Vol. 7, No. 3, p. 217226, September 2003
Biomineralization of a poorly crystalline Fe(III) oxide, akaganeite, by an anaerobic Fe(III)-reducing bacterium (Shewanella alga) isolated from marine environment
ABSTRACT: Formation of Fe(II)-containing mineral through microbial processes may play an important role in iron and car- bon geochemistry in subsurface environments. Fe(III)-reducing bacteria form Fe(II)-containing minerals such as siderite, magne- tite, vivianite, and green rust using iron oxides. A psychrotolerant Fe(III)-reducing bacterium, Shewanella alga (PV-4), was used to examine the reduction and biomineralization of a poorly crystal- line iron oxide, akaganeite (ββββ-FeOOH), in the absence of a soluble electron shuttle, anthraquinone disulphonate (AQDS), under dif- ferent atmospheric compositions as well as in HCO3-
buffered medium (30 to 210 mM). Iron biomineralization was also exam- ined under different growth conditions such as incubation time, electron donors, and electron acceptors. The Fe(III)-reducing bac- terium, PV-4, reduced akaganeite, Fe(III)-citrate, and Co(III)- EDTA using lactate or H2 as an electron donor. The iron biomin- eralization of Fe(III) oxide, akaganeite−as it undergos reduction by an iron reducing bacterium−is a complex process influenced by biogeochemical factors including microorganisms, bicarbonate buffer concentration, atmospheric composition, electron donors/
acceptors, incubation time, and Eh/pH. From this research we found that microorganisms do participate in the formation of diverse iron minerals and that microbial iron biomineralization may affect Fe and C biogeochemistry in subsurface environments.
Key words: iron(III)-reducing bacteria, biomineralization, magnetite, siderite, carbon cycle
1. INTRODUCTION
Iron(III)-reducing bacteria can reduce crystalline and amorphous iron oxides including amorphous iron oxyhy- droxide, goethite, and magnetite by using organic com- pounds or hydrogen as an electron donor (Lovley, 1993;
Roden and Zachara, 1996; Dong et al., 2000; Roh and Moon, 2001). In subsurface environment, amorphous, poorly crys- talline, and crystalline Fe(III) oxides are ubiquitous as weath- ering products of Fe-containing minerals (Childs, 1992;
Zachara et al., 2001). Iron-reducing bacteria can induce reduction of amorphous and poorly crystalline iron oxides and transform it to Fe(II)-containing minerals such as mag- netite (Fe3O4), siderite (FeCO3) and vivianite [Fe3(PO4)2· 8H2O] (Zhang et al., 1997, 1998; Fredrickson et al., 1998;
Roh and Moon, 2001; Zachara et al., 2002). A previous
research showed that amorphous iron oxyhydroxides are more easily reduced than crystalline iron oxide under the same conditions (Zachara et al., 2001). In addtion, the coprecipitated or adsorbed Ni2+ inhibited the bioreduction of amorphous hydrous iron oxide by Shewanella putrefa- ciens (CN 32) at 30oC (Fredrickson et al., 2001). However, Co-substituted magnetite was formed by Thermoanaero- bacter ethanolicus (TOR-39) using iron hydroxide and Co- EDTA as an electron acceptor at 65oC (Roh et al., 2001).
The microbial reduction of Fe(III) oxide and its transfor- mation into Fe(II)-containing minerals can provide us with important information such as the most optimal redox con- dition, carbon cycle, and fate of metals or organic matter on the biogeochemistry of both pristine and contaminated soils and sediments. The mobilization and immobilization of metal and organic contaminants in anaerobic soils and sed- iments were found to be indirectly affected by the microbial reduction of Fe(III) oxide and iron biomineralizaton (Urru- tia et al., 1998; Roh et al., 2001). We also found that micro- bial reduction of iron oxide can result in mobilization of adsorbed metals and radionuclides (Chapelle, 1994; Jenne, 1997; Lovley, 1991, 1993) or immobilization of adsorbed metals and radionuclides by formation of metal incorpo- rated iron minerals such as metal-substituted magnetite (Roh et al., 2001).
Iron reducing bacteria have been isolated from diverse natural environments, which include mesophilic (25−35oC;
BrY, Shewanella alga), psychrotolerant (0−37ºC; W-3-6-1, Shewanella baltica), and thermophilic (40−70oC; X514, Thermoanaerobacter ethanolicus) bacteria (Rosselló-Mora et al., 1994; Roh and Moon, 2001; Roh et al., 2002). Although microbial activity in the natural subsurface environment has certain implications on modern and ancient geological set- tings (Liu et al., 1997; Zhang et al., 1996, 1997, 1998; Roh and Moon, 2001), there has been only a few studies on bio- geochemical conditions controlling iron biomineralization by psychrotolerant Fe(III)-reducing bacteria.
Microbial formations of magnetite and siderite using amorphous hydrous ferric oxide (Fredrickson et al., 1998) and crystalline Fe oxides (Roden and Zachara, 1996; Dong et al., 2000; Liu et al., 2001; Kukkadapu et al., 2001; Zachara Sang Han Lee
Insung Lee*
Yul Roh
}
School of Earth and Environmental Sciences, Seoul National University, Seoul 151-747, Korea Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA*Corresponding author: [email protected]
et al., 2002) in the presence of exogenous electron carrier substances such as humic acids (i.e., anthraquinone-2, 6-disul- fonate, a humic acid analog) have been examined extensively.
However, little is known about the microbial formation of magnetite and siderite using a crystalline Fe(III) oxide with- out an exogeneous electron carrier. Therefore the objective of this study is to identify the biogeochemical factors (i.e., bicarbonate concentration, pCO2, electron donors/acceptors) that control microbial formation of Fe(II)-containing min- erals in the absence of an exogeneous electron carrier using a poorly crystalline Fe(III) oxide, akaganeite, as an electron acceptor.
2. MATERIALS AND METHODS 2.1. Source of Organism
In this study, we examined the microbial formation of Fe(II)-containing minerals using a psychrotolerant Fe(III)- reducing bacterium (PV-4, Shewanella alga) isolated from the water column near a hydrothermal vent off the coast of Hawaii.
The psychrotolerant iron reducing bacterium, Shewanella alga (PV-4), is a gram negative, rod-shaped, facultative anaerobe that grows well either aerobically in the presence of oxygen or anaerobically coupled to the reduction of various metals including Fe(III), Co(III), and Mn(IV) (Stapleton et al., 2003).
A previous study (Stapleton et al., 2003) showed that the psychrotolerant iron reducing bacterium (Shewanella alga, PV-4) has a maximum temperature growth at 28oC and which in turn brings about reduction in Fe(III)-citrate using organic compounds at the temperature within the range of 0−37oC. Psychrophiles show optimum growth at tempera- tures of below 15oC and have upper temperature limits of below 20oC, whereas psychrotolerant microorganisms are capable of growth at or close to 0oC, show optimum growth above 15oC and a maximum growth above 20oC (Morita, 1975).
2.2. Growth Condition
The growth medium was prepared by first dissolving and adding the following ingredients (g/l) into deionized water:
2.5 NaHCO3, 0.08 CaCl2·2H2O, 1.0 NH4Cl, 0.2 MgCl2·6H2O, 10.0 NaCl, 1.0 MOPS (3-(N-Morpholino) propanesulfonic acid), 0.1 ml of rezazurin (0.1%), 0.5 yeast extract, 10 ml of Oak Ridge National Laboratory (ORNL) trace metals (x 10) and 1ml of ORNL vitamin solutions (x 10) (Phelps et al., 1989).
The trace metal solution contained (g/l): 1500 nitrilotriace- tic acid, 200 FeCl2·4H2O, 100MgCl2·6H2O, 20 sodium tung- state, 100 MnCl2·4H2O, 100 CoCl2·6H2O, 1000 CaCl2·2H2O, 50 ZnCl2, 2 CuCl2·2H2O, 5 H3BO3, 10 sodium molybdate, 1000 NaCl, 17 Na2SeO3, and 24 NiCl2·2H2O. The vitamin solution contained (g/L) 0.02 biotin, 0.02 folic acid, 0.1 B6 (pyridoxine) HCl, 0.05 B1 (thiamine) HCl, 0.05 B2 (ribo- flavin), 0.05 nicotinic acid (niacin), 0.05 pantothenic acid,
0.001 B12 (cyanobalamine) crystalline, 0.05 PABA (P-ami- nobenzoic acid), and 0.05 lipoic acid (thioctic). Then, the medium was adjusted to have a pH value of 7.9 and boiled on a hot plate under a stream of N2. After boiling, it was cooled to room temperature with a stream of N2 gas. 10 mM of medium was transferred into 26 ml pressure tubes under a stream of N2 or CO2,and then sealed using butyl rubber stopper and aluminum caps. The prepared media were then sterilized at 120oC and 16 psi for about 20 min. The final pH and Eh of media were 7.9 and 20 mV, respectively. No exogenous electron carrier substance (i.e., anthraquinone disulfonate) or reducing agents (i.e., cysteine) was added to the medium.
Microbial reduction and mineralization of a poorly crys- talline Fe(III) oxide, akaganeite (β-FeOOH), was investi- gated. The akaganeite was prepared as follows: NaOH solution (10 M) was slowly added into a FeCl2·6H2O solution (0.4 M) to precipitate Fe(OH)2 by gravity only and with rapid stirring at pH 7.0 (Roh et al., 2001). The suspension was aerated overnight by magnetic stirring to ensure uniform oxidation. The Fe(III) phases formed were washed three times in deionized water and centrifuged after each wash- ing. A final solution of the iron oxide was flushed with N2
and then autoclaved for microbiological use. X−ray diffrac- tion (XRD) analysis showed that the autoclaved Fe(III) phase was mainly poorly crystalline akaganeite (β-FeOOH).
Cell growth was determined by directly counting cells, which were stained with 0.1% acridine orange, and cell numbers were measured using an epifluorescent microscope (Kirch- man et al., 1982).
2.3. Geochemical Factors on Metal Reduction and Iron Biomineralization
A variety of biogeochemical factors, including electron donors/acceptors, different atmospheric composition, and different bicarbonate concentration, on iron biomineraliza- tion using a psychrotolerant Fe(III)-reducing bacterium (Shewanella alga, PV-4) were examined to determine their influence on iron mineral formation,. To assess the capability of iron reduction and biomineralization using a psychrotoler- ant Fe(III)-reducing bacterium (Shewanella alga, PV-4), various electron acceptors such as ferric citrate (20 mM), akaganeite (~70 mM), hematite (~50 mM), Co(III)-EDTA (1 mM), and potassium chromate (0.5 mM) were examined at 25oC using lactate (10 mM) as an electron donor. To determine the range of growth substrates, the psychrotoler- ant Fe(III)-reducing bacterium (Shewanella alga, PV-4) was inoculated into anaerobic medium with lactate (10 mM), acetate (20 mM), pyruvate (10 mM), glucose (10 mM) or H2 (H2−CO2 as an 80:20 ratio) as an electron donor and an akaganeite (70 mM) as an electron acceptor and then incu- bated at 25oC in the dark. All of the experiments were per- formed under strict anaerobic condition.
To determine the effect of incubation time on iron biom- ineralization, cell generation, Eh-pH changes, and ferrous iron concentration, the psychrotolerant iron-reducing bacte- rium was inoculated into anaerobic medium containing 30 mM bicarbonate buffer concentration using akaganeite as an electron accepter and lactate as an electron donor and then was incubated up to 21 days. The effect of bicarbonate buffer concentration (210 mM) and incubation time on iron biomineralization was also examined using the Fe(III)-reducing bacterium with lactate (10 mM) as an electron donor and akaganeite as an electron acceptor and then was incubated up to 21-day incubation. To understand the effect of head- space atmosphere on iron biomineralization, the anaerobic media were prepared under two different headspace atmo- spheres including N2 (100%) and CO2 (100%) using aka- ganeite (~70 mM) as an electron acceptor and lactate (10 mM) as an electron donor. The effect of a foreign ion, Co(III)- EDTA, on iron biomineralization was examined using aka- ganeite (70 mM) plus Co(III)-EDTA (2.5 mM) as an elec- tron acceptor and lactate (10 mM) as an electron donor.
Abiotic controls were established for each experiment.
2.4. Mineralogical and Geochemical Characterization To examine the chemical conditions of metal reduction and mineral formation by the psychrotolerant Fe(III)-reduc- ing bacterium (Shewanella alga, PV-4), subsamples (1 ml) of bacterial cultures and abiotic controls were taken from the culture bottles at different times and measured for redox potential (Eh) and pH at room temperature in an anaerobic chamber. Fe(II) concentrations (0.5 N HCl, soluble) were determined by measuring the absorbance at 562 nm on a Hewlett-Packard model 8453 spectrophotometer following the ferrozine method (Stookey, 1970) using anaerobic water for sample dilution.
A JSM-35CF (JEOL, Ltd, Tokyo, Japan) scanning electron microscope (SEM) with an energy-dispersive X-ray (EDX) detector was used for the analysis of the morphology, min- eralogy, and chemistry of the iron mineral phases precipi- tated or transformed by the psychrotolerant Fe(III)-reducing bacterium (Shewanella alga, PV-4). Their mineralogical compositions of the precipitated or transformed phases were determined by using X-ray diffraction (XRD) analysis. All XRD analyses were performed on an XDS 2000 diffracto- meter (40 kV, 35 mV; Scintag, Inc., Sunnyvale, CA) equipped with Co-Kα radiation. To understand the morphology of the transformed crystalline iron minerals, mineralogical char- acteristics of the precipitates were also examined by trans- mission electron microscopy (TEM). Culture media containing bacterial cells, organic matter, and inorganic solids (mag- netite and akaganeite) were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate (Zhang et al., 1998). After washing with a HEPES buffer and an alcohol-water solution (1:1 ratio), the samples were dehydrated with propylene oxide and
embedded in a low-viscosity, thermally curing epoxy resin.
Ultra-thin sections (70 to 80 nm) were cut from resin blocs with a diamond knife and transferred to a 30-mesh Form- var-coated Cu TEM grid for image analysis on an FZ 2000 TEM (JEOL, Ltd.) equipped with an EDX detector.
The redox potential (Eh) and pH were measured at room temperature in an anaerobic chamber during the time course experiments of the biomineraloization processes. In per- forming the pH measurements, we used a combination of pH electrode and an ORION EA 920 expandable ion ana- lyzer (Orion Research, Beverly, MA), standardized with pH buffer 7 and the appropriate buffer of either pH 4 or 10. Eh val- ues were measured using platinum microelectrodes (Micro- electrodes, Inc., Londonderry, NH). The probe was placed directly into the sample tube and equilibrated for at least 5 min before recording the value.
3. RESULTS
3.1. Solution Chemistry and Microbial Growth
Table 1 shows the changes of pH, Eh, Fe(II) concentra- tions, and cell generation during the microbial reduction of akaganeite using lactate (10 mM) as an electron donor using 30 mM bicarbonate buffer concentration under a N2 atmo- sphere. Reddish-brown colored akaganeite (β-FeOOH) trans- formed into black colored magnetic minerals during the biomineralization processes that was carried out for 21 days. The measured pH and Eh were found to be decreased from 8.4 to 7.3 and from −30 mV to −300 mV gradually during the incubation, respectively (Table 1). The measurement of Eh and pH values were plotted on Eh-pH stability for lapi- docrosite, magnetite, and siderite in the Fe−water−CO2 sys- tem at 25oC and 1 atm total pressure (Fig. 1). Measured Eh and pH were consistent with the thermodynamic stability of magnetite. The effect of incubation time on microbial growth rate and Fe(III) reduction rates was examined with the psy- chrotolerant Fe(III)-reducing bacterium (Table 1). The cell density increased from 4.12×106 cells/ml at time zero to
Table 1. The variation of Eh, pH, cell number, and Fe(II) concen- tration during time course experiments using akaganetite (70 mM) as an electron acceptor and lactate (10 mM) as an electron donor by a psychrotolerant iron-reducing bacterium (Shewanella alga, PV-4) under a N2 atmosphere.
Incubation time pH Eh (mV) Cell (ml-1) Fe(II) (mM)
0-day 8.40 −30 4.12×106 0.1
1-day 8.32 −150 1.01×107 0.5 2-day 8.11 −180 1.30×107 0.8
7-day 7.90 −200 4.01×107 5.2
10-day 7.81 −210 4.15×107 8.1 14-day 7.59 −270 2.00×108 15.0 21-day 7.37 −300 3.98×107 21.2
2.00×108 cells/ml at 14-day incubation at 25oC. And then the cell density was decreased from 2.00×108 cells/ml at 14-day incubation to 3.98×107 cells/ml at 21-day incubation.
Then, the Fe(II) concentration was increased from 0.1 mM at time zero to 21.2 mM at 21-day incubation. The cell growth rate was correlated with Fe(II) concentration during the time course experiments for microbial transformation of akaganeite.
Shewanella alga (PV-4) reduced akaganeite using lactate as an electron donor and consequently, produced black-colored magnetic minerals under a N2 atmosphere. But magnetic minerals did not form in control tubes containing reddish- brown colored magnetite precursor, akaganeite, in absence of cells.
3.2. Effect of Electron Donors and Electron Acceptors We found that the psychrotolerant Fe(III)-reducing bac- terium (Shewanella alga, PV-4) can use lactate or H2 as an electron donor in reducing akaganeite, Fe(III)-citrate, and Co(III)-EDTA as electron acceptors. However, the psychro- tolerant Fe(III)-reducing bacterium (Shewanella alga, PV-4) did not reduce akaganeite using glucose or acetate as an electron donor. When Co(III)-EDTA as an electron acceptor with lactate (10 mM) or hydrogen as an electron donor was inoculated into anaerobic media, the psychrotolerant Fe(III)- reducing bacterium (Shewanella alga, PV-4) caused the pur- ple colored Co(III)-EDTA to change into colorless Co(II) at 25oC. The psychrotolerant Fe(III)-reducing bacterium also caused the reduction of brown colored Fe(III)-citrate into colorless Fe(II) using lactate or H2 as an electron donor.
However, the psychrotolerant Fe(III)-reducing bacterium
(Shewanella alga, PV-4) could not reduce both potassium chromate (0.5 mM) and hematite even by using either lac- tate or hydrogen as an electron donor in the N2 headspace.
3.3. Effect of Incubation Time
X-ray diffraction analyses (Fig. 2) of iron minerals formed during time course experiment using akaganeite (β-FeOOH) as an electron acceptor under 30 mM bicarbonate buffer by the psychrotolerant Fe(III)-reducing bacterium (Shewanella alga, PV-4) showed that magnetite peaks appeared after 10 days of incubation at 25oC. This first evidence of magnetite peaks agreed with visual observation of the biomineralization pro- cesses in which the reddish-brown colored magnetite pre- cursor, akaganeite, turned black. After 14 days of incubation, the transformed minerals turned completely black, and at the same time the magnetite (Fe3O4) peaks were observed to be dominant in the diffraction pattern (Fig. 2). X−ray dif- fraction analyses showed that the magnetite precursor was predominantly akaganeite (β-FeOOH). Furthermore, we could not observe Fe(III) reduction or magnetite formation occur- ring in the control tubes.
Incase of X−ray diffraction analyses (Fig. 3) of iron min- erals that formed using akaganeite as an electron acceptor and lactate (10 mM) as an electron donor under 210 mM bicarbonate concentration, magnetite peaks appeared after Fig. 1. Eh-pH stability fields for lepidocrosite, magnetite, siderite
in the water−Fe−CO2 at 25oC and 1 atm total pressure. Measured Eh and pH were plotted: A=Time zero, B=1-day; C=2-day; D=7- day; E=10-day; F=14-day; G=21-day.
Fig. 2. XRD patterns of iron minerals formed by the psychrotol- erant iron reducing bacterium (Shewanella alga, PV-4) using 30 mM bicarbonate buffer under a N2 atmosphere for 21 days. A:
Akaganeite; M: Magnetite.
10 days of incubation and siderite (FeCO3) peaks appeared after 14 days of incubation at 25oC. In addition, the reddish- brown colored akaganeite changed to brownish-black col- ored phases and a mixture of magnetite and siderite was the principal reduced iron minerals, as determined from X−ray diffraction analysis, under 210 mM bicarbonate buffer.
X−ray diffraction analysis of iron minerals formed under 30mM bicarbonate buffer and 210 mM bicarbonate buffer after 21 days of incubation indicates that the psychrotolerant Fe(III)-reducing bacterium formed mainly magnetite as a dominant iron mineral under 30 mM bicarbonate buffer and formed mainly siderite with magnetite as dominant Fe(II)- containing minerals under the 210 mM bicarbonate buffer.
3.4. Effect of Bicarbonate Concentration and pCO2
The Fe(III)-reducing bacterium (Shewanella alga, PV-4) formed a mixture of magnetite and siderite using an anaer- obic medium buffered with NaHCO3 (30−210 mM) under a N2 atmosphere (Fig. 4). XRD analysis based on peak inten- sities showed that siderite is a dominant iron mineral with increased bicarbonate buffer concentration (210 mM). Micro- bial Fe(III) reduction using 30 mM bicarbonate buffered medium under N2 atmosphere predominantly formed mag- netite (Fig. 4). Iron minerals formed under bicarbonate
buffer concentration ranging from 60 mM to 150 mM were siderite, goethite, and magnetite. Iron minerals formed by the psychrotolerant Fe(III)-reducing bacterium (Shewanella alga, PV-4) are predominantly magnetite (Fe3O4) under a N2 (100%) atmosphere and siderite (FeCO3) under a CO2
(100%) atmosphere (Fig. 5).
3.5. Effect of a Foreign Ion, Co(III)-EDTA
The psychrotolerant Fe(III)-reducing bacterium (Shewanella Alga, PV-4) reduced a poorly crystalline iron oxide, aka- ganeite, and formed magnetite in the presence of Co(III)- EDTA using lactate as an electron donor under a N2 atmo- sphere (Fig. 6B). Time course experiments showed that a foreign ion, Co(III)-EDTA, did not affect magnetite forma- tion compared to that in the absence of a foreign ion, Co(III)-EDTA (Fig. 2). However, cell generation rate during the microbial reduction of akaganeite in the presence of Co(III)- EDTA (Fig. 6A) was slower than cell generation rate during the microbial reduction of akaganeite without Co(III)-EDTA (Table 1). In the presence of a foreign ion, Co(III)-EDTA, the cell den- sity increased from 3.34×107 cells/ml at time zero to 1.78×108 cells/ml at 31-day incubation at 25oC.
3.6. Bacteria, Magnetite and Siderite Morphology Transmission electron microscopy showed that rod-shaped Fig. 3. XRD patterns of iron minerals formed by the psychrotol-
erant iron reducing bacterium (Shewanella alga, PV-4) using 210 mM bicarbonate buffer under a N2 atmosphere for 21 days. A:
Akaganeite; M: Magnetite; S: Siderite.
Fig. 4. XRD patterns of iron minerals formed by the psychrotol- erant iron reducing bacterium (Shewanella alga, PV-4) in 30 mM to 210 mM bicarbonate buffered medium for 2 month of incuba- tion. M: Magnetite; S: Siderite; G: Goethite.
bacteria (Shewanella alga, PV-4) reduced Fe(III)-citrate and precipitated iron phases using lactate as an electron donor (Fig. 7A). Transmission electron microscopy showed this bacterium also reduced akaganeite using lactate (10 mM) as an electron donor under 30 mM bicarbonate buffered media and a N2 atmosphere and formed microcrystalline magnetite crystals (Fig. 7B). Scanning electron microscopy analysis (Fig. 8A) of iron minerals formed using akaganeite under the 210 mM bicarbonate buffered media under a N2 atmo- sphere showed that rhombohedral siderite crystals formed by this bacterium. These siderite crystals are similar to sid- erite formed by Geobacter metallireducens (GS-15) and found in natural samples. The siderite crystals co-existed with microcrystalline magnetite crystals (Fig. 8B). Energy dispersive X-ray analysis of the iron minerals including magnetite and siderite showed that the crystalline phases contained significant quantity of Fe (Fig. 8C, D).
4. DISCUSSION
4.1. Environmental Factors in Iron Biomineralization During the biomineralization processes, measured Eh decreased from −30 mV at time zero to −300 mV at 21-day incubation and pH decreased from 8.4 to 7.37 at 21-day incubation (Table 1). Measured pH and Eh in the anaerobic medium during the iron biomineralization processes were observed to be consistent with the thermodynamic stability of magnetite. Fe(II) concentration increased from 0.1 mM
at time zero to 21.2 mM at 21-day incubation and cell num- bers increased from 4.12×106 cells/ml to 2×108 cells/ml during the iron biomineralization processes. These results indicate that the iron reduction and formation of Fe(II)-con- taining minerals appear to be coupled with microbial growth as verified by the following evidences: (i) no iron reduction or formation of Fe(II)-containing minerals was observed when no living cells were added; (ii) the production of Fe(II) was concomitant with cell growth; (iii) magnetite for- mation was observed when non-fermentative substrates, such as lactate and H2, were used as electron donors; and (iv) microbial processes changed Eh and pH in anaerobic media and the measured Eh and pH values were consistent with the thermodynamic stability of magnetite.
Further research on the biological reduction and iron biomineralization processes need to be conducted as there is Fig. 5. XRD patterns of iron minerals formed by the psychrotol-
erant iron reducing bacterium (Shewanella alga, PV-4) under a N2
atmosphere and a CO2 atmosphere. M: Magnetite; S: Siderite.
Fig. 6. Cell generation during biomineralization of magnetite using akaganeite plus Co(III)-EDTA using lactate as an electron donor for 32-day incubation (A). XRD pattern of iron mineral formed using akaganeite plus Co(III)-EDTA using lactate as an electron donor at 32-day incubation (B).
still insufficient understanding of this subject (Lovley, 1991, 1993; Zhang et al., 1998). In particular, the mechanism by which the bacterium splits hydrogen or other organic com- pound is not known with certainty and the specific meta- bolic pathways of the resulting electrons have not been identified. However, by placing the overall mineralizing action into the context of the Eh-pH diagram (Fig. 1), cell generation, and Fe(II) concentration (Table 1), the effect of the microbial processes on the external thermodynamics and kinetics can be evaluated. And you can expect that the microbial oxidation of organic compounds or H2 coupled with reduction of Fe(III) oxide, akaganeite (β-FeOOH), will release Fe(II) ions in the anaerobic medium to form Fe(II)- containing minerals. Amorphous iron oxide and poorly crystalline iron oxide are ubiquitous and can be used as electron acceptors in the recent sediment and marine envi- ronments (Karlin and Levi, 1983).
From this study, we found that geochemical factors in iron biomineralization include incubation time, electron accep- tors, electron donors, atmospheric composition, and biocar- bonate buffer concentration. In addition, we found that microbial Fe(III) reduction and iron biomineralization could be important processes for organic matter oxidation in anaerobic subsur- face environments. Previous studies have shown that organic compounds such as lactate, formate, and pyruvate are potentially available in terrestrial subsurface environments (Walker, 1984; Lovley, 1991). And, the microbial reduction of akaganeite with oxidation of organic compounds such as lactate can be expected to release Fe(II) ions in subsurface environments (Lovley, 1993; Fredrickson et al., 1998). The Shewanella culture (PV-4) can couple the oxidation of H2
to the reduction of Fe(III) oxide such as akaganeite. Hydro- gen gas is generated from anaerobic decomposition of organic matter as well as from geochemical processes in subsurface Fig. 7. TEM analysis of precipitate formed by microbial reduction of Fe(III)-citrate (A) and akaganeite (B).
Fig. 8. Scanning electron micrographs (A, B) and energy dispersive X−ray analysis (C, D) of siderite and magne- tite formed by a psychrotolerant iron reducing bacterium (Shewanella alga, PV-4) using 210 mM bicarbonate buffered medium.
environments and probably constitutes a sustainable source of energy for subsurface biosphere ecosystems today (Ped- erson, 2000). It has been proposed that prior to the devel- opment of life on Earth, there was production of Fe(III) and hydrogen could be produced by hydrothermal systems (Cai- rins-Smith et al., 1992; Nealson and Myers, 1990). The oxi- dation of hydrogen and organic compounds coupled to the reduction of Fe(III) species by Shewanella alga provides a biological model for geochemical reactions on Early Earth.
This information could contribute to understand the poten- tial of life processes on extraterrestrial bodies, where subsurface environments is capable of sustaining life under otherwise adverse contemporary conditions (McKay et al., 1996).
A crystalline Fe(III) oxide, hematite, was reducible by Shewanella putrefaciens strain CN 32 with a soluble elec- tron shuttle, AQDS (Roden and Zachara, 1996). However, this study showed that Shewanella alga (PV-4) could not reduce hematite without a soluble electron shuttle, AQDS.
In addtion, we found that the formation of magnetite and siderite that were biologically facilitated by using a poorly crystalline iron oxide, akaganeite, as an electron acceptor, does not require any addition of exogenous electron carrier sub- stance, humic acid (e.g., AQDS) or a reducing agent (cystene).
This study showed that a foreign ion, Co(III)-EDTA, does not affect microbial reduction and formation of magnetite using akaganeite as an electron acceptor without a soluble electron shuttle, AQDS. The pyschrotolerant Fe(III)-reduc- ing bacterium (Shewanella alga, PV-4) may reduce Co(III)- EDTA and akaganetite together and the reduced Co(II) may be incorporated or adsorbed into magnetite crystal (Roh et al., 2001). Therefore further research is necessary to con- firm the fate of Co(III)-EDTA in the presence of akaganeite during the biomineralization processes by Shewanella alga.
Microbial formation of magnetite using akganetite in the presence of other metals using a organic compound, lactate, indicates that biomineralization processes may influence the biogeochemical cycles of carbon, iron, and other metals in subsurface environments.
Atmospheric composition and buffer concentrations appear to affect the mineralogical composition of iron minerals formed by Fe(III)-reducing bacteria. High bicarbonate buffer (i.e., 210 mM) and Fe2+, as promoted by microbial activity, seem to favor siderite (FeCO3) formation (Rajan et al., 1996;
Mortimer and Coleman, 1997; Fredrickson et al., 1998).
Siderite formation is generally associated with the bacterial respiration of organic matter coupled with dissimilatory Fe reduction (Suess, 1979; Pye et al., 1990), even though there is little understanding on the in-situ geochemical conditions (Pye et al., 1990).
4.2. Geochemical Implications of Iron Biomineralization The ability of Fe(III)-reducing bacteria to reduce crystal- line Fe(III)-oxide, akaganeite, and to form magnetite and
siderite has far-reaching implications on microbial processes in subsurface sediments where Fe(III) associated with crys- talline and poorly crystalline iron oxides may represent the largest mass of electron acceptor. Microbial formation of carbonate minerals (i.e., siderite) and iron oxides (i.e., magnetite) may play an important role in trace metal immobilization because metals (i.e., Co, Cr) are readily reduced and incorporated into the magnetite and siderite crystals when the Fe(III)-reducing bacteria formed magne- tite and siderite (Fredrickson et al., 2001; Roh et al., 2001).
The ferrous iron and Fe(II)-containing minerals generated by the Fe(III)-reducing bacteria can chemically reduce multivalent metals such as U(VI), Cr(VI), and Tc(VII) as well as can abiotically reduce nitroaromatics and chlori- nated solvents.
Siderite is frequently observed as diagentic precipitation in recent aquatic and geologic sediments (Pye et al., 1990;
Mortimer et al., 1997). Its formation is generally associated with the bacterial respiration of organic matter coupled with the microbial iron(III) reduction (Suess, 1979; Pye et al., 1990). In this study, we found that the atmosphere and bicarbonate buffer concentration in conjunction with iron biomineralization processes exhibited profound influences on Fe carbonate formation. In addition, siderite formation is generally associated with the bacterial respiration of organic matter coupled with microbial Fe(III) reduction with the conditions of reducing environment, CO2 atmosphere, and high alkalinity (Suess, 1979; Pye et al., 1990; Fredrickson et al., 1998). Given the abundance of Fe in anaerobic sed- imentary systems, the capacity of Fe(III)-reducing bacteria to precipitate siderite using iron oxides and dissolved Fe(II) ion species could have a significant impact on carbon sequestration.
5. CONCLUSIONS
Geochemical factors in iron biomineralization include atmospheric composition, bicarbonate buffer concentration, incubation time, and electron donors/acceptors. These parame- ters were found to have profound influences on the types of Fe(II)-containing minerals. The presence of high bicarbon- ate in the aqueous phase and a large reservoir of CO2(g) to maintain aqueous bicarbonate concentration are important factors that allow the microbial reduction of iron oxide and formation of siderite in subsurface environments. Therefore, the microbially induced formation of siderite may occur naturally when such a ligand and appropriate electron donors are in sufficient concentration. Given the abundance of Fe minerals and CO2 in anaerobic sedimentary systems, Fe mineralization process by Fe-reducing bacterium can provide us an important information about the sequestration of CO2. Biomineraliza- tion processes may play an important role in biogeochemical cycling of metals and carbon in diverse subsurface environ- ments.
ACKNOWLEDGMENTS: This study was supported through SEES by BK21 program, Ministry of Education, Korea and Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. We are grateful to Sue Carroll for her help in connection with cell counting and ferrous iron analysis.
REFERENCES
Cairins-Smith, A.G., Hall, A.J. and Russell, M.J, 1992, Mineral the- ories of the origin of life and an iron sulfide example. Origins of Life Evolution, Biosph, 22, 161−180.
Chapelle, F.J., 1994, Groundwater microbiology and geochemistry.
New York: John Wiley & Sons, Inc.
Childs, C.W., 1992, Ferrihydrite: A review of structure, properties, and occurrence in relation to soils. Z Pfanzenernbhr Bndenk, 155, 441−448.
Dong, H., Fredrickson, J.K., Kennedy, D.W., Zachara, J.M., Kukk- adapu, R.K. and Onstott, T.C., 2000, Mineral transformations associated with the microbial reduction of magnetite. Chemical Geology, 169, 299−318.
Emerson, S., 1976, Early diagenesis in anaerobic lake sediments:
chemical equilibria in interstitial waters. Geochimica et Cosmo- chimica Acta, 40, 925−934.
Fredrickson, J.K., Zachara, L.N., Kennedy, D.W., Dong, H., Onstott, T.C., Hinman, N.W. and Li, S., 1998, Biogenic iron mineraliza- tion accompanying the dissimilatory reduction of hydrous ferric oxide by a groundwater bacterium. Geochimica et Cosmochim- ica Acta, 62, 3239−3257.
Fredrickson, J.K., Zachara, J.M., Kukkadau, R.K., Gorby, Y.A., Smith, S.C. and Brown, C.F, 2001, Biotransformation of Ni-sub- stituted hydrous ferric oxide by an Fe(III)-reducing bacterium.
Environmental Science & Technology, 35, 703−712.
Jenne, E.A., 1977, Trace element sorption by sediments and soil-sites and processes. In: Chappel, K.P., (ed.), Symposium on molyb- denum in the environment. Marcel Dekker, New York, p. 425− 553.
Karlin, R. and Levi, S., 1983, Diagenesis of magnetic minerals in recent hemipelagic sediments. Nature, 303, 327−330.
Kirchman, D., Sigda, J., Kapuscinski, R. and Mitchell, R., 1982, Sta- tistical analysis of the direct count method for enumeration bac- teria. Applied Environmental Microbiology, 44, 376−382.
Kukkadapu, R.k., Zachara, J.M., Smith, S.C., Fredrickson, J.K. and Liu, C.X., 2001, Dissimilatory bacterial reduction of Al-substi- tuted goethite in subsurface sediments. Geochimica et Cosmo- chimica Acta, 65, 2913−2924.
Liu, S., Zhou, J., Zhang, C.L., Cole, D.R., Gajdarziska-Josifovska, M. and Phelps, T.J., 1997, Thermophilic Fe(III)-reducing bacteria from the deep subsurface: The evolutionary implications. Sci- ence, 277, 1106−1109.
Liu, C.X., Kota, S., Zachara, J.M., Fredrickson, K.K. and Brinkman, C.K., 2001, Kinetic analysis of the bacteria reduction of goethite.
Environmental Science & Technology, 35, 2482−2490.
Lovley, D.R., 1991, Dissimilatory Fe(III) and Mn(IV) reduction.
Microbialogical Review, 55, 259−287.
Lovley, D.R., 1993, Dissimilatory metal reduction. Annual Reviews of Microbiology, 47, 263−290.
Lovley, D.R., 1995, Bioremediation of organic and metal contaminants with dissimilatory metal reduction. Journal of Industrial Micro- biology, 14, 85−93.
Mckay, D.S., Gibson, E.K., Thomas-Keprta, K.L., Vai, H., Romanek,
C.S., Clemett, S.J., Maechling, C.R. and Zare, R.N., 1996. Search for past life on Mars: Possible relic biogenic activity in Martian meteorite ALH84001. Science, 273, 924−930.
Morita, R.Y., 1975, Psychrophilic bacteria. Bacteriology Review, 39, 144−167.
Mortimer, R.J.G. and Coleman, M.L., 1997, Microbial influence on the oxygen isotopic composition of diagenetic siderite. Geochim- ica et Cosmochimica Acta, 6, 1705−1711.
Mortimer, R.J.G., Coleman, M.L. and Rae, J.E., 1997, Effect of bac- teria on the elemental composition of early diagenetic siderite:
implications for paleoenvironmental interpretations. Sedimentol- ogy, 44, 759−765.
Nealson, K.H. and Myers, C.R., 1990, Iron reduction by bacteria: A potential role in the genesis of banded iron formation. American Journal of Science, 290A, 35−45.
Pedersen, K., 2000, Exploration of deep intraterrestrial microbial life:
current perspectives. FEMS Microbiology Letters, 185, 9−16.
Phelps, T.J., Raione, E.G, White, D.C. and Fliermans, C.B., 1989, Microbial activity in deep subsurface environments. Geomicro- biology Journal, 7, 79−91.
Rajan, S., Mackenzie, F.T. and Glenn, C.R., 1996, A thermodynamic model for water column precipitation of siderite in the Plio-Pleis- tocene Black Sea. American Journal of Science, 296, 506−548.
Roden, E.E. and Zachara, J.M., 1996, Microbial reduction of crys- talline Fe(III) oxides: Influence of oxide surface area and potential for cell growth. Environmental Science and Technology, 30, 1618−1628.
Roh, Y., Lauf, R.J., McMillan, A.D., Zhang, C., Rawn, C.J., Bai, J.
and Phelps, T.J., 2001, Microbial synthesis and the characteriza- tion of some metal-doped magnetite. Solid State Communica- tions, 118, 529−534.
Roh, Y. and Moon, H.S., 2001, Iron reduction by a psychrotolerant Fe(III)-reducing bacterium isolated from ocean sediment. Geo- sciences Journal, 5, 183−190.
Roh, Y., Liu, S.V., Li, G., Huang, H., Phelps, T.J. and Zhou, J., 2002, Isolation and Characterization of Metal-reducing Thermoanaer- obacter strains from deep subsurface environments of the Piceance Basin, Colorado. Applied and Environmental Microbiology, 68, 6013−6020.
Rosselló-Mora, R.A., Caccavo, F.Jr., Osterlehner, K., Springer, N., Springer, N., Spring, S., Schüler, D., Ludwig, W., Amann, R., Vanncanneyt, M. and Schleifer, K.H., 1994, Isolation and taxo- nomic characterization of a halotolerant, facultatively iron-reduc- ing bacterium. Systematic and Applied Microbiology, 17, 569−573.
Rye, K., Dickson, A.D., Schiavon, N., Coleman, M.L. and Cox, M., 1990, Formation of siderite-Mg-calcite-iron sulfide concretions in intertidal marsh and sandflat sediments, north Norfolk, England.
Sedimentology, 37, 325−343.
Stookey, L.L., 1970, Ferrozine-a new spectrophotometric reagent for iron. Analytical Chemistry, 42, 779−781.
Suess, E., 1979, Mineral phases formed in anoxic sediments by microbial decomposition of organic matter. Geochimica et Cos- mochimica Acta, 43, 339−352.
Stapleton, R.D.Jr., Sabree, Z.L., Palumbo, A.V., Moyer, C., Devol, A., Roh, Y. and Jhou, J., 2003, Metabolic capabilities and dis- tribution of Shewanella isolates from diverse marine environ- ments. Aquatic and Microbial Ecology. (in review)
Urrutia, M.M., Roden, E.E., Fredrickson, J.M. and Zachara, J.M., 1998, Microbial and surface chemistry controls on reduction of synthetic Fe(III) oxide minerals by the dissimilatory iron-reduc- ing bacterium Shewanella alga. Geomicrobiology Journal, 15,
269−291.
Walker, J.C.G., 1984, Suboxic diagenesis in banded iron formation.
Nature, 309, 340−342.
Zachara, J.M., Fredrickson, J.K., Smith, S.C. and Gassman, P.L., 2001, Solubilization of Fe(III) oxide-bound trace metals by a dis- similatory Fe(III) reducing bacterium. Geochimica et Cosmochimica Acta, 65, 65−73.
Zachara, J.M., Kukkadapu, R.K., Fredrickson, J.K., Gorby, Y.A. and Smith, S.C., 2002, Biomineralization of poorly crystalline Fe(III) oxides by dissimilatory metal reducing bacteria (DMRB).
Geomicrobiology Journal, 19, 179−207.
Zhang, C., Liu, S., Logan, J., Mazumer, R. and Phelps, T.J., 1996, Enhancement of Fe(III), Co(III), and Cr(VI) reduction at elevated
temperatures and by a thermophilic bacterium. Applied Bio- chemistry and Biotechnology, 57/58, 923−932.
Zhang, C., Liu, S., Phelps, T.J., Cole, D.R., Horita, J., Fortier, S.M., Elless, M. and Valley, J.W., 1997, Physiochemical, mineralogical, and isotopic characterization of magnetite-rich iron oxides formed by thermophilic iron-reducing bacteria. Geochimica et Cosmo- chimica Acta, 61, 4621−4632.
Zhang, C., Vali, H., Romanek, C.S., Phelps, T.J. and Liu, S., 1988, Formation of single-domain magnetite by a thermophilic bacte- rium. American Mineralogist, 83, 1409−1418.
Manuscript received June 21, 2003 Manuscript accepted August 30, 2003
