2001 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, 2000 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, so 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.

The original objectives of this research project were 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 [AFB]); and (5) use the findings from (1) through (4) to identify key rhizospheric processes that regulate metal bioavailability.

Progress Summary:

Humic Interactions in Plant-Metal Uptake. Background information on the role of soil OM, such as plant root exudates and especially soil humic substances (HS), in heavy metal accumulation by plants is grossly lacking, yet necessary for understanding heavy metal mobilization by plants from soils. Our experiments to date indicate that HS+Cd treatment of Chinese spring variety of wheat plants did not attenuate-and even slightly increased-the accumulation of some transition metals and Cd in roots (see Figure 1); this is contrary to the role of HS as a competitive chelator for metal ions. Despite this enhanced accumulation of the toxic metal Cd, the presence of HS stimulated plant growth, improved metal ion ligand (MIL) production, and alleviated part of the Cd-induced growth inhibition. It is likely that some HS component(s) may directly mediate metal ion (e.g., Cd, Zn, Ni) uptake via their cotransport with metal ions into wheat roots. Also, the siderophore 2'-DMA (see Figure 1), under Cd treatment, was not associated with enhanced uptake of Ni, Cu, Zn, Mn, Cd itself, and not even Fe. This questions whether such siderophores actually function as "siderophores" when under Cd stress.

Figure 1. Comparison of Exudate MIL Concentration Versus Metal Content in Wheat Roots Under a Combination of Soil Humate (HS, 5 ppm) and Cd (5 ppm) Treatments. All MILs declined drastically in exudation while accumulation of transition metals in roots was elevated with Cd or Cd+HS treatments. The presence of 5 ppm Cd in growth media resulted in a nearly 1,000-fold accumulation in wheat roots.

Metal ions may be stabilized internally in the plant by enhanced MIL-such as amino acids, organic acids, and phytochelatins (PC)-accumulation in wheat roots and shoots (data not shown). We also observed release of substances chemically identifiable as HS (data not shown) back into the root exudates, consistent with HS uptake by plants. This agrees with the high affinity of HS for Cd(II), as observed earlier in this project by pyrolysis-gas chromatography mass spectrometry (GCMS) (Higashi, et al., 1998).

In view of the importance of HS in metal ion sequestration in soils and intimate connection to plant-microbe interactions, we have previously reported our efforts in developing concerted measurements by pyro-GCMS, NMR, FT-IR, and other biophysical methodologies for characterizing molecular structure motifs of HS that are likely to be involved in metal ion interaction (Higashi, et al., 1998; Fan, et al., 2000; Fan and Lane, 2000). This year, through combining natural abundance carbon isotope ratio measurement by combustion isotope-ratio mass spectrometry (cIRMS) with pyrolysis-GCMS, we are developing the ability to track the turnover kinetics of various HS structural fragments based on their 13C (see Figure 2). This capability will open the door for probing the chemical mechanism of humification and reveal those substructures of HS that become recalcitrant to turnover by interacting with metal ions. This knowledge is required for designing and implementing metal remediation via both stabilization and mobilization strategies.

Figure 2. Pyrolysis-GC-cIRMS Analysis of Soil Beneath Plants Flushed With 12C-enriched CO2. Pyrolysis is performed in inert helium gas, so it is distinct from combustion analysis. The three channels-12C channel, 13C channel, and ratio (13C)-are shown on their own ordinate scales and offset for illustrative purposes. On the ratio (13C) channel, it is clear that some peaks are (+), and some are (-) relative to an arbitrary null line. The sharp trio of pulses at the beginning and end of the chromatogram (seen as negative peaks in the ratio channel) are reference CO2 infusions, which provide data for properly scaling the sample 13C. The data shown here is only for illustration of the method; for precise quantification of 13C, minor data adjustments are needed.

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 (Lazo, et al., 1995; Kagi, et al., 1993; Bremner and Beattie, 1990). Notable examples include the metallothioneins (MTs) and phytochelatins (PCs, also known as class III metallothioneins). MTs are found largely in the animal kingdom, and PCs are present mainly in vascular plants, algae, and fungi. The synthesis of both types of peptides is highly responsive to exposure to heavy metals such as Cd (Kaegi and Schaeffer, 1988).

Phytochelatins have the general chemical structure (-Glu-Cys)2-11-Gly and are synthesized by a consecutive transfer of -glutamyl cysteinyl units to glutathione (-Glu-Cys-Gly) via the action of PC synthase (Rauser, 1990). Although Gly is the most common C-terminal amino acid, -Ala and Ser have been noted to replace Gly at the C terminus (Rauser, 1990; Mehra and Winge, 1991; Grill, et al., 1986). Intermediate and variable molecular weights, with the lack of a convenient chromophore, has made PC analysis a laborious task by conventional methods. Similar problems also apply to the analysis of MTs. Vertebrate MTs typically contain approximately 60 amino acid residues, of which about 30 percent are cysteine. Although the cysteine content and position are highly conserved, the overall amino acid composition of MTs can differ by 2-25 percent (Kagi, et al., 1993), which makes it challenging to analyze different MTs and their isoforms at a trace level.

Thus, we recently developed a method that combined fluorescent tagging of thiol-rich peptides by bromobimane with SDS-PAGE, confirmed by 2-D NMR and liquid chromatography (LC)-tandem MS analyses (confirmations not shown). This method allowed a fast and simultaneous assay of both phytochelatins (PC) and metallothionein-like proteins, as illustrated for wheat, rice, and clam in Figure 3. For both wheat and rice, Cd treatments induced a large accumulation of PC (<3.5 kD SH-rich peptides) but not any other SH-rich proteins in root and shoot. The PC accumulation (see Figure 3, lanes 6 and 8) was sensitive to Cd accumulation only. These peptides exhibited a high affinity for Cd and Pb, as revealed by chelator competition and capillary electrophoresis coupled with inductively coupled plasma (ICP)-MS (in collaboration with Dr. V. Majidi, Los Alamos National Laboratory; data not shown). These results suggest the role of PCs in sequestering heavy metals, particularly in roots where much higher amounts of Cd were accumulated than in shoots. 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.

Figure 3. SDS-PAGE of Bromobimane-Tagged SH-Rich Peptides and Proteins From Wheat, Rice, and Clam Tissues. Lanes 1, MW standards; 2, rabbit metallothionein (MT); 3-5, clam MT extracts; 6, Cd-treated and 7, control wheat root extracts; 8, Cd-treated rice shoots. The MW standards are shown in a white background as they are not fluorescent.

Our results indicate that both SH-rich peptides and non-peptidic MIL may be important to heavy metal accumulation in plant tissues. They may contribute toward a plant's ability to tolerate high levels of metal accumulation (see in Figure 1) and may influence subsequent fate of metals in the humification process. In addition, plant roots accumulated a high amount of metals that were not mediated by the exudation of phytosiderophores or small water-soluble MIL. This raises the question on the role of macromolecular components in the rhizosphere (e.g., HS and extracellular biopolymeric substances) in mediating heavy metal uptake. Moreover, the metals sequestered by roots will not be practical to remove from soils by simple harvest. From the phytoremediation standpoint, the long-term fate of these metals including bioavailability and leachability will need to be addressed. Depending on their speciation (e.g., formation of PC complexes) and interaction with soil OM and minerals, pollutant metals may be "entombed" with aging (Cunningham and Ow, 1996). Such aging process, if understood, may be enhanced to stabilize metal ion movement and transport in the root and vadose zones.

Results of Long-Term Soil Incubation Pots for Experiments. As described in last year's report, we have designed a soil aging experimental system capable of stable operation for several months, and used it to conduct an experiment to investigate the effect of organic amendments on soil OM transformation and heavy metal leaching. An agricultural soil (Ag) low in Cd and Pb concentrations and a Cd-Pb contaminated soil from McClellan AFB were amended with wheat straw and cow manure. These soils were allowed to age for 25 weeks, during which microbial activities (as CO2 and N emissions), and metal ion leaching were monitored. Chemical profiles of aging soil OM also were acquired using pyrolysis-GCMS after 9 and 25 weeks. Initial analysis indicated that NH3 emission was higher in manure-amended soils (both Ag and AFB) than the wheat straw-amended counterparts, while the opposite was observed for the total amounts of leached organic carbon. The enhanced C leaching with wheat straw treatment may be correlated with the somewhat higher Cd leaching from this treatment.

With regards to organic matter profiles, the pyrolysis-GCMS revealed a cacophony of detailed changes, as expected. When the markers for peptide bonds (reconstructed ion chromatograms, or RIC, for masses 79, 93, and 103 Da) were directly examined, all soils showed increases, which were less for the AFB than the Ag soil experiments (data not shown). One interpretation of the increases of peptides is that it is an effect of an increase in microbial biomass; however, at this juncture, it is not possible to assign the relative peptide amounts in live microbes versus OM.

In contrast, lignin residues (revealed through the RIC of 124, 135, and 151 Da; see Figure 4) are interpretable as OM, because microbes do not produce lignin structures. Both wheat straw and manure amendments added intense lignin markers to the soils. As illustrated in Figure 4 for a pair of experiments, 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.

Figure 4. Pyrolysis-GC-MS 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.

Continued Stable Isotope Labeling of Soil. Last year, we initiated collaboration with Dr. Robin Brigmon at the Department of Energy's (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. As reported last year, using SRS soils, we have begun preparation of 13C- and 15N-labeled OM so that the turnover kinetics of various organic matter markers can be followed. In this experiment, 13C-glucose and 15N-nitrate were incubated with SRS soils to generate 13C and 15N-labeled HS.

This aging experiment has continued into this year for a total 34 weeks. Pyrolysis-GCMS analysis of the soils indicates that various substructures of HS (polysaccharides, peptides, lignin) have been strongly labeled (e.g., >90 percent isotopic substitution in peptide backbone carbons, data not shown). These labeled soils will be useful for following the turnover rate of HS structures, thereby allowing their recalcitrant properties to be characterized as a function of chemical class.

Production of Cd-Contaminated Plant Material. The above plant-metal-MIL studies 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 to address this, as reported last year, we started a larger-scale hydroponic production of wheat contaminated with several levels of Cd exposure, including controls. This year, the production of this material was completed, the shoot and root tissues freeze-dried, ground, and stored at -70°C. Using the new analysis method for thiol-rich peptides (see above), we established the PC contents of these tissues.

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 the 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. This long-term experiment is still underway; Figures 5-7 show selected element leachate results thus far, with the interim results summarized in the legends. The standard pyrolysis-GCMS can quantify isotopic abundances for highly labeled material. To track lower levels, we use the pyrolysis-GC-cIRMS.

Figure 5. Total Cadmium Mobilized by Periodic Leaching of Stable Isotope-Labeled Soils Spiked With Cd-Bioaccumulated Wheat Root and Amendments as Noted on the x-Axis. Initial Cd concentrations averaged 2.4 mg/kg (range = 1.6-3.4 mg/kg). The chitin and bone meal treatments caused increased Cd leaching over the control, and the addition of pine shavings decreased Cd losses. As expected, Cd leaching diminished rapidly over time, with the highest losses measured in the first four leachings (days 1-22).

Figure 6. Total Phosphorus Leached From Amended Soils. Results are shown for stable isotope 13C-labeled soils for simplification, 15N-labeled soils produced similar leachates. The bone meal amendment added P to levels (ca. 1 g/kg), 5-10 times higher than all other treatments. Some of this phosphorus was leached out early in the incubation. Wheat straw and chitin amendments, which did not initially have quantifiably more P than control, also caused elevated losses of P. Note that rates of metals loss from all treatments seem to stabilize at the same value by the end of the 5-month incubation, and levels of P remain elevated in some treatments.

Figure 7. Copper Leached From Amended Soils. As in the previous figure, results are shown for 13C-labeled soils only; results for 15N-labeled soils were similar. Both Cu and Ni (data not shown) were leached more readily from the bone meal, wheat straw, and chitin amended soils, though the initial concentrations of these metals were invariant with treatment. Note the time lag in the loss of all reported elements from the chitin-amended soils.

In summary:

· 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, phytosiderophores and other exuded MIL were surpressed, yet the uptake of transition metals Fe, Ni, Cu, Zn, and Mn were enhanced.

· The stable soil pot system successfully developed and tested last year, was used to generate 13C and 15N-labeled HS in 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 brought online.

· 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.

· Analyses of soil and leachates for elemental profile, isotope-enriched soil OM structure, PC, and microbial communities (by collaboration) will be conducted. Interim results show interesting element leaching patterns.

Bremner I, Beattie HH. Metallothionein and the Trace Minerals. Annual Review of Nutrition 1990;10:63-84.

Cunningham SK, Ow DW. Promises and prospects of phytoremediation. Plant Physiology 1996;110:715-719.

Fan TW-M, Lane AN. NMR in the Plant-Soil Environment. In: Encyclopedia of Analytical Chemistry, John Wiley and Sons, New York 2000, pp. 4082-4108.

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 and Technology 2000;34(9):1636-1646.

Grill E, Gekeler W, Winnacker E-L, Zenk HH. 1986 FEBS 205:47-50.

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;123:911-918.

Kaegi JHR, Schaeffer A. Biochemistry 1988;27:8509-8515.

Käegi JHR, Suzuki KT, Imura N, Kimura M, eds. Metallothionein III: Biological Roles and Medical Implications. Birkhèauser Verlag, Basel, Switzerland 1993, pp. 29-55.

Lazo JS, Pitt BR. Metallothioneins and cell death by anticancer drugs. Annual Review of Pharmacology and Toxicology 1995;35:635-653.

Mehra RK, Winge DR. Journal of Cellular Biochemistry 1991;45:30-40.

Rauser WE. Phytochelatins. Annual Review in Biochemistry 1990;59:61-86.

Future Activities:

As this was the final year of the project, the latest soil experiment will be completed, the final report to the U.S. Environmental Protection Agency will be written, and at least two manuscripts are planned for submission to peer-reviewed journals.

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, 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
  • 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
  • Final Report