TECHNICAL ARTICLE

Enzymatically and Ultraviolet-Labile Phosphorus in HumicAcid Fractions From Rice Soils

Zhongqi He,' Daniel C. 0/k. 2 C. Wayne Honeycutt,' and Ann-Marie Fortuna3

Abstract: Humic acid (HA) is an important soil component that canimprove nutrient availability and impact other important chemical, bio-logical. and physical properties of soils. We investigated the lability ofphosphorus (P) in the mobile HA and calcium huinate fractions of fourrice soils as measured by orthophosphate-releasing enzymatic hydrolysisand CV irradiation. Enzymatic hydrolysis releases hydrolyzable organicP. and CV irradiation abiotically releases P by breaking down phosphate-HA complexes. Less than 25% of the P in these fractions was detected 01

the soluble orthophosphate form (detectable soluble P 1 ). Enzymatic in-cubtition increased detectable soluble P, to 60% of total P. Treatment byCV irradiation alone released an additional 5% to 20% of humic-boundP compared with the untreated fractions. However, treatment by both CVirradiation and enzymatic hydrolysis released 0% to 14% of humie-bound P more than the amount of organic P that was enzymaticallyreleased from nonirradiated samples. The smaller amounts of P thatwere released from some humic fractions by both CV irradiation andenzymatic hydrolysis than by CV irradiation alone indicated some over-lap between CV-degradable organic P and enzymatically labile organicP. Generally. about one half to two thirds of humic P in these rice soilswas labile. These data demonstrated that either assumption that no P inhumie fractions was labile P or all P in htimic fractions was labile is nottrue. The lability of humie-bound P should be experimentally evaluatedas reported in this work.Key words: 1-lumic substances. phosphatasc. phosphorus, rice soil,soil organic matter.

(Soil .Sci 2009;174: 91-87)

Phosphorus (P) is an essential element for all forms of life. Itis involved in the biosynthesis of diverse cellular conipo-

nents, including nucleic acids, proteins, lipids, and sugars. Phos-phorus has long been known to be present in natural organicmatter (NOM) from various sources and is found mainly in thehuinic fractions (Stevenson. 1982). However. information OTtthe bioavailabilily or lability of NOM-bound P is very limited,although such information is critical for understanding the roleof NOM in P cycling and nutrition (He et al.. 2006b; Mahieuet al.. 2000tt).

He et al. (2006b) demonstrated that biotic and abiotic re-lease of P in NOM can be investigated by phosphatase hydro-lysis and CV irradiation, respectively. Phosphatase activity is asignificant mechanism for plant and microbial use of organic Pin soil environments because phosphatase hydrolysis breaksthe P-0-C bond, thereby converting oi'gailic P compounds intobioavailable inorganic phosphate (Tarafdar and Claassen, 1988;

'USDA-ARS. New England Plant. Soil, and Water Laboratory, Orono. ME04469. Dr. He is corresponding author. E-mail: Zhongqi.IIe(dars. usda.goCSDA-ARS. National Soil Filth Lab. Ames, IA.iDepa ilment of Crop and Soil Sciences, Washin gton State University.Pullman, WA.Reccicd May 21, 2008. and in revised form December 2, 2008.Accepted for publication December 3. 2008.Copyright s 2009 by Lippincott Williams & Wilkins, Inc.ISSN: 0038-075X1)01: l0.1097!SS.0b013C3l81981dC5

Soil Science Volume 174, Number 2, February 2009

Yadav and Tarafdar, 2003: Senwo et al., 2007). Thus, phospha-tase hydrolysis of organic P in extracts of environmental samplesprovides an estimate of hydrolyzable (labile) organic P fractionsin the environment (Pant ci al.. 1994, 2002; He et al., 2007).

Ultraviolet irradiation is an abiotic mechanism for releas-ing inorganic P from complex P compounds in environmentalsamples (Franeko and Heath. 1979; Wetzel et al., 1995). Ultra-violet light induced the release of P from Fe( lil)-P compoundsthrough photoreduction, thus suggesting that sunlight couldplay a role in maintaining labile P in soils (Pant and Warman,2000). Hens and Merckx (2001) reported the presence of OM-metal-P complexes in colloidal solutions of sandy soils thatcould he released by fluoride addition. UV irradiation, andphosphatase hydrolysis. Ultraviolet irradiation also abioticallydegraded some P fractions associated with purified hunlic sub-stances into soluble inorganic P or more easily hydrolyzableorganic P (He et al., 2006b).

To address growing concerns of soil nutrient supply andsystem sustainability. long-term field trials of multiple ricecropping systems have been carried out in the Philippines (01ket al., 2000). To investigate the structure-function relations ofsoil 0M in nutrient cycling, two humic fractions (i.e.. mobilehumic acid [MHA] and calcium humate [Cal IA]) were extractedfrom soils collected from these field trials. These fractions aredistinguished based on their binding with soil Ca. The MHAfraction is not bound by Ca and is enriched in relatively younglabile materials (01k, 2006), including diester P (Mahieu et al..2000a), arnide N (Mahieu et al.. 2000b), and lignin-derivedphenols (01k et al., 2002). The CaHA fraction is bound to soilCa, and it is composed of relatively older more humiliedmaterials. In these studies, both fractions were more responsivethan was total soil OM in quantity and chemical nature to recentmanagement for tropical lowland rice soils, and N fertilizercycled more quickly through both fractions than total soil OMduring one rice growing season (01k et al., 2006). These state-merits are much more true for the MHA fraction than for theCaHA fraction. Hence. these distinct fractions appear to containrelatively labile materials, which encourage the study of thechemical forms of their nutrient stocks.

Therefore, we hypothesized that not all P associated withthese humic fractions were equally labile, and furthermore, thedegrees of biotically and abiotically labile P could be measuredby phosphatase hydrolysis and CV irradiation, respectively. In-formation obtained in this work would be helpful in answeringthe question whether humic matter calTies P in its molecule inadequate amounts for plant growth (Tan. 2003).

MATERIALS AND METHODSHumk Samples

The field trials that were sampled for this study involvedcontinuous cropping of irrigated lowland rice and were locatedat the International Rice Research Institute in Los Baiios,Laguna. Philippines. They were described in detail by 01k et al.(2000). Briefly, the field soils were collected from the triple-cropped Long-term Continuous Cropping Experiment (3-crop),

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Hu i a! Soil Science Volume .174, Number 2, February 2009

the double-cropped Long-Term Fertility Experiment (2-crop),and a rain-fed rice field that had not been flood irrigated forseveral years before sampling (dryland). In the 2-crop and 3-crop fields, soils receiving nonlimiting application rates ofNPKfertilizer were sampled (NPK and High N, respectively), and inthe 2-crop field, soil that did not receive NPK fertilizer was alsosampled (control). Soil types have not been mapped within thesefields, but all soil were of clay or silty clay texture and wereslightly acid (pH 6.2-6.8) (Mahieu et al., 2000a).

Details of the extraction of humic fractions from thesesoils were in Mahieu et al. (2000a). Briefly, the MHA was ex-racted by overnight incubation of the soil in 0.25 MNaOH. The

soil residues were then decalcified by repeated 0.2 M hydro-chloric acid washings until the pH of the washing supernatantwas less than 2.0. The CaHA fraction was extracted from thewashed soil residues by another overnight extraction in 0.25 MNaOH. Both fractions were then purified of soil and saltcontaminants through HF washing and dialysis in acid and watersolutions, and finally they were lyophilized. In these field soils,the MHA fraction contained 9% to 13% of total soil C, and the(aHA fraction contained 6% to 8%.

Humic fractions of each of the 2-crop replicate fields wereused in this study. However, composite fractions of replicate 3-crop or dryland fields were analyzed in duplicate in the labo-ratory because of the limited amounts of available material.Thus, the S.D. of the data for 2-crop fields represented thefield variation within treatments, and those of 3-crop anddryland fields demonstrated the variability in the laboratorymeasurements.

Stock solutions of these field replicate (2-crop) orcomposite (3-crop and dryland) humic fractions were made in0.05 MNaOH based on 10 mg of dry matter L' stock solutionand kept at 4 C until use. Part of the stock solutions was usedfor analysis of P and other selected elements by inductivelycoupled plasma-atomic emission spectroscopy, and the resultswere listed in Table 1.

Enzymatic HydrolysisPotato phosphatase, type IV-S acid phosphatase (EC

3.1.3.2), and fungal phytase (EC 3.1.3.8) from Aspergillus/icuuin were purchased from Sigma (St Louis, MO). Lyophi-lized nuclease P1 (EC 3.1.30.1) from Penicilliuni citrinuni waspurchased from Roche Diagnostics Corporation (Indianapolis,

IN). One unit (U) of enzyme activity was defined as the lib-eration of 1.0 !J.mol/L of the relevant product from appropri-ate substrates at appropriate incubation conditions based on thesupplier's information. The stock solutions of potato phospha-tases (10 U mL') and phytase (7 U mL) were dispensed inmicrocentrifhge vials in I mL each and stored at -20 C untiluse. Nuclease P1 was purchased in bottles containing 1 or 5 mg,and the buffer was directly added into the bottle to obtain anactivity concentration of 960 U mL - . Enzyme solutions werestored per manufacturer's instructions.

All enzymatic incubations were carried out at 37 C for20 h in a refrigerator-shaker (250 r.p.m.). Each incubation mix-ture (0.180 ml-) contained 0.045 mL of an enzyme-bufferworking solution (final concentration 100 mmol/L sodium ace-tate buffer, pH 5.0, potato phosphatase 0.25 U mL, fungalphytase 0.25 U mL ,and nuclease P1 5 U mL_t, separately orin combination) and an appropriate amount of a stock humicfraction based on 10 mg P Li incubation mixture (He et al..2004, 2006b). Sufficient distilled water was included in the in-cubation mixture to make the final volume of 0.180 mL. Aftera 20-h incubation interval at 37 C, soluble inorganic P (P)in these mixtures was determined as later described. Recentcross examination of the solution 31 P-NMR spectra of poultrylitter extracts before and after enzymatic incubation indicatedthat the enzymatic hydrolysis of relevant organic P species wascompleted under the established experimental conditions (Heet al., 2008).

UV IrradiationFor UV irradiation treatments, 1.0 to 1.2 mL of humic stock

solutions (pH 5.0) was placed in a closed I .5-mL quartz cuvette(1-cm light path with 4.5 cm high). A Spectroline 1 ISC- l shortwave UV pencil lamp (254 nm, 4.5 mW cm 2 , 5.5-cm high,Spectronics Corporation, Westbury, NY) was placed near thecuvette (1 cm away), and the sample was irradiated for 4 It at22 C (He et al., 2006b). The irradiated samples were thensubjected to enzymatic hydrolysis as previously described.

Soluble Inorganic P Determination andDesignation of Labile P Forms

Soluble inorganic orthophosphate (soluble P3 in the re-action mixtures was directly quantified by a modified molybde-num blue method (He and Honeycutt, 2005). Specifically, 50 or

TABLE 1. Selected Elemental Contents in Humic Fractions Used in This Study

Sample P CaMg Al FeMn Zn

MHA2-Crop control2CropNPKa3CrophighNbDrylandb

CaHA2-Crop control"2CropNPKc3-Crop-high-N"Drylandt'

2.08 0.160.09 0.01