Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue11.Nov2000:
Organic Geochemistry 31 (2000) 1095±1102
www.elsevier.nl/locate/orggeochem
Production rates of C37 alkenones determined by 13C-labeling
technique in the euphotic zone of Sagami Bay, Japan
Junko Hamanaka a,*, Ken Sawada b, Eiichiro Tanoue a
a
Institute for Hydrospheric-Atmospheric Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
b
Department 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 C37 alkenones in the euphotic zone of Sagami Bay, Japan were determined by the 13Clabeling technique in conjunction with gas chromatography±mass spectrometry. The maximum in the alkenone production rate (C37:2 plus C37:3 alkenones) 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 dayÿ1 at this depth. Temperature,
0
based on the newly-produced alkenone unsaturation index (Uk37), was close to in situ temperatures at the depth where
alkenones production was active. # 2000 Elsevier Science Ltd. All rights reserved.
0
Keywords: Alkenone; 13C-labeling technique; Compound-speci®c production rate; Production rate based-Uk37; 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 (family Gephyrocapsaceae and Isochrysidaceae; 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 paleothermometer for reconstructing past sea surface temperatures (e.g. Brassell et al., 1986a,b; Prahl and
* Corresponding author. Tel.: +81-52-789-3475; fax: +8152-789-3436.
E-mail address: [email protected] u.ac.jp
(J. Hamanaka).
Wakeham, 1987). There have been, however, few studies
concerning the productivity, the stability and the relationship between production and vertical ¯ux of alkenones in surface water environments.
The purposes of this study were to evaluate the production rates of C37 alkenones (alkadien-2-one and
alkatrien-2-one: C37:2 and C37:3 alkenones, respectively)
using a 13C-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 (35 N, 139 200 E) 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.
PII: S0146-6380(00)00118-2
1096
J. Hamanaka et al. / Organic Geochemistry 31 (2000) 1095±1102
(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 seawater (each 9 l) were taken for determination of concentrations and natural 13C atom% of suspended
particles. Duplicate samples of seawater (each 9 l) for
incubations were transferred into 9 l acid-cleaned polycarbonate 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 sediment 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 precombusted (450 C, 4 h) glass ®ber ®lters (Whatman GF/
F) and stored at ÿ20 C until analysis. Zooplankton
swimmers in traps were carefully picked out.
Water temperature, salinity and irradiance of photosynthetically available radiation (PAR) were measured by
the OCTOPUS system (Ishimaru et al., 1984). Concentrations of nutrients and chlorophyll a (Chl a) were measured
by an Autoanalyser AA-2 (Technicone) and a Turner
Designs ¯uorometer after extraction with N,N-dimethylformamide (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-C21:0 fatty 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 Na2SO4 and
evaporated to dryness. Lipids were transesteri®ed with
3±5% (w/v) HCl-methanol at 85 C 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 150 to
320 C at 5 C minÿ1 and then maintained at 320 C 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 was 3% (1 S.D.,
n=10).
2.3.
13
C-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. The 13C atom% of each alkenone was determined by GC±MS (Varian 3400 chromatograph 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, 230 C; mass scan, m/z 500±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
340 C. The 13C atom% of each alkenone was calculated
from the relative ratios of isotopic ion peaks to the quasimolecular ion peak according to Kouchi (1982). Discrimination of 13C was not considered in this study. The
precision of the 13C atom%, based on GC±MS measurement of alkenone in the samples with a natural 13C
ratio, was 0.02 at.% (1 S.D., n=7). The accuracy of the
measurement of the enrichment 13C atom% in the present
study was determined by measurement of ®ve 13C-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
13
C 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 lÿ1 dayÿ1)=(aisÿans)/
(aicÿans) AlkC/t, where ais is 13C atom% in the alkenone in the incubated sample, ans is 13C atom% of the
alkenone in the natural sample, aic is 13C atom% in the
13
C-enriched inorganic carbon, AlkC is the carbon concentration of the alkenone at the end of incubation (ng
C lÿ1), and t is the duration of incubation (day).
3. Results and discussion
3.1. Physicochemical conditions at study site
Sagami Bay is located on the southeastern coast of
Honshu, the main island of Japan and has a wide mouth
J. Hamanaka et al. / Organic Geochemistry 31 (2000) 1095±1102
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 Kuroshio region are mixed with coastal waters. Fig. 1 shows
water temperature, salinity, density, PAR and concentrations of Chl a and 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. 1 mmol
lÿ1. Water temperature was 19.9 C at the surface and
gradually decreased to 15.8 C 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 Institute, unpub. results), the seasonal halocline around 20 m
(Fig. 1a) had been developed at least two weeks previous to the present observation with the increase of the
1097
in¯ow of coastal water (Furushima and Sugimoto,
1994).
3.2. Alkenone-speci®c production rate determined by the
13
C-labeling technique
Mass spectra of the C37:2 and C37:3 alkenones in suspended particles were obtained by GC±MS (Fig. 2). The
base peaks at m/z 532 and 530 are the quasi-molecular
ions of the C37:2 and C37:3 alkenones, respectively. The
relative intensity of the isotope peak at m/z 533 for the
C37:2 alkenone 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
13
C (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/z 531) of the C37:3 alkenone in
the incubated sample from 5 m depth also increased
during incubation and the intensity in the incubated
Fig. 1. Vertical pro®les of (a) water temperature, practical salinity scale and sigma-t, (b) PAR and concentrations of Chl a and nitrate
plus nitrite. Stippled shading indicates depth of
www.elsevier.nl/locate/orggeochem
Production rates of C37 alkenones determined by 13C-labeling
technique in the euphotic zone of Sagami Bay, Japan
Junko Hamanaka a,*, Ken Sawada b, Eiichiro Tanoue a
a
Institute for Hydrospheric-Atmospheric Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
b
Department 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 C37 alkenones in the euphotic zone of Sagami Bay, Japan were determined by the 13Clabeling technique in conjunction with gas chromatography±mass spectrometry. The maximum in the alkenone production rate (C37:2 plus C37:3 alkenones) 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 dayÿ1 at this depth. Temperature,
0
based on the newly-produced alkenone unsaturation index (Uk37), was close to in situ temperatures at the depth where
alkenones production was active. # 2000 Elsevier Science Ltd. All rights reserved.
0
Keywords: Alkenone; 13C-labeling technique; Compound-speci®c production rate; Production rate based-Uk37; 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 (family Gephyrocapsaceae and Isochrysidaceae; 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 paleothermometer for reconstructing past sea surface temperatures (e.g. Brassell et al., 1986a,b; Prahl and
* Corresponding author. Tel.: +81-52-789-3475; fax: +8152-789-3436.
E-mail address: [email protected] u.ac.jp
(J. Hamanaka).
Wakeham, 1987). There have been, however, few studies
concerning the productivity, the stability and the relationship between production and vertical ¯ux of alkenones in surface water environments.
The purposes of this study were to evaluate the production rates of C37 alkenones (alkadien-2-one and
alkatrien-2-one: C37:2 and C37:3 alkenones, respectively)
using a 13C-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 (35 N, 139 200 E) 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.
PII: S0146-6380(00)00118-2
1096
J. Hamanaka et al. / Organic Geochemistry 31 (2000) 1095±1102
(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 seawater (each 9 l) were taken for determination of concentrations and natural 13C atom% of suspended
particles. Duplicate samples of seawater (each 9 l) for
incubations were transferred into 9 l acid-cleaned polycarbonate 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 sediment 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 precombusted (450 C, 4 h) glass ®ber ®lters (Whatman GF/
F) and stored at ÿ20 C until analysis. Zooplankton
swimmers in traps were carefully picked out.
Water temperature, salinity and irradiance of photosynthetically available radiation (PAR) were measured by
the OCTOPUS system (Ishimaru et al., 1984). Concentrations of nutrients and chlorophyll a (Chl a) were measured
by an Autoanalyser AA-2 (Technicone) and a Turner
Designs ¯uorometer after extraction with N,N-dimethylformamide (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-C21:0 fatty 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 Na2SO4 and
evaporated to dryness. Lipids were transesteri®ed with
3±5% (w/v) HCl-methanol at 85 C 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 150 to
320 C at 5 C minÿ1 and then maintained at 320 C 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 was 3% (1 S.D.,
n=10).
2.3.
13
C-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. The 13C atom% of each alkenone was determined by GC±MS (Varian 3400 chromatograph 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, 230 C; mass scan, m/z 500±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
340 C. The 13C atom% of each alkenone was calculated
from the relative ratios of isotopic ion peaks to the quasimolecular ion peak according to Kouchi (1982). Discrimination of 13C was not considered in this study. The
precision of the 13C atom%, based on GC±MS measurement of alkenone in the samples with a natural 13C
ratio, was 0.02 at.% (1 S.D., n=7). The accuracy of the
measurement of the enrichment 13C atom% in the present
study was determined by measurement of ®ve 13C-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
13
C 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 lÿ1 dayÿ1)=(aisÿans)/
(aicÿans) AlkC/t, where ais is 13C atom% in the alkenone in the incubated sample, ans is 13C atom% of the
alkenone in the natural sample, aic is 13C atom% in the
13
C-enriched inorganic carbon, AlkC is the carbon concentration of the alkenone at the end of incubation (ng
C lÿ1), and t is the duration of incubation (day).
3. Results and discussion
3.1. Physicochemical conditions at study site
Sagami Bay is located on the southeastern coast of
Honshu, the main island of Japan and has a wide mouth
J. Hamanaka et al. / Organic Geochemistry 31 (2000) 1095±1102
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 Kuroshio region are mixed with coastal waters. Fig. 1 shows
water temperature, salinity, density, PAR and concentrations of Chl a and 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. 1 mmol
lÿ1. Water temperature was 19.9 C at the surface and
gradually decreased to 15.8 C 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 Institute, unpub. results), the seasonal halocline around 20 m
(Fig. 1a) had been developed at least two weeks previous to the present observation with the increase of the
1097
in¯ow of coastal water (Furushima and Sugimoto,
1994).
3.2. Alkenone-speci®c production rate determined by the
13
C-labeling technique
Mass spectra of the C37:2 and C37:3 alkenones in suspended particles were obtained by GC±MS (Fig. 2). The
base peaks at m/z 532 and 530 are the quasi-molecular
ions of the C37:2 and C37:3 alkenones, respectively. The
relative intensity of the isotope peak at m/z 533 for the
C37:2 alkenone 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
13
C (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/z 531) of the C37:3 alkenone in
the incubated sample from 5 m depth also increased
during incubation and the intensity in the incubated
Fig. 1. Vertical pro®les of (a) water temperature, practical salinity scale and sigma-t, (b) PAR and concentrations of Chl a and nitrate
plus nitrite. Stippled shading indicates depth of