Marine ecosystems represent a rich source of macromolecules with unique physico-chemical characteristics . In this sense, polysaccharides extracted from marine macroalgae are receiving increasing attention due to their diversity, biocompatibility and structural features not found in any other organism .
Species of the genus Ulva are the most abundant and cosmopolitan macroalgae in the Chlorophyta Division being able to adapt across diverse geo-climatic conditions with high productivity and opportunistic growth. Ulva cultivation is increasing worldwide due to its potential as functional foods, feed and biofuel  . Although polysaccharides from the red (carrageenan and agar) and brown (alginate) macroalgae have been used in the food industry, polysaccharides from green macroalgae remain largely unexploited. The main polysaccharide of Ulva species is ulvan, corresponding to 29% of dry weight with a promising technological application.
Ulvan is homogeneously distributed within the intercellular space and in the fibrillar wall   being responsible for maintaining the osmolar stability and protecting the thallus from marine bacterial attack  . Ulvan is composed of variable amounts of rhamnose, glucuronic acid, iduronic acid, xylose and sulfate  . This sulfated heteropolysaccharide is built on sequences of two major repeating disaccharides unities designated as ulvanobiuronic acid 3-sulfate type A (A3s) [→4)-β-D-GlcA-(1 → 4)-α-L-Rha 3S-(1→] and type B (B3s) [→4)-α-L-IdoA-(1 → 4)-α-L-Rha 3s(1→]  . Minor sulfated residues with xylose (O-2 sulfated or not) denominated ulvanobiose (U3s) [→4)-β-d-GlcA-(1-2)-α-D-Xyl-(1→] can also occur in place of uronic acids .
With the growing interest in novel and renewable polymers, ulvan has drawing attention with studies showing its potential as antioxidant  , antiviral  , anticancer   among others   . The presence of the remarkable rare sugars, rhamnose and iduronic acid, similar to mammalian glycosaminoglycans   single out ulvan from other algal polysaccharides. L-rhamnose used in a variety of anti-aging cosmetics   is specifically recognized by a number of mammalians lectins . Iduronic acid, which has never been identified in algal polysaccharides   is required in the synthesis of heparin analogs being used against respiratory syncytial virus infection and antithrombotic activities  . Currently, this substance is obtained through several steps that could be avoided using ulvan  .
Although the biotechnological applications of ulvan are promising, structural variations may occur due to ecophysiological factors acting on Ulva  . The commercial use of polysaccharides requires ulvan with predictive structure and functional properties, which could be obtained by the controlled cultivation of Ulva. To determine the potential of cultivated Ulva for ulvan production in this work we characterized (FTIR and NMR) the ulvan extracts from Ulva fasciata Delile (Chlorophyta) after in vitro biomass cultivation in nutrient enriched water and compared it against ulvan from biomass collected in an oligotrophic natural environment, to enhance potential structural differences.
2. Material and Methods
2.1. Algal Material
Healthy thalli of Ulva fasciata were collected in the intertidal zone at Prainha Beach, Arraial do Cabo/RJ Brazil (22˚57'40"S/042˚01'13"W) rinsed with local seawater and transported to laboratory inside coolers. Individuals were cleaned with distilled water for further removal of sediment and macroscopic epibionts. The species-level identification as U. fasciata was determined by molecular studies (barcoding using tuƒA markers) . Voucher specimens were deposited in the Institute of Bioscience Herbarium, at the University of São Paulo, Brazil (SPF-57877). Part of the fresh biomass (SEA) was oven dried at 50˚C until constant weight and stored in desiccator until ulvan extraction. The remaining material was used in the cultivation experiment (CULT). In sequence biomass from natural environment and cultivation experiment (SEA and CULT, respectively) were used for ulvan extraction (Figure 1).
2.2. Ulva Cultivation
To ensure that all individuals presented comparable initial physiological conditions, U. fasciata thalli underwent a seven-day acclimatization period to the laboratory conditions followed by a three-day starvation period prior to the cultivation. For acclimatization, individuals were kept in sterilized seawater enriched with von Stosch culture medium  in a temperature-controlled room at 24.0 ± 1.0˚C, 70 μmol photons m−2∙s−1 photosynthetically active radiation (PAR) and 12 hours light photoperiod. For starvation, individuals weighing approximately 3.5 g were placed in 2.8 L Erlenmeyer flasks filled UV-sterilized natural seawater (nutrient concentration—0.5 µM NH4, 0.03 µM NO2, 0.41 µM NO3 and 0.09 µM PO3− 4).
For the cultivation, starved individuals weighting 3.0 ± 0.11 g were cultivated in 3 liters Erlenmeyer flasks (n = 4) filled with seawater enriched with 200 µM of ammonium (NH4Cl), 8 µM of nitrate (NaNO3), 12 µM of phosphate (Na2HPO4∙12H2O),
Figure 1. Schematic representation of steps to ulvan extraction and characterization.
salts and vitamins . The nutrient concentrations were chosen with reference to the mean maximum nutrient concentration after a five years monitoring of one of the points of an important Brazilian bay  with potential for Ulva cultivation. The same light, photoperiod and temperature conditions of the acclimatization and starvation period were maintained. The experiment lasted five days.
2.3. Ulvan Extraction
After the cultivation, individuals were washed with distilled water to remove salts and oven dried at 50˚C until constant weight. The polysaccharide was extracted according to method described by . Dried algal biomass (SEA and CULT) were grinded into a powder, suspended in ultrapure water (Milli-q®) (100 ml/10g) and autoclaved at 120˚C for 40 min. The supernatant was centrifuged at 10,000 g and 4˚C for 10 min (Eppendorf centrifuge 5810 r). Ulvan was precipitated with three volumes of ultrapure ethanol (Merck®), cooled at – 20˚C for 48 hours and further centrifuged at 3500 g at 4˚C for 5 min. The recovered pellet (ulvan) was freeze-dried. Ulvan extraction yield was 16.29% ± 0.93% calculated using formula proposed by :
where, We is the dry ulvan weight extracted and Wf is the macroalgae dry weight.
2.4. Fourier-Transform Infrared Spectroscopy (FT-IR) Analysis
Ulvan (SEA and CULT) infrared spectra with Fourier transform (FT-IR) were recorded on a spectrophotometer (IR Prestige_21, Shimadzu) at room temperature. The FT-IR spectra were obtained in the transmission mode at 400-4000 cm−1. The transmission spectra were recorded using KBr (Merck®) pellets containing 2.5 mg of ulvan powder.
2.5. Nuclear Magnetic Resonance Spectroscopy
NMR analyses were performed using a Varian VNMRSYS 500 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) at 37˚C. Proton and carbon operating frequencies were 499.77 and 125.68 MHz, respectively. 1H NMR spectra were recorded with a 90 degree observe pulse width (pw = 90 µs), a 2.04 s acquisition time and a 1 s relaxation delay. A total of 32 scans were performed for each sample. For 13C NMR, a 90 degree pulse was used (pw = 90 µs), 1.04 s acquisition time, 2 s relaxation delay and a total of 114.624 scans were collected. Ulvan samples (2% w/v) were dissolved in D2O 99.99% (Sigma-Aldrich®). 1H and 13C NMR chemical shifts were expressed in parts per million (ppm).
3. Results and Discussion
FTIR and NMR spectroscopy are rapid and non-destructive analysis that provide fundamental information on ulvan polysaccharide structure . In this work, such techniques showed that polysaccharides from natural environment (SEA) and cultivated U. fasciata (CULT) are mainly constituted of rhamnose, iduronic and glucuronic acid, sulfate and, in the case of SEA, xylose.
IR Spectra of SEA and CULT ulvan are presented in Figure 2 with the signals assignment provided by comparison with published data    . CULT and SEA spectra presented all the characteristics peaks described in literature   , confirming that the extracted polysaccharides are ulvan. According to FTIR spectra, there was no visible difference between the two ulvan extracts and those reported in the literature   .
The absorption band at around 3300 cm−1 was attributed to a stretching of hydroxyl groups (O-H). Signal observed at approximately 2937 cm−1 is due to C-H stretching vibration and is characteristic of polysaccharides . Bands of carboxylate groups of uronic acid with similar intensities are present in both spectra at around 1651 and 1435 cm−1. The absorptions between 1147 and 848 cm−1 are known as the fingerprint region for ulvan, being the most important absorptions. At 983 cm−1 signal is characteristic of the vibration of glycosidic bonds and at 848 cm−1 corresponds to the bending vibration of C-O-S of sulfate in axial position. Peaks bellow 900 cm−1 in both ulvan samples are characteristic of the presence of sulfate.
The presence, degree and distribution of the sulfate groups are important in determining the biological activity of ulvan . CULT ulvan presented stronger signals of sulfate groups than SEA between 1159 and 625 cm−1. Previous studies have found that the antioxidant activity and regulation of physiological stress by ulvan is related to it is sulfate content  . This result suggests that CULT ulvan could have a higher antioxidant potential.
The 13C NMR spectra are shown in Figure 3, with typical signals of ulvan
Figure 2. Infrared spectra of ulvan from Ulva fasciata from natural environment (SEA) and cultivated in controlled conditions (CULT) between 400 and 4000 cm−1.
Figure 3. 13C NMR spectra (ppm) of ulvan from Ulva fasciata collected in natural environment (SEA) and cultivated in vitro (CULT). Residue A = rhamnose, B = glucuroni acid, C = iduronic acid and D = xylose.
structure attributed by comparison with published data    . Carbon of rhamnose and glucuronic acid that constitutes the type A ulvanobiuronic acid (A3s) and the chemical shifts attributed to rhamnose 3-sulfate linked to iduronic acid in type B ulvanobiuronicacid (B3s) were identified, confirming that the extracted polysaccharides are mainly composed of repeated sequences of these two disaccharides. Signals in the resonance region corresponding to carbon rings (70.06 - 80.28 ppm and 69.60 - 79.88 ppm in CULT and SEA, respectively), C-6 methyl group of rhamnose at around 19 ppm and carboxyl signal of uronic acid at approximately 177 ppm are present. Further assigned signals of anomeric carbons (99.28 - 104.62 ppm) were observed in SEA.
1H NMR spectra are presented in Figure 4 and the signals associated to each hydrogen atom are assigned according to reference data    . Anomeric protons of rhamnose and iduronic acid are present between 4.28 - 5.13 ppm. Proton chemical shifts of rhamnose are shown at 1.30 ppm. Glucuronic acid chemical shifts are between 3.36 and 3.79 ppm. Signals of xylose are indicated at 3.20 and 5.27 ppm at SEA ulvan.
Noise observed in the 13C NMR spectra is related to sample dilution increased by the high molecular weight of the polymer and solution viscosity. Ulvan molecular weight can vary from 1.8 × 105 to 2 × 106 depending on extraction methods, specie and polydispersity of the samples  . According to  ulvan
Figure 4. 1H NMR spectra (ppm) of ulvan from Ulva fasciata collected in natural environment (SEA) and cultivated in vitro (CULT). Residue A = rhamnose, B = glucuroni acid, C = iduronic acid and D = xylose.
extracted with temperatures between 80˚C - 90˚C, close to the used in this study, tend to present higher molecular weight.
In 13C and 1H NMR spectra of SEA ulvan we could observe signals of xylose, suggesting the presence of ulvanobiose (U3s) in this ulvan structure. In CULT we could not detect U3s, but peak characteristic to C-1 of rhamnose in A3s disaccharide (4.82 ppm) was present . According to  during the active growth of Ulva the macroalgae tends to synthesize more ulvanobiuronic acid type A, with the production of ulvanobiose being developmentally regulated. In this study, U. fasciata presented an average growth rate of 5.7% day−1 and active nutrient uptake throughout the cultivation experiment (data not shown), an indication that individuals had not reach their growth plateau when collected.
In this study both ulvan presented similar global structure, but ulvan from cultivated U. fasciata presented stronger signals of sulfate and ulvan from natural environment had signals of xylose. Future studies with purification and sugar quantification procedures could help elucidate the fine structure of both ulvan samples and assess the efficacy of CULT ulvan for different applications such as antioxidant.
The production of ulvan with predictive structure and in necessary amounts is one of the hindrances for the ulvan market development. The results gathered here show that ulvan from cultivated U. fasciata is similar to those reported in literature and could be a source for obtaining this polysaccharide. By controlling abiotic conditions ulvan production could be maximized meeting commercial requirements.
We are grateful to the reviewers who helped improve this manuscript. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001, and the Brazilian National Council of Technological and Scientific Development (CNPq)—Productivity Fellowship (YYV-301939/2019-9).
 Wang, H.M.D., Chen, C.C., Huynh, P. and Chang, J.S. (2015) Exploring the Potential of Using Algae in Cosmetics. Bioresource Technology, 184, 355-362.
 Wells, M.L., Potin, P., Craigie, J.S., Raven, J.A., Merchant, S.S., Helliwell, K.E., Smith, A.G., Camire, M.E. and Brawley, S.H. (2017) Algae as Nutritional and Functional Food Sources: Revisiting Our Understanding. Journal of Applied Phycology, 29, 949-982.
 Tziveleka, L.A., Ioannou, E. and Roussis, V. (2019) Ulvan, a Bioactive Marine Sulphated Polysaccharide as a Key Constituent of Hybrid Biomaterials: A Review. Carbohydrate Polymers, 218, 355-370.
 Kidgell, J.T., Magnusson, M., de Nys, R. and Glasson, C.R.K. (2019) Ulvan: A Systematic Review of Extraction, Composition and Function. Algal Research, 39, 101422.
 Chi, Y., Li, H., Wang, P., Du, C., Ye, H., Zuo, S., Guan, H. and Wang, P. (2020) Structural Characterization of Ulvan Extracted from Ulva clathrata Assisted by an Ulvan Lyase. Carbohydrate Polymers, 229, Article No.: 115497.
 Costa, C., Alves, A., Pinto, P.R., Sousa, R.A., Borges da Silva, E.A., Reis, R.L. and Rodrigues, A.E. (2012) Characterization of Ulvan Extracts to Assess the Effect of Different Steps in the Extraction Procedure. Carbohydrate Polymers, 88, 537-546.
 Qi, H., Zhang, Q., Zhao, T. and Chen, R. (2005) Antioxidant Activity of Different Sulfate Content Derivatives of Polysaccharide Extracted from Ulva pertusa (Chlorophyta) in Vitro. International Journal of Biological Macromolecules, 37, 195-199.
 Li, W., Jiang, N., Li, B., Wan, M., Chang, X., Liu, H., Zhang, L., Yin, S., Qi, H. and Liu, S. (2018) Antioxidant Activity of Purified Ulvan in Hyperlipidemic Mice. International Journal of Biological Macromolecules, 113, 971-975.
 Hardouin, K., Bedoux, G., Burlot, A.S., Donnay-Moreno, C., Bergé, J.P., Nyvall-Collén, P. and Bourgougnon, N. (2016) Enzyme-Assisted Extraction (EAE) for the Production of Antiviral and Antioxidant Extracts from the Green Seaweed Ulva armoricana (Ulvales, Ulvophyceae). Algal Research, 16, 233-239.
 Lopes, N., Ray, S., Espada, S.F., Bomfim, W.A., Ray, B., Faccin-Galhardi, L.C., Linhares, R.E.C. and Nozawa, C. (2017) Green Seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) Derived Sulphated Polysaccharides Inhibit Herpes Simplex Virus. International Journal of Biological Macromolecules, 102, 605-612.
 Thanh, T.T.T., Quach, T.M.T., Nguyen, T.N., Vu Luong, D., Bui, M.L. and Van Tran, T.T. (2016) Structure and Cytotoxic Activity of Ulvan Extracted from Green Seaweed Ulva lactuca. International Journal of Biological Macromolecules, 93, 695-702.
 Shao, P., Pei, Y., Fang, Z. and Sun, P. (2014) Effects of Partial Desulfation on Antioxidant and Inhibition of DLD Cancer Cell of Ulva fasciata Polysaccharide. International Journal of Biological Macromolecules, 65, 307-313.
 del Rocío Quezada-Rodríguez, P. and Fajer-ávila, E.J. (2017) The Dietary Effect of Ulvan from Ulva Clathrata on Hematological-Immunological Parameters and Growth of Tilapia (Oreochromis niloticus). Journal of Applied Phycology, 29, 423-431.
 Adrien, A., Bonnet, A., Dufour, D., Baudouin, S., Maugard, T. and Bridiau, N. (2017) Pilot Production of Ulvans from Ulva sp. and Their Effects on Hyaluronan and Collagen Production in Cultured Dermal Fibroblasts. Carbohydrate Polymers, 157, 1306-1314.
 Pageon, H., Azouaoui, A., Zucchi, H., Ricois, S., Tran, C. and Asselineau, D. (2019) Potentially Beneficial Effects of Rhamnose on Skin Ageing: An in Vitro and in Vivo Study. International Journal of Cosmetic Science, 41, 213-220.
 Alban, S., Schauerte, A. and Franz, G. (2002) Anticoagulant Sulfated Polysaccharides: Part I. Synthesis and Structure-Activity Relationships of New Pullulan Sulfates. Carbohydrate Polymers, 47, 267-276.
 Hallak, L.K., Collins, P.L., Knudson, W. and Peeples, M.E. (2000) Iduronic Acid-Containing Glycosaminoglycans on Target Cells Are Required for Efficient Respiratory Syncytial Virus Infection. Virology, 271, 264-275.
 Bindschadler, P., Adibekian, A., Grünstein, D. and Seeberger, P.H. (2010) De Novo Synthesis of Differentially Protected L-Iduronic Acid Glycosylating Agents. Carbohydrate Research, 345, 948-955.
 Robic, A., Rondeau-Mouro, C., Sassi, J. F., Lerat, Y. and Lahaye, M. (2009) Structure and Interactions of Ulvan in the Cell Wall of the Marine Green Algae Ulva rotundata (Ulvales, Chlorophyceae). Carbohydrate Polymers, 77, 206-216.
 Robic, A., Sassi, J.F., Dion, P., Lerat, Y. and Lahaye, M. (2009) Seasonal Variability of Physicochemical and Rheological Properties of Ulvan in Two Ulva Species (Chlorophyta) from the Brittany Coast. Journal of Phycology, 45, 962-973.
 Martins, N.T. (2016) Physiological Responses of Ulva fasciata Delile (Ulvales, Chlorophyta): Comparison of Two Populations from Thermally Distinct Sites from Brazilian Coast. Msc Dissertation, Federal University of Sao Paulo, Sao Paulo.
 Ursi, S. and Plastino, E.M. (2001) Crescimento in Vitro de Linhagens de Coloracao Vermelha e Verde Clara de Gracilaria Birdiae (Gracilariales, Rhodophyta) em Dois Meios de Cultura: Análise de Diferentes Estádios Reprodutivos. Revista Brasileira de Botanica, 24, 587-594.
 Reis, R.P., Carvalho Junior, A.A., Facchinei, A.P., Calheiros, A.C.S. and Castelar, B. (2018) Direct Effects of Ulvan and a Flour Produced from the Green Alga Ulva fasciata Delile on the Fungus Stemphylium solani Weber. Algal Research, 30, 23-27.
 Yaich, H., Ben Amira, A., Abbes, F., Bouaziz, M., Besbes, S., Richel, A., Blecker, C., Attia, H. and Garna, H. (2017) Effect of Extraction Procedures on Structural, Thermal and Antioxidant Properties of Ulvan from Ulva lactuca Collected in Monastir Coast. International Journal of Biological Macromolecule, 105, 1430-1439.
 Tako, M., Tamanaha, M., Tamashiro, Y. and Uechi, S. (2015) Structure of Ulvan Isolated from the Edible Green Seaweed, Ulva pertusa. Advances in Bioscience and Biotechnology, 6, 645-655.
 Qi, H., Zhao, T., Zhang, Q., Li, Z., Zhao, Z. and Xing, R. (2005) Antioxidant Activity of Different Molecular Weight Sulfated Polysaccharides from Ulva pertusa Kjellm (Chlorophyta). Journal of Applied Phycology, 17, 527-534.
 Alves, A., Caridade, S.G., Mano, J.F., Sousa, R.A. and Reis, R.L. (2010) Extraction and Physico-Chemical Characterization of a Versatile Biodegradable Polysaccharide Obtained from Green Algae. Carbohydrate Research, 345, 2194-2200.
 Robic, A., Bertrand, D., Sassi, J. and Lerat, Y. (2009) Determination of the Chemical Composition of Ulvan, a Cell Wall Polysaccharide from Ulva Spp. (Ulvales, Chlorophyta) by FT-IR and Chemometrics. Journal of Applied Phycology, 21, 451-456.
 Tabarsa, M., You, S. and Hashem, E. (2018) Water-Soluble Polysaccharides from Ulva intestinalis: Molecular Properties, Structural Elucidation and Immunomodulatory Activities. Journal of Food and Drug Analysis, 26, 599-608.
 Leiro, J.M., Castro, R., Arranz, J.A. and Lamas, J. (2007) Immunomodulating Activities of Acidic Sulphated Polysaccharides Obtained from the Seaweed Ulva rigida C. Agardh. International Immunophasmacology, 7, 879-888.
 Qi, H. and Sun, Y. (2015) Antioxidant Activity of High Sulfate Content Derivative of Ulvan in Hyperlipidemic Rats. International Journal of Biological Macromolecules, 76, 326-329.
 Lahyae, M., Cimadevilla, E.A., Kuhlenkamp, R., Lognon, V. and Dion, P. (1999) Chemical Composition and 13C NMR Spectroscopic Characterization of Ulvans from Ulva (Ulvales, Chlorophyta). Journal of Applied Phycology, 11, 1-7.
 Lahaye, M. (1998) NMR Spectroscopic Characterization of Oligosaccharides from Two Ulva Rigida Ulvan Samples (Ulvales, Chlorophyta) Degraded by a Lyase. Carbohydrate Research, 314, 1-12.
 Alves, A., Sousa, R.A. and Reis, R.L. (2013) In Vitro Cytotoxicity Assessment of Ulvan, a Polysaccharide Extracted from Green Algae. Phytotherapy Research, 27, 1143-1148.
 Van Tran, T.T., Truong, H.B., Tran, N.H.V., Quach, T.M.T., Nguyen, T.N., Bui, M.L., Yuguchi, Y. and Thanh, T.T.T. (2018) Structure, Conformation in Aqueous Solution and Antimicrobial Activity of Ulvan Extracted from Green Seaweed Ulva reticulata. Natural Product Research, 32, 2291-2296.