Characterisation of soil organic phosphorus in NaOH-EDTA extracts: A comparison of 31P NMR spectroscopy and enzyme addition assays

Abstract

The identification and quantification of different soil organic phosphorus (P) compounds is crucial for a better understanding of soil P dynamics. The aim of this study was to compare two commonly used characterisation methods: 31P NMR spectroscopy and enzyme addition assays (EAAs). The same 0.25 M NaOH and 0.05 M ethylenediaminetetraacetic acid (EDTA) extracts of ten temperate and tropical topsoils under arable crops or permanent grassland were analysed by each method. Additionally, the substrate specificity of the used enzymes was verified through 31P NMR analysis of one enzyme-treated soil extract. Finally, the molecular weight distribution of organic P was characterised using gel filtration chromatography. NaOH-EDTA extractable organic P ranged from 7 to 1108 mg P kg−1 soil. Using 31P NMR spectroscopy, six organic P species in the mono- and diester region plus orthophosphate, pyrophosphate and phosphonates were detected. Deconvolution of 31P NMR spectra was not possible for two soils due to poor signal to noise ratio. Using EAAs, inositol phosphate-like P was identified as the largest enzyme-labile organic P class in most soils, followed by monoester-like P and DNA-like P. Corresponding classes of organic P determined by 31P NMR and EAAs were established and concentrations were found to agree well in general. However, repeatability was higher for 31P NMR spectroscopy than for EAAs. 31P NMR spectroscopy on an enzyme-treated extract showed that each enzyme acted on the anticipated organic P class, although treatment with phytase caused the appearance of a new and yet unidentified peak in the monoester region. Gel filtration chromatography of alkaline extracts revealed the presence of high-molecular weight organic P (>5 kDa) which had a 1:1 relationship with enzyme-stable P. For both methods, advantages and drawbacks with respect to required sample pre-treatment, analysis time and cost and the total number of identifiable compounds are discussed. While EAAs are suitable for a quick and coarse characterisation of larger sample numbers, 31P NMR is more robust and allows a more detailed quantification of P forms.