Lipidomic signature of the green macroalgae Ulva rigida farmed in a sustainable integrated multi-trophic aquaculture

Ulva species, green macroalgae, are widely distributed across the globe, being one of the most heavily traded edible seaweeds. Nonetheless, although this genus has been largely used in scientific studies, its lipidome remains rather unexplored. The present study sheds light over the lipid profile of Ulva rigida produced in a land-based integrated multi-trophic aquaculture (IMTA) system using liquid chromatography coupled to high-resolution mass spectrometry for molecular lipid species identification. The lipidome of U. rigida revealed the presence of distinct beneficial n-3 fatty acids for human health, namely alpha-linoleic acid (ALA) and docosapentaenoic acid (DPA). A total of 87 molecular species of glycolipids, 58 molecular species of betaine lipids, and 57 molecular species of phospholipids were identified in the lipidome of U. rigida including some species bearing PUFA and with described bioactive properties. Overall, the present study contributes to the valorization and quality validation of sustainably farmed U. rigida.


Introduction
Edible macroalgae are a good source of beneficial compounds for human health that display distinct functional properties that stimulate interest to number of high-value chains (e.g., medical, nutraceutical, and cosmeceutical) (Holdt and Kraan 2011;Leal et al. 2013;Abreu et al. 2014;Rajauria 2015;Roohinejad et al. 2016). Ulva spp. have long been listed in FAO as one of the main macroalgae for commercial use (Naylor 1976). These popular green seaweeds can be used fresh, dried, or in liquid extracts, either for direct or processed consumption worldwide (McHugh 2003;Barriga et al. 2017). Popularly known in the human food market as sea lettuce, Ulva belongs to class Ulvophyceae and can be found in marine and brackish waters, being widely distributed across the globe. Ulva species are well adapted to aquaculture production and can be successfully cultured by using an integrated multitrophic aquaculture (IMTA) framework (Bolton et al. 2008;Msuya and Neori 2008;Marinho et al. 2013;Shpigel et al. 2017). This innovative and sustainable culture approach mimics the natural ecosystem of species from different trophic levels, associating the production of fed species (e.g., finfish) with other extractive organisms, namely marine invertebrates and/or algae, that incorporate organic and inorganic compounds resulting from the metabolism of fed species, as well as from uneaten feed. Overall, IMTA promotes a balanced production framework that is environmentally sustainable and viable from an economic point of view (Barrington et al. 2009;Chopin et al. 2012). The culture of seaweeds under an IMTA approach allows the removal of excess nutrients, namely phosphorus and nitrogen, from wastewater (Neori 2008;Lawton et al. 2013), while enhancing quality and stability of seaweeds biomass and their biochemical profile (Abreu et al. 2014).
Ulva species are consumed directly as Bsea vegetables^and used as a food and feed ingredient. They are also recognized as an important source of valuable polysaccharides (such as ulvans) and oligosaccharides rich in functional groups that bind important microelements for human and animal nutrition (Lahaye and Robic 2007;Stengel et al. 2011;Berri et al. 2016;Wijesekara et al. 2017). However, to date, the lipid profile of Ulva spp. is still poorly studied at molecular level and few articles have reported their lipid characterization (Takahashi et al. 2002;Rozentsvet and Nesterov 2012;Ragonese et al. 2014), with most studies solely describing their fatty acid (FA) profile (van Ginneken et al. 2011;Ragonese et al. 2014;Kendel et al. 2015). While lipids may represent from 1 to 3% of the whole algal dry matter, they do display an important nutritional value, with emphasis into polyunsaturated fatty acids (PUFAs) from the n-3 (e.g., alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid) and n-6 (linoleic acid, gamma-linolenic acid, and arachidonic acid) (Kumari et al. 2010). As essential PUFAs are not synthesized by humans, they need to be obtained through diet to provide energy and other health benefits (e.g., reduce the risk of coronary disease and blood cholesterol) (Ginzberg et al. 2000;Simopoulos 2008;Kendel et al. 2015). Furthermore, PUFAs are also precursors of important mediators that play a key-role in inflammation and regulation of immunity (Calder 2001). These biomolecules mostly occur in their esterified form in polar lipids, namely phospholipids (PLs) and glycolipids. This feature enhances the nutritional properties of these classes of polar lipids. Additionally, glycolipids isolated from macroalgae have already been described as displaying bioactive proprieties, namely antitumoral (Ohta et al. 1998;Eitsuka et al. 2004), antiinflammatory (Banskota et al. 2013(Banskota et al. , 2014, antimicrobial (El Baz et al. 2013;Parveez Ahamed et al. 2017), and antiviral activity (Wang et al. 2007).
The potential added value of macroalgal polar lipids has received a new momentum with the advent of mass spectrometrybased approaches which have already been employed to provide an in-depth characterization of lipidomic signatures of different macroalgae, namely Chondrus crispus , Codium tomentosum, Gracilaria sp., and Porphyra dioica (da Costa et al. 2015(da Costa et al. , 2017(da Costa et al. , 2018. The aim of the present study is to analyze the lipidome of Ulva rigida from a land-based IMTA system using liquid chromatography-high-resolution mass spectrometry-based approach. The data presented will contribute to promote ongoing efforts in the responsible, controlled, and sustainable production of high-value macroalgae.

Biomass
The fresh biomass of Ulva rigida was produced by ALGAplus (production site located at Ria de Aveiro coastal lagoon, mainland Portugal, 40°36′ 43″ N, 8°40′ 43″ W) in an IMTA system, harvested in November 2016 (batch U1.4616.L). The ALGAplus IMTA system is composed of fish organic certified production units (seabass and seabream) and the seaweed land-based tank system. The water flows from the fish units, to the seaweed tanks, and then to the exit channel that discharges clean water into the coastal lagoon. Seaweeds are cultivated using exclusively water input from the fish farm (nothing is added to the water). Stocking densities and water flows are manipulated in each season to achieve optimal biomass yields and/or specific biomass quality traits (i.e., chemical composition, color). After being harvested, all biological samples were cleaned to remove epiphytic foreign matters, washed with seawater that is sequentially filtered up to 25 μm, and then sterilized by UV and Ozone treatment. The samples were then frozen at − 80°C, freeze-dried, and stored at − 80°C until lipid extraction.

Moisture and ash determination
Moisture was determined by drying freeze-dried samples (250 mg × five replicates) in crucibles on an oven at 105°C for 15 h. For ash determination, the dried biomass in the crucibles was first pre-incinerated for 20 min using a heating plate and then placed in a muffle furnace at 575°C for 6 h.

Nitrogen determination and protein estimation
Nitrogen content of freeze-dried samples (2 mg × five replicates) was obtained by elemental analysis on a Leco Truspec-Micro CHNS 630-200-200 elemental analyzer at combustion furnace temperature 1075°C and afterburner temperature 850°C. Nitrogen was detected using thermal conductivity. The protein content was estimated from the nitrogen determination using two nitrogen-protein conversion factors, 6.25 and 5 (Angell et al. 2016).

Total lipid extraction
Freeze-dried samples were homogenized in a mortar and pestle to obtain small-sized flakes. A biomass of 250 mg of macroalgae was mixed with 2.5 mL of CH 3 OH and 1.25 mL of CHCl 3 in a glass PYREX tube and homogenized by vortexing for 2 min. After incubation in ice on rocking platform shaker (Stuart equipment, Bibby Scientific, UK) for 2.5 h, the mixture was centrifuged (Selecta JP Mixtasel, Spain) for 10 min at 2000 rpm and the organic phase was collected in a new glass tube. The biomass residue was re-extracted twice with 2 mL of MeOH and 1 mL of CHCl 3 . To wash the lipid extract and induce phase separation, 2.3 mL of Milli-Q water was added to the final organic phase, following by centrifugation for 10 min at 2000 rpm. The organic lower phase was collected in a new glass tube, dried under nitrogen stream. Lipid extracts were then transferred to amber vials, dried again, weighed, and stored at − 20°C. Lipid content was estimated as dry weight percentage.

Fatty acid analysis by gas chromatography-mass spectrometry
Fatty acid methyl esters (FAMEs) were prepared using a methanolic solution of potassium hydroxide (2.0 M) . A volume of 2 μL of hexane solution containing FAMEs was analyzed by gas chromatography-mass spectrometry (GC-MS) on a GC system (Agilent Technologies, USA) equipped with a DB-FFAP column with the following specifications: 30 m long, 0.32 mm internal diameter, and 0.25 μm film thickness (J & W Scientific, USA). The GC equipment was connected to an Agilent 5973 Network Mass Selective Detector operating with an electron impact mode at 70 eV and scanning the range m/z 50-550 in a 1 s cycle in a full scan mode acquisition. The oven temperature was programmed from an initial temperature of 80°C for 3 min, a linear increase to 160°C at 25°C min −1 , followed by linear increase at 2°C min −1 to 210°C, then at 30°C min −1 to 250°C, standing at 250°C for 10 min. The injector and detector temperatures were 220 and 280°C, respectively. Helium was used as the carrier gas at a flow rate of 1.4 mL min −1 . FA identification was performed considering the retention times and MS spectra of FA standards (Supelco 37 Component Fame Mix, Sigma-Aldrich), and by MS spectrum comparison with chemical databases (Wiley 275 library and AOCS lipid library). The relative amounts of FAs were calculated by the percent area method with proper normalization, considering the sum of all areas of identified FAs.

Lipid extract fractionation
Isolation of polar lipids from pigments was performed using a modification of Pacetti's method (da Costa et al. 2017). A sample of lipid extract (5 mg) was dissolved in 600 μL of chloroform and transferred to a glass column with 500 mg of silica gel (40-60 μm, 60 Å, Åcros Organics) followed by sequential elution with 5 mL of chloroform, 12 mL of ether diethyl ether:acetic acid (98:2), 7 mL of acetone:methanol (9:1 v/v), and 10 mL of methanol. Fractions 1 and 2, corresponding to neutral lipids and pigments, were discarded. Fractions 3 and 4, rich in glycolipids and in phospholipids plus betaines, respectively, were recovered, dried under nitrogen, and stored at − 20°C prior to analysis by Hydrophilic interaction liquid chromatography mass spectrometry HILIC-ESI-MS.

Hydrophilic interaction liquid chromatography mass spectrometry
Lipid extracts and fraction were analyzed by hydrophilic interaction liquid chromatography HILIC (Ascentis Si column, 15 cm × 1 mm, 3 μm, Sigma-Aldrich) on a high-performance LC (HPLC) system (Thermo Scientific AccelaTM) with a autosampler coupled online to a Q-Exactive mass spectrometer with Orbitrap technology. Mobile phase A consisted of 25% water, 50% acetonitrile, and 25% methanol, with 1 mM ammonium acetate in relation to the water volume, and mobile phase B consisted of 60% acetonitrile and 40% methanol, with the same amount of ammonium acetate in mobile phase A. The solvent gradient, flow rate through column, and conditions used for acquisition of full scan LC-MS spectra and LC-MS/MS spectra in both positive and negative ion modes were the same as previously described (da Costa et al. 2015;Melo et al. 2015). Initially, 0% of mobile phase A was held isocratically for 8 min, followed by a linear increase to 60% of mobile phase A within 7 min, and a maintenance period of 15 min, returning to the initial conditions in 10 min. A volume of 5 μL of each sample, containing 10 μg (10 μL) of lipid extract in CHCl 3 , 4 μL of phospholipid standards mix (dMPC-0.02 μg, dMPE-0.02 μg, NPSM-0.02 μg, LPC-0.02 μg, dPPI-0.08 μg, dMPG-0.012 μg, dMPS-0.04 μg), and 86 μL of eluent B, was introduced into the Ascentis Si column HPLC Pore column (15 cm × 1 mm, 3 μm, Sigma-Aldrich) with a flow rate of 40 μL min −1 at 30°C. The mass spectrometer with Orbitrap technology was operated in simultaneous positive (electrospray voltage 3.0 kV) and negative (electrospray voltage − 2.7 kV) modes with high resolution with 70,000 and AGC target of 1 × 10 6 , the capillary temperature was 250°C, and the sheath gas flow was 15 U. In MS/MS experiments, a resolution of 17,500 and AGC target of 1 × 10 5 was used and the cycles consisted in one full scan mass spectrum and ten data-dependent MS/MS scans were repeated continuously throughout the experiments with the dynamic exclusion of 60 s and intensity threshold of 1 × 10 4 . Normalized collision energy (CE) ranged between 25, 30, and 35 eV. Data acquisition was performed using the Xcalibur data system (V3.3, Thermo Fisher Scientific). The identification of molecular species of polar lipids was based on the assignment of the molecular ions observed in LC-MS spectra, typical retention time, mass accuracy, and LC-MS/ MS spectra interpretation that allows to confirm the identity of the polar head group and the fatty acyl chains for most of the molecular species.

Results
The total lipid content of the U. rigida was estimated by gravimetry of the lipid extracts. Also, samples were analyzed for the contents of moisture and ash, proteins, and carbohydrates and other compounds (estimated by difference). The mean moisture content (expressed as percentage of freeze-dried sample weight) of U. rigida was 6.41 ± 0.84, which was Although the factor 6.25 is the most commonly used indirect nitrogen-to-protein conversion factor, studies have been shown that the protein content of seaweed is overestimated by applying factor 6.25 (Hardouin et al. 2016). Angell et al. (2016) proposed the use of a universal nitrogen-to-protein conversion factor of 5 for determination of the protein content of seaweeds. Thus, both factors were used. Using factor 6.25 for protein estimation, the protein content (%DW) was 17.75 ± 0.492, and the content of carbohydrates and other compounds (%DW) was 53.25. Considering factor 5, the protein content decreased to 14.20 ± 0.393, while the content of carbohydrates and other compounds increased to 56.80. The fatty acids (FAs) profile of U. rigida revealed the presence of saturated FAs (SFAs) such as 14:0, 16:0, 18:0, and 22:0; monounsaturated FAs (MUFAs) such as 16:1 and 18:1; and PUFAs such as 16:4, 18:3, 18:4, 20:4, 20:5, and 22:5, as detailed in Table 1. The FA profile showed 16:0 and 18:0 as the most abundant with relative abundance of 43.41 and 19.30%, respectively. It is also noteworthy the abundance of the PUFAs 16:4 (n-3) (3.76%), 18:3 (n-3) (4.45%), 18:4 (n-3) (8.82%), and 22:5 (n-3) (3.76%).
Polar lipid profile evaluated by HILIC-LC-MS and HILIC-LC-MS/MS allowed the identification at molecular level of glycolipids, betaine lipids, and phospholipids in U. rigida. This lipidomic approach allowed the identification, in the case of glycolipids, of the acidic glycolipid sulfoquinovosyl diacylglycerol (SQDG) and it lyso form sulfoquinovosyl monoacylglycerol (SQMG), as well as the neutral glycolipid d i g a l a c t o s y l d i a c y l g l y c e r o l ( D G D G ) a n d monogalactosyldiacylglycerol (MGDG). SQDGs and SQMGs were identified as negative [M − H] − ions in the LC-MS spectra. Overall, 20 molecular species of SQDG and 5 molecular species of SQMG (Table 2 and Fig. 1) were identified. The most abundant SQDG was assigned as SQDG (34:1) at m/z 819.5, identified as SQDG (18:1/16:0), while the most abundant SQMG was detected at m/z 555.3 and corresponded to SQMG (16:0) (Fig. 1). Typical fragmentation of SQMG and SQDG species observed in LC-MS/MS spectra as [M − H] − ions showed the product ion at m/z 225.0, corresponding to the anion of the sulfoquinovosyl polar head group that confirmed the presence of sulfoglycolipids, as seen in the LC-MS/MS spectra of SQMG at m/z 555.3 (Fig. 1b) and SQDG at m/z 819.5 (Fig. 1d). Furthermore, product ions corresponding to the neutral loss of fatty acyl chains as carboxylic acid (RCOOH) can be identified and confirm the composition of fatty acyl chains. SQMG species exhibit only one neutral loss of one fatty acid R 1 COOH (El Baz et al. 2013;da Costa et al. 2015;Melo et al. 2015). LC-MS/MS spectrum of SQMG (16:0) at m/z 555.3 shows the neutral loss of palmitic acid (− 16:0 R 1 COOH, 256 Da) that leads to the formation of the product ion at m/z 299.0 (Fig.  1b). LC-MS/MS spectrum at m/z 819.5, corresponding to SQDG (18:1/16:0), shows the loss of two fatty acyl chains R 1 COOH and R 2 COOH, that correspond to the neutral loss of 18:1 RC 1 OOH (− 282 Da) and the neutral loss of palmitic acid 16:0 R 2 COOH (− 256 Da) with formation of the product ions at m/z 537.3 and 563.3, respectively (Fig. 1d).  (Table 3 and Fig. 2). The representative LC-MS spectra of MGDG and DGDG classes are shown in Fig. 2, as well as the LC-MS/MS spectra of the most abundant species of each class. The predominant MGDG were detected at m/z 760.5. The DGDG were similarly predominate at m/z 932.6 and 936.7, representative spectrum in Fig. 2 (Table 5).
The LC-MS/MS spectra of PG (Fig. 4a) and LPG species allowed to confirm their polar head by the presence of the product ion at m/z 171.0, corresponding to [C 3 H 7 O 2 OPO 3 H] − . On the other hand, the polar head of PI (Fig. 4b) and LPI is observed at m/z 241.0, corresponding to an inositol-1,2-cyclic phosphate anion (C 6 H 10 O 5 PO 3 ] − . The carboxylate anions R 1 COO − and R 2 COO − allowed the identification of fatty acyl chains (Murphy 2015

Discussion
To the best knowledge of the authors, the present study represents the first in-depth characterization of lipidomic signature of the green macroalga U. rigida. Ulva rigida screened in the present work was produced in a land-based IMTA system, with this culture approach being considered as a sustainable and environmentally friendly approach to produce seaweeds and provide high-grade safe biomass. When compared to the harvesting of seaweeds from the wild, this production system has as main advantages the production of high biomass loads under controlled and replicable conditions, a less variable biochemical profile that allows product standardization, as well as the implementation of mandatory traceability protocols for seaweeds and seaweed-based-products targeting premium markets (Ridler et al. 2007;Chopin et al. 2012). Fatty acids profile identified was similar with that reported for the same species (Ak et al. 2014) and for other species belonging to the genus Ulva, namely Ulva lactuca, Ulva rotundata, Ulva clathrata, and Ulva intestinalis (Fleurence et al. 1994;Peña-Rodríguez et al. 2011;van Ginneken et al. 2011;Rozentsvet and   Nesterov 2012). As the PUFAs reported in the present study are essential FAs for humans, U. rigida can be an affordable dietary source of these FAs (Li et al. 2009;Cottin et al. 2011). There are several studies that defend an ideal n-6/n-3 ratio. While n-3 PUFAs exhibit anti-inflammatory and antioxidant activity, improve the cardiac system, and prevent breast cancer (Mozaffarian et al. 2005;Siriwardhana et al. 2012;Fabian et al. 2015), n-6 PUFAs tend to promote tumor growth and inflammatory processes (Patterson et al. 2011). One of the important dietary factors in the obesity prevention is a balanced n-6/n-3 ratio of 1-2/1 (Simopoulos 2016). Therefore, the consumption of n-6 FAs should be lower than n-3, in order to avoid several diseases including depressive disorder (Okuyama et al. 1997;Husted and Bouzinova 2016). In addition, lower n-6/n-3 ratio was associated with decreased risk of breast cancer in women (Simopoulos 2008). In this context, U. rigida had a relative abundance of n-6 and n-3 PUFAs of 1.51 and 21.77%, respectively. Therefore, its n-6/n-3 ratio is lower than 1, highlighting the potential health-promoting properties of this macroalgae for human consumption. Although n-6/n-3 ratios are known to vary between species and growth condition, to the authors best knowledge, U. rigida farmed using a sustainable land-based IMTA approach described in the present study displayed the lowest n-6/n-3 ratio report so far for Ulva spp. (van Ginneken et al. 2011;Kendel et al. 2015). This finding confirms the added value of algal biomass originating from land-based IMTA, as a higher contents in n-3 fatty acids are commonly associated with health-promoting benefits for consumers (Simopoulos 2002).
Identified FAs are esterified into lipid molecules such as glycolipids, betaine lipids, and phospholipids (PLs). The glycolipids detected include sulfolipids and galactolipids which together represented the most abundant structural compounds of chloroplast membranes (Hölzl and Dörmann 2007) with up to 87 molecular species being identified in U. rigida.
There are several studies that demonstrated glycolipids bioactivity from different algae species, such as antiviral, antibacterial, and antitumoral activity (Plouguerné et al. 2014;Blunt et al. 2016). Wang et al. (2007) described the antiviral activity attributed to SQDG (32:0) from the green macroalgae Caulerpa racemosa. Furthermore, El Baz et al. (2013) analyzed the SQMG (16:0) as antitumor and antimicrobial activity. Other authors demonstrated the inhibitory effect of SQDG and DGDG from the brown macroalgae Sargassum horneri suggesting the use of these compounds like chemotherapy agents (Hossain et al. 2005). It is also reported that seaweeds with an abundant presence of PUFAs in their composition proved to display anti-inflammatory activity by inhibiting nitric oxide release by macrophages (Banskota et al. 2013;Lopes et al. 2014). Betaine lipids (DGTS and MGTS) represent a group of polar lipids low studied to date and few studies have characterized their profile in seaweeds (da Costa et al. 2015(da Costa et al. , 2017Melo et al. 2015). Some species of DGTS identified in U. rigida have already been reported in green microalgae like Chlamydomonas reinhardtii and chlorarachniophytes (Vieler et al. 2007;Roche and Leblond 2010). It has been suggested that DGTS has the same function as PC due to their similar zwitterionic structure. Moreover, they are interchangeable with each other in their roles within the cell (Riekhof et al. 2005). Organisms that contain a high level of DGTS display either an absence of PC or its presence is very low (Dembitsky and Rezanka 1995;Kunzler and Eichenberger 1997). Furthermore, van Ginneken et al. (2017) revealed that Ulva sp. uses a mechanism rarely reported in euckaryotes, as it applies the biochemical pathway to produce DGTS that can replace PC in seaweed cell wall (Klug and Benning 2001). It was suggested that the high DGTS/PC ratio occur communlyin in species of the genus Ulva.
Regarding PLs, their beneficial effects have been studied since the early 1900s (Küllenberg de Gaudry et al. 2012). The positive effect of PLs is supported by several studies that showed an improvement of the pharmacokinetics of some drugs when associated with PLs compounds, and a reduction of side effects of some drugs when administered together, namely indomethacin (NSAID) (Dial et al. 2006;Lichtenberger et al. 2009). Their cytoprotective effects and anti-fibrogenic potential have already been highlighted (Gundermann et al. 2011). Moreover, PLs from marine organisms have shown a remarkable effect in the regulation of the blood lipid profile in patients suffering from hyperlipidemia (Bunea et al. 2004). PLs beneficial dietary effect is the result of their interaction with cellular membranes influencing a vast number of signaling processes and also the effect of their fatty acid composition. The great advantage of these molecules is related with the ability of their esterified n-3 FAs to compensate n-3 FA deficiency in a more efficient way than other n-3 FA supplements (e.g., as triacylglycerides or as free FAs). Thus, PLs from foodstuff are major supplies of n-3 PUFAs for living systems (Jannace et al. 1992). Furthermore, the antioxidant potential of PG found in U. rigida could be explored (Banskota et al. 2014).
Traditionally, the study of algal lipids has targeted fatty acids analysis through GC-MS or GC-FID (Marshall et al. 2002). However, the overall information acquired through these techniques is limited and solely refers to fatty acids, which in living systems are mostly linked to polar lipids. In the last decade, with the advent of mass spectrometry, the commercialization of new devices with higher sensitivity,  resolution, and sample screening speed, such as Orbitrap ant Q-TOF instruments, allowed to gain a more in-depth knowledge of lipids. The used of liquid chromatography (LC) online with mass spectrometry is nowadays an advanced and promising approach to study lipids in living systems. The LC-MS platforms allows to identify and quantify molecular structural details in one single run over very short periods of time (Maciel et al. 2016). In 1 LC-MS run, more than 200 lipid species from different lipid classes are routinely identified and quantified. Lipid species identification is based on the ions in MS and, in the case of high-resolution MS, through confirmation of mass accuracy. The structural details are confirmed by MS/MS data of each molecular species, namely through the analysis of typical ion fragments. In recent years, this lipidomic approach has been successfully used to unravel the lipidome of seaweeds (da Costa et al. 2015(da Costa et al. , 2017(da Costa et al. , 2018Melo et al. 2015) and has become a powerful tool to screen for high value lipid species with potential biotechnological applications.

Conclusion
The mass spectrometry-based approach employed in the present study allowed the identification of 202 molecular species of polar lipids shared between glycolipids, betaine lipids, and phospholipids, most of them confirmed by their fatty acids composition. The knowledge of lipid composition of U. rigida from a sustainable land-based IMTA system comes to inspire future studies of valorization of this seaweed, as its aquaculture production under controlled conditions will continue to increase as it offers consumers a safer and more standardized product, from an organoleptically (industry communication) and biochemical point of view. Moreover, the present study may also serve to stimulate the consumption of U. rigida produced under controlled conditions, as its lipidome displays a number of molecular species with beneficial bioactive properties that may also foster new biotechnological applications. project GENIALG-Genetic diversity exploitation for innovative macro-alga biorefinery (ANR-15-MRSE-0015) funded by European Union's Horizon 2020 Framework Programme. Tânia Melo is grateful for her Post-Doc grant (BPD/UI 51/5388/2017) funded by RNEM. This work is a contribution of the Marine Lipidomics Laboratory and was also supported by the Integrated Programme of SR&TD BSmart Valorization of Endogenous Marine Biological Resources Under a Changing Climate( Centro-01-0145-FEDER-000018), co-funded by Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund.