Biotechnology Letters, Vol 20, No 7, July 1998, pp.

643–646

Characterization of an extracellular
flocculating substance produced by a
planktonic cyanobacterium, Anabaena
sp.
Chang Won Choi, Soon-Ae Yoo, In-Hye Oh and Sang Ho Park1
Department of Biology, Pai Chai University, Taejon 302–735, and 1Korea Research Institutue of Bioscience and
Biotechnology, KIST, Yusong, Taejon 305–600, Korea. E-mail: choicw@woonam.paichai.ac.kr

Two planktonic cyanobacteria, Anabaena sp. N1444 and Anabaena sp. PC-1, and a green eukaryotic alga, Scene-
desmus sp., produced extracellular flocculants. The flocculant of Anabaena PC-1, when purified, was found to be a
macromolecular polysaccharide consisting of neutral sugars, uronic acids, and proteins, but not keto acids,
hexosamines nor fatty acids. The flocculant bound a cationic dye, Alcian Blue, indicating it to be polyanionic. The
flocculating activity was high under acidic conditions, slightly enhanced by the addition of salts and metals, and
increased to about 40% upon heating at 100 °C for 7 min. The flocculant could flocculated various inorganic and
organic compounds in solution.

Introduction cultures at 30 °C in inorganic BG-11 media (Allen, 1968)

Flocculants are used for sedimentation of colloidal sub-
stances and cellular materials and thus applied in a wide
range of industrial fields such as clarification of tap water,
dredging and industrial processes. A variety of micro-
organisms, including fungi (Nakamura et al. 1976; Nam et
al. 1996), and bacteria (Kurane et al., 1986; Shimofuruya et
al., 1996; Toeda and Kurane, 1991) produce various floc-
culating substances. Cyanobacteria also produce exopoly-
saccharides (Plude et al., 1991) and flocculants (Bar-Or and
Shilo, 1987) and therefore play an important role in this
field. One example is the excretion of a potent macro-
molecular flocculant by the benthic cyanobacterium Phor-
midium sp. J-1. (Bar-Or and Shilo, 1987). Planktonic
cyanobacteria produce extracellular polysaccharides (Wang
and Tischer, 1973), but there is little information on the
flocculating activities from these bacteria. In this study we
described that planktonic cyanobacteria produced some
flocculants capable of aggregating kaolin. Furthermore, we
have purified and characterized the properties of a floccu-
lant from Anabaena sp. PC-1.
Materials and methods
Cyanobacterial strains and growth conditions
Four planktonic cyanobacteria Anabaena sp. N1444, Ana-
baena sp. PC-1, Anabaena cylindrica, Microcystis sp. and a
green eukaryotic alga Scenedesmus sp. were from the culture
collection of our department, Pai Chai University, Korea.
The cyanobacteria were grown in axenic 100 ml batch
under ambient air and with rotary shaking. Light was
supplied by white fluorescent tubes at a photosynthetic
photon flux density of 10 mmol photons m-2s-1. Actively
growing cells were inoculated into the flasks to provide
initial cell densities of approximately 1.0 3 104–105 cells/
ml. To determine the cell growth, samples were taken at
weekly intervals starting after inoculation. An aliquot of
samples was serially diluted, spread onto BG-11 agar
plates and the plates were then incubated under light for
2–3 weeks at 30 °C. The colonies which appeared on plates
were counted.
Purification of the flocculant
Four weeks after inoculation, cyanobacterial culture from
the flask was centrifuged at 10,000 g for 10 min and
cetyltrimethylammonium bromide (Sigma) was added at
0.2% to the culture supernatant solution. The precipitate
was collected by centrifugation, dissolved in 0.15 M NaCl
and kept overnight for complete solubilization. Two volu-
mes of cold ethanol was added and the precipitate was
separated by centrifugation. The resulting pellet was
washed with ethanol, dried and dissolved in distilled water.
The resulting solution was then applied to a column of
Sephacryl S200 column (36.5 3 2.5 cm) pre-equilibrated
with distilled water. The column was then eluted with
distilled water. Each fraction (2.1 ml) was collected and
determined for the flocculating activity.

© 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 7 ⋅ 1998 643

C. Won Choi et al.
Table 1 The flocculating activities of two planktonic
cyanobacteria and a green eukaryotic alga
Kaolin binding Age of culture
Strain activity (%) (weeks)
Anabaena N1444 61 2
Anabaena PC-1 83 4
Scenedesmus sp. 46 7
Determination of the flocculating activity by kaolin
clay
Kaolin powder was suspended in distilled water at 5 g/l
and the solution was adjusted pH to 3 with HCl. Each
sample was added to 3 ml of this suspension in a test tube.
The final volume of the mixture was made up to 5 ml with
distilled water, and the mixture was vortexed. The floccu-
lating activity was measured at 550 nm and calculated by
the equation described by Shimofuruya et al.(1996).
Determination of Alcian Blue binding activity
The Alcian Blue binding assay was performed with a
minor modification by the method of Bar-Or and Shilo
(1988). Alcian Blue (Sigma) was dissolved in 0.5 M acetic
acid to give 1 mg/ml. After adding 850 ml of 0.5 M acetic
acid to a 100 ml sample, the solution was mixed. Then
50 ml of the Alcian Blue solution was added, and the
solution mixed again. After standing overnight the solu-
tion was centrifuged at 15,000 g for 30 min, and then the
absorbancy of the supernatant was read at 610 nm.
Analytical methods
The protein concentration was determined by Bio-Rad dye
reagent. Neutral sugar determination was done by the
method of Dubois (1956) using glucose as a standard.
Uronic acids were evaluated by the carbazole method
(Taylor and Buchanan-Smith 1992). Keto acids and amino
sugars were assayed by the method of Katsuki et al. (1971)
and of Reissig et al. (1955), respectively. For estimation of
fatty acid, the flocculants were hydrolyzed with 0.5 M
KOH for 90 min at 50°C, then extracted with petroleum
ether. The solvent extraction was evaporated, and the
residue was weighed.
Results and discussion
Four planktonic cyanobacteria and a green eukaryotic alga
were screened for production of extracellular flocculants. As
shown in Table 1, three strains except Anabaena cylindrica
and Microcystis sp. were found to have kaolin binding
activities during the cultivation. Therefore, this result
indicated that certain planktonic cyanobacteria could pro-
duce extracellular flocculants.
Since relatively high level of flocculant was detected in the
644 Biotechnology Letters ⋅ Vol 20 ⋅ No 7 ⋅ 1998
Figure 1 Growth of and production of extracellular floc-
culants by Anabaena sp. PC-1. The cultivation was done
with BG-11 medium under the conditions described in
Materials and Methods. 100 ul of each sample was
evaluated for kaolin binding activity.
Kaolin binding activity (j); cyanobacterial growth (d).
culture supernatants of Anabaena PC-1, the flocculant of
Anabaena PC-1 was further characterized. Growth and
extracellular flocculant production of Anabaena PC-1 are
shown in Fig.1. The flocculating activity steadily increased
until 4 weeks and then slightly decreased. The decrease of
flocculating activity in later stage of cultivation might be
due to a partial degradation of the product in cyanobacter-
ial culture. In addition, it was also observed that the liquid
culture medium became viscous with the cyanobacterial
growth indicating of the excretion of extracellular mate-
rials.
The purified flocculant solution showed high viscosity and
somewhat insoluble in cold water but highly soluble in salt
solution. The elution profile of gel-filtration showed that
the flocculating activity appeared at the void volume.
Furthermore, the active fractions were positive with the
phenol-sulfric reagent, indicating it to be a macromo-
lecular polysaccharide. The flocculant consisted of 63%
neutral sugars, 5% uronic acids, and 12% proteins of the
total weight of the flocculant, but not keto acids, hex-
osamines nor fatty acids. The remaining component is
under investigation. Alcian Blue is a cationic dye with a
high affinity for polyanions along with sulfates (Ramus,
1977). This reagent has been thus used for an indirect
assay for calculation of flocculant concentrations and for
comparison of the anion densities of different flocculants
(Bar-Or and Shilo, 1987; Ramus, 1977). When increasing
amounts of the flocculant were added to a constant amount

Figure 2 Binding of Alcian Blue by the flocculant.
Figure 3 Effect of pH on flocculating activity.
The pH of the suspended kaolin solution was adjusted
by the addition of 0.1 M HCl or 0.1 M NaOH and the
kaolin binding activity was measured.
of Alcian Blue, the flocculating activity was increased
curvilineraly (Fig. 2). Therefore, the flocculant of Anabaena
PC-1 may be polyanionic and/or sulfated polysaccharides.
The effect of pH on flocculating activity was examined. As
shown in Fig.3, the maximum flocculating activity was
observed at pH 2.0 and the activity was gradually
decreased until the pH reached to 10. It has been described
that the addition of various salts and metals induces the
effective flocculation of microbial flocculants (Toeda and
Kurane, 1991). However, the kaolin binding activity of the
Extracellular flocculants from cyanobacterium
Figure 4 Effect of heat treatment on flocculating activ-
ity.
The flocculant solution was boiled in a water bath for
indicated time and the kaolin binding activity was
measured.
flocculant was slightly enhanced by NaCl, NaNO3,
MgSO4, CaCl2 and LiCl at 0.001 to 1.0 M. The highest
activity (12% increase) appeared in 0.1M ZnSO4 solution.
Thermal stability of flocculant was also examined. 0.1%
solution of flocculant was heated at 100°C and the residual
activity was measured (Fig. 4). The flocculant maintained
its stability and its flocculating activity increased to about
40% upon heating at 100°C for 7 min. This result is
similar to the previous reports that the flocculants from
Aspergillus sp. (Nam et al., 1996), and Rhodococcus ery-
thropolis (Kurane et al., 1986) are heat-stable and increase
their flocculating activities rapidly during heating.
The flocculating activity was assayed against a variety of
suspended organic and inorganic compounds and chroma-
tographic resins. The materials tested were active carbon,
silica, aluminum oxide, bentonite, chitin, chitosan, pow-
dered agar and cellulose, DEAE, Dowex-50W, phosphate
cellulose, and Sephadex G-25. The flocculant from Ana-
baena PC-1 could flocculated all materials in aqueous
solution to some degrees in its activity depending on the
compounds. Therefore, the flocculant from Anabaena PC-1
seems to have a fairly broad range of substrate specificity.
In this study, we demonstrate that the planktonic cyano-
bacterium Anabaena PC-1 produces a flocculant. As this
flocculant has a broad substrate specificity, rapid flocculat-
ing activity and thermal stability, it may be exploited for
Biotechnology Letters ⋅ Vol 20 ⋅ No 7 ⋅ 1998 645
C. Won Choi et al.

many potential uses, such as wastewater treatment and
clarification of solar ponds, etc.
Acknowledgements
This work was financially supported by the Basic Science
Research Institute Program, Ministry of Education, 1996,
Project No. BSRI-96-4444.
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Received:- 11 March 1998

Revisions requested:- 8 April 1998
Revisions received:- 12 May 1998
Accepted:- 14 May 1998

646 Biotechnology Letters ⋅ Vol 20 ⋅ No 7 ⋅ 1998