Improving Fucoxanthin Production in Mixotrophic Culture of Marine Diatom Phaeodactylum tricornutum by LED Light Shift and Nitrogen Supplementation

Abstract

Fucoxanthin (Fx), a kind of primary carotenoids in brown seaweeds and diatoms, has attractive efficacy in human's healthcare including loss weight, the prevention of diabetes and Alzheimer's disease. Marine diatom Phaeodactylum tricornutum is now realized as a promising producer for commercial Fx production due to its higher content of Fx than brown seaweeds with easily artificial cultivation and Fx extraction. In the present study, to improve Fx production in P. tricornutum, the mixotrophic cultures were applied to optimize initial cell density, light intensity, light regime and nitrogen supplementation. The results showed that the higher initial cell density (1 × 10⁷ cells mL^-1) and lower light intensity (20 μmol m^-2 s^-1) were favorable for biomass production and Fx accumulation. The maximal Fx content [16.28 mg g^-1 dry weight (DW)] could be achieved under blue light (BL), but the highest biomass concentration (5.53 g L^-1) could be attained under red: blue light (R: B, 6:1) in the batch culture. A novel two-phase culture approach was developed to increase the biomass concentration to the highest value (6.52 g L^-1) with the maximal productivity of Fx (8.22 mg L^-1 d^-1) through light shift from R:B ratio (6:1) in phase 1 to R:B ratio (5:1) by enhancing BL and tryptone addition in phase 2. The content and intracellular amount of Fx were also increased 8% and 12% in phase 2 compared to phase 1. The expression levels analysis revealed that genes encoding phytoene synthase (PSY), zeaxanthin epoxidase (ZEP), and fucoxanthin-chlorophyll-protein b (FCPb) were upregulated significantly, with downregulation of the gene encoding violaxanthin de-epoxidase (VDE), leading to the improvement of Fx in phase 2. The present study demonstrated the two-phase culture strategy could promote Fx productivity through enhancing biomass production and increasing Fx content, indicating that strengthening BL coupled with adding tryptone were effective to facilitate Fx production by mixotrophic cultivation of marine diatom P. tricornutum.

Keywords: Phaeodactylum tricornutum; fucoxanthin; light regime; qRT-PCR; two-phase culture.

FIGURE 1

Effects of initial cell density and light intensity on cell growth (A,B), biomass concentration and fucoxanthin content (C,D) in the mixotrophic growth of P. tricornutum. Significant differentiation level was set at * p < 0.05 and ** p < 0.01 compared with initial cell density of 1 × 10⁷ cells mL^{– 1} and light intensity of 20 μmol m^{– 2} s^{– 1}, respectively.

FIGURE 2

The light spectrum of white light (WL) and various red: blue lights (1:0, 6:1, 1:1, 1:2, 0:1) (A) and light regimes in the two-phase culture (B).

FIGURE 3

The cell growth (A), biomass (B) and glycerol concentration (C), fucoxanthin production (D) at white light (WL) and various red: blue lights (1:0, 6:1, 1:1, 1:2, 0:1). Significant differentiation level was set at * p < 0.05 and ** p < 0.01 compared with white light (WL).

FIGURE 4

Effects of light shift and nitrogen supplementation on cell growth (A,C) and fucoxanthin production (B,D) in two phase of two batch cultivation. In batch 1 (A,B), the mixotrophic cells grew at R: B light (6:1) for 6 days in phase 1, then light shifted to pure blue light (BL) with (+) nitrogen supplementation in phase 2. Nitrogen source was presented as urea (U), tryptone (T) and their mixture at a ratio (T:U = 1:1, N mol). Significant differentiation level was set at ** p < 0.01 compared with blue light (BL) shift. In batch 2 (C,D), blue light was strengthened solely to form R: B lights (5:1, 3:1, 1:1), or shifted to pure blue light (BL) or pure green light (GL), respectively, with (+) tryptone (T) addition in phase 2. Significant differentiation level was set at * p < 0.05 and ** p < 0.01 compared with blue light shift with tryptone addition (BL + T) in batch 2.

FIGURE 5

Bioprocess analysis of R: B (5:1) + T group in the batch 2. Production and nutrient consumption (A,B) in two-phase culture, fucoxanthin content and intercellular amount (C), genes expression levels in fucoxanthin biosynthesis pathway on the 6th, 8th, 10th, 12th days in phase 2 (D). Significant differentiation level was set with * p < 0.05 and ** p < 0.01 compared with that at 6th day in phase 1, respectively. The color boxes indicate the expression values of log₂FC (8th/6th), log₂FC (10th/6th), and log₂FC (12th/6th).