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Aqueous Geochemistry Research and Development
Biocatalysis Studies

Rates of Microbially-mediated Ferrihydrite and MnO₂ Dissolution: Evidence for Slower Reaction Rates due to Decreased Surface Reduction Sites and Increased Dissolved Metals

Brent A. Meyer and Lisa L. Stillings

Ferrihydrite and MnO₂ are important minerals that control the mobility of metals in the environment. This is because of their high surface areas and the affinity of metals for adsorption sites at their surface. These are not stable minerals, however; they can be dissolved through chemical reduction (e.g., in wetland and lake sediments) and they can transform to more stable phases. The purpose of this work is to identify factors that influence the rate of reductive dissolution of these phases by using reaction rate modeling. The kinetics of reduction of ferrihydrite and manganese oxide by a dissimilatory Fe³⁺-oxide-reducing bacteria (Milltown Enrichment Culture, MEC) under anoxic conditions were studied. Microbial reduction was monitored by measuring the production of Fe²⁺ or Mn²⁺ over time in sealed batch reactors containing ferrihydrite or manganese oxide, respectively. We developed a kinetic model combining first-order and zero-order expressions. By accounting for Fe²⁺ adsorption and ferrihydrite dissolution, we determined that dissolved Fe²⁺ tolerance was a stronger influence on microbial growth than ferrihydrite concentration. The presence of adsorbed arsenic on ferrihydrite greatly reduced the production of Fe²⁺ indicating that microbial growth was limited. The initial rate of Mn²⁺ production was found to be considerably greater than the initial rate of Fe²⁺ production from an equal concentration of oxide.

The Problem

The geochemical literature contains many examples of thermodynamic disequilibrium under anoxic conditions. For example, in sediments of the Milltown Reservoir, near Missoula, MT, As(V) and hydrous ferric oxide (HFO), were observed in a reducing environment, at >65cm deep in the reservoir sediments. (Moore, 1994)

What processes control HFO dissolution and As(V) reduction and therefore arsenic mobility?

Hypotheses

Based on previous studies of metal reduction in similar environmental systems (Brock, 1997; Aurilio et al., 1994), we hypothesized that microbial Fe(III) reduction would occur much faster than abiotic Fe(III) reduction and therefore would be the process that controlled the mobility of iron under anoxic conditions.
Most anaerobic iron- and arsenic-reducing bacteria derive energy for reproduction and cell metabolism from the transfer of electrons from an organic electron donor to an electron acceptor (Brock, 1997). We therefore hypothesized that increasing the amount of available electron donor or electron acceptor would increase the rate of microbial growth and Fe(III) reduction.
We hypothesized that the adsorption of cations and anions onto HFO would interfere with the electron transfer process and that the rate of Fe(III) reduction would decrease as the amount of adsorbed ions increased.
Previous studies like that of Laverman et al., 1995, have shown that iron-reducing bacteria can also reduce As(V) and other redox sensitive metals. In addition, the transformation of Fe(III) to Fe(II) yields more energy than the transformation of As(V) to As(III). Based on this, we hypothesized that the MEC would sequentially reduce both Fe(III) and As(V). As(V) reduction would occur only after the Fe(III) source was exhausted.

Methods

Bacterial Culture

In order to test our hypotheses, we used a culture isolated from anoxic zone sediments extracted from the Milltown Reservoir (Markwiese, 1998). The Milltown Reservoir Culture (MEC) contains a fermentor capable of transforming lactate to acetate and an as yet unclassified acetate utilizing iron-reducing bacterium (Fig. 1).

Microcosm experiments

SEM image of an iron-reducing bacterium from the Milltown Enrichment Culture grown with lactate.

Figure 1. SEM image of an iron-reducing bacterium from the Milltown Enrichment Culture (MEC) grown with lactate. View large version of bacterium photo.

The effect of varying environmental conditions on the microbial reduction of HFO and the mobility of adsorbed As(V) was investigated by studying the kinetics of reductive dissolution of synthetic HFO in three batch-reactor experiments. Growth medium, containing HFO as an electron acceptor (EA) and acetate as an electron donor (ED), was dispensed into 500-ml septum sealed serum bottles. Each bottle was inoculated with an enrichment culture (MEC) containing an anaerobic Fe-reducing bacterium obtained from sediments at Milltown Reservoir near Missoula, MT. Each enrichment culture grew for at least 600 hours and exhibited both exponential and stationary growth. Microbial reduction was monitored by measuring the production of dissolved Fe(II).

Kinetic Model for the Microbial Reduction of HFO
Fe(II) production was modeled by:

Return to Results place 1x = X_s(1−e^-ke^t)−[k_L(e^-ke^t)]+(k_L/k_e) (1)

where
x is the total Fe(II) concentration (mM) at t,
k_e is the exponential production rate constant (hr^-1),
X_s is the total Fe(II) concentration (mM) at the time of transition between exponential and stationary growth,
t is the time since inoculation minus lag time, and
k_L is the stationary (linear) production rate constant (mM hr^-1).

Maximum production rate is equal to
R_max= k_eX_s+k_L (2)Return to Results place 2

Results

In the following plots, Fe(II) is the product of the microbially catalyzed reductive dissolution of ferrihydrite:

8Fe(OH)₃ + CH₃COO^- + 17H⁺ MEC over right arrow 8Fe⁺ + 2CO₂ + 22H₂O (3)
Ferrihydrite acetate

The concentration of Fe(II) produced by microbial growth and reduction of HFO is used to calculate the kinetic rate constants k_e and k_L (eqn. 1Equation 1 ). Greater concentrations of Fe(II) represent greater values of production, R_max (eqn. 2Equation 2 )

Total Fe(III) Production Model Increasing HFO Concentrations, graph of Fe(II) concentration versus Time.
Figure 2a. Variation in Fe(II) production, as a function of the concentration of electron acceptor (HFO; concentrations in the legend are mM) in the system. The system with the greatest concentration of electron acceptor (30mM HFO and 10mM) acetate produced the most Fe(II) and exhibited the greatest R_max. View large version of Figure 2aReturn to Conclusions

Total Fe(II) Production Model Increasing Acetate Concentrations, graph of Fe(II) Concentration versus Time
Figure 2b. Variation in Fe(II) production, as a function of the concentration of electron donor (acetate) in the system (concentrations in the legend are mM). Fe(II) production did not vary between the systems, therefore the concentration of electron donor did not affect the reaction rate constants. View large version of Figure 2bReturn to Conclusions

Varying Fe(II) Production due to the presence of As(V), graph of Fe(II) Concentration versus Time
Figure 3. The red, blue, and green lines repeat the modeled data shown in Fig. 2a (concentrations in the legend are mM). Triangular symbols are additional data, representing the concentration of Fe(II) produced when 1 mM of As is added to the system. It is clear that adsorption of As(V) to the HFO surface substantially reduced production of Fe(II), although the exponential rate constant, k_e, was not affected. View large version of Figure 3

Conclusions

No Fe(II) production was observed in representative blank and kill controls that were run concurrent with growth experiments. We accept hypothesis #1 that microbial Fe(III) (HFO) reduction occurred at a much faster rate than abiotic Fe(III) reduction.
Increasing the concentration of electron acceptor (HFO) increased the rate of microbial growth and Fe(II) production (Fig. 2aFigure 2a), but increasing the concentration of electron donor had no effect on Fe(II) production (Fig. 2bFigure 2b). Half of hypothesis #2 could be accepted.
Addition of 1mM of As(V) to the system led to complete adsorption of As(V) to the HFO surface, and substantially decreased the concentration of Fe(II) produced, hence decreasing the rate of the reductive dissolution reaction (in Fig. 3Figure 3, compare the red line to the triangular symbols). We accept hypothesis 3 that an increase in the concentration of ions adsorbed to the HFO surface will decrease the rate of HFO dissolution.
We saw no evidence of As(V) release from the HFO surface, and no As(III) in solution. However our experiments did not run long enough to completely dissolve the HFO in the system, so we cannot completely evaluate hypothesis #4. However under the conditions of our experiments we did not see As(V) desorption and/or reduction.

We modeled Fe(II) production in our experiments with the kinetic model presented in Equations 1 and 2, and the constants extracted from data in Figs. 2aFigure 2a,bFigure 2b. (Table 1).

Table 1. Constants for the kinetic model of Fe(II) production.
Experiment	k_e (mM^-1)	t_s (hr)	t_lag (hr)	X_s (mM FeII)	k_L (mM FeII / hr)	R_max (mM FeII / hr)
MEC-Fe-10:10	0.0159	147	76	0.60	0.0010	0.011
MEC-Fe-20:10	0.0207	122	77	1.08	0.0019	0.024
MEC-Fe-30:10	0.0224	118	77	1.68	0.0029	0.040

References

Aurilio, A.C., R.P. Mason and H.F. Hemond (1994) Speciation and fate of arsenic in three lakes of the Aberjona watershed. Environ. Sci. Technol. 28, 577-585.

Brock, T. D. (1996) Biology of Microorganisms 8th ed. Prentice-Hall, Inc. New Jersey.

Johnson, D. H. and M. E. Q. Pilson (1972) Spectrophotometric determination of Arsenite, Arsenate, and Phosphate in Natural Waters. Anal. Chim. Acta, 58, 289-299.

Laverman, A.M., J. Switzer-Blum, J.K. Schaefer, E.I.P. Phillips, D.R. Lovley and R.S. Oremland (1995) Growth of strain SES-3 with arsenate and other electron acceptors. Appl. Environ. Microbiol. 61, 3356-3561.

Lovley, D. R. and E. J. P. Phillips (1986) Organic Matter Mineralization with Reduction of Ferric Iron in Anaerobic Sediments. Appl. Environ. Microbial, 31, 171-177.

Markwiese, J. (1998) Microbial Ecology of Fe(III)-Reducing Bacteria: Hydrocarbon Bioremediation, Heavy Metal Mobilization and Heavy Metal Toxicity, Ph.D., Department of Zoology and Physiology, University of Wyoming, Laramie.

Moore, J.N. (1994) Contaminant mobilization resulting from redox pumping in a metal-contaminated river-reservoir system. ACS Sym. Ser. 237, 451- 471.

Skougstad, M.W., M.J. Fishman, L.C. Friedman, D.E. Erdmann and S.D. Duncan (eds.) (1979) Methods for Analysis of Inorganic Substances in Water and Fluvial Sediments. U.S. Geological Survey Open-file Report 78-679. USGS, Reston, VA.

Products

Journal Articles, Review Volumes, and Theses

Meyer, B.A., 2004, Critical factors limiting microbial Fe³⁺- and Mn⁴⁺-oxide reduction: oxide surface area, dissolved concentration of reduced ion, and arsenic adsorption: M.S. Thesis, University of Nevada, Reno, 312 p. (Lisa Stillings, advisor)

Abstracts, Presentations, and Posters for Professional Technical Meetings

Meyer, B.A., and Stillings, L.L., 2002, Release and co-reduction of As(V) as a function of microbial reductive dissolution of ferrihydrite [abs.]: Geological Society of America 2002 Annual Meeting, Denver, Colorado, October 27-30, 2002, Abstracts with Programs, v. 34, no. 6, p. 493. (Abstract and poster) Available online at http://gsa.confex.com/gsa/2002AM/finalprogram/abstract_45088.htm.

Meyer, B.A., and Stillings, L.L., 2003, The effect of ion adsorption on microbial dissimilatory iron-reduction and the mobility of adsorbed As(V) [abs.]: American Geophysical Union (AGU) Fall Meeting, San Francisco, California, December 13-17, 2004: Eos Trans. AGU, v. 84, no. 46, Fall Meet. Suppl., Abstract B32A-0374. (Abstract and poster) Available online at http://www.agu.org/cgi-bin/SFgate/SFgate?&listenv=table&multiple=1&range=1&directget=1&application=fm03&database=%2Fdata%2Fepubs%2Fwais%2Findexes%2Ffm03%2Ffm03&maxhits=200&=%22B32A-0374%22.

Meyer, B.A., and Stillings, L.L., 2004, Critical factors limiting microbial Fe(III)- and Mn(IV)-oxide reduction: oxide surface area, dissolved concentration of product ion, and arsenic adsorption [abs.]: Geological Society of America 2004 Annual Meeting, Denver, Colorado, November 7-10, 2004, Abstracts with Programs, v. 36, no. 5, p. 88. (Abstract and poster) Available online at http://gsa.confex.com/gsa/2004AM/finalprogram/abstract_80301.htm.

Acknowledgements

Brent Meyer conducted this research for his Masters Degree in Hydrological Sciences, awarded by the University of Nevada, Reno, in December, 1994. Stillings gratefully acknowledges and thanks the National Science Foundation POWRE program for providing funding for Brent Meyer's graduate student stipend, laboratory equipment and supplies, NSF # 9806121. The USGS Aqueous Geochemistry Research Project is gratefully acknowledged, also, for additional funding for laboratory equipment and supplies.

Collaborators

Photo of Brent Meyer and Lisa Stillings
Brent Meyer (MS in Hydrological Sciences, University of Nevada, Reno) and his thesis advisor, Dr. Lisa Stillings (USGS) at Meyer's post-thesis defense party. October, 1994. View large version of Meyer & Stillings photo

Photo of Colberg, Meyer, and Stillings.
Dr. Patricia Colberg, Dept. Zoology and Physiology, University of Wyoming, with Meyer and Stillings. The MEC microbial culture used in these experiments was isolated and enriched in Colberg's laboratory. View large version of Colberg, Meyer, & Stillings photo

Contact Information

Lisa Stillings
Mackay School of Earth Sciences and Engineering
Mail Stop 176
University of Nevada, Reno
Reno, NV 89557-0047
Phone: (775) 784-5803
Email: stilling

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Rates of Microbially-mediated Ferrihydrite and MnO2 Dissolution: Evidence for Slower Reaction Rates due to Decreased Surface Reduction Sites and Increased Dissolved Metals