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Geochimica et Cosmochimica Acta 189 (2016) 96109
www.elsevier.com/locate/gca

Eect of salinity on 2H/1H fractionation in lipids from

continuous cultures of the coccolithophorid Emiliania huxleyi
Julian P. Sachs , Ashley E. Maloney, Josh Gregersen, Christopher Paschall
School of Oceanography, University of Washington, Box 355351, Seattle, WA 98195, USA
Received 29 March 2016; accepted in revised form 25 May 2016; Available online 4 June 2016

Abstract
Salinity and temperature dictate the buoyancy of seawater, and by extension, ocean circulation and heat transport. Yet
there remain few widely applicable proxies for salinity with the precision necessary to infer all but the largest hydrographic
variations in the past. In the last decade the hydrogen isotope composition (2H/1H or d2H) of microalgal lipids has been
shown to increase systematically with salinity, providing a foundation for its use as a paleosalinity proxy. Culture and eld
studies have indicated a wide range of sensitivities for this response, ranging from about 0.63.3 ppt
1 depending on the
lipid, location and/or culturing conditions. Lacking in these studies has been the controlled conditions necessary to isolate
the response to salinity while keeping all other growth parameters constant. Here we show that the hydrogen isotope composition of lipids in the marine coccolithophorid Emiliania huxleyi grown in chemostats increased by 1.6 0.3 ppt
1
(p < 0.05) in eight individual alkenones and by 2.0 0.1 ppt
1 (p < 0.05) in three individual fatty acids over the salinity
range 20 42 ppt. Hydrogen isotope ratios of phytol and the sterol 24-methyl-cholest-5,22-dien-3b-ol (brassicasterol) also
increased with salinity but correlations were weaker than for the acetogenic lipids. For eight individual alkenones, linear
regression analyses of the fractionation factors on salinity yielded slopes of 1.22.2 ppt
1. This sensitivity of d2Halkenone
to salinity is 4571% of that previously reported for E. huxleyi, which can be attributed to the fact that previous experiments
were performed with batch cultures in which growth rates and other parameters diered between salinity treatments. The
underlying cause of this response to salinity remains unknown, but may result from changes in (1) the proportion of lipid
hydrogen derived from NADPH versus water, (2) the proportion of lipid hydrogen derived from NADPH from Photosystem
I versus the oxidative pentose phosphate pathway (and other metabolic sources), or (3) the d2H value of intracellular water.
2016 Elsevier Ltd. All rights reserved.
Keywords: Hydrogen isotopes; Hydrogen isotope fractionation; Lipid biomarkers; Marine phytoplankton; Continuous cultures;
Emiliania huxleyi; Coccolithophorid; Alkenones; Fatty acids; Salinity

1. INTRODUCTION
The salinity of seawater inuences ocean circulation and
on glacial-interglacial timescales is dictated by regional precipitation, evaporation, runo, and sea ice, and globally by
continental ice volume. Notwithstanding the importance of
this parameter in paleoceanography and paleoclimatology

Corresponding author. Tel.: +1 (206) 221 5630.

E-mail address: jsachs@uw.edu (J.P. Sachs).

http://dx.doi.org/10.1016/j.gca.2016.05.041
0016-7037/ 2016 Elsevier Ltd. All rights reserved.

there remain few reliable means to reconstruct it in the geologic record. Presently the best estimates of past ocean
salinity are reconstructed from the residuals of oxygen isotope
measurements
(d18O = (18O/16O)sample/(18O/16OVSMOW)
1, where VSMOW is Vienna Standard Mean
Ocean Water) in planktonic foraminifera after correcting
for the temperature and global ice volume signals, yielding
estimates for d18Owater (Schmidt, 1999). The derived
d18Owater values are then typically converted to salinity
based on the assumption of a constant relationship between
d18Owater and salinity. This method often has large

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

uncertainties owing to a spatially and temporally varying

relationship between water d18O and salinity (Schmidt,
1999; Rohling, 2000; Leduc et al., 2009; LeGrande and
Schmidt, 2011; Holloway et al., 2016) and the inherent
imprecision of paleotemperature estimates (Rohling,
2007). Other methods for determining salinity in the past,
such as microfossil transfer functions (de Vernal et al.,
1994, 2000, 2005; Solignac et al., 2004; Hassan et al.,
2009; Verleye and Louwye, 2010; Dickson et al., 2014;
Wu et al., 2015) or Ba/Ca ratios in planktonic foraminifera
(Weldeab et al., 2007) can yield good results but are limited
in their applicability by the need for local calibrations and
the assumption of stationarity (Telford, 2006).
Following development of techniques to measure the
hydrogen isotopic composition of individual lipids
separated by gas chromatography by Sessions et al.
in the late 1990s (Sessions et al., 1999, 2001) it became
possible to measure the hydrogen isotope composition
(d2H = (2H/1H)sample/(2H/1H)VSMOW
1) of lipid biomarkers
from marine phytoplankton. Alkenones, C36C39 methyl
and ethyl ketones, abundant in the cosmopolitan coccolithophorids Emiliania huxleyi and Gephyrocapsa oceanica
and unique to just a small number of prymnesiophytes
(Volkman et al., 1980, 1998; Marlowe et al., 1984a,b,
1990), became promising targets for directly reconstructing
d2Hwater (Englebrecht and Sachs, 2005; Pahnke et al., 2007).
Even though a large vital eect resulted in lipids from
phytoplankton being depleted in 2H relative to environmental water by some 200400 (Sessions et al., 1999), d2Hlipid
was perfectly correlated (R2 > 0.99) with d2Hwater in all
lipids measured from a wide diversity of cultured freshwater
and marine phytoplankton (Englebrecht and Sachs, 2005;
Schouten et al., 2006; Zhang and Sachs, 2007).
With publication of the seminal work by Schouten et al.
(2006) demonstrating that d2Halkenone values from batch
cultures of E. huxleyi and G. oceanica increased systematically by about 3 for every part-per-thousand increase in
salinity, Rohling (2007) proposed the use of alkenone d2H
measurements in concert with paired planktonic foraminiferal d18O and Mg/Ca-paleotemperature values to reconstruct ocean salinity with appropriate assumptions about
the slope of the local meteoric water line and the deuterium
excess (d = d2H
8*d18O) value of surface water. van der
Meer et al. (2007) applied a similar approach using alkenone paleotemperature estimates in place of foraminiferal
Mg/Ca to reconstruct Holocene salinity changes in the
Black Sea. They noted however that large uncertainties
resulted in part from indeterminate and potentially timevarying local meteoric-water-line-slopes and deuterium
excess values, but most importantly from uncertainty in
the aalkenone-water versus salinity relationship (van der
Meer et al., 2007). Subsequent eld calibrations with multiple phytoplankton and cyanobacterial lipids from globally
distributed saline lakes and a large estuary conrmed the
systematic and linear decrease in 2H/1H fractionation with
increasing salinity, but showed this relationship to be closer
to 1 ppt
1, about 1/3 of the value reported by Schouten
et al. (2006) (Sachse and Sachs, 2008; Sachs and Schwab,
2011; Nelson and Sachs, 2014a,b). Leduc et al. (2013)
used this revised estimate of the salinity eect on 2H/1H

97

fractionation in microalgal lipids to evaluate Holocene

salinity and water isotopic variations in the equatorial east
Atlantic Gulf of Guinea (Leduc et al., 2013).
Because environmental and physiological parameters
other than salinity have since been shown to inuence
2
H/1H fractionation in microalgal lipids, such as growth
rate (Schouten et al., 2006; X. Zhang et al., 2009; Sachs
and Kawka, 2015), light intensity (van der Meer et al.,
2015), growth stage (Wolhowe et al., 2009; Heinzelmann
et al., 2015b), and temperature (Z.H. Zhang et al., 2009;
Sachs, 2014), there is a need for culture experiments with
phytoplankton growing under steady-state conditions, with
constant growth rate, light, temperature and nutrient
regimes, so that the 2H/1H fractionation response to salinity can be isolated and quantied. Though several studies
have reported H isotopic changes as a function of salinity
in cultured Prymnesiophytes of the order Isochrysidales,
which contains the marine coccolithophores E. huxleyi
and G. oceanica and the coastal haptophyte Isochrysis galbana (Schouten et al., 2006; Chivall et al., 2014a,b; Mboule
et al., 2014; Heinzelmann et al., 2015a), this is the rst to do
so using the chemostat technique (Monod, 1949; Novick
and Szilard, 1950). This culturing method allows the
microalgae to be maintained at steady state in exponential
growth phase, with a stable population, and constant nutrients, water chemistry and light. In this study chemostat cultures of E. huxleyi were grown at six dierent salinities
between 20 and 42 ppt. A constant growth rate of 0.96
0.04 div d
1 was maintained in each culture and across
salinity treatments by supplying nitrate and phosphate at
a molar ratio of two (N/P = 2, compared to the optimal
ratio of 16). The results conrm a systematic linear decrease
in 2H/1H fractionation as salinity increases, averaging 1.6
0.3 ppt
1 in eight alkenones and 2.0 0.1 ppt
1 in
three fatty acids.
2. METHODS
2.1. Culture methods
Nitrogen-limited chemostats of E. huxleyi (CCMP 374)
were used to obtain cells from the widest salinity range possible while maintaining the same temperature (19 1 C),
irradiance (225 54 lmol m
2 s
1), nutrient regime
(adjusted non-silica f/2 to obtain N:P 2:1 (N/2 media)),
and growth rate. Nine chemostats were grown at 20, 22
(n = 2), 27, 32 (n = 3), 37, and 42 ppt and were initiated
with starter cultures that had been acclimated to each salinity level for at least 6 generations. The average growth rate
achieved for all chemostat cultures was 0.96 0.04 div d
1
(Fig. 1). Chemostat steady state conditions were maintained for 36 days followed by 78 days of subsampling
for daily in vitro uorescence (IVF), cell density, cell size,
chlorophyll a (Chl a) (Table EA1), and water isotopes
(Tables 1 and 2). Cell density and cell size were measured
on a Beckman Coulter Z2 particle counter calibrated with
10 lm polystyrene beads (Beckman Coulter). Chl a subsamples were taken by ltering 5 mL of culture and immediately freezing the lter (
20 C). Three samples from each
culture were analyzed for Chl a according to standard

98

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

methods (UNESCO 1994). Further details of the culture

conditions are given in Table EA2. Culture water was subsampled for water isotope analysis in 4 mL glass vials that
were taped shut to prevent evaporation. At the completion
of 78 days of steady state, each 5 L culture was harvested
onto a pre-combusted Whatman 142 mm diameter 0.7 lm
GF/F lter and immediately frozen at
20 C until lipid
extraction.
2.2. Lipid analyses

Fig. 1. Average growth rate in starter cultures and continuous

chemostats. Open squares are the average growth rates
(k = ln(N1/N0)/(T1
T0)/0.6931) for 150 mL semi-continuous
batch cultures grown in 250 mL asks used as starter cultures.
Error bars reect the standard deviation of 6 non-consecutive
generations after the acclimation period. Starter cultures grown
outside the salinity range 2042 ppt were not successful. Blue
diamonds are the average division rate for the 5 L chemostat
cultures grown in 12 L carboys; error bars reect slight variability
in pumping speed and culture volume during the last 68 days
of steady state. The dashed line represents the target division
rate (l/0.6931 = 1.0 div d
1) set by the target pumping speed
(2.4 mL/min) in continuous cultures.

Frozen lters were freeze-dried and cut into

~0.5  0.5 cm pieces for extraction. Lipids were extracted
by an Accelerated Solvent Extractor (ASE-200, Dionex
Corp.) using 9:1 dichloromethane:methanol (DCM:MeOH)
at 1500 psi and 100 C for three 5-min cycles. Total lipid
extracts (TLEs) were saponied with 1 N KOH at 60 C
for 12 h followed by phase separations with hexane. The
saponied TLE was puried using two-step column chromatography. Polar and non-polar lipids were rst separated
on a solid phase of 500 mg aminopropyl silica gel (Supelco)
and 8 mL of 3:1 DCM:isopropyl alcohol to elute the nonpolar fraction (containing the sterol, phytol, and alkenones), followed by 6 mL 4% acetic acid in diethyl ether
to elute the polar fraction containing fatty acids. The
non-polar fraction was then further separated on 1.0 g 5%

Table 1
Hydrogen isotope ratios and per-cell concentrations of fatty acids, phytol and brassicasterol from continuous cultures of E. huxleyi grown at
six salinities between 20 and 42 ppt. All d2H values and concentrations are averages of 3 measurements. Concentration values have a 1-sigma
uncertainty of approximately 13%.
Salinity (ppt)
Lab ID
Water
Brassicasterol

Phytol

C14:0

C16:0

C18:1

d2H
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD

20
1

22
2

22
3

27
1

32
5

32
6

32
10

37
6

42
9

70.7
0.08
0.31

345
2.5
0.704
0.0024
0.09

408
1.0
0.638
0.001
1.03

280
1.6
0.775
0.0017
0.38

284
2.8
0.770
0.0030
0.85

207
1.0
0.853
0.0011

74.5
0.08
0.25

353
1.9
0.699
0.0019
0.12

403
0.8
0.646
0.001
1.08

299
1.9
0.758
0.0021
0.39

280
1.8
0.778
0.0020
0.79

227
2.0
0.835
0.0021

74.4
0.02
0.30

354
3.5
0.698
0.0035
0.07

389
4.8
0.660
0.005
1.08

300
1.3
0.756
0.0014
0.36

291
0.7
0.766
0.0008
0.78

240
2.6
0.821
0.0028

75.8
0.11
0.24

365
2.6
0.687
0.0026
0.09

393
3.0
0.657
0.003
0.81

294
0.5
0.764
0.0006
0.26

295
3.3
0.763
0.0036
0.48

217
1.0
0.847
0.0011

74.4
0.18
0.23

359
2.8
0.692
0.0027
0.08

410
2.8
0.638
0.003
1.29

293
1.5
0.764
0.0016
0.41

271
3.4
0.787
0.0037
0.92

215
0.1
0.848
0.0001

73.6
0.13
0.09

355
2.3
0.696
0.0023
0.05

420
2.3
0.626
0.002
0.43

283
1.8
0.774
0.0020
0.14

289
3.8
0.767
0.0041
0.28

215
2.0
0.847
0.0022

67.3
0.06
0.25

351
2.4
0.696
0.0023
0.04

368
2.5
0.677
0.002
1.02

273
1.6
0.780
0.0018
0.31

285
1.6
0.766
0.0017
0.51

209
3.3
0.848
0.0036

74.0
0.25
0.41

344
4.6
0.709
0.0046
0.11

380
4.1
0.670
0.004
1.39

261
2.4
0.798
0.0026
0.38

266
0.9
0.793
0.0010
0.84

195
2.3
0.869
0.0025

73.7
0.17
0.11

336
1.9
0.717
0.0019
0.04

354
2.1
0.698
0.002
0.29

249
1.9
0.811
0.0020
0.10

234
2.4
0.827
0.0026
0.22

183
1.5
0.882
0.0016

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

99

Table 2
Hydrogen isotope ratios and per
cell concentrations of alkenones from continuous cultures of E. huxleyi grown at six salinities between 20
and 42 ppt. All d2H values and concentrations are averages of 3 measurements. Concentration values have a 1-sigma uncertainty of
approximately 13%.
Salinity (ppt)
Lab ID
Water
C37:2 Me

C37:3 Me

C38:2 Me

C38:3 Me

C38:2 Et

C38:3 Et

C39:2 Et

C39:3 Et

d2H
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD
[CL] pg cell
1
d2H
SD
a
SD

20
1

22
2

22
3

27
1

32
5

32
6

32
10

37
6

42
9

70.7
0.08
0.87

247
4.2
0.810
0.0045
0.49

267
1.8
0.788
0.0019
0.08

242
0.3
0.815
0.0003
0.16

260
0.5
0.796
0.0006
0.21

257
2.6
0.799
0.0028
0.16

262
1.7
0.794
0.0018
0.04

253
1.6
0.804
0.0018
0.04

261
2.3
0.795
0.0025

74.5
0.08
0.97

241
2.9
0.820
0.0032
0.61

256
1.4
0.804
0.0015
0.14

244
3.5
0.817
0.0038
0.27

262
2.2
0.797
0.0023
0.42

254
2.4
0.807
0.0026
0.29

265
1.0
0.794
0.0011
0.08

254
2.2
0.806
0.0024
0.06

260
2.7
0.800
0.0029

74.4
0.02
0.90

240
0.8
0.822
0.0009
0.40

262
4.9
0.797
0.0053
0.07

243
3.3
0.818
0.0035
0.19

260
2.6
0.800
0.0028
0.20

255
3.0
0.805
0.0032
0.20

264
1.7
0.795
0.0018
0.05

255
3.4
0.805
0.0036
0.05

263
4.2
0.796
0.0045

75.8
0.11
0.63

239
4.4
0.823
0.0048
0.29

257
4.3
0.803
0.0046
0.07

232
3.8
0.831
0.0041
0.17

257
3.4
0.803
0.0036
0.21

250
1.9
0.812
0.0021
0.20

258
0.8
0.803
0.0009
0.05

247
2.3
0.815
0.0025
0.05

261
0.7
0.800
0.0007

74.4
0.18
0.81

236
3.1
0.826
0.0033
0.54

251
3.5
0.809
0.0037
0.10

238
2.6
0.824
0.0018
0.18

256
3.1
0.804
0.0044
0.32

253
0.3
0.808
0.0004
0.21

251
0.7
0.809
0.0002
0.06

248
3.5
0.813
0.0028
0.05

247
4.1
0.814
0.0044

73.6
0.13
0.45

240
1.7
0.821
0.0019
0.24

242
4.6
0.818
0.0050
0.12

235
3.3
0.826
0.0035
0.27

253
4.5
0.807
0.0048
0.39

248
3.7
0.812
0.0040
0.29

255
2.6
0.804
0.0028
0.08

248
4.4
0.811
0.0047
0.07

252
4.5
0.808
0.0049

67.3
0.06
0.58

227
1.8
0.829
0.0019
0.36

245
2.2
0.810
0.0024
0.06

220
4.0
0.837
0.0043
0.11

244
3.0
0.811
0.0032
0.23

239
1.9
0.816
0.0021
0.16

241
3.1
0.814
0.0033
0.04

231
2.1
0.824
0.0023
0.04

240
1.9
0.815
0.0021

74.0
0.25
0.84

219
1.6
0.844
0.0017
0.43

238
4.3
0.823
0.0046
0.06

203
3.5
0.860
0.0038
0.12

234
4.3
0.827
0.0064
0.24

229
2.5
0.833
0.0027
0.19

233
0.9
0.828
0.0009
0.05

223
2.8
0.840
0.0031
0.05

229
2.2
0.832
0.0024

73.7
0.17
0.38

215
2.6
0.847
0.0028
0.14

233
2.3
0.828
0.0024
0.04

198
2.8
0.866
0.0030
0.12

230
4.1
0.832
0.0045
0.25

234
3.4
0.826
0.0037
0.26

239
1.0
0.822
0.0011
0.04

222
2.5
0.839
0.0027
0.05

233
2.6
0.828
0.0028

H2O deactivated silica gel 60 (4063 lm, BDH Chemicals).

Hydrocarbons were eluted with 10 mL hexane, ketones
with 6 mL of DCM:hexane (1:1), alcohols with 8 mL ethyl
acetate:hexane (1:4), followed by a polar fraction with 6 mL
methanol.
The aminopropyl gel polar fraction was methylated by
dissolving the samples in 1 mL hexane with 2 mL 10:1
MeOH:acetyl chloride and heating for 12 h at 60 C followed by phase separation with hexane. The silica gel alcohol fraction was acetylated by dissolving in 20 lL of
pyridine plus 20 lL of acetic anhydride and heating at
70 C for 0.5 h. Lipid identications were made by comparing sample mass spectra generated by gas chromatographymass spectrometry (GCMS) to previously published EI

spectra and laboratory standards. Identication analyses

were performed using an Agilent GC 6890N connected to
an Agilent quadrupole MSD5975 detector and equipped
with an Agilent 7683 autosampler, a split-splitless injector
operated in splitless mode, and a VF-17 ms capillary column (60 m  0.25 mm  0.25 lm, Agilent) used with He
carrier gas at 1.5 mL/min with the oven ramped from
110 C to 150 C at 15 C/min, then ramped to 320 C at
6 C/min and held for 24 min (Table EA3).
Quantications were made with gas chromatographyame ionization (GC-FID) by comparing analyte peak
area to a known amount of 5a-cholestane added to
the sample. Analyses were performed on an Agilent
6890N GC equipped with an Agilent 7683 autosampler, a

100

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

programmable temperature vaporization inlet operated in

splitless mode, and an Agilent DB-5 ms capillary column
(60 m  0.32 mm  0.25 lm) used with He carrier gas
(1.3 mL/min). The oven temperature held at 110 C for
4 min, increased from 110 C to 150 C at 15 C/min and
then increased to 320 C at 6 C/min and held for 28 min
(Table EA4).
2.3. Water isotope measurements
d2H and d18O values of water were measured six times
per sample (the rst three measurements were discarded
to avoid memory eects) with a Picarro L2130-i Isotopic
Liquid Water Analyzer in high precision mode and normalized to VSMOW using lab standards. The average d2H precision of the samples was 0.19.
2.4. Lipid 2H/1H measurements
Lipid d2H values were measured with a gas-chromatograph
coupled to an isotope-ratio mass spectrometer (GC-IRMS).
Lipid samples were injected by Triplus autosampler
(Thermo Scientic) into a Trace GC Ultra II (Thermo
Scientic) gas chromatograph operated in splitless mode
at 325 C. Eluting compounds were pyrolyzed in a
1400 C ceramic reactor and the resultant H2 gas
introduced into a Thermo DELTA V PLUS IRMS
(Thermo Scientic). For polar fractions the GC was
equipped with an Agilent DB-5 ms capillary column
(60 m  0.32 mm  0.25 lm, 1.5 mL/min He) to achieve
baseline separation between fatty acid methyl esters. The
oven was programmed to hold at 120 C for 10 min,
increase to 180 C at 20 C/min, increase to 325 C at
3 C/min, and hold 325 C for 11 min. The GC was
equipped with an Agilent VF-17 ms capillary column

(60 m  0.25 mm  0.25 lm, 1.1 mL/min He) for acetylated alcohol fractions containing the sterol and phytol.
The oven was programmed to hold 120 C for 10 min,
increase to 220 C at 20 C/min, increase to 300 C at
2 C/min, increase to 325 C at 10 C/min, and hold 325 C
for 5 min. The GC was equipped with an Agilent VF200 ms capillary column (60 m  0.25 mm  0.25 lm,
1.5 mL/min He) for ketone fractions containing the alkenones.
As shown in Fig. 2, Baseline separation of all 8 alkenones
was achieved using GC methods adapted from Longo
et al. (2013). The oven was programmed to hold 120 C
for 1 min, increase to 255 C at 15 C/min, increase to
300 C at 1 C/min, increase to 320 C at 10 C/min, and
hold 325 C for 10 min. External standards of known
hydrogen isotopic composition including nC26-alkane
(
54.9, CAS # 630-01-3), nC32-alkane (
212.4, CAS
# 544-85-4), nC38-alkane (
102.6, CAS # 7194-85-6)
and nC44-alkane (
199.9, CAS # 7098-22-8) (Dr. Arndt
Schimmelmann, Indiana University, http://pages.iu.edu/
~aschimme/les/list%20of%20n-alkanes.pdf) were injected
throughout each run and used to correct lipid data as
described in detail in (Nelson and Sachs, 2014a). Fatty
acids were corrected for methylation and sterol for acetylation by mass balance. The precision of d2H measurements
of external standards through the course of this study was
3.6. Detailed parameters for GC-irMS runs are provided
in Table EA5.
3. RESULTS
3.1. Cellular lipid concentrations
Per cell concentrations of the 13 lipids analyzed varied
between 0.04 and 1.4 pg cell
1 with no statistically signicant
co-variations with salinity (i.e., 0.1 < p < 0.4) (Tables 1 and 2).

Fig. 2. Chromatograms from GC-irMS for E. huxleyi chemostat culture ESN42.9 grown at a salinity of 42 ppt. Baseline separation of all 8
alkenones was routinely achieved. The top panel is the ratio of m/z 3 (2H1H) to m/z 2 (1H1H). The middle panel is the intensity of the m/z 2
ion. The bottom panel is the intensity of the m/z 3 ion. The peak identications are shown in the middle panel along with the peak areas. Peaks
less than 12 Vs were rerun at higher concentrations to obtain accurate d2H values. All cultures had similar chromatograms.

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

101

not vary systematically with salinity (Fig. 3). This equates

to a temperature of 18.1 1.2 C using the Prahl et al.
(1988) Uk
37 temperature calibration (Prahl et al., 1988). This
compares favorably to a growth temperature of 19 1 C
for all cultures.
3.2. d2H values of lipids

Fig. 3. Uk
37 values and associated temperatures using the Prahl
et al. (1988) temperature calibration were unaected by salinity.
They averaged 0.654 0.042 and 18.1 1.2 C (N = 9), respectively. The latter compares favorably to the actual temperature at
which the cultures were maintained, 19 1 C (black arrow).

Fatty acids were between 0.1 and 1.4 pg cell
1 with myristic
acid (C14:0) generally in highest abundance, followed by
oleic acid (C18:1) and palmitic acid (C16:0). Per cell concentrations of phytol were between 0.04 and 0.12 pg cell
1 while
those of 24-methyl-cholest-5,22-dien-3b-ol (brassicasterol)
were between 0.09 and 0.41 pg cell
1.
Of the four pairs of alkenone isomers (C37Me, C38Me,
C38Et, C39Et) the C37 methyl alkenones were in highest
abundance, with C37:2Me between 0.38 and 0.97 pg cell
1
and C37:3Me between 0.14 and 0.61 pg cell
1. Concentrations of C38:3Et, the next most abundant alkenone, were
between 0.16 and 0.29 pg cell
1, while those of C38:2Et
were between 0.2 and 0.42 pg cell
1. In next highest abundance C38:3Me varied in concentration between 0.11 and
0.27 pg cell
1, followed by C38:2Me which varied between
0.04 and 0.14 pg cell
1. In lowest abundance were the C39
ethyl alkenones, which varied between 0.04 and
0.08 pg cell
1.
The alkenone unsaturation ratio commonly used
for sea surface temperature reconstructions, Uk
37 (=C37:2/
(C37:2 + C37:3)) averaged 0.654 0.042 (N = 9) and did

Fatty acid d2H values were between
240 and
183 in

C18:1 and between
300 and
234 in C14:0 and C16:0
(Table 1). When accounting for the d2H values
of water in the growth media, the fractionation factor
a (= (d2Hlipid + 1000)/(d2Hwater + 1000)) was between
0.821 and 0.882 for C18:1 and between 0.756 and 0.827
for C14:0 and C16:0. Fractionation factors for all three
fatty acids were positively correlated with salinity at the
95% condence level, with regression slopes averaging
1.99 0.08 ppt
1 (Table 3 and Fig. 4).
d2H values of phytol and brassicasterol were between

420 to
354 and
365 to
336, respectively
(Table 1). Fractionation factors (alipid-water) were between
0.6260.698 and 0.6870.717 for phytol and brassicasterol,
respectively (Table 1). Although 2H/1H fractionation
decreased with salinity in both lipids neither regression
was signicant at the 95% condence level (i.e., p = 0.10
and 0.19 for phytol and brassicasterol, respectively)
(Table 3).
The d2H values of all eight alkenones were depleted
relative to water in the culture media by 134212
(i.e., 0.866 > a > 0.788) and positively correlated with salinity at the 95% condence level (Tables 2 and 3, Fig. 4). d2H
in 7 of the 8 alkenones (C37:2Me, C37:3Me, C38:3Me,
C38:2Et, C38:3Et, C39:2Et, C39:3Et) increased by 1.2
1.7 ppt
1, while in C38:2Me d2H increased by 2.2 ppt
1.
Tri-unsaturated alkenones were 2H-depleted relative to diunsaturated alkenones by about 1020, C37Me alkenones
were 2H-enriched relative to C38Et alkenones of the same
saturation state by about 36, and both of the former were
2
H-enriched relative to C38Me and C39Et alkenones of the

Table 3
Linear regression statistics for a as a function of salinity in 13 E. huxleyi lipids. All regressions were signicant at the 95% level (i.e., p < 0.05)
except for phytol (p = 0.10) and brassicasterol (p = 0.19).
Lipid

R2

p level

Slope (ppt
1)

Slope SE (ppt
1)

Intcpt

Intcpt SE

C37:2Me
C37:3Me
C38:2Me
C38:3Me
C38:2Et
C38:3Et
C39:2Et
C39:3Et
Alkenones (N = 8)

0.80
0.89
0.80
0.86
0.75
0.85
0.81
0.87

0.7847
0.7621
0.7663
0.7615
0.7765
0.7612
0.7676
0.7593

0.00808
0.00647
0.01297
0.00734
0.00814
0.00736
0.00937
0.00757

9
9
9
9
9
9
9
9

0.68
0.54
0.63

0.00053
0.00071
0.00055

0.7149
0.7201
0.7938

0.01599
0.02143
0.01665

9
9
9

Sterol
Phytol

0.23
0.34

0.00142
0.00159
0.00225
0.00159
0.00124
0.00155
0.00169
0.00171
0.00163
0.00029
0.00205
0.00202
0.0019
0.00199
0.00008
0.00059
0.00178

0.00027
0.00021
0.00043
0.00024
0.00027
0.00024
0.00031
0.00025

C14:0
C16:0
C18:1
Fatty acids (N = 3)

0.0011
0.0001
0.0012
0.0003
0.0024
0.0004
0.0009
0.0002
Avg
SD
0.0059
0.0243
0.0104
Avg
SD
0.1928
0.0992

0.00041
0.00094

0.6824
0.6039

0.01245
0.02846

9
9

(1.63)
(0.29)

(1.99)
(0.08)

102

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

Fig. 4. Hydrogen isotope fractionation (a) factors for 8 alkenones, 3 fatty acids, one sterol, and phytol from chemostats of E. huxleyi. Three
separate chemostat experiments were conducted at 32 ppt, and two at 22 ppt salinity. All values are averages of 3 measurements (i.e., N = 3
for all analyses). Error bars represent the propagated error for the lipid and water and are smaller than the symbol in some cases. The d2H
dierence between water and lipid, the per mil enrichment factor e (= (1
a)*1000), is shown on the right axis.

same saturation state by about 416 (Fig. 4). These ndings are consistent with those reported for suspended particulate alkenones from the Chesapeake Bay estuary (Schwab
and Sachs, 2009, 2011).
4. DISCUSSION
4.1. Mechanisms responsible for greater 2H/1H fractionation
at higher salinity
Before the application of continuous cultures, prior
explanations for the decrease in 2H/1H fractionation in
microalgal lipids with increasing salinity in culture and eld
studies fell into two main categories. The rst and most
widely discussed in the literature calls upon an increase in
the d2H value of intracellular water by one or more of several possible mechanisms as salinity increases, and the subsequent transfer of this 2H-enrichment to all lipids either
directly from water or indirectly via reduced nicotinamide
adenine dinucleotide phosphate (NADPH) or nicotinamide
adenine dinucleotide (NADH) (Sachse and Sachs, 2008;
Sachs and Schwab, 2011; Heinzelmann et al., 2015a). This
explanation is attractive because it can account for the fact
that an increase in alipid-water is observed in all lipid classes
from both prokaryotes and eukaryotes as salinity increases
(Sachse and Sachs, 2008). The second explanation called
upon a decrease in growth rate, and consequent increase
in alipid-water, as salinity increased (Sachs and Schwab,
2011). The continuous culture results reported here rule
out the possibility that growth rate changes are the sole
cause of the fractionation response to salinity since they
were held constant across treatments.

At the most basic level the hydrogen in lipids can be considered to derive from two sources, NAD(P)H and intracellular water. Although strictly speaking acetogenic lipids
such as fatty acids and alkenones derive about 50% of their
hydrogen directly from NAD(P)H, 25% from cell water,
and 25% from acetyl-CoA (Saito et al., 1980; Robins
et al., 2003; Schmidt et al., 2003; X. Zhang et al., 2009),
the acetyl hydrogens in the latter ultimately derive from
glyceraldehyde 3-phosphate (GAP), via pyruvate, which
receives its hydrogen from water and NADPH. Similarly,
isoprenoid lipids derive 2040% of their hydrogen directly
from NADPH and the remainder from either acetyl-CoA
(MVA pathway) or pyruvate and GAP (DOXP/MEP pathway) (Sessions et al., 2002), and therefore from water and
NADPH.
NADP+ is reduced by ferredoxin-NADP+ reductase
in Photosystem 1 (PS1) of photosynthesis, resulting
in highly 2H-depleted NADPH (approximately
600
(Luo et al., 1991; Schmidt et al., 2003)). It can also be
reduced by glucose-6-phosphate dehydrogenase and
6-phosphogluconate in the cytosolic oxidative pentose
phosphate pathway (OPP), resulting in NADPH with a
higher d2H value than that from photosynthesis because it
acquires hydride from hexose metabolites rather than
photooxidized water (Hayes, 2001; Schmidt et al., 2003).
The relative proportions of hydrogen from intracellular
water and NADPH on the one hand, and NADPH from
either PS1 or OPP on the other, can be expressed with
the simple mass balance model:
d2 H lipid f  d2 H NADPH =PS1  x d2 H NADPH =opp 1
x
1
f  d2 H water

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

where f is the fraction of hydrogen in lipids that comes from

NADPH and x is the fraction of that NADPH produced in
PS1 of photosynthesis (Fig. 5) (Sachs and Kawka, 2015).
According to this model, when fractionation factors for
PS1- and OPP-derived NADPH are assumed to be 0.4
and 0.75, respectively, and 50% of the hydrogen in
lipids is assumed to come from NADPH and intracellular
water (Schmidt et al., 2003; X. Zhang et al., 2009),
alipid-water = 0.8 (Sachs and Kawka, 2015), close to the
average for alkenones and fatty acids in our E. huxleyi
cultures (Fig. 4). Based on this simple model higher salinity
would result in higher lipid d2H values if it caused (1) a
decrease in the proportion of hydrogen in lipids coming
from NADPH relative to water (f) (2) a decrease in the
proportion of NADPH coming from PS1 relative to
OPP (x), or (3) an increase in d2Hwater. These three mechanisms are discussed below.
4.1.1. Mechanism 1
The proportion of hydrogen in lipid precursors that
derives from water (i.e., 1
f in Eq. 1) versus NAD(P)H is
likely to be sensitive to environmental conditions such as
salinity to the extent that such conditions inuence central
metabolic pathways of the cell. For instance, the methyl
group of pyruvate likely derives more of its hydrogen from
water when it is synthesized via malic enzyme acting on
malate from the TCA Cycle, compared to the methyl group
of pyruvate that comes from pyruvate kinase during glycolysis (Buchanan et al., 2015). Pyruvate that is generated
from fumarate and malate gets at least two of three hydrogens directly from (relatively 2H-enriched) cell water
(Fig. 6a), while pyruvate generated from GAP during glycolytic reactions may only get one (Fig. 6b). Thus a simple
increase in the fraction of pyruvate for lipids that is derived
from malic enzyme in the TCA cycle versus pyruvate kinase
in glycolysis would be expected to cause an increase in
d2Hlipid. Feakins and Sessions (2010) came to a similar
conclusion in a study of 2H/1H fractionation in leaf wax
n-alkanes from plants capable of crassulacean acid metabolism (CAM) (Feakins and Sessions, 2010). Evidence from

Fig. 5. Model of hydrogen isotopic relationships giving rise to

observed d2H values of lipids in E. huxleyi cells. f is the fraction of
hydrogen in lipids that comes from NADPH, 1
f is the fraction
of hydrogen in lipids that comes from water, x is the fraction of
hydrogen in lipids derived from PS1 of photosynthesis, 1
x is the
fraction of hydrogen in lipids derived from the OPP pathway.
Figure adapted from (Sachs and Kawka, 2015).

103

higher plants indicates that increased malic enzyme gene

expression and activity may play a role in the response to
salt (Fushimi et al., 1994; Farias de Aragao et al., 1997;
Sun et al., 2003; Cheng and Long, 2007; Liu et al., 2007).
Given that regulation of these enzymes is sensitive to a multitude of intra- and extracellular conditions (Smith et al.,
2012; Bender et al., 2014) it seems plausible that higher
ionic strength of extracellular water could alter this balance
in phytoplankton as well.
4.1.2. Mechanism 2
As pointed out by several researchers, a second possible
cause of lipid 2H-enrichment at higher salinity would be
increased proportions of metabolically produced NAD(P)
H relative to photosynthetic NADPH (X. Zhang et al.,
2009; Osburn et al., 2011; Rai et al., 2013; Dirghangi and
Pagani, 2013a,b; Gamarra and Kahmen, 2015; Sachs and
Kawka, 2015; Heinzelmann et al., 2015a). Increased expression of genes encoding for enzymes involved with OPP have
been observed in a diatom culture grown at high salinity
(Cheng et al., 2014) and increased activity of OPP enzymes
has been observed in bacterial cells (Danevcic and Stopar,
2011) and other micro- and macro-algae grown at high
salinity (Liska et al., 2004; Danevcic and Stopar, 2011;
Rai et al., 2013, 2014; Huan et al., 2014). Whether NAD
(P)H production via OPP or other enzymes increases relative to that via PS1 in E. huxleyi at higher salinity remains
to be demonstrated.
4.1.3. Mechanism 3
A third mechanism by which lipids may become
enriched in 2H as salinity increases is via an increase in d2Hwater. Kreuzer-Martin et al. demonstrated that intracellular water from both prokaryotic and heterotrophic cells can
dier isotopically from extracellular water (Kreuzer-Martin
et al., 2006; Kreuzer et al., 2012). Reduced transport of
environmental water into cells, resulting in increased recycling of intracellular water, was previously hypothesized
as a mechanism for decreased apparent 2H/1H fractionation in microalgal lipids at higher salinities (Sachse and
Sachs, 2008; Sachs and Schwab, 2011; Heinzelmann et al.,
2015a). Decreasing transport of environmental water across
the cell membrane can be achieved by increasing the osmolality of the cytoplasm (e.g., by enhanced osmolyte production) (Bisson and Kirst, 1995; Yancey, 2005) or via
transcriptional down-regulation of aquaporins (Hasegawa
et al., 2000; Boursiac et al., 2005; Horie et al., 2011).
Another means by which intracellular water might
become 2H-enriched is by an increase in the exudation of
organic hydrogen from cells. E. huxleyi and other phytoplankton taxa can exude up to 76% of their photosynthetically xed carbon depending on growth conditions and
species (Van Oostende et al., 2013; Thornton, 2014), with
higher values associated with sub-optimal growth conditions (Zlotnik and Dubinsky, 1989; Alcoverro et al., 2000;
Abdullahi et al., 2006) such as high salinity (Marsalek
and Rojckova, 1996; Liu and Buskey, 2000; Abdullahi
et al., 2006; Zhang et al., 2011; Aslam et al., 2012;
Steele et al., 2014). With a = 0.4 (Luo et al., 1991) the
photosynthetic oxidation of water ought to leave the

104

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

Fig. 6. Schematic outlining hydrogen sources for the methyl group of pyruvate from dierent pathways. In (a) at least 2 of 3 methyl
hydrogens are from intracellular water when pyruvate is synthesized via malic enzyme with malate derived from the TCA cycle: the enzyme
fumarase adds 1 hydrogen from water and then the synthesis of pyruvate via malic enzyme adds another hydrogen from cell water. In (b) at
least 1 of 3 methyl hydrogens come from cell water during glycolytic production of pyruvate: glyceraldehyde 3-phosphate generated from
glucose during glycolysis follows several steps in conversion to phosphoenoylpyruvate and maintains the methylene group with 2 hydrogens
originally from glucose. The synthesis of pyruvate from phosphoenolpyruvate via the enzyme pyruvate kinase adds one hydrogen from the cell
water and should therefore be relatively more 2H-depleted compared to malate-derived pyruvate.

intracellular (or at least the plastidial) water pool enriched

in 2H. If highly 2H-depleted NADP-H from PS1 is exported
from the cell by exudates it could result in 2H-enrichment of
cell water.
4.2. Relationship of chemostat results to other culture studies
The only two other culture studies reporting on the eect
of salinity on 2H/1H fractionation in E. huxleyi found a sensitivity of aalkenone-water to salinity of 2.1 ppt
1 (Mboule
et al., 2014) and 3.3 ppt
1 (Schouten et al., 2006). Both
of those studies were performed with non-steady-state
batch cultures and reported d2H values only for the combined di- and tri-unsaturated C37Me alkenones. The sensitivity of aalkenone-water to salinity in our chemostats was 1.4
(0.3) and 1.6 (0.2) ppt
1, respectively, for C37:2Me
and C37:3Me (Table 3), both signicantly lower than for
either of the batch culture studies. Because growth rate
has been shown to exert a strong 2H/1H fractionation
response of 2438 (div d
1)
1 in alkenones from continuous cultures of E. huxleyi (Sachs and Kawka, 2015), and
it was uncontrolled in the batch experiments, a direct comparison with the results from chemostat is not possible.
Rather we note that all the culture experiments with E. huxleyi and other alkenone producing prymnesiophytes
(Mboule et al., 2014; Chivall et al., 2014a; Heinzelmann
et al., 2015a) indicate a decrease in 2H/1H fractionation
with increasing salinity and interpret the results from chemostats reported here as representing the 2H/1H fractionation response directly attributable to salinity in E. huxleyi.

4.3. Lower sensitivity of alipid-water to salinity in isoprenoid

lipids
The slopes of alipid-water vs salinity for sterols from E.
huxleyi (0.6 ppt
1 for 24-methyl-cholest-5,22-dien-3b-ol)
cultures were lower than those for acetogenic lipids in the
same cultures that averaged 1.7 0.3 ppt
1 in (N = 11,
consisting of 8 alkenones and 3 fatty acids) (Fig. 4). The
slope of alipid-water vs. salinity for brassicasterol was statistically lower at p < 0.05 (1-tailed t-test, 16 df) than that for 7
of 8 alkenones (all except C37:2Me and C38:2Et) and all 3
fatty acids. Because alipid-water for phytol in E. huxleyi, the
only other isoprenoid lipid analyzed, was similar to that
for acetogenic lipids (i.e., 1.8 vs 1.7 ppt
1; Table 3) it cannot be concluded that the lower sensitivity of alipid-water to
salinity characterizes isoprenoid lipids in general, however
the dierence in (alipid-water vs salinity) slopes between phytol and brassicasterol was not statistically dierent at
p < 0.05 (1-tailed t-test, 16 df).
Sterols can be synthesized by two independent pathways
in phytoplankton: the mevalonate (MVA) pathway that
operates in the cytosol and is present in all eukaryotes
and the methylerythritol 4-phosphate (MEP) pathway that
operates in the chloroplast of plants and many algae
(Dubey et al., 2003; Eisenreich et al., 2004; Lohr et al.,
2012). While cyanobacteria and the majority of primary
endosymbiotic phototrophs (e.g., chlorophytes) studied to date
use the MEP pathway exclusively for isoprenoid synthesis, most
secondary endosymbiotic microalgae, including E. huxleyi,
maintain the use of both pathways (Lohr et al., 2012).

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

Even though the two isoprene synthetic pathways are

localized in dierent cellular compartments (i.e., the plastid
for MEP and the cytosol for MVA) they operate simultaneously to provide IPP for the wide range of isoprenoidal primary and secondary metabolites throughout the cell
(Rodrguez-Concepcion, 2006; Hemmerlin et al., 2012). In
addition to multiple signaling pathways and feedbacks regulating the production of IPP by one or the other pathway
there is shuttling, or cross-talk, of IPP across the plasmid
membrane (Hemmerlin et al., 2003; Schuhr et al., 2003;
Bartram et al., 2006; Rodrguez-Concepcion, 2006;
Skorupinska-Tudek et al., 2008; Lohr et al., 2012;
Mendoza-Poudereux et al., 2015). It is now established that
the pathway from which terpenoid precursors derive and
the extent of their transfer between cell compartments is
dynamically controlled in plants and that this control is
post-translational, responding to a wide range of extraand intra-cellular stimuli (Bartram et al., 2006;
Hemmerlin, 2013) such as those brought on by light, temperature or water stress (Rodrguez-Concepcion, 2006;
Vickers et al., 2009; Hemmerlin, 2013). Although to our
knowledge it has not yet been demonstrated it seems reasonable to hypothesize that the proportion of IPP incorporated into microalgal sterols from the MVA and MEP
pathways can be modulated in response to osmotic stress.
Zhang and Sachs (2007) and Z.H. Zhang et al. (2009)
hypothesized that cross-talk of isoprenoid precursors
between the chloroplast and the cytosol was responsible
for the greater 2H/1H fractionation in 24-methyl-cholesta5,24(28)-dien-3b-ol from continuous cultures of Thalassiosira pseudonana grown at high vs. low growth rates in
response to nitrogen deprivation (Zhang and Sachs, 2007;
Z.H. Zhang et al., 2009). They reasoned that a large hydrogen isotope eect associated with the nal reduction step in
IPP synthesis via the MEP pathway would result in terpene
precursors that were depleted in 2H compared to those
derived from the MVA pathway. A similar mechanism
might explain the lower sensitivity of alipid-water to salinity
in sterols from E. huxleyi compared to acetogenic lipids
that have just one primary synthesis pathway. If less
MEP-derived IPP (or more MVA-derived IPP) ends up in
sterols at low salinity as compared to high salinity it would
be expected to increase the d2H values of sterols thus produced, and vice versa. Alternatively, the fact that the highest d2H values in brassicasterol, and thus minimum 2H/1H
fractionation, occur at the two highest and lowest salinities
(Fig. 4) would be consistent with an optimal salinity of 27
32 for the coastal strain of E. huxleyi we cultured (CCMP
374 from the Gulf of Maine) and osmotic-stress-induced
repression of IPP synthesis from MEP or activation of
IPP synthesis from MVA outside that range. This hypothesis could be evaluated with cultures of a chlorophyte since
they use the MEP pathway exclusively for the synthesis of
sterols (Schwender et al., 2001).
4.4. Implications for paleoclimate reconstructions
With these chemostat results there is now strong evidence that increasing salinity, independent of all other environmental parameters, causes an increase in the hydrogen

105

isotopic composition of alkenones from the coccolithophorid E. huxleyi of 1.6 0.3 ppt
1 (Fig. 4). This
sensitivity of alkenone d2H to salinity is about half the
3.03.3 ppt
1 value reported by Schouten et al. (2006).
Because it is derived from chemostat cultures it is suggested
that the lower sensitivity be used in paleosalinity reconstructions, such as those proposed by Rohling (2007), van
der Meer et al. (2007) and Leduc et al. (2013). In addition,
owing to the range of salinity sensitivities for eight dierent
alkenones, which ranged from 1.2 to 2.2 ppt
1 in this
study, it is suggested that the d2H values of individual alkenones be used in paleosalinity calculations, rather than mixtures of alkenones (as recommended in (van der Meer et al.,
2013)).
An additional outcome of this study is conrmation that
there is no relationship between salinity and alkenonederived temperature estimates. Based on the Prahl et al.
(1988) calibration, Uk
37-derived temperatures averaged
0.654 0.042 and 18.1 1.2 C, respectively, for the 9 cultures across the 22 ppt range of salinity (Fig. 3). This value
was similar to the 19 1 C temperature at which the cultures were maintained. Paleotemperature reconstructions
from alkenone unsaturation ratios are therefore expected
to be unaected by salinity changes, at least where
E. huxleyi is the primary alkenone producer.
5. CONCLUSION
With the use of chemostat cultures the direct eect of
salinity on the d2H values of alkenones, fatty acids, phytol,
and a sterol in E. huxleyi has been determined. d2H values
of alkenones increased linearly with salinity by 1.6
0.3 ppt
1 over the salinity range 2042 ppt. Seven of
the eight alkenones synthesized by this species (C37:2Me,
C37:3Me, C38:3Me, C38:2Et, C38:3Et, C39:2Et, C39:3Et)
had d2H values that increased with salinity by 1.2
1.7 ppt
1, while one (C38:2Me) had d2H values that
increased by 2.2 ppt
1, all signicant at the 95% condence level. Tri-unsaturated alkenones were 2H-depleted
relative to di-unsaturated alkenones by about 1020,
C37Me alkenones were 2H-enriched relative to C38Et alkenones of the same saturation state by about 36, and
both of the former were 2H-enriched relative to C38Me
and C39Et alkenones of the same saturation state by about
416.
d2H values of all three fatty acids produced by this species (myristic acid, C14:0; palmitic acid, C16:0; oleic acid,
C18:1) also increased linearly with salinity, by 2.0
0.1 ppt
1 (p < 0.05). Neither phytol nor brassicasterol
had a statistically signicant change in d2H with salinity.
Possible causes of the greater enrichment of 2H in lipids
at higher salinity include an increased proportion of hydrogen from cellular water at the expense of NAD(P)H,
increased proportions of metabolically-produced NAD(P)
H relative to photosynthetic NADPH, and an increase in
the d2H value of intracellular water.
Irrespective of the specic mechanism underlying the
salinity response there is now a wealth of empirical evidence
from culture and eld studies to support the use of
d2Halkenone values to reconstruct paleosalinities. Doing so

106

J.P. Sachs et al. / Geochimica et Cosmochimica Acta 189 (2016) 96109

will require either an independent estimate of d2Hwater or

potentially the measurement of two dierent lipid d2H
values so that both variables, salinity and d2Hwater, can be
determined.
ACKNOWLEDGMENTS
This material is based upon work supported by the National
Science Foundation under Grant No. OCE-1027079 (J.P.S.). We
would like to thank P. MacCready for lending lab space, V. Armbrust and A. Ingalls for lending equipment, K. Hemeon for assisting with culture maintenance and cell counts, J. Huynh for lipid
purication assistance, and A. Morello for analyzing chlorophyll.
We are grateful for conversations with V. Armbrust, M. Behrenfeld, M. Wolhowe, and O. Kawka that improved this manuscript,
and for the comments of reviewer S. Feakins, two anonymous
reviewers, and Associate Editor A. Pearson.

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.gca.2016.05.041.
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