Method for rapidly detecting AOC in water based on flow cytometry and application of method

Abstract

The invention relates to the technical field of water quality detection, and discloses a method for rapidly detecting AOC in water based on a flow cytometry and application of the method. The method comprises the following steps: (1) respectively and sequentially carrying out low-nutrition domestication and culture on two test bacteria P17 and NOX to obtain a P17 bacterial solution and an NOX bacterial solution; (2) making a standard curve by successively inoculating two test bacteria of P17 and NOX; and (3) determining the concentration of AOC in the water sample: determining the concentration of AOC in the water sample by using the KP17 and the KNOX. The method provided by the invention is obviously superior to a common nonlinear discrete trend in a traditional P17 / NOX plate counting method, ensures reliable association between strain growth and cell counting under different carbon concentration conditions, and has good reproducibility and anti-interference capability.

Description

Technical Field

[0001] The present invention relates to the technical field of water quality detection, and in particular to a method for quickly detecting AOC in water based on flow cytometry and an application thereof. Background Art

[0002] Assimilable organic carbon (AOC) refers to organic carbon in water that can be directly utilized by heterotrophic bacteria and converted into building blocks for their own organisms. It is an important indicator for measuring the potential for microbial regrowth and biostability in drinking water. The concept was first proposed by Dutch scholar VanderKooij and is now widely used to assess the microbial control capacity of drinking water plant effluent and distribution systems. Compared with traditional indicators such as total organic carbon (TOC) and biodegradable organic carbon (BDOC), AOC more directly reflects the content of organic matter in water that is readily absorbed and utilized by microorganisms. It is a key parameter for assessing the risk of "secondary contamination" in drinking water and controlling regrowth in pipe networks.

[0003] The current standard method for detecting AOC primarily uses two reference strains: Fluorescent Pseudomonas P17 and Spirillum strain NOX. These are cultured in solutions of varying concentrations of acetic acid and carbon, followed by plate counts to establish a correlation between carbon concentration and bacterial growth. Although widely used, this method still has the following limitations: Long detection cycles. Traditional methods typically require incubation periods exceeding three days, sometimes as long as 7–14 days, making them inadequate for rapid monitoring and emergency response in water plants. Counting errors are significant. Plate counts are complex, subject to subjective errors in colony identification, and suffer from poor parallelism and reproducibility. Operational efficiency is low. Relying on manual culture and plating procedures is time-consuming and labor-intensive, making high-throughput testing difficult. The detection limit is also insufficient. In drinking water samples with low AOC concentrations, traditional methods have low sensitivity, making it difficult to accurately identify microbial risk signals. Furthermore, they are susceptible to interference from contaminants. Plate counts lack specific staining or gating mechanisms, making them susceptible to background bacteria, impacting strain purity and accuracy.

[0004] The advancement of flow cytometry in environmental microbial detection, with its advantages such as high throughput, automated counting, and cell staining and identification, has provided new approaches for AOC detection. However, despite previous studies attempting to apply flow cytometry to drinking water AOC assessment, a comprehensive, standardized, yield-based flow cytometric AOC determination process applicable to actual water sample testing is currently lacking. In particular, a systematic, integrated approach is still lacking for key steps such as strain culture adaptation, sample pretreatment, staining optimization, standard curve construction, and concentration back-calculation.

[0005] The applicant's research group conducted a preliminary study (Xu Xinyuan, Li Weiying, Zhou Yanyan, et al. Comparison of Assimilable Organic Carbon (AOC) Detection Methods [J] Water Purification Technology, 2019, 38 (8): 47-52) to compare the plate coating method, flow cytometry + pure bacterial inoculation, and flow cytometry + indigenous bacterial inoculation methods for detecting AOC. The results showed that compared with the plate coating method, the AOC standard curve measured by flow cytometry had a better linear relationship, the test results were stable, and all bacteria in the water could be counted, overcoming the defect of the traditional plate coating method that only live and culturable bacteria in the water could be detected. However, the flow cytometry + pure bacterial inoculation method still had the problem of inconsistent adaptability to characteristic water samples and low ecological relevance.

[0006] Therefore, developing a rapid AOC determination method suitable for low-concentration drinking water samples, which is simple to operate, highly sensitive, and has a short detection cycle, has important engineering application value and promotion prospects. Summary of the Invention

[0007] The purpose of the present invention is to overcome the problems of the AOC detection method in the prior art, such as long time consumption, poor repeatability, insufficient sensitivity and inconsistent adaptability.

[0008] In order to achieve the above object, the first aspect of the present invention provides a method for rapid detection of AOC in water based on flow cytometry, comprising the following steps: (1) The two test bacteria, P17 and NOX, were subjected to low-nutrient acclimation and culture respectively to obtain P17 bacterial solution and NOX bacterial solution; (2) Preparation of standard curve: S1, first inoculating the P17 bacterial solution into a standard solution of acetic acid with different concentrations, culturing for 3-4 days to obtain a culture solution I, sampling and staining with SYBR Green I dye and PI dye, and then counting by flow cytometry to obtain the concentration of viable P17 bacteria in the culture solution I; S2. The culture fluid I is pasteurized and then inoculated with the NOX bacterial solution. After culturing for 3-4 days, a culture fluid II is obtained. A sample is taken and stained with SYBR Green I dye and PI dye, and then counted using flow cytometry to obtain the concentration of viable NOX bacteria in the culture fluid II. S3. Linearly fit the P17 viable bacteria concentration and the NOX viable bacteria concentration obtained under the different concentrations of the carbon acetate standard solution to obtain their respective standard curves. The slope of the standard curve is taken as the yield coefficient. and ; (3) Determination of AOC concentration in water samples: using the and stated Determination of AOC concentration in water samples.

[0009] The second aspect of the present invention provides application of the method described in the first aspect to detecting the AOC content in water, wherein the concentration of AOC in the water is in the range of 20-150 μg / L.

[0010] Compared with the prior art, the present invention has at least the following advantages: The method provided by the present invention can significantly shorten the detection time (can be completed in as fast as 24-48 hours); the operation process is standardized and batch testing is possible; it is applicable to low-concentration water samples and has a lower detection limit; it has high sensitivity, strong repeatability, and a rapid response to microbial contamination; it is particularly suitable for drinking water safety, biological stability assessment and water operation risk warning. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 These are the standard curves of the P17 strain and the NOX strain in the preferred embodiment provided by the present invention. DETAILED DESCRIPTION

[0012] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0013] As mentioned above, the first aspect of the present invention provides a method for rapid detection of AOC in water based on flow cytometry, comprising the following steps: (1) The two test bacteria, P17 and NOX, were subjected to low-nutrient acclimation and culture respectively to obtain P17 bacterial solution and NOX bacterial solution; (2) Preparation of standard curve: S1, first inoculating the P17 bacterial solution into a standard solution of acetic acid with different concentrations, culturing for 3 days to obtain a culture solution I, sampling and staining with SYBR Green I dye and PI dye, and then counting by flow cytometry to obtain the concentration of viable P17 bacteria in the culture solution I; S2. The culture fluid I is pasteurized and then inoculated with the NOX bacterial solution. After culturing for 4 days, a culture fluid II is obtained. A sample is taken and stained with SYBR Green I dye and PI dye, and then counted using flow cytometry to obtain the concentration of viable NOX bacteria in the culture fluid II. S3. Linearly fit the P17 viable bacteria concentration and the NOX viable bacteria concentration obtained under the different concentrations of the carbon acetate standard solution to obtain their respective standard curves. The slope of the standard curve is taken as the yield coefficient. and ; (3) Determination of AOC concentration in water samples: using the and stated Determination of AOC concentration in water samples.

[0014] Preferably, the concentration of the SYBR Green I dye is 4 μg / mL.

[0015] Preferably, the concentration of the PI dye is 400 μg / mL.

[0016] It should be noted that in the present invention, the P17 is Fluorescent Pseudomonads P17, which belongs to Gram-negative Pseudomonas, has a rod-shaped body, a diameter of about 0.6 μm, a light yellow colony, and is suitable for aerobic conditions at around 25°C; the NOX is Spirillum strain NOX, which belongs to a slender spiral bacterium, has a diameter of about 0.25 μm, a milky white colony, and is suitable for aerobic growth at around 25°C.

[0017] Preferably, in step (1), the two test bacteria P17 and NOX are purchased from the American ATCC bacterial culture library.

[0018] Preferably, in step (1), the two test bacteria, P17 and NOX, are revived before the low-nutrient acclimation and the cultivation, comprising: The two test bacteria, P17 and NOX, were inoculated into 20 mL of beef extract peptone medium at 25° C. for culture, wherein the medium inoculated with P17 was cultured for 48 hours, and the medium inoculated with NOX was cultured for 72 hours; then, they were inoculated into fresh slant culture medium, cultured at 25° C. for 48 hours, and then stored at 4° C., and the shelf life was no more than 6 months.

[0019] Preferably, in step (1), the low-nutrient acclimation and cultivation operations include: A loopful of each of the resuscitated P17 and NOX strains was inoculated into tap water that had been high-temperature calcined and then autoclaved, and cultured at 25°C for 7 days to complete acclimatization, thereby enhancing the growth adaptability of P17 and NOX in a low-nutrient environment. The high-temperature calcination temperature was 450°C and the time was 5 hours. Then, 100 μL of each of the low-nutrient-acclimated P17 and NOX bacterial cultures was inoculated into 50 mL of a sodium acetate solution containing 2 mg / L of carbon. Culture was then performed at 25°C for 7 days until the plateau phase, yielding the P17 and NOX bacterial cultures. This preferred method is more conducive to maintaining bacterial activity and establishing a standard curve.

[0020] Preferably, in step (1), the prepared P17 bacterial solution and the NOX bacterial solution are stored at 4° C. in the dark for a period of no more than 3 months.

[0021] Preferably, the method of the present invention further comprises, in step (3), after completing the low-nutrient acclimation and the culturing, and before preparing the standard curve, determining the viable cell concentrations of the corresponding bacterial species in the P17 bacterial solution and the NOX bacterial solution, comprising: sampling the P17 bacterial solution and the NOX bacterial solution respectively, staining them with SYBR Green I dye and PI dye, and then performing on-machine analysis using a flow cytometer, wherein the injection speed is set to medium speed, and the injection is stopped when the cumulative number of detected cells reaches 500,000 events, and the P17 viable cell concentration in the P17 bacterial solution and the NOX viable cell concentration in the NOX bacterial solution are obtained by gate counting analysis; Then, based on the required inoculation concentration, the obtained P17 live cell concentration in the P17 bacterial solution and / or the NOX live cell concentration in the NOX bacterial solution are used to calculate the volume of the P17 bacterial solution and / or NOX bacterial solution required for the subsequent preparation of the standard curve and / or the determination of the AOC concentration in the water sample.

[0022] Preferably, in step (2) and step (3), in the preparation of the standard curve and in the determination of the AOC concentration in the water sample, the inoculation concentration of the P17 bacterial solution and / or the inoculation of the NOX bacterial solution is 1×10 4 cells / mL.

[0023] Preferably, in steps S1 and S2, the culturing is performed in a constant temperature box at 25°C.

[0024] Preferably, in the present invention, all operations for staining using SYBR Green I dye and PI dye independently include: adding 5 μL of SYBR Green I dye and 5 μL of PI dye to 500 μL of culture medium or bacterial liquid to be stained, shaking and mixing in the dark, and incubating at 25°C for 20 min.

[0025] Preferably, in step (2), the carbon acetate standard solutions of different concentrations are prepared by a method comprising the following steps: 1 mL, 2 mL, 3 mL, and 4 mL of the sodium acetate mother solution with a concentration of 2000 μgC / L were taken, and diluted to 40 mL with phosphate buffer to prepare carbonic acid solutions with concentrations of 50 μgC / L, 100 μgC / L, 150 μgC / L, and 200 μgC / L, respectively; At the same time, two 40 mL ultrapure water blank samples were prepared; 100 μL of a 100-fold diluted mineral salt solution was added to each concentration of carbon acetate solution and the two blank water samples, respectively, and the mixture was autoclaved at 121° C. for 20 min to obtain the carbon acetate standard solutions of different concentrations and two control groups; The mineral salt solution is a mixture obtained by dissolving 85.5 mg of KH2PO4, 383.5 mg of NaCl, and 722.0 mg of KNO3 in 500 mL of ultrapure water.

[0026] According to a preferred embodiment, the method of the present invention further comprises: in step (3), the determination of the AOC concentration in the water sample comprises the following steps: SS1. The P17 bacterial solution was first inoculated into the water sample and cultured for 3 days. After sampling, the sample was stained with SYBR Green I dye and PI dye, and the concentration of P17 viable bacteria in the water sample was obtained by flow cytometry. ; SS2. After pasteurizing the water sample incubated with the P17 test bacteria in step SS1, the NOX bacterial solution was inoculated and cultured for 4 days. After sampling, the sample was stained with SYBR Green I dye and PI dye, and the concentration of viable NOX bacteria in the water sample was obtained by flow cytometry, which was recorded as ; SS3. Calculate the AOC concentrations corresponding to P17 and NOX in the water sample according to the following formulas: and : , ; ; in: 、 are the viable bacterial concentrations of P17 and NOX in the control group, respectively.

[0027] Preferably, the blank water sample is ultrapure water.

[0028] Preferably, when determining the AOC concentration in the water sample, three parallel replicates are set for each water sample, and the average value is finally taken as the detection result to ensure the repeatability and stability of the method.

[0029] Further preferably, the method of the present invention further comprises: in step (3), before determining the AOC concentration in the water sample, pre-treating and sterilizing the water sample to eliminate residual chlorine interference, remove suspended particles and microorganisms, and establish sterile culture conditions.

[0030] More preferably, the steps of pre-treating and sterilizing include: First, the free chlorine content in the water sample is determined using a DPD method or a residual chlorine colorimetric reagent, and then an aqueous solution of sodium thiosulfate is added at a molar ratio of Cl2:Na2S2O3 of 1:1.2 for neutralization; Then filter through a 0.45 μm microporous filter membrane, take the filtrate into a sterile glass conical flask, seal the bottle mouth and put it into a high-pressure sterilizer, sterilize it at 121°C for 20 minutes, cool it to 20-28°C, and measure the AOC concentration within 24 hours.

[0031] According to a preferred embodiment, in step (2), Origin software is used to perform linear regression processing on the experimental data to improve the accuracy of curve fitting.

[0032] As mentioned above, the second aspect of the present invention provides the application of the method described in the first aspect to detecting the AOC content in water, where the concentration of AOC in the water is in the range of 20-150 μg / L.

[0033] The present invention is described in detail below by way of examples. Unless otherwise specified, the raw materials used are all common commercially available products.

[0034] Test bacteria: P17: purchased from the ATCC strain library in the United States and stored as freeze-dried powder; NOX: purchased from the ATCC bacterial culture bank in the United States and stored as freeze-dried powder; SYBR Green I dye: SYBR® Green I Nucleic Acid Stain 10000X, purchased from Thermo Fisher Scientific, CAS No. 163795-75-3, Catalog No. S7563; PI dye: Propidium Iodide, purchased from Sigma, CAS No. 25535-16-4, product No. P4170.

[0035] Prepare mineral salt solution: Weigh 85.5 mg of KH2PO4, 383.5 mg of NaCl, and 722.0 mg of KNO3 and dissolve them in 500 mL of ultrapure water to obtain a mineral salt solution.

[0036] Prepare carbonyl acetate standard solutions of different concentrations: 1 mL, 2 mL, 3 mL, and 4 mL of the sodium acetate mother solution with a concentration of 2000 μgC / L were taken, and diluted to 40 mL with phosphate buffer to prepare carbonic acid solutions with concentrations of 50 μgC / L, 100 μgC / L, 150 μgC / L, and 200 μgC / L, respectively; At the same time, two 40 mL ultrapure water blank samples were prepared; 100 μL of 100-fold diluted mineral salt solution was added to each concentration of carbon acetate solution and the two blank water samples, respectively. After high-pressure sterilization at 121°C for 20 min, carbon acetate standard solutions of different concentrations and two control groups were obtained.

[0037] Water sample: water sample from the water supply network.

[0038] Example 1 This example is used to illustrate the method for rapid detection of AOC in water based on flow cytometry provided by the present invention, which is performed according to the following steps: (1) The two test bacteria, P17 and NOX, were revived, acclimated, and cultured in sequence to obtain P17 bacterial solution and NOX bacterial solution, including: The two test bacteria, P17 and NOX, were inoculated into 20 mL of beef extract peptone medium at 25°C for 48 hours and 72 hours, respectively. The two test bacteria, P17 and NOX, were then inoculated into fresh slant medium, cultured at 25°C for 48 hours, and then stored at 4°C. Then, a loopful of each of the resuscitated P17 and NOX strains was inoculated into tap water that had been high-temperature calcined and then autoclaved, and cultured at 25°C for 7 days to complete acclimatization, thereby enhancing the growth adaptability of P17 and NOX in a low-nutrient environment. The high-temperature calcination temperature was 450°C and the time was 5 hours. Then, 100 μL of the low-nutrient acclimated P17 and NOX bacterial solutions were inoculated into 50 mL of a sodium acetate solution with a carbon concentration of 2 mg / L, respectively, and cultured at 25°C for 7 days until the plateau phase, to obtain the P17 bacterial solution and the NOX bacterial solution, which were then stored at 4°C in the dark. Determine the viable cell concentration of the corresponding bacterial species in P17 and NOX bacterial solutions: The P17 bacterial solution and the NOX bacterial solution were sampled separately, stained with SYBR Green I dye and PI dye, and then analyzed on a flow cytometer, wherein the injection speed was set to medium speed, and the injection was stopped when the cumulative number of detected cells reached 500,000 events. The P17 viable cell concentration in the P17 bacterial solution and the NOX viable cell concentration in the NOX bacterial solution were obtained by gate counting analysis; (2) Preparation of standard curve: S1, inoculate the P17 bacterial solution in different concentrations of carbon acetate standard solution, the inoculation concentration is 1×10 4cells / mL, cultured in a constant temperature box at 25°C for 3 days to obtain culture medium I, sampled 500 μL, added 5 μL of SYBR Green I dye and 5 μL of PI dye, incubated at 25°C for 20 min in the dark, and then counted by flow cytometry to obtain the P17 viable bacterial concentration (cells / mL) in the culture medium I, recorded as N P17 ; S2, the culture solution I was pasteurized and then inoculated with the NOX bacterial solution at a concentration of 1×10 4 cells / mL, cultured in a constant temperature box at 25°C for 4 days to obtain culture medium II, sampled 500 μL, added 5 μL of SYBR Green I dye and 5 μL of PI dye, incubated at 37°C for 20 min in the dark, and then counted by flow cytometry to obtain the NOX viable bacteria concentration (cells / mL) in the culture medium II, recorded as N NOX ; S3, respectively performing linear fitting on the P17 viable bacteria concentration and the NOX viable bacteria concentration obtained under the different concentrations of the acetic acid carbon standard solution to obtain respective standard curves; Figure 1 The standard curves of the P17 strain and the NOX strain are shown. To improve the accuracy of curve fitting, the experimental data were processed by linear regression using Origin software; The results showed that the regression coefficients (R²) of the two standard curves were 0.86 and 0.96 respectively; both strains P17 and NOX showed good linear responses in the range of 0-200 μgC / L: The P17 regression equation is: Y=980X+39000; The NOX regression equation is: Y=830X+54000; The yield coefficients of different concentrations of carbon acetate standard solutions were calculated according to the following formula: and : ; Wherein, N0 is the viable cell concentration in the blank control (cells / mL); C P17 、C NOX is the known carbon concentration in the carbon acetate standard solution (μgC / L); Get the yield coefficient and They are 3.42×10 6 cells•(μg C / L) -1 and 1.02×10 7cells•(μg C / L) -1 ; (3) Pre-treat and sterilize the water sample: First, the free chlorine content in the water sample was determined using a residual chlorine colorimetric reagent, and then an aqueous solution of sodium thiosulfate was added at a molar ratio of Cl2:Na2S2O3 of 1:1.2 for neutralization; Then filter through a 0.45 μm microporous membrane, take the filtrate into a sterile glass triangular flask, seal the bottle mouth and place it in an autoclave, sterilize at 121°C for 20 minutes, cool to 25°C and measure the AOC concentration: SS1, inoculate the P17 bacterial solution in the water sample to be tested and culture for 3 days (inoculation concentration is 1×10 4 cells / mL), 500 μL of the sample was added with 5 μL of SYBR Green I dye and 5 μL of PI dye, the mixture was shaken in the dark and incubated at 37°C for 20 min for staining, and the concentration of P17 viable bacteria in the water sample was obtained by flow cytometry and recorded as ; SS2: After the water sample incubated with the P17 test bacteria in step SS1 was pasteurized, the NOX bacterial solution was inoculated and cultured for 4 days (inoculation concentration was 1×10 4 cells / mL), 500 μL of the sample was added with 5 μL of SYBR Green I dye and 5 μL of PI dye, the mixture was shaken in the dark and incubated at 37°C for 20 min for staining, and the concentration of viable NOX bacteria in the water sample was obtained by flow cytometry and recorded as ; SS3. Calculate the AOC concentrations of P17 and NOX in the water sample according to the following formulas: and : , ; ; in: 、 are the average values ​​of the viable bacterial concentrations of P17 and NOX in the two control groups, respectively; According to the required inoculation concentration, the obtained P17 live cell concentration in the P17 bacterial solution and the NOX live cell concentration in the NOX bacterial solution are used to calculate the volume of the P17 bacterial solution or NOX bacterial solution required for preparing a standard curve and determining the AOC concentration in the water sample; When measuring the AOC concentration in the water sample, three parallel replicates were set for each water sample, and the average value was finally taken as the test result. That is, the AOC content in the water sample measured in this embodiment was 67±12 μg C / L.

[0039] Comparative Example 1 This comparative example was carried out using a method similar to that of Example 1, except that in step (1), the low-nutrient acclimation conditions were different, specifically: A loopful of each of the resuscitated P17 and NOX strains was inoculated into water samples from the water supply network and cultured at 25°C for 7 days to complete acclimation. The high-temperature calcination temperature was 450°C for 5 hours. Then, 100 μL of the low-nutrient acclimated P17 and NOX bacterial solutions were inoculated into 50 mL of a sodium acetate solution with a carbon concentration of 2 mg / L, respectively, and cultured at 25°C for 7 days until the plateau phase, to obtain the P17 bacterial solution and the NOX bacterial solution, which were then stored at 4°C in the dark. Finally, the AOC content in the water sample was measured to be 61±24μg C / L.

[0040] Comparative Example 2 This comparative example adopts the method of flow cytometry + pure bacteria inoculation to detect AOC in the previous research of the applicant's research group (Xu Xinyuan, Li Weiying, Zhou Yanyan, et al. Comparison of assimilable organic carbon (AOC) detection methods [J] Water Purification Technology, 2019, 38 (8): 47-52) to detect water samples; Finally, the AOC content in the water sample was measured to be 65±18μg ​​C / L.

[0041] Comparative Example 3 This comparative example was carried out using a method similar to that of Example 1, except that, when preparing the standard curve and determining the AOC concentration in the water sample, the P17 bacterial solution and the NOX bacterial solution were simultaneously inoculated into different concentrations of acetic acid carbon standard solutions or pretreated and sterilized water samples and cultured for 7 days; Finally, the AOC content in the water sample was measured to be 65±20μg C / L.

[0042] The above results show that the method provided by the present invention is significantly superior to the nonlinear discrete trend commonly seen in the traditional P17 / NOX plate count method, ensuring a reliable correlation between strain growth and cell counts under different carbon concentration conditions, and all data points are distributed within the confidence band (e.g. Figure 1 The 95% confidence interval (shaded) demonstrates the excellent reproducibility and anti-interference capabilities of the detection system. The standard curve is a linear equation, allowing AOC concentrations to be directly inferred from cell counts without the need for complex modeling and calculations, making it easy to deploy in routine operations at water plants or laboratories.

[0043] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for rapid detection of AOC in water based on flow cytometry, characterized in that: The following steps are involved: (1) The two test bacteria, P17 and NOX, were subjected to low-nutrient acclimation and culture respectively to obtain P17 bacterial solution and NOX bacterial solution; (2) Preparation of standard curve: S1, first inoculating the P17 bacterial solution into a standard solution of acetic acid with different concentrations, culturing for 3-4 days to obtain a culture solution I, sampling and staining with SYBR Green I dye and PI dye, and then counting by flow cytometry to obtain the concentration of viable P17 bacteria in the culture solution I; S2. The culture fluid I is pasteurized and then inoculated with the NOX bacterial solution. After culturing for 3-4 days, a culture fluid II is obtained. A sample is taken and stained with SYBR Green I dye and PI dye, and then counted using flow cytometry to obtain the concentration of viable NOX bacteria in the culture fluid II. S3. Linearly fit the P17 viable bacteria concentration and the NOX viable bacteria concentration obtained under the different concentrations of the carbon acetate standard solution to obtain their respective standard curves. The slope of the standard curve is taken as the yield coefficient. and ; (3) Determination of AOC concentration in water samples: using the and stated Determination of AOC concentration in water samples.

2. The method according to claim 1, characterized in that In step (1), the low-nutrient acclimation and cultivation operations include: One loopful of each of the resuscitated P17 and NOX strains was inoculated into tap water that had been calcined at high temperature and then autoclaved. The cells were cultured at 25°C for 7 days to complete acclimatization, thereby enhancing the growth adaptability of P17 and NOX in a low-nutrient environment. The calcination temperature was 450°C for 5 hours. Then, 100 μL of the low-nutrient acclimated P17 and NOX bacterial solutions were inoculated into 50 mL of a sodium acetate solution with a carbon concentration of 2 mg / L, and cultured at 25° C. for 7 days until the plateau phase, to obtain the P17 bacterial solution and the NOX bacterial solution.

3. The method according to claim 1 or 2, characterized in that The method further comprises, in step (3), after completing the low-nutrient acclimation and the culturing, and before preparing the standard curve, determining the viable cell concentrations of the corresponding bacterial species in the P17 bacterial solution and the NOX bacterial solution, comprising: sampling the P17 bacterial solution and the NOX bacterial solution respectively, staining them with SYBRGreen I dye and PI dye, and then performing on-machine analysis using a flow cytometer, wherein the injection speed is set to medium speed, and the injection is stopped when the cumulative number of detected cells reaches 500,000 events, and the P17 viable cell concentration in the P17 bacterial solution and the NOX viable cell concentration in the NOX bacterial solution are obtained by gate counting analysis; Then, based on the required inoculation concentration, the obtained P17 live cell concentration in the P17 bacterial solution and / or the NOX live cell concentration in the NOX bacterial solution are used to calculate the volume of the P17 bacterial solution and / or NOX bacterial solution required for the subsequent preparation of the standard curve and / or the determination of the AOC concentration in the water sample.

4. The method according to claim 1 or 2, characterized in that In step (2) and step (3), in the preparation of the standard curve and in the determination of the AOC concentration in the water sample, the inoculation concentration of the P17 bacterial solution and / or the inoculation of the NOX bacterial solution is 1×10 4 cells / mL.

5. The method according to claim 1 or 2, characterized in that in, All staining operations using SYBR Green I dye and PI dye independently include: adding 5 μL of SYBR Green I dye and 5 μL of PI dye to 500 μL of culture medium or bacterial liquid to be stained, shaking and mixing in the dark, and incubating at 25°C for 20 minutes.

6. The method according to claim 1 or 2, characterized in that In step (2), the carbon acetate standard solutions of different concentrations are prepared by a method comprising the following steps: Take 1 mL, 2 mL, 3 mL, and 4 mL of the sodium acetate mother solution with a concentration of 2000 μgC / L, dilute it to 40 mL with phosphate buffer, and prepare carbon acetate standard solutions with concentrations of 50 μgC / L, 100 μgC / L, 150 μgC / L, and 200 μgC / L, respectively; At the same time, two 40 mL ultrapure water blank samples were prepared; 100 μL of a 100-fold diluted mineral salt solution was added to each concentration of carbon acetate solution and the two blank water samples, respectively, and the mixture was autoclaved at 121° C. for 20 min to obtain the carbon acetate standard solutions of different concentrations and two control groups; The mineral salt solution is a mixture obtained by dissolving 85.5 mg of KH2PO4, 383.5 mg of NaCl, and 722.0 mg of KNO3 in 500 mL of ultrapure water.

7. The method according to claim 1 or 2, characterized in that The method further includes: in step (3), the determination of the AOC concentration in the water sample includes the following steps: SS1. The P17 bacterial solution was first inoculated into the water sample and cultured for 3 days. After sampling, the sample was stained with SYBR Green I dye and PI dye, and the concentration of P17 viable bacteria in the water sample was obtained by flow cytometry. ; SS2. The water sample incubated with the P17 test bacteria was pasteurized and then inoculated with the NOX bacterial solution for 4 days. The sample was then stained with SYBR Green I dye and PI dye, and the concentration of viable NOX bacteria in the water sample was obtained by flow cytometry and recorded as ; SS3. Calculate the AOC concentrations corresponding to P17 and NOX in the water sample according to the following formulas: and : ， ； ； in: 、 are the viable bacterial concentrations of P17 and NOX in the control group, respectively.

8. The method according to claim 1 or 2, characterized in that The method further includes: in step (3), before determining the AOC concentration in the water sample, pre-treating and sterilizing the water sample to eliminate residual chlorine interference, remove suspended particles and microorganisms, and establish sterile culture conditions.

9. The method according to claim 8, characterized in that The steps of pretreatment and sterilization include: First, the free chlorine content in the water sample is determined using a DPD method or a residual chlorine colorimetric reagent, and then an aqueous solution of sodium thiosulfate is added at a molar ratio of Cl2:Na2S2O3 of 1:1.2 for neutralization; Then filter through a 0.45 μm microporous filter membrane, take the filtrate into a sterile glass conical flask, seal the bottle mouth and put it into a high-pressure sterilizer, sterilize it at 121°C for 20 minutes, cool it to 20-28°C, and measure the AOC concentration within 24 hours.

10. Application of the method according to any one of claims 1 to 9 in detecting the AOC content in water, characterized in that: In the water body, the concentration of AOC ranged from 20 to 150 μg / L.

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