An Eco-Friendly Extraction and Purification Approach for Obtaining Active Ingredients for Cosmetics from Two Marine Brown Seaweeds.

Brown seaweeds are attracting attention due to their richness in bioactive compounds, in particular, their phlorotannins. We present here a case study of two Fucales, Ascophyllum nodosum and Halidrys siliquosa, sustainably collected, to produce active polyphenols for the cosmetics sector. Phenolic contents of crude extracts, obtained by Accelerated Solvent Extraction (ASE), were more elevated in H. siliquosa at 100.05 mg/g dry weight (DW) than in A. nodosum (29.51 mg/g DW), considering 3 cycles with cell inversion. The temperature of extraction for a high phenolic content and high associated antioxidant activities close to positive controls was 150 °C for both algae and the use of only one cycle was enough. A semi-purification process using Solid-phase Extraction (SPE) was carried out on both ASE crude extracts (one per species). The majority of phlorotannins were found in the ethanolic SPE fraction for A. nodosum and the hydroethanolic one for H. siliquosa. The SPE process allowed us to obtain more concentrated fractions of active phenolic compounds (×1.8 and 2 in A. nodosum and H. siliquosa, respectively). Results are discussed in regard to the exploitation of seaweeds in Brittany and to the research of sustainable processes to produce active natural ingredients for cosmetics.

Mycosporine-like Amino Acids in Palmaria palmata (Rhodophyta): Specific Implication of Usujirene in Photoprotection.

The effect of UV radiation on the accumulation of mycosporine-like amino acids (MAAs) and pigments was investigated on red macroalga Palmaria palmata cultivated for 21 days. The data were combined with the effect of NaNO3 to further investigate the synthesis of these nitrogenous compounds. A progressive decrease in both total MAA and pigment contents was observed, with a positive effect of nitrate supply. Usujirene was the only MAA exhibiting a significantly increasing content when exposed to UV radiation, changing from 9% to 24% of the total MAA's contribution, with no variation observed with NaNO3. This suggests a specific induction or synthesis pathway of usujirene for photoprotection, while the synthesis of other MAAs could have been limited by an insufficient amount of UV radiation and/or irradiance. The photoprotective ability of some MAAs could have been impacted by nitrogen starvation over time, resulting in a limited synthesis and/or potential use of MAAs as a nitrogen source for red macroalgae. The data confirmed the multiple effects of environmental factors on the synthesis of MAAs while providing new insights into the specific synthesis of usujirene, which could find an application in the cosmetics sector as natural sunscreen or an anti-ageing agent.

Predominant heterotrophic diazotrophic bacteria are involved in Sargassum proliferation in the Great Atlantic Sargassum Belt.

Since 2011, the Caribbean coasts have been subject to episodic influxes of floating Sargassum seaweed of unprecedented magnitude originating from a new area "the Great Atlantic Sargassum Belt" (GASB), leading in episodic influxes and mass strandings of floating Sargassum. For the biofilm of both holopelagic and benthic Sargassum as well as in the surrounding waters, we characterized the main functional groups involved in the microbial nitrogen cycle. The abundance of genes representing nitrogen fixation (nifH), nitrification (amoA), and denitrification (nosZ) showed the predominance of diazotrophs, particularly within the GASB and the Sargasso Sea. In both location, the biofilm associated with holopelagic Sargassum harboured a more abundant proportion of diazotrophs than the surrounding water. The mean δ15N value of the GASB seaweed was very negative (-2.04‰), and lower than previously reported, reinforcing the hypothesis that the source of nitrogen comes from the nitrogen-fixing activity of diazotrophs within this new area of proliferation. Analysis of the diversity of diazotrophic communities revealed for the first time the predominance of heterotrophic diazotrophic bacteria belonging to the phylum Proteobacteria in holopelagic Sargassum biofilms. The nifH sequences belonging to Vibrio genus (Gammaproteobacteria) and Filomicrobium sp. (Alphaproteobacteria) were the most abundant and reached, respectively, up to 46.0% and 33.2% of the community. We highlighted the atmospheric origin of the nitrogen used during the growth of holopelagic Sargassum within the GASB and a contribution of heterotrophic nitrogen-fixing bacteria to a part of the Sargassum proliferation.

Kinetics of metal and metalloid concentrations in holopelagic Sargassum reaching coastal environments.

Since 2011, the Caribbean Islands have experienced unprecedented stranding of a pelagic brown macroalgae Sargassum inducing damages for coastal ecosystems and economy. This study measures the kinetics of metal trace elements (MTE) in Sargassum reaching different coastal environments. In July 2021, over a period of 25 days, fixed experimental floating cages containing the three Sargassum morphotypes (S. fluitans III and S. natans I and VIII) were placed in three different coastal habitats (coral reef, seagrass, and mangrove) in Guadeloupe (French West Indies). Evolution of biomasses and their total phenolic content of Sargassum reveals that environmental conditions of caging were stressful and end up to the death of algae. Concentrations of 19 metal(loid) trace elements were analyzed and three shapes of kinetics were identified with the MTE that either concentrate, depurate, or remains stable. In the mangrove, evolution of MTE was more rapid than the two other habitats a decrease of the As between 70 and 50 µg g-1 in the mangrove. Sargassum natans I presented a different metal composition than the two other morphotypes, with higher contents of As and Zn. All Sargassum morphotype are rapidly releasing the metal(oid)s arsenic (As) when they arrive in studied coastal habitats. In order to avoid the transfer of As from Sargassum to coastal environments, Sargassum stranding should be avoided and their valorization must take into account their As contents.

Trace metal content from holopelagic Sargassum spp. sampled in the tropical North Atlantic Ocean: Emphasis on spatial variation of arsenic and phosphorus.

We document for the first time, the spatial distribution at basin scale (North tropical Atlantic Ocean) of As, P and trace metal (TM) concentrations in the three morphotypes belonging to the two holopelagic species Sargassum natans and S. fluitans and three morphotypes: S. natans VIII, S. natans I and S. fluitans III. These samples collected in the North equatorial current (NEC) and in the subtropical Sargasso Sea (sSS) (∼25°N, 60°W) were also compared to coastal samples collected downwind Guadeloupe Island and on the strand of Martinique (mangrove and beach). Along the studied zonal oceanic transect, the highest values of As (range 120-240 µg g-1, dry weight, dw) were found in the sSS area where primary production is highly limited by phosphorus. At these stations, the P content of Sargassum spp. was minimal (range 500-1000 µg g-1, dw) as well as the content in Cd and Zn known for their nutrient-like oceanic behaviors and distributions very similar to P. This illustrates for the first time in the natural environment, the higher bioaccumulation of arsenic in Sargassum spp. in P-limiting conditions which is due to the competition in the phosphate transporter between arsenate and phosphate. As compared to samples collected at sea, the Sargassum spp. collected in the strand of Martinique had (1) lower As concentrations (typical range 30-45 µg g-1, dw) and (2) much higher Al, Fe, Mn, Cr and Co concentrations, showing a certain ability of Sargassum spp. to be depurated of its As content in the coastal zone following competitive exchange with terrigenous metals.

Phlorotannin and Pigment Content of Native Canopy-Forming Sargassaceae Species Living in Intertidal Rockpools in Brittany (France): Any Relationship with Their Vertical Distribution and Phenology?

Five native Sargassaceae species from Brittany (France) living in rockpools were surveyed over time to investigate photoprotective strategies according to their tidal position. We gave evidences for the existence of a species distribution between pools along the shore, with the most dense and smallest individuals in the highest pools. Pigment contents were higher in lower pools, suggesting a photo-adaptive process by which the decreasing light irradiance toward the low shore was compensated by a high production of pigments to ensure efficient photosynthesis. Conversely, no xanthophyll cycle-related photoprotective mechanism was highlighted because high levels of zeaxanthin rarely occurred in the upper shore. Phlorotannins were not involved in photoprotection either; only some lower-shore species exhibited a seasonal trend in phlorotannin levels. The structural complexity of phlorotannins appears more to be a taxonomic than an ecological feature: Ericaria produced simple phloroglucinol while Cystoseira and Gongolaria species exhibited polymers. Consequently, tide pools could be considered as light-protected areas on the intertidal zone, in comparison with the exposed emerged substrata where photoprotective mechanisms are essential.

Short-term effects of increased CO2, nitrate and temperature on photosynthetic activity in Ulva rigida (Chlorophyta) estimated by different pulse amplitude modulated fluorometers and oxygen evolution.

Short-term effects of pCO2 (700-380 ppm; High carbon (HC) and Low carbon (LC), respectively) and nitrate content (50-5 µM; High nitrogen (HN) and Low nitrogen (LN), respectively on photosynthesis were investigated in Ulva rigida (Chlorophyta) under solar radiation (in-situ) and in the laboratory under artificial light (ex-situ). After six days of incubation at ambient temperature (AT), algae were subjected to a 4 °C temperature increase (AT+4 °C) for 3 d. Both in-situ and ex-situ maximal electron transport rate (ETRmax) and in situ gross photosynthesis (GP), measured by O2 evolution, presented highest values under HCHN, and lowest under HCLN, across all measuring systems. Maximal quantum yield (Fv/Fm), and ETRmax of photosystem (PS) II [ETR(II)max] and PSI [ETR(I)max], decreased under HCLN at AT+4 °C. Ex situ ETR was higher than in situ ETR. At noon, Fv/Fm decreased (indicating photoinhibition), whereas ETR(II)max and maximal non-photochemical quenching (NPQmax) increased. ETR(II)max decreased under AT+ 4 °C in contrast to Fv/Fm, photosynthetic efficiency (α ETR) and saturated irradiance (EK). Thus, U. rigida exhibited a decrease in photosynthesis under acidification, changing LN, and AT+4 °C. These results emphasize the importance of studying the interaction between environmental parameters using in-situ versus ex-situ conditions, when aiming to evaluate the impact of global change on marine macroalgae.

A New Protocol Using Acidification for Preserving DMSP in Macroalgae and Comparison with Existing Protocols.

Dimethylsulfoniopropionate (DMSP) plays many important physiological and ecological roles in macroalgae. The most common method to measure DMSP is by gas chromatography analysis of the dimethylsulfide (DMS) produced after NaOH hydrolysis (pH > 12). Storage of DMS, however, is not recommended for more than a week. We investigated if acidification can be a suitable method to preserve DMSP in macroalgal samples over three months of storage, compared to widely used protocols such as drying and freezing at -20°C. The DMSP content of green (Ulva sp. and Ulva compressa), red (Chondrus crispus), and brown (Bifurcaria bifurcata) macroalgae were analyzed: 24 h after NaOH addition (control values); and after acidification (0.2 mol · L HCl-1 ) for 24 h of fresh material, followed by NaOH addition for 24 h. These values were compared to measurements after 3-month storage of samples that had been either dried in a heater (60°C for a night, and storage at room temperature), or frozen at -20°C, or kept in 0.2 mol · L HCl-1 . There was no significant difference between DMSP measurements on freshly collected material and after acidification of the samples, whether 24 h later or after 3 months of storage. This was in contrast to 3-month storage protocols involving overnight drying at 60°C (75-98% DMSP loss), and to a lesser degree freezing at -20°C (37-80% DMSP loss). We thus advise to acidify macroalgal samples for preservation over long periods of time rather than drying or freezing, when assaying DMSP content.

From In Situ to satellite observations of pelagic Sargassum distribution and aggregation in the Tropical North Atlantic Ocean.

The present study reports on observations carried out in the Tropical North Atlantic in summer and autumn 2017, documenting Sargassum aggregations using both ship-deck observations and satellite sensor observations at three resolutions (MSI-10 m, OLCI-300 m, VIIRS-750 m and MODIS-1 km). Both datasets reported that in summer, Sargassum aggregations were mainly observed off Brazil and near the Caribbean Islands, while they accumulated near the African coast in autumn. Based on in situ observations, we propose a five-class typology allowing standardisation of the description of in situ Sargassum raft shapes and sizes. The most commonly observed Sargassum raft type was windrows, but large rafts composed of a quasi-circular patch hundreds of meters wide were also observed. Satellite imagery showed that these rafts formed larger Sargassum aggregations over a wide range of scales, with smaller aggregations (of tens of m2 area) nested within larger ones (of hundreds of km2). Match-ups between different satellite sensors and in situ observations were limited for this dataset, mainly because of high cloud cover during the periods of observation. Nevertheless, comparisons between the two datasets showed that satellite sensors successfully detected Sargassum abundance and aggregation patterns consistent with in situ observations. MODIS and VIIRS sensors were better suited to describing the Sargassum aggregation distribution and dynamics at Atlantic scale, while the new sensors, OLCI and MSI, proved their ability to detect Sargassum aggregations and to describe their (sub-) mesoscale nested structure. The high variability in raft shape, size, thickness, depth and biomass density observed in situ means that caution is called for when using satellite maps of Sargassum distribution and biomass estimation. Improvements would require additional in situ and airborne observations or very high-resolution satellite imagery.

Photo-protective compounds in red macroalgae from Brittany: Considerable diversity in mycosporine-like amino acids (MAAs).

To cope with the biotic and abiotic stresses experienced within their environment, marine macroalgae have developed certain defence mechanisms including the synthesis of photo-protective molecules against light and particularly harmful UV radiation. The aim of this study was to screen selected red algae, a highly diverse phylogenetic group, for the production of photo-protective molecules. The pigment content and composition (i.e. chlorophyll-a, phycobiliproteins and carotenoids) and the composition of mycosporine-like amino acids (MAAs) were studied in 40 species of red macroalgae collected in Brittany (France), at two distinct periods (i.e. February and July 2017). A high inter-specific variability was demonstrated in terms of pigment content and MAA composition. Twenty-three potential MAAs were detected by HPLC, and six were identified by LC-MS (i.e. shinorine, palythine, asterina-330, porphyra-334, usurijene and palythene). This is the first study to report on the composition of pigments and MAAs in a diverse group of red seaweeds from Brittany, including some species for which the MAA composition has never been studied before. Nevertheless, the results suggested that some species of red algae are more likely to cope with high levels of light radiation since those species such as Bostrychia scorpioides, Porphyra dioica, Gracilaria vermiculophylla and Vertebrata lanosa are living in environments exposed to higher levels of irradiation, and had various MAAs in addition to their photo-protective pigments.

Connecting marine productivity to sea-spray via nanoscale biological processes: Phytoplankton Dance or Death Disco?

Bursting bubbles at the ocean-surface produce airborne salt-water spray-droplets, in turn, forming climate-cooling marine haze and cloud layers. The reflectance and ultimate cooling effect of these layers is determined by the spray's water-uptake properties that are modified through entrainment of ocean-surface organic matter (OM) into the airborne droplets. We present new results illustrating a clear dependence of OM mass-fraction enrichment in sea spray (OMss) on both phytoplankton-biomass, determined from Chlorophyll-a (Chl-a) and Net Primary Productivity (NPP). The correlation coefficient for OMss as a function of Chl-a increased form 0.67 on a daily timescale to 0.85 on a monthly timescale. An even stronger correlation was found as a function of NPP, increasing to 0.93 on a monthly timescale. We suggest the observed dependence is through the demise of the bloom, driven by nanoscale biological processes (such as viral infections), releasing large quantities of transferable OM comprising cell debris, exudates and other colloidal materials. This OM, through aggregation processes, leads to enrichment in sea-spray, thus demonstrating an important coupling between biologically-driven plankton bloom termination, marine productivity and sea-spray modification with potentially significant climate impacts.

Marine Algae: a Source of Biomass for Biotechnological Applications.

Biomass derived from marine microalgae and macroalgae is globally recognized as a source of valuable chemical constituents with applications in the agri-horticultural sector (including animal feeds and health and plant stimulants), as human food and food ingredients as well as in the nutraceutical, cosmeceutical, and pharmaceutical industries. Algal biomass supply of sufficient quality and quantity however remains a concern with increasing environmental pressures conflicting with the growing demand. Recent attempts in supplying consistent, safe and environmentally acceptable biomass through cultivation of (macro- and micro-) algal biomass have concentrated on characterizing natural variability in bioactives, and optimizing cultivated materials through strain selection and hybridization, as well as breeding and, more recently, genetic improvements of biomass. Biotechnological tools including metabolomics, transcriptomics, and genomics have recently been extended to algae but, in comparison to microbial or plant biomass, still remain underdeveloped. Current progress in algal biotechnology is driven by an increased demand for new sources of biomass due to several global challenges, new discoveries and technologies available as well as an increased global awareness of the many applications of algae. Algal diversity and complexity provides significant potential provided that shortages in suitable and safe biomass can be met, and consumer demands are matched by commercial investment in product development.

Spectrophotometric Assays of Major Compounds Extracted from Algae.

This chapter describes spectrophotometric assays of major compounds extracted from microalgae and macroalgae, i.e., proteins, carbohydrates, pigments (chlorophylls, carotenoids, and phycobiliproteins) and phenolic compounds. In contrast to other specific analytical techniques, such as high pressure liquid chromatography (HPLC) or mass spectrometry (MS), commonly applied to purified extracts to reveal more detailed composition and structure of algal compound families, these assays serve as a first assessment of the global contents of extracts.

Chlorophyll a fluorescence responses of temperate Phaeophyceae under submersion and emersion regimes: a comparison of rapid and steady-state light curves.

The potential of algae to acclimate to environmental stress is commonly assessed using chlorophyll a fluorescence, with changes in parameters of photosynthesis versus irradiance (P/E) curves measured either as rapid light curves (RLC) or steady-state light curves (LC). Here, effects of emersion on primary photosynthesis of four brown macroalgae (Ascophyllum nodosum, Fucus serratus, Sargassum muticum, Laminaria digitata) were compared by applying both RLC and LC. When LC were used, photosynthetic performance was enhanced during emersion in A. nodosum and F. serratus as shown by increases in q(P), rETR(max) and E(k). By contrast, emersion had no impact on photosynthetic parameters of S. muticum and L. digitata. Relative changes in the NPQ-rETR relationship were reduced in A. nodosum, F. serratus and S. muticum, but remained unaffected in L. digitata. As none of the species developed their potential NPQ(max), corresponding values could not be determined from RLC. Using RLC, observed photosynthetic performance of F. serratus and L. digitata was reduced upon emersion, whilst values for NPQ(max) were enhanced. Only results derived from LC provide evidence for a potential physiological adaptation of brown macroalgae to their natural habitat; it is recommended using the LC protocol to detect environmental impacts on photosynthesis.

Profiling phlorotannins in brown macroalgae by liquid chromatography-high resolution mass spectrometry.

INTRODUCTION: Phlorotannins, phenolic compounds produced exclusively by Phaeophyceae (brown algae), have recently been associated with a wide variety of beneficial bioactivities. Several studies have measured the total phenolic content in extracts from various species, but little characterisation of individual phlorotannin components has been demonstrated. OBJECTIVE: The purpose of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) based method for rapid profiling of phlorotannins in brown algae. METHODOLOGY: Phlorotannin-enriched extracts from five phaeophyceaen species were analysed by ultrahigh-pressure liquid chromatography (UHPLC) operating in hydrophilic interaction liquid chromatography (HILIC) mode combined with high resolution mass spectrometry (HRMS). The method was optimised using an extract of Fucus vesiculosus; separation was achieved in less than 15 min. The basic mobile phase enhanced negative-ion electrospray ionisation (ESI), and generated multiply charged ions that allowed detection of high molecular weight phlorotannins. RESULTS: The phlorotannin profiles of Pelvetia canaliculata, Fucus spiralis, F. vesiculosus, Ascophyllum nodosum and Saccharina longicruris differed significantly. Fucus vesiculosus yielded a high abundance of low molecular weight (< 1200 Da) phlorotannins, while P. canaliculata exhibited a more evenly distributed profile, with moderate degrees of polymerisation ranging from 3 to 49. HRMS enabled the identification of phlorotannins with masses up to 6000 Da using a combination of accurate mass and ¹³C isotopic patterns. CONCLUSION: The UHPLC-HRMS method described was successful in rapidly profiling phlorotannins in brown seaweeds based on their degree of polymerisation. HILIC was demonstrated to be an effective separation mode, particularly for low molecular weight phlorotannins.

Algal chemodiversity and bioactivity: sources of natural variability and implications for commercial application.

There has been significant recent interest in the commercial utilisation of algae based on their valuable chemical constituents many of which exhibit multiple bioactivities with applications in the food, cosmetic, agri- and horticultural sectors and in human health. Compounds of particular commercial interest include pigments, lipids and fatty acids, proteins, polysaccharides and phenolics which all display considerable diversity between and within taxa. The chemical composition of natural algal populations is further influenced by spatial and temporal changes in environmental parameters including light, temperature, nutrients and salinity, as well as biotic interactions. As reported bioactivities are closely linked to specific compounds it is important to understand, and be able to quantify, existing chemical diversity and variability. This review outlines the taxonomic, ecological and chemical diversity between, and within, different algal groups and the implications for commercial utilisation of algae from natural populations. The biochemical diversity and complexity of commercially important types of compounds and their environmental and developmental control are addressed. Such knowledge is likely to help achieve higher and more consistent levels of bioactivity in natural samples and may allow selective harvesting according to algal species and local environmental conditions for different groups of compounds.

Impacts of ambient salinity and copper on brown algae: 2. Interactive effects on phenolic pool and assessment of metal binding capacity of phlorotannin.

The aim of this study was to establish in laboratory experiments a quantitative link between phenolic pool (production, composition and exudation) in Ascophyllum nodosum and Fucus vesiculosus and their potential to bind metals. Additionally, the copper binding capacity of purified phlorotannin was investigated. A reduction in salinity decreased total phenolic contents, altered phenolic composition by increasing proportion of cell-wall phenolics, and also increased phenolic exudation of the two seaweed species. After 15 days at a salinity of 5, the inhibition of photosynthesis observed previously for A. nodosum coincided with the high exudation of phenolic compounds into the surrounding water of the seaweed tips which resulted in a significant reduction of phenolic contents. Increased copper concentration also reduced total phenolic contents, changed phenolic composition (increase in proportion and level of cell-wall phenolics), and positively affected phenolic exudation of A. nodosum and F. vesiculosus. A decrease in salinity enhanced the copper toxicity and caused the earlier impact on the physiology of seaweed tips. An involvement of phlorotannins in copper binding is also demonstrated; purified phlorotannins from A. nodosum collected from a site with little anthropogenic activity contained all four metals tested. When placed in copper-enriched water, as for the seaweed material, copper contents of the phenolics increased, zinc and cadmium contents decreased, but no change in chromium content was observed. The use of cell-wall phenolic content as biomarker of copper contamination seems promising but needs further investigation.

Impacts of ambient salinity and copper on brown algae: 1. Interactive effects on photosynthesis, growth, and copper accumulation.

The effect of copper enrichment and salinity on growth, photosynthesis and copper accumulation of two temperate brown seaweeds, Ascophyllum nodosum and Fucus vesiculosus, was investigated in laboratory experiments. A significant negative impact of reduced salinity on photosynthetic activity and growth was observed for both species. After 15 days at a salinity of 5, photosynthesis of A. nodosum was entirely inhibited and growth ceased at a salinity of 15. Increased copper concentration negatively affected photosynthetic activity of A. nodosum and F. vesiculosus resulting in chlorosis and reduced seaweed growth; 5 mg L⁻¹ copper caused an inhibition of the photosynthesis and the degradation of seaweed tips. Under reduced salinity, copper toxicity was enhanced and caused an earlier impact on the physiology of seaweed tips. After exposure to copper and different salinities for 15 days, copper contents of seaweeds were closely related to copper concentration in the water; seaweed copper contents reached their maximum after 1 day of exposure; contents only increased again when additional, free copper was added to the water. At high water copper concentrations or low salinity, or a combination of both, copper content of A. nodosum decreased. By contrast, copper content of F. vesiculosus increased, suggesting that different binding sites or uptake mechanisms exist in the two species. The results suggest that when using brown seaweeds in biomonitoring in situ, any change in the environment will directly and significantly affect algal physiology and thus their metal binding capacity; the assessment of the physiological status of the algae in combination with the analysis of thallus metal content will enhance the reliability of the biomonitoring process.