International Journal of Future Biotechnology (2012), 1(2), 1-4

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ISSN 2319 1031(Online)

BIOACTIVITY OF PULCHERRIMIN ISOLATED FROM

Bacillus subtilis SU-10 GROWN IN FeSO4 RICH MEDIUM
Jayalakshmi R1, Bavanilatha M1, Narendrakumar G1 and Antony V.Samrot1*
1
Department of Biotechnology, Sathyabama University
Jeppiaar Nagar, Sholinganallur, Chennai 600 119, Tamil Nadu, India.

ABSTRACT
Presence of iron salts is found to influence the production of pigments in some microorganisms. Bacillus sp
are known to produce pigments in the presence of FeSO4 or any ferrous ions in the medium. In this study,
Bacillus subtilis SU-10 was isolated from garden soil of Sathyabama University which was found to produce
pigment in FeSO4 rich medium. The pigment was isolated and characterized by GC analysis. The pigment
was also subjected for bioactivity such as antibacterial and cytotoxicity assay. The pigment did not show any
interesting bioactivity.
INTRODUCTION
Iron salts are found to influence the effect of
growth pattern of the microorganisms. Excess iron
presence can inhibit yields of diphtheria toxin (Uffen
and Canale-Parola,1966), Shigella dysenteriae type 1
toxin
(Canale-Parola,1963),
and
Pseudomonas
aeruginosa toxin A. iron content in the medium were
found influence a number of bacteria and yeasts to
produce extracellular pigments (Van derwalt, 1952).
Thiobacillus ferrooxidans inoculated into FeSO4
solution was found to produce ferric oxide sulphate
Fe2(SO4)3 under aerobic conditions (Boiko et al., 2004).
Bacillus subtilis produces pulcherrimin pigment when it
is cultured in iron containing medium (Kupfer et al.,
1967). Pulcherrimin are salt of pulcherriminic acid (2,5diisobutyl-3,6-dihydrox-ypyrazine-1, 4-dioxide or a
tautomeric form of this compound). Kupfer (1967)
found that the formation of pulcherrimin is a nonenzymatic reaction and it derives from pulcherriminic
acid produced in the medium and it reacts with
available of iron in the medium (Gondry et al., 2009)
and has no readily apparent effect on cell metabolism.
MacDonald (1963) found biosynthesis of the
pulcherriminic acid in C. pulcherrima from L- leucine
and has cyclo-L-leucyl-L-leucyl as an intermediate.
Growing aerobic spore forming bacteria produce
pulcherrimin in media containing amino acids and a
carbohydrate such as starch (Canale-parola,1963;
Kupfer et al., 1967; Uffen and Canaleparola,1966,1969). The requirement for a carbohydrate
suggested the possibility that Bacillus species
synthesized pulcherriminic acid via a metabolic route
Identification of microorganism

different from that present in C. pulcherrima (Cryle et

al., 2010; Gondry et al., 2009). Strong antagonistic
activities
of
pulcherrimin
isolated
from
Metschnikowia pulcherrima (a yeast) was found
against human pathogens like Proteus vulgaris,
Escherichia coli, Candida albicans, C.parapsilosis,
C.krusei and Trichosporon mucoides were
determined (Turkel and Ener, 2009). In this study,
pulcherrimin was isolated from Bacillus subtilis SU10 and the bioactivity of the pulcherrimin was also
assessed.
MATERIALS AND METHODS
Isolation of the bacterial strain
The bacterial strain was isolated from the garden soil
of Sathyabama University, Chennai, TamilNadu-600
119, India. Soil sample collected was inoculated into
the medium containing FeSO4.7H2O (5 g/l), Na2H
PO4 (12.8 g/l), KH2 PO4(3 g/l), NaCl (5 g/l),
NH4Cl(1 g/l), MgSO4.7H2O (2.4 g/l), glucose (10
g/l) and left undisturbed for two weeks at room
temperature. After two weeks 1ml of sample was
used for the serial dilution and pour plated on above
selective iron rich medium with agar (20 g/l) and
incubated at 370C for 3 to 4 days. The organism
which grown on the iron rich medium and produced
pigment was selected and inoculated into 50ml of
same iron rich liquid medium. These procedures were
repeated three times in order to stimulate an
enrichment culture.

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International Journal of Future Biotechnology (2012), 1(2), 1-4

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Identification of microorganism was done by

performing routine biochemical tests and 16S rRNA
sequencing.
Isolation of pigment
The pigment produced by the organism was isolated by
scrapping the cells grown on the FeSO4 containing
solid medium. The pigment was extracted out of the
cells by cold extraction method using ethanol. The
pigment thus extracted was dried and used for further
analysis.
Gas chromatography
The pigment isolated was subjected for GC analysis
Antimicrobial susceptibility assay
Invitro antimicrobial susceptibility assay for the
pigment was done by following conventional Nathans
agar well diffusion method and Micro broth dilution
method. The strains include the standard ATCC strain
was used as the control for this study. Klebsiella sp,
Staphylococcus aureus, E.coli and Pseudomonas sp.
Antioxidant activity
Antioxidant activity of the pigment was assessed by
DPPH free radical scavenging activity.
Cytotoxicity of pigment
Cytotoxicity of pigment was done against vero cells and
Hep2 cell lines following MTT assay.
RESULT AND DISCUSSION
The organism was isolated from the garden soil from
Sathyabama University, Chennai 600119, Tamil
Nadu, India. The organism was found to grow produce
pigment FeSO4 containing solid medium and liquid
medium. The organism was subjected for biochemical
and 16SrRNA sequencing and found to be Bacillus
subtilis and designated as Bacillus subtilis SU-10
(Genbank accession number: GU902972) (Figure 1a
and 1b).

ISSN 2319 1031(Online)

Figure 1b Growth of Bacillus cereus on FeSO4 rich liquid

medium.

The organism was found to produce higher

concentration of reddish brown pigment in FeSO4
containing solid medium than in the liquid broth. The
pigment was isolated from the organism grown in
solid medium by cold extraction method and utilized
for the further studies.The pigment did not show any
activity against the bacterial isolates used for
antibacterial activity. Whereas, Turkel and Ener
(2009) have found pulcherrimin isolated from the
Metschnikowia pulcherrima (a yeast) to have
antibacterial activity. Increasing the concentration of
the pigment increased antioxidant activity which was
assessed by DPPH free radical scavenging activity
(Figure 2). When the pigment produced was subjected
for cytotoxicity analysis, it showed 50% cytotoxicity
(IC50) at 0.3 mg/ml concentration against Vero cells
and Hep2 cells. It is believed that the pigment was
toxic to both normal cells (vero cells) and cancer cells
(Figure 3). The pigment isolated was subjected for
GC analysis and the spectrum of the pigment
confirmed that the produced pigment is pulcherrimin
(Figure 4). When the pigment produced was subjected
for cytotoxicity analysis, it showed 50% cytotoxicity
(IC50) at 0.3 mg/ml concentration against Vero cells
and Hep2 cells. It is believed that the pigment was
toxic to both normal cells (vero cells) and cancer cells
(Figure 3). The pigment isolated was subjected for
GC analysis and the spectrum of the pigment
confirmed that the produced pigment is pulcherrimin
(Figure 4).

Figure 1A Growth of Bacillus cereus on FeSO4 rich medium

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International Journal of Future Biotechnology (2012), 1(2), 1-4

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ISSN 2319 1031(Online)

50
47

46

45

%
D
P
P
HS
c
a
v
e
n
g
in
ga
c
tiv
ity

40

35

30

29
27

Pigment + Hep2 cells

Pigment + Vero cells

25
23

22

20

15

10

5.4

4.6

0.676

0
5

2.5

1.25

0.625

0.6

0.5

0.3

0.15

Concentration (mg/ml)

Figure 2 Percentage DPPH scavenging activity of pigment against Vero cells and Hep2 cells
120

100
97
87

85
80

%
c
y
to
to
x
ic
ity

77
74
68
65
60

Pigment + Vero
Pigment + Hep2

59
54
51

40
36
27
20

0
0.15

0.3

0.625

1.25

2.5

Concentration (mg/ml)

Figure 3 Cytotoxicity assay of pigment against Vero and Hep2 cells

Figure 4 GC chromatogram of pigment produced by Bacillus subtilis SU-10.

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International Journal of Future Biotechnology (2012), 1(2), 1-4

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CONCLUSION

The pigment produced by Bacillus subtilis SU-10 found 5. Uffen RL, Canale-Parola E (1972). Synthesis
of
to have antioxidant property but it was found to be toxic
Pulcherriminic acid by Bacillus subtilis. J
to cancer cells (Hep2) and normal cells (Vero) at the
Bacteriol. 111(1):86-93.
concentration of 0.3mg/ml. The pigment produced by 6. Canale-Parola, E. 1963. A red pigment produced
Bacillus subtilis SU-10 was found to be pulcherrimin,
by aerobic spore forming bacteria. Arch.
which was confirmed by Gas chromatography analysis
Mikrobiol. 46:-414- 427.
7. Kupfer, D. G., Uffen, R. L. and Canale-Parola,
REFERENCE:
E.(1967).The role of iron and molecular oxygen
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and biochemical characterization of the cytochrome
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P450
CypX (CYP134A1) from
Bacillus 9. Uffen, R. L., and E. Canale-Parola. 1966.
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Temperature dependent pigment production by
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12:590-593.
a Family of tRNA-dependent peptide bond-forming 10. Uffen, R. L., and E. Canale-Parola. 1969.
Enzymes. Nat Chem Biol 5:414420.
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4. Turkel S and Ener B (2009). Isolation and
Bacillus cereus var
alesti. Zeitsch. Allg.
characterization of new Metschnikowia pulcherrima
Mikrobiol. 9:231-233.
strains as producers of the antimicrobial pigment 11. van der Walt, J. P. 1952. On the yeast Candida
pulcherrimin. Z Natur forsch C; 64(5-6):405-10.
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Delft.

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