*2.4. Growth and Carotenogenesis*

## 2.4.1. Biomass Accumulation and Pigment Composition

In order to evaluate the potential of *H. pluvialis* BM1 for biotechnology the isolate was cultivated in a closed bubble-column photobioreactor as described in the "Experimental" Section (see also Supplementary Figure S2d). The "green" cell cultures reached a maximum cell density of 1.6 × 106 mLƺ1 (37 mg·mLƺ1 Chl, 6 mg·mLƺ1 Car, ca. 1.0 g·Lƺ1 of cell dry weight, DW) in 5–7 days corresponding to the specific growth rate, ΐ = 0.095 dayƺ1 at the exponential phase (Supplementary Figure S2). At the exponential growth phase, the culture was comprised, to a considerable extent, by dividing "green" cells (Figure 2). The cell suspension was bright green in color due to low (ca. 0.18 ± 0.01) Car/Chl ratio (by weight). The Car at this growth phase were represented exclusively by primary carotenes and xanthophylls, there was no detectable presence of Ast (see Section 2.4.3 below and Figure 6). After 10 day of cultivation, accumulation of astaxanthin was detected and the Car/Chl ratio gradually increased, apparently, due to nitrate depletion in the medium. 

## 2.4.2. Stress-Induced Astaxanthin Accumulation

To induce the massive accumulation of Car, the "green" cells of *H. pluvialis* BM1 were transferred to the stressful conditions mimicking, to a certain extent, the nutrient deficiency and excessive solar irradiation in their natural habitat. Specifically, the cells were washed, resuspended in distilled water, and exposed to irradiance one order of magnitude higher than that optimal to the "green cells" (see the "Experimental" section). Under these conditions, most of the cells displayed a rapid induction of Ast biosynthesis and turned into non-motile "red" cells (Supplementary Figure S2a). 

The induction of carotenogenesis occurred in the background of a sharp decline of Chl content. As a result, the shape of the absorption spectra of extracts from the "red" cells was governed by Ast absorption (Supplementary Figure S2b). Notably, the cells of *H. pluvialis* BM1 even after prolonged stress exposure always retained a certain amount of Chl; only dead colorless cells possessed no detectable Chl content. On the whole, the dynamics of stress-induced Car accumulation displayed by BM1 was similar to that recorded in known *H. pluvialis* strains [20]. 

High performance liquid chromatography (HPLC) analysis (see Section 2.4.4 below) confirmed that nearly 99% of total Car in the "red" cells were represented by Ast reaching 5.0%–5.5% DW by the 6th day of stress (corresponding to a Car/Chl of 13.0 ± 0.1). After the 6th day of stress exposition, the Ast content declined sharply (Supplementary Figure S2c). This process was manifested by a massive appearance of bleached cells. 

2.4.3. Salinity Effects on the Growth of the "Green Cells"

The abrupt changes of salinity characteristic of the habitat of BM1 (see Section 2.1) suggest an increased ability to acclimate to this factor in the microalga under investigation. To obtain a preliminary estimation of BM1 salinity tolerance, we cultivated the microalga under salinity similar to that of the rock bath water (25‰), which is typically below the White Sea water salinity (29‰) because of dilution with rainwater. 

It was found that the increase in salinity *per se* did not trigger a decline in Chl accumulation by the culture (Figure 5a) or an increase in Car accumulation over Chl (Figure 5b) typical of the stress-induced carotenogenic response. During first 5–7 days, the kinetics of growth on the saline medium did not differ significantly from that on the medium lacking NaCl (see Section 2.4.1 above and Supplementary Figure S1). Only a limited accumulation of Ast was detected in the cultures grown at 25‰ NaCl (insert in Figure 5c). 

**Figure 5.** Effects of 25‰ NaCl on (**a**) chlorophyll accumulation; ( **b**) carotenoid-to-chlorophyll ratio; and (**c**) normalized absorption spectra of the cell dimethyl sulfoxide (DMSO) extracts of *H. pluvialis* BM1 "green" cell culture. The spectra for (*1*) initial culture (Day 0) as well as those recorded after one day (*<sup>2</sup>*, *2*<sup>ȝ</sup>) and five days (*<sup>3</sup>*, *3*<sup>ȝ</sup>) of cultivation in the medium containing (*2*<sup>ȝ</sup>, *3*<sup>ȝ</sup>) or lacking (*<sup>2</sup>*, *3*) NaCl are shown. Insert: different absorbance spectra of the extract spectra presented in the panel (**c**). Note a positive peak in the green region indicative of a limited accumulation of astaxanthin by the fifth day of cultivation in the presence of NaCl. 

**Figure 6.** Pigment composition of *H. pluvialis* BM1 cells at different cultivation stages (**a**) The "green" cells (high performance liquid chromatography (HPLC)); (**b**) The "red" cells (thin-layer chromatography (TLC) + HPLC). 

At the same time, it is known that the addition of 0.8% NaCl (8‰) to the medium normally causes a cessation of growth of *H. pluvialis* [7,20]. In view of these facts, the new strain *H. pluvialis* BM1 seems to have a higher tolerance to salinity stress although a more detailed investigation of the limits and possible side effects of its 

salinity tolerance is necessary. Nevertheless, this finding may be important for the biotechnology of Ast production in the areas with a limited supply of fresh water suggesting the possibility of cultivation of "green" cells of *H. pluvialis* BM1 in brackish water. 

## 2.4.4. Stress-Induced Changes in Pigment and Fatty Acid Composition

Under conditions conducive to rapid growth of the culture, the pigment composition of *H. pluvialis* BM1 was typical of green algae thylakoid membranes [21] including Chl *a* and *b* as well as primary Car; only trace quantities of Ast esters were detected (Figure 6a). 

Thin-layer chromatographical (TLC) separation of the extracts from the "red" cells yielded five pigment fractions (Fractions I–V, Figure 6b). The absorption spectra of all fractions resembled those of pure Ast ( *Ώ*max = 490). Incubation in air at room temperature for 10–15 min resulted in the long-wave shift of the maximum to 494 nm typical for astacene, an oxidation product of Ast [22]. The fast conversion of the pigment from the Fraction I to astacene suggests that the Fraction I contained non-esterified (free) Ast. The bulk (ca. 70%) of the Ast in the "red" cells was found in the Fractions II ( *R*f = 0.30–0.34) and III ( *R*f = 0.35–0.37). The Fractions IV ( *R*f = 0.43– 0.46) and V ( *R*f = 0.54–0.56) were substantially less abundant. Free Ast (the Fraction I) comprised less than 3.5% of the total Ast. 


**Table 1.** Fatty acid composition (*mas.-%*) of esterified carotenoid fractions of *H. pluvialis* BM1 "red" cells. 

\* not detected; \*\* unsaturation index (a) 6,9,12,15–18:4, 20:0, 22:0, 24:0 also were present; the concentration of each was 0.6%–0.8%; (b) also contained 0.5% of 20:0 FA; (c) 12:0, 20:0 and 22:0 were present, the concentration each was 0.7%–0.9%; (d) also contained 7,10,13– 16:3—1.9%, 4,7,10,13–16:3—2.3% and 20:0, 22:0, 24:0, the concentration of each was 0.1%– 0.2%. 

The HPLC-diode-array detector (DAD) analysis of the Fraction I as obtained by TLC confirmed the presence of free Ast. The HPLC elution profile of the Fractions II and III pigments contained eight major peaks ( *R*t 8–10 min). One could suggest that these peaks correspond to individual molecular species of Ast monoesters. The Fraction V harbored a large number of nonpolar compounds containing Ast chromophore, most probably Ast fatty acid diesters. 

Notably, accumulation of Ast in *H. pluvialis* BM1 cells took place along with a significant increase in neutral lipids in cytoplasmic lipid droplets as evidenced by vital staining of the "red" cells with Nile Red. The gas chromatography-mass spectrometry (GC-MS) fatty acid analysis (Table 1) demonstrated that the fatty acid profile of the Ast monoesters was dominated by palmitic (16:0), oleic ( ̇9–18:1), and linoleic ( ̇9,12–18:2) acids; it was similar to that of the known *H. pluvialis* strains [11,12,23]. Interestingly, The FA from the diester fraction (V) possessed a nearly twofold higher unsaturation index in comparison with those from the monoester fractions (II–IV). Taking into account the strong differences in FA composition of the mono- and diesters of Ast in *H. pluvialis* BM1, one may speculate that (i) their FA originate from different pools and (ii) the enzymes responsible for biosynthesis of the different classes of Ast esters possess a different substrate specificity. 
