1. Isolation and Characterization of
Prodigiosin from Soil Bacterium
Under the guidance of-
•Dr. K. Kumanan (External guide; Dean, Faculty of Basic Sciences,
TANUVAS, Madras Veterinary College, Chennai.)
•Mrs. G. Dhanavathy (Internal guide; Assistant Professor,
Department of Biotechnology, SRM University, Chennai.)
Efforts by-
• Aditya Sharma (1091210015)
• Aditya Rishi (1091210038)
• Ashley Abraham (1091210040)

2. Introduction
• Serratia marcescens is a gram-negative, rod-shaped, motile bacterium belonging to
the family Enterobacteriaceae.
• S.marcesescens are facultative anaerobes that produce a red to dark pink pigment
called prodigiosin which is an alkaloid secondary metabolite with a unique tripyrrole
chemical structure.
• It has been reported to exhibit anti-bacterial, anti-fungal, anti-neoplastic, anti-
proliferative, anti-oxidant , immunosuppressive and anti-malarial activities.
• DNA staining ability of prodigiosin has the potential to substitute the chemically
synthesized , carcinogenic, conventional stains like ethidium bromide.
Plate.1: Prodigiosin((5( (3-methoxy- 5-pyrrol-2-ylidene-pyrrol-2-ylidene) -methyl) -2-
methyl-3-pentyl-1H- pyrrole)
Nat. Rev. Microbiol., Williamson et al, 2006

3. Review of Literature
• Serratia marcescens is known to produce more concentration of prodigiosin when compared to other
strains like Pseudomonas , Streptomyces, Pseudoalteromonas sp., Haella chejuensis and Vibrio sp.(Song
et al., 2006; Blake et al. 1990)
• The regular liquid media currently being used for prodigiosin biosynthesis is nutrient broth .(Pryce and
Terry, 2000)
• The optimum production of prodigiosin was at 28◦C after 48 hrs. (Siva et al.,2012; Darshan and
Manomani, 2015)
• Colour change of prodigiosin extract to red in acidified sol. and yellow/tan in alkaline sol. gives a
positive presumptive test for prodigiosin (Gerber and Lechevalier, 1976).
• Thin layer chromatography of prodigiosin was carried in hexane-methanol mobile phase, Rf value
deduced to be 0.8 (Rahul et al., 2016).
• HPLC of prodigiosin was carried in orthophosphoric acid-acetonitrile mobile phase, retention time
observed was 10.424 min (Chaudhari et al., 2014).
• The Serratia marcescens prodigiosin showed bactericidal and bacteriostatic effect showing promising
antimicrobial activity.(Lapenda et al.,2014)
• Prodiginines have been described as pro-apoptotic anticancer compounds and have been shown to induce
cellular stresses such as cell cycle arrest, DNA damage and a change of intracellular pH (pHi), all of
which can induce apoptosis (Darshan and Manonmani, 2015).
• Prodigiosin is shown to intercalate into dsDNA. In the presence of copper, it promotes oxidative cleavage
of the DNA. (Melvin et al., 2002)
• In in-vitro and cultured cells, prodigiosin-DNA interaction is shown to prefer alternating base pairs but
with no discrimination between AT and GC sequences; dual abolition of topoisomerase I and II activity
and as a consequence, DNA cleavage. (Montaner et al., 2005)

4. Objectives
• To isolate the pigment prodigiosin from soil bacterium Serratia marcescens.
• To characterize prodigiosin.
• To test prodigiosin for its anti-microbial activity.
• To test prodigiosin for its anti-neoplastic activity.
• To test prodigiosin for its DNA staining ability.

5. Methodology
Pigment production and extraction
Estimation of prodigiosin by UV spectrophotometry
Characterization of pigment using TLC and HPLC
MTT assay performed on A-72, Hep-2 and Vero cell lines
Biochemical analysis (presumptive test) for preliminary confirmation
Determination of DNA staining ability by agarose gel electrophoresis
In-vitro determination of anti-microbial activity using well diffusion method

6. Results
Plate.2: Serratia marcescens
culture in nutrient broth
Plate.3: Serratia marcescens streaked
nutrient agar plates showing discrete
colonies
Plate.4: Prodigiosin pigment isolated from
S.marcescens in 4% acidified ethanol
Plate.5: 2 ml of lyophilized prodigiosin pigment
isolated in 4% acidified ethanol
Plate.6: Colour change of prodigiosin extract to red in acidified sol. A
and yellow in alkaline sol., showing a positive presumptive test for
prodigiosin.
T1: Prodigiosin extract acidified with a
drop of conc. HCl.
T2: Prodigiosin pigment isolated from S.
marcescens in 95% methanol (extract).
T3: Prodigiosin extract alkalinized
with a drop of conc. ammonia sol.

7. Results
Prodigiosin unit/cell = [OD499 – (1.381 x OD620)] x 1000
OD 620
Serratia marcescens was found to produce 1516.9 prodigiosin units/cell in nutrient broth.
Estimation of prodigiosin by UV spectrophotometer:
Isolated prodigiosin was estimated using the following equation (Mekhael and
Yousif, 2008) : OD499 – pigment absorbance
OD620 – bacterial cell absorbance
1.381 – constant
Prodigiosin unit/cell =
[0.823 – (1.381 x 0.284)] x 1000
0.284
= 1516.9 units/cell
T1 T2 T3
OD499 (pigment absorbance) OD620 (bacterial cell absorbance)
0.807 0.274
0.820 0.288
0.842 0.291
Average = 0.823 Average = 0.284
Fig.1: HPLC analysis of prodigiosin showing a retention time of 10.757 min. (closely corresponding to the
retention time of 10.424 min.(Chaudhari et al., 2014))

8. Results
Plate.7: Thin layer
chromatography of pigment
prodigiosin in hexane:methanol
(1:2) mobile phase , exhibiting an
Rf of 0.81 (In agreement with Rf
of 0.8 deduced by Rahul et al.
(2015) in the exact solvent system)
Plate.8: Assay showing the zone of inhibition at 25 µg, 50 µg and 75 µg concentration of the pigment
prodigiosin against few bacterial strains
Streptococcus aureus Salmonella enterica Escherichia coli Staphylococcus agalactiae

9. Results
0
5
10
15
20
25
Streptococcus
aureus
Staphylococcus
agalactiae
Salmonella enterica Eschirechia coli
Zoneofinhibitioninmm
Bacterial species
Antimicrobial activity of varying prodigiosin concentration – A
comparison
Control
25 µg
50 µg
75 µg
Fig.2: Antimicrobial
activity of varying
prodigiosin
concentration - A
comparison.
Fig.3: MTT assay of A-72cells indicating cytotoxicity of
prodigiosin. Increased cell death was noticed when the
concentration of prodigiosin was increased
Fig.4: MTT assay of Hep-2 cells indicating cytotoxicity of
prodigiosin. Increased cell death was noticed when the
concentration of prodigiosin was increased

10. Results
Fig.5: MTT assay of Vero cells indicating cytotoxicity of prodigiosin. Increased
cell death was noticed when the concentration of prodigiosin was increased
Fig.6: A
comparison
showing specific
neoplasm directed
cytotoxicity of
prodigiosin
Cancer cells contain 3.5 fold
higher concentration of
Cu(II) than non-malignant
cells
The electron rich bipyrrole
chromophore of permeable
prodigiosin oxidizes itself
In turn reduces stable
Cu(II) to unstable Cu(I)
Cu(I) then inhibits
phosphorylation of
enzymes- topoisomerase I
& II , hence inactivating
the enzymes
This causes super-coiling of
DNA leading to its
fragmentation

11. Results
5 µg EtBr
2 µg prodigiosin 3 µg prodigiosin 5 µg prodigiosin 10µg prodigiosin4µg prodigiosin
20 µg prodigiosin 30 µg prodigiosin 60 µg prodigiosin50 µg prodigiosin40µg prodigiosin
L1: 1 kb DNA ladder
L2: Staphylococcus
agalactiae DNA
L3: Escherichia coli DNA
L4*: Love bird DNA
Plate.9: Electrophoresis of DNA
samples in 0.8% agarose gel
stained with increasing
concentration of prodigiosin

12. Summary
•Nutrient broth was found to be the optimum medium to produce prodigiosin. (1516.9
prodigiosin units/cell in nutrient broth , greater than that produced in triolein , maltose
containing medium and dextrose).
•The natural red pigment produced by S. marcescens demonstrated a retention factor
and retention time corresponding to prodigiosin reported in the literature in addition to
a positive presumptive test.
•The clinical isolates evaluated in our study- Streptococcus aureus, Staphylococcus
agalactiae, Salmonella enterica and Eschirechia coli, exhibited relevant sensitivity to
prodigiosin as significant inhibition zones were achieved.
•A dose-dependent decrease in the number of viable cells was observed in all the cell
lines studied . However, we did not observe a significant decrease in the viability of the
non-malignant Vero cells when compared with the malignant cell lines. Hence, it can be
inferred that prodigiosin possesses specific neoplasm directed cytotoxicity .
•It was observed that prodigiosin starts staining DNA at a concentration as low as 3 µg.
However, effective staining was observed at a range between 10 µg and 40 µg. At 50
µg concentration, agarose gel electrophoresis showed DNA fragmentation, due to the
positive correlation between cytotoxicity and DNA cleavage.

13. References
• Chaudhari V, Gosai H, Raval S and Kothari V. (2014). Effect of certain natural products and organic
solvents on quorum sensing in Chromobacterium violaceum. Asian Pac J Trop Med, Suppl1,S201-S211.
• Darshan N and Manonmani HK. (2015) .Prodigiosin and its potential applications. Journal of Food
Science and Technology. Vol. 52, p5397-5407.
• Elahian F, Moghimi B, Dinmohammadi F, Ghamghami M, Hamidi M and Mirzaei SA. (2013). The
anticancer agent prodigiosin is not a multidrug resistance protein substrate. DNA and Cell Biology.
Vol32, p90-97.
• Lapenda JC, Silva PA, Vicalvi MC , Sena K X and Nascimento S C. (2015). Antimicrobial activity of
prodigiosin isolated from Serratia marcescens UFPEDA 398. World journal of Microbiology and
Biotechnology. Vol31,p399–406.
• Mekhael R and Yousif S Y. (2009) . The role of red pigment produced by Serratia marcescens as
antibacterial and plasmid curing agent. J.Duhok Univ. Vol12,p268-274.
• Melvin M, Tomlinson J, Saluta G, Kucera G, Lindquist N and Manderville R. (2000). Double-strand
DNA cleavage by copper prodigiosin. Journal of American Chemical Society. Vol.122,p6333-6334.
• Montaner B, Navarro S, Piqué M, Vilaseca M, Martinell M, Giralt E, Gil J, Pérez-Tomás R (2000).
Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell
lines. Vol.131, 585-593.
• Rahul S, Chandrashekhar, Hemant B, Bipinchandra S, Mouray E, Grellier P and Satish P. (2015). In vitro
antiparasitic activity of microbial pigments and their combination with phytosynthesized metal
nanoparticles .Parasitology International.
• Song MJ, Bae J, Lee DS, Kim CH, Kim JS, Kim SW and Hong SI. (2006). Purification and
characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. Journal of
Bioscience and Bioengineering, Vol. 101, p157–161.