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

Physiological responses of lipids in and oceanica (Haptophyceae) to growth status and their implications for alkenone paleothermometry

Masanobu Yamamoto a,*, Yoshihiro Shiraiwa b, Isao Inouye b

aDepartment 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 of E. huxleyi and G. oceanica strains isolated from a water sample of the NW Paci®c were examined using an isothermal batch culture K0 system. In both E. huxleyi and G. oceanica the unsaturation index U37 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 K0 K0 U37. 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 n- fatty 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. K0 K oceanica can be e€ectively distinguished using the U37-U38Et diagram. # 2000 Elsevier Science Ltd. All rights reserved.

K0 Keywords: Alkenones; U37; Paleotemperature; n-Fatty acids; Long-chain alkenes; Sterols; Batch culture; Emiliania huxleyi; Gephyr- ocapsa oceanica; Coccolithophorids

1. Introduction Gephyrocapsa (Family Gephyrocapsae) and and (Family Isochrysidaceae) (Marlowe et al., Alkenone paleothermometry was proposed in the 1984; Volkman et al., 1995). In recent classi®cation sys- mid-1980s (Brassell et al., 1986; Prahl and Wakeham, tems, the former two genera are often classi®ed into the 1987), and has been widely applied to the assessment of Family (e.g. Jordan and Kleijne, late Quaternary changes in sea surface temperature 1994). Although the phylogenetic relationship between (reviewed by Brassell, 1993; MuÈ ller et al., 1998). Long the Isochrysidaceae and Noelaerhabdaceae was uncer- chain alkenones are biolipids in a speci®c group of tain, the monophyly of Emiliania, Gephyrocapsa and Iso- algae (Volkman et al., 1980), and until now chrysis was recently con®rmed using 18SrDNA sequence they were reported exclusively from Emiliania and analysis (Edvandersen et al., 2000). In open marine environments, alkenones are thought to be produced by Emiliania and Gephyrocapsa exclusively (Marlowe et al., * Corresponding author. Fax:+81-298-61-3666. 1984, 1990). The function and biosynthetic pathways of E-mail address: [email protected] (M. Yamamoto). 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 dependent on growth status and show intraspeci®c response of the unsaturation degree of C37 alkenones to variability. K K0 growth temperature. The unsaturation degree is expres- There are large variations of U37 and U37-temperature K K0 sed as the unsaturation indices U37 and U37 (Brassell et relationships among both cultured strains and ®eld al., 1986; Prahl and Wakeham, 1987), which are de®ned samples, along with biases due to alkenone production K as U37=([C37:2Me][C37:4Me])/([C37:2Me]+[C37:3Me]+ depth, seasonal temperature change, water column K0 [C37:4Me]) and U37=[C37:2Me]/([C37:2Me]+[C37:3Me]), degradation and sedimentary alteration. For this rea- K K0 where [C37:2Me], [C37:3Me] and [C37:4Me] are the con- son, the correlation between the U37 or U37 from core centrations of di-, tri- and tetra-unsaturated C37 alke- top sediments and the measured temperature of the nones, respectively. Early studies demonstrated a linear overlying surface water (core-top calibration) has been relationship between alkenone unsaturation indices and assessed for each region (e.g. Sikes et al., 1991; Rosell- growth temperature in a batch culture experiment with Mele et al., 1995; Pelejero and Grimalt, 1997; Sonzogni E. huxleyi (strain 55a) from the NE Paci®c (Prahl and et al., 1997; MuÈ ller et al., 1998; Herbert et al., 1998; Wakeham, 1987; Prahl et al., 1988), and this calibration TEMPUS Project Members, 1998). This is the typical has been used for assessing paleo-sea surface temperature. way of assessing paleoceanographic proxies. However, it K It remains to be resolved why, or by what mechanism, does not clarify what cause the variations of U37-and K0 alkenone unsaturation indices and growth temperature U37-temperature relationships in cultured strains and are correlated. In general, membrane lipids change their ®eld samples, or why these relationships show regional degree of unsaturation in response to varying growth variation. Answers to these questions would improve temperatures in order to maintain ¯uidity and rigidity of the simply empirical core-top calibration, and could the membrane. It is speculated that alkenones have the minimize the errors in the application of alkenone same function (Brassell et al., 1986). paleothermometry. To augment the future application After the initial calibration by Prahl and coworkers, of alkenone thermometry, there is thus a need for fur- K Volkman et al. (1995) found that the U37-temperature ther investigations of processes ranging from alkenone relationship in JBO2, a G. oceanica strain from the SW production to alkenone burial. Paci®c, di€ered from that suggested by Prahl's calibra- In this study we examined the physiological responses tion, especially in the range of temperatures lower than of alkenone unsaturation indices to changes in growth 20C. Sawada et al. (1996) reported that EH2, an E. status of E. huxleyi and G. oceanica isolated from a K huxleyi strain from the SW Paci®c, exhibits a U37-tem- water sample of the NW Paci®c using an isothermal perature relationship similar to that of strain JB02 (G. batch culture system. Our comparison of the con- oceanica), whereas GO1, a G. oceanica strain from the centration and compositional changes of alkenones and Mutsu Bay is similar to strain 55a (E. huxleyi). Conte et other lipids over the growth periods of these strains K al. (1998) demonstrated large variations in U37-tem- should help to clarify the physiological factors control- perature relationships among E. huxleyi and G. oceanica ling alkenone unsaturation indices. strains from various locations. These variations in cul- tured strains account for the range of variation of the K0 U37 in the particulate organic matter in water-column 2. Experiments samples from numerous locations (e.g. Conte et al., 1992; Conte and Eglinton, 1993; Sikes and Volkman, 2.1. Samples and culture experiments 1993; Ternois et al., 1997; Sawada et al., 1998). Conte et al. (1995) found that replicate isothermal Both E. huxleyi (E1A) and G. oceanica (G1A) strains cultures of the same strain showed signi®cant variability were collected o€ Ishigaki Island in the NW Paci®c in their biomarker pro®les, indicating that their synth- (24220N, 124200E) during March 1998 in conjunction esis ratios are in¯uenced by environmental and/or phy- with the CREST2 program. The measured temperature siological variables in addition to temperature. and salinity of the surface water at the sampling loca- Recently, Epstein et al. (1998) and Conte et al. (1998) tion were 23.12C and 34.75 psu, respectively, at the K0 demonstrated the changes of U37 with varying growth time of the sampling. A unialgal culture of E. huxleyi phase in batch culture experiments on strains of E. (E1A) was established by dilution of the seawater sam- huxleyi. They considered that nitrate de®ciency a€ects ple. G. oceanica appeared mixed with E. huxleyi in a K0 U37. Popp et al. (1998) used a continuous culture system crude culture. A single cell of G. oceanica was isolated K0 (chemostat culture) and found that the U37 values were using a micropipette, and was used to establish a uni- signi®cantly lower than those in batch culture systems, algal culture (G1A). For both species, taxonomic iden- K0 and that the U37 of the non-calcifying strain decreased ti®cation was con®rmed by scanning electron slightly with increasing growth rate, while the calcifying microscopy. strain showed no systematic change. These results sug- For stock cultures, both species were grown in a 100- K K0 gest that the U37- and U37-temperature relationships are ml Erlenmeyer ¯ask containing 50 ml of the ESM-nat- M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811 801 ural seawater medium (Okaichi et al., 1982) under a 16- and passed through the detector twice at a scan speed of h light/8-h dark regime. Cultures were gently shaken by 0.17 cm/s before use. Approximately 0.3 mg of sample hand once a day to avoid settling at the bottom of ¯ask. was dissolved in 50±100 ml of dichloromethane, and a 4± For experimental cultures, a small portion of the algal 10 ml aliquot was applied using a 5-ml microsyringe. culture, usually at the late logarithmic phase, was After spotting, the rods were conditioned for 10 min at a transferred to a 500-ml Sakaguchi ¯ask containing 300 constant humidity of 65%, and subsequently suspended ml of the arti®cial seawater, Marine Art SF (Senju for 10 min in a developing tank. Four di€erent solvent Pharmaceutical Co., Japan), enriched with modi®ed systems were used to obtain four chromatograms per ESM, in which soil extract was replaced by 10 nmol/L rod (modi®ed after Parrish, 1987). The ®rst chromato- sodium selenite, as reported by Danbara and Shiraiwa gram was obtained after 20 min of development in (1999). Cultures were illuminated continuously by hexane:diethyl ether (96:1) by scanning the range of 1.5±

¯uorescent lamps and shaken by hand once daily. The 10 cm from the origin to detect hydrocarbons (Rf: 0.74) temperature and the light intensity during both the and alkenones and alkenoates (Rf: 0.27±0.44). The sec- stock and experimental cultures were 19Æ0.5C and 30 ond was obtained after 20 min of development in mmol/m2/s, respectively. Although the growth of E. hexane:diethyl ether:acetic acid (60:17:0.15) by scanning huxleyi is similar under continuous illumination and L/ the range of 1±10 cm to detect triacylglycerols (Rf: 0.48) D cycle (Price et al., 1998), it has not yet been estab- and sterols (Rf: 0.25). The third was obtained after a 6 lished how a light/dark regime, and particularly the min development in acetone by scanning the range of 1± darkness component, a€ects alkenone production. For 10 cm to detect chloroplast components such as pig- this reason, the culturing was conducted under con- ments and glycolipids (Rf: 0.94). The last was obtained tinuous light. after a 20 min development in chloroform:methanol:- Packed cell volume (PCV) was determined by cen- water (80:15:2) by full scanning to detect phospholipids trifugation of 5 ml of a suspension of cells in a (Rf: 0.22±0.97). After each development, rods were dried hematocrit tube with a scale from 0 to 10 ml for 10 min at 60C for 5 min. Lipid classes were quanti®ed using at 2000 rpm (Sekino and Shiraiwa, 1994; Danbara and FID calibration curves. The calibration curves were Shiraiwa, 1999). Throughout the culture period, the obtained by analyzing standard compounds in the same growth rate (Kg) was calculated at each sampling inter- manner as above. The standards included 1-eicosene val according to the equation: Kg (ml-PCV/ml/d)=1/ (GL Science Co., Tokyo, Japan) as a representative of (t2t1)log(PCV2/PCV1), where t1 and t2 are culture hydrocarbons, n-hexadecan-3-one (SIGMA Chemical times (day), and PCV1 and PCV2 are packed cell Co., St Louis, MO, U.S.A.) for ketones, 1,2-dipalmi- volumes (ml/ml) at time t1 and t2, respectively. For the toyl-3-oleoyl-rac-glycerol (SIGMA Chemical) for tria- estimation of chlorophylls the algal pellet obtained by cylglycerols, cholesterol (GL Science) for sterols and l- centrifugation was suspended in the seawater medium. a-phosphatidylcholine (SIGMA Chemical) for phos- The algal suspension was disrupted ®ve times by a pholipids. l-a-Phosphatidylcholine was also used for sonicator, and then chlorophylls were extracted with the quanti®cation of chloroplast components because of acetone. The concentration of chlorophyll was deter- the lack of an authentic standard. The standard devia- mined according to Je€rey and Humphrey (1975). After tions in 15 duplicate analyses averaged 5.9% of the the cultivation, the samples were collected at intervals concentration. on pre-combusted GF/F ®lters and stored frozen at An aliquot of the lipid extract was separated into 20C for subsequent lipid analysis. three fractions [F1: 3 ml of hexane:toluene (3:1); F2: 4 ml of toluene; F3: 3 ml of toluene:methanol (3:1)] by

2.2. Analytical column chromatography (SiO2 with 5% distilled water; i.d., 5.5 mm; length, 45 mm). n-C24D50 and n-C36H74 Lipids were extracted by ®ve, 5-min rounds of ultra- were added as internal standards into the F1 (alkenes) sonication with 5 ml of dichloromethane-methanol and F2 (alkenones and alkenoates) fractions, respec- (6:4), then concentrated and passed through a short bed tively. of Na2SO4 to remove water. Another aliquot of the lipid extract was saponi®ed An aliquot of the extracted lipid was analyzed by thin with 1 ml of 0.5 mol/l KOH/methanol at 100C for 2 h layer chromatography-¯ame ionization detection (TLC- under nitrogen gas in a vacuum tube. The volume of the FID) for the determination of lipid class compositions. saponi®ed lipids was reduced, supplemented with 2 ml The analysis was conducted using an Iatroscan MK5 of distilled water, and then extracted with hexane-die- TLC-FID analyzer (Iatron Laboratories Inc., Tokyo, thyl ether (85:15) ®ve times. After the water phase was Japan). The ¯ame ionization detector was operated at a acidi®ed by adding 35% HCl, liberated lipids were hydrogen ¯ow-rate of 150 ml/min, an air ¯ow-rate of extracted with hexane-diethyl ether (85:15) ®ve times. 2000 ml/min, and a scan speed of 0.40 cm/s. Silica gel The combined extract was reduced in volume, passed

SIII Chromarods were developed with polar solvents, 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 next 6 days (phase B, corresponding to the late of toluene; S3: 3 ml of toluene-methanol (3:1)] by SiO2 logarithmic or linear phase), and retardingly afterwards column chromatography. The S3 fraction was methy- (phase C, corresponding to the retarding or stationary  lated with 14% BF3-methanol at 80 C for 15 min under phase). After day 10 at phase C, the chlorophyll con- nitrogen gas in a vacuum tube. The methylated fraction centration remained constant. This suggests that the was supplemented with 1 ml of distilled water, and increase in PCV might have been due mainly to calci®- extracted with toluene ®ve times. The extracted lipids cation, rather than to an increase in organic mass, since were condensed, passed through a short bed of Na2SO4, a limitation in nitrate and phosphorus is known to and separated into three fractions [M1: 3 ml of hex- increase the number of coccoliths per cell (Paasche, ane:toluene(3:1);M2:4mloftoluene;M3:3mloftoluene- 1998). G. oceanica grew exponentially during the ®rst 11 methanol (3:1)] by SiO2 column chromatography. n- days (logarithmic phase), and retardingly thereafter C24D50 was added as an internal standard into the M2 (stationary phase). The packed cell volume of G. ocea- (fatty acids) and M3 (sterols and a part of polyunsaturated nica was several times as large as that of E. huxleyi over fatty acids) fractions. Prior to gas chromatographic analy- the corresponding period. Acidi®cation treatment of sis, the M3 fraction was silylated with BSTFA [N,O-bis(- both species showed that the cell volume of their spher- trimethylsilyl)-tri¯uoroacetamide):pyridine (1:1)] at 70C oplasts was almost the same (data not shown). This for 30 min. indicates that the G. oceanica strain has fewer but much Gas chromatography was conducted using a Hewlett larger coccoliths than the E. huxleyi strain. Packard 5890 series II gas chromatograph (GC) with on-column injection and electron pressure control sys- 3.2. Lipid classes tems and a ¯ame ionization detector (FID). Samples were dissolved in hexane. Helium was used as a carrier The lipid contents of the E. huxleyi and G. oceanica gas, and the ¯ow velocity was maintained at 30 cm/s. strains obtained by TLC-FID varied within 7.4±13.2 mg/ The column used was a Chrompack CP-Sil5CB (length, ml-PCV and 3.4±13.3 mg/ml-PCV, respectively (Fig. 2a 60 m; i.d., 0.25 mm; thickness, 0.25 mm). For the ana- and b). The lipid content of both strains decreased lyses of F1, M2 and M3 fractions, the oven temperature rapidly during the earlier phase. The major lipid classes was programmed from 70 to 130 at 20C/min, from 130 were alkenones and alkenoates, chloroplast components to 310Cat4C/min., and then held at 310C for more (mainly pigments and glycolipids), and phospholipids, than 20 min. For the F2 fraction, the oven temperature and together these three classes made up more than was programmed from 70 to 310Cat20C/min and 84% of the total lipids (Fig. 2c and d). The minor lipid then held at 310C for 40 min. The standard deviations classes, which made up less than 8% of total lipids, K0 in 5 duplicate analyses averaged 0.008 for U37 and 7.5% included hydrocarbons, triacylglycerols and sterols. In of the concentration for C37 alkenones. G. oceanica, the relative abundance of chloroplast com- Gas chromatography±mass spectrometry was con- ponents decreased, while alkenones and alkenoates ducted using a Hewlett Packard 5973 gas chromato- increased, with growth period. The relative abundance graph±mass selective detector with on-column injection of triacylglycerols in E. huxleyi decreased with growth and electron pressure control systems and a Quadrupole period, in contrast to the increase in triacylglycerol mass spectrometer. The GC column and the oven tem- concentrations previously observed in many other mar- perature and carrier pressure programs were the same as ine microalgal species (Berkalo€ and Kadar, 1975; described above. The mass spectrometer was run in the Lichtle and Dubacq, 1984; Kuwata et al., 1993). full scan ion-monitoring mode (m/z 50±650). Electron impact spectra were obtained at 70 eV. Identi®cation of 3.3. Individual lipids compounds was achieved by comparison of their mass spectra and retention times with those of standards and The contents of total lipids obtained by GC-FID those in the literature (e.g. de Leeuw et al., 1980; (Fig. 2e and f) were in agreement with those obtained by Rechka and Maxwell, 1988). 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 3. Results times) than those measured by TLC-FID due to the lack of a suitable quantitative standard of similar chain 3.1. Packed cell volume lengths for calibration (Brown et al., 1993). The con- tents of alkenes and sterols by GC-FID di€ered from Growth curves and growth rates of E. huxleyi and G. those by TLC-FID due to the low quantitative accuracy oceanica are shown in Fig. 1. E. huxleyi grew for low-loading components in the TLC-FID analysis. exponentially during the ®rst 3 days (phase A, corre- The low triacylglycerol content indicated that most of sponding to the logarithmic phase), linearly during the the fatty acids identi®ed by GC-FID were the M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811 803

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 abundance ratios of C38:2 ethyl alkenoate to total phospholipids. alkenoates (EE/K ratio) and to C37MK (EE/K37 ratio) increased abruptly at day 8 (Fig. 3e). Both the EE/K

3.4. Alkenones and alkenoates and EE/K37 ratios changed in parallel, indicating that

Both the E. huxleyi and G. oceanica strains contain common alkenones and alkenoates (Brassell, 1993; Table 1 Conte et al., 1994). Alkenones identi®ed in the present Paleotemperature indices referred to in this paper study were C37:23 methyl alkenones (C37:23MK), Index Equation Ref.a C38:23 methyl alkenones (C38:23MK), C38:23 ethyl UK [C MK]- [C MK]/([C MK] 1 alkenones (C38:23EK) and C39:23 ethyl alkenones 37 37:2 37:4 37:2 (C EK). Alkenoates identi®ed were C methyl +[C37:3MK]+[C37:4MK]) 39:23 37:23 K0 U37 [C37:2MK]/([C37:2MK]+[C37:3MK]) 2 alkenoates and C38:2 ethyl alkenoate (EE). Concentra- K U38Me [C38:2MK]/([C38:2MK]+[C38:3MK]) 3 tion and unsaturation indices are given in Table 1. K U38Et [C38:2EK]/([C38:2EK]+[C38:3EK]) 4 The alkenone content of E. huxleyi (strain E1A) var- UK [C EK]/([C EK]+[C EK]) ied between 1.43 and 2.65 mg/ml-PCV, and showed a 39Et 39:2 39:2 39:3 K37 [C37:2MK]+[C37:3MK]+[C37:4MK] 5 decreasing trend with growth period (Fig. 2e). The K38Me [C38:2MK]+[C38:3MK] abundance ratios of lower to higher homologues of K38Et [C38:2EK]+[C38:3EK] alkenones (K37/K38Me,K37/K38Et,K37/K39Et and K38Et/ K39Et [C39:2EK]+[C39:3EK] K39Et ratios) reached maximums at days 3 and 4 (Fig. EE/K37 [C38:2EE]/([C37:2MK]+ 5 K K K [C MK]+[C MK]) 3a). The unsaturation indices (U37, U38Me, U38Et and 37:3 37:4 K EE/K [C38:2EE]/(K37+K38Me+ U39Et) varied in parallel within the range of about 0.2, K +K ) and decreased in two steps at days 8 and 16 (Fig. 3c). 38Et 39Et K The U37 ranged from 0.50 to 0.68, and the variation was a 1: Brassell et al. (1986), 2: Prahl and Wakeham (1987), 3: 0.17, corresponding to a temperature di€erence of 5.1C Conte and Eglinton (1993), 4: Conte et al. (1998), 5: Prahl et al. when the equation of Prahl et al. (1988) is applied. The (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 con- centrations 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 and C38 homologs were not detected in either strain. alkenone homologs did not have a pronounced in¯u- The alkene content of E. huxleyi decreased over the ®rst ence on the EE/ K37 ratio. 9 days and was nearly constant thereafter, while that of The alkenone content of G. oceanica (strain G1A) G. oceanica showed a di€erent trend that peaked at day varied between 0.80 and 1.71 mg/ml-PCV, and reached a 9 (Fig. 4). maximum at day 9 (Fig. 2f). The abundance ratio of lower to higher homologues of alkenones peaked at day 3.6. Fatty acids 5, decreased until day 13, and was nearly constant thereafter (Fig. 3b). The unsaturation indices varied in The n-fatty acids detected in the E. huxleyi and G. parallel within a range of about 0.13, and were at their oceanica strains included C12±C22 and C36 homologues. K0 minimum at day 9 (Fig. 3d). The U37 ranged from 0.45 The results of the TLC-FID analysis indicated that most to 0.56, and the variation (0.11) corresponded to a tem- of the n-C12±n-C22 fatty acids were the constituents of perature di€erence of 3.1C by application of the equa- glycolipids and phospholipids. They were identi®ed as tion of Prahl et al. (1988). The EE/K and EE/K37 ratios 12:0, 13:0, 14:0, 15:0, 16:0, 16:1(n-7), 17:0, 18:0, 18:1(n- increased abruptly at day 8 (Fig. 3f). 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-

3.5. Alkenes fatty acids. The n-C36 homologue comprised 36:2 and 36:3 compounds, which originated from the hydrolysis Alkenes detected in E. huxleyi and G. oceanica strains of alkenoates. Identi®cation of these compounds was include n-C21:6, n-C31:12 and n-C33:24 alkenes. The C37 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/ K0 K K K K39Et ratios) (a and b), unsaturation indices (U37, 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 5-en-3b-ol to total sterol decreased over the ®rst 9 days well as with data from the literature (Volkman et al., (Fig. 5e and f). 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 poly- 4. Discussion unsaturated n-C18 fatty acids (C18:0/C18:2,C18:0/C18:3 K0 and C18:0/C18:4 ratios) showed variations parallel to 4.1. Changes of U37 and lipid compositions with growth those of the C16/C18 ratio (Fig. 5a±d). period

3.7. Sterols Previous studies have demonstrated the changes of K0 U37 with culture age in several batch-cultured strains of 24-Methylcholesta-5,22E-dien-3b-ol and cholest-5-en- E. huxleyi (Conte et al., 1998; Epstein et al., 1998). The 3b-ol were detected in both the E. huxleyi and G. ocea- present study showed that similar changes occur for a G. nica strains. The results of the TLC-FID analysis oceanica strain. In the previous studies, the E. huxleyi indicated that most of these sterols existed in a free strains from the Iceland Basin, a Norwegian fjord and K0 form. The sterol contents of both strains changed in the Sargasso Sea showed changes of U37 in di€erent contrast to the abundance ratios of saturated to poly- growth phases (Conte et al., 1998; Epstein et al., 1998). K0 unsaturated n-C18 fatty acids (Fig. 5c±f). In both E. In contrast, the U37 did not change in the strains from huxleyi and G. oceanica, the abundance ratio of cholest- the NE Paci®c, the SW Paci®c or the SW Indian Ocean 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 di€erent strains of E. huxleyi. Our results showed that E. et al., 1998). Time series examinations by Epstein et al. huxleyi and G. oceanica strains from the same water K0 (1998) and in this study demonstrated more detailed sample demonstrated di€erent patterns of U37, most K0 variations of U37 changes in di€erent species and likely suggesting that genotypic variation is a major fac- strains. An E. huxleyi strain (CCMP372) from the Sar- tor a€ecting the pattern of physiological responses of K0 K0 gasso Sea showed lower U37 values in the logarithmic U37. K0 phase than in the stationary phase (Epstein et al., 1998). In our experiment, the range of U37 in the cultured In contrast, our E. huxleyi strain from the NW Paci®c strains of E. huxleyi was 0.50±0.68 (Fig. 3c), and the K0 showed a decreasing trend in U37 with growth period variation was 0.17, corresponding to a temperature (Fig. 3c), and our G. oceanica strain showed a minimum variation of more than 5C according to the equation of K0 U37 value in the late logarithmic phase (Fig. 3d). Prahl et al. (1988). This range is similar to that for the K0 Sawada et al. (1995) speculated that the variation of U37 of E. huxleyi (0.44±0.69) obtained from published K0  physiological responses of U37 to temperature among culture calibration equations at 19 C (Prahl et al., 1988; strains from di€erent locations was likely caused by the Sawada et al., 1996; Conte et al., 1998). The range of K0 adaptation of strains to the geographical environment U37 in the cultured strains of G. oceanica was 0.45±0.56 K0 from where the strain was sampled. This speculation (Fig. 3d). This range falls within that for the U37 of G. was based on the observation that two E. huxleyi strains oceanica (0.41±0.63) obtained from published culture from di€erent locations showed a di€erent dependence calibration equations at 19C (Volkman et al., 1995; K of U37 on temperature (Sawada et al., 1996). There are, Sawada et al., 1996; Conte et al., 1998). The combined K0 however, genotypic variations in E. huxleyi (Young and range for the U37 of E. huxleyi and G. oceanica in our Westbroek, 1991; van Bleijswijk et al., 1991). It, there- experiment (0.45±0.68) was much larger than the range K0 fore, cannot be ruled out that intraspeci®cally genotypic of U37 (0.64±0.70) obtained from published core-top di€erences may be responsible for the variation of phy- calibration equations at 19C (Sikes et al., 1991; Rosell- K0 siological responses of U37 to temperature among the Mele 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. 5,22 5 C28Á =24-methylcholesta-5,22E-dien-3b-ol, C27Á =cholest-5-en-3b-ol. et al., 1997; Herbert et al., 1998; MuÈ ller et al., 1998), but of microalgae, rapid uptake of nutrients and a high is similar to the range of scatter in individual measure- proportion of the produced organic matter sinking K0 ments of U37 values in large core-top data sets (e.g. through the water column (Eppley and Peterson, 1979). MuÈ ller et al., 1998). These ®ndings suggest that the var- The condition after upwelling-induced blooming resem- K0 iation of the U37-temperature relationship in the open bles that after the late logarithmic phase of batch cul- ocean can be attributed at least partly to the deviation ture (Takahashi et al., 1986, Hama et al., 1988). of nonthermal e€ects observed in culture experiments. Therefore, in the open marine environment, or at least Conte et al. (1998) indicated that the abundance in upwelling regions, the alkenone distributions pro- ratios of total alkenoates to total alkenones in sediments duced after the late logarithmic or linear phase in batch are approximately equal to those of E. huxleyi in the late cultures are more likely to be similar to those exported logarithmic and stationary phases, suggesting that the through the water column. However, the oligotrophic late logarithmic or stationary phase is more typical of oceans, such as the subtropical central gyre, are char- that found in the marine environment. The present acterized by constant growth of microalgae and trace study also indicates that the EE/K and EE/K37 ratios but constant levels of nutrients (Eppley and Peterson, after day 9 [average values 0.16 (n=5) and 0.18 (n=5) 1979; Goldman, 1980). In such regions, the early or mid in E. huxleyi and G. oceanica, respectively] are approxi- logarithmic phase might be a better representation of mately equal to those of the late Quaternary California the microalgal physiological status in such environ- margin sediments from upwelling region from an ments. The EE/K and EE/K37 ratios may be useful for upwelling region (av. 0.13 in ODP Site 1014, n=74; av. understanding the relationship between the type of 0.16 in Site 1016, n=93; Yamamoto and Tanaka, in marine environment and its corresponding growth prep.). Upwelling regions, where nutrients are supplied phase of the alga in batch culture. For the exact deter- massively and temporally, are characterized by blooms mination of paleotemperature, further culture studies 808 M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811 will be needed to calibrate the temperature dependence Sterol contents changed in contrast to the abundance K0 of U37 using a value in the appropriate growth phase. ratios of saturated to polyunsaturated n-C18 fatty acids K0 This study demonstrated a minimum value of U37 at (Fig. 5e and f). Sterols serve as a membrane stabilizer day 9 in the culture of E. huxleyi and G. oceanica strains ®lling the matrix of acyl chains of lipid bilayers (Alberts from the NW Paci®c (Fig. 3c and d), although E. hux- et al., 1994). The increase of cis-unsaturation of mem- leyi showed a subsequent minimum at day 17, while brane lipids requires sterols as a stabilizing material to Epstein et al. (1998) reported a minimum at day 7 in an maintain the rigidity and strengthen the membrane. E. huxleyi strain from the Sargasso Sea. Both studies The proportion of cholest-5-en-3b-ol to total sterol K0 indicate that the U37 minimum occurs at the later stage decreased with growth period (Fig. 5e and f). Volkman of the logarithmic phase. In our experiments, the decline et al. (1981) reported that cholest-5-en-3b-ol is more K0 of U37 started when the concentrations of the cells abundant in the motile cells than the sessile cells of E. exceeded about 0.2 ml-PCV/ml-medium both in E. hux- huxleyi. Microscopic observation in the present study leyi and G. oceanica (Fig. 1). Cell concentration and the showed that all the cells existed in the sessile form. This, culture environment change relatively slowly during early along with the result that both E. huxleyi and G. ocea- exponential growth, but very rapidly during late expo- nica showed the same trends in sterol composition, nential growth, so therefore the cell physiology could implies that the changes of sterol composition depend exhibit some changes before the onset of the stationary on the changes of growth status as well as the life cycle. phase (Darley, 1982). Epstein et al. (1998) suggested that The degree of unsaturation increased both in alke- K0 nitrate depletion decreases U37 by unknown mechanisms. nones and n-fatty acids at almost the same period in the Limitations in nitrate, as well as phosphorus, concentra- later stage of the logarithmic phase, implying that a tions is known to a€ect coccolith formation and cell common factor was involved in the formation of unsa- replication in batch and chemostat cultures (Paasche, turation. Consequently, examination into the factors 1998). Therefore, changes in alkenone production may be controlling the formation of n-fatty acid unsaturation a€ected by changes in cellular metabolism. should provide clues to understanding the physiological K0 In this study, the U37 decrease at day 8 occurred in factors controlling alkenone unsaturation indices. association with the continuous decreases of the abun- Known factors a€ecting the unsaturation of plant n- dance ratios of lower to higher homologues of fatty acids include temperature (Sato and Murata, alkenones (K37/K38Me,K37/K38Et,K37/K39Et and K38Et/ 1980), O2 (Harris and James, 1969; Rebeille et al., 1980), K39Et ratios) around day 8 and the rapid increases of the CO2 (Sato, 1989; Tsuzuki et al., 1990; Revill et al., abundance ratio of C38:2 ethyl alkenoate to total 1999), and nutrient concentrations (Chuecas and Riley, alkenoates (EE/K ratio) and C37 alkenones (EE/K37 1969; Kuwata et al., 1993). Among these factors, it is ratio) at day 8 (Fig. 3). These associated changes can be possible that the de®ciencies of CO2 and/or nutrients attributed to the physiological response of lipids for occurred after the later stage of the logarithmic phase in maintaining their ¯uidity in the isothermal culture, since our experiment. Recently, Revill et al. (1999) reported higher homologues have higher melting points in most that although the unsaturation and carbon number of n- cases, and unsaturation formation decreases the melting fatty acids in E. huxleyi increased with decreasing aqu- K0 point (Larsson and Quinn, 1994). This tendency, how- eous CO2 concentration, the U37 did not change K0 ever, might not be generalized. After day 11, U37 gra- signi®cantly, suggesting that CO2 de®ciency is less likely dually increased, but this change was not associated to have an e€ect on the alkenone unsaturation. There with any increase in the abundance ratios of lower to are contradictory reports on the e€ect of nutrient higher homologues of alkenones or to any decreases in concentrations. Chuecas and Riley (1969) showed that the EE/K or EE/K37 ratios (Fig. 3). This should result in cultured microalgae produced more abundant poly- the increase of melting point of bulk alkenones and unsaturated fatty acids under nutrient-rich conditions. alkenoates, which cannot be simply explained by the But this ®nding presumably re¯ects the high proportion physiological response mentioned above. of membrane lipids to storage lipids in the algae grown Parallel changes of carbon numbers and unsaturation in nutrient-rich medium, because membrane lipids are degree were observed in n-fatty acids (Fig. 5). The richer in polyunsaturated fatty acids than storage lipids. decrease of C16/C18 ratio was accompanied with In contrast, Kuwata et al. (1993) found that, both in decreases in the abundance ratios of saturated to poly- neutral and polar lipids of a diatom Chaetoceros pseu- unsaturated n-C18 fatty acids (C18:0/C18:2,C18:0/C18:3 docurvisetus, polyunsaturated n-fatty acids were more and C18:0/C18:4 ratios). TLC-FID analysis indicated that highly accumulated in resting spores and resting cells most of the n-fatty acids detected were the constituents that grew under condition of nutrient depletion than in of membrane lipids such as glycolipids and phospholi- vegetative cells that grew in a nutrient-sucient med- pids. The associated changes were attributed to the ium. They speculated that the diatom consumes the physiological response of lipids for maintaining the excess ATP and NAD(P)H produced under nutrient ¯uidity of membranes. 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 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 these Gephyrocapsa species have an alkenone composition similar to that of the present E. huxleyi.

5. Conclusions

K0 U37 changed with growth status both in E. huxleyi Fig. 6. A plot of batch-cultured E. huxleyi (solid circle) and G. and G. oceanica from the NW Paci®c. The patterns of K0 k oceanica (solid square) on the U37-U38Et diagram. these changes were di€erent, suggesting that the geno- typic variation is a major factor in determining the pat- K0 avoid lethal photochemical damage. We consider that tern of physiological responses of U37. the nutrient depletion is more likely to cause the The C38:2 ethyl alkenoate/total alkenone (EE/K) and increase in unsaturation of alkenones and n-fatty acids, C38:2 ethyl alkenonate/C37 alkenone (EE/K37) ratios as suggested by Epstein et al. (1998), although further increased abruptly at day 8 (the later stage of the loga- studies will be needed on the physiological e€ects of rithmic phase) in both strains. These ratios should be

CO2 and nutrients on the alkenone and n-fatty acid useful in clarifying the relationship between the marine unsaturation. environment and its corresponding growth phase of In the culture of G. oceanica, the relative proportions batch culture. K0 of alkenones and alkenoates to total lipids increased Changes of U37 were associated with those of the gradually after day 6, while the relative abundances of unsaturation indices of C38 and C39 alkenones, the phospholipids and chloroplast lipids decreased during abundance ratios of lower to higher homologues of the same period (Fig. 2d). This indicates that the meta- alkenones, the abundance ratios of saturate to poly- bolically-active membrane lipids decreased due to the unsaturated n-fatty acids, EE/K and EE/K37 ratios and changes of metabolic balance. It also implies that alke- sterol contents. These associated changes are attributed nones are metabolically-inactive, and behave as a sto- partly to the physiological response of lipids for main- rage lipid or a byproduct of metabolism, although this is taining their ¯uidity. still speculative and remains to be proven. The increase in the degree of unsaturation both in alkenones and n-fatty acids at almost the same period 4.2. Di€erences between E. huxleyi and G. oceanica implies that a common factor, possibly nutrient deple- tion, a€ects lipid unsaturation.

C37/C38 alkenone and EE/K37 ratios have been pro- The present results indicate that E. huxleyi and G. K0 posed to distinguish the contribution of E. huxleyi to oceanica can be e€ectively distinguished using the U37- K sedimentary alkenones from that of G. oceanica (Volk- U38Et diagram. man et al., 1995; Sawada et al., 1996). The present ®ndings, and those of Conte et al. (1998), indicate that these ratios are a€ected by the growth phase of batch Acknowledgements culture rather than species di€erence. Conte et al. (1998) demonstrated that both species can be distinguished on We thank Ms. Kazuko Hino (Geological Survey of K K the U38Me-U38Et diagram. In the present study, both spe- Japan) for her analytical assistance in the laboratory cies were better distinguished on the diagram similar to and Dr. Ken Sawada (University of Tsukuba) for his K K that in Conte et al., (1998), i.e., the U37- U38Et diagram valuable input. Special thanks are due to Dr. Hajime (Fig. 6). Unexpectedly, most California margin sedi- Kayanne and Mr. Kouji Hata (University of Tokyo) for ment samples from the late Miocene to Recent (ODP their help with the sampling, and to Drs. Yuichiro Sites 1014 and 1016) are distributed in the region of E. Tanaka and Masatoshi Komiya (GSJ) for their useful huxleyi, not G. oceanica (Yamamoto and Tanaka, comments. The helpful reviews by Drs. Bonnie L. unpublished data). This indicates that, even though the Epstein and Hanno Kinkel and the editorial comments 810 M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811 by Dr. John K. Volkman improved the quality of this Edvandersen, B., Eikrem, W., Green, J.C., Andersen, R.A., manuscript. This study was ®nancially supported by the Moon-van der Staay, S.Y., Medlin, L.K., 2000. Phylogenetic Science and Technology Agency of Japan. reconstructions of the Haptophyta inferred from 18S ribo- somal DNA sequences and available morphological data. Associate EditorÐJ. Volkman Phycologia 39, 19±35. Eppley, R.W., Peterson, B.J., 1979. Particulate organic matter ¯ux and planktonic new production in the deep ocean. Nat- ure 282, 677±680. References Epstein, B.L., D'Hondt, S., Quinn, J.G., Zhang, J., Hargraves, P.E., 1998. An e€ect of dissolved nutrient concentrations on Alberts, B., Bray, D., Lewis, J., Ra€, M., Roberts, K., Watson, alkenone-based temperature estimates. Paleoceanography J.D., 1994. Molecular Biology of the Cell, 3rd Edition. Gar- 13, 122±126. land Publishing, New York. Goldman, J.C., 1980. Physiological processes, nutrient avail- Berkalo€, C., Kader, J.C., 1975. Variations of the lipid com- ability, and the concept of relative growth rate in marine position during the formation of cysts in the green alga Pro- phytoplankton ecology. In: Falkowski, P.G. (Ed.), Primary tosiphon botryodes. Phytochemistry 14, 2353±2355. Production in the Sea. Plenum Press, New York, pp. 179±193. van Bleijswijk, J., van der Wal, P., Kempers, R., Veldhuis, M., Hama, T., Handa, N., Takahashi, M., Whitney, F., Wong, Young, J.R., Muyzer, G. et al., 1991. Distribution of two C.S., 1988. Change in distribution patterns of photo- types of Emiliania huxleyi () in the north- synthetically incorporated C during phytoplankton bloom in west Atlantic region as determined by immuno¯uorescence controlled experimental ecosystem. Journal of Experimental and coccolith morphology. Journal of Phycology 27, 566±570. Marine Biology and Ecology 120, 39±56. Brassell, S.C., 1993. Applications of biomarkers for delineating Harris, P., James, A.T., 1969. The e€ect of low temperatures on marine paleoclimatic ¯uctuations during the Pleistocene. In: fatty acid biosynthesis in plants. Biochemical Journal 112, Engel, M.H., Macko, S.A. (Eds.), Organic Geochemistry, 325±330. Chapter 34, Plenum Press, New York, pp. 699±738. Herbert, T.D., Schu€ert, J.D., Thomas, D., Lange, C., Wein- Brassell, S.C., Eglinton, G., Marlowe, I.T., P¯aumann, U., heimer, A., Peleo-Alampay, A., Herguera, J.-C., 1998. Depth Sarnthein, M., 1986. Molecular stratigraphy: a new tool for and seasonality of alkenone production along the California climatic assessment. Nature 320, 129±133. margin inferred from a core top transect. Paleoceanography Brown, M.R., Dunstan, G.A., Je€rey, S.W., Volkman, J.K., 13, 263±271. Barrett, S.M., LeRoi, J.-M., 1993. The in¯uence of irradiance Je€rey, S.W., Humphrey, G.F., 1975. New spectrophotometric

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