Production of carbohydrates, lipids and polyunsaturated fatty acids (PUFA) by the polar marine microalga Chlamydomonas malina RCC2488

Polar microalgae that are highly productive in cold climates can produce large amounts of biomass and polyunsaturated fatty acids (PUFA). The polar Chlamydomonas malina RCC2488, grows at low temperatures and produces high amounts of lipids, which are mainly composed of PUFA. However, not much is known about its phylogenetic relationship with other strains within the order Chlamydomonadales and the optimum growth conditions for maximum biomass productivity have not yet been identified. In this study, a phylogenetic analysis was performed to determine the closest relatives of C. malina within the Chlamydomonadales order. To select the best growth conditions for maximum biomass productivities in cultivations performed at 8 °C, different salinities (0–80) and light intensities (70–500 μmol photons m s) were tested, using bubble column and flat-panel photobioreactors. The effect of nitrogen limitation was tested to determine if C. malina can accumulate energy reserve metabolites (carbohydrates and lipids). Phylogenetic analysis confirmed that C. malina, which belongs to the Chlamydomonales order, is closely related to the psychrophilics Chlamydomonas sp. UWO 241 and Chlamydomonas sp. SAG 75.94, as well as to the mesophilic C. parkeae MBIC 10599. The highest biomass (527mg L day), lipid (161.3 mg L day) and polyunsaturated fatty acids (PUFA; 85.4 mg L day) productivities were obtained at a salinity of 17.5, light intensity of 250 μmol photons m s and nitrogen replete conditions. Strikingly, the marine C. malina can grow even in fresh water, but the biomass productivity was reduced. While the intracellular lipid content remained unchanged under nitrogen deprivation, the carbohydrate content increased (up to 49.5% w/w), and the protein content decreased. The algal lipids were mainly comprised of neutral lipids, which were primarily composed of PUFA. Chlamydomonas malina RCC2488 is a polar marine microalga suitable for high biomass, carbohydrate, lipid and PUFA productivities at low temperatures.


Introduction
Polyunsaturated fatty acids (PUFA) have gained interest in the pharmaceutical industry due to its beneficial properties for human and animal health [1][2][3]. PUFA used for human nutrition are mainly obtained from fish oil [3,4]. However, obtaining PUFA from fish have several limitations, such as possible depletion of the resource, contamination with heavy metals, variability in the oil composition and quality, unpleasant odor, and environmental negative impacts like degradation of marine habitats [3][4][5][6]. On the other hand, through the food chain, fish obtain PUFA from microalgae via bioaccumulation [4,7]. PUFA from microalgae can be a sustainable, environmentally friendly, and a "vegetarian and vegan" alternative [2,8,9]. Moreover, its production can be enhanced by using polar or cold adapted microalgae, which are the ideal candidate due to its naturally occurring high T Chlamydomonas malina RCC2488 (called C. malina hereafter) is a polar microalga isolated in 2009 from the Beaufort Sea of the Arctic Ocean [18]. In our previous study, C. malina had high levels of PUFA when cultivated at 8°C compared to 15°C [17]. However, the phylogenetic relationship of C. malina within the Chlamydomonadales order and its best growth conditions for obtaining desired biomass and metabolites are yet to be investigated. In this study, we performed a phylogenetic analysis of the 18S rRNA gene sequence of C. malina and available Chlamydomonas strains. We report the performance of C. malina under different experimental conditions, such as salt concentrations, light intensities, and nitrogen stress, to identify the optimal growth conditions at which this microalga produces commercially important metabolites, such as lipids, carbohydrates, and PUFA.

Strain
The polar marine microalgae Chlamydomonas malina RCC2488 was obtained from the Roscoff Culture Collection, France (RCC). This strain was isolated from the Beaufort Sea, within the Arctic Ocean, at latitude 69°48 N and longitude 138°26E [18]. The strain has been maintained in k/2 media at 4°C, and transferred to fresh media every 4 weeks at the culture collection station (http://roscoff-culture-collection.org/rccstrain-details/2488).

Phylogenetic analyses
The sequence of the 18S ribosomal RNA gene of C. malina strain was obtained from the Roscoff Culture Collection with accession number JN934686. 18S rRNA related genes were identified by BLASTN searches against GenBank and NCBI, using C. malina 18S rRNA gene sequence as a query (JN934686). Sequences were aligned by Muscle [19]; all gaps and missing data were eliminated. The evolutionary history was inferred by constructing a phylogenetic tree using the Maximum Likelihood method based on the Tamura-Nei Model [20]. Evolutionary analyses were conducted in MEGA7 [21].

Culture conditions
Stock cultures were maintained on agar plates containing f/2 medium [22]. Inocula for all experiments were cultured in 250 mL Erlenmeyer shake-flasks (100 rpm) containing 100 mL of f/2 at 8°C with an irradiance of~120 μmol photons m −2 s −1 (Phillips TLD 840 fluorescence lamps) and ambient levels of CO 2 . For the medium preparation, we used seawater from the North Atlantic shoreline of Bodø (Norway) containing a salinity approximately of 35. The f/2 medium comprised the following macronutrients (in mM): NaNO 3  For salinity experiments, C. malina was cultured in f/2 medium and 120 μmol photons m −2 s −1 for 10 days at salinities 0, 17.5, 35, and 80, using combinations of fresh water (salinity: 0) and seawater (salinity: 35) or supplementing with NaCl for higher salinity (80). For testing of different light intensities and nitrogen stress, a salinity 17.5 was used. For light intensity experiments, C. malina was grown in f/2 medium for 10 days at 70, 120, 250 and 500 μmol photons m −2 s −1 . For nitrogen stress experiments, C. malina was pre-cultured in f/2 medium and 120 μmol photons m −2 s −1 until the middle logarithmic phase was reached (day 5). At this growth stage, cells were collected by centrifugation, washed twice with water (salinity:17.5), and re-suspended in f/2 medium with NaNO 3 (F2 + N) or without NaNO 3 (F2eN) at a biomass concentration of 1.5 g DCW L −1 . The nitrogen stress experiments denote the presence or absence of NaNO 3 , referred as only nitrogen hereafter. Cultures were grown for five days at a light intensity of 250 μmol photons m −2 s −1 . After five days, cells were harvested by centrifugation (2000 g, t = 5 min), washed with 0.5 M ammonium formate, centrifuged again (2000 g, t = 5 min), and the pellets were stored at −70°C for further analyses.

Cultivation in photobioreactors
Salinity and nitrogen stress experiments were conducted in bubble column photobioreactors (Table 1) designed by Hulatt et al. [16]. Briefly, the arrangement consisted of glass tubes (Friedel, Oslo, Norway) measuring 35 mm internal diameter with 300 mL of working volume, fitted with sealed silicon stoppers and autoclaved as complete units at 121°C for 20 min. The medium was autoclaved separately in 1 L flasks. Filtered air (0.2 μm, Acrodisc ® PTFE filters, Pall Corporation, USA) containing 1% CO 2 was supplied to each photobioreactor at a flow of 1 vvm (300 mL min −1 ) using a rotameter (Omega, Manchester, UK). A mass flow control system (GMS-150, Photon Systems Instruments, Czech Republic) was used to control the CO 2 concentration by mixing it with compressed air. These photobioreactor systems were placed in a temperature-controlled environment chamber at 8°C (Termaks AS, Bergen, Norway) fitted with nine fluorescent lamps (cool daylight, 36 W, Phillips) illuminated from one side at a light intensity of 120 μmol photons m −2 s −1 .
For a more uniformity of the photosynthetic photon flux density (PPFD), the light intensity experiments were carried out in autoclaved flat-panel photobioreactors (Algaemist-S, Ontwikkelwerkplaats, Wageningen UR, The Netherlands) ( Table 1), fully described previously [23]. The measurement of the PPFD impinging on the front of the cultivation vessel was performed using a Li-Cor189 2π quantum sensor (LI-COR Biosciences, Lincoln, NE, USA), accordingly to Suzuki et al. [9]. The working volume was 380 mL with a light path of 14 mm. The cultures were aerated at 1 vvm with 0.2 μm filtered air mixed with 1% CO 2 . Continuous light intensity of 70, 120, 250 and 500 μmol photons m −2 s −1 was provided by warm-white LEDs. The cultivation temperature of 8°C ± 0.5°C was controlled by the Algaemist software. Initial pH was 7 without control along the cultivation period. All experiments were carried out in triplicate for 10 days.

Growth measurements
The growth of C. malina was measured by the optical density, which was calibrated against the dry weight. Samples of the culture (0.5-1 mL) were collected daily to measure the absorbance at 750 nm in a 1 cm micro-cuvette using a spectrophotometer (Hach-Lange DR3900, Hach, International). The DCW was evaluated gravimetrically by filtrating 5-10 mL of culture through a pre-dried and pre-weighed

Lipid, protein, and carbohydrate analyses
Total lipids from C. malina were extracted using organic solvents, and fatty acid methyl esters (FAMEs) from triacylglycerols (TAGs) and polar lipids were identified and quantified by gas chromatography (GC) as previously described by Breuer et al. [24], with some modifications. Briefly, 10 mg of freeze-dried microalgal biomass was weighed using a precision balance (MX5, Mettler-Toledo, USA), then the lipids were extracted and gravimetrically quantified. For the extraction, a mix of chloroform:methanol (2:2.5 v/v) containing an internal standard (Tripentadecanoin, C15:0 Triacylglycerol, Sigma-Aldrich, USA) was added to the samples to extract the lipids, and then the cells were disrupted using a bead mill (Bertin technologies, Precellys Evolution, France, 0.1 mm glass beads). Methanol from the solution containing the extracted lipids was separated by adding an aqueous solution of Tris buffer (6 g L −1 Tris, 58 g L −1 NaCl, pH 7.5). The chloroform phase containing the lipids was removed and dried under a stream of nitrogen. To determine the fatty acid composition, lipid samples were chemically derivatized to fatty-acid methyl-esters (FAMEs) using 5% H 2 SO 4 in methanol and heated at 70°C for 3 h. Methanolic H 2 SO 4 from the solution containing the FAMEs was separated by adding a mix of hexane:H 2 O (1:1). Finally, samples containing the FAMEs and hexane were placed into chromatographic vials. The obtained organic phase was analyzed in a GC fitted with a Flame Ionization Detector (Scion 436, Bruker, USA) and an Agilent CP-Wax 52 CB column (Agilent Technologies, USA) using splitless injector. To identify and quantify the most common FAMEs, external Supelco® 37-component standards (Sigma-Aldrich, USA) were used. Blanks were included in the extraction process to eliminate background trace peaks.
For carbohydrate determination, samples were hydrolyzed with HCl to yield simple sugars, and the resultant monosaccharides were quantified using the phenol-sulphuric acid method [25].
For protein determination, lysis buffer (60 mM Tris pH 9, 2% sodium dodecyl sulfate) was added to 10 mg of freeze-dried biomass samples prior to cells disruption in bead milling system as described before, and then protein content was determined using the Lowry method [26].

Calculations
The cellular growth kinetic and productivity were calculated accordingly with a 4-parameter logistic function [23] using the following equation: where C x is the dry weight (g L −1 ) at time t (days), ϕ1 is the lowest asymptote (minimum C x ), ϕ2 is the upper asymptote (maximum C x ), ϕ3 is t at 0.5ϕ2 (the inflection point), and ϕ4 is the scale parameter [23]. From the previous equation, the volumetric productivity was calculated between two time points, accordingly with Eq. (2): where P is the productivity (g L −1 day −1 ), C x.i and C x.i-1 are the concentrations of the biomass (g L −1 ) at two time points, and t i and t i-1 are the time of cultivation (days).

Statistical analysis
For all treatments, the normal distribution of data was confirmed using Saphiro-Wilk test, and the homogeneity of the variance between treatments was validated using Brown-Forsythe test. For salinity and light intensity treatments, one-way analysis of variance (ANOVA) and post-hoc Tukey's multiple comparison test were used. For nitrogen stress treatments, a t-test was applied. P values smaller than 0.05 were considered statistically significant.

Phylogenetic analysis
Previous results indicated that C. malina belongs to the order Chlamydomonales [18], we confirm this result (Fig. S1). We constructed a phylogenetic tree by the comparison of the sequences of 18S rRNA gene of isolate C. malina and only the Chlamydomonales order ( Fig. 1). In this tree, the C. malina strain is placed in a lineage closely related to C. parkeae, within the Moewusinia clade. Chlamydomonas malina is closely related to Chlamydomonas sp. UWO 241 (UWO 241 hereafter, score = 2983 bit, identity = 99%), Chlamydomonas sp. SAG 75.94 (score = 2942 bit, identity = 99%) with a strong bootstrap (BS = 100). All the sequences form clusters with bootstrap support of  no less than 50%. Previous phylogenetic studies indicated that this microalga cluster together with C. raudensis CCAP 11/131 and C. parkeae within the Moewusii clade [18]. It is clear that C. malina belongs to the Chlamydomonadales order, mainly composed by freshwater flagellates within the Chlorophyceae [18]. However, C. malina in the present phylogenetic analysis appeared more closely related to UWO 241 and C. parkeae MBIC10599 than to C. raudensis that is being grouped in a sister clade (Fig. 1). The reason for this discrepancy is that UWO 241 was earlier misidentified as C. raudensis CCAP 11/131 [27]. But recent studies on phylogenetic analysis of nuclear and plastid DNA sequences revealed that UWO 241 is in fact closely affiliated to the marine strain C. parkeae SAG 24.89 and strongly differs from C. raudensis SAG 49.72 [28].

Effect of salinity
Highest biomass concentration (5.02 g DCW L −1 ) and productivity (480 mg DCW L −1 d −1 ) were attained at salinities of 17.5 and 35 (p > 0.05, Figs. 2A and B). The growth and maximal biomass concentration decreased 5-fold at the highest salinity tested (80) ( Figs. 2A  and B). Chlamydomonas malina RCC2488 is a marine microalga, originated from the Beaufort Sea located in the Arctic Ocean [18]. There, during late summer when most phytoplankton blooms are occurring, salinities shift between 28 and 32 due to freshwater inflow from rivers and melting ice [18,29,30]. Chlamydomonas malina may have adapted to these salinity shifts, explaining the high growth performance at salinities ≤ 35. As demonstrated in C. malina closest relative, UWO 241, one possible explanation of the salinity tolerance of C. malina is the modulation of the redox signal (redox state of plastoquinone pool), affecting later the expression of genes responsible of high salinity acclimation [15,31]. After 7 days of cultivation, cells entered into stationary phase. This condition, could be due to a limitation or depletion of essential nutrients, inhibitory compounds formation, or high cell densities that cause limited nutrient/gas transfer rates due to mixing issues, and limited light penetration into the cultivation that causes cell self-shading.
Protein content in treatments from 0 to 35 had similar values (26.1-27.6% of DCW, p > 0.05, Fig. 2C). A lower protein content was found in cells cultivated at salinity 80 as compared to 17.5 (p < 0.05), probably as a result of a lower metabolic activity.
The carbohydrate content in cells cultivated at salinity ≤ 35 was not statistically different (24.5-26.1% of DCW; p > 0.05). Carbohydrate accumulation up to 33.2% of DCW was stimulated by high salinities, resulting in a linear trend from salinities 17.5 to 80 (Fig. S2).
In the case of total lipid content, the statistical analysis (one-way  , p < 0.01, Fig. 3A). In general, the TAG pool comprised the major lipid fraction in all treatments, ranging from 230.1 to 267.4 mg TAG g DCW −1 . Specifically, into this TAG pool, cells cultivated at salinity ≤ 35 mainly synthesized PUFA (hexadecatetraenoic acid, C16:4n-3 and α-linolenic acid, C18:3n-3) (Fig. 3B  and 8A). Maximal PUFA productivity of 65.1 mg PUFA L −1 day −1 was obtained in cells cultivated at salinities 17.5 and 35 (p < 0.05, Fig. 3B). Interestingly, the high biomass productivity at salinity ≤ 35 did not compromise the cellular total lipid content as being usually found for microalgae [32,33]. For the ongoing experiments, a salinity of 17.5 was chosen.

Effect of light intensity
The highest biomass concentration of 5.3 g L −1 and overall productivity of 527.4 mg DCW L −1 day −1 was found at 250 μmol photons m −2 s −1 (p < 0.05, Figs. 4A and B). Strikingly, C. malina could grow at low but also at high light intensities, suggesting that this microalga may tolerate high light intensities such as 500 μmol photons m −2 s −1 or even higher. The ability of C. malina to grow at high light intensities was not found in UWO 241, which did not grow above light intensities of 250 μmol photons m −2 s −1 [15,34]. Probably, one reason could be that UWO 241 lacks PsbS, which is a protein that plays a key role in photoprotection [31], however the presence of this protein in C. malina is unknown. Therefore, for further studies, it would be an interesting phenomenon to investigate the effect of even higher light intensities on the physiology, metabolism, and genome of C. malina. Nevertheless, the high performance of C. malina in a broad range of light intensities may be a consequence of living in the Arctic, where this microalga had to adapt to low light levels occurring during winter and to high light irradiances during summer [10]. Adaptations to light variabilities include evolution to a structurally and functionally distinct photosynthetic apparatus, and augmented light-harvesting apparatus [10,27]. It has been demonstrated that some polar algae from the genera Chlamydomonas and Chloromonas are able to produce various secondary carotenoids, such as astaxanthin when they are exposed to high light conditions [12,35]. These carotenoids are known to play a role in stress response such as shielding the photosystem against excessive irradiation [36]. Consequently, a detailed study of pigments content in C. malina is suggested. After 7 days of cultivation, cells entered into stationary phase, possibly due to the effects previously mentioned in Section 3.2.
The content of carbohydrate and lipid had no significant differences (p > 0.05) among all light intensities, suggesting that light intensities, ranging from 70 to 500 μmol photons m −2 s −1 , did not have an effect on the synthesis of energy reserve metabolites in C. malina. This response is contradictory to what has been observed with other microalgal strains. For example, diminution of the protein and carbohydrate content but an increase in the lipid content are typical responses to high light intensity in several strains [37][38][39][40][41]. Nevertheless, in all treatments the lipid and carbohydrate content were relatively high for cells taken in the mid exponential phase of growth. As discussed below, probably another stressor, such as temperature, induced high lipid and carbohydrate synthesis. The highest light intensity (500 μmol photons m −2 s −1 ) induced higher content of polar lipids (p < 0.01; Fig. 5A), specifically SFA (C14:0, C16:0 and C18:0; Fig. 8B) and MUFA (C16:1n-7, C18:1n-9, C18:1n-7; Fig. 8B). This increase in the polar lipids pool was probably due to remodeling or relocation of membrane lipids in response to the high light intensity [39,42,43]. For example, the glycolipid digalactosyl diacylglycerol (DGDG) is known to stabilize, structurally and functionally, the chloroplast which led to cell survival [39,42,43]. Photoprotection is another function of polar lipids like monogalactosyl diacylglycerol (MGDG) at high light intensities [39,42,43]. Indeed, high levels of membrane lipids were also found in UWO 241 (C. malina closest relative), where polar lipids were mainly composed of MGDG, DGDG, and sulfoquinovosyldiacylglycerol [31]. In all treatments, lipids were mainly TAG with similar composition (Fig. 5B; p > 0.05). This TAG fraction comprises primarily of PUFA such as C16:4n-3 and C18:3n-3 (Fig. 8B). The maximal total PUFA productivity of 83.8 mg PUFA L −1 day −1 was found in the treatments at light intensities of 120 and 250 μmol photons m −2 s −1 (p > 0.05).

Nitrogen stress
Nitrogen stress condition was applied to C. malina cells to test if energy reserve metabolites can be accumulated. Due to a halt in cell division, the final biomass concentration and productivity of cells maintained under nitrogen deprivation (F2eN) were~2 times lower than cultures at replete nitrogen conditions (F2 + N, Figs. 6A and B, p < 0.0001, t-test). Cells reached stationary phase after one day under nitrogen deplete conditions and after two days under nitrogen replete conditions. Possibly causes are described in Section 3.2. Nitrogen deprivation stimulated the accumulation of carbohydrates (up to 49.5% DCW, p < 0.01, t-test, Fig. 6C), at protein synthesis expenses (12% DCW decrease, p < 0.01, Fig. 6C). One of the most preferred strategies to increase reserve metabolites production in several microalgal species is a nutrient stress, like nitrogen deprivation [44]. When nitrogen availability is limited, cells synthesize energy storage compounds such as carbohydrates, while reducing nitrogen-containing compounds such as proteins [45]. Hence, this strategy was effective for carbohydrate but not for lipid accumulation. The lipid content had no significant differences among the treatments (p > 0.05; Fig. 6C). Nevertheless, the lipid content of C. malina is relatively high in both treatments (average 32.5% of DCW), compared with other Chlamydomonas strains, such as the mesophilic C. reinhardtii, that can accumulate up to 19% at nutrient replete conditions [46]. This high lipid content has been reported in other polar and mesophilic algal species as an essential player for temperature acclimation and adaptation [47][48][49]. Consequently, a possible explanation is that this polar algal strain was already under a temperature stress at 8°C, which induced lipid accumulation in all treatments tested in this study. Although, we advise additional studies at lower temperatures to support this observation. The total PUFA productivity of cells cultivated in F2 + N was 76.9 mg PUFA L −1 day −1 , while the PUFA productivity of cells maintained in F2eN was substantially lower (40.9 mg PUFA L −1 day −1 ). In both cases, PUFA was mainly comprised of C16:4n-3 and C18:3n-3 (Fig. 8C). The proportion of total polar lipids and total TAG was similar in both treatments (p > 0.05, Figs. 7 and 8C). The PUFA content in the TAG pool (Fig. 7B) was similar in F2 + N and F2eN (p > 0.05). However, the PUFA content in the polar lipid pool was significantly higher in F2eN (p < 0.05) (Figs. 7A and 8C). The effect of low temperature on the membrane lipid composition is well-known [50,51], like the contribution of C16:4n-3 in the transition from liquid-crystalline to gel phase [31]. This phenomenon has been also observed in the psychrophilic UWO 241, which contains high levels of C16:4n-3 [52]. Nevertheless, the combined effect of low temperature and nitrogen deprivation on the  membrane lipid composition is not yet understood and requires further studies.

Chlamydomonas malina for biotechnology
Currently, commercial microalgal production is limited to mesophilic organisms with optimal temperature productivities of around 24-40°C [16,[53][54][55][56]. However, in colder climates, this approach is unfeasible due to higher production cost inherent to energy expenses for heating or seasonal production times limited to summer months. In addition, we found that some microalgae have a high productivity and are rich in omega-3-fatty acids [16]. In this context, polar and coldadapted microalgae, such as C. malina, are efficient alternatives for biomass and metabolite production in cold climates [9,16]. In the present study, we optimized the cultivation of a polar microalga. Our results indicate that the biomass productivities obtained from C. malina are comparable with the biomass productivities of mesophilic and other polar and cold-adapted microalgae [9,16,44]. It is noteworthy that the metabolic rates (measured here as biomass productivity) of polar and cold-adapted microalgae were not reduced due to low temperatures, as suggested by several studies [57][58][59]. This statement probably is only applicable to mesophilic microalgae that cannot grow below thermal optimum temperature [16]. Taken together, optimized C. malina can be potentially employed for cultivation in cold climates with high biomass productivities. The metabolites produced by C. malina have the potential to be used in biotechnological applications. For example, PUFA are essential nutrients for a balanced human and animal diet, and their nutraceutical and pharmaceutical applications have been extensively reviewed [60][61][62]. Biofuels can be produced from neutral lipids (TAGs) and carbohydrates [8,63,64]. Proteins from C. malina can be marketed as human health food or as animal feed [65][66][67][68], but prior protein quality analysis is suggested. Further studies to determine a possible production of high-value pigments, like lutein and astaxanthin from C. malina, would be desired and recommended to evaluate the potential use of this strain in food, nutraceutical, pharmaceuticals and cosmetics applications [69][70][71][72], among others.

Conclusions
The arctic green alga Chlamydomonas malina RCC2488 is a novel polar microalga that belongs to the Chlamydomonadales order, closely related to the well-studied Antarctic strain Chlamydomonas sp. UWO 241. Both strains share similar physiological features such as tolerance to a wide range of salinities, high lipid content composed mainly by C16:4n-3 and C18:3n-3. However, C. malina tolerate high light intensities, a trait that was not found for UWO 241. Chlamydomonas malina achieved maximum productivities of biomass (527 mg L −1 day −1 ), lipids (161.3 mg L −1 day −1 ) and PUFA (85.4 mg L −1 day −1 ) under nitrogen replete conditions at salinity 17.5 and a light intensity of 250 μmol photons m −2 s −1 . Nitrogen deprivation triggered the accumulation of carbohydrates in cells (up to 49.5% w/w) at the expense of proteins but without compromising lipid biosynthesis. Chlamydomonas malina is a polar microalga suitable for biomass, lipid, PUFAs and carbohydrate production at 8°C with potential biotechnological applications.

Author contributions
Rene Wijffels designed the research in the workpackage in the A2F project which resulted in this manuscript. Based on that framework Daniela Morales-Sánchez designated the study and collected the data. Daniela Morales-Sánchez and Peter Schulze performed the bioreactor experiments and executed lipid and fatty acid analysis. Daniela Morales-Sánchez, Peter Schulze, Rene Wijffels and Kiron Viswanath contributed to manuscript drafting, discussion and critical revision of the article for important intellectual content.

Funding
This work was funded by the Research Council of Norway's BIONAER Programme and is part of the Algae2Future project (267872).

Declaration of competing interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. No conflicts, informed consent, or human or animal rights are applicable to this study.