ARTICLE IN PRESS

Bioresource Technology xxx (2006) xxx–xxx

Characterization of a novel bioflocculant, p-KG03,

from a marine dinoflagellate, Gyrodinium impudicum KG03
Joung Han Yim, Sung Jin Kim, Se Hun Ahn, Hong Kum Lee *

Korea Polar Research Institute, KORDI, Ansan P.O. Box 29, Ansan, Seoul 425 600, South Korea

Received 25 July 2005; received in revised form 19 December 2005; accepted 19 December 2005

Abstract

The flocculating activity of an exopolysaccharide, p-KG03, produced by a marine dinoflagellate Gyrodinium impudicum KG03 was
investigated. The p-KG03 was a highly sulfated exopolysaccharide that showed strong antiviral activity against encephalomyocarditis
virus (EMCV) and immunostimulating activity by NK cell activation. For the industrial applications of p-KG03, as the bioflocculant
agent, p-KG03 showed that more than 90% of the flocculating activity in kaolin suspension occurred at concentrations of 0.5 mg/l with
the maximum at 1.0 mg/l. However, flocculation decreased from 2.5 mg/l. The flocculation rate increased linearly with concentration and
was higher than that observed in commercial products such as polyacrylamide (
1.0 mg/l) or zooglan (
3.0 mg/l). The p-KG03 was an
effective flocculant under acidic conditions (pH 3–6) and over a wide temperature range (4–90
C). The presence of cations did not enhance
flocculating activity. The average molecular mass, as determined by gel filtration chromatography, was about 1.87 · 103 KDa. Galactose
was the main sugar in p-KG03, which also contained uronic acid (2.9%, w/w) and sulfate groups (10.3%, w/w). The infrared spectrum of p-
KG03 showed absorption bands of carboxylate groups. Thermogravimetric analysis indicated a degradation temperature (Td) of 250
C.
Several other properties of p-KG03 such as intrinsic viscosity, the rheological behavior, consistency index (k) and flow behavior index (g)
were also studied.
 2006 Elsevier Ltd. All rights reserved.

Keywords: Bioflocculant; Gyrodinium impudicum; Marine dinoflagellate; Rheological properties; Sulfated exopolysaccharide

1. Introduction flocculants (polyacrylamide, polyethyleneimine), and natu-

Exopolysaccharides from marine microorganisms such
as Zoogloea sp., Pseudomonas sp., Cyanothece sp. and
Altrermonas maleolii have been investigated as potential
new biomaterials (Philippis et al., 1993; Raguenes et al.,
1996). Bioflocculation is a dynamic process resulting from
the synthesis of extracellular polymers by living cells. A
variety of flocculants, including inorganic flocculants (poly-
aluminium chloride and aluminium sulphate), organic
Abbreviations: k, consistency index; g, flow behavior index; gapp, apparent
viscosity; gr, relative viscosity; gsp, specific viscosity; gred, reduced viscos-
ity; gInh, inherent viscosity
*
Corresponding author. Tel.: +82 31 500 4511; fax: +82 31 500 4510.
E-mail address: hklee@kopri.re.kr (H.K. Lee).
ral flocculants or bioflocculants (gelatin, chitosan guar
gum and microbial flocculants) have been widely used in
chemical and mineral industrial fields such as tap water
production, wastewater treatment, dredging, downstream
processing, fermentation, and food industries (Koizumi
et al., 1991). Although chemical flocculants have been
widely used because of their effective flocculating activity
and low cost, they have neurotoxic and carcinogenic mono-
mers, and their use is restricted (Vanhoric and Mones,
1983). In recent years, the use of microbial flocculants
has been promoted as a solution to environmental prob-
lems because their intermediates are harmless and biode-
gradable. The rheological behavior of exopolysaccharides
reflects the ordered conformation and consequent intermo-
lecular interactions usually adapted in aqueous solutions.

0960-8524/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.12.021
 ARTICLE IN PRESS
2 J.H. Yim et al. / Bioresource Technology xxx (2006) xxx–xxx
Rheological properties provide significant information
regarding the optimization of formulations on the basis
of the relationship between microstructure and physical
properties, and objective textural analysis of the commer-
cial products (Cuvelier and Launay, 1986). Gyrodinium
impudicum, a harmful red-tide dinoflagellate, is known to
produce a mucous exopolysaccharide (Park and Park,
1999), and its sulfated polysaccharide, p-KG03, extracted
from G. impudicum, is a selective inhibitor of in vitro viral
replication, especially EMCV (Yim et al., 2004). Herein,
some of the biochemical and rheological characteristics of
purified p-KG03 produced by this strain for biotechnolog-
ical applications are reported.
2. Methods
2.1. Strain and production conditions
The microalgal strain KG03 was originally isolated from
the marine red-tide microalga, G. impudicum, which origi-
nated from a seawater sample that was collected in the
coastal region of Kang-Gu, Korea. This strain was main-
tained in f/2 culture medium at 22.5
C under a light inten-
sity of approximately 55 lmol/m2/s and a 14-h light:10-h
dark illumination cycle. Bacterium-free cultures of G. impu-
dicum were obtained by the modified phototaxis behavior
method and antibiotic treatment (Yim and Lee, 2004).
The bacterium-free strain of G. impudicum was designated
KG03 and was used in the following experiments. It was
grown aseptically in a 10-l photoreactor. The production
conditions were 1% CO2, 50 ml/min airflow, production
medium (M-KG03) at 22.5
C, light intensity of approxi-
mately 150 lmol/m2/s and a 16-h light:8-h dark illumina-
tion cycle (Yim et al., 2003).
2.2. Purification of bioflocculant
Cells were removed from the culture medium by centri-
fugation at 12,000g for 30 min at 4
C. The polysaccharides
were separated from the supernatant by the addition of two
volumes of ethanol and precipitation at 4
C for 24 h. The
precipitated polysaccharides were collected by filtration
(Whatmann GFF filter), dissolved in deionized water,
and lyophilized. Protein concentration of crude p-KG03
was 2.58 ± 0.31% by Lowry method (1951). For the exclu-
sion of protein, crude p-KG03 was treated by protease
(500 unit/l) at 37
C for 3 h, dialyzed against deionized
water and lyophilized. Crude p-KG03 was dissolved in
deionized water and reprecipitated by addition of a 10%
solution of cetylpyridinium chloride (CPC). The precipi-
tated CPC-polysaccharide complex was collected by centri-
fugation at 10,000g for 20 min at 4
C and re-dissolved in a
10% NaCl solution. The precipitated polysaccharide was
recovered by addition of three volumes of ethanol. The
extracted polysaccharide was dissolved in deionized water,
dialyzed twice against deionized water and lyophilized.
Further fractionation and purification were achieved using
gel chromatography on a Sepharose 4B column (Sigma,
USA), followed by elution with a 0.4 M NaCl buffer. Each
fraction was analyzed using the phenol–sulfuric acid
method (Dubois et al., 1956) and protein contamination
was monitored at A280. The main peak was 50–57 fractions
(data not shown). The fractions of the main peak were
collected, dialyzed using Viva-Flow (Sartorius, Germany),
and lyophilized.
2.3. Measurement of flocculating activity
Assays of flocculating activity were performed according
to the Kurane methods of using a suspension of kaolin clay
as a test material with minor modifications (Kurane et al.,
1986). The kaolin clay (300 mesh, Junsei Chemical Co.,
Japan) was suspended in distilled water at a concentration
of 5000 mg/l (kaolin suspension). In a test tube, 19.2 ml of
kaolin suspension was added and mixed with 0.4 ml of 5%
CaCl2 solution. To this mixture, 0.4 ml of the test p-KG03
concentrated solution was added, vortexed for 30 s, and
allowed to stand for 5 min at room temperature. One milli-
liter of the supernatant (A) was carefully removed from
the upper layer. The absorbance of the upper phase was
measured at 550 nm using a spectrophotometer (Shimadzu,
UV-VIS2410PC, Japan). A control solution (B) was pre-
pared in the same manner, except that 0.4 ml of distilled
water was addition to the suspension instead of the p-KG03
solution, and the absorbance was measured. Flocculating
activity (%) was defined and calculated as [(B  A)/B] · 100.
2.3.1. Flocculation of various suspended solids
The flocculating activities of various inorganic solid sus-
pensions, including activated carbon, solid clay, Ca(OH)2,
and aluminum oxide were studied, with kaolin solution
(5 g/l) being replaced by the various solid suspensions
(5 g/l). A soil suspension was prepared by adding 500 g
of soil to 1 l of distilled water. After stirring and letting
the mixture stand for 3 min, the upper phase was used
for the flocculation test.
2.3.2. Effects of various salts, pH, and temperature
on flocculating activity
To examine the effects of salts, flocculant tests were con-
ducted using the method described above, except that the
CaCl2 solution was replaced by various metal salt solutions,
and the flocculating activity was measured. Solutions of
KCl, NaCl, MgCl2, CaCl2 Æ 2H2O, FeSO4 Æ 7H2O and
FeCl3 Æ 6H2O were used as cations sources. To investigate
the effect of pH on flocculating activity, the pH of the kaolin
suspension was adjusted using HCl and NaOH in the pH
range of 3–11. The effects of temperature were examined
using kaolin and bioflocculant at various temperatures.
2.4. Analytical procedures
For the analysis of sugar components, purified p-KG03
was hydrolyzed with 2 M H2SO4 for 3 h at 100
C in a
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sealed glass tube. After cooling, the solution was neutral-
ized with CaCO3. The mixture was filtered, and the solu-
tion was passed through an exchange column (Amberlite
IR-120-H+ resin), and then concentrated. Thin-layer chro-
matography (TLC) and high-performance liquid chroma-
tography (HPLC) analyzed the neutral sugar content of
the p-KG03. TLC was performed using silica gel plates
(60F254S TLC; Merck, Germany). The plates were devel-
oped in two steps: the first separation was developed in
5 cm of a mixture of n-butanol:acetic acid:water (3:1:1),
and the second separation was developed in 10 cm of a
mixture of ethyl acetate:pyridine:water:acetic acid (35:35:
25:5). The chromatogram was developed using an orci-
nol–sulphuric acid reagent. HPLC was carried out with
Hewlett–Packard equipment and an evaporative light scat-
tering detector (ELSD). Waters carbohydrate analysis
column was used at 35
C with acetonitrile:water (80:20,
v/v) as the solvent. The peak molar masses (Mpk) were esti-
mated at 35
C by gel-permeation chromatography (GPC)
using the Hewlett–Packard equipment, the RI detector,
and Showdex-GPC 804, 805, and 860 M columns, with
0.4 M NaCl as the elution buffer. The GPC was calibrated
using pulluan standards (Shodex, Japan). The sulfate con-
tent was confirmed after hydrolysis of p-KG03, using a
Shimadzu HIC-6A Ion Chromatograph with a Shimpack
IC-A1 column (4.6 · 100 mm), a conductivity detector,
and 2.5 mM phthalic acid that contained 2.4 mM Tris–
aminomethethane (pH 4) at 40
C at a flow rate of 1.0
ml/min. The uronic acid concentration was determined
using the meta-hydroxydiphenyl method (Blumenkrantz
and Asboe-Hansen, 1973). Infrared (IR) spectra were ana-
lyzed on KBr pellets using a Nicolet Magna 550 FT-IR
spectrophotometer. Analysis of the elements contained in
p-KG03 was obtained using an elemental analyzer
(EA1108 Elemental analyzer, Carlo-Erba Inc., USA). The
degradation temperature of the biopolymer was achieved
using a TGA Q50 (TA Instruments-Waters LLC, Dela-
ware, USA). An accurately weighed sample of the polymer
(20–25 mg) was heated from 30 to 900
C at a rate of
10
C/min under a constant flow of air. The ultra-structure
of p-KG03 was observed using scanning electro-micros-
copy (JSM-5410LV, JEOL, Japan).
2.5. Rheological characterization of the bioflocculant
To measure the intrinsic viscosity [g] of each p-KG03
solution in 0.1 M NaCl solution, we used Huggins and
Kraemer plots of relative viscosity and specific viscosity
against concentrations in the range of 0.01–1.0 dl/g, with
measurements made on an Ubbelohde capillary viscometer
(536 13/Ic, SCHOTT-GERÄTE, Hofheim, Germany) at
25
C. After measuring the flow time of p-KG03 against
0.1 M NaCl solution, relative viscosity (gr), specific visco-
sity (gsp), reduced viscosity (gred), and inherent viscosity
(gInh) were calculated (Swenson, 1963).
To investigate steady shear flow properties, the lyo-
philized crude p-KG03 was dissolved in deionized water.
3
Agitation was maintained overnight at 4
C. The concen-
tration of the p-KG03 stock solution was determined using
the phenol–sulfuric acid method. This stock solution was
diluted to the desired concentration (ranging from 10 to
0.01 g/l) and homogenized. All polysaccharide solutions
were centrifuged at 10,000g for 10 min to remove air bub-
bles prior to measurements. Apparent viscosity measure-
ments of the solutions were achieved using a rotational
spindle viscometer LTV fitted with a small sample adaptor
(DV-III, Brookfield, USA). The measurement of apparent
viscosity was performed at different shear rates for each
concentration (0.01%, 0.02%, 0.1%, 0.2%, and 0.5%, w/v)
of p-KG03 and xanthan gum fluids. Spindle SC4-32 was
measured from 0.28 to 56.0 and SC4-18 from 1.32 to
132 s1. The pH of the two polysaccharide solutions was
controlled using 1.0 M HCl and 1.0 M NaOH, and the
effects of temperature and heat treatment were investigated
over ranges of 20–90
C and 121–25
C. We also investi-
gated the effect of the addition of different salts (NaCl
and CaCl2) over a range of concentrations (0.25–5% w/v
in a constant volume of 1% p-KG03) on the rheological
properties of p-KG03.
2.6. Statistical analyses
The results of experiments performed in triplicate are
shown with the values expressed as the means ± SE.
3. Results and discussion
3.1. Assay of flocculating activity
3.1.1. Effect of flocculant concentration
The typical flocculation curve of p-KG03 showed the
relationship between the concentration of p-KG03 and its
flocculating activity in kaolin suspension (Fig. 1). More
than 90% of flocculant activity was observed in the range
100
Flocculating activity (%)
80
60
40
20
p-KG03, Flocculating activity
0
0 1 2 3 4
p-KG03 concentration (mg/L)
Fig. 1. Effects of bioflocculant concentration on flocculating activity (%).
Flocculating reactions were performed at different concentrations in
kaolin suspension.
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4 J.H. Yim et al. / Bioresource Technology xxx (2006) xxx–xxx
of 0.5–2.5 mg/l, with the maximum at 1.0 mg/l
(90.5 ± 1.1%). The increase in flocculating rate of p-
KG03 with concentration was linear. Flocculating activity
decreased at p-KG03 concentrations (2.5 mg/l, 90.2 ±
0.9%) because sedimentation of flocculated particles was
inhibited by the viscosity generated at high concentrations
(4.0 mg/l, 0.6 ± 1.81). The biopolymer flocculants cause
aggregation of particles and cells by bridging and charge
neutralization. Bridging occurs if the biopolymeric floccu-
lant extends from the particle’s surface into the solution
for a distance greater than the distance over which the
interparticle repulsion acts. In this case, the biopolymer
can adsorb to other particles to form flocs. This mechanism
explains flocculation by neutral or like-charged biofloccu-
lants (Levy et al., 1992). Therefore, the concentration of
bioflocculant was in the range of a proportional response
(Gomoiu and Catley, 1996). The flocculating activities of
polyacrylamide (PAM) and zooglan were highest at 1.0
and 3.0 mg/l, respectively (Suh et al., 1997). The p-KG03
was effective in flocculating reactions at similar concentra-
tions to other polysaccharides.
3.1.2. Flocculation of various suspended solids
The spectrum of flocculating activity for various sus-
pended solids was studied in inorganic suspended solu-
tions. The suspensions of kaolin clay, active carbon,
aluminium oxide and Ca(OH)2 could be flocculated
effectively in the presence of Ca2+, the flocculant activities
(%) in suspensions containing 1.0 mg/l of p-KG03 were
93.6 ± 4.3%, 61.1 ± 1.4%, 83.4 ± 6.3% and 91.2 ± 5.1%,
respectively. For the bioflocculant of Bacillus licheniformis,
suspensions of kaolin, active carbon, and soil solid were
effectively flocculated in suspensions containing 3.7 mg/l
(Shih et al., 2001). The flocculating activity of REA-11
raw product from Corynebacterium glutamican was 12%,
16% and 19% for polyaluminium chloride, aluminium
sulfate, and polyacrylamide, respectively (He et al., 2002).
3.1.3. Effect of various salts, pH, and temperature
on flocculating activity
The effects of the pH of the reaction mixture on kaolin-
flocculating activity were investigated at bioflocculant con-
centrations of 1.0 mg/l. Flocculant activity was maintained
at high levels under acidic conditions (pH range of approx-
imately 3–6), but dropped when the pH was greater than 6.
Maximum flocculant activity was observed at pH 4. The
flocculant of Streptomyces griseus was active in acidic con-
ditions ranging from pH 2 to 6, with the maximum activity
observed at pH 4 (Shimofuruya et al., 1996). The maximum
activity of the flocculant of Enterobacter sp. BY-29 was
observed at pH 3, and the activity decreased with increas-
ing pH. Effective flocculation of kaolin occurred between
temperatures of 4–90
C (Yokoi et al., 1996). The floccu-
lants of Bacillus sp. PY-90 and the flocculant of S. griseus
were heat-labile (Shimofuruya et al., 1996).
The flocculant activity was not enhanced by the addition
of any cations, including Ca2+, and high concentrations of
Ca2+ led to a decrease in the flocculating activity (data not
shown). Cations stimulate flocculating activity by neutral-
izing and stabilizing the residual negative charge of func-
tional groups and by forming bridges between particles.
The role of bivalent and trivalent cations is to increase
the initial adsorption of biopolymers on suspended parti-
cles by decreasing the negative charge on both the poly-
mer and the particle (Levy et al., 1992). Like p-KG03,
the flocculating activity of Citrobacter sp. TKF04 was
not enhanced by the addition of any cations including
Ca2+ (Fujita et al., 2000). These results suggested that this
bioflocculant could be successfully used in the clarification
of a wide range of waters and/or wastewaters under various
environmental conditions.
3.2. Physiochemical characterizations of bioflocculant
Crude p-KG03 was purified by Sepharose 4B column
chromatography, eluted using 0.4 N NaCl buffer, and pro-
duced a single peak. The yield was 43.0 ± 2.36% of the
crude preparation, and the protein content was 2.58 ±
0.31%. The compositional analysis of p-KG03 by TLC
revealed only a single spot (Rf = 0.41), which resembled
with that of galactose (Rf = 0.40). HPLC analysis of the
p-KG03 hydrolysates showed the presence of galactose.
The molecular weight of p-KG03 was determined by
gel permeation chromatography as approximately 1.87 ·
103 kDa. The sulfate content of p-KG03 was estimated to
be 10.2 ± 1.24% w/w. The uronic acid concentration of
p-KG03, as measured by the carbazole–sulfuric acid
method, was 2.96 ± 0.12%. On the UV spectrum, the max-
imum adsorption wavelength of p-KG03 was 205.6 nm.
From the infrared spectrum of p-KG03, the polysaccha-
ride’s chemical group characteristics were analyzed as fol-
lows: –OH stretching at 3400 cm1, C–H stretching at
2900 cm1, stretching vibration of the carboxylate group
at 1650 cm1, symmetric CH bending at 1340 cm1,
S@O stretching at 1250 cm1, C–O–C antisymmetrical
stretching at 1000 cm1, and CO2 in air at 2400 cm1,
which was similar to typical polysaccharide IR spectra.
The IR spectrum of the polymer evidenced the presence
of carboxyl groups that could serve as binding sites for
divalent cations. The carboxyl groups could also work as
functional moieties to generate new and/or modified poly-
saccharide variants using different approaches, including
polymer engineering or novel formulation designing, by
linking this polysaccharide with starch and/or other syn-
thesized polymers (Ha et al., 1991). The elemental analysis
of p-KG03 showed that the proportions of carbon, hydro-
gen, nitrogen and sulfur were 32.2 ± 2.41%, 3.9 ± 0.11%,
0.52 ± 0.04% and 10.3 ± 1.24%, respectively. The ultra-
structure of p-KG03 was a fibrous type (Fig. 2).
The pyrolysis characterization of p-KG03 was invested
using thermogravimetric analysis (TGA). A degradation
temperature (Td) of 250
C was determined from the
TGA curve for the bioflocculant p-KG03. The biofloccu-
lant showed an initial weight loss due to moisture content
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J.H. Yim et al. / Bioresource Technology xxx (2006) xxx–xxx 5

Fig. 2. Microphotograph of the biopolymer p-KG03 from Gyrodinium impudicum strain KG03 showing the surface morphology under different
concentrations. (A) 1%; (B) 0.5%; (C) 0.1%.

between 40 and 230
C. The initial moisture content in the
sample was caused by the increased level of carboxyl
groups in the polysaccharide. This was because the higher
the carboxyl content, the greater the affinity of the polysac-
charide for water molecules (Kumar and Anand, 1998).
The decline in weight above 230
C was attributed to the
degradation of p-KG03. The onset of decomposition
occurred at 260
C, and the recorded mass loss was 10%.
The weight of p-KG03 dramatically decreased around
310
C, and thereafter decreased gradually.
3.3. Intrinsic viscosity [g]
The [g] of p-KG03 was determined by capillary viscosity
at concentrations ranging from 0.01% to 0.1% (w/v). The
tests conducted at high concentrations and different shear
rates suggested that under these conditions, the solution
viscosity was essentially Newtonian (Wang et al., 1997).
The results for the reduced and inherent viscosity tests
are shown in Fig. 3. The intrinsic viscosity of p-KG03
was 12.1 and 12.5 dl/g with 0.1 M NaCl solution, as calcu-
lated using the Huggins equation and Kraemer equation,
respectively. Using the Huggins equation, the intrinsic vis-
cosity of p-KG03 was found to be higher than that of xan-
than gum (10.8 dl/g). The intrinsic viscosity of p-KG03 was
30
Huggins equation
25 Y =115.88x + 12.105, R 2 = 0.99
dl/g)
12.1, which implied that solutions of p-KG03 had viscous
effects at low concentrations.
3.4. Steady shear flow property of bioflocculant
Most non-Newtonian fluids without residual tension are
represented by the Power-law equation, where the apparent
viscosity (ga) is
ga ¼ krn1 ;
where k is the consistency index of the fluid, and g is the
flow behavior index.
In this model, the parameter g constitutes a physical
property that characterizes its degree of non-Newtonian
behavior, and when g < 1, the fluid is pseudoplastic. The
parameter k indicates the relative viscosity or thickness of
a fluid; the higher the value of k, the thicker or more
viscous is the fluid. The p-KG03 solution showed charac-
teristic non-Newtonian fluid property behavior (Fig. 4).
In aqueous dispersions of p-KG03 1.0%, the consistency
index (k) and flow behavior index (g) were 2172 and 0.52,
respectively (xanthan gum: 2769 and 0.23; Table 1). The
p-KG03 solution was a pseudoplastic fluid under the
Power-law model. Steady shear flow properties of p-
KG03 were influenced by the concentration of salt, pH,
temperature, and ionic compounds. The shear stress of p-
20 180
p-KG03, 0.1% (D/cm 2 )
Xanthan, 0.1% (D/cm 2 ) 160

p-KG03, 0.1% (cp)

140
Shear stress (D/cm2)

Xanthan, 0.1% (cp)

Apparent viscosity (cp)

20 15
inh,

120
dl/g) or (

15 100
10
80
red,

10
60
(

5
5 40
Kraemer equation
2
Y = -3.0052x + 12.563, R = 0.98 20
0 0 0
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0 20 40 60 80 100 120 140
p-KG03 concentration (dl/g) Shear rate (1/sec)

Fig. 3. Intrinsic viscosity of p-KG03 fluid calculated using reduced Fig. 4. Variation in shear stress and apparent viscosity of p-KG03 0.1%
viscosity and inherent viscosity equations. (w/v) fluid with xanthan gum 0.1% (w/v) at 25
C.
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6 J.H. Yim et al. / Bioresource Technology xxx (2006) xxx–xxx

Table 1 KG03 decreased rapidly with increasing shear rate at all

Consistency index (k) and flow behavior index (g) of p-KG03 and other concentrations (Fig. 5A). The pH dependency of p-KG03
polysaccharides
is shown in Fig. 5B. The shear stress of p-KG03 was similar
Polysaccharide (1.0%) Consistency Flow behavior under acidic and alkaline conditions (pH 4–10), and was
index (k) index (g)
considered stable under changes in pH. The effects of differ-
Xanthan gum 2000–3000 0.29 ent salts on the steady shear-flow properties of p-KG03 are
Guar gum 2000–3000 0.17
Sodium-alginate 200–700 0.48
shown in Fig. 5C and D. The salts used were NaCl in the
Locust bean gum 2000–3000 0.78 range of 0.25–5.0% (w/v) and CaCl2 in the range of 0.25–
p-KG03 2172 0.52 2.0% (w/v) in a constant volume of 0.5% p-KG03 fluid.

100 40
p-KG03, 0.1%
p-KG03, 0.25%
80 p-KG03, 0.5%

Shear stress (D/cm2)

Shear stress (D/cm2)

p-KG03, 1.0% 30

60
20 pH 4
40 pH 5
pH 6
pH 7
10
20 pH 8
pH 9
pH 10
0 0
0 20 40 60 80 100 0 20 40 60 80 100 120 140
(A) Shear rate (1/sec) (B) Shear rate (1/sec)

40 40
Shear stress (D/cm2)

30 30
Shear stress (D/cm2)

20 NaCl, 0% 20
CaCl2 , 0%
NaCl, 0.25%
CaCl2 , 0.25%
NaCl, 0.5%
NaCl, 1.0% CaCl2 , 0.5%
10 NaCl, 2.0% 10 CaCl2 , 1.0%
NaCl, 5.0% CaCl2 , 2.0%

0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
(C) Shear rate (1/sec) (D) Shear rate (1/sec)

40 40

25 oC
40 oC
Shear stress (D/cm2)

Shear stress (D/cm 2)

30 30
50 oC
o
60 C

20 20 p-KG03, 0.5%
Xanthan gum, 0.5%
Xanthan gum, 0.5% + p-KG03, 0.5%
Gellan gum, 0.5%
10 10 Gellan gum, 0.5% + p-KG03, 0.5%

0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
(E) Shear rate (1/sec) (F) Shear rate (1/sec)

Fig. 5. Comparisons of shear stress and shear rate of p-KG03 fluid. (A) Concentration, (B) pH, (C) NaCl, (D) CaCl2, (E) temperature, (F) mixed with
xanthan gum.
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The shear stress of p-KG03 was similar with the addition
of NaCl and CaCl2, and was thus stable to salts. The nat-
ure of the interaction of the counter-ion (Na+ and Ca2+)
with the charged site on the macromolecular backbone is
important in determining the polyelectrolyte solution
properties (Pasika, 1977). Effects of rheological properties
of p-KG03 according to temperature are shown in
Fig. 5E. The shear stress of p-KG03 decreased with
increased temperatures of between 25 and 60
C, and seems
to be somewhat temperature-dependent. The effect of the
addition of xanthan gum is shown in Fig. 5F. The shear
stress of p-KG03 increased when mixed with xanthan gum.
4. Conclusions
G. impudicum, a red-tide microalga, produced mucous
exopolysaccharide, the flocculating activity of p-KG03
was high in range of 0.5–2.5 mg/l and especially effective
in the flocculating reaction at low concentration, compared
with other bioflocculant. Thus, p-KG03 could be used as
bioflocculant in various industrial processes, which how-
ever would need further investigations.
Acknowledgements
This study was supported by a grant to HKL from the
MarineBio 21 Program (PM05050) of the Korean Ministry
of Maritime Affairs and Fisheries.
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