Application of Crabutella saccharivorans in degradation of aromatic hydrocarbon

Abstract

The invention relates to the technical field of industrial and domestic sewage treatment, and discloses an application of Crabutella saccharivorans in degradation of aromatic hydrocarbon. The saccharophilous clabutella is a saccharophilous clabutella belonging to a bruceae family, namely ATCC19623 (Attenuated Colorful Culture Collection Center 19623). The aromatic hydrocarbon is benzene series, and the benzene series comprise benzene, methylbenzene, ethylbenzene and xylene. The culture conditions of the Crabutella saccharophila are as follows: the temperature is 15-40 DEG C, the pH value is 7-8, and the inoculum size is 5-15%. According to the application of the Crabutella saccharivorans in degradation of the aromatic hydrocarbon, the Crabutella saccharivorans is inoculated into sewage, benzene series in the sewage can be effectively decomposed, the degradation rate reaches 50% or above, and the Crabutella saccharivorans can be used for preparing microbial preparations for degrading the sewage.

Description

Technical Field

[0001] The present invention relates to the technical field of industrial and domestic sewage treatment, and in particular to the application of Crabtreea saccharophila in the degradation of aromatic hydrocarbons. Background Art

[0002] In recent years, benzene series compounds, as an important class of organic compounds, are widely present in nature and human production activities. Benzene series compounds are not only important raw materials for many chemical products, but also widely used in the fields of fuels, pharmaceuticals, and pesticides. However, due to the wide use of benzene series compounds and the stability of their chemical properties, they remain in the environment for a long time, posing a potential threat to the ecosystem and human health. Benzene series compounds refer to organic compounds containing a benzene ring structure, such as benzene, toluene, ethylbenzene, xylene, etc. Their main sources include the extraction and processing of petroleum and coal, fuel combustion, industrial production waste gas, wastewater discharge, etc. After entering the environment, benzene series compounds often exist in the air, water, and soil in the form of gaseous, dissolved, or adsorbed states.

[0003] The benzene ring in the molecular structure of benzene series compounds gives them high chemical stability and hydrophobicity, resulting in a long degradation period in the environment. In addition, some polycyclic aromatic hydrocarbons (PAHs) have carcinogenic, mutagenic, and reproductive toxicity, becoming important pollutants in environmental risks. For example, the concentration of benzene series compounds detected in industrial wastewater or domestic wastewater is often relatively high, and these compounds cause long-term harm to the human body through accumulation in drinking water or the food chain. For benzene series compound pollution, scientists have developed a variety of treatment technologies. Biological treatment technologies use the metabolic action of microorganisms to degrade benzene series compounds and convert them into harmless products such as carbon dioxide and water. Compared with other technologies, biological treatment has the characteristics of economy, high efficiency, and environmental friendliness. By screening strains with high efficiency in degrading benzene series compounds and applying bioaugmentation technology, the removal efficiency of benzene series compounds can be significantly improved. Recent research shows that developing characteristic strains with high degradation efficiency for aromatic hydrocarbons and optimizing their application conditions in actual sewage treatment are important directions for dealing with benzene series compound pollution. In addition, studying the toxicity of intermediate products during the degradation process of benzene series compounds and their interactions with environmental factors are also important topics for achieving the complete removal of pollutants. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of Crabtreea saccharophila in the degradation of aromatic hydrocarbons. When Crabtreea saccharophila is inoculated into sewage, it can effectively decompose benzene series compounds in the sewage, and the degradation rate reaches more than 50%, and it can be used to prepare microbial preparations for degrading sewage.

[0005] To achieve the above purpose, the present invention provides the application of Crabtreea saccharophila in the degradation of aromatic hydrocarbons, and the Crabtreea saccharophila is Crabtreea saccharophila of the family Brucellaceae, genus ATCC19623.

[0006] Further, the aromatic hydrocarbon is a benzene series compound.

[0007] Further, the culture conditions of the Crabtreea saccharophila are a temperature of 15 - 40 °C, a pH value of 7 - 8, an inoculum amount of 5 - 15%, and a benzene series compound concentration of 100 mg / L.

[0008] Further, during application, the degradation rate of the aromatic hydrocarbon by the Crabtreea saccharophila is greater than 45%.

[0009] The present invention also provides an application of the Crabtreea saccharophila in degrading benzene series compounds in sewage, and the Crabtreea saccharophila is Crabtreea saccharophila of the family Brucellaceae, genus ATCC19623.

[0010] The present invention also provides a microbial preparation for degrading benzene series compounds, and the active ingredient of the microbial preparation includes Crabtreea saccharophila of the family Brucellaceae, genus ATCC19623.

[0011] The present invention also provides a method for degrading benzene series compounds in sewage by the Crabtreea saccharophila, inoculating the Crabtreea saccharophila into the sewage to degrade the benzene series compounds in the sewage.

[0012] The advantages and positive effects of the application of the Crabtreea saccharophila in degrading aromatic hydrocarbons according to the present invention are as follows:

[0013] 1. By inoculating the Crabtreea saccharophila into the sewage, the benzene series compounds in the sewage can be effectively decomposed, and the degradation rate reaches more than 50%, and it can be used for preparing a microbial preparation for degrading sewage.

[0014] The technical solution of the present invention will be further described in detail below with reference to the drawings and examples. Description of the Drawings

[0015] Figure 1 It is a curve showing the change of the benzene series compound degradation rate with the culture days in Example 1 of the present invention;

[0016] Figure 2 It is a curve showing the change of the benzene series compound degradation rate with the temperature in Example 1 of the present invention;

[0017] Figure 3 It is a curve showing the change of the benzene series compound degradation rate with the pH in Example 1 of the present invention;

[0018] Figure 4 It is a curve showing the change of the benzene series compound degradation rate with the substrate concentration in Example 1 of the present invention;

[0019] Figure 5 It is a curve showing the change of the benzene series compound degradation rate with the inoculum amount in Example 1 of the present invention. Detailed Embodiments

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention pertains.

[0022] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally determined according to national standards. The experimental instruments, equipment, and reagents not indicated as sources in the following embodiments are all commercially available raw materials.

[0023] The equipment used in each step of the following embodiments is all conventional equipment. If there is no corresponding national standard, it shall be carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0024] Unless otherwise defined or explained, all the professional and scientific terms used in the present invention have the same meanings as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0025] The preservation number of Crabtreea saccharophila is ATCC19623, and it was purchased from Mingzhou Biology in April 2024.

[0026] Example 1

[0027] Cultivation and detection of Crabtreea saccharophila:

[0028] (1) Preparation of the Crabtreea saccharophila seed solution:

[0029] Inoculate Crabtreea saccharophila (purchased from Mingzhou Biology, with the preservation number of the strain being ATCC19623) in a liquid medium containing benzene series compounds, and culture it at 25°C until the logarithmic phase to prepare a seed solution;

[0030] The formulation of the liquid medium containing benzene series compounds is as follows: 3.5 g / L of Na2HPO4·2H2O, 1.0 g / L of KH2PO, 0.5 g / L of (NH4)2SO4, 0.2 g / L of CaCl2, 0.1 g / L of MgCl2·6H2O, 50 mf / L of toluene, 50 mg / L of benzene, 50 mg / L of ethylbenzene, 50 mg / L of xylene, and 1.0 mL / L of trace elements (trace elements: 0.5 g / L of EDTA, 0.2 g / L of FeSO4·7H2O, 0.08 g / L of MnSO4·H2O, 0.3 g / L of boric acid, 0.2 g / L of CoCl2·6H2O, 0.01 g / L of CuCl2·2H2O, 0.01 g / L of NiCl2·H2O, 0.02 g / L of Na2MoO4·2H2O).

[0031] (2) Optimization of strain conditions:

[0032] The seed liquid cultured to the logarithmic phase was inoculated into 100 mL of PBSA liquid medium under different single-variable conditions and cultured under different conditions. The culture temperatures were 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C respectively; the pH values were 3, 5, 6, 7, 7.5, 8, 9, and 11 respectively; the initial concentrations of benzene series compounds were 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L respectively, and the inoculation amount of the bacterial liquid was 1%, 2%, 5%, 10%, 15%, and 20%. It was cultured at a rotation speed of 150 r / min.

[0033] (3) Enrichment and elution of benzene series compounds after degradation by the strain:

[0034] 15 mL was taken out from the medium cultured in step (2), centrifuged for 20 min, and the supernatant was extracted with an activated solid-phase small column (0asis HLB 6 mL, 500 mg). For the remaining slurry, 2 mL of methanol was added, and then ultrasonicated three times, 10 min each time. After centrifugation again, the supernatant was diluted with 10 mL of deionized water and finally extracted with the same solid-phase small column.

[0035] Activation (pretreatment) of the solid-phase small column: A. Activate the C18 small column with 5 mL of methanol at a flow rate of 2 ml / min;

[0036] B. Activate the C18 small column with 10 mL of chromatographic pure water at a flow rate of 2 mL / min;

[0037] Elution of the target analyte: Elute the column with 10 mL of methanol at a flow rate of 15 mL / min;

[0038] Then, dry the extract with N2 and dissolve it with acetone;

[0039] Finally, filter it into an amber liquid vial with a 0.22 μm filter head for HPLC analysis to be determined.

[0040] Liquid chromatography conditions for HPLC analysis:

[0041] Column temperature: 30 °C; Mobile phase: methanol: water = 3:1; Flow rate: 1 mL / min; Retention time: 2.5 min.

[0042] The culture results are as shown in Figure 1 、 2 、3, and 4.

[0043] It can be seen from Figure 1 that as the culture time prolongs, the degradation rate of aromatic hydrocarbons gradually increases. However, after the eighth day, as time goes on, the increase in the degradation rate of aromatic hydrocarbons is not significant. Therefore, the optimal culture time is 8 d.

[0044] It can be seen from Figure 2 that as the temperature increases, the degradation rate of Crabtreea saccharophila first increases and then decreases. When the temperature is relatively high, the activity of Crabtreea saccharophila will be affected, resulting in a decrease in the degradation rate. The optimal degradation temperature is 30 °C.

[0045] It can be seen from Figure 3 that as the pH increases, the degradation rate of Crabtreea saccharophila first increases and then decreases. When the pH is relatively high, the activity of Crabtreea saccharophila is inhibited, resulting in a decrease in the degradation rate. The optimal degradation pH is 7.5.

[0046] It can be seen from Figure 4 that as the substrate concentration increases, the degradation rate of Crabtreea saccharophila shows a decreasing trend. The optimal degradation condition is that the concentration of benzene series compounds is 100 mg / L.

[0047] It can be seen from Figure 5 that as the inoculation amount increases, the degradation rate of Crabtreea saccharophila shows an increasing trend. However, when the inoculation amount is greater than 10%, the increase in the degradation rate of Crabtreea saccharophila is not significant. Therefore, to save resources, the optimal inoculation amount is 10%.

[0048] In summary, under the optimal culture conditions (temperature 30 °C, pH 7.5, concentration of benzene series compounds 100 mg / L, inoculation amount 10%) for 8 d, the degradation rates of Crabtreea saccharophila for benzene series compounds are as follows: toluene reaches 50.82%, ethylbenzene reaches 49.18%, benzene reaches 46.75%, and xylene reaches 45.52%.

[0049] (4) Water body disinfection:

[0050] After using Crabtreea saccharophila to degrade pollutants in water, water disinfection is required to ensure that the water quality meets safety standards and to prevent residual pathogenic bacteria from affecting water safety. Crabtreea saccharophila is a pathogenic bacterium with strong vitality. Therefore, after water treatment, effective disinfection measures must be taken to ensure that the pathogenic bacteria in the water are completely inactivated. As a strong oxidant, ozone (O3) can effectively inactivate pathogenic bacteria by destroying the cell walls, proteins, or nucleic acids of microorganisms. Ozone disinfection can not only quickly inactivate Crabtreea saccharophila in water, but also remove the color, odor, and organic matter in water, improving water quality. After ozone decomposes, the generated oxygen is harmless, so no harmful disinfection by-products will be left.

[0051] 1) Equipment selection and inspection:

[0052] The ozone generator needs to ensure normal operation and be able to generate sufficient ozone concentration according to the required flow rate. Common ozone generators use the corona discharge method or the ultraviolet method to generate ozone, and the equipment should meet the scale requirements of the treated water body. At the same time, select a suitable bubble generator, usually a fine pore bubble generator or a microbubble generator, so that the ozone gas can be injected into the water in the form of smaller bubbles, increasing the contact time between the water body and the ozone gas. Ozone generators usually require an external air or pure oxygen supply. During the water quality treatment process, it is recommended to use pure oxygen to generate ozone because pure oxygen can improve the ozone generation efficiency.

[0053] 2) Application parameters (Table 1):

[0054] Table 1 Parameter settings

[0055]

[0056] Example 2

[0057] Practical application of Crabtreea 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 culture it to the logarithmic phase according to the seed solution obtained in step (1) of Example 1. The inoculation amount is 10%. At room temperature, treat for 8 days, and detect the benzene series compounds according to the method in Example 1. Finally, the toluene concentration is 3.15 mg / L, the ethylbenzene concentration is 3.44 mg / L, the benzene concentration is 3.87 mg / L, and the xylene concentration is 4.12 mg / L. The degradation rate of this strain to the benzene series compounds can reach up to 62%. It shows that Crabtreea saccharophila can effectively decompose the benzene series compounds in sewage and can be used to prepare microbial preparations for degrading sewage.

[0059] Therefore, the present invention adopts the above application of Crabtreea saccharophila in degrading aromatic hydrocarbons. When Crabtreea saccharophila is inoculated into sewage, it can effectively decompose benzene series substances in the sewage, and the degradation rate reaches more than 50%, and it can be used to prepare microbial agents for degrading sewage.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of Crabtreea saccharophila in degrading aromatic hydrocarbons, characterized in that: The Crabtreea saccharophila is Crabtreea saccharophila of the family Brucellaceae, ATCC19623.

2. Use of Crabtreea saccharophila according to claim 1 in degrading aromatic hydrocarbons, characterized in that: The aromatic hydrocarbon is a benzene series substance, and the benzene series substance includes benzene, toluene, ethylbenzene, and xylene.

3. Use of a Crabtree-positive sugar-loving bacterium according to claim 1 in degrading aromatic hydrocarbons, characterized in that: The culture conditions of the Crabtreea saccharophila are a temperature of 15 - 40 °C, a pH value of 7 - 8, an inoculation amount of 5 - 15%, and a benzene series substance concentration of 100 mg / L.

4. Use of Crabtree-positive Saccharomyces cerevisiae according to claim 1 in degrading aromatic hydrocarbons, characterized in that: During application, the degradation rate of the aromatic hydrocarbon by the Crabtreea saccharophila is greater than 45%.

5. Application of Crabtreea saccharophila in degrading benzene series compounds in sewage, characterized in that: The Crabtreea saccharophila is Crabtreea saccharophila of the family Brucellaceae, ATCC19623.

6. A microbial preparation for degrading benzene series compounds, characterized in that: The active ingredient of the microbial preparation includes Crabtreea saccharophila of the family Brucellaceae, ATCC19623.

7. Method for Klebsiella oxytoca to degrade benzene series compounds in sewage, characterized in that: The Crabtreea saccharophila is inoculated into sewage to degrade the benzene series substances in the sewage.

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