The Influence of Growth Rate on 2H/1H Fractionation in Continuous Cultures of the Coccolithophorid Emiliania huxleyi and the Diatom Thalassiosira pseudonana

The hydrogen isotope (2H/1H) ratio of lipids from phytoplankton is a powerful new tool for reconstructing hydroclimate variations in the geologic past from marine and lacustrine sediments. Water 2H/1H changes are reflected in lipid 2H/1H changes with R2 > 0.99, and salinity variations have been shown to cause about a 1‰ change in lipid δ2H values per unit (ppt) change in salinity. Less understood are the effects of growth rate, nutrient limitation and light on 2H/1H fractionation in phytoplankton. Here we present the first published study of growth rate effects on 2H/1H fractionation in the lipids of coccolithophorids grown in continuous cultures. Emiliania huxleyi was cultivated in steady state at four growth rates and the δ2H value of individual alkenones (C37:2, C37:3, C38:2, C38:3), fatty acids (C14:0, C16:0, C18:0), and 24-methyl cholest-5,22-dien-3β-ol (brassicasterol) were measured. 2H/1H fractionation increased in all lipids as growth rate increased by 24‰ to 79‰ (div d-1)-1. We attribute this response to a proportional increase in the fraction of NADPH from Photosystem I (PS1) of photosynthesis relative to NADPH from the cytosolic oxidative pentose phosphate (OPP) pathway in the synthesis of lipids as growth rate increases. A 3-endmember model is presented in which lipid hydrogen comes from NADPH produced in PS1, NADPH produced by OPP, and intracellular water. With published values or best estimates of the fractionation factors for these sources (αPS1 = 0.4, αOPP = 0.75, and αH2O = 0) and half of the hydrogen in a lipid derived from water the model indicates αlipid = 0.79. This value is within the range measured for alkenones (αalkenone = 0.77 to 0.81) and fatty acids (αFA = 0.75 to 0.82) in the chemostat cultures, but is greater than the range for brassicasterol (αbrassicasterol = 0.68 to 0.72). The latter is attributed to a greater proportion of hydrogen from NADPH relative to water in isoprenoid lipids. The model successfully explains the increase in 2H/1H fractionation in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol from marine centric diatom T. pseudonana chemostat cultures as growth rate increases. Insensitivity of αFA in those same cultures may be attributable to a larger fraction of hydrogen in fatty acids sourced from intracellular water at the expense of NADPH as growth rate increases. The high sensitivity of α to growth rate in E. huxleyi lipids and a T. pseudonana sterol implies that any change in growth rate larger than ~0.15 div d-1 can cause a change in δ2Hlipid that is larger than the analytical error of the measurement (~5‰), and needs to be considered when interpreting δ2Hlipid variations in sediments.

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title: Impact of the Messinian Salinity Crisis on Black Sea hydrology - Insights from hydrogen isotopes analysis on biomarkers
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title: Biogeochemistry of the stable hydrogen isotopes
abstract: The fractionation of H isotopes between the water in the growth medium and the organically bonded H from microalgae cultured under conditions, where light intensity and wavelength, temperature, nutrient availability, and the H isotope ratio of the water were controlled, is reproducible and light dependant. All studies were based either on the H isotope ratios of the total organic H or on the lipids, where most of the H is firmly bonded to C. H bonded into other macromolecules, proteins, carbohydrates and nucleic acids, does not exchange with water, when algae are incubated in water enriched with deuterium. Only after the destruction of quaternary H bonds are labile hydrogens in macromolecules free to exchange with water. By growing algae (18 strains), including blue-green algae, green algae and diatoms, in continuous light, the isotope fractionations in photosynthesis were reproducibly −93 to −178 %. depending on the organism tested. This fractionation was not temperature dependent. Microalgae grown in total darkness with an organic substrate did not show the isotope fractionation seen in cells grown in light. In both light- and dark-grown algae, however, additional depletion of deuterium (−30 to −60%.) in cellular organic matter occurs during the metabolism of carbohydrates to form lipids. Plants from several natural populations also fractionated isotopes during photosynthesis by an average of −90 to −110%. In addition, the organically bonded H in nonsaponifiable lipids was further fractionated by −80%. from that in saponifiable lipids, isolated from two geographically distinct populations of marsh plants. This difference between H isotope ratios of these two groups of lipids provides an endogenous isotopic marker.

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abstract: Recent works have investigated use of the hydrogen isotopic composition of C37 alkenones (δDK37s, lipid biomarkers of certain haptophyte microalgae, as an independent paleosalinity proxy. We discuss herein the factors impeding the success of such an application and identify the potential alternative use of δDK37s measurements as a proxy for non-thermal, physiological stress impacts on the U37K' paleotemperature index. Batch-culture experiments with the haptophyte Emiliania huxleyi (CCMP 1742) were conducted to determine the magnitude and variability of the isotopic contrasts between individual C37 alkenones. Further experiments were conducted with Emiliania huxleyi (CCMP 1742) andGephyrocapsa oceanica (PZ3-1) to determine whether, and to what extent, δDK37s varies between the physiological extremes of nutrient-replete exponential growth and nutrient-depleted senescence. Emiliania huxleyi was observed to exhibit an isotopic contrast between di- and tri-unsaturated C37 alkenones (αK37:3-K37:2≈0.97) that is nearly identical to that reported recently by others for environmental samples. Furthermore, this contrast appears to be constant with growth stage. The consistency of the offset across different growth stages suggests that a single, well-defined value for αK37:3-K37:2 may exist and that its use in an isotope mass-balance will allow accurate determination of δD values for individual alkenones without having to rely on time- and labor-intensive chemical separations. The isotopic fractionation between growth medium and C37 alkenones was observed to increase dramatically upon the onset of nutrient-depletion-induced senescence, suggesting that δDK37s may serve as an objective tool for recognizing and potentially correcting, at least semi-quantitatively, for the effects of nutrient stress on U37K' temperature records.

title: Inverse relationship between D/H fractionation in cyanobacterial lipids and salinity in Christmas Island saline ponds
abstract: Sediments from 28 saline and hypersaline (salinity 13.6–149.2) ponds on Christmas Island (Kiritimati), in the Central tropical Pacific Ocean, were investigated for the effect of salinity on the D/H ratios of lipid biomarkers. Hydrogen isotope ratios (expressed as δD values) of total lipid extracts, and individual hydrocarbons heptadecane, heptadecene, octadecane, octadecene, diploptene, and phytene from cyanobacteria, became increasingly enriched in deuterium as salinity increased, spanning a range of 100‰, while lake water δD values spanned a range of just 12‰. Net D/H fractionation between lipids and source water thus decreased as salinity increased. Isotope fractionation factors (αlipid–water) were strongly correlated with salinity, and increased in all compound classes studied by up to 0.0967 over a salinity range of 136. Differences in the hydrogen isotopic composition of lipids derived from three biosynthetic pathways (acetogenic, mevalonate, and non-mevalonate) remained similar irrespective of the salinity. This suggests that the mechanism responsible for the observed αlipid–water–salinity relationship originates prior to the last common biosynthetic branching point, the Calvin Cycle. We propose that a decrease in the exchange of intra- and extra-cellular (ambient) water resulting from down-regulation or closure of water channels (aquaporins) within cyanobacterial cell membranes, and subsequent isotopic enrichment of the intracellular water, likely resulting from metabolic reactions. These findings imply that caution must be exercised when attempting to reconstruct source water δD values using lipid δD values from environments that may have experienced salinity variations. On the other, hand our results can be used to establish a paleo-salinity proxy based on lipid δD, if additional constraints on source water δD values can be made.

title: Hydrogen isotopes in dinosterol from the Chesapeake Bay estuary
abstract: The hydrogen isotope ratio of the dinoflagellate sterol dinosterol (4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol) was measured in suspended particles and surface sediments from the Chesapeake Bay estuary in order to evaluate the influence of salinity on hydrogen isotope fractionation. D/H fractionation was found to decrease by 0.99 ± 0.23‰ per unit increase in salinity over the salinity range 10–29 PSU, a similar decrease to that observed in a variety of lipids from hypersaline ponds on Christmas Island (Kiribati). We hypothesize that the hydrogen isotopic response to salinity may result from diminished exchange of water between algal cells and their environment, lower growth rates and/or increased production of osmolytes at high salinities. Regardless of the mechanism, the consistent sign and magnitude of dinosterol δD response to changing salinity should permit qualitative to semi-quantitative reconstructions of past salinities from sedimentary dinosterol δD values.

title: The influence of salinity on D/H fractionation in alkenones from saline and hypersaline lakes in continental North America
abstract: We present a preliminary assessment of the hydrogen isotopic composition of individual alkenones (C 37–39 di-, tri- and tetra-unsaturated methyl- and ethyl-ketones) purified from surface lake sediment from sites spanning a range of salinities from 20 to 133 ppt. Combining these measurements with measurements of the hydrogen isotopic composition of modern lake water, we estimate the magnitude of D/H fractionation across our sample set and observe reduced sensitivity to salinity compared to observations from Emiliania huxleyi and Gephyrocapsa oceanica . This lends support to the hypothesis that D/H fractionation during biosynthesis is less sensitive to changes in salinity in alkenone producers from continental interior sites than in producers from open marine environments. We also observe stronger correlations between the tetra-unsaturated alkenone δD values with water δD values, and associated α values with salinity than we do for the di- and tri-unsaturated compounds, and suggest that this may result from increased number of lacustrine haptophyte species producing the di- and tri-unsaturated as compared to the tetra-unsaturated.

title: The influence of salinity on D/H fractionation in dinosterol and brassicasterol from globally distributed saline and hypersaline lakes
abstract: Salinity, growth rate, growth stage, nutrient limitation and temperature have all been shown to influence the magnitude of D/H fractionation in algal lipids through laboratory and field studies. Of these factors, salinity has been studied most extensively in the field, but to date all such investigations have focused on transect studies within specific and isolated environments. Here we test the relationship between salinity and the magnitude of D/H fractionation in algal lipids through paired analyses of sedimentary and particulate lipid and water hydrogen isotope values at a wide range of continental and coastal lake sites spanning salinities from 0 to 117 ppt. Our results demonstrate broad consistency between D/H fractionations in dinosterol and brassicasterol with those obtained from previous work, with salinity changes of 1 ppt resulting in lipid δD changes of 0.7–1‰. Although our results also show variability in D/H fractionation between sites that is not related to salinity, the fact that any relationship emerges above the influences of other factors suggests that the salinity effect is dominant for some lipids in the majority of saline to hypersaline environments. This improved understanding of D/H fractionation in dinosterol and brassicasterol synthesis supports the use of these compounds as paleohydrologic indicators. When combined with D/H measurements from a second lipid or oxygen isotope measurements from carbonate, quantitative reconstructions of salinity and lake water isotope changes are possible. Extending the number of algal lipids within which a consistent relationship between D/H fractionation and salinity has been identified also supports the notion that the relationship is widespread among unicellular photoautotrophs.

title: Stable Hydrogen Isotope Fractionations during Autotrophic and Mixotrophic Growth of Microalgae.
abstract: Isotope effects, studied with precision isotope ratio mass spectrometry, have been used to locate critical steps in the H metabolism of plants. By manipulating the growth conditions of versatile microalgae, the discrimination of H isotopes between water in the growth medium and the organically bonded H in carbohydrates from these microalgae was -100 to -120 per thousand and was regulated by both the light and the dark reactions of photosynthesis. Photosynthetic electron transport discriminated against the heavy isotope of H and formed a pool of reductant available for biosynthesis that was enriched in the light isotope. Growth in red or white light activated phosphoglyceric acid reduction and H isotope discrimination, when H was fixed into organic matter. An additional fractionation of -30 to -60 per thousand occurred during the biosynthesis of proteins and lipids and was associated with glycolysis. This fractionation paralleled the isotope effect seen in carbohydrate metabolism, indicating that H metabolism in photosynthesis was coupled with that in dark biosynthetic reactions via the pool of reductant, probably NADPH.

title: Systematics of 2H patterns in natural compounds and its importance for the elucidation of biosynthetic pathways
abstract: The relative global 2H-content of natural plant products is correlated to that of the primary hydrogen source, i.e. water, to the site of their biosynthesis (C3-, C4- and CAM-plants; chloroplasts, cytosol), and to their biosynthetic pathways. A relative global 2H-content sequence can be established in the order phenylpropanoids > carbohydrates > bulk material > hydrolysable lipids > steroids. A detailed analysis of the 2H-patterns of the main groups of secondary compounds reveals regularities, in that they are correlated to the primary precursors and to the origin of hydrogen from four main pools with the mean δ2H-values [‰]V−SMOW: leaf H2O ∼+30; carbohydrates ∼−70; NADPH ∼−250; flavoproteins ∼−350. Aside from the 2H-discrimination between these pools, kinetic isotope effects on defined reactions only become effective in connection with metabolic branching events. So, the 2H-pattern of natural aromatic compounds can be correlated to the 2H-pattern of the precursor carbohydrates and a reduction step in the course of the shikimic acid pathway, furthermore to the implication of the NIH-shift. The pattern of aromatic compounds from the polyketide is different from that of the shikimate pathway. The alternating 2H-abundance of fatty acid chains is caused by the origin of their hydrogen atoms from carbohydrates and from NADPH, directly or via a flavoprotein, respectively. This is similar for isoprenoids, and the natural 2H-patterns permit their assignment to the mevalonate or non-mevalonate biosynthetic pathway. Generally, the correlations and regularities of the 2H-patterns of organic compounds found are a new reliable tool for the elucidation of biosynthetic pathways and origin assignments.

title: Large D/H variations in bacterial lipids reflect central metabolic pathways
abstract: Large hydrogen-isotopic (D/H) fractionations between lipids and growth water have been observed in most organisms studied to date. These fractionations are generally attributed to isotope effects in the biosynthesis of lipids, and are frequently assumed to be approximately constant for the purpose of reconstructing climatic variables. Here, we report D/H fractionations between lipids and water in 4 cultured members of the phylum Proteobacteria, and show that they can vary by up to 500‰ in a single organism. The variation cannot be attributed to lipid biosynthesis as there is no significant change in these pathways between cultures, nor can it be attributed to changing substrate D/H ratios. More importantly, lipid/water D/H fractionations vary systematically with metabolism: chemoautotrophic growth (approximately −200 to −400‰), photoautotrophic growth (−150 to −250‰), heterotrophic growth on sugars (0 to −150‰), and heterotrophic growth on TCA-cycle precursors and intermediates (−50 to +200‰) all yield different fractionations. We hypothesize that the D/H ratios of lipids are controlled largely by those of NADPH used for biosynthesis, rather than by isotope effects within the lipid biosynthetic pathway itself. Our results suggest that different central metabolic pathways yield NADPH—and indirectly lipids—with characteristic isotopic compositions. If so, lipid δD values could become an important biogeochemical tool for linking lipids to energy metabolism, and would yield information that is highly complementary to that provided by 13C about pathways of carbon fixation.

title: Oxygen and Hydrogen Isotope Fractionation during Cellulose Metabolism in Lemna gibba L.
abstract: Lemna gibba L. B3 was grown under heterotrophic, photoheterotrophic, and autotrophic conditions in water having a variety of hydrogen and oxygen isotopic compositions. The slopes of the linear regression lines between the isotopic composition of water and leaf cellulose indicated that under the three growth conditions about 40, 70, and 100% of oxygens and carbon-bound hydrogens of cellulose exchanged with those of water prior to cellulose formation. Using the equations of the linear relationships, we estimated the overall fractionation factors between water and the exchanged oxygen and carbon bound-hydrogen of cellulose. At least two very different isotope effects must determine the hydrogen isotopic composition of Lemna cellulose. One reflects the photosynthetic reduction of NADP, while the second reflects exchange reactions that occur subsequent to NADP reduction. Oxygen isotopic composition of cellulose apparently is determined by a single type of exchange reaction with water. Under different growth conditions, variations in metabolic fluxes affect the hydrogen isotopic composition of cellulose by influencing the extent to which the two isotope effects mentioned above are recorded. The oxygen isotopic composition of cellulose is not affected by such changes in growth conditions.

title: Fractionation of Carbon and Hydrogen Isotopes in Biosynthetic Processes
abstract: This review is concerned with the isotopic relationships between organic compounds produced by a single organism, specifically their enrichments or depletions in 13C relative to total-biomass carbon. These relationships are biogeochemically significant because



1. An understanding of biosynthetically controlled, between-compound isotopic contrasts is required in order to judge whether plausibly related carbon skeletons found in a natural mixture might come from a single source or instead require multiple sources.

2. An understanding of compound-to-biomass differences must underlie the interpretation of isotopic differences between individual compounds and total organic matter in a natural mixture.



My approach is pedagogic. The coverage is meant to be thorough, but the emphases and presentation have been chosen for readers approaching this subject as students rather than as research specialists. In common with the geochemists in my classes, many readers of this paper may not be very familiar with biochemistry and microbiology. I have not tried to explain every concept from those subjects and I have not inserted references for points that appear in standard texts in biochemistry or microbiology. Among such books, I particularly recommend the biochemistry text by Garrett and Grisham (1999) and the microbiology text by Madigan et al. (2000). The biochemistry text edited by Zubay (1998) is also particularly elegant and detailed. White (1999) has written a superb but condensed text on the physiology and biochemistry of prokaryotes.

A schematic overview of the relevant processes is shown in . Plants and other autotrophs fix CO2. Animals and other heterotrophs utilize organic compounds. If the assimilated carbon is a small molecule (like CO2, CH4, or acetate), significant isotopic fractionation is likely to accompany the fixation or assimilation of C. Such fractionations establish the isotopic relationship between an organism and its carbon source. Those associated …

title: Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells
abstract: Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

title: Metabolic processes account for the majority of the intracellular water in log-phase Escherichia coli cells as revealed by hydrogen isotopes.
abstract: It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.

title: Culture of Phytoplankton for Feeding Marine Invertebrates
abstract: These pages describe relatively simple and reliable methods for the culture of marine phytoplankton species useful for feeding marine invertebrates. The methods suffice for the most fastidious algae now routinely cultivable, and simplifications indicated for less demanding species are easily made; for example, omission of silicate for plants other than diatoms. Certain modifications of techniques, ancillary methods, and precautions will be treated briefly because questions often arise concerning them, but documentation will be minimal and hopefully restricted to publications readily available.

title: Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran.
abstract: Bacteria-free clones of the small centric diatom Cyclotella nana Hustedt were isolated, three from estuarine localities, one from Continental Shelf waters, and one from the Sargasso Sea. Detonula confervacea was isolated from Narragansett Bay. Morphology of all clones was studied with the light and electron microscopes. Morphological differences between clones of C. nana do not at present warrant separating any as distinct species.Clones of C. nana require only vitamin B12; D. confervacea has no vitamin requirement.Growth of the estuarine clones of C. nana was unaffected by salinity down to 0.5‰ and increased with temperature to 25 °C. The Shelf clone grew more rapidly at salinities above 8‰ and at temperatures between 10° and 20 °C. The Sargasso Sea clone did not survive below 15 °C or 17.5‰, while D. confervacea did not survive at temperatures above 15° or at salinities below 8‰. The physiological differences between clones correspond roughly to the conditions obtaining in nature where each was collected.

title: Hydrogen isotope fractionation in freshwater and marine algae: II. Temperature and nitrogen limited growth rate effects
abstract: Zhang and Sachs [Hydrogen isotope fractionation in freshwater algae: I. Variations among lipids and species. Organic Geochemistry 38, 582–608, 2007] demonstrated that algal lipid δD values track water δD values with high fidelity (R2 > 0.99), but that D/H fractionation varied among lipids and algal species. Here we report on the influence of temperature and nitrogen limitation on D/H fractionation in lipids from cultured microalgae. Two species of freshwater green algae, Eudorina unicocca and Volvox aureus, were grown in batch culture at 15 °C and 25 °C. Increased D/H fractionation of 2−4‰/°C occurred at the higher temperature in all lipids analyzed. The marine diatom Thalassiosira pseudonana was grown in continuous culture under nitrogen replete (NR) and nitrogen limited (NL) conditions, resulting in a growth rate that was 4.4 fold lower under the latter conditions. The fatty acid content of NL cells was approximately 4 fold higher than in NR cells, whereas the sterol content was similar in both. While sterols from the NL culture were enriched in deuterium by 37‰ relative to the NR culture, fatty acids from both cultures had similar δD values, implying that D/H fractionation during isoprenoid (branched) lipid synthesis is affected by nitrogen limitation, but D/H fractionation during acetogenic (linear) lipid synthesis is not. Cross-talk of the precursor isopentenyl diphosphate between the cytosolic MVA and plastidic DOXP/MEP synthetic pathways is a plausible mechanism for the observed D/H differences between isoprenoid and acetogenic lipids. This preliminary study highlights the need to consider both the type of lipid and potential changes in growth conditions in paleoenvironmental studies using lipid D/H ratios.

title: The effect of frozen storage of open-ocean seawater samples on the concentration of dissolved phosphate and nitrate
abstract: Filtered seawater samples which were stored at −20°C in containers of different surface area to volume ratios were analysed for the effects of surface adsorption of nitrate and phosphate during storage for a maximum of 24 months. The results for nitrate showed no significant change in concentration over the duration of the experiment. However, the concentration of phosphate steadily decreased in samples stored for longer than 4 months. The loss of phosphate after this time cannot be explained at present, but this study showed that it was not due to the effects of surface adsorption.

title: The measurement of D/H ratio in alkenones and their isotopic heterogeneity.
abstract: Previous paleoenvironmental studies reported the δD values of a mixture of coeluting alkenones. Here, we present a semi-preparative normal-phase high-performance liquid chromatography–mass spectrometry (NP-HPLC–MS) method for purifying long chain (C37 and C38) unsaturated methyl and ethyl ketones (alkenones) on the basis of chain length and degree of unsaturation. The method was applied to purify alkenones in suspended particles and surface sediments from a site in Chesapeake Bay, eastern USA. The hydrogen isotopic composition of di- and triunsaturated C37 and C38 alkenones differed significantly on the basis of chain length and the degree of unsaturation, demonstrating the importance of gas chromatography–isotope ratio-mass spectrometry (GC–irMS) analysis of individual alkenones for accurate paleoenvironmental reconstruction. Constant fractionation factors between alkenones with different chain length but the same degree of unsaturation ( α C 37 : 2 – C 38 : 2 and α C 37 : 3 – C 38 : 3 = 1.01 ) and those with the same chain length but different degree of unsaturation ( α C 37 : 2 – C 37 : 3 and α C 38 : 2 – C 38 : 3 = 0.97 ) in all samples suggest that the values may represent hydrogen isotope fractionation associated with elongation and desaturation during alkenone biosynthesis.

title: Studies on Fat Accumulation by Algae
abstract: The generally held view that fat accumulation is characteristic of certain classes of algae is based on inadequate evidence. In this investigation the fatty acid, unsaponifiable lipoid, total cell nitrogen, and hydrolysable polysaccharide contents of the following algae have been determined at various stages during growth in pure culture: Chlorella vulgaris, Scotiella sp., Euglena gracilis var. bacillaris, Tribonema aequale, Monodus subterraneas, Navicula pelliculosa, Porphyridium cruentum, Anabaena cylindrica, and Oscillatoria sp. Using the results obtained, the capacities of the algae to accumulate fat have been compared in the following ways : (i) on a percentage dry-weight basis ; (2) by means of the differential growth factor relating the fat content of cultures to dry weight of algal material ; (3) by consideration of the proportion which fats form of the total reserve material (taken as fat plus hydrolysable polysaccharide) as a function of total cell nitrogen content. Species belonging to the same class have been found to resemble each other in the relative amounts of crude protein, fats, and hydrolysable polysaccharide which they contain. Marked difference's in composition between species belonging to different classes have been found, but it is concluded that there are no funda mental differences in the physiological relations of fat accumulation in the algae belonging to the Chlorophyceae, Euglenineae, Xanthophyceae, and Bacillario phyceae. Representatives of the Rhodophyceae and Myxophyceae, although they may under certain circumstances have moderately high fat contents, appear to differ in that in them fat accumulation is not associated with low cell nitrogen contents as it is in the other algae examined. No well-defined correlations between the unsaponifiable lipoid and either the fatty acid or total cell nitrogen contents of the algae have been found. Euglena gracilis var. bacillaris has been found to have a rather higher content of un saponifiable lipoid than the other algae.

title: PHYTOPLANKTON LIPIDS: INTERSPECIFIC DIFFERENCES AND EFFECTS OF NITRATE, SILICATE AND LIGHT‐DARK CYCLES 1
abstract: The lipid content of various phytoplankton species was measured in response to nitrogen and silicon limitation and over the cell cycle in synchronized cultures. In a survey of 30 species it was found that during log‐phase growth, green algae contained an average of 17.1% total lipids (% of total dry weight), whereas diatoms contained an average of 24.5%. Nitrogen deprivation for 4 to 9 days resulted in 2‐ to 3‐fold increases in the lipid content of green algae, whereas both increases and decreases were noted in diatoms, depending on the species. The greatest lipid content measured in the study was 72% in Monallantus salina (strain GSB Sticho) which had been deprived of nitrogen for 9 days. Nitrate replenishment in a nitrogen starved culture of Oocystis polymorpha Groover & Bold showed that the excess cellular lipids do not rapidly disappear during recovery, until cell division occurs.

title: Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low‐cost photobioreactor
abstract: Thirty microalgal strains were screened in the laboratory for their biomass productivity and lipid content. Four strains (two marine and two freshwater), selected because robust, highly productive and with a relatively high lipid content, were cultivated under nitrogen deprivation in 0.6‐L bubbled tubes. Only the two marine microalgae accumulated lipid under such conditions. One of them, the eustigmatophyte Nannochloropsis sp. F&M‐M24, which attained 60% lipid content after nitrogen starvation, was grown in a 20‐L Flat Alveolar Panel photobioreactor to study the influence of irradiance and nutrient (nitrogen or phosphorus) deprivation on fatty acid accumulation. Fatty acid content increased with high irradiances (up to 32.5% of dry biomass) and following both nitrogen and phosphorus deprivation (up to about 50%). To evaluate its lipid production potential under natural sunlight, the strain was grown outdoors in 110‐L Green Wall Panel photobioreactors under nutrient sufficient and deficient conditions. Lipid productivity increased from 117 mg/L/day in nutrient sufficient media (with an average biomass productivity of 0.36 g/L/day and 32% lipid content) to 204 mg/L/day (with an average biomass productivity of 0.30 g/L/day and more than 60% final lipid content) in nitrogen deprived media. In a two‐phase cultivation process (a nutrient sufficient phase to produce the inoculum followed by a nitrogen deprived phase to boost lipid synthesis) the oil production potential could be projected to be more than 90 kg per hectare per day. This is the first report of an increase of both lipid content and areal lipid productivity attained through nutrient deprivation in an outdoor algal culture. The experiments showed that this marine eustigmatophyte has the potential for an annual production of 20 tons of lipid per hectare in the Mediterranean climate and of more than 30 tons of lipid per hectare in sunny tropical areas. Biotechnol. Bioeng. 2009;102: 100–112.

title: PRODUCTION AND CELLULAR LOCALIZATION OF NEUTRAL LONG‐CHAIN LIPIDS IN THE HAPTOPHYTE ALGAE ISOCHRYSIS GALBANA AND EMILIANIA HUXLEYI 1
abstract: Isochrysis galbana Parke, Emiliania huxleyi (Lohm.) Hay and Mohler, and some related prymnesiophyte algae produce as neutral lipids a set of polyunsaturated long‐chain (C37–39) alkenones, alkenoates, and alkenes (PULCA). These biomarkers are widely used for paleothermometry, but the biosynthesis and cellular location of these unique lipids remain largely unknown. By staining with the fluorescent lipophilic dye Nile Red, we found that I. galbana and E. huxleyi, like many other algae, package their neutral lipid into cytoplasmic vesicles or lipid bodies. We found that these lipid bodies increase in abundance under nutrient limitation and disappear under prolonged darkness and show that this pattern correlates well with the concentration of PULCA as measured by TLC. In addition, we show that lipid vesicles purified by sucrose density gradient centrifugation consist predominantly of PULCA. We also found significant pools of neutral lipid associated with chloroplasts, and PULCA component profiles in lipid vesicles and chloroplasts are similar. Examination of cell ultrastructure shows conspicuous cytoplasmic and chloroplast lipid bodies, and we suggest that PULCA may be synthesized in chloroplasts and then exported to cytoplasmic lipid bodies for storage and eventual metabolism. Our results connect and extend prior observations of lipid bodies and membrane‐unbound PULCA in I. galbana and E. huxleyi, as well as the behavior of PULCA during nutrient and light stress.

title: Genetic and physiological influences on the alkenone/alkenoate versus growth temperature relationship in Emiliania huxleyi and Gephyrocapsa oceanica
abstract: Selected warm and cold water strains of the coccolithophorid Emiliania huxleyi and the closely related species Gephyrocapsa oceanica were cultured under controlled temperature conditions to assess genetic and physiological variability in the alkenone/alkenoate vs. temperature relationship. Differences in the strains’ growth rates over the 6–30°C experimental temperature range were small but consistent with their cold or warm water origins. E. huxleyi and G. oceanica had similar alkenone/alkenoate biochemistry, justifying the extension of alkenone stratigraphy to sediments predating the appearance of E. huxleyi. These species could not be distinguished by C38/C37 alkenone or alkenoate/alkenone ratios as previously suggested (Volkman et al. 1995; Sawada et al. 1996) but given samples from a range of temperatures may be distinguished by a plot of the C38 ethyl vs. C38 methyl unsaturation ratios (U38EtK and U38MeK, respectively).

Biochemical responses to temperature and the C37 alkenone-based (U37K′) temperature calibrations differed significantly among the strains. The U37K′ temperature calibration was nonlinear for five of the six strains examined. A reduction in slope of the calibration at temperatures 21°C suggests the cell’s alkenone-based adaptation to temperature is limited at the extremes of its growth temperature range. The unsaturation ratios of the C38 methyl and ethyl alkenones (U38MeK and U38EtK) varied similarly with temperature and were strongly intercorrelated.

The experiments also documented an influence of cell physiological state on both alkenone and alkenoate composition and on alkenone unsaturation. Cells in late logarithmic and stationary growth had significantly increased abundance of alkenoates and C38 ethyl alkenones relative to C38 methyl alkenone abundance. In some strains the unsaturation ratios of both C37 and C38 alkenones also significantly decreased when cells entered the late log phase.

Comparison of culture results with field data indicates that the average physiological state of alkenone-synthesizers in the open ocean differs from cultured cells growing under exponential growth and appears to be more similar to cells in late log or stationary growth phases. Differences in alkenone/alkenoate ratios between cultured cells and sediments underlying waters of a similar temperature most probably reflect a difference in cell physiology between cultured cells and oceanic populations and not greater diagenetic losses of alkenones relative to alkenoates, as previously suggested (Prahl et al. 1995).

Our experiments confirm that biogeographical variations observed in the alkenone vs. temperature relationship in natural waters reflect, at least in part, differences in genetic makeup and physiological status of the local alkenone-synthesizing populations. Hence, alkenone-based paleo sea surface temperature estimates are subject to errors, albeit small, which arise from genetic differences between modern-day and paleo-populations. The reduction in slope of the U37K′ temperature calibration for most strains at T >24°C indicate that linear U37K′ temperature calibrations (e.g., Prahl et al. 1988) which are currently used to estimate paleo SST, and which are poorly constrained at higher temperatures, probably underestimate the magnitude of SST change for tropical and subtropical regions.

title: An effect of dissolved nutrient concentrations on alkenone‐based temperature estimates
abstract: The ratio of 37-carbon diunsaturated to diunsaturated and triunsaturated alkenones (UK′37) produced by some haptophytes is widely used as a proxy for past sea surface temperatures. However, our isothermal culturing experiments with Emiliania huxleyi clone CCMP372 show UK′37 values to also vary with nutrient availability and cell division rate. These results provide a reasonable explanation for large isothermal variation in UK′37 values of single coccolithophorid strains grown in culture. They also suggest that alkenone-based estimates of past sea surface temperatures may have been influenced by dissolved nutrient concentrations as well as by temperature.

title: Impact of salinity and growth phase on alkenone distributions in coastal haptophytes
abstract: Batch cultures of Isochrysis galbana (strain CCMP 1323) and Chrysotila lamellosa (strain CCMP 1307) were grown at salinity values of ca. 10 to ca. 35 and the alkenone distributions determined for different growth phases. U37K' values decreased slightly with salinity for C. lamellosa but were largely unaffected for I. galbana, except during the decline phase. The values decreased with incubation time in both species. The proportion of C37:4, used as a proxy for salinity, increased in both species at 0.16-0.20% per salinity unit, except during the stationary phase for I. galbana. C37:4 was much more abundant in C. lamellosa (30-44%) than in I. galbana (4-12%). Although our results suggest that salinity has a direct effect on alkenone distribution, growth phase and species composition should also have a marked impact, complicating the use of the distributions as a proxy for salinity in the marine environment.

title: Hydrogen isotope fractionation during lipid biosynthesis by Haloarcula marismortui
abstract: We studied the controls on the fractionation of hydrogen isotopes during lipid biosynthesis by Haloarcula marismortui, a halophilic archaea, in pure culture experiments by varying organic substrate, the hydrogen isotope composition (D/H) of water, temperature, and salinity. Cultures were grown on three substrates: succinate, pyruvate and glycerol with known hydrogen isotope compositions, and in water with different hydrogen isotopic compositions. All culture series grown on a particular substrate show strong correlations between δDarchaeol and δDwater. However, correlations are distinctly different for cultures grown on different substrates. Our results indicate that the metabolic pathway of substrate exerts a fundamental influence on the δD value of lipids, likely by influencing the D/H composition of NADPH (nicotinamide adenine dinucleotide phosphate), the reducing agent that contributes hydrogen to carbon atoms during lipid biosynthesis. Temperature and salinity have smaller, but similar effects on δDlipid, primarily due to the way temperature and salinity influence growth rate, as well as temperature effects on the activity of enzymes.

title: The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants1[W]
abstract: The availability of nitrogen varies greatly in the ocean and limits primary productivity over large areas. Diatoms, a group of phytoplankton that are responsible for about 20% of global carbon fixation, respond rapidly to influxes of nitrate and are highly successful in upwelling regions. Although recent diatom genome projects have highlighted clues to the success of this group, very little is known about their adaptive response to changing environmental conditions. Here, we compare the proteome of the marine diatom Thalassiosira pseudonana (CCMP 1335) at the onset of nitrogen starvation with that of nitrogen-replete cells using two-dimensional gel electrophoresis. In total, 3,310 protein spots were distinguishable, and we identified 42 proteins increasing and 23 decreasing in abundance (greater than 1.5-fold change; P < 0.005). Proteins involved in the metabolism of nitrogen, amino acids, proteins, and carbohydrates, photosynthesis, and chlorophyll biosynthesis were represented. Comparison of our proteomics data with the transcriptome response of this species under similar growth conditions showed good correlation and provided insight into different levels of response. The T. pseudonana response to nitrogen starvation was also compared with that of the higher plant Arabidopsis (Arabidopsis thaliana), the green alga Chlamydomonas reinhardtii, and the cyanobacterium Prochlorococcus marinus. We have found that the response of diatom carbon metabolism to nitrogen starvation is different from that of other photosynthetic eukaryotes and bears closer resemblance to the response of cyanobacteria.

title: Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability
abstract: Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium‐replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.

title: Transcriptional responses of three model diatoms to nitrate limitation of growth
abstract: Diatoms are among the most diverse groups of phytoplankton in the ocean. Despite their widely recognized influence on ocean ecosystems and global biogeochemistry, little is known about the impact of this diversity on large-scale processes. Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. The three species shared 5,583 clusters of orthologous genes based on OrthoMCL clustering of publically available diatom genomes. These clusters represent 30-54% of the predicted genes in each diatom genome. Less than 5% of genes within these core clusters displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling. Characterization of these finely tuned responses will help to better predict which types of diatoms will bloom under which sets of environmenta

title: Emiliania huxleyi endures N-limitation with an efficient metabolic budgeting and effective ATP synthesis
abstract: BackgroundGlobal change will affect patterns of nutrient upwelling in marine environments, potentially becoming even stricter regulators of phytoplankton primary productivity. To better understand phytoplankton nutrient utilization on the subcellular basis, we assessed the transcriptomic responses of the life-cycle stages of the biogeochemically important microalgae Emiliania huxleyi to nitrogen-limitation. Cells grown in batch cultures were harvested at ‘early’ and ‘full’ nitrogen-limitation and were compared with non-limited cells. We applied microarray-based transcriptome profilings, covering ~10.000 known E. huxleyi gene models, and screened for expression patterns that indicate the subcellular responses.ResultsThe diploid life-cycle stage scavenges nitrogen from external organic sources and -like diatoms- uses the ornithine-urea cycle to rapidly turn over cellular nitrogen. The haploid stage reacts similarly, although nitrogen scavenging is less pronounced and lipid oxidation is more prominent. Generally, polyamines and proline appear to constitute major organic pools that back up cellular nitrogen. Both stages induce a malate:quinone-oxidoreductase that efficiently feeds electrons into the respiratory chain and drives ATP generation with reduced respiratory carbon throughput.ConclusionsThe use of the ornithine-urea cycle to budget the cellular nitrogen in situations of limitation resembles the responses observed earlier in diatoms. This suggests that underlying biochemical mechanisms are conserved among distant clades of marine phototrophic protists. The ornithine-urea cycle and proline oxidation appear to constitute a sensory-regulatory system that monitors and controls cellular nitrogen budgets under limitation. The similarity between the responses of the life-cycle stages, despite the usage of different genes, also indicates a strong functional consistency in the responses to nitrogen-limitation that appears to be owed to biochemical requirements. The malate:quinone-oxidoreductase is a genomic feature that appears to be absent from diatom genomes, and it is likely to strongly contribute to the uniquely high endurance of E. huxleyi under nutrient limitation.

title: The fire of life
abstract: The instant Application deals with a process for the manufacture of methacrylonitrile by the catalytic ammonoxidation of isobutylene at elevated temperature, in gaseous phase, in the presence of a catalyst consisting of the elements tellurium, cerium, molybdenum, of one or more of the elements selected from the group comprising lithium, sodium, potassium rubidium cesium and moreover of oxygen chemically combined with the above cited elements.

title: Invariant scaling relationships for interspecific plant biomass production rates and body size
abstract: The allometric relationships for plant annualized biomass production (“growth”) rates, different measures of body size (dry weight and length), and photosynthetic biomass (or pigment concentration) per plant (or cell) are reported for multicellular and unicellular plants representing three algal phyla; aquatic ferns; aquatic and terrestrial herbaceous dicots; and arborescent monocots, dicots, and conifers. Annualized rates of growth G scale as the 3/4-power of body mass M over 20 orders of magnitude of M (i.e., G ∝ M3/4); plant body length L (i.e., cell length or plant height) scales, on average, as the 1/4-power of M over 22 orders of magnitude of M (i.e., L ∝ M1/4); and photosynthetic biomass Mp scales as the 3/4-power of nonphotosynthetic biomass Mn (i.e., Mp ∝ Mn3/4). Because these scaling relationships are indifferent to phylogenetic affiliation and habitat, they have far-reaching ecological and evolutionary implications (e.g., net primary productivity is predicted to be largely insensitive to community species composition or geological age).

title: Salinity dependent hydrogen isotope fractionation in alkenones produced by coastal and open ocean haptophyte algae
abstract: The hydrogen isotope fractionation in alkenones produced by haptophyte algae is a promising new proxy for paleosalinity reconstructions. To constrain and further develop this proxy the coastal haptophyte Isochrysis galbana and the open ocean haptophyte alga Emiliania huxleyi were cultured at different salinities. The fractionation factor, αalkenones–water, ranged between 0.853 and 0.902 for I. galbana and 0.789 and 0.822 for E. huxleyi. The results show a strong linear correlation between the fractionation factor α and salinity for E. huxleyi, in agreement with earlier studies, but also for I. galbana. Both haptophytes show the same response to changes in salinity, represented by the slopes of the α–salinity relationship (∼0.002 per salinity unit). This suggests that the same process, in both coastal as well as open ocean haptophytes, is responsible for reducing fractionation with increasing salinity. However, there is a significant difference in absolute isotope fractionation between E. huxleyi and I. galbana, i.e. E. huxleyi produces alkenones which are 90‰ more depleted in D under the same culturing conditions than I. galbana. Our data suggest that the δD of alkenones can be used to reconstruct relative shifts in paleosalinity in coastal as well as open ocean environments with careful consideration of species composition and other complicating factors especially in coastal regions.

title: A new method for estimating growth rates of alkenone‐producing haptophytes
abstract: National Science Foundation grants OCE-9986306 and OCE-0094637 (B.N.P., R.R.B., ), OCE-0094272 (B.N.P.), OCE-9986306 and OCE-0094329 (F.G.P.). 
SOEST contribution

title: Review of alkenone calibrations (culture, water column, and sediments)
abstract: I review the status of alkenone unsaturation parameters as proxies for growth temperature and the potential for pCO2 estimates using the carbon isotopic composition of alkenones. Culture studies allow investigators to manipulate parameters that could influence the relative proportions of C37 and C38 ketones in producing organisms and to assess the possible influence of genetic variability on the calibration of unsaturation to growth temperature. Culture studies suggest large variability in the relation of unsaturation indices to growth temperature and hint that growth rate may play a role along with temperature in controlling unsaturation. Water column studies match the unsaturation index measured in particulate matter to ambient temperature, providing a snapshot of unsaturation-temperature relationship. In comparison to culture work, assemblages of regional water column data sets give more consistent relations of the unsaturation index to temperature, although the argument can be made for regional rather than basin-wide or global calibrations. A much simpler picture emerges from analyzing a global array of core top sediments. Regressions show that the sedimentary unsaturation indices follow a linear trend that is best correlated to mean annual temperature in the upper 10 m of the overlying water column. The regression is indistinguishable from the original Prahl et al. [1988] culture calibration of the U37k′ index to growth temperature. Exceptions occur at high latitudes, where the effects of highly seasonal production and the possible influence of salinity may affect the unsaturation index in sediments. Core top analyses also fail to reveal the variability of other alkenone parameters (for example, the ratio of C37 to C38 ketones) which have been found in culture studies. Much of the variance that appears in culture and water column calibrations is thus apparently removed in sediments, perhaps by temporal averaging. The promising paleo-pCO2 approach based on the isotopic composition of alkenones awaits the arrival of a significant number of core top and stratigraphic studies that will determine the extent to which physiological factors compromise the alkenone paleobarometer.

title: Alkenones as paleoceanographic proxies
abstract: Alkenone-derived estimates of temperature and CO2 are contributing to our understanding of Earth's climate history. In order to increase confidence in alkenone-based climate proxies we recommend the following actions. First, the sedimentary component or fraction containing alkenones should be identified in order to assess the impact of horizontal advection and vertical mixing on alkenone-derived temperature and CO2 estimates. Second, differential mixing rates of alkenone-containing particles and sand-sized foraminifera should be quantified by independent dating of the two phases. Until that is accomplished, apparent temporal offsets of climate proxies in the two phases should be interpreted cautiously. Third, the stability of the unsaturation ratio and carbon isotopic compositions of alkenones during all phases of diagenesis should be confirmed. Both field and laboratory observations are required. Fourth, future alkenone investigations should be coupled with other paleoclimate proxy measurements at high-deposition-rate-sites in a variety of oceanographic settings. In upwelling regions and in the vicinity of river plumes, salinity and nutrient proxies should be measured since changes in these parameters may affect alkenone biosynthesis.

title: Ecology and biogeochemistry of alkenone production at Station ALOHA
abstract: In our study of Station ALOHA, a site representative of the oligotrophic North Pacific subtropical gyre (NPSG), alkenone content and composition were examined in (1) sinking particulate materials collected in sediment traps moored at 2800 m during 2 year-long deployments (1992–1993 and 2000–2001) and (2) suspended particulate materials collected throughout the euphotic zone in two contrasting seasons (winter and summer). Each year, sediment trap records documented pronounced alkenone export events in both seasons. The winter event appears triggered by eddy diffusive mixing of nutrients into the upper water column and the release of light limitation on alkenone-producing photoautotrophs growing in the deep chlorophyll maximum layer (DCML). The summer event originates apparently from a stratified euphotic depth well above the DCML where others have now documented the occurrence of N2 fixation-based ‘new production’. Although our time series record is yet limited, it is likely that the absolute magnitude of alkenone export and its quantitative importance in winter relative to summer is an ecological feature of the NPSG which can oscillate significantly on decadal time scales in response to climatic forcing. Despite the significant seasonal and interannual differences in alkenone export noted in our study, findings suggest that paleotemperature assessments based on analysis of the alkenone unsaturation index U37K' measured in underlying sediments would likely be unimpaired by the higher frequency temporal variability in the surface water ecology of alkenone-producers. When translated using a widely accepted calibration equation (U37K'=0.034 T+0.039), U37K' values in average sediment trap particles are consistent with the mean annual sea-surface temperature at Station ALOHA.

title: Global temperature calibration of the alkenone unsaturation index (UK′37) in surface waters and comparison with surface sediments
abstract: In this paper, we compile the current surface seawater C37 alkenone unsaturation (UK′37) measurements (n = 629, −1 to 30°C temperature range) to derive a global, field-based calibration of UK′37 with alkenone production temperature. A single nonlinear “global” surface water calibration of UK′37 accurately predicts alkenone production temperatures over the diversity of modern-day oceanic environments and alkenone-synthesizing populations (T = −0.957 + 54.293(UK′37) − 52.894(UK′37)2 + 28.321(UK′37)3, r2 = 0.97, n = 567). The mean standard error of estimation is 1.2°C with insignificant bias in estimated production temperature among the different ocean regions sampled. An exception to these trends is regions characterized by strong lateral advection and extreme productivity and temperature gradients (e.g., the Brazil-Malvinas Confluence). In contrast to the surface water data, the calibration of UK′37 in surface sediments with overlying annual mean sea surface temperature (AnnO) is best fit by a linear model (AnnO = 29.876(UK′37) − 1.334, r2 = 0.97, n = 592). The standard error of estimation (1.1°C) is similar to that of the surface water production calibration, but a higher degree of bias is observed among the regional data sets. The sediment calibration differs significantly from the surface water calibration. UK′37 in surface sediments is consistently higher than that predicted from AnnO and the surface water production temperature calibration, and the magnitude of the offset increases as the surface water AnnO decreases. We apply the global production temperature calibration to the coretop UK′37 data to estimate the coretop alkenone integrated production temperature (coretop IPT) and compare this with the overlying annual mean sea surface temperature (AnnO). We use simple models to explore the possible causes of the deviation observed between the coretop temperature signal, as estimated by UK′37, and AnnO. Our results indicate that the deviation can best be explained if seasonality in production and/or thermocline production as well as differential degradation of 37:3 and 37:2 alkenones both affect the sedimentary alkenone signal.

title: Further evaluation of long-chain alkenones as indicators of paleoceanographic conditions
abstract: Cultures of the marine coccolithophorid, Emiliania huxleyi, were grown in the laboratory at five temperatures (8°, 10°, 15°, 20°, 25°C) and monitored by capillary gas chromatography for their long-chain, unsaturated lipid compositions. The long-chain lipids of this plant comprise a series of C37, C38 and C39 di-, tri- and, in cells grown below 15°C, tetra-unsaturated methyl and ethyl ketones and a methyl and ethyl ester of a di-unsaturated C36 fatty acid. Systematic changes in the degree of unsaturation and in the overall carbon chain length distribution of the alkenones and in the proportion of fatty acid esters relative to alkenones are noted as a function of growth temperature. We present temperature calibrations for these changes in the lipid composition of laboratory cultures and compare these results with the compositions of this biomarker series measured in a variety of sediments accumulating beneath warm (⩾25°C) and cold (⩽12°C) surface waters in the tropical and temperate North Pacific Ocean, respectively. The comparisons demonstrate 1) this series of biomarkers is deposited in these oceanic sediments with minimal evidence of alteration to its original composition and 2) the strain of E. huxleyi used in this laboratory calibration is representative of the “average” marine phytoplankton supplying this novel series of biomarkers to contemporary sediments in these two environments and a wide variety of other oceanic environments. The long-chain alkenones constitute a major component (8.0 ± 2.9%) of the total organic carbon content of living cells of E. huxleyi. The high cellular abundance of these compounds appears to be relatively constant and independent of the growth temperature of the plant. These biomarkers provide a well-designed and useful geochemical tool for assessing variations not only in surface water temperatures but potentially also in the productivity of an important group of marine phytoplankton in oceans of the recent and distant past.