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Journal of Environmental Quality 32:694-701 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Waste Management

Polyvalent Cation Effects on myo-Inositol Hexakis Dihydrogenphosphate Enzymatic Dephosphorylation in Dairy Wastewater

Thanh H. Dao*

USDA-ARS, Animal Manure and By-Products Laboratory, BARC-East, 10300 Baltimore Ave., Beltsville, MD 20705

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

Received for publication March 11, 2002.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Information is needed on organic polyphosphates such as myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate or phytate (IP6) contribution to the sources and sinks of dissolved phosphorus (PO4–P) in the soil–manure–water system. Effects of Na+, Ca2+, Al3+, and Fe3+ and cation to IP6-P mole ratios on the enzymatic dephosphorylation of IP6 were studied to determine controlling mechanisms of dephosphorylation and persistence in manure. Phytate- and PO4–P were analyzed by high-performance liquid chromatography. Phytate dephosphorylation by Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8 decreases by 50 ± 3.6 and 40 ± 4% at pH 4.5 and 6, respectively, as Ca2+ concentrations increase and cation to IP6-P mole ratios reach 6:6. Polyanionic IP6 has a high affinity for Al3+ and Fe3+ and reductions in dephosphorylation average 27 and 32% at a cation to IP6-P mole ratio of 1:6 for Al3+ and Fe3+, respectively, while reaching more than 99% at a mole ratio of 6:6. A phytase-hydrolyzable phosphorus (PHP) fraction is native to ruminant animal manure and is proportional to total solids (TS) concentration in 1 to 100 g L-1 suspensions. Added phytase, in effect, increases water-extractable P content of manure and the risk of environmental P dispersion. As the bioavailability and ecological effect of IP6-P appear to be regulated not only by pH-controlled enzyme activity but also by the associated counterions, the differential protective effects of cations influence the accuracy of manure PHP fraction estimates and increase phytate resistance to enzymatic dephosphorylation that may lead to its persistence in manure.

Abbreviations: IP6, phytate • PHP, phytase-hydrolyzable phosphorus • TS, total solids


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
PHYTIC ACID (myo-inositol-1,2,3,5/4,6 hexakis dihydogen phosphate) typically accounts for 60 to 90% of the stored reserves of P and myo-inositol in grains that include wheat (Triticum aestivum L.), rice (Oryza sativa L.), corn (Zea mays L.), soybean [Glycine max (L.) Merr.], and mung bean [Vigna radiata (L.) R. Wilczek var. radiata] (Scott and Loewus, 1986). Developing seeds synthesize and store large quantities of calcium, magnesium, and potassium phytate, together referred to as phytin. These salts are typically stored in the aleurone layer, scutellum, cotyledon, or endosperm (Bergman et al., 2000). Phytic acid is also found in fibers and other organs of reproduction such as mature pollen of a wide range of plant species (Jackson et al., 1982). Forages and grains are fed to livestock in various proportions, depending on stages of growth and energy requirements of their life cycle and production goals. Rumen microflora and intestinal mucosa phytases (myo-inositol hexakis dihydrogenphosphate phophohydrolase) catalyze the hydrolysis of feed grain and fiber IP6 to myo-inositol and PO4–P, and P absorption occurs primarily in the small intestine. However, swine (Sus scrofa domesticus) and poultry (Gallus gallus) lack the phytase enzymes to break down IP6 and excrete most of this form of organic polyphosphate (Harper et al., 1997; Zyla et al., 2000). Ruminant livestock (Bos taurus), thought to be able to utilize IP6-P because of microbial hydrolytic activity in the rumen, can excrete a large portion of IP6-P depending on animal performance levels. As a result, substantial amounts of P stored in grains are essentially not available to the animal and contribute to water pollution rather than animal productivity (Jongbloed and Kemme, 1990; Cromwell et al., 1993).

Phytate contribution to sources of environmental PO4–P and eutrophication through runoff and soil erosion from manure-amended soils may be environmentally significant should IP6 persist in animal excreta. Remediation strategies or technologies to reduce excessive P levels in manure and P-loaded soils depend on our understanding of the dephosphorylation of organic polyphosphates of fiber and grains that are the major components of manure. Increasing interest on post-excretion treatments has been shown in strategies to chemically bind or remove suspended particulate and dissolved P in manure before it is applied to fields (Dao, 1999; Codling et al., 2000; Dao et al., 2001; Smith et al., 2001; Dao and Daniel, 2002). Many agricultural fields in the United States near confined animal feeding operations (CAFOs) are loaded with nutrients, in particular P (Zhang et al., 2002). The runoff and eroded soils from those fields carry dissolved and particulate nutrients to water bodies even if no additional manure is applied.

The six HPO2-4 moieties give the IP6 molecule the potential of 12 coordinate ligands for complexing cations. Phytate complexes monovalent cations and stoichiometrically forms monomeric salts such as sodium IP6 (Pierce, 1985). With polyvalent cations, the possibility of inter- and intramolecular bonding exists and results in the formation of both monomeric and polymeric compounds (William and Pierce, 1982). The polymeric compounds have high molecular weight and are probably insoluble in aqueous solutions and wastewater. The extent to which polyvalent cations influence IP6 physical state depends on (i) the cation, (ii) cation concentration or mole ratio of cation to IP6-P, and (iii) the interaction of other cations present (Vohra et al., 1965). The biological dephosphorylation of organic P should be well understood because IP6 could act as a reserve pool to control the solution concentration of dissolved P in manure suspensions or in manure-amended soils. Manure treatments to sequester dissolved P will be more effective when the exact proportions of inorganic to organic P in manure under different production methods and feeding regimes are well characterized. By the same token, biological nutrient reduction strategies and recovery of PO4–P by precipitation in animal manure, such as the crystallization of struvite (Mg–NH4–PO4) (Greaves et al., 1999) or the reversible sorption by zeolite-based anion exchangers (Dao, 2003) requires a nearly complete release of orthophosphate. Therefore, the improved knowledge of the control mechanisms of enzymatic dephosphorylation of organic polyphosphates will allow the optimization of biological and environmental conditions to ensure the success of such a remediation approach. The objectives of this study were to (i) develop an improved understanding of the dephosphorylation of IP6 by extracellular phytase and (ii) evaluate the effects of counterions to polyanionic IP6 and particulate content of dairy wastewater on dephosphorylation to determine controlling mechanisms of IP6 persistence in manure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
NOTES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sodium IP6 isolated from corn and preparations of fungal phytase (3-phytase EC 3.1.3.8; CAS no. 37288-11-2) isolated from A. ficuum were obtained from Sigma Chemical1 (St. Louis, MO). The phytase preparation specific activity was 3.5 units mg-1, where one unit liberates 1 µmol of PO4–P from 4.2 x 10-2 M IP6 per minute at pH 2.5 and 37°C.

The effect of solution pH on enzyme activity was determined with 100-mL aliquots of a solution of 1.5 mM IP6-P. The pH of these aliquots was adjusted with a 5.5 M HCl or 10 M NaOH solution to achieve IP6 solutions with pH ranging from 2 to 10. Aliquots of a 3-phytase EC 3.1.3.8 stock were also added to attain a final enzyme activity of 0.05 units mL-1. The jars containing the mixture were agitated on a gyratory shaker (250 rpm). Five-milliliter aliquots of the IP6 and enzyme mixtures were then periodically taken to determine IP6-P and PO4–P concentrations. The dephosphorylation reaction was stopped by adjusting the solution pH to 8 and boiling in a 100°C water bath for 10 min. Each pH treatment was replicated three times.

Effect of Counterions and pH
The effects of Na+, Ca2+, Al3+, and Fe3+ on the dephosphorylation of IP6 were determined in polycarbonate jars containing 100-mL aliquots of a stock solution of sodium IP6 in a pH 4.5 acetate buffer. Appropriate volumes of 5 mM stock solutions of CaCl2, AlCl3, and FeCl3 and deionized water were added to attain cation to IP6-P mole ratios of 1:6, 3:6, and 6:6. An additional set of cation–IP6-P samples where solution pH was adjusted to pH 6 was also prepared to measure the effects of cation solubility or the effective concentrations of dissociated Ca2+, Al3+, and Fe3+ on the dephosphorylation of IP6. Five-milliliter aliquots were periodically taken at t = 0, 15, and 30 min, 1, 3, and 6 h, and periodically up to 5 d after enzyme addition. Solution concentrations of IP6-P and PO4–P were determined after adjusting solution pH to 8 with a 10 M NaOH stock solution and boiling in a 100°C water bath for 10 min. Each treatment was replicated three times.

Effects of Total Solids and Counterions in Manure Suspensions
Bulk batches of reconstituted dairy manure were prepared from freshly collected feces and acidified urine from Holstein cows housed in free stalls on the USDA-ARS Beltsville Dairy Research Facility (Dao and Daniel, 2002). The ratio of feces to urine was 1.6 to 1. The reconstituted manure was homogenized in a blender at 2500 rpm and autoclaved at 100 kPa and 120°C for two 20-min sessions to deactivate native phytase enzyme activity. The pH was adjusted to 4.5 before the initiation of the study. Tap water was added to prepare manure suspensions containing from 1 to 100 g L-1 of total solids. Three mole ratios of Ca2+, Al3+, and Fe3+ to IP6-P were established as previously described. The final pH of the suspension was readjusted to pH 4.5 with a 5.5 M HCl solution. Five-milliliter aliquots were periodically taken at t = 0, 15, and 30 min, and periodically up to 5 d after enzyme addition.

The aliquots were re-acidified to pH 2 before P analysis to dissolve all forms of PO4–P. Dissolved P concentrations of suspension aliquots were determined chromatographically with a Waters 2690 LC system, equipped with UV (Model PDA 996) and electrical conductivity (Model W432) detectors (Waters Corporation, Milford, MA). An anion-exchange column (IC-Pak HC; Waters) 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. Electrical conductivity was used to detect and quantify solution PO4–P concentrations. Phytate P was analyzed by reverse-phase pair-ion chromatography with a 3.9 x 150 mm Nova-Pak C-18 column (Waters) according to a procedure described by Sandberg and Ahderinne (1986), with the exception that concurrent UV detection at 254 nm and electrical conductivity were used to quantify IP6-P.

Dephosphorylation experiments in buffers and manure wastewaters were established according to a randomized complete block design with cation to IP6-P mole ratio treatments replicated three times. Differences in treatment main effects and interactions were detected following analysis of variance and the Tukey HSD test at the 0.05 probability level with the Statistical Analysis System (SAS Institute, 1989). Phosphate P concentrations as a function of time elapsed since enzyme addition were fitted to kinetic models using regression and least-square minimization curve fitting methods (TableCurve 2D; Jandel Scientific Software, 1996). The comparison of linearized first-order kinetic equation parameters (i.e., the slope or the kinetic rate constants k and Cmax for each cation and pH treatment) was also made according to SAS procedures outlined in a previous work (Dao et al., 1982).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Molybdate-Reactive Phosphorus versus Dissolved Phosphorus by Anion Chromatography
In preliminary dissolved P measurements in mixed solutions of PO4–P and IP6-P, we observed that molybdenum-reactive P determination by colorimetry appeared to overestimate dissolved P in the presence of IP6 (Table 1). In a series of PO4–P and IP6-P standard solutions in the concentration range normally encountered in the calibration curves for molybdenum-reactive P (American Public Health Association, 1998), we found that the greater the sample IP6 concentration, the greater the deviation from the calculated target PO4–P concentration (Table 1). Relative errors of measurements also were higher at the low PO4–P concentration range. One explanation for the higher molybdenum-reactive P estimates was that molybdate apparently interacted with the phosphate ester groups of IP6 to simulate the phosphomolybdate–antimony complex. Reduction of the complex by ascorbic acid yielded the blue color that was quantified as PO4–P. In addition, the introduction of enzyme in the reaction mixture added proteins that formed insoluble compounds during the colorimetric determination of molybdate-reactive P (Kirkbright et al., 1971). The reduction in spectral absorbance caused by the insoluble protein–phosphomolybdate complexes can be minimized by dimethyl sulfoxide (Shand and Smith, 1997). Although modification of the samples and reagent stream could be made, the two P chemical species were principal components of our samples. We chose to measure PO4–P and IP6-P by high-performance liquid chromatography (HPLC) methods because concentrations of PO4–P determined by anion chromatography were unaffected by the presence of IP6-P in the 0.016 to 0.16 mM PO4–P range (Table 1). The HPLC procedures were not themselves immune to analytical interferences. The intermediate myo-inositol mono-, di-, and triphosphates formed during the dephosphorylation of IP6 are not easily separated from PO4–P by anion chromatography (Skoglund et al., 1997) and are included in the PO4–P fraction. However, these intermediary products are probably further dephosphorylated to PO4–P. Precipitated insoluble Ca-, Al-, or Fe-IP6 also are not detected by the paired-ion HPLC method and IP6 dephosphorylation would be overestimated, compared with the measurements of PO4–P. In subsequent discussion of the experimental results, we are presenting the data of PO4–P concentrations measured by anion chromatography. The accumulation of the reaction end product was used to describe the dephosphorylation of IP6 to minimize the confounding effects of mixtures of monomeric and polymeric IP6 in the presence of polyvalent ions and the differences in their detection.


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  		Table 1. Dissolved P analysis in the presence of various proportions of phytate (IP6) by the ascorbic acid–molybdenum blue and high-performance anion chromatographic methods.

 
Effect of pH and Counterion on Enzyme Activity
At 20°C, fungal phytase EC 3.1.3.8 catalysis of the dephosphorylation of IP6 was a function of solution acidity between 2 and 10 (Fig. 1) . At pH <= 8, the reaction showed two maxima, one at pH of approximately 1.9 and another at 5.8 during the initial stages of dephosphorylation. The fungal phytase initiated the dephosphorylation of IP6 at the 3 position of the myo-inositol ring and eventually fully dephosphorylated the substrate molecule (Nayini and Markakis, 1986). More than 80% of the dephosphorylation reaction was completed by 24 h in all solutions at pH less than 8. One explanation for the initial differences in enzyme activity was pH-dependent changes in the molecular conformation of IP6 (Bauman et al., 1999). Phosphorus-31 nuclear magnetic resonance spectroscopy studies showed that IP6 undergoes inversion between the 1-axial/5-equilateral conformation and the 5-axial/1-equilateral conformation as a function of solution pH. In our study, these pH-dependent conformational changes may have affected enzyme binding and the kinetics of dephosphorylation. The fungal phytase activity was completely inhibited at solution pH 8 and above, and these experimental conditions were applied to deactivate the enzyme and stop the enzymatic reaction, in addition to heating at 100°C.



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  		Fig. 1. Effect of pH on the catalytic activity of Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8 and the accumulation of the phytate (IP6) dephosphorylation reaction end-product PO4–P at selected times after enzyme addition. Error bars indicate mean ± standard deviation.

 
The dephosphorylation of the Na salt of IP6 at pH 4.5 was described by a first-order decay rate model (Fig. 2) :

where y = disappearance of IP6-P (mM), C = instantaneous IP6-P concentration, t = time (min), yo = 0.129 (±0.079) mM, a = 1.309 mM (95% confidence limits = 1.048 and 1.569), and b = 0.00651 mM min-1 (95% confidence limits = 0.00294 and 0.01007 with P > |t| = 0.00425), with an r2 = 0.962. The accumulation of PO4–P as a function of time following enzyme addition approximated and mirrored the IP6-P disappearance. The Cmax and rate of accumulation for PO4–P was in agreement within the experimental error of the initial IP6-P concentration: y = 0.194 + 1.342[1 - e(0.00608t)], where y = appearance of PO4–P (mM) and t = time (min), with a coefficient of determination of 0.970.



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  		Fig. 2. Effect of counterions and their concentrations on the dephosphorylation of phytate (IP6) by Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8 at pH 4.5. Solid lines represent best-fitted first-order kinetic rate equations.

 
Divalent Ca2+ did not affect the dephosphorylation of IP6 at a mole ratio of 1:6, in comparison with Na+ (Fig. 2 and Table 2). Calcium IP6 is completely soluble at low mole ratios of Ca to IP6-P, in agreement with the work of Kaufman and Kleinberg (1971). However, the dephosphorylation of IP6 was significantly reduced when Ca2+ increased, and calcium IP6 begins to precipitate as colloidal particles as the mole ratio of Ca to IP6-P increases to 6 to 6 (i.e., 6 to 1 of Ca2+ to IP6). Phosphate accumulation Cmax decreased by 50 ± 3.6 and 40 ± 4% at pH 4.5 and 6, respectively (Table 2). Divalent Ca2+ did not completely inhibit dephosphorylation to the degree that Al3+ and Fe3+ did because Ca-IP6 was still soluble at pH of approximately 4 (Wise and Gilburt, 1981).


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  		Table 2. First-order kinetic rate model parameter Cmax for the dephosphorylation of phytate (IP6){dagger} by fungal phytase EC 3.1.3.8 as affected by pH, counterions, and counterion mole ratios at 20°C.

 
As a strong ligand, IP6 has a high affinity for Al3+ and Fe3+. Dephosphorylation of IP6 was progressively inhibited as Al3+ and Fe3+ concentrations or mole ratios increased (Table 2). Reductions in PO4–P accumulation averaged 27 and 32% at Al3+ and Fe3+ at the 1:6 cation to IP-P mole ratio or concentrations of 0.25 mM. Inhibition in dephosphorylation reached levels of more than 80 and 99% for both Al3+ and Fe3+ at the 3:6 and 6:6 mole ratios, respectively.

It was suggested that increased intermolecular bonding by the trivalent cations increased the concentrations of insoluble polymeric Al and Fe IP6, limiting the hydrolytic activity of the enzyme (Fig. 2 and 3) .



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  		Fig. 3. Effect of counterions and their concentrations on the dephosphorylation of phytate (IP6) by Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8 at pH 6. Solid lines represent best-fitted first-order kinetic rate equations.

 
Phytate was more susceptible to enzymatic dephosphorylation at pH 6 as Cmax values were higher than those at pH 4.5 for Al and Fe to IP6-P mole ratios <= 3:6 (Table 2). As the suspension pH was raised, more metal hydroxide species [i.e., M(OH)2+, M(OH)2+, and M(OH)3, where M = Al and Fe] and consequently less Al(H2O)63+ or Fe(H2O)63+ aquo-metal ions would exist in solution. Calculations of metal speciation were made with MINTEQ2A Version 4.0 to verify this hypothesis (data not shown) (USEPA, 1999). The pH of minimum solubility of metal hydroxide precipitates was shown to be about 6.3 and 8 for freshly precipitated Al- and Fe-hydroxide, respectively (VanBenschoten and Edzwald, 1990). Mechanistically, the formation of more amorphous Al(OH)3 and Fe(OH)3 would reduce the effective concentrations of Al3+ and Fe3+ to react with IP6 and more dissolved IP6 remained in dissociated forms to undergo dephosphorylation at pH 6.0, compared with the pH 4.5 treatment. As the re-acidification of the samples to pH 2 before P determinations would dissolve any Ca-, Al-, or Fe-phosphates, the inhibition of dephosphorylation by Al3+ or Fe3+ was attributed primarily to the sequestration and conformational inaccessibility of IP6 in insoluble forms.

Effect of Suspension Total Solids
Incubation of the dairy manure suspensions that did not receive exogenous IP6-P increased solution PO4–P by up to 2.5 mM with the addition of phytase EC 3.1.3.8 (Table 3). The unamended manure contained a phytase-hydrolyzable phosphorus (PHP) fraction that was proportional to the suspension TS concentrations. Although we purposely limited the native enzyme activity by autoclaving, the added Aspergillus phytase was able to hydrolyze a net average of 2.2 mmol PHP kg-1 dairy manure solids. Therefore, the results suggested that added phytases and potentially native manure phytases will hydrolyze excreted IP6. The enzymatic process, in effect, increases dissolved inorganic P content of stockpiled manure to contribute to the temporal and spatial variability of manure dissolved P. The implications for managing manure from livestock or poultry that were fed rations containing phytases are that such manures should be land-applied close to the time of crop needs. Otherwise, high levels of water-soluble manure P will present undue risks of potential transfer to runoff and off-site transport.


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  		Table 3. Effects of cation and manure suspension total solids (TS) on PO4–P accumulation{dagger} from the dephosphorylation of phytate (IP6) in dairy wastewater.

 
Calcium did not interfere with the dephosphorylation of added IP6, and for that matter, with the native manure PHP fraction at any of the Ca to IP6-P mole ratios and suspension TS concentrations (Table 3). However, the partial inhibition previously observed in pH 4.5 buffers at a Ca to IP6-P mole ratio of 6:6 was not evident in any of the manure suspensions. The results suggested that the negatively charged manure particulates interacted with Ca2+ ions, lowering the effective Ca to IP6-P mole ratio to less than the targeted 6:6, and, therefore, their ability to complex and form insoluble calcium phytate.

Trivalent Al3+ progressively inhibited the dephosphorylation of IP6 at cation to IP6-P mole ratios at all TS levels. When we accounted for the native PHP background, reduction in net PO4–P accumulation averaged 30% at the 1:6 Al to IP6-P mole ratio and increased to more than 90% at mole ratios of 3:6 and higher. Apparently, Fe3+ was even more efficient than Al3+ and Ca 2+ in inhibiting the dephosphorylation of IP6 in suspensions containing >= 30 g L-1 TS. The added Fe3+ also reduced the mineralization of the manure native PHP fraction at cation to IP6-P mole ratios >= 3:6. The high affinity of IP6 for Fe3+ and low solubility of iron IP6 were the basis of early analytical methods for determining IP6 with FeCl3 (Young, 1936; Holt, 1955). In previous studies, Fe3+ and Al3+ salts were effective in aggregating dairy manure solids, and that efficacy was shown to increase with manure TS concentration (Dao and Daniel, 2002). Ferric chloride was more efficient than an Al3+ salt at interbridging and aggregating manure suspended particles. Increased manure particle aggregation suggested sorption and coprecipitation of IP6-P because PHP was shown to decrease with decreasing suspended solid concentrations (Table 3).

These results also showed that the differential protective effects of cations would have a large influence on the accuracy of estimates of the native enzyme-hydrolyzable fraction in manure. Manure has been found to contain elevated concentrations of trace minerals, as mineral supplements are routinely added to livestock feeds to minimize potential deficiency or suppress disease (Zhang et al., 2002). Minerals such as Ca, P, K, Mg, and S, and trace minerals such as Fe, As, Se, Cr, Cu, and Zn are added to feeds to meet dietary requirements of livestock. Dairy manure averaged 26 g Ca, 1.5 g Al, and 1.8 g Fe L-1 in a random survey of Midwestern U.S. farms (Combs et al., 1998). Therefore, these conditions are favorable for IP6 to exist as salts of dietary metals in dairy manure. Our experimental results suggested that IP6 persistence was likely as these metal phytates were less susceptible to dephosphorylation. Furthermore, a substantial amount of the feed organic P was not available to the animal and was excreted in ruminant livestock manure.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The effects of polyvalent cations were an important consideration in our attempts to develop an enzymatic assay for plant-derived polyphosphates, in particular myo-inositol phosphates, and increase our understanding of phytase behavior in manure and phosphate effects on forms and fate of organic polyphosphate compounds in animal manure. Polyanionic IP6 has a high affinity for cations and forms insoluble precipitates. Dephosphorylation was increasingly inhibited as counterion concentration increased to cation to IP6-P mole ratios of 6:6 in the order of Fe3+ > Al3+ > Ca2+, and is a potential mechanism by which IP6 persists in animal manure. A phytase-hydrolyzable P fraction was native to dairy manure suspensions and the experimental results suggested that added phytases would increase water-extractable P concentration in manure under favorable biological and environmental conditions. The differential protective effects of cations would influence the accuracy of PHP estimates in manure. Increased manure TS also decreased IP6 susceptibility to dephosphorylation, as a synergistic interaction existed between manure TS and Al3+ and Fe3+ concentrations to increase manure particulate aggregation and sequestration of IP6.

Thus, the differences in susceptibility to hydrolysis between phytate salts suggested that the bioavailability and potential ecological effect of IP6-P were regulated by pH-controlled enzyme activity and by counterions associated with IP6. Phosphorus immobilization strategies and remedial methods for sequestering excess inorganic dietary P should include conditions that favor the inhibition of dephosphorylation of organic IP6 in manure. Whereas the size of the pool of immobilized mineral phosphates will depend on their solubility product (Ksp) and nonbiological factors such as temperature and ionic strength, changes in the biological environmental conditions will be less controllable and more likely will vary widely across handling and storage environments. These latter changes can alter intracellular and exozyme activity, and therefore the manure water-soluble P pool size and environmental dispersion of PO4–P.


   ACKNOWLEDGMENTS
 
The author sincerely acknowledges the technical assistance of Lynne Heighton and Tingting Wong in the course of this study.


   NOTES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 NOTES
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
1 Mention of trade or manufacturer names is made for information only and does not imply an endorsement, recommendation, or exclusion by the USDA Agricultural Research Service. Back


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


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