Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis.

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

A Novel Algicidal Bacterium, Microbulbifer sp. YX04, Triggered Oxidative Damage and Autophagic Cell Death in Phaeocystis globosa, Which Causes Harmful Algal Blooms.

Phaeocystis globosa causes severe marine pollution by forming harmful algal blooms and releasing hemolytic toxins and is therefore harmful to marine ecosystems and aquaculture industries. In this study, Microbulbifer sp. YX04 exerted high algicidal activity against P. globosa by producing and secreting metabolites. The algicidal activity of the YX04 supernatant was stable after exposure to different temperatures (-80 to 100°C) and pH values (4 to 12) for 2 h, suggesting that algicidal substances could temporarily be stored under these temperature and pH value conditions. To explore the algicidal process and mechanism, morphological and structural changes, oxidative stress, photosynthesis, autophagic flux, and global gene expression were investigated. Biochemical analyses showed that the YX04 supernatant induced reactive oxygen species (ROS) overproduction, which caused lipid peroxidation and malondialdehyde (MDA) accumulation in P. globosa. Transmission electron microscopy (TEM) observation and the significant decrease in both maximum photochemical quantum yield (Fv/Fm) and relative electron transfer rate (rETR) indicated damage to thylakoid membranes and destruction of photosynthetic system function. Immunofluorescence, immunoblot, and TEM analyses indicated that cellular damage caused autophagosome formation and triggered large-scale autophagic flux in P. globosa. Transcriptome analysis revealed many P. globosa genes that were differentially expressed in response to YX04 stress, most of which were involved in photosynthesis, respiration, cytoskeleton, microtubule, and autophagosome formation and fusion processes, which may trigger autophagic cell death. In addition to P. globosa, the YX04 supernatant showed high algicidal activity against Thalassiosira pseudonana, Thalassiosira weissflogii, Skeletonema costatum, Heterosigma akashiwo, and Prorocentrum donghaiense. This study highlights multiple mechanisms underlying YX04 supernatant toxicity toward P. globosa and its potential for controlling the occurrence of harmful algal blooms. IMPORTANCEP. globosa is one of the most notorious harmful algal bloom (HAB)-causing species, which can secrete hemolytic toxins, frequently cause serious ecological pollution, and pose a health hazard to animals and humans. Hence, screening for bacteria with high algicidal activity against P. globosa and studies on the algicidal characteristics and mechanism will contribute to providing an ecofriendly microorganism-controlling agent for preventing the occurrence of algal blooms and reducing the harm of algal blooms to the environment. Our study first reported the algicidal characteristic and mechanism of Microbulbifer sp. YX04 against P. globosa and demonstrated that P. globosa shows different response mechanisms, including movement ability, antioxidative systems, photosynthetic systems, gene expression, and cell death mode, to adapt to the adverse environment when algicidal compounds are present.

Effect of KNO3 on Lipid Synthesis and CaCO3 Accumulation in Pleurochrysis dentata Coccoliths with a Special Focus on Morphological Characters of Coccolithophores.

Pleurochrysis genus algae are widely distributed in ocean waters. Pleurochrysis sp. algae are popularly known for its coccolithophores. Calcium carbonate (CaCO3) shells are major components of the coccolithophore, and they are key absorbers of carbondioxide. In this study, we have reported the effects of potassium nitrate (KNO3) concentration on calcium accumulation and total lipid, carbohydrate and protein contents of Pleurochrysis dentata. Results obtained from complexometric titration and scanning electron microscopy analysis showed higher rates of CaCO3 accumulation on Pleurochrysis dentata cell surface. We have also observed that overall cell size of Pleurochrysis dentata reached maximum when it was cultured at 0.75 mmol L-1 of KNO3. During 10 days of Pleurochrysis dentata culture total lipids and carbohydrate contents decreased, with slightly increased protein content. Results obtained from Fourier-Transform Infrared Spectroscopy (FTIR) also reported an increase in protein and decrease in lipids and carbohydrate contents, respectively. Similarly, Pleurochrysis dentata cultured at 1 mmol L-1 concentration of KNO3 exhibited the lowest carbohydrate (21.08%) and highest protein (32.87%) contents. Interestingly, Pleurochrysis dentata cultured without KNO3 exhibited 33.61% of total lipid content which reduced to a total lipid content of 13.67% when cultured at 1 mmol L-1 concentration of KNO3. Thus, culture medium containing higher than 1 mmol L-1 of KNO3 could inhibit the cell size of Pleurochrysis dentata and CaCO3 accumulation in shells but it could promote its cell growth. For the first time we have reported a relatively complete coccolith structure devoid of its protoplast. In this study, we have also described about the special planar structure of Pleurochrysis dentata CaCO3 shells present on its inner tube of the R unit and parallel to the outer tube of the V unit which we named it as "doornail structure". We believe that this doornail structure provides structural stability and support to the developing coccoliths in Pleurochrysis dentata. Also, we have discussed about the "double-disc" structure of coccoliths which are closely arranged and interlocked with each other. The double-disc structure ensures fixation of each coccolith and objecting its free horizontal movement and helps in attaining a complementary coccolith structure.

Relationship between coccolith length and thickness in the coccolithophore species Emiliania huxleyi and Gephyrocapsa oceanica.

Coccolith mass is an important parameter for estimating coccolithophore contribution to carbonate sedimentation, organic carbon ballasting and coccolithophore calcification. Single coccolith mass is often estimated based on the ks model, which assumes that length and thickness increase proportionally. To evaluate this assumption, this study compared coccolith length, thickness, and mass of seven Emiliania huxleyi strains and one Gephyrocapsa oceanica strain grown in 25, 34, and 44 salinity artificial seawater. While coccolith length increased with salinity in four E. huxleyi strains, thickness did not increase significantly with salinity in three of these strains. Only G. oceanica showed a consistent increase in length with salinity that was accompanied by an increase in thickness. Coccolith length and thickness was also not correlated in 14 of 24 individual experiments, and in the experiments in which there was a positive relationship r2 was low (<0.4). Because thickness did not increase with length in E. huxleyi, the increase in mass was less than expected from the ks model, and thus, mass can not be accurately estimated from coccolith length alone.

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis.

Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton, especially in oligotrophic surface waters. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques (FIB-SEM, nanoSIMS, Synchrotron X-ray fluorescence), we show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. The multiplication of plastids can lead to a 38-fold increase of the total plastid volume in a cell. Subcellular mapping of nutrients (nitrogen and phosphorous) and their stoichiometric ratios shows that symbiotic algae are impoverished in phosphorous and suggests a higher investment in energy-acquisition machinery rather than in growth. Nanoscale imaging also showed that the host supplies a substantial amount of trace metals (e.g., iron and cobalt), which are stored in algal vacuoles at high concentrations (up to 660 ppm). Sulfur mapping reveals a high concentration in algal vacuoles that may be a source of antioxidant molecules. Overall, this study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.

High concentration of Phaeocystis globosa reduces the sensitivity of rotifer Brachionus plicatilis to cadmium: Based on an exponential approach fitting the changes in some key life-history traits.

Most coastal waters are at risk of heavy metal pollution, and the biomass of primary producer phytoplankton always fluctuates, which usually causes zooplankton to be exposed in different concentrations of food and heavy metal. Phytoplankton abundance and heavy metal may interact on zooplankton. Therefore, to assess the definite interactive way, in this study we investigated the combined effects of different cadmium (Cd) levels and Phaeocystis globosa concentrations on some key life-history traits of the rotifer Brachionus plicatilis. Results showed that the Cd level and P. globosa concentration had a significant interaction on the key life-history parameters of the rotifer. Mid-level algal concentrations (5-36 × 104 cells mL-1) had an apparent effect on brood production and the number of rotifers producing offspring at high Cd level. The time to first reproduction exponentially decreased with increasing P. globosa concentrations under any Cd levels and then subsequently reached a constant value. With increasing P. globosa concentration, the total number of offspring exponentially increased and then reached the asymptotic value; the survival time under any Cd levels exponentially decreased with the increasing P. globosa concentration and subsequently tended to be a constant value. Without Cd, the low P. globosa concentration only decreased the reproduction of rotifers. However, the extreme low P. globosa concentration (1-3 × 104 cells mL-1) under higher Cd level (0.0354 mM) completely inhibited the reproduction and also shorten the survival time. Higher Cd level decreased the asymptotic total offspring per rotifer and survival time. High concentration of P. globosa can reduce the sensitivity of rotifer to heavy metal. However, the negative effects could not be eliminated completely by the increasing P. globosa concentration. The findings indicated that ecotoxicological studies on the toxicity of heavy metal need to consider the effects of food concentrations, which contributes to understanding the diverse tolerance of zooplankton to heavy metals.

The requirement for calcification differs between ecologically important coccolithophore species.

Coccolithophores are globally distributed unicellular marine algae that are characterized by their covering of calcite coccoliths. Calcification by coccolithophores contributes significantly to global biogeochemical cycles. However, the physiological requirement for calcification remains poorly understood as non-calcifying strains of some commonly used model species, such as Emiliania huxleyi, grow normally in laboratory culture. To determine whether the requirement for calcification differs between coccolithophore species, we utilized multiple independent methodologies to disrupt calcification in two important species of coccolithophore: E. huxleyi and Coccolithus braarudii. We investigated their physiological response and used time-lapse imaging to visualize the processes of calcification and cell division in individual cells. Disruption of calcification resulted in major growth defects in C. braarudii, but not in E. huxleyi. We found no evidence that calcification supports photosynthesis in C. braarudii, but showed that an inability to maintain an intact coccosphere results in cell cycle arrest. We found that C. braarudii is very different from E. huxleyi as it exhibits an obligate requirement for calcification. The identification of a growth defect in C. braarudii resulting from disruption of the coccosphere may be important in considering their response to future changes in ocean carbonate chemistry.

Temperature effects on sinking velocity of different Emiliania huxleyi strains.

The sinking properties of three strains of Emiliania huxleyi in response to temperature changes were examined. We used a recently proposed approach to calculate sinking velocities from coccosphere architecture, which has the advantage to be applicable not only to culture samples, but also to field samples including fossil material. Our data show that temperature in the sub-optimal range impacts sinking velocity of E. huxleyi. This response is widespread among strains isolated in different locations and moreover comparatively predictable, as indicated by the similar slopes of the linear regressions. Sinking velocity was positively correlated to temperature as well as individual cell PIC/POC over the sub-optimum to optimum temperature range in all strains. In the context of climate change our data point to an important influence of global warming on sinking velocities. It has recently been shown that seawater acidification has no effect on sinking velocity of a Mediterranean E. huxleyi strain, while nutrient limitation seems to have a small negative effect on sinking velocity. Given that warming, acidification, and lowered nutrient availability will occur simultaneously under climate change scenarios, the question is what the net effect of different influential factors will be. For example, will the effects of warming and nutrient limitation cancel? This question cannot be answered conclusively but analyses of field samples in addition to laboratory culture studies will improve predictions because in field samples multi-factor influences and even evolutionary changes are not excluded. As mentioned above, the approach of determining sinking rate followed here is applicable to field samples. Future studies could use it to analyse not only seasonal and geographic patterns but also changes in sinking velocity over geological time scales.

Simulation Method Linking Dense Microalgal Culture Spectral Properties in the 400-750 nm Range to the Physiology of the Cells.

This work describes a method to model the optical properties over the (400-750 nm) spectral range of a dense microalgal culture using the chemical and physical properties of the algal cells. The method was based on a specific program called AlgaSim coupled with the adding-doubling method: at the individual cell scale, AlgaSim simulates the spectral properties of one model, three-layer spherical algal cell from its size and chemical composition. As a second step, the adding-doubling method makes it possible to retrieve the total transmittance of the algal medium from the optical properties of the individual algal cells. The method was tested by comparing the simulated total transmittance spectra for dense marine microalgal cultures of Isochrysis galbana (small flagellates) and Phaeodactylum tricornutum (diatoms) to spectra measured using an experimental spectrophotometric setup. Our study revealed that the total transmittance spectra simulated for the quasi-spherical cells of Isochrysis galbana were in good agreement with the measured spectra over the whole spectral range. For Phaeodactylum tricornutum, large differences between simulated and measured spectra were observed over the blue part of the transmittance spectra, probably due to non-spherical shape of the algal cells. Prediction of the algal cell density, mean size and pigment composition from the total transmittance spectra measured on algal samples was also investigated using the reversal of the method. Mean cell size was successfully predicted for both species. The cell density was also successfully predicted for spherical Isochrysis galbana, with a relative error below 7%, but not for elongated Phaeodactylum tricornutum with a relative error up to 26%. The pigments total quantity and composition, the carotenoids:chlorophyll ratio in particular, were also successfully predicted for Isochrysis galbana with a relative error below 8%. However, the pigment predictions and measurements for Phaeodactylum tricornutum showed large discrepancies, with a relative error up to 88%. These results give strong support for the development of a promising tool providing rapid and accurate estimations of biomass and physiological status of a dense microalgal culture based on only light transmittance properties.

Phosphorus starvation induces membrane remodeling and recycling in Emiliania huxleyi.

Nutrient availability is an important factor controlling phytoplankton productivity. Phytoplankton contribute c. 50% of the global photosynthesis and possess efficient acclimation mechanisms to cope with nutrient stress. We investigate the cellular response of the bloom-forming coccolithophore Emiliania huxleyi to phosphorus (P) scarcity, which is often a limiting factor in marine ecosystems. We combined mass spectrometry, fluorescence microscopy, transmission electron microscopy (TEM) and gene expression analyses in order to assess diverse cellular features in cells exposed to P limitation and recovery. Early starvation-induced substitution of phospholipids in the cells' membranes with galacto- and betaine lipids. Lipid remodeling was rapid and reversible upon P resupply. The PI3K inhibitor wortmannin reduced phospholipid substitution, suggesting a possible involvement of PI3K- signaling in this process. In addition, P limitation enhanced the formation and acidification of membrane vesicles in the cytoplasm. Intracellular vesicles may facilitate the recycling of cytoplasmic content, which is engulfed in the vesicles and delivered to the main vacuole. Long-term starvation was characterized by a profound increase in cell size and morphological alterations in cellular ultrastructure. This study provides cellular and molecular basis for future ecophysiological assessment of natural E. huxleyi populations in oligotrophic regions.

Coordinated regulation of nitrogen supply mode and initial cell density for energy storage compounds production with economized nitrogen utilization in a marine microalga Isochrysis zhangjiangensis.

Lipids and carbohydrates are main energy storage compounds (ESC) of microalgae under stressed conditions and they are potential feedstock for biofuel production. Yet, the sustainable and commercially successful production of ESC in microalgae needs to consider nitrogen utilization efficiency. Here the impact of different initial cell densities (ICDs) on ESC accumulation in Isochrysis zhangjiangensis under two nitrogen supply modes (an initially equal concentration of nitrogen per-cell in the medium (N1) and an equal total concentration of nitrogen in the culture system (N2)) were investigated. The results demonstrated that the highest ESC yield (1.36gL(-1)) at N1, which included a maximal nitrogen supply in the cultivation system, and the highest ESC content (66.5%) and ESC productivity per mass of nitrogen (3.28gg(-1) (N) day(-1)) at N2, were all obtained under a high ICD of 8.0×10(6)cellsmL(-1). Therefore I. zhangjiangensis qualifies for ESC-enriched biomass production with economized nitrogen utilization.

Coccolithophore biomineralization: New questions, new answers.

Coccolithophores are unicellular phytoplankton that are characterized by the presence intricately formed calcite scales (coccoliths) on their surfaces. In most cases coccolith formation is an entirely intracellular process - crystal growth is confined within a Golgi-derived vesicle. A wide range of coccolith morphologies can be found amongst the different coccolithophore groups. This review discusses the cellular factors that regulate coccolith production, from the roles of organic components, endomembrane organization and cytoskeleton to the mechanisms of delivery of substrates to the calcifying compartment. New findings are also providing important information on how the delivery of substrates to the calcification site is co-ordinated with the removal of H(+) that are a bi-product of the calcification reaction. While there appear to be a number of species-specific features of the structural and biochemical components underlying coccolith formation, the fluxes of Ca(2+) and a HCO3(-) required to support coccolith formation appear to involve spatially organized recruitment of conserved transport processes.

Validation of algal viability treated with total residual oxidant and organic matter by flow cytometry.

Algal cell growth after starch and oxidant treatments in seawater species (Isochrysis galbana and Phaeodactylum tricornutum) and freshwater species (Selenastrum capricornutum and Scenedesmus obliquus) were evaluated by flow cytometry with fluorescein diacetate (FDA) staining to determine algal viability. Growth of algal cell was found to be significantly different among groups treated with NaOCl, starch and/or sodium thiosulfate, which are active substance (Total Residual Oxidant; TRO as Cl2), organic compound to meet efficacy testing standard and neutralizer of TRO by Ballast Water Management Convention of International Maritime Organization, respectively. The viability of algal cell treated with TRO in starch-add culture of 5days after treatment and neutralization was decreased significantly. ATP contents of the treated algal cells corresponded to the FL1 fluorescent signal of flow cytometry with FDA staining. I. galbana was the most sensitive to TRO-neutralized cultures during viability analysis.

Coordinated response of photosynthesis, carbon assimilation, and triacylglycerol accumulation to nitrogen starvation in the marine microalgae Isochrysis zhangjiangensis (Haptophyta).

The photosynthetic performance, carbon assimilation, and triacylglycerol accumulation of Isochrysis zhangjiangensis under nitrogen-deplete conditions were studied to understand the intrinsic correlations between them. The nitrogen-deplete period was divided into two stages based on the photosynthetic parameters. During the first stage, carbon assimilation was not reduced compared with that under favorable conditions. The marked increase in triacylglycerols and the variation in the fatty acid profile suggested that triacylglycerols were mainly derived from de novo synthesized acyl groups. In the second stage, the triacylglycerol content continued increasing while the carbohydrate content decreased from 44.0% to 26.3%. These results indicated that the intracellular conversion of carbohydrates to triacylglycerols occurred. Thus, we propose that sustainable carbon assimilation and incremental triacylglycerol production can be achieved by supplying appropriate amounts of nitrogen in medium to protect the photosynthetic process from severe damage using the photosynthetic parameters as indicators.

Identification of carbohydrates as the major carbon sink of the marine microalga Isochrysis zhangjiangensis (Haptophyta) and optimization of its productivity by nitrogen manipulation.

Microalgae represent a potential feedstock for biofuel production. During cultivation under nitrogen-depleted conditions, carbohydrates, rather than neutral lipids, were the major carbon sink of the marine microalga Isochrysis zhangjiangensis (Haptophyta). Carbohydrates reached maximum levels of 21.2 pg cell(-1) on day 5, which was an increase of more than 7-fold from day 1, while neutral lipids simultaneously increased 1.9-fold from 4.0 to 7.6 pg cell(-1) during the ten-day nitrogen-depleted cultivation. The carbohydrate productivity of I. zhangjiangensis was improved by optimization of the nitrate supply mode. The maximum carbohydrate concentration was 0.95 g L(-1) under batch cultivation, with an initial nitrogen concentration of 31.0 mg L(-1), which was 2.4-fold greater than that achieved under nitrogen-depleted conditions. High performance liquid chromatography (HPLC) analysis showed that the accumulated carbohydrate in I. zhangjiangensis was composed of glucose. These results show that I. zhangjiangensis represents an ideal carbohydrate-enriched bioresource for biofuel production.

Evolutionary responses of a coccolithophorid Gephyrocapsa oceanica to ocean acidification.

The ongoing ocean acidification associated with a changing carbonate system may impose profound effects on marine planktonic calcifiers. Here, we show that a coccolithophore, Gephyrocapsa oceanica, evolved in response to an elevated CO2 concentration of 1000 µatm (pH reduced to 7.8) in a long-term (∼670 generations) selection experiment. The high CO2 -selected cells showed increases in photosynthetic carbon fixation, growth rate, cellular particulate organic carbon (POC) or nitrogen (PON) production, and a decrease in C:N elemental ratio, indicating a greater upregulation of PON than of POC production under the ocean acidification condition. Cells from the low CO2 selection process shifted to high CO2 exposure showed an enhanced cellular POC and PON production rates. Our data suggest that the coccolithophorid could adapt to ocean acidification with enhanced assimilations of carbon and nitrogen but decreased C:N ratios.

Photochemical production and behavior of hydroperoxyacids in heterotrophic bacteria attached to senescent phytoplanktonic cells.

The photooxidation of cellular monounsaturated fatty acids was investigated in senescent phytoplanktonic cells (Emiliania huxleyi) and in their attached bacteria under laboratory controlled conditions. Our results indicated that UV-visible irradiation of phytodetritus induced the photooxidation of oleic (produced by phytoplankton and bacteria) and cis-vaccenic (specifically produced by bacteria) acids. These experiments confirmed the involvement of a substantial singlet oxygen transfer from senescent phytoplanktonic cells to attached bacteria, and revealed a significant correlation between the concentration of chlorophyll, a photosensitizer, in the phytodetritus and the photodegradation state of bacteria. Hydroperoxyacids (fatty acid photoproducts) appeared to be quickly degraded to ketoacids and hydroxyacids in bacteria and in phytoplanktonic cells. This degradation involves homolytic cleavage (most likely induced by UV and/or transition metal ions) and peroxygenase activity (yielding epoxy acids).

Effects of CO2 enrichment and nutrients supply intermittency on batch cultures of Isochrysis galbana.

Aiming at enhanced performance to increase economic feasibility of microalgae based processes, Isochrysis galbana was grown in three modes of cultivation: batch, intermittent fed batch and semi-continuous. The batch mode was conducted under two regimes of aeration: conventional aeration and CO2 enriched aeration (5% v/v in air). Increased biomass productivity without significant impact on lipid accumulation was observed for CO2 enriched aeration relatively to cultivation aerated with air only. The intermittent fed batch cultivation policy was proven to be useful for lipid accumulation, increasing the lipid content by 19.8%. However, the semi-continuous mode resulted in higher productivity due to increased biomass concentration; the biomass productivity reached 0.51 g/(Ld). Fluorescence measurements were performed; the calculated low electron transport rate showed the need to increase the irradiance. The results showed that I. galbana can be grown in semi-continuous condition at high levels of biomass productivity.

Release of dissolved carbohydrates by Emiliania huxleyi and formation of transparent exopolymer particles depend on algal life cycle and bacterial activity.

The coccolithophore Emiliania huxleyi plays a pivotal role in the marine carbon cycle. However, we have only limited understanding of how its life cycle and bacterial interactions affect the production and composition of dissolved extracellular organic carbon and its transfer to the particulate pool. We traced the fate of photosynthetically fixed carbon during phosphate-limited stationary growth of non-axenic, calcifying E. huxleyi batch cultures, and more specifically the transfer of this carbon to bacteria and to dissolved high molecular weight neutral aldoses (HMW NAld) and extracellular particulate carbon. We then compared the dynamics of dissolved carbohydrates and transparent exopolymer particles (TEP) between cultures of non-axenic and axenic diploid E. huxleyi. In addition, we present the first data on extracellular organic carbon in (non-axenic) haploid E. huxleyi cultures. Bacteria enhanced the accumulation of dissolved polysaccharides and altered the composition of dissolved HMW NAld, while they also stimulated the formation of TEP containing high densities of charged polysaccharides in diploid E. huxleyi cultures. In haploid E. huxleyi cultures we found a more pronounced accumulation of dissolved carbohydrates, which had a different NAld composition than the diploid cultures. TEP formation was significantly lower than in the diploid cultures, despite the presence of bacteria. In diploid E. huxleyi cultures, we measured a high level of extracellular release of organic carbon (34-76%), retrieved mainly in the particulate pool instead of the dissolved pool. Enhanced formation of sticky TEP due to bacteria-alga interactions, in concert with the production of coccoliths, suggests that especially diploid E. huxleyi blooms increase the efficiency of export production in the ocean during dissolved phosphate-limited conditions.

Viral infection of Phaeocystis globosa impedes release of chitinous star-like structures: quantification using single cell approaches.

Phaeocystis globosa is an ecologically important bloom-forming phytoplankton, which sequesters substantial amounts of inorganic carbon and can form carbon-enriched chitinous star-like structures. Viruses infecting P. globosa (PgVs) play a significant regulatory role in population dynamics of the host species. However, the extent to which viruses alter host physiology and its carbon assimilation on single cell level is still largely unknown. This study demonstrates for the first time the impact of viral infection on carbon assimilation and cell morphology of individual axenic P. globosa cells using two single cell techniques: high resolution nanometre-scale Secondary-Ion Mass Spectrometry (nanoSIMS) approach and atomic force microscopy (AFM). Up until viral lysis (19 h post infection), the bulk carbon assimilation by infected P. globosa cultures was identical to the assimilation by the non-infected cultures (33 µmol C l(-1)). However, single cell analysis showed that viral infection of P. globosa impedes the release of star-like structures. Non-infected cells transfer up to 44.5 µmol C l(-1) (36%) of cellular biomass in the form of star-like structures, suggesting a vital role in the survival of P. globosa cells. We hypothesize that impediment of star-like structures in infected P. globosa cells may inactivate viral infectivity by forming flocculants after cell lysis. Moreover, we show that substantial amounts of newly produced viruses (≈ 68%) were attached to P. globosa cells prior to cell lysis. Further, we speculate that infected cells become more susceptible for grazing which provides potential reasons for the sudden disappearance of PgVs in the environment. The scenarios of enhanced grazing is at odds to the current perspective that viral infections facilitates microbial mediated processes by diverting host material away from the higher trophic levels.