Soil and litter phosphorus-31 nuclear magnetic resonance spectroscopy: extractants, metals, and phosphorus relaxation times

Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy is an excellent tool with which to study soil organic P, allowing quantitative, comparative analysis of P forms. However, for 31P NMR to be tative, all peaks must be completely visible, and in their correct relative proportions. There must be no line broadening, and adequate delay times must be used to avoid saturation of peaks. The objective of this study was to examine the effects of extractants on delay times and peak saturation. Two samples (a forest litter and a mineral soil sample) and three extractants (0.25 M NaOH, NaOH plus Chelex (Bio-Rad Laboratories, Hercules, CA), and NaOH plus EDTA) were used to determine the differences in the concentration of P and cations solubilized by each extractant, and to measure spin-lattice (T1) relaxation times of P peaks in each extract. For both soil and litter, NaOH-Chelex extracted the lowest concentrations of P. For the litter sample, T1 values were short for all extractants due to the high Fe concentration remaining after extraction. For the soil sample, there were noticeable differences among the extractants. The NaOH-Chelex sample had less Fe and Mn remaining in solution after extraction than the other extractants, and the longest delay times used in the study, 6.4 s, were not long enough for quantitative analysis. Delay times of 1.5 to 2 s for the NaOH and NaOH-EDTA were adequate. Line broadening was highest in the NaOH extracts, which had the highest concentration of Fe. On the basis of these results, recommendations for future analyses of soil and litter samples by solution 31P NMR spectroscopy include: careful selection of an extractant; measurement of paramagnetic ions extracted with P; use of appropriate delay times and the minimum number of scans; and measurement of T1 values whenever possible.     

Comparison of phosphorus forms in wet and dried animal manures by solution phosphorus-31 nuclear magnetic resonance spectroscopy and enzymatic hydrolysis

Both enzymatic hydrolysis and solution (31)P nuclear magnetic resonance (NMR) spectroscopy have been used to characterize P compounds in animal manures. In this study, we comparatively investigated P forms in 0.25 M NaOH/0.05 M EDTA extracts of dairy and poultry manures by the two methods. For the dairy manure, enzymatic hydrolysis revealed that the majority of extracted P was inorganic P (56%), with 10% phytate-like P, 9% simple monoester P, 6% polynucleotide-like P, and 18% non-hydrolyzable P. Similar results were obtained by NMR spectroscopy, which showed that inorganic P was the major P fraction (64-73%), followed by 6% phytic acid, 14 to 22% other monoesters, and 7% phosphodiesters. In the poultry manure, enzymatic hydrolysis showed that inorganic P was the largest fraction (71%), followed by 15% phytate-like P and 1% other monoesters, and 3% polynucleotide-like P. NMR spectroscopy revealed that orthophosphate was 51 to 63% of extracted P, phytic acid 24 to 33%, other phosphomonoesters 6 to 12%, and phospholipids and DNA 2% each. Drying process increased orthophosphate (8.4% of total P) in dairy manure, but decreased orthophosphate (13.3% of total P) in poultry manure, suggesting that drying treatment caused the hydrolysis of some organic P to orthophosphate in dairy manure, but less recovery of orthophosphate in poultry manure. Comparison of these data indicates that the distribution patterns of major P forms in animal manure determined by the two methods were similar. Researchers can utilize the method that best fits their specific research goals or use both methods to obtain a full spectrum of manure P characterization.     

Phosphorus speciation in manure-amended alkaline soils

Two common manure storage practices are stockpiles and lagoons. The manure from stockpiles is applied to soils in solid form, while lagoon manure is applied as a liquid. Soil amendment with manure in any form introduces a significant amount of phosphorus (P) that exists in both organic and inorganic forms. However, little is known about P speciation in manure stored under different conditions, or the subsequent forms when applied to soils. We used solution (31)P nuclear magnetic resonance (NMR) spectroscopy and conventional P fractionation and speciation methods to investigate P forms in dairy manure and liquid lagoon manure, and to study how long-term amendment with these manures influenced surface and subsurface soil P speciation. Our results show that the P forms in solid and lagoon manure are similar. About 30% of the total P was organic, mostly as orthophosphate monoesters. On a dry weight basis, total P was much higher in the solid manure. In the manure-amended soils the total P concentrations of the surface soils were similar, regardless of manure type. Total P in the subsurface soil was greater in the lagoon-manure-amended soil than the solid-manure-amended subsurface soil. However, the fraction of organic P was greater in the subsurface of the solid-manure-amended soil. The NMR results indicate that the majority of organic P in the soils is phytic acid, which is enriched in the surface soils compared with the subsurface soils. These results provide insight into P speciation and dynamics in manure-amended soils that will further increase our understanding on how best to manage manure disposal on soils.     

Phosphorus forms and chemistry in the soil profile under long-term conservation tillage: a phosphorus-31 nuclear magnetic resonance study

In many regions, conservation tillage has replaced conventional tilling practices to reduce soil erosion, improve water conservation, and increase soil organic matter. However, tillage can have marked effects on soil properties, specifically nutrient redistribution or stratification in the soil profile. The objective of this research was to examine soil phosphorus (P) forms and concentrations in a long-term study comparing conservation tillage (direct drilling, "No Till") and conventional tillage (moldboard plowing to 20 cm depth, "Till") established on a fine sandy loam (Orthic Humo-Ferric Podzol) in Prince Edward Island, Canada. No significant differences in total carbon (C), total nitrogen (N), total P, or total organic P concentrations were detected between the tillage systems at any depth in the 0- to 60-cm depth range analyzed. However, analysis with phosphorus-31 nuclear magnetic resonance spectroscopy showed differences in P forms in the plow layer. In particular, the concentration of orthophosphate was significantly higher under No Till than Till at 5 to 10 cm, but the reverse was true at 10 to 20 cm. Mehlich 3-extractable P was also significantly higher in No Till at 5 to 10 cm and significantly higher in Till at 20 to 30 cm. This P stratification appears to be caused by a lack of mixing of applied fertilizer in No Till because the same trends were observed for pH and Mehlich 3-extractable Ca (significantly higher in the Till treatment at 20 to 30 cm), reflecting mixing of applied lime. The P saturation ratio was significantly higher under No Till at 0 to 5 cm and exceeded the recommended limits, suggesting that P stratification under No Till had increased the potential for P loss in runoff from these sites.     

Multispecies and Multiscale Conservation Planning: Setting Quantitative Targets for Red‐Listed Lichens on Ancient Oaks

Abstract: Species occurrence in a habitat patch depends on local habitat and the amount of that habitat in the wider landscape. We used predictions from empirical landscape studies to set quantitative conservation criteria and targets in a multispecies and multiscale conservation planning effort. We used regression analyses to compare species richness and occurrence of five red‐listed lichens on 50 ancient oaks (Quercus robur; 120–140 cm in diameter) with the density of ancient oaks in circles of varying radius from each individual oak. Species richness and the occurrence of three of the five species were best explained by increasing density of oaks within 0.5 km; one species was best explained by the density of oaks within 2 km, and another was best predicted by the density of oaks within 5 km. The minimum numbers of ancient oaks required for “successful conservation” was defined as the number of oaks required to obtain a predicted local occurrence of 50% for all species included or a predicted local occurrence of 80% for all species included. These numbers of oaks were calculated for two relevant landscape scales (1 km² and 13 km²) that corresponded to various species responses, in such a way that calculations also accounted for local number of oaks. Ten and seven of the 50 ancient oaks surveyed were situated in landscapes that already fulfilled criteria for successful conservation when the 50% and 80% criteria, respectively, were used to define the level of successful conservation. For cost‐efficient conservation, oak stands in the landscapes most suitable for successful conservation should be prioritized for conservation and management (e.g., grazing and planting of new oaks) at the expense of oak stands situated elsewhere.     

An examination of spin-lattice relaxation times for analysis of soil and manure extracts by liquid state phosphorus-31 nuclear magnetic resonance spectroscopy

Phosphorous (P)-31 nuclear magnetic resonance (NMR) spectroscopy is used in the analysis of P forms in extracts of soils and manures for environmental and agronomic purposes. Quantitative spectra require knowledge about spin-lattice relaxation times (T1) to ensure adequate delays between pulses. This paper determined T1 values of P forms in reconstituted (0.2 g in 0.7 mL(-1)) samples of freeze-dried 0.25 M NaOH plus 50 mM EDTA extracts of eight diverse soils (Aquept, Dystrochrept x 2, Hapludand, Rendoll, Udand, Haplostoll, and Orthod), three different manures (dairy cattle, deer, and sheep), and one epiphyte moss. Total concentrations in the reconstituted samples ranged from 5 to 175 mg Fe mL(-1), 2 to 62 mg Mn mL(-1), and 72 to 837 mg P mL(-1). Values of T1 for orthophosphate monoesters, orthophosphate diesters, and pyrophosphate varied from 0.42 to 1.69 s in soils and from 0.89 to 2.59 s in manures and the epiphyte. In contrast, T(1) for orthophosphate varied from 0.78 to 1.94 s in soils and 1.45 to 5.82 s in manures and the epiphyte. For quantitative 31P NMR, delay times should be three to five times the T1 value, translating to delays of 3 to 5 s for soils and up to 25 s for manures. If the required delay is too long then strategies such as adding paramagnetics could shorten T1, provided this does not increase line-broadening too much. A regression relationship was obtained between orthophosphate T1 values and the ratio of P concentration to Fe and Mn concentration on a w/v basis (r2= 0.97, P < 0.001), and between the T1 for all other compound classes and the ratio of P to Fe and Mn (r2= 0.70, P < 0.01). By combining measurement of Fe, Mn, and P in the reconstituted extract and these relationships, T1 can be estimated and the appropriate delay time used. If T1 is not considered and the delay time is too short, some peaks will be under- or over-represented and the relative distribution of P forms not quantitative.     

Establishing a linkage between phosphorus forms in dairy diets, feces, and manures

Effective manure management to efficiently utilize organic wastes without causing environmental degradation requires a clear understanding of the transformation of P forms from diet to manure. Thus, the objective of this study was to establish quantitative relationships between P forms in diets, feces, and manures collected from U.S. Northeastern and Mid-Atlantic commercial dairy farms. Total P in diets ranged from 3.6 to 5.3 g kg(-1) dry matter, while the feces had higher P than diets (5.7-9.5 g kg(-1)) and manures had lower P (2.5-8.9 g kg(-1)) than feces. The farms with total dietary P of 4.8 to 5.3 g P kg(-1) had twofold higher concentrations of phytic acid (1647-2300 mg P kg(-1)) than farms with 3.6 to 4.0 g dietary P kg(-1) (844-1100 mg P kg(-1)). Much of the phytic acid in diets was converted to inorganic orthophosphate in the rumen as indicated by a reduction in phytic acid percentage from diets (32%) to feces (18%). The proportion of orthophosphate diesters (phospholipids, deoxyribonucleic acid [DNA]) was twice as high in feces (6.2-10%) as diets (2.4-5.3%) suggesting the excretion of microbial residues in feces. Phosphonates (aminoethyl phosphonates and phosphonolipids) were not seen in diets but were detected in feces and persisted in manures, which suggests a microbial origin. These organic compounds (phytic acid, phospholipids, DNA) were decomposed on storage of feces in slurry pits, increasing orthophosphate in manures by 9 to 12% of total P. These results suggest that reducing dietary P and typically storing feces in dairy farms will result in manure with similar chemical forms (primarily orthophosphate: 63-77%) that will be land applied. Thus, both the reduction of dietary P and storage of manure on farm are important for controlling solubility and bioavailability of P forms in soils and waters.     

A comparison of phosphorus speciation and potential bioavailability in feed and feces of different dairy herds using 31p nuclear magnetic resonance spectroscopy

An experiment was conducted to examine how potential phosphorus (P) bioavailability (inferred from speciation) differs in feed and feces collected in spring from four dairy herds representing different management systems: (i) total confinement with cows fed total mixed ration (TMR), (ii) total confinement with TMR plus P mineral supplement, (iii) a hybrid of confinement with TMR and pastoral grazing, and (iv) predominantly grazing with supplemental grains. A treatment was included that air dried feces to simulate conditions after dung deposition. Wet chemical techniques and solution (31)P nuclear magnetic resonance spectroscopy ((31)P-NMR) were used to identify P concentrations and compounds present in water (a surrogate for P in overland flow), dilute acid (0.012 M HCl, an estimate of P utilization by cattle), or NaOH-EDTA (a solution that maximizes the organic P extraction) extracts of feed and feces. In general, P concentration in feces paralleled P in feed. Air drying feces decreased water-extractable P by 13 to 61% largely due to a decrease in orthophosphate, whereas NaOH-EDTA-extractable P increased by 18 to 48%. Analysis of dilute HCl was unsuccessful due to orthophosphate precipitation when pH was adjusted to 12 for (31)P-NMR. In water extracts, more P was in bioavailable diester-P forms, undetectable by colorimetry, than in NaOH-EDTA extracts. In feed, orthophosphate dominated (46-70%), but myo-IHP varied with feed (<10% in forage samples but 43% in a TMR sample). The proportion of myo-IHP decreased in feces compared with feed via mineralization but decreased less in systems with a greater proportion of available P input (e.g., orthophosphate and phospholipids). Feed and drying effect the concentrations and forms of P in feces and their potential impact on soil and water quality. Although bioavailable P in feces from pasture-based and confined systems can be similar in spring, dung-P is distributed on a lower kg P ha(-1) rate in grazing systems. The best method to mitigate P loss from feces is to decrease P in feed.