PHYTOTHERAPY RESEARCH

Phytother. Res. 18, 754757 (2004)

754 Published online in Wiley InterScience (www.interscience.wiley.com).
G. OZDEMIR ET AL. DOI: 10.1002/ptr.1541

Antibacterial Activity of Volatile Component

and Various Extracts of Spirulina platensis

Guven Ozdemir1,*, N. Ulku Karabay 1, Meltem Conk Dalay 2 and Baris Pazarbasi 3
1
Ege University, Faculty of Science, Department of Biology, 35100 Bornova, Izmir, Turkey
2
Ege University, Faculty of Engineering, Department of Bioengineering, 35100 Bornova, Izmir, Turkey
3
Celal Bayar University, Faculty of Science and Literature, Department of Biology, 45040 Manisa, Turkey

The methanol, dichloromethane, petroleum ether, ethyl acetate extracts and volatile components of Spirulina
platensis were tested in vitro for their antimicrobial activity (four Gram-positive, six Gram-negative bacteria
and Candida albicans ATCC 10239). GC-MS analysis of the volatile components of S. platensis resulted in
the identication of 15 compounds which constituted 96.45% of the total compounds. The volatile components
of S. platensis consisted of heptadecane (39.70%) and tetradecane (34.61%) as major components. The
methanol extract showed more potent antimicrobial activity than dichloromethane, petroleum ether, ethyl
acetate extracts and volatile components. Copyright 2004 John Wiley & Sons, Ltd.

Keywords: Spirulina platensis; volatile components; extracts; antimicrobial activity.

INTRODUCTION MATERIALS AND METHODS

It is generally considered that compounds produced
naturally, rather than synthetically, will be biodegraded
more easily and will therefore be more environment-
ally acceptable. This assumption is based on the known
diversity and adaptability of microorganisms, which can
degrade most of the substances occurring naturally on
this planet. Up to now, extracts from thousands of plant
species have been screened for their activity against
pathogenic microorganisms. But relatively few were
found to be sufciently active in addition to being non-
toxic to man (Gilliver and Osborn, 1949). Cyanobacteria
are potential sources of high value chemicals and phar-
maceuticals. The cyanobacterium S. platensis has been
used for the past 20 years as a model organism in many
studies on the cultivation of algal biomass as a source
of protein and chemicals (Chen and Zhang, 1997). It
also exhibits a variety of biological properties such as
the reduction of hypercholesterolemia, enhancement of
immune function and prevention of oral cancer. Stud-
ies have shown that calcium spirulan (Ca-SP), a sulfated
polysaccharide chelating calcium, isolated from S.
platensis inhibited the replication of several enveloped
viruses, including herpes simplex virus type I (HSV-1),
human cytomegalovirus, measles virus, inuenza A
virus and human immunodeciency virus-1 (HIV-1)
(Mishima et al., 1998). There is a growing interest in
the area of research on the positive effects of S. platensis
on human health.
The aim of the study reported here was to investig-
ate the antimicrobial activity of various extracts and
volatile components of S. platensis cultivated in dif-
ferent growth conditions against some microorganisms.
* Correspondence to: Dr G. Ozdemir, Ege University, Faculty of Science,
Department of Biology, 35100 Bornova, Izmir, Turkey.
E-mail: gozdemir@sci.ege.edu.tr
Contract/grant Sponsor: Ege University Research Foundation.
Copyright 2004 John Wiley & Sons, Ltd.
Copyright 2004 John Wiley & Sons, Ltd.
Production and preparation of material. S. platensis was
obtained from the Ege University Culture Collection.
S. platensis was cultivated under photoautotrophic
growth conditions. Zarrouk medium was used for culti-
vation. The initial pH was adjusted to 9.5, and was found
to increase only slightly during cultivation. The culture
was illuminated at a light intensity of 80 mEm2s1. The
culture temperature was maintained at 30 0.1 C,
while the culture was agitated at 300 rpm. S. platensis
were grown until the late exponential phase of growth
(i.e. at 108 h). S. platensis material was collected and
freeze-dried.
Preparation of various extracts of S. platensis. Freeze-
dried S. platensis samples were pulverized. 15 g of pul-
verized S. platensis sample was extracted as reported
by Khan et al. (1988) and Vlachos et al. (1996) in 150 mL
methanol, ethanol, chloroform, acetone and hexane for
24 h using a soxhlet apparatus (yield 13%, 10.6%, 4%,
11.8%, 2.4%, respectively). The resulting extracts of
S. platensis from these different solvents were kept at
+4 C until use.
Extraction of the volatile components of S. platensis.
For the volatile compounds, the dried cyanobacteria
(50 g) were subjected to hydrodistillation for 4 h using
a Clevenger-type apparatus. The distillate was diluted
with ethyl acetate and the volume was reduced 100-
fold prior to analysis.
GC-MS analysis. The steam-distilled components
were analysed by GC and GC/MS. A HP 6890 gas
chromatograph equipped with a FID and a 5 m 0.2 mm
HP-1 capillary column (0.33 m coating) was employed
for the GC analysis. GC/MS analysis was performed on
a HP 5973 mass selective detector coupled with a HP
6890 gas chromatograph, equipped with a HP-1 capil-
lary column. The column temperature was programmed
Received
Phytother. Res. 14 May(2004)
18, 754757 2003
Accepted 4 June 2004
 ANTIBACTERIAL ACTIVITY OF SPIRULINA PLATENSIS 755

from an initial temperature of 70 C to a nal tempera-
ture of 280 C at 10 C/min. The injector temperature
was 150 C (1 L injection size), whereas the detector
temperature was 250 C. The carrier gas was helium
(2 mL/min). Identication of the individual components
was performed by comparison of mass spectra with
literature data and by a comparison of their reten-
tion indices (RI) relative to a C8-C32 n-alkanes mixture
(Adams, 1995). A computerized search was carried out
using the Wiley 275 L. GC/MS library and ARGEFAR
GC/MS library created with authentic samples.
Test microorganisms. In vitro antibacterial studies were
carried out against ten bacteria strains (Streptococcus
faecalis ATCC 8043, Bacillus subtilis ATCC 6633,
Staphylococcus aureus ATCC 6538, S. epidermidis
ATCC 12228, Pseudomonas aeruginosa ATCC 27853,
Enterobacter aerogenes CIP 6069, E. cloacae ATCC
13047, Escherichia coli ATCC 11230, Salmonella
typhimurium CCM 583, Proteus vulgaris ATCC6897)
and one yeast strain (Candida albicans ATCC 10239)
which were obtained from the Microbiology Depart-
ment Culture Collection of Ege University, Faculty
of Science. The bacteria strains were inoculated on
nutrient broth (Oxoid) and incubated for 24 h at 30
0.1 C, while the yeast strain was inoculated on malt
extract broth (Oxoid) and incubated for 48 h. Adequate
amounts of autoclaved Muller Hinton Agar (Oxoid)
and Malt Extract Agar were dispensed into sterile plates,
and allowed to solidify under aseptic conditions. The
counts of bacteria strains and yeast strain were adjusted
to yield approximately 1.0 1071.0 108 mL1 and 1.0
1051.0 106 mL1, respectively, using the standard
McFarland counting method. 0.1 mL of the test organ-
isms was inoculated with a sterile swab on the surface
of appropriate solid medium in plates. Tobramycin discs
(10 g/disc) and nystatin discs (30 g/disc) were used
as positive controls. The plates containing the bacteria
cultures and yeast culture were incubated at 25 0.1 C
and 30 0.1 C, respectively, for 1 h.
Antimicrobial testing. Antimicrobial activities of the
different S. platensis extracts were measured by the
paper disk diffusion method (Collins and Lyne, 1989;
Bradshaw, 1992). Briey, sterile, 6 mm diameter lter
paper discs (Schleicher and Schl, Nr 2668, Germany)
were impregnated with 2030 L of four different
concentrations (1, 2, 4, 8 mg disc1) of the respec-
tive S. platensis extracts and dried in an oven at 50
1 C. The agar plates inoculated with the test organ-
isms were incubated for 1 h before placing the extract
impregnated paper discs on the plates. Following
this, the sterile discs impregnated with the different
extracts were placed on the agar plates. The bacterial
plates were incubated at 30 0.1 C for 48 h while the
yeast plates were incubated at 25 0.1 C for 72 h.
After incubation, all plates were observed for zones of
growth inhibition, and the diameters of these zones were
measured in millimeters. All tests were performed
under sterile conditions in duplicate and repeated three
times.
RESULTS AND DISCUSSION
Chemical composition of the volatile compounds
The GC-MS method was used to determine the
composition of S. platensis volatile compounds. Fifteen
compounds were identied, constituting 96.45% of
the total component. The components are listed in
Table 1. The major component was heptadecane
(39.7%). Heptadecane has been reported to be a
common major volatile component in many other
cyanobacteria species.
Antimicrobial activity of the volatile component and
various extracts of S. platensis using disc diffusion
method
The antibacterial activities are reported in Tables 2
and 3.
In many studies, it has been shown that Spirulina has
a positive effect on human health. The high proportion
of linoleic acids found in Spirulina stimulates the
synthesis of prostaglandin (PGE1), a hormone that af-
fects the level of cholesterol in the blood (Fox, 1993).
Others have shown that Spirulina strengthens the

Table 1. Volatile components of S. platensis (GC-MS analysis)

R t (min) Compound Area (%)

9.78 Tetradecane 34.61

11.06 -Ionene 0.46
13.08 Pentadecane 3.20
13.84 2-Hexadecene 1.77
14.36 Hexadecane 2.18
14.58 Hexadecanenitrile (palmitonitril) 1.85
15.20 6(Z), 9(E) Heptadecadiene 1.18
15.31 8 Heptadecene 1.06
15.60 Heptadecane 39.70
17.17 Neophytadiene 2.05
17.57 Pentadecanenitrile 2.15
17.70 9,12-Octadecadienoic acid (linoleic acid) 1.01
17.94 Hexadecanoic acid, methyl ester (palmitic acid) 0.84
18.64 Isophytol 1.14
19.94 Phytol 3.25
Total 96.45

Copyright 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 754757 (2004)
756 G. OZDEMIR ET AL.

Table 2. Antimicrobial activity of S. platensis volatile components

Inhibition zone (mm)a

g/disc Standard
Microorganism G 0.0075 0.015 Tob Nys

Streptococcus faecalis ATCC 8043 + 7 9 nt

Bacillus subtilis ATCC 6643 + 8 24 nt
Staphylococcus aureus 6538/P + 7.5 16 nt
Staphylococcus epidermidis ATCC 12228 + 8 7 nt
Enterobacter aerogenes CIP 6069 8 14 nt
Enterobacter cloacae ATCC 13047 7 13 nt
Escherichia coli ATCC 11230 7 8 10 nt
Pseudomonas aeroginosa ATCC 27853 7 12 nt
Salmonella typhimurium CCM 583 9 10 nt
Proteus vulgaris ATCC 6897 7 9 13 nt
Candida albicans ATCC 10239 7 nt 18

a
Zone of inhibition, including the diameter of the filter paper disc (6 mm); mean value of three independent experiments; Tob,
tobramycin (10 g/disc); Nys, nystatin (30 g/disc); nt, not tested; G, gram reaction; , no activity.

Table 3. Antimicrobial activity of S. platensis extracts

Diameter of zone of inhibition (mm)

Methanol Dichloromethane Petroleum ether Ethyl acetate
extract (mg/mL) extract (mg/mL) extract (mg/mL) extract (mg/mL)
Microorganism 1 2 4 8 1 2 4 8 1 2 4 8 1 2 4 8

S. faecalis ATCC 8043 13 13 14 14

B. subtilis ATCC 6643 7 7 8 8 7
S. aureus 6538/P 7 7 7 7 7
S. epidermidis ATCC 12228 13 14 15 16 7 8
E. aerogenes CIP 6069 7 7 7 9 10
E. cloacae ATCC 13047 7 7 8 9 7 8
E. coli ATCC 11230 7 7 7 7 8
P. aeroginosa ATCC 27853 7 7 7 7
S. typhimurium CCM 583 7 7 8 9 7 7 7 8
P. vulgaris ATCC 6897 9 9 10 10 8 9 7 7 7 8
C. albicans ATCC 10239 10 11 12 13

defence mechanism of living organisms by augmenting
the functions of T lymphocyte cells. Furthermore,
Spirulina is used for AIDS and cancer treatments,
because of its well-known antitumoral characteristics
and it has been also shown that Spirulina can slow
the process of skin and stomach cancer (Manoj and
Venkataraman, 1992).
There is a growing interest in isolating antimicrobial
substances from cyanobacteria. In different studies, the
antimicrobial effects of Nostoc (Bloor and England,
1991), Phormidium (Fish and Codd, 1994), Anabaena,
Oscillatoria, Pseudoanabaena, Synechocystis (Kreitlow
et al., 1999), Oscillatoria anguistisima and Calothrix
parietina (Issa, 1999) extracts on some pathogenic
organisms have been reported. They have also re-
ported that the extracts extracted in different solvents
were effective against both Gram-positive and Gram-
negative organisms. This is in agreement with our
ndings, since the S. platensis extracts extracted in dif-
ferent solvents had similar effects on both types of
organism used in this study.
The methanol extracts of S. platensis (comparable
to tobramycin (10 g disc1), especially against Strepto-
coccus faecalis, Staphylococcus epidermidis and Candida
Copyright 2004 John Wiley & Sons, Ltd.
albicans) showed more potent antimicrobial activity
than the ethanol, hexane, acetone and chloroform
extracts.
Generally, when compared with the standard,
tobramycin, all extracts, except the methanol extracts,
exhibited low antimicrobial activity. The methanol and
ethanol extracts showed antifungal activity against
Candida albicans but less than that of the standard
nystatin.
The results obtained show a spectrum of antimicro-
bial activities that provide support to some traditional
uses of S. platensis extracts.
CONCLUSIONS
The volatile components of S. platensis, inhibited
the growth of the tested microorganisms. The meth-
anol extract (comparable to 10 g/disc tobramycin,
especially against S. faecalis, S.epidermidis and C.
albicans) showed more potent antimicrobial activity than
dichloromethane, petroleum ether, ethyl acetate extracts
and volatile components. In general, when compared
Phytother. Res. 18, 754757 (2004)
 ANTIBACTERIAL ACTIVITY OF SPIRULINA PLATENSIS
with tobramycin, all extracts except the methanol
extract, exhibited low antimicrobial activity. The metha-
nol extract showed antifungal activity against C. albicans
but less than that of nystatin. The results obtained indi-
cate a spectrum of antimicrobial activities that provide
support to some of the traditional uses of S. platensis
and show that the cyanobacteria have a potential use in
pharmaceutical sciences.
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Acknowledgements
This study was designed and performed in Ege University, Faculty of
Science, Department of Biology and Ege University, Center for Drug
R&D and Pharmacokinetic Applications. The authors would like to
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Phytother. Res. 18, 754757 (2004)