CN115386496A

CN115386496A - Flagellates capable of ingesting microcystis aeruginosa and degrading microcystins and application

Info

Publication number: CN115386496A
Application number: CN202211166439.2A
Authority: CN
Inventors: 徐虹; 陈双双; 谢婉馨; 杨龙凤
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2022-11-25
Anticipated expiration: 2042-09-23
Also published as: CN115386496B

Abstract

The invention discloses a flagellate capable of ingesting microcystis aeruginosa and degrading microcystin and application thereof, in particular to a Poteriospella flagellate Poteriosporium lacustris NY1 belonging to Poteriospella genus, which is obtained by separating and screening from a south-first reservoir where water bloom is exploded in Nanjing county of Zhongzhou province in Fujian province. The dinoflagellate NY1 can quickly ingest microcystis aeruginosa and efficiently degrade microcystins, so that the microcystins are prevented from being released into a water body to cause secondary pollution, and the effect is stable in a certain temperature, illumination and pH range. The flagellate NY1 treats the harmful cyanobacterial bloom, thereby not only avoiding secondary pollution caused by the release of algal toxins to a water body, but also reducing the interference to the aquatic ecological environment, and having wide application prospect.

Description

Flagellates capable of ingesting microcystis aeruginosa and degrading microcystins and application

Technical Field

The invention relates to dinoflagellates, in particular to dinoflagellates capable of ingesting microcystis aeruginosa and degrading microcystin and application thereof. Belongs to the technical field of water treatment.

Background

With eutrophication of water and global warming, cyanobacterial bloom frequently erupts, resulting in deterioration of lake water quality and imbalance of water ecological system. Toxic microcystis aeruginosa is a typical dominant algal species with outbreak of water bloom, and after algal toxins released by the toxic microcystis aeruginosa enter fishes, mammals and human bodies along food chains, diseases such as liver, digestive system, nervous system and the like, such as liver cancer, are induced, so that the method for controlling and removing the water bloom of the microcystis aeruginosa is not slow enough.

Compared with a chemical method and a physical method, the biological algae control method is considered to be an environment-friendly method, the method cannot cause secondary pollution of the water body, the organisms are easy to culture in a large scale, the cost is low, and the method has a wide application prospect. Biological methods include allelopathy of plants, predation of zooplankton, algicidal action of microorganisms, and phagocytosis of some phycobionts. Common biological methods include the algicidal action of microorganisms, allelochemical action of plants and predation action of zooplankton. However, when the conventional biological method is used for treating harmful cyanobacterial bloom, especially microcystis aeruginosa, microcystin is released, water body is polluted, human health is threatened, and certain ecological potential safety hazards still exist if the conventional biological method is used for treating the harmful cyanobacterial bloom. Therefore, it is particularly urgent to find a safe and efficient biological control method for effectively controlling the microcystis aeruginosa bloom.

Phycobionts, mainly dinoflagellates, are important components of the micro-food ring, and can feed on bacteria, algae, organic debris and the like, playing an important role in mass transfer and energy flow of the ecosystem. Dinoflagellates have their unique advantages over other algicidal microorganisms. On the one hand, the dinoflagellates can efficiently eliminate algal cells. For example, the brown dinoflagellate Ochromonas sp can directly swallow microcystis aeruginosa and indirectly release allelopathyThe substance inhibits the growth and reproduction of Microcystis aeruginosa. The whole ditch algae Teleaula amphioxeia can effectively eliminate the initial cell density of 5.4 multiplied by 10 ³ -1.5×10 ⁷ cell/mL of Synechococcus, and clearance increased with increasing initial algal cell number. On the other hand, dinoflagellates have extremely strong tolerance to microcystins and can even degrade microcystins. For example, chrysophyceae Monas sp can degrade three types of microcystins MC-LR, MC-RR and MC-YR simultaneously. ZX1 makes microcystis aeruginosa cell number from 4.3X 10 in 40 hr ⁶ cells/mL is reduced to 1X 10 ⁴ cells/mL, the phycotoxin concentration decreased from 114. Mu.g/L to 19.72. Mu.g/L. Initial cell numbers of 3X 10 were almost completely cleared by the dinoflagellate O.gloeopora YZ1 within 10 days ⁵ cells/mL microcystis aeruginosa, and completely degrades the initial concentration of 50 mug/L algal toxin. In addition, the dinoflagellates have the advantage of wide algae feeding range, and one dinoflagellate can often feed various algae with similar forms. The Phaeoflagellate P.malhamensis CMBB-1KY432752 can swallow various types of microalgae such as chlorella, scenedesmus acutus, chlamydomonas, nannochloropsis, synechocystis, and the like. Manage different algal cells such as microcystis aeruginosa, diatom and green algae are observed in the chlamydomonas persicae sp. In conclusion, the phycotrophic organisms have great potential in the control of harmful algal blooms, and it would be an ideal way to control the population number of algae by controlling the population number of the phycotrophic organisms.

Currently, the genus Trichophyton (Ochromonas), the genus Trichophyton (Poterioochromonas), the genus Trichophyton (Monas), the genus Pleurocystis (Parapymonas), etc. have been confirmed to have the ability to swallow, and there is no report on the swallowing effect of Trichophyton sp. Although researchers explored the nature of dinoflagellates' feeding and ability to degrade algal toxins, ecological safety with respect to their control of water bloom has never been evaluated.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a dinoflagellate which can ingest microcystis aeruginosa and degrade microcystin and application thereof.

In order to realize the purpose, the invention adopts the following technical scheme:

1. a flagellate (Poteriosporium lactistis) NY1 capable of ingesting microcystis aeruginosa and degrading microcystin is preserved in China center for type culture collection (CGMCC) at 2022 years, 8 months and 17 days, and the preservation addresses are as follows: in the Wuhan university school of eight paths 299 # in Wuchang area of Wuhan city, hubei province, the preservation number is CCTCC NO: m20221293.

2. The application of the dinoflagellate in preparing algaecide.

Preferably, the algaecide comprises the following algaecide species: blue algae and green algae.

Further preferably, the blue algae comprises microcystis aeruginosa and synechocystis, and the green algae comprises Platymonas subcordiformis and chlorella vulgaris.

3. The use of the aforementioned dinoflagellates for the control of harmful algal blooms.

4. An algaecide contains the culture solution of dinoflagellate as effective component.

5. The application of the dinoflagellate in reducing the content of microcystin in water is provided.

6. A degradation agent for reducing microcystin in water contains the culture solution of dinoflagellate as effective component.

The invention has the beneficial effects that:

the invention separates and screens a strain of Poteriosporium flagellata Poteriosporium lacustris NY1 from a first reservoir in south Jing county of Zhangzhou, fujian province, wherein the strain is heterotrophic flagellate, and the existence of freshwater algae such as microcystis aeruginosa, chlorella vulgaris, platymonas subcordiformis and Synechocystis is maintained by directly swallowing and digesting the freshwater algae.

The dinoflagellate NY1 can quickly ingest microcystis aeruginosa and efficiently degrade microcystins, so that the microcystins are prevented from being released into a water body to cause secondary pollution, and the effect is stable in a certain temperature, illumination and pH range. NY1 is added into the culture solution of Microcystis aeruginosa in the lag phase and the exponential phase, can remove algae cells, degrade algae toxins, reduce the release amount of soluble organic matters in a short time, and reduce the toxicity of water on bright photobacterium, daphnia magna and grass carp fries. The flagellate NY1 treats the harmful cyanobacterial bloom, thereby not only avoiding the secondary pollution caused by the release of algal toxins into water, but also reducing the interference to aquatic ecological environment, and therefore, the method has wide application prospect.

Drawings

FIG. 1 shows the content of phycophyta in the clarified solution at a scale of 20 μm.

FIG. 2 identification of the phycobiont Poteriosporium lactisteris NY1. Wherein, a is a phylogenetic evolutionary tree constructed based on an 18S rRNA sequence, b is an optical microscope picture of NY1, c is a scanning electron microscope picture of NY1, and d is a transmission electron microscope picture of NY1. Wherein M is mitochondria and N is cell nucleus; FV is food bubble.

FIG. 3 light microscopy (a-d) and transmission microscopy (e-h) for NY1 feeding microcystis aeruginosa. Wherein M is mitochondria and N is cell nucleus; FV is food bubble.

FIG. 4 initial cell number 5X 10 ³ cells/mL(a)，1×10 ⁴ cells/mL (b) and 3X 10 ⁴ cells/mL (c) of NY1 treatment the number of cells of Microcystis aeruginosa and NY1 was varied.

FIG. 5 shows the efficiency of NY1 in degrading microcystins at different inoculation ratios.

FIG. 6 the effect of temperature on NY1 ingestion of Microcystis aeruginosa and degradation of algal toxins. Wherein, the picture a is the color change of the microcystis aeruginosa culture solution, the picture b is the cell number change of the microcystis aeruginosa, the picture c is the clearance rate of NY1 to the microcystis aeruginosa, and the picture d is the total microcystin content change.

FIG. 7 is the effect of light intensity on NY1 ingestion of Microcystis aeruginosa and degradation of algal toxins. Wherein, the picture a is the color change of the microcystis aeruginosa culture solution, the picture b is the cell number change of the microcystis aeruginosa, the picture c is the clearance rate of NY1 to the microcystis aeruginosa, and the picture d is the total microcystin content change.

FIG. 8 the effect of pH on NY1 ingestion of Microcystis aeruginosa and degradation of algal toxins. Wherein, the picture a is the color change of the microcystis aeruginosa culture solution, the picture b is the cell number change of the microcystis aeruginosa, the picture c is the clearance rate of NY1 to the microcystis aeruginosa, and the picture d is the total microcystin content change.

FIG. 9NY1 shows the efficiency of eliminating Microcystis aeruginosa and degrading microcystin at different growth stages. Grey arrows represent day 0 addition of NY1; black arrows represent day 10 addition of NY1.

FIG. 10 variation of dissolved organic concentration during the course of algae removal for NY1. Group T0 represents the treatment of Microcystis aeruginosa by adding NY1 on day 0; group T10 represents treatment of Microcystis aeruginosa with NY1 addition at day 10.

FIG. 11 acute toxicity of supernatant after algae removal of NY1 to Photobacterium brightens.

FIG. 12 acute toxicity of supernatant after algae removal by NY1 to Daphnia magna.

FIG. 13 acute toxicity of supernatant after algae removal by NY1 to grass carp fry.

Preservation information

And (3) classification and naming: dinoflagellate

Latin literature name: poteriospumella lactisteri

The name of the depository: china typical microbiological culture collection center (CCTCC)

The address of the depository: eight-path 299 Wuhan university school interior in Wuhan city Wuchang area in Hubei province

The preservation date is as follows: 8/17/2022

Detailed Description

The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.

1. Isolation culture and identification of phycotrophic organisms NY1

(1) Collecting water sample from south reservoir with cyanobacterial bloom outbreak in Nanjing county of Zhangzhou, fujian province, filtering the water sample with a filter membrane with the aperture of 5 mu m, taking 2mL of filtrate, and adding the filtrate into 20mL of microcystis aeruginosa culture solution in logarithmic phase (the number of algae cells is about 6 multiplied by 10) ⁶ cells/mL) at a temperature of 25 ℃ and an illumination intensity of 100. Mu. Mol m ^-2 s ^-1 Culturing in an incubator with the light cycle of 12/12h (light/dark). After 7 days, the color of the culture broth was observed to change from green to light yellow and finally to clear. In order to eliminate the contingency of the experiment, the lysis solution containing the phycophagous organisms is transferred to the microcystis aeruginosa culture solution for a plurality of timesIn (5), the color of the culture broth finally became clear.

(2) Purifying the algae-eating organisms by a double-layer plate method. First, a lower layer medium (BG 11 medium + agar with a mass concentration of 1.2%) was prepared, cooled for 20min, and poured into a glass petri dish. Subsequently, an upper layer medium (BG 11 medium + agar 0.7% by mass) was prepared, and the temperature was lowered to 40 ℃. Uniformly mixing 4mL of upper layer culture medium, 100 mu L of lysate containing phycobionts and 1mL of microcystis aeruginosa, pouring the mixture into a glass plate containing the solidified lower layer culture medium, and setting the mixture into a treatment group; 4mL of the upper agar medium, 100. Mu.L of the lysate containing an algal organism, and 1mL of the BG11 medium were mixed and poured onto another glass plate containing the coagulation lower medium, and set as a control group. Placing the double-layer agar plate at 25 deg.C and illumination intensity of 100 μmol m ^-2 s ^-1 In an incubator with the illumination period of 12/12h (illumination/darkness), after 7 days of culture, algal plaques with different diameters are formed on the algal plates.

(3) Picking up single algae plaque to 20mL Microcystis aeruginosa culture solution in logarithmic growth phase (the number of algae cells is about 6 × 10) ⁶ cells/mL) at 25 ℃ under an illumination intensity of 100. Mu. Mol m ^-2 s ^-1 Culturing in incubator with light cycle of 12/12h (light/dark) for 7 days until the culture solution becomes clear, and observing with optical microscope to find that clear solution contains large amount of transparent phycophyta (figure 1). And (4) taking the clarified liquid, repeating the steps (2) and (3) for multiple times, continuously pouring the double-layer culture medium flat plate for multiple times, and obtaining a relatively pure phycobiont NY1 after the system is stable. Culturing the algae-eating microorganism NY1 in a liquid BG-11 culture medium, and adding microcystis aeruginosa every 5-7 days to feed the algae-eating microorganism NY1.

(4) 150mL of algal lysate containing phycobionts was aspirated, and centrifuged at 4,500rpm for 5min to collect cells. Extracting the whole genome of the phycobiont, amplifying the 18S rRNA sequence, connecting the PCR product by a T vector, and sending the PCR product to Xiamen platinum end company for sequencing. The sequences were then uploaded to NCBI databases (http:// blast. NCBI. Nlm. Nih. Gov/blast. Cgi) for alignment after primers were removed using DNAstar, and phylogenetic trees were constructed using the Neighbor-Joining method of MEGA7.0 software. As a result, as shown in FIG. 2, the phycobiont NY1 has a similarity of 99.83% to Poteriosperma lactis Yongseokyo 072317C3, and can be determined to belong to the genus Poteriosperma of Phaeophyta (Ochrophyta), chrysophyceae (Chrysophyceae), chromotales (Chromolinales), and Dinobryaceae (Dinobryaceae), and is named as Poteriosperma lactisteris NY1. The 18S rRNA sequence of NY1 was uploaded to NCBI database, genBank accession No. MZ707558.

(5) The cell morphology of NY1 was observed by means of an optical microscope, a scanning electron microscope and a transmission electron microscope, and as shown by b-d in FIG. 1, the NY1 cells were oval or circular, about 15-20 μm in length and about 10-12 μm in width. The cells were transparent, colorless and had two smooth flagella of almost equal length at the cell base. The internal structure of the cell is loose, has single or multiple vacuolated structures similar to food vacuoles, and the nucleus coated with a double-layer membrane and multiple mitochondria are clearly visible but do not contain chloroplasts.

2. Observation of different stages of NY1 ingestion of Microcystis aeruginosa

(1) 5mL of lysate containing NY1 was added to 500mL of Microcystis aeruginosa culture medium in logarithmic growth phase (algal cell count of about 6X 10) ⁶ cells/mL), after 0, 24, 48, 72h, respectively, 100mL was sampled, and cells were collected by centrifugation at 4,500rpm for 5 min.

(2) And observing different stages of the NY1 ingestion of the microcystis aeruginosa under an optical microscope and a transmission electron microscope. As can be seen from FIG. 3, at the stage of non-ingestion, NY1 is small in volume and does not contain algal cells; in the initial stage of ingestion, NY1 swims rapidly in water to increase the probability of contact with algal cells, then directly contacts algal cells through the cell membrane and engulfs the entire algal cell into the cell; in the middle stage of ingestion, the microcystis aeruginosa is gradually digested in the food bubble of NY1, the cytoskeleton and the cell wall of the microcystis algae are damaged, and the cells are not regular and full; at the end of ingestion, the cell structure of the microcystis aeruginosa becomes loose and the contents flow out and disperse in the NY1 food bubbles; it was concluded that NY1 achieves the effect of clearing algae cells by direct uptake of whole algae cells followed by digestion within their food vacuoles.

3. Measuring the feeding efficiency of NY1 to different freshwater algae

(1) The algae species used in the experiment: platymonas subcordiformis (Platymonas subcordiformis CCMA-418), chlorella vulgaris (Chlorella vulgaris CCMA-410), synechocystis sp.PCC 6803), microcystis aeruginosa (Microcystis aeruginosa PCC7806, PCC1752, and PCC 7820) were all purchased from the institute of aquatic biology, chinese academy of sciences. Culturing algae in glass triangular flask at 25 deg.C in BG11 liquid medium under illumination intensity of 100 μmol m ^-2 s ^-1 The light cycle was 12/12h (light/dark).

(2) Culturing different algae to logarithmic growth phase, subpackaging the algae liquid into 12-hole plates, subpackaging each hole with 4mL, and placing in a light incubator for adaptation for 24h. Addition of 1X 10 to treatment group ⁴ cells/mL NY1, control without any treatment, each set of 3 flat biological replicates. The cell count is measured by a cell counter, and the clearance rate of NY1 to the algae cells is calculated according to the following formula: clearance (%) = (C) ₀ -C _t )÷C ₀ X 100%, wherein C ₀ Algal cell number of 0h, C _t The number of algal cells corresponding to the treatment time was obtained.

(3) As shown in table 1, except for m.aeruginosa PCC7806, NY1 showed very strong feeding ability to m.aeruginosa PCC1752, m.aeruginosa PCC7820 and synechocystis sp.pcc6803, and the clearance rate to algal cells after 48 hours of treatment was 98%. In addition to cyanobacteria, NY1 also has high clearance rates for chlorella c.vulgaris CCMA-410 and physosiphon subcordiformis CAMA-418 of the chlorophyta, 78.69% and 71.82%, respectively. The NY1 has wide ingestion algae spectrum and high clearing efficiency, and has great potential in controlling the harmful algal blooms caused by the freshwater algae.

TABLE 1 clearance of NY1 over 48h for different algae

4. Determination of the growth rate of NY1 and the rate of ingestion of Microcystis aeruginosa cells

(1) Respectively culturing Microcystis aeruginosa to 5 × 10 ⁶ 、6×10 ⁶ 、7×10 ⁶ 、8×10 ⁶ 、9×10 ⁶ 、1×10 ⁷ 、2×10 ⁷ 、3×10 ⁷ 、4×10 ⁷ And 5X 10 ⁷ cells/mL. Subpackaging the algae liquid into 12-hole plates, subpackaging 4mL per hole, and placing in a light incubator for 24h. Adding 5 × 10 of algae culture solution ⁴ cells/mL NY1, with 3 biological replicates per group. mu.L of the samples were taken at 0, 12, 24, 36, 48, 60 and 72h, respectively, and the cell counts of NY1 and Microcystis aeruginosa cells were determined by means of a cytometer.

(2) The specific growth rate μ of NY1 cells was calculated as follows: μ = (lgN) _t -lgNt ₀ )÷(t-t ₀ ) Wherein Nt is the number of algae cells corresponding to late exponential t, N ₀ Is an exponential prophase t ₀ The corresponding number of algae cells, t is the time corresponding to the later exponential phase, t ₀ The corresponding time in the early stage of the index. Passage time G was calculated as follows: g = ln2 ÷ μ, where μ is specific to growth rate.

(3) As can be seen from Table 2, NY1 has high ingestion rate and good algae removal effect on Microcystis aeruginosa, and the initial cell number is 5X 10 ⁴ cell/mL NY1 can effectively eliminate the cell number of 4 multiplied by 10 in a short time ⁶ -5×10 ⁷ cell/mL Microcystis aeruginosa. The higher the initial cell number ratio of NY1 and microcystis aeruginosa, the higher the clearance rate of NY1 to algae cells, and when the initial cell number ratio is more than 1; when the initial cell number ratio is less than 1, the time required for the clearance to reach 90% is 72 hours. In addition, at a ratio of 1 ^-1 And 6.974h, indicating that this initial cell number ratio is most favorable for rapid proliferation of the NY1 cell population.

TABLE 2 specific growth rate, passage time and algal cell clearance of NY1

Kinetics curve of NY1 ingestion of Microcystis aeruginosa

(1) Microcystis aeruginosa was inoculated into 50mL of BG11 medium and cultured to logarithmic growth phase (about 6X 10 cell number) ⁶ cells/mL). Adding 6 × 10 of the culture solution ³ 、1×10 ⁴ And 3X 10 ⁴ cells/mL NY1, so that the initial cell number ratio for NY1 and Microcystis aeruginosa were 1, 1000,1 and 600 and 1, respectively, 20 μ L were sampled at 0, 12, 24, 36, 48, 60, 72, 84 and 96h and the two cell numbers were determined by means of a cytometer.

(2) The results are shown in FIG. 4, in which the number of Microcystis aeruginosa cells in the control group is gradually increased and 3X 10 cells are added ⁴ cell/mL NY1 (1. When the number of NY1 cells is 1X 10 ⁴ cells/mL (1 ³ When cells/mL (1. In addition, the number of NY1 cells showed a tendency to increase and then decrease. When the algae cells are sufficient, NY1 has enough food sources to maintain the survival of the algae cells, and the cell number is rapidly increased; once the algal cells are completely eliminated, NY1 dies gradually due to lack of food, and the number of cells is significantly reduced, because NY1 has no chloroplast, cannot be autotrophic through photosynthesis, and mainly depends on external food for growth.

6. Detecting the content of microcystin

(1) Inoculating Microcystis aeruginosa into 50mLBG11 culture medium, and culturing to logarithmic growth phase (cell number is about 6 × 10) ⁶ cells/mL). Adding 6 × 10 of culture solution into the algae culture solution ³ 、1×10 ⁴ And 3X 10 ⁴ cells/mL NY1, so that the initial cell ratio of NY1 and Microcystis aeruginosa is 1, 1000,1, 600 and 1.

(2) 1mL of sample is crushed by an ultrasonic cell crusher (voltage is 100W, working time is 5s, interval time is 5s, circulation is 60 times), the crushed lysate is 7,000-8,000rpm, centrifugation is carried out for 8-10min, and supernatant is taken as the algae toxin solution to be detected. All supernatants in the control group and 0 th and 24 th supernatants in the treatment group were diluted 400-fold with 0.1mol/L PBS, and the remaining supernatants were diluted 100-fold with 0.1mol/L PBS.

(3) The detection of the content of the microcystin is carried out according to the operation instruction of a microcystin enzyme-linked immunosorbent assay kit (Beacon, cat. # 20-0068). The degradation rate of the microcystin is calculated by the following formula: degradation rate (%) = (M) ₀ -M _t )÷M ₀ X 100%, wherein M ₀ An algal toxin content of 0 h; m is a group of _t The algal toxin content for the corresponding treatment time.

(4) As shown in FIG. 5, initially, the concentrations of intracellular and extracellular microcystins in each culture system were 203.99 and 24.51. Mu.g/L, respectively. After 72h of culture, the intracellular and extracellular algal toxin concentrations in the control group were increased to 305.76 and 67.83. Mu.g/L, respectively; in each treatment group, the microcystin concentration decreased with the decrease of algal cells, and the total microcystin degradation rates at the ratio of 1.

7. The efficiency of NY1 to ingest microcystis aeruginosa and degrade algal toxins under different environmental factors

(1) Inoculating Microcystis aeruginosa into 50mLBG11 culture medium, and culturing to logarithmic growth phase (cell number is about 6 × 10) ⁶ cells/mL). Addition of 1X 10 to treatment group ⁴ cells/mL NY1, such that the ratio of the initial cell number of NY1 to the microcystis aeruginosa is 1 600, whereas no NY1 is added to the control group, with 3 biological replicates per group set up.

(2) After treatment under different environmental conditions, 20. Mu.L of the suspension was sampled every 12h in a sterile operating station and the cell counts of NY1 and Microcystis aeruginosa were determined by means of a cell counter. 1mL of the sample was taken at intervals of 24 hours, and the total microcystin content was measured with reference to the above 6- (1).

(3) Influence of different temperatures on the NY1 effect: temperatures of 15, 20, 25, 30 and 35 ℃ were set, respectively. In the culture medium with pH value of 8 and illumination intensity of 50-100 μmol m ^-2 s ^-1 Culturing under the condition of 12/12h of light cycle (light/dark). KnotAs shown in FIG. 6, when the culture medium of algae to which no NY1 was added and the culture medium of algae to which NY1 was added were maintained at the same temperature, the color of the latter became remarkably lighter and the number of algal cells decreased remarkably. In the first 48h, when the temperature range is 30-35 ℃, the clearance rate of NY1 to microcystis aeruginosa reaches up to 90.29 percent; while the clearance rate is as low as 14.83% when the temperature is 15-20 ℃. However, by 72h, almost all algal cells in the treated groups were cleared by NY1, and the clearance rate was higher than 97%. The variation trend of the algal toxin concentration is consistent with that of the algal cell number, after the algal toxin is cultured for 72 hours at the temperature of 15-35 ℃, the total algal toxin concentration in the control group is about 280 mu g/L, the total algal toxin concentration in the treatment group is lower than 5.50 mu g/L, and the degradation rate is higher than 97%. The result shows that NY1 is affected in the early stage of temperature change, once the NY1 adapts to environmental change, the NY1 can quickly eat food, and finally completely eliminates algae cells and degrades algae toxins.

(3) Influence of different light intensities on the NY1 effect: the light intensities were set to 0, 25, 50, 100 and 200. Mu. Mol m, respectively ^- ² s ^-1 Culturing under the conditions of pH value of the culture medium of 8, temperature of 25 ℃ and light cycle of 12/12h (light/dark). As can be seen from FIG. 7, the light intensity range is 0 to 200. Mu. Mol m ^-2 s ^-1 And in the time, NY1 shows higher clearance rate and degradation rate, compared with a control group without NY1, the color of the algae culture solution in the treatment group added with NY1 is obviously lightened, the number of algae cells is obviously reduced, and the clearance rate is higher than 90% at 48 h. Meanwhile, the concentration of the microcystins in each culture solution under different illumination intensities is detected, and the total concentration of the microcystins in a control group is about 235.96 mu g/L at 72 hours; the concentration of the phycotoxin in the treatment group is about 8.63 mug/L, and the degradation rate reaches 98.77%. Experiments show that the particle size is between 0 and 200 mu mol m ^-2 s ^-1 Under the illumination intensity, the efficiencies of NY1 for ingesting microcystis aeruginosa and degrading algal toxins are not influenced, and the biological process of ingesting and degrading NY1 is presumed to be independent of light.

(4) Effect of different pH on NY1 effect: the pH values were set to 6, 7, 8, 9, and 10, respectively. At the temperature of 20-27 ℃, the illumination intensity is 50-100 mu mol m ^-2 s ^-1 Culturing under the condition of 12/12h of light cycle (light/dark). As can be seen from FIG. 8, whenWhen the pH value is 6-10, in the control group without NY1, the growth state of the microcystis aeruginosa is good, and the growth speed of cells has no obvious difference; after the NY1 is added to treat the verdigris microcapsules, the reduction rate of the number of algae cells in each treatment group has no obvious difference, and the clearance rate of the NY1 to the algae cells is higher than 98% and the degradation rate of the microcystins is higher than 97% in 72 hours. Experiments show that when the pH value is 6-10, the efficiency of NY1 for ingesting microcystis and degrading microcystin is not affected, which shows that NY1 can tolerate the pH range of 6-10, and cells can rapidly adjust self mechanism to adapt to pH change.

Efficiency of NY1 in eliminating microcystis aeruginosa in different growth stages and degrading microcystin

(1) Microcystis aeruginosa was inoculated into 3L BG11 medium, and the initial cell count was determined to be 2.01X 10 ⁶ cells/mL. 3 groups were constructed, respectively, and group 1 (T0 group) was processed: adding 1 × 10 of the culture solution to the culture solution on day 0 ⁴ cells/mL NY1; treatment group 2 (T10 group): adding 3.8 × 10 of the culture solution to the culture solution at day 10 ⁴ cells/mL NY1; control groups were not supplemented with NY1 and 3 flat biological replicates were set per group.

(2) mu.L of the sample was taken every 2 days, and the algal cell count was determined by means of a cytometer. 1mL of the sample was taken every 2 days, and the total microcystin content was determined with reference to the above 6 (1).

(3) As shown in FIG. 9, NY1 was added to the algal cells at the lag phase (day 0) and the exponential phase (day 10). At day 0, the cell density of Microcystis aeruginosa was 2.01X 10 ⁶ cells/mL, microcystin concentration of 53.15 μ g/L, and the number of algae cells in 96h after NY1 is added is reduced to 2.70 × 10 ⁵ cells/mL, clearance rate up to 87.24%; the concentration of the phycotoxin is reduced to 10.32 mu g/L, and the degradation rate is 80.58 percent. At day 10, the cell density of Microcystis aeruginosa was 7.67X 10 ⁶ cells/mL, microcystin concentration of 416.52 μ g/L, and the number of algae cells in 96h after NY1 is added is reduced to 1.77 × 10 ⁵ cells/mL, clearance rate up to 97.69%; the concentration of the phycotoxin is reduced to 28.74 mu g/L, and the degradation rate is 93.10 percent. By day 18, the algal cells in both treatment groups were almost completely eliminated and the algal toxin concentration was below 2.10. Mu.g/L. The above experimental results show thatNo matter the NY1 is put in the lag phase or the exponential phase of the microcystis aeruginosa, the number of the microcystin aeruginosa cells can be obviously reduced, the content of microcystin is reduced, and the bloom outbreak of the blue algae can be effectively controlled.

9.NY1 cleaning the change of the concentration of the soluble organic matters in the process of microcystis

(1) The experimental group was set according to the above 8 (1).

(2) Soaking a 40mL brown silicon boronized glass bottle in 2M HCl for 24h to remove residual inorganic salts, washing for three times by using single distilled water and double distilled water respectively, and drying for later use. The dried brown silicon boronized glass bottle and the 0.45 mu m acetate fiber filter membrane are wrapped by tinfoil paper and then are placed in a muffle furnace to be calcined for 4 hours at 450 ℃ so as to remove organic carbon on the tube wall. 10mL of each algae liquid is respectively taken, filtered by an acetate fiber filter membrane and collected into a brown silicon boronized glass bottle.

(3) Each sample was examined using a fluorescence spectrophotometer, and three-dimensional fluorescence spectrum scanning was performed using a 1cm quartz cuvette with ultrapure water as a blank. The slit width of the excitation monochromator was 10nm and the slit width of the emission excitation monochromator was 10nm. Excitation wavelength (Ex) range from 220-450nm, step-by-step 5nm; the emission wavelength (Em) ranged from 250-550nm, stepped at 5nm.

(4) As shown in FIG. 10, in the control group, as the culture time of Microcystis aeruginosa increases, 3 distinct fluorescence peaks gradually appear, which are distributed in the fluorescence region III (Ex/Em, 200-250/380-550), the fluorescence region IV (Ex/Em, 250-450/280-380) and the fluorescence region V (Ex/Em, 250-450/380-550) and are fulvic acids, soluble microbial products and humic acids produced by algae cells respectively; in the T0 group, since the initial algae cell number is small and NY1 is rapidly removed in a short time, soluble organic matters in a water body can hardly be detected; in the T10 group, the algae cells grow slowly in the first 6 days, released extracellular soluble organic matters are less, and no characteristic fluorescence peak appears, after a large number of algae cells are swallowed by NY1 after 10 days, a part of soluble organic matters are released, and a fluorescence peak appears in the water body, and the peak is mainly concentrated in an IV area to represent that the water body contains a large number of soluble microbial products. The experimental results show that the humic acid, fulvic acid and other components in the two treatment groups are obviously reduced compared with the control group, and the reason is presumed that the soluble organic substances released to the outside of the cells are reduced because NY1 directly swallows the microcystis and decomposes most macromolecular substances in the cells.

10.NY1 for eliminating the biotoxicity of water bodies in the process of microcystis aeruginosa

10.1NY1 eliminates the toxicity of water bodies to bright photobacterium after microcystis aeruginosa is removed

(1) Set up 5 groups respectively: a positive control group (BG 11 culture medium), a negative control group (microcystis aeruginosa lysate), a microcystis aeruginosa culture solution without NY1, and a microcystis aeruginosa culture solution with NY1 added at day 0 and day 10 respectively, wherein each group is provided with 3 flat biological repetitions.

(2) 1mL of sample is collected on 0 th, 6 th, 12 th and 18 th days respectively, the collected sample is crushed by an ultrasonic cell crusher (voltage is 100W, working time is 5s, interval time is 5s, circulation is performed for 60 times), the lysate is centrifuged for 10min at 6,000rpm, and the supernatant is taken as a liquid to be detected.

(3) The P.lucidum was activated and cultured to log phase. Respectively taking 450 mu L of liquid to be detected, putting the liquid into a 1.5mL centrifuge tube, adding 50 mu L of 30% NaCl solution to ensure that the final salinity of the sample is 3%, and taking double distilled water with the salinity of 3% as a blank control group.

(3) For each 1 sample tube tested, 1 control tube was tested simultaneously. Add 10. Mu.L of Leuconostoc into each sample tube and react for 15min immediately with time, and measure the fluorescence of the sample tube using chemiluminescence apparatus. The relative luminous efficiency calculation formula is as follows: relative luminescence (%) = (sample luminescence ÷ control luminescence) × 100%

(4) As a result, as shown in FIG. 11, BG11 medium had no inhibitory effect on the luminescence intensity of Photobacterium brightens; the lysate of the microcystis aeruginosa after ultrasonic crushing has strong toxicity to the bright photobacterium, and the relative luminous rate of the photobacterium is only 10 percent; in the culture solution of the microcystis aeruginosa without adding NY1, algal cells continuously release algal toxins in the growth process, the luminous intensity of the bright photobacterium is gradually inhibited, and the relative luminous rate in 6 th to 18 th days is lower than 50 percent; in the T0 group, the algal cells were rapidly cleared by NY1 on day 0, so that the luminous intensity of the Photobacterium brightens was not suppressed for 18 days; in the T10 group, at day 6, the water body contains the algae toxins, the relative luminous rate of the photobacterium is 47.16%, at days 12-18, the algae toxins in the water body are degraded by NY1, the toxicity is relieved, and the luminous intensity of the photobacterium is not obviously different from that of a BG11 culture medium. The NY1 can reduce the toxicity of water to the photobacterium brightens after algae removal.

10.2NY1 eliminates the toxicity of microcystis aeruginosa on daphnia magna in water

(2) All groups of liquids were dispensed into 50mL beakers, each beaker was dispensed into 20mL, 3 replicates were set for each group. And (3) placing about 26 fleas with good life status and active swimming in each beaker, observing the status of the fleas for 24, 48 and 72 hours respectively and counting the number of the surviving fleas. The mortality rate of daphnia magna is calculated as follows: mortality (%) = (D) ₀ –D _t )÷D ₀ X 100%, wherein D ₀ For 0h of Daphnia magna population, D _t The number of daphnia magna corresponding to the treatment time.

(3) As shown in fig. 12, in BG11 medium, daphnia magna has a certain natural mortality rate, and the mortality rate in 72 hours is 25.03%; the toxicity of the algae lysate to the daphnia magna is extremely strong, so that the death rate of the daphnia magna within 72 hours is up to 98.72 percent; the microcystis aeruginosa releases less algal toxins into the water body in the first 12 days, the death rate of the daphnia magna is only 24.62%, but the algal toxins in the water body are continuously accumulated when the microcystis aeruginosa grows to the 18 th day, so that the death rate of the daphnia magna is increased to 68.12%; however, the growth status of Daphnia magna in the T0 and T10 treatment groups was good and there were essentially no acute toxicity symptoms, and the mortality rate of Daphnia magna was 30.34% and 25.67% on day 18, respectively, indicating that NY1 reduced the toxicity of the water body after algae removal, thereby reducing the mortality rate of Daphnia magna.

10.3NY1 eliminates toxicity of microcystis aeruginosa on grass carp fry

(1) Set up 5 groups respectively: a positive control group (BG 11 culture medium), a negative control group (microcystis aeruginosa lysate), a microcystis aeruginosa culture solution without NY1, and a microcystis aeruginosa culture solution with NY1 added at the 0 th day and the 10 th day respectively, wherein 3 flat biological repeats are arranged in each group.

(2) The liquid of all groups is subpackaged in 300mL of preservation boxes, each preservation box is subpackaged in 200mL, and each group is provided with 3 parallels. About 51 grass carp fries with good life state and active swimming are placed in each beaker, water and feed are not changed during the experiment, and the dead fries are cleaned in time. Observing the states of the grass carp fries at 24h, 48h and 72h respectively, counting the number of the survival grass carp fries, and calculating the death rate.

(3) The result is shown in figure 13, the juvenile fish in BG11 medium swim normally, without obvious poisoning character, the natural mortality rate is 14.38%; the algae lysate shows extremely high fish toxicity, and the juvenile fish does not swim any more at first, the body is curled, and finally all the juvenile fish die; the microcystis aeruginosa culture solution without NY1 shows a certain fish toxicity to grass carp, and when the algae cells grow to 18 days, a large amount of toxic substances in water are accumulated, so that the death rate of juvenile fish reaches 45.64%; however, in the T0 and T10 treatment groups added with NY1, only a very small part of fish fries have toxic symptoms, and the death rate of juvenile fish in 18 days is lower than 28.02%, which shows that the NY1 can reduce the toxicity of water bodies after algae removal, thereby reducing the death rate of juvenile fish.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto, and various modifications and changes that can be made by those skilled in the art without inventive efforts based on the technical solutions of the present invention are within the scope of the present invention.

Claims

1. A flagellate (Poteriosporium lacustris) NY1 capable of ingesting microcystic aeruginosa and degrading microcystin is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation time of 2022 years, 8 months and 17 days, and the preservation addresses are as follows: in the Wuhan university school of eight paths 299 # in Wuchang area of Wuhan city, hubei province, the preservation number is CCTCC NO: m20221293.

2. Use of the dinoflagellate of claim 1 in the preparation of an algaecide.

3. The use of claim 2, wherein the algaecide is of the algaecide species comprising: blue algae and green algae.

4. The use according to claim 3, wherein said cyanobacteria comprises Microcystis aeruginosa, synechocystis aeruginosa, and said green algae comprises Platymonas subcordiformis, chlorella vulgaris.

5. Use of the dinoflagellates as claimed in claim 1 for the control of harmful algal blooms.

6. An algaecide comprising the culture solution of dinoflagellate as claimed in claim 1 as an active ingredient.

7. Use of the dinoflagellate of claim 1 for reducing a level of microcystin in water.

8. A degrading agent for reducing microcystin in water, which contains the culture solution of dinoflagellate as claimed in claim 1 as an active ingredient.