Characterizing soil organic nitrogen using advanced molecular analytical techniques
Soil organic N (SON) comprises 90% of all N in surface soils, yet as much as half remains in forms which are chemically unknown or, at best, poorly understood. Analytical methods such has pyrolysis field-ionization mass spectrometry (Py-FIMS) and 15N cross polarization magic-angle spinning nuclear magnetic resonance (CPMAS-NMR) spectroscopy are widely used for the characterization of SON; however, these methods have limitations which contribute to the gaps in our understanding of SON chemistry. For example, Py-FIMS may produce heat-induced secondary compounds, and 15N-NMR may lack sensitivity and resolution for experiments at natural 15N abundance. X-ray absorption near edge structure (XANES) spectroscopy probes the bonding environment of individual elements. The application of this technique to complex environmental samples such as soil is still in its infancy, but early studies suggest that this technique may help resolve SON molecular structure. This dissertation sought to develop and apply synchrotron-based N and C K-edge XANES spectroscopy to the study of soil and soil extracts to determine the structures in which SON is bound. In these studies, Py-FIMS was coupled with XANES as a corroboratory technique. Initial methodological development resulted in a calibration method whereby N2 gas generated in ammonium-containing salts was used to calibrate a soft X-ray beamline at the N K-edge. Although XANES can produce secondary compound artifacts, contrary to early assertions that it is a non-destructive technique, it was shown in a second study that beam-induced decomposition can be minimized by moving the beam to a fresh spot between scans. Three applied studies exploring SON composition were conducted. These studies followed a spatial gradient ranging from the landscape scale, through a rhizosphere study, and ended with a study of glomalin-related soil protein (GRSP). Glomalin-related soil protein is a persistent soil glycoprotein of arbuscular mycorrhizal origin (AMF) implicated in aggregation and long-term C and N storage. Nitrogen and C K-edge XANES and Py-FIMS were used in all studies, and GRSP was further characterized using proteomics techniques. Soil organic N composition was largely controlled by topographic position, and to a lesser degree, by cultivation. Divergent (i.e., water shedding) positions were enriched in carbohydrates and low molecular weight lignins, whereas convergent, depressional and level positions showed enrichment in lipid-type compounds. These differences were attributed to tillage-induced redistribution of soil, and water movement from upper to lower slope positions. Nitrogen XANES revealed a unique form of organic N, identified as N-bonded aromatics, particularly in the divergent positions. Rhizosphere soil was enriched in higher molecular weight lipid-type materials and depleted in low molecular weight polar compounds. This was attributed to increased input of fresh plant material and higher microbial turnover in the rhizosphere. Nitrogen-bonded aromatics also were detected in the rhizosphere. The GRSP extracts were characterized as mostly proteinaceous, but also contained many co-extracted, non-protein compounds. Despite being previously described as a glycoprotein, only weak carbohydrate signals were observed. Proteomics-based assessment of GRSP showed no homology to any proteins of AMF origin, instead showing homology with thioredoxin and with heat-stable soil proteins. This may be because protein databases do not yet contain glomalin-related sequences, or that glomalin is homologous to non-AMF soil proteins. This dissertation demonstrated that N XANES is a sensitive and novel method for characterizing SON, and can be used complementarily with other analytical techniques such as Py-FIMS and proteomics. The continued development of XANES will provide a useful tool for SOM research into the future.
synchrotron, analytical chemistry, organic carbon, organic nitrogen, soil, pyrolysis, proteomics, glomalin
Doctor of Philosophy (Ph.D.)