Method for measuring chlorophyll content of microcystis aeruginosa in water

Abstract

The invention discloses a method for measuring the content of chlorophyll a of microcystis aeruginosa in water, and belongs to the technical field of environmental monitoring and water treatment. The method comprises the following steps: filtering algae in water by adopting a mixed cellulose ester membrane, soaking the filtered filter membrane in absolute ethyl alcohol, then carrying out ultrasonic treatment under the conditions of ice bath and darkness, centrifuging the obtained extracting solution to obtain supernate, measuring the absorbance of the supernate by using an ultraviolet spectrophotometer by taking absolute ethyl alcohol as a reference, and calculating the content of the algae in the water according to the absorbance of the supernate. And calculating the content of chlorophyll a in the algae. The method has the advantages of accurate and stable determination result, good reproducibility, safety, environmental protection, short test time, less required equipment and low test cost. The method is expected to be widely applied to conventional online monitoring of surface water quality, water eutrophication evaluation and the like in the future.

Description

Technical Field

[0001] The present invention belongs to the technical fields of environmental monitoring and water treatment, and particularly relates to a method for measuring the content of chlorophyll a of Microcystis aeruginosa in water. Background Art

[0002] Microcystis aeruginosa is a common cyanobacterium that easily multiplies in large numbers in eutrophic water bodies, forming algal blooms. Chlorophyll a is an important photosynthetic pigment in algal photosynthesis, and its content is an important indicator reflecting the biomass and growth status of Microcystis aeruginosa, which is of great significance for evaluating the degree of water eutrophication and studying the health status of the water ecosystem. Accurately measuring the content of chlorophyll a of Microcystis aeruginosa in water helps to deeply understand the growth law, photosynthesis mechanism of Microcystis aeruginosa and its role in the water ecosystem. At the same time, it also provides key technical support for the monitoring, early warning and treatment of water eutrophication. For example, by long-term monitoring of the change of chlorophyll a content, the growth trend of Microcystis aeruginosa in water can be detected in time, and corresponding prevention and control measures can be taken in advance to prevent the harm caused by algal bloom outbreaks to the water ecosystem, drinking water safety and human health.

[0003] Chlorophyll a belongs to synthetic natural low-molecular organic compounds, which are insoluble in water and soluble in organic solvents such as acetone, ethanol, chloroform, etc. At present, there are various methods for measuring the content of chlorophyll a of Microcystis aeruginosa in water, such as spectrophotometry, fluorescence spectrophotometry, high performance liquid chromatography, etc. These methods have their own advantages and disadvantages, and need to be selected and optimized according to specific scenarios in practical applications. The more commonly used one is the grinding-acetone extraction spectrophotometry. Its principle is based on the absorption characteristics of chlorophyll for light of specific wavelengths, and the content of chlorophyll a is calculated by measuring the absorbance. This method filters the water sample with an acetate fiber filter membrane to intercept the algae. After the filter membrane is dried at low temperature for 6-8 h, it is ground and extracted with 90% acetone multiple times, centrifuged to take the supernatant, and the content of chlorophyll a is measured and calculated with a spectrophotometer. However, there are also some problems in the actual application of this method. On the one hand, the pretreatment process is cumbersome. Especially the traditional manual grinding extraction not only takes a long time, but also may lead to the degradation of chlorophyll. During the repeated addition of solvents and transfer process, the solvent is easily adhered to the wall of the grinder, resulting in the loss of chlorophyll and affecting the accuracy of the results. On the other hand, during the extraction of chlorophyll a, the organic solvent acetone is highly toxic and volatile, which will potentially threaten the health of operators and the environment. It can be seen that the existing spectrophotometry is easily affected by background interference, resulting in errors in the measurement results. Therefore, it is urgent to optimize the existing spectrophotometry to improve the accuracy, efficiency and safety of detecting the content of chlorophyll a in algae. Summary of the Invention

[0004] To address the deficiencies in existing methods, the present invention proposes a method for measuring the chlorophyll a content of Microcystis aeruginosa in water. This method mainly improves the pigment extraction method by changing the types and concentrations of extraction agents and assisting with additional means, thereby enhancing the efficiency of detecting the chlorophyll a content in algae. This method is safe, harmless, highly accurate in results, and easy to operate.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for measuring the chlorophyll a content of Microcystis aeruginosa in water, comprising the following steps: filtering the algae in water using a mixed cellulose ester membrane, soaking the filtered membrane in absolute ethanol, then performing ultrasonic treatment under ice bath and dark conditions to fully extract the chlorophyll in the algal cells into absolute ethanol, centrifuging the obtained extract to obtain the supernatant, using absolute ethanol as a reference, and measuring the absorbance of the supernatant at 630, 645, 663, and 750 nm with a UV spectrophotometer to calculate the chlorophyll a content in the algae.

[0007] Further, the pore size of the mixed cellulose ester membrane is 0.45 μm.

[0008] Further, the specific operation steps of the ultrasonic treatment include: adopting an intermittent method of pausing for 1 minute every 2 minutes of ultrasonic treatment and treating for 1 hour at a power of 40 kHz and 100 W.

[0009] Further, the specific operation steps of the centrifugation are: centrifuging at a speed of 4000 r / min for 10 minutes.

[0010] Further, before filtering the algae in water using a mixed cellulose ester membrane, a magnesium carbonate suspension with a mass concentration of 1% is added to the water containing algae.

[0011] Furthermore, the volume ratio of the magnesium carbonate suspension to the water containing algae is 0.1∶100.

[0012] Further, the chlorophyll a content is calculated according to the following formula:

[0013]

[0014] In the formula: Chl-a—the concentration of chlorophyll a in algae (μg / L);

[0015] V1—the fixed volume of the extract in the centrifuge tube (mL);

[0016] D 750 、D 663 、D 645 、D 630 —the absorbance values measured at different characteristic wavelengths;

[0017] V—the volume of the filtered algal solution (L);

[0018] δ—the optical path length of the cuvette (cm).

[0019] Application of the method provided by the present invention in the field of on-line monitoring of surface water quality.

[0020] Application of the method provided by the present invention in the field of evaluation of water eutrophication.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] (1) The present invention proposes a technical method for measuring the content of chlorophyll a in Microcystis aeruginosa in water, which is easy to operate, especially suitable for rapid batch detection of a large number of samples in the laboratory, and can be used for real-time on-line monitoring of the content of chlorophyll a in Microcystis aeruginosa.

[0023] (2) Grinding is not required, which can greatly save manpower and reduce the loss of chlorophyll caused by repeated transfer of the extract, further ensuring the scientific reliability of the extraction process.

[0024] (3) Using absolute ethanol as the extractant, it has low toxicity, low volatility, and little harm to human safety, meeting the requirements of green environmental protection and pollution-free.

[0025] (4) The extract can be placed at room temperature without special storage in the refrigerator, which is easy to operate and reduces energy consumption.

[0026] (5) The ultrasonic technology can break algal cells more fully, accelerate the release of chlorophyll, extract more completely, and reduce the extraction time. By calibrating the wavelength of the ultraviolet spectrophotometer, preheating before use, and taking the average value of multiple measurements for each sample, not only the measurement process is optimized, but also the accuracy and reliability of the measurement results are effectively improved. Description of the Drawings

[0027] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 is the operation flow chart for measuring the content of chlorophyll a in Microcystis aeruginosa in water according to the present invention;

[0029] Figure 2 is the breakthrough curve showing the concentration of Microcystis aeruginosa in terms of the content of chlorophyll a under the experimental conditions of a specific filter column in Application Example 2 of the present invention; a is filter material 1, and b is filter material 2. Detailed Embodiments

[0030] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0035] An embodiment of the present invention provides a method for measuring the content of chlorophyll a in Microcystis aeruginosa in water, including the following steps: filtering the algae in water with a mixed cellulose ester membrane, soaking the filtered membrane in absolute ethanol, performing ultrasonic treatment under ice bath and dark conditions to fully release the chlorophyll in the algal cells into absolute ethanol, centrifuging the obtained extract to obtain the supernatant, using absolute ethanol as a reference, measuring the absorbance of the extract at 630, 645, 663, and 750 nm with a UV spectrophotometer, and calculating the content of chlorophyll a in the algae.

[0036] In the following preferred embodiment, the pore size of the mixed cellulose ester membrane is 0.45 μm.

[0037] In the following preferred embodiment, the specific operation steps of the ultrasonic treatment include: pausing for 1 minute every 2 minutes of ultrasonic treatment, and the total ultrasonic treatment time is 1 hour.

[0038] In the following preferred embodiment, the specific operation steps of the centrifugation are as follows: centrifuge at a speed of 4000 r / min for 10 min.

[0039] In the following preferred embodiment, before filtering algae in water with a mixed cellulose ester membrane, a magnesium carbonate suspension with a mass concentration of 1% is added to the water containing algae. The volume ratio of the magnesium carbonate suspension to the water containing algae is 0.1:100.

[0040] Exemplarily, the present invention is implemented by the following method:

[0041] (I) Enrichment treatment of algae samples

[0042] (1) Preparation of algal solution: Take out 100 mL of Microcystis aeruginosa solution in the logarithmic growth phase for use.

[0043] (2) Concentration of algal solution: Add 0.1 mL of magnesium carbonate suspension with a mass concentration of 1% to 100 mL of algal solution, shake well to prevent chlorophyll from being destroyed due to the acidic environment and play a role in protecting chlorophyll. Place a 0.45 μm pore size mixed cellulose ester membrane on the suction filtration device, pour in 100 mL of algal solution for suction filtration. During suction filtration, the negative pressure should not be too large, and end the suction filtration when the water sample just completely passes through the filter membrane. Blot the remaining moisture with filter paper. Three filter membranes are required for each water sample for three parallel experiments. Take down the filter membrane that has been fully enriched with algae after suction filtration, cut it into pieces and put it into a 10 mL centrifuge tube for use.

[0044] (II) Extraction of chlorophyll

[0045] Place the filter membrane that has been fully enriched with algae in step (I) into a 10 mL stoppered centrifuge tube, add 10 mL of absolute ethanol for soaking, and then adopt an intermittent ultrasonic method of ultrasonicating for 2 min and pausing for 1 min. Perform ultrasonic crushing treatment at 40 kHz and 100 W power in the dark, and place an ice pack in the ultrasonic cleaner to reduce the damage to chlorophyll caused by the heat released during the ultrasonic process through ice bath ultrasonic, with a constant temperature of 4 °C and a total ultrasonic treatment time of 1 h to obtain an extract.

[0046] (III) Calculation of chlorophyll a content

[0047] (1) Centrifuge to obtain supernatant: Centrifuge the extract obtained in step (II) with a high-speed centrifuge. The centrifugation conditions are set as: rotation speed 4000 r / min, time 10 min, to obtain a supernatant.

[0048] (2) Calculation of chlorophyll a content: Use absolute ethanol as the blank sample, zero the ultraviolet spectrophotometer, take the supernatant after centrifugation, and pour it into a quartz cuvette with a light path of 1 cm. Measure the absorbance values at the characteristic wavelengths of 630 nm, 645 nm, 663 nm, and 750 nm respectively. Note that if the absorbance value at 750 nm is greater than 0.005, it is necessary to filter it in time with a polytetrafluoroethylene organic phase needle filter with a pore size of 0.45 μm and then measure it.

[0049] The chlorophyll a content is calculated according to Equation 1-1 below:

[0050]

[0051] Where: Chl-a—the concentration of algal chlorophyll a (μg / L);

[0052] V1—the fixed volume of the extract in the centrifuge tube (mL);

[0053] D 750 、D 663 、D 645 、D 630 —the absorbance values measured at different characteristic wavelengths;

[0054] V—the volume of the filtered algal solution (L);

[0055] δ—the light path of the cuvette (cm).

[0056] The method provided by the present invention has accurate and stable measurement results, good reproducibility; is safe and environmentally friendly; has a short test time, requires few equipment, and has low test costs. It is expected to be widely used in the future for the routine on-line monitoring of surface water quality and the evaluation of water eutrophication.

[0057] In the present invention, the "room temperature" mentioned refers to 20-30 °C unless otherwise specified.

[0058] All raw materials used in the present invention are obtained by purchasing on the market.

[0059] The technical solution of the present invention is further described below through examples.

[0060] Figure 1 This is the operation flow chart for measuring the chlorophyll a content of Microcystis aeruginosa in water according to the present invention.

[0061] Example 1

[0062] A method for measuring the chlorophyll a content of Microcystis aeruginosa in water, comprising the following steps:

[0063] (I) Enrichment treatment of algal samples

[0064] (1) Preparation of algal solution: Take out 100 mL of Microcystis aeruginosa solution in the logarithmic growth phase for later use;

[0065] (2) Concentration of algal solution: Add 0.1 mL of magnesium carbonate suspension with a mass concentration of 1% to 100 mL of algal solution, shake well to prevent chlorophyll from being destroyed due to the acidic environment and play a role in protecting chlorophyll. Place a 0.45 μm pore size mixed cellulose ester membrane on the suction filtration device, pour 100 mL of algal solution for suction filtration. During suction filtration, the negative pressure should not be too large, and end the suction filtration when the water sample just completely passes through the filter membrane. Blot the remaining water with filter paper. Prepare 3 filter membranes for each water sample for 3 parallel experiments. Take down the filter membrane that has been fully enriched with algae after suction filtration, cut it into pieces and put it into a 10 mL centrifuge tube for later use;

[0066] (II) Extraction of chlorophyll

[0067] Place the filter membrane that has fully enriched algae in step (I) into a 10 mL stoppered centrifuge tube, add 10 mL of absolute ethanol, and then perform ultrasonic fragmentation treatment in an intermittent ultrasonic mode of ultrasonic for 2 min and pause for 1 min. Conduct the treatment in the dark at a frequency of 40 kHz and a power of 100 W, and place an ice pack in the ultrasonic cleaner to reduce the damage to chlorophyll caused by the heat released during ultrasonic through ice bath ultrasonic. Keep the constant temperature at 4 °C, and the total ultrasonic treatment time (including the pause time) is 1 h to obtain the extract.

[0068] (III) Calculation of chlorophyll a content

[0069] (1) Centrifuge and take the supernatant: Use a high-speed centrifuge to centrifuge the extract obtained in step (II). Set the centrifuge conditions as: rotation speed 4000 r / min, time 10 min to obtain the supernatant;

[0070] (2) Calculation of chlorophyll a content: Use absolute ethanol as the blank sample to zero the ultraviolet spectrophotometer. After centrifugation, take the supernatant and pour it into a quartz cuvette with an optical path of 1 cm, and measure the absorbance values at the characteristic wavelengths of 630 nm, 645 nm, 663 nm, and 750 nm respectively.

[0071] The chlorophyll a content is calculated according to the following formula 1-1:

[0072]

[0073] In the formula: Chl-a—the concentration of algal chlorophyll a (μg / L);

[0074] V1—the fixed volume of the extract in the centrifuge tube (mL);

[0075] D 750 、D 663 、D 645 、D630 —Absorbance values measured at different characteristic wavelengths;

[0076] V—Volume of the filtered algal solution (L);

[0077] δ—Optical path of the cuvette (cm).

[0078] Comparative Example 1

[0079] Same as Example 1, except that in step (ii), the total ultrasonic treatment time is 30 min.

[0080] Comparative Example 2

[0081] Same as Example 1, except that in step (ii), absolute ethanol is replaced with ethanol with a volume concentration of 90% in equal volume.

[0082] Comparative Example 3

[0083] Same as Example 1, except that in step (ii), absolute ethanol is replaced with ethanol with a volume concentration of 75% in equal volume.

[0084] Comparative Example 4

[0085] Same as Example 1, except that in step (ii), ultrasonic treatment is replaced with shaking and crushing treatment. The specific steps are as follows: The filter membrane completely enriched with algae in step (i) is placed in a 10 mL stoppered centrifuge tube, 10 mL of absolute ethanol is added, and then it is shaken and crushed in a constant temperature oscillator at 200 r / min and 4 °C for 1 h to obtain an extract.

[0086] Comparative Example 5

[0087] Same as Example 1, except that in step (ii), ultrasonic treatment is replaced with shaking and crushing treatment. The specific steps are as follows: The filter membrane completely enriched with algae in step (i) is placed in a 10 mL stoppered centrifuge tube, 10 mL of absolute ethanol is added, and then it is shaken and crushed in a constant temperature oscillator at 200 r / min and 4 °C for 30 min to obtain an extract.

[0088] Comparative Example 6

[0089] Same as Example 1, except that in step (ii), ultrasonic treatment is not performed. The specific steps are as follows: The filter membrane completely enriched with algae in step (i) is placed in a 10 mL stoppered centrifuge tube, 10 mL of absolute ethanol is added, and it is refrigerated in a refrigerator (4 °C) for 1 h to obtain an extract.

[0090] Comparative Example 7

[0091] Same as Example 1, except that in step (ii), ultrasonic treatment is not carried out, and the soaking time in absolute ethanol is 30 min. The specific steps are as follows: Place the filter membrane with completely enriched algae in step (i) into a 10 mL stoppered centrifuge tube, add 10 mL of absolute ethanol, and refrigerate in a refrigerator (4 °C) for 30 min to obtain an extract.

[0092] Control Group 1

[0093] A method for measuring the chlorophyll a content of Microcystis aeruginosa in water, comprising the following steps:

[0094] (i) Enrichment treatment of algal samples

[0095] (1) Preparation of algal solution: Take out 100 mL of Microcystis aeruginosa solution in the logarithmic growth phase for use.

[0096] (2) Concentration of algal solution: Add 0.1 mL of a 1% magnesium carbonate suspension to 100 mL of the algal solution, shake well to prevent chlorophyll from being destroyed due to the acidic environment and play a role in protecting chlorophyll. Place a 0.45 μm pore size mixed cellulose ester membrane on the suction filtration device, pour in 100 mL of the algal solution for suction filtration. The negative pressure during suction filtration should not be too large, and end the suction filtration when the water sample just completely passes through the filter membrane. Blot the remaining moisture with filter paper. Three filter membranes are required for each water sample for three parallel experiments. Remove the filter membrane with completely enriched algae after suction filtration, cut it into pieces and put it into a 10 mL centrifuge tube for use.

[0097] (ii) Extraction of chlorophyll

[0098] (1) Preparation of acetone solution: Measure 90 mL of acetone with a 100 mL measuring cylinder and place it in a 250 mL beaker, add 10 mL of deionized water to obtain an acetone solution with a volume concentration of 90%.

[0099] (2) Cut the filter membrane with completely enriched algae in step (i) into pieces and put them into a mortar. Add 2 mL of the acetone solution with a volume concentration of 90% to the mortar and grind thoroughly until the filter membrane is ground into pieces. Then transfer the extract to a 10 mL stoppered centrifuge tube, rinse the mortar with the acetone solution with a volume concentration of 90%, and transfer the rinsing solution into the centrifuge tube. After multiple rinses, make up the volume of the liquid in the centrifuge tube to 10 mL with the acetone solution with a volume concentration of 90%, and seal it with a sealing film. After shaking vigorously, place it in a 4 °C refrigerator and extract chlorophyll in the dark for 20 h to fully extract chlorophyll. Shake it once at intervals, and shake it a total of 3 times.

[0100] (iii) Calculation of chlorophyll a content

[0101] (1) Centrifugation to obtain supernatant: The extraction solution fully extracted in step (2) is re - diluted to 10 mL with acetone solution with a volume concentration of 90% at room temperature, and then the solution is centrifuged using a high - speed centrifuge. The centrifugation conditions are set as follows: rotation speed 4000 r / min, time 10 min, to obtain the supernatant;

[0102] (2) Calculation of chlorophyll a content: Acetone solution with a volume concentration of 90% is used as a blank sample to zero the ultraviolet spectrophotometer. After centrifugation, the supernatant is taken and poured into a quartz cuvette with an optical path of 1 cm, and the absorbance values at characteristic wavelengths of 630 nm, 645 nm, 663 nm, and 750 nm are measured respectively.

[0103] The accuracy and reproducibility of this method are verified by comparing different methods. The results of chlorophyll a content measured by different methods in Specific Example 1, Comparative Examples 1 - 7, and Control Group 1 are shown in Table 1.

[0104] Table 1 Comparison of chlorophyll a content measured by different methods

[0105]

[0106]

[0107] As shown in the results of Table 1, extending the extraction time helps the extraction of chlorophyll a, and the extraction effect is: 1 h > 30 min. Under the same extraction time, changing the treatment method is beneficial to the extraction of chlorophyll a, and the extraction effect is: ultrasonic > shaking > simple refrigeration. Under the optimal extraction time and treatment method, the influence of the extractant concentration on the extraction effect is further explored. The higher the extractant concentration, the better the extraction effect, specifically: absolute ethanol > 90% ethanol > 75% ethanol. In summary, under various treatment methods, the treatment effect of "absolute ethanol - soaking for 1 h - ultrasonic" in Example 1 is the best. The result is relatively close to that of the currently most commonly used acetone low - temperature refrigeration method for 20 h (Control Group 1). The method adopted in Example 1 of the present invention greatly shortens the sample treatment time while ensuring the accuracy of the detection results. Moreover, the methods provided by the present invention do not require manual grinding, which can save labor, and at the same time can reduce the loss of chlorophyll caused by transferring the extraction solution, and the result reproducibility is good. Ethanol is used as the extractant, which is safe and non - toxic to the human body.

[0108] Application Example 1

[0109] According to the method of Example 3, it is used to detect the chlorophyll a content in actual natural water bodies. Taking the Qingnian Lake in the Beiyangyuan Campus of Tianjin University as an example, the specific steps are as follows:

[0110] Take 100 mL of water sample from the Youth Lake for standby, add 0.1 mL of magnesium carbonate suspension with a mass concentration of 1%, and shake well. Place a 0.45 μm pore size mixed cellulose ester membrane on the suction filtration device, pour 100 mL of algal solution for suction filtration. After suction filtration, place the filter membrane in a 10 mL stoppered centrifuge tube, directly soak the filter membrane that has intercepted the algae with 10 mL of absolute ethanol, perform ultrasonic fragmentation treatment at a frequency of 40 kHz and a power of 100 W, adopt an intermittent ultrasonic method of pausing for 1 minute every 2 minutes of ultrasonic treatment, add an ice bag for ice bath treatment, place it in the dark for extraction for 1 hour and then take it out. The obtained extract is centrifuged at a speed of 4000 r / min for 10 minutes, take the supernatant, use a quartz cuvette with an optical path of 1 cm, and use absolute ethanol as a blank control to measure the absorbance values at wavelengths of 630, 647, 664, and 750 nm respectively. Calculate the chlorophyll a content according to formula (1-1). Prepare 3 filter membranes for each water sample for 3 parallel experiments, and the results are shown in Table 2.

[0111] Table 2 Measured chlorophyll a content in the water of the Youth Lake in Application Example 1

[0112]

[0113] It can be seen from the results in Table 2 that the content of chlorophyll a in the lake water is 29.132 μg / L, exceeding the specified 10 μg / L in the eutrophication judgment standard, and special attention should be paid to the risk of algal blooms.

[0114] Application Example 2

[0115] According to the method provided in Example 1, study the migration behavior of Microcystis aeruginosa in a certain filter material. The specific steps are as follows:

[0116] 1) Preparation of solution: Prepare a Microcystis aeruginosa solution with a concentration of 2×10 6 cells / mL, and the background solution is 0.1 mM KCl solution;

[0117] 2) Column migration experiment: Fill filter material 1 (quartz sand) and filter material 2 (manganese sand) with a size of 30 - 35 mesh into a filter column with an inner diameter of 1.6 cm and a length of 15 cm by wet packing method, with a pH of 6.5 - 7.5 and a temperature of room temperature. Send the prepared Microcystis aeruginosa solution to the filter column at a constant flow rate of 5 mL / min. After continuously introducing it for 14 minutes, rotate the four-way valve and introduce the background solution, continuously introduce it for 14 minutes, and collect the water samples from the outlet of the filter column every 56 s;

[0118] 3) Concentration of water sample: Add 0.0046 mL of magnesium carbonate suspension with a mass concentration of 1% to 4.6 mL of water sample, shake well to reduce the destruction of chlorophyll and protect chlorophyll; use a filter membrane with a pore size of 0.45 μm to perform suction filtration on the water sample. After suction filtration, remove the filter membrane that has been fully enriched with algae, fold it in half and place it in a 10 mL centrifuge tube for later use;

[0119] 4) Extraction of chlorophyll a: Cut the filter membrane into pieces and put them into a centrifuge tube, add anhydrous ethanol to make the volume up to 10 mL and seal it with a sealing film. Perform ultrasonic fragmentation treatment at a frequency of 40 kHz and a power of 100 W. Pause for 1 minute every 2 minutes of ultrasonic treatment, and at the same time perform ice bath. After extracting in the dark for 1 hour, take it out;

[0120] 5) Calculation of chlorophyll a content: Re - dilute the fully extracted extract with anhydrous ethanol to 10 mL at room temperature. Centrifuge and take the supernatant, with the parameters set as: 4000 r / min, 10 min. Use anhydrous ethanol as a blank sample to zero the ultraviolet spectrophotometer. Pour the centrifuged supernatant into a quartz cuvette with a light path of 1 cm, and measure the absorbance at wavelengths of 630 nm, 645 nm, 663 nm, and 750 nm respectively.

[0121] The chlorophyll content is calculated using formula (1 - 1).

[0122] Substitute the absorbances measured at the characteristic wavelengths of each water sample into formula (1 - 1) to calculate the chlorophyll a content of Microcystis aeruginosa in the effluent water samples at each time node. The experimental results are shown in Figure 2 .

[0123] From Figure 2 it can be seen that the method provided in Example 1 of the present invention can accurately and quickly measure the chlorophyll a content of algae, and the repeatability and stability of the measurement results are good.

[0124] In summary, the method proposed by the present invention can be used to measure the chlorophyll a content of Microcystis aeruginosa, and in particular, it can be used for research on algal biomass represented by chlorophyll a in conventional water quality indicators in the future. Through improvement, it is expected to achieve on - line monitoring of chlorophyll a.

[0125] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for measuring the chlorophyll a content of Microcystis aeruginosa in water, characterized in that, It includes the following steps: filtering algae in water with a mixed cellulose ester membrane, soaking the filtered membrane in absolute ethanol, then performing ultrasonic treatment under ice bath and dark conditions, centrifuging the obtained extract to obtain the supernatant, measuring the absorbance of the supernatant with a UV spectrophotometer, and calculating the content of chlorophyll a in the algae.

2. The method for measuring the chlorophyll a content of Microcystis aeruginosa in water according to claim 1, wherein The pore size of the mixed cellulose ester membrane is 0.45 μm.

3. The method for measuring the chlorophyll a content of Microcystis aeruginosa in water according to claim 1, characterized in that, The specific operation steps of the ultrasonic treatment include: adopting an intermittent mode of pausing for 1 minute every 2 minutes of ultrasonic treatment, and treating for 1 hour at a power of 40 kHz and 100 W.

4. The method for measuring the chlorophyll a content of Microcystis aeruginosa in water according to claim 1, wherein The specific operation steps of the centrifugation are: centrifuging at a rotation speed of 4000 r / min for 10 minutes.

5. The method for measuring the chlorophyll a content of Microcystis aeruginosa in water according to any one of claims 1 to 4, characterized in that, Before filtering algae in water with a mixed cellulose ester membrane, adding a magnesium carbonate suspension with a mass concentration of 1% to the water containing algae.

6. The method for measuring the chlorophyll a content of Microcystis aeruginosa in water according to claim 5, characterized in that, The volume ratio of the magnesium carbonate suspension to the water containing algae is 0.1∶100.

7. The method for measuring the chlorophyll a content of Microcystis aeruginosa in water according to claim 1, wherein The content of chlorophyll a is calculated according to the following formula: In the formula: Chl-a—the concentration of chlorophyll a in algae (μg / L); V1—the fixed volume of the extract in the centrifuge tube (mL); D 750 、D 663 、D 645 、D 630 —Absorbance values measured at different characteristic wavelengths; V—the volume of the filtered algae solution (L); δ—the optical path of the cuvette (cm).

8. Application of the method according to any one of claims 1-7 in the field of on-line monitoring of surface water quality.

9. Application of the method according to any one of claims 1-7 in the field of evaluation of water eutrophication.

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