Study on Newly Isolated Dysmorphococcus Strains from Reunion Island as Potential Sources of High-Value Carotenoids

Abstract

Certain secondary carotenoids, such as astaxanthin and canthaxanthin, are of growing economic interest in the fields of human nutrition, food, health and cosmetics, as well as feed and aquaculture, particularly due to their numerous biological activities, such as their remarkable antioxidant properties. The present study was devoted to assessing, in a photobioreactor, the feasibility of cultivating newly isolated Dysmorphococcus strains from the biodiversity of Reunion Island for the production of these valuable xanthophylls. The results showed that all these strains were capable of producing and accumulating canthaxanthin and astaxanthin in response to environmental stresses. Among them, a strain which presented interesting morphological, genetic and biochemical properties as compared to the other Dysmorphococcus strains was further cultivated in a 3 L benchtop photobioreactor and was found to produce maximum carotenoid-rich biomass concentrations and productivities of about 4 g L^-1 dw and 0.055 g L^-1 d^-1 dw, respectively. We also found that the biomass contained up to 1.2 mg g^-1 dw of canthaxanthin and 0.7 mg g^-1 dw of different forms of astaxanthin, mainly astaxanthin monoesters. The productivity of these carotenoids was found to be lower than those observed for other microalgal species previously reported, and we suggested that further optimizations with respect to the cultivation and the carotenogenesis induction processes are needed to improve productivities and to make this locally isolated Dysmorphococcus strain useful for future commercial production of natural canthaxanthin and astaxanthin.

Keywords: Dysmorphococcus; astaxanthin; biodiversity; canthaxanthin; new application; photobioreactor; reunion island.

Figure 1

A map of Reunion Island; photographs of sample collection locations concerning the Dysmorphococcus strains isolated during our previous study: (1) Bras-Rouge river, Cilaos, for the strains’ CBRs; (2) Terre-Sainte, Saint-Pierre, for the strain M8.

Figure 2

Microphotographs of some monoclonally isolated Dysmorphococcus strains: (a) CBR1; (b) CBR2; (c) CBR5; (d) CBR3; (e) CBR4; (f,g) CBR11; (h) M8. (a,b) Phase contrast; (c–h) bright field. (a,c,e–h) Scale bar 25 µm; (b,d) scale bar 50 µm. (a,b) Arrows show parental cell walls after the release of daughter cells; (f,g) arrows show cell clusters.

Figure 3

Growth kinetics of the Dysmorphococcus strains CBR11 and CBR1 cultivated in batch mode with the 3N-BBM medium under increasing photosynthetically active photon flux density (PPFD): (a) CBR11 in a 2 L glass reactor; (b) CBR11 in a 3 L benchtop photobioreactor; (c) CBR1 in a 3 L benchtop photobioreactor; (d) comparison between the three cultures where cell densities are expressed in a logarithmic scale. Bars represent standard deviations on 4 replicates of the same sample; arrow with (*) represents a harvest of 500 mL of culture replaced by fresh medium.

Figure 4

Photographs of the culture of the Dysmorphococcus strain CBR11 cultivated in a 3 L benchtop photobioreactor with the 3N-BBM medium: (a) after 13 days; (b) after 77 days.

Figure 5

HPTLC chromatograms of pigment extracts obtained from the isolated Dysmorphococcus strains: (a) under transmitted white light illumination; (b) under reflected 366 nm light illumination; (c) densitogram for the transmitted white light illumination. Tracks 1: carotenoid standards; 2: astaxanthin esters from H. lacustris reference extract; 3: CBR1; 4: CBR2; 5: CBR3; 6: CBR4; 7: CBR5; 8: CBR7; 9: CBR11; 10: M8.

Figure 6

HPLC chromatograms of pigment extracts obtained from the isolated Dysmorphococcus strains: a: CBR1; b: CBR2; c: CBR3; d: CBR4; e: CBR5; f: CBR7; g: CBR11; h: M8. Peak 11 corresponded to that of Table 2. Detection with DAD at 480 nm.

Figure 7

HPTLC chromatograms of pigment extracts obtained from the isolated Dysmorphococcus strain CBR11: (a) under transmitted white light illumination; (b) under reflected 366 nm light illumination; (c) densitogram for the transmitted white light illumination. Track 1: carotenoid standards; track 2: astaxanthin esters from H. lacustris reference extract; track 3: during the vegetative green stage; tracks 4 and 5: during the aplanospore orange stage before and after saponification, respectively. The lines on the densitogram indicate R_F of canthaxanthin (left) and free astaxanthin (right).

Figure 8

HPLC chromatograms of pigment extracts obtained from the Dysmorphococcus strain CBR11: (a,d) in the vegetative green stage; (b,e) in the aplanospore orange stage; (c,f) in the aplanospore orange stage after saponification; (a–c) detection with DAD at 480 nm; (d–f) detection in a 3D field mode.

Figure 9

LC-MS chromatograms of the extract obtained from the orange biomass of the isolated Dysmorphococcus strain CBR11: (a) free astaxanthin; (b) canthaxanthin; (c) astaxanthin monoester. The ratios m/z of the positive ions [M + H]⁺ corresponding to each peak are indicated to the right of the chromatograms.

Figure 10

Molecular structure of all-trans-canthaxanthin [31].