Optimization of the Bead Concentration of the Culture Medium

In the literature the bead concentration is usually reported either as a number of beads per ml of culture medium or as a ratio of the volumes of the beads and the culture medium. In this review the ratio of the volume of the beads to the volume of the culture medium is mostly used.

As can be seen from Table 2, a wide range of bead concentrations have been used in the experiments investigating the effect of bead concentration on the nutrient removal. Both Abdel Hameed (2007) and Tam and Wong (2000) determined 1:3 beads to culture medium ratio to be optimal for nutrient removal. In his experiment, Abdel Hameed (2007) cultivated C. vulgaris immobilized in beads with initial algal concentration of 1.5  10⁶ cells/bead in ratios of 1:3, 1:2, 1:1, and 2:1 in primary treated wastewater for 48 hours. Treatments with ratios of 1:3 and 1:2 removed ammonia significantly more effectively than treatments with higher ratios, and also reached the highest nitrate removals. In the treatments with high bead concentrations, the removal of N-compounds was most likely affected by the self-shading effects caused by the large number of beads. The phosphate removal on the other hand was not significantly affected by the self-shading effects or bead concentration, and all the treatments were equally effective reaching about 95% phosphate removal. Between the most effective treatments, 1:3 beads to culture medium ratio was selected to be the most ideal and economical solution for wastewater treatment.

The experiment by Tam and Wong (2000) was closely similar to that of Abdel Hameed (2007) as C. vulgaris immobilized in beads at concentration of 10⁶ cells/bead was cultivated in synthetic wastewater that simulate settled domestic wastewater. Unlike Abdel Hameed (2007), Tam and Wong (2000) tested also lower bead concentrations than the optimal 1:3 beads to culture medium ratio, as the tested ratios ranged from 1:9 to 1:1.8. However, the treatments with low bead concentrations did not contain enough algal biomass for efficient nutrient removal as after 24-hour treatment both ammonium and phosphate concentrations in the treatment with the lowest beads to culture medium ratio (1:9) were significantly higher compared to other treatments. Similarly as Abdel Hameed (2007), Tam and Wong (2000) observed that in phosphate removal the differences between the other treatments were minor, and in ammonium removal the treatment with 1:3 beads to culture medium ratio performed best, reaching 100% ammonia removal (Table 2). Some of the beads in the treatments with high bead concentrations did not stay suspended in the culture medium during the cultivation and settled to the bottom of the bioreactor, reducing the surface to volume ratio of the beads and the efficiency of the light utilization. This in addition to the self-shading effects had a negative

impact on the N-removal of the treatments with high bead concentrations. Tam and Wong (2000) also observed that algae have minor contribution in the COD (chemical oxygen demand) removal. The differences between the treatments with blank beads and different concentrations of algal beads in COD removal were insignificant, which indicates that the major removal mechanism of organic matter was adsorption onto the alginate matrix.

In the experiment by Oluwole et al. (2019) the bead concentration of 40 g of beads per litre of culture medium was observed to exhibit the best ammonium removal out of the three tested concentrations (20, 40, and 80 g/l) after 43-hour cultivation in synthetic wastewater. However, also the other bead concentrations were able to remove ammonium effectively. Microalga C.

vulgaris was used also in this experiment. Mollamohammada et al. (2020) came to conclusion that from the range of 1:15 to 1:1, 1:7 was optimal beads to culture medium ratio for nitrate removal (Table 2). The result of Mollamohammada et al. (2020) was quite different from those of Abdel Hameed (2007) and Tam and Wong (2000), which was probably due to the differences in the experimental set-ups between the studies. In the experiment by Mollamohammada et al.

(2020) most of the nutrients, that are needed for algal growth, were incorporated into the alginate matrix of the beads, and the culture medium consisted of deionized water enriched with nitrate and glucose instead of wastewater that was used in the other experiments. In addition, different algal species was used (C. sorokiniana) and the experiment was considerably longer than the other experiments, lasting 16 days. In this experiment it was also observed that the pH, the concentration of dissolved oxygen, and the conductivity of the culture medium were higher in the treatments with high bead concentrations. The rise of the conductivity of the culture medium was mostly caused by the calcium and chloride ions originating from the alginate beads.

Whitton et al. (2016) approached the topic of bead concentration from a different point of view.

Based on the experimental results from cultivating suspended cultures of S. obliquus and C.

vulgaris in several wastewaters with different phosphate concentrations, the cell concentrations required for optimal phosphate remediation (under 0.1 mg P/l) from each initial concentration as well as the required retention times were calculated. Then the cell concentrations were converted to bead concentrations for beads with initial algal concentration of 10⁶ cells/bead.

The obtained bead concentrations for the remediation of different initial phosphate concentrations (1, 5, and 10 mg P/l) ranged from 1:12.5 to 2.1:1 for S. obliquus and from 1:9 to 1.2:1 for C. vulgaris, and the required retention time for the treatment with most of these

bead concentrations was under one day. To achieve short retention times, high bead concentrations were needed, but the authors considered 1:1 to be the highest possible bead concentration for practical reasons. With this bead concentration the phosphate removal could be performed in 1.5-2.5 hours with both of the studied algal species from the initial concentration of 1 mg P/l. For the treatment of initial concentration of 5 mg P/l, retention times of 6.3 and 3.5 h would be needed for S. obliquus and C. vulgaris, respectively. According to the authors, immobilized microalgae seem to be a promising technology for tertiary wastewater treatment as short retention times could be achieved with influent phosphate concentrations common at this stage of wastewater treatment. In the experiment by Tam and Wong (2000), the initial phosphate-phosphorus concentration of 5.7 mg/l declined near zero in just four hours with the bead concentration of 1:1.8. The same algal species (C. vulgaris) and initial algal concentration were used both in this experiment and in the calculation by Whitton et al. (2016), which implies that the calculations were accurate. However, if also N-compounds are targeted in the algal treatment, the beads to culture medium ratio of 1:1, which Whitton et al. (2016) used, would be unideal as both Abdel Hameed et al. (2007) and Tam and Wong (2000) showed that high bead concentrations decreased the efficiency of the removal of N-compounds.

Among the experiments that focused on bead concentrations, the experiment by Tam and Wong (2000) was the only one that measured algal growth in addition to nutrient removal. The authors observed that self-shading effects had a negative impact not only on the nutrient removal, but also on the number of algal cells within the beads. The treatments with highest bead concentrations (1:1.8 and 1:2.25) were most affected by the self-shading effects. In these treatments the algal growth was minimal, and the cell numbers were maintained in the initial 10⁶ cells/bead throughout the 48-hour cultivation. In the treatments with lower bead concentrations algal growth was observed. The cell number of the treatment with lowest bead concentration (1:9) doubled during the cultivation. The results from the chlorophyll content analysis were similar as the results from the cell counts: the increase in chlorophyll content was greatest in low bead concentrations and lowest in high bead concentrations.

Table 2. Description of the experiments studying the effect of the bead concentration on the algal growth and nutrient removal.

Reference Cultivation

conditions Culture medium Bead preparation Microalgal

species Treatment

time Bead number Nutrient removal Algal growth

Tam and Wong,

all the nutrients used for algal cultivation, except nitrate