Escherichia coli DH5 alpha-B and application thereof
By using Escherichia coli DH5α-B in a specific fermentation medium, the problem of low degradation efficiency of chlorinated paraffin in the prior art is solved, and efficient degradation of short-chain and medium-chain chlorinated paraffin is achieved, which has significant environmental governance application value.
Patent Information
- Application Number
- CN202510180730.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art is inefficient in degrading medium-chain and long-chain chlorinated paraffin, especially the degradation strains of medium-chain chlorinated paraffin are fewer, and the degradation cycle of existing strains is relatively long, making it difficult to meet the needs of environmental governance.
Escherichia coli DH5α-B was used. This strain was identified and named through molecular biology and has good performance in degrading chlorinated paraffin. It can achieve a degradation rate of short-chain chlorinated paraffin in 20 days and a degradation rate of medium-chain chlorinated paraffin. This strain is fermented and degraded in a specific fermentation medium.
Escherichia coli DH5α-B significantly improves the degradation efficiency of chlorinated paraffin, especially in a short period of time, showing high efficiency in the degradation of medium-chain chlorinated paraffin, and has huge environmental governance application prospects.
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Figure CN119875945A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, in particular to Escherichia coli DH5α-B and applications thereof. Background Art
[0002] Chlorinated paraffins (CPs) are complex mixtures formed by direct chlorination of n-alkane raw material free radicals. CPs are also called polychlorinated alkanes (PCAs), and the degree of chlorination is usually 30-70% (mass fraction). According to the length of the carbon chain, CPs can be divided into short chain (SCCPs, C10-C13), medium chain (MCCPs, C14-C17) and long chain (LCCPs, C18-C30).
[0003] Chlorinated paraffins are flame retardant, low volatile, and insulating, and are widely used in many fields. However, chlorinated paraffins are also toxic pollutants that are difficult to degrade and are widely distributed in various environmental media such as the atmosphere, organisms, water, soil, and sediments, and have even been detected in polar regions. Compared with MCCPs and LCCPs, SCCPs have the most special physical and chemical properties, the greatest potential for environmental release, and the "triple effects" of carcinogenicity, teratogenicity, and mutagenicity. They are the most toxic and were eventually included in the list of controlled persistent organic pollutants (POPs) in Annex A of the Stockholm Convention on Persistent Organic Pollutants in 2017.
[0004] Therefore, it is necessary to reduce and remove chlorinated paraffins in the environment. The removal methods of chlorinated paraffins mainly include physical and chemical methods and biological removal methods. Among them, biological removal, as the most economical method, bioadsorbs or biodegrades pollutants. It is considered to be one of the most reliable methods for removing organic pollutants in the environment and is highly favored by researchers. It is generally divided into bacterial degradation method, bacterial adsorption method, plant absorption method and other theoretically feasible methods (fungi, algae and animals). SCCPs-degrading bacteria that can use SCCPs as carbon sources and energy sources have been identified, but the disadvantage is that their effective degradation cycle is longer (such as 30-100 days), and there are fewer strains that can effectively degrade MCCPs. Summary of the invention
[0005] Based on the technical problems existing in the background technology, the present invention proposes an Escherichia coli DH5α-B and an application thereof. The Escherichia coli DH5α-B of the present invention has good performance in degrading chlorinated paraffins. After 20 days of degradation, the degradation rate of SCCPs can reach 53.3%, and the degradation rate of MCCPs can reach 49.9%.
[0006] The invention provides Escherichia coli DH5α-B, whose preservation number is CCTCCNO:M 20242005.
[0007] The inventor isolated a strain from the sludge of the sewage outlet in Huoqiu County, Anhui Province, which was identified as Escherichia coli by molecular biology and named Escherichia coli DH5α-B. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on September 19, 2024, with the deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the deposit number is CCTCC NO:M 20242005.
[0008] The invention also proposes the application of the Escherichia coli DH5α-B in degrading chlorinated paraffin.
[0009] Preferably, the chlorinated paraffin is at least one of short-chain chlorinated paraffin and medium-chain chlorinated paraffin.
[0010] The chlorine content of the above short-chain chlorinated paraffin is ≤60%, more preferably ≤50%.
[0011] The chlorine content of the above medium-chain chlorinated paraffin is less than 45%, more preferably ≤40%.
[0012] The present invention also provides a method for degrading chlorinated paraffin, comprising the following steps: fermenting and degrading the above-mentioned Escherichia coli DH5α-B in a fermentation medium containing chlorinated paraffin.
[0013] Preferably, in the fermentation medium containing chlorinated paraffin, the concentration of chlorinated paraffin is 45-55 mg / L; more preferably 50 mg / L.
[0014] Preferably, the formula of the fermentation medium is: 1L of water contains 28-32g sucrose, 45-55mg chlorinated paraffin, 2-3g ammonium sulfate, 1-2g potassium dihydrogen phosphate, 1-2g dipotassium hydrogen phosphate, 0.8-1.2g citric acid, 0.6-0.8g magnesium sulfate heptahydrate, 0.08-0.12g ferrous sulfate heptahydrate, 1.5-2.5mg biotin, and the pH is 7.0-7.2.
[0015] Preferably, the formula of the fermentation medium is: 30 g sucrose, 50 mg paraffin wax, 2.5 g ammonium sulfate, 1.5 g potassium dihydrogen phosphate, 1.5 g dipotassium hydrogen phosphate, 1 g citric acid, 0.7 g magnesium sulfate heptahydrate, 0.1 g ferrous sulfate heptahydrate, 2 mg biotin in 1 L of water, and the pH is 7.0.
[0016] Preferably, the fermentation temperature is 35-37°C.
[0017] Preferably, during the fermentation process, the dissolved oxygen content of the fermentation liquid is maintained at 23-25%.
[0018] Preferably, during the fermentation process, the pH of the fermentation broth is maintained at 7.0-7.2.
[0019] Preferably, during the fermentation process, the reducing sugar content in the fermentation broth is maintained at not less than 0.4%.
[0020] The above water is sterile water.
[0021] Beneficial effects:
[0022] The present invention isolates an Escherichia coli DH5α-B from the sludge of a sewage outlet in Huoqiu County, Anhui Province. It is found that the Escherichia coli DH5α-B has good performance in degrading short-chain chlorinated paraffins and medium-chain chlorinated paraffins, and has great application prospects in promoting environmental governance and the like. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is an electron micrograph of Escherichia coli DH5α-B strain staining. DETAILED DESCRIPTION
[0024] The technical solutions of the present invention are described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.
[0025] Example 1
[0026] 1. Screening and Isolation of Escherichia coli DH5α-B
[0027] Take the sludge from the sewage outlet of Huoqiu County, Anhui Province, mix 1g of the sludge sample with 10mL of sterile saline, take 1mL of the suspension and dilute it on the solid culture medium, culture it at 37℃ for 2-3 days, separate the colonies after they grow, pick the colonies and streak them on a new solid plate, culture it at 37℃, and after new colonies grow, continue to streak them on a new solid plate, and repeat this step until colonies with a single morphology grow on the solid culture medium.
[0028] The solid culture medium used above is the same, all containing: 10g of tryptone, 5g of yeast extract, 15g of agar and 10g of sodium chloride, 20mg of chlorinated paraffin (the weight ratio of short-chain chlorinated paraffin to medium-chain chlorinated paraffin is 1:1) in 1L of water, pH 7.0, and sterilized at 121°C for 15min.
[0029] Take the single strain obtained in step 1 and perform crystal violet staining and morphological observation. The results are as follows Figure 1 shown.
[0030] Figure 1 This is an electron micrograph of Escherichia coli DH5α-B strain staining.
[0031] Depend on Figure 1 It can be seen that the strain is in the shape of a short rod, with both ends being blunt and approximately club-shaped.
[0032] Molecular biological identification was carried out on the above-mentioned single strain, and it was finally determined that the above-mentioned single strain was Escherichia coli, which was named Escherichia coli DH5α-B. It was deposited in the China Center for Type Culture Collection (CCTCC) on September 19, 2024, with the preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation number is CCTCC NO:M 20242005.
[0033] Example 2
[0034] A method for degrading chlorinated paraffin comprises the following steps:
[0035] Escherichia coli DH5α-B was inoculated into LB liquid culture medium and cultured at 37°C with shaking for 48 hours to obtain an activation solution;
[0036] The activation solution was inoculated into the seed culture medium (1L seed culture medium contains: 28g sucrose, 8g yeast extract, 5.5g potassium dihydrogen phosphate, 4.5g dipotassium hydrogen phosphate, 1.2g magnesium sulfate heptahydrate, 40mg ferrous sulfate heptahydrate, 0.2mg biotin, pH 7.0) at a 10% inoculation rate, and cultured at 37°C with shaking until OD600 is 8.2, and the seed solution is obtained;
[0037] Inoculating the seed liquid into the fermentation medium at an inoculum rate of 10%, adjusting the temperature to 35-37° C. and the dissolved oxygen content to 23-25%, and fermenting, and during the fermentation process, maintaining the pH of the fermentation liquid at 7.0 by adding ammonia water, maintaining the reducing sugar content in the fermentation liquid at not less than 0.4% by adding sucrose, and defoaming by adding a defoaming agent;
[0038] The formula of the fermentation medium is as follows: 1L of water contains: 30g sucrose, 50mg chlorinated paraffin (short-chain chlorinated paraffin or medium-chain chlorinated paraffin), 2.5g ammonium sulfate, 1.5g potassium dihydrogen phosphate, 1.5g dipotassium hydrogen phosphate, 1g citric acid, 0.7g magnesium sulfate heptahydrate, 0.1g ferrous sulfate heptahydrate, 2mg biotin, and the pH is 7.0.
[0039] The chlorine content of the short-chain chlorinated paraffin to be degraded is ≤60%, and the chlorine content of the medium-chain chlorinated paraffin to be degraded is <45%.
[0040] The fermentation broths at different fermentation times were tested and the degradation rates of chlorinated paraffins were calculated. The results are shown in Table 1.
[0041] Table 1 Degradation rate of chlorinated paraffin
[0042] Time (day) Degradation rate of short-chain chlorinated paraffins (%) Degradation rate of medium-chain chlorinated paraffin (%) 5 11.8 11.5 10 25.1 23.6 15 39.2 36.8 20 53.3 49.9
[0043] It can be seen from Table 1 that the present invention has a good degradation rate for both short-chain chlorinated paraffins and medium-chain chlorinated paraffins within just 20 days.
[0044] Comparative Example 1
[0045] Escherichia coli ATCC 25922 was fermented according to the method of Example 2. It was found that short-chain chlorinated paraffins and medium-chain chlorinated paraffins were not degraded after 20 days of fermentation.
[0046] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. An Escherichia coli DH5α-B, whose deposit number is CCTCC NO:M20242005.
2. Use of the Escherichia coli DH5α-B as claimed in claim 1 in degrading chlorinated paraffin.
3. The application according to claim 2, characterized in that: The chlorinated paraffin is at least one of short-chain chlorinated paraffin and medium-chain chlorinated paraffin.
4. A method for degrading chlorinated paraffin, characterized in that: The method comprises the following steps: fermenting and degrading the Escherichia coli DH5α-B as claimed in claim 1 in a fermentation medium containing chlorinated paraffin.
5. The method for degrading chlorinated paraffin according to claim 4, characterized in that: In the fermentation medium containing chlorinated paraffin, the concentration of chlorinated paraffin is 45-55 mg / L.
6. The method for degrading chlorinated paraffin according to claim 4 or 5, characterized in that: The formula of the fermentation medium is as follows: 1L of water contains 28-32g of sucrose, 45-55mg of chlorinated paraffin, 2-3g of ammonium sulfate, 1-2g of potassium dihydrogen phosphate, 1-2g of dipotassium hydrogen phosphate, 0.8-1.2g of citric acid, 0.6-0.8g of magnesium sulfate heptahydrate, 0.08-0.12g of ferrous sulfate heptahydrate, 1.5-2.5mg of biotin, and the pH is 7.0-7.
2.
7. The method for degrading chlorinated paraffin according to any one of claims 4 to 6, characterized in that: The fermentation temperature is 35-37℃.
8. The method for degrading chlorinated paraffin according to any one of claims 4 to 7, characterized in that: During the fermentation process, the dissolved oxygen content of the fermentation liquid was maintained at 23-25%.
9. The method for degrading chlorinated paraffin according to any one of claims 4 to 8, characterized in that: During the fermentation process, the pH of the fermentation liquid was maintained at 7.0-7.
2.
10. The method for degrading chlorinated paraffin according to any one of claims 4 to 9, characterized in that: During the fermentation process, the reducing sugar content in the fermentation liquid is maintained at not less than 0.4%.