PREPARATION OF THE BEADS .1 Bead Preparation Procedure

The beads were prepared as described by Dos Santos et al. (2002), with slight modifications.

1.8% (w/v) alginate solution was used for preparing the beads. 0.25 g of Na-alginate and 14 ml of liquid (deionized water, tap water, and algal suspension) was used for the preparation of

Aquaculture wastewater:

initial PO₄^3-, NO₃^-, NO₂ -and NH₃concentrations

Algal stock:

initial algal concentration

After 4.5 days:

final PO₄^3-, NO₃^-, NO₂^-and NH₃concentrations

After 6 days:

final algal concentration Cultivation

Preparation of beads

beads for each experimental unit, which resulted in 1:10.7 beads to culture medium ratio (v:v) when 150 ml of culture medium was used in the cultivations. For the preparation of beads for the bead concentration experiment, the amount of Na-alginate was varied to produce different number of beads for each treatment. To prepare the beads, Na-alginate was added to deioinised water and left on a magnetic stirrer overnight to homogenize and form a gel-like mixture. When preparing beads for the bead concentration experiment, 64% of the total added liquid was added at this point, whereas with other experiments 82% of the liquid was added. With most of the experiments a large batch of Na-alginate was prepared and divided to smaller sections for the treatments based on the weight of the mixture. The beakers that were used for the division of the Na-alginate mixture were not identical, and thus the amount of Na-alginate residues on the beaker surface and consequently the amount of Na-alginate that ended up in the beads may vary slightly between the treatments.

For each treatment approximately 140 ml of algal stock was centrifuged (Eppendorf Centrifuge 5430, 5000 rpm, 4 min) by removing the supernatant after the first round of centrifugation, refilling the tube, and repeating the centrifugation twice. For the treatments of algal concentration experiment the volume of centrifuged algal stock was varied to prepare beads with different algal concentrations. After centrifugation the algal pellet was resuspended, and the volume of the suspension was adjusted with tap water to the volume that was supposed to be added to Na-alginate. Algal suspension was then added to Na-alginate mixture and kept on a magnetic stirrer until homogenized.

The beads were prepared by adding the algae-Na-alginate mixture to 1.5% (w/v) CaCl2 solution dropwise through a syringe. All beads except the beads for the bead size experiment were prepared using a 60 ml single use plastic syringe (Terumo) with an inner diameter of the tip approximately 2 mm. Even though similar syringes were used for the preparation of the beads, the size of the beads produced with different syringes varied in some cases. The syringe was set up 11-18 cm above the CaCl2 solution, which was stirred with a magnetic stirrer. Algae-Na-alginate mixture was poured into the syringe and let drop through the tip under gravity flow.

The preparation process of the beads is presented in Figure 2. The formed Ca-alginate beads were round and had a diameter of approximately 3.4 mm (Figure 3). After all the beads were formed, they were kept in CaCl2 solution for 20-40 min and then rinsed thoroughly under running tap water. The beads for each treatment were weighed and divided to two experimental

units based on the weight. Detailed descriptions of the beads for each treatment are presented in Table 4.

Table 4. Description of the treatments and initial pollutant concentrations.

Treatment Description of the

concentration half of the medium bead concentration

concentration medium bead concentration 1.0  0.5

(1.2  0.5) 1:10.7 (1.1) High bead

concentration double the medium bead concentration

concentration medium algal concentration 2.3  0.0

(3.5  0.0) 1:10.7 (1.5) Suspended algal cells non-immobilized algal cells (4.1  0.8) -

Blank beads beads without algae - 1:10.7 (1.4)

Control aquaculture

wastewater wastewater without the

addition of algae - -

Figure 2. Preparation process of the beads. In the picture beads for the bead concentration experiment are being prepared. From left to right are the beads for low, medium, and high bead concentration treatments.

Figure 3. A close-up picture of the algal beads.

4.4.2 Preparation of the Beads for the Bead Concentration Experiment

To prepare the beads for the bead concentration experiment, a batch of Na-alginate was prepared and divided to three parts in a way that high bead concentration treatment received double and low bead concentration treatment half of the volume of the Na-alginate in medium

bead concentration treatment. The weight of Na-alginate and the volume of liquid that were used for the preparation of the beads for one experimental unit in low, medium, and high bead concentration treatments were 0.12 g and 7 ml, 0.25 g and 14 ml, and 0.49 g and 28 ml, respectively. Beads to culture medium ratios (v:v) and bead concentrations in beads/ml for treatments with low, medium, and high bead concentration were 1:21.4 and 0.6, 1:10.7 and 1.1, and 1:5.4 and 2.5, respectively. Initial algal concentrations in the treatments with low, medium, and high bead concentration were 1.9 ± 0.9, 1.0 ± 0.5, and 0.5 ± 0.2  10⁵ cells/bead, respectively. In cells/ml of culture medium the algal concentration was 1.2  0.5  10⁵ in each treatment.

4.4.3 Preparation of the Beads for the Bead Size Experiment

To prepare the beads for the bead size and algal concentration experiments, a batch of Na-alginate was prepared and divided to six equal parts. A 60 ml single use plastic syringe was used to prepare the beads for the small bead treatment. To prepare the beads for medium and large bead treatments, two dropping tools were prepared by cutting the top parts of two Pasteur pipettes open and adjusting the tip diameters to approximately 3 and 5 mm. Some of the large and medium-sized beads contained air bubbles due to the difficulty to maintain a constant flow of Na-alginate in a narrow Pasteur pipette. The diameters and weights of small, medium and large beads were 3.4, 4.0, and 4.7 mm, and 27, 36, and 59 mg, respectively. The lengths of the diameters were determined by taking a picture of the beads next to a ruler (Figure 4), measuring the bead diameters in the picture, and counting the lengths based on the ratio of the lengths of the ruler and the picture. The weights were determined by dividing the weight of the beads with the number of the beads. Initial algal concentrations in the treatments with small, medium, and large beads were 2.3 ± 0.0, 3.5 ± 0.0, and 4.7 ± 0.0  10⁵ cells/bead, respectively. In cells/ml of culture medium the algal concentration was 3.5  0.0  10⁵ in each treatment.

Figure 4. Small, medium, and large bead next to a ruler.

4.4.4 Preparation of the Beads for the Algal Concentration Experiment

To prepare the beads for the bead size and algal concentration experiments, a batch of Na-alginate was prepared and divided to six equal parts. To prepare beads with low, medium, and high algal concentration, approximately 70, 140, and 275 ml of algal stock were centrifuged and added to Na-alginate mixtures as described earlier. The initial algal concentrations in low, medium, and high algal concentration treatments were 1.2  0.0 (1.8  0.0), 2.3  0.0 (3.5  0.0), and 5.1  0.0 (6.9  0.0)  10⁵ cells/bead (cells/ml of culture medium).

4.4.5 Preparation of the Beads for the Mg Addition Experiment

To prepare the beads for low, medium and high Mg concentration treatments, three batches of Na-alginate were prepared. To enrich the beads with different concentrations of Mg, 5, 10, and 20 mg of magnesium sulfate (MgSO4  7 H2O) was dissolved to deionized water before the addition of Na-alginate. The amount of added MgSO4  7 H2O was selected in a way that the amount of MgSO4  7 H2O in the experimental units of the high Mg concentration treatment was close to the amount of MgSO4  7 H2O in 150 ml (the volume of culture medium in this experiment) of BBM (11 mg). The concentrations of MgSO4  7 H2O in the beads in low, medium, and high Mg concentration treatment were 179, 357, and 714 mg/l, respectively. For some reason the beads of the low Mg concentration treatment were clearly smaller compared to the beads of the other treatments. Initial algal concentrations in the treatments with low, medium, and high Mg concentration were 1.9 ± 0.4, 2.7 ± 0.6, and 2.8 ± 0.6  10⁵ cells/bead, respectively. In cells/ml of culture medium the algal concentration was 4.1  0.8  10⁵ in each treatment.

To prepare the algal beads without Mg and blank beads without algae a batch of Na-alginate was prepared and divided to two equal parts. Instead of algal suspension, tap water was added to Na-alginate mixture for the preparation of blank beads. Initial algal concentration of the algal bead treatment in cells/bead was 2.9  0.6  10⁵ and in cells/ml of culture medium 4.1  0.8  10⁵.