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
baDepartment of Mineral and Fuel Resources, Geological Survey of Japan, 1-1-3 Higashi, Tsukuba, Ibaraki 305-8567, Japan
bInstitute 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 ofE. huxleyi andG.
oceanica strains isolated from a water sample of the NW Paci®c were examined using an isothermal batch culture
system. In both E. huxleyiandG. oceanica the unsaturation index U37K
0
changed during the growth period, but the eects of this change were dierent. 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
U37K
0
. Changes ofU37K
0
were associated with those of the unsaturation indices of C38and C39alkenones, the abundance
ratios of lower to higher homologues of alkenones, the abundance ratios of saturated to polyunsaturatedn-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
n-fatty acids increased at day 8, possibly due to nutrient depletion. The ethyl alkenoate/total alkenone and ethyl alkenoate/C37alkenone 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. huxleyiandG.
oceanicacan be eectively distinguished using theU37K
0
-U38EtK diagram.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Alkenones;U37K
0
; Paleotemperature;n-Fatty acids; Long-chain alkenes; Sterols; Batch culture;Emiliania huxleyi; Gephyr-ocapsa 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
Gephyrocapsa (Family Gephyrocapsae) and Chrysotila
andIsochrysis(Family Isochrysidaceae) (Marlowe et al.,
1984; Volkman et al., 1995). In recent classi®cation sys-tems, 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 uncer-tain, the monophyly ofEmiliania,Gephyrocapsaand
Iso-chrysiswas recently con®rmed using 18SrDNA sequence
analysis (Edvandersen et al., 2000). In open marine environments, alkenones are thought to be produced by
EmilianiaandGephyrocapsaexclusively (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. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 0 8 0 - 2
www.elsevier.nl/locate/orggeochem
* Corresponding author. Fax:+81-298-61-3666.
Alkenone paleothermometry uses the physiological response of the unsaturation degree of C37alkenones to
growth temperature. The unsaturation degree is expres-sed as the unsaturation indicesU37K andU37K
0
(Brassell et al., 1986; Prahl and Wakeham, 1987), which are de®ned as U37K=([C37:2Me]ÿ[C37:4Me])/([C37:2Me]+[C37:3Me]+
[C37:4Me]) and U37K
0
=[C37:2Me]/([C37:2Me]+[C37:3Me]),
where [C37:2Me], [C37:3Me] and [C37:4Me] are the
con-centrations of di-, tri- and tetra-unsaturated C37
alke-nones, 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 theU37K-temperature
relationship in JBO2, aG. oceanicastrain from the SW Paci®c, diered from that suggested by Prahl's calibra-tion, especially in the range of temperatures lower than 20C. Sawada et al. (1996) reported that EH2, an E.
huxleyistrain from the SW Paci®c, exhibits aU37K
-tem-perature relationship similar to that of strain JB02 (G.
oceanica), whereas GO1, a G. oceanicastrain from the
Mutsu Bay is similar to strain 55a (E. huxleyi). Conte et al. (1998) demonstrated large variations in U37K
-tem-perature relationships amongE. huxleyiandG. oceanica
strains from various locations. These variations in cul-tured strains account for the range of variation of the
U37K
0
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 synth-esis ratios are in¯uenced by environmental and/or phy-siological variables in addition to temperature. Recently, Epstein et al. (1998) and Conte et al. (1998) demonstrated the changes of U37K
0
with varying growth phase in batch culture experiments on strains of E.
huxleyi. They considered that nitrate de®ciency aects
U37K
0
. Popp et al. (1998) used a continuous culture system (chemostat culture) and found that theU37K
0
values were signi®cantly lower than those in batch culture systems, and that the U37K
0
of the non-calcifying strain decreased slightly with increasing growth rate, while the calcifying strain showed no systematic change. These results sug-gest that theU37K- andU37K
0
-temperature relationships are
dependent on growth status and show intraspeci®c variability.
There are large variations ofU37KandU37K
0
-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 rea-son, the correlation between theU37K orU37K
0
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; Rosell-Mele 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 U37K-and
U37K
0
-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 fur-ther 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 con-centration and compositional changes of alkenones and other lipids over the growth periods of these strains should help to clarify the physiological factors control-ling alkenone unsaturation indices.
2. Experiments
2.1. Samples and culture experiments
BothE. huxleyi(E1A) andG. oceanica(G1A) strains
were collected o Ishigaki Island in the NW Paci®c (24220N, 124200E) during March 1998 in conjunction
with the CREST2program. The measured temperature
and salinity of the surface water at the sampling loca-tion were 23.12C 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
sam-ple. G. oceanica appeared mixed with E. huxleyi in a
crude culture. A single cell ofG. oceanicawas isolated using a micropipette, and was used to establish a uni-algal culture (G1A). For both species, taxonomic iden-ti®cation was con®rmed by scanning electron microscopy.
ESM-nat-ural seawater medium (Okaichi et al., 1982) under a 16-h lig16-ht/8-16-h dark regime. Cultures were gently s16-haken 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 190.5C and 30 mmol/m2/s, respectively. Although the growth of E.
huxleyiis similar under continuous illumination and L/
D cycle (Price et al., 1998), it has not yet been estab-lished how a light/dark regime, and particularly the darkness component, aects alkenone production. For this reason, the culturing was conducted under con-tinuous light.
Packed cell volume (PCV) was determined by cen-trifugation of 5 ml of a suspension of cells in a hematocrit tube with a scale from 0 to 10ml 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
inter-val 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 timet1andt2, 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 deter-mined according to Jerey and Humphrey (1975). After the cultivation, the samples were collected at intervals on pre-combusted GF/F ®lters and stored frozen at
ÿ20C for subsequent lipid analysis.
2.2. Analytical
Lipids were extracted by ®ve, 5-min rounds of ultra-sonication with 5 ml of dichloromethane-methanol (6:4), then concentrated and passed through a short bed of Na2SO4to remove water.
An aliquot of the extracted lipid was analyzed by thin layer chromatography-¯ame ionization detection (TLC-FID) 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,
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±100ml 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 dierent solvent systems were used to obtain four chromatograms per rod (modi®ed after Parrish, 1987). The ®rst chromato-gram 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
sec-ond 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 pig-ments 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 60C 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-dipalmi-toyl-3-oleoyl-rac-glycerol (SIGMA Chemical) for tria-cylglycerols, cholesterol (GL Science) for sterols andl -a-phosphatidylcholine (SIGMA Chemical) for phos-pholipids. l-a-Phosphatidylcholine was also used for
the quanti®cation of chloroplast components because of the lack of an authentic standard. The standard devia-tions 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 (SiO2with 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, respec-tively.
Another aliquot of the lipid extract was saponi®ed with 1 ml of 0.5 mol/l KOH/methanol at 100C for 2 h
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 methy-lated with 14% BF3-methanol at 80C 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 hex-ane:toluene (3:1); M2: 4 ml of toluene; M3: 3 ml of toluene-methanol (3:1)] by SiO2 column chromatography.
n-C24D50 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 analy-sis, the M3 fraction was silylated with BSTFA [N,O-bis(-trimethylsilyl)-tri¯uoroacetamide):pyridine (1:1)] at 70C
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 sys-tems 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, 0.25mm). For the ana-lyses of F1, M2 and M3 fractions, the oven temperature was programmed from 70 to 130 at 20C/min, from 130
to 310C at 4C/min., and then held at 310C for more
than 20 min. For the F2 fraction, the oven temperature was programmed from 70 to 310C at 20C/min and
then held at 310C for 40 min. The standard deviations
in 5 duplicate analyses averaged 0.008 forU37K
0
and 7.5% of the concentration for C37alkenones.
Gas chromatography±mass spectrometry was con-ducted using a Hewlett Packard 5973 gas chromato-graph±mass selective detector with on-column injection and electron pressure control systems and a Quadrupole mass spectrometer. The GC column and the oven tem-perature 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 ofE. huxleyiandG.
oceanica are shown in Fig. 1. E. huxleyi grew
exponentially during the ®rst 3 days (phase A, corre-sponding 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 con-centration 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. oceanicagrew exponentially during the ®rst 11 days (logarithmic phase), and retardingly thereafter (stationary phase). The packed cell volume ofG. ocea-nicawas several times as large as that ofE. huxleyiover the corresponding period. Acidi®cation treatment of both species showed that the cell volume of their spher-oplasts was almost the same (data not shown). This indicates that theG. oceanicastrain has fewer but much larger coccoliths than theE. huxleyistrain.
3.2. Lipid classes
The lipid contents of theE. huxleyiandG. oceanica
strains obtained by TLC-FID varied within 7.4±13.2mg/
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
com-ponents decreased, while alkenones and alkenoates increased, with growth period. The relative abundance of triacylglycerols in E. huxleyidecreased with growth period, in contrast to the increase in triacylglycerol concentrations previously observed in many other mar-ine microalgal species (Berkalo and Kadar, 1975; Lichtle and Dubacq, 1984; Kuwata et al., 1993).
3.3. Individual lipids
constituents of membrane lipids such as glycolipids and phospholipids.
3.4. Alkenones and alkenoates
Both theE. huxleyi andG. 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 C38:2ethyl alkenoate (EE).
Concentra-tion and unsaturaConcentra-tion indices are given in Table 1. The alkenone content ofE. huxleyi(strain E1A) var-ied 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/K39Etand K38Et/
K39Et ratios) reached maximums at days 3 and 4 (Fig.
3a). The unsaturation indices (U37K, U38MeK , U38EtK and
U39EtK ) varied in parallel within the range of about 0.2,
and decreased in two steps at days 8 and 16 (Fig. 3c). TheU37K ranged from 0.50 to 0.68, and the variation was
0.17, corresponding to a temperature dierence of 5.1C
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/K37ratio)
increased abruptly at day 8 (Fig. 3e). Both the EE/K and EE/K37ratios changed in parallel, indicating that Fig. 1. Changes of packed cell volume (PCV, circle), the sum of chlorophyllsaandc(Chla+c, triangle) and the calculated growth rate (kg) of batch-culturedE. huxleyi(strain E1A) andG. oceanica(strain G1A). Open symbols indicate the values of the previous
stock cultures inoculated into the experimental culture.
Table 1
Paleotemperature indices referred to in this paper
Index Equation Ref.a U37K [C37:2MK]- [C37:4MK]/([C37:2MK]
+[C37:3MK]+[C37:4MK])
1
U37K
0
[C37:2MK]/([C37:2MK]+[C37:3MK]) 2 U38MeK [C38:2MK]/([C38:2MK]+[C38:3MK]) 3 U38EtK [C38:2EK]/([C38:2EK]+[C38:3EK]) 4 U39EtK [C39:2EK]/([C39:2EK]+[C39:3EK])
K37 [C37:2MK]+[C37:3MK]+[C37:4MK] 5
K38Me [C38:2MK]+[C38:3MK]
K38Et [C38:2EK]+[C38:3EK]
K39Et [C39:2EK]+[C39:3EK]
EE/K37 [C38:2EE]/([C37:2MK]+
[C37:3MK]+[C37:4MK])
5 EE/K [C38:2EE]/(K37+K38Me+
K38Et+K39Et)
a 1: Brassell et al. (1986), 2: Prahl and Wakeham (1987), 3:
the relative decrease of C37alkenones (K37) to the other
alkenone homologs did not have a pronounced in¯u-ence on the EE/ K37ratio.
The alkenone content of G. oceanica (strain G1A) varied between 0.80 and 1.71mg/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 minimum at day 9 (Fig. 3d). TheU37K
0
ranged from 0.45 to 0.56, and the variation (0.11) corresponded to a tem-perature dierence of 3.1C by application of the
equa-tion of Prahl et al. (1988). The EE/K and EE/K37ratios
increased abruptly at day 8 (Fig. 3f).
3.5. Alkenes
Alkenes detected inE. huxleyiandG. oceanicastrains includen-C21:6,n-C31:1ÿ2andn-C33:2ÿ4alkenes. The C37
and C38 homologs were not detected in either strain.
The alkene content ofE. huxleyidecreased over the ®rst 9 days and was nearly constant thereafter, while that of
G. oceanicashowed a dierent trend that peaked at day
9 (Fig. 4).
3.6. Fatty acids
The n-fatty acids detected in the E. huxleyi andG.
oceanicastrains included C12±C22and C36homologues.
The results of the TLC-FID analysis indicated that most of then-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(n -7), 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)
n-fatty 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
standard mixtures (SUPELCO, Bellefonte, USA), as well as with data from the literature (Volkman et al., 1989). Then-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 poly-unsaturated 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/C18ratio (Fig. 5a±d).
3.7. Sterols
24-Methylcholesta-5,22E-dien-3b-ol and cholest-5-en-3b-ol were detected in both theE. huxleyiandG. ocea-nica 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 poly-unsaturated n-C18 fatty acids (Fig. 5c±f). In both E.
huxleyi and G. oceanica, the abundance ratio of
cholest-5-en-3b-ol to total sterol decreased over the ®rst 9 days (Fig. 5e and f).
4. Discussion
4.1. Changes ofU37K
0
and lipid compositions with growth period
Previous studies have demonstrated the changes of
U37K
0
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 aG.
oceanica strain. In the previous studies, the E. huxleyi
strains from the Iceland Basin, a Norwegian fjord and the Sargasso Sea showed changes of U37K
0
in dierent growth phases (Conte et al., 1998; Epstein et al., 1998). In contrast, theU37K
0
did not change in the strains from the NE Paci®c, the SW Paci®c or the SW Indian Ocean
Fig. 3. Changes in the abundance ratios of lower to higher homologues of alkenones (K37/K38Me, K37/K38Et, K37/K39Etand K38Et/
K39Etratios) (a and b), unsaturation indices (U37K
0
,U38MeK ,U38EtK andU39EtK ) (c and d), and the abundance ratios of C38:2ethyl alkenoate
to total alkenones (EE/K) and C37MK (EE/K37) (e and f) in batch-culturedE. huxleyiandG. oceanica. The range bars for a sample
(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 variations of U37K
0
changes in dierent species and strains. AnE. huxleyistrain (CCMP372) from the Sar-gasso Sea showed lower U37K
0
values in the logarithmic phase than in the stationary phase (Epstein et al., 1998). In contrast, our E. huxleyistrain from the NW Paci®c showed a decreasing trend in U37K
0
with growth period (Fig. 3c), and ourG. oceanicastrain showed a minimum
U37K
0
value in the late logarithmic phase (Fig. 3d). Sawada et al. (1995) speculated that the variation of physiological responses of U37K
0
to temperature among strains from dierent 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 twoE. huxleyistrains from dierent locations showed a dierent dependence ofU37K on temperature (Sawada et al., 1996). There are,
however, genotypic variations inE. huxleyi(Young and Westbroek, 1991; van Bleijswijk et al., 1991). It, there-fore, cannot be ruled out that intraspeci®cally genotypic dierences may be responsible for the variation of phy-siological responses of U37K
0
to temperature among the
dierent strains ofE. huxleyi. Our results showed thatE.
huxleyi and G. oceanica strains from the same water
sample demonstrated dierent patterns of U37K
0
, most likely suggesting that genotypic variation is a major fac-tor aecting the pattern of physiological responses of
U37K
0
.
In our experiment, the range ofU37K
0
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 5C according to the equation of
Prahl et al. (1988). This range is similar to that for the
U37K
0
ofE. huxleyi (0.44±0.69) obtained from published
culture calibration equations at 19C (Prahl et al., 1988;
Sawada et al., 1996; Conte et al., 1998). The range of
U37K
0
in the cultured strains ofG. oceanicawas 0.45±0.56 (Fig. 3d). This range falls within that for theU37K
0
ofG.
oceanica (0.41±0.63) obtained from published culture
calibration equations at 19C (Volkman et al., 1995;
Sawada et al., 1996; Conte et al., 1998). The combined range for theU37K
0
ofE. huxleyiandG. oceanicain our
experiment (0.45±0.68) was much larger than the range of U37K
0
(0.64±0.70) obtained from published core-top calibration equations at 19C (Sikes et al., 1991;
Rosell-Mele et al., 1995; Pelejero and Grimalt, 1997; Sonzogni
et al., 1997; Herbert et al., 1998; MuÈller et al., 1998), but is similar to the range of scatter in individual measure-ments of U37K
0
values in large core-top data sets (e.g. MuÈller et al., 1998). These ®ndings suggest that the var-iation of the U37K
0
-temperature relationship in the open ocean can be attributed at least partly to the deviation of nonthermal eects 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 ofE. huxleyiin 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/K37ratios
after day 9 [average values 0.16 (n=5) and 0.18 (n=5)
inE. huxleyiandG. oceanica, respectively] are
approxi-mately 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 resem-bles that after the late logarithmic phase of batch cul-ture (Takahashi et al., 1986, Hama et al., 1988). Therefore, in the open marine environment, or at least in upwelling regions, the alkenone distributions pro-duced 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 char-acterized 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 environ-ments. The EE/K and EE/K37ratios 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 deter-mination of paleotemperature, further culture studies
Fig. 5. Changes in the C16/C18ratio (a and b) ofn-fatty acids, the abundance ratios of saturated to polyunsaturatedn-C18fatty acids
(C18:0/C18:2, C18:0/C18:3and C18:0/C18:4ratios, c and d) and sterol concentrations (e and f) in batch-culturedE. huxleyiandG. oceanica.
will be needed to calibrate the temperature dependence ofU37K
0
using a value in the appropriate growth phase. This study demonstrated a minimum value ofU37K
0
at day 9 in the culture ofE. huxleyiandG. oceanicastrains from the NW Paci®c (Fig. 3c and d), althoughE. hux-leyi 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
indicate that theU37K
0
minimum occurs at the later stage of the logarithmic phase. In our experiments, the decline of U37K
0
started when the concentrations of the cells exceeded about 0.2ml-PCV/ml-medium both inE.
hux-leyiandG. oceanica(Fig. 1). Cell concentration and the
culture environment change relatively slowly during early exponential growth, but very rapidly during late expo-nential 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 nitrate depletion decreasesU37K
0
by unknown mechanisms. Limitations in nitrate, as well as phosphorus, concentra-tions is known to aect coccolith formation and cell replication in batch and chemostat cultures (Paasche, 1998). Therefore, changes in alkenone production may be aected by changes in cellular metabolism.
In this study, theU37K
0
decrease at day 8 occurred in association with the continuous decreases of the abun-dance ratios of lower to higher homologues of alkenones (K37/K38Me, K37/K38Et, K37/K39Etand K38Et/
K39Etratios) 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, how-ever, might not be generalized. After day 11,U37K
0
gra-dually 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/K37ratios (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 poly-unsaturated n-C18 fatty acids (C18:0/C18:2, C18:0/C18:3
and C18:0/C18:4ratios). TLC-FID analysis indicated that
most of then-fatty acids detected were the constituents of membrane lipids such as glycolipids and phospholi-pids. 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 polyunsaturatedn-C18fatty 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 ofcis-unsaturation of mem-brane 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 ofE.
huxleyi. Microscopic observation in the present study
showed that all the cells existed in the sessile form. This, along with the result that bothE. huxleyiandG. ocea-nica 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 alke-nones andn-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 unsa-turation. 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 aecting the unsaturation of plant
n-fatty 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
n-fatty acids inE. huxleyiincreased with decreasing aqu-eous CO2 concentration, the U37K
0
did not change signi®cantly, suggesting that CO2de®ciency is less likely
to have an eect on the alkenone unsaturation. There are contradictory reports on the eect of nutrient concentrations. Chuecas and Riley (1969) showed that cultured microalgae produced more abundant poly-unsaturated 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 diatomChaetoceros
pseu-docurvisetus, polyunsaturated n-fatty acids were more
avoid lethal photochemical damage. We consider that the nutrient depletion is more likely to cause the increase in unsaturation of alkenones andn-fatty acids, as suggested by Epstein et al. (1998), although further studies will be needed on the physiological eects of CO2 and nutrients on the alkenone and n-fatty acid
unsaturation.
In the culture ofG. oceanica, the relative proportions of alkenones and alkenoates to total lipids increased gradually after day 6, while the relative abundances of phospholipids and chloroplast lipids decreased during the same period (Fig. 2d). This indicates that the meta-bolically-active membrane lipids decreased due to the changes of metabolic balance. It also implies that alke-nones are metabolically-inactive, and behave as a sto-rage lipid or a byproduct of metabolism, although this is still speculative and remains to be proven.
4.2. Dierences betweenE. huxleyiandG. oceanica
C37/C38 alkenone and EE/K37 ratios have been
pro-posed to distinguish the contribution of E. huxleyi to sedimentary alkenones from that of G. oceanica (Volk-man et al., 1995; Sawada et al., 1996). The present ®ndings, and those of Conte et al. (1998), indicate that these ratios are aected by the growth phase of batch culture rather than species dierence. Conte et al. (1998) demonstrated that both species can be distinguished on theU38MeK -U38EtK diagram. In the present study, both
spe-cies were better distinguished on the diagram similar to that in Conte et al., (1998), i.e., the U37K- U38EtK diagram
(Fig. 6). Unexpectedly, most California margin sedi-ment samples from the late Miocene to Recent (ODP Sites 1014 and 1016) are distributed in the region ofE.
huxleyi, not G. oceanica (Yamamoto and Tanaka,
unpublished data). This indicates that, even though the
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
Gephyr-ocapsa muellerae and small Gephyrocapsa (<3 mm),
including G. ericsonii and G. apera, were dominant alkenone-producing species throughout these periods (Tanaka and Yamamoto, unpublished data). We spec-ulate that theseGephyrocapsaspecies have an alkenone composition similar to that of the presentE. huxleyi.
5. Conclusions
U37K
0
changed with growth status both in E. huxleyi
andG. oceanicafrom the NW Paci®c. The patterns of
these changes were dierent, suggesting that the geno-typic variation is a major factor in determining the pat-tern of physiological responses ofU37K
0
.
The C38:2ethyl alkenoate/total alkenone (EE/K) and
C38:2 ethyl alkenonate/C37 alkenone (EE/K37) ratios
increased abruptly at day 8 (the later stage of the loga-rithmic phase) in both strains. These ratios should be useful in clarifying the relationship between the marine environment and its corresponding growth phase of batch culture.
Changes of U37K
0
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 saturate to poly-unsaturatedn-fatty acids, EE/K and EE/K37ratios and
sterol contents. These associated changes are attributed partly to the physiological response of lipids for main-taining their ¯uidity.
The increase in the degree of unsaturation both in alkenones andn-fatty acids at almost the same period implies that a common factor, possibly nutrient deple-tion, aects lipid unsaturation.
The present results indicate that E. huxleyi and G.
oceanicacan be eectively distinguished using theU37K
0
-U38EtK diagram.
Acknowledgements
We thank Ms. Kazuko Hino (Geological Survey of Japan) for her analytical assistance in the laboratory and Dr. Ken Sawada (University of Tsukuba) for his valuable input. Special thanks are due to Dr. Hajime Kayanne and Mr. Kouji Hata (University of Tokyo) for their help with the sampling, and to Drs. Yuichiro Tanaka and Masatoshi Komiya (GSJ) for their useful comments. The helpful reviews by Drs. Bonnie L. Epstein and Hanno Kinkel and the editorial comments
Fig. 6. A plot of batch-culturedE. huxleyi(solid circle) andG. oceanica(solid square) on theU37K
0
by Dr. John K. Volkman improved the quality of this manuscript. This study was ®nancially supported by the Science and Technology Agency of Japan.
Associate EditorÐJ. Volkman
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