The molecular size continuum of soil organic phosphorus and its chemical associations

The chemical nature of most organic P (Porg) in soil remains ‘unresolved’ but is accounted for by a broad signal in the phosphomonoester region of solution 31P nuclear magnetic resonance (NMR) spectra. The molecular size range of this broad NMR signal and its molecular structure remain unclear. The aim of this study was to elucidate the chemical nature of Porg with increasing molecular size in soil extracts combining size exclusion chromatography (SEC) with solution 31P NMR spectroscopy. Gel-filtration SEC was carried out on NaOH-EDTA extracts of four soils (range 238-1135 mg Porg/kgsoil) to collect fractions with molecular sizes of <5, 5-10, 10-20, 20-50, 50-70, and >70 kDa. These were then analysed by NMR spectroscopy.

Organic P was detected across the entire molecular size continuum from <5 to >70 kDa. Concentrations of Porg in the >10kDa fraction ranged from 107 to 427 mg P/kgsoil and exhibited on average three to four broad signals in the phosphomonoester region of NMR spectra. These broad signals were most prominent in the 10-20 and 20-50 kDa fractions, accounting for on average 77 % and 74 % of total phosphomonoesters, respectively. Our study demonstrates that the broad signal is present in all investigated molecular size fractions and comprises on average three to four components of varying NMR peak line width (20 to 250 Hz). The stereoisomers myo- and scyllo-inositol hexakisphosphates (IP6) were also present across multiple molecular size ranges but were predominant in the 5-10 kDa fraction. The proportion of IP associated with large molecular size fractions >10 kDa was on average 23 % (SD=39 %) of total IP across all soils. These findings suggest that stabilisation of IP in soil includes processes associated with the organic phase.