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Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation
Bioresource Technology 102 (2011) 82–87
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Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation Ya-Ling Cheng a, Yu-Chuan Juang a, Guan-Yu Liao a, Pei-Wen Tsai a, Shih-Hsin Ho b, Kuei-Ling Yeh b, Chun-Yen Chen b,c, Jo-Shu Chang b,c,d, Jhy-Chern Liu e, Wen-Ming Chen f, Duu-Jong Lee a,* a
Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan c Sustainable Environment Research Center, National Cheng Kung University, Tainan 701, Taiwan d Center for Biosciences and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan e Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan f Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan b
a r t i c l e
i n f o
Article history: Received 23 March 2010 Received in revised form 19 April 2010 Accepted 24 April 2010
Keywords: Algae Harvesting Ozone Flotation Algogenic organic matters
a b s t r a c t The Scenedesmus obliquus FSP-3, a species with excellent potential for CO2 capture and lipid production, was harvested using dispersed ozone flotation. While air aeration does not, ozone produces effective solid–liquid separation through flotation. Ozone dose applied for sufficient algal flotation is similar to those used in practical drinking waterworks. The algae removal rate, surface charge, and hydrophobicity of algal cells, and fluorescence characteristics and proteins and polysaccharides contents of algogenic organic matter (AOM) were determined during ozonation. Proteins released from tightly bound AOM are essential to modifying the hydrophobicity of bubble surfaces for easy cell attachment and to forming a top froth layer for collecting floating cells. Humic substances in the suspension scavenge dosed ozone that adversely affects ozone flotation efficiency of algal cells. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Efficient harvesting of the biomass from cultivation froth is essential for mass production of biodiesel from microalgae (Cooney et al., 2009; Ramanan et al., 2010; Jorquera et al., 2010). Harvesting utilizes solid–liquid separation processes, such as centrifugation, filtration, flotation, and sedimentation (Levin et al., 1962; Grima et al., 2003; Henderson et al., 2008a). Algae harvesting can be costly (Dismukes et al., 2008; Reijnders, 2008) since the mass fractions in a culture broth are generally low, while the cells generally have a negative charge and excess algogenic organic matter (AOM) to retain stability in a dispersed state. Dissolved air flotation (DAF) effectively separated algae when cationic surfactants were present (Van Vuuren and Van Duuren, 1965; Funk et al., 1968; Bare et al., 1975; Liu et al., 1999). Ozone is a strong oxidant that preferentially oxidizes double bonds and electron-rich aromatic rings into oxygenated functional groups such as aldehydic, ketonic, and especially carboxylic groups (Von Gunten, 2003; Beltrán, 2004; Li et al., 2008). Particle separation in water can be enhanced by oxidation using ozone, chlorine, chloride dioxide, permanganate, or chloramines (Jekel, 1998). Betzer et al. (1980) investigated the use of ozone-induced * Corresponding author. Tel.: +886 2 33663028; fax: +886 2 23623040. E-mail address: [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.04.083
flotation for algae recovery and effluent treatment. They determined that dosed ozone produced froth; the settlement of froth yielded settled sludge with 2–7% w/w solids. Benoufella et al. (1994) eliminated the Microcystis strain of cyanobacteria using an ozoflotation process. Mass cultivation of algae requires a high overflow rate, which promotes flotation during which algae move upward instead of sedimentation during which algae move downward (Koopman and Lincoln, 1983; Edzwald, 1993). Flotation has advantages of flexible operation and small footprint when compared with coagulation–flocculation and sedimentation (Liu et al., 1999). Biofuel production from algal cells requires the extraction of lipids from cultivated cells; this can be achieved by cells lysis with ozone. Furthermore, the addition of chemicals should be minimized to prevent contamination of extracted products. This study utilized dispersed ozone flotation for harvesting and cell lysis of Scenedesmus obliquus FSP-3, a species that has a high potential for CO2 capture and lipid production (Mandal and Mallick, 2009). (Note: In parallel study the S. obliquus FSP-3 was shown to fix CO2 at a rate of 256 g/m3 d, a value higher than most data reported in literature.) The algae removal rate, surface charge, and hydrophobicity of algal cells, fluorescence characteristics, and proteins and polysaccharides content in AOM were recorded during ozonation tests with no chemicals added. Experimental findings are discussed.
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2. Methods 2.1. Cultivation of algae The microalgal strain used in this work was obtained from freshwater in southern Taiwan. The microalga was identified as S. obliquus FSP-3 based on its morphology and by 23S rDNA sequence matching (GeneBank accession number DQ396875.1). Modified Detmer’s medium (DM) was employed to grow a pure culture of S. obliquus FSP-3. The medium was as follows (g/L): Ca(NO3)24H2O, 1.00; KH2PO4, 0.26; MgSO47H2O, 0.55; KCl, 0.25; FeSO47H2O, 0.02; EDTA2Na, 0.2; H3BO3, 0.0029; ZnCl2, 0.00011; MnCl24H2O, 0.00181; (NH4)6Mo7O244H2O, 0.000018; and CuSO45H2O, 0.00008. After pre-culturing in 200 ml flasks, the S. obliquus cells were inoculated into a 20-L photobioreactor with an inoculum size of 15 mg/L. S. obliquus FSP-3 was grown at 28 °C for 12 d under a light intensity of roughly 60 lmol/m2 s (illuminated by a TL5 fluorescent lamp) in the glass photobioreactor with an agitation speed of 300 rpm. Carbon dioxide (20%) was fed into the culture continuously as the sole carbon source. The CO2 feeding rate was 4 L/min during 12-d cultivation. The time-course profile of algae growth shows that the cumulative biomass production and overall biomass production rate of S. obliquus FSP-3 reached 1610 mg/L and 136 mg/L d, respectively. 2.2. Ozonation dispersed flotation Batch flotation tests were performed in a flotation column with an inside diameter of 5 cm and maximum depth of 47 cm. A porous glass diffuser with a nominal pore diameter of 30–60 lm was placed at the column bottom, just above the gas inlet port. The froth collection port was located at the column top. Liquid samples were withdrawn from the sampling port just above the porous glass diffuser. A T-408 ozone generator (Welsbach, CA, USA) was used to produce ozone from oxygen, which was the flotation gas. The gas pressure leaving the ozone generator was 0.65 kg cm 2 and the flow rate was 0.6 l min 1. No chemicals were added during all flotation tests. 2.3. Algae cell isolation and AOM extraction The original algal samples were centrifuged at 7000g at 4 °C for 15 min and the supernatant was obtained. Organic substances in the supernatant were loosely bound AOM (LBAOM). Residues were re-suspended to the original volume using Milli-Q water and centrifuged again at 7000g at 4 °C for 15 min with decanted supernatant. Half of the residues were then re-suspended in phosphate buffered saline (PBS) (pH 7.4) to the original volume, and were considered ‘‘washed” in this study. The remaining residue was re-suspended in Milli-Q water to its original volume and extracted using formaldehyde (37%, 0.06 ml) + ultrasound (120 W, 5 min) + NaOH (50 ml, 4 °C, 1 h) (Adav and Lee, 2008). The supernatant was recovered via centrifugation (5000g, 4 °C, 10 min). The organic matter in the recovered supernatant was tightly bound AOM (TBAOM). 2.4. Modified BATH test A test, a modified version of the bacterial adhesion to hydrocarbons test (BATH) hydrophobicity test (Rosenberg et al., 1980) was performed. Absorbance at 680 nm was determined for the original algal suspension and for liquid samples collected from the flotation column. In total, 4 ml of the algal sample was placed in a test tube with an inner diameter of 13 mm. Hexane (1 ml) was added and shaken for 10 s, and allowed to settle for 30 min. Carefully, 3 ml
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was obtained from the bottom layer in the test tube and its absorbance at 680 nm was again determined. The extractability of the hexane layer on organic substances in the algal suspension was calculated as (original absorbance absorbance of the water layer)/(original absorbance) 100%. 2.5. Excitation–emission matrix fluorescence spectroscopy analysis The excitation–emission matrix (EEM) spectra of collected samples were determined using luminescence spectrometry (Cary Eclipse, Varian Inc., CA, USA). The EEM spectra were obtained by scanning a sample for both excitation and emission wavelengths at 200–550 nm. The blank EEM spectrum obtained for double-distilled water was utilized to ensure the accuracy of scanned samples. For all measurements, excitation and emission slits were maintained at 5 nm, and a 290-nm emission cutoff filter was employed to eliminate second-order Raleigh light scattering. The EEM spectra are the elliptical shapes of contours, where the X-axis represents the emission spectra at 200–550 nm and the Y-axis is the excitation wavelength at 200–550 nm. In total, 40 contour lines, as the third dimension, are shown for each EEM spectra to represent fluorescence intensity at an interval of 5. 2.6. Chemical analysis The dry weight of, and volatile suspended solids (VSS) in, collected samples were determined using Standard Methods (APHA, 1998). The size and zeta potential of fine particles in collected samples were determined using a Zeta sizer (Nano-ZS) (Malvern Co., UK). In some tests, 10 or 20 mg/L sodium dodecyl sulfate (SDS) was dosed prior to zeta potential measurements. The organic carbon content (dissolved organic carbon (DOC)), polysaccharides (PS) and protein (PN) content for LBAOM were determined using a total organic carbon (TOC) analyzer (model 1030, O.I. Corporation, Texas, USA). The PS content in samples was determined using the Anthrone method with glucose as the standard. The PN content was determined using a modified Lowry method with bovine serum albumin as the standard. Washed algal suspensions, before and after ozonation–flotation, were filtered to collect cells for scanning electron microscopy (SEM) (S-2400, Hitachi Ltd., Tokyo, Japan) with the Ronatec system for energy dispersive spectroscopy (EDS) microanalysis. All SEM observations were conducted following fixing with 2.5% glutaraldehyde for 2 h, and dehydration via successive passages through 30%, 50%, 75%, 85%, 90%, 95% and 100% ethanol, followed by critical drying in a critical point dryer (HCP-2) (Hitachi Ltd., Tokyo, Japan).
3. Results and discussion 3.1. Ozone flotation Figs. S1 and S2 of Supplementary Materials show SEM micrographs for original algal cells and aggregates collected at the column top, and a photograph of the froth layer for the algal suspension ozonated for 4 min. The original algae were 4 2 lm ellipsoids. The collected froth liquor contained aggregates of injured cells sized 10–12 lm (in parallel tests using particle sizer, the mean size of aggregates was 10–30 lm). Hence, dispersed ozone flotation induced algae aggregation; this experimental finding is in agreement with analytical results obtained by Jekel (1994). Strong turbulence existed in the ozone flotation column in all tests; hence, the suspension in the column was assumed mixed completely. In the control test, simple air aeration did not cause algal cells to float to the column top. Fig. 1 shows residual turbidity
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DF=1, [O3]=98 mg/L
-5
DF=3, [O3]=98 mg/L
800
DF=1, [O3]=63 mg/L
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Residual Turbidity ( NTU )
0
Washed, [O 3]=63 mg/L
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Ozone dosed ( mg O3 / mg Biomass ) Fig. 1. Residual turbidity in flotation column. DF: dilution ratio; washed: algae cells in PBS solution; and dashed line: NTU0 = 4800 * O3,i (in mg/mg biomass).
in the column (original and 100% or 200% in dilution) under three ozone concentrations. In total, four batches of cultivated algal suspensions were tested, yielding data fluctuation. With an ozone dosage exceeding a threshold value, turbidities of algal suspensions declined over time. For example, 0.16–0.20 mg O3/mg biomass was needed to initiate turbidity removal for original suspensions (initial turbidity (NTU0) = 800–1000 NTU). In total, around 0.52 mg O3/mg biomass was required to reduce turbidity by 95%. With 100% diluted (2) samples (NTU0 = 400 NTU), roughly 0.09 mg O3/mg biomass was needed to initiate turbidity removal and roughly 0.36 mg O3/mg biomass was consumed to reach turbidity removal of 95%. With 200% diluted (3) samples (NTU0 = 240 NTU), the threshold O3 was 0.05 mg/mg biomass and the 95% removal dosage was 0.22 mg/mg biomass. The following general linear correlation existed between NTU0 and the threshold ozone dosage (O3,i): NTU0 (in NTU) = 4800 * O3,i (in mg/mg biomass). Additionally, concentrated algal suspension consumed more ozone than diluted suspension at the same fraction of turbidity removal. Conversely, the turbidity of washed algal cells (in PBS) started declining immediately after ozonation started. No threshold ozone dosage existed. With an initial 400 NTU, roughly 0.07 mg O3/mg biomass was needed to remove 95% of turbidity, only about 15% of that was needed for unwashed samples with the same NTU0.
0.2
0.4
0.6
0.8
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Ozone dosed ( mg O3 / mg Biomass) Fig. 2. Zeta potentials for algae particles as functions of dosed ozone. Same legends as in Fig. 1. Zeta potentials for original suspension were 6 to 7 mV, and those for 2 samples and washed cells were 13 mV.
About 50–80% of cells in the original algal suspension or 40% of washed cells left an aqueous phase after mixing in the modified BATH tests (Fig. 3). Ozonation first increased and then decreased the extractability of algal cells. The extractability for un-diluted samples peaked at 0.4–0.5 mg O3/mg biomass, while that for washed cells peaked at roughly 0.05 mg O3/mg biomass. A foam layer formed between the aqueous and n-hexane layers, while most cells that left an aqueous phase did not move to an oil phase, but remained within the foam layer. In all BATH tests, no cells dissolved into an oil phase, regardless of ozone dosage or mixing time. 3.3. AOM in samples The DOC contents in LBAOM and TBAOM for the original suspensions were measured at 51.7 ± 0.73 and 199.5 ± 2.9 mg-C/L, respectively. The TBAOM contained almost four times the quantity of DOC in the LBAOM. In total, six characteristic EEM peaks were identified for the original and ozonated samples (shown later). The EEM peaks in the ellipse were centered at Ex/Em = 350/460 nm and 320/415 nm, and were likely attributable to humic-like compounds (HA1 and
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3.2. Characteristics of ozonated samples 80
Extractability ( % )
The zeta potentials for original suspensions were 6 to 7 mV; that for the 100% dilution sample and washed cells was 13 mV (Fig. 2). Hence, algal cells were negatively charged. Dilution or washing reduced ionic strength, thereby reducing the zeta potentials of algal cells. Ozonation gradually reduced the zeta potentials of the original and diluted samples. In other words, ozonation increased the negative charge of cell wall surfaces. For example, adding 0.2 mg O3/mg biomass reduced zeta potentials of algal cells from 7 to 11 mV, while adding additional 0.2 mg O3/mg biomass further reduced these potentials to around 15 mV. Conversely, adding <0.02 mg O3/mg biomass reduced the zeta potentials of washed cells to 27 from 13 mV. The zeta potentials of the original algal cells were not affected by the presence of SDS (data not shown), demonstrating that the algal cells have hydrophilic surfaces. In the presence of SDS, the zeta potentials of ozonated cells dropped by only 3 mV. Ozonation did not significantly increase surface hydrophobicity of algal cells.
60
40
20
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0.4
0.6
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Ozone dosed ( mg O3 / mg Biomass ) Fig. 3. Extractability by hydrocarbon layer using modified BATH test. Same legends as in Fig. 1.
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HA2), and that centered at Ex/Em = 260/460 nm was due to fulviclike compounds (FA1) (Chen et al., 2003). Moreover, the three peaks (PR1–PR3) centered at Ex/Em = 280/360 nm, 255/300 nm, and 230/365 nm were likely attributable to protein-like compounds (Chen et al., 2003). Fig. 4 shows the EEM fluorescence peaks of the original algal suspension. Strong HA1 and FA1 peaks suggested the presence of humic substances in the suspension. Figs. S3 and S4 of Supplementary Materials show the EEM plots for LBAOM and TBAOM, respectively. The EEM pattern for LBAOM resembled that for the original suspension, indicating that LBAOM was primarily comprised of humic-like and fulvic-like substances. Conversely, the TBAOM was principally composed of protein-like substances. Fig. S5 of Supplementary Materials shows the EEM fluorescence plots for algal suspensions under ozonation. Fig. 5 shows the EEM peak intensities as functions of ozone dosage. The peak intensities of HA1 and FA1 for original algal suspensions decreased as ozone dosage increased (Fig. 5). The HA2 peak increased in intensity until dosed ozone reached 0.11 mg/mg biomass, and then decreased when ozone dosage was further increased. The peak intensity of PR1 increased as ozone dosage increased, and peaked at 0.22 mg O3/mg biomass. Moreover, the intensity of PR2 peaked at 0.43 mg O3/mg biomass. Fig. 6 shows the PS and PN contents in dissolved organic matter in ozonated samples. The PN content increased, peaked, and then decreased as ozone dosage increased. Conversely, PS content in dissolved organic matter was almost constant under ozonation.
14
HA1 FA1 HA2 PR1 PR2
Intensity ( a. u. )
12 10 8 6 4 2 0 0.0
0.2
0.4
0.6
Ozone dosed ( mg O3 / mg Biomass ) Fig. 5. Intensities of EEM peaks for original algal suspension at [O3] = 98 mg/L. Characteristic peak values were extracted from EEM plots in Fig. S5.
between the solids in raw water (up to 15 NTU), so the total ozone dose is low; while those in fermentation froth is high (up to 900 NTU), which acquires high ozone dose. Hence, ozone disperse flotation is regarded a potential candidate for mass harvesting of unicellular algae such as the S. obliquus FSP-3 investigated herein. Algal cells have negatively charged hydrophilic surfaces, accounting for why simple air aeration does not yield flotation. Conversely, ozone-dispersed flotation efficiently separated S. obliquus FSP-3 from the broth suspension, particularly when the suspension comprised washed cells (Fig. 1). That is, the interaction between algal cells and bubbles was enhanced by ozonation. Ozonation can break down natural organic matter (NOM) molecules into hydrophilic low-molecular-weight fragments (Nawrocki et al., 2003). Chheda and Grasso (1994) demonstrated that ozonation reduced the stability of Na-montmorillonite particles coated with NOM in river water. They noted that adsorption of NOM on Na-M makes particles increasingly hydrophilic, while ozonation partially dealuminized Na-M and transformed coated NOM to increase particle surface hydrophobicity, thereby destabilizing particles. Grasso and Weber (1988) noted that ozonation
3.4. Discussion Ozonation is commonly regarded an expensive process for water and wastewater treatment. Ozone doses of 0.4–0.8 mg O3/ mg C were applied in seven US waterworks as preoxidants for destabilizing particles and removing precursors of total organic carbon (TOC) and trihalomethane (THM) (Chang and Singer, 1991). The ozone dose noted herein to sufficient harvesting of S. obliquus FSP-3 was about noted 0.2–0.5 mg/mg biomass, close or even lower than that adopted in practical water works. Such a conclusion seems controversial to the common understanding that ozone is an expensive process and should be used only in a pretreatment stage. One should take into account the difference
Excitation wavelength (nm)
400 -4 -2 0 2 4 6 8 10 12 14 12.0 13.5
HA1
350
HA1 300
PR1 PR2
FA1
250
PR3
200 250
300
350
400
450
500
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Emission wavelength ( nm ) Fig. 4. EEM plot for original algal suspension. HA1–HA2, FA1 and PR1–PR3 denote respectively the characteristic peaks noted for humic-like substances, fulvic-acid-like substance and protein-like substances in the present study.
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60 DF=1; [ O 3 ] = 63mg / L DF=1; [ O 3 ] = 98mg / L
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DF=1; [ O 3 ]= 135 mg / L DF=2; [ O 3 ]=
PN (mg/L)
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30
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60
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Ozone dosed ( mg O3 / mg Biomass) Fig. 6. Contents of proteins (a) and polysaccharides (b) as functions of dosed ozone. Symbols in (b) are defined the same as in (a).
increases polarity, cleaves aromatic rings, and decreases the apparent molecular weights of compounds in tested humic acids. Conversely, these authors determined that ozonation decreases polarity, creates long aliphatics, and increases the apparent molecular weights of organic compounds in a natural water samples collected from the Huron River (Michigan, USA). Henderson et al. (2008b) demonstrated that AOM extracted from four algae species (Chlorella vulgaris, Microcystis aeruginosa, Asterionella formosa and Melosira sp.) were dominated by hydrophilic polysaccharides and hydrophobic proteins with low specific UV absorbance (SUVA) and negative zeta potentials. Air bubbles in nearly neutral aqueous solutions have negatively charged hydrophobic surfaces. The S. obliquus cells carried negative charges; ozonation further reduced their surface charges (Fig. 2). In particular, <20 mg/L O3 effectively reduced the zeta potentials of washed cells to 27 from 13 mV. Hence, the electrostatic interaction mechanisms unlikely interpreted the noted enhanced algae flotation. Although divalent ions (Ca2+ and Mg2+) were present in the present system, the proposed Ca–organic substance complexation model by Chandrakanth and Amy (1996) cannot interpret the more negative surface charge of algal cells after ozonation. Extractability measured using the modified BATH test indicated that the hydrophobicity of algal cells increased after ozonation (Fig. 3). However, the BATH test assessed the hydrophobicity of algal cell wall surfaces based on the degree of preferential dissolution of algal cells into hydrocarbon layers. Experimental observations
demonstrate that cells (and the lysed) in this study were concentrated in the foam layer, and did not dissolve into the oil layer. Moreover, the zeta potentials in the presence of 10 or 20 mg/L SDS decreased slightly for ozonated cells. Therefore, the hydrophobicity of algal cell walls was not markedly increased following ozonation. Aerated bubbles can be modified by adding chemicals. Han et al. (2006) produced positively charged bubbles by adding cationic metal salts or cationic polymer for flotation. Henderson et al. (2008a, 2009), who modified bubbles using cationic surfactants, demonstrated that bubbles with surfaces modified by chemicals with both a hydrophobic long tail and a hydrophilic high charge head achieve sufficient algal removal without upstream coagulation and flocculation. Reckhow et al. (1986) demonstrated that ozone can lyse algae and liberate biopolymers, which then act as coagulating polymers. Ma and Liu (2002), who pre-oxidized algae with potassium ferrate, speculated that biopolymers from injured algae cells can be present as coagulant aids. In this study, ozone preferentially degraded humic-like substances (HA1 and FA1 (Fig. 5)) in suspensions, and then released protein-like substances (PR1) bound to algae cell surfaces. We propose that the released protein-like substances have surface activity once released that can bind with bubble surfaces and thereby modify bubble characteristics. That the released proteins carried a positive charge in the neutral solution is unlikely. Rather, the proteins bound bubbles make bubble surfaces increasingly hydrophilic, facilitating easy cell attachment. Since humic-like substances were removed from washed cells, ozone directly reacted with algal cells to release proteins; thus, flotation was achieved at a much lower ozone dose than that for unwashed cells (Fig. 1). A cultivation strategy for minimal production of humic-like substances is recommended for easy harvesting via dispersed ozone flotation process. Prolonged idle stage after cultivation is commonly adopted for enhancing lipid production, which may be also yield excess humic substances. A compromise between lipid production enhancement and humic substance secretion should be done for selecting optimal cultivation strategy. 4. Conclusions The S. obliquus FSP-3 was harvested using dispersed ozone flotation process. Simple air aeration failed to yield algal flotation, while ozone produced flotation efficiently. The ozone dose required to harvesting of S. obliquus FSP-3 ranged 0.2–0.5 mg/mg biomass. Ozonation produced algal cells with more negatively charged and slightly increasingly hydrophobic surfaces compared with intact cells. Proteins released from cell lysis were regarded surfactants to make bubble surface increasingly hydrophilic to yield effective bubble–cell collision and formation of top froth layer for cell collection. Humic substances in suspension, however, exhaust dosed ozone, whose formation should be minimized in cultivation stage. Acknowledgements Authors are grateful to Dr. Yuehwa Yu of National Taiwan University for providing ozonation equipments. The authors gratefully acknowledge financial supports from Taiwan’s Ministry of Economical Affairs (98-EC-17-A-10-S2-0066) and Taiwan’s National Science Council (NSC 96-2628-E-006-004-MY3, NSC 97-2218-E006-005 and NSC 98-3114-E-006-012). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.04.083.
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Henderson, R.K., Baker, A., Parsons, S.A., Jefferson, B., 2008b. Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Water Res. 42, 3435–3445. Henderson, R.K., Parson, S.A., Jefferson, B., 2009. The potential for using bubble modification chemicals in dissolved air flotation algae removal. Sep. Sci. Technol. 44, 1923–1940. Jekel, M.R., 1994. Flocculation effects of ozone. Ozone-Sci. Eng. 16, 55–66. Jekel, M.R., 1998. Effects and mechanisms involved in preoxidation and particle separation processes. Water Sci. Technol. 37, 1–7. Jorquera, O., Kiperstok, A., Sales, E.A., Embirucu, M., Ghirardi, M.L., 2010. Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors. Bioresour. Technol. 101, 1406–1413. Koopman, B.L., Lincoln, E.P., 1983. Autoflotation of algae fro high rate pond effluent. Aquaculture Waste 5, 231–246. Levin, G.V., Clendenning, J.R., Gibor, A., Bogar, F.D., 1962. Harvesting of algae by froth flotation. Appl. Microbiol. 10, 169–175. Li, J., Liu, H., Zhao, X., Qu, J., Liu, R., Ru, J., 2008. Effect of preozonation on the characteristic transformation of fulvic acid and its subsequent trichloromethane formation potential: presence or absence of bicarbonate. Chemosphere 71, 1639–1645. Liu, J.C., Chen, M.Y., Ju, Y.H., 1999. Separation of algal cells from water by column flotation. Sep. Sci. Technol. 34, 2259–2272. Ma, J., Liu, W., 2002. Effectiveness and mechanism of potassium ferrate (VI) preoxidation for algae removal by coagulation. Water Res. 36, 871–878. Mandal, S., Mallick, N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84, 281–291. Nawrocki, J., S´wietlik, J., Raczyk-Stanislawiak, U., Dabrowska, A., Bilozor, S., Ilecki, W., 2003. Influence of ozonation conditions on aldehyde and carboxylic acid formation. Ozone – Sci. Eng. 25, 53–62. Ramanan, R., Kannan, K., Deshkar, A., Yadav, R., Chakrabarti, T., 2010. Enhanced algal CO2 sequestration through calcite deposition by Chlorella sp. and Spirulina platensis in a mini-raceway pond. Bioresour. Technol. 101, 2616–2622. Reckhow, D.A., Singer, P.C., Trusell, R.R., 1986. Proc. American Water Works Association, Annual Conference, Denver, CO. Reijnders, L., 2008. Do biofuels from microalgae beat biofuels from terrestrial plant? Trends Biotechnol. 26, 349–350. Rosenberg, M., Gutnick, D., Rosenberg, E., 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbiol. Lett. 9, 29–33. Van Vuuren, L.R.J., Van Duuren, F.A., 1965. Removal of algae from wastewater maturation pond effluent. J. Water Pollut. Control Fed. 37, 1256–1262. Von Gunten, U., 2003. Ozonation of drinking water: part I. Oxidation kinetics and product formation. Water Res. 37, 1443–1467.
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Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation Ya-Ling Cheng a, Yu-Chuan Juang a, Guan-Yu Liao a, Pei-Wen Tsai a, Shih-Hsin Ho b, Kuei-Ling Yeh b, Chun-Yen Chen b,c, Jo-Shu Chang b,c,d, Jhy-Chern Liu e, Wen-Ming Chen f, Duu-Jong Lee a,* a
Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan c Sustainable Environment Research Center, National Cheng Kung University, Tainan 701, Taiwan d Center for Biosciences and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan e Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan f Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan b
a r t i c l e
i n f o
Article history: Received 23 March 2010 Received in revised form 19 April 2010 Accepted 24 April 2010
Keywords: Algae Harvesting Ozone Flotation Algogenic organic matters
a b s t r a c t The Scenedesmus obliquus FSP-3, a species with excellent potential for CO2 capture and lipid production, was harvested using dispersed ozone flotation. While air aeration does not, ozone produces effective solid–liquid separation through flotation. Ozone dose applied for sufficient algal flotation is similar to those used in practical drinking waterworks. The algae removal rate, surface charge, and hydrophobicity of algal cells, and fluorescence characteristics and proteins and polysaccharides contents of algogenic organic matter (AOM) were determined during ozonation. Proteins released from tightly bound AOM are essential to modifying the hydrophobicity of bubble surfaces for easy cell attachment and to forming a top froth layer for collecting floating cells. Humic substances in the suspension scavenge dosed ozone that adversely affects ozone flotation efficiency of algal cells. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Efficient harvesting of the biomass from cultivation froth is essential for mass production of biodiesel from microalgae (Cooney et al., 2009; Ramanan et al., 2010; Jorquera et al., 2010). Harvesting utilizes solid–liquid separation processes, such as centrifugation, filtration, flotation, and sedimentation (Levin et al., 1962; Grima et al., 2003; Henderson et al., 2008a). Algae harvesting can be costly (Dismukes et al., 2008; Reijnders, 2008) since the mass fractions in a culture broth are generally low, while the cells generally have a negative charge and excess algogenic organic matter (AOM) to retain stability in a dispersed state. Dissolved air flotation (DAF) effectively separated algae when cationic surfactants were present (Van Vuuren and Van Duuren, 1965; Funk et al., 1968; Bare et al., 1975; Liu et al., 1999). Ozone is a strong oxidant that preferentially oxidizes double bonds and electron-rich aromatic rings into oxygenated functional groups such as aldehydic, ketonic, and especially carboxylic groups (Von Gunten, 2003; Beltrán, 2004; Li et al., 2008). Particle separation in water can be enhanced by oxidation using ozone, chlorine, chloride dioxide, permanganate, or chloramines (Jekel, 1998). Betzer et al. (1980) investigated the use of ozone-induced * Corresponding author. Tel.: +886 2 33663028; fax: +886 2 23623040. E-mail address: [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.04.083
flotation for algae recovery and effluent treatment. They determined that dosed ozone produced froth; the settlement of froth yielded settled sludge with 2–7% w/w solids. Benoufella et al. (1994) eliminated the Microcystis strain of cyanobacteria using an ozoflotation process. Mass cultivation of algae requires a high overflow rate, which promotes flotation during which algae move upward instead of sedimentation during which algae move downward (Koopman and Lincoln, 1983; Edzwald, 1993). Flotation has advantages of flexible operation and small footprint when compared with coagulation–flocculation and sedimentation (Liu et al., 1999). Biofuel production from algal cells requires the extraction of lipids from cultivated cells; this can be achieved by cells lysis with ozone. Furthermore, the addition of chemicals should be minimized to prevent contamination of extracted products. This study utilized dispersed ozone flotation for harvesting and cell lysis of Scenedesmus obliquus FSP-3, a species that has a high potential for CO2 capture and lipid production (Mandal and Mallick, 2009). (Note: In parallel study the S. obliquus FSP-3 was shown to fix CO2 at a rate of 256 g/m3 d, a value higher than most data reported in literature.) The algae removal rate, surface charge, and hydrophobicity of algal cells, fluorescence characteristics, and proteins and polysaccharides content in AOM were recorded during ozonation tests with no chemicals added. Experimental findings are discussed.
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2. Methods 2.1. Cultivation of algae The microalgal strain used in this work was obtained from freshwater in southern Taiwan. The microalga was identified as S. obliquus FSP-3 based on its morphology and by 23S rDNA sequence matching (GeneBank accession number DQ396875.1). Modified Detmer’s medium (DM) was employed to grow a pure culture of S. obliquus FSP-3. The medium was as follows (g/L): Ca(NO3)24H2O, 1.00; KH2PO4, 0.26; MgSO47H2O, 0.55; KCl, 0.25; FeSO47H2O, 0.02; EDTA2Na, 0.2; H3BO3, 0.0029; ZnCl2, 0.00011; MnCl24H2O, 0.00181; (NH4)6Mo7O244H2O, 0.000018; and CuSO45H2O, 0.00008. After pre-culturing in 200 ml flasks, the S. obliquus cells were inoculated into a 20-L photobioreactor with an inoculum size of 15 mg/L. S. obliquus FSP-3 was grown at 28 °C for 12 d under a light intensity of roughly 60 lmol/m2 s (illuminated by a TL5 fluorescent lamp) in the glass photobioreactor with an agitation speed of 300 rpm. Carbon dioxide (20%) was fed into the culture continuously as the sole carbon source. The CO2 feeding rate was 4 L/min during 12-d cultivation. The time-course profile of algae growth shows that the cumulative biomass production and overall biomass production rate of S. obliquus FSP-3 reached 1610 mg/L and 136 mg/L d, respectively. 2.2. Ozonation dispersed flotation Batch flotation tests were performed in a flotation column with an inside diameter of 5 cm and maximum depth of 47 cm. A porous glass diffuser with a nominal pore diameter of 30–60 lm was placed at the column bottom, just above the gas inlet port. The froth collection port was located at the column top. Liquid samples were withdrawn from the sampling port just above the porous glass diffuser. A T-408 ozone generator (Welsbach, CA, USA) was used to produce ozone from oxygen, which was the flotation gas. The gas pressure leaving the ozone generator was 0.65 kg cm 2 and the flow rate was 0.6 l min 1. No chemicals were added during all flotation tests. 2.3. Algae cell isolation and AOM extraction The original algal samples were centrifuged at 7000g at 4 °C for 15 min and the supernatant was obtained. Organic substances in the supernatant were loosely bound AOM (LBAOM). Residues were re-suspended to the original volume using Milli-Q water and centrifuged again at 7000g at 4 °C for 15 min with decanted supernatant. Half of the residues were then re-suspended in phosphate buffered saline (PBS) (pH 7.4) to the original volume, and were considered ‘‘washed” in this study. The remaining residue was re-suspended in Milli-Q water to its original volume and extracted using formaldehyde (37%, 0.06 ml) + ultrasound (120 W, 5 min) + NaOH (50 ml, 4 °C, 1 h) (Adav and Lee, 2008). The supernatant was recovered via centrifugation (5000g, 4 °C, 10 min). The organic matter in the recovered supernatant was tightly bound AOM (TBAOM). 2.4. Modified BATH test A test, a modified version of the bacterial adhesion to hydrocarbons test (BATH) hydrophobicity test (Rosenberg et al., 1980) was performed. Absorbance at 680 nm was determined for the original algal suspension and for liquid samples collected from the flotation column. In total, 4 ml of the algal sample was placed in a test tube with an inner diameter of 13 mm. Hexane (1 ml) was added and shaken for 10 s, and allowed to settle for 30 min. Carefully, 3 ml
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was obtained from the bottom layer in the test tube and its absorbance at 680 nm was again determined. The extractability of the hexane layer on organic substances in the algal suspension was calculated as (original absorbance absorbance of the water layer)/(original absorbance) 100%. 2.5. Excitation–emission matrix fluorescence spectroscopy analysis The excitation–emission matrix (EEM) spectra of collected samples were determined using luminescence spectrometry (Cary Eclipse, Varian Inc., CA, USA). The EEM spectra were obtained by scanning a sample for both excitation and emission wavelengths at 200–550 nm. The blank EEM spectrum obtained for double-distilled water was utilized to ensure the accuracy of scanned samples. For all measurements, excitation and emission slits were maintained at 5 nm, and a 290-nm emission cutoff filter was employed to eliminate second-order Raleigh light scattering. The EEM spectra are the elliptical shapes of contours, where the X-axis represents the emission spectra at 200–550 nm and the Y-axis is the excitation wavelength at 200–550 nm. In total, 40 contour lines, as the third dimension, are shown for each EEM spectra to represent fluorescence intensity at an interval of 5. 2.6. Chemical analysis The dry weight of, and volatile suspended solids (VSS) in, collected samples were determined using Standard Methods (APHA, 1998). The size and zeta potential of fine particles in collected samples were determined using a Zeta sizer (Nano-ZS) (Malvern Co., UK). In some tests, 10 or 20 mg/L sodium dodecyl sulfate (SDS) was dosed prior to zeta potential measurements. The organic carbon content (dissolved organic carbon (DOC)), polysaccharides (PS) and protein (PN) content for LBAOM were determined using a total organic carbon (TOC) analyzer (model 1030, O.I. Corporation, Texas, USA). The PS content in samples was determined using the Anthrone method with glucose as the standard. The PN content was determined using a modified Lowry method with bovine serum albumin as the standard. Washed algal suspensions, before and after ozonation–flotation, were filtered to collect cells for scanning electron microscopy (SEM) (S-2400, Hitachi Ltd., Tokyo, Japan) with the Ronatec system for energy dispersive spectroscopy (EDS) microanalysis. All SEM observations were conducted following fixing with 2.5% glutaraldehyde for 2 h, and dehydration via successive passages through 30%, 50%, 75%, 85%, 90%, 95% and 100% ethanol, followed by critical drying in a critical point dryer (HCP-2) (Hitachi Ltd., Tokyo, Japan).
3. Results and discussion 3.1. Ozone flotation Figs. S1 and S2 of Supplementary Materials show SEM micrographs for original algal cells and aggregates collected at the column top, and a photograph of the froth layer for the algal suspension ozonated for 4 min. The original algae were 4 2 lm ellipsoids. The collected froth liquor contained aggregates of injured cells sized 10–12 lm (in parallel tests using particle sizer, the mean size of aggregates was 10–30 lm). Hence, dispersed ozone flotation induced algae aggregation; this experimental finding is in agreement with analytical results obtained by Jekel (1994). Strong turbulence existed in the ozone flotation column in all tests; hence, the suspension in the column was assumed mixed completely. In the control test, simple air aeration did not cause algal cells to float to the column top. Fig. 1 shows residual turbidity
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DF=1, [O3]=98 mg/L
-5
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Ozone dosed ( mg O3 / mg Biomass ) Fig. 1. Residual turbidity in flotation column. DF: dilution ratio; washed: algae cells in PBS solution; and dashed line: NTU0 = 4800 * O3,i (in mg/mg biomass).
in the column (original and 100% or 200% in dilution) under three ozone concentrations. In total, four batches of cultivated algal suspensions were tested, yielding data fluctuation. With an ozone dosage exceeding a threshold value, turbidities of algal suspensions declined over time. For example, 0.16–0.20 mg O3/mg biomass was needed to initiate turbidity removal for original suspensions (initial turbidity (NTU0) = 800–1000 NTU). In total, around 0.52 mg O3/mg biomass was required to reduce turbidity by 95%. With 100% diluted (2) samples (NTU0 = 400 NTU), roughly 0.09 mg O3/mg biomass was needed to initiate turbidity removal and roughly 0.36 mg O3/mg biomass was consumed to reach turbidity removal of 95%. With 200% diluted (3) samples (NTU0 = 240 NTU), the threshold O3 was 0.05 mg/mg biomass and the 95% removal dosage was 0.22 mg/mg biomass. The following general linear correlation existed between NTU0 and the threshold ozone dosage (O3,i): NTU0 (in NTU) = 4800 * O3,i (in mg/mg biomass). Additionally, concentrated algal suspension consumed more ozone than diluted suspension at the same fraction of turbidity removal. Conversely, the turbidity of washed algal cells (in PBS) started declining immediately after ozonation started. No threshold ozone dosage existed. With an initial 400 NTU, roughly 0.07 mg O3/mg biomass was needed to remove 95% of turbidity, only about 15% of that was needed for unwashed samples with the same NTU0.
0.2
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Ozone dosed ( mg O3 / mg Biomass) Fig. 2. Zeta potentials for algae particles as functions of dosed ozone. Same legends as in Fig. 1. Zeta potentials for original suspension were 6 to 7 mV, and those for 2 samples and washed cells were 13 mV.
About 50–80% of cells in the original algal suspension or 40% of washed cells left an aqueous phase after mixing in the modified BATH tests (Fig. 3). Ozonation first increased and then decreased the extractability of algal cells. The extractability for un-diluted samples peaked at 0.4–0.5 mg O3/mg biomass, while that for washed cells peaked at roughly 0.05 mg O3/mg biomass. A foam layer formed between the aqueous and n-hexane layers, while most cells that left an aqueous phase did not move to an oil phase, but remained within the foam layer. In all BATH tests, no cells dissolved into an oil phase, regardless of ozone dosage or mixing time. 3.3. AOM in samples The DOC contents in LBAOM and TBAOM for the original suspensions were measured at 51.7 ± 0.73 and 199.5 ± 2.9 mg-C/L, respectively. The TBAOM contained almost four times the quantity of DOC in the LBAOM. In total, six characteristic EEM peaks were identified for the original and ozonated samples (shown later). The EEM peaks in the ellipse were centered at Ex/Em = 350/460 nm and 320/415 nm, and were likely attributable to humic-like compounds (HA1 and
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3.2. Characteristics of ozonated samples 80
Extractability ( % )
The zeta potentials for original suspensions were 6 to 7 mV; that for the 100% dilution sample and washed cells was 13 mV (Fig. 2). Hence, algal cells were negatively charged. Dilution or washing reduced ionic strength, thereby reducing the zeta potentials of algal cells. Ozonation gradually reduced the zeta potentials of the original and diluted samples. In other words, ozonation increased the negative charge of cell wall surfaces. For example, adding 0.2 mg O3/mg biomass reduced zeta potentials of algal cells from 7 to 11 mV, while adding additional 0.2 mg O3/mg biomass further reduced these potentials to around 15 mV. Conversely, adding <0.02 mg O3/mg biomass reduced the zeta potentials of washed cells to 27 from 13 mV. The zeta potentials of the original algal cells were not affected by the presence of SDS (data not shown), demonstrating that the algal cells have hydrophilic surfaces. In the presence of SDS, the zeta potentials of ozonated cells dropped by only 3 mV. Ozonation did not significantly increase surface hydrophobicity of algal cells.
60
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Ozone dosed ( mg O3 / mg Biomass ) Fig. 3. Extractability by hydrocarbon layer using modified BATH test. Same legends as in Fig. 1.
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HA2), and that centered at Ex/Em = 260/460 nm was due to fulviclike compounds (FA1) (Chen et al., 2003). Moreover, the three peaks (PR1–PR3) centered at Ex/Em = 280/360 nm, 255/300 nm, and 230/365 nm were likely attributable to protein-like compounds (Chen et al., 2003). Fig. 4 shows the EEM fluorescence peaks of the original algal suspension. Strong HA1 and FA1 peaks suggested the presence of humic substances in the suspension. Figs. S3 and S4 of Supplementary Materials show the EEM plots for LBAOM and TBAOM, respectively. The EEM pattern for LBAOM resembled that for the original suspension, indicating that LBAOM was primarily comprised of humic-like and fulvic-like substances. Conversely, the TBAOM was principally composed of protein-like substances. Fig. S5 of Supplementary Materials shows the EEM fluorescence plots for algal suspensions under ozonation. Fig. 5 shows the EEM peak intensities as functions of ozone dosage. The peak intensities of HA1 and FA1 for original algal suspensions decreased as ozone dosage increased (Fig. 5). The HA2 peak increased in intensity until dosed ozone reached 0.11 mg/mg biomass, and then decreased when ozone dosage was further increased. The peak intensity of PR1 increased as ozone dosage increased, and peaked at 0.22 mg O3/mg biomass. Moreover, the intensity of PR2 peaked at 0.43 mg O3/mg biomass. Fig. 6 shows the PS and PN contents in dissolved organic matter in ozonated samples. The PN content increased, peaked, and then decreased as ozone dosage increased. Conversely, PS content in dissolved organic matter was almost constant under ozonation.
14
HA1 FA1 HA2 PR1 PR2
Intensity ( a. u. )
12 10 8 6 4 2 0 0.0
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Ozone dosed ( mg O3 / mg Biomass ) Fig. 5. Intensities of EEM peaks for original algal suspension at [O3] = 98 mg/L. Characteristic peak values were extracted from EEM plots in Fig. S5.
between the solids in raw water (up to 15 NTU), so the total ozone dose is low; while those in fermentation froth is high (up to 900 NTU), which acquires high ozone dose. Hence, ozone disperse flotation is regarded a potential candidate for mass harvesting of unicellular algae such as the S. obliquus FSP-3 investigated herein. Algal cells have negatively charged hydrophilic surfaces, accounting for why simple air aeration does not yield flotation. Conversely, ozone-dispersed flotation efficiently separated S. obliquus FSP-3 from the broth suspension, particularly when the suspension comprised washed cells (Fig. 1). That is, the interaction between algal cells and bubbles was enhanced by ozonation. Ozonation can break down natural organic matter (NOM) molecules into hydrophilic low-molecular-weight fragments (Nawrocki et al., 2003). Chheda and Grasso (1994) demonstrated that ozonation reduced the stability of Na-montmorillonite particles coated with NOM in river water. They noted that adsorption of NOM on Na-M makes particles increasingly hydrophilic, while ozonation partially dealuminized Na-M and transformed coated NOM to increase particle surface hydrophobicity, thereby destabilizing particles. Grasso and Weber (1988) noted that ozonation
3.4. Discussion Ozonation is commonly regarded an expensive process for water and wastewater treatment. Ozone doses of 0.4–0.8 mg O3/ mg C were applied in seven US waterworks as preoxidants for destabilizing particles and removing precursors of total organic carbon (TOC) and trihalomethane (THM) (Chang and Singer, 1991). The ozone dose noted herein to sufficient harvesting of S. obliquus FSP-3 was about noted 0.2–0.5 mg/mg biomass, close or even lower than that adopted in practical water works. Such a conclusion seems controversial to the common understanding that ozone is an expensive process and should be used only in a pretreatment stage. One should take into account the difference
Excitation wavelength (nm)
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Emission wavelength ( nm ) Fig. 4. EEM plot for original algal suspension. HA1–HA2, FA1 and PR1–PR3 denote respectively the characteristic peaks noted for humic-like substances, fulvic-acid-like substance and protein-like substances in the present study.
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60 DF=1; [ O 3 ] = 63mg / L DF=1; [ O 3 ] = 98mg / L
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Ozone dosed ( mg O3 / mg Biomass) Fig. 6. Contents of proteins (a) and polysaccharides (b) as functions of dosed ozone. Symbols in (b) are defined the same as in (a).
increases polarity, cleaves aromatic rings, and decreases the apparent molecular weights of compounds in tested humic acids. Conversely, these authors determined that ozonation decreases polarity, creates long aliphatics, and increases the apparent molecular weights of organic compounds in a natural water samples collected from the Huron River (Michigan, USA). Henderson et al. (2008b) demonstrated that AOM extracted from four algae species (Chlorella vulgaris, Microcystis aeruginosa, Asterionella formosa and Melosira sp.) were dominated by hydrophilic polysaccharides and hydrophobic proteins with low specific UV absorbance (SUVA) and negative zeta potentials. Air bubbles in nearly neutral aqueous solutions have negatively charged hydrophobic surfaces. The S. obliquus cells carried negative charges; ozonation further reduced their surface charges (Fig. 2). In particular, <20 mg/L O3 effectively reduced the zeta potentials of washed cells to 27 from 13 mV. Hence, the electrostatic interaction mechanisms unlikely interpreted the noted enhanced algae flotation. Although divalent ions (Ca2+ and Mg2+) were present in the present system, the proposed Ca–organic substance complexation model by Chandrakanth and Amy (1996) cannot interpret the more negative surface charge of algal cells after ozonation. Extractability measured using the modified BATH test indicated that the hydrophobicity of algal cells increased after ozonation (Fig. 3). However, the BATH test assessed the hydrophobicity of algal cell wall surfaces based on the degree of preferential dissolution of algal cells into hydrocarbon layers. Experimental observations
demonstrate that cells (and the lysed) in this study were concentrated in the foam layer, and did not dissolve into the oil layer. Moreover, the zeta potentials in the presence of 10 or 20 mg/L SDS decreased slightly for ozonated cells. Therefore, the hydrophobicity of algal cell walls was not markedly increased following ozonation. Aerated bubbles can be modified by adding chemicals. Han et al. (2006) produced positively charged bubbles by adding cationic metal salts or cationic polymer for flotation. Henderson et al. (2008a, 2009), who modified bubbles using cationic surfactants, demonstrated that bubbles with surfaces modified by chemicals with both a hydrophobic long tail and a hydrophilic high charge head achieve sufficient algal removal without upstream coagulation and flocculation. Reckhow et al. (1986) demonstrated that ozone can lyse algae and liberate biopolymers, which then act as coagulating polymers. Ma and Liu (2002), who pre-oxidized algae with potassium ferrate, speculated that biopolymers from injured algae cells can be present as coagulant aids. In this study, ozone preferentially degraded humic-like substances (HA1 and FA1 (Fig. 5)) in suspensions, and then released protein-like substances (PR1) bound to algae cell surfaces. We propose that the released protein-like substances have surface activity once released that can bind with bubble surfaces and thereby modify bubble characteristics. That the released proteins carried a positive charge in the neutral solution is unlikely. Rather, the proteins bound bubbles make bubble surfaces increasingly hydrophilic, facilitating easy cell attachment. Since humic-like substances were removed from washed cells, ozone directly reacted with algal cells to release proteins; thus, flotation was achieved at a much lower ozone dose than that for unwashed cells (Fig. 1). A cultivation strategy for minimal production of humic-like substances is recommended for easy harvesting via dispersed ozone flotation process. Prolonged idle stage after cultivation is commonly adopted for enhancing lipid production, which may be also yield excess humic substances. A compromise between lipid production enhancement and humic substance secretion should be done for selecting optimal cultivation strategy. 4. Conclusions The S. obliquus FSP-3 was harvested using dispersed ozone flotation process. Simple air aeration failed to yield algal flotation, while ozone produced flotation efficiently. The ozone dose required to harvesting of S. obliquus FSP-3 ranged 0.2–0.5 mg/mg biomass. Ozonation produced algal cells with more negatively charged and slightly increasingly hydrophobic surfaces compared with intact cells. Proteins released from cell lysis were regarded surfactants to make bubble surface increasingly hydrophilic to yield effective bubble–cell collision and formation of top froth layer for cell collection. Humic substances in suspension, however, exhaust dosed ozone, whose formation should be minimized in cultivation stage. Acknowledgements Authors are grateful to Dr. Yuehwa Yu of National Taiwan University for providing ozonation equipments. The authors gratefully acknowledge financial supports from Taiwan’s Ministry of Economical Affairs (98-EC-17-A-10-S2-0066) and Taiwan’s National Science Council (NSC 96-2628-E-006-004-MY3, NSC 97-2218-E006-005 and NSC 98-3114-E-006-012). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.04.083.
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