Abstract
The diatom Phaeodactylum tricornutum is known to accumulate polyunsaturated fatty acids (PUFAs), especially omega-3 fatty eicosapentaenoic acid (EPA) 20:5n3, which is health beneficial and are essential in human and animal diets. The main genes involved in the biosynthesis of EPA have been reported, but the effect of light and other environmental factors on transcription of such genes is poorly understood. This paper evaluates the transcription of six genes related to EPA production, fatty acid methyl esters (FAMEs), and pigments as a result of growing P. tricornutum under 150 and 750 μmol photons m−2 s−1 irradiance up to 72 h. Regarding transcription levels, all genes displayed a wide variation range in time. Under 24 h of exposure to 750 μmol photons m2 s−1, transcription of PTD6, PTD15, ELO6_b1, and ELO6_b2 significantly increased, whereas 72 h exposure led PTD6 transcript levels to decrease significantly, and PTD5α and ELO6_b1 1 to increase. The 72-h evaluation showed that the 150 μmol photons m−2 s−1 irradiance resulted in a 4-fold increase pigments (chlorophyll and carotenoids) production, as well as in PUFAs (16.59%) production, including EPA (5.72%). Conversely, the 750 μmol photons m−2 s−1 irradiance resulted in increased saturated fatty acids (SFAs) and decreased PUFA concentrations in the FAME distribution. The irradiance variation had a major effect on microalgal metabolism as a result of membranes remodeling, PUFA synthesis rerouting, SFA, and pigments. Such information could be utilized for improving pigments, EPA, and other PUFA syntheses from P. tricornutum.
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.Avoid common mistakes on your manuscript.
Introduction
Global efforts to improve sustainability have led to many studies on the economic viability of the use of microalgae for different biotechnological purposes, e.g., biofuel production, bioremediation, food, and animal feed. Besides, the production of high-value pharmaceuticals and nutraceuticals derived from microalgae can also be pursued (Li et al. 2014; Neumann et al. 2018).
In this scenario, the marine diatom Phaeodactylum tricornutum stands out mainly because it can accumulate up to 30% of EPA of the total fatty acids (Qiao et al. 2016), which is one of the most valuable omega-3 fatty acids. PUFAs can be classified in families like omega-3 (ω-3) or omega-6 (ω-6) fatty acids, depending on where the first unsaturation is located in the long fatty acid chain. The ω-3 fatty acids are associated with human health benefits because they act as precursors for anti-inflammatory eicosanoids, are cellular membrane components, and are an essential nutrient for newborns and infants (Luchtman and Song 2013; Ulmann et al. 2017). For these reasons, they are considered nutraceuticals and are often added to food supplements.
In addition to the importance for humans, ω-3 are considered essential nutrients for the larvae development of many aquatic organisms like fish, shrimp, and bivalves (Marquez et al. 2019). Therefore, this industry has a strong demand for ω-3, which is largely obtained from marine fish (Shah et al. 2018). Considering that consumption of these supplements will increase, and their natural sources are becoming scarce, it is imperative to develop alternative sources of ω-3 (Mühlroth et al. 2013; Li et al. 2014).
Fatty acid synthesis in P. tricornutum begins in the chloroplast, where long chains are produced and then transported to the endoplasmic reticulum for the PUFA synthesis. This pathway requires energy and is catalyzed by desaturases and elongases that catalyze the double bond formation and elongation in aliphatic carbon chains, thus producing EPA (Arao and Yamada 1994; Zulu et al. 2018).
Domergue et al. (2002) cloned and characterized the desaturases genes PDT5 and PDT6, and demonstrated their front-end activity in P. tricornutum. Based on this information, subsequent work demonstrated that the activity of these enzymes, through overexpression in this organism, exhibited a significant increase in EPA production, indicating that gene expression and transcription can be limiting factors in PUFA pathway (Peng et al. 2014; Zhu et al. 2017). The influence of stress exposure on transcription of fatty acid desaturases and fatty acid composition has also been analyzed in some studies, including nitrate starvation of Isochrysis galbana (Huerlimann et al. 2014), increased salinity in Tetraselmis sp. (Adarme-Vega et al. 2014), and the nitrogen/phosphorus ratio in P. tricornutum (Lopes et al. 2019), but the effects of light changes on P. tricornutum gene expression as related to lipids composition are not well understood yet.
Evaluating the transcriptome of P. tricornutum exposed to high light with 500 μmol photons m−2 s−1, Nymark et al. (2009) described the molecular mechanisms of capacity to adapt the light harvesting complexes (LHC) in response to the new environmental condition. The LHC are a complex structures of proteins and pigment molecules in the thylakoid membrane, which in low light improves energy capturing, and in excess of light rapidly induces thermal dissipation of excess absorbed energy (Lepetit et al. 2012; Buck et al. 2019). Among pigments, the most abundant carotenoid is fucoxanthin that forms the protein complexes called fucoxanthin-chlorophyll a/c-binding antenna protein (FCP) which are peripheral antennae of the photosystems (Domingues et al. 2012).
Associated with human and animal health benefits, fucoxanthin is used in dietary supplement form acting as antioxidant, anti-obesity, anti-inflammatory, antidiabetic, and antihypertensive agents, and can be used as an additive in the feed industry (Peng et al. 2011; Wang et al. 2018). PUFAs and fucoxanthin have high commercial value, and their production by microalgae can leverage environmental benefits, although challenges to the production of algal biomass must be overcome to attain large-scale production (Wang et al. 2018). A possibility is applying metabolic engineering, as shown in Dinamarca et al. (2017), where lipid biosynthesis was increased, especially triacylglycerols (TAGs) after overexpression of diacylglycerol acyltransferase in P. tricornutum. Hence, there is a need to understand the microalgae metabolism and identify new targets for genetic manipulation with new techniques like CRISPR (Nymark et al. 2016).
Based on the literature review, in order to improve the lipid metabolism gene response understanding, it is necessary to identify factors involved in gene expression modulation, especially those linked to environmental conditions, such as light intensity. In such context, this study focused on analyzing the differential expression of desaturases and elongases under two irradiance conditions. In this way, modulations in the gene expression ratio for fatty acid composition and the effect on the synthesis of pigments in P. tricornutum were investigated.
Material and methods
Culture and growth analyses
The microalga Phaeodactylum tricornutum strain CCAP1055/1 was cultivated in 4 L of f/2 medium (Guillard and Ryther 1962) plus silica and was acclimated at 22 °C, 200 μmol photons m−2 s−1 at constant irradiance white fluorescent lamps, monitored using Li-250A light meter (Li-COR) and supplemented with 0.5% CO2 (v/v) for 7 days. The biomass was harvested by 5 min centrifugation at 2950×g and washed 1× in Guillard f/2 medium-plus silica. The cells were resuspended at turbidity equivalent to 100 mg dry weight, according to Lopes et al. (2019), estimated indirectly through the absorbance read at 700 nm in UV-Vis spectrophotometer (Thermo Fisher Scientific), according to the equation: Biomass (g L−1) = ABS700 × 0.0022, R2 = 0.994 and fractionated into six borosilicate flasks with 2 L of medium. Three independent replicas used in each of the treatments at constant irradiances of 150 and 750 μmol photons m−2 s−1, supplemented with 0.5% CO2 (v/v) at 22 °C. To avoid self-shading, cultures were partly replaced with fresh medium once every 24 h until turbidity equivalent to approximately 100 mg dry weight. At the sampling periods (0, 12, 24, and 72 h), 30 mL were taken to measure turbidity, pigments and gene expression.
Pigments analyses
The pigments were extracted according to Strickland and Parsons (1972), for the photosynthetic pigments chlorophylls a (Chl a), c (c1 + c2); content was calculated indirectly according to Jeffrey and Humphrey (1975), for carotenoids (fucoxanthin, diadinoxanthin, and β-carotene) according to Carreto and Catoggio (1977), through absorbance readings at 480, 510, 664, and 630 nm.
RNA extraction and qPCR analysis
For gene expression, the cells were obtained by centrifugation for 5 min at 3500×g and resuspended in 1 mL of RNA Later solution. Extraction was performed with the TRIzol reagent, and the concentration was estimated at 260 nm, with purity checked at ratios Abs260/Abs280 and Abs260/Abs230. The transcription of the desaturase, PDT5α, PDT5β, PDT6, PTD15; and elongase, ELO6_b1, ELO6_b2, genes were evaluated, with primers described by Lopes et al. (2019). For reference genes, RPS and H4 were used as baseline expressions, synthesizing the 30S ribosomal subunit and Histone H4, respectively, according to Siaut et al. (2007). Amplifications were performed with a StepOne thermal cycler with a Power SYBR Green RNA-to-Ct 1-Step, according to the manufacturer’s instructions. In each run, RT-PCR efficiency was checked for each primer pair by constructing a standard curve from serial dilutions from a pool of all samples, and assays were carried out in triplicates and all reagents and equipment at Thermo Fisher Scientific. For statistical analysis, the equation of Livak and Schmittgen (2001) was used (2−ΔΔCT method).
Lipid analysis
At the beginning and end of the cultivation, 1 L of cultured cells were withdrawn from each treatment at 0 and 72 h and was centrifuged at 3500×g for 10 min at 4 °C, and lyophilized to obtain biomass to analyze their fatty acid profile.
Lipids were extracted from the dry biomass using a solvent-based extraction method, according to Borges et al. (2016). Dry biomass (0.5 g) was placed into test tubes (in three replicates) with 1.5 mL of a chloroform-methanol (2:1 v/v) mixture. The solution was exposed to ultrasound (Quimis USC-1400/40 kHz) for 20 min. The mixture was then centrifuged for 2 min at 2000×g. The lipid extraction process was repeated three times for each tube. The liquid phase was transferred to previously weighed flasks. Afterward, the solvent was evaporated under vacuum in a rotary evaporator, and the flasks were then reweighed. The total lipid fraction was determined based on the differences in flask weights, and the lipid content was calculated based on the percentage of the initial dry weight.
The derivatization of the lipid fraction of microalgae was carried out according to Lemões et al. (2016), in which the sample containing the lipid fraction (300 mg) was placed in a test tube, and a mixture of 3 mL of boron trifluoride/methanol was added. The mixture was heated in a water bath at 70 °C for 20 min. For recovery of the FAMEs, the mixture derived was washed in a separatory funnel with 15 mL of hexane and 20 mL of distilled water. The organic and aqueous phases were then separated, the organic phase containing the fatty esters was dried, and the solvent was evaporated at 50 °C.
Analyses were performed using a Shimadzu GCMS-QP2010 Plus chromatographic system equipped with a split/split less injector coupled with a mass detector. The operating temperatures of the detector were 280 °C for the interface and 230 °C for the source. Detection was performed using a full scan from m/z 30 to m/z 500 with a scan time of 0.20 s. The ionization mode electron impact was at 70 eV. The operating conditions of the chromatograph were as follows: injector 250 °C, column 80 °C (initial temperature, 0 min), followed by a gradient of 10 °C min−1 to 180 °C and then 7 °C min−1 to the final temperature of 330 °C, gas flow 1.3 mL min−1, pressure 88.5 kPa, average linear velocity 42 cm s−1, and 1 mL injection volume with the split ratio 1:100. A crossbond 5% dimethyl polysiloxane diphenyl 95% column (30 m × 0.25 mm × 0.25 μm; Restek) was used. The fatty acid methyl esters were identified by comparison with known standards (Supelco 37 Component FAME Mix) and were quantified using the standardized areas method.
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 7.00 for Windows. The data were assessed for normality with the Kolmogorov-Smirnov test. The data were compared by one-way analysis of variance (ANOVA) with replicate post hoc comparisons made using the Tukey’s test for lipid content and growth. For pigments and gene expression, the Mann-Whitney test one-tailed was applied. A significance of P < 0.05 was applied to all statistical tests performed. Results are presented as mean ± standard deviation (n = 3).
Results
This study analyzed the influence of light on physiological responses of P. tricornutum exposed to up to 72 h to irradiation conditions of 150 and 750 μmol photons m−2 s−1. Concerning biomass production, no statistically significant differences was observed between the light irradiances up to 72 h of cultivation, as shown in Fig. 1. Regarding the photosynthetic pigments, there was a significant difference among treatments. At the lower irradiance, an increase was observed in the synthesis of Chl a, c (c1 + c2) and carotenoids, especially after 72 h, and the highest increase was observed in Chl a 15.37 ± 3.54 to 67.19 ± 6.18 mg g−1, as well as fucoxanthin 6.58 ± 1.25 to 26.79 ± 4.36 mg g−1 (Fig. 2).
The EPA biosynthesis pathway in P. tricornutum involves the transcription of six main gene products (four desaturation steps and one elongation step), which were quantified in this work, as presented in Fig. 3. Among the four desaturase genes analyzed (PTD5α, PTD5β, PTD6, and PTD15), in the first 12 h of exposure, the genes most negatively affected by high irradiance were PTD5α, PTD5β, and PTD15. The opposite was found at 24 h because the genes PTD6 and PTD15 presented a significant increase in expression. After 72 h of exposure at 750 μmol photons m−2 s−1, PTD5α was more than one-fold higher, and there was a significant reduction in transcription of PTD6 in the treatments. With regard to the elongase gene expression, there was no significant variation in the first 12 h of the highest irradiance; however, after 24 h of exposure at 750 μmol photons m−2 s−1 irradiance, a significant increase was found in ELO6_b1, ELO6_b2, and at 72 h, only the transcript level of ELO6_b1 was increased.
Figure 4 presents comparative data about the fatty acid profiles, the most significant change was observed for saturated fatty acids (SFAs), which increased 16.57% in the treatment with higher light intensity (750 μmol photons m−2 s−1), and the principal fatty acid was 16:0 (palmitic acid) with 36.72% of the FAMEs. The monounsaturated fatty acid (MUFAs) concentrations were similar above 30% of total fatty acids, but significant difference was observed for synthesis of 16:1 (palmitoleic acid).
Concerning PUFA biosynthesis, it was possible to verify the presence of C22:6 docosahexaenoic acid (DHA) in both treatments, and its content was not significantly altered by the light intensity. Regarding EPA, the variations in concentrations were 24.26% in the lower, and 18.54% (w/w) in the higher irradiance, a variation of 5.72% was detected, which suggests that its synthesis was negatively affected by excess light conditions. In contrast, an increase of 16.59% in PUFAs was observed at the lower light intensity (150 μmol photons m−2 s−1). Many fatty acid compounds of 18 carbon atoms were observed in low concentrations in both treatments, and a few were observed only in traces such as 15:0 pentadecanoic, 20:0 arachidonic, and 22:0 arachidic acid, as shown by Table S1.
Discussion
It is well known that changes in environmental conditions, like nitrogen restriction, can affect the modulation of lipid biosynthesis (Huerlimann et al. 2014; Remmers et al. 2017). Light plays a central role in photosynthetic organisms; therefore, in this work, the microalgae P. tricornutum was evaluated for its physiological response to different light intensities, 150 and 750 μmol photons m−2 s−1, in gene expression ratio, fatty acids biosynthesis, biomass production, and synthesis of photosynthetic pigments which were analyzed because of their high potential as nutraceuticals and therefore economic importance.
Phaeodactylum tricornutum is a marine organism well adapted to changes in light availably in environment that can rapidly acclimate to the established laboratory conditions, being resilient to sudden changes of irradiance due to photoacclimation mechanisms, variable light environments characteristic of turbulent waters (Nymark et al. 2009; Domingues et al. (2012)). It was possible to observe in Fig. 1. the minor differences in growth could be related to dilution made at 24 h of exposure, to avoid self-shading, which may have caused unbalanced growth. An increase in growth rate in high irradiance was also observed in previous reports such as Chrismadha and Borowitzka (1994) using 56 to 1712 μmol photons m−2 s−1 and Nogueira et al. (2015) using 50 a 600 μmol photons m−2 s−1.
Regarding the photosynthetic pigments such as chlorophyll a, c and fucoxanthin, it was observed a significant increase at lowest irradiance. The photosynthetic capacity depends on a specific range of light intensities on diatoms, which may provide different mechanisms and strategies for optimizing the photosynthetic rate for optimum exploitation of available photons (Heydarizadeh et al. 2017). Thus, it responds to irradiance conditions that include adjustments in the amount and proportion of chlorophyll a, c, and fucoxanthin, as well as the size of the photosynthetic unit, which alters the carbon assimilation capacity in photoadaptive response, also reported by Heydarizadeh et al. (2019) and Gundermann et al. (2019).
The high irradiance treatment was observed to inhibit the synthesis of photosynthetic pigments when photocatalytic stress light conditions occur, specialized chloroplast structures lose the ability to absorb excess excitation and a photoprotection mechanism, non-photochemical quenching (NPQ), responsible for the dissipation of light in the form of heat comes to action, which was reported by Nymark et al. (2009), Domingues (2012) and Remmers et al. (2017). However, the diadinoxanthin content presented the lowest variation between the irradiances used in this study, since this pigment is more related to the xanthophyll cycle (conversion of diadinoxanthin into diatoxanthin) and associated with protection of photo-oxidative damage (Domingues 2012; Lepetit et al. 2012). Buck et al. (2019) obtained genetic modification in P. tricornutum with range of NPQ and verified, based on physiological effect of high light, the diatoxanthin and proteins LHC1/2/3 act mediated inducible thermal energy dissipation.
The analysis conducted in this study allowed observing changes in the profile of fatty acids: SFA, MUFA, and PUFA. Qiao et al. (2016) studied lipid synthesis in P. tricornutum and did not find significant differences in growth and lipid content when altering several variables such as salinity (15, 20, 28, and 35 ppt) and light intensity (50, 100, and 150 μmol photons m−2 s−1) and only found significant differences under nitrogen starvation stress (1.24 mg L−1). Remmers et al. (2017) evaluated luminosities of 60 to 750 μmol photons m−2 s−1 and obtained the variation of 25.5 to 18%, respectively, of EPA in membrane lipid fraction in P. tricornutum cultivated in systems 16:8 h light/dark cycles and nitrogen starvation.
During photosynthesis light directly influences lipid synthesis, especially PUFAs that are essential for membrane synthesis and maintenance, as well as for cell division (Li et al. 2014). According to these authors, at low growth rates, an SFA (16:0) and MUFA (16:1) accumulation occurs, which is used to store the excess carbon accumulated during photosynthesis. Here, we showed that even with similar growth, the increase in light intensity increased SFA, while it decreased PUFAs. The importance of EPA in the structure of thylakoid membranes and photosynthesis dynamics can be confirmed by the high proportion of this fatty acid found in glycerolipids strongly associated with photosynthesis and plastid membranes. In P. tricornutum, according to Abida et al. (2015), EPA is the major fatty acid in monogalactosyl diacylglycerides (MGDG) and phosphatidylcholine (PC)—approximately 40% and 35% of the total associated fatty acids, respectively—two of the most abundant glycerolipids together with sulfoquinovosyl diacylglyceride (SQDG), which are responsible for maintaining membrane fluidity across all chloroplast membranes during the low light at photosynthesis.
We verified the gene expression for FA synthesis, and a summary of the results is presented in Fig. 5. EPA biosynthesis begins with 18:1 (oleic acid) and can occur in the ω-3 pathway with 18:3 (α-linolenic acid) or in the ω-6 pathway with 18:2 (linoleic acid), forming 18:4 (stearidonic acid) or 18:3 (gamma-linolenic acid), respectively. Although these lipid fractions were not detectable in FAME analyses, the findings demonstrate a possible limitation in the substrate availability in both pathways. We observed low expression of PTD6 at high irradiance for this enzyme, especially after 72 h of exposure, which could be associated with a decrease of EPA, indicating that the synthesis can be controlled at the beginning of the route. In a study with a similar approach with a variation of the N/P ratio, Lopes et al. (2019) verified that desaturases PTD6 and PTD5 alpha were modulated by changes in N/P ratio, but the EPA levels were unaffected by these conditions.
No difference between treatments was found on the PTD5β gene, with its product catalyzing the first step of unsaturation of 18:1 (oleic acid) and the last desaturation step of 20:4 (ETA) to 20:5 (EPA), while its ortholog PTD5α showed an increase in the transcript. The same was observed for gene ElO6_b1, which is responsible for elongation of C18:4 (stearidonic acid) into C20:4 and C18:3 into C20:3 in both alternative pathways. This enzyme was significantly affected by light, being more expressed at 72 h of exposure to high irradiance, while the same was not observed for ELO6_b2, which remained at the same transcription level, reinforcing the understanding of different mechanisms of regulation that contribute to the activity of this limiting step under different environmental conditions. These data show that the regulation of unsaturation and elongation is strongly related to adjustments to environmental stresses.
The methyl-end desaturase Delta 15 is responsible for interconversion between the two pathways, ω-6 and ω-3, and has been identified in many organisms, though not in mammals (Lee et al. 2016). For this reason, mammals must acquire essential fatty acids such as LA and ALA (ω-3) from foods or nutritional supplements, in order to synthesize longer chain ω-3 fatty acids such as EPA and DHA (Plourde and Cunnane 2007). In contrast, a significant difference was observed, which could be related to more efficient interconversion, in response to a change in the natural environment. This hypothesis is based on the fluidics of membranes at lower temperatures that are signaled by light for marine algae since EPA synthesis can be produced from lipid precursors from both the ω-3 and ω-6 pathways.
The genes responsible for the synthesis pathway were analyzed, and important differences were observed: Light significantly affected the lipids profile, while lower light intensity was observed to be more favorable to PUFA synthesis, especially EPA. For future research, it would be interesting to verify other genes, especially Phatr2_9316 and PtFAD6, and enzymes ∆9 and ∆12 (respectively), which are responsible for the formation of 18:1 and 18:2 and represent the starting point of EPA synthesis. It would be interesting to create conditions for overexpression of these genes and determine if it is possible to obtain more precursors of PUFA synthesis.
Conclusions
The physiological effect in P. tricornutum exposed for up to 72 h at two irradiances, 150 and 750 μmol photons m−2 s−1 was investigated. The highest production in carotenoids and pigments syntheses occurred at the lowest irradiance, with 67.19 and 26.79 mg g−1 of chlorophyll a and fucoxanthin, respectively.
Analysis of the gene expression responsible for the synthesis of PUFAs indicated a considerable variation in the transcript level of such genes, which hindered the association of their modulation. Furthermore, analysis of the formed products indicated that the lipid profile was strongly altered by this important environmental factor. The MUFA content was similar for both treatments. Under 750 μmol photons m−2 s−1 exposure, the SFA content was 45.6% and PUFAs 23.92%, in contrast to 29.04 and 40.51% under lower irradiance. A significant increase in PUFA synthesis was achieved in the 150 μmol photons m−2 s−1 light treatment, resulting in 5.73% increase in the most important fatty acid for this study, EPA.
Although microalgae have been shown to be suitable for many biotechnological applications, such potential could be substantially enhanced by maximizing the MUFA, SFA, and PUFA yield. In this way, the economic viability of industrial cultivation is expected to be clearly demonstrated.
The key conclusion is that a clear step to reach that objective has been taken in this study by showing that cultivation in 150 μmol photons m−2 s−1 constant luminosity is the most viable path to obtain fucoxanthin and EPA. Additionally, PTD6 expression could be correlated to EPA increase, so that it was possible to reroute P. tricornutum metabolism in order to increase the molecules of interest by altering environmental conditions.
References
Abida H, Dolch L-J, Meï C, Villanova V, Conte M, Block MA, Finazzi G, Bastien O, Tirichine L, Bowler C, Rébeillé F, Petroutsos D, Jouhet J, Maréchal E (2015) Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum. Plant Physiol 167:118–136
Adarme-Vega TC, Thomas-Hall SR, Lim DKY, Schenk PM (2014) Effects of long-chain fatty acid synthesis and associated gene expression in microalga Tetraselmis sp. Mar Drugs 12:3381–3398
Arao T, Yamada M (1994) Biosynthesis of polyunsaturated fatty acids in the marine diatom, Phaeodactylum tricornutum. Phytochemistry 35:1177–1181
Borges L, Caldas S, D’Oca MGM, Abreu PC (2016) Effect of harvesting processes on the lipid yield and fatty acid profile of the marine microalga Nannochloropsis oculata. Aquacult Rep 4:164–168
Buck JM, Sherman J, Bártulos CR, Serif M, Halder M, Henkel J, Falciatore A, Lavaud J, Gorbunov MY, Kroth PG, Falkowski PG, Lepetit B (2019) Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum. Nat Commun 10:4167
Carreto JI, Catoggio JA (1977) An indirect method for the rapid estimation of carotenoid contents in Phaeodactylum tricornutum: possible application to other marine algae. Mar Biol 40:109–116
Chrismadha T, Borowitzka MA (1994) Effect of cell density and irradiance on growth, proximate composition and eicosapentaenoic acid production of Phaeodactylum tricornutum grown in a tubular photobioreactor. J Appl Phycol 6:67–74
Dinamarca J, Levitan O, Kumaraswamy KG, Lun DS, Falkowsk P (2017) Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis. J Phycol 53:405–414
Domergue F, Lerchl J, Zähringer U, Heinz E (2002) Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem 269:4105–4113
Domingues N, Matos AR, Marques JS, Cartaxana P (2012) Response of the diatom Phaeodactylum tricornutum to photooxidative stress resulting from high light exposure. PLoS One 7:0038162
Guillard RRL, Ryther JH (1962) Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239
Gundermann K, Wagner V, Mittag M, Büchel C (2019) Fucoxanthin-chlorophyll protein complexes of the centric diatom Cyclotella meneghiniana differ in Lhcx1 and Lhcx6_1 content. Plant Physiol 179:1779–1795
Heydarizadeh P, Boureba W, Zahedi M, Huang B, Moreau B, Lukomska E, Couzinet-Mossion A, Wielgosz-Collin G, Martin-Jézéquel V, Bougaran G, Marchand J, Schoefs B (2017) Response of CO2-starved diatom Phaeodactylum tricornutum to light intensity transition. Phil Trans R Soc B 372. https://doi.org/10.1098/rstb.2016.0396
Heydarizadeh P, Veidl B, Huang B, Lukomska E, Wielgosz-Collin G, Couzinet-Mossion A, Bougaran G, Marchand J, Schoefs B (2019) Carbon orientation in the diatom Phaeodactylum tricornutum: The effects of carbon limitation and photon flux density. Front Plant Sci https://doi.org/10.3389/fpls.2019.00471
Huerlimann R, Steinig EJ, Loxton H, Zenger KR, Jerry DR, Heimann K (2014) Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO). Gene 545:36–44
Jeffrey SW, Humphrey GF (1975) New spectrophotometric equation for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanz 167:191–194
Lee JM, Lee H, Kang S, Park WJ (2016) Fatty acid desaturases, polyunsaturated fatty acid regulation, and biotechnological advances. Nutrients 8(1):E23
Lemões JS, Sobrinho R, Farias SP, Moura RR, Primel EG, Abreu PC, Martins AF, D’Oca MGM (2016) Sustainable production of biodiesel from microalgae by direct transesterification. Sust Chem Pharm 3:33–38
Lepetit B, Goss R, Jakob T, Wilhelm C (2012) Molecular dynamics of the diatom thylakoid membrane under different light conditions. Photosynth Res 111:245–257
Li HY, Lu Y, Zheng JW, Yang WD, Liu JS (2014) Biochemical and genetic engineering of diatoms for polyunsaturated fatty acid biosynthesis. Mar Drugs 12:153–166
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408
Lopes RG, Cella H, Mattos JJ, Marques MRF, Soares AT, Antoniosi NRF, Derner RB, Rörig LR (2019) Effect of phosphorus and growth phases on the transcription levels of EPA biosynthesis genes in the diatom Phaeodactylum tricornutum. Braz J Bot 42:13–22
Luchtman DW, Song C (2013) Cognitive enhancement by omega-3 fatty acids from childhood to old age: findings from animal and clinical studies. Neuropharmacology 64:550–565
Marquez A, Lodeiros C, Loor A, Revilla J, Da Costa F, Sonnenholzner S (2019) Microalgae diet for juveniles of Spondylus limbatus. Aquacult Int 27:323–335
Mühlroth A, Li K, Røkke G, Winge P, Olsen Y, Hohmann-Marriott MF, Vadstein O, Bones AM (2013) Pathways of lipid metabolism in marine algae, co-expression network, bottlenecks and candidate genes for enhanced production of EPA and DHA in species of Chromista. Mar Drugs 11:4662–4697
Neumann U, Louis S, Gille A, Derwenskus F, Schmid-Staiger U, Briviba K, Bischoff SC (2018) Anti-inflammatory effects of Phaeodactylum tricornutum extracts on human blood mononuclear cells and murine macrophages. J Appl Phycol 30:2837–2846
Nogueira DPK, Silva AF, Araújo OQF, Chaloub RM (2015) Impact of temperature and light intensity on triacylglycerol accumulation in marine microalgae. Biomass Bioenergy 72:280–287
Nymark M, Valle K, Brembu T, Hancke K, Winge P, Andresen K, Johnsen G (2009) An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum Tricornutum. PLoS One 4:e7743
Nymark M, Sharma A, Sparstad T, Bones AM, Winge P (2016) A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6:24951
Peng J, Yuan J-P, Wu C-F, Wang J-H (2011) Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: metabolism and bioactivities relevant to human health. Mar Drugs 9:1806–1828
Peng KT, Zheng CN, Xue J, Chen XY, Yang WD, Liu JS, Bai W, Li HY (2014) Delta 5 fatty acid desaturase upregulates the synthesis of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum. J Agric Food Chem 62:8773–8776
Plourde M, Cunnane SC (2007) Extremely limited synthesis of long chain polyunsaturates in adults: implications for their dietary essentiality and use as supplements. Appl Physiol Nutr Metab 32:619–634
Qiao H, Cong C, Sun C, Li B, Wang J, Zhang L (2016) Effect of culture conditions on growth, fatty acid composition and DHA/EPA ratio of Phaeodactylum tricornutum. Aquaculture 452:311–317
Remmers IM, Martens DE, Wijffels RH, Lamers PP (2017) Dynamics of triacylglycerol and EPA production in Phaeodactylum tricornutum under nitrogen starvation at different light intensities. PloS One 12(4):e0175630
Shah MR, Lutzu GA, Alam A, Sarker P, Chowdhury MAK, Parsaeimehr A, Liang Y, Daroch M (2018) Microalgae in aquafeeds for a sustainable aquaculture industry. J Appl Phycol 30:197–213
Siaut M, Heijde M, Mangogna M, Montsant A, Coesel S, Allen A, Manfredonia A, Angela F, Bowler C (2007) Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406:23–35
Strickland JDH, Parsons TR (1972) A practical handbook of seawater analysis, 2nd edn. Bull. Fish. Res Bd Can. 167:310 pp
Ulmann L, Blanckaert V, Mimouni V, Andersson MX, Schoefs B, Chenais B (2017) Microalgal fatty acids and their implication in health and disease. Mini-Rev Med Chem 17:1112–1123
Wang H, Zhang Y, Chen L, Liu T (2018) Combined production of fucoxanthin and EPA from two diatom strains Phaeodactylum tricornutum and Cylindrotheca fusiformis cultures. Bioprocess Biosyst Eng 41:1061–1071
Zhu BH, Tu CC, Shi HP, Yang GP, Pan KH (2017) Overexpression of endogenous delta-6 fatty acid desaturase gene enhances eicosapentaenoic acid accumulation in Phaeodactylum tricornutum. Process Biochem 57:43–49
Zulu NN, Zienkiewicz K, Vollheyde K, Feussner I (2018) Current trends to comprehend lipid metabolism in diatoms. Prog Lipid Res 70:1–16
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM1
(DOCX 27 kb)
Rights and permissions
About this article
Cite this article
Conceição, D., Lopes, R.G., Derner, R.B. et al. The effect of light intensity on the production and accumulation of pigments and fatty acids in Phaeodactylum tricornutum. J Appl Phycol 32, 1017–1025 (2020). https://doi.org/10.1007/s10811-019-02001-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10811-019-02001-6