Aquaculture denitrification microcapsule and its preparation method and application

Abstract

The present invention discloses an aquaculture denitrification microcapsule, a preparation method, and an application thereof, and relates to the fields of aquaculture and biotechnology. The Lactobacillus casei provided by the present invention can secrete acidic metabolites, which can inhibit pathogenic Vibrio, helping to reduce the occurrence of diseases in aquaculture. Lactobacillus casei has the ability to efficiently degrade ammonia nitrogen and nitrite nitrogen in the aquaculture environment, and can sustainably achieve efficient cleaning of the aquaculture environment. In addition, a denitrification microcapsule is provided, which creates a more favorable microenvironment for the efficient denitrification strain, thereby more stably exerting repeated denitrification performance, and the denitrification microcapsules can be reused.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] The present 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, total aquaculture production in the world and China has continued to rise in recent years, reaching 87.5 million tons and 55.6546 million tons, respectively, in 2020. The proportion of aquaculture production from feed-fed aquaculture has increased annually, leading to rapid development of the aquatic feed industry. As the aquaculture industry continues to expand, the pursuit of high yields has led to the widespread use of high-protein and high-fat feeds, resulting in low feed utilization rates for aquatic animals. Large amounts of leftover bait and feces lead to increased endogenous pollution (nitrogen compounds) in the aquaculture environment, frequent diseases, and serious harm to the physiological health of aquatic animals. Therefore, the development of efficient and clean technologies for green, sustainable aquaculture environments has become a pressing scientific issue.

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

[0006] Microencapsulation technology is an emerging bioprocessing technology. It utilizes membrane-forming natural or synthetic polymers as wall materials to effectively encapsulate a core material containing functional ingredients, creating a stable microcapsule structure. While this technology has experienced rapid development in recent years, it still faces drawbacks such as high costs for core and wall materials, complex preparation processes, and limited universal applicability to 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 current aquaculture biological microcapsule products such as high cost, complex preparation process, poor biocompatibility, and susceptibility to environmental influences (low C / N, etc.).

[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] A highly efficient denitrifying strain (GSVWA6) was isolated from a shrimp aquaculture pond in Guangdong Province. 16S rRNA sequencing revealed the highest similarity between this strain and Lactobacillus casei, leading to its identification as Lactobacillus casei. The strain was deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on January 22, 2024. The strain was designated GSVWA6, classified as Lacticaseibacillus casei GSVWA6, and identified as viable.

[0012] On the one hand, lactobacillus casei provided by the invention can secrete acidic metabolites, and acidic metabolites can suppress pathogenic vibrio, contribute to reducing the generation of disease in aquaculture; On the other hand, lactobacillus casei has efficient denitrification performance, can be converted into non-toxic substances by the ammonia and nitrite nitrogen in water body and other aquaculture metabolites, can be converted into bacterium self composition (a part can be converted into non-toxic gases such as nitrogen and discharge aquaculture water body, and a part can be converted into bacterium self composition). Therefore, lactobacillus casei provided by the invention has the performance that obviously suppresses pathogenic vibrio and efficient denitrification, is prepared aquaculture denitrification microcapsule as main body by lactobacillus casei and modified biochar, can be used for the efficient removal of nitrogen pollutants in aquaculture environment or artificial wetland.

[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 present invention is highly effective in degrading ammonia nitrogen and nitrite nitrogen in aquaculture environments, enabling sustainable and efficient cleaning of aquaculture environments. The microcapsules prepared using Lactobacillus casei have a lifespan of over 21 days, providing technical support for ecological purification technologies in aquaculture. This technology aligns with the concept of green and healthy development of aquaculture and has high application value and market prospects.

[0016] After a large number of experiments, it was found that under storage conditions of 4°C, microbial microcapsules containing 4% biochar content were able to achieve a 90% removal rate of ammonia nitrogen and nitrite within 40 days. Within a 60-day test period, microbial microcapsules containing 4% biochar content were able to achieve a removal rate of more than 85% of ammonia nitrogen and nitrite. In terms of the number of viable bacteria, microbial microcapsules containing 4% biochar content were able to maintain a viable bacteria count of 80% or more within 30 days, and a viable bacteria count of 60% or more within a 60-day test period. In comparison, the storage stability of microbial microcapsules containing 8% biochar content was improved compared to that of microbial microcapsules containing 4% biochar content. Microbial microcapsules containing 8% biochar content were able to achieve a 90% removal rate of ammonia nitrogen and nitrite within the entire 60-day test period.

[0017] Furthermore, in terms of viable bacterial counts, microbial microcapsules containing 4% and 8% biochar maintained 90% or higher viable bacterial counts within 30 days, and 70% or higher viable bacterial counts within a 60-day test period. This suggests that higher biochar content provides a more suitable microenvironment for denitrifying microorganisms, significantly improving their adaptability to the environment, thereby better utilizing their nitrogen removal performance and potentially unlocking greater potential for application in mid- and late-stage aquaculture environments.

[0018] It should be noted that for rapid denitrification, those skilled in the art could readily consider using microbial microcapsules with reduced biochar content (e.g., less than 4%), which is also within the scope of the present invention. Furthermore, if the biochar content is less than 4%, the denitrification effect will be somewhat affected.

[0019] Constructed wetlands, a unique, composite ecological wastewater treatment technology composed of plants, substrates, and microorganisms, have demonstrated significant application advantages in the deep treatment of wastewater treatment plant tailwater in developing regions, becoming a common and emerging method for deep wastewater purification. To achieve this deep wastewater purification, those skilled in the art can use the Lactobacillus casei provided by the present invention for wastewater denitrification.

[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 needed, without being limited to the form of microcapsules or bacterial agents.

[0022] In other embodiments, to improve the denitrification effect, those skilled in the art can easily conceive 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 comprising the aforementioned Lactobacillus casei. The bacterial agent may be in the form of, but not limited to, freeze-dried bacterial powder, bacterial liquid, fermentation precipitate, fermentation supernatant, or bacterial residue processed by a filter press or other equipment.

[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 sequentially arranged from the inside to the outside.

[0027] This invention uses natural polymer materials, sodium alginate and chitosan, as microcapsule wall materials, offering the advantage of low cost. The novel aquaculture denitrification microcapsules offer low cost, reducing overall costs by over 30%, and possess high application value and market prospects. The inventors have discovered that replacing the chitosan and sodium alginate layers with other materials reduces the ammonia nitrogen degradation rate.

[0028] Denitrification microcapsules feature a robust three-layer structure of sodium alginate, chitosan, and sodium alginate. Their small particle size, large surface area, and high mass transfer efficiency facilitate the removal of nitrogen pollutants from aquaculture water. They create a microbial environment ideal for the growth and reproduction of beneficial bacteria, mitigating the effects of environmental factors such as low C / N ratios in aquaculture environments. They demonstrate universal microbial compatibility and highly efficient denitrification.

[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, providing certain advantages in transportation, storage, and use. Replacing gelatin and silica with other substances may result in a decrease in the ammonia nitrogen degradation rate.

[0031] In summary, the present invention uses chitosan, sodium alginate, and silica as the wall materials of the microcapsules, and gelatin and modified biochar as the core materials to prepare a new type of microcapsule material. The mechanical strength of the wall material and the adsorption performance of the core material are improved, the universality of microorganisms is enhanced, and sufficient carbon source and suitable attachment space are provided for Lactobacillus casei, thereby improving the denitrification performance of Lactobacillus casei by more than 2 times.

[0032] Biochar, a densely packed, porous, and carbon-rich material produced by carbonizing biomass in a low-oxygen environment, has recently garnered widespread attention due to its potential applications in greenhouse gas nitrous oxide (NO) emission reduction, soil remediation, and pollutant adsorption. Biochar is easy to prepare, requires no activation, and costs only about one-sixth 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 at a low cost. Modified biochar significantly increases the pH of the material and significantly improves the carrier's compatibility with various bacterial strains. It has been shown to have high biocompatibility with various strains, including 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, a boric acid solution containing 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 6-8% calcium chloride by weight of water is 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 microcapsules 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 a 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, the microbial microcapsules with a 4% biochar content can maintain a viable bacteria count of 80% or more within 30 days, and maintain a viable bacteria count of 60% or more within a 60-day test period. In comparison, the storage stability of the 8% biochar microcapsules has been improved compared to the 4% biochar content microbial microcapsules. The 8% biochar content microbial microcapsules can achieve a 90% removal effect of ammonia nitrogen and nitrite within the entire test period of 60 days. From this, it can be inferred that a higher biochar content can provide a more suitable microenvironment for denitrification microorganisms, which can significantly improve the adaptability of denitrification microorganisms 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 alternative embodiment, the modified biochar is prepared by combined alkali and magnesium ion modification of the biochar. Alkali modification can etch the microporous structure of the biochar, converting it into mesoporous or macroporous structures, and Mg ion modification can make the biochar more appealing 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 will readily appreciate 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 prepare 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 silica as an auxiliary material, 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, which is low in cost. Modified rice husk biochar significantly increases 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 are prepared using a three-step coating method, resulting in microcapsule particles with a robust three-layer structure of sodium alginate, chitosan, and sodium alginate. The biochar-based microcapsules have a small particle size, large specific surface area, and high mass transfer efficiency, facilitating the removal of nitrogen pollutants from aquaculture water.

[0049] In a preferred embodiment of the present invention, the preparation method further comprises liquefying the gel microspheres having a sodium alginate layer and a chitosan layer, followed by freeze-drying to produce denitrified microcapsules. The term "liquefaction" specifically refers to the conversion of sodium alginate into calcium alginate by reacting with a calcium chloride solution, which then instantaneously forms a chitosan microcapsule membrane on the surface of the microgel beads through an ion complexation reaction. The calcium in the calcium alginate gel is then displaced with a sodium citrate solution, liquefying the microcapsules 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 effect.

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

[0052] Vacuum freeze-drying involves freezing a bacterial suspension below the triple point and then applying a vacuum. This allows free water inside and outside the cells to sublime directly into gas while frozen, achieving the goal of removing moisture. This technology preserves most of the active ingredients in the microcapsules. The components form a stable solid skeleton upon freezing, which largely retains its original shape after the water sublimates. The porous structure of the product exhibits excellent solubility, rehydration properties, and rehydration rate. Vacuum freeze-drying can remove over 95% of the water in the microcapsules, enabling long-term storage at room or higher temperatures while remaining lightweight 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 microorganisms caused by external harsh environmental conditions, thereby increasing the survival rate of 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 ratio of chitosan to 1% acetic acid is 0.5%-2%. For example, the mass ratio 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, 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 a 1.5% chitosan solution is used for coating.

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

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

[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 modification 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 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 water into non-toxic substances. Some of them can be converted into non-toxic gases such as nitrogen and discharged from the aquaculture water body, and some 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) The Lactobacillus casei provided by the present invention has the effectiveness of efficiently degrading ammonia nitrogen and nitrite nitrogen in the breeding environment, and can sustainably achieve efficient cleaning of the aquaculture environment. The microcapsule prepared based on Lactobacillus casei has a service life of more than 21 days, providing technical support for aquaculture ecological purification technology, conforming to the green and healthy development technology concept of aquaculture, and having extremely high application value and market prospects. In addition, the denitrification microcapsules provided by the present invention create a more favorable microenvironment for the efficient denitrification strain, thereby more stably exerting the repeated denitrification performance and being able to be reused.

[0072] (4) The provided denitrification microcapsules (or biochar microcapsules) 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 a 60-day test period, the denitrification microcapsules with a 4% biochar content achieved a removal rate of more than 85% for ammonia nitrogen and nitrite, while the denitrification microcapsules with an 8% biochar content achieved a removal rate of 90% for ammonia nitrogen and nitrite throughout the entire 60-day test period. Both the 4% and 8% biochar content denitrification microcapsules were able to 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 following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0075] Figure 1 This is a dynamic statistical result 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 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 Figure 1 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] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, 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 invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[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 person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (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); and PCR: The Polymerase Chain Reaction. 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] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

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

[0085] Example 1

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

[0087] (1) Separation and purification

[0088] 90 mL of ammonia nitrogen and nitrite nitrogen enrichment culture medium were respectively prepared and placed in 250 mL conical flasks. After sterilization in a sterilizer at high temperature and high pressure, 10 mL of pond water sample was added to the culture medium, which was then placed in a constant temperature shaking incubator to ensure culture at 28°C and 160 rpm. 50 mL of enrichment culture medium in the conical flask was replaced every day for a total of 72 h of enrichment culture.

[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 the specific bacteria suspension were 10 -4 , 10 -5 , 10 -6 100 μL of each bacterial suspension was spread onto the surface of ammonia nitrogen and nitrite nitrogen solid screening medium and incubated in a constant temperature incubator maintained at 28°C until a sufficient number of colonies grew. Colonies of varying color, properties, and size were selected and transferred to 2216E agar solid medium for streak culture until a single colony was obtained. The culture was then preserved in 25% glycerol in a seed tube and stored in a -80°C ultra-low temperature freezer in preparation for further research.

[0090] The preserved strains were activated and cultured in 2216E liquid culture medium for 24 h, and then inoculated into ammonia nitrogen and nitrite nitrogen degradation culture medium at a 5% inoculum size. The cultures were cultured at 28°C and 160 rpm for 48 h. The ammonia nitrogen and nitrite nitrogen removal abilities of the strains were determined by hypobromite oxidation method and naphthylethylenediamine spectrophotometry, respectively.

[0091] (2) Identification

[0092] DNA from denitrifying strains was extracted using a freeze-thaw cycle. Several denitrifying strains were inoculated into 2216E liquid medium and activated for 24 hours. After streaking onto 2216E solid medium, individual colonies were cultured until they formed. Using a sterilized toothpick, a single colony was picked into a 1.5ml centrifuge tube containing 50µL of sterile ultrapure water. After three freeze-thaw cycles, the tube was centrifuged at 12,000 rpm and 4°C for 10 minutes to obtain the bacterial DNA template for subsequent PCR amplification. The PCR reaction system and conditions are shown in Table 2-1.

[0093] The PCR amplification products were sent to Bioengineering (Shanghai) Co., Ltd. for sequencing. The obtained nucleotide sequences were further compared online by BLAST (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. The deposit address is: Wuhan University, Wuhan, China. The name of the deposited 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 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 inoculum size of 3.0% of the weight of the liquid medium. The culture was shaken at 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 ml 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 a 1% acetic acid solution and stir to dissolve in a water bath at 60°C to prepare 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 and stirred in a constant temperature water bath at 100°C to dissolve to prepare a sodium alginate solution. The mass percentage of sodium alginate to water was 1.5%.

[0106] ③ Preparation of modified rice husk biochar: First, 10g of unmodified rice husk biochar was soaked in 100mL of NaOH (1mol·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 soaked 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, with the total mass percentage of gelatin and silicon dioxide in water being 2%, and a high-efficiency denitrification lactic acid strain (bacterial activity is (1-3)×10 8 CFU / g), and rice husk biochar was added at a mass ratio of 4% to ultrapure water, mixed evenly, and then added dropwise to a 2% calcium chloride boric acid saturated solution using a syringe. The solidification time was 12 h, and then the microspheres were rinsed with ultrapure water and sieved with 100-150 mesh to obtain preliminary gel microspheres.

[0108] ⑤ The gel microspheres were placed in a 1.5% chitosan solution for the first coating, and the coating time was 10 minutes. After coating, they were washed three times with ultrapure water to remove the residual chitosan solution on the surface. The microcapsules with the initial coating were placed in a 0.15% low-concentration sodium alginate solution for the second coating, and the coating time was 10 minutes. After washing and sieving with 100-150 mesh, double-layer membrane gel microspheres were 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 them through 100-150 mesh, and vacuum freeze-dry them 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%, the film forming time of the first and second coatings in step (5) is 8 minutes, and 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%, the concentration of the raw material chitosan used in the first coating in step (5) is 2%, and 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%, the concentration of the raw material chitosan used in the first coating in step (5) is 2%, and 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] Orthogonal design experiments revealed that, in 4% biochar-based microbial microcapsules, the microcapsules exhibited the best physical performance when the sodium alginate addition was 1.5%, the chitosan addition was 1.5%, and the film-forming time was 10 minutes. In 8% biochar-based microbial microcapsules, the microcapsules exhibited the best physical performance when the sodium alginate addition was 1%, the chitosan addition was 2%, and the film-forming time was 10 minutes. The optimal solutions, determined by comprehensively evaluating the four performance indicators of bacterial loading, moisture content, water retention rate, and specific surface area, were Examples 2 and 7.

[0129] The results of the orthogonal experiment 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 results of the orthogonal experiment 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 level table of each factor 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 (with biochar addition ratios of 4% and 8%, respectively) were taken to treat 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 hours. The ammonia nitrogen and nitrite degradation rates of the microbial microcapsules were measured, and the number of viable microorganisms in the microcapsules was determined by the plate count method.

[0140] The experimental results are as follows Figure 1 、 Figure 2 The results show that, under storage conditions of 4°C, microbial microcapsules containing 4% biochar achieved a 90% removal of both ammonia nitrogen and nitrite within 40 days. Within the 60-day experimental period of this study, microbial microcapsules containing 4% biochar achieved over 85% removal of both ammonia nitrogen and nitrite. In terms of viable bacterial counts, microbial microcapsules containing 4% biochar maintained 80% or more of their viable bacterial counts within 30 days and 60% or more of their viable bacterial counts within the 60-day experimental period of this study. However, with extended storage, the biological activity of highly efficient denitrifying bacteria exhibited a significant lag period, potentially leading to increased denitrification time and reduced 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] Furthermore, in terms of viable bacterial counts, microbial microcapsules containing both 4% and 8% biochar maintained 80% or more of the viable bacteria within 30 days, and maintained 70% or more of the viable bacteria over the 60-day experimental period. This suggests that a higher biochar content provides a more suitable microenvironment for denitrifying microorganisms, significantly improving their ability to adapt to the environment, thereby better utilizing their nitrogen removal performance and potentially unlocking greater potential for application in mid- and late-stage aquaculture environments.

[0143] Experimental Example 3

[0144] The repeated denitrification efficiency of the 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 conditions of a C / N ratio of 10, a temperature of 28°C, and an oscillation rate of 160 r / min, 50 ml of simulated wastewater with a concentration of 10 mg / L of ammonia nitrogen and 10 mg / L of nitrite nitrogen were treated with the microbial microcapsules. Every 6 hours, 2.5 ml of water samples were taken to determine the degradation rates of ammonia nitrogen and nitrite nitrogen. When the degradation rate exceeded 95%, the microbial microcapsules were removed, rinsed with 0.9% saline, and transferred to another conical flask containing 50 ml of simulated wastewater with a concentration of 10 mg / L of ammonia nitrogen and nitrite nitrogen. The above steps were repeated. The microbial microcapsules were tested for their sustained removal of ammonia nitrogen and nitrite nitrogen, as well as their reusability.

[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 used as a cycle of experimental 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 a removal effect of 80% or more in the five test cycles of this experiment, and both the microbial microcapsules with 4% and 8% biochar content could achieve a 90% ammonia nitrogen and nitrite removal effect within three 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 pathogens by Lactobacillus paracasei.

[0152] The results of inhibition zone test refer to Figure 5As shown, the Oxford cup method was used. Two strains of denitrifying bacteria were inoculated into 2216E liquid culture medium and activated under constant temperature and 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. 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. The plate was then placed in a constant temperature incubator and cultured at 28°C for 48 hours. The appearance of an inhibition zone around the Oxford cup was continuously observed. If an inhibition zone appeared, it indicated that there was a possible antagonistic or inhibitory effect between the two strains. If no inhibition zone appeared, it indicated that there was no antagonistic effect between the two strains.

[0153] The results showed that this strain had strong inhibitory activity against 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 flask. Incubate at 28℃ and 160rpm. Take 2.5ml of water sample every 24h to measure the degradation rate of total nitrogen.

[0157] Results reference Figure 6 The results show that the addition of biochar significantly increases the mass transfer performance and specific surface area of ​​the microcapsules. Compared to free, efficient denitrifying bacteria, biochar-based microcapsules provide a safer and more stable living environment for the strains, ensuring their proper function. An 8% biochar content provides more attachment sites for the efficient denitrifying bacteria. Furthermore, the increased biochar content significantly enhances adsorption, resulting in higher total nitrogen removal efficiency. It is important to note that the total nitrogen content of the free denitrifying bacteria decreased over the 72-hour experimental period, while the 4% and 8% biochar-based microcapsules reached their lowest total nitrogen content at 48 hours and then increased slightly within 72 hours. This is presumably due to the larger pores in the biochar-based microcapsules, which release minimal adsorbed nitrogen. However, overall, the denitrification efficiency of the biochar-based microcapsules was 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 24 hours. Then, it was added to conical flasks containing 100ml of ammonia nitrogen and nitrite nitrogen degradation culture medium at an inoculation ratio of 5%. After culturing for 48 hours under different environmental conditions, a certain amount of culture medium was taken and centrifuged at 4000rpm for 10 minutes. The supernatant was retained and its ammonia nitrogen and nitrite nitrogen contents were determined. Three parallel sets were set for each group.

[0160] The experimental environment was set at 28°C, pH 8.3, C / N ratio 5, an initial nitrogen concentration of 10 mg / L, and a constant temperature shaker speed of 160 rpm / min. Salinity levels were set at 10‰, 20‰, 30‰, 40‰, 50‰, and 60‰.

[0161] The results showed that when the environmental salinity was maintained at 20-60‰, the strain was sensitive to NH4 + -N and NO2 - The degradation rates of -N were all above 80%. Salinity below 20‰ significantly inhibited the growth of the strain, further improving the denitrification efficiency. At a salinity of 40‰, the strain had the highest degradation rates of ammonia nitrogen and nitrite nitrogen. As the salinity increased, the removal efficiency of ammonia nitrogen and nitrite nitrogen gradually decreased. Therefore, this Lactobacillus casei has a strong salinity tolerance and can maximize the denitrification performance of the strain when the salinity of the water environment is 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 microcapsules prepared by the present invention are mainly based on lactic acid bacteria and biochar, and the total production cost thereof is reduced by more than 30%, and the microcapsules have extremely high application value and market prospects.

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

[0167] Furthermore, the Lactobacillus casei and its microcapsules provided by the present invention are highly effective in degrading ammonia nitrogen and nitrite nitrogen in aquaculture environments. Their useful life is over 21 days, providing technical support for ecological purification technologies in aquaculture. This supports the concept of green and healthy development of aquaculture and possesses high application value and market prospects.

[0168] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A Lactobacillus casei ( Lacticaseibacillus 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.

4. The use according to claim 2, characterized in that The application in the denitrification includes at least one of the following uses: Application in the preparation of denitrification microcapsules and in the preparation of denitrification bacterial agents.

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

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

7. The denitrification microcapsule according to claim 6, characterized in that The lactobacillus casei is loaded on the gel microspheres, and the wall material outside the gel microspheres comprises a chitosan layer and a sodium alginate layer which are sequentially arranged from the inside to the outside.

8. The denitrification microcapsule according to claim 7, characterized in that 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.

9. The denitrification microcapsule according to claim 8, characterized in that The modified biochar with a water mass percentage of 4% to 8%, Lactobacillus casei with a water mass percentage of 3% to 5%, sodium alginate with a water mass percentage of 0.5% to 2%, gelatin with a water mass percentage of 1% to 2%, silicon dioxide with a water mass percentage of 1% to 2%, and a boric acid solution with a water mass percentage of 6% to 8% calcium chloride are mixed.

10. The denitrification microcapsule according to claim 8, characterized in that The modified biochar with a water mass percentage of 4% to 8%, Lactobacillus casei with a water mass percentage of 3% to 5%, sodium alginate with a water mass percentage of 0.5% to 2%, gelatin with a water mass percentage of 1-2%, silicon dioxide with a water mass percentage of 1-2%, and boric acid solution with a water mass percentage of 7% calcium chloride are mixed.

11. The denitrification microcapsule according to claim 8, characterized in that The modified biochar is prepared by jointly modifying biochar with alkali and Mg ions; The biochar is sourced from at least one of rice husks, straw and reeds.

12. The denitrification microcapsule according to claim 11, characterized in that The straw is selected from at least one of rice straw, corn straw and sesame straw.

13. The denitrification microcapsule according to claim 7, characterized in that Each gram of the denitrification microcapsules contains (1-3)×10 8 CFU of Lactobacillus casei.

14. The denitrification microcapsule according to claim 13, characterized in that Each gram of the denitrification microcapsules contains 2.5×10 8 CFU of Lactobacillus casei.

15. A method for preparing the denitrification microcapsules according to any one of claims 6 to 14, characterized in that: It includes the following steps: Lactobacillus casei is embedded in the wall material.

16. The method for preparing denitrification microcapsules according to claim 15, characterized in that: The method of embedding Lactobacillus casei in the wall material includes: 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 prepare 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.

17. The method for preparing denitrification microcapsules according to claim 16, characterized in that: The preparation method further comprises: liquefying the gel microspheres having the sodium alginate layer and the chitosan layer, and freeze-drying the gel microspheres to obtain denitrification microcapsules.

18. The method for preparing denitrification microcapsules according to claim 17, characterized in that: Liquefy in 0.055 mol / L sodium citrate solution for 4-6 minutes; after washing, pass through a 100-150 mesh sieve and vacuum freeze-dry for 24-36 hours.

19. The method for preparing denitrification microcapsules according to claim 16, characterized in that: 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 water were mixed and then added dropwise to a boric acid solution of calcium chloride; the mixture was cured for 10-12 hours and passed through a 100-150 mesh sieve; 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%; The time for coating in chitosan solution is 6-10 minutes; The gel microspheres coated with the chitosan layer are placed in a sodium alginate solution containing 0.15-0.2% by mass for coating.

20. The method for preparing denitrification microcapsules according to claim 19, characterized in that: The gel microspheres coated with the chitosan layer are placed in a sodium alginate solution containing 0.15% by mass and coated for 6-10 minutes.

21. The method for preparing denitrification microcapsules according to claim 16, characterized in that: 4% of modified biochar by weight of water, 5% of Lactobacillus casei by weight of water, 1.5% 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 1.5% chitosan solution is used for coating.

22. The method for preparing denitrification microcapsules according to claim 16, wherein: 8% modified biochar, 5% Lactobacillus casei, 1% sodium alginate, 1-2% gelatin, and 1-2% silicon dioxide were mixed with water; and 2% chitosan solution was used for coating.

23. The method for preparing denitrification microcapsules according to claim 16, characterized in that: 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; every 10g of the biochar to be modified is mixed with a 0.1M strong alkaline solution; The strong alkaline solution is sodium hydroxide or potassium hydroxide solution; 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.

24. The method for preparing denitrification microcapsules according to claim 23, characterized in that: Every 5 g of alkali-modified biochar was mixed with 0.025 M MgCl2 solution for modification.

25. The method for preparing denitrification microcapsules according to claim 23, characterized in that: Every 5 g of alkali-modified biochar was mixed with 0.025 M MgCl2 solution and modified for 10-12 h.

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