Raellia XZW-1 and application thereof in degrading organic pollutants

Abstract

The application of Raellia XZW-1 in degrading organic pollutants belongs to the field of microbial technology. The application provides Raellia XZW-1, and the preservation number of the Raellia XZW-1 is CCTCC No: M 2022557. The application also provides the application and application method of the Raellia XZW-1. The Raellia XZW-1 can grow and reproduce by taking chlorobenzene as carbon source and energy source, and can efficiently degrade chlorobenzene. In addition, the Raellia XZW-1 can also take 1,2-dichlorobenzene, toluene and 1,2-dimethylbenzene as carbon source and energy source, and can efficiently degrade 1,2-dichlorobenzene, toluene and 1,2-dimethylbenzene.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Rhesus oryzae XZW-1 and its application in the degradation of organic pollutants. Background Technology

[0002] Volatile organic compounds (VOCs) are important precursors to PM2.5 and ozone, seriously affecting ambient air quality. With the increasing levels of SO2 and NO... x With effective control measures in place, VOCs emission reduction has become a key focus and crucial aspect of air pollution control in recent years. Chlorobenzenes (CBs) are widely used in the production processes of dyes, plastics, fragrances, pharmaceuticals, and organic synthesis, and are a significant component of chlorine-containing VOCs industrial waste gases. CBs are chemically stable and possess carcinogenic, mutagenic, and teratogenic effects, making them a class of hydrophobic and persistent organic pollutants with extremely high ecological and environmental risks. They are listed as priority pollutants by the US EPA, seriously threatening the ecological environment and human health. Therefore, conducting research on CBs waste gas purification is of significant practical importance.

[0003] Biological purification of VOCs utilizes microorganisms to convert VOCs in waste gas into simple inorganic substances (CO2, H2O, etc.) and cellular components through metabolic activities. It has advantages such as low cost, high removal efficiency, and no secondary pollution, and has become one of the research hotspots in the field of air pollution control technology.

[0004] Currently, many strains capable of degrading chlorobenzene have been discovered. For example, Zhang Shih-han et al. domesticated and isolated a strain of *Bacillus angustifolius* ZJUTCB-1 with highly efficient chlorobenzene degradation capabilities from activated sludge in a wastewater treatment plant (*Bacillus angustifolius* ZJUTCB-1 with highly efficient chlorobenzene degradation and its application, Publication No.: CN109370945A). Under aerobic conditions, it achieved a 100% removal efficiency for chlorobenzene at initial concentrations of 347.31-2604.80 μmol / L. Therefore, screening, isolating, and using targeted domestication methods to obtain highly efficient chlorobenzene-degrading strains for VOCs, and studying their biological and degradation characteristics, can provide technical support and theoretical basis for the biological treatment of chlorobenzene-related VOCs waste gas. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for Ralstonia solanacearum XZW-1 and its application in the degradation of organic pollutants. The Ralstonia solanacearum XZW-1 of this invention can achieve efficient degradation of chlorobenzene using chlorobenzene as both a carbon source and energy source, which is of great significance for the engineering application of biological purification of chlorobenzene.

[0006] This invention is specifically achieved through the following technical solutions:

[0007] The first aspect of this invention provides a strain of Ralstonia sp. XZW-1, which was deposited at the China Center for Type Culture Collection on May 6, 2022, with accession number CCTCC No: M 2022557, and the address of the depository is Wuhan University, Wuhan, China. The microbiological classification name is Ralstonia sp.

[0008] The aforementioned Ralstonia species was isolated from activated sludge at a wastewater treatment plant in Zhejiang Province. It is an aerobic, Gram-negative bacterium, with colonies that are small, dot-like, milky white, opaque, plump, smooth, and moist, easily picked up, and grow along the streaks. After 16S rDNA sequencing, it was identified as belonging to the Ralstonia genus and named Ralstoniasp. XZW-1.

[0009] The Ralstonia solanacearum XZW-1 strain of this invention can grow and reproduce using chlorobenzene as a carbon and energy source, efficiently degrades chlorobenzene, and can adapt to a wide range of temperatures and pH values. Furthermore, Ralstonia solanacearum XZW-1 can also grow and reproduce using 1,2-dichlorobenzene and toluene as carbon and energy sources, and efficiently degrade 1,2-dichlorobenzene and toluene.

[0010] A second aspect of the present invention provides the application of Rhesus rhizome XZW-1 in the degradation of organic pollutants.

[0011] Furthermore, the organic pollutant is one or more of chlorobenzene, 1,2-dichlorobenzene, toluene, and 1,2-xylene.

[0012] A third aspect of the present invention provides a method for degrading organic pollutants using Rhesus rhamnoides XZW-1, comprising the following steps:

[0013] a. Inoculate the Rhesus xZW-1 strain onto a solid slant culture medium and incubate at 30–35°C for 24–48 h to obtain slant cells;

[0014] b. Inoculate the slant bacterial cells into an inorganic salt culture medium and incubate at 25–30°C for 24–36 hours to obtain OD. 600 =0.1~0.2% bacterial solution;

[0015] c. Mix the bacterial solution and organic pollutants to prepare a mixed solution;

[0016] d. The mixture is subjected to a degradation reaction at 20–40°C.

[0017] Furthermore, the solid slant culture medium in step a comprises the following components in parts by weight: yeast extract 0.50–1.00 g / L, soluble starch 0.50–1.00 g / L, MgSO4 0.50–1.00 g / L, tryptone 0.50–1.00 g / L, glucose 0.50–1.00 g / L, sodium pyruvate 0.30–0.60 g / L, K2HPO4 0.45–0.90 g / L, agar 18–20 g / L, and deionized water as the solvent.

[0018] Furthermore, the inorganic salt culture medium in step b comprises the following components in parts by weight: Na2HPO4 4.0–5.0 g / L, KH2PO4 0.8–1.2 g / L, (NH4)2SO4 2.3–2.8 g / L, MgSO4 0.18–0.23 g / L, CaCl2 0.022–0.24 g / L, and trace element stock solution 0.8–1.2 mL / L, with deionized water as the solvent.

[0019] Furthermore, the pH of the inorganic salt culture medium in step b is 4.0–10.0.

[0020] Furthermore, the organic pollutant in step c is one or more of chlorobenzene, 1,2-dichlorobenzene, toluene, and 1,2-xylene.

[0021] Furthermore, the concentration of organic pollutants in step c is 20–200 mg / L.

[0022] Furthermore, the trace element mother liquor comprises the following components: FeSO4 0.8–1.2 g / L, CuSO4 0.015–0.025 g / L, H3BO3 0.013–0.015 g / L, MnSO4 0.08–0.13 g / L, ZnSO4 0.08–0.13 g / L, Na2MoO4 0.015–0.025 g / L, and CoCl2 0.015–0.025 g / L, with deionized water as the solvent.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The Rhesus oryzae XZW-1 of the present invention can use chlorobenzene, 1,2-dichlorobenzene and / or toluene as carbon and energy sources to achieve efficient degradation of chlorobenzene and 1,2-dichlorobenzene. The degradation rate of chlorobenzene can reach more than 99% in 12 hours, the degradation rate of 1,2-dichlorobenzene can reach more than 95%, and the degradation rate of toluene can reach more than 50%.

[0025] (2) The Rhesus xZW-1 of the present invention can adapt to a wide range of temperature and pH, and can grow and reproduce at 20-40℃ and pH 6.0-8.0 and degrade chlorobenzene to a certain extent. Attached Figure Description

[0026] Figure 1 Transmission electron microscopy image of Rhesus 1XZW-1;

[0027] Figure 2 Phylogenetic tree of Rhesus xZW-1;

[0028] Figure 3 The graph shows the cell growth and chlorobenzene degradation curves of Rhesus genus XZW-1.

[0029] Figure 4 The effects of different pH values ​​on the chlorobenzene degradation performance (Figure A), growth (Figure B), and mineralization (Figure C) of Ralstonia solanacearum XZW-1 in culture medium;

[0030] Figure 5 The effects of different temperatures on the chlorobenzene degradation efficiency (Figure A), mineralization efficiency (Figure B), and growth (Figure C) of Ralstonia solanacearum XZW-1.

[0031] Figure 6 The degradation performance of Rhesus oryzae XZW-1 on chlorobenzene at different initial concentrations was investigated.

[0032] Figure 7 The growth curves of Rhesus oryzae XZW-1 at different initial chlorobenzene concentrations are shown. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments.

[0034] The components of the culture media used in the following examples are as follows:

[0035] Inorganic salt culture medium: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, anhydrous CaCl2 0.023g / L, trace element stock solution 1mL / L, pH 7.0, solvent is deionized water;

[0036] The composition of the trace element mother liquor is as follows: FeSO4·7H2O 1.0 g / L, CuSO4·5H2O 0.02 g / L, H3BO3 0.014 g / L, MnSO4·4H2O 0.10 g / L, ZnSO4·7H2O 0.10 g / L, Na2MoO4·2H2O 0.02 g / L, CoCl2·6H2O 0.02 g / L, and the solvent is deionized water.

[0037] R2A solid slant culture medium: yeast extract 0.50 g / L, soluble starch 0.50 g / L, MgSO4·7H2O 0.50 g / L, tryptone 0.50 g / L, glucose 0.50 g / L, sodium pyruvate 0.30 g / L, K2HPO4·3H2O 0.45 g / L, agar 18 g / L, solvent is deionized water, pH is natural.

[0038] Example 1: Isolation, purification and identification of Rhesus oryzae XZW-1

[0039] (1) Isolation and purification of Rhesus oryzae XZW-1

[0040] Activated sludge was collected from a wastewater treatment plant in Zhejiang Province. The lower layer of sludge after settling was mixed with inorganic salt culture medium at a ratio of 1:2 (v / v). 3L of the mixture was introduced into a 5L sludge acclimation tank. Chlorobenzene was used as the carbon and energy source. After acclimation and cultivation at room temperature for nearly 20 days, 5mL of sludge was taken from the acclimation tank and added to a shake flask containing 50mL of inorganic salt culture medium. It was found that the acclimated sludge could stably degrade 20mg / L of chlorobenzene per day in the shake flask, with a degradation rate of 80-90% (shake flask experimental conditions: 30℃, 160r / min).

[0041] Sludge samples from the shake flasks were transferred in batches of 1 and 2 mL to other shake flasks for further degradation performance testing. It was found that the transferred sludge could stably degrade 20 mg / L chlorobenzene per day in the shake flasks. This sludge sample was then further transferred and enriched for 6 generations (1 mL each time). The sludge enriched for 6 generations was then processed at 10... -1 ~10 -6 The bacteria were plated at multiples, single colonies were picked, and chlorobenzene degradation performance was determined using chlorobenzene as a substrate. After isolation and purification, a strain XZW-1 with chlorobenzene degradation activity was obtained.

[0042] (2) Identification of Rhesus oryzae XZW-1

[0043] The colonies of strain XZW-1 are small, dot-like, milky white, opaque, plump, smooth, and moist, easily picked up, and the bacterial growth follows the streaks. A transmission electron microscope image of Ralstonia solanacearum XZW-1 is shown below. Figure 1 As shown.

[0044] The physiological and biochemical characteristics of strain XZW-1 are: aerobic, Gram-negative.

[0045] 16S rDNA sequencing of strain XZW-1 was performed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA of strain XZW-1 (SEQ ID NO.1) has the Genebank accession number ON377124.

[0046] The 16S rDNA sequence of strain XZW-1 was uploaded to the Ezbiocloud.net website and compared with standard strains on the website. A phylogenetic tree was constructed using the Neighbor-Joining method with MEGA-X software, and evaluated using the Bootstrap method (repeated 1000 times). The constructed phylogenetic tree is shown below. Figure 2 Thus, strain XZW-1 was identified as belonging to the genus Ralstonia and named Ralstonia sp. XZW-1. It was deposited at the China Center for Type Culture Collection (CCTCC) on May 6, 2022, with accession number CCTCC No: M 2022557, address: Wuhan University, Wuhan, China, 430072, China.

[0047] Example 2: Large-scale culture of Rhesus pyrenella XZW-1

[0048] After expanding the culture of Rhesus oryzae XZW-1, a bacterial suspension containing Rhesus oryzae XZW-1 was obtained. The specific process is as follows:

[0049] 1) Slant culture: Rhesus xZW-1 was inoculated into R2A solid slant medium and cultured at 30℃ for 48h to obtain slant cells.

[0050] 2) Expanded culture: Using an inoculation loop, pick up the slant cells obtained in step 1) and inoculate them into an inorganic salt medium. Incubate at 30°C for 24 hours to obtain OD. 600 =0.1 bacterial solution.

[0051] Example 3: Performance of Rhesus xZW-1 in degrading chlorobenzene

[0052] The bacterial culture prepared in Example 2 was inoculated into 50 mL of fresh inorganic salt medium (pH = 7) containing 50 mg / L chlorobenzene, so that the initial bacterial concentration was expressed as OD0.05. 600 The value was calculated as 0.01. Two parallel samples and one blank control group without inoculation were designed. The samples were incubated in a shaker at 30℃ and 160 rpm. Samples were taken every 3–6 hours to determine the chlorobenzene degradation rate. A portion of the bacterial culture was also extracted using a 5 mL syringe to determine the bacterial cell OD value. The results are shown in the table below. Figure 3 .

[0053] from Figure 3 It can be seen that the bacterial cell concentration gradually increases with the extension of time, reaching its maximum after 12 hours of culture (approximately OD). 600 =0.06), and the chlorobenzene degradation rate reached over 99%. This indicates that Rhesus oryzae XZW-1 can utilize chlorobenzene as a carbon source and energy source for growth and reproduction, and has a stable and efficient ability to degrade chlorobenzene.

[0054] Example 4: Effect of initial pH on the degradation of chlorobenzene by Rhesus oryzae XZW-1

[0055] The inorganic salt culture medium was adjusted to different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) using 1 mol / L NaOH or 1 mol / L H2SO4 aqueous solution. The bacterial suspension prepared in Example 2 was inoculated into 50 mL of fresh inorganic salt culture medium containing 50 mg / L chlorobenzene. After adjusting the pH, the initial bacterial concentration was determined by OD0.05. 600 The value was calculated to be 0.01. Two parallel samples and one blank control group without inoculation were designed. The samples were cultured in a constant temperature shaker at 30℃ and 160 r / min for 12 h. Samples were taken after 12 h to measure the chlorobenzene degradation rate and OD in the reaction solution. 600 Values ​​and CO2 values, results are shown in [link to results]. Figure 4 .

[0056] from Figure 4 It can be seen that within the pH range of 6.0 to 8.0, Ralstonia solanacearum XZW-1 can grow and reproduce using chlorobenzene as both a carbon and energy source, and degrade chlorobenzene to a certain extent; within the pH range of 7.0 to 8.0, Ralstonia solanacearum XZW-1 exhibits a high degradation rate (over 99%) for chlorobenzene. Furthermore, the growth rate and CO2 production of Ralstonia solanacearum XZW-1 show a trend consistent with the chlorobenzene degradation rate.

[0057] Example 5: Effect of temperature on the degradation of chlorobenzene by Rhesus oryzae XZW-1

[0058] The bacterial culture prepared in Example 2 was inoculated into 50 mL of fresh inorganic salt medium (pH = 7) containing 50 mg / L chlorobenzene, so that the initial bacterial concentration was expressed as OD0.05. 600 The value was calculated as 0.01. Each sample was placed in a shaker at 20℃, 25℃, 30℃, 35℃, and 40℃ for constant temperature shaking (shaking speed 160 r / min). Two parallel samples and one blank control group without inoculation were designed for each temperature. Samples were taken after 12 h of incubation, and the chlorobenzene degradation rate and OD in the reaction solution were measured. 600 Values ​​and CO2 values, results are shown in [link to results]. Figure 5 .

[0059] from Figure 5 It can be seen that within the temperature range of 20-40℃, Ralstonia solanacearum XZW-1 can grow and reproduce using chlorobenzene as a carbon source and energy source, and degrade chlorobenzene to a certain extent; within the temperature range of 25-30℃, Ralstonia solanacearum XZW-1 has a high degradation rate of chlorobenzene (above 99%), and the growth rate and CO2 value of Ralstonia solanacearum XZW-1 also show a trend consistent with the degradation rate of chlorobenzene.

[0060] Example 6: Effect of substrate concentration on the degradation of chlorobenzene by Rhesus xanthipes XZW-1

[0061] Different concentrations of the substrate chlorobenzene (initial concentrations of 20, 50, 100, 150, and 200 mg / L) were added to fresh inorganic salt culture medium, and the bacterial suspensions prepared in Example 2 were inoculated accordingly, so that the initial bacterial concentration was expressed as OD0. 600 The value was calculated as 0.01. Two parallel samples and one blank control group without inoculation were designed. The samples were cultured in a shaker at 30℃ and 160 r / min, and samples were taken periodically to determine the OD of Ralstonia solanacearum XZW-1. 600 The concentrations of chlorobenzene and chlorobenzene are shown in the following figures. Figure 6 and Figure 7 .

[0062] from Figure 6 and Figure 7 It can be seen that when the concentration of chlorobenzene is 20-200 mg / L, Rhesus oryzae XZW-1 can multiply rapidly within 24 hours and almost completely degrade chlorobenzene after 24 hours; as the concentration of chlorobenzene increases, the time for complete degradation also increases to some extent.

[0063] Example 7: Degradation capacity of Rhesus oryzae XZW-1 for different carbon source substrates

[0064] Add all substrates except chlorobenzene (substrate concentrations are shown in Table 1) to fresh inorganic salt culture medium, and inoculate with the bacterial suspension prepared in Example 2, so that the initial cell concentration is expressed as OD0. 600 The value was calculated as 0.01. The cells were cultured under isothermal shaking at 30℃ and 160 r / min for 24 h, and the OD was measured afterward. 600 The degradation rates were measured, and the results are shown in Table 1. It was found that Ralstonia solanacearum XZW-1 degraded 1,2-dichlorobenzene and toluene to varying degrees.

[0065] Table 1. Degradation capacity of Rhesus genus XZW-1 for different carbon source substrates

[0066]

[0067] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Rhesus bacteria ( Ralstonia sp.)XZW-1, its accession number is CCTCC No: M 2022557.

2. The application of Ralstonia solanacearum XZW-1 as described in claim 1 in the degradation of organic pollutants, wherein the organic pollutants are one or more of chlorobenzene, 1,2-dichlorobenzene, toluene, and 1,2-xylene.

3. The method for degrading organic pollutants using Ralstonia solanacearum XZW-1 according to claim 1, wherein the organic pollutant is one or more of chlorobenzene, 1,2-dichlorobenzene, toluene, and 1,2-xylene, characterized in that... Includes the following steps: a. Inoculate the Rhesus xZW-1 strain onto a solid slant culture medium and incubate at 30–35°C for 24–48 h to obtain slant cells; b. Inoculate the slant bacterial cells into an inorganic salt culture medium and incubate at 25–30°C for 24–36 h to obtain OD. 600 =0.1~0.2% bacterial solution; c. Mix the bacterial solution and organic pollutants to prepare a mixed solution; d. The mixture is subjected to a degradation reaction at 20–40°C.

4. The method as described in claim 3, characterized in that... The solid slant culture medium in step a consists of the following components in parts by weight: yeast extract 0.50–1.00 g / L, soluble starch 0.50–1.00 g / L, MgSO4 0.50–1.00 g / L, tryptone 0.50–1.00 g / L, glucose 0.50–1.00 g / L, sodium pyruvate 0.30–0.60 g / L, K2HPO4 0.45–0.90 g / L, agar 18–20 g / L, and deionized water as the solvent.

5. The method as described in claim 3, characterized in that... The inorganic salt culture medium in step b consists of the following components in parts by weight: Na₂HPO₄ 4.0–5.0 g / L, KH₂PO₄ 0.8–1.2 g / L, (NH₄)₂SO₄ 2.3–2.8 g / L, MgSO₄ 0.18–0.23 g / L, CaCl₂ 0.022–0.24 g / L, and trace element stock solution 0.8–1.2 mL / L, with deionized water as the solvent; The trace element mother liquor is composed of the following components: FeSO4 0.8–1.2 g / L, CuSO4 0.015–0.025 g / L, H3BO3 0.013–0.015 g / L, MnSO4 0.08–0.13 g / L, ZnSO4 0.08–0.13 g / L, Na2MoO4 0.015–0.025 g / L, and CoCl2 0.015–0.025 g / L, with deionized water as the solvent.

6. The method as described in claim 3 or 5, characterized in that... The pH of the inorganic salt culture medium in step b is 4.0 to 10.

0.

7. The method as described in claim 3, characterized in that... The concentration of organic pollutants in step c is 20–200 mg / L.

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