Energy-®ltering transmission electron microscopy (EFTEM)
and electron energy-loss spectroscopy (EELS) investigation of
clay±organic matter aggregates in aquatic sediments
Yoko Furukawa
Naval Research Laboratory, Sea¯oor Sciences Branch, Stennis Space Center, MS 39529, USA
Abstract
High resolution (<10 nm) transmission electron microscopy (TEM), energy-®ltering TEM (EFTEM), and electron energy loss spectroscopy (EELS) have been used for the direct microstructural imaging and analysis of clay-organic matter aggregates in ®ne-grained aquatic sediments from Jourdan River Estuary, MS, USA. EFTEM and EELS allow rapid, high-resolution spatial mapping and analysis of light elements such as carbon. The study area sediments are comprised of discrete organic matter masses and aggregates of clay plates. Clay aggregates often include organic mat-ter. The comparison of clay aggregate images obtained by the TEM bright-®eld technique and the EFTEM carbon mapping technique shows that carbon within clay domains has spatial features that are in the same size scale as the features of individual clay plates within the aggregates (<20 nm). These intimate spatial associations suggest that organic matter in clay aggregates is either closely attached to the surfaces of individual clay plates or structurally incorporated into clay crystals. Organic matter within clay aggregates does not appear to exist as discrete or massive masses that ®ll the pore spaces within the clay aggregates. These intimate associations, whether they are due to che-mical interaction or physical sequestration, should aect the reactivity of organic matter during early diagenesis. The EELS spectra of clay aggregates show that organic matter always coexists with calcium, suggesting that Ca-containing smectite, rather than Ca-poor clay minerals such as kaolinite or illite, is preferentially associated with organic matter in the study area sediments. Further research is required to determine whether this is due to the sources and depositional history or physico-chemical properties of dierent clay minerals. Published by Elsevier Science Ltd.
Keywords:Energy-®ltering transmission electron microscopy; EFTEM; Electron energy-loss spectroscopy; EELS; Sediment micro-fabric
1. Introduction
A large fraction of organic matter (OM) in ®ne-grained siliciclastic aquatic sediments is believed to be associated with clay mineral particles (e.g. Keil et al., 1994; Mayer, 1994a,b; Bergamaschi et al., 1997; Hedges and Oades, 1997; Keil and Cowie, 1999). Direct obser-vations of aquatic sediments and their precursor marine snow at micron and sub-micron scales using transmis-sion electron microscopy techniques have revealed close spatial relationships between OM and clay particles and aggregates (Heissenberger et al., 1996; Leppart et al., 1996; Ransom et al., 1997; Lienemann et al., 1998). Such close spatial associations are also observed in the lithi®ed counterparts (Boussa®r et al., 1995). On the other hand, associations at nanometer scales have been
inferred through characterization of bulk sediments as well as size- and density-fractionated sediments. Keil et al. (1994) found that more than 90% of organic matter within the sediment samples from the Washington con-tinental margin is inseparable from mineral phases by physical means. Other studies have observed linear cor-relations between bulk OM content and measured available surface area in aquatic sediments from diverse sedimentary environment (Keil et al., 1994; Mayer, 1994a,b; Hedges and Keil, 1995). Mayer (1994b) showed through N2sorption experiments that small pores of 10
nm or smaller account for most of the surface area in ®ne-grained siliciclastic marine sediments.
These bulk characterizations have led to a hypothesis that a certain fraction of sedimentary OM may be phy-sically protected from diagenetic remineralization and
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preserved due to its microstructural location within sedimentary clay domains (Hedges and Oades, 1997). Kubicki et al. (1997) suggested that clay±OM associa-tions primarily occur at the clay edges. At the same time, pores of <10 nm diameter (i.e. ``mesopores'' in Mayer, 1994b), are abundant at such clay edges (Mayer, 1999). Such small pores are also commonly seen within clay aggregates of marine sediments in which individual clay plates are aggregated in face-to-face, face-to-edge, or random arrangements (Bennett et al., 1991). Organic molecules located within a mesopore may have less interaction with pore ¯uid constituents due to low per-meability, and as a result, may be more likely to be preserved during early diagenesis than organic mole-cules existing outside a mesopore. The mesopores are also physically too small to accommodate organic car-bon-hydrolyzing enzymes whereas they are large enough to accommodate ordinary OM molecules (Mayer, 1999). This mesopore protection hypothesis has important implications for the mechanistic and quantitative treat-ment of OM degradation kinetics. In early diagenetic studies, OM degradation rates would have to be quan-ti®ed not only in terms of the chemical reactivity of the organic compounds but also in terms of the micro-structures. The spatial arrangement of clay plates and OM is also an important parameter in geotechnical stu-dies because it aects the rheological properties of sedi-ments (Bennett et al., 1991).
Despite the scienti®c importance and strong sugges-tions from the indirect characterizasugges-tions, the actual nanometer-scale spatial relationship between clay aggregates and sedimentary OM has not been well understood. This is primarily due to technical limita-tions: transmission electron microscopy (TEM), the pri-mary tool for the microstructural studies of aquatic sediments, has not been able to routinely produce nan-ometer-scale images of phases that are comprised of only light elements such as carbon. When heavy metal stains are employed in order to make organic carbon ``visible'' to electron beams by creating bonding between organic constituents and electron-dense elements, they reduce the image resolution. Moreover, it is question-able whether or not a sucient amount of stain solution permeates into the mesopores, the potential sites for the close associations between clay surfaces and OM. The greater part of the clay surface area is considered to be contained in such small pore spaces (Mayer, 1994b). Consequently, the existing TEM studies of clay±OM associations in aquatic sediments and precursor marine snow have focused on microstructural features that are generally no less than 50 to 100 nm (Heissenberger et al., 1996; Ransom et al., 1997; Lienemann et al., 1998). Such resolution is insucient for resolving features most rele-vant to the mesopore protection hypothesis (i.e. <10 nm). Energy-®ltering TEM (EFTEM), a new frontier in electron microscopy, allows the high resolution spatial
mapping of elements, including light elements such as carbon. By using EFTEM, one can obtain high-resolu-tion images created by beam electrons that have been through known amounts of energy loss. The minimum amount of energy loss experienced by TEM beam elec-trons due to the excitation of inner shell elecelec-trons is element speci®c (Egerton, 1986). Consequently, a spatial distribution of a given element within the specimen can be created by using all electrons that have experienced the amount of energy loss equivalent to the required excitation energy for the element of interest. The theo-retical spatial resolution limit has been considered and determined to be well below a few nm (e.g. Krivanek et al., 1995).
Electron energy-loss spectroscopy (EELS), having been utilized to study degradation products of terrestrial plants (Watteau et al., 1996), is another technique pre-viously underutilised for the study of aquatic sediments. In this technique, the electron energy loss is directly investigated using the two-dimensional plots of electron energy vs. electron counts, rather than by forming two-dimensional spatial images. The plots can be used to qualitatively identify elements that coexist within a given spatial area.
This study investigates spatial arrangements and the chemistry of the clay±OM association in aquatic sedi-ment samples from the Jourdan River Estuary near the town of Bay St. Louis, MS, USA. EFTEM is used in conjunction with high-resolution TEM in order to spatially resolve the intimate associations between OM and clay mineral aggregates at scales relevant to the sizes of indivi-dual clay plates and associated mesopores. EELS is used to obtain aggregate- and individual particle-scale chemistry of the clay±OM association. Pore water and solid phase chemistry is also presented to allow us to discuss the sour-ces and fates of OM in the study site sediments.
2. Methods
Sediments from one of the tube cores were sliced into 1 cm-thick sections and centrifuged to yield pore water samples within 6 h of coring. The pore water samples
were kept frozen in tightly capped high-density polyethylene bottles until the time of aqueous phase analysis (of chloride by AgNO3 titration, ammonia by
spectrophotometry, and sulfate by liquid ion chroma-tography; Gieskes et al., 1991), which took place within two weeks. The solids, after discarding plant fragments visible to the unassisted eyes, were kept frozen until the time of solid phase analysis (of total organic carbon and total organic nitrogen by Carlo Elba CNS analy-zer, and bulk mineralogy by X-ray powder diraction), which took place within three months. The other tube core was kept refrigerated until the time of 1 cm-reso-lution grain size (by sieving and pipette analysis; Folk, 1974) and porosity (by standard techniques; Lambe, 1951) analyses, which took place within one week of coring.
Samples for the TEM investigations were taken as sub-samples of the slab-type sediment core, which col-lected sediments from zero to 20 cm below the water± sediment interface. The slab cores were kept in a refrig-erator until the time of sub-sampling. Within 24 h of coring, the slab was laid horizontally, opened, and a small amount of sub-sample was taken using a mini-corer (Lavoie et al., 1996) for the subsequent embed-ding, ultrathin-sectioning, and TEM study. The sub-sample was located at 9 cm below the water±sediment interface where the sediment was free of visible grass roots and fragments. The color (anoxic black) and
tex-ture (®ne-grained, cohesive, and away from burrows) were also criteria in choosing the representative sub-sample. The grain size and geochemical analyses, shown in Figs. 2±4, indicate that the BSL6 sediments do not exhibit any detectable abrupt change or boundary downcore, and thus TEM samples may be taken from any given depth to represent the continuum of the sedi-mentary processes at the site.
In order to spatially map the distribution of organic carbon, the sub-sample was embedded in elemental sul-fur rather than resin. Although resin is the conventional medium for embedding aquatic sediments, it is a carbon compound and overwhelms carbon energy loss signals from organic carbon under EFTEM and EELS. The sub-sample was air dried at room temperature, mixed with 99.98% purity powdered elemental sulfur (Aldrich Chemical Company), heated to 130C for approxi-mately 20 min in an oven, and cooled to form an amorphous mass at room temperature. The resulting small block of sulfur-embedded sample particles was ultramicrotomed using Leica Ultracut to the thickness of 60 to 100 nm and the ultrathin-sections were moun-ted on 2000-mesh Cu grids. The extremely ®ne mesh better supports the ultrathin-sectioned beam sensitive sulfur-embedded samples.
Although essential for the carbon mapping, the ele-mental sulfur embedding medium employed in this study is disadvantageous in terms of preservation of some of the microstructural features. The most rigorous preparation of ultrathin-sections, which involves pore ¯uid replacement by progressively concentrated alcohol solutions and ultimately by resin, is considered to embed marine sediments without microfabric distor-tions (Baerwald et al., 1991). Consequently, another mini-core was taken from the same slab core at the same depth and embedded in resin following the ¯uid repla-cement sequence. The microwave protocol (Lavoie et al., 2000) was employed for the ¯uid replacement sequence and resin curing in order to hasten the process. The microfabric sample in resin block was ultrathin-sectioned using Leica Ultracut to the thickness of 70 nm, mounted on ordinary 200 mesh Cu grids, and stained using lead citrate and uranyl acetate solutions (Hayat, 1970).
The ultrathin-sections were examined with a JEOL 3010 transmission electron microscope (TEM) at the Naval Research Laboratory, Stennis Space Center, MS, USA, operated at 300 kV in conjunction with Gatan Image Filter (GIF 2000) which allows EELS and
EFTEM imaging. A comprehensive review of the theory and application of EFTEM and EELS can be found in Reimer (1995). A brief description pertinent to this study follows. GIF allows energy selection (``®ltration'') of beam electrons by bending the electron paths with a magnetic prism. The bending angle is a function of energy. When a TEM beam electron is transmitted through a thin material, it may cause various excitations by transferring (losing) energy to the material, and the minimum energy transfer (loss) required for one type of excitation, ionization of inner shell electrons, is element speci®c. GIF takes advantage of this element speci®c energy loss, selected by the magnetic prism, to map ele-mental distributions by projecting images only using electrons that have lost certain amounts of energy, rather than using all transmitted electrons as in ordinary bright-®eld imaging. GIF can also create EELS spectra by plotting the energy loss vs. number of electrons. In case of carbon, the minimum element-speci®c energy loss (i.e. inner shell loss edge) is at 284 eV, and the majority of energy loss occurs within several eV above that value, following which the incident of energy loss gradually decreases with energy. In this study, a 5 eV wide energy selecting slit was set to transmit electrons that have lost 285 to 290 eV in order to map the spatial distribution of carbon. Some of the energy loss between 285 and 290 eV results from background, which includes energy loss due to other elements whose inner shell loss edges occur much before 284 eV. In order to calculate the background contribution and subtract from the image created by the 285±290 eV electrons, two more energy-®ltered images are collected at 245±250 eV and 265±270 eV. The background decay with energy is cal-culated from these two images and extrapolated to 285± 290 eV in order to subtract the background contribu-tions. The background modeling based on the power law is carried out by the GIF 2000 software package.
The typical imaging and spectroscopy protocol employed during this study is described below. First, the ultrathin-sections are scanned under the ordinary TEM bright-®eld mode in order to determine the areas of interest for further high-resolution imaging. Once an area is selected, a high-resolution bright-®eld image is recorded using a CCD camera (Gatan 694 Slow-Scan Camera). Next, a quick electron energy loss spectrum (EELS) of the area is acquired in order to calibrate the GIF energy selecting slit. Finally, three energy-®ltered images of the area with the energy ranges speci®ed above are recorded using the same CCD camera. A high-quality EELS spectrum is recorded for a 30 nm-diameter circular region that includes all spatial features such as clay sheet faces, clay edges, and intra-aggregate pore spaces after an aggregate for analysis is randomly selected. The EELS spectra show the smooth back-ground decay of the amount of electrons which experi-enced the energy loss as a function of the energy level. Fig. 3. Total organic carbon (TOC, wt.%) and atomic ratio of
The energy resolution of electron energy loss in the experimental con®guration used in this study is con-sidered to be better than 2 eV. The bright-®eld images were taken at the exposure time of 0.01 to 0.1 s, whereas the energy-®ltered images required much longer expo-sure, typically around 60 s, due to the small number of electrons that experience 245±295 eV energy loss.
Areas for the high-resolution bright ®eld imaging and energy-®ltered imaging were selected to represent clay domains commonly observed in the study area samples. Moreover, clay mineral particles that have been ultra-thin-sectioned perpendicular to their cleavage planes were preferentially chosen because such orientation is ideal for the observation of both types of clay mineral surfaces, cleavage surfaces and sheet edges.
The resin-mounted ultrathin-sections were also observed using TEM in order to investigate the clay aggregate structures and pore space geometry.
3. Sediment characterization results
X-ray powder diraction of the study site sediments reveals that illite, smectite, and kaolinite clays are pre-sent. Also present are quartz and plagioclase feldspar. No carbonate phase was detected. Based on the grain
size analysis, the site sediments are characterized as silty or clayey mud (Fig. 2). Depth pro®les of the total organic carbon (TOC) and organic carbon to organic nitrogen atomic ratio (C/N) are shown in Fig. 3, for both BSL6 and BSL4. The C/N values of BSL6 are generally at the high end of the range expected for soil OM, suggesting that the OM in the sediments is a mix-ture of soil organic matter transported by Jourdan River and some in situ input of decaying marsh plant matter. Compared to BSL4, which shows a high TOC and strong correlation between TOC and C/N, BSL6 has received relatively small amount of in situ input of marsh plant matter. The pore water pro®les of ammonia and sulfate (Fig. 4) indicate active anoxic remineraliza-tion of OM in the study area sediments.
4. TEM results and discussion
Bright-®eld images of six clay aggregates and their corresponding carbon elemental maps by EFTEM are shown in Fig. 5. Note that only those aggregates that yielded carbon signals are shown. Areas represented by the carbon maps do not exactly match, and are some-times slightly smaller than, their bright ®eld counter-parts due to the inevitable small amount of specimen
drift during the acquisition of images. The elemental maps can only be calculated from areas that are inclu-ded in all three consecutive energy-®ltered images.
Side-by-side comparisons of the bright-®eld images and carbon maps reveal the intimate spatial relationship between individual clay plates and organic carbon even at these scales of 20 nm or less. Organic carbon signals often mimic the nm-scale features observed in the bright-®eld images, such as the orientation of platy clay particles and pore spaces that occur between sheets within the clay domains, suggesting that OM in these aggregates is intimately associated with individual clay plates either by attaching to the clay surfaces or by incorporating into the plate structure. None of the
car-bon maps represent organic matter as discrete masses lacking spatial features of 20 nm or less. The images do not yield quantitative information regarding the asso-ciation of OM with dierent types of clay aggregate features: one cannot quantitatively compare the amount of OM associated with cleavage surfaces on the exterior of aggregates, clay sheet edges, and cleavage surfaces located inside the aggregates from these images. How-ever, presence of the coinciding platy features in the carbon maps and bright-®eld images indicates that organic carbon in these aggregates is closely associated with the clay mineral plates of <20 nm.
For example, Fig. 5(a) shows a pair of bright-®eld and carbon map images of a portion of clay aggregate.
On the left in the bright-®eld image, dark areas represent individual clay plates whereas bright areas represent either the elemental sulfur matrix or holes in the ultra-thin section. The carbon map on the right, on the other hand, shows areas from which carbon signals are detected as bright areas and areas with little or no carbon signal as dark areas. Presumably the carbon signals come from intrinsic organic carbon. A comparison of these images reveals that organic carbon is very closely associated with individual clay plates at the scale of 20 nm or less.
One would expect carbon signals to come from all parts of a clay aggregate if OM were to exist as massive binding material. Instead, in these samples shown in Fig. 5, carbon signals mimic the features of individual clay plates such as their orientations and plate thickness. The spatial resolution of these images is not sucient to determine whether OM in these samples is either closely attached to the surfaces of individual clay plates or incorporated into plate structures. Likely mesopore locations such as at the edge to face junctions of play plates [indicated by arrows in Fig. 5(a)] do not seem to have particularly large concentration of OM. On the other hand, other locations with possible concentration of mesopores, plate edges [indicated by arrows in Fig. 5(b) and (c)], seem to yield strong carbon signals.
EELS spectra obtained from 10 separate clay aggre-gates are shown in Fig. 6. Note that all high-quality EELS spectra obtained during the course of investiga-tion are shown regardless of the presence or absence of the carbon edge. The spectra indicate that not all clay aggregates contain organic carbon, and some aggregates contain more carbon than others. These observations also show that when the carbon edge (®lled arrow, begins at284 eV and slowly tapers o after reaching maximum at 295 eV) is present, calcium edge (gray
arrow, at350 eV) is also present except for one case (Fig. 6a). A possible explanation is that OM in the study area sediments is more likely to be associated with Ca-rich smectite clays rather than Ca-poor phases such as illite and kaolinite. Whether this association is due to the transport and depositional history, the chemical properties, or nature of mesopores in dierent types of clay minerals cannot be determined from the data available at this point.
Typical TEM images of the resin-embedded and stained ultrathin-sections are represented in Fig. 7. Numerous micron- to submicron-sized clay aggregates (CA) and possible polysaccharide networks (P) can be seen in the low resolution image (a) within the highly open (i.e. porous) microfabric. The dimensions of these
clay aggregates and constituent individual clay plates are very similar to those observed during the EFTEM imaging in elemental-sulfur embedded samples. The gray areas represent pore spaces. The white patches are ultrathin-sectioning artifacts due to sediment grains being plucked out by the slicing knife. The high-resolu-tion image of the interior of a clay aggregate shows the platy stacking features that are also commonly seen in the sulfur-embedded ultrathin-sections.
5. Conclusion
Earlier studies of mineral-OM association microfabric in marine sediments and marine snow (Heissenberger et al., 1996; Ransom et al., 1997; Lienemann et al., 1998) have revealed the intimate micron to sub-micron scale spatial associations between mineral particles and OM. The present study further shows that the close spatial associations are still intact even at scales of <20 nm. This is evident by the <20 nm features that appear in both bright-®eld and carbon-map images, including the coinciding orientation of platy carbon masses and clay plates. This must result from close microstructural associations between OM and individual clay plates at those scales. OM does not exist as discrete masses larger than the size of individual clay plates when it is asso-ciated with clay aggregates.
Some previous studies have argued that pore spaces smaller than 10 nm might be the preferred sites for the preservation of organic carbon through early diagenesis (Mayer, 1994a,b; Hedges and Keil, 1995). Presumably,
such small pore spaces physically protect OM from direct interaction with carbon-hydrolyzing enzymes and pore water constituents essential for OM remineraliza-tion. This argument has been indirectly supported by the previous observation of bulk sediments, that show (1) a linear relationship between measured surface area and OM content (Keil et al., 1994; Mayer, 1994a,b; Hedges and Keil, 1995), and (2) the greater part of the surface area being accounted for by pore spaces smaller than 10 nm (Mayer, 1994b). Whereas the present study does not yield direct evidence regarding this argument, such as the direct observation of OM residing within a 10 nm pore, it does provide a framework to support the association. The coinciding <20 nm features in the sets of carbon maps and bright-®eld images (Fig. 5) posi-tively indicate that OM does not exist as discrete masses of size larger than the size of individual clay particle in thec-axis direction (50 nm or less) when it is associated
with clay aggregates. Also, there seems to be a relatively high concentration of OM at the edge of clay plates, where a high concentration of mesopores is expected.
This study shows that EFTEM and EELS are appro-priate tools for the investigations of clay±OM associa-tions in aquatic sediments at the scales relevant to the physical and chemical interactions between OM, clay aggregates, and OM-hydrolyzing enzymes. Further stu-dies combining this technique with bulk sedimentary characterizations, size and density fractionation techni-ques, and TEM-assisted mineralogical techniques are planned to determine the controlling variables for the preservation and remineralization of sedimentary OM.
Acknowledgements
Discussions with G. Cody and L. Mayer determined the course of this study. I thank R. Mang, A. Reed, and C. Vaughan for their assistance in collecting the ®eld samples, M Tuel for C/N analysis, and A. Falster for XRD. J. Watkins assisted me in preparing the TEM samples. T. Daulton provided support for the TEM instruments. This study was funded by Dr. D. Lavoie as a sub-component to her NRL 6.1 Core Research, and by ONR 322GG (Dr. J. Kravitz, Program Manager).
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