INTRODUCTION
Fucoxanthin is a key light-harvesting pigment found in the photosynthetic apparatus of brown algae, diatoms, and golden algae (Bai et al. 2022, Marcolin et al. 2024). In addition to its vital role in photosynthesis, fucoxanthin has been shown to possess a range of biological activities, including potent antioxidant, anti-inflammatory, and anti-tumor effects. These properties have led to its recognition for significant health benefits, such as the prevention and treatment of liver steatosis, obesity, diabetes, and cardiovascular diseases (Koller et al. 2014, Maeda et al. 2018, McClure et al. 2018, Din et al. 2022).
Commercially, fucoxanthin is primarily extracted from brown seaweed species like Laminaria japonica (kelp) and Undaria pinnatifida (Wang et al. 2021, Din et al. 2022). The global production of purified fucoxanthin is estimated at approximately 2,000 kg annually, with the bulk of it used in nutraceuticals aimed at weight management and liver fat reduction (Wang et al. 2020a, Mohibbullah et al. 2022). This market is valued at around US$ 200 million in 2024 (Budiarso et al. 2025).
Despite the ongoing cultivation and wild harvesting of seaweeds, the fucoxanthin content in seaweed biomass remains inherently low, typically ranging from 0.01 to 0.1% on a dry weight (DW) basis (Wang et al. 2021, Li et al. 2023). The low fucoxanthin content necessitates complex and expensive extraction and purification processes. As a result, fucoxanthin is sold at a high price—between US$ 45,000 and 55,000 per kilogram (Hu Personal Communication 2025)—making it a costly ingredient and limiting its use to niche markets. Thus, there is a critical need for alternative, more cost-effective production sources.
Diatoms have emerged as promising alternatives for fucoxanthin production. These organisms grow much faster than seaweeds and can accumulate fucoxanthin levels (1–3% of DW) that are one or two orders of magnitude higher than those found in brown seaweeds (McClure et al. 2018, Wang et al. 2021, Leong et al. 2022). Additionally, unlike seaweed farming, which allows for only one harvest per year, diatoms cultured in photobioreactors (PBRs) can be harvested weekly, potentially offering year-round production. In theory, mass cultivation of diatoms should enable significantly larger fucoxanthin yields with reduced production cost compared to traditional seaweed farming.
The diatom Phaeodactylum tricornutum is a well-established model organism for basic research (Budiarso et al. 2025, Wang and Hu 2025) and is emerging as a promising system for large-scale fucoxanthin production (McClure et al. 2018, Wang et al. 2022). Despite these advantages, large-scale, commercially viable cultivation of P. tricornutum for fucoxanthin production has not yet been realized. Key challenges remain, including the need for more robust algal strains, scalable and cost-effective PBRs, and optimized LED-based artificial lighting regimes that promote both enhanced algal growth and fucoxanthin biosynthesis under large-scale conditions.
This study addresses these challenges through a multifaceted approach. First, we evaluated the effects of varying light intensities and wavelengths of LED light on the growth and fucoxanthin content of P. tricornutum in glass column PBRs. Second, we used atmospheric and room temperature plasma (ARTP) mutagenesis to generate a heterogenous mutant library from the wild type (WT) strain. Third, the mutant library was subjected to adaptive evolution in a turbidostat under continuous cultivation conditions to select for strains with improved robustness and optimized fucoxanthin productivity. Finally, we developed and tested novel 200-L and 10,000-L PBR designs using both the WT strain and the superior mutant consortia. The results of this multifaceted approach are expected to lay the foundation for the future commercial development of P. tricornutum and other microalgae-based fucoxanthin production.
MATERIALS AND METHODS
Strains and culture conditions
The P. tricornutum UTEX640 used in this study was obtained from UTEX Culture Collection of Algae, the University of Texas at Austin, USA, and the algal strain was preserved in the laboratory using the F/2 culture medium with 10× nitrogen in both agar plate and liquid culture (He et al. 2022). To stepwise scale up cultivation of P. tricornutum, P. tricornutum colonies from an agar plate (Fig. 1A) were picked up and transferred into a triangular flask (Fig. 1B) and column PBRs (Fig. 1C). The cultivation of algal cells in both plate and triangular flask were conducted under cool white fluorescence light at an intensity of 30 μmol photons m−2 s−1 and maintained a temperature at 20°C. Effects of light quality (i.e., 450, 530, 600, 660, and 730 nm) and intensity (i.e., 5, 10, 25, 50, 100, 200, and 300 μmol photons m−2 s−1) on P. tricornutum growth and fucoxanthin production were investigated in a laboratory glass column photoreactor and further validated in both 200 L and 10,000 L PBRs (200 L PBR and 10,000 L PBR) with internal illumination by a LED lighting system (Fig. 1E & F). The 200 L PBR, measuring 500 mm (length) × 400 mm (width) × 1.4 m (depth), contained a number of red LED tubes (λ = 660 nm). Each LED tube had a power output of 75 W. The 10,000 L PBR, with dimensions of 1.8 m (length) × 2.4 m (width) × 2.6 m (depth), was installed a number of red LED tubes (λ = 660 nm), each with a power output of 100 W (Fig. 1F). By using the glass column turbidostat culture system (Fig. 1D), mutant consortia exhibiting enhanced biomass and fucoxanthin production were screened and further validated in both 200 L PBR and 10,000 L PBR (Fig. 1E & F). The culture system was maintained under continuous illumination at 20 ± 1°C and aerated with compressed air containing 1% CO2 at a rate of 0.1 vvm (volume of air per volume of culture medium per minute).
In this work, photosynthetically active radiation was measured as photosynthetic photon flux density using a spectrometer (HiPoint HR - 450; HiPoint, Kaohsiung, Taiwan) with a CMOS linear image sensor, which captured spectral data from 380 to 780 nm at 1 nm resolution. The sensor was oriented normal to the light source and measurements were taken at multiple positions to approximate the incident light field. Nevertheless, volumetric heterogeneity in light distribution persisted as a fundamental constraint in scaled PBRs, particularly under high cell densities and increasing reactor volume.
Growth analysis method
Biomass concentration was measured by a DW method (Zhu and Lee 1997). The 47 mm GF/C filtration membrane (aperture 1.2 μm Whatman GF/C; Whatman, Little Chalfont, UK) was first placed at 105°C and dried to a constant weight (W0). A certain amount (volume V) of algal culture was filtered through a vacuum pump onto the pre-weighed GF/C filtration membrane, and then the biomass was washed with 50 mL of 0.5 M NH4HCO3 to remove any attached salts. Then the membrane with algal biomass on it was dried at 105°C to a constant weight (W1).
Biomass concentration (B, g L−1) is calculated as:
, where “V” represents the volume of algal culture sampled.
Biomass productivity (BP, g L−1 d−1) is calculated as follows:
, where “B0” represents the initial biomass concentration on day 0 and “Bt” represents the biomass concentration on day t.
Fucoxanthin measurement
Cellular fucoxanthin content was determined by high performance liquid chromatography (HPLC) (He et al. 2022). Centrifuged the algae solution at 3,000 ×g for 5 min, and washed it with normal saline three times to remove the salt attached to the algae, and then freeze-dried (FreeZone12; Labconco, Kansas City, MO, USA). Weighed 10 mg of freeze-dried algal powder, dissolved it in a mixture of 1mL methanol and dichloromethane (methanol : dichloromethane = 3 : 1), put glass beads (G8772-500 g; Sigma, St. Louis, MO, USA) for shock grinding for 1 min, shaked well, and centrifuged at 10,000 rpm for 10 min. Transferred the supernatant to a 10 mL brown volumetric bottle, and the above steps were repeated three times until the supernatant was colorless. Then fixed the volume to 10 mL, shaked it well, and filtered the extract through 0.22 μm microporous membrane for chromatographic analysis.
Waters 2695 HPLC system (Waters, Milford, MA, USA) with Symmetry C18 chromatographic column was used for the filtrate analysis. The mobile phase was eluted by gradient elution of dichloromethane, methanol, acetonitrile and water, and the gradient elution procedure was shown in Table 1. The flow rate was 1 mL min−1, the sample size was 10 μL, the column temperature was 35°C, and the detection wavelength was 450 nm. The samples were quantified by external standard curve.
Construction of Phaeodactylum tricornutum mutant library
Monoclonal algal colonies grown on solid F/2-enriched culture medium were inoculated into a 50 mL flask containing 20 mL of culture medium and cultured for 10 d at 20°C under continuous illumination at a light intensity of 15 μmol photons m−2 s−1 and shaking at 110 rpm. After 7 d, the algal culture reached the logarithmic growth phase and was centrifuged at 3,000 ×g for 5 min. The cells were then resuspended in F/2 enriched culture medium to adjust a high cell concentration of 2.14 × 108 cells mL−1. Glycerol (80%) was added to the concentrated algal suspension to a final concentration of 5% (v/v, 80% glycerol : concentrated algal suspension = 1 : 15), resulting in a cell density of 2 × 108 cells mL−1.
A volume of 10 μL of the concentrated algal suspension mentioned above was pipetted onto a metal slide and spread evenly across the surface using a pipette tip. A total of 10 metal slides were prepared in this manner. The prepared slides were individually placed into the Atmospheric and Room Temperature Plasma Mutagenesis Breeding Machine (ARTP-M; Tmaxtree Biotechnology, Wuxi City, China) device for mutagenesis under the following conditions: a mutagenesis time of 40 seconds, helium gas flow rate of 10 mL min−1, power of 120 W, and an operating distance of 2 mm.
After ARTP-M induced mutagenesis, the metal slides with the algal suspension were immediately transferred into EP tubes containing 1 mL of F/2-enriched culture medium. The algal cells were gently resuspended from the slides into the medium using a pipette. The EP tubes were then incubated at 20°C under continuous illumination at 15 μmol photons m−2 s−1 and shaking at 110 rpm for 16 h to reactivate the cells. Subsequently, the algal cultures were transferred to 50 mL flasks containing 10 mL of F/2 enriched liquid culture medium for further cultivation, resulting in a mixed mutant library.
Enrichment screening of fast-growing mutants of Phaeodactylum tricornutum
The mixed mutants were cultured in the 700 mL glass columns in a continuous culture mode. The stable cell concentration (2.0 < OD730 < 2.5) of the culture was realized by a turbidostat controller (Fig. 1D). After 80 d of uninterrupted cultivation, the enrichment in fast-growing mutants in the libraries was assessed.
Data analysis
The data in this work were expressed as means ± standard errors, based on three replicates. Prior to ANOVA, the assumptions of normality and homogeneity of variances were verified using the Shapiro-Wilk test and Levene’s test, respectively. Statistical analysis was performed using SPSS ver. 24.0 (IBM Corp., Armonk, NY, USA), employing one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test to assess distinctions, with significance set at p < 0.05. The degrees of freedom, F-value, and corresponding p-value were presented in Supplementary Table S1.
RESULTS
Effects of different light qualities on biomass and fucoxanthin production in Phaeodactylum tricornutum
The effects of light quality on biomass and fucoxanthin production in the WT strain of P. tricornutum were investigated in glass columns (5.0 cm in inner diameter, 700 mL of culture volume) over a 6-d cultivation period, starting with an initial cell concentration of 0.25 g L−1 (Fig. 2). Five light qualities were tested, namely 450, 530, 600, 660, and 730 nm, while a cool white fluorescence light served as a control. Regardless of the light quality, the light intensity was maintained at 100 μmol photons m−2 s−1. The results showed that a two-day lag phase, indicative of algal adaptation to the new culture environment, preceded exponential growth, after which cell concentration and BP increased steadily (Fig. 2A & B). On day 6, the maximum biomass concentration of 1.96 g L−1 and the maximum BP of 0.29 g L−1 d−1 were obtained under 660 nm light, while the lowest values occurred at 730 nm (Fig. 2A & B). The culture pH (initially 8.15) decreased after aeration with 1% CO2-enriched air bubbles but gradually rose in parallel with the increase in biomass concentration, stabilizing at 6.30 under 730 nm and a range from 6.88 to 7.06 under other wavelengths (Fig. 2C). The fucoxanthin content (% DW) increased significantly by day 6 compared to day 3 under all light conditions, peaking at 1.76% DW under 730 nm (Fig. 2D). However, the highest fucoxanthin concentration (29.08 mg L−1) and productivity (4.18 mg L−1 d−1) were achieved at 660 nm on day 6, significantly surpassing other treatments (p < 0.05) (Fig. 2E & F). Thus, 660 nm was identified as the optimal wavelength for achieving maximum biomass and fucoxanthin productivity in P. tricornutum.
Effects of different intensities of 660 nm red light on biomass and fucoxanthin production in Phaeodactylum tricornutum
After identifying 660 nm red light as the optimal light quality for P. tricornutum, the next objective was to determine which intensity of red light could further enhance growth, fucoxanthin content, and productivity. To this end, a culture experiment was conducted using a series of red light intensities (i.e., 5, 10, 25, 50, 100, 200, and 300 μmol photons m−2 s−1), with a control group maintained under cool white fluorescent light (100 μmol photons m−2 s−1). Minimal growth was observed at 5 μmol photons m−2 s−1, while cultures exposed to other intensities exhibited varying growth rates. Growth increased with light intensity from 10 to 200 μmol photons m−2 s−1 but declined significantly at 300 μmol photons m−2 s−1, indicating photosynthetic photoinhibition at this intensity. On day five, biomass concentration and productivity were highest at 100 and 200 μmol photons m−2 s−1, reaching 2.09 and 2.18 g L−1, respectively, with corresponding biomass productivities of 0.37 and 0.39 g L−1 d−1 (p > 0.05) (Fig. 3A & B). Biomass declined outside this optimal intensity range (Fig. 3A & B).
The cellular fucoxanthin content (% DW) showed an inverse correlation with light intensity, peaking at 2.27% DW under 5 μmol photons m−2 s−1 and decreasing to 0.72% DW at 300 μmol photons m−2 s−1 on day five (Fig. 3C). Fucoxanthin concentration (mg L−1) increased significantly across all light conditions by day 5, reaching maxima of 30.44 mg L−1 at 50 μmol photons m−2 s−1 and 28.77 mg L−1 at 100 μmol photons m−2 s−1, with corresponding fucoxanthin productivities of 4.85 and 4.73 mg L−1 d−1 (p > 0.05) (Fig. 3D & E). Considering the greater energy efficiency and cost-effectiveness of lower light intensities, 50 μmol photons m−2 s−1 was identified as the optimal condition for fucoxanthin production in P. tricornutum.
Determination of a culture period for maximum fucoxanthin production in Phaeodactylum tricornutum
To determine the culture period during which maximum fucoxanthin production can be sustained under optimal light quality (660 nm) and intensity (50 μmol photons m−2 s−1), the cultivation period was extended from the previous 5 to 10 d. As shown in Fig. 4, the cells grew relatively slowly for the first 3 d, after which the biomass concentration increased, reaching 2.34 g L−1 by day 10. The maximum BP of 0.21 g L−1 d−1 was achieved by day 7, followed by stabilization (Fig. 4A & B). A linear correlation (y = 0.046 + 1.937x, R2 = 0.9857, Pearson’s r = 0.9928) between biomass DW and OD730 absorbance was established, enabling rapid biomass estimation (Fig. 4C). The fucoxanthin content peaked at 1.79% on day 5 and then gradually declined afterward (Fig. 4D). By day 10, the fucoxanthin concentration reached 31.18 mg L−1, with maximum productivity of 3.35 mg L−1 d−1 occurring on day 7 (Fig. 4E & F). Under these conditions, the P. tricornutum biomass stabilized without entering a decline phase, while the fucoxanthin productivity remained consistently high, demonstrating significant potential for industrial applications.
Optimization of light intensity in a newly designed 200 L PBR
To advance the mass culture of P. tricornutum for fucoxanthin production, a pilot-scale 200 L PBR was developed. The PBR comprised a culture vessel (measuring 59 cm in length, 49 cm in width, and 110 cm in height, an interior LED light system, an aeration system, and a cooling system. To determine the optimal light intensity—measured as electricity consumption per cubic meter of culture volume (m3)—for biomass and fucoxanthin production in the 200 L PBR, three light regimes were compared: Regime 1: Illumination with 0.45 kW m−3 for the first 2 d, followed by an increase to 0.9 kW m−3 for the following 2 d. Regime 2: Gradual increases to 0.45, 0.9, 1.35, and 1.8 kW m−3 on days 1, 2, 3, and 4, respectively. Regime 3: Higher increments to 0.9, 1.35, 1.8, and 2.25 kW m−3 on days 1, 2, 3, and 4, respectively. When light intensity was concerned, the energy levels of 0.45, 0.9, 1.35, 1.8, and 2.25 kW m−3 corresponded to approximately 200, 400, 600, 800, and 1,000 μmol photons m−2 s−1, respectively, measured at the surface of individual tubes (12 tubes in total inside the 200 L PBR).
At an inoculation density of 0.17 g L−1, the biomass of P. tricornutum reached 0.48, 0.63, and 0.71 g L−1 within 4 d (Fig. 5A), with corresponding biomass productivities of 0.08, 0.11, and 0.14 g L−1 d−1 for regimes 1, 2, and 3, respectively (Fig. 5B). By day 4, the fucoxanthin content in cultures exposed to light regimes 1, 2, and 3 decreased from 1.38 to 1.21, 1.03, and 0.83% (of DW), respectively (Fig. 5C). These changes correspond to reductions of 12.32, 25.36, and 39.86% compared to the initial levels. Light regime 1 showed significantly higher fucoxanthin content compared to regimes 2 and 3 (p < 0.05). However, regimes 2 and 3 resulted in greater fucoxanthin productivities (1.09 and 0.97 mg L−1 d−1, respectively) than regime 1 (0.80 mg L−1 d−1) (Fig. 5D). These results demonstrated that light regime 1 was preferred for mass cultivation of P. tricornutum as it maintained significantly higher fucoxanthin content, thereby ensuring a more sustainable and high-quality product yield in the 200 L PBR and perhaps larger PBRs of this design.
In the 200 L PBR, optimization of illumination revealed that Regime 1 resulted in higher cellular fucoxanthin content, whereas Regimes 2 and 3 achieved somewhat higher fucoxanthin productivity. This suggests that a gradual or elevated light-increasing strategy enhances total fucoxanthin production per unit volume and time, attributable to improved biomass accumulation under elevated light intensities in the later cultivation phase. However, the higher irradiance levels in Regimes 2 and 3 also made metabolic shifts that reduce fucoxanthin content at the higher light intensities (McClure et al. 2018, Wang et al. 2021). Despite the higher productivity under more intense light regimes, Regime 1 was selected for further scale-up due to its superior cellular pigment content and presumably healthier physiological status of the cells, both essential for successful expansion to larger-scale cultivation for an extended period of culture.
Development of superior Phaeodactylum tricornutum mutant consortia by ARTP technique
ARTP is a novel physical mutagenesis technique based on the principles of radio-frequency atmospheric pressure glow discharge. By ionizing inert gases (e.g., helium) in a high-frequency electric field, ARTP generates plasma containing highly reactive species (such as excited helium atoms, atomic oxygen, and hydroxyl radicals) under atmospheric pressure and room temperature conditions (Zhang et al. 2015). These reactive species induce various types of DNA damage (e.g., strand breaks, base oxidation) in microbial, plant, and animal cells (Wang et al. 2020b, 2023, Huang et al. 2021, Zhang et al. 2023). Applying this technique to P. tricornutum resulted in numerous mutant strains. To obtain mutants with desirable traits, such as enhanced photosynthetic growth potential and/or increased fucoxanthin production, a pool of P. tricornutum heterogenous mutants was inoculated into the glass columns under optimal culture conditions (e.g., F/2 culture medium with 10× nitrogen, 660 nm red light, and 50 μmol photons m−2 s−1). A turbidostat culture mode was adopted, maintaining an appropriate constant culture turbidity (ca. 1.0–1.6 OD730) by adjusting the average growth rate of the mixed P. tricornutum mutants through automated control of culture medium supply and dilution rate.
The primary goal of turbidostat culture was to enrich mutants with the improved traits. After eight months of continuous turbidostat cultures, the ARTP-induced P. tricornutum mutant consortia underwent 845 medium feedings, surpassing the WT control’s feedings (530) (Fig. 6A). This indicated potential enrichment of fast-growing mutants. Batch culture assessments in the glass columns confirmed these findings. In the glass columns with 700 mL of culture illuminated by 660 nm red light at 50 μmol photons m−2 s−1, the mutant consortia and WT strain achieved biomass concentrations of 2.21 and 1.91 g L−1, respectively, after 7 d (Fig. 6B). The BP (0.28 g L−1 d−1) of the mutant consortia significantly exceeded that of the WT (0.23 g L−1 d−1, p < 0.05) (Fig. 6C). While day-7 fucoxanthin content was comparable between the mutants (1.80%) and WT (1.72%, p > 0.05) (Fig. 6D), the mutant consortia demonstrated significantly higher fucoxanthin concentration (39.85 vs. 30.90 mg L−1, p < 0.05) (Fig. 6E) and productivity (5.03 vs. 3.67 mg L−1 d−1, p < 0.05) than those of the WT (Fig. 6F). Overall, the mutant consortia outperformed the WT in growth and fucoxanthin production in the 700 mL column PBR.
Evaluation of mass culture of the consortia of Phaeodactylum tricornutum mutants in 200 L PBR and 10,000 L PBR
The growth performance of the mutant consortia was further evaluated in the 200 L PBR. Based on the optimized light quality and intensity for the 200 L PBR, Light Regimen 1 was employed, namely with light intensities set at 0.45 kW m−3 for days 1–2 and 0.90 kW m−3 for days 3–4. The results were shown in Fig. 7. After a 4-d cultivation period, the mutant consortia achieved a biomass concentration of 0.60 g L−1, representing a 25% increase over the WT (0.48 g L−1) (Fig. 7A), with a BP of 0.12 g L−1 d−1 (Fig. 7B). On day 4, the mutant consortia exhibited a 20.66% higher fucoxanthin content (1.46% DW) compared to the WT (1.21% DW) (Fig. 7C). Additionally, the mutant consortia demonstrated significantly higher fucoxanthin concentration (8.68 mg L−1) and productivity (1.80 mg L−1 d−1) compared to the WT (5.81 and 0.80 mg L−1 d−1, respectively; p < 0.05) (Fig. 7D & E). In summary, the mutant consortia outperformed the WT in biomass and fucoxanthin production in the 200 L PBR.
Building upon the design and principles of the 200 L PBR, a scaled-up 10,000 L PBR of the similar design was developed. The light power was maintained at 0.45 kW m−3 for days 1–2 and 0.90 kW m−3 for days 3–4, consistent with the 200 L PBR. Both the WT and the mutant consortia were cultivated in the 10,000 L PBR (Fig. 8). The initial inoculation densities were 0.15 g L−1 for the WT and 0.12 g L−1 for the mutant consortia. After 4 d, the biomass concentrations reached 0.50 g L−1 for the WT and 0.59 g L−1 for the mutant consortia (Fig. 8A), with corresponding productivities of 0.09 and 0.12 g L−1 d−1, respectively (Fig. 8B). Compared to the WT, the mutant consortia demonstrated an 18.00% increase in biomass concentration and a 33.33% increase in productivity (p < 0.05). On day 4, the fucoxanthin content was 1.10% for the WT and 1.23% for the mutant consortia (p < 0.05), with concentrations of 5.49 mg L−1 and 7.29 mg L−1, respectively (Fig. 8C & D). Fucoxanthin productivity was 0.95 mg L−1 d−1 for the WT and 1.47 mg L−1 d−1 for the mutant consortia (Fig. 8E), with the mutant consortia significantly exceeding the WT (p < 0.05). In summary, the mutant consortia showed significant advantages in both biomass and fucoxanthin production over the WT in the 10,000 L PBR, highlighting its potential for scalable industrial applications.
To assess long-term culture stability, both WT and mutant strain consortia were serially passaged in 200 L PBRs. In the semi-continuous culture system, each batch was cultivated for 4 d, after which approximately 80% of the culture suspension was harvested. The remaining culture volume, along with fresh growth medium, was used as the inoculum for the next cycle. Over the course of 23 consecutive batches (over 3 months), the mutant strain consortia maintained stable growth without any signs of deterioration (red curve). In contrast, the WT strain showed a significant reduction in growth rate after 3 batches and almost complete growth arrest by batch 5 (black curve) (Fig. 9).
DISCUSSION
Although the biological functions and commercial value of fucoxanthin from P. tricornutum have been well recognized, large-scale outdoor cultivation of organism has not been commercialized. This stems from two major obstacles, i.e., the lack of suitable large-scale PBRs for sustainable cultivation of P. tricornutum, and the changing environments constantly under outdoor conditions during the day and throughout the year make mass culture of the wildtype strain of P. tricornutum unstable and poor fucoxanthin productivity. For instance, Song et al. (2018) reported a low biomass of 0.178 g L−1 after 5 d in an 8,000 L open consortia, while Pereira et al. (2021) achieved 200 kg of biomass over 118 d in a 125 m3 PBR, with a very low average fucoxanthin content of 0.4%. To overcome the first obstacle, the novel LED-powered 200 L and 10,000 L PBRs were developed, of which the 10,000 L PBR is the largest single unit of LED-based closed PBR reported for mass culture of P. tricornutum (Table 2). Although mass culture of microalgae on artificial light is generally much more costly compared to that powered by sunlight, the former sometimes could be a preferred option for selected algal strains for sustainable production of a specific product (Blanken et al. 2013), such as P. tricornutum for fucoxanthin production. In this study, these LED-based PBRs demonstrated the ability to achieve sustainable cultivation of P. tricornutum during the day and throughout the year. The content and productivity of fucoxanthin in P. tricornutum obtained in this work were among the highest in the literature (Table 2).
To solve the second problem, instead of utilizing solar irradiance, a 660 nm monowavelength red light was employed as the light source for P. tricornutum culture. It was found that compared to blue light, green light, yellow light, and far-red light, the red light of 660 nm not only increased algal growth but also fucoxanthin content and thus productivity of this organism (Fig. 2). These findings are in agreement with previous studies using the same organism (Zang et al. 2015, Zhang et al. 2022) and several other species (Gaytán-Luna et al. 2016, Zhang et al. 2021).
Light intensity of a given wavelength light is another important factor affecting algal growth and fucoxanthin production. The highest fucoxanthin content of 2.27% was obtained by providing the cultures with red light of 5 μmol photons m−2 s−1 (Fig. 3C). The pigment content decreased gradually as the light intensity increased to 300 μmol photons m−2 s−1 (Fig. 3C). The effect of light intensity on the cellular fucoxanthin content observed in this study was consistent with the previous reports (Xia et al. 2013, Guo et al. 2016, McClure et al. 2018). For example, Guo et al. (2016) reported fucoxanthin concentrations of 1.08% at 10 μmol photons m−2 s−1, declining to 0.62% at 40 μmol photons m−2 s−1. Similarly, McClure et al. (2018) observed higher fucoxanthin concentrations at 100 μmol photons m−2 s−1 (42.8 ± 19.5 mg g−1) compared to 210 μmol photons m−2 s−1 (9.9 ± 4.2 mg g−1). The discrepancies in terms of exact fucoxanthin content may be due to the differences in algal strains, culture vessels, and operational protocols adopted by the individual studies.
The optimal fucoxanthin productivity observed under 660 nm red light at moderate intensity (e.g., 50–100 μmol m−2 s−1) can be attributed to its efficient stimulation of photosynthetic activities and downstream metabolic regulation. Red light at the moderate light intensity aligns closely with the absorption peaks of chlorophyll a, maximizing photon capture and electron transport rates while minimizing energy loss (Blanken et al. 2013, Schulze et al. 2014, Tsaballa et al. 2023). This enhanced energy supply is believed to upregulate the methylerythritol phosphate pathway, thereby potentially increasing precursor availability for fucoxanthin biosynthesis (Chenge-Espinosa et al. 2018). Concurrently, the moderate light intensity avoids oversaturating the photosystems, thereby avoiding the photoinhibitory damage (Li et al. 2022). Furthermore, the specific light regime determined in this study promotes a balanced allocation of resources between growth and fucoxanthin biosynthesis. While high light intensity or shorter-wavelength light (e.g., blue light) exacerbates photodamage through reactive oxygen species generation and activates energy-dissipating mechanisms, red light at optimized intensity sustains photosynthetic efficiency without imposing severe oxidative stress (Kommareddy and Anderson 2003, Fu et al. 2013, Sharma et al. 2020). This favorable condition allows the cells to invest more carbon into light harvesting pigments like fucoxanthin. The synergy between enhanced photosynthetic output and controlled light conditions under red illumination thus creates an ideal environment for high fucoxanthin productivity.
In addition to employing the improved PBRs coupled with optimized light quality and intensity to enhance the growth and fucoxanthin production of P. tricornutum, this study also utilized ARTP-based mutagenesis to further increase fucoxanthin yield. This study subjected the ARTP-generated P. tricornutum mutant consortia to adaptive evolution under optimal growth conditions in the turbidostats for over a year. The significantly higher number of medium feedings in the turbidostat-cultured mutant consortia suggested a substantial increase in BP relative to the wild type, consistent with the enrichment of fast-growing phenotypes in the mutant consortia. This enhanced growth directly supported the improved fucoxanthin production observed, as a higher biomass output provides greater amount for fucoxanthin. The feeding count data thus serve as a robust proxy for both sustained growth and overall productivity in the turbidostat system. Previous studies have used chemical mutagens such as ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) to generate P. tricornutum mutants, resulting in a 69.3% increase in fucoxanthin content (approximately 1.69%) compared to the WT (Yi et al. 2018). Similarly, another study reported that an EMS-induced P. tricornutum mutant exhibited a 35% increase in fucoxanthin content relative to the WT (Yi et al. 2016). However, these results were obtained from single P. tricornutum mutant strain, where only a single desirable mutant was selected and cultured. In contrast, this study subjected the ARTP-based mutagenesis coupled with the adaptive evolution selection approach. The resulting mutant consortia with enrichment of fast-growing phenotypes, resulted in 15–20% increase in BP compared to the WT strain across scales.
Previous efforts to improve fucoxanthin production in genetically modified P. tricornutum mutants were limited to laboratory-scale experiments (Table 2). In this study, however, the superior performance and higher fucoxanthin productivity of the ARTP-generated P. tricornutum mutant consortia were demonstrated in pilot-scale 200 L and 10,000 L PBRs, bringing P. tricornutum-based fucoxanthin production closer to commercial viability. The highest BP of the mutant consortia was observed on day 1 in the 10,000 L PBR (Fig. 8B), likely attributable to the low initial biomass density, which facilitated greater light penetration and nutrient availability. As cell concentration increased, light availability to individual algal cells became limited, resulting in reduced photosynthetic productivity. Notably, the culture stability observed in large-scale cultures of the mutant consortia over extended periods (more than one year) highlights the effectiveness and robustness of using the consortia of enriched superior mutants for the cultivation of P. tricornutum and potentially other commercially valuable microalgae.
Although the mutant consortia exhibited stable phenotypic productivity over 1 year, it is important to note that strains derived from random mutagenesis remain genetically heterogeneous and may be prone to potential long-term instability. The absence of phenotypic reversion in this study is promising but does not eliminate the risk associated with non-targeted mutations. Therefore, while the mutant consortia serve as a valuable proof-of-concept, industrial deployment necessitates the development of clonal lines. Future work will focus on isolating the most promising mutants from the consortia to perform detailed genomic and molecular analyses. This includes targeted expression profiling (e.g., quantitative polymerase chain reaction or RNA sequencing) of key genes involved in the methylerythritol phosphate pathway and carotenoid biosynthesis in response to light quality and intensity, to elucidate the mechanistic links between genotype and phenotype. Further validation of the long-term performance of the mutant consortia at larger scales will also be conducted to ensure that the mutant consortia remains stable and continues to outperform the WT or any single mutant in terms of both productivity and stability over extended cultivation periods.
The development of the 200 L and 10,000 L internally illuminated PBRs was to address key engineering challenges in light distribution, mixing efficiency, and CO2 delivery in the culture vessels. Red LED tubes (λ = 660 nm) were strategically positioned (e.g., all the LED tubes were 15 cm apart from one another) to ensure uniform illumination and minimize gradient formation, thereby reducing photolimitation and photoinhibition. The aeration system, supplying air enriched with 1% CO2 at 0.1 vvm, enhanced both gas-liquid mass transfer and fluid mixing, preventing CO2 depletion and cell settling. Combined with precise temperature control at 20 ± 1°C, these conditions supported consistent metabolic activity and high fucoxanthin productivity across scales. The reactor geometry and internal light configuration helped maintain comparable light exposure per unit volume, contributing to reproducible performance between the 200 L and 10,000 L systems. This integrated approach demonstrates the scalability of PBR designs that concurrently optimize light, gas transfer, and mixing, aligning with established scale-up principles for phototrophic cultivation. While this integrated approach demonstrates scalable modular PBR design aligning with established principles for phototrophic cultivation, further quantification of key scale-up parameters, such as mixing efficiency, mass transfer coefficient, and nutrient distribution homogeneity, will be essential to fully characterize performance in the 10,000 L and ever larger scale PBR of this design for improved culture performance and its industrial applicability.
The demonstrated scalability of P. tricornutum cultivation in internally illuminated PBRs up to 10,000 L presents a technically viable pathway for fucoxanthin production. To robustly evaluate its industrial feasibility, a techno-economic analysis (TEA) is indispensable. Major operational costs originate from energy-intensive illumination using red LEDs and gas exchange systems, while capital investment is dominated by customized PBR with integrated optical and control systems. Additional expenditures include cooling, culture media recycling, labor, and maintenance. Key strategies for improving economic sustainability include enhancing photon conversion efficiency through spectral and intensity modulation, adopting renewable energy, implementing media recycling, and advancing automation to reduce labor dependency. A comprehensive TEA that incorporates upstream and downstream processing costs, alongside market analysis of fucoxanthin value, is necessary to validate commercial competitiveness. Such an assessment would bridge the gap between laboratory-scale success and industrially relevant biomanufacturing, providing critical insights into the financial practicality of large-scale microalgal fucoxanthin production.
This study demonstrates enhanced biomass and fucoxanthin production in P. tricornutum through optimized light regimes combined with a novel PBR design. The introduction of a P. tricornutum mutant consortia, composed of ARTP mutagenesis-generated mutants enriched for fast-growing phenotypic characteristics through adaptive evolution, further increased fucoxanthin productivity compared to the WT strain. The fucoxanthin productivities obtained from cultures maintained in 700 mL glass columns, 200 L PBRs, and 10,000 L PBRs in this study are among the highest reported in the literature (Table 2). To build on this work, large-scale cultivation of P. tricornutum mutant consortia in an industrial setting will further evaluate their superiority and sustainability compared to the WT strain. Additionally, the isolation of individual superior mutants, genomic characterization, and long-term monitoring will be conducted to ensure the reliability and scalability of the mutant consortia for large-scale production.
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