2000 Progress Report: Dependence of Metal Ion Bioavailability on Biogenic Ligands and Soil Humic Substances

EPA Grant Number: R825960
Title: Dependence of Metal Ion Bioavailability on Biogenic Ligands and Soil Humic Substances
Investigators: Higashi, Richard M. , Fan, Teresa W-M. , Lane, Andrew N.
Institution: University of California - Davis
EPA Project Officer: Lasat, Mitch
Project Period: January 1, 1998 through December 31, 2001
Project Period Covered by this Report: January 1, 1999 through December 31, 2000
Project Amount: $345,816
RFA: EPA/DOE/NSF/ONR - Joint Program On Bioremediation (1997) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management


Organic matter (OM) can strongly affect metal ion binding to soil and sediment. In fact, production of a major form of OM-low molecular weight organic ligands-is the principal mechanism by which plants and microbes acquire metal ions. Consequently, the chemistry of biogenic OM is the key to understanding mechanisms of bioavailability for bioremediation purposes.

The complex interaction between metal ions, biogenic ligands, and humic substances must be understood to engineer the proper organisms and conditions for bioremediation of metal ion contamination. The objectives of this research project are to: (1) determine the sorption behavior of metal ions on isolated humic substances in the presence of biogenic and synthetic ligands; (2) conduct a subset of experiments from (1) as longer-term aging experiments; (3) investigate the properties of isolated humic substances that are involved in (1) and (2); (4) assess the relationship of (1) and (2) to metal ion bioavailability to vascular plants, including evaluation of soils from a federal demonstration site (McClellan Air Force Base); and (5) use the findings from these objectives to identify key rhizospheric processes that regulate metal bioavailability.

Progress Summary:

Plant Exudates-Metal Interactions. The purpose of this set of experiments is to examine the differences between exuded and internal metal ion ligand (MIL) among wheat genotypes, which are potentially major organo-metal forms that can enter the soil. Understanding the biological parameters that can influence the uptake of metals by plants and resulting bioformation of these organo-metal forms is a critical step in understanding metal-humic interactions in real systems. Briefly stated, the study had multiple findings that provided entirely new information to the field. These were: (1) production of the principal exuded MIL involved in transition metal uptake, 2'DMA, was not involved in uptake of Cd2+, which was contrary to expectations; (2) no other exuded MIL appeared to be associated with uptake of Cd2+; and (3) metals such as Zn co-accumulated with Cd, despite the absence of production of 2'DMA that is normally required for uptake. In contrast, as predictable from the literature, we found the major internal MIL, phytochelatin (PC), to be highly correlated with Cd uptake.

Establishing Long-Term Soil Incubation Pots for Experiments. We started a trial to establish the necessary setup and conditions for long-term (months) soil experiments. In particular, the aim was to test a setup where we could incubate soil for months in small pots with controlled air and water leachings. If successful, this system would be used for stable-isotope labeling brownfield McClellan Air Force Base (AFB) and agricultural University of California-Davis Long Term Research in Agriculture Sustainability test site (LTRAS) soil OM with 13C and 15N using labeled materials, and for following the fate of Cd-contaminated plant material (e.g., PC-bound Cd). For this trial, AFB and LTRAS soils were amended using unlabeled wheat straw and cow manure.

Soil was set up in the funnel portion of an airtight filter funnel system, with 100 ml per minute of air passed over it. The setup is shown in Figure 1. The inlet air was humidified to prevent soil dryout, and made NH3 and CO2-free. First, compressed air was passed through both aqueous boric acid and KOH bubblers to remove any NH3 and CO2, followed by a -20°C trap to remove any oil vapors and moisture to ensure proper functioning of rotameters assigned to each chamber. Each rotameter was followed by bubbling through water to add moisture, thus preventing soil dryout. The outlet air was bubbled through boric acid and KOH solutions to trap NH3 and CO2, respectively, for analysis. The evolved NH3 and CO2 in the traps were determined by ion selective electrode and ion chromatography, respectively.

Figure 1. Airtight Soil Chambers. These were developed for long-term incubation and leaching experiments. Air scrubbers (not shown) removed water, CO2, and NH3 from inlet air, which was then humidified in the bottles shown to prevent drying of soil. Exit air was routed through CO2 and NH3 traps for analysis. Periodically, the soils were leached to the bottom chamber. The whole system is sited within a temperature-controlled plant growth chamber. This system served the threefold purpose of analytical development, initial survey of soil amendment effects, and trial runs before committing the expensive stable isotopes for incorporation into soil OM.

The soil also was leached periodically with water to determine leachability changes in Cd and other elements. Nanopure-grade water was added to soil from a sealed reservoir and collected using vacuum in a sealed vessel below the soil funnel; this procedure minimized contamination of the air and soil. Elements were analyzed by energy dispersive X-ray fluorescence spectroscopy (ED-XRF) using high sensitivity-thin film techniques (Meltzer and King, 1991). After 2 weeks, the system seemed to stabilize in terms of soil moisture, leachate elemental composition, and CO2 and NH3 generation (data not shown). Pyrolysis-gas chromatography-mass spectrometry (GCMS) analysis of soils after 9 weeks revealed multiple differences in wheat straw degradation and/or incorporation into the OM; for example, there appeared to be over 80 percent decline of lignoid constituents in the LTRAS soil, while the AFB soil showed essentially no change. This result is shown in Figure 2. We detected no difference in Cd leaching among the experiments, which remained small at 9 weeks. We continued the experiments for a total of 25 weeks. Analysis of the final samples were conducted in the first quarter of 2001, and reported in the next annual report.

Figure 2. Pyrolysis-GCMS Chromatograms of Lignin Markers in Soil. Similar masses of soil samples were directly loaded into the instrument and analyzed. Note that the patterns qualitatively look the same. However, as indicated by the ordinate scale, the lignin has declined by over 90 percent in the Ag soil, while there was very little change in the AFB soil. This difference is possibly due to differences in microbial communities in the two soils, despite receiving the same organic amendment (wheat) as the major carbon source.

Stable Isotope Labeling of Soil. Following the successful testing of the soil chambers (see above), we set up similar soil chamber experiments with the purpose of stable-isotope labeling of soil OM. This labeled soil would be used as the base material in experiments aimed at tracking turnover of soil organic constituents in relation to mobility (as measured by leachability) of Cd (see section below). We initiated collaboration with Dr. Robin Brigmon at the Department of Energy (DOE) Savannah River Site (SRS) on uncovering age markers in soil OM that are associated with heavy metal sequestration in soils. This information would be valuable towards evaluating metal ion stability in contaminated field sites, and directing bioengineering efforts in stabilizing metals and radionuclides at these sites. The SRS soil was chosen over the AFB soil we had previously used, due to the extensive work available on the SRS sites, including phytoremediation, microbioremediation, and microbial community assessments.

The soil chambers were set up and conducted as described above, except that universally-labeled 13C-glucose and 15N-nitrate were amended to soil chambers instead of wheat straw, and headspace GCMS was used to determine the air-trapped 13C/12C and 15N/14N ratios, while leached 13C and 15N was determined using combustion isotope radio mass spectrometry (cIRMS). The stable-isotope 13C and 15N experiments were conducted separately in order to simplify subsequent pyrolysis-GCMS analysis; thus 13C glucose experiments also were supplemented with unlabeled nitrate, while 15N nitrate experiments were supplemented with unlabeled glucose.

After 8 weeks, we analyzed the soil using pyrolysis-GCMS, and we noted several interesting observations. First, the incorporation of isotope in both 13C and 15N experiments were readily evident in most peaks, indicating extensive incorporation of the isotopes in a wide range of soil OM constituents. Secondly, examination of a peak (methyl indole) representing the peptide bond atoms showed two distinct pools of peptidic materials. For the 13C experiment, those with all carbons labeled and those with no carbons labeled. The interpretation of the pyrolysis-GCMS pattern is explained in Figure 3. This indicates that partial carbon incorporation into peptide bonds was nonexistent under these conditions, such that incorporation of glucose carbons into peptides were an all-or-none process. This also indicates that, after many weeks, there remains a pool of peptidic material that does not turn over in this system. This is consistent with our earlier finding of considerable pools of peptidic material remaining in natural soil humics (Fan, et al., 2000), contrary to the former assumptions, based on the absence of direct evidence, that peptidic material will be rapidly turned over in soil.

Figure 3. Analysis of 13C Labeling in Soil OM by Pyrolysis-GCMS. Pyrolysis-GCMS was used to determine the extent of labeling in the soil; the example shown here is for peptidic groups. Pyrolysis-GCMS causes the peptidic group to form indole, C8NH10, with an expected natural-abundance isotopic ion at m/z 117. Depending on how many of the carbons were labeled, we expected a distribution of peaks from m/z 117 -> 125. The top panel plots single ion traces in this range: it was approximately 50 percent unlabeled (tallest peak in black; resistant to exchange) and 40 percent fully-labeled (next tallest peak in light blue; rapidly incorporated). The mass spectrum of this peak (bottom panel) clearly shows this labeling of peptidic structures at both extremes (i.e., primarily at m/z 117 (not labeled) and m/z 125 (all carbons labeled)).

Production of Cd-Contaminated Plant Material. The plant-metal-MIL study (Fan, et al., 2001) raised the following issue for this project: because it is generally accepted that most of the internal Cd in plants is chelated to PCs, this raises the question of the fate of this organo-metal complex as shoots and roots degrade in the soil. To prepare for experiments that address this, we started a larger-scale hydroponic production of wheat plants contaminated with several levels of Cd exposure, including controls.

Fan TWM, Higashi RM, Lane AN. Chemical characterization of a chelator-treated soil humate by solution-state multinuclear two-dimensional NMR with FTIR and pyrolysis-GCMS. Environmental Science and Technology 2000;34:1636-1646.

Fan TWM, Baraud F, Higashi RM. Genotypic influence on metal ion mobilization and sequestration via metal ion ligand production by wheat. In: Nuclear Site Remediation. Eller PG, Heineman WR, eds. American Chemical Society, Washington, DC, 2001, pp. 417-431.

Meltzer C, King BS. Trace element analysis of solutions at the PPB level. In: Advances in X-Ray Analysis. Barret CS, ed. New York, Plenum Press: pp. 41-55.

Schultz LF, Young TM, Higashi RM. Sorption-desorption behavior of phenanthrene elucidated by pyrolysis GCMS studies of soil organic matter. Environmental Toxicology and Chemistry 1999;18:1710-1719.

Future Activities:

Analyses of the final samples from long-term brownfields soil experiment will be completed in the first quarter of 2001. The stable-isotope labeling of soil, which started at the end of this period, will continue for an undetermined time; the duration will depend on the results of periodic analyses for istopic incorporation of 13C and 15N into soil OM. There will be continued studies on humic interactions of plant-metal uptake; in particular, they will focus on one or two strains of wheat, rather than comparisons of various strains as described here. Lastly, because PCs appear to be a major internal chelator of Cd in plants, the fate of the PCs and PC-Cd complexes when plant material humifies in soil will be examined. This will start with development of methods to efficiently analyze for PC in soils.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 28 publications 8 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Fan TW-M, Higashi RM, Lane AN. Chemical characterization of a chelator-treated soil humate by solution-state multinuclear two-dimensional NMR with FTIR and pyrolysis-GCMS. Environmental Science & Technology 2000;34(9):1636-1646. R825960 (1999)
R825960 (2000)
R825960 (Final)
R825433 (Final)
R825433C007 (Final)
  • Abstract: ACS-Abstract
  • Supplemental Keywords:

    soil, sediments, adsorption, chemical transport, heavy metals, bioremediation, environmental chemistry., Scientific Discipline, Geographic Area, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Contaminated Sediments, Remediation, Environmental Chemistry, State, Fate & Transport, Bioremediation, West Coast, fate, fate and transport, sorption, biogenic ligands, humic substances, NMR spectroscopy, pyrolysis GCMS, rhizospheric, contaminated sediment, soils, adsorption, chemical transport, vascular plants, metal ion bioavailability, FTIR microspectroscopy, wheat, California, phytoremediation, sediments, soil humic substances, biogenic organic matter, heavy metals, metal compounds, metals, fluoroescence spectrophotometry, duckweed

    Progress and Final Reports:

    Original Abstract
  • 1998
  • 1999 Progress Report
  • Final Report