Application of clavibacter michiganensis in degrading aromatic hydrocarbon
By optimizing the culture conditions of Saccharophilic Crabtree in sewage, the problem of difficult degradation of benzene series compounds was solved, a degradation rate of more than 50% was achieved, and microbial preparations for sewage treatment were prepared, thereby improving water quality safety.
Patent Information
- Application Number
- CN202510469743.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2045-04-15
AI Technical Summary
Existing technologies are unable to efficiently degrade benzene series compounds in the environment, resulting in their long-term residues in the environment, posing a threat to the ecosystem and human health.
Saccharophila Crabtree (Brucellaceae genus ATCC19623) was used to degrade aromatic hydrocarbons in sewage. The culture conditions were optimized as follows: temperature 15-40℃, pH 7-8, inoculation size 5-15%, and BTEX concentration 100 mg/L, achieving efficient degradation of BTEX.
Under optimal conditions, Saccharophila Crabtree Bacillus can achieve a benzene degradation rate of more than 50%, and can be used to prepare microbial preparations for sewage treatment to improve water quality safety.
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Figure CN120247278B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of industrial and domestic sewage treatment, and in particular to application of clavibacter glycolates in degradation of aromatic hydrocarbons. BACKGROUND
[0002] In recent years, benzene series as an important class of organic compounds, widely exist in nature and human production activities. Benzene series is not only an important raw material for many chemical products, but also widely used in the fields of fuel, medicine and pesticide. However, due to the wide use of benzene series and its chemical stability, it leads to long-term residue in the environment, which poses a potential threat to the ecosystem and human health. Benzene series refers to organic compounds containing benzene ring structure, such as benzene, toluene, ethylbenzene, xylene, etc. Its main sources include oil and coal mining and processing, fuel combustion, industrial production waste gas, wastewater discharge, etc. After benzene series enters the environment, it often exists in the air, water and soil in gaseous, dissolved or adsorbed state.
[0003] The benzene ring in the molecular structure of benzene series makes it have high chemical stability and hydrophobicity, resulting in a long degradation period in the environment. In addition, some polycyclic aromatic hydrocarbons (PAHs) have carcinogenicity, mutagenicity and reproductive toxicity, becoming important pollutants in environmental risk. For example, the concentration of benzene series detected in industrial wastewater or domestic wastewater is often high, and these compounds accumulate through drinking water or food chain to cause long-term harm to the human body. In view of benzene series pollution, scientists have developed various treatment technologies, and biological treatment technology utilizes the metabolic action of microorganisms to degrade benzene series, which is converted into harmless products such as carbon dioxide and water. Compared with other technologies, biological treatment has the characteristics of economic efficiency and environmental friendliness. By screening strains with high degradation efficiency of benzene series and applying biological strengthening technology, the removal efficiency of benzene series can be significantly improved. Recent studies have shown that developing characteristic strains with high degradation efficiency of aromatic hydrocarbons and optimizing their application conditions in actual wastewater treatment are important directions for dealing with benzene series pollution. In addition, studying the toxicology of intermediates in the benzene series degradation process and the interaction with environmental factors is also an important task to achieve complete removal of pollutants. SUMMARY
[0004] The purpose of the present application is to provide an application of clavibacter glycolates in degradation of aromatic hydrocarbons, which can effectively decompose benzene series in sewage by inoculating clavibacter glycolates into the sewage, and the degradation rate reaches more than 50%, and can be used for preparing microbial preparation for degrading sewage.
[0005] To achieve the above purpose, the application provides an application of clavibacter glycolates in degradation of aromatic hydrocarbons, wherein the clavibacter glycolates is clavibacter glycolates genus ATCC19623 of brucellaceae.
[0006] Further, the aromatic hydrocarbon is benzene series.
[0007] Further, the culture condition of the Kluyveromyces sp. is temperature 15-40℃, pH value 7-8, inoculation amount 5-15%, and benzene series concentration 100mg / L.
[0008] Further, in application, the degradation rate of the Kluyveromyces sp. in degrading aromatic hydrocarbon is greater than 45%.
[0009] The application further provides an application of Kluyveromyces sp. in degrading benzene series in sewage, wherein the Kluyveromyces sp. is Brucellaceae Kluyveromyces sp. ATCC19623.
[0010] The application further provides a microbial preparation for degrading benzene series, wherein the effective component of the microbial preparation comprises Brucellaceae Kluyveromyces sp. ATCC19623.
[0011] The application further provides a method for Kluyveromyces sp. to degrade benzene series in sewage, wherein the Kluyveromyces sp. is inoculated into the sewage to degrade the benzene series in the sewage.
[0012] The application provides a Kluyveromyces sp. in degrading aromatic hydrocarbon.
[0013] 1. The Kluyveromyces sp. is inoculated into the sewage to effectively decompose the benzene series in the sewage, and the degradation rate is greater than 50%, and the Kluyveromyces sp. can be used to prepare a microbial preparation for degrading sewage.
[0014] The technical solutions of the application are further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 Figure 1 is a curve of the degradation rate of benzene series in Example 1 of the application with the change of culture days;
[0016] Figure 2 Figure 2 is a curve of the degradation rate of benzene series in Example 1 of the application with the change of temperature;
[0017] Figure 3 Figure 3 is a curve of the degradation rate of benzene series in Example 1 of the application with the change of pH value;
[0018] Figure 4 Figure 4 is a curve of the degradation rate of benzene series in Example 1 of the application with the change of substrate concentration;
[0019] Figure 5 Figure 5 is a curve of the degradation rate of benzene series in Example 1 of the application with the change of inoculation amount. DETAILED DESCRIPTION
[0020] The technical solutions of the present application are further illustrated by the accompanying drawings and examples.
[0021] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those with ordinary skills in the art to which the present application pertains.
[0022] Based on the examples in the present application, all other examples obtained by those with ordinary skills in the art without making creative efforts shall fall within the scope of protection of the present application. The experimental methods not specified in the following examples are generally determined according to the national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.
[0023] The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is performed according to the general international standard, conventional conditions, or according to the conditions recommended by the manufacturer.
[0024] Unless otherwise defined or explained, all professional and scientific terms used in the present application have the same meanings as those familiar to those with ordinary skills in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.
[0025] The Clostridium saccharoperbutylacetonicum has the preservation number ATCC19623 and was purchased from Mingzhou Biology in April 2024.
[0026] Example 1
[0027] Culture and detection of Clostridium saccharoperbutylacetonicum:
[0028] (1) Preparation of Clostridium saccharoperbutylacetonicum seed solution:
[0029] The Clostridium saccharoperbutylacetonicum (purchased from Mingzhou Biology, with the strain preservation number ATCC19623) was inoculated in a benzene series liquid culture medium and cultured at 25°C to the logarithmic phase to prepare a seed solution;
[0030] The liquid medium containing benzene series is formulated as follows: Na2HPO4·2H2O 3.5 g / L, KH2PO 1.0 g / L, (NH4)2SO4 0.5 g / L, CaCl2 0.2 g / L, MgCl2·6H2O 0.1 g / L, toluene 50 mf / L, benzene 50 mg / L, ethylbenzene 50 mg / L, dimethylbenzene 50 mg / L, trace elements 1.0 mL / L (trace elements: EDTA 0.5 g / L, FeSO4·7H2O 0.2 g / L, MnSO4·H2O 0.08 g / L, boric acid 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuCl2·2H2O 0.01 g / L, NiCl2·H2O 0.01 g / L, Na2MoO4·2H2O 0.02 g / L).
[0031] (2) Optimization of strain conditions:
[0032] The seed liquid cultured to the logarithmic phase is inoculated into 100 mL PBSA liquid medium under different single variable conditions, and cultured under different conditions. The culture temperature is 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C, respectively. The pH value is 3, 5, 6, 7, 7.5, 8, 9, and 11, respectively. The initial benzene series concentration is 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L, respectively. The amount of inoculated bacterial liquid is 1%, 2%, 5%, 10%, 15%, and 20%. The culture is carried out at a speed of 150 r / min.
[0033] (3) Enrichment and elution of benzene series after degradation by the strain:
[0034] 15 mL of the culture medium after step (2) is taken out and centrifuged for 20 min. The supernatant is extracted with an activated solid-phase cartridge (0asis HLB 6 mL, 500 mg). The remaining slurry is added with 2 mL of methanol, and then ultrasonicated three times for 10 min each time. The supernatant is diluted with 10 mL of deionized water, and finally extracted with the same solid-phase cartridge.
[0035] Activation (pretreatment) of the solid-phase cartridge: A. 5 mL of methanol is used to activate the C18 cartridge at a flow rate of 2 ml / min;
[0036] B. 10 mL of chromatographic grade pure water is used to activate the C18 cartridge at a flow rate of 2 mL / min;
[0037] Elution of the target analyte: 10 mL of methanol is used to elute the column at a flow rate of 15 mL / min;
[0038] Then, the extract is blown dry with N2, and then dissolved with acetone;
[0039] Finally, filter to the amber liquid phase vial with 0.22 μm filter head, and then measure HPLC.
[0040] Liquid chromatography conditions of HPLC analysis:
[0041] Column temperature 30℃; mobile phase is methanol: water = 3:1; flow rate 1 mL / min; retention time is 2.5 min.
[0042] The culture results are shown in Figure 1 、 2 , 3, 4.
[0043] As can be seen from Figure 1 , with the extension of culture time, the degradation rate of aromatic hydrocarbon gradually increases, but after the eighth day, with the extension of time, the degradation rate of aromatic hydrocarbon increases little, so the optimal culture time is 8d.
[0044] As can be seen from Figure 2 , with the increase of temperature, the degradation rate of Klabochia sugare first increases and then decreases, when the temperature is high, the activity of Klabochia sugare will be affected, leading to the decrease of degradation rate, the optimal degradation temperature is 30℃.
[0045] As can be seen from Figure 3 , with the increase of pH, the degradation rate of Klabochia sugare first increases and then decreases, when the pH is high, the activity of Klabochia sugare is inhibited, leading to the decrease of degradation rate, the optimal degradation pH is 7.5.
[0046] As can be seen from Figure 4 , with the increase of substrate concentration, the degradation rate of Klabochia sugare shows a decreasing trend, the optimal degradation condition is benzene series concentration 100mg / L.
[0047] As can be seen from Figure 5 , with the increase of inoculation amount, the degradation rate of Klabochia sugare shows a increasing trend, but when the inoculation amount is greater than 10%, the degradation rate of Klabochia sugare increases little, so in order to save resources, the optimal inoculation amount is 10%.
[0048] As can be seen from the above, under the optimal culture conditions (temperature 30℃, pH 7.5, benzene series concentration 100mg / L, inoculation amount 10%), Klabochia sugare is cultured for 8d, the degradation rate of Klabochia sugare to benzene series is 50.82% for toluene, 49.18% for ethylbenzene, 46.75% for benzene, and 45.52% for dimethylbenzene.
[0049] (4) Water disinfection:
[0050] After the degradation of pollutants in water by Klabochia saccharophila, water disinfection is needed to ensure that the water quality meets safety standards and to avoid the impact of residual pathogenic bacteria on water safety. Klabochia saccharophila is a pathogenic bacterium with strong vitality, so effective disinfection measures must be taken after water treatment to ensure that the pathogenic bacteria in the water are completely inactivated. As a strong oxidizing agent, ozone (O3) can effectively inactivate pathogenic bacteria by damaging the cell wall, protein, or nucleic acid of microorganisms. Ozone disinfection not only rapidly inactivates Klabochia saccharophila in water, but also removes color, odor, and organic matter in water, improving water quality. The oxygen generated after ozone decomposition is harmless, so there are no harmful disinfection by-products left.
[0051] 1) Equipment selection and inspection:
[0052] The ozone generator needs to ensure normal operation and can generate sufficient ozone concentration according to the required flow. Common ozone generators use corona discharge or ultraviolet methods to generate ozone, and the equipment should meet the scale requirements of the treated water. At the same time, a suitable bubble generator is selected, usually a fine-bubble generator or a micro-bubble generator, which can inject ozone gas into the water in the form of small bubbles, increasing the contact time between the water and the ozone gas. The ozone generator usually needs external air or pure oxygen supply. During water quality treatment, it is recommended to use pure oxygen to generate ozone, as pure oxygen can improve the efficiency of ozone generation.
[0053] 2) Application parameters (Table 1):
[0054] Table 1 Parameter settings
[0055]
[0056] Example 2
[0057] Practical application of Klabochia saccharophila:
[0058] Take 100 mL of lake water from the Youth Lake of Tianjin University, add 5 mg / L of toluene, ethylbenzene, benzene, and xylene to the obtained lake water sample, and then inoculate the seed liquid obtained in step (1) of Example 1 to the logarithmic phase with an inoculation amount of 10%. After 8 days of treatment at room temperature, detect the benzene series according to the method in Example 1. The final concentrations of toluene, ethylbenzene, benzene, and xylene are 3.15 mg / L, 3.44 mg / L, 3.87 mg / L, and 4.12 mg / L, respectively. The highest degradation rate of the strain on the benzene series can reach 62%. This shows that Klabochia saccharophila can effectively decompose benzene series in sewage and can be used to prepare microbial preparations for degrading sewage.
[0059] Therefore, the application adopts the above-mentioned application of the Crabtree-positive Kluyvera bacteria in degrading aromatic hydrocarbons, inoculates the Kluyvera bacteria into sewage, can effectively decompose benzene series in the sewage, the degradation rate reaches more than 50%, and can be used for preparing a microbial preparation for degrading sewage.
[0060] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.
Claims
1. An application of Saccharophila Crabtree in the degradation of aromatic hydrocarbons, characterized by: The saccharophilic Crabtree bacteria are of the Brucella family and belong to the genus Saccharophilic Crabtree ATCC19623. The aromatic hydrocarbons are benzene series, which include benzene, toluene, ethylbenzene and xylene.
2. The use of Saccharophila Crabtree in the degradation of aromatic hydrocarbons according to claim 1, characterized in that: The culture conditions of the saccharophilic Crabtree bacteria are as follows: a temperature of 15-40° C., a pH value of 7-8, an inoculation amount of 5-15%, and a benzene series concentration of 100 mg / L.
3. The use of Saccharophila Crabtree in the degradation of aromatic hydrocarbons according to claim 1, characterized in that: When applied, the degradation rate of aromatic hydrocarbons by Saccharophila Crabtree was greater than 45%.
4. An application of Saccharophilus Crabtree in the degradation of benzene series in sewage, characterized by: The Saccharophila Crabtree is Brucella genus ATCC19623; the benzene series are benzene, toluene, ethylbenzene and xylene.
5. A method for degrading benzene series in sewage using Saccharophila Crabtree, characterized in that: Saccharophilic Crabtree Bacteria is inoculated into sewage to degrade benzene series in the sewage; the Saccharophilic Crabtree Bacteria is Brucella family Saccharophilic Crabtree Bacteria genus ATCC19623; the benzene series are benzene, toluene, ethylbenzene and xylene.