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a USDA-ARS, New England Plant, Soil, and Water Lab., Orono, ME 04469
b Geological and Environmental Sciences, Stanford Univ., Stanford, CA 94305
c Biological and Agricultural Engineering, Univ. of Arkansas, Fayetteville, AR 72701
d Dep. of Plant and Soil Sciences, Univ. of Delaware, Newark, DE 19716
* Corresponding author (Zhongqi.He{at}ars.usda.gov)
Received for publication December 19, 2006.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND IMPLICATIONSREFERENCES |
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Abbreviations: NMR, nuclear magnetic resonance
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND IMPLICATIONSREFERENCES |
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Enzymatic hydrolysis is another method used to identify organic P species in the environment. This method has been used to characterize hydrolyzable organic P in soils (Pant et al., 1994; Shand and Smith, 1997; Pant and Warman, 2000; Turner et al., 2002; He et al., 2004a, 2004b), manures (He et al., 2004c, 2006b, 2006c), and waters (Herbes et al., 1975; Toor et al., 2003). Unlike 31P NMR, enzymatic hydrolysis cannot classify all organic P in a sample. However, because organic P must first be hydrolyzed to inorganic P for plant uptake, enzymatic hydrolysis can provide an estimate of hydrolyzable, and thus bioavailable, organic P in manure and soil (Seeling and Jungk, 1996; Otani and Ae, 1999), which is not possible to obtain with NMR analysis. If appropriate phosphatases are used, specific forms of hydrolyzable organic P can be identified and quantified by the difference in inorganic P detected after incubation in the presence and absence of phosphatase (He and Honeycutt, 2001; Turner et al., 2002; Toor et al., 2003; He et al., 2004c, 2006b, 2006c). He et al. (2004b, 2006c) have developed a phosphatase hydrolysis method to characterize manure and soil P. In this enzymatic approach, organic P extracted from manure or soil is incubated at pH 5.0 (100 mM sodium acetate buffer) with either (i) acid phosphatase from potato; (ii) acid phosphatases from potato and nuclease P1; or (iii) phosphatases from potato and wheat germ plus nuclease P1. Each of the enzymes shows optimal activity at 37°C and near pH 5. The P differentially released by these three enzymes is designated as simple monoester P, polynucleotide-like P, and phytate-like P, respectively.
For any methods that attempt to characterize Po in sample extracts, concerns exist about the influence of sample preparation on extracted P forms. Manures are usually dried and ground for easier handling and to improve sample heterogeneity (Ajiboye et al., 2004; Vadas and Kleinman, 2006; Dail et al., 2007). However, drying samples before extraction has been shown to alter P extractability, e.g., reducing water-extractable P from poultry manure (Sistani et al., 2001), but increasing water-extractable P from dairy manure (Ajiboye et al., 2004; Vadas and Kleinman, 2006). These changes may occur through degradation of P forms or through changes in P bonding to the sample matrix (Turner, 2005). If P forms are degraded, this could influence the results of both P NMR and phosphatase hydrolysis. However, if bonding mechanisms alone are altered, this may affect phosphatase hydrolysis but not P forms revealed by P NMR.
Both 31P NMR spectroscopy and phosphatase hydrolysis are valuable tools for P characterization. However, there have been no reports comparing data obtained by the two methods on a single extract of manure or soil, or comparing the effects of sample drying before extraction on the results from both methods. Thus, it is not clear to what degree the P forms identified by the two methods are similar, or may be affected by pre-extraction treatment. Therefore, in this work, we analyzed the P forms in NaOH-EDTA extracts of fresh and dried dairy and poultry manure by 31P NMR spectroscopy, quantitatively comparing the similarities and differences between the two methods. Such information is essential for cross comparison and application of the two sets of P data reported, and will aid researchers in choosing the method most applicable to their future research goals.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND IMPLICATIONSREFERENCES |
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2 kg per cow). The samples were mixed and a composite 20-kg sample was taken. The cows were fed a diet of total mixed ration that had 4.26 mg total P kg–1 dry matter. Total P in the dried and ground manure was 6.88 g kg–1. Fresh poultry manure (mixture of feces, spilled feed, without bedding) was collected from a Central Maine layer farm after a single annual cleaning. Five grab samples of manure were removed from inside the poultry house, composited, and homogenized (Dail et al., 2007). A subsample of the fresh dairy and poultry manures was homogenized at room temperature. A part of the fresh homogenized manure sample was dried at 65°C, ground, and sieved through a 2-mm screen. Both fresh and dried samples were then stored at –20°C until use. Therefore, in this study, the terms "fresh" and "dried" refer to the initial different treatment conditions. The dry matter content of the poultry manure was 416 g kg–1 (41.6%), while dairy manure was 150 g kg–1 (15%). Total P, Ca, Mg, K, Fe, and Al contents in dried, ground, and sieved manures were determined, in duplicate, by microwave digestion with concentrated HNO3 followed by inductively coupled plasma–optical emission spectroscopy (ICP–OES) (USEPA, 1986). Typically, the various chemical constituents in this manure, as shown in Table 1, are similar to the other dairy manures produced on Northeastern and Mid-Atlantic dairy farms (Toor et al., 2005). Total C was analyzed, in duplicate, by dry combustion (LECO CNS-2000, LECO Corp., St. Joseph, MI) (Table 1).
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10 mL) was freeze-dried and used for 31P NMR analysis. Total recovery of P was 99 to 103% in dairy manure while in the poultry manure only 65 to 71% of P was removed with NaOH-EDTA extraction (Table 2). Therefore, we conducted an additional extraction of poultry litters with 1 M HCl. The poultry litter residues from NaOH-EDTA extraction were washed with 10 mL of deionized water and supernatant was discarded after centrifugation. Washed poultry litter residues were extracted with 1 M HCl for 16 h. After centrifuging, 1 mL of the HCl extracts was diluted to 10 mL by slowly adding 8 mL of 400 mM un-buffered sodium acetate and 1 mL of deionized water. The final pH of the diluted extracts was adjusted to 5.0. These pH-adjusted extracts were analyzed for P forms with NMR and enzymatic hydrolysis. The remainder of the sample was freeze-dried.
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Compounds were identified by their chemical shifts (ppm) relative to an external orthophosphate standard. The spectra were processed with NUTS software (Acorn NMR, Livermore, CA, 2000), using automated peak analysis and spectral integration, based on total peak area (Cade-Menun, 2005), after standardizing the orthophosphate peak to 6 ppm. Spectra were processed with 7 Hz and 1 Hz line broadening, using automated baseline-correction and peak-picking routines in the processing software. Peak assignments were based on previous work (Turner et al., 2003b; Cade-Menun, 2005).
The inorganic P forms detected in these samples included orthophosphate (6.0 ppm), pyrophosphate (–4.3 ppm), and polyphosphate (–8 to –22 ppm). Within the organic P forms, peaks for two phosphonates were detected: aminoethyl phosphonates at 20.1 ppm and phosphonolipids at 18.3 ppm (Cade-Menun, 2005). In the orthophosphate monoesters region, peaks were observed for phytate (myo-inositol hexakisphosphate) in most samples. The C2 peak of phytate was clearly visible in all spectra (Fig. 1 ), and was used to calculate total phytate concentration (Turner, 2004). Peaks for other orthophosphate monoesters were seen at 5.2, 5.0, 4.8, 4.7, 4.2, 3.6, 3.6, 3.3, and 2.9 ppm. These compounds may include other inositol phosphates, mononucleotide, and sugar phosphates, and may also include degradation products produced during extraction (Turner et al., 2003b), which were not specifically identified. Orthophosphate diester species were separated into phospholipids (1.7, 1.3, 1.1, 0.0, 0.5, 0.1, and –0.2 ppm), deoxyribonucleic acid (DNA) at –0.7 ppm, and other orthophosphate diesters (–1.0, –1.5 ppm).
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For enzymatic incubations, the diluted and pH-adjusted extracts were further diluted 4- to 15-fold with distilled water or sodium acetate buffer to keep a final sodium acetate buffer concentration of 100 mM at pH 5.0. All enzymatic incubations were performed at 37°C for 1 h in a refrigerator-shaker (250 rpm). Each incubation mixture (1.0 mL) contained 0.8 mL of extract and 0.2 mL of enzyme-buffer working solution (acid phosphatase type IV-S from potato 0.25 U, acid phosphatase from wheat germ 0.25 U, and nuclease P1 5 U mL–1, separately or in combination) (He et al., 2004b). Controls omitting either the enzyme or substrates were included.
Soluble inorganic P in the incubation mixtures was determined by molybdate blue method, using 0.2 mL of each mixture (He and Honeycutt, 2005). This method is modified from an early method (Dick and Tabatabai, 1977) by changing the measuring wavelength to 850 nm and addition of 2% sodium dodecyl sulfate. It is worth noting that this method is developed for accurate determination of orthophosphate, whereas other molybdenum blue methods determine a loosely defined "molybdenum-reactive P" which may include some labile organic P and condensed inorganic phosphate (Dick and Tabatabai, 1977; He et al., 2006a). Thus, in this work, soluble inorganic P determined in the enzymatic hydrolysis was also referred to as orthophosphate for the comparison with the 31P NMR data. Three types of enzymatically hydrolyzable organic P forms were quantified based on previous substrate specificity studies (He et al., 2004b, 2006b): (i) simple monoester P, which was inorganic P released by potato phosphatase alone; (ii) polynucleotide P, which was the difference between inorganic P determined after incubation with potato phosphatase + nuclease P1, and potato phosphatase alone; and (iii) phytate-like P, which was defined as the net increase in inorganic P determined after incubation with potato phosphatase + nuclease P1 + wheat germ phosphatase, compared to inorganic P determined in step (ii). In the HCl extracts, only total monoester P was determined by incubating pH-adjusted extracts with potato phosphatase + wheat germ phosphatase, as a previous investigation demonstrated that monoester P (mainly phytate P) was the major organic P form in poultry litter (He et al., 2006b). Total P in each fraction was determined after persulfate-sulfuric acid digestion (He et al., 2006c). Non-hydrolyzable organic P was calculated as the difference between total P and the sum of other identified P forms as described above.
Statistical Analyses
Genstat 4.2, fifth edition (Lawes Agricultural Trust, Rothamsted, UK) was used to calculate means and standard deviations for the manure samples. The least significant difference (LSD) test using one-way analysis of variance in Genstat 4.2 was performed on the wet or dry manures to test for significant differences (P < 0.05) between P forms in these manures. A correlation coefficient was generated in the Correlation Analysis Tool of Microsoft Excel to compare the two sets of data of NaOH-EDTA extracts yielded by enzymatic hydrolysis and NMR analysis.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND IMPLICATIONSREFERENCES |
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Based on McDowell et al. (2006), it is possible that the delay time used for these NMR experiments was too short, particularly for the poultry manure samples, which had very high ratios of P/(Fe+Mn). However, increasing the delay times to 15 s or more was not practical with respect to machine time or cost. In addition, while McDowell et al. (2006) found that increasing the delay from 0.1 to 15 s for a sheep dung sample improved spectral resolution and altered peak areas, the differences in relative percents was small (1–2%). Given that our delay time (4.3 s) was much longer than the shortest delay time used by McDowell et al. (2006), we assumed that changes in peak areas for our samples with a longer delay time would be similar or smaller.
Drying dairy manures resulted in changes in P forms (Fig. 2 ). For example, drying significantly increased orthophosphate by 560 mg kg–1 or 8.4% and significantly decreased many organic P forms by 8.4% (other monoesters, 7.6%; DNA, 0.5%; other diesters, 0.3%). In contrast, drying poultry manure caused a significant decrease in orthophosphate (2310 mg kg–1 or 13.3%) while the phytic acid and other monoesters increased by 9.6 and 5.3%, respectively (14.9% total increase). These differences appear to be related to the chemical nature of the manures. Because there was no change in P recovery after drying the dairy manure and the increase in orthophosphate was directly proportional to the decrease in organic P forms, drying appears to hydrolyze non-phytate monoesters and some orthophosphate diesters to orthophosphate. These other non-phytate orthophosphate monoesters most likely include sugar phosphates, which are more labile in soils than phytic acid (Condron et al., 2005). McDowell and Stewart (2005) also observed a reduction in labile P forms and increase in the relative proportion of orthophosphate after drying manure from field-grazed cattle, although their P recoveries were lower than ours (57% for fresh and 48% for dried manure).
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Phosphorus Forms Identified in Manure Extracts by Enzymatic Hydrolysis
Using phosphatase hydrolysis, P in NaOH-EDTA extracts was classified into five groups (Table 5). For dairy manure, inorganic P was significantly greater in dry than wet manure, while organic P significantly decreased by drying. However, NaOH-EDTA solution extracted less inorganic P from dried poultry manure than from wet manure. No significant difference was observed in the organic P forms between the dry and wet samples of poultry manure although total organic P was slightly higher in dry (2700 mg kg–1) than wet (2604 mg kg–1) poultry manure. The majority of the hydrolyzable organic P was present in the monoester forms in both manures. However, dairy manure contained almost equal amounts of phytate and simple monoester P. Interestingly, polynucleotide-like P was significantly greater in wet compared with dry dairy manure, which clearly shows that the drying process hydrolyzed polynucleotide P. One possible explanation for this observation is that polynucleotide-like P originated from biomass of microbes present in manures before and/or after excretion whereas other P forms are more or less related to dietary P (Toor et al., 2005). These results are consistent with the P-NMR results, and also suggest that differing responses to drying are due to differences in manure chemistry.
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In the HCl extracts, enzymatic hydrolysis revealed that 6066 mg kg–1 (92%) of the wet poultry manure was inorganic P, 200 mg kg–1 (3%) was monoester P, and 310 mg kg–1 (4.7%) was non-hydrolyzable organic P (Table 5). In the HCl extracts of dry poultry manure, 5303 mg kg–1 (84%) was inorganic P, 973 mg kg–1 (16%) was monoester P, and no non-hydrolyzable organic P was detected. These data indicate that there was considerable inorganic and organic P in both wet and dry poultry manure after NaOH-EDTA extraction. A prior paper (Turner, 2004) recommended NaOH-EDTA extraction as a standard method for complete manure P characterization. Our data imply that researchers must be aware that NaOH-EDTA may not be effective to extract all P in high Ca manure as numerous factors discussed in the Recovery Section would affect the recovery. Therefore, the general applicability of the NaOH-EDTA procedure should be modified to account for the specific conditions of the poultry manures studied.
Comparison and Scope of Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy and Enzymatic Hydrolysis to Identify Phosphorus Forms in Animal Manures
Although not all the P forms identified by the two methods were the same, there were four similar forms: orthophosphate or inorganic P, phytate-like P, other or simple orthophosphate monoesters, and polynucleotide-like or DNA P. There was no significant difference in the amount of inorganic P in wet and dry dairy manure measured via the two methods. However, there was a significant difference between the amount of inorganic P measured in wet and dry poultry manure (Fig. 3
). Total organic P identified by NMR was significantly greater for all manure types than by enzymatic hydrolysis. Phytic acid in dairy manure showed inconsistent trends: no significant difference between the methods for wet manure, but lower phytic acid by enzymatic hydrolysis in dry manure. In poultry manure, enzymatic hydrolysis identified significantly less phytic acid than NMR for both wet and dry extractions. Similarly, enzymatic hydrolysis detected significantly less other monoesters in the extracts of both dairy and poultry manures than NMR. The lower contents of phytic acid and other monoesters with enzymatic hydrolysis suggest that not all these forms were hydrolyzed with added enzymes. The percentage of polynucleotide-like P determined by enzymatic hydrolysis was greater than DNA determined by NMR analysis. This may be attributed to the endonucleotical cleavage of the P-O bonds present in both DNA and RNA by nuclease phosphatase. NP does not directly cleave the P-O bond in Po compounds, but instead endonucleolytically cleaves polynucleotide bonds in RNA and DNA to produce mononucleotides. In addition, commercial nuclease phosphatase contains other phosphatase, such as 3'-AMPase and protein 3'-nucleotidase, as impurities, which may have hydrolyzed other orthophosphate diesters thus causing greater amount of polynucleotides than NMR (He et al., 2004b, 2006c).
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CONCLUSIONS AND IMPLICATIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND IMPLICATIONSREFERENCES |
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Comparison of the two sets of data indicated that the distribution patterns of major P forms in animal manure determined by the two methods were similar with a correlation coefficient of 0.93. Although it is acceptable to compare the information on P distribution obtained by the two methods, some discretion should be applied as NMR generally identified greater amounts of phytic acid and other monoesters than enzymatic hydrolysis. As each method had its advantages and disadvantages, and the P forms detected by the two methods are not always the same due to different identities and/or stability to hydrolysis, we recommend researchers use a method to best fit their specific research goals (i.e., use NMR for more information on P forms or enzymatic hydrolysis for information on bioavailable P fractions) or use both methods to obtain a full spectrum of P characterization.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND IMPLICATIONSREFERENCES |
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REFERENCES |
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