Examining marine particulate organic matter at sub-micron scales using scanning transmission X-ray microscopy and carbon X-ray absorption near edge structure spectroscopy
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 (>50%) of carbon in sinking POM. The presence of “analytically uncharacterized” 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 compounds are homogenously distributed within samples, 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 heterogeneity 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-spectroscopy methods such as infrared and X-ray microscopy are increasingly used as tools to investigate problems in environmental chemistry and biochemistry (Myneni, 2002, Dumas and Miller, 2003, Miller et al., 2003). These spectromicroscopy methods can generate information on functional group distributions within samples with enough spatial and chemical resolution to minimize “signal averaging” problems, and with minor sample handling (e.g., no derivatization or extraction). Infrared microscopy is diffraction limited to spatial resolutions of roughly 3–10 μm, 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) spectroscopy to examine the potential for identifying particle compositions and mapping particle heterogeneity. This approach allows us to examine the composition of POM on a particle-by-particle basis. Although there are limitations in examining large particles (>200 μm diameter particles were not analyzed), and particles need to be dried and briefly heated to embed them in S° prior to sectioning, our approach examines all particles without extraction or derivatization and thus provides an overall view of particle compositions.
Section snippets
Background on X-ray microscopy
X-ray absorption edges arise when an incident X-ray photon exceeds the threshold energy needed to completely remove (ionize) an electron from an inner-shell orbital. The result is a step-like rise (the “edge” in 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
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 °C) a mixture of fine S° grains with 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
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 analyzed. Although material from four traps was analyzed, 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
Overall distributions
The roughly 60 particles from Arabian Sea sediment 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 compositions, 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
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 characteristic of carboxylic acids formed the highest
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, stimulating discussions and
References (59)
- et al.
Extracting information from sequences of spatially resolved EELS spectra using multivariate statistical analysis
Ultramicroscopy
(1999) - et al.
Calculation of the 13C NMR chemical shift of ether linkages in lignin derived geopolymers: constraints on the preservation of lignin primary structure with diagenesis
Geochimica et Cosmochimica Acta
(1999) - et al.
The application of soft X-ray microscopy to the in-situ analysis of sporinite in coal
International Journal of Coal Geology
(1996) Formation and clay mineral reactions of melanoidins
Geochimica et Cosmochimica Acta
(1978)- et al.
The molecularly-uncharacterized component of nonliving organic matter in natural environments
Organic Geochemistry
(2000) - et al.
Carbon K-shell excitation-spectra of linear and branched alkanes
Journal of Electron Spectroscopy and Related Phenomena
(1987) - et al.
Radiation damage of materials due to high-energy ion irradiation
Nuclear Instruments & Methods in Physics Research. Section B, Beam Interactions with Materials and Atoms
(2002) - et al.
Particulate organic carbon fluxes: compilation of results from the 1995 US JGOFS Arabian Sea Process Study
Deep-Sea Research. Part 2. Topical Studies in Oceanography
(1998) - et al.
Composition and flux of particulate amino acids and chloropigments in equatorial Pacific seawater and sediments
Deep-Sea Research. Part 1. Oceanographic Research Papers
(2000) - et al.
Cluster analysis of soft x-ray spectromicroscopy data
Ultramicroscopy
(2004)