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Published in Agron. J. 96:1188-1195 (2004).
© American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA

PHOSPHORUS MANAGEMENT

Ligands and Phytase Hydrolysis of Organic Phosphorus in Soils Amended with Dairy Manure

Thanh H. Dao*

USDA-ARS, AMBL, BARC-East, Beltsville, MD 20705-2350

* Corresponding author (thdao{at}anri.barc.usda.gov).

Received for publication December 15, 2003.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
Information about orthophosphate and organic phosphorus (P) desorption in soil amended with animal manure is needed because a build-up of P following repeated manure applications poses risks of offsite dissolved P discharges. An enzymatic hydrolysis and soil P desorption study was conducted to develop a novel ligand-based phytase-hydrolyzable P (PHP) method and quantify water-extractable uncomplexed and complexed P forms (i.e., bioactive P) in Unicorn and Christiana soils (Typic Hapludults). Extractability of inorganic P with ligands alone (LEP) increased from 2 and 22 mg kg–1 (0.3 and 3.1 ± 0.16% total P) up to sevenfold with increasing ligand (LIGND) concentrations in the order of CDTA = EDTA > DTPA >> oxalate. The LIGNDs nullified the benefits of using an anion sink in soil P desorption measurements. Soil organic P was hydrolyzed in situ by Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8, corroborating the presence of myo-inositol phosphomonoesters including IP6. Adding CDTA and EDTA increased phytases' efficacy and that of the PHP assay for measuring previously inaccessible complexed organic P; soil PHP increased from 2.3 and 6.4% to peak at 32.0 ± 0.7 and up to 40.8 ± 0.2% of total P in Unicorn and Christiana soils, respectively. In long-term manure-amended Christiana silt loam under orchardgrass–red clover stands, the LIGND-PHP method showed that repeated manure applications resulted in soil storage of unextractable complexed PHP and a buildup of inorganic LEP. Accumulation of LEP increases risks of mobilization of bioactive P in the top 10 cm of these soils.

Abbreviations: CDTA, 1,2-cyclohexane diamino-tetraacetate • DTPA, diethylenetriamine-N, N, N', N'', N''-pentaacetate • EDTA, ethylenediamine-N, N, N', N'-tetraacetate • LIGND, other, non-IP6 organic ligand • IP, myo-inositol phosphate monoesters • IP6, myo-inositol 1, 2, 3, 5/4, 6-hexakis dihydrogenphosphate • LEP, inorganic ligand-extractable P • PHP, phytase-hydrolyzable P • WEP, water-extractable P • ZVMA, zerovalent metal alloy


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
IN AREAS OF INTENSIVE animal agriculture, soils treated with large amounts of manure accumulate and show elevated inorganic and organic P contents (Zhang et al., 2002). Animal manures are major sources of organic P (Peperzak et al., 1959; Gerritse and Eksteen, 1978; Dao, 2003a). The organic P fraction is composed principally of myo-inositol phosphomonoesters (IP), and in smaller proportion, phospholipids and orthophosphate diesters. myo-Inositol hexakis dihydrogenphosphate (IP6) is excreted in animal manure as swine and poultry lack the phytase enzymes to break down IP6 (Harper et al., 1997). Manure from ruminant livestock, thought able to utilize IP6-P because of microbial hydrolytic activity in the rumen and intestinal mucosa, also contains a large phytase-hydrolyzable P pool (Dao, 2003a; Dao et al., personal communication, 2004). The persistence and excretion of IP6 and other IPs in dairy manure are attributed to the protective effect of IP6 complexation with polyvalent cations that reduces dephosphorylation. As a result, substantial amounts of dietary IP6-P of feed grains are excreted, contributing to water pollution rather than animal growth and development.

In soils amended with animal manure, IP6 and other organic P are added to the soil P pool, in addition to inorganic PO4–P. The need for detailed organic P characterization studies is critical as orthophosphate mono- and diesters have been observed to move through soils, posing risks of water quality degradation via soil subsurface transport (Wild and Oke, 1966; Gerritse and Eksteen, 1978; Chardon et al., 1997). myo-Inositol hexakis dihydrogenphosphate and lower phosphomonoesters of inositol are the most abundant identifiable organic P compounds, and may make up to 50% of total soil organic P (Cosgrove, 1962; Wild and Oke, 1966). The phosphomonesters have been identified by 31P nuclear magnetic resonance (NMR) spectroscopy, using alkaline extractants such as 0.5 M NaOH or 0.25 M NaOH–0.05 M Na2EDTA (Newman and Tate, 1980; Cade-Menun et al., 2002; Koopmans et al., 2003). Although such methods yield structural identity and chemical classes information, current NMR techniques measure total orthophosphate monoesters and diesters in the soil extracts. Alterations in chemical form may occur because of the high alkalinity of the extraction medium, i.e., hydrolysis of orthophosphate diesters (Leinweber et al., 1997).

Extracellular enzymes have been used in the characterization of organic P because substrate specificity and variety of phosphatases provide an analytical approach to determining organic P forms in animal manure (He and Honeycutt, 2001; Dao, 2003a), soil extracts (Hino, 1989; Shand and Smith, 1997), and leachates (Toor et al., 2003). Current enzymatic methods have been hampered by a relatively low recovery of soil P (Shand and Smith, 1997; Hayes et al., 2000); for example, Hayes et al. (2000) reported <2% of soil total P (6.2 mg kg–1) was desorbed and hydrolyzed in a 50-mM citric acid extract and much less (0.3 mg kg–1) in a water extract. Soil P recovery depended upon the solvent strength as well as the form of the extracted organic P because most reported enzymatic methods were based on a specific soil extraction scheme. Subsequently, various phosphatase enzymes were added to the extracts to hydrolyze P-containing compounds, yielding orthophosphate as a measure of the parent compound content in soil.

The enzymatic intermediates and end products of IP6 dephosphorylation are also highly reactive, with many biogeochemical sinks in soils contributing to the low recovery of hydrolyzed P. Alternative ion sinks of various chemical nature have been used to measure the labile P fraction in soil following manure additions (Abrams and Jarrell, 1992), P fertilizer application (van Diest et al., 1960), or to determine the soil microbial P fraction (Myers et al., 1999). Ion sinks materials have included ion-exchange resins (Amer et al., 1955) and iron oxide–impregnated paper (Sharpley, 1993). Although these materials successfully quantified the soil labile P pool, their retention capacity or the saturation point of exchange sites is relatively limited.

Complexed and insoluble inositol phosphates are relatively resistant to enzymatic hydrolysis as complexed and polymeric compounds are formed via inter- and intra-molecular bonding with polyvalent cations, limiting the availability of these substrates to phytases (Dao, 2003a). Organic anions were reported to facilitate the enzymatic release of PO4–P in Spodosols; differences in efficiency of ligand exchange existed between organic anions, as oxalate was more effective than formate, for example (Fox et al., 1990). Frequently studied organic anions have been low molecular weight, naturally occurring aliphatic organic acids (Fox and Comerford, 1990; Jones, 1998). Large polydentate ligands (i.e., CDTA, EDTA, and DTPA) were found to decouple IP6 and associated counterions via a ligand exchange process (Dao, 2004). These LIGNDs, thereby reduced the inhibitory effect of Ca2+, Al3+, and Fe3+ on the dephosphorylation of IP6 in dairy manure suspensions. Whether these ligands function in a similar manner to increase the recovery of soil IP-P with phytase enzymes in manure-amended soils is the subject of this study; and if the ligands are effective, could a more accurate measurement of the organic PHP fraction be obtained when used with an anion sink for the PO4–P released to the soil suspension? A strong or irreversible sink is needed to compete against the high affinity of the soil mineral phase for PO4–P and estimate the true size of the pool of organic enzyme-hydrolyzable P. Therefore, this study was conducted with the objectives of (i) evaluating alternative ion sinks for PO4–P in soils for an in situ soil P desorption and PHP analytical procedure, and (ii) determining the effects of LIGNDs, ion sinks, and dairy manure management practices on various fractions of P in soil that are present as water-extractable and insoluble complexed P in orchardgrass–red clover stands.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
The soils used in the study included a Christiana silt loam (fine, kaolinitic, mesic Typic Paleudult) from an orchardgrass (Dactylis glomerata L.)–red clover (Trifolium pratense L.) pasture (Prince George's County, Maryland) that had received no dairy manure for the last 7 yr, and an Unicorn sandy loam (coarse-loamy, mixed, semiactive, mesic Typic Hapludult) from a farm located in Queen Anne County, Maryland. The field was cropped to a soybean [Glycine max (L.) Merr.]–wheat (Triticum aestivum L.) rotation the year of sampling. Bulk samples were collected from the 0- to 10-cm depth, air-dried, and crushed to pass a 2-mm sieve before P analyses. Selected soil properties are presented in Table 1.


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  		Table 1. Selected properties of the soils used in the experiments.

 
Additional samples of the Christiana soil were collected from a field study located on a Christiana–Beltsville soil association. The replicated field plots (ca. 2.5 ha each) were managed as permanent orchardgrass–red clover stands that received annual applications of dairy manure at rates of 0, 15, and 30 kg P ha–1 in the spring since 1996. Core samples were obtained from the 0- to 10-cm depth, air-dried, crushed, passed through a 2-mm sieve, and stored at 4°C until determination of various bioactive P fractions.

Ion Sinks
Sorbent materials that were evaluated as anion sinks included a zerovalent aluminum–copper alloy (ZVMA) and a waste stream by-product of the manufacture of synthetic zeolite at the W.R. Grace, Curtis Bay plant, near Baltimore, MD. Bulk samples of the wet filter cake were obtained from the dewatering process of the manufacturing plant's waste stream. The ZVMA was obtained from Sigma-Aldrich Chemicals (St. Louis, MO).

Phosphorus retention capacity of the sorbents was determined by equilibrating each material with known volumes of standard solutions of PO4–P and IP6-P, ranging in concentrations between 0 and 10 mM. Solutions (1:20, w/v) of PO4–P or IP6-P were added to the tubes containing 0.5-g samples of the sorbents. The mixtures and P standard solutions were agitated for 16 h on a gyratory shaker at 20°C. After centrifugation at 7000 x g for 15 min, aliquots of equilibrium solutions containing IP6 were hydrolyzed by phytase EC 3.1.3.8 to release PO4–P. Then, PO4–P concentrations of all supernatant solutions were determined by the phosphomolybdate–ascorbic acid method (Am. Public Health Assoc., 1998). The amount of sorbed PO4–P or IP6-P was calculated as the difference between amounts of P in control tubes containing no sorbent and those in tubes containing sorbents at the end of the equilibration period (Dao, 2003b).

The sorbents in a powder form were embedded in a polyacrylamide gel, following the polymerization reaction (O'Farrell, 1975). Cylindrical capsules of 15 mm in diameter and 20 mm in height were formed. Total P analysis of spent sorbent capsules was performed using a modified potassium persulfate digestion procedure (Nelson, 1987; Bowman, 1989). In summary, reagent-grade K2S2O8 was added to the sorbent capsules along with 10 mL of 5.5 M H2SO4. The mixture was brought up to a boil at 180°C for 0.5 h and the temperature of digestion was raised and maintained at 350°C for another 1 h. Deionized water was added to dilute to a final volume of 35 mL. Total P was determined according to the phosphomolybdate–ascorbic acid procedure (Am. Public Health Assoc., 1998). Total acid-digest P content of soil samples was determined similarly.

Water-Extractable Phosphorus
Water-extractable P concentrations were determined in soil–water suspensions with and without sorbent capsules. Deionized water was added to polycarbonate jars containing 0.7-g samples of each soil to attain a soil/solution ratio of 1:100 (w/v). The suspensions were agitated at 250 rpm for 1 h at room temperature. Aliquots of the solution-phase were centrifuged at 7000 x g, and the supernatant P concentration determined by a high-performance liquid chromatographic (HPLC) method for anionic species (Erkelens et al., 1986). A Waters 2690 LC system, equipped with UV (Model PDA 996) and electrical conductivity (Model W432) detectors (Waters Corp., Milford, MA) was used. An anion-exchange column (IC-Pak HC, Waters Corp., Milford, MA) and pre-column were used to separate and quantify PO4–P. The eluent was a borate–gluconate (2%)–acetonitrile (12%) solution, adjusted to pH 8.5, and pumped at a flow rate of 1.5 mL min–1.

Inorganic Ligand–Extractable Phosphorus
Polydentate ligands studied included CDTA, DTPA, EDTA, and oxalate. The acid forms of the LIGNDs were converted to the Na+ forms by titration with 10 M NaOH before preparing the LIGND stock solutions (pH 4.5). All other reagents were also adjusted to pH 4.5. Aliquots of 5 mM stock solutions of the LIGNDs were added to polycarbonate jars containing deionized water and 0.7-g samples of soil to obtain LIGND concentrations of 0, 0.05, 0.15, 0.3, and 0.5 mM and a final soil/solution ratio of 1:100 (w/v). Soil suspensions LEP concentrations were determined with and without ZVMA capsules to determine the soil complexed inorganic P pool. The mixtures were agitated at 250 rpm for 2 h to minimize the hydrolysis of any co-extracted organic P species. Aliquots of the solution-phase were centrifuged at 7000 x g, and the supernatant P concentration determined by liquid chromatography as previously described.

Phytase-Hydrolyzable Phosphorus
The soil PHP fraction was determined in the same soil–EDTA suspension used to measure soil LEP concentrations. The 2-mL volume of supernatants removed for the measurements of LEP was replaced with 2 mL of a phytase EC 3.1.3.8 stock. Aliquots of were added to attain a final enzyme activity of 0.05 unit mL–1. The soil–enzyme aqueous mixtures were agitated on a gyratory shaker at 250 rpm at 20°C. After a 24-h equilibration period, solution-phase concentrations of PO4–P were determined after boiling an aliquot of the solution-phase in a 100°C water bath for 10 min and centrifugation at 7000 x g for 15 min. Aliquots of the supernatant were used to determine P concentrations by liquid chromatography as previously described.

The soil P batch desorption experiments were established according to a randomized complete block design with LIGND and LIGND levels replicated three times. Differences in treatment main effects and interactions were detected following analysis of variance and the Duncan multiple range test at the 0.05 probability level using the Statistical Analysis System (SAS Inst., 1996).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
SUMMARY AND CONCLUSIONS
 REFERENCES
 
Phosphate Ion Sinks
Phosphate sorption onto ZVMA reached an initial maximum (Smax = 45 ± 0.6 mmol kg–1) but then Ce/S increased rapidly with Ce exceeding 2 mM (Fig. 1A) . This behavior was indicative of P removal from solution by a precipitation reaction, accounting for the steep slope of the Langmuir isotherm at Ce > 2 mM. Phosphate sorption on the synthetic zeolite by-product (Smax = 51 ± 6.3 mmol kg–1), along with ZVMA, were best described by the Langmuir sorption model:

where Ce = solution-phase P (mmol L–1), S = sorbed-phase P (mmol kg–1), b = sorption maximum (mmol kg–1), and Kb = constant related to bonding energy (L mmol–1).



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  		Fig. 1. (A) Retention of orthophosphate and (B) myo-inositol 1, 2, 3, 5/4, 6-hexakis dihydrogenphosphate on two ion sink materials as described by the Langmuir sorption model. Error bars represent standard deviation (n = 6).

 
Phytic acid sorption on ZVMA also best fitted the Langmuir model, with a Smax of 3 mmol kg–1. It was strongly sorbed onto the synthetic zeolite by-product without reaching a maximum (Fig. 1B). The two materials made effective ion sinks for both P forms; however, we were most successful at embedding ZVMA uniformly in a polyacrylamide gel. The formed capsules were stable and contained no initial P. The ZVMA capsule was a practically infinite sink for dissolved P in the two soils (Fig. 2) . Recovery of solution-phase WEP was approximately two and six times larger when ZVMA was used alone in the control soil–water–ZVMA mixture of Christiana or Unicorn soils, respectively. The ZVMA proved useful in assessing dissolved PO4–P desorption from mineral soils, particularly in soils of low P saturation index. Because of the high reactivity of inorganic orthophosphate, the many biochemical sinks for PO4–P in soil would lead to the underestimation of the quantity of WEP desorbed from the soil exchange as well as that derived from the hydrolysis of soil organic P forms. The ZVMA functions as a sink that binds soluble P, in a manner similar to other anion sinks used in the literature (for example, FeCl3 impregnated on filter paper). In this case, the surface aluminum- and zinc-hydroxyl groups coordinatively interact with phosphate to sequester the anion. The low water solubility of aluminum phosphate, for example variscite (AlPO4·2H2O) (log K25°C = –2.5) and zinc phosphate, for example hydrated hopeite (Zn3[PO4]2·4H2O) (log K25°C = 3.8) would favor phosphate sorption onto the ZVMA and the formation of solid phosphates (Lindsay, 1979).



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  		Fig. 2. Effects of zerovalent aluminum–copper alloy (ZVMA), organic ligands, and ZVMA-ligand combinations on desorption of inorganic complexed P in a Unicorn sandy loam and Christiana silt loam. Error bars represent standard deviation (n = 12).

 
Ligand Effects on Soil Phosphorus Desorption
In both soils, adding EDTA, CDTA, and DTPA increased desorbed P concentrations of the solution phase, over those of the control (no-LIGND) treatment (Fig. 2). Oxalate did not appreciably change the extractability of soil P by water, at concentrations up to 0.5 mM. Fox et al. (1990) observed low desorption of P from an Ultic Haplaquod in a 0.1 mM oxalate extracting solution, being no greater than in water or a 1.0 mM formate solution; then, soil P desorption increased 10-fold when they used a 1.0 mM oxalate extracting solution. Oxalate attained a critical solution concentration and became more effective at exchanging with phosphate. In our study, EDTA, CDTA, and DTPA very effectively desorbed soil P by maintaining exchangeable P in solution upon complexing and sequestering polyvalent cationic species such as soil Ca2+, Mg2+, or Al3+. The LIGNDs dissolved near-edge bound calcium, magnesium, or aluminum phosphates, forming coordination complexes with the released metals preventing reprecipitation of P (Lan et al., 1995; Dao, 2003a). The observed increased desorption of soil P in Unicorn and Christiana is similar to those induced by organic anions in the release of P from Al–P surfaces in kaolinite and forested or cultivated Spodosols (Nagarajah et al., 1968; Fox and Comerford, 1990).

These LIGNDs also induced the decoupling of polyvalent cations and IP6, hydrolysis, and release PO4–P from the resulting uncomplexed form, including the other five inositol phosphomonoesters (Dao, 2004). Because HPLC was used to quantify solution-phase PO4–P, the increase in desorbed P was associated with the mobilization of complexed inorganic phosphates. The ZVMA appeared to work in conjunction with all four ligands to sequester desorbed P in Unicorn soil. However, the effects of ZVMA and LIGNDs were not complementary or additive. In fact, more soil P was desorbed when CDTA was used alone in the Christiana soil. It was postulated that these anthropogenic LIGNDs interfered with the binding of PO4–P onto ZVMA. While ZVMA performed well alone or with oxalate in measuring soil P desorption, the three synthetic LIGNDs competed with dissolved P for adsorption sites and nullified the benefits of using the phosphate sink with these strong LIGNDs. Any ZVMA-bound P would be re-mobilized via a ligand exchange.

Desorbed LEP increased with increasing LIGND concentrations when used by themselves, in the order of CDTA = EDTA > DTPA >> oxalate (Fig. 3) . Both EDTA and CDTA induced the highest level of desorption of soil LEP at concentrations ≥0.3 mM, yielding four to seven times more soil P (20 and 80 mg kg–1) than deionized water (2.3 and 22 mg kg–1) in Unicorn and Christiana soils, respectively. The difference in EDTA and CDTA efficacy to mobilize complexed P was observed in previous studies of IP6 dephosphorylation in dairy manure (Dao, 2004). The difference was associated with pH and its effect on LIGND speciation and resulting charge concentrations in the equilibrating solution phase. The fraction of major anionic species of organic ligands varied with solution pH based on their thermodynamic dissociation constants. Examples of computation of LIGND speciation showed that CDTA exists primarily as CDTA– (8.9%) and CDTA2– (88.8%) at pH 4.5 (Dao, 2004). Meanwhile, EDTA is fully dissociated and 96.5% is present as the trivalent EDTA3– at pH 4.5 and at a higher charge concentration than CDTA. Although a slight pH difference exists between soils (Table 1), EDTA was more effective at exchanging for LEP than CDTA in Unicorn soil while CDTA was the more effective ligand in Christiana soil. No clear cause was found to explain this difference between the soils. Differences may exist in the type and concentration of soil cations, and the degree of base saturation of the soils to affect the ligand exchange process.



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  		Fig. 3. Extractability of inorganic complexed P as affected by concentrations of organic ligands in Unicorn and Christiana soils. Error bars represent standard deviation (n = 3).

 
Ligand Effects on Soil Phytase-Hydrolyzable Phosphorus
Both Christiana and Unicorn soils have a large P fraction that was hydrolyzed by phytase EC 3.1.3.8 (Fig. 4) . In soil suspensions amended with phytases alone without any added LIGND, PHP concentrations increased from 2 ± 0.8 to 12 ± 0.2 (2.3% of total P) and 22 ± 0.8 to 46 ± 1.8 mg kg–1 (6.4%) in the Unicorn and Christiana soils, respectively. The use of an enzymatic system showed that the source substrates were biologically active or susceptible to biodegradation. Furthermore, the phytase-based assay corroborated the presence of myo-inositol phosphomonoesters that include IP6 in the organic P fraction of these soils.



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  		Fig. 4. Effects of organic ligands on the recovery of phytase-hydrolyzable P fraction in Unicorn and Christiana soils. Error bars represent standard deviation (n = 3).

 
Adding the three anthropogenic ligands, CDTA and EDTA in particular, significantly enhanced the PHP assay's effectiveness; the LIGND–PHP fraction peaked at about 32.0 ± 0.7 and reached upward of 40.8 ± 0.2% of soil total P in Unicorn and Christiana soils, respectively. The increased hydrolysis and release of solution PO4–P translated to about a 2- and 15-fold increase in the recovery of soil total P, respectively. The LIGNDs decoupled organic P and their counterions to cause organic P forms to be more accessible to phytase enzymes. Previous work has shown that it was the concentration of IP6, decoupled from polyvalent counterions, that was the limiting factor in the dephosphorylation of the organic P, and not the activity of the enzymes nor the lack of inositol phosphate substrates (Dao, 2003a). The LIGND–PHP procedure was even more effective in the fractionation of P in organic matrices, accounting for 69 ± 14.6% of the total P content of a diverse set of 107 dairy manure samples (Dao, 2004; Dao et al., personal communication, 2004). Although the highest recovery of soil bioactive P was about 40% of soil total P, the procedure recovered a greater proportion than the Mehlich-3 method (Fig. 4 and Table 2). Further work to relate bioactive P to plant or other biological responses of major biosystems remains to be done and to validate a biological index.


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  		Table 2. Selected soil P fractions in the 0- to 10-cm depth of Christiana silt loam following seven annual dairy manure applications to a permanent orchardgrass–red clover stand.

 
Organic ligands were observed to increase the release of P to the soil solution, from 10 to 30% of soil total P in the A horizon and 30 to 70% in the Bh horizon of an Ultic Haplaquod (Fox et al., 1990). To our knowledge, no other enzymatic methods of soil P were in situ hydrolysis methods, and they only rely on soil extraction and treatment of the extracts. No direct comparison of the results of this study to others is made because the soil-water extractable P fraction is a small fraction of soil total P. However, free and immobilized phosphatases have been shown to hydrolyze between 40 to 75% of extracted P in soil aqueous extracts (Pant and Warman, 2000) and up to 88% of the soil leachate P under an Udic Haplustept treated with dairy wastewater is enzyme-labile (Toor et al., 2003).

In this study, in situ measures of the organic PHP fraction in these two soils were within limits of reported estimates of soil organic P released in 0.5 M NaOH or 0.25 M NaOH–0.05 M Na2EDTA extracts (Newman and Tate, 1980; Cade-Menun et al., 2002). The correspondence of the enzyme-labile PHP fraction with alkaline-extractable P warrants further investigation. However, the differences in harshness of the two extraction solutions made little difference in their ability to extract soil organic P. The LIGNDs have the advantage of being able to decouple and replace organic P forms sorbed on mineral surfaces, in intra-aggregates, or inter-laminar space of soil particles (Stumm, 1986). They induce dissolution of near-surface metal phosphates and orthophosphate monoesters by coordinative bonding to compensate for the reduced ability to dissolve the soil organic C matrix. In addition, the LIGNDs maintain mobilized P in solution by forming stable complexes with polyvalent cations. This solution-phase sequestration mechanism prevents the re-immobilization of the released P by reactive surface functional groups of soil particles such as silanol groups and metal hydroxyl groups of these mineral soils.

Soil Bioactive Phosphorus following Repeated Applications of Dairy Manure
Manure amendment and loading rates influenced soil chemical properties and desorption of soil bioactive P fractions (Fig. 5) . In a permanent grass stand, addition of dairy manure to the soil surface over the years increased soil total organic C, storing manure C in the 0- to 10-cm depth of the Christiana soil (Table 2). In addition, manure P accumulated in the near-surface zone. There were significant increases in soil total P, but also in WEP and Mehlich 3-P (i.e., plant-available P) fractions, compared with those of the untreated soil. These levels were characteristic of high-P soils that pose risks of impairment of the quality of surface waters (Zhang et al., 2002). The potential for offsite P loss and the adverse environmental impacts are directly related to the P buildup.



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  		Fig. 5. Bioactive P relative concentrations in the 0- to 10-cm depth of Christiana silt loam in an orchardgrass–red clover stand after seven annual surface applications of dairy manure at three levels of P.

 
At the 0.5 mM concentration, EDTA and CDTA, followed by DTPA, desorbed large quantities of bioactive P in untreated and manure-amended soils, between 22 and 40.8%, compared with 6.6 and 8.6% of soil total P by water alone and the 0.5 mM oxalate solution, respectively. The LIGND x manure rate interaction was statistically significant and only the three anthropogenic LIGNDs were able to separate the effects of manure loading rate, showing that the 30 kg ha–1 treatment was significantly different from the 0 and 15 kg ha–1 treatments.

The increase in solution-phase P following the addition of phytase EC 3.1.3.8 with each of the anthropogenic ligands, and to a smaller extent with oxalate, corroborated the feasibility of using the LIGND-PHP method for determining the bioactive P fraction and the impact of dairy manure management on soil P (Fig. 6) . The procedure yielded 8 to 15 times more bioactive P with LIGNDs than without added LIGND; and it was able to differentiate the effects of manure loading rate. The method also yielded an insight into the distribution of accumulated P forms that are water-desorbable and potentially bioactive. Upon normalizing WEP, LEP, and PHP to the total bioactive P fraction for each LIGND, the experimental results suggested that the LIGND mobilized complexed inorganic phosphates, increasing LEP from almost nothing to between 20 and 30% of the total extracted bioactive P. The mobilization of this LEP pool is controlled by the solubility of mineral phosphates that have a wide range of solubility characteristics (i.e., Ksp) as these compounds are likely in a metastable solid state. The PHP also made up a greater proportion of the desorbed P (48–55%) as LIGNDs mobilized additional hydrolyzable organic P from a pool of complexed organic P forms that was inaccessible to phytases.



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  		Fig. 6. Distribution of the water-extractable inorganic and phytase-hydrolyzable P fractions of the 0- to 10-cm depth of Christiana silt loam in an orchardgrass–red clover stand after seven annual surface applications of dairy manure at three levels of P.

 
Across manure rate treatments, the proportion of WEP, LEP, and PHP appeared to shift with an increase in LEP and a slight decrease in the percentage of PHP for the 30 kg P ha–1 treatment, in the EDTA and CDTA extracts. This buildup of LEP likely resulted from a direct addition of PO4–P native to manure. While soil PHP concentrations continued to increase with increasing manure loading, the decline in their proportion of total bioactive P occurred because organic P added with manure was either (i) continually being dephosphorylated and sequestered as complexed mineral LEP, or (ii) increasingly unextractable by water or cannot be mobilized by even these strong LIGNDs to be dephosphorylated by extracellular phytases, or phosphatases in general. The latter reason is a more plausible scenario as sequestration and storage of organic PHP, in particular IP6, can come about because of the high insolubility of complexed forms and resistance to hydrolysis by phytases (Dao, 2003a).


   SUMMARY AND CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
REFERENCES
 
Desorption of soil WEP forms plays an important role in plant nutrition, transfer to runoff, and potential environmental transport, particularly in manure nutrient-laden soils. Enzyme hydrolysis and desorption experiments showed that soils amended with manure P have a large quantity of water-extractable uncomplexed and complexed forms that are referred to as bioactive P. The LIGND-based method is simple to use, yielding valuable insights into the distribution of bioactive P forms that are water-desorbable. In addition to increasing the desorption of inorganic LEP, the LIGNDs mobilized additional enzyme-hydrolyzable P from a pool of complexed organic P forms that was previously inaccessible to phytase enzymes. In long-term manure-amended soils under a permanent orchardgrass–red clover stand, the LIGND-PHP method showed that soils treated with large amounts of animal manure accumulated inorganic and organic P. While soil PHP concentrations continue to increase with manure loading, the results suggested that added organic P was increasingly unextractable and was not mobilized by EDTA or CDTA to be dephosphorylated by extracellular phytases. This decrease in the PHP proportion of extracted P indicated a shift in the partitioning and storage of these P forms in heavily manured soils. Therefore, the practice of repeated applications of animal manure to permanent pastures and grasslands caused an accumulation of soil P in the near-surface zone. The use of an enzymatic method showed that the organic P substrates were biologically active, and land application of this class of P compounds added to the risk of mobilization of bioactive P and transfer to runoff water during rainfall. The simplicity of the LIGND-PHP procedure may increase the practicality of widespread measurements of composition and P bioavailability in soil to rapidly develop mitigation practices to detect soil conditions favorable to and prevent potential offsite discharges of bioactive P from high-P soils or soils amended with animal manure.


   ACKNOWLEDGMENTS
 
The author sincerely acknowledges the technical assistance of Guy Stone and Mai Le during the conduct of the study.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 
The mention of trade or manufacturer names is made for information only and does not imply an endorsement, recommendation, or exclusion by the USDA-ARS.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 REFERENCES
 




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