Rhodococcus ruber ZYH-ZY and application thereof in degradation of organic pollutants

Abstract

The invention is applicable to the technical field of organic pollutant degradation, and provides rhodococcus ruber, which is named as rhodococcus ruber ZYH-ZY, is preserved in the China Center for Type Culture Collection on February 29, 2024, and has the preservation number of CCTCC NO: M 2024359. The invention further provides application of the rhodococcus ruber in degradation of organic pollutants. The rhodococcus ruber ZYH-ZY provided by the invention can degrade 2-methylpentane, 3-methylpentane and normal hexane by taking 2-methylpentane, 3-methylpentane and normal hexane as a carbon source and an energy source, especially 2-methylpentane and 3-methylpentane, the degradation rate of 2-methylpentane in 48 hours can reach 99% or above, the degradation rate of 3-methylpentane can reach 80% or above, and the rhodococcus ruber ZYH-ZY can be used for degrading 2-methylpentane, 3-methylpentane and normal hexane in 48 hours. The n-hexane degradation rate can reach 60% or above; the Rhodococcus ruber can adapt to a wide pH range, can grow and breed when the pH is 4.0-9.0, and can degrade 2-methylpentane to a certain extent.

Description

Technical Field

[0001] The invention belongs to the technical field of organic pollutant degradation, and in particular relates to a Rhodococcus erythrorhizium and an application thereof in degrading organic pollutants. Background Art

[0002] The petrochemical industry plays a pivotal role in the development of the national economy. The atmospheric pollutants emitted by the petrochemical industry mainly include SO2, NO x And volatile organic compounds (VOCs), among which VOCs are of various types, highly toxic and harmful. They are important precursors to the formation of ozone and secondary organic particulate matter, and are also the culprits of regional environmental problems such as photochemical smog and haze. They seriously threaten human health and ecological security and have become the focus of prevention and control in the petrochemical industry.

[0003] For example, 2-Methylpentane (2-Methylpentane), also known as isohexane, is a typical volatile organic compound that is widely used in petroleum refining, chemical raw materials, synthetic rubber and other petrochemical industries. It is highly toxic and irritating. After human contact, it will cause burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea and vomiting, which is harmful to human health and has certain harm to the environment such as the atmosphere and soil.

[0004] Using microbial metabolism to degrade and transform VOCs has the advantages of low cost, high removal efficiency, and no secondary pollution. This is also a current research hotspot in air pollution control. Therefore, it is of great significance to find a microorganism that can degrade organic pollutants. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a Rhodococcus erythrocyte to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is implemented as follows: a red coccus, named as red coccus ZYH-ZY, is deposited in the China Center for Type Culture Collection, with a preservation date of February 29, 2024, and a preservation number of CCTCC NO: M 2024359.

[0007] Preferably, the 16S rDNA sequence of Rhodococcus erythrocytes ZYH-ZY is as shown in SEQ ID NO.1.

[0008] Another object of an embodiment of the present invention is to provide a use of the above-mentioned Rhodococcus in degrading organic pollutants.

[0009] Preferably, the organic pollutant is one or more of 2-methylpentane, 3-methylpentane and n-hexane.

[0010] Preferably, the steps include:

[0011] (1) inoculating the Rhodococcus erythrorhizium into a solid culture medium, culturing at 25-30° C. for 48-72 hours, and obtaining planar bacteria;

[0012] (2) inoculating the planar bacteria into an inorganic salt culture medium, culturing at 25-30° C. for 24 h, and obtaining a bacterial solution with an OD600 of 0.013;

[0013] (3) Mix the bacterial solution and organic pollutants to make a mixed solution, and place the mixed solution in an environment of 20 to 40°C to carry out the degradation process.

[0014] Preferably, in step (1), the solid culture medium comprises the following ingredients: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 18-20 g / L agar, and the solvent is deionized water.

[0015] Preferably, in step (2), the inorganic salt culture medium comprises the following components: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl 0.2033 g / L, FeCl3 0.0162 g / L, CaCl 0.011 g / L, trace element mother solution 5 ml / L, and the solvent is deionized water.

[0016] Preferably, the trace element mother solution comprises the following components: CuSO4 0.05 g / L, FeSO4 1.0 g / L, MnSO4 0.1 g / L, Na2MoO4 0.02 g / L, CoCl2 0.02 g / L, H3BO3 0.014 g / L, ZnSO4 0.1 g / L, and the solvent is deionized water.

[0017] Preferably, in step (2), the pH value of the inorganic salt culture medium is 4.0 to 9.0.

[0018] Preferably, in step (3), the concentration of the organic pollutants is 15 to 75 mg / L.

[0019] A Rhodococcus erythrocyte provided in an embodiment of the present invention can use 2-methylpentane, 3-methylpentane and n-hexane as carbon sources and energy sources to achieve the degradation of 2-methylpentane, 3-methylpentane and n-hexane, especially 2-methylpentane and 3-methylpentane. The degradation rate of 2-methylpentane can reach more than 99% in 48 hours, the degradation rate of 3-methylpentane can reach more than 80%, and the degradation rate of n-hexane can reach more than 60%;

[0020] At the same time, the red coccus can adapt to a wide pH range, can grow and reproduce at pH 4.0-9.0 and degrade 2-methylpentane to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A colony morphology photograph of Rhodococcus erythrocytes ZYH-ZY on LB medium provided in Example 1 of the present invention;

[0022] Figure 2 A transmission electron microscope image (0.2 μm and 0.5 μm) of Rhodococcus erythrocytes ZYH-ZY provided in Example 1 of the present invention;

[0023] Figure 3 A phylogenetic tree diagram of Rhodococcus erythrocytes ZYH-ZY provided in Example 1 of the present invention;

[0024] Figure 4 The degradation rate of 2-methylpentane by Rhodococcus erythrocytes ZYH-ZY at different pH values ​​for 48 hours provided in Example 3 of the present invention;

[0025] Figure 5 A bar graph showing the effect of biomass changes of Rhodococcus erythrocytes ZYH-ZY at different pH values ​​after 48 hours provided in Example 3 of the present invention;

[0026] Figure 6 The degradation curve of Rhodococcus erythrocytes ZYH-ZY to 2-methylpentane of different initial concentrations provided in Example 4 of the present invention;

[0027] Figure 7 This is a bar graph showing the effect of Rhodococcus erythrocytes ZYH-ZY provided in Example 4 of the present invention on the biomass change after 62.5 hours of exposure to 2-methylpentane at different initial concentrations. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0030] Example 1

[0031] The separation, purification and identification of Rhodococcus erythrocytes ZYH-ZY specifically include the following steps:

[0032] (1) Isolation and purification of Rhodococcus erythrocytes ZYH-ZY

[0033] Activated sludge was collected from a sewage treatment plant in Zhejiang Province. The lower sludge after standing was mixed with inorganic salt culture medium at a ratio of 1:2 (v / v). 3L of the mixture was inoculated into a 5L sludge acclimation tank. 2-methylpentane was used as a substrate as a carbon source and energy source. The acclimation was carried out at room temperature. After nearly 20 days, 5mL of sludge was taken from the acclimation tank and added into a shake flask containing 50mL of inorganic salt culture medium. It was found that the acclimated sludge could stably degrade 27mg / L 2-methylpentane per day in the shake flask, and the degradation rate could reach more than 90% (shake flask experimental conditions: 30℃, 160r / min), and the acclimated sample was obtained.

[0034] 1 and 2 mL of the sludge sample in the shake flask were transferred to other shake flasks for further performance testing. It was found that the transferred sludge could stably degrade 27 mg / L 2-methylpentane per day in the shake flask. The sludge sample was transferred for enrichment for 6 generations (1 mL each time), and then the sludge after 6 generations of enrichment was transferred for 10 -1 ~10 -6 The strain was plated at multiple times, and a single colony was picked. The degradation activity was determined using 2-methylpentane as a substrate, and the strain was isolated and purified to obtain a strain ZYH-ZY with 2-methylpentane degradation activity.

[0035] (2) Identification of Rhodococcus erythrocytes ZYH-ZY

[0036] The colony morphology of strain ZYH-ZY is as follows Figure 1 As shown, it is orange-yellow, round and opaque, with a rough and dry surface, easy to pick up, and the fungus grows along the streaks;

[0037] Transmission electron microscopy images of strain ZYH-ZY are shown in Figure 2. Figure 2 As shown;

[0038] The physiological and biochemical characteristics of strain ZYH-ZY are: aerobic, Gram stain positive;

[0039] The strain ZYH-ZY was subjected to 16S rDNA sequencing, which was completed by Zhejiang Tianke High-Tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology). The 16S rDNA sequence of the strain ZYH-ZY is shown in SEQ ID NO.1, and the Genbank accession number is NR_026185.1. The 16S rDNA sequence of the strain ZYH-ZY 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 3As shown, the strain ZYH-ZY was determined to be of the genus Rhodococcus, named Rhodococcus ruber ZYH-ZY, and deposited in the China Center for Type Culture Collection on February 29, 2024, with a deposit number of CCTCCNO: M 2024359, address: Wuhan University, Wuhan, China, Postal Code 430072.

[0040] Example 2

[0041] The expanded culture of Rhodococcus erythrocytes ZYH-ZY specifically includes the following steps:

[0042] 1) Planar culture medium: inoculate Rhodococcus ZYH-ZY into a solid culture medium, culture at 30° C. for 72 h, and obtain a slant cell, wherein the solid culture medium comprises the following components: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 18-20 g / L agar, and the solvent is deionized water;

[0043] 2) Expanding the culture: Using an inoculation loop, pick the planar bacteria obtained in step 1) and inoculate them into an inorganic salt medium, culture at 30° C. for 24 h to obtain a bacterial solution with an OD600 of 0.013, wherein the inorganic salt medium comprises the following components: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl 0.2033g / L, FeCl30.0162g / L, CaCl0.011g / L, trace element mother solution 5ml / L, the solvent is deionized water, the trace element mother solution includes the following components: CuSO40.05g / L, FeSO41.0g / L, MnSO40.1g / L, Na2MoO40.02g / L, CoCl20.02g / L, H3BO30.014g / L, ZnSO40.1g / L, the solvent is deionized water.

[0044] Example 3

[0045] Analysis of the effect of initial pH on the degradation of 2-methylpentane by Rhodococcus erythrocytes ZYH-ZY:

[0046] (1) Using 1 mol / L NaOH aqueous solution or 1 mol / L H2SO4 aqueous solution to adjust fresh inorganic salt culture medium to different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0), wherein the inorganic salt culture medium comprises the following components: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl 0.2033 g / L, FeCl3 0.0162 g / L, CaCl 0.011 g / L, trace element mother solution 5 ml / L, the solvent is deionized water, and the trace element mother solution comprises the following components: CuSO4 0.05 g / L, FeSO4

[0047] 1.0g / L, MnSO40.1g / L, Na2MoO40.02g / L, CoCl20.02g / L, H3BO30.014g / L, ZnSO40.1g / L, the solvent is deionized water;

[0048] (2) Take the bacterial solution prepared in Example 2 and inoculate it into 50 mL of inorganic salt medium so that the initial bacterial cell concentration is OD 600 The inorganic salt medium is the inorganic salt medium whose pH value has been adjusted in step (1), and the medium contains 54 mg / L 2-methylpentane;

[0049] (3) Design two parallel samples and a blank control group without inoculation of the strain, and culture them in a constant temperature shaker at 30°C and 160 r / min. Take samples after 48 h of culture, measure the concentration of 2-methylpentane, and calculate the degradation rate of 2-methylpentane. The results are as follows: Figure 4 As shown, the OD of Rhodococcus ZYH-ZY was measured at the same time. 600 Value, the result is Figure 5 shown.

[0050] according to Figure 4 and Figure 5 It can be seen that in the range of pH = 4.0 to 9.0, Rhodococcus ZYH-ZY can grow and reproduce using 2-methylpentane as a carbon source and energy source, and degrade 2-methylpentane to a certain extent;

[0051] In the pH range of 6.0 to 7.0, Rhodococcus ZYH-ZY has a high degradation rate (more than 95%) for 2-methylpentane, and the growth amount of Rhodococcus ZYH-ZY also shows a trend consistent with the degradation rate of 2-methylpentane.

[0052] Example 4

[0053] Analysis of the effect of substrate concentration on the degradation of 2-methylpentane by Rhodococcus erythrocytes ZYH-ZY:

[0054] (1) Add different concentrations of substrate 2-methylpentane to fresh inorganic salt culture medium, so that the initial concentration of the substrate is 15 mg / L, 30 mg / L, 45 mg / L, 60 mg / L, and 75 mg / L, respectively. The inorganic salt culture medium comprises the following components: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl 0.2033 g / L, FeCl3 0.0162 g / L, CaCl 0.011g / L, trace element mother solution 5ml / L, the solvent is deionized water, the trace element mother solution includes the following components: CuSO40.05g / L, FeSO41.0g / L, MnSO40.1g / L, Na2MoO40.02g / L, CoCl20.02g / L, H3BO30.014g / L, ZnSO40.1g / L, the solvent is deionized water;

[0055] (2) The bacterial solution prepared in Example 2 was inoculated into the inorganic salt medium in step (1) to make the initial bacterial cell concentration measured by OD 600 It is calculated as 0.013;

[0056] (3) Design two parallel samples, culture them in a shaker at 30°C and 160 r / min, and take samples regularly to measure the 2-methylpentane concentration and the OD of Rhodococcus erythrocytes ZYH-ZY. 600 The degradation rate of 2-methylpentane was calculated. The results are as follows Figure 6 , Figure 7 shown.

[0057] according to Figure 6 and Figure 7 It can be seen that when the concentration of 2-methylpentane is 15 mg / L, Rhodococcus ZYH-ZY can multiply rapidly within 24 hours and can almost completely degrade 2-methylpentane after 40 hours; as the concentration of 2-methylpentane increases, the complete degradation time also increases to a certain extent.

[0058] Example 5

[0059] Analysis of the degradation ability of Rhodococcus erythrocytes ZYH-ZY on different carbon source substrates:

[0060] (1) 3-Methylpentane and n-hexane were added as substrates to a fresh inorganic salt medium, and then the bacterial solution prepared in Example 2 was inoculated to make the initial bacterial concentration 0.013 in terms of OD600. The inorganic salt medium included the following components: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl 0.2033 g / L, FeCl3 0.0162 g / L, CaCl 0.011g / L, trace element mother solution 5ml / L, the solvent is deionized water, the trace element mother solution includes the following components: CuSO40.05g / L, FeSO41.0g / L, MnSO40.1g / L, Na2MoO40.02g / L, CoCl20.02g / L, H3BO30.014g / L, ZnSO40.1g / L, the solvent is deionized water;

[0061] (2) The culture was carried out in a shaker at 30°C and 160 r / min. After 48 h of culture, the concentration of the two substrates and the OD of Rhodococcus ZYH-ZY were measured. 600 The values ​​were calculated and the substrate degradation rate was shown in Table 1.

[0062] Table 1

[0063]

[0064] According to Table 1, it can be seen that Rhodococcus erythrocytes ZYH-ZY also have different degrees of degradation ability for 3-methylpentane and n-hexane.

[0065] In summary, the Rhodococcus ZYH-ZY separated and purified in the embodiment of the present invention can be applied to the degradation of organic pollutants, such as 2-methylpentane, 3-methylpentane, n-hexane, especially 2-methylpentane. The degradation rate of 2-methylpentane in 48 hours can reach more than 99%. At the same time, the Rhodococcus ZYH-ZY can adapt to a wide pH range and has degradation ability at pH 4.0 to 9.0. When the pH is 6.0 to 7.0, the Rhodococcus ZYH-ZY reproduces faster, which can achieve rapid degradation of 2-methylpentane.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Rhodococcus sp., characterized in that: It is named Rhodococcus erythrocytes ZYH-ZY and deposited in the China Center for Type Culture Collection on February 29, 2024, with the deposit number CCTCC NO:M 2024359.

2. The Rhodococcus according to claim 1, characterized in that The 16S rDNA sequence of the Rhodococcus erythrocytes ZYH-ZY is shown in SEQ ID NO.

1.

3. Use of the Rhodococcus erythrocytes as claimed in claim 1 or 2 in degrading organic pollutants.

4. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 3, characterized in that: The organic pollutant is one or more of 2-methylpentane, 3-methylpentane and n-hexane.

5. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 3, characterized in that: The following steps are involved: (1) inoculating the Rhodococcus erythrocytes into a solid culture medium, culturing at 25-30° C. for 48-72 hours, and obtaining planar bacteria; (2) inoculating the planar bacteria into an inorganic salt culture medium, culturing at 25-30° C. for 24 h, and obtaining a bacterial solution with an OD600 of 0.013; (3) Mix the bacterial solution and organic pollutants to make a mixed solution, and place the mixed solution in an environment of 20 to 40°C to carry out the degradation process.

6. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 5, characterized in that: In step (1), the solid culture medium comprises the following components: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 18-20 g / L agar, and the solvent is deionized water.

7. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 5, characterized in that: In step (2), the inorganic salt culture medium includes the following components: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl 0.2033 g / L, FeCl3 0.0162 g / L, CaCl 0.011 g / L, trace element mother solution 5 ml / L, and the solvent is deionized water.

8. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 7, characterized in that: The trace element mother solution includes the following components: CuSO4 0.05 g / L, FeSO4 1.0 g / L, MnSO4 0.1 g / L, Na2MoO4 0.02 g / L, CoCl2 0.02 g / L, H3BO3 0.014 g / L, ZnSO4 0.1 g / L, and the solvent is deionized water.

9. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 5, characterized in that: In step (2), the pH value of the inorganic salt culture medium is 4.0 to 9.

0.

10. The use of Rhodococcus erythrocytes in degrading organic pollutants according to claim 5, characterized in that: In step (3), the concentration of the organic pollutants is 15 to 75 mg / L.

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