Barbara J. Cade-Menun

A comparison of soil extraction procedures for 31P NMR spectroscopy

The effect of extractants on phosphorus determination by 31 P NMR spectroscopy was examined using five forest floor samples. The extractants used were: 0.25 M NaOH, 1:6 soil to Chelex in water, 1:6 soil to Chelex in 0.25 M NaOH, and a 1:1 mix of 0.5 M NaOH and O.1 M EDTA. The broadest peaks were produced by the NaOH + EDTA extraction. However, NaOH + EDTA extracts contained the highest percentage of total phosphorus and the greatest diversity of P forms. These extracts were the only ones to show peaks for polyphosphates. Metals analysis indicated that NaOH + EDTA maintained Mn in solution, which seemed to be responsible for the line broadening. The sharpest peaks, with the best separation, were produced with Chelex + NaOH, and these were improved further by increasing the pH with NaOH prior to NMR analysis. Chelex + NaOH extracted 23 to 35% of the total soil P, Chelex in water extracted 10 to 13%, NaOH alone extracted 22 to 34%, and NaOH + EDTA extracted 71 to 90%. This work suggests that, because the extractant used will affect the P forms, care must be taken when interpreting studies of P cycling in soils using 31 P NMR spectroscopy and when comparing studies using different extractants.

Extraction of soil organic phosphorus.

Organic phosphorus is an important component of soil biogeochemical cycles, but must be extracted from soil prior to analysis. Here we critically review the extraction of soil organic phosphorus, including procedures for quantification, speciation, and assessment of biological availability. Quantitative extraction conventionally requires strong acids and bases, which inevitably alter chemical structure. However, a single-step procedure involving sodium hydroxide and EDTA (ethylenediaminetetraacetate) is suitable for most soils and facilitates subsequent speciation by nuclear magnetic resonance spectroscopy. Analysis of extracts by molybdate colorimetry is a potential source of error in all procedures, because organic phosphorus is overestimated in the presence of inorganic polyphosphates or complexes between inorganic phosphate and humic substances. Sequential extraction schemes fractionate organic phosphorus based on chemical solubility, but the link to potential bioavailability is misleading. Research should be directed urgently towards establishing extractable pools of soil organic phosphorus with ecological relevance.

Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy

Phosphorus nuclear magnetic resonance ((31)P-NMR) spectroscopy has advanced our knowledge of organic phosphorus (P) in soils and environmental samples more than any other technique. This paper reviews the use of (31)P-NMR spectroscopy for soil, water and other environmental samples. The requirements for a successful solid-state or solution (31)P-NMR experiment are described, including experimental set-up, sample preparation, extractants, experimental conditions, and post-experimental processing. Next, the literature on solid-state and solution (31)P-NMR spectroscopy in environmental samples is reviewed, including papers on: methods; P transformations; agricultural, forest and natural ecosystem soil studies; humic acid and particle size separations; manure, compost and sludge studies; and water research, including freshwater, estuary and marine studies. Future research needs are also discussed as well as suggestions to improve results, such as increased standardization among research groups.

Characterization of organic phosphorus in leachate from a grassland soil

The degree of eutrophication in fresh water ecosystems may be influenced by the forms of phosphorus (P) leached from agricultural systems. Physico-chemical fractionation of P in leachate from a grassland soil carried out over a two year period indicated that the majority of the P loss from the Lismore soil occurred in unreactive particulate (55‐ 76%) P forms. 31 P nuclear magnetic resonance analysis of a selected leachate sample indicated that unreactive P was mainly comprised of monoester and diester forms of organic P. The presence of phosphomonoesterase (20 ‐ 200 mg p nitrophenol l 21 h 21 ) and phosphodiesterase (68 mg bis-p nitrophenol l 21 h 21 ) activity in leachate resulted in hydrolysis of 10 ‐ 21% of total unreactive P (TUP), indicating that some of the monoesters and diesters can be eventually hydrolyzed into inorganic P forms during P transport. Enzyme hydrolysis showed that 23% of the TUP was present as labile monoester P (LMP), followed by 20% as inositol hexakisphosphate (IHP) and 14% as diesters (phospholipids and nucleic acids). The findings of this study suggest that LMP, IHP and diesters are an important component of organic P leaching from the grassland soil. q 2003 Elsevier Ltd. All rights reserved.

Selective phosphorus regeneration of sinking marine particles: evidence from 31P-NMR

Phosphorus (P) regeneration and transformation in the oceanic water column and in marine sediments depends on the chemical nature of the sinking particulate P pool. For the first time, we have characterized the molecular composition of this pool, in various oceanic settings and water depths, using 31 P nuclear magnetic resonance (NMR) spectroscopy. Both inorganic P (orthophosphate, pyrophosphate, and polyphosphate) and organic P compounds (orthophosphate monoesters, orthophosphate diesters, and phosphonates) were identified. The inorganic P is present predominantly as orthophosphate with small amounts (<10%) of pyro- and polyphosphates. These inorganic compounds may be at least partially of biological origin. The relatively high abundance of inorganic P suggests that considerable transformation from the organic to the inorganic pool occurs in the water column. Some of this inorganic P may be present in association with mineral phases (apatite, clays, and oxyhydroxides) and thus may not be bioavailable. The distribution of organic P compounds in the sinking particulate matter pool is generally similar in composition to phytoplankton and significantly different than in the dissolved organic matter (DOM) pool. Results indicate that in most oceanic regions the majority of P regeneration occurs at very shallow depths. However, in the Ross Sea, a significant fraction of organic P is exported to depth below the euphotic zone. Hydrolysis of P compounds continues throughout the water column as indicated by a decrease in total particulate P with depth and a relative decrease in the organic P fraction at some sites. Orthophosphate monoesters dominate the organic P pool at all locations, followed by orthophosphate diesters. Phosphonates are present in a few samples but never contribute more than 6% of total extractable P compared to 25% abundance in the dissolved organic P (DOP) pool. This work shows that considerable spatial and temporal variability in the molecular composition of sinking particulate P exists. A more systematic study is needed to assess the different environmental parameters that affect the composition of particulate P and result in this variability. D 2003 Elsevier Science B.V. All rights reserved.

Characterization of plant-derived water extractable organic matter by multiple spectroscopic techniques.

Water extractable organic matter (WEOM) derived from fresh- or early-stage decomposing soil amendment materials may play an important role in the process of organic matter accumulation. In this study, eight WEOM samples extracted with a 40:1 (v/w) water to sample ratio from alfalfa (Medicago sativa L.), corn (Zea mays L.), crimson clover (Trifolium incarnatum L.), hairy vetch (Vicia villosa L.), lupin (Lupinus albus L.), soybean (Glycine max L. Merr.), wheat (Triticum aestivum L.), and dairy manure were investigated using ultraviolet (UV)–visible, Fourier transform infrared (FT-IR), solution 31P nuclear magnetic resonance (NMR), and solid state 13C NMR spectroscopies. UV–visible and FT-IR spectra of the plant-derived WEOM samples were typical for natural organic matter, but possessed less humic-like characteristics than dairy manure-derived WEOM. Solution 31P NMR spectra indicated that WEOM samples extracted from alfalfa, corn, and soybean shoots contained both orthophosphate and monoester P. Of the monoester P in WEOM from soybean shoot, 70% was phytate P. WEOM from crimson clover, hairy vetch, lupin, and wheat shoots contained orthophosphate only. The solid-state 13C NMR spectra of the seven plant-derived WEOM samples indicated that they all were primarily composed of sugars, amino acids or peptides, and low molecular mass carboxylic acids. Carbohydrates were dominant components with very few aromatics present in these samples. In addition, WEOM from crimson clover and lupin, but not other three leguminous plant WEOM samples, contained significant asparagine. On the other hand, WEOM from corn and wheat contained less amino acids or peptides. The spectra of WEOM of dairy manure revealed the presence of significant amounts of nonprotonated carbons and lignin residues, suggesting humification of the manure-derived WEOM. Significant carbohydrates as well as aromatics were present in this WEOM. The P and C bonding information for these WEOM samples may be useful for understanding the effects of WEOM on soil nutrient availability to plants.

Investigation of soil legacy phosphorus transformation in long-term agricultural fields using sequential fractionation, P K-edge XANES and solution P NMR spectroscopy.

Understanding legacy phosphorus (P) build-up and draw-down from long-term fertilization is essential for effective P management. Using replicated plots from Saskatchewan, Canada, with P fertilization from 1967 to 1995 followed by either P fertilization or P cessation (1995-2010), soil P was characterized in surface and subsurface layers using sequential fractionation, P K-edge X-ray absorption near-edge structure (XANES) and solution (31)P nuclear magnetic resonance (P NMR) spectroscopy. Legacy P from a 28-year build-up was sufficient for 15 years of wheat cultivation, resulting in no significant differences in crop yield in 2010. In surface soils, soil test (Olsen) P decreased significantly in unfertilized plots compared with 1995, which was reflected in declining aluminum (hydr)oxide-associated inorganic P by fractionation and XANES. Furthermore, XANES analysis revealed a decrease of calcium-associated P in 2010-unfertilized soils at both depths and an increase of Fe (hydr)oxides-associated P in the 2010-fertilized and -unfertilized surface soils relative to the 1995 soils. Increased total organic P and orthophosphate diesters by P NMR and accumulated inositol hexaphosphate by XANES were observed in surface soils with P fertilization cessation. In subsurface soils, few legacy P transformations were detected. These results provide important information about legacy P to improve agricultural sustainability while mitigating water quality deterioration.

Phosphorus-31 nuclear magnetic resonance spectroscopy transect study of poultry operations on the Delmarva Peninsula.

Nonpoint source phosphorus (P) pollution into the Chesapeake Bay watershed from poultry operations contributes to the algal blooms, hypoxia, anoxia, and fish kill events that occur there most years. A major source of soluble, bioavailable P species is poultry litter, which is used as a crop fertilizer on fields adjacent to the tributaries of the Bay. A potentially significant source of orthophosphate in the litter is the heavily phosphorylated compound myo-inositol hexakisphosphate (phytate), which is indigestible by poultry and thus becomes a major component of their excreta. Phytate evaluation in environmental samples is expensive; hence, its impact is not captured in standard farmer-friendly eutrophication potential guides, like Delawares Phosphorus Site Index. In this transect study of two poultry operations on the Delmarva Peninsula, we measured the incidence of all P compounds using solution 31P nuclear magnetic resonance (NMR) spectroscopy and extracts, relating them to relevant geochemical properties. The contribution of phytate to the overall pool of P declined from around 50% in manures to between 2 and 13% in down-gradient soils and sediments, corresponding to a rise in the relative proportion of orthophosphate (increasing from 39% to 65-88%). The results show that the large pool of phytate P spread onto croplands during standard operating practice at poultry farms on the Delmarva Peninsula does not appear to accumulate; rather, phytate decreases in down-gradient locations, most likely due to transport off-site and/or through in situ biological activity.

Phosphorus forms in conventional and organic dairy manure identified by solution and solid state p-31 NMR spectroscopy.

Organic dairy production has increased rapidly in recent years. Organic dairy cows (Bos taurus) generally eat different diets than their conventional counterparts. Although these differences could impact availability, utilization, and cycling of manure nutrients, little such information is available to aid organic dairy farmers in making nutrient and manure management decisions. In this study, we comparatively characterized P in organic and conventional dairy manure using solution and solid state (31)P NMR spectroscopic techniques. Phosphorus in both types of dairy manure was extracted with water, Na acetate buffer (100 mmol L(-1), pH 5.0) plus 20 mg Na dithionite mL(-1), or 0.025 mol L(-1) NaOH with 50 mmolL(-1) EDTA. Solution NMR analysis revealed that organic dairy manure contained about 10% more inorganic phosphate than conventional dairy manure. Whereas organic dairy manure did contain slightly more phytate P, it contained 30 to 50% less monoester P than conventional dairy manure. Solid state NMR spectroscopy revealed that mono-, di-, and trivalent metal P species with different stabilities were present in the two dairy manures. Conventional dairy manure contained relatively higher contents of soluble inorganic P species and stable metal phytate species. In contrast, organic dairy manure contained more Ca and Mg species of P. These results indicate that P transformation rates and quantities should be expected to differ between organic and conventional dairy manures.

The inositol phosphates in soils and manures: Abundance, cycling, and measurement

Giles, C. D., Cade-Menun, B. J. and Hill, J. E. 2011. The inositol phosphates in soils and manures: Abundance, cycling, and measurement. Can. J. Soil Sci. 91: 397-416. This review focuses on recent advances in understanding the origins, abiotic and biotic cycling, and measurement of inositol phosphates (IPx) in manures and soils. With up to eight orthophosphates bound to inositol via ester linkages, this class of compounds has the potential to be unavailable to enzymatic hydrolysis when sorbed or in complex with soil metals, limiting the release of phosphorus (P) for uptake by plants. However, hydrolysis of IPx by microbial phytases in aquatic environments could result in a potent source of the eutrophication agent orthophosphate. This review discusses the forms and stereoisomers of IPx that have been identified in environmental samples. Next, it discusses the various techniques used to identify IPx, including extraction and concentration, separation techniques such as electrophoresis, spectroscopic methods such as phosphorus-31 nuclear magnetic resonance spectroscopy (31P-NMR), mass spectrometry and X-ray absorption near-edge structure (XANES), and enzymatic techniques, such as enzyme hydrolysis (EH). Recent advances in knowledge about abiotic and biotic factors controlling the cycling of IPx in soil, manure and water are summarised, including soil characteristics affecting IPx sorption, transportation processes, and the microbial production and degradation of IPx. Finally, areas for future research focus are discussed.