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Department of Plant, Soil, and Environmental Sciences, University of Maine, Orono, ME 04469
* Corresponding author (erich{at}maine.edu).
Received for publication February 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: A285, molar absorptivity at 285 nm • Bmax, copper binding capacity • CB, carboxylic acid content • cKCu, pH-dependent conditional stability constant • LMW, low molecular weight • TA, total acidity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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One fraction of the soluble C pool receiving increased attention is the LMW (
1 kDa) fraction that includes aliphatic, aromatic, and amino acids. Though LMW organic acids comprise 10% of total soluble C, their high solubility and metal complexation capacity may disproportionately affect soil processes (Fox et al., 1990). Low molecular weight organic acids are probably building blocks of fulvic and humic acids and play a key role in nutrient uptake, mineral weathering, and soil genesis, and the alleviation of metal-induced plant root and aquatic organism toxicity (Huang and Schnitzer, 1986; Nigam et al., 2001). Distribution of LMW organic acids in the soil is influenced by microbial activity, vegetation, soil moisture, and clay content and, in agricultural systems, by the adoption of management practices such as reduced tillage and green manure or compost application (Bolan et al., 1994).
It has been suggested that land management practices encouraging the application or buildup of organic residues may contribute to the complexation and mobilization of soil metals (Evangelou and Marsi, 2001). For example, in agricultural soils, increased Cu solubility has been observed at pH 5.0 to 6.5 and has been correlated with the complexation of Cu by LMW carbon (Karathanasis, 1999). In such soils, elevated Cu burdens often result from the use of fungicides such as Bordeaux mix (Holmgren et al., 1993). Bordeaux mix (CuSO4 + lime) has traditionally been applied in vineyards and orange [Citrus sinensis (L.) Osbeck] groves and can be applied to field crops such as potatoes (Solanum tuberosum L.). The mean U.S. agricultural soil copper burden is 50 mg Cu kg-1 soil, and may reach, in some locations, an order of magnitude higher (Holmgren et al., 1993). The use of Bordeaux mix is also increasing as European Union directives both encourage the adoption of organic production practices and allow higher fungicide-derived soil Cu burdens than are permitted through the land application of sewage sludge (Flores-Velez et al., 1996). Copper burdens as high as 1500 and 500 mg Cu kg-1 soil have been measured in French vineyard and Kenyan coffee (Coffea spp.) plantation soils, respectively (Flores-Velez et al., 1996).
One potential concern with soils containing high copper burdens is the formation of LMW soluble Cu complexes, the small size of which may render them mobile in soil solution. Such mobility may have implications for plant root development, and there may be percolation through soils or runoff of Cu complexes into adjacent water bodies. Guggenberger et al. (1994) studied hydrophobic and hydrophilic fractions of soil solution dissolved C. They concluded that the hydrophilic fraction had a higher Cu complexation capacity than the hydrophobic fraction and that, due to its relative soil mobility, probably played a significant role in Cu leaching. Kuiter and Mulder (1993) suggested that the LMW fraction of forest litter layer and humus horizon dissolved C extracts were responsible for 50 to 99% versus 30 to 60% of Cu-binding capacity, respectively. Romkens et al. (1999) cautioned that the significant role that LMW acids play in the complexation and transport of soil Cu should not be overlooked.
The principal objective of this study was to monitor plant material decomposition for evidence of humification-related transformations within the bulk and the LMW fraction of the soluble C pool. While the humification-related stabilization of C has traditionally influenced research into the C dynamics of undisturbed ecosystems, the stabilization of soil C is also relevant for agricultural systems following organic or conservation-oriented production. Aromaticity was probed as a measure of humification through the Folin–Ciocalteau reagent, fluorescence spectroscopy, and absorbance at 285 nm (A285). This wavelength was chosen because 
bond electron transitions occur at this wavelength for both simple and complex aromatic compounds (Chin et al., 1994). Fluorescence fingerprints may likewise aid in differentiating source and molecular structure of complex environmental mixtures (Senesi et al., 1991). A second objective was to determine whether plant material–derived soluble C could desorb and complex resin-bound Cu and to assess how early-stage humification affected Cu complexation capacity. As both aromaticity and polymerization have been correlated with the soluble C pool's affinity for metal pollutants (Chin et al., 1994), Cu complexation capacity provides a relevant means of assessing the environmental effects of early-stage humification.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Incubation
An 8-wk incubation was performed in 250-mL polyethylene screw-top bottles. Acid-washed silica sand (80 g) was mixed with soil (2.55 g) plus plant material at a concentration of 2.0 g C incubation bottle-1. Mixed sand, plant material, and soil were moistened to 20% by weight with deionized water (crimson clover) or deionized water containing N, P, and K as described above (wheat straw). There were two replicates of each amendment plus two blanks (sand plus soil with no amendment). Bottles were maintained in a 22°C incubator loosely capped to permit O2 entry, weighed every 2 d to determine water content, and remoistened when necessary. At Days 7, 14, 28, 42, and 56, bottle contents were extracted for 30 min with deionized water (125 mL) on a wrist-action shaker, centrifuged for 30 min at 3000 x g, and vacuum-filtered (90 kPa) through Nylaflo 0.2-µm nylon membrane filters (Pall Corporation, Ann Arbor, MI). Material collected on the filters was returned to the incubation bottles. Blanks were extracted at the same time intervals. A Time 0 extraction involved separately prepared bottles that were destructively sampled. These bottles were prepared as for the incubation and extracted following the same protocol.
Analyses
Supernatant analyses included pH (ROSS combination electrode; Thermo Orion, Beverly, MA), electrical conductivity (Model 2052 conductivity meter; VWR International, West Chester, PA), and water-extractable C (TOC-500; Shimadzu, Kyoto, Japan). Cations (Al, Ca, Fe, K, and Mg) and P were measured with inductively coupled plasma–atomic emission spectrometry (Model 975, Plasma AtomCorp; or TJA IRIS 1000, Thermo Elemental, Franklin, MA). A 5-mL aliquot of each extract was diluted to 3 mM C to measure absorbance at 285 nm (Spectronic 2000; Bausch & Lomb, Rochester, NY). To collect the LMW fraction, a 50-mL aliquot of each vacuum-filtered aqueous extract was pressure-filtered (using N2 gas at 0.35 MPa) through a 50-mL Diaflo stirred cell fitted with a YM1 1000 MWCO ultrafiltration membrane (Millipore, Billerica, MA). Filtration efficiency can be compromised by the overconcentration of solution retentate (Wershaw and Aiken, 1985), and filtration was deemed operationally complete when 60% of the extract (i.e., 30 mL) had passed through the cell.
A 10-mL aliquot of both ultrafiltration permeate and the bulk (i.e., not ultrafiltered) extract were analyzed fluorimetrically (F-4500 fluorimeter; Hitachi, Tokyo, Japan). Samples were prepared with standardized carbon concentration (3 mM), ionic strength (10 mM using KCl), and pH (5.5). Samples containing less than 3 mM C were not analyzed. Excitation-emission matrices were generated to allow examination of changes in aromatic peak location over time and between plant materials. Instrument parameters were: EX and EM slits, 5 nm; response time, 8 s; and scan speed, 1200 nm min-1. Aliquots (5 mL) of the ultrafiltration permeate and the bulk sample were analyzed for soluble phenolic compounds using the Folin–Ciocalteau reagent with a ferulic acid standard (Blum et al. 1992).
Acidity and Cu complexation capacity of organic ligands were examined for both materials at Time 0 and following a separate 7-d incubation. Incubations and extractions were performed in 250-mL polyethylene screw-top bottles following the already defined protocol. Potentiometric titrations were conducted with extracts diluted to either 10 or 20 mM C in 50 mL of total solution volume using CO2–free deionized water (Ohno and Cronan, 1997). Extracts were bubbled continuously with N2 to further minimize CO2 contamination, and before titration were brought to pH 3.0 (HCl) and equilibrated for 15 min. Titrations were undertaken with 0.048 M standardized NaOH dispensed in either 0.1- or 0.05-mL aliquots. Extract ionic strength was standardized at 10 mM using 1.0 M KCl. A 0.01 M KCl solution was titrated over the experimental range as a blank correction, and all results were corrected for the dissociation of protons from phosphate. Extracts were titrated between pH 3.0 to 11.0 to assess total acidity with carboxylic acid content defined operationally as titratable acidity at pH 7.0. Average ionization constants were determined with a correction made for titration outside the range of pH 4.0 to 10.0 (Albert and Serjeant, 1988).
The copper binding capacity (Bmax) and pH-dependent conditional stability constant (cKCu) for Cu–water-extractable C complexes were calculated for both bulk extracts and the extract LMW fraction using the equilibrium ion exchange method (EIM) (Luster et al., 1994, 1996). This method assumes that in low ionic strength solutions the nonlinear portion of adsorption isotherms may be used in analysis (Luster et al., 1994). A Bio-Rad (Hercules, CA) 50W X-8 200-mesh strong cation exchange resin was converted to Na+ form and used as a soil proxy. Using CuNO3, Cu solutions were prepared at six concentrations ranging from 3.0 µM to 1.0 mM. Water-extractable C concentration (25 mg L-1 C) and background solution ionic strength (0.01 M using NaNO3) were standardized.
Sixty milligrams of Na+–form resin, appropriately measured aliquots of Cu solution, and NaNO3 were weighed into 60-mL polyethylene screw-top bottles and equilibrated for 1 h at 21°C on a tabletop shaker. Total solution volume was brought to 30 mL and total C concentration to 25 mg L-1 with the addition of water-extractable C aliquots. Solution pH was standardized at pH 6.0 (NaOH or HNO3) and bottles were reequilibrated on a shaker table in a 4°C incubator for 24 h. Solutions were filtered (0.2 µm) following incubation to separate resin from the filtrate and the filtrate was analyzed for Cu using inductively coupled plasma–atomic emission spectrometry (TJA IRIS 1000). Initial Cu–resin equilibrium was determined through a reference isotherm (i.e., resin + Cu solution + NaNO3) that allowed subsequent determination of Cu redistribution in the presence of organic ligand. Studies examining the interaction between water-extractable C and purifying resins have calculated C sorption concentrations reaching 15% (Ohno and Cronan, 1997). This suggests that a desalting pretreatment for fresh crop residue extracts may alter bulk water-extractable C composition and bias experimental results; therefore, no desalting pretreatment was used in this study.
Copper binding capacity was modeled as involving the noncompetitive interaction of two binding site classes: (i) a strong binding (L1) class defined by inner-sphere complexation and (ii) a weak binding (L2) class defined by outer-sphere complexation. The model equation is defined as:
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Spectrophotometric molar absorptivity (A285) values at Time 0 were approximately 67 L mol-1 C cm-1 for both wheat straw and crimson clover and increased to approximately 330 L mol-1 C cm-1 for both materials over the incubation (Fig. 3)
. The greatest incremental increase for both materials occurred during the first 7 d of the incubation. This increase probably resulted in part from the initial degradation of labile, nonhumic components. The term A285 may be converted to an estimate of percent aromaticity (
) via the regression equation of Chin et al. [i.e., = 0.05(A285) + 6.74, r2 = 0.90, with defined as percent C in the 110- to 160-ppm shift region of 13C cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR)] (Chin et al., 1994). When calculated here over 8 wk, decomposition resulted in aromaticity increase from approximately 10 to 23% for both plant materials. Published values (generated via the same regression equation) to which these numbers may be compared include 23% aromaticity (A285 = 330 L mol-1 C cm-1) for chestnut (Castanea spp.) leaf litter and 23 and 13% aromaticity (i.e., A285 = 308 and 150 L mol-1 C cm-1, respectively) for river water and Antarctic lake water fulvic acid, respectively (Brunner et al., 1996; Chin et al., 1994). Values of A285 exceeding 220 L mol-1 C cm-1 (18% aromaticity) may signal the presence of fulvic-like refractory organic compounds (Rosten and Cellot, 1995).
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ex/em = 270–280/335–350) was present in Week 0 scans of both materials with a peak intensity that was higher for crimson clover than wheat straw (Fig. 4)
. Peak A, disappearing after Time 0 for wheat straw and after Week 1 for crimson clover, has been identified as the aromatic amino acid tryptophan (Brunner et al., 1996). Fluorophore B (ex/em = 250–260/440–460), present throughout the incubation in wheat straw and crimson clover bulk water-extractable C, has not been positively identified. A humic acid peak has been recorded at ex/em = 270/460, though the scan was conducted at pH 10.0 (Wolfbeis, 1985). Fluorophore C (ex/em = 310–315/435–445), present only in Time 0 extracts of both materials, has been observed in leaf and needle extracts and correlated with the peak location of hydroxybenzoic and hydroxycinnamic acids, and/or coumarins or flavonoids (Brunner et al., 1996; Wolfbeis, 1985). Fluorophore D (ex/em = 325/435–445), appearing at Week 1 and present in all subsequent scans, has been correlated with both terrestrial and aquatic fulvic and humic acids and does not represent a reflection of the Fluorophore B peak (Senesi et al., 1991; Blum et al., 1992). Yang et al. (1994) observed both Fluorophores C and D in a study of pine needles and forest O horizons and noted that as excitation peaks shift to longer wavelengths for extended conjugated structures the shift witnessed between needle and O horizon scans probably resulted from increasing humification of simple aromatic monomers. This view is supported by fluorescence scans generated from C extracted from fresh plant residues and animal manures (Ohno and Crannell, 1996). With these materials, ex increased from 310 nm [(crimson clover and hairy vetch (Vicia villosa Roth subsp. villosa)] to 349 nm (poultry and cattle manure) with em remaining unchanged (435–450 nm). If Fluorophores C and D thus correspond to simple and humified aromatic structures, respectively, then the excitation wavelength shift between Time 0 and Week 1 suggests the inception of monomer polymerization.
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bonds were still present at Day 60, the compounds involved had shifted from simple aromatics toward those with a more complex, humified structure.
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In the present incubation, the measured increase in phenolic acidity for both wheat straw and crimson clover is consistent with the increase in relative aromaticity as measured by A285 and the peak excitation wavelength shift from Fluorophore C to D. The apparent concentration of titratable acidity in the LMW fraction may be interpreted in several ways. While humification involves a dynamic transformation toward higher molecular weight components, this process also involves breakdown steps in which simple molecules are enzymatically cleaved from larger compounds. These molecules may exist ephemerally as intermediate, acidic products before the reactive formation of humic structures. A second possibility involves the identification of low molecular weight fulvic acids (Fox, 1995). As humification progresses through low molecular weight polymerization reactions involving acidic functional groups, TA may increase with the structural complexity and stabilization that accompanies polymerization. That the increase in acidity was more pronounced in the LMW fraction than the bulk extract suggests both the scale invariance of the polymerization process and that the early stages of humification are initially more visible in lower molecular weight components.
It is difficult to define what percentage of the increase in titratable acidity resulted from the generation of acidic breakdown products and LMW fulvic acids and what resulted from the degradation of labile, nontitratable components. The removal of such nontitratable materials would clearly play a role in the acidity increase measured, though to what extent it is the explanation is uncertain. In the context of defining early-stage humification, however, the distinction between LMW polymerization and simple component degradation may prove arbitrary. These two processes, operating in tandem, may represent complementary facets of the same dynamic transformation occurring in the water-extractable C pool during the early stages of humification.
Copper Complexation
In analyzing binding capacity data, multiple factors clearly influence both the magnitude of Cu-ligand stability constants (log cKCu) and maximum ligand binding capacity (Bmax). At Time 0, stability constants for both complexes (L1 and L2) were greater for wheat straw bulk extract than for crimson clover (Table 2). Following incubation, stability constants decreased for wheat straw and increased for crimson clover, such that ultimate Week 1 strong binding values (log cKCu1) were similar between materials.
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Conditional stability constants for wheat straw and crimson clover water-extractable C were compared with published values to explore the functional group composition of each class of binding complex. Relevant published stability constants include log cKCu = 7.5 for Cu complexation with catechol or histidine, log cKCu = 6.9 for a juniper leaf litter extract, and log cKCu = 6.2 to 7.0 for Cu complexation with a peat-derived humic acid (Luster et al., 1996; Taga et al., 1991). All values were calculated at pH 5.5 to 6.0 and with 0.1 to 0.01 M ionic strength. An electron spin resonance study of juniper (Juniperus spp.) leaf litter extract suggested that strong binding involved catecholates and amino acids (Luster et al., 1996). Infrared spectroscopic analysis of Cu–humic acid complexes suggested that strong binding was predominantly a function of carboxyl group content (Taga et al., 1991).
For crimson clover, the greater Time 0 Bmax1 (relative to wheat straw) was potentially a function of a higher concentration of aromatic amino N, greater TA and CB, and/or higher initial percentage of Folin–Ciocalteau reactive soluble phenolic compounds (i.e., 6.8 versus 3.4% of bulk water-extractable C). The relative decrease in crimson clover Bmax1 over the 7-d incubation was potentially affected by the diminishing concentration of aromatic amino N. As this decrease was coupled with increases in both humification and titratable acidity, it may correspond to a change in the relative participation of various strong binding sites. This suggests that while the maximum binding capacity of N-bearing sites is higher, binding affinity may increase with O-dominated geometry. For wheat straw, while aromatic amino N was present in the Time 0 extract, the concentration was low, and this suggests that a larger percentage of Time 0 strong Cu binding involved O. The dominance of O-bearing sites may explain the greater Cu binding affinity of wheat straw water-extractable C.
A clear pattern was not discerned for weak binding capacity. If the anomalously high value for crimson clover Time 0 is excluded, bulk extract stability constants are log cKCu = 4.6 to 5.8. Values in this range are consistent with published values including log cKCu = 4.9 to 5.6 for weak, hydroxyl-mediated Cu–humic acid binding and log cKCu = 4.1 for a Cu–juniper leaf litter complex (Taga et al., 1991; Luster et al., 1996). This latter value was characterized by the researcher as atypical for a Cu-inner sphere complex with known model compounds (Luster et al., 1996). All values were calculated at pH 5.5 to 6.0 and with 0.1 to 0.01 M ionic strength.
Discussion exists as to whether metals preferentially bind to the LMW fraction. Vulkan et al. (2002) analyzed the solution-phase speciation of metals extracted from soils amended with sewage sludge and concluded both that the majority (91%) of solution-phase Cu was complexed and that the predominant complexing agent was LMW (<1 kDa) carbon. While comparisons in the current experiment are not strictly between distinct molecular weights, the strong binding capacity of the LMW fraction may be analyzed relative to the bulk extract at a standardized C concentration. For crimson clover, at Time 0 the LMW fraction strongly bound 28 µmol Cu mmol-1 CB versus 58 µmol Cu mmol-1 CB for the bulk extract. Following incubation, binding capacity per LMW carboxylic acid site increased to 48 µmol Cu mmol-1 CB with binding capacity per bulk extract charge site decreasing slightly to 53 µmol Cu mmol-1 CB. This increase represents an increase in the relative contribution of LMW binding sites to the total Cu complexation capacity of crimson clover water-extractable C. For wheat straw, at Time 0 the LMW fraction strongly bound 22 µmol Cu mmol-1 CB versus 34 µmol Cu mmol-1 CB for the bulk extract. While binding capacity per charge site increased absolutely for the LMW wheat straw extract (to 36 µmol Cu mmol-1 CB) following incubation, the relative percent contribution to bulk extract charge did not change. For both materials, however, the LMW fraction was responsible for at least 64% of total strong binding capacity (as defined per mmol CB) following incubation. As the contribution of the LMW fraction to total water-extractable C concentration decreased significantly over this time interval (from 71 to 15% for wheat straw and 62 to 33% for crimson clover) this suggests that the LMW fraction is indeed a disproportionately effective strong metal complexing agent.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Both wheat straw and crimson clover water-extractable C complexed resin-bound Cu. The potential inner-sphere binding capacity of the plant residue extracts was 0.11 to 0.55 mmol Cu bound g-1 C with the higher values associated with the LMW fraction of water-extractable C. For the bulk water-extractable C there was no consistent pattern of increasing binding capacity associated with the increasing structural complexity observed after incubation. For the LMW fraction, maximum strong binding capacities increased with increasing structural complexity due to decomposition. Crimson clover residue generated water-extractable C containing a significant fraction of LMW C even after 8 wk of decomposition. The continuing presence of LMW C during crimson clover decomposition and the relatively strong Cu binding capacity of this fraction suggest that crimson clover residue has the potential to enhance soil Cu mobility.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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