Environ. Sci. Technol. 2007, 41, 2437-2444
Kinetic Analysis of Microbial Reduction of Fe(III) in Nontronite D E B P . J A I S I , † H A I L I A N G D O N G , * ,† A N D CHONGXUAN LIU‡ Department of Geology, Miami University, Oxford, Ohio 45056, and Pacific Northwest National Laboratory, Richland, Washington 99352
Microbial reduction of structural Fe(III) in nontronite (NAu-2) was studied in batch cultures under non-growth condition using Shewanella putrefaciens strain CN32. The rate and extent of structural Fe(III) reduction was examined as a function of electron acceptor [Fe(III)] and bacterial concentration. Fe(II) sorption onto NAu-2 and CN32 surfaces was independently measured and described by the Langmuir expression with the affinity constant (log K) of 3.21 and 3.30 for NAu-2 and bacteria, respectively. The Fe(II) sorption capacity of NAu-2 decreased with increasing NAu-2 concentration, suggesting a particle aggregation effect. An empirical equation for maximum sorption capacity was derived from the sorption isotherms as a function of NAu-2 concentration. The total reactive surface concentration of Fe(III) was proposed as a proxy for the “effective” or bioaccessible Fe(III) concentration. The initial rate of microbial reduction was first-order with respect to the effective Fe(III) concentration. A kinetic biogeochemical model was assembled that incorporated the first-order rate expression with respect to the effective Fe(III) concentration, Fe(II) sorption to cell and NAu-2 surfaces, and the empirical equation for maximum sorption capacity. The model successfully described the experimental results with variable NAu-2 concentration. The initial rate of microbial reduction of Fe(III) in NAu-2 increased with increasing cell concentration from 102 up to ∼108 cells/mL, and then leveled off with further increase. A saturation-type kinetics with respect to cell concentration was required to describe microbial reduction of Fe(III) in NAu-2 as a function of cell concentration. Overall, our results indicated that the kinetics of microbial reduction of Fe(III) in NAu-2 can be modeled at variable concentration of key variables (clay and cell concentration) by considering the surface saturation, Fe(II) production, and its sorption to NAu-2 and cell surfaces.
Introduction Clay minerals are ubiquitous in natural sediments, soils, and sedimentary rocks. Many clay minerals contain iron in their structures (1-5), and the oxidation state of iron can be cycled between ferrous (+2) and ferric (+3). The iron oxidation state can affect many clay mineral properties including swelling, cation exchange capacity, cation fixation capacity, surface area, clay mineral-organic matter interactions, surface acidity, and redox properties (1, 6). These properties fundamentally * Corresponding author phone: 513-529-2517; fax: 513-529-1542; e-mail:
[email protected]. † Miami University. ‡ Pacific Northwest National Laboratory. 10.1021/es0619399 CCC: $37.00 Published on Web 02/24/2007
2007 American Chemical Society
affect the extent and rate of a number of environmental processes, including nutrient cycling, plant growth, contaminant migration, organic matter maturation, and petroleum production (4, 5). Many studies have shown that bacteria are able to reduce structural Fe(III) in smectite (3-8) and illite (4), thus influencing their chemical and physical properties and the fate and transport of organic (9) and inorganic contaminants in sediments (10, 11). However, none has explored the kinetics of microbial reduction of Fe(III) in clay minerals to develop models that can be used qualitatively or semiquantitatively to describe the reduction process in terms of microbial growth or substrate (electron acceptor or donor) consumption (12). Such kinetic models may be useful to understand clay mineral properties in the presence of Fe(III)-reducing bacteria in anoxic subsurface environments and to study the effects of this redox reaction on other environmental processes. Mathematical models have been developed to describe microbial reduction of ferric iron in goethite and iron oxyhydroxides (13-18). These models have treated the complex biochemical network of electron transfer from electron donor to acceptor [i.e., Fe(III)] as a catalytic “species” without considering its internal structure. Microbial reduction of iron oxides is first-order with respect to its surface area, and saturation type kinetics with respect to electron donor concentration has been proposed (14). The rate and extent of microbial reduction of iron oxides are affected by sorption and precipitation of biogenic Fe(II) that decrease Fe(III) surface area available for microbial attachment and reaction free energy that thermodynamically regulates reductive dissolution (8, 14, 15, 17-20). The applicability of such macroscopic models to bioreduction of structural Fe(III) in clay minerals is unknown. For bioreduction of solid-state Fe(III) in mineral structure, bioavailability of Fe(III) is difficult to estimate due to the poorly understood interfacial electrontransfer reactions between bacteria and Fe(III) in clay minerals and the fate of dissolved constituents and modified clay minerals. This research focuses on the kinetics of microbial reduction of structural Fe(III) in a clay mineral nontronite (NAu-2) [M+0.72(Si7.55Al0.45)(Fe3.83Mg0.05)O20(OH)4, where M may be Ca, Na, K (21)]. The objective of this study was to establish phenomenological relationships between microbial reduction rates and electron acceptor and cell concentrations in a simplified microcosm system. The ultimate goal was to provide a better understanding of microbial reduction of Fe(III) in nontronite and influence of biogenic Fe(II) on microbial activities. A simplified system was achieved by using a single electron donor and a single electron acceptor and by performing experiments with a pure culture of Shewanella putrefaciens CN32 under non-growth conditions. This bacterium has been used extensively in microbial reduction studies and its physiological/metabolic properties and interactions with Fe(III) in solid minerals has been reviewed recently (22). The non-growth conditions, i.e., in the absence of externally supplied growth nutrients, were used to isolate the reduction process from growth-related cellular effects (23) and to mimic natural environments.
Materials and Methods Mineral Preparation. Mineral nontronite (NAu-2) was purchased from the Source Clays Repository, IN. NAu-2 samples were thoroughly ground, saturated with NaCl, and sonicated in an ultrasonic water bath. NAu-2 suspension in distilled water was centrifuged to obtain a size fraction of 0.02-0.5 µm. This fraction was repeatedly washed until no VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Cl- was detected by the silver nitrate test. This size fraction was chosen as a representative for a range of clay mineral sizes commonly present in soils and sediments (24). The above-treated NAu-2 sample was characterized with direct current plasma (DCP) emission spectroscopy, chemical extraction, X-ray diffraction (XRD), and transmission electron microscopy (TEM) (25). The treated NAu-2 was pure without any other mineral phases and contained 23.4% total iron in its structure with almost all (99.8%) iron as Fe(III) (21, 25). The BET surface area of the 0.02-0.5 µm size fraction was measured with a Coulter SA3100 surface area analyzer, and it was 33.5 m2/g. Bacteria Culture and Media Preparation. CN32 cells were routinely cultured aerobically in tryptic soy broth (30 g/L) from the stock culture kept in 40% glycerol at -80 °C. After harvesting in TSB at mid to late log growth phase, CN32 cells were washed three times in bicarbonate buffer (2 g/L of NaHCO3, and 0.1 g/L of KCl). For Fe(II) sorption experiments the cells were washed and suspended in 30 mM PIPES (1,4piperazine diethanesulfonic acid) buffer instead of bicarbonate buffer to avoid potential precipitation of siderite. Bacterial Reduction Experiments. Reduction experiments were performed with Fe(III) in NAu-2 as the sole electron acceptor and lactate as the sole electron donor in the presence of CN32 cells. Control experiments were identical to the reduction experiments except that no cells were added. All experiments were performed in triplicates with 20 mL final volume in Balch tubes. The concentration of NAu-2 used in the experiments varied from 2.5 to 10 mg/ mL and cell concentration from 1 × 102 to 1 × 1010 cells/mL with a constant lactate concentration of 20 mM (except in the test of the reaction order of the reduction rate with respect to lactate, in which case it varied from 2.5 to 40 mM). The experimental tubes were purged with N2:CO2 gas mix (80:20) and sealed with thick butyl rubber stoppers, and then incubated at 30 °C with shaking at 60 rpm. All treatments and sampling were carried out under sterile and anaerobic conditions. Analyses of Fe(II) and Cell Counts. At selected time points cell-mineral suspensions were sampled, mixed anaerobically with 1N Ultrex HCl at 1:1 ratio, and equilibrated for 24 h to analyze total Fe(II) [Fe(II)tot] concentration by Ferrozine assay (26). The aqueous [Fe(II)aq] concentration was measured in supernatants obtained by filtering cell-mineral suspension through 0.2 µm filter followed by Ferrozine assay. The rate and extent of microbial reduction of NAu-2 was monitored by measuring Fe(II) production with time. The cell number was counted at different times (colony forming unit, CFU). pH was buffered at 7, but ionic strength (initially at 25 mM) might have slightly increased due to some reductive dissolution of NAu-2. Scanning Electron Microscopy. The Zeiss Supra VP FEG SEM microscope at an accelerating voltage of 10-15 keV was used to analyze the cell-NAu-2 spatial relationship. The mineral-cell suspension was initially fixed in 2.5% glutaraldehyde for at least 1 day, followed by dehydration with critical point drying (CPD) and coating with a 20 nm gold layer. Fe(II) Sorption Experiments. The Fe(II) sorption experiments were performed to determine equilibrium distribution of Fe(II) between aqueous and solid phases in three environments: NAu-2 only, cells only, NAu-2 and cell mixture. The deionized water (used to prepare PIPES buffer, NAu-2 and Fe(II) stock solutions) was initially purged overnight with O2-free N2 and CO2 gases and was then transferred into a container in a strict oxygen trap (27) for removal of a trace amount of oxygen for at least 9 days. This procedure reduces the oxygen level by a factor of about 1001000. The strict oxygen trap was housed inside an anaerobic chamber (96% N2 and 4% H2). The NAu-2 stock solution was prepared and stored in the chamber for 2 days, but the Fe(II) 2438
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stock solution was prepared from FeCl2 inside the chamber before the experiment and was kept in a dark, Al-foil wrapped container. Fe(II) sorption onto NAu-2 was studied at an NAu-2 concentration of 1-30 mg/mL with a range of Fe(II) concentrations (0.01-50 mM). Similarly, Fe(II) sorption onto CN32 cell surfaces was studied at a cell concentration of 1 × 106 to 1 × 109 cells/mL with a range of Fe(II) concentrations (0.01-25 mM). Except for certain experiments designed to examine time-course kinetics of Fe(II) sorption onto NAu-2 and CN32 surfaces, all experimental tubes were equilibrated for 24 h with continuous mixing (60 rpm) at 30 °C. The aqueous and total Fe(II) concentrations were determined as described above. The concentration of sorbed Fe(II) was calculated as the difference between the total added [Fe(II)tot] and the aqueous [Fe(II)aq] concentration at the end of the experiment. The buffer strength (30 mM PIPES) and pH (7) was kept constant in all Fe(II) sorption experiments.
Results and Discussion Microbial Reduction of Fe(III) in Nontronite. Fe(III) reduction under non-growth condition persisted for more than a year (Figure 1). The reduction rate decreased after an initial phase of fast reduction (ca. 15% of total Fe(III) or ∼90% of the observed extent of reduction by 80 days), despite the presence of a significant amount of residual Fe(III) in the suspensions. The extent of reduction did not increase significantly beyond 80 days. The existence of such a prolonged enzymatic activity (for as long as 80 days) has been previously reported in the bioreduction of subsurface material containing Fe(III)-oxides (28). Slow or no Fe(III) bioreduction in oxides and clay minerals after an initial fast reduction has been attributed to the inhibitory effects of sorbed Fe(II) (onto mineral and cell surfaces) on enzymatic electron transfer (15, 16, 29, 30) and/or thermodynamic constraints (14). Alternatively, cells might not be viable in prolonged incubations. Calculations using equilibrium sorption isotherms (see below) showed that, by the end of the experiment, about 65 and 61% of the total sorption sites were blocked by sorbed Fe(II) on CN32 cells and NAu-2 surfaces, respectively (Supporting Information Figure S1). This level of biogenic Fe(II) sorption to cell and NAu-2 surfaces significantly reduced reactive surface sites for bacterial engagement and was also assumed here responsible for cessation of Fe(III) reduction at the later stage (30) (Figure 1). The measured Fe(II)aq concentration (Figure 1, inset) was insignificant as compared to Fe(II)tot (less than 1% of Fe(II)tot). Fe(II) Sorption. Fe(II) sorption onto NAu-2 reached equilibrium within 11 h (Supporting Information Figure S2) and the majority of sorption (57-60%) occurred within 10 min. Fe(II) sorption onto cell surfaces was also fast with approximately 75% of FE(II) sorption occurring within 10 min (Supporting Information Figure S3). In both sorption experiments equilibrium was reached well before 24 h. The equilibrium concentration of Fe(II) sorbed onto NAu-2 increased with increasing NAu-2 concentration. However, Fe(II) sorption normalized to NAu-2 or Fe(III) concentration (in NAu-2) (Figure 2) decreased with increasing NAu-2 concentration. Such type of decreased Fe(II) sorption at higher sorbent concentration has been reported previously for goethite and was ascribed to the particle aggregation effect (14). This aggregation effect (Supporting Information Figure S4) may be facilitated by sorbed Fe(II) through cation bridging (31) and high clay concentration.
FIGURE 1. Production of biogenic Fe(II) and consumption of Fe(III) in NAu-2 as a function of time. Inset: Change of aqueous Fe(II) concentration with time. Fe(II)aq concentration was much lower than the 0.5 N HCl extractable Fe(II)tot and it accounted for only about 1% (or less) of Fe(II)tot. Experiments were performed with 20 mM of lactate, 21 mM of Fe(III) in NAu-2 at a cell concentration of 1 × 108 cells/mL.
FIGURE 2. The sorption of Fe(II) onto NAu-2 (Fe(III) concentration normalized) fitted to the Langmuir isotherm. The maximum sorption capacity and other parameters are listed in Table 1. The inset showed the fitted maximum sorption capacity of NAu-2, expressed as a dimensionless sorption capacity, as a function of Fe(III) concentration in NAu-2 (eq 2). The measured equilibrium sorption data were fitted according to the Langmuir type of sorption isotherm as
Fe(II)NAu-2 )
qmax Fe(II)aq [b + Fe(II)aq]
(1)
Where Fe(II)NAu-2 is the sorbed Fe(II) concentration [mmol of Fe(II)/mmol of Fe(III) in NAu-2], Fe(II)aq is the equilibrium aqueous concentration of Fe(II) (mmol/L), qmax is the maximum sorption capacity [mmol of Fe(II)/mmol of Fe(III) in NAu-2], and b ) R-1 (R is the affinity constant). Parameters qmax and b were obtained by fitting eq 1 to the experimental data in Figure 2 in two steps. At first, each isotherm curve in Figure 2 (i.e., with a specific NAu-2 concentration) was independently used to estimate parameters qmax and b using the least-square method. The estimated b values (Table 1) were similar for all isotherms with an average value of 0.615
( 0.001, indicating that the affinity constant did not change with NAu-2 concentration. With this common affinity constant, eq 1 was used to refit each isotherm by fixing b value and only adjusting qmax. The estimated maximum sorption capacity of NAu-2 from the second step was reported in Table 1. The maximum sorption capacity for NAu-2 was higher (about 2-4 times) than that for iron oxides. For example, at a similar concentration of NAu-2 (10 mg/mL, 33.5 m2/g) and goethite (8.9 mg/mL, 60.2 m2/g), our fitted maximum sorption capacity was 0.181 mmol Fe(II)/mmol Fe(III) or 0.77 mmol/g (Table 1), compared with 0.25 mmol/g for goethite (14, 29). The estimated maximum sorption capacity decreased with increasing NAu-2 concentration (Figure 2 inset and Table 1) and was empirically related to Fe(III) concentration as follows:
qmax )
c d + Fe(III)
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TABLE 1. Parameters of Langmuir Isotherms and Sorption Site Calculation for the Sorption of Fe(II) onto NAu-2 and Shewanella putrefaciens CN32 Surfacesa NAu-2 concentration
1 mg/mL
2 mg/mL
5 mg/mL
10 mg/mL
20 mg/mL
30 mg/mL
affinity constant ()1/b) R2 qmax (maximum sorption capacity) in mmol of Fe(II)/mmol of Fe(III) in NAu-2 in mmol/g of NAu-2 R2
1.63((0.09) 0.98
1.62((0.09) 0.99
1.58((0.09) 0.99
1.66((0.09) 0.98
1.60((0.11) 0.97
1.64((0.08) 0.96
0.50((0.01) 2.09((0.05) 0.98
0.35((0.01) 1.46((0.05) 0.98
0.22((0.01) 0.92((0.04) 0.97
0.18((0.01) 0.77((0.05) 0.98
0.14((0.02) 0.60((0.07) 0.97
0.10((0.01) 0.44((0.04) 0.95
NAu-2 NAu-2 or cell concentration adsorbed Fe(II), mmol/g ions of Fe(II)/g surface area, m2/g no. of Fe(II) sites/nm2 a
1 mg/mL
5 mg/mL
10 mg/mL
20 mg/mL
2.09 1.26 × 1021 33.50 38
0.92 5.53 × 1020 33.50 17
0.77 4.60 × 1020 33.50 14
0.60 3.61 × 1020 33.50 11
CN32 1 × 108 cells/mL 0.09 5.53 × 1019 0.19 299
Note: For the convenience of comparison with published literature values, the values of qmax were also presented in unit of mmol/g.
where c (8.3 ( 0.2) and d (12.3 ( 0.3) were estimated by fitting eq 2 to the qmax data in Table 1 and the inset of Figure 2 (R2 ) 0.925). This relationship (eq 2) was used to account for the sorption capacity change as a function of NAu-2 concentration in the subsequent microbial reduction experiments. The experimental data of Fe(II) sorption to CN32 cells were also fitted using the Langmuir isotherm. The best-fit data showed a maximum sorption capacity of 4.13 mmol/ 1012 cells and an affinity constant of 3.3. These values were close to previously reported values (4.19 and 3.29 mmol per 1012 cells (18)). On the dry weight basis, the maximum sorption capacity of Fe(II) on CN32 cells was 0.09 mmol per g of cells (assuming 45 g/1012 cells (32)), whereas it was 0.44-2.09 mmol/g on NAu-2 (depending on its concentration). Based on the cell and NAu-2 concentrations used in this study, these data indicated that Fe(II) sorption to cells was minor relative to NAu-2. Due to low cell concentration (c.a. 1 × 105 cells/g dry weight of agricultural soil) (33) in natural environments, NAu-2 surfaces may serve as a primary Fe(II) sink. When cells and NAu-2 were co-present in the system, measured Fe(II) sorption was slightly higher than but close to the predictions from the numerical summation of individual sorptions onto NAu-2 and cell surfaces (Supporting Information Figure S5). Fe(III) Reduction at Variable NAu-2 Concentration. To test the order of the bioreduction reaction with respect to lactate (CH3CHOHCOO-) concentration, additional experiments were performed with variable lactate concentrations (1.25-40 mM) at a constant NAu-2 and cell concentration (5 mg/mL and 1 × 108 cells/mL, respectively). The test using the van Hoff’s plot (34) showed approximately zero-order for this range of lactate concentration. All our experiments had excess lactate concentration (20 mM) relative to the amount of Fe(III) present in our system so that only the electron acceptor [structural Fe(III) in NAu-2] or cell concentration was rate limiting. Biogenic Fe(II) consisted of solidassociated Fe(II) (via sorption to cell and NAu-2 surfaces) and aqueous phase Fe(II). Solid Fe(II)-bearing phases such as siderite were not observed because of the very low aqueous Fe(II) concentration (e2% of the 0.5 N HCl extraction) (consistent with XRD and TEM results, 25). The initial rate of Fe(III) bioreduction (determined within 48 h of bioreduction) increased linearly with increasing initial NAu-2 surface site concentration [qmax Fe(III)]. The result was consistent with previous observations for Fe(III) oxides that microbial reduction rate was first-order with respective to surface site concentration (14, 15, 35), and suggested that microbial reduction of structural Fe(III) in NAu-2 also 2440
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followed the first-order rate with respect to NAu-2 surface site concentration:
dFe(III) ) - vmax X[qmax Fe(III)] dt
(3)
where vmax is the maximum bioreduction rate, and X is the cell concentration. Because Fe(II) sorption on NAu-2 was expected to reduce the number of surface sites that were otherwise available for microbial attachment and reduction, the bioreduction rate expression (eq 3), which was derived from the early phase of bioreduction when sorbed Fe(II) was negligible, needs adjustment to account for surface sites that were blocked by Fe(II) sorption:
dFe(III) ) - vmax XSfree dt
(4)
where Sfree is the surface site concentration of Fe(III) (mM) that is the difference between the total number of surface sites minus the number of surface sites blocked by Fe(II). The Sfree is mathematically expressed as the following:
Sfree ) qmax Fe(III) - aFe(II)NAu-2
(5)
where Fe(II)NAu-2 (mM) is Fe(II) sorbed onto NAu-2 surfaces at time t, and a is the stoichiometric ratio of moles of NAu-2 surface sites blocked by one mole of sorbed Fe(II). Fe(III) is the total Fe(III) remaining in the system at time t. Combined eqs 4 and 5 become eq 3 when there is no sorbed Fe(II) at time zero. Combining eqs 2, 4, and 5 yields the following:
[
]
dFe(III) c ) - νmax X Fe(III) - aFe(II)NAu-2 dt {d + Fe(III)} (6) Other equations required to model Fe(III) reduction include (1) Mass balance of Fe in the system:
Fe(III) ) Fe(III)o - Fe(II)tot
(7)
where Fe(III)0 is the initial Fe(III) concentration, and Fe(II)tot is the total Fe(II) concentration at time t. (2) Mass balance of Fe(II):
Fe(II)tot ) Fe(II)aq + Fe(II)cell + Fe(II)NAu-2
(8)
where Fe(II)cell is the sorbed Fe(II) concentration on cell surfaces. The sorbed Fe(II) on cell and NAu-2 surfaces is
the literature (15, 37)) and the experiments were performed without any nutrients, the variation of the measured cell concentration (CFU counts) was within a factor of 3 (Supporting Information Figure S6). Thus, in the modeling, the cell concentration (X) was kept constant. The parameter νmax was independently estimated from the inset of Figure 3 by linear regression using eq 3. The estimated νmax was 2.76 × 10-5 ((3 × 10-7) hr-1 (for 1 × 108 cells). Parameter a was the only fitting parameter that was estimated by minimizing the least-square errors between the model calculated and measured Fe(II)tot concentrations (Figure 3). The fitted a value was similar for different initial NAu-2 concentrations with an average value of 2.2 ( 0.3. The good agreement between the model calculated and measured Fe(II)tot with variable initial Fe(III) concentration indicated that the model satisfactorily described the experimental results (Figure 3).
TABLE 2. Reactions for the Fe(II) Sorption and Fe(III) Reduction 1. Fe(II) sorption to NAu-2 Fe(II)aq + NAu-2 ) [NAu-2-Fe2+] 2. Fe(II) sorption to cells Fe(II)aq + Cell ) [Cell-Fe2+] 3.a reduction of Fe(III) in NaAu-2 and oxidation of lactate (39) 1.044 [Fe(III)3.83]NAu-2 + CH3CHOHCOO- + 2H2O ) 1.044[Fe(II)3.83]-3.83NAu-2 + CH3COO- + HCO3-+5H+ 4. rate of Fe(III) reduction (dFe(III))/(dt) ) - νmax X[qmax Fe(III) - aFe(II)NAu-2] 5. Fe(II)tot ) Fe(III)0 - Fe(III) 6. Fe(II) speciation Fe(II)tot ) Fe(II)aq + Fe(II)cell + Fe(II)NAu-2 a This reaction is written to illustrate the general reaction between lactate oxidation and Fe(III) reduction in NAu-2 without consideration of speciation of biogenic Fe(II). In reality, a certain fraction of biogenic Fe(II) is released from the NAu-2 structure and becomes available for sorption onto NAu-2 and cell surfaces.
described by eq 1 with parameters listed in Table 1. The initial (t ) 0) conditions are as follows:
Fe(III) ) Fe(III)0 and Fe(II)tot ) 0
(9)
The model described by eqs 6-8, together with two Fe(II) sorption equations (Table 2) with Fe(II) speciation as per sorption isotherm (eq 1), were simultaneously solved under the initial condition (eq 9) using an ordinary differential equation solver package (STELLA 6.0, High Performance Systems, Inc., NH) to simulate the kinetics of microbial reduction of Fe(III) in NAu-2 as a function of NAu-2 concentration (Figure 3). All involved reactions and rate expressions are tabulated in Table 2. In the model simulation, Fe(II)tot was calculated individually for each time point during Fe(III) reduction. Since the initial cell density was high (1 × 108 cells/mL or 39.8 mM based on the conversion factor from
Parameter a (in eqs 5 and 6) was used to account for the effective blocking of NAu-2 surface sites by sorbed Fe(II) that masked solid-phase Fe(III) and reduced the pool of bioavailable Fe(III). Since the effect of Fe(II) sorption on the electron acceptor availability for further bioreduction cannot be explicitly quantified, several authors (14, 16) assumed 1:1 stoichiometric ratio (i.e., a ) 1) for the blockage of available Fe(III) in goethite by sorbed Fe(II). In other studies, the value of a ranged from 1.5 to 3.0 to account for Fe(II) sorption and siderite precipitation (15) or the Fe(II)/Fe(III) ratio in magnetite. Because of the relatively low Fe(III) concentration in NAu-2 (23.4%) compared to iron oxides and extensive inhibition effects of sorbed Fe(II) on the extent of Fe(III) reduction (30), a higher value of a (a) 2.2 ( 0.3), the inhibition factor, appears reasonable for NAu-2. Fe(III) Reduction at Variable Cell Concentration. The initial rate of reduction was also examined with a variable initial cell concentration (Figure 4), while the Fe(III) concentration in NAu-2 was kept constant at 21 mM. The initial rate of reduction increased with increasing cell concentration until the cell concentration of 108 cells/mL was reached, after which point, the initial rate of reduction leveled off. These
FIGURE 3. Experimental and modeling results for reduction of Fe(III) in NAu-2 by Shewanella putrefaciens CN32. The experiments were performed at constant lactate (20 mM), bacterial concentration (1 × 108 cells/mL), and variable Fe(III) concentrations. The inset shows the initial rate of reduction as a function of initial surface site concentration of Fe(II) (product of dimensionless sorption capacity and initial Fe(III) concentration) as described in eqs 3 or 4. The rate followed a first-order relationship with respect to free Fe(III) surface sites on NAu-2. VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Initial NAu-2 Fe(III) reduction rate as a function of cell concentration. The dotted line is an illustrative saturation line and not a model fit. The inset shows the same reduction rates after normalized to cell concentration with two line segments with different slopes. Experiments were performed with 20 mM lactate and 21 mM Fe(III) in NAu-2 at different cell concentrations.
FIGURE 5. Experimental and modeling results for Fe(III) reduction in NAu-2 as a function of cell concentration. Experiments were performed with 20 mM lactate and 21 mM Fe(III) in NAu-2 at different cell concentrations. The inset shows the estimated vmax as a function of cell concentration. data suggest that cell concentration did not linearly affect the reduction rate, but rather exhibited a saturation type behavior. The observed saturation point (i.e., ∼108 cells/mL) closely matched the calculated cell concentration required for cells to saturate NAu-2 surfaces based on the measured BET surface area of NAu-2 (Table 1) and published CN32 cell size (14) according to the method used by Roden and Zachara (19) (ca. about 1.7 × 108/mL cells at the NAu-2 concentration of 5 mg/mL). When cell concentration was higher than the saturation point, the excess cells probably remained floated in solution with a limited chance to contact Fe(III) sites for bioreduction, due to the limited surface sites available on NAu-2 (Supporting Information Figure S7). Additionally, the number of Fe(III) centers on clay surface that were in contact with a cell should decrease with increasing cell concentration because the cell might be in partial contact with other cells. Such a decreased contact would result in a decreased unit cell activity of Fe(III) reduction. Such a phenomenon has been previously discussed (36). High cell concentrations could also help coagulate/aggregate the clay particles into larger clusters, effectively decreasing the bioavailable surface area of NAu-2 (31). With previously determined parameters including a (except vmax), we applied the model described in Table 2 to 2442
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estimate vmax in eq 6 by matching the model calculated and measured Fe(II)tot for each Fe(II)tot production curve as a function of time (Figure 5). The measured cell concentration was used for each curve in the model calculations. The estimated vmax has a similar trend as the initial rate with increasing cell concentration (Figure 5 inset). The rate of microbial reduction of electron acceptors or oxidation of electron donors is commonly assumed to be a linear function of cell concentration under both growth and non-growth conditions (e.g., 14, 38). Our results, however, indicated that such an assumption was not applicable to describe microbial reduction of structural Fe(III) in NAu-2. This effect likely resulted from a limited number of Fe(III) centers on NAu-2 surfaces (Figure S7) as described above. This result was consistent with that of Burgos et al., (2003) (35) who suggested that all cells are not equally active in competing for solid-phase electron acceptor due to limited surface sites. More work is required to quantify the mechanism of electron transfer to solid-state electron acceptor. Nonetheless, our results suggest that a nonlinear, saturationtype microbial reduction rate as a function of cell concentration is required to describe Fe(III) reduction with variable cell concentration, even under non-growth conditions.
This study demonstrated that the bioreduction of structural Fe(III) in clay minerals is first-order with respect to the reactive surface concentration of Fe(III) and saturation-type kinetics with respect to bacterial cell concentration. With these rate relationships, the relative importance of various parameters that control microbial reduction of Fe(III) in clay minerals can be systematically evaluated. Our results also provide conceptual and/or numerical bases to predict the geomicrobiological behavior of this important group of minerals.
Acknowledgments This research was supported by a grant from National Science Foundation (EAR-0345307) to H.D. and the Clay Minerals Society (CMS) and Geological Society of America (GSA) student research grants to D.J. The research was also supported by a grant from US-DOE, Environmental Remediation Science Program (ERSP) to C.L. We thank Dr. Jonathan Levy for his suggestion during manuscript preparation. We are grateful to three anonymous reviewers for their constructive criticisms which significantly improved the quality of the manuscript.
Supporting Information Available Additional details are shown in Figures S1-S7 This material is available free of charge via the Internet at http:// pubs.acs.org.
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Received for review August 11, 2006. Revised manuscript received December 23, 2006. Accepted January 19, 2007. ES0619399
