CN115386496B - Dinoflagellates capable of feeding microcystis aeruginosa and degrading microcystin and application thereof - Google Patents
Dinoflagellates capable of feeding microcystis aeruginosa and degrading microcystin and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
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- C12N1/12—Unicellular algae; Culture media therefor
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C12R2001/89—Algae ; Processes using algae
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Abstract
The invention discloses a dinoflagellate capable of feeding microcystis aeruginosa and degrading microcystin and application thereof, in particular relates to a dinoflagellate Poteriospumella lacustris NY1 of Poteriospumella, which is separated and screened from a south reservoir of water bloom in Nanjing county of Zhangzhou, fujian province. The dinoflagellate NY1 can not only rapidly ingest microcystis aeruginosa, but also efficiently degrade microcystin, avoid secondary pollution caused by release of microcystin to water, and has stable effect in certain temperature, illumination and pH range. The dinoflagellate NY1 is used for treating harmful cyanobacterial bloom, so that secondary pollution caused by release of algae toxins to a water body is avoided, and the interference to aquatic ecological environment is reduced, and therefore, the dinoflagellate NY1 has a wide application prospect.
Description
Technical Field
The invention relates to dinoflagellates, in particular to dinoflagellates capable of feeding microcystis aeruginosa and degrading microcystin and application thereof. Belongs to the technical field of water treatment.
Background
With eutrophication of water and global warming, blue algae bloom frequently occurs, which results in deterioration of lake water quality and unbalance of water area ecological system. Among them, toxic microcystis aeruginosa is a typical dominant species of water bloom, and when the toxins released by them enter fish, mammals and human bodies along the food chain, they induce diseases of liver, digestive system and nervous system, such as liver cancer, so that the method for controlling and removing microcystis aeruginosa water bloom is indistinct.
Compared with chemical methods and physical methods, the biological algae treatment method is considered as an environment-friendly means, the method does not cause secondary pollution to the water body, and the method is easy for large-scale cultivation of organisms, has low cost and has wide application prospect. Biological methods include plant allelopathy, zooplankton predation, microbial algicidal action, phagocytosis by some algae-eating organisms, and the like. The biological methods currently in common include the algicidal action of microorganisms, the chemosensory action of plants, and the predatory action of zooplankton. However, when the conventional biological method is used for treating harmful cyanobacterial bloom, especially microcystic aeruginosa algal bloom, microcystic toxins are often released to pollute water bodies, the human health is threatened, and certain ecological potential safety hazards still exist if the biological method is used for treating the harmful cyanobacterial bloom. Therefore, the requirement for a safe and efficient biological control method for effectively controlling microcystis aeruginosa bloom is urgent.
Algae-eating organisms mainly comprising dinoflagellates are important components in micro-food loops, can eat bacteria, algae, organic debris and the like, and play an important role in mass transfer and energy flow of a ecological system. Compared with other algae-treating microorganisms, the dinoflagellate has unique advantages. In one aspect, dinoflagellates are capable of efficiently removing algal cells. For example, the Ochromonas sp can directly ingest microcystis aeruginosa and indirectly release allelochemicals to inhibit the growth and reproduction of microcystis aeruginosa. The whole-groove algae Teleaulax amphioxeia can effectively remove the initial cell density of 5.4X10 3 -1.5×10 7 cell/mL of Synechococcus, and the clearance rate increased with the increase of the initial algal cell number. On the other hand, dinoflagellates not only have extremely strong tolerance to microcystins, but also can degrade microcystins. For example, dinoflagellate Monas sp is capable of degrading three types of microcystins, MC-LR, MC-RR and MC-YR simultaneously. Poterochromonas sp.ZX1 of Pythium gracile produced by microcystis aeruginosa cell number of 4.3X10 in 40 hours 6 The cell/mL is reduced to 1X 10 4 The concentration of the algae toxin is reduced from 114 mug/L to 19.72 mug/L. The initial cell number of the dinoflagellate O.gloeopara YZ1 is almost completely cleared within 10 days to be 3 multiplied by 10 5 Microcystis aeruginosa at cells/mL and completely degraded the initial concentration of 50 μg/L of algal toxin. In addition, the dinoflagellate has the advantage of wide algae feeding range, and one dinoflagellate can always feed various algae with similar forms. The Pythium gracile P.malhamensis CMBB-1KY432752 can swallow various microalgae such as chlorella, sphaerotheca gracilis, chlamydomonas, nannochloropsis and Synechocystis. Manage different algae cells such as microcystis aeruginosa, diatom and green algae are observed in the cells of Polytillella polis sp, which shows that the dinoflagellate has swallowing effect on various algae. In summary, algae-eating organisms have great potential in controlling harmful algal bloom, and it would be an ideal approach if the population number of algae could be controlled by controlling the population number of algae-eating organisms.
At present, the genus Brown algae (Ochromonas), genus Brown algae (Poterriooochromonas), genus trichomonas (Monas), genus near-cyst algae (Paraphysiomonas), and the like have been confirmed to have swallowing ability, and no report has been made about the swallowing effect of Poteriosphaella on harmful blue algae. Although researchers explore the feeding characteristics of dinoflagellates and the ability to degrade algal toxins, the ecological safety of controlling water bloom has never been assessed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a dinoflagellate capable of feeding microcystis aeruginosa and degrading microcystin and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
1. the dinoflagellate Poteriospumella lacustris NY1 which can ingest microcystis aeruginosa and degrade microcystin is preserved in China center for type culture collection, the preservation time is 2022, 8 months and 17 days, and the preservation address is: in the eight-path 299-grade university of Wuhan in Wuchang district of Wuhan, hubei province, the preservation number is CCTCC NO: m20221293.
2. The application of the dinoflagellate in preparing the algicide.
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 Fucus vesiculosus and Chlorella.
3. The application of the dinoflagellate in controlling harmful algal bloom.
4. An algicide contains the culture solution of dinoflagellate as effective component.
5. The application of the dinoflagellate in reducing the microcystin content in water.
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 Poteriospumella flagelliforme Poteriospumella lacustris NY1 from a reservoir in the south Jing county of Zhangzhou, fujian province, and NY1 is heterotrophic flagelliforme which can maintain itself to survive by directly swallowing and digesting light water algae such as microcystis aeruginosa, chlorella, flat algae of heart shape and synechocystis.
The dinoflagellate NY1 can not only rapidly ingest microcystis aeruginosa, but also efficiently degrade microcystin, avoid secondary pollution caused by release of microcystin to water, and has stable effect in certain temperature, illumination and pH range. NY1 is put into the microcystis aeruginosa culture solution in the delay period and the exponential phase, so that algae cells can be cleared in a short time, algae toxin can be degraded, the release amount of soluble organic matters can be reduced, and the toxicity of water bodies to luminous bacillus, daphnia magna and grass carp fries can be reduced. The dinoflagellate NY1 is used for treating harmful cyanobacterial bloom, so that secondary pollution caused by release of algae toxins to a water body is avoided, and the interference to aquatic ecological environment is reduced, and therefore, the dinoflagellate NY1 has a wide application prospect.
Drawings
The algae-eating organisms in the clarified liquid of FIG. 1 are in a scale of 20. Mu.m.
FIG. 2 identification of algae-eating organisms Poteriospumella lacustris NY1. Wherein a is a phylogenetic tree constructed based on an 18SrRNA sequence, b is an optical microscope image of NY1, c is a scanning electron microscope image of NY1, and d is a transmission electron microscope image of NY1. Wherein M is mitochondria and N is nucleus; FV is a food bubble.
FIG. 3 Process of feeding Microcystis aeruginosa by NY1 under light microscope (a-d) and transmission microscope (e-h). Wherein M is mitochondria and N is nucleus; FV is a food bubble.
FIG. 4 initial cell number 5X 10 3 cells/mL(a)，1×10 4 cells/mL (b) and 3X 10 4 cell number change of microcystis aeruginosa and NY1 under NY1 treatment of cells/mL (c).
FIG. 5 shows the efficiency of degradation of microcystins by NY1 at different inoculation ratios.
FIG. 6 effect of temperature on Ny1 feeding microcystis aeruginosa and degrading algal toxins. Wherein, graph a shows the color change of the microcystis aeruginosa culture solution, graph b shows the cell number change of microcystis aeruginosa, graph c shows the clearance rate of NY1 to microcystis aeruginosa, and graph d shows the total microcystin content change.
FIG. 7 effect of light intensity on Ny1 feeding microcystis aeruginosa and degrading algal toxins. Wherein, graph a shows the color change of the microcystis aeruginosa culture solution, graph b shows the cell number change of microcystis aeruginosa, graph c shows the clearance rate of NY1 to microcystis aeruginosa, and graph d shows the total microcystin content change.
FIG. 8 effect of pH on Ny1 feeding microcystis aeruginosa and degrading algal toxins. Wherein, graph a shows the color change of the microcystis aeruginosa culture solution, graph b shows the cell number change of microcystis aeruginosa, graph c shows the clearance rate of NY1 to microcystis aeruginosa, and graph d shows the total microcystin content change.
FIG. 9NY1 shows the efficiency of microcystis aeruginosa removal and microcystin degradation over different growth periods. Grey arrows represent day 0 addition of NY1; black arrows represent addition of NY1 on day 10.
FIG. 10NY1 shows the change in concentration of soluble organics during algae removal. Group T0 represents day 0 where NY1 was added to treat Microcystis aeruginosa; group T10 represents day 10 where microcystis aeruginosa was treated with NY1.
FIG. 11 acute toxicity of supernatant after NY1 algae removal to Protobacterium brightens.
FIG. 12 acute toxicity of the supernatant after NY1 algae removal to daphnia magna.
FIG. 13 acute toxicity of supernatant after algae removal of NY1 to grass carp fries.
Preservation information
Classification naming: dinoflagellate (Botrytis cinerea)
Latin Wen Xueming: poteriospumella lacustris
Preservation unit name: china center for type collection of microorganisms (CCTCC)
Deposit unit address: eight paths of 299 Wuhan university school in Wuhan City of Hubei province
Preservation date: 2022 8.17.
Detailed Description
The invention is further illustrated in the following figures and examples, which are provided for the purpose of illustration only and are not intended to be limiting.
1. Isolated culture and identification of algae-eating organism NY1
(1) Collecting water sample from a reservoir of south Jingxian county of Zhangzhou, fujian, filtering with 5 μm-pore filter membrane, adding 2mL filtrate into 20mL microcystis aeruginosa culture solution in logarithmic phase (algae cell number is about 6×10) 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 a light period of 12/12h (light/dark). After 7 days the culture was observed to change from green to pale yellow and finally to clear. To exclude the contingency of the experiment, lysates containing algae-eating organisms were transferred several times consecutively into microcystis aeruginosa broth, and the final broth color became clear.
(2) The algae-eating organisms are purified by adopting a double-layer flat plate method. First, a lower medium (BG 11 medium+1.2 mass% agar) was prepared, cooled for 20 minutes, and poured into a glass culture dish. Subsequently, an upper medium (BG 11 medium+0.7 mass% agar) was prepared and the temperature was allowed to drop to 40 ℃. Uniformly mixing 4mL of upper culture medium, 100 mu L of lysate containing algae-eating organisms and 1mL of microcystis aeruginosa, pouring the mixture into a glass plate containing coagulated lower culture medium, and setting the mixture into a treatment group; after mixing 4mL of the upper agar medium, 100. Mu.L of the lysate containing the algae-eating organisms and 1mL of BG11 medium, the mixture was poured into another glass plate containing the solidified lower medium and set as a pairAnd (5) group illumination. The double-layer agar plate is placed at 25 ℃ and the illumination intensity is 100 mu mol m -2 s -1 After culturing in an incubator with a light period of 12/12h (light/dark) for 7 days, algal plaques with different diameters are formed on the algal plates.
(3) Picking up single algae plaque into 20mL microcystis aeruginosa culture solution in logarithmic phase (algae cell number is about 6×10) 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 a light cycle of 12/12h (light/dark) for 7 days until the algae culture solution becomes clear, and observing in an optical microscope that the clear solution contains a large amount of transparent algae-eating organisms (figure 1). And (3) taking clear liquid, repeating the steps (2) and (3) for a plurality of times, continuously pouring the double-layer culture medium plate for a plurality of times, and obtaining the relatively pure algae-eating organism NY1 after the system is stable. Algae-eating microorganism NY1 is cultured in liquid BG-11 medium, and microcystis aeruginosa is added every 5-7 days to feed algae-eating microorganism NY1.
(4) 150mL of algae lysate containing algae-eating organisms was aspirated, and cells were collected by centrifugation at 4,500rpm for 5 min. Extracting the whole genome of the algae-eating organism and amplifying the 18S rRNA sequence, and connecting the PCR product by a T vector and then sending the PCR product to Xiamen platinum Rayleigh company for sequencing. The sequences were aligned with DNAstar removal of primers and uploaded to NCBI database (http:// blast. NCBI. Nlm. Nih. Gov/blast. Cgi) and phylogenetic tree was constructed using the Neighbor-Joing method of MEGA7.0 software. As a result, as shown in FIG. 2 a, the similarity between the algae-eating organism NY1 and Poteriospumella lacustris Yongseonkyo072317C3 was as high as 99.83%, and it was judged that it belongs to the Poteriosphaella genus of Phaeophyta (Ochrophyta), chrysophyceae (Chrysophyceae), chromorpha (Chromorphines), conyzophyceae (Dinobryaceae), and was named Poteriospumella lacustris NY1. The 18S rRNA sequence of NY1 was uploaded to NCBI database under GenBank accession number 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 a result, as shown in FIGS. 2 b-d, the NY1 cells were oval or circular in shape, about 15-20 μm long and about 10-12 μm wide. The cells are transparent and free of pigment bodies, and two smooth flagella with almost equal length are arranged at the cell base. The cell internal structure is loose, has a single or multiple vacuolated structures similar to food bubbles, and the nucleus and multiple mitochondria coated by the double-layer membrane are clearly visible, but do not contain chloroplasts.
2. Different phases of feeding of Ny1 into Microcystis aeruginosa were observed
(1) 5mL of the lysate containing NY1 was added to 500mL of microcystis aeruginosa culture broth in logarithmic growth phase (the number of algal cells was about 6X 10) 6 cells/mL), 100mL was sampled after 0, 24, 48, 72h, and cells were collected by centrifugation at 4,500rpm for 5 min.
(2) Different phases of NY1 feeding microcystis aeruginosa were observed under an optical microscope and transmission electron microscopy. As can be seen from fig. 3, during the non-feeding phase, NY1 is smaller in volume and the cells do not contain algae cells; during the initial stages of ingestion, NY1 rapidly moves in water to increase the probability of contact with algal cells, then directly contacts the algal cells through cell membranes, and engulfs the whole algal cells into the cells; in the middle stage of ingestion, microcystis aeruginosa is gradually digested in NY1 food bubbles, the microcystis cytoskeleton and cell walls are destroyed, and cells are no longer regular and full; at the end of ingestion, the cell structure of microcystis aeruginosa becomes loose and the contents flow out and disperse in the food bubbles of NY1; from this, it is inferred that NY1 achieves the effect of eliminating algal cells by directly taking whole algal cells, followed by digestion in its food bubbles.
3. Determination of the feeding efficiency of NY1 on different fresh algae
(1) Algae species used in the experiments: the flat algae (Platymonas subcordiformis CCMA-418), chlorella (Chlorella vulgaris CCMA-410), synechocystis sp.PCC6803, microcystis aeruginosa (Microcystis aeruginosa PCC7806, PCC1752 and PCC 7820) are all purchased from the institute of aquatic life of the national academy of sciences. Culturing algae in glass triangular flask at 25deg.C with BG11 liquid culture medium and illumination intensity of 100 μm -2 s -1 The illumination period was 12/12h (light/dark).
(2) Culturing different algae to logarithmic growth phase, subpackaging the algae liquid into 12-hole plates, subpackaging 4mL each hole, and placing in an illumination incubator for adaptation for 24h. Addition of 1X 10 to treatment group 4 cells/mL NY1, controlGroups were not treated and 3 plain biological replicates were set for each group. The algae cell number is measured by means of a cell counter, and the clearance rate of NY1 to algae cells is calculated as follows: clearance (%) = (C) 0 -C t )÷C 0 X 100%, where C 0 Number of algae cells of 0h, C t The number of algal cells corresponding to the treatment time.
(3) The results are shown in Table 1, with the exception of M.aerobosa PCC7806, NY1 exhibited very strong feeding habits for M.aerobosa PCC1752, M.aerobosa PCC7820 and synecholysis sp.PCC6803, and after 48h treatment, the clearance rate for algae cells reached 98%. Besides cyanobacteria, NY1 also has higher clearance rates of 78.69% and 71.82% for chlorella C.vulgaris CCMA-410 and P.subtorodiformi CAMA-418 of Chlorella. The method shows that the NY1 has wider algae ingestion spectrum and high removal efficiency, and has great potential in controlling harmful water bloom caused by the fresh water algae.
TABLE 1 clearance of NY1 to different algae within 48h
4. Determination of growth Rate of NY1 and Rate of feeding Microcystis aeruginosa cells
(1) Culturing Microcystis aeruginosa to 5×10 respectively 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. And subpackaging the algae liquid into 12-hole plates, subpackaging 4mL of algae liquid into each hole, and placing the algae liquid into an illumination incubator for adaptation for 24 hours. Adding 5×10 to the algae culture solution 4 cells/mL NY1, 3 biological replicates per group were set. 20. Mu.L were sampled at 0, 12, 24, 36, 48, 60 and 72 hours, respectively, and the cell numbers of NY1 and Microcystis aeruginosa were determined by means of a cytometer.
(2) The specific growth rate μ of NY1 cells is calculated as follows: mu= (lgN) t -lgNt 0 )÷(t-t 0 ) Wherein Nt is the number of algae cells corresponding to the later period t of the index, N 0 Is the exponential prophase t 0 Corresponding algae cell number, t is the time corresponding to the later period of the index, t 0 The time corresponding to the exponential prophase. The passage time G was calculated as follows: g=ln2++μ, where μ is the growth rate.
(3) As is clear from Table 2, NY1 has a high feeding rate and a good algae removal effect on microcystis aeruginosa, and the initial cell number is 5×10 4 cell/mL of NY1 was effective in removing 4X 10 cells in a short period of time 6 -5×10 7 cell/mL of microcystis aeruginosa. The higher the initial cell number ratio of NY1 to microcystis aeruginosa is, the higher the clearance rate of NY1 to algae cells is, and when the initial cell number ratio is more than 1:200, the time for the clearance rate to reach 90% is 24 hours; when the ratio of the initial cell number is less than 1:1000, 72 hours are required for the clearance to reach 90%. In addition, at a ratio of 1:200, the NY1 specific growth rate was the largest and the passage time was the shortest, respectively 0.099h -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
Kinetic profile of Ny1 feeding microcystis aeruginosa
(1) Microcystis aeruginosa was inoculated into 50mL BG11 medium and cultured to logarithmic growth phase (cell number about 6×10) 6 cells/mL). Adding 6×10 of the culture solution to the algae 3 、1×10 4 And 3X 10 4 cells/mL of NY1 were such that the initial cell number ratios of NY1 and Microcystis aeruginosa were 1:1000,1:600 and 1:200, respectively, 20. Mu.L was sampled at 0, 12, 24, 36, 48, 60, 72, 84 and 96h, respectively, and both cell numbers were determined by means of a cytometer.
(2) As shown in FIG. 4, the number of microcystis aeruginosa cells in the control group was gradually increased, and 3X 10 cells were added 4 When cells/mL of NY1 (1:200), the number of algae cells is obviously reduced within 24 hours, and the clearance rate reaches 92.82 percent. When the number of NY1 cells is 1×10 4 cells/mL (1:600) and 6X10) 3 At cell/mL (1:1000), the solution was clearedThe time for the removal rate to reach 90% is 48h and 72h respectively, which shows that NY1 can rapidly and efficiently remove microcystis aeruginosa, and the removal rate has concentration-dependent effect. In addition, the number of cells of NY1 showed a tendency to increase and decrease. When algal cells are abundant, NY1 has enough food sources to sustain its survival, and the cell number increases rapidly; once the algae cells are completely cleared, NY1 gradually dies due to lack of food, and the cell number is significantly reduced, because NY1 itself does not have chloroplasts, cannot autotrophy through photosynthesis, and mainly depends on external food for growth.
6. Detection of microcystin content
(1) Inoculating Microcystis aeruginosa into 50mLBG11 medium, and culturing to logarithmic phase (cell number is about 6X10) 6 cells/mL). Adding 6×10 of the culture solution to the algae 3 、1×10 4 And 3X 10 4 cells/mL of NY1, so that the initial cell number ratio of NY1 to microcystis aeruginosa is 1:1000,1:600 and 1:200 respectively, and 1mL of algae liquid is taken at 0, 24, 48 and 72 hours respectively.
(2) 1mL of the sample is crushed by an ultrasonic cell crusher (voltage 100W, working time 5s, interval time 5s and circulation 60 times), the crushed lysate is centrifuged at 7,000-8,000rpm for 8-10min, and the supernatant is taken as the solution to be measured for the algae toxin. All supernatants in the control group and supernatants at 0 and 24h 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 algae toxin content is carried out according to a microcystin ELISA kit (Beacon, cat. #)
20-0068). The microcystin degradation rate is calculated as follows: degradation rate (%) = (M 0 -M t )÷M 0 X 100%, where M 0 An algal toxin content of 0 h; m is M t The content of the algae toxin is corresponding to the treatment time.
(4) As a result, as shown in FIG. 5, initially, the intracellular and extracellular microcystins were present in the respective culture systems at 203.99 and 24.51. Mu.g/L, respectively. After 72h of incubation, the intracellular and extracellular algal toxin concentrations in the control group increased to 305.76 and 67.83 μg/L, respectively; in each treatment group, the concentration of the microcystin decreases with the decrease of the algae cells, and the total microcystin degradation rates at the ratios of 1:200, 1:600 and 1:1000 are 93.88%, 93.20% and 93.47% respectively at 72h, which indicates that NY1 can efficiently degrade the algae toxins without accumulating the algae toxins in vivo.
7. Efficiency of NY1 feeding microcystis aeruginosa and degrading algal toxins under different environmental factors
(1) Inoculating Microcystis aeruginosa into 50mLBG11 medium, and culturing to logarithmic phase (cell number about 6X10) 6 cells/mL). Addition of 1X 10 to treatment group 4 cells/mL of NY1 was such that the initial cell number ratio of NY1 to microcystis aeruginosa was 1:600, whereas the control group was not supplemented with NY1, and 3 biological replicates were set per group.
(2) After treatment under different environmental conditions, 20 μl was sampled every 12h in a sterile operating table and the cell numbers of NY1 and microcystis aeruginosa were determined by means of a cytometer. 1mL of the sample was sampled every 24 hours, and the total microcystin content was measured with reference to the above 6- (1).
(3) Effect of different temperatures on NY1 action effect: the temperatures were set at 15, 20, 25, 30 and 35 c, respectively. At pH of 8, illumination intensity of 50-100 μm -2 s -1 Culturing under light cycle of 12/12h (light/dark). As a result, as shown in FIG. 6, when the algae culture solution to which NY1 was not added and the algae culture solution to which NY1 was added were at the same temperature, the color of the latter was significantly reduced, and the number of algae cells was significantly reduced. In the first 48 hours, when the temperature range is 30-35 ℃, the clearance rate of NY1 to microcystis aeruginosa is up to 90.29%; and the clearance rate is as low as 14.83% when the temperature is 15-20 ℃. However, by 72 hours, almost all algal cells in the treatment group were cleared by NY1 at each temperature, and the clearance was higher than 97%. The concentration of the algae toxins is consistent with the variation trend of the algae cell number, after the algae toxins are cultured for 72 hours at the temperature of 15-35 ℃, the total algae toxin concentration in the control group is about 280 mug/L, the total algae toxin concentration in the treatment group is lower than 5.50 mug/L, and the degradation rate is higher than 97%. The results show that NY1 is affected early in the temperature change, and once it adapts to the environmental changes, it rapidly feeds, eventually completely eliminating algal cells and degrading algal toxins.
(3) Different light intensity pairsEffect of NY1 action effect: the light intensities were set to 0, 25, 50, 100 and 200. Mu. Mol m - 2 s -1 Culturing under the condition that the pH value of the culture medium is 8, the temperature is 25 ℃ and the illumination period is 12/12h (illumination/darkness). As can be seen from FIG. 7, the illumination intensity ranges from 0 to 200. Mu. Mol m -2 s -1 When the algae culture solution is used, the NY1 has higher clearance rate and degradation rate, and compared with a control group without the NY1, the color of the algae culture solution in a treated group with the NY1 is obviously lightened, the number of algae cells is obviously reduced, and the clearance rate is higher than 90% at 48 hours. Meanwhile, detecting the concentration of the microcystins in each culture solution under different illumination intensities, wherein the total microcystins in the control group are about 235.96 mug/L at 72 hours; the concentration of the algae toxin in the treatment group is about 8.63 mug/L, and the degradation rate reaches 98.77 percent. Experiments show that the molecular weight of the catalyst is 0-200 mu mol m -2 s -1 The efficiency of Ny1 feeding microcystis aeruginosa and degrading algal toxins is not affected, and the biological process of Ny1 feeding and degrading is presumed to be independent of light.
(4) Effect of different pH on NY1 action effect: the pH was set to 6, 7, 8, 9, 10, respectively. At 20-27deg.C, the illumination intensity is 50-100 μm -2 s -1 Culturing under light cycle of 12/12h (light/dark). As can be seen from FIG. 8, when the pH value is in the range of 6-10, the growth state of microcystis aeruginosa is good and the growth speed of cells is not obviously different in the control group without NY1; after the NY1 is added to treat the copper green microcapsule, the reduction rate of the algae cell number in each treatment group is not obviously different, and the clearance rate of the NY1 to the algae cell is higher than 98% and the degradation rate to the microcystin is higher than 97% at 72h. Experiments show that when the pH value is 6-10, the efficiency of feeding microcystis and degrading algae toxins is not affected by NY1, which indicates that the NY1 can tolerate the pH range of 6-10, and the cells can quickly adjust own mechanisms to adapt to pH change.
Efficiency of NY1 in eliminating microcystis aeruginosa and degrading microcystin different growth periods
(1) The initial cell number was measured to be 2.01X10 when microcystis aeruginosa was inoculated into 3L BG11 medium 6 cells/mL. 3 groups were constructed separately, treatment group 1 (T0 group): 1X 10 was added to the algal culture solution on day 0 4 cells/mL NY1; treatment group 2 (T10 group): 3.8X10 additions to the algae culture on day 10 4 cells/mL NY1; control group was not supplemented with NY1, and 3 plain biological replicates were set per group.
(2) 20. Mu.L was sampled every 2 days, and the algal cell number was measured by means of a cytometer. 1mL of the sample was sampled every 2 days, and the total microcystin content was measured by referring to the above 6 (1).
(3) As a result, as shown in FIG. 9, NY1 was administered in the lag phase (day 0) and the exponential phase (day 10) of algal cell growth, respectively. At day 0, the cell density of Microcystis aeruginosa was 2.01X10 6 The concentration of microcystin in cells/mL was 53.15. Mu.g/L, and after addition of NY1, the number of algal cells was reduced to 2.70X10 s in 96 hours 5 The cell/mL has a clearance rate of 87.24%; the concentration of the algae toxin is reduced to 10.32 mug/L, and the degradation rate is 80.58 percent. At day 10, the cell density of microcystis aeruginosa was 7.67×10 6 The concentration of microcystin in cells/mL is 416.52 mug/L, and after NY1 is added, the number of algae cells in 96h is reduced to 1.77×10 5 The cell/mL has a clearance rate of 97.69%; the concentration of the algae toxin is reduced to 28.74 mug/L, and the degradation rate is 93.10 percent. By day 18 the algae cells in both treatment groups were almost completely cleared, with algae toxin concentrations below 2.10 μg/L. The experimental results show that NY1 can obviously reduce the number of microcystis aeruginosa cells and reduce the microcystin content no matter being put in the delay period or the exponential period of microcystis aeruginosa, and effectively control the cyanobacterial bloom outbreak.
Concentration variation of soluble organics during microcystis removal by NY1
(1) The experimental group was set according to 8 (1) above.
(2) The 40mL brown borosilicate glass bottle was soaked in 2M HCl for 24h to remove residual inorganic salts, washed three times with single distilled water and double distilled water respectively, and dried for later use. The dried brown borosilicate glass bottle and the 0.45 mu m acetate fiber filter membrane are wrapped by tinfoil paper and are placed in a muffle furnace for calcination at 450 ℃ for 4 hours to remove organic carbon on the tube wall. Respectively taking 10mL of each group of algae liquid, and filtering and collecting the algae liquid into a brown silicon boride glass bottle through an acetate fiber filter membrane.
(3) Each sample was detected using a fluorescence spectrophotometer, and three-dimensional fluorescence spectrum scanning was performed using ultrapure water as a blank and a 1cm quartz cuvette. The slit width of the excitation monochromator was 10nm, and the slit width of the emission excitation monochromator was 10nm. The excitation wavelength (Ex) ranges from 220-450nm, the step is 5nm; the emission wavelength (Em) ranges from 250-550nm, stepping to 5nm.
(4) As shown in FIG. 10, in the control group, 3 distinct fluorescence peaks gradually appear with the prolongation of the culture time of Microcystis aeruginosa, and are distributed in fluorescence regions III (Ex/Em, 200-250/380-550), IV (Ex/Em, 250-450/280-380) and V (Ex/Em, 250-450/380-550), respectively, furilic acids, soluble microbial products and humic substances produced by algal cells; in the T0 group, since the initial algal cell number is small and NY1 is rapidly cleared in a short time, the soluble organic matters in the water body are hardly detected; in the T10 group, algae cells slowly grow in the first 6 days, released extracellular soluble organic matters are less, characteristic fluorescent peaks do not appear, after a large number of algae cells are swallowed by NY1 after 10 days, a part of soluble organic matters are released, a fluorescent peak appears in a water body, the peak is mainly concentrated in an IV region, and a large number of soluble microorganism products are contained in the water body. In summary, the experimental results show that compared with the control group, the humic acid, fulvic acid and other components in the two treatment groups are obviously reduced, and presumably, the NY1 directly swallows the microcystis and is decomposed into most macromolecular substances in the cells, so that the release of soluble organic substances outside the cells is reduced.
NY1 eliminates biotoxicity of water body in microcystis aeruginosa process
10.1NY1 the toxicity of water body to luminous bacillus after removing microcystis aeruginosa
(1) Setting 5 groups respectively: positive control (BG 11 medium), negative control (microcystis aeruginosa lysate), microcystis aeruginosa broth without NY1 added, microcystis aeruginosa broth with NY1 added on day 0 and day 10 respectively, each set with 3 flat biological replicates.
(2) 1mL of sample was collected on days 0, 6, 12 and 18, the collected sample was crushed by an ultrasonic cell crusher (voltage 100W, working time 5s, interval time 5s, 60 cycles), and the lysate was centrifuged at 6,000rpm for 10min, and the supernatant was taken as the test solution.
(3) The luminous bacilli were activated and cultured to log phase. Respectively taking 450 mu L of the liquid to be detected in a 1.5mL centrifuge tube, adding 50 mu L of 30% NaCl solution to make the final salinity of the sample be 3%, and taking double distilled water with the salinity of 3% as a blank control group.
(3) For every 1 sample tube to be measured, 1 control tube is measured simultaneously. 10. Mu.L of the luminous bacillus was added to each sample tube, and the reaction was immediately timed for 15min, and the fluorescence value of the sample tube was measured by using a chemiluminescent apparatus. The relative luminous efficacy is calculated as follows: relative luminous efficiency (%) = (sample luminosity +.control luminosity) ×100%
(4) As shown in FIG. 11, BG11 medium had no inhibitory effect on the luminescence intensity of P.brightly; the toxicity to the luminous bacillus in the cracking liquid after the ultrasonic breaking of the microcystis aeruginosa is extremely strong, and the relative luminous rate of the luminous bacillus is only 10%; in the microcystis aeruginosa culture solution without NY1, algae cells continuously release algae toxin in the growth process, the luminous intensity of the luminous bacillus is gradually inhibited, and the relative luminous rate in the 6 th to 18 th days is lower than 50%; in the T0 group, since the algae cells were rapidly cleared by NY1 on day 0, the luminous intensity of the luminous bacillus was not inhibited within 18 days; in the T10 group, at the 6 th day, the water body contains algae toxins, the relative luminous rate of the luminous bacillus is 47.16%, at the 12 th to 18 th days, the algae toxins in the water body are degraded by NY1, the toxicity is relieved, and the luminous intensity of the luminous bacillus is not obviously different from that of the BG11 culture medium. The result shows that NY1 can reduce the toxicity of water body to luminous bacillus after algae removal.
10.2NY1 the toxicity of water body to daphnia magna after removing microcystis aeruginosa
(1) Setting 5 groups respectively: positive control (BG 11 medium), negative control (microcystis aeruginosa lysate), microcystis aeruginosa broth without NY1 added, microcystis aeruginosa broth with NY1 added on day 0 and day 10 respectively, each set with 3 flat biological replicates.
(2) All groups of liquids were dispensed into 50mL beakers, each beaker containing 20mLEach group was set with 3 parallels. And placing 26 daphnia magna with good life state and active swimming in each beaker, observing the states of the daphnia magna at 24, 48 and 72 hours respectively, and counting the number of surviving daphnia magna. The mortality calculation formula for daphnia magna is as follows: mortality (%) = (D) 0 -D t )÷D 0 X 100%, where D 0 To treat 0h of daphnia, D t The number of daphnia corresponding to the treatment time.
(3) As shown in FIG. 12, in BG11 medium, daphnia magna had a natural mortality rate of 25.03% within 72 hours; the toxicity of the algae lysate to the daphnia magna is extremely strong, leading to the death rate of the daphnia magna within 72h up to 98.72%; microcystis aeruginosa releases less algae toxin into the water body within the first 12 days, the death rate of daphnia magna is only 24.62%, but when the microcystis aeruginosa grows to 18 days, the algae toxin in the water body is continuously accumulated, and the death rate of daphnia magna is increased to 68.12%; however, the T0 and T10 treated groups showed good daphnia growth, substantially no acute toxicity symptoms, and the daphnia mortality rates at day 18 were 30.34% and 25.67%, respectively, indicating that NY1 reduced water toxicity after algae removal, thereby reducing daphnia mortality.
10.3NY1 the toxicity of water body to grass carp fries after removing microcystis aeruginosa
(1) Setting 5 groups respectively: positive control (BG 11 medium), negative control (microcystis aeruginosa lysate), microcystis aeruginosa broth without NY1 added, microcystis aeruginosa broth with NY1 added on day 0 and day 10 respectively, each set with 3 flat biological replicates.
(2) All groups of liquid are respectively packaged in 300mL of preservation boxes, each preservation box is respectively packaged in 200mL, and each group is provided with 3 parallel. 51 grass carp fries with good life state and active swimming are placed in each beaker, water and feeding are not changed during the experiment, and dead fries are cleaned in time. The status of grass carp fries was observed and the number of surviving grass carp fries was counted at 24, 48, 72 hours, respectively, and the mortality was calculated.
(3) As shown in fig. 13, the juvenile fish in the BG11 culture medium swim normally without obvious poisoning characteristics, and the natural mortality rate is 14.38%; the algae lysate shows extremely high fish toxicity, the juvenile fish does not swim any more, the body curls, and finally all the juvenile fish die; the microcystis aeruginosa culture solution without NY1 shows a certain fish toxicity to grass carp, and when algae cells grow for 18 days, toxic substances in the water body are accumulated in a large amount, 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 fries have poisoning symptoms, and the death rate of the fries is lower than 28.02% within 18 days, which indicates that after the algae removal of NY1, the toxicity of the water body can be reduced, so that the death rate of the fries is reduced.
While the foregoing description of the embodiments of the present invention has been presented with reference to the drawings, it is not intended to limit the scope of the invention, but rather, various modifications or variations can be made by those skilled in the art without the need of inventive effort on the basis of the technical solutions of the present invention.
Claims (5)
1. The dinoflagellate Poteriospumella lacustris NY1 capable of feeding microcystis aeruginosa and degrading microcystin is characterized in that the dinoflagellate Poteriospumella lacustris NY is preserved in China center for type culture collection, the preservation time is 2022, 8 months and 17 days, and the preservation address is: in the eight-path 299-grade university of Wuhan in Wuchang district of Wuhan, hubei province, the preservation number is CCTCC NO: m20221293.
2. Use of the dinoflagellate of claim 1 in the preparation of an algaecide; the algae removing agent is used for removing algae such as blue algae, green algae such as microcystis aeruginosa and synechocystis, and green algae such as Fucus vesiculosus and Chlorella.
3. The use of the dinoflagellate of claim 1 in the control of harmful algal bloom; the harmful algal bloom is blue algae and green algae, the blue algae is microcystis aeruginosa or synechocystis, and the green algae is flat heart alga or chlorella.
4. An algicide, characterized in that the effective component is the dinoflagellate culture algae liquid of claim 1.
5. A degradation agent for reducing microcystin in water is characterized in that the effective component is the dinoflagellate culture solution of claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111826318A (en) * | 2020-07-30 | 2020-10-27 | 中冶华天工程技术有限公司 | Microcystis aeruginosa dissolving bacterium and application thereof |
| CN114854725A (en) * | 2021-02-04 | 2022-08-05 | 目立康株式会社 | Algaecide and algaecide method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111826318A (en) * | 2020-07-30 | 2020-10-27 | 中冶华天工程技术有限公司 | Microcystis aeruginosa dissolving bacterium and application thereof |
| CN114854725A (en) * | 2021-02-04 | 2022-08-05 | 目立康株式会社 | Algaecide and algaecide method |
Non-Patent Citations (2)
| Title |
|---|
| Intraspecific Variation in Protists: Clues for Microevolution from Poteriospumella lacustris (Chrysophyceae);Stephan Majda等;Genome Biol Evol;第11卷(第9期);第2492-2504页 * |
| 绿色鞭毛藻类的新种及中国新记录种;陈珊等;武汉植物学研究;第20卷(第3期);第191-198页 * |
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