## 3 Results and Discussion
### Carotenoid Profile in the B. aggregatus BM5/15 Biomass at the Induction Stage  
In the cells of microalgae, primary and secondary carotenoids are distinguished. In the first case, pigments are coupled with photosynthetic apparatus. Their amount is limited by and under strong control of the cell. In the second case, carotenoids are synthesized independently on photosynthetic apparatus. Therefore, pigment level is not limited by cell photosynthetic capacity, and their content reaches high yield [32; 36; 94]. Under the inductive conditions, pigment content in the _B. aggregatus_ BM5/15 cells sharply raised (Figure 2b,d). The culture demonstrated signs of secondary carotenoids accumulation (see above). A total of 11 pigment fractions were obtained from the extracts of _B. aggregatus_ BM5/15 cultivated under the inductive conditions for five days (Figure 3a). Based on the \(R_{f}\) and absorption spectra [67] the following pigments were observed in the total extract: \(\beta\)-carotene, adonirubin, adonixanthin, echinenone, canthaxanthin, estaxanthin, primary xanthophylls (violaxanthin, zeaxanthin, antheraxanthin, neoxanthin, and lutein), pheophytins, and chlorophylls (Table 2). Astaxanthin, adonixanthin and \(\beta\)-carotene were predominant pigments in the cells, their fractions were ca. 48, 23 and 13%, respectively. Astaxanthin, adonixanthin and adonirubin, possessing hydroxyl group(s), might be deposited in the cells of _B. aggregatus_ BM5/15 mainly in the form of FA esters. Formation of esters with FA makes it possible to accumulate carotenoids in the hydrophobic environment of lipid globules, which play the role of a cell compartment for storage of lipophilic molecules [91; 95; 96]. Diesters represented the majority of astaxanthin forms in _B. aggregatus_ BM5/15. It is in accordance with previous data on the pigment profile of _B. minor_ under inductive conditions [70]. In primary work on _B. aggregatus_ BM5/15 with non-optimized method of carotenoid synthesis induction [53], similar results had been obtained. Astaxanthin and \(\beta\)-carotene were also predominant, but \(\beta\)-carotene fraction was higher and most of astaxanthin was in the form of monoesters. Thus, it would be possible to modulate carotenoid composition of _B. aggregatus_ BM5/15 by variation of the induction method. \begin{table}
\begin{tabular}{c c c} \hline \hline \(R_{f}\) & **Pigment** & **Carotenoid Content (Mas.-\% of Other Carotenoids)** \\ \hline
0.93 & \(\beta\)-carotene & 13.1 \\ \hline
0.80 & Adonixanthin diesters & 2.4 \\ \hline
0.59 & Echinene & 1.2 \\ \hline
0.56 & Astaxanthin diesters & 31.1 \\ \hline
0.44 & Adonirubin esters & 8.0 \\ \hline
0.38 & Canthaxanthin & 2.1 \\ \hline
0.24 & Astaxanthin monoesters & 16.8 \\ \hline
0.18 & Adonirubin monoesters & 20.5 \\ \hline
0.15 & Free ketocarotonoids 1 & 1.4 \\ \hline
0.04 & Primary xanthophylls 2 & 3.4 \\ \hline \hline \end{tabular}  
* Free axtaxanthin and adonixanthin and, possibly, products of their oxidation, 2 Xanthophyll associated with photosynthetic apparatus: zeaxanthin, unidexanthin, roaxanthin and lutein.  
\end{table} 
Table 2: Fractions of carotenoids obtained after separation of the extracts from the cells of _Bracteacoccus agregatus_ BM5/15 (IPPAS C-2045) cultivated for five days under the inductive conditions by thin layer chromatography. Values of the retardation factor (\(R_{f}\)), pigment name based on its \(R_{f}\) value and absorbance spectrum, as well as its mass fraction in the total extract are provided. Figure 3: Carotenoid composition of _Bracteacoccus agregatus_ BM5/15 (IPPAS C-2045) at the inductive stage of cultivation (5 th day). (**a**) Representation results of separation of chloroform pigment extracts from the \(B\). _aggregatus_ BM5/15 cells after five days of cultivation under the inductive conditions. Values of the retardation factor are presented for each pigment fraction. (**b**) Putative pathways of xanthophyll synthesis in \(B\). _aggregatus_ BM5/15 based on its pigment profile.