Chemical characterization of soil organic P using 31P nuclear magnetic resonance (NMR) spectroscopy relies on the correct assignment of resonances. We examined eight Australian soils and identified the main orthophosphate monoester peaks by spiking model organic P compounds (α‐ and β‐glycerophosphate, ethanolamine phosphate, phytate, scyllo‐inositol hexakisphosphate, and choline phosphate) into NaOH–ethylenediaminetetraacetic acid (EDTA) soil extracts. For five of the soils, the strongest monoester resonances were identified as being due to phytate, while for the other three soils, the strongest resonances were identified as being due to α‐ and β‐glycerophosphate. Importantly, the appearance of spectra dominated by phytate and those dominated by glycerophosphate were deceptively similar because the separation between the two strongest phytate resonances was very similar to the separation between the α‐ and β‐glycerophosphate resonances. We believe this may have resulted in the misidentification of these species in some previous studies. Identification of these species is hindered by the sensitivity of their chemical shifts to pH and electrolyte concentration. Our spiking methodology, in which we add only enough of the compound to approximately double the native concentration, overcomes this problem. We also investigated the alkaline hydrolysis of phospholipids as a likely source of α‐ and β‐glycerophosphate. We found that the rate of phospholipid hydrolysis was dependant on NaOH concentration and that the presence of glycerophosphate resonances in soil extracts probably results from the extraction and redissolution procedures rather than the presence of native glycerophosphate in the soils. We also identified an intermediate diester product that may have previously been misidentified as the phospholipid itself.

Phosphorus (P) is an essential element for life, an innate constituent of soil organic matter, and a major anthropogenic input to terrestrial ecosystems. The supply of P to living organisms is strongly dependent on the dynamics of soil organic P. However, fluxes of P through soil organic matter remain unclear because only a minority (typically <30%) of soil organic P has been identified as recognizable biomolecules of low molecular weight (e.g., inositol hexakisphosphates). Here, we use (31)P nuclear magnetic resonance spectroscopy to determine the speciation of organic P in soil extracts fractionated into two molecular weight ranges. Speciation of organic P in the high molecular weight fraction (>10 kDa) was markedly different to that of the low molecular weight fraction (<10 kDa). The former was dominated by a broad peak, which is consistent with P bound by phosphomonoester linkages of supra-/macro-molecular structures, whereas the latter contained all of the sharp peaks that were present in unfractionated extracts, along with some broad signal. Overall, phosphomonoesters in supra-/macro-molecular structures were found to account for the majority (61% to 73%) of soil organic P across the five diverse soils. These soil phosphomonoesters will need to be integrated within current models of the inorganic-organic P cycle of soil-plant terrestrial ecosystems.

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myo-Inositol hexakisphosphate (phytate) is widely regarded as an abundant form of soil organic phosphorus (P) in many soils. Its abundance is believed to be because of its resistance to microbial degradation. We examined the fate of phytate added to a calcareous soil as a solution at a concentration of 58 mg P kg −1 , with and without the addition of wheat straw. The soil was incubated for 13 weeks, with phytate concentrations determined at 0, 1, 4, 7 and 13 weeks using NaOH-EDTA soil extraction followed by 31 P nuclear magnetic resonance (NMR) spectroscopy. The phytate concentration declined rapidly, with 18% (phytate + wheat straw) and 12% (phytate) of the initial phytate remaining after 13 weeks. This coincided with an increase in the proportion of orthophosphate relative to total NaOH-EDTA extractable P (from 65 to 81%) and a small increase in α-and β-glycerophosphate concentration, providing evidence for the microbial degradation of phytate. The decrease in phytate concentration was consistent with a first-order decay with a half-life for phytate of 4-5 weeks. This study demonstrates that in the calcareous soil examined, phytate was not highly stable, but a potentially biologically available form of P. In order to quantify the concentration of P species, we developed an improved method of spectral deconvolution. This method accounted for a broad signal (3.5-6.5 ppm) in the monoester region of the spectra that represented up to 23% of the total extractable P. We found that when this broad signal was not included, phytate concentrations were over-estimated by up to 54%.