Bioresource Technology 102 (2011) 923–927

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Coagulation/flocculation-based removal of algal–bacterial biomass from

piggery wastewater treatment
Ignacio de Godos a,b,c,1,2,3, Héctor O. Guzman a,1, Roberto Soto a,1, Pedro A. García-Encina b,2, Eloy Becares c,3,
Raúl Muñoz b,⇑, Virginia A. Vargas a,1
a
Center of Biotechnology. Universidad Mayor de San Simón, Campus Universitario, s/n Cochabamba, Bolivia
b
Department of Chemical Engineering and Environmental Technology, Universidad de Valladolid, Paseo del Prado de la Magdalena s/n, 47011 Valladolid, Spain
c
Department of Biodiversity and Environmental Management, Universidad de León, Campus Vegazana, 24071 León, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Two conventional chemical coagulants (FeCl3 and Fe2(SO4)3) and five commercial polymeric flocculants
Received 23 July 2010 (Drewfloc 447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 and Chitosan) were comparatively
Received in revised form 9 September 2010 evaluated for their ability to remove algal–bacterial biomass from the effluent of a photosynthetically
Accepted 9 September 2010
oxygenated piggery wastewater biodegradation process. Chlorella sorokiniana, Scenedesmus obliquus,
Available online 17 September 2010
Chlorococcum sp. and a wild type Chlorella, in symbiosis with a bacterial consortium, were used as model
algal–bacterial consortia. While the highest biomass removals (66–98%) for the ferric salts were achieved
Keywords:
at concentrations of 150–250 mg L1, dosages of 25–50 mg L1 were required for the polymer flocculants
Coagulation
Flocculation
to support comparable removal efficiencies. Process efficiency declined when the polymer flocculant was
Harvesting overdosed. Biomass concentration did not show a significant impact on flocculation within the concen-
Microalgae tration range tested. The high flocculant requirements herein recorded might be due to the competition
Piggery wastewater of colloidal organic for the flocculants and the stationary phase conditions of biomass.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction ventional treatment technologies such as activated sludge systems

Microalgae can play a key role in the quest for sustainable
wastewater treatment in the 21st century. These photosynthetic
microorganisms furnish the O2 needed by bacteria to mineralize
organic matter, enhance nutrients removal and provide the highest
pathogen removal efficiencies among biological wastewater treat-
ments (Ruiz-Marin et al., 2010; Schumacher et al., 2003; Wang
et al., 2010). Microalgae-based treatments are powered by sun-
light, which reduces the energy input to the process. This lower en-
ergy consumption, together with the inherent assimilation of CO2
during microalgal growth (photosynthesis), mitigates a significant
part of the greenhouse gas emissions associated to wastewater rec-
lamation. Another important advantage of this technology is the
production of a valuable microalgal biomass, which can be used
for biofuel or biofertilizer production (Mulbry et al., 2005). Finally,
it is important to stress that stabilization and high rate algae ponds
(the most widespread systems for microalgae-based wastewater
treatment) exhibit a simpler construction and operation than con-
⇑ Corresponding author. Tel.: +34 983184934; fax: +34 983423013.
E-mail address: mutora@iq.uva.es (R. Muñoz).
1
Tel.: +591 4 4542895; fax: +591 4 4542895.
2
Tel.: +34 983184934; fax: +34 983423013.
3
Tel.: +34 987291568; fax: +34 987291563.
or anaerobic digesters.
Microalgae-based treatments can also play a key role in sustain-
able farming: the high nutrients requirements of the vegetable
crops needed to support animal growth are supplied by microalgal
biofertilizers produced from livestock effluent treatment. In this
context, the potential of algal–bacterial photobioreactors as nutri-
ents-recovery systems and of microalgal biomass as slow-release
fertilizer have been consistently proven (Olguín et al., 2003; de
Godos et al., 2009; Mulbry et al., 2005). In addition, microalgae
from bioremediation process have been successfully used as a high
quality protein source in animal nutrition (Zepka et al., 2010).
However, despite the above mentioned advantages, the implemen-
tation of this technology is often hampered by the cost-effective-
ness of microalgae harvesting. Hence, the presence of freely-
suspended microalgae (characterized by low settling velocities) al-
ways challenges the removal efficiencies of COD and nutrients in
algal–bacterial photobioreactors, despite the merits of paddle-
wheel mixing in high rate algal ponds (HRAPs) on algal–bacterial
floc formation. Unfortunately, freely-suspended species such as
Chlorella and Scenedesmus are ubiquitous in wastewater treatment
ponds due to their high tolerance to contaminated environments
(Canovas et al., 1996). Therefore, the development of cost-effective
methods for microalgae removal in photosynthetically oxygenated
wastewater treatment processes is crucial in order to guarantee
consistent treatment efficiencies.

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.09.036
924 I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Microalgae harvesting can be carried out by centrifugation, fil-
tration and coagulation/flocculation (Molina-Grima et al., 2003).
While centrifugation presents prohibitive energy costs in the con-
text of wastewater treatment (low added value processes), filtra-
tion technologies are only useful for the recovery of relative large
species such as Spirulina. Low-cost filter presses often fail to har-
vest small microalgae such as Chlorella or Scenedesmus. Coagula-
tion/flocculation processes can however provide high microalgal
biomass recoveries at reasonable costs (Molina-Grima et al.,
2003). These processes are based on the addition of chemicals
capable of inducing the aggregation of individual microalgal cells.
Thus, while coagulants neutralize or invert electrical repulsions be-
tween microalgal cells, flocculants promote the formation of cell
aggregates by creating bridges between the neutralized microal-
gae. This harvesting technique has been successfully tested in
aquaculture, biofuels production, wastewater treatment and re-
moval of microalgae in fresh water reservoirs (Buelna et al.,
1990; Danquah et al., 2008; Knuckey et al., 2006; Henderson
et al., 2008). However, most of the studies conducted to date as-
sessed the potential of conventional aluminum or ferric salts for
microalgae removal and little attention has been given to the
new generation of high-performance polymeric flocculants.
In this study, the ability of two chemical flocculants (FeCl3 and
Fe2(SO4)3) and five commercial polymeric flocculants (Drewfloc
447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 and
Chitosan) to remove algal–bacterial biomass from piggery waste-
water treatment was evaluated. Three axenic species (Chlorella
sorokiniana, Scenedesmus obliquus and Chlorococcum sp.) and a wild
type microalgal consortium isolated from a stabilization pond, in
symbiosis with a bacterial consortium, were used as model al-
gal–bacterial consortia to evaluate the performance of the coagu-
lants/flocculants. Therefore, the main goal of our study was to
address the sedimentation of effluents containing free living mic-
roalgae, which is the worst case scenario and represents a rather
common situation in algal–bacterial processes.
2. Methods
2.1. Microorganisms and culture conditions
C. sorokiniana 211/8 k and S. obliquus were obtained from the
Culture Collection of Algae and Protozoa of the SAMS Research Ser-
vices (Argyl, Scotland). A strain of the species Chlorococcum was
isolated from ‘‘Laguna Colorada’ (Potosí, Bolivia) in the mineral salt
medium (MSM) previously described by Muñoz et al. (2003). NaH-
CO3 was added as carbon source at a final concentration of
700 mg L1. A microalgae consortium mainly composed of Chlorella
strains (from now on referred as Chlorella consortium) was isolated
from a stabilization pond treating piggery wastewater (de Godos
et al., 2010). The microalgae inocula were prepared in 500 mL E-
flasks filled with 200 mL of MSM enriched with 700 mg L1 of NaH-
CO3. All inocula were incubated at room temperature (25 ± 2 °C)
under continuous magnetic stirring (300 rpm) and illumination
(3000 lux) (TLC Philips, Chile, 18 W).
The bacterial consortium used for organic matter mineraliza-
tion was obtained from Cochabamba Wastewater Treatment Plant
(Bolivia).
2.2. Piggery wastewater
Piggery wastewater was obtained from the main collector of a
pig farm in Tiquipaya, (Cochabamba, Bolivia) and stored at 4 °C.
Prior to experimentation, wastewater was centrifuged for 10 min
at 6000 rpm. Therefore, only the soluble fraction of the wastewater
was used for the biodegradation test.
2.3. Flocculants
Flocudex CS-5000, Flocusol CM-78 and Drewfloc 447 were sup-
plied by Lamirsa Laboratorios, S.A., (Barcelona, Spain). Chemifloc
CV-300 was supplied by Chemipol S.A (Terrassa, Spain). Chitosan,
FeCl3 and Fe2(SO4)3 were purchased from Sigma–Aldrich (Spain).
Stocks solutions of 2000 mg L1 were prepared for each flocculant
prior to experimentation. Chitosan was dissolved in a 1% acetic
acid solution.
2.4. Piggery wastewater biodegradation in a fed-batch
photobioreactor
A magnetically stirred 5-L glass tank photobioreactor was ini-
tially filled with 2800 mL of 20-folds diluted centrifuged wastewa-
ter and inoculated with 40 mL of the tested microalgae (one per
batch) and 10 mL of bacterial inoculum. An aliquot of 150 mL of
centrifuged wastewater was then added to the photobioreactor
on the second day of cultivation resulting in 3000 mL of approxi-
mately10-folds diluted wastewater. The photobioreactor was oper-
ated at room temperature under continuous magnetic agitation
and illumination at 300 rpm and 3000 lux, respectively. Liquid
samples of 10 mL were daily drawn for the determination of cul-
ture absorbance at 550 nm (OD550) and pH. Biodegradation tests
were allowed to run until steady values for OD550 and pH were re-
corded, and the cultivation broth readily used in the coagulation/
flocculation tests. The concentration of the soluble COD and N–
NHþ 4 was measured at the beginning and the end of each
cultivation.
2.5. Coagulation/flocculation tests
Coagulation/flocculation tests were conducted in 100 mL glass
beakers filled with 40 mL of each algal–bacterial broth under mag-
netic stirring (300 rpm). The performance of each flocculant was
evaluated at 0 (control tests), 5, 25, 50, 100, 150 and 250 mg L1.
Following the addition of the flocculant, stirring was maintained
for 1 min and the tests allowed settling for 10 min in the absence
of stirring. A liquid sample of 1 mL was then drawn for OD550 anal-
ysis at 1 cm below the surface of the treated algal–bacterial broth.
Biomass removal was calculated based on OD550 values recorded in
the control tests (thus considering natural settling). All tests were
carried out in duplicate.
2.6. Influence of biomass concentration on removal efficiency
The influence of biomass concentration on the efficiency of the
coagulation/flocculation process was assessed using C. sorokiniana
as model microalgae in the algal–bacterial consortium, and Chem-
ifloc CV-300 and Drewfloc-447 as model flocculants. In order to
avoid interfering matrix effects, fresh algal–bacterial biomass from
the photobioreactor was concentrated by centrifugation or diluted
with the supernatant resulting from centrifugation. Using this pro-
cedure, two folds concentrated (namely 2:1 test) and two folds di-
luted (namely 1:2 test) algal–bacterial broths were prepared in the
same treated piggery wastewater matrix. Chemifloc CV-300 and
Drewfloc-447 were tested at 25 mg L1, which was the optimum
concentration for biomass removal by Chemifloc CV-300 according
to the previous coagulation/flocculation tests.
2.7. Analytical procedures
OD550 and pH were measured in a Lambda 25 UV/visible spec-
trophotometer (Perkin Elmer, USA) and a pH-probe (Thermo Scien-
tific Orion, USA), respectively. COD and N–NHþ 4 were measured
according to Standard Methods (Eaton et al., 2005).
 I. de Godos et al. / Bioresource Technology 102 (2011) 923–927 925

3. Results and discussion
3.1. Piggery wastewater biodegradation in a fed-batch
photobioreactor
S. obliquus, C. sorokiniana, Chlorococcum sp. and the Chlorella
consortium were capable of supporting piggery wastewater bio-
degradation as shown by the increase in culture absorbance con-
comitant with COD and NHþ 4 removal. The overall COD
concentration (considering the wastewater amendment performed
by the 2nd day of experimentation) in each fed-batch cultivation
(202 ± 12 mg L1) decreased by 66%, 49%, 78% and 65% in the
tests conducted with the Chlorella consortium, S. obliquus, Chloro-
coccum sp. and C. sorokiniana, respectively. Likewise, the overall
N–NH4+ concentration decreased from 55 ± 1 mg L1 to 12, 10, 11
and 11 mg L1 at the end of the biodegradation tests in the systems
inoculated with the Chlorella consortium, S. obliquus, Chlorococcum
sp. and C. sorokiniana, respectively, resulting in N–NHþ 4 -RE of 77%,
81%, 80% and 79%. These final COD and N–NHþ 4 -REs were in agree-
ment with previous studies conducted by the authors using the
soluble fraction of piggery wastewater (de Godos et al., 2010).
Based on the fact that the removal mechanisms supporting the
above described COD and NHþ 4 removal are the same as those re-
corded in outdoors full scale photobioreactors and the fact that
the initial characteristics of our pre-treated wastewater are similar
to those of domestic wastewater, the treated effluent from our
batch photobioreactors can be regard as representative.
Final OD550 ranging from 0.43 to 0.66 was recorded in the tests
supplied with S. obliquus, C. sorokiniana and Chlorococcum sp. after
approximately 100 h of cultivation. However, the Chlorella consor-
tium achieved an OD550 of 1.3 by the 4th day of experimentation.
Despite all biodegradation tests reported were conducted sequen-
tially using the same piggery wastewater batch, the test carried out
with the Chlorella consortium was the first one in the series. The
gradual deterioration of piggery wastewater even at 4 °C is not a
rare phenomenon and it has been consistently observed in our
lab (de Godos et al., 2009). The cultivation broth resulting from
the Chlorella consortium test probably presented a higher buffer
capacity that the other tests. This fact was supported by the lower
pH values recorded at the end of the biodegradation tests com-
pared to the other tests (9.88 vs. 10.47) and by the maintenance
of higher pH values in the presence of 250 mg L1 of FeCl3 and
Fe2(SO4)3 (5.7–6 compared to 3.3–3.7), although this hypothesis
shall have been supported by alkalinity measurements. Finally, it
must be stressed that a higher buffer capacity prevents microalgae
NH3-mediated inhibition, which could have promoted an extended
microalgal autotrophic growth in this particular test.
3.2. Coagulation/flocculation tests
The different nature and concentration of the coagulants/flocc-
ulants tested, together with the differences in the properties of the
microalgae evaluated, resulted in a wide range of biomass removal
efficiencies (Tables 1–4). Biomass settling in the absence of coagu-
lant/flocculant (control tests) was negligible regardless of the mic-
roalgae tested (data not shown).
Both FeCl3 and Fe2(SO4)3 presented their maximum removal
efficiencies at the highest concentrations tested (>100 mg L1).
Overall, concentrations below 50 mg L1 exerted little effect on
the removal of biomass regardless of the microalgae evaluated
(RE < 20%). Thus, biomass removals higher than 90% were achieved
in the tests supplied with 250 mg L1 of FeCl3 and Fe2(SO4)3 in the

Table 1
RE of algal–bacterial biomass (%) in tests conducted with Chlorella consortium.

Concentration (mg L1) 5 25 50 100 150 250

Floccculant–Coagulant FeCl3 1±3 4±4 9±6 10 ± 5 14 ± 7 98 ± 1
Fe2(SO4)3 2±2 2±2 7±4 7±4 12 ± 5 90 ± 10
Chitosan 2±1 58 ± 8 18 ± 3 11 ± 3 13 ± 3 15 ± 5
Flocusol CM-78 23 ± 4 73 ± 4 93 ± 5 94 ± 1 91 ± 2 77 ± 1
Drewfloc 447 33 ± 1 89 ± 4 99 ± 1 93 ± 3 76 ± 1 74 ± 2
Chemifloc CV-300 29 ± 3 86 ± 17 94 ± 1 84 ± 1 76 ± 3 66 ± 1
Flocudex CS-5000 30 ± 4 95 ± 2 86 ± 1 76 ± 2 60 ± 3 56 ± 1

Table 2
RE of algal–bacterial biomass (%) in tests conducted with S. obliquus.

Concentration (mg L1) 5 25 50 100 150 250

Floccculant–Coagulant FeCl3 4±1 1±5 1±4 95 ± 3 14 ± 9 26 ± 2
Fe2(SO4)3 6±0 5±8 14 ± 2 96 ± 4 98 ± 1 87 ± 6
Chitosan 3±7 20 ± 15 3±7 1±3 0±0 1±0
Flocusol CM-78 33 ± 4 72 ± 5 83 ± 5 41 ± 16 54 ± 6 22 ± 9
Drewfloc 447 73 ± 9 56 ± 2 57 ± 10 40 ± 8 42 ± 9 37 ± 1
Chemifloc CV-300 73 ± 14 84 ± 2 64 ± 5 67 ± 1 52 ± 9 56 ± 4
Flocudex CS-5000 34 ± 18 61 ± 2 19 ± 1 10 ± 11 26 ± 5 4±2

Table 3
RE of algal–bacterial biomass (%) in tests conducted with Chlorococcum sp.

Concentration (mg L1) 5 25 50 100 150 250

Floccculant–Coagulant FeCl3 0±2 15 ± 14 24 ± 15 63 ± 15 90 ± 8 79 ± 10
Fe2(SO4)3 16 ± 14 6±8 11 ± 11 32 ± 10 87 ± 3 86 ± 10
Chitosan 23 ± 16 38 ± 1 13 ± 9 0±9 12 ± 6 8±2
Flocusol CM-78 62 ± 6 88 ± 2 92 ± 0 76 ± 1 74 ± 1 44 ± 1
Drewfloc 447 66 ± 6 88 ± 10 88 ± 10 72 ± 14 47 ± 2 63 ± 2
Chemifloc CV-300 46 ± 6 91 ± 4 71 ± 2 82 ± 17 77 ± 7 81 ± 17
Flocudex CS-5000 11 ± 7 79 ± 3 78 ± 14 56 ± 5 38 ± 9 11 ± 8
926 I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Table 4
RE of algal–bacterial biomass (%) in tests conducted with C. sorokiniana.
Concentration (mg L1) 5 25
Floccculant–Coagulant FeCl3 2±5
Fe2(SO4)3 0±4
Chitosan 5±4
Flocusol CM-78 33 ± 2
Drewfloc 447 32 ± 2
Chemifloc CV-300 31 ± 7
Flocudex CS-5000 15 ± 1
Chlorella consortium test (Table 1). The maximum REs for C. soroki-
niana were also recorded at 250 mg L1 (66 ± 0 for FeCl3 and
98 ± 1% for Fe2(SO4)3) (Table 4). No significant differences in RE
(86%) were observed in Chlorococcum sp. cultivation broth at
150 and 250 mg L1 for and Fe2(SO4)3 (Table 3). The removal of S.
obliquus–bacteria biomass exhibited its maximum efficiency at
100 mg FeCl3 L1 and 150 Fe2(SO4)3 mg L1. However, in the partic-
ular case of FeCl3, an increase in concentrations of up to 150 and
250 mg L1 reduced severely these removals to 14 ± 9% and
26 ± 2%, respectively. These results were not in agreement with
those previously reported by Sukenic et al. (1988), who observed
biomass-REs of up to 90% in Chlorella stigmatophora cultures using
25 mg L1 of FeCl3. Likewise, Jiang et al. (1993) reported Anabaena
flosaquae and Asterionella formosa removals ranging from 63–74%
using FeCl3 at 58 mg L1. The differences in the nature of the aque-
ous matrices might explain this apparent mismatch, since most of
the experiments conducted so far with ferric salts were carried out
on clean media (i.e. reservoir water or synthetic media). In our par-
ticular case, the high concentrations of colloidal organic matter
present in the diluted piggery wastewater probably decreased floc-
culant efficiency, which might explain the higher requirements of
ferric salts recorded. The need for high coagulant dosages due to
the presence of organic matter have been previously described in
microalgal cultures (Jiang et al., 1993). In addition, the algal growth
phase must be taken into account in a coagulation/flocculation
process as reported by Tenney (1969), who observed higher floccu-
lant requirements dosages when the algal biomass from batch cul-
tures was in stationary phase. This phenomenon was directly
linked to the accumulation of extracellular organic matter, which
acts as a protective colloid. In this scenario, microalgae from fed-
batch tests harvested in the stationary growth phase would be less
susceptible to precipitate with ferric salts than continuous cultures
reported by other authors. It is also important to stress that differ-
ent biomass REs were recorded at similar Fe concentration depend-
ing on the salt applied. Thereby, when FeCl3 and Fe2(SO4)3 were
applied at concentrations of 150 mg L1(corresponding to 52 and
42 mg L1 of Fe, respectively), remarkable differences in biomass
REs were observed in S. obliquus: 14 ± 9% vs. 98 ± 1% and C. soroki-
niana 5 ± 2% vs. 93 ± 1%. Iron being the active element in the coag-
ulation–flocculation process, other factors such as pH or ionic
strength could have affected process efficiency. Finally, it must
be highlighted that the addition of these chemical coagulants grad-
ually decreased the pH from 10–10.5 in the control tests to 3–3.7 at
250 mg L1.
Chitosan is a natural flocculant commonly used in wastewater
treatment for suspended solid separation. Its low cost and non-
toxic nature make it one of the preferred flocculants in microal-
gae-based biotechnologies (Lersutthiwong et al., 2009). However,
in our particular case, chitosan presented the lowest biomass-REs
among the flocculants tested. Despite the best flocculant perfor-
mance was always recorded at 25 mg L1 for all microalgae evalu-
ated, the removals achieved were lower than 40% for C. sorokiniana,
Chlorococcum sp. and S. obliquus, and 58 ± 8% for the Chlorella Con-
sortium. These algal–bacterial biomass removals were below those
previously reported, which might be due to the interaction of col-
50 100 150 250
2±2 4±2 3±2 5±2 66 ± 0
0±0 0±2 0±2 93 ± 1 98 ± 1
30 ± 11 28 ± 4 16 ± 2 17 ± 4 20 ± 4
78 ± 4 83 ± 1 76 ± 3 69 ± 5 62 ± 0
55 ± 3 59 ± 8 63 ± 3 54 ± 4 57 ± 4
84 ± 3 75 ± 15 73 ± 9 69 ± 13 72 ± 1
62 ± 6 35 ± 6 40 ± 0 35 ± 4 38 ± 0
loidal organic matter with chitosan. Hence, Sukenic et al. (1988) re-
corded almost complete C. stigmatophora recoveries using
2.5 mg chitosan L1 compared to maximum REs of 58% at
25 mg L1. When Chitosan was applied at 50–250 mg L1 no
enhancement in biomass removal was observed. This deterioration
in the flocculation process at increasing chitosan concentrations
was in agreement with the observations of Sukenic et al. (1988)
and Buelna et al. (1990), who recorded a decrease in the efficiency
of flocculation when Chitosan was applied above its optimum dos-
ages (10 and 20 mg L1, respectively). This deterioration was likely
the result of the repulsive forces established when microalgal cells
were covered by an excess of flocculant (Danquah et al., 2008;
Pushparaj et al., 1992). Due to the presence of acetic acid in the
chitosan stock solution the pH steadily decreased at increasing
chitosan dosages down to pH 3.7.
When polyacrylamide-based flocculants such as Flocusol CM-
78, Drewfloc 447, Chemifloc CV-300 and Flocudex CS-5000 were
dosed, low concentrations (5–50 mg L1) were needed to remove
most of the algal–bacterial biomass. Thus, Flocusol CM-78 exhib-
ited its maximum biomass removal at 50 mg L1 for S. obliquus,
Chlorococcum sp. and C. sorokiniana biomass (83–92%). However,
an increase in the concentration of this flocculant for these algal–
bacterial consortia brought about a decrease in flocculation perfor-
mance likely due to repulsion forces (Tables 2–4). When applied to
the Chlorella consortium, the optimum polymer concentration was
100 mg L1, which resulted in biomass removals of 94 ± 1% (Ta-
ble 1). On the other hand, when Drewfloc-447 was supplied to S.
obliquus, Chlorococcum sp., and the Chlorella consortium an opti-
mum performance was recorded at 5, 25 and 50 mg L1, support-
ing REs of 73 ± 9%, 88 ± 10% and 99 ± 1%, respectively. However,
C. sorokiniana required concentrations of 100 mg L1 to achieve
its maximum RE (63 ± 4%). Biomass removals ranging from 84%
to 91% were recorded at 25 mg Chemifloc CV-300 L1 regardless
the microalgae tested and further increases in flocculant dosage re-
sulted in a severe deterioration in the flocculation performance. Fi-
nally, Flocudex CS-5000 exhibited the best performance (average
RE of 74 ± 16% for the four microalgae cultures) when supplied at
25 mg L1. However, the removal efficiency of this polymer se-
verely decreased at increasing concentrations (likely due to repul-
sion forces), reaching removals of 11 ± 8% and 4 ± 2% in S. obliquus
and Chlorococcum sp. tests, respectively, at 250 mg L1. Neither
Flocusol, Drewfloc, Chemifloc nor Flocudex caused a significant
variation in the pH of the algal–bacterial broths. When using poly-
mers, flocculation occurs by polymer attachment to the surface of
the algae (negatively charged due to the ionization of their func-
tional groups) at one or more sites, and by subsequent bridging
among cells, thus creating three-dimensional algal-polymer matri-
ces (Tenney et al., 1969; Uduman et al., 2010). This interaction
mainly depends on polymer properties such as coil size, charge
density and degree of branching (for nonlinear polymers). All poly-
mers used in this work were cationic polyacrylamides since anio-
nic and non-ionic polymers often exhibit a poor performance due
to it neutral or negative charge (Uduman et al., 2010).
Process economics in this harvesting technique are governed by
both the optimal dosage of chemical and their cost. For instance, if
 I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Table 5
Removal of algal–bacterial biomass (%) at different concentrations of biomass with
Drewfloc-447 and Chemifloc CV-300.
Biomass dilution 1:2 1:1
Drewfloc-447 73 ± 10 56 ± 3
Chemifloc CV-300 74 ± 1 84 ± 3
we consider the Chlorella consortium as model algal–bacterial bio-
mass, ferric chloride would be the most economic option. Never-
theless, taken into account the slight difference of price between
FeCl3 and Chemifloc CV-300 (0.1 and 0.13 € for each m3 of algal–
bacterial biomass effectively treated, respectively) and the fact that
polyamides do not alter the pH of treated effluent, Chemifloc CV-
300 can be considered as the most suitable option. In addition, re-
cent studies highlighted the benefits of using organic flocculant on
the subsequent utilization of the solid fraction removed. In this
context, (González et al., 2008) showed that the use of Chemifloc
CV-300 did not affect the anaerobic biodegradation of the solid
fraction removed.
3.3. Influence of biomass concentration on removal efficiency
The experimental data obtained in this test revealed that bio-
mass concentration did not have a severe impact on flocculant per-
formance. Possibly due to different optimum dosage for Drewfloc-
447 and Chemifloc CV-300 (Table 4), while the REs of Drewfloc-447
slightly increased from 56 ± 3% to 73 ± 10% when decreasing bio-
mass concentrations by a factor of 2, the REs of Chemifloc CV-
300 decreased from 84% to 74% for the same decrease in biomass
concentration. Hence, this study showed that an increase in al-
gal–bacterial biomass concentration during the flocculation pro-
cess conducted with Drewfloc 447 and Chemifloc CV-300 did not
result in a sustained deterioration or enhancement of biomass re-
moval (Table 5). These results confirmed previous studies carried
out by Tenney et al. (1969) who reported that biomass flocculation
does not present a linear correlation between flocculant dosage
and biomass concentration. This empirical finding was further con-
firmed by the consistent flocculation pattern present in the four
microalgal–bacterial consortium tested, despite the differences in
culture absorbance pointed out at the beginning of the discussion
section.
Finally, this study showed that the coagulation/flocculation pro-
cess for the colonial green microalgae Chlorococcum sp. was similar
to that of the free cell microalgae. Despite the higher size of the
flocks formed with Chlorococcum sp. the flocculant requirements
to carry out biomass precipitation were comparable to those sup-
plied for the precipitation of free cell microalgae.
4. Conclusions
Microalgae harvesting using commercial polymers required
lower dosages than conventional coagulation based on ferric salts.
In addition, biomass concentration did not show a significant im-
pact on flocculation performance within the concentration range
tested. However, the concentrations required were up to one order
of magnitude higher than those reported in literature for compara-
ble removal efficiencies, which highlights the impact of the matrix
on the flocculation process. Hence, while low flocculant concentra-
tions are often reported in reservoir water or synthetic inorganic
media, the high flocculant requirements herein recorded might
be attributed to the competition of colloidal organic matter for
the flocculants and the stationary phase conditions of biomass.
927
Acknowledgements
This research was supported by the Spanish International Coop-
2:1 eration Agency (A/023287/09 project) and the Regional Govern-
56 ± 3 ment of Castilla y León (GR76 project). The Spanish Ministry for
83 ± 5 Science and Innovation (RYC-2007-01667 contract and the project
CONSOLIDER-NOVEDAR CSD 2007-00055) is also gratefully
acknowledged.
References
Buelna, G., Bhattari, K.K., de la Noüe, J., Taiganides, E.P., 1990. Evaluation of various
flocculants for the recovery of algal biomass grown on pig-waste. Biol. Wastes
31 (3), 211–222.
Canovas, S., Picot, B., Casellas, C., Zulki, H., Dubois, A., Bontoux, J., 1996. Seasonal
development of phytoplankton and zooplankton in a high rate algae pond.
Water Sci. Technol. 33 (7), 199–206.
Danquah, M.K., Ang, L., Uduman, N., Moheimani, N., Forde, G.M., 2008. Dewatering
of Microalgal culture for biodiesel production: exploring polymer flocculation
and tangential flow filtration. J. Chem. Technol. Biotechnol. 84 (7), 1078–1083.
Eaton, A.D., Clesceri, L.S., Greenberg, A.E., 2005. Standard Methods for the
Examination of Water and Wastewater, 21st ed. American Public Health
Association/American Water Works Association/Water Environment
Federation, Washington, DC, USA.
de Godos, I., González, C., García-Encina, P., Bécares, E., Muñoz, R., 2009.
Simultaneous nitrification–denitrification, phosphorous and carbon removal
during pre-treated swine slurry degradation in a tubular biofilm
photobioreactor. Appl. Microbiol. Biotechnol. 82 (1), 187–194.
de Godos, I., Vargas, V.A., Blanco, S., García, M.C., Soto, R., García-Encina, P.A.,
Becares, E., Muñoz, R., 2010. A comparative evaluation of microalgae for the
degradation of piggery wastewater under photosynthetic oxygenation.
Bioresour. Technol. 101 (14), 5150–5158.
González, C., León-Cofreces, C., García Encina, P.A., 2008. Different pretreatments for
the biodegradability in swine manure. Bioresour. Technol. 99, 8710–8714.
Henderson, R., Parsons, S.A., Jefferson, B., 2008. The impact of algal properties and
pre-oxidation on solid–liquid separation of algae. Water Res. 42, 1827–2845.
Jiang, J.Q., Graham, N.J.D., Harward, C., 1993. Comparison of polyferric sulphate with
other coagulants for the removal of algae-derived organic matter. Water. Sci.
Technol. 27 (11), 221–230.
Knuckey, R.M., Brown, M.R., Robert, R., Frampton, D.M.F., 2006. Production of
microalgal concentrates by flocculation and their assessment as aquaculture
feeds. Aquacul. Eng. 35, 300–313.
Lersutthiwong, P., Suttikarn, S., Powtongsook, S., 2009. Optimization of chitosan
flocculation for removal of phytoplankton in shrimp ponds. Aquacul. eng. 41,
188–193.
Molina-Grima, E., Belarbi, E.-H., Ancién Fernández, F.G., Robles-Medina, A., Chisti, Y.,
2003. Recovery of microalgal biomass and metabolites: process options and
economics. Biotechnol. Adv. 20, 491–515.
Mulbry, W., Kebede-Westhead, E., Pizarro, C., Sikora, L., 2005. Recycling of manure
nutrients: use of algal biomass from dairy manure treatment as a slow release
fertilizer. Bioresour. Technol. 96, 451–458.
Muñoz, R., Guieysse, B., Mattiasson, B., 2003. Phenanthrene biodegradation by an
algal-bacterial consortium in two-phase partitioning bioreactors. Appl.
microbiol. Biotechnol. 61, 261–267.
Olguín, E.J., Galicia, S., Mercado, G., Pérez, T., 2003. Annual productivity of Spirulina
(Arthrospira) and nutrients removal in a pig wastewater recycling process under
tropical conditions. J. Appl. Phycol. 15, 249–257.
Pushparaj, B., Pelosi, E., Torzillo, G., Matterassi, R., 1992. Microbial biomass recovery
using synthetic cationic polymer. Bioresour. Technol. 43, 59–62.
Ruiz-Marin, A., Mendoza-Espinosa, L.G., Stephenson, T., 2010. Growth and nutrient
removal in free and immobilized green algae in batch and semi-continuous
cultures treating real wastewater. Bioresour. Technol. 101, 58–64.
Schumacher, G., Blume, T., Sekoulov, I., 2003. Bacteria reduction and nutrient
removal in small wastewater treatments plants by algal biofilm. Water Sci.
Technol. 47, 195–202.
Sukenic, A., Bilanovic, D., Shelef, G., 1988. Flocculation of microalgae in brackish and
seawater. Biomass 15, 187–198.
Tenney, M.W., Echelberger, W.F., Schuessler, R.G., Pavoni, J.L., 1969. Algal
flocculation with synthetic organic polyelectrolytes. Appl. Microbiol. 18, 965–
971.
Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering
microalgal cultures: A major bottleneck to algae-based fuels. J. Renew. Sustain.
Energy 2, 012701.
Wang, L., Li, Y., Chen, P., Min, M., Chen, Y., Zhu, J., Ruan, R., 2010. Anaerobic digested
dairy manure as a nutrient supplement for cultivation of oil-rich green
microalgae Chlorella sp. Bioresour. Technol. 101, 2623–2628.
Zepka, L.Q., Jacob-Lopes, E., Goldbeck, R., Souza-Soares, L.A., Queiroz, M.I., 2010.
Nutritional evaluation of single-cell protein produced by Aphanothece
microscópica Nägeli. Bioresour. Technol. 101, 7107–7111.