Biochemical characterization and potential valorization of the invasive seaweed Rugulopteryx okamurae

doi:10.21203/rs.3.rs-4831629/v1

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Biochemical characterization and potential valorization of the invasive seaweed Rugulopteryx okamurae

https://doi.org/10.21203/rs.3.rs-4831629/v1

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The invasion of the brown seaweed Rugulopteryx okamurae is altering the equilibrium and reducing the biodiversity of marine coastal ecosystems of southern Europe, with negative economic consequences for fishermen, tourism and governments that regularly remove tons of arrivals from the coast. However, the large amount of biomass produced also implies an opportunity for the valorization of these seaweeds, for example, in the form of biofertilizer and/or biostimulant for agricultural use.

In this study, we characterized the biochemical composition of Rugulopteryx okamurae and aqueous extracts elaborated from them. Their annual compositional variation was studied by measuring the concentration of carbohydrates, lipids, proteins, minerals, phenolic compounds, and antioxidant capacity, together with metals and micro/macro elements, in samples collected in two consecutive years from southern Spain.

The composition of the algae varied between years for macro/micronutrients, metals, proteins, polyphenols and lipids (mainly terpenes), while polysaccharides (fucoidan, laminarin and alginate) remained in similar concentration. The seaweed composition highlighted for the terpene concentration mainly in 2023. The aqueous extracts significantly increased fucoidan and laminarin content respect to the seaweed. In all, the biochemical characterization conducted suggests that Rugulopteryx okamurae is valuable as a biofertilizer due to its mineral content, while the extract composition (sulfur, polyphenolics, fucoidan, laminarin, and glycolipids) suggests its potential use as biostimulant. Thus, this study proposes Rugulopteryx okamurae as a valuable waste for agricultural proposals.

valorization

fucoidan

laminarin

glycolipids

biostimulant

biofertilizer

The arrival of Rugulopteryx okamurae (Div. Phaeophyta, O. Dictyotales) to the Strait of Gibraltar is one of the most dramatic examples of invasive seaweed proliferation(Navarro-Barranco et al. 2019; García-Gómez et al. 2020). This species, native to the Pacific Ocean (Agatsuma et al. 2005), was first detected in 2002 in the Mediterranean coasts of France, appearing in the Strait of Gibraltar in 2015, where it quickly spread into habitats ranging from 0 to 50 m depth (Altamirano-Jeschke et al. 2016; Muñoz et al. 2019). Since then, this seaweed expanded in the region, being responsible for numerous episodes of massive wrack deposits all over the Atlantic coasts of Tarifa, Algeciras, and North Africa coast, as well as on the Mediterranean shores of Andalusia (Muñoz et al. 2019). In 2019, more than 10.000 tons of this seaweed were removed from the shores of Málaga, Cadiz and Ceuta, highlighting its unprecedented invasive potential in European waters (Ministerio Español para la Transición Ecológica y el Reto demográfico 2022; García-Gómez et al. 2020; Sempere-Valverde et al. 2021).

One of the main strategies that have been proposed to deal with the large amounts of biomass accumulated on beaches is their valorization for agricultural purposes. Seaweeds have been historically applied to agricultural soils as soil organic amendments, although their use has progressively shifted to the application of different extracts, especially aqueous extracts, with microbicide, nematicide, insecticide, biofertilizer, biostimulating, and soil conditioning function (Battacharyya et al. 2015). Extracts are considered safe to the environment and thus compatible with organic farming practices, implying a great market potential. In fact, the market of seaweed biostimulants in the US accounted for 758 million dollars in 2021, and roughly accounted for 29,8–33,5% of the global plant biostimulants market, which was estimated to be 2,540 million dollars during last year (EBIC, 2023). Moreover, with the application of the new Fertilizing Products Regulation in July 2022, which allows the placement of plant biostimulants in the European Union Single Market (EU (2019)), the use and economic importance of seaweed-based biostimulants is expected to keep increasing in the coming years. In one study, Zarraonaindia et al. (2023) demonstrated the capacity of aqueous extract of Rugulopteryx okamurae to induce the activation of several secondary metabolite pathways in vines and promoting the abundance of beneficial microbiota involved in grapevine protection, suggesting the potential valorization of Rugulopteryx okamurae as a biostimulant.

The use of seaweed extracts as biostimulants relies on their composition, which consists of a diverse range of bioactive compounds that stimulate plant growth and defense responses against pathogens. These molecules include organic and inorganic compounds such as polysaccharides, polyphenols, phytohormones, lipids, minerals, and other different components. Polysaccharides have been singled out as one of the most bioactive agents for biostimulation and against fungal infection (Mamede et al. 2023; Raja and Vidya 2023). For example, Ascophylum nodosum extracts, a brown seaweed rich in fucoidans, have been shown to have a positive effect on vegetative growth, ripeness and grape quality, while contributing to mitigate abiotic stress, improving grapevine defenses, and promoting soil health (Shukla et al. 2019). However, one of the main limitations of the use of uncultivated seaweeds for manufacturing biostimulants, particularly at commercial scale, is the variability of their chemical composition, which depends on the geographic area of collection, season, and environmental condition(Stengel et al. 2011; Manns et al. 2017; Agarwal et al. 2021; Fleurence 2022). Thus, it is crucial to fully understand how the composition of seaweeds vary across space and time to ensure their proper valorization. This aspect is completely unknown for Rugulopteryx okamurae.

In the current study, we characterized the biochemical composition (elemental content, total carbohydrates, total proteins, total lipids, total polyphenols and the antioxidant capacity) of Rugulopteryx okamurae as well as of their aqueous extracts during two consecutive years. In addition, we conducted a deeper analysis of carbohydrates (fucoidan, laminarin, alginates) and lipids (fatty acids and polar factions) to gain further understanding on the potential of Rugulopteryx okamaurae as a biofertilizer and/or biostimulant in agriculture.

2.1. Seaweed collection and extract elaboration

The collection of Rugulopteryx okamurae took place in Punta Carnero (Cadiz, Spain, 36º04’14’’N, 5º25’50”W), in April 2022 (RO22) and April 2023 (RO23). Seaweeds were washed with tap water to remove epiphytes and salt, drained and frozen at -80 ºC and freeze-dried (Cryodos, Telstar, Spain). Lyophilized samples were grounded into a fine powder. An aqueous extract was produced from each alga. Briefly, 50 g of freeze-died algae were combined with 500 mL of Milli-Q-water, heated for 2 hours at 70ºC in a shaker, twice. The aqueous solution was frozen and freeze-dried (Zarraonaindia et al. 2023).

2.2. Elemental composition

Total carbon, nitrogen, and sulphur contents were determined from lyophilized seaweed and extracts using an elemental analyzer (CNHS-932, LECO, St. Joseph, MI, USA) in the Research Support Central Services of the University of Cadiz. Macro-and microelements, many of which are plant nutrients, were determined on lyophilized samples by a calibrated X-ray fluorescence equipment (Bruker, CTX800). We measured the concentration of plant primary (C, N, P, and S) and secondary (Ca, K, Mg) macronutrients; micronutrients such as silicon (Si), chlorine (Cl), copper (Cu), manganese (Mn), iron (Fe), nickel (Ni), zinc (Zn), and molybdenum (Mo); non-essential nutrients such as selenium (Se) and non-nutrients such as bromide (Br), titanium (Ti), rubidium (Rb), strontium (Sr), barium (Ba), aluminum (Al), cadmium (Cd), Chromium (Cr), mercury (Hg), lead (Pb) and arsenic (As).

2.3. Determination of ash, total carbohydrates, total lipids and total proteins

The ash content was measured by heating the sample overnight in a furnace (Nabertherm, Germany) at 525°C, and the content was determined gravimetrically. Total carbohydrate content was determined following the method previously described by Dubois et al., (1956). In short, 5 mg of lyophilized alga were combined with 5 ml of H₂SO₄ (1M). The mix was shaking (1h, 100 ºC) and subsequently centrifugated (5 min, 4500rpm). From the above liquid phase, 100 µL were diluted in 900 µL of milliQ-water. After that, 25 µL of phenol 81% were added, vortex and left to incubate in darkness for 5 minutes. Following this, 2.5 mL sulfuric acid 98% were incorporated, vortexed and left to incubate in darkness for 30 minutes. Absorbance was read at 490 nm in the plate-reader Multiskan Skyhigi (Thermo Scientific, Singapore) in rounded bottom 96-wells. The concentration of sugars was determined using a standard curve prepared ranging from 0 to 1 mg mL^− 1 of D-glucose.

Total lipids were extracted using the methodology from Folch et al. (1957) with slight modifications by (Christie 2003). In brief, 250 mg of dry seaweed powder were homogenised with a 1600 MiniG® homogenizer (Spex, NJ, USA) in ice-cold chloroform/methanol (2:1, v:v). The non-lipid and lipid layers were separated by adding 0.88% KCl (w/v). The upper aqueous layer was discarded, while the lower organic layer was dried under oxygen-free nitrogen. The lipid content was determined gravimetrically after drying overnight in a vacuum desiccator.

Total proteins were calculated using the nitrogen-to-protein conversion factor of 6.25 for seaweeds (Jones 1931).

2.4. Total polyphenols determination and antioxidant capacity

For the determination of Total Phenolic Compounds (TPC) 60 mg of lyophilized sample was extracted with 6 ml of methanol:water (80:20, v/v) shaking during 24h (20ºC) at darkness. Subsequently, samples were centrifugated (4.500g, 15 min, 4ºC) (Digicen 20-R, Spain). Liquid phase was concentrated in a rotavapor (Heidolph, Germany) and the solid was dissolved in water (10 mg mL^− 1). Then, the TPC was determined using the Folin–Ciocalteu method according to Singleton & Rossi, 1965, (Singleton and Rossi 1965) with modifications. Briefly, 10 µl of sample was mixed with 230 µl of MilliQ water and 15 µL of Folin–Ciocalteu phenol reagent (Sigma-Aldrich, St. Louis, USA). The mixtures were incubated for 3 min in darkness, and then 45 µL of sodium carbonate 35% were added. Samples were mixed again and incubated for 1 hour at room temperature in darkness. After that, absorbances were measured at 765 nm (Lambda, Perking-Elmer, Korea). Galic acid was used as standard for quantification. The results were expressed as µg GAE/mg of lyophilized seaweed.

The Antioxidant capacity (AC) of the samples was evaluated through the FRAP assay according to (Benzie and Strain 1996). In short, 0.5 gr of lyophilized seaweed was mixed with 12 mL of phosphate buffer during 5 min. Samples were then centrifuged (10.000 rpm, 10 min, 4ºC). The liquid phase (5 µL) was measured following the FRAP method (Benzie and Strain 1996). Ascorbic acid was used as standard for quantification. The results were expressed as mg ascorbic acid equivalent (AEE)/g of lyophilized seaweed.

2.5. Fucoidan, alginate and laminarin extraction and determination

Fucoidan was extracted as described by Fletcher et al. (2017) with minor modifications. Briefly, 1 g of sample was left overnight stirring with 10 mL of 85% ethanol at room temperature (22ºC). Subsequently, samples were centrifuged, removing the supernatant, followed by addition of 10 ml of ethanol, centrifugation, and supernatant removal. After that, 10 mL acetone were added, samples were centrifuged, and the supernatant was again removed. The pellet was left for 2h to dry until constant weight. Afterwards, 0.3 g were weighted and moved to a glass tube were 7.5 mL of HCl (0.1M) were added. Samples were then incubated at 80 ºC under constant shaking for 4 hours. Then, they were left to cool, centrifuged and the supernatant was transferred to a new tube where the pH was neutralized using a pH meter (CRISON®, Spain) and 6 mL of CaCl₂ were added. Samples were stored at 4ºC overnight for alginate precipitation. A new centrifugation step was performed, and the supernatant was transferred to a clean tube where ethanol was added to a final concentration of 40%. Samples were left at 4ºC for 4 hours for laminarin precipitation. Following this, samples were centrifuged again, and the supernatant was transferred to a new tube where ethanol was added till a 70% concentration was achieved. Samples were then stored at 4ºC for at least 4 hours. Finally, a last centrifugation step was employed to remove the supernatant and the extracted fucoidan was left to dry until constant weigh for 24 hours. Finally, precipitates were weighted and alginate, laminarin and fucoidan content were weighted and determined gravimetrically.

2.6. Lipids extraction and determination

A solid-phase extraction was performed on total lipid (see 2.3 section) using silica cartridges (ExtraBond500 mg, 3 mL, Scharlab, Spain) to obtain the lipid fraction, which includes neutral lipids, glycolipids, and other polar lipids. These three fractions were obtained by successively eluting with methylterbutyl ether, acetone:methanol (9:1, v:v), and methanol. Fractions were weighed for quantification after the solvents were evaporated.

Lipid classes were separated after applying known quantities from the extracts on High Performance Thin Layer Chromatography (HPTLC) 20x10 cm silica gel 60 plates (Merck, Darmstadt, Germany). A semiautomatic TLC Sampler Linomat 5 (CAMAG, Muttenz, Switzerland) was used to apply the samples to a TLC chamber. The polar solvent used in this study was a mixture of methyl acetate, isopropanol, chloroform, methanol, and 0.25% KCl (25:25:25:10:4, v:v:v:v:v), as described by (Mock and Kroon 2002). The neutral solvent used was a mixture of toluene, ethyl acetate, and formic acid (139:83:8, v:v:v), as described by Jesionek et al. (2015). Quantification was achieved by scanning densitometry (CAMAG TLC Scanner 2) after staining of plates with cupric acetate and charring at 160°C for 10 min. Identification of phosphorous containing lipids was achieved by thoroughly spraying with themolybdenum reagent of Dittmer and Lester, (1964).

To extract Fatty Acids (FA), lipids were subject to acid catalysed transmethylation for 16 hours at 50°C, using toluene and sulphuric acid 1% (1:2, v:v) in methanol according to (Christie 2003). Before transmethylation, tricosanoic acid (23:0) was added to the lipid extract as an internal standard. The resulting fatty acid methyl esters (FAME) were purified on cartridge columns (Clean-Up Silica, 6mL). FAME were separated and quantified using a Shimadzu GC 2010 (Shimadzu, Kyoto, Japan) gas chromatograph equipped with a flame-ionization detector (250°C) and a fused silica capillary column SUPRAWAX- 280 (15 m × 0.1 mm I.D.). Hydrogen was used as carrier gas and the oven initial temperature was 100°C for 0.5 min., followed by an increase at a rate of 20°C min^− 1 to a final temperature of 250°C for 8 min. Individual FAME were identified by reference to authentic standards.

2.7. Statistical analysis

One factor analysis of variances (ANOVA) was performed to test the null hypothesis of values not varying between independent groups (p-value > 0.05). When significant differences among groups were found, Fisher Least Significant Test (LSD) was employed by using Statistix version 9.0 (Analytical Software, Tallahassee, FL, USA). and GraphPad 8 was employed for graphs (Graphpad Software, Inc. La Jolla, CA, USA) software.

3.1. Elements, minerals and metals

Seaweed C and N contents were higher in 2022, while the C/N relationship and S content were constant between years (Table 1). Seaweed mineral composition varied between years with significant differences found for K, Ca, Cl, Ti, Fe, Mn, Br, Sr and Cu (Table 1).

The aqueous extract from 2022 showed higher amount for CNS than the extract from 2023. When comparing seaweed vs extracts an enrichment in S and a higher C/N relation was found for the extracts, which are within the desirable range for agricultural purposes. Regarding mineral composition, the extracts were found to be rich in macroelements such as P and K and microelements such as Mg and Cl. Zn and As heavy metals were quantified in the samples. High quantities of As were found in both seaweeds and extracts (30–40 mg/kg and 90 mg/kg respectively). Other heavy metals were under detection limits: Cd (1 mg/kg), Hg (1 mg/kg), Pb (1 mg/kg), as well as under the allowed concentrations.

Table 1

Elemental composition (%), minerals and metals (mg/kg lyophilized) of *Rugulopteryx okamurae* seaweed and aqueous extracts.
	RO22	RO23	RU22	RU23
Elemental C content	41.4 ± 0.3 A	34.2 ± 0.5 B	30.6 ± 0.2 C	21.3 ± 0.2 D
Elemental N content	2.2 ± 0.0 A	1.8 ± 0.0 B	0.9 ± 0.0 C	0.8 ± 0.0 D
Relation C/N	19.0 ± 0.2 C	19.1 ± 0.3 C	33.4 ± 0.1 A	26.8 ± 0.6 B
Elemental S content	1.3 ± 0.0 C	1.3 ± 0.0 C	2.14 ± 0.0 A	1.8 ± 0.0 B
P	1244 ± 55.2 B	1039.2 ± 50.8 B	1669.8 ± 157.8 A	1095.8 ± 61.0 B
K	23882 ± 96.5 C	33207 ± 1097.0 B	36719 ± 3548.0 A	38993 ± 2503.9 A
Ca	16631 ± 33.1 A	8843.8 ± 1563.7 B	9049.7 ± 799.6 B	3103.2 ± 123.3 C
Mg	3776.0 ± 1960.0 B	3314.3 ± 858.1 B	5638.7 ± 729.1 A	3418.2 ± 566.7 B
Al	2166.1 ± 487.3	2633.3 ± 821.6	< LOD	< LOD
Si	9203.0 ± 253.1 A	11065.0 ± 3862.2 A	921.6 ± 99.8 B	2891.5 ± 202.1 B
Cl	1810.0 ± 323.0 D	42870.0 ± 1012.1 B	36720.0 ± 3548.0 C	63749.0 ± 2839.9 A
Ti	6.0 ± 3.0 B	14.0 ± 6.3 A	< LOD	< LOD
Fe	1094.5 ± 0.7B	1636.7 ± 7.1 A	164.5 ± 44.5 C	445.5 ± 58.9 B
Mn	35 ± 0.6 A	18.2 ± 4.1 B	16.7 ± 1.9 B	19.5 ± 1.6 B
Br	263.0 ± 4.3 D	680.0 ± 45.4 B	619.3 ± 26.6 C	1218.5 ± 5 A
Rb	17.00 ± 4.6 C	22.3 ± 3.0 C	31.3 ± 1.9 B	41.0 ± 2.3 A
Sr	1110.0 ± 7.1 A	1001.8 ± 6.7 B	358.8 ± 8.9 C	222.0 ± 5.4 D
As	32.0 ± 3.4 B	40.0 ± 3.8 B	89.1 ± 4.6 A	87.6 ± 4.8 A
Cu	6.0 ± 2.1 B	9.5 ± 2.8 A	5.6 ± 1.2 B	7.0 ± 0.8 B
Zn	22.0 ± 2.1 A	25.8 ± 4.9 A	7.2 ± 1.2 B	6.2 ± 1.7 B

Values are expressed as average ± standard deviation (n = 3). RO22, Rugulopteryx okamurae collected in 2022; RO23, Rugulopteryx okamurae collected in 2023; RU22, Rugulopteryx okamurae aqueous extract from the seaweed in 2022; RU23 Rugulopteryx okamurae aqueous extract from the seaweed in 2023. Letters represent significant differences (ANOVA, LSD Test, p < 0.05). Cr, Ni, Se, Ba, Mo, Cd, Hg and Pb were under detection limit.

3.2. Biochemical composition

Rugulopteryx okamurae seaweed contained high quantities of ashes (33%), together with 8–9% total carbohydrates, 8–10% lipids, 11–13% proteins, 2–4% total polyphenols (TPC) and 6–8% antioxidant activity (AC), which suggested low variability in the biochemical composition between years although proteins and TPC significantly differed with years (Table 2).

Regarding the aqueous extract, ashes, carbohydrates, lipids, proteins, TPC and AC accounted for 27–29%, 9–13%, 4–6%, 5–6%, 3–7% and 8–13% respectively. In overall, the extraction efficiency of most of these compounds was higher for 2022 seaweed samples than for 2023 samples. The aqueous extraction was particularly efficient for the recovery of carbohydrates and total polyphenols. Expectedly, lipids recovery was low due to their insolubility in the solvent of choice, water, as happened for proteins (Table 2). Finally, antioxidant capacity was in concordance with TPC concentration, increasing in the extract samples respect to the seaweed, especially in 2022 (Table 2).

Table 2

Biochemical composition and antioxidant capacity of *Rugulopteryx okamurae* and its aqueous extracts.
	RO22	RO23	RU22	RU23
Ashes (%)	33.4 ± 0.2 A	33.9 ± 1.8 A	27.0 ± 1.5 B	28.7 ± 3.4 B
Total Carbohydrates (%)	7.9 ± 1.1 B	8.3 ± 0.8 B	12.4 ± 0.0 A	8.5 ± 0.0 B
Total Lipids (%)	8.8 ± 0.8 A	10.0 ± 0.5 A	5.8 ± 0.3 B	4.3 ± 1.3 B
Total Proteins (%)	13.6 ± 0.1 A	11.2 ± 0.2 B	5.7 ± 0.0 C	5.1 ± 0.2 D
TPC (mg GAE /g lyophilized)	3.5 ± 0.3 B	1.9 ± 0.2 C	6.7 ± 0.7 A	3.4 ± 0.3 B
AC (mg AAE/g lyophilized)	7.9 ± 0.6 B	6.9 ± 0.7 B	12.5 ± 0.7 A	7.9 ± 0.8 B
Values are expressed as average ± standard deviation (n = 9). RO22, Rugulopteryx okamurae collected in 2022; RO23, Rugulopteryx okamurae collected in 2023; RU22, Rugulopteryx okamurae aqueous extract from the seaweed in 2022; RU23 Rugulopteryx okamurae aqueous extract from the seaweed in 2023. TPC, Total Polyphenol content; AC, Antioxidant Capacity. Letters represent significant differences (ANOVA, LSD Test, p < 0.05)

3.3. Polysaccharide composition

The analysis conducted here identified fucoidan, laminarin and alginate in both seaweeds and extracts. The Rugulopteryx okamurae seaweed contained around 1% of fucoidan (Fig. 1.A), 0.5% of laminarin (Fig. 1.B) and 6% of alginate (Fig. 1.C), indicating minimal fluctuations between 2022 and 2023 years, likely due to consistent environmental conditions (data not shown). When compared to the seaweed, the extracts were enriched in fucoidan (3.4-folds for 2022 and 1.5-folds for 2023; Fig. 1A) and laminarin (5.3-folds for 2022 and 4.3-folds for 2023; Fig. 1B). In contrast, alginates decreased from raw seaweed to extract by 1.7-folds and 1.4-folds for 2022 and 2023 samples, respectively (Fig. 1C), suggesting that the extraction process conducted may not be optimal for this compound. The polysaccharide extraction process from seaweed to the extract resulted more efficient in 2022 than in 2023, as described before for elemental compounds, which should be considered together with the annual composition variation of the seaweed.

3.4. Lipid composition

Despite the total lipid content not differing between seaweeds collected in 2022 and 2023 (Table 2), significant differences were observed in the lipid fractions, including neutral lipids, glycolipids, and other polar lipids (Fig. 2). Around 40–60% of the compounds were identified and quantified depending on the fractions (Table 3). Notably, the neutral fraction increased significantly in seaweed 2023 (Fig. 2A), doubling in comparison to the previous year, which was mainly attributed to an increase in terpenes (Table 3). Additionally, fucosterol and the carotenoid pigment fucoxanthin were found within the neutral fraction with significant differences between years (Table 3). In contrast, a decrease of about 50% in the glycolipids fraction was observed for seaweed 2023 respect to 2022, particularly regarding sulfoquinovosyldiacylglycerides (SQDG), digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG) (Table 3, Fig. 2B). Other polar lipids showed similar values in both years (Fig. 2C). Only betaine was identified in this last fraction, mainly in Rugulopteryx okamurae seaweed from 2023 (data not shown).

Table 3. Lipid compounds identified and quantified (mg/g dw) in the different lipid fractions of Rugulopteryx okamurae and its aqueous extracts.

	RO22	RO23	RU23	RU23
Neutral fraction
Fucoxantine	nd	8.0±0.8	nd	nd
Fucosterol	11.0±1.4 A	7.3±0.4 B	6.9±0.9 B	5.7±0.3 B
Terpenes	8.6±0.6 B	29.4±0.2 A	nd	2.2±1.6 C
Glycolipid fraction
SQDG	4.5±0.5 A	3.0±0.1 B	4.3±1.0 A	1.9±0.1 B
MGDG	4.1±0.2 A	3.0±0.2 B	2.3±0.5 B	1.0±0.1 D
DGDG	3.6±1.2 A	2.7±0.1 AB	4.0±1.2 A	1.3±0.3 B

nd, not detected; SQDG, sulfoquinovosyldiacylglycerols; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol. Values are expressed as average and standard deviation (SD) (n = 3). RO22, Rugulopteryx okamurae collected in 2022; RO23, Rugulopteryx okamurae collected in 2023; RU22, Rugulopteryx okamurae aqueous extract from the seaweed in 2022; RU23 Rugulopteryx okamurae aquose extract from the seaweed in 2023. Letters represent significant differences (ANOVA, LSD Test, p<0.05).

The analysis of fatty acids in Rugulopteryx okamurae revealed that saturated fatty acids (SFA) were the most abundant, accounting for 37–46% of the lipid profile (Table 4). Monounsaturated fatty acids (MUFA) were the second most abundant at 30%, followed by polyunsaturated fatty acids (PUFA) at 14–19%. The main fatty acid present in Rugulopteryx okamurae was palmitic acid (16:0) and oleic acid (18:1n9), which accounted for 24–30% and 12–15% of the total fatty acid content, respectively. Regarding PUFA, it is noteworthy that the fatty acid profile includes n-3 PUFA eicosanoid acid (EPA, 20:5n-3), linolenic acid (18:3n-3), and stearidonic acid (18:4n-3), as well as n-6 PUFA arachidonic acid (ARA, 20:4n-6) and linoleic acid (18:2n-6). The fatty acid profile of the seaweed collected in 2023 showed higher levels of n-6 polyunsaturated fatty acids, and lower levels of SFA and oleic acid (18:1n-9).

Importantly, in R.okamurae extracts the most reduced faction was the neutral lipid fraction followed by de glycolipid fraction, while other polar lipid fraction increased mainly in 2023 extract (Fig. 2). In fact, terpenes were importantly reduced (13-fold in 2023 samples, Table 3), which caused qualitative changes in the lipid profile of the extracts. Fatty acids, on the other hand, showed similar quantities on seaweed and extract (Table 4).

Table 4

Fatty acids composition (% respect total fatty acids) of *Rugulopteryx okamurae* and its aqueous extracts.
	RO22	RO23	RU22	RU23
14:0	5.7 ± 0.1 B	6.5 ± 0.3 A	4.3 ± 0.5 B	5.9 ± 0.8 A
15:0	0.9 ± 0.0 A	0.8 ± 0.1 B	0.6 ± 0.0 C	-
16:0	29.5 ± 0.6 B	24.0 ± 0.7 C	30.2 ± 1.2 B	31.5 ± 1.6 A
17:0	0.6 ± 0.0 AB	0.2 ± 0.0 C	1.0 ± 0.1 A	0.2 ± 0.4 BC
18:0	4.7 ± 0.2 C	3.1 ± 0.3 D	6.3 ± 0.4 B	7.7 ± 0.4 A
20:0	3.3 ± 0.2 AB	2.5 ± 0.0 C	3.3 ± 0.3 B	3.8 ± 0.6 A
24:0	0.4 ± 0.0 A	0.3 ± 0.0 A	0.5 ± 0.0 A	0.4 ± 0.4 A
SFA	45.0 ± 0.9 B	37.3 ± 0.9 C	45.9 ± 2.0 B	49.6 ± 1.7 A
14:1	0.4 ± 0.0 BC	0.3 ± 0.0 C	0.74 ± 0.0 B	1.6 ± 0.4 A
16:1n9	-	-	0.3 ± 0.3	-
16:1n7	6.2 ± 0.2 B	7.1 ± 0.2 A	2.3 ± 0.3 C	1.8 ± 0.3 D
16:1n5	5.4 ± 0.0 B	7.2 ± 0.2 A	3.5 ± 0.3 D	5.2 ± 0.3 C
17:1	0.3 ± 0.0 B	0.9 ± 0.1A	0.3 ± 0.0 B	-
18:1n9	15.0 ± 0.1 B	12.4 ± 0.2 C	17.3 ± 1.0 A	17.5 ± 1.7 A
18:1n5	1.0 ± 0.1 B	1.2 ± 0.0 A	0.5 ± 0.0 C	0.9 ± 0.0 B
18:1n7	0.8 ± 0.0 C	0.8 ± 0.0 C	1.2 ± 0.1 B	1.9 ± 0.2A
20:1n9	0.1 ± 0.1 A	-	0.2 ± 0.1 A	-
20:1n7	0.2 ± 0.0	-	-	-
MUFA	29.5 ± 0.1 A	29.9 ± 1.0 A	26.4 ± 1.4 B	28.5 ± 2.1 AB
18:2n6	1.8 ± 0.0 A	1.8 ± 0.1 A	2.6 ± 0.7 A	2.5 ± 0.8 A
18:3n6	0.2 ± 0.0 A	0.1 ± 0.0 B	2.2 ± 0.3 A	-
20:2n6	0.1 ± 0.0 B	-	0.2 ± 0.2 A	-
20:3n6	0.4 ± 0.0 B	0.6 ± 0.0 A	0.20 ± 0.0 C	-
20:4n6	3.01 ± 0.1 C	5.9 ± 0.1 A	1.97 ± 0.3 D	3.0 ± 0.3 B
22:4n6	0.8 ± 0.1 C	1.6 ± 0.0 B	1.6 ± 0.0 D	2.0 ± .0.3A
n-6 PUFA	6.5 ± 0.1 C	10.6 ± 0.1 A	7.0 ± 0.6 C	6.6 ± 0.6 C
18:3n3	1.8 ± 0.0 B	1.4 ± 0.0 C	2.1 ± 0.2 A	1.1 ± 0.1 D
18:4n3	1.4 ± 0.0 B	2.2 ± 0.0 A	1.2 ± 0.2 C	1.0 ± 0.2 C
20:4n3	0.8 ± 0.0 AB	1.1 ± 0.0A	0.5 ± 0.0 BC	0.4 ± 0.4 C
20:5n3	1.6 ± 0.0 C	2.3 ± 0.0 A	1.6 ± 0.3 BC	1.9 ± 0.2 B
21:5n3	1.6 ± 0.3 B	0.9 ± 0.7 B	2.9 ± 0.3 AB	4.6 ± 2.3 A
22:5n3	1.6 ± 0.1 B	0.3 ± 0.0 C	2.9 ± 0.3 A	0.3 ± 0.5 C
22:6n3	-	-	0.4 ± 0.4	-
n-3 PUFA	8.6 ± 0.4 A	8.2 ± 0.7 A	11.0 ± 0.6 A	9.3 ± 2.9 A
16:4n1	0.1 ± 0.0 AB	0.2 ± 0.3 AB	0.4 ± 0.3 A	-
18:4n1	-	-	0.3 ± 0.1	-
22:2	-	0.2 ± 0.2	-	-

Values are expressed as average ± standard deviation (n = 3). SFA. saturated fatty acids; MUFA. Total monosaturated fatty acids; PUFA. total polyunsaturated fatty acids. RO22. RO22, Rugulopteryx okamurae collected in 2022; RO23, Rugulopteryx okamurae collected in 2023; RU22, Rugulopteryx okamurae aqueous extract from the seaweed in 2022; RU23 Rugulopteryx okamurae aqueous extract from the seaweed in 2023. Letters represent significant differences (ANOVA. LSD Test. p < 0.05).

The effects of seaweed-based plant biostimulant vary depending on the type of seaweed used, the spatiotemporal source of the seaweed raw material, the conditions in the areas where the seaweed is collected, and the manufacturing process, including the temperature and pH conditions of the extraction process (Gaubert et al. 2019; Staykov et al. 2020; Goñi et al. 2021; López-Hortas et al. 2023).With all this in mind, the present work attempts to transform the threat of Rugulopteryx okamurae into an opportunity for agriculture throughout its valorization, contributing to circular bioeconomy. Ours results demonstrate that Rugulopteryx okamurae is susceptible of being valorized for agricultural proposes.

We evaluated the elemental composition (CNS and minerals) as well as biochemical composition of Rugulopteryx okamurae across two consecutive years. Our results showed C/N ratios around 19 in the seaweed for both years, which is consistent with values found in other brown seaweeds and indicative of nitrogen limitation (Sheppard et al. 2023). This relationship was improved in the aqueous seaweed extracts, in which C/N ranged from 27 to 34, values that are close to those recommended for fertilizers (Cole et al. 2016). Moreover, both seaweed and extracts had a rich mineral composition, which is interesting regarding agriculture since minerals are decisive for plant growth and development, suggesting this variation should be taken with care if this seaweed is intended to be used in agriculture. The seaweed resulted rich in K, Ca, Mg and Fe, this last is highlighting as iron combats chlorosis in vines, giving healthier greener foliage, and increasing sugar levels in berries and therefore alcohol levels in wines. Meanwhile K is crucial for grapevine growth and fruit development (Visconti et al. 2024). Special attention should be given to As, which showed high concentration in seaweeds and specially in the extracts, with values higher than those permitted for fertilizers (40 mg/kg, EU2019/1009). These high values were previously described for Rugulopteryx okamurae by Cebrián-Lloret et al., (2024), and seems to be characteristic for brown seaweed as Laminaria digitata, Laminaria hyperborea and Ascophyllum nodosum (Lorenzo et al. 2017; Garcia-Vaquero et al. 2021).

The biochemical composition of the seaweed was consistent with previous data reported by Vega et al. (2022) and Lopez-Hortas (2023) for Rugulopteryx okamurae collected in a nearby location, as well as for the observations made by Cebrian et al. (2024) when collecting the seaweed in Granada. However, there was a notable disparity in carbohydrate values, with Cebrian et al. (2024) reporting values close to 60%, while Vega et al. (2022) and the current study showing values around 8%. The differences could be attributed to the extraction and determination techniques, as Cebrian et al. utilized an acid methanolysis extraction and anion exchange chromatography for determination, while our study and Vega et al. (2022) employed the Dubois method. Regarding extracts, they were enriched mainly in sulfur, total carbohydrates and TPC, which was the goal of the extraction process since seaweed biostimulation properties in plants are mainly attributed to sulphated polysaccharides (EBIC, 2023) and therefore highlights the potential usage of the aqueous extract of Rugulopteryx okamurae for agriculture purposes. The high TPC values point to further characterization of phenolic profile of the algae in future studies. Other brown algae are described as a source of pholorotannins, a family of phenolic compounds with huge potential applications due to their biological properties (Ismail et al. 2023).

The polysaccharides content in seaweeds is species highly variable, with values ranging from 4–76% dw (Gullón et al. 2021). Brown seaweeds, in particular, can contain 30–50% of carbohydrates on dry weight, of which 19% possibly been alginates, while fucoidan and laminarin would be both present at 11% (Manns et al. 2017; Fernández-Medina et al. 2022). Among brown algae, Ascophyllum nodosum stands out as one of the most well-studied in terms of composition and is regarded as one of the primary sources for the commercial production of bioactive compounds (EBIC, 2023). Its benefits are mainly attributed to its polysaccharide composition: around 7% fucoidan (Fletcher et al. 2017), laminarin around 4% (Shukla et al. 2019) and alginate 6–22% (Ummat et al. 2024). The putative presence of polysaccharides such as fucoidan (1%), laminarin (0.5%) and alginates (6%) in Rugulopetryx okamurae seaweed would be indicative of the suitability of this algae for crop biostimulation, considering the previous attribution of bioactivity to these polysaccharides (Mamede et al. 2023). Few data are available regarding polysaccharide composition of Rugulopteryx okamurae. Cebrian-Lloret et al reported the alginate up to 32% (Cebrián-Lloret et al. 2024), by using acid methanolysis and high-performance anion exchange chromatography with pulsed amperometric detection. This is the first work in which the fucoidan and laminarin content have been reported for Rugulopteryx Okamurae. The aqueous extracts significantly increased the concentration of fucoidan (up to 3.5 folds) and especially of laminarin (up to 5.5 folds), which have been described as key compounds for biostimulants in plants (Shukla et al. 2019; Pagliarani et al. 2020).

Lipid content in the commonly used seaweeds does not exceed 5% of the dry biomass. Brown seaweed such as Ascophyllum nodosum or Rugulopteryx okamurae contain up to 4% and 8% of lipids on dry weight respectively (Lorenzo et al. 2017; Vega et al. 2022), which is in agreement with our data. Lipids play a key role in pathogen (or elicitor) recognition and in inciting host defence responses. (De Corato et al. 2017) demonstrated that high contents of fatty acids in marine brown seaweed may have a role in fungal treatment. In addition, it is now known that plant lipids play central roles in the biosynthesis of cutin, the stimulation of signalling pathways that trigger different immune responses, and the reprogramming of defence-related genes (Mehta et al. 2021). Among lipids, sterols and glycolipids have attracted great interest for their ability to control plant diseases (Melo et al. 2015). Our result show that glycolipids were the main fraction in 2022 seaweed, in contrast with 2023 seaweed where neutral lipid were the majoritarian. Both fractions contain interesting functional lipid such as glycolipids SQDG, DGDG and MGDG or neutral lipids such as fucosterol, fucoxantine and terpenes. Terpenes were described for the first time in Rugulopteryx okamurae by Casal-Porras et al. (2021) (Casal-Porras et al. 2021), who reported the quantitative significance of these compounds and identified dilkamural as the primary terpene. The value reported by these authors was similar to the determination of terpenes in the algae harvested in 2023 in the present study. Dilkamural is a potent toxic compound for herbivore and its potential as antifungal should be checked. As for fatty acids profile, in general seaweeds are rich in n3 and n-6 PUFAs. For Ascophyllum nodosum 25%, 31% and 44% of SFA, MUFA and PUFA have been reported (Lorenzo et al. 2017), meanwhile our data show a different distribution, 40%, 30% and 20% for SFA, MUFA and PUFA respectively, similar to values reported by Cebrian et al., (2024) (Cebrián-Lloret et al. 2024) for PUFA. Rugulopteryx okamurae present significant amounts of n3 PUFA and low n6/n3 ratio, indicating a good balance between essential fatty acids. As expected, the aqueous extract had lower amount of lipids and major lipid classes, particularly terpenes. Additionally, the fatty acid profile of the extract exhibited minimal differences compared to that of the seaweeds. Nevertheless, certain lipid classes, such as glycolipids SQGD and DGDG, retained their content in the extract.

The current study highlights the significant potential of Rugulopteryx okamurae for agricultural applications based on its composition, and therefore it is a first step for the valorization of its biomass. The novelty of the work lies in i) the determination of fucoidan, laminarin, SQDG, DGDG, MGDG and fucoxantine for the first time in Rugulopteryx okamurae, ii) the assessment of the potential of both seaweed and extracts as biofertilizer and/or biostimulant iii) the establishment of compositional variation between years, while assessing the seasonal variability of the algae, besides the annual one, would be necessary. Our data show important yearly variations in seaweed composition mainly in lipids and minerals, while elemental and biochemical composition as well as polysaccharides show lower variations. The macro (potassium and calcium) and micronutrient (iron and manganese) composition imply seaweed potential use as biofertilizer, although C/N ratio should be adjusted. The presence and abundance of various polysaccharides (mainly fucoidan, laminarin) and glycolipids support the capacity of the aqueous extracts, as plant biostimulant with possible antifungal properties. The arsenic content must be checked before using any of them (seaweed or extract) in agriculture, and developing suitable methods to reduce it, when necessary, would be advisable. Notwithstanding the data are reported, further research is required to fully understand the potential of this waste of biological source and, thus, future studies should determine how current extraction methods can be optimized to reap the greatest benefit from this invasive and still poorly characterized seaweed.

Ethical Approval

Not applicable

Funding

Funding for this research work come from MCIN/AEI/ 10.13039/501100011033 (PID2020-112644RR-C21 and -C22) and by FEDER co-finance at 80%, within the Operational Program for Andalusia 2021–2027 (AVA23.INV2023.008).

Author Contribution

Conceptualization, E. C-V. and I.H-C..; methodology, J.J. C-G, M.J. J-H and E.Z.; validation, R.O-H, B.P. and I. Z; formal analysis, J.J. C-G. and I. H-C.; investigation, J.J. C-G, M.J. J-H, and E.Z.; data curation,R. O-H, I. H-C, I.Z.. and E. C-V.; writing—original draft preparation, E. C-V.; writing—review and editing, R.O-H and I-Z.; visualization and supervision, B.P. and I. H-C; project administration, E. C-V; funding acquisition, B.P and E. C-V. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

This research work is supported by MCIN/AEI/ 10.13039/501100011033 (PID2020-112644RR-C21 and -C22) and by FEDER co-finance at 80%, within the Operational Program for Andalusia 2021–2027 (AVA23.INV2023.008). We thank the Strait of Gibraltar Natural Park for issuing the necessary permits for harvesting Rugulopteryx okamurae.

Data Availability

The data presented in this study are available on request from the corresponding author

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Biochemical characterization and potential valorization of the invasive seaweed Rugulopteryx okamurae

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1. Seaweed collection and extract elaboration

2.2. Elemental composition

2.3. Determination of ash, total carbohydrates, total lipids and total proteins

2.4. Total polyphenols determination and antioxidant capacity

2.5. Fucoidan, alginate and laminarin extraction and determination

2.6. Lipids extraction and determination

2.7. Statistical analysis

3. Results

3.1. Elements, minerals and metals

3.2. Biochemical composition

3.3. Polysaccharide composition

3.4. Lipid composition

4. Discussion

5. Conclusions

Declarations

Ethical Approval

Funding

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1