Method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds and use thereof

Abstract

The present application belongs to the technical field of microorganism detection, and relates to a detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds and application. In view of the fact that there is no color developing system for the targeted detection of the degradation ability of nitrogen-containing heterocyclic compounds in the prior art, and the traditional absorbance detection has the technical problems of false positive and low accuracy, the detection method provided in the present application comprises the following steps: inoculating a to-be-detected bacterium into a culture medium containing nitrogen-containing heterocyclic compounds as the only carbon source, preparing a bacterial suspension, and setting the initial OD 600 of the bacterial suspension to 0.4-0.6; performing a color developing reaction on the bacterial suspension and a color developing agent WST-8 or XTT to obtain a reaction solution, and setting the initial concentration of the color developing agent in the reaction solution to 250-350 mg / L; and determining the absorbance difference of the reaction solution by using a double-wavelength spectrophotometry method, so as to characterize the degradation ability of the to-be-detected bacterium to the nitrogen-containing heterocyclic compounds, and the present method has high accuracy. The present application also provides the application of the method in screening nitrogen-containing heterocyclic compound degrading bacteria.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a detection method for the ability of microorganisms to degrade nitrogenous heterocyclic compounds and application. BACKGROUND

[0002] Nitrogenous heterocyclic compounds (NHCs) are a typical class of refractory organic pollutants, which are widely present in coal chemical, pharmaceutical and pesticide wastewater. Nitrogenous heterocyclic compounds are chemically stable and have a wide range of biological toxicity. They can be discharged into natural water bodies and soil environments with wastewater, posing potential risks to the ecological system and human health.

[0003] Compared with traditional technologies such as membrane separation, adsorption and advanced oxidation processes, biological treatment of nitrogenous heterocyclic compound wastewater has the advantages of low cost, large treatment capacity, high treatment efficiency and effective prevention of secondary pollution, and thus becomes a key technology for treating nitrogenous heterocyclic compound wastewater. At present, it has been found that some microorganisms have 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 degradation rate information in practical application, and there is a lack of a simple, rapid and alternative detection method for quantitatively evaluating the ability of microorganisms to degrade specific nitrogenous heterocyclic compounds.

[0004] In recent years, high-throughput colorimetric technology based on microplate has been widely used in the study of microbial carbon source metabolism or electron acceptor utilization ability, providing an efficient means for the detection of functional microorganisms. However, this kind of technology still lacks a directional detection strategy for the degradation ability of specific pollutants (such as nitrogenous heterocyclic compounds), and the traditional absorbance determination method is difficult to distinguish the color reaction caused by pollutant degradation and basic metabolism of microorganisms, which is easy to appear false positive and affects the accuracy of the results.

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

[0006] 1. Problems to be solved

[0007] In view of the fact that there is no color developing system for targeted detection of the degradation ability of nitrogen-containing heterocyclic compounds in the prior art, and the traditional absorbance detection is prone to interference from basic metabolism, false positives, low accuracy and long cycle, the present application provides a method for detecting the degradation ability of nitrogen-containing heterocyclic compounds by microorganisms, which is based on a double-wavelength absorbance detection method of color developing agent WST-8 or XTT, and is used for evaluating the degradation ability of microorganisms to nitrogen-containing heterocyclic compounds, and has the advantages of high throughput, strong directionality, high accuracy and short cycle. Meanwhile, the present application also provides an application of the method for detecting the degradation ability of nitrogen-containing heterocyclic compounds by microorganisms in screening nitrogen-containing heterocyclic compound-degrading bacteria, and an application in the process of nitrogen-containing heterocyclic compound pollution treatment.

[0008] 2. Technical solutions

[0009] To achieve the above-mentioned purposes, the technical solutions provided are as follows:

[0010] The method for detecting the degradation ability of nitrogen-containing heterocyclic compounds by microorganisms comprises the following steps:

[0011] The test bacteria are inoculated into a culture medium containing nitrogen-containing heterocyclic compounds as the sole carbon source to prepare a bacterial suspension, and the initial OD of the bacterial suspension is 0.4-0.6; 600

[0012] The bacterial suspension is subjected to color developing reaction with color developing agent WST-8 or XTT to obtain a reaction solution, and the initial concentration of WST-8 or XTT in the reaction solution is 250 mg / L-350 mg / L;

[0013] The absorbance difference of the reaction solution is determined by a double-wavelength spectrophotometry, and the absorbance difference is used to represent the degradation ability of the test bacteria to nitrogen-containing heterocyclic compounds.

[0014] WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) is a water-soluble tetrazolium salt.

[0015] XTT (2,3-diphenyl-5-(2,4-disulfophenyl)-2H-tetrazolium) is a water-soluble tetrazolium salt.

[0016] Preferably, the color developing reaction is carried out in a 96-well plate, the volume ratio of the bacterial suspension to the color developing agent is 1:1, the addition amount of the bacterial suspension is 100 μL, the addition amount of the color developing agent is 100 μL, and the total volume of each well is 200 μL.

[0017] Preferably, the initial concentration of nitrogen-containing heterocyclic compounds in the reaction solution is 100 mg / L.

[0018] ​Furthermore, the dual wavelengths are 470 nm and 630 nm respectively, the absorbance difference is ΔOD, and ΔOD=OD 470 -OD 630 .

[0019] OD 470 OD (optical density at 470 nm) is used to measure the results of a specific color reaction because the product generated in the color reaction has a clear absorption peak at 470 nm; 630 (Optical density at 630 nm) is used as background correction, as this wavelength is usually not absorbed by the colorimetric product. By measuring at a wavelength that does not absorb light, interference signals caused by turbidity, bubbles, or other nonspecific scattering and absorption can be corrected. Calculate OD 470 -OD 630 The difference between the two values ​​can effectively subtract background interference and retain the actual absorption signal of the target substance. This method improves the accuracy and sensitivity of the measurement and reduces the errors caused by environmental factors, experimental conditions, and the instrument itself.

[0020] Furthermore, the nitrogen-containing heterocyclic compound is any one of pyrridine, pyrrole or benzothiazole.

[0021] Furthermore, the color development reaction is carried out under conditions of constant temperature and in the dark for 20 to 48 hours.

[0022] Preferably, when the nitrogen-containing heterocyclic compound is pyridine, the reaction is carried out at a constant temperature in the dark for 30 to 36 hours; when the nitrogen-containing heterocyclic compound is pyrrole, the reaction is carried out at a constant temperature in the dark for 40 to 48 hours; when the nitrogen-containing heterocyclic compound is benzothiazole, the reaction is carried out at a constant temperature in the dark for 20 to 24 hours.

[0023] Preferably, the constant temperature is 30°C.

[0024] Furthermore, the method further comprises: 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.

[0025] Preferably, the degradation rate is: the residual concentration of the nitrogen-containing heterocyclic compound in the reaction solution is measured by high performance liquid chromatography, and the degradation rate % = (initial concentration - residual concentration) / initial concentration × 100%.

[0026] Furthermore, the culture medium with nitrogen-containing heterocyclic compounds as the sole carbon source is MSM culture medium, and the MSM culture medium includes: 57.3 mg / L NH4Cl, 13.2 mg / L KH2PO4, 15 mg / L MgSO4, 16.5 mg / L CaCl2, and 0.4 mL / L trace elements.

[0027] Further, the microelements 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.

[0028] Further, the to-be-tested bacteria are single strains or combinations of multiple strains.

[0029] In one experiment, the degradation ability of single strains and combinations of multiple strains (combination bacteria) on different pollutants is evaluated at the same time. Compared with the traditional shake flask experiment which needs to verify the degradation effect of different to-be-tested bacteria step by step, the present method can quickly lock single bacteria or combination bacteria with strong degradation ability, has high accuracy and greatly reduces the experimental workload, and improves the screening efficiency.

[0030] Further, the to-be-tested bacteria are Rhodococcus pyridinivorans strain Rho48, classified as Rhodococcus pyridinivorans, and preserved in the China General Microbiological Culture Collection Center on December 4, 2024, with a preservation number of CGMCC No.32892.

[0031] The application of the detection method of the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, the application of the method in screening nitrogen-containing heterocyclic compound degrading bacteria, or the application of the method in the process of nitrogen-containing heterocyclic compound pollution treatment.

[0032] 3. Beneficial effects

[0033] Compared with the prior art, the technical scheme provided by the present application has the following beneficial effects:

[0034] (1) The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds of the present application can realize the detection of 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 color developing agent, and combining a color developing reaction, thereby avoiding false positive results caused by simple metabolic activity and achieving high accuracy. Microorganisms can metabolize nitrogen-containing heterocyclic compounds as the sole carbon source, and in this process, the microorganisms rely on intracellular or cell surface electron transfer enzymes (such as NADH / NADPH-dependent dehydrogenase) to reduce specific color developing agents (such as WST-8, XTT) to orange-yellow water-soluble formazan products. The product has a characteristic absorption peak, which facilitates quantitative analysis in a microplate by optical density detection at a specific wavelength, and can indirectly reflect the degradation ability of microorganisms under the condition of using nitrogen-containing heterocyclic compounds as the sole carbon source. The absorbance difference is determined by a dual-wavelength method, which eliminates the influence of background noise. The greater the absorbance difference, the stronger the degradation ability of the microorganisms. The use of a 96-well plate combined with a dual-wavelength absorbance method for high-throughput detection reduces the experimental period and labor input, has the advantages of simple operation, high sensitivity, and strong adaptability, and provides reliable technical support for the rapid screening of high-efficiency degradation strains in the biological treatment of nitrogen-containing heterocyclic organic pollutants.

[0035] (2) The detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds of the present application further comprises determining 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. The standard curve shows that the absorbance difference is significantly positively correlated with the degradation rate (R 2 >0.99), and in the actual pollution treatment process, real-time monitoring of the degradation efficiency is crucial for optimizing reaction conditions and regulating the degradation process. This linear relationship makes real-time monitoring possible, so that reaction conditions can be adjusted in a timely manner based on the monitoring results to improve the degradation efficiency.

[0036] (3) The application of the detection method for the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds of the present application. The detection method of the present application can be applied to the screening of nitrogen-containing heterocyclic compound-degrading bacteria, which can be single strains or combinations of multiple strains. Traditional methods for screening degrading bacteria are time-consuming and labor-intensive. The detection method of the present application can quickly and accurately screen strains with the ability to degrade nitrogen-containing heterocyclic compounds by determining the absorbance difference, thereby shortening the screening period and improving the screening efficiency. It can also be applied to the treatment of nitrogen-containing heterocyclic compound pollution. In the pollution treatment process, the detection method of the present application can monitor the effect of microorganisms in degrading nitrogen-containing heterocyclic compounds in real time. By detecting key indicators such as the degradation rate during the degradation process, the treatment plan can be adjusted in a timely manner. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the pyridine degradation rate-absorbance standard curve;

[0038] Figure 2 The pyrrole degradation rate-absorbance standard curve is drawn;

[0039] Figure 3 The benzothiazole degradation rate-absorbance standard curve is drawn;

[0040] Figure 4 The pyridine degradation rate-absorbance correlation curve when the initial concentration of WST-8 is 300 mg / L is drawn;

[0041] Figure 5 The pyridine degradation rate-absorbance correlation curve when detection is performed using a single wavelength (OD 470 ) is drawn;

[0042] Figure 6 The pyridine degradation rate-absorbance scatter plot when detection is performed using a tetrazolium violet color reagent is drawn. DETAILED DESCRIPTION

[0043] For a further understanding of the present application, reference will be made to the following examples.

[0044] The present application will be further described below in conjunction with specific examples.

[0045] It should be noted that the terms such as "upper", "lower", "left", "right", "middle" and the like cited in the present specification are merely for the convenience of clear description, and are not intended to limit the scope of implementation, and the change or adjustment of the relative relationship is also regarded as the scope of implementation of the present application without substantial change in technical content.

[0046] 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 this application belongs; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0047] Unless otherwise specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are all conventional products that can be purchased on the market, unless otherwise specified.

[0048] As used herein, the term "about" is used to provide flexibility to a given term, measurement, or value. The degree of flexibility of a particular variable can be readily determined by one of skill in the art.

[0049] As used herein, the term "at least one of" is intended to mean one or more of the listed items. For example, "at least one of A, B, and C" includes only A, only B, only C, and combinations thereof.

[0050] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of a range, but also to include all the individual numerical values or sub-ranges

[0051] The instruments, reagents and strains used in the detection method of the microbial degradation ability of nitrogen-containing heterocyclic compounds in the following examples and comparative examples are as follows:

[0052] 1. Instruments

[0053] BioTek Synergy H1 multifunctional microplate reader (Agilent, USA), 1260 Infinity II high performance liquid chromatograph (Agilent, USA), 96-well microplate.

[0054] 2. Reagents

[0055] Pyridine, pyrrole and benzothiazole were purchased from Macklin Biochemical Technology Co., Ltd.; color reagent WST-8 was purchased from Shanghai Yuye Biological Technology Co., Ltd.; color reagent XTT was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.; and the rest of the reagents were domestic analytical pure.

[0056] 3. Strains

[0057] The strain with nitrogen-containing heterocyclic compound degradation ability identified through previous laboratory screening was Rhodococcus pyridinivorans Rho48, which was preserved in the China General Microbiological Culture Collection Center on December 4, 2024, at No. 3, Beichen West Road, Chaoyang District, Beijing, with the preservation number of CGMCC No. 32892. Hereinafter referred to as Rho48.

[0058] Example 1

[0059] The detection method of the microbial degradation ability of nitrogen-containing heterocyclic compounds in this example was optimized and included the following steps:

[0060] I. Solution and medium configuration

[0061] Solution preparation: 1 g / L pyridine stock solution, 1 g / L XTT color reagent, and 2 g / L WST-8 color reagent were accurately prepared with deionized water, respectively. The solutions were sterilized by filtration using a 0.22 μm pore size membrane and stored at 4 °C in the dark until use.

[0062] Trace element stock solution preparation: MnCl2·4H2O 250 mg, ZnSO4·7H2O 100 mg, CuSO4·5H2O 50 mg, CoCl2·6H2O 25 mg, Na2MoO4·2H2O 50 mg, FeSO4·7H2O 200 mg, and deionized water were added to 1000 mL.

[0063] MSM medium preparation: NH4Cl 57.3 mg, KH2PO4 13.2 mg, MgSO4 15 mg, CaCl2 16.5 mg, and trace element stock solution 0.4 mL were added to 1000 mL of deionized water.

[0064] II. Optimization and establishment of detection method for microbial degradation of nitrogen-containing heterocyclic compounds

[0065] (1) Color development principle

[0066] Microorganisms can metabolize nitrogen-containing heterocyclic compounds (pyridine, pyrrole, and benzothiazole) as the sole carbon source. In this process, microorganisms rely on intracellular or cell surface electron transfer enzymes (such as NADH / NADPH-dependent dehydrogenase) to reduce specific color reagents (such as WST-8, XTT) to orange-yellow water-soluble formazan products. The product has a characteristic absorption peak, which facilitates quantitative analysis in a microplate by optical density detection at a specific wavelength, and can indirectly reflect the degradation ability of microorganisms under the condition of using nitrogen-containing heterocyclic compounds as the sole carbon source. The increase in absorbance difference indicates an increase in degradation ability. By measuring the change in absorbance, the degradation activity of the strain can be indirectly reflected.

[0067] (2) Optimization of color reagent type and concentration

[0068] Experimental group: After activation, the pure strain Rho48 was inoculated in LB medium and incubated overnight at 37 °C and 150 r / min in a shaker until it reached the logarithmic growth phase. The bacterial suspension was centrifuged at 8000 rpm for 3 min, and the supernatant was discarded. The bacterial cells were washed 3 times with PBS buffer and collected. The bacterial cells were inoculated into MSM medium with pyridine as the sole carbon source to obtain a bacterial suspension. The initial OD 600 of the bacterial suspension was set to 0.5.

[0069] The color developing agent XTT and WST-8 mother liquor were respectively diluted with deionized water to a final concentration of 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, and 700 mg / L.

[0070] The bacterial suspension and the color developing agent (WST-8 or XTT) were inoculated in a 96-well plate at a volume ratio of 1:1, and the bacterial suspension was added to each well of the 96-well plate at an amount of 100 μL, and the color developing agent was added at an amount of 100 μL, so that the total volume of each well was 200 μL. The initial concentration of pyridine in the color developing reaction system was 100 mg / L, and each color developing agent concentration was set with three biological replicates. The reaction was carried out at 30°C in the dark for 36 h, and the OD 470 and OD 630 of the reaction solution were measured by an enzyme-labeled instrument. 470 630

[0071] The blank control group: set control group 1 without pyridine (only containing bacterial bodies, MSM medium and color developing agent); control group 2 without bacterial bodies (only containing pyridine, MSM medium and sterile color developing agent). Among them, the color developing agent concentration, pyridine concentration, initial OD 600 and detection method of the bacterial suspension were consistent with those of the experimental group.

[0072] Table 1 Influence of color developing agent type and concentration on ΔOD

[0073]

[0074] As shown in Table 1, in the two color developing agent systems, the ΔOD value of the experimental group showed a growth trend with the color developing agent concentration. In the WST-8 system, the ΔOD reached 0.256 at 500 mg / L, and then increased slightly to 0.272 (an increase of 6.3%) at 700 mg / L, and the sensitivity was close to the plateau. In the XTT system, the ΔOD reached 0.247 only when the concentration 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 could obtain higher response at a lower concentration. In addition, the single-well color developing cost of WST-8 was only about 25% of that of XTT, and the cost performance was better.

[0075] ​​Two groups of blank controls set under the same conditions also showed that the specificity of the color reaction was good. In control group 1 (no pyridine), only containing bacterial bodies and color developing agent, the ΔOD value was much lower than the corresponding experimental group, for example, the ΔOD of WST-8 system at 500 mg / L in 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 bacterial body to the color developing agent was weak; in control group 2 (no bacterial body), only containing pyridine and color developing agent, the ΔOD was generally lower than that of control group 1, such as WST-8 at 500 mg / L was only 0.007, indicating that there was basically no direct reaction between pyridine and color developing agent. Therefore, the color developing signal mainly comes from the metabolic activity of the bacterial body in the presence of pyridine, and the ΔOD can effectively reflect the degradation capacity of microorganisms to pollutants.

[0076] In summary, considering the color developing sensitivity, background interference and economy, it is determined that WST-8 at 500 mg / L is the best color developing agent concentration, and the bacterial suspension and color developing agent are mixed in a volume ratio of 1:1 in the actual reaction system to obtain the reaction solution, that is, the initial concentration of WST-8 in the reaction solution is 250 mg / L, which is used for subsequent experiments.

[0077] (3) Optimization of initial inoculum size

[0078] In order to further optimize the initial inoculum size of the bacterial suspension in the color developing reaction system and ensure good color developing effect and stability of the reaction system, different initial OD 600 values of the bacterial suspension were set for comparison experiments.

[0079] Experimental group: after activation, the pure bacteria Rho48 were inoculated in LB medium and placed in a shaking bed at 37 ℃ and 150 r / min for overnight amplification culture until they reached the logarithmic growth phase to obtain bacterial liquid; the bacterial liquid was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial body was washed with PBS buffer for 3 times; the bacterial body was inoculated into MSM medium with pyridine as the only carbon source to obtain bacterial suspension;

[0080] The bacterial suspension and color developing agent WST-8 (concentration of 500 mg / L) were inoculated in a 96-well plate in a volume ratio of 1:1, the bacterial suspension was added to each well of the 96-well plate at an amount of 100 μL, the color developing agent was added at an amount of 100 μL, so that the total volume of each well was 200 μL, the initial concentration of pyridine in this color developing reaction system was 100 mg / L, and the concentration of WST-8 color developing agent was 250 mg / L, which was cultured at 30 ℃ in the dark. The initial OD 600 of the bacterial suspension was set as 0.1, 0.3, 0.5, 0.7 and 1.0 respectively. Each inoculum size was set in triplicate, and the OD 470 and OD 630 were measured after 12 h, 24 h and 36 h of reaction respectively, and the ΔOD value was calculated.

[0081] Blank control group: Set control group 1 without pyridine (only containing bacteria, MSM medium and color developing agent); control group 2 without bacteria (only containing pyridine, MSM medium and color developing agent sterile liquid). Among them, the concentration of color developing agent, the concentration of pyridine, the initial OD of bacterial suspension 600 And the detection method is the same as the experimental group.

[0082] Table 2 Effect of initial inoculation amount on ΔOD

[0083]

[0084] The results are shown in Table 2, and the initial OD of the bacterial suspension 600 Too low (such as 0.1, 0.3), the reaction system ΔOD grows slowly, and the color developing sensitivity is not high, which is not conducive to the efficiency and sensitivity of high-throughput screening. While the initial OD of the bacterial suspension 600 Too high (such as 0.7, 1.0), the system color develops too fast, which leads to the rapid depletion of color developing agent, and the ΔOD value appears a platform period or even decreases after 12 h, which affects the accuracy of the linear relationship between degradation rate and ΔOD. Considering the reaction speed, color developing degree and system stability, the initial OD of the bacterial suspension 600 0.4~0.6 is selected, among which the initial OD of the bacterial suspension 600 0.5 is the best inoculation amount condition.

[0085] The data of the control group shows that the ΔOD value always maintains at a low level, and the change amplitude is small with the increase of the initial inoculation amount, among which the difference of ΔOD between control group 1 and the experimental group gradually increases with the increase of the initial inoculation amount, which further illustrates that the color developing reaction is mainly due to the metabolic activity of the bacteria under the induction of pyridine, rather than non-specific background reaction.

[0086] In the best color developing reaction system, the initial OD of the bacterial suspension 600 0.5, the initial concentration of pyridine in the reaction solution is 100 mg / L, and the initial concentration of WST-8 color developing agent is 250 mg / L.

[0087] Example 2

[0088] The detection method of the microbial degradation of nitrogen-containing heterocyclic compounds in this embodiment and the drawing of the pyridine degradation rate-absorbance standard curve include the following steps:

[0089] I. Solution and medium preparation: same as example 1.

[0090] II. Drawing of pyridine degradation rate-absorbance standard curve

[0091] After activation, the pure bacteria Rho48 was inoculated in LB medium and placed in a 37 °C, 150 r / min shaker for overnight expansion culture until it reached the logarithmic growth phase to obtain the bacterial liquid; the bacterial liquid was centrifuged at 8000 rpm for 3 min, and the supernatant was discarded, and the bacterial body was washed with PBS buffer for 3 times; the bacterial body was inoculated into the culture medium with pyridine as the only carbon source to obtain the bacterial suspension, and the initial OD 600 of the bacterial suspension was set to 0.5.

[0092] A 500 mg / L WST-8 solution was prepared with deionized water, and the bacterial suspension and the chromogenic agent were inoculated in a 96-well plate at a volume ratio of 1:1 to obtain the reaction liquid, and the initial concentration of pyridine in the reaction liquid was 100 mg / L, and the initial concentration of WST-8 chromogenic agent was 250 mg / L, and the reaction was carried out at 30 °C for 36 h in the dark.

[0093] The OD 470 and OD 630 of the reaction liquid were measured by an enzyme marker at different degradation reaction time points 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 30 h, and 36 h, respectively. After the absorbance determination at each time point, a destructive sampling method was used, and 2.4 mL of reaction liquid (from 12 wells) was taken from the 96-well plate at each time point, and then filtered through a 0.22 μm organic phase filter membrane and transferred to a liquid phase vial (three parallel samples). The concentration of pyridine in the filtered reaction liquid was determined by HPLC, and the chromatographic column used was an Eclipse Plus C18 chromatographic column, with a sample injection volume of 20 μL and a column temperature of 30 °C; an ultraviolet detector (DAD) was used, and the detection wavelength was set to 253 nm; methanol and water were used as the mobile phase, with a volume ratio of 60:40 (v / v), and a flow rate of 1.0 mL / min.

[0094] Meanwhile, blank controls were set, blank control 1 did not contain pyridine (only contained bacterial body, MSM medium and chromogenic agent); blank control 2 did not contain bacterial body (only contained pyridine, MSM medium and sterile liquid of chromogenic agent).

[0095] Data processing:

[0096] ① ΔOD calculation: for the absorbance data of each well of the 96-well plate, the ΔOD value was calculated according to the formula: ΔOD = OD 470 - OD 630 .

[0097] ② Biological parallel processing: for each degradation system (containing 3 parallel wells), the outliers were removed (Grubbs test, α = 0.05), and the arithmetic mean of ΔOD was taken as the final characterization value of the system.

[0098] 3. Pyridine degradation rate calculation: According to the residual concentration of pyridine in each well measured by high performance liquid chromatography, the degradation rate was calculated: degradation rate = (initial concentration - residual concentration) / initial concentration x 100%.

[0099] 4. Correlation analysis between ΔOD and pyridine degradation rate: Linear fitting was performed with ΔOD value as independent variable and degradation rate as dependent variable, and the correlation curve of ΔOD and pyridine degradation rate was drawn to evaluate the linear relationship between them and the goodness of fit (R2), as shown in Figure 1 .

[0100] The standard curve showed that ΔOD was significantly positively correlated with degradation rate (R 2 > 0.99).

[0101] III. Accuracy verification

[0102] To verify the accuracy of the method, the reaction liquid at 6 h, 14 h and 28 h was selected respectively, and the ΔOD was measured as 0.091, 0.109 and 0.152, respectively. The predicted degradation rate calculated by the standard curve was 17.72%, 29.84% and 58.78%, respectively. At the same time, the degradation rate calculated by the pyridine concentration directly measured by HPLC was 16.93%, 30.31% and 58.24%, respectively. Comparing the results obtained by the two methods, the error was less than 5%, which proved that the detection method of microbial degradation of nitrogen-containing heterocyclic compounds in this embodiment had high accuracy.

[0103] Example 3

[0104] The detection method of microbial degradation of nitrogen-containing heterocyclic compounds in this embodiment, the preparation of pyrrole degradation rate-absorbance standard curve, includes the following steps:

[0105] I. Solution and medium preparation

[0106] Solution preparation: 1 g / L pyrrole stock solution and 2 g / L WST-8 color developing agent were accurately prepared with deionized water, respectively. The solutions were filtered and sterilized using 0.22 μm pore size membrane, and stored at 4 °C in the dark until use.

[0107] Medium preparation: same as Example 1.

[0108] II. Preparation of pyrrole degradation rate-absorbance standard curve

[0109] After activation, the pure bacteria Rho48 was inoculated in LB medium and placed in a 37 °C, 150 r / min shaker for overnight expansion culture until it reached the logarithmic growth phase to obtain the bacterial liquid; the bacterial liquid was centrifuged at 8000 rpm for 3 min, and the supernatant was discarded, and the bacterial body was washed with PBS buffer for 3 times; the bacterial body was inoculated into the culture medium with pyrrole as the only carbon source to obtain the bacterial suspension, and the initial OD 600 of the bacterial suspension was set to 0.5.

[0110] A 500 mg / L WST-8 solution was prepared with deionized water, and the bacterial suspension and the chromogenic agent were inoculated in a 96-well plate at a volume ratio of 1:1 to obtain the reaction liquid, and the initial concentration of pyrrole in the reaction liquid was 100 mg / L, and the initial concentration of WST-8 chromogenic agent was 250 mg / L, and the reaction was carried out at 30 °C for 48 h in the dark.

[0111] The OD 470 and OD 630 of the reaction liquid were measured by an enzyme marker 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, respectively. After the absorbance determination at each time point, a destructive sampling method was used, and 2.4 mL of reaction liquid (12 wells of reaction liquid) was taken from the 96-well plate at each time point, and then filtered through a 0.22 μm organic phase filter membrane and transferred to a liquid phase vial (three parallel samples). The concentration of pyrrole in the filtered reaction liquid was determined by HPLC, and the chromatographic column used was an Eclipse Plus C18 chromatographic column, and the sample volume was set to 20 μL, and the column temperature was 30 °C; an ultraviolet detector (DAD) was used, and the detection wavelength was set to 210 nm; methanol and water were used as the mobile phase, and the volume ratio was set to 70:30 (v / v), and the flow rate was 1.0 mL / min.

[0112] Meanwhile, blank controls were set, blank control 1 did not contain pyrrole (only contained bacterial body, MSM culture medium and chromogenic agent); blank control 2 did not contain bacterial body (only contained pyrrole, MSM culture medium and sterile liquid of chromogenic agent).

[0113] Data processing:

[0114] ① ΔOD calculation: for the absorbance data of each well of the 96-well plate, the ΔOD value was calculated according to the formula: ΔOD = OD 470 -OD 630 .

[0115] ② Biological parallel processing: for each degradation system (containing 3 parallel wells), the outlier value was removed (Grubbs test, α = 0.05), and the arithmetic mean of ΔOD was taken as the final characterization value of the system.

[0116] ③Pyrrole degradation rate calculation: According to the residual concentration of pyrrole in each well measured by high performance liquid chromatography, the degradation rate was calculated: degradation rate = (initial concentration - residual concentration) / initial concentration x 100%.

[0117] ④ΔOD and pyrrole degradation rate correlation analysis: Linear fitting was performed with ΔOD value as independent variable and degradation rate as dependent variable, and the correlation curve of ΔOD and pyrrole degradation rate was drawn to evaluate the linear relationship between them and the goodness of fit (R²), as shown in Figure 2

[0118] The standard curve shows that ΔOD is significantly positively correlated with degradation rate (R 2 >0.99).

[0119] III. Accuracy verification

[0120] To verify the accuracy of the method, the reaction liquid at 6 h, 14 h and 28 h was selected respectively, and the ΔOD was measured as 0.096, 0.110 and 0.123, respectively. The predicted degradation rate calculated by the standard curve was 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 were 26.78%, 46.02% and 66.20%, respectively. Comparing the results obtained by the two methods, the error is less than 5%, which proves that the detection method of microbial degradation of nitrogen-containing heterocyclic compounds in this embodiment has high accuracy.

[0121] Example 4

[0122] The detection method of microbial degradation of nitrogen-containing heterocyclic compounds in this embodiment, the preparation of benzothiazole degradation rate-absorbance standard curve, includes the following steps:

[0123] I. Solution and medium preparation

[0124] Solution preparation: 1 g / L benzothiazole stock solution and 2 g / L WST-8 color developing agent were accurately prepared with deionized water, respectively. The solutions were filtered and sterilized using 0.22 μm pore size membrane, and stored at 4 ℃ in the dark until use.

[0125] Medium preparation: same as Example 1.

[0126] II. Preparation of benzothiazole degradation rate-absorbance standard curve

[0127] ​After activation, the pure bacteria Rho48 was inoculated in LB medium and placed in a 37 °C, 150 r / min shaker for overnight expansion culture until it reached the logarithmic growth phase to obtain the bacterial liquid; the bacterial liquid was centrifuged at 8000 rpm for 3 min, and the supernatant was discarded, and the bacterial body was washed with PBS buffer for 3 times; the bacterial body was inoculated into the medium with benzothiazole as the sole carbon source to obtain the bacterial suspension, and the initial OD 600 of the bacterial suspension was set to 0.5.

[0128] A 500 mg / L WST-8 solution was prepared with deionized water, and the bacterial suspension and the chromogenic agent were inoculated in a 96-well plate at a volume ratio of 1:1 to obtain the reaction liquid, and the initial concentration of benzothiazole in the reaction liquid was 100 mg / L, and the initial concentration of WST-8 chromogenic agent was 250 mg / L, and the reaction was carried out at 30 °C for 20 h in the dark.

[0129] The OD 470 and OD 630 of the reaction liquid were measured by an enzyme marker at different degradation time points of 0 h, 2 h, 4 h, 8 h, 12 h, 16 h and 20 h, respectively. 470 After the absorbance determination at each time point, a destructive sampling method was used, and 2.4 mL of reaction liquid (12 wells of reaction liquid) was taken from the 96-well plate at each time point, and then 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 liquid was determined by HPLC, and the chromatographic column used was an Eclipse Plus C18 chromatographic column, and the sample volume was set to 20 μL, and the column temperature was 30 °C; an ultraviolet detector (DAD) was used, and the detection wavelength was set to 254 nm; methanol and water were used as the mobile phase, and the volume ratio was set to 70:30 (v / v), and the flow rate was 1.0 mL / min.

[0130] Meanwhile, blank controls were set, blank control 1 did not contain benzothiazole (only contained bacterial body, MSM medium and chromogenic agent); blank control 2 did not contain bacterial body (only contained benzothiazole, MSM medium and sterile liquid of chromogenic agent).

[0131] Data processing:

[0132] ① ΔOD calculation: for the absorbance data of each well of the 96-well plate, the ΔOD value was calculated according to the formula: ΔOD = OD 470 -OD 630 .

[0133] ② Biological parallel processing: for each degradation system (containing 3 parallel wells), the outliers were removed (Grubbs test, α = 0.05), and the arithmetic mean of ΔOD was taken as the final characterization value of the system.

[0134] ③Benzothiazole degradation rate calculation: 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 x 100%.

[0135] ④ΔOD and benzothiazole degradation rate correlation analysis: linear fitting was performed with ΔOD value as independent variable and degradation rate as dependent variable, and the correlation curve of ΔOD and benzothiazole degradation rate was drawn to evaluate the linear relationship between them and the goodness of fit (R Figure 3 ).

[0136] The standard curve shows that ΔOD is significantly positively correlated with degradation rate (R 2 >0.99).

[0137] III. Accuracy verification

[0138] To verify the accuracy of the method, the reaction liquid at 6 h, 10 h and 14 h was selected respectively, and the ΔOD was measured as 0.263, 0.409 and 0.528, respectively. The predicted degradation rate calculated by the standard curve was 37.02%, 60.85% and 80.27%, respectively. At the same time, the degradation rate calculated by the benzothiazole concentration directly measured by HPLC was 38.23%, 58.92% and 82.78%, respectively. Comparing the results obtained by the two methods, the error is less than 5%, which proves that the detection method of the microbial degradation of nitrogen-containing heterocyclic compounds in this embodiment has high accuracy.

[0139] Comparative Example 1

[0140] The detection method of the microbial degradation of nitrogen-containing heterocyclic compounds in this comparative example is basically the same as that in Example 2, except that the WST-8 chromogenic agent concentration is 300 mg / L.

[0141] The experimental results are shown in Figure 4 At a WST-8 concentration of 300 mg / L, the ΔOD signal is generally low, and the signal-to-noise ratio is poor, resulting in a decrease in the linear correlation between ΔOD and pyridine degradation rate, and the goodness of fit (R 2 ) of the standard curve is only 0.79. In contrast, at a WST-8 concentration of 500 mg / L, the ΔOD signal intensity is moderate and the background interference is small, and the R 2 reaches 0.99, which is significantly better than Comparative Example 1. The WST-8 chromogenic agent concentration of 500 mg / L has a more excellent linear relationship of the standard curve, the fitting result is more accurate, and the system stability is higher.

[0142] Comparative Example 2

[0143] 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 a single wavelength (OD 470 ) were tested, and the OD of the reaction solution was measured using an enzyme marker at different degradation reaction time points: 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 30 h, and 36 h. 470 For each degradation system (including 3 parallel wells), after removing outliers (Grubbs test, α = 0.05), OD 470 The arithmetic mean is taken as the final characterization value of the system. 470 Perform linear fitting for the independent variable and degradation rate as the dependent variable, and draw OD 470 The correlation curve between the two was used to evaluate the linear relationship and goodness of fit (R²).

[0144] The experimental results are as follows Figure 5 As shown, when using a single wavelength (OD 470 ) was used for detection, the linear correlation between OD value and pyridine degradation rate decreased, and the goodness of fit of the standard curve (R 2 ) is only 0.73. In contrast, when dual wavelength detection is used, the standard curve R 2 Reaching 0.99, it is significantly better than comparative example 2. Using dual wavelength (ΔOD=OD 470 -OD 630 ) The detection has a better linear relationship of the standard curve, more accurate fitting results and higher system stability.

[0145] Comparative Example 3

[0146] 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 developer used for detection is tetrazolium violet (MTT). Unlike WST-8 or XTT, the formazan product generated by this color developer after accepting electrons during microbial metabolism is a water-insoluble purple crystal. It needs to be dissolved by adding an organic solvent (such as DMSO) before optical density measurement. The operation process is more complicated and is not conducive to achieving rapid and continuous absorbance monitoring in a microplate.

[0147] The experimental results are as follows Figure 6 As shown in the figure, when using tetrazolium violet as the colorimetric 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 colorimetric 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. Detection using WST-8 color developer has higher sensitivity and more accurate fitting results.

[0148] The above described embodiments only express the preferred embodiments of the present application, which are described in more detail and in more specifically, but can not be understood as the limitation of the patent scope of the present application. It should be noted that for the ordinary skilled in the art, several modifications, improvements and substitutions can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, characterized by: The method comprises the following steps: The bacteria to be tested are inoculated into a culture medium containing the nitrogen-containing heterocyclic compound as the sole carbon source to prepare a bacterial suspension, the initial OD of the bacterial suspension being 0.4-0.

6. 600 0.4-0.

6. The color developing agent WST-8 or XTT is used to develop color of the bacterial suspension 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; The absorbance difference of the reaction liquid is determined by a dual-wavelength spectrophotometry, the dual-wavelengths are 470 nm and 630 nm respectively, the absorbance difference is ΔOD, the ΔOD = OD 470 -OD 630 , and the absorbance difference is used to characterize the degradation capacity of the bacteria to be detected to the nitrogen-containing heterocyclic compound. The absorbance difference of the reaction solution is measured at different reaction time points to establish a standard working curve between the absorbance difference and the degradation rate; The nitrogen-containing heterocyclic compound is any one of pyridine, pyrrole or benzothiazole, the reaction time of the pyridine is 0 h to 36 h, the reaction time of the pyrrole is 0 h to 48 h, and the reaction time of the benzothiazole is 0 h to 20 h.

2. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 1, characterized by: The MSM medium containing the nitrogen-containing heterocyclic compound as the sole carbon source comprises NH4Cl 57.3 mg / L, KH2PO4 13.2 mg / L, MgSO4 15 mg / L, CaCl2 16.5 mg / L and trace elements 0.4 mL / L.

3. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 2, characterized by: The trace elements comprise 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 and FeSO4·7H2O 200 mg / L.

4. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 3, characterized by: The bacteria to be detected are single strains or a combination of multiple strains.

5. The method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds according to claim 4, characterized by: The test bacteria is Rhodococcus pyridinovorans strain Rho48, which is classified and named as Rhodococcus pyridinivorans Rhodococcus pyridinovorans, and was preserved in the China General Microbiological Culture Collection Center on December 4, 2024, with a preservation number of CGMCC No. 32892.

6. Use of a method for detecting the ability of microorganisms to degrade nitrogen-containing heterocyclic compounds, characterized in that: The method according to any one of claims 1-5 is applied to screening of the nitrogen-containing heterocyclic compound degrading bacteria or to pollution treatment of the nitrogen-containing heterocyclic compound.

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