Search

Astaxanthin is a red orange xanthophyll carotenoid produced mainly by microalgae but which can also be chemically synthesized. As demonstrated by several studies, this lipophilic molecule is endowed with potent antioxidant properties and is able to modulate biological functions. Unlike synthetic astaxanthin, natural astaxanthin (NAst) is considered safe for human nutrition, and its production is considered eco-friendly. The antioxidant activity of astaxanthin depends on its bioavailability, which, in turn, is related to its hydrophobicity. In this study, we analyzed the water-solubility of NAst and assessed its protective effect against oxidative stress by means of different approaches using a neuroblastoma cell model. Moreover, due to its highly lipophilic nature, astaxanthin is particularly protective against lipid peroxidation; therefore, the role of NAst in counteracting ferroptosis was investigated. This recently discovered process of programmed cell death is indeed characterized by iron-dependent lipid peroxidation and seems to be linked to the onset and development of oxidative-stress-related diseases. The promising results of this study, together with the “green sources” from which astaxanthin could derive, suggest a potential role for NAst in the prevention and co-treatment of chronic degenerative diseases by means of a sustainable approach.

The use of multi-wavelength spectrofluorometers for the fast detection of algal taxa, based on chlorophyll

The use of multi-wavelength spectrofluorometers for the fast detection of algal taxa, based on chlorophyll a (Chl-a) emission spectra, has become a common practice in freshwater water management, although concerns about their accuracy have been raised. Here, inter-laboratory comparisons using monoalgal cultures have been performed to assess the reliability of different spectrofluorometer models, alongside Chl-a extraction methods. Higher Chl-a concentrations were obtained when using the spectrofluorometers than extraction methods, likely due to the poor extraction efficiencies of solvents, highlighting that traditional extraction methods could underestimate algal or cyanobacterial biomass. Spectrofluorometers correctly assigned species to the respective taxonomic group, with low and constant percent attribution errors (Chlorophyta and Euglenophyceae 6–8%, Cyanobacteria 0–3%, and Bacillariophyta 10–16%), suggesting that functioning limitations can be overcome by spectrofluorometer re-calibration with fresh cultures. The monitoring of a natural phytoplankton assemblage dominated by Chlorophyta and Cyanobacteria gave consistent results among spectrofluorometers and with microscopic observations, especially when cell biovolume rather than cell density was considered. In conclusion, multi-wavelength spectrofluorometers were confirmed as valid tools for freshwater monitoring, whereas a major focus on intercalibration procedures is encouraged to improve their reliability and broaden their use as fast monitoring tools to prevent environmental and public health issues related to the presence of harmful cyanobacteria.

Macroalgae produce several allelopathic substances, including polyunsaturated aldehydes (PUAs), which may inhibit photosynthesis and growth rates of other algal species, and grazing. Additionally, macroalgal structural complexity is an important factor in determining abundance patterns and size structure of epiphytic organisms. In this study the PUAs production of two Mediterranean macroalgae, Dictyopteris polypodioides, (DP, Phaeophyceae, Dictyotales) and Cystoseira compressa (CC, Phaeophyceae, Fucales), was characterized to clarify the relationships between the meiobenthic and microphytobenthic communities. Results showed a higher PUAs production and a diverse qualitative profile for DP, which reported long-chain compounds (i.e. C14-C16) as main aldehydes, than CC, with the short-chain C6:2 as the main compound, as well as variability among sampling times. A clear separation of the meiofauna and microphytobenthos assemblages was found for the macroalgae, but with different temporal trends. Dissimilarities were due to five microalgal orders, namely Naviculales, Lyrellales, Gonyaulacales (i.e. Ostreopsis), Bacillariales, and Licmophorales, and to the meiofaunal groups nematodes, copepods, and copepod nauplii, which were more abundant on DP than on CC. Results indicate that macroalgal complexity is a major determinant of the meiofaunal community structure (accounting for 26% of the variation), rather than PUAs production itself (17%). PUAs effects seem species-specific, thus affecting some grazers instead of the entire community. Conversely, microphytobenthos affected the meiofauna assemblages, particularly harpacticoids, confirming the role of these organisms as the primary food source of all marine food chain producers. Since PUAs are produced also by several epiphytic diatoms, the understanding of their effects on the community structure and on the relationships among taxa in the field is complicated and requires further in-depth investigations in simplified systems (i.e. microcosms).

Chemical interactions between macroalgae and other organisms play an important role in determining species compositions and dominance patterns, and can explain the widespread success of some species in establishing their predominant populations in a specific coastal area. Allelopathy could act as a self-regulatory strategy of the algal community, being not only a succession regulator but also an active mechanism maintaining the species diversity especially in a delimited environment, such as the benthic ecosystem. Polyunsaturated aldehydes (PUAs) are among the most studied allelopathic compounds and are commonly released into the aquatic envi-ronment by different phytoplankton species in response to environmental stressors (e.g. wounding, grazing, or competition for nutrients). Diatom-released PUAs were observed to affect phytoplankton community dynamics and structure, and showed inhibitory effects on the reproduction and development of marine invertebrates. As for macroalgae, there are only a few reports that attest to the production of PUAs, and mostly refer to Ulva spp. In this study, the production of PUAs by several Mediterranean macroalgae was investigated at different sampling times, aiming at providing the first evidence of potential allelochemical activity. Results highlighted the potential production by macroalgae of a variety of aldehydes, among which some have never reported so far. Some species (i.e. D. polypodioides and U. cf. rigida) were found to produce higher PUAs amounts than others, and even a wider variety of structures (e.g. length of the carbon chain); these species might exert strong effects on epiphytic species or other organisms of the benthic community, especially considering the differential sensitivities of the various taxa. A high dPUA concentration (order of μM) potentially due to the release of PUAs by algal species was found, and might affect the population dynamics of the epiphytic organisms (e.g. microalgae, meiofauna), of grazers, as well as of the microbial community.

Three novel hydrophobic deep eutectic solvents (DESs) based on oleic acid and terpenes (thymol, DL-menthol, and geraniol) were prepared, characterized, and used to extract astaxanthin from the microalga Haematococcus pluvialis without any pre-treatment of the cells. The three DES were composed of Generally Recognized As Safe (GRAS) and edible ingredients. All the tested DESs gave astaxanthin recovery values of about 60 and 30% in 6 h if applied on freeze-dried biomass or directly on algae culture, respectively. The carotenoid profile was qualitatively identical to what was obtained by using traditional organic solvents, regardless of the DES used; the monoesters of astaxanthin with C18-fatty acids were the main compounds found in all the carotenoid extracts. The thymol:oleic acid DES (TAO) could preserve astaxanthin content after prolonged oxidative stress (40% of the astaxanthin initially extracted was still present after 13.5 h of light exposure), thanks to the superior antioxidant properties of thymol. The capacity of improving astaxanthin stability combined with the intrinsic safety and edibility of the DES components makes the formulation astaxanthin-TAO appealing for the food ingredients/additives industry.

The ability of microalgae to preserve viable in coastal sediments as resting forms provides a reservoir of biodiversity and a useful tool to determine species spreadings. This study represents the first port baseline survey on dinoflagellate cysts, investigated in nine Adriatic ports during a cross border project. 40 dinoflagellate taxa were detected. The assemblages resulted in all ports dominated by Lingulodinium polyedra and Alexandrium minutum/affine/tamutum group. General separation to the western and eastern side of the Adriatic regarding cysts assemblage composition, partially abundance, was observed. Six taxa were detected as non-indigenous species for the Adriatic. Two taxa are included in the list of harmful aquatic organisms, indicating the potential threat of ballast waters in the Adriatic. Potential spreading of taxa by general circulation and ballast waters, intra- and extra-Adriatic was investigated. The entering in to force of the ballast waters management regulations should enhance prospects to minimize future harmful impacts.

The ability of microalgae to sequester carbon and at the same time synthesise valuable compounds with potential applications in nutraceutical, pharmaceutical and cosmetic industries makes them attractive for commercial deployment, especially in view of a blue bioeconomy. Among microalgae, the diatom Phaeodactylum tricornutum is considered as an important potential source of omega-3 polyunsaturated fatty acids, such as eicosapentanoic acid, an essential polyunsaturated fatty acid with anti-inflammatory and antimicrobial properties. The aim of this study was to perform the Life Cycle Assessment of the cultivation of P. tricornutum - at semi-industrial scale in photobioreactor - for the production of high-quality bioactive compounds comparing synthetic carbon dioxide supply to a supply with waste carbon dioxide from a biogas upgrading process hypothesizing industrial symbiosis network. The effect of renewable energy use instead of the European electricity mix was also examined. Primary data on the production process, including the stages of cleaning and sterilisation, cultivation, harvesting and freeze-drying, were used. The midpoint impact categories recommended in the ILCD Handbook were used for performing the impact assessment. A sensitivity analysis was also performed on algal productivity, culture medium recirculation factor and amount of solvents per cleaning cycle. Firstly, results indicate in general cultivation and freeze-drying as the most contributing stages to the impacts. Secondly, they demonstrate in the comparative assessment that the use of carbon dioxide from the biogas upgrading is a feasible and attractive alternative to the synthetic one, as it allows for the improvement of the environmental performance of the production process in all the analysed impact categories. Finally, sensitivity analysis suggests that the environmental performance could be further improved by acting on other key factors, such as electricity source, nutrients culture medium and cleaning solutions.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

The production of polyunsaturated aldehydes (PUAs) has been reported by many planktonic diatoms, where they have been implicated in deleterious effects on copepod reproduction and growth of closeby microbes or suggested as infochemicals in shaping plankton interactions. This study investigates the production of PUAs by diatoms commonly occurring in the microphytobenthic communities in temperate regions: Tabularia affinis , Proschkinia complanatoides and Navicula sp. Results highlight the production of PUAs by the three benthic diatoms during stationary and decline phases, with intracellular concentrations from 1.8 to 154.4 fmol cell −1 , which are within the range observed for planktonic species. The existence of a large family of PUAs, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal, was observed. Since particulate and dissolved PUAs were positively correlated, together with cell lysis, equivalent concentrations may be released during late growth stages, which may affect benthic invertebrates grazing on them and other microalgae.

The study evaluated the growth of Desmodesmus communis on column photobioreactor and its thermochemical treatment by catalytic pyrolysis using HZSM-5 zeolite. D. communis showed good results in terms of growth (0.05g L−1 d−1). Analytical pyrolysis of original algae and derived bio-oil mixed with zeolite was used as a screening method in order to gather information on the cracking process. Preparative pyrolysis on bench scale reactor was performed on algae biomass over a zeolite bed at 1:10 ratio (wt/wt). Py-GC–MS of biomass/catalyst mixture showed that the denitrogenation/deoxygenation increased with increasing zeolite load from 1:5 to 1:20 ratio and became significant at 1:10 ratio. The composition observed by analytical pyrolysis was featured by the predominance of alkylated monoaromatic hydrocarbons. The scaling-up to bench scale confirmed the results obtained with analytical pyrolysis in terms of monoaromatic hydrocarbons. However, low yield of catalytic oil (8% by weight) was observed.

Plastid-encoded rbcL and psbA sequences from branched, Caribbean Sea Lithophyllum specimens indicate that four species are present, not one. Short (263 base pairs) rbcL sequences from an isolectotype of L. kaiseri (Gulf of Suez) and the holotypes of L. congestum, L. daedaleum and L. platyphyllum (Caribbean Sea) show that L. congestum and L. daedaleum are conspecific with L. kaiseri, the last having nomenclatural priority. Lithophyllum platyphyllum, currently considered a synonym of L. congestum, is recognised as a valid species. Lithophyllum stictaeforme, originally described from the Mediterranean Sea, is not conspecific with L. kaiseri (as L. congestum) as previously suggested. Lithophyllum neocongestum sp. nov. and L. pseudoplatyphyllum sp. nov. are proposed. Together with L. platyphyllum, these three branched species are so far endemic to the Caribbean Sea. This is the first report, documented by DNA sequence data, of a coralline species (L. kaiseri) widespread through the tropical Indo-West Pacific Oceans, Red Sea and Caribbean Sea.

In the last decade Ostreopsis cf. ovata blooms have been among the most intense along the entire Mediterranean coast, leading to ecological and human health problems, that are associated with the toxins (palytoxin-like compounds) produced by these algal cells. These compounds are secondary metabolites, whose rates of synthesis depend on the metabolism of their precursors. In general, growth dynamics and toxicity of dinoflagellates reflect the physiological status of the organism. The aim of the present study was to investigate the cellular production of the main biochemical compounds likely involved in the growth and toxicity dynamics of O. cf. ovata during exponential to the late stationary phase in batch cultures of an Adriatic strain. Removal of major nutrients from the medium was monitored along with concentration, biovolume and production of the main cellular components (e.g. polysaccharides, proteins, lipids and toxins). Nutrient uptake, as well as toxin production rates were calculated in the different growth periods. Nutrients (N and P) were completely depleted when cells entered stationary phase and the greatest net toxin production rate (RTOX) occurred during the first days of growth. The various palytoxins reported a relative abundance quite stable during the different growth phases, while the total toxin cellular amount increased along the growth curve. Total and extracellular released polysaccharides, as well as the lipid content increased greatly during the stationary phase, while proteins were mainly produced by cells during the exponential phase. The continuous release of polysaccharides could facilitate cell aggregation and the formation of the benthic community during algal blooms. The trend of production of the main cellular compounds in O. cf. ovata and the growth dynamics of this species lead us to hypothesize that the fast growth of this dinoflagellate, associated with the rapid use of environmental resources (nutrients, and phosphates in particular), may be an ecological/ adaptive strategy which could favor this organism in competition with other species.

International audience; Lithophyllum species in the Mediterranean Sea function as algal bioconstructors, contributing to the formation of biogenic habitats such as coralligenous concretions. In such habitats, thalli of Lithophyllum, consisting of crusts or lamellae with entire or lobed margins, have been variously referred to as either one species, L. stictiforme, or two species, L. stictiforme and L. cabiochiae, in the recent literature. We investigated species diversity and phylogenetic relationships in these algae by sequencing three markers (psbA and rbcL genes, cox2,3 spacer), in conjunction with methods for algorithmic delimitation of species (ABGD and GMYC). Mediterranean subtidal Lithophyllum belong to a well‐supported lineage, hereby called the L. stictiforme complex, which also includes two species described from the Atlantic, L. lobatum and L. searlesii. Our results indicate that the L. stictiforme complex consists of at least 13 species. Among the Mediterranean species, some are widely distributed and span most of the western and central Mediterranean, whereas others appear to be restricted to specific localities. These patterns are interpreted as possibly resulting from allopatric speciation events that took place during the Messinian Salinity Crisis and subsequent glacial periods. A partial rbcL sequence from the lectotype of L. stictiforme unambiguously indicates that this name applies to the most common subtidal Lithophyllum in the central Mediterranean. We agree with recent treatments that considered L. cabiochiae and L. stictiforme conspecific. The diversity of Lithophyllum in Mediterranean coralligenous habitats has been substantially underestimated, and future work on these and other Mediterranean corallines should use identifications based on DNA sequences.

Phaeodactylum tricornutum is considered a promising source of polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (EPA). In this study, P. tricornutum cultivation using waste products from anaerobic digestion (i.e. liquid digestate and CO2) was tested and scaled-up in closed and open prototype systems. The chemical composition of algal biomass was evaluated to optimize the lipid content. Algal productivity and composition, especially in terms of PUFAs, were not modified by the use of waste CO2. Digestate led to a lower protein (24%) content than medium (36–37%), without affecting lipid amount (about 37%). Algal and EPA productivity were nearly two-fold higher by using photobioreactors (0.075 g biomass L−1 day−1 and 1.62 mg EPA g−1 day−1) than open ponds, which are more influenced by environmental conditions. This study highlights that economic and environmental benefits could be achieved by using waste CO2 and liquid digestate from anaerobic digestion for microalgae cultivation.

We thank the University of Bologna (RFO program) for funding. The microalga Haematococcus pluvialis is capable of accumulating natural astaxanthin when subjected to external stress and shifted towards a red-cyst phase, characterized by a robust and multilayered cell wall. In the present study, still-unexplored solvents were applied for extracting astaxanthin from H. pluvialis, directly from algae culture and without any pre-treatment of the cells. Among the tested solvents, some of them (e.g. ethyl acetate and 2-methyltehydrofuran) gave excellent astaxanthin recovery (>80%) in a short time (30 min) and others (e.g. isoamyl acetate, well known as a human-compatible solvent and already in use as a food additive) gave an astaxanthin recovery close to 90% in 1 hour and thus they are exploitable in the natural astaxanthin market. Almond oil is proved to be able to extract astaxanthin and keep H. pluvialis alive, without affecting the algal photosynthetic activity, providing the possibility to milk and regeneratively cultivate H. pluvialis and avoid an uneconomical loss of biomass.

The feasibility of the green alga Desmodesmus communis for biomass production was investigated, firstly testing different nitrogen forms in the growth medium and the effect of CO2-enriched air supply, secondarily scaling up the cultivation system in 70 L photobioreactors (PBRs). Maximum nitrogen uptake rate obtained in the performed kinetic experiment was higher for ammonium than for nitrate (188.0 vs 11.7 μmol g−1 h−1); however, D. communis cultured in PBRs with only aeration grew faster with nitrate reaching a biomass yield (1.23 g L−1) and a productivity (0.036 g L−1 day−1) about twofold higher than with ammonium, which caused a pH decrease in the medium affecting the algal growth. CO2 supply allowed algal growth optimization, maintaining a high productivity with both nitrogen sources, slightly higher with nitrate (0.050 vs 0.038 g L−1 day−1). Additionally, nitrate-supplied cells showed higher lipids (19.0 vs 9.4%) and proteins (33.0 vs 27.2%) values than those grown with ammonium. The semi-continuous scaled-up cultivation performed for 5 months attests the potential utilization of this species for valuable algal biomass production exploitable in various industrial applications.

Flocculation induced by pH decrease is here presented as a solution to pre-concentrate algal cultures of species belonging to Scenedesmaceae and preserve the integrity of the biomass, while maintaining the photosynthetic capacity of non-harvested cells. The low-pH flocculation method gives highly effective results exclusively by the strength of the acid (hydrochloric or formic) used for the treatment, and is applicable to cultures having high cell densities (> 2 g L− 1). High flocculation efficiencies (> 90%) are obtained also by using heterogeneous acids, such as solid acid carbons from naturally available compounds, which are proposed as cheap recyclable materials to be exploited for this purpose. Additionally, the potential reuse of the filtered medium (sterilized or not) and of the supernatant (containing non-harvested cells) is investigated to avoid great water losses and to re-inoculate new cultures.

Ostreopsis blooms regularly occur in many Mediterranean coastal areas in late summer-autumn. In the northern Adriatic Sea, Ostreopsis blooms affect diatom-dominated microphytobenthic communities. In this study, the effects of the filtrates of some diatom species, both benthic (Tabularia affinis, Proschkinia complanatoides and Navicula sp.) and planktonic (Thalassiosira sp. and Skeletonema marinoi) on cell morphology, cytological features and growth of O. cf. ovata were investigated. Our results showed a marked decrease of O. cf. ovata growth when cells were exposed to all diatom filtrates tested. The highest inhibitions were observed for exposures to P. complanatoides and Navicula sp. filtrates (92.5% and 80%, respectively) and increased with the age of diatom culture. Moreover, a clear DNA degradation and abnormal forms of O. cf. ovata cells (83.8% of the total) were found at the highest concentrations using Navicula sp. filtrate after 10 days of the inoculum.

Lithophyllum byssoides is a comm on coralline alga in the intertidal zone of Mediterranean coasts, where it produces biogenic concretions housing a high algal and invertebrate biodiversity. This species is an ecosystem engineer and is considered a target for conservation efforts, but designing effective conservation strategies currently is impossible due to lack of information abo ut its population structure. The morphological and molecular variation of L. byssoides was investigated using morphoanatomy and DNA sequences (psbA and cox2,3) obtained from populations at 15 localities on the Italian and Croatian coasts. Lithophyllum byssoides exhibited a high number of haplotypes (31 psbA haplotypes and 24 cox2,3 haplotypes) in the central Mediterranean. The psbA and cox2,3 phylogenies were congruent and showed seven lineages. For most of these clades, the distribution was limited to one or a few localities, but one of them (clade 7) was widespread across the central Mediterranean, spanning the main biogeographic boundaries recognized in this area. The central Mediterranean populations formed a lineage separate from Atlantic samples; psbA pairwise divergences suggested that recognition of Atlantic and Mediterranean L. byssoides as different species may be appropriate. The central Mediterranean haplotype patterns of L. byssoides were interpreted as resulting from past climatic events in the hydrogeological history of the Mediterranean Sea. The high haplotype diversity and the restricted spatial distribution of the seven lineages suggest that individual populations should be managed as independent units.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

Diatoms have been shown to produce and release a wide range of secondary metabolites that mediate interactions between individuals of different species. Among these compounds, different types of fatty acid derived long-chained polyunsaturated aldehydes (PUAs) have been related to multiple functions such as intra- or interspecific signals and adverse effect on the reproduction of marine organisms. Several studies have reported changes on growth, cell membrane permeability, flow cytometric properties and cell morphology in phytoplankton organisms exposed to PUAs, but little information is available on the effect of these compounds on benthic microalgae. Ostreopsis cf. ovata is a toxic benthic dinoflagellate which causes massive blooms along the Mediterranean coasts typically during the late summer period. In this study the effects of three toxic PUAs known to be produced by several algae (2 E ,4 E -decadienal, 2 E ,4 E -octadienal and 2 E ,4 E -heptadienal) on the growth, cytological features and cell morphology of O. cf. ovata were investigated. Our results show a clear decrease of O . cf. ovata growth with longer-chain molecules than with shorter-chain ones, confirmed also by EC 50 values calculated at 48 h for 2 E ,4 E -decadienal and 2 E ,4 E -octadienal (6.6 ± 1.5, 17.9 ± 2.6 μmol L −1 respectively) and at 72 h for 2 E ,4 E -heptadienal (18.4 ± 0.7 μmol L −1 ). Moreover, morphological analysis highlighted up to 79% of abnormal forms of O. cf. ovata at the highest concentrations of 2 E ,4 E -decadienal tested (9, 18 and 36 μmol L −1 ), a gradual DNA degradation and an increase of lipid droplets with all tested PUAs. Further studies are needed to better clarify the interactions between diatoms and O . cf. ovata , especially on bloom-forming dynamics.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & O. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value

A new procedure based on switchable polarity solvents (SPS) was proposed for lipid extraction of wet algal samples or cultures, thereby circumventing the need for an energy intensive drying step and facilitating easy recovery of the lipids from the extraction liquid. Lipids were extracted by using N,N-dimethylcyclohexylamine (DMCHA) and recovered by adding CO2, thereby switching DMCHA into a hydrogen carbonate ammonium salt and resulting in the formation of a separate liquid lipid phase.

Bacteria associated to benthic dinoflagellate Ostreopsis cf. ovata cultures were removed to assess their effects on algal growth and toxins' production. Bacteria were removed using an antibiotic cocktail (streptomycin, ciprofloxacin, gentamicin and penicillin G). The actual axenic status of antibiotic treated cultures and bacterial growth in control cultures were assessed by epifluorescence microscopy using SYBR gold dye. The removal of bacteria unaffected algal growth, except conferring a higher cell number at mid stationary phase. Toxin profile and quantification of each toxin (PLTX, OVTX-a, -b, -c, -d, -e) were performed by HR LC-MS on both cell pellet and growth medium extracts. No changes in toxins' profile nor in cell and extra-cellular toxins' concentrations were found between bacteria-free and control cultures at the early stationary phase. Whereas, in late stationary phase axenic cultures showed significant lower cell toxins' concentrations and higher extra-cellular toxins' values, thoug...

This article describes the toxic effects of imidazolium ionic liquids bearing alkyl (BMIM), monoethoxy (MOEMIM), and diethoxy (M(OE)(2)MIM) side chains toward two marine diatoms, Skeletonema marinoi and Phaeodactylum tricornutum. MOEMIM and M(OE)(2)MIM cations showed a lower inhibition of growth and photosynthetic efficiency with respect to their alkyl counter part, with both algal Species. However, a large difference in sensitivity was found between S. marinoi and P. tricornutum, the first being much more Sensitive to the action of ionic liquids than the second one. The effects of salinity on BMIM Cl toxicity toward S. marinoi revealed that a decrease from salinity 35 to salinity 15 does not influence the biological effects toward the alga. Finally, Fourier transform infrared (FT-IR) microscopy of alga cells after ionic liquids exposure allowed us to detect an alteration of the organic cellular components related. to silica uptake and organization. On the basis of these results, the different behavior of the two diatom species can be tentatively ascribed to different silica uptake and organization in outer cell walls

Since the 1990s red-tide blooms of Fibrocapsa japonica have frequently been observed in European waters; despite their recurrence also in the Adriatic coastal areas, they have never been conclusively linked to ichthyotoxic events. Since the toxicity of F. japonica is still under debate and its effects differ among the strains, in this study all the compounds previously postulated as involved in the toxic mechanism, such as brevetoxins, fatty acids and ROS, were screened for Adriatic strains. Whole algal extracts were analyzed for brevetoxin PbTx-2, which was not found, and for a qualitative and quantitative analysis of fatty acids. The fatty acid profile evidenced the presence of PUFAs, with considerable amounts of 18:4n-3, 20:4n-6, and 20:5n-3, as already found in different strains, but with a lower amount of arachidonic acid (20:4n-6). None of the PUFAs was released in the extracellular medium. Different toxicological assays (Vibrio fischeri, Artemia sp., haemolysis of fish erythrocytes) were performed using algal cellular, extracellular or subcellular samples as well as fractions of the algal extract, in order to identify the toxic compounds. The tested cultures were found to inhibit V. fischeri bioluminescence, to affect Artemia nauplii viability and to have haemolytic effects comparable to those previously observed in different strains. The solid phase extraction (SPE) of the Adriatic F. japonica extracts resulted in a high concentration of PUFAs in two main fractions, which caused mortality in Artemia nauplii and inhibition of V. fischeri bioluminescence. Fish (Dicentrarchus labrax) assays were also performed, reporting mortality after long exposure times and the cause of fish death was investigated. A significant increase of H2O2 in the tanks where sea basses were exposed to F. japonica was obtained, indicating that the presence of fish stimulates H2O2 production by algal cells and leading to the hypothesis of H2O2 involvement in F. japonica toxicity. The presence of oxidative stress in the exposed fish was confirmed by the increased malondialdehyde concentration in their gills. Overall these results indicate that since F. japonica cells can easily cling to fish gills, due to their high polysaccharide production, it is presumable that high PUFA amounts are released in loco, as a result of cell breakage, and that exert their toxicity together with ROS. This study shows that the Adriatic strains of F. japonica can be harmful to higher level organisms. Nevertheless a high cell density and a long lasting bloom are necessary to cause severe damage to fish gills or death for crustaceans.

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata are usually associated with shallow and calm coastal waters, characterized by low nutrient concentrations. The algal cells typically cover the benthic substrates, such as the macroalgal and invertebrate communities and rocks, forming a mucilaginous film. Data reported on O. cf. ovate toxin production observed under both field and culture conditions show high variability in terms of toxic profile and cellular content; little is known about the environmental and physiological aspects which regulate the toxin dynamics. In this study, O. cf. ovata physiology was investigated using batch cultures supplied with nutrient concentrations similar to those found in the Adriatic Sea during the recurrent blooms and the observed cellular dynamics were compared with those found in a culture grown under optimal conditions, used as a reference. Data on the cellular C, N and P content during the growth highlighted a possible important role of the cellular nutritional status in regulating the toxin production that resulted to be promoted under specific intervals of the C:N and C:P ratios. The variable toxicity found for 0. cf. ovata in various geographic areas could be related to the different in situ prevalent environmental conditions (e.g., nutrient concentrations) which affect the cellular elemental composition and carbon allocation. The obtained results strongly suggest that in the environment toxin production is steadily sustained by a low and constant nutrient supply, able to maintain appropriate cellular C:N (>12) or C:P (>170) ratios for a long period. These results explain to some extent the variability in toxicity and growth dynamics observed in blooms occurring in the different coastal areas. (C) 2016 Elsevier B.V. All rights reserved.

Pyrrolidinium imides are considered among the most promising electrolytes for the development of novel and sustainable portable energy devices. Because of this widespread potentiality, a risk scenario of an erroneous disposal of ionic liquids-based batteries in the environment has to be taken into account. In the present study, some of the best energy performing pyrrolidinium-based ionic liquids were evaluated in terms of persistence in aquatic environments and hazard toward freshwater organisms (crustacean Daphnia magna and unicellular green alga Raphidocelis subcapitata). The examined ionic liquids were not aerobically biodegradable (biodegradation less than 5% in 28 days), but they demonstrated low toxicity toward algae and crustaceans, according to the standard bioassay end points (EC50 > 100 mg L-1). However, ionic liquids were able to alter the cellular morphology of R subcapitata and an increased amount of proteins (30%) was observed in the exposed cells, suggesting an inhibition of cellular division.

Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies have identified a number of palytoxin-like compounds, termed ovatoxins, along with trace amounts of putative palytoxin as the causative agents of the O. cf. ovata -related human sufferings. So far, any risk assessment for ovatoxins as well as establishment of their allowance levels in seafood has been prevented by the lack of pure toxins. The present paper reports on the isolation, NMR-based structural determination, and preliminary mouse lethality evaluation of ovatoxin-a, the major toxic compound contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will open the double prospect of fully evaluating its toxicity and preparing reference standards to be employed in LC/MS quantitative analyses. Elucidation of ovatoxin-a's complex structure will ultimately herald the understanding of the molecular bases of ovatoxins bioactivity.

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported.

In the Mediterranean Sea, blooms of Ostreopsis cf. ovata and Ostreopsis siamensis have become increasingly frequent in the last decade and O. cf. ovata was found to produce palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, -d and -e), a class of highly potent toxins. The environmental conditions seem to play a key role in influencing the abundance of Ostreopsis spp. High cell densities are generally recorded in concomitance with relatively high temperature and salinity and low hydrodynamics conditions. In this study the effects of temperature and salinity on the growth and toxicity of an Adriatic O. cf. ovata isolate were investigated. The highest growth rates of the Adriatic strain were recorded for cultures grown at 20 °C and at salinity values of 36 and 40, in accordance with natural bloom surveys. Toxicity was affected by growth conditions, with the highest toxin content on a per cell basis being measured at 25 °C and salinity 32. However, the highest total toxin content on a per litre basis was recorded at 20 °C and salinity 36, since under such conditions the growth yield was the highest. O. cf. ovata had lethal effects on Artemia nauplii and juvenile sea basses, and produced haemolysis of sheep erythrocytes. A comparison between haemolysis neutralization assay and HR LC-MS results showed a good correlation between haemolytic effect and total toxin content measured through HR LC-MS. Considering the increasing need for rapid and sensitive methods to detect palytoxin in natural samples, the haemolytic assay appears a useful method for preliminary quantification of the whole of palytoxin-like compounds in algal extracts.

Since the end of the 1990s the occurrence of blooms of the benthic dinoflagellates Ostreopsis spp. is spreading in many tropical and temperate regions worldwide, sometimes causing benthonic biocenosis suffering and occasional human distress. Ostreopsis ovata has been found to produce palytoxin-like compounds, a class of highly potent toxins. As general, the highest abundances of Ostreopsis spp. are recorded during warmer periods characterized by high temperature, salinity, and water column stability. Moreover, as these cells are easily resuspended in the water column, the role of hydrodynamism in the blooms development and decline has been highlighted. The environmental conditions appear, therefore, to be one of the main factors determining the proliferation of these species as testified by several field surveys. Laboratory studies on the effect of environmental parameters on growth and toxicity of O. ovata are rather scarce. With regard to the effects of temperature, culture results indicate that different strains blooming along Italian coasts displayed different optima, in accordance to blooming periods, and that higher toxin levels correlated with best growth conditions. Additionally, in relation to an Adriatic strain, cell growth positively correlated with the increase in salinity, while toxicity was lowest at the highest salinity value (i.e. 40). For the same strain, both nitrogen and phosphorus limitation determined a decrease in cell toxicity showing different behaviour with respect to many other toxic dinoflagellates.

Alexandrium ostenfeldii is a widespread toxic dinoflagellate that has recently bloomed across the Adriatic Sea, seriously threatening both shellfish consumers and aquacultures. In 2007 we reported on preliminary studies carried out on field samples and cultures of A. ostenfeldii. At the time, along with three major spirolides - among which 27-hydroxy-13,19-didesmethyl spirolide C (3) proved to be a novel compound - a number of new minor spirolides were detected. Unfortunately, for all of them only Mass Spectrometry-based structural hypotheses could be ventured due to their very small amount. In the present paper we report on isolation and High Resolution Mass Spectrometry- and NMR-based structural elucidation of two of those minor spirolides detected in our previous study.

Fibrocapsa japonica resting cysts were detected in coastal sediments of the Marche region (northern Adriatic Sea, Italy), where summer blooms regularly occur. Sampling was carried out along 18 transects (2 stations each) during May–June 2007. The identification and counting of raphidophyte cysts were carried out under the inverted microscope and molecular analyses were applied to sediment samples to confirm species-specific identification. Raphidophyte cysts were recorded in almost all stations with maximum abundance of 9 ± 5 and 12 ± 3 cysts g−1 dw for cysts of F. japonica and undetermined Raphidophyceae, respectively. Cyst formation was induced in F. japonica monoclonal cultures under different conditions: shadow-optimal temperature and dark-low temperature treatment; both experiments were performed at two salinity values (30 and 35). Cyst formation (preliminary cysts) was observed in both experiments, although with different encystment rates. However, only a further permanence in the dark at 15 °C in microplates led to the formation of mature cysts, probably supported by the possibility to adhere to solid surfaces. Before pre-cyst formation, vegetative cells showed a cytoplasmatic “brown body”.

New polyethoxylated tertiary amines were synthesised and applied as Switchable Hydrophilicity Solvents (SHS) in the extraction and recovery of lipids from algal cultures, bypassing harvesting and de-watering steps. The eco-toxicological profile of the new amines was explored by evaluating their potential in inhibiting algal growth, and deepened by measuring the acute toxicity towards the crustacean Daphnia magna and the ready biodegradability in water. Among the synthesised amines, the one bearing two ethoxy units, named amine 2, showed the best combination in terms of lipids' extractive performances and toxicity; therefore it could be considered as a good alternative to N,N-dimethylcyclohexylamine (DMCHA), at the moment the most studied and effective SHS. © 2014 The Royal Society of Chemistry.

Algal–bacterial interactions play a major role in shaping diversity of algal associated bacterial communities. Temporal variation in bacterial phylogenetic composition reflects changes of these complex interactions which occur during the algal growth cycle as well as throughout the lifetime of algal blooms. Viruses are also known to cause shifts in bacterial community diversity which could affect algal bloom phases. This study investigated on changes of bacterial and viral abundances, bacterial physiological status, and on bacterial successional pattern associated with the harmful benthic dinoflagellate Ostreopsis cf. ovata in batch cultures over the algal growth cycle. Bacterial community phylogenetic structure was assessed by 16S rRNA gene ION torrent sequencing. A comparison between bacterial community retrieved in cultures and that one co‐occurring in situ during the development of the O. cf. ovata bloom from where the algal strain was isolated was also reported. Bacterial community growth was characterized by a biphasic pattern with the highest contributions (~60%) of highly active bacteria found at the two bacterial exponential growth steps. An alphaproteobacterial consortium composed by the Rhodobacteraceae Dinoroseobacter (22.2%–35.4%) and Roseovarius (5.7%–18.3%), together with Oceanicaulis (14.2‐40.3%), was strongly associated with O. cf. ovata over the algal growth. The Rhodobacteraceae members encompassed phylotypes with an assessed mutualistic‐pathogenic bimodal behavior. Fabibacter (0.7%–25.2%), Labrenzia (5.6%–24.3%), and Dietzia (0.04%–1.7%) were relevant at the stationary phase. Overall, the successional pattern and the metabolic and functional traits of the bacterial community retrieved in culture mirror those ones underpinning O. cf. ovata bloom dynamics in field. Viral abundances increased synoptically with bacterial abundances during the first bacterial exponential growth step while being stationary during the second step. Microbial trends also suggest that viruses induced some shifts in bacterial community composition.