|Other Abstract||The accumulation of secondary carotenoids and biosynthesis of astaxanthin in Scenedesmus obliquus were analyzed and morphological characters, ultramicrostructture, physiological changes of algae cells during the accumulation and degradation of secondary carotenoids were observed. The in vivo functional aspect of astaxanthin was also discussed. The main results were reported as follows:
A two-step cultivation for the inducing of carotenoids accumulation was applied and the optimal cultural conditions were adjusted at 0.75g/L of nitrogen concentration, 180μmol•m-2•s-1 of illumination intensity, temperature of 35℃, 0.4g/L of salt concentration and pH 7. The analysis of HPLC-MS and TLC showed Scenedesmus obliquus cells contained two kinds of carotenoids: primary carotenoids including neoxanthin and lutein, and secondary carotenoids including echinenone, canthaxanthin, 3’-hydroxyechinenone, adonirubin, adonixanthin and astaxanthin. The biosynthetic pathway of astaxanthin in algal cell started from β-carotene which first converted to canthaxanthin, then canthaxanthin and 3’-hydroxyechinenone formed. Canhtaxanthin could converse to astaxanthin via adonirubin, and 3’-hydroxyechinenone could converse to both adonirubin and adonixanthin. In the final step, a hydroxylation or oxygenation introduced to adonirubin or adonixanthin to form astaxanthin. There were three proposed pathways of astaxanthin biosynthesis. In this accumulation course, the contents of chlorophyll, lutein and neoxanthin decreased.
The ingredients of cell pigments changed remarkably while the stress conditions were removed. At first, secondary carotenoids degraded and free astaxanthin could not be detected anymore, also the precursors such as echinenone, canthaxanthin and adonirubin all hydrolyzed. At the same time, primary carotenoid and chlorophyll became the main part of pigments again. As far as the metabolic map of astaxanthin, three possible pathways were concluded: one part of free astaxanthin was esterized to become mono or di-esters, another part of astaxanthin was utilized by the cell with other secondary carotenoids by some certain forms, and the residual part conversed to lutein to take part in photosynthesis, which was similar to xanthophyll cycle.
The cellular activity of photosynthesis was the most distinct change during the biosynthesis of astaxanthin. In 48 hours, the oxygen evolution rate drastically decreased from 39μmol• O2• mg-1 chla• h-1 to 5.21 μmol• O2• mg-1 chla• h-1 and the variable fluorescence Fv/Fm also declined from 0.72 to 0.26, while the respiration rate increased from 18.24μmol• O2• mg-1 chla• h-1 to 38.40 μmol• O2• mg-1 chla• h-1. The decrease in photosynthesis could continue with the standing of stress conditions, but a reverse movement could happen once the cultural conditions changed into appropriate state. With the degradation of secondary carotenoids for 72 hours, the oxygen evolution rate resumed to 54.02μmol• O2• mg-1 chla• h-1 which was even higher than that of initial level under stress. The variable fluorescence Fv/Fm also increased to 0.64 while the respiration rate evidently dropped to 3.98μmol• O2• mg-1 chla• h-1. These contrary photosynthetical changes of algal cells indicated secondary carotenoid and astaxanthin have a screening function to protect cell against high irradiation by the absorption of photosynthetic light.
The observation results of LM revealed some significante changes during the accumulation of astaxanthin. The coenobium alga was composed of four or eight spindly unicells under normal state, however, the number of unicells and dual-cell coenobium increased and the shape of cell changed into circular or elliptic ones with the deposition of secondary carotenoids. The color of cells also turned from green to brown or orange. The transmission electron microscope photos showed a mass of lipid body came into being and held the most of inner space. The nucleus and chloroplast were wrapped by lipid body and diverted to the central or side of the cell, but their shape and structure both kept integrity. The results also showed the morphological changes were reversible which influenced strongly by environmental conditions, and secondary carotenoids played a physical protection role for organelle especial the chloroplast.
The growth and multiplication of algal cells both impacted by the induced conditions with the formation of secondary carotenoids. In 48 hours, cell dry weight increased by 18.9% and was higher than that of cells under normal growth. The contents of protein and RNA also both declined compared to those of normal cells, and the numbers of cells, the contents of DNA both decreased. However, these changes could recovery to well-balanced state with the removing of stress. These results suggested the cell growth was restrained by the induced conditions. Furthermore, the bigger volume and increased dry weight of cells mostly ascribed to the accumulation of secondary carotenoids.|