Method for detecting capability of microorganisms in degrading nitrogen-containing heterocyclic compound and application of method

Abstract

The invention belongs to the technical field of microbiological detection, and relates to a method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds and application of the method. The technical problems that in the prior art, a color developing system for directional detection of the degradation capacity of a nitrogen-containing heterocyclic compound is lacked, and traditional absorbance detection has false positive and low accuracy are solved. The detection method provided by the invention comprises the following steps: inoculating bacteria to be detected into a culture medium taking a nitrogen-containing heterocyclic compound as a unique carbon source to prepare a bacterial suspension with initial OD600 of 0.4-0.6; the bacterial suspension and a color developing agent WST-8 or XTT are subjected to a color developing reaction, a reaction solution is obtained, and the initial concentration of the color developing agent in the reaction solution is 250-350 mg / L; the absorbance difference value of the reaction liquid is measured by adopting a dual-wavelength spectrophotometric method and is used for representing the degradation capability of the bacteria to be detected on the nitrogen-containing heterocyclic compound, and the method is high in accuracy. The invention also provides an application of the method in screening nitrogen-containing heterocyclic compound degrading bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and specifically, relates to a method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds and its application. Background Art

[0002] Nitrogenous heterocyclic compounds (NHCs) are a class of typical refractory organic pollutants, which are widely present in coal chemical, pharmaceutical and pesticide wastewaters. Nitrogenous heterocyclic compounds have stable chemical properties and extensive biological toxicity, and can enter natural water bodies and soil environments with wastewater discharge, posing potential risks to the ecosystem and human health.

[0003] Compared with traditional technologies such as membrane separation, adsorption and advanced oxidation processes, the biological method for treating wastewater containing nitrogenous heterocyclic compounds has the advantages of low cost, large treatment capacity, high treatment efficiency and effective avoidance of secondary pollution, thus becoming a key technology for treating wastewater containing nitrogenous heterocyclic compounds. At present, some studies have found that some microorganisms have a certain degradation ability for nitrogenous heterocyclic compounds, and the degradation rate can be evaluated by traditional methods such as chromatographic analysis technology. However, the existing methods often rely on complex instruments or cannot quickly obtain the degradation rate information in practical applications, and there is a lack of a simple, rapid alternative detection method for quantitatively evaluating the ability of microorganisms to degrade specific nitrogenous heterocyclic compounds.

[0004] In recent years, the high-throughput colorimetric technology based on microplates has been widely used in the study of the ability of microorganisms to metabolize carbon sources or utilize electron acceptors, providing an efficient means for the detection of functional microorganisms. However, this type of technology still lacks a targeted detection strategy for the degradation ability of specific pollutants (such as nitrogenous heterocyclic compounds), and the traditional absorbance measurement method is difficult to distinguish the color reaction caused by pollutant degradation from the basic metabolism of microorganisms, resulting in false positives and affecting the accuracy of the results.

[0005] Therefore, there is an urgent need to establish an absorbance detection method with the advantages of directionality, accuracy and high throughput for quickly evaluating the degradation ability of microorganisms to nitrogenous heterocyclic compounds. Summary of the Invention

[0006] 1. Problems to be Solved In view of the lack of a colorimetric system for the directional detection of the degradation ability of nitrogen-containing heterocyclic compounds in the prior art, and the problems that traditional absorbance detection is susceptible to basal metabolism interference, has false positives, low accuracy, and a long cycle. The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds provided in this application is based on a dual-wavelength absorbance detection method using the colorimetric reagents WST-8 or XTT, and is used to evaluate the degradation ability of microorganisms to nitrogen-containing heterocyclic compounds, with the advantages of high throughput, strong directionality, high accuracy, and short cycle. At the same time, this application also provides the application of the detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in screening nitrogen-containing heterocyclic compound-degrading bacteria, as well as its application in the process of treating nitrogen-containing heterocyclic compound pollution.

[0007] 2. Technical Solution To achieve the above object, the technical solution provided is as follows: A detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, comprising the following steps: Inoculate the test bacteria into a medium with a nitrogen-containing heterocyclic compound as the sole carbon source to prepare a bacterial suspension, and the initial OD 600 of the bacterial suspension is 0.4 - 0.6; Perform a color reaction on the bacterial suspension with the colorimetric reagent WST-8 or XTT to obtain a reaction solution; the initial concentration of WST-8 or XTT in the reaction solution is 250 mg / L - 350 mg / L; Use dual-wavelength spectrophotometry to measure the absorbance difference of the reaction solution, and the absorbance difference is used to characterize the degradation ability of the test bacteria to nitrogen-containing heterocyclic compounds.

[0008] WST-8 (sodium 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) is a water-soluble tetrazolium salt.

[0009] XTT (2,3-diphenyl-5-(2,4-disulfophenyl)-2H-tetrazolium) is a water-soluble tetrazolium salt.

[0010] Preferably, the color reaction is carried out in a 96-well plate, the volume ratio of the bacterial suspension to the colorimetric reagent is 1:1, the addition amount of the bacterial suspension is 100 μL, the addition amount of the colorimetric reagent is 100 μL, and the total volume of each well is 200 μL.

[0011] Preferably, the initial concentration of the nitrogen-containing heterocyclic compound in the reaction solution is 100 mg / L.

[0012] Furthermore, the two wavelengths are 470 nm and 630 nm respectively, the absorbance difference is ΔOD, and the ΔOD = OD 470 - OD 630 .

[0013] OD 470 (Optical density at a wavelength of 470 nm) is used to measure the result of a specific color reaction because in the color reaction, the generated product has an obvious absorption peak at 470 nm; OD 630 (Optical density at a wavelength of 630 nm) is used as background correction. This wavelength is usually the region where the color reaction product does not absorb light. By measuring at a wavelength where light is not absorbed, interference signals caused by turbidity, bubbles, or other non-specific scattering and absorption can be corrected. Calculate OD 470 - OD 630 The difference can effectively subtract the background interference and retain the actual absorption signal of the target substance. This method improves the accuracy and sensitivity of the measurement and reduces errors caused by environmental factors, experimental conditions, and the instrument itself.

[0014] Further, the nitrogen-containing heterocyclic compound is any one of pyridine, pyrrole, or benzothiazole.

[0015] Further, the conditions of the color reaction are to react in the dark for 20 h to 48 h under a constant temperature condition.

[0016] Preferably, when the nitrogen-containing heterocyclic compound is pyridine, react in the dark for 30 h to 36 h under a constant temperature condition; when the nitrogen-containing heterocyclic compound is pyrrole, react in the dark for 40 h to 48 h under a constant temperature condition; when the nitrogen-containing heterocyclic compound is benzothiazole, react in the dark for 20 h to 24 h under a constant temperature condition.

[0017] Preferably, the constant temperature is 30 °C.

[0018] Further, the method further includes: measuring the absorbance difference of the reaction solution at different reaction time points and establishing a standard working curve between the absorbance difference and the degradation rate.

[0019] Preferably, the degradation rate is: measuring the residual concentration of the nitrogen-containing heterocyclic compound in the reaction solution by high performance liquid chromatography, degradation rate % = (initial concentration - residual concentration) / initial concentration × 100%.

[0020] Further, the medium with the nitrogen-containing heterocyclic compound as the sole carbon source is the MSM medium, and the MSM medium includes: NH4Cl 57.3 mg / L, KH2PO4 13.2 mg / L, MgSO4 15 mg / L, CaCl2 16.5 mg / L, trace elements 0.4 mL / L.

[0021] Further, the trace elements include: 250 mg / L of MnCl2·4H2O, 100 mg / L of ZnSO4·7H2O, 50 mg / L of CuSO4·5H2O, 25 mg / L of CoCl2·6H2O, 50 mg / L of Na2MoO4·2H2O, and 200 mg / L of FeSO4·7H2O.

[0022] Further, the bacteria to be tested are a single strain or a combination of multiple strains.

[0023] In one experiment, the degradation capabilities of a single strain and a combination of multiple strains (combined bacteria) on different pollutants are evaluated simultaneously. Compared with the traditional shake-flask experiment that requires step-by-step verification of the degradation effects of different bacteria to be tested, this method can quickly identify the single bacterium or combined bacterium with stronger degradation ability, with high accuracy and a significant reduction in the experimental workload, improving the screening efficiency.

[0024] Further, the bacteria to be tested are Rhodococcus pyridinivorans strain Rho48, taxonomically named Rhodococcus pyridinivorans, and was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on December 4, 2024, with the deposit number CGMCC No. 32892.

[0025] Application of the method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, application of the method in screening nitrogen-containing heterocyclic compound-degrading bacteria, or application in the process of treating nitrogen-containing heterocyclic compound pollution.

[0026] 3. Beneficial effects Adopting the technical solution provided by the present invention, compared with the existing well-known technologies, it has the following beneficial effects: (1)The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in the present invention can detect the degradation ability of nitrogen-containing heterocyclic compounds by constructing a culture system with nitrogen-containing heterocyclic compounds as the sole carbon source, using WST-8 or XTT as a chromogenic agent, and combining with the chromogenic reaction, avoiding false positive results caused by simple metabolic activity and having high accuracy; microorganisms can metabolize nitrogen-containing heterocyclic compounds as the sole carbon source. During this process, microorganisms rely on electron transfer enzymes (such as NADH / NADPH-dependent dehydrogenases) inside or on the cell surface to reduce a specific chromogenic agent (such as WST-8, XTT) to an orange-yellow water-soluble formazan product. This product has characteristic absorption peaks, facilitating quantitative analysis through the detection of the optical density of specific wavelengths in a microplate, and can indirectly reflect the degradation ability of microorganisms under the condition of using nitrogen heterocyclic compounds as the sole carbon source. The double-wavelength method is used to measure the difference in absorbance of the reaction solution, eliminating the influence of background noise. The greater the difference in absorbance, the stronger the degradation ability of microorganisms. Using a 96-well plate combined with the double-wavelength absorbance method for high-throughput detection reduces the experimental period and human input, and has the advantages of simple operation, sensitive detection, and strong adaptability, providing a reliable technical support for the rapid screening of highly efficient degradation strains in the biological treatment of nitrogen-containing heterocyclic organic pollutants.

[0027] (2)The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in the present invention further includes measuring the difference in absorbance of the reaction solution at different reaction time points and establishing a standard working curve between the difference in absorbance and the degradation rate. The standard curve shows that the difference in absorbance and the degradation rate are significantly positively correlated (R 2 >0.99). In the actual pollution treatment process, it is crucial to be able to monitor the degradation efficiency in real time for optimizing reaction conditions and regulating the degradation process. This linear relationship makes real-time monitoring possible, so that the reaction conditions can be adjusted in a timely manner according to the monitoring results to improve the degradation efficiency.

[0028] (3)The application of the detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in the present invention. The detection method of the present invention can be applied to screening nitrogen-containing heterocyclic compound-degrading bacteria, and the degrading bacteria are a single strain or a combination of multiple strains. The traditional method for screening degrading bacteria is often time-consuming and laborious. The detection method of the present invention can quickly and accurately screen out strains with the ability to degrade nitrogen-containing heterocyclic compounds by measuring the difference in absorbance, shortening the screening period and improving the screening efficiency. It can also be applied to the process of treating nitrogen-containing heterocyclic compound pollution. During the pollution treatment process, the detection method of the present invention can monitor the effect of microorganisms degrading nitrogen-containing heterocyclic compounds in real time. By detecting key indicators (such as the degradation rate) during the degradation process of microorganisms, the treatment plan can be adjusted in a timely manner. Description of the Drawings

[0029] Figure 1 is the standard curve of pyridine degradation rate - absorbance; Figure 2 is the standard curve of pyrrole degradation rate - absorbance; Figure 3 is the standard curve of benzothiazole degradation rate - absorbance; Figure 4 is the correlation curve of pyridine degradation rate - absorbance when the initial concentration of WST - 8 is 300 mg / L; Figure 5 is the correlation curve of pyridine degradation rate - absorbance when detected using a single wavelength (OD 470 ); Figure 6 is the scatter plot of pyridine degradation rate - absorbance when detected using a tetrazolium violet color reagent. Detailed implementation mode

[0030] To further understand the content of the present invention, the present invention will be described in detail in combination with embodiments.

[0031] The following further describes the present application in combination with specific embodiments.

[0032] It should be noted that the terms such as "upper", "lower", "left", "right", "middle" cited in this specification are only for the convenience of narration and are not used to limit the scope of implementation. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope of implementation of the present application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0034] For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0035] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. Those skilled in the art can easily determine the degree of flexibility of a specific variable. As used herein, the term "at least one of..." is intended to be synonymous with "one or more of...". For example, "at least one of A, B, and C" clearly includes only A, only B, only C, and their respective combinations. Concentrations, amounts, and other numerical data may be presented herein in a range format. It should be understood that such range formats are used solely for convenience and brevity and should be interpreted flexibly as including not only the values explicitly recited as the limits of the range but also all individual values or sub-ranges subsumed within the stated range as if each value and sub-range were explicitly recited. For example, a numerical range of from about 1 to about 4.5 should be interpreted as including not only the explicitly recited limits of 1 to about 4.5 but also the individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that recite only one value, such as "less than about 4.5", which should be interpreted as including all of the above values and ranges. Additionally, this interpretation should apply regardless of the breadth of the range or feature being described. The instruments, reagents, and strains used in the detection methods for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in the following examples and comparative examples are as follows: 1. Instruments BioTek Synergy H1 multi-functional microplate reader (Agilent Technologies, USA), 1260 Infinity II high-performance liquid chromatography (Agilent Technologies, USA), 96-well microplates.

[0036] 2. Reagents Pyridine, pyrrole, and benzothiazole were all purchased from Macklin Biochemical Technology Co., Ltd.; the chromogenic reagent WST-8 was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the chromogenic reagent XTT was purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the remaining reagents were all domestic analytical grade.

[0037] 3. Strains Strains with the ability to degrade nitrogen-containing heterocyclic compounds identified by preliminary laboratory screening, Rhodococcus pyridinivorans Rho48, was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on December 4, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC No. 32892. Hereinafter referred to as Rho48.

[0038] Example 1 The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in this example has an optimized detection method, including the following steps: I. Preparation of solutions and culture media Solution preparation: Prepare a 1 g / L pyridine stock solution, a 1 g / L XTT chromogenic reagent, and a 2 g / L WST-8 chromogenic reagent accurately with deionized water respectively. Filter-sterilize the solutions with a 0.22 μm pore size membrane and store them in the dark at 4 °C until use.

[0039] Preparation of trace element stock solution: 250 mg of MnCl2·4H2O, 100 mg of ZnSO4·7H2O, 50 mg of CuSO4·5H2O, 25 mg of CoCl2·6H2O, 50 mg of Na2MoO4·2H2O, 200 mg of FeSO4·7H2O, and add deionized water to 1000 mL.

[0040] Preparation of MSM medium: 57.3 mg of NH4Cl, 13.2 mg of KH2PO4, 15 mg of MgSO4, 16.5 mg of CaCl2, 0.4 mL of trace element stock solution, and add deionized water to 1000 mL.

[0041] II. Optimization and establishment of the detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds (1) Color development principle Microorganisms can use nitrogen-containing heterocyclic compounds (pyridine, pyrrole, and benzothiazole) as the sole carbon source for metabolism. During this process, microorganisms rely on electron transfer enzymes (such as NADH / NADPH-dependent dehydrogenases) inside or on the cell surface to reduce specific color developers (such as WST-8, XTT) to orange-yellow water-soluble formazan products. This product has characteristic absorption peaks, facilitating quantitative analysis through the detection of the optical density of light with a specific wavelength in a microplate, and can indirectly reflect the degradation ability of microorganisms under the condition of using nitrogen heterocyclic compounds as the sole carbon source. The greater the increase in the absorbance difference, the greater the degradation ability. By measuring the change in absorbance, the degradation activity of the strain can be indirectly reflected.

[0042] (2) Optimization of the type and concentration of color developers Experimental group: Inoculate the activated pure strain Rho48 into LB medium, place it in a shaker at 37 °C and 150 r / min for overnight amplification culture until it reaches the logarithmic growth phase to obtain a bacterial solution; centrifuge the bacterial solution at 8000 rpm for 3 min, discard the supernatant, and wash it 3 times with PBS buffer to collect the bacterial cells; inoculate the bacterial cells into MSM medium with pyridine as the sole carbon source to obtain a bacterial suspension, and set the initial OD 600 of the bacterial suspension to 0.5.

[0043] Dilute the mother solutions of the color developers XTT and WST-8 with deionized water into solutions with final concentrations of 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, and 700 mg / L respectively.

[0044] The bacterial suspension and the chromogenic reagent (WST-8 or XTT) were inoculated into a 96-well plate at a volume ratio of 1:1. The addition amount of the bacterial suspension in each well of the 96-well plate was 100 μL, and the addition amount of the chromogenic reagent was 100 μL, so that the total volume of each well was 200 μL. The initial concentration of pyridine in this chromogenic reaction system was 100 mg / L. Three biological replicates were set for each chromogenic reagent concentration. The reaction was carried out in the dark at 30 °C for 36 h, and the OD of the reaction solution was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 470 and OD 630 ,and calculate ΔOD (OD 470 - OD 630 ).

[0045] Blank control group: Control group 1 was set without pyridine (only containing bacteria, MSM medium and chromogenic reagent); Control group 2 was without bacteria (only containing pyridine, MSM medium and chromogenic reagent without sterile solution). Among them, the chromogenic reagent concentration, pyridine concentration, initial OD of the bacterial suspension 600 and the detection method were the same as those in the experimental group.

[0046] Table 1 Effects of chromogenic reagent types and concentrations on ΔOD

[0047] As can be seen from Table 1, in the two chromogenic reagent systems, the ΔOD value of the experimental group showed an increasing trend with the increase of the chromogenic reagent concentration. Among them, in the WST-8 system, the ΔOD reached 0.256 at 500 mg / L, and then only slightly increased to 0.272 at 700 mg / L (an increase of 6.3%), and the sensitivity was close to the plateau. While in the XTT system, the ΔOD reached 0.247 only when it was increased to 700 mg / L, which was still slightly lower than the sensitivity of WST-8 at 500 mg / L, indicating that WST-8 can obtain a higher response at a lower concentration. In addition, the single-well chromogenic cost of WST-8 was only about 25% of that of XTT, and the cost performance was better.

[0048] The two groups of blank controls set under the same conditions also showed good specificity of the chromogenic reaction. In control group 1 (without pyridine), only bacteria and chromogenic reagent were contained, and its ΔOD value was much lower than that of the corresponding experimental group. For example, in the WST-8 system at 500 mg / L, the ΔOD of control group 1 was only 0.074, and the ΔOD of the corresponding experimental group was 0.256, indicating that the non-specific reduction of the chromogenic reagent by bacteria itself was weak; in control group 2 (without bacteria), only pyridine and chromogenic reagent were contained, and its ΔOD was generally lower than that of control group 1. For example, in the WST-8 system at 500 mg / L, it was only 0.007, indicating that there was basically no direct reaction between pyridine and the chromogenic reagent. Therefore, the chromogenic signal mainly originated from the metabolic activity of bacteria in the presence of pyridine, and ΔOD could effectively reflect the degradation ability of microorganisms to pollutants.

[0049] In summary, considering the color development sensitivity, background interference, and economy comprehensively, it is determined that the optimal concentration of WST-8 for use as a color developer is 500 mg / L. In the actual reaction system, the bacterial suspension and the color developer are mixed at a volume ratio of 1:1 to obtain a reaction solution, that is, the initial concentration of WST-8 in the reaction solution is 250 mg / L, which is used for subsequent experiments.

[0050] (3)Optimization of the initial inoculum size To further optimize the initial inoculum size of the bacterial suspension in the color development reaction system and ensure that the reaction system has good color development effects and stability, different initial OD values of the bacterial suspension were set for comparison experiments. 600 values for comparison experiments.

[0051] Experimental group: The activated pure bacteria Rho48 were inoculated into LB medium and placed in a shaker at 37 °C and 150 r / min for overnight amplification culture until it reached the logarithmic growth phase to obtain a bacterial solution; the bacterial solution was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the cells were washed 3 times with PBS buffer, and the cells were collected; the cells were inoculated into MSM medium with pyridine as the sole carbon source to obtain a bacterial suspension; The bacterial suspension and the color developer WST-8 (concentration 500 mg / L) were inoculated into a 96-well plate at a volume ratio of 1:1. The addition amount of the bacterial suspension in each well of the 96-well plate was 100 μL, and the addition amount of the color developer was 100 μL, so that the total volume of each well was 200 μL. The initial concentration of pyridine in this color development reaction system was 100 mg / L, and the concentration of the WST-8 color developer was 250 mg / L, and it was cultured in the dark at 30 °C. The initial OD of the bacterial suspension 600 was set to 0.1, 0.3, 0.5, 0.7, and 1.0 respectively. Three parallels were set for each inoculum size, and OD 470 and OD 630 were measured after 12 h, 24 h, and 36 h of reaction respectively, and the ΔOD value was calculated.

[0052] Blank control group: Control group 1 without pyridine (only containing cells, MSM medium, and color developer) was set; Control group 2 without cells (only containing pyridine, MSM medium, and color developer without sterile solution). Among them, the color developer concentration, pyridine concentration, initial OD of the bacterial suspension 600 and the detection method were the same as those in the experimental group.

[0053] Table 2 Effects of the initial inoculum size on ΔOD

[0054] The results are shown in Table 2. When the initial OD of the bacterial suspension 600 is too low (such as 0.1, 0.3), the ΔOD of the reaction system increases slowly, the color development sensitivity is not high, which is not conducive to the efficiency and sensitivity of high-throughput screening. And when the initial OD of the bacterial suspension600 When it is too high (such as 0.7, 1.0), the system develops color too quickly, resulting in rapid depletion of the color reagent. The ΔOD value reaches a plateau or even decreases after 12 h, affecting the accuracy of the linear relationship between the degradation rate and ΔOD. Considering the reaction rate, color development degree, and system stability comprehensively, the initial OD of the bacterial suspension is selected. 600 is 0.4 - 0.6, where the initial OD of the bacterial suspension 600 is 0.5 as the optimal inoculation amount condition.

[0055] The data of the control group show that the ΔOD value always remains at a low level and changes little with the increase of the initial inoculation amount. The ΔOD difference between control group 1 and the experimental group gradually widens with the increase of the initial inoculation amount, further indicating that the color reaction mainly stems from the metabolic activity of the bacteria induced by pyridine, rather than non-specific background reactions.

[0056] In the optimal color reaction system, the initial OD of the bacterial suspension 600 is 0.5, the initial concentration of pyridine in the reaction solution is 100 mg / L, and the initial concentration of the WST-8 color reagent is 250 mg / L.

[0057] Example 2 The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in this example, and the drawing of the pyridine degradation rate - absorbance standard curve, includes the following steps: I. Preparation of solutions and media: The same as in Example 1.

[0058] II. Drawing of the pyridine degradation rate - absorbance standard curve Take the activated pure bacterium Rho48 and inoculate it into LB medium, place it in a shaker at 37 °C and 150 r / min for overnight amplification culture until it reaches the logarithmic growth phase to obtain a bacterial solution; centrifuge the bacterial solution at 8000 rpm for 3 min, discard the supernatant, and wash it 3 times with PBS buffer to collect the bacteria; inoculate the bacteria into the medium with pyridine as the sole carbon source to obtain a bacterial suspension, and set the initial OD of the bacterial suspension 600 to be 0.5.

[0059] Prepare a 500 mg / L WST-8 solution with deionized water, inoculate the bacterial suspension and the color reagent into a 96-well plate according to a volume ratio of 1:1 to obtain a reaction solution. The initial concentration of pyridine in the reaction solution is 100 mg / L, and the initial concentration of the WST-8 color reagent is 250 mg / L. React in the dark at 30 °C for 36 h.

[0060] At different degradation reaction time points of 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 30 h, and 36 h, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD of the reaction solution 470 and OD630 After the absorbance measurement is completed at each time point, a destructive sampling method is adopted. At each time point, 2.4 mL of the reaction solution (from 12 wells) is aspirated from the 96-well plate and filtered through a 0.22 μm organic phase filter membrane and transferred to a liquid phase vial (three sets of parallel samples). The concentration of pyridine in the filtered reaction solution is determined by HPLC. The chromatographic column used is an Eclipse Plus C18 column, with an injection volume of 20 μL and a column temperature of 30 °C; an ultraviolet detector (DAD) is used, with a detection wavelength set at 253 nm; methanol and water are used as the mobile phase, with a volume ratio of 60:40 (v / v) and a flow rate of 1.0 mL / min.

[0061] At the same time, blank controls are set. Blank control 1 does not contain pyridine (only contains bacteria, MSM medium and chromogenic agent); blank control 2 does not contain bacteria (only contains pyridine, MSM medium and sterile chromogenic agent solution).

[0062] Data processing: ①Calculation of ΔOD: For the absorbance data of each well in the 96-well plate, calculate the ΔOD value according to the formula: ΔOD = OD 470 - OD 630 .

[0063] ②Biological parallel processing: For each degradation system (including 3 parallel wells), after removing outliers (Grubbs test, α = 0.05), take the arithmetic mean of ΔOD as the final characterization value of the system.

[0064] ③Calculation of pyridine degradation rate: According to the pyridine residual concentration measured by high performance liquid chromatography for each well, calculate the degradation rate: Degradation rate = (initial concentration - residual concentration) / initial concentration × 100%.

[0065] ④Correlation analysis between ΔOD and pyridine degradation rate: Using the ΔOD value as the independent variable and the degradation rate as the dependent variable for linear fitting, draw the correlation curve of ΔOD and pyridine degradation rate, and evaluate the linear relationship and goodness of fit (R²) between the two, as Figure 1 shown.

[0066] This standard curve shows that ΔOD and the degradation rate are significantly positively correlated (R 2 > 0.99).

[0067] III. Accuracy verification To verify the accuracy of this method, reaction solutions at three time points of 6 h, 14 h, and 28 h were selected respectively, and the measured ΔOD values were 0.091, 0.109, and 0.152. The predicted degradation rates calculated through the standard curve were 17.72%, 29.84%, and 58.78% respectively. At the same time, the degradation rates were calculated using the pyridine concentrations directly measured by HPLC, and the obtained results were 16.93%, 30.31%, and 58.24%. By comparing the results obtained by the two methods, the errors were all less than 5%, which proved that the detection method for the ability of microbial degradation of nitrogen heterocyclic compounds in this example had high accuracy.

[0068] Example 3 For the detection method of the ability of microbial degradation of nitrogen heterocyclic compounds in this example, the drawing of the pyrrole degradation rate - absorbance standard curve includes the following steps: I. Preparation of solutions and culture media Solution preparation: Prepare a 1 g / L pyrrole stock solution and a 2 g / L WST-8 color reagent accurately with deionized water respectively. Filter and sterilize the solutions with a 0.22 μm pore size membrane, and store them in the dark at 4 °C until use.

[0069] Culture medium preparation: The same as in Example 1.

[0070] II. Drawing of the pyrrole degradation rate - absorbance standard curve Inoculate the activated pure strain Rho48 into LB medium, place it in a shaker at 37 °C and 150 r / min for overnight amplification culture until it reaches the logarithmic growth phase to obtain a bacterial solution; centrifuge the bacterial solution at 8000 rpm for 3 min, discard the supernatant, and wash it 3 times with PBS buffer to collect the bacterial cells; inoculate the bacterial cells into the medium with pyrrole as the sole carbon source to obtain a bacterial suspension, and set the initial OD of the bacterial suspension 600 to be 0.5.

[0071] Prepare a 500 mg / L WST-8 solution with deionized water, inoculate the bacterial suspension and the color reagent into a 96-well plate according to a volume ratio of 1:1 to obtain a reaction solution. The initial concentration of pyrrole in the reaction solution is 100 mg / L, and the initial concentration of the WST-8 color reagent is 250 mg / L. React in the dark at 30 °C for 48 h.

[0072] Measure the OD of the reaction solution using an enzyme-linked immunosorbent assay (ELISA) reader at different degradation time points of 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 30 h, 36 h, and 48 h 470 and OD 630, after the absorbance measurement is completed at each time point, a destructive sampling method is adopted. At each time point, 2.4 mL of the reaction solution (the reaction solutions of 12 wells) is aspirated from the 96-well plate, and after filtering it through a 0.22 μm organic phase filter membrane, it is transferred to a liquid phase vial (three parallel samples). The concentration of pyrrole in the filtered reaction solution is determined by HPLC. The chromatographic column used is an Eclipse Plus C18 chromatographic column, with an injection volume of 20 μL and a column temperature of 30 °C; a UV detector (DAD) is used, and the detection wavelength is set at 210 nm; methanol and water are used as the mobile phase, with a volume ratio of 70:30 (v / v) and a flow rate of 1.0 mL / min.

[0073] At the same time, blank controls are set. Blank control 1 does not contain pyrrole (only contains bacteria, MSM medium, and chromogenic agent); blank control 2 does not contain bacteria (only contains pyrrole, MSM medium, and sterile chromogenic agent solution).

[0074] Data processing: ① Calculation of ΔOD: For the absorbance data of each well in the 96-well plate, calculate the ΔOD value according to the formula: ΔOD = OD 470 - OD 630 .

[0075] ② Biological parallel processing: For each degradation system (including 3 parallel wells), after excluding outliers (Grubbs test, α = 0.05), take the arithmetic mean of ΔOD as the final characterization value of this system.

[0076] ③ Calculation of pyrrole degradation rate: According to the pyrrole residual concentration measured by high performance liquid chromatography for each well, calculate the degradation rate: Degradation rate = (initial concentration - residual concentration) / initial concentration × 100%.

[0077] ④ Correlation analysis between ΔOD and pyrrole degradation rate: Using the ΔOD value as the independent variable and the degradation rate as the dependent variable for linear fitting, draw the correlation curve between ΔOD and pyrrole degradation rate, and evaluate the linear relationship and goodness of fit (R²) between the two, as Figure 2 shown.

[0078] This standard curve shows that ΔOD and the degradation rate are significantly positively correlated (R 2 > 0.99).

[0079] III. Accuracy verification To verify the accuracy of this method, the reaction solutions at three time points of 6 h, 14 h, and 28 h were selected, and the measured ΔOD was 0.096, 0.110, and 0.123, respectively. The predicted degradation rates calculated by the standard curve were 27.65%, 46.72%, and 64.43%, respectively. At the same time, the degradation rate was calculated by directly measuring the pyrrole concentration by HPLC, and the results obtained were 26.78%, 46.02%, and 66.20%. Comparing the results obtained by the two methods, the errors were less than 5%, proving that the detection method of the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in this embodiment has high accuracy.

[0080] Example 4 The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in this embodiment, and drawing a benzothiazole degradation rate-absorbance standard curve, comprises the following steps: 1. Solution and culture medium preparation Solution preparation: 1 g / L benzothiazole stock solution and 2 g / L WST-8 color developer were accurately prepared with deionized water, and the solutions were sterilized by filtration using a 0.22 μm pore size membrane and stored at 4 °C in the dark until use.

[0081] Culture medium configuration: same as in Example 1.

[0082] 2. Drawing of the benzothiazole degradation rate-absorbance standard curve The activated pure bacteria Rho48 was inoculated into LB medium and placed in a shaker at 37 °C and 150 r / min for overnight amplification culture until it reached the logarithmic growth phase to obtain a bacterial solution; the bacterial solution was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the cells were washed three times with PBS buffer to collect the cells; the cells were inoculated into a culture medium with benzothiazole as the only carbon source to obtain a bacterial suspension, and the initial OD of the bacterial suspension was set to 600 is 0.5.

[0083] A 500 mg / L WST-8 solution was prepared with deionized water, and the bacterial suspension and the color developer were inoculated in a 96-well plate at a volume ratio of 1:1 to obtain a reaction solution. The initial concentration of benzothiazole in the reaction solution was 100 mg / L, and the initial concentration of WST-8 color developer was 250 mg / L. The reaction was carried out at 30 °C in the dark for 20 h.

[0084] The OD of the reaction solution was measured by an enzyme marker at different degradation time points: 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 20 h. 470 and OD 630, after the absorbance was measured at each time point, a destructive sampling method was adopted. At each time point, 2.4 mL of the reaction solution (from 12 wells) was aspirated from the 96-well plate, filtered through a 0.22 μm organic phase filter membrane, and transferred to a liquid phase vial (three parallel samples). The concentration of benzothiazole in the filtered reaction solution was determined by HPLC using an Eclipse Plus C18 column with an injection volume of 20 μL, a column temperature of 30 °C; a UV detector (DAD) was used with a detection wavelength of 254 nm; methanol and water were used as the mobile phase with a volume ratio of 70:30 (v / v) and a flow rate of 1.0 mL / min.

[0085] At the same time, blank controls were set. Blank control 1 contained no benzothiazole (only cells, MSM medium, and color reagent); blank control 2 contained no cells (only benzothiazole, MSM medium, and sterile color reagent solution).

[0086] Data processing: ① Calculation of ΔOD: For the absorbance data of each well in the 96-well plate, the ΔOD value was calculated according to the formula: ΔOD = OD 470 - OD 630 .

[0087] ② Biological parallel processing: For each degradation system (including 3 parallel wells), after excluding outliers (Grubbs test, α = 0.05), the arithmetic mean of ΔOD was taken as the final characterization value of the system.

[0088] ③ Calculation of benzothiazole degradation rate: According to the residual concentration of benzothiazole in each well measured by high performance liquid chromatography, the degradation rate was calculated: Degradation rate = (initial concentration - residual concentration) / initial concentration × 100%.

[0089] ④ Correlation analysis between ΔOD and benzothiazole degradation rate: Linear fitting was performed with the ΔOD value as the independent variable and the degradation rate as the dependent variable, and the correlation curve between ΔOD and benzothiazole degradation rate was plotted to evaluate the linear relationship and goodness of fit (R²) between the two, as Figure 3 shown.

[0090] The standard curve showed a significant positive correlation between ΔOD and the degradation rate (R 2 > 0.99).

[0091] III. Accuracy verification To verify the accuracy of this method, reaction solutions at three time points of 6 h, 10 h, and 14 h were selected respectively, and the measured ΔOD values were 0.263, 0.409, and 0.528. The predicted degradation rates calculated through the standard curve were 37.02%, 60.85%, and 80.27% respectively. At the same time, the degradation rates were calculated using the benzothiazole concentration directly measured by HPLC, and the obtained results were 38.23%, 58.92%, and 82.78%. Comparing the results obtained by the two methods, the errors were all less than 5%, which proved that the detection method for the ability of microbial degradation of nitrogen heterocyclic compounds in this example had high accuracy.

[0092] Comparative Example 1 The detection method for the ability of microbial degradation of nitrogen heterocyclic compounds in this comparative example was basically the same as that in Example 2, except that the concentration of the WST-8 color reagent was 300 mg / L.

[0093] The experimental results are as Figure 4 shown. When the concentration of WST-8 was 300 mg / L, the ΔOD signal was generally low and the signal-to-noise ratio was poor, resulting in a decrease in the linear correlation between ΔOD and the pyridine degradation rate. The goodness of fit (R 2 ²) of the standard curve was only 0.79. In contrast, under the condition of a WST-8 concentration of 500 mg / L, the ΔOD signal intensity was moderate, the background interference was small, and R 2 ² reached 0.99, which was significantly better than that in Comparative Example 1. The 500 mg / L WST-8 color reagent concentration had a more excellent linear relationship of the standard curve, the fitting result was more accurate, and the system stability was higher.

[0094] Comparative Example 2 The detection method for the ability of microbial degradation of nitrogen heterocyclic compounds in this comparative example was basically the same as that in Example 2, except that single wavelength (OD 470 ) was used for detection. At different degradation reaction time points of 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 30 h, and 36 h, the OD 470 of the reaction solution was measured using an enzyme-linked immunosorbent assay reader. For each degradation system (including 3 parallel wells), after excluding the outliers (Grubbs test, α = 0.05), the arithmetic mean of OD 470 was taken as the final characterization value of the system. Using OD 470 as the independent variable and the degradation rate as the dependent variable for linear fitting, the correlation curve between OD 470 and the pyridine degradation rate was plotted to evaluate the linear relationship and goodness of fit (R²) between the two.

[0095] The experimental results are as Figure 5 shown. When using single wavelength (OD 470When detection is carried out, the linear correlation between the OD value and the pyridine degradation rate decreases, and the goodness of fit of the standard curve (R 2 is only 0.73. In contrast, when detection is carried out using dual wavelengths, the standard curve R 2 reaches 0.99, which is significantly better than Comparative Example 2. Using dual wavelengths (ΔOD = OD 470 - OD 630 ) for detection has a more excellent linear relationship of the standard curve, more accurate fitting results, and higher system stability.

[0096] Comparative Example 3 The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds in this comparative example is basically the same as that in Example 2, except that the color reagent used for detection is tetrazolium violet (MTT). Different from WST-8 or XTT, the formazan product generated after accepting electrons during the microbial metabolism of this color reagent is a purple crystal insoluble in water, and it needs to be dissolved by adding an organic solvent (such as DMSO) before the optical density can be measured. The operation process is more complex and is not conducive to rapid and continuous absorbance monitoring in a microplate.

[0097] The experimental results are as Figure 6 shown. When using tetrazolium violet as the color reagent for detection, the ΔOD signal is generally low and there is no obvious upward trend within 36 h. In contrast, when using WST-8 as the color reagent for detection, the ΔOD signal intensity is moderate, and the goodness of fit of the standard curve (R 2 ) can reach 0.99, which is significantly better than Comparative Example 3. Using WST-8 as the color reagent for detection has higher sensitivity and more accurate fitting results.

[0098] The above-described embodiments only represent the preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications, improvements, and substitutions can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. A method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, characterized in that: Comprising the following steps: Inoculate the bacteria to be tested into a medium with a nitrogen-containing heterocyclic compound as the sole carbon source to prepare a bacterial suspension, and the initial OD of the bacterial suspension 600 is 0.4 to 0.6; Carrying out a color reaction on the bacterial suspension with a chromogenic agent WST-8 or XTT to obtain a reaction solution; the initial concentration of WST-8 or XTT in the reaction solution is 250 mg / L to 350 mg / L; Measuring the difference in absorbance of the reaction solution by double-wavelength spectrophotometry, and the difference in absorbance is used to characterize the degradation ability of the test bacterium to the nitrogen-containing heterocyclic compound.

2. The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 1, characterized in that: The two wavelengths are 470 nm and 630 nm respectively, the absorbance difference is ΔOD, and ΔOD = OD 470 - OD 630 .

3. The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 2, characterized in that: The nitrogen-containing heterocyclic compound is any one of pyridine, pyrrole or benzothiazole.

4. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 3, characterized in that: The conditions for the color reaction are to react in the dark for 20 h to 48 h under constant temperature conditions.

5. The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 4, wherein: The method further includes: measuring the difference in absorbance of the reaction solution at different reaction time points, and establishing a standard working curve between the difference in absorbance and the degradation rate.

6. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to any one of claims 1-5, characterized in that: The medium with the nitrogen-containing heterocyclic compound as the sole carbon source is the MSM medium, and the MSM medium includes: NH4Cl 57.3 mg / L, KH2PO4 13.2 mg / L, MgSO4 15 mg / L, CaCl2 16.5 mg / L, trace elements 0.4 mL / L.

7. The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 6, characterized in that: The trace elements include: MnCl2·4H2O 250 mg / L, ZnSO4·7H2O 100 mg / L, CuSO4·5H2O 50 mg / L, CoCl2·6H2O 25 mg / L, Na2MoO4·2H2O 50 mg / L, FeSO4·7H2O 200 mg / L.

8. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 7, characterized in that: The test bacterium is a single strain or a combination of multiple strains.

9. The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 8, characterized in that: The test bacterium is Rhodococcus pyridinivorans strain Rho48, taxonomically named Rhodococcus pyridinivorans, and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 4, 2024, with the deposit number CGMCC No. 32892.

10. Use of a method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, characterized in that: Use of the method according to any one of claims 1-9 in screening nitrogen-containing heterocyclic compound-degrading bacteria, or use in the process of treating nitrogen-containing heterocyclic compound pollution.

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