A method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS

Abstract

The present invention relates to the field of analytical technology, and discloses a method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS. The present invention obtains a test solution by sequentially extracting, purifying and extracting the test object; then standard sample configuration is performed: reasonable chromatographic conditions and mass spectrometry conditions are set, and finally the AHLs content is calculated. Compared with existing methods, this method has the characteristics of more thorough extraction, higher sensitivity, lower detection limit, high recovery rate, simple operation, etc., and is suitable for 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 a new quality control method for sewage treatment plants and biogas projects.

Description

Technical Field

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

[0002] AHLs (acylhomoserine lactones) are a class of signaling molecules that facilitate intercellular communication in Gram-negative bacteria and play a crucial role in regulating bacterial behavior and metabolism. In anaerobic digestion and activated sludge systems, the presence of AHLs significantly influences sludge granulation, biofilm formation, and methanogenic activity, and can improve overall system performance by regulating microbial behavior and metabolism.

[0003] As a model of high-density microbial communities, activated sludge exhibits extensive intercellular communication. Current research on the regulatory role of quorum sensing in this system focuses primarily on biofilm and granular sludge formation. Related studies have demonstrated that quorum sensing significantly influences biofilm formation in various wastewater biological treatment reactors, such as sequencing batch biofilm reactors, anammox biofilm reactors, and membrane bioreactors. During granular sludge formation, the content of signaling molecules increases as sludge particle size increases; a decrease in the concentration of quorum sensing signaling molecules leads to granule disintegration 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 stage relies on the collaborative and symbiotic cooperation of different functional microorganisms. Some studies have found that quorum sensing mediated by AHLs can balance the diverse microbial populations during anaerobic digestion, and high methane production is associated with numerous AHL genes. In a study on the accelerated healing of anaerobic granular sludge after temperature shock, Lv et al. found that AHLs increased protein levels in tightly bound EPS and the abundance of hydrophobic functional groups in EPS. Notably, application of 10 μM AHLs significantly enhanced the enrichment of eutrophic methanogens. Furthermore, researchers have investigated the regulation of AHLs by microbial density to enhance the synergistic effects of acetogens and methanogens in anaerobic digestion. They found that interspecific regulation of syntrophic propionate oxidizers and hydrogenotrophic methanogens is mediated by 3-oxo-C6-HSL and C10-HSL, respectively. The type of AHLs influences the acclimated anaerobic microbial community.

[0005] In summary, AHLs may influence microbial interactions, metabolic activity, and product synthesis during anaerobic digestion and activated sludge treatment. Therefore, rapid and accurate detection of the composition, content, and variation of AHLs in anaerobic fermentation products and activated sludge is crucial for studying quorum sensing behaviors and mechanisms. Current methods for detecting AHLs in activated sludge have significant flaws. For example, Luo Meng et al. detected AHLs in activated sludge using high-speed centrifugation as a pretreatment to separate the water and matrix, and then collected the supernatant for analysis. This centrifugation caused the granular and flocculent sludge to settle to the bottom, ignoring AHLs adsorbed or bound to EPS, rendering them undetectable and resulting in low AHL values in the sample. Sun et al. improved this centrifugation pretreatment by performing high-speed centrifugation on the activated sludge, collecting the supernatant, and then re-dissolving the bottom sludge to extract the extracellular polymeric substances. The extraction method involves heating the activated sludge in an 80°C water bath for 30 minutes, followed by centrifugation and supernatant extraction. Current AHL detection studies have consistently shown that samples for AHL detection should be promptly frozen and analyzed within a short period of time. Our laboratory research has shown that AHLs standard working solution degrades and decreases in content after storage at 4°C for 24 hours. Excessively high temperatures during the extraction of extracellular polymeric substances can lead to some loss of AHLs. Furthermore, there are relatively few reports on the detection of AHLs in anaerobic fermentation products. Anaerobic fermentation substrates are more complex than activated sludge, making detection more challenging. Therefore, relevant studies have employed the same methods used for 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-MS / MS to address 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 ratios.

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

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

[0009] (1) Sample processing: The analyte is sequentially extracted, purified, and extracted to obtain the test solution;

[0010] (a) Extraction: Vortex the analyte, acetonitrile, homogenizer, and EDTA, then sonicate to obtain a mixture; freeze the mixture, vortex with anhydrous magnesium sulfate and disodium citrate, and centrifuge to complete the extraction and obtain a supernatant; the ratio of analyte, acetonitrile, EDTA, 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: The supernatant of the solution obtained in step (a) was mixed with a dehydrating agent (anhydrous magnesium sulfate) and two adsorbents, and then centrifuged. The supernatant was dried with nitrogen and redissolved 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: The purified solution is subjected to solid phase extraction, spin-dried, and second reconstitution to obtain a test solution;

[0014] The solid phase extraction comprises installing a polyethylene-diethylene glycol solid phase extraction column in a solid phase extraction instrument, activating the column with 5-10 mL of methanol, balancing with 2-10 mL of ultrapure water, washing with a 15-30 mmol / L sodium acetate solution, loading the purified solution obtained in step (b), and finally eluting with a 5 mL×6 methanol-isopropanol mixed solution (methanol-isopropanol volume ratio of 5:1);

[0015] (2) Standard preparation: 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 a 50 mg / L mixed standard stock solution of nine 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 has been verified to be free of the analyte through multiple verifications;

[0017] (3) Chromatographic conditions and mass spectrometry conditions: The mass spectrometry conditions are: electrospray ionization 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 in water containing 0.1-0.3 mM ammonium acetate; mobile phase B: 0.05% formic acid in methanol 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 minutes; the centrifugal speed is 4000 to 5000 r / min, and the centrifugation time is 3 to 10 minutes; the drying is carried out in a 30 to 40°C water bath with nitrogen; the reagent used for the first re-dissolution is 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 methanol solution, and the mass fraction of the methanol solution is 20-40%.

[0023] In the present invention, the preparation of a 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 initially grinding 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 one year, and seal for storage. The blank matrix is processed according to the sample processing steps to obtain a blank matrix solution, and the blank matrix solution is analyzed by LC-MS / MS. The blank matrix solution does not detect the AHLs target substance 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 amount of analyte residue 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] This invention provides a method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MS / MS. This method offers high sensitivity, high recovery, and simple operation. It overcomes the issues of other methods, such as the inability to extract extracellular polymeric substances or the high temperature required. The method is applicable to both activated sludge and anaerobic fermentation products. It provides a new approach and approach for studying the quorum sensing effects of AHLs in activated sludge and anaerobic fermentation products, and can also serve as a quality control tool for sewage treatment plants and biogas plants. 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0029] Figure 1 This 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as 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 spectrometry (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, Phenomenon, USA); distilled water (Watson's); standards: 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 (Aiger, China, PE2006-2); tail-capped C18 adsorbent (Phenomenon, USA, 04G-4348); primary and secondary amine adsorbent (Phenomenon, USA, 04G-4610); homogeneous proton (Agilent, USA, 5982-9313).

[0037] The blank matrix solution used in the following examples was prepared as follows: 30 kg of anaerobic fermentation product and activated sludge were collected, baked at 60°C for 10 days, and initially ground with a mortar until the sample completely passed through a 30-mesh sieve. The sample was then baked at 105°C for 36 hours. Finally, the sample was ground with an agate ball mill at 300 rpm for 30 minutes until it completely passed through a 100-mesh sieve. The sample was then collected in a sealed bag, aged for at least one year, and sealed for storage. The blank matrix solution was then processed according to the sample processing steps to obtain a blank matrix solution, which was then analyzed by LC-MS / MS.

[0038] Example 1

[0039] This example measured and verified 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 ionization 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 ionization 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, leading 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 9 AHLs are shown in Figure 2 The secondary mass spectra of 9 AHLs are shown in Figure 3 The linear relationships of the 9 AHLs are shown in Table 3. The results showed that the standard curve had a good linear relationship in the range of 0.02 to 5 μg / L, and 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 limits of detection and quantification of nine AHLs in activated sludge and anaerobic fermentation products:

[0054] (1) Preparation of blank matrix solution: 30 kg of anaerobic fermentation products and activated sludge were collected and baked at 60 ° C for 10 days. The samples were initially ground with a mortar until they could completely pass through a 30-mesh sieve. The samples were then baked at 105 ° C for 36 hours. Finally, the samples were ground with an agate ball mill at 300 r / min for 30 minutes until they could completely pass through a 100-mesh sieve. The samples were collected in a sealed bag and aged for more than 1 year. The bags were sealed and stored. The blank matrix was processed through the sample processing steps to obtain a blank matrix solution, which was analyzed by LC-MSMS. The ion flow diagram 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, R 2 The software calculated the signal-to-noise ratio (S / N) for the nine AHLs matrix standards (0.5-50 ng / L) at the lowest detectable concentration. The results were shown in Table 4, with a S / N ratio of 3 times the detection limit and a S / N ratio of 10 times the quantification limit.

[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, with the detection limit and quantification limit being less than 5 ng / kg and 17 ng / kg, respectively.

[0060] Example 3

[0061] This example measured the recoveries and precisions of nine AHLs in activated sludge and anaerobic fermentation products:

[0062] (1) Weigh 10 g of activated sludge or anaerobic fermentation product into a 50 mL centrifuge tube, add 0.1 mL of a mixed standard solution of 9 AHLs at 0.5 μg / L and 5 μg / L, and prepare a blank matrix solution. Add two ceramic homogenizers, add 10 mL of acetonitrile and 0.5 g of EDTA, mix well, vortex for 1 min, then ultrasonicate in a water bath at 200 W for 20 min, vortex for 1 min, remove and add 5 g of anhydrous magnesium sulfate and 2 g of sodium acetate, vortex for 1 min, and centrifuge at 5000 r / min for 5 min. Pipette all the supernatant (about 8 mL) and transfer it to a 15 mL centrifuge tube. Add 80 mg of magnesium sulfate, 20 mg of tail-capped C18 adsorbent, and 20 mg of primary and secondary amine, vortex for 1 min, and centrifuge at 5000 r / min for 5 min. Accurately transfer 6 mL of supernatant and blow to dryness with nitrogen at 30 °C. Add 5 mL of 25 mM sodium acetate solution, ultrasonicate for 15 min, vortex for 1 min, and re-dissolve before loading onto the column. A polyethylene-vinyl chloride solid phase extraction column was connected to the top of the solid phase extractor, and the flow rate was controlled at 2 s / drop with negative pressure. First, 5 mL of methanol was used to activate the column, and then 10 mL of ultrapure water was used to equilibrate the column. The column was dried under negative pressure for 2 min, and the test solution was added and loaded at a loading speed of 3 s / drop. The column was then washed with 5 mL of 25 mM sodium acetate and dried under negative pressure for 2 min. Finally, 5 mL × 6 times of a mixed solution of methanol and isopropanol (methanol and isopropanol volume ratio of 5:1) were used for elution. The eluate was dried on a parallel vacuum evaporator at 35 ° C, re-dissolved with 1 mL of 30% methanol by vortexing for 1 min, and loaded for analysis.

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

[0064] The present embodiment compares the extraction effect of extraction agent acetonitrile and ethyl acetate during sample extraction, and the result shows that acetonitrile recovery is high, and purification effect is good, and ethyl acetate purification effect is poor, and matrix effect is enhanced. Cleaning agent C18 and primary secondary amine exceed 50mg, will have an impact on recovery, and 20mg is the minimum addition amount that does not affect recovery. Different solid phase extraction columns (PEP-2, C8 and weak cation exchange column) are studied, and the result shows that PEP-2 recovery is higher. The influence of different elution solvents on recovery (the ratio of acetonitrile, methanol, isopropanol, acetone and mixed reagent thereof) is studied, and the result shows that methanol elution is relatively reasonable, and adding a small amount of isopropanol to mix with methanol in eluent can improve C 14 The recovery rate of -HCL. The volume of eluent was studied and it was found that when the eluent was 30mL, complete elution was possible, but when the volume was reduced, C10-HSL and C14-HSL could not be completely eluted.

[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 ranged from 77.64% to 93.72%, and those in anaerobic fermentation products ranged from 72.48% to 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, the AHLs of five activated sludges and five anaerobic fermentation products were measured, which were respectively from actual production and laboratory simulation systems. The applicability of the method can be verified through the detection.

[0071] (1) Sample information:

[0072] Activated Sludge A: This SBR reactor uses synthetic wastewater simulating biogas slurry as its influent. The volume ratio of activated sludge to influent is 1:1, with 2L of influent and effluent per day. The SBR operating parameters are: hydraulic retention time of 48 hours, temperature of 28°C, and one cycle per day. Each cycle (24 hours) consists of: influent (10 minutes) - idle (230 minutes) - aeration (960 minutes) - sedimentation (230 minutes) - effluent (10 minutes).

[0073] Activated sludge B: Synthetic wastewater with propionic acid as the sole carbon source at a concentration of 10 g COD L⁻¹ was used as the influent, and anaerobic digested sludge from swine manure was used as the inoculum. A semi-continuous test was used with an organic loading 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 obtained 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: Initial aerobic activated sludge volume was 2.0 L, and the amount of pig farm biogas added was 2.0 L; the sludge inoculum was 12 g / L, the hydraulic retention period was 2 days, and the dissolved oxygen concentration was 4.0 mg / 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 the synthetic wastewater, and ammonium sulfate was added to adjust the NH4 + -N to about 300mg / L, and the COD concentration was maintained at 480-642mg / L by adding organic matter. The operating parameters of the SBR were: feeding time 10min, standing time 110min, aeration time 480min, settling time 110min, discharge time 10min, HRT 24h, and 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 the raw material for anaerobic digestion and digested at 30°C;

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

[0079] Anaerobic fermentation product 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: Return sludge collected from the secondary sedimentation tank of a wastewater treatment plant in Guangzhou, China, was allowed to settle at 4°C for 24 hours, and the supernatant was decanted to obtain concentrated sludge. The inoculum sludge was obtained from an anaerobic digester at a treatment plant in Zhongshan, China. The anaerobic digester operated at a temperature of 36–38°C, with a sludge retention time of 20–25 days.

[0081] Anaerobic digester E: Cow manure was collected from a cattle farm. Chicken manure was diluted with water to a TS concentration of approximately 15%. It was then sieved through a 2mm sieve to remove debris such as feathers and sand before use. The anaerobic digester had a hydraulic retention period of 20 days (i.e., 500g of feed per day, once per day), an influent total solids concentration of 8%, and a digestion temperature of 40°C.

[0082] (2) Sample testing:

[0083] Weigh 2g of activated sludge or anaerobic fermentation product into a 50mL centrifuge tube and prepare a blank matrix solution. Add two ceramic homogenizers, 10mL of acetonitrile, and 0.3g of EDTA, mix thoroughly, and vortex for 1 minute. Sonicate in a waterbath at 150W for 25 minutes, vortex for 1 minute, remove the supernatant, add 4g of anhydrous magnesium sulfate and 1.5g of disodium citrate, vortex for 1 minute, and centrifuge at 5000 rpm for 5 minutes. Transfer the entire supernatant (approximately 8mL) to a 15mL centrifuge tube, add 50mg of magnesium sulfate, 20mg of tail-capped C18 adsorbent, and 20mg of primary and secondary amine, vortex for 1 minute, and centrifuge at 5000 rpm for 5 minutes. Accurately transfer 6mL of supernatant to a 30°C nitrogen purge until nearly dry. Add 5mL of 20mM sodium acetate solution, sonicate for 15 minutes, and vortex for 1 minute to reconstitute the solution before loading onto the column. Connect the polyethylene-vinyl chloride 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 and load it at a speed of 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 parallel vacuum evaporator at 30℃, re-dissolved with 1mL of 20% methanol by vortexing for 1min, and loaded for analysis.

[0084] (3) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, dilute it stepwise to 1, 5, 20, 50, 100, and 200 ng / L using blank matrix solution, and use LC-MS / MS for analysis.

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

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

[0087]

[0088] Example 5

[0089] This example measures nine 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 prepare a blank matrix solution. Add two ceramic homogenizers, 15mL of acetonitrile, and 0.7g of EDTA, mix thoroughly, and vortex for 2 minutes. Sonicate in a waterbath at 250W for 15 minutes. Vortex for 2 minutes. Remove and add 6g of anhydrous magnesium sulfate and 4g of disodium citrate, vortex for 1 minute, and centrifuge at 4000 rpm for 10 minutes. Transfer 10mL of the supernatant to a 15mL centrifuge tube, add 100mg of magnesium sulfate, 50mg of tail-capped C18 adsorbent, and 50mg of primary and secondary amine, vortex for 1 minute, and centrifuge at 4000 rpm for 10 minutes. Accurately remove 8mL of the supernatant and purge with nitrogen at 40°C until nearly dry. Add 5mL of 30mM sodium acetate solution, sonicate for 15 minutes, and vortex for 1 minute to reconstitute the solution before loading onto the column. Connect the polyethylene-vinyl chloride 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 equilibrate the column with 10mL of ultrapure water, dry it under negative pressure for 2min, add the test solution and load it at a speed of 3s / drop. Then wash the column with 5mL of 30mM sodium acetate and dry it under negative pressure for 2min. Finally, elute with 5mL×6 methanol-isopropanol mixed solution (methanol-isopropanol volume ratio 5:1). Dry the eluate on a parallel vacuum evaporator at 40℃, re-dissolve it with 1mL of 20% methanol by vortexing for 1min, and load it for analysis.

[0093] (3) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, dilute it stepwise to 1, 5, 20, 50, 100, and 200 ng / L using blank matrix solution, and use LC-MS / MS for analysis.

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

[0095] Table 7 AHLs content 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for quantitatively analyzing AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS, characterized in that: The steps include: (1) Sample processing: The analyte is sequentially extracted, purified, and extracted to obtain the test solution; (a) Extraction: Vortex the analyte, acetonitrile, homogenizer, and EDTA, then sonicate to obtain a mixture; freeze the mixture, vortex with anhydrous magnesium sulfate and disodium citrate, and centrifuge to complete the extraction and obtain a supernatant; the ratio of analyte, acetonitrile, EDTA, 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. The supernatant is dried with nitrogen and redissolved 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; The dehydrating agent is magnesium sulfate, adsorbent 1 is a capped C18 adsorbent, and adsorbent 2 is a primary or secondary amine adsorbent; Step (b) The reagent used for the first re-dissolution is sodium acetate solution, and the concentration of the sodium acetate solution is 15 to 30 mmol / L; (c) Secondary purification: The purified solution is subjected to solid phase extraction, spin-dried, and second reconstitution to obtain a test solution; The solid phase extraction comprises installing a polyethylene-diethylene glycol solid phase extraction column in a solid phase extraction instrument, activating the column with 5 to 10 mL of methanol, balancing with 2 to 10 mL of ultrapure water, washing with a 15 to 30 mmol / L sodium acetate solution, loading the purified solution obtained in step (b), and finally eluting with a 5 mL×6 methanol-isopropanol mixed solution, wherein the volume ratio of methanol to isopropanol is 5:1; The reagent used for the second re-dissolution is a methanol solution, and the mass fraction of the methanol solution is 20-40%; (2) Standard preparation: 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 a 50 mg / L mixed standard stock solution of nine 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 has been verified to be free of the analyte through multiple verifications; (3) Chromatographic conditions and mass spectrometry conditions: The mass spectrometry conditions are: electrospray ionization 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 in water containing 0.1-0.3 mM ammonium acetate; mobile phase B: 0.05% formic acid in methanol 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 quantitatively determining AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS according to claim 1, characterized in that: The mixing time in step (b) is 1 to 2 minutes; the centrifugal speed is 4000 to 5000 r / min, and the centrifugal time is 3 to 10 minutes; and the drying is carried out by passing nitrogen in a 30 to 40° C. water bath.

4. The method for quantifying AHLs in anaerobic fermentation products and activated sludge based on LC-MSMS according to claim 1, characterized in that: The spin drying temperature of the parallel vacuum evaporator in the step (c) is 30-40°C.

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