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USDA-ARS, Northwest Irrigation and Soils Research Laboratory, 3793N. 3600E., Kimberly, ID 83341
* Corresponding author (bturner{at}ifas.ufl.edu).
Received for publication April 28, 2003.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: NMR, nuclear magnetic resonance
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The development of robust techniques for identifying P compounds in animal manures is a fundamental prerequisite to understanding the effects of dietary manipulations on animals and the environment. The first comprehensive evaluation of manure P composition was reported by Peperzak et al. (1959), who used a fractionation procedure to determine that phytic acid (measured as acid-soluble organic P) dominated the organic P component of a wide range of manures, with relatively small proportions of phospholipids and DNA. Comparable results were obtained by subsequent authors using similar procedures (Gerritse and Vriesema, 1984; Barnett, 1994). Various chromatographic techniques are routinely employed for the specific determination of phytic acid in feeds (Phillippy and Bland, 1988; Du
ková et al., 2000; Raboy et al., 2000), but are rarely applied to animal manures because phosphates exist in complexes with mineral and organic compounds that confound separation by conventional procedures. Sample analysis by chromatography is rapid compared with spectroscopic techniques, but errors can arise through poor recoveries from chromatography columns, matrix interference with standards, and misidentification of compounds (Kemme et al., 1999; Turner et al., 2002). In most cases, a secondary technique, such as solution 31P NMR spectroscopy, is required to identify the separated fractions (Kemme et al., 1999; Raboy et al., 2000). Recently, an enzyme hydrolyzable P technique was employed to characterize functional P groups in chemically extracted fractions of swine and cattle manures (He and Honeycutt, 2001). This relatively low-cost method has promise as a routine laboratory procedure for manure P characterization, although large proportions of the total P remain unidentified.
Spectroscopic techniques are less frequently employed for manure P speciation. The value of solid-state 31P NMR spectroscopy remains limited at present due to poor spectral resolution (Frossard et al., 2002), but solution 31P NMR spectroscopy has numerous benefits over chromatographic and other procedures, and potentially offers the most convenient method of P characterization. In particular, it allows the simultaneous identification of multiple P compounds in the complex matrices of manure extracts with minimal sample handling (Condron et al., 1997). The technique has been widely applied to soils during the previous two decades, and was advanced recently by improvements in the extraction procedure, signal identification, and the understanding of compound degradation during extraction and analysis (Cade-Menun and Preston, 1996; Makarov et al., 2002; Turner et al., 2003a). Importantly, the inclusion of EDTA in the alkaline extraction solution markedly improves P recovery from soils (Bowman and Moir, 1993), but this has not been tested for animal manures.
Solution 31P NMR spectroscopic procedures have been developed for quantification of phytic acid in food (O'Neill et al., 1980), animal feed (Kemme et al., 1999), and sewage sludge (Hinedi et al., 1989). Of the few studies using solution 31P NMR spectroscopy to analyze animal manure, Leinweber et al. (1997) identified orthophosphate monoesters and diesters in NaOH extracts of swine slurry, while Crouse et al. (2000) characterized functional P groups in NaOH–EDTA extracts of turkey litter. Spectral resolution was relatively poor in these studies, and this represents perhaps the greatest limitation of the technique. In particular, poor resolution in the orthophosphate monoester region of the spectra often precludes accurate quantification of phytic acid, the dominant organic P compound in most manures.
Given the current interest in the P composition of animal manures, there is an urgent requirement for a comprehensive study of their analysis by solution 31P NMR spectroscopy. The aim of this work was to address this by investigating the optimal extraction conditions for different types of animal manures, the potential degradation of P compounds during extraction and analysis, and the optimal NMR machine parameters for quantitative spectroscopy. The ultimate aim was to recommend a standard procedure for the extraction and analysis of animal manures by solution 31P NMR spectroscopy.
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MATERIALS AND METHODS |
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Solution Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy
Manures (2.00 ± 0.01 g) were extracted with 40 mL of NaOH–EDTA for 4 h at 20°C. Three extraction solutions were used, containing different concentrations of NaOH (0.15, 0.25, and 0.50 M) with a constant concentration of 50 mM EDTA. Extracts were centrifuged and aliquots (2 mL) taken for chemical analysis as described above. The remainder of the extracts was then frozen rapidly at –80°C, lyophilized, and ground to a fine powder. Immediately before NMR spectroscopy, each freeze-dried extract (approximately 100 mg) was redissolved in 0.9 mL of 1 M NaOH and 0.1 mL of D2O (for signal lock) and transferred to a 5-mm NMR tube. The pH of the redissolved samples varied slightly depending on the initial extractant concentration, with averages for 0.15, 0.25, and 0.50 M NaOH extracts of the three manures being 13.71 ± 0.06, 13.78 ± 0.07, and 13.99 ± 0.06, respectively.
Solution 31P NMR spectra were obtained using a Bruker (Billerica, MA) Avance DRX 500 MHz spectrometer operating at 202.456 MHz for 31P and 500.134 MHz for 1H. We used a 5-µs pulse (45°), a delay time of 5.0 s, an acquisition time of 0.8 s, and broadband proton decoupling for all samples. The number of scans required to give an acceptable signal-to-noise ratio varied among the manures depending on total P concentration and extract properties, being 1500 to 5500 for broiler litter extracts, 10200 to 11000 for swine manure extracts, and 11000 to 14000 for cattle manure extracts. The relatively long delay time used here (5 s) allowed sufficient spin–lattice relaxation between scans for P compounds in these extracts with low paramagnetic ion concentrations (see Discussion). This was determined for selected samples by acquiring a set number of scans at different delay times up to 10 s.
Temperature was regulated at 20°C to minimize degradation of P compounds and ensure consistent signal intensities (Cade-Menun et al., 2002; Turner et al., 2003a). To allow comparison among extracts of each manure type, spectra were plotted using the line broadening necessary to produce acceptable resolution for the least-resolved spectrum of the three different NaOH concentrations. These values were 0.5 Hz for the broiler litter extracts, 1 Hz for the swine manure extracts, and 4 Hz for the cattle manure extracts. Where these values concealed resolution, additional spectra were plotted using less line broadening.
Chemical shifts of signals were determined in ppm relative to 85% H3PO4 and assigned to individual P compounds or functional groups based on literature reports (Turner et al., 2003a). Signal areas were calculated by integration and P concentrations calculated by multiplying the proportion of the total spectral area assigned to a specific signal by the total P concentration (mg P g–1 dry manure) in the original extract. In well-resolved spectra, concentrations of phytic acid were determined by the same procedure by summing the areas of the four signals at approximately 5.95, 5.06, 4.70, and 4.54 ppm occurring in the ratio 1:2:2:1. In spectra where signals from phytic acid overlap with those from other monoesters, phytic acid concentrations can be calculated by multiplying the signal from the phosphate at the C-2 position on the inositol ring (occurring at approximately 5.95 ppm) by six, because this signal is often well-resolved from the orthophosphate and other monoester signals (O'Neill et al., 1980). Therefore, phytic acid concentrations were also calculated using this technique for comparison. Mean polyphosphate chain lengths were calculated from the concentrations of end groups (approximately –4 ppm) and mid-chain groups (–18 to –21 ppm) using the formula:
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It is difficult to estimate the error in NMR spectroscopy without acquiring replicate spectra, but for manure and feed samples, analytical error has been estimated to be approximately 5% for larger signals and 10% for smaller signals (Leinweber et al., 1997; Kemme et al., 1999). Differences in extraction efficiency with time and solution chemistry were investigated using analysis of variance procedures in SAS Version 8.0 (SAS Institute, 1999).
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RESULTS |
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Effect of Extraction Time on Phosphorus Recovery
Most total P in the manures was extracted after 1 h, although maximum recovery occurred between 4 and 16 h (e.g., for 0.5 M NaOH + 50 mM EDTA; Fig. 2)
. The differences in recovery between the 4- and 16-h extraction times were not significantly different (P > 0.05), so a 4-h extraction time was selected for all further experiments to minimize degradation of alkali-labile P compounds. The increase in P recovery during longer extraction times was mainly accounted for by the unreactive P fraction, with most of the reactive P being recovered during the first hour (data not shown). Calcium recovery followed a similar trend to P, but there were no significant increases in the recovery of Al, Fe, or Mn with increasing extraction time (data not shown).
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For the cattle manure, line broadening was similar for the different NaOH concentrations, although resolution was slightly improved in the 0.15 M NaOH + 50 mM EDTA extract (Fig. 6). For example, this was the only cattle manure extract in which the C-2 phosphate signal from phytic acid was clearly visible.
Differences in Phosphorus Composition between Manure Types
The three manures differed in their P composition (Table 4, Fig. 3). The broiler litter contained mainly orthophosphate and orthophosphate monoesters, with only traces of phospholipids and pyrophosphate, and no detectable polyphosphates or DNA (Fig. 4). In contrast, the cattle and swine manures were dominated by orthophosphate (Fig. 3). Cattle manure contained the richest P composition, including considerable proportions of orthophosphate monoesters, orthophosphate diesters (both DNA and phospholipids), and polyphosphates (both pyro- and polyphosphate). Indeed, these were the only extracts in which polyphosphate end-groups were visible (Fig. 6). Swine manure also contained orthophosphate monoesters, orthophosphate diesters, and traces of pyro- and polyphosphates. The high P concentrations in these extracts meant that most of the lower-concentration compounds were clearly detectable in the spectra (Fig. 5).
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Signals from DNA, pyrophosphate, and orthophosphate monoesters did not change with increasing NaOH concentrations (Table 4; Fig. 4, 5, and 6), although there appeared to be some loss of the orthophosphate monoester signals at 4.69 and 5.06 ppm in the 0.25 and 0.5 M NaOH extracts of cattle manure (Fig. 6). Interestingly, DNA was apparently not extracted from swine manure by 0.15 M NaOH + 50 mM EDTA, only appearing in the spectrum of the 0.25 M NaOH + 50 mM EDTA extract (Fig. 5).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The extraction solution also markedly influenced results from solution 31P NMR spectroscopy, because the concentration of NaOH affected both spectral resolution and the apparent P composition of the manures. The only significant difference in solution chemistry among the different NaOH extracts was the concentration of paramagnetic ions, because almost no Fe and Mn were present in the 0.5 M NaOH + 50 mM EDTA extracts. This seems a likely explanation of differences in spectral resolution, because paramagnetic ions are known to influence line broadening, and even relatively small concentrations can reduce spectral quality (O'Neill et al., 1980). However, this does not account for the spectra of cattle manure extracts, because resolution was similar for all three NaOH concentrations, despite a decrease in paramagnetic ion concentration in the strongest NaOH extract. Spectral resolution can be influenced by sample pH (Crouse et al., 2000), but this was ruled out because the redissolved extracts analyzed here were all at pH of >13.7, which is sufficiently high to eliminate such effects. A more likely explanation is the more viscous nature of the cattle manure extract compared with the swine manure and broiler litter extracts, because increases in viscosity can also increase line broadening (Nanny et al., 1997). This phenomenon requires further investigation, because similar improvements in spectral resolution would significantly enhance the spectroscopic analysis of P in a range of environmental samples, notably soils and sediments.
Improvements in spectral resolution by extraction with more concentrated NaOH solution were compromised by the loss of signals from phospholipids and polyphosphates. These compounds were quantitatively unimportant in broiler litter, and to a lesser extent in the swine manure (Peperzak et al., 1959; Barnett, 1994), but constituted relatively large proportions of the total organic P in the cattle manure. Degradation of phospholipids was expected in the stronger alkaline solutions (Turner et al., 2003a) and was reported previously in swine slurry (Leinweber et al., 1997). However, all extracts were redissolved in 0.9 M NaOH for NMR spectroscopy, suggesting that any alkali-induced degradation should have been consistent across all samples irrespective of the strength of the initial extract. For polyphosphates, degradation is minimal in 0.25 M NaOH + 50 mM EDTA extracts of soils and sediments, because metal chelation by EDTA precludes chemical hydrolysis (Hupfer et al., 1995; Turner et al., 2003a). Therefore, changes in polyphosphate signals with increasing NaOH concentration are more likely to be explained by changes in solubility. In particular, the marked decreases in metal concentrations in stronger alkaline solutions indicate possible coprecipitation with polyphosphates. However, pyrophosphate concentrations in the more alkaline cattle manure extracts increased in proportion with decreases in polyphosphate concentrations (Table 4), suggesting that at least some degree of degradation was involved. It should be noted that significant quantities of polyphosphates have not previously been reported in manures, but their presence clearly demonstrates that simple classification of extractable P into inorganic and organic fractions based on molybdate colorimetry can be misleading.
A large difference in phytic acid concentrations was detected between swine manure and broiler litter, but this was not unexpected. Poultry litters typically contain considerable proportions of phytic acid (Barnett, 1994), while the value of 4.5 to 5% of the total P reported here for swine manure is similar to that of 4% reported by Kemme et al. (1999). However, despite the small concentrations of phytic acid in the swine manure, the well-resolved spectrum ensured that it was quantified with some confidence. Clearly, the NMR procedure is sensitive enough to detect low concentrations of organic P compounds (e.g., approximately 100 µg P g–1 dry manure), yet robust enough to achieve this in the complex matrices of manure extracts.
It would also be possible to quantify phytic acid in the 0.25 M NaOH extract of the broiler litter, but overlapping signals from phytic acid and orthophosphate or other monoester signals mean there is a much greater chance of introducing error. This problem can be overcome by calculating phytic acid concentrations using the signal from the C-2 phosphate of phytic acid (O'Neill et al., 1980). Phytic acid concentrations in the swine manure and broiler litter extracts were similar when calculated based on the sum of all phytic acid signals or the C-2 phosphate alone. This suggests that phytic acid can be quantified with confidence even in relatively poorly resolved spectra providing that signals from orthophosphate and the C-2 phosphate are sufficiently resolved.
None of the spectra of cattle manure extracts were sufficiently resolved in the orthophosphate monoester region to permit the quantification of phytic acid. This may be overcome by pretreating extracts with hypobromite oxidation before NMR spectroscopy (Irving and Cosgrove, 1981), or by using spectral deconvolution software to separate phytic acid signals from complex spectra (Turner et al., 2003b). However, the cattle manure appeared to contain only small concentrations of phytic acid, because the signal from the C-2 phosphate of phytic acid was small and only visible in the spectrum of the 0.15 M NaOH + 50 mM EDTA extract. This was not unexpected, because pasture plants contain little phytic acid compared with the grains fed to swine and broilers (Peperzak et al., 1959). It was noted that signals from phytic acid appeared at chemical shifts slightly upfield of those measured in soil extracts (Turner et al., 2003a). Again, these were not due to differences in pH in the redissolved extracts, but may have been caused by differences in solution chemistry, because manure extracts contained large concentrations of Ca, but low concentrations of paramagnetic ions, compared with soil extracts.
Selection of an appropriate delay time is important to ensure quantitative analysis by allowing all P nuclei to fully relax between scans (Cade-Menun et al., 2002). In soil extracts, the high concentrations of paramagnetic ions allow relatively short delay times (<1 s) to be used, because the magnetic properties of Fe and Mn help P nuclei to relax more rapidly after excitation than would otherwise be expected if paramagnetics were not present (Wilson, 1987). This minimizes the machine time required to obtain acceptable signal-to-noise ratios. However, the low concentrations of paramagnetic ions in the manure extracts analyzed here necessitated relatively long delay times of at least 5 s for quantitative analysis, which meant that several hours of machine time were required to obtain suitable spectra. It may be possible to decrease this by adding small concentrations of lanthanide shift reagents or paramagnetic ions to the NMR tube to help P nuclei relax more rapidly (Nanny et al., 1997), although O'Neill et al. (1980) reported that there is a small and narrow range of optimum paramagnetic ion concentrations above which any advantage of decreased spin–lattice relaxation time is compromised by increased line-broadening.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ková, D., M. Marounek, and P. B
ezina. 2000. Determination of phytic acid in feeds and faeces of pigs and poultry by capillary isotachophoresis. J. Sci. Food Agric. 81:36–41.Related articles in JEQ:
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