The algicidal bacterium Vibrio brasiliensis H115 was isolated from the seawater of Dameisha Bay (Shenzhen, China), and subsequently cultured in 2216E medium (5 g/L peptone, 1 g/L yeast extract, 0.1 g/L ferric phosphate, pH 7.6–7.8) for 24 h (25°C, 200 rpm).

Akashiwo sanguinea used in this study was provided by the Algal Culture Collection of the Institute of Hydrobiology at Jinan University (Guangzhou, China). The culture was maintained in modified f/2 medium (Lananan et al., 2013; Zhang et al., 2014) at 20 ± 2°C with light (3500 l ×, 12:12 light-dark cycle) (Sun et al., 2016). The algal culture was incubated for 24 h to achieve its exponential growth phase before use.

The preparation of immobilized bacteria was conducted as reported previously with slight modifications (Idris and Suzana, 2005). Briefly, Vibrio brasiliensis H115 culture (10 mL) was centrifuged (8,000 rpm, 10 min) and the collected pellets were mixed thoroughly with 3% (m/v) sterilized SA (Solarbio, Beijing, China) solution (10 mL). The mixture was then dropped into a beaker with 4% (m/v) CaCl₂ (Macklin, Shanghai, China) using a pipette with sterile tips (1 mL). The solution was stirred slowly for 24 h at 4°C to complete the bead formation process. The cell density in the bead was 7.4 × 10⁸ cell/g. The beads were washed with sterile water and stored at 4°C before use.

The initial fermentation was conducted by inoculating 1 g of the immobilized beads into a flask (n = 3) with 100 mL of modified 2216E medium (with an extra 10 g/L of sorbitol and 20 g/L of peptone) and incubating at 40°C and 200 rpm for up to 26 h. The fermentation broth was collected at different time points (0, 4, 8, 14, 18, 22, 24, and 26 h) for the detection of OD₆₀₀ and then centrifuged (10,000 g, 10 min). The OD₆₀₀ in the fermentation broth was used as a proxy for the cell density of H115 in the beads. The supernatants were collected and filtered to get the cell-free fermentation broth. The algicidal efficiency of the cell-free fermentation broth against A. sanguinea was detected by co-incubating the cell-free fermentation broth (1 mL) with A. sanguinea (100 mL) for 10 min. A control experiment was added by co-incubating 1 mL of sterile modified 2216E with 100 mL A. sanguinea for 10 min to test if the modified 2216E medium has any effect on A. sanguinea. Algicidal efficiency was calculated as described by Zhang et al. (2018):Algicidal efficiency (%) = 100 × (N₀−Nt)/N₀, where N₀ and Nt represent the cell density of A. sanguinea measured at 0 h and different sampling time (t), respectively. Bioassays were conducted in triplicate. The cell number of the A. sanguinea was counted using a microscope (Olympus IX51, Tokyo, Japan) with a hemocytometer after staining with Lugol’s reagent (Wang Y. et al., 2020).

After the initial fermentation, the immobilized beads were collected and washed with sterile distilled water three times before inoculating into 100 mL of optimized 2216E medium for repeated batch fermentations. Repeated batch fermentation was conducted three times as above to determine the algicidal activity of the beads and evaluate their stability.

After 14 h of fermentation, the fermentation broth was collected and centrifuged (10,000 g, 10 min), the supernatants were collected and filtered to get the cell-free supernatants. The cell-free supernatants were sprayed (5 r/min) at 180°C using a spray drier (Bioq-8005, Huihetang Bioengineering Equipment Co., China) to get the powder of fermentation broth. The powder was then added into 100 mL A. sanguinea culture at different concentrations (0.1–1.5 g/L) and the algicidal efficiency was calculated after 24 h exposure. A. sanguinea culture without the addition of powder was set as the control and three replicates were conducted. The cell number of the A. sanguinea was counted at 0 and 24 h as above to calculate the algicidal efficiency.

The dried powder was then used for the preparation of floating algicidal microcapsules. The preparation of the floating microcapsules was modified from the previous study (Yang and Wang, 2014). Firstly, solution A (100 mL) was prepared by mixing CaCO₃ (Macklin, Shanghai, China) with 2% of SA (3:4 ratio) and solution B (100 mL) was made by mixing 3% citric acid (Macklin, Shanghai, China) with 3% CaCl₂. The dried powder was added into solution A with a concentration of 1.7 g/L and mixed thoroughly, the mixture was then dropped into a beaker with solution B using a pipette with sterile tips (1 mL). The solution was then stirred (200 rpm) to obtain the SA beads. The SA beads were washed with sterile water three times and air-dried. The SA beads were then dispersed in ethylcellulose (EC, M70, Macklin, Shanghai, China) solution (2%, w/v) and stirred for 20 min to acquire EC coating. The beads were then washed with ethanol (100%) once and then washed with sterile water three times. The SA-EC beads were then dried at 40°C and stored at 4°C before use.

To determine an optimum preparation condition for the SA-EC microcapsules, single factor effect experiments were conducted to examine the effect of different factors on the physical characteristics of the SA-EC microcapsules. The microcapsules were prepared with different concentrations of SA (1.0, 2.0, 3.0, 4.0, and 5.0%), CaCO₃: SA ratios (0, 1:4, 1:2, 3:4, and 1:1), concentrations of CaCl₂ (1.0, 2.0, 3.0, 4.0, and 5.0%), concentrations of citric acid (1.0, 2.0, 3.0, 4.0, and 5.0%), concentrations of EC (0, 1.0, 2.0, 3.0, 4.0, and 5.0%), and different crosslinking time (10, 20, 30, 40, and 50 min). When conducting the single factor effect experiments, except for the variables, the basic experimental conditions were 3% SA, CaCO₃: SA of 3:4, 3% CaCl₂, 3% citric acid, 2% EC, and crosslinking for 30 min.

The diameter of the microcapsules was obtained by calculating the average diameter of fifty microcapsules, which was measured with a Vernier caliper (He et al., 2015).

To estimate the floating ability of the microcapsules, fifty microcapsules were added into 100 mL of seawater and incubated with shaking (50 rpm) for 4 days, and the number of the floating microcapsules was counted for the determination of floating ability.

Crystal violet solution was used as a middle marker to assess the permeability of the microcapsules. Approximately, 0.7 g of microcapsules were immersed in 50 mL of crystal violet solution (with an absorbance of about 1.0) and incubated for 4 h (n = 3). Samples (3 mL) were taken at 0 and 4 h to measure the crystal violet absorbance (A₅₇₀) using a spectrophotometer (UV-8000S, METASH, Shanghai, China). The permeability of beads was estimated based on the difference between the two OD values as follows: Permeability (%) = 100 × (A₀−A)/A₀, where A₀ and A represent the absorbance of crystal violet solution measured at 0 and 4 h, respectively. All experiments were conducted in triplicate.

The algicidal activity of the optimized SA-EC microcapsules was tested by inoculating 1 g of microcapsules into 100 mL of A. sanguinea culture. The concentration of algicidal powder in the microcapsules was 1.7 g/L. Samples were collected at 4, 8, 16, and 24 h to count the algal cell numbers and calculate the algicidal efficiency. Microcapsules without powder were used as control.

The maximum photosynthetic quantum yield (QY) of PSII (Fv/Fm) and the relative electron transport rate (rETR) were also analyzed using a chlorophyll fluorometer (Water-PAM, WALZ, Germany). Prior to fluorescence measurement, samples were dark-acclimated at room temperature for 20 min. The untreated cell-free A. sanguinea culture was used as blank when determining the Fv/Fm and rETR.

Statistical analyses were performed in R (v. 3.6.0). At least three replicates were conducted in all experiments, data were presented as mean ± standard error. Significant changes (p < 0.05) of algicidal efficiency over time/concentration of algicidal powder were analyzed by one-way ANOVA. To test the significant difference (p < 0.05) of measured variables (algicidal efficiency, Fv/Fm and rETR) between control and treatment groups over time, data were analyzed by two-way ANOVA with time and treatment as independent variables and followed by Tukey’s HSD post hoc tests.

Repeated batch fermentation of immobilized bacteria has been widely applied for the production of secondary metabolites. Studies have shown that the productivity of secondary metabolites of microorganisms, such as penicillin (Rani et al., 2004), gibberellic acid (Meleigy and Khalaf, 2009), cyclosporin A (Suvase et al., 2010) and lovastatin (Porcel et al., 2008) can be enhanced during repeated fermentations. In this study, the algicidal activity of the cell-free fermentation broth was significantly improved using repeated batch fermentation. The algicidal efficiency of the initial fermentation was compared to the repeated batch fermentation of the immobilized strain H115 (Figure 1). Figure 1A shows the growth of H115 in the fermentation broth during the initial fermentation and the corresponding algicidal activity of the cell-free fermentation broth. The growth of H115 in the fermentation broth was used as a proxy for the cell density of H115 in the beads. Results showed that both OD₆₀₀ and algicidal efficiency did not change significantly at the first 8 h (p = 0.33 and 0.40, respectively), indicating a lag phase. The OD₆₀₀ of fermentation broth increased significantly (p < 0.05) and reached 4.37 ± 0.02 at 24 h, while the algicidal efficiency reached 100% (Figure 1A). The lag phase in the initial fermentation can be explained by the direct addition of immobilized beads without pre-activation. For the repeated fermentation, the average OD₆₀₀ increased significantly from 0.43 ± 0.01 to 4.35 ± 0.08 in 16 h (p < 0.05, Figure 1B). The average algicidal efficiency of cell-free fermentation broth increased significantly (p < 0.05), and an algicidal efficiency of 100% was reached at 14 h (Figure 1B). To test the effect of modified 2216E medium on A. sanguinea, a control experiment was conducted by inoculating 1 mL of sterile modified 2216E with 100 mL A. sanguinea for 10 min. Results showed that the number of the algal cells did not change significantly before and after co-incubation with modified 2216E medium (p = 0.80), indicating that the modified 2216E medium has no algicidal effect on A. sanguinea. These results indicated that the fermentation time of immobilized beads can be greatly shortened during repeated batch fermentations, which is of great significance for increasing the productivity of the algicidal compounds.

The cell-free fermentation broth of H115 was collected and spray dried to get the fermentation powder. Figure 2 shows that the algicidal efficiency increased significantly with an increase in the concentration of fermentation powder (p < 0.05) at the concentration ranges of 0-1.0 g/L. The maximum algicidal efficiency (100%) was achieved when the concentration of the fermentation powder reached 1.0 g/L of algal culture. This could be explained by the dose-response relationship between algicidal compounds and algae cells, that is the maximal effect of algal cell lysis can be achieved at a certain dose of algicidal compounds (Tilney et al., 2014; Wang Y. et al., 2020). A similar trend was found in the study of Wang Y. et al. (2020), where the EC₅₀ and EC₉₀ values of algicidal compounds to A. sanguinea were 0.68 and 1.43 g/L, respectively. Karlodinium veneficum also exhibited a typical dose response in photochemical inhibition and cell density with increasing IRI-160AA, with an average EC₅₀ of 7.9% (v/v) IRI-160AA (Tilney et al., 2014). Using fermentation powder as core material, floating microcapsules were successfully prepared with SA as wall material and EC as the coating material. Floating microcapsules have been widely applied for the encapsulation and controlled release of the drugs in drug delivery systems (Ma et al., 2008; Ahmed et al., 2016; Selvakumaran et al., 2016). However, studies that explore the potential application of floating microcapsules in other areas are sparse. Yang and Wang prepared sodium alginate/hydroxypropyl methylcellulose (SA/HPMC) microcapsules that can float in hai-hong wine (Yang and Wang, 2014). Immobilized beads with algicidal bacteria (Kang et al., 2012; Jung et al., 2013; Wang and Coyne, 2020) were shown to improve the algicidal efficiency for the potential control of HABs. However, to the best of our knowledge, this is the first report of the preparation of floating microcapsules with active algicidal compounds. When compared with the algicidal bacterial based immobilized beads, the floating microcapsules containing algicidal powder have advantages. The active algicidal compounds in microcapsules released slowly and sustainedly from the microcapsules, which enables efficient contact between the active algicidal compound and algae.

The algicidal activity of the microcapsules was tested by co-incubating the microcapsules with A. sanguinea for up to 24 h. Results showed that the algicidal efficiency of the microcapsules increased significantly (p < 0.05) over time from 18.71 ± 1.37% (4 h) to 100% (24 h) (Figure 4), which were significantly higher (p < 0.05) than that of the control (microcapsules without algicidal powder). For the control, an algicidal efficiency of 15.44 ± 0.53% was observed after 24 h of incubation. These results indicated that the algicidal powder was slowly released into the algal culture through the pores of the microcapsule and then acted on the algal cells. At present, the immobilization of algicidal bacteria and their potential application in controlling HABs have been studied (Jung et al., 2013; Sun et al., 2015; Wang and Coyne, 2020). To our best knowledge, this is the first report on floating microcapsules based on algicidal powder. In our study, the floating microcapsules achieved a sustained release of the active algicidal compounds and facilitated sufficient contact between the algicidal powder and A. sanguinea.

Fv/Fm represents the photochemical efficiency of algal cells, and rETR represents the relative photosynthetic electron transport rate of algal cells. Figure 5 shows the changes of Fv/Fm and rETR during incubation. Fv/Fm in control groups experienced significant decreases over time (Figure 5A, p < 0.05). For the microcapsules treatment group, the Fv/Fm value decreased significantly from 0.3 ± 0.007 to 0.08 ± 0.005 (p < 0.05). The difference between these two groups was significant (p < 0.05). The changes of rETR over time during incubation for the control group was not significant (Figure 5B, p = 0.1). However, for the microcapsules treatment group, rETR decreased significantly over time from 53.76 ± 1.57 to 12.27 ± 0.56 (p < 0.05). The difference between these two groups was significant (p < 0.05). It is reported that lower Fv/Fm values infer inactivation or down-regulation of PSII reaction centers (Tilney et al., 2014). These results suggested that the addition of floating microcapsules had significant effects on the photosynthetic efficiency and capacity of A. sanguinea (Zhang et al., 2018). A similar phenomenon was observed for the algicidal bacteria Paracoccus sp. Y42 and Pseudoalteromonas S1 when co-incubated with the target algae (Sun et al., 2016; Zhang et al., 2018). Photosynthesis provides primary metabolites and energy for algae. It has been reported that some active algicidal substances inhibit the growth of algae by destroying the photosynthetic pigments, blocking the respiratory chain, and reducing the assimilation products (Tilney et al., 2014; Zhang et al., 2018). Under stress conditions, the photosynthetic apparatus and the transmission of photosynthetic electron of A. sanguinea could be affected (Zhang et al., 2020), thus significant fluctuations in Fv/Fm and rETR values could be observed. The inhibition of both Fv/Fm and rETR indicated that the algicidal compounds released from microcapsules disrupted the photosynthetic apparatus of A. sanguinea.