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Article

Enhanced Production of Bioactive Polyunsaturated Fatty Acids and Pigments in Rhodosorus marinus: Optimization of Thermal and Photic Stress Conditions

by
Wen-Ping Cheng
1,†,
Han-Yang Yeh
2,†,
Yen-Ling Chen
1,3,
Yi-Jung Chen
1,
Fat-Tin Agassi Sze
1,4,
Chi-Cheng Huang
1,
Fan-Hua Nan
1,
Ming-Chih Fang
5,* and
Meng-Chou Lee
1,6,*
1
Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan
2
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung City 912301, Taiwan
3
Aquaculture Division, Fisheries Research Institute, Ministry of Agriculture, Keelung City 202008, Taiwan
4
Algaetech Key Laboratory, Algaetech (Hong Kong) Co., Ltd., Kowloon, Hong Kong, China
5
Department of Food Science, National Taiwan Ocean University, Keelung City 202301, Taiwan
6
Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 202301, Taiwan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Mar. Drugs 2026, 24(2), 78; https://doi.org/10.3390/md24020078
Submission received: 9 January 2026 / Revised: 31 January 2026 / Accepted: 10 February 2026 / Published: 13 February 2026
(This article belongs to the Special Issue Innovations in Marine Algal Biotechnology: From Bioprocessing to Applications)
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Abstract

The marine unicellular red alga Rhodosorus marinus is a promising source of the valuable phycobiliprotein phycoerythrin and essential omega-3 polyunsaturated fatty acids (PUFAs), yet the environmental triggers for their optimal co-production remain to be fully elucidated. This study was conducted to investigate the effects of thermal and photic stress in terms of maximizing the yield of these high-value bioactive compounds. R. marinus was cultivated under a range of temperatures (18–24 °C) and light intensities (100–335 µmol photons m−2 s−1) to assess its physiological and biochemical responses, particularly focusing on lipid accumulation. This study investigates the effects of thermal (18–24 °C) and photic (100–335 µmol photons m−2 s−1) stress on the concurrent production of the valuable phycobiliprotein, phycoerythrin (PE), and essential omega-3 polyunsaturated fatty acids (PUFAs) in the marine red microalga Rhodosorus marinus. Fatty acid profiles were quantified using gas chromatography (GC), while pigment content was assessed via spectrophotometry. Statistical analyses, including one-way ANOVA and Tukey’s post hoc test, were employed to determine the significance of environmental effects. Our results demonstrate that a mild hypothermic condition of 18 °C significantly enhanced the production of eicosapentaenoic acid (EPA) compared to higher temperatures. Conversely, cell density was maximized at 22 °C. Under the 18 °C thermal regime, lower light intensities (100–185 µmol photons m−2 s−1) promoted a superior synthesis of both bioactive lipids and pigments. In conclusion, the strategic application of mild hypothermia combined with moderate light intensity is an effective approach to substantially boost the metabolic yield of high-value compounds in R. marinus, highlighting its potential as a sustainable source for nutraceutical and pharmaceutical applications.

1. Introduction

The marine biosphere represents the largest reservoir of biodiversity on Earth, offering an unparalleled source of chemical diversity for drug discovery and the nutraceutical industry [1,2]. Algae play a crucial role in marine ecosystems and are a significant source of bioactive compounds [3,4]. Through highly efficient photosynthesis, algae fix carbon dioxide (CO2) and assimilate inorganic nutrients, making an indispensable contribution to environmental sustainability challenges such as mitigating eutrophication, wastewater treatment, and addressing global climate change [5,6,7]. Microalgae, for instance, can fix CO2 at rates 10–50 times higher than terrestrial plants, with reported fixation rates reaching 1–4 g L−1 day−1 [8]. Furthermore, their efficiency in nutrient removal from wastewater is remarkable, with removal rates of up to 99.6% for nitrogen and 100% for phosphorus [9].
Among the bioactive substances synthesized by algae, photosynthetic pigments and lipids have garnered widespread attention due to their notable pharmacological activities. The photosynthetic pigment system primarily consists of chlorophylls and phycobiliproteins (PBPs) [10]. PBPs, water-soluble accessory pigments, transfer energy to reaction centers in Cyanobacteriota and Rhodophyta [11,12]. Notably, Phycoerythrin (PE) is considered a high-value compound due to its intense fluorescence, high stability, and diverse biological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects [13,14]. Specifically, the red microalga Rhodosorus marinus has been identified as an excellent source rich in B-phycoerythrin (B-PE) [15,16]. Phycoerythrin (PE) derivatives, such as B-phycoerythrin (B-PE), are extensively employed as a natural food colorant and nutritional supplement, primarily owing to their distinctive spectral properties and robust antioxidant capacity; moreover, B-PE has demonstrated significant anti-neoplastic effects in vitro by effectively inhibiting the proliferation of Graffi and myeloma cells. Furthermore, C-phycoerythrin (C-PE) exhibits considerable therapeutic potential, effectively mitigating systemic hypertension and acute kidney injury associated with chronic kidney disease (CKD) via the attenuation of oxidative and endoplasmic reticulum stress [17,18].
In addition to pigments, microalgae are also efficient “biofactories” for the synthesis of polyunsaturated fatty acids (PUFAs) [19]. Microalgae are pivotal organisms in the marine environment capable of synthesizing the essential fatty acids linoleic acid (LA, 18:2n-6) and alpha-linolenic acid (ALA, 18:3n-3) from 18:0-acyl carrier protein (ACP). Subsequently, LA and ALA are bioconverted into the long-chain polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) through the sequential action of fatty acyl elongases and desaturases, which are then accumulated within the cells [20,21]. The clinical benefits of these PUFAs for human health are well-established, with sufficient daily intake of marine omega-3 significantly reducing the risk of chronic diseases such as cardiovascular disorders [22]. Specifically, Porphyridium purpureum demonstrate remarkable metabolic flexibility, capable of accumulating high levels of B-phycoerythrin (0.193 ± 0.002 g/L) and long-chain poly-unsaturated fatty acids (EPA yield up to 0.072 g/L), depending on the specific cultivation conditions and medium composition employed [23]. However, these production levels remain suboptimal for large-scale commercial applications, necessitating the optimization of cultivation parameters to enhance metabolite yields. Furthermore, the combination of microalgae-derived PUFAs with postbiotics has emerged as a novel strategy to modulate the gut microbiota–adipose tissue axis, thereby alleviating systemic inflammation and metabolic disorders associated with obesity [24,25]. The physiological response of microalgae to environmental changes, such as fluctuations in temperature, light intensity, or nutrient supply, triggers the adjustment or degradation of their stored lipid and pigment metabolic pathways as a survival mechanism [26,27]. While previous research has explored the accumulation of bioactive compounds under various environmental conditions [16,28], a comprehensive study is still lacking that aims to simultaneously optimize the concurrent production of both phycoerythrin and PUFAs in R. marinus under controlled environmental stress, and to correlate the physiological changes with the biochemical yields.
This study aims to systematically investigate the impact of environmental factors (such as temperature and light) on the growth kinetics and biochemical composition of R. marinus. The ultimate goal of this research is to identify conditions that significantly enhance the productivity of both phycobiliproteins and PUFAs, thereby enhancing the application potential of these high-value bioactive compounds in the pharmaceutical and nutraceutical fields.

2. Results

2.1. Effects of Temperature on Growth, Physiology, and Fatty Acid Composition

2.1.1. Effects of Temperature on Growth and Physiological Parameters

R. marinus was cultured in basal medium under the following conditions: four temperature regimes (18 ± 1, 20 ± 1, 22 ± 1, and 24 ± 1 °C), initial pH of 8.0 ± 0.2, photoperiod of 12:12 h (light: dark), and air flow rate of 5 L/min. Cultures were inoculated at 105 cells/mL and maintained for 14 days, with samples collected daily.
The experimental results demonstrated that the highest cell numbers of R. marinus were observed when cultured at 22 °C, followed by 18 °C and 20 °C, with the lowest cell numbers recorded at 24 °C (p < 0.05). After 14 days of cultivation, the Relative Index of Chlorophyll (RIChl) was highest at 18 °C, followed by 20 °C and 22 °C, and lowest at 24 °C (p > 0.05). The Relative Index of Phycoerythrin (RIPE) was observed to be higher at 18 °C and 20 °C, and lower at 22 °C and 24 °C (p < 0.05) (Figure 1).
On the 14th day of cultivation, the lowest temperature (18 °C) yielded the highest maximum photochemical quantum yield (Fv/Fm) value, followed by 20 °C, 22 °C, and 24 °C (p < 0.05). The maximum electron transport rate (ETRmax) showed a similar trend, with 18 °C offering a better ETRmax, while lower values were recorded at 20 °C, 22 °C, and 24 °C (p < 0.05) (Figure 2).

2.1.2. Effects of Temperature on Fatty Acid Composition

Algae were harvested on the 14th day of cultivation for fatty acid analysis. Within the studied temperature range, the main fatty acid components identified were C20:5, C16:0, C18:1, C18:2, and C16:1. Among these, the content of C20:5 exhibited significant variations across different temperature conditions. The concentration of C20:5 significantly decreased as the temperature increased from 18 °C to 24 °C (p < 0.05). The contents of other fatty acids, C16:0, C18:1, C18:2, and C16:1, did not show significant differences between 18 °C, 20 °C, 22 °C, and 24 °C (p > 0.05) (Table 1). Total fatty acids, total triglycerides, polyunsaturated fatty acids (PUFAs), and total fat contents showed no significant differences between groups (Supplementary Figure S2).
Considering the combined effects on growth performance, chlorophyll fluorescence, and fatty acids, a cultivation temperature of 18 °C was ultimately selected for the subsequent light intensity experiment.

2.2. Effects of Light Intensity on Growth, Physiology, and Fatty Acid Changes

2.2.1. Effects of Light Intensity on Growth and Physiological Parameters

Over the 14 day cultivation period, all light intensities supported cell growth, with no significant differences in cell numbers observed among the groups (p > 0.05). However, RIChl and RIPE were highest at 100 µmol photons m−2 s−1, followed by 185 and 245 µmol photons m−2 s−1, with the lowest values observed at 335 µmol photons m−2 s−1 (p < 0.05) (Figure 3).
Other photosynthetic parameters, such as Fv/Fm, were highest at 100 µmol photons m−2 s−1, followed by 185 µmol photons m−2 s−1, with the lowest values observed at 245 and 335 µmol photons m−2 s−1 (p < 0.05). Conversely, ETRmax did not show significant differences across 100, 185, 245, and 335 µmol photons m−2 s−1 (p > 0.05) (Figure 4).

2.2.2. Effects of Light Intensity on Fatty Acid Composition

Algal samples cultivated under light intensities of 100, 185, 245, and 335 µmol photons m−2 s−1 were collected on the 14th day for fatty acid analysis. Within the studied light intensity range, the primary fatty acids identified were C20:5, C16:0, C18:1, and C18:2. The highest content of C20:5 was observed under 185 µmol photons m−2 s−1 cultivation conditions, followed by 100, 335, and 245 µmol photons m−2 s−1 (p < 0.05). The highest contents of C16:0, C18:1, and C18:2 were all found at 100 µmol photons m−2 s−1 (p < 0.05) (Table 2). Total fatty acids, total triglycerides, PUFAs, and total fat contents were also highest at 100 µmol photons m−2 s−1 (p < 0.05), with C20:5 (EPA) content peaking at both 100 and 185 µmol photons m−2 s−1 (Supplementary Figure S3). It should be noted that while statistical analysis demonstrated significant differences among the tested conditions, these findings represent observed conditions that promote enhanced metabolite production rather than validated optimal parameters determined through formal optimization procedures (e.g., response surface methodology).

3. Discussion

3.1. Effects of Temperature and Light Intensity on Growth and Physiological Parameters

To quantitatively compare the growth dynamics, the specific growth rates (μ) were calculated from the exponential phase of cell growth under different temperatures. The maximum specific growth rate (μmax) was observed at 22 °C (μ = 0.22 day−1), which was notably higher than the rates recorded at 18 °C (μ = 0.17 day−1) and 24 °C (μ = 0.18 day−1). This suggests that 22 °C is the optimal thermal condition for the exponential growth phase of this culture. This value is comparable to the rates reported for other red microalgae, such as Porphyridium purpureum (μ = 0.20 day−1) under similar optimized conditions [29]. While the final cell density was highest at 22 °C, the lower temperature of 18 °C, despite a slightly lower growth rate, proved more effective for the accumulation of target bioactive compounds. Algal growth rates are significantly influenced by environmental factors, with light and temperature being primary determinants of chemical reaction rates and cellular component structure. These factors impact enzymatic activity, membrane fluidity, electron transport chain efficiency, and other metabolic mechanisms [30]. Previous research indicates that increasing light intensity enhances the growth of microalgae under light-limiting conditions, as microalgae generally prefer higher light intensities until a photo-saturation point. Consequently, providing sufficient light intensity promotes photosynthesis within algal cells and facilitates the accumulation of physiologically active substances [31,32].
Generally, algal growth rates increase with rising temperature or light intensity within suitable ranges. However, different algal species exhibit varying degrees of adaptability to environmental conditions, which indirectly affects their growth performance, physiological parameters, and the accumulation of bioactive substances. For instance, Nannochloropsis sp. and Isochrysis galbana grew optimally at 14 °C and 20 °C, respectively, while Rhodella maculata performed best at 26 °C [33]. This study revealed that R. marinus samples cultivated at 22 °C surpassed other groups in terms of cell density over time. No significant difference was observed between the 18 °C and 20 °C samples, suggesting that these two temperatures yield similar growth results for R. marinus. The optimal cultivation temperature was further evaluated by correlating cell density with physiological parameters such as Fv/Fm, ETRmax, and the target fatty acid EPA.
R. marinus exhibited slow growth rates under light intensities ranging from 20 to 60 µmol photons m−2 s−1, but cell numbers increased significantly at 100 µmol photons m−2 s−1 [16]. These results suggest that the growth of R. marinus is inhibited under low light conditions, and that 100 µmol photons m−2 s−1 is within the light-limiting region. This study showed that no significant differences in cell numbers were observed among the various light intensities at 18 °C and 100–335 µmol photons m−2 s−1 (p > 0.05), indicating that the light saturation range for R. marinus is between 100 and 335 µmol photons m−2 s−1. This experiment demonstrated that R. marinus can grow and maintain a stable physiological state at 18 °C and under light intensities ranging from 100 to 185 µmol photons m−2 s−1.

3.2. Effects of Temperature and Light Intensity on Photosynthetic Pigments

Chlorophyll plays a pivotal role in photosynthesis by absorbing and transferring light energy and maintaining algal cell metabolism [34]. At lower temperatures, chlorophyll synthesis increases to accommodate light absorption under cold conditions. However, higher temperatures inhibit chlorophyll synthesis, leading to instability in photosynthesis and cellular metabolism [35]. In this study, R. marinus showed a relatively higher RIChl at 18 °C compared to 22 °C and 24 °C. Correlation analysis revealed a strong positive correlation between RIChl and RIPE at 18 °C, 20 °C, and 22 °C (r = 0.82–0.98, p < 0.05) (Supplementary Table S1), indicating a close relationship between chlorophyll and phycoerythrin under specific temperatures. Previous research confirms that temperature influences algal photosynthesis and cellular metabolism, thereby regulating the rates of chlorophyll synthesis and degradation. Interestingly, the EPA content in samples cultivated at 18 °C was positively correlated with the RIChl, suggesting a link between temperature and EPA synthesis, and that EPA is a component of chloroplast membranes [36,37,38].
Light intensity is another factor that influences RIChl. Studies have found that R. marinus exhibits higher RIChl under low light intensity, with decreases significantly under higher intensities. Previous research shows that the photoperiod affects the chlorophyll content of Ankistrodesmus falcatus. When cultured under a 12L:12D or 18L:6D photoperiod, its chlorophyll content was significantly higher compared to an experimental group without light [32]. Bonente et al. (2012) demonstrated that chlorophyll content of Chlamydomonas reinhardtii was inversely proportional to light intensity at 20, 60, and 400 μmol photons m−2 s−1 [31]. The chlorophyll a content of Rhodomonas salina was inversely proportional to light intensity under light conditions varying from 20 to 250 μmol photons m−2 s−1 [39]. Numerous studies have shown that algae have an increased demand for light energy under low light intensity conditions, which promotes chlorophyll accumulation. Under high light intensity conditions, algae tend to reduce chlorophyll content to protect cells from photodamage. The results of the present study are thus consistent with the findings of previous studies.
Previous studies have demonstrated that temperature significantly influences the synthesis and accumulation of phycoerythrin. Algae typically enhance phycoerythrin synthesis at lower temperatures, a physiological adaptation to optimize light harvesting under environmental light intensity and temperature stress. Conversely, elevated temperatures often lead to the inhibition of phycoerythrin synthesis [40]. Our temperature experiments revealed that R. marinus exhibited higher RIPE values when cultured at 18 °C and 20 °C, with a notable decrease observed at 22 °C and 24 °C. This aligns with observations that the proportion of phycoerythrin within the phycobiliproteins of Colaconema sp. is temperature-dependent [40]. Similarly, Porphyridium purpureum displayed a higher phycoerythrin content at 20 °C compared to 25 °C [41], and Rhodomonas sp. showed its lowest phycoerythrin content at 32 °C when compared to 20 °C and 26 °C [42].
In the present investigation, R. marinus achieved its highest RIPE at 18 °C under a light intensity of 100 μmol photons m−2 s−1 (p < 0.05). Interestingly, a negative correlation was identified between the growth rate of R. marinus and its RIPE, indicating that increased light intensity corresponded to a reduction in RIPE content within algal cells. This observation is consistent with prior research suggesting that microalgae augment the concentration of pigments in their reaction centers and light-harvesting antennae under low light conditions to maximize photon absorption and sustain optimal cell growth [34,43]. Furthermore, our correlation analysis demonstrated a strong positive correlation (r = 0.96–0.99, p < 0.05) (Supplementary Table S2) between RIChl and RIPE across light intensities ranging from 100 to 335 μmol photons m−2 s−1. This suggests that within the light saturation region, R. marinus actively regulates the content of both chlorophyll and phycoerythrin to maintain the efficiency of its reaction center. These findings are in agreement with previous studies [40,44].
In summary, the RIPE of R. marinus exhibited a gradual decrease with increasing light intensity. We hypothesize that at a high light intensity of 335 μmol photons m−2 s−1, R. marinus may downregulate phycoerythrin synthesis as a protective mechanism to prevent the generation of excessive energy, thereby mitigating photooxidative damage. These observations are consistent with findings from previous investigations [40,45]. Phycoerythrin, a prominent phycobiliprotein (PBP), is recognized for its diverse biological properties, including antioxidant, antitumor, and photosensitive activities, and its utility as a fluorescent marker in pharmacological applications [46]. Leveraging these intrinsic characteristics, the present study aimed to elucidate the optimal culture conditions for enhancing the RIPE of R. marinus, consequently augmenting its application value and future biotechnological potential.

3.3. Effects of Temperature and Light Intensity on Photosynthetic Efficiency

Fv/Fm has been widely recognized as a robust indicator of photosynthetic organism fitness, directly reflecting the photosynthetic efficiency of Photosystem II (PSII). Under optimal conditions, Fv/Fm typically remains stable; however, it decreases significantly under various environmental stresses [47]. When microalgae are exposed to saturating light intensities, further increases in irradiance do not proportionally enhance photosynthetic rates. Instead, excessive light can induce photooxidative damage, leading to a decline in photosynthetic efficiency, a phenomenon termed photoinhibition [48]. In the present study, a higher Fv/Fm was observed in R. marinus cultured at 18 °C, indicative of high PSII efficiency and a robust capacity for converting light energy into chemical energy. Conversely, lower Fv/Fm values were recorded at 22 °C and 24 °C, suggesting a reduction in photosynthetic efficiency. Correlation analysis revealed a strong negative correlation between cell numbers and Fv/Fm (r = −0.92 to −0.98, p < 0.05) (Supplementary Table S1), as well as between cell numbers and ETRmax (r = −0.86 to −0.90, p < 0.05) (Supplementary Table S1) at 20 °C, 22 °C, and 24 °C. This suggests that while elevated temperatures promoted algal cell growth, the photosynthetic parameters Fv/Fm and ETRmax did not increase synchronously. Based on these photosynthetic responses, we infer that temperatures between 20 °C and 24 °C constituted a stress environment for R. marinus, leading to a diminished photosynthetic capacity. The significant decline in Fv/Fm when algae were exposed to temperatures exceeding their tolerance threshold is consistent with previous observations [49].
The maximum electron transport rate (ETRmax), determined through light curves, serves as a critical indicator of algal photosynthetic efficiency [50]. Our temperature experiments demonstrated that photosynthetic capacity was better maintained at 18 °C compared to 20 °C, 22 °C, and 24 °C, although this did not directly correlate with the observed cell growth rate. It has been proposed that during photosynthesis, light energy is converted into ATP and NADPH by the PSII reaction center. When algae experience stress due to temperature and light intensity, leading to an energy imbalance, they regulate the redox state via the PSII electron transport chain to adapt to environmental challenges [34]. For instance, Chlamydomonas reinhardtii possesses a regulatory mechanism for light exposure: under excessive light, algal cells dissipate excess light energy as heat to reduce photosynthetic efficiency and prevent photodamage [31,51]. Similarly, Rhodomonas salina achieved its highest ETRmax at a low light intensity of 75 µmol photons m−2 s−1 [52], which aligns with the optimal ETRmax response observed in our experiment at a low light intensity of 100 µmol photons m−2 s−1. This collectively indicates that microalgae employ sophisticated regulatory mechanisms for light absorption to sustain photosynthetic efficiency. The present study thus demonstrates that the PSII of R. marinus exhibits superior photosynthetic performance under culture conditions of 18 °C and light intensities ranging from 100 to 185 µmol photons m−2 s−1.
In conclusion, algal photosynthesis is intricately linked to optimal growth temperatures. Optimal temperatures facilitate efficient light absorption and electron transfer processes during photosynthesis, thereby enhancing overall photosynthetic efficiency. Conversely, excessively high temperatures can induce oxidative stress and photoinhibition during photosynthesis, resulting in a reduction in Fv/Fm. In the current investigation, a similar phenomenon was observed under consistent light exposure durations: higher light intensities led to lower Fv/Fm values, signifying a physiological response by the algae to protect their cellular machinery from damage.

3.4. Effects of Temperature and Light Intensity on Fatty Acid and Selective Enrichment of EPA and PUFA Composition

Triacylglycerol (TAG) biosynthesis is generally synthesized via two primary pathways: (1) de novo fatty acid synthesis, which involves the CDP–choline pathway where fatty acids are directly incorporated into TAG [53]; and (2) the conversion of pre-existing polar glycerolipids [54,55]. TAG synthesized through the de novo pathway is typically characterized by a high content of 16:0 and 18:1 fatty acids. In contrast, TAG derived from cell membranes exhibits a higher proportion of long-chain polyunsaturated fatty acids (LC-PUFA), such as C20:5 (EPA) [56,57].
Chloroplasts, mitochondria, and the endoplasmic reticulum serve as the primary sites for fatty acid and lipid synthesis in plants [58,59]. This intricate process necessitates two key enzyme systems: acetyl-CoA carboxylase and fatty acid synthase [60]. In plants, acetyl-CoA carboxylase exists in two molecular forms: a multi-protein complex and a multifunctional protein. Fatty acid synthase, requiring a multi-protein complex, facilitates the covalent attachment and extension of the growing fatty acyl chain to an acyl carrier protein (ACP), ultimately yielding saturated fatty acids such as C16 palmitic acid and C18 stearic acid [60]. Subsequent to the formation of LC-PUFA, cells undergo elongation, desaturation, and further modifications. For instance, stearic acid, in conjunction with stearoyl-ACP, Δ9 desaturase, and Δ12 desaturase, is converted into linoleic acid, which is then further transformed into α-linolenic acid by Δ15 desaturase. Additionally, some algae and bacteria produce EPA and DHA via the polyketide synthase (PKS) pathway [61].
When microalgal growth is inhibited and photosynthetic capacity is reduced, microalgae tend to store carbon in the form of lipids or carbohydrates [62]. Temperature variations can significantly influence the degree of unsaturation of fatty acids [60]. This study demonstrated that R. marinus primarily accumulated C16:0, C18:1, C18:2, C20:4, and C20:5 across different temperatures, with C16:0 and C20:5 being the most abundant. A notable observation was the decrease in the proportion of C20:5 with increasing temperature, while the proportion of C16:0 exhibited an inverse trend. Elevated temperatures may inhibit the fatty acid synthesis pathway for fatty acids shorter than 20 carbons, but not those longer than 20 carbons, potentially due to reduced activity of Δ17 desaturase [45].
Polyunsaturated fatty acids (PUFAs) are crucial for maintaining cell membrane structure and stability. At lower temperatures, high levels of PUFAs contribute to preserving membrane fluidity [63]. Different species exhibit varying capacities to accumulate EPA under cold conditions. For example, the cold-water species Chaetoceros brevis efficiently accumulates EPA at 7 °C. Consequently, PUFAs are considered essential bioactive substances for microalgal survival in cold environments. Among PUFAs, EPA and other LC-PUFAs show diverse accumulation capacities across species. Temperate species such as Thalassiosira weissflogii and Fibrocapsa japonica efficiently accumulate EPA at 16 °C [64]. In Porphyridium cruentum, EPA content decreased from 13.5 mg g−1 to 3.1 mg g−1 when the temperature increased from 20 °C to 30 °C [5], and the proportion of EPA to total fatty acids declined from 6.75 ± 0.81% to 6.36 ± 1.94% when temperature increased from 0 °C to 25 °C [65]. Similarly, in Nannochloropsis salina, the proportion of EPA decreased with increasing temperature from 13 °C to 33 °C, while the proportions of C16:0 and C16:1 increased. This inverse relationship between unsaturated fatty acid content and temperature suggests that R. marinus enhances EPA content at 18 °C to both increase membrane fluidity and facilitate essential processes such as photosynthesis. Algae preferentially accumulate unsaturated fatty acids at low temperatures to maintain cellular function and membrane fluidity, which is considered a key adaptive mechanism to cold temperatures [61].
Regarding the impact of light intensity on PUFAs, studies have indicated that in Chlorella vulgaris cultured under a 12L:12D cycle, the proportion of PUFAs to total fatty acids decreased from 26.63% to 20.99% as light intensity increased from 37.5 to 100 μmol photons m−2 s−1 [66]. Analogously, in Rhodomonas salina, PUFA content was higher at 20 μmol photons m−2 s−1 compared to 250 μmol photons m−2 s−1, suggesting that elevated light intensity led to a decrease in PUFAs and an increase in saturated fatty acids, potentially due to photooxidation [35]. Interestingly, our results contradict these findings, as the highest light intensity group (335 μmol photons m−2 s−1) exhibited a nearly 50% proportion of PUFAs to total fatty acids. We hypothesize that R. marinus may have increased PUFA production to counteract the environmental stress induced by high light intensity. Despite the higher PUFA content in the highest light intensity group, the overall lipid content was significantly lower compared to the lowest light intensity group (100 μmol photons m−2 s−1). PUFAs contribute to maintaining cell membrane adaptability to changes in light conditions. Our observations of both increases and decreases in PUFAs under high light conditions in different algal strains suggest that various algal species possess diverse capacities to adapt to light. This aligns with previous research demonstrating that different algal species exhibit varied responses to light stress [67].
Porphyridium cruentum exhibited a maximum EPA content of 7.9 mg g−1 at 30 °C and 187 μmol photons m−2 s−1 [5]. Porphyridium purpureum reached a maximum EPA content of 7.00 mg g−1 at 25 °C and 280 μmol photons m−2 s−1 [68]. Notably, our results showed a similar maximum EPA content of 7.45 mg g−1 at 18 °C and 185 μmol photons m−2 s−1. Significantly, none of Porphyridium cruentum, Porphyridium purpureum, and R. marinus produce DHA, suggesting that the pathway for the production, elongation, desaturation, and further modification of LC-PUFA is conserved among these unicellular red algae.
Red algae typically contain elevated levels of C20 PUFAs, with EPA being particularly abundant, and some red algae also contain arachidonic acid (AA). Both of these are recognized as essential fatty acids for animal growth [69]. In the present study, the highest EPA content was 7.45 ± 0.28 mg g−1, constituting 33.30 ± 0.63% of total fatty acids. This can be favorably compared to other microalgae and macroalgae such as Chaetoceros sp. (2.84 mg g−1 EPA), Amphora coffeaformis (2.74 mg g−1), Cryptomonas sp. (2.64 mg g−1), and Rhodomonas sp. (0.38–1.63 mg g−1) [70,71]. The proportion of EPA to total fatty acids was also observed in other algae, such as Porphyra sp. from Korea and Japan (20.9 ± 26.54%), Porphyra sp. from China (10.4 ± 7.46%), Undaria pinnatifia (13.2 ± 0.66%), and Laminaria sp. (16.2 ± 8.90%). Collectively, these results underscore that the high EPA content in R. marinus distinguishes it from many other algal species in terms of fatty acid composition [72]. Furthermore, as a marine microalga, the physiological responses of R. marinus are likely sensitive to variations in salinity. Salinity stress has been shown to influence membrane fluidity, osmotic balance, and the subsequent synthesis of compatible solutes and fatty acids in other rhodophytes [73,74]. Although this study maintained a constant salinity of 33‰, future work should explore how different salinity levels interact with temperature and light stress to modulate the production of PUFAs and phycoerythrin, which could reveal new strategies for optimizing yields in aquaculture settings.
A limitation of this study is that nutrient concentrations (e.g., nitrate and phosphate) in the culture medium were not monitored during the 14-day cultivation period. Nutrient depletion, particularly in the later stages of cultivation, could act as an additional stressor, potentially affecting both cell growth and the biosynthesis of fatty acids and pigments. The interplay between physical stressors (temperature and light) and nutrient availability represents a critical aspect that warrants further investigation. Notably, nitrogen limitation is a well-documented trigger for lipid accumulation in microalgae, as cells redirect carbon flux from protein synthesis towards storage lipid synthesis. However, this often occurs at the expense of growth and the production of nitrogen-containing compounds such as phycoerythrin. Therefore, a comprehensive understanding of how nutrient dynamics correlate with growth phases and metabolite partitioning is essential for developing an optimized cultivation strategy.

4. Materials and Methods

4.1. Microalgal Strain Preservation and Cultivation

The algal specimens used in this experiment were isolated, purified, and cultured by the algae laboratory at National Taiwan Ocean University. The algae were then identified as R. marinus using optical microscopy, transmission electron microscopy, and by comparison the sequences of the rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) gene and 16S ribosomal DNA. R. marinus was cultivated at 22 ± 1 °C and 33‰ salinity with a PG medium [63,64]. The initial pH of the medium was adjusted to 8.0 ± 0.2. Light was provided at 100 μmol photons m−2 s−1 using LED T8 (Everlight, Taipei, Taiwan) with a 12L:12D photoperiod. Filtered air was bubbled into the culture at a rate of 5 L/min.

4.2. Effects of Temperature on Growth, Physiology, and Fatty Acid Changes

Experiments were carried out using 1 L Erlenmeyer flasks containing 1000 mL of PG medium and an initial cell density of 5 × 105 cells mL−1. The cultures were aerated with a continuous air supply at 5 L/min and maintained on a 12L:12D photoperiod. All sampling was conducted at the same time each day to ensure consistency. The cultures were incubated at 18 °C, 20 °C, 22 °C, and 24 °C for 14 days in triplicate. The light source was LED T8 white light (100 μmol photons m−2 s−1, Everlight, Taiwan). The required light intensities were measured using a spectrometer (MK350N Premium, UPRtek, Miaoli, Taiwan). Cell numbers, relative index of chlorophyll (RIChl), and relative index of phycoerythrin (RIPE) were assessed using Attune™ NxT flow cytometry (Thermo Fisher Scientific, Taipei, Taiwan) every two days. Fv/Fm and maximum electron transport rate (ETRmax) were assessed using a phytoplankton analyzer (PHYTO-PAM, Walz, Effeltrich, Germany) every two days. On day 14 of the experiment, algal cells were collected, centrifuged at 11,000 rpm, freeze-dried, and stored for fatty acid analysis.

4.3. Effects of Light Intensities on Growth, Physiology, and Fatty Acid Changes

Based on the results of the temperature experiment, 18 °C was selected for the subsequent light intensity experiment. Experiments were carried out using 1 L Erlenmeyer flasks containing 1000 mL of PG medium and an initial cell density of 5 × 105 cells mL−1. The cultures were incubated in triplicate at light intensities of 100, 185, 245, and 335 µmol photons m−2 s−1 for 14 days. Light source was LED white strip lights (ALOHA, Taipei, Taiwan), and adjusted to the required light intensities under the instruction of a spectrometer. Cell numbers, RIChl, RIPE, Fv/Fm, and ETRmax were assessed every two days. Algal cells were collected, centrifuged, and freeze-dried at the 14th days for further analysis.

4.4. Determination of Algae Growth

1 mL of the R. marinus culture was collected from each Erlenmeyer flask. A 1 mL syringe with 21G needle was used to aspirate the culture multiple times to separate aggregated algal cells. The sample was diluted 5-fold and analyzed using Attune™ NxT acoustic focusing flow cytometer at a flow rate of 100 μL min−1 to assess changes in cell numbers, RIChl, and RIPE under various growth conditions. Data for each parameter were obtained during a single analysis. The X-axis represents forward scatter (FSC), indicating cell size, while the Y-axis represents side scatter (SSC), indicating cell complexity. R. marinus cells were gated in the R1 region based on FSC and SSC to reflect relative cell count. Simultaneously, cells emitting high-intensity fluorescence signals in the channels with 695/40 nm and 574/26 nm filters were gated in the R2 and R3 regions, respectively, under excitation from a blue laser (488 nm, 50 mW) [16] (Supplementary Figure S1).
The numbers in the gated regions R1, R2, and R3 were calculated as follows:
Cell numbers (cells mL−1) = R1
Relative index of chlorophyll, RIChl (%) = R2/R1 × 100%
Relative index of phycoerythrin, RIPE (%) = R3/R1 × 100%

4.5. Determination of Chlorophyll Fluorescence

The photosynthetic efficiency of algal cells was determined by chlorophyll fluorescence measurements using a PHYTO-PAM phytoplankton analyzer to measure the parameters photosynthetic efficiency (Fv/Fm), relative electron transport rate (rETR), and maximum electron transport rate (ETRmax). A 1 mL aliquot of algal cell suspension was incubated in the dark for 20 min before measurement. The maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm) was calculated using the following equation [75]:
Fv/Fm = (Fm − F0)/Fm
where F0 represents the minimum fluorescence yield after dark adaptation, and Fm denotes the maximum fluorescence yield measured with a 0.2 s saturation pulse at an intensity of 3500 μmol photons m−2 s−1 using red light at a wavelength of 655 nm.
rETR was calculated as follows:
rETR = Yield × PAR × 0.5 × 0.84 (µmol electrons m−2 s−1)
In the light response curve, the maximum rETR is known as ETRmax. Yield denotes the effective quantum yield of PSII; PAR stands for photosynthetically active radiation; 0.5 indicates that half of the quanta of the incident PAR are distributed to PS II; and the factor 0.84 is the ETR factor [76,77,78].

4.6. Determination of Fatty Acid Compositions

The fatty acid composition was determined according to the method described by Lu et al. [79], with minor modifications. Screened algae powder (50 mg) was placed in a 10 mL brown vial with Teflon-lined lid. Hexane 1 mL and 0.5 mL of the internal standard (methyl heptadecanoate, 500 ppm in hexane) were added. The vial was sonicated at 80 °C for 20 min, then the solvent was removed by a gentle nitrogen purge. The residue was saponified by 1 mL of NaOH-MeOH (1 M) and heated at 80 °C for 15 min (vortex for 1 min every 5 min). Then, 1 mL of 30% boron trifluoride was added and heated at 110 °C for 15 min. After cooling, 6 mL of saturated sodium chloride solution was added and vortexed for 1 min. The fatty acid methyl ester was extracted by 1 mL of hexane and vortexed for 1 min. The vial was centrifuged at 490× g for 3 min, and then the supernatant 0.5 mL was transferred into a 1.5 mL vial with a small amount of anhydrous sodium sulfate.
An Agilent gas chromatograph with a flame ionization detector (GC/FID) Agilent 8860 GC system (Santa Clara, CA, USA) equipped with an Agilent 7650A autosampler was used for fatty acid analysis. Separation was performed on an Rt-2560 GC capillary column (100 m × 0.25 mm × 0.20 μm; RESTEK, Bellefonte, PA, USA) using nitrogen as the carrier gas at a flow rate of 1.0 mL/min. The temperature program for the chromatography column was as follows: the initial temperature was 150 °C, which was held for 2 min; it was then heated at 10 °C/min to 190 °C and held for 0 min; Then heated at 3 °C/min to 220 °C, held for 5 min; and finally heated at 10 °C/min to a final temperature of 245 °C, held for 15 min.
The content of each fatty acid in the sample, Wx (mg), was determined using the following equation:
(Ax × Rx × Wis × FFAx)/(Ais × Ws) = Wx (mg/mg)
Ax: Peak area of each fatty acid methyl ester;
Ais: Peak area of the internal standard methyl ester;
FFax: Conversion factor of each fatty acid methyl ester to its corresponding fatty acid;
Rx: Relative response factor of each fatty acid methyl ester to the internal standard methyl ester in the FID (referred to AOCS Ce 1h-05);
Wis: Amount of internal standard added;
Ws: Sample weight;
Wx: Content of each fatty acid in the sample.

4.7. Statistical Analysis

All data were processed using SPSS Statistics 22 (IBM, New York, NY, USA) for one-way ANOVA and Tukey’s post hoc test to assess intergroup differences. All experimental results were expressed as mean ± standard deviation (n = 3). A p-value < 0.05 was considered statistically significant. Pearson’s correlation analysis was used to investigate the associations among all variables. Significance testing was used to determine the statistical significance of these relationships.

5. Conclusions

This study successfully demonstrates that the optimal physiological performance and accumulation of bioactive substances (including Fv/Fm, ETRmax, Relative Index of Chlorophyll, Relative Index of Phycoerythrin, polyunsaturated fatty acids, and lipids) in R. marinus occurred at 18 °C and a light intensity of 100 µmol photons m−2 s−1. Notably, high levels of EPA were accumulated at 18 °C and light intensities of 100 to 185 µmol photons m−2 s−1, indicating that R. marinus demonstrated photoacclimation up to a certain light saturation point. However, further research is needed to investigate the potential photoinhibition effects associated with higher light intensities. To build upon these findings, future research should investigate the effects of nutrient stress and varying salinity concentrations on the concurrent production of PE and PUFAs in R. marinus. Crucially, such studies should include the simultaneous monitoring of key nutrient dynamics to establish a clearer correlation between nutrient consumption, growth phases, and the synthesis of high-value metabolites. Additionally, the identified cultivation conditions should be validated through larger-scale experiments, and the potential production of other bioactive compounds, including carbohydrates, proteins, carotenoids, and phytohormones, should be evaluated to maximize the value of R. marinus biomass for nutraceutical and pharmaceutical applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/md24020078/s1, Figure S1: Flow cytometry of R. marinus; Figure S2: The effects of temperature (18, 20, 22 and 24 °C) on (A) contents and (B) compositions of fatty acids of R. marinus over 14 days cultivation. Flow cytometry of R. marinus; Figure S3: The effects of light intensity (100, 185, 245, and 335 µmol photons m−2 s−1) on (A) contents and (B) compositions of fatty acids of R. marinus over 14 days cultivation; Table S1: The correlation coefficients for cell numbers, RIChl, RIPE, Fv/Fm and ETRmax of R. marinus over a 14 days cultivation; Table S2: The correlation coefficients for cell numbers, RIChl, RIPE, Fv/Fm and ETRmax of R. marinus over a 14 days cultivation. It includes one file containing the following: statistical analyses, flow cytometry results, and the distribution of various fatty acids under different culture conditions.

Author Contributions

W.-P.C.: Investigation, Formal analysis, Resources, Writing—Original Draft; H.-Y.Y.: Conceptualization, Methodology, Writing—Original Draft; Y.-L.C.: Conceptualization, Writing—Editing; Y.-J.C.: Conceptualization; F.-T.A.S.: Methodology; C.-C.H.: Methodology; F.-H.N.: Methodology; M.-C.F.: Methodology; M.-C.L.: Visualization, Writing—Review and Editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Taiwan’s National Science and Technology Council (NSTC-113-2321-B-019-001) and a National Taiwan Ocean University internal research subsidy.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

Fat-Tin Agassi Sze is employed by Algaetech Key Laboratory, Algaetech (Hong Kong) Co., Limited, and the other authors declare that there are no potential conflicts of interest. Algaetech Key Laboratory, Algaetech (Hong Kong) Co., Limited had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
PUFAsPolyunsaturated fatty acids
LC-PUFALong-chain polyunsaturated fatty acid
EPAEicosapentaenoic acid
DHADocosahexaenoic acid
ARAArachidonic acid
FAsFatty acids
TGTriglycerides
PBPsPhycobili proteins
PEPhycoerythrin
B-PEB-phycoerythrin
R-PER-phycoerythrin
C-PEC-phycoerythrin
ACPAcyl carrier protein
ETRmaxElectron transport rate maximum
RIChlChlorophyll relative index
RIPEPhycoerythrin relative index

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Marinedrugs 24 00078 g001
Figure 1. The effects of temperature (18 °C, 20 °C, 22 °C, and 24 °C) on (A) cell numbers, (B) the chlorophyll relative index (RIChl), and (C) the phycoerythrin relative index (RIPE) of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Figure 1. The effects of temperature (18 °C, 20 °C, 22 °C, and 24 °C) on (A) cell numbers, (B) the chlorophyll relative index (RIChl), and (C) the phycoerythrin relative index (RIPE) of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Marinedrugs 24 00078 g001
Marinedrugs 24 00078 g002
Figure 2. The effects of temperature (18 °C, 20 °C, 22 °C, and 24 °C) on (A) Fv/Fm and (B) ETRmax of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Figure 2. The effects of temperature (18 °C, 20 °C, 22 °C, and 24 °C) on (A) Fv/Fm and (B) ETRmax of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Marinedrugs 24 00078 g002
Marinedrugs 24 00078 g003
Figure 3. The effects of light intensity (100, 185, 245, and 335 µmol photons m−2 s−1) on (A) cell numbers, (B) the chlorophyll relative index (RIChl), and (C) the phycoerythrin relative index (RIPE) of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Figure 3. The effects of light intensity (100, 185, 245, and 335 µmol photons m−2 s−1) on (A) cell numbers, (B) the chlorophyll relative index (RIChl), and (C) the phycoerythrin relative index (RIPE) of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Marinedrugs 24 00078 g003
Marinedrugs 24 00078 g004
Figure 4. The effects of light intensity (100, 185, 245 and 335 µmol photons m−2 s−1) on (A) Fv/Fm and (B) ETRmax of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Figure 4. The effects of light intensity (100, 185, 245 and 335 µmol photons m−2 s−1) on (A) Fv/Fm and (B) ETRmax of R. marinus over 14 days of cultivation. Data are presented as the mean ± SD. Significant differences (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3) are indicated by different letters.
Marinedrugs 24 00078 g004
Table 1. The effects of temperature (18 °C, 20 °C, 22 °C, and 24 °C) on the contents of fatty acids of R. marinus over 14 days of cultivation.
Table 1. The effects of temperature (18 °C, 20 °C, 22 °C, and 24 °C) on the contents of fatty acids of R. marinus over 14 days of cultivation.
Fatty Acid
(mg g−1) DCW		18 °C20 °C22 °C24 °C
C20:5 (EPA)5.82 (5.0%) a5.04 (7.3%) a5.09 (9.6%) a3.80 (8.4%) b
C16:01.68 (152.4%) a5.38 (11.5%) a6.58 (23.2%) a6.38 (13.6%) a
C18:13.65 (1.4%) a3.33 (7.2%) a3.92 (12.0%) a3.86 (9.6%) a
C18:21.59 (1.3%) a1.76 (7.9%) a2.79 (35.5%) a2.32 (9.9%) a
Total FAs18.78 (0.2%) a18.97 (0.5%) a23.10 (1.2%) a19.13 (0.5%) a
Total PUFAs13.53 (2.7%) a12.63 (8.3%) a14.53 (11.9%) a11.74 (10.5%) a
FAs: fatty acids; PUFAs: polyunsaturated fatty acids; DCW: dry cell weight; RSD%: relative standard deviation percentage. Data are presented as mean (RSD%) with statistical significance indicated by different letters (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3).
Table 2. The effects of light intensity (100, 185, 245, and 335 µmol photons m−2 s−1) on the contents of fatty acids of R. marinus over 14 days of cultivation.
Table 2. The effects of light intensity (100, 185, 245, and 335 µmol photons m−2 s−1) on the contents of fatty acids of R. marinus over 14 days of cultivation.
Fatty Acid
(mg g−1) DCW		μmol Photons m−2 s−1
100185245335
C16:07.68 (8.3%) a5.64 (5.5%) b5.90 (2.7%) ab5.31 (3.4%) b
C18:15.14 (3.1%) a3.94 (3.8%) b4.69 (1.1%) ab4.68 (3.2%) ab
C20:5 (EPA)6.91 (1.0%) ab7.45 (3.8%) a6.23 (1.3%) c6.39 (4.1%) bc
Total FAs28.16 (0.2%) a22.38 (0.3%) b23.14 (0.1%) b22.17 (0.1%) b
Total PUFAs19.67 (3.2%) a16.20 (5.6%) b16.73 (1.7%) b16.61 (2.4%) b
FAs: fatty acids; PUFAs: polyunsaturated fatty acids; DCW: dry cell weight; RSD%: relative standard deviation percentage. Data are presented as mean (RSD%) with statistical significance indicated by different letters (p < 0.05, one-way ANOVA and Tukey’s post hoc test, n = 3).
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Cheng, W.-P.; Yeh, H.-Y.; Chen, Y.-L.; Chen, Y.-J.; Sze, F.-T.A.; Huang, C.-C.; Nan, F.-H.; Fang, M.-C.; Lee, M.-C. Enhanced Production of Bioactive Polyunsaturated Fatty Acids and Pigments in Rhodosorus marinus: Optimization of Thermal and Photic Stress Conditions. Mar. Drugs 2026, 24, 78. https://doi.org/10.3390/md24020078

AMA Style

Cheng W-P, Yeh H-Y, Chen Y-L, Chen Y-J, Sze F-TA, Huang C-C, Nan F-H, Fang M-C, Lee M-C. Enhanced Production of Bioactive Polyunsaturated Fatty Acids and Pigments in Rhodosorus marinus: Optimization of Thermal and Photic Stress Conditions. Marine Drugs. 2026; 24(2):78. https://doi.org/10.3390/md24020078

Chicago/Turabian Style

Cheng, Wen-Ping, Han-Yang Yeh, Yen-Ling Chen, Yi-Jung Chen, Fat-Tin Agassi Sze, Chi-Cheng Huang, Fan-Hua Nan, Ming-Chih Fang, and Meng-Chou Lee. 2026. "Enhanced Production of Bioactive Polyunsaturated Fatty Acids and Pigments in Rhodosorus marinus: Optimization of Thermal and Photic Stress Conditions" Marine Drugs 24, no. 2: 78. https://doi.org/10.3390/md24020078

APA Style

Cheng, W.-P., Yeh, H.-Y., Chen, Y.-L., Chen, Y.-J., Sze, F.-T. A., Huang, C.-C., Nan, F.-H., Fang, M.-C., & Lee, M.-C. (2026). Enhanced Production of Bioactive Polyunsaturated Fatty Acids and Pigments in Rhodosorus marinus: Optimization of Thermal and Photic Stress Conditions. Marine Drugs, 24(2), 78. https://doi.org/10.3390/md24020078

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