African Journal of Microbiology Research Vol. 6(10), pp.

2574-2579, 16 March, 2012

Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR12.152
ISSN 1996-0808 2012 Academic Journals

Full Length Research Paper

Antibacterial activity of crude extracts of cyanobacteria

Phormidium and Microcoleus species
Sirikul Thummajitsakul1,2*, Kun Silprasit3 and Siriporn Sittipraneed4
1
Mahidol University, Kanchanaburi Campus, 199 Moo 9 Saiyok District, Kanchanaburi 71150, Thailand.
2
Division of Health Promotion, Faculty of Health Science, Srinakharinwirot University Ongkharak,
Nakhon-Nayok 26120, Thailand.
3
Faculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Bangkok, 10110 Thailand.
4
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Accepted 20 February, 2012

Cyanobacteria is a group of prokaryotic organisms that occurred originally in the world. Thailand is well
known as an area of a high biological diversity. In our study, eighteen cyanobacteria from the natural
area were isolated and screened for their antibacterial activities. Of these, two isolated cyanobacteria
(NCI1 and NCI4) with positive antibacterial activities were identified by their morphology and 16S rRNA
sequences as Phormidium and Microcoleus group in family Oscillatoriaceae. The ethanol extracts of
Phormidium sp. and Microcoleus sp. at various concentrations (0.2, 0.06, 0.03 and 0.015 g/ml) showed
the antibacterial activity against Streptococcus enteritidis and Escherichia coli on the media. Each
cyanobacterial extract showed more inhibition zones against S. enteritidis and E. coli by increasing
their concentrations, especially those of Phormidium extracts. At the highest concentration 0.2 g/ml,
the maximum diameter of inhibition were observed in the extracts of Phormidium sp. and Microcoleus
sp., 11 and 10 mm against S. enteritidis, and 10 and 10 mm against E. coli, respectively. As results, the
extracts of Phormidium sp. and Microcoleus sp. showed potentially interesting antibacterial activities
against the two pathogens. Therefore, the two cyanobacteria may be useful in various applications and
used as basic knowledge for further investigations.

Key words: Phormidium sp., Microcoleus sp., antibacterial activity.

INTRODUCTION

Cyanobacteria are morphologically diverse group of
Gram-negative eubacteria. It is able to perform oxygenic
photosynthesis and used as important food for other
organisms. Moreover, it is widely found in various
locations such as pond, soil, rock, bark, sea and fresh
water (Carr and Whitton, 1982; Castenholz and
Waterbury, 1989). Cyanobacteria are several potential
*Corresponding author. E-mail: sirikult@swu.ac.th. Tel: +66-85-
2243708
Abbreviations: NCI1, Natural cyanobacteria Isolate 1; NCI4,
natural cyanobacteria Isolate 4.
benefits to study on bioactive compounds from these
organisms. Although, antibacterial, antiviral, algicide,
antifungal and cytotoxic activities have been many
researched in these organisms (Rao, 1994; Issa, 1999;
Pushparaj, 1999; Schlegel et al., 1999; Schaeffer, 2000),
the properties of the bioactive compounds are still not
completely understood (Inderjit and Dakshini, 1994).
However, cyanobacteria is able to produce many
bioactive compounds both intra- and extracellular to
survive in extreme environmental sources (Dvornyk and
Nevo, 2003; Kulik, 1995; Kreitlow et al., 1999; Patterson
et al., 1994). Some properties of bioactive compounds
from several cynabacteria are useful in medicine and
agriculture applications (Borowitzka, 1995; Kulik, 1995),
such as cryptophycin 1 agent with anticancer (Moore,

1996; Patterson et al., 1991). The discovery of several
bioactive compounds demonstrated the important
development of new organic agricultures because using
the natural compounds can be self-degraded and less
toxic than chemicals (Saxena and Pandey, 2001).
However, there are limited studies on certain
cyanobacteria such as Phormidium and Microcoleus.
Nowadays, the valuable natural resources are still
remaining abundance in Thailand. Therefore, the survey
of cyanobacteria from the natural regions may increase
the opportunities to find cyanobacteria with antibacterial
activity. Our goal is to determine antibacterial activity of
cyanobacterias isolated from the natural regions and
phylogenetic analysis of 16S rRNA sequences.
MATERIALS AND METHODS
Isolation and culture conditions
Samples were collected from natural different locations in Bangkok
and Kanchanaburi provinces of Thailand and carried to the
laboratory and kept under 4C until use. Samples were cultured
directly in BG-11 agar media and isolated by using streak plate
method (Richmond, 1986). Each isolated cyanobacteria was
cultured in a 500 ml flask containing 200 ml of BG-11 medium
without shaking, for 20-40 days incubated at room temperature and
illumination at 3000 lux with a white continuous light.
Preparation of cyanobacterial extracts
Cyanobacterial cells were dried at 60C, and then the cells were
grinded in sterile tubes. The cells were mixed with 80% ethanol for
1 ml with shaking for 5 min and kept in room temperature for 8 h.
After that, the solvent was removed by incubation at 60C and
redissolved in water (ratio 0.2, 0.06, 0.03 and 0.015 g/ml) and kept
at 4C until use for further assay.
Antibacterial test of cyanobacterial extracts
The antibacterial activities of cyanobacterial extracts were assayed
by agar disc diffusion (Bauer et al., 1966). Gram-positive bacteria,
Streptococcus enteritidis and Gram-negative bacteria, Escherichia
coli were used to as tested pathogens. Filter paper disks (6 mm)
were saturated with 10 l of each extract and placed on nutrient
agar plates with a lawn of the tested pathogens. Plates were
incubated at 37C for 24-28 h and inhibition zones were
determined. Ampicilins (100 g) was used as positive control. The
plates were incubated at 37C for 24-28 h and the inhibition zones
were measured as described above.
Deoxyribonucleic acid (DNA) extractions
Genomic DNA was extracted from isolated cyanobacteria cells
following standard methods (Sambrook et al., 2001) with slight
adjustments. Then, DNA was checked by running on a 0.7%
agarose gel (Sambrook et al., 2001).
PCR amplification and 16S rRNA sequencing
The purified DNA was used as a template for PCR amplification.
Thummajitsakul et al. 2575
The 16S rRNA gene was amplified using specific primers CYA359F
and CYA781R (Nbel et al., 1997). The 50 l of mixtures contained
1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 M of each primer, 0.1 mg/ml
bovine serum albumin, 1.5 U Taq polymerase, 1 buffer (Promega)
and 15 ng DNA. The PCR was carried out by an initial
predenaturation at 95C for 5 min, followed by 35 cycles consisting
1 min at 94C denaturation, 1 min at 60C and 30 sec at 72C. The
final extension was 7 min at 72C. The PCR products were checked
on 1% agarose gel by comparing with 100 bp standard DNA
marker.
PCR products were purified by PCR purification kit (QIAGEN)
and directly sequenced at Macrogen Inc., Korea. The nucleotide
sequences (partial 16S ribosomal RNA sequences) were analyzed
by using BLAST program at http://www.ncbi.nlm.nih.gov and
aligned using BioEdit program version 7.0.9.0 (Hall, 1999).
Phylogenetic analyses
Phylogenetic tree was constructed using the neighbor-joining
method which is implemented MEGA 4.0.2 (Tamura at al., 2007).
The tree topology was evaluated by 10,000 resampling bootstrap.
RESULTS
In the present study, eighteen cyanobacterias from the
natural areas were isolated and screened for their anti-
bacterial activities. Of these, two genera of cyanobacteria
(NCI1 and NCI4) with positive antibacterial activities
obtained from each fresh water pond in Bangkok and
Kanchanaburi province of Thailand, respectively.
Cyanobacteria, NCI1 and NCI4 were basically identified
by external morphology according to Peerapornpisal
(2005) and Anagnostidis and Komrek (1988). It indi-
cated that these two isolates were belonged to the family
Oscillatoriaceae (Figure 1). Likewise, they were
confirmed by 16S rRNA sequences. The 16S rRNA
sequences of the two isolates were analyzed by using
BLASTN program (www.ncbi.nlm.nih.gov) that provides
the highest similarity scores for cyanobacteria. It was
found that the 16S rRNA sequences of NCI1 and NCI4
cyanobacteria resemble to Phormidium sp. for 98 and
100% in Microcoleus sp., respectively. 16SrRNA
sequences of Phormidium sp. and Microcoleus sp. were
aligned with those of other cyanobacteria from the
GenBank by using BioEdit 7.0.4.1 (data not shown).
Phylogenetic tree was constructed using the neighbor-
joining method which is implemented MEGA 4.0.2 and
supported by 10,000 bootstrap replications. The result
indicated that the NCI1 and NCI4 could be recognized to
phylogenetically deferent clusters. The NCI1 and NCI4
were recognized in group Phormidium sp. and
Microcoleus sp., respectively (Figure 2). The effect of
ethanol extractions of cyanobacteria Phormidium sp. and
Microcoleus sp. on the inhibition of tested pathogens was
shown in Table 1. The results showed that ethanol
extracts of Phormidium sp. and Microcoleus sp. at
various concentrations 0.2, 0.06, 0.03 and 0.015 g/ml
exhibited the antibacterial activities against
Streptococcus enteritidis and E. coli. Inhibition activities
2576 Afr. J. Microbiol. Res.

Figure 1. Light photomicrographs of NCI1 (A) and NCI4 (B) corresponding to Phormidium sp.
and Microcoleus sp., respectively.

Figure 2. Neighbor-joining phylogenetic tree based on analysis of cyanobacterial 16S rRNA

sequences with other sequences obtained from GenBank Phormidium sp. and Microcoleus
sp. were underlined and asterisked, respectively. Numbers near the point at nodes indicate
bootstrap values.
 Thummajitsakul et al. 2577

Table 1. Antibacterial activity of Phormidium sp. and Microcoleus sp. extracted with 80% ethanol (diameter of inhibition zone in mm).

Pathogen bacteria
Species Phormidium sp. (g/ml) Microcoleus sp. (g/ml)
0.015 0.03 0.06 0.2 0.015 0.03 0.06 0.2
Streptococcus enteritidis 8 8 10 11 7 9 10 10
Escherichia coli 8 8 9 10 7 7 8 10

Figure 3. Antibacterial activity of Phormidium sp. and Microcoleus sp.

of the two cyanobacterial extracts were more effective at
high concentration against the tested pathogens at the
low concentration, especially those of the Phormidium
extracts (Figure 3). At the highest concentration 0.2 g/ml,
the maximum diameter of inhibition was noticed in the
extracts of Phormidium sp. and Microcoleus sp., 11 and
10 mm against Streptococcus enteritidis, and 10 and 10
mm against Escherichia coli, respectively. As results, the
extracts of Phormidium sp. and Microcoleus sp. showed
potentially interesting antibacterial activities against the
tested pathogens.
DISCUSSION
Cyanobacteria are groups of photosynthetic eubacteria
with diversity, approximately 7500 species (Chapman
and Chapman, 1975). In the present study, eighteen
cyanobacterias of Thailand were selected from the
natural area. Among them, two genera of cyanobacterias
(NCI1 and NCI4) which are shown in positive
antibacterial activities were obtained. Although,
cyanobacteria are widely distributed in various natural
regions (Carr and Whitton, 1982; Castenholz and
Waterbury, 1989), there are limitations in the study of
microorganisms from the environments because some of
them are not able to grow on synthesized mediums in the
laboratory. Therefore, the selection and study of
microorganisms from natural sources may provide less
species (Embry and Stackebrandt, 1997).
In the present study, NCI1 and NCI4 with positive
antibacterial activity were first identified by morphology
and confirmed by 16S rRNA sequence analysis. The 16S
rRNA sequences of the two isolates were analyzed by
using BLASTN program and aligned with other
cyanobacteria sequences from the GenBank. Honda et
al. (1999) reported about the phylogenetic analysis of
several cyanobacteria from 16S rRNA sequences.
In this study, phylogenetic tree was constructed using
the neighbor-joining method which is implemented MEGA
4.0.2 and supported by 10,000 bootstrap replication. The
result showed that, NCI1 and NCI4 were recognized in
2578 Afr. J. Microbiol. Res.
group Phormidium sp. and Microcoleus sp., respectively.
The species Phormidium and Microcoleus demonstrated
the effective extracts for antibacterial activity against
Streptococcus enteritidis and Escherichia coli. The
extracts of Phormidium sp. shows highest inhibition zone
to Streptococcus enteritidis (11 mm) at 0.2 g/ml and
Streptococcus enteritidis showed more sensitivity to the
cyanobacteria extracts than those of Escherichia coli.
Moreover, the cyanobacterial extracts showed more
effective inhibition activities by increasing their concen-
tration. It was reported that correlation between the
extracts concentration and the inhibition zone sizes in the
logarithm revealed the linear relationship (Crosby, 1991).
For other cyanobacteria species, Anabaena sp., it have
been reported that antibacterial properties against S.
aureus, E. coli, P. aeruginosa, S. typhi and K.
pneumoniae (Kaushik et al., 2009). It was imply that the
extracts of cyanobacteria may present diverse bioactive
compounds responsible for the antibacterial activity
(Kempf and Bremer, 1998; Schwartz et al., 1990). More-
over, many bioactive compounds may be excreted into
environment due to stress to survival of cyanobacteria
(Martin and Maris, 1995; Nicholson et al., 2000; Soltani et
al., 2006). It hypothesized that bioactive compounds can
damage microorganisms by attacking the cytoplasmic
membrane and penetrate the cell (Lampe et al., 1998;
Maillard, 2002; Ultee et al., 2000). The bioactive
compounds were able to disturb the Gram-posi-tive
bacteria by destabilizing the cytoplasmic membrane and
led to cell death (Ultee et al., 2000). In the Gram-negative
bacteria, the outer membrane of the bacteria showed the
hydrophilic surface on the side chains of lipopoly-
saccharides which restrict the entrance of hydrophobic
substances to the cellular membrane (Bergsson, 2005).
However, certain molecules can destabilize the
lipopolysaccharide layer, such as terpenoid and phenolic
compounds (Helander et al., 1998).
In the present study, it was concluded that the extracts
of Phormidium and Microcoleus species indicated the
potential of antibacterial activity against S. enteritidis and
E. coli. Therefore, the basic knowledge may useful in
various applications such as pharmaceutics and
agricultures, and for further investigations.
ACKNOWLEDGMENTS
This research project is supported by Mahidol University.
We would like to thank Mahidol University Kanchanaburi
campus for providing facilities and support. We also
thank Dr. Wuttinun Raksajit for increasing our
understanding of the cyanobacteria species suggestions.
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