]. Chang et al. has reported
3 mg/ml of prodigiosin when dextrose was used in
the medium[15]. It was also reported that glucose
and sorbitol had a repressive effect on prodigiosin
synthesis [16].
Effect of pH on the production of
prodigiosin
The pH of the fermentation was varied from 5.0 to
9.0 to study its effect on prodigiosin production. At
very low and very high pH i.e. at 5.0 and 9.0, very
low prodigiosin production was observed (Fig. 2).
This may be due to the reason that the Serratia
marcescens MTCC 4822 strain grows best only
near neutral pH. Any change in media alkalinity or
acidity hinders the prodigiosin production.
Prodigiosin production of 1383.91 unit/cell was
maximum at pH 7. These results suggested that the
optimal pH for prodigiosin production with the
MTCC 4822 strain was in the range of 6.8 ±2.
Similarly, in an another studied the influence of
initial pH of the growth medium on prodigiosin
production and found that the yield was maximum
at pH 7 [9].
Effect of temperature on the production
of prodigiosin
The effect of temperature on prodigiosin production
was studied by cultivating the bacterial strain in
media at temperature range of 20-40°C. Prodigiosin
production increased with temperature until 28°C
and then decreased gradually above 30°C (Fig.3).
At very high temperature like 37°C and above there
was complete block of the production of
prodigiosin. Optimal growth for prodigiosin from
Serratia has been reported at 28-30°C [9]. The
results suggested the maximum prodigiosin
production in batch culture of Serratia marcescens
MTCC 4822 at 28°C was 1382.25 unit/cell and it’s
a temperature dependent phenomenon, slight
increase and decrease in the optimal temperature
directly influence the production of the biopigments
in Serratia. In a similar, study it has been reported
that Serratia marcescens produce more prodigiosin
at 28oC at pH 7 and the rate was reduced as the
temperature increases. Similarly it has been
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[17]. A complete block in
prodigiosin was observed in most of the basically
used media tested at 37°C [12] .
Effect of different salt concentration on
prodigiosin production
NaCl was added to the fermentation media at
different concentration of 0.5-2.5%. It is evident
from (Fig. 4). that the increase in the concentration
of NaCl hinders the production of prodigiosin. It
has been observed that at salt concentration 0.5%
support the maximum yield of1379.05 unit/cell.
After that as the salt concentration increased the
pigment production also decreased, this might be
due to the influence of increase salt concentration

4. Int.J.Adv.Microbiol.Health.Res.2018; 2(1):17-23
20
on cellular metabolism and process and hinder the
production of prodigiosin production. It has been
reported that the optimal prodigiosin production
was observed at 0.75% NaCl containing nutrient
broth [18].
Effect of different incubation period on
prodigiosin production
The fermentation media was prepared from the
above set of parameters i.e. maltose concentration
of 2 (%, w/v), salt concentration 0.5 (%, w/v),
having pH 7.0 was incubated with stationary
conditions at 28°C and samples were taken at
regular intervals of 24h. From the results it can be
observed that there was continuous increase in the
prodigiosin production of as a function of
incubation period (Fig.5). Prodigiosin production
increased up to 96 hrs and beyond this incubation
period, it decreased, which may be due to
exhaustion of nutrients, and metabolic changes in
the medium. Prodigiosin production with the
Serratia marcescens MTCC 4822 strain was found
to be 1390 unit/cell with a biomass content of 3.45
g L-1 at 96 hrs. Similarly, Samrot et al. 2011
studied the effect of incubation period ranged from
24 to 90 h for the production of prodigiosin by
Serratia marcescens[18]. They observed that at
incubation period of 72 hrs produced maximum
prodigiosin. The amount of produced prodigiosin
was 1616.8 and 1539.9 unit/cell with the extraction
of ethanol: HCl and ethanol: acetone respectively.
CONCLUSION
In the present study it has been investigated that
Serratia marcescens MTCC 4822 has good
potential for Prodigiosin production. Among the
screened media components, maltose was the best
carbon source for the production for this strain. The
fermentation media supplemented with maltose
(2%) and NaCl (0.5%) at pH 6.8-7.0 incubated at
28°C gave maximum prodigiosin production (1390
unit/cell) with the biomass content of 3.45 g L-1
after 96 hrs of incubation period. Prodigiosin, a red
pigment, produced by bacterial species Serratia
marcescens, belongs to the family of tripyrrole was
found to exhibit antibacterial, antimycotic,
immunomodulating, anti-tumor and anti-malarial
properties. A lot of attention is now paid to the
biotechnological synthesis of the colours through
the microorganisms. Plant cell and tissue culture,
microbial fermentation and gene manipulation have
been investigated with respect to the production of
biopigments. However, extensive safety testing of
such products is required before they are given
clearance as safe food additives or other
applications.
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Fig.1. Effect of different carbon sources on prodigiosin production of Serratia marcescens MTCC
4822
1377.5
1378
1378.5
1379
1379.5
1380
1380.5
1381
1381.5
1382
Glucose Maltose Sucrose Fructose
Prodigiosinproductionunit/cell
Carbon sources

6. Int.J.Adv.Microbiol.Health.Res.2018; 2(1):17-23
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Fig.2. Effect of different pH on prodigiosin production of Serratia marcescens MTCC 4822
Fig.3. Effect of different temperature on prodigiosin production of Serratia marcescens MTCC
4822
1050
1100
1150
1200
1250
1300
1350
1400
1450
5 6 7 8 9
Prodigiosinproductionunit/cell
pH
0
200
400
600
800
1000
1200
1400
1600
20 28 30 35 37 40
ProdigisionproductionUnit/cell
Temperature (°C)

7. Int.J.Adv.Microbiol.Health.Res.2018; 2(1):17-23
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Fig.4. Effect of different salt concentration on prodigiosin production of Serratia marcescens MTCC
4822
Fig.5. Effect of incubation period on prodigiosin production of Serratia marcescens MTCC 4822
0
200
400
600
800
1000
1200
1400
1600
0.5 1 1.5 2 2.5
ProdigiosinProductionunit/cell
NaCl concentration (%w/v)
0
0.5
1
1.5
2
2.5
3
3.5
4
0
200
400
600
800
1000
1200
1400
1600
24 48 72 96 120 144
BiomassYieldgL-1
ProdigiosinProductionunit/cell
Incubation Period (h)
Prodigiosin
production
How to cite this article:
Prasad B. Effect of Various Substrate and Process Parameters on the Production of Prodigiosin by
Serratia marcescens MTCC 4822. Int. J. Adv.Microbiol.Health.Res., 2018; 2(1):17-23.
Source of Financial Support: Nil, Conflict of interest: Nil
Corresponding Author: Bhairav Prasad
Assistant Professor, College of Health Science, Vidya Jyoti
Institution, Derabassi, SAS Nagar Mohali, Punjab (India).
E-mail: bhairavmicro@gmail.com