CN104004745A - Immobilized microorganism oil-spilling repairing agent and preparation method - Google Patents

Abstract

The invention discloses an immobilized microbial oil spill restoration agent, which is characterized in that: the concentration of microorganisms embedded in the immobilized microbial oil spill restoration agent is 5×10 ⁷ to 2×10 ⁸ cell/g, and the carrier group Ingredients include mussel shells, sodium alginate, CaCl ₂ . The immobilized microbial oil spill restoration agent of the present invention can degrade petroleum hydrocarbons up to 82-94% in an environment with a salinity of 5-55‰, a temperature of 10-30°C, and a pH of 6.5-9.0. In comparison, its degradation rate is increased by 39%-47%, and its tolerance to the environment is enhanced.

Description

A kind of immobilized microbial oil spill restoration agent and preparation method thereof

technical field

The invention relates to a bioremediation technology for petroleum product pollution. the

Background technique

With the development of offshore oil development and marine transportation, oil spill accidents caused by blowouts, oil spills from transport ships, shipwrecks, and oil pipeline leaks have occurred frequently. After the oil spill accident, although some surface oil can be removed by physical methods (such as fence recovery, etc.) and chemical methods (spraying dispersant, etc.), the remaining petroleum hydrocarbons remain in the marine environment for a long time, which is harmful to marine ecosystems and Human health will cause huge persistent damage (Pelletier E, Delille D, Delille B. Crude oil bioremediation in sub-Antarctic intertidal sediments: chemistry and toxicity of oiled residues [J]. Mar. Environ. Res., 2004, 57( 4): 311-327.). the

At present, the biological remediation technology of oil pollution is mainly based on microbial remediation (Xia Wenxiang, Lin Haitao, Zhang Ying, etc. Pollution control technology of marine oil spills [J]. Journal of Qingdao Institute of Architecture and Engineering, 2004, 25(1): 54 -57.). Researchers at home and abroad have found that there are more than 200 kinds of hydrocarbon-degrading microorganisms growing in seawater and freshwater, including bacteria, fungi and algae, and their density and reproduction speed are closely related to the degree of oil pollution in water areas (Fingas M. The Basic of Oil Spill Clean up (2nd edition) [M]. USA: Lewis Publishers, 2001, 39-50.). Microbial treatment technology is an internationally recognized new technology for efficient repair of oil pollution. Based on the fact that microorganisms can adapt to various complex ecological environments, their reproductive and metabolic capabilities are extremely strong, they can quickly degrade various toxic substances in oil, and they are cheap and environmentally friendly. Friendly and many other advantages (Harayama S, Kasai Y, Hara A. Microbial communities in oil-contaminated seawater [J]. Curr. Opin. Biotechnol., 2004, 15(3): 205-214.). Using this technology to deal with oil spills that cannot be removed by physical and chemical methods is the best way to restore the ecological environment, and it has great scientific significance and application value for protecting my country's marine ecological environment and maintaining the sustainable development of marine resources. the

Since the 1970s, adding appropriate microorganisms to control oil pollution has been considered a powerful strategy (Atlas R M.Petroleum biodegradation and oil spill bioremediation[J].Mar.Pollut.Bull., 1995, 31(4- 12): 178-182.), successful examples in field tests such as: using Alpha BioSea to deal with the crude oil spill on the coast of Texas in 1990, and using Terra-zyme (TM) to deal with the crude oil spill at Nakhodka port in Japan in 2000 ( Maki H, Hirayama N, Hiwatari T, et al. Crude oil bioremediation field experiment in the sea of Japan[J].Mar.Pollut.Bull.,2003,47(1-6):74-77.), 1989 From Xia to 1991, the application of nutrients and high-efficiency hydrocarbon-degrading bacteria was used to treat the pollution caused by oil tanker leakage in Exxon Valdez Prince Bay, Alaska, and achieved very obvious results (Atlad R M. Bioremediation of Petroleum Pollutants [J]. Int. Biodeter. Biodegrad., 1995, 35(3):317-327.). the

However, in the process of microbial remediation of marine oil spills, there are problems such as serious loss of effective strains and a sharp drop in remediation efficiency (De-Bashan L E, Bashan Y. Immobilized microalgae for removing pollutants: Review of practical aspects[J].Bioresour . Technol., 2010, 101(6): 1611-1627.). Therefore, aiming at the unique treatment environment of marine oil spill restoration, this patent adopts the microorganism immobilization technology. Compared with other bioremediation technologies, immobilized microorganism technology can maintain high density and high activity of microorganisms, reduce or eliminate the loss of microorganisms, high treatment efficiency, strong tolerance to the environment, easy control of the reaction, and can effectively improve the efficiency of the target area. Remediation efficiency of marine oil spill pollution microbial agents (Takeno K, Yamaoka Y, Sasaki K. Treatment of oil-containing sewage wastewater using immobilized photosynthetic bacteria [J]. World J. Microbiol. Biotechnol., 2005, 21 (8-9 ):1385-1391.). the

As a kind of immobilization method, embedment immobilization technology, compared with adsorption method, cross-linking method, covalent bonding method and other immobilization methods, because the preparation method is simple and easy to operate, the reaction conditions are mild and easy to realize, and the microorganisms are not easy to leak. Good stability and reusability, and high microbial activity and cell capacity (Bao Mutai, Gong Yuanjiao, Li Yiming. The role of immobilized microorganisms in degrading oily sewage[J]. Journal of Wuhan University (Science Edition), 2010 , 56(1):109-144.). Sodium alginate-CaCl ₂ embedding and polyvinyl alcohol-H ₃ BO ₃ embedding are the most commonly used embedding and immobilization methods (Massalha N, Basheer S, Sabbah I. Effect of adsorption and bead size of immobilized biomass on the rate of biodegradation of phenol at high concentration levels[J].Ind.Eng.Chem.Res.,2007,46(21):6820-6824.). Compared with the sodium alginate-CaCl2 embedding method, although the immobilized preparation prepared by polyvinyl alcohol-H ₃ BO ₃ has higher mechanical strength and longer service life, it is difficult for polyvinyl alcohol to form into balls due to the strong agglomeration effect. Moreover, the saturated boric acid solution used for cross-linking polyvinyl alcohol is highly toxic to microorganisms and will significantly reduce the activity of microorganisms after immobilization. Although the sodium alginate-CaCl ₂ embedding method has mild reaction conditions, the embedding material is non-toxic to organisms, and the embedded cells have high activity, the preparation has low mechanical strength and poor mass transfer.

Contents of the invention

The technical problem to be solved in the present invention is to overcome the existing defects, and provide a non-biological toxicity, high microbial activity after curing, high mechanical strength, and good mass transfer properties. Immobilized microbial oil spill restoration agent;

Another object of the present invention is to provide a preparation method of the above-mentioned immobilized microbial oil spill restoration agent. the

The purpose of the present invention is specifically achieved through the following technical solutions:

An immobilized microbial oil spill remediation agent, the concentration of microorganisms embedded in the immobilized microbial oil spill remediation agent is 5×10 ⁷ to 2×10 ⁸ cell/g, and its carrier components include mussel shells, sodium alginate , CaCl ₂ .

As a preferred option, the above-mentioned immobilized microbial oil spill restoration agent, the percentage of each carrier component in the immobilized microbial oil spill restoration agent is: mussel shell 1.2wt%, sodium alginate 6wt%, CaCl ₂ 0.3-0.8wt%, The rest is water.

The preparation method of the above-mentioned immobilized microbial oil spill restoration agent uses sodium alginate-CaCl ₂ -modified mussel shells as a carrier for embedding, and the specific steps are:

1) mussel shell modification treatment;

2) Mixing an appropriate amount of modified mussel shells with the seed bacterial liquid at a mass ratio of 1: (2 to 5) for adsorption until the adsorption is saturated to obtain modified mussel shells for adsorbing bacteria;

3) Add sodium alginate solution to the modified mussel shells adsorbing bacteria obtained in step 2), mix well, inject into _CaCl2 solution with ball forming equipment, disperse into balls, crosslink for 6-18 hours, and obtain immobilized Microbial microspheres.

Preferably, in said step 1), the mussel shell modification treatment steps are as follows:

a. Clean, sieve, dry and crush the raw mussel shells, pass through a 40-80 mesh sieve, and set aside;

b. Weigh the crushed mussel shells in step a, add the citric acid solution with a concentration of 2-5wt%, stir and mix well, and soak the mixed material solution at room temperature for about 1.0-2.0h, wherein the mussel shells The mass ratio with citric acid solution is 1:(1～3);

c. Put the impregnated material liquid into the muffle furnace for activation, take it out and cool it;

d. Crushing the obtained activated sample through a 120-150 mesh sieve to obtain the modified mussel shell. the

Further preferred,

In the step b, the concentration of citric acid solution is 2.5wt%; the mass ratio of mussel shells and citric acid solution is 1:2;

The activation temperature in step c is 300-600° C., the heating rate from room temperature to the required activation temperature is 40-60° C./min, and the temperature is kept for 15-30 minutes. ; The preferred activation conditions are: the activation temperature is 550°C, the heating rate is 50°C/min, and the temperature is kept for 30min. the

Preferably, Bacillus brevis D-1 is used as the petroleum hydrocarbon-degrading bacterium in the seed liquid, and the concentration of the seed liquid is (5-8)×10 ⁹ cell/g, among which, the optimal concentration of the seed liquid is 6× 10 ⁹ cells/g.

Preferably, the concentration of sodium alginate used is 4wt%-8wt%, the optimum concentration is 6wt%; the concentration of _CaCl2 solution is 2.5wt%-3.5wt%, the optimum concentration is 3wt%.

Preferably, in the step 3), the mass ratio of modified mussel shell: sodium alginate is 1.2:6. When injecting, the sodium alginate and the mussel shell carrier are injected into the calcium chloride solution in a spherical shape, and the calcium chloride slowly enters the microspheres, forms calcium alginate with sodium alginate, and is fixed in the microspheres. the

The immobilized microbial microspheres obtained in the step 3) are washed with physiological saline for 2 to 4 times, and the finished product is obtained. the

The concentration of microorganisms embedded in the immobilized microbial oil spill restoration agent prepared by testing is 5×10 ⁷ ~ 2×10 ⁸ cell/g, at a salinity of 5-55‰, a temperature of 10-30°C, and a pH of 6.5 In the environment of ~9.0, the degradation rate of petroleum hydrocarbons can reach 82-94%, compared with free microorganisms, the degradation rate is increased by 39%-47%, and the tolerance to the environment is enhanced.

The immobilized microbial microspheres prepared by the present invention are compared with other immobilized microbial microspheres without modified mussel shells and common inorganic activated carbon, and the comparison results are shown in the following table:

Table 1 Performance comparison of different immobilized microbial microspheres (petroleum hydrocarbon concentration 3g/L)

As can be drawn from Table 1, the immobilized microbial microspheres added with modified mussel shells of the present invention are greatly improved in mechanical strength and mass transfer performance, and the embedded cells have higher activity. The degradation rate can reach 94%. the

Beneficial effects of the present invention:

The immobilized microbial oil spill restoration agent of the present invention adds modified mussel shells to the sodium alginate-calcium chloride embedded immobilization carrier for the first time, which improves the mechanical strength of the preparation, and due to the porous structure of the modified mussel shells, it can be greatly improved. Improve its mass transfer, make nutrients more easily absorbed by microorganisms, and greatly improve the activity and degradation efficiency of microorganisms. Compared with the general adsorbent-activated carbon, the modified mussel shells have more extensive sources of raw materials, lower prices, and better performance of immobilized microorganisms, and the reuse of discarded mussel shells solves environmental problems and realizes waste disposal. Recycling of mussel shells. the

The invention uses sodium alginate-calcium chloride-modified mussel shells as the preparation method of the immobilized microbial oil spill restoration agent embedded and immobilized, and improves the mechanical strength and mass transfer of the preparation by adding the modified mussel shells It has the characteristics of simple process, low cost of raw materials, and mild reaction conditions. The prepared repair agent microorganisms are not easy to leak, have good stability and reusability, and have high microbial activity and cell capacity, and can degrade petroleum hydrocarbons. The rate can reach 82-94%. the

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:

Fig. 1 is a photograph of the appearance of the embedded and immobilized microbial microspheres (diameter 3-5 mm) of the present invention. the

Detailed ways

The preferred embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention. the

Example 1:

A preparation method of an immobilized microbial oil spill restoration agent using sodium alginate-calcium chloride-modified mussel shells as an embedded immobilization material, the specific steps are:

1. Preparation of modified mussel shells

1) Cleaning, sieving, drying and pulverizing the raw mussel shells, passing through an 80-mesh sieve, and setting aside;

2) Weigh the mussel shells crushed above, and add the citric acid solution with a mass fraction of 2.5%, wherein the mass ratio of the mussel shells to the citric acid solution is 1:2, stir and mix, and mix the mixed feed solution in Immerse at room temperature for about 1.5h;

3) Put the impregnated material liquid into a high-temperature muffle furnace, raise it from room temperature to the required activation temperature of 550° C., and keep the temperature at a rate of 50° C./min for 30 minutes. the

4) The activated sample is taken out from the muffle furnace and cooled;

5) Pulverize the obtained activated sample through a 140-mesh sieve to obtain modified mussel shells. the

2. The petroleum hydrocarbon degrading bacteria used

The petroleum hydrocarbon-degrading bacteria used for embedding is Bacillus brevus D-1 screened from a certain marine sample, in an environment with a salinity of 25-35‰, a temperature of 15-25°C, and a pH of 7.5-8.5 The degradation rate of crude oil (concentration 1-5g/L) can reach 35-55%. the

3. Embedding

1) Mix the modified mussel shells with the seed bacteria liquid at a mass ratio of 1:3 for adsorption until the adsorption is saturated, and prepare the modified mussel shells for adsorbing bacteria;

2) Add sterilized sodium alginate solution (concentration of sodium alginate is 6wt%) to the above-mentioned modified mussel shells adsorbing bacteria, wherein the mass ratio of modified mussel shells: sodium alginate is 1.2:6, mix well Afterwards, inject into CaCl _solution (concentration is 3wt%) with sphere-forming equipment and disperse into spheres, crosslink 16h, obtain immobilized microspheres, the mass percentage concentration of modified mussel shells accounted for immobilized microspheres is 1.3% ;

3) Rinse the obtained immobilized microbial microspheres with physiological saline for 2 to 4 times to obtain a finished product, the shape of which is shown in Figure 1;

4) Finally, the prepared immobilized microbial oil spill remediation agent is stored at 0-4°C for future use. the

The concentration of microorganisms embedded in the immobilized microbial oil spill remediation agent prepared by testing is 2×10 ⁸ cell/g, in an environment with a salinity of 5-55‰, a temperature of 10-30°C, and a pH of 6.5-9.0 The degradation rate of petroleum hydrocarbons reaches 94%.

Example 2:

A preparation method of an immobilized microbial oil spill restoration agent using sodium alginate-calcium chloride-modified mussel shells as an embedded immobilization material, the specific steps are:

1. Preparation of modified mussel shells

1) Cleaning, screening, drying and pulverizing the raw mussel shells, passing through a 40-mesh sieve, and setting aside;

2) Take the mussel shells that have been crushed, and add them to a citric acid solution with a mass fraction of 5%, wherein the mass ratio of the mussel shells to the citric acid solution is 1:1, stir and mix, and put the mixed feed solution in Immerse at room temperature for about 2.0h;

3) Put the impregnated material liquid into a high-temperature muffle furnace, raise it from room temperature to the required activation temperature of 300° C., and keep the temperature at a rate of 60° C./min for 30 minutes. the

4) The activated sample is taken out from the muffle furnace and cooled;

5) Pulverize the obtained activated sample through a 150-mesh sieve to obtain modified mussel shells. the

2. The petroleum hydrocarbon degrading bacteria used

The petroleum hydrocarbon-degrading bacteria used for embedding is Bacillus brevus D-1 screened from a certain marine sample, in an environment with a salinity of 25-35‰, a temperature of 15-25°C, and a pH of 7.5-8.5 The degradation rate of crude oil (concentration 1-5g/L) can reach 35-55%. the

3. Embedding

1) Adsorption is carried out by mixing an appropriate amount of modified mussel shells and seed bacteria liquid at a mass ratio of 1:5, until the adsorption is saturated, and preparing modified mussel shells for adsorbing bacteria;

2) Add sterilized sodium alginate solution (the concentration of sodium alginate is 8wt%) to the above-mentioned modified mussel shells adsorbing bacteria, wherein the mass ratio of modified mussel shells: sodium alginate is 1.2:6, mix well Afterwards, inject into CaCl _solution (concentration is 2.5wt%) with sphere-forming equipment and disperse into balls, crosslink 6h, obtain immobilized microbial microspheres, the mass percentage concentration of modified mussel shells accounted for immobilized microspheres is 0.8 %;

3) Rinse the obtained immobilized microbial microspheres with normal saline for 2 to 4 times to obtain the finished product;

4) Finally, the prepared immobilized microbial oil spill remediation agent is stored at 0-4°C for future use. the

The concentration of microorganisms embedded in the immobilized microbial oil spill remediation agent prepared by testing is 1×10 ⁸ cell/g, in an environment with a salinity of 5-55‰, a temperature of 10-30°C, and a pH of 6.5-9.0 The degradation rate of petroleum hydrocarbons is 89.8%.

Example 3:

A preparation method of an immobilized microbial oil spill restoration agent using sodium alginate-calcium chloride-modified mussel shells as an embedded immobilization material, the specific steps are:

1. Preparation of modified mussel shells

1) Cleaning, sieving, drying and pulverizing the raw mussel shells, passing through an 80-mesh sieve, and setting aside;

2) Take the crushed mussel shells and add them to a citric acid solution with a mass fraction of 2%, wherein the mass ratio of the mussel shells to the citric acid solution is 1:3, stir and mix, and put the mixed feed solution in Immerse at room temperature for about 1.0h;

3) Put the impregnated material liquid into a high-temperature muffle furnace, raise it from room temperature to the required activation temperature of 600° C., and keep the temperature at a rate of 50° C./min for 15 minutes. the

4) The activated sample is taken out from the muffle furnace and cooled;

5) Pulverize the obtained activated sample through a 120-150 mesh sieve to obtain the modified mussel shell. the

2. The petroleum hydrocarbon degrading bacteria used

The petroleum hydrocarbon-degrading bacteria used for embedding is Bacillus brevus D-1 screened from a certain marine sample, in an environment with a salinity of 25-35‰, a temperature of 15-25°C, and a pH of 7.5-8.5 The degradation rate of crude oil (concentration 1-5g/L) can reach 35-55%. the

3. Embedding

1) Adsorption is carried out by mixing an appropriate amount of modified mussel shells and seed bacteria liquid at a mass ratio of 1:3 until the adsorption is saturated, and preparing modified mussel shells for adsorbing bacteria;

2) Add sterilized sodium alginate solution (concentration of sodium alginate is 4wt%) to the above-mentioned modified mussel shells adsorbing bacteria, wherein the mass ratio of modified mussel shells: sodium alginate is 1.2:6, mix well Afterwards, inject into CaCl ₂ solution (concentration is 3wt%) with sphere-forming equipment and disperse into ball, cross-link 18h, obtain immobilized microbial microsphere, the mass percent concentration that modified mussel shell accounts for immobilized microsphere is 1.6% ;

3) Rinse the obtained immobilized microbial microspheres with normal saline for 2 to 4 times to obtain the finished product;

4) Finally, the prepared immobilized microbial oil spill remediation agent is stored at 0-4°C for future use. the

The concentration of microorganisms embedded in the immobilized microbial oil spill remediation agent prepared by testing is 9.7×10 ⁷ cell/g, in an environment with a salinity of 5-55‰, a temperature of 10-30°C, and a pH of 6.5-9.0 The degradation rate of petroleum hydrocarbons is 93.4%.

Example 4:

A preparation method of an immobilized microbial oil spill restoration agent using sodium alginate-calcium chloride-modified mussel shells as an embedded immobilization material, the specific steps are:

1. Preparation of modified mussel shells

1) Cleaning, sieving, drying and pulverizing the raw mussel shells, passing through an 80-mesh sieve, and setting aside;

2) Weigh the mussel shells crushed above, and add the citric acid solution with a mass fraction of 2%, wherein the mass ratio of the mussel shells to the citric acid solution is 1:2.5, stir and mix, and mix the mixed feed solution in Immerse at room temperature for about 2.0h;

3) Put the impregnated material liquid into a high-temperature muffle furnace, raise it from room temperature to the required activation temperature of 600° C., and keep the temperature at a rate of 50° C./min for 30 minutes. the

4) The activated sample is taken out from the muffle furnace and cooled;

5) Pulverize the obtained activated sample through a 150-mesh sieve to obtain modified mussel shells. the

2. The petroleum hydrocarbon degrading bacteria used

The petroleum hydrocarbon-degrading bacteria used for embedding is Bacillus brevus D-1 screened from a certain marine sample, in an environment with a salinity of 25-35‰, a temperature of 15-25°C, and a pH of 7.5-8.5 The degradation rate of crude oil (concentration 1-5g/L) can reach 35-55%. the

3. Embedding

1) Adsorption is carried out by mixing an appropriate amount of modified mussel shells and seed bacteria liquid at a mass ratio of 1:5, until the adsorption is saturated, and preparing modified mussel shells for adsorbing bacteria;

2) Add sterilized sodium alginate solution (concentration of sodium alginate is 5.5wt%) to the above-mentioned modified mussel shells adsorbing bacteria, wherein the mass ratio of modified mussel shells: sodium alginate is 1.2:6, mix After homogenization, inject into _CaCl2 solution (concentration is 3.5wt%) with sphere-forming equipment and disperse into balls, crosslink 10h, obtain immobilized microbial microspheres, the mass percentage concentration of modified mussel shells accounted for immobilized microspheres is 1.4%;

3) Rinse the obtained immobilized microbial microspheres with normal saline for 2 to 4 times to obtain the finished product;

4) Finally, the prepared immobilized microbial oil spill remediation agent is stored at 0-4°C for future use. the

The concentration of microorganisms embedded in the immobilized microbial oil spill restoration agent prepared by testing is 9.2×10 ⁷ cell/g, in an environment with a salinity of 5-55‰, a temperature of 10-30°C, and a pH of 6.5-9.0 The degradation rate of petroleum hydrocarbons reaches 92.8%.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still understand the foregoing embodiments The recorded technical solutions are modified, or some of the technical features are equivalently replaced. All within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., should be included within the protection scope of the present invention. the

Claims (10)

1. an immobilized microorganism oil spilling renovation agent, is characterized in that: in described immobilized microorganism oil spilling renovation agent, the microorganism concn of embedding is 5 × 10 ⁷～2 × 10 ⁸cell/g, its carrier component comprises mussel shell, sodium alginate, CaCl ₂.

2. immobilized microorganism oil spilling renovation agent according to claim 1, is characterized in that: the per-cent that each carrier component accounts for immobilized microorganism oil spilling renovation agent is: mussel shell 1.2wt%, sodium alginate 6wt%, CaCl ₂0.3-0.8wt%, all the other are water.

3. a preparation method for immobilized microorganism oil spilling renovation agent, is characterized in that: with sodium alginate-CaCl ₂-modification mussel shell carries out embedding as carrier, and concrete steps are:

1) mussel shell modification;

2) by mussel shell after appropriate modification with plant daughter bacteria liquid by 1:(2～5) mass ratio mixes and adsorbs, until adsorb saturatedly, obtains adsorbing the modification mussel shell of bacterium;

3) to step 2) in add sodium alginate soln in the modification mussel shell of the absorption bacterium that obtains, after mixing, be injected into CaCl with balling-up equipment ₂in solution, disperse balling-up, crosslinked 6～18h, being fixed microbe microsphere.

4. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 3, is characterized in that: described step 1) in, mussel shell modification step is as follows:

A, raw material mussel shell cleaning, sieve are chosen, be dried, are pulverized, mistake 40～80 mesh sieves, for subsequent use;

B, take the mussel shell crushing in step a, join the citric acid solution that concentration is 2～5wt%, stirring and evenly mixing, at room temperature floods approximately 1.0～2.0h by the feed liquid mixing, and wherein, the mass ratio of mussel shell and citric acid solution is 1:(1～3);

C, the feed liquid of having flooded is put into muffle furnace activate, take out cooling;

D, the sample after the activation making was pulverized to 120～150 mesh sieves, obtained modification mussel shell.

5. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 4, is characterized in that: in described step b, citric acid solution concentration is 2.5wt%; The mass ratio of mussel shell and citric acid solution is 1:2.

6. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 4, it is characterized in that: the activation temperature in described step c is 300～600 DEG C, the temperature rise rate that rises to required activation temperature from room temperature is 40～60 DEG C/min, insulation 15～30min; Preferably activation condition is: activation temperature is 550 DEG C, and temperature rise rate is 50 DEG C/min, insulation 30min.

7. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 3, is characterized in that: described kind of daughter bacteria liquid is using Brevibacillus brevis D-1 as petroleum hydrocarbon degradation bacterium, and seed bacterial concentration is (5～8) × 10 ⁹cell/g, wherein, seed bacterial concentration is preferably 6 × 10 ⁹cell/g.

8. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 3, is characterized in that: the concentration of described use sodium alginate is 4wt%～8wt%, and preferred concentration is 6wt%; CaCl ₂strength of solution is 2.5wt%～3.5wt%, and preferred concentration is 3wt%.

9. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 3, is characterized in that: described step 3) in, modification mussel shell: the mass ratio of sodium alginate is 1.2:6.

10. the preparation method of immobilized microorganism oil spilling renovation agent according to claim 3, is characterized in that: described step 3) in normal saline flushing 2～4 times for the immobilized microorganism microballoon that obtains, be finished product.