Escherichia coli T0P10-B and application thereof
By using Escherichia coli TOP10-B to degrade chlorinated paraffin in fermentation medium, the problem of low degradation efficiency of medium-chain chlorinated paraffin in the prior art was solved, and a rapid and efficient degradation effect was achieved, which had important environmental governance application value.
Patent Information
- Application Number
- CN202510180728.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-04-15
AI Technical Summary
The prior art has low efficiency in degrading medium-chain chlorinated paraffin (MCCPs), and has a long effective degradation cycle, making it difficult to meet the needs of environmental governance.
A strain of Escherichia coli TOP10-B was proposed, which was able to rapidly degrade short-chain chlorinated paraffin (SCCPs) and medium-chain chlorinated paraffin (MCCPs) in fermentation medium, with degradation rates reaching 51.8% and 48.1% within 20 days.
Escherichia coli T0P10-B significantly shortens the degradation cycle of chlorinated paraffin, achieves a high degradation efficiency, and has important application prospects for environmental governance.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to an Escherichia coli T0P10-B and an application thereof. Background Art
[0002] Chlorinated paraffins (CPs) are industrial mixed organic chemical products composed of alkanes of different chain lengths and degrees of chlorination. m H (2m+2-n) Cl n (where m = 10-30, n = 1-17), chlorine content is between 30-72%. According to the length of its carbon chain, it can be divided into three categories, namely short-chain chlorinated paraffins (C10-C13, shortchain chlorinated paraffins, SCCPs), medium-chain chlorinated paraffins (C14-C17, medium chain chlorinated paraffins, MCCPs) and long-chain chlorinated paraffins (C18-C30, Longchain chlorinated paraffins, LCCPs).
[0003] Chlorinated paraffins have the characteristics of low volatility, good flame retardancy, good electrical insulation, and low cost. Therefore, they are used as important flame retardants and plasticizers and are widely used in industrial production. Studies have shown that SCCPs are biotoxic and bioaccumulative, and the shorter the carbon chain, the stronger the toxicity of the chlorinated paraffin. Currently, chlorinated paraffins have been detected in a variety of organisms. Several strains of SCCPs-degrading bacteria that can use SCCPs as carbon sources and energy sources have been identified, but the disadvantage is that the effective degradation cycle is relatively long (such as 30-100 days), and there are fewer strains that can effectively degrade MCCPs. Summary of the invention
[0004] Based on the technical problems existing in the background technology, the present invention proposes an Escherichia coli T0P10-B and its application. The Escherichia coli T0P10-B of the present invention has good performance in degrading chlorinated paraffins. After 20 days of degradation, the SCCPs degradation rate can reach 51.8%, and the MCCPs degradation rate can reach 48.1%.
[0005] The invention provides an Escherichia coli T0P10-B, whose preservation number is CCTCC NO:M 20242003.
[0006] 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 T0P10-B. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on September 19, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO:M 20242003.
[0007] The present invention also proposes the use of the Escherichia coli T0P10-B in degrading chlorinated paraffin.
[0008] Preferably, the chlorinated paraffin is at least one of short-chain chlorinated paraffin and medium-chain chlorinated paraffin.
[0009] The chlorine content of the above short-chain chlorinated paraffin is ≤60%, more preferably ≤50%.
[0010] The chlorine content of the above medium-chain chlorinated paraffin is less than 45%, more preferably ≤40%.
[0011] The present invention also provides a method for degrading chlorinated paraffin, comprising the following steps: fermenting and degrading the above-mentioned Escherichia coli T0P10-B in a fermentation medium containing chlorinated paraffin.
[0012] Preferably, in the fermentation medium containing chlorinated paraffin, the concentration of chlorinated paraffin is 45-55 mg / L; more preferably 50 mg / L.
[0013] 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.
[0014] 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.
[0015] Preferably, the fermentation temperature is 35-37°C.
[0016] Preferably, during the fermentation process, the dissolved oxygen content of the fermentation liquid is maintained at 23-25%.
[0017] Preferably, during the fermentation process, the pH of the fermentation broth is maintained at 7.0-7.2.
[0018] Preferably, during the fermentation process, the reducing sugar content in the fermentation broth is maintained at not less than 0.4%.
[0019] The above water is sterile water.
[0020] Beneficial effects:
[0021] The present invention isolates an Escherichia coli T0P10-B from the sludge of a sewage outlet in Huoqiu County, Anhui Province, and finds that the strain 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
[0022] Figure 1 This is an electron micrograph of Escherichia coli T0P10-B strain staining. DETAILED DESCRIPTION
[0023] 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.
[0024] Example 1
[0025] 1. Screening and Isolation of Escherichia coli T0P10-B
[0026] 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.
[0027] 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.
[0028] Take the single strain obtained in step 1 and perform crystal violet staining and morphological observation. The results are as follows Figure 1 shown.
[0029] Figure 1This is an electron micrograph of Escherichia coli T0P10-B strain staining.
[0030] 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.
[0031] Molecular biological identification was performed on the above-mentioned single strain, and it was finally determined that the above-mentioned single strain was Escherichia coli, and it was named Escherichia coli T0P10-B. It was deposited in the China Center for Type Culture Collection (CCTCC) on September 19, 2024, with the preservation address of Wuhan University, Wuhan, China, and the preservation number is CCTCC NO:M 20242003.
[0032] Example 2
[0033] A method for degrading chlorinated paraffin comprises the following steps:
[0034] Escherichia coli T0P10-B was inoculated into LB liquid culture medium and cultured at 37° C. with shaking for 48 hours to obtain an activation solution;
[0035] 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 OD 600 is 8.2, and the seed solution is obtained;
[0036] 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;
[0037] 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.
[0038] 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%.
[0039] 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.
[0040] Table 1 Degradation rate of chlorinated paraffin
[0041] Time (day) Degradation rate of short-chain chlorinated paraffins (%) Degradation rate of medium-chain chlorinated paraffin (%) 5 11.5 11.3 10 24.4 23.0 15 38.2 35.6 20 51.8 48.1
[0042] 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.
[0043] Comparative Example 1
[0044] 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.
[0045] 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 T0P10-B, whose deposit number is CCTCC NO:M20242003.
2. Use of the Escherichia coli TOP10-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 T0P10-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%.