Properties of neutral phosphate buffer extractable organic matter in soils revealed using size exclusion chromatography and fractionation with polyvinylpyrrolidone

A neutral phosphate buffer (NPB) extraction method has been used to estimate the amount of available N in soil. However, the properties of soil NPB-extractable organic N have not been fully elucidated. The purpose of the present study was to characterize the properties of organic matter in the NPB extracts of soils. The NPB extracts were obtained from three soil samples, and the organic matter in the extracts was separated into three fractions according to its solubility in acid and adsorption onto polyvinylpyrrolidone (PVP). High-performance size exclusion chromatography (HPSEC) with ultraviolet (UV) and fluorescence detections was applied to the NPB extracts and their fractions. The HPSEC analysis of the NPB extract revealed the presence of a single broad peak, irrespective of the detection methods. The broad peak was identified as humic substances using the on-flow measurements of UV absorption spectra and fluorescence emission spectra. Among the fractions, the PVP-non-adsorbed fulvic acid (FA) fraction accounted for the largest proportion of organic C or N in the NPB extract, followed by the PVP-adsorbed FA and humic acid (HA) fractions. The peak of humic substances was observed for all fractions using HPSEC with the on-flow measurement of UV absorption and fluorescence emission spectra. The molecular weight of the humic substances varied with each fraction. When the Coomassie Blue-reactive substances (CBRS) were quantified using a Bradford protein assay, they were detected in the NPB extract and almost half were distributed in the PVP-non-adsorbed FA fraction. However, humic substances were considered to be the main constituents of CBRS in the soil NPB extract because of their reactivity with Coomassie Blue and the absence of proteinaceous materials. Furthermore, an incubation experiment revealed that the organic matter available to microorganisms was included in the HA and PVP-non-adsorbed FA fractions. Based on the HPSEC analysis of the NPB extracts and their fractions, it was observed that the humic substances in the NPB extract, particularly in the HA and PVP-non-adsorbed FA fractions, were available to microorganisms.