Changes in holopelagic Sargassum spp. biomass composition across an unusual year.

The year 2021 marked a decade of holopelagic sargassum (morphotypes Sargassum natans I and VIII, and Sargassum fluitans III) stranding on the Caribbean and West African coasts. Beaching of millions of tons of sargassum negatively impacts coastal ecosystems, economies, and human health. Additionally, the La Soufrière volcano erupted in St. Vincent in April 2021, at the start of the sargassum season. We investigated potential monthly variations in morphotype abundance and biomass composition of sargassum harvested in Jamaica and assessed the influence of processing methods (shade-drying vs. frozen samples) and of volcanic ash exposure on biochemical and elemental components. S. fluitans III was the most abundant morphotype across the year. Limited monthly variations were observed for key brown algal components (phlorotannins, fucoxanthin, and alginate). Shade-drying did not significantly alter the contents of proteins but affected levels of phlorotannins, fucoxanthin, mannitol, and alginate. Simulation of sargassum and volcanic ash drift combined with age statistics suggested that sargassum potentially shared the surface layer with ash for ~50 d, approximately 100 d before stranding in Jamaica. Integrated elemental analysis of volcanic ash, ambient seawater, and sargassum biomass showed that algae harvested from August had accumulated P, Al, Fe, Mn, Zn, and Ni, probably from the ash, and contained less As. This ash fingerprint confirmed the geographical origin and drift timescale of sargassum. Since environmental conditions and processing methods influence biomass composition, efforts should continue to improve understanding, forecasting, monitoring, and valorizing sargassum, particularly as strandings of sargassum show no sign of abating.

Environmentally benign alginate extraction and fibres spinning from different European Brown algae species.

Applications of natural fibres are expanding, and sustainable alternatives are needed to support this growing demand. We investigated the production of fibres using alginates from Saccharina latissima (SAC), Laminaria digitata (LAM), Sacchoriza polyschides (SACC), and Himanthalia spp. (HIM). After extraction (3 % w/v biomass) using a sustainable protocol based on citric acid, crude alginate represented 61-65 % of the biomass dry weight for SAC and LAM, and 34-41 % for SACC and HIM when experiments were performed at small scale (1.5 g of starting material). Interestingly, scaling-up extraction (60 g of starting material) decreased yields to 26-30 %. SAC and LAM alginates had the highest M/G (mannuronic acid/guluronic acid) ratios and molecular weights when compared to those from SACC and HIM (M/G:1.98 and 2.23, MW: 302 and 362 kDa, vs 1.83 and 1.86, 268 and 168 kDa). When the four types of alginates were tested for spinning fibres cross-linked with CaCl2, only SAC and LAM alginates produced fibres. These fibres showed no clumps or cracks under stretching action and presented a similar Young's modulus (2.4 and 2.0 GPa). We have demonstrated that alginate extracted from S. latissima and L. digitata can be successfully spun into functional fibres cross-linked with CaCl2.

In silico identification of bacterial seaweed-degrading bioplastic producers.

There is an urgent need to replace petroleum-based plastic with bio-based and biodegradable alternatives. Polyhydroxyalkanoates (PHAs) are attractive prospective replacements that exhibit desirable mechanical properties and are recyclable and biodegradable in terrestrial and marine environments. However, the production costs today still limit the economic sustainability of the PHA industry. Seaweed cultivation represents an opportunity for carbon capture, while also supplying a sustainable photosynthetic feedstock for PHA production. We mined existing gene and protein databases to identify bacteria able to grow and produce PHAs using seaweed-derived carbohydrates as substrates. There were no significant relationships between the genes involved in the deconstruction of algae polysaccharides and PHA production, with poor to negative correlations and diffused clustering suggesting evolutionary compartmentalism. We identified 2 987 bacterial candidates spanning 40 taxonomic families predominantly within Alphaproteobacteria, Gammaproteobacteria and Burkholderiales with enriched seaweed-degrading capacity that also harbour PHA synthesis potential. These included highly promising candidates with specialist and generalist specificities, including Alteromonas, Aquisphaera, Azotobacter, Bacillus, Caulobacter, Cellvibrionaceae, Duganella, Janthinobacterium, Massilia, Oxalobacteraceae, Parvularcula, Pirellulaceae, Pseudomonas, Rhizobacter, Rhodanobacter, Simiduia, Sphingobium, Sphingomonadaceae, Sphingomonas, Stieleria, Vibrio and Xanthomonas. In this enriched subset, the family-level densities of genes targeting green macroalgae polysaccharides were considerably higher (n=231.6±68.5) than enzymes targeting brown (n=65.34±13.12) and red (n=30.5±10.72) polysaccharides. Within these organisms, an abundance of FabG genes was observed, suggesting that the fatty acid de novo synthesis pathway supplies (R)-3-hydroxyacyl-CoA or 3-hydroxybutyryl-CoA from core metabolic processes and is the predominant mechanism of PHA production in these organisms. Our results facilitate extending seaweed biomass valorization in the context of consolidated biorefining for the production of bioplastics.

Pelagic Sargassum events in Jamaica: Provenance, morphotype abundance, and influence of sample processing on biochemical composition of the biomass.

Pelagic Sargassum species have been known for centuries in the Sargasso Sea of the North Atlantic Ocean. In 2011, a new area concentrating high biomass of these brown algae started developing in the Tropical Atlantic Ocean. Since then, massive and recurrent Sargassum influxes have been reported in the Caribbean and off the coast of Western Africa. These Sargassum events have a major negative impact on coastal ecosystems and nearshore marine life, and affect socio-economic sectors, including public health, coastal living, tourism, fisheries, and maritime transport. Despite recent advances in the forecasting of Sargassum events, and elucidation of the seaweed composition, many knowledge gaps remain, including morphotype abundance during Sargassum events, drift of the seaweeds in the months prior to stranding, and influence of sample processing methods on biomass biochemical composition. Using seaweeds harvested on the coasts of Jamaica in summer of 2020, we observed that S. fluitans III was the most abundant morphotype at different times and sampling locations. No clear difference in the geographical origin, or provenance, of the Sargassum mats was observed. The majority of Sargassum backtracked from both north and south of Jamaica experienced ambient temperatures of around 27 °C and salinity in the range of 34-36 psu before stranding. We also showed that cheap (sun) compared to expensive (freeze) drying techniques influence the biochemical composition of biomass. Sun-drying increased the proportion of phenolic compounds, but had a deleterious impact on fucoxanthin content and on the quantities of monosaccharides, except for mannitol. Effects on the content of fucose containing sulfated polysaccharides depended on the method used for their extraction, and limited variation was observed in ash, protein, and fatty acid content within most of the sample locations investigated. These observations are important for the storage and transport of the biomass in the context of its valorisation.

A Novel Protein from Ectocarpus sp. Improves Salinity and High Temperature Stress Tolerance in Arabidopsis thaliana.

Brown alga Ectocarpus sp. belongs to Phaeophyceae, a class of macroalgae that evolved complex multicellularity. Ectocarpus sp. is a dominant seaweed in temperate regions, abundant mostly in the intertidal zones, an environment with high levels of abiotic stresses. Previous transcriptomic analysis of Ectocarpus sp. revealed several genes consistently induced by various abiotic stresses; one of these genes is Esi0017_0056, which encodes a protein with unknown function. Bioinformatics analyses indicated that the protein encoded by Esi0017_0056 is soluble and monomeric. The protein was successfully expressed in Escherichia coli,Arabidopsis thaliana and Nicotiana benthamiana. In A. thaliana the gene was expressed under constitutive and stress inducible promoters which led to improved tolerance to high salinity and temperature stresses. The expression of several key abiotic stress-related genes was studied in transgenic and wild type A. thaliana by qPCR. Expression analysis revealed that genes involved in ABA-induced abiotic stress tolerance, K+ homeostasis, and chaperon activities were significantly up-regulated in the transgenic line. This study is the first report in which an unknown function Ectocarpus sp. gene, highly responsive to abiotic stresses, was successfully expressed in A. thaliana, leading to improved tolerance to salt and temperature stress.

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp.

BACKGROUND: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.

Biomass composition of the golden tide pelagic seaweeds Sargassum fluitans and S. natans (morphotypes I and VIII) to inform valorisation pathways.

Massive strandings of the pelagic brown algae Sargassum have occurred in the Caribbean, and to a lesser extent, in western Africa, almost every year since 2011. These events have major environmental, health, and economic impacts in the affected countries. Once on the shore, Sargassum is mechanically harvested and disposed of in landfills. Existing commercial applications of other brown algae indicate that the pelagic Sargassum could constitute a valuable feedstock for potential valorisation. However, limited data on the composition of this Sargassum biomass was available to inform on possible application through pyrolysis or enzymatic fractionation of this feedstock. To fill this gap, we conducted a detailed comparative biochemical and elemental analysis of three pelagic Sargassum morphotypes identified so far as forming Atlantic blooms: Sargassum natans I (SnI), S. fluitans III (Sf), and S. natans VIII (SnVIII). Our results showed that SnVIII accumulated a lower quantity of metals and metalloids compared to SnI and Sf, but it contained higher amounts of phenolics and non-cellulosic polysaccharides. SnVIII also had more of the carbon storage compound mannitol. No differences in the content and composition of the cell wall polysaccharide alginate were identified among the three morphotypes. In addition, enzymatic saccharification of SnI produced more sugars compared to SnVIII and Sf. Due to high content of arsenic, the use of pelagic Sargassum is not recommended for nutritional purposes. In addition, low yields of alginate extracted from this biomass, compared with brown algae used for industrial production, limit its use as viable source of commercial alginates. Further work is needed to establish routes for future valorisation of pelagic Sargassum biomass.

Low Mannitol Concentrations in Arabidopsis thaliana Expressing Ectocarpus Genes Improve Salt Tolerance.

Mannitol is abundant in a wide range of organisms, playing important roles in biotic and abiotic stress responses. Nonetheless, mannitol is not produced by a vast majority of plants, including many important crop plants. Mannitol-producing transgenic plants displayed improved tolerance to salt stresses though mannitol production was rather low, in the µM range, compared to mM range found in plants that innately produce mannitol. Little is known about the molecular mechanisms underlying salt tolerance triggered by low concentrations of mannitol. Reported here is the production of mannitol in Arabidopsis thaliana, by expressing two mannitol biosynthesis genes from the brown alga Ectocarpus sp. strain Ec32. To date, no brown algal genes have been successfully expressed in land plants. Expression of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase genes was associated with the production of 42.3-52.7 nmol g-1 fresh weight of mannitol, which was sufficient to impart salinity and temperature stress tolerance. Transcriptomics revealed significant differences in the expression of numerous genes, in standard and salinity stress conditions, including genes involved in K+ homeostasis, ROS signaling, plant development, photosynthesis, ABA signaling and secondary metabolism. These results suggest that the improved tolerance to salinity stress observed in transgenic plants producing mannitol in µM range is achieved by the activation of a significant number of genes, many of which are involved in priming and modulating the expression of genes involved in a variety of functions including hormone signaling, osmotic and oxidative stress, and ion homeostasis.

Isoprostanoid Profiling of Marine Microalgae.

Algae result from a complex evolutionary history that shapes their metabolic network. For example, these organisms can synthesize different polyunsaturated fatty acids, such as those found in land plants and oily fish. Due to the presence of numerous double-bonds, such molecules can be oxidized nonenzymatically, and this results in the biosynthesis of high-value bioactive metabolites named isoprostanoids. So far, there have been only a few studies reporting isoprostanoid productions in algae. To fill this gap, the current investigation aimed at profiling isoprostanoids by liquid chromatography -mass spectrometry/mass spectrometry (LC-MS/MS) in four marine microalgae. A good correlation was observed between the most abundant polyunsaturated fatty acids (PUFAs) produced by the investigated microalgal species and their isoprostanoid profiles. No significant variations in the content of oxidized derivatives were observed for Rhodomonas salina and Chaetoceros gracilis under copper stress, whereas increases in the production of C18-, C20- and C22-derived isoprostanoids were monitored in Tisochrysis lutea and Phaeodactylum tricornutum. In the presence of hydrogen peroxide, no significant changes were observed for C. gracilis and for T. lutea, while variations were monitored for the other two algae. This study paves the way to further studying the physiological roles of isoprostanoids in marine microalgae and exploring these organisms as bioresources for isoprostanoid production.

The genome of Ectocarpus subulatus - A highly stress-tolerant brown alga.

Brown algae are multicellular photosynthetic stramenopiles that colonize marine rocky shores worldwide. Ectocarpus sp. Ec32 has been established as a genomic model for brown algae. Here we present the genome and metabolic network of the closely related species, Ectocarpus subulatus Kützing, which is characterized by high abiotic stress tolerance. Since their separation, both strains show new traces of viral sequences and the activity of large retrotransposons, which may also be related to the expansion of a family of chlorophyll-binding proteins. Further features suspected to contribute to stress tolerance include an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defence compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. Overall, E. subulatus has mainly lost members of gene families down-regulated in low salinities, and conserved those that were up-regulated in the same condition. However, 96% of genes that differed between the two examined Ectocarpus species, as well as all genes under positive selection, were found to encode proteins of unknown function. This underlines the uniqueness of brown algal stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies.

Engineering Mannitol Biosynthesis in Escherichia coli and Synechococcus sp. PCC 7002 Using a Green Algal Fusion Protein.

The genetic engineering of microbial cell factories is a sustainable alternative to the chemical synthesis of organic compounds. Successful metabolic engineering often depends on manipulating several enzymes, requiring multiple transformation steps and selection markers, as well as protein assembly and efficient substrate channeling. Naturally occurring fusion genes encoding two or more enzymatic functions may offer an opportunity to simplify the engineering process and to generate ready-made protein modules, but their functionality in heterologous systems remains to be tested. Here we show that heterologous expression of a fusion enzyme from the marine alga Micromonas pusilla, comprising a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase, leads to synthesis of mannitol by Escherichia coli and by the cyanobacterium Synechococcus sp. PCC 7002. Neither of the heterologous systems naturally produce this sugar alcohol, which is widely used in food, pharmaceutical, medical, and chemical industries. While the mannitol production rates obtained by single-gene manipulation were lower than those previously achieved after pathway optimization with multiple genes, our findings show that naturally occurring fusion proteins can offer simple building blocks for the assembly and optimization of recombinant metabolic pathways.

Isoprostanoids quantitative profiling of marine red and brown macroalgae.

With the increasing demand for direct human and animal consumption seaweed farming is rapidly expanding worldwide. Macroalgae have colonized aquatic environments in which they are submitted to frequent changes in biotic and abiotic factors that can trigger oxidative stress (OS). Considering that isoprostanoid derivatives may constitute the most relevant OS biomarkers, we were interested to establish their profile in two red and four brown macroalgae. Seven phytoprostanes, three phytofuranes, and four isoprostanes were quantified through a new micro-LC-MS/MS method. The isoprostanoid contents vary greatly among all the samples, the ent-16(RS)-9-epi-ST-Δ14-10-PhytoF and the sum of 5-F2t-IsoP and 5-epi-5F2t-IsoP being the major compounds for most of the macroalgae studied. We further quantified these isoprostanoids in macroalgae submitted to heavy metal (copper) exposure. In most of the cases, their concentrations increased after 24 h of copper stress corroborating the original hypothesis. One exception is the decrease of ent-9-L1-PhytoP content in L. digitata.

Meneco, a Topology-Based Gap-Filling Tool Applicable to Degraded Genome-Wide Metabolic Networks.

Increasing amounts of sequence data are becoming available for a wide range of non-model organisms. Investigating and modelling the metabolic behaviour of those organisms is highly relevant to understand their biology and ecology. As sequences are often incomplete and poorly annotated, draft networks of their metabolism largely suffer from incompleteness. Appropriate gap-filling methods to identify and add missing reactions are therefore required to address this issue. However, current tools rely on phenotypic or taxonomic information, or are very sensitive to the stoichiometric balance of metabolic reactions, especially concerning the co-factors. This type of information is often not available or at least prone to errors for newly-explored organisms. Here we introduce Meneco, a tool dedicated to the topological gap-filling of genome-scale draft metabolic networks. Meneco reformulates gap-filling as a qualitative combinatorial optimization problem, omitting constraints raised by the stoichiometry of a metabolic network considered in other methods, and solves this problem using Answer Set Programming. Run on several artificial test sets gathering 10,800 degraded Escherichia coli networks Meneco was able to efficiently identify essential reactions missing in networks at high degradation rates, outperforming the stoichiometry-based tools in scalability. To demonstrate the utility of Meneco we applied it to two case studies. Its application to recent metabolic networks reconstructed for the brown algal model Ectocarpus siliculosus and an associated bacterium Candidatus Phaeomarinobacter ectocarpi revealed several candidate metabolic pathways for algal-bacterial interactions. Then Meneco was used to reconstruct, from transcriptomic and metabolomic data, the first metabolic network for the microalga Euglena mutabilis. These two case studies show that Meneco is a versatile tool to complete draft genome-scale metabolic networks produced from heterogeneous data, and to suggest relevant reactions that explain the metabolic capacity of a biological system.

The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea.

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

Host-microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures.

Like most eukaryotes, brown algae live in association with bacterial communities that frequently have beneficial effects on their development. Ectocarpus is a genus of small filamentous brown algae, which comprises a strain that has recently colonized freshwater, a rare transition in this lineage. We generated an inventory of bacteria in Ectocarpus cultures and examined the effect they have on acclimation to an environmental change, that is, the transition from seawater to freshwater medium. Our results demonstrate that Ectocarpus depends on bacteria for this transition: cultures that have been deprived of their associated microbiome do not survive a transfer to freshwater, but restoring their microflora also restores the capacity to acclimate to this change. Furthermore, the transition between the two culture media strongly affects the bacterial community composition. Examining a range of other closely related algal strains, we observed that the presence of two bacterial operational taxonomic units correlated significantly with an increase in low salinity tolerance of the algal culture. Despite differences in the community composition, no indications were found for functional differences in the bacterial metagenomes predicted to be associated with algae in the salinities tested, suggesting functional redundancy in the associated bacterial community. Our study provides an example of how microbial communities may impact the acclimation and physiological response of algae to different environments, and thus possibly act as facilitators of speciation. It paves the way for functional examinations of the underlying host-microbe interactions, both in controlled laboratory and natural conditions.

Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp.

The sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology.