Rhodococcus sp. yh-1 and application and aromatic hydrocarbon pollution degradation agent

Abstract

The present application relates to the field of microbial technology, in particular to a strain of Rhodococcus sp YH-1, its application and aromatic hydrocarbon pollution degradation agent, the Rhodococcus sp YH-1 is preserved in China typical culture preservation center on April 30, 2024, and the preservation number is CCTCC NO:M2024845.The Rhodococcus sp YH-1 of the present application has good degradation performance to benzene, toluene, chlorobenzene and o-dichlorobenzene.When degrading for 96h, the degradation rate of Rhodococcus sp YH-1 to 1000mg / L toluene, chlorobenzene and o-dichlorobenzene all reaches more than 50%;The degradation rate to benzene with concentration of 20-1000mg / L reaches more than 90%.The results show that Rhodococcus sp YH-1 has good degradation effect to aromatic hydrocarbon, and it has high practical application value and broad development prospect in repairing aromatic hydrocarbon contaminated soil, purifying aromatic hydrocarbon contaminated air and treating aromatic hydrocarbon wastewater.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of Rhodococcus erythropolis YH-1, and its application and aromatic hydrocarbon pollution degrading agent. BACKGROUND

[0002] Benzene is a common organic compound, and its main sources include processes such as petroleum refining, coal coking, chemical production, and combustion emissions. It is a basic raw material for the synthesis of fuels, plastics, pharmaceuticals, rubbers, and chemicals. Although benzene has a wide range of applications in industrial production and daily life, long-term exposure can cause poisoning and harm to the human body, especially affecting the blood system and nervous system; long-term exposure to high concentrations of benzene can increase the risk of leukemia and other blood system tumors; some studies have shown that benzene can cause damage to the reproductive system and affect fertility. Moreover, benzene is a volatile organic compound, and the release of benzene during combustion emissions or industrial production can cause air pollution, and the discharge of benzene into soil or water can have a negative impact on the ecosystem and threaten biodiversity.

[0003] The treatment methods for aromatic hydrocarbon pollution can generally be divided into physical, chemical, and biological methods. Among them, the physical treatment method mainly includes adsorption and membrane separation. Adsorption uses adsorbents (such as activated carbon, adsorption resin, zeolite, metal-organic framework, etc.) to adsorb aromatic hydrocarbons from water or gas, and then removes or recovers them; membrane separation uses membrane technology to separate and purify aromatic hydrocarbons in water, removing pollutants through membrane permeation and separation. The physical method is relatively simple to operate, has low technical threshold, and has lower cost compared to other technologies, but it does not change the chemical properties of the pollutants and can only transfer the pollution, with very low treatment efficiency, which is prone to secondary pollution, and often requires the joint action of chemical and biological methods to achieve treatment effect. Chemical treatment methods involve oxidation and reduction. Oxidation uses chemical oxidizing agents (such as hydrogen peroxide, ozone, etc.) to oxidize and decompose aromatic hydrocarbons into non-toxic products; reduction reaction converts aromatic hydrocarbons into less toxic substances. Chemical methods can quickly decompose pollutants and have a wide range of applications, but they may generate byproducts, increasing the difficulty of treatment, and require a large amount of chemical agents during treatment, which is costly and energy-intensive. Biological treatment methods include biodegradation and biosorption. Biodegradation uses microorganisms to decompose aromatic hydrocarbons into non-toxic or less toxic substances; biosorption uses the adsorption capacity of microorganisms or plants to remove aromatic hydrocarbons from water, which has the lowest negative impact on the environment, is safe, economical, and efficient.

[0004] Although biological treatment is an environmentally friendly method, it faces technical and reaction condition limitations, degradation product treatment, and other technical challenges when dealing with difficult-to-degrade and toxic compounds such as aromatic hydrocarbons, which also results in a relatively small number of such methods, and the treatment effect often does not meet the expected effect.

[0005] Therefore, it is of great significance to continuously research and optimize the biological treatment method to obtain a more efficient, sustainable and environmentally friendly method for biodegrading aromatic hydrocarbons, so as to solve the environmental and health problems caused by aromatic hydrocarbon pollution and promote the development and progress of related technologies. SUMMARY

[0006] In order to solve the above problems, the application provides a Rhodococcus ruber YH-1 and its application and an aromatic hydrocarbon pollution degradation agent.

[0007] In order to achieve the above purpose, the application provides the following technical scheme:

[0008] The application provides a Rhodococcus ruber YH-1, which is preserved in the China Center for Type Culture Collection on April 30, 2024, and the preservation number is CCTCC NO:M2024845.

[0009] The application also provides the application of the Rhodococcus ruber YH-1 in degrading aromatic hydrocarbons.

[0010] Preferably, the aromatic hydrocarbons include one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

[0011] The application also provides an aromatic hydrocarbon pollution degradation agent containing the Rhodococcus ruber YH-1.

[0012] Preferably, the content of the Rhodococcus ruber YH-1 in the aromatic hydrocarbon pollution degradation agent is 1.5x10 7 CFU / mL.

[0013] The application also provides the application of the aromatic hydrocarbon pollution degradation agent in degrading aromatic hydrocarbons.

[0014] Preferably, the aromatic hydrocarbons include one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

[0015] The application has the following beneficial effects:

[0016] The Rhodococcus ruber YH-1 has good degradation performance on benzene, toluene, chlorobenzene and o-dichlorobenzene. When the degradation time is 96h, the degradation rate of the Rhodococcus ruber YH-1 on 1000mg / L of toluene, chlorobenzene and o-dichlorobenzene all reaches more than 50%, and the degradation rate on benzene with a concentration of 20-1000mg / L reaches more than 90%. The results show that the Rhodococcus ruber YH-1 has good degradation effect on aromatic hydrocarbons, and has high practical application value and broad development prospect in repairing aromatic hydrocarbon contaminated soil, purifying aromatic hydrocarbon contaminated air and treating aromatic hydrocarbon wastewater. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0018] Figure 1 A chart showing that the process medium for degrading benzene by the Rhodococcus ruber YH-1 in a 50mL serum bottle becomes turbid over time is provided in the present application;

[0019] Figure 2 A colony morphology and SEM chart of the benzene-degrading strain YH-1 is provided in the present application, wherein A is the colony morphology and B is the SEM;

[0020] Figure 3 A growth and degradation characteristic chart of the benzene-degrading strain YH-1 for degrading benzene is provided in the present application.

[0021] BIOLOGICAL PRESERVATION

[0022] The Rhodococcus ruber YH-1, Latin name Rhodococcus ruber YH-1, was preserved in the China Center for Type Culture Collection on April 30, 2024, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO:M2024845. DETAILED DESCRIPTION

[0023] The present application provides a Rhodococcus ruber YH-1, which was preserved in the China Center for Type Culture Collection on April 30, 2024, and the preservation number is CCTCC NO:M2024845.

[0024] The present application also provides the application of the Rhodococcus ruber YH-1 in degrading aromatic hydrocarbons. In the present application, the aromatic hydrocarbons preferably include one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

[0025] The application further provides the aromatic hydrocarbon pollution degradation agent containing the Rhodococcus sp YH-1. 7 CFU / mL.

[0026] The application further provides the application of the aromatic hydrocarbon pollution degradation agent in degrading aromatic hydrocarbon.

[0027] The Rhodococcus sp YH-1 has the degradation ability to aromatic hydrocarbon, and is screened, separated and purified from groundwater of a paper mill in Shandong when benzene is used as the only carbon source and energy material. 7 The Rhodococcus sp YH-1 has the degradation ability to aromatic hydrocarbon, and is screened, separated and purified from groundwater of a paper mill in Shandong when benzene is used as the only carbon source and energy material.

[0028] In order to further illustrate the application, the application is described in detail in combination with examples below, but they should not be understood as the limitation to the protection scope of the application.

[0029] Example 1

[0030] Screening, separation and identification of the Rhodococcus sp YH-1:

[0031] The Rhodococcus sp YH-1 having the degradation ability to aromatic hydrocarbon is screened, separated and purified from groundwater of a paper mill in Shandong when benzene is used as the only carbon source and energy material.

[0032] The SSDM inorganic salt culture medium used for screening was prepared as follows: 0.2 g ammonium chloride (NH4Cl), 7.95 g sodium chloride (NaCl), 0.77 g magnesium chloride hexahydrate (MgCl2·6H2O), 1.05 g magnesium sulfate heptahydrate (MgSO4·7H2O), 0.076 g calcium chloride (CaCl2), 0.22 g potassium chloride (KCl), 0.01 g sodium bicarbonate (NaHCO3), 0.026 g sodium bromide (NaBr), and 0.25 g dipotassium hydrogen phosphate (K2HPO4) were weighed and mixed, then subjected to reverse osmosis (RO). Make up the volume to 1L with water, and add 1mL of trace elements per 1L of culture medium: Dissolve 0.15g zinc sulfate heptahydrate (ZnSO4·7H2O), 0.26g manganese sulfate monohydrate (MnSO4·H2O), 0.03g cobalt chloride hexahydrate (CoCl2·6H2O), 4.5g ferrous sulfate heptahydrate (FeSO4·7H2O), 0.02g nickel chloride hexahydrate (NiCl2·6H2O), 0.01g copper chloride (CuCl2), 0.1g sodium molybdate dihydrate (Na2MoO4·2H2O), and 0.06g boric acid (H3BO3) in 1L of RO water, adjust the pH to 7.0-7.5, and obtain the inorganic salt culture medium liquid. Then, dispense the inorganic salt culture medium liquid, seal it with sealing film, and sterilize it in an autoclave at 121℃ for 20min. After cooling, store it for later use.

[0033] Benzene was used as the acclimation substrate, with concentrations of 20 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L, and 1200 mg / L, respectively. First, 10 mL of groundwater was added to an inorganic salt culture medium containing 20 mg / L benzene compounds. After acclimation at 30°C for 7 days, a 5% inoculum was transferred to the next concentration for further acclimation, and so on, gradually increasing the concentration. After acclimation, the final concentration of bacterial culture was diluted 10 times. -1 ~10 -5 Take 100 μL of each culture and spread it onto an inorganic salt roller solid medium. Then, spread benzene evenly on the surface of the solid medium, seal it, and incubate it in a constant temperature incubator at 30℃ for 3 days. Select the viable strains, and then pick a single colony into a 50 mL serum bottle containing 10 mL of inorganic salt medium, adding benzene as the sole carbon source. Incubate at 30℃ and a shaking speed of 150 rpm. -1 Incubate under dark conditions until turbidity appears, then repeat the above operation three times; pick a single bacterium and place it in LB liquid medium at 30°C and a shaking speed of 150 rpm. -1 Under the condition of being cultured in the dark, after the turbidity is reached, the bacterial solution is aspirated and streaked onto an LB solid medium plate. The above operation is repeated 4 times to obtain the benzene-degrading bacteria YH-1.

[0034] The target fragment was obtained by extracting the genome of the strain YH-1, and primers were designed for PCR amplification. The amplification product was sent to a general biological company for sequencing. According to the 16S rRNA sequence provided by the general biological sequencing, homologous comparison was carried out in the NCBI database, and the species of the screened aromatic hydrocarbon degrading bacteria were identified. The amplification primers in the PCR process can be selected from a pair of universal primers, which are as follows:

[0035] Upstream primer 27F (SEQ ID No. 1): 5' AGTTTGATCMTGGCTCAG-3';

[0036] Downstream primer 1492R (SEQ ID No. 2): 5'-GGTTACCTTGTTACGACTT-3'.

[0037] The whole amplification system is as follows:

[0038] Genome: 1 μL

[0039] ddH2O: 20 μL

[0040] dNTPMix: 1 μL

[0041] 2×PhantaMax Buffer: 2.5 μL

[0042] Phanta Max Super-Fidelity DNA Polymerase: 1 μL

[0043] Primer: 1 μL each

[0044] The PCR amplification conditions are as follows: 95℃ pre-denaturation for 10s, 95℃ denaturation for 15s, 56℃ annealing for 15s, 72℃ extension for 20s, 30 cycles, and 72℃ repair extension for 5min.

[0045] The sequencing result is as follows (SEQ ID No. 3):

[0046]

[0047] According to the 16S rRNA sequence provided by general bio-sequence, homologous comparison analysis was carried out in the NCBI database, and it was found that the strain was most similar to Rhodococcus ruber strain TH-22, with a similarity of 100%. Therefore, the phenol-degrading bacteria screened in the application should belong to the genus Rhodococcus, and is named Rhodococcus ruber YH-1. It was preserved in the China Center for Type Culture Collection (CCTCC) on April 30, 2024, with a preservation number of CCTCC NO: M2024845 and a preservation address of Wuhan University, Wuhan, China.

[0048] Example 2

[0049] Study on the degradation characteristics of the strain under different pH (6-11) conditions

[0050] Luria-Bertani culture medium formula: sodium chloride (NaCl) 10 g / L, yeast extract 5 g / L, tryptone 10 g / L.

[0051] After the single-bacterium YH-1 was placed in 50 mL of LB medium and activated for 48 h, 9000 r·min -1 was used for centrifugation for 5 min, and the supernatant was resuspended with SSDM inorganic salt medium to wash away the carbon sources such as yeast powder in the LB medium. The above operation was repeated twice, with a volume of 10 mL. The absorbance was measured at a wavelength of 600 nm, and 50 mL of serum bottle was added. The serum bottle contained 30 mL of inorganic salt medium containing 1000 mg / L of benzene, and the initial bacterial content in the degradation system was 0.8 x 10 6 CFU / mL. To prevent benzene volatilization, a butyl plug was inserted and an aluminum cap was clamped. The culture was incubated at 30℃, 150 r·min -1 , in the dark, and the pH values were changed to 6, 7, 8, 9, 10, and 11, respectively. Samples were taken every 12 h, and the concentration of benzene and the absorbance at a wavelength of 600 nm were measured.

[0052] The degradation characteristics results are shown in Figure 3 . YH-1 can tolerate 1000 mg / L of benzene, and the degradation rate reaches 98.5% within 96 h. YH-1 can grow well under the growth condition of pH = 10.0 and has the best degradation effect. During the degradation process, the SSDM inorganic salt medium gradually becomes turbid, as shown in Figure 1 .

[0053] Example 3

[0054] Study on the degradation of the strain to other aromatic hydrocarbons (toluene, chlorobenzene, o-dichlorobenzene):

[0055] 10mLSSDM inorganic salt medium was placed in a 50mL serum bottle, and the bacterial liquid was added to adjust the bacterial content to 1.5×10 7 CFU / mL, aromatic hydrocarbons (toluene, chlorobenzene, o-dichlorobenzene) were added as the sole carbon source, the final concentration of aromatic hydrocarbons was 1000mg / L, and a butyl plug was immediately inserted and the aluminum cap was tightly pressed. Under the conditions of temperature 30℃, pH=10, shaking speed 150r·min -1 -1, the aromatic hydrocarbon concentration was determined every 24h.

[0056] Figure 2 The colony morphology of the benzene-degrading strain YH-1 provided by the present application is shown in Table 1, which shows that YH-1 completely degrades 1000mg / L benzene within 96h, and toluene, chlorobenzene and o-dichlorobenzene are degraded by about 50%, indicating that the benzene-degrading strain YH-1 has good substrate broad-spectrum property.

[0057] Table 1 Study on the degradation ability of YH-1 to different substrates

[0058]

[0059]

[0060] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A strain of Rhodococcus rubescens ( Rhodococcus ruber YH-1, characterized in that, The Rhodococcus ruber YH-1 was preserved in China Center for Type Culture Collection on April 30, 2024, and the preservation number is CCTCC NO: M2024845.

2. The use of the Rhodococcus ruber YH-1 in claim 1 in degrading aromatic hydrocarbons. The aromatic hydrocarbons are one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

3. An aromatic hydrocarbon contamination degrading agent, characterized by comprising: Containing the Rhodococcus ruber YH-1 in claim 1.

4. The aromatic hydrocarbon contamination degrader according to claim 3, characterized by, The content of Rhodococcus ruber YH-1 in the aromatic hydrocarbon pollution degradation agent is 1.5 x 10 7 CFU / mL.

5. The use of the aromatic hydrocarbon pollution degrading agent in claim 3 or 4 in degrading aromatic hydrocarbons. The aromatic hydrocarbons are one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

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