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: kanchariya_p@kkumail.com, thesom@kku.ac.th

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