Determination of algae in eutrophic lakes
First, the purpose of the experiment
The eutrophic lake is polluted by water, especially nitrogen and phosphorus, which causes the algae to grow vigorously. The concentration of chlorophyll a representing algae in such water bodies is often greater than 10 micrograms per liter. In this experiment, the eutrophication of algae was determined by measuring the concentration of chlorophyll a in different water bodies.
Second, equipment and supplies
1. Spectrophotometer (wavelength selection is greater than 750 nm, accuracy is 0.5-2 nm).
2. Cuvette (1cm; 4cm).
3, desktop centrifuge (3500r / min)
4, centrifuge tube (15ml with scale and stopper); refrigerator
5. Homogenizer or small mortar.
6, Chua filter; filter membrane (0.45 micrograms, diameter 47mm).
7, vacuum pump (maximum pressure does not exceed 300kpa).
8. MgCO3 suspension: lg MgCO3 fine powder was suspended in 100 ml of distilled water.
9. 90% acetone solution: 90 parts acetone + 10 parts distilled water.
10, water sample: two different levels of pollution of the lake water sample each 2L.
Third, methods and steps
1. According to the sampling method of phytoplankton, the lake and the reservoir sample 500ml, and the pond 300ml. The sampling point and water collection time are the same as "phytoplankton".
2. Cleaning glass instruments: The glass instruments used in the whole experiment should be cleaned with detergent, especially the decomposition of chlorophyll a caused by acidic conditions.
3. Filter the water sample; install the filter on the Chua filter, and measure 50-500 ml of each test water sample under reduced pressure. Add 0.2 ml of MgCO3 suspension before the water sample remains a few milliliters, shake until the water sample is drained. The addition of MgCO3 can increase the retention of algae cells on the membrane while preventing the decomposition of chlorophyll a during extraction. If the filtered algae filter membrane cannot be extracted immediately, it should be placed in a desiccator and stored in a dark place (4 ° C) in a dark place for a maximum of 48 hours. 4. Extraction; place the filter in a homogenizer or small mortar, add 2-3 ml of 90% acetone solution, and homogenize to break the algae cells. Then, the homogenate was transferred to a graduated centrifuge tube with a pipette, rinsed twice with 5 ml of 90% acetone, and finally 90% acetone was added to the centrifuge tube to make the total volume in the tube 10 ml. Plug the plug tightly and put a light shield on the outside of the tube, shake it fully, and put it in the refrigerator for 18-24 hours.
5. Centrifugation: After the extraction is completed, place the centrifuge tube on a tabletop centrifuge at 3500r/min, centrifuge for 10min, remove the centrifuge tube, transfer the supernatant into the graduated centrifuge tube with a pipette, plug the stopper, and centrifuge at 3500r/min for 10min. . Record the volume of the extract correctly.
6. Determination of optical density: Algae chlorophyll a has its unique absorption spectrum (663 nm), so its content can be measured by spectrophotometry. The extract was transferred to a 1 cm cuvette with a pipette, and the optical density value (OD) of the extract was measured at a wavelength of 750, 663, 645, and 630 nm with a 90% acetone solution as a blank. Note: The OD663 value for sample extraction is required to be between 0.2 and 1.0. If it is not within this range, the cuvette should be exchanged or the amount of filtered water should be changed. When the OD663 is less than 0.2, a wider cuvette should be used or the amount of water should be increased. When the OD663 is greater than 1.0, the extract can be diluted or the water sample can be reduced by excessive filtration, and a 1cm cuvette colorimetric color can be used.
7. Calculation of chlorophyll a concentration: subtract the optical density value (OD750) at 750 nm from the optical density value (OD663 OD645 OD630) of the sample extract at 663, 645, and 630 nm, which is a non-selective Substrate light absorption correction value. The formula for calculating chlorophyll a concentration is as follows:
(1) The concentration of chlorophyll a in the sample extract is Ca: Ca (μg/L) = 11.64 (OD663-OD750) - 2.16 (OD645-OD750) + 0.1 (OD630-OD750)
(1)  The concentration of chlorophyll a in water samples is: chlorophyll a (μg/L) = Ca × v / (V × L)
(2) Chlorophyll a (μg/L) = Ca × v / V × L
Ca: Chlorophyll a concentration in sample extract (μg/L)
v : 90% acetone extract volume (ml)
V: volume of filtered water sample (L)
L: cuvette width (cm)
This is a traditional chemical method for the detection of chlorophyll a. Now there are some simple instrumental methods. For example, the ChloroTech121 series handheld chlorophyll analyzer of Amno Laboratory in the United States has a low detection limit, which can reach ppb level. Simultaneous determination of chlorophyll a and turbidity in two channels enabled turbidity data to provide a correction function for chlorophyll a data.
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This article was published by Yingnuo Instruments (Shanghai) Co., Ltd.
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