Introduction

The proliferation of Microcystis aeruginosa leads to bloom and adversely affects the environment. Plants can control the excessive reproduction of M. aeruginosa by releasing allelochemicals (Xiang et al. 2015; Wang et al. 2017a, b, Qian et al. 2018). When evaluating the allelopathic potential of Pontederia cordata to M. aeruginosa, we found that small amounts of P. cordata planting water that had not been filtered to remove microorganisms could quickly kill M. aeruginosa, indicating the possible presence of algae-killing organisms in the planting water.

Algae-inhibiting organisms include mainly viruses, bacteria, microfungi, amoebae, ciliates, eukaryotic microalgae, and protozoa (Van Wichelen et al. 2016). Among them, there have been fewer studies on fungi and viruses, and most studies are on algae-inhibiting bacteria. Most of these bacteria inhibit the growth of algae by releasing chemicals, and a high density of bacteria is often required to noticeably inhibit algal growth (Guo et al. 2015; Lin et al. 2016). Myxobacteria can lyse algae cells directly when they come into contact, but the living conditions of myxobacteria are relatively harsh (Shilo 1970; Daft et al. 2010). Phagocytosis of cyanobacteria by protozoan ciliates and amoebae has also been reported (Van Wichelen et al. 2016), while phagocytosis by flagellates, mainly mixotrophic flagellates of the Chrysophyta and Cryptophyta, has been reported even more often (Zhang and Watanabe 2001; Touloupakis et al. 2016; Princiotta et al. 2019).

Mixotrophic flagellates have strong environmental adaptability, grow rapidly, can feed a variety of algae, can degrade Microcystins, and favor more diverse phytoplankton community (Baek et al. 2009; Wilken et al. 2010; Ma et al. 2018a, b; Zhang et al. 2020). Flagellates can prey on algae cells, resulting in a rapid decrease in their biomass, while bacteria tend to inhibit the growth of algae cells. Flagellates are also important predators of bacteria (Isaksson et al. 1999, Boenigk and Arndt 2002). In the presence of a large number of flagellates, bacteria are unlikely to reach a density that can inhibit M. aeruginosa.

In nature, plants and microorganisms always coexist and influence each other. Plants affect the community structure of the surrounding microorganisms by releasing organic carbon and allelochemicals. Studies have found that more algae-lysing bacteria can be isolated from water bodies with aquatic plants than from water bodies without aquatic plants, indicating that plants may stimulate the growth of algae-lysing bacteria (Kojima et al. 2016; Sakami et al. 2017). The presence of benthic fauna also stimulates plants to produce more allelochemicals, thus enhancing their allelopathic and algae-inhibiting ability (Zuo et al. 2015, 2016). Therefore, the main purpose of this article is to isolate and identify algae-inhibiting microorganisms and to study their growth and feeding characteristics. This will lay the foundation for further research on the synergistic inhibition of flagellates and allelochemicals and the evaluation of the effects of plants on the growth of flagellates.

Methods and materials

Experimental materials

The toxin-producing cyanobacteria M. aeruginosa (FACHB-915) used in the experiment were provided by Freshwater Algae Culture Collection at the Institute of Hydrobiology, China.

Obtaining of P.cordata planting water

P.cordata was collected from the water body of the Sculpture Park in Wuhu City (31.34°N, 118.41°E). The P.cordata collected in the field was washed, and three stalks (wet weight 118.41 ± 30 g) were planted in 1 L beakers with water culture baskets. They were respectively added to 500 mL BG11 medium and cultured in light incubators for 20 days. The planting water was merged and filtered to remove physical particles. Use 100 mL of 10 times concentrated BG11 medium to supplement nutrition, and then use ultrapure water to make the volume to 1.5 L for use.

Comparison of algae inhibition by allelochemicals and microorganisms in planting water

A sterile 0.22 μm cellulose acetate membrane filter was used to filter the planting water. The filtrate containing only allelochemicals made up the allelochemicals treatment (group A), and the organisms remaining on the filter membrane made up the biological treatment (group B). The unfiltered planting water was used as a mixed treatment (group A + B) that contained both allelochemicals and organisms. Twenty milliliters of planting water from the different groups was added to sterilized Erlenmeyer flask of 50 mL. Additionally, 20 mL BG11 medium was added to an Erlenmeyer flask as the control group (CK). M. aeruginosa suspension (5 mL) with a density of 1 × 106 cells/mL was added to each group above. Three replicates were set up for each group and cultured in a light incubator with a light intensity of 2000 Lux and a temperature of 25 ± 1 °C. The cell density of M. aeruginosa was detected every 24 h, and the inhibition rate was calculated.

M. aeruginosa contains phycocyanin, which has its largest fluorescence peak at 650 nm. Fluorescence is not influenced by bacteria or P. malhamensis. The fluorescence intensity(y) has a good linear relationship with the density of M. aeruginosa (y = 0.554x − 0.858 R2 = 0.998) and can be used to quickly characterize the biomass of M. aeruginosa (Bastien et al. 2011). The fluorescence intensity was detected by excitation (EX) light 610 and emission (EM) light 650 with a microplate reader (Spark 10 M, Tecan, Switzerland).

The inhibition rate was calculated as follows: IRt (%) = (N0-Nt)/N0 × 100, where t is time, IRt is inhibition rate at day t, Nt is the mean value of EM 650 of M. aeruginosa in the treatment group at day t, and N0 is the mean value of EM 650 of M. aeruginosa in the control group at day t (Qian et al. 2019).

Isolation and identification of predatory flagellate based on cell morphology and phylogenetic analysis

After the algae inhibition experiment, the liquid was concentrated by centrifugation, and a large number of swimming flagellates were observed. Using the gradient dilution separation method, the flagellates were diluted to one cell per drop with M. aeruginosa in the logarithmic phase (3 × 105 cells/mL), cultured in a 96-well plate under illumination, and observed and shaken gently every day. After a period of co-cultivation, if the liquid in the well became transparent, it indicated that M. aeruginosa had been preyed upon and that was a monoclonal algicidal microorganism in the well. In contrast, if M. aeruginosa grew normally, the liquid in the well remained green. The purified flagellates were subjected to the above process, but M. aeruginosa was replaced with autoclaved wheat grain medium. Finally, the purified flagellates were extracted and preserved in wheat grain medium for later experiments.

The purified flagellate was observed and photographed under the light microscope (Leica DMi8) and transmission electron microscope (TEM). The TEM methods were essentially the same as those described in (Gong et al. 2015).

Purified flagellates were sent to be sequenced (Sangon, China). DNA was extracted and amplified using universal forward primer SSU-F (5′-ACCTGGTTGATCCTGCCAGT-3′) and reverse primer SSU-R (5′-TCACCTACGGAAACCTTGT-3′) (Wang et al. 2006). For the temperature cycling, pre-denaturation at 94 °C for 4 min was followed by 30 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min, and finally an elongation at 72 °C for 10 min. Products of polymerase chain reaction were purified ligated into a pMD ®18-T vector transformed into competent cell ligation product transformation, blue-white screening, plasmid extraction, and sequencing. The sequences were submitted to GeneBank (Accession No. MN022220.1). Alignment and arrangement were conducted by MEGA 7.0; the phylogenetic tree was constructed with 26 sequences of representative chrysophytes by the neighbor-joining method.

Growth of P. malhamensis in different media

P. malhamensis growth was studied in the following mediums. (1) M. aeruginosa was initially cultivated on BG11 medium. (2) The logarithmic phase of M. aeruginosa: M. aeruginosa was cultivated for 6 days, starting with an initial density of 1.5 × 106 cells/mL (3) The decline phase of M. aeruginosa: M. aeruginosa was cultivated for 30 days and then diluted to 1.5 × 106 cells/mL. (4) Culture solution of M. aeruginosa: M. aeruginosa cultures in the logarithmic growth phase were collected by centrifugation at 4000 rpm for 5 min. The supernatant was collected and filtered with a 0.22 μm filter membrane for later use. (5) Wheat grain medium: One hundred wheat seeds were added to 1 L ddH2O, autoclaved and saved for later use.

P. malhamensis was inoculated onto the above mentioned growth media at 50 mL, and the initial density reached 1 × 105 cells/ml. Three replicates were set up for each group and cultured in a light incubator with a light intensity of 2000 Lux (light: dark = 14:10) and a temperature of 25 ± 1 °C.

One milliliter of the culture liquid was sampled every 24 h and fixed with Lugol’s solution. The density of P. malhamensis cells was counted by a hemocytometer, and the specific growth rate was calculated as follows: μ = ln (Mt/M0)/t, where M0 and Mt represent the cell density (cells/mL) of P. malhamensis at the beginning and at time t, respectively (Ma et al. 2018a, b).

Effects of environmental factors on the growth and feeding behavior of P. malhamensis

Environmental factors including light intensity, temperature, and the pH values of the growth medium were tested as follows:

  1. (1)

    Light intensity experiment: Three different light intensities were tested: 0 Lux, 2000 Lux, and 10,000 Lux, with a light period of 14 h:10 h (light: dark). The experiment was conducted in three light incubators (PRX-350B, Saifu Apparatus, China). During the cultivation, the temperature was 25 ± 1 °C, and the medium pH was not adjusted artificially.

  2. (2)

    Temperature experiment: three different temperatures of 20 ± 1 °C, 25 ± 1 °C, and 30 ± 1 °C were tested. The experiment was conducted in three temperature-controlled incubators. The light intensity was 2000 Lux and a photoperiod of 14 h:10 h (light: dark). Medium pH was not artificially adjusted during the cultivation.

  3. (3)

    Medium pH experiment: four different medium pH values of 6.0 ± 0.2, 7.0 ± 0.2, 8.0 ± 0.2, and 9.0 ± 0.2 were tested. Medium pH was measured and adjusted every day with 1 mM HCl or 1 mM NaOH, as appropriate, using a pH meter (PHS-3C, Leici, China). The light intensity was 2000 Lux and a photoperiod of 14 h:10 h (light: dark). The temperature was 25 ± 1 °C.

For each treatment, the co-culture was incubated in triplicate 100 mL flasks with 50 mL M. aeruginosa in the logarithmic growth phase. Initial inoculation concentrations of P. malhamensis and M. aeruginosa were 1 × 104 cells/mL and 1–3 × 106 cells/m L. The cell densities of M. aeruginosa and P. malhamensis were detected every 24 h, with reference to 2.2 and 2.4.

Effects of M. aeruginosa growth phase and density on P. malhamensis growth and grazing

P. malhamensis feeding on M. aeruginosa in different growth phase

M. aeruginosa cultures employed were brought to exponential phase in continuous cultures. M. aeruginosa cultures in the decline phase were cultured for 1 month. During the experiment, the pH of the exponential phase culture was measured to be 9. In order to eliminate the influence of pH on grazing, the initial pH of the culture in the decline phase was adjusted to 9 and 7 with 1 M hydrochloric acid. P. malhamensis was inoculated into the above three algal suspensions (50 mL) at a density of 1 × 104 cells/mL. The experiments were carried out in a light incubator as described in “Comparison of algae inhibition by allelochemicals and microorganisms in planting water.” Three replicates were set up for each group, and the biomass of M. aeruginosa was measured every day.

Effects of M. aeruginosa density on the growth and grazing of P. malhamensis

M. aeruginosa in the logarithmic phase was centrifuged at 4000 rpm, and the precipitated cells were resuspended in BG11 to an initial algal density of 5 × 107 cells/mL. Then, M. aeruginosa suspensions were prepared at densities of 5000, 2500, 1250,625, 312, 156, and 78 × 104 cells/mL by twofold gradient dilution. The 50 mL M. aeruginosa suspensions mentioned above were combined with P. malhamensis at a density of 2 × 104 cells/mL. The initial density ratios of M. aeruginosa to P. malhamensis were 2500:1, 1250:1600:1300:1150:1, 70:1, and 35:1. The densities of M. aeruginosa and P. malhamensis were detected every 2 days after co-culturing for 8 days. The culture conditions are the same as those described in “Comparison of algae inhibition by allelochemicals and microorganisms in planting water.”

Statistics

The significance test was carried out by one-way ANOVA by IBM SPSS Inc.22.

Results

Comparison of inhibitory effects of allelochemicals and microorganisms in planting water

Figure 1 shows that the allelochemicals and microorganisms in the planting water had a good inhibitory effect on algae. The mixture of allelochemicals and microorganisms inhibited algae faster than allelochemicals or microorganisms alone. With the extension of the culture time, the inhibitory effect of microorganisms on algae became stronger than that of allelochemicals. After 3 days of culture, the inhibition rate of microorganisms was 86.35%, the inhibition rate of allelochemicals was only 48.25%, and that of the mixed group was 97.47%, indicating that there may be a synergistic algae inhibition effect between allelochemicals and microorganisms.

Fig. 1
figure 1

Inhibition effect of allelochemicals and microorganisms on algae in planting water. Significant differences are indicated by different lowercase letters (one-way ANOVA, P < 0.05). Error bars represent the standard errors of the mean, n = 3. The following is the same

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Identification of an unknown predatory flagellate

A monoclonal flagellate was successfully isolated and purified. Most flagellates are spherical or oval, and their cells are deformable. Most flagellates are 10–12 μm in width, but some of the newly divided offspring are narrower. After phagocytosis of M. aeruginosa, the cells were irregularly spherical and could be enlarged. The flagellate isolated in this study had two flagella of different lengths, and the long flagellum was approximately 1–1.5 times the length of the body. Most flagellate cells contained two peripheral flaky yellowish pigmented components. In the grazing experiments, we did not observe the formation of M. aeruginosa colonies, but we often observed the formation of flagellate colonies. In addition, round objects without cellular structures were often observed, and they tended to cluster together (Fig. 2).

Fig. 2
figure 2

a–c Cells fixed with Lugol’s solution in order to more easily photograph the flagella; c shows a dividing cell. d Living cells of different shapes and sizes. e–g Phagocytosis and digestion of M. aeruginosa. h, i Round objects with no cell structure. j Flagellate clusters. Bar = 5 µm

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Predation and digestion

Under the microscope, we observed flagellates swimming around the M. aeruginosa cells and touching them with a flagellum. The flagellates moved the water by vibrating and rotating their flagella, and the cells of M. aeruginosa followed the currents alongside the flagellate cells. Then, the M. aeruginosa cells rotated rapidly between the two flagella and were brought into the flagellates by phagocytic vacuoles. The cell wall and cell membrane of the ingested M. aeruginosa cells were separated, the cell contents were gradually absorbed by the flagellates, and the cell wall shrank into small black particles (Fig. 3).

Fig. 3
figure 3

a–c The ultrastructure of the unknown flagellate and the digestion process of M. aeruginosa. d–f The digestion process. e is a local magnification of d in which the cell wall and contents of M. aeruginosa began to separate (arrow). f The cell wall and protoplast of M. aeruginosa in the digestive vesicle had become seriously separated, and the protoplast shrank into a mass, indicating that digestive enzymes had hydrolyzed and digested the cell contents through the cell wall. FV (food vacuole), CP (chloroplast), MT (mitochondria) N (nucleus), M (Microcystis aeruginosa), Scale bars, a,d,f = 1 μm; b,c,e = 500 nm

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To further confirm the morphological results, the predatory flagellate was also characterized by the gene sequence of its 18S rDNA. The 18 s sequence length is 1661 bp, NCBI accession number: MN022220.1. Blast results showed that it was 99.46% identical to that of Poterioochromonas malhamensis SAG933.1c (Gene Bank No., EF165114). The coverage is 100% (Fig. 4). So the isolated flagellate can be identified as Chrysophyceae, Ochromonadales, and Ochromonadaceae Poterioochromonas malhamensis yzs924.

Fig. 4
figure 4

The 18S rDNA sequence phylogenetic tree constructed using the neighbor-joining method indicates the evolutionary status of this flagellate (Poterioochromonas malhamensis), the number on the branch point indicates bootstrap support values, and the scale indicates the genetic distance

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The growth of P. malhamensis in different media

Fig. 5 shows that P. malhamensis rapidly proliferated in the wheat grain medium. The density increased from the initial 1 × 105 cells/mL to 1.33 × 106 cells/mL in 1 day. The generation time was 5–6 h, and the growth rate reached 2.56 day−1. After 4 days of inoculation, the growth rate and generation time gradually decreased, and the growth rate was 0.71 day−1 at 5 days.

Fig. 5
figure 5

Growth rate of P. malhamensis in different growth media. BG11 BG11 medium, CS culture solution in the logarithmic growth phase of M. aeruginosa, DP the decline phase of M. aeruginosa, LP the log phase of M. aeruginosa, WM wheat grain medium

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The growth rates of P. malhamensis in the logarithmic phase (LP) and the decline phase (DP) were not significantly different, indicating that M. aeruginosa in different growth states had no effect on the growth of P. malhamensis. P. malhamensis could also grow in M. aeruginosa culture (cell-free) and reached a density of 2–4 × 105 cells/mL The growth rate was 0.26/day, close to that in the DP and LP groups.

In the first 2 days after inoculation into BG11 medium, the P. malhamensis cell density decreased and then gradually increased. After 1 month, the cell density had reached 6 × 105 cells/mL, and the specific growth rate was 0.06 d−1. P. malhamensis was stored in sterilized wheat grain medium and grew in heterotrophic mode; in contrast, BG11 medium lacks organic nutrients, which caused part of the P. malhamensis to die. After 2 days, P. malhamensis gradually changed from organic heterotrophic mode to autotrophic mode, at which point the cell density began to increase again.

Effects of light, temperature, and pH on the growth and grazing of P. malhamensis

As shown in Fig. 6a, P. malhamensis grazed on M. aeruginosa under both light and dark conditions. Under dark conditions, the removal of M. aeruginosa was faster than that under light conditions in the first 2 days but did not continue after the second day. Under light conditions (2000 and 10,000 Lux), the removal of M. aeruginosa was slower in the first 2 days but continued after the second day. After 4 days, the biomass of M. aeruginosa had been reduced to a very low level, and M. aeruginosa cells could hardly be found under the microscope. Figure 6b shows that there were no significant differences in the biomass of P. malhamensis under 2000 and 10,000 Lux illumination. However, the biomass of P. malhamensis under light conditions was significantly higher than that under dark conditions. In summary, different light intensities had no significant effect on the growth or grazing of P. malhamensis, but continuous dark conditions were not conducive to the growth and grazing of P. malhamensis.

Fig. 6
figure 6

Effect of light intensity (a, b), temperature (c, d), and pH (e, f) on the growth and grazing of P. malhamensis

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As shown in Fig. 6c, the removal of M. aeruginosa at different temperatures was similar at 1 day and 4 days, but the removal of M. aeruginosa was the lowest at 20 °C at 2 days and 3 days. As shown in Fig. 7d, the biomass of P. malhamensis at 30 °C was higher than that at 20 °C and 25 °C. Therefore, 30 °C was the temperature that was the most beneficial to P. malhamensis and growth and grazing.

Fig. 7
figure 7

Measured pH values in the different groups on each day

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Figure 6e shows that when the pH was maintained at 9, the biomass of M. aeruginosa gradually increased and could not be reduced, indicating that P. malhamensis may not be able to graze or digest M. aeruginosa at pH 9. When the pH was controlled at 6, 7, or 8, P. malhamensis grazed M. aeruginosa, and the grazing speed decreased with increasing pH value. As shown in Fig. 6f, the density of P. malhamensis decreased with increasing pH on the 2nd day, but the differences in P. malhamensis density were not significant among pH values of 6, 7, and 8 after the 3rd day, possibly because of food restrictions. When the pH was maintained at 9, P. malhamensis was almost undetectable under the microscope.

As shown in Fig. 7, when the pH was controlled at 6, the daily measured pH value increased to approximately 7, and when the pH was controlled at 9, the measured pH was reduced to approximately 8, indicating that the pH tended to adjust to reach similar values (7 and 8). In summary, a low pH is more beneficial to the growth and grazing of P. malhamensis. When the pH is continuously controlled at 9, P. malhamensis cannot survive.

Effects of M. aeruginosa growth phase and density on P. malhamensis growth and grazing

As shown in Fig. 8, M. aeruginosa in the logarithmic growth phase could be grazed, and its biomass was reduced to an extremely low value, but M. aeruginosa in the decline growth phase was not easily grazed by P. malhamensis. The biomass of M. aeruginosa in the decline phase decreased in the first 3 days but no longer decreased after three days.

Fig. 8
figure 8

Effect of M. aeruginosa growth phase on the grazing of P. malhamensis

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Though M. aeruginosa in the decline phase could not be completely grazed by P. malhamensis, the initial pH value affected the final density of M. aeruginosa. Compared with that in the initial pH 9 group, the M. aeruginosa biomass in the initial pH 7 group decreased faster and eventually remained at a lower level; this may have occurred because low pH is conducive to the growth and grazing of P. malhamensis (Fig. 6e). The results in Fig. 6f show that P. malhamensis could not grow at pH 9, but the pH had to be continuously artificially maintained at 9. As Fig. 7 shows, the pH of the culture decreased to approximately 8 when the initial pH was 9. Therefore, adjusting the initial pH to 9 does not inhibit P. malhamensis feeding on M. aeruginosa.

As shown in Fig. 9a, when the initial density of M. aeruginosa was lower than 2.5 × 107 cells/mL, the density of M. aeruginosa gradually decreased. The M. aeruginosa density was reduced to extremely low values after 7 days in all experimental treatments except the S0 group.

Fig. 9
figure 9

Population dynamics of M. aeruginosa (a) and P. malhamensis (b) in different inoculation proportions (M. aeruginosa: P. malhamensis) with the same concentration of P. malhamensis (1 × 104 cells/mL). The M. aeruginosa densities were as follows: S0: 5 × 107 cells/mL; S1: 2.5 × 107 cells/mL; S2: 1.25 × 107 cells/mL; S3: 6.25 × 106 cells/mL; S4: 3 × 106 cells/mL; S5: 1.50 × 106 cells/mL; S6: 7.5 × 105 cells/mL

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The initial density of M. aeruginosa was 5 × 107 cells/mL; then, it was diluted by 50%. However, the density of P. malhamensis obviously did not decrease by 50% (Fig. 9b).

In the S0 group, the density of P. malhamensis first increased and then decreased after the 4th day. Initially, the high-density M. aeruginosa provided ample food for P. malhamensis, causing the flagellate population to grow rapidly. After 4 days, the density of the flagellate in this group gradually decreased. One possible reason for this decrease is that the pH (Fig. 6) or growth phase (Fig. 8) of M. aeruginosa changed, which caused grazing resistance in P. malhamensis. As a result, the density of M. aeruginosa during this period showed almost no reduction (Fig. 9a).

Comparing the density of P. malhamensis among the different groups revealed that the differences between S2, S3, S4, S5, and S6 were larger than the differences between S3, S4, S5, and S6. In the S6 group, the initial density of M. aeruginosa was only 7.5 × 105 cells/mL, but the density of P. malhamensis still reached 2.05 × 106 cells/mL. This result shows that the density of P. malhamensis is less affected by M. aeruginosa densities within the ranges in the S3–S6 groups.

During the experimental period, the density of P. malhamensis in the S1 group increased gradually. In addition, the density of P. malhamensis in the S1 group was much higher than that in the S2–S6 groups because the S1 group had sufficient food, and M. aeruginosa exhibited no resistance to grazing by P. malhamensis.

Discussion

Caron et al. (1993) studied the effect of light on the uptake rate of Dinobryon cylindricum. The results showed that the uptake rate of algae at light intensities > 150 μEm−2 s−1 was approximately 5–10 bacteria alga−1 h−1, while that of D. cylindricum cultured under 30 μEm−2 s−1 continuous light was consistently low (1.8 bacteria alga−1 h−1). D. cylindricum cultured in the dark quickly stopped ingesting bacteria. In contrast, the results of this paper (Fig. 6a) show that in the first 2 days of the experiment, P. malhamensis ingested M. aeruginosa faster in the dark than in the light; that is, light had a negative effect on the feeding rate. Holen (1999) found that in 120 min after adding bacteria, light had a negative effect on the feeding rate of Poterioochromonas malhamensis on bacteria. After incubation for 15 min under high-intensity light, 29% of flagellates ingested FLB (fluorescently labeled bacteria), while under low light, 39% of flagellates ingested FLB; 53% of flagellates ingested FLB in the dark.

P. malhamensis could not continue to graze on M. aeruginosa under dark conditions in the last two days of this experiment, while P. malhamensis did continue to graze on M. aeruginosa under light conditions. These results indicate that the feeding and digestion processes of P. malhamensis on M. aeruginosa may depend on light or on photosynthetic products. Zhang and Watanabe (2001) found that phagotrophic populations of P. malhamensis were incapable of growth in continuous darkness for longer than 5 days and were light-dependent for phagotrophy. Li et al. (2000) found that phagocytosis by the dinoflagellate Gyrodinium galatheanum (Braarud) Taylor might be light-dependent and that photosynthesis-derived metabolites were needed to support its growth. Strom (2001) found that relative to complete darkness, bright light (900 µmol photons m−2 s−1) resulted in a 40-fold increase in food vacuole loss rates (a proxy for digestion) in the heterotrophic dinoflagellate Noctiluca scintillans when fed phytoplankton prey. However, light had no effect on the vacuole loss rate when N. scintillans was fed heterotrophic (nonpigmented) prey. One possible mechanism to explain this difference is the photo oxidative breakdown of ingested organic matter in these nearly transparent grazers. Klein et al. (1986) measured algal pigments during grazing experiments with the protozoan Oxyrrhis marina Dujardin on Rhodomonas sp. and found that the rates of chlorophyll and carotene degradation were the highest in the light.

Can predatory flagellates live without light? Holen (1999) found that Poterioochromonas malhamensis could grow in DYIV + glucose medium without light, and the growth rate was similar to that under light, indicating that light was not necessary for osmotic nutrition. However, when predatory flagellates survive through prey consumption, light may also be necessary. Andersson et al. (1989) studied Ochromonas sp. that were grown on E. coli and transferred to an inorganic salt medium. Ochromonas sp. survived without additional bacteria as food in the light but died quickly under dark conditions, and only 1% of the cells remained intact at the end of the experiment. In our research (Fig. 6a), the density of P. malhamensis under dark conditions was lower than that under light conditions after 4 days of culture, but P. malhamensis did not die in large numbers, which may have been due to the secretion of dissolved organic carbon by M. aeruginosa. In the first 2 days of the experiment, the phagocytosis of M. aeruginosa by P. malhamensis and the growth of P. malhamensis may have depended on the dissolved organic carbon secreted by M. aeruginosa. In the continuously dark environment, M. aeruginosa could not continue to secrete dissolved organic carbon, so P. malhamensis could not remove the remaining M. aeruginosa cells and could not continue to divide and proliferate.

Mixotrophic Chrysophyta can often adapt to extreme environments and exhibit a wide range of adaptations to pH due to their variety of nutrient acquisition methods. Some flagellates of the genus Mallomonas can even reach high abundances in environments with pH values of 4 (Roijackers 1988). Moser and Weisse (2011) found that there are flagellates in acidic lakes that are the main algae in acidic lakes. This study shows that P. malhamensis can live at pH values between 6 and 9, which is a suitable pH environment for the growth of most aquatic organisms. Guo (2008) found that the phagocytic rate of Poterioochromonas sp. decreased with increasing pH within a pH range of 6–10 and that there was a strong negative correlation between the phagocytic rate and pH. The growth performance of Poterioochromonas sp. was the best in the pH range of 7–8, and its algal phagocytosis rate was also high within this range. Touloupakis et al. (2016) found that flagellates could not grow at a pH of 11. Moreover, P. malhamensis was very sensitive to pH shock, resulting in a rapid loss of motility followed by cell lysis within a few hours when the pH was suddenly increased to 11. The tolerance range of P. malhamensis is usually between 6 and 10, but an alkaline environment is not conducive to its growth. This may be because the digestive enzymes in Ochromonas cells, such as amylase, show higher activity under weakly acidic conditions (Tarayre et al. 2014). Many plants, especially floating plants and ecological floating beds, can reduce the pH value in water (Hu et al. 2010). Higher temperature and pH are beneficial to cyanobacteria (Yang et al. 2018), and the reproduction of cyanobacteria promotes an increase in the water pH, which inhibits the growth of P. malhamensis. Therefore, in the competition between predatory flagellates and M. aeruginosa, the presence of plants benefits P. malhamensis by adjusting the environmental pH.

P. malhamensis has three nutrition modes: photo autotrophy, phagocytosis, and osmosis. Of these, osmosis results in the fastest growth. In the wheat grain medium, the growth rate of P. malhamensis on the first day was 2.56 ± 0.12 day−1, and the generation time was approximately 5–6 h. Röderer (1986) reported that Poterioochromonas malhamensis grows rapidly under heterotrophic conditions (Bacto-tryptone, yeast extract, and d-glucose, each 0.1% (wt/vol) in double-distilled water); the generation time was approximately 5–6 h. Zhang et al. (2016) reported that the growth rate of Ochromonas sp. reached 2.66 ± 0.36/day when 150 mg/L glucose was added to the BG11 medium. These studies suggest that a growth rate of 2.6 day−1 may be the maximum growth rate of Ochromonas and Poterioochromonas. However, when bacteria or algae were used as food for phagocytosis, the growth rate could not reach this value (Zhang and Watanabe 2001; Zhang et al. 2009; Ma et al. 2018a, b). This may be because phagocytosis consumes energy not only to take in prey and form food vacuoles but also to digest prey. Osmotic nutrients can be absorbed and utilized directly, which is more efficient. At the same time, flagellates can achieve higher cell density when acquiring nutrients through osmosis. In this study, the density of P. malhamensis reached more than 107 cells/mL in the wheat grain medium, but P. malhamensis could not reach this density when M. aeruginosa was provided as prey, even when the density of M. aeruginosa reached 5 × 107 cells/mL (Fig. 9a). When Chlorella was used as prey, even if the density of Chlorella reached 4 × 108 cells/ml, the maximum density of P. malhamensis was only approximately 4 × 106 cells/ml, regardless of whether the experiment was performed in a 250-mL flask or at the larger scale of greenhouse (Ma et al. 2018a, b). Guo (2008) found that when Poterioochromonas sp.DO2004 was fed on M. aeruginosa, the cell density of the flagellate could not reach 107 cells/ml. Zhang et al. (2016) reported that the growth rate and maximum cell density of the flagellate Ochromonas sp. could be increased by adding glucose alone to an inorganic nutrient medium.

In this paper, it was found that when the initial density of M. aeruginosa was 5 × 107 cells/mL (M.aeruginosa: P. malhamensis = 2500:1), P. malhamensis could not remove all of the M. aeruginosa, and M. aeruginosa maintained a stable density. There are three possible explanations for this process. First, the high density of M. aeruginosa leads to a higher pH value in the environment, which inhibits the grazing of P. malhamensis on M. aeruginosa. Second, M.aeruginosa may change under these conditions to resist P. malhamensis grazing, and many studies have reported the phenomenon of resistance in bacteria and algae to grazing by flagellates(Jousset 2012; Ma et al. 2019). It has been reported in the literature that M.aeruginosa can form colonies under grazing pressure (Wang et al. 2010) but that Poterioochromonas malhamensis can still feed on smaller M.aeruginosa colonies (Kim and Han 2007). However, we did not observe that M. aeruginosa formed colonies during these experiments, indicating that M.aeruginosa may resist grazing by other means. Third explanation may be that M.aeruginosa produces a certain substance that inhibits feeding by flagellates. Microcystis aeruginosa can produce allelochemicals and inhibit phytoplankton such as cyanobacteria (Microcystis wesenbergii), green algae (Scenedesmus quadricauda, Chlorella pyrenoidosa), and diatom (Cyclotella meneghiniana) (Yang et al. 2014; Wang et al. 2017a, b). Studies have shown that micro-plastics and green algae can inhibit flagellate feeding (Zhang et al. 2018; Kong et al. 2021). Chlorella could still be grazed even if its density reached 4 × 108 cells/ml (Ma et al. 2018a, b), which is an unfortunate result for the high-density cultivation of Chlorella. Exploring the reasons why high-density M. aeruginosa resists grazing may be beneficial for controlling the influence of flagellates when cultivating Chlorella at high density.

In summary, environmental factors and the density and growth of prey affect the growth and grazing of P. malhamensis.