Mud flat petroleum hydrocarbon degradation immobilized pellet as well as preparation method and application thereof
By employing encapsulation techniques and other technological means, the technical bottlenecks existing in current technologies have been overcome, achieving efficient oil pollution control.
Patent Information
- Application Number
- CN202410689511.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-30
- Publication Date
- 2025-12-02
AI Technical Summary
In existing microbial remediation methods, microbial agents are prone to antagonistic and competitive relationships with wild microbial communities in the natural environment, resulting in their inability to survive and making it difficult to achieve industrial application. Furthermore, the treatment effect of a single microbial species is limited, and the composition and ratio of immobilization materials affect mass transfer performance and mechanical strength, which need to be optimized.
Microspheres encapsulated with petroleum-degrading bacteria are used to form immobilized microspheres with rich pore structures through a coating shell. Combining the SI-JHS, C04-38, Tust-DM21 and SJDQ-112 bacterial strains with materials such as sodium alginate, polyvinyl alcohol, activated carbon, chitosan, and calcium carbonate, immobilized microspheres with high mechanical strength and mass transfer performance are formed, avoiding competition between the bacterial agent and wild bacterial populations.
It achieves efficient degradation of petroleum by composite degrading bacteria in contaminated areas, solving the problem of petroleum pollution. It has high mechanical strength, wear resistance and biodegradability, making it suitable for industrial application and overcoming the technical bottlenecks in existing technologies.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of petroleum hydrocarbon degradation technology, specifically to a petroleum hydrocarbon degradation immobilized microsphere for tidal flats, its preparation method, and its application. Background Technology
[0002] In recent years, with the vigorous exploitation of seabed oil resources and the increase in crude oil trade, marine oil spills have become frequent, putting unprecedented pressure on the marine environment. Regardless of whether the oil spill occurs on the ocean surface or the seabed, even after physical and chemical methods are used to remove most of the oil, some non-recoverable oil will remain in the ocean, harming the marine natural ecosystem.
[0003] Bioremediation is the most economical and effective means of ecological restoration. Currently, microbial remediation methods mainly fall into two categories: biostimulation and microbial agent application. Both methods involve directly adding microbial agents to the contaminated area. However, because these agents, when released into the natural environment, easily interact with wild microbial communities, they can antagonize or compete with them, leading to their failure to survive and hindering their industrial application.
[0004] Microbial immobilization technology has been widely used in the purification and treatment of organic pollutants due to its outstanding advantages such as resistance to environmental shocks, high removal rates, and recyclability. Common methods for immobilizing microorganisms include encapsulation, adsorption, and covalent bonding. Encapsulation involves embedding microbial agents in a porous carrier, resulting in good mass transfer performance and mechanical strength. However, on the one hand, the treatment effect of a single microbial species is often limited, necessitating the exploration of synergistic effects between different species to improve the degradation of petroleum hydrocarbons; on the other hand, the composition and ratio of the immobilization material directly affect the mechanical strength, mass transfer performance, breakage rate, and wear resistance coefficient of the immobilization carrier, requiring further optimization of the immobilization material and encapsulation conditions. Summary of the Invention
[0005] The purpose of this invention is to provide immobilized microspheres for petroleum hydrocarbon degradation in tidal flats, their preparation method, and applications. These immobilized microspheres not only have good degradation effects but also ensure sphere formation, high mechanical strength, mass transfer performance, breakage rate, and wear resistance. They can degrade petroleum hydrocarbons, solve petroleum pollution problems, and can be industrialized, providing an efficient and feasible solution for the treatment of petroleum hydrocarbon pollution in tidal flats.
[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides an immobilized microsphere, comprising:
[0008] Microspheres containing petroleum-degrading bacteria and a coating shell on at least one of the microspheres, the coating shell having a porous structure;
[0009] The microspheres are prepared from an encapsulating agent, a cross-linking agent, and the petroleum composite degrading bacteria; the petroleum composite degrading bacteria are composed of SI-JHS, CO4-38, Tust-DM21, and SJDQ-112; the encapsulating agent includes sodium alginate, polyvinyl alcohol, and activated carbon.
[0010] The raw materials for preparing the coating shell include chitosan and calcium carbonate; the porous structure on the coating shell is generated by reaction in an inorganic acid.
[0011] This invention involves mixing petroleum-degrading bacteria and a cross-linking agent, then adding an encapsulating agent to obtain microspheres. At least one microsphere is then coated with a shell and placed in hydrochloric acid, creating permeable pores on the shell, resulting in immobilized microspheres with a rich pore structure. This abundant pore structure increases the specific surface area of the immobilized microspheres, allowing the petroleum-degrading bacteria inside to fully contact the petroleum hydrocarbons in the polluted tidal flat environment, thereby degrading the petroleum hydrocarbons and solving the petroleum pollution problem. These immobilized microspheres possess advantages such as high mechanical strength, wear resistance, and resistance to biodegradation. They prevent interference from the external environment on the petroleum-degrading bacteria and maintain a high concentration of these bacteria within the petroleum-polluted area, enabling efficient degradation of petroleum hydrocarbons. Furthermore, this invention avoids the antagonistic and competitive relationships that can arise between the petroleum-degrading bacteria and wild-type bacteria when directly introduced into the polluted environment. This allows for the industrial application of petroleum-degrading bacteria, providing an efficient and feasible solution for the remediation of petroleum hydrocarbon pollution in tidal flats.
[0012] The SI-JHS screening method of this invention was used to collect sediment from the tidal flats at the mouth of the Bohai Sea. The strain was identified as *Pseudoalteromon* sp. and preserved at the Tianjin Institute of Seawater Desalination and Comprehensive Utilization, State Oceanic Administration. This strain has been disclosed in "Screening, Identification and Degradation Activity of Marine Petroleum Degrading Bacteria" (Zhang Aijun, Hao Jianan, Yang Bo, et al. Screening, Identification and Degradation Activity of Marine Petroleum Degrading Bacteria [J]. Chemical Industry and Engineering, 2015(32):1). The applicant promises to make this strain publicly available within twenty years from the date of this patent application. Under optimal growth conditions, with a salinity of 3.5%, pH of 7.0, and temperature of 35°C, the petroleum degradation efficiency of this strain is 75.71%.
[0013] The C04-38 strain of this invention was screened from seabed sediments in the oil-polluted waters of Dalian Xingang, and belongs to the genus *Vagococcus*, preserved at the Institute of Marine Microbiology, Dalian Minzu University. This strain has been disclosed in "Isolation, Identification and Degradation Performance Analysis of Anaerobic Microorganisms for Marine Petroleum Degradation" (Zhang Mei, Chen Chao, Liu Qiu. Isolation, Identification and Degradation Performance Analysis of Anaerobic Microorganisms for Marine Petroleum Degradation [J]. Green Technology, 2019(4):3). The applicant promises to make this strain publicly available within twenty years from the date of this patent application. The C04-38 strain exhibits a high degradation rate of 44.31% for petroleum.
[0014] The Tust-DM21 strain used in this invention was screened from the waste oil collection area of an offshore oil exploration vessel in the Bohai Bay. It belongs to the genus *Acinetobacter* and is preserved at the China Marine Microbial Culture Collection Center. This strain has been disclosed in "Analysis of Key Genes of Petroleum-Degrading Bacteria of the Genus *Acinetobacter* and Research on Petroleum Hydrocarbon Degradation" (Yang Jie. Analysis of Key Genes of Petroleum-Degrading Bacteria of the Genus *Acinetobacter* and Research on Petroleum Hydrocarbon Degradation [D]. Tianjin University of Science and Technology, 2018). The applicant promises to make this strain publicly available within twenty years from the date of this patent application. Degradation rate determination showed that after 144 hours, this strain achieved a degradation rate of 97.5% for medium-chain alkanes, 98.5% for long-chain alkanes, 81% for cyclic hydrocarbons, and 87% for aromatic hydrocarbons. It demonstrated excellent degradation ability for all components of petroleum and showed strong degradation effects on short, medium, and long-chain alkanes.
[0015] The SJDQ-112 strain used in this invention was screened from tropical marine environments and belongs to the genus *Vibrioproteolyticus*, preserved in the Key Laboratory of Environmental Toxicology, Hainan University. This strain has been previously disclosed in "Screening of Petroleum-Degrading Bacteria in Tropical Waters of my country and Their Application in Bioremediation" (Su Zengjian. Screening of Petroleum-Degrading Bacteria in Tropical Waters of my country and Their Application in Bioremediation [D]. China University of Mining and Technology (Beijing), 2019). The applicant promises to make this strain publicly available within twenty years from the date of this patent application. Degradation rate testing showed that this strain achieved a 37.9% degradation rate of petroleum hydrocarbons in a marine environment after 7 days.
[0016] In the aforementioned immobilized microspheres, the colony count ratio of viable bacteria SI-JHS, CO4-38, Tust-DM21, and SJDQ-112 in the petroleum composite degrading bacteria is (1-3):(5-8):3:(10-15), including but not limited to 1:5:3:10, 2:5:3:12, and 3:8:3:15. In the petroleum composite degrading bacteria composed of strains with the above ratio, each strain has a synergistic effect, can stably exist inside the immobilized microspheres, and can fully contact petroleum hydrocarbons in the polluted tidal flat environment, thereby fully degrading petroleum hydrocarbons and solving the petroleum pollution problem. More preferably, the petroleum composite degrading bacteria can exist in the form of a bacterial solution with a viable bacteria concentration of 1×10⁻⁶. 8 cfu / mL ~5×10 8 cfu / mL, such as 1×10 8 cfu / mL, 3×10 8 cfu / mL or 5×10 8 CFU / mL; when using the above concentration of petroleum-degrading composite bacteria, the petroleum-degrading composite bacteria account for 50% to 80% of the total mass of the encapsulating agent, such as 50%, 70%, or 80%. The total mass of the encapsulating agent refers to the mass of sodium alginate, polyvinyl alcohol, and activated carbon solid phase. The above-mentioned concentration and ratio of live bacteria in the petroleum-degrading composite bacteria can be fully immobilized in microspheres, thereby obtaining immobilized microspheres with excellent petroleum hydrocarbon degradation effect. Specifically, the petroleum-degrading composite bacteria can be obtained by culturing different single-bacterial bacterial solutions at sufficient concentrations in a constant-temperature shaker for 16-18 hours, and then mixing the bacterial solutions in different proportions to obtain a composite bacterial system. SI-JHS was cultured in an inorganic salt medium at 35°C and 160 rpm in a constant temperature shaker; C04-38 was cultured in a modified artificial seawater medium at 30°C in a constant temperature incubator; Tust-DM21 was cultured in liquid LB medium at 26°C and 130 rpm in a constant temperature shaker; and SJDQ-112 was cultured in liquid enrichment medium (SLB) at 25°C and 160 rpm in a constant temperature shaker. Each culture medium was obtained according to the formulation of the culture medium described in the published literature of the above strains.
[0017] In the aforementioned immobilized microspheres, the mass ratio of sodium alginate, polyvinyl alcohol, and activated carbon in the encapsulating agent is (20-40):(40-60):10, such as 20:40:10, 30:50:10, or 40:60:10; the activated carbon includes any one of cassava charcoal, coconut charcoal, and coconut shell fiber; the particle size of the activated carbon is 20μm-60μm. Activated carbon can increase the density and mechanical strength of the immobilized microspheres. The immobilized microspheres prepared by the encapsulating agent composed of sodium alginate, polyvinyl alcohol, and activated carbon in the above-mentioned mass ratio of the present invention have the advantages of high mechanical strength, wear resistance, biodegradability resistance, and good mass transfer performance.
[0018] In the aforementioned immobilized microspheres, the crosslinking agent is CaCl2; the crosslinking agent can fully react with sodium alginate in the encapsulation agent to obtain microspheres with superior mechanical properties. The crosslinking agent CaCl2 of this invention reacts at the molecular level with the groups on the surface of the petroleum-degrading bacteria, causing the petroleum-degrading bacteria to bind with CaCl2, and subsequently crosslink with the encapsulation agent, thus immobilizing the petroleum-degrading bacteria on the microspheres. Further, in preparing the microspheres, the crosslinking agent exists in solution form with a mass concentration of 1% to 3%, such as 1%, 2%, or 3%; the molar ratio of CaCl2 to sodium alginate in the encapsulation agent is 1:1.
[0019] In the aforementioned immobilized microspheres, the coating shell is made of chitosan solution and calcium carbonate. The chitosan exists in solution form with a mass concentration of 0.75%–1%, such as 0.75%, 0.8%, or 1%. The calcium carbonate has a particle size of 2 μm–8 μm. The amount of calcium carbonate used per 1 mL of the chitosan solution is 0.04 g–0.1 g, such as 0.04 g. This invention, by adding calcium carbonate to the chitosan solution, obtains a coating shell solution capable of coating at least one (e.g., more than two) microspheres. The pores between the microspheres are filled by the coating shell solution, forming a composite microsphere with at least one microsphere as the inner layer and a coating shell as the outer layer. Using chitosan and calcium carbonate to prepare the coating shell can further improve the wear resistance and biodegradability of the immobilized microspheres. It can also act as a pore-forming component to form microsphere pores under the action of inorganic acids, facilitating contact between petroleum hydrocarbon-degrading bacteria and petroleum hydrocarbons and helping to maintain the long-term activity of the petroleum hydrocarbon-degrading bacteria.
[0020] In the aforementioned immobilized microspheres, the inorganic acid is hydrochloric acid or sulfuric acid; the concentration of the inorganic acid is 0.15% w / v to 0.20% w / v, such as 0.15% w / v, 0.18% w / v, or 0.20% w / v. In this invention, w / v refers to g / mL. This invention involves placing the composite microspheres in hydrochloric acid, which dissolves the calcium carbonate particles with a diameter of 2–8 μm in the coating layer, resulting in immobilized microspheres with a rich porous structure. This rich porous structure increases the specific surface area of the immobilized microspheres, allowing the petroleum-degrading bacteria inside the immobilized microspheres to fully contact the petroleum hydrocarbons in the polluted tidal flat environment, thereby degrading the petroleum hydrocarbons and solving the petroleum pollution problem.
[0021] Secondly, the present invention provides a method for preparing the immobilized microspheres, comprising the following steps:
[0022] (1) The crosslinking agent is added to the petroleum composite degrading bacteria to obtain a first mixture;
[0023] (2) Dissolve or disperse the embedding agent in water to prepare an embedding agent dispersion, and drop the embedding agent dispersion into the first mixture for cross-linking to obtain the microspheres;
[0024] (3) Prepare a coating shell mixture formed by chitosan solution and calcium carbonate, immerse the microspheres in the coating shell mixture, and stir to coat at least one microsphere to obtain a second mixture;
[0025] (4) The second mixture is dropped into the stirred inorganic acid. After the small balls float, they are separated to obtain the immobilized small balls.
[0026] In the above preparation method, in step (2), the crosslinking temperature is 4 to 8°C (e.g., 4°C, 6°C or 8°C), and the time is 18 to 30 hours (e.g., 18 hours, 24 hours or 30 hours);
[0027] In step (3), the stirring speed is 500-800 r / min (e.g., 500 r / min, 700 r / min, 800 r / min), and the time is 13-15 min (e.g., 15 min, 14 min or 13 min);
[0028] In step (4), the second mixture is added dropwise to an inorganic acid at a stirring speed of 10-15 r / min (e.g., 10 g / min, 13 g / min or 15 g / min) at a rate of 10-15 g / min (10 r / min, 14 r / min or 15 r / min).
[0029] In this invention, the embedding agent dispersion is dripped into a mixed solution of cross-linking agent of petroleum composite degrading bacteria, so that the embedding agent and cross-linking agent react to obtain microspheres; then, a coating shell mixture is used to coat multiple microspheres to form composite microspheres with a coating shell layer; then, hydrochloric acid is slowly dripped into the composite microspheres until all the composite microspheres float up, then they are separated and washed with distilled water 2 to 3 times to obtain immobilized microspheres with rich pore structure.
[0030] Thirdly, the present invention provides the application of the immobilized microspheres described in any of the above claims in the degradation of petroleum hydrocarbons in tidal flats.
[0031] Fourthly, the present invention provides a petroleum composite degrading bacteria, including SI-JHS, CO4-38, Tust-DM21 and SJDQ-112 with a live colony ratio of (1-3):(5-8):3:(10-15), including but not limited to 1:5:3:10, 2:5:3:12 and 3:8:3:15.
[0032] The present invention has the following beneficial effects:
[0033] This invention utilizes a composite microbial strain of SI-JHS, CO4-38, Tust-DM21, and SJDQ-112 as the petroleum-degrading composite bacteria. These bacteria are encapsulated within the microspheres using an encapsulating agent and a cross-linking agent. A shell structure is formed on the outside of the microspheres using chitosan and calcium carbonate, and a porous structure is created using inorganic acids. The various bacterial strains exhibit synergistic effects, and the abundant porous structure increases the specific surface area of the immobilized microspheres, allowing for sufficient contact between the petroleum-degrading composite bacteria inside the microspheres and the petroleum hydrocarbons in the polluted tidal flat environment. This process degrades the petroleum hydrocarbons, thus solving the petroleum pollution problem. Furthermore, the immobilized microspheres possess advantages such as high mechanical strength, wear resistance, and resistance to biodegradation. This invention's immobilized microspheres can degrade petroleum hydrocarbons, solve petroleum pollution problems, and can be industrially applied, providing an efficient and feasible solution for the remediation of petroleum hydrocarbon pollution in tidal flats. Detailed Implementation
[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0035] Unless otherwise specified, the methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0036] Example 1
[0037] This embodiment provides a microsphere for petroleum hydrocarbon degradation and immobilization in tidal flats, comprising: microspheres embedded with petroleum composite degrading bacteria and a coating shell covering at least one of the microspheres, wherein the coating shell has a porous structure.
[0038] The microspheres are prepared from an encapsulating agent, a cross-linking agent, and petroleum-degrading bacteria.
[0039] The petroleum-degrading compound bacteria consisted of SI-JHS, C04-38, Tust-DM21, and SJDQ-112, with a colony count ratio of 1:5:3:10, and the viable cell concentration of each compound bacteria was 1×10⁻⁶. 8 cfu / mL.
[0040] The embedding agent is made by uniformly mixing sodium alginate, polyvinyl alcohol and activated carbon in a mass ratio of 20:40:10. The activated carbon is cassava charcoal with a particle size of 20-60μm. Petroleum composite degrading bacteria account for 50% of the total mass of the embedding agent (solid).
[0041] The crosslinking agent is a 1% (w / w) CaCl2 solution, wherein the molar ratio of CaCl2 to sodium alginate is 1:1.
[0042] The raw materials for preparing the coating shell include chitosan and calcium carbonate.
[0043] The coating shell is made by adding 0.2g of calcium carbonate to 5mL of 0.75% chitosan solution, and the calcium carbonate has a particle size of 2-8μm.
[0044] The porous structure on the coating shell is formed by the reaction in hydrochloric acid at a concentration of 0.15% w / v.
[0045] The above-mentioned method for preparing immobilized microspheres specifically includes the following operations:
[0046] Sodium alginate and polyvinyl alcohol were dissolved in 100 mL of water, with the concentration of sodium alginate being 0.2 g / mL and the concentration of polyvinyl alcohol being 0.4 g / mL. Then, activated carbon (0.1 g / mL) was dispersed in the same 100 mL of water. The solution was then placed in an autoclave and sterilized at 121°C for 20 min to remove bacteria. After cooling to room temperature, the embedding agent dispersion was obtained.
[0047] 100 mL of a 1% CaCl2 solution was placed in an autoclave and sterilized at 121°C for 20 min to remove contaminants. After cooling to room temperature, 5 mL of a petroleum-degrading compound bacteria (SI-JHS, CO4-38, Tust-DM21, and SJDQ-112 colony count ratio of 1:5:3:10) with a viable bacterial concentration of 1×10⁻⁶ cells / mL was added. 8 The petroleum composite degrading bacteria account for 50% of the mass of the encapsulating agent. The first mixture of petroleum composite degrading bacteria and cross-linking agent is obtained by thorough mixing.
[0048] Take 5 mL of the encapsulating agent dispersion and add it dropwise into the first mixture of the above-mentioned petroleum composite degrading bacteria and cross-linking agent. Cross-link at 4℃ for 18 h, filter out the microspheres, rinse twice with distilled water, and then soak them in 5 mL of a coating shell mixture consisting of 0.75% chitosan solution containing 0.2 g calcium carbonate. Stir at 500 r / min for 15 min to obtain the second mixture.
[0049] The second mixed solution was then added dropwise at a rate of 10 g / min to a 0.15% w / v hydrochloric acid solution that was stirred at a rate of 10 r / min. After the microspheres floated up, they were filtered and separated. After washing twice with distilled water, the immobilized microspheres were obtained.
[0050] Example 2
[0051] This embodiment provides a microsphere for petroleum hydrocarbon degradation and immobilization in tidal flats, comprising: microspheres embedded with petroleum composite degrading bacteria and a coating shell covering at least one microsphere, the coating shell having a porous structure;
[0052] The microspheres are prepared from an encapsulating agent, a cross-linking agent, and petroleum-degrading bacteria.
[0053] The petroleum-degrading compound bacteria consisted of SI-JHS, CO4-38, Tust-DM21, and SJDQ-112, with a colony count ratio of 2:5:3:12. The viable cell concentration of each petroleum-degrading compound bacteria was 3 × 10⁻⁶. 8 cfu / mL.
[0054] The embedding agent is prepared by uniformly mixing sodium alginate, polyvinyl alcohol, and activated carbon in a mass ratio of 30:50:10. The activated carbon is cassava charcoal with a particle size of 20-60 μm. Petroleum-degrading bacteria account for 70% of the total mass of the embedding agent.
[0055] The crosslinking agent is a 2% (w / w) CaCl2 solution, wherein the molar ratio of CaCl2 to sodium alginate is 1:1;
[0056] The raw materials for preparing the coating shell include chitosan and calcium carbonate.
[0057] The coating shell is made by adding 0.4g of calcium carbonate to 5mL of 0.8% chitosan solution, and the calcium carbonate has a particle size of 2-8μm.
[0058] The porous structure on the coating shell is formed by the reaction in hydrochloric acid at a concentration of 0.18% w / v.
[0059] The above-mentioned method for preparing immobilized microspheres includes the following steps:
[0060] Sodium alginate and polyvinyl alcohol were dissolved in 100 mL of water, with the concentration of sodium alginate being 0.3 g / mL and the concentration of polyvinyl alcohol being 0.05 g / mL. Then, activated carbon was dispersed in the above aqueous solution at a concentration of 0.1 g / mL. The solution was then placed in an autoclave and sterilized at 121°C for 20 min to remove impurities. After cooling to room temperature, the embedding agent dispersion was obtained.
[0061] 100 mL of a 2% CaCl2 solution was placed in an autoclave and sterilized at 121°C for 20 min to remove contaminants. After cooling to room temperature, 5 mL of a petroleum-degrading compound bacteria (SI-JHS, CO4-38, Tust-DM21, and SJDQ-112 colony ratio of 2:5:3:12, with a viable bacterial concentration of 3 × 10⁻⁶) was added. This compound had been cultured in a constant-temperature shaker for 16-18 h. 8The petroleum composite degrading bacteria account for 70% of the mass of the encapsulating agent (cfu / mL), and the first mixture of petroleum composite degrading bacteria and cross-linking agent is obtained by thorough mixing.
[0062] Take 5 mL of the encapsulating agent dispersion and add it dropwise to the first mixture of the above-mentioned petroleum composite degrading bacteria and cross-linking agent. Cross-link at 6℃ for 24 h, filter out the microspheres, rinse three times with distilled water, and then soak them in 5 mL of a coating shell mixture consisting of 0.8% chitosan solution containing 0.4 g calcium carbonate. Stir at 700 r / min for 14 min to obtain the second mixture.
[0063] The second mixture was then added dropwise at a rate of 13 g / min to a 0.18% w / v hydrochloric acid solution that was stirred at a rate of 14 r / min. After the microspheres floated up, they were filtered and separated. After washing with distilled water three times, the immobilized microspheres were obtained.
[0064] Example 3
[0065] This embodiment provides a microsphere for petroleum hydrocarbon degradation and immobilization in tidal flats, comprising: microspheres embedded with petroleum composite degrading bacteria and a coating shell covering at least one of the microspheres, wherein the coating shell has a porous structure;
[0066] The microspheres are prepared from an encapsulating agent, a cross-linking agent, and petroleum-degrading bacteria.
[0067] The petroleum-degrading compound bacteria consisted of SI-JHS, C04-38, Tust-DM21, and SJDQ-112, with a colony count ratio of 3:8:3:15. The viable cell concentration of each strain in the petroleum-degrading compound bacteria was 5 × 10⁻⁶. 8 cfu / mL.
[0068] The embedding agent is made by uniformly mixing sodium alginate, polyvinyl alcohol, and activated carbon in a mass ratio of 40:60:10. The activated carbon is coconut shell fiber with a particle size of 20-60 μm. Petroleum-degrading bacteria account for 80% of the total mass of the embedding agent.
[0069] The crosslinking agent is a 3% CaCl2 solution, wherein the molar ratio of CaCl2 to sodium alginate is 1:1.
[0070] The raw materials for preparing the coating shell include chitosan and calcium carbonate.
[0071] The coating shell is made by adding 0.5g of calcium carbonate to 5mL of 1% chitosan solution, and the calcium carbonate has a particle size of 2-8μm.
[0072] The porous structure on the coating shell is formed by the reaction in hydrochloric acid at a concentration of 0.20% w / v.
[0073] The above-mentioned method for preparing immobilized microspheres includes the following operations:
[0074] Sodium alginate and polyvinyl alcohol were dissolved in 100 mL of water, with the concentration of sodium alginate being 0.4 g / mL and the concentration of polyvinyl alcohol being 0.6 g / mL. Then, activated carbon was dispersed in the above aqueous solution at a concentration of 0.1 g / mL. The solution was then placed in an autoclave and sterilized at 121°C for 20 min to remove contaminants. After cooling to room temperature, the embedding agent dispersion was obtained.
[0075] 100 mL of a 3% CaCl2 solution was placed in an autoclave and sterilized at 121°C for 20 min to remove contaminants. After cooling to room temperature, 5 mL of a petroleum-degrading compound bacteria (SI-JHS, CO4-38, Tust-DM21, and SJDQ-112 in a mass ratio of 3:8:3:15, with a viable bacterial concentration of 5 × 10⁻⁶) was added. 8 The petroleum composite degrading bacteria account for 80% of the mass of the encapsulating agent. The first mixture of petroleum composite degrading bacteria and cross-linking agent is obtained by thorough mixing.
[0076] Take 5 mL of the embedding agent and add it dropwise into the first mixture of the above-mentioned petroleum composite degrading bacteria and cross-linking agent. Cross-link at 8°C for 30 h. Filter out the microspheres and rinse them 3 times with distilled water. Then, soak them in 5 mL of a coating shell solution composed of 1% chitosan solution containing 0.5 g of calcium carbonate and stir at 800 r / min for 13 min to obtain the second mixture.
[0077] The second mixture was then added dropwise at a rate of 15 g / min to a 0.20% w / v hydrochloric acid solution that was stirred at a rate of 15 r / min. After the microspheres floated up, they were filtered and separated. After washing with distilled water three times, the immobilized microspheres were obtained.
[0078] Comparative Example 1
[0079] Compared with Example 2, the embedding agent is composed of sodium alginate and polyvinyl alcohol in a mass ratio of 30:50, and the remaining components and preparation methods are the same as in Example 2.
[0080] Comparative Example 2
[0081] Compared with Example 2, the concentration of the crosslinking agent CaCl2 was 5%, and the remaining components and preparation methods were the same as in Example 2.
[0082] Comparative Example 3
[0083] Compared with Example 2, the coating solution was 0.8% chitosan by mass, and the remaining components and preparation methods were the same as in Example 2.
[0084] Comparative Example 4
[0085] Compared with Example 2, the petroleum composite degrading bacteria single strain SI-JHS, the other components and preparation methods are the same as in Example 2.
[0086] Comparative Example 5
[0087] Compared with Example 2, the petroleum composite degrading bacteria were SI-JHS and CO4-38 with a colony count ratio of 2:5, and the remaining components and preparation methods were the same as in Example 2.
[0088] Comparative Example 6
[0089] Compared with Example 2, the petroleum composite degrading bacteria were SI-JHS, CO4-38 and Tust-DM21 with a colony count ratio of 2:5:3, and the remaining components and preparation methods were the same as in Example 2.
[0090] Comparative Example 7
[0091] Compared with Example 2, no petroleum-degrading bacteria were added, and the remaining components and preparation methods were the same as in Example 2.
[0092] Verification Example 1
[0093] The immobilized microspheres from Examples 1-3 and Comparative Examples 1-3 were evaluated for their pelletizing effect, mechanical strength, mass transfer performance, breakage rate, and wear resistance coefficient. The measurement methods are as follows:
[0094] 1) Spherical formation effect: Observe whether the spherical formation is uniform after the embedding agent is dropped into calcium chloride and cross-linked for 24 hours, and whether there are any unformed sphericals or tailing phenomena.
[0095] 2) Take out 3 immobilized microspheres from each of the prepared microspheres, place them on an electronic balance, zero the balance, and slowly press the microspheres. Use the reading of the electronic balance when the microsphere breaks as an indicator of the mechanical strength of the microsphere.
[0096] 3) Mass transfer performance: Prepare a 2% methylene blue ethanol solution, add 30 drops of the prepared methylene blue ethanol solution to 500 mL of distilled water, and place 50 immobilized microspheres into equal volumes of the solution. After 48 h, measure the absorbance and compare it with the original methylene blue solution.
[0097] 4) Breakage rate: 50 immobilized microspheres were placed in a 250mL conical flask containing 100mL of seawater and shaken in a constant temperature shaker at 25℃ and 170r / min for 48h. The breakage rate of the immobilized microspheres was calculated after that.
[0098] 5) Wear resistance coefficient: Add 50 mL of seawater and 10 g of sand (passed through a 60-mesh sieve) to each conical flask. Place 20 immobilized microspheres in a shaker for 3 days. Measure the change in diameter of the immobilized microspheres before and after treatment using vernier calipers. Compare the wear resistance coefficients of the immobilized microspheres prepared in Examples 1-3 and Comparative Examples 1-3. The calculation formula is: Wear resistance coefficient = R末 / R 初 , where R 末 This represents the average particle size of the immobilized spheres after 3 days in a shaker; similarly, R... 初 This represents the average particle size of the immobilized spheres before the experiment.
[0099] The results are shown in Table 1.
[0100] Table 1. Performance of immobilized microspheres
[0101] Sphere formation effect Mechanical strength / g Mass transfer performance Breakage rate (%) wear resistance coefficient Example 1 Rules of small balls 89 58.3％ 10 0.89 Example 2 Rules of small balls 130 68.2％ 4 0.96 Example 3 Rules of small balls 135 70.2％ 3 0.98 Comparative Example 1 Irregular small ball 70 56.7％ 185 0.79 Comparative Example 2 Irregular small ball 150 53.1％ 0 1 Comparative Example 3 Irregular small ball 138 62.1％ 3 0.92
[0102] As shown in Table 1, the immobilized microspheres prepared in Examples 1-3 of this invention have better sphericity, mechanical strength, mass transfer performance, breakage rate and wear resistance coefficient than those in Comparative Examples 1-3.
[0103] Verification Example 2
[0104] 1. Assessment of the degradation rate of petroleum hydrocarbons in seawater
[0105] The immobilized microspheres prepared in Examples 1-3 and Comparative Examples 1-7 were subjected to petroleum hydrocarbon degradation tests.
[0106] The specific method is as follows: The immobilized microspheres obtained in Examples 1-3 and Comparative Examples 1-7 were used as experimental groups and placed into conical flasks containing 50 mL of petroleum salt culture medium, with approximately 0.05 g of petroleum (petroleum concentration of 1000 mg / L) added to each flask; the immobilized microspheres obtained in Comparative Example 7 were used as the control group (CK1); and sterile petroleum salt culture medium without immobilized microspheres was used as the blank group (C0). The composition of the petroleum salt culture medium was: 2 g of KNO3, 1 g of MgSO4·7H2O, 1 g of petroleum, and 1000 mL of seawater.
[0107] The conical flasks of Examples 1-3, Comparative Examples 1-7, and the blank group were placed in a constant temperature shaker at 160 rpm and 25°C for 7 days. Then, the residual petroleum content of each group was determined using an infrared spectrometer, and the absolute petroleum degradation rate after 7 days was calculated according to formulas (I) and (II).
[0108] Petroleum degradation rate = (C0-C1) / C0 Equation (I);
[0109] Absolute degradation rate of petroleum = Petroleum degradation rate - 7-day degradation rate of control group (Equation II);
[0110] Where C0 represents the petroleum concentration of the sample before degradation, and C1 represents the petroleum concentration of the sample after degradation.
[0111] 2. Prepare the actual concentration (C) x Standard curve of standard concentration (C0)
[0112] Weigh 0.1 g of petroleum into a 100 mL volumetric flask and dilute to volume with CCl4 to prepare a 1000 mg / L petroleum solution. Add 0 mL, 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL of the prepared 1000 mL petroleum solution to a series of 50 mL volumetric flasks, respectively, and dilute to volume with CCl4 to obtain petroleum solutions with concentrations of 0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L. Determine the concentration values using an infrared spectrophotometer. The standard curve obtained is y = 5.3208x - 2.7829, with a linear correlation coefficient R0. 2 It is 0.9999.
[0113] Linear coefficients R 2 >0.999, ensuring accurate and reliable petroleum concentration results, with spiked recovery rate controlled between 95% and 105%.
[0114] 3. Determination of petroleum degradation rate
[0115] Hydrochloric acid was added dropwise to the solutions of the experimental groups (Examples 1-3, Comparative Examples 1-6), the control group (Comparative Example 7), and the blank group after 7 days of degradation to make the pH of the petroleum salt culture medium less than or equal to 2. The solutions were then poured into a separatory funnel. The conical flask was washed repeatedly with 50 mL of CCl4 until all the petroleum was dissolved. The solution was then transferred to the separatory funnel, and about 20 g of NaCl was added. The mixture was shaken thoroughly for 2 minutes until the petroleum was uniformly dissolved, and the stopcock was opened frequently to release air. The mixture was then allowed to stand and separate into layers. The extract was then poured into a 50 mL volumetric flask through a glass frosted funnel containing about 10 mm of anhydrous sodium sulfate. The anhydrous sodium sulfate layer was then thoroughly washed with CCl4 and the volume was adjusted to the mark. The mixture was shaken well, and the concentration of petroleum in the samples was determined using an infrared spectrophotometer according to the standard curve method described above. The degradation rate was calculated according to formulas (I) and (II).
[0116] The experimental results are shown in Table 2.
[0117] Table 2. Petroleum Degradation Effect
[0118]
[0119] Table 2 shows that Examples 1-3 exhibited the best petroleum hydrocarbon degradation effect, while Comparative Examples 1-7 showed a less effective degradation effect than Examples 1-3. This indicates that the petroleum-degrading bacteria in Examples 1-3 of this invention have a synergistic effect, enhancing each other's petroleum hydrocarbon degradation efficiency. Although Comparative Examples 4-6 also used compound degrading bacteria, single bacteria, two bacteria, or three bacteria, none of them achieved the petroleum hydrocarbon degradation effect of the petroleum-degrading bacteria of this invention. Furthermore, the pelleting effect also plays an important role in petroleum decomposition.
[0120] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A type of immobilized petroleum hydrocarbon degradation microsphere for tidal flats, characterized in that, include: Microspheres containing petroleum-degrading bacteria and a coating shell containing at least one of the microspheres, the coating shell having a porous structure; The microspheres are prepared from an encapsulating agent, a cross-linking agent, and the petroleum composite degrading bacteria; the petroleum composite degrading bacteria are composed of SI-JHS, CO4-38, Tust-DM21, and SJDQ-112; the encapsulating agent includes sodium alginate, polyvinyl alcohol, and activated carbon. The raw materials for preparing the coating shell include chitosan and calcium carbonate; the porous structure on the coating shell is generated by reaction in an inorganic acid.
2. The immobilized microsphere according to claim 1, characterized in that: The viable colony ratio of SI-JHS, CO4-38, Tust-DM21 and SJDQ-112 in the petroleum composite degrading bacteria is (1-3):(5-8):3:(10-15); The viable bacterial concentration of the petroleum composite degrading bacteria is 1×10⁻⁶. 8 cfu / mL ~5×10 8 cfu / mL; the petroleum-degrading composite bacteria account for 50% to 80% of the total mass of the encapsulating agent.
3. The immobilized microsphere according to claim 1 or 2, characterized in that: In the embedding agent, the mass ratio of sodium alginate, polyvinyl alcohol and activated carbon is (20-40):(40-60):10; The activated carbon includes any one of cassava charcoal, coconut charcoal, and coconut shell fiber. The activated carbon has a particle size of 20 μm to 60 μm.
4. The immobilized microsphere according to claim 3, characterized in that: The crosslinking agent is CaCl2, and the molar ratio of CaCl2 to sodium alginate is 1:1; In the preparation of the microspheres, the crosslinking agent exists in solution form with a mass concentration of 1% to 3%.
5. The immobilized microsphere according to claim 4, characterized in that: The coating shell is made of chitosan solution and calcium carbonate, wherein the mass concentration of the chitosan solution is 0.75% to 1%, and the amount of calcium carbonate used per 1 mL of the chitosan solution is 0.04 g to 0.1 g. The calcium carbonate has a particle size of 2μm to 8μm. The mass ratio of sodium alginate to chitosan is 1:(1.5-3.75).
6. The immobilized microsphere according to claim 1, characterized in that: The inorganic acid is hydrochloric acid or sulfuric acid; The inorganic acid exists in the form of an aqueous solution with a concentration of 0.15% w / v to 0.20% w / v.
7. The method for preparing immobilized microspheres according to any one of claims 1-6, comprising the following steps: (1) The crosslinking agent is added to the petroleum composite degrading bacteria to obtain a first mixture; (2) Dissolve or disperse the embedding agent in water to prepare an embedding agent dispersion, and drop the embedding agent dispersion into the first mixture for cross-linking to obtain the microspheres; (3) Prepare a coating shell mixture formed by chitosan solution and calcium carbonate, immerse the microspheres in the coating shell mixture, and stir to coat at least one microsphere to obtain a second mixture; (4) The second mixture is dropped into the stirred inorganic acid. After the small balls float, they are separated to obtain the immobilized small balls.
8. The preparation method according to claim 7, characterized in that: In step (2), the crosslinking temperature is 4–8°C and the time is 18–30 h; In step (3), the stirring speed is 500-800 r / min and the time is 13-15 min; In step (4), the second mixture is added dropwise to the inorganic acid at a rate of 10-15 g / min and a stirring speed of 10-15 r / min.
9. The application of the immobilized microspheres according to any one of claims 1-6 in the degradation of petroleum hydrocarbons in tidal flats.
10. A petroleum-degrading compound microorganism, comprising SI-JHS, CO4-38, Tust-DM21 and SJDQ-112 with a live colony ratio of (1-3):(5-8):3:(10-15).