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].