Aquaculture denitrification microcapsule as well as preparation method and application thereof

Abstract

The invention discloses an aquaculture denitrification microcapsule as well as a preparation method and application thereof, and relates to the technical field of aquaculture and biology. The lactobacillus casei provided by the invention can secrete acid metabolites, and the acid metabolites can inhibit pathogenic vibrio, so that the occurrence of diseases in aquaculture can be reduced. Lactobacillus casei has the efficacy of efficiently degrading ammonia nitrogen and nitrite nitrogen in the aquaculture environment, and can continuously realize efficient cleaning of the aquaculture environment. In addition, the invention also provides a denitrification microcapsule which creates a more favorable microenvironment for the efficient denitrification strain, so that the repeated denitrification performance is more stably exerted, and the denitrification microcapsule can be repeatedly utilized.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims the priority of Chinese patent application No. 2024102186066, filed with the Chinese Patent Office on February 28, 2024, and entitled “A kind of aquaculture denitrification microcapsule and its preparation method and application”, the entire contents of which are incorporated by reference in this application. Technical Field

[0003] The invention relates to the fields of aquaculture and biotechnology, and in particular to an aquaculture denitrification microcapsule and a preparation method and application thereof. Background Art

[0004] According to FAO statistics, the total output of aquaculture in the world and China has continued to increase in recent years, reaching 87.5 million tons and 55.6546 million tons in 2020, respectively. The proportion of feeding aquaculture output in the total aquaculture output has increased year by year, and the aquatic feed industry has developed rapidly. As the scale of aquaculture continues to expand, due to the pursuit of high yields, aquatic feeds are mostly high-protein and high-fat feeds, and the feed utilization rate of aquatic animals is not high. A large amount of leftover bait and feces leads to increased endogenous pollution (nitrogen compounds) in the breeding environment, and diseases occur frequently, seriously endangering the physiological health of aquatic animals. Therefore, green, sustainable, efficient and clean technology for aquaculture environments has become a scientific issue that urgently needs to be studied.

[0005] At present, aquaculture pollution treatment technologies are divided into physical, chemical and biological technologies. Biotechnology is valued for its advantages such as being green, safe and low-cost. Among them, microecological agents have the functions of cleaning water quality and inhibiting pathogens in the breeding environment, and have become an essential application technology in aquaculture. However, in actual production practice, due to the sole use of microbial agents, the survival rate of beneficial bacteria is low, the stability of their function is poor, and they are easily affected by the environment (low C / N, etc.), making it difficult to achieve ideal water purification and treatment effects.

[0006] Microencapsulation technology is an emerging bioprocessing technology in recent years. It uses natural or synthetic polymer materials that can form a film as wall materials to effectively embed the core material of functional components to form a stable microcapsule structure. In recent years, microencapsulation technology has developed rapidly, but it still has defects such as high cost of core and wall materials, complex preparation process, and poor universality of microorganisms.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide an aquaculture denitrification microcapsule and its preparation method and application to solve the technical problems of high cost, complex preparation process, poor biocompatibility and susceptibility to environmental influences (low C / N, etc.) of current aquaculture biological microcapsule products.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a Lactobacillus casei, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2024151.

[0011] An efficient denitrification strain (GSVWA6) was isolated from the shrimp farming pond environment in Guangdong Province. After 16s rRNA comparison, the strain had the highest similarity with Lactobacillus casei, so it was identified as Lactobacillus casei. It was deposited in the China Center for Type Culture Collection on January 22, 2024, and the deposit address is: Wuhan University, Wuhan, China. The name of the deposited strain is GSVWA6, the classification name is Lacticaseibacillus casei GSVWA6, and the identification result is survival.

[0012] On the one hand, Lactobacillus casei provided by the invention can secrete acidic metabolites, and acidic metabolites can suppress pathogenic vibrio, and contribute to reducing the occurrence of diseases in aquaculture; On the other hand, Lactobacillus casei has efficient denitrification performance, and aquaculture metabolites such as ammonia and nitrite nitrogen in the water body can be converted into non-toxic substances, and can be converted into bacteria own components (a part can be converted into non-toxic gases such as nitrogen and discharged from the aquaculture water body, and a part can be converted into bacteria own components). Therefore, Lactobacillus casei provided by the invention has the performance of obviously suppressing pathogenic vibrio and efficient denitrification, and Lactobacillus casei and modified biochar are used as the main body to prepare aquaculture denitrification microcapsules, which can be used for the efficient removal of nitrogen pollutants in aquaculture environment or artificial wetlands.

[0013] In a second aspect, the present invention provides the use of Lactobacillus casei in denitrification.

[0014] In a preferred embodiment of the present invention, denitrification is aquaculture denitrification or artificial wetland denitrification.

[0015] The Lactobacillus casei provided by the invention has the effect of efficiently degrading ammonia nitrogen and nitrite nitrogen in the breeding environment, and can sustainably realize efficient cleaning of the aquaculture environment. The microcapsule prepared based on Lactobacillus casei has a service life of more than 21 days, provides technical support for aquaculture ecological purification technology, conforms to the technical concept of green and healthy development of aquaculture, and has extremely high application value and market prospects.

[0016] After a large number of experiments, under storage conditions of 4°C, microbial microcapsules containing 4% biochar content can achieve a 90% removal effect of ammonia nitrogen and nitrite within 40 days. Within a 60-day test period, microbial microcapsules with 4% biochar content can achieve a removal effect of more than 85% of ammonia nitrogen and nitrite. In terms of the number of viable bacteria, microbial microcapsules with 4% biochar content can maintain 80% or more of the number of viable bacteria within 30 days, and maintain 60% or more of the number of viable bacteria within a 60-day test period. In comparison, the storage stability of 8% biochar microcapsules has been improved compared to 4% biochar content microbial microcapsules. 8% biochar content microbial microcapsules can achieve a 90% removal effect of ammonia nitrogen and nitrite within 60 days during the entire test period.

[0017] In addition, in terms of the number of live bacteria, the microbial microcapsules with 4% and 8% biochar content can maintain 90% or more of the number of live bacteria within 30 days, and maintain 70% or more of the number of live bacteria within a 60-day test period. It can be inferred that a higher biochar content can provide a more suitable microenvironment for denitrification microorganisms, which can significantly improve the ability of denitrification microorganisms to adapt to the environment, thereby better exerting nitrogen removal performance and exerting greater application potential in the mid- and late-stage aquaculture environment.

[0018] It should be noted that, in order to rapidly denitrify, those skilled in the art can easily think of setting microbial microcapsules with reduced biochar content (such as less than 4% biochar content), which is also within the scope of protection of the present invention. In addition, if the biochar content is less than 4%, the denitrification effect will be affected to a certain extent.

[0019] As a unique composite ecological sewage treatment technology composed of plants, substrates, microorganisms, etc., artificial wetlands have outstanding application advantages in the deep treatment of tailwater from sewage treatment plants in developing regions, and have gradually become one of the emerging methods commonly used for deep purification of sewage. In order to achieve deep purification of sewage, those skilled in the art can use the Lactobacillus casei provided by the present invention for denitrification of sewage.

[0020] In an optional embodiment, the use in denitrification includes at least one of the following uses:

[0021] Application in the preparation of denitrification microcapsules and application in the preparation of denitrification bacterial agents. In other embodiments, those skilled in the art can directly place Lactobacillus casei in an environment requiring denitrification as required, without being limited to the form of microcapsules or bacterial agents.

[0022] In other embodiments, in order to improve the denitrification effect, those skilled in the art can easily think of combining the Lactobacillus casei provided by the present invention with other nitrifying bacteria and denitrifying bacteria. Nitrifying bacteria and denitrifying bacteria have denitrification properties and can convert ammonia and nitrite in water into non-toxic substances.

[0023] In a third aspect, the present invention provides a denitrifying bacterial agent, which includes the above-mentioned Lactobacillus casei. The forms of the bacterial agent include but are not limited to: freeze-dried bacterial powder, bacterial liquid, fermentation precipitate, fermentation supernatant, bacterial residue treated by equipment such as filter press, etc.

[0024] In a fourth aspect, the present invention provides a denitrification microcapsule, comprising: Lactobacillus casei and a wall material for encapsulating the Lactobacillus casei.

[0025] In the present invention, denitrification microcapsules are synonymous with biochar-based microbial microcapsules.

[0026] In a preferred embodiment of the present invention, Lactobacillus casei is loaded on the gel microspheres, and the wall material outside the gel microspheres includes a chitosan layer and a sodium alginate layer arranged in sequence from the inside to the outside.

[0027] The present invention uses natural polymer materials sodium alginate and chitosan as microcapsule wall materials, which have the advantage of low price. The new aquaculture denitrification microcapsule has low cost, and the total cost is reduced by more than 30%, which has extremely high application value and market prospects. The inventor found that if the chitosan layer and the sodium alginate layer are replaced by other substances, the ammonia nitrogen degradation rate will be reduced.

[0028] The denitrification microcapsule has a three-layer stable structure of sodium alginate-chitosan-sodium alginate. It has a small particle size, a large specific surface area, and a high mass transfer efficiency, which is conducive to the removal of nitrogen pollutants in the aquaculture water environment. The denitrification microcapsule creates a micro space suitable for the growth and reproduction of beneficial bacteria, which can reduce the impact of environmental factors such as low C / N in the aquaculture environment, and has microbial universality and high-efficiency denitrification performance.

[0029] In an optional embodiment, the gel microspheres loaded with Lactobacillus casei are prepared by mixing modified biochar, Lactobacillus casei, sodium alginate, gelatin, silicon dioxide, water and a boric acid solution of calcium chloride and solidifying the mixture.

[0030] The addition of silica as an auxiliary material improves the mechanical strength of the wall material, which has certain advantages in transportation, storage and use. If gelatin and silica are replaced with other substances, the ammonia nitrogen degradation rate may be reduced.

[0031] In summary, the present invention adopts chitosan, sodium alginate and silicon dioxide as the wall materials of the microcapsule, and adopts gelatin and modified biochar as the core materials to prepare a new type of microcapsule material, which improves the mechanical strength of the wall material and the adsorption performance of the core material, enhances the universality of microorganisms, provides sufficient carbon source and suitable attachment space for Lactobacillus casei, and improves the denitrification performance of Lactobacillus casei by more than 2 times.

[0032] Biochar is a type of carbon-rich, densely packed, lamellar, porous environmental functional material produced by carbonizing biomass in a low-oxygen environment. In recent years, biochar has shown great potential for application in greenhouse gas N2O emission reduction, soil improvement and remediation, and pollutant adsorption, and has received widespread attention. Biochar is easy to prepare, does not require activation, and costs only about 1 / 6 of activated carbon.

[0033] The present invention uses modified biochar as a slow-release carbon source, replacing traditional carbon sources such as fructose and glucose, which is low-cost. The modified biochar significantly increases the pH of the material and the universality of the carrier's bacterial species. It has been proven to have high biocompatibility for a variety of strains such as Bacillus and yeast, and has great application prospects.

[0034] By modifying the biochar, the porosity of the biochar is increased. The modified biochar has a large specific surface area and high mass transfer efficiency, which is beneficial to the removal of nitrogen pollutants in the aquaculture water environment.

[0035] In an optional embodiment, 4% to 8% modified biochar by weight of water, 3% to 5% Lactobacillus casei by weight of water, 0.5% to 2% sodium alginate by weight of water, 1% to 2% gelatin by weight of water, 1% to 2% silicon dioxide by weight of water, and 6% to 8% calcium chloride by weight of water in a boric acid solution are mixed.

[0036] In an optional embodiment, 4% to 8% modified biochar by weight of water, 3% to 5% Lactobacillus casei by weight of water, 0.5% to 2% sodium alginate by weight of water, 1-2% gelatin by weight of water, 1-2% silicon dioxide by weight of water, and 7% calcium chloride by weight of water in a boric acid solution are mixed.

[0037] The content of modified biochar in the microcapsule is, for example, selected from 4%, 5%, 6%, 7% or 8%. After a large number of experiments, under storage conditions of 4°C, microbial microcapsules containing 4% biochar content can achieve a 90% removal effect of ammonia nitrogen and nitrite within 40 days. Within a 60-day test period, microbial microcapsules with 4% biochar content can achieve a removal effect of more than 85% of ammonia nitrogen and nitrite. In terms of the number of viable bacteria, microbial microcapsules with 4% biochar content can maintain 80% or more of the number of viable bacteria within 30 days, and maintain 60% or more of the number of viable bacteria within a 60-day test period. In contrast, the storage stability of 8% biochar microcapsules has been improved compared to 4% biochar content microbial microcapsules, and 8% biochar content microbial microcapsules can achieve a 90% removal effect of ammonia nitrogen and nitrite within 60 days during the entire test period. It can be inferred that a higher biochar content can provide a more suitable microenvironment for denitrifying microorganisms, which can significantly improve the ability of denitrifying microorganisms to adapt to the environment, thereby better exerting nitrogen removal performance and exerting greater application potential in the mid- and late-stage aquaculture environment.

[0038] In an optional embodiment, the modified biochar is a modified biochar prepared by combined modification of biochar with alkali and Mg ions. Alkali modification can be used (alkali modification can etch the microporous structure of biochar to make it mesoporous or macroporous), and Mg ion modification can be used (Mg ion modification can make biochar more affinity to microorganisms).

[0039] In an optional embodiment, the source of biochar is at least one of rice husk, straw and reed. In addition, those skilled in the art can easily imagine that other plant tissues can also form biochar, thereby being used to prepare microcapsules.

[0040] In an optional embodiment, the straw is selected from at least one of rice straw, corn straw and sesame straw. Preferably, the straw is selected from rice straw (rice husk material is selected from rice husk with light weight, dense pores and better air permeability).

[0041] In an optional embodiment, each gram of denitrification microcapsules contains (1-3)×10 8 In an optional embodiment, each gram of denitrification microcapsules contains 2.5×10 8 CFU Lactobacillus casei.

[0042] In a fifth aspect, the present invention provides a method for preparing denitrification microcapsules, comprising the following steps: embedding Lactobacillus casei in a wall material.

[0043] The method for embedding Lactobacillus casei in the wall material specifically comprises:

[0044] preparing modified biochar, then mixing the modified biochar with Lactobacillus casei, sodium alginate, gelatin, silicon dioxide, water and a boric acid solution of calcium chloride, and solidifying the mixture to obtain gel microspheres;

[0045] Then, the gel microspheres are coated with a chitosan solution so that a chitosan layer is formed on the outer wall of the gel microspheres;

[0046] Then, the gel microspheres coated with the chitosan layer are coated with a sodium alginate solution to form a sodium alginate layer on the outer wall of the chitosan layer.

[0047] The raw materials used in the microcapsule preparation method provided by the present invention are low in cost. Natural polymer materials sodium alginate and chitosan are used as microcapsule wall materials, which are low in price. The mechanical strength is improved by adding silicon dioxide auxiliary materials, which has certain advantages in transportation, storage and use. Modified biochar is used as a slow-release carbon source, replacing traditional carbon sources such as fructose and glucose, and has low cost. Modified rice husk biochar significantly improves the pH of the material and significantly improves the universality of the carrier's bacterial strains. It has been verified that various strains such as Bacillus and yeast have high biocompatibility and have great application prospects.

[0048] Biochar-based microbial microcapsules were prepared by a three-step coating method to form microcapsule particles with a three-layer stable structure of sodium alginate-chitosan-sodium alginate. The biochar-based microcapsule particles have a small particle size, a large specific surface area, and a high mass transfer efficiency, which is beneficial to the removal of nitrogen pollutants in the aquaculture water environment.

[0049] In a preferred embodiment of the present invention, the preparation method further comprises: liquefying the gel microspheres having the sodium alginate layer and the chitosan layer, and preparing the denitrification microcapsules by freeze drying. The term "liquefaction" has a clear meaning that the sodium alginate is converted into calcium alginate by the calcium chloride solution, and a layer of chitosan microcapsule membrane is instantly formed on the surface of the microgel beads through ion complexation reaction, and then the calcium in the calcium alginate gel is replaced by the sodium citrate solution, so that the microcapsule is liquefied into a liquid environment.

[0050] The inner core of the microcapsule is liquefied with sodium citrate, which provides a relatively transparent and stable internal liquid environment for the efficient denitrification strain, which is conducive to its growth and reproduction and the performance of efficient denitrification.

[0051] In an optional embodiment, the liquefaction is carried out in a 0.055 mol / L sodium citrate solution for 4-6 min; after washing, the mixture is sieved through a 100-150 mesh sieve and vacuum freeze-dried for 24-36 h.

[0052] Vacuum freeze drying is to freeze the bacterial suspension in a low temperature environment below the triple point and then evacuate it. The free water inside and outside the cells is directly sublimated into gas in the frozen state, achieving the purpose of removing moisture. This technology can retain most of the effective ingredients in the microcapsule; each component forms a stable solid skeleton when frozen, and the solid skeleton basically maintains its original shape after the water sublimates, and the porous structure product has excellent solubility, rehydration and rehydration rate; vacuum freeze drying can remove more than 95% of the water in the microcapsule, so that the product can be stored for a long time at room temperature or higher temperature, and it is light and easy to transport.

[0053] In other embodiments, in view of the impact of external environments such as freezing, drying, and storage on the survival rate of microorganisms, technicians in this field can easily think of adding suitable protective agents to the bacterial suspension to reduce or avoid damage to the microorganisms caused by external harsh environmental conditions, thereby increasing the survival rate of the microorganisms and providing possibilities for their commercial application.

[0054] In a preferred embodiment of the present invention, modified biochar accounting for 4% to 8% of the mass percentage of water, Lactobacillus casei accounting for 3% to 5% of the mass percentage of water, sodium alginate accounting for 0.5% to 2% of the mass percentage of water, gelatin accounting for 1% to 2% of the mass percentage of water, and silicon dioxide accounting for 1% to 2% of the mass percentage of water are mixed with water, and then added dropwise to a boric acid solution of calcium chloride; solidified for 10-12 hours, and passed through a 100-150 mesh sieve.

[0055] In an optional embodiment, the gel microspheres are coated by placing them in a chitosan solution, wherein the chitosan solution is chitosan placed in a 1% acetic acid solution, and the mass percentage of chitosan to 1% acetic acid is 0.5%-2%. For example, the mass percentage of chitosan to 1% acetic acid is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.5%, 1.8% or 2%.

[0056] In an optional embodiment, the coating time in the chitosan solution is 6-10 minutes, for example, the coating time is 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0057] In an optional embodiment, the gel microspheres coated with the chitosan layer are placed in a sodium alginate solution containing 0.15-0.2% by mass for coating; in an optional embodiment, the gel microspheres coated with the chitosan layer are placed in a 0.15% sodium alginate solution for coating for 6-10 minutes.

[0058] In an optional embodiment, 4% of modified biochar, 5% of Lactobacillus casei, 1.5% of sodium alginate, 1-2% of gelatin, 1-2% of silicon dioxide and water are mixed; and 1.5% chitosan solution is used for coating;

[0059] In an optional embodiment, 8% modified biochar accounting for 5% of water mass, 5% of Lactobacillus casei accounting for 1% of water mass, 1% of sodium alginate accounting for 1-2% of water mass, 1-2% of water mass of gelatin, and 1-2% of water mass of silicon dioxide are mixed with water; and 2% chitosan solution is used for coating.

[0060] In the 4% biochar-based microbial microcapsules, when the sodium alginate addition was 1.5%, the chitosan addition was 1.5%, and the film-forming time was 10 minutes, the microcapsules had the most outstanding physical performance indicators, the microcapsule mass and microcapsule diameter were moderate, the microcapsule bacterial load was higher, the microcapsule water content was high, the microcapsule water retention rate was higher, and the microcapsule specific surface area was larger. In the 8% biochar-based microbial microcapsules, when the sodium alginate addition was 1%, the chitosan addition was 2%, and the film-forming time was 10 minutes, the microcapsules had the most outstanding physical performance indicators, the microcapsule bacterial load was higher, the microcapsule water content was high, the microcapsule water retention rate was higher, and the microcapsule specific surface area was larger.

[0061] In a preferred embodiment of the present invention, the modified biochar is prepared as follows: the biochar to be modified is modified in a strong alkaline solution, washed and dried, and the dried product is then placed in a MgCl2 solution for modification, washed and dried;

[0062] In an optional embodiment, every 10 g of the biochar to be modified is mixed with a 0.1 M strong alkali solution.

[0063] In an optional embodiment, the strong alkaline solution is a sodium hydroxide or potassium hydroxide solution; if the modified solution is changed to an acidic solution, the modification effect will be reduced.

[0064] In an optional embodiment, the biochar is modified in a strong alkaline solution for 10-15 hours, then washed until the biochar is neutral, and dried to obtain alkali-modified biochar;

[0065] In an optional embodiment, every 5 g of alkali-modified biochar is mixed with a 0.025 M MgCl2 solution for modification;

[0066] In an optional embodiment, every 5 g of alkali-modified biochar is mixed with 0.025 M MgCl2 solution for modification for 10-12 hours.

[0067] The present invention has the following beneficial effects:

[0068] (1) The Lactobacillus casei provided by the present invention can secrete acidic metabolites, which can inhibit pathogenic Vibrio and help reduce the occurrence of diseases in aquaculture.

[0069] (2) Lactobacillus casei has high denitrification performance and can convert aquaculture metabolites such as ammonia and nitrite in the water into non-toxic substances. Some of them can be converted into the bacteria's own components. Part of them can be converted into non-toxic gases such as nitrogen and discharged from the aquaculture water, and part of them can be converted into the bacteria's own components.

[0070] Therefore, the Lactobacillus casei provided by the present invention has obvious performance of inhibiting pathogenic Vibrio and highly efficient denitrification. The aquaculture denitrification microcapsules prepared with Lactobacillus casei and modified biochar as main components can be used for highly efficient removal of nitrogen pollutants in aquaculture environments or artificial wetlands.

[0071] (3) Lactobacillus casei provided by the present invention has the effect of efficiently degrading ammonia nitrogen and nitrite nitrogen in the breeding environment, and can sustainably realize the efficient cleaning of the aquaculture environment. The microcapsule prepared based on Lactobacillus casei has a service life of more than 21 days, which provides technical support for aquaculture ecological purification technology, conforms to the green and healthy development technology concept of aquaculture, and has high application value and market prospects. In addition, the denitrification microcapsule provided by the present invention creates a more favorable microenvironment for the efficient denitrification strain, thereby more stably exerting the repeated denitrification performance and can be reused.

[0072] (4) The denitrification microcapsules (or biochar microcapsules) provided have small particle size, large specific surface area, and high mass transfer efficiency, which is beneficial to the removal of nitrogen pollutants in the aquaculture water environment.

[0073] (5) The provided denitrification microcapsules (or biochar microcapsules) have high storage stability at 4°C. During the 60-day test period, the denitrification microcapsules with 4% biochar content can achieve a removal effect of more than 85% of ammonia nitrogen and nitrite, and the denitrification microcapsules with 8% biochar content can achieve a removal effect of 90% of ammonia nitrogen and nitrite within the entire test period of 60 days. The denitrification microcapsules with 4% and 8% biochar content can maintain a viable bacterial count of 90% or more within 30 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0075] Figure 1 This is a dynamic statistical result diagram of the removal effect of ammonia nitrogen and nitrite by microbial microcapsules with 4% biochar content within 60 days under 4℃ storage conditions;

[0076] Figure 2 This is a dynamic statistical result diagram of the removal effect of ammonia nitrogen and nitrite by microbial microcapsules with 8% biochar content within 60 days under 4°C storage conditions;

[0077] Figure 3 The dynamic statistical results of the removal of ammonia nitrogen and nitrite by microbial microcapsules with 4% and 8% biochar content in 5 cycles;

[0078] Figure 4 It is a phylogenetic tree diagram;

[0079] Figure 5 This is the result of strain compatibility experiment;

[0080] Figure 6 Statistical results of the degradation rate of total nitrogen in shrimp culture pond water by microcapsules with 4% and 8% biochar content within 72 hours. DETAILED DESCRIPTION

[0081] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0082] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0083] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0084] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0085] Example 1

[0086] This example provides a separation, purification, and identification process for a highly efficient denitrifying lactic acid strain, as follows:

[0087] (1) Separation and purification

[0088] Prepare 90 mL of ammonia nitrogen and nitrite nitrogen enrichment culture medium respectively and place them in 250 mL conical flasks. After sterilization in a sterilizer with high temperature and high pressure, add 10 mL of pond water sample to the culture medium, and then place it in a constant temperature shaking incubator to ensure culture at 28°C and 160 rpm. Replace 50 mL of enrichment culture medium in the conical flask every day, and carry out enrichment culture for a total of 72 hours.

[0089] After the enrichment culture was completed, 1 ml of the enrichment solution was diluted to a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The concentrations of 10 -4 , 10 -5 , 10 -6 100 μL of bacterial suspension was applied on the surface of ammonia nitrogen and nitrite nitrogen solid screening culture medium, and placed in a constant temperature incubator to ensure 28°C culture until a certain number of colonies grew. Select colonies of different colors, properties, and sizes and transfer them to 2216E agar solid culture medium for streak culture until a single colony is obtained. Use 25% glycerol to preserve the seeds in seed tubes and store them in a -80°C ultra-low temperature refrigerator in preparation for the next step of research.

[0090] The preserved strains were activated and cultured in 2216E liquid culture medium for 24 hours, then inoculated into ammonia nitrogen and nitrite nitrogen degradation medium at a 5% inoculation rate, and cultured at 28°C and 160 rpm for 48 hours. The ammonia nitrogen and nitrite nitrogen removal capabilities of each strain were determined by hypobromite oxidation method and naphthylethylenediamine spectrophotometry, respectively.

[0091] (2) Identification

[0092] The DNA of denitrifying strains was extracted by repeated freezing and thawing. After several denitrifying strains were inoculated into 2216E liquid medium for activation for 24 hours, they were streaked on 2216E solid medium and cultured until single colonies grew. The single colonies were picked into a 1.5ml centrifuge tube containing 50uL sterile ultrapure water with a high-temperature sterilized toothpick. After repeated freezing and thawing for 3 times, the tube was centrifuged at 12000rpm and 4℃ for 10 minutes to obtain bacterial DNA templates for subsequent PCR amplification. The PCR reaction system and conditions are shown in Table 2-1.

[0093] The PCR amplification product was sent to Bioengineering (Shanghai) Co., Ltd. for sequencing. The obtained nucleotide sequence was further determined by BLAST online comparison (http: / / www.ncbi.nlm.nih.gov / BLAST / Blast.cgi) to further determine the species information of the strain, and two efficient denitrification strains were comprehensively selected to construct a phylogenetic evolutionary tree by the Neighbor-joining method. One of the isolated lactic acid bacteria was deposited in the China Center for Type Culture Collection on January 22, 2024, and the deposit address is: Wuhan University, Wuhan, China. The name of the preserved strain is GSVWA6, and the classification name is Lacticaseibacillus casei GSVWA6. The identification result is survival, and the deposit number is: CCTCC NO: M2024151.

[0094] PCR reaction system and reaction conditions

[0095]

[0096] The 16s rRNA sequence of lactic acid bacteria is shown in SEQ ID NO.1, and the phylogenetic tree diagram is shown in Figure 4 shown.

[0097] The sequence shown in SEQ ID NO.1 is as follows:

[0098]

[0099] Example 2

[0100] This embodiment provides a method for preparing biochar-based microbial microcapsules.

[0101] (1) Activation culture of efficient denitrification lactic acid strains

[0102] The high-efficiency denitrification strain of Example 1 was inoculated into the MRS medium at an inoculation amount of 3.0% of the weight of the liquid medium. The medium was cultured under shaking conditions of 28.0°C, pH 7.5, and a rotation speed of 160 r / min for 24 h to obtain a seed solution. The microbial strain was Lactobacillus casei, and each milliliter of the seed solution contained 1×10 8 CFU bacteria.

[0103] (2) Preparation of biochar-based microbial microcapsules

[0104] ① Chitosan solution: Place chitosan in 1% acetic acid solution, maintain a constant temperature of 60°C in a water bath, and stir to dissolve to obtain a chitosan solution. The mass percentage of chitosan to 1% acetic acid is 1.5%.

[0105] ② Sodium alginate solution: Sodium alginate was placed in ultrapure water, stirred and dissolved in a constant temperature water bath at 100°C to obtain a sodium alginate solution. The mass percentage of sodium alginate to water was 1.5%.

[0106] ③ Preparation of modified rice husk biochar: First, 10 g of unmodified rice husk biochar was soaked in 100 mL of NaOH (1 mol·L -1 ) solution, filtered after 12 hours and washed with distilled water until the biochar was neutral and dried at 70℃ to obtain NaOH-modified biochar. Then 5g of NaOH-modified biochar was immersed in 50mL 0.5mol·L -1 MgCl2 solution for 12 hours, repeat the above steps of filtering, rinsing and drying to obtain NaOH + Mg 2+ Combined modified rice husk biochar.

[0107] ④ 1.5% sodium alginate solution (the mass ratio of sodium alginate to water is 1.5:100), gelatin solution (2% concentration), and silicon dioxide were mixed evenly, the total mass percentage of gelatin and silicon dioxide in water was 2%, and 5% of the water mass was added with a high-efficiency denitrification lactic acid strain (bacterial activity was (1-3)×10 8 CFU / g), and rice husk biochar was added in a mass ratio of 4% to ultrapure water, mixed evenly and then added dropwise to a 2% calcium chloride boric acid saturated solution with a syringe, solidified for 12 hours, then rinsed with ultrapure water and sieved with 100-150 mesh to obtain preliminary gel microspheres.

[0108] ⑤ Place the gel microspheres in a 1.5% chitosan solution for the first coating, the coating time is 10 minutes, wash them three times with ultrapure water after coating to remove the residual chitosan solution on the surface, place the initially coated microcapsules in a 0.15% low concentration sodium alginate solution for a second coating, the coating time is 10 minutes, and after washing and sieving with 100-150 mesh, double-layer membrane gel microspheres are obtained.

[0109] ⑥ Place the double-layer membrane gel microspheres in a 0.055 mol / L sodium citrate solution to liquefy the spheres for 5 minutes. After liquefaction, wash them three times with ultrapure water, sieve through 100-150 mesh, and vacuum freeze-dry for 24.0 hours to obtain biochar-based microbial microcapsules, which are stored at 4°C for later use.

[0110] Example 3

[0111] Compared with Example 2, the only difference is that the concentration of the microcapsule raw material - sodium alginate in step (4) is 1%, and the film forming time of the first and second film coatings in step (5) is 8 minutes. The other preparation conditions and steps are the same.

[0112] Example 4

[0113] Compared with Example 2, the only difference is that the concentration of the microcapsule raw material - sodium alginate in step (4) is 1%, and the concentration of the raw material chitosan used in the first coating in step (5) is 2%. The other preparation conditions and steps are the same.

[0114] Example 5

[0115] Compared with Example 2, the only difference is that in step (4), rice husk biochar is added at a mass ratio of 8% to ultrapure water, and the other preparation conditions and steps are the same.

[0116] Example 6

[0117] Compared with Example 5, the only difference is that the concentration of the raw material chitosan used in the first coating in step (5) is 2%, the time for the first coating and the second coating is 6 minutes, and the other preparation conditions and steps are the same.

[0118] Example 7

[0119] Compared with Example 5, the only difference is that the concentration of the microcapsule raw material - sodium alginate in step (4) is 1%, and the concentration of the raw material chitosan used for the first coating in step (5) is 2%. The other preparation conditions and steps are the same.

[0120] Experimental Example 1

[0121] The mass, microcapsule diameter, microcapsule bacterial loading, microcapsule moisture content, microcapsule water retention rate, and microcapsule specific surface area of ​​the microcapsules prepared in Examples 2-7 were measured respectively.

[0122] The results are shown in Tables 1 and 2.

[0123] Table 1 Performance parameters of microcapsules of Examples 2-4 at 4% biochar content.

[0124]

[0125] Table 2 Performance parameters of microcapsules of Examples 5-7 at 8% biochar content.

[0126]

[0127]

[0128] Through orthogonal design experiments, the results show that in 4% biochar-based microbial microcapsules, when the sodium alginate addition is 1.5%, the chitosan addition is 1.5%, and the film-forming time is 10 minutes, the microcapsules have the best physical performance indicators. In 8% biochar-based microbial microcapsules, when the sodium alginate addition is 1%, the chitosan addition is 2%, and the film-forming time is 10 minutes, the microcapsules have the best physical performance indicators. The best solutions are Example 2 and Example 7, which are comprehensively measured by the four performance indicators of bacterial load, water content, water holding capacity, and specific surface area.

[0129] The orthogonal experimental results of 4% biochar-based microbial microcapsules are shown in the following table:

[0130]

[0131] Note: "+" indicates average properties, "++" indicates good properties, and "+++" indicates excellent properties.

[0132] The orthogonal experimental results of 8% biochar-based microbial microcapsules are shown in the following table:

[0133]

[0134] Note: "+" indicates average properties, "++" indicates good properties, and "+++" indicates excellent properties.

[0135] The table of factor levels in the orthogonal experiment is as follows:

[0136]

[0137] Experimental Example 2

[0138] The storage stability of biochar-based microbial microcapsules was tested.

[0139] After the biochar-based microbial microcapsules were stored at 4°C for 5, 10, 20, 30, 40, 50 and 60 days, 10 g of the microbial microcapsules prepared in Example 2 and Example 7 (the biochar addition ratio was 4% and 8%, respectively) were taken to treat the simulated wastewater with ammonia nitrogen and nitrite nitrogen concentrations of 10 mg / L, and the treatment was carried out at a temperature of 28°C and an oscillation rate of 160 r / min for 72 h. The ammonia nitrogen and nitrite degradation rates of the microbial microcapsules were determined, and the number of live microorganisms in the microcapsules was determined by the plate counting method.

[0140] The experimental results are as follows Figure 1 , Figure 2 As shown in the figure, the results show that under the storage condition of 4℃, the microbial microcapsules with 4% biochar content can achieve 90% removal of ammonia nitrogen and nitrite within 40 days. In the 60-day test period of this study, the microbial microcapsules with 4% biochar content can achieve more than 85% removal of ammonia nitrogen and nitrite. In terms of the number of viable bacteria, the microbial microcapsules with 4% biochar content can maintain 80% or more of the number of viable bacteria within 30 days, and maintain 60% or more of the number of viable bacteria within the 60-day test period of this experiment. However, with the extension of storage time, the biological activity of efficient denitrification bacteria has an obvious lag period, which may lead to an increase in denitrification time and a decrease in efficiency.

[0141] In comparison, the storage stability of 8% biochar microbial microcapsules has been improved compared with 4% biochar content microbial microcapsules. 8% biochar content microbial microcapsules can achieve 90% removal of ammonia nitrogen and nitrite within the entire test period of 60 days.

[0142] In addition, in terms of the number of live bacteria, the microbial microcapsules with 4% and 8% biochar content can maintain 80% or more of the live bacteria within 30 days, and maintain 70% or more of the live bacteria within the 60-day test period of this test. It can be inferred that a higher biochar content can provide a more suitable microenvironment for denitrification microorganisms, which can significantly improve the ability of denitrification microorganisms to adapt to the environment, thereby better exerting nitrogen removal performance and exerting greater application potential in the mid- and late-stage aquaculture environment.

[0143] Experimental Example 3

[0144] The repeated denitrification efficiency of denitrification microcapsules was tested.

[0145] Take 10g of biochar-based microbial microcapsules prepared in Example 2 and Example 7 (the biochar addition ratio is 4% and 8% respectively, and the bacterial solution addition amount is 2.5ml (OD 600=1), under the conditions of C / N of 10, temperature of 28°C, and oscillation rate of 160r / min, 50ml of simulated wastewater with ammonia nitrogen and nitrite nitrogen concentration of 10mg / L were treated respectively, and 2.5ml of water sample was taken every 6h to determine the degradation rate of ammonia nitrogen and nitrite nitrogen. When the degradation rate was greater than 95%, the microbial microcapsules were taken out, washed with 0.9% physiological saline, and transferred to another conical flask containing 50ml of simulated wastewater with ammonia nitrogen and nitrite nitrogen concentration of 10mg / L, and the above steps were repeated. The continuous removal ability of microbial microcapsules for ammonia nitrogen and nitrite nitrogen and the reusability were determined.

[0146] Ammonia nitrogen simulated wastewater: glucose 0.5g, K2HPO4·3H2O 0.05g, MgSO4·7H2O 0.5g, FePO4·4H2O0.01g, (NH4)2SO4 0.0481g, 0.45μm filter membrane filtered seawater (salinity 30‰) 1L, pH 7.5. NH4 + -N 10mg / L, C / N=10.

[0147] Nitrite nitrogen simulated wastewater: glucose 0.5g, K2HPO4·3H2O 0.05g, MgSO4·7H2O0.5g, FePO4·4H2O 0.01g, NaNO2 0.05g, 0.45μm filter membrane filtered seawater (30‰) 1L, pH 7.5. - -N 10mg / L, C / N=10.

[0148] In previous preliminary experiments, microcapsules with 4% and 8% biochar content were able to degrade ammonia nitrogen and nitrite nitrogen by 95% or more within 72 hours of experiment time, so 72 hours was taken as a cycle of experiment time.

[0149] The experimental results are as follows Figure 3 As shown, the results showed that the microbial microcapsules with 4% biochar content could achieve 80% or more removal effect in 5 test cycles of this experiment, and both the 4% and 8% biochar content microcapsules could achieve 90% ammonia nitrogen and nitrite removal effect within 3 cycles. Compared with the microcapsules with 4% biochar content, the microcapsules with 8% biochar content could create a more favorable microenvironment for the efficient denitrification strains, thereby more stably exerting the repeated denitrification performance.

[0150] Experimental Example 4

[0151] This experimental example conducted an experiment on the inhibition of pathogenic bacteria by Lactobacillus paracasei.

[0152] The results of the inhibition zone test refer to Figure 5As shown, the Oxford cup method was used to inoculate two strains of denitrifying bacteria into 2216E liquid culture medium and activate them under constant temperature aerobic conditions at 28°C and 160rpm for 24 hours. 100 μL of the bacterial solution of one of the strains was spread on the solid culture medium, and the sterilized Oxford cup was placed on the coated 22216E plate with sterilized tweezers. 100 μL of the bacterial solution of the other strain was added to the Oxford cup, and then the plate was placed in a constant temperature incubator and cultured at 28°C for 48 hours. The inhibition zone around the Oxford cup was continuously observed. If an inhibition zone appeared, it proved that there was a possible antagonism or inhibition between the two strains. If no inhibition zone appeared, it proved that there was no antagonism between the two strains.

[0153] The results showed that this strain had a strong inhibitory effect on pathogenic bacteria (Vibrio parahaemolyticus and Vibrio harveyi).

[0154] Experimental Example 5 (Actual Degradation Experiment)

[0155] Water sample of Penaeus vannamei culture pond: salinity 30‰, pH=8, TN=21mg / L.

[0156] Use a 0.45μm filter membrane to screen out large particles in aquaculture pond water, tail water and domestic sewage samples, take 10g of microbial microcapsules and the same amount of single bacteria and add them to a 100mL sample conical bottle, culture it at 28℃ and 160rpm, take 2.5ml of water sample every 24h, and measure the degradation rate of total nitrogen.

[0157] Results reference Figure 6 As shown in the figure, the results show that the addition of biochar can greatly increase the mass transfer performance and specific surface area of ​​the microcapsules. Compared with free high-efficiency denitrifying bacteria, biochar-based microcapsules provide a safer and more stable living environment for the strains, which can ensure the normal performance of the high-efficiency denitrifying strains. The 8% biochar content provides more attachment sites for the high-efficiency denitrifying strains. In addition, the increase in the biochar content also plays a great adsorption advantage, thus having a higher total nitrogen removal efficiency. It should be noted that the total nitrogen content of the free denitrifying bacteria showed a downward trend within the 72h test period, while the 4% and 8% biochar-based microcapsules reached the lowest total nitrogen content at 48h, and the total nitrogen content increased slightly within 72h. It is speculated that because there are more pores in the biochar-based microcapsules, a very small amount of adsorbed nitrogen will be released, but the overall denitrification efficiency of the biochar-based microcapsules is significantly higher than that of the free denitrifying bacteria.

[0158] Experimental Example 6

[0159] Lactobacillus casei was inoculated into MRS liquid culture medium and activated under constant temperature and aerobic conditions at 28°C and 160rpm for 24h. Then, it was added into conical flasks containing 100ml ammonia nitrogen and nitrite nitrogen degradation culture medium according to an inoculation ratio of 5%. After culturing for 48h under different environmental conditions, a certain amount of culture solution was taken and centrifuged at 4000rpm for 10min. The supernatant was retained and its ammonia nitrogen and nitrite nitrogen contents were determined. Three parallels were set for each group.

[0160] The environmental factors set in the experiment were temperature 28°C, pH=8.3, C / N=5, initial nitrogen concentration 10 mg / L, constant temperature shaker speed set to 160 rpm / min. Salinity was set to 10‰, 20‰, 30‰, 40‰, 50‰, and 60‰ respectively.

[0161] The results showed that when the environmental salinity was maintained at 20-60‰, the strains were sensitive to NH4 + -N and NO2 - -N degradation rates were higher than 80%, and salinity below 20‰ would significantly inhibit the growth of the strain, thereby further significantly improving the denitrification efficiency. When the salinity was 40‰, the ammonia nitrogen and nitrite nitrogen degradation rates of the strain were the highest. As the salinity increased further, the removal efficiency of ammonia nitrogen and nitrite nitrogen gradually decreased. Therefore, this Lactobacillus casei had a strong salinity tolerance, and the denitrification performance of the strain could be maximized when the salinity of the water environment was maintained at 40‰ (p<0.05).

[0162] Salt tolerance test of strains

[0163]

[0164] In summary, the Lactobacillus casei screened by the present invention meets the requirements of the "Regulations on the Administration of Feed and Feed Additives" (2013) issued by my country, can release acidic metabolites, significantly inhibit pathogenic Vibrio, and has the effect of efficient denitrification. It has broad application prospects.

[0165] The novel aquaculture denitrification microcapsule prepared by the invention mainly based on lactic acid bacteria and biochar can reduce the total production cost by more than 30%, and has extremely high application value and market prospect.

[0166] The present invention strengthens the mechanical strength of the wall material by adding silicon dioxide, adopts modified rice husk biochar (core material), effectively improves the pH of the biochar, significantly increases the specific surface area, creates a micro space suitable for the growth and reproduction of beneficial bacteria, can reduce the influence of environmental factors such as low C / N in an aquaculture environment, has microbial universality and efficient denitrification performance; a strain of Lactobacillus casei (lactic acid bacteria) screened from a shrimp breeding environment is selected as a functional microorganism, which can secrete acidic metabolites and has the performance of significantly inhibiting pathogenic Vibrio and efficiently denitrifying.

[0167] In addition, the Lactobacillus casei and microcapsules provided by the present invention have the effect of efficiently degrading ammonia nitrogen and nitrite nitrogen in the breeding environment. Its use period is more than 21 days, providing technical support for aquaculture ecological purification technology, conforming to the technical concept of green and healthy development of aquaculture, and having extremely high application value and market prospects.

[0168] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Lactobacillus casei, characterized in that It is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2024151.

2. the application of Lactobacillus casei as claimed in claim 1 in denitrification.

3. The use according to claim 2, characterized in that: The denitrification is aquaculture denitrification or artificial wetland denitrification; Preferably, the application in denitrification includes at least one of the following uses: Application in the preparation of denitrification microcapsules and application in the preparation of denitrification bacterial agents.

4. A denitrifying bacterial agent, characterized in that: The method comprises the Lactobacillus casei according to claim 1.

5. A denitrification microcapsule, characterized in that: It includes: The Lactobacillus casei and the wall material for embedding the Lactobacillus casei according to claim 1.

6. The denitrification microcapsule according to claim 5, characterized in that: The Lactobacillus casei is loaded on the gel microspheres, and the wall material outside the gel microspheres includes a chitosan layer and a sodium alginate layer arranged in sequence from the inside to the outside; Preferably, the gel microspheres loaded with Lactobacillus casei are prepared by mixing modified biochar, Lactobacillus casei, sodium alginate, gelatin, silicon dioxide, water and a boric acid solution of calcium chloride and curing; Preferably, 4% to 8% of modified biochar by weight of water, 3% to 5% of Lactobacillus casei by weight of water, 0.5% to 2% of sodium alginate by weight of water, 1% to 2% of gelatin by weight of water, 1% to 2% of silicon dioxide by weight of water, and 6% to 8% of calcium chloride by weight of water are mixed; Preferably, 4% to 8% of modified biochar by weight of water, 3% to 5% of Lactobacillus casei by weight of water, 0.5% to 2% of sodium alginate by weight of water, 1% to 2% of gelatin by weight of water, 1% to 2% of silicon dioxide by weight of water, and 7% of calcium chloride by weight of water are mixed; Preferably, the modified biochar is a modified biochar prepared by combined modification of biochar with alkali and Mg ions; Preferably, the source of the biochar is at least one of rice husk, straw and reed; Preferably, the straw is selected from at least one of rice straw, corn straw and sesame straw; Preferably, each gram of the denitrification microcapsules contains (1-3)×10 8 CFU of Lactobacillus casei; Preferably, each gram of the denitrification microcapsules contains 2.5×10 8 CFU of Lactobacillus casei.

7. A method for preparing the denitrification microcapsules according to any one of claims 5 to 6, characterized in that: It includes the following steps: Lactobacillus casei is embedded in the wall material.

8. The method for preparing denitrification microcapsules according to claim 7, characterized in that: The method of embedding Lactobacillus casei in the wall material comprises: preparing modified biochar, then mixing the modified biochar with Lactobacillus casei, sodium alginate, gelatin, silicon dioxide, water and a boric acid solution of calcium chloride, and solidifying the mixture to obtain gel microspheres; Then, the gel microspheres are coated with a chitosan solution so that a chitosan layer is formed on the outer wall of the gel microspheres; Then, the gel microspheres coated with the chitosan layer are coated with a sodium alginate solution to form a sodium alginate layer on the outer wall of the chitosan layer; Preferably, the preparation method further comprises: liquefying the gel microspheres having the sodium alginate layer and the chitosan layer, and freeze-drying to obtain denitrification microcapsules; Preferably, the liquefaction is carried out in a 0.055 mol / L sodium citrate solution for 4-6 min; after washing, the filtrate is passed through a 100-150 mesh sieve and vacuum freeze-dried for 24-36 h.

9. The method for preparing denitrification microcapsules according to claim 8, characterized in that: Mix 4% to 8% of modified biochar, 3% to 5% of Lactobacillus casei, 0.5% to 2% of sodium alginate, 1% to 2% of gelatin, 1% to 2% of silicon dioxide and water, and then add the mixture dropwise to a calcium chloride boric acid solution; solidify for 10 to 12 hours, and pass through a 100 to 150 mesh sieve; Preferably, the gel microspheres are placed in a chitosan solution for coating, wherein the chitosan solution is chitosan placed in a 1% acetic acid solution, and the mass percentage of the chitosan to the 1% acetic acid is 0.5%-2%; Preferably, the coating time in the chitosan solution is 6-10 minutes; Preferably, the gel microspheres coated with the chitosan layer are placed in a sodium alginate solution containing 0.15-0.2% by mass for coating; preferably, the gel microspheres coated with the chitosan layer are placed in a sodium alginate solution containing 0.15% by mass for coating for 6-10 minutes; Preferably, 4% of modified biochar, 5% of Lactobacillus casei, 1.5% of sodium alginate, 1-2% of gelatin, 1-2% of silicon dioxide and water are mixed; 1.5% chitosan solution is used for coating; Preferably, 8% of modified biochar by weight of water, 5% of Lactobacillus casei by weight of water, 1% of sodium alginate by weight of water, 1-2% of gelatin by weight of water, 1-2% of silicon dioxide by weight of water and water are mixed; and 2% chitosan solution is used for coating.

10. The method for preparing denitrification microcapsules according to claim 8, characterized in that: The preparation method of the modified biochar is as follows: modifying the biochar to be modified in a strong alkaline solution, washing and drying, and then placing the dried product in a MgCl2 solution for modification, washing and drying; Preferably, every 10 g of the biochar to be modified is mixed with a 0.1 M strong alkaline solution; Preferably, the strong alkaline solution is sodium hydroxide or potassium hydroxide solution; Preferably, the biochar is modified in a strong alkaline solution for 10-15 hours, then washed until the biochar is neutral, and dried to obtain alkali-modified biochar; Preferably, every 5 g of alkali-modified biochar is mixed with 0.025 M MgCl2 solution for modification; Preferably, every 5 g of alkali-modified biochar is mixed with 0.025 M MgCl2 solution for modification for 10-12 h.

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