Rhodococcus YZ-1 and its application in degradation of organic pollutants

Abstract

The present invention belongs to the technical field of organic pollutant treatment, and specifically relates to a strain of Rhodococcus YZ-1 with high efficiency in pentane degradation. The Rhodococcus of the present invention is named Rhodococcus ( Rhodococcus sp.) YZ-1, which was deposited in China Center for Type Culture Collection on July 13, 2022, with a deposit number of CCTCC No: M 20221106. The Rhodococcus sp. of the present invention ( Rhodococcus Rhodococcus sp. YZ-1 can grow and reproduce using pentane as its sole carbon and energy source, adapts to a wide range of environmental temperatures and pH values, and can efficiently degrade pentane at initial concentrations up to 600 mg / L. Furthermore, the bacterium can also use n-hexane, cyclohexane, 2-methylpentane, and 3-methylpentane as carbon and energy sources, efficiently degrading n-hexane, 2-methylpentane, and 3-methylpentane. The Rhodococcus sp. YZ-1 provided by the present invention will play an important role in the practical treatment of pentane, n-hexane, cyclohexane, 2-methylpentane, and 3-methylpentane waste gas and wastewater.

Description

Technical Field

[0001] The present invention relates to a strain of Rhodococcus ( Rhodococcus sp . )YZ-1, belongs to the technical field of organic pollutant degradation. Background Art

[0002] Pentane ( n -Pentane, C5H 12 Pentane is a colorless, transparent liquid with a faint minty aroma. It is commonly used as a solvent, a foaming agent in the plastics industry, in the manufacture of artificial ice, an anesthetic, and in the synthesis of amyl alcohol. However, it has certain biological toxicity, primarily causing mild irritation to the eyes and respiratory tract, leading to mild dermatitis. High concentrations can cause mild irritation to the eyes and respiratory mucosa, narcosis, and even temporary loss of consciousness, posing a health hazard.

[0003] Waste gas generated during wastewater treatment in industries like the petrochemical industry often contains organic pollutants such as alkane and hexane. Currently, conventional treatment methods for these organic waste gases include physical and chemical methods such as adsorption, absorption, and high-temperature incineration. However, these traditional methods are costly, energy-intensive, and produce high carbon emissions. Therefore, the development of green, efficient, and low-carbon waste gas treatment methods is urgent and has become a hot topic and a challenge in environmental pollution research.

[0004] Biological methods that utilize microbial degradation have the advantages of mild reaction conditions, no secondary pollution, and low cost. Most microorganisms can simultaneously degrade multiple organic pollutants and have strong environmental adaptability. However, there are no reports of efficient degradation strains that can use pentane as the sole carbon and energy source, hindering the efficient treatment of pentane-containing waste gas by biological methods. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides Rhodococcus ( Rhodococcus sp . ) YZ-1 and its application in degrading organic pollutants, the Rhodococcus ( Rhodococcus sp . ) YZ-1 can achieve efficient degradation of organic pollutants, especially pentane, n-hexane, cyclohexane, 2-methylpentane and 3-methylpentane.

[0006] First, the present invention provides Rhodococcus ( Rhodococcus sp . )YZ-1, deposited in China Center for Type Culture Collection, the deposit number is CCTCC No: M 20221106, the deposit date is July 13, 2022, and the deposit address is Wuhan University, Wuhan, China, postal code 430072.

[0007] Secondly, the present invention also proposes the Rhodococcus ( Rhodococcus sp . YZ-1 was obtained by collecting activated sludge from a petrochemical enterprise's wastewater treatment plant, separating and purifying it. Its 16S rDNA has a Genebank accession number of OP389141. Its colony characteristics are as follows: The bacteria are oval, measuring (0.5-0.8) μm x (0.8-1.5) μm, are spore-free, and lack flagella. The colonies are light pink, round, opaque, smooth, moist, and easily lifted. They grow along streaked lines and are Gram-positive.

[0008] Again, the present invention also proposes the Rhodococcus ( Rhodococcus sp . ) Application of YZ-1 in the degradation of organic pollutants, wherein the organic pollutants include one or more of pentane, n-hexane, cyclohexane, 2-methylpentane and 3-methylpentane.

[0009] As a preferred application, the Rhodococcus ( Rhodococcus sp . ) YZ-1 was added into inorganic salt culture medium containing organic pollutants to carry out degradation reaction.

[0010] As a further preference, the conditions of the degradation reaction are: pH 4-10, temperature 20-40°C.

[0011] As a further preference, the initial concentration of the organic pollutants in the inorganic salt culture medium is 100-600 mg / L.

[0012] As further preferred, the Rhodococcus ( Rhodococcus sp . ) YZ-1 was added in the form of bacterial suspension.

[0013] As further preferred, the bacterial suspension is prepared according to the following steps:

[0014] (1) Slant culture: Rhodococcus ( Rhodococcus sp . ) YZ-1 was activated and inoculated into R2A solid medium and cultured at 30°C for 24-36 h to obtain slant bacteria;

[0015] (2) Preparation of bacterial suspension: Pick an inoculation loop of colonies from the slant in step (1) and inoculate it into an inorganic salt culture medium. Add pentane with an initial concentration of 200 mg / L and culture at 30°C and 160 rpm for 24 to 36 hours to obtain a bacterial suspension.

[0016] As further preferred, the R2A solid culture medium comprises: yeast powder 0.50 g / L, soluble starch 0.50 g / L, MgSO4·7H2O 0.05 g / L, peptone 0.50 g / L, glucose 0.50 g / L, sodium pyruvate 0.30 g / L, K2HPO4·3H2O 0.45 g / L, agar 15-20 g / L, pH about 7.2, and the solvent is deionized water.

[0017] As a further preference, the inorganic salt culture medium includes: Na2HPO4 4.5 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.023 g / L, trace element mother solution 1 mL / L, pH is about 7.0, and the solvent is deionized water.

[0018] As a further preference, the trace element mother solution comprises: FeSO4·7H2O 1.0 g / L, CuSO4·5H2O0.02 g / L, H3BO3 0.014 g / L, MnSO4·4H2O 0.10 g / L, ZnSO4·7H2O 0.10 g / L, Na2MoO4·2H2O0.02 g / L, and CoCl2·6H2O 0.02 g / L, and the solvent is deionized water.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention provides a highly efficient pentane-degrading bacterium, Rhodococcus ( Rhodococcus sp . ) YZ-1, a non-spore, non-flagellated Gram-positive bacterium that can grow with pentane as the sole carbon and energy source and efficiently degrade the substrate. In addition, Rhodococcus ( Rhodococcus sp . ) YZ-1 also has a certain degradation effect on other organic pollutants such as n-hexane;

[0021] (2) The Rhodococcus provided by the present invention ( Rhodococcus sp.) YZ-1 has a good degradation effect. Under the condition of low initial bacterial amount, it can produce excellent degradation effect on pentane with an initial concentration of up to 600 mg / L.

[0022] (3) The Rhodococcus provided by the present invention ( Rhodococcus sp.) YZ-1 has a wide range of optimal growth temperature and pH. 600 OD 600=0.01, the temperature is 25~35°C, and the pH is 6.0~8.0, pentane can be well degraded, and the degradation rate can reach more than 70%;

[0023] (4) Using the Rhodococcus provided by the present invention ( Rhodococcus sp.) YZ-1 has the advantages of simple operation, stable operation, low cost, mild reaction conditions and environmental friendliness for the degradation of organic pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention, the following is a brief introduction to the accompanying drawings:

[0025] Figure 1 For Rhodococcus ( Rhodococcus sp . ) Transmission electron microscopy image of YZ-1;

[0026] Figure 2 For Rhodococcus ( Rhodococcus sp.) Phylogenetic tree of YZ-1;

[0027] Figure 3 For Rhodococcus ( Rhodococcus sp.) YZ-1 bacterial growth and pentane degradation curve;

[0028] Figure 4 The effects of different pH values ​​of culture medium on Rhodococcus ( Rhodococcus sp . ) Bar graphs showing the effects of pentane degradation (A), growth (B), and mineralization (C) on YZ-1.

[0029] Figure 5 The effects of different temperatures on Rhodococcus Rhodococcus sp.) Bar graphs showing the effects of pentane degradation (A), growth (B), and mineralization (C) of YZ-1;

[0030] Figure 6 For Rhodococcus ( Rhodococcus sp . ) Degradation curves of YZ-1 to different initial concentrations of pentane;

[0031] Figure 7 For Rhodococcus ( Rhodococcus sp . ) Growth curves of YZ-1 at different initial pentane concentrations. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0033] The material composition of the inorganic salt culture medium in the following implementation is as follows: the inorganic salt culture medium composition is: Na2HPO44.5 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.023 g / L, trace element mother solution 1 mL / L, pH 7.0, solvent is deionized water; the material composition of the trace element mother solution 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, the solvent was deionized water.

[0034] Example 1: Rhodococcus ( Rhodococcus sp . ) Isolation, purification and identification of YZ-1

[0035] (1) Rhodococcus Rhodococcus sp . ) Isolation and purification of YZ-1

[0036] Activated sludge was collected on-site from a wastewater treatment station of a petrochemical enterprise in Zhejiang Province. After standing and settling for 2 hours, the supernatant and suspended impurities were removed, leaving only sludge with fine particles. The lower sludge after standing for 2 hours was mixed with inorganic salt culture medium at a ratio of 1:2 (v / v). 1.5 L of the mixture was then transferred to a 2 L sludge acclimation tank and acclimated at room temperature (25°C) using pentane as the sole carbon source and energy source in the entire system.

[0037] During the sludge acclimation experiment, the acclimation tanks were maintained with stable aeration. After stopping aeration each day and allowing the tanks to rest for 30 minutes, the pH of the upper layer of the acclimation tanks was measured and maintained at around 7.0. Half of the upper layer of culture medium was removed daily and replaced with fresh inorganic salt culture medium. Substrate was added daily after pentane was completely degraded, bringing the substrate concentration back to 126 mg / L. One month later, 10 mL of the sludge-water mixture was added to a shake flask containing 50 mL of sterilized inorganic salt culture medium. The acclimated sludge was found to steadily degrade 50 mg / L of pentane per day in the shake flask (shake flask experimental conditions: 30°C, 160 rpm), thus obtaining the acclimated sample.

[0038] 5 mL of the acclimated sample in the shake flask was transferred to other shake flasks for further performance testing, and pentane with an initial concentration of 50 mg / L was added. The culture was carried out at 30°C and 160 r / min. The substrate concentration was tested after 24 hours. It was found that the acclimated sample after transfer could stably degrade 95% or more of pentane in the shake flask within 48 hours. The sludge sample was further transferred and enriched into a shake flask containing 50 mL of inorganic salt culture medium, and pentane with an initial concentration of 50 mg / L was added. The transfer was carried out 6 times in total (5 mL for the first 3 times, 2 mL for the fourth time, and 1 mL for the last two times). The bacterial solution sample after 6 passages and enrichment was then cultured according to the conditions of 10 -1 ~10 -6 After multiple dilution, the plate was spread and cultured in a constant temperature incubator at 30°C for 2-3 days. Single colonies on the plate were picked and separated by streaking to obtain pure colonies.

[0039] (2) Rhodococcus Rhodococcus sp . ) Identification of YZ-1

[0040] Please refer to Figure 1 , Rhodococcus ( Rhodococcus sp . YZ-1 has morphological characteristics: its body is oval, measuring (0.5-0.8) μm x (0.8-1.5) μm, and it lacks spores and flagella. Its colonies are light pink, round, opaque, smooth, moist, and easily raised, growing along streaked lines. Furthermore, strain YZ-1 is aerobic and Gram-positive.

[0041] The obtained strain was entrusted to Zhejiang Tianke High-tech Development Co., Ltd. for PCR amplification and sequencing, and the 16S rDNA sequencing results were as follows (SEQ ID NO. 1, Genebank accession number OP389141):

[0042]

[0043] The sequencing results were uploaded to the Ezbiocloud.net website and compared with the standard strains on the website. Then, MEGA9.05 software was used to construct the bacterial phylogenetic tree using the Neighbor-Joining method. Finally, the Bootstrap method (repeated 1000 times) was used for evaluation. The constructed phylogenetic tree is shown in the figure. Figure 2 , thus confirming that the strain was Rhodococcus sp . , named Rhodococcus ( Rhodococcus sp . ) YZ-1, deposited in China Center for Type Culture Collection, deposit date: July 13, 2022, deposit number: CCTCC No: M 20221106 YZ-1, deposit address: Wuhan University, Wuhan, China, Postal Code 430072.

[0044] Example 2: Rhodococcus hodococcus sp . ) Expanded cultivation of YZ-1

[0045] (1) Slant culture: Rhodococcus ( Rhodococcus sp . ) YZ-1 was inoculated into R2A solid slant medium and cultured at 30°C for 24 h to obtain slant cells. The R2A solid medium consisted of 0.50 g / L yeast extract, 0.50 g / L soluble starch, 0.05 g / L MgSO4·7H2O, 0.50 g / L peptone, 0.50 g / L glucose, 0.30 g / L sodium pyruvate, 0.45 g / L K2HPO4·3H2O, and 15 g / L agar. The pH was approximately 7.2, and the solvent was deionized water.

[0046] (2) Preparation of bacterial suspension: Pick an inoculation loop of colony from the slant in step (1) and inoculate it into inorganic salt culture medium, add pentane with an initial concentration of 200 mg / L, and culture at 30°C and 160 r / min for 24 h to obtain a bacterial suspension. The inorganic salt culture medium is composed of: Na2HPO4 4.5 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O0.2 g / L, CaCl2 0.023 g / L, trace element mother solution 1 mL / L, pH 7.0, and the solvent is deionized water; the material composition of the trace element mother solution 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, CoCl2·6H2O0.02 g / L, and the solvent was deionized water.

[0047] Example 3: Rhodococcus ( Rhodococcus Degradation characteristics of pentane by YZ-1

[0048] Pentane was used as a Rhodococcus sp . ) is the sole carbon and energy source for YZ-1. The bacterial suspension prepared by the method of Example 2 was taken and inoculated into 50 mL of inorganic salt culture medium to make the initial bacterial concentration (measured in OD 600 The initial concentration of pentane was 0.01, and pentane was added at an initial concentration of 100 mg / L. The cells were cultured in a shaker at 30°C and 160 r / min. Samples were taken every 3 hours to determine the pentane degradation rate. At the same time, a portion of the bacterial solution was withdrawn with a 5 mL syringe to determine the bacterial OD value. The results are shown in the figure. Figure 3 During the experiment, two parallel samples and a blank control group without inoculation of the strain were designed. As time went on, the bacterial concentration gradually increased. At 30 h, the bacterial concentration reached a maximum of approximately OD 600 =0.303, the pentane degradation rate reached 92%. This embodiment shows that Rhodococcus ( Rhodococcus sp . ) YZ-1 can use pentane as the sole carbon source and energy source for growth and reproduction, and has the ability to degrade pentane stably and efficiently.

[0049] Example 4: Expansion of Rhodococcus sp. YZ-1

[0050] This embodiment differs from Embodiment 2 only in the following ways, and the remaining identical parts will not be described again in detail.

[0051] (1) Slant culture: The slant culture time is 36 h, and the agar content in the R2A solid medium is 20 g / L;

[0052] (2) Preparation of bacterial suspension: The culture time is 36 hours.

[0053] Example 5: Effect of pH on Rhodococcus Rhodococcus Effect of YZ-1 on the degradation of pentane

[0054] The inorganic salt medium was adjusted to different pH values ​​(specifically 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) using 1 mol / L NaOH aqueous solution or 1 mol / L H2SO4 aqueous solution. The bacterial suspension prepared by the method of Example 2 was inoculated into 50 mL of inorganic salt medium to make the initial bacterial cell concentration (measured in OD 600 The initial concentration of pentane was 0.01, and then pentane was added at an initial concentration of 100 mg / L. The cells were cultured in a shaker at 30°C and 160 r / min. After 30 hours of culture, samples were taken to measure the pentane degradation rate and bacterial OD in the reaction solution. 600 Value and CO2 value, see the results Figure 4 During the experiment, two parallel samples and a blank control group without inoculation of the strain were designed. Figure 4 It can be seen that with the increase of pH, Rhodococcus ( Rhodococcus sp.) YZ-1 showed a trend of increasing first and then decreasing in the degradation rate of pentane. Rhodococcus sp.) YZ-1 showed a degradation rate of more than 60% for pentane, especially when the pH was 6 or 7. Rhodococcus sp . ) YZ-1 has the best effect on pentane degradation, with a degradation rate of more than 85%. Rhodococcus sp . ) The growth and CO2 production of YZ-1 showed a consistent trend with the pentane degradation rate.

[0055] Example 6: Effect of temperature on Rhodococcus Rhodococcus sp . ) Effect of YZ-1 on pentane degradation

[0056] Take the bacterial suspension prepared by the method of Example 2 and inoculate it into 50 mL of inorganic salt culture medium to make the initial bacterial concentration (measured in OD 600 The initial concentration of pentane was 0.01, and then pentane was added at an initial concentration of 100 mg / L. Each sample was placed in a shaker at 20°C, 25°C, 30°C, 35°C, and 40°C for constant temperature shaking (the shaker speed was 160 r / min). After 30 hours of incubation, samples were taken to measure the pentane degradation rate in the reaction solution and the bacterial liquid OD.600 Value and CO2 value, see the results Figure 5 During the experiment, two parallel samples and a blank control group without inoculation of the strain were designed. Figure 5 It can be seen that with the increase of temperature, Rhodococcus ( Rhodococcus sp.) YZ-1 showed a trend of increasing first and then decreasing in the degradation rate of pentane. In the temperature range of 20℃~40℃, Rhodococcus ( Rhodococcu s sp.) YZ-1 had a degradation rate of more than 60% for pentane, especially at 30℃, Rhodococcus ( Rhodococcus sp. ) YZ-1 has the best effect on pentane degradation, with a degradation rate of more than 85%. Rhodococcus sp.) YZ-1 also showed the same trend in growth and CO2 production as the pentane degradation rate.

[0057] Example 7: Effect of substrate concentration on the activity of Rhodococcus Rhodococcus sp . ) Effect of YZ-1 on pentane degradation

[0058] Take the bacterial suspension prepared by the method of Example 2 and inoculate it into 50 mL of inorganic salt culture medium to make the initial bacterial concentration (measured in OD 600 The initial concentration of pentane was 100, 200, 300, 400, 500, and 600 mg / L. The mixture was placed in a shaker at 30°C and 160 r / min for incubation. Samples were taken regularly to measure the pentane concentration and the activity of Rhodococcus ( Rhodococcus sp . ) YZ-1 bacterial solution OD 600 Value, see the result Figure 6 、 7 During the experiment, two parallel samples and a blank control group without inoculation of the strain were designed. Figure 6 、 7 It can be seen that with the increase of initial substrate concentration, Rhodococcus ( Rhodococcus sp . ) YZ-1 did not have much effect on the degradation of pentane, so it can be inferred that pentane is less toxic to the strain and the strain has a strong tolerance to the concentration of pentane. However, when the reaction time exceeds 50h, Rhodococcus ( Rhodococcus sp . ) The degradation ability of YZ-1 on pentane gradually weakened under closed conditions.

[0059] Example 8: Rhodococcus ( Rhodococcus sp . ) Degradation ability of YZ-1 on different carbon source substrates

[0060] Take the bacterial suspension prepared by the method of Example 2 and inoculate it into 50 mL of inorganic salt culture medium to make the initial bacterial concentration (measured in OD 600 The initial concentration of n-hexane, cyclohexane, 2-methylpentane, and 3-methylpentane was 100 mg / L. The culture was carried out in a shaker at 30°C and 160 r / min. The substrate degradation rate in the bottle was measured at 24 and 72 hours. The results are shown in Table 1. Rhodococcus sp . ) YZ-1 can degrade n-hexane, cyclohexane, 2-methylpentane and 3-methylpentane to varying degrees.

[0061] Table 1. Rhodococcus Rhodococcus sp . ) Degradation ability of YZ-1 on different carbon source substrates

[0062]

Claims

1. Rhodococcus Rhodococcus sp. )YZ-1, deposited in China Center for Type Culture Collection, the deposit number is CCTCC No: M 20221106, the deposit date is July 13, 2022, and the deposit address is Wuhan University, Wuhan, China, Postal Code 430072.

2. The Rhodococcus according to claim 1 ( Rhodococcus sp. ) The application of YZ-1 in the degradation of organic pollutants is characterized by: The organic pollutant is selected from one or more of pentane, n-hexane, cyclohexane, 2-methylpentane and 3-methylpentane.

3. The use according to claim 2, characterized in that The application is to use Rhodococcus ( Rhodococcus sp. ) YZ-1 is added to an inorganic salt culture medium containing organic pollutants to perform a degradation reaction; the conditions of the degradation reaction are: pH = 4~10, temperature 20~40°C.

4. The use according to claim 2, characterized in that The organic pollutant is pentane.

5. The use according to claim 3, characterized in that The initial concentration of the organic pollutants in the inorganic salt culture medium is 100-600 mg / L.

6. The use according to claim 3, characterized in that The Rhodococcus ( Rhodococcus sp. ) YZ-1 was added in the form of bacterial suspension.

7. The use according to claim 6, characterized in that The bacterial suspension was prepared according to the following steps: (1) Slant culture: Rhodococcus ( Rhodococcus sp. ) YZ-1 was activated and inoculated into R2A solid medium and cultured at 30°C for 24-36 h to obtain slant bacteria; (2) Preparation of bacterial suspension: Pick a colony from the slant and inoculate it into an inorganic salt culture medium. Add pentane with an initial concentration of 200 mg / L and culture at 30°C and 160 r / min for 24-36 hours to obtain a bacterial suspension.

8. The use according to claim 7, characterized in that The R2A solid culture medium includes: 0.50 g / L yeast powder, 0.50 g / L soluble starch, 0.05 g / L MgSO4·7H2O, 0.50 g / L peptone, 0.50 g / L glucose, 0.30 g / L sodium pyruvate, 0.45 g / L K2HPO4·3H2O, 15-20 g / L agar, pH is about 7.2, and the solvent is deionized water.

9. The use according to any one of claims 3 or 7, characterized in that The inorganic salt culture medium includes: Na2HPO4 4.5 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.023 g / L, and trace element mother solution 1 mL / L, with a pH of 7.0, and the solvent is deionized water.

10. The use according to claim 9, characterized in that The trace element mother solution comprises: 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, and CoCl2·6H2O 0.02 g / L, and the solvent is deionized water.

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