Immobilized microbial biotype seed carrier product and method for preparing the same

Abstract

The application discloses a kind of immobilized microbial biological seed carrier product and its preparation method, preparation method includes the following steps: first, diatomite is used to adsorb petroleum hydrocarbon-degrading bacteria, then vacuum freeze-drying into powdered bacterium agent, secondly, powdered bacterium agent is mixed with coating agent high molecular water-absorbing resin, bentonite, polyvinyl alcohol, gibberellic acid uniformly, with seed and coating agent raw material 1:1 is coated, color rape seed is placed in the rotating disc of coating machine with uniform speed, quantitative binder solution is sprayed, it is mixed granulation under the rotation speed of 90r / min, continuous alternate operation several times to the coating agent is evenly wrapped on rape seed, finally idle 2min to increase strength, obtain seed carrier immobilized microbial product.The carrier material is green and environment-friendly, low in cost, simple in operation, and can be mass-produced, and lays a certain foundation for strengthening plant-microbe collaborative remediation.

Description

Technical Field

[0001] This invention relates to the field of petroleum hydrocarbon pollution control technology, and in particular to a biological seed carrier product for immobilized microorganisms and its preparation method. Background Technology

[0002] In recent years, with the rapid development of global industrialization and urbanization, people's consumption of petroleum and related products has also increased significantly. During the extraction, transportation, processing, and utilization of petroleum, due to factors such as inadequate management, incomplete combustion, and leaks from aging pipelines, oil spills into the soil are almost inevitable. Petroleum hydrocarbon pollutants are recalcitrant compounds and have been listed as priority pollutants for control.

[0003] In recent years, extensive technological research and development has been conducted to address the remediation of petroleum hydrocarbon-contaminated sites. Utilizing the inherent characteristics of plants and microorganisms, and their synergistic effects, phytoremediation of petroleum hydrocarbon-contaminated soils can effectively reduce the time required for bioremediation and significantly improve remediation efficiency. However, the complexity and volatility of environmental factors within ecosystems make phytoremediation challenging to implement.

[0004] A search revealed Chinese patent application number CN104946620A, which discloses a microbial immobilization agent and its preparation method for remediating petroleum hydrocarbon-contaminated saline-alkali soil. The agent uses biochar as a carrier and adds sodium alginate to form immobilized bacterial balls for preservation. The mixture is prepared by mixing bacterial suspension, biochar, and sodium alginate in a ratio of 100:5:2 (v / w / w). This invention has good stability and can significantly improve the degradation efficiency of petroleum hydrocarbons in saline-alkali soil.

[0005] Chinese patent application number CN115491372A discloses an immobilized bacterial agent for polycyclic aromatic hydrocarbon degrading strains and its preparation method. The brewer's grain biochar prepared by this invention is very conducive to the attachment and growth of microorganisms inside it and the continuous performance of pollutant degradation efficiency. It also has a good protective effect on the loaded microorganisms and improves the resistance of microorganisms to adverse external environments.

[0006] However, existing reports mainly focus on the removal of target pollutants by different biochar-adsorbed and immobilized microorganisms, with limited research on the synergistic remediation of plants and immobilized microorganisms. Given the extremely complex soil pollution environment, the survival and growth of microorganisms and plants in such conditions is a highly challenging task. Therefore, there is an urgent need to develop a product based on bio-seed carriers for immobilized microorganisms that can be applied to complex and variable petroleum hydrocarbon contaminated sites, improve remediation efficiency, and be environmentally friendly. Summary of the Invention

[0007] The purpose of this invention is to address the deficiencies in the existing technology by proposing a bio-type seed carrier product for immobilized microorganisms and its preparation method.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A bio-type seed carrier product with immobilized microorganisms comprises the following raw materials: adhesive, water-retaining agent, plant growth regulator, filler material, microorganisms and water.

[0010] Furthermore, the adhesive is polyvinyl alcohol, the water-retaining agent is superabsorbent polymer, the plant growth regulator is gibberellic acid, the filler material is diatomaceous earth and bentonite, and the microorganism is petroleum hydrocarbon-efficient degrading bacteria.

[0011] Furthermore, the following percentage-by-weight parts of raw materials are specifically used:

[0012] The adhesive is specifically polyvinyl alcohol, with a mass percentage of 3% to 5%;

[0013] The water-retaining agent is specifically a superabsorbent polymer, with a mass percentage of 1% to 10%.

[0014] The plant growth regulator is specifically gibberellic acid, with a mass percentage of 0.03% to 0.075%.

[0015] The filling material is diatomaceous earth, in which the ratio of bentonite is 4:1; 3:2; 2:3;

[0016] The remainder is water.

[0017] Furthermore, the following percentage-by-weight parts of raw materials are specifically used:

[0018] Adhesive: 5% polyvinyl alcohol;

[0019] Water-retaining agent: 5% superabsorbent polymer;

[0020] Plant growth regulator: gibberellic acid 0.03%;

[0021] Filler material: Diatomaceous earth: Bentonite in a ratio of 4:1;

[0022] The remainder is water.

[0023] Furthermore, the effective viable count of the highly efficient petroleum hydrocarbon degrading bacteria is greater than 1×10⁻⁶. 9 CFU / mL.

[0024] A method for preparing a biological seed carrier product, based on the above-mentioned immobilized microorganism biological seed carrier product, includes the following steps:

[0025] S1. Select plump, undamaged rapeseed seeds, wash them with distilled water, disinfect them, and then rinse them three times with distilled water. After that, soak the disinfected rapeseed seeds in gibberellic acid for 0.5 hours.

[0026] S2. Large-scale culture of petroleum hydrocarbon degrading bacteria: Petroleum hydrocarbon degrading bacteria are fermented at high density.

[0027] S3. Loading of petroleum hydrocarbon degrading bacteria: The cultured petroleum hydrocarbon degrading bacteria solution is centrifuged, washed, and resuspended to obtain a bacterial suspension, which is then mixed with diatomaceous earth. The mixture is placed in a constant temperature shaker for adsorption.

[0028] S4. Preparation of powdered bacterial agent: After adsorption by shaking, the mixture is placed in a vacuum freeze dryer for freeze drying to obtain a dry and active powdered bacterial agent.

[0029] S5. Coating: Mix the powdered microbial agent with other coating agents evenly, put the rapeseed seeds into a coating machine with a uniform rotation speed, spray in the adhesive, then add the mixed coating agent, and finally spray the adhesive and add the mixed coating agent. Repeat this alternating operation several times to obtain a product of immobilized microorganisms based on a biological seed carrier.

[0030] S6. The immobilized microbial product based on the biological seed carrier obtained after S5 treatment is dried at room temperature.

[0031] Furthermore, in step S3, the parameters of the constant temperature oscillator are set to a rotation speed of 150 r / min, a temperature of 30℃, and an oscillation adsorption time of 2 h.

[0032] Furthermore, in step S4, the mixture is placed in a vacuum freeze dryer. It should first be pre-frozen for 1 hour to lock in the moisture, and then vacuum dried for 24 hours.

[0033] Furthermore, in step S5, the rotation speed of the coating machine is 90 r / min.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] The preparation of the immobilized microbial bio-seed carrier product in this invention is based on the principle of seed coating. An adhesive, water-retaining agent, plant growth regulator, and filler material are mixed and granulated using a coating machine. This coating agent coats the seeds, increasing their weight and enabling better mechanical sowing. Simultaneously, the synergistic effect between the components provides better nutrition to rapeseed during its early growth stages and throughout the entire growth process, and enhances the rapeseed's resistance to petroleum hydrocarbon pollution.

[0036] The seed carrier product of this invention uses colored rapeseed seeds. Rapeseed is a common oil crop in my country. Colored rapeseed is ornamental, and its oil composition is similar to that of diesel oil, making it an ideal raw material for biodiesel. This lays a foundation for the subsequent resource utilization of the plant.

[0037] The seed carrier immobilized microorganisms of this invention are Acinetobacter, a species of petroleum hydrocarbon-specific degrading bacteria that maintains high activity and can significantly promote the biodegradation of petroleum hydrocarbons.

[0038] This invention combines seed coating and immobilized microorganism technology, which can effectively improve the stress resistance of rapeseed seeds. The coating layer on the outside of the rapeseed can protect the seeds from the toxic effects of petroleum pollutants during germination, and also provide space for the microorganisms immobilized on it to grow and reproduce in the early stage of entering the soil, thus inhibiting the competition from native microorganisms.

[0039] The raw materials used in this invention are low-cost, environmentally friendly, and can be mass-produced. They can also effectively improve the efficiency of plant-microbe synergistic remediation and have potential application value in bioremediation. Attached Figure Description

[0040] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0041] Figure 1 This is a schematic diagram illustrating the observation of microbial load and the determination of viable bacteria count on the carrier in an embodiment of the present invention;

[0042] Figure 2: 2a and 2b: Scanning electron microscope images of microorganisms supported on a single diatomaceous earth in an embodiment of the present invention;

[0043] Figure 3: 3a-3d: Schematic diagrams of viable cell count detection on seed carriers in embodiments of the present invention. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0045] In the following examples, unless otherwise specified, the reagents are standard reagents and can be purchased from any standard reagent manufacturer or distributor.

[0046] Example 1

[0047] A bio-type seed carrier product for immobilized microorganisms, comprising the following percentage parts by weight of raw materials:

[0048] Adhesive: 3% polyvinyl alcohol;

[0049] Water-retaining agent: 1% superabsorbent polymer;

[0050] Filler material: Diatomaceous earth: Bentonite = 4:1;

[0051] Plant growth regulator: gibberellic acid 0.075%

[0052] The remainder is water.

[0053] It should be noted that the added filler materials, diatomaceous earth and bentonite, are coated in a 4:1 ratio, resulting in a low shedding rate and high uniformity. Diatomaceous earth alone is not easily used to form uniform pellets, and an appropriate amount of bentonite can improve the coating uniformity. Furthermore, diatomaceous earth possesses a natural "molecular sieve" pore structure, is porous, has a large specific surface area, and contains numerous hydroxyl groups and hydrogen bonds on its surface. These chemical bonds also exist within the numerous micropores of diatomaceous earth, giving it good adsorption properties and providing an environment conducive to the survival of microorganisms.

[0054] In addition, a method for preparing a biotype seed carrier product of immobilized microorganisms includes the following steps:

[0055] Colored rapeseed pretreatment: Select plump, undamaged rapeseed seeds, wash them with distilled water, disinfect them, and then rinse them three times with distilled water; then soak the disinfected rapeseed seeds in gibberellic acid for 0.5 hours.

[0056] in:

[0057] ① Weigh out 10g of colored rapeseed seeds;

[0058] ② Add an appropriate amount of distilled water and wash three times with distilled water to remove surface dust;

[0059] ③ Add 2% sodium hypochlorite solution for disinfection, and sterilize on a magnetic stirrer for 10 minutes, then rinse three times with distilled water;

[0060] ④ Soak the seeds in 0.075% gibberellic acid for 0.5 hours, then rinse with distilled water, remove the seeds and dry the surface of the seeds with filter paper.

[0061] Coating colored rapeseed seeds:

[0062] ① Ensure that there is no moisture remaining on the beaker, iron spoon, or other utensils;

[0063] ② Weigh polyvinyl alcohol, add distilled water, dissolve at 60℃, and allow to cool. Weigh the remaining raw materials for the coating agent (superabsorbent polymer, diatomaceous earth, and bentonite), weigh them separately in order, and place them into beakers one by one;

[0064] ③ Coating process: The seed and coating agent raw materials are coated at a 1:1 ratio. 10g of soaked seeds are placed in the rotating disc of a coating machine with a uniform rotation speed. A certain amount of adhesive solution is sprayed in to moisten the surface of the colored rapeseed seeds without them sticking together. The remaining evenly mixed coating material is then added to the coating machine to cover the surface of the colored rapeseed seeds. The mixture is granulated at a speed of 90r / min. This process is repeated several times until the coating agent is evenly coated on the rapeseed seeds. Finally, the remaining material is added to promote drying and flowability. The machine is then idle for 2 minutes to increase strength.

[0065] ④ Place the coated seeds at room temperature to dry;

[0066] ⑤ Select evenly coated seeds from the dried seeds and place them in a petri dish lined with two layers of filter paper. Add 2 mL of distilled water and conduct a germination experiment with a cycle of 7 days. Place 20 seeds in each petri dish, and perform three replicates for each example.

[0067] ⑥ Conduct coating quality inspections on the remaining coated seeds, such as testing physical indicators like pass rate, seed content, single seed rate, breakage rate, and disintegration rate.

[0068] It should be noted that the optimal concentration and ratio of raw materials were screened through orthogonal experiments. The seed carrier preparation method of this invention can effectively solve the survival problem of plants and microorganisms in complex and variable petroleum hydrocarbon contaminated sites, and improve the stress resistance of plants and microorganisms. At the same time, it can also enhance the synergistic remediation ability of plants and microorganisms in petroleum hydrocarbon contaminated soil. This invention has good application prospects in the field of petroleum hydrocarbon pollution control.

[0069] Meanwhile, the seed carrier immobilized microorganisms product prepared by this invention has advantages such as low cost, environmental friendliness, simple operation, and the ability to be mass-produced. Furthermore, tests on some physical properties verify that the product meets the required standards.

[0070] Example 2: A method for preparing a bio-based seed carrier

[0071] The difference between this embodiment and Embodiment 1 is that:

[0072] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0073] Adhesive: 3% polyvinyl alcohol;

[0074] Water-retaining agent: 5% superabsorbent polymer;

[0075] Filler material: Diatomaceous earth: Bentonite = 3:2;

[0076] Plant growth regulator: gibberellic acid 0.043%

[0077] The remainder is water.

[0078] Other parameters and operations are the same as in Example 1.

[0079] Example 3: A method for preparing a biological seed carrier product.

[0080] The difference between this embodiment and Embodiment 1 is that:

[0081] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0082] Adhesive: 3% polyvinyl alcohol;

[0083] Water-retaining agent: 10% superabsorbent polymer;

[0084] Filler material: Diatomaceous earth: Bentonite = 2:3;

[0085] Plant growth regulator: gibberellic acid 0.03%

[0086] The remainder is water.

[0087] Other parameters and operations are the same as in Example 1.

[0088] Example 4: A method for preparing a biological seed carrier product.

[0089] The difference between this embodiment and Embodiment 1 is that:

[0090] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0091] Adhesive: 4% polyvinyl alcohol;

[0092] Water-retaining agent: 1% superabsorbent polymer;

[0093] Filler material: Diatomaceous earth: Bentonite = 3:2;

[0094] Plant growth regulator: gibberellic acid 0.03%

[0095] The remainder is water.

[0096] Other parameters and operations are the same as in Example 1.

[0097] Example 5: A method for preparing a biological seed carrier product.

[0098] The difference between this embodiment and Embodiment 1 is that:

[0099] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0100] Adhesive: 4% polyvinyl alcohol;

[0101] Water-retaining agent: 5% superabsorbent polymer;

[0102] Filler material: Diatomaceous earth: Bentonite = 2:3;

[0103] Plant growth regulator: gibberellic acid 0.075%

[0104] The remainder is water.

[0105] Other parameters and operations are the same as in Example 1.

[0106] Example 6: A method for preparing a biological seed carrier product.

[0107] The difference between this embodiment and Embodiment 1 is that:

[0108] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0109] Adhesive: 4% polyvinyl alcohol;

[0110] Water-retaining agent: 10% superabsorbent polymer;

[0111] Filler material: Diatomaceous earth: Bentonite = 4:1;

[0112] Plant growth regulator: gibberellic acid 0.043%

[0113] The remainder is water.

[0114] Other parameters and operations are the same as in Example 1.

[0115] Example 7: A method for preparing a biological seed carrier product.

[0116] The difference between this embodiment and Embodiment 1 is that:

[0117] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0118] Adhesive: 5% polyvinyl alcohol;

[0119] Water-retaining agent: 1% superabsorbent polymer;

[0120] Filler material: Diatomaceous earth: Bentonite = 2:3;

[0121] Plant growth regulator: gibberellic acid 0.043%

[0122] The remainder is water.

[0123] Other parameters and operations are the same as in Example 1.

[0124] Example 8: A method for preparing a biological seed carrier product.

[0125] The difference between this embodiment and Embodiment 1 is that:

[0126] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0127] Adhesive: 5% polyvinyl alcohol;

[0128] Water-retaining agent: 5% superabsorbent polymer;

[0129] Filler material: Diatomaceous earth: Bentonite = 4:1;

[0130] Plant growth regulator: gibberellic acid 0.03%;

[0131] The remainder is water.

[0132] Other parameters and operations are the same as in Example 1.

[0133] Example 9: A method for preparing a biological seed carrier product.

[0134] The difference between this embodiment and Embodiment 1 is that:

[0135] The raw materials and their mass percentages for the bio-type seed carrier product of the immobilized microorganisms are as follows:

[0136] Adhesive: 5% polyvinyl alcohol;

[0137] Water-retaining agent: 10% superabsorbent polymer;

[0138] Filler material: Diatomaceous earth: Bentonite = 3:2;

[0139] Plant growth regulator: gibberellic acid 0.075%

[0140] The remainder is water.

[0141] Other parameters and operations are the same as in Example 1.

[0142] Example 10: A bio-type seed carrier product for immobilized microorganisms and its preparation method.

[0143] 1. Based on the analysis of the results of Examples 1-9, the raw material combination used is as follows:

[0144] 5% polyvinyl alcohol, 5% superabsorbent polymer, 0.03% gibberellic acid, diatomaceous earth: bentonite in a ratio of 4:1.

[0145] 2. Ensure that there is no moisture remaining on the beaker, iron spoon, or other utensils.

[0146] 3. Weigh polyvinyl alcohol, add distilled water, dissolve at 60℃, and let cool; weigh diatomaceous earth and put it into a 50mL conical flask; weigh the raw materials (superabsorbent polymer and bentonite) in the remaining coating agent components, weigh them separately in order, and put them into beakers one by one;

[0147] 4. Loading of petroleum-degrading bacteria: After high-density fermentation, Acinetobacter spp. were centrifuged, washed, and resuspended to obtain a bacterial suspension. The OD was adjusted to 1. Diatomaceous earth, a component of the coating agent, was used to load the bacterial agent. The bacterial agent and diatomaceous earth were mixed at a ratio of 5:1. The mixture was placed in a constant temperature shaker at 150 r / min and 30℃ for 2 h for adsorption.

[0148] 5. Preparation of powdered bacterial agent: After adsorption by shaking, the mixture is placed in a vacuum freeze dryer, pre-frozen for 1 hour, and then freeze-dried under vacuum for 24 hours to obtain a dry and active powdered bacterial agent;

[0149] 6. Seed carrier immobilization process: Mix the powdered microbial agent with other coating materials, using a seed-to-coating agent ratio of 1:1 for coating treatment. Place 10g of soaked seeds in a coating machine turntable with uniform rotation speed, spray a measured amount of adhesive solution to moisten the surface of the colored rapeseed seeds without them sticking together, take the uniformly mixed coating material and put it into the coating machine to cover the surface of the colored rapeseed seeds, mix and granulate at a speed of 90r / min, and repeat the process several times until the coating agent is evenly coated on the rapeseed seeds. Finally, add the remaining material to promote drying and flowability, and run idle for 2 minutes to increase strength.

[0150] 7. Place the seed carrier immobilized microorganisms in a room temperature environment to dry;

[0151] 8. The dried product was examined using a scanning electron microscope to observe the microbial load, and the product was also tested for viable bacteria count using a flow cytometer.

[0152] To better understand the technical solution of the present invention, the following description is provided in conjunction with experiments and accompanying drawings.

[0153] The evaluation scheme is as follows: 1. Evaluation of seed carrier germination indicators

[0154] Seeds coated in Examples 1-9 were used to determine the germination rate, germination potential, and germination index of the coated seeds, as shown in Table 1.

[0155]

[0156]

[0157] Table 1: Germination index determination of different seed carrier formulations in orthogonal experiments

[0158] The results of the orthogonal experiment showed that among the seed-coated seeds of Examples 1-9, the germination potential, germination rate, and germination index of the 8th treatment group were the highest. Based on the actual values ​​of each index in the 9 treatment groups, the optimal concentration levels of the four coating materials were determined. The combination of 5% polyvinyl alcohol as a binder, 5% superabsorbent polymer as a water-retaining agent, 0.03% gibberellic acid as a plant growth regulator, and diatomaceous earth:bentonite in a ratio of 4:1 as a filler material showed the best germination vitality.

[0159] Evaluation of seed physical performance indicators on carrier

[0160] The coated seeds from Examples 1-9 were used to determine the qualification rate, seed content, single seed rate, disintegration rate, and breakage rate of the coated seeds.

[0161] (1) Pass rate: Seeds with a surface coating area greater than 80% are considered qualified. The mass of the pelleted seeds after coating treatment is weighed, and the unqualified coated seeds are picked out. The weight of the unqualified coated seeds is weighed to obtain the pass rate. Pass rate (%) = (Weight of tested seeds - Weight of unqualified coated seeds / Weight of tested seeds) × 100.

[0162] (2) Seed content: Weigh the coated seeds after pelleting, remove the empty pellets, and weigh the seeds after removing the empty pellets to obtain the seed content. Seed content (%) = (Weight of test seeds - Weight of empty pellets / Weight of test seeds) × 100.

[0163] (3) Single seed rate: Weigh the coated seeds after pelleting, pick out the clumped rapeseed seeds, and weigh the seeds after removing the clumped seeds to obtain the single seed rate. Single seed rate (%) = (weight of test seeds - weight of clumped seeds / weight of test seeds) × 100.

[0164] (4) Disintegration rate: 100 pelleted seeds were randomly selected and neatly placed on filter paper in a 7cm diameter culture dish, with the seeds separated from each other. 5mL of tap water was slowly added along the wall of the culture dish, and the disintegration of the pelleted seeds was observed within 1 minute. The shedding of the powder layer was considered disintegration.

[0165] (5) Breakage rate: The pelleted seeds are packed in a self-sealing bag and dropped naturally from a height of 100cm. The percentage of pelleted seeds that break out is calculated out of the total number of seeds tested.

[0166] Table 2. Determination of physical properties of different seed carrier formulations in orthogonal experiments.

[0167]

[0168]

[0169] Table 2

[0170] The results showed that Examples 1-9 had no significant differences in pass rate, seed content, and single seed rate, all meeting the standards. However, there were significant differences in disintegration rate and breakage rate. Disintegration rate refers to the degree to which coated seeds naturally loosen after absorbing water at a certain rate. This characteristic directly reflects the ecological respiration of seeds before germination and the ease with which seedlings emerge from the shell. It also indicates the aeration and water permeability of the coating material used and whether it affects the germination vigor of the seeds. Examples 8 and 9 achieved a disintegration rate of 100%, indicating that the mass percentage and proportion of the coating material used were good. Breakage rate reflects adhesive strength and is an important indicator for subsequent seed carrier storage and transportation. Factors affecting this indicator mainly include the type and concentration of the adhesive, the material ratio and particle size, and the rationality of the coating process. In Examples 1-9, as the concentration of the adhesive polyvinyl alcohol increased, the breakage rate of the coated seeds decreased. When the polyvinyl alcohol concentration was 5%, Examples 7, 8, and 9 all showed low breakage rates, laying a foundation for subsequent seed carrier storage and transportation.

[0171] 3. Microbiological index testing of products immobilized with seed carriers

[0172] Take the product listed in Implementation 10, such as Figure 1 As shown, the microbial load was observed and the number of viable bacteria on the carrier was determined.

[0173] Depend on Figures 2a-2b As can be seen, diatomaceous earth is mainly disc-shaped, and its surface exhibits abundant, uniformly and orderly arranged micropores with unobstructed flow, providing numerous adsorption sites and a large adsorption area. This provides a basis for microbial attachment.

[0174] As shown in Figure 3: Figures 3a-3d As shown, the specific viable cell count of microorganisms on the seed carrier is shown in [reference needed].

[0175] Table 3.

[0176]

[0177] Table 3

[0178] The above test results show that there are an average of 2.84 × 10⁻⁶ seeds per mg of coating material. 6 The detection results of the petroleum hydrocarbon-degrading bacteria on the carrier enable quantitative assessment of the microorganisms on the carrier. The seed carrier-immobilized microorganisms product described in this invention can be better applied in the field of petroleum hydrocarbon contaminated soil remediation, and has broader application prospects.

[0179] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A bio-type seed carrier product for immobilized microorganisms, characterized in that, The following raw materials are used: adhesives, water-retaining agents, plant growth regulators, filler materials, microorganisms, and water; The adhesive is polyvinyl alcohol, the water-retaining agent is superabsorbent polymer, the plant growth regulator is gibberellic acid, the filler material is diatomaceous earth and bentonite, and the microorganism is petroleum hydrocarbon highly efficient degrading bacteria. The following percentages by weight of raw materials are used: Adhesive: 5% polyvinyl alcohol; Water-retaining agent: 5% superabsorbent polymer; Plant growth regulator: gibberellic acid 0.03%; Filler material: Diatomaceous earth: Bentonite in a ratio of 4:1; The remainder is water; The effective viable count of the petroleum hydrocarbon high-efficiency degrading bacteria is greater than 1×10⁻⁶. 9 CFU / mL.

2. A method for preparing a biological seed carrier product, the biological seed carrier product of immobilized microorganisms according to claim 1, characterized in that, Includes the following steps: The method specifically includes the following steps: S1. Select plump, undamaged rapeseed seeds, wash them with distilled water, disinfect them, and then rinse them three times with distilled water. After that, soak the disinfected rapeseed seeds in gibberellic acid for 0.5 hours. S2. Large-scale culture of petroleum hydrocarbon degrading bacteria: Petroleum hydrocarbon degrading bacteria are fermented at high density. S3. Loading of petroleum hydrocarbon degrading bacteria: The cultured petroleum hydrocarbon degrading bacteria solution is centrifuged, washed, and resuspended to obtain a bacterial suspension, which is then mixed with diatomaceous earth. The mixture is placed in a constant temperature shaker for adsorption. S4. Preparation of powdered bacterial agent: After adsorption by shaking, the mixture is placed in a vacuum freeze dryer for freeze drying to obtain a dry and active powdered bacterial agent. S5. Coating: Mix the powdered microbial agent with other coating agents evenly, put the rapeseed seeds into a coating machine with a uniform rotation speed, spray in the adhesive, then add the mixed coating agent, and finally spray the adhesive and add the mixed coating agent. Repeat this alternating operation several times to obtain a product of immobilized microorganisms based on a biological seed carrier. S6. The immobilized microbial product based on the biological seed carrier obtained after S5 treatment is dried at room temperature.

3. The method for preparing the biological seed carrier product according to claim 2, characterized in that, In step S3, the parameters of the constant temperature oscillator are set to 150 r / min rotation speed and 30℃ temperature, and the oscillation adsorption time is 2h.

4. The method for preparing the biological seed carrier product according to claim 2, characterized in that, In step S4, the mixture is placed in a vacuum freeze dryer. It should be pre-frozen for 1 hour to lock in the moisture, and then vacuum dried for 24 hours.

5. The method for preparing the biological seed carrier product according to claim 2, characterized in that, In step S5, the coating machine rotates at 90 r / min.

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