Exploring Ultrasound, Microwave and Ultrasound-Microwave Assisted Extraction Technologies to Increase the Extraction of Bioactive Compounds and Antioxidants from Brown Macroalgae.

This study aims to determine the influence of (1) ultrasound-assisted extraction (UAE), (2) microwave-assisted extraction (MAE) and (3) a combination of ultrasound-microwave-assisted extraction (UMAE) on the yields of fucose-sulphated polysaccharides (FSPs), total soluble carbohydrates and antioxidants extracted from A. nodosum. Scanning electron microscopy (SEM) was used to evaluate the influence of the extraction technologies on the surface of macroalgae while principal component analysis was used to assess the influence of the extraction forces on the yields of compounds. UMAE generated higher yields of compounds compared to UAE and MAE methods separately. The maximum yields of compounds achieved using UMAE were: FSPs (3533.75 ± 55.81 mg fucose/100 g dried macroalgae (dm)), total soluble carbohydrates (10408.72 ± 229.11 mg glucose equivalents/100 g dm) and phenolic compounds (2605.89 ± 192.97 mg gallic acid equivalents/100 g dm). The antioxidant properties of the extracts showed no clear trend or extreme improvements by using UAE, MAE or UMAE. The macroalgal cells were strongly altered by the application of MAE and UMAE, as revealed by the SEM images. Further research will be needed to understand the combined effect of sono-generated and microwave-induced modifications on macroalgae that will allow us to tailor the forces of extraction to target specific molecules.

Light-induced dynamic structural color by intracellular 3D photonic crystals in brown algae.

Natural photonic crystals are responsible for strong reflectance at selective wavelengths in different natural systems. We demonstrate that intracellular opal-like photonic crystals formed from lipids within photosynthetic cells produce vivid structural color in the alga Cystoseira tamariscifolia. The reflectance of the opaline vesicles is dynamically responsive to environmental illumination. The structural color is present in low light-adapted samples, whereas higher light levels produce a slow disappearance of the structural color such that it eventually vanishes completely. Once returned to low-light conditions, the color re-emerges. Our results suggest that these complex intracellular natural photonic crystals are responsive to environmental conditions, changing their packing structure reversibly, and have the potential to manipulate light for roles beyond visual signaling.

High-density genetic map and identification of QTLs for responses to temperature and salinity stresses in the model brown alga Ectocarpus.

Deciphering the genetic architecture of adaptation of brown algae to environmental stresses such as temperature and salinity is of evolutionary as well as of practical interest. The filamentous brown alga Ectocarpus sp. is a model for the brown algae and its genome has been sequenced. As sessile organisms, brown algae need to be capable of resisting the various abiotic stressors that act in the intertidal zone (e.g. osmotic pressure, temperature, salinity, UV radiation) and previous studies have shown that an important proportion of the expressed genes is regulated in response to hyposaline, hypersaline or oxidative stress conditions. Using the double digest RAD sequencing method, we constructed a dense genetic map with 3,588 SNP markers and identified 39 QTLs for growth-related traits and their plasticity under different temperature and salinity conditions (tolerance to high temperature and low salinity). GO enrichment tests within QTL intervals highlighted membrane transport processes such as ion transporters. Our study represents a significant step towards deciphering the genetic basis of adaptation of Ectocarpus sp. to stress conditions and provides a substantial resource to the increasing list of tools generated for the species.

Phototaxis and chemotaxis of brown algal swarmers.

Brown algae exhibit three patterns of sexual reproduction: isogamy, anisogamy, and oogamy. Unicellular swarmers including gametes and zoospores bear two heterogenous flagella, an anterior flagellum with mastigonemes (fine tripartite hairs) and a posterior one. In seawater, these flagellates usually receive physico-chemical signals for finding partners and good habitats. It is well known that brown algal swarmers change their swimming direction depending on blue light (phototaxis), and male gametes do so, based on the sex pheromones from female gametes (chemotaxis). In recent years, the comparative analysis of chemotaxis in isogamy, anisogamy, and oogamy has been conducted. In this paper, we focused on the phototaxis and chemotaxis of brown algal gametes comparing the current knowledge with our recent studies.

Stress Tolerance of the Endemic Antarctic Brown Alga Desmarestia anceps to UV Radiation and Temperature is Mediated by High Concentrations of Phlorotannins.

The endemic Antarctic brown macroalga Desmarestia anceps is strongly shade-adapted, but shows also a high capacity to cope with different environmental stressors, e.g. UV radiation and temperature. Therefore, this species colonizes wide depth gradients, which are characterized by changing environmental conditions. In this study, we examine whether the different physiological abilities allowing D. anceps to grow across a wide depth range is determined by high levels of phlorotannins. Photosynthesis, measured by PAM-fluorometry, the contents of soluble phlorotannins, antioxidant capacities of field grown were analyzed in response to different conditions of radiation (PAR and PAR + UV) and temperature (2, 7 and 12°C). The results show that maximal quantum of fluorescence (Fv /Fm ) decreased with increasing doses of UV radiation, but remained unaffected by temperature. High levels of soluble phlorotannins were detected and confirmed by microscopic observation revealing the abundance of large physodes. Exposure to UV radiation and elevated temperature showed that phlorotannins were not inducible by UV but increased at 12°C. ROS scavenging capacity was positively correlated with the contents of phlorotannins. In general, highest contents of phlorotannins were correlated with the lowest inhibition of Fv /Fm in all experimental treatments, highlighting the UV-protective role of these compounds in D. anceps.

Seasonal biochemical and photophysiological responses in the intertidal macroalga Cystoseira tamariscifolia (Ochrophyta).

Seasonal changes in the biochemistry and photophysiology of the brown macroalga Cystoseira tamariscifolia was analyzed in southern Spain. Total carbon and nitrogen contents, phenolic compounds, antioxidant and photosynthetic activities were seasonally determined over two years. Carbon, nitrogen and photoprotective phenolic contents were higher in winter and spring than in summer and autumn. Antioxidant levels were highest in spring and we found a positive correlation between phenolic content and antioxidant activity (EC50). Photosynthetic capacity (ETRmax) and photosynthetic efficiency (αETR) were also highest in spring, and there was a positive correlation between ETRmax and the amount of phenols present. Increased irradiance in spring enhanced algal productivity, antioxidant activity and the production of photoprotective compounds but in summer nutrient depletion due to thermal stratification of coastal waters reduced photosynthetic activity and the photoprotective capacity of C. tamariscifolia. Electron microscopy showed that phenols occurred in the cytoplasm of cortical cells inside physodes. Spring would be the best period to harvest C. tamariscifolia to extract photoprotectors and antioxidants for potential commercial uses, although the environmental impacts would need to be carefully assessed.

Lack of Physiological Depth Patterns in Conspecifics of Endemic Antarctic Brown Algae: A Trade-Off between UV Stress Tolerance and Shade Adaptation?

A striking characteristic of endemic Antarctic brown algae is their broad vertical distribution. This feature is largely determined by the shade adaptation in order to cope with the seasonal variation in light availability. However, during spring-summer months, when light penetrates deep in the water column these organisms have to withstand high levels of solar radiation, including UV. In the present study we examine the light use characteristics in parallel to a potential for UV tolerance (measured as content of phenolic compounds, antioxidant activity and maximum quantum yield of fluorescence) in conspecific populations of four Antarctic brown algae (Ascoseira mirabilis, Desmarestia menziesii, D. anceps and Himantothallus grandifolius) distributed over a depth gradient between 5 and 30 m. The main results indicated that a) photosynthetic efficiency was uniform along the depth gradient in all the studied species, and b) short-term (6 h) exposure to UV radiation revealed a high tolerance measured as chlorophyll fluorescence, phlorotannin content and antioxidant capacity. Multivariate analysis of similarity indicated that light requirements for photosynthesis, soluble phlorotannins and antioxidant capacity are the variables determining the responses along the depth gradient in all the studied species. The suite of physiological responses of algae with a shallower distribution (A. mirabilis and D. menziesii) differed from those with deeper vertical range (D. anceps and H. grandifolius). These patterns are consistent with the underwater light penetration that defines two zones: 0-15 m, with influence of UV radiation (1% of UV-B and UV-A at 9 m and 15 m respectively) and a zone below 15 m marked by PAR incidence (1% up to 30 m). These results support the prediction that algae show a UV stress tolerance capacity along a broad depth range according to their marked shade adaptation. The high contents of phlorotannins and antioxidant potential appear to be strongly responsible for the lack of clear depth patterns in light demand characteristics and UV tolerance.

Ocean acidification modulates the response of two Arctic kelps to ultraviolet radiation.

The combined effects of ocean acidification and ultraviolet radiation (UVR) have been studied in the kelps Alaria esculenta and Saccharina latissima from Kongsfjorden (Svalbard), two major components of the Arctic macroalgal community, in order to assess their potential to thrive in a changing environment. Overall results revealed synergistic effects, however with a different amplitude in the respective species. Changes in growth, internal N, C:N ratio, pigments, optimum quantum yield (Fv/Fm) and electron transport rates (ETR) following CO2 enrichment and/or UVR were generally more pronounced in S. latissima than in A. esculenta. The highest growth rates were recorded under simultaneous CO2 enrichment and UVR in both species. UVR-mediated changes in pigment content were partially prevented under elevated CO2 in both species. Similarly, UVR led to increased photosynthetic efficiency (α) and ETR only if CO2 was not elevated in A. esculenta and even under high CO2 in S. latissima. Increased CO2 did not inhibit external carbonic anhydrase (eCA) activity in the short-term but in the mid-term, indicating a control through acclimation of photosynthesis rather than a direct inhibition of eCA by CO2. The higher benefit of simultaneous CO2 enrichment and UVR for S. latissima respect to A. esculenta seems to involve higher C and N assimilation efficiency, as well as higher ETR, despite a more sensitive Fv/Fm. The differential responses shown by these two species indicate that ongoing ocean acidification and UVR could potentially change the dominance at lower depths (4-6m), which will eventually drive changes at the community level in the Arctic coastal ecosystem. These results support an existing consideration of S. latissima as a winner species in the global change scenario.

Antioxidant marine algae phlorotannins and radioprotection: a review of experimental evidence.

Radiation has been widely used for cancer therapy in human medicine. However, the side effects of radiation are problematic and can limit its application. Radiation generates reactive oxygen species, leading to cell death via multiple signaling pathways. The blocking of certain signaling cascades using antioxidants represents a compensatory therapy of radiation-induced tissue injury. Although synthetic chemicals have been investigated in recent decades, anti-oxidants from natural resources have been searched for continuously. Among them, phlorotannins from marine algae, including Ecklonia cava, have been shown to protect cells from radiation-induced injury as well as oxidative stress. In the present review, the radioprotective capacity of phlorotannins derived from marine algae and the mechanisms involved are discussed.

Vulnerability and acclimation to increased UVB radiation in three intertidal macroalgae of different morpho-functional groups.

The vulnerability and acclimation to increased UVB radiation in three macroalgae of different morpho-functional groups collected in the Mediterranean coastal waters were evaluated. The algae were submitted for 7 days to increased (PAB+) and decreased (PAB-) UVB radiation. The thickness and morphology influenced the response to increased UVB radiation, being Cystoseira tamariscifolia the less vulnerable algae followed by Ellisolandia elongata. The highest resistance to increased UVB radiation in C. tamariscifolia was related to the accumulation of polyphenols and high antioxidant activity, whereas E. elongata was due to its high reflectance. Finally, Ulva rigida suffered the highest photoinhibition under PAB+ culture. The latest species presented 10 times lower polyphenol content and antioxidant activity than C. tamariscifolia. The three species showed different acclimation patterns to the changes of UVB radiation related to the morphology, photosynthetic activity, accumulation of photoprotectors and antioxidant activities. The ecological implications of the UVB variations on macroalgae are discussed.

Photoacclimation responses of the brown macroalga Sargassum Cymosum to the combined influence of UV radiation and salinity: cytochemical and ultrastructural organization and photosynthetic performance.

The photoacclimation responses of the brown macroalga Sargassum cymosum were studied to determine its cytochemical and ultrastructural organization, as well as photosynthetic pigments and performance. S. cymosum was cultivated in three salinities (30, 35 and 40 psu) under four irradiation treatments: PAR-only, PAR + UVA, PAR + UVB and PAR + UVA + UVB. Plants were exposed to PAR at 70 µmol photons m(-2) s(-1), PAR + UVB at 0.35 W m(-2) and PAR +UVA at 0.70 W m(-2) for 3 h per day during 7 days in vitro. Growth rate was not significantly affected by any type of radiation or salinity. The amount of pigments in S. cymosum was significantly influenced by the interaction of salinity and radiation treatments. Compared with PAR-only, UVR treatments modified the kinetics patterns of the photosynthesis/irradiance curve. After exposure to UVR, S. cymosum increased cell wall thickness and the presence of phenolic compounds. The number of mitochondria increased, whereas the number of chloroplasts showed few changes. Although S. cymosum showed insensitivity to changes in salinity, it can be concluded that samples treated under four irradiation regimes showed structural changes, which were more evident, but not severe, under PAR + UVB treatment.

Comparison of transcriptome under red and blue light culture of Saccharina japonica (Phaeophyceae).

Saccharina japonica is one of the most important economic seaweeds. Several aspects such as photosynthesis in Saccharina lives are affected by blue light, the predominant light spectrum in the habitat. In this study, transcriptome profiling of S. japonica by next generation sequencing technology generated 55,102 qualified transcripts and 40.5 % transcripts were assigned to functional annotation. Expression of a large proportion of genes has been previously reported to be regulated by blue light, taking dark as control. However, by comparison among white, blue and red light, the significantly differentially expressed gene tags (DEGs) accounted for only 6.75 % of the identified sequences. It indicated that light-regulated gene expression in kelps is not a specific blue-light response. Unexpectedly, red light had more extensive effects on the transcriptomic activity than blue light did, since the most (68.4 %) DEGs were red light-regulated and only 17.5 % were specifically regulated by blue light. Some of the DEGs with the highest mRNA levels under blue light are not blue light-upregulated but red light-downregulated. The extensive regulation on gene expression under red light together with the abundant presence of phytochrome-like protein gene tags in S. japonica indicated their significant roles in the lives of brown algae. By highlighting the photosynthetic metabolism, blue light is more efficient than red light in triggering the pigment biosynthesis, light reaction and carbon fixation, revealing a molecular basis for rapid growth of kelps, since most of the time blue light is predominant in their habitat.

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.

Transcriptome sequencing and comparative analysis of Saccharina japonica (Laminariales, Phaeophyceae) under blue light induction.

BACKGROUND: Light has significant effect on the growth and development of Saccharina japonica, but there are limited reports on blue light mediated physiological responses and molecular mechanism. In this study, high-throughput paired-end RNA-sequencing (RNA-Seq) technology was applied to transcriptomes of S. japonica exposed to blue light and darkness, respectively. Comparative analysis of gene expression was designed to correlate the effect of blue light and physiological mechanisms on the molecular level. PRINCIPAL FINDINGS: RNA-seq analysis yielded 70,497 non-redundant unigenes with an average length of 538 bp. 28,358 (40.2%) functional transcripts encoding regions were identified. Annotation through Swissprot, Nr, GO, KEGG, and COG databases showed 25,924 unigenes compared well (E-value <10(-5)) with known gene sequences, and 43 unigenes were putative BL photoreceptor. 10,440 unigenes were classified into Gene Ontology, and 8,476 unigenes were involved in 114 known pathways. Based on RPKM values, 11,660 (16.5%) differentially expressed unigenes were detected between blue light and dark exposed treatments, including 7,808 upregulated and 3,852 downregulated unigenes, suggesting S. japonica had undergone extensive transcriptome re-orchestration during BL exposure. The BL-specific responsive genes were indentified to function in processes of circadian rhythm, flavonoid biosynthesis, photoreactivation and photomorphogenesis. SIGNIFICANCE: Transcriptome profiling of S. japonica provides clues to potential genes identification and future functional genomics study. The global survey of expression changes under blue light will enhance our understanding of molecular mechanisms underlying blue light induced responses in lower plants as well as facilitate future blue light photoreceptor identification and specific responsive pathways analysis.

Phlorotannin production and lipid oxidation as a potential protective function against high photosynthetically active and UV radiation in gametophytes of Alaria esculenta (Alariales, Phaeophyceae).

Radiation damage can inter alia result in lipid peroxidation of macroalgal cell membranes. To prevent photo-oxidation within the cells, photoprotective substances such as phlorotannins are synthesized. In the present study, changes in total fatty acids (FA), FA composition and intra/extracellular phlorotannin contents were determined by gas chromatography and the Folin-Ciocalteu method to investigate the photoprotective potential of phlorotannins to prevent lipid peroxidation. Alaria esculenta juveniles (Phaeophyceae) were exposed over 20 days to high/low photosynthetically active radiation (PAR) in combination with UV radiation (UVR) in the treatments: PAB (low/high PAR + UV-B + UV-A), PA (low/high PAR + UV-A) or low/high PAR only. While extracellular phlorotannins increased after 10 days, intracellular phlorotannins increased with exposure time and PA and decreased under PAB. Interactive effects of time:radiation wavebands, time:PAR dose as well as radiation wavebands:PAR dose were observed. Low FA contents were detected in the PA and PAB treatments; interactive effects were observed between time:high PAR and PAB:high PAR. Total FA contents were correlated to extra/intracellular phlorotannin contents. Our results suggest that phlorotannins might play a role in intra/extracellular protection by absorption and oxidation processes. Changes in FA content/composition upon UVR and high PAR might be considered as an adaptive mechanism of the A. esculenta juveniles subjected to variations in solar irradiance.

Induction of phlorotannins during UV exposure mitigates inhibition of photosynthesis and DNA damage in the kelp Lessonia nigrescens.

Phlorotannins of brown algae are multifunctional compounds with putative roles in herbivore deterrence, antioxidation and as primary cell wall components. Due to their peripheral localization and absorption at short wavelengths, a photoprotective role is suggested. We examined the induction of phlorotannins by artificial UV radiation in the intertidal kelp Lessonia nigrescens and whether they attenuate the inhibition of photosynthesis and DNA damage, two major detrimental effects of UV. The soluble and cell wall-bound fractions of phlorotannins were quantified in blades collected in summer and winter. Major findings were that (1) the synthesis of phlorotannins (both forms) was induced by UV only in summer; (2) the induction was fast (within 3 days); and (3) there was a positive relationship between of the contents of insoluble phlorotannins and the suppression of photoinhibition and DNA damage, measured as formation of cyclobutane pyrimidine dimers and 6-4 photoproducts. Overall, the photoprotective role of phlorotannins appears to respond to an interplay between the external UV stimulus, seasonal acclimation and intrinsic morpho-functional processes. In summer, when algae are naturally exposed to high UV irradiances, soluble phlorotannins are induced, while their transition to insoluble phlorotannins could be related with the growth requirements, as active blade elongation occurs during this season.

Regulation and function of xanthophyll cycle-dependent photoprotection in algae.

The xanthophyll cycle represents one of the important photoprotection mechanisms in plant cells. In the present review, we summarize current knowledge about the violaxanthin cycle of vascular plants, green and brown algae, and the diadinoxanthin cycle of the algal classes Bacillariophyceae, Xanthophyceae, Haptophyceae, and Dinophyceae. We address the biochemistry of the xanthophyll cycle enzymes with a special focus on protein structure, co-substrate requirements and regulation of enzyme activity. We present recent ideas regarding the structural basis of xanthophyll cycle-dependent photoprotection, including different models for the mechanism of non-photochemical quenching of chlorophyll a fluorescence. In a dedicated chapter, we also describe the unique violaxanthin antheraxanthin cycle of the Prasinophyceae, together with its implication for the mechanism of xanthophyll cycle-dependent heat dissipation. The interaction between the diadinoxanthin cycle and alternative electron flow pathways in the chloroplasts of diatoms is an additional topic of this review, and in the last chapter we cover aspects of the importance of xanthophyll cycle-dependent photoprotection for different algal species in their natural environments.

Interactive effects of radiation, temperature and salinity on different life history stages of the Arctic kelp Alaria esculenta (Phaeophyceae).

To estimate the potential effects of climate change on polar marine macroalgae, studies on interactive stress effects of multiple climate-related parameters are essential. Interactions of temperature, radiation and salinity on two different life history stages of Alaria esculenta (L.) Greville from the Kongsfjord (Spitsbergen) were investigated for the first time within this study. Adult macroscopic sporophytes of A. esculenta were exposed to different temperatures between 4 and 21 degrees C combined with artificial irradiation conditions [photosynthetically active radiation, ultraviolet (UV) radiation: UV-A/UV-B, first experiment] and with different salinities [34, 28, 20 practical salinity units (p.s.u.) second experiment]. Effects of photosynthetic activity were determined by measuring variable chlorophyll fluorescence of photosystem II. Germination success of young microscopic zoospores of A. esculenta was studied under multifactorial stress. Zoospore suspensions were exposed to the three different salinities and irradiation conditions at four temperatures between 2 and 16 degrees C. Overall, A. esculenta exhibited a highly stage-specific susceptibility towards the experimental treatments. In both experiments using sporophytes, photosynthetic activity showed significant temperature effects and only very few significant radiation and salinity effects. Microscopic stages of A. esculenta were shown to be more sensitive than the adult macroscopic stages, since germination capacity of zoospores was significantly affected by temperature and salinity changes, and interactions of both. These results suggest that multiple stress factors interact synergistically. Temperature seems to be a predominant environmental parameter for the kelp A. esculenta. Overall, A. esculenta proved to be relatively tolerant and adaptable to increasing temperature and UV radiation, as well as to diluted salinities, but only up to a specific limit.

Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus.

Development of the sporophyte and gametophyte generations of the brown alga E. siliculosus involves two different patterns of early development, which begin with either a symmetric or an asymmetric division of the initial cell, respectively. A mutant, immediate upright (imm), was isolated that exhibited several characteristics typical of the gametophyte during the early development of the sporophyte generation. Genetic analyses showed that imm is a recessive, single-locus Mendelian factor and analysis of gene expression in this mutant indicated that the regulation of a number of life-cycle-regulated genes is specifically modified in imm mutant sporophytes. Thus, IMM appears to be a regulatory locus that controls part of the sporophyte-specific developmental programme, the mutant exhibiting partial homeotic conversion of the sporophyte into the gametophyte, a phenomenon that has not been described previously.

Regulation of gamete release in the economic brown seaweed Hizikia fusiforme (Phaeophyta).

Gamete release is an essential event in artificial seeding of the economic brown seaweed, Hizikia fusiforme. Mass egg release occurred in the dark, with few eggs being discharged in the light. Release of eggs was elicited with eight practical salinity units (one PSU identical with 1 g sea salts l(-1)) and was inhibited by salinity levels >32 PSU. Egg release was optimal at 23 degrees C, and was decreased by 72% in agitated seawater compared to unstirred seawater. Inhibitors of photosynthesis and ions channels suppressed egg release, indicating that this process was physiologically associated with photosynthetic activity and ion transport.