Marine diatom P. tricornutum CCMP 1327 was kindly provided by Dr. Hanhua Hu in Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. The seed culture was applied in 250-mL Erlenmeyer flasks containing 100-mL modified f/2 medium (Guillard, 1975) under mixotrophic condition. Temperature at 20°C was setup with continuous illumination of 10 μmol m^–2 s^–1 under white LED light and rotating speed of 150 r/min in shaking incubator. The modified f/2 medium contained (per liter): 20 g sea salt, 9.20 g glycerol (0.10 mol L^–1), 10 mg NaH₂PO₄.H₂O, 30 mg Na₂SiO₃.9H₂O, 3.15 mg FeCl₃.6H₂O, 4.36 mg Na₂EDTA.2H₂O, 9.80 μg CuSO₄.5H₂O, 6.30 μg Na₂MoO₄.2H₂O, 22 μg ZnSO₄.7H₂O, 10 μg CoCl₂.6H₂O, 180 μg MnCl₂.4H₂O. In addition, 1.17 g L^–1 of tryptone and 0.30 g L^–1 of urea (1:1, N mol/N mol) were added into the medium to final concentration of total nitrogen (TN) at 0.02 mol L^–1. The medium was autoclaved at 121°C for 20 min followed by urea addition using stock solution filtered through 0.45 μm membrane. The seed culture in late logarithmic phase was harvested by centrifugation and re-suspended in the medium above as the inoculum for subsequent experiments.

A series of initial cell densities (1 × 10⁶, 4 × 10⁶, 7 × 10⁶, 1 × 10⁷ cells mL^–1) were investigated by adding the inoculum and culturing for 14 days. Then, various light intensities (10, 20, 30, 40, 50, 100, 150, 200 μmol m^–2 s^–1) were evaluated at initial cell density of 1 × 10⁷ cells mL^–1 by cultivation for 12 days. The other culture conditions were same as the seed culture. Cell density, glycerol and nitrogen concentration were measured every 2 days during the cultivation. Biomass, content and volumetric concentration of Fx were measured at the end of cultivation.

All data were performed in biological triplicates and presented as mean ± standard deviation (SD). Origin V9.0 software was used to plot figures. The statistical analysis was performed by one-way analysis of variance (ANOVA) and LDS t-test with SPSS V22.0 software. Significant differentiation level were set at ^∗p < 0.05 and ^∗∗p < 0.01 by compared with control group.

The effects of initial cell density was shown in Figure 1A. The rapid increase of cell growth were observed in the first 8 days but slowed down in the next 6 days. With the increase of initial cell density, the average specific growth rate of cells was significantly decreased (p < 0.01) (Table 2). The highest final cell density (6.72 × 10⁷ cells mL^–1) and the maximal biomass concentration (3.73 g L^–1) were achieved at the highest initial cell density (1 × 10⁷ cells mL^–1) (Figures 1A,C). Even though the highest content (18.61 mg g^–1) and intercellular amount of Fx (1.23 pg cell^–1) were attained at 4 × 10⁶ cells mL^–1 (Figure 1C and Table 2), the highest volumetric concentration of Fx reached to 59.66 mg L^–1 at the highest initial cell density (1 × 10⁷ cells mL^–1) (Table 2). Therefore, for improving biomass and Fx production in a shorter time, the highest initial cell density was the option in the following experiments.

To further promote the cell growth under high cell density, the effects of light intensity were evaluated. Interestingly, the increase of light intensity had no obviously negative effect on biomass production from 30 to 200 μmol m^–2 s^–1 (Figure 1D), but an inhibiting effect on cell density was observed (Figure 1B). The highest biomass concentration (4.80 g L^–1) and Fx content (16.03 mg g^–1) could be reached at light intensity of 20 μmol m^–2 s^–1 (Figure 1D). The content and intercellular amount of Fx significantly decreased when the light intensity exceeded 30 μmol m^–2 s^–1, which was consist with the previous studies. For instance, the Fx content reached the maximal value at 13.5 μmol m^–2 s^–1 (Gomez-Loredo et al., 2016) and dropped from 7.50 mg g^–1 to 1.10 mg g^–1 when the light intensity increased from 30 to 180 μmol m^–2 s^–1 in P. tricornutum (Wang H. et al., 2018). However, the cell growth and biomass production were both decreased with the increase of light intensity under autotrophic condition, which may be explained by the photo-inhibition (Wang H. et al., 2018). Besides, Fx and Chl a contents were significantly reduced but fucoxanthin-to-chlorophyll a ratio (Fx/Chl a) was significantly increased from 20 μmol m^–2 s^–1 in this study (p < 0.05) (Table 2). Since Chl a exists not only in FCP, but also in free Chl a (Nymark et al., 2009), the increase of Fx/Chl a ratio indicated that free Chl a degraded more than that in FCP, resulting in higher degradation of Chl a in total Chl a than Fx when cells exposed to high light. Similarly, both of Fx and Chl a contents decreased sharply with the increase of light intensity in autotrophic P. tricornutum (Nur et al., 2019; Conceicao et al., 2020). The transcriptome analysis indicated that the genes encoding enzymes in biosynthesis of GGPP, which is the precursor of both Chl a and Fx, were downregulated upon high light intensity in P. tricornutum, leading to the reduction of Chl a and Fx (Nymark et al., 2009). When cells were exposed to high light, NPQ was activated to convert excess light energy into heat energy (Nymark et al., 2009). In diatoms, Ddx cycle plays a critical role in NPQ, which protects cell from high-light damage (Hao et al., 2018). Under high light, the contents of Ddx and Fx were lower than low light (Conceicao et al., 2020), which due to the upregulation of VDE promoted the conversion of Ddx to Dtx and violaxanthin to zeaxanthin, leading to the decline of Fx (Nymark et al., 2009). Therefore, the dim light at 20 μmol m^–2 s^–1 is favorable for cell growth and Fx accumulation under mixotrophic condition without photo inhibition.

The light spectrums of WL and various red: blue lights (R: B, 0:1, 6:1, 1:1, 1:2, 1:0) were shown in Figure 2A. The wavelength of WL was ranged from 402 nm to 760 nm, and the peak wavelength of blue and red light was 452 nm and 636 nm, respectively. As shown in Figure 3A, the cells grew rapidly under red: blue lights and the highest biomass concentration (5.53 g L^–1) with productivity (351.39 mg L^–1 d^–1) could be achieved at R: B light (6:1), which was 1.22- and 1.38-fold higher than WL (p < 0.01) (Figure 3B and Table 3). Even though the biomass productivity in R: B light (6:1) and R: B light (1:2) were similar (Table 3), which might due to the nutritional limitation in the late phase of culture, the former in biomass productivity was 4.12% higher than the later. Similarly, the previous study indicated that the biomass was significantly higher at the mix of red and blue light than the fluorescent WL in autotrophic culture of P. tricornutum, but it was similar in the group between R:B (70:30) and R:B (30:70), which was consist with this study (Sirisuk et al., 2018). The increase of biomass in the first 6 days (Figure 3B) was due to the prior uptake of tryptone and urea as carbon and nitrogen source, which explained the slow consumption of glycerol in this period (Figure 3C). The maximum average glycerol consumption rate during 12-days cultivation reached to 327.78 mg L^–1 d^–1, while the highest biomass yield per TN consumed (Y_X/TN) reached to 28.49 mg mg^–1 at R: B light (6:1) (Table 3). Moreover, the highest Fx content (16.28 mg g^–1) and Chl a content (32.68 mg g^–1) were attained under BL (Figure 3D and Table 3), which were 12% and 36% higher than WL (p < 0.01), respectively. These results were similar with that the Fx and Chl a contents were significantly higher under BL than WL in autotrophic P. tricornutum and Coscinodiscus granii (Sirisuk et al., 2018; Su, 2019). However, the biomass concentration (3.63 g L^–1) under BL was the lowest one (Figure 3B) in this study. Compared to WL, the results indicated that R: B light (6:1) could promote the cell growth and biomass production, but BL has positive effect on Fx accumulation.

The similar reports could be found that P. tricornutum produced more biomass under higher proportion of red light in mixture light under autotrophic condition (Sirisuk et al., 2018), but higher contents of xanthophyll cycle pigments (including Fx) were obtained under BL (Jungandreas et al., 2014). The light qualities, red, green, and blue light, played a vital role in regulation of carbon flow distribution. For example, the shift from BL to red light increased the intermediates of glycolysis and promoted the accumulation of carbohydrates. On the contrary, the shift of red light to BL led to the accumulation of amino acids and tricarboxylic acid (TCA) cycle intermediates, as well as biosynthesis of proteins (Jungandreas et al., 2014). The previous study indicated that the expression of photosynthesis-related nuclear genes were light quality-independent, while the energy transfer efficiency, photo protection and PSII repair related genes were highly dependent on light quality, especially BL (Valle et al., 2014). Similar results were observed in another research, in which compared to red light, BL performed larger pool size of xanthophyll cycle pigments and higher value of NPQ and de-epoxidation state [DES = Ddt/(Ddx + Ddt)], which meant BL showed more capacity of photo protection (Costa et al., 2013). BL not only influenced the genes expression to regulate metabolism, but also directly regulate the activities of specific enzymes, like nitrate reductase (Azuara and Aparicio, 1983). The significant reduction of C/N ratio in cells under BL indicated that it performed higher nitrogen assimilation in cells compared to red light (Jungandreas et al., 2014). This phenomenon was similar in our study, the nitrogen consumption rate was increased with higher proportion of BL in the mixed light except R:B (1:1) (Table 3). For the reason why TN consumption rate at R:B (1:1) was higher than at (1:2), it might be due to that the nitrogen source used in this study was the mixture of tryptone and urea. Perhaps more TN was used as carbon source at R: B (1:1), resulting in the higher TN consumption rate with the lower consumption rate of glycerol in this group. Interestingly, when cells were exposed to WL, BL, and red light, respectively, after dark treatment, the transcript levels of PSY, PDS, ZEP, and FCPb increased immediately upon blue and WL, while the expression levels were much lower in response to red light (Coesel et al., 2008). Additionally, transcriptome analysis indicated that the transcripts under BL were enriched in Fx-related expressed sequence tag (EST) database (Coesel et al., 2008), and the expression of PSY, PDS, ZDS, and ZEP3 were enhanced, resulting in the promotion of Fx production (Costa et al., 2013; Valle et al., 2014). Therefore, choosing R: B light (6:1) as the optimal light in phase 1 and BL as inducing light in phase 2 were carried out in further experiments.

Feeding substrates in batch culture is a common approach in microalgal cultivation which benefits biomass and bioactive compounds production (Ceron-Garcia et al., 2013; Lu et al., 2018). Fx exists in FCP, and sufficient nitrogen is essential not only for biomass production, but also for Fx accumulation (McClure et al., 2018; Wang H. et al., 2018; Nur et al., 2019). Thus, the effects of light shift and nitrogen supplementation in phase 2 were evaluated in two batch cultures.

In the batch 1, the mixotrophic cells were cultivated at R:B light (6:1) for 6 days in phase 1, then light shifted to pure BL for induction with or without supplementation of nitrogen [tryptone, T; urea, U; or the mixture, T:U = 1:1 (N mol)] in different groups in phase 2. As shown in Figure 4A, the culture groups of BL + T or BL+ the mixture in phase 2 could observe the promotion of cell growth compared to BL group, but BL + U inhibited the cell growth as well as Fx production (Figure 4B). The final cell density and biomass concentration reached to the highest value of 1.30 × 10⁸ cells mL^–1 and 5.80 g L^–1 by tryptone addition (Figures 4A,B), which were 23% and 58% higher than the control group (BL) (p < 0.05), respectively. The highest biomass productivity achieved 373.61 mg L^–1 d^–1 under tryptone addition, which was significant higher than BL + U and the control group (Table 4). In contrast, the previous study showed that successive supplementation of 0.01 mol L^–1 urea in mixotrophic growth of P. tricornutum led to 5.37-fold higher biomass, while the biomass was reduced when urea concentration exceeded 0.01 mol L^–1 with 0.10 mol L^–1 glycerol (Garcia et al., 2005). This phenomenon was caused by that the cells prioritized to utilize amino acids in tryptone as carbon and nitrogen source in phase 1, while the higher urea residual after urea addition in phase 2 exceed the optimum concentration, leading to the negative impact on cell division and Fx production in this study. As shown in Figure 4B and Table 4, the highest content (13.21 mg g^–1) and volumetric concentration (76.58 mg L^–1) of Fx were attained by tryptone addition in phase 2, with the consumption rate of glycerol (233.33 mg L^–1 d^–1) and TN (13.69 mg L^–1 d^–1). Besides, the intercellular amount of Fx was significantly increased up to 0.61 pg cell^–1 in BL + T group in phase 2, which was 1.42-fold higher than the control group (BL) (Table 4). Compared to the group of 12-days cultivation under R: B light (6:1) (Figures 3B,D), the biomass, Fx content and volumetric concentration were increased by 5%, 7%, and 12% under the two-phase culture in batch 1 (Figure 4B), respectively.

In the batch 2, to verify the inducing effect on Fx production, after cultivated at R: B light (6:1) for 6 days in phase 1, various light shifts were evaluated in phase 2 compared with the control group (BL + T). Among them, BL was enhanced solely to form R: B lights (5:1, 3:1, 1:1), or light shifted to pure green light (GL). All of the groups were combined with tryptone addition in phase 2. The light spectrums were shown in Figure 2B. Interestingly, the cell growth showed obvious difference from the 6th day in phase 2 but reached to a similar level of final cell density (Figure 4C), leading to a same specific growth rate in all groups (Table 4). However, the nutrition consumption rates were different (Table 4), which affected the metabolic flux of nutrients converted to intercellular components (carbohydrates, proteins, lipids etc.) in different groups, resulting in the difference of biomass concentrations. The highest biomass concentration (6.52 g L^–1), which was 17% higher than the control group (BL + T) (p < 0.01), was achieved with the maximum productivity (402.78 mg g^–1 L^–1) in R: B (5:1) + T group in phase 2 (Figure 4D and Table 4). Glycerol consumption rate and Y_X/TN were consistently increased with the increasing biomass, and reached to the highest value in R: B (5:1) + T group in phase 2 (Table 4), respectively. Compared to the control group (BL + T) in phase 2, even though the Fx content was lower in R:B (5:1) + T group (p < 0.05), the intercellular amount of Fx was similar in the two groups (Table 4) but the volumetric concentration of Fx (84.48 mg L^–1) achieved the highest level (p < 0.05) (Figure 4D). Therefore, the shift to R: B (5:1) with tryptone addition in phase 2 was the best option to encourage more biomass production and accumulation of intercellular Fx, leading to an increasing volumetric concentration of Fx.

It is noteworthy that the cell density was increased rapidly in GL + T in phase 2 (Figure 4C), but the lowest biomass concentration was observed (Figure 4D). Similarly in the previous report, the biomass entered stable phase and the lipid content increased by 53% and 29% when light shifted from R:B (50:50) (Sirisuk et al., 2018) and BL (Jung et al., 2019) to green light, respectively. Additionally, the content and intercellular amount of Fx were the lowest in GL + T compared to other groups, resulting in the lowest volumetric concentration of Fx (Figure 4D and Table 4). It was reported that carotenoids content was reduced after 3-days green light exposure (Jung et al., 2019), and the genes encoding LCYB, ZEP1, and ZEP 2 performed high initial transcription levels, then balanced out the difference after 24 h exposure to green light (Valle et al., 2014), which explained the reduction of Fx under green light. Additionally, the previous study indicated that Chl a was more inhibited compared to carotenoids under green light induction (Jung et al., 2019). These results explained the increase of Fx/Chl a ratio under green light in Table 4. Therefore, shifting to green light was neither the option for biomass nor for Fx production.

To further understand the physiological response and regulation mechanism of cells in R: B (5:1) + T group in the batch 2, the biomass and Fx production, nutrient consumption were analyzed in the two-phase culture. The cell samples were taken from the culture in phase 2 at four time points (6th, 8th, 10th, and 12th days) to evaluate the Fx content and key genes expression in Fx biosynthesis pathway.

As shown in Figure 5A, the biomass increased slowly with low biomass productivity (208.33 mg L^–1 d^–1) in phase 1, and then the cells utilized glycerol more quickly for rapid growth from 4th day, which might be due to the exhaustion of available amino acids in tryptone as carbon source of prior utilization. With the rapid consumption of glycerol in phase 2, the biomass productivity reached to 597.22 mg L^–1 d^–1, which was 2.87-fold higher than phase 1 (p < 0.01) (Figures 5A,B). The glycerol consumption rate achieved 533.33 mg L^–1 d^–1 in phase 2, resulting the 223% increase in the final biomass concentration (Figures 5A,B). However, the consumption rate of TN was lower in phase 2 (Figure 5B), which might be caused by a slow urea consumption in the medium since 6th day. The addition of tryptone provided sufficient organic carbon and nitrogen for biomass production in phase 2, while the Fx content and intercellular amount reached to the highest level (13.26 mg g^–1 and 0.95 pg cell^–1) on the 8th and 12th day, respectively (Figure 5C). At the end of cultivation, the content and intercellular amount of Fx increased by 8% and 12% compared to day 6 (p < 0.05) (Figure 5C). Through enhancing biomass concentration and Fx content in phase 2, the Fx productivity increased to 8.22 mg L^–1 d^–1, which was the highest level ever reported so far (Table 5).

It is known that biosynthesis of Fx involved in methylerythritol phosphate (MEP) pathway, IPP pathway and Fx formation (Bertrand, 2010). However, the final steps of Fx formation were not know completely so far (Lohr and Wilhelm, 2001; Dambek et al., 2012; Figure 5D). The DXS and PSY are two key rate-limiting enzymes to control the biosynthesis of Fx. The previous study indicated that the overexpression of DXS and PSY could raise the content of Fx by 2.40- and 1.80-fold in P. tricornutum, respectively (Eilers et al., 2016). Under the mixotrophic condition, glycerol consumption could supply abundant G3P (Villanova et al., 2017), which is the substrate of DXS and carbon skeleton of carotenoids biosynthesis. In this study, the expression of DXS was almost stable from 6th to 10th day, but decreased 1.46-fold on the 12th day compared to the 6th day (Figure 5D), suggesting that the BL strengthening with tryptone addition induced more carbon flux to TCA cycle and protein biosynthesis rather than pigments formation (Jungandreas et al., 2014). In contrast, the expression of PSY was 0.70-fold downregulated on the 8th day and significantly upregulated (|log₂fold change| > 2, p < 0.01) from the 10th day. Similarly, the previous report indicated that BL induction was proved to upregulate the expression of PSY under autotrophic condition in P. tricornutum (Coesel et al., 2008). One possible reason for the delay of PSY response was that the cells need time to adapt to light shift (Jungandreas et al., 2014), the other possibly reason relate to the expression of genes encoding ZEP. There were three types of ZEP identified in P. tricornutum (Bowler et al., 2008), and the expression levels of ZEP1, ZEP2, and ZEP3 were 4.87-, 2.06-, and 2.28-fold upregulated on the 8th day compared to 6th day, which not only contributed to the improvement of Fx content on the 8th day, but also accelerated the conversion from zeaxanthin to violaxanthin, leading to the promotion of PSY expression level from 10th day. Additionally, the expression level of FCPb was 1.70-fold higher on the 8th day than the 6th day but returned to the initial level from 10th day. However, the expression of VDE performed an opposite pattern, in which VDE transcript level changed slightly from 6th to 10th day and was 4.29-fold decreased on the 12th day (p < 0.01). In the previous report, the accumulation of Fx was not synchronized with the abundance of PSY transcripts, while the Fx content at the stationary phase was correlated with the amounts of PSY transcripts at the exponential phase (Kadono et al., 2015). Therefore, even though the intercellular amount of Fx increased slightly on the 8th day, the Fx content on the 8th day in phase 2 was significantly improved which depended on the upregulation of ZEPs and FCPb. And the reduction of intercellular amount and content of Fx on the 10th day might be due to the downregulation of PSY on the 8th day and to initial transcript level of FCPb on the 10th day. More importantly, the continued upregulation of PSY and ZEPs with downregulation of VDE contributed to the final Fx accumulation (both of intercellular amount and content) on the 12th day (Figure 5C), which proved that the option of R: B (5:1) + T in phase 2 was beneficial for enhancing Fx production in two-phase culture in the batch 2.

It was noteworthy that the expression of ZEPs were significantly upregulated (| log₂fold change| > 2, p < 0.01) when the culture shifted to phase 2. Among them, ZEP1 was the most sensitive gene in response to the BL induction (Figure 5D). A similar phenomenon was observed in the previous study, in which the increase of ZEP1 transcript level was over 50-fold higher than ZEP2 and ZEP3 under BL induction after dark treatment (Coesel et al., 2008). Since violaxanthin cycle and Ddx cycle were two xanthophyll cycles in diatoms participated in NPQ (Lavaud et al., 2003), BL and light intensity play vital roles in NPQ (Bertrand, 2010; Costa et al., 2013). One possible explanation was that P. tricornutum did not have the specific enzymes of Dtx epoxidase/diadinoxanthin de-epoxidase (DEP/DDE) in Ddx cycle, and the enzymes of ZEP/VDE in violaxanthin cycle played the same role instead (Bowler et al., 2008). So the ZEP1 regulated the transformation from zeaxanthin to violaxanthin and ZEP3 regulated the conversion from Dtx to Ddx (Nymark et al., 2009), leading to different response pattern of the ZEP1 and ZEP3 under BL. The another possible explanation was that ZEP1 and ZEP2 were suggested to be classified into category that contained light-harvesting complex and enzymes for pigments synthesis, while ZEP3 was classified as enzyme involved in photo-protection (Nymark et al., 2013; Valle et al., 2014). In this study, the high cell density (Figure 4C) resulted in less light exposure to individual cell during the 6th to 12th day, and ZEP3 might drive the conversion of violaxanthin to zeaxanthin and Ddx to Dtx in low light.

In a word, the expression levels of key genes involving Fx biosynthesis (PSY, ZEPs, FCPb, and VDE) were significantly regulated by BL strengthening and tryptone addition, which had positive effects on Fx accumulation, leading to the a great improvement of Fx production.