Study on Selecting Characterization Parameters of Viable Algae Cells Number in Ballast Water Based on Chlorophyll Fluorescence Kinetics

Photosynthetic fluorescence parameter measurement method Measuring instruments: FRRf (Fast repetition rate fluorometer, Chelsea Technologies Group) measures photosynthetic fluorescence parameters under different experimental conditions.FRR fluorescence technology applies a series of subsaturated excitation pulses (flashes) at microsecond intervals to induce fluorescence transients. This method is very flexible and allows for the generation of flashes of single turn (ST) and multiple turn (MT). By applying a tightly spaced and evenly spaced flash of light, when in the saturation phase, the algae's light system, PSII, is completely switched off and the fluorescence increases from F0 to Fm or F' to Fm'. With the ST method, the saturation phase can also be calculated for σPSII. Methods for measuring viable algae cells numberTake 1 mL of algae sample, add 5 μL of 1 mmol·L-1FDA and 10 μL of 250 μmol·L-1CMFDA, and mix well. The final working concentration is 5μmol·L-1 and 2.5 μmol·L-1, staining protected from light for 10 min, then perform microscopic examination. Preparation of FDA stock: weighing 0.1 g of FDA powder, dissolving it with 5mL of dimethyl sulfoxide (DMSO), so as to obtain a concentration of 50 mmol·L-1 FDA preparation solution. Take 980 μL of DMSO and use it to dissolve 20 μL of the above preparation solution to prepare a concentration of 1 mmol·L-1 FDA stock solution. Preparation of CMFDA stock: Taking 50 μg of CMFDA dissolve in 430 μL DMSO to obtain a concentration of 250 μmol·L-1 of CMFDA stock. Measuring instrument: upright fluorescence microscope Nikon Ni-U and 0.1 mL plankton count box. Measurement principle: FDA and CMFDA are membrane permeable dyes, which are inherently non-fluorescent, but once they enter the viable algae cell, non-specific esterase will lyse the molecules of the stain, resulting in impermeable fluorescence products, and green fluorescence can be observed under blue light excitation. Since FDA and CMFDA have similar emission spectra, they can be used simultaneously to enhance the stability of green fluorescence emitted by viable algae cells. CDOM solution concentration measurementmethodCDOM (Colored dissolved organic matter), it is a colored soluble organic that is dissolved in water, fluorescent, and can be measured by optical instruments. Measuring instrument: Fluorescence Spectrophotometer F-7000. The width of the excitation and emission gap is 5nm, the excitation wavelength is 200nm to 435nm, the interval is 5nm, and the emission wavelength is 250nm-600nm, and the fluorescence spectrum is obtained at a 1nm interval, and the Three-dimensional Fluorescence Spectrum of Milli-Q ultrapure water is subtracted to correct the Raman scattering of water. Measuring principle: at a concentration of less than 75mg·L-1, thefluorescence intensity of CDOM aqueous solution (λex/λem=320/410nm) has a good linear relationship with the concentration (r2=0.9881). Dead cells fluorescence interferes with the experimentSimulate dead cells fluorescence environment of the ballast water tank by formulating different concentrations of dead cell fluid and determine whether the photosynthetic fluorescence parameters are affected by dead cells fluorescence by observing the change of photosynthetic fluorescence parameters. After filtering the original solution of Dunaliella salina with a membrane of 0.22 μm, the viable algae cells on the membrane were backwashed with seawater to obtain a sample containing only viable cells. The viable cell samples of Dunaliella salina are heated in a water bath at 50°C for 30 min to inactivate to obtain a sample containing only dead cells. According to the dilution multiples of 0, 2, 4, 8, 16, the dead cell sample is diluted with seawater, 25mL of dead cell sample under each dilution multiplier, and then a volume of 25mL and other quantities of Dunaliella salina viable cells samples are added, so as to obtain samples with the same viable algae cells number and a gradient change in the number of dead cells. Among them, dead Dunaliella salina cells number is used to anchor the fluorescence of dead algae cells. The number of dead algae cells was obtained by upright fluorescence microscopy Nikon Ni-U. The Fm, F0, Fv, and [RCII] values of the sample were measured by FRRf (Fast repetition rate fluorometer, Chelsea TechnologiesGroup).CDOM fluorescence interference experimentSimulate the change of CDOM concentration in ballast water by controlling the concentration of algal filtrate, and determine whether the photosynthetic fluorescence parameters are affected by CDOM concentration by observing the change of photosynthetic fluorescence parameters. CDOM solution is obtained from a filtrate of a very high chlorophyll concentration algae mixture in the decaying phase of laboratory culture for more than 2 months. Filter using a 0.2 μm mixed fibroidid membrane, filtering twice to ensure that all algal cells are filtered out. Immediately after filtration, CDOM solution is used. According to the dilution multiples of 0, 2, 4, 8, 16, the CDOM solution is diluted with seawater, the CDOM sample under each dilution multiple is taken 25mL, and then a volume of 25mL is added to viable cells of Dunaliella salina, so as to obtain all samples with the same viable algae cells number and a gradient change in CDOM concentration. The fluorescence intensity of algal filtrate is determined by F-7000 fluorescence spectrophotometer, and then the CDOM concentration of algae filtrate is calculated. Comparative experiments with multiple fluorescence disturbances Viable algae cells stock solution preparation: After filtering the original solution of Dunaliella salina with a 0.22 μm membrane, the viable algal cells on the membrane are backwashed with seawater to obtain a sample containing only viable cells. The above viable cell stock solution of Dunaliella salina was diluted in proportion to the dilution of dead cell solution, CDOM solution, random mixture of the two and seawater(controlgroup), respectively. And the dilution multipleswere 0, 2, 4, 8 and 16. Experimental study of DCMU stressconditionsIn six 25mL samples of Dunaliella salina stock solution, 1 mL of DCMU samples with concentrations of 5, 10, 20, 40, 50μg·L-1 and 1mL seawater (control sample) were added, respectively. After standing for 10min, the photosynthetic fluorescence parameters were measured by FRRf (Fast repetition rate fluorometer, Chelsea Technologies Group). The above-mentioned data processing software is origin and spss.