Available online at www.sciencedirect.com
Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
www.elsevier.com/locate/gca
Structural identification of long-chain polyamines associated
with diatom biosilica in a Southern Ocean sediment core
Maxime C. Bridoux, Anitra E. Ingalls *
School of Oceanography, Box 355351, University of Washington, Seattle, WA 98195, USA
Received 11 November 2009; accepted in revised form 12 April 2010; available online 20 April 2010
Abstract
Long-chain polyamines (LCPAs) constitute a new family of natural organic compounds that have recently been isolated
and characterized from the biosilicified cell walls of diatom cultures. To date, diatom-specific polyamines have not been investigated from the marine environment and their fate in the environment is entirely unknown. Here, we report a series of LCPAs
in a diatom frustule-rich sediment core (TNO57-13 PC4), originating from the Atlantic sector of the Southern Ocean and
spanning from the Holocene to the Last Glacial Maximum (LGM). Liquid chromatography with electrospray ionization mass
spectrometry (LC–ESI–MS) revealed a complex mixture of linear polyamines with at least 28 individual molecular species.
Ion trap mass fragmentation studies, combined with high resolution Time of Flight (TOF) mass spectrometry showed that
the polyamine pool consisted of a series of N-methylated propylamine compounds attached to a putrescine moiety, with individual LCPAs varying in chain length and degree of methylation. The structural similarity between LCPAs extracted from the
diatom-rich sediment core and those extracted from the frustules of cultured diatoms suggests that sedimentary LCPAs are
derived from diatom frustules. We hypothesize that these intrinsically labile organic molecular fossils are protected from diagenesis by encapsulation within the frustule. These compounds constitute a new class of biomarkers that could potentially be
indicators of diatom species distribution. Isotopic analysis of LCPAs could be used to improve age models for sediment cores
that lack calcium carbonate and to improve current interpretations of diatom-based paleoproxies, including diatom-bound
nitrogen isotopes.
Ó 2010 Elsevier Ltd. All rights reserved.
1. INTRODUCTION
Diatoms are single celled eukaryotes responsible for
approximately 20% of the photosynthesis on earth (Nelson
et al., 1995). They produce an intricately nanopatterned
skeleton (or frustule) composed of amorphous hydrated silica (SiO2) and an associated organic template (Hecky et al.,
1973; Weiner and Erez, 1984; Kröger et al., 2000). Diatom
frustules display on their surface a homogeneously distributed network of pores organized at the nano to micrometer
scale. In the marine environment, the majority of diatom
frustules dissolve in the water column after cell death. How-
*
Corresponding author.
E-mail address: [email protected] (A.E. Ingalls).
0016-7037/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.gca.2010.04.010
ever, a significant fraction of frustules settle to the sea floor
where they can be buried in marine sediments. These sedimentary frustules can retain their highly organized porous
structures (Fig. 1).
Diatoms are widely used in paleoceanographic studies
because they record past climate variations via various organic and inorganic frustule based proxies (SchneiderMor et al., 2005). Such paleoproxies include isotopes of
biosilica (d18Odiatom, d30Sidiatom) (De La Rocha et al.,
1998; De La Rocha, 2006; Swann and Leng, 2009) as well
as elemental (C/N, C/Si, N/Si) and isotopic (15N, 13C) analysis of organic matter encapsulated within the frustule (Sigman et al., 1999; Crosta et al., 2002, 2005; Jacot Des
Combes et al., 2008; Robinson and Sigman, 2008). Proxies
relying on this so called “diatom-bound organic matter” are
possible because the organic template for silicification
LCPA identification in Southern Ocean sediments
4045
Fig. 1. SEM images showing the intricate structural features of fossil diatom frustules of Fragilariopsis curta (A and B) and Fragilariopsis
kerguelensis (C and D) from sediment core TNO57-13 PC4 (519–521 cm, 10, 200 years).
escapes consumption by heterotrophs and sinks to the seafloor, where it is thought to be preserved on geological
timescales (Sigman et al., 1999). The extent to which diatom-bound organic matter is protected from decomposition
is currently unknown and ultimately depends on the nature
of its association with silica (Christiansen et al., 2006; Brunner and Lutz, 2007; Abramson et al., 2009). While recent
work has aimed to characterize the organic matter protected by frustules, this characterization is still incomplete
(King, 1974; Ingalls et al., 2004, 2006, 2010; Hatte et al.,
2008).
LCPAs are comprised of N-methylated derivatives of
polypropyleneimine units (PPI) attached to putrescine,
ornithine, spermine or spermidine moieties. While LCPAs
are considered relatively rare, bacterial and archaeal
sources of LCPAs have been identified, predominantly in
extremophiles (Oshima, 1979; Carteni-Farina et al., 1985;
Oshima et al., 1987; Hamana et al., 2003; Knott, 2009).
In addition, LCPAs have been isolated and characterized
from the HF and NH4F digested frustules of various marine diatom species (Kröger et al., 2000; Sumper et al.,
2005; Sumper and Lehmann, 2006) as well as from marine
sponge spicules (Matsunaga et al., 2007). The LCPAs in
diatoms are the longest linear polyamines found in nature
and, along with proteins, constitute the main organic
framework of the biosilica matrix in all diatom species
studied so far (Kröger et al., 2000). In diatoms, LCPAs
display species-specific structural characteristics, suggesting their involvement in morphogenesis of the nanopatterned biosilica cell wall (Pohnert, 2002; Foo et al.,
2004; Sumper, 2004; Sumper and Kröger, 2004; Sumper
and Brunner, 2006). Biosynthesis of these biomarkers by
diatoms is likely the major pathway to their formation
in the environment.
Structural differences among LCPAs include varying
chain length, degree of methylation, relative position of secondary and tertiary amines and the presence/absence of
quaternary ammonium groups (Fig. 2) (Sumper and Brunner, 2008). The first silica-associated LCPAs to be identified
were extracted from the cell wall of Cylindrotheca fusiformis
(Fig. 2, structure I) and consist of 6–11 N-methyl-propylamine repeat units (Kröger et al., 1997). LCPAs isolated
from the marine diatom Coscinodiscus asteromphalus were
highly methylated, with only four secondary amine groups
remaining in each polyamine (Fig. 2, structure III). The
LCPA pool isolated from the frustules of C. granii was
not methylated and contained only primary and secondary
amines (Fig. 2, structure IV) (Sumper and Lehmann, 2006).
The polyamines of the marine diatom Thalassiosira pseudonana revealed the presence of unusual quaternary ammonium functionalities (Fig. 2, structure II) (Sumper et al.,
2005).
To date, LCPAs from diatoms have only been carefully
studied in cultures. Their occurrence and distribution in the
water column as well as their fate in sediments is currently
unknown. However, biogenic silica (in the form of opal)
makes up an important part of marine sediments, particularly in the Southern Ocean (Broecker and Peng, 1982; Nelson et al., 1995). Also, sedimentary diatom-bound organic
compounds, released via HF dissolution of frustules in
the lab, are inaccessible to solvents, bleach and strong acid
(Ingalls et al., 2003, 2010), probably due to the protective
function of the silicified cell wall. As a result, frustulebound organic matter constitutes a main pool of organic
4046
M.C. Bridoux, A.E. Ingalls / Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
I
R
N
R
R
N
N
N
R = H, CH3
CH 3
CH3
n
R
II
N
N
N
N
N
N
N
N
N
N
N
N
N
N
III
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
N
N
H
H
N
H
N
IV
HN
N
H
HN
N
H
H
N
N
H
H
N
N
H
H
N
NH2
H
N
H
N
H
N
H
N
H
N
H
N
H
N
NH2
Fig. 2. Representative structures of long-chain polyamines (LCPAs) extracted from the frustules of: I, Nitzschia angularis (Kröger et al.,
2000); II, Thalassiosira pseudonana (Sumper et al., 2005); III, Coscinodiscus asteromphalus (Sumper and Lehmann, 2006) and IV,
Coscinodiscus granii (Sumper and Lehmann, 2006).
matter preserved in Southern Ocean sediments (Ingalls
et al., 2003).
Prior analysis of diatom intrinsic organic matter by LC–
ESI–MS suggested the presence of a series of compounds
with nominal masses corresponding to those of LCPAs
(Ingalls et al., 2004). In that study, LCPAs were not routinely detected in samples and they were not unambiguously
identified because the LCPA fraction did not retain on the
C8 reversed phase column used. Also, LCPAs co-eluted
with other components in the column void volume, likely
suppressing ionization of LCPAs. Later, Hatte et al.
(2008) reported isolating and radiocarbon dating an organic mixture composed of polyamines and silaffin proteins
from frustules isolated from various Southern Ocean sediment cores. In that study, no analysis of the organic compounds in the extract was performed and so the
assumption that the mixture contained polyamines was
not tested.
Given the importance of diatom-dominated, high latitude, high nutrient low chlorophyll regions (e.g., Southern
Ocean and Subarctic North Pacific) in global climate (Sigman and Boyle, 2000; Anderson et al., 2009), reconstructing
the history of these environments is of great interest. In particular, studies that rely on diatom microfossils for reconstructing marine chronologies (Ingalls et al., 2004; Hatte
et al., 2008), past sea-surface temperature (Shemesh et al.,
1993), sea-ice cover (Schneider-Mor et al., 2005); nutrient
utilization (Sigman et al., 1999; Robinson et al., 2004,
2005; Robinson and Sigman, 2008), and past CO2 concen-
trations in surface waters (Rosenthal et al., 2000; Crosta
et al., 2005), could benefit from an investigation of the
molecular and isotopic composition of LCPAs.
In this paper, we describe the isolation and mass spectral
characterization of LCPAs from an Antarctic diatomaceous ooze sediment core sample retrieved from the Atlantic sector of the Southern Ocean: TNO57-13PC4. Our
results demonstrate the occurrence of LCPAs throughout
the Holocene and the Last Glacial Maximum.
2. MATERIAL AND METHODS
2.1. Sample locations
The sediment core TNO57-13PC4 (53.2°S 5.1°E) was
retrieved from the Atlantic Sector of the Southern Ocean,
in 1996, south of the present day Polar front, aboard the
R.V. Thompson. The core was kept refrigerated at 4 °C at
Lamont Doherty Earth Observatory, freeze-dried and
stored at room temperature in our lab until analysis. This
sediment core consists mainly of diatoms, and a mixture
of clays and ice-rafted debris. The following sub-samples
were analyzed for LCPAs: 101–103 (89.39 wt% opal),
139–141 (84.80 wt% opal), 265–267 (93.20 wt% opal),
519–521 (86.91 wt% opal), 609–611 (86.96 wt% opal),
799–801 (87.41 wt% opal), 859–861 (59.84 wt% opal),
918–920 cm (48.01 wt% opal) (wt% opal values were determined by S. Kanfoush and the data were kindly provided
by D. Hodell).
LCPA identification in Southern Ocean sediments
2.2. Diatom cleaning procedure and extraction of LCPAs
Diatoms were chemically cleaned of exogenous organic
material following a protocol adapted from Ingalls et al.
(2010). In brief, sediments were sequentially extracted three
times each in 6 mL MeOH, then 6 mL DCM:MeOH and finally in 6 mL DCM, in Teflon centrifuge tubes. Samples
were vortexed, sonicated (5 min in an ultrasonic bath)
and centrifuged at 12,000 g for 5 min between each solvent
extraction step to form a pellet. The pellet was then rinsed
with milli-Q water and acid hydrolyzed in 6 N HCl for 20 h
at 110 °C. The cleaned frustules were rinsed with milli-Q
water until neutral pH was obtained and the cleaned sediment was freeze-dried. Concentrated HF (3 mL per gram
opal) was added to 1–2 g freeze-dried opal, to release organic compounds incorporated within the hydrated silica
matrix. HF treatment resulted in complete dissolution of
the sample. The resulting solution was then concentrated
to dryness using a CentriVap. The dried residue containing
LCPAs was dissolved in 1 mL milli-Q water, filtered
through a 0.2-lm Teflon syringe filter and subjected to liquid chromatography mass spectrometry (LC-MS).
2.3. Liquid chromatography mass spectrometry
LCMS analyses were performed on an Agilent HP1100
chromatographic system controlled by Chemstation Version B.0.03 (Hewlett Packard, Palo Alto, CA, USA). The
chromatographic system was coupled to an Agilent XCT
ion trap equipped with an electrospray ionization source
operated in the positive ionization mode. Due to their high
polarity and degree of positive charge, long-chain polyamines display poor retention on most reverse phase columns. This can be avoided by using the ion pairing agent
heptafluorobutyric acid (HFBA), which allows analysis of
underivatized long-chain polyamines (Häkkinen et al.,
2007; Sanchez-Lopez et al., 2009). Chromatographic separation of LCPAs was carried out using a Zorbax Eclipse
XDB C18 column (4.6 mm id 150 mm, 5 lm) maintained
at 25 °C, at a flow rate of 0.75 ml min 1 with a gradient of
0.1% heptafluorobutyric acid (HFBA) in water (solvent A)
and 0.1% HFBA in acetonitrile (solvent B). The solvent
program was as follows: 0% B at time zero, 20% B by
7 min, 50% B by 9 min, 80% B by 17 min and held until
23 min. The column was re-equilibrated in solvent A for
10 min between injections. LCMS settings were as follow:
capillary voltage 3500 V, nebulizer gas (N2) pressure
60 psi, dry gas (N2) flow 11 l/min, dry temperature
350 °C. The MS scanned from 50 to 2000 m/z. Fragmentation experiments (MSn) were performed using a fragmentation voltage of 1.00 V. Accurate-mass measurements of
long-chain polyamines were obtained using a quadrupole
orthogonal Time of Flight (TOF) mass spectrometer (QTOF, Synapte HDMS/Waters Corp, Milford, USA) using
the same solvent program as described above. The cone
voltage was set at 25 V and N2 was the nebulising gas.
The MS scanned from 50 to 2000 m/z. Synthetic LCPA (linear PPI) standards were kindly provided by Prof. Henning
Menzel (Braunschweig University, Germany) and were prepared by polymerization of 1,3-oxazine, followed by aque-
4047
ous sodium hydroxide hydrolysis of the prepolymers
(Menzel et al., 2003).
2.4. Scanning electron microscopy
Samples of freeze-dried diatom particles were sprinkled
onto carbon adhesive tapes and sputter coated with approximately 5 nm of Pt (SPI Module Sputter Coater, SPI Supplies, Inc., West Chester, PA). They were then imaged
using a JSM7000 SEM scanning microscope (JEOL USA,
Inc., Peabody, MA) operating at 10 kV.
3. RESULTS AND DISCUSSION
3.1. LCPA isolation and chemical characterization
Identification of diatom-bound polyamines in cleaned,
HF digested sediments was based on chromatographic
retention times, mass spectrometry, fragmentation studies,
accurate-mass measurements, as well as comparisons of
environmental samples with cultures and laboratory synthesized LCPAs. The total ion chromatogram of the
cleaned sediment core TNO57-13 (799–801 cm) HF extract
shows one major peak eluting at a retention time of 12 min
(Fig. 3). UV and Visible spectra showed no absorption during the elution of this peak. ESI mass spectra, averaged
over the polyamine peak width, reveal a complex mixture
of protonated molecular ions between m/z 401.6 and
1069.0, characteristic of a series of long-chain polyamines
(Fig. 4). This mixture is composed of 10 major compounds
with a series of m/z values (401.6, 472.7, 543.8, 614.9, 685.9,
757.0, 828.0, 899.0, 970.0 and 1041.0) that differ by 71 mass
units, a mass difference corresponding to LCPAs that vary
in the number of repeating N-methyl-propylamine units.
These molecular species constitute the most abundant components of quintuplets of mass peaks separated by 14 mass
units, suggesting five degrees of methylation. Extracted ion
chromatograms of selected LCPAs show that although all
polyamines co-elute in one chromatographic peak, the elution order is related to the length of the aliphatic chain
(Fig. 5).
The main molecular species of the sedimentary polyamine population were selected for more detailed structural
analysis, using tandem mass spectrometry. For this experiment, the m/z 757.0 ion (Fig. 4) was isolated in the ion trap
and fragmented by collision-induced dissociation (CID).
Previous studies show that fragmentation of LCPAs only
occurs at the C–N bond and preferentially at the site of a
positively charged nitrogen atom (Sumper and Lehmann,
2006). The resulting fragmentation of m/z 757.0 produced
a highly regular pattern, displaying two series of fragments
shifted by 14 mass units (Fig. 6A). Molecular species from
each series of fragments are again separated by 71 mass
units (m/z 214.2, 285.3, 356.5, 427.5, 498.7, 569.8, 640.8),
confirming the presence of a series of N-methylated propyleneimine units. A similar fragmentation pattern was observed in studies of LCPAs isolated from the frustules of
various diatom cultures (Kröger et al., 2000; Sumper and
Lehmann, 2006). The fragmentation pattern indicated a
series of 9 methyl propylamine units attached to the amine
4048
M.C. Bridoux, A.E. Ingalls / Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
2.5
Σ LCPAs
Intensity x107
2.0
1.5
1.0
0.5
0.0
5
10
15
20
Retention time (min)
25
30
Fig. 3. Base peak chromatogram (positive ion, ion pairing RP-LCMS) showing the LCPA peak from sediment core TNO57-13PC4 (799–
801 cm).
757.0
∇
771.0
100
685.9
∇
0
400
800
970.0
∇
984.0
998.0
913.1
∇
942.0 927.0
956.0
871.0
885.0
856.0
899.0
814.0
600
842.0
∇
800.0
729.0
529.7
628.9
632.9
657.9
600.8
586.9
∇
458.7
∇
401.6
557.7
472.7
713.9
∇
743.0
699.9
671.9
543.8
50
486.6
% intensity
∇
785.0
828.0
614.9
1041.0
∇
1000
m/z
Fig. 4. Electrospray ionization mass spectra of the LCPA peak from Fig. 3, averaged over the peak width. Each peak in the mass spectra
represents a singly charged positive ion. Triangles above selected masses indicate pseudomolecular ions whose masses differ by 71 units.
group of a di-methylated putrescine building block
(Fig. 6B). This proposed structure, contains one secondary
amine group at each extremity of the polyamine chain, on
the putrescine moiety and on the terminal propylamine
group. This structure is also confirmed by the presence of
two unusual fragments at m/z 671.9 and 685.9 (Fig. 6).
These fragments can only occur by internal proton transfer
from a secondary amine group adjacent to the cleavage site
(Sumper and Lehmann, 2006). The presence of these two
fragments is therefore indicative of two terminal secondary
amines. Fragmentation of m/z 671.9 (Fig. 6C) produced a
somewhat different product ion spectra where the major
fragments are separated by 71 mass units (214.1, 285.3,
356.5, 427.6, 498.6, 569.7) confirming the presence of a series of N-methylated propyleneimine units but, no other
fragments corresponding to m/z 14 units were detected.
The fragmentation pattern indicated a series of 7 methyl
propylamine units attached to the amine group of a methylated putrescine building block (Fig. 6D). This proposed
structure, contains one primary and one secondary amine
group at each extremity of the polyamine chain, on the
putrescine moiety and on the terminal propylamine group,
respectively. Also, only one fragment corresponding to the
proton transfer from a secondary amine group adjacent to
the cleavage site is observed, at m/z 600.7, in agreement
with the proposed structure in Fig. 6D.
In order to validate the mass spectral fragmentation
experiments conducted on sedimentary LCPAs, a suite of
non-methylated LCPA standards (PPIs) were analyzed by
direct infusion electrospray ionization mass spectrometry.
The full scan mass spectrum of the LCPA standards
(Fig. 7A) indicates that the standard mixture consists of a
series of linear LCPAs with 7–12 repeating degrees of polymerization (Pn = 7–12 PPI units). Fragmentation of m/z
532.8 (Pn = 9) produced a spectrum characterized by a regular fragmentation pattern, dominated by two series of
fragments shifted by 17 mass units (Fig. 7B) corresponding
to an amine group. Molecular species from each series were
separated by 57 mass units (i.e., m/z 172.1, 229.2, 286.3,
343.4, 400.5, 457.6), confirming the presence of a series of
N-propyleneimine units. A similar fragmentation pattern
was observed from the unmethylated LCPA pool extracted
from sponge spicules (Matsunaga et al., 2007) and can be
explained by a linear PPI structure with Pn = 9 (Fig. 7B).
Similar fragmentation analysis was performed with the
other most abundant polyamine species present in the sediment core mass spectra (Fig. 8). All compounds displayed
product ion spectra that were analogous to the one
LCPA identification in Southern Ocean sediments
4049
Intensity
x107
3
2
1
BPC – all MS
0
0.8
EIC m/z = 472.7
0.4
0
2
EIC m/z = 614.9
1
0
EIC m/z = 685.9
2
1
0
1.5
EIC m/z = 757.0
0.5
EIC m/z = 828.0
x106
1
0.5
0
0.8
EIC m/z = 1041.0
0.4
0
10
11
12
13
14
Time [min]
Fig. 5. Base peak chromatogram (BPC) and extracted ion chromatograms (EIC) of the major LCPA masses observed.
observed for m/z 757.0 (Fig. 6A). The LCPA population,
extracted from the sediment core TNO57-13, consists of a
series of N-methyl propyleneimine repeat units attached
to the amine group of a putrescine moiety. The general
structure of the LCPA family observed in the sediment core
shows that the two terminal amine groups of the linear
polyamine chain can be substituted by up to two methyl
groups (Fig. 8). In summary, each quintuplet of mass peaks
separated by 14 mass units observed in the ESI mass spectra
(Fig. 4) corresponds to the general structure displayed in
Fig. 8, where the 14 mass unit differences among species
correspond to different degrees of methylation on the two
terminal amine groups.
Further confirmation of polyamine structures came
from accurate-mass analysis of individual LCPAs in the
519–521 cm depth interval, using high resolution Q-TOF
(quadrupole-Time of Flight) mass spectrometry. Accurate-mass measurements observed for the major protonated
LCPA species includes a total of 26 molecular formulas
(Table 1). All reported masses are within ±10 ppm of those
expected from the proposed structure (Table 1). Each
molecular formula matches our structural interpretation
based on fragmentation studies. For example, the most
abundant species within the quadruplets of peaks were observed at: 614.6527 (C34H80N9); 685.7252 (C38H89N10);
756.8000 (C42H98N11) and 827.8773 (C46H107N12); these
molecular formulas support our interpretation of the MS/
MS data. Accurate-mass measurements also provided further supporting data for the varying degrees of methylation
(Table 1). For example, the quintuplet mass series:
657.6951, 671.7118, 685.7252, 699.7408 and 713.7625, all
separated by 14 mass units had the following calculated formulae: C36H85N10, C37H87N10, C38H89N10, C39H91N10,
C40H93N10; confirming that each single mass peak within
a quintuplet corresponds to varying degrees of methylation
and that the series of peaks separated by 71 units differ in
their number of (N-methyl) propyleneimine repeats.
3.2. Oceanographic significance
LCPAs have been found in diatom cell walls (e.g., Kröger et al., 2000) and sponge spicules (Matsunaga et al.,
2007), although their presence in other silicified organisms
such as radiolarian and silicified flagellates is likely. Diatom
frustules are considered to be the dominant source of biogenic silica flux to the seafloor in the vicinity of TNO5713PC4 (e.g., Shemesh et al., 2002; Anderson et al., 2009).
The high degree of structural similarity between the LCPAs
in our sediment core and LCPAs previously reported from
diatom cultures is strong support for the hypothesis that
polyamines in TNO57-13PC4 derive from diatom frustules.
Despite this, it is still possible that other groups (radiolaria
or sponges) contribute to the LCPA pool. The detection of
these intrinsically labile compounds (Höfle, 1984) in solvent
extracted and acid hydrolyzed Holocene to Last Glacial
Maximum (LGM) age sediments suggests a high degree
of steric protection from diagenesis. In his biosilicification
model, Sumper (2002) postulated that an emulsion of organic nanodroplets containing LCPAs was produced within
the silica depositing vesicles. As silica polymerizes and precipitates around the LCPAs, there is potential for them to
become incorporated, or “encapsulated” within the newly
synthesized frustule. This encapsulation would afford the
LCPAs physical protection so long as the silica remains.
In-vitro experiments have recently shown that natural and
synthetic LCPAs (along with silaffins) induce silica precipi-
4050
M.C. Bridoux, A.E. Ingalls / Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
100
A
685.9
2
MS : 757.0
569.8
% intensity
671.9
654.9
50
640.8
498.7
441.5
356.5
583.8
512.7
427.5
285.3
370.5
214.2
299.3
228.2
0
200
300
B
400
500
640
H
N
600
700
m/z
671
569
N
N
498
427
N
356
N
228
285
N
299
214
N
370
441
N
N
512
N
583
N
H
654
685
100
498.6
C
2
MS : 671.9
569.7
% intensity
427.6
285.3
356.5
214.1
50
600.7
0
100
200
D
2HN
569
N
300
498
N
400
427
N
214
500
m/z
356
N
285
285
N
356
600
214
N
427
700
N
498
N
N
H
569
600
Fig. 6. Product ion spectra obtained by collision-induced dissociation of (A) the [M+H]+ 757.0 and (C) [M+H]+ 671.9 species from TNO5713PC4 (see mass signal in Fig. 4). Note that for m/z 757.0, two series of ions separated by 14 units are detected. Molecular species from each
series differ by 71 units (see triangles above mass peaks). (B) and (D): proposed structures corresponding to m/z 757.0 and 671.9 species,
respectively. The cleavage positions leading to the observed product ion spectrum (A and C) are depicted by arrows, and the corresponding
molecular masses are indicated.
tation when added to a metastable solution of monosilicic
acid (Kröger et al., 2000; Noll et al., 2002; Bernecker
et al., 2010). The protonated amino groups of LCPAs
may serve as binding sites for silicic acid (Coradin et al.,
2002). Also, X-ray studies revealed that silica gel and polyamines interact at the molecular level and appear to be
hybridized through hydrogen bonding (Mitzutani et al.,
1998). In this scenario, silica dissolution in the water col-
umn would be the primary control on LCPA loss from
the diatom-bound organic carbon and nitrogen pool. It is
also possible that LCPAs exert some control over silica dissolution and high concentrations of LCPAs could impart
some protection from dissolution to heavily silicified
diatoms.
In opal rich regions such as the Southern Ocean,
approximately half of the biogenic opal formed in the
LCPA identification in Southern Ocean sediments
532.8
A
475.8
1.5
589.9
546.8
Intens. X 10
7
489.8
603.9 646.9
660.9
1.0
432.8
703.9
418.8
717.9
0.5
0.0
400
500
B
700
343.4
286.3
MS 532.8 →
2000
600
m/z
800
400.5
229.2
2
Intens.
4051
457.6
172.1
417.6
1000
360.5
303.4
246.2
189.1
0 100
2HN
H
N
200
300
m/z
400
500
189.1
246.2
303.4
360.5
417.6
H
N
H
N
H
N
H
N
H
N
H
N
172.1
229.2
286.3
343.4
400.5
H
N
N H2
457.6
Fig. 7. Full scan mass spectra (A) and product ion spectra (B) of the m/z 532.8 species obtained from a polypropyleneimine (PPI) standard
mixture.
euphotic zone is dissolved in the upper 100 m of the water
column, while only 15–25% percent arrives in marine sediments (Nelson et al., 1995). Thus, LCPAs are likely to be
released into the water column, along with dissolving frustules. In surface waters, frustule-bound C and N comprise a
negligible fraction of the total C and N pools (Ingalls et al.,
2003), but in deep waters and sediments where biomineral
bound C and N is significant and the majority of DOM is
considered refractory (Williams and Druffel, 1987), LCPAs
released during frustule dissolution could provide a readily
available source of nitrogen-rich organic matter for microbial heterotrophs (Höfle, 1984). While current methods do
not allow for the quantification of LCPA, they could represent a major fraction of diatom-bound organic matter. This
model of physical protection of organic matter by frustules
supports earlier studies that have used 13C NMR spectroscopy of sinking marine particles to suggest that the protective action of the inorganic matrix in particles resulted in
minimal changes in organic matter composition with depth,
despite extensive loss of carbon (Hedges et al., 2001). Only
a few published studies have reported the distribution and
fate of polyamines in marine environments and all of them
focused on common short chain polyamines, such as cadaverine, putrescine, spermidine and spermine (Höfle, 1984;
Lee and Jørgensen, 1995; Nishibori et al., 2003), which
are likely to be the metabolic precursors of LCPAs in diatoms (Knott et al., 2007). Others report the uptake and degradation of smaller polyamines (putrescine and spermidine)
by marine algae (Badini et al., 1994). These studies suggest
that LCPAs could be a good carbon and nitrogen source
for microbial populations.
3.3. Paleoceanographic applications
Only seven diatom species have had their LCPA compositions analyzed. While these species display entirely distinct
LCPA populations, it is likely that specific LCPAs are
shared among species, even if the overall LCPA patterns remain species specific (Kröger et al., 2000; Sumper et al.,
2005). This study represents the first attempt to examine
LCPAs in mixed assemblages and across a major climate
transition. We were interested in determining if changes in
LCPA composition co-occur with changes in diatom species distributions and if LCPA abundance per gram opal
varies over time. In order to assess variability in the source
and composition of LCPAs in our study site, we analyzed
LCPAs in several depth intervals of the sediment core
TNO57-13PC4 and compared their distribution to microscopic diatom counts, opal (biosilica) flux and opal concentration (wt%).
4052
M.C. Bridoux, A.E. Ingalls / Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
X106
214.2
A
1.5
5
401.6 →
285.3
143.2
Intens
X105
6
214.2
B
472.7 →
285.3
4
157.2
228.2
1.0
3
157.1
356.5
370.5
228.3
143.2
299.3
299.4
2
387.5
401.5
0.5
1
0.0
100
200
400
300
Intens
3
472.6
C
5
543.8 →
4
214.2
2
285.3 356.5
400
543.7
614.9 →
3
512.7 529.7
427.6
356.4
441.6
214.1
370.4 498.7
228.2
299.3
285.3
2
427.5
1
100
200
300
400
500
X105
100
200
300
400
500
X105
E
614.8
498.7
685.9 →
3
Intens
300
0
0
370.2
2
427.6
299.2
285.3
214.2
441.6
5
569.8
583.8
4
356.5
228.2
600.8
512.7
3
2
1
0
200
D
299.4
157.1
1
458.7
441.6
370.5
228.2
4
100
X105
X105
4
0
1
100
200
300
400
500
600
0
F
614.8
757.0 →
685.8
569.8 600.8
441.6
498.7
512.7
427.6
299.3 370.5
356.5
228.2
285.3
214.2
100
200
m/z
300
400
500
583.8
600
671.9
700
m/z
R
N
R
R = H, CH 3
R
N
N
CH3
CH3
N
n
R
Fig. 8. Product ion spectra produced by collision-induced dissociation of selected ions observed in the mass spectra (Fig. 4). (A, m/z = 401.6;
B, m/z = 472.7; C, m/z = 543.8; D, m/z = 614.9; E, m/z = 685.9; F, m/z = 757.0). The generic structure of LCPAs is shown on the bottom
with the putrescine moiety in bold.
The lack of LCPA quantification standards and the coelution of all LCPAs in one broad chromatographic peak
do not allow us to accurately quantify LCPAs at this time.
Nevertheless, changes in the relative abundance of LCPAs
between samples can offer some insight into how their relative concentration and distribution change overtime. Open
ocean assemblage of Fragilariopsis kerguelensis (approximately 60%, see Fig. 1) and Thalassiosira lentiginosa
(approximately 10%) dominate the sediment core throughout the Holocene (Crosta et al., 2002). Upon the transition
to the LGM, these species become much less abundant
(approximately 30%) with a concomitant increase in Chaetoceros spores (30%) (Nielsen, 2004). Species counts are not
weighted for their contribution to total biosilica and since
F. kerguelensis and T. lentiginosa are large and thickly silicified, they are likely to represent an even higher portion of
LCPA identification in Southern Ocean sediments
4053
Table 1
Accurate-mass of the major ions observed in the electrospray Q-TOF mass spectrum of protonated long-chain polyamines isolated from
sediment core TNO57-13PC4 (519–521 m depth).
Observed mass
Calculated mass
458.4893
472.5049
529.5634
543.5797
557.5941
586.6219
600.6381
614.6527
628.6693
657.6951
671.7118
685.7252
699.7408
713.7625
742.7865
756.8000
770.8159
784.8393
813.8598
827.8773
841.8847
856.8921
884.9297
898.9478
912.9625
984.0399
458.4910
472.5067
529.5645
543.5802
557.5958
586.6224
600.6380
614.6537
628.6693
657.6959
671.7115
685.7272
699.7428
713.7585
742.7850
756.8007
770.8163
784.8320
813.8585
827.8742
841.8898
856.9007
884.9320
898.9477
912.9633
984.0368
a
Errora (ppm)
3.7
3.8
2.1
0.9
3.0
0.9
0.2
1.6
0.0
1.2
0.4
2.9
2.9
5.6
2.0
0.9
0.5
9.3
1.6
3.7
6.1
6.5
2.6
0.1
0.9
3.2
Suggested formula
C25H60N7
C26H62N7
C29H69N8
C30H71N8
C31H73N8
C32H76N9
C33H78N9
C34H80N9
C35H82N9
C36H85N10
C37H87N10
C38H89N10
C39H91N10
C40H93N10
C41H96N11
C42H98N11
C43H100N11
C44H102N11
C45H105N12
C46H107N12
C47H109N12
C47H111N13
C49H114N13
C50H116N13
C51H118N13
C55H127N14
The error is given as the ppm difference between the calculated and the measured m/z.
total biosilica, and LCPAs, than is reflected by frustule
counts alone. At this point, lack of data for LCPAs in the
diatom species present in our core prevent the assignment
of individual LCPAs to specific diatoms. The LCPA distribution likely reflects the contribution of various preserved
diatom species. Overall, each depth interval investigated
showed a similar group of polyamines, dominated by m/z
401.6, 472.7, 543.8, 614.9, 685.9, 757.0, 828.0, 899.0, 970.0
and 1041.0 (Fig. 4). Depth interval 519–521 cm displayed
a higher mass range, with apparent molecular species up
to m/z 1041.3 and 1112.4 (Fig. 9). Also, depth intervals
859–861 and 918–920 cm are characterized by a more simple distribution, with fewer masses observed (Fig. 9). The
observed shift in the LCPA composition in the two deepest
samples corresponds to the transition to the LGM (see Stuiver et al., 1998; Shemesh et al., 2002 for details of the age
model for TNO57-13PC4) that was accompanied by a dramatic decrease in the relative abundance of F. kerguelensis
and concomitant increase in Chaetoceros spores (Nielsen,
2004). These data suggest that the LCPA composition is
broadly sensitive to the species distributions. A more detailed quantitative study that employs improved analytical
methods and examines LCPAs in the various source organisms will be needed to pursue these observations further.
To investigate relative LCPA concentrations downcore,
extracted ion chromatograms, corresponding to the most
abundant polyamine species (m/z 401.6, 472.7, 543.8,
614.9, 685.9, 757.0, 828.0, 899.0, 970.0 and 1041.0;
Fig. 5) were integrated and the corresponding peak areas
were normalized to the dry weigh of opal that was HF digested. We use LCPA peak area per gram opal as a proxy
for LCPA abundance per gram opal. While our sample
resolution is too low to determine the exact timing of
changes in LCPA/g opal, we found generally low values
in the upper and lower parts of the core and high values
in the middle section (Fig. 10). Diatom assemblages do
not change markedly between 0 and 700 cm depth interval, covering the last 14,000 years. So, this change in
LCPA peak area per gram opal is not likely to be due
to changes in diatom species abundances. Instead, this
interval of high LCPA/g opal co-occurs with a period of
increased opal flux, between 10,000 and 17,000 years ago
(Anderson et al., 2009). This increase in biogenic opal flux
may have resulted from increased silicic acid availability
due to changes in ocean circulation. Abundant silicic acid
allows diatoms unlimited access to silica, and may result
in highly silicified diatoms that are well preserved. This
condition may also result in biogenic opal with higher
concentrations of LCPAs. These finding are consistent
with findings that C/Si and N/Si ratios of biosilica change
with time and may be suggestive of the nutrient status of
diatoms (Crosta et al., 2002).
LCPAs incorporated into and protected by the silicified
frustule in living diatoms likely constitute a pristine carbon
and nitrogen archive related to the original primary photosynthate. As such, LCPAs constitute an ideal material for
the development of new paleoceanographic proxies for
studies of chronologies and past nutrient cycling. Several
4054
M.C. Bridoux, A.E. Ingalls / Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
6
A
614.9
543.8
519 - 521 cm
685.9
757.0
Intens. x10 6
472.7
4
828.0
401.6
899.1
970.1 1041.3
2
330.5
1112.4
0
400
1.5
B
600
800
1000
472.7
859 - 861 cm
401.6
Intens. x10 6
543.7
614.8
685.9
1.0
0.5
0.0
400
8
C
472.7
600
543.7
800
1000
918 - 920 cm
671.9
614.8
Intens. x10 5
6
401.6
4
2
0
400
600
800
1000
m/z
Fig. 9. LC-ESI full scan mass spectra displaying the LCPA distribution at various depth intervals in the core TNO57-13PC4. (A) 519–521 cm,
(B) 859–861 cm and (C) 918–920 cm.
studies have noted the presence of “diatom-bound organic
matter” in sediments, and many researchers have isolated
this pool of organic matter in order to use its elemental
and isotopic composition to reconstruct past productivity
and nutrient utilization in the ocean (Shemesh et al.,
1993; Sigman et al., 1999; Rosenthal et al., 2000; Crosta
et al., 2002; Robinson et al., 2004, 2005; Brunelle et al.,
2007; Robinson and Sigman, 2008). Specifically, sediment
cores from the Southern Ocean have revealed a change in
the bulk N isotopic composition at the last glacial/interglacial transition, with higher d15N values (1–4&) in the glacial period (Last Glacial Maximum) compared with the
Holocene (Francois et al., 1992, 1997). This glacial/interglacial change in d15N values is generally mirrored in diatombound d15N values (d15NDB) and is hypothesized to document a higher degree of nitrate utilization in Antarctic surface waters during the LGM (Sigman et al., 1999; Robinson
et al., 2004, 2005). However, d15NDB has not been well
characterized. Variability in the molecular composition between samples from glacial interglacial time periods, prepared using different protocols, containing divergent
species assemblages or among members of the same species
growing under different conditions have all been raised as
potential confounding factors that have tempered confidence in these measurements (Shemesh et al., 2002; Robinson et al., 2005; Brunelle et al., 2007; Jacot Des Combes
et al., 2008). The origin of discrete components of heterogeneous organic matter can be investigated by compoundspecific isotope analysis of source-specific biomarkers (Eglinton et al., 1997; Sachs and Repeta, 1999; Ingalls and Pearson, 2005; Higgins et al., 2010). Therefore, isotopic analysis
of LCPAs could be used to enhance current interpretations
of diatom-based paleoproxies, including diatom-bound
nitrogen isotopes.
LCPA identification in Southern Ocean sediments
LCPA EIC peak area (g dw
0.0
4.0e+4
8.0e+4
-1
4055
opal)
1.2e+5
1.6e+5
720
1,830
200
m/z 401
6,930
m/z 472
m/z 543
400
9,600
m/z 614
10,290
m/z 685
m/z 756
600
m/z 827
m/z 898
14,500
m/z 969
800
m/z 1040
29,500
1000
Calibrated Years
Depth (cm)
Fig. 10. Semiquantitative LCPA abundance vs depth and calibrated age (years) in the core TNO57-13PC4. LCPA abundance is expressed as
the integrated extracted ion chromatogram (EIC) signal area normalized to cleaned opal dry weight. Core chronology is from Stuiver et al.
(1998).
4. SUMMARY
A suite of 28 biomineral-bound long-chain polyamines
has been found in a diatom-rich sediment core from the
Atlantic Sector of the Southern Ocean (TNO57-13PC4).
Ion trap fragmentation studies combined with high resolution TOF mass spectrometry demonstrate that all polyamine species detected were N-methylated propylamines
attached to a putrescine moiety with individual species
varying in chain length and degree of methylation. The
presence of these compounds throughout the Holocene
age core indicates that LCPAs are protected from diagenetic transformations by their tight association with the
frustule. The similarity in polyamine species distribution
throughout the Holocene section of the core agrees with
the relative uniformity of diatom assemblages, strongly
dominated by open ocean assemblages of F. kerguelensis
and T. lentiginosa. Semiquantitative downcore profiles
demonstrate that the abundance of LCPAs per gram of
opal change with depth and are highest when opal fluxes
to the sediment are also high.
Our results provide evidence that LCPAs constitute an
important carbon and nitrogen archive linked to the original primary photosynthate. We suggest that LCPAs represent a new class of biomarkers that could potentially be an
important source of nitrogen-rich dissolved organic matter
in parts of the deep ocean, and may be indicators of diatom
species distributions. LCPA concentrations and isotopic
values in paleoceanographic studies should provide new
diatom-based paleoceanographic proxies.
ACKNOWLEDGMENTS
This work was supported by grants from the NSF OCE0525829
(A.E.I.), The Comer Foundation and start-up funds from the University of Washington. We thank T. Guilderson and D. Hodell, under NSF OCE-0350418 for providing samples from core TNO57-
13PC4, S. Kanfoush for the wt% biosilica data. The SEM work
was conducted with the help of H. Fong at the shared experimental
facilities of Genetically Engineered Materials Science and Engineering Center, University of Washington. TOF-MS analyses were
performed at the Mass Spectrometry Facility in the Department of
Medicinal Chemistry with the help of D. Whittington. Synthetic
long-chain polyamine standards were kindly provided by H. Menzel of the Braunschweig University, Germany. We also thank J.
Sachs, S. Wakeham, S. Nielsen and two anonymous reviewers for
their valuable comments.
REFERENCES
Abramson L., Wirick S., Lee C., Jacobsen C. and Brandes J. A.
(2009) The use of X-ray spectromicroscopy to investigate the
distribution and composition of organic matter in a diatom
frustule and a biomimetic analog. Deep-Sea Res. II 56, 1369–
1380.
Anderson R. F., Ali S., Bradtmiller L. I., Nielsen S. H. H., Fleisher
M. Q., Anderson B. E. and Burckle L. H. (2009) Wind-driver
upwelling in the Southern Ocean and the deglacial rise in
atmospheric CO2. Science 323, 1443–1448.
Badini L., Pistocchi R. and Bagni N. (1994) Polyamine transport in
the seaweed Ulva rigida (Chlorophyta). J. Phycol. 30, 599–605.
Bernecker A., Wieneke R., Riedel R., Seibt M., Geyer A. and
Steinem C. (2010) Tailored synthetic polyamines for controlled
biomimetic silica formation. J. Am. Chem. Soc. 132(3), 1023–
1031.
Broecker W. S. and Peng T.-H. (1982) Tracers in the Sea. LamontDoherty Geological Observatory, Palisades, NY.
Brunelle B. G., Sigman D. M., Cook M. S., Keigwin L. D., Haug
G. H., Plessen B., Schettler G. and Jaccard S. L. (2007)
Evidence from diatom-bound nitrogen isotopes for Subarctic
Pacific stratification during the last ice age and a link to North
Pacific denitrification changes. Paleoceanography 22, PA1215.
doi:10.1029/2005PA001205.
Brunner E. and Lutz K. (2007) Solid state NMR in biomimetic
silica formation and silica biomineralization. In Handbook of
4056
M.C. Bridoux, A.E. Ingalls / Geochimica et Cosmochimica Acta 74 (2010) 4044–4057
Biomineralization: Biomimetic, Bioinspired Chemistry (eds. Peter
Behrens and Edmund Baeuerlein). Wiley-VCH Verlag GmbH
& Co.
Carteni-Farina M., Porcelli M., Cacciapuoti G., De Rosa M.,
Gambacorta A., Grant W. D. and Ross H. N. M. (1985)
Polyamines in halophilic archaebacteria. FEMS Microbiol.
Lett. 28, 323–327.
Christiansen S. C., Hedin N., Epping J. D., Janicke M. T., del Amo
Y., Demarest M., Brzezinski M. and Chmelka B. F. (2006)
Sensitivity considerations in polarization transfer and filtering
using dipole–dipole couplings: implications for biomineral
systems. Solid-State NMR 29, 170–182.
Coradin T., Durupthy O. and Livage J. (2002) Interactions of
amino-containing peptides with sodium silicate and colloidal
silica: a biomimetic approach of silicification. Langmuir 18,
2331–2336.
Crosta X., Shemesh A., Salvignac M. E., Gildor H. and Yam R.
(2002) Late quaternary variations of elemental ratios (C/Si and
N/Si) in diatom bound–bound organic matter from the
Southern Ocean. Deep-Sea Res. II 49, 1939–1952.
Crosta X., Crespin J., Billy I. and Ther O. (2005) Major factors
controlling Holocene d13Corg changes in a seasonal sea-ice
environment, Adelie Land, East Antarctica. Global Geochem.
Cycles 19, GB4029. doi:10.1029/2004GB002426.
De La Rocha C. L., Brzezinski M. A., De Niro M. J. and Shemesh
A. (1998) Silicon-isotope composition of diatoms as an indicator of past oceanic change. Nature 395, 680–683.
De La Rocha C. L. (2006) Opal-based isotopic proxies of
paleoenvironmental conditions. Global Biogeochem. Cycles 20,
GB4S09. doi:10.1029/2005GB002664.
Eglinton T. I., Benitez-Nelson B. C., Pearson A., McNichol A. P.,
Bauer J. E. and Druffel E. R. (1997) Variability in radiocarbon
ages of individual organic compounds from marine sediments.
Science 277, 796–799.
Foo C. W. P., Huang J. and Kaplan D. L. (2004) Lessons from
seashells: silica mineralization via protein templating. Trends
Biotechnol. 22(11), 577–585.
Francois R., Altabet M. A. and Burckle L. H. (1992) Glacial to
interglacial changes in surface nitrate utilization in the Indian
sector of the Southern Ocean as recorded by sediment d15N.
Paleoceanography 7, 589–606. doi:10.1029/92PA01573.
Francois R., Altabet M. A., Yu E. F., Sigman D. M., Bacon M. P.,
Frank M., Bohrman G., Bareille G. and Labeyrie L. D. (1997)
Contribution of Southern Ocean surface water stratification to
low atmospheric CO2 concentrations during the last glacial
period. Nature 389, 929–935.
Hamana K., Tanaka T., Hosoya R., Niitsu M. and Itoh T. (2003)
Cellular polyamines of the acidophilic, thermophilic and
thermoacidophilic archaebacteria, Acidilobus, Ferroplasma,
Pyrobaculum, Pyrococcus, Staphylothermus, Thermococcus,
Thermodiscus and Vulcanisaeta. J. Gen. Appl. Microbiol. 49,
287–293.
Häkkinen M. R., Keinänen T. A., Vepsäläinen J., Khomutov A.
R., Alhonen L., Jänne J. and Auriola S. (2007) Analysis of
underivatized polyamines by reversed phase liquid chromatography with electrospray tandem mass spectrometry. J. Pharmaceut. Biomed. 45, 625–634.
Hatte C., Hodgins G., Jul A. J. T., Bishop B. and Tesson B. (2008)
Marine chronology based on 14C dating on diatoms proteins.
Mar. Chem. 109, 143–151.
Hecky R. E., Mopper K., Kilham P. and Degens E. T. (1973) The
amino acid and sugar composition of diatom cell-walls. Mar.
Biol. 19, 323–331.
Hedges J. I., Baldock J. A., Gelinas Y., Lee C., Peterson M. and
Wakeham S. G. (2001) Evidence for non-selective preservation of
organic matter in sinking marine particles. Nature 409, 801–804.
Higgins M. B., Robinson R. S., Carter S. J. and Pearson A. (2010)
Evidence from chlorine nitrogen isotopes for alternating
nutrient regimes in the eastern Mediterranean Sea. Earth
Planet. Sci. Let. 290, 102–107.
Höfle M. G. (1984) Degradation of putrescine and cadaverine in
seawater cultures by marine bacteria. Appl. Environ. Microbiol.
47, 843–849.
Ingalls A. E., Lee C., Wakeham S. G. and Hedges J. I. (2003) The
role of biominerals in the sinking flux and preservation of
amino acids in the Southern Ocean along 170°W. Deep-Sea Res.
II 50, 713–738.
Ingalls A. E., Anderson R. F. and Pearson A. (2004) Radiocarbon
dating of diatom-bound organic compounds. Mar. Chem. 92,
91–105.
Ingalls A. E. and Pearson A. (2005) Ten years of compoundspecific radiocarbon analysis. Oceanography 18(3), 18–31.
Ingalls A. E., Liu Z. and Lee C. (2006) Seasonal trends in the
pigment and amino acid compositions of sinking particles in
biogenic CaCO3 and SiO2 dominated regions of the Pacific
sector of the Southern Ocean along 170°W. Deep-Sea Res. I 53,
836–859.
Ingalls A. E., Whitehead K. and Bridoux M. C. (2010) Tinted
windows: the presence of UV-absorbing compounds embedded
in the frustules of marine diatoms. Geochim. Cosmochim. Acta
74, 104–115.
Jacot Des Combes H., Esper O., De La Rocha C. L., Abelman A.,
Gersonde R., Yam R. and Shemesh A. (2008) Diatom d13C,
d15N, and C/N since the Last Glacial Maximum in the
Southern Ocean: potential impact of species composition.
Paleoceanography 23, PA4209. doi:10.1029/2008PA001589.
King K. J. (1974) Preserved amino-acids from silicified protein in
fossil Radiolaria. Nature 252, 690–692.
Knott J. M., Romer P. and Sumper M. (2007) Putative spermine
synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine. FEBS
Lett. 581, 3081–3086.
Knott J. M. (2009) Biosynthesis of long-chain polyamines by
crenarchaeal polyamine synthases from Hyperthermus butylicus
and Pyrobaculum aerophilum. FEBS Lett. 583, 3519–3524.
doi:10.1016/j.febslet.2009.10.014.
Kröger N., Deutzmann R. and Sumper M. (1997) Polycationic
peptides from diatom biosilica that direct silica nanosphere
formation. Science 286, 1129–1132.
Kröger N., Deutzmann R., Bergsdorf C. and Sumper M. (2000)
Species-specific polyamines from diatoms control silica morphology. Proc. Natl. Acad. Sci. 97(26), 14133–14138.
Lee C. and Jørgensen N. O. G. (1995) Seasonal cycling of
putrescine and amino acids in relation to biological production
in a stratified coastal pond. Biogeochemistry 29, 8–17.
Matsunaga S., Sakai R., Jimbo M. and Kamiya H. (2007) Long
chain polyamines (LCPAs) from marine sponge: possible
implication in spicule formation. Chem. Biochem. 8, 1729–1735.
Menzel H., Horstmann S., Behrens P., Barnreuther P., Krueger I.
and Jahns M. (2003) Chemical properties of polyamines with
relevance to the biomineralization of silica. Chem. Commun. 24,
2994–2995.
Mitzutani T., Nagase H., Fujiwara N. and Ogoshi H. (1998) Silicic
acid polymerization catalyzed by amines and polyamines. Bull.
Chem. Soc. Japan 71, 2017–2022.
Nelson D. M., Treguer P., Brzezinski M. A., Leynaert A. and
Queguiner B. (1995) Production and dissolution of biogenic
silica in the ocean: revised global estimates, comparison with
regional data and relationship to biogenic sedimentation.
Global Biogeochem. Cycles 9, 359–372.
Nielsen S.H.H. (2004) Southern Ocean climate variability. Thesis,
University of Tromsø.
LCPA identification in Southern Ocean sediments
Nishibori N., Matuyama Y., Uchida T., Moriyama T., Ogita Y.,
Oda M. and Hirota H. (2003) Spatial and temporal variations
in free polyamine distributions in Uranouchi Inlet, Japan. Mar.
Chem. 83, 307–314.
Noll F., Sumper M. and Hampp N. (2002) Nanostructure of
diatom silica silica surfaces and of biomimetic analogs. Nano
Lett. 2, 91–95.
Oshima T. (1979) A new polyamine, thermospermine, 1,12diamino-4,8-diazadodecanone, from an extreme thermophile.
J. Biol. Chem. 254(18), 8720–8722.
Oshima T., Hamasaki N., Senshu M., Kakinuma K. and Kuwajima I. (1987) A new naturally occurring polyamine containing a
quaternary ammonium nitrogen. J. Biol. Chem. 262(25), 11979–
11981.
Pohnert G. (2002) Biomineralization in diatoms mediated through
peptide- and polyamine-assisted condensation of silica. Angew.
Chem. Int. Ed. 41(17), 3167–3169.
Robinson R. S., Brunelle B. G. and Sigman D. M. (2004)
Revisiting nutrient utilization in the glacial Antarctic: evidence
from a new method for diatom-bound N isotopic analysis.
Paleoceanography 19, PA3001. doi:10.1029/2003PA000996.
Robinson R., Sigman D. M., DiFiore P. J., Rohde M. M.,
Mashiotta T. A. and Lea D. W. (2005) Diatom-bound 15N/14N:
new support for enhanced nutrient consumption in the ice age
subantarctic. Paleoceanography 20, PA3003. doi:10.1029/
2004PA001114.
Robinson R. S. and Sigman D. M. (2008) Nitrogen isotopic
evidence for a poleward decrease in surface nitrate within the
ice age Antarctic. Quatern. Sci. Rev. 27, 1076–1090.
Rosenthal Y., Dahan M. and Shemesh A. (2000) Southern Ocean
contributions to glacial–interglacial changes of atmospheric
pCO2: an assessment of carbon isotope records in diatoms.
Paleoceanography 15(1), 65–75.
Sachs J. P. and Repeta D. (1999) Oligotrophy and nitrogen fixation
during eastern Mediterranean sapropel events. Science
286(5449), 2485–2488.
Sanchez-Lopez J., Camanes G., Flors V., Vicent C., Pastor V.,
Vicedo B., Cerezo M. and Garcia-Agustin P. (2009) Underivatized polyamine analysis in plant samples by ion pair LC
coupled with electrospray tandem mass spectrometry. Plant
Physiol. Biochem. 47, 592–598.
Schneider-Mor A., Yam R., Bianchi C., Kunz-Pirrung M.,
Gersonde R. and Shemesh A. (2005) Diatom stable isotopes,
sea ice presence and sea surface temperature records of the past
640 ka in the Atlantic sector of the Southern Ocean. Geophys.
Res. Lett. 32(10), L10704.1–L10704.4.
4057
Shemesh A., Macko S., Charles C. D. and Rau G. (1993) Isotopic
evidence for reduced productivity in the Glacial Southern
Ocean. Science 262, 407–410.
Shemesh A., Hodell D., Costra X., Kanfoush S., Charles C. and
Guilderson T. (2002) Sequence of events during the last
deglaciation in Southern Ocean sediments and Antarctic ice
cores.
Paleoceanography
17(4),
1056.
doi:10.1029/
2000PA000599.
Sigman D. M., Altabet M. A., Francois R., McCorkle D. C. and
Gaillard J.-F. (1999) The isotopic composition of diatombound nitrogen in Southern Ocean sediments. Paleoceanography 14(2), 118–134.
Sigman D. M. and Boyle E. A. (2000) Glacial/interglacial
variations in atmospheric carbon dioxide. Nature 407, 859–869.
Stuiver M., Reimer P. J. and Braziunas T. F. (1998) High-precision
radiocarbon age calibration for terrestrial and marine samples.
Radiocarbon 40(3), 1127–1151.
Sumper M. (2002) A phase separation model for the nanopatterning of diatom biosilica. Science 295, 2430–2433.
Sumper M. (2004) Biomimetic patterning of silica by long-chain
polyamines. Angew. Chem. Int. Ed. 43, 2251–2254.
Sumper M. and Kröger N. (2004) Silica formation in diatoms: the
function of long chain polyamines and silaffins. J. Mater. Chem.
14, 2059–2065.
Sumper M., Brunner E. and Lehmann G. (2005) Biomineralization
in diatoms: characterization of novel polyamines associated
with silica. FEBS Lett. 579, 3765–3769.
Sumper M. and Brunner E. (2006) Learning from diatoms: nature’s
tools for the production of nanostructured silica. Adv. Funct.
Mater. 16, 17–26.
Sumper M. and Lehmann G. (2006) Silica pattern formation in
diatoms: species-specific polyamine biosynthesis. Chem. Biochem. 7, 1419–1427.
Sumper M. and Brunner E. (2008) Silica biomineralization in
diatoms: the model organism Thalassiosira pseudonana. Chem.
Biochem. 9, 1187–1194.
Swann G. E. A. and Leng M. J. (2009) A review of diatom d18O in
paleoceanography. Quatern. Sci. Rev. 28, 384–398.
Weiner S. and Erez J. (1984) Organic matrix of the shell of the
foraminifers, Heterostegina depressa. J. Foram. Res. 14, 206–
212.
Williams P. M. and Druffel E. R. M. (1987) Radiocarbon in
dissolved organic matter in the central North Pacific Ocean.
Nature 330, 246–248.
Associate editor: Jack J. Middelburg