Ashlea L. Doolette

A quantitative assessment of phosphorus forms in some Australian soils

Solution 31P nuclear magnetic resonance (NMR) spectroscopy is the most common technique for the detailed characterisation of soil organic P, but is yet to be applied widely to Australian soils. We investigated the composition of soil P in 18 diverse Australian soils using this technique. Soils were treated with a mixture of sodium hydroxide–ethylenediaminetetra-acetic acid (NaOH-EDTA), which resulted in the extraction of up to 89% of total soil P. It was possible to identify up to 15 well-resolved resonances and one broad signal in each 31P NMR spectrum. The well-resolved resonances included those of orthophosphate, α- and β-glycerophosphate, phytate, adenosine-5′-monosphosphate, and scyllo-inositol phosphate, as well as five unassigned resonances in the monoester region and two unassigned resonances downfield (higher ppm values) of orthophosphate. The majority of 31P NMR signal in the NaOH-EDTA extracts was assigned to orthophosphate, representing 37–90% of extractable P. Orthophosphate monoesters comprised the next largest pool of extractable P (7–55%). The most prominent resonances were due to phytate, which comprised up to 9% of total NaOH-EDTA extractable P, and α- and β-glycerophosphate, which comprised 1–5% of total NaOH-EDTA extractable P. A substantially greater portion of organic P (2–39% of total NaOH-EDTA extractable P) appeared as a broad peak in the monoester P region; we propose that this is due to P found in large, ‘humic’ molecules. Orthophosphate diesters (1–5% of total NaOH-EDTA extractable P) and pyrophosphate (1–5% of total NaOH-EDTA extractable P) were minor components of P in all soil extracts. These results suggest that organic P in large humic molecules represents the second most abundant form of NaOH-EDTA extractable soil P (behind orthophosphate). Furthermore, small P-containing compounds, such as phytate, represent a much smaller proportion of soil P than is commonly assumed.

Soil Organic Phosphorus Speciation Using Spectroscopic Techniques

The most commonly used differentiation of soil phosphorus (P) is between inorganic and organic forms, despite the fact that this is only the beginning of soil P speciation. Forms of inorganic and organic soil P include a large range of specific P compounds, and spectroscopic techniques can offer the best potential for determining the speciation of soil organic P. The focus of this chapter is to summarise the relative merits of three spectroscopic techniques: solution and solid state 31P nuclear magnetic resonance (NMR), and X-ray absorption near-edge structure (XANES). We aim to provide current and potential end-users of these techniques the ability to compare these methods on the basis of four criteria: sample preparation, sensitivity, resolution and quantitation.

Complex Forms of Soil Organic Phosphorus–A Major Component of Soil Phosphorus

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.

Identification of RNA Hydrolysis Products in NaOH-EDTA Extracts using 31P NMR Spectroscopy

Ribonucleic acid (RNA) is the most abundant form of organic phosphorus (P) in plant and microbial biomass and is therefore expected to be present in materials such as soils, sediments, composts, and manures. Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy is increasingly used to characterize organic P in these materials, usually following extraction into a mixture of sodium hydroxide and ethylenediaminetetraacetic acid (NaOH-EDTA). Under these alkaline conditions, RNA is hydrolysed, providing a distinctive pattern or “fingerprint” in the 31P NMR spectrum. Complete assignment of the eight ribonucleotides produced was achieved using a spiking approach. The near coincidence in chemical shift of β-glycerophosphate and two of the ribonucleotide peaks complicates quantification of RNA concentrations when phospholipids are also present, but an approach based on quantifying signal in the most well-separated ribonucleotide peaks is suggested.

Quantitative analysis of 31P NMR spectra of soil extracts – dealing with overlap of broad and sharp signals

Solution 31P NMR analysis following extraction with a mixture of sodium hydroxide and ethylenediaminetetraacetic acid is the most widely used method for detailed characterization of soil organic P. However, quantitative analysis of the 31P NMR spectra is complicated by severe spectral overlap in the monoester region. Various deconvolution procedures have been developed for the task, yet none of these are widely accepted or implemented. In this mini‐review, we first describe and compare these varying approaches. We then review approaches to similar issues of spectral overlap in biomedical science applications including NMR‐based metabolic profiling and analyzing 31P magnetic resonance spectra of ex vivo and in vivo intact tissues. The greater maturity and resourcing of this biomedical research means that a wider variety of approaches has been developed. Of particular relevance are approaches to dealing with overlap of broad and sharp signals. Although the existence of this problem is still debated in the context of soil analyses, not only is it well‐recognized in biomedical applications, but multiple approaches have been developed to deal with it, including T2 editing and time‐domain fitting. Perhaps the most transferable concept is the incorporation of ‘prior knowledge’ in the fitting of spectra. This is well established in biomedical applications but barely touched in soil analyses. We argue that shortcuts to dealing with overlap in the monoester region 31P NMR soil spectra are likely to be found in the biomedical literature, although some degree of adaptation will be necessary. Copyright

The composition of organic phosphorus in soils of the Snowy Mountains region of south-eastern Australia

Few studies have considered the influence of climate on organic phosphorus (P) speciation in soils. We used sodium hydroxide–ethylenediaminetetra-acetic acid (NaOH–EDTA) soil extractions and solution 31P nuclear magnetic resonance spectroscopy to investigate the soil P composition of five alpine and sub-alpine soils. The aim was to compare the P speciation of this set of soils with those of soils typically reported in the literature from other cold and wet locations, as well as those of other Australian soils from warmer and drier environments. For all alpine and sub-alpine soils, the majority of P detected was in an organic form (54–66% of total NaOH–EDTA extractable P). Phosphomonoesters comprised the largest pool of extractable organic P (83–100%) with prominent peaks assigned to myo- and scyllo-inositol hexakisphosphate (IP6), although trace amounts of the neo- and d-chiro-IP6 stereoisomers were also present. Phosphonates were identified in the soils from the coldest and wettest locations; α- and β-glycerophosphate and mononucleotides were minor components of organic P in all soils. The composition of organic P in these soils contrasts with that reported previously for Australian soils from warm, dry environments where inositol phosphate (IP6) peaks were less dominant or absent and humic-P and α- and β-glycerophosphate were proportionally larger components of organic P. Instead, the soil organic P composition exhibited similarities to soils from other cold, wet environments. This provides preliminary evidence that climate is a key driver in the variation of organic P speciation in soils.

Organic phosphorus speciation in Australian Red Chromosols: stoichiometric control

Organic phosphorus (P) plays an important role in the soil P cycle. It is present in various chemical forms, the relative amounts of which vary among soils, due to factors including climate, land use, and soil type. Few studies have investigated co-variation between P types or stoichiometric correlation with the key elemental components of organic matter– carbon (C) and nitrogen (N), both of which may influence P pool structure and dynamics in agricultural soils. In this study we determined the organic P speciation of twenty Australian Red Chromosols soils, a soil type widely used for cropping in Australia. Eight different chemical forms of P were quantified by 31P NMR spectroscopy, with a large majority (>90%) in all soils identified as orthophosphate and humic P. The strongest correlations (r2 = 0.77–0.85, P < 0.001) between P types were found among minor components: (i) between two inositol hexakisphosphate isomers (myo and scyllo) and (ii) between phospholipids and RNA (both detected as their alkaline hydrolysis products). Total soil C and N were correlated with phospholipid and RNA P, but not the most abundant P forms of orthophosphate and humic P. This suggests an influence of organic matter content on the organic P pool consisting of phospholipid and RNA, but not on inositol P or the largest organic P pool in these soils – humic P.

Use of 33P to trace in situ the fate of canola below-ground phosphorus, including wheat uptake in two contrasting soils

Our understanding of the contribution of crop root residues to phosphorus (P) cycling is mainly derived from studies using excavated roots re-introduced to soil. This study aims to quantify total below-ground P (BGP) of mature canola in situ and to estimate directly the proportion accessed by subsequent wheat. 33P-Labelled phosphoric acid was fed by stem wick to canola (Brassica napus) grown in sand or loam in pots. Shoots were removed from all plants at maturity. Half of the pots were destructively sampled. After a 3-week fallow, wheat was grown for 5 weeks in the remaining undisturbed pots. At canola maturity, 23–36% of the 33P was partitioned in recovered roots and 34–40% in the soil. More 33P was recovered in the loam than the sand. Within the soil, 6–10% of the fed 33P was present in resin P and 3–5% was in hexanol-released P pools. Ratios of shoot P : BGP (8 : 1 in sand and 15 : 1 in loam) were much narrower than those of shoot P : recovered root P (17 : 1 in sand and 39 : 1 in loam). A greater proportion and amount of the mature canola BG33P was recovered by wheat grown in the loam (26%, 2.6 mg/plant) than in the sand (21%, 1.5 mg/plant). The majority of canola BG33P remained in the bulk soil. Input of P below-ground by mature canola and subsequent P benefit to wheat was greater in loam than sand. The P from canola below-ground residues contributed up to 20% of P uptake in wheat during the first 5 weeks of growth. Longer term benefits of P from below-ground residues require investigation.

Improving Sensitivity of Solution 31P NMR Analysis in Australian Xeralfs

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy is widely used to identify and quantify phosphorus (P) forms in soil. This study aimed to determine whether narrowing the soil to extractant sodium hydroxide–ethylenediaminetetraacetic acid (NaOH-EDTA) ratio from 1:20 to values as low as 1:4 would improve sensitivity of solution 31P NMR spectroscopy without degrading resolution or quantitation. Four Australian soils were tested using four ratios. The narrowest ratio of 1:4 gave the best quality NMR spectra in terms of signal-to-noise ratio. Peak resolution was not degraded on narrowing the ratio. There was no clear effect of narrowing the extraction ratio on extraction efficiency or the distribution of signal among chemical shift regions (orthophosphate, monoester P, diester P, and pyrophosphate). We conclude that a ratio of 1:4 improved NMR analysis for these particular soils and should be considered for other soils, particularly low-P soils, where NMR sensitivity is limiting.

Facile decomposition of phytate in the solid-state: Kinetics and decomposition pathways

GRAPHICAL ABSTRACT ABSTRACT The thermal decomposition of myo-inositol hexakisphosphate (phytate) in the solid state and in aqueous solution was investigated using 31P NMR spectroscopy. The progression of phytate decomposition was consistent with facile thermal dephosphorylation resulting in a cascading mixture of lower order inositol phosphates with similar, but not identical, trajectories in the solid state and in aqueous solution. In both states, phytate decomposition was well described by a simple exponential decay function, consistent with first order kinetics. At 60°C the reaction proceeded more rapidly in the solid state (k = 2.03 × 10−4 min−1; t1/2 = 2.4 days) than in aqueous solution (pH 3.84; k = 0.387 × 10−4 min−1; t1/2 = 12.4 days). In the solid state at 80°C (k = 19.6 × 10−4 min−1; t1/2 = 5.9 h) and 95°C (k = 106 × 10−4 min−1; t1/2 = 65.4 min) phytate loss also closely followed an exponential decay function. We hypothesise the rapid thermal decomposition is due to an impurity present in the phytate source that acts as a catalyst for facile transfer of the phospho (H2PO3 / HPO3- / PO32−) group from phosphate esters to a range of oxygen functionalities present (water, alcohol groups, phosphate group). These reactions have important implications for understanding the stability of phytate under environmental conditions.