Karenia mikimotoi was obtained from the Laboratory of Microalgae Research, Ocean University of China, Qingdao, China. The axenic algae were cultured at 25°C in sterile f/2 medium (Lananan et al., 2013) prepared with 0.45-μm filtered natural seawater, with a light intensity of approximately 80 μmol photons m^–2 s^–1 under a 12 h:12 h light:dark cycle. The axenic culture of K. mikimotoi was tested by culturing on the plates and microscopy method. Cell numbers were counted using a hemocytometer under a light microscope (CX21FS1; Olympus, Tokyo, Japan).

The strain P. homiensis O-4 was previously isolated from seawater (Zheng et al., 2018; Ding et al., 2021). The 16S rRNA gene sequence of the strain was deposited in GenBank (MG457257). Seawater was obtained from Luxun Park, Qingdao, China. The bacterial strain was cultured in 2216E agar medium (peptone 5 g, yeast extraction 1 g, ferric phosphorous acid 0.1 g, agar 10 g, pH 7.6–7.8, fixed capacity to 1 L using sterile seawater) at 25°C for 72 h. For the algicidal test, 1-, 3-, and 5-ml amounts of the bacterial solutions (with volume ratios of 1%, 3%, and 5%, respectively) were each inoculated into the 100-ml flask containing exponentially growing K. mikimotoi algal cultures. The algal cells were fixed with Lugol’s iodine. The algicidal activity was monitored by counting the cell numbers using a microscope. Algicidal activity by O-4 was calculated according to the following equation: algicidal activity (%) = (1–Tt/Ct) × 100%, where T and C are the concentrations of algal cells in the treatment and control groups, respectively, and t is the incubation time. All experiments were performed in triplicate.

To assess the mode of action in the algae inhibition of K. mikimotoi, the cell-free filtrate was used: the bacterial culture after 72-h cultivation was centrifuged at 15,000 × g for 10 min at 4°C and passed through a 0.22-μm membrane filter (Merck Millipore, Darmstadt, Germany). The remaining cell pellets in the bottle were washed twice with a sterile 2216E medium. The cells were resuspended in f/2 medium with the same concentration (3%), shaken, and then labeled as O-4 cells. The control group comprised an algae culture supplemented with a 3% sterile 2216E medium.

The contents of chlorophyll a (Chl a) and carotenoid (Car) were analyzed after the 3 and 5% strain O-4 treatment. Algal cells were collected via centrifugation (5,000 rpm, 20 min) after 0, 4, 8, 12, 24, and 48 h of culture. Algal pigments were extracted using 85% acetone solution in the dark at 4°C for 24 h, followed by centrifugation (5,000 rpm, 10 min). The absorbance of the supernatant was measured at the wavelengths of 470, 645, and 663 nm (Gan and Lian, 2021). The absorbance of 85% acetone was used as the control. The formula of photosynthetic pigment is as follows:

where Ca, Cb, and Cc represent the concentrations of chlorophyll a, chlorophyll b, and total carotenoid.

The maximum photochemical quantum yield of photosystem II is Fv/Fm, representing the maximum photosynthetic potential. The chlorophyll fluorescence parameters of the treated algae cells were measured using a water pulse amplitude modulation chlorophyll fluorescence analyzer (Walz, Germany). Algal cells were dark-adapted for 15 min before the experiment. The algal fluorescence was detected using a measuring light (0.01 μmol photons m^–2 s^–1) with a saturation pulse (0.8 s, 3,500 μmol photons m^–2 s^–1). The maximum photochemical quantum yield of the photosystem (Fv/Fm) was used to indicate the efficiency in light energy conversion during photosynthesis.

The intracellular ROS level within K. mikimotoi was measured using a ROS detection kit (Biyuntian, Shanghai, China) with slight modifications. The methods were as follows: (1) after 4, 8, 12, 24, 36, and 48 h of culture, 40 ml of algal solution was centrifuged at 4°C, and the supernatant was immediately discarded to collect algal cells; (2) DCFH-DA probe dye was added, and the mixture was incubated at 37°C for 30 min; (3) the mixture was centrifuged (at 1,000 rpm, 5 min), and the algal cells were washed with PBS; (4) the mixture was centrifuged again to settle the solution, and the supernatant was discarded to obtain the algal cells; (5) after resuspending the cells using PBS, the fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a flow cytometer (Novocyte 2040R, ACEA, United States).

Bacterial filtrates of O-4 were inoculated in the exponential phase axenic K. mikimotoi cultures until the concentration of the bacterial solution reached 3 and 5%. An axenic 2216E medium of the same volume was added separately to act as a control. After co-culture for 0, 6, 12, 24, and 48 h, algal cells were collected using centrifugation (10,000 rpm, 20 min), followed by washing with PBS (50 mM, pH 7.4). The cell disruption was assessed using an ultrasonic cell disruption system (200 W, 5 s; 10 s, five times at less than below 4°C). The extracting solution was centrifuged for 15 min at 10,000 × g, and the supernatant was used in the cell membrane permeability analysis. Lipid peroxidation levels were measured by assessing the malondialdehyde level following the methods by Dogru et al. (2008).

The crude protease solution was also obtained as described previously (Ding et al., 2018). After co-culture for 0, 6, 12, 24, and 48 h, the algal cell suspension was centrifuged at 4°C for 20 min (10,000 rpm min^–1). The supernatants were discharged, and the algal cells were collected. The algal cells were washed with PBS (0.05 mol L^–1, pH 7.8) and transferred to a test tube. Under ice-bath conditions, the algal cells were crushed by ultrasound for 5 min (5 s, interval of 10 s, 200 W). An amount of 1.5 ml of supernatant was absorbed into the Eppendorf tube and centrifuged again at 4°C for 15 min. The supernatants obtained after centrifugation comprised the crude protease solution to be measured. The SOD, CAT, and POD activities in the algal cells were measured following the manufacturer’s instructions (Jiancheng, Nanjing, China). Glutathione peroxidase was measured using a GPx assay kit (Biyuntian, Shanghai, China).

Glutathione (GSH) was determined following the instructions in a GSH assay kit (Jiancheng, Nanjing, China). Glutathione oxidized (GSSG) was measured using a GSSG assay kit (Solarbio, Beijing, China). Ascorbic acid (AsA) and dehydroascorbic acid (DHA) were determined via the method described in Ding et al. (2018) as follows. Supernatants of 200 μl were prepared (as described above), then 200 μl of dithiothreitol (5 mmol L^–1) and 500 μl of potassium phosphate of buffer solution (100 mmol L^–1, pH 7.4) were added to the test tube, and the mixture was incubated at 25°C for 10 min. Then, 100 μl of N-ethylmaleimide, 400 μl of trichloroacetic acid (0.61 mol L^–1), 400 μl of phosphoric acid (0.8 mol L^–1), 400 μl of 2,2’-bipyridine (0.5 mol L^–1), and 200 μl of ferric chloride (3 mol L^–1) were added to the mixture. They were set in the water bath and incubated at 55°C for 10 min. The absorbance of each tube was measured at a wavelength of 525 nm. The total ascorbic acid content could then be calculated. In the above process, the dithiothreitol and N-ethylmaleimide were not added, and alternatively, the double steaming water and the reduced ascorbic acid content could be measured. The DHA content was the difference between total AsA and AsA content.

The data were presented as means, and their standard errors were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL, United States).

The effects of O-4 on the growth of K. mikimotoi are presented in Figure 1. The results indicate that the algicidal activity by strain O-4 against K. mikimotoi cells was concentration-dependent. When compared to the control group, the addition of 1% O-4 exhibited no algicidal activity (p > 0.05), and within 120 h, the growth of K. mikimotoi slightly increased. In the treatment groups receiving 3 and 5% O-4 bacterial culture, there was significant growth inhibition (p < 0.05). The 3% O-4 caused 83% of the cells to be lysed after the treatment duration of 48 h. In the 3% O-4 treatments, 94% of the cells were lysed after 120 h. The 5% bacterial concentration of O-4 exhibited the strongest algicidal activity, leaving no visible intact algal cells after 120 h. The results suggest that the algicidal effects are enhanced with an increased bacterial concentration and treatment duration. Due to the strong algicidal activity in the 3 and 5% O-4 bacterial cultures, they were used in the subsequent stages of our evolving research program.

The bacterial cells and cell-free filtrate were separated and inoculated into K. mikimotoi cultures to explore the action of the algicidal mode of O-4. The results are provided in Figure 2. The additional bacterial cells did not significantly inhibit the growth of K. mikimotoi. However, both the cell-free filtrate and bacterial culture caused a significant inhibitory effect on K. mikimotoi (p < 0.05). The cell-free filtrate of O-4 reduced the algal cell density, and the algicidal activity against K. mikimotoi was 88.6% after 60 h. These results imply that the algicidal activity by O-4 is indirect.

To investigate the stress caused by O-4 on the algae, we determined the Chl a, carotenoid content, and maximum quantum yield of photosystem (PS) II [variable fluorescence (Fv)/maximum fluorescence (Fm)]. Figures 3A,B demonstrate that the pigment contents of the algal cells treated with O-4 were significantly lower than that in the control group after 48 h (p < 0.01). After 12 h, the contents of both Chl a and carotenoids in the 3% O-4 treatment groups decreased to 32% (Chl a) and 57% (carotenoids), respectively, compared to the control. After 48 h, the pigment content was reduced by approximately 86% (Chl a) and 60% (carotenoid) compared to the control. Moreover, after 48 h, the reduction in Chl a and carotenoid contents in the 5% O-4 treatment groups was approximately 90% (Chl a) and 91% (carotenoids) with respect to the control. These results indicate that O-4 is capable of damaging pigments in algal cells.

The Fv/Fm ratio was evaluated to determine the photosynthetic status of the algal cells after the 3 and 5% O-4 treatments (Figure 4). The Fv/Fm values decreased significantly relative to untreated cells after 12 h (p < 0.05). As the treatment duration progressed, the Fv/Fm values continued to decrease. At 48 h, when compared to the Fv/Fm values of the control group, the 3% treatment group was 3.1-fold lower (p < 0.05), and the 5% treatment group was 6.2-fold lower (p < 0.01. These results demonstrate that the photosynthetic capacity was inhibited in the treated cells (Figure 4).

An ROS level analysis explored the oxidative stress caused by O-4 in K. mikimotoi cells. Figure 5 shows a slight increase in DCF fluorescence intensity in the control group. Conversely, the DCF fluorescence intensity was significantly increased in algal cells treated with O-4 (p < 0.05, Figure 5). The ROS levels were significantly increased after 8 h of exposure in both treatment groups containing O-4, with ROS levels 3.8-fold (3% O-4) and 4.8-fold (5% O-4) higher than the control group. However, the ROS levels in both treatment groups began decreasing after 12 h of exposure, and at 48 h, the ROS content of algal cells in both treatment groups was maintained at a low level compared to the control.

Figure 6 illustrates the effects of O-4 on lipid peroxidation in the algal cells. Algae cells exposed to O-4 exhibited a pronounced increase in MDA content. The MDA levels in all the treated groups were higher than those in the controls (p < 0.05), and MDA content increased with exposure duration and increased concentrations of O-4. At 48 h, the MDA levels were 2.4 (3% O-4 group) and 3.5 (5% O-4 group) times higher than the control group.

We investigated the physiological defense responses induced by exposure to the algicidal O-4 by determining the representative enzymatic activities within cells, including SOD, CAT, POD, and GPx (Figures 7A–D). Figure 7A demonstrates that SOD activity increased as the exposure duration increased relative to the control in all treatment groups (p < 0.05). The SOD activity initially decreased slightly until 48 h of exposure in the 5% group. The CAT values were significantly higher in the 3% O-4 treatment groups when compared to the control (Figure 7B); the values were 1.2-fold higher after 12 h, 1.78-fold after 24 h, and 6.36-fold after 48 h (p < 0.05). The CAT values were significantly higher in the 5% O-4 treatment groups when compared to the control; the values were 1.51-fold higher (p < 0.05) after 12 h, 4.82-fold (p < 0.05) after 24 h, and 17.0-fold after 48 h (p < 0.01). The POD values of 3% O-4 treatment group were slightly lower than the control groups after 6-h and 12-h exposure but increased significantly with exposure duration and additional ratios (Figure 7C), reaching a peak at 48 h (p < 0.05). The GPx activity had similar results to the POD activity (Figure 7D). When algal cells were exposed to 3 and 5% O-4 bacterial cultures for 48 h, the activity values were 2.79 and 3.67 times higher than the control, respectively (p < 0.05).

Antioxidant non-enzymatic activities (including GSH, GSSG, AsA, and DHA) were assayed to analyze the algal cell protective responses against O-4 (Figures 8A–D). Figure 8A shows that GSH was stimulated by O-4 (p < 0.05). The only exception was the 3% treatment group after 12 h. The GSH content in the treatment groups compared to the control groups increased from 498 to 634 μmol L^–1 (3%) and 511 to 909 μmol L^–1 (5%) within 48 h. The GSSG contents of the algae in both treatment groups increased significantly with increasing O-4 concentrations and treatment duration and remained higher than the control group over 48 h (p < 0.05, Figure 8B). The AsA levels in K. mikimotoi cells were similar to the GSSG results (Figure 8C). At 48 h, the AsA levels were 1.5-fold (3% O-4) and 1.66-fold (5% O-4) higher than the control group. Figure 8D graphically demonstrates the effects of the strain O-4 on DHA in K. mikimotoi cells. After 12 h, the 3% O-4 group exhibited a stimulatory effect on DHA. Within 48 h, there was a significant inhibitory effect on the DHA in the 5% O-4 treatment group (p < 0.05).