Bioresource Technology 102 (2011) 923–927

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Bioresource Technology

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Coagulation/flocculation-based removal of algal–bacterial biomass frompiggery 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, Boliviab Department of Chemical Engineering and Environmental Technology, Universidad de Valladolid, Paseo del Prado de la Magdalena s/n, 47011 Valladolid, Spainc 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:Received 23 July 2010Received in revised form 9 September 2010Accepted 9 September 2010Available online 17 September 2010

Keywords:CoagulationFlocculationHarvestingMicroalgaePiggery wastewater

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.09.036

⇑ Corresponding author. Tel.: +34 983184934; fax: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.

Two conventional chemical coagulants (FeCl3 and Fe2(SO4)3) and five commercial polymeric flocculants(Drewfloc 447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 and Chitosan) were comparativelyevaluated for their ability to remove algal–bacterial biomass from the effluent of a photosyntheticallyoxygenated piggery wastewater biodegradation process. Chlorella sorokiniana, Scenedesmus obliquus,Chlorococcum sp. and a wild type Chlorella, in symbiosis with a bacterial consortium, were used as modelalgal–bacterial consortia. While the highest biomass removals (66–98%) for the ferric salts were achievedat concentrations of 150–250 mg L�1, dosages of 25–50 mg L�1 were required for the polymer flocculantsto support comparable removal efficiencies. Process efficiency declined when the polymer flocculant wasoverdosed. Biomass concentration did not show a significant impact on flocculation within the concen-tration range tested. The high flocculant requirements herein recorded might be due to the competitionof colloidal organic for the flocculants and the stationary phase conditions of biomass.

� 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 sustainablewastewater treatment in the 21st century. These photosyntheticmicroorganisms furnish the O2 needed by bacteria to mineralizeorganic matter, enhance nutrients removal and provide the highestpathogen removal efficiencies among biological wastewater treat-ments (Ruiz-Marin et al., 2010; Schumacher et al., 2003; Wanget 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 significantpart of the greenhouse gas emissions associated to wastewater rec-lamation. Another important advantage of this technology is theproduction of a valuable microalgal biomass, which can be usedfor 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 wastewatertreatment) exhibit a simpler construction and operation than con-

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+34 983423013.

or anaerobic digesters.Microalgae-based treatments can also play a key role in sustain-

able farming: the high nutrients requirements of the vegetablecrops needed to support animal growth are supplied by microalgalbiofertilizers produced from livestock effluent treatment. In thiscontext, the potential of algal–bacterial photobioreactors as nutri-ents-recovery systems and of microalgal biomass as slow-releasefertilizer have been consistently proven (Olguín et al., 2003; deGodos et al., 2009; Mulbry et al., 2005). In addition, microalgaefrom bioremediation process have been successfully used as a highquality 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 inalgal–bacterial photobioreactors, despite the merits of paddle-wheel mixing in high rate algal ponds (HRAPs) on algal–bacterialfloc formation. Unfortunately, freely-suspended species such asChlorella and Scenedesmus are ubiquitous in wastewater treatmentponds due to their high tolerance to contaminated environments(Canovas et al., 1996). Therefore, the development of cost-effectivemethods for microalgae removal in photosynthetically oxygenatedwastewater treatment processes is crucial in order to guaranteeconsistent treatment efficiencies.

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 largespecies 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 microalgalbiomass recoveries at reasonable costs (Molina-Grima et al.,2003). These processes are based on the addition of chemicalscapable 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 cellaggregates by creating bridges between the neutralized microal-gae. This harvesting technique has been successfully tested inaquaculture, 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; Hendersonet al., 2008). However, most of the studies conducted to date as-sessed the potential of conventional aluminum or ferric salts formicroalgae removal and little attention has been given to thenew generation of high-performance polymeric flocculants.

In this study, the ability of two chemical flocculants (FeCl3 andFe2(SO4)3) and five commercial polymeric flocculants (Drewfloc447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 andChitosan) to remove algal–bacterial biomass from piggery waste-water treatment was evaluated. Three axenic species (Chlorellasorokiniana, Scenedesmus obliquus and Chlorococcum sp.) and a wildtype microalgal consortium isolated from a stabilization pond, insymbiosis 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 toaddress the sedimentation of effluents containing free living mic-roalgae, which is the worst case scenario and represents a rathercommon situation in algal–bacterial processes.

2. Methods

2.1. Microorganisms and culture conditions

C. sorokiniana 211/8 k and S. obliquus were obtained from theCulture Collection of Algae and Protozoa of the SAMS Research Ser-vices (Argyl, Scotland). A strain of the species Chlorococcum wasisolated from ‘‘Laguna Colorada’ (Potosí, Bolivia) in the mineral saltmedium (MSM) previously described by Muñoz et al. (2003). NaH-CO3 was added as carbon source at a final concentration of700 mg L�1. A microalgae consortium mainly composed of Chlorellastrains (from now on referred as Chlorella consortium) was isolatedfrom a stabilization pond treating piggery wastewater (de Godoset al., 2010). The microalgae inocula were prepared in 500 mL E-flasks filled with 200 mL of MSM enriched with 700 mg L�1 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 apig farm in Tiquipaya, (Cochabamba, Bolivia) and stored at 4 �C.Prior to experimentation, wastewater was centrifuged for 10 minat 6000 rpm. Therefore, only the soluble fraction of the wastewaterwas 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). ChemiflocCV-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 L�1 were prepared for each flocculantprior to experimentation. Chitosan was dissolved in a 1% aceticacid solution.

2.4. Piggery wastewater biodegradation in a fed-batchphotobioreactor

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 perbatch) and 10 mL of bacterial inoculum. An aliquot of 150 mL ofcentrifuged wastewater was then added to the photobioreactoron 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 agitationand illumination at 300 rpm and 3000 lux, respectively. Liquidsamples of 10 mL were daily drawn for the determination of cul-ture absorbance at 550 nm (OD550) and pH. Biodegradation testswere 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–NHþ4 was measured at the beginning and the end of eachcultivation.

2.5. Coagulation/flocculation tests

Coagulation/flocculation tests were conducted in 100 mL glassbeakers filled with 40 mL of each algal–bacterial broth under mag-netic stirring (300 rpm). The performance of each flocculant wasevaluated at 0 (control tests), 5, 25, 50, 100, 150 and 250 mg L�1.Following the addition of the flocculant, stirring was maintainedfor 1 min and the tests allowed settling for 10 min in the absenceof 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 inthe control tests (thus considering natural settling). All tests werecarried out in duplicate.

2.6. Influence of biomass concentration on removal efficiency

The influence of biomass concentration on the efficiency of thecoagulation/flocculation process was assessed using C. sorokinianaas model microalgae in the algal–bacterial consortium, and Chem-ifloc CV-300 and Drewfloc-447 as model flocculants. In order toavoid interfering matrix effects, fresh algal–bacterial biomass fromthe photobioreactor was concentrated by centrifugation or dilutedwith 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 thesame treated piggery wastewater matrix. Chemifloc CV-300 andDrewfloc-447 were tested at 25 mg L�1, which was the optimumconcentration for biomass removal by Chemifloc CV-300 accordingto 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–NHþ4 were measuredaccording 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-batchphotobioreactor

S. obliquus, C. sorokiniana, Chlorococcum sp. and the Chlorellaconsortium were capable of supporting piggery wastewater bio-degradation as shown by the increase in culture absorbance con-comitant with COD and NHþ4 removal. The overall CODconcentration (considering the wastewater amendment performedby the 2nd day of experimentation) in each fed-batch cultivation(�202 ± 12 mg L�1) decreased by 66%, 49%, 78% and 65% in thetests conducted with the Chlorella consortium, S. obliquus, Chloro-coccum sp. and C. sorokiniana, respectively. Likewise, the overallN–NH4

+ concentration decreased from 55 ± 1 mg L�1 to 12, 10, 11and 11 mg L�1 at the end of the biodegradation tests in the systemsinoculated with the Chlorella consortium, S. obliquus, Chlorococcumsp. and C. sorokiniana, respectively, resulting in N–NHþ4 -RE of 77%,81%, 80% and 79%. These final COD and N–NHþ4 -REs were in agree-ment with previous studies conducted by the authors using thesoluble fraction of piggery wastewater (de Godos et al., 2010).Based on the fact that the removal mechanisms supporting theabove described COD and NHþ4 removal are the same as those re-corded in outdoors full scale photobioreactors and the fact thatthe initial characteristics of our pre-treated wastewater are similarto those of domestic wastewater, the treated effluent from ourbatch photobioreactors can be regard as representative.

Final OD550 ranging from 0.43 to 0.66 was recorded in the testssupplied with S. obliquus, C. sorokiniana and Chlorococcum sp. afterapproximately 100 h of cultivation. However, the Chlorella consor-tium achieved an OD550 of 1.3 by the 4th day of experimentation.

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

Concentration (mg L�1) 5 25

Floccculant–Coagulant FeCl3 1 ± 3 4Fe2(SO4)3 2 ± 2 2Chitosan 2 ± 1 58Flocusol CM-78 23 ± 4 73Drewfloc 447 33 ± 1 89Chemifloc CV-300 29 ± 3 86Flocudex CS-5000 30 ± 4 95

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

Concentration (mg L�1) 5 2

Floccculant–Coagulant FeCl3 4 ± 1Fe2(SO4)3 6 ± 0Chitosan 3 ± 7 2Flocusol CM-78 33 ± 4 7Drewfloc 447 73 ± 9 5Chemifloc CV-300 73 ± 14 8Flocudex CS-5000 34 ± 18 6

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

Concentration (mg L�1) 5 25

Floccculant–Coagulant FeCl3 0 ± 2 15Fe2(SO4)3 16 ± 14 6Chitosan 23 ± 16 38Flocusol CM-78 62 ± 6 88Drewfloc 447 66 ± 6 88Chemifloc CV-300 46 ± 6 91Flocudex CS-5000 11 ± 7 79

Despite all biodegradation tests reported were conducted sequen-tially using the same piggery wastewater batch, the test carried outwith the Chlorella consortium was the first one in the series. Thegradual deterioration of piggery wastewater even at 4 �C is not arare phenomenon and it has been consistently observed in ourlab (de Godos et al., 2009). The cultivation broth resulting fromthe Chlorella consortium test probably presented a higher buffercapacity that the other tests. This fact was supported by the lowerpH values recorded at the end of the biodegradation tests com-pared to the other tests (9.88 vs. 10.47) and by the maintenanceof higher pH values in the presence of 250 mg L�1 of FeCl3 andFe2(SO4)3 (5.7–6 compared to 3.3–3.7), although this hypothesisshall have been supported by alkalinity measurements. Finally, itmust be stressed that a higher buffer capacity prevents microalgaeNH3-mediated inhibition, which could have promoted an extendedmicroalgal 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 themicroalgae evaluated, resulted in a wide range of biomass removalefficiencies (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 removalefficiencies at the highest concentrations tested (>100 mg L�1).Overall, concentrations below 50 mg L�1 exerted little effect onthe removal of biomass regardless of the microalgae evaluated(RE < 20%). Thus, biomass removals higher than 90% were achievedin the tests supplied with 250 mg L�1 of FeCl3 and Fe2(SO4)3 in the

50 100 150 250

± 4 9 ± 6 10 ± 5 14 ± 7 98 ± 1± 2 7 ± 4 7 ± 4 12 ± 5 90 ± 10± 8 18 ± 3 11 ± 3 13 ± 3 15 ± 5± 4 93 ± 5 94 ± 1 91 ± 2 77 ± 1± 4 99 ± 1 93 ± 3 76 ± 1 74 ± 2± 17 94 ± 1 84 ± 1 76 ± 3 66 ± 1± 2 86 ± 1 76 ± 2 60 ± 3 56 ± 1

5 50 100 150 250

1 ± 5 1 ± 4 95 ± 3 14 ± 9 26 ± 25 ± 8 14 ± 2 96 ± 4 98 ± 1 87 ± 60 ± 15 3 ± 7 1 ± 3 0 ± 0 1 ± 02 ± 5 83 ± 5 41 ± 16 54 ± 6 22 ± 96 ± 2 57 ± 10 40 ± 8 42 ± 9 37 ± 14 ± 2 64 ± 5 67 ± 1 52 ± 9 56 ± 41 ± 2 19 ± 1 10 ± 11 26 ± 5 4 ± 2

50 100 150 250

± 14 24 ± 15 63 ± 15 90 ± 8 79 ± 10± 8 11 ± 11 32 ± 10 87 ± 3 86 ± 10± 1 13 ± 9 0 ± 9 12 ± 6 8 ± 2± 2 92 ± 0 76 ± 1 74 ± 1 44 ± 1± 10 88 ± 10 72 ± 14 47 ± 2 63 ± 2± 4 71 ± 2 82 ± 17 77 ± 7 81 ± 17± 3 78 ± 14 56 ± 5 38 ± 9 11 ± 8

Table 4RE of algal–bacterial biomass (%) in tests conducted with C. sorokiniana.

Concentration (mg L�1) 5 25 50 100 150 250

Floccculant–Coagulant FeCl3 2 ± 5 2 ± 2 4 ± 2 3 ± 2 5 ± 2 66 ± 0Fe2(SO4)3 0 ± 4 0 ± 0 0 ± 2 0 ± 2 93 ± 1 98 ± 1Chitosan 5 ± 4 30 ± 11 28 ± 4 16 ± 2 17 ± 4 20 ± 4Flocusol CM-78 33 ± 2 78 ± 4 83 ± 1 76 ± 3 69 ± 5 62 ± 0Drewfloc 447 32 ± 2 55 ± 3 59 ± 8 63 ± 3 54 ± 4 57 ± 4Chemifloc CV-300 31 ± 7 84 ± 3 75 ± 15 73 ± 9 69 ± 13 72 ± 1Flocudex CS-5000 15 ± 1 62 ± 6 35 ± 6 40 ± 0 35 ± 4 38 ± 0

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Chlorella consortium test (Table 1). The maximum REs for C. soroki-niana were also recorded at 250 mg L�1 (66 ± 0 for FeCl3 and98 ± 1% for Fe2(SO4)3) (Table 4). No significant differences in RE(�86%) were observed in Chlorococcum sp. cultivation broth at150 and 250 mg L�1 for and Fe2(SO4)3 (Table 3). The removal of S.obliquus–bacteria biomass exhibited its maximum efficiency at100 mg FeCl3 L�1 and 150 Fe2(SO4)3 mg L�1. However, in the partic-ular case of FeCl3, an increase in concentrations of up to 150 and250 mg L�1 reduced severely these removals to 14 ± 9% and26 ± 2%, respectively. These results were not in agreement withthose previously reported by Sukenic et al. (1988), who observedbiomass-REs of up to 90% in Chlorella stigmatophora cultures using25 mg L�1 of FeCl3. Likewise, Jiang et al. (1993) reported Anabaenaflosaquae and Asterionella formosa removals ranging from 63–74%using FeCl3 at 58 mg L�1. The differences in the nature of the aque-ous matrices might explain this apparent mismatch, since most ofthe experiments conducted so far with ferric salts were carried outon clean media (i.e. reservoir water or synthetic media). In our par-ticular case, the high concentrations of colloidal organic matterpresent in the diluted piggery wastewater probably decreased floc-culant efficiency, which might explain the higher requirements offerric salts recorded. The need for high coagulant dosages due tothe presence of organic matter have been previously described inmicroalgal cultures (Jiang et al., 1993). In addition, the algal growthphase must be taken into account in a coagulation/flocculationprocess 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 directlylinked to the accumulation of extracellular organic matter, whichacts as a protective colloid. In this scenario, microalgae from fed-batch tests harvested in the stationary growth phase would be lesssusceptible to precipitate with ferric salts than continuous culturesreported 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 wereapplied at concentrations of 150 mg L�1(corresponding to 52 and42 mg L�1 of Fe, respectively), remarkable differences in biomassREs 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 ionicstrength could have affected process efficiency. Finally, it mustbe 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 at250 mg L�1.

Chitosan is a natural flocculant commonly used in wastewatertreatment 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-REsamong the flocculants tested. Despite the best flocculant perfor-mance was always recorded at 25 mg L�1 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 thosepreviously reported, which might be due to the interaction of col-

loidal organic matter with chitosan. Hence, Sukenic et al. (1988) re-corded almost complete C. stigmatophora recoveries using2.5 mg chitosan L�1 compared to maximum REs of 58% at25 mg L�1. When Chitosan was applied at 50–250 mg L�1 noenhancement in biomass removal was observed. This deteriorationin the flocculation process at increasing chitosan concentrationswas in agreement with the observations of Sukenic et al. (1988)and Buelna et al. (1990), who recorded a decrease in the efficiencyof flocculation when Chitosan was applied above its optimum dos-ages (10 and 20 mg L�1, respectively). This deterioration was likelythe result of the repulsive forces established when microalgal cellswere covered by an excess of flocculant (Danquah et al., 2008;Pushparaj et al., 1992). Due to the presence of acetic acid in thechitosan stock solution the pH steadily decreased at increasingchitosan dosages down to pH 3.7.

When polyacrylamide-based flocculants such as Flocusol CM-78, Drewfloc 447, Chemifloc CV-300 and Flocudex CS-5000 weredosed, low concentrations (5–50 mg L�1) were needed to removemost of the algal–bacterial biomass. Thus, Flocusol CM-78 exhib-ited its maximum biomass removal at 50 mg L�1 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 tothe Chlorella consortium, the optimum polymer concentration was100 mg L�1, 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 L�1, support-ing REs of 73 ± 9%, 88 ± 10% and 99 ± 1%, respectively. However,C. sorokiniana required concentrations of 100 mg L�1 to achieveits maximum RE (63 ± 4%). Biomass removals ranging from 84%to 91% were recorded at 25 mg Chemifloc CV-300 L�1 regardlessthe 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 (averageRE of 74 ± 16% for the four microalgae cultures) when supplied at25 mg L�1. 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. obliquusand Chlorococcum sp. tests, respectively, at 250 mg L�1. NeitherFlocusol, Drewfloc, Chemifloc nor Flocudex caused a significantvariation in the pH of the algal–bacterial broths. When using poly-mers, flocculation occurs by polymer attachment to the surface ofthe algae (negatively charged due to the ionization of their func-tional groups) at one or more sites, and by subsequent bridgingamong cells, thus creating three-dimensional algal-polymer matri-ces (Tenney et al., 1969; Uduman et al., 2010). This interactionmainly depends on polymer properties such as coil size, chargedensity 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 dueto it neutral or negative charge (Uduman et al., 2010).

Process economics in this harvesting technique are governed byboth the optimal dosage of chemical and their cost. For instance, if

Table 5Removal of algal–bacterial biomass (%) at different concentrations of biomass withDrewfloc-447 and Chemifloc CV-300.

Biomass dilution 1:2 1:1 2:1

Drewfloc-447 73 ± 10 56 ± 3 56 ± 3Chemifloc CV-300 74 ± 1 84 ± 3 83 ± 5

I. de Godos et al. / Bioresource Technology 102 (2011) 923–927 927

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 betweenFeCl3 and Chemifloc CV-300 (0.1 and 0.13 € for each m3 of algal–bacterial biomass effectively treated, respectively) and the fact thatpolyamides 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 onthe subsequent utilization of the solid fraction removed. In thiscontext, (González et al., 2008) showed that the use of ChemiflocCV-300 did not affect the anaerobic biodegradation of the solidfraction 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-447slightly 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 biomassconcentration. 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 notresult in a sustained deterioration or enhancement of biomass re-moval (Table 5). These results confirmed previous studies carriedout by Tenney et al. (1969) who reported that biomass flocculationdoes not present a linear correlation between flocculant dosageand biomass concentration. This empirical finding was further con-firmed by the consistent flocculation pattern present in the fourmicroalgal–bacterial consortium tested, despite the differences inculture absorbance pointed out at the beginning of the discussionsection.

Finally, this study showed that the coagulation/flocculation pro-cess for the colonial green microalgae Chlorococcum sp. was similarto that of the free cell microalgae. Despite the higher size of theflocks formed with Chlorococcum sp. the flocculant requirementsto carry out biomass precipitation were comparable to those sup-plied for the precipitation of free cell microalgae.

4. Conclusions

Microalgae harvesting using commercial polymers requiredlower 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 rangetested. However, the concentrations required were up to one orderof magnitude higher than those reported in literature for compara-ble removal efficiencies, which highlights the impact of the matrixon the flocculation process. Hence, while low flocculant concentra-tions are often reported in reservoir water or synthetic inorganicmedia, the high flocculant requirements herein recorded mightbe attributed to the competition of colloidal organic matter forthe flocculants and the stationary phase conditions of biomass.

Acknowledgements

This research was supported by the Spanish International Coop-eration Agency (A/023287/09 project) and the Regional Govern-ment of Castilla y León (GR76 project). The Spanish Ministry forScience and Innovation (RYC-2007-01667 contract and the projectCONSOLIDER-NOVEDAR CSD 2007-00055) is also gratefullyacknowledged.

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