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Marine Chemistry 92
Examining marine particulate organic matter at sub-micron scales
using scanning transmission X-ray microscopy and carbon X-ray
absorption near edge structure spectroscopy
Jay A. Brandesa,*, Cindy Leeb, Stuart Wakehamc, Michael Petersond, Chris Jacobsene,
Sue Wiricke, George Codyf
aMarine Science Institute, The University of Texas at Austin, 750 Channel View Drive, Port Aransas, TX 78373, United StatesbMarine Sciences Research Center, Stony Brook University, Stony Brook, NY 11794-5000, United States
cSkidaway Institute of Oceanography, 10 Science Circle, Savannah, GA 31411, United StatesdSchool of Oceanography, University of Washington, Seattle, WA 98195, United StateseDepartment of Physics, SUNY-Stony Brook, Stony Brook, NY 11794, United States
fGeophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Road NW, Washington, DC 20015, United States
Received 27 October 2003; received in revised form 4 May 2004; accepted 30 June 2004
Available online 3 October 2004
Abstract
Marine sinking particulate organic matter (POM) represents the link between surface primary production and burial of
organic matter in marine sediments. As such, the nature of this material has been the subject of numerous studies attempting
to characterize its composition. The results of these studies have shown that a significant proportion of POM is not
recognizable as known compounds, and that the proportion of uncharacterized material increases with age/depth/diagenesis.
However, few studies have examined the spatial heterogeneity of this material. This study uses a new tool, scanning
transmission X-ray microscopy (STXM), together with carbon X-ray absorption near edge structure (C-XANES), to examine
POM collected from sediment traps deployed at one location in the Arabian Sea as part of the JGOFS program. The results
indicate that POM is composed primarily of four distinct phases: protein, an aliphatic rich phase, a carboxylic-acid-rich
phase, and a phase with complex unsaturated and quinone character. This last phase may be a condensation product between
carbohydrates and proteins, or from degraded plant pigments. Many particles consisted of a single chemical phase; however,
in particles with mixed compositions, individual domains retained distinctive chemical signatures at the instrument’s
resolution limit (50 nm). All major chemical phases were observed in sediment trap particles from 531 to 3369 m depth,
supporting the hypothesis that non-selective degradation dominates particle remineralization, and that overall particle
compositions are determined by near surface processes. Only one particle, out of more than 60 examined, exhibited soot-like
0304-4203/$ - s
doi:10.1016/j.m
* Correspon
E-mail addr
(2004) 107–121
ee front matter D 2004 Elsevier B.V. All rights reserved.
archem.2004.06.020
ding author. Tel.: +1 361 7496756.
ess: [email protected] (J.A. Brandes).
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121108
composition. The lack of a significant black carbon/soot component may be attributable to sampling during the winter
monsoon period.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Organic Geochemistry; Sediment trap; X-ray microscopy; Arabian Sea; Black carbon; Algaenan
1. Introduction
The nature of organic matter leaving the euphotic
zone and its fate have been the subject of much
debate (Lee et al., in press). Wakeham et al. (1997)
were able to identify many compounds directly by
HPLC, GC, and GC/MS, yet, their work pointed to
an inability to identify the majority (N50%) of carbon
in sinking POM. The presence of banalyticallyuncharacterizedQ material in sinking POM (Hedges
et al., 2000) has led to a number of hypotheses about
the nature of this material. Sources may include:
algaenan or waxy insoluble material (Hwang and
Druffel, 2003), soot or ash-derived black carbon
(Deuser et al., 1983), transparent exopolymers
formed from polysaccharides (Chin et al., 1998;
Passow et al., 2001; Lee et al., in press), or organic
matter that has been rendered unidentifiable by
internal polymerization/crosslinking (Hedges, 1978;
Qian et al., 1992). Attempting to reconcile between
these competing hypotheses is difficult as different
methods examine only selective fractions of POM.
There is also the unstated assumption that com-
pounds are homogenously distributed within sam-
ples, but there is very little information to support or
refute this.
One reason that spatial information content of
samples has been largely ignored in biogeochemistry
is that the analytical techniques most commonly used
in this field do not lend themselves to acquiring this
information. Studies of cells using various microscopy
techniques (Mitchell, 2001; Sommer and Franke,
2002; Jamin et al., 2003) serve as reminders that
organic matter is produced with chemical heteroge-
neity on nanoscales. Although the composition of
individual structures may be tremendously altered by
diagenesis, it is not a given that these structures will
be homogenized. In special cases, plant cell structures
have been shown to survive burial for hundreds of
millions of years, with some features still identifiable
by their distinct chemical signatures (Boyce et al.,
2002).
Electromagnetic-radiation-based micro-spectro-
scopy methods such as infrared and X-ray micro-
scopy are increasingly used as tools to investigate
problems in environmental chemistry and biochem-
istry (Myneni, 2002; Dumas and Miller, 2003; Miller
et al., 2003). These spectromicroscopy methods can
generate information on functional group distribu-
tions within samples with enough spatial and
chemical resolution to minimize bsignal averagingQproblems, and with minor sample handling (e.g., no
derivatization or extraction). Infrared microscopy is
diffraction limited to spatial resolutions of roughly
3–10 Am, while X-ray microscopy has a theoretical
resolution limit of less than 10 nm, with current
instruments limited to about 30 nm (Jacobsen and
Kirz, 1998).
Using marine particulate organic matter (POM)
from the Arabian Sea, we have applied scanning
transmission X-ray microscopy (STXM) together
with concurrent measurements of carbon X-ray
absorption near edge structure (C-XANES) spectro-
scopy to examine the potential for identifying
particle compositions and mapping particle hetero-
geneity. This approach allows us to examine the
composition of POM on a particle-by-particle basis.
Although there are limitations in examining large
particles (N200 Am diameter particles were not
analyzed), and particles need to be dried and briefly
heated to embed them in S8 prior to sectioning, our
approach examines all particles without extraction or
derivatization and thus provides an overall view of
particle compositions.
2. Background on X-ray microscopy
X-ray absorption edges arise when an incident X-
ray photon exceeds the threshold energy needed to
Table 1
Approximate energy ranges for primary absorption peaks at the
carbon 1s edge
eV Functionality
284 quinone
284.9–285.5 unsaturated/aromatic
285.8–286.4 aromatic CUOH
286.6 ketone CMO
287.1–287.4 aliphatic
287.7–288.6 carbonyl
289.3–289.5 alcohol
290.3–290.6 carbonate
Sources: Francis and Hitchcock (1992), Hitchcock et al. (1986
1992), Hitchcock and Ishii (1987), Hitchcock and Stohr (1987), and
Urquhart and Ade (2002).
Fig. 1. Schematic representation of X-ray absorption near-edge
structure or XANES spectroscopy. The first peak represents the
primary XANES resonance (e.g., a 1s–k* transition), followed by
the absorption edge. Transitions can be from a low level occupied
orbital (n=1, 2, or 3) to either an unoccupied molecular orbital
(represented by the first arrow) or to the continuum (second arrow),
where electrons are fully ionized by absorption of X-ray energy.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121 109
completely remove (ionize) an electron from an inner-
shell orbital. The result is a step-like rise (the bedgeQin X-ray absorption near edge structure spectroscopy)
in the absorption cross section. Photons with energies
just below the ionization edge (280–300 eV in the
case of carbon 1s electrons) can promote core
electrons into a variety of bound states that correspond
to unoccupied or partially occupied molecular orbitals
(Fig. 1). These near edge absorption bands are
sensitive indicators of the local chemical bonding
environment surrounding the atom in question (Stohr,
1992). In the case of C (1s) XANES, the absorption
spectrum can be used to estimate the abundances of
different functional groups, subject to the caveat that
carbon in different electronic environments may have
vastly different absorption coefficients, and in many
cases absorption bands from different organic func-
tional groups overlap.
Extensive C-XANES studies of polymers and other
well-characterized materials provide generally robust
peak assignment criteria. In the case of carbon K-shell
electrons, the near edge absorption bands span from
280 to 295 eV and correspond to transitions to both
unoccupied k* (antibonding) and low lying j*orbitals. As a general rule, the peak width is given
by the lifetime of the photoexcited transition. Tran-
sitions at lower energies relative to ionization edge
have longer lifetimes, and since the 1s–k* transitions
occur at lower energy than do 1s–j* transitions, the
1s–k* transitions tend to have a narrow band width.
Conjugated k orbitals present multiple unoccupied k*orbitals to which the photoexcited electron can be
promoted. In general, however, these higher energy
1s–k* transitions are considerably weaker than the
lowest energy 1s–k* transition. Table 1 lists the peak
energy assignments for common carbon functional
groups. The lowest absorption bands, occurring at
around 284–285 eV, correspond to molecules with
unusually low energy k* states, e.g., quinones.
Aromatic carbon that is bonded to hydrogen or
reduced carbon exhibits a strong 1s–k* transition at
~285 eV. With the addition of electron withdrawing
elements in the ring (e.g., pyridinic nitrogen) or
substituted on the ring (e.g., carbonyl or oxygen) the
binding energy of the 1s electron is increased shifting
the 1s–k* transitions of aromatic carbon to higher
energies, e.g., ~286 eV for carbonyl and nitrogen, and
~287 eV for oxygen (e.g., phenols). In the case of
carboxyl functional groups, the effect of two oxygens
results in a shift of the 1s–k* transition up to 288.5
eV; carboxyamide’s 1s–k* transition lies slightly
lower at 288.2 eV (see discussions by Urquhart and
Ade, 2002). Saturated carbon exhibits a relatively
strong absorption band near 287.5–288 eV, when
substituted with oxygen (e.g., ethers and alcohols),
this transition shifts up to ~289.5 eV. Although an
overlap exists between different functional groups
(most notably in the 286–289 region), in marine
,
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121110
organic matter one can use other information, e.g.,
previous NMR studies, to rule out the presence of
significant aldehyde or ketone functionalities (Hedges
et al., 2001).
Spectra at the full 30–50 nm resolution limit of
present-day X-ray microscopes can be obtained
either by using cross-correlation methods for post-
facto alignment (Jacobsen et al., 2000), or laser-
interferometer scanning stage control (Kilcoyne et
al., 2003), to acquire a large series of images over a
range of energies spanning the XANES spectral
region. This stack of images yields a data set with
spatial dimensions X and Y, and energy dimension E.
All of the particle data presented in this paper were
collected from such three-dimensional data sets. By
taking the ratio of data from images collected below
and at a particular absorption edge, one can map the
concentration of specific functional groups across a
sample. For example, calculation of the ratios of
image maps from 285 and 280 eV yields a map of
the distribution of unsaturated carbon within a
sample, while a ratio of 298 eV (above the XANES
regions) to 280 eV yields a carbon density or optical
density map.
Because we obtain data with 100–200 spectral
points, and several tens of thousands of spatial
locations, multivariate statistical analysis methods are
used to conduct detailed sample analysis. Principal
component analysis (PCA) provides a means of
highlighting successive themes of correlation of
spectra among the (X, Y, E) data (King et al.,
1989; Bonnet et al., 1999; Osanna and Jacobsen,
2000), and to reject poor correlations which are
dominated by photon statistics noise only. We have
used PCA as a method of generating a noise-filtered,
orthogonal space for cluster analysis of these data
(Jacobsen et al., 2003; Lerotic et al., 2004). By
grouping pixels together based on their spectroscopic
signature and with PCA noise-filtering, one can
obtain spectra from representative regions in the
sample with a vastly improved signal to noise ratio.
At the stage of development employed here, cluster
analysis has not always succeeded in finding few-
pixel regions with different spectroscopic signatures,
although efforts to improve this in future work are
underway. This paper therefore makes use of both
cluster analysis and user-selected spectromicroscopy
analysis.
3. Methods
Archived sediment trap samples from the Arabian
Sea JGOFS program were analyzed from station M4
(531, 814, 2222, and 3369 m). The collection
methodologies are described in Lee et al. (1998) and
Wakeham et al. (2002). Small subsamples (~1 mg)
were embedded in reagent grade elemental sulfur by
briefly heating (~120 8C) a mixture of fine S8 grainswith the samples on a clean glass slide until the
mixture melted into a drop. The drop was cooled,
removed from the slide with a razor, and glued to an
epoxy mount using cyanoacrylate glue. The drop was
then subsampled into 70–100 nm thick sections using
a Leica Ultra-Cutk ultramicrotome and a diamond
knife. Sections were transferred to silica monoxide
supported TEM grids for storage and analysis. Images
and stacks were collected at the scanning transmission
X-ray microscope located on beamline X1-A (out-
board branch) at the National Synchrotron Light
Source in Brookhaven National Laboratory (Winn et
al., 2000). Full details on the microscope design,
operation and capabilities are given in Feser et al.
(2000) and Feser et al. (2001).
4. Results
Rather than present spectral libraries of possible
model organic compounds present in sinking POM,
we present below representative spectra found in an
investigation of more than 60 POM particles ana-
lyzed. Although material from four traps was ana-
lyzed, the number of particles observed ranged from 7
(531 m trap) to 20 (2222 m trap). The limited number
of observations, necessitated by an average analysis
time of 2 h per observation (one to five particles per
observation), makes combining all observations the
best choice for the overview analysis presented here.
Particle chemical identifications are based upon peak
positions (Table 1) and relative intensities. The spectra
shown in this paper are in units of optical density,
which is the negative of the logarithm of the ratio of
transmitted to incident flux, �ln(I/Io). As a reference,
a 110-nm-thick, 2 g/cm3 carbon film would have an
optical density of 1.0 at 300 eV. The x-axis is plotted
as X-ray energy in electronvolts. The following
organic matter types were observed.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121 111
4.1. Protein
Proteins exhibit both a 285 eV peak, due to the
presence of aromatic amino acids phenylalanine,
tyrosine and tryptophan, and a strong 288.2 eV peak
(Fig. 2A). The 288.2 eV peak is due to the presence of
amide-linked carbonyl carbon, and is shifted down in
energy by 0.2–0.3 eV from the 1s–k* transition of
carboxylic acids. As noted below, protein may also be
present in several of the other particle types identified.
However, for the purposes of identifying the majority
chemical composition, the presence of a 288–288.2
eV peak was considered diagnostic for the dominance
of protein within a particle.
4.2. Material with a strongly acidic nature
In many particles with a dominant absorption peak
in the 288–289 eV region, the peak center was
observed at 288.5 eV, characteristic of carboxylic
acids (Table 1; Fig. 2B). This material exhibited
consistent spectra, namely a small peak in the 285–
286 eV region followed by a primary peak centered at
288.5 eV. The small 285 eV peak indicates the
presence of either aromatic or olefinic carbon. The
relatively broad nature of the 288.5 eV peak, with
significant energy in the 288.0–288.3 eV range likely
records some protein content. A protein spectrum is
overlaid in Fig. 2b to illustrate the spectral differences
between these particle chemistries. The molar absorp-
tion coefficient of aliphatic carbon is much weaker
than that of carbonyl carbon; nonetheless, there is a
significant absorption component in the 287–288 eV
region, as well as some spread of the 288.5 eV energy
into the 289.5 eV region. Material containing a very
prominent and narrow 288.5 eV peak and consisting
of long, thin bstringsQ was found at 531 and 814 m
depths. Spectrally and structurally, this material
strongly resembled lipid-rich cell walls, although an
absolute confirmation of its source could not be made.
All acid-rich particles typically had a 288.5 eV peak
that was 20–30% greater in intensity than the 290–300
eV range.
4.3. Aliphatic particles
Some particles were notable for lack of a strong
peak in the 287–288 eV region, instead exhibiting a
rise/plateau spectrum in this energy range. This
suggests a chemical composition dominated by
aliphatic carbon (Table 1). This material also con-
tained significant energy absorption in the 286 and
287 eV ranges, but little or no absorption in the
aromatic region. The high C/O ratio and presence of
peaks in the ketone/aldehyde region suggest a waxy or
paraffinic nature of this material.
4.4. Carbohydrates
Sinking particulate organic matter contains a
significant proportion of carbohydrates (Wakeham et
al., 1997). Carbohydrates are dominated by a 289.5
eV absorption peak (Cody et al., 1996; Cody and
Saghi-Szabo, 1999) (Fig. 2D). There is also a minor
286.5 eV peak that may be an artifact of radiation
damage or diagenesis. Although we used techniques
designed to minimize this problem (scanning from
low to high energy range, using minimal dwell times
and imaging the particle as few times as possible prior
to spectral analysis) carbohydrates (as well as other
compounds) are susceptible to radiation damage
(Foster et al., 1992; Cazaux, 1997; Cherezov et al.,
2002; Ito et al., 2002; Ravelli et al., 2002), and this
damage appears as both a decrease in the 289.5 eV
absorption (mass loss) and an increase in the 286.5 eV
absorption. The increase at 286.5 eV is presumably
due to the formation of aromatic heterocycles like
furans (Newbury et al., 1986). The formation of
phenolic carbon is unlikely as there is an insignificant
component present at 285 eV.
4.5. Calcium carbonate
Carbonates exhibit a very characteristic strong
absorption peak at 290.5 eV (Fig. 2E) (Urquhart and
Ade, 2002). In pure CaCO3, only a few other minor
absorption peaks are observed (Fig. 2E), but in our
sediment trap samples, the presence of organic matter
was often detected intermixed with the mineral.
4.6. Particles with quinone/unsaturated character
There were a significant number of particles
exhibiting strong absorbance in the 284–286 eV range
associated with unsaturated or aromatic carbon (Fig.
2F). This organic matter is distinguished by several
Fig. 2. Carbon XANES spectra of different classes of marine particulate matter. (A) Protein, (B) acid-rich (with protein spectrum as a
comparison), (C) aliphatic-rich, (D) carbohydrate, (E) CaCO3, (F) quinone/unsaturated, (G) soot/black carbon, and (H) non-carbon mineral.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121112
Fig. 3. Particle chemistry histogram. Dark bards represent particles
observed without any sign of a CaCO3 peak at 290.5 eV, while
speckled bars represent particles with a CaCO3 peak.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121 113
spectral features: (1) presence of a significant 285 eV
peak with components at 284 and 286 eV associated
with unsaturated carbon, phenols, quinone, and
polynuclear aromatic carbon (Horsley et al., 1985;
Francis and Hitchcock, 1992; Hitchcock et al., 1992;
Hitchcock and Mancini, 1994); (2) significant absorp-
tion in the 287–288 eV range associated with aliphatic
carbon; (3) a lack of a strong absorption peak in the
288 or 289 eV range. Some particles do exhibit small
peaks in the 288.5 and 289.5 region, suggesting that
they also may contain organic acids and alcohol
moieties.
4.7. Black carbon/soot
The presence of soot or combustion products in
sinking POM has been observed (Schmidt and Noack,
2000). Of the ~60 particles that we have examined
from station M4 in the Arabian Sea, only one has
unambiguously fit the profile of a combustion source
particle (Fig. 2G). This particle contained a large and
well-defined 285 eV peak, followed by peaks in the
288.5 and 287.5 eV range. This spectrum is character-
istic of combustion-derived soot (see spectra of NIST
standard soot at http://xray1.physics.sunysb.edu/data/
spectra.php), with the exception of a minor 288.5 peak
that may be the result of adsorbed marine carbon or
UV induced oxidation.
4.8. Non-carbon minerals and elements
Mineral grains and SiO2 frustules have bflatQspectra with no absorption peaks present (Fig. 2H).
The presence of mineral in organic matter is
expressed by a uniform increase in optical density,
which is especially noticeable in the region below the
C edge (b283 eV). Thus, if a particle contains a
significant amount of mineral, the optical density will
be shifted upwards by N0.5 (compare the baseline in
Fig. 2D with that in Fig. 2A for an example). The
only common element with an absorption peak in the
280–300 eV range is potassium, this exhibits 2sYk*shell doublet peaks at 295 and 297 eV. This energy
range lies above the region of interest for organic
carbon; however, it can complicate efforts to deter-
mine particle densities based upon techniques that
integrate the region immediately above the absorption
edge at ~290 eV.
5. Discussion
5.1. Overall distributions
The roughly 60 particles from Arabian Sea sedi-
ment traps examined by C-XANES were dominated
by acid-rich (~25%), aliphatic (15%), protein-rich
(15%), and quinone/aromatic (10%) compositions
(Fig. 3). In many cases, particles consisted of
adjoining regions with different chemical composi-
tions, such as carbohydrate/acid, protein/acid, and
algaenan/acid; in several cases, particles consisted of
three or more dominant chemical types distributed
non-homogenously (denoted as Mix, Fig. 3). Our
estimate of particle chemical distributions is prelimi-
nary; without detailed spectral deconvolution within
each region, we cannot quantify exact chemical
proportions. The presence of a noticeable (N0.1
optical density) peak at 290.5 eV on organic particles
was considered diagnostic for the presence of CaCO3.
Most of the CaCO3-associated material fell into the
acidic particle category (Fig. 3). This is consistent
with the known preferential association of acidic
amino acids and other compounds with CaCO3. The
presence of protein in some of the acid-rich particles
Fig. 4. Carbon optical density maps (ratio of absorbance from 295 to
300 eV to absorbance from 280 to 283 eV) of cell wall remnants
High carbon concentration is shown in bright areas of this optica
density image. Samples from M4, Arabian Sea, 813 m depth.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121114
is also likely, as many of these particles contained
non-trivial (N0.05 optical density) 285 eV peaks
associated with aromatic amino acids (although
unsaturated fatty acids and other olefins are also a
possibility). Thus, our estimate of the number of
protein-containing particles is an underestimate. The
same is true for the aliphatic class; without spectral
deconvolution, it is impossible to assign a proportion
of this composition to particles containing a signifi-
cant amount of protein or acidic materials.
In three cases, we observed carboxylic acid-rich
linear or branched structures (two each in the 814 m
samples, one in the 531 m samples). Examples of
two of these are given in Fig. 4. The strong peak at
288.5 eV in this material is consistent with the
presence of fatty acids. This material may include
the remnants of cell walls, and indeed in Fig. 4A, a
series of linked 4–5 Am length cells is presented.
This material appears devoid of debris or other
cellular remains.
Most particles examined appear to be composed
of only a single chemical phase. About a quarter of
particles consisted of two or more closely inter-
mingled phases. This suggests that the processes of
digestion and aggregation that lead to POM for-
mation do not normally homogenize or mix this
material on tens of nanometer scales, although
smaller scale mixing is possible. Principal compo-
nent analysis on one mixed particle indicated that,
although some of the components appear primarily
to be variations in thickness of the same material
(e.g., Fig. 5D and E), there are at least four
chemically distinct phases present. The material
mapped in Fig. 5C, D, and E are dominated by
aliphatic components, although the outer edges
appear to have some added acidic or proteinaceous
character. There is also evidence of carbohydrate
(Fig. 5G,H), CaCO3 intermixed at a sub-micron scale
with acidic or proteinaceous organic material (Fig.
5I), and inorganic, presumably siliceous, particles
(Fig. 5J). The carbohydrate-rich material appears
more closely associated with the non-CaCO3 con-
taining particles, while the other fractions appear to
be randomly distributed.
In the case of the three-component system shown
in Fig. 6, an inorganic core is surrounded by an
aliphatic-rich layer, followed by a transition to a
proteinaceous/acidic outer layer. The presence of
.
l
defined peaks in the background spectrum shown in
Fig. 6C indicates that the PCA software has integrated
some regions with organic matter in them. However,
the intensity of such peaks is very small (compare the
maximum peak intensity in Fig. 6C at ~0.08 optical
density, with that in Fig. 6D at ~1.2 O.D.). Thus,
although some organic matter has been integrated into
the average spectrum, it is not a major component and
represents very thin regions around the periphery of
the particles in Fig. 6. There is also a small CaCO3
dominated particle in the upper central portion of the
image (Fig. 6G). Even in cases where compounds are
Fig. 5. Optical density and cluster analysis maps of sediment trap sample. Sample was collected from station M4, 2222 m depth. (A) Carbon
optical density map (ratio of absorption integrated from 295 to 298 eV divided by intensity integrated from 280 to 283 eV). (B) Principal
component analysis (PCA) map. Spectra in graphs C–J correspond to integrated signal intensity from regions denoted in color from PCA maps.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121 115
mixed, we observed distinct chemical bzonesQ withinparticles (Figs. 5 and 6). It is interesting to speculate
whether the presence of heterogeneous chemical
bdomainsQ within particles might lead to entanglement
or encapsulation of reactive compounds that could
make it difficult for enzymes to recognize organic
matter. For example, in Fig. 6, there exists a gradient
between acid-rich and aliphatic-rich compositions
within a single particle.
5.2. Particle character
Our results suggest that marine sinking POM
from the Arabian Sea is a mixture of phases,
Fig. 6. Sample from station M4, Arabian Sea, 814 m depth. (A) Cluster analysis map. (B) Carbon optical density map (ratio of absorbance from
295 to 300 eV to absorbance from 280 to 283 eV). (C) Map and spectra of background. (D) Map and spectra of non-carbonaceous material. (E)
Map of amino acid/carboxylic acid rich organic matter. (F) Map of aliphatic-rich material. (G) Map of calcium carbonate.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121116
including several that could be difficult to analyze by
liquid or gas chromatography. One such phase could
be algaenans. The suggestion that algaenans and
other waxy or aliphatic cross-linked phases are
abundant in sinking POM (Hwang and Druffel,
2003) is supported by the presence of a significant
component of aliphatic-rich particles (15%, Fig. 3).
If one includes all particles containing some fraction
of this material, the proportion rises to N20%. While
recognizing that particle abundances do not neces-
sarily correlate to particle mass, this still suggests
that this material is a major component of the
sinking POM. The lack of a strong 288 or 289 eV
peak in this material indicates that it is extremely
oxygen poor, and thus hydrophobic. As the oscillator
strength of carboxyl groups is quite high, even a
small amount of COOH functionality would result in
a noticeable 288.5 eV peak, which was not observed
in most cases (Fig. 2C).
The most intriguing particle composition category
is the quinone/aromatic type (Fig. 2F). This is a
significant particle category (10%, 15% if mix
particles are included). The major cellular biochem-
icals: protein, carbohydrate, lipids, and nucleic acids,
do not exhibit an absorption peak in the 284–285 eV
(quinone) region. However, certain photosynthetic
reaction centers use quinonic moieties for electron
transport, and certain condensation reactions can also
produce quinones. The dissimilarity in spectra
between chlorophyll a (Fig. 7) and the samples
Fig. 7. Carbon XANES spectra of (A) chlorophyll a and (B)
melanoidin produced by the reaction of lysine with glucose.
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121 117
indicates that this POM category is not simply
undegraded chlorophyll or even a simple degradation
product of this pigment (e.g., phaeophorbide). Only
minor amounts of pigments and their intact degrada-
tion products are found in sinking POM (Welsch-
meyer and Lorenzen, 1985; Lee et al., 2000;). Another
possible reaction pathway that could produce quinone
moieties is the reaction between proteins and carbo-
hydrates, the Maillard reaction (Hedges, 1978).
Several studies have shown that this reaction is
capable of providing a wide range of polynuclear
aromatic, quinone and unsaturated compounds (e.g.,
Olsson et al., 1977, 1978; Miller et al., 1984). Indeed,
C-XANES of the reaction product of lysine and
glucose (e.g., melanoidin) contains significant spectral
content in the 284–285 eV range (Fig. 7A), but is
again, not spectrally identical to this POM class.
There is also a spectral feature at around 286.3–287.0
in Fig. 2F that may correspond to a peak at 286.4 eV
in the melanoidin spectrum. Without further work, it
is not possible to distinguish between these two
sources, or to rule out another pathway that can
produce this material.
Surprisingly, a particle class that does not appear
to form a major component of sinking POM in
Arabian Sea samples is soot/black carbon (BC). In
this study, we observed only one such particle (out
of N60); if combustion products made up a signifi-
cant percentage of sinking POM, then one might
expect to observe many more. The quinone/aromatic
fraction described above may be ruled out as soot or
combustion derived BC based upon two observa-
tions: (1) Most of that particle class had a
morphology inconsistent with soot, being composed
of strings and membrane-like structures; and (2) soot
and graphitic material does not, in our experience,
exhibit a significant sub-285 eV component. It must
be noted that the Arabian Sea is a marine produc-
tivity dominated regime, and that samples from sites
closer to urban population centers may contain larger
amounts of soot and ash derived black carbon. These
samples were also taken during the winter monsoon
period, when prevailing winds come from Somalia.
The presence of significant amounts of soot and
combustion products in aerosols originating from the
Indian subcontinent (Dickerson et al., 2002; Menon
et al., 2002) would presumably generate a greater
abundance of black carbon in particles collected
when winds from the NE are prevalent. It also may
be possible that very small aerosol particles of BC
would be overlooked by our method (although stack
analysis targeting aromatic rich particles was done
for each data set). However, very small particles
would not sink unless adsorbed or otherwise attached
to larger particles.
Given the high proportion of protein identified in
sinking POM by Wakeham et al. (1997) and Lee et
al. (2000), it is not surprising to see protein as a
major particle composition class (15%, or 27%
including mixtures, Fig. 3). Some particles within
the acid-rich particle class (25%, Fig. 3) also
undoubtedly contain significant amounts of protein,
based upon broadening of the primary peak in the
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121118
288.0–288.3 region. It must be noted that other,
non-protein, compounds can give spectra quite
similar to the acid-rich type particles shown in
Fig. 2B. For example, peptidoglycan and chitin both
have primary absorption peaks centered at 288.5 eV.
The chief spectral differences between pure pepti-
doglycan and the particle spectra in Fig. 2B are that
peptidoglycan exhibits no absorption in the aromatic
region (285 eV) and has a significant 289.5 eV
peak associated with alcohol (peptidoglycan, chitin
and other model compound spectra can be found at
the X1A online spectral library http://xray1.physics.
sunysb.edu/data/spectra.php). The lack of strong
289.5 eV peaks in the acidic particles indicates that
either alcohol functionalities are lost early in
diagenesis or that the material began with a lower
alcohol/carbon ratio than typical for carbohydrates.
5.3. Implications for the alteration of sinking POM
Given the results presented above, one can begin
to put different results in the context of the large
body of work examining POM decomposition.
Hedges et al. (2001) suggested that the overall
chemical composition of sinking POM was consis-
tent with passage through the water column.
Although we did not measure enough particles to
make definitive statements about the effect of early
diagenesis upon particle composition (less than 15
particles were measured at each depth), we did
observe all of the major particle chemical categories
at each depth. This would suggest that each particle
type is labile to some extent, as there will be a
significant loss of material between the 531 and
3369 m traps (Lee et al., 1998). In addition, even
material that would presumably be extremely labile,
such as proteins, is present at depths where over 2/3
of the total POM flux has been remineralized
(Wakeham et al., 1997; Lee et al., 1998). Thus, the
overall pattern of particle chemistry, including the
presence of presumably highly altered material,
appears to be set early on in the lifespan of POM.
The large proportion of analytically uncharacteriz-
able material in deep-water POM is consistent with
the presence of waxy, balgaenanQ-like material
(Hwang and Druffel, 2003) as well as heavily
degraded, presumably crosslinked quinone/aromatic
containing organic matter possibly derived from
reactions between proteins and carbohydrates. The
apparent absence of soot or black carbon in this
marine organic matter, as opposed to previous
studies (Masiello and Druffel, 1998; Middelburg et
al., 1999; Tsapakis et al., 2003), may be due to a
lack of input of these materials during the sample
period, or it may be an artifact of the chemical and
physical isolation procedures used.
Many questions still remain and new ones are
raised by this work. Chief among these is why
different compound classes appear to be remineralized
with equal alacrity by heterotrophs. When one
considers the spectrum of particle character, from cell
walls and protein-rich particles to waxy/algaenan-like
and highly condensed quinone/aromatic containing
molecules, the expectation would be that some
material would be removed/remineralized preferen-
tially, consistent with the results obtained by molec-
ular-level studies (Wakeham et al., 1997; Lee et al., in
press). Again, we cannot make definitive statements
as to particle class abundance with depth, but clearly,
given the limited subset of data presented here, we can
state that no one class is completely removed by
diagenesis at depth. It is not possible, given the
overall mass losses experienced by sinking POM, to
state that any of the chemical types presented in Fig. 2
is immune from degradation. A plausible explanation
is provided by Armstrong et al. (2002), where material
that reaches the deep sea is dominated by mineral-
ballasted organic matter. It may be hypothesized that
all of the organic matter particle types presented here
are susceptible to degradation, but at a rate slow
enough so that particles with mineral ballast can reach
the deeper ocean with their initial and overall
chemistries intact.
6. Conclusions
The use of synchrotron-radiation-based soft X-ray
spectromicroscopy has enabled the examination of
individual particle compositions from sediment trap
samples with minimal sample alteration. Particles
collected in traps from 531, 814, 2222, and 3369 m
depths from station M4 in the Arabian Sea JGOFS
program (Lee et al., 1998) showed a variety of
individual particle phases, but were dominated by
four types. Particles with strong 288.5 eV peaks
J.A. Brandes et al. / Marine Chemistry 92 (2004) 107–121 119
characteristic of carboxylic acids formed the highest
percentage of material, but protein, waxy, and
quinone/aromatic particle chemistries were also
observed. Intact cell walls were observed in trap
samples down to a depth of 814 m, but not seen
below that depth. All other particle classes were
observed down to the lowermost trap samples at
3369 m depth. The results suggest that much of the
bmolecularly uncharacterizedQ fraction described by
Wakeham et al. (1997) and Hedges et al. (2000) is
composed of aliphatic-rich and quinone/unsaturated
material. We hypothesize that this latter particle
composition is the result of Maillard-type reactions
between proteins and carbohydrates and or pigments
that take place early in the particle’s history. Only 1
out of more than 60 particles fit the spectral profile
of soot or other bblack carbonQ combustion derived
materials, suggesting that this material is not a major
component of sinking organic matter in the Arabian
Sea during the winter monsoon period. The presence
of all four major particle classes in even the deepest
trap samples also suggests that no one particle
composition is particularly susceptible to degrada-
tion, rather, the survival of individual particles may
be a balance between slow degradation of all particle
classes and sinking rates influenced by the presence
or absence of inorganic minerals. Future work
involving detailed spectral deconvolution or blineshapingQ, as well as examination of more particles
collected from different regions and seasons, will
provide a more complete assessment of particle
chemical compositions.
Acknowledgements
This work was supported by NSF grants OCE-
0221295 and OCE-0118036 (JAB), OCE-9310364
(SGW), and OCE-9312694 (CL). The National
Synchrotron Light Source is a Department of Energy
supported facility. The authors wish to thank Patty
Garlough and Paul Haberstroh (UT) for assistance in
sample handling and sample analysis, and Mirna
Lerotic (SUNY) for assistance in PCA and cluster
analysis. This work is dedicated to the memory of
John Hedges, who provided encouragement, stim-
ulating discussions and samples for this project.
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