Improved production of lutein and β -carotene by thermal and light intensity upshifts in the marine microalga Tetraselmis sp. CTP4

The industrial microalga Tetraselmis sp. CTP4 is a promising candidate for aquaculture feed, novel food, cos- meceutical and nutraceutical due to its balanced biochemical pro ﬁ le. To further upgrade its biomass value, carotenogenesis was investigated by testing four environmental factors, namely temperature, light intensity, salinity and nutrient availability over di ﬀ erent growth stages. The most important factor for carotenoid induction in this species is a su ﬃ cient supply of nitrates leading to an exponential growth of the cells. Furthermore, high temperatures of over 30°C compared to lower temperatures (10 and 20°C) induced the accumulation of carotenoids in this species. Remarkably, the two di ﬀ erent branches of carotenoid synthesis were regulated depending on di ﬀ erent light intensities. Contents of β -carotene were 3-fold higher under low light intensities (33 μ molm − 2 s − 1 ) while lutein contents increased 1.5-fold under higher light intensities (170 and 280 μ molm − 2 s − 1 ). Nevertheless, highest contents of carotenoids (8.48 ± 0.47mgg − 1 DW) were found upon a thermal upshift from 20°C to 35°C after only two days at a light intensity of 170 μ molm − 2 s − 1 . Under these conditions, high contents of both lutein and β -carotene were reached accounting for 3.17 ± 0.18 and 3.21 ± 0.18mgg − 1 DW, respectively. This study indicates that Tetraselmis sp. CTP4 could be a sustainable source of lutein and β -carotene at locations where a robust, euryhaline, thermotolerant microalgal strain is required.


Introduction
In recent years, consumer interest in healthy food and natural products has been growing due to a rising awareness that a healthy nutrition can increase life expectancy. Evidence has been put forward that ingredients used as nutraceuticals and cosmeceuticals may confer additional health and medical benefits such as a decreased risk for chronic disease or cancer [1]. In turn, this has led to research efforts for the development of food supplements and cosmetics from natural sources. Microalgae are promising biological resources that meet these market needs due to their well-balanced biochemical profile as well as antioxidant, anticancer and anti-inflammatory activities [2]. Furthermore, microalgae can be cultivated in bioreactors or ponds placed on non-arable land, for example, deserts or shorelines, thus not competing with plant production. Moreover, they also display higher productivities than land crops [3].
One important group of bioactive compounds produced by microalgae are carotenoids. These molecules play essential roles as accessory light harvesting pigments and photoprotective agents, being also structural components of the light harvesting complexes of microalgae [4]. Their typical polyene structure is responsible for their lipophilic character, and the absence or presence of oxygenated groups defines two different carotenoid sub-classes, namely carotenes (e.g., αand β-carotene) and xanthophylls (e.g., lutein and violaxanthin), respectively. Furthermore, because of the presence of conjugated double bonds in their chemical structure, carotenoids not only have different colors, ranging from yellow to red, but also display antioxidant activities [5]. Carotenoids can scavenge reactive oxygen species (ROS), a well-known group of chemicals that can cause oxidative stress [6]. Because of their diverse roles, and aside from the genetics of a particular strain, carotenogenesis in microalgae is highly influenced by the environmental conditions to which the microalgal cells are exposed. Thus, culture conditions such as light intensity, nutrient availability, temperature and salinity may influence carotenoid contents. In the case of the chlorophytes Dunaliella salina and Haematococcus pluvialis, carotenogenesis is induced by high light intensity and/or nutrient depletion [7]. Under these conditions, the overproduction of these lipophilic carotenoids depends on triacylglycerol synthesis, which form lipid droplets and serve as a sink for carotenoid deposition [8]. However, other microalgae do not accumulate carotenoids in lipid droplets, but in the thylakoid membranes as carotenogenesis usually depends on growth-promoting conditions rather than on abiotic stress. Microalgae belonging to the genus Tetraselmis are known for their great variety in carotenoids such as β-carotene, lutein and violaxanthin [9]. Even though Tetraselmis biomass is usually used in aquaculture feeds, recently the European Union approved the human consumption of biomass from a microalga of this genus as "novel food" [10]. Moreover, the inclusion of microalgal products in the human diet has been suggested, not only because of their comparable levels of carotenoids with those of vegetables, but also due to their antioxidant and cell-repairing activities against human lung cancer [11,12].
Recently, a robust marine microalga Tetraselmis sp. CTP4 was isolated displaying high growth rates and the ability to outcompete contaminants [13]. Remarkably, this microalga can withstand temperatures ranging from 5 to 40°C, being able to grow in wastewater as well as seawater with salinities of up to 75‰ (data not shown). All these properties allowed for the successful scale-up to industrial photobioreactors of this robust microalgal strain (Pereira et al., 2018). In addition, because this microalga becomes predominantly unflagellated at later growth stages, a low-cost harvesting step in a settling tank driven by natural sedimentation was also implemented [14]. Furthermore, the wet biomass of this microalga showed significant amounts of extractable carotenoids when using acetone and glass-bead milling (L. Schüler, L. Barreira, J. Varela, unpublished results).
In this study, carotenoid profiles of Tetraselmis sp. CTP4 were investigated under four abiotic growth factors, namely light intensity, temperature, salinity, nitrate repletion or depletion during different growth stages. Even though few studies have focused on the induction of carotenoids by abiotic stress factors in microalgae belonging to this genus, this is the first thorough report dissecting the interaction of these growth conditions [15][16][17][18][19]. The aim of this study is to provide knowledge of how carotenoid biosynthesis is regulated in Tetraselmis microalgae and where carotenoids accumulate in this chlorophyte. Furthermore, a cultivation strategy is suggested to obtain carotenoidrich biomass, which could be applicable not only in aquaculture but also as novel food or cosmeceutical.

Organism, standard growth conditions and harvesting
The microalga Tetraselmis sp. CTP4 was isolated from Ria Formosa in Portugal as described in Pereira et al. (2016). Standard growth conditions were established in previous reports [13,20]. Briefly, initial microalgal cultures were grown in 5-L reactors (Ø = 12 cm) using sterilized Atlantic seawater from the shoreline of Faro, Portugal  harvested by centrifugation (5000 g, 5 min); pellets and supernatants were kept frozen at −20°C until further analysis.

2.2.
Culture conditions for abiotic stress on carotenoid induction 2.2.1. Effect of temperature and salinity Cultures were inoculated in 5-L reactors (Ø = 13 cm) containing different salinities (5, 20 and 35‰), which were achieved by diluting the seawater with distilled water. After supplementation with MAM, cultures were grown at 5, 20 or 30°C with a PFD of 100 μmol m −2 s −1 until early stationary phase.

Effect of light intensity and nitrate availability
This experiment was performed in 100-mL glass photobioreactors (Ø = 3 cm), filled up with culture to a volume of 80 mL. All cultures were inoculated with a biomass concentration of 0.3 g L −1 and grown in seawater with a salinity of 35‰ supplemented with MAM without nitrate. A solution of NaNO 3 was added to half of the cultures to achieve a final concentration of 97 mg N- whereas the other half were incubated without additional nitrate (N-, nitrogen depleted). During this growth period, three different PFDs were applied: 33, 170 and 280 μmol m −2 s −1 . Cultures were incubated at 30°C and samples were taken after 2, 5 and 10 days.

Effect of short-term induction at high temperature
This experiment was performed under the same conditions as the previous experiment with the following modifications. All cultures were inoculated with a biomass concentration of 0.6 g L −1 and were incubated at 35°C using two different PFDs: 33 and 170 μmol m −2 s −1 . Samples were taken after 2 and 5 days.

Microscopy
For microscopy, samples were taken from cultures grown at high Influence of temperature and salinity on carotenoid content of Tetraselmis sp. CTP4. Cultures were inoculated in 5-L reactors containing different salinities of 5, 20 and 35‰ and grown at 5, 20 and 30°C with a light intensity of 100 μmol m −2 s −1 until stationary phase (n = 3, average ± SD). Carotenoid extracts were analyzed by HPLC and neoxanthin (A), violaxanthin (B), lutein (C) and β-carotene (D) were quantified. Different letters over the bars indicate significant differences (p < 0.05) using two-way ANOVA with post hoc Tukey HSD test. temperature (35°C) under nitrogen repletion or nitrogen depletions during a period of 5 days. Images were acquired with a Zeiss AXIOM-AGES Z2 microscope, equipped with a coollSNApHQ2 camera and AxioVision software version 4.8 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany), using a 100× magnification. For fluorescence imaging, a Zeiss 38 HE filter set (ex. 470/40 nm, em. 525/50 nm) was used (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and the transmitted light images were acquired using differential interference contrast (DIC). Images were treated using Image J software (Research Service Branch, NIH, Bethesda, MD).

Determination of growth by optical density and dry weight
To monitor cell growth and biomass concentration, the optical density was measured in 96-well plates at 750 nm (OD 750 ) and the dry weight was determined as described previously [13,20]. Briefly, dry weight (DW) was determined by filtering a 2-ml sample of each culture through a pre-washed and pre-weighed glass microfiber filter (0.47 μm), washed with 20 mL of ammonium formate (31.5 g L −1 ) and dried at 60°C until constant weight. A significant correlation (r ≥ 0.96, p < 0.01) between OD 750 and DW was found using biomass from different growth conditions (n = 55). For the calculation of the DW used in carotenoid extraction, the following Eq. (1) was used: (1)

Determination of nitrate concentration
Nitrate concentration was determined by the UV spectrophotometric method as described in [20].

Carotenoid extraction and quantification
Carotenoids were extracted from wet biomass by resuspending a biomass sample of about 2 mg in 3 mL of acetone under dimmed light to avoid oxidation. After the addition of 0.7 g of glass beads (425-600 μm), the tubes were vortexed using an IKA Vortex Genius 3 at maximum speed for 2 min to lyse the cells. To collect the supernatant, the samples were centrifuged for 8 min at 8000 g. The extraction procedure was repeated until both the pellet and the supernatant became colorless. After evaporation of acetone under a gentle nitrogen flow, the extracts were resuspended in 700 μL of methanol and filtrated through 0. 22

Data treatment
The carotenoid production was calculated as follows: Results were analyzed using SPSS (release 25.0, SPSS Inc., Chicago, IL) software without data treatment. Data was evaluated for normality (Shapiro-Wilk) and homogeneity of variances (Levene test). For the comparison of means (ANOVA), the Tukey HSD test for equal variances with a confidence interval of 95% was performed.

Carotenoid profile of Tetraselmis sp. CTP4
The major carotenoids of the chlorophyte Tetraselmis sp. CTP4 were violaxanthin, lutein and β-carotene as detected by HPLC analysis of acetone extracts (Fig. 1A). Under the conditions used, microalgal cells also contained detectable amounts of neoxanthin, zeaxanthin and αcarotene. These results thus suggest that this strain has two active branches of the carotenoid biosynthetic pathway (Fig. 1B): one giving rise to α-carotene and lutein, and a second one leading to the biosynthesis of β-carotene and other high-value xanthophylls [22]. Both branches depend upon different lycopene cyclases that give rise to different intermediates [23]. For example, the branch yielding β- carotene is known to generate zeaxanthin via a reaction of hydroxylation. This xanthophyll can be converted to antheraxanthin and then to violaxanthin by different reversible reactions, which are usually referred to as "the violaxanthin cycle" [4]. Therefore, the detection of violaxanthin in the biomass suggests that this photoprotective pathway may be active in this strain, as appears to be the case for many chlorophytes [24]. Interestingly, even though neoxanthin can be synthesized by the direct isomerization of violaxanthin, it was only found at minor concentrations in Tetraselmis sp. CTP4.

Effect of temperature and salinity on carotenoid contents
In order to understand what environmental conditions favored the induction of carotenogenesis in this microalga, the first abiotic factors addressed were temperature and salinity (Fig. 2). A temperature of 20°C and a salinity of 35‰ corresponded to "standard growth conditions" as used in previous studies on this strain [13,20], under which the cells had contents of neoxanthin, violaxanthin, lutein and β-carotene of 0.5, 0.76, 1.64, and 2.4 mg g −1 DW, respectively (Fig. 2). These amounts are similar or even higher than those reported in other Tetraselmis strains [9,12], except for one species that was isolated from tropical waters, which had lutein and β-carotene contents of 4.8 and 51.4 mg g −1 DW, respectively [18].
At 20°C, salinity downshifts to as low as 5‰ did not significantly affect the carotenoid contents. A decrease in temperature to 10°C, however, resulted in an overall 2-fold decrease in all pigments, regardless of the salinity, as compared to standard growth conditions (Fig. 2). At low temperatures, the nutrient uptake rate decreases and thus the metabolism slows down leading to lower amounts of carotenoids [25]. Indeed, lower growth rates of cultures grown at 10°C (data not shown) were observed. Remarkably, when carotenoid contents in cells cultivated at 30°C are compared, significant differences can be observed between cultures grown at different salinities for carotenoids produced by the β-carotene branch within the carotenoid biosynthetic pathway (Figs. 1B and 2). Remarkably, at this temperature, neoxanthin and violaxanthin contents increased about 2fold with increasing salinity, reaching a maximum of 1.01 ± 0.06 mg g −1 DW and 1.58 ± 0.16 at 35‰, respectively ( Fig. 2A, B). In the case of β-carotene, cells at a salinity of 5‰ showed a 2-fold decrease in the content of this pigment as compared to standard growth conditions. Conversely, at a salinity of 35‰, cells increased their β-carotene content to a maximum of 3.37 ± 0.36 mg g −1 DW   6. Autofluorescence and carotenoid contents of Tetraselmis sp. CTP4 cells under different abiotic stressors. After incubation of the cultures for 5 days at 35°C, neoxanthin (neo), violaxanthin (vio), lutein (lut) and β-carotene (β-car) were analyzed. Furthermore, of the same samples, images were taken using differential interference contrast (DIC) and filter set 38 HE (ex. 470/40 nm, em. 525/50 nm) for autofluorescence. Scale bar = 5 μm. (Fig. 2D). The increase in temperature, however, did not result in significant differences in lutein contents (Fig. 2C). Nevertheless, at a salinity of 35‰ and 30°C, lutein content reached the maximum of 2.15 ± 0.25 mg g −1 DW. An increase in lutein contents with temperature has been observed in other microalgae such as Chlorella sorokiniana, Muriellopsis sp. and Scenedesmus almeriensis [26][27][28]. Xanthophylls are structural components in the thylakoid membrane and may help to stabilize the membranes upon thermal upshifts to maintain membrane fluidity, which is important for molecule exchange or the function of light harvesting complexes [29]. Furthermore, high salinity and heat stress are responsible for the accumulation of ROS, which can be scavenged by β-carotene and lutein [6]. In this study, the combined application of a temperature and salinity upshift led to the highest total carotenoid contents in Tetraselmis sp. CTP4 (8.11 ± 0.61 mg g −1 DW).

Effect of light intensity and nitrogen availability on carotenoid contents
As previous experiments have shown that a temperature of 30°C and a salinity of 35‰ are optimal for carotenoid induction, these parameters were maintained constant. Regarding carotenoid contents, the most decisive factor was nitrogen availability, as cells under nitrogen repletion showed on average total carotenoid contents 2.5-fold higher than those of cells under nutrient depletion (Fig. 4). Enhanced carotenoid contents under nitrogen repletion is in agreement with studies on other Tetraselmis species [16][17][18]. The importance of, for example, nitrogen availability for carotenogenesis may be due to the connection with the synthesis of proteins of the light harvesting complex that bind, among others, to carotenoids [24]. Even though this microalga accumulated high lipid contents of up to 30% of its DW under nitrogen depletion, this condition did not promote the accumulation of carotenoids [13]. On the contrary, the observed decreased contents of carotenoids under nitrogen depletion may be the result of reduced metabolic and photosynthetic activity of the cells. Interestingly, the contents of lutein in N-starving cells levelled at about 0.95 mg g −1 DW throughout the experiment (Fig. 4C). This result suggests that this xanthophyll is perhaps one of the most important carotenoids, particularly when this alga is under stress. Lutein has been proposed to be an important photoprotective pigment with a scavenging role in cells under these conditions [21]. When comparing different microalgal cultures grown at different light intensities, total carotenoid contents were 1.5-fold higher in cells at a low light intensity (33 μmol m −2 s −1 ) compared to those under a higher light intensity (170 μmol m −2 s −1 ). The induction of carotenoids under low light conditions could be due to a larger chloroplast and the need for a more efficient light utilization [30]. The xanthophylls neoxanthin and violaxanthin were found to be up to 3-fold higher in cells grown under these lower light conditions than under higher light (Fig. 4B). Furthermore, the content of β-carotene was up to 3-fold higher in cells at low light (33 μmol m −2 s −1 ) as compared to those under higher light intensities, most probably due to its role as a light harvesting pigment under this lower light intensity (Fig. 4D). Lutein contents, however, were 1.5-fold higher under higher light intensities (170 and 280 μmol m −2 s −1 ) than under lower light (Fig. 4C) in cells under nutrient repletion. Conversely, this was not observed in cells under nutrient starvation.
Taken together, these results seem to highlight once more the role of lutein as a photoprotective pigment in actively growing Tetraselmis cells. However, the observed increase in the intracellular lutein levels is apparently inhibited in starved microalgal cultures. These observations are in agreement with studies on C. sorokiniana (3.1 mg lutein g −1 DW at 690 μmol m −2 s −1 ) and Muriellopsis sp. (0.51 pg lutein cell −1 at 460 μmol m −2 s −1 ), where lutein contents increased with increasing light intensity in actively growing cultures [26,28].
Furthermore, the incubation period influenced the carotenoid contents. Already after 2 days of incubation at a light intensity of 33 μmol m −2 s −1 β-carotene content reached a maximum of 3.31 ± 0.16 mg g −1 DW, followed by a significant decrease after 5 and 10 days of incubation to 1.98 ± 0.2 mg g −1 DW (Fig. 4D). A similar response was observed for violaxanthin and neoxanthin contents, which were highest 2 days after a downshift of the light intensity to 33 μmol m −2 s −1 (0.87 ± 0.09 mg g −1 DW and 0.52 ± 0.03 mg g −1 DW, respectively). Lutein contents at low light intensities of 33 μmol m −2 s −1 increased with increasing incubation time reaching 1.53 ± 0.25 mg g −1 DW after 10 days (Fig. 4C). These contents at low light intensities are similar to those obtained previously (Fig. 2), which leads to the assumption that the actual light intensity in the larger photobioreactors (5 L) was sensed as low light by the cells. The observed increase in carotenoid levels after 2 days may be a short-term acclimation response to the new growth conditions. At low light, βcarotene is apparently the most important pigment due to its double role in light harvesting and 1 O 2 quenching, while at higher light intensities lutein becomes the main carotenoid. After 5 days of incubation, the cells seemed to have become acclimated to the new environment, ceasing carotenoid biosynthesis, so that most of the carbon (and energy) is used in growth and cell division, resulting in the observed drop in carotenoid contents. After 10 days, however, when the cultures reach high cell concentrations and nitrates became limiting (Fig. 3), carotenoid contents tend to rebound, in particular at the highest light intensity used, probably due to their role in photoprotection. Highest contents of lutein were observed at this time point, reaching a maximum of 2.24 ± 0.02 mg g −1 DW at higher light intensities of 280 μmol m −2 s −1 . In a previous study on lutein production by Scenedesmus obliquus, the highest contents of this xanthophyll (4.75 ± 1.69 mg g −1 DW) were also observed at the beginning of nitrogen depletion at a light intensity of 150 μmol m −2 s −1 [31].
Taken together, these results point out a possible combination of high temperature of 35°C and a light intensity of 170 μmol m −2 s −1 under which Tetraselmis sp. CTP4 can simultaneously produce high amounts of lutein and β-carotene. Remarkably, this was possible despite their biosynthesis being differently regulated in response to a changing environment in the previous experiments. Furthermore, these higher contents of carotenoids were achieved in a very short period of only two days. Thus, in combination with the easy harvesting step of this microalga, this process becomes more economical and Tetraselmis sp. CTP4 may be considered a novel source of carotenoids. However, further studies are necessary to examine the production of carotenoids in industrial photobioreactors.
Microscopic observation of the cultures grown at a PFD of 33 μmol m −2 s −1 and 170 μmol m −2 s −1 under nitrogen repletion and depletion sampled on day 5 revealed the localization of carotenoids and lipids inside cells (Fig. 6). Cells under the higher light intensity and under nitrate depletion contained round intracellular structures corresponding to lipid droplets as reported previously [13,14]. As expected, total carotenoid content was low (2.37 ± 0.11 mg g −1 DW), of which lutein accounted for > 50% (1.44 ± 0.09 mg g −1 DW). Under nitrate repletion conditions, however, almost no lipid droplets are visible; the carotenoid autofluorescence is diffuse throughout the cell. The total carotenoid content was 2.6-fold higher than under depletion conditions (6.15 ± 0.54 mg g −1 DW, Fig. 6), of which β-carotene accounted for > 50% (3.07 ± 0.2 mg g −1 DW). Conversely, at a PFD of 170 μmol m −2 s −1 , the autofluorescence becomes more intense at specific spots inside the cell, becoming less diffuse. Furthermore, the carotenoids seem to be present only in the chloroplast, following its typical U-shape [14]. Compared with the lower light intensity, the total carotenoid content was lower (4.31 ± 0.54 mg g −1 DW) while lutein content increased 2-fold (2.35 ± 0.18 mg g −1 DW). On closer examination of the overlay of DIC and autofluorescence micrographs, cells under nitrogen depletion appear to accumulate carotenoids outside of the lipid bodies, unlike Haematococcus pluvialis and Dunaliella salina, which accumulate astaxanthin and β-carotene in lipid bodies [32,33]. These findings strongly suggest that lutein and β-carotene are present in the thylakoid membrane rather than in lipid droplets in this species [7], which may explain why Tetraselmis cells do not accumulate carotenoids under the exact same conditions that favor lipid production. Therefore, it would be important to improve this microalga by selection of carotenoid-hyperproducing mutants, which may be able to accumulate carotenoids inside the lipid bodies.

Conclusion
This study evaluated the carotenoid profiles of Tetraselmis sp. CTP4 under a wide range of abiotic stress factors. A two-stage cultivation strategy could maximize the production of lutein, violaxanthin and βcarotene; during the first stage high biomass concentrations would be attained, and in the second stage, pigments would be induced by thermal and light upshifts. Although Tetraselmis microalgae are mainly used in aquaculture as feed, Tetraselmis sp. CTP4 could be an interesting candidate for the production of violaxanthin, lutein and β-carotene for nutraceutical and/or cosmeceutical applications, in particular when thermotolerant, robust microalgae are needed, such as the southern part of Portugal.

Contributions
The conception and design of the current manuscript was carried out by all authors. Data acquisition and analysis was primarily performed by Lisa Schüler (LS). Tamára Santos (TS), Hugo Pereira (HP), Paulo Duarte (PD) and Katkam Gangadhar (KG) assisted during the data collection and assembly of the data. Cláudia Florindo played an important role in data acquisition using fluorescence microscopy. Peter Schulze (PS) and Luísa Barreira (LB) analyzed the data with their statistical expertise. LS and João Varela (JV) performed the initial drafting of the article, and subsequent versions were drafted and reviewed by LS, PS, HP, LB and JV. All authors contributed to the final approval of the article.