2.2 Analysis of physiological parameters
In this study, photosynthesis, respiration, P accumulation, fluorescence, intra- and extracellular proteins and polysaccharides were determined. For photosynthesis, the algal cells were measured for Fv/Fm and rETRmax using a multiple excitation wavelength phytoplankton & photosynthesis analyzer (Phyto-PAM-II Modular Version, USA) after four days of incubation. The plate reader (SpectraMax i3X, USA) was used to measure OD680 in each sample and then the number of cells was calculated [23]. For respiration rate, standard gas was evenly injected into a saline bottle for 2 min, and then the culture was sealed in darkness for 1 h. Gas chromatography (GC, Agilent GC-7890B, USA) was used to analyze the concentration of CO2 in a 5 mL bottle with a 10 mL syringe [24].
Another aliquot of the algal sample was centrifuged (5000 rpm, 5 min) and the supernatant was filtered via a 0.45 μm membrane for the P measurement by ICP-MS (Perkin Elmer NexION 2000, USA). In addition, for the fluorescence analysis of dissolved EPS from algal cells, the excitation wavelength (Ex) and emission wavelength (Em) were set to 200~500 nm with a step size of 10 nm, the scanning speed was 6000 nm/min, with a spectral bandwidth of 3 nm (SHIMADZU RF-6000, Japan). The pellet was extracted with the equal volume of methanol. After re-suspension, the algal solution was placed in a refrigerator at 4 oC for 24 h. Then absorbance of chlorophyll a (Chl a ), chlorophyll b (Chl b ) and carotenoids were measured using the plate reader (SpectraMax i3X, USA) [25].
For measurements of intracellular and extracellular proteins and polysaccharides, the suspension of C. reinhardtii was filtered directly through 0.45 μm PES membrane (soluble proteins and polysaccharides (Ps)). The samples left on the filter membrane were resupsended, heated at 50 oC for 2 h and homogenized by sonication (total proteins and polysaccharides (Pt)). Then, they were filtered through PES membrane (soluble and cell-surface bound proteins and polysaccharides (Ps+c)). The cell-surface bound proteins and polysaccharides was defined as the difference between the Ps+c and the Ps. The content of intracellular proteins and polysaccharides (Pi) was the contrast between the Pt and Ps+c. The proteins and polysaccharides contents were measured by bicinchoninic acid method (BCA) and the phenol sulfuric acid digestion method, respectively [22].