In the ALS x-ray microscope, image contrast stems from the photoelectric absorption of x rays, and hence, element- and functional-group-specific images can be obtained at high spectral resolution for organic molecules. Although significant information on the structure of HSs has been obtained previously by using electron microscopy, nuclear magnetic resonance, and infrared spectroscopy, until now, direct evidence for how structural transformations depend on the sample's origin (soil vs. water), solution chemistry, and substrate mineralogy has not been documented. Furthermore, because of the structural changes associated with sample drying, the structures obtained by using techniques that require dried samples cannot be correlated with solution chemistry.

To test the influence of these parameters, the researchers conducted x-ray microscopy experiments on HSs isolated from water and soil samples. The observations were made on samples in solution under varying chemical conditions, including pH (2-12), ionic strength (0.01-2 M NaCl or CaCl₂), organic molecule concentration (0.03-10 g/L carbon), and complexing cations (1 mM Ca⁺² or Fe⁺³). Studies were also conducted in the presence of common soil minerals such as goethite, calcite, and clays (kaolinite, montmorillonite). To correlate the structures of the isolated HSs with those of undisturbed natural samples, the researchers also examined "pristine" soil organic molecules that were not extracted from the surrounding soil matrix.

The researchers found a great deal of structural variety, including sheets and globular configurations, thread- and net-like shapes, and small, uniform aggregates. The results indicate that the microstructures of humic substances are different under different solution chemical conditions, in contrast to the previously held belief that the molecules simply form rings in acidic or strong electrolyte solutions and elongate in dilute alkaline solutions. Also, HSs from soil and water require different chemical conditions to assume a particular configuration, reflecting the comparatively lower solubility, higher aromatic carbon content, and low carboxyl content of soil materials. The presence of minerals added further complexity: the composition and thickness of HS coatings on the mineral surfaces were found to depend on pH and HS concentration and origin (soil or water). The latter observations were made possible by surface-sensitive photoemission spectroscopy methods available at the ALS. The structures observed in pristine soil samples were similar to the organomineral structures seen in the isolated soil samples, suggesting that the results obtained from the latter can be applied to HSs in soils and sediments, but not necessarily to those in solution.

Does Organic Matter Really Matter?

The study of humic substances--organic matter left over from plant and animal decomposition--doesn't sound very glamorous. Nevertheless, scientists from a long list of diverse fields are finding that detailed knowledge of humic substances and their structures is the key to understanding a wide variety of significant environmental phenomena.

Biologists, chemists, geologists, soil scientists, and a host of other multidisciplinary specialists are interested in humic substances because this organic matter can affect soil fertility, mineral weathering, and water acidity; are involved in the transport, sequestration, and mitigation of contaminants; and may even have an impact on atmospheric chemistry through the carbon cycle, in which carbon is constantly recycled between plants, animals, soil, air, and water. These are just a few examples of humic substances at work in the environment.

Despite such diverse and far-reaching effects, humic substances are still very poorly understood. Systematic studies of the structures of these substances are the first step toward understanding how they interact with other elements and compounds. Such knowledge will be necessary before we can hope to predict and possibly control the impact of chemical and biological changes in the environment.

Effect of solution pH. A) Globular and ring-like structures in acidic solution. B) Uniform, small aggregates in alkaline solution.

The observed changes in microstructure can modify the exposed surface area and alter the functional group chemistry of the HSs, affecting, for example, protonation and cation complexation. Systematic structural studies with high-resolution in-situ x-ray microscopy is the first step toward understanding, predicting, and possibly controlling the chemical interactions of HSs in nature.

Effect of cation presence. C) Thread-like structures with divalent cations. D) Globular and thread-like structures with trivalent cations. (Scale bar = 500 nm.)

Research conducted by S.C.B. Myneni (Princeton University, Berkeley Lab), J.T. Brown, W. Meyer-Ilse (Berkeley Lab), and G.A. Martinez (University of Puerto Rico).

Research Funding: Laboratory Directed Research and Development program, Berkeley Lab; Office of Basic Energy Sciences (BES), U.S. Department of Energy. Operation of the ALS is supported by BES.

Publication about this research: S.C.B. Myneni, J.T. Brown, G.A. Martinez, and W. Meyer-Ilse, "Imaging of Humic Substance Macromolecular Structures in Water and Soils," Science 286, 1335-1337 (1999).