CN114958706A - Method for improving capacity of bacillus for removing red tide algae cells - Google Patents
Method for improving capacity of bacillus for removing red tide algae cells Download PDFInfo
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- CN114958706A CN114958706A CN202210375643.9A CN202210375643A CN114958706A CN 114958706 A CN114958706 A CN 114958706A CN 202210375643 A CN202210375643 A CN 202210375643A CN 114958706 A CN114958706 A CN 114958706A
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention belongs to the technical field of harmful red tide prevention and control, and particularly relates to a method for improving the capacity of bacillus for removing red tide algae cells. The bacillus is mixed with clay to enrich the bacillus on the surface of the clay, thereby improving the capacity of the bacillus to remove red tide algae cells. According to the method, the biomass and the algae removal capacity of the bacillus can be improved by co-culturing the clay and the bacillus; meanwhile, the premixed suspension obtained after co-culturing the bacillus and the clay is dried to be made into a powdery product, and the active bacillus capable of being stored for a long time is obtained. The method does not produce secondary pollution in the using process, and is simple to operate, economic and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of harmful red tide prevention and control, and particularly relates to a method for improving the capacity of bacillus for removing red tide algae cells.
Background
The red tide is an ecological abnormal phenomenon that micro plankton in the sea rapidly proliferate or gather to cause the damage of the structure and the function of an ecological system, and belongs to typical marine ecological disasters. The occurrence of red tide not only seriously damages the offshore ecological environment, but also seriously restricts the development of marine economy, and even seriously threatens the safety of marine nuclear power. In recent years, red tide has a global expansion situation, and the damage range and degree are increased year by year. Therefore, it is more important to find more effective and safer red tide treatment methods.
The fundamental principle of treating the red tide is to reduce the algae density of the red tide organisms, inhibit the growth and diffusion of the red tide organisms and reduce the harmful effect caused by the outbreak of the red tide; based on this principle, treatment methods can be classified into physical methods, chemical methods, biological methods, and mineral flocculation methods. The physical method mainly reduces the number of algae cells by manual salvage, mechanical stirring, ultrasonic crushing, air floatation, ultraviolet irradiation and other methods, and the use cost of the physical method is higher. Chemical methods refer to killing algae cells in a body of water using chemical action or reducing the number of algae cells by means of chemical flocculation. The common algicide adopts the algicide principle that strong oxidation effect or biotoxicity effect is utilized, and chemical reagents are easy to cause secondary pollution in the using process and damage other organisms in the environment. The biological method is to control the proliferation of red tide organisms and even kill the red tide organisms by using the functions of competing, predating or secreting allelochemicals and the like by using other organisms in the marine ecosystem, such as viruses, fungi, bacteria, aquatic animals, aquatic plants and the like; compared with chemical and physical methods, the biological method has the advantages of lower cost, environmental friendliness and the like. At present, algae-lysing bacteria are easy to reproduce, are widely distributed in natural water, have certain ecological advantages and have strong algae-lysing capability, and become a research hotspot for red tide treatment. The algicidal bacteria can crack the algae cells by means of direct attack, indirect secretion of algicidal substances and the like, so that the algae cells die, and the number of red tide organisms can be continuously reduced. The algae-lysing bacteria are very abundant in species, including pseudoalteromonas, vibrio, bacillus, alteromonas mairei, coccus, etc. The bacillus can remove algae, is commonly used as probiotics and widely applied to aquaculture industry, and is expected to become beneficial algae removal bacteria to be applied to red tide treatment; however, most of related researches are limited in a laboratory, and when the research is actually applied to a complex water body environment, the effect is unstable, the action time is long, and the survival and the effectiveness are difficult to ensure. The mineral flocculation method is to utilize natural clay minerals to treat red tide, and the main reasons for the effect are as follows: the clay mineral has hydrophilicity, larger specific surface area and strong capacity of adsorbing solid, gas, liquid and substances dissolved in the liquid, so that excessive nutrient substances such as nitrogen, phosphorus and the like in a water body can be adsorbed and settled to destroy the living environment of algae cells; ② the clay mineral can be attached on the surface of the algae cell to flocculate and settle the algae cell to inactivate the algae cell. However, the flocculation ability of natural clay mineral is low, and Shu Shiming et al propose a method for surface modification of natural clay mineral and develop modified clay with high flocculation ability, thus greatly improving the application value of the method.
As mentioned above, the algicidal bacteria are easy to reproduce, have high algae removal effect and low cost, can continuously control red tide organisms, and have certain ecological advantages, but have the defects of poor response and difficult survival and the like in the practical application process; the clay mineral can flocculate and settle algae cells quickly. Therefore, the invention utilizes the advantages of the algae removal materials to obtain the novel composite algae removal material which has strong removal capability and can be applied in large scale.
The invention content is as follows:
aiming at the defects in the technology, the invention provides a method for improving the capacity of bacillus for removing red tide algae cells based on co-culture of bacillus and clay.
In order to realize the purpose, the invention adopts the technical scheme that:
a method for improving the capacity of bacillus to remove red tide algae cells is characterized in that bacillus is selected and mixed with clay, so that the surface of the clay is enriched with the bacillus, and the capacity of the bacillus to remove the red tide algae cells is improved.
Further, the bacillus-containing culture solution is mixed with clay, and co-cultured after mixing to obtain suspension, so that the red tide removing capability of the bacillus can be improved; wherein, when the culture solution containing the bacillus is mixed with the clay, the proportion of the clay to the bacillus is as follows according to the mass (g) of the clay: cell number (cells) was measured and controlled at 1: 5X 10 5 -2×10 10 。
And drying the suspension to enable the liquid to be solidified to prepare the product. Further, the liquid is solidified to prepare the product by freeze drying, centrifugal spray drying and other modes.
The suspension mixed with the clay and the culture solution containing the bacillus is aged and cultured for 8-24h at the shaking frequency of 0-300rpm (preferably 150-250rpm) and the temperature of 10-40 ℃ so as to promote the bacillus to fully grow and propagate under the action of the clay and to be aggregated on the surface of the clay.
The bacillus is one or more of bacillus amyloliquefaciens, bacillus subtilis, bacillus brevis, bacillus cereus and bacillus pumilus.
The bacillus is inoculated into LB culture medium, 2216E culture medium or nutrient broth culture medium to obtain a culture solution containing the bacillus.
The clay particles are one or more than one of aluminosilicate minerals and/or one or more than one of modified aluminosilicate minerals.
The clay particles are aluminosilicate minerals such as kaolin, bentonite, sepiolite, attapulgite and the like; may be a mixture of the above clays; the modified clay may be a modified derivative of the above clay, for example, a modified clay modified with an inorganic cation containing iron, aluminum, or the like, or an organic flocculant such as chitosan, PAM, or the like.
The invention has the advantages that:
according to the invention, the algicidal bacteria (bacillus) and the clay with strong emergency property are combined for use, so that the removal effect of the algicidal bacteria and the survival rate in practical application are improved, and the algicidal bacteria can be used as a red tide treatment technology with both acute and ecological advantages; further, the following steps are carried out:
according to the invention, the probiotic bacillus commonly used in aquaculture is co-cultured with the clay to obtain the premixed suspension, so that the maximum biomass of the bacillus can be improved, the bacillus can be promoted to gather and concentrate on the surface of the clay, the local concentration of the bacillus and secretion thereof in the algae removal process is improved, and the algae removal capability of the bacillus is further improved. After the suspension is made into a powdery product through drying, the activity of the bacillus in long-term storage can be ensured, the bacillus is easier to store and transport, the bacillus is simpler and more convenient to use, has no secondary pollution, has certain ecological advantages, and has better application prospect.
Description of the drawings:
FIG. 1 is a graph showing the effect of different kinds of clay on the biomass (b) and the removal ability (a) of Bacillus amyloliquefaciens according to the present invention.
FIG. 2 is a graph showing the effect of co-culturing Bacillus with clay for different periods of time on the biomass (b) and removability (a) of Bacillus amyloliquefaciens according to an embodiment of the present invention.
FIG. 3 is a flow chart of the co-cultivation of Bacillus and Clay and liquid drying process provided by the embodiment of the present invention.
FIG. 4 is an electron microscope image of a product prepared by co-culturing Bacillus amyloliquefaciens and clay provided by an embodiment of the present invention.
FIG. 5 is a diagram showing the removal effect of different red tide algae on the product prepared by co-culturing the Bacillus amyloliquefaciens and clay provided by the embodiment of the invention.
FIG. 6 is a graph showing the effect of kaolin on the removal of different Bacillus bacteria according to an embodiment of the present invention.
The specific implementation mode is as follows:
the following examples are presented to further illustrate embodiments of the present invention, and it should be noted that the embodiments described herein are intended to illustrate and explain the present invention and are not intended to limit the present invention.
The invention adopts the co-culture of the bacillus and the clay, has simple equipment and less process flow, does not need to add other chemical reagents, does not need other special treatment and does not produce secondary pollution.
Both the bacillus and the original source of clay used in the examples described below are commercially available.
Example 1
Respectively weighing 0.5g of kaolin, montmorillonite and bentonite, respectively adding into a 250mL triangular conical flask, sterilizing with high-temperature steam at 121 deg.C for 30min, oven drying at 80 deg.C for 1h, sterilizing under ultraviolet lamp for 20min, and cooling to room temperature to obtain three parts of sterile clay.
0.5g of different sterile clays were added to 20mL of sterile LB liquid medium, respectively, and inoculated with 10g of a solution 6 Putting cells/mL of bacillus amyloliquefaciens into an LB liquid culture medium, and placing the cells/mL of bacillus amyloliquefaciens into a constant-temperature shaking culture box (30 ℃, 200rpm) for shaking culture for 24 hours to obtain a co-culture suspension of the bacillus in different clays; the other group is a bacillus amyloliquefaciens bacterial liquid group without adding clay: the inoculation amount is 10 6 cells/mL are inoculated into a sterile LB liquid medium, and the rest culture conditions are consistent with those of a clay-added group except that no clay is added; while clay alone was used as a control.
Wherein, the bacillus amyloliquefaciens is purchased from Qingdao root-sourced organism Limited, and is separated and purified by a conventional method according to the record in the specification to obtain the purified bacillus amyloliquefaciens. Kaolin clay was obtained from Bintang PUSPITA bumiwopa, Indonesia, and montmorillonite and bentonite from Aladdin.
The steps for separating and purifying the purified bacillus amyloliquefaciens by the conventional method are as follows: and culturing the purchased strain for more than 24 hours by using an LB solid plate culture medium in an incubator at the temperature of 30 ℃, streaking the bacillus amyloliquefaciens if no mixed bacteria grow, and placing the strain in a constant-temperature incubator to grow for 24 hours after streaking is finished to obtain the purified target strain.
Collecting original dinoflagellate solution (with cell density of about 1 × 10) of east China sea in exponential growth phase 8 cells/L) to 50mL of colorimetric cylinder, and adding the bacillus amyloliquefaciens and clay co-culture suspension, the bacillus amyloliquefaciens bacterial solution group and the clay into the colorimetric cylinder respectively, wherein the clay group alone enables the clay in the colorimetric cylinder to be concentrated finallyThe degree is 0.075g/L, and the addition volume of the bacillus amyloliquefaciens bacterial liquid group and the bacillus amyloliquefaciens and clay co-culture suspension group is the same, specifically 0.15 mL.
After the above-mentioned components were left standing for 24 hours, the algae removal rate was calculated (see FIG. 1 a).
Counting the bacillus amyloliquefaciens and clay co-culture suspension (microorganism composite clay group) and the bacillus amyloliquefaciens bacterial liquid (microorganism group without clay) by adopting a dilution coating flat plate counting method; the dilution coating plate counting method comprises the following steps: absorbing 1mL of fermentation broth (microorganism composite clay group, microorganism group without clay), adding into 9mL of sterile physiological saline, and shaking for 5s on vortex oscillator to obtain 10 -1 Diluting the solution again from 10 -1 Taking 1mL of the diluted solution, adding into 9mL of sterile physiological saline to obtain 10 -2 Dilution, or the like, to 10 -6 、10 -7 Take 10 -6 、10 -7 0.1mL of the dilution was applied to an LB solid plate, and the plate was incubated at 30 ℃ in an incubator for 1d until a clear colony grew, and the number of colonies was counted (see FIG. 1 b).
The results in fig. 1 show that the clay (kaolin, montmorillonite, bentonite) can increase the biomass of bacillus amyloliquefaciens (fig. 1a) and can increase the removal effect on prorocentrum donghaiense (fig. 1 b).
Example 2
The co-cultivation method described in example 1 was followed, using 25g/L clay and 10 6 cells/mL Bacillus amyloliquefaciens co-culture suspension liquid with different time (0h, 8h, 12h and 24h) and the Bacillus amyloliquefaciens bacterial liquid without clay.
The algae removal efficiency of the two groups of prorocentrum donghaiense at different culture times was tested using the algae removal and biomass counting experiments described in example 1 (see figure 2).
The result shows that the algae removal effect is better when the bacterial biomass of the clay and the bacillus amyloliquefaciens in the co-culture suspension is more along with the increase of the co-culture time, wherein the biomass is the highest and the algae removal effect is the best in 24 h.
Example 3
The flow chart of the preparation process of bacillus co-culture with clay and liquid drying as shown in figure 3 is as follows:
placing the purchased strain in an LB solid plate culture medium in an incubator at 30 ℃ for more than 24h, streaking the bacillus amyloliquefaciens if no infectious microbes grow, placing the strain in a constant-temperature incubator for 24h after streaking, dipping mature target strains, streaking the strains in a plate in a Kirschner flask (50 mL of LB solid culture medium is filled in each Kirschner flask), culturing the strains for 48h at 30 ℃ until clear colonies grow out, and paving the strains in the solid culture medium in the Kirschner flask for later use.
Culturing according to LB culture medium, 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 25g/L kaolin, weighing the required raw materials, putting into a 50L fermentation tank, filling 35L liquid, adding water to about 32L after air consumption (partial condensed water is generated after sterilization), starting stirring, mixing uniformly, and adjusting pH to 7.0 with NaOH.
And sterilizing and cooling the culture medium of the fermentation tank to 30 ℃, sucking 100mL of sterile normal saline into a Kirschner bottle, scraping the bacillus amyloliquefaciens bacterial colony from the Kirschner bottle into the sterile normal saline, and transferring the normal saline with the bacillus amyloliquefaciens bacterial liquid into a 50L fermentation tank for fermentation culture. The culture conditions were: 30 ℃, stirring speed 300rpm, sterile air flux 1: 1, the pot pressure is 0.05 Mpa. After fermenting for 24h, stopping the tank and spray drying the co-culture suspension.
Drying a suspension sample (fermentation broth after the fermentation is stopped after 24 hours) co-cultured by bacillus and clay by using a centrifugal spray drying tower, wherein the operation process is as follows: firstly, opening an air inlet; secondly, starting a preheating program to enable the temperature of air at the air inlet to reach 175 ℃; thirdly, when the temperature of the wind reaches 175 ℃, the atomizer is opened, and the frequency of the atomizer is 40 Hz; fourthly, when the temperature of the air inlet is 175 ℃ and the temperature of the air outlet is 90 ℃, the feeding pump is started, the machine is firstly debugged by running water, and after water is fed for a period of time, when the temperature of the air inlet is stabilized to 175 ℃ again and the temperature of the air outlet is stabilized to about 90 ℃, fermentation liquor can be fed; collecting the powder product at a discharge port after the machine operates for 1 hour, and drying and storing; when the machine runs, people need to pay attention to the running condition of the machine in real time.
Example 4
The powdery product of the bacillus amyloliquefaciens composite clay obtained in the above example was fixed with a conductive adhesive and sprayed with gold. The solidification condition of the microorganisms on the surface of the bacillus amyloliquefaciens composite clay is observed by using a scanning electron microscope (S-3400N, Hitachi, JP). As shown in fig. 4, the clay was able to locally enrich bacillus amyloliquefaciens.
Example 5
Collecting prorocentrum donghaiense solution (with cell density of about 1 × 10) in exponential growth phase 8 cells/L), Karenia mikimotoi algae liquid (algae cell density about 5X 10) 7 cells/L), red tide Isochrysis algae liquid (algae cell density about 1 × 10) 8 cells/L) were added to 50mL colorimetric tubes, respectively, and thereto were added the co-culture suspension of bacillus amyloliquefaciens obtained in example 1 and clay (kaolin), kaolin, and polyaluminum chloride-Modified Clay (MCI) respectively, at a final clay concentration of 0.2g/L in each system; after standing for 48h, the algae removal rate was calculated (see FIG. 5)
The result shows that the bacillus composite clay has higher algae removal effect on prorocentrum donghaiense, Karenia mikimotoi and red tide isocurvularia.
Example 6
The co-cultivation process described in example 1 was followed, respectively 10 6 cells/mL of different bacilli (bacillus pumilus, bacillus subtilis and bacillus cereus) are added into a culture medium LB containing 25g/L of kaolin/not containing kaolin, and the mixture is placed in a constant-temperature shaking incubator (30 ℃, 200rpm) for shaking culture for 24 hours to obtain a co-culture solution and a bacillus liquid which is cultured independently (without adding kaolin).
An algae removal experiment was performed using the algae removal method described in example 1 (fig. 6). The results show that the kaolin can improve the removal effect of different bacilli (bacillus pumilus, bacillus subtilis and bacillus cereus) on the prorocentrum donghaiense.
Claims (7)
1. A method for improving the capacity of bacillus to remove red tide algae cells is characterized by comprising the following steps: the bacillus is mixed with clay to enrich the bacillus on the surface of the clay, thereby improving the capacity of the bacillus to remove red tide algae cells.
2. The method of claim 1, wherein the bacillus is capable of removing red tide algae cells, and wherein: mixing the culture solution containing the bacillus with clay, mixing and co-culturing to obtain suspension, namely the red tide removing capability of the bacillus can be improved; wherein, when the culture solution containing the bacillus is mixed with the clay, the proportion of the clay to the bacillus is as follows according to the mass (g): cell number (cells) was measured and controlled at 1: 5X 10 5 -2×10 10 Within the range.
3. The method of claim 2, wherein the bacillus is capable of removing red tide algae cells, and wherein: and drying the suspension to enable the liquid to be solidified to prepare the product.
4. The method of claim 2, wherein the bacillus is capable of removing red tide algae cells, and wherein: the suspension obtained by mixing the clay and the culture solution containing the bacillus is aged and cultured for 8-24h at the oscillation frequency of 0-300rpm and the temperature of 10-40 ℃, so that the bacillus is promoted to fully grow and propagate under the action of the clay and to be gathered on the surface of the clay.
5. The method for enhancing the ability of a bacillus to remove red tide algae cells according to any one of claims 1-4, wherein: the bacillus is one or more of bacillus amyloliquefaciens, bacillus subtilis, bacillus brevis, bacillus cereus and bacillus pumilus.
6. The method of claim 5, wherein the bacillus is capable of removing red tide algae cells, and wherein: the bacillus is inoculated into LB culture medium, 2216E culture medium or nutrient broth culture medium to obtain a culture solution containing the bacillus.
7. The method of any one of claims 1 to 4, wherein the bacillus has the ability to remove red tide algae cells, and wherein: the clay particles are one or more than one of aluminosilicate minerals and/or one or more than one of modified aluminosilicate minerals.
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| CN202210375643.9A CN114958706A (en) | 2022-04-11 | 2022-04-11 | Method for improving capacity of bacillus for removing red tide algae cells |
| PCT/CN2023/076672 WO2023197743A1 (en) | 2022-04-11 | 2023-02-17 | Method for improving ability of bacillus species to remove red tide algae cells |
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| WO2023197743A1 (en) * | 2022-04-11 | 2023-10-19 | 中国科学院海洋研究所 | Method for improving ability of bacillus species to remove red tide algae cells |
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| CN103114050A (en) * | 2012-10-23 | 2013-05-22 | 中国水产科学研究院南海水产研究所 | Halobacillus kuroshimensis HSQAY1 with capacity of dissolving skeletonema costatum and application thereof |
| CN108517307A (en) * | 2018-05-22 | 2018-09-11 | 清华大学深圳研究生院 | A kind of method that modified clay couples use inhibition algal grown with algal control bacterium |
| CN108559722A (en) * | 2018-05-22 | 2018-09-21 | 清华大学深圳研究生院 | A kind of method that modified clay couples use inhibition algal grown with algal control bacterium |
| CN113321318A (en) * | 2021-06-02 | 2021-08-31 | 中国科学院海洋研究所 | Method for improving efficiency of clay in treating harmful algal blooms based on microbial composite modification |
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| JP2932107B2 (en) * | 1991-02-28 | 1999-08-09 | メルシャン株式会社 | Red tide control agent containing microorganisms as active ingredient |
| KR101099075B1 (en) * | 2009-08-31 | 2011-12-26 | 다이나믹(주) | Water soluble free amine chitosan with algicidal activity against harmful algal bloom species induced red tide, anti-red tide agent including the same as an active ingredient and removal method of red tide using the same |
| CN103725728B (en) * | 2013-12-12 | 2015-07-29 | 浙江大学 | The preparation method of a kind of Bacillamide compound and Bacillamide precursor |
| CN114958706A (en) * | 2022-04-11 | 2022-08-30 | 中国科学院海洋研究所 | Method for improving capacity of bacillus for removing red tide algae cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114050A (en) * | 2012-10-23 | 2013-05-22 | 中国水产科学研究院南海水产研究所 | Halobacillus kuroshimensis HSQAY1 with capacity of dissolving skeletonema costatum and application thereof |
| CN108517307A (en) * | 2018-05-22 | 2018-09-11 | 清华大学深圳研究生院 | A kind of method that modified clay couples use inhibition algal grown with algal control bacterium |
| CN108559722A (en) * | 2018-05-22 | 2018-09-21 | 清华大学深圳研究生院 | A kind of method that modified clay couples use inhibition algal grown with algal control bacterium |
| CN113321318A (en) * | 2021-06-02 | 2021-08-31 | 中国科学院海洋研究所 | Method for improving efficiency of clay in treating harmful algal blooms based on microbial composite modification |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023197743A1 (en) * | 2022-04-11 | 2023-10-19 | 中国科学院海洋研究所 | Method for improving ability of bacillus species to remove red tide algae cells |
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