*3.6. Analytical Methods*

Chlorophyll fluorescence was measured with an FMS2 fluorometer (Hansatech, Norfolk, UK). Cells grown to an exponential phase (30 μg of chlorophyll a) were loaded onto a glass-fiber filter, and the filter was placed on the leaf clip. For determination of the PSII operating efficiency (Y(II)), cells (without dark-adaptation) were exposed to stepwise-increasing actinic light (from 1 to 900 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1) for 20 s at each light intensity, and a saturating flash (3000 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1, 0.7 s duration) was applied to measure Fm'. Y(II) was calculated as (Fm'–Fs)/Fm'. The cellular chlorophyll and carotenoid contents were determined spectrophotometrically as described [53,54].

The cell density was determined by measuring OD680 using UV-spectrophotometer (Shimadzu, Kyoto, Japan) or dry cell weight (DCW) every 24 h. DCW was only determined for large-scale cultivation by filtering aliquots of samples using pre-weighed filter paper. Then, the cell suspensions (10 mL) were filtered with GF/F glass microfiber filters (Whatman, Cambridge, UK) and dried at 105 ◦C overnight. DCW was determined by the difference between the mass of the biomass-containing filter paper and that of pre-weighed filter paper.

In order to identify the accurate amount of intracellular astaxanthin using a high performance liquid chromatography (HPLC) system, algal cells were collected by centrifuging the culture fluid at 3000 rpm for 10 min at 4 ◦C, and the astaxanthin in cell pellet was extracted using a homogenizer (TissueLyser II, Qiagen, Hilden, Germany) in the presence of methanol and glass beads [55]. Then, the homogenized lysates were saponified with 0.01 M KOH to convert the esterified astaxanthin into free forms.

After saponification, the astaxanthin concentration of each sample was determined by HPLC system equipped with two LC-10AD pumps and SPD-10 UV-Vis detector (Shimadzu, Kyoto, Japan). The extracts were separated using a 250 × 4.6 mm HS-303 hydrosphere C18 column (YMC, Kyoto, Japan). The mobile phase consisted of solvents A (dichloromethane: methanol: acetonitrile: water, 5.0: 85.0: 5.5: 4.5, *v/v*) and solvent B (dichloromethane: methanol: acetonitrile: water, 22.0: 28.0: 45.5: 4.5, *v/v*). For the effective separation of astaxanthin, a linear gradient system was used: 0% B for 8 min, a linear gradient from 0 to 100% B for 12 min, and 100% B for 50 min. The flow rate was set as 1.0 mL min−<sup>1</sup> and the peaks were measured at 480 nm [50].
