Production rates of C
37
alkenones determined by
13
C-labeling
technique in the euphotic zone of Sagami Bay, Japan
Junko Hamanaka
a,*, Ken Sawada
b, Eiichiro Tanoue
aaInstitute for Hydrospheric-Atmospheric Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
bDepartment of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
Received 22 February 2000; accepted 17 August 2000 (returned to author for revision 9 May 2000)
Abstract
The production rates of C37alkenones in the euphotic zone of Sagami Bay, Japan were determined by the 13
C-labeling technique in conjunction with gas chromatography±mass spectrometry. The maximum in the alkenone pro-duction rate (C37:2plus C37:3alkenones) was observed at 5 m depth and the speci®c production rate, calculated from
the rate of production relative to standing stock in suspended particles, was 0.64 dayÿ1at this depth. Temperature,
based on the newly-produced alkenone unsaturation index (Uk0
37), was close to in situ temperatures at the depth where
alkenones production was active.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Alkenone;13C-labeling technique; Compound-speci®c production rate; Production rate based-Uk0
37; Sinking ¯ux; Euphotic zone
1. Introduction
Long-chain (C37±C39) unsaturated ketones (alkenones)
have generally been found in particulate organic matter in seawater and marine sediments, and derive from some Haptophycean algae (familyGephyrocapsaceae and Iso-chrysidaceae; e.g. Volkman et al., 1980, 1995; Marlowe et al., 1984, 1990). Since this group of algae, particularly
Emiliania huxleyi, comprises one of the main groups of calcifying organisms, there has been much interest in the production and downward ¯ux in terms of the carbon cycle in the ocean (e.g. Holligan et al., 1993; Sikes and Fabry, 1994). The abundance of alkenones in marine sediments has been used for reconstructing past changes in the productivity of the alkenone-producing algae (e.g. Prahl et al., 1989; Jasper and Gagosian, 1993; Martinez et al., 1996). Alkenones are also widely used as a paleo-thermometer for reconstructing past sea surface tem-peratures (e.g. Brassell et al., 1986a,b; Prahl and
Wakeham, 1987). There have been, however, few studies concerning the productivity, the stability and the rela-tionship between production and vertical ¯ux of alke-nones in surface water environments.
The purposes of this study were to evaluate the pro-duction rates of C37 alkenones (alkadien-2-one and
alkatrien-2-one: C37:2and C37:3alkenones, respectively)
using a13C-labeling technique in conjunction with gas
chromatography±mass spectrometry (GC±MS) in natural assemblages of alkenone-producing algae in the euphotic zone. From comparison of alkenone-speci®c productivity, sinking ¯ux obtained using surface sediment traps and standing stock, we discuss production and vertical transport of alkenones in a surface water column.
2. Experimental
2.1. Field experiments
Sampling and ®eld experiments were conducted at a central site in Sagami Bay (35N, 139200E) during
the cruise of the R.V. Tansei Maru (Ocean Research Institute, University of Tokyo) on 31 May±6 June 1995
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 1 1 8 - 2
www.elsevier.nl/locate/orggeochem
* Corresponding author. Tel.: 52-789-3475; fax: +81-52-789-3436.
(KT-95-8). The site is located ca. 20 km o Cape Kawana on the Izu Peninsula at a water depth of ca. 1500 m.
Seawater samples were collected from depths of 5, 10, 25, 40 and 60 m with Niskin water samplers equipped with Te¯on-coated stainless-steel springs at 04:30 (local time) on 1 June. A surface water sample (0±1 m depth) was collected using a plastic bucket. Samples of sea-water (each 9 l) were taken for determination of con-centrations and natural 13C atom% of suspended
particles. Duplicate samples of seawater (each 9 l) for incubations were transferred into 9 l acid-cleaned poly-carbonate bottles and then spiked with 13C-labeled
sodium bicarbonate at a concentration of 7.0 at.% of the dissolved inorganic carbon in the samples. The bottles were suspended at each corresponding depth along a ¯oating array (in situ incubation). Cylinder-type sedi-ment traps (size; 6.5 cm inner diameter, 62 cm height) were placed at depths of 15, 30, 45 and 65 m on the same array as used in the incubation experiment. No poisons or preservatives were used. The ¯oating array was set at 07:30 (local time) on 1 June and recovered at 05:30 (local time) on 2 June. All samples were ®ltered on to pre-combusted (450C, 4 h) glass ®ber ®lters (Whatman GF/
F) and stored at ÿ20C until analysis. Zooplankton
swimmers in traps were carefully picked out.
Water temperature, salinity and irradiance of photo-synthetically available radiation (PAR) were measured by the OCTOPUS system (Ishimaru et al., 1984). Concentra-tions of nutrients and chlorophylla(Chla) were measured by an Autoanalyser AA-2 (Technicone) and a Turner Designs ¯uorometer after extraction with N,N -dimethyl-formamide (Suzuki and Ishimaru, 1990), respectively.
2.2. Lipid extraction and gas chromatographic (GC) analysis
Lipids were extracted in chloroform/methanol (2:1, v/ v) using high energy ultrasonication. Henicosanoic acid (n-C21:0fatty acid) was added as internal standard prior
to extraction. The extraction was repeated three times, and the lipid-containing chloroform fraction was separated from non-lipid components by washing with Milli-Q water. The lipid extract was passed through Na2SO4and
evaporated to dryness. Lipids were transesteri®ed with 3±5% (w/v) HCl-methanol at 85C for 2.5 h, and the
products were extracted into hexane.
GC analysis was performed on a Shimadzu GC-9A chromatograph equipped with a ¯ame ionization detector (FID) and a fused silica capillary column (50 m0.32 mm i.d. CPSil5CB, Chrompack) as described elsewhere (Sawada et al., 1998). Helium was used as the carrier gas. The oven temperature was programmed from 150to
320C at 5C minÿ1and then maintained at 320C for 30
min. Identi®cation of C37 alkenones was con®rmed by
mass spectra and retention times of these compounds obtained from a cultured sample of E. huxleyi (strain
EH2). The yield obtained from the exogenously added henicosanoic acid was 973% (average1 S.D., n=3) and precision of GC measurement was3% (1 S.D.,
n=10).
2.3. 13C-labeling technique
The production rates of speci®c organic compounds were determined by the 13C-labeling technique as
described elsewhere (Hama et al., 1987, 1993) and described here only brie¯y. The13C atom% of each
alke-none was determined by GC±MS (Varian 3400 chroma-tograph coupled to a Finnigan MAT SSQ-7000 mass spectrometer). Chemical ionization (CI) with isobutane as the reagent gas was used to obtain the quasi-molecular ion peak. The following analytical conditions were used: electron energy, 200 eV; emission current, 300 mA; ion
source temperature, 230C; mass scan,m/z500±600 per
0.5 s. The GC conditions were the same as mentioned above. Measurement of alkenones required that the transfer line between the systems of GC and MS be held at 340C. The13C atom% of each alkenone was calculated
from the relative ratios of isotopic ion peaks to the quasi-molecular ion peak according to Kouchi (1982). Dis-crimination of13C was not considered in this study. The
precision of the 13C atom%, based on GC±MS
mea-surement of alkenone in the samples with a natural13C
ratio, was0.02 at.% (1 S.D.,n=7). The accuracy of the measurement of the enrichment13C atom% in the present
study was determined by measurement of ®ve13C-enriched
fatty acid standards (13C abundance from 1.98 to 4.73
at.%) prepared by diluting 13C labeled [1-13C]palmitic
acid (99.3 at.%, mass Trace) with palmitic acid with a natural 13C ratio (1.11 at.%). The mean value of the
atom% of each enrichment standard was determined within 3% (1 S.D.,n=5) of the theoretical values.
The alkenone production rate was calculated from
13C atom% and the concentration of each alkenone
according to Hama et al. (1987), who applied the method to measure amino acid production rate. The carbon production rate of each alkenone was calculated as follows: production rate (ng C lÿ1dayÿ1)=(a
isÿans)/
(aicÿans)AlkC/t, whereaisis13C atom% in the
alke-none in the incubated sample,ansis13C atom% of the
alkenone in the natural sample,aicis13C atom% in the 13C-enriched inorganic carbon, AlkC is the carbon
con-centration of the alkenone at the end of incubation (ng C lÿ1), andtis the duration of incubation (day).
3. Results and discussion
3.1. Physicochemical conditions at study site
that opens southward to the Kuroshio region in the North Paci®c. The water mass of the bay is strongly in¯uenced by the Kuroshio Current and the warm and oligotrophic oceanic water originating from the Kur-oshio region are mixed with coastal waters. Fig. 1 shows water temperature, salinity, density, PAR and con-centrations of Chl aand nitrate plus nitrite at the site. The euphotic zone, de®ned as the 1% level of surface PAR, extended to 32 m depth. Chl a concentrations were high (3.8 mg lÿ1) in the upper 5 m and gradually
decreased with increasing depth. The nitrate plus nitrite concentration in the euphotic zone was low at ca. 1mmol
lÿ1. Water temperature was 19.9C at the surface and
gradually decreased to 15.8C at 70 m, and the water of
the upper euphotic zone was characterized by low salinity and low density. Judging from the data from time-series research (Kanagawa Prefectural Fisheries Research Insti-tute, unpub. results), the seasonal halocline around 20 m (Fig. 1a) had been developed at least two weeks pre-vious to the present observation with the increase of the
in¯ow of coastal water (Furushima and Sugimoto, 1994).
3.2. Alkenone-speci®c production rate determined by the
13C-labeling technique
Mass spectra of the C37:2and C37:3alkenones in
sus-pended particles were obtained by GC±MS (Fig. 2). The base peaks atm/z532 and 530 are the quasi-molecular ions of the C37:2and C37:3alkenones, respectively. The
relative intensity of the isotope peak atm/z533 for the C37:2alkenone was 0.380 in the natural sample from 5 m
depth (Fig. 2a) and increased to 0.556 in the incubated sample at the same depth due to the incorporation of
13C (Fig. 2b). The relative intensity in the incubated
sample from 25 m depth was, on the other hand, increased slightly to 0.410 (Fig. 2c). The relative intensity of the isotope peak (m/z531) of the C37:3alkenone in
the incubated sample from 5 m depth also increased during incubation and the intensity in the incubated
sample from 25 m depth was minimally increased (Fig. 2d±f). The13C atom% of the C
37:2and C37:3alkenones
in the incubated sample at 5 m depth was calculated to be 3.2 and 2.9 atom%, respectively. The values were 1.3 and 1.2 atom%, respectively, for the sample incubated at 25 m depth.
The production rates of the C37alkenones were
cal-culated from their concentrations and13C atom% in the
incubated samples (Fig. 3a and Table 1). The maxima in
the C37:2 and C37:3 alkenone production rates were
observed at 5 m depth and the summed rate (C37:2plus
C37:3alkenones) was 269 ng lÿ1dayÿ1. The value rapidly
decreased even within the euphotic zone to a very low value of 1.4 ng lÿ1dayÿ1at 25 m depth. Maximum Chla
concentrations were observed over the upper 5 m (Fig. 1b), indicating that the depth of the highest production rate of C37alkenones did not coincide with that of Chla.
The Chlaconcentration at 25 m was about two-thirds that at 5 m (Fig. 1b), whereas the C37 alkenones
con-centration at 25 m was about one-tenth that at 5 m and the C37 alkenones production rate at 25 m was about
one two-hundredth the rate at 5 m (Fig. 3a and b). These facts suggest that alkenones were produced in a narrow layer in the upper euphotic zone, particularly around 5 m depth.
It is noteworthy that the vertical pro®les of the C37
alkenones concentrations in suspended particles were dierent from the production rates (Fig. 3a and b). The concentrations in suspended particles ranged from 36 to 420 ng lÿ1and exhibited two maxima. The peak at 5 m
coincided with the maximum in alkenones production rate and Chl aconcentration, while the peak at 40 m depth was located below the euphotic zone.
Speci®c production rates of C37alkenones were
cal-culated from the rate of production relative to standing stock in suspended particles (Table 1). The maximum value of the speci®c production rate was 0.64 dayÿ1at 5
m depth, corresponding to the depth of active alkenone production (Fig. 3a), while the rate was less than 0.04 dayÿ1 below 25 m. The speci®c production rate at a
given depth can be considered to be related to the activity of alkenone-producing algae at that depth. Table 1 indicates that alkenone-producing algae were most active at 5 m depth and newly produced alkenones were mainly distributed in the upper euphotic zone. Although the origin of the alkenones distributed below 25 m is unclear at present, they probably derived from detritus or senescent cells of alkenone-producing algae.
3.3. Analyses combined with the sediment trap experiment
Sinking particles collected by sediment traps were analyzed for vertical ¯uxes of C37 alkenones (Fig. 3c).
The largest summed vertical ¯ux was 0.62 mg mÿ2
dayÿ1, which occurred at 15 m depth, just below the
alkenone productive layer and in the seasonal halocline. The value rapidly decreased by more than a factor of two from 15 m to the base of the euphotic zone (30 m) and then remained relatively constant from 30 to 65 m, ranging from 0.18 to 0.22 mg mÿ2dayÿ1.
It is dicult to know the accurate vertical ¯ux out of the euphotic zone because there have been growing concerns about the uncertainties of ¯uxes measured with sediment traps in the upper ocean (e.g. Buesseler,
Fig. 2. Partial CI mass spectra of C37:2and C37:3alkenones
1991; Gardner, 1995). However, a ®rst-order approx-imation of the comparison between the production and vertical ¯ux of the C37alkenones is possible. We
eval-uated the sinking rate of C37alkenones at the base of
the euphotic zone relative to the standing stock in the
euphotic zone, assuming the dynamics of alkenones were in steady state, and de®ned it as the speci®c sinking rate in the present study. The speci®c sinking rate from the euphotic zone was tentatively calculated to be 0.04 dayÿ1. The value is one order of magnitude lower than
the value of the speci®c production rate in the euphotic zone (Table 1), indicating that the majority of the newly produced alkenones were not vertically transported. The value was similar to that of bulk POC (Table 1, footnote e). Thus, the rate of removal of alkenone from the euphotic zone was not dierent from the value of bulk POC, whereas the alkenone-speci®c production rate was about four times higher than the value of bulk POC. Indirect evidence indicated that the newly produced alkenones were actively degraded on a daily basis as compared with bulk POC. The present observation veri®es the previous suggestion that alkenones are not stable relative to other organic constituents of sus-pended particles in a surface water column (Conte et al., 1992; Freemann and Wakeham, 1992; Ternois et al., 1997; Sawada et al., 1998).
3.4. Relationship between production rate and Uk0
37
The alkenone unsaturation index (Uk0
37) is calculated
for suspended particles using the equation of Prahl and Wakeham (1987), i.e. Uk0
37=[C37:2]/([C37:2]+[C37:3]),
where [C37:2] and [C37:3] are the concentrations of C37:2
and C37:3alkenones, respectively. In this study, we also
calculated the Uk0
37 value from the production rates of
C37:2and C37:3alkenones determined by the13C-labeling
Fig. 3. Vertical pro®les of C37alkenones: (a) production rate, (b) concentration in suspended particles and (c) downward ¯ux. The
production rate at 10 m depth is missing due to the limitation of GC±MS measurement. Stippled shading indicates depth of <1% light penetration.
Table 1
Production rate, speci®c production rate of C37alkenones and
PR-Uk0
37in the euphotic zone
Depth (m) Production rate
(ng lÿ1dayÿ1)
Speci®c production ratea(dayÿ1)
PR-Uk0 37b
0 24.8 0.36 0.81
5 268.9 0.64 0.62
10 ndc ± ±
25 1.4 0.04 0.78
Euphotic zone-integratede
3.45d 0.54 0.63
a Production rate of the speci®c compound/standing stock of
the corresponding compound.
b [C
37:2Prod]/([C37:2Prod]+[C37:3Prod]), where [C37:2Prod]
and [C37:3Prod] are the production rates of C37:2 and C37:3
alkenones (ng lÿ1 dayÿ1or mg mÿ2dayÿ1), respectively. The
error of the PR-Uk0
37value with GC and GC±MS measurements
was0.02.
c Not detected. d mg mÿ2dayÿ1.
e Euphotic zone-integrated speci®c production rate and
sinking rate of bulk POC were 0.14 and 0.04 dayÿ1, respectively
technique; this is de®ned as `production rate based-Uk0 37
(PR-Uk0
37)'. PR-Uk 0
37 values varied with depth, ranging
from 0.62 to 0.81 even in the euphotic zone (Table 1) and the euphotic zone-integrated value was 0.63. For conversion of this value into growth temperature, 10 reported regression lines of Uk0
37 temperature
calibra-tions, were derived from culture experiments using nine strains of alkenone-producing algae under logarithmic growth phase, were applied (Table 2). The value at 5 m depth was estimated to be 202C, virtually identical to
the in situ temperature of 19.8C (Fig. 1a and Table 2).
However, the values at 0 and 25 m depths did not agree with the in situ temperature and were consistently high (Table 2).
Since the PR-Uk0
37value was calculated from the newly
produced alkenones during the period of incubation and was independent of alkenone composition of the bulk cells and detritus of alkenone-producing algae, the variability of the value only depends on the physiology of the algae, in terms of the biosynthesis of alkenones. It has been reported from culture experiments that Uk0
37
values varied due to dierences in stage of the growth cycle or growth conditions; however, a relationship between Uk0
37 values and algal physiological conditions
seems to be complicated. For example, the batch culture experiments suggested that Uk0
37 value can be strongly
aected by variations in the growth status of cells: the Uk0
37value of some strains ofE. huxleyifor cells in their
logarithmic growth phase or at high growth rate was low as compared with the value for their stationary
growth phase or low growth rate [0.20 lower shown by Epstein et al. (1998)], whereas the value decreased by as much as 0.2 when cells entered the late logarithmic growth phase in some strains ofE. huxleyi(Conte et al., 1998). On the other hand, a small change in Uk0
37value
can result from changes in nutrient-limited growth rate in chemostat cultures (Popp et al., 1998).
In the natural environment, it is dicult to know the in situ physiological state of alkenone-producing algae. However, it is suggested from the alkenone-speci®c production rate that alkenone-producing algae were active at 5 m but inactive at 0 and 25 m (Table 1). The fact that temperature estimated from the PR-Uk'
37values
agreed with in situ temperature at 5 m, but not at 0 and 25 m indicates that the regression lines of Uk0
37-based
temperature obtained from culture experiments can be applied to natural assemblages of alkenone-producing algae. For accurate estimation of in situ temperature using regression lines of Uk0
37-based temperature obtained from
culture experiments, consideration of the speci®c produc-tion rate of alkenones and the PR-Uk0
37value is useful.
Fig. 4 shows vertical pro®les of Uk0
37values in suspended
and sinking particles. Values in suspended particles varied widely from 0.47 to 0.63 through the water column. The values in suspended particles do not correspond to the PR-Uk0
37values and the dierence between Uk 0
37and
PR-Uk0
37value was 0.05 even at the alkenone productive depth
(5 m). It is reasonable to assume that detritus of alkenone-producing algae contributed to the suspended particles because the alkenone-speci®c production rate was 0.64
Table 2 PR-Uk0
37based temperature calculated by regressions obtained from the cultures ofEmiliana huxleyiandGephyrocapsa oceanicastrains
harvested in log-phase
PR-Uk0
37based temperature (C)
0 m 5 m 25 m Regression line of Uk0
37vs. growth temp. Refs.
E. huxleyi
23.2 17.5 22.3 0.033T+0.043 Prahl and Wakeham (1987)
22.7 17.1 21.8 0.034T+0.039 Prahl et al. (1988)
23.8 19.8 23.2 0.151+0.004T+0.001T2 Conte et al. (1998)
23.4 19.8 22.9 0.053+0.009T+0.001T2 Conte et al. (1998)
27.6 23.9 27.0 0.025+0.001T+0.001T2 Conte et al. (1998)
26.5 20.2 25.5 0.014+0.03T Conte et al. (1998)
22 19 21 1.322ÿ0.283T+0.020T2ÿ0.0004T3
(1.322ÿ0.283T+0.02028T2
ÿ0.00039T3?)
Conte et al. (1998)
G. oceanica
27.2 23.3 26.6 0.049Tÿ0.52 Volkman et al. (1995)
23.1 18.8 22.4 0.044Tÿ0.204 Sawada et al. (1996)
22 19 21 1.470ÿ0.241T+0.015T2
ÿ0.0003T3
In situ CTD temperature (C)
dayÿ1at 5 m. The water in the upper euphotic zone had
been in¯uenced by the in¯ow of low salinity coastal water as mentioned above (Fig. 1). Some of the alke-nones in suspended particles might be transported lat-erally into the study site along the in¯ow of coastal water masses. On the other hand, Uk0
37values in sinking
particles are close to the value of PR-Uk0
37at 5 m depth
and are relatively constant through the surface water column. The results indicate that alkenones, entrapped in sinking particles at the depth where alkenones pro-duction was active, were vertically transported through the surface water column.
4. Conclusions
The production rates of C37 alkenones in natural
assemblages of alkenone-producing algae were deter-mined by the13C-labeling technique in conjunction with
GC±MS in the euphotic zone of Sagami Bay, Japan. The maxima in the C37:2and C37:3alkenones production
rates (C37:2plus C37:3alkenones: 269 ng lÿ1dayÿ1) were
observed at 5 m and the value rapidly decreased to low values of 1.4 ng lÿ1dayÿ1even within the euphotic zone
(above 32 m depth). The alkenone-speci®c production rate, calculated from the rate of production relative to standing stock in suspended particles, ranged from 0.04 to 0.64 dayÿ1in the euphotic zone and was highest at 5
m depth. From the comparison between the alkenone-speci®c production rate and sinking loss rate determined by sediment traps, it was concluded that alkenones were not stable relative to other organic constituents in sus-pended particles in the euphotic zone. We calculated the Uk0
37value from the production rates of alkenones and
de®ned it as `production rate based-Uk0
37 (PR-Uk 0 37)5.
Temperature based on the PR-Uk0
37value using reported
regression lines obtained from culture experiments under logarithmic growth phase was close to the in situ temperature at 5 m depth, where the highest speci®c production rate was recorded. The Uk0
37values in sinking
particles collected by sediment traps through the surface water column were rather closer to the PR-Uk0
37 at the
alkenones-productive depth than the Uk0
37values in
sus-pended particles in the euphotic zone. It is suggested that the newly produced alkenones entrapped in sinking particles were preferentially transported through the surface water column.
Acknowledgements
We are grateful to N. Handa, T. Hama, T. Nishida, the ocers and crew of the R.V.Tansei Maru(KT-95-8) for support during the observations, and T. Saino for giv-ing the sediment trap samples, and T. Tanaka and Y. Mino for nutrient and Chl a analysis. Kanagawa Prefectural Fisheries Research Institute kindly provided the ocea-nographic data set of time-series research in Sagami Bay. S. McElroy helped in editing an early version of this manuscript. We acknowledge two anonymous referees for constructive comments and J.R. Maxwell for editorial help. Partial support of this study was pro-vided by a Grant-in-Aid from MESC (nos 10440161 and 11878088), Japan.
Associate EditorÐJ. Maxwell
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