Final 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 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. It is for this reason that the chemistry of biogenic organic matter is the key to understanding mechanisms of bioavailability for bioremediation purposes.

Therefore, 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. This led to the original objectives for this project, which were: (1) to determine the sorption behavior of metal ions on isolated humic substances in the presence of biogenic and synthetic ligands; (2) to conduct a subset of experiments from (1) as longer-term aging experiments; (3) to investigate the properties of isolated humic substances that are involved in (1) and (2); (4) to 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 (AFB)); and (5) to use findings from (1) thru (4) to identify key rhizospheric processes that regulate metal bioavailability.

Summary/Accomplishments (Outputs/Outcomes):

Isolated Humic Studies. Studies of bioavailability of soil pollutants such as Cd2+ to plants involves a complex mixture of other metals (nutrients), inorganic anions, and a suite of biogenic ligands such as organic acids. A major organic phase involved in both organic and metal pollutant retention includes humic substances (HS). Understanding the chemistry imposed by these complex mixtures is crucial, and therefore is a major focus of our effort.

Using pyrolysis-gas chromatography mass spectrometry (GCMS) and 2-dimensional nuclear magnetic response spectrometry (NMR), we uncovered kinetic and structural interactions of certain biogenic ligands (Cd2+ and a soil HS), which limit the formation of ligand-Cd complexes. This, in turn, might affect bioavailability of metal ions to plants. A combination of new HS isolation methods, several 2-dimensional NMR techniques, and pyrolysis-GCMS sketched a mobile nature for the peptidic side groups, but a relatively rigid structure for lignoid moeities. This means that the peptidic moeities may be more accessible to waterborne contaminants than the lignoid groups.

Plant Exudates-Metal Interactions. The purpose of this set of experiments was to examine the differences between exuded and internal metal ion organic ligands (MIL) among wheat genotypes, which are potential 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. The study had multiple findings that were entirely new information to the field.

We found that the production of the principal exuded MIL involved in transition metal uptake, 2'DMA, was not involved in uptake of Cd2+, which contradicted our expected results. No other exuded MIL appeared to be associated with uptake of Cd2+. Metals such as Zn and Fe co-accumulated with Cd, despite the absence of production of 2'DMA that is normally required for uptake. In contrast, as predicted from the literature, we found the major internal MIL, PC, to be highly correlated with Cd uptake.

Humic Interactions in Plant-Metal Uptake. Background information on the role of soil OM, such as plant root exudates and especially soil HS, in heavy metal accumulation by plants is grossly lacking, yet necessary for understanding heavy metal mobilization by plants from soils.

HS interactions with plant metal uptake showed that HS did not attenuate-and even slightly increased-the accumulation of some transition metals and Cd in roots. HS also stimulated plant growth, improved MIL production, and alleviated part of the Cd-induced growth inhibition. Under Cd treatment-with or without HS-phytosiderophores and other exuded MIL were suppressed, yet the uptake of transition metals (Fe, Ni, Cu, Zn, and Mn) were enhanced.

Role of Thiol-Rich Peptides. Metal-binding thiol (SH)-rich peptides are ubiquitous in organisms and have been recognized to serve critical cellular functions including storage of essential metals, detoxification of heavy metals, and protection against oxidative damage.

Lacking a convenient screening technique, we developed a method that combined fluorescent tagging of thiol-rich peptides by bromobimane with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), confirmed by 2-dimensional NMR and liquid chromatography (LC)-tandem mass spectrometry (MS) analyses. This method allowed a fast and simultaneous assay of both phytochelatins (PC) and metallothionein-like proteins. Our results indicated that both SH-rich peptides and non-peptidic MIL may be important to sequestering heavy metals in plant tissues. When plants die or their shoots are harvested, the plant root litter decomposes, and the fate of these metal-PC complexes in soils will need to be understood to address metal stabilization or remobilization.

Establishing Long-Term Soil Incubation Chambers for Experiments. The aim was to test a setup where we could incubate soil for months in small chambers with controlled air and water leachings. If successful, this system would be used for stable-isotope labeling of soil OM with 13C and 15N, and for following the fate of Cd-contaminated plant material (e.g., PC-bound Cd). For this trial, McClellan AFB and University of California-Davis Long Term Research in Agriculture Sustainability Test Site (LTRAS) soils were amended using unlabeled wheat straw and cow manure.

These soils were allowed to age for a total of 25 weeks, during which, microbial activities (CO2 and NH3 emissions), and metal ion leaching were monitored. Final analysis indicated that NH3 emission was higher in manure-amended soils (both Ag and AFB) than in the wheat straw-amended counterparts. The opposite was observed for the total amounts of leached organic carbon.

Pyrolysis-GCMS revealed a cacophony of detailed changes, as expected. Lignin residues are interpreted as OM, because microbes do not produce lignin structures. Both wheat straw and manure amendments added intense lignin markers to the soils. The lignin marker intensity declined over 90 percent for the Ag soil, but not for the AFB soil. One possible interpretation of this result is that the Cd-contaminated AFB soil failed to promote the lignin-degraders, despite the fact that both soils received the same major organic carbon source. Thus, this approach is poised to provide comparative biogeochemical information, with regards to techniques that probe the microbial community.

Stable Isotope Labeling of Soil. Following the successful testing of the soil chambers, 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 next section below). The stable soil chamber system was then used over a period of 34 weeks to generate 13C and 15N-labeled HS in the Department of Energy Soil River Site (SRS) soil for use in soil OM turnover measurements. Analytical methods for isotope-enriched soil OM structure, PC analysis, and collaboration to examine microbial communities were made available online. Partial carbon incorporation into peptide bonds was not included in the soil labeling experiment, 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.

Stable-Isotope Labeled SRS Soil Column Experiment With Cd-Bioaccumulated Wheat Root Powder and Organic Amendments. The above sections were brought together to conduct a unique experiment. The stable isotope-labeled SRS soils, spiked with a Cd-bioaccumulated wheat root as the contamination source (which also serves as a source of PC-bound Cd) were used in soil-aging microcolumn experiments with the following amendments: cellulose (polysaccharide), wheat straw (lignocellulose/silicate), pine shavings (lignocellulose/phenolic), chitin (nitrogenous polysaccharide), and bone meal (divalent cation phosphate). The reasons for these amendments are as follows: (1) cellulose is a major single-substance terrestrial OM input to soil from plant matter; (2) lignocellulose/silicate are representative of grassy and other plant material; (3) lignocellulose/phenolic are representative of pine forest inputs; (4) chitin is representative of insect and fungal OM inputs to soil; and (5) phosphate from bone meal is intended to provide a biological source of divalent cation binder for comparison.

Soil OM turnover experiments by aging with organic amendments are underway, using Cd-bioaccumulated roots as the contaminant source in 13C and 15N-labeled SRS soil microcolumns. Results show a strong dependence of element leaching on amendments.

Journal Articles on this Report : 4 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
  • Journal Article Fan TW-M, Lane AN, Shenker M, Bartley JP, Crowley D, Higashi RM. Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS. Photochemistry 2001;57(2):209-221. R825960 (2001)
    R825960 (Final)
    R825433 (Final)
    R825433C007 (Final)
  • Abstract from PubMed
  • Full-text: OSTI-Full Text PDF
  • Abstract: ScienceDirect-Abstract
  • Journal Article Fan TW-M, Lane AN, Higashi RM. An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins. Phytochemical Analysis 2004;15(3):175-183. R825960 (Final)
    R828676 (Final)
    R828676C003 (2003)
  • Abstract from PubMed
  • Abstract: Wiley Online
  • Journal Article Higashi RM, Fan TW-M, Lane AN. Association of desferrioxamine with humic substances and their interaction with cadmium(II) as studied by pyrolysis-gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Analyst 1998;123(5):911-918. R825960 (1999)
    R825960 (Final)
    R825433 (Final)
    R825433C007 (Final)
  • Abstract: RSC-Abstract
  • Supplemental Keywords:

    soil, sediment, 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
  • 2000 Progress Report