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Isolation and Characterization of Algicidal
Bacterium against the Toxic Cyanobacterium
Microcystis Aeruginosa
Kanchariya Phankhajon and Theerasak Somdee Department of Microbiology/Faculty of Science/Khon Kaen University, Thailand
Email: [email protected], [email protected]
Abstract—Aquatic bacteria were isolated from eutrophic
lake in Khon Kaen Province, Thailand. One hundred and
eighty eight bacterial isolates were screened for anti-
cyanobacterial activity against the toxic cyanobacterium
Microcystis aeruginosa by well-diffusion and liquid-culture
methods. The result showed that five isolates (KKU-A3,
KKU-A6, KKU-C3, KKU-D4 and KKU-E5) were capable of
inhibiting M. aeruginosa. The strain KKU-A3 showed the
strongest anti-Microcystis by both methods. The pH optimal
conditions for the growth of isolate KKU-A3 were found to
be pH 8. Glucose and casein were found to be the most
suitable carbon and nitrogen sources, respectively. On the
basis of morphological, cultural, physiological and chemical
studies indicated that strain KKU-A3 belonged to the genus
Streptomyces.
Index Terms—Anti-Microcystis, Microcystis aeruginosa,
biological control
I. INTRODUCTION
Blooms of freshwater cyanobacteria are widespread in
eutrophic lakes and reservoirs throughout the world. One
of the most common and widespread bloom-forming
cyanobacteria is Microcystis aeruginosa. They can
produce hepatotoxic cyclic peptide, known as
microcystins. The toxins cause a serious water quality
problems and also pose threaten human and animal health
[1]. The direct removal and growth inhibition of the
bloom is the application of chemical algicides such as
copper sulfate, but this approach is potentially damaging
to the environment. Even where chemical treatments have
no immediate damaging effects on lakes there is a risk of
accumulation of microcystins released into the
environment from lysed cells [2]. Therefore, research on
the application of biological control agents, especially
algicidal bacteria is an alternative approach to eliminate
cyanobacterial population. The algicidal bacteria isolated
from water ecosystems have been assigned to the genera
Myxococcus, Alcaligenes, Pseudomonas, Streptomyces
and Bacillus [3]-[7]. The aim of the present study was to
isolate a strongest anti-Microcystis bacterium to inhibit M.
aeruginosa growth and to investigate some characteristics
of this bacterium.
II. MATERIALS AND METHODS
A. Cyanobacterial Species
M. aeruginosa strain KKU-13 were previously isolated
from Chulabhorn Dam, Chaiyaphum Province, Thailand.
The cultures were grown in MLA medium in a shaking
incubator at 25C and 150 rpm with fluorescent tubes and
under a light intensity of 20 µmol protons m-2
s-1
with a
12/12 h light/dark cycle. After incubation for 7 day,
culture of M. aeruginosa is typically in exponential
growth phase.
B. Isolation and Screening of Anti-Microcystis Bacteria
Bacterial strains were isolated from eutrophic lakes of
Khon Kaen province, Thailand by the double-layer algal-
lawn method [8]. Briefly, culture of M. aeruginosa were
grown in MLA broth for 7 days and harvested by
centrifugation. The culture pellet was mixed with the
MLA soft agar medium. The cyanobacterial lawn was
cultivated for 7 days at 25C under illumination (20 µmol
protons m-2
s-1
). A volume of 0.1 mL of lake water taken
during a bloom of M. aeruginosa was spread-plated onto
cyanobacterial lawn. After incubation for 5 days, the anti-
Microcystis bacteria showed as the clear zone of
inhibition on the lawn were isolated by serial streaked on
nutrient agar (NA) plates. Each bacterial isolates were
cultured on NA plates.
C. Anti-Microcystis Activity Test of Isolated Bacteria on
M. aeruginosa
The anti-Microcystis activity of isolated bacteria was
investigated by agar well diffusion method [9] and a
liquid-culture method [10]. In the first method, culture of
M. aeruginosa was grown in MLA medium for 7 days
and harvested by centrifugation at 5,000 rpm at 4C for
20 min. The culture pellet was mixed with the MLA agar
medium and poured onto a plate. After incubation under
illumination (20 µmol protons m-2
s-1
) for 7 day, the well
was cut with sterile cork borer (6 mm in diameter) and
100 µL of 48-h bacterial culture in starch casein broth
were transferred into each well of the algal lawn. The
inoculated plates were incubated at 25C under
illumination for 5 days. The anti-cyanobacterial activity
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 216doi: 10.12720/jolst.1.4.216-219
Manuscript received August 31 2013; revised November 15, 2013.,
was determined by measuring the diameter of the clear
zone formed in the algal lawn. Bacterial strains that
yielded a clear zone with width >10 mm were selected as
the first candidates.
In the replace this as liquid-culture method, the M.
aeruginosa were grown in MLA medium in a shaking
incubator at 25C and 150 rpm with fluorescent tubes and
under a light intensity of 20 µmol protons m-2
s-1
with a
12/12 h light/dark cycle for 5 days. The cyanobacterial
culture was diluted with sterile MLA broth until the
culture had an optical density at 750 nm was 0.1 (OD750 =
0.1). A volume of 45 mL M. aeruginosa culture was
inoculated with 5 mL of 48-h bacterial culture. Co-
cultures of M. aeruginosa and bacterial culture were
inoculated under the same condition for 7 days. The
growth of M. aeruginosa was monitored by measuring
the changes in chlorophyll-a concentration compared
with the control. The chlorophyll-a was determined as
described by Mu et al [11].
D. Characterization of Anti-Microcystis Strain
The micro-morphological observations were carried
out using light microscope and scanning electron
microscope (SEM). Under light microscope, the
bacterium was observed by gram staining [12]. For SEM
observation, the bacterium was cultured for 14 days on
agar medium and then fixed overnight with osmium
tetroxide vapor, freeze-dried, mounted on the stub,
sputter-coated with gold palladium and examined under a
scanning electron microscope.
Cultural features of strain KKU-A3 were characterized
by the International Streptomyces Project (ISP). Various
biochemical tests performed for the identification of the
potent isolates include the ability of KKU-A3 to nitrate
reduction, fermentation of citrate, liquefaction of gelatin,
hydrolysis of casein, hydrolysis of starch and urease.
E. Determination of Different Carbon, Nitrogen Sources
and pH for Growth
Strain KKU-A3 was cultured on basal medium at 30C
for 7-14 day. Basal medium was consisted of (g/L):
soluble starch 10.0, casein 0.3, K2HPO4 0.2, KNO3 2.0,
NaCl 2.0, CaCO3 0.02, FeSO4.7H2O 0.01,
MgSO4.7H2O 0.05, agar 20 [13]. The culture was grown
a 250-mL erlenmeyer flask containing 100 mL of basal
medium broth and incubated at 30C in an incubator
shaker at a shaking speed of 150 rpm for 7 days. Samples
were taken every-day for determination of growth.
Glucose, fructose, sucrose, xylose and lactose were
added to the basal medium supplemented with soluble
starch in order to study the effect of carbohydrate carbon
sources. Yeast extract, peptone, skim milk, malt extract
and ammonium nitrate, were added to the basal medium
supplemented with casein in order to study the effect of
nitrogen source. For the experiments investigating the
initial pH effect, the pH of 5.0-10.0 was used. Triplicate
samples were analyzed for cell dry weight. The culture
broth was centrifuged at 8,000 rpm at 4C for 20 min.
Harvested biomass was washed twice with distilled water
and then dried at 90°C to constant weight. The biomass
was determined gravimetrically.
III. RESULTS AND DISCUSSION
A. Screening of Anti-Microcystis Bacterium
A total of 188 bacterial strains were isolated from
eutrophic lake, Khon Kaen, Thailand. Among them, five
isolates showed anti-cyanobacterial activity against M.
aeruginosa. Strain KKU-A3 showed the strongest anti-
Microcystis activity from both the agar well diffusion and
liquid-culture method (Table I).
TABLE I. ANTI-CYANOBACTERIAL EFFECT OF BACTERIAL STRAINS
ON M. AERUGINOSA
Isolate Well diffusion clear zone (mm) Liquid-culture
KKU-A3 23 +++
KKU-A6 16 +
KKU-C3 16 +
KKU-D4 19 ++
KKU-E5 14 +
Liquid-culture assay was determined by measurement of chlorophyll-a
concentration after 7 days. Growth inhibition was compared with the control. +++, >85% inhibition of total; ++, >60% inhibition of total; and
+, >40% inhibition of total.
B. Characterization of the Anti-Microcystis Strain
TABLE II. CULTURE CHARACTERISTICS OF BACTERIAL STRAIN KKU-A3 ON ISP MEDIA AFTER 14 DAYS OF INCUBATION AT 30C
ISP media Growth Aerial mycelium Substrate mycelium
Tryptone-yeast extract (ISP1) Good White Yellow
Yeast extract-malt extract (ISP2) Good White Yellow
Oat meal (ISP3) Good White Yellow
Inorganic salt-starch(ISP4) Good White-yellow Yellow Glycerol asparagine (ISP5) Modulate White-yellow Yellow
Peptone-yeast extract-iron (ISP6) Poor - Yellow-brown
Tyrosine (ISP7) Good White Yellow-brown
The characteristics of strain KKU-A3 were compared
with those of the known species of bacteria described in
Bergey‘s manual of determinative bacteriology [14]. The
isolate was determined to be gram-positive and formation
of hard colony surfaces on agar plates. Fig. 1 shows a
scanning electron micrograph of aerial and vegetative
mycelia. Each mature spore chain was rectiflexible and
consisted of 10 or more spore. Strain KKU-A3 grew on
ISP media 1, 2, 3, 4, 5, 6 and 7, showing a typical
bacteria appearance with white to yellow aerial mycelium,
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 217
while that of the substrate mycelium was yellow-brown
(Table II). The physiological and biochemical
characteristics of KKU-A3 are shown in Table III. The
isolate was able to grow in all the examined carbon
sources (Fig.2). It produced highest biomass (5.49
mg/mL) in glucose supplemented medium. Of all the
tested nitrogen sources, casein was found to be the best
nitrogen sources for growth (Fig 3). Casein provided
highest biomass yield (5.52 mg/mL) and optimal pH was
found to be 8.0 (Fig. 4) after incubation for 5 day.
Figure 1. Scanning electron microphotograph of bacterial strain KKU-A3
0
1
2
3
4
5
6
0 1 2 3 4 5 6 7 8
Bio
mass (
mg/m
L)
Time (days)
Starch Glucose Fructose
Sucrose Xylose Lactose
Figure 2. Effect of carbon sources on cell growth of isolate KKU-A3
at 30C
0
1
2
3
4
5
6
0 1 2 3 4 5 6 7 8
Bio
mass (
mg/m
L)
Time (days)
Casein Yeast extract
Peptone Skim milk
Malt extract Ammonium nitrate
Figure 3. Effect of nitrogen sources on cell growth of isolate KKU-A3
at 30C
0
1
2
3
4
5
6
0 1 2 3 4 5 6 7 8
Bio
mas
s (m
g/m
L)
Time (days)
pH 5 pH 6 pH 7
pH8 pH 9 pH 10
Figure 4. Effect of pH on cell growth of isolate KKU-A3 at 30C
TABLE III. BA3
Characteristics Results
Gram‗s strain Gram Positive
Hydrolysis of starch +
Hydrolysis of casein +
Liquefaction of gelatin +
Nitrate reduction +
Fermentation of citrate
Urease +
Plus sign positive response, minus sign negative/no response
IV. CONCLUSION
One hundred and eighty eight isolates of bacterial
strains were isolated from eutrophic lake, Khon Kaen,
Thailand. Strain KKU-A3 showed the strongest anti
cyanobacterial activity against M. aeruginosa by two
methods: agar well diffusion and liquid-culture methods.
Glucose and casein were found to be the best carbon and
nitrogen sources respectively, while pH was found to be
8.0 for optimum production of growth. On the basis of
morphological, physiological and biochemical
characteristics of bacterial strain KKU-A3 were similar to
the genus Streptomyces.
ACKNOWLEDGMENT
This work was supported by The National Research
University (NRC) and Incubator Researcher‘s Project,
Khon Kaen University, Khon Kaen, Thailand.
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Kanchariya Phankhajon. I graduated Bachelor
degree in microbiology from Khon Kaen University. Now, I‘m studying Master degree in
microbiology at Khon Kaen University, Thailand.
The research area is algicidal bacterium against the toxic cyanobacterium Microcystis aeruginosa.
Assistant Prof. Dr. Theerasak Somdee completed his PhD at Massey University, New
Zealand in microbiology. The research areas are cyanobacterial bloom and cyanotoxins, especially
in biocontrol of toxic cyanobacteria and
cyanotoxins. Also, he is interested in bioactive compounds from cyanobacteria.
Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013
2013 Engineering and Technology Publishing 219