Method for eliminating algal blooms and algal toxins of alexandrium alexandrium by improving modified clay

Abstract

The invention relates to the related technical field of prevention and control of harmful algal blooms, in particular to a method for eliminating algal blooms and algal toxins of alexandrium alexandrium by improving modified clay. Bacillus subtilis S3, kaolin and potassium monopersulfate are used in cooperation; a bacillus subtilis S3 bacterial suspension with algae-lysing capacity, sterilized kaolin and potassium monopersulfate are pretreated according to a certain proportion and then are uniformly sprayed on the surface of a toxic algal bloom water body so as to remove alexandrium alexandrium and degrade paralytic shellfish poison. According to the method disclosed by the invention, alexandrium cells in the water body can be cracked in the flocculating settling process, and paralytic shellfish poison released into the water body can be quickly degraded. The method has the advantages that the formation of sporocysts in the algae-lysing process can be reduced, algal toxins released by algae cell rupture in the water body and the algae-lysing process can be effectively degraded, and the method is safe and non-toxic and conforms to the concept of green sustainable development.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of harmful algal bloom prevention and control, and in particular to a method for improving modified clay to eliminate alexandrium algae blooms and algal toxins. BACKGROUND

[0002] Harmful algal blooms (HABs) are an ecological anomaly phenomenon that the explosive proliferation of phytoplankton, macroalgae or cyanobacteria in water bodies threatens the ecological system and human health. In recent years, harmful algal blooms have shown a trend of increasing in outbreak size and duration, and evolution of outbreak causes to dinoflagellates and toxic algae, which seriously threatens the safety of marine ecological system and sustainable development of coastal economy. In particular, some harmful algal bloom-causing algae species can also produce algal toxins, which not only affect biological growth, but also enter the human body through the food chain, causing diarrhea, paralysis and even death, and thus have adverse effects on the marine ecological system, human health and coastal economy. Therefore, it is more important to seek efficient and safe methods for toxic and harmful algal bloom control.

[0003] The methods for controlling harmful algal blooms mainly include physical method, chemical method, biological method and mineral flocculation method. The physical method mainly reduces the density of algae by manual salvage, but this method has high cost. The chemical method kills or flocculates algae cells through chemical action, but this method is prone to secondary pollution. The biological method controls algal blooms through competitive action, among which, algae-lysing bacteria become a research hotspot for algal bloom control due to their wide distribution and strong algae-lysing ability, but this method is slow in effect. The natural mineral flocculation method has the characteristics of green safety, and has been applied in Japan and South Korea, but has problems such as low efficiency, large dosage and serious sedimentation. The modified clay method changes the surface electric property of clay, so that the negative repulsion between the original natural clay and red tide organisms is changed to positive attraction, which greatly improves the flocculation and algae removal efficiency. Among them, the lower dosage of oxidized modified clay can also degrade algal toxins, but it is easy to stimulate alexandrium algae to produce cysts and cannot effectively degrade intracellular toxins. SUMMARY

[0004] The purpose of the present application is to provide a method for improving modified clay to eliminate alexandrium algae blooms and algal toxins, so as to solve the problem in the prior art that oxidized modified clay can degrade algal toxins, but is easy to leave cysts and has limited degradation effect on intracellular toxins.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solution: a method for improving modified clay to eliminate alexandrium algae blooms and algal toxins, the modified clay is composed of Bacillus subtilis S3, kaolin and oxidizing agent potassium peroxymonosulfate (PMS), and the specific method comprises the following steps:

[0006] Step S1, inoculate the commercial Bacillus strain in sterile LB liquid medium, and culture at room temperature with a rotation speed of 150 r / min for 24 h to obtain Bacillus subtilis S3 bacterial suspension with algal dissolving ability;

[0007] Step S2, after crushing the kaolin natural mineral, sterilize it at 121℃ for 30 min, dry it in an oven at 80℃ for 1 h, and sterilize it under ultraviolet light for 20 min. After cooling to room temperature, obtain the sterilized kaolin;

[0008] Step S3, add activated Bacillus subtilis S3 bacterial suspension and sterilized medium to the sterilized kaolin, shake well, and place in a constant temperature shaking incubator. Culture at room temperature with a shaking frequency of 150 r / min for 24 h to allow the microorganisms to fully contact the clay particles and aggregate and solidify on their surface;

[0009] Step S4, use Bacillus subtilis S3 bacterial suspension with algal dissolving ability, sterilized kaolin, and potassium monopersulfate together by uniformly spraying on the surface of the algal bloom water body to achieve flocculation and settling of Pacific Alexandrium algae cells and simultaneous efficient reduction of the content and toxicity of paralytic shellfish toxins (PSTs) released after the algae cells are broken.

[0010] More specifically, the particle size of the kaolin is 4-8 µm, and the main chemical components of the kaolin are Al2O3 and SiO2.

[0011] More specifically, the oxidizing agent potassium monopersulfate (PMS) has a chemical formula of 2KHSO5 5·KHSO4·K2SO4, and the effective component of the oxidizing agent potassium monopersulfate (PMS) is potassium monopersulfate, with a content of ≥47% by mass fraction.

[0012] Preferably, the amount of Bacillus subtilis S3 bacterial suspension used for maturation in step S3 is not less than 0.7% of the volume of the sterilized medium, and the maturation concentration of kaolin is 20 g / L. The maturation culture is carried out until the density of Bacillus subtilis S3 bacteria is not less than 1×10 9 cells / L.

[0013] More specifically, the Alexandrium is Pacific Alexandrium, the algal toxin produced by the Pacific Alexandrium is paralytic shellfish toxins (PSTs), and the paralytic shellfish toxins (PSTs) include saxitoxin (STX) and its derivatives.

[0014] Preferably, the volume ratio of the Bacillus subtilis S3 suspension during application is 0.5% (v / v), the concentration of the kaolin during application is 0.1 g / L, and the concentration of the oxidant potassium persulfate (PMS) during application is 70 mg / L.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. By effectively combining commercial Bacillus subtilis S3 with kaolin, microorganisms can aggregate, concentrate, and solidify on the clay surface, increasing the local concentration of microorganisms in the aquatic environment and maintaining their biological activity. This enhances the ability of modified clay to dissolve harmful algal blooms. Since the spores of Bacillus subtilis S3 have the ability to resist oxidative damage, low concentrations of PMS (0 mg / L–100 mg / L) have little effect on algal dissolution. At the same time, since Bacillus subtilis S3 can destroy the integrity of algal cells and release intracellular substances, the combined use of PMS and Bacillus subtilis S3 can effectively reduce the toxin content and toxicity inside and outside algal cells, thereby reducing the environmental risk of PSTs.

[0017] 2. By combining microorganisms with clay minerals and using oxidants simultaneously, the method can rapidly lyse algal cells while efficiently reducing the content and toxicity of toxins released into the water. The method is simple to operate and has a high removal effect, removing more than 80% of Pacific Alexandrium and PSTs in 3 hours, effectively mitigating the ecological harm and human health risks of toxic algal blooms. Attached Figure Description

[0018] The graph shows the algicidal effect, PSTs content reduction effect, and PSTs toxicity reduction effect of the method for improving the algicidal and detoxifying effect of modified clay provided in Example 1 of the present invention after 3 hours of treatment.

[0019] The graph shows the overall, intracellular, and extracellular PSTs content of the algal solution system provided in Example 1 of the present invention and the algal solution system treated by the method of the present invention for 3 hours.

[0020] The diagram shows the algal dissolution rate after adding Bacillus subtilis S3, kaolin, and PMS to the algal solution provided in Comparative Example 1 of the present invention, and after treatment with the method described in Example 1 for 3 hours.

[0021] The graph shows the reduction rate of PSTs content and the reduction rate of PSTs toxicity after adding Bacillus subtilis S3, kaolin, and PMS to the algal solution of Alexandrium paclitaxum provided in Comparative Example 1 of the present invention, and after treatment with the method described in Example 1 for 3 hours.

[0022] The PSTs content reduction rate and PSTs toxicity reduction rate graphs of Pacific Alexandrium algae liquid provided for the present application comparative example 2 after adding kaolin and algicidal bacteria, oxidized modified clay, algicidal bacteria and PMS and treating for 3h according to the method described in example 1.

[0023] The PSTs content reduction rate and PSTs toxicity reduction rate graphs of Pacific Alexandrium algae liquid provided for the present application comparative example 2 after adding kaolin and algicidal bacteria, oxidized modified clay, algicidal bacteria and PMS and treating for 3h according to the method described in example 1. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] Please refer to Figures 1-6 The present application provides a technical solution: a method for improving the elimination of Alexandrium algal blooms and algal toxins by modified clay, which is composed of Bacillus subtilis S3, kaolin and oxidizing agent potassium peroxymonosulfate (PMS). The specific method comprises the following steps:

[0026] Step S1, inoculate the commercial Bacillus strain into sterile LB liquid medium, and culture in a shaking incubator at room temperature and a rotation speed of 150r / min for 24h to obtain Bacillus subtilis S3 bacterial suspension with algicidal ability;

[0027] Step S2, after crushing the kaolin natural mineral, sterilize it at 121℃ for 30min, then dry it in an oven at 80℃ for 1h, and sterilize it under ultraviolet light for 20min. After cooling to room temperature, obtain the sterilized kaolin;

[0028] Step S3, add activated Bacillus subtilis S3 bacterial suspension and sterilized culture medium to the sterilized kaolin, shake well and place it in a constant temperature shaking incubator. Culture and mature it at room temperature for 24h at a shaking frequency of 150r / min, so that the microorganisms and clay particles are in full contact and aggregate and solidify on the surface.

[0029] Step S4, the Bacillus subtilis S3 bacteria suspension with algicidal ability, sterilized kaolin and potassium monopersulfate are used together by uniformly spraying on the surface of the algal bloom water body to achieve flocculation and settlement and lysis and breakage of the Alexandrium tamarense algal cells, and simultaneously efficiently reduce the content and toxicity of paralytic shellfish toxins (PSTs) released in the water body and after the breakage of the algal cells.

[0030] Further, the particle size of the kaolin is 4-8 µm, and the main chemical components of the kaolin are Al2O3 and SiO2.

[0031] Further, the chemical formula of the oxidant potassium monopersulfate (PMS) is 2KHSO5·5·KHSO4·K2SO4, and the effective component of the oxidant potassium monopersulfate (PMS) is potassium monopersulfate, and the content is ≥47% by mass fraction.

[0032] Further, the amount of the Bacillus subtilis S3 bacteria suspension used for maturation in step S3 is not less than 0.7% of the volume of the sterilized culture medium, and the maturation concentration of the kaolin is 20 g / L, and the maturation culture is performed until the density of the Bacillus subtilis S3 bacteria is not less than 1×10 9 cells / L.

[0033] Further, the Alexandrium tamarense is Alexandrium tamarense, and the algal toxin produced by the Alexandrium tamarense is paralytic shellfish toxins (PSTs), and the paralytic shellfish toxins (PSTs) include saxitoxin (STX) and its derivatives.

[0034] Further, the volume ratio of the Bacillus subtilis S3 bacteria suspension when used is 0.5% (v / v), the concentration of the kaolin when used is 0.1 g / L, and the concentration of the oxidant potassium monopersulfate (PMS) when used is 70 mg / L.

[0035] Example 1

[0036] The high-temperature sterilized kaolin and the Bacillus subtilis S3 bacteria suspension with a volume ratio of 0.7% (v / v) are added to the LB culture medium, uniformly shaken, and placed in a room temperature shaking incubator for maturation at a room temperature and a shaking frequency of 150 r / min for 24 h to obtain a microbial composite modified clay system, wherein the microbial density is 1.3*10 10 cells / L, and the PMS is added to the ultrapure water and uniformly shaken to prepare a PMS stock solution.

[0037] The Alexandrium tamarense algal liquid in the late exponential growth phase (algal cell density is about 10.87*10 3cells / ml) into 50ml colorimetric tubes, then add microbial composite modified clay system and PMS stock solution, so that the final density of S3 in the algal liquid system is 1x10 6 cells / L, the final concentration of kaolin is 0.1g / L, the final concentration of PMS is 70mg / L, shake well and stand for 3h, then take samples to determine the number of intact algal cells and the content of PSTs toxin, and calculate the number of lysed algal cells, i.e. the algal lysis rate, according to the following formula:

[0038] Algal lysis rate (%) = (N ck -N t ) / N ck *100%

[0039] Wherein, N t is the number of intact algal cells at time t in the experimental group, N ck is the number of intact algal cells at time t in the control group, and the intact algal cells include vegetative cells and temporary cysts.

[0040] Toxicity calculation: according to the toxicity equivalent factor (Table 1), the amount of each component of PSTs toxin in each sample is converted into the amount of STX-equivalent (STX-equ. nmol), and the toxicity is calculated by addition.

[0041] Table 1 Toxicity factors of common paralytic shellfish toxins

[0042] PSTs STX GTX1 GTX4 GTX2 GTX3 GTX5 C1 C2 Toxic factors 1 0.99 0.73 0.36 0.64 0.06 0.006 0.096

[0043] As can be seen from , after adding Bacillus subtilis S3, kaolin and PMS to the algal liquid of Pacific Alexandrium, the algal cell lysis rate reaches 88.96% after 3h of treatment, the PSTs degradation rate reaches 93.40%, and the PSTs detoxification rate reaches 94.53%. According to As can be seen from -1 , the PSP in the control group is dominated by GTX1 (2468MU·µmol -1 ) with high toxicity, while after treatment, C2 (239MU·µmol -1 ) with low toxicity is dominant, indicating that the method can efficiently lyse and break algal cells, degrade intracellular and extracellular algal toxins, and at the same time, after removal, the PSTs with high proportion in the overall system are converted from GTX1 with high toxicity to C2 with low toxicity, thereby effectively reducing the toxicity of PSTs and the environmental risk of Pacific Alexandrium algal bloom.

[0044] Comparative Example 1

[0045] According to the use method and concentration described in Example 1, Bacillus subtilis S, kaolin and PMS were added to the algal liquid, and at the same time, according to the input amount of Bacillus subtilis S3, kaolin and PMS described in Example 1, the three factors were added to the algal liquid as a control.

[0046] From It can be found that the addition of kaolin alone to the algal liquid does not have an algal dissolution ability, the addition of Bacillus subtilis S3 or PMS alone can dissolve and break 55.41% and 72.22% of algal cells respectively in 3h, but after using the method described in Example 1, the algal dissolution rate can reach 88.96% in the same time, and at the same time, from It can be found that kaolin and Bacillus subtilis S3 do not have a detoxification effect, the addition of PMS alone can remove 59.95% of PSTs, and after 3h of treatment using the method described in Example 1, 93.40% of PSTs can be removed, and the toxicity is reduced by 94.53%. Therefore, compared with separate use, the simultaneous use according to the method described in Example 1 can effectively improve the algal dissolution and detoxification effect of each other, and achieve simultaneous high-efficiency algal dissolution and detoxification.

[0047] Comparative Example 2

[0048] According to the use method and concentration described in Example 1, Bacillus subtilis S3, kaolin and PMS were added to the algal liquid, and at the same time, 0.7% (v / v) of Bacillus subtilis S3 and 0.1g / L of kaolin were added to the algal liquid to prepare a matured liquid, 0.1g / L of kaolin and 70mg / L of PMS were used to prepare an oxidized modified clay, and 0.7% (v / v) of Bacillus subtilis S3 and 70mg / L of PMS were added to the algal liquid as a control.

[0049] From It can be found that the addition of kaolin and algal dissolution bacteria to the algal liquid after maturation can have a higher algal dissolution effect, compared with the treatment method described in Example 1, the use of oxidized modified clay and algal dissolution bacteria and PMS has a lower algal dissolution effect, and the algal dissolution bacteria are all below 80%, and at the same time, combined , the algal dissolution bacteria and kaolin have a good algal dissolution effect, but do not have a detoxification function, and even increase the overall toxin content and toxicity, the use of oxidized modified clay and algal dissolution bacteria and PMS can simultaneously dissolve and detoxify, but the algal dissolution and detoxification effect is lower, and according to the method described in Example 1, the algal dissolution and detoxification effect can reach 80% and above in 3h, achieving the effect of 1+1>2.

[0050] In summary, the modified clay of the present application is composed of bacillus subtilis S3, kaolin and oxidant potassium monopersulfate (PMS), in the use process, first, the bacillus subtilis S3 bacterial suspension is mixed with kaolin and aged for 24 hours, in the aging liquid, the bacterial density and the kaolin concentration can be adjusted according to the density of the algal bloom to be removed, and then the oxidant PMS is uniformly sprayed to the algal bloom outbreak area.

[0051] The present application takes the Pacific Alexandrium as the research object which can produce paralytic shellfish poison, and based on bacillus subtilis S3, kaolin and PMS to treat the Pacific Alexandrium, uses microscope examination technology to determine the algal cell lysis rate, and uses high performance liquid chromatography tandem mass spectrometry to determine the degradation effect of PSTs.

[0052] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for improving the elimination of Alexandrium blooms and algal toxins using modified clay, characterized in that, The modified clay is composed of Bacillus subtilis S3, kaolin, and the oxidant potassium peroxymonosulfate (PMS). The specific method includes the following steps: Step S1: Inoculate a commercial Bacillus strain into sterile LB liquid medium and culture it in a shaker at room temperature and a speed of 150 r / min for 24 h to obtain a Bacillus subtilis S3 bacterial suspension with algicidal ability. Step S2: After crushing the natural mineral kaolin, sterilize it with high-temperature steam at 121℃ for 30 minutes, dry it in an oven at 80℃ for 1 hour, sterilize it under ultraviolet light for 20 minutes, and cool it to room temperature to obtain sterilized kaolin. Step S3: Add activated Bacillus subtilis S3 bacterial suspension and sterilized culture medium to the sterilized kaolin, shake well and place in a constant temperature shaking incubator. Cultivate and mature at room temperature for 24 hours with a shaking frequency of 150 r / min to allow the microorganisms to fully contact the clay particles and aggregate and solidify on their surface. Step S4: The three components, Bacillus subtilis S3 bacterial suspension with algicidal ability, sterilized kaolin, and potassium persulfate, are used together and sprayed evenly on the surface of the algal bloom water to achieve flocculation, sedimentation, lysis and breakage of Alexandrium algae cells in the Pacific Ocean. At the same time, this effectively reduces the content and toxicity of paralytic shellfish toxins (PSTs) in the water and released after the algal cells rupture.

2. The method for improving the elimination of Alexandrium blooms and algal toxins using modified clay according to claim 1, characterized in that, The kaolin has a particle size of 4-8µm, and its main chemical components are Al2O3 and SiO2.

3. The method for improving the elimination of Alexandrium blooms and algal toxins using modified clay according to claim 1, characterized in that, The oxidant potassium persulfate (PMS) has the chemical formula 2KHSO5·KHSO4·K2SO4, and the active ingredient of the oxidant potassium persulfate (PMS) is potassium persulfate, with a mass fraction of ≥47%.

4. The method for improving the elimination of Alexandrium blooms and algal toxins using modified clay according to claim 1, characterized in that, In step S3, the amount of Bacillus subtilis S3 bacterial suspension used for maturation shall not be less than 0.7% of the sterilized culture medium volume, the kaolin maturation concentration shall be 20 g / L, and the maturation culture shall be carried out until the Bacillus subtilis S3 bacterial density is not less than 1 × 10⁻⁶. 9 cells / L.

5. The method for improving the elimination of Alexandrium blooms and algal toxins using modified clay according to claim 1, characterized in that, The Alexandrium is Pacific Alexandrium, and the algal toxins produced by Pacific Alexandrium are paralytic shellfish toxins (PSTs), including Saxitoxin (STX) and its derivatives.

6. The method for improving the elimination of Alexandrium blooms and algal toxins using modified clay according to claim 1, characterized in that, The Bacillus subtilis S3 bacterial suspension was applied at a volume ratio of 0.5% (v / v), the kaolin clay was applied at a concentration of 0.1 g / L, and the oxidant potassium persulfate (PMS) was applied at a concentration of 70 mg / L.

Images