Biological agent and method and system for preparing biological agent

Abstract

The invention relates to the technical field of microorganisms and environmental protection, and discloses a biological agent and a method and system for preparing the biological agent, and the biological agent comprises a wall material and a core material embedded in the wall material; wherein the core material comprises slow-release oxygen microspheres and aerobic functional bacteria, the slow-release oxygen microspheres are prepared from a raw material composition containing chitosan, sodium alginate, an activating agent, persulfate, an emulsifying agent, a dispersing agent and a cross-linking agent through an emulsification cross-linking method, the emulsifying agent is a biological surfactant, and the cross-linking agent is glyceraldehyde. According to the biological agent provided by the invention, the slow-release oxygen microspheres and the aerobic functional bacteria are embedded in the wall material at the same time, and the raw materials and the preparation method of the slow-release oxygen microspheres are designed, so that the finally prepared biological agent can give full play to the function of the functional bacteria, and when the biological agent is applied to harmless biodegradation of organic pollutants, the degradation efficiency is high. In addition, the biological agent is high in degradability, green, free of secondary pollution and beneficial to engineering application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and environmental protection, and particularly relates to a biological bacterial agent and a method and system for preparing the biological bacterial agent. BACKGROUND

[0002] In the process of oil exploitation, storage, transportation, processing and use, due to accidents, maintenance, improper operation and leakage, etc., various organic pollutants enter the soil and groundwater, causing pollution and damage to the ecological environment; at the same time, the organic pollutants enter the biological chain through migration and diffusion and finally endanger human life and health. Microbial degradation technology has been widely concerned due to its economic efficiency, simple operation, greenness and no secondary pollution, etc., and the immobilization technology of microbial bacterial agent has become a research hotspot in recent years due to its strong mechanical properties, high biological concentration, strong stress resistance and good stability.

[0003] Compared with anaerobic functional strains, aerobic functional strains have the advantages of high degradation efficiency, short degradation period and easy expansion, but the amount of dissolved oxygen in soil and groundwater is small, which limits the treatment capacity of the oxygen functional strain, resulting in low degradation efficiency of the organic pollutants, thereby limiting the large-scale application of the aerobic functional strain.

[0004] Chinese patent application CN103951152A discloses a composite preparation capable of continuously reducing riverway sediment and a preparation method thereof. The first preparation loaded with degradation functional bacteria and the second preparation loaded with oxygen release are simply mixed and then put into the water to treat the organic pollutants. However, on the one hand, the patent uses calcium peroxide-based oxygen release material, and after being put into the water, the pH is too high, which affects the activity of the functional bacteria; on the other hand, the water flow is disturbed, so that the two preparations are dispersed far away, which cannot guarantee the demand of the functional bacteria for dissolved oxygen, thereby limiting the degradation efficiency of the functional bacteria. SUMMARY

[0005] The present application aims to overcome the problems of the prior art, i.e., the pH is too high after the composite bacterial agent containing the oxygen release preparation and the functional bacterial preparation is put into the water, and the combination of the oxygen release preparation and the biological preparation in the composite bacterial agent is not tight enough, resulting in poor degradation efficiency of the composite bacterial agent, and provides a biological bacterial agent and a method and system for preparing the biological bacterial agent.

[0006] In order to achieve the above-mentioned purpose, the present application provides a biological bacterial agent, which comprises a wall material and a core material embedded in the wall material.

[0007] The core material comprises oxygen release microspheres and aerobic functional bacteria, and the oxygen release microspheres are prepared by an emulsification cross-linking method using a raw material composition containing chitosan, sodium alginate, an activator, a persulfate, an emulsifier, a dispersant and a cross-linking agent, wherein the emulsifier is a biological surfactant, and the cross-linking agent is glycerol aldehyde.

[0008] Preferably, the particle size of the biological agent is 50-500 μm.

[0009] Preferably, the aerobic functional bacteria are oil-degrading bacteria capable of degrading petroleum hydrocarbons.

[0010] Preferably, the particle size of the slow-release oxygen microspheres is 0.5-3 μm.

[0011] Preferably, in the raw material composition, the molecular weight of the chitosan is 300-400 kDa, and the degree of deacetylation is 70-90%;

[0012] Preferably, the activator is biochar.

[0013] Preferably, the persulfate salt is selected from one or more of sodium persulfate, potassium persulfate and ammonium persulfate.

[0014] Preferably, the emulsifier is selected from one or more of rhamnolipid, trehalose lipid and sophorolipid.

[0015] Preferably, the dispersant is vegetable oil containing sodium dodecyl sulfate.

[0016] Preferably, in the raw material composition, the mass ratio of the chitosan, the activator and the persulfate salt is 2-10:1:2-10.

[0017] Preferably, in the raw material composition, the volume ratio of the emulsifier and the dispersant is 1-30:100.

[0018] Preferably, in the raw material composition, the mass of the sodium alginate to the total volume of the emulsifier and the dispersant is 1 g:100-500 mL.

[0019] Preferably, in the raw material composition, the amount ratio of the activator to the dispersant is 1 g:50-300 mL.

[0020] Preferably, the material of the wall material is selected from one or more of sodium alginate, polyvinyl alcohol and chitosan.

[0021] The second aspect of the present application provides a method for preparing a biological agent, which comprises the following steps:

[0022] (1) mixing the aerobic functional bacteria, the slow-release oxygen microspheres, the wall material raw material and water to obtain a dispersed phase solution;

[0023] (2) injecting the dispersed phase solution into a calcium chloride solution or a calcium lactate solution and stirring;

[0024] The slow-release oxygen microspheres are prepared by an emulsification cross-linking method from a raw material composition containing chitosan, sodium alginate, an activator, a persulfate, an emulsifier, a dispersant, and a cross-linking agent, wherein the emulsifier is a biological surfactant, and the cross-linking agent is glycerol aldehyde.

[0025] Preferably, step (2) specifically comprises: after the dispersed phase solution forms the bacterial beads with a particle size of 50-500 μm through the microfluidic unit, the bacterial beads are injected into a calcium chloride solution or a calcium lactate solution and stirred.

[0026] Preferably, the microfluidic unit comprises a microfluidic pressure pump and an injection device, wherein the pressure of the microfluidic pressure pump is 500-5000 mbar, and the pore diameter of the capillary dropper of the injection device is 0.17-0.41 mm.

[0027] Preferably, in step (1), the aerobic functional bacteria are added in the form of a bacterial suspension or an immobilized bacterial agent.

[0028] Preferably, when the aerobic functional bacteria are added in the form of a bacterial suspension, the use amount ratio of the slow-release oxygen microspheres, the bacterial suspension, and the wall material raw material is 0.5-3 g:10-30 ml:1.5-3.5 g, and the OD value of the bacterial suspension is 0.2-0.5. 600

[0029] Preferably, when the aerobic functional bacteria are added in the form of an immobilized bacterial agent, the mass ratio of the use amount of the slow-release oxygen microspheres, the immobilized bacterial agent, and the wall material raw material is 0.5-3:1-5:1.5-3.5.

[0030] Preferably, the wall material raw material is selected from one or two or more of sodium alginate, polyvinyl alcohol, and chitosan.

[0031] Preferably, the slow-release oxygen microspheres are prepared according to the following procedures:

[0032] A1, mixing an emulsifier and a dispersant to obtain an oil phase solution;

[0033] A2, mixing an activator, a persulfate, and a chitosan dilute acid solution to obtain an aqueous phase solution;

[0034] A3, mixing a sodium alginate aqueous solution and the oil phase solution to obtain a preliminary emulsion;

[0035] A4, mixing the preliminary emulsion and the aqueous phase solution to obtain a final emulsion;

[0036] A5, adding the final emulsion into a cross-linking agent to perform a cross-linking reaction to obtain a reaction liquid;

[0037] A6, performing solid-liquid separation on the reaction liquid, and then washing the obtained solid phase to obtain slow-release oxygen microspheres. ​

[0038] Preferably, in step A2, the chitosan dilute acid solution contains chitosan and a dilute acid solution, wherein the pH of the dilute acid solution is 3-5.

[0039] Preferably, in the chitosan dilute acid solution, the content of chitosan is 2-5 wt%.

[0040] Preferably, the acid in the dilute acid solution is selected from one or more than two of glacial acetic acid, lactic acid, malic acid and ascorbic acid.

[0041] Preferably, in step A3, the mass concentration of sodium alginate in the sodium alginate aqueous solution is 1.5-3.5%, and the volume ratio of the sodium alginate aqueous solution to the oil phase solution is 1:4-7.

[0042] Preferably, in step A4, the volume ratio of the water phase solution to the primary emulsion is 1:4-7.

[0043] The third aspect of the present application provides a biological bacterial agent prepared by the method described above.

[0044] The fourth aspect of the present application provides a system for preparing the biological bacterial agent described above, which comprises:

[0045] The system comprises a microfluidic unit and a cross-linking coagulation unit, wherein the microfluidic unit comprises a microfluidic pressure pump, an injection device and a high-voltage power supply, the microfluidic pressure pump is connected with the injection device, the injection device comprises a capillary dropper, one end of the high-voltage power supply is connected with the capillary dropper through a high-voltage power supply positive connection line, and the other end is immersed into a cross-linking agent liquid in the cross-linking coagulation unit through a high-voltage power supply negative connection line;

[0046] The dispersed phase solution is injected into the cross-linking coagulation unit after forming the bacterial beads with a particle size of 50-500 μm through the capillary dropper, and the calcium chloride solution or calcium lactate solution in the cross-linking coagulation unit is mixed with the bacterial beads.

[0047] The dispersed phase solution contains aerobic functional bacteria, oxygen microspheres, wall material raw materials and water.

[0048] Preferably, the cross-linking coagulation unit comprises:

[0049] A shell, in which the cross-linking agent solution and the bacterial beads from the microfluidic unit are mixed;

[0050] A stirring device arranged in the shell for stirring the materials in the shell;

[0051] A heating device for heating the materials in the shell; and

[0052] A filter screen is arranged at the bottom of the shell for trapping biological bacteria.

[0053] The biological bacteria provided by the application, the slow-release oxygen microspheres and the aerobic functional bacteria are embedded in the wall material at the same time. After being put into a deep micro-aerobic environment with pollution, the slow-release oxygen microspheres can ensure the demand of the aerobic functional bacteria for dissolved oxygen, thereby prolonging the life of the functional bacteria and improving the activity of the functional bacteria. Meanwhile, sodium alginate is a poly-anionic polysaccharide extracted from marine organisms, and has good biological viscosity and biodegradability. Traditional emulsification methods use Span 80 as an emulsifier and glutaraldehyde as a crosslinking agent, and Span 80 and glutaraldehyde can reduce the biological activity of the bacteria. However, in the preparation of the slow-release oxygen microspheres, a biological surfactant is used as an emulsifier, and glycerol is used as a crosslinking agent. Through the addition of sodium alginate, a bacteria product with high biological activity is prepared, and the viable bacterial load of the bacteria product is greatly increased. Therefore, the biological bacteria can fully play the role of the functional bacteria, and when the biological bacteria are applied to the harmless biodegradation of organic pollutants, the degradation efficiency is high. In addition, by selecting biodegradable and non-toxic reagents as raw materials, the biodegradability of the biological bacteria is strong, and the biological bacteria are green and do not cause secondary pollution, which is beneficial to engineering application.

[0054] The method for preparing the biological bacteria provided by the application realizes the simultaneous embedding of the slow-release oxygen microspheres and the aerobic functional bacteria through the design of the experimental steps and the raw materials and the preparation method of the slow-release oxygen microspheres.

[0055] The system for preparing the biological bacteria provided by the application can accurately control the size of the bacteria beads and the thickness of the wall material based on microfluidic technology, so that the size of the prepared biological bacteria is small (50-500 μm), thereby increasing the substrate reaction area and improving the mass transfer, and further greatly improving the degradation efficiency of the prepared biological bacteria on pollutants. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a structural schematic diagram of an embodiment of the system for preparing the biological bacteria provided by the application;

[0057] Figure 2 is a particle size comparison diagram of the biological bacteria products prepared by example 1 and example 5 of the application;

[0058] Figure 3 is a diagram of local microorganisms in the capsule of the biological bacteria A prepared by example 1 of the application;

[0059] Figure 4 is an oxygen release performance diagram of the slow-release oxygen microspheres A prepared by preparation example 1 of the application;

[0060] Figure 5 is an oxygen release performance diagram of the slow-release oxygen microspheres B prepared by preparation example 2 of the application;

[0061] Figure 6 is the oxygen release performance chart of the bio-agent product prepared by the present application embodiment 1-4.

[0062] Explanation of reference signs

[0063] 11-injection device, 12-microfluidic pressure pump, 13-high voltage power supply, 111-pushing device, 112-dispersed phase injection inlet, 113-capillary dropper, 131-high voltage power supply positive connection line, 132-high voltage power supply negative connection line;

[0064] 21-stirring device, 22-heating device, 23-filter screen, 24-waste liquid funnel, 25-waste liquid valve, 26-waste liquid pipe, 27-housing;

[0065] 31-dispersed phase storage pool; 32-injection pump. DETAILED DESCRIPTION

[0066] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0067] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and the values are approximate values and should be understood as including values approximately near these ranges and values within these ranges. For ranges of values, the endpoints of the ranges are included in the ranges, and the endpoints and individual points within the ranges are included in the ranges, and the ranges are inclusive of the individual points.

[0068] The present application provides a bio-agent, which comprises a wall material and a core material embedded in the wall material; wherein the core material comprises oxygen-releasing microspheres and aerobic functional bacteria, the oxygen-releasing microspheres are prepared by an emulsification cross-linking method from a raw material composition containing chitosan, sodium alginate, an activator, a persulfate, an emulsifier, a dispersant and a cross-linking agent, the emulsifier is a biosurfactant, and the cross-linking agent is glycerol aldehyde.

[0069] In a preferred embodiment, the dispersant is a plant oil containing sodium dodecyl sulfate.

[0070] The biological agent, the slow-release oxygen microspheres and the aerobic functional bacteria are embedded in the wall material at the same time, which can ensure the demand of the aerobic functional bacteria for dissolved oxygen after being put into the treatment site, thereby prolonging the life of the functional bacteria and improving the activity of the functional bacteria; meanwhile, the sodium alginate is a poly anionic polysaccharide extracted from marine organisms, which has good biological viscosity and biodegradability; the traditional emulsification method uses Span80 as an emulsifier, liquid paraffin as a dispersant and glutaraldehyde as a crosslinking agent, and the above Span80, liquid paraffin and glutaraldehyde can reduce the biological activity of the bacterial agent, while the biological surfactant is used as an emulsifier, the plant oil containing sodium dodecyl sulfate is used as a dispersant and the glycerol is used as a crosslinking agent in the preparation of the slow-release oxygen microspheres, and the bacterial agent product with high biological activity is prepared by adding sodium alginate, thereby greatly increasing the viable bacterial load of the bacterial agent product. Therefore, the biological agent can fully play the role of the functional bacteria, and has high degradation efficiency when being applied to the harmless biodegradation of organic pollutants. In addition, the biodegradability of the biological agent is strong, green and free of secondary pollution by selecting biodegradable and non-toxic reagents as raw materials, thereby being beneficial to engineering application.

[0071] The present application does not limit the specific type of the aerobic functional bacteria, which can be selected according to actual needs. In a specific embodiment, the aerobic functional bacteria are petroleum-degrading bacteria capable of degrading petroleum hydrocarbons, and the biological agent can be applied to the harmless biodegradation of organic pollutants by selecting petroleum-degrading bacteria as the aerobic functional bacteria, thereby effectively solving the problem of organic pollution of soil and groundwater and having a wide application prospect.

[0072] The present application also does not limit the specific selection of the petroleum-degrading bacteria, which can be obtained by self-selection or be a commercially available product, as long as it can degrade organic pollutants. In a specific embodiment, the petroleum-degrading bacteria are one or two or more of Cellulomonas sp. A, Pseudomonas sp. E and Cellulomonas sp. D, wherein the preservation number of Cellulomonas sp. A is GDMCC No: 62343, the preservation number of Pseudomonas sp. E is GDMCC No: 62339 and the preservation number of Cellulomonas sp. D is GDMCC No: 62340. The degradation range of the above petroleum-degrading bacteria is 15-40℃ and pH=6.5-10.5.

[0073] In the present application, the biological agent is in a capsule shape. The present application does not limit the size of the biological agent, which can be in millimeter level (e.g. 1-10 mm) or in micron level (e.g. 10-500 μm). In a preferred embodiment, the particle size of the biological agent is 50-500 μm, which can increase the reaction area of the substrate and the loading amount of the functional bacteria, thereby greatly improving the pollutant degradation efficiency.

[0074] In a preferred embodiment, the particle size of the slow-release oxygen microspheres is 0.5-3 μm.

[0075] The present application does not limit the specific parameters of chitosan in the raw material composition, and any chitosan commonly used in the art can be selected. In a specific embodiment, the molecular weight of chitosan in the raw material composition is 300-400 kDa, and the degree of deacetylation is 70-90%.

[0076] The present application also does not limit the specific selection of the activating agent in the raw material composition, which can be any activating agent commonly used in the art. In a specific embodiment, the activating agent is biochar.

[0077] In the present application, the persulfate salt is used as an oxidizing agent. Further, the persulfate salt can be selected from one or more than two of sodium persulfate, potassium persulfate and ammonium persulfate.

[0078] In the present application, the emulsifier in the raw material composition is a biosurfactant, which can be rhamnolipid, trehalose lipid or sophorolipid. In a preferred embodiment, the emulsifier is selected from one or more than two of rhamnolipid, trehalose lipid and sophorolipid.

[0079] The present application does not limit the amount of each component in the raw material composition, which can be designed according to the actual needs. In a preferred embodiment, the mass ratio of chitosan, activating agent and persulfate salt in the raw material composition is 2-10:1:2-10.

[0080] In a preferred embodiment, the volume ratio of the emulsifier and dispersant in the raw material composition is 1-30:100.

[0081] In a preferred embodiment, the mass of sodium alginate in the raw material composition is 1 g, and the total volume of the emulsifier and dispersant is 100-500 mL.

[0082] In a preferred embodiment, the sodium alginate in the raw material composition is prepared into an aqueous sodium alginate solution before being added, so that the sodium alginate and other raw material components can be more easily mixed.

[0083] In a preferred embodiment, the ratio of the amount of the activator to the dispersant in the raw material composition is 1g: 50-300mL.

[0084] In order to facilitate the mixing of chitosan with other raw material components, in a preferred embodiment, the chitosan is first mixed with a dilute acid solution and then added. The pH of the dilute acid solution is 3-5. The present application does not limit the specific type of the dilute acid solution, which can be a dilute acid solution commonly used in the art, for example, an acetic acid solution, a lactic acid solution, a malic acid solution or an ascorbic acid solution.

[0085] In a preferred embodiment, the wall material is selected from one or more than two of sodium alginate, polyvinyl alcohol and chitosan, which are readily available and low in cost.

[0086] The present application also provides a method for preparing the bio-agent as described above, which comprises the following steps:

[0087] (1) mixing the aerobic functional bacteria, the oxygen-releasing microspheres, the wall material raw materials and water to obtain a dispersed phase solution;

[0088] (2) injecting the dispersed phase solution into a calcium chloride solution or a calcium lactate solution and stirring;

[0089] The oxygen-releasing microspheres are prepared from a raw material composition containing chitosan, sodium alginate, an activator, a persulfate, an emulsifier, a dispersant and a crosslinking agent by an emulsification crosslinking method, the emulsifier is a biological surfactant, and the crosslinking agent is glycerol aldehyde.

[0090] The method for preparing the bio-agent provided by the present application realizes the simultaneous embedding of the oxygen-releasing microspheres and the aerobic functional bacteria by designing the experimental steps and the raw materials and preparation method of the oxygen-releasing microspheres, prolongs the life of the functional bacteria and improves the activity of the functional bacteria, which is beneficial to the engineering application.

[0091] The present application does not limit the specific operation procedure of step (2), which can be a commonly used "dropping pill method" in the art. In order to make the degradation efficiency of the prepared bio-agent higher, in a preferred embodiment, step (2) specifically comprises: after the dispersed phase solution is formed into bacteria beads with a particle size of 50-500μm by a microfluidic unit, the bacteria beads are injected into a calcium chloride solution or a calcium lactate solution and stirred, so that the size of the prepared bio-agent is small, thereby increasing the substrate reaction area, improving the functional bacteria loading capacity and further greatly improving the pollutant degradation efficiency.

[0092] The present application does not limit the specific implementation of the microfluidic unit to form the bacteria beads with a particle size of 50-500 μm, and can be performed according to the conventional operation in the art. In a preferred embodiment, the microfluidic unit comprises a microfluidic pressure pump and an injection device, wherein the pressure of the microfluidic pressure pump is 500-5000 mbar, and the pore size of the capillary dropper of the injection device is 0.17-0.41 mm. By adjusting the pressure of the microfluidic pressure pump and the pore size of the capillary dropper, the size of the formed bacteria beads can be controlled to be 50-500 μm.

[0093] In a specific embodiment, in step (1), the aerobic functional bacteria are added in the form of a bacterial suspension or an immobilized bacterial agent.

[0094] In a preferred embodiment, when the aerobic functional bacteria are added in the form of a bacterial suspension, the dosage ratio of the slow-release oxygen microspheres, the bacterial suspension, and the wall material raw material is 0.5-3 g: 10-30 ml: 1.5-3.5 g, and the OD value of the bacterial suspension is 0.2-0.5. 600

[0095] In a preferred embodiment, when the aerobic functional bacteria are added in the form of an immobilized bacterial agent, the mass ratio of the dosage of the slow-release oxygen microspheres, the immobilized bacterial agent, and the wall material raw material is 0.5-3: 1-5: 1.5-3.5. The present application does not limit the specific preparation method of the immobilized bacterial agent, as long as it is loaded with aerobic functional bacteria.

[0096] In a specific embodiment, the method further comprises preparing an immobilized bacterial agent according to the following procedure: fermenting a bacterial solution containing aerobic functional bacteria to obtain a fermented bacterial solution, mixing the fermented bacterial solution with biochar at an inoculation amount of 5-10%, adding a liquid medium, stirring and mixing at 30-37°C for 20-24 h, then filtering and drying to obtain the immobilized bacterial agent. More specifically, the particle size of the biochar can be 80-100 mesh, the stirring speed can be 100-200 rpm, and the formula of the liquid medium can be: yeast extract 5 g / L, tryptone 10 g / L, and sodium chloride 10 g / L.

[0097] In a preferred embodiment, in step (1), the wall material raw material is selected from one or more of sodium alginate, polyvinyl alcohol, and chitosan.

[0098] The slow-release oxygen microspheres in step (1) can be prepared by the above-mentioned raw materials according to the conventional emulsification cross-linking method in the art. In a preferred embodiment, the slow-release oxygen microspheres are prepared according to the following procedure:

[0099] Step A1, mixing the emulsifier and the dispersant to obtain an oil phase solution;

[0100] ​Step A2, mixing the activator, persulfate and chitosan dilute acid solution to obtain an aqueous phase solution;

[0101] Step A3, mixing the sodium alginate aqueous solution and the oil phase solution to obtain a primary emulsion;

[0102] Step A4, mixing the primary emulsion and the aqueous phase solution to form a final emulsion.

[0103] Step A5, adding the final emulsion into a crosslinking agent to perform a crosslinking reaction to obtain a reaction liquid;

[0104] Step A6, performing solid-liquid separation on the reaction liquid, and then washing the obtained solid phase to obtain the slow-release oxygen microspheres.

[0105] In a preferred embodiment, the preparation method of the chitosan dilute acid solution in step A2 comprises: dissolving chitosan in a dilute acid solution with a pH of 3-5 to obtain the chitosan dilute acid solution. Further preferably, the content of chitosan in the chitosan dilute acid solution is 2-5 wt%.

[0106] Further preferably, the acid in the dilute acid solution is selected from one or more than two of glacial acetic acid, lactic acid, malic acid and ascorbic acid, and specifically, for example, can be a glacial acetic acid solution, a lactic acid solution, a malic acid solution or an ascorbic acid solution.

[0107] In a preferred embodiment, in step A3, the mass concentration of sodium alginate in the sodium alginate aqueous solution is 1.5-3.5%, and the volume ratio of the use amount of the sodium alginate aqueous solution to the oil phase solution is 1:4-7.

[0108] Preferably, in step A4, the volume ratio of the use amount of the aqueous phase solution to the primary emulsion is 1:4-7.

[0109] In a preferred embodiment, step A5 specifically comprises: adding the final emulsion dropwise into a crosslinking agent to form a crosslinking system to perform a crosslinking reaction to obtain a reaction liquid; wherein the volume concentration of the crosslinking agent in the crosslinking system is 0.01%-2%.

[0110] The application further provides a biological microbial agent prepared by the method as described above.

[0111] The application further provides a system for preparing the biological microbial agent as described above, which will be described in combination with Figure 1The system comprises a microfluidic unit and a cross-linking coagulation unit, wherein the microfluidic unit comprises a microfluidic pressure pump, an injection device and a high-voltage power supply, the microfluidic pressure pump is connected with the injection device, the injection device comprises a capillary dropper, one end of the high-voltage power supply is connected with the capillary dropper through a high-voltage power supply positive electrode connecting line, and the other end is immersed into a liquid in the cross-linking coagulation unit through a high-voltage power supply negative electrode connecting line;

[0112] The dispersed phase solution is injected into the cross-linking coagulation unit after forming the bacterial beads with a particle size of 50-500 μm through the capillary dropper, and the calcium chloride solution or the calcium lactate solution in the cross-linking coagulation unit is mixed with the bacterial beads.

[0113] The dispersed phase solution contains aerobic functional bacteria, oxygen-releasing microspheres, wall material raw materials and water.

[0114] In the system, the size of the high-efficiency biological bacterial beads can be controlled by adjusting the pressure of the microfluidic pressure pump and the aperture of the capillary dropper; the thickness of the wall material of the high-efficiency biological bacterial beads can be controlled by adjusting the voltage of the high-voltage electric field, so as to quantitatively control the mass transfer property.

[0115] In a specific embodiment, the injection device further comprises a pushing device and a dispersed phase injection inlet, and the microfluidic pressure pump pushes the pushing device of the injection device through outputting a specific pressure, so as to accurately control the flow rate.

[0116] In a preferred embodiment, the cross-linking coagulation unit comprises:

[0117] A shell, in which the cross-linking agent solution is mixed with the bacterial beads from the microfluidic unit;

[0118] A stirring device arranged in the shell and used for stirring the materials in the shell;

[0119] A heating device used for heating the materials in the shell; and

[0120] A filter screen arranged at the bottom of the shell and used for retaining the biological bacterial agents.

[0121] The application does not limit the specific arrangement of the stirring device, as long as the materials in the shell can be stirred. In a specific embodiment, the stirring device is fixedly arranged on the inner wall of the shell. More specifically, the stirring device comprises a stirring motor and a stirring paddle, and the stirring motor is used for providing power to the stirring paddle.

[0122] In a specific embodiment, the heating device is arranged on the inner wall of the shell.

[0123] In a specific embodiment, the cross-linking coagulation unit further comprises a waste liquid funnel, a waste liquid valve and a waste liquid pipe connected in sequence, and the waste liquid funnel is arranged at the lower side of the filter screen of the shell.

[0124] In a specific embodiment, the system further comprises a dispersed phase injection unit for providing a dispersed phase solution. More specifically, the dispersed phase injection unit comprises a dispersed phase storage tank and a syringe pump, which are connected in sequence, and the dispersed phase liquid enters the pushing device through the dispersed phase injection inlet by the action of the syringe pump.

[0125] The application will be described in detail below through examples, but the protection scope of the application is not limited thereto. The experimental materials used in the following examples are all conventional commercially available products, unless otherwise specified.

[0126] In the following examples and comparative examples, the dispersant is olive oil (manufacturer: Aldrich, model: O108685), which contains sodium dodecyl sulfate (manufacturer: Aldrich, model: S108347).

[0127] The emulsifier is rhamnolipid (manufacturer: Shaanxi Ruide, model: 25CS-95).

[0128] Preparation Example 1

[0129] This preparation example is used to illustrate the slow-release oxygen microspheres and the preparation method thereof.

[0130] (1) Selection of medicaments

[0131] Chitosan dilute acid solution: 2.0wt% chitosan (molecular weight 300-400kDa, degree of deacetylation 90%) was dissolved in lactic acid (pH=4, aqueous solution volume fraction, 2.0%); 1.5% sodium alginate aqueous solution: sodium alginate (1.5%) was dissolved in deionized water; emulsifier: rhamnolipid; dispersant: olive oil containing sodium dodecyl sulfate (mass ratio of sodium dodecyl sulfate to olive oil 1:10); activator: biochar; persulfate: sodium persulfate; cross-linking agent: glyceraldehyde.

[0132] (2) Preparation method of oil phase solution: 1ml of emulsifier was taken into 100ml of dispersant, stirred at 400rpm for 20min to obtain the oil phase solution.

[0133] (3) Preparation method of aqueous phase solution: 0.5g of activator and 2g of persulfate were taken into the chitosan dilute acid solution to obtain 100ml of aqueous phase solution.

[0134] (4) Emulsification method: 25 ml of the sodium alginate aqueous solution was slowly added to 100 ml of the oil phase solution, and stirring was performed at 400 rpm for 20 min to form a primary emulsion, and then 31 ml of the aqueous phase solution was added to form a final emulsion.

[0135] (5) Cross-linking method: the final emulsion was slowly and uniformly added to a cross-linking agent to form a cross-linking system for cross-linking reaction (the cross-linking agent accounted for 0.01% of the volume of the entire cross-linking system solution), and stirring was performed at 500 rpm for 30 min, and then the cross-linking solidification was washed with anhydrous ethanol to obtain the oxygen-releasing solid-phase microspheres A (average particle size: 0.5-3 um).

[0136] Preparation Example 2

[0137] The present preparation example is used to illustrate the oxygen-releasing microspheres and the preparation method thereof.

[0138] (1) Selection of medicaments

[0139] Chitosan dilute acid solution: 5.0 wt% chitosan (molecular weight: 300-400 kDa, degree of deacetylation: 90%) was dissolved in an ascorbic acid (pH = 4, aqueous solution volume fraction: 2.0%); 3.5 wt% sodium alginate aqueous solution: sodium alginate (3.5%) was dissolved in deionized water; emulsifier: sophorolipid; dispersant: olive oil containing sodium dodecyl sulfate (mass ratio of sodium dodecyl sulfate to vegetable oil: 10:1); activator: biochar; persulfate: sodium persulfate; cross-linking agent: glyceraldehyde.

[0140] (2) Preparation method of the oil phase solution: 10 ml of the emulsifier was added to 100 ml of the dispersant, and stirring was performed at 800 rpm for 20 min to obtain the oil phase solution.

[0141] (3) Preparation method of the aqueous phase solution: 1 g of the activator and 5 g of the persulfate were placed in the chitosan dilute acid solution to obtain 100 ml of the aqueous phase solution.

[0142] (4) Emulsification method: 25 ml of the sodium alginate aqueous solution was slowly added to 150 ml of the oil phase solution, and stirring was performed at 800 rpm for 20 min to form a primary emulsion, and then 25 ml of the aqueous phase solution was added to form a final emulsion.

[0143] (5) Cross-linking method: the final emulsion was slowly and uniformly added to a cross-linking agent to form a cross-linking system for cross-linking reaction (the cross-linking agent accounted for 2.0% of the volume of the entire cross-linking system solution), and stirring was performed at 800 rpm for 20 min, and then the cross-linking solidification was washed with anhydrous ethanol to obtain the oxygen-releasing solid-phase microspheres B (average particle size: 0.5-3 um).

[0144] Example 1

[0145] The present example is used to illustrate the biological bacterial agent and the preparation method thereof.

[0146] The aerobic functional bacteria selected strain is Cellulomonas sp. A, and its preservation number is GDMCC No: 62343. The embodiment is implemented in the system shown in Figure 1

[0147] (1) Preparation of the dispersed phase solution: 0.5 g of the oxygen-releasing solid-phase microspheres A, 10 ml of the aerobic functional bacteria liquid (OD 600 = 0.5), and 2 g of sodium alginate are mixed and dissolved in deionized water to prepare a 100-ml solution;

[0148] (2) The voltage of the high-voltage electric field is 20 kV;

[0149] (3) The crosslinking agent in the crosslinking and solidification unit is a 2%-mass-concentration calcium lactate aqueous solution;

[0150] (4) Preparation step: The dispersed phase solution is injected into the dispersed phase storage tank and enters the injection device via the injection pump. The pressure of the microfluidic pressure pump is adjusted to 5000 mbar, the capillary pipette aperture is 0.17 mm, the average size of the bacteria beads is 50-250 um, the dispersed phase enters the crosslinking and solidification unit, and the waste liquid is filtered and discharged after 1 h of stirring at 150 rpm. The bacteria beads are placed on the filter screen, dried at 25℃ for 2 h, and then the biological bacteria agent A is collected.

[0151] Example 2

[0152] The embodiment is used to illustrate the biological bacteria agent and the preparation method thereof.

[0153] The aerobic functional bacteria selected strain is Cellulomonas sp. A, and its preservation number is GDMCC No: 62343. The embodiment is implemented in the system shown in Figure 1

[0154] (1) Preparation of the dispersed phase solution: 0.5 g of the oxygen-releasing solid-phase microspheres A, 10 ml of the aerobic functional bacteria liquid (OD 600 = 0.5), and 2 g of sodium alginate are mixed and dissolved in deionized water to prepare a 100-ml solution;

[0155] (2) The voltage of the high-voltage electric field is 20 kV;

[0156] (3) The crosslinking agent in the crosslinking and solidification unit is a 2%-mass-concentration calcium lactate aqueous solution;

[0157] ​​(4) Preparation step: the dispersed phase solution is injected into the dispersed phase storage tank, and enters the injection device through the injection pump; the pressure of the microfluidic pressure pump is adjusted to 500 mbar, the capillary droplet aperture is 0.41 mm, the average size of the bacterial beads is 200-500 um, the dispersed phase enters the cross-linking coagulation device, stirring at 150 rpm for 1 h, after the waste liquid is filtered and discharged, the bacterial beads are placed on the filter screen, dried at 25℃ for 2 h, and the biological bacterial agent B is collected.

[0158] Example 3

[0159] According to the method described in Example 1, the implementation is carried out, and the difference is that the preparation of the dispersed phase solution is as follows: 0.5 g of slow-release oxygen solid-phase microspheres A, 1 g of immobilized bacterial agent, and 2 g of sodium alginate are mixed and dissolved in deionized water to prepare a 100 ml solution; wherein the immobilized bacterial agent is prepared according to the following procedure: the aerobic functional bacteria are fermented and cultured to obtain a fermentation broth with OD 600 = 1, then mixed with biochar (80-100 mesh) at a 10% inoculation amount, then add liquid medium (yeast extract 5 g / L, tryptone 10 g / L and sodium chloride 10 g / L) to completely immerse the biochar, cultivate at 35℃ with a rotation speed of 135 rpm for 24 h, and finally filter and dry to obtain the immobilized bacterial agent.

[0160] The average size of the bacterial beads is 50-250 um, and the biological bacterial agent C is finally prepared.

[0161] Example 4

[0162] According to the method described in Example 1, the implementation is carried out, and the difference is that the preparation of the dispersed phase solution is as follows: 0.5 g of slow-release oxygen solid-phase microspheres A, 5 g of immobilized bacterial agent, and 2 g of sodium alginate are mixed and dissolved in deionized water to prepare a 100 ml solution; wherein the immobilized bacterial agent is prepared according to the following procedure: the aerobic functional bacteria are fermented and cultured to obtain a fermentation broth with OD 600 = 1, then mixed with biochar at a 10% inoculation amount, then add liquid medium (yeast extract 5 g / L, tryptone 10 g / L and sodium chloride 10 g / L) to completely immerse the biochar, cultivate at 35℃ with a rotation speed of 135 rpm for 24 h, and finally filter and dry to obtain the immobilized bacterial agent.

[0163] The average size of the bacterial beads is 200-500 um, and the biological bacterial agent D is finally prepared.

[0164] Example 5

[0165] According to the method described in Example 1, the implementation is carried out, and the difference is that the microfluidic technology equipment is not used, and the conventional drop pill method is used to obtain the biological bacterial agent E.

[0166] Conventional drop pill method: After the bacterial liquid and the gelling agent are mixed, they are stored in a burette or syringe. Through natural gravity or manual squeezing, the droplets fall into the cross-linking agent, and then they are washed and filtered for later use.

[0167] Comparative Example 1

[0168] The method described in Example 4 was followed, except that no slow-release oxygen microspheres were added during the preparation of the biological agent. Thus, biological agent F was finally prepared in this comparative example.

[0169] Test Example 1

[0170] (1) The biological microbial agent products prepared in Example 1 and Example 5 were observed, and the results were as follows: Figures 2-3 As shown, Figure 2 A is a photograph of the biological agent E prepared in Example 5. Figure 2 B is a photograph of the biological agent A prepared in Example 1.

[0171] The results showed that the particle size of the biological agent A prepared based on microfluidic technology was 50-250 μm, while the particle size of the biological agent E prepared based on the conventional pellet method was 3-4 mm.

[0172] (2) The bacterial load and yield of the biological agents prepared in Examples 1-5 were measured.

[0173] The test method for the bacterial load Log (CFU / g) is as follows: take 2g of the biological agent, add 20ml of sterile water, shake it with ultrasound (power 100w, 10s / 10s, frequency 20kHz, 5min), press 10 -2 , 10 -4 , 10 -6 , 10 -8 Scale dilutions were performed and the colony counts were recorded after constant temperature incubation for 24 hours. The calculation formula is as follows:

[0174]

[0175] Where y is the number of viable bacteria after dilution;

[0176] n is the dilution factor;

[0177] v is the total liquid volume;

[0178] m is the mass of the biological agent.

[0179] The bacterial load and yield of the biological agents prepared in Examples 1-5 are shown in Table 1.

[0180] Table 1

[0181] Number Bacterial load Yield Example 1 48 x 10 8 CFU / g 18.7 g / min Example 2 30 x 10 8 CFU / g 16.9 g / min Example 3 50 x 10 8 CFU / g 17.2 g / min Example 4 31 x 10 8 CFU / g 17.8 g / min Example 5 4 x 10 8 CFU / g 0.95 g / min

[0182] As shown in Table 1, the bacterial load and yield of the biological inoculant prepared in Examples 1-4 are much higher than that of Example 5, indicating that the use of microfluidic technology in the preparation of the biological inoculant can greatly improve the bacterial load and yield of the prepared biological inoculant.

[0183] Test Example 2

[0184] Two cylindrical reactors were prepared, a control group and an experimental group, and each group was treated as follows: a cylindrical reactor containing 1 L of high-temperature sterilized pure water was used, a pH probe and a dissolved oxygen (DO) analysis probe were inserted into the reactor, and a sealing plug was used for sealing. 0.2 g of Na2SO3 and 0.05 g of CoCl2 were added to the dosing port, and the dosing port was closed. When the dissolved oxygen DO in the reactor was removed to 0 mg / L and stabilized for 30 min. The control group did not add the slow-release oxygen solid-phase microspheres A, and the experimental group added 5 g of the slow-release oxygen solid-phase microspheres A. The pH value was measured within 24 h at 25°C, and the DO value was measured within 168 h. The test results are shown in Table 2. Figure 4

[0185] The pH value and DO value of the slow-release oxygen solid-phase microspheres B were measured by the same method as above, and the test results are shown in Table 3. Figure 5

[0186] The test results show that the pH of the slow-release oxygen microspheres prepared in Preparation Examples 1-2 is stable at 6.7-7.2, and the DO value is stable at about 4 mg / L at 180 h, and the dissolved oxygen concentration is maintained at a high level for a long time.

[0187] Test Example 3

[0188] The test method is the same as that of Test Example 2. 20 g of the biological inoculant products (A, B, C, D) prepared in Examples 1-4 were added after being cut, and the DO value and pH value were measured at 15°C (at this temperature, the growth of living bacteria is slow, which can eliminate the influence of living bacteria on the consumption of dissolved oxygen). The test results are shown in Table 4. Figure 6

[0189] The test results show that the pH of the biological inoculants A and C is stable at about 7.0, and the DO value is stable at about 1.8 mg / L after 60 h; the pH of the biological inoculants B and D is stable at about 6.8, and the DO value is stable at about 2.4 mg / L after 60 h; the dissolved oxygen content is positively correlated with the content of the slow-release oxygen medicament, and the slow-release oxygen medicament in the inoculant product provides dissolved oxygen for microorganisms, which can provide an electron acceptor for stimulating the degradation activity of functional microorganisms in the inoculant beads and indigenous functional microorganisms.

[0190] Test Example 4

[0191] Test method:

[0192] (1) Test medium: 100 mL of inorganic salt medium + 2 g of petroleum. ​​​

[0193] (2) The bio-inoculants A, B, C, D, F prepared from Examples 1-4 and Comparative Example 1 were inoculated into the test medium in step (1) at a mass ratio of 3%.

[0194] (3) The culture was incubated at 37°C, 135 rpm for 15 days, and then the concentration of petroleum hydrocarbon (C10-C40) in the culture solution was detected and the degradation rate of petroleum hydrocarbon was calculated.

[0195] Test results: The results showed that the degradation rates of petroleum hydrocarbon of the bio-inoculants A, B, C, D prepared from Examples 1-4 were 82.4%, 86.8%, 85.4, and 90.3%, respectively, while the degradation rate of petroleum hydrocarbon of the bio-inoculant F prepared from Comparative Example 1 was 75.1%.

[0196] Comparing Example 4 with Comparative Example 1, it can be seen that the degradation rate of petroleum hydrocarbon of Example 4 (bio-inoculant D) was increased by about 20%, which indicated that the addition of the slow-release oxygen microspheres could greatly improve the degradation effect of the prepared bio-inoculant on petroleum hydrocarbon.

[0197] Test Example 5

[0198] This test example was used to illustrate the degradation effect of the bio-inoculant on petroleum hydrocarbon in the surface soil (0-20 cm).

[0199] Test method:

[0200] (1) Petroleum contaminated soil experiment: depth 0-20 cm, pH = 7.8.

[0201] (2) The bio-inoculants A, B, C, D, F prepared from Examples 1-4 and Comparative Example 1 were applied at a mass ratio of 3% of the dry weight of the soil, and the water content of the soil was 22%. The soil was plowed once a day. The experimental period was 30 days. After 30 days, the concentration of petroleum hydrocarbon (C10-C40) in the soil was detected, and the degradation rate of petroleum hydrocarbon was calculated.

[0202] Test results: The results showed that the degradation rates of petroleum hydrocarbon of the bio-inoculants A, B, C, D prepared from Examples 1-4 were 63.8%, 67.2%, 67.5, and 75.1%, respectively, while the degradation rate of petroleum hydrocarbon of the bio-inoculant F prepared from Comparative Example 1 was 50.2%.

[0203] Comparing Example 4 with Comparative Example 1, it can be seen that the degradation rate of petroleum hydrocarbon of Example 4 (bio-inoculant D) was increased by about 50%.

[0204] Test Example 6

[0205] This test example was used to illustrate the degradation effect of the bio-inoculant on petroleum hydrocarbon in the deep soil (20-40 cm).

[0206] Test method:

[0207] (1) Petroleum contaminated soil experiment: depth 20-40 cm, pH = 7.8.

[0208] (2) The bio-inoculants A, B, C, D, F prepared from Examples 1-4 and Comparative Example 1 were applied at 3% of the dry weight of the soil, and the soil moisture content was 22%. The soil was tilled once a day. The experiment lasted for 30 days. After 30 days, the concentration of petroleum hydrocarbons (C10-C40) in the soil was detected, and the petroleum hydrocarbon degradation rate was calculated.

[0209] Test results: The results showed that the petroleum hydrocarbon degradation rates of the bio-inoculants A, B, C, D prepared from Examples 1-4 were 51.7%, 55.6%, 54.4%, and 61.4%, respectively, while the petroleum hydrocarbon degradation rate of the bio-inoculant F prepared from Comparative Example 1 was 30.7%.

[0210] Comparing Example 4 with Comparative Example 1, it can be seen that the petroleum hydrocarbon degradation rate of Example 4 (bio-inoculant D) was increased by about 100%.

[0211] Test Example 7

[0212] The bio-inoculant D prepared from Example 4 and the bio-inoculant F prepared from Comparative Example 1 were tested as follows.

[0213] Test method: The permeable reaction wall was set perpendicular to the direction of groundwater flow, penetrating through the entire aquifer, with a length that could capture most of the pollution plume. Activated carbon and bio-inoculant product (mass ratio 5:1) were mixed, in which the activated carbon adsorbed most of the oil, and the bio-inoculant product overcame the groundwater environment, fully contacted the oil in the groundwater, and decomposed it. The activated carbon and bio-inoculant D were mixed to obtain bio-inoculant D group, and the activated carbon and bio-inoculant F were mixed to obtain bio-inoculant F group.

[0214] Test results: After 50 days, the petroleum hydrocarbon (C10-C40) degradation rate of the bio-inoculant D group was 55.3%, which had high engineering application value. The petroleum hydrocarbon degradation rate of the bio-inoculant F group was only 24.4%.

[0215] Test Example 8

[0216] Test method: (1) Petroleum contaminated soil experiment: salinity 1.5%, pH = 8.5.

[0217] (2) The bio-inoculants A, B, C, D, F prepared from Examples 1-4 and Comparative Example 1 were applied at 3% of the dry weight of the soil, and the soil moisture content was 22%. The soil was tilled once a day. The experiment lasted for 30 days. After 30 days, the concentration of petroleum hydrocarbons (C10-C40) in the soil was detected, and the petroleum hydrocarbon degradation rate was calculated.

[0218] Test results: The results show that the degradation rates of petroleum hydrocarbon of the biological fungicide A, B, C and D prepared in Examples 1-4 are 35.8%, 38.9%, 39.1% and 43.7% respectively. The degradation rate of petroleum hydrocarbon of the biological fungicide F prepared in Comparative Example 1 is only 20.6%.

[0219] Comparing Example 4 with Comparative Example 1, it can be seen that the degradation rate of petroleum hydrocarbon of the biological fungicide D is increased by about 112%. It can be seen that the biological fungicide product prepared in the present application has good stress resistance and high degradation rate.

[0220] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as the disclosed content of the present application and fall within the protection scope of the present application.

Claims

1. A biological agent, characterized in that: The biological agent includes a wall material and a core material embedded in the wall material; The core material includes slow-release oxygen microspheres and aerobic functional bacteria. The slow-release oxygen microspheres are prepared by an emulsification and cross-linking method from a raw material composition containing chitosan, sodium alginate, an activator, persulfate, an emulsifier, a dispersant and a cross-linking agent. The emulsifier is a biosurfactant and the cross-linking agent is glyceraldehyde.

2. The biological agent according to claim 1, characterized in that The particle size of the biological agent is 50 to 500 μm; Preferably, the aerobic functional bacteria are petroleum-degrading bacteria capable of degrading petroleum hydrocarbons.

3. The biological agent according to claim 1, characterized in that The particle size of the slow-release oxygen microspheres is 0.5 to 3 μm.

4. The biological agent according to claim 1, characterized in that In the raw material composition, the molecular weight of the chitosan is 300-400 kDa, and the degree of deacetylation is 70-90%; Preferably, the activating agent is biochar; Preferably, the persulfate is selected from one or more of sodium persulfate, potassium persulfate and ammonium persulfate; Preferably, the emulsifier is selected from one or more of rhamnolipids, trehalolipids and sophorolipids; Preferably, the dispersant is vegetable oil containing sodium lauryl sulfate.

5. The biological agent according to claim 1, characterized in that In the raw material composition, the mass ratio of the chitosan, the activator and the persulfate is 2-10:1:2-10; Preferably, in the raw material composition, the volume ratio of the emulsifier to the dispersant is 1 to 30:100; Preferably, in the raw material composition, the ratio of the mass of the sodium alginate to the total volume of the emulsifier and the dispersant is 1 g: 100-500 mL; Preferably, in the raw material composition, the dosage ratio of the activator to the dispersant is 1 g: 50 to 300 mL.

6. The biological agent according to claim 1, characterized in that The wall material is made of one or more selected from the group consisting of sodium alginate, polyvinyl alcohol and chitosan.

7. A method for preparing a biological agent, characterized in that: The method comprises the following steps: (1) mixing aerobic functional bacteria, slow-release oxygen microspheres, wall material raw materials and deionized water to obtain a dispersed phase solution; (2) injecting the dispersed phase solution into a calcium chloride solution or a calcium lactate solution and stirring; The slow-release oxygen microspheres are prepared from a raw material composition containing chitosan, sodium alginate, an activator, persulfate, an emulsifier, a dispersant and a cross-linking agent by an emulsification and cross-linking method. The emulsifier is a biosurfactant and the cross-linking agent is glyceraldehyde.

8. The method according to claim 7, characterized in that Step (2) specifically includes: the dispersed phase solution is formed into bacterial beads with a particle size of 50 to 500 μm through a microfluidic unit, and then injected into a calcium chloride solution or a calcium lactate solution and stirred.

9. The method according to claim 8, characterized in that The microfluidic unit includes a microfluidic pressure pump and an injection device, wherein the pressure of the microfluidic pressure pump is 500-5000 mbar, and the aperture of the capillary dropper of the injection device is 0.17-0.41 mm.

10. The method according to claim 7, characterized in that In step (1), the aerobic functional bacteria are added in the form of a bacterial suspension or an immobilized bacterial agent; Preferably, when the aerobic functional bacteria are added in the form of bacterial suspension, the dosage ratio of the slow-release oxygen microspheres, bacterial suspension and wall material raw materials is 0.5-3g:10-30ml:1.5-3.5g, and the OD of the bacterial suspension is 600 Value = 0.2 to 0.5; Preferably, when the aerobic functional bacteria are added in the form of an immobilized bacterial agent, the mass ratio of the slow-release oxygen microspheres, the immobilized bacterial agent, and the wall material raw material is 0.5-3:1-5:1.5-3.5; Preferably, the wall material raw materials are selected from one or more of sodium alginate, polyvinyl alcohol and chitosan.

11. The method according to claim 7, characterized in that The slow-release oxygen microspheres are prepared according to the following steps: A1. Mixing an emulsifier and a dispersant to obtain an oil phase solution; A2, mixing the activator, persulfate and chitosan dilute acid solution to obtain an aqueous solution; A3, mixing the sodium alginate aqueous solution with the oil phase solution to obtain a primary emulsion; A4, mixing the primary emulsion with the aqueous phase solution to obtain a final emulsion; A5, adding a cross-linking agent to the final emulsion to carry out a cross-linking reaction to obtain a reaction solution; A6. The reaction solution is subjected to solid-liquid separation, and the obtained solid phase is washed to obtain slow-release oxygen microspheres.

12. The method according to claim 11, characterized in that In step A2, the chitosan dilute acid solution contains chitosan and a dilute acid solution, wherein the pH of the dilute acid solution is 3 to 5; Preferably, the chitosan content in the chitosan dilute acid solution is 2 to 5 wt%; Preferably, the acid in the dilute acid solution is selected from one or more of glacial acetic acid, lactic acid, malic acid and ascorbic acid; Preferably, in step A3, the mass concentration of sodium alginate in the sodium alginate aqueous solution is 1.5-3.5%, and the volume ratio of the sodium alginate aqueous solution to the oil phase solution is 1:4-7; Preferably, in step A4, the volume ratio of the aqueous phase solution to the primary emulsion is 1:4-7.

13. A biological agent prepared by the method according to any one of claims 7 to 12.

14. A system for preparing the biological agent according to any one of claims 1 to 6 and 13, characterized in that: The system includes a microfluidic unit and a cross-linking and coagulation unit, wherein the microfluidic unit includes a microfluidic pressure pump, an injection device and a high-voltage power supply, the microfluidic pressure pump is connected to the injection device, the injection device includes a capillary dropper, one end of the high-voltage power supply is connected to the capillary dropper via a high-voltage power supply positive electrode connection line, and the other end is immersed in the liquid in the cross-linking and coagulation unit via a high-voltage power supply negative electrode connection line; The dispersed phase solution is formed into bacterial beads with a particle size of 50 to 500 μm through the capillary dropper and then injected into the cross-linking and coagulation unit, and the calcium chloride solution or calcium lactate solution in the cross-linking and coagulation unit is mixed with the bacterial beads; The dispersed phase solution contains aerobic functional bacteria, slow-release oxygen microspheres, wall material raw materials and water.

15. The system according to claim 14, wherein: The cross-linking and coagulation unit comprises: a shell, wherein a crosslinker solution and bacterial beads from the microfluidic unit are mixed in the shell; A stirring device, the stirring device is provided in the shell and is used to stir the material in the shell; a heating device, the heating device being used to heat the material in the housing; and The filter screen is arranged at the bottom of the shell and is used to intercept the biological bacteria agent.

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