Separation of acid-soluble constituents of soil humic acids by dissolution in alkaline urea solution and precipitation with acid

Abstract

Background: Humic substances are considered to be composed of relatively small, heterogeneous molecules bound by weak linkages. The dissociation of acid-soluble constituents from soil humic acids (HAs) during the preparative polyacrylamide gel electrophoresis in the presence of concentrated urea has been previously demonstrated. Moreover, the dissociation of acid-soluble constituents has been attributed to the action of concentrated urea. The aim of this study was to investigate the effects of concentrated urea on the dissociation of acid-soluble constituents of soil HAs. Results: Three types of soil HAs were solubilized in 0.1 M NaOH containing 7 M urea and precipitated after 16 h by acidifying the samples to pH 1.0. The acid-soluble constituents were separated from the dark-colored precipitates by concentrated urea treatment and accounted for 16–45 % of the total organic carbon in HAs. Approximately half of the acid-soluble constituents was recovered in the DAX-8-adsorbed fraction. The humification degree of the DAX-8-adsorbed fraction was considerably lower than that of the corresponding unfractionated HA. In contrast, the humification degree of the precipitated fraction increased due to the separation of acid-soluble constituents. The molecular sizes of the DAX-8-adsorbed and DAX-8-non-adsorbed fractions, estimated by high-performance size exclusion chromatography, were similar and smaller than the precipitated fraction. Three-dimensional excitation-emission matrix fluorescence spectroscopy revealed that the acid-soluble constituents exhibited fluorescence similar to that of fulvic acid (FA), added to which the DAX-8-non-adsorbed fraction exhibited protein-like fluorescence. Diffuse reflectance infrared Fourier transform spectroscopy showed that the DAX-8-adsorbed fraction contained proteinous moieties and the DAX-8-non-adsorbed fraction was rich in proteinous and polysaccharide moieties. Conclusions: The present findings suggest that soil HAs are formed by the molecular associations between dark-colored acid-insoluble constituents, FA-like acid-soluble constituents, protein-like constituents, and polysaccharides bound by weak linkages.