An axenic strain of C. sorokiniana IAM C-212 made axenic by the addition of antibiotics (^{Watanabe et al., 2005}) and one of its bacterial symbionts (CSSB-3) isolated from the contaminated strain were used. Strain CSSB-3 (AB167383) showed 98.6% 16S rRNA gene sequence similarity to Microbacterium trichotecenolyticum (Y17240), belonging to the high-G+C Gram-positive bacteria (^{Watanabe et al., 2005}).

Preculture of C. sorokiniana was carried out according to the method described by ^{Ogbonna et al. (1995)}. Subculturing was carried out every 4 weeks by transferring 5 mL of the precultured broth into a 500-mL Erlenmeyer flask containing 200 mL MM4N medium composed of the following salts (per litre of distilled water): 5.0 g KNO₃, 1.25 g KH₂PO₄, 0.1 g K₂HPO₄, 2.5 g MgSO₄?7H₂O, 1.8 g of NaCl, 0.04 g FeSO₄?7H₂O, 0.03 g EDTA and 1 mL A₅ solution (containing, per litre of distilled water, 2.86 g H₃BO₄, 1.81 g MnCl₂?4H₂O, 0.22 g ZnSO₄?7H₂O, 0.08 g CuSO₄?5H₂O and 0.021 g Na₂MoO₄?2H₂O), and incubating on a rotary shaker (120 r.p.m.) at 30 ?C under continuous illumination (50 ?mol m^?2 s^?1). The preculture was done by inoculating Roux flasks containing 1 L MM4N medium with 10 mL of the stock culture. It was then incubated at 37 ?C for 2 weeks under continuous illumination (360 ?mol m^?2 s^?1). Aeration and mixing were achieved by sparging air enriched with 5% CO₂ through a glass-ball filter (Kinoshita Rika, Tokyo, Japan), which was inserted to the bottom of the Roux flask, at 0.3 v v^?1 min^?1 (vvm).

Chlorella culture broth was prepared by cultivating C. sorokiniana under photoautotrophic conditions for 2 weeks. The culture broth was centrifuged (5000 g, for 15 min at 4 ?C), and the supernatant was filtered through a sterile 0.2-?m pore size filter (Millex GV, Millipore, Billerica, MA). Five grams of Chelex-100 cation exchange resin (Nippon Bio-Rad Laboratories, Tokyo, Japan) was added to 100 mL of this filtrate. It was gently shaken for 1 h to remove cations from the filtrate. Residual metals in Chelex-treated filtrate were assayed using a sequential plasma spectrometer (ICPS-8100, Shimazu, Shiga, Japan). They comprised the following (mg L^?1): Na, <0.1; K, <0.1; Mg, 0.1; Ca, <0.05; Mn, <0.05; Fe, 0.2; Cu, <0.1; Zn, 0; Mo, <0.05.

The metals used in aggregation tests were aqueous solutions of KCl, NaCl, MgCl₂?6H₂O, CaCl₂?2H₂O and SrCl₂?6H₂O. Each chloride salt was dissolved in MilliQ water at various concentrations. Five hundred microlitres of the filtered Chlorella culture broth and required volumes of the metal solutions were mixed to yield the desired final concentrations (0, 1, 2, 5, 10, 20, 50 mM). After incubation at 25 ?C for 15 min, the mixtures were centrifuged at 12 000 g for 10 min at 4 ?C. The pellet was washed five times in 1 mL of each concentration of the metals and then suspended in 500 ?L of 50 mM EDTA. The total amount of carbohydrate in the pellet was determined using the phenol/sulphuric acid method with glucose as a standard (^{Dubois et al., 1956}). Results are expressed in mg equivalent of glucose per litre of Chlorella culture broth. These experiments were performed in triplicate and the results are expressed as the mean?SE.

Flocculated C. sorokiniana culture was examined after addition of 50 mM CaCl₂ to the medium. The precultured C. sorokiniana (10 mL) was inoculated to a Roux flask containing 490 mL MM4N medium and incubated at 37 ?C for 10 days under continuous illumination. The light intensity, aeration and mixing were achieved as described above. After 3 days of cultivation, 10 mL of 2.5 M CaCl₂ aqueous solution was added to the culture broth through a 0.2-?m pore size filter. A culture to which 10 mL of distilled water was added was used as the control. Growth of C. sorokiniana was monitored by cell counting under a microscope, after addition of 50 mM EDTA to disintegrate cell flocculation. The amount of dissolved polysaccharide in C. sorokiniana culture broth was determined as follows. Algal cells were removed by centrifugation and 3 mL of the supernatant was obtained. Seven millilitres of cold 100% ethanol was added to the supernatant, and this was then incubated overnight at 4 ?C. After incubation, the mixture was centrifuged at 8000 g for 10 min to precipitate the polysaccharides. The polysaccharide pellet was washed twice with 80% (v/v) ethanol and finally with 100% ethanol. After evaporating the residual alcohol under vacuum, the polysaccharide pellet was dissolved in an appropriate volume of distilled water to adjust the sugar concentration. Determination of the total saccharide content was examined using the phenol/sulphuric acid method with glucose as a standard.

The symbiont was precultured in diluted Luria-Bertani (DLB) medium (per litre distilled water: 1.0 g peptone, 0.5 g yeast extract and 1.0 g NaCl, pH 6.8) at 30 ?C for 24 h and used after washing in sterile saline solution (0.85% NaCl).

Chlorella sorokiniana was cultured axenically in a 500-mL Roux flask as described above and incubated at 37 ?C. After 3 days of cultivation, 10 mL of washed cultures (1.0 ? 10⁶ cells mL^?1) of symbiotic bacteria (CSSB-3) was inoculated to C. sorokiniana culture followed by addition of 10 mL of 2.5 M CaCl₂ solution to yield a final concentration of 50 mM. The resulting microbial floc was incubated for 7 days under light conditions. The final cell concentration of C. sorokiniana in the culture broth was determined by cell counting. The experimental conditions are detailed in Table 1. These experiments were performed in triplicate.

Effects of flocculated symbiont on growth of C. sorokiniana were evaluated via ANOVA. Significant results were subjected to Tukey?Kramer's test for multiple comparisons between groups. The calculations were carried out using MICROSOFT EXCEL 2003 with STATCEL2 as add-in software (OMS Publishing, Tokorozawa, Japan).

Two hundred and fifty microlitres of Chlorella?symbiont complex culture was fixed using 250 ?L of 0.2 M sodium cacodylate, 250 ?L of 4% (v/v) glutaraldehyde and 250 ?L of 0.2 M phosphate buffer (per litre distilled water: 5.28 g NaH₂PO₄?H₂O and 43.46 g Na₂HPO₄? 7H₂O, pH 7.4) and again fixed with 250 ?L of 0.2 M sodium cacodylate and 4% osmic acid, and 250 ?L of 0.2 M phosphate buffer (pH 7.4). The fixed sample was dehydrated sequentially with 50%, 70%, 80%, 90% and 100% (v/v) ethanol. Finally, 100% ethanol was exchanged with 2-methyl-2-propanol. This sample was freeze-dried (VD-800F, Taitec, Tokyo, Japan), and coated with Pt ? Pd (E102, Hitachi, Tokyo, Japan). The sample was observed with a field-emission scanning electron microscope (JSM-6330F, JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV. Micrographs were taken at ? 10 000 magnification.

Chlorella culture broth was used for isolation of a propionate-degrading bacterium. The culture broth contained extracellular organic carbon (EOC), which is excreted during C. sorokiniana culture (^{Watanabe et al., 2005}). The filtered culture broth was mixed with an equal volume of photoautotrophic medium, and the pH was adjusted to 6.0 with 1 N HCl. One gram of soil sample was incubated in EOC medium containing 1.0 g L^?1 sodium propionate at 30 ?C for 3 days. Thereafter, 1 mL of the culture medium was transferred into fresh EOC medium containing 5.0 g L^?1 sodium propionate and incubated at 30 ?C for 24 h. Bacterial cells were isolated from the resultant culture by spreading and cultivating on an EOC agar plate (2.0% agar) containing 5.0 g L^?1 propionate for 3 days. A propionate-degrading bacterial strain (PDS1) was obtained from this procedure.

Strain PDS1 was characterized based on 16S rRNA gene sequence analysis. DNA was extracted from samples using the Instagene matrix (Bio-Rad) according to the manufacturer's protocol. Amplification of the 16S rRNA gene was carried out using universal primers 27F and 1492R (primers are listed in Table 2). The PCR products were sequenced directly on a CEQ 2000 DNA sequencer using a CEQ Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman Coulter, Fullerton, CA). The 16S rRNA gene sequence primers 27F, 341F, 518R, 530F, 764F, 911R, 1100R, 1390R and 1492R were used. Sequences of all primers in this study are shown in Table 2. The sequences obtained were compared with known sequences using the FASTA service.

PDS1 was precultured in DLB medium with shaking at 30 ?C for 24 h. This bacterial cell suspension was added to fresh DLB medium containing 1 g L^?1 propionate at an A_{580 nm} of 0.02, and incubated on a rotary shaker (200 r.p.m.) at 30 ?C. Propionate concentrations were measured on a liquid chromatograph equipped with Inertsil ODS-3 column (GL Science, Tokyo, Japan) and Intelligent UV/VIS detector (UV-970; Jasco Company, Tokyo, Japan). The oven temperature was maintained at 40 ?C, while 20 mM phosphate buffer (containing 2.72 g KH₂PO₄ L^?1; pH adjusted to 3.0 with H₃PO₄) was used for elution at a flow rate of 1.0 mL min^?1.

The effect of propionate concentration on axenic C. sorokiniana culture was examined. Five hundred microlitres of the precultured C. sorokiniana (containing 5.0 ? 10⁷ cells mL^?1) was inoculated into 4.5 mL MM4N medium containing the desired concentration of propionate in a Monod tube. It was cultured at 30 ?C for 3 days on a monod-type shaker (Monosin II A, Taitec, Saitama, Japan) under a light intensity of 200 ?mol m^?2 s^?1. The C. sorokiniana cell concentration was determined after 3 days of cultivation. This experiment was performed four times and the results were expressed as the mean values.

An appropriate volume of the precultured C. sorokiniana was taken to obtain 2.5 ? 10⁷ cells, and the volume was adjusted to 450 ?L with MM4N medium. Fifty microlitres of the washed PDS1 culture (containing 2.5 ? 10⁹ cells) was mixed with the Chlorella culture and 500 ?L of 500 mM CaCl₂ solution was added for flocculation. The microbial floc obtained was inoculated to 4 mL MM4N medium containing 125 mg L^?1 sodium propionate. The Chlorella cell concentration was determined on the 3rd day by cell counting. The experimental conditions are detailed in Table 3. These experiments were performed in triplicate. Effects of flocculated PDS1 on C. sorokiniana cell growth were evaluated via ANOVA and significant results were subjected to Tukey?Kramer's test for multiple comparisons between groups. Calculations were carried out using MICROSOFT EXCEL 2003 with STATCEL2 as add-in software (OMS Publishing).

The amount of saccharide precipitated from the culture broth increased with increase in the concentration of calcium and strontium chlorides. Maximum amounts of saccharide were achieved when 20 mM of Sr²⁺ or 50 mM of Ca²⁺ was added to the culture broth (Fig. 1). There were no saccharide precipitates when NaCl, KCl or MgCl₂ was added to the culture.

Addition of CaCl₂ solution resulted in flocculation of C. sorokiniana. Each floc comprised tens of C. sorokiniana cells within a gelatinous matrix. The Chlorella-flocs were observed even 7 days after cation addition (Fig. 2). When CaCl₂ solution was added to the culture, the amount of polysaccharide in the C. sorokiniana culture broth decreased from 2.02 to 0.89 mg L^?1 (Fig. 3). In the control culture, the quantity of polysaccharide in the culture broth increased linearly, reaching 8.34 mg L^?1 on day 10. In the Ca²⁺ addition culture, only 3.45 mg L^?1 of polysaccharide was produced by day 10. The quantity of polysaccharide in the culture with CaCl₂ was always lower than those in cultures without addition of CaCl₂. In the flocculated C. sorokiniana culture, cell numbers increased from an initial value of 2.85 ? 10⁸ to 5.26 ? 10⁸ cells mL^?1 at the end of the culture. However, this value was lower than the value obtained in the control experiment (7.69 ? 10⁸ cells mL^?1).

Coflocculated C. sorokiniana and its symbiont were studied as an example of artificial symbiotic microorganisms. Chlorella sorokiniana and its symbiont (CSSB-3) were coflocculated within the gel matrix produced by C. sorokiniana in a medium containing CaCl₂. These flocs were observed immediately after addition of CaCl₂. Electron microscopic observation revealed that C. sorokiniana and the symbiont were coflocculated within the algal sheath even 1 week after the addition of CaCl₂ (Fig. 4).

The number of C. sorokiniana cells was counted 7 days after addition of symbionts and CaCl₂. The C. sorokiniana cell number obtained in the coflocculated culture (SBCa, mean 7.14 ? 10⁸ cells mL^?1) was significantly (P <0.01) higher than the number obtained in the control culture (Control, mean 5.39 ? 10⁸ cells mL^?1). It was also higher than the number of C. sorokiniana obtained in the mixed culture (SB, mean 6.24 ? 10⁸ cells mL^?1, P <0.05; Fig. 5).

The results of axenic cultivation of C. sorokiniana in the medium containing sodium propionate showed that propionate inhibited growth of C. sorokiniana (Fig. 6). A complex of C. sorokiniana and propionate-degrading bacterium that can grow in the presence of propionate was therefore constructed. Screening for propionate-degrading bacteria was done using the EOC medium containing sodium propionate. The bacterial strain isolated (PDS1) was able to degrade 94% of the propionate in the medium within 24 h (Fig. 7). PDS1 grew in EOC medium with or without propionate, but it did not grow in the photoautotrophic medium containing propionate as the sole carbon source (data not shown). Identification of the bacteria isolated was carried out by 16S rRNA gene sequence analysis. Strain PDS1 (AB302851) showed 99.9% similarity to Microbacterium oxydans (AJ717357) belonging to the Actinobacteria.

A coflocculated complex of C. sorokiniana and PDS1 was constructed by addition of CaCl₂ to a mixture of PDS1 and C. sorokiniana. This floc was cultured in a medium containing propionate, and C. sorokiniana cell number was counted on the third day. In axenic culture (PP), average cell C. sorokiniana number was 8.9 ? 10⁶ cells mL^?1, which is significantly lower (P <0.01) than the 15.1 ? 10⁶ cells mL^?1 obtained in the control culture without addition of propionate (Control; Fig. 8). By contrast, in the coflocculated culture of C. sorokiniana and PDS1 (PDSCa), mean cell number (13.9 ? 10⁶ cells mL^?1) was higher than that obtained in axenic culture (PP). There was no significant difference between the cell concentrations obtained in coflocculated culture (PDSCa) and control cultures (Control). At the end of the coflocculated culture, the propionate concentration in the medium decreased below the measurement limits (15 mg L^?1) of our HPLC system. However, there was no significant decrease in the concentration of propionate in the control culture (data not shown). There was no significant difference in the cell concentrations of C. sorokiniana in the axenically flocculated culture (PPCa) and axenic culture (PP).