阅读说明：本技术 一种活性污泥碱性磷酸酶活性测定方法 (Method for determining activity of alkaline phosphatase in activated sludge ) 是由 赵林 龙莎 于 2019-10-09 设计创作，主要内容包括：本发明公开一种活性污泥碱性磷酸酶活性测定方法,主要步骤为：(1)标准曲线绘制、(2)样品测定和(3)碱性磷酸酶活性计算；所述步骤(2)样品测定分为酶反应和测试分析两部分。酶反应具体步骤为：取5mL活性污泥混合液,加2mL甲苯,37℃下200r/min水浴振荡15min后,加入10mL 0.5％磷酸苯二钠,仔细摇匀后,37℃下140r/min水浴振荡培养16h；测试分析具体步骤为：将酶反应液全部转移至50mL比色管,加蒸馏水定容至50mL,使甲苯在刻度线以上,摇匀并静置5min使液面分层,取下层反应液,亲水PTFE滤膜过滤；吸取3mL滤液于50mL比色管中,每管加入5mL pH9.4硼酸盐缓冲液和200μL氯代二溴对苯醌亚胺试剂,显色后稀释至刻度,摇匀；30min后在分光光度计上610nm处比色。(The invention discloses a method for measuring the activity of alkaline phosphatase in activated sludge, which mainly comprises the following steps: (1) drawing a standard curve, (2) measuring a sample and (3) calculating the activity of alkaline phosphatase; the sample determination in the step (2) is divided into two parts of enzyme reaction and test analysis. The enzyme reaction comprises the following specific steps: taking 5mL of activated sludge mixed solution, adding 2mL of toluene, oscillating in a water bath at 37 ℃ and 200r/min for 15min, adding 10mL of 0.5% disodium phenyl phosphate, carefully shaking up, and oscillating and culturing in a water bath at 37 ℃ and 140r/min for 16 h; the specific steps of the test analysis are as follows: transferring all enzyme reaction liquid to a 50mL colorimetric tube, adding distilled water to a constant volume of 50mL, enabling toluene to be above a scale mark, shaking up and standing for 5min to enable the liquid level to be layered, taking down the reaction liquid at the lower layer, and filtering with a hydrophilic PTFE filter membrane; 3mL of filtrate is sucked into a 50mL colorimetric tube, 5mL of pH9.4 borate buffer solution and 200 mu L of chloro dibromo-p-benzoquinone imine reagent are added into each tube, and the tube is diluted to scale after color development and shaken up; after 30min the colour was taken at 610nm on a spectrophotometer.)

1. The method for measuring the alkaline phosphatase activity of the activated sludge is characterized by comprising three parts, namely (1) drawing a standard curve, (2) measuring a sample and (3) calculating the alkaline phosphatase activity;

the sample determination in the step (2) is divided into two parts of enzyme reaction and test analysis:

wherein the enzyme reaction comprises the following specific steps: placing 5mL of the activated sludge mixed solution into a 50mL triangular flask with a plug, adding 2mL of toluene, oscillating in a water bath at 37 ℃ for 15min at 200r/min, adding 10mL of 0.5% disodium phenyl phosphate (prepared by a pH9.4 borate buffer solution), carefully shaking uniformly, and performing oscillation culture in a water bath at 37 ℃ for 140r/min for 16 h;

the method comprises the following specific steps of: transferring all enzyme reaction liquid to a 50mL colorimetric tube, adding distilled water to a constant volume of 50mL, enabling toluene to be above a scale mark, shaking up and standing for 5min to enable the liquid level to be layered, taking down the reaction liquid at the lower layer, and filtering with a 0.45-micrometer hydrophilic PTFE filter membrane; 3mL of filtrate is sucked into a 50mL colorimetric tube, 5mL of pH9.4 borate buffer solution and 200 mu L of chloro dibromo-p-benzoquinone imine reagent are added into each tube, and the tube is diluted to scale after color development and shaken up; after 30min the colour was taken at 610nm on a spectrophotometer.

2. The method for measuring the activity of the activated sludge alkaline phosphatase according to claim 1, wherein the standard curve in the step (1) is drawn as follows:

1g of redistilled phenol is dissolved in 1L of distilled water to obtain 1g/L of phenol stock solution. Taking 10mL of phenol stock solution, and diluting the phenol stock solution to 1L by using distilled water to obtain 10mg/L of phenol working solution;

weighing 0.125g of chloro dibromo-p-benzoquinone imine, dissolving in 10mL of 96% ethanol solution, storing in a brown bottle at 4 ℃, and using the stored yellow solution before the brown color is changed;

taking 1, 3, 5, 7, 9, 11 and 13mL of phenol working solution, putting the working solution into a 50mL colorimetric tube, adding 5mL of pH9.4 borate buffer solution and 200 mu L of chloro dibromo-p-benzoquinone imine reagent into each colorimetric tube, diluting to scale after color development, and shaking up;

after 30min, carrying out color comparison at 610nm on a spectrophotometer;

and (3) drawing by taking the phenol concentration as an abscissa and the absorbance as an ordinate to obtain a standard curve, and performing linear regression on the standard curve to obtain a standard equation.

3. The method for determining activated sludge alkaline phosphatase activity according to claim 1, wherein in the step (3), the alkaline phosphatase activity is calculated by:

alkaline phosphatase activity is expressed in milligrams of phenol released in 1g (dry weight) of activated sludge after 16 h. The absorbance at 610nm is obtained through the sample determination in the previous step, and the absorbance is substituted into a standard equation to obtain the phenol concentration c; and substituting the phenol concentration, the sludge concentration and other indexes of the activated sludge sample into a formula (1), and calculating to obtain the activated sludge alkaline phosphatase activity.

c-concentration of phenol, mg. mL, according to the Standard equation ^-1；

V-color development liquid volume, 50 mL;

ts-division multiple, 50/3;

m-sludge dry weight, g;

m is 5mL multiplied by sludge concentration MLSS;

t－16h。

Technical Field

The invention belongs to the technical field of microbial activity determination, and particularly relates to a method for determining the activity of alkaline phosphatase of activated sludge in a wastewater biological treatment process.

Background

Phosphatase is a kind of phosphate ester hydrolase, and the main forms are alkaline phosphatase, acid phosphatase and phosphodiesterase, which are widely present in nature, such as human body, animals and plants, soil, sediments, sludge, etc. Alkaline phosphatase is a non-specific phosphate hydrolase, has high activity under alkaline conditions, can catalyze the hydrolysis of phosphate and the degradation of polyphosphate, participates in the metabolic activity of organic matters such as protein, polysaccharide, lipid, nucleic acid and the like, and plays an important role in the phosphorus metabolism in nature.

The biological sewage treating process mainly uses active sludge as main component. After the organophosphorus compounds in the sewage are hydrolyzed by extracellular alkaline phosphatase, the organophosphorus compounds can be absorbed and utilized by microorganisms. Therefore, the alkaline phosphatase activity of the activated sludge can be used for expressing the capacity of the activated sludge for decomposing organic phosphorus. Understanding the activity of the activated sludge alkaline phosphatase is helpful for further understanding the biodegradation process and the biological phosphorus removal process of organic matters, thereby providing an idea for upgrading and modifying the sewage treatment process. However, most of the current studies are directed to soil, water, sediments, etc., and only a few studies focus on the alkaline phosphatase activity of the wastewater treatment process. The method for measuring the alkaline phosphatase activity is provided aiming at soil samples in the early stage, however, the properties and the compositions of the soil and the activated sludge are different, and a standard method for measuring the alkaline phosphatase activity of the activated sludge is not provided at present.

Disclosure of Invention

The invention provides a method for measuring the activity of alkaline phosphatase in activated sludge, which refers to the method for measuring the activity of the alkaline phosphatase in soil, but is modified according to the characteristics of the activated sludge, so that the operability and the accuracy of the activity measurement of the alkaline phosphatase in the activated sludge are improved.

The technical scheme of the invention is that the method for measuring the activity of the alkaline phosphatase of the activated sludge comprises three parts, namely drawing a standard curve, measuring a sample and calculating the activity of the alkaline phosphatase.

(1) And (6) drawing a standard curve. Weighing 1g of redistilled phenol and dissolving in 1L of distilled water to obtain 1g/L of phenol stock solution. 10mL of phenol stock solution is diluted to 1L by distilled water to obtain 10mg/L of phenol working solution. 0.125g of dibromop-benzoquinone chloride imine chloride is weighed and dissolved in 10mL of 96 percent ethanol solution, and the solution is stored in a brown bottle and stored at 4 ℃, and the stored yellow solution can be used before being browned. Taking 1, 3, 5, 7, 9, 11 and 13mL of phenol working solution, placing the phenol working solution into a 50mL colorimetric tube, adding 5mL of pH9.4 borate buffer solution and 200 mu L of chlorodibromide p-benzoquinone imine reagent into each colorimetric tube, diluting to scale after color development, and shaking up. After 30min the colour was taken at 610nm on a spectrophotometer. And (5) drawing by taking the phenol concentration as an abscissa and the absorbance as an ordinate to obtain a standard curve. And performing linear regression on the standard curve to obtain a standard equation.

(2) And (4) sample determination. The sample determination is divided into two parts of enzyme reaction and test analysis.

Wherein the enzyme reaction comprises the following specific steps: placing 5mL of the activated sludge mixed solution into a 50mL triangular flask with a plug, adding 2mL of toluene, oscillating in a water bath at 37 ℃ for 15min at 200r/min, adding 10mL of 0.5% disodium phenyl phosphate (prepared by a pH9.4 borate buffer solution), shaking uniformly, and performing oscillation culture in a water bath at 37 ℃ for 140r/min for 16 h.

The specific steps of the test analysis are as follows: transferring all the enzyme reaction solution to a 50mL colorimetric tube, adding distilled water to a constant volume of 50mL, enabling toluene to be above a scale mark, shaking up and standing for 5min to enable the liquid level to be layered, taking down the reaction solution at the lower layer, and filtering with a 0.45-micrometer hydrophilic PTFE filter membrane. 3mL of the filtrate was taken out of a 50mL colorimetric tube, 5mL of pH9.4 borate buffer solution and 200. mu.L of chlorodibromobenzoquinoneimine reagent were added to each tube, and the mixture was diluted to the scale after color development and shaken up. After 30min the colour was taken at 610nm on a spectrophotometer.

(3) Calculation of alkaline phosphatase Activity

Alkaline phosphatase activity is expressed in milligrams of phenol released in 1g (dry weight) of activated sludge after 16 h. And (5) measuring the sample in the previous step to obtain the absorbance at 610nm, and substituting the absorbance into a standard equation to obtain the phenol concentration c. And substituting the phenol concentration, the sludge concentration and other indexes of the activated sludge sample into a formula (1), and calculating to obtain the activated sludge alkaline phosphatase activity.

c-concentration of phenol, mg. mL, according to the Standard equation ^-1；

V-color development liquid volume, 50 mL;

ts-division multiple, 50/3;

m-sludge dry weight, g;

m is 5mL multiplied by sludge concentration MLSS;

t－16h。

advantageous effects

The invention provides a method for measuring alkaline phosphatase activity of activated sludge by referring to a method for measuring the activity of soil phosphatase, which aims at the characteristics of the activated sludge, provides specific guidance for an enzyme reaction system, reaction conditions, reaction time, analysis steps and a calculation mode, and the standard deviation of the measurement result of the alkaline phosphatase activity is less than or equal to 0.433 mg.g ^-1·h ^-1。

The method for measuring the activity of the alkaline phosphatase of the activated sludge is easy to operate, good in accuracy and high in repeatability.

Drawings

FIG. 1 is a standard curve.

Detailed Description

The present invention will be described in detail with reference to specific examples. The examples are intended to better enable those skilled in the art to better understand the present invention and are not intended to limit the present invention in any way. The specific implementation method comprises three steps: standard curve drawing, sample determination and alkaline phosphatase activity calculation

(1) And (6) drawing a standard curve. Weighing 1g of redistilled phenol and dissolving in 1L of distilled water to obtain 1g/L of phenol stock solution. 10mL of phenol stock solution is diluted to 1L by distilled water to obtain 10mg/L of phenol working solution. 0.125g of dibromop-benzoquinone chloride imine chloride is weighed and dissolved in 10mL of 96 percent ethanol solution, and the solution is stored in a brown bottle and stored at 4 ℃, and the stored yellow solution can be used before being browned. Taking 1, 3, 5, 7, 9, 11 and 13mL of phenol working solution, placing the phenol working solution into a 50mL colorimetric tube, adding 5mL of pH9.4 borate buffer solution and 200 mu L of chlorodibromide p-benzoquinone imine reagent into each colorimetric tube, diluting to scale after color development, and shaking up. After 30min the colour was taken at 610nm on a spectrophotometer and the results are shown in Table 1.

TABLE 1 Absorbance of Standard Curve

Plotting the phenol concentration as abscissa and the absorbance as ordinate to obtain a standard curve, the results are shown in FIG. 1:

performing linear regression on the standard curve to obtain a standard equation:

y＝252.68x+0.0021 (2)

R ²＝0.9998

wherein x is the phenol concentration (mg/mL) and y is the absorbance.

(2) And (4) sample determination. The sample determination is divided into two parts of enzyme reaction and test analysis.

Wherein the enzyme reaction comprises the following specific steps: placing 5mL of the activated sludge mixed solution into a 50mL triangular flask with a plug, adding 2mL of toluene, oscillating in a water bath at 37 ℃ for 15min at 200r/min, adding 10mL of 0.5% disodium phenyl phosphate (prepared by a pH9.4 borate buffer solution), shaking uniformly, and performing oscillation culture in a water bath at 37 ℃ for 140r/min for 16 h.

The specific steps of the test analysis are as follows: transferring all the enzyme reaction solution to a 50mL colorimetric tube, adding distilled water to a constant volume of 50mL, enabling toluene to be above a scale mark, shaking up and standing for 5min to enable the liquid level to be layered, taking down the reaction solution at the lower layer, and filtering with a 0.45-micrometer hydrophilic PTFE filter membrane. 3mL of the filtrate was taken out of a 50mL colorimetric tube, 5mL of pH9.4 borate buffer solution and 200. mu.L of chlorodibromobenzoquinoneimine reagent were added to each tube, and the mixture was diluted to the scale after color development and shaken up. After 30min the colour was taken at 610nm on a spectrophotometer.

4 samples of activated sludge from different sources were taken and assayed according to the enzymatic reaction and assay procedures described above, and two replicates were set up, with the results shown in Table 2.

(3) Calculation of alkaline phosphatase Activity

Alkaline phosphatase activity is expressed in milligrams of phenol released in 1g (dry weight) of activated sludge after 16 h. The absorbance at 610nm was obtained by the sample measurement in the previous step, and this was substituted into the standard equation (2), to obtain the phenol concentration c. And (3) respectively substituting the phenol concentration, the sludge concentration and other indexes of the 4 parts of activated sludge samples into a formula (3), and calculating to obtain the alkaline phosphatase activity of each of the 4 parts of activated sludge samples.

c-according to the standard equationThe obtained phenol concentration, mg. mL ^-1；

V-color development liquid volume, 50 mL;

ts-division multiple, 50/3;

m-sludge dry weight, g;

m is 5mL multiplied by sludge concentration MLSS;

t－16h。

the results are shown in Table 2, and the alkaline phosphatase activities of 4 activated sludge samples were 5.31, 9.65, 6.15 and 5.63mg g ^-1·h ^-1The standard deviation of the result is less than or equal to 0.433mg g ^-1·h ^-1. The method for measuring the activity of the alkaline phosphatase of the activated sludge provided by the invention has the advantages of good accuracy and high repeatability.

TABLE 2 activated sludge sample alkaline phosphatase Activity

a: mean ± standard deviation;

b, expressed as milligrams of phenol released in 1g of activated sludge after 16 h.

It should be understood that the embodiments and examples discussed herein are illustrative only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.