Microorganisms capable of degrading nitrogen-containing heterocyclic compounds and their application in wastewater treatment

Abstract

The present invention discloses a microorganism for degrading nitrogen-containing heterocyclic compounds and its application in wastewater treatment, belonging to the technical field of wastewater treatment. The microorganism is Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 has a broad spectrum and can degrade and mineralize a variety of nitrogen-containing heterocyclic compounds including indole, quinoline, benzothiazole or pyridine. It can also be combined with Rhodococcus erythrocytes ( Rhodococcus red ) and Rho4, the two can form a composite bacterial community, which can further improve the degradation and mineralization effect of nitrogen-containing heterocyclic compounds, especially indole. Whether Rho48 is used alone or in combination with Rho4, it can degrade and mineralize at least three nitrogen-containing heterocyclic compounds including quinoline, benzothiazole or pyridine, making it more selective in treating wastewater containing nitrogen-containing heterocyclic compounds, especially complex polluted wastewater containing multiple nitrogen-containing heterocyclic compounds.

Description

Technical Field

[0001] The invention belongs to the technical field of wastewater treatment, and in particular relates to microorganisms capable of degrading nitrogen-containing heterocyclic compounds and applications thereof in wastewater treatment. Background Art

[0002] Nitrogen-containing heterocyclic compounds refer to organic compounds that contain nitrogen atoms in their molecules and participate in the formation of a ring structure as a heteroatom. They mainly include five-membered nitrogen-containing heterocyclic compounds such as pyrrole, imidazole, pyrazole and thiazole, six-membered nitrogen-containing heterocyclic compounds such as pyridine, pyrimidine and pyrazine, benzo-heterocyclic compounds such as indole, quinoline, benzothiazole and benzimidazole, and heterocyclic compounds such as purine and pteridine. Among them, pyridine, indole, quinoline, benzothiazole, etc. are difficult-to-degrade organic pollutants commonly found in industrial wastewater such as coking wastewater. These nitrogen-containing heterocyclic compounds pose a serious threat to the environment and human health due to their teratogenic, carcinogenic and mutagenic properties.

[0003] Common treatment processes for nitrogen-containing heterocyclic compounds include: catalytic wet oxidation, biodegradation and physical and chemical methods. Among them, the biodegradation method uses the metabolism of different types of microorganisms to degrade nitrogen-containing heterocyclic compounds into inorganic substances, thereby achieving the removal of pollutants. The biodegradation method is economical and efficient, has no secondary pollution and has relatively mild action conditions. It is currently an effective way to remove and transform nitrogen-containing heterocyclic compounds. In recent years, some functional strains with significant ability to degrade nitrogen-containing heterocyclic compounds have been isolated and cultured, such as Rhodococcus, Burkholderia, Pseudomonas, Bacillus and Paracoccus. These degradation strains are considered to be potential resources for enhanced removal of nitrogen-containing heterocyclic compounds.

[0004] However, when using functional strains to treat nitrogen-containing heterocyclic compounds, it was found that the functional strains were specific to nitrogen-containing heterocyclic compounds, and a single degradation strain could not degrade multiple types of nitrogen-containing heterocyclic compounds at the same time, or it could degrade a small number of nitrogen-containing heterocyclic compounds. Bacillus When treating the combined pollution of indole, quinoline, pyridine and 3-methylindole, sp. L1 had no obvious degradation effect on pyridine and 3-methylindole. This may be because the degradation strain has limited metabolic pathways and can only degrade one or a few specific nitrogen-containing heterocyclic compounds.

[0005] In actual industrial production processes, nitrogen-containing heterocyclic compounds in wastewater are often not isolated. Therefore, it is particularly important to provide more microorganisms that can degrade nitrogen-containing heterocyclic compounds to adapt to different wastewater treatments. Summary of the invention

[0006] 1. Problem to be solved

[0007] On the one hand, the present invention aims at the technical problem that the microorganisms that degrade nitrogen-containing heterocyclic compounds have substrate (nitrogen-containing heterocyclic compounds) specificity, which leads to the limitation of the treatment of nitrogen-containing heterocyclic compound wastewater, and provides a new microorganism Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48, a broad-spectrum microorganism capable of degrading at least three nitrogen-containing heterocyclic compounds including quinoline, benzothiazole or pyridine.

[0008] On the other hand, the present invention is also directed to Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 is not effective enough in degrading indole, and another microorganism Rhodococcus erythrorhizium that degrades nitrogen-containing heterocyclic compounds is provided ( Rhodococcus red ) Rho4, the two microorganisms were used in combination to form a composite bacterial community to provide more metabolic pathways and improve the degradation effect of indole.

[0009] 2. Technical solution

[0010] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0011] The present invention provides a pyridinophilic rhodococcus ( Rhodococcus pyridinivorans ) Rho48, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), the deposit date is December 4, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32892; the pyridinophilic Rhodococcus ( Rhodococcus pyridinivorans ) Rho48 was selected from the activated sludge of the membrane bioreactor of Wuxi Municipal Wastewater Treatment Plant. It has a broad spectrum and can degrade a variety of nitrogen-containing heterocyclic compounds including indole, quinoline, benzothiazole or pyridine, among which quinoline, benzothiazole and pyridine have very high degradation efficiency.

[0012] The present invention also provides the above-mentioned Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans )Application of Rho48 in the treatment of nitrogen-containing heterocyclic compound wastewater, Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 can degrade nitrogen-containing heterocyclic compounds in wastewater.

[0013] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes any one or more of indole, quinoline, benzothiazole, and pyridine.

[0014] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes any one or more of quinoline, benzothiazole, and pyridine.

[0015] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes quinoline or benzothiazole or pyridine.

[0016] Furthermore, the above application also includes the use of Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 combination, Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC) on December 4, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32891; Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 can form a complex bacterial community, which can further improve the degradation effect of nitrogen-containing heterocyclic compounds in wastewater.

[0017] Furthermore, in the above application, the combined use includes: preparing Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 into bacterial suspensions respectively; then mixing the bacterial suspension of Rhodococcus pyridinii Rho48 and the bacterial suspension of Rhodococcus erythrocytes Rho4 and adding them to the wastewater, or adding the bacterial suspension of Rhodococcus pyridinii Rho48 and the bacterial suspension of Rhodococcus erythrocytes Rho4 to the wastewater respectively; the bacterial suspension of Rhodococcus pyridinii Rho48 and the bacterial suspension of Rhodococcus erythrocytes Rho4 are added to the wastewater respectively, and the present invention does not limit the order of addition, and the bacterial suspension of Rhodococcus pyridinii Rho48 can be added first, or the bacterial suspension of Rhodococcus erythrocytes Rho4 can be added first, or both can be added at the same time.

[0018] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes any one or more of indole, quinoline, benzothiazole, and pyridine.

[0019] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes indole; compared with the degradation by a single microorganism, the combined use can significantly improve the degradation and mineralization effect of indole, presumably because Rhodococcus pyridinophilus ( Rhodococcus pyridinium-eating ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 forms a complex bacterial community, exerts a synergistic effect, and provides more metabolic pathways.

[0020] Furthermore, in the above application, the wastewater includes industrial wastewater such as coking wastewater, printing and dyeing wastewater, pharmaceutical wastewater and / or rubber wastewater.

[0021] The present invention also provides a kind of Rhodococcus rubrum ( Rhodococcus red ) Rho4, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), the deposit date is December 4, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32891; the red coccus ( Rhodococcus red) Rho4 was screened from the activated sludge of the membrane bioreactor of Wuxi Municipal Wastewater Treatment Plant. Rhodococcus red ) Rho4 can degrade nitrogen-containing heterocyclic compounds such as indole, quinoline, benzothiazole and / or pyridine to a certain extent; it is combined with Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 can further improve the degradation and mineralization effect of nitrogen-containing heterocyclic compounds indole, quinoline, benzothiazole and / or pyridine, especially the nitrogen-containing heterocyclic compound indole, which can play a role in the degradation and mineralization of Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) The synergistic effect of Rho4 significantly improved the degradation and mineralization of indole compared with the degradation by a single microorganism.

[0022] The present invention also provides the above-mentioned Rhodococcus ( Rhodococcus red ) Application of Rho4 in treating wastewater containing nitrogen heterocyclic compounds, which includes the use of Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 and Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) and Rho48 to degrade nitrogen-containing heterocyclic compounds in wastewater.

[0023] Furthermore, in the above application, the combined use includes: preparing Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 into bacterial suspensions respectively; then mixing the bacterial suspension of Rhodococcus pyridinii Rho48 and the bacterial suspension of Rhodococcus erythrocytes Rho4 and adding them to the wastewater, or adding the bacterial suspension of Rhodococcus pyridinii Rho48 and the bacterial suspension of Rhodococcus erythrocytes Rho4 to the wastewater respectively.

[0024] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes any one or more of indole, quinoline, benzothiazole, and pyridine.

[0025] Furthermore, in the above application, the nitrogen-containing heterocyclic compound includes indole.

[0026] Furthermore, in the above application, the wastewater includes industrial wastewater such as coking wastewater, printing and dyeing wastewater, pharmaceutical wastewater and / or rubber wastewater.

[0027] The present invention also provides the above-mentioned Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Application of Rho4 in the preparation of microbial agents.

[0028] The present invention also provides a microbial agent, which comprises: Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans )Rho48.

[0029] The present invention also provides the use of the above-mentioned microbial agent in treating wastewater containing nitrogen heterocyclic compounds.

[0030] Furthermore, the above application includes: preparing Rhodococcus pyridinii Rho48 into a bacterial suspension; and then adding it into wastewater, and Rhodococcus pyridinii Rho48 can degrade nitrogen-containing heterocyclic compounds in the wastewater.

[0031] The present invention also provides a microbial agent, which comprises: Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4, Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 separates and packages.

[0032] The present invention also provides the use of the above-mentioned microbial agent in treating wastewater containing nitrogen heterocyclic compounds.

[0033] Furthermore, the above application includes: preparing Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 into bacterial suspensions respectively; then mixing the Rhodococcus pyridinii Rho48 bacterial suspension and the Rhodococcus erythrocytes Rho4 bacterial suspension and adding them to wastewater; or adding the Rhodococcus pyridinii Rho48 bacterial suspension and the Rhodococcus erythrocytes Rho4 bacterial suspension to wastewater respectively, Rhodococcus pyridinii ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 can form a complex bacterial community, which can further improve the degradation effect of nitrogen-containing heterocyclic compounds in wastewater.

[0034] The present invention also provides a method for treating wastewater containing nitrogen heterocyclic compounds, which comprises: adding the above-mentioned Rhodococcus pyridinophilus Rho48 to the wastewater.

[0035] Furthermore, in the above method, Rhodococcus pyridinii Rho48 is prepared into a bacterial suspension; and then added into the wastewater, Rhodococcus pyridinii Rho48 can degrade nitrogen-containing heterocyclic compounds in the wastewater.

[0036] Furthermore, in the above method, the nitrogen-containing heterocyclic compound includes any one or more of indole, quinoline, benzothiazole, and pyridine.

[0037] Furthermore, in the above method, the nitrogen-containing heterocyclic compound includes any one or more of quinoline, benzothiazole, and pyridine.

[0038] Furthermore, in the above method, the wastewater includes industrial wastewater such as coking wastewater, printing and dyeing wastewater, pharmaceutical wastewater and / or rubber wastewater.

[0039] The present invention also provides a method for treating wastewater containing nitrogen heterocyclic compounds, which comprises: adding the above-mentioned Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 to the wastewater.

[0040] Furthermore, the above application includes: preparing Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 into bacterial suspensions respectively; then mixing the Rhodococcus pyridinii Rho48 bacterial suspension and the Rhodococcus erythrocytes Rho4 bacterial suspension and adding them to wastewater; or adding the Rhodococcus pyridinii Rho48 bacterial suspension and the Rhodococcus erythrocytes Rho4 bacterial suspension to wastewater respectively, Rhodococcus pyridinii ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 can form a complex bacterial community, which can further improve the degradation effect of nitrogen-containing heterocyclic compounds in wastewater.

[0041] Furthermore, in the above method, the nitrogen-containing heterocyclic compound includes any one or more of indole, quinoline, benzothiazole, and pyridine.

[0042] Furthermore, in the above method, the nitrogen-containing heterocyclic compound includes indole.

[0043] Furthermore, in the above method, the wastewater includes industrial wastewater such as coking wastewater, printing and dyeing wastewater, pharmaceutical wastewater and / or rubber wastewater.

[0044] Furthermore, in the above application or method, the preparation of the suspension of Rhodococcus pyridinophilus Rho48 includes: inoculating Rhodococcus pyridinophilus Rho48 into LB liquid culture medium, and culturing at 35-40°C and 150-250 rpm for 24-48 h.

[0045] Furthermore, in the above application or method, the preparation of the Rhodococcus Rho4 bacterial suspension includes: inoculating Rhodococcus Rho4 into LB liquid culture medium, and culturing at 35-40°C and 150-250 rpm for 24-48 h.

[0046] Furthermore, the preparation of the above-mentioned Rhodococcus pyridinophilus Rho48 bacterial suspension or the Rhodococcus erythrophilus Rho4 bacterial suspension was carried out under the culture conditions of 37° C. and 180 rpm for 36 h.

[0047] Further, in the above application or method, the inoculation biomass of Rhodococcus pyridinophilus Rho48 or Rhodococcus erythrophilus Rho4 is: the inoculation biomass is OD 600 =0.05~0.1.

[0048] Furthermore, in the above application or method, the inoculation biomass ratio of Rhodococcus pyridinophilus Rho48 or Rhodococcus erythrophilus Rho4 is 1:1.

[0049] Furthermore, in the above application or method, the temperature for treating the nitrogen-containing heterocyclic compound wastewater is 35-40°C; the pH is 6.5-7.5; the rotation speed is 150-250 rpm; and the treatment time is 0-72 h.

[0050] Furthermore, in the above application or method, the temperature for treating the nitrogen-heterocyclic compound wastewater is 37°C; the pH is 6.5-7.5; the rotation speed is 180 rpm; and the treatment time is 24-72 h.

[0051] Furthermore, in the above application or method, the processing time is 48 to 72 hours.

[0052] 3. Beneficial effects

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The microorganism for degrading nitrogen-containing heterocyclic compounds provided by the present invention and its application in wastewater treatment, wherein the microorganism for degrading nitrogen-containing heterocyclic compounds is Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 has a broad spectrum and can degrade and mineralize a variety of nitrogen-containing heterocyclic compounds including indole, quinoline, benzothiazole or pyridine. In particular, it has very high degradation and mineralization efficiency for quinoline, benzothiazole and pyridine, with a degradation efficiency close to 100%.

[0055] (2) The microorganism for degrading nitrogen-containing heterocyclic compounds provided by the present invention and its application in wastewater treatment, wherein the microorganism for degrading nitrogen-containing heterocyclic compounds is Rhodococcus erythrocytes ( Rhodococcus red ) Rho4, the Rhodococcus erythrocytes ( Rhodococcus red ) Rho4 is able to degrade and mineralize nitrogen-containing heterocyclic compounds such as indole, quinoline, benzothiazole and / or pyridine to a certain extent. In addition, it was combined with Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) and Rho48, the two can form a composite bacterial community, which can further improve the degradation and mineralization effect of nitrogen-containing heterocyclic compounds indole, quinoline, benzothiazole and / or pyridine, with a degradation rate of up to 98%; especially the nitrogen-containing heterocyclic compound indole, which can play a role in the degradation of Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red ) The synergistic effect of Rho4 significantly improved the degradation and mineralization of indole compared with the degradation and mineralization of a single microorganism.

[0056] (3) The microorganisms for degrading nitrogen-containing heterocyclic compounds provided by the present invention and their use in wastewater treatment, whether it is Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 alone or Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus red) Rho4 can be used in combination to degrade and mineralize at least three nitrogen-containing heterocyclic compounds including quinoline, benzothiazole or pyridine, making the treatment of nitrogen-containing heterocyclic compound wastewater, especially the complex polluted wastewater containing multiple nitrogen-containing heterocyclic compounds, more selective. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is the treatment result of a single nitrogen-containing heterocyclic compound by the single strain Rhodococcus pyridinophilus Rho48 and the single strain Rhodococcus erythrophilus Rho4 provided by the present invention.

[0058] Figure 2 This is the degradation result of a single nitrogen-containing heterocyclic compound using the combination of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 provided by the present invention.

[0059] Figure 3 This is the mineralization result of a single nitrogen-containing heterocyclic compound using the combination of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 provided by the present invention.

[0060] Figure 4 This is the treatment result of the combined use of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 provided by the present invention on the combined pollution of nitrogen-containing heterocyclic compounds (indole 10 mg / L+quinoline 10 mg / L+pyridine 10 mg / L).

[0061] Figure 5 This is the result of treating the combined pollution of nitrogen-containing heterocyclic compounds (indole 25 mg / L + quinoline 25 mg / L) by using the combination of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 provided by the present invention. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with specific embodiments.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0064] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0065] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0066] As used herein, the term "any one or more of" is intended to be synonymous with "one or more of." For example, "any one or more of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0067] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is used only for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly stated as the range limit, but also including all individual numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly stated.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly stated, but also including individual numbers (such as 2,3,4) and sub-ranges (such as 1 to 3, 2 to 4, etc.).The same principle is applicable to the range of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this interpretation should be applied.

[0068] Example 1

[0069] This example provides the isolation and identification of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4.

[0070] (1) Isolation of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4

[0071] The matrix used to enrich strains that degrade nitrogen-containing heterocyclic compounds was taken from the activated sludge of the membrane bioreactor of the Wuxi Municipal Wastewater Treatment Plant. The enriched bacterial liquid was obtained through a gradient enrichment strategy (increasing the concentration of the nitrogen-containing heterocyclic compound benzothiazole while reducing the concentration of glucose).

[0072] The obtained enriched bacterial solution was diluted to 10 with sterile ultrapure water. -4 ~10 -7, 100 μL of diluted bacterial solution was evenly spread on an inorganic salt solid medium with glucose as the sole carbon source, and cultured in a 37°C constant temperature incubator for 5-7 days, and single colonies of different morphologies were picked and streaked on the solid medium for multiple times to obtain purified strains. Among them: the composition of the inorganic salt medium is: glucose 281 mg / L, NH4Cl 57.3 mg / L, KH2PO413.2 mg / L, MgSO4•7H2O 15 mg / L, CaCl2•2H2O 16.5 mg / L, MnCl2•4H2O 100 μg / L, FeSO4•7H2O 80 μg / L, ZnSO4•7H2O 40 μg / L, CuSO4•5H2O 20 μg / L, Na2MoO4•2H2O 20 μg / L, CoCl2•6H2O 10 μg / L.

[0073] The purified strain was inoculated into LB liquid medium and cultured in a constant temperature shaker at 37°C and 180 rpm for standby use. The composition of LB medium is: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0074] (2) Identification of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4

[0075] The purified strain culture fluid was subjected to PCR amplification using universal primers for bacterial 16S rRNA gene sequences, wherein the primer sequences were:

[0076] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 1);

[0077] 1492R: 5'-TACGGYTACCTTGTTAYGACTT-3' (SEQ ID NO. 2);

[0078] PCR reaction system: 12.5 μL 2 × EasyTaq ® PCR SuperMix (Quanshijin, China), 10.5 μL sterile enzyme-free water, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), and 1 μL bacterial solution;

[0079] The PCR program was set as follows: pre-denaturation at 95°C for 8 min; denaturation at 95°C for 20 s, annealing at 55°C for 25 s, and extension at 72°C for 70 s, for a total of 32 cycles; and extension at 72°C for 5 min.

[0080] The PCR products were purified using an agarose gel DNA purification kit (Takara, Japan) and sequenced by 16S rDNA. The resulting nucleotide sequences were shown as SEQ ID NO.3 and SEQ ID NO.4, respectively. The sequences were compared by BLAST homology in the NCBI-nr database.

[0081] The results showed that the isolated strains were Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) and Rhodococcus erythrocytes ( Rhodococcus ruber ), respectively named in the present invention as Rhodococcus pyridinophilus ( Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus ruber ) Rho4. The above-mentioned Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 have been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on December 4, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit numbers CGMCC No. 32892 and CGMCC No. 32891 respectively.

[0082] Sequence SEQ ID NO.3 (identified as Rhodococcus pyridinophilus):

[0083]

[0084] Sequence SEQ ID NO.4 (identified as Rhodococcus erythrorhizium):

[0085]

[0086] Example 2

[0087] This example provides a degradation experiment of a single nitrogen-containing heterocyclic compound by a single strain of Rhodococcus pyridinophilus Rho48, comprising the following steps:

[0088] (1) Preparation of Rhodococcus pyridinophilus Rho48 bacterial suspension

[0089] The pyridinophilic Rhodococcus Rho48 was inoculated into LB liquid medium sterilized by high temperature and high pressure, and cultured in a constant temperature shaker at 37°C and 180 rpm. After 36 h, the strain was cultured to the logarithmic phase, and 8000× the bacterial solution was taken out. g Centrifuge for 2 min, discard the supernatant, and resuspend the precipitated bacteria in PBS (phosphate buffer solution). Repeat the above operation 3 times; finally, resuspend the precipitated bacteria in an inorganic salt culture medium (same as in Example 1) to obtain a suspension of Rhodococcus pyridinophilus Rho48 for use.

[0090] (2) Degradation of single nitrogen-containing heterocyclic compounds

[0091] In an inorganic salt medium (same as in Example 1), 10 mg / L of a single nitrogen-containing heterocyclic compound, namely indole, quinoline, benzothiazole and pyridine, was first added to simulate nitrogen-containing heterocyclic compound wastewater, and then the prepared suspension of Rhodococcus pyridinophilus Rho48 was inoculated to ensure that the biomass after inoculation was OD 600 =0.05~0.1, and finally a 100 mL degradation system was established, and the pH of the system was adjusted between 6.5~7.5.

[0092] The degradation conditions were 37°C, aerobic conditions were formed on a magnetic stirrer at a speed of 180 rpm, and the treatment time was 48 h.

[0093] Each experimental group was replicated three times, and samples were taken at 0 h and 48 h to detect the concentrations of nitrogen-containing heterocyclic compounds and total organic carbon (TOC).

[0094] The concentrations of the above nitrogen-containing heterocyclic compounds were measured by high performance liquid chromatography (HPLC, 1260 Infinity Ⅱ, Agilent Technologies, USA), the detector type was an ultraviolet light source (DAD-3000, Agilent Technologies, USA), the chromatographic column was a C18 column (4.6 × 250 mm, 5 μm, Agilent Technologies, USA), and the detection conditions were shown in Table 1 below:

[0095] Table 1

[0096]

[0097] TOC concentration was determined in accordance with the “Determination of Total Organic Carbon in Water Quality—Combustion Oxidation-Non-Dispersive Infrared Absorption Method” (HJ 501-2009).

[0098] (3) Results analysis

[0099] The experimental results are as follows Figure 1 As shown in the figure, the degradation rate of quinoline, benzothiazole and pyridine by Rhodococcus pyridinophilus Rho48 is as high as about 98%, and the mineralization rate of benzothiazole and pyridine is above 50%, which shows that it has a certain broad-spectrum and high-efficiency treatment performance; however, the treatment performance of indole is weak as a whole, and its degradation rate and mineralization rate are both below 20%.

[0100] Example 3

[0101] This example provides a degradation experiment of a single nitrogen-containing heterocyclic compound by a single strain of Rhodococcus erythrorhizium Rho4, comprising the following steps:

[0102] (1) Preparation of Rhodococcus erythrocytes Rho4 bacterial suspension

[0103] Rhodococcus erythrocytes Rho4 were inoculated into LB liquid medium sterilized by high temperature and high pressure, and cultured in a constant temperature shaker at 37°C and 180 rpm. After 36 h, the strain was cultured to the logarithmic phase, and 8000× the bacterial solution was taken out. g Centrifuge for 2 min, discard the supernatant, and resuspend the precipitated bacteria in PBS (phosphate buffer solution). Repeat the above operation 3 times; finally, resuspend the precipitated bacteria in an inorganic salt culture medium (same as in Example 1) to obtain a suspension of Rhodococcus erythrocytes Rho4 for use.

[0104] (2) Degradation of single nitrogen-containing heterocyclic compounds

[0105] In an inorganic salt medium (same as in Example 1), 10 mg / L of a single nitrogen-containing heterocyclic compound, namely indole, quinoline, benzothiazole and pyridine, was first added to simulate nitrogen-containing heterocyclic compound wastewater, and then the prepared Rhodococcus Rho4 bacterial suspension was inoculated to ensure that the biomass after inoculation was OD 600 =0.05~0.1, and finally a 100 mL degradation system was established, and the pH of the system was adjusted between 6.5~7.5.

[0106] The degradation conditions were 37°C, aerobic conditions were formed on a magnetic stirrer at a speed of 180 rpm, and the treatment time was 48 h.

[0107] Each experimental group was replicated three times, and samples were taken at 0 h and 48 h to detect the concentrations of nitrogen-containing heterocyclic compounds and total organic carbon (TOC).

[0108] The detection method of nitrogen-containing heterocyclic compounds and TOC concentration is the same as that in Example 2.

[0109] (3) Results analysis

[0110] The experimental results are as follows Figure 1 As shown, the degradation performance and mineralization ability of Rhodococcus erythrocytes Rho4 for the above four nitrogen-containing heterocyclic compounds are relatively low, especially in the pyridine group, where the degradation rate is still below 40% and the mineralization rate is also below 20%.

[0111] Example 4

[0112] This example provides a degradation experiment of a single nitrogen-containing heterocyclic compound using a combination of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4, comprising the following steps:

[0113] Preparation of single strain bacterial suspension: The preparation of the bacterial suspension of Rhodococcus pyridinophilus Rho48 was the same as in Example 2; the preparation of the bacterial suspension of Rhodococcus erythrophilus Rho4 was the same as in Example 3.

[0114] (2) Degradation of single nitrogen-containing heterocyclic compounds

[0115] Different from the inoculation of a single strain of Rhodococcus pyridinii Rho48 bacterial suspension or Rhodococcus erythrocytes Rho4 bacterial suspension in Example 2 or 3, in this example, a suspension of Rhodococcus pyridinii Rho48 and a suspension of Rhodococcus erythrocytes Rho4 were inoculated in each simulated nitrogen-containing heterocyclic compound wastewater (the same as in Example 2 or 3), and Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 were inoculated in equal proportions, and the inoculated biomass was ensured to be at OD 600 =0.05~0.1, and the other compositions in the system, degradation conditions, and detection of nitrogen-containing heterocyclic compounds and TOC concentrations are the same as those in Example 2 or Example 3.

[0116] (3) Results analysis

[0117] The results of the combined use of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 on the degradation of a single nitrogen-containing heterocyclic compound are shown in Figure 2. Figure 2 As shown, within the degradation time of 48 h, the degradation rates of single nitrogen-containing heterocyclic compounds indole, quinoline, benzothiazole and pyridine were all over 98%. Compared with the degradation effect of single strains in Example 2 and Example 3, the combined use further improved the degradation effect of nitrogen-containing heterocyclic compounds indole, quinoline, benzothiazole and / or pyridine, and had a broader spectrum and more efficient treatment capacity, which could meet the demand for nearly 100% removal of more nitrogen-containing heterocyclic compounds.

[0118] The mineralization results of single nitrogen-containing heterocyclic compounds using the combination of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 are shown in Figure 2. Figure 3As shown, the mineralization rate of quinoline is about 50%, and the mineralization rates of indole, benzothiazole and pyridine can reach up to about 84%. Compared with the treatment of single strain, the mineralization effect is also significantly improved.

[0119] The above results show that the combination of Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 can effectively achieve the co-conversion of the nitrogen-containing heterocyclic compound parent and the intermediates produced during the degradation process. This result may be due to the formation of a composite bacterial community of Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 in the wastewater, which exerts a synergistic effect. In particular, compared with the low degradation and low mineralization of indole by single strains Rho48 or Rho4, the degradation rate and mineralization rate of the combined use have been significantly improved.

[0120] Example 5

[0121] This embodiment provides a method for treating wastewater contaminated with nitrogen-containing heterocyclic compounds by using a combination of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4.

[0122] In this embodiment, in order to evaluate the treatment capability of the composite bacterial community on the composite pollution of nitrogen-containing heterocyclic compounds in wastewater, this embodiment simulates the composite pollution of nitrogen-containing heterocyclic compounds in coking wastewater.

[0123] In this embodiment, the preparation of simulated coking wastewater includes: first adding various nitrogen-containing heterocyclic compounds to an inorganic salt culture medium (same as in Example 1), and the final simulated coking wastewater is:

[0124] A: 10 mg / L indole + 10 mg / L quinoline + 10 mg / L pyridine;

[0125] B: 25 mg / L indole + 25 mg / L quinoline.

[0126] The treatment of wastewater contaminated by nitrogen-containing heterocyclic compounds by combining Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 includes the following steps:

[0127] Preparation of single strain bacterial suspension: The preparation of the bacterial suspension of Rhodococcus pyridinophilus Rho48 was the same as in Example 2; the preparation of the bacterial suspension of Rhodococcus erythrophilus Rho4 was the same as in Example 3.

[0128] Degradation of nitrogen-containing heterocyclic compound composite pollution Referring to Example 4, a suspension of Rhodococcus pyridinii Rho48 and a suspension of Rhodococcus erythrocytes Rho4 were inoculated in simulated coking wastewater, and Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 were inoculated in equal proportions, and the inoculated biomass was ensured to be at OD 600=0.05~0.1, the other compositions in the system, the degradation conditions and the detection of nitrogen-containing heterocyclic compounds and TOC concentrations are the same as those in Example 2 or Example 3, and the treatment time is extended to 72 h; samples are taken at 0 h, 24 h, 48 h and 72 h to detect the concentrations of nitrogen-containing heterocyclic compounds and TOC.

[0129] Results Analysis The combined use of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 treated the combined pollution of three nitrogen-containing heterocyclic compounds in simulated coking wastewater A. Figure 4 As shown in the figure, indole and quinoline were completely degraded within 48 hours; pyridine was completely degraded within 72 hours, and the overall mineralization effect was close to 50% within 72 hours; the treatment results of the combined pollution of two nitrogen-containing heterocyclic compounds in the simulated coking wastewater B are shown in the figure. Figure 5 As shown in the figure, after the concentration of indole and quinoline increased to 25 mg / L, the composite bacterial community was still able to achieve nearly 100% degradation of indole and quinoline within 72 h.

[0130] The above experimental results show that Rhodococcus pyridinii Rho48 and Rhodococcus erythrocytes Rho4 in the present invention have good application potential for treating complex pollution of nitrogen-containing heterocyclic compounds in actual wastewater. Rhodococcus pyridinii Rho48 can be used alone or in combination with Rhodococcus erythrocytes Rho4, and can efficiently degrade and mineralize complex pollution of multiple nitrogen-containing heterocyclic compounds.

Claims

1. Application of Rhodococcus pyridinophilus Rho48 in treating wastewater containing nitrogen heterocyclic compounds, characterized in that: The application includes the following steps: Rhodococcus pyridinivorans ) Rho48 and Rhodococcus erythrocytes ( Rhodococcus ruber ) Rho4 is used in combination; the pyridinophilic Rhodococcus ( Rhodococcus pyridinivorans ) Rho48, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is December 4, 2024, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32892; the Rhodococcus rubrum ( Rhodococcus ruber ) Rho4 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 4, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.32891; the nitrogen-containing heterocyclic compound is any one or more of indole, quinoline, benzothiazole, and pyridine.

2. The use according to claim 1, characterized in that: The applications include: The bacterial suspensions of Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 were prepared respectively; Then, the bacterial suspension of Rhodococcus pyridinophilus Rho48 and the bacterial suspension of Rhodococcus erythrophilus Rho4 were added into the wastewater.

3. The use according to claim 2, characterized in that: The bacterial suspension of Rhodococcus pyridinophilus Rho48 and the bacterial suspension of Rhodococcus erythrophilus Rho4 are added into the wastewater: The bacterial suspension of Rhodococcus pyridinophilus Rho48 and the bacterial suspension of Rhodococcus erythrophilus Rho4 are first mixed and then added into the wastewater; or The bacterial suspension of Rhodococcus pyridinophilus Rho48 and the bacterial suspension of Rhodococcus erythrophilus Rho4 are added into the wastewater respectively.

4. A microbial agent, characterized in that: The microbial agent comprises: Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 as described in claim 1.

5. A method for treating wastewater containing nitrogen heterocyclic compounds, characterized in that: The method comprises: adding the Rhodococcus pyridinophilus Rho48 and Rhodococcus erythrophilus Rho4 described in claim 1 to wastewater; the nitrogen-containing heterocyclic compound is any one or more of indole, quinoline, benzothiazole and pyridine.

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