A material for enhancing microbial degradation of petroleum hydrocarbon pollutants and a preparation process thereof

Abstract

The present application relates to the technical field of microbial degradation material, and particularly relates to a material for enhancing microbial degradation of petroleum hydrocarbon pollutants and a preparation process thereof; the material comprises the following components in percentage by mass: dry earthworm powder: 20-25%; centipede grass: 15-20; modified rotifer powder: 0.5-1%; repairing agent: 1-2%; fatty acid hydroxylase: 0.5-1%; and degradation bacteria: the rest. The preparation process comprises the following steps: S1, preparation of microbial powder; S2, preparation of carrier; and S3, preparation of degradation material. The degradation material of the present application adds the centipede grass and modified rotifer powder, promotes the diversity and abundance of soil microorganisms, can improve the adsorption capacity and biodegradation efficiency of microorganisms to petroleum hydrocarbon, the preparation process improves the wettability of the degradation material, increases the contact area and time of the reaction, can more quickly transport the active ingredients in the soil, increases the adsorption sites of the carrier material, and enhances the rate of microbial degradation of petroleum hydrocarbon.

Description

Technical Field

[0001] This invention relates to the field of petroleum hydrocarbon degradation technology, specifically to a material for enhancing microbial degradation of petroleum hydrocarbon pollutants and its preparation process. Background Technology

[0002] Oil well blowouts, leaks during oil transportation, storage, and processing have resulted in large-scale oil-contaminated soil.

[0003] Microbial degradation technology refers to the use of microbial enzyme activity to transform toxic and harmful substances into non-toxic and harmless substances through certain treatments. Due to the strong adaptability and decomposition metabolic capabilities of microorganisms, microorganisms in petroleum-contaminated soils have evolved the ability to degrade petroleum hydrocarbons.

[0004] Since there are many types of petroleum hydrocarbons, a single microbial strain usually cannot degrade all components of petroleum hydrocarbons. Therefore, in order to solve the above problems, this invention designs a material and its preparation process to enhance the microbial degradation of petroleum hydrocarbon pollutants, thereby improving the microbial degradation efficiency. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a material for enhancing the microbial degradation of petroleum hydrocarbon pollutants and its preparation process.

[0006] A material for enhancing the microbial degradation of petroleum hydrocarbon pollutants, comprising the following components by mass percentage:

[0007] Earthworm powder: 20-25%;

[0008] Cynodon dactylon: 15-20%;

[0009] Modified rotifer powder: 0.5-1%;

[0010] Repair agent: 1-2%;

[0011] Fatty acid hydroxylase: 0.5–1%;

[0012] Degrading bacteria: Balance;

[0013] The degrading bacteria are a composite degrading bacteria composed of Azotobacter chrysophyte, Pseudomonas fluorescens, and Aeromonas hydrophila in a mass ratio of 1 to 1.5:1:1.

[0014] Furthermore, the concentration of *Azotobacter chrysophagus* in the degrading bacteria is 1–8 × 10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the fluorescent Pseudomonas was 1.5–5.5 × 10⁻⁶. 9 cfu·mL -1The bacterial concentration of the aforementioned Aeromonas hydrophila is 1.5–5.5 × 10⁻⁶. 9 cfu·mL -1 .

[0015] Note: If the concentration of degrading bacteria is too low, the degradation rate may be slow because fewer microbial cells are insufficient to generate enough metabolic activity to effectively break down petroleum hydrocarbons. If the concentration of degrading bacteria is too high, it may lead to nutrient deficiency, as microorganisms consume nutrients while decomposing petroleum hydrocarbons. In this case, even with a large number of microorganisms present, their metabolic activity will be limited due to the lack of necessary nutritional support, thus affecting the degradation efficiency.

[0016] Furthermore, the repair agent, by weight, comprises: 1-2 parts maltose, 8-12 parts mannitol, 5-15 parts dipotassium hydrogen phosphate, 18-25 parts ferric chloride, 8-15 parts bentonite, and 0.1-0.3 parts polyamine.

[0017] Note: Maltose and mannitol can serve as carbon sources, providing energy and nutrients to microorganisms to aid their growth and maintain metabolic activities. Dipotassium hydrogen phosphate can act as a nutritional supplement, providing essential phosphorus, which helps microorganisms synthesize cell structures and energy transfer molecules. Ferric chloride can act as a flocculant to help aggregate petroleum hydrocarbon particles, facilitating their decomposition by microorganisms. Bentonite may fix petroleum hydrocarbons through adsorption, reducing their mobility in the environment, while also providing a surface for microbial attachment. Polyamine compounds can serve as nutrients, influencing microbial behavior and metabolism.

[0018] Furthermore, the preparation method of the modified rotifer powder is as follows:

[0019] First, the rotifers are ground into powder, and then the powder is pretreated under a low-voltage electric field with an output voltage of 1500-2500V, a current of 0.2-0.4mA, and a treatment temperature of -15 to -5℃.

[0020] The pretreated powder was soaked in the modification solution at a solid-liquid ratio of 1g:20-30mL. After soaking at -40 to -30MPa and 30 to 35℃ for 10 to 20 minutes, protease was added. The solid-liquid ratio of the protease to the modification solution was 1g:40-50mL. The pressure was adjusted to 65 to 75MPa and the temperature to 50 to 55℃ for high-pressure homogenization for 15 to 20 minutes.

[0021] After high-pressure homogenization, the powder is filtered and then vacuum-dried at 70–80°C to obtain modified rotifer powder.

[0022] The modified solution is a mixture of sodium bisulfite solution and sodium pyrophosphate solution in a mass ratio of 1:0.6 to 0.8, wherein the mass concentration of sodium bisulfite solution is 15 to 25% and the mass concentration of sodium pyrophosphate solution is 5 to 8%.

[0023] Explanation: Low-voltage electric field pretreatment increases the surface area of ​​rotifer powder, improves porosity, and activates bioactive components, which helps improve the efficiency and effectiveness of subsequent modification processes. Immersing the pretreated earthworm powder in the modification solution forms a modified layer on the surface of the rotifer powder, exhibiting better lipophilicity, thereby improving the adsorption and degradation efficiency of petroleum hydrocarbons. Adding protease during the modification process allows for the biocatalytic decomposition of proteins, producing small peptides and amino acids. These small molecules typically have better solubility and bioavailability, improving the adsorption capacity and biodegradation efficiency of rotifer powder for petroleum hydrocarbons, exposing more active sites, and thus enhancing its interaction with petroleum hydrocarbons. High-pressure homogenization helps to more effectively disperse the small molecules produced by enzymatic hydrolysis, making them easier to contact with petroleum hydrocarbons, thereby improving degradation efficiency.

[0024] A preparation process for a material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in any of the above claims includes the following steps:

[0025] S1. Preparation of microbial powder:

[0026] The degrading bacteria were prepared into a bacterial solution, and then the bacterial solution was mixed with fatty acid hydroxylase at the ratio described above at a speed of 1500-2000 rpm for 20-30 min. Then, the mixture was spray-dried with an inlet temperature of 120-150℃ and an outlet temperature of 60-100℃ to obtain microbial powder.

[0027] S2. Preparation of the carrier:

[0028] The remaining components are mixed at 1500-2000 rpm in the specified proportion for 50-60 minutes to obtain a mixture. The mixture is then placed on a shaping plate for shaping treatment. The shaping treatment method is as follows: the shaping plate is placed horizontally in a vacuum chamber and vacuumed at 25-35°C and 0.8-1 MPa for 25-30 minutes; then the vacuumed mixture is vulcanized at 150-160°C and 10-12 MPa for 1.5-2 hours. After cooling to room temperature, the mixture is separated from the shaping plate to obtain the carrier.

[0029] S3, Preparation of degradable materials;

[0030] Next, the microbial powder was co-deposited on the carrier to obtain the degradation material. The temperature of the co-deposition reaction was 155-175℃, the stirring rate was 80-90 rpm, and the reaction time was 14-24 h.

[0031] Furthermore, the surface of the shaping plate has a mesh structure.

[0032] Explanation: The network structure increases the water contact angle of the mixture, thereby increasing its wettability. Materials with strong wettability can more effectively contact pollutants in the soil, increasing the contact area and time of the reaction, thus improving the degradation efficiency. Furthermore, its anisotropic characteristics increase the adsorption sites of the carrier material, improve the binding between the carrier material and the microbial powder, thereby enhancing the compactness of the degradation material and improving the degradation efficiency.

[0033] Further, the preparation method of the bacterial solution of the degrading bacteria is as follows: First, the degrading bacteria are inoculated onto an activation medium for activation culture at a temperature of 25-30℃ for 25-28 hours to obtain activated strains; the activated strains are then inoculated into a seed culture medium with a fermentation liquid fraction of 15-20%, the pH is controlled at 6.0-7.0, the temperature is 25-30℃, the rotation speed is 140-150 r / min, and the culture is carried out for 17-19 hours to obtain seed liquid; finally, the seed liquid is loaded into a fermentation medium with a fermentation liquid fraction of 15-20%, and cultured on a shaker at 25-30℃ for 11-13 hours at a rotation speed of 140-150 r / min to obtain the bacterial solution of the degrading bacteria.

[0034] Note: Degrading bacteria that have been activated, seed cultured, and then fermented have more vigorous cell metabolism and can begin to degrade petroleum hydrocarbons more quickly. This pretreatment step also helps the strains establish adaptations to specific environmental conditions, such as tolerance and competitiveness, which is crucial for working effectively in complex petroleum-polluted environments.

[0035] Further, the components of the activation culture medium include: 10-15 g / L glucose, 4-6 g / L MgCl2, 0.2-0.3 g / L MgSO4·7H2O, 0.1-0.3 g / L KCl, 0.1-0.2 g / L (NH4)2SO4, 6-7 g / L Ca3(PO4)2, 12-16 g / L agar, pH 7.0;

[0036] The seed culture medium comprises: 120–140 mg / L phenanthrene, 6–9 g / L peptone, 1–2 g / L potassium dihydrogen phosphate, 1–2 g / L dipotassium hydrogen phosphate, pH 7.0;

[0037] The fermentation medium comprises: 9–12 g / L peptone, 4–6 g / L yeast extract, 8–10 g / L NaCl, and pH 7.0.

[0038] Note: The components of the above culture medium can restore the growth and metabolic activity of degrading bacteria, prepare a sufficient number of active strains to optimize their growth rate and biomass accumulation, and promote the synthesis of target metabolites.

[0039] Compared with existing petroleum hydrocarbon degradation materials, the beneficial effects of this invention are:

[0040] (1) The degradation material of the present invention contains bermudagrass and modified rotifer powder. Bermudagrass is a plant that can grow in petroleum-contaminated soil. It can promote the diversity and abundance of soil microorganisms through its root secretions, thereby enhancing the soil's ability to degrade petroleum hydrocarbons. Modified rotifer powder is rich in protein and other nutrients. After modification, it can improve the adsorption capacity and biodegradation efficiency of microorganisms for petroleum hydrocarbons, expose more active sites, and thus enhance their interaction with petroleum hydrocarbons.

[0041] (2) In the preparation process of the degradation material of the present invention, microbial powder is co-deposited on the carrier material. The co-deposition reaction can enable microorganisms to form a biofilm on the surface of the carrier material. Such a structure helps to protect microorganisms from adverse environmental factors, while providing more attachment points and increasing the activity and stability of microorganisms. Different kinds of microorganisms can coexist in the co-deposited material, forming a mutually beneficial symbiotic relationship and jointly promoting the degradation of petroleum hydrocarbons. Moreover, the co-deposited material can be more easily separated from the polluted environment. Furthermore, since the microorganisms are fixed on the carrier, these materials can be reused to a certain extent, reducing the cost of remediation.

[0042] (3) The network structure carrier material prepared by the present invention can improve the wettability of the degradation material, increase the contact area and time of the reaction, and can more quickly transport active ingredients in the soil, thus accelerating the degradation rate of pollutants. Moreover, the network structure increases the anisotropic characteristics of the degradation material. The carrier material usually has a porous structure and a large specific surface area, thereby increasing the adsorption sites of the carrier material. This helps to adsorb petroleum hydrocarbon pollutants, increase the contact opportunities between microorganisms and pollutants, and improve the degradation efficiency. Attached Figure Description

[0043] Figure 1 This is a comparison chart of the results of the first investigation into the degradation materials of this invention;

[0044] Figure 2 This is a comparison chart of the results of the second investigation into the preparation process of this invention;

[0045] Figure 3 This is a surface structure diagram of the shaping plate used in the preparation process of this invention. Detailed Implementation

[0046] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0047] Example 1: A material for enhancing microbial degradation of petroleum hydrocarbon pollutants, comprising the following components by mass percentage:

[0048] Earthworm powder: 23%;

[0049] Bermuda grass: 18;

[0050] Modified rotifer powder: 0.8%;

[0051] Repair agent: 1.5%;

[0052] Fatty acid hydroxylase: 0.8%;

[0053] Degrading bacteria: Balance;

[0054] The degrading bacteria are a composite degrading bacteria composed of *Azotobacter chrysophagus*, *Pseudomonas fluorescens*, and *Aeromonas hydrophila* in a mass ratio of 1.2:1:1, with the *Azotobacter chrysophagus* having a cell concentration of 4 × 10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the fluorescent Pseudomonas was 3 × 10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the aforementioned Aeromonas hydrophila is 3 × 10⁻⁶. 9 cfu·mL -1 ;

[0055] The repair agent, by weight, comprises: 1.5 parts maltose, 10 parts mannitol, 10 parts dipotassium hydrogen phosphate, 21 parts ferric chloride, 11 parts bentonite, and 0.2 parts polyamine;

[0056] The modified rotifer powder is prepared as follows:

[0057] First, the rotifers are ground into powder, and then the powder is pretreated under a low-voltage electric field with an output voltage of 2000V, a current of 0.3mA, and a treatment temperature of -10℃.

[0058] The pretreated powder was soaked in the modification solution at a solid-liquid ratio of 1g:25mL. After soaking at -35MPa and 32℃ for 15min, protease was added. The solid-liquid ratio of the protease to the modification solution was 1g:45mL. The pressure was adjusted to 70MPa and the temperature to 53℃ for high-pressure homogenization for 18min.

[0059] After high-pressure homogenization, the powder was filtered and then vacuum dried at 75°C to obtain modified rotifer powder.

[0060] The modified solution is a mixture of sodium bisulfite solution and sodium pyrophosphate solution in a mass ratio of 1:0.7, wherein the mass concentration of sodium bisulfite solution is 20% and the mass concentration of sodium pyrophosphate solution is 6.5%.

[0061] Example 2: The preparation process of the material for enhancing microbial degradation of petroleum hydrocarbon pollutants described in Example 1 includes the following steps:

[0062] S1. Preparation of microbial powder:

[0063] The degrading bacteria were prepared into a bacterial solution. The preparation method of the bacterial solution was as follows: First, the degrading bacteria were inoculated onto an activation medium and activated at 28°C for 27 hours to obtain an activated strain. The activated strain was then inoculated into a seed culture medium with a fermentation liquid fraction of 18%, with the pH controlled at 6.5, the temperature at 28°C, and the rotation speed at 145 r / min for 18 hours to obtain a seed liquid. Finally, the seed liquid was placed into a fermentation medium with a fermentation liquid fraction of 18% and cultured on a shaker at 28°C for 12 hours at a rotation speed of 145 r / min to obtain the bacterial solution of the degrading bacteria.

[0064] The components of the activation medium include: 13 g / L glucose, 5 g / L MgCl2, 0.25 g / L MgSO4·7H2O, 0.2 g / L KCl, 0.15 g / L (NH4)2SO4, 6.5 g / L Ca3(PO4)2, 14 g / L agar, pH 7.0;

[0065] The seed culture medium comprises: 130 mg / L phenanthrene, 8 g / L peptone, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate, pH 7.0;

[0066] The fermentation medium comprises: 10 g / L peptone, 8 g / L yeast extract, 9 g / L NaCl, pH 7.0;

[0067] The bacterial culture and fatty acid hydroxylase were then mixed at 1750 rpm for 25 min according to the ratio in Example 1, and then spray-dried at an inlet temperature of 135°C and an outlet temperature of 80°C to obtain microbial powder.

[0068] S2. Preparation of the carrier:

[0069] The remaining components were mixed at 1750 rpm for 55 minutes according to the proportions of Example 1 to obtain a mixture. The mixture was then placed on a shaping plate for shaping treatment, such as... Figure 3As shown, the surface of the shaping plate has a mesh structure. The shaping process is as follows: the shaping plate is placed horizontally in a vacuum chamber and vacuumed for 28 minutes at 30°C and 0.9MPa; then the mixture after vacuuming is vulcanized at 155°C and 11MPa for 1.8 hours. After cooling to room temperature, the mixture is separated from the shaping plate to obtain the carrier.

[0070] S3, Preparation of degradable materials;

[0071] Next, the microbial powder was co-deposited on the carrier to obtain the degradation material. The co-deposition reaction was carried out at a temperature of 160°C, a stirring rate of 85 rpm, and a reaction time of 19 h.

[0072] Example 3: This example differs from Example 1 in that it is a material for enhancing the microbial degradation of petroleum hydrocarbon pollutants, comprising the following components by mass percentage:

[0073] Earthworm powder: 20%;

[0074] Cynodon dactylon: 15%;

[0075] Modified rotifer powder: 0.5%;

[0076] Repair agent: 1%;

[0077] Fatty acid hydroxylase: 0.5%;

[0078] Degrading bacteria: Balance.

[0079] Example 4: This example differs from Example 1 in that it is a material for enhancing the microbial degradation of petroleum hydrocarbon pollutants, comprising the following components by mass percentage:

[0080] Earthworm powder: 25%;

[0081] Bermuda grass: 20%;

[0082] Modified rotifer powder: 1%;

[0083] Repair agent: 2%;

[0084] Fatty acid hydroxylase: 1%;

[0085] Degrading bacteria: Balance.

[0086] Example 5: This example differs from Example 1 in that the degrading bacteria are a composite degrading bacteria composed of *Azotobacter chrysophagus*, *Pseudomonas fluorescens*, and *Aeromonas hydrophila* in a mass ratio of 1:1:1; and the concentration of *Azotobacter chrysophagus* in the degrading bacteria is 1×10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the fluorescent Pseudomonas was 5.5 × 10⁻⁶.9 cfu·mL -1 The bacterial concentration of the aforementioned Aeromonas hydrophila was 5.5 × 10⁻⁶. 9 cfu·mL -1 .

[0087] Example 6: This example differs from Example 1 in that the degrading bacteria are a composite degrading bacteria composed of *Azotobacter chrysophagus*, *Pseudomonas fluorescens*, and *Aeromonas hydrophila* in a mass ratio of 1.5:1:1; the concentration of *Azotobacter chrysophagus* in the degrading bacteria is 8 × 10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the fluorescent Pseudomonas was 1.5 × 10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the aforementioned Aeromonas hydrophila is 1.5 × 10⁻⁶. 9 cfu·mL -1 .

[0088] Example 7: This example differs from Example 1 in that the repair agent, by weight, includes: 1 part maltose, 8 parts mannitol, 5 parts dipotassium hydrogen phosphate, 18 parts ferric chloride, 8 parts bentonite, and 0.3 parts polyamine.

[0089] Example 8: This example differs from Example 1 in that the repair agent, by weight, includes: 2 parts maltose, 12 parts mannitol, 15 parts dipotassium hydrogen phosphate, 25 parts ferric chloride, 15 parts bentonite, and 0.1 parts polyamine.

[0090] Example 9: The difference between this example and Example 1 is that the rotifers are first ground into powder, and then the powder is pretreated under a low-voltage electric field with an output voltage of 1500V, a current of 0.2mA, and a treatment temperature of -15℃.

[0091] Example 10: The difference between this example and Example 1 is that the rotifers are first ground into powder, and then the powder is pretreated under a low-voltage electric field with an output voltage of 2500V, a current of 0.4mA, and a treatment temperature of -5℃.

[0092] Example 11: The difference between this example and Example 1 is that the pretreated powder was soaked in the modified solution at a solid-liquid ratio of 1g:20mL for 10min at -40MPa and 30℃.

[0093] Example 12: The difference between this example and Example 1 is that the pretreated powder was soaked in the modified solution at a solid-liquid ratio of 1g:30mL for 20min at -30MPa and 35℃.

[0094] Example 13: The difference between this example and Example 1 is that the solid-liquid ratio of the protease to the modified solution is 1g:40mL, and the pressure is adjusted to 65MPa and the temperature to 50℃ for high-pressure homogenization for 15min.

[0095] Example 14: The difference between this example and Example 1 is that the solid-liquid ratio of the protease to the modified solution is 1g:50mL, and the pressure is adjusted to 75MPa and the temperature to 55℃ for high-pressure homogenization for 20min.

[0096] Example 15: This example differs from Example 1 in that the modified solution is a mixture of sodium bisulfite solution and sodium pyrophosphate solution with a mass ratio of 1:0.6, wherein the mass concentration of sodium bisulfite solution is 25% and the mass concentration of sodium pyrophosphate solution is 5%.

[0097] Example 16: This example differs from Example 1 in that the modified solution is a mixture of sodium bisulfite solution and sodium pyrophosphate solution with a mass ratio of 1:0.8, the mass concentration of sodium bisulfite solution is 15%, and the mass concentration of sodium pyrophosphate solution is 8%.

[0098] Example 17: This example differs from Example 2 in that the preparation method of the degrading bacteria culture is as follows: First, the degrading bacteria are inoculated onto an activation medium for activation culture at 25°C for 25 hours to obtain activated strains; the activated strains are then inoculated into a seed culture medium with a fermentation liquid fraction of 15%, with the pH controlled at 6.0, the temperature at 25°C, and the rotation speed at 140 r / min for 17 hours to obtain seed liquid; finally, the seed liquid is placed into a fermentation medium with a fermentation liquid fraction of 15% and cultured on a shaker at 25°C for 11 hours at a rotation speed of 140 r / min to obtain the degrading bacteria culture.

[0099] Example 18: This example differs from Example 2 in that the preparation method of the degrading bacteria culture is as follows: First, the degrading bacteria are inoculated onto an activation medium for activation culture at 30°C for 28 hours to obtain activated strains; the activated strains are then inoculated into a seed culture medium with a fermentation liquid fraction of 20%, with the pH controlled at 7.0, the temperature at 30°C, and the rotation speed at 150 r / min for 19 hours to obtain seed liquid; finally, the seed liquid is placed into a fermentation medium with a fermentation liquid fraction of 20% and cultured on a shaker at 30°C for 13 hours at a rotation speed of 150 r / min to obtain the degrading bacteria culture.

[0100] Example 19: This example differs from Example 2 in that the components of the activation culture medium include: 10 g / L glucose, 4 g / L MgCl2, 0.2 g / L MgSO4·7H2O, 0.1 g / L KCl, 0.1 g / L (NH4)2SO4, 6 g / L Ca3(PO4)2, 12 g / L agar, pH 7.0;

[0101] The seed culture medium comprises: 120 mg / L phenanthrene, 6 g / L peptone, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, pH 7.0;

[0102] The fermentation medium comprises: 9 g / L peptone, 4 g / L yeast extract, 8 g / L NaCl, and pH 7.0.

[0103] Example 20: This example differs from Example 2 in that the components of the activation culture medium include: 15 g / L glucose, 6 g / L MgCl2, 0.3 g / L MgSO4·7H2O, 0.3 g / L KCl, 0.2 g / L (NH4)2SO4, 7 g / L Ca3(PO4)2, 16 g / L agar, pH 7.0;

[0104] The seed culture medium comprises: 140 mg / L phenanthrene, 9 g / L peptone, 2 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, pH 7.0;

[0105] The fermentation medium comprises: 12 g / L peptone, 6 g / L yeast extract, 10 g / L NaCl, and pH 7.0.

[0106] Example 21: The difference between this example and Example 2 is that the bacterial solution and fatty acid hydroxylase are mixed at 1500 rpm for 20 minutes.

[0107] Example 22: The difference between this example and Example 2 is that the bacterial solution and fatty acid hydroxylase are mixed at 2000 rpm for 30 minutes.

[0108] Example 23: The difference between this example and Example 2 is that the inlet temperature of the spray dryer is 120°C and the outlet temperature is 60°C.

[0109] Example 24: The difference between this example and Example 2 is that the inlet temperature of the spray dryer is 150°C and the outlet temperature is 100°C.

[0110] Example 25: This example differs from Example 2 in that the remaining components are mixed at 1500 rpm for 50 minutes to obtain a mixture.

[0111] Example 26: This example differs from Example 2 in that the remaining components are mixed at 2000 rpm for 60 minutes to obtain a mixture.

[0112] Example 27: The difference between this example and Example 2 is that the shaping plate is placed horizontally in a vacuum chamber and vacuumed for 25 minutes at 25°C and 0.8MPa.

[0113] Example 28: The difference between this example and Example 2 is that the shaping plate is placed horizontally in a vacuum chamber and vacuumed for 30 minutes at 35°C and 1MPa.

[0114] Example 29: The difference between this example and Example 2 is that the mixture after vacuuming is vulcanized at 150°C and 10MPa for 1.5 hours.

[0115] Example 30: This example differs from Example 2 in that the mixture after vacuuming is vulcanized at 160°C and 12MPa for 2 hours.

[0116] Example 31: The difference between this example and Example 2 is that the temperature of the co-deposition reaction is 155°C, the stirring rate is 80 rpm, and the reaction time is 14 h.

[0117] Example 32: The difference between this example and Example 2 is that the temperature of the co-deposition reaction is 175°C, the stirring rate is 90 rpm, and the reaction time is 24 h.

[0118] Experimental Example: The description of this experimental example is based on the scheme described in Example 2, and aims to illustrate the practical application effect of the present invention.

[0119] Experimental design: 1 kg of the degradation material prepared in Example 2 was added to 100 kg of petroleum hydrocarbon-contaminated soil for remediation experiment. The degradation rate of petroleum hydrocarbon was detected after 30 days.

[0120] 1. To investigate the effects of the component content of the degradable material and the preparation parameters of the modified rotifer powder on the degradation rate.

[0121] The difference between Comparative Example 1 and Example 1 is that the degradable material does not include modified rotifer powder;

[0122] The difference between Comparative Example 2 and Example 1 is that no pretreatment of the rotifer powder was performed;

[0123] The difference between Comparative Example 3 and Example 1 is that no protease was added during the modification process;

[0124] Depend on Figure 1The results showed that the degradation rates of Control Example 1 (lacking modified rotifer powder), Control Example 2 (lacking pretreatment), and Control Example 3 (lacking protease) were significantly lower than those of Examples 1, 3-16. This indicates that modified rotifer powder significantly improves the degradation efficiency of the degradation material, and that the pretreatment and addition of protease in the preparation of modified rotifer powder help to enhance its effect.

[0125] Comparing Examples 1 and 3-16, it can be seen that if the proportion of modified rotifer powder is too small or too large, the proportion of Azotobacter chrysophagus in the degrading bacteria is too small or too large, the proportion of polyamine in the remediation agent is too small or too large, the parameters of rotifer powder pretreatment are too small or too large, the parameters of modified soaking are too small or too large, the parameters of high-pressure homogenization are too small or too large, and the parameters of sodium bisulfite solution in the modified solution are too small or too large, Junhui will reduce the degradation rate of petroleum hydrocarbons. The degradation rate of Example 10 is comparable to that of Example 1, but it consumes more energy. Therefore, from an economic point of view, the parameters of Example 1 are relatively better.

[0126] 2. Investigate the effects of various parameters in the preparation process on the degradation rate.

[0127] The difference between Comparative Example 4 and Example 1 is that the shaping plate has a flat surface;

[0128] Depend on Figure 2 The results show that the structure of the carrier material in Comparative Example 4 becomes flat, reducing wettability and adsorption sites. Therefore, compared with Examples 2 and 17-32, the degradation rate of petroleum hydrocarbons is significantly reduced. This indicates that the network structure of the carrier material has a certain enhancing effect on the degradation of petroleum hydrocarbons.

[0129] Furthermore, comparing Examples 2 and 17-32, it can be seen that excessively small or large parameters in bacterial solution preparation, excessively small or large parameters in culture medium components, excessively small parameters in the mixing of bacterial solution and enzyme, excessively small or large parameters in spray drying, excessively small parameters in the mixing of mixtures, excessively small or large parameters in vacuuming, excessively small or large parameters in sulfidation, and excessively small or large parameters in co-deposition reaction will all reduce the degradation rate of petroleum hydrocarbons. In Example 22, the mixing parameters of bacterial solution and enzyme are larger, and in Example 26, the mixing parameters of mixtures are larger, thus slightly improving the degradation rate of petroleum hydrocarbons. However, from an economic point of view, the parameters in Example 2 are relatively better.

[0130] In summary, the degradation material prepared by this invention can achieve a degradation rate of over 72% for petroleum hydrocarbons, significantly enhancing the efficiency of microbial degradation of petroleum hydrocarbons.

Claims

1. A material for enhancing microbial degradation of petroleum hydrocarbon pollutants, characterized in that, By mass percentage, it includes the following components: Earthworm powder: 20-25%; Bermuda grass: 15-20%; Modified rotifer powder: 0.5~1%; Repair agent: 1~2%; Fatty acid hydroxylase: 0.5~1%; Degrading bacteria: Balance; The degrading bacteria are a composite degrading bacteria composed of Azotobacter chrysophagus, Pseudomonas fluorescens, and Aeromonas hydrophila in a mass ratio of 1 to 1.5:1:

1. The modified rotifer powder is prepared as follows: First, the rotifers are ground into powder, and then the powder is pretreated under a low-voltage electric field with an output voltage of 1500~2500V, a current of 0.2~0.4mA, and a treatment temperature of -15~-5℃. The pretreated powder was soaked in the modification solution at a solid-liquid ratio of 1g:20~30mL. After soaking at -40~-30MPa and 30~35℃ for 10~20min, protease was added. The solid-liquid ratio of the protease to the modification solution was 1g:40~50mL. The pressure was adjusted to 65~75MPa and the temperature to 50~55℃ for high-pressure homogenization for 15~20min. After high-pressure homogenization, the powder is filtered and then vacuum-dried at 70-80°C to obtain modified rotifer powder. The modified solution is a mixture of sodium bisulfite solution and sodium pyrophosphate solution with a mass ratio of 1:0.6~0.8, wherein the mass concentration of sodium bisulfite solution is 15~25% and the mass concentration of sodium pyrophosphate solution is 5~8%.

2. The material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in claim 1, characterized in that, The concentration of *Azotobacter chrysophagus* in the degrading bacteria is 1~8×10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the fluorescent Pseudomonas was 1.5~5.5×10⁻⁶. 9 cfu·mL -1 The bacterial concentration of the aforementioned Aeromonas hydrophila is 1.5~5.5×10⁻⁶. 9 cfu·mL -1 .

3. The material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in claim 1, characterized in that, The repair agent, by weight, comprises: 1-2 parts maltose, 8-12 parts mannitol, 5-15 parts dipotassium hydrogen phosphate, 18-25 parts ferric chloride, 8-15 parts bentonite, and 0.1-0.3 parts polyamine.

4. The preparation process of a material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Preparation of microbial powder: The degrading bacteria were prepared into a bacterial solution, and then the bacterial solution was mixed with fatty acid hydroxylase at a ratio of 1500~2000 rpm for 20~30 min. Then, the solution was spray-dried with an inlet temperature of 120~150℃ and an outlet temperature of 60~100℃ to obtain microbial powder. S2. Preparation of the carrier: The remaining components are mixed in proportion at 1500~2000 rpm for 50~60 min to obtain a mixture. The mixture is placed on a shaping plate for shaping treatment. The shaping treatment method is as follows: the shaping plate is placed horizontally in a vacuum chamber and vacuumed at 25~35℃ and 0.8~1MPa for 25~30 min; then the vacuumed mixture is vulcanized at 150~160℃ and 10~12MPa for 1.5~2 h. After cooling to room temperature, the mixture is separated from the shaping plate to obtain the carrier. S3. Preparation of degradable materials: Next, the microbial powder is co-deposited on the carrier to obtain the degradation material. The temperature of the co-deposition reaction is 155~175℃, the stirring rate is 80~90rpm, and the reaction time is 14~24h.

5. The preparation process of a material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in claim 4, characterized in that, The surface of the shaping plate has a mesh structure.

6. The preparation process of a material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in claim 4, characterized in that, The method for preparing the bacterial culture of the degrading bacteria is as follows: First, the degrading bacteria are inoculated onto an activation medium for activation culture at a temperature of 25-30℃ for 25-28 hours to obtain an activated strain; the activated strain is then inoculated into a seed culture medium with a fermentation liquid fraction of 15-20%, the pH is controlled at 6.0-7.0, the temperature is 25-30℃, the rotation speed is 140-150 r / min, and the culture is carried out for 17-19 hours to obtain the seed culture; Finally, the seed culture was loaded into a fermentation medium with a fermentation liquid fraction of 15-20%, and cultured on a shaker at 25-30℃ for 11-13 hours at a speed of 140-150 r / min to obtain the bacterial culture of the degrading bacteria.

7. The preparation process of a material for enhancing microbial degradation of petroleum hydrocarbon pollutants as described in claim 6, characterized in that, The components of the activation medium include: 10-15 g / L glucose, 4-6 g / L MgCl2, 0.2-0.3 g / L MgSO4·7H2O, 0.1-0.3 g / L KCl, 0.1-0.2 g / L (NH4)2SO4, 6-7 g / L Ca3(PO4)2, 12-16 g / L agar, pH=7.0; The seed culture medium comprises: 120-140 mg / L phenanthrene, 6-9 g / L peptone, 1-2 g / L potassium dihydrogen phosphate, 1-2 g / L dipotassium hydrogen phosphate, pH=7.0; The fermentation medium comprises: 9-12 g / L peptone, 4-6 g / L yeast extract, 8-10 g / L NaCl, and pH=7.0.

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