Method for quantifying AHLs in anaerobic fermentation product and activated sludge based on LC-MSMS (Liquid Chromatography-Mass Spectrometry-Mass Spectrometry)

Abstract

The invention relates to the technical field of analysis, and discloses a method for quantifying AHLs in anaerobic leavening and activated sludge based on LC-MSMS (Liquid Chromatography-Mass Spectrometry-Mass Spectrometry). The method comprises the following steps: sequentially extracting, purifying and extracting a to-be-detected object to obtain a to-be-detected solution; and then preparing a standard substance: setting reasonable chromatographic conditions and mass spectrum conditions, and finally calculating the content of AHLs. Compared with an existing method, the method has the advantages of being more thorough in extraction, higher in sensitivity, lower in detection limit, high in recovery rate, easy to operate and the like, and is suitable for two matrixes of activated sludge and anaerobic fermentation products. A new method and thought are provided for studying the quorum sensing effect of AHLs of activated sludge and anaerobic fermentation products, and a brand new quality control means can be provided for sewage treatment plants and biogas projects.

Description

Technical Field

[0001] The invention relates to the field of analytical technology, and in particular to a method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS. Background Art

[0002] AHLs (acyl homoserine lactones) are a class of signaling molecules that promote intercellular communication of Gram-negative bacteria. They play an important role in regulating bacterial behavior and metabolic processes. In anaerobic digestion systems and activated sludge systems, the presence of AHLs has a significant effect on sludge granulation, biofilm formation, and methanogenic activity, and can improve the overall performance of the system by regulating the behavior and metabolic processes of microorganisms.

[0003] As a model of high-density microbial communities, activated sludge has extensive intercellular communication. At present, the research on the regulatory role of quorum sensing on its system mainly focuses on biofilm formation and granular sludge formation. Related studies have shown that quorum sensing has a significant effect on the formation of biofilms in different wastewater biological treatment reactors, such as sequencing batch biofilm reactors, Anammox biofilm reactors, membrane bioreactors, etc. In the process of granular sludge formation, as the sludge particle size increases, the content of signal molecules also increases; the decrease in the concentration of quorum sensing signal molecules leads to the disintegration of particles or smaller particle size. In addition, studies on the exogenous addition of quorum sensing signal molecules in the system have shown that the addition of sensing signal molecules is conducive to the formation of granular sludge. For example, 3OC6-HSL significantly enhances the adhesion and growth ability of bacteria, while the addition of 3OC-HSL and C6-HSL helps to increase the biomass growth rate, the activity of nitrifying bacteria and the production of extracellular proteins, promoting the formation of nitrifying granular sludge; the exogenous addition of 40μL AHLs significantly increased the PS (14-36%) and PN (7-16%) contents in flocculent sludge, and the adhesion of microorganisms was significantly enhanced.

[0004] The anaerobic digestion process can be divided into four stages: hydrolysis, acidification, acetogenesis, and methanogenesis, each of which relies on the synergy and symbiosis of different functional microorganisms. Some studies have found that AHLs-mediated quorum sensing can balance various microbial populations during anaerobic digestion, and high methane production is associated with many AHLs genes. In a study on the accelerated healing of anaerobic granular sludge after temperature shock, Lv et al. found that AHLs increased the protein level in tightly bound EPS and the abundance of hydrophobic functional groups in EPS. It is worth noting that the application of 10 μM AHLs significantly enhanced the enrichment of eutrophic methanogens. In addition, the researchers also studied the regulation of AHLs by microbial density to enhance the synergy of acetobacteria and methanogens in anaerobic digestion. They found that interspecific regulation of syntrophic propionate oxidizing bacteria and hydrogenotrophic methanogens was mediated by 3-oxo-C6-HSL and C10-HSL, respectively. The type of AHLs affects the domesticated anaerobic microbial community.

[0005] In summary, AHLs may affect the interaction, metabolic activity and product synthesis of microbial communities during anaerobic digestion and activated sludge treatment. Therefore, rapid and accurate detection of the composition, content and change law of AHLs in anaerobic fermentation products and activated sludge is an important basis for studying the behavior and mechanism of quorum sensing. The current methods for detecting AHLs in activated sludge have major defects: for example, Luo Meng detected AHLs in activated sludge. In the pretreatment of this method, high-speed centrifugation was used to separate water and matrix, and the supernatant was taken for detection. Centrifugation allowed granular sludge and flocculent sludge to sink to the bottom, ignoring the AHLs adsorbed or bound to EPS, making them undetectable, resulting in low AHLs values ​​in the sample. Sun improved the pretreatment of the above centrifugation separation: high-speed centrifugation of activated sludge, collection of supernatant, and redissolution of the bottom sludge to extract the extracellular polymers of the sludge. The extraction method was to heat in a water bath at 80°C for 30 minutes, and then centrifuge to obtain the supernatant. At present, relevant AHLs detection studies have shown that AHLs detection samples should be frozen and stored in time and analyzed in a short time. Our laboratory study showed that the AHLs standard working solution degraded and its content decreased after being stored at 4°C for 24 hours. If the temperature is too high when extracting extracellular polymers, AHLs may be lost to a certain extent. In addition, there are few reports on the detection of AHLs in anaerobic fermentation products. The matrix of anaerobic fermentation products is more complex than that of activated sludge, and the detection is more difficult. The relevant research used the same method as activated sludge. Summary of the invention

[0006] In view of this, the present invention provides a method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS to solve the defects of existing detection methods, namely, using centrifugation to obtain supernatant for detection or high-temperature extraction of extracellular polymers of activated sludge and anaerobic fermentation products, which leads to low AHLs values ​​of samples and distorted proportions.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for quantitatively analyzing AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS comprises the following steps:

[0009] (1) Sample processing: The sample to be tested is sequentially extracted, purified and extracted to obtain a test solution;

[0010] (a) Extraction: vortex the analyte, acetonitrile, homogenizer and ethylenediaminetetraacetic acid, and then sonicate to obtain a mixed solution; freeze the mixed solution, vortex and vortex with anhydrous magnesium sulfate and disodium citrate, and then centrifuge to complete the extraction and obtain a supernatant; the amount ratio of the analyte, acetonitrile, ethylenediaminetetraacetic acid, anhydrous magnesium sulfate and disodium citrate is 2-10 g: 8-15 mL: 0.3-0.7 g: 4-6 g: 1-4 g;

[0011] (b) Purification: taking the supernatant of the solution obtained in step (a), mixing it with a dehydrating agent (anhydrous magnesium sulfate) and two adsorbents, and then centrifuging, taking the supernatant, drying it with nitrogen, and redissolving it for the first time, to obtain a purified solution;

[0012] The amount ratio of the supernatant, dehydrating agent, adsorbent 1 and adsorbent 2 in step (b) is 5-10 mL: 50-100 mg: 20-50 mg: 20-50 mg;

[0013] (c) secondary purification: subjecting the purified solution to solid phase extraction, spin drying and secondary re-dissolution in sequence to obtain a test solution;

[0014] The solid phase extraction is to install a polyethylene-vinyl solid phase extraction column in a solid phase extraction instrument, use 5-10 mL of methanol to activate the small column, balance with 2-10 mL of ultrapure water, wash with 15-30 mmol / L sodium acetate solution, load the purified solution obtained in step (b), and finally elute with 5 mL×6 methanol-isopropanol mixed solution (methanol-isopropanol volume ratio 5:1);

[0015] (2) Preparation of standard substances: C4-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL, C14-HSL, 3-oxo-C14-HSL, 3-OH-C6-HSL, and 3-OH-C10-HSL were prepared into 50 mg / L mixed standard stock solutions of 9 AHLs; the mixed standard concentration for establishing the mass spectrometry method was 50 μg / L, and the standard curve concentrations were 0.001 to 300 μg / L;

[0016] The solvent used in the preparation of the AHLs mixed standard stock solution is a blank matrix solution obtained by pre-treatment of anaerobic fermentation products or aerobic granular sludge, and the blank matrix is ​​verified to contain no analyte through multiple verifications;

[0017] (3) Chromatographic conditions and mass spectrometry conditions: The mass spectrometry conditions are: electrospray ion source, curtain gas 30-50 psi, collision gas Medium, ionization voltage 4400-4600 V, temperature 430-450° C., spray gas 50-55 psi, auxiliary heating gas 55-60 psi;

[0018] The chromatographic conditions are as follows: mobile phase A: 0.05% formic acid aqueous solution containing 0.1-0.3 mM ammonium acetate; mobile phase B: 0.05% formic acid methanol solution containing 0.1-0.3 mM ammonium acetate; the chromatographic column is a biphenyl column; the column temperature is 30-50° C.; the injection volume is 5-20 μL;

[0019] (4) Calculate the AHLs content.

[0020] Preferably, in step (a), the mixing time is 1 to 2 minutes; the ultrasonic power is 150 to 250 W, and the ultrasonic time is 15 to 25 minutes; the centrifugal speed is 4000 to 5000 r / min, and the centrifugal time is 3 to 10 minutes.

[0021] Preferably, in step (b), adsorbent 1 is a capped C18 adsorbent, and adsorbent 2 is a primary or secondary amine adsorbent; the mixing time is 1 to 2 min; the centrifugal speed is 4000 to 5000 r / min, and the centrifugal time is 3 to 10 min; the drying is carried out by passing nitrogen in a 30 to 40° C. water bath; the reagent used for the first re-dissolution is a sodium acetate solution, and the concentration of the sodium acetate solution is 15 to 30 mmol / L.

[0022] Preferably, the parallel vacuum evaporator spin drying temperature in step (c) is 30-40°C; the reagent used for the second re-dissolution is a methanol solution, and the mass fraction of the methanol solution is 20-40%.

[0023] In the present invention, the preparation of the blank matrix solution includes the following steps: collecting 30 kg of anaerobic fermentation products and aerobic granular sludge, baking at 60°C for 10 days, and grinding with a grinder until the sample can completely pass through a 30-mesh sieve. Continue baking at 105°C for 36 hours, and finally grind with an agate ball mill at 300r / min for 30 minutes until it can all pass through a 100-mesh sieve, collect in a sealed bag, age for more than 1 year, and seal for storage. The blank matrix is ​​treated by the sample treatment step to obtain a blank matrix solution, and the blank matrix solution is analyzed by LC-MSMS. The blank matrix solution does not detect the AHLs target, and can be used as a matrix blank solution for detecting AHLs.

[0024] Calculation results: AHLs quantification was calculated according to the following formula:

[0025] Wherein, ω is the residual amount of the analyte in the sample (ng / kg); ρ1 is the mass concentration of the analyte in the matrix-matched standard working solution (ng / L); A is the mass chromatogram peak area of ​​the analyte in the sample solution; As is the mass chromatogram peak area of ​​the analyte in the matrix-matched standard working solution; V refers to the volume of the extract (mL); and m refers to the mass of the sample (g).

[0026] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS. The method has the characteristics of high sensitivity, high recovery rate, simple operation, etc., and solves the problems of other methods such as failure to extract extracellular polymers or high temperature heating. The method is applicable to two matrices, activated sludge and anaerobic fermentation products. It provides new methods and ideas for studying the group sensing effect of AHLs in activated sludge and anaerobic fermentation products, and can also provide quality control means for sewage treatment plants and biogas fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 It is the extracted ion current chromatogram of the 9 AHLs described in Example 1; Note: the numbers in the figure represent the XIC peaks of the following substances: 1, C4-HSL, 2, 3-OH-C6-HSL, 3, C6-HSL, 4, 3-OH-C10-HSL, 5, C8-HSL, 6, C10-HSL, 7, C12-HSL, 8, 3-oxo-C14-HSL, 9, C14-HSL.

[0030] Figure 2 This is the standard curve of the 9 AHLs described in Example 1.

[0031] Figure 3 This is the secondary mass spectra of the 9 AHLs described in Example 1.

[0032] Figure 4 This is the extracted ion current chromatogram of the blank matrix solution of the anaerobic fermentation product described in Example 2;

[0033] Figure 5 This is the extracted ion current chromatogram of the activated sludge blank matrix solution described in Example 2. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0036] The key instruments and reagents used in the following examples are: ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (U.S. AB SCIEX QTRAP 6500); methanol, formic acid, ammonium formate (mass spectrometry grade, Thermo Fisher Scientific, USA); chromatographic column (Kinetex Biphenyl, 2.1×100 mm×1.7 μm, Phenomet Corporation, USA); distilled water (Watsons Company); standard substances: C4-HSL, 3-OH-C10-HSL, 3-oxo-C14-HSL, 3-OH-C6-HSL, C8-HSL, C6-HSL, C10-HSL, C14-HSL, C12-HSL (Merck, USA); polyethylene-divinylbenzene solid phase extraction column (China Agilent, PE2006-2); tailed C18 adsorbent (Phonometa Corporation, USA, 04G-4348); primary and secondary amine adsorbent (Phonometa Corporation, USA, 04G-4610); homogeneous proton (Agilent, USA, 5982-9313).

[0037] The preparation of the blank matrix solution used in the following examples includes the following steps: 30 kg of anaerobic fermentation products and activated sludge are collected, baked at 60° C. for 10 days, and ground with a grinder until the sample can completely pass through a 30-mesh sieve. Continue baking at 105° C. for 36 hours, and finally grind with an agate ball mill at 300 r / min for 30 minutes until it can completely pass through a 100-mesh sieve, collect in a sealed bag, age for more than 1 year, and seal for storage. The blank matrix is ​​treated by the sample treatment step to obtain a blank matrix solution, and the blank matrix solution is analyzed by LC-MSMS.

[0038] Example 1

[0039] This example measures and verifies 9 AHLs standard solutions:

[0040] (1) The standard stock solution was diluted to a 10 μg / L working standard solution, and then the working standard solution was diluted with 20% methanol to a concentration of 0.02 μg / L, 0.05 μg / L, 0.1 μg / L, 0.5 μg / L, 1 μg / L, and 5 μg / L, and detected using a high performance liquid chromatography-triple quadrupole mass spectrometer.

[0041] (2) Mass spectrometry conditions: Electrospray ion source, curtain gas 40 psi, collision gas Medium, ionization voltage 4500 V, temperature 450°C, spray gas 55 psi, auxiliary heating gas 60 psi. The mass spectrometry parameters and ion parameters of the nine HSLs are shown in Table 1. Chromatographic conditions: Mobile phase A: 0.05% formic acid-water (containing 0.2 mM ammonium acetate); Mobile phase B: 0.05% formic acid-methanol (containing 0.2 mM ammonium acetate); Column temperature: 40°C; Injection volume: 5 μL. The gradient elution program is shown in Table 2.

[0042] Table 1 Mass spectrometry parameters and ion parameters of 9 AHLs

[0043]

[0044]

[0045] Table 2 Gradient elution program

[0046] Time (min) Flow rate (mL / min) A(％) B(％) 0 0.25 70 30 3 0.25 10 90 8 0.25 10 90 8.1 0.25 70 30 10 0.25 70 30

[0047] (3) Through detection and analysis, the nine AHLs were completely separated, without the appearance of tailing peaks, front peaks, inverted peaks, broad peaks, and no baseline noise or drift.

[0048] The extracted ion chromatograms of 9 AHLs are shown in Figure 1 The standard curves of the 9 AHLs are shown in Figure 2 The secondary mass spectra of the nine AHLs are shown in Figure 3 The linear relationship of the 9 AHLs is shown in Table 3. The results show that the standard curve concentration has a good linear relationship in the range of 0.02-5 μg / L, R 2 The above data show that the method of this embodiment has the advantages of accuracy, good linearity, high sensitivity and short detection time.

[0049] Table 3 Linear relationships of 9 AHLs

[0050]

[0051]

[0052] Example 2

[0053] This example determined the detection limits and quantification limits of 9 AHLs in activated sludge and anaerobic fermentation products:

[0054] (1) Preparation of blank matrix solution: Collect 30 kg of anaerobic fermentation products and activated sludge, bake at 60°C for 10 days, and grind with a mortar until the sample can completely pass through a 30-mesh sieve. Continue baking at 105°C for 36 hours, and finally grind with an agate ball mill at 300 r / min for 30 minutes until it can completely pass through a 100-mesh sieve. Collect in a sealed bag, age for more than 1 year, and seal for storage. Treat the blank matrix through the sample treatment steps to obtain a blank matrix solution, and analyze the blank matrix solution by LC-MSMS. The ion flow chart of the blank matrix solution of anaerobic fermentation products is shown in Figure 4 , the ion current diagram of the activated sludge blank matrix solution is shown in Figure 5 .

[0055] (2) Preparation of standard curve: The standard stock solution was diluted with methanol to a 5 μg / L working standard solution, and then the working standard solution was diluted with blank matrix solution to a concentration of 0.5 ng / L, 2 ng / L, 5 ng / L, 20 ng / L, and 50 ng / L, and detected by LC-MSMS.

[0056] (3) The matrix standard curve has a good linear relationship in the range of 0.5 to 50 ng / L. 2 The value was above 0.998, and the concentrations of 9 AHLs matrix standards (0.5-50 ng / L) were analyzed at the lowest concentration that could be detected, and the signal-to-noise ratio (S / N) was calculated by the software. 3 times the signal-to-noise ratio was used as the detection limit of the method, and 10 times the signal-to-noise ratio was used as the quantification limit of the method. The calculation results are shown in Table 4.

[0057] Table 4 Detection limits and quantification limits of 9 AHLs in activated sludge and anaerobic fermentation products

[0058]

[0059] The results showed that the method had high sensitivity, and the detection limit and quantification limit were less than 5ng / kg and 17ng / kg, respectively.

[0060] Example 3

[0061] This example measured the sample spike recovery and precision of 9 AHLs in activated sludge and anaerobic fermentation products:

[0062] (1) Weigh 10g of activated sludge or anaerobic fermentation product into a 50mL centrifuge tube, add 0.1mL of 0.5μg / L and 5μg / L mixed standard solutions of 9 kinds of AHLs, and prepare a blank matrix solution at the same time. Add 2 ceramic homogenizers, add 10mL of acetonitrile and 0.5g of EDTA to mix, vortex for 1min using a vortex shaker, then ultrasonicate in a water bath at 200W for 20min, vortex for 1min, take out and add 5g of anhydrous magnesium sulfate and 2g of sodium acetate, vortex for 1min, and centrifuge at 5000r / min for 5min. Pipette all the supernatant (about 8mL) and transfer it to a 15mL centrifuge tube, add 80mg of magnesium sulfate, 20mg of tailed C18 adsorbent, and 20mg of primary and secondary amine, vortex for 1min, and centrifuge at 5000r / min for 5min. Accurately transfer 6mL of supernatant and blow it to near dryness with nitrogen at 30℃. Add 5mL of 25mM sodium acetate solution, ultrasonicate for 15min, vortex for 1min to dissolve and load on the column. Connect the polyethylene-vinyl acetate solid phase extraction column to the top of the solid phase extractor, and use negative pressure to control the flow rate at 2S / drop. First, use 5mL of methanol to activate the column, then balance the column with 10mL of ultrapure water, dry it under negative pressure for 2min, add the test solution for loading, and the loading speed is 3s / drop. Then wash the column with 5mL of 25mM sodium acetate, dry it under negative pressure for 2min, and finally elute with 5mL×6 times of a mixed solution of methanol and isopropanol (the volume ratio of methanol and isopropanol is 5:1). The eluate is dried on a parallel vacuum evaporator at 35℃, re-dissolved with 1mL of 30% methanol by vortexing for 1min, and loaded for analysis.

[0063] (2) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, dilute it step by step to 1, 5, 20, 50, and 200 ng / L using blank matrix solution, and use LC-MSMS for sample injection and analysis.

[0064] In the present embodiment, the extraction effects of the extractants acetonitrile and ethyl acetate were compared during the sample extraction process. The results showed that acetonitrile had a high recovery rate and good purification effect, while ethyl acetate had a poor purification effect, which enhanced the matrix effect. The purifier C18 and primary and secondary amines exceeding 50 mg will affect the recovery rate, and 20 mg is the minimum addition amount that does not affect the recovery rate. Different solid phase extraction columns (PEP-2, C8 and weak cation exchange columns) were studied, and the results showed that the PEP-2 recovery rate was higher. The effects of different elution solvents on the recovery rate (the ratio of acetonitrile, methanol, isopropanol, acetone and their mixed reagents) were studied, and the results showed that methanol elution was more reasonable. Adding a small amount of isopropanol and methanol to the eluent can improve C 14 The recovery rate of -HCL. The volume of the eluent was studied, and when the eluent was 30mL, it could be completely eluted, and the reduction of the volume could not completely elute C10-HSL and C14-HSL.

[0065] The recoveries and precisions of the nine AHLs in activated sludge and anaerobic fermentation products are shown in Table 5. The recoveries of the nine AHLs in activated sludge were 77.64-93.72%, and those in anaerobic fermentation products were 72.48-114.40%.

[0066] Table 5 Recovery and precision of 9 AHLs in activated sludge and anaerobic fermentation products

[0067]

[0068]

[0069] Example 4

[0070] In this example, AHLs of five activated sludges and five anaerobic fermentation products were measured, which were from actual production and laboratory simulation systems, respectively. The scope of application of the method can be verified through the detection.

[0071] (1) Sample information:

[0072] Activated sludge A: The influent of this SBR reactor is synthetic wastewater that simulates biogas slurry. The volume ratio of activated sludge to influent is 1:1, and the influent and effluent are 2L per day. The operating parameters of SBR are: hydraulic retention time is 48h, temperature is 28℃, and one cycle is run every day. Each cycle (24h) is divided into: influent (10min) - idle (230min) - aeration (960min) - sedimentation (230min) - effluent (10min);

[0073] Activated sludge B: synthetic wastewater with propionic acid as the only carbon source was used as influent, with a concentration of 10 g COD L-1, and anaerobic digestion sludge of pig manure was used as inoculation sludge. A semi-continuous test was used, with an organic load of 2.5 g COD L-1d-1, a hydraulic retention time of 4 days, and a test temperature of 35°C;

[0074] Activated sludge C: The inoculated activated sludge was taken from a sewage treatment plant in Chongzhou. The operating parameters of the SBR were as follows: hydraulic retention time was 48 h, and the operating cycle (24 h) was feeding (10 min) - idling (110 min) - aeration (360 min) - sedimentation (360 min) - aeration (360 min) - sedimentation (230 min) - slag discharge (10 min);

[0075] Activated sludge D: The initial aerobic activated sludge was 2.0L, and the amount of pig farm biogas added was 2.0L; the sludge inoculation amount was 12g / L, and the hydraulic retention period was 2d. The dissolved oxygen concentration was 4.0mg / L;

[0076] Activated sludge E: SBR was inoculated with 1L activated sludge, and the ratio of influent volume to activated sludge volume was 1:1; sodium bicarbonate was used to adjust the alkalinity of synthetic wastewater, and ammonium sulfate was added to adjust NH4 + -N to about 300mg / L, and the COD concentration was maintained at 480-642mg / L by adding organic matter. The operating parameters of SBR are: feeding time 10min, standing time 110min, aeration time 480min, sedimentation time 110min, discharge time 10min, HRT 24h, aeration rate 0.8L / min.

[0077] Anaerobic fermentation product A: corn straw and cow dung were mixed in a ratio of 1:3 (w / w) as anaerobic digestion raw materials and anaerobic digestion was carried out at 30°C;

[0078] Anaerobic fermentation product B: kitchen waste and inoculum sludge were added in a ratio of 1:2, and intermittent anaerobic digestion was carried out at mesophilic temperature (37 ± 1 °C) in a serum bottle with a working volume of 400 mL, and the headspace was filled with N2 for 3 to 5 min;

[0079] Anaerobic fermentation C: A series of batch anaerobic digestion experiments were conducted in 160 mL serum bottles with a working volume of 100 mL, adding 50 mL of inoculum sludge, glucose and nutrient stock solutions to a final concentration of 2 g / L, respectively;

[0080] Anaerobic fermentation product D: The return sludge collected from the secondary sedimentation tank of a sewage treatment plant in Guangzhou, China was left to stand at 4°C for 24 hours, and the supernatant was poured to obtain concentrated sludge. The inoculum sludge was obtained from the anaerobic digester of a treatment plant in Zhongshan, China. The operating temperature of the anaerobic digester was 36-38°C, and the sludge retention time was 20-25 days.

[0081] Anaerobic fermentation product E: Cow dung was taken from a cattle farm. Chicken dung was diluted with water to a TS mass concentration of about 15%, and was sieved with a 2mm sieve to remove feathers, sand and other debris before use. The hydraulic retention period of anaerobic digestion was 20 days (i.e., 500g of material was fed and discharged every day, once a day), the total solid mass concentration of the sludge was 8%, and the digestion temperature was 40°C.

[0082] (2) Sample testing:

[0083] Weigh 2g of activated sludge or anaerobic fermentation product into a 50mL centrifuge tube, and make a blank matrix solution at the same time. Add 2 ceramic homogenizers, add 10mL acetonitrile, 0.3g EDTA and mix well, use a vortex shaker to vortex for 1min, then ultrasonicate in a 150w water bath for 25min, vortex for 1min, take out and add 4g anhydrous magnesium sulfate, 1.5g disodium citrate vortex for 1min, and centrifuge at 5000r / min for 5min. Absorb all the supernatant (about 8mL) and transfer it to a 15mL centrifuge tube, add 50mg magnesium sulfate, 20mg tailed C18 adsorbent, 20mg primary secondary amine vortex for 1min, and centrifuge at 5000r / min for 5min. Accurately transfer 6mL of supernatant to 30℃ nitrogen and blow it to near dryness. Add 5mL 20mM sodium acetate solution, ultrasonicate for 15min, vortex for 1min to dissolve and wait for column loading. Connect the polyethylene-vinyl acetate solid phase extraction column to the top of the solid phase extractor, and use negative pressure to control the flow rate at 2s / drop. First, use 5mL of methanol to activate the column, then balance the column with 10mL of ultrapure water, dry it under negative pressure for 2min, add the test solution to load, the loading speed is 3s / drop, then wash the column with 5mL of 20mM sodium acetate, dry it under negative pressure for 2min, and finally elute with 5mL×4 methanol-isopropanol mixed solution (methanol-isopropanol volume ratio 5:1). The eluate is dried on a 30℃ parallel vacuum evaporator, re-dissolved with 1mL of 20% methanol by vortexing for 1min, and loaded for analysis.

[0084] (3) Preparation of standard curve: Pipette an appropriate amount of AHLs mixed standard and dilute it step by step to 1, 5, 20, 50, 100, and 200 ng / L using blank matrix solution. Then, analyze it using LC-MSMS.

[0085] (4) The mass spectrometry data were quantitatively analyzed using MultiQuant software. The contents of AHSL in different activated sludge and anaerobic fermentation samples are shown in Table 6.

[0086] Table 6 Contents of AHLs in different activated sludge and anaerobic fermentation samples

[0087]

[0088] Example 5

[0089] This example is to determine 9 AHLs in different types of anaerobic fermentation products and activated sludge. The experimental parameter range is different from that of Example 4, and the test results are similar to those of Example 4:

[0090] (1) Sample information: Same as Example 4 (1)

[0091] (2) Sample testing:

[0092] Weigh 10g of activated sludge or anaerobic fermentation product into a 50mL centrifuge tube, and make a blank matrix solution at the same time. Add 2 ceramic homogenizers, add 15mL acetonitrile, 0.7g EDTA and mix well, use a vortex shaker to vortex for 2min, then ultrasonicate in a 250w water bath for 15min, vortex for 2min, take out and add 6g anhydrous magnesium sulfate, 4g disodium citrate, vortex for 1min, and centrifuge at 4000r / min for 10min. Transfer 10mL of supernatant to a 15mL centrifuge tube, add 100mg magnesium sulfate, 50mg tailed C18 adsorbent, 50mg primary secondary amine, vortex for 1min, and centrifuge at 4000r / min for 10min. Accurately transfer 8mL of supernatant to 40℃ nitrogen and blow until almost dry. Add 5mL 30mM sodium acetate solution, ultrasonicate for 15min, vortex for 1min to dissolve and wait for column loading. Connect the polyethylene-vinyl acetate solid phase extraction column to the top of the solid phase extractor, and use negative pressure to control the flow rate at 2s / drop. First, use 10mL of methanol to activate the column, then balance the column with 10mL of ultrapure water, dry it under negative pressure for 2min, add the test solution to load, and the loading speed is 3s / drop. Then wash the column with 5mL of 30mM sodium acetate, dry it under negative pressure for 2min, and finally elute with 5mL×6 methanol-isopropanol mixed solution (methanol-isopropanol volume ratio 5:1). The eluate is dried on a parallel vacuum evaporator at 40℃, re-dissolved with 1mL of 20% methanol by vortexing for 1min, and loaded for analysis.

[0093] (3) Preparation of standard curve: Pipette an appropriate amount of AHLs mixed standard and dilute it step by step to 1, 5, 20, 50, 100, and 200 ng / L using blank matrix solution. Then, analyze it using LC-MSMS.

[0094] (4) The mass spectrometry data were quantitatively analyzed using MultiQuant software. The contents of AHSL in different activated sludge and anaerobic fermentation samples are shown in Table 7.

[0095] Table 7 Contents of AHLs in different activated sludge and anaerobic fermentation samples

[0096]

[0097]

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for quantitative determination of AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS, characterized in that: The steps include: (1) Sample processing: The sample to be tested is sequentially extracted, purified and extracted to obtain a test solution; (a) Extraction: vortex the analyte, acetonitrile, homogenizer and ethylenediaminetetraacetic acid, and then sonicate to obtain a mixed solution; freeze the mixed solution, vortex and vortex with anhydrous magnesium sulfate and disodium citrate, and then centrifuge to complete the extraction and obtain a supernatant; the amount ratio of the analyte, acetonitrile, ethylenediaminetetraacetic acid, anhydrous magnesium sulfate and disodium citrate is 2-10 g: 8-15 mL: 0.3-0.7 g: 4-6 g: 1-4 g; (b) Purification: taking the supernatant of the solution obtained in step (a), mixing it with a dehydrating agent and two adsorbents, and then centrifuging, taking the supernatant, drying it with nitrogen, and redissolving it for the first time, to obtain a purified solution; The amount ratio of the supernatant, dehydrating agent, adsorbent 1 and adsorbent 2 in step (b) is 5-10 mL: 50-100 mg: 20-50 mg: 20-50 mg; (c) secondary purification: subjecting the purified solution to solid phase extraction, spin drying and secondary re-dissolution in sequence to obtain a test solution; The solid phase extraction is to install a polyethylene-vinyl solid phase extraction column in a solid phase extraction instrument, use 5-10 mL of methanol to activate the small column, balance with 2-10 mL of ultrapure water, wash with 15-30 mmol / L sodium acetate solution, load the purified solution obtained in step (b), and finally elute with 5 mL×6 methanol-isopropanol mixed solution (methanol-isopropanol volume ratio 5:1); (2) Preparation of standard substances: C4-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL, C14-HSL, 3-oxo-C14-HSL, 3-OH-C6-HSL, and 3-OH-C10-HSL were prepared into 50 mg / L mixed standard stock solutions of 9 AHLs; the mixed standard concentration for establishing the mass spectrometry method was 50 μg / L, and the standard curve concentrations were 0.001 to 300 μg / L; The solvent used in the preparation of the AHLs mixed standard stock solution is a blank matrix solution obtained by pre-treatment of anaerobic fermentation products or aerobic granular sludge, and the blank matrix is ​​verified to contain no analyte through multiple verifications; (3) Chromatographic conditions and mass spectrometry conditions: The mass spectrometry conditions are: electrospray ion source, curtain gas 30-50 psi, collision gas Medium, ionization voltage 4400-4600 V, temperature 430-450° C., spray gas 50-55 psi, auxiliary heating gas 55-60 psi; The chromatographic conditions are as follows: mobile phase A: 0.05% formic acid aqueous solution containing 0.1-0.3 mM ammonium acetate; mobile phase B: 0.05% formic acid methanol solution containing 0.1-0.3 mM ammonium acetate; the chromatographic column is a biphenyl column; the column temperature is 30-50° C.; the injection volume is 5-20 μL; (4) Calculate the AHLs content.

2. The method for quantitatively determining AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS according to claim 1, characterized in that: In the step (a), the mixing time is 1 to 2 minutes; the ultrasonic power is 150 to 250 W, and the ultrasonic time is 15 to 25 minutes; the centrifugal speed is 4000 to 5000 r / min, and the centrifugal time is 3 to 10 minutes.

3. The method for quantitative determination of AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS according to claim 1, characterized in that: In the step (b), adsorbent 1 is a capped C18 adsorbent, and adsorbent 2 is a primary or secondary amine adsorbent; the mixing time is 1 to 2 minutes; the centrifugal speed is 4000 to 5000 r / min, and the centrifugal time is 3 to 10 minutes; the drying is carried out by passing nitrogen in a 30 to 40° C. water bath; the reagent used for the first re-dissolution is a sodium acetate solution, and the concentration of the sodium acetate solution is 15 to 30 mmol / L.

4. The method for quantitative determination of AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS according to claim 1, characterized in that: In the step (c), the spin drying temperature of the parallel vacuum evaporator is 30-40° C.; the reagent used for the second re-dissolution is a methanol solution, and the mass fraction of the methanol solution is 20-40%.

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