- Email: [email protected]
flocculation-based removal of algal–bacterial biomass from piggery wastewater treatment
Bioresource Technology 102 (2011) 923–927
Contents lists available at ScienceDirect
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 b c
Center of Biotechnology. Universidad Mayor de San Simón, Campus Universitario, s/n Cochabamba, Bolivia Department of Chemical Engineering and Environmental Technology, Universidad de Valladolid, Paseo del Prado de la Magdalena s/n, 47011 Valladolid, Spain 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
Article history: Received 23 July 2010 Received in revised form 9 September 2010 Accepted 9 September 2010 Available online 17 September 2010 Keywords: Coagulation Flocculation Harvesting Microalgae Piggery wastewater
a b s t r a c t 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 comparatively evaluated for their ability to remove algal–bacterial biomass from the effluent of a photosynthetically oxygenated piggery wastewater biodegradation process. Chlorella sorokiniana, Scenedesmus obliquus, 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 at concentrations of 150–250 mg L1, dosages of 25–50 mg L1 were required for the polymer flocculants to support comparable removal efficiencies. Process efficiency declined when the polymer flocculant was overdosed. Biomass concentration did not show a significant impact on flocculation within the concentration range tested. The high flocculant requirements herein recorded might be due to the competition of colloidal organic for the flocculants and the stationary phase conditions of biomass. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction 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 treatments (Ruiz-Marin et al., 2010; Schumacher et al., 2003; Wang et al., 2010). Microalgae-based treatments are powered by sunlight, which reduces the energy input to the process. This lower energy consumption, together with the inherent assimilation of CO2 during microalgal growth (photosynthesis), mitigates a significant part of the greenhouse gas emissions associated to wastewater reclamation. 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. 1 2 3
E-mail address: [email protected] (R. Muñoz). Tel.: +591 4 4542895; fax: +591 4 4542895. Tel.: +34 983184934; fax: +34 983423013. Tel.: +34 987291568; fax: +34 987291563.
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.09.036
ventional treatment technologies such as activated sludge systems or anaerobic digesters. Microalgae-based treatments can also play a key role in sustainable 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 nutrients-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 implementation of this technology is often hampered by the cost-effectiveness of microalgae harvesting. Hence, the presence of freelysuspended microalgae (characterized by low settling velocities) always challenges the removal efficiencies of COD and nutrients in algal–bacterial photobioreactors, despite the merits of paddlewheel 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.
924
I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Microalgae harvesting can be carried out by centrifugation, filtration and coagulation/flocculation (Molina-Grima et al., 2003). While centrifugation presents prohibitive energy costs in the context of wastewater treatment (low added value processes), filtration technologies are only useful for the recovery of relative large species such as Spirulina. Low-cost filter presses often fail to harvest small microalgae such as Chlorella or Scenedesmus. Coagulation/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 between microalgal cells, flocculants promote the formation of cell aggregates by creating bridges between the neutralized microalgae. This harvesting technique has been successfully tested in aquaculture, biofuels production, wastewater treatment and removal 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 assessed 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 wastewater 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 algal–bacterial consortia to evaluate the performance of the coagulants/flocculants. Therefore, the main goal of our study was to address the sedimentation of effluents containing free living microalgae, 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 Services (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). NaHCO3 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 Eflasks filled with 200 mL of MSM enriched with 700 mg L1 of NaHCO3. 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 mineralization was obtained from Cochabamba Wastewater Treatment Plant (Bolivia).
2.3. Flocculants Flocudex CS-5000, Flocusol CM-78 and Drewfloc 447 were supplied 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 initially filled with 2800 mL of 20-folds diluted centrifuged wastewater 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 approximately10-folds diluted wastewater. The photobioreactor was operated 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 culture absorbance at 550 nm (OD550) and pH. Biodegradation tests were allowed to run until steady values for OD550 and pH were recorded, 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 magnetic 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 analysis 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 Chemifloc 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 procedure, two folds concentrated (namely 2:1 test) and two folds diluted (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.
2.2. Piggery wastewater 2.7. Analytical procedures 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.
OD550 and pH were measured in a Lambda 25 UV/visible spectrophotometer (Perkin Elmer, USA) and a pH-probe (Thermo Scientific Orion, USA), respectively. COD and N–NHþ 4 were measured according to Standard Methods (Eaton et al., 2005).
925
I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Despite all biodegradation tests reported were conducted sequentially 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 compared 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. 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 biodegradation as shown by the increase in culture absorbance concomitant with COD and NHþ removal. The overall COD 4 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, Chlorococcum 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 agreement 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 recorded 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 consortium achieved an OD550 of 1.3 by the 4th day of experimentation.
3.2. Coagulation/flocculation tests The different nature and concentration of the coagulants/flocculants 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 coagulant/flocculant (control tests) was negligible regardless of the microalgae 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) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
5
25
50
100
150
250
1±3 2±2 2±1 23 ± 4 33 ± 1 29 ± 3 30 ± 4
4±4 2±2 58 ± 8 73 ± 4 89 ± 4 86 ± 17 95 ± 2
9±6 7±4 18 ± 3 93 ± 5 99 ± 1 94 ± 1 86 ± 1
10 ± 5 7±4 11 ± 3 94 ± 1 93 ± 3 84 ± 1 76 ± 2
14 ± 7 12 ± 5 13 ± 3 91 ± 2 76 ± 1 76 ± 3 60 ± 3
98 ± 1 90 ± 10 15 ± 5 77 ± 1 74 ± 2 66 ± 1 56 ± 1
5
25
50
100
150
250
4±1 6±0 3±7 33 ± 4 73 ± 9 73 ± 14 34 ± 18
1±5 5±8 20 ± 15 72 ± 5 56 ± 2 84 ± 2 61 ± 2
1±4 14 ± 2 3±7 83 ± 5 57 ± 10 64 ± 5 19 ± 1
95 ± 3 96 ± 4 1±3 41 ± 16 40 ± 8 67 ± 1 10 ± 11
14 ± 9 98 ± 1 0±0 54 ± 6 42 ± 9 52 ± 9 26 ± 5
26 ± 2 87 ± 6 1±0 22 ± 9 37 ± 1 56 ± 4 4±2
Table 2 RE of algal–bacterial biomass (%) in tests conducted with S. obliquus. Concentration (mg L1) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
Table 3 RE of algal–bacterial biomass (%) in tests conducted with Chlorococcum sp. Concentration (mg L1) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
5
25
50
100
150
250
0±2 16 ± 14 23 ± 16 62 ± 6 66 ± 6 46 ± 6 11 ± 7
15 ± 14 6±8 38 ± 1 88 ± 2 88 ± 10 91 ± 4 79 ± 3
24 ± 15 11 ± 11 13 ± 9 92 ± 0 88 ± 10 71 ± 2 78 ± 14
63 ± 15 32 ± 10 0±9 76 ± 1 72 ± 14 82 ± 17 56 ± 5
90 ± 8 87 ± 3 12 ± 6 74 ± 1 47 ± 2 77 ± 7 38 ± 9
79 ± 10 86 ± 10 8±2 44 ± 1 63 ± 2 81 ± 17 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) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
5
25
50
100
150
250
2±5 0±4 5±4 33 ± 2 32 ± 2 31 ± 7 15 ± 1
2±2 0±0 30 ± 11 78 ± 4 55 ± 3 84 ± 3 62 ± 6
4±2 0±2 28 ± 4 83 ± 1 59 ± 8 75 ± 15 35 ± 6
3±2 0±2 16 ± 2 76 ± 3 63 ± 3 73 ± 9 40 ± 0
5±2 93 ± 1 17 ± 4 69 ± 5 54 ± 4 69 ± 13 35 ± 4
66 ± 0 98 ± 1 20 ± 4 62 ± 0 57 ± 4 72 ± 1 38 ± 0
Chlorella consortium test (Table 1). The maximum REs for C. sorokiniana 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 particular 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 aqueous 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 particular case, the high concentrations of colloidal organic matter present in the diluted piggery wastewater probably decreased flocculant 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 flocculant requirements dosages when the algal biomass from batch cultures 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 fedbatch 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 different biomass REs were recorded at similar Fe concentration depending 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. sorokiniana 5 ± 2% vs. 93 ± 1%. Iron being the active element in the coagulation–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 gradually 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 nontoxic nature make it one of the preferred flocculants in microalgae-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 performance was always recorded at 25 mg L1 for all microalgae evaluated, the removals achieved were lower than 40% for C. sorokiniana, Chlorococcum sp. and S. obliquus, and 58 ± 8% for the Chlorella Consortium. These algal–bacterial biomass removals were below those previously reported, which might be due to the interaction of col-
loidal organic matter with chitosan. Hence, Sukenic et al. (1988) recorded 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 dosages (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 CM78, 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 exhibited 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 performance 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% (Table 1). On the other hand, when Drewfloc-447 was supplied to S. obliquus, Chlorococcum sp., and the Chlorella consortium an optimum performance was recorded at 5, 25 and 50 mg L1, supporting 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 resulted in a severe deterioration in the flocculation performance. Finally, 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 severely decreased at increasing concentrations (likely due to repulsion 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 polymers, flocculation occurs by polymer attachment to the surface of the algae (negatively charged due to the ionization of their functional groups) at one or more sites, and by subsequent bridging among cells, thus creating three-dimensional algal-polymer matrices (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 polymers used in this work were cationic polyacrylamides since anionic 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
2:1
Drewfloc-447 Chemifloc CV-300
73 ± 10 74 ± 1
56 ± 3 84 ± 3
56 ± 3 83 ± 5
we consider the Chlorella consortium as model algal–bacterial biomass, ferric chloride would be the most economic option. Nevertheless, 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 CV300 can be considered as the most suitable option. In addition, recent 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 biomass concentration did not have a severe impact on flocculant performance. Possibly due to different optimum dosage for Drewfloc447 and Chemifloc CV-300 (Table 4), while the REs of Drewfloc-447 slightly increased from 56 ± 3% to 73 ± 10% when decreasing biomass concentrations by a factor of 2, the REs of Chemifloc CV300 decreased from 84% to 74% for the same decrease in biomass concentration. Hence, this study showed that an increase in algal–bacterial biomass concentration during the flocculation process conducted with Drewfloc 447 and Chemifloc CV-300 did not result in a sustained deterioration or enhancement of biomass removal (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 confirmed 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 process 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 supplied 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 impact 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 comparable removal efficiencies, which highlights the impact of the matrix on the flocculation process. Hence, while low flocculant concentrations 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 Cooperation Agency (A/023287/09 project) and the Regional Government of Castilla y León (GR76 project). The Spanish Ministry for 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.
Contents lists available at ScienceDirect
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 b c
Center of Biotechnology. Universidad Mayor de San Simón, Campus Universitario, s/n Cochabamba, Bolivia Department of Chemical Engineering and Environmental Technology, Universidad de Valladolid, Paseo del Prado de la Magdalena s/n, 47011 Valladolid, Spain 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
Article history: Received 23 July 2010 Received in revised form 9 September 2010 Accepted 9 September 2010 Available online 17 September 2010 Keywords: Coagulation Flocculation Harvesting Microalgae Piggery wastewater
a b s t r a c t 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 comparatively evaluated for their ability to remove algal–bacterial biomass from the effluent of a photosynthetically oxygenated piggery wastewater biodegradation process. Chlorella sorokiniana, Scenedesmus obliquus, 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 at concentrations of 150–250 mg L1, dosages of 25–50 mg L1 were required for the polymer flocculants to support comparable removal efficiencies. Process efficiency declined when the polymer flocculant was overdosed. Biomass concentration did not show a significant impact on flocculation within the concentration range tested. The high flocculant requirements herein recorded might be due to the competition of colloidal organic for the flocculants and the stationary phase conditions of biomass. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction 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 treatments (Ruiz-Marin et al., 2010; Schumacher et al., 2003; Wang et al., 2010). Microalgae-based treatments are powered by sunlight, which reduces the energy input to the process. This lower energy consumption, together with the inherent assimilation of CO2 during microalgal growth (photosynthesis), mitigates a significant part of the greenhouse gas emissions associated to wastewater reclamation. 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. 1 2 3
E-mail address: [email protected] (R. Muñoz). Tel.: +591 4 4542895; fax: +591 4 4542895. Tel.: +34 983184934; fax: +34 983423013. Tel.: +34 987291568; fax: +34 987291563.
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.09.036
ventional treatment technologies such as activated sludge systems or anaerobic digesters. Microalgae-based treatments can also play a key role in sustainable 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 nutrients-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 implementation of this technology is often hampered by the cost-effectiveness of microalgae harvesting. Hence, the presence of freelysuspended microalgae (characterized by low settling velocities) always challenges the removal efficiencies of COD and nutrients in algal–bacterial photobioreactors, despite the merits of paddlewheel 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.
924
I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Microalgae harvesting can be carried out by centrifugation, filtration and coagulation/flocculation (Molina-Grima et al., 2003). While centrifugation presents prohibitive energy costs in the context of wastewater treatment (low added value processes), filtration technologies are only useful for the recovery of relative large species such as Spirulina. Low-cost filter presses often fail to harvest small microalgae such as Chlorella or Scenedesmus. Coagulation/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 between microalgal cells, flocculants promote the formation of cell aggregates by creating bridges between the neutralized microalgae. This harvesting technique has been successfully tested in aquaculture, biofuels production, wastewater treatment and removal 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 assessed 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 wastewater 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 algal–bacterial consortia to evaluate the performance of the coagulants/flocculants. Therefore, the main goal of our study was to address the sedimentation of effluents containing free living microalgae, 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 Services (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). NaHCO3 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 Eflasks filled with 200 mL of MSM enriched with 700 mg L1 of NaHCO3. 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 mineralization was obtained from Cochabamba Wastewater Treatment Plant (Bolivia).
2.3. Flocculants Flocudex CS-5000, Flocusol CM-78 and Drewfloc 447 were supplied 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 initially filled with 2800 mL of 20-folds diluted centrifuged wastewater 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 approximately10-folds diluted wastewater. The photobioreactor was operated 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 culture absorbance at 550 nm (OD550) and pH. Biodegradation tests were allowed to run until steady values for OD550 and pH were recorded, 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 magnetic 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 analysis 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 Chemifloc 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 procedure, two folds concentrated (namely 2:1 test) and two folds diluted (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.
2.2. Piggery wastewater 2.7. Analytical procedures 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.
OD550 and pH were measured in a Lambda 25 UV/visible spectrophotometer (Perkin Elmer, USA) and a pH-probe (Thermo Scientific Orion, USA), respectively. COD and N–NHþ 4 were measured according to Standard Methods (Eaton et al., 2005).
925
I. de Godos et al. / Bioresource Technology 102 (2011) 923–927
Despite all biodegradation tests reported were conducted sequentially 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 compared 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. 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 biodegradation as shown by the increase in culture absorbance concomitant with COD and NHþ removal. The overall COD 4 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, Chlorococcum 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 agreement 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 recorded 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 consortium achieved an OD550 of 1.3 by the 4th day of experimentation.
3.2. Coagulation/flocculation tests The different nature and concentration of the coagulants/flocculants 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 coagulant/flocculant (control tests) was negligible regardless of the microalgae 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) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
5
25
50
100
150
250
1±3 2±2 2±1 23 ± 4 33 ± 1 29 ± 3 30 ± 4
4±4 2±2 58 ± 8 73 ± 4 89 ± 4 86 ± 17 95 ± 2
9±6 7±4 18 ± 3 93 ± 5 99 ± 1 94 ± 1 86 ± 1
10 ± 5 7±4 11 ± 3 94 ± 1 93 ± 3 84 ± 1 76 ± 2
14 ± 7 12 ± 5 13 ± 3 91 ± 2 76 ± 1 76 ± 3 60 ± 3
98 ± 1 90 ± 10 15 ± 5 77 ± 1 74 ± 2 66 ± 1 56 ± 1
5
25
50
100
150
250
4±1 6±0 3±7 33 ± 4 73 ± 9 73 ± 14 34 ± 18
1±5 5±8 20 ± 15 72 ± 5 56 ± 2 84 ± 2 61 ± 2
1±4 14 ± 2 3±7 83 ± 5 57 ± 10 64 ± 5 19 ± 1
95 ± 3 96 ± 4 1±3 41 ± 16 40 ± 8 67 ± 1 10 ± 11
14 ± 9 98 ± 1 0±0 54 ± 6 42 ± 9 52 ± 9 26 ± 5
26 ± 2 87 ± 6 1±0 22 ± 9 37 ± 1 56 ± 4 4±2
Table 2 RE of algal–bacterial biomass (%) in tests conducted with S. obliquus. Concentration (mg L1) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
Table 3 RE of algal–bacterial biomass (%) in tests conducted with Chlorococcum sp. Concentration (mg L1) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
5
25
50
100
150
250
0±2 16 ± 14 23 ± 16 62 ± 6 66 ± 6 46 ± 6 11 ± 7
15 ± 14 6±8 38 ± 1 88 ± 2 88 ± 10 91 ± 4 79 ± 3
24 ± 15 11 ± 11 13 ± 9 92 ± 0 88 ± 10 71 ± 2 78 ± 14
63 ± 15 32 ± 10 0±9 76 ± 1 72 ± 14 82 ± 17 56 ± 5
90 ± 8 87 ± 3 12 ± 6 74 ± 1 47 ± 2 77 ± 7 38 ± 9
79 ± 10 86 ± 10 8±2 44 ± 1 63 ± 2 81 ± 17 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) Floccculant–Coagulant
FeCl3 Fe2(SO4)3 Chitosan Flocusol CM-78 Drewfloc 447 Chemifloc CV-300 Flocudex CS-5000
5
25
50
100
150
250
2±5 0±4 5±4 33 ± 2 32 ± 2 31 ± 7 15 ± 1
2±2 0±0 30 ± 11 78 ± 4 55 ± 3 84 ± 3 62 ± 6
4±2 0±2 28 ± 4 83 ± 1 59 ± 8 75 ± 15 35 ± 6
3±2 0±2 16 ± 2 76 ± 3 63 ± 3 73 ± 9 40 ± 0
5±2 93 ± 1 17 ± 4 69 ± 5 54 ± 4 69 ± 13 35 ± 4
66 ± 0 98 ± 1 20 ± 4 62 ± 0 57 ± 4 72 ± 1 38 ± 0
Chlorella consortium test (Table 1). The maximum REs for C. sorokiniana 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 particular 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 aqueous 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 particular case, the high concentrations of colloidal organic matter present in the diluted piggery wastewater probably decreased flocculant 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 flocculant requirements dosages when the algal biomass from batch cultures 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 fedbatch 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 different biomass REs were recorded at similar Fe concentration depending 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. sorokiniana 5 ± 2% vs. 93 ± 1%. Iron being the active element in the coagulation–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 gradually 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 nontoxic nature make it one of the preferred flocculants in microalgae-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 performance was always recorded at 25 mg L1 for all microalgae evaluated, the removals achieved were lower than 40% for C. sorokiniana, Chlorococcum sp. and S. obliquus, and 58 ± 8% for the Chlorella Consortium. These algal–bacterial biomass removals were below those previously reported, which might be due to the interaction of col-
loidal organic matter with chitosan. Hence, Sukenic et al. (1988) recorded 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 dosages (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 CM78, 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 exhibited 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 performance 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% (Table 1). On the other hand, when Drewfloc-447 was supplied to S. obliquus, Chlorococcum sp., and the Chlorella consortium an optimum performance was recorded at 5, 25 and 50 mg L1, supporting 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 resulted in a severe deterioration in the flocculation performance. Finally, 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 severely decreased at increasing concentrations (likely due to repulsion 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 polymers, flocculation occurs by polymer attachment to the surface of the algae (negatively charged due to the ionization of their functional groups) at one or more sites, and by subsequent bridging among cells, thus creating three-dimensional algal-polymer matrices (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 polymers used in this work were cationic polyacrylamides since anionic 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
2:1
Drewfloc-447 Chemifloc CV-300
73 ± 10 74 ± 1
56 ± 3 84 ± 3
56 ± 3 83 ± 5
we consider the Chlorella consortium as model algal–bacterial biomass, ferric chloride would be the most economic option. Nevertheless, 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 CV300 can be considered as the most suitable option. In addition, recent 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 biomass concentration did not have a severe impact on flocculant performance. Possibly due to different optimum dosage for Drewfloc447 and Chemifloc CV-300 (Table 4), while the REs of Drewfloc-447 slightly increased from 56 ± 3% to 73 ± 10% when decreasing biomass concentrations by a factor of 2, the REs of Chemifloc CV300 decreased from 84% to 74% for the same decrease in biomass concentration. Hence, this study showed that an increase in algal–bacterial biomass concentration during the flocculation process conducted with Drewfloc 447 and Chemifloc CV-300 did not result in a sustained deterioration or enhancement of biomass removal (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 confirmed 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 process 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 supplied 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 impact 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 comparable removal efficiencies, which highlights the impact of the matrix on the flocculation process. Hence, while low flocculant concentrations 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 Cooperation Agency (A/023287/09 project) and the Regional Government of Castilla y León (GR76 project). The Spanish Ministry for 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.