Environ. Sci. Technol. 2000, 34, 4917-4922
Bioavailability of Chelated and Soil-Adsorbed Copper to Methylosinus trichosporium OB3b JOHN D. MORTON, KIM F. HAYES, AND JEREMY D. SEMRAU* Environmental and Water Resources Engineering Program, Department of Civil and Environmental Engineering, University of Michigan, 1351 Beal Avenue, Ann Arbor, Michigan 48109-2125
The effect of chelated and soil-adsorbed copper on whole-cell soluble methane monooxygenase activity in Methylosinus trichosporium OB3b was measured to assess the bioavailability of these forms of copper. Strongly binding chelating agents (Log K > 16) added to the growth medium at a concentration of 20 µM were able to reduce copper bioavailability. As these chelating agents limited equilibrium binding of copper by cell-produced copper binding ligands and limited accumulation by passive uptake mechanisms, it appears the ability of the cells’ copper uptake system to sequester copper in a bioavailable form is compromised by these competing ligands. Copper sorbed as surface complexes on montmorillonite clay, γ-alumina, ferrihydrite, and goethite were bioavailable. However, with increasing concentration of surface sites, bioavailability decreased, indicating the competitive advantage for copper binding provided by high concentrations of binding sites limited the ability of the cells’ copper uptake system to accumulate copper in a bioavailable form. Such information on the bioavailability of copper can enhance the utility of methanotrophs for in situ biodegradation of priority pollutants. As these cells’ ability to degrade chlorinated solvents changes dramatically with varying copper bioavailability, these studies provide data that can be used to optimize methanotrophic mediated bioremediation efforts.
Introduction Metals play important roles in microbial activity and can significantly affect bioremediation strategies. As such, it is important to determine metal bioavailability, i.e., the amount of any given metal that can be taken up by microorganisms such that a physiological response is observable, e.g., differential gene expression and/or enzymatic activity. One important example of a metal-dependent microbial process that is significantly influenced by metal bioavailability is the activity and expression of methane monooxygenase (MMO) in methanotrophs. Methanotrophs, bacteria that utilize methane as their sole source of carbon and energy, can express two forms of MMO depending on the amount of copper available. At low copper-to-biomass ratios (less than 0.89 µmoles copper per g cell dry weight), a cytoplasmic, or * Corresponding author phone: (734)764-6487; fax: (734)763-2275; e-mail:
[email protected]. 10.1021/es001330m CCC: $19.00 Published on Web 10/21/2000
2000 American Chemical Society
soluble methane monooxygenase (sMMO) is synthesized while a membrane-associated or particulate methane monooxygenase (pMMO) is found at high copper-to-biomass ratios (1). Because of this difference, measurement of sMMO/ pMMO can be used as an indication of decreasing bioavailability with sMMO and pMMO activity either decreasing or increasing respectively with increasing copper bioavailability. Understanding copper bioavailability to methanotrophs is necessary to optimize methanotrophic-mediated bioremediation of chlorinated solvents. Although both forms of MMO can degrade priority pollutants such as trichloroethylene (TCE), they do so at much different rates, with sMMOexpressing cells typically degrading TCE 10-100 times faster than cells expressing pMMO, although pMMO-expressing cells may be able to degrade TCE to lower levels due to a higher apparent affinity for TCE (2-5). While it is known that the type of complexing agent to which copper is bound can regulate bioavailability to methanotrophs (6, 7), the effect of the chelating agent binding affinity for copper on its bioavailability to methanotrophs has not been systematically studied. Furthermore, understanding the bioavailability of copper adsorbed to reactive soil particles is also necessary to enhance in situ bioremediation attempts using methanotrophs. Of particular significance are the adsorption reactions between copper and the surface hydroxyl sites on soil oxides and clay minerals. Transition metals such as copper have been shown to form strong, inner-sphere complexes on these sites (8-16). Given the prevalence and strength of these complexes, understanding their bioavailability is an important step to understanding the activity of methanotrophs in situ. Along with the speciation and distribution of copper in different systems, another significant aspect of metal bioavailability is how cells bind and sequester metals. For methanotrophs, a protein with a strong affinity for copper, referred to as the copper binding compound/cofactor (CBC), has been found in the spent medium of Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath (1719). The CBC appears to sequester copper in a siderophorelike fashion where the metal binding ligand is excreted during growth and subsequently reinternalized (18). With its strong affinity for copper and apparent role in sequestering extracellular copper, the CBC may be a key factor determining the bioavailability of different forms of copper. As methanotrophs have been extensively examined for pollutant degradation, here we present systematic studies examining the bioavailability of copper in the presence of different chelating agents and soils to a representative methanotroph, Methylosinus trichosporium OB3b. In these studies, increased copper bioavailability has been assessed by monitoring changes in sMMO activity and in particular reduction in its activity as copper is known to repress sMMO expression as well as reduce its activity (20, 21). The affinity of the copper binding ligands excreted during growth was also measured and compared to the copper formation constants of the added chelators. Based on these findings, a simple equilibrium model of copper speciation was developed to describe the binding of copper by the CBC in the presence of soils and chelating agents.
Materials and Methods Growth Medium Preparation. M. trichosporium OB3b was grown in M2M medium as previously described (7). To study the effect of the chelating agent copper binding affinity, the 1100 µM of pyrophosphate present in M2M medium was replaced by 20 µM of either pyrophosphate (PP) (copper VOL. 34, NO. 23, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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chelate formation constant log K ) 10.4), nitrilotriacetic acid (NTA) (log K ) 13.1), diethylene triamine (DEN) (log K ) 15.9), ethylene glycol (β-aminoethyl ether) N, N, N′, N′tetraacetic acid (EGTA) (log K ) 17.5) or ethylenediamine tetraacetic acid (EDTA) (log K ) 18.8). Soil Oxide and Clay Preparation. “Cheto” Ca-montmorillonite from Apache Co. Arizona was obtained from the Clay Mineral Society Repository (University of Missouri, Columbia, MO), and pretreated to remove organic and inorganic impurities as described by Kunze and Dixon (22). The clay stock was sterilized by γ-irradiation. 0.05 micron γ-alumina was obtained from Buehler (Lake Bluff, IL). The γ-alumina was sterilized by autoclaving as a dry powder and then acid washed with ultrapure 0.1 M HNO3 (Fisher Optima) to remove any trace copper. It was then rinsed and stored in sterile water with 0.1 M NaNO3. Ferrihydrite (Fe(OH)3 (am)) and goethite (R-FeOOH) were prepared as described by Cornell and Schwertmann (23) and sterilized by autoclaving. The hydroxyl site surface areas for the selected soil constituents were assumed to be 92 m2/g for montmorillonite (24), 50 m2/g for goethite (23) and 650 m2/g for ferrihydrite (25). The surface area of γ-alumina was measured to be 100 m2/g using BET. Assuming a surface hydroxyl site density between 1 and 5 sites/nm2, the surface hydroxyl site concentration for the soils were estimated to be 153-764 µmol/g for montmorillonite, 166-830 µmol/g for γ-alumina, 83-415 µmol/g for goethite and 1080-5400 µmol/g for ferrihydrite. Copper Adsorption to Soils Constituents. To measure whole-cell sMMO activity in the presence of soil-adsorbed copper, M2M medium was prepared without pyrophosphate and the soil constituents and copper were added to the growth medium after autoclaving. The pH of the medium was adjusted to 6.8 using either NaOH or HNO3. Copper was then allowed to adsorb to the soil constituents for 24 h (rotation at 270 rpm, 30 °C) after which time the growth medium was inoculated with M. trichosporium OB3b. To avoid iron growth limitation, the iron concentration was increased from 1 to 10 µM in the presence of γ-alumina and montmorillonite. No iron was added to the media prepared with the iron oxides ferrihydrite and goethite. All of the added copper was adsorbed to the oxides and clay as confirmed by measuring the copper concentration in the supernatant using Atomic Absorption Spectrophotometry (AAS). Whole-Cell sMMO Enzyme Activity Assays. sMMO activity in M. trichosporium OB3b was measured by monitoring the production of naphthol from naphthalene as described earlier (7, 26). For experiments with soil constituents, standards for the naphthol measurement were run in the growth media in the presence and absence of each of the soil constituents to account for interferences. Biomass was measured as OD600 and converted to protein using a predetermined relationship derived from linear regression analysis. As with the naphthol measurements, OD600 was measured in the presence and absence of each soil constituent to account for interference. Titration of Spent Media and Cells with Copper. Spent media were prepared by growing cells in M2M medium with 0.04 µM copper but no pyrophosphate to limit interferences during copper titration of the biogenic extracellular ligands. After growth to an OD600 of 0.3, the cells were separated by centrifugation (7650 x g; 20 min) and the supernatant (spent media) removed for titration. Copper titrations were performed by placing the spent medium in a well-mixed 50 mL polycarbonate reactor maintained at pH ) 6.8 and 30 °C. Copper was added in small increments and monitored for free copper with an ion selective electrode (ISE). Uninoculated medium was also titrated for comparison with the spent media. For all titrations, the amount of copper associated with the copper binding ligands (Cu-L) was calculated as the 4918
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difference between the total copper added and the measured free copper (Cu2+) in solution. The total ligand concentration was determined using Scatchard plots (27). The amount of free ligand (L-) for each titration point was calculated as the difference between the total ligand concentration and the copper bound ligand (Cu-L). The conditional formation constant at pH ) 6.8 (K*) was then calculated for each free copper concentration using the equation: K* ) [Cu-L] /[Cu2+][L-] and the average taken as the formation constant for a given ligand. Copper Accumulation by Inactivated Cells. These experiments were run similarly to those described earlier (7). After growth, cells were washed in PIPES buffer (5 mM; 0.02 M NaNO3) and inactivated using acetylene (28). The cells were then resuspended in growth media with 0.12 µM copper prepared in the same way as in the sMMO activity experiments. After incubating the cells for 24 h in the presence of acetylene, the cells were centrifuged and copper accumulation was determined as the difference between the total copper added to the media and the amount of copper measured in the supernatant by AAS.
Results and Discussion Whole-Cell sMMO Activity in the Presence of Chelated Copper. The trends of whole-cell sMMO activity in the presence of different chelating agents are shown in Figure 1A. Consistent with earlier work (7), whole-cell sMMO activity decreased with increasing total copper-to-biomass ratios, indicating increased copper bioavailability. However, the trend was different depending on the chelating agent as evident from the steepness of the slope of the decrease in sMMO activity. For growth media with 20 µM of PP, NTA and DEN, whole-cell sMMO activity slope decreased more substantially with increasing copper-to-biomass ratios than for growth media with 20 µM of EGTA and EDTA. To compare the bioavailability of copper in the presence of different chelating agents, the x-intercept or maximum total copper-to-biomass ratio with measurable sMMO activity of the linear regression analyses shown in Figure 1A are plotted in Figure 1B. The higher the maximum total copperto-biomass ratio with whole-cell sMMO activity, the lower the proportion of total copper that was bioavailable. As the trends for the systems with PP, NTA and DEN were statistically indistinguishable, one linear regression analysis was used for these trends. It should be noted for these data sets the points above 2 µmoles copper/g protein, where some low, but positive sMMO activity measurements were made, were not included in the regression analysis as these points were statistically indistinguishible from zero. As can be seen in Figure 1B, in the presence of 20 µM NTA, PP, and DEN, wholecell sMMO activity was not significant above 1.6 µmoles Cu/g protein while in the presence of 20 µM EGTA and EDTA whole-cell sMMO activity was observed up to 3.14 and 5.2 µmol/g protein, respectively. This indicates a large proportion of the copper in the presence of EGTA and EDTA did not affect sMMO activity and therefore was less bioavailable. From these results, it appears that copper bioavailability was constant in the presence of chelating agents with a copper formation constant Log K
