South As. J. Biol. Sci.

1(2)2011:87 98

Research Article (ISSN 2249 6599)

Antimicrobial activity of Spirulina platensis and Aphanothece sp. on selected

clinical bacterial isolates and its Antioxidant activity
S.S.Sudha, R.Karthic, J.Rengaramanujam and Athulya
P.G and Research Dept of Microbiology,
Dr.N.G.P. Arts and Science College, Kalapatti,
Coimbatore, Tamilnadu, India.

Abstract
The screening of antioxidant and antimicrobial potentiality of Spirulina platensis and
Aphanothece sp against multidrug resistant pathogens were investigated using standard
microbiological techniques and antioxidant is determined by FRAP and DPPH. The intracellular
and extra cellular extracts were tested by agar well diffusion method for activity against
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp, Proteus sp and Embedobacter
sp. isolated from clinical samples. The susceptibility patterns of the test isolates against the
extract intracellular and extra cellular was determined. The result showed that no significant
antibacterial activity by Aphanothece sp, in contrast intracellular extracts of S.platensis produce
antibacterial activity against above mentioned strains and S.platensis have more antioxidant
activity than Aphanothece sp. The observed antibacterial effects were believed to be due to the
presence of alkaloids, tannins, and flavonoids identified in the extracts. The results apparently
justified their use in the treatment of infections.
Key words: Spirulina platensis, Aphanothece sp, alkaloids, tannins, and flavonoids.

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Introduction
Several studies have focused on physiological properties of some valuable antiviral or
antioxidant compounds in blue green alga Spirulina1,2. The occurrence of many compounds
possess antioxidant activity in biological systems in higher plants is well documented, while in
microalgae are less documented3. The presence of certain substances act as antioxidant or free
radical scavenger may protect the body from the consequences of oxidative stress. Thus,
antioxidants play an important role in the protection of cells against oxidative damage caused
by ROS4,5. Among algal species, Spirulina has been reported to prevent oxidative damage by
scavenging free radicals and active oxygen, and hence can indirectly reduce cancer formation in
human body. In this respect, the increased consumption of foods characterized by free radical
scavenging activity, leads up to a doubling of protection against many common types of cancer
formulation6. Spirulina platensis or its extracts show therapeutic properties, such as the ability to
prevent the incidence of cancers, decrease blood cholesterol levels, stimulate the immunological
system, reduce the nephrotoxicity of pharmaceuticals and toxic metals and provide protection
against the harmful effect of radiation

7,8

. Although extremely effective, antibiotics are able to

induce resistance in bacteria. For 450 years, bacterial resistance has been the main factor
responsible for the increase of morbidity, mortality and health care costs of bacterial infections.
The defense mechanism against antibiotics is widely present in bacteria (e.g. Pseudomonas,
Klebsiella, Enterobacter, Acinetobacter, Salmonella, Staphylococcus, Enterococcus and Streptococcus) and
became a world health problem9. Some Spirulina species exhibit antibacterial activity10. Spirulina
as many other cyanobacteria species have the potential to produce a large number of
antimicrobial substances, so they are considered as suitable organisms for exploitation as
biocontrol agents of plant pathogenic bacteria and fungi

11

. Other studies have found a

significant coupling of cyanobacterial primary production and bacterial secondary production,

particularly during the development and cessation of blooms including Aphanothece sp

12,13

According to the authors, the cyanobacterial exudates represented a striking proportion of

bacterial organic carbon supply. Antimicrobial compounds found in cyanobacterial exudates
include polyphenols, fatty acids, glycolipids, terpenoids, alkaloids, and a variety of yet to be
described bacteriocins14. The present work was conducted to study the antioxidant and
antimicrobial activities as well as the phytonutrients of Spirulina platensis and Aphanothece sp.
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Materials and methods

Collection of cyanobacterial samples
Cyanobacteria found as mat in the water, flakes on the dried sediment and on
pneumatophores were collected and transport to laboratory in sterile polythene bags as well as
in plastic vials containing sterile ASN III medium. The samples were transferred to Erlenmeyer
flasks containing the ASN III medium for further identification.
Cyanobacterial identification
The collected samples were observed by microscopic and macroscopic view and
identification of cyanobacteria was made by using the standard taxonomic manuals of Geitler 15
and Desikachary16.
Spirulina platensis
The blue green algae, Spirulina platensis was obtained from the OFERR Nallayan Research
Centre, Chennai and the cultures are grown in ASN III media had kept in room temperature
near north facing window in natural light.
Growth conditions
Large-scale cultivation on ASN III medium containing different concentration of Nacl,
Mgcl2, Kcl, NaNo3, K2HPo4.3H2O, MgSo4.7H2O, CaCl2.2H2O, Citric acid, Ferric ammonium
citrate, EDTA, Na2CO3.H2O, Trace elements at pH 7.5 were done in five aquariums (16 L, each).
The cultures were gassed with air containing 0.3% CO2 (v/v), and continuously illuminated
with two cool white fluorescent lamps (40 W each, Philips). The culture temperature was
maintained at 28 2 C.
Growth measurements
Algal growth was spectophotometrically measured as described by Payer (22). The
calculated biomass (the average of three experiments) was used to obtain maximum specific
growth rates (mmax) from the log phase of the growth curves by exponential regression.
Productivities was calculated from the equation P= (Xi -X0) / ti, where P = productivity (mg L1

day-1), X0 =initial biomass density (mg L- 1), Xi = biomass density at time i (mg L-1) and ti =time

interval (h) between X0 and Xi17 .

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Drying
The harvested algal biomass were collected in aseptic plastic bags and subjected to
drying under direct sunlight. After complete drying, the dried algal biomass was placed in hot
air oven at 600C for one hour. Until further use, the algal biomass was stored under refrigerated
condition (40C).
Preparation of algal extracts
Freshly dried Spirulina platensis and Aphanothece sp was mixed with ethanol (150ml
solvent/100g of Algae) in soxhlet apparatus and extracted for 60 minutes. The extracts were
filtered and the solvent was removed by air drying. The extracts were stored in an airtight glass
bottles in a refrigerator for the analysis of phytonutrients and other findings.
Phytochemical Analysis
Qualitative analysis of phytonutrients of algal extracts: Qualitative analysis of phytonutrients
was done for ethanolic extract 18 of Aphanothece sp and Spirulina platensis.
Test for carbohydrates
A small quantity of extract was dissolved separately in 5 ml of distilled water and
filtered. The filtrate was tested to detect the presence of carbohydrates.
Molischs test: To 2ml of algal extract, 2 ml of Molischs reagent was added. Then, 2 ml of
concentrated sulfuric acid was added along the sides of the test tubes. Disappearance in color on
the addition of excess solution indicated the presence of carbohydrates.
Benedicts test: To 0.5 ml of extract, 5 ml of Benedicts reagent was added. The mixture is then
boiled for 5 minutes. Presence of a bluish green precipitate indicated the presence of
carbohydrates.
Test for Glycosides: To 2ml of algal extract 1ml of aqueous NaOH solution was added. The
appearance of a yellow color indicated the presence of glycosides.

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Test for Proteins and Amino acids

Ninhydrin test A small quantity extract solution was boiled with 0.2% solution of ninhydrin.
Purple color indicated the presence of free amino acids.
Test for Phytosterols and Triterpenoids
Salkowski test: To 2 ml of the algal extract, 1 ml of concentrated sulfuric acid added.
Chloroform was added along the sides of the test tube. A red color produced in the chloroform
layer indicated the presence of Phytosterols or if it is yellow in color at the lower layer indicated
the presence of triterpenoids.
Test for Flavanoids
Zinc hydrochloride reduction test: The extract was treated with mixture of zinc dust and
concentrated hydrochloric acid. Red color indicated the presence of flavanoids.
Test for Alkaloids: A small portion of the solvent free extract was stirred separately with a few
drops of dilute hydrochloric acid and filtered. The filtrate was tested with Mayers reagent
(Potassium mercuric iodide solution). The cream precipitate indicates the presence of alkaloids.
Test for Tannins
Gelatin test: To 5 ml of algal extract, few drops of 1 % lead acetate were added. Absence of a
yellow or red precipitate indicated the absence of tannins.
Test for Saponins: To 5 ml of the algal extract, a drop of sodium bicarbonate was added. It was
then shaken vigorously and kept undisturbed for 3 minutes. Appearance of a honey comb like
froth indicated the presence of saponins.
Estimation of In-vitro antioxidant activity
In these experiment, two complementary methods of free scavenging activity; DPPH radical
scavenging test and FRAP were used.
DPPH assay
1 mg extract powder was dissolved in 1 ml of 50% ethanol solution to obtain 1000 g/ ml
sample solution. 1000 g/ml solutions were series diluted into 1 g/ml, 5 g/ml, 10 g/ml, 20
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g/ml, 50 g/ml, 100 g/ml, 200 g/ml, 500 g/ml, and 1000 g/ml with 50% ethanol. In each
reaction, the solutions were mixed with 1 ml of 0.1 mM 1,1-Diphenyl-2- picrylhydrazyl (DPPH),
0.45 ml of 50mM Tris-HCl buffer (pH 7.4), and 0.05 ml samples at room temperature for 30 min.
50% ethanol solution was used as control. The reduction of the DPPH free radical was measured
by reading the absorbance at 517 nm. DPPH, a purple-colored, stable free radical is reduced to
the yellow-colored diphenyl picrylhydrazine when antioxidants are added. L-ascorbic acid and
(+)-catechin were used as positive controls. The inhibition ratio (percent) was calculated from
the following equation: % inhibition = [(absorbance of control absorbance of test
sample)/absorbance of control] x 100%. The antioxidant activity of each sample was expressed
in terms of IC50 (micromolar concentration required to inhibit DPPH radical formation by
50%), calculated from the inhibition curve.
FRAP assay
The procedure described by Benzie and Strain was followed17 . The principle of this
method is based on the reduction of a ferric-tripyridyltriazine complex to its ferrous colored
form in the presence of antioxidants. The FRAP assay measures the change in absorbance at 593
nm owing to the formation of a blue colored FeII-tripyridyltriazine compound from the colorless
oxidized FeIII form by the action of electron donating antioxidants. The FRAP reagent consist
of 300 mM acetate buffer (3.1 g sodium acetate + 16 mL glacial acetic acid, made up to 1 L with
distilled water; pH = 3.6), 10 mM TPTZ in 40 mMHCl and 20 mM FeCl3.6H2O in the ratio of
10:1:1. Briefly 50 L of sample supernatant was added to 1.5 mL freshly prepared and pre warmed
(37C) FRAP reagent in a test tube and incubated at 37C for 10 min. The absorbance of the blue
colored complex was read against reagent blank (1.5 mL FRAP reagent+50 L distilled water) at
593 nm. For construction of the calibration curve, five concentrations of FeSO4 7H2O (1000,
750, 500, 250 and 125 mol L-1) were used and the absorbencies were measured as sample
solution. The data was expressed as mole ferric ions reduced to ferrous form per liter (FRAP
value).
Antimicrobial activity of crude algal extract
Test organisms
18 hours incubated peptone broth of clinical isolated cultures (equilibrated to Mcfarland
standard turbidity scale 10 5 -10 6cfu/ml for bacteria) of Staphylococcus aureus, Escherichia coli,
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Pseudomonas aeruginosa, Klebsiella sp, Proteus sp and Embedobacter sp, were used in the antibacterial
studies. The test strains are collected from KMCH of Kovai Medical Centre.
Preparation of the crude algal extract
The algal culture was filtered and the filtrate was used as the extracellular extract. The
biomass obtained was grinded and used as the intracellular extract.
Agar well diffusion technique
Sterile Mueller Hinton agar plates were prepared and swabbed with the clinical isolated
cultures. Using a sterile cork borer, wells were cut in the Mueller Hinton agar plates.
Intracellular extract, extracellular extract were added into their respective wells. The
appropriate standard antibiotic discs for each organism were placed on the plates. The plates
were subjected to incubation at 37C for 24 hours. After incubation, the zones of inhibition were
examined.

Result and Discussion

Spirulina is a microscopic blue-green alga in the shape of a spiral coil, living both in sea
and fresh water. Spirulina is the common name for human and animal food supplements
produced primarily from two species of cyanobacteria: Arthrospira platensis, and Arthrospira
maxima19. Antimicrobially active lipids and active fatty acids are present in a high concentration
in this alga 20. It was hypothesised that lipids kill microorganisms by leading to disruption of the
cellular membrane21 as well as bacteria, fungi and yeasts because they can penetrate the
extensive meshwork of peptidoglycan in the cell wall without visible changes and reach the
bacterial membrane leading to its disintegration. This can probably be explained by the strong
fabric of the cell wall of Gram-positive bacteria, which maintain their structure in spite of
substantial hydrostatic turgor pressure within the bacteria22. Among the collected
cyanobacterial culture sample from muthupet mangrove
Aphanothece sp plays an predominant role. It is under the 45X of the microscope,
Aphanothece were visualized as green to olive green colored ovoid cells. The cells were embeded in
mucilage and there was no sheath around the cell and the cells of the Aphanothece plated on the
ASN III medium containing 0.5% agar and incubated at room temperature showed asymmetric
mucilaginous spreading growth .The color of the algae in nature as well as in the culture was
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dirty brown due to Phycoerythrin pigment. So the Aphanothece sp choosen for examination of
various parameters with S.platensis. The results obtained from the present study concerning the
antimicrobial activity of Spirulina platensis and Aphanothece sp crude extract against different species of
bacteria are recorded in table 1.
Table: 1 Antibacterial activity of Aphanothece and Spirulina

Microorganism
Escherichia coli

Effect of algal crude extracts

(Zone of inhibition diameter in mm)
Spirulina platensis
Aphanothece sp
Crude extract
Crude extract
11
R

Klebsiella pneumonia

Pseudomonas aeruginosa

Antibiotics
(Inhibition zone
diameter in mm)
17((cf))
13(cf)
12(ce)

R
Proteus sp

14(ce)
R

Empedobacter sp

10

13(t)
R

Staphylococcus aureus

15(cf)
R

It is clear from study that the diameter of the inhibition zone depends mainly on the
crude extracts used and the tested bacteria. The phytochemical characters of the two algae
(S.platensis and Aphanothece sp) were investigated are summerized in Table 2. Carbohydrates,
alkaloids, flavonoids, glycosides, proteins and saponins were present in S.platensis and
carbohydrates, terpenes, flavonoids, tannins were present in Aphanothece sp. The crude extract of
Spirulina platensis showed maximum antimicrobial activity of 11.0 mm against Escherichia coli and a
adequate activity of 6.0 mm against Proteus sp, 7mm against Klebsiella pneumonia,9mm against
Pseudomonas aeroginosa,10 mm against Empedobacter sp and 8mm against Staphylococcus aureus. All the
tested microorganisms were resistant to crude extracts of Aphanothece sp and the crude extracts
of S.platensis was better antimicrobial than Aphanothece sp extracts.

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Table 2. Preliminary phytochemical analysis of ethanolic extract of the cyanobacterium

Spirulina and Aphanothece
Constituents
Carbohydrates

Test

Spirulina

Aphanothece

Molischs test

++

++

Benedicts test

++

++

Glycosides

Killer-killanis test

++

--

Amino acids/Protein

Ninhydrin test

++

--

Terpenes and steroids

Salkowski test

--

++

Flavonoids

Zinc hydrochloride reduction test

++

Alkaloids

Meyers test

--

Phenolics /Tannins

Gelatin Test

--

Saponins

Frothing test

++

--

Key: - = Negative (absent); + = Positive (slightly present); ++ = Positive (moderately present)

Antioxidant activity says chemical assay based on the removal of stable DPPH free
radical has been shown to indicate the presence of reducing compounds in terms of hydrogen
donation capacity in the test system. Purple color of the freshly prepared DPPH solution fades
when an antioxidant compound is present in the test solution, to quench DPPH; the
antioxidant molecule provides a hydrogen atom or donates an electron, to DPPH23. The table 2
shows the results of the DPPH free radical scavenging activity, the absorbance of DPPH
solution changed when the algal extracts were added to the solution. In the present study the
antioxidant activity of the S.platensis sample at concentration of 15mg/ml exhibited about
51.94% DPPH scavenging activity with IC50 value is 14.43 and Aphanothece sp sample at a
concentration of 20mg exhibited 16.31% DPPH scavenging activity with IC50 Value is 61.31.
The FRAP assay showing the result for S.platensis is 1400(micromoles/mg) and Aphanothece sp is
720 (micromoles /mg).
It is intended that the present work will contribute to an understanding and determining
the phytonutrients, antimicrobial and antioxidant factors of S.platensis and Aphanothece sp.
Because antimicrobial metabolites of algae are of special interest in the development of new
harmless environment.

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Table 3: The DPPH and FRAP assay

Algae species

Spirulina

Aphanothece

Sample
concentration

% of DPPH
Scavenged

0.3

0.8

0.6

0.17

1.5

3.75

3.0

10.90

6.0

21.18

15.0

51.94

0.4

Negative

0.8

Negative

2.0

0.18

4.0

3.80

8.0

8.92

20.0

16.31

FRAP

1400(micromoles/mg)

720 (micromoles /mg)

Acknowledgement
The authors are grateful to University Grant Commission (UGC) for the financial
support given to the present study under the Major Research Project programmed entitled
Investigation on crude and bioactive compounds from marine micro algae for preventing
Coronary Heart Disease (Sanction No. F. 38-56/2009 (SR) dated on 19.12.2009). The authors
are thankful to the management of Dr.N.G.P. Arts and Science College for their encouragement
and support.

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