Lipids of different phytoplankton groups differ in sensitivity to degradation: Implications for carbon export.

The future of life on Earth depends on how the ocean might change, as it plays an important role in mitigating the effects of global warming. The main role is played by phytoplankton. Not only are phytoplankton the base of the oceans' food web, but they also play an important role in the biological carbon pump (BCP), the process of forming organic matter (OM) and transporting it to the deep sea, representing a sink of atmospheric CO2 . Lipids are considered important vectors for carbon sequestration. A change in the phytoplankton community composition as a result of ocean warming is expected to affect the BCP. Many predictions indicate a dominance of small at the expense of large phytoplankton. To gain insight into interplay between the phytoplankton community structure, lipid production and degradation, and adverse environmental conditions, we analyzed phytoplankton composition, particulate organic carbon (POC) and its lipid fraction in the northern Adriatic over a period from winter to summer at seven stations with a gradient of trophic conditions. We found that at high salinity and low nutrient content, where nanophytoplankton prevailed over diatoms, the newly fixed carbon is substantially directed toward the synthesis of lipids. Lipids produced by nanophytoplankton, coccolithophores, and phytoflagellates, are more resistant to degradation than those produced by diatoms. The difference in lipid degradability is discussed as a difference in the size of the cell phycosphere. We hypothesize that the lipids of nanophytoplankton are less degradable due to the small phycosphere with a poorer bacterial community and consequently a lower lipid degradation rate compared with diatoms. The lipid chemical composition of the different phytoplankton groups could have a different susceptibility to degradation. Results suggest a successful lipid carbon sink of nanophytoplankton and, thus, a negative feedback on global warming.

Reconstructed membrane vesicles from the microalga Dunaliella as a potential drug delivery system.

The aim of this biophysical study is to characterize reconstructed membrane vesicles obtained from microalgae in terms of their morphology, properties, composition, and ability to transport a model drug. The reconstructed vesicles were either emptied or non-emptied and exhibited a non-uniform distribution of spherical surface structures that could be associated with surface coat proteins, while in between there were pore-like structures of up to 10 nm that could contribute to permeability. The reconstructed vesicles were very soft and hydrophilic, which could be attributed to their composition. The vesicles were rich in proteins and were mostly derived from the cytoplasm and chloroplasts. We demonstrated that all lipid classes of D. tertiolecta are involved in the formation of the reconstructed membrane vesicles, where they play fundamental role to maintain the vesicle structure. The vesicles appeared to be permeable to calcein, impermeable to FITC-ovalbumin, and semipermeable to FITC-concanavalin A, which may be due to a specific surface interaction with glucose/mannose units that could serve as a basis for the development of drug carriers. Finally, the reconstructed membrane vesicles could pave a new way as sustainable and environmentally friendly marine bioinspired carriers and serve for studies on microtransport of materials and membrane-related processes contributing to advances in life sciences and biotechnology.

How do open coastal fire episodes' impact sea surface microlayer neuston communities?

Human-induced climate change is expected to increase the frequency and severity of vegetation fires. The Mediterranean region is considered particularly prone to fire episodes in summer. It is well known that pyrogenic particles are an important source of external nutrients for the marine environment, especially in oligotrophic areas. In this study, the plankton components of the sea surface layers were integrated to evaluate, for the first time, their dynamics over six months and their response to fire events in a typical coastal area of the Adriatic Sea. Concentrations of nutrients and organic compounds, together with plankton communities were significantly higher in the sea surface microlayer (SML, < 1 mm thick), than in the underlying water from 1 m depth. The piconeuston community and chlorophyll a responded with extreme abundance and concentration to the most intense fire event that enriched the SML with NH4+. Phytoneuston abundance increased with a delay of 2 weeks, while diversity indices decreased slightly after the fire events. The large abundances of the studied piconeuston parameters could be explained by the high availability of organic compounds and the immediate availability of NH4+, while the phytoneuston community responded to an increased NO3- concentration, triggered by the fire events. We confirmed that fast-acting marine heterotrophs are important members of biogeochemical cycles associated with fire events and that, together with phytoplankton, they are unavoidable parameters to detect environmental changes.

Effects of high temperature and nitrogen availability on the growth and composition of the marine diatom Chaetoceros pseudocurvisetus.

The assimilation of inorganic nutrients by phytoplankton strongly depends on environmental conditions such as the availability of nitrogen and temperature, especially warming. The acclimation or adaptation of different species to such changes remains poorly understood. Here, we used a multimethod approach to study the viability and physiological and biochemical responses of the marine diatom Chaetoceros pseudocurvisetus to different temperatures (15, 25, and 30 °C) and different N:P ratios. Nitrogen limitation had a greater effect than high temperature on cell growth and reproduction, leading to a marked elongation of setae, decreased phosphorus assimilation, increased lipid accumulation, and decreased protein synthesis. The elongation of setae observed under these conditions may serve to increase the surface area available for the uptake of inorganic and/or organic nitrogen. In contrast, high temperatures (30 °C) had a stronger effect than nitrogen deficiency on cell death, nitrogen assimilation, chlorophyll a accumulation, the cessation of setae formation, and cell lipid remodelling. Significant changes in thylakoid lipids were observed in cells maintained at 30 °C, with increased levels of digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol. These changes may be explained by the role of galactolipids in thylakoid membrane stabilization during heat stress.

Bioprospecting of Microalgae Isolated from the Adriatic Sea: Characterization of Biomass, Pigment, Lipid and Fatty Acid Composition, and Antioxidant and Antimicrobial Activity.

Marine microalgae and cyanobacteria are sources of diverse bioactive compounds with potential biotechnological applications in food, feed, nutraceutical, pharmaceutical, cosmetic and biofuel industries. In this study, five microalgae, Nitzschia sp. S5, Nanofrustulum shiloi D1, Picochlorum sp. D3, Tetraselmis sp. Z3 and Tetraselmis sp. C6, and the cyanobacterium Euhalothece sp. C1 were isolated from the Adriatic Sea and characterized regarding their growth kinetics, biomass composition and specific products content (fatty acids, pigments, antioxidants, neutral and polar lipids). The strain Picochlorum sp. D3, showing the highest specific growth rate (0.009 h-1), had biomass productivity of 33.98 ± 0.02 mg L-1 day-1. Proteins were the most abundant macromolecule in the biomass (32.83-57.94%, g g-1). Nanofrustulum shiloi D1 contained significant amounts of neutral lipids (68.36%), while the biomass of Picochlorum sp. D3, Tetraselmis sp. Z3, Tetraselmis sp. C6 and Euhalothece sp. C1 was rich in glycolipids and phospholipids (75%). The lipids of all studied microalgae predominantly contained unsaturated fatty acids. Carotenoids were the most abundant pigments with the highest content of lutein and neoxanthin in representatives of Chlorophyta and fucoxanthin in strains belonging to the Bacillariophyta. All microalgal extracts showed antioxidant activity and antimicrobial activity against Gram-negative E. coli and S. typhimurium and Gram-positive S. aureus.

Morpho-physiological adaptations of Leptocylindrus aporus and L. hargravesii to phosphate limitation in the northern Adriatic.

The northern Adriatic is highly productive and shallow area characterized by numerous spatio-temporal gradients (e.g. nutrients, salinity, temperature). It is strongly influenced by numerous freshwater inputs, mainly from Po river. Its current systems as well as Po river, generates gradients of phosphate availability with an expressed N/P imbalance and phosphate limitation. A number of recent studies characterized these gradients as major factors affecting abundance and composition of microphytoplankton communities. Focus of this study is on two Leptocylindrus species, Leptocylindrus aporus (F.W. French & Hargraves) D. Nanjappa & A. Zingone 2013 and Leptocylindrus hargravesii D. Nanjappa & A. Zingone 2013. Species belonging to Leptocylindrus genus are frequently observed and have high abundances and also high contributions to the microphytoplankton community in this area. We focused on their morphological and physiological responses to phosphate limitation in situ and also performed in vitro experiments. In this study we report data on species specific growth rates under phosphorus (P) deplete and P rich conditions, localization and characteristics of alkaline phosphate activity, phosphate uptake rates as well as their morphological differences in P deplete versus P rich conditions. Our in vitro experiments showed that both Leptocylindrus species morphologically reacted similarly to phosphorus depletion and showed significantly elongated pervalvar axis in P depleted conditions if compared to P rich conditions. Also average chain lengths increased when in P depleted conditions. Two previously mentioned adaptations indicate their tendency to increase cellular surface areas available for alkaline phosphatase. Chlorophyll fluorescence of both species significantly decreased in P depleted medium. Although both species morphologically reacted similarly, our experiment demonstrated significant differences in physiological reactions to P depleted conditions.

Global warming and oligotrophication lead to increased lipid production in marine phytoplankton.

Earth temperature is rising and oligotrophication is becoming apparent even in coastal seas. In this changing environment, phytoplankton use carbon and nutrients to form important biomolecules, including lipids. However, the link between lipid production and changing environment is still unexplored. Therefore, we investigated the phytoplankton lipid production in the diatom Chaetoceros pseudocurvisetus cultures under controlled temperatures ranging from 10 to 30 °C and nutrient regimes mimicking oligotrophic and eutrophic conditions. Results were compared to plankton community's lipid production in the northern Adriatic at two stations considered as oligotrophic and mesotrophic during an annual monthly sampling. In order to gain detailed information on the investigated system, we supplemented lipid data with chlorophyll a concentrations, phytoplankton taxonomy, cell abundances and nutrient concentration along with hydrographic parameters. We found enhanced particulate lipid production at higher temperatures, and substantially higher lipid production in oligotrophic conditions. Enhanced lipid production has two opposing roles in carbon sequestration; it can act as a retainer or a sinker. Lipid remodeling, including change in ratio of phospholipids and glycolipids, is more affected by the nutrient status, than the temperature increase. Triacylglycerol accumulation was observed under the nitrogen starvation.

Insights into the life strategy of the common marine diatom Chaetoceros peruvianus Brightwell.

Chaetoceros peruvianus is a marine diatom species with circumglobal distribution. While frequently observed, it appears never to dominate the marine phytoplankton community hence it can be characterized as a rather opportunistic, generalistic species. Here we present ecological interpretations from a long-term data set on marine microphytoplankton in the northern Adriatic Sea, where the abundancies and relative contributions of C. peruvianus were observed along a set of steep ecological gradients. Limited supply of dissolved inorganic phosphate was identified as the driving ecological factor for this ecosystem. In parallel C. peruvianus was cultivated in monoclonal cultures and its morphological and physiological reaction to replete and phosphorus depleted medium was analysed. C. peruvianus reacted to phosphorus depletion by an increase in cell height and length as well as thickness and length of setae. This morphological reaction included an increase in cellular volume and calculated carbon content. Additionally, it represents the transition between two described morphological varieties, C. peruvianus and C. peruvianus var. robusta. C. peruvianus showed a significant induction of extracellular alkaline phosphatase activity if grown in phosphate depleted medium. Microscopical analysis demonstrated this activity to be located exclusively on the setae of the cells.

Copper-phospholipid interaction at cell membrane model hydrophobic surfaces.

Detailed investigation of Cu (II) binding with natural lipid phosphatidylglycerol (PG) in aqueous solution was carried out by voltammetric measurements at the mercury drop electrode, complemented by monolayer studies in a Langmuir trough and electrophoretic measurements, all used as models for hydrophobic cell membranes. Penetration of copper ions into the PG layer was facilitated by the formation of hydrophilic Cu-Phenanthroline (Phen) complex in the subphase, followed by the mixed ligand Cu-Phen-PG complex formation at the hydrophobic interface. Electrophoretic measurements indicated a comparatively low abundance of the formed mixed ligand complex within the PG vesicles, resulting it the zeta potential change of +0.83mV, while monolayer studies confirmed their co-existence at the interface. The Cu-Phen-PG complex was identified in the pH range from 6 to 9. The stoichiometry of the complex ([PhenCuOHPG]), as well as its stability and kinetics of formation, were determined at the mercury drop electrode. Cu-Phen-PG reduces quasireversibly at about -0.7V vs. Ag/AgCl including reactant adsorption, followed by irreversible mixed complex dissociation, indicating a two-electron transfer - chemical reaction (EC mechanism). Consequently, the surface concentration (γ) of the adsorbed [PhenCuOHPG] complex at the hydrophobic electrode surface was calculated to be (3.35±0.67)×10-11molcm-2. Information on the mechanism of Cu (II) - lipid complex formation is a significant contribution to the understanding of complex processes at natural cell membranes.

Improved separation and analysis of glycolipids by Iatroscan thin-layer chromatography-flame ionization detection.

We demonstrate improved power of Iatroscan thin layer chromatography/flame ionization detection (TLC-FID) technique for analysis of complex marine lipid mixture by developing protocol for the separation and analysis of glycolipids including sulfoquinovosyldiacylglycerols (SQDG), monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG). We have modified the common protocol used so far for the analysis of lipid classes by replacing the elution step which uses pure acetone for the elution of acetone mobile polar lipids, with the elution step containing chloroform-acetone (72:28, v:v) for separation of MGDG and DGDG. To separate SQDG from the complex lipid matrix we introduced solvent mixture acetone-chloroform-methanol-formic acid (33:33:33:0.6, v:v:v:v). Quantification of glycolipid classes was performed after calibration with glycolipid standards for the masses between 0.2 and 2.7-5.0µg. With this new protocol we have successfully separated three glycolipids from the complex particulate lipid mixture of the seawater samples. Such an approach extends the power of existing protocol for the analysis of lipids which altogether ensure detection and quantification of 18 lipid classes what was demonstrated on seawater samples. This enables to gain a very broad system overview of the particularly complex environments as are seas, oceans and freshwaters.

Adaptation of marine plankton to environmental stress by glycolipid accumulation.

A systematic investigation of non-phosphorus containing glycolipids (GL) was conducted in the northern Adriatic Sea during two years at two stations with different nutrient loads. GL concentration varied both spatially and temporally, with values of 1.1-21.5 µg/L and 0.4-44.7 µg/L in the particulate and the dissolved fraction, respectively. The highest concentrations were measured during summer in surface waters and at the more oligotrophic station, where GL yields (% of total lipids) were often higher than 20% and 50% in the particulate and dissolved fractions, respectively. To obtain more insight into factors governing GL accumulation autotrophic plankton community structure (pico-, nano- and microplankton fractions), chlorophyll a, heterotrophic bacteria and nutrient concentrations were measured together with hydrographic parameters and sunlight intensity. During the investigated period smaller autotrophic plankton cells (pico- and followed by nanoplankton) prevailed in abundance over larger cells (microplankton), which were found in large numbers in freshened surface samples. Several major findings resulted from the study. Firstly, during PO4 limitation, particularly at the oligotrophic station, enhanced glycolipid instead of phospholipid accumulation takes place, representing an effective phosphate-conserving mechanism. Secondly, results suggest that at seawater temperatures >19 °C autotrophic plankton considerably accumulate GL, probably to achieve thermal stability. Thirdly, high sunlight intensities seem to influence increased GL accumulation; GL possibly plays a role in cell mechanisms that prevent/mitigate photooxidation. And finally, substantial accumulation of GL detected in the dissolved fraction could be related to the fact that GL do not contain biologically relevant elements, like phosphorus, which makes them an unattractive substrate for enzyme activity. Therefore, substantial portion of CO2 could be removed from the atmosphere in P-limited regions during summer via its capture by plankton and conversion to GL.

A method for characterization of sea surface microlayer based on monolayer properties in presence and absence of phospholipids.

Electrochemical impedance spectroscopy, ac voltammetry and fractal analysis were used to characterize model compounds, compound mixtures and extracted samples of sea surface microlayer (ssm) und underlying water (ulw). The reasons for carrying out this work were to use the scientific basis of these characterizations in future on-line analytical procedures of ssm. The mercury (Hg) drop electrode uncoated and coated with a monolayer of dioleoyl phosphatidylcholine (DOPC) was used as an experimental basis for investigation of the major sea surface film forming material. Firstly, the interaction of the uncoated and DOPC coated Hg electrode with model water insoluble compounds of increasing polarity was investigated. The compounds studied in order of increasing polarity were: nonadecane, stearic acid, cholesterol and cardiolipin. Subsequently the electrochemical response of the system to different ssm extracts was compared to signals observed with model compounds to demonstrate method selectivity. From the electrochemical results, it is observed that both the molecular structure and polarity of the investigated compounds have a role in their interaction with the uncoated and DOPC coated electrode. In the fractal analysis the increase above 2 of fractal dimension D imparted to the DOPC layer is related to the degree of apolarity of the additive model compound. Consistent with this, the more apolar hexane extracted ssm 2 imparts a fractal dimension D value of 2.45 when incorporated in DOPC layers. The electrochemical response to the ssm and ulw follows that characteristic of sterol compounds.