Effect of different flocculants on the flocculation performance of microalgae, Chaetoceros calcitrans, cells
ABSTRACT The possibility of using flocculation technique for the separation of microalgae, Chaetoceros calcitrans, biomass from the culture broth was investigated. The flocculation experiments were conducted in 500 mL beaker using culture broth obtained from 10 L photobioreactor. The harvesting efficiency of 90 and 60% was obtained in flocculation without flocculants conducted for 10 days at 27°C (in light and dark) and 4°C (dark), respectively. Harvesting efficiency higher than 90% with short settling time was achieved by adjusting the culture pH to 10.2 using either sodium hydroxide (NaOH) or potassium hydroxide (KOH). Improved cell viability (> 80%) and settling time with a slight improvement of flocculation efficiency was achieved by the addition of polyelectrolytes flocculant (Magnafloc® LT 27 and LT 25). However, the flocculants were only functioned when the pH of the microalgae culture was pre-adjusted to a certain value that promotes cells entrapment and surface charge neutralization prior to flocculation process. The flocculation efficiency and cell viability obtained in flocculation with Magnafloc® (LT 25 and LT 27) was comparable to that obtained in flocculation with chitosan. When chitosan and Magnafloc® (LT 25 and LT 27) were used as flocculants, the highest flocculation efficiency of C. calcitrans cells was observed at pH 8 and 10.2, respectively. Substantial increased in sedimentation rate was observed with increasing flocculants dosage though the flocculation efficiency and cell viability were not significantly varied. © 2009 Academic Journals. Cited By (since 1996): 2, Export Date: 14 June 2011, Source: Scopus
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ABSTRACT: Chemical flocculation is considered to be a reliable and economical means for the harvesting of microalgae. Organic cationic polymers can induce efficient flocculation of freswater microalgae at low dosages (between 1 and 10 mg litre−1). However, the high salinity of the marine environment was found to inhibit flocculation with polyelectrolytes. This phenomenon was studied with three different cationic polymers. Inhibition of flocculation was diminished at reached salinity levels, and effective flocculation was attained at salinity levels lower than 5 g litre−1. The reduced effectiveness of cationic polymers to induce microalgae flocculation in sea and brackish water is primarily attributed to the effect of medium ionic strength on the configuration and dimension of the polymer, as indicated by changes in the intrinsic viscosity. At high ionic strength, the polymer shrinks to its smallest dimensions, and fails to bridge between algal cells.Biomass. 01/1988;
- [show abstract] [hide abstract]
ABSTRACT: Laboratory experiments were conducted to compare the effectiveness of chitosan (by-product derived from shrimp and crab shells), Zetag 63 and CF 400 (hydrolyzed polyacrylamide) as flocculants to concentrate a mixed culture of chlorophyceae dominated by Chlorella sp. The algae were grown in a high-rate algal pond (HRAP) fed with dilute pig-waste. Algal sedimentation rates were measured in the laboratory.For a pH range of 6·0–9·0, flocculation efficiency of 95–100% was obtained at 20 mg/liter chitosan and 5 mg/liter Zetag 63. The optimum range of initial biomass concentration for maximum algal recovery was found to be 100–200 mg dry weight algae/liter.Biological Wastes. 01/1990;
- [show abstract] [hide abstract]
ABSTRACT: The focused beam reflectance measurement (FBRM), also known as scanning laser microscopy (SLM), was used as a real-time monitor to study the flocculation and reflocculation of clay suspensions under different shear conditions in the presence of single polymer, dual polymer, microparticle and poly(ethylene oxide)/phenolformaldehyde (PEO/PFR) flocculation systems. For initial flocculation, the high molecular weight PEO and cationic polyacrylamide (CPAM) produced larger flocs than others. However, reflocculation of clay suspensions formed by these non- or low-charged polymers was insignificant after the initial flocs were broken under high shear force. In contrast, high charge density polymers, such as poly(diallyldimethylammonium chloride) (PDADMAC), do not form large initial flocs, but they showed significant reflocculation ability under a continuous shear condition. It is concluded that high flocculation can be obtained by effective polymer bridging, but high reflocculation can only be induced by high electrostatic attractive forces between suspended particles.Journal of Colloid and Interface Science 11/2004; 278(1):139-45. · 3.17 Impact Factor
African Journal of Biotechnology Vol. 8 (21), pp. 5971-5978, 2 November, 2009
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals
Full Length Research Paper
Effect of different flocculants on the flocculation
performance of microalgae, Chaetoceros calcitrans,
Zuharlida Tuan Harith1, Fatimah Mohd Yusoff1, Mohd Shamzi Mohamed2, Mohamed Shariff
Mohamed Din3 and Arbakariya B. Ariff1,2*
1Laboratory of Industrial Biotechnology, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor,
2Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia,
43400 Serdang, Selangor, Malaysia.
3Faculty of Veterinary Medicine, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Accepted 19 June, 2009
The possibility of using flocculation technique for the separation of microalgae, Chaetoceros calcitrans,
biomass from the culture broth was investigated. The flocculation experiments were conducted in 500
mL beaker using culture broth obtained from 10 L photobioreactor. The harvesting efficiency of 90 and
60% was obtained in flocculation without flocculants conducted for 10 days at 27ºC (in light and dark)
and 4ºC (dark), respectively. Harvesting efficiency higher than 90% with short settling time was
achieved by adjusting the culture pH to 10.2 using either sodium hydroxide (NaOH) or potassium
hydroxide (KOH). Improved cell viability (> 80%) and settling time with a slight improvement of
flocculation efficiency was achieved by the addition of polyelectrolytes flocculant (Magnafloc® LT 27
and LT 25). However, the flocculants were only functioned when the pH of the microalgae culture was
pre-adjusted to a certain value that promotes cells entrapment and surface charge neutralization prior
to flocculation process. The flocculation efficiency and cell viability obtained in flocculation with
Magnafloc® (LT 25 and LT 27) was comparable to that obtained in flocculation with chitosan. When
chitosan and Magnafloc® (LT 25 and LT 27) were used as flocculants, the highest flocculation efficiency
of C. calcitrans cells was observed at pH 8 and 10.2, respectively. Substantial increased in
sedimentation rate was observed with increasing flocculants dosage though the flocculation efficiency
and cell viability were not significantly varied.
Key words: Flocculation, microalgae, Chaetoceros calcitrans, polyelectrolyte, chitosan.
Microalgae are widely used in aquaculture as live feeds
for several aquaculture species such as molluscs, fish,
penaeid prawn larvae and rotifers (Brown, 2002).
Intensive cultivation for production of large quantities of
microalgae biomass requires a proper harvesting techni-
que. One of the problems in large scale productions of
microalgae is the development of efficient downstream
processing to enable efficient separations of cells from
*Corresponding author. E-mail: email@example.com.
Tel.: 603 8946 7591. Fax: 603 89467510.
culture broth as well as to maintain their viability and
bioactivity prior to use in the field. The process options
and economics of the different methods for the recovery
of microalgal biomass have been reviewed by Molina
Grima et al. (2003). Different production systems require
a proper selection and optimization of the harvesting
method and these must accommodate to the requirement
of the final form of the algal cells and the subsequent pro-
cessing steps of the harvesting and formulation methods.
Flocculation or sedimentation occurs when particles in
liquid settle to the bottom of the tank due to gravitational
force and fluid drag force. This method of solid-liquid
separation is preferred for harvesting large cells like
5972 Afr. J. Biotechnol.
microalgae due to its low costs compared to other
methods such as centrifugation and filtration (Bilanovic et
al., 1988). The flocculation efficiency of microalgae can
be improved by the use of flocculants (Tilton et al., 1972;
Bilanovic et al., 1988). Flocculation occurs when solid
particles aggregate into large but loose particles resulting
from the interaction of the flocculants with the surface
charge of the suspended solid and subsequent coales-
cing of these aggregates into large flocs that settle out of
suspension (Knuckey et al., 2006). This process has
been extensively used in the industry to remove suspen-
ded solids such as clarification of waste water treatment
(Mahvi and Razavi, 2005), clarification of drinking water,
colour removal in paper making industry and mineral
processing (Yoon and Deng, 2004).
Flocculation process has been applied in the harvesting
of microalgal biomass (Gualteri et al., 1988; D’Souza et
al., 2002; Knuckey et al., 2006). Algae flocculated using
aluminium sulphate has been fed to common carp,
Cyprinus carpia since 1970 (Sandbank and Hepher,
1978). Substantially high flocculation efficiency (> 80%)
of marine microalgae was obtained when ferric chloride
was used as flocculant (Sukenik et al., 1988). In addition,
alum and lime can also be used as flocculants to con-
centrate C. calcitrans, Skeletonema costatum and
Tetraselmis chuii but not Isochrysis sp. (Millamena et al.,
1990). Mineral coagulants such as alum and ferric chlo-
ride might be toxic to animals when consumed due to
high concentration of residual aluminum and iron in the
biomass harvested (Buelna et al., 1990). Therefore, the
choices of flocculants are crucial and several alternatives
have been studied as replacement to mineral coagulants.
Polyelectrolytes are known as effective flocculants and
have been used in the improvement of flocculation. They
are usually high in molecular weight and water soluble
organic compound, which can be anionic, cationic or
nonionic (Baraniak et al., 2004; Voisin and Vincent, 2003;
Kim et al., 2001). Magnafloc® is one of the well known
brand of polyelectrolytes flocculants. Chitosan, which is
an organic cationic polymer, has also been used as a
flocculating agent in the treatment of wastewater and
food industry. It is a ?-N-acetyl-D-glucosamine polyca-
tionic polymer extracted from crustaceans exoskeletons
(Gualteri et al., 1988). Chitosan remains insoluble in
water, alkali solution and alcohol but easily dissolved in
dilute acids. Reduced flocculation efficiency of micro-
algae cells in salt water system has been reported
(Heasman et al., 2001). However, the assessment of
chitosan as a potential flocculant in concentrating algae
need to be explored due to its low toxicity, ease of manu-
facturing and low working dosage. Recently, Knuckey et
al. (2006) claimed that the adjustment of the microalgae
culture pH by adding base to promote precipitation and en-
trapment of cells increased the harvesting efficiency sub-
stantially. This simple method could be an attractive choice
because it is non-toxic to the cells and it also eliminates the
use of mineral coagulants. However, this method was
only tested to small number of microalgal strain.
The objective of this study was to compare the floccu-
lation efficiencies of different types of flocculants in
harvesting microalgae, C. calcitrans, from culture broth
obtained from the cultivation in the laboratory scale
photobioreactor (10 L working volume). The effects of
culture pH, flocculation conditions and flocculant dosage
on the flocculation efficiency were also investigated.
MATERIALS AND METHODS
Microalgae and cultivation method
Microalgae, C. calcitrans, obtained from Aquatic Animal Health Unit,
Faculty of Veterinary, Universitiy Putra Malaysia was used through-
out this study. The microalgae was cultivated in 10 L photobio-
reactor using Conway medium (Walne, 1966) at 29 ppt salinity and
with the addition of 0.02 g/L of silica. The temperature within the
photobioreactor was regulated at 20 ± 2ºC by air-conditioning and
aeration was provided by air bubbling through sparger. The pH of
the medium was maintained at 8 ± 0.2 by sparging with a mixture of
air and carbon dioxide (CO2) at a ratio of 97:3. Cultures were grown
under continuous illumination by white fluorescent (4500-5000 lux)
during day and night time. Cells were harvested at late logarithma-
tic growth phase (after 6 days of cultivation) for subsequent use in
the flocculation experiments.
All flocculation or sedimentation experiments of C. calcitrans cells
were carried out using 500 mL beaker (diameter = 85 mm and
height = 120 mm). Initially, the microalgae cultures (pH 8) were
allowed to settle at 3 different conditions;
(i) 27oC (in the dark),
(ii) 27oC (with light) and
(iii) 4ºC (in the dark) without flocculants.
Samples were taken everyday for a period of 15 days at 4 cm of
above the base of the beaker for evaluation of flocculation effi-
ciency. The viability of C. calcitrans cells was determined after 15
days of flocculation.
Subsequently, the effect of culture pH on the flocculation
efficiency was carried out by adjusting the culture pH ranging from
pH 10 to pH 10.6 using either 5 M sodium hydroxide (NaOH) or 5 M
potassium hydroxide (KOH). The bases were added to the culture
at high mixing rate provided by agitation using magnetic bar stirrer
(38 mm), agitated at 200 rpm to allow for steady increase and
homogeneity in pH. When the required pH was reached, slower
mixing (50 rpm) was applied for 2 min, followed by quiet settling
under gravity for flocculation process. At the end of flocculation (4
h), surface water was siphoned off and the flocs were collected for
In the subsequent experiments, the effect of polyelectrolytes floc-
culants was studied by adding different dosages of Magnafloc®LT
25, Magnafloc®LT 27 (Ciba Specialty Chemicals, Switzerland) and
chitosan (SIGMA, United States) to the culture where the pH was
previously adjusted to the required value according to method as
described earlier. Magnafloc® LT 25 and LT 27) evaluated in this
study have been approved for waste water treatment process in
United Kingdom by the drinking water inspectorate and the Scottish
office as proved by the manufacturer. It is recommended for use as
coagulants aid in clarification and filtration process (Mahvi and
Razavi, 2005) suggesting that these polyelectrolyes are safe to be
used in microalgae cells separation.
Stock solution of these polyelectrolytes were prepared by dis-
Figure 1. Profiles of flocculation efficiency of C.
calcitrans during sedimentation without flocculant at three
different culture conditions. Initial pH was 8 and not
adjusted prior to the flocculation experiment. Symbols
represent (?) 4 ºC (dark); (?) 27 ºC (dark); (?) 27 ºC with
(light). Arrow bars indicate standard deviation of three
solving 0.5 g/L Magnafloc® LT 25 and Magnafloc® LT 27 in distilled
water followed by extensive stirring. Flocculants were added to the
culture, followed with vigorous mixing (200 rpm) for 1 min and sub-
sequently with slow mixing (50 rpm) for another 2 min. Then, the
stirrer was removed from the culture to allow the flocculation under
gravity. At the end of flocculation (4 h), surface water was siphoned
off and the flocs were collected for analysis.
For comparison, flocculations using chitosan as a flocculant were
also carried at different pH values (pH 5 to pH 10) and dosages (10
mg/L to 150 mg/L). The pH of microalgae culture was dropped from
8 to 3.14 after the addition of chitosan, due to its acidic charac-
teristic. NaOH was used to adjust the culture pH to the required
value. Stock solution of chitosan was prepared by dissolving 1 mg/
mL of chitosan flakes in 1% (v/v) acetic acid followed by extensive
sonication (Model no. Branson 3510, United States) until the flakes
was totally dissolved.
For staining procedure, 20 mL of samples were treated with 1 mL of
1% (w/v) stock solution of Evan’s blue. The samples were allowed
to stand at room temperature for at least 30 min and cells were
observed microscopically under light microscope (Leica DMLB,
Germany). The dead cells were stained blue due to the penetration
of the stain through the cell wall whereas the viable cells would
retain their natural colour due to intact cell wall. Cell numbers were
counted using haemacytometer. The percentage of viable cells was
calculated using equation (1);
Harith et al. 5973
Flocculation or harvesting efficiency
The flocculation efficiency was evaluated by comparing the remain-
ing cell density in the clear region with the concentration before
treatment. The flocculation or harvesting efficiency (%) was calcu-
lated using equation (2).
Flocculation/harvest efficiency (%)
Where Ci is the concentration of cell in suspension before treatment
and Cf is the final concentration of cells in suspension.
Determination of the flocculation or sedimentation rate
During the flocculation experiments, the flocculation or sediment-
tation rate was estimated by the observation of the displacement of
the upper interface of the cell suspension with time (in sec) through
the naked eyes. This means that the movement of the layer bet-
ween the clear solution with the layer of high density cell suspen-
sion toward the bottom of the beaker during the flocculation was
monitored on the attached scale (in mm), which was termed as
sediment height. The maximum flocculation or sedimentation rate
was determined from the slope of the plot of sediment height versus
flocculation time. Determination of maximum sedimentation rate is
necessary for an appropriate comparison of the sedimentation rate
without the effect of cell accumulation at the bottom of the beaker
(López et al., 1996).
RESULTS AND DISCUSSION
Flocculation without flocculant at different conditions
The flocculation of C. calcitrans culture without flocculant
at different flocculation conditions is shown in Figure 1. In
all cases, flocculation efficiency was increased gradually
with time and reached maximum after about 8 days.
However, increased in flocculation efficiency with time
was higher for flocculation carried out at 27ºC as com-
pared to 4ºC. The effect of providing light and total dark-
ness gave not significant effect on the flocculation effi-
ciency. The maximum flocculation efficiency obtained at
27ºC (either in the dark or with light) was about 91%,
while the value obtained for flocculation at 4ºC was only
about 70%. Fluctuation of flocculation efficiency was ob-
served when it reached maximum values for flocculation
at 4ºC. On the other hand, very stable flocculate at
steady-state was observed for flocculation at 27ºC though
a slight increase in flocculation efficiency from day 8
(91%) to 94% at day 15 were observed.
Figure 2 shows the results for the viability of C. calci-
trans obtained after 15 days of flocculation without floc-
culant at different conditions. The highest viability (80.8 ±
3.19%) was obtained for sedimentation at 4ºC and
significantly reduced for sedimentation at 27ºC. It is also
interesting to note that the viability during sedimentation
at 27ºC was higher in culture without light (79.98 ±
2.93%) as compared to culture with light (63.53 ± 9.91%).
5974 Afr. J. Biotechnol.
Figure 2. Viability of C. calcitrans cells after 15 days of
flocculation process without flocculant at three different
culture conditions. Initial pH was 8 and not adjusted prior to
the flocculation experiment.
Microscopic examination of sedimented C. calcitrans
cells after 15 days of sedimentation indicate that the cells
were in the singly form without aggregation occurred.
Reduced sedimentation rate of C. calcitrans cells is
mainly due to it low cell dry weight (15 ?g) and very fine
cell size (4.0 x 3.4 µm) (Neil and O’Connor, 1991).
Heasman et al. (2001) reported that 100% of harvesting
efficiency of C. calcitrans cells was only achieved after
162 h of sedimentation time. The morphology of the cell
also affect the sedimentation rate due to presence of
spike and internal biochemical changes, such as gas and
lipid content that promote buoyancy in the absence of
light. The presence of spike was also observed on physi-
cal observation of C. calcitrans cells during sedimentation
process, suggesting that the cells were viable with
changes in the internal biochemical. In this study, it was
found that reduction in temperature and darkness aimed
at reduction the metabolic activity of the cells did not
enhance the sedimentation process but im-proved the
cells viability. Heasman et al. (2001) reported that less
than 20% of Tetraselmis sp. (T. Iso) cells retained its
viability after 14 days of storage. It is important to note
that C. calcitrans could maintain high viability after 15
days of storage in chilling condition.
Flocculation with pH adjustment
Figure 3 shows the flocculation or harvesting efficiency of
C. calcitrans cultures with pH adjustments prior to floccu-
lation process. With a slight increase in culture pH from
10 to 10.2, the harvesting efficiency was increased al-
most 2 times. At pH below 10, very low separation occurs
after 4 h of flocculation process. When the pH was adjus-
ted up to 10 using KOH, the efficiency was about 35%
and increased to 78% when NaOH was used. Less quan-
Figure 3. Flocculation efficiency of C. calcitrans cells in
flocculation with pH adjustment using KOH and NaOH.
Symbols represent; () KOH; and ( ) NaOH.
tity of NaOH was required to adjust the pH of the
microalgae culture to pH above 10 as compared to KOH.
However, slightly higher flocculation efficiency was ob-
served when KOH is used compared to NaOH, where the
harvesting efficiency of 98% was obtained at pH 10.2 and
above. Further increment of pH did not show further im-
provement in the flocculation efficiency. It is worthy to
note that the additional bases tend to increase the preci-
pitation and formation of loose flocs.
The use of pH adjustment did not enhance the floccu-
lation of some microalgae species. For example, the
harvesting efficiency of Nannocloropsis oculata and Iso-
chrysis sp. (T-iso) after pH adjustment was less than 30%
(Knuckey et al., 2006). During the initial stage of floccu-
lation process, when the pH of medium was increased,
the small particles aggregated and slowly settled due to
gravitational force. The cells formed large loose and
dense packed aggregates that settled under gravitational
force. Once the fine capture achieved equilibrium, further
addition of flocculant might lead to the formation of larger
aggregates. This in turn might cause higher settling rates
with minimal addition of flocculants (Owen et al., 2002).
Adding additional bases to the culture medium did not im-
prove the flocculation efficiency but increased in precipi-
tation and formation of loose flocs.
Flocculation with polyelectrolytes and pH adjustment
The changes in flocculation efficiency, cell viability and
sedimentation rate of C. calcitrans at different pH adjust-
ment with NaOH followed by addition of Magnafloc® LT
25 is given in Figure 4. The flocculation efficiency was
increased drastically from 13 to 82% after 4 h when the
pH was adjusted from 8 to 10. Further increase in floccu-
lation efficiency to 92% was observed with increasing
Figure 4. Change in flocculation efficiency, cells viability and
sedimentation rate for C. calcitrans flocculated at different pH
adjustment using NaOH followed by addition of 0.1 mg/L
Magnafloc®LT 25. Symbols represent; (
efficiency; ( ) cells viability; and (?) sedimentation rate.
pH to 10.2 and slightly increases to 98% with increasing
pH to 10.3 and 10.6. On the other hand, the
sedimentation rate was
increasing pH from 10 to 10.2 and remains at the same
rate with further increment in pH to 10.6. At higher pH
(10.2 - 10.6) the flocs of the microalgae cells became
less dense, thus slowly settled. It is important to note that
the viability of the microalgae cells gradually reduced with
increment of pH from 8 to 10.6. The viability at pH 8, pH
10 and pH 10.2 was 98, 78 and 68%, respectively.
Although reduction in cell viability was observed at pH
10.2, this pH value was chosen as the preferred pH for
the flocculation process due to substantial enhancement
in sedimentation rate. Higher pH, ranging from 10.6 to
11, was reported in the literature as the preferred pH for
the flocculation of microalgae (Heasman et al., 2001).
Effect of polyelectrolyte flocculant dosage on
Figure 5 and 6 shows the flocculation/ harvesting efficien-
cy, viability and maximum settling velocity for C. calci-
trans harvested at optimal pH adjustment (pH 10.2)
followed by addition of Magnafloc®LT 25 and Magnafloc®
LT 27, respectively. For Magnafloc®LT 25, flocculation
efficiency did not show significant difference with increas-
ing dosage. However, the sedimentation rate was increa-
sed gradually with increasing dosage of Magnafloc®LT
25. The cells viability was not changed with increasing
dosage. Almost similar results were also observed for
flocculation with Magnafloc®LT 27. However, the viability
of C. calcitrans improved with the addition of Magnafloc®
LT 27 as compared to culture without flocculant, though
reduced drastically with
Harith et al. 5975
Figure 5. Changes in flocculation efficiency, cells viability and
sedimentation rate for C. calcitrans flocculated at different
dosage of Magnafloc®LT 25 after pH adjustment at 10.2 using
NaOH. Symbols represent: (
( ) cells viability; and (?) sedimentation rate. The
sedimentation rate for control was too low and not measured.
) flocculation efficiency;
the viability was not changed with increasing dosage. The
flocculation efficiency was comparable to Magnafloc®LT
25. Interestingly, for both cases, the sedimentation rate
was substantially increased with increasing dosage of the
If the sedimentation rate shall be considered, higher
dosage of both flocculants shall be used. Higher sedi-
mentation rate resulted in shorter flocculation time. When
adjusting the pH of cultures, a buffering region was en-
countered at pH between pH 10.2 and 10.5. Within this
region, a precipitate formed entrapped the microalgae
cells. The formation of this precipitate was independent of
the presence of cells. This precipitate was closely to neu-
tral buoyancy and hence settled slowly (Knuckey et al.,
2006). Addition of polyelectrolytes at this stage acts as
enhancer by increasing the flocs size and sedimentation
rate by promoting the bridging, binding and strengthening
of the algal flocs. Besides that, the physical properties of
the flocculants such as molecular weight and functionality
play an important role in the performance of the floccula-
tion process. Nevertheless, the use of synthetic polymer
as flocculants is known to negatively affect the water eco-
system. Therefore, recommendation for maximum per-
missible concentration was set for flocculants residues.
However, Magnafloc®LT 25 and Magnafloc®LT 27 have
been approved for use by the drinking water inspec-
torate in United Kingdom and are classified as non
Effect of pH adjustment of flocculation efficiency
The flocculation of C. calcitrans with the addition of 20
mg/L of chitosan into the culture followed by pH adjust-
5976 Afr. J. Biotechnol.
Figure 6. Changes in flocculation efficiency, cells viability and
sedimentation rate for C. calcitrans flocculated at different
dosage of Magnafloc®LT 27 after pH adjustment at 10.2 using
NaOH. Symbols represent; (
() cells viability; and (?) sedimentation rate. The
sedimentation rate for control was too low and not measured.
) flocculation efficiency;
ment at different values was given in Figure 7. Due to
acidic characteristic of chitosan solution, the pH of the
culture reduced to around 3.14 after the addition of
chitosan. Microscopic examination showed cells in good
shape although the colour of cells were slightly greenish
due to excretion of pigments from the cells. However, the
cells retained their integrity and viability (> 80%). Floccu-
lation efficiency was drastically increased with increasing
pH from 5 to 8. Flocculation efficiency obtained at pH 5
was only about 50% and increased to 83% at pH 8. The
flocculation efficiency was measured after 4 h of floccu-
lation process. A slight reduction in flocculation efficiency
(76-77%) was observed with further increment in pH to 9
and 10. Cells viability was not significantly changed with
pH but the highest viability (81%) was obtained at pH 8.
Chitosan’s behavior is affected by pH. In acidic
condition, it exists as linear chain due to -NH2 groups
carrying positive charge and thus closely spaced. The
positively charged -NH2 and -NH3+ group repel each other
and during this condition, chitosan remain dispersed.
With an alkaline pH, the positive charge gradually dis-
appear (neutralization point at pH 7.9) and chitosan tends
to coil and precipitate (Gualteri et al., 1988). Chitosan
and C. calcitrans interact with each other through elec-
trostatic interaction. Chitosan attached to the negatively
charged algal surface via its positive charged group.
Bridges were formed between algal cells when the chain
had sufficient length to bind more than one cell. There-
fore, during acidic condition the degree of flocculation is
very weak. In alkaline pH, the positive charge was neu-
tralized and the highest neutralizing point was approxi-
mately achieved at pH 8. At this pH, C. calcitrans cells
have the highest negative charge, thus the flocculation
efficiency was enhanced.
Figure 7. Changes in flocculation efficiency and cells viability
of C. calcitrans flocculated with 20 mg/L of chitosan followed
with pH adjustment at different values. Symbols represent:
() cells viability; and () flocculation efficiency.
Effect of chitosan dosage on flocculation efficiency
The flocculation of C. calcitrans with different dosages of
chitosan into the culture followed by pH adjustment at 8 is
shown in Figure 8, which also include data for flocculation
without pH adjustment after addition of chitosan. For both
cases, flocculation efficiency was increased almost 2
times with increasing dosage from 10 to 20 mg/L. Floccu-
lation efficiency was not significantly increased with in-
creasing dosage of chitosan up to 150 mg/L. However,
the flocculation efficiency for system with pH adjustment
to 8 was about 2 times higher than those obtained in
flocculation without pH adjustment.
In the flocculation of Rhodomonas baltica using chito-
san as flocculant, more than 75% flocculation efficiency
was obtained at 80 mg/L of chitosan (Lubian et al., 1989).
On the other hand, lower dosage of chitosan (40 mg/L)
was required for several Tetraselmis species and very
high dosage (150 mg/L) was required for flocculation of
Chaetoceros muelleri (Heasman et al., 2001). Gualteri et
al. (1988) reported that the flocculation efficiency (96 -
98%) of Euglena gracilis was enhanced using 200 mg/L
of chitosan followed by pH adjustment at 7.5.
The flocculation efficiency of microalgae for separation of
cells from the culture broth was greatly influenced by the
pH. Comparable flocculation efficiency (more than 90%)
was obtained in flocculation with Magnafloc® (LT 25 and
LT 27) to those obtained in flocculation using chitosan.
Enhanced sedimentation rate was the obvious advantage
of flocculation with flocculant, which significantly reduced
Harith et al. 5977
Figure 8. Changes in flocculation efficiency of C. calcitrans flocculated at different dosages
of chitosan followed with pH adjustment to 8 using NaOH. Symbol represent; (
adjusted to 8 after the addition of chitosan; and (
) pH was
) pH was not adjusted after the addition
the sedimentation time. Although chitosan is more envi-
ronmental friendly compared to polyelectrolyte floccu-
lants, it may not be economical for microalgae separation
from culture broth due to higher price of chitosan. Effi-
cient flocculation process that can maintain high cell
viability could be a method of choice due to rapid,
inexpensive and simple method for harvesting large
quantity of microalgae cells such as C. calcitrans from the
culture broth prior to commercial formulation.
The authors would like to acknowledge the Johor Satellite
Biotechnology Project, Malaysia, for the funding support
(grant number: BSP(J)BTK/004(4)) during this study pe-
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