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

<div><p>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. <i>Emiliania huxleyi</i> was cultivated in steady state at four growth rates and the δ²H value of individual alkenones (C_37:2, C_37:3, C_38:2, C_38:3), fatty acids (C_14:0, C_16:0, C_18:0), 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 (α_PS1 = 0.4, α_OPP = 0.75, and α_H2O = 0) and half of the hydrogen in a lipid derived from water the model indicates α_lipid = 0.79. This value is within the range measured for alkenones (α_alkenone = 0.77 to 0.81) and fatty acids (α_FA = 0.75 to 0.82) in the chemostat cultures, but is greater than the range for brassicasterol (α_{brassicasterol} = 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 <i>T</i>. <i>pseudonana</i> chemostat cultures as growth rate increases. Insensitivity of α_FA 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 <i>E</i>. <i>huxleyi</i> lipids and a <i>T</i>. <i>pseudonana</i> sterol implies that any change in growth rate larger than ~0.15 div d^-1 can cause a change in δ²H_lipid that is larger than the analytical error of the measurement (~5‰), and needs to be considered when interpreting δ²H_lipid variations in sediments.</p></div

Concentration of lipids as a function of growth rate in <i>E</i>. <i>huxleyi</i> cultures.

<p>Concentrations presented in ng lipid per mL of culture media. (A) Alkenones and brassicasterol. (B) Fatty acids.</p

Lipid concentrations in <i>T</i>. <i>pseudonana</i> chemostat cultures.

<p>Lipid concentrations in <i>T</i>. <i>pseudonana</i> chemostat cultures.</p

Fractionation factor for C₃₇ alkenones as a function of growth rate in <i>E</i>. <i>huxleyi</i> and <i>G</i>. <i>oceanica</i> cultures.

<p>The presented results are from the following sources: <i>E</i>. <i>huxleyi</i> data: continuous cultures from this study (solid green circles), batch cultures from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref010" target="_blank">10</a>] (open brown circles) and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref036" target="_blank">36</a>] (open blue circles). <i>G</i>. <i>oceanica</i> data: batch cultures from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref010" target="_blank">10</a>] (open brown squares) and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref023" target="_blank">23</a>] (open purple squares). All data are from C₃₇ methyl alkenones. The C_37:2 and C_37:3 alkenones were measured and plotted separately in this study, whereas they were combined and measured together in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref010" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref036" target="_blank">36</a>].</p

Lipid concentrations in <i>E</i>. <i>huxleyi</i> chemostat cultures.

<p>Lipid concentrations in <i>E</i>. <i>huxleyi</i> chemostat cultures.</p

Growth conditions for continuous cultures of <i>E</i>. <i>huxleyi</i> and <i>T</i>. <i>pseudonana</i>.

<p>Growth conditions for continuous cultures of <i>E</i>. <i>huxleyi</i> and <i>T</i>. <i>pseudonana</i>.</p

Hydrogen isotope fractionation in 24-methyl-cholesta-5,24(28)-dien-3β-ol and three fatty acids as a function of growth rate in <i>T</i>. <i>pseudonana</i> chemostat cultures.

<p>Open symbols are results reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.ref037" target="_blank">37</a>]. Fractionation factors (α) decreased in the sterol, indicating greater ²H/¹H fractionation between lipids and extracellular water, as growth rates increased by 20‰ (div d^-1)^-1, and nearly constant in C_14:0, C_16:0 and C_16:1 fatty acids.</p

Hydrogen isotope ratios and fractionation factors in <i>E</i>. <i>huxleyi and T</i>. <i>pseudonana</i> chemostat cultures.

<p>Hydrogen isotope ratios and fractionation factors in <i>E</i>. <i>huxleyi and T</i>. <i>pseudonana</i> chemostat cultures.</p

Model of hydrogen isotopic relationships giving rise to observed δ²H values of lipids in <i>E</i>. <i>huxleyi</i> cells.

<p><i>f</i> is the fraction of hydrogen in lipids that comes from NADPH, 1-<i>f</i> is the fraction of hydrogen in lipids that comes from water, <i>x</i> is the fraction of hydrogen in lipids derived from PS1 of photosynthesis, <i>1-x</i> is the fraction of hydrogen in lipids derived from the OPP pathway.</p

Sensitivity of the ²H/¹H fractionation factor, α, to intracellular hydrogen source.

<p>The fractionation factor, α, can respond to both (A) <i>f</i>, the fraction of lipid hydrogen derived from NADPH versus intracellular water, and (B) <i>x</i> the fraction of NADPH-derived hydrogen in lipids that comes from photosynthesis as opposed to the OPP pathway. δ²H values of NADPH/PS1 and NADPH/OPP are set at -600‰ and -250‰, respectively. Intracellular water δ²H is set at 0‰. The shaded areas in (A) indicate the range of α values measured for 3 lipid classes (fatty acids, FA-green; alkenones-red; brassicasterol-purple) in our <i>E</i>. <i>huxleyi</i> continuous cultures (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141643#pone.0141643.t003" target="_blank">Table 3</a>). The slope of the relationship would increase if less than half of the NADPH-derived hydrogen in lipids came from photosynthesis (i.e., <i>x</i> < 0.5 In (B) it is assumed that half of the hydrogen in lipids is from NADPH and half from water (i.e., <i>f</i> = 0.5). A greater fraction of NADPH from photosynthesis (higher <i>x</i>) results in lower α values since photosynthetically produced hydride is ²H -depleted relative to NADPH produced via OPP in the cytosol. In (B) the sensitivity of α to changes in <i>x</i> decreases if either δ²H _NADPH/PS1 > -600‰ or δ²H _NADPH/OPP < -250‰.</p