Dissolution of Al-substituted goethites by an aerobic Pseudomonas mendocina var. bacteria

Geochimica et Cosmochimica Acta, Vol. 64, No. 8, pp. 1363–1374, 2000 Copyright © 2000 Elsevier Science Ltd Printed in the USA. All rights reserved 0016-7037/00 $20.00 ⫹ .00

Pergamon

PII S0016-7037(99)00404-4

Dissolution of Al-substituted goethites by an aerobic Pseudomonas mendocina var. bacteria P. A. MAURICE,1,* Y.-J. LEE,2 and L. E. HERSMAN3 1

Department of Civil Engineering & Geological Sciences, University of Notre Dame, Notre Dame, IN 46556. 2 Department of Geology, Kent State University, Kent, OH, 44242 USA 3 Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545 USA (Received July 13, 1999; accepted in revised form November 1, 1999)

Abstract—Goethite particles in soil environments often contain Al3⫹ substituted for Fe3⫹ in octahedrally coordinated sites. Al substitution has been shown to alter mineral stability and abiotic dissolution rates. This study focused on the effects of Al substitution (to 8.8 mol%) on synthetic goethite dissolution by an aerobic Pseudomonas mendocina var. bacteria. In contrast to dissimilatory iron reducing bacteria (DIRB), this bacteria is not capable of using Fe as a terminal electron acceptor for oxidative phosphorylation, and hence only requires ␮M concentrations of Fe for metabolism. Pure and substituted goethites were reacted with microorganisms in Fe-limited growth media wherein the only source of Fe was the solid phase, so that microbial populations could only grow by obtaining Fe through mineral dissolution. Because at least some Fe was taken up by the bacteria, we could not measure Fe release rates directly from dissolved Fe concentrations. Rather, we relied upon microbial growth measurements as indirect indicators of mineral dissolution. Increasing Al substitution resulted in particles with progressively decreasing mean particle length and aspect ratios, as well as fewer domains, as measured by atomic-force microscopy (AFM); but with increasing structural order as determined by XRD line widths. Experiments conducted in the dark at 22°C, exposed to the atmosphere, showed that maximum microbial population did not correlate with particle specific surface area, which is in contrast with previous studies using DIRB. Maximum microbial population increased a small amount with increasing Al content of the goethites, in contrast with several previous investigations of abiotic dissolution. Because dense biofilms formed, we were unable to use AFM to observe mineral dissolution features. AFM imaging suggested that more highly substituted goethites formed denser aggregates, and previous investigations have shown that aggregate structure is important for microbial attachment, which is prerequisite for dissolution. Hence, effects of Al substitution on aggregate structure is a focus of ongoing research. Copyright © 2000 Elsevier Science Ltd sythe et al., 1998; Hersman et al., in prep.) has shown that the P. mendocina var. is able to achieve substantial growth in the presence of hematite, goethite, or ferrihydrite as compared to growth in FeEDTA solution, under aerobic conditions. Research has also shown that microbial growth increases with increasing concentration of Fe(III)(hydr)oxide, although not in a linear fashion (Hersman et al., 1996; Hersman et al., in prep.). Both of these observations indicate that the bacteria are capable of obtaining Fe from the mineral phases, and hence in promoting Fe(III)(hydr)oxide dissolution. Analysis of metabolic products has shown significant differences between growth on mineralogic Fe sources as compared to FeEDTA, suggesting the production of extracellular metabolites capable of enhancing Fe acquisition (Hersman et al., 1996; Hersman et al., in prep.). Quantification of dissolution rates in the presence of siderophores produced by these microorganisms is the subject of ongoing investigations (e.g., Hersman et al., 1995; Kraemer et al., 1999). At the present time, the mechanism(s) by which the P. mendocina var. enhance Fe(III)(hydr)oxide dissolution remain unknown; there is evidence for primarly non-reductive processes although some ferrous Fe has also been observed in solution (Hersman et al., 1996; Hersman et al., in prep.). The research reported here focuses on microbially mediated dissolution (referred to hereafter as microbial dissolution) of pure and Al-substituted goethites. Soil goethites commonly exhibit substitution of octahedrally coordinated Fe3⫹ by the

1. INTRODUCTION

Fe is an important nutrient required by most life forms; yet, in aerobic environments, dissolved Fe concentrations tend to be extremely low except in the presence of significant concentrations of organic complexing or chelating agents. Schwertmann (1991) pointed out that because the solubility products of common soil Fe(III)(hydr)oxides (Fe3⫹) 䡠 (OH)3 ⱕ 10⫺37, the availability of Fe in aerobic soils must be governed by dissolution rate. Microorganisms living in aerobic environments therefore are faced with the need to acquire Fe under conditions of low solubility. In order to better understand the rates and mechanisms of microbially enhanced Fe(III)(hydr)oxide dissolution in aerobic environments, we are studying dissolution by an aerobic Pseudomonas mendocina var. Unlike commonly studied (e.g., Arnold et al., 1988; Arnold et al., 1990; Lovley, 1991; Lovley et al., 1986; Nealson and Meyers, 1992; Zachara et al., 1998) dissimilatory iron reducing bacteria (DIRB), the aerobic P. mendocina var. is not capable of using Fe as a terminal electron acceptor for oxidative phosphorylation (Hersman et al., 1996; Forsythe et al., 1998); hence, the P. mendocina var. bacteria require only ␮M quantities of Fe, compared to the mM concentrations used by DIRB. Previous research by our group (Hersman et al., 1996; For* Author to whom correspondence should be addressed (pmaurice@ nd.edu). 1363

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Maurice et al. Table 1. Characteristics of synthetic goethite samples used in this study.

Al content (mol%) 0 1.0 1.9 2.6 3.5 4.2 6.2 8.8 a b

BET surface area (m2 g-1) 33.4 36.0 34.7 30.0 36.5 35.6 30.9 26.9

AFM particles measured (no.) 23 25 27 20 19 21 15 20

Mean particle length (nm)a

Mean particle aspect ratio b

2195 (1116) 1812 (961) 1161 (663) 1125 (546) 761 (290) 616 (218) 420 (155) 379 (81)

19:1(30:1) 15:1 11:1 10:1 8:1 6:1 5:1 4:1

Comments multidomainic multidomainic 兩 兩 兩 V few domains 1–2 domains

Length parallel to c axis, standard deviation in parentheses. Ratio in parentheses represents aspect ratio for single domain particles. Aspect ratio is defined as the ratio of particle length to particle width.

smaller Al3⫹ cation (e.g., Fitzpatrick and Schwertmann, 1982; Tardy and Nahon, 1985; Schwertmann, 1991). Tardy and Nahon (1985) showed that the Al contents of goethites in laterites range from 0 to 30 mole%. Schulze and Schwertmann (1984) demonstrated that goethite needles become shorter and less multidomainic in character as Al substitution increases. Schwertmann (1984) suggested that Al substitution results in particles with fewer structural defects due to a slower crystallization rate. Mun˜oz (1988) observed a narrowing of XRD line widths with increasing Al substitution, suggesting some increase in structural order with increasing Al substitution. Systematic changes in particle micromorphology, multidomainicity (i.e., the presence of numerous parallel subunits or domains), and X-ray diffraction (XRD) peak positions suggest that Al is contained within the goethite lattice structure, most probably in a random fashion (e.g., Schwertmann and Cornell, 1991). At the present time, however, we do not know the exact details of how the Al is distributed. Schwertmann (1991) reported a substantial decrease in goethite dissolution rate in 6 M HCl at 25°C with increasing Al substitution (to ⬃10 mol%). Most of the dissolution appeared to occur along domain boundaries, and number of domains decreased with Al substitution. Torrent et al. (1987) observed a similar Al-content/dissolution rate trend for dissolution in dithionite-citrate-bicarbonate. However, Lim-Nunez and Gilkes (1987) did not observe such a trend, upon dissolution in 1 M HCl. Al substitution also appears to affect hematite/goethite relative stabilities, but different results have been reported in laboratory versus field studies (Cornell and Schwertmann, 1996). Given that the morphology, microtopography, multidomainic character, structural order, stability, and abiotic dissolution rates of goethite change with Al substitution, it is reasonable to expect that microbial dissolution rates also will be affected. Hence, an understanding of rates and mechanisms of bacterially mediated dissolution must take into consideration the widespread Al substitution of goethite in soils. The purpose of this research was to determine the effects of Al substitution on goethite dissolution by an aerobic P. mendocina var. bacteria. A series of Al substituted goethites (0 – 8.8 mol%) was synthesized, characterized, and reacted with the P. mendocina var. bacteria to determine effects of Al substitution on microbial growth (as related to mineral dissolution). Batch dissolution experiments in 0.001 M oxalate, pH 3, 22°C also were conducted, to test for the presence of highly soluble oxalate-extractable phases (ferrihydrite surface coating or ad-

sorbed Fe; Schwertmann and Cornell, 1991) which may form during goethite synthesis, and because oxalate is the most common simple organic ligand in soils (e.g., Fox and Comerford, 1990). As described below, maximum microbial population increased with increasing Al substitution; hence, mineralogic variability of the type commonly observed in soil environments can be expected to alter rates of microbially mediated dissolution processes. 2. MATERIALS AND METHODS

2.1. Mineralogic Samples A series of pure and Al substituted goethite (␣-FeOOH) samples was prepared according to methods (section 5.2.2; FeNO3 and AlNO3 in KOH) described by Schwertmann and Cornell (1991). The Fe content was measured by flame atomic absorption (AA) and the Al content by cation exchange chromatography (Al-CEC, Sutheimer and Cabaniss, 1995) on digested samples; mol% Al substitution data are presented in Table 1. The highest Al concentration (8.8 mol%) is close to the reported maximum amount (10 –12 mol%) that will be taken up in goethites grown at high pH, because of the increased solubility of Al (Schwertmann and Cornell, 1991). Qualitative X-ray diffraction (XRD) analysis (Rigaku X-ray powder diffractometer D/MAX-2 TBX ␪/␪, Cu K␣ radiation) showed that each sample is monomineralic to the detection limit of the XRD analysis (approximately 5%). XRD analysis also showed peak shifts with increasing Al substitution towards higher angle, especially at high 2␪ (Fig. 1). As described by Schwertmann and Cornell (1991), isomorphous substitution of Al for Fe leads to slightly smaller unit cell edge lengths, resulting in XRD peak shifts to higher angles. The peak shifts, in turn, provide evidence that the Al is incorporated into the goethite structure. Relative peak intensities in these diffractograms cannot be used to infer degree of structural order/disorder because as described by Brown (1980) and Schulze and Schwertmann (1984), changing particle morphology results in changing preferred orientation with increasing Al substitution. However, decreased line widths with with increased Al substitution suggest increased structural order (‘crystallinity’); a similar decrease was reported by Muno˜z (1988). BET surface area measurements (i.e., surface area determined from gas adsorption isotherms, Brunauer et al., 1938) were conducted with a model SA3100 volumetric sorption analyzer from Coulter Instruments, Inc. using N2 adsorption at ⫺195°C. Prior to adsorption measurements, mineral samples were degassed at 120°C for 14 h under reduced pressure of approximately 10⫺4 Torr. This degassing temperature was used because at temperatures higher than 140°C, apparent phase changes were observed for some samples (samples changed color). Resulting BET surface areas are presented in Table 1.

2.2. Bacterial Sample The microorganism used in our experiments is a bacteria isolated from surface soil samples collected at the Nevada Test Site. The

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Fig. 1. X-ray diffractograms of pure and Al-substituted goethites. Schwertmann and Cornell (1991) hypothesized that shift to higher angles occurs because of replacement of Fe3⫹ by the smaller Al3⫹, leading to slightly smaller unit cell edge lengths. All samples appear to be monomineralic (only goethite) at the limits of detection of the XRD (⬃5%). Decrease in line widths for Al-substituted samples suggests increase in structural order (crystallinity).

individual bacteria are rod-shaped, approximately 2 ␮m long, 0.5–1 ␮m wide, and typically have one polar flagellum. Using comparisons of 16s rRNA gene total sequence, the bacteria has been aligned at the species level (0.78% difference) to P. mendocina (MIDI Labs, Newark, DE). In addition, it tested positive for catalase, oxidase, and nitrate reduction (assimilatory), but negative for fermentation and Fe and sulfate reduction. Although this microbe is not capable of using Fe as a terminal electron acceptor for oxidative phosphorylation, it has been shown to reduce Fe during growth on hematite under overall aerobic conditions (Hersman et al., 1996).

2.3. Microbial Promoted Goethite Dissolution Experiments A modification (different concentrations in growth medium) of the procedure described by Hersman et al. (1996) and Forsythe et al. (1998) was used for batch microbial dissolution. The growth medium consisted of the following (g L⫺1): K2HPO4, 0.5; NH4Cl, 1.0; MgSO4 䡠 7H2O, 0.2; CaCl2, 0.05; succinic acid disodium salt anhydrous (C4H4Na2O4), 5; trace elements, 0.125 ml (0.005 g MnSO4 䡠 H2O, 0.0065 g CoSO4.7H2O, 0.0023 g CuSO4, 0.0033 g ZnSO4 and 0.0024 g MoO3 per 100 mL distilled, deionized water); H2O, 1.0 L; pH ⫽ 7.1 (not buffered; pH generally increases to ⬃8 in experiments of this type). The medium purposely contained too little Fe to support rich microbial growth, so that in order for the bacteria to obtain Fe, they must promote goethite dissolution. Previous experiments reported by Hersman et al. (1996) showed that the succinate medium, itself, did not promote Fe(III)(hydr)oxide dissolution. 12 mg of each goethite sample was added to acid-washed polycarbonate flasks containing 30 mL of growth medium. Following sterilization by autoclave, the flasks were inoculated with 110 ␮L of an overnight culture of P. mendocina var. having an optical density of 0.515 absorbance units at 600 nm wave number. These cultures were then incubated at room temperature, in the dark, while shaking at 50 rpm. Reaction flasks were loosely covered to allow for equilibration with the atmosphere. Controls lacking Fe particles, and uninoculated Fe particle controls also were prepared for comparison. At various time intervals, 1 mL samples were removed from the top of the reaction vessels by sterile pipet, and microbial population was determined by absorbance at 600 nm. In the presence of the bacteria, all of the goethite settled to the bottom of the flasks within a few hours, so that goethite

particles did not interfere with absorbance measurements. This was reflected in the low values of the first absorbance measurements made (see 3.4 below). In collecting samples for analysis, we were careful to avoid the dense goethite/microbial biofilms which formed at the bottom of the flasks (see Forsythe et al., 1998). AFM imaging of samples collected in this way and then filtered onto polycarbonate membranes revealed bacteria but no goethite needles. While absorbance measures only cells in suspension and not those attached to the mineral surface, Lowry protein analysis (Lowry et al., 1951) determined that there was no significant difference between protein in the cells in the broth alone and the total protein of cells in the broth plus those cells attached to mineral surfaces. Absorbance was then correlated to colony forming units, by the dilution-pour-plate method (see Standard Methods et al., 1971).

2.4. Oxalate-Promoted Dissolution Experiment An oxalate-promoted dissolution (referred to hereafter as ‘oxalate dissolution’) experiment was performed in order to test for the presence of a highly reactive surface precipitate or adsorbed Fe (see below), as suggested by AFM images of the 8.8% goethite, and for comparison of dissolution behavior. We chose 0.001 M oxalate concentration because oxalate is often present at millimolar concentrations in aerobic soils (e.g., Fox and Comerford, 1990). Goethite samples were dissolved in 0.001 M oxalic acid solution at pH3 in batch reactors at 22°C. 12 milligrams of goethite (pure, 1.0%, 4.2%, and 8.8% Al substitution, chosen to represent a range of Al substitutions) were added to 2 mL of Milli Q deionized water in 30 mL polypropylene centrifuge tubes. The tubes had been washed in 0.1 N HCl and rinsed repeatedly in deionized water prior to use. MilliQ deionized water was used for all solutions. The goethite suspensions were then placed in an ultrasonic bath for 2 sec. to promote disaggregation without breaking crystals apart along domain boundaries. After ultrasonification, 23 mL of 0.0011 M oxalic acid solution (concentration needed to make a 0.001 M oxalate final concentration; pH adjusted to 3.0 using 0.01 N NaOH and HNO3) were added to each centrifuge tube. One sample blank, in which no goethite particles were present, was also made. Separate centrifuge tubes were set up for each of the following time periods: 2 h, 6 h, 12 h, and 24 h (2 replicates per sample per each time period plus additional replicates for pH measurements). After preparation, all of the tubes were placed on a shaker table at 22°C in the dark. Separate tubes were removed at

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Fig. 2. Example of Height mode (a) and Amplitude mode (b) TMAFM images of 5.5% Al substituted goethite on muscovite. While the Height mode image provides data in the z-direction (height or depth), the Amplitude mode image provides more easily interpretable surface microtopography. The full z-range scale for (a) (white to black) is 1.2 ␮m.

the above time intervals. Samples were centrifuged for 20 min. at 13000 rpm, and the solution was decanted and filtered through 0.1 ␮m Nuclepore威 polycarbonate membrane filters. The solutions were analyzed for Fe by graphite-furnace AA (Perkin Elmer model 5100 PC equipped with an automatic sampler). Although we planned Al analysis by HP-CEC (Sutheimer and Cabaniss, 1995), we were unable to complete the analyses because of a mechanical problem in the HP-CEC unit at the time of the experiment. Standards for graphite-furnace AA analysis were made up in 0.001 M oxalate using Fisher Scientific brand analytical AA standards. Samples were also analyzed for dissolved organic carbon content using a Shimadzu TOC5000 organic carbon analyzer.

2.5. Atomic Force Microscopy Samples were prepared for AFM imaging by suspending 1 mg of goethite in a beaker containing 10 mL MilliQ deionized water, and placing the beaker in an ultrasonic bath for 2 sec. A droplet of the resulting suspension was then applied to a freshly cleaved muscovite (mica) basal-plane surface, and allowed to air dry under protective cover. Goethite samples were imaged in air using a Digital Instruments Inc. Multimode Nanoscope III Atomic Force Microscope in the Department of Geology at Kent State University. A description of AFM and its application to environmental particles can be found in Maurice et al. (1996) and Maurice and Lower (1998). Tapping-mode AFM (TMAFM) was used throughout. In most instances, two data types were collected simultaneously: Height mode and Amplitude mode. While Height mode gives more quantitative topographic information (height of sample’s z axis), Amplitude mode gives data that are essentially the first derivative of the Height mode data, highlighting changes in microtopography and resulting in an image that is more easily interpretable. An example of these two types of images is shown in Figure 2, which is from 5.5% Al goethite. The z-axis data are not directly related to topography in Amplitude mode and hence are not reported in additional images shown below. Z-axis data from Height mode images collected simultaneously were used to measure particle dimensions. We used specialized Tesp etched Si probes (tips) with 125 ␮m cantilever length and resonant frequency in the range of 284 –355 kHz. Following reaction, biofilm-containing goethite aggregates were transferred to clean glass slides in the manner described by Forsythe et al. (1998) and allowed to air dry prior to TMAFM imaging. While air drying likely alters biofilm properties, soil environments often are characterized by

periodic wet/dry cycles. Comparison with in-solution epifluorescence imaging of biofilm structure is provided by Forsythe et al. (1998). 3. RESULTS AND DISCUSSION

3.1. AFM of Unreacted Samples Figure 3a– e presents examples of Amplitude-mode TMAFM images of pure and Al-substituted goethites. Particle dimensions measured from corresponding Height-mode images are summarized in Table 1. As shown in Figure 2, above, particles often were aggregated, intergrown, twinned, and/or contained parallel striations along the c-axis. As described by Cornell and Schwertmann (1996 and refs. therein), TEM (including high resolution TEM, HRTEM) analysis has shown that goethite crystals grown in alkaline media frequently consist of parallel domains or intergrowths extending along the c-axis and stacked along the a and b-axis. Individual domain widths measured by TEM range from 10 –30 nm. The TEM images showed that the domain lengths also varied, so that goethite particles often contained irregular ends consisting of (021) faces. Comparison of our AFM images with the TEM images (Cornell and Schwertmann, 1996 and refs. therein) suggests that while some fine striations may be steps, many, and particularly the most prominent, are likely the surface expressions of domain boundaries; hence, many of the goethite crystals have have multidomainic character. The complex intergrowths, etc. shown in Figure 2 made measurement of particle lengths difficult; hence, we averaged a number of measurements per particle. In agreement with XRD analysis, the morphologies of particles imaged by AFM were appropriate for goethite. Only needles were observed; no platelets (hematite) or clusters of amorphous-looking material were detected. As shown in Figure 3a, the pure goethite sample contains acicular, highly multi-domainic crystals exhibiting a broad distribution of particle lengths, from several hundred

Bacterially mediated Al-goethite dissolution

Fig. 3. Amplitude-mode TMAFM images in air of pure (a,b), 1.9% (c), 3.5% (d), and 8.8% (e) Al substituted goethites deposited the basal-plane surface of muscovite mica as a substrate for imaging. The ends of particles have shapes that are likely altered to some degree by the AFM tip shape. Schwertmann and colleagues have observed sharper terminations on goethite, consisting of (021) faces (See Cornell and Schwertmann, 1996).

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nanometers (nm) to as many as 7 microns (␮m); mean particle length ⫽ 2195 nm, and mean aspect ratio ⫽ 19:1. Here, the aspect ratio is defined as the ratio of crystal length to crystal width. Individual particles commonly exhibit numerous striations parallel to the c-axis (lengthwise); the more prominent striations likely represent domain boundaries (Fig. 3b). Schwertmann (1984) showed that goethite crystals of this sort are dominated by (110) faces. Some particles contain only a single domain; these particles have aspect ratios as high as 30:1. Needle cross-sections are not circular, so that we measured needle width (x–y direction on AFM images) as well as depth (z direction on AFM images) and averaged the two measurements for use in determining aspect ratios. In performing measurements of particle length and width, we took into consideration the finite shape of the AFM tip, which alters particle edges in AFM images (See Maurice and Lower, 1998). AFM measurements showed that as Al substitution increased, the needle cross-section (perpendicular to c) became narrower in one direction and thicker in the other (i.e., more platey). This agrees with previous results reported by Schwertmann (1984) based on TEM imaging of Al goethites. The goethite needles were found to preferentially sit on the thicker prismatic crystal faces, which would lead to preferred orientation in XRD analysis. Figure 3c– e show TMAFM images of goethite samples with Al substitution. Representative samples at 1.9%, 3.5%, and 8.8% Al substitution are shown (c– e, respectively). As summarized in Table 1, particle length, aspect ratio, and multidomainic character decrease with increasing Al content. Particle size distributions also become narrower. The most highly substituted sample (8.8%; Fig. 3e) contains particles only on the order of several hundred nm in length, with mean length ⫽ 379 nm and mean aspect ratio ⫽ 4:1. Most particles are monodomainic. The surface microtopography of the 8.8% Al goethite needles is not crisp; rather, many of the needles have slightly rough, rounded microtopography which might represent adsorbed Fe and/or a thin coating of precipitate (see section 3.3, below). Measurements made from AFM images are in good agreement with TEM results by Schulze and Schwertmann (1984), who described a decrease in length from 1246 nm for a pure goethite to 358 nm for a goethite with 9.7 mol% Al substitution. According to Parks (1990), particle specific surface area (As), increases as particle or grain size decreases and with increasing intricacy of particle shape, according to the relationship: As ⫽ ks/pd, where ks is a shape factor, p is the specific gravity, and d is the particle size. For the goethites used in our experiments, As (as measured by the BET method) was not found to correlate well with particle length (e.g., linear R2 ⫽ 0.401). This result is in agreement with the data of Schulze and Schwertmann (1984) on Al substituted goethites. Using their data from 0 to 9.7 mol% Al, we calculate no correlation between particle length and BET measured As (e.g., linear R2 ⫽ 0.104). Geometrical specific surface areas (Asg) were calculated from AFM measurements (Table 1), by assuming a cylindrical shape for goethite, correcting for the difference in Fe and Al atomic weights,

and neglecting surface microtopography. Asg generally decreased in a linear fashion with decreasing particle length, but with a linear correlation of only R2 ⫽ 0.809 (data not shown here) reflecting the simultaneously changing aspect ratios and deviations from cylindrical shape. No correlation was observed between calculated Asg and BET measured As (R2 ⫽ 0.217; data not shown here). These observations suggest that surface microtopography, which is a component of the shape factor, ks, may play an important role in determining BET measured As. As % Al substitution increases and number of domains per particle decreases, there are fewer striations at the surfaces of particles. It is also possible that particle aggregation leads to errors in BET measurements as well as geometrically estimated surface area. Visual inspection, optical microscopy, and scanning electron microscopy showed that the goethite samples contained numerous aggregates, and that the aggregates appeared to be denser and more numerous for the more Al-rich samples. This was reflected in the difficulty of AFM imaging; the more Al-rich samples tended to show numerous artifacts related to tip-sample shape interactions. Such artifacts are common when particles are aggregated and hence do not lie flat (Maurice and Lower, 1998). 3.2. AFM Observations of Samples Reacted with Bacteria Visual inspection of samples following reaction with bacteria showed that many goethite aggregates formed in the reaction vessels, and that aggregates were coated and interconnected with biofilm material. This is in agreement with previous observations reported by Forsythe et al. (1998), based not only on visual inspection but also on in-situ (in solution) epifluorescence imaging and ex-situ (in vacuum and air, respectively) scanning electron microscopy (SEM) and AFM. AFM (Fig. 4) revealed that the biofilm material was similar to that described in detail by Forsythe et al. (1998), and that it contained individual and aggregated goethite particles as well as bacterial bodies, all embedded in an amorphous looking organic material through which a network of fibrous attachment features was intertwined. Because the goethite particles were coated with biofilm material, observation of potential dissolution features (as per Grantham and Dove, 1996; Grantham et al., 1997) was not possible. The dense biofilm material also made it impossible to determine whether bacteria attached preferentially to particular goethite surface structures, or vice versa; in many cases, individual bacterial bodies were larger than individual goethite particles. In the experiments performed here, we saw no apparent differences in cell size during the initial growth phase. 3.3. Oxalate-Promoted Dissolution Dissolution of Fe(III)(hydr)oxides by simple organic ligands such as oxalate has been shown to be surface controlled (e.g., Zhang et al., 1985; Stumm et al., 1985; Sulzberger et al., 1989; Maurice et al., 1995). Stumm and his colleagues have proposed that non-reductive, ligand-promoted dissolution of Fe(III) (hydr)oxides proceeds by the following steps: 1. A fast inner-sphere adsorption step wherein an Fe(III)organic surface complex is formed;

Bacterially mediated Al-goethite dissolution

Fig. 4. TMAFM Amplitude mode image in air of biofilm formed upon 3 days reaction of 1.9% Al substituted goethite with the P. mendocina var. bacteria. The biofilm material was deposited on a clean glass slide and allowed to air dry prior to imaging. The biofilm consists of a thin (20 –200 nm thick) film of organic material in which microorganisms and goethite particles are embedded. Some salts (primarily NaCl, determined by SEM/EDS) which precipitated on drying also are present, imparting a rough texture to the glass substrate surface.

2. A slow step wherein the Fe(III)-ligand complex detaches from the surface; and 3. A fast re-protonation of metal-hydroxide surface sites. Step (2), which is the slowest step, controls the overall rate of reaction. Our experiments were conducted in the dark to prevent potential reductive dissolution, which proceeds by a similar but more complex mechanism. As shown in Figure 5, samples with 0, 1.0, and 4.2% Al substitution showed similar Fe release behavior in 0.001 M

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oxalate at pH 3, 22°C. There is an initial very rapid dissolution (to 2 h), as commonly observed in batch dissolution experiments (e.g., Berner 1981; Chin and Mills 1991; Stillings et al. 1995). By 6 h reaction time, an apparent steady-state Fe release rate is achieved, ⬃0.6 ⫾ 0.1 ␮g Fe m⫺2 h⫺1. Cornell et al. (1974) and Cornell et al., (1976) reported initial Fe release rates from a range of synthetic goethites (no Al substitution) in 0.5 M HCl at 20°C of 0.3–7.5 ␮g Fe m⫺2 h⫺1; hence, our rates are within the range of rates reported by those authors. The 8.8% Al substituted goethite showed significantly greater initial (within first 2 h) dissolution, but the apparent steady-state Fe release rate (at ⱖ6 h) was approximately the same as for the other samples. This suggests to us that the 8.8% Al goethite contained highly reactive Fe associated with the solid phase, most probably as adsorbed Fe or a thin precipitate (no ferrihydrite observed by XRD, no small clusters of rounded particles typical of ferrihydrite observed by AFM, low overall surface area), but that once this highly reactive Fe dissolved, dissolution proceeded with the same Fe release rate as observed for the other pure and Al substituted goethites. As discussed in section 3.1 above, AFM imaging of the the 8.8% Al goethite prior to reaction revealed a thin patchy film on the surfaces of individual particles, suggesting the presence of a surface coating. Because we did not measure Al release, we could not calculate Al-goethite overall dissolution rates or rate constants. Oxalate adsorption correlated well with %Al substitution (Figure 5b). Mun˜oz (1988) observed enhanced sulfate adsorption on Al-substituted goethites, and suggested that this enhanced sorption could be due to changes in goethite point of zero charge and/or to different densities of adsorption sites as related to crystallographic changes. The effects of Al substitution on goethite point of zero charge have not been well documented at the present time. Initial Fe release was not found to correlate with oxalate adsorption density (at 2 h), which is not surprising considering that initial dissolution generally is complex. Our results, which show no effect of Al substitution on the

Fig. 5. (a) Dissolution of pure and Al substituted goethites in 0.001M oxalic acid, pH 3 22°C. Each data point represents the mean of two replicate experiments. (b) Oxalate adsorption to goethite as a function of Al substitution, at 2 h reaction time (R2 ⫽ 0.978).

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Fig. 6. Microbial population (measured by absorbance at 600 nm, related to cell count) over time for a control sample (no particles present), a pure goethite, and several Al substituted goethites. Not all samples are shown here because the figure would be too cluttered if all were shown. In general, maximum growth tended to occur earlier, and to attain a higher absorbance (correlates with cell count) as Al content increased. This indicates increasing microbial dissolution with increasing Al content. Each point represents the mean of 3 samples. By way of comparison, growth in 30 ␮M FeEDTA achieved a maximum of 1.90 absorbance units at day 1 and growth in 0.24 ␮M FeEDTA achieved a maximum of 1.06 absorbance units at day 4.

Fe release rate from goethite in 0.001 M oxalate at pH 3, 22C, contrast with the results of Schwertmann (1984) and Schwertmann (1991) which show a decrease in dissolution rate with increasing Al content in 6 M HCl (Schwertmann also measured dissolution rate from Fe release). He attributed the trend primarily to high reactivity of less ordered parts of the goethite crystals, which he termed ‘interdomainic material,’ in the less Al-substituted samples. Our results suggest that oxalate dissolution is not as sensitive to the effects of Al substitution as is dissolution in 6 M HCl. However, our results agree with previous results by Cornell and Schindler (1987) showing no effect of Al substitution on photochemical dissolution of goethites in oxalate at pH 2.6. In contrast to Schwertmann’s experiments, our experiments focused only on initial dissolution. Our study did not investigate long-term or extensive dissolution which might probe more thoroughly the effects of Al substitution on reactivity as related to structure. 3.4. Microbial Growth Curves Figure 6 shows microbial growth curves (growth measured by absorbance at 600 nm; 1.0 absorbance ⫽ 1.4 ⫻ 109 cells mL⫺1 as determined by dilution-pour-plate) over time for the control (bacteria only, no goethite), pure goethite, and several Al-substituted goethites. Not all of the Al substituted goethites are shown in this figure because overlapping growth curves make the data difficult to visualize. The bacteria showed substantial growth in the presence of all of the goethite samples. The growth curves peak and then decrease; the latter decrease is likely due to consumption of nutrients and buildup of toxic byproducts as is characteristic of many batch microbial exper-

Fig. 7. Maximum microbial population (absorbance at 600 nm) normalized to goethite As (in meters squared per gram) plotted against Al content. There is a clear increase in maximum microbial population (indicating enhanced mineral dissolution) with increasing Al content. The intial, high peak value for the 8.8% goethite sample (*) is not in line with other values. In this and subsequent figures, value with * is the initial high peak for the 8.8% goethite sample. Maximum microbial population corrected for the control in this and subsequent figures.

iments. The overall trend (see Fig. 6 as well as Fig. 7) was one of greater peak absorbance, representing greater maximum population size because absorbance is proportional to cell counts, with increased Al content. All but the most highly substituted goethite showed maximum population size at about 4 d reaction time; the 8.8% goethite showed maximum population size at only ⬃2 d. The 8.8% Al goethite growth curve consisted of a distinct, high initial peak, which decreased to a broad peak similar to the peaks observed on the other samples. Both the oxalate dissolution experiments and AFM imaging suggested the presence of a thin, highly reactive precipitate and/or adsorbed Fe on the surfaces of the 8.8% Al sample. Like oxalate, the bacteria apparently were able to access this highly reactive Fe easily, and once depleted, mineral dissolution was similar to that observed in the other samples. Although Al is toxic to many forms of life, the P. mendocina var. bacteria apparently are not inhibited by the presence of Al, at least when supplied in mineralogic form (substituted in goethite). At least some bacterial siderophores have been shown to have Alcomplexing capabilities (e.g., Evers et al., 1989), although we have not established whether this is the case for siderophores produced by the P. mendocina var. No correlation is observed when peak absorbance (related to maximum population size) is plotted against particle BET measured As (nor calculated Asg, data not shown here) for the 8 goethite samples (Fig. 8). This is in contrast with previous investigations involving effects of surface area on dissolution by DIRB (e.g., Roden and Zachara, 1996). Theoretical considerations suggest that oxides, which generally dissolve by surface-controlled dissolution mechanisms (e.g., Berner, 1978; Stumm et al., 1985), should exhibit increased dissolution rates with increased As, if other factors are held constant. For the Al goethites, however, a variety of characteristics change simultaneously (see Table 1).

Bacterially mediated Al-goethite dissolution

Fig. 8. Maximum microbial population (units of absorbance at 600 nm) versus particle surface area (in meters squared per gram) for pure and Al substituted goethites. As shown in this plot, there was no correlation between maximum microbial population and particle surface area, for the 8 goethite samples.

In this study, we have used microbial growth as an indirect means of monitoring mineral dissolution. This is possible because growth can be controlled and limited by the amount of Fe added to the medium. By comparing cell growth in a medium containing Fe(III)(hydr)oxide as the only source of Fe to growth curves for cells grown in various concentrations of FeEDTA, one can estimate the amount of Fe the bacteria dissolved from the Fe(III)(hydr)oxide. We found maximum population size of 1.90 absorbance units at day 1 in 30 ␮M (1676 ␮g L⫺1) FeEDTA and of 1.06 absorbance units at day 4 in 0.24 ␮M (13.4 ␮g L⫺1) FeEDTA. These results are in keeping with observations of Hersman et al. (in prep.) who found that bacterial growth is shifted towards smaller overall peak absorbances (smaller maximum population size) and delayed time periods as available Fe concentration decreases or as Fe is supplied in a less readily accessible form. We hypothesize that the shifts result because the bacteria have to exert more energy or perform more work (e.g., biofilm formation, siderophore production) to obtain sufficient Fe for growth. Ongoing work aims at determining a work function related to Fe supply. With the exception of the initial high peak for 8.8% Al-goethite, maximum absorbances for the goethites used in our experiments (Fig. 6) are similar to or slightly less than observed in 0.24 ␮M FeEDTA. Therefore, by analogy, the overall initial goethite Fe release rates to 4 d are on the order of a few tenths of a ␮g Fe m⫺2 hr⫺1, although they may potentially vary by as much as an order of magnitude. 3.5. Effects of Al Substitution on Bacterially Mediated Dissolution When normalized to As, peak absorbance (maximum population size) as related to mineral dissolution is shown to increase a small amount with increasing Al content (Fig. 7). In

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Fig. 9. Maximum microbial population (absorbance at 600 nm) normalized to goethite As (in meters squared per gram) plotted against mean particle length (in nanometers). There is an increase in maximum population size (indicating enhanced mineral dissolution) with decreasing particle size and decreasing particle aspect ratio (data not shown here). Linear correlation coefficient neglecting *, R2 ⫽ 0.758.

Figure 7 as well as Figure 9, data for the 8.8% Al goethite are plotted twice; once for the early, high peak (probably resulting from dissolution of a highly reactive surface coating) and once for the broader peak (probably resulting from dissolution of the Al goethite particles). Data from the broader peak are in line with data from the other samples shown in this figure; linear correlation coefficient, R2 ⫽ 0.749. However, 7 of the 8 samples (neglecting sample with 1.85% Al) show a strong linear correlation coefficient, R2 ⫽ 0.936. AFM images did not reveal any properties of the 1.85% Al sample which might account for the fact that this sample is significantly off the general trend. Why does microbial dissolution rate increase with increasing Al content? This question is difficult to answer because as discussed above, Al substitution affects several mineral characteristics: mineral composition (Al/Fe), multidomainic character, structural order, particle length, and particle aspect ratio. Quantification of defect densities is important because surface controlled, ligand-promoted dissolution of (hydr)oxide minerals is thought to occur preferentially at so-called reactive surface sites (e.g., steps, pits, kinks, dislocations, point defects; see Maurice et al., 1995 and refs. therein), many of which would be associated with structural defects (see section 3.3., above). Unfortunately, the dense biofilm covering on our samples and the probable low dissolution rate precluded us from using AFM to determine directly whether or not bacterial dissolution occurred preferentially along domain boundaries or at other sites. Schwertmann (1984) and Schwertmann, (1991) showed that domain boundaries increased inorganic dissolution. In our experiments, the particles with fewer domain boundaries (higher Al substitution) actually dissolved more quickly (more microbial growth). Structural order/disorder is also important for Fe(hydr)oxide dissolution. Stumm et al. (1985) showed that 0.001 M oxalate dissolved the more disordered phase ferrihydrite to a much

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greater extent than the more ordered phases goethite or hematite, and that this effect was stronger at pH 3 than at pH 5. Systematic shifts in XRD peak positions suggest that Al substitutes into the goethite lattice (Schwertmann and Cornell, 1991, and other refs.), although defect structures were not observed in the bulk of the lattice, using HRTEM (Mann et al., 1985). The effects of Al on structural order/disorder are open to some debate. On the one hand, Schwertmann (1984) and Schwertmann (1991) suggested that Al substitution results in goethites with fewer structural defects (referring to less ordered portions of the crystals within interdomains) because of slower crystal growth rates. On the other hand, Cornell and Schwertmann (1996) state that Al substituted goethites, particularly soil goethites, often have poor crystallinity. Finally, Mann et al. (1985) suggest, based on high resolution transmission electron microscopy (HRTEM), that Al does not induce defects in the bulk crystal. Infra-red (IR) spectroscopy has been applied but it is subject to spurious results when dealing with small Fe(III)(hydr)oxide particles. Indeed, Cornell and Schwertmann (1996) point out that it can be difficult to resolve various effects (e.g., particle size, Al substitution, ‘crystallinity’) on IR spectra. Mun˜oz (1988) observed narrowing of XRD peaks with increasing Al content of goethites grown similarly to those used in our experiments, indicating increased crystallinity (structural order) with increased Al substitution. We also observed peak narrowing with Al substitution. If structural order does indeed increase with increasing Al substitution, then our observed microbial dissolution trends are counter to structural order/ disorder considerations. However, it should be noted that our experiments most likely probed only initial dissolution, which would likely only dissolve material at or near the goethite surface; we do not currently know how surface structure relates to bulk crystalline properties of these goethites. As described in section 3.3. above, Fe release from goethite in 0.001 M oxalate at pH 3, 22°C was not sensitive to Al content in our samples. Moreover, oxalate is a good complexing ligand for both Al3⫹ and Fe3⫹, and for this reason may not be sensitive to Al substitution per se. While it is possible that our observations of increased microbial dissolution with increased Al substitution could result from the presence of Al sites (if Al substituted sites represent high-energy surface reactive sites), we are unable to evaluate this possibility at the current time because we do not know how the Al is distributed at and near the surface or the mechanism(s) of dissolution. Mineral dissolution rates often increase with decreasing particle size. When other factors are held constant, small, highsurface-area particles generally tend to be fast reacting (Parks, 1990). We observed increased microbial growth/microbial dissolution with decreasing particle length (Fig. 9; R2 ⫽ 0.758) and decreasing aspect ratio (R2 ⫽ 0.705; data not shown here). However, this particle length-dissolution trend cannot be explained by typical particle length-As relationships, because in our system, As does not correlate inversely with particle length. There are three main types of surface hydroxyl groups present on goethite: coordinated to one underlying Fe atom (A), coordinated to two Fe atoms (B), and coordinated to three Fe atoms (C). Type A groups appear to be the most reactive (e.g., Lewis and Farmer, 1986). Schwertmann and his colleagues have shown that most goethite crystals are dominated by pris-

matic (110) faces which contain all three types of hydroxyl sites, and that they are terminated by (021) faces which contain only types A and B sites (Schwertmann and Cornell, 1996 and refs. therein). Barron´ and Torrent (1995) reported that (110) faces contain 3.0 A groups nm⫺2 whereas (021) faces contain 8.2 A groups nm⫺2. Our AFM analysis shows that particle aspect ratios decrease with increasing Al substitution, which should lead to a greater relative importance of (021) faces and hence a greater proportion of the reactive type A sites. This could help to explain at least in part the observed trend of increased microbial dissolution with increased Al substitution, because dissolution is likely surface controlled. Mun˜oz (1988) showed that the unit cell parameters of goethite change slightly with Al content, but the effects of changing unit cell parameters on surface hydroxyl group density and reactivity, while likely small, has not yet been evaluated. Attachment of microorganisms to mineral surfaces has been reported as essential for Fe(III) (hydr)oxide dissolution to occur (Arnold et al., 1988). We speculate that perhaps the bacteria preferred shorter, lower aspect-ratio crystals as attachment and dissolution sites (and vice versa; i.e., shorter Al substituted goethites might have sorbed more readily to bacteria) either because of the individual crystal morphologies/aspect ratios, including differences in which faces are presented to solution; because of changes in surface charge density or density of reactive surface sites; or because of the particular aggregation behavior of small crystals. Observations of increased anionic adsorption with increased Al substitution in goethite (Muno˜z, 1988 and oxalate data, above) suggest that organic ligands produced by the bacteria may sorb more readily to the Al goethites and that negatively charged microorganisms also may attach more easily. However, Little et al. (1997) showed that exopolysaccharides (EPS) produced by bacteria may sometimes overcome surface charge effects on microbial attachment, so that mineral surface charge is not always a good indicator of likely microbial attachment. Microorganisms are well known to be gregarious; they live well in communities and tend to colonize surfaces (e.g., Little, et al., 1997). Successful colonization often involves formation of large aggregates, and adhesion is necessary for the aggregate formation. Although we did not include a detailed study of particle aggregation in our experiments, optical microscopy, scanning electron microscopy (SEM), and AFM imaging (on dried samples) suggested the presence of dense aggregates in the more Al rich goethites. Indeed, it was difficult to obtain good AFM images on the more Al-rich samples because dense aggregates led to significant AFM tip-sample interaction artifacts (for an explanation of such artifacts, see Maurice and Lower, 1998). The aggregation behavior of the Al goethites in the presence of the bacteria, and the role of aggregation in microbial attachment and microbially mediated dissolution is an area ripe for further study. 4. CONCLUSIONS

Dissolution of goethite by the strict aerobe P. mendocina var. increased with increasing Al substitution, decreasing particle length, aspect ratio, and number of domains per particle, and increasing structural order (crystallinity). There was a small increase in maximum microbial population size with Al

Bacterially mediated Al-goethite dissolution

substitution, but by comparison with Fe-EDTA data described above, the observed differences in maximum population size could potentially relate to as much as an order of magnitude difference in concentration of Fe release. In contrast to previous studies involving DIRB (e.g., Roden and Zachara, 1996), As (whether measured by BET or calculated geometrically) did not appear to be an important factor controlling microbially mediated dissolution. At the current time, fundamental questions remain as to the exact nature of Al substitution in goethite; the effects of substitution on crystal structure and defect density at the particle surfaces; and the effects of substitution on particle aggregation kinetics and aggregate structure. These questions need to be addressed if we are to determine the mechanistic reasons behind the observed dissolution trends. For experiments such as ours that probe only initial dissolution which probably does not proceed deep into the structure, we need to know more about how surface properties relate to bulk properties. It is interesting that the P. mendocina var. growth increased with increasing Al substitution because many soil goethites are Al substituted (e.g., Tardy and Nahon, 1985; Trolard and Tardy, 1987). Perhaps the preference exhibited by the bacteria for Al substituted goethites is ultimately the result of evolutionary forces to take advantage of the characteristics of particles in real-world aerobic soil environments. Acknowledgments—We thank J. Forsythe (LANL) for help in setting up the microbial growth experiments and E. Carlson (KSU) for help with XRD analysis. We thank the following Kent State University graduate students for assistance with analytical measurements: M. Manecki, K. Namjesnik-Dejanovic, M. Pullin, and Q. Zhou. S. Cabaniss and G. Sposito provided valuable discussion. S. Kraemer, S. Lower, and U. Schwertmann kindly reviewed early drafts of this manuscript. This research was funded by the Department of Energy, Basic Energy Sciences Program (DEFGO2-96ER14668). REFERENCES Arnold R. G., DiChristina T. J., and Hoffman M. R. (1988) Reductive dissolution of Fe(III) oxides by Pseudomonas sp. 200. Biotech. and Bioengr. 32, 1081–1096. Arnold R. G., Hoffman M. R., DiChristina T. J., and Picardal F. W. (1990) Regulation of dissimilatory Fe(III) reduction activity in Shewanella putrifaciens. Appl. Env. Microbiol. 56, 2811. Barron´ V. and Torrent J. (1995) Surface hydroxyl configuration of different crystal faces of hematite and goethite. J. Colloid Interface Sci. 177, 407– 410. Berner R. A. (1978) Rate control of mineral dissolution under earth surface conditions. Amer. J. Sci. 278, 1235–1252. Berner R. A. (1980) Early diagenesis. Princeton University Press, Princeton, New Jersey. Berner R. A. (1981) Kinetics of weathering and diagenesis. In Kinetics of Geochemical Processes, (ed. A. C. Lasaga and R. J. Kirkpatrick), pp. 69 –110, Mineralogical Society of America. Brown G. (1980) Associated minerals. In Crystal structures of clay minerals and their X-ray identification. (ed. G. W. Brindley and G. Brown), pp. 361– 410, Mineralogical Society. Brunauer S., Emmett P.H., and Teller E. (1938) Adsorption of gases in multimolecular layers. J. Phys. Chem. 60, 309 –319. Chin P. F. and Mills G. L. (1991) Kinetics and mechanisms of kaolinite dissolution: Effects of organic ligands. Chem. Geol. 90, 307–317. Cornell R. M., Posner A. M., and Quirk J. P. (1974) Crystal morphology and the dissolution of goethite. J. Inorg. Nucl. Chem. 36, 1937–1946. Cornell R. M., Posner A. M., and Quirk J. P. (1976) Kinetics and mechanisms of the acid dissolution of goethite (␣-FeOOH). J. Inorg. Nucl. Chem. 38, 563–567.

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