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Published in J. Environ. Qual. 33:349-357 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Waste Management

Organic Ligand Effects on Enzymatic Dephosphorylation of myo-Inositol Hexakis Dihydrogenphosphate in Dairy Wastewater

Thanh H. Dao*

USDA Agricultural Research Service, Animal Manure and By-Products Laboratory, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705

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

Received for publication December 27, 2002.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Animal manure contains partially digested feed fiber and grains where phosphorus (P) is bound in organic compounds that include myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate or phytic acid (IP6). Information is needed on the effects of other (non-IP6) organic ligands (LIGND) on the enzymatic dephosphorylation of IP6, which is a potential source of dissolved orthophosphate P (PO4–P) in the soil–manure–water system. The effects of 1,2-cyclohexane diamino-tetraacetate (CDTA), diethylenetriamine-N,N,N',N'',N''-pentaacetate (DTPA), ethylenediamine-N,N,N',N'-tetraacetate (EDTA), oxalate (OXA), and phthalate (PHTH) and LIGND to IP6 molar ratio and charge concentration ratio on IP6 dephosphorylation were studied to determine controlling mechanisms of IP6 persistence in manure. Solution PO4–P concentrations were analyzed by ion chromatography as the phosphomolybdate–ascorbic acid method partly includes IP6-P. Uncomplexed IP6 dephosphorylation by Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8 at pH 4.5 and 6 is unaffected by the presence of LIGNDs. As the concentrations of Ca2+, Al3+, or Fe3+ increase, dephosphorylation is reduced. Their inhibitory effect lessens in the presence of LIGNDs, in the following order: CDTA = EDTA > DTPA >> OXA ≥ PHTH. Whether CDTA or EDTA is the most effective LIGND depends upon the acidity of the suspension and LIGND charge concentration, reducing the inhibitory effect of polyvalent counterions to the point of promoting the hydrolysis of a manure phytase-hydrolyzable phosphorus (PHP) fraction that is otherwise unavailable. Therefore, ligand-induced changes increase the mobilization and dephosphorylation of complexed organic P, above and beyond the simple dissolution of inorganic phosphates. An analytical method for potentially bioavailable PHP in animal manure should include a LIGND as extracting reagent. Also, potential LIGNDs in an organic carbon–rich dairy wastewater may increase the release of PHP and environmental dispersion of PO4–P.

Abbreviations: CDTA, 1,2-cyclohexane diamino-tetraacetate • DTPA, diethylenetriamine-N,N,N',N'',N''-pentaacetate • EDTA, ethylenediamine-N,N,N',N'-tetraacetate • IP6, myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate • LIGND, other (non-IP6) organic ligand • OXA, oxalate • PHP, phytase-hydrolyzable P • PHTH, phthalate


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
PHYTIC ACID typically accounts for 60 to 90% of the stored reserves of P and myo-inositol in grains of a number of agronomic crops. Forages that are fed to livestock in various proportions with grains, depending upon stages of growth and energy requirements, also contain IP6. myo-Inositol hexakis dihydrogenphosphate is largely indigestible to swine (Sus scrofa domesticus) and poultry (Gallus gallus) and is excreted in manure (Council for Agricultural Science and Technology, 2002). A large phytase-hydrolyzable fraction is also found native to manure of cattle (Bos taurus), although rumen microflora possess necessary enzymes to break down IP6 (Dao, 2003a). myo-Inositol hexakis dihydrogenphosphate is dephosphorylated by phytases (myo-inositol hexakis dihydrogenphosphate phosphohydrolases) and phosphatases in plants, soil, water, and animal manure (Nayini and Markakis, 1986; Hino, 1989; Bergman et al., 2000; Dao, 2003a). Added Aspergillus ficuum (Reichardt) Henn. phytase EC 3.1.3.8 was reported able to hydrolyze a net average of 2.2 mmol P kg–1 dairy manure solids. The enzymatic process, in effect, increases dissolved inorganic P content of stockpiled manure that is temporally and spatially highly variable (Dao, 2003a).

The polydentate ligand IP6 has a maximum coordination number of 12, complexes monovalent cations, and stoichiometrically forms monomeric salts such as sodium IP6. With polyvalent cations, the possibility of inter- and intra-molecular bonding exists and results in the formation of both monomeric and polymeric compounds (William and Pierce, 1982). As a strong ligand, IP6 has a high affinity for Al3+, Fe3+, and Ca2+. Dephosphorylation of IP6 is progressively inhibited by these cations when cation to IP6-P mole ratios increase to the theoretical 6 to 6 or higher. Increased intermolecular bridging by the trivalent cations increases the concentrations of insoluble Al- and Fe-IP6, limiting the hydrolytic activity of phytases. As such, complexed, insoluble inositol phosphate species persist in dairy manure (Dao, 2003a).

Organic anions have been extensively studied for their ability to inhibit mineral precipitation, in particular, mineral phosphates such as dicalcium phosphate dihydrate (Freche et al., 1992) or hydroxyapatite (Inskeep and Silvertooth, 1988) in the management of P fertilization and plant uptake. Organic anions play a critical role in the bioavailability of P in the rhizosphere of a number of plant species (Hinsinger, 2001). Excreted in lower amounts than carbohydrates, the organic anions include a variety of products of the citric acid cycle, bearing one or more carboxylate functional groups. In the soil, commonly identified low molecular weight organic anions include formate, acetate, propionate, oxalate, and citrate that are formed during microbial metabolism and decomposition of plant residues (Rovira and McDougall, 1967). Soil organic matter also contributes aliphatic acids, phenols, phenolic acids, and fulvic and humic substances that can act as LIGNDs (Ritchie et al., 1982; Haynes and Mokolobate, 2000). These humic matter ligands are large and have multiple functional groups to bind and control the speciation of cations. Animal manure and wastewater also contain large amounts of organic matter that probably generate complex LIGNDs (Ma et al., 2001). Mechanisms of PO4–P mobilization in dairy wastewater may include ligand exchange (Jones and Darrah, 1994), complexation of counterions (Earl et al., 1979), and solubilization (Kirk et al., 1999). In addition, organic anions have been extensively studied for their ability to inhibit mineral precipitation in the process of P removal from domestic and animal wastewaters (Sharma et al., 1992; House, 1999; van der Houwen and Valsami-Jones, 2001). Extensive research is focused on understanding the conditions for the nucleation of calcium phosphates. Organic ligands have been shown to inhibit the precipitation of dicalcium phosphate dihydrate, tricalcium phosphate, octacalcium phosphate, or hydroxyapatitite by adsorbing onto active growth sites of the seeding materials. Phospho–citrate complexes have been identified as inhibitors of the precipitation reaction (Sharma et al., 1992).

There is increasing interest on post-excretion treatments of manure to chemically bind or remove P in manure before it is applied to fields (Dao, 1999, 2003b; Schuiling and Andrade, 1999; Dao et al., 2001; Dao and Daniel, 2002). A number of chemical products and by-products containing polyvalent cations such as Al3+ in alum [Al2(SO4)3 · 18 H2O] or drinking water treatment residuals rich in Al3+ and Fe3+ have been used to immobilize and reduce manure and soil soluble P. Such approaches may alleviate nonpoint-source discharge risks posed by repeated land applications and the nutrient-laden conditions that exist on many agricultural fields near confined animal feeding operations (Zhang et al., 2002). The runoff and sediments from these fields carry dissolved and particulate nutrients to surface waters, resulting in impaired water quality (Correll, 1998). Remediation strategies or technologies to reduce bioavailable P levels in manure, such as the crystallization of magnesium ammonium phosphate (Ohlinger et al., 1999; Schuiling and Andrade, 1999) or the recovery of PO4–P by ion-exchange sorbents (Dao, 2003b), also depend on the understanding of the dephosphorylation of organic polyphosphates. The extent of organic P mineralization may influence the overall P removal efficiency of post-excretion treatment of animal manure and will allow the optimization of biological and environmental conditions to ensure the success of such remediation strategies. This study was conducted to (i) develop an improved understanding of the solution-phase chemistry of dephosphorylation of IP6 by extracellular phytase and (ii) determine the effects of naturally occurring and synthetic LIGNDs of increasing structural complexity on potential mechanisms of IP6 persistence in dairy manure.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
LIGNDs and Speciation Computations
Sodium IP6 isolated from corn (Zea mays L.) and preparations of fungal phytase (3-phytase EC 3.1.3.8) isolated from Aspergillus ficuum were of the same sources as previously described (Dao, 2003a). In summary, the commercial phytase preparation's 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 min at pH 2.5 and 37°C. The activity and substrate affinity of the enzyme preparation was determined using reagent-grade sodium IP6 at different pH as previously described (Dao, 2003a). Detailed kinetic studies were conducted in several standard solutions buffered in the pH range of 2 to 10 at 20°C. Although the commercial preparation may contain other phytases, acidic and/or alkaline phosphatases, the specific experimental conditions for IP6 dephosphorylation were optimized and shown to hydrolyze exogenous IP6 at a specific kinetic rate. 3-Phytase EC 3.1.3.8 converts myo-inositol hexakis dihydrogenphosphate to 1-D-myo-inositol 1,2,4,5,6-pentakis dihydrogenphosphate (IP5) and orthophosphate (McKelvie et al., 1995). The 3-phytase EC 3.1.3.8 preparation continues to hydrolyze and release orthophosphate from IP5 and lower myo-inositol phosphates, with the results being similar to those of Nayini and Markakis (1986), McKelvie et al. (1995), and Dao (2003a). The recovery of 1.5 mM IP6-P was quantitative when enzymatically hydrolyzed and detected as PO4–P by 24 h at 20°C using high-performance liquid chromatography (HPLC) methods. While the preparation is a lyophilized crude extract of Aspergillus ficuum phytases and may have high affinity for IP6, the enzymes appear to be primarily nonspecific phosphomonoesterases that can hydrolyze and release orthophosphate from model IP6 (McKelvie et al., 1995).

Polydentate ligands studied included CDTA, DTPA, EDTA, oxalic acid, and phthalic acid. The acid forms were converted to the Na+ forms (except for potassium hydrogen phthalate) by titration with 10 M NaOH before preparing the LIGND stock solutions.

The effects of LIGNDs on the dephosphorylation of IP6 were determined in the presence of Ca2+, Al3+, and Fe3+ on both a LIGND to IP6 (i) molar ratio and (ii) charge concentration ratio basis. At first, deionized water and appropriate volumes of 5 mM stock solutions of CaCl2, AlCl3, FeCl3, and Na-IP6 were added to attain cation to IP6-P mole ratios of 1:6, 3:6, and 6:6 and induce various degree of inhibition of the dephosphorylation of IP6. Then, aliquots of 5 mM LIGND were combined with the cation–IP6 mixtures to attain a final concentration of 0.25, 0.75, and 1.5 mM LIGND, or LIGND to IP6 molar ratios of 1:1, 3:1, and 6:1, respectively.

As coordination number and molecular weight of the LIGNDs varied widely, the effect of charge concentration ratio was also determined. Results of computations of ionic species fractions are presented in Table 1. Published LIGNDs and IP6 dissociation constants (Ka) were used to derive equivalent molar concentrations for LIGND to IP6 charge concentration ratios of 1:1, 2:1, and 4:1 at each pH level (Evans et al., 1982; Dawson et al., 1986, p. 402–404). The pH of 5 mM LIGNDs stock solutions was adjusted to either 4.5 or 6 with a 5.5 M HCl or 10 M NaOH solution. All other reagents were also adjusted to either pH 4.5 or 6, before reacting in a background of IP6 and counterions, to attain appropriate LIGND to IP6 mole or charge density. Aliquots of a 3-phytase EC 3.1.3.8 stock were added to attain a final enzyme activity of 0.05 unit mL–1. The mixtures were equilibrated at 20°C on a gyratory shaker (250 rpm) for 24 h. Each treatment was replicated three times. After the equilibration period, solution-phase concentrations of PO4–P were determined by HPLC, after boiling in a 100°C water bath for 10 min to stop the enzymatic reaction. Chromatographic conditions are described in a following section.


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  		Table 1. Fraction of major anionic species{dagger} (HxA) of organic ligands at two solution pH values.

 
Counterions and LIGNDs in Manure Suspensions
Bulk batches of reconstituted dairy manure were prepared from freshly collected feces and acidified urine from holstein cows as described in previous work (Dao and Daniel, 2002). In summary, 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 of the manure was adjusted to 6.0 before the initiation of the study. Tap water was added to prepare manure suspensions containing 100 g L–1 of total solids. Three mole ratios of Ca2+, Al3+, and Fe3+ to IP6-P and control treatments receiving no enzyme and no polyvalent counterions were established as previously described. The final pH of the suspension was readjusted to pH 6 with a 5.5 M HCl or 10 M NaOH solution, as appropriate. On a molar basis, LIGND to IP6 mole ratios of 1:1, 3:1, and 6:1 (i.e., 0.25, 0.75, and 1.5 mM of LIGND to the 0.25 mM IP6, respectively) were established. Aliquots of a 3-phytase EC 3.1.3.8 stock were also added to attain a final enzyme activity of 0.05 unit mL–1. Each treatment unit was replicated three times. Jars containing the mixture were agitated on a gyratory shaker at 250 rpm.

After a 24-h equilibration period, solution-phase concentrations of PO4–P were determined after boiling in a 100°C water bath for 10 min. The suspensions were centrifuged at 7000 x g for 20 min. The supernatant was filtered through membranes with 0.45-µm openings. Aliquots of the supernatant were re-acidified to pH 2 before P analysis to dissolve all forms of PO4–P based on a HPLC method for anionic species (Erkelens et al., 1986). Dissolved P concentrations of suspension aliquots were determined chromatographically using a Waters (Milford, MA) 2690 LC system, equipped with UV (Model PDA 996) and electrical conductivity (Model W432) detectors. 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.

Dephosphorylation experiments in buffers and manure wastewaters were established according to a randomized complete block design with cation to IP6-P mole ratio and LIGND to IP6 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 using the Statistical Analysis System (SAS Institute, 1996).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Effect of pH and LIGND to IP6 Ratios on Phytase Activity
The dephosphorylation of IP6 by fungal phytase EC 3.1.3.8 is complete and appears unaffected by the LIGNDs in the absence of Al3+ (Fig. 1A) , Ca2+, or Fe3+ (data not shown). The observation holds true, whether the reaction medium pH is either 4.5 or 6. There are contradictory reports of organic ligands inhibiting phytase activity; EDTA, OXA, and citrate suppress yeast (Saccharomyces cerevisiae) phytases whereas they do not affect the activity of wheat (Triticum aestivum L.) bran phytase (Nayini and Markakis, 1986). In this study, EDTA and the other LIGNDs did not appear to interfere with the hydrolytic activity of EC 3.1.3.8 at molar concentrations between 6.1 and up to 22.6 mM for EDTA and PHTH, respectively, in buffers and in dairy manure suspensions as discussed in a subsequent section.



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  		Fig. 1. Effect of non-IP6 organic ligands (LIGND), LIGND to myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate (IP6) mole ratios, and suspension pH on the dephosphorylation of IP6 by Aspergillus ficuum phytase EC 3.1.3.8 at four levels of IP6 counterion. (A) 0, (B) 0.25, (C) 0.75, and (D) 1.5 mM Al3+. Error bars indicate mean ± standard deviation.

 
Phytic acid is a strong polyanionic ligand and has a high affinity for Al3+ (or Fe3+ and Ca2+, data not shown), in agreement with previous work (Dao, 2003a). Dephosphorylation of IP6 alone is progressively inhibited as solution-phase Al3+ concentration increases (Fig. 1B, 1C, and 1D). Competitively with IP6, the LIGNDs induce a partial to complete reversal of the inhibitory effect of Al3+. The LIGNDs segregate themselves in three groups, where CDTA and EDTA make up the effective group, OXA and PHTH are in the inefficient group, and DPTA is of intermediate efficacy. Differences in efficacy between LIGNDs are more evident at pH 4.5 on the LIGND to IP6 mole ratio basis. The CDTA and EDTA ligands are able to completely reverse the inhibition of IP6 hydrolysis at all three Al3+ amendment rates. In the solution-phase, CDTA and EDTA are present primarily as divalent H2 CDTA2– and trivalent HEDTA3–, respectively. They are apparently comparable in reactivity and ability to decouple counterion and IP6 (Table 1).

At pH 6, overall IP6 resistance to dephosphorylation is greater than that at 4.5 and the LIGNDs are not as effective at pH 6 as they are at pH 4.5. Overall, EDTA is much more effective than CDTA, particularly at Al3+ amendment rates exceeding 0.75 mM (Fig. 1C and 1D). Charge concentrations for EDTA4– and chelating ability exceed those of CDTA, which theoretically exists as H2CDTA2– and HCDTA3–. Reinforcing that point, EDTA is the only ligand that provides relief from the inhibition of the dephosphorylation reaction when Al3+ rate is 1.5 mM. At the higher pH, Al3+ exists as Al(OH)2+ and Al(OH)3 (aq) as predicted by MinteqA2 (USEPA, 1999). In addition to complexation, amorphous metal hydroxides can sorb and protect IP6 from enzymatic hydrolysis. Hydroxyl functional groups react with IP6, as follows:

It was noted that OXA, commonly used to chelate Fe and Al, is only able to partially reverse the inhibition of dephosphorylation at the 0.25 mM Al3+ treatment and OXA to IP6 molar ratio of ≥3:1. The ligand is ineffective at higher Al3+ amendment rates. Oxalate is even less effective at pH 6, where it should exist as the fully dissociated unprotonated conjugate base (Fig. 2B) . This is also true for OXA and PHTH when they are added on the LIGND to IP6 charge ratio basis (Fig. 2B and 3B) .



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  		Fig. 2. Effect of non-IP6 organic ligands (LIGND), LIGND to myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate (IP6) charge ratios, and equivalent LIGND to IP6 mole ratios on the dephosphorylation of IP6 by Aspergillus ficuum phytase EC 3.1.3.8 at four levels of IP6 counterion. (A) 0, (B) 0.25, (C) 0.75, and (D) 1.5 mM Al3+ at suspension pH 4.5. Error bars indicate mean ± standard deviation.

 


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  		Fig. 3. Effect of non-IP6 organic ligands (LIGND), LIGND to myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate (IP6) charge ratios, and equivalent LIGND to IP6 mole ratios on the dephosphorylation of IP6 by Aspergillus ficuum phytase EC 3.1.3.8 at four levels of IP6 counterion. (A) 0, (B) 0.25, (C) 0.75, and (D) 1.5 mM Al3+ at suspension pH 6. Error bars indicate mean ± standard deviation.

 
LIGND to IP6 Charge Concentration Ratio
Overall, the capacity to overcome the inhibitory effect of Al3+ increases linearly with LIGND to IP6 charge concentration ratios in the 1 to 4 range (Fig. 2 and 3). The results of fitting a linear model and comparisons of rates of reduction of the inhibitory effect of Al3+ (i.e., regression line slopes) are presented in Table 2. In the absence of Al3+, all five LIGNDs have no effect on IP6 hydrolysis. All slopes of the regression lines relating PO4–P accumulation and LIGND concentration are nearly zero and not different from the control no-LIGND treatment. Increasing Al3+ to IP6-P molar ratios progressively inhibits the dephosphorylation reaction, and increasing LIGND charge concentration appears to reverse and reduce IP6 resistance to enzymatic hydrolysis.


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  		Table 2. Slopes of linear regression equations describing the reversal of the inhibition of dephosphorylation of myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate (IP6) by Al3+ as a function of other (non-IP6) organic ligands (LIGND) to IP6 charge concentration ratio (CR).

 
The LIGNDs possess two (OXA and PHTH), four (CDTA and EDTA), or five (DTPA) carboxylate functional groups. Apparently only EDTA, CDTA, and DTPA are the most able and consistent ligands to dissociate Al3+ and IP6 and increase hydrolysis of the latter. The differences in efficacy of CDTA and EDTA previously noted between pH 4.5 and 6 are reconfirmed when studied and explained from a LIGND to IP6 charge concentration basis (Fig. 2 and 3). Therefore, excess charge is needed to decouple and mobilize complexed IP6, and to attain the LIGND to IP6 charge concentration ratios studied, equivalent molar concentration ratios have to be between 1.5- and 12-fold that of IP6 (Fig. 2 and 3). The equivalent molar concentration for PHTH is highest because of its high molecular weight. One should note that there is a limit to increasing charge concentration to overcome the inhibitory effect of polyvalent counterions as the entropy of the IP6–counterion–LIGND–counterion system increases. Dephosphorylation will be reduced because of biophysical as well as chemical interference to the activity of the phytase enzyme.

Efficacy of LIGNDS in Dairy Wastewaters
Dairy manure contains a phytase-hydrolyzable phosphorus (PHP) fraction that is readily released as bioavailable PO4–P to the manure suspension. The experimental results are presented as net concentration relative to the no-added-cations and no-LIGND treatments (Fig. 4) . In addition, amending these batches of dairy manure with the LIGNDS, without adding phytase EC 3.1.3.8, yields a constant background of 0.73 ± 0.053 mM PO4–P (data not shown).



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  		Fig. 4. Effects of non-IP6 organic ligands (LIGND) on the inhibition of myo-inositol 1,2,3,5/4,6-hexakis dihydrogenphosphate (IP6) dephosphorylation by Aspergillus ficuum phytase EC 3.1.3.8 by polyvalent counterions in 100 g total solid L–1 dairy manure suspension. The IP6 counterion concentrations ranged from 0 to 1.5 mM. (A) 0, (B) 0.25, (C) 0.75, and (D) 1.5 mM Ca2+, Al3+, and Fe3+. Dashed lines represent relative concentration of PO4–P in untreated manure suspensions that had no added counterion and no added IP6.

 
In manure suspensions amended with only exogenous IP6, in the absence of added polyvalent counterion (Fig. 4A), CDTA and EDTA additions increase the release of PO4–P over and above the added P. The DTPA ligand also appears effective in promoting the enzymatic hydrolysis of manure organic P at a DTPA to IP6 mole ratio of ≥3:1. These results suggest that these LIGNDs increase the mobilization and dephosphorylation of complexed organic P, native to the manure, and not merely the dissolution of inorganic phosphates because of their chelating property, as there was no detectable effect of the LIGNDS by themselves without added phytase EC 3.1.3.8 in unamended manure.

Increasing polyvalent Al3+ and Fe3+ concentrations in the manure suspension increases the inhibition of IP6 dephosphorylation (Fig. 4B, 4C and 4D). Divalent Ca2+ did not reduce the dephosphorylation of added IP6 because calcium is a known co-factor of the dephosphorylation reaction (Nayini and Markakis, 1986). The ligands EDTA, CDTA, and to some extent, DTPA, maintain their activity in dairy manure suspensions as they are able to reverse the inhibitory effect of Al3+ and Fe3+ at trivalent cation to IP6 and LIGND to IP6 molar ratios of ≤3:1, and Fe3+ to IP6 molar ratio of 6:1 (1.5 mM Fe3+). The predominant anionic forms of the LIGNDs are EDTA4–, H2CDTA2–, HCDTA3–, and H2DTPA2–. No difference in reactivity of these LIGND forms has been detected in either Al3+– or Fe3+–amended manure. Again, PHTH and OXA have little or no effect on the reaction in manure.

In this study, the dephosphorylation of added or manure-native inositol phosphates is indistinguishable. However, the increased recovery of PO4–P following phytase addition, over and above the quantity of added P, reaffirms the existence and extent of IP6 presence in dairy manure (Peperzak et al., 1959; Reeves et al., 2002; Dao, 2003a). The experimental results also imply that LIGNDs are important ingredients in an enzymatic assay for determining the potentially hydrolyzable organic P or the PHP fraction in manure. A LIGND to IP6 molar ratio of 3:1 appears necessary when polyvalent counterion to IP6 ratio is expected to be ≤3:1 (Fig. 4C). The results presented in Fig. 4A suggest that the PHP fraction was actually 40% higher than that measured without using EDTA, CDTA, and DTPA. That fraction is even larger when the inhibitory effect of trivalent cations that shielded complexed IP6 from hydrolysis is removed by the LIGNDs (Fig. 4B, 4C, and 4D).

The implications for managing manure from livestock or poultry that were fed rations containing phytase enzymes are that such manures can be high in IP6-derived dissolved P, in addition to mineral dietary P. As stored manure ages, microbial metabolic by-products and degradation products of feed materials are released to the manure suspension. Polyphenols, tannins, humate, fulvate, and other macromolecules of microbial metabolism are potential ligands. Anthropogenic CDTA, EDTA, and DTPA and similar organic ligands would be competitive at displacing and chelating the counterions associated with manure IP6. The mobilization of the polyphosphate leads to greater susceptibility to enzymatic dephosphorylation.


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The effects of organic ligands were an important consideration in our research to develop an accurate assay for plant-derived organic P, in particular myo-inositol phosphates, and increase our understanding of phytase behavior in manure and its effect on forms and fate of organic polyphosphate compounds. Polyanionic IP6 has a high affinity for polyvalent cations and forms insoluble compounds, resulting in progressive reduction in dephosphorylation as counterion concentration increases. Organic ligands are shown to have the ability to overcome the inhibitory effect of polyvalent cations that increases linearly with LIGND to IP6 charge concentration ratios. The LIGNDs differ in their competitiveness to dissociate and mobilize complexed IP6, with synthetic EDTA and CDTA being the most effective ones under the study conditions. In dairy manure, LIGNDs also reduce the inhibitory effect of polyvalent cations, to the point of promoting the hydrolysis of the manure PHP fraction that is otherwise unaccessible. Therefore, the experimental results suggested that organic anions mobilize complexed IP6 and possibly other inositol phosphates, and that LIGNDs are necessary ingredients of a method for determining the PHP fraction of manure. The LIGND to IP6 charge concentration ratio must be ≥3 to 1 to overcome the inhibitory effect of polyvalent cations, particularly when the manure contains high concentrations of trivalent cations. In animal wastewaters, metabolic byproducts with cation-chelating properties can change the biological environmental conditions and environment of extracellular phytases. Ligand-induced changes in IP6-P bioavailability may alter manure dissolved P pool size.


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


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




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