31P NMR Reveals Predominance of Small Molecules in Organic Phosphorus within NaOH-EDTA Soil Extracts

Organic phosphorus (P) plays a crucial role in maintaining the health and productivity of soils. Understanding the composition of organic phosphorus in soils is thus relevant to a range of disciplines, spanning from agricultural sciences to ecology. Over the past few decades, efforts have been directed towards characterizing and quantifying various soil organic P compounds and determining their turnover rates. Despite these efforts, the precise nature of soil organic P remains unclear, particularly that of orthophosphate monoesters, which dominate ³¹P NMR spectra of NaOH-EDTA extracts globally.

Typically, the monoester region of 1D ³¹P NMR spectra appears as a series of sharp signals "sitting" on a broad background where the broad background can account for a substantial part of the monoester region. This is prompting questions about how to integrate and identify these signals and to what extent this fraction may be ecologically important. To investigate this monoester background, we employed 1D ³¹P NMR and 2D ¹H-³¹P NMR¹, along with ³¹P transverse relaxation (T₂)² measurements to calculate intrinsic linewidths. We related this linewidth to molecular weight to unveil the nature of the observed background. Analysing seven soils from different ecosystems, we observed linewidths ranging from 0.5 to 3 Hz for both resolved monoester signals and the background. This suggests that the background comprises numerous, possibly exceeding 100, sharp signals associated with small (<1.5 kDa) organic P molecules.

Organic P in the form of nucleic acids, phospholipids, P-containing metabolites, and phosphorylated proteins dominate the P content of live leaves, leaf litter and microbial tissues. Furthermore, P-containing metabolites are exuded by roots and are present in a vast array of organisms. Evidence that the background potentially can contain a large number of small metabolites is thus not surprising and may account for an important part of the organic P pool given that the background accounts for about 55% of the monoester region. Our findings warrant further research specifically addressing to what extent this pool may play for plant and microbial P nutrition.

We provide recommendations for treating ³¹P NMR spectra to accurately quantify phosphomonoester species, representing a crucial step in linking observed P speciation to its bioavailability. Our findings align with previous ³¹P NMR studies detecting background signals in soil-free samples and new evidence suggesting that alkali-soluble soil organic matter consists of self-assemblies of small organic compounds mimicking large molecules.

¹Vestergren, J.; Vincent, A. G.; Jansson, M.; Persson, P.; Ilstedt, U.; Gröbner, G.; Giesler, R.; Schleucher, J. High-Resolution Characterization of Organic Phosphorus in Soil Extracts Using 2D ¹H–³¹P NMR Correlation Spectroscopy. Environmental Science & Technology 2012, 46 (7), 3950–3956. https://doi.org/10.1021/es204016h.

²Vincent, A. G.; Schleucher, J.; Gröbner, G.; Vestergren, J.; Persson, P.; Jansson, M.; Giesler, R. Changes in Organic Phosphorus Composition in Boreal Forest Humus Soils: The Role of Iron and Aluminium. Biogeochemistry 2012, 108 (1), 485–499. https://doi.org/10.1007/s10533-011-9612-0.