We aim to study the influence of photosynthetic activity change on chlorophyll fluorescence yield and explore the error source of measuring algae chlorophyll a concentration by living fluorescence method, so as to provide an important basis for the subsequent development of on-site accurate detection method of algae chlorophyll concentration in water based on living fluorescence method.

We use ACT2&FastOcean FRRF algal fluorescence meter (CTG Company, UK) to measure the photosynthetic activity parameter F_v/F_m. After 15 min dark adaptation of the algal sample (the photosynthetic reaction center of the algal sample is fully open), we selecte an excitation light source of 450 nm (the characteristic excitation wavelength of Chlorella pyrenoidosa) and measure the chlorophyll fluorescence induction curve. According to the basic parameters of the curve, the maximum photochemical quantum yield F_v/F_m is calculated. We use HJ897-2017 water quality determination of chlorophyll aspectrophotometry to measure the concentration of chlorophyll aof sample algae. In the experiment, the Hitach7000 fluorescence spectrophotometer is used to measure the three-dimensional fluorescence spectrum of Chlorella pyrenoidosa, as shown in Fig. 1(a). The characteristic fluorescence spectrum region of Chlorella pyrenoidosa (excitation wavelength range of 380-500 nm and emission wavelength range of 660-750 nm) is selected, and the fluorescence intensity of living algae is obtained by fluorescence region integration method. The fluorescence yield, namely the fluorescence intensity per unit of chlorophyll a, is further obtained. Three groups of parallel samples are allocated for the experiment, and the final test results are taken as the average value, so as to study the change rule between photosynthetic activity and fluorescence yield of Chlorella pyrenoidosa.

Under the DCMU toxicity stress, the chlorophyll fluorescence yield and photosynthetic activity of Chlorella pyrenoidosa with different mass concentrations have the opposite trend under the DCMU stress. With the increase in DCMU mass concentration, F_v/F_m value changes from 0.605 to 0.229, showing a gradual downward trend, with a decline of 62%; η value changes from 245 (μg·L^-1)^-1 to 678 (μg·L^-1)^-1, showing a gradual upward trend, with an increase of 177%. The variation trend of each group of experiments is the same; the repeatability is high, and the data fluctuation range is within 5%. The variation relationship between photosynthetic activity and fluorescence yield is independent of algae mass concentration. Therefore, there is a significant negative correlation between photosynthetic activity and fluorescence yield. Chlorophyll fluorescence yield and photosynthetic activity of Chlorella pyrenoidosa cultured in different mass concentrations for two hours under different light intensities show a reverse trend. With the increase in light intensity, F_v/F_m value gradually increases, and η is gradually decreasing. The changing trend has nothing to do with the algae mass concentration. When the light intensity increases from 5600 to 44800 lx, the photosynthetic activity value decreases from 0.563 to 0.388, with an average decrease of about 26%. The chlorophyll fluorescence yield increases from 241 (μg·L^-1)^-1 to 453 (μg·L^-1) ^-1, with an average increase of 80%. In addition, the changing trend of each group of experiments is the same, with high repeatability, and the data fluctuation range is within 10%. In the temperature experiment, the chlorophyll fluorescence yield and photosynthetic activity of Chlorella pyrenoidosa samples with different mass concentrations also show the opposite trend, and the fluctuation range of each group of repeated experimental data is within 3%. With the increase in temperature, F_v/F_m value rises slowly and then decreases rapidly, and η trend of change has nothing to do with the mass concentration of algae. At 5-25 ℃, F_v/F_m increases from 0.566 to 0.605, with an average increase of 7%, and η changes from 253 (μg·L^-1)^-1 to 284 (μg·L^-1)^-1, an average decrease of 2%. At 25-50 ℃, F_v/F_m decreases from 0.605 to 0.376, with an average decrease of 38%, and η rises from 284 (μg·L^-1)^-1 to 473 (μg·L^-1)^-1, an average increase of 91%. By simulating three environmental conditions to change the photosynthetic activity of Chlorella pyrenoidosa, the effect of the change of photosynthetic activity on the change of fluorescence yield is studied. The results show that the change range of photosynthetic activity of Chlorella pyrenoidosaa is 0.229-0.605, and the change range of fluorescence yield is 235-668 (μg·L^-1)^-1. There is a negative correlation between photosynthetic activity and fluorescence yield of Chlorella pyrenoidosa. The linear fitting results between photosynthetic activity and fluorescence yield measured under three environmental conditions are y=-1073x+866, y=-1012x+1018, and y=-1354x+827, and the linear goodness R² between the two is above 0.91. It can be seen that the change of photosynthetic activity is an important factor affecting the change of fluorescence yield, and it is the error source of inaccurate measurement of in vivo fluorescence method.

The photosynthetic activity of Chlorella pyrenoidosa is changed by toxic stress, illumination, and temperature control, and three different growth environments are simulated to study the influence of changes in the photosynthetic activity of algae on the change of chlorophyll fluorescence yield. The results show that in three different growth environments, with the change of photosynthetic activity, the chlorophyll fluorescence yield of algae changes. The photosynthetic activity of algae is an important factor affecting the yield of chlorophyll fluorescence. With the increase in photosynthetic activity, the yield of chlorophyll fluorescence shows a downward trend. There is an obvious negative correlation between the two, and the correlation coefficient R² can reach more than 0.91. This study is an in vivo fluorescence method, which can provide an experimental basis for the subsequent improvement of the measurement accuracy of the in vivo fluorescence method.