Slightly-changed tsukamurella LXY and application thereof
By separating and identifying a slightly changing bacteria LXY, the strain can use tinane as the only carbon source and energy growth and degrade the tin anxle efficiently, solving the problem of difficult degradation in existing technologies, and realized the tin anxane, and realized the tin alumane. The stable and efficient degradation provides the application foundation of biological method purification.
Patent Information
- Application Number
- CN202410735928.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-07
- Publication Date
- 2025-01-28
AI Technical Summary
No microbial strains that can efficiently degrade nonane and its analogues have been found in the prior art, resulting in nonane being present in the environment for a long time and pose a threat to human health and ecological security.
By separating and identifying a slightly changing bacteria LXY, the strain is a Gram -positive bacteria that requires oxygen and non -mortar spores, which can grow with tin alumane as the only carbon source and energy growth, and highly degrade the tinane. The strain is vaccinated in a wastewater or inorganic salt culture medium containing tensumane, and the pH value and temperature are controlled to achieve high -efficiency degradation of tinane.
The strain can stabilize and efficiently degrade the alkane, and has a strong ability to adapt to temperature and pH, and can maintain high -efficiency degradation performance under different conditions. In addition, the strain can also degrade the degradation, providing the engineering application basis containing virtic or alkane wastewater and exhaust gas.
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Figure CN119372079A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biodegradation, and in particular relates to a Tsukamurella variegata LXY and an application thereof. Background Art
[0002] Nonane is a saturated aliphatic hydrocarbon with the general properties of alkanes, including high chemical stability and flammability. As a non-polar organic solvent, this compound is commonly used in coatings, inks, cosmetics, rubber, resins and other industries. In addition, n-nonane is also widely used in laboratories as a solvent, standard substance and internal standard substance and other reagents. However, long-term exposure to nonane may cause skin irritation such as dryness, itching, redness and swelling. Inhalation of nonane may cause respiratory irritation symptoms such as headache, eye pain, difficulty breathing, coughing, etc. Inhalation of high concentrations of nonane may cause dizziness, loss of consciousness and even suffocation.
[0003] Due to the wide application of nonane, it is necessary to study the efficient degradation of nonane in the environment for human health. The biological method uses the metabolic capacity of microorganisms to degrade pollutants and convert them into harmless substances, thereby purifying the pollutants. Biological development has the advantages of being green, safe, and free of secondary pollution. If we can screen and separate microorganisms that can efficiently degrade nonane and degrade nonane into substances that are non-toxic to the human body and the environment, this will have far-reaching significance for maintaining human health and ecological safety.
[0004] However, no efficient degradation strains that can use nonane and its analogs as carbon and energy sources have been found so far. Summary of the invention
[0005] In view of the problems existing in the prior art, the object of the present invention is to design and provide a technical solution for Tsukamurella variegata LXY and its application.
[0006] In order to achieve the above objectives, this application specifically adopts the following technical solutions:
[0007] The first aspect of the present invention provides Tsukamurella paurometabola LXY for degrading nonane. The strain has been deposited in the China Center for Type Culture Collection on May 30, 2024, with a deposit number of CCTCC NO: M 20241105, and an address of 430072, Wuhan University, Wuhan, China.
[0008] The above-mentioned Tsukamurella paurometabola was isolated from the activated sludge of a chemical plant in Ningbo, Zhejiang. It is an obligate aerobic Gram-positive bacterium with large colonies, white, opaque, irregular and rough surfaces, and bacterial mosses growing along the streaks. After 16S rDNA sequencing, it was determined to be Tsukamurella paurometabola LXY and was named Tsukamurella paurometabola LXY.
[0009] The second aspect of the present invention provides a bacterial agent containing the above-mentioned Tsukamurella subspecies LXY.
[0010] The third aspect of the present invention provides the use of the above-mentioned Tsukamurella mutans LXY or the above-mentioned bacterial agent in degrading nonane.
[0011] Furthermore, in this application, the bacterial solution obtained by inoculating the Tsukamurella mutans LXY into wastewater containing nonane or culturing inorganic salts containing nonane is used as an enzyme source, and a degradation reaction is carried out in an inorganic salt culture medium at pH = 5.0-9.0 and 20°C-40°C.
[0012] Further, the inorganic salt culture medium concentration composition in the application is as follows: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.023g / L, trace element mother solution 1.0mL / L, pH 7.0, and the solvent is deionized water; the trace element mother solution concentration composition is as follows: FeSO4·7H2O 1.0g / L, CuSO4·5H2O 0.02g / L, H3BO3 0.014g / L, MnSO4·4H2O0.10g / L, ZnSO4·7H2O 0.10g / L, Na2MoO4·2H2O 0.02g / L, CoCl2·6H2O 0.02g / L, the solvent is deionized water.
[0013] Further, the initial concentration of nonane in the inorganic salt medium in the application is 72-360 mg / L, and the amount of the enzyme source is based on the OD 600 Counted as 0.01.
[0014] The fourth aspect of the present invention provides the use of the above-mentioned Tsukamurella mutans LXY or the above-mentioned bacterial agent in degrading octane.
[0015] Furthermore, in the application, the bacterial solution obtained by inoculating the Tsukamurella mutans LXY into wastewater containing octane or culturing inorganic salts containing octane is used as an enzyme source, and a degradation reaction is carried out in an inorganic salt culture medium at pH = 5.0-9.0 and 20°C-40°C.
[0016] Further, the inorganic salt culture medium concentration composition in the application is as follows: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.023g / L, trace element mother solution 1.0mL / L, pH 7.0, and the solvent is deionized water; the trace element mother solution concentration composition is as follows: FeSO4·7H2O 1.0g / L, CuSO4·5H2O 0.02g / L, H3BO3 0.014g / L, MnSO4·4H2O0.10g / L, ZnSO4·7H2O 0.10g / L, Na2MoO4·2H2O 0.02g / L, CoCl2·6H2O 0.02g / L, the solvent is deionized water.
[0017] Further, the initial concentration of octane in the inorganic salt medium in this application is 65-360 mg / L, and the amount of the enzyme source is based on the OD 600 Counted as 0.01.
[0018] The beneficial effects of the present invention are mainly reflected in: the present invention provides a highly efficient nonane-degrading bacterium, which is an aerobic, non-spore-forming, non-motile Gram-positive bacillus, and can grow with nonane as the sole carbon source and energy source while efficiently degrading the substrate; the strain can also degrade octane; the present invention lays a foundation for the engineering application of biological purification of wastewater and waste gas containing nonane or octane. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is the phylogenetic tree of Tsukamurella serrata LXY;
[0020] Figure 2 This is the bacterial growth and nonane degradation curve of Tsukamurella variegata LXY;
[0021] Figure 3 The bar graphs show the effects of different pH values of culture medium on the nonane degradation (Figure A), growth (Figure B), and mineralization (Figure C) of Tsukamurella variegata LXY.
[0022] Figure 4 The bar graphs show the effects of different temperatures on nonane degradation (Figure A), growth (Figure B), and mineralization (Figure C) of Tsukamurella serrata LXY.
[0023] Figure 5 This is the degradation curve of Tsukamurella mutans LXY to nonane with different initial concentrations;
[0024] Figure 6 is the maximum OD of Tsukamurella variegata LXY at different initial concentrations 600 value. DETAILED DESCRIPTION
[0025] The present invention will be further described below in conjunction with the embodiments.
[0026] The components of each culture medium used in the following examples are as follows:
[0027] Inorganic salt culture medium: Na2HPO4·12H2O 4.5g / L, KH2PO41.0 g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, anhydrous CaCl20.023 g / L, trace element mother solution 1mL / L, pH 7.0, and the solvent is deionized water; the components of the trace element mother solution are as follows: FeSO4·7H2O 1.0g / L, CuSO4·5H2O0.02g / L, H3BO3 0.014g / L, MnSO4·4H2O 0.10g / L, ZnSO4·7H2O 0.10g / L, Na2MoO4·2H2O 0.02g / L, CoCl2·6H2O0.02g / L, and the solvent is deionized water;
[0028] R2A solid slant culture medium: yeast powder 0.50 g / L, soluble starch 0.50 g / L, MgSO4·7H2O0.50 g / L, tryptone 0.50 g / L, glucose 0.50 g / L, sodium pyruvate 0.30 g / L, K2HPO4·3H2O0.45 g / L, agar 18 g / L, and the solvent is deionized water.
[0029] Example 1: Isolation and identification of Tsukamurella serrata LXY
[0030] (1) Sample collection and acclimatization
[0031] Activated sludge from a chemical plant in Ningbo, Zhejiang was collected on site and domesticated and enriched with nonane as the sole carbon source and energy source. After several months, the enriched activated sludge was inoculated into a 250mL sealed saline bottle containing 50mL of inorganic salt culture medium, and nonane was used as the sole carbon source and energy source for further cultivation and enrichment. The experiment required a constant temperature (30±1℃) and was carried out under aerobic conditions.
[0032] (2) Strain isolation and identification
[0033] The mixed bacterial solution enriched by multiple passages in a saline bottle was diluted and spread, and a single colony was picked according to the differences of the bacterial community. After multiple streaking separation and purification of the single colony, it was inoculated into an inorganic salt culture solution with nonane as the only carbon source and energy source to test the degradation activity. Pure bacteria with nonane degradation ability were selected, further separated and purified, and strain LXY with nonane degradation activity was obtained.
[0034] The strain LXY was subjected to 16S rDNA sequencing, and the sequencing work was completed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA of the strain LXY is shown in SEQ ID NO.1.
[0035] The 16S rDNA sequence of strain LXY was uploaded to the Ezbiocloud.net website, compared with the standard strains on the website, and the bacterial phylogenetic tree was constructed using the Neighbor-Joining method using MEGA-X software and evaluated using the Bootstrap method (repeated 1000 times). The constructed phylogenetic tree is shown in Figure 1 , thus determining that the strain was Tsukamurella and named it Tsukamurella paurometabolaLXY.
[0036] Strain LXY is an obligate aerobic Gram-positive bacterium with large colonies, white, opaque, irregularly shaped, rough surfaces, and bacterial lawns growing along streaks.
[0037] The strain LXY was deposited in the China Center for Type Culture Collection on May 30, 2024, with the deposit number: CCTCC NO: M 20241105, address: Wuhan University, Wuhan, China, Postal Code 430072.
[0038] Example 2: Preparation of seed solution of Tsukamurella variegata LXY strain
[0039] 1) Slant culture: Inoculate Tsukamurella mutans LXY-2 into slant R2A solid culture medium and culture at 30°C for 48 h to obtain slant cells.
[0040] 2) Expanded culture: Use an inoculation loop to pick the slant bacteria obtained in step 1) and inoculate them into R2A liquid culture medium, culture at 30°C for 46 hours, and obtain the OD 600 =1 bacterial solution.
[0041] Example 3: Performance of Tsukamurella mutans LXY in degrading nonane
[0042] Using nonane as the sole carbon source for Tsukamurella spp. LXY, the bacterial solution prepared by the method of Example 2 was inoculated into 50 mL of fresh inorganic salt medium containing 288 mg / L nonane to make the initial bacterial cell concentration (measured in OD 600 The pH value was 0.01 and 7. Two parallel samples and a blank control group without inoculation were designed. The samples were placed in a shaker at 30°C and 160 r / min for incubation. The nonane degradation rate was determined every 2 to 5 hours, and a portion of the bacterial solution was drawn out with a 5 mL syringe to determine the bacterial OD. 600 Value, see the result Figure 2 .
[0043] from Figure 2 It can be seen that with the extension of time, the bacterial concentration gradually increased, and at 22.5h, the bacterial concentration reached the maximum (about OD 600 =0.09). This example shows that Tsukamurella variegata LXY can use nonane as the only carbon source and energy source for growth and reproduction, and has the ability to degrade nonane stably and efficiently.
[0044] Example 4: Effect of initial pH on the degradation of nonane by Tsukamurella mutans LXY
[0045] The inorganic salt medium was adjusted to different pH values (5.0, 6.0, 7.0, 8.0, 9.0) using 1 mol / L NaOH aqueous solution or 1 mol / L H2SO4 aqueous solution; the bacterial solution prepared in Example 2 was inoculated into 50 mL of fresh pH-adjusted inorganic salt medium containing 288 mg / L nonane, and the initial bacterial cell concentration was expressed as OD 600 The sample was shaken and cultured in a constant temperature shaker at 30°C and 160 r / min for 22.5 h. The nonane degradation rate and OD in the reaction solution were measured. 600 Value and CO2 value, see Figure 3 .
[0046] from Figure 3 It can be seen that: in the range of pH = 5.0 ~ 9.0, all microorganisms can degrade nonane; as the pH increases from 5.0 to 9.0, the nonane degradation rate is limited only at pH = 5.0, and Tsukamurella serrata LXY has a strong adaptability to pH.
[0047] Example 5: Effect of temperature on the degradation of nonane by Tsukamurella variegata LXY
[0048] The bacterial solution prepared in Example 2 was inoculated into 50 mL of fresh inorganic salt medium (pH = 7) containing 288 mg / L nonane, and the initial bacterial cell concentration was OD 600 =0.01. Each sample was placed in a shaker at 20°C, 25°C, 30°C, 35°C, and 40°C for constant temperature shaking culture (the shaker speed was 160r / min). Two parallel samples and a blank control group without inoculation were designed at each temperature. Samples were taken after 12 hours of culture to measure the nonane degradation rate and OD 600 Value and CO2 value, see Figure 4 .
[0049] from Figure 4It can be seen that: within the temperature range of 20-40°C, Tsukamurella subvar LXY can grow and reproduce with nonane as the sole carbon source and energy source, and degrade nonane to a certain extent; within the temperature range of 25-35°C, Tsukamurella subvar LXY has a high degradation rate of nonane (above 99%), which shows that Tsukamurella subvar LXY has a strong ability to adapt to temperature.
[0050] Example 6: Effect of substrate concentration on the degradation of nonane by Tsukamurella variegata LXY
[0051] Different concentrations of substrate nonane were added to fresh inorganic salt culture medium to make the initial substrate concentrations 72, 144, 216, 288, and 360 mg / L, respectively. The bacterial solution prepared in Example 2 was inoculated to make the initial bacterial concentration measured by OD 600 0.01. Design two parallel samples and a blank control group without inoculation. Cultivate in a shaker at 30°C and 160 r / min, and take samples regularly to measure the nonane concentration and the OD of Tsukamurella variegata LXY 600 The results are shown in Figure 5 and Figure 6 .
[0052] from Figure 5 and Figure 6 It can be seen that when the nonane concentration is 72-360 mg / L, Tsukamurella spp. LXY can complete rapid reproduction within 18 hours and can almost completely degrade nonane after 18 hours; with the increase of nonane concentration, the complete degradation time also increases to a certain extent. Tsukamurella spp. LXY has a strong ability to degrade 72-360 mg / L nonane.
[0053] Example 7: Octane degradation ability of Tsukamurella variegata LXY
[0054] Octane was added to the fresh inorganic salt medium to make the initial concentration of octane 71.8 mg / L, and the bacterial solution prepared in Example 2 was inoculated to make the initial bacterial concentration measured in OD 600 The culture was performed in a shaker at 30°C and 160 r / min, and the OD was measured after 12 h of culture. 600 The results are shown in Table 1.
[0055] Table 1 Degradation ability of Tsukamurella variegata LXY on different carbon source substrates
[0056]
[0057] As can be seen from Table 1, Tsukamurella solani LXY can also use octane as the only carbon source and energy source to achieve efficient degradation.
[0058] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.
[0059] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.
Claims
1. Tsukamurella paurometabola LXY that degrades nonane, whose deposit number is: CCTCC M 20241105.
2. A bacterial agent containing the Tsukamurella serrata LXY according to claim 1.
3. Use of the Tsukamurella mutans LXY as claimed in claim 1 or the bacterial agent as claimed in claim 2 in degrading nonane.
4. The use according to claim 3, characterized in that The bacterial solution obtained by inoculating the Tsukamurella solani LXY into wastewater containing nonane or culturing inorganic salt containing nonane is used as an enzyme source, and a degradation reaction is carried out in an inorganic salt culture medium at pH = 5.0-9.0 and 20°C-40°C.
5. The use according to claim 4, characterized in that The inorganic salt culture medium has a concentration composition as follows: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.023g / L, trace element mother solution 1.0mL / L, pH 7.0, and the solvent is deionized water; the trace element mother solution has a concentration composition as follows: FeSO4·7H2O 1.0g / L, CuSO4·5H2O 0.02g / L, H3BO3 0.014g / L, MnSO4·4H2O 0.10g / L, ZnSO4·7H2O 0.10g / L, Na2MoO4·2H2O 0.02g / L, and CoCl2·6H2O 0.02g / L, and the solvent is deionized water.
6. The use according to claim 4, characterized in that The initial concentration of nonane in the inorganic salt medium is 72-360 mg / L, and the amount of the enzyme source is based on the OD 600 Counted as 0.
01.
7. Use of the Tsukamurella mutans LXY as claimed in claim 1 or the bacterial agent as claimed in claim 2 in degrading octane.
8. The use according to claim 7, characterized in that The bacterial solution obtained by inoculating the Tsukamurella solani LXY into wastewater containing octane or culturing inorganic salt containing octane is used as an enzyme source, and a degradation reaction is carried out in an inorganic salt culture medium at pH = 5.0-9.0 and 20°C-40°C.
9. The use according to claim 8, characterized in that, The inorganic salt culture medium has a concentration composition as follows: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.023g / L, trace element mother solution 1.0mL / L, pH 7.0, and the solvent is deionized water; the trace element mother solution has a concentration composition as follows: FeSO4·7H2O 1.0g / L, CuSO4·5H2O 0.02g / L, H3BO3 0.014g / L, MnSO4·4H2O 0.10g / L, ZnSO4·7H2O 0.10g / L, Na2MoO4·2H2O 0.02g / L, and CoCl2·6H2O 0.02g / L, and the solvent is deionized water.
10. The use according to claim 8, characterized in that The initial concentration of octane in the inorganic salt medium is 65-360 mg / L, and the amount of the enzyme source is based on the OD 600 Counted as 0.01.