CN107884349B - Determination method of superoxide anion free radicals in microorganisms - Google Patents
Determination method of superoxide anion free radicals in microorganisms Download PDFInfo
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- G—PHYSICS
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Abstract
The invention discloses a method for measuring superoxide anion free radicals in a microorganism body, which comprises the following steps: firstly, extracting a microorganism sample, making a standard curve, then setting a test group, a control group and a blank group, respectively measuring absorbance values of the biological sample, then obtaining the difference value of the absorbance of the three groups as the absorbance value of the microorganism in the test group, then obtaining the concentration value of superoxide anion free radicals in the microorganism by using the standard curve and a formula, and then calculating the O in the organism of unit mass according to the formula2 ‑.Content, finishing the determination of superoxide anion free radicals in the microorganism; according to the invention, a carbonic acid buffer solution with the pH of 9.5-10.5 is used, and compared with a phosphate buffer solution with the pH of 7.8 and a Tris-HCl buffer solution with the pH of 7.6 which are used conventionally, a system to be detected detects O2 ‑.The sensitivity of the method is improved by nearly 10 times, and the method has the advantages of good specificity, simple operation, short time consumption and easy popularization.
Description
Technical Field
The invention relates to a determination method of superoxide anion free radicals in microorganisms, belonging to the technical field of biological purification treatment of wastewater and waste gas.
Background
Superoxide anion radical (O)2 -.) Belongs to an active oxygen group and has stronger oxidability. O is2 -.Ubiquitous in microbial systems, when organisms are subjected to pollutant stress, the production of reactive oxygen species in vivo will be greatly increased; when it exceeds the antioxidant defense capacity of the organism, the cellular components of the organism, such as lipids, proteins, nucleic acids, etc., will be in O2 -.Irreversible damage occurs, thereby interfering with cellular metabolism or causing cell death, and superoxide anion radicals are therefore considered to be an important pathway and mechanism for the toxicity of contaminants. In addition, studies have shown that reactive oxygen species are involved in cell signal transduction and induction of physiological responses, and are an important class of signaling molecules of cells.
The active oxygen life in living body is very short, such as the survival time of superoxide anion free radical in water solution is 1s, and the life of hydroxyl free radical is only 10-6s, and additionally very low concentration of reactive oxygen species in the microorganismMaking its qualitative and quantitative detection extremely difficult. Common methods for measuring superoxide anion radicals include NBT (nitrotetrazolium chloride blue), cytochrome, pyrogallol, epinephrine, and hydroxylamine oxidation. After the cytochrome C with oxidation activity is reduced by superoxide anion free radicals, ferrous cytochrome with strong absorption at the wavelength of 550nm is formed, and the cytochrome C can be used for measuring the superoxide anion free radicals; however, the presence of other reducing substances (such as reducing enzymes) in the cytochrome C reduction system can cause significant interference with the test results. NBT is reduced to generate water-insoluble blue dimethyl (Diformazan) under the action of superoxide anion free radicals, and the maximum absorption wavelength of the NBT is 560 nm; however, dimethyl is insoluble in water, and precipitates appear after long-term storage, so that the dimethyl is difficult to apply to the dynamic research process of determining the superoxide anion free radicals along with the time extension. The traditional hydroxylamine oxidation method has low content of O2 -.The detection is difficult, the layering of the n-butanol extraction liquid and the reaction medium is not obvious, the test effect is influenced by the self-oxidation of hydroxylamine, and the like. The superoxide anion free radical can also rapidly react with 1, 2-dihydroxy benzene-3, 5-disulfonic acid sodium (Tiron) to generate a spin adduct called as a Tiron semiquinone free radical, and can be detected by an electron paramagnetic resonance spectrometer (EPR) at room temperature; the EPR spin trapping technology requires strict experimental conditions and requires expensive analysis and test instruments such as EPR, thereby limiting the popularization and application of the technology.
The content of superoxide anion free radicals in a microorganism is low, and the concentration of oxygen radicals required by the traditional hydroxylamine oxidation method is relatively high; in addition, many factors interfere with the superoxide anion radical test, and the measurement result is sometimes unstable. At present, no effective method for measuring the concentration of superoxide anion free radicals in microorganisms exists, so that the development of a method for measuring superoxide anion free radicals in microorganisms, which is simple and convenient to operate and has high stability, is of great significance.
Disclosure of Invention
The invention aims to provide a method for measuring superoxide anion free radicals in a microorganism, which is used for measuring the concentration of the superoxide anion free radicals in the microorganism on the basis of the principle that hydroxylamine can react with the superoxide anion free radicals in the microorganism to generate pink azo compounds.
As hydroxylamine hydrochloride is subjected to autoxidation, the hydroxylamine hydrochloride is added as a blank group in the test, so that the problem of high measurement result caused by the autoxidation of hydroxylamine hydrochloride can be effectively solved. Secondly, the group without hydroxylamine hydrochloride is taken as a control group, so that residual pigment and endogenous NO can be effectively eliminated2-The interference of (2). Thus, during the measurement, O2-.Actual a530 values-absorbance for test set-control set-blank set.
The invention sets a test group, a control group and a blank group, respectively adds carbonic acid buffer solution into the test group, the control group and the blank group, respectively adds hydroxylamine hydrochloride solution into the test group and the blank group, adds distilled water with the same volume into the control group, then simultaneously places the three groups into a constant temperature water bath, then adds supernatant into the test group and the control group, adds carbonic acid buffer solution with the same volume into the blank group, respectively shakes uniformly, then stands in the water bath, then sequentially adds sulfanilamide and N-1-naphthyl ethylenediamine into the test group, the control group and the blank group, respectively, develops color in the water bath, adds extractant into the solution after the three groups develop color, respectively carries out centrifugal treatment after fully mixing uniformly, then respectively takes upper extraction phase, respectively measures absorbance of the test group, the control group and the blank group at 540nm, calculates difference of the measured absorbance values, taking the difference as y and substituting the y into a standard curve equation, and calculating the x value which is NO in the test group corresponding to 540nm2 -Concentration, then calculating the O in the organism per unit mass according to a formula2 -.And (4) measuring the content of the superoxide anion free radicals in the microorganisms.
A method for measuring superoxide anion free radicals in microorganisms specifically comprises the following operations:
(1) centrifuging an activated sludge suspension with biomass greater than 800mg/L in a microorganism sample for 8-10 min under the condition of 10000-12000 r/min, taking 1-2 g of the centrifuged bottom microorganism sample, repeatedly sampling and accumulating to 1-2 g when the centrifuged bottom microorganism sample is less than 1g, then adding a carbonic acid buffer solution with pH of 9.5-10.5 into the microorganism sample, adding 3.5-4.5 mL of the mixed solution into each 1g of the microorganism sample, then placing the mixed solution into an ice water bath for ultrasonic crushing, finally centrifuging and taking a supernatant, wherein the ultrasonic crushing time is 2-3 min, and the centrifugal rotating speed is 10000-120000 r/min when the supernatant is taken centrifugally;
(2) and (3) standard curve preparation: with NaNO2Preparing more than 7 groups of NO with the concentration of 0-30 mu mol/L2 -Respectively diluting the standard solutions to 200mL in constant volume, then adding carbonic acid buffer solution with pH of 9.5-10.5 and hydroxylamine hydrochloride solution with concentration of 50-100 mmol/L into more than 7 blank test tubes, wherein the adding amount of the carbonic acid buffer solution is 0.5-1 mL per 1g of microorganism sample, the adding amount of the hydroxylamine hydrochloride solution is 0.1-0.3 mL per 1g of microorganism sample, then placing each group of solutions in a constant-temperature water bath at 25-35 ℃ for heating for 10-15 min, and then adding 0.5mL of NO with different concentrations into each group of solutions2 -Shaking the standard solution uniformly, standing the solution in a constant-temperature water bath at the temperature of 25-35 ℃ for 18-22 min, sequentially adding a sulfanilamide solution and an N-1-naphthyl ethylenediamine solution into each group of solution, wherein the sulfanilamide solution and the N-1-naphthyl ethylenediamine solution are obtained by respectively dissolving an aminobenzenesulfonamide solid and an N-1-naphthyl ethylenediamine solid in an acetic acid solution with the concentration of 10-13 mol/L, the concentrations of the sulfanilamide solution and the N-1-naphthyl ethylenediamine solution are respectively 55-60 mmol/L and 5-10 mmol/L, 0.8-1 mL of sulfanilamide solution and N-1-naphthyl ethylenediamine solution are added into each 1g of microbial sample, then placing the microbial sample in the water bath for developing for 20-30 min, and then adding an extracting agent into each group of solution, mixing, centrifuging, collecting upper extract phase, measuring absorbance at 540nm, and taking the measured absorbance value as y value to obtain NO2-Taking the concentration as a value x, and obtaining a standard curve equation after linear fitting, wherein the volume of the added extracting agent is 0.8-1.2 times of the volume of each group of solution before adding, and the extracting agent is dichloromethane, trichloromethane or toluene;
(3) setting a test group, a control group and a blank group, adding carbonic acid buffer solutions into the three groups respectively, shaking up, adding hydroxylamine hydrochloride solutions into the test group and the blank group, adding distilled water with the same volume into the control group, simultaneously placing the three groups in a constant-temperature water bath at 25-35 ℃ for heating for 10-15 min, then adding 0.5mL of supernatant into the test group and the control group, adding carbonic acid buffer solutions with the same volume into the blank group, shaking up respectively, standing in the constant-temperature water bath at 25-35 ℃ for 18-22 min, then sequentially adding sulfanilamide solution and N-1-naphthyl ethylenediamine solution into the test group, the control group and the blank group respectively, and developing in the water bath for 20-30 min, wherein the concentration and the adding amount of each solution are the same as those in the step (2);
(4) adding an extracting agent into the three groups of colored solutions obtained in the step (3), fully and uniformly mixing, then respectively carrying out centrifugal treatment, then respectively taking upper extraction phases, and measuring the absorbance of a test group, a control group and a blank group at 540nm, wherein the used extracting agent is the same as that in the step (2), and the time of the centrifugal treatment is 3-5 min;
(5) and (3) calculating the difference of the absorbance values measured in the step (4), and then substituting the difference into the standard curve equation in the step (1) to obtain NO in the test group corresponding to the 540nm position2 -Concentration, then calculating the O in the organism per unit mass according to a formula2 -.Content, namely completing the determination of superoxide anion free radicals in the microorganism, wherein the formula is
Wherein
Is the value of x in units of. mu. mol. L-1，V1The volume of the sample to be tested is the test group, and the unit is mL and V2Centrifuging the test group obtained in the step (2) after ultrasonic crushing to obtain the total volume of supernatant, wherein the unit is mL, and rho is the concentration of the solid matter of sludge in a sample to be tested of the test group and is mg.L-1，m1The mass of the microorganism sample after centrifugation in step (2) of the test group is given in g, m2The mass of the sediment in g after sonication and centrifugation in step (2) of the test group.
The standard curve is established, the addition amount of the same reagent in different test tubes is consistent with that of the test sample.
The invention has the advantages and technical effects that:
1. according to the invention, a carbonic acid buffer solution with the pH of 9.5-10.5 is used, and compared with a phosphate buffer solution with the pH of 7.8 and a Tris-HCl buffer solution with the pH of 7.6 which are used conventionally, a system to be detected detects O2 -.The sensitivity of (a) is improved by nearly 10 times.
2. The extracting agent is toluene, trichloromethane or dichloromethane, when dichloromethane is used as the extracting agent, the extraction phase is positioned on the upper layer of the liquid phase of the test system and is obviously layered with the raffinate phase, and the absorbance value of the system to be tested is the highest.
3. The method for determining superoxide anion free radicals by using the sulfanilamide and the N-1-naphthyl ethylenediamine has the advantages of good specificity, simple operation, short time consumption and easy popularization.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited to the above-described examples.
Example 1: the method for measuring the superoxide anion free radicals in the microbial sample after hydrogen phosphide acclimation specifically comprises the following operations:
taking 20mL of suspended microorganism domesticated by phosphine, wherein the biomass is about 5000mg/L, centrifuging for 8min under the condition of 10000r/min, taking 1g of microorganism sample at the bottom of a centrifuge tube, adding 3.5mL of carbonic acid buffer solution with the pH value of 9.5, placing the microorganism sample in an ice water bath, carrying out ultrasonic crushing for 2min, centrifuging for 10min under 10000r/min, and taking supernatant for experimental determination;
and (3) standard curve equation formulation: with NaNO28 groups of NO were formulated at concentrations of 3, 6, 9, 12, 15, 18, 21 and 24. mu. mol/L2 -Taking 8 test tubes as standard solutions, respectively adding 0.5mL of carbonic acid buffer solution with pH of 9.5, then adding 0.1mL of hydroxylamine hydrochloride solution with concentration of 70mmol/L, then placing each group of solutions in a constant temperature water bath at 30 ℃ for heating for 12min, and then adding 0.5mL of solution with concentration of 3, 6, 9, 12, 15, 18, 21 and 24 mu mol/L into each group of solutionsNO2 -Shaking the standard solution, standing in 30 deg.C constant temperature water bath for 20min, sequentially adding 0.8mL of 58mmol/L sulfanilamide solution (dissolved in 10mol/L acetic acid) and 0.8mL of 7mmol/L N-1-naphthylethylenediamine solution (dissolved in 10mol/L acetic acid) into each group of solutions, placing in 30 deg.C water bath for color development for 25min, adding dichloromethane as extractant into each group of solutions, adding volume 1 times of the total volume of the above solutions, centrifuging respectively, centrifuging at 10000r/min for 3min, collecting upper extract phase, measuring absorbance of each group at 540nm, taking the measured absorbance value as y value, and collecting NO of each group2 -Taking the concentration as a value x, and obtaining a standard curve equation y of 0.0184x +0.1909 (R of 0.9994) after linear fitting;
setting a test group, a control group and a blank group, adding carbonic acid buffer solution into the three groups respectively, shaking up, adding hydroxylamine hydrochloride solution into the test group and the blank group, adding distilled water with the same volume into the control group, simultaneously placing the three groups in a constant-temperature water bath at 30 ℃ for heating for 12min, then adding 0.5mL of supernatant into the test group and the control group, adding carbonic acid buffer solution with the same volume into the blank group, shaking up respectively, standing for 20min in the constant-temperature water bath at 30 ℃, then sequentially adding sulfanilamide solution and N-1-naphthyl ethylenediamine solution into the test group, the control group and the blank group respectively, developing for 25min in the water bath, wherein the concentration and the adding amount of each solution are the same as the step (2), then adding pigment dichloromethane into the three groups of solutions, centrifuging at a high speed for 3min under the condition of 10000r/min, respectively taking three groups of pink upper layer extract phases and measuring absorbance at 540nm to obtain three groups A530The difference is 0.258, the standard curve equation is substituted to obtain x, and then the formula is used
The oxygen radical concentration of the test group is calculated to be 0.311 mu mol g-1。
Example 2: the method for determining the superoxide anion free radical in the microorganism sample of the municipal sewage treatment plant specifically comprises the following operations:
taking 20mL of microbial suspension of an urban sewage treatment plant, wherein the biomass is about 6800mg/L, centrifuging for 9min under the condition of 11000r/min, taking 1.5g of a microbial sample at the bottom of a centrifuge tube, adding 6mL of carbonic acid buffer solution with the pH value of 10.00, placing the microbial sample in an ice water bath for ultrasonic crushing for 2.5min, centrifuging for 10min under the condition of 11000r/min, and taking supernatant for experimental determination.
And (3) standard curve equation formulation: with NaNO27 groups of NO with the concentration of 4, 8, 12, 16, 20, 24 and 28 mu mol/L are prepared2 -Taking 7 test tubes as standard solutions, respectively adding 1.5mL of carbonic acid buffer solution with pH of 10.0, then adding 0.45mL of hydroxylamine hydrochloride solution with concentration of 100mmol/L, then placing each group of solutions in a constant-temperature water bath with temperature of 35 ℃ for heating for 10min, and then adding 0.5mL of NO with concentration of 4, 8, 12, 16, 20, 24 and 28 mu mol/L into each group of solutions2 -Shaking the standard solution, standing in 35 deg.C constant temperature water bath for 18min, sequentially adding 1.5mL of sulfanilamide solution (dissolved in 11mol/L acetic acid) with concentration of 55mmol/L and 1.5mL of N-1-naphthyl ethylenediamine solution (dissolved in 11mol/L acetic acid) with concentration of 5mmol/L into each group of solutions, placing in 35 deg.C water bath for color development for 20min, adding toluene as extractant into each group of solutions, adding volume of 0.8 times of the total volume of the above solutions, centrifuging, respectively collecting upper extraction phase, measuring absorbance at 540nm, taking the measured absorbance as y value, and taking NO of each group as y value2 -Taking the concentration as a value x, and obtaining a standard curve equation y which is 0.0115x +0.1622(R which is 0.9997) after linear fitting;
setting a test group, a control group and a blank group, adding carbonic acid buffer solution into the three groups respectively, shaking up, adding hydroxylamine hydrochloride solution into the test group and the blank group, adding distilled water with the same volume into the control group, placing the three groups in a constant-temperature water bath at 35 ℃ simultaneously, heating for 10min, then adding 0.5mL of supernatant into the test group and the control group, adding carbonic acid buffer solution with the same volume into the blank group, shaking up respectively, standing in the constant-temperature water bath at 35 ℃ for 18min, and then sequentially adding para-ammonia into the test group, the control group and the blank group respectivelyPerforming color development on the phenylsulfonamide solution and the N-1-naphthyl ethylenediamine solution in a water bath at 35 ℃ for 20min, wherein the concentration and the adding amount of each solution are the same as those in the step (2), adding toluene into the three groups of solutions, performing high-speed centrifugation for 4min under the condition of 10000r/min, respectively taking the pink upper layer extract phases of the three groups, measuring the absorbance at 540nm, and obtaining three groups A530The difference is 0.209, substitute the standard curve to obtain x, and then use the formula
The oxygen radical concentration of the test group is calculated to be 0.102 mu mol g-1。
Example 3: the method for determining the superoxide anion free radical in the microorganism sample of the coking wastewater treatment plant specifically comprises the following operations:
taking 20mL of microorganism suspension of a coking wastewater treatment plant, wherein the biomass is about 4500mg/L, centrifuging for 10min under the condition of 12000r/min, taking 2g of microorganism sample at the bottom of a centrifuge tube, adding 8mL of carbonic acid buffer solution with the pH value of 10.00, placing the microorganism sample in an ice water bath, carrying out ultrasonic crushing for 2.5min, centrifuging for 10min at 11000r/min, and taking supernatant for experimental determination;
and (3) standard curve equation formulation: with NaNO2NO concentrations of 3, 6, 9, 12, 15, 18, 21 and 24. mu. mol/L were formulated2 -Taking 8 test tubes as standard solutions, respectively adding 1mL of carbonic acid buffer solution with pH of 10.0, then adding 0.3mL of hydroxylamine hydrochloride solution with concentration of 80mmol/L, then placing each group of solutions in a constant-temperature water bath at 25 ℃ for heating for 15min, and then adding 0.5mL of NO with concentration of 3, 6, 9, 12, 15, 18, 21 and 24 mu mol/L into each group of solutions2 -Shaking the standard solution, standing in a constant temperature water bath at 25 deg.C for 22min, sequentially adding 0.9mL of 60mmol/L sulfanilamide solution (dissolved in 13mol/L acetic acid) and 0.9mL of 10mmol/L N-1-naphthylethylenediamine solution (dissolved in 13mol/L acetic acid) into each group of solutions, placing in a water bath at 25 deg.C for color development for 30min, adding dichloromethane as extractant into each group of solutions, adding volume of 1.1 times of the total volume of the above solutions, centrifuging respectively, and centrifuging at 10000r/minCentrifuging at high speed for 5min, respectively collecting upper extract phase, measuring absorbance at 540nm, taking the measured absorbance value as y value, and measuring NO of each group2 -Taking the concentration as a value x, and obtaining a standard curve equation y which is 0.0201x +0.1045(R which is 0.9992) after linear fitting;
setting a test group, a control group and a blank group, adding carbonic acid buffer solution into the three groups respectively, shaking up, adding hydroxylamine hydrochloride solution into the test group and the blank group, adding distilled water with the same volume into the control group, simultaneously placing the three groups in a constant-temperature water bath at 25 ℃ for heating for 15min, then adding 0.5mL of supernatant into the test group and the control group, adding carbonic acid buffer solution with the same volume into the blank group, shaking up respectively, standing for 22min in the constant-temperature water bath at 25 ℃, then sequentially adding sulfanilamide solution and N-1-naphthyl ethylenediamine solution into the test group, the control group and the blank group respectively, developing for 30min in the water bath at 25 ℃, wherein the concentration and the adding amount of each solution are the same as in the step (2), then adding dichloromethane into the three groups, centrifuging at a high speed for 5min under the condition of 10000r/min, respectively taking pink upper layer extraction phases of the three groups, and measuring the absorbance at 540nm, find out three groups A530The difference is 0.279, the standard curve is substituted to obtain x, and then the formula is used
The oxygen radical concentration of the test group is calculated to be 0.478 mu mol g-1。
Example 4: the method for determining the superoxide anion free radical in the microorganism sample of the municipal sewage treatment plant specifically comprises the following operations:
taking 20mL of microbial suspension of an urban sewage treatment plant, wherein the biomass is about 6300mg/L, centrifuging for 10min under the condition of 12000r/min, taking 2g of microbial sample at the bottom of a centrifuge tube, adding 9mL of carbonic acid buffer solution with the pH value of 10.50, placing the microbial sample in an ice water bath, carrying out ultrasonic crushing for 3min, centrifuging for 10min under 12000r/min, and taking supernatant for experimental determination;
and (3) standard curve equation formulation: with NaNO28 groups of NO were formulated at concentrations of 3, 6, 9, 12, 15, 18, 21 and 24. mu. mol/L2 -Taking 8 test tubes as standard solutions, respectively adding 2mL of carbonic acid buffer solution with pH of 9.5, then adding 0.6mL of hydroxylamine hydrochloride solution with concentration of 50mmol/L, then placing each group of solutions in a constant-temperature water bath at 30 ℃ for heating for 10min, and then adding 0.5mL of NO with concentration of 3, 6, 9, 12, 15, 18, 21 and 24 mu mol/L into each group of solutions2 -Shaking the standard solution, standing in 30 deg.C constant temperature water bath for 22min, sequentially adding 2mL of 58mmol/L sulfanilamide solution (dissolved in 12mol/L acetic acid) and 2mL of 7mmol/L N-1-naphthyl ethylenediamine solution (dissolved in 12mol/L acetic acid) into each group of solutions, placing in 30 deg.C water bath for color development for 30min, adding chloroform as extractant 1 times of the total volume of the above solutions, mixing, centrifuging at 10000r/min for 3.2min, collecting upper extraction phase, measuring absorbance at 540nm, and taking the measured absorbance as y value2 -Taking the concentration as a value x, and obtaining a standard curve equation y of 0.0104x +0.1922(R of 0.9997) after linear fitting;
setting a test group, a control group and a blank group, adding carbonic acid buffer solution into the three groups respectively, shaking up, adding hydroxylamine hydrochloride solution into the test group and the blank group, adding distilled water with the same volume into the control group, simultaneously placing the three groups in a constant-temperature water bath at 30 ℃ for heating for 10min, then adding 0.5mL of supernatant into the test group and the control group, adding carbonic acid buffer solution with the same volume into the blank group, shaking up respectively, standing for 22min in the constant-temperature water bath at 30 ℃, then sequentially adding sulfanilamide solution and N-1-naphthyl ethylenediamine solution into the test group, the control group and the blank group respectively, developing for 30min in the water bath at 30 ℃, wherein the concentration and the adding amount of each solution are the same as the step (2), then adding trichloromethane into the three groups, centrifuging at a high speed for 3.2min under the condition of 10000r/min, respectively taking pink upper layers of the three groups, extracting phases and measuring the absorbance at 540nm, find out three groups A530The difference is 0.301, the x is obtained by substituting the standard curve and the formula
The oxygen radical concentration of the test group is calculated to be 0.589 mu mol g-1。
Example 5: the method for measuring the superoxide anion free radical in the microorganism sample in the denitrification trickling filter comprises the following specific operations:
taking 20mL of microorganism suspension in a denitrification trickling filter, wherein the biomass is about 8200mg/L, centrifuging for 10min under the condition of 12000r/min, taking 2g of microorganism sample at the bottom of a centrifuge tube, adding 8.5mL of carbonic acid buffer solution with the pH value of 10.00, placing the microorganism sample in an ice water bath, carrying out ultrasonic crushing for 3min, centrifuging for 10min under 12000r/min, and taking supernatant for experimental determination.
And (3) standard curve equation formulation: with NaNO28 groups of NO with the concentration of 3, 6, 9, 12, 15, 18, 21 and 24 mu mol/L are prepared2 -Taking 8 test tubes as standard solutions, respectively adding 1mL of carbonic acid buffer solution with pH of 10.0, then adding 0.2mL of hydroxylamine hydrochloride solution with concentration of 90mmol/L, then placing each group of solutions in a constant-temperature water bath with temperature of 35 ℃ for heating for 10min, and then adding 0.5mL of NO with concentration of 3, 6, 9, 12, 15, 18, 21 and 24 mu mol/L into each group of solutions2 -Shaking the standard solution, standing in 35 deg.C constant temperature water bath for 18min, sequentially adding 2mL of 58mmol/L sulfanilamide solution (dissolved in 10mol/L acetic acid) and 2mL of 7mmol/L N-1-naphthylethylenediamine solution (dissolved in 10mol/L acetic acid) into each group of solutions, placing in 35 deg.C water bath for color development for 24min, adding dichloromethane as extractant in 1.2 times of the total volume of the above solutions, mixing well, centrifuging at 10000r/min for 3min, collecting upper extraction phase, measuring absorbance at 540nm, and taking the measured absorbance as y value2 -Taking the concentration as a value x, and obtaining a standard curve equation y which is 0.0122x +0.1751 (R which is 0.9998) after linear fitting;
setting test group, control group and blank group, adding carbonic acid buffer solution into the three groups respectively, shaking, adding hydroxylamine hydrochloride solution into the test group and blank group, adding the same volume of hydroxylamine hydrochloride into the control groupDistilled water, simultaneously placing the three groups in a constant-temperature water bath at 35 ℃ for heating for 10min, then adding 0.5mL of supernatant into a test group and a control group, adding carbonic acid buffer solution with the same volume into a blank group, shaking up respectively, standing for 18min in the constant-temperature water bath at 35 ℃, then sequentially adding sulfanilamide solution and N-1-naphthyl ethylenediamine solution into the test group, the control group and the blank group respectively, then developing for 24min in the water bath at 35 ℃, wherein the concentration and the adding amount of each solution are the same as those in the step (2), then adding trichloromethane into the three groups of solutions, centrifuging at high speed for 3min under the condition of 10000r/min, respectively taking pink upper layer extract phases of the three groups, measuring absorbance at 540nm, and obtaining three groups A530The difference is 0.256, and the difference is substituted into the standard curve to obtain x, and then the formula is used
The oxygen radical concentration of the test group is calculated to be 0.325 mu mol g-1。
Claims (4)
1. A method for measuring superoxide anion free radicals in microorganisms is characterized by comprising the following steps:
(1) centrifuging an activated sludge suspension with biomass more than 800mg/L in a microorganism sample for 8-10 min under the condition of 10000-12000 r/min, taking 1-2 g of the centrifuged bottom microorganism sample, repeatedly sampling and accumulating to 1-2 g when the centrifuged bottom microorganism sample is less than 1g, then adding a carbonic acid buffer solution with pH of 9.5-10.5 into the microorganism sample, adding 3.5-4.5 mL of the mixed solution into each 1g of the microorganism sample, then placing the mixed solution into an ice water bath for ultrasonic crushing, and finally centrifuging to take a supernatant;
(2) and (3) standard curve preparation: with NaNO2Preparing more than 7 groups of NO with the concentration of 0-30 mu mol/L2 -Taking more than 7 blank test tubes, respectively adding a carbonic acid buffer solution with the pH value of 9.5-10.5 and a hydroxylamine hydrochloride solution with the concentration of 50-100 mmol/L, wherein the adding amount of the carbonic acid buffer solution is 0.5-1 mL for every 1g of microorganism sample, the adding amount of the hydroxylamine hydrochloride solution is 0.1-0.3 mL for every 1g of microorganism sample, and then placing each group of solutions in a constant temperature of 25-35 DEG CHeating in warm water bath for 10-15 min, and then adding 0.5mL of NO with different concentrations into each group of solution2 -Shaking the standard solution uniformly, standing the solution for 18-22 min in a constant-temperature water bath at the temperature of 25-35 ℃, sequentially adding a sulfanilamide solution and an N-1-naphthyl ethylenediamine solution into each group of solution, wherein the sulfanilamide solution and the N-1-naphthyl ethylenediamine solution are obtained by respectively dissolving sulfanilamide solid and N-1-naphthyl ethylenediamine solid into 10-13 mol/L acetic acid solution, the concentrations of the sulfanilamide solution and the N-1-naphthyl ethylenediamine solution are respectively 55-60 mmol/L and 5-10 mmol/L, 0.8-1 mL of sulfanilamide solution and N-1-naphthyl ethylenediamine solution are added into each 1g of microbial sample, then placing the microbial sample into the water bath for developing for 20-30 min, then adding an extracting agent into each group of solution, mixing, centrifuging, collecting upper extract phase, measuring absorbance at 540nm, and taking the measured absorbance value as y value to obtain NO2-Taking the concentration as an x value, and obtaining a standard curve equation after linear fitting;
(3) setting a test group, a control group and a blank group, adding carbonic acid buffer solutions into the three groups respectively, shaking up, adding hydroxylamine hydrochloride solutions into the test group and the blank group, adding distilled water with the same volume into the control group, simultaneously placing the three groups in a constant-temperature water bath at 25-35 ℃ for heating for 10-15 min, then adding 0.5mL of supernatant into the test group and the control group, adding carbonic acid buffer solutions with the same volume into the blank group, shaking up respectively, standing in the constant-temperature water bath at 25-35 ℃ for 18-22 min, then sequentially adding sulfanilamide solution and N-1-naphthyl ethylenediamine solution into the test group, the control group and the blank group respectively, and developing in the water bath for 20-30 min, wherein the concentration and the adding amount of each solution are the same as those in the step (2);
(4) adding an extracting agent into the three groups of colored solutions obtained in the step (3), fully and uniformly mixing, respectively performing centrifugal treatment, respectively taking upper extraction phases, and measuring the absorbance of a test group, a control group and a blank group at 540nm, wherein the used extracting agent is the same as the step (2);
(5) the absorbance values measured in the step (4) are subjected to difference calculation and then are carried into the stepThe standard curve equation of step (2) is used to obtain NO in the test group corresponding to 540nm2 -Concentration, then calculating the O in the organism per unit mass according to a formula2 - .Content, namely completing the determination of superoxide anion free radicals in the microorganism, wherein the formula is
Wherein
NO in the test group corresponding to 540nm2 -The concentration of the active ingredients in the mixture is,V 1 to determine the volume of the sample to be tested in the test group,V 2 centrifuging the test group in the step (2) after ultrasonic crushing to obtain the total volume of the supernatant,
for the test group to test the concentration of the sample sludge solids,m 1 to determine the mass of the microbiological samples after centrifugation in step (2) of the test group,m 2 the mass of the sediment after ultrasonication and centrifugation in step (2) of the test group;
the extractant in the step (2) is dichloromethane, trichloromethane or toluene.
2. The method for measuring superoxide anion radicals in microorganisms according to claim 1, wherein: the ultrasonic crushing time in the step (1) is 2-3 min, and the centrifugal rotating speed is 10000-120000 r/min when the supernatant is obtained through centrifugation.
3. The method for measuring superoxide anion radicals in microorganisms according to claim 1, wherein: and (3) adding an extracting agent in the step (2) and then centrifuging for 3-5 min.
4. The method for measuring superoxide anion radicals in microorganisms according to claim 1, wherein: and (3) adding the extracting agent in the step (2) in a volume which is 0.8-1.2 times of the volume of each group of solution before adding.
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