Microbial remediation agent for degradation of marine oil pollution and preparation method thereof

Abstract

The present application relates to the technical field of marine oil pollution degradation, in particular to a microbial remediation agent for marine oil pollution degradation and a preparation method thereof, which is used to solve the problem that the existing biological remediation method is difficult to play an effective degradation role in the complex marine environment; the microbial remediation agent takes hydrophobic microspheres coated carbon fixed bacteria agent and modified composite particles as main raw materials, combines the dual degradation mechanisms of microbial degradation and photocatalytic degradation, significantly improves the degradation effect and efficiency of oil pollutants, has good stability, prolongs the service life, and the selected materials have excellent environmental protection performance and will not cause secondary pollution to the marine environment, so it has a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of marine oil pollution degradation technology, specifically to a microbial remediation agent for marine oil pollution degradation and its preparation method. Background Technology

[0002] With the continuous development of offshore oil extraction, transportation, and maritime shipping, marine oil pollution incidents occur frequently, causing serious damage to marine ecosystems. Hydrocarbon compounds in petroleum (such as alkanes and aromatics) are persistent, toxic, and bioaccumulative, affecting the survival and reproduction of marine life, disrupting the marine food chain, and ultimately threatening human health.

[0003] Currently, the main methods for treating marine oil pollution include physical methods (such as oil booms and absorbent pads), chemical methods (such as dispersants and solidifying agents), and bioremediation. While physical and chemical methods can reduce oil pollution in the short term, they suffer from problems such as incomplete treatment and the potential for secondary pollution. Bioremediation utilizes the metabolic activity of microorganisms to degrade petroleum hydrocarbons into harmless carbon dioxide and water. It boasts advantages such as low cost, high efficiency, and environmental friendliness, making it a promising remediation technology with broad application prospects.

[0004] However, petroleum-degrading microorganisms in the natural environment are few in number and have low activity, and their survival rate is low in the complex marine environment. They are easily washed away by water currents, and petroleum hydrocarbons have poor dispersibility in water, resulting in insufficient contact with microorganisms and making it difficult for them to exert an effective degradation effect. Therefore, developing a microbial remediation agent for the degradation of marine oil pollution and its preparation method is of great practical significance.

[0005] To address the aforementioned technical shortcomings, a solution is proposed. Summary of the Invention

[0006] In order to overcome the above-mentioned technical problems, the purpose of this invention is to provide a microbial remediation agent for the degradation of marine oil pollution and its preparation method, which solves the problem that existing bioremediation methods are difficult to play an effective degradation role in complex marine environments.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A microbial remediation agent for the degradation of marine oil pollution comprises the following components in parts by weight:

[0009] The mixture consists of 35-55 parts of hydrophobic microspheres coated with carbon-fixed bacterial agent, 12-18 parts of modified composite particles, 5-7 parts of ammonium nitrate, 2-3 parts of dipotassium hydrogen phosphate, 1-2 parts of ferrous sulfate, 5 parts of polysorbate-803, 2-4 parts of xanthan gum, 20-25 parts of deionized water, and 40-50 parts of anhydrous ethanol.

[0010] In a preferred embodiment of the present invention, the hydrophobic microsphere-coated carbon-immobilized bacterial agent is prepared by the following steps:

[0011] Step a1: Crush the dried reed stalks through an 80-100 mesh sieve, then soak them in a potassium hydroxide solution for 10-12 hours. After that, place them in a vacuum drying oven and dry them at 80-85℃ for 2-3 hours. Then, place them in a tube furnace and purge them with nitrogen. Then, calcine them at a heating rate of 5-7℃ / min to 450-460℃ for 40-50 minutes. After that, calcine them at 650-700℃ for 2-3 hours. Finally, cool them in the furnace to obtain reed charcoal.

[0012] Step a2: Inoculate the mixed bacterial culture into LB medium and incubate at 35-37℃ and 110-130 r / min for 48-72 h. Then centrifuge and dilute with physiological saline to 1×10⁻⁶. 9 Mixed bacterial culture with CFU / mL;

[0013] Step a3: Add sodium alginate, chitosan, and acetic acid solution to a three-necked flask equipped with a stirrer and thermometer. Stir the reaction at 25-30℃ and 200-300 r / min for 20-30 min. Then add reed charcoal and mixed bacterial solution and continue stirring for 2-3 h. Then add calcium chloride solution dropwise while stirring, controlling the dropping rate to 1-3 drops / s. After the addition is complete, continue stirring for 3-5 h. Then add octyl glycidyl ether and continue stirring for 1-2 h. After the reaction is complete, filter the reaction product under vacuum. Wash the filter cake 2-3 times with anhydrous ethanol and distilled water, and then freeze-dry under vacuum to obtain hydrophobic microsphere-coated carbon-fixed bacterial agent.

[0014] In a preferred embodiment of the present invention, the ratio of reed straw to potassium hydroxide solution in step a1 is 3g:15-20mL.

[0015] In a preferred embodiment of the present invention, the mass fraction of the potassium hydroxide solution in step a1 is 8-12%.

[0016] In a preferred embodiment of the present invention, the ratio of the mixed bacterial strain and LB culture medium in step a2 is 0.8-1g:10g.

[0017] In a preferred embodiment of the present invention, the mixed bacterial strains in step a2 are a mixture of *Hydrocotyle vulgaris*, *Hydrocotyle spp.*, *Hydrocotyle oleifera*, and *Hydrocotyle spp.* in a mass ratio of 1:1:1:1.5-2:1.5-2.

[0018] In a preferred embodiment of the present invention, the ratio of sodium alginate, chitosan, acetic acid solution, reed charcoal, mixed bacterial solution, calcium chloride solution and octyl glycidyl ether in step a3 is 1.5-2.5g: 2-3g: 50-55mL: 0.5-0.9g: 30-60mL: 120-150mL: 0.9-1.7g.

[0019] In a preferred embodiment of the present invention, the chitosan in step a3 has a molecular weight of 100 kDa and a degree of deacetylation of 95%; the acetic acid solution has a mass fraction of 1-2%; and the calcium chloride solution has a mass fraction of 5-6%.

[0020] In a preferred embodiment of the present invention, the modified composite particles are prepared by the following steps:

[0021] Step b1: Add sodium tungstate dihydrate and deionized water to a three-necked flask equipped with a stirrer, thermometer, and constant pressure dropping funnel. Stir the reaction for 10-15 min at a temperature of 20-25℃ and a stirring rate of 200-300 r / min. Then, add hydrochloric acid solution dropwise while stirring, controlling the dropping rate to 1-3 drops / s. After the addition is complete, continue stirring for 20-30 min. Then, add ammonium oxalate and continue stirring for 10-15 min. Then, add titanium dioxide and continue stirring for 30-50 min. Then, transfer the mixture to a reaction vessel and hydrothermally react at a temperature of 120-180℃ for 10-12 h. After the reaction is complete, cool the reaction product to room temperature, then centrifuge. Wash the precipitate 3-5 times with distilled water, then place it in a vacuum drying oven and dry it at a temperature of 60-65℃ for 3-5 h to obtain composite particles.

[0022] Step b2: Add silane coupling agent KH-560, acetic acid, and ethanol solution to a three-necked flask equipped with a stirrer and thermometer. Stir the reaction at 20-25℃ and 200-300 r / min for 30-50 min. Then add the composite particles and continue stirring for 10-20 min. After that, raise the temperature to 70-80℃ and continue stirring for 5-7 h. After the reaction is complete, cool the reaction product to room temperature, centrifuge it, and then wash the precipitate 3-5 times with anhydrous ethanol. Then place it in a vacuum drying oven and dry it at 50-55℃ for 3-4 h to obtain the modified composite particles.

[0023] In a preferred embodiment of the present invention, the ratio of sodium tungstate dihydrate, deionized water, hydrochloric acid solution, ammonium oxalate and titanium dioxide in step b1 is 0.7-1.3g: 50-55mL: 15-20mL: 0.6-0.7g: 1g.

[0024] In a preferred embodiment of the present invention, the molar concentration of the hydrochloric acid solution in step b1 is 3-5 mol / L; the titanium dioxide is NTR-606 titanium dioxide.

[0025] In a preferred embodiment of the present invention, the ratio of the amount of silane coupling agent KH-560, acetic acid, ethanol solution and composite particles in step b2 is 0.5-1.9g: 2-3mL: 60-70mL: 5g.

[0026] In a preferred embodiment of the present invention, the volume fraction of the ethanol solution in step b2 is 80-90%.

[0027] As a preferred embodiment of the present invention, a method for preparing a microbial remediation agent for the degradation of marine oil pollution includes the following steps:

[0028] Step 1: Weigh out the following components according to weight: 35-55 parts of hydrophobic microsphere-coated carbon immobilizing agent, 12-18 parts of modified composite particles, 5-7 parts of ammonium nitrate, 2-3 parts of dipotassium hydrogen phosphate, 1-2 parts of ferrous sulfate, 5 parts of polysorbate-803, 2-4 parts of xanthan gum, 20-25 parts of deionized water, and 40-50 parts of anhydrous ethanol. Set aside for later use.

[0029] Step 2: Add the hydrophobic microsphere-coated carbon immobilizing agent, modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer. Mix for 30-60 minutes at a temperature of 20-25℃ and a stirring rate of 500-1000 r / min. Then dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This invention discloses a microbial remediation agent for the degradation of marine oil pollution and its preparation method. The agent is prepared by mixing hydrophobic microspheres coated with carbon-immobilized bacteria, modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol, followed by spray drying. This microbial remediation agent combines multiple microbial degradation and photocatalytic degradation mechanisms, significantly improving the degradation effect and efficiency of oil pollutants. It also exhibits good stability, extending its service life. Furthermore, the selected materials possess excellent environmental performance, preventing secondary pollution of the marine environment and demonstrating broad application prospects.

[0032] In the preparation of the microbial remediation agent, a hydrophobic microsphere-coated carbon immobilization agent was first prepared. Reed straw was activated with potassium hydroxide and then calcined to form biochar, yielding reed char. Subsequently, various bacterial strains with good degradation capabilities for petroleum hydrocarbons were cultured to obtain a mixed bacterial solution. Then, using sodium alginate and chitosan as the coating layer and reed char as the carrier, the reed char adsorbed and loaded various bacterial strains. A dense sodium alginate-chitosan coating layer was then formed through calcium chloride cross-linking. This layer was then modified through a chemical reaction with the epoxy groups on octyl glycidyl ether, introducing a large number of long carbon chains into the coating layer, resulting in hydrophobic microsphere-coated carbon. Immobilized microbial agents: By adsorbing and coating multiple strains of bacteria, active microorganisms are firmly immobilized, effectively resisting seawater erosion. This not only prevents the loss of active microorganisms but also protects them from external toxins, maintaining their activity and achieving long-term repair effects. It also provides a stable living environment for microorganisms, enabling them to multiply rapidly and produce enzymes, thereby enhancing their tolerance and degradation capabilities. Furthermore, the long carbon chains achieve lipophilicity, adsorbing petroleum pollutants and enriching them on the surface of the hydrophobic microsphere-coated carbon-immobilized microbial agent. This improves the contact efficiency between microorganisms and petroleum pollutants, achieving highly efficient degradation of marine oil pollution.

[0033] In the process of preparing the microbial remediation agent, a modified composite particle was also prepared. Using sodium tungstate dihydrate as a raw material, tungsten trioxide was formed on the surface of titanium dioxide using a hydrothermal synthesis method to obtain the composite particle. Then, the composite particle was modified using the silane coupling agent KH-560. The siloxane on the silane coupling agent KH-560 was hydrolyzed to form silanol, which was then grafted onto the surface of the composite particle. Simultaneously, epoxy groups were introduced, resulting in the modified composite particle. This modified composite particle utilizes the epoxy groups to enable it to react with the amino and hydroxyl groups on the sodium alginate-chitosan coating layer, thereby grafting onto the surface of the hydrophobic microsphere-coated carbon-fixed bacterial agent. This allows for sufficient contact with the petroleum pollutants enriched on the surface of the hydrophobic microsphere-coated carbon-fixed bacterial agent. Furthermore, both titanium dioxide and tungsten trioxide possess excellent photocatalytic capabilities; their combination enhances the photocatalytic performance, thus enabling the efficient photocatalytic degradation of petroleum pollutants. Attached Figure Description

[0034] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0035] Figure 1 This is a schematic diagram illustrating the degradation effect of the microbial remediation agents of Examples 1-3 and Comparative Examples 1-4 on dibenzothiophene.

[0036] Figure 2 This is a schematic diagram illustrating the degradation effect of the microbial remediation agents of Examples 1-3 and Comparative Examples 1-4 on anthracene.

[0037] Figure 3This is a schematic diagram illustrating the degradation effect of the microbial remediation agents of Examples 1-3 and Comparative Examples 1-4 on pyrene. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1:

[0040] This embodiment describes a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0041] Step S1: Crush 3g of dried reed stalks through an 80-mesh sieve, then soak them in 15mL of 8% potassium hydroxide solution for 10h, then place them in a vacuum drying oven and dry them at 80℃ for 2h, then place them in a tube furnace and purge them with nitrogen, then calcine them at 450℃ for 40min at a heating rate of 5℃ / min, then calcine them at 650℃ for 2h, and then cool them with the furnace to obtain reed charcoal;

[0042] Step S2: Inoculate 0.8g of the mixed bacterial culture into 10g of LB medium and incubate at 35℃ and 110r / min for 48h. Then centrifuge and dilute with physiological saline to 1×10⁻⁶. 9 A mixed bacterial culture of CFU / mL; the mixed bacterial strains are a mixture of *Hydrocotyle vulgaris*, *Hydrocotyle spp.*, *Hydrocotyle oleracea*, and *Hydrocotyle spp.* in a mass ratio of 1:1:1:1.5:1.5.

[0043] Step S3: Add 1.5g sodium alginate, 2g chitosan with a molecular weight of 100kDa and a degree of deacetylation of 95%, and 50mL of 1% acetic acid solution to a three-necked flask equipped with a stirrer and thermometer. Stir and react for 20min at 25℃ and a stirring rate of 200r / min. Then add 0.5g reed charcoal and 30mL mixed bacterial solution and continue stirring and reacting for 2h. Then add dropwise to 120mL of 5% calcium chloride solution while stirring, controlling the dropping rate to 1 drop / s. After the addition is complete, continue stirring and reacting for 3h. Then add 0.9g octyl glycidyl ether and continue stirring and reacting for 1h. After the reaction is complete, vacuum filter the reaction product. Wash the filter cake twice with anhydrous ethanol and distilled water, and then freeze-dry it under vacuum to obtain hydrophobic microsphere-coated carbon-fixed bacterial agent.

[0044] Step S4: Add 0.7g of sodium tungstate dihydrate and 50mL of deionized water to a three-necked flask equipped with a stirrer, thermometer, and constant pressure dropping funnel. Stir the reaction at 20℃ and 200r / min for 10min. Then, while stirring, add 15mL of 3mol / L hydrochloric acid solution dropwise at a rate of 1 drop / s. After the addition is complete, continue stirring for 20min. Then, add 0.6g of ammonium oxalate and continue stirring for 10min. Then, add 1g of titanium dioxide NTR-606 and continue stirring for 30min. Then, transfer the mixture to a reaction vessel and hydrothermally react at 120℃ for 10h. After the reaction is complete, cool the reaction product to room temperature, centrifuge, wash the precipitate three times with distilled water, and then place it in a vacuum drying oven and dry at 60℃ for 3h to obtain composite particles.

[0045] Step S5: Add 0.5g of silane coupling agent KH-560, 2mL of acetic acid, and 60mL of 80% ethanol solution to a three-necked flask equipped with a stirrer and thermometer. Stir the reaction at 20℃ and 200r / min for 30min. Then add 5g of composite particles and continue stirring for 10min. Then raise the temperature to 70℃ and continue stirring for 5h. After the reaction is completed, cool the reaction product to room temperature, centrifuge, wash the precipitate three times with anhydrous ethanol, and then place it in a vacuum drying oven and dry at 50℃ for 3h to obtain modified composite particles.

[0046] Step S6: Weigh out 35 parts of hydrophobic microsphere-coated carbon fixative, 12 parts of modified composite particles, 5 parts of ammonium nitrate, 2 parts of dipotassium hydrogen phosphate, 1 part of ferrous sulfate, 3 parts of polysorbate-80, 2 parts of xanthan gum, 20 parts of deionized water, and 40 parts of anhydrous ethanol according to the following weight proportions, and set aside for later use.

[0047] Step S7: Add the hydrophobic microsphere-coated carbon immobilizing agent, modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer and mix for 30 minutes at a temperature of 20°C and a stirring rate of 500 r / min. Then, dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0048] Example 2:

[0049] This embodiment describes a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0050] Step S1: Crush 3g of dried reed stalks through a 90-mesh sieve, then soak them in 18mL of 10% potassium hydroxide solution for 11h. After that, place them in a vacuum drying oven and dry them at 82℃ for 2.5h. Then place them in a tube furnace and purge them with nitrogen. Then calcine them at 455℃ for 45min at a heating rate of 6℃ / min. After that, calcine them at 680℃ for 2.5h. After that, cool them with the furnace to obtain reed charcoal.

[0051] Step S2: Inoculate 0.9g of the mixed bacterial culture into 10g of LB medium and incubate at 36℃ and 120r / min for 60h. Then centrifuge and dilute with physiological saline to 1×10⁻⁶. 9 A mixed bacterial culture of CFU / mL; the mixed bacterial strains are a mixture of *Hydrocotyle vulgaris*, *Hydrocotyle spp.*, *Hydrocotyle oleracea*, and *Hydrocotyle spp.* in a mass ratio of 1:1:1:1.8:1.8;

[0052] Step S3: Add 2g sodium alginate, 2.5g chitosan with a molecular weight of 100kDa and a degree of deacetylation of 95%, and 52mL of acetic acid solution with a mass fraction of 1.5% to a three-necked flask equipped with a stirrer and thermometer. Stir and react for 25min at a temperature of 28℃ and a stirring rate of 250r / min. Then add 0.7g reed charcoal and 45mL mixed bacterial solution and continue stirring and reacting for 2.5h. Then add dropwise to 135mL of calcium chloride solution with a mass fraction of 5.5% while stirring, controlling the dropping rate to 2 drops / s. After the addition is complete, continue stirring and reacting for 4h. Then add 1.3g octyl glycidyl ether and continue stirring and reacting for 1.5h. After the reaction is complete, filter the reaction product under vacuum. Wash the filter cake twice with anhydrous ethanol and distilled water, and then freeze-dry under vacuum to obtain hydrophobic microsphere-coated carbon-fixed bacterial agent.

[0053] Step S4: Add 1g of sodium tungstate dihydrate and 52mL of deionized water to a three-necked flask equipped with a stirrer, thermometer, and constant pressure dropping funnel. Stir the reaction at 22℃ and 250r / min for 12min. Then, while stirring, add 18mL of 4mol / L hydrochloric acid solution dropwise at a rate of 2 drops / s. After the addition is complete, continue stirring for 25min. Then, add 0.65g of ammonium oxalate and continue stirring for 12min. Then, add 1g of titanium dioxide NTR-606 and continue stirring for 40min. Then, transfer the mixture to a reaction vessel and hydrothermally react at 150℃ for 11h. After the reaction is complete, cool the reaction product to room temperature, centrifuge, wash the precipitate four times with distilled water, and then place it in a vacuum drying oven and dry at 62℃ for 4h to obtain composite particles.

[0054] Step S5: Add 1.2g of silane coupling agent KH-560, 2.5mL of acetic acid, and 65mL of 85% ethanol solution to a three-necked flask equipped with a stirrer and thermometer. Stir the reaction at 22℃ and 250r / min for 40min. Then add 5g of composite particles and continue stirring for 15min. Then raise the temperature to 75℃ and continue stirring for 6h. After the reaction is completed, cool the reaction product to room temperature, centrifuge, wash the precipitate four times with anhydrous ethanol, and then place it in a vacuum drying oven and dry at 52℃ for 3.5h to obtain modified composite particles.

[0055] Step S6: Weigh out 40 parts by weight of hydrophobic microsphere-coated carbon immobilizing agent, 15 parts by weight of modified composite particles, 6 parts by weight of ammonium nitrate, 2.5 parts by weight of dipotassium hydrogen phosphate, 1.5 parts by weight of ferrous sulfate, 4 parts by weight of polysorbate-804, 3 parts by weight of xanthan gum, 22 parts by weight of deionized water and 45 parts by weight of anhydrous ethanol, and set aside.

[0056] Step S7: Add the hydrophobic microsphere-coated carbon immobilizing agent, modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer and mix for 45 minutes at a temperature of 22°C and a stirring rate of 800 r / min. Then, dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0057] Example 3:

[0058] This embodiment describes a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0059] Step S1: Crush 3g of dried reed stalks through a 100-mesh sieve, then soak them in 20mL of 12% potassium hydroxide solution for 12h. After that, place them in a vacuum drying oven and dry them at 85℃ for 3h. Then place them in a tube furnace and purge them with nitrogen. Then calcine them at 460℃ for 50min at a heating rate of 7℃ / min. After that, calcine them at 700℃ for 3h. After that, cool them with the furnace to obtain reed charcoal.

[0060] Step S2: Inoculate 1g of the mixed bacterial culture into 10g of LB medium and incubate at 37℃ and 130r / min for 72h. Then centrifuge and dilute with physiological saline to 1×10⁻⁶. 9 A mixed bacterial culture of CFU / mL; the mixed bacterial species is a mixture of *Hydrocotyle vulgaris*, *Hydrocotyle spp.*, *Hydrocotyle oleifera*, and *Hydrocotyle spp.* in a mass ratio of 1:1:1:2:2;

[0061] Step S3: Add 2.5g sodium alginate, 3g chitosan with a molecular weight of 100kDa and a degree of deacetylation of 95%, and 55mL of 2% acetic acid solution to a three-necked flask equipped with a stirrer and thermometer. Stir and react for 30min at 30℃ and a stirring rate of 300r / min. Then add 0.9g reed charcoal and 60mL mixed bacterial solution and continue stirring and reacting for 3h. Then add dropwise to 150mL of 6% calcium chloride solution while stirring, controlling the dropping rate to 3 drops / s. After the addition is complete, continue stirring and reacting for 5h. Then add 1.7g octyl glycidyl ether and continue stirring and reacting for 2h. After the reaction is complete, vacuum filter the reaction product. Wash the filter cake three times with anhydrous ethanol and distilled water, and then freeze-dry it under vacuum to obtain hydrophobic microsphere-coated carbon-fixed bacterial agent.

[0062] Step S4: Add 1.3g of sodium tungstate dihydrate and 55mL of deionized water to a three-necked flask equipped with a stirrer, thermometer, and constant pressure dropping funnel. Stir the reaction at 25℃ and 300r / min for 15min. Then, while stirring, add 20mL of 5mol / L hydrochloric acid solution dropwise at a rate of 3 drops / s. After the addition is complete, continue stirring for 30min. Then, add 0.7g of ammonium oxalate and continue stirring for 15min. Then, add 1g of titanium dioxide NTR-606 and continue stirring for 50min. Then, transfer the mixture to a reaction vessel and hydrothermally react at 180℃ for 12h. After the reaction is complete, cool the reaction product to room temperature, centrifuge, wash the precipitate 5 times with distilled water, and then place it in a vacuum drying oven and dry at 65℃ for 5h to obtain composite particles.

[0063] Step S5: Add 1.9g of silane coupling agent KH-560, 3mL of acetic acid, and 70mL of 90% ethanol solution to a three-necked flask equipped with a stirrer and thermometer. Stir the reaction at 25℃ and 300r / min for 50min. Then add 5g of composite particles and continue stirring for 20min. Then raise the temperature to 80℃ and continue stirring for 7h. After the reaction is completed, cool the reaction product to room temperature, centrifuge, wash the precipitate 5 times with anhydrous ethanol, and then place it in a vacuum drying oven and dry at 55℃ for 4h to obtain modified composite particles.

[0064] Step S6: Weigh out 55 parts of hydrophobic microsphere-coated carbon fixative, 18 parts of modified composite particles, 7 parts of ammonium nitrate, 3 parts of dipotassium hydrogen phosphate, 2 parts of ferrous sulfate, 5 parts of polysorbate-80, 4 parts of xanthan gum, 25 parts of deionized water, and 50 parts of anhydrous ethanol according to the following weight proportions, and set aside for later use.

[0065] Step S7: Add the hydrophobic microsphere-coated carbon immobilizing agent, modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer and mix for 60 minutes at a temperature of 25°C and a stirring rate of 1000 r / min. Then, dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0066] Comparative Example 1:

[0067] This comparative example illustrates a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0068] Step S1: Weigh out 7 parts ammonium nitrate, 3 parts dipotassium hydrogen phosphate, 2 parts ferrous sulfate, 5 parts polysorbate-80, 4 parts xanthan gum, 25 parts deionized water, and 50 parts anhydrous ethanol according to the following weight proportions, and set aside.

[0069] Step S2: Add ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water and anhydrous ethanol to a mixer and mix for 60 minutes at a temperature of 25°C and a stirring rate of 1000 r / min. Then dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0070] Comparative Example 2:

[0071] This comparative example illustrates a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0072] Step S1: Crush 3g of dried reed stalks through a 100-mesh sieve, then soak them in 20mL of 12% potassium hydroxide solution for 12h. After that, place them in a vacuum drying oven and dry them at 85℃ for 3h. Then place them in a tube furnace and purge them with nitrogen. Then calcine them at 460℃ for 50min at a heating rate of 7℃ / min. After that, calcine them at 700℃ for 3h. After that, cool them with the furnace to obtain reed charcoal.

[0073] Step S2: Inoculate 1g of the mixed bacterial culture into 10g of LB medium and incubate at 37℃ and 130r / min for 72h. Then centrifuge and dilute with physiological saline to 1×10⁻⁶. 9 A mixed bacterial culture of CFU / mL; the mixed bacterial species is a mixture of *Hydrocotyle vulgaris*, *Hydrocotyle spp.*, *Hydrocotyle oleifera*, and *Hydrocotyle spp.* in a mass ratio of 1:1:1:2:2;

[0074] Step S3: Add 2.5g sodium alginate, 3g chitosan with a molecular weight of 100kDa and a degree of deacetylation of 95%, and 55mL of 2% acetic acid solution to a three-necked flask equipped with a stirrer and thermometer. Stir and react for 30min at 30℃ and a stirring rate of 300r / min. Then add 0.9g reed charcoal and 60mL mixed bacterial solution and continue stirring and reacting for 3h. Then add dropwise to 150mL of 6% calcium chloride solution while stirring, controlling the dropping rate to 3 drops / s. After the addition is complete, continue stirring and reacting for 5h. Then add 1.7g octyl glycidyl ether and continue stirring and reacting for 2h. After the reaction is complete, vacuum filter the reaction product. Wash the filter cake three times with anhydrous ethanol and distilled water, and then freeze-dry it under vacuum to obtain hydrophobic microsphere-coated carbon-fixed bacterial agent.

[0075] Step S4: Weigh out 55 parts of hydrophobic microsphere-coated carbon immobilizing agent, 7 parts of ammonium nitrate, 3 parts of dipotassium hydrogen phosphate, 2 parts of ferrous sulfate, 5 parts of polysorbate-80, 4 parts of xanthan gum, 25 parts of deionized water, and 50 parts of anhydrous ethanol according to the following weight proportions, and set aside for later use.

[0076] Step S5: Add hydrophobic microspheres coated with carbon immobilizing agent, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water and anhydrous ethanol to a mixer and mix for 60 minutes at 25°C and 1000 r / min. Then dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0077] Comparative Example 3:

[0078] This comparative example illustrates a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0079] Step S1: Add 1.3g of sodium tungstate dihydrate and 55mL of deionized water to a three-necked flask equipped with a stirrer, thermometer, and constant pressure dropping funnel. Stir the reaction at 25℃ and 300r / min for 15min. Then, while stirring, add 20mL of 5mol / L hydrochloric acid solution dropwise at a rate of 3 drops / s. After the addition is complete, continue stirring for 30min. Then, add 0.7g of ammonium oxalate and continue stirring for 15min. Then, add 1g of titanium dioxide NTR-606 and continue stirring for 50min. Then, transfer the mixture to a reaction vessel and hydrothermally react at 180℃ for 12h. After the reaction is complete, cool the reaction product to room temperature, centrifuge, wash the precipitate 5 times with distilled water, and then place it in a vacuum drying oven and dry at 65℃ for 5h to obtain composite particles.

[0080] Step S2: 1.9g of silane coupling agent KH-560, 3mL of acetic acid, and 70mL of 90% ethanol solution were added to a three-necked flask equipped with a stirrer and thermometer. The mixture was stirred at 25℃ and 300r / min for 50min. Then, 5g of composite particles were added and the mixture was stirred for another 20min. The mixture was then heated to 80℃ and stirred for another 7h. After the reaction was completed, the reaction product was cooled to room temperature and centrifuged. The precipitate was then washed five times with anhydrous ethanol and placed in a vacuum drying oven at 55℃ for 4h to obtain the modified composite particles.

[0081] Step S3: Weigh out 18 parts of modified composite particles, 7 parts of ammonium nitrate, 3 parts of dipotassium hydrogen phosphate, 2 parts of ferrous sulfate, 5 parts of polysorbate-80, 4 parts of xanthan gum, 25 parts of deionized water and 50 parts of anhydrous ethanol according to the following weight proportions, and set aside.

[0082] Step S4: Add the modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer and mix for 60 minutes at a temperature of 25°C and a stirring rate of 1000 r / min. Then, dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0083] Comparative Example 4:

[0084] This comparative example illustrates a method for preparing a microbial remediation agent for the degradation of marine oil pollution, comprising the following steps:

[0085] Step S1: Weigh out 55 parts by weight of mixed bacterial culture, 18 parts by weight of titanium dioxide NTR-60618 parts, 7 parts by weight of ammonium nitrate, 3 parts by weight of dipotassium hydrogen phosphate, 2 parts by weight of ferrous sulfate, 5 parts by weight of polysorbate-80, 4 parts by weight of xanthan gum, 25 parts by weight of deionized water, and 50 parts by weight of anhydrous ethanol, and set aside; the mixed bacterial culture is a mixture of sea spirulina, sea bacillus, cyclohexylella oleracea, spirochetes and alkyl consuming bacteria in a mass ratio of 1:1:1:2:2;

[0086] Step S2: Add the mixed bacterial culture, titanium dioxide NTR-606, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer and mix for 60 minutes at a temperature of 25°C and a stirring rate of 1000 r / min. Then dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

[0087] Natural seawater taken from near Xiaomaidao Island in Qingdao was filtered through a 0.22μm water film before use to remove small particles and most bacteria. Then, 10mg of dibenzothiophene, 10mg of anthracene, or 10mg of pyrene was added to 100mL of natural seawater. After stirring evenly, 2g of the microbial remediation agent for marine oil pollution degradation from Examples 1-3 and Comparative Examples 1-4 was added respectively. The mixture was cultured for 30 days at a temperature of 30℃ and a stirring rate of 160r / min. During this period, xenon lamps (PLS-SEXE300) were used to simulate sunlight for illumination during four time periods each day: 8:00-9:00, 10:00-11:00, 12:00-13:00, and 14:00-15:00.

[0088] The concentrations A0 and A1 of dibenzothiophene, anthracene, or pyrene during the initial and intermediate stages of the reaction were determined using a UV-Vis spectrophotometer, in mg / L. The degradation rate η was calculated using η = (A0 - A1) / A0 × 100%. The test results are as follows: Figures 1-3 As shown.

[0089] See Figures 1-3 As shown, based on the comparison between Examples 1-3 and Comparative Examples 1-4, it can be seen that adding hydrophobic microsphere-coated carbon immobilizing agent and modified composite particles can significantly improve the degradation effect of microbial remediation agent on dibenzothiophene, anthracene and pyrene, thus indicating that microbial remediation agent containing hydrophobic microsphere-coated carbon immobilizing agent and modified composite particles has excellent marine oil pollution degradation ability.

[0090] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0091] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in this application, they should all fall within the protection scope of the present invention.

Claims

1. A microbial remediation agent for the degradation of marine oil pollution, characterized in that, Includes the following components by weight: The mixture consists of 35-55 parts of hydrophobic microspheres coated with carbon-fixed bacterial agent, 12-18 parts of modified composite particles, 5-7 parts of ammonium nitrate, 2-3 parts of dipotassium hydrogen phosphate, 1-2 parts of ferrous sulfate, 3-5 parts of polysorbate-80, 2-4 parts of xanthan gum, 20-25 parts of deionized water, and 40-50 parts of anhydrous ethanol. The hydrophobic microsphere-coated carbon-immobilized bacterial agent is prepared by the following steps: Step a1: Crush and sieve the dried reed stalks, then soak them in potassium hydroxide solution, then dry and calcine them, and then cool them in the furnace to obtain reed charcoal; Step a2: Inoculate the mixed bacterial strain into LB medium and culture, then centrifuge and dilute with physiological saline to obtain a mixed bacterial solution; the mixed bacterial strain is a mixture of *Hylocereus undatus*, *Hylocereus piscyl*, *Hylocereus piscyl*, and *Hylocereus piscyl* in a mass ratio of 1:1:1:1.5-2:1.5-2. Step a3: Sodium alginate, chitosan and acetic acid solution are stirred and reacted. Then, reed charcoal and mixed bacterial solution are added and stirred and reacted. Then, calcium chloride solution is added dropwise while stirring. After the addition is completed, stirring and reacting are continued. Octyl glycidyl ether is added and stirring and reacting are continued. After the reaction is completed, the reaction product is vacuum filtered. The filter cake is washed and freeze-dried under vacuum to obtain hydrophobic microsphere-coated carbon immobilized bacterial agent. The modified composite particles are prepared by the following steps: Step b1: Sodium tungstate dihydrate and deionized water are stirred and reacted. Then, hydrochloric acid solution is added dropwise while stirring. After the addition is complete, ammonium oxalate and titanium dioxide are added in sequence and the reaction is continued with stirring. Then, a hydrothermal reaction is carried out. After the reaction is completed, the reaction product is cooled, centrifuged, and the precipitate is washed and dried to obtain composite particles. Step b2: The silane coupling agent KH-560, acetic acid and ethanol solution were stirred and reacted. Then the composite particles were added and the reaction was continued. After the reaction was completed, the reaction product was cooled, centrifuged, and the precipitate was washed and dried to obtain the modified composite particles.

2. The microbial remediation agent for the degradation of marine oil pollution according to claim 1, characterized in that, In step a1, the ratio of reed straw to potassium hydroxide solution is 3g:15-20mL; the mass fraction of potassium hydroxide solution is 8-12%.

3. The microbial remediation agent for the degradation of marine oil pollution according to claim 1, characterized in that, The ratio of the mixed bacterial strain and LB culture medium used in step a2 is 0.8-1g:10g.

4. The microbial remediation agent for the degradation of marine oil pollution according to claim 1, characterized in that, In step a3, the ratio of sodium alginate, chitosan, acetic acid solution, reed charcoal, mixed bacterial solution, calcium chloride solution, and octyl glycidyl ether is 1.5-2.5g: 2-3g: 50-55mL: 0.5-0.9g: 30-60mL: 120-150mL: 0.9-1.7g; the chitosan has a molecular weight of 100kDa and a degree of deacetylation of 95%; the acetic acid solution has a mass fraction of 1-2%; and the calcium chloride solution has a mass fraction of 5-6%.

5. A microbial remediation agent for the degradation of marine oil pollution according to claim 1, characterized in that, In step b1, the ratio of sodium tungstate dihydrate, deionized water, hydrochloric acid solution, ammonium oxalate, and titanium dioxide is 0.7-1.3g: 50-55mL: 15-20mL: 0.6-0.7g: 1g; the molar concentration of the hydrochloric acid solution is 3-5mol / L; and the titanium dioxide is NTR-606 titanium dioxide.

6. The microbial remediation agent for the degradation of marine oil pollution according to claim 1, characterized in that, In step b2, the ratio of the silane coupling agent KH-560, acetic acid, ethanol solution, and composite particles is 0.5-1.9g: 2-3mL: 60-70mL: 5g; the volume fraction of the ethanol solution is 80-90%.

7. A method for preparing a microbial remediation agent for the degradation of marine oil pollution, characterized in that, The preparation of the microbial remediation agent for marine oil pollution degradation as described in any one of claims 1-6 includes the following steps: Step 1: Weigh out the following components by weight: 35-55 parts of hydrophobic microsphere-coated carbon immobilizing agent, 12-18 parts of modified composite particles, 5-7 parts of ammonium nitrate, 2-3 parts of dipotassium hydrogen phosphate, 1-2 parts of ferrous sulfate, 3-5 parts of polysorbate-80, 2-4 parts of xanthan gum, 20-25 parts of deionized water, and 40-50 parts of anhydrous ethanol. Set aside for later use. Step 2: Add the hydrophobic microsphere-coated carbon immobilizing agent, modified composite particles, ammonium nitrate, dipotassium hydrogen phosphate, ferrous sulfate, polysorbate-80, xanthan gum, deionized water, and anhydrous ethanol to a mixer. Mix for 30-60 minutes at a temperature of 20-25℃ and a stirring rate of 500-1000 r / min. Then dry the mixture in a spray dryer to obtain a microbial remediation agent for the degradation of marine oil pollution with an average particle size of 1 mm.

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