Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue9.2000:

Organic Geochemistry 31 (2000) 799±811
www.elsevier.nl/locate/orggeochem

Physiological responses of lipids in Emiliania huxleyi and
Gephyrocapsa oceanica (Haptophyceae) to growth status and
their implications for alkenone paleothermometry
Masanobu Yamamoto a,*, Yoshihiro Shiraiwa b, Isao Inouye b
a

Department of Mineral and Fuel Resources, Geological Survey of Japan, 1-1-3 Higashi, Tsukuba, Ibaraki 305-8567, Japan
b
Institute of Biological Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan
Received 4 January 2000; accepted 7 June 2000
(returned to author for revision 11 April 2000)

Abstract
The physiological responses of alkenone unsaturation indices to changes in growth status of E. huxleyi and G.
oceanica strains isolated from a water sample of the NW Paci®c were examined using an isothermal batch culture
0
system. In both E. huxleyi and G. oceanica the unsaturation index UK
37 changed during the growth period, but the

e€ects of this change were di€erent. This suggests that genotypic variation rather than the adaptation of the strains to
the geographical environment of the sampling location is a major factor in determining the physiological responses to
0
K0
UK
37 . Changes of U37 were associated with those of the unsaturation indices of C38 and C39 alkenones, the abundance
ratios of lower to higher homologues of alkenones, the abundance ratios of saturated to polyunsaturated n-fatty acids,
the abundance ratio of ethyl alkenoate to alkenones, and sterol contents. These associations might be attributable to
the physiological response of lipids for maintaining their ¯uidity. The degree of unsaturation both in alkenones and nfatty acids increased at day 8, possibly due to nutrient depletion. The ethyl alkenoate/total alkenone and ethyl
alkenoate/C37 alkenone ratios increased abruptly at day 8 in both strains. These ratios should be useful in clarifying the
relationship between the marine environment and its corresponding growth phase of batch culture. E. huxleyi and G.
0
K
oceanica can be e€ectively distinguished using the UK
37 -U38Et diagram. # 2000 Elsevier Science Ltd. All rights reserved.
0

Keywords: Alkenones; UK
37 ; Paleotemperature; n-Fatty acids; Long-chain alkenes; Sterols; Batch culture; Emiliania huxleyi; Gephyrocapsa oceanica; Coccolithophorids

1. Introduction
Alkenone paleothermometry was proposed in the
mid-1980s (Brassell et al., 1986; Prahl and Wakeham,
1987), and has been widely applied to the assessment of
late Quaternary changes in sea surface temperature
(reviewed by Brassell, 1993; MuÈller et al., 1998). Long
chain alkenones are biolipids in a speci®c group of
haptophyte algae (Volkman et al., 1980), and until now
they were reported exclusively from Emiliania and

* Corresponding author. Fax:+81-298-61-3666.
E-mail address: yamamoto@gsj.go.jp (M. Yamamoto).

Gephyrocapsa (Family Gephyrocapsae) and Chrysotila
and Isochrysis (Family Isochrysidaceae) (Marlowe et al.,
1984; Volkman et al., 1995). In recent classi®cation systems, the former two genera are often classi®ed into the
Family Noelaerhabdaceae (e.g. Jordan and Kleijne,
1994). Although the phylogenetic relationship between
the Isochrysidaceae and Noelaerhabdaceae was uncertain, the monophyly of Emiliania, Gephyrocapsa and Isochrysis was recently con®rmed using 18SrDNA sequence
analysis (Edvandersen et al., 2000). In open marine

environments, alkenones are thought to be produced by
Emiliania and Gephyrocapsa exclusively (Marlowe et al.,
1984, 1990). The function and biosynthetic pathways of
these compounds, however, remain unknown.

0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00080-2

800

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

Alkenone paleothermometry uses the physiological
response of the unsaturation degree of C37 alkenones to
growth temperature. The unsaturation degree is expresK0
sed as the unsaturation indices UK
37 and U37 (Brassell et
al., 1986; Prahl and Wakeham, 1987), which are de®ned
as UK
37=([C37:2Me]ÿ[C37:4Me])/([C37:2Me]+[C37:3Me]+

0
[C37:4Me]) and UK
37 =[C37:2Me]/([C37:2Me]+[C37:3Me]),
where [C37:2Me], [C37:3Me] and [C37:4Me] are the concentrations of di-, tri- and tetra-unsaturated C37 alkenones, respectively. Early studies demonstrated a linear
relationship between alkenone unsaturation indices and
growth temperature in a batch culture experiment with
E. huxleyi (strain 55a) from the NE Paci®c (Prahl and
Wakeham, 1987; Prahl et al., 1988), and this calibration
has been used for assessing paleo-sea surface temperature.
It remains to be resolved why, or by what mechanism,
alkenone unsaturation indices and growth temperature
are correlated. In general, membrane lipids change their
degree of unsaturation in response to varying growth
temperatures in order to maintain ¯uidity and rigidity of
the membrane. It is speculated that alkenones have the
same function (Brassell et al., 1986).
After the initial calibration by Prahl and coworkers,
Volkman et al. (1995) found that the UK
37-temperature
relationship in JBO2, a G. oceanica strain from the SW

Paci®c, di€ered from that suggested by Prahl's calibration, especially in the range of temperatures lower than
20 C. Sawada et al. (1996) reported that EH2, an E.
huxleyi strain from the SW Paci®c, exhibits a UK
37-temperature relationship similar to that of strain JB02 (G.
oceanica), whereas GO1, a G. oceanica strain from the
Mutsu Bay is similar to strain 55a (E. huxleyi). Conte et
al. (1998) demonstrated large variations in UK
37-temperature relationships among E. huxleyi and G. oceanica
strains from various locations. These variations in cultured strains account for the range of variation of the
0
UK
37 in the particulate organic matter in water-column
samples from numerous locations (e.g. Conte et al.,
1992; Conte and Eglinton, 1993; Sikes and Volkman,
1993; Ternois et al., 1997; Sawada et al., 1998).
Conte et al. (1995) found that replicate isothermal
cultures of the same strain showed signi®cant variability
in their biomarker pro®les, indicating that their synthesis ratios are in¯uenced by environmental and/or physiological variables in addition to temperature.
Recently, Epstein et al. (1998) and Conte et al. (1998)
0

demonstrated the changes of UK
37 with varying growth
phase in batch culture experiments on strains of E.
huxleyi. They considered that nitrate de®ciency a€ects
0
UK
37 . Popp et al. (1998) used a continuous culture system
0
(chemostat culture) and found that the UK
37 values were
signi®cantly lower than those in batch culture systems,
0
and that the UK
37 of the non-calcifying strain decreased
slightly with increasing growth rate, while the calcifying
strain showed no systematic change. These results sugK0
gest that the UK
37- and U37 -temperature relationships are

dependent on growth status and show intraspeci®c

variability.
K0
There are large variations of UK
37 and U37 -temperature
relationships among both cultured strains and ®eld
samples, along with biases due to alkenone production
depth, seasonal temperature change, water column
degradation and sedimentary alteration. For this reaK0
son, the correlation between the UK
37 or U37 from core
top sediments and the measured temperature of the
overlying surface water (core-top calibration) has been
assessed for each region (e.g. Sikes et al., 1991; RosellMele et al., 1995; Pelejero and Grimalt, 1997; Sonzogni
et al., 1997; MuÈller et al., 1998; Herbert et al., 1998;
TEMPUS Project Members, 1998). This is the typical
way of assessing paleoceanographic proxies. However, it
does not clarify what cause the variations of UK
37-and
0
UK

37 -temperature relationships in cultured strains and
®eld samples, or why these relationships show regional
variation. Answers to these questions would improve
the simply empirical core-top calibration, and could
minimize the errors in the application of alkenone
paleothermometry. To augment the future application
of alkenone thermometry, there is thus a need for further investigations of processes ranging from alkenone
production to alkenone burial.
In this study we examined the physiological responses
of alkenone unsaturation indices to changes in growth
status of E. huxleyi and G. oceanica isolated from a
water sample of the NW Paci®c using an isothermal
batch culture system. Our comparison of the concentration and compositional changes of alkenones and
other lipids over the growth periods of these strains
should help to clarify the physiological factors controlling alkenone unsaturation indices.

2. Experiments
2.1. Samples and culture experiments
Both E. huxleyi (E1A) and G. oceanica (G1A) strains
were collected o€ Ishigaki Island in the NW Paci®c

(24 220 N, 124 200 E) during March 1998 in conjunction
with the CREST2 program. The measured temperature
and salinity of the surface water at the sampling location were 23.12 C and 34.75 psu, respectively, at the
time of the sampling. A unialgal culture of E. huxleyi
(E1A) was established by dilution of the seawater sample. G. oceanica appeared mixed with E. huxleyi in a
crude culture. A single cell of G. oceanica was isolated
using a micropipette, and was used to establish a unialgal culture (G1A). For both species, taxonomic identi®cation was con®rmed by scanning electron
microscopy.
For stock cultures, both species were grown in a 100ml Erlenmeyer ¯ask containing 50 ml of the ESM-nat-

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

ural seawater medium (Okaichi et al., 1982) under a 16h light/8-h dark regime. Cultures were gently shaken by
hand once a day to avoid settling at the bottom of ¯ask.
For experimental cultures, a small portion of the algal
culture, usually at the late logarithmic phase, was
transferred to a 500-ml Sakaguchi ¯ask containing 300
ml of the arti®cial seawater, Marine Art SF (Senju
Pharmaceutical Co., Japan), enriched with modi®ed
ESM, in which soil extract was replaced by 10 nmol/L

sodium selenite, as reported by Danbara and Shiraiwa
(1999). Cultures were illuminated continuously by
¯uorescent lamps and shaken by hand once daily. The
temperature and the light intensity during both the
stock and experimental cultures were 190.5 C and 30
mmol/m2/s, respectively. Although the growth of E.
huxleyi is similar under continuous illumination and L/
D cycle (Price et al., 1998), it has not yet been established how a light/dark regime, and particularly the
darkness component, a€ects alkenone production. For
this reason, the culturing was conducted under continuous light.
Packed cell volume (PCV) was determined by centrifugation of 5 ml of a suspension of cells in a
hematocrit tube with a scale from 0 to 10 ml for 10 min
at 2000 rpm (Sekino and Shiraiwa, 1994; Danbara and
Shiraiwa, 1999). Throughout the culture period, the
growth rate (Kg) was calculated at each sampling interval according to the equation: Kg (ml-PCV/ml/d)=1/
(t2ÿt1)log(PCV2/PCV1), where t1 and t2 are culture
times (day), and PCV1 and PCV2 are packed cell
volumes (ml/ml) at time t1 and t2, respectively. For the
estimation of chlorophylls the algal pellet obtained by
centrifugation was suspended in the seawater medium.

The algal suspension was disrupted ®ve times by a
sonicator, and then chlorophylls were extracted with
acetone. The concentration of chlorophyll was determined according to Je€rey and Humphrey (1975). After
the cultivation, the samples were collected at intervals
on pre-combusted GF/F ®lters and stored frozen at
ÿ20 C for subsequent lipid analysis.
2.2. Analytical
Lipids were extracted by ®ve, 5-min rounds of ultrasonication with 5 ml of dichloromethane-methanol
(6:4), then concentrated and passed through a short bed
of Na2SO4 to remove water.
An aliquot of the extracted lipid was analyzed by thin
layer chromatography-¯ame ionization detection (TLCFID) for the determination of lipid class compositions.
The analysis was conducted using an Iatroscan MK5
TLC-FID analyzer (Iatron Laboratories Inc., Tokyo,
Japan). The ¯ame ionization detector was operated at a
hydrogen ¯ow-rate of 150 ml/min, an air ¯ow-rate of
2000 ml/min, and a scan speed of 0.40 cm/s. Silica gel
SIII Chromarods were developed with polar solvents,

801

and passed through the detector twice at a scan speed of
0.17 cm/s before use. Approximately 0.3 mg of sample
was dissolved in 50±100 ml of dichloromethane, and a 4±
10 ml aliquot was applied using a 5-ml microsyringe.
After spotting, the rods were conditioned for 10 min at a
constant humidity of 65%, and subsequently suspended
for 10 min in a developing tank. Four di€erent solvent
systems were used to obtain four chromatograms per
rod (modi®ed after Parrish, 1987). The ®rst chromatogram was obtained after 20 min of development in
hexane:diethyl ether (96:1) by scanning the range of 1.5±
10 cm from the origin to detect hydrocarbons (Rf: 0.74)
and alkenones and alkenoates (Rf: 0.27±0.44). The second was obtained after 20 min of development in
hexane:diethyl ether:acetic acid (60:17:0.15) by scanning
the range of 1±10 cm to detect triacylglycerols (Rf: 0.48)
and sterols (Rf: 0.25). The third was obtained after a 6
min development in acetone by scanning the range of 1±
10 cm to detect chloroplast components such as pigments and glycolipids (Rf: 0.94). The last was obtained
after a 20 min development in chloroform:methanol:water (80:15:2) by full scanning to detect phospholipids
(Rf: 0.22±0.97). After each development, rods were dried
at 60 C for 5 min. Lipid classes were quanti®ed using
FID calibration curves. The calibration curves were
obtained by analyzing standard compounds in the same
manner as above. The standards included 1-eicosene
(GL Science Co., Tokyo, Japan) as a representative of
hydrocarbons, n-hexadecan-3-one (SIGMA Chemical
Co., St Louis, MO, U.S.A.) for ketones, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (SIGMA Chemical) for triacylglycerols, cholesterol (GL Science) for sterols and la-phosphatidylcholine (SIGMA Chemical) for phospholipids. l-a-Phosphatidylcholine was also used for
the quanti®cation of chloroplast components because of
the lack of an authentic standard. The standard deviations in 15 duplicate analyses averaged 5.9% of the
concentration.
An aliquot of the lipid extract was separated into
three fractions [F1: 3 ml of hexane:toluene (3:1); F2: 4
ml of toluene; F3: 3 ml of toluene:methanol (3:1)] by
column chromatography (SiO2 with 5% distilled water;
i.d., 5.5 mm; length, 45 mm). n-C24D50 and n-C36H74
were added as internal standards into the F1 (alkenes)
and F2 (alkenones and alkenoates) fractions, respectively.
Another aliquot of the lipid extract was saponi®ed
with 1 ml of 0.5 mol/l KOH/methanol at 100 C for 2 h
under nitrogen gas in a vacuum tube. The volume of the
saponi®ed lipids was reduced, supplemented with 2 ml
of distilled water, and then extracted with hexane-diethyl ether (85:15) ®ve times. After the water phase was
acidi®ed by adding 35% HCl, liberated lipids were
extracted with hexane-diethyl ether (85:15) ®ve times.
The combined extract was reduced in volume, passed
through a short bed of Na2SO4, and separated into

802

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

three fractions [S1: 3 ml of hexane:toluene (3:1); S2: 4 ml
of toluene; S3: 3 ml of toluene-methanol (3:1)] by SiO2
column chromatography. The S3 fraction was methylated with 14% BF3-methanol at 80 C for 15 min under
nitrogen gas in a vacuum tube. The methylated fraction
was supplemented with 1 ml of distilled water, and
extracted with toluene ®ve times. The extracted lipids
were condensed, passed through a short bed of Na2SO4,
and separated into three fractions [M1: 3 ml of hexane:toluene (3:1); M2: 4 ml of toluene; M3: 3 ml of toluenemethanol (3:1)] by SiO2 column chromatography. nC24D50 was added as an internal standard into the M2
(fatty acids) and M3 (sterols and a part of polyunsaturated
fatty acids) fractions. Prior to gas chromatographic analysis, the M3 fraction was silylated with BSTFA [N,O-bis(trimethylsilyl)-tri¯uoroacetamide):pyridine (1:1)] at 70 C
for 30 min.
Gas chromatography was conducted using a Hewlett
Packard 5890 series II gas chromatograph (GC) with
on-column injection and electron pressure control systems and a ¯ame ionization detector (FID). Samples
were dissolved in hexane. Helium was used as a carrier
gas, and the ¯ow velocity was maintained at 30 cm/s.
The column used was a Chrompack CP-Sil5CB (length,
60 m; i.d., 0.25 mm; thickness, 0.25 mm). For the analyses of F1, M2 and M3 fractions, the oven temperature
was programmed from 70 to 130 at 20 C/min, from 130
to 310 C at 4 C/min., and then held at 310 C for more
than 20 min. For the F2 fraction, the oven temperature
was programmed from 70 to 310 C at 20 C/min and
then held at 310 C for 40 min. The standard deviations
0
in 5 duplicate analyses averaged 0.008 for UK
37 and 7.5%
of the concentration for C37 alkenones.
Gas chromatography±mass spectrometry was conducted using a Hewlett Packard 5973 gas chromatograph±mass selective detector with on-column injection
and electron pressure control systems and a Quadrupole
mass spectrometer. The GC column and the oven temperature and carrier pressure programs were the same as
described above. The mass spectrometer was run in the
full scan ion-monitoring mode (m/z 50±650). Electron
impact spectra were obtained at 70 eV. Identi®cation of
compounds was achieved by comparison of their mass
spectra and retention times with those of standards and
those in the literature (e.g. de Leeuw et al., 1980;
Rechka and Maxwell, 1988).

3. Results
3.1. Packed cell volume
Growth curves and growth rates of E. huxleyi and G.
oceanica are shown in Fig. 1. E. huxleyi grew
exponentially during the ®rst 3 days (phase A, corresponding to the logarithmic phase), linearly during the

next 6 days (phase B, corresponding to the late
logarithmic or linear phase), and retardingly afterwards
(phase C, corresponding to the retarding or stationary
phase). After day 10 at phase C, the chlorophyll concentration remained constant. This suggests that the
increase in PCV might have been due mainly to calci®cation, rather than to an increase in organic mass, since
a limitation in nitrate and phosphorus is known to
increase the number of coccoliths per cell (Paasche,
1998). G. oceanica grew exponentially during the ®rst 11
days (logarithmic phase), and retardingly thereafter
(stationary phase). The packed cell volume of G. oceanica was several times as large as that of E. huxleyi over
the corresponding period. Acidi®cation treatment of
both species showed that the cell volume of their spheroplasts was almost the same (data not shown). This
indicates that the G. oceanica strain has fewer but much
larger coccoliths than the E. huxleyi strain.
3.2. Lipid classes
The lipid contents of the E. huxleyi and G. oceanica
strains obtained by TLC-FID varied within 7.4±13.2 mg/
ml-PCV and 3.4±13.3 mg/ml-PCV, respectively (Fig. 2a
and b). The lipid content of both strains decreased
rapidly during the earlier phase. The major lipid classes
were alkenones and alkenoates, chloroplast components
(mainly pigments and glycolipids), and phospholipids,
and together these three classes made up more than
84% of the total lipids (Fig. 2c and d). The minor lipid
classes, which made up less than 8% of total lipids,
included hydrocarbons, triacylglycerols and sterols. In
G. oceanica, the relative abundance of chloroplast components decreased, while alkenones and alkenoates
increased, with growth period. The relative abundance
of triacylglycerols in E. huxleyi decreased with growth
period, in contrast to the increase in triacylglycerol
concentrations previously observed in many other marine microalgal species (Berkalo€ and Kadar, 1975;
Lichtle and Dubacq, 1984; Kuwata et al., 1993).
3.3. Individual lipids
The contents of total lipids obtained by GC-FID
(Fig. 2e and f) were in agreement with those obtained by
TLC-FID within the errors caused by the di€erence of
techniques. The contents of total alkenones and
alkenoates by GC-FID were consistently lower (0.7
times) than those measured by TLC-FID due to the lack
of a suitable quantitative standard of similar chain
lengths for calibration (Brown et al., 1993). The contents of alkenes and sterols by GC-FID di€ered from
those by TLC-FID due to the low quantitative accuracy
for low-loading components in the TLC-FID analysis.
The low triacylglycerol content indicated that most of
the fatty acids identi®ed by GC-FID were the

803

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

Fig. 1. Changes of packed cell volume (PCV, circle), the sum of chlorophylls a and c (Chl a+c, triangle) and the calculated growth
rate (kg) of batch-cultured E. huxleyi (strain E1A) and G. oceanica (strain G1A). Open symbols indicate the values of the previous
stock cultures inoculated into the experimental culture.

constituents of membrane lipids such as glycolipids and
phospholipids.
3.4. Alkenones and alkenoates
Both the E. huxleyi and G. oceanica strains contain
common alkenones and alkenoates (Brassell, 1993;
Conte et al., 1994). Alkenones identi®ed in the present
study were C37:2ÿ3 methyl alkenones (C37:2ÿ3MK),
C38:2ÿ3 methyl alkenones (C38:2ÿ3MK), C38:2ÿ3 ethyl
alkenones (C38:2ÿ3EK) and C39:2ÿ3 ethyl alkenones
(C39:2ÿ3EK). Alkenoates identi®ed were C37:2ÿ3 methyl
alkenoates and C38:2 ethyl alkenoate (EE). Concentration and unsaturation indices are given in Table 1.
The alkenone content of E. huxleyi (strain E1A) varied between 1.43 and 2.65 mg/ml-PCV, and showed a
decreasing trend with growth period (Fig. 2e). The
abundance ratios of lower to higher homologues of
alkenones (K37/K38Me, K37/K38Et, K37/K39Et and K38Et/
K39Et ratios) reached maximums at days 3 and 4 (Fig.
K
K
3a). The unsaturation indices (UK
37, U38Me, U38Et and
K
U39Et) varied in parallel within the range of about 0.2,
and decreased in two steps at days 8 and 16 (Fig. 3c).
The UK
37 ranged from 0.50 to 0.68, and the variation was
0.17, corresponding to a temperature di€erence of 5.1 C
when the equation of Prahl et al. (1988) is applied. The

abundance ratios of C38:2 ethyl alkenoate to total
alkenoates (EE/K ratio) and to C37MK (EE/K37 ratio)
increased abruptly at day 8 (Fig. 3e). Both the EE/K
and EE/K37 ratios changed in parallel, indicating that
Table 1
Paleotemperature indices referred to in this paper
Index

Equation

Ref.a

UK
37

[C37:2MK]- [C37:4MK]/([C37:2MK]
+[C37:3MK]+[C37:4MK])
[C37:2MK]/([C37:2MK]+[C37:3MK])
[C38:2MK]/([C38:2MK]+[C38:3MK])
[C38:2EK]/([C38:2EK]+[C38:3EK])
[C39:2EK]/([C39:2EK]+[C39:3EK])
[C37:2MK]+[C37:3MK]+[C37:4MK]
[C38:2MK]+[C38:3MK]
[C38:2EK]+[C38:3EK]
[C39:2EK]+[C39:3EK]
[C38:2EE]/([C37:2MK]+
[C37:3MK]+[C37:4MK])
[C38:2EE]/(K37+K38Me+
K38Et+K39Et)

1

0

UK
37
UK
38Me
UK
38Et
UK
39Et
K37
K38Me
K38Et
K39Et
EE/K37
EE/K

2
3
4
5

5

a
1: Brassell et al. (1986), 2: Prahl and Wakeham (1987), 3:
Conte and Eglinton (1993), 4: Conte et al. (1998), 5: Prahl et al.
(1988).

804

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

Fig. 2. The total lipid contents (a and b) and lipid class compositions (c and d) obtained by TLC-FID and the individual lipid concentrations measured by GC-FID (e and f) in batch-cultured E. huxleyi and G. oceanica.

the relative decrease of C37 alkenones (K37) to the other
alkenone homologs did not have a pronounced in¯uence on the EE/ K37 ratio.
The alkenone content of G. oceanica (strain G1A)
varied between 0.80 and 1.71 mg/ml-PCV, and reached a
maximum at day 9 (Fig. 2f). The abundance ratio of
lower to higher homologues of alkenones peaked at day
5, decreased until day 13, and was nearly constant
thereafter (Fig. 3b). The unsaturation indices varied in
parallel within a range of about 0.13, and were at their
0
minimum at day 9 (Fig. 3d). The UK
37 ranged from 0.45
to 0.56, and the variation (0.11) corresponded to a temperature di€erence of 3.1 C by application of the equation of Prahl et al. (1988). The EE/K and EE/K37 ratios
increased abruptly at day 8 (Fig. 3f).
3.5. Alkenes
Alkenes detected in E. huxleyi and G. oceanica strains
include n-C21:6, n-C31:1ÿ2 and n-C33:2ÿ4 alkenes. The C37

and C38 homologs were not detected in either strain.
The alkene content of E. huxleyi decreased over the ®rst
9 days and was nearly constant thereafter, while that of
G. oceanica showed a di€erent trend that peaked at day
9 (Fig. 4).
3.6. Fatty acids
The n-fatty acids detected in the E. huxleyi and G.
oceanica strains included C12±C22 and C36 homologues.
The results of the TLC-FID analysis indicated that most
of the n-C12±n-C22 fatty acids were the constituents of
glycolipids and phospholipids. They were identi®ed as
12:0, 13:0, 14:0, 15:0, 16:0, 16:1(n-7), 17:0, 18:0, 18:1(n7), 18:1(n-9), 18:2(n-6), 18:3(n-3), 18:3(n-6), 18:4(n-3),
20:0, 20:2, 20:3, 20:4(n-6), 20:5(n-3), 22:0, 22:6(n-3) nfatty acids. The n-C36 homologue comprised 36:2 and
36:3 compounds, which originated from the hydrolysis
of alkenoates. Identi®cation of these compounds was
achieved by comparison with authentic and natural

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

805

Fig. 3. Changes in the abundance ratios of lower to higher homologues of alkenones (K37/K38Me, K37/K38Et, K37/K39Et and K38Et/
0
K
K
K
K39Et ratios) (a and b), unsaturation indices (UK
37 , U38Me, U38Et and U39Et) (c and d), and the abundance ratios of C38:2 ethyl alkenoate
to total alkenones (EE/K) and C37MK (EE/K37) (e and f) in batch-cultured E. huxleyi and G. oceanica. The range bars for a sample
(E. huxleyi at day 7) indicate the standard deviations of triplicate analysis. The deviations of some indices are so small that the bars
cannot be indicated.

standard mixtures (SUPELCO, Bellefonte, USA), as
well as with data from the literature (Volkman et al.,
1989). The n-fatty acid content of both strains decreased
rapidly during the ®rst 4 and 6 days (Fig. 2e and f). In
both strains, the abundance ratios of saturated to polyunsaturated n-C18 fatty acids (C18:0/C18:2, C18:0/C18:3
and C18:0/C18:4 ratios) showed variations parallel to
those of the C16/C18 ratio (Fig. 5a±d).

5-en-3b-ol to total sterol decreased over the ®rst 9 days
(Fig. 5e and f).

3.7. Sterols

Previous studies have demonstrated the changes of
0
UK
37 with culture age in several batch-cultured strains of
E. huxleyi (Conte et al., 1998; Epstein et al., 1998). The
present study showed that similar changes occur for a G.
oceanica strain. In the previous studies, the E. huxleyi
strains from the Iceland Basin, a Norwegian fjord and
0
the Sargasso Sea showed changes of UK
37 in di€erent
growth phases (Conte et al., 1998; Epstein et al., 1998).
0
In contrast, the UK
37 did not change in the strains from
the NE Paci®c, the SW Paci®c or the SW Indian Ocean

24-Methylcholesta-5,22E-dien-3b-ol and cholest-5-en3b-ol were detected in both the E. huxleyi and G. oceanica strains. The results of the TLC-FID analysis
indicated that most of these sterols existed in a free
form. The sterol contents of both strains changed in
contrast to the abundance ratios of saturated to polyunsaturated n-C18 fatty acids (Fig. 5c±f). In both E.
huxleyi and G. oceanica, the abundance ratio of cholest-

4. Discussion
0

4.1. Changes of UK
37 and lipid compositions with growth
period

806

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

Fig. 4. Changes in the concentrations (a and b) and C31/C33 homologous ratio (c and d) of polyunsaturated alkenes in batch-cultured
E. huxleyi and G. oceanica.

(Prahl and Wakeham, 1987; Sawada et al., 1996; Conte
et al., 1998). Time series examinations by Epstein et al.
(1998) and in this study demonstrated more detailed
0
variations of UK
37 changes in di€erent species and
strains. An E. huxleyi strain (CCMP372) from the Sar0
gasso Sea showed lower UK
37 values in the logarithmic
phase than in the stationary phase (Epstein et al., 1998).
In contrast, our E. huxleyi strain from the NW Paci®c
0
showed a decreasing trend in UK
37 with growth period
(Fig. 3c), and our G. oceanica strain showed a minimum
0
UK
37 value in the late logarithmic phase (Fig. 3d).
Sawada et al. (1995) speculated that the variation of
0
physiological responses of UK
37 to temperature among
strains from di€erent locations was likely caused by the
adaptation of strains to the geographical environment
from where the strain was sampled. This speculation
was based on the observation that two E. huxleyi strains
from di€erent locations showed a di€erent dependence
of UK
37 on temperature (Sawada et al., 1996). There are,
however, genotypic variations in E. huxleyi (Young and
Westbroek, 1991; van Bleijswijk et al., 1991). It, therefore, cannot be ruled out that intraspeci®cally genotypic
di€erences may be responsible for the variation of phy0
siological responses of UK
37 to temperature among the

di€erent strains of E. huxleyi. Our results showed that E.
huxleyi and G. oceanica strains from the same water
0
sample demonstrated di€erent patterns of UK
37 , most
likely suggesting that genotypic variation is a major factor a€ecting the pattern of physiological responses of
0
UK
37 .
0
In our experiment, the range of UK
37 in the cultured
strains of E. huxleyi was 0.50±0.68 (Fig. 3c), and the
variation was 0.17, corresponding to a temperature
variation of more than 5 C according to the equation of
Prahl et al. (1988). This range is similar to that for the
0
UK
37 of E. huxleyi (0.44±0.69) obtained from published
culture calibration equations at 19 C (Prahl et al., 1988;
Sawada et al., 1996; Conte et al., 1998). The range of
0
UK
37 in the cultured strains of G. oceanica was 0.45±0.56
0
(Fig. 3d). This range falls within that for the UK
37 of G.
oceanica (0.41±0.63) obtained from published culture
calibration equations at 19 C (Volkman et al., 1995;
Sawada et al., 1996; Conte et al., 1998). The combined
0
range for the UK
37 of E. huxleyi and G. oceanica in our
experiment (0.45±0.68) was much larger than the range
0
of UK
37 (0.64±0.70) obtained from published core-top
calibration equations at 19 C (Sikes et al., 1991; RosellMele et al., 1995; Pelejero and Grimalt, 1997; Sonzogni

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

807

Fig. 5. Changes in the C16/C18 ratio (a and b) of n-fatty acids, the abundance ratios of saturated to polyunsaturated n-C18 fatty acids
(C18:0/C18:2, C18:0/C18:3 and C18:0/C18:4 ratios, c and d) and sterol concentrations (e and f) in batch-cultured E. huxleyi and G. oceanica.
C28
5,22=24-methylcholesta-5,22E-dien-3b-ol, C27
5=cholest-5-en-3b-ol.

et al., 1997; Herbert et al., 1998; MuÈller et al., 1998), but
is similar to the range of scatter in individual measure0
ments of UK
37 values in large core-top data sets (e.g.
MuÈller et al., 1998). These ®ndings suggest that the var0
iation of the UK
37 -temperature relationship in the open
ocean can be attributed at least partly to the deviation
of nonthermal e€ects observed in culture experiments.
Conte et al. (1998) indicated that the abundance
ratios of total alkenoates to total alkenones in sediments
are approximately equal to those of E. huxleyi in the late
logarithmic and stationary phases, suggesting that the
late logarithmic or stationary phase is more typical of
that found in the marine environment. The present
study also indicates that the EE/K and EE/K37 ratios
after day 9 [average values 0.16 (n=5) and 0.18 (n=5)
in E. huxleyi and G. oceanica, respectively] are approximately equal to those of the late Quaternary California
margin sediments from upwelling region from an
upwelling region (av. 0.13 in ODP Site 1014, n=74; av.
0.16 in Site 1016, n=93; Yamamoto and Tanaka, in
prep.). Upwelling regions, where nutrients are supplied
massively and temporally, are characterized by blooms

of microalgae, rapid uptake of nutrients and a high
proportion of the produced organic matter sinking
through the water column (Eppley and Peterson, 1979).
The condition after upwelling-induced blooming resembles that after the late logarithmic phase of batch culture (Takahashi et al., 1986, Hama et al., 1988).
Therefore, in the open marine environment, or at least
in upwelling regions, the alkenone distributions produced after the late logarithmic or linear phase in batch
cultures are more likely to be similar to those exported
through the water column. However, the oligotrophic
oceans, such as the subtropical central gyre, are characterized by constant growth of microalgae and trace
but constant levels of nutrients (Eppley and Peterson,
1979; Goldman, 1980). In such regions, the early or mid
logarithmic phase might be a better representation of
the microalgal physiological status in such environments. The EE/K and EE/K37 ratios may be useful for
understanding the relationship between the type of
marine environment and its corresponding growth
phase of the alga in batch culture. For the exact determination of paleotemperature, further culture studies

808

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

will be needed to calibrate the temperature dependence
0
of UK
37 using a value in the appropriate growth phase.
0
This study demonstrated a minimum value of UK
37 at
day 9 in the culture of E. huxleyi and G. oceanica strains
from the NW Paci®c (Fig. 3c and d), although E. huxleyi showed a subsequent minimum at day 17, while
Epstein et al. (1998) reported a minimum at day 7 in an
E. huxleyi strain from the Sargasso Sea. Both studies
0
indicate that the UK
37 minimum occurs at the later stage
of the logarithmic phase. In our experiments, the decline
0
of UK
37 started when the concentrations of the cells
exceeded about 0.2 ml-PCV/ml-medium both in E. huxleyi and G. oceanica (Fig. 1). Cell concentration and the
culture environment change relatively slowly during early
exponential growth, but very rapidly during late exponential growth, so therefore the cell physiology could
exhibit some changes before the onset of the stationary
phase (Darley, 1982). Epstein et al. (1998) suggested that
0
nitrate depletion decreases UK
37 by unknown mechanisms.
Limitations in nitrate, as well as phosphorus, concentrations is known to a€ect coccolith formation and cell
replication in batch and chemostat cultures (Paasche,
1998). Therefore, changes in alkenone production may be
a€ected by changes in cellular metabolism.
0
In this study, the UK
37 decrease at day 8 occurred in
association with the continuous decreases of the abundance ratios of lower to higher homologues of
alkenones (K37/K38Me, K37/K38Et, K37/K39Et and K38Et/
K39Et ratios) around day 8 and the rapid increases of the
abundance ratio of C38:2 ethyl alkenoate to total
alkenoates (EE/K ratio) and C37 alkenones (EE/K37
ratio) at day 8 (Fig. 3). These associated changes can be
attributed to the physiological response of lipids for
maintaining their ¯uidity in the isothermal culture, since
higher homologues have higher melting points in most
cases, and unsaturation formation decreases the melting
point (Larsson and Quinn, 1994). This tendency, how0
ever, might not be generalized. After day 11, UK
37 gradually increased, but this change was not associated
with any increase in the abundance ratios of lower to
higher homologues of alkenones or to any decreases in
the EE/K or EE/K37 ratios (Fig. 3). This should result in
the increase of melting point of bulk alkenones and
alkenoates, which cannot be simply explained by the
physiological response mentioned above.
Parallel changes of carbon numbers and unsaturation
degree were observed in n-fatty acids (Fig. 5). The
decrease of C16/C18 ratio was accompanied with
decreases in the abundance ratios of saturated to polyunsaturated n-C18 fatty acids (C18:0/C18:2, C18:0/C18:3
and C18:0/C18:4 ratios). TLC-FID analysis indicated that
most of the n-fatty acids detected were the constituents
of membrane lipids such as glycolipids and phospholipids. The associated changes were attributed to the
physiological response of lipids for maintaining the
¯uidity of membranes.

Sterol contents changed in contrast to the abundance
ratios of saturated to polyunsaturated n-C18 fatty acids
(Fig. 5e and f). Sterols serve as a membrane stabilizer
®lling the matrix of acyl chains of lipid bilayers (Alberts
et al., 1994). The increase of cis-unsaturation of membrane lipids requires sterols as a stabilizing material to
maintain the rigidity and strengthen the membrane.
The proportion of cholest-5-en-3b-ol to total sterol
decreased with growth period (Fig. 5e and f). Volkman
et al. (1981) reported that cholest-5-en-3b-ol is more
abundant in the motile cells than the sessile cells of E.
huxleyi. Microscopic observation in the present study
showed that all the cells existed in the sessile form. This,
along with the result that both E. huxleyi and G. oceanica showed the same trends in sterol composition,
implies that the changes of sterol composition depend
on the changes of growth status as well as the life cycle.
The degree of unsaturation increased both in alkenones and n-fatty acids at almost the same period in the
later stage of the logarithmic phase, implying that a
common factor was involved in the formation of unsaturation. Consequently, examination into the factors
controlling the formation of n-fatty acid unsaturation
should provide clues to understanding the physiological
factors controlling alkenone unsaturation indices.
Known factors a€ecting the unsaturation of plant nfatty acids include temperature (Sato and Murata,
1980), O2 (Harris and James, 1969; Rebeille et al., 1980),
CO2 (Sato, 1989; Tsuzuki et al., 1990; Revill et al.,
1999), and nutrient concentrations (Chuecas and Riley,
1969; Kuwata et al., 1993). Among these factors, it is
possible that the de®ciencies of CO2 and/or nutrients
occurred after the later stage of the logarithmic phase in
our experiment. Recently, Revill et al. (1999) reported
that although the unsaturation and carbon number of nfatty acids in E. huxleyi increased with decreasing aqu0
eous CO2 concentration, the UK
37 did not change
signi®cantly, suggesting that CO2 de®ciency is less likely
to have an e€ect on the alkenone unsaturation. There
are contradictory reports on the e€ect of nutrient
concentrations. Chuecas and Riley (1969) showed that
cultured microalgae produced more abundant polyunsaturated fatty acids under nutrient-rich conditions.
But this ®nding presumably re¯ects the high proportion
of membrane lipids to storage lipids in the algae grown
in nutrient-rich medium, because membrane lipids are
richer in polyunsaturated fatty acids than storage lipids.
In contrast, Kuwata et al. (1993) found that, both in
neutral and polar lipids of a diatom Chaetoceros pseudocurvisetus, polyunsaturated n-fatty acids were more
highly accumulated in resting spores and resting cells
that grew under condition of nutrient depletion than in
vegetative cells that grew in a nutrient-sucient medium. They speculated that the diatom consumes the
excess ATP and NAD(P)H produced under nutrient
depletion by inducing unsaturation in lipids in order to

M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811

809

source of alkenones before the middle Quaternary was
solely the genera Gephyrocapsa and Reticulofenestra
(Marlowe, 1984; 1990), the alkenone producers have
previously had an alkenone composition similar to that
of the present E. huxleyi. Nannofossil assemblages at
Site 1014 during the last 140 ka indicate that Gephyrocapsa muellerae and small Gephyrocapsa (