From model organism to application: Bacteria-induced growth and development of the green seaweed Ulva and the potential of microbe leveraging in algal aquaculture.

The marine green macroalga Ulva (Chlorophyta, Ulvales), also known as sea lettuce, coexists with a diverse microbiome. Many Ulva species proliferate in nature and form green algal blooms ("green tides"), which can occur when nutrient-rich wastewater from agricultural or densely populated areas is flushed into the sea. Bacteria are necessary for the adhesion of Ulva to its substrate, its growth, and the development of its blade morphology. In the absence of certain bacteria, Ulva mutabilis develops into a callus-like morphotype. However, with the addition of the necessary marine bacteria, the entire morphogenesis can be restored. Surprisingly, just two bacteria isolated from U. mutabilis are sufficient for inducing morphogenesis and establishing the reductionist system of a tripartite community. While one bacterial strain causes algal blade cell division, another causes the differentiation of basal cells into a rhizoid and supports cell wall formation because of a low concentration of the morphogen thallusin (below 10-10 mol/L). This review focuses on the research conducted on this topic since 2015, discusses how U. mutabilis has developed into a model organism in chemical ecology, and explores the questions that have already been addressed and the perspectives that a reductionist model system allows. In particular, the field of systems biology will achieve a comprehensive, quantitative understanding of the dynamic interactions between Ulva and its associated bacteria to better predict the behavior of the system as a whole. The reductionist approach has enabled the study of the bacteria-induced morphogenesis of Ulva. Specific questions regarding the optimization of cultivation conditions as well as the yield of raw materials for the food and animal feed industries can be answered in the laboratory and through applied science. Genome sequencing, the improvement of genetic engineering tools, and the first promising attempts to leverage macroalgae-microbe interactions in aquaculture make this model organism, which has a comparatively short parthenogenetic life cycle, attractive for both fundamental and applied research. The reviewed research paves the way for the synthetic biology of macroalgae-associated microbiomes in sustainable aquacultures.

Copper Is Accumulated as Copper Sulfide Particles, and Not Bound to Glutathione, Phytochelatins or Metallothioneins, in the Marine Alga Ulva compressa (Chlorophyta).

To analyze the mechanism of copper accumulation in the marine alga Ulva compressa, it was cultivated with 10 µM of copper, with 10 µM of copper and increasing concentrations of a sulfide donor (NaHS) for 0 to 7 days, and with 10 µM of copper and a concentration of the sulfide acceptor (hypotaurine) for 5 days. The level of intracellular copper was determined as well as the level of glutathione (GSH) and phytochelatins (PCs) and the expression of metallothioneins (UcMTs). The level of intracellular copper in the algae treated with copper increased at day 1, slightly increased until day 5 and remained unchanged until day 7. The level of copper in the algae cultivated with copper and 100 or 200 µM of NaHS continuously increased until day 7 and the copper level was higher in the algae cultivated with 200 µM of NaHS compared to 100 µM of NaHS. In contrast, the level of intracellular copper decreased in the algae treated with copper and hypotaurine. The level of intracellular copper did not correlate with the level of GSH or with the expression of UcMTs, and PCs were not detected in response to copper, or copper and NaHS. Algae treated with copper and with copper and 200 µM of NaHS for 5 days were visualized by TEM and the elemental composition of electrondense particles was analyzed by EDXS. The algae treated with copper showed electrondense particles containing copper and sulfur, but not nitrogen, and they were mainly located in the chloroplast, but also in the cytoplasm. The algae treated with copper and NaHS showed a higher level of electrondense particles containing copper and sulfur, but not nitrogen, and they were located in the chloroplast, and in the cytoplasm. Thus, copper is accumulated as copper sulfide insoluble particles, and not bound to GSH, PCs or UcMTs, in the marine alga U. compressa.

Biochemical characterization of a new ulvan lyase and its applicability in utilization of ulvan and preparation of ulva oligosaccharides.

Ulva is globally distributed specie and has a high economic value. Ulvan is one of the main active substances in Ulva, which has a variety of biological properties. Ulvan lyase degrades ulvan through a ß-elimination mechanism which cleaves the ß-glycosidic bond between Rha3S and GlcA or IdoA. The complex monosaccharide composition of ulvan makes it promising for use in food and pharmaceutical applications. This thesis explores a putative ulvan lyase from Alteromonas sp. KUL_42. We expressed and purified the protein, performed a series of characterizations and signal peptide had been removed. The results showed that the protein molecular weight of ULA-2 was 53.97 kDa, and it had the highest catalytic activity at 45 °C and pH 8.0 in Tris-HCl buffer. The Km and Vmax values were 2.24 mg · mL-1 and 2.048 µmol · min-1 · mL-1, respectively. The activity of ULA-2 was able to maintain more than 80% at 20 ~ 30 °C. ESI-MS analysis showed that the primary end-products were mainly disaccharides to tetrasaccharides. The study of ULA-2 enriches the ulvan lyase library, promotes the development and high-value utilization of Ulva resources, and facilitates further research applications of ulvan lyase in ulva oligosaccharides.

Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Metal-free carbocatalysts derived from macroalga biomass (Ulva lactuca) for the activation of peroxymonosulfate toward the remediation of polycyclic aromatic hydrocarbons laden marine sediments and its impacts on microbial community.

Potential toxic chemicals, specifically, polycyclic aromatic hydrocarbons (PAHs), are major sediment contaminants. Herein, green seaweed (Ulva lactuca) was used as a feedstock and pyrolyzed at temperature in the range between 300 and 900 °C. The metal-free carbocatalyst (GSBC) for peroxymonosulfate (PMS) activation to degrade PAHs contaminated sediments was studied. The effects of GSBC‒PMS treatment on microbial community abundance was studied as well. The pyrolysis temperature of GSBC preparation affected the PMS activation performance. Results show that GSBC700 exhibited remarkable catalytic characteristics in PAHs degradation by effective activation of PMS. The results also demonstrated that the sulfate radical-carbon-driven advanced oxidation processes (SR-CAOP) reaction achieved 87% and apparent rate constant (kobs) of 6.3 × 10-2 h-1 of total PAHs degradation in 24 h at 3.3 g/L of GSBC, PMS dose of 1 × 10-4 M, and pH 3.0. The degradation of 2-, 3-, 4-, 5-, and 6-ring PAHs was 84, 83, 83, 80, and 89%, respectively. The synergetic effect established between GSBC and PMS enhanced the formation of ROSs, namely, SO4-, HO, and 1O2, which were major species contributing to PAHs degradation. The synergistic effect of π‒π stacking structure and graphitization of GSBC formed electron shuttle, which contributed to PAHs degradation performance. Microbial community structure analyses in the GSBC‒PMS treated sediments showed that the relative abundance of Lactobacillus_rhamnosus species, most of which belonged to the Lactobacillus genus and Firmicutes phylum, which aided in continuing PAHs biodegradation post GSBC‒PMS treatment. Therefore, GSBC can be a promising carbocatalyst produced via biomass-to-biochar conversion as biowaste-to-energy source used in the SR-CAOP-mediated process for sediment remediation.

Seaweed Extracts: A Promising Source of Antibiofilm Agents with Distinct Mechanisms of Action against Pseudomonas aeruginosa.

The organization of bacteria in biofilms is one of the adaptive resistance mechanisms providing increased protection against conventional treatments. Thus, the search for new antibiofilm agents for medical purposes, especially of natural origin, is currently the object of much attention. The objective of the study presented here was to explore the potential of extracts derived from three seaweeds: the green Ulva lactuca, the brown Stypocaulon scoparium, and the red Pterocladiella capillacea, in terms of their antibiofilm activity against P. aeruginosa. After preparation of extracts by successive maceration in various solvents, their antibiofilm activity was evaluated on biofilm formation and on mature biofilms. Their inhibition and eradication abilities were determined using two complementary methods: crystal violet staining and quantification of adherent bacteria. The effect of active extracts on biofilm morphology was also investigated by epifluorescence microscopy. Results revealed a promising antibiofilm activity of two extracts (cyclohexane and ethyl acetate) derived from the green alga by exhibiting a distinct mechanism of action, which was supported by microscopic analyses. The ethyl acetate extract was further explored for its interaction with tobramycin and colistin. Interestingly, this extract showed a promising synergistic effect with tobramycin. First analyses of the chemical composition of extracts by GC-MS allowed for the identification of several molecules. Their implication in the interesting antibiofilm activity is discussed. These findings suggest the ability of the green alga U. lactuca to offer a promising source of bioactive candidates that could have both a preventive and a curative effect in the treatment of biofilms.

Balancing Damage via Non-Photochemical Quenching, Phenolic Compounds and Photorespiration in Ulva prolifera Induced by Low-Dose and Short-Term UV-B Radiation.

The Yellow Sea green tide (YSGT) is the world's largest transregional macroalgal blooms, and the causative species Ulva prolifera (U. prolifera) suffers from ultraviolet-b radiation (UVBR) during the floating migration process. Previous study confirmed that U. prolifera displayed a wide variety of physiological responses characterized as acclimation to UVBR, while the response mechanisms against low-dose and short-term radiation (LDSTR) are not clear. A study with photosynthetically active radiation (PAR) and UVBR was designed: normal light (NL: 72 µmol photons m-2 s-1), NL+0.3 (UVBR: 0.3 W·m-2), and NL+1.6 (UVBR: 1.6 W·m-2). The results showed that high-dose UVBR inhibited photosynthesis in thalli, especially under long-term exposure, while a variety of physiological responses were observed under LDSTR. The inhibition of photosynthesis appeared to be ameliorated by the algae under LDSTR. Further analysis showed that U. prolifera achieved balancing damage by means of non-photochemical quenching (NPQ), accumulation of phenolic compounds coupled with the ASA-GSH cycle involved in the antioxidant process and enhanced photorespiratory metabolism under LDSTR. This study provides new insights into the balancing damage mechanisms of U. prolifera under LDSTR, enabling the thalli to adapt to the light conditions during the long duration and distance involved in floating migration.

Stereoselective Total Synthesis of (-)-Thallusin for Bioactivity Profiling.

Chemical mediators are key compounds for controlling symbiotic interactions in the environment. Here, we disclose a fully stereoselective total synthesis of the algae differentiation factor (-)-thallusin that utilizes sophisticated 6-endo-cyclization chemistry and effective late-stage sp2 -sp2 -couplings using non-toxic reagents. An EC50 of 4.8 pM was determined by quantitative phenotype profiling in the green seaweed Ulva mutabilis (Chlorophyte), underscoring this potent mediator's enormous, pan-species bioactivity produced by symbiotic bacteria. SAR investigations indicate that (-)-thallusin triggers at least two different pathways in Ulva that may be separated by chemical editing of the mediator compound structure.

Synthesis and release of fatty acids under the interaction of Ulva pertusa and Heterosigma akashiwo by stable isotope analysis.

Symbiosis of marine algae is inevitable in the marine environment, and species may occur interaction on the growth. In this study, the macroalgae Ulva pertusa and marine microalgae Heterosigma akashiwo were selected as target species to study the interaction mechanism between them. After the 8 days of co-cultivation, the inhibition on growth was observed for both of U. pertusa and H. akashiwo. Eight fatty acids in U. pertusa was detected, with the significant decrease in contents of polyunsaturated fatty acids (PUFAs) especially for C18:2, C18:3n-3 and C18:3n-6. Twelve fatty acids in H. akashiwo was detected, with the significant change for PUFAs. PUFA concentrations in the co-culture group were less than those in the mono-culture. Meanwhile the principal component analysis was conducted to insight into the interaction between U. pertusa and H. akashiwo by fatty acids content and carbon stable isotope ratio of fatty acids (δ13CFAs). Fatty acid content could not distinguish mono and co-culture. However, δ13CFAs could distinguish not only the culture time of algae, but also the living environment of algae. In addition, this study combined fatty acids content and δ13CFAs to explore the release of fatty acids by algae into the seawater. The C18:3n-3 was identified as the allelochemical released by U. pertusa to inhibit the growth of H. akashiwo. The ratio of δ13CFAs in seawater decreased. This study provides a theoretical basis for the symbiosis of marine algae, and a new method of compound-specific stable carbon isotopes was used to better explore the metabolism of fatty acids in algae.

How Ulva lactuca can influence the impacts induced by the rare earth element Gadolinium in Mytilus galloprovincialis? The role of macroalgae in water safety towards marine wildlife.

Rare earth elements (REEs) are gaining growing attention in environmental and ecotoxicological studies due to their economic relevance, wide range of applications and increasing environmental concentrations. Among REEs, special consideration should be given to Gadolinium (Gd), whose wide exploitation as a magnetic resonance imaging (MRI) contrast agent is enhancing the risk of its occurrence in aquatic environments and impacts on aquatic organisms. A promising approach for water decontamination from REEs is sorption, namely through the use of macroalgae and in particular Ulva lactuca that already proved to be an efficient biosorbent for several chemical elements. Therefore, the present study aimed to evaluate the toxicity of Gd, comparing the biochemical effects induced by this element in the presence or absence of algae. Using the bivalve species Mytilus galloprovincialis, Gd toxicity was evaluated by assessing changes on mussels' metabolic capacity and oxidative status. Results clearly showed the toxicity of Gd but further revealed the capacity of U. lactuca to prevent injuries to M. galloprovincialis, mainly reducing the levels of Gd in water and thus the bioaccumulation and toxicity of this element by the mussels. The results will advance the state of the art not only regarding the effects of REEs but also with regard to the role of algae in accumulation of metals and protection of aquatic organisms, generating new insights on water safety towards aquatic wildlife and highlighting the possibility for resources recovery.

Ulva compressa from Copper-Polluted Sites Exhibits Intracellular Copper Accumulation, Increased Expression of Metallothioneins and Copper-Containing Nanoparticles in Chloroplasts.

In order to analyze the mechanisms involved in copper accumulation in Ulva compressa, algae were collected at control sites of central and northern Chile, and at two copper-polluted sites of northern Chile. The level of intracellular copper, reduced glutathione (GSH), phytochelatins (PCs), PC2 and PC4, and transcripts encoding metallothioneins (MTs) of U. compressa, UcMT1, UcMT2 and UcMT3, were determined. Algae of control sites contained around 20 µg of copper g-1 of dry tissue (DT) whereas algae of copper-polluted sites contained 260 and 272 µg of copper g-1 of DT. Algae of control sites and copper-polluted sites did not show detectable amounts of GSH, the level of PC2 did not change among sites whereas PC4 was increased in one of the copper-polluted sites. The level of transcripts of UcMT1 and UcMT2 were increased in algae of copper-polluted sites, but the level of UcMT3 did not change. Algae of a control site and a copper-polluted site were visualized by transmission electron microscopy (TEM) and the existence of copper in electrodense particles was analyzed using energy dispersive x-ray spectroscopy (EDXS). Algae of copper-polluted sites showed electrodense nanoparticles containing copper in the chloroplasts, whereas algae of control sites did not. Algae of a control site, Cachagua, were cultivated without copper (control) and with 10 µM copper for 5 days and they were analyzed by TEM-EDXS. Algae cultivated with copper showed copper-containing nanoparticles in the chloroplast whereas control algae did not. Thus, U. compressa from copper-polluted sites exhibits intracellular copper accumulation, an increase in the level of PC4 and expression of UcMTs, and the accumulation of copper-containing particles in chloroplasts.

Effect of different cooking methods on sea lettuce (Ulva rigida) volatile compounds and sensory properties.

BACKGROUND: The effect of different cooking methods (boiling, vacuum and steamed cooking) on the volatile compound content and sensory properties of sea lettuce (Ulva rigida) seaweed was assessed. Sea lettuce was cooked at three different temperatures (50, 70 and 100 °C) for three different lengths of time (5, 10 and 15 min). Various statistical techniques were employed in order to establish any possible changes. RESULTS: The different cooking methods modified significantly both the volatile compound content and the sensory properties of sea lettuce seaweed. In general, the cooked samples had lower concentrations of several volatile compounds than the control sample, mainly aldehydes. Regarding sensory analysis, the cooked samples exhibited lower values for various aroma descriptors such as seaside and seaweed, whereas descriptor scores such as cooked fish, salty dry fish and crustacean increased. No clear statistical differences were found between different cooking lengths of time and temperature levels with regard to both volatile compounds and sensory properties. CONCLUSIONS: In the cooking of sea lettuce seaweeds, the main sensory changes and modifications in their volatile content took place during the first minutes of cooking and at medium cooking temperatures. © 2020 Society of Chemical Industry.

Copper-induced concomitant increases in photosynthesis, respiration, and C, N and S assimilation revealed by transcriptomic analyses in Ulva compressa (Chlorophyta).

BACKGROUND: The marine alga Ulva compressa is the dominant species in copper-polluted coastal areas in northern Chile. It has been shown that the alga tolerates micromolar concentrations of copper and accumulates copper at the intracellular level. Transcriptomic analyses were performed using total RNA of the alga cultivated with 10 µ M copper for 0, 1, 3 and 5 days using RNA-seq in order to identify processes involved in copper tolerance. RESULTS: The levels of transcripts encoding proteins belonging to Light Harvesting Complex II (LHCII), photosystem II (PSII), cytochrome b6f, PSI, LHCI, ATP synthase and proteins involved in repair of PSII and protection of PSI were increased in the alga cultivated with copper. In addition, the level of transcripts encoding proteins of mitochondrial electron transport chain, ATP synthase, and enzymes involved in C, N and S assimilation were also enhanced. The higher percentages of increase in the level of transcripts were mainly observed at days 3 and 5. In contrast, transcripts involved protein synthesis and degradation, signal transduction, and replication and DNA repair, were decreased. In addition, net photosynthesis and respiration increased in the alga cultivated with copper, mainly at days 1 to 3. Furthermore, the activities of enzymes involved in C, N and S assimilation, rubisco, glutamine synthase and cysteine synthase, respectively, were also increased, mainly at days 1 and 3. CONCLUSIONS: The marine alga U. compressa tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance.

Extensive Variations in Diurnal Growth Patterns and Metabolism Among Ulva spp. Strains.

Green macroalgae of the genus Ulva play a key role in coastal ecosystems and are of increasing commercial importance. However, physiological differences between strains and species have yet to be described in detail. Furthermore, the strains of Ulva used in aquaculture usually originate from opportunistic collection in the wild without prior selection of best performing strains. Hence, efforts are required to detect the potential variability in growth and metabolic accumulation between Ulva strains and ultimately select the best performing strains under given environmental conditions. Here, the growth, physiological, and metabolic characteristics of 49 laminar Ulva spp. strains were investigated using a custom-made high-throughput phenotyping platform, enzymatic assays, and gas chromatography-mass spectrometry. We found large natural variation for a wide range of growth and metabolic characteristics, with growth rates varying from 0.09 to 0.37 mg.mg-1d-1 among strains. Ulva spp. possess a unique diurnal growth pattern and primary metabolism compared with land plants, with higher growth rates during the night than during the light period. Starch and sucrose only contributed on average 35% of the carbon required to sustain Ulva's night growth. Nitrates accumulated during the night in Ulva tissues, and nitrate accumulation and consumption was positively correlated with growth. In addition, we identified six amino acids as possible biomarkers for high growth in Ulva The large variability in growth and metabolite accumulation recorded among morphologically similar Ulva strains justifies future efforts in strain selection for increasing biomass, metabolite yields, and nutrient removal in the growing aquaculture industry.

Photosynthetic and biochemical traits change in the green-tide-forming macroalga Ulva pertusa during sporulation1.

The physiological and biochemical changes in the green macroalga Ulva pertusa during the progression of sporulation have been characterized. The transition from the vegetative to the sporulation stage was accompanied by an increase in chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid content, as well as an increase in DPPH scavenging and responsiveness to diphenylamine. However, oxygen evolution and maximum electron transport rate decreased. The discrepancy between photosynthetic performance and pigment content might relate to the self-shading of spores within a sporangium. Spore-forming U. pertusa thalli were low-light-adapted, due to an increase in the number of photosynthetic units. Decreased electron transport during sporulation might trigger sporulation, as for some cyanobacteria and other Ulva spp., via oxidization of the plastoquinone pool and cyclic phosphorylation, thus producing ATP to generate carbon and nitrogen skeletons required for spores. It is thus concluded that carotenoids function both in spore initiation and/or maturation and in their photoprotection.

Metagenomic Analysis of the Effect of Enteromorpha prolifera Bloom on Microbial Community and Function in Aquaculture Environment.

Enteromorpha prolifera blooms considerably affected coastal environments in recent years. However, the effects of E. prolifera on microbial ecology and function remained unknown. In this study, metagenomic sequencing was used to investigate the effect of E. prolifera bloom on the microbial communities and functional genes in an aquaculture environment. Results showed that E. prolifera bloom could significantly alter the microbial composition and abundance, and heterotrophic bacteria comprised the major groups in the E. prolifera bloom pond, which was dominated by Actinomycetales and Flavobacteriales. The study indicated that viruses played an important role in shaping the microbial community and diversity during E. prolifera bloom. These viruses affected various dominant microbial taxa (such as Rhodobacteraceae, Synechococcus, and Prochlorococcus), which produced an obvious impact on potential nutrient transformation. Functional annotation analysis indicated that E. prolifera bloom would considerably shift the metabolism function by altering the structure and abundance of the microbial community. E. prolifera bloom pond had the low ability of potential metabolic capabilities of nitrogen, sulfur, and phosphate, whereas promoted gene abundance of genetic information processing. These changes in the microbial community and function could produce serious effect on aquaculture ecosystem.

A Supercomplex, of Approximately 720 kDa and Composed of Both Photosystem Reaction Centers, Dissipates Excess Energy by PSI in Green Macroalgae Under Salt Stress.

The thylakoid membranes of plants play a critical role in electron transfer and energy fixation, and are highly dynamic. So far, studies on the thylakoid membranes have mainly focused on microalgae and higher plants, yet very little information is available on the macroalgal thylakoids. Here, we studied the structure and organization of the thylakoid membranes in Ulva prolifera, a representative species of the green macroalgae. We found that U. prolifera had few but long loosely stacked membranes which lack the conventional grana found in higher plants. However, the thylakoid membrane complexes demonstrate lateral heterogeneity. Moreover, we found a supercomplex composed of PSII, light-harvesting complex II (LHCII) and PSI from U. prolifera under salt stress. The supercomplex is approximately 720 kDa, and includes the two important photoprotection proteins, the PSII S subunit (PsbS) and the light-harvesting complex stress-related protein (LhcSR), as well as xanthophyll cycle pigments (violaxanthin, antheraxanthin and zeaxanthin). Time-resolved fluorescence analysis suggested that, in the supercomplex, excitation energy could efficiently be transferred from PSII to PSI, even when PSII was inhibited, a function which disappeared when the supercomplex was incubated in mild detergent. We suggest that the supercomplex might be an important mechanism to dissipate excess energy by PSI in green macroalgae under salt stress.

In Vitro and In Silico Approaches to Generating and Identifying Angiotensin-Converting Enzyme I Inhibitory Peptides from Green Macroalga Ulva lactuca.

A protein extract was generated from the macroalga Ulva lactuca, which was subsequently hydrolysed using the food-grade enzyme papain and angiotensin-converting Enzyme I and renin inhibitory peptides identified using a combination of enrichment strategies employing molecular weight cutoff filtration and mass spectrometry analysis. The generated hydrolysates with the most promising in vitro activity were further purified using preparative RP-HPLC and characterised. The 1 kDa hydrolysate (1 kDa-UFH), purified and collected by preparative RP-HPLC at minutes 41‒44 (Fr41‒44), displayed statistically higher ACE-I inhibitory activities ranging from 96.91% to 98.06%. A total of 48 novel peptides were identified from these four fractions by LC-MS/MS. A simulated gastrointestinal digestion of the identified peptide sequences was carried out using in silico enzyme cleavage simulation tools, resulting in 86 peptide sequences that were further assessed for their potential activity, toxicity and allergenicity using multiple predictive approaches. All the peptides obtained in this study were predicted to be non-toxic. However, 28 out of the 86 novel peptides released after the in silico gastrointestinal digestion were identified as potential allergens. The potential allergenicity of these peptides should be further explored to comply with the current labelling regulations in formulated food products containing U. lactuca protein hydrolysates.

The effect of salinity and alkalinity on growth and the accumulation of copper and zinc in the Chlorophyta Ulva fasciata.

Copper and zinc accumulation in macroalgae is a complex issue. While these metals exist as micronutrients and can serve to add nutritional value to the macroalgae when consumed by both plants and animals, elevated levels of the metals can reduce growth or even kill the algae. Many water parameters can influence the toxicity of the metals, though past studies have rarely isolated individual water parameters. This study aimed to independently determine the effects that salinity and alkalinity have on the growth and accumulation of these two metals on the macroalgae Ulva fasciata, distinguishing the effects of salinity and alkalinity as whole parameters from the collective effects of the water different constituents. The effect of salinity was determined using sodium chloride additions rather than seawater dilution, as performed in past studies, while alkalinity was tested using sodium bicarbonate additions to artificial seawater. The results of the study reinforce previous findings that copper is very toxic to macroalgae, even at low concentrations (50 µg L-1) though the effects of zinc remain inconclusive at 50 µg L-1, since the experiment was conducted over only a two-week trial period. The research suggests that salinity and alkalinity have no significant effect on the toxicity of copper to the growth of the macroalgae, but alkalinity significantly reduced copper and increased zinc accumulation in U. fasciata. The results of this study warrant further research in the field to investigate which other components of seawater and macroalgae reduce metal toxicity in the macroalgae. Additionally, these findings suggest the need for further refinement of toxicity models when adapted to macroalgae.

Phytoremediation potential of Ulva ohnoi (Chlorophyta): Influence of temperature and salinity on the uptake efficiency and toxicity of cadmium.

Ulva ohnoi is a green macroalga with fast growth and high rates of nitrogen and phosphorus absorption. Recently, this species has been recorded in several places with record green tide formation in some of them. Using molecular tools, we herein report the first occurrence of this species in Brazil and demonstrate its potential for phytoremediation in typical environmental concentrations of Cd (0.625-15 µg L-1). Similarly, the effects of physicochemical parameters (salinity and temperature) on the toxicity and uptake efficiency of this species were evaluated. Molecular analysis of two sequences (1141 bp) obtained corroborates another 34 sequences for U. ohnoi obtained from GenBank. The addition of Cd in the medium affected photosynthetic parameters and reduced growth rate. U. ohnoi showed resistance to Cd when cultivated at 18 °C, S15 and 18-25 °C, S35, at concentrations between 0.625 and 2.5 µg. L-1 of Cd; yet, positive growth rate was maintained. Dose-dependent accumulation was observed in all combinations of factors used with a maximum value of 4.20 µg Cd per gram of dry seaweed at 15 µg. L-1 of Cd at 18 °C and S35. Maximum value of the concentration factor was 81.3 ±â€¯1.1% of Cd added at the concentration of 0.625 µg. L-1 to S15 and 18 °C. Our results demonstrate the potential of using U. ohnoi in the phytoremediation of Cd in saltwater or brackish water.