Journal of Bioscience and Bioengineering

VOL. 118 No. 1, 29e33, 2014

www.elsevier.com/locate/jbiosc

Characterization of the flocculating agent from the spontaneously flocculating

microalga Chlorella vulgaris JSC-7

Md. Asraful Alam,1 Chun Wan,1 Suo-Lian Guo,1 Xin-Qing Zhao,1, * Zih-You Huang,2 Yu-Liang Yang,3
Jo-Shu Chang,4 and Feng-Wu Bai1

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China,1 Institute of Biochemical Sciences, National Taiwan University, Taipei 106,
Taiwan,2 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan,3 and Department of Chemical Engineering, National Cheng Kung University,
Tainan 701, Taiwan4

Received 30 July 2013; accepted 24 December 2013

Available online 4 February 2014
High cost of biomass recovery is one of the bottlenecks for developing cost-effective processes with microalgae,
particularly for the production of biofuels and bio-based chemicals through biorefinery, and microalgal biomass re-
covery through cell flocculation is a promising strategy. Some microalgae are naturally flocculated whose cells can be
harvested by simple sedimentation. However, studies on the flocculating agents synthesized by microalgae cells are still
very limited. In this work, the cell flocculation of a spontaneously flocculating microalga Chlorella vulgaris JSC-7 was
studied, and the flocculating agent was identified to be cell wall polysaccharides whose crude extract supplemented at
low dosage of 0.5 mg/L initiated the more than 80% flocculating rate of freely suspended microalgae C. vulgaris CNW11
and Scenedesmus obliquus FSP. Fourier transform infrared (FTIR) analysis revealed a characteristic absorption band at
1238 cmL1, which might arise from P]O asymmetric stretching vibration of PO2 L phosphodiester. The unique cell wall-
associated polysaccharide with molecular weight of 9.863103 g/mol, and the monomers consist of glucose, mannose and
galactose with a molecular ratio of 5:5:2. This is the first time to our knowledge that the flocculating agent from
C. vulgaris has been characterized, which could provide basis for understanding the cell flocculation of microalgae and
breeding of novel flocculating microalgae for cost-effective biomass harvest.
Ó 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Chlorella vulgaris; Spontaneous flocculation; Biomass harvest; Flocculating agent; Cell wall polysaccharides]

Microalgae are potential biomass sources for biorefinery to
produce biofuels and bio-based products due to their advantages,
such as not competing for arable land with agricultural production,
high efficiency in capturing solar energy, and mitigating CO2
emissions compared to terrestrial plants (1). In addition to mass
cultivation, biomass recovery of tiny microalgal cells presents
another challenge, since the biomass density is extremely low in
open culture systems that are suitable for microalgae culture at low
cost for the biorefinery purpose. For example, dry cell weight
(DCW) achieved with raceway ponds is only 0.14 g/L (2), which
consequently produces large amount of culture broth.
Currently, filtration using various filters such as plate-and-frame
pressure filters, rotary-drum filters and vacuum filters has been
applied in microalgae dewatering with a long history, and is suit-
able for small scale cultures with closed photobioreactors for value-
added products, in which much higher biomass density is achieved,
and the quantity of culture broth is much smaller. Nowadays, more
efficient membrane-based technologies have been developed for
microalgae dewatering (3). On the other hand, centrifugation with
disk stack centrifuge is applied for microalgae harvest when more
culture broth is processed, but significant capital investment on the
* Corresponding author. Tel.: þ86 411 8470 6319; fax: þ86 411 8470 6329.
E-mail address: xqzhao@dlut.edu.cn (X.-Q. Zhao).
equipment and energy consumption on its operation are needed
(4,5). Apparently, these technologies are not practical for microalgal
biomass recovery at larger scales for the purpose of biorefinery.
Biomass recovery by cell sedimentation through the flocculation
of microalgal cells does not require significant capital investment,
and energy consumption associated with the process is negligible,
making it one of the most economically competitive strategies for
microalgal biomass harvest at large scales (6). Although microalgal
cells can be flocculated by chemicals such as Al3þ, Fe3þ and poly-
electrolytes (6,7), large amount of wastewater discharged from
these processes is not suitable for microalgae cultivation, and the
chemical flocculation also raises environmental concerns on the
wastewater treatment.
To date, several spontaneously flocculating microalgae have
been discovered, such as Ankistrodesmus falcatus, Tetraselmis sue-
cica, Scenedesmus obliquus and Ettlia texensis (8e10), the cells of
which can settle down from culture broth for easy biomass recov-
ery, which enables a novel strategy in microalgae harvesting via cell
self-flocculation. It was reported that water-soluble extract of ma-
rine microalga Skeletonema marinoi induced flocculation of Nan-
nochloropsis oculata; however, it was not clear which specific
compound might be responsible for the flocculation (11). No doubt,
elucidation of the mechanism underlying the spontaneous floccu-
lation of those flocculating microalgal species and studies of the
flocculating agents will benefit genetic modifications of non-

1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2013.12.021
30 ALAM ET AL.
flocculating microalgal strains with the flocculation phenotype for
cost-effective biomass recovery by sedimentation. Unfortunately,
flocculating agents produced by microalgae are so far not well
characterized.
In the present study, the spontaneous flocculation of Chlorella
vulgaris JSC-7 was studied, and the flocculating agent was charac-
terized. Experimental results indicated that cell wall poly-
saccharide was responsible for the flocculation of the species,
which lay a basis for further understanding of their biosynthetic
pathways and identifying genes encoding key enzymes for the
biosynthesis of the flocculating agents in order to engineer non-
flocculating microalgal strains with the flocculation phenotype for
cost-effect biomass recovery.
MATERIALS AND METHODS
Microalgal strains and culture conditions The self-flocculating microalgal
strain C. vulgaris JSC-7 used in this work was isolated from fresh water in Southern
Taiwan. The modified Bold’s Basal Medium with 3-fold nitrogen supplementation
(3N-BBM) was used for the growth of C. vulgaris JSC-7. The composition of the 3N-
BBM medium (g/L) was: NaNO3, 0.75; CaCl2, 0.025; MgSO4$7H2O, 0.075;
K2HPO4$3H2O, 0.075; KH2PO4, 0.175, NaCl, 0.025; EDTA, 0.0045; FeCl3, 0.00058;
MnCl2$4H2O, 0.00046; ZnCl2, 0.00003; CoCl2$6H2O, 0.00004; Na2MoO4, 0.0003. The
pH of the medium was adjusted to 6.9. Non-flocculating microalgae strains
C. vulgaris CNW11 and S. obliquus FSP were selected and cultivated with BG11 me-
dium (12) and DM medium (13), respectively, to test the function of the flocculating
agents isolated from C. vulgaris JSC-7.
All the algal strains were cultivated at 28 C under a 13/11 h light/dark cycle with
an average illumination of 25.0 mmol/m2/s.
Biomass density measurement and morphology of C. vulgaris JSC-
7 Microalgal biomass density was measured by optical density at 690 nm with
a spectrophotometer (Varioskan Flash, ThermoFisher, USA). Scanning electron mi-
croscopy (SEM, Quanta 450, FEI, USA) was used to observe the morphology of the
flocculation of C. vulgaris JSC-7.
Evaluation of microalgal flocculation After two weeks cultivation, the
cultures of the three microalgal strains were harvested and centrifuged at 6000 rpm
for 10 min. The cell pellets were washed with distilled water and re-suspended to
the final OD690 of w0.9. Three experiments were performed to evaluate the floc-
culation efficiency of microalgae: the flocculating C. vulgaris JSC-7 (experiment A),
the non-flocculating C. vulgaris CNW11 and S. obliquus FSP (experiment B), and the
mixture of C. vulgaris JSC-7 with C. vulgaris CNW11 and S. obliquus FSP (experiment
C), respectively, at volumetric ratios of 1:5 and 1:2. All these experiments were
performed in duplicate.
For the flocculation assay, microalgae cultures were shaken to disperse the cells,
and 10 mL cell suspension was transferred into a test tube to measure OD690 by
sampling 200 mL cell suspensions at the depth of 4 cm from the top surface, which
was designated as A. After the test tubes were rested for 30 min, another 200 mL cell
suspension was sampled to measure OD690, which was designated as B. The floc-
culation efficiency F was calculated by the equation
F ¼ ð1  B=AÞ  100% (1)
(14) Evaluation of microalgal flocculation was performed at room temperature, and
no metal ion was added during flocculation experiment unless otherwise specified.
The same method was also used to measure the flocculating effect of the purified
flocculating agents from C. vulgaris JSC-7 on the flocculation of C. vulgaris CNW11
and S. obliquus FSP. To obtain better understanding of flocculating characteristics of
flocculating agents, effects of metal ions, pH, and temperature on flocculation effi-
ciency of freely suspended strain C. vulgaris CNW11 were investigated. The pH value
was adjusted to the range of 4e10 using 5 mM HCl or NaOH. The temperature was
controlled in water bath from 15 to 50 C and 120 C in autoclave for 20 min. Metal
salts of FeCl3, AlCl3, CaCl2 and KCl at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and
1.0 mg/L were tested as cationic coagulants, while FeCl3, AlCl3, and KCl at concen-
trations of 5e25 mM were tested with the addition of 0.5 mg/L purified flocculating
agent.
Purification and characterization of the flocculating agents from C. vulgaris
JSC-7 The culture of C. vulgaris JSC-7 was centrifuged at 6000 g for 10 min to
separate cells from the supernatant. While cell wall-bound polysaccharides were
extracted by pronase treatment and ethanol precipitation following the methods
described previously (15), two volumes of ethanol were added into the supernatant
to precipitate the flocculating agents in the culture broth.
Both fractions were dialyzed overnight with MD34 (GE, USA), after which they
were dissolved in TE buffer (pH 8.0) and upload to a DE52 anion exchange cellulose
column (Whatman, UK) previously equilibrated with TE buffer (pH 8.0), and then
eluted with 0.1 M NaCl solution at a flow rate of 0.5 mL/min. The eluted fractions
were lyophilized to obtain the purified compound for further characterization.
J. BIOSCI. BIOENG.,
The total sugar content of the flocculating agents was determined by the phenol-
sulfuric acid method using glucose as the standard (16), and their molecular weight
was analyzed by gel permeation chromatography (GPC, 1200 Series, Agilent Tech-
nology, USA) with the eluent of ddH2O at the rate of 0.5 ml min1 and chitosan as the
standard. Elemental analysis was carried out using an elemental analyzer (varioEL
III, Elementar, Germany). The monomer composition of the polysaccharides was
analyzed using gas chromatography/mass spectrometry (GC/MS) according to the
procedure described elsewhere (17). The FTIR spectra of the flocculating agent were
obtained with a FTIR spectrometer (ThermoFisher NEXUS) in the frequency range of
400e4000 cm1. Phosphate ion was detected and concentrated using ion chroma-
tography (ICS-5000, Dionex, USA) with the eluent of KOH at the rate of 1.0 ml min1
using PO4 3 solution as the standard (18). EPS sample was diluted in ddH2O before
detecting the phosphate ion.
RESULTS AND DISCUSSION
Flocculation of C. vulgaris JSC-7 In shake flask culture,
C. vulgaris JSC-7 cells settled down for biomass recovery due to
their spontaneous flocculation. Therefore, the flocculation effi-
ciency of C. vulgaris JSC-7 and its ability of flocculating freely sus-
pended C. vulgaris CNW11 and S. obliquus FSP cells were further
studied (Table 1).
Compared to the non-flocculating C. vulgaris CNW11 and
S. obliquus FSP, C. vulgaris JSC-7 exhibited much higher flocculation
efficiency of 76%. On the other hand, the supplementation of
C. vulgaris JSC-7 culture into that of C. vulgaris CNW11 and S. obliquus
FSP facilitated their flocculation, making their flocculation effi-
ciencies increased to 68.3% and 62.7%, respectively, from 25.6% and
28.1% observed with the original cultures of the microalgal strains.
Moreover, it was found that the flocculation efficiency of the non-
flocculating microalgae was higher when more C. vulgaris JSC-7
culture was supplemented. Similar observations were also reported
elsewhere (9,19) using the flocculating E. texensis, A. falcatus and
S. obliquus to harvest the non-flocculating C. vulgaris.
Many studies reported that increasing pH could induce the
flocculation of various microalgae cells (20). We thus adjusted the
pH value ranging from 6 to 10 using HCl and NaOH to investigate
the flocculation efficiency of C. vulgaris JSC-7, but no significant
difference was observed, since slightly higher flocculation effi-
ciency of 76% was at pH 7.0, while 74% and 75% were observed at pH
6.0 and 10.0, respectively, suggesting that the naturally occurred
flocculation of C. vulgaris JSC-7 was not affected by the pH varia-
tions. It has been generally accepted that the mechanisms of yeast
flocculation involve the binding of cell surface flocculins (glyco-
proteins) with sugar residues of the flocculins on the surface of
surrounding cells (21,22). We thus further investigated the cell
surface components for flocculating agents produced by the floc-
culating microalga.
Purification and characterization of the flocculating agent of
C. vulgaris JSC-7 Scanning electron microscopy (SEM) visualiza-
tion revealed that C. vulgaris JSC-7 cells were attached to each other
(Fig. 1), while no attachment was observed in the freely-suspended
C. vulgaris CNW11 and S. obliquus FSP. The aggregation observed
TABLE 1. Flocculation rate of self-flocculation and freely suspended microalgae.
Algae strains OD at 690 nm Flocculation
efficiency (%)
A B
C. vulgaris JSC-7 0.891 0.221 76.3
C. vulgaris CNW11 0.880 0.654 25.6
S. obliquus FSP 0.890 0.639 28.1
C. vulgaris JSC-7: C. vulgaris CNW11 at 1:5 0.862 0.562 34.8
C. vulgaris JSC-7: C. vulgaris CNW11 at 1:2 0.885 0.280 68.3
C. vulgaris JSC-7: S. obliquus FSP at 1:5 0.921 0.542 41.2
C. vulgaris JSC-7: S. obliquus FSP at 1:2 0.889 0.332 62.7
A, homogenized cell suspension; B, sedimentation after 30 min. Samples were
withdrawn from a depth of 4 cm at the top surface to measure the optical density at
690 nm.
VOL. 118, 2014
FIG. 1. SEM images of C. vulgaris JSC-7 cells.
with the SEM image suggests that flocculation of C. vulgaris JSC-7
might be associated with the substance presenting on the cell
wall, which was explored by fractioning the crude extract of this
microalga for cell wall-bound substance. In order to support this
speculation, crude extract was also obtained from the supernatant,
but no flocculation activity was observed when supplemented
into the non-flocculating C. vulgaris CNW11 and S. obliquus FSP.
Indeed, the cell wall-bound fraction induced flocculation of the
freely-suspended C. vulgaris CNW11 and S. obliquus FSP, and color
reaction with phenol-sulfuric acid test provided clues that the
flocculating agent might be polysaccharides. A total of 47 mg
flocculating agent was extracted from 4 L C. vulgaris culture broth
with the culture OD690 of 0.7.
Effects of the purified agent on the flocculation of C. vulgaris
CNW11 and S. obliquus FSP The dosage effect of the flocculating
agent on the flocculation of C. vulgaris CNW11 and S. obliquus FSP
was investigated (Fig. 2). As can be seen, the cell wall-bounded
flocculating agent of C. vulgaris JSC-7 showed impressive
flocculation capability with the freely suspended microalgae
C. vulgaris CNW11 and S. obliquus FSP. All the aggregates settled
down in the suspension within 1 h when 0.3 mg/L of the
microalgal flocculating agent was added. The flocculation
FIG. 2. Dosage effects on flocculation rate of the freely suspended microalgae C. vulgaris
CNW11 and S. obliquus FSP using purified flocculating agents from C. vulgaris JSC-7.
SPONTANEOUS FLOCCULATION OF C. VULGARIS 31
efficiency reached about 85% per hour with a flocculating agent
dose of as low as 0.5 mg/L. Effect of metal ions on the flocculation
efficiency of C. vulgaris CNW11 was investigated, and the results
are shown in Fig. 3a. When purified flocculating agent was tested
for flocculating the C. vulgaris CNW11 cells, it was found that a
dosage of 0.5 mg/L resulted in a flocculation efficiency of more
than 80%. In contrast, only 42.1% flocculation efficiency was
achieved when 1.0 mg/L FeCl3 was used to flocculate the
microalgae cells, and other metal ions (Al3þ, Ca2þ, Kþ) showed
much lower flocculation efficiencies (Fig. 3a), which suggests that
the flocculating agent produced by C. vulgaris JSC-7 is much more
efficient than these chemical flocculants. The maximum
flocculation efficiency (93%) was achieved when 5 mM Fe3þ was
added together with the flocculating agents (Fig. 3b). For a better
understanding of flocculation induced by purified flocculating
agent, effects of pH, and temperature were further investigated.
When the pH of the culture was ranged from 4 to 10, the
maximum flocculation efficiency of 80% was observed at pH 7.0
(Fig. 3c). No notable change of flocculation efficiency was
observed when temperature was adjusted from 15 C to 50 C
(Fig. 3d). Our studies demonstrated that cell wall-associated
polysaccharide is responsible for the cell flocculation of C. vulgaris
JSC-7. In our previous study (8), we have found that the activities
of flocculating agents purified from S. obliquus AS-6-1 was also pH
and temperature dependent. Both flocculating agents from
C. vulgaris JSC-7 and S. obliquus AS-6-1 are sensitive to high
temperature, when treated at 121 C for 20 min, about 50%
flocculating efficiency was remained. For the dosage effect,
addition of 0.3e0.5 mg/L purified flocculating agent resulted in
efficient flocculation; similar dosage was reported using the
flocculating agent from S. obliquus AS-6-1 (8). In another study
(23), artificially synthesized polyacrylamide grafted starch (St-g-
PAM) was used to harvest Chlorella sp. CB4. Although the
optimized dosage is similar to that in our study (0.8 mg/L), it was
found that this artificial flocculent showed good effect on cell
harvest only at high pH (pH 10.5), where the maximum 85.84% of
microalgae recovery was achieved. However, when pH value was
lower than 10.5, the flocculating efficiency of the artificial
flocculent was very week, only less than 50% cells were recovered.
Therefore, the advantage of flocculating agent produced by
C. vulgaris JSC-7 is that high pH is not required to achieve cell
flocculation, thus it will not affect the quality of the end product,
and medium can also be reused which will partly compensate the
production cost of the flocculating agents. When chitosan was
investigated to harvest C. sorokiniana (24), the optimal dosage was
determined to be 5 mg/L at pH 6, whereas 10 mg/L chitosan was
needed when pH was increased to 7. In our experiment, the
flocculating activity using flocculent from C. vulgaris JSC-7 is not
so sensitive to pH (Fig. 3c). The present study demonstrates that
cell wall associated polysaccharides might lead to the flocculation
of C. vulgaris JSC-7, and also suggests that such polysaccharides
might be responsible for assisting the flocculation of non-
flocculating microalgae cells as well.
Characterization of the flocculating agents and mechanistic
speculation on the flocculation of the microalgal
cells Studies on the cell wall components using FTIR spectra
for C. vulgaris JSC-7 and C. vulgaris CNW11 revealed several differ-
ences. As shown in Fig. 4, FTIR analysis shows that a broadly-
stretched intense peak around 3400 cm1, which is a
characteristic of hydroxyl groups, and the occurrence of a
relatively strong absorption around 1645 cm1 is attributed to the
characteristics of carboxyl (25), which is presented in both the
flocculating and non-flocculating microalgal cells. The broad
stretch of CeOeC and CeO at 1000e1200 cm1 corresponded to
the presence of carbohydrate and polysaccharide (26). The FTIR
32 ALAM ET AL. J. BIOSCI. BIOENG.,

FIG. 3. Factors affecting the flocculation efficiency of freely suspended strain C. vulgaris CNW11. (a) Comparison of the flocculation efficiency of the purified flocculating agent with
that of various metal ions. (b) Effect of metal ions on flocculating efficiency. Control, no addition of any metal ion. (c) Effects of pH on the flocculating efficiency. (d) Effects of
temperature on the flocculating efficiency.

spectrum of C. vulgaris JSC-7 exhibits two bands at 1647 cm1 and
1534 cm1, whereas C. vulgaris CNW11 shows single band in
1625 cm1. The medium stretch and bend in 1650e1580 cm1 are
attributed to NeH stretching (26). It is still unclear whether the
difference at 1647 cm1 contributes to the flocculation of
C. vulgaris JSC-7; however, it is very clear that C. vulgaris JSC-7
has a characteristic adsorption peak at 1238 cm1. The region
between 1250 and 1200 cm1 corresponds to >P]O asymmetric
stretching frequencies that is also observed in the cyanobacteria
Calothrix sp. and brown alga Padina tetrastromatica (27,28), but
has never been reported so far in green microalgae. The
phosphodiester functional group in Calothrix sp. and
P. tetrastromatica displayed strong chelating properties and was
proposed to have a crucial role in metal chelation. C. vulgaris JSC-
7 shows the characteristic band at 1238 cm1, suggesting that the
cell wall-associated polysaccharides might contain PO2 
phosphodiester. The presence of phosphate ion was further
detected and quantified to be 0.038%. In addition, elemental
analysis of the flocculating agent from C. vulgaris JSC-7 reveals
the presence of carbon, hydrogen, nitrogen and sulfate.
GCeMS analysis shows that the monomers consist of glucose,
mannose and galactose with a molecular ratio of 5:5:2. In addition,
this polysaccharide is white in color and water soluble. Based on
the calibration curve of the elution retention time of various stan-
dards on GPC, the molecular weight of the flocculating agent is
estimated to be 9.86  103 g/mol. A summary of the properties of
the natural flocculating agent extracted from C. vulgaris JSC-7 was
depicted in Table 2.
Based on the above observations, we supposed that the major
functional groups such as hydroxyl and carboxyl groups in the

TABLE 2. General properties of the flocculating agent extracted from

C. vulgaris JSC-7.

Properties Descriptions

Functional groups CeH, COOH, P]O, CeC, CeO, CeOeP

MW 9.86  103 g/mol
Nitrogen cc. 7.23%
Carbon cc. 40.43%
Sulfate cc. 1.10%
Hydrogen cc. 6.56%
Phosphate cc. 0.038%.
Color White
Solubility Water soluble
Thermal stability Until cc. 220 C
FIG. 4. FTIR spectra of the cell wall polysaccharides from the flocculating C. vulgaris JSC- Melting temperature Non-meltable
7 (line A) and non-flocculating C. vulgaris CNW11 (line B).
VOL. 118, 2014
polysaccharides of C. vulgaris JSC-7 might serve as binding sites
during the flocculation process, and the high molecular weight was
estimated to involve more adsorption points and stronger bridging
for the flocculating phenotype. In the previous report in our group,
cell wall-associated polysaccharide from self-flocculating
S. obliquus AS-6-1 was characterized (8). It was found that five
monosaccharides (glucose, mannose, galactose, rhamnose and
fructose) were present as constituent monosaccharide in S. obliquus
AS-6-1, whereas flocculating agents from C. vulgaris JSC-7 in this
study were revealed to contain glucose, mannose and galactose,
suggesting the different molecular architecture and characteristics
for different flocculating microalgae. Although flocculating micro-
algae E. texensis was also studied for easy cell harvest, the chemical
nature of the extracellular polymeric substances (EPS) proposed to
be involved in self-flocculation was not further elucidated (10). The
results in this study presented the second example of character-
ization of flocculating agents from spontaneously flocculating
microalgae.
So far biosynthesis of polysaccharide in microalgae is very
poorly studied. Our studies provided the first example of phos-
phodiester-containing polysaccharide in microalga. Phosphogly-
cans are widespread in nature, and presence of phosphodiester-
linked oligopolysaccharide repeating units is reported in bacteria,
yeast, Leishmania protozoan parasites, as well as in animals (29).
Bacterial polysaccharide is synthesized using precursors including
nucleoside diphosphate sugars, nucleoside diphosphate sugar acids
and nucleoside diphosphate sugar derivatives (30). It will be
interesting to further study how phosphodiester functional group is
incorporated into EPS in C. vulgaris JSC-7.
Collection of microalgal biomass from culture systems
contribute greatly to the operation cost of the overall process. The
unique cell wall polysaccharides in C. vulgaris JSC-7 induced floc-
culation of freely-suspended microalgal cells. The low dosage
(0.5 mg/L) of the natural flocculating agent from C. vulgaris JSC-7
required for the flocculation is an additional advantage, and in the
meantime it does not interfere with the product quality of the
harvested algal biomass. Selection and breeding of flocculating
microalgae will benefit low cost production of microalgal products.
Further studies on metabolic engineering of special polysaccharide
biosynthesis will shed light on the modulation of microalgal cell
flocculation. To our knowledge, this is the first report on the floc-
culating agent produced by a self-flocculating Chlorella sp.
ACKNOWLEDGMENTS
This work was supported by financial support from National
Basic Research Program of China (2011CB200905). This research
was also financially supported in part by the Headquarters of Uni-
versity Advancement at the National Cheng Kung University, which
is sponsored by Taiwan’s Ministry of Education. Md. Asraful Alam is
grateful to Chinese Government Scholarship Council (CSC) for his
Ph.D. scholarship.
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