Emiliania Huxleyi Cells Research Articles

A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis.

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

Influence of physiological states of Emiliania huxleyi cells on their lipids and associated molecular isotopic compositions during microbial degradation

The goal of this study was to examine the potential effect of physiological states of phytoplankton cells on their lipids (phytol, sterols, alkenones, and fatty acids) and associated stable carbon isotopic compositions during microbial degradation. Cells of Emiliania huxleyi were cultivated using batch cultures with the same medium but over different growth stages: exponential growth phase (culture-1) and stationary phase (culture-2), respectively. There were remarkable differences in cellular lipid contents and relative compositions between these two cultures: ~8 times higher lipid content with higher percentages of poly- and mono-unsaturated fatty acids, and alkenones but lower percentages of saturated fatty acids, phytol, and sterols in culture-2 than in culture-1. Dead phytoplankton cells (killed by freezing) from the two cultures were incubated in the dark with addition of natural seawater (containing natural microbial communities) under aerobic conditions at 17°C for about one month. Most phytoplankton fatty acids (except for 16:0) from the two cultures were almost completely degraded while most neutral lipids (phytol, sterols and alkenones) (except for 27Δ5 from culture-1) were partially degraded with relatively higher percentages of these compounds remaining in culture-2 than in culture-1. Along with degradation of lipids, their stable carbon isotopic compositions varied differently among compound classes: positive shifts (+1.7‰ to +3.9‰) in δ13C values for fatty acids; little change for sterols; and for alkenones, either little change in most cases or a negative shift (−3‰) in one case (37:2 alkenone in culture-1). Moreover, relatively larger isotopic shifts (~+1.5‰ each) for fatty acids 16:0 and 18:1(ω9) were observed in culture-1 compared to those in culture-2. The results from these experiments imply that the variability in isotopic compositions of phytoplankton lipid compounds during microbial degradation is critically affected by physiological states of phytoplankton cells due to heterogeneity in isotopic compositions of cellular lipids and differential reactivities of various lipid compounds bound in different intracellular components, which are synthesized through different cell growth phases.

Phenotypic diversity of diploid and haploid Emiliania huxleyi cells and of cells in different growth phases revealed by comparative metabolomics

In phytoplankton a high species diversity of microalgae co-exists at a given time. But diversity is not only reflected by the species composition. Within these species different life phases as well as different metabolic states can cause additional diversity. One important example is the coccolithophore Emiliania huxleyi. Diploid cells play an important role in marine ecosystems since they can form massively abundant algal blooms but in addition the less abundant haploid life phase of E. huxleyi occurs in lower quantities. Both life phases may fulfill different functions in the plankton. We hypothesize that in addition to the functional diversity caused by this life phase transition the growth stage of cells can also influence the metabolic composition and thus the ecological impact of E. huxleyi. Here we introduce a metabolomic survey in dependence of life phases as well as different growth phases to reveal such changes. The comparative metabolomic approach is based on the extraction of intracellular metabolites from intact microalgae, derivatization and analysis by gas chromatography coupled to mass spectrometry (GC-MS). Automated data processing and statistical analysis using canonical analysis of principal coordinates (CAP) revealed unique metabolic profiles for each life phase. Concerning the correlations of metabolites to growth phases, complex patterns were observed. As for example the saccharide mannitol showed its highest concentration in the exponential phase, whereas fatty acids were correlated to stationary and sterols to declining phase. These results are indicative for specific ecological roles of these stages of E. huxleyi and are discussed in the context of previous physiological and ecological studies.

The 2008 Emiliania huxleyi bloom along the Patagonian Shelf: Ecology, biogeochemistry, and cellular calcification

AbstractCoccolithophore blooms are significant contributors to the global production and export of calcium carbonate (calcite). The Patagonian Shelf is a site of intense annual coccolithophore blooms during austral summer. During December 2008, we made intensive measurements of the ecology, biogeochemistry, and physiology of a coccolithophore bloom. High numbers of Emiliania huxleyi cells and detached coccoliths (>1 × 103 mL−1 and >10 × 103 mL−1, respectively), high particulate inorganic carbon concentrations (>10 mmol C m−2), and high calcite production (up to 7.3 mmol C m−2 d−1) all characterized bloom waters. The bloom was dominated by the low‐calcite‐containing B/C morphotype of Emiliania huxleyi, although a small (<10 µm) Southern Ocean diatom of the genus Fragilariopsis was present in almost equal numbers (0.2–2 × 103 mL−1). Estimates of Fragilariopsis contributions to chlorophyll, phytoplankton carbon, and primary production were >30%, similar to estimates for E. huxleyi and indicative of a significant role for this diatom in bloom biogeochemistry. Cell‐normalized calcification rates, when corrected for a high number of nonactive cells, were relatively high and when normalized to estimates of coccolith calcite indicate excessive coccolith production in the declining phase of the bloom. We find that low measures of calcite and calcite production relative to other blooms in the global ocean indicate that the dominance of the B/C morphotype may lead to overall lower calcite production. Globally, this suggests that morphotype composition influences regional bloom inventories of carbonate production and export and that climate‐induced changes in morphotype biogeography could affect the carbon cycle.

Calcification state of coccolithophores can be assessed by light scatter depolarization measurements with flow cytometry

Coccolithophores are important primary producers and a dominant group of calcifying organisms in the ocean. Calcification state depends on genetic, physiological and environmental factors. We show that flow cytometric measurement of the depolarization forward scattered light using a Brewster's Window Analyzer can be used to quantify the degree of calcification of coccolithophores at the single-cell level. Calcite-containing particles or cells were distinguished from non-calcified particles or cells by high values of forward scatter light with polarization orthogonal to that of the laser. Forward scatter polarization state varied strongly and linearly with the number of attached coccoliths per coccosphere when Emiliania huxleyi cells were first completely decalcified and then allowed to rebuild coccospheres. Cells of the heavily calcified E. huxleyi R-morphotype strain NZEH were also grown in different extracellular Ca2+ concentrations, forming complete coccospheres that contained similar numbers of attached coccoliths but varied in total calcite mass. Forward scatter polarization state varied strongly and linearly with coccosphere calcite mass. In contrast, forward scatter polarization state of detached coccoliths did not vary significantly with calcite weight, although forward scatter and side scatter did. Treatments had relatively minor effects on forward scatter, side scatter and forward scatter polarization state of decalcified cells, suggesting that depolarization of forward scatter light from E. huxleyi cells might be linearly determined, to a first approximation, by the ratio of surface calcite to organic protoplast. We suggest that flow cytometric measurement of forward scatter depolarization provides a potentially valuable method for analysis of calcification state of individual cells.

Comparative study of UV and visible light induced degradation of lipids in non-axenic senescent cells of Emiliania huxleyi

Irradiation of killed non-axenic cells of Emiliania huxleyi (Prymnesiophyceae) by PAR and UVR resulted in a strong photodegradation of most of the unsaturated lipid components (chlorophylls, unsaturated fatty acids and brassicasterol) of this haptophyte. This degradation, which mainly involves type II (i.e. singlet oxygen-mediated) photosensitized oxidation processes, was induced by both PAR and UVR. Alkenones were the sole unsaturated components of E. huxleyi unaffected by photodegradation processes. This particular recalcitrance was attributed to the trans configuration of their double bonds, which is less reactive toward singlet oxygen than the classical cis configuration of the double bonds of the other lipids. UVR exposure also induced photosensitized stereomutation ( cis-trans isomerization) of the double bonds of some lipids (e.g. monounsaturated fatty acids and chlorophyll phytyl side-chain) and of some of their oxidation products affording (after reduction) some compounds (e.g. 9-hydroxyoctadec- cis-10-enoic and 10-hydroxyoctadec- cis-8-enoic acids arising from oleic acid oxidation and 11-hydroxyoctadec- cis-12-enoic and 12-hydroxyoctadec- cis-10-enoic acids arising from cis-vaccenic acid oxidation) sufficiently specific to act as tracers of UVR-induced photodegradation in situ. A strong photodegradation of specific bacterial monounsaturated fatty acids (e.g. cis-vaccenic and 11-MeC 18:1ω12 acids) was also observed and attributed to the migration of singlet oxygen from phytodetritus to the attached heterotrophic bacteria.

Biomarkers derived from heterolytic and homolytic cleavage of allylic hydroperoxides resulting from alkenone autoxidation

Laboratory incubation of alkenone mixtures with tert-butyl hydroperoxide and di- tert-butyl nitroxide (radical initiator) in hexane, as a means to simulate alkenone autoxidation processes, rapidly led to the formation of allylic hydroperoxides, whose presence was recently demonstrated in Emiliania huxleyi cells. After incubation in seawater and subsequent reduction with NaBH 4 (to reduce residual hydroperoxides before analysis), these reaction products quickly disappeared and were replaced by complex mixtures of n-alcohols, fatty acids, alkyldiols and hydroxyacids. Methyl alkenones produced saturated n-alkan-1-ols and fatty acids ranging from C 13 to C 16 and two series of C 13−C 16 (ω-1)-hydroxyacids and (1,ω-1)-diols. Ethyl alkenones also afforded C 13−C 16 saturated n-alkan-1-ols and fatty acids, accompanied by the production of C 14−C 17 (ω-2)-hydroxyacids and (1,ω-2)-diols. Deuterium labelling allowed us to show that most of the n-alkan-1-ols, hydroxyacids and alkyldiols resulted from the reduction during the NaBH 4 treatment of the corresponding aldehydes, ketoxyacids and ketoxyaldehydes formed from heterolytic or homolytic cleavages of allylic hydroperoxyl groups resulting from the oxidation of the double bonds of di- and triunsaturated alkenones. Amongst these products, the (ω-1)- and (ω-2)-hydroxyacids formed after NaBH 4 reduction of the (ω-1)- and (ω-2)-ketoxyacids were selected as potential biomarkers for alkenone autoxidation. Re-examination of lipid extracts of post-bloom seawater particulate matter samples from the DYFAMED station in the Ligurian Sea (where strong autoxidative alteration of the lipid distributions had previously been detected) showed the presence of significant amounts of 12-hydroxytetradecanoic, 13-hydroxytetradecanoic, 14-hydroxyhexadecanoic and 15-hydroxyhexadecanoic acids thus providing good evidence that these autoxidative processes occur in natural samples.

Effects of intracellular structural associations on degradation of algal chloropigments in natural oxic and anoxic seawaters

To understand the effects of intracellular structural associations on degradation of algal chloropigments, we conducted a series of microcosm experiments by incubating Emiliania huxleyi cells (a marine haptophyte) in natural oxic and anoxic seawaters collected from a stratified water column in the Cariaco Basin. The incubated cell detritus were sequentially treated with two buffer solutions to separate pigment components into soluble and insoluble fractions. By using non-denaturing gel electrophoresis, several chlorophyll-complexes, free chlorophyll, and another unknown chlorophyll-containing component were further separated from the soluble fraction. The chlorophyll-complexes included those bound with high molecular weight core-proteins (CP-I and CP43+CP47) and low molecular weight polypeptides (LHC-I and LHC-II) in the cellular photosystems PS-I and PS-II. Overall pigment recovery from these fractions and gel bands was well equivalent to the total amount from direct acetone extraction of the cells. We followed the time-dependent concentration changes of chlorophyll-a (Chl-a), phaeophorbide-a (Ppb-a), and phaeophytin-a (Ppt-a) in all fractions and complexes to estimate the degradation rate constants of chloropigments in natural oxic and anoxic seawaters. Our experimental results demonstrated that the intracellular structural associations had important influences on degradation of chloropigments under different redox conditions. In general, total Chl-a degraded faster (∼4X) in oxic seawater than in anoxic seawater. However, the rate differences between oxic and anoxic conditions varied among the fractions and complexes. Degradation rate constants of Chl-a in soluble fraction were much higher (>10X) than those in insoluble fraction under both oxic and anoxic conditions. Chl-a bound with the complexes in PS-II appeared to be more reactive (∼2X) than that in PS-I under oxic conditions but the difference in degradation rate constants between two photosystems became indistinguishable under anoxic conditions. Variations of Ppb-a in different fractions and complexes during incubation showed different patterns, implying that cellular Chl-a could degrade through two different pathways: (1) internal degradation into Ppb-a within insoluble pool and polypeptide complexes; and (2) release first from protein complexes and followed by external degradation.

Biochemical degradation of algal fatty acids in oxic and anoxic sediment–seawater interface systems: effects of structural association and relative roles of aerobic and anaerobic bacteria

To examine microbially mediated degradation of algal fatty acids in marine environments, we conducted a series of microcosm experiments by incubating Emiliania huxleyi cells in simulated oxic/anoxic sediment–water interface systems. Variations in concentration of fatty acids, lipid-degrading enzyme (lipase) activity, and bacterial abundance over 2-month incubations were followed to determine degradation rate constants of major algal fatty acids and responses of bacteria. In the cell-spiked experiments, fatty acids bound in the membrane and intracellular components were separated to examine effects of structural association of fatty acids on their degradation. Experimental results showed that algal fatty acids generally degraded faster (2–4×) under oxic than under anoxic conditions. Most membrane fatty acids seemed to more readily degrade than intracellular ones under anoxic conditions but the two classes degraded at similar rates under oxic conditions. Ratios of oxic to anoxic degradation rate constants were generally higher for intracellular fatty acids than for membrane fatty acids, implying that oxygen might play a more critical role in intracellular fatty acid degradation. Most algal fatty acids degraded almost completely under oxic conditions while a significant fraction (10–40%) of initially added algal fatty acids remained after 2 months under anoxic conditions. By contrast, variations in bacterial abundance during incubations were apparently greater under anoxic conditions compared to oxic conditions, suggesting that the function and relative effectiveness of aerobic vs. anaerobic bacteria rather than total bacterial abundance control biochemical degradation of algal fatty acids. Variations in potential lipase activity followed the same pattern as bacterial abundance in oxic and anoxic systems, indicating that bacteria might be a major source for lipase in these experimental systems. Bacteria-specific fatty acids varied differently during incubations and were not directly linked to bacterial abundance.

Isoforms of Glutamine Synthetase in the Marine Coccolithophorid Emiliania huxleyi (Prymnesiophyceae)

Two isoenzymes of glutamine synthetase (EC 6.3.1.2), GS1 and GS2, have been purified from cells of Emiliania huxleyi using Cibacron blue dye ligand chromatography and gel filtration, separated by ion-exchange chromatography on Mono-Q and partly characterized. Each enzyme is a homohexamer with a molecular mass of 402 kDa for GS1 and 501 kDa for GS2. The molecular mass of the subunits of GS1 and GS2 was estimated to be 61 and 78 kDa, respectively. As in higher plants, GS1 is slightly more thermostable than GS2 and much less stimulated by thiols than GS2. For these reasons, GS1 was designated as the cytosolic enzyme and GS2 as the chloroplastic one. Although the Kms for NH2OH are about the same, GS2 possesses a much higher affinity for glutamine than GS1. As in bacteria, ATP appears to play an important role in the allosteric regulation of GS2. l-Ala and CTP are potent inhibitors of GS1 activity. CTP, carbamoyl-phosphate and l-Ala exert a cumulative inhibitory effect on GS1 activity. GS2 is also inhibited to some extent by l-Ala and l-His. NH2-terminal sequence analysis of GS2 did not show any homology with bacteria, cyanobacteria or higher plants.

Stability of long-chain alkenones in senescing cells of Emiliania huxleyi: effect of photochemical and aerobic microbial degradation on the alkenone unsaturation ratio (U k′37)

Alkenones were photooxidized in pyridine solution of haematoporphyrin and in killed cells of Emiliania huxleyi. These ketones seem to be protected from photochemical damage in phytodetritus; this photochemical recalcitrance has been attributed to their chemical structure and localization in the cells. These results indicate that U k′ 37 ratios are not likely to be strongly photochemically modified during senescence of Emiliania huxleyi (increase limited to 0.04 U k′ 37 unit). In contrast, the major sterol of this algae (24α-methylcholesta-5,22 E-dien-3β-ol) appeared to be very sensitive to photochemical processes. Non-sterile incubations of broken cells of Emiliania huxleyi showed a strong biodegradation of alkenones, indicating that aerobic microbial processes must play an important role during senescence of this species. However, this degradation takes place without dramatic variations in U k′ 37 values (decrease limited to 0.05 U ′ 37 unit). Several mechanisms are proposed in order to explain this strong and weakly selective biodegradation.

Grazing in the heterotrophic dinoflagellate Oxyrrhis marina: size selectivity and preference for calcified Emiliania huxleyi cells

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 10:307-313 (1996) - doi:10.3354/ame010307 Grazing in the heterotrophic dinoflagellate Oxyrrhis marina: size selectivity and preference for calcified Emiliania huxleyi cells Hansen FC, Witte HJ, Passarge J Selective grazing on nanophythoplankton by the heterotrophic dinoflagellate Oxyrrhis marina was investigated in laboratory experiments. O. marina was offered 2 mixtures of differently sized algae: (1) Micromonas pusilla (2 um Equivalent Spherical Diameter), Emiliania huxleyi (5 um ESD), and Tetraselmis suecica (7 um ESD); and (2) Nannochloris sp. (2 um ESD), Isochrysis galbana (5 um ESD), Rhodomonas sp. (8 um ESD), and Tetraselmis sp. (10 um ESD). O. marina grazed selectively on the larger algal species (ESD &GT= 7 um), and also selected for the larger cells within each species. O. marina discriminated against E. huxleyi. To test this selection, O. marina was offered mixtures of equal concentrations of the similarly sized algae E. huxleyi and I. galbana. In these mixtures, O. marina preferred the calcified E. huxleyi cells over noncalcified E. huxleyi cells and discriminated against I. galbana. This result is also interpreted in terms of size-selective grazing. Protozoa . Food selection . Prey size . Microphytoplankton . Coccoliths Full text in pdf format PreviousExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 10, No. 3. Publication date: June 27, 1996 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1996 Inter-Research.

Carbon isotope fractionation by Emiliania huxleyi

The ratio of l*C : 13C was measured for Emiliania huxleyi cells grown under a range of irradiances in batch culture. Based on the assumption that HCO, was the carbon source, the maximum discrimination against 13C in the decalcified E. huxleyi cells was 24.6?& at 4.8 mol photons m-* d-l and the minimum discrimination was 17.9?& at 0.5 mol photons m-* d-l. In calcified cells, the range of 13C isotope discrimination was markedly lower (12-217~). In both calcified and decalcified cells, the carbon isotopic composition of and carbon isotopic discrimination by E. huxleyi was a significant linear function of irradiance from 0.5 to 4.8 mol photons m-* d- I. The relationship between isotopic discrimination and irradiance in E. huxleyi provides new evidence that the 613C of the oceanic POC samples should not be treated simply as a function of sea surface temperature or [CO,],,. We propose that the P3C of E. huxleyi was controlled by irradiance through variation in the intracellular HC03- -derived CO2 supply which influenced the discrimination of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) against 13C.

ROLE OF THE LIGHT‐DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

ABSTRACTProduction of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light‐dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early‐stage, coccolith‐production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient‐rich water by increasing the sinking rate of the cells.