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a Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716
b Department of Soil Science, University of Saskatchewan, Saskatoon, Canada S7N 5A8
* Corresponding author (gurpal{at}udel.edu)
Received for publication July 16, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: HAPC, high available phosphorus corn • LC, linear combination • NORC, normal corn • NPP, non-phytate phosphorus • XANES, X-ray absorption near edge structure spectroscopy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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At present, from an agronomic standpoint only total P is typically measured in manures before land application because the usual goal is to know the amount of manure P added relative to crop P uptake or removal. Total P measurements, however, provide no information on the chemical forms of P present in manures or the impacts of modifying diets and other manure management practices on the fate, plant availability, and mobility of manure P in soils. There are other methods available to characterize the forms of P in manures; the majority involve some form of chemical fractionation such as sequential extraction with various acids or bases (Dou et al., 2000; Sharpley and Moyer, 2000). These methods can separate manure P into fractions of differing solubility, but cannot identify exact inorganic or organic P species present.
Recently, there has been an increased interest in the use of new analytical methods such as 31P nuclear magnetic resonance (NMR), enzymatic hydrolysis, and synchrotron-based XANES spectroscopy to characterize the organic and inorganic species of P present in manures and manured soils. Solution 31P NMR has been successively used to characterize organic P in manures, providing insights into the dynamics and availability of manure organic P (Toor et al., 2003, 2004a). The major disadvantage of this method is that specific mineral forms of P (e.g., Al-P, Ca-P, Fe-P), which can be as much as 80% of manure total P, cannot be identified (Dou et al., 2002; Ebeling et al., 2002; Toor et al., 2004a; Turner, 2004). This is because solution state NMR sample preparation uses a chemical extractant, typically NaOH–EDTA, that dissolves all P minerals, thus only allowing for quantification of the total concentration of inorganic P present. Solid state 31P NMR can be used to study manure P without extraction and dissolution of P minerals. However, interferences due to complexation of P with paramagnetic cations such as Fe, Al, and Mn can limit the applicability of this method (Hunger et al., 2004). Enzymatic hydrolysis is another method to identify organic P species in manures and has been successfully used for P speciation in dairy and swine manures (He et al., 2004a; He et al., 2004b).
X-ray absorption near edge structure spectroscopy is a new analytical method that may help improve our ability to characterize inorganic P in manures. While XANES analysis is not cost or time effective for routine analysis, it can be invaluable in assessing the success in chemical fractionation at predicting shifts in P speciation in manures that may accompany changes in animal diet. It has been successfully used to identify P minerals in soils (Beauchemin et al., 2003) and poultry manures (Peak et al., 2002). Beauchemin et al. (2003) reported that calcium phosphates in the form of hydroxyapatite and octacalcium phosphate were present in all soils, ranging from 11 to 59 and 24 to 53% of total P, respectively. The remaining P was adsorbed on ferrihydrite (17–60%), goethite (15–23%), aluminum hydroxide (18–27%), and alumina (16–34%). Peak et al. (2002) found that in unamended poultry litter samples, P was present as weakly bound inorganic and organic forms and as dicalcium phosphate. In contrast, in alum-amended poultry litter, P was adsorbed on amorphous aluminum hydroxide surfaces.
Understanding the effects of modifying poultry diets to reduce P excretion on the forms of P in manures and manure-amended soils is needed given the increased use of this management practice to protect and improve water quality. Consequently, our objective in this study was to use chemical fractionation and XANES spectroscopy as methods to characterize P speciation in broiler litters and turkey manures produced from normal diets and diets including reduced amounts of NPP and phytase or in combination with low phytic acid corn.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Broiler Litters
Broiler litter was generated in three consecutive 53-d floor pen trials. In each trial, there were six dietary treatments with 10 replications of 46 chicks each. Details of the diet formulation can be found in J.T. Sims et al. (unpublished data, 2000). In brief, chickens were fed two different types of corn: normal corn (NORC) or high available phosphorus corn (HAPC). Normal corn had 0.27% total P (phytic acid: 0.19% [70% of total], NPP: 0.08% [30% of total]). High available P corn was developed using the low phytic acid 1-1 (lpal-1) allele of the corn LPA1 gene, and contained 0.27% total P (phytic acid: 0.10% [37% of total], NPP: 0.17% [63% of total]) (Raboy et al., 2000; Raboy and Gerbasi, 1996). These two types of corn were amended with either the normal amount of NPP or reduced concentrations of NPP and microbial phytase (Natuphos; BASF, Mount Olive, NJ). The reduction of NPP in these diets resulted in nonsignificant effects on broiler growth, health, and bone development (J.T. Sims et al., unpublished data, 2000).
The details of diets formulated with NORC are as follows: (i) NORC diet = 0.43% NPP, (ii) NORC–0.1% NPP + phytase = 0.33% NPP and phytase, and (iii) NORC–0.2% NPP + phytase = 0.23% NPP and phytase. Another set of three diets formulated with HAPC were: (i) HAPC diet = 0.43% NPP, (ii) HAPC–0.1% NPP + phytase = 0.33% NPP and phytase, and (iii) HAPC–0.2% NPP + phytase = 0.23% NPP and phytase. Phytase was added at 600 U kg–1 of diet in the NORC and HAPC diets. The NPP concentrations in diets were adjusted with defluorinated P, and limestone was used to maintain the same Ca concentrations in all diets. The approximate diet composition was: corn (NORC or HAPC = 64%), soybean meal (29%), poultry fat (2.2%), poultry by-product meal (1.5%), defluorinated P (0.4–1.5%), and limestone (0.6–1.5%). At the initiation of the first trial, each pen received 7.6 cm (3 in) of fresh pine sawdust as a bedding material. Litter removed after end of three trials was dried at 55°C, ground, and sieved (0.8 mm) before analyses.
Turkey Manures
Turkey manure was produced from three diets fed to four replications of seven birds each: (i) normal (0.70% NPP), (ii) P deficient (0.36% NPP), and (iii) P deficient and phytase (0.36% NPP and 600 U phytase kg–1) (Maguire et al., 2003). The normal diet had 0.28% CaCO3, while a greater concentration of CaCO3 (0.80%) was added in P deficient and phytase diets to maintain a similar concentration of Ca (1.2%) in all diets. To maintain a higher concentration of NPP in the normal diet, monocalcium phosphate (MCP) was added at 1.38%, while it was only 0.15% in other diets. Each diet was fed to turkeys between 8 and 15 d of age, and manure was collected (no bedding material was used). The manure was dried at 55°C, ground, and sieved (0.8 mm) before analyses. The information about effect of dietary modification on turkey health and performance can be found in Angel et al. (2002)
Litter and Manure Chemical Analysis
Total P in litters and manures was determined by microwave digestion using concentrated HNO3 followed by inductively coupled plasma–optical emission spectroscopy (ICP–OES) (USEPA, 1986). We also sequentially extracted 0.2 g of manure with 40 mL each of deionized H2O, 0.5 M NaHCO3, 0.1 M NaOH, and 1 M HCl followed by filtration through a 0.45-µm membrane filter paper using a slightly modified Sharpley and Moyer (2000) method, which is adapted from the soil P fractionation scheme of Hedley et al. (1982) and Tiessen et al. (1983). The P concentration in all extracts was determined by ICP–OES and residual P was calculated as the difference between total P separately measured with microwave digestion and the sum of sequentially extracted P fractions.
X-Ray Absorption Near Edge Structure Spectroscopy
Phosphorus K-edge XANES spectroscopy of all litters and manures was conducted at the Canadian Synchrotron Radiation Facility (CSRF) at the University of Wisconsin-Madison Synchrotron Radiation Center (SRC) using a double crystal monochromator (DCM) beamline. Beamline configuration and experimental parameters were set up as recommended by previous researchers (Sarret et al., 1999) using the CSRF DCM. Air-dried, ground samples were analyzed by placing a small amount of sample on a piece of graphite tape and transferring it into the ultra high vacuum (UHV) chamber. The beamline was calibrated using P powder to an edge energy of 2145 eV in the total electron yield (TEY) spectrum. For XANES experiments, the storage ring was operated at either 800 MeV or 1 GeV. The CSRF DCM is a UHV beamline, so it was possible to collect both fluorescence yield (FY; via a multichannel plate detector) and TEY (current yield) on powder samples. Total electron yield probes only the surface of samples, while FY provides information on bulk sample properties. The TEY data were used for quantitative analysis because it is not as subject to self-absorption distortions of the spectra. These distortions caused by self-absorption typically decrease the intensity of the white line peak (Khare et al., 2004) and could make linear combination (LC) fitting over the full spectral range somewhat difficult to do quantitatively. Multiple scans (3–5) were averaged (after correcting for edge shifts, if necessary) to improve signal to noise. The P XANES spectra were analyzed using WinXAS v. 2.3 (Ressler, 1998). All spectra were background subtracted, and normalized over the energy range of interest (2145–2199 eV) to an edge jump of 1.0. As a test of variance in the XANES data, individual scans were compared with the averaged final scan after background subtraction and normalization. The variance of individual scans from the published "averaged" scan was less than 0.2% over the 2145 to 2190 eV range. Manure samples were then fit using a variety of P reference standards (discussed below) and the LC approach of WinXAS 2.3. WinXAS allows users to visually inspect the quality of fit by showing individual components, the final total fit, and the residual along with the raw data being analyzed.
Phosphorus Standards for X-Ray Absorption Near Edge Structure Spectroscopy
Initially, many standards were also compared with the manure samples, including ammonium and sodium phosphate salts, Fe(II) and Fe(III) phosphate minerals, calcium phosphate minerals (monetite, hydroxylapatite, fluoroapatite, calcium phosphate tribasic), and aluminum phosphate minerals (variscite). The maximum number of standards chosen for LC-XANES fitting was based on running principal component analysis on the set of nine manure samples (six broiler and three turkey) discussed in this paper. It was found that four components were needed to describe this data set. The P standards needed to obtain a good fit to the litter and manure samples used in this study were dicalcium phosphate (monetite phase), hydroxylapatite, phytic acid, and aqueous phosphate. The P XANES spectra of these standards are shown in Fig. 1
. While there is little difference in spectral features between aqueous phosphate and phytic acid, the intensity of the white line peak is decreased in phytic acid and the shape of the oxygen oscillation is somewhat changed.
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The choice of aqueous phosphate as a standard for fitting in these dried and evacuated manure samples deserves further discussion. It is true that the total amount of residual water available for aqueous phosphate formation is fairly small in these samples. However, it is not possible to quantitatively distinguish aqueous phosphate from phosphate adsorbed on minerals with P XANES alone as there are no distinctive features to separate the reference adsorption samples from aqueous phosphate (Peak et al., 2002). Researchers have shown with attenuated total reflectance Fourier transform infrared (ATR-FTIR) (Hug, 1997) that drying sulfate adsorption samples on hematite caused a shift from one adsorption complex (monodentate inner-sphere) to another (bidentate inner-sphere). While this certainly is expected to have some effect on the rate of desorption from these manures, we believe that it is justified to lump residual aqueous phosphate and weakly bound (adsorbed) phosphate into one group. These species are all expected to correlate strongly with water-soluble P levels in the manures, and all have XANES spectra that are very similar to aqueous phosphate. For this reason, we represented "free and weakly bound phosphate" with the aqueous phosphate standard. There is of course some uncertainty in this assignment, but no more suitable single standard is obtainable and adding multiple standards to the fit with extremely similar spectra was undesirable. Goodness of fit was evaluated by examining the residuals in WinXAS's fit routine. Visual inspection of the residual was done to verify that no important structural features were being missed or overestimated by the standards, and a maximum acceptable residual value of 1.5 over the entire 2145 to 2199 eV range was chosen. In fact, all the final fits had a residual between 0.8 and 1.0.
Phosphorus XANES spectra of the aqueous phosphate sample were collected at pH 7.5. At this pH, H2PO–4 and HPO2–4 are present in approximately equal conditions. This pH is similar to the final pH of the manure samples analyzed in this study. However, shifts in protonation state of phosphate do slightly change the shape of the XANES spectra of aqueous phosphate samples, so there is some uncertainty associated with this standard even if one assumes that the "aqueous phosphate" contribution in the LC-XANES fitting is purely due to entrained free aqueous phosphate.
Overall, though, we feel that the aqueous phosphate standard represents a useful P pool of aqueous and weakly bound phosphate reasonably well. The absolute amounts of weakly bound and free aqueous phosphate may differ somewhat from the LC-XANES results using an ideal standard, but the trends should still be correct and of use in determining phosphate lability in the manures.
Statistics
Descriptive statistics and one-way ANOVA were performed using GenStat 4.2 (Lawes Agricultural Trust, 2000) to calculate means and standard deviations, and to test for significant differences of total P for broiler litters and turkey manures produced from different diets at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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For diets using NORC, reducing NPP by 0.1% and using phytase increased the concentration and percentage of H2O-P in litter, relative to the normal diet, while decreasing litter HCl-P and residual P. This suggests that phytase was more effective at converting phytic acid P in diets to easily digestible forms of P than anticipated and that the reduction in NPP used (0.1%) was not adequate to compensate for this and prevent excess soluble P in the diet, relative to broiler nutritional requirements. Thus, while this diet did reduce litter total P, relative to the normal diet, more of the excreted P was readily soluble in H2O than the NORC diet where concentration and percentage of excreted P as phytic acid (HCl-P and residual P) were higher. Decreasing NPP in the diet by 0.2% and using phytase reduced the concentration, but again increased the percentage, of H2O-P compared with litter from the NORC diet. Thus, in this diet, increase in litter H2O-P percentage was due to phytase use was more closely balanced by reductions in litter H2O-P concentration that were caused by decreases in dietary NPP.
The relative distribution of chemical fractions of litter P in diets formulated with HAPC was similar to those in litters from NORC diets (Table 1). Use of HAPC instead of NORC (without phytase) reduced litter total P, again reflected mainly by decreases in HCl-P. As with the NORC diets, increases in the concentration and percentage of litter H2O-P were observed in the HAPC and phytase generated litters. The higher values for H2O-P were probably due to the more digestible forms of P in low phytic acid corn. This would increase absorption of P by the broilers during digestion of feed but also increase the percentage of excreted P found in H2O-extractable form. Reducing NPP and using phytase in combination with HAPC decreased the concentration of H2O-P, and HCl-P, relative to the HAPC diet. For the HAPC diet with a 0.1% reduction in NPP, the use of phytase again increased the percentage of H2O-P in litters (77%), compared with the HAPC (67%) and NORC (56%) diets. As with NORC diets, this suggests that further reductions in dietary NPP would result in reduced H2O-P concentrations in litters. We did observe such a decrease in the diet that combined HAPC, a 0.2% reduction in NPP, and phytase, where the concentration of litter H2O-P was reduced by 42% (7.8 g kg–1) compared with the HAPC diet (13.5 g kg–1). Furthermore, the percentage of litter H2O-P in this diet (69%) was very similar to the HAPC diet, less than all other diets using phytase, but still greater than the NORC diet.
In general, chemical fractionation results suggest that inclusion of phytase in diets is more effective at reducing litter total P than litter soluble P and will increase the percentage, but not necessarily the concentration of soluble forms of litter P. Accurate manipulation of dietary NPP and phytase concentrations can clearly reduce litter H2O-P concentrations. Reductions in total P are probably due to reduced excretion of phytic acid and other recalcitrant organic and mineral forms of litter P (HCl-P and residual P). However, chemical analysis alone cannot identify the insoluble mineral forms of P or explain the sources of litter H2O-P. Therefore, we next used XANES spectroscopy to further characterize litter P speciation.
The XANES spectroscopic data showed that the percentage of aqueous phosphate was three- to fivefold lower (13–18%) than H2O-P (56–77%) in all broiler litters (Table 2, Fig. 2 and 3) . This suggests that all the P extracted with H2O and measured by ICP–OES was not simply orthophosphate dissolved in the H2O phase of the litters. Instead, much of the litter H2O-P was probably adsorbed on the surfaces of solid phases in litters or present as readily soluble P minerals such as dicalcium phosphate, which dissolved during the H2O extraction. We found a significant positive correlation (r = 0.85, significant at the 0.001 probability level) between litter H2O-P and litter dicalcium phosphate, suggesting that most of the P extracted with H2O originated from the dissolution of dicalcium phosphate. Further, the proportion of dicalcium phosphate determined by XANES was similar (64–79%) to H2O-P (56–77%) for all litters except for that generated using the HAPC–0.2% NPP + phytase diet. This HAPC–0.2% NPP + phytase sample seems to be a bit anomalous in that it could be fit acceptably well using only dicalcium phosphate. However, it is also possible to fit these spectra quite well to HAPC–0.2% NPP + phytase sample (see the fit results in Fig. 3) with a mixture of 53% dicalcium phosphate, 11% hydroxylapatite, and 35% phytic acid. If that fit result (which has a slightly higher residual than purely dicalcium phosphate) is used, then the speciation of phosphate actually makes more sense in the following discussion about Ca to P ratio and H2O-P effects on P speciation for all manures (both broiler and turkey). It is reasonable to use a mixture that contains organic P in the fitting because all other samples show 10 to 20% contributions from that species making it unlikely that this component would be completely absent. However, this suggests that the XANES fitting is not 100% conclusive for this particular sample (HAPC–0.2% NPP + phytase). This could be because the hydroxylapatite content is low enough to make the fitting less reliable (11%), or it could be that the raw spectrum was somewhat noisier because the total amount of P in this sample was less than the others (due to diet), or that at this intermediate Ca to P ratio range octacalcium phosphate is actually the stable phase and not hydroxylapatite. This would be in agreement with thermodynamics, but adding a fifth component to the LC-XANES fitting for this sample only would be problematic. This is especially true because the XANES spectra of octacalcium phosphate are expected to be fairly similar in features to that of hydroxylapatite (Hesterberg et al., 1999).
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The P XANES spectra for NORC litters were similar to dicalcium phosphate and phytic acid, with some slight variations noted with changes in diet (Fig. 2 and 3). In contrast to the chemical fractionation results for litter H2O-P, the percentage of aqueous phosphate determined by XANES was similar for unamended and reduced NPP and phytase litters. The percentage of dicalcium phosphate, however, did increase when NPP was reduced and phytase included in diets (Table 2). For example, dicalcium phosphate was 65% for NORC, 72% for NORC–0.1% NPP + phytase, and 80% for NORC–0.2% NPP + phytase litters. A similar trend was seen for HAPC generated litters (Fig. 2 and 3), with the exception of the HAPC–0.2% NPP + phytase litter where no aqueous phosphate was found but a small contribution (11%) from hydroxylapatite was predicted with LC-XANES fits. This decrease in the amount of aqueous phosphate from XANES analysis is supported by significantly lower concentrations of H2O-P (<7.8 g kg–1) in this litter compared with other broiler litters (all of which were >11.1 g kg–1). The greater percentages of dicalcium phosphate in the reduced NPP and phytase litters are attributed to the hydrolysis of phytic acid to inorganic phosphate during digestion, which was then precipitated in litters as dicalcium phosphate. This is supported by lower phytic acid concentrations in reduced NPP and phytase generated litters. For example, in NORC litter, phytic acid was 20%, which decreased in NORC–0.1% NPP + phytase (12%), and NORC–0.2% NPP + phytase (7%). A similar trend can be seen for the HAPC generated litters. A strong positive correlation (r = 0.96, significant at the 0.001 probability level) observed between HCl-P (4.1–21.7%) and phytic acid (7–20%) suggests that HCl mainly extracts phytic acid in broiler litters. This contention is supported by recent NMR studies of litter P speciation (Maguire et al., 2004; McGrath, 2004).
Turkey Manure
Modifying turkey diets by reducing NPP from 0.70% (normal diet) to 0.36% and including phytase to hydrolyze phytic acid, reduced manure total P by 40% (Table 3). Manure from a P deficient diet (decreased NPP, without phytase) had 35% less total P than that produced from the normal diet.
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Calcium Effects on Phosphorus Forms in Broiler Litters and Turkey Manures
X-ray absorption near edge structure spectroscopy results indicated that shifts from dicalcium phosphate to hydroxylapatite were occurring in some of the samples. Accordingly, examining the Ca to P ratio in litters and manures may provide insight on the controlling factor in P speciation for these samples. The relationship between different chemical species (from XANES analysis) and total Ca to P ratio and H2O-P levels is seen in Fig. 4
. There was no apparent relationship between Ca to P ratio and phytic acid concentration for these samples. However, the relative proportion of other P species (aqueous phosphate, dicalcium phosphate, hydroxylapatite) clearly depended on the litter or manure Ca to P ratio. At Ca to P ratios less than 2.0 in wastes, aqueous phosphate can be observed in the broiler litter and normal turkey manure samples and dicalcium phosphate is the only mineral phase present. At Ca to P ratios of greater than 2.0 (turkey manures), however, hydroxylapatite can be observed whereas aqueous phosphate contributions disappear. This is in good agreement with calcium phosphate chemistry. Hydroxylapatite is far less soluble than dicalcium phosphate, so when this phase controls P solubility then solution P levels are expected to be very low. When the more soluble dicalcium phosphate phase controls solubility then more aqueous phosphate will be present in the litters and manures. The XANES results are strengthened by the fact that it is well known that different phases limit calcium phosphate solubility as the Ca to P ratio is changed. For example, at Ca to P ratios of 1.0 dicalcium phosphate dominates, at Ca to P ratios of 1.5 tricalcium phosphate is dominant, and at Ca to P ratios of 1.67 and above hydroxylapatite is the stable phase (Ben-Nissan et al., 1995).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The detection of higher amounts of P as dicalcium phosphate (>65%) in broiler litters and normal turkey manure can be used to reduce mineral P fertilizer addition to soils by considering that a part of P in manures is present in the soluble form (e.g., dicalcium phosphate), and adding the remainder of P from the fertilizers. In this case, broiler litters regardless of diet and normal turkey manure will result in higher P availability in soils on application because dicalcium phosphate was the dominant mineral controlling solubility of P. In contrast, there was a mix of hydroxylapatite and dicalcium phosphate in reduced NPP and phytase-amended turkey manures. This suggests that these dietary modifications for turkey manures resulted in reducing solubility of P by changing P forms in manure from being more soluble (dicalcium phosphate) to less soluble (hydroxylapatite), which will result in less P in solution in neutral soils.
In general, Ca seems to be the dominant cation affecting the formation of P minerals in manures. For example, for all broiler litters, the total Ca to P ratio was <2.0, and the P mineral observed was dicalcium phosphate. In reduced NPP and phytase turkey manures, total Ca to P ratio was >2.0, and a mix of hydroxylapatite and dicalcium phosphate was observed. This is because P was reduced in diets but levels of Ca were similar, which means that a higher amount of Ca was available to react with P to form hydroxylapatite in turkey manures. There is clear evidence from XANES that hydroxylapatite formation at Ca to P ratios above 2.0 plays an important role in reducing H2O-P levels. Therefore, one of the management strategies to reduce P solubility in manures may involve manipulation of manures to produce a Ca to P ratio of 2.0 or more.
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ACKNOWLEDGMENTS |
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REFERENCES |
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), dicalcium phosphate is represented with a dashed line (- - -), phytic acid is represented with a dotted line (• • •), aqueous phosphate is represented by open triangles (
), and hydroxylapatite is represented with a plus (+). Of special interest is the appearance of hydroxylapatite in the HAPC–0.2% non-phytate phosphorus (NPP)+ phytase sample (middle row) and in the P deficient as well as P deficient plus phytase turkey samples (bottom row). The presence of this species can be explained by differences in the Ca to P ratios of these manure samples.