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Microbial preparation of metal-substituted magnetite nanoparticles
Journal of Microbiological Methods 70 (2007) 150 – 158 www.elsevier.com/locate/jmicmeth
Microbial preparation of metal-substituted magnetite nanoparticles Ji-Won Moon a , Yul Roh b , Robert J. Lauf a , Hojatollah Vali c , Lucas W. Yeary d,e , Tommy J. Phelps a,⁎ b
a Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA Faculty of Earth System and Environmental Sciences, Chonnam National University, Gwangju, 500-757, Republic of Korea c Department of Anatomy and Cell Biology, McGill University, Montreal, QC Canada H3A 2B2 d Engineering Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA e Inorganic and Integration Technologies, Corning, NY 14831, USA
Received 27 November 2006; received in revised form 3 April 2007; accepted 13 April 2007 Available online 29 April 2007
Abstract A microbial process that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular metal (M)substituted magnetite nanoparticles using akaganeite and dopants of dissolved form has previously been reported. The objectives of this study were to develop methods for producing M-substituted magnetite nanoparticles with a high rate of metal substitution by biological processes and to identify factors affecting the production of nano-crystals. The thermophilic and psychrotolerant iron-reducing bacteria had the ability to form Msubstituted magnetite nano-crystals (MyFe3−yO4) from a doped precursor, mixed-M iron oxyhydroxide, (MxFe1−xOOH, x ≤ 0.5, M is Mn, Zn, Ni, Co and Cr). Within the range of 0.01 ≤ x ≤ 0.3, using the mixed precursor material enabled the microbial synthesis of more heavily substituted magnetite compared to the previous method, in which the precursor was pure akaganeite and the dopants were present as soluble metal salts. The mixed precursor method was especially advantageous in the case of toxic metals such as Cr and Ni. Also this new method increased the production rate and magnetic properties of the product, while improving crystallinity, size control and scalability. © 2007 Elsevier B.V. All rights reserved. Keywords: Metal-substituted magnetite; Mixed-metal precursor; Shewanella sp. strain PV-4; Thermoanaerobacter sp. TOR-39
1. Introduction Nanometer-sized magnetite could have diverse applications including magnetic targeting (drug delivery and radionuclide therapy), magnetic nanoparticles as contrast agents for magnetic resonance imaging, diagnostics, immunoassays, molecular biology, RNA- and DNA-purification, cell separation and purification, cell adhesion research, hyperthermia (Moroz et al., 2002a,b), and magnetic ferrofluids for magnetocaloric pumps (Love et al., 2004, 2005). Microbial iron reduction has been intensively studied to understand biogeochemical processes including the cycling of metal, carbon, nitrogen, phosphate and sulfur in natural and contaminated subsurface environments (Lovley et al., 1987; Lovley, 1995; Liu et al., 1997; Fredrickson et al., 1998, 2001) and as mechanisms for producing copious nm-sized magnetites (Roh et al., 2001; Yeary et al., 2005). Lowenstam (1981) has classified ⁎ Corresponding author. Tel.: +1 865 574 7290; fax: +1 865 576 3989. E-mail address: [email protected] (T.J. Phelps). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.04.012
the biomineralization as either biologically induced mineralization or boundary-organized biomineralization. The latter process applies to situations such as the formation of magnetic crystals within the cells of magnetotactic bacteria as reported by Blakemore (1975). These bacteria typically contain particles having a narrow size distribution around 40–50 nm and characteristic morphologies, enveloped by a membrane (Balkwill et al., 1980; Gorby et al., 1988). In the former process, bacteria may change the environment of their outer membrane, creating electrochemical conditions favorable for magnetite precipitation. It is unlikely to be associated with an organic matrix and produces a broad size-distributed magnetite (Frankel, 1987). A microbial process, that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular M-substituted magnetite nanoparticles using akaganeite and dopants in soluble form, has been previously reported (Roh et al., 2001). Microbially facilitated magnetite synthesis may have numerous advantages. Biological processes can effectively produce ferrite particles of well-defined nanometer size and fine scaled crystallographic
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morphology while incorporating selected metals into the magnetite (MyFe3−yO4) structure, where M was Co, Cr, Mn and Ni (Roh et al., 2001). It has been calculated that the dissimilatory Fe(III)-reducing bacteria used can produce 5000-fold more magnetite per unit biomass than magnetotactic bacteria (Frankel, 1987). Fermentative production of nanoparticles would also be an environmentally benign and fairly economical manufacturing process, which could easily be scaled to the quantities needed for industrial production. To synthesize M-substituted magnetite, Roh et al. (2001) used pure akaganeite (β-FeOOH) along with a stock solution of a dopant metal salt, from which the bacteria produced Msubstituted magnetite. The process developed by Roh et al. (2001) has some drawbacks; 1) some dopants are fairly toxic in the form of soluble M salts and 2) the dopant may be distributed non-uniformly in the magnetite structure because of environmental changes or solubility issues that might reduce dopant concentration during crystal growth. Overall, both yield and product quality may suffer from the effects of soluble M salts. In this study, mixed-M precursors were used in place of pure akaganeite and soluble metal salts (Fig. 1). The purpose of the present study was to develop a procedure for synthesizing M- substituted magnetites by microbially facilitated processes while enhancing the dopant concentration, the production rate, the magnetite properties, the mineralogical and crystallographical quality (phase purity), size and morphology control and the scalability of the process in a less toxic environment. 2. Materials and methods 2.1. Source of microorganisms The microbial formation of iron minerals with thermophilic (Thermoanaerobacter sp. TOR-39, Liu et al., 1997) and
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psychrotolerant (Shewanella sp. strain PV-4, Roh et al., 2006) bacteria were examined. These Fe(III)-reducing bacteria were isolated from sediments and water collected from a variety of environments including deep subsurface sediments (TOR-39) or the water column near a hydrothermal vent off the coast of Hawaii (PV-4). These Fe(III)-reducing bacteria were used to synthesize magnetite and metal-substituted ferrite nanoparticles under anaerobic conditions. 2.2. Media preparation and incubation The culture medium contained the following ingredients (g/L): 2.5 NaHCO3, 0.08 CaCl2·2H2O, 1.0 NH4Cl, 0.2 MgCl2·6H2O, 1–10.0 NaCl, 7.2 HEPES (hydroxyethylpiperazine-N′-2ethanesulfonic acid), 0.5 yeast extract, 0.1 mL of 0.1% resazurin, 10 mL of Oak Ridge National Laboratory (ORNL) trace minerals and 1 mL of ORNL vitamin solutions (Phelps et al., 1989). No exogeneous electron carrier substance (i.e., anthraquinone disulfonate) or reducing agent (i.e., cysteine) was added to the anaerobic medium. The trace mineral solution contained (mg/L): 1500 Nitrilotriacetic acid, 200 FeCl2·4H2O, 100 MgCl2·6H2O, 20 sodium tungstate, 100 MnCl2·4H2O, 100 CoCl2·6H2O, 1000 CaCl2·2H2O, 50 ZnCl2, 2 CuCl2·2H2O, 5 H3BO3, sodium molybdate, 1000 NaCl, 17 Na2SeO3, 24 NiCl2·6H2O. 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 (riboflavin), 0.05 nicotinic acid (niacin), 0.05 pantothenic acid, 0.001 B12 (cyanobalamine) crystalline, 0.05 PABA (P-aminobenzoic acid), and 0.05 lipoic acid (thioctic). The dissolved basal medium was boiled with N2 purging and cooled with continuous N2 purging. For serum vial tests, 50 mL aliquots were dispensed into 160 mL serum bottles with N2 purging and had a final pH of about 8.0–8.2. Each bottle was
Fig. 1. Comparison of procedures for the microbial synthesis of the transitional metal-substituted magnetite between traditional and new precursor methods.
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sealed with a butyl rubber stopper (Bellco Glass, Inc.) and an aluminum crimp seal. The incubation for microbial synthesis of M-substituted magnetites in duplicates was initiated with the addition of 10 mM of glucose, 15 mM of MOPS titrated to pH 7.8, about 40 mM of mixed-M precursor and 1 mL of mid-log growth which fermented for one day with 10 mM glucose as electron donor without electron acceptor for preparing the inoculum of TOR-39. For PV-4 samples, 10 mM of lactate, about 40 mM of mixed-M precursor and 1 mL of mid-log growth of metal reducing PV-4, that was incubated using 10 mM lactate and 10 mM ferric citrate as the electron donor and electron acceptor for 5 days, were used per 50 mL of media in a serum bottle. The incubation was at 65 °C for 30 days for TOR-39 and room temperature for 75 days for PV-4. Control samples received an equal volume of pure akaganeite were also incubated. To get pure magnetite, the headspace was N2 only, and the medium had a low bicarbonate concentration, because metalreducing bacteria could also facilitate conversion of CO2 into sparingly soluble carbonate minerals such as siderite (FeCO3) under a CO2 atmosphere or bicarbonate buffer (N 30 mM) (Roh et al., 2001). 2.3. New methods for magnetite precursor preparation The mixed-M precursors were prepared as follows: NaOH solution (10 M) was added drop wise into a mixed solution of total 0.4 M of FeCl3·6H2O and metal chloride (hydrate) like MnCl2·4H2O, NiCl2·6H2O, CoCl2·6H2O, CrCl3·6H2O, ZnCl2 and CuCl2·2H2O with rapid stirring and stopped at pH 7.00. The mixtures containing metals were established at specific cation mole fraction (CMF) up to 0.5 (0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5) keeping 0.4 M total cation concentration irrespective of metal species. Each suspension was aerated overnight by magnetic stirring, ensuring homogeneous oxidation of the precursors. Fe(III) phases formed were washed with deionized water at an end-to-end shaker for 15 min and centrifuged at 4000 rpm for 30 min after each washing. This process was repeated five times. A final solution of about 0.4 M MxFe1−xOOH (M: Mn, Ni, Co, Zn, Cr and Cu) was flushed with N2 and stored under N2 headspace for one month. The solution was centrifuged and washed again, then stored anaerobically at room temperature under N2 headspace. X-ray diffraction analysis showed that the Fe(III)-phase was mainly poorly crystalline akaganeite (β-FeOOH). 2.4. Chemical and mineralogical study of M-substituted magnetites After the scheduled incubation time, duplicate bottles were removed from the incubator and transferred to an anaerobic (N2: H2 = 97:3) chamber (Coy Laboratory Products, Inc., Ann Arbor, MI). The final pH and Eh of bacterial cultures and controls were measured in the anaerobic chamber using a combination of pH electrode (Orion 815600) and Eh electrode (Tiod 9180BN) with Orion EA-920 Expandable ion analyzer (ThermoOrion, Beverly, MA). The probe was placed directly into the serum bottle and
equilibrated for at least 10 min before recording the value. After the incubation, dopant incorporation into the magnetite was monitored by measuring acid-extractable ferrous ion and metal concentrations from the final medium subsamples. Acidextractable Fe(II) concentrations were determined by measuring the absorbance at 510 nm on a UV-spectrophotometer (Hewlett Packard 8453) by the 1,10-phenanthroline method (APHA/ AWWA/WEF, 1992) with 0.5 M HCl solution for extraction and dilution. The other subsamples were diluted into 1% nitric acid solution, then, analyzed for total iron and metal concentrations in the final medium using ICP-MS (Inductively Coupled PlasmaPolyScan Iris Spectrometer, Thermo Jarrell Ash, Franklin, MA). Monitoring increases in microbial biomass was accomplished using the BCA™ protein assay (Pierce, Rockford, IL) using bicinchoninic acid. Subsamples were removed at 0, 1, 2, 3 and 7 days with washing–centrifuging twice using phosphate buffered saline solution containing the ingredients (g/L); 1.18 Na2HPO4, 0.22 NaH2PO4·H2O and 8.5 NaCl and colorimetric detection at 562 nm (UV-spectrophotometer, Hewlett Packard 8453). Quantitation of total protein was standardized to albumin. Solid mineral phases from the reduction experiments were washed by repeated cycles of centrifugation and redispersion in deionized water five times, collected by freeze drying and stored under N2 gas. Magnetic susceptibility was measured for screening magnetization using a magnetic susceptibility meter (SM-30, Terraplus, Inc., CO), which uses a measuring field on the order of 10− 6 SI. The solid slurry was smeared on a glass slide with ethyl alcohol for acquiring the mineralogical composition of the precipitated or synthesized phases using an Xray diffractometer (XRD, PAD V, Scintag, Inc., Sunnyvale, CA) equipped with Cu–Kα radiation at 45 kV/40 mA, with a scan rate of 2–3° 2θ/min. Average crystallite size was derived from XRD result using Scherrer equation (Wilson, 1962). Size distribution was measured by dynamic light scattering (3000 HS Zetasizer, Malvern Instruments, U.K.). The samples were diluted in deionized water and sonicated for 8 min with Darvan 821A, a dispersant (Burke et al., 1998), then filtered through a 0.2 μm syringe filter. Filtration insured that agglomerated particles and bacterial cells remaining after washing would not be counted in the size analysis. A scanning electron microscope (SEM, XL30-FEG, Phillips) with energy dispersive X-ray analysis (EDX) was used for the analysis of morphology, mineralogy and chemistry of the solid mineral phases. Transmission electron microscopy (TEM, FX 2000, JEOL, Japan) was used to study the morphology of the precipitated crystallites of iron minerals (Zhang et al., 1998). 3. Results and discussion 3.1. Synthesis of metal-substituted magnetites Based on the preliminary incubation of a Zn-mixed precursor (ZnxFe1−xOOH, x = 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5), the iron-reducing bacterium TOR-39 reduced Fe(III) in Znmixed akaganeite and produced black Zn-substituted magnetic particles (ZnyFe3−yO4) which could be recognized as distinct from brown non-magnetic akaganeite by observing magnetic attraction
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to a hand-held magnet after 30 day incubation (Fig. 2A). Precipitates at 0.4 and 0.5 CMF remained brownish and nonmagnetic (Fig. 2B). The limit of maximum mixing amounts of mixed-M precursors which could produce magnetites lay between 0.3 and 0.4. This indicated that mixed-M precursors could be microbially synthesized to magnetite with the magnetite structure having about 1/3 of divalent and 2/3 of trivalent cations. The range of CMF was similar for other metals (Mn, Co and Ni) except Cr (0.05–0.1), which is a well known toxic element. All samples except the whole Cu-series and the highly Cr-mixed precursor (Cr0.1Fe0.9OOH) showed magnetic response to a hand magnet, even though there was different magnetic strength according to bacteria species and the type and amount of substituted elements. Fig. 3 illustrates the representative changes in pH and Eh of the final medium after 30 days incubation of TOR-39 and 75 days of PV-4 with various Ni-mixed precursors. The other series of mixed precursors exhibited similar trends. The initial pH and Eh of the medium was 8.0–8.2 and between − 260 and − 70 mV wobbling due to degassing differences for media
Fig. 2. Test for the maximum substitution of metal for microbial transformation of magnetite. (A) Photograph after 30 days incubation of Zn-substituted magnetites. Black precipitates exhibiting magnetic attraction from 0.2 and 0.3 and brownish unreacted Zn-mixed akaganeites from 0.4 and 0.5 CMF. (B) Magnetic susceptibility change of precipitate after 30 days incubation with Zn-mixed precursors and TOR-39.
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Fig. 3. Final pH and Eh of the medium that produced Ni-substituted magnetites from NixFe1−xOOH, where x = 0.02, 0.1, 0.2 and 0.3. Straight lines: pure magnetite as a control.
preparation. All samples after incubation with TOR-39 showed a pH range between 7.1–7.25 except for Cu-mixed precursors (7.7–7.8), while all pH values of PV-4 incubated samples ranged from 7.8 to 8.2. Mixed-M precursors could be synthesized to M-substituted magnetites above the pH of control, pure magnetite irrespective of thermophilic or psychrotolerant species. Although additional 15 mM of MOPS (pH 7.8) was added to TOR-39 media, pH dropped to around 7.1 due to organic acid and CO2 formed by glucose fermentation (Zhang et al., 1996). By contrast, PV-4 kept the pH at around 8.0 without MOPS buffer. The samples containing NixFe1−xOOH at 0.2 and 0.3 CMF exhibited a pH similar to the initial media pH at around 8.2 and this lack of pH change was attributed to the absence of microbial activity by PV-4. The pH change can be associated with the presence of sizable microbial activity of samples containing precursors including low amounts of divalent ions, because this microbial reductive activity leads to produce available reduced ferrous ions to incorporate into the magnetite structure. The pH of the PV-4 media exhibited more variation than TOR-39 media (Fig. 3). It is likely concluded that the limited and constrained incubation condition (65 °C) of TOR-39 media prevented variation in duplicate samples. Even though the Eh of the final medium decreased to between − 370 and − 260 mV for both TOR-39 and PV-4, a comparison between species cannot be made to each control sample due to the gap of the initial Eh values followed by degassing and media preparation. Final concentrations of acid (0.5 M HCl)-extractable ferrous ion in the final medium substantiated microbial reduction according to the amounts of mixed metal (Data not shown). Ferrous ion concentration in the TOR-39 culture (i.e., Co-mixed precursors 0.22–0.49 mM) was more stable than that of PV-4 (i.e., 0.02–3.26 mM). This identical trend among mixed metals
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Fig. 4. Metal concentration in the final medium with TOR-39 at 65 °C for 30 days.
such as Zn, Co, Ni and Mn was likely caused by the fact that TOR-39, which could start to make magnetite within 1–2 days, left a small amount of ferrous ion in the medium after 30 days. In contrast, PV-4, which required up to 3 months for magnetite accumulation, could not use a significant amount of reduced iron in the medium. Similarly, Li et al. (2004) found that the concentration of acid-extractable ferrous ion peaked at the lepidocrocite phase among the sequence of ferrihydrite–intermediate phase–lepidocrocite–magnetite transformation using
GS-15. All the samples except Cu-mixed precursors and Cr0.1 precursor incubated with TOR-39 had lower ferrous ion concentrations than did control samples containing pure precursor. This indirectly indicated that most of the reduced ferrous ions were incorporated into the M-substituted magnetite because the mixed-M precursor would have a lower mole fraction of iron than that of pure akaganeite. This trend was also reflected in the metal concentration in the final medium (Fig. 4). The concentrations of Cr were below the detection limit because of the low CMF of Cr-mixed precursor. The fact that concentrations of Zn were predominantly lower than Mn, Co and Ni was affected by the high free energy of formation of franklinite than any other M-substituted magnetite and even pure magnetite (Moon et al., 2007). The common trend was increasing metal concentration along with the increased CMF in the precursor, however, Mn concentration decreased due to the precipitation of rhodochrosite as a subsidiary mineral phase. Theoretical concentration of metals was 10.8 mM at 0.3 CMF. Only Ni-mixed precursor at 0.2 and 0.3 CMF exceeded 0.3 mM due to the incongruent incorporation into the magnetite structure (Moon et al., 2007). 3.2. Mineralogical investigation of synthesized M-substituted magnetite Fig. 5 shows XRD patterns of the M-substituted magnetites (Mn, Co, Cr and Ni) by metal reducing bacteria TOR-39 and
Fig. 5. X-ray diffraction patterns of the M-substituted magnetites synthesized from mixed-M magnetite precursors at different CMF of (A) MnxFe1−xOOH, x = 0.05, 0.1, 0.2 and 0.3, (B) and (C) CoxFe1−xOOH and NixFe1−xOOH, x = 0.02, 0.1, 0.2 and 0.3, (D) CrxFe1−xOOH, x = 0.02, 0.05 and 0.1. Abbreviations: A, mixed-M akaganeite; M, M-substituted magnetite; R, rhodochrosite; ⁎, not oxalate treated.
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PV-4 with various mixed-M precursors having different mixing levels. Up to x = 0.3 of Mn, Zn, Co and Ni and 0.05 of Cr-mixed precursors formed M-substituted magnetite using TOR 39 and the mixed-M precursor. Rhodochrosite (MnCO3) was precipitated from MnxFe1−xOOH precursors (0.1, 0.2 and 0.3 CMF) irrespective of bacterial species. It was observed that PV-4 could not make M-substituted magnetites with Ni0.2, Ni0.3 and Zn0.3 due to toxicity. XRD analyses of minerals formed by
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thermophilic and psychrotolerant bacteria under a N2 atmosphere showed that M-substituted magnetites (MyFe3−yO4) were predominantly formed during the microbial Fe(III) reduction of mixed-M precursors (MxFe1−xOOH). Metal incorporation into magnetite was ascertained by SEM and EDX analysis (Moon et al., 2007) and the morphology of precipitated single crystallites of M-substituted magnetites was examined by TEM analysis (Fig. 6). Substituted magnetites
Fig. 6. Transmission electron microscopic images and crystallite size analysis of individual magnetite grains using MxFe1−xOOH precursor (where M was Zn, Co, Mn and Ni, respectively) with TOR-39 for 4 weeks at 65 °C.
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Table 1 Comparison of maximum mixing concentration of metals for microbially reductive production from precursor to magnetite Traditional method a
New precursor method b
b1 mM of Cr Using 1.0 mM of K2CrO2− 4 with FeOOH b10 mM of Co Using 10 mM of Co(III)-EDTA with FeOOH N2 mM of Ni Using 2 mM of NiCl2 with FeOOH
N1.8 mM of Cr Using 36 mM of Cr0.05Fe0.95 O(OH) N10.8 mM of Co Using 36 mM of Co0.3Fe0.7 O(OH) N10.8 mM of Ni Using 36 mM of Ni0.3Fe0.7O(OH)
a Minimum concentration suppressing microbial activity (values from Roh et al., 2001). b Maximum concentration in which strain can survive and make magnetite in this study.
with Zn, Co, Mn and Ni exhibited averaged crystallite size of 25, 51, 53 and 75 and median size of 22, 46, 49 and 74 nm, respectively. The Zn- and Mn-substituted magnetites showed a narrow size distributions indicating these elements were congruently incorporated into magnetite crystal structure. Congruent incorporation was supported by comparing the metallic cation mole fraction among mixed-M precursors, M-substituted magnetite and final medium concentrations (Moon et al., 2007). Zn exhibited greater incorporation than Co or Ni. The average size of Co-substituted magnetite was similar to that of Mn-substituted magnetite but it had wide size distribution. The large crystallite size of Ni-substituted magnetite was likely due to incongruent incorporation of Ni into the magnetite structure as reflected by; final medium had higher Ni cation mole fraction than those of mixed precursor and substituted magnetite compared to Zn or Co (Moon et al., 2007) and similar size to pure magnetite (median 85.2 nm by XRD, data not shown). 3.3. Advantages of new method Table 1 shows that the first critical advantage of this new mixed precursor method was to increase the permissible levels
of substituted magnetites with toxic metals. In the new method, mixed precursor, MxFe1−xOOH (where, x N at least 0.3 for Mn, Ni, Co, Zn and 0.05 for Cr) was converted to M-substituted magnetite at higher substitution levels than could be achieved by the previous method (Roh et al., 2001) using pure magnetite precursor as akaganeite phase and soluble metal salts. The microbially induced magnetites incorporate metals in amounts that could be toxic to the bacteria if the metal ions were added in the salt form; Cr (1.8), Co (1.1) and Ni (5.4). The advantage of this method was dramatically improved in the case of toxic metals such as Ni and Cr. Advantage of the mixed precursor method was confirmed by the comparing microbial biomass as measured by total protein amount using BCA protein assay (Fig. 7). Zn-substituted magnetites, irrespective of methods produced were within the tested range of Zn from 1.8 mM to 7.2 mM incubated with TOR-39 at 65 °C. After two day incubations each set had an identical trend; traditional method exhibited almost constant or slight decreases compared to 2 day concentration, while the new mixed method exhibited continuous increase. Therefore the predominant difference between two methods was confirmed by P value 0.0053 for statistical differences after 2 days of incubation. In contrast to Zn, toxic Ni-substituted magnetites were produced throughout the range of 1.8–7.2 mM for new method but only as high as 1 mM for traditional method (Data not shown). Samples of 1 mM of Ni salt exhibited similar biomass to Ni at 1.8 mM (0.02 CMF) for the mixed precursor method and produced magnetite. Though growth occurred with Ni salts at 4 and 7 mM the precipitates remained dark brown with a very weak magnetic response. In contrast, the mixed precursor method produced magnetite at 7.2 mM. Again, the new mixed precursor method was much more effective in the case of toxic metals. Zinc-substituted magnetites transformed from both 36 mM of Zn0.15Fe0.85OOH and FeOOH with about 6 mM of dissolved ZnCl2 were compared under the same conditions. The runproduct using the Zn-mixed precursor collected after 30 hours showed a stronger magnetic signature than the pure akaganeite with zinc chloride solution (Fig. 8A). Microbial production of magnetite, ascertained by semi-quantitative analysis of mineral phases using relative intensity (Chung, 1974), was complete in
Fig. 7. Comparison of microbial activity of TOR-39 incubated at 65 °C represented by total protein using the BCA protein assay. Each value was from triplicates and standardized to an albumin standard.
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Fig. 8. The comparative study with TOR-39 incubated at 65 °C for (A) magnetic susceptibility (10− 6 SI), (B) quantitative analysis between magnetite and precursor, (C) crystallite size, (D) crystallinity with height, area and FWHM and (E) size distribution by zetasizer. Symbols: solid, magnetite from 36 mM Zn0.15Fe0.85OOH and open, magnetite from FeOOH + ZnCl2 6 mM.
one week when using the new mixed precursor method as compared to three weeks when Zn2+ was added as a salt (Fig. 8B). The mass of magnetite among precipitates from the new method increased faster than that of the traditional method. Detailed examination of the XRD pattern for crystallite size using the Scherrer equation and crystallinity by height, area and full width at half maximum (FWHM) indicated fast growth of crystallite and good crystal structure with sharper peaks of Znsubstituted magnetite from Zn-mixed precursor than those produced by the traditional method (Figs. 8C and D). Zn-mixed precursors enabled a faster growth rate of the magnetite with an average size of 40 nm after 2 weeks and 100 nm after 3 weeks incubation in contrast to 22 and 25.7 nm, respectively, using the salt and pure akaganeite method, as determined by the zetasizer analysis. Based on the asymmetry with long tails toward large size in log-scale, large aggregate particles were not dispersed even after the application of a dispersant and sonication.
However, the higher precipitate accumulation rate with the new method was in good agreement with those calculated by the XRD. The traditional method's 10 to 20 nm sized magnetites exhibited limitation in size even after 4 weeks incubation. This new method enabled microbial synthesis of magnetite to substitute larger amounts of dopants such as toxic elements and led to faster phase transformation rates and magnetization with good crystal habit and large crystallite size after short incubation times. Generally, microbial reduction can produce magnetite particles of well-defined size (typically tens of nanometers) and crystallographic morphology. Microbially synthesized Msubstituted magnetite from the new mixed precursor method is another alternative to enhance magnetite properties like size control, capacity for substitution of toxic metals and magnetic response. Finally, the new method can expand the application of microbial induced substituted magnetite according to the flexibility of wide range of substitution amounts.
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Acknowledgements This research was supported by the Defense Advanced Research Projects Agency (DARPA) Biomagnetics Program under Contract 1868-HH43-X1 and the US Department of Energy's (DOE) Office of Fossil Energy with Student support provided by the DOE Environmental Molecular Science Initiative. Oak Ridge National Laboratory (ORNL) is managed by UT-Battelle, LLC, for the US DOE under Contract DE-AC0500OR22725. J.-W. Moon is supported by the Post-doctoral Fellowship Program of Korea Science and Engineering Foundation and in part by an appointment to the ORNL Postdoctoral Research Associates Program administered jointly by the Oak Ridge Institute for Science and Education and ORNL. References American Public Health Association-American Water Works Association-Water Environment Federation, 1992. Standard methods for the examination of water and wastewater18 Edition. Washington, D.C. Balkwill, D.L., Maratea, D., Blakemore, R.P., 1980. Ultrastructure of a magnetotactic Spirillum. J. Bacteriol. 141, 1399–1408. Blakemore, R.P., 1975. Magnetotactic bacteria. Science 190, 377–379. Burke, M., Greenwood, R., Kendall, K., 1998. Experimental methods for measuring the optimum amount of dispersant for seven Sumitomo alumina powders. J. Mater. Sci. 33, 5149–5156. Chung, F.H., 1974. Quantitative interpretation of X-ray diffraction patterns of mixtures: II. adiabatic principle of X-ray diffraction analysis of mixtures. J. Appl. Crystallogr. 7, 519–525. Frankel, R.B., 1987. Microbial metabolism—anaerobes pumping iron. Nature 330, 208–209. Fredrickson, J.K., Zachara, J.M., Kennedy, D.W., Dong, H., Onstott, T.C., Hinman, N.W., Li, S., 1998. Biogenic iron mineralization accompanying the dissimilatory reduction of hydrous ferric oxide by a groundwater bacterium. Geochim. Cosmochim. Acta 62, 3239–3257. Fredrickson, J.K., Zachara, J.M., Kukkadau, R.K., Gorby, Y.A., Smith, S.C., Brown, C.F., 2001. Biotransformation of Ni-substituted hydrous ferric oxide by an Fe(III)-reducing bacterium. Environ. Sci. Technol. 35, 703–712. Gorby, Y.A., Beveridge, T.J., Blakemore, R.P., 1988. Characterization of the bacterial magnetosome membrane. J. Bacteriol. 170, 834–841. Li, Y.-L., Zhang, C.L., Vali, H., Cole, D.R., Phelps, T.J., 2004. Mossbauer spectroscopy of extracellular tabular magnetite formed during microbial iron reduction. Geochim. Cosmochim. Acta 69, A461.
Liu, S., Zhou, J., Zhang, C., Cole, D.R., Gajdarziska, M., Phelps, T.J., 1997. Thermophilic Fe(III)-reducing bacteria from the deep subsurface: the evolutionary implication. Science 277, 1106–1109. Love, L.J., Jansen, J.F., McKnight, T.E., Roh, Y., Phelps, T.J., 2004. A magnetocaloric pump for microfluidic applications. IEEE Trans. Nanobioscience 3, 101–110. Love, L.J., Jansen, J.F., McKnight, T.E., Roh, Y., Phelps, T.J., Yeary, L.W., Cunningham, G.T., 2005. Ferrofluid field induced flow for microfluidic applications. IEEE/ASME Trans. Mechatron. 10, 68–76. Lovley, D.R., 1995. Bioremediation of organic and metal contaminants with dissimilatory metal reduction. J. Ind. Microbiol. 14, 85–93. Lovley, D.R., Stolz, J.F., Nord Jr., G.L., Phillips, E.J.P., 1987. Anaerobic production of magnetite by a dissimilatory iron-reducing microorganism. Nature 330, 252–254. Lowenstam, H.A., 1981. Minerals formed by organisms. Science 211, 1126–1131. Moon, J.-W., Yeary, L.W., Rondinone, A.J., Rawn, C.J., Kirkham, M.J., Roh, Y., Love, L.J., Phelps, T.J., 2007. Magnetic response of microbially synthesized transition metal- and lanthanide-substituted nano-sized magnetites. Magn. Magn. Mater. 313, 283–292. Moroz, P., Jones, S.K., Gray, B.N., 2002a. Magnetically mediated hyperthermia: current status and future directions. Int. J. Hypertherm. 18, 267–284. Moroz, P., Jones, S.K., Gray, B.N., 2002b. Tumor response to arterial embolization hyperthermia and direct injection hyperthermia in a rabbit liver tumor model. J. Surg. Oncol. 80, 149–156. Phelps, T.J., Raione, E.G., White, D.C., Fliermans, C.B., 1989. Microbial activity in deep subsurface environments. Geomicrobiol. J. 7, 79–91. Roh, Y., Lauf, R.J., McMillan, A.D., Zhang, C., Rawn, C.J., Bai, J., Phelps, T.J., 2001. Microbial synthesis and the characterization of metal-substituted magnetites. Solid State Commun. 118, 529–534. Roh, Y., Gao, H., Vali, H., Kennedy, D.W., Yang, Z.K., Gao, W., Dohnalkova, A.C., Stapleton, R.D., Moon, J.-W., Phelps, T.J., Fredrickson, J.K., Jizhong Zhou, J., 2006. Metal reduction and iron biomineralization by a psychrotolerant Fe(III)-reducing bacterium, Shewanella sp. strain PV-4. Appl. Environ. Microbiol. 72, 3236–3244. Wilson, A.J.C., 1962. On variance as a measure of line broadening in diffractometry: general theory and small particle size. Proc. Phys. Soc. 80, 286–294. Yeary, L.W., Moon, J.-W., Love, L.J., Thompson, J.R., Raw, C.J., Phelps, T.J., 2005. Magnetic properties of biosynthesized magnetic nanoparticles. IEEE Trans. Magn. 41, 4384–4389. Zhang, C., Liu, S., Logan, J., Mazumder, R., Phelps, T.J., 1996. Enhancement of Fe(III), Co(III), and Cr(VI) reduction at elevated temperatures and by a thermophilic bacterium. Appl. Biochem. Biotech. 57/58, 923–932. Zhang, C., Vali, H., Romanek, C.S., Phelps, T.J., Liu, S.V., 1998. Formation of single-domain magnetite by a thermophilic bacterium. Am. Mineral. 83, 1409–1418.
Microbial preparation of metal-substituted magnetite nanoparticles Ji-Won Moon a , Yul Roh b , Robert J. Lauf a , Hojatollah Vali c , Lucas W. Yeary d,e , Tommy J. Phelps a,⁎ b
a Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA Faculty of Earth System and Environmental Sciences, Chonnam National University, Gwangju, 500-757, Republic of Korea c Department of Anatomy and Cell Biology, McGill University, Montreal, QC Canada H3A 2B2 d Engineering Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA e Inorganic and Integration Technologies, Corning, NY 14831, USA
Received 27 November 2006; received in revised form 3 April 2007; accepted 13 April 2007 Available online 29 April 2007
Abstract A microbial process that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular metal (M)substituted magnetite nanoparticles using akaganeite and dopants of dissolved form has previously been reported. The objectives of this study were to develop methods for producing M-substituted magnetite nanoparticles with a high rate of metal substitution by biological processes and to identify factors affecting the production of nano-crystals. The thermophilic and psychrotolerant iron-reducing bacteria had the ability to form Msubstituted magnetite nano-crystals (MyFe3−yO4) from a doped precursor, mixed-M iron oxyhydroxide, (MxFe1−xOOH, x ≤ 0.5, M is Mn, Zn, Ni, Co and Cr). Within the range of 0.01 ≤ x ≤ 0.3, using the mixed precursor material enabled the microbial synthesis of more heavily substituted magnetite compared to the previous method, in which the precursor was pure akaganeite and the dopants were present as soluble metal salts. The mixed precursor method was especially advantageous in the case of toxic metals such as Cr and Ni. Also this new method increased the production rate and magnetic properties of the product, while improving crystallinity, size control and scalability. © 2007 Elsevier B.V. All rights reserved. Keywords: Metal-substituted magnetite; Mixed-metal precursor; Shewanella sp. strain PV-4; Thermoanaerobacter sp. TOR-39
1. Introduction Nanometer-sized magnetite could have diverse applications including magnetic targeting (drug delivery and radionuclide therapy), magnetic nanoparticles as contrast agents for magnetic resonance imaging, diagnostics, immunoassays, molecular biology, RNA- and DNA-purification, cell separation and purification, cell adhesion research, hyperthermia (Moroz et al., 2002a,b), and magnetic ferrofluids for magnetocaloric pumps (Love et al., 2004, 2005). Microbial iron reduction has been intensively studied to understand biogeochemical processes including the cycling of metal, carbon, nitrogen, phosphate and sulfur in natural and contaminated subsurface environments (Lovley et al., 1987; Lovley, 1995; Liu et al., 1997; Fredrickson et al., 1998, 2001) and as mechanisms for producing copious nm-sized magnetites (Roh et al., 2001; Yeary et al., 2005). Lowenstam (1981) has classified ⁎ Corresponding author. Tel.: +1 865 574 7290; fax: +1 865 576 3989. E-mail address: [email protected] (T.J. Phelps). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.04.012
the biomineralization as either biologically induced mineralization or boundary-organized biomineralization. The latter process applies to situations such as the formation of magnetic crystals within the cells of magnetotactic bacteria as reported by Blakemore (1975). These bacteria typically contain particles having a narrow size distribution around 40–50 nm and characteristic morphologies, enveloped by a membrane (Balkwill et al., 1980; Gorby et al., 1988). In the former process, bacteria may change the environment of their outer membrane, creating electrochemical conditions favorable for magnetite precipitation. It is unlikely to be associated with an organic matrix and produces a broad size-distributed magnetite (Frankel, 1987). A microbial process, that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular M-substituted magnetite nanoparticles using akaganeite and dopants in soluble form, has been previously reported (Roh et al., 2001). Microbially facilitated magnetite synthesis may have numerous advantages. Biological processes can effectively produce ferrite particles of well-defined nanometer size and fine scaled crystallographic
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morphology while incorporating selected metals into the magnetite (MyFe3−yO4) structure, where M was Co, Cr, Mn and Ni (Roh et al., 2001). It has been calculated that the dissimilatory Fe(III)-reducing bacteria used can produce 5000-fold more magnetite per unit biomass than magnetotactic bacteria (Frankel, 1987). Fermentative production of nanoparticles would also be an environmentally benign and fairly economical manufacturing process, which could easily be scaled to the quantities needed for industrial production. To synthesize M-substituted magnetite, Roh et al. (2001) used pure akaganeite (β-FeOOH) along with a stock solution of a dopant metal salt, from which the bacteria produced Msubstituted magnetite. The process developed by Roh et al. (2001) has some drawbacks; 1) some dopants are fairly toxic in the form of soluble M salts and 2) the dopant may be distributed non-uniformly in the magnetite structure because of environmental changes or solubility issues that might reduce dopant concentration during crystal growth. Overall, both yield and product quality may suffer from the effects of soluble M salts. In this study, mixed-M precursors were used in place of pure akaganeite and soluble metal salts (Fig. 1). The purpose of the present study was to develop a procedure for synthesizing M- substituted magnetites by microbially facilitated processes while enhancing the dopant concentration, the production rate, the magnetite properties, the mineralogical and crystallographical quality (phase purity), size and morphology control and the scalability of the process in a less toxic environment. 2. Materials and methods 2.1. Source of microorganisms The microbial formation of iron minerals with thermophilic (Thermoanaerobacter sp. TOR-39, Liu et al., 1997) and
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psychrotolerant (Shewanella sp. strain PV-4, Roh et al., 2006) bacteria were examined. These Fe(III)-reducing bacteria were isolated from sediments and water collected from a variety of environments including deep subsurface sediments (TOR-39) or the water column near a hydrothermal vent off the coast of Hawaii (PV-4). These Fe(III)-reducing bacteria were used to synthesize magnetite and metal-substituted ferrite nanoparticles under anaerobic conditions. 2.2. Media preparation and incubation The culture medium contained the following ingredients (g/L): 2.5 NaHCO3, 0.08 CaCl2·2H2O, 1.0 NH4Cl, 0.2 MgCl2·6H2O, 1–10.0 NaCl, 7.2 HEPES (hydroxyethylpiperazine-N′-2ethanesulfonic acid), 0.5 yeast extract, 0.1 mL of 0.1% resazurin, 10 mL of Oak Ridge National Laboratory (ORNL) trace minerals and 1 mL of ORNL vitamin solutions (Phelps et al., 1989). No exogeneous electron carrier substance (i.e., anthraquinone disulfonate) or reducing agent (i.e., cysteine) was added to the anaerobic medium. The trace mineral solution contained (mg/L): 1500 Nitrilotriacetic acid, 200 FeCl2·4H2O, 100 MgCl2·6H2O, 20 sodium tungstate, 100 MnCl2·4H2O, 100 CoCl2·6H2O, 1000 CaCl2·2H2O, 50 ZnCl2, 2 CuCl2·2H2O, 5 H3BO3, sodium molybdate, 1000 NaCl, 17 Na2SeO3, 24 NiCl2·6H2O. 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 (riboflavin), 0.05 nicotinic acid (niacin), 0.05 pantothenic acid, 0.001 B12 (cyanobalamine) crystalline, 0.05 PABA (P-aminobenzoic acid), and 0.05 lipoic acid (thioctic). The dissolved basal medium was boiled with N2 purging and cooled with continuous N2 purging. For serum vial tests, 50 mL aliquots were dispensed into 160 mL serum bottles with N2 purging and had a final pH of about 8.0–8.2. Each bottle was
Fig. 1. Comparison of procedures for the microbial synthesis of the transitional metal-substituted magnetite between traditional and new precursor methods.
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sealed with a butyl rubber stopper (Bellco Glass, Inc.) and an aluminum crimp seal. The incubation for microbial synthesis of M-substituted magnetites in duplicates was initiated with the addition of 10 mM of glucose, 15 mM of MOPS titrated to pH 7.8, about 40 mM of mixed-M precursor and 1 mL of mid-log growth which fermented for one day with 10 mM glucose as electron donor without electron acceptor for preparing the inoculum of TOR-39. For PV-4 samples, 10 mM of lactate, about 40 mM of mixed-M precursor and 1 mL of mid-log growth of metal reducing PV-4, that was incubated using 10 mM lactate and 10 mM ferric citrate as the electron donor and electron acceptor for 5 days, were used per 50 mL of media in a serum bottle. The incubation was at 65 °C for 30 days for TOR-39 and room temperature for 75 days for PV-4. Control samples received an equal volume of pure akaganeite were also incubated. To get pure magnetite, the headspace was N2 only, and the medium had a low bicarbonate concentration, because metalreducing bacteria could also facilitate conversion of CO2 into sparingly soluble carbonate minerals such as siderite (FeCO3) under a CO2 atmosphere or bicarbonate buffer (N 30 mM) (Roh et al., 2001). 2.3. New methods for magnetite precursor preparation The mixed-M precursors were prepared as follows: NaOH solution (10 M) was added drop wise into a mixed solution of total 0.4 M of FeCl3·6H2O and metal chloride (hydrate) like MnCl2·4H2O, NiCl2·6H2O, CoCl2·6H2O, CrCl3·6H2O, ZnCl2 and CuCl2·2H2O with rapid stirring and stopped at pH 7.00. The mixtures containing metals were established at specific cation mole fraction (CMF) up to 0.5 (0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5) keeping 0.4 M total cation concentration irrespective of metal species. Each suspension was aerated overnight by magnetic stirring, ensuring homogeneous oxidation of the precursors. Fe(III) phases formed were washed with deionized water at an end-to-end shaker for 15 min and centrifuged at 4000 rpm for 30 min after each washing. This process was repeated five times. A final solution of about 0.4 M MxFe1−xOOH (M: Mn, Ni, Co, Zn, Cr and Cu) was flushed with N2 and stored under N2 headspace for one month. The solution was centrifuged and washed again, then stored anaerobically at room temperature under N2 headspace. X-ray diffraction analysis showed that the Fe(III)-phase was mainly poorly crystalline akaganeite (β-FeOOH). 2.4. Chemical and mineralogical study of M-substituted magnetites After the scheduled incubation time, duplicate bottles were removed from the incubator and transferred to an anaerobic (N2: H2 = 97:3) chamber (Coy Laboratory Products, Inc., Ann Arbor, MI). The final pH and Eh of bacterial cultures and controls were measured in the anaerobic chamber using a combination of pH electrode (Orion 815600) and Eh electrode (Tiod 9180BN) with Orion EA-920 Expandable ion analyzer (ThermoOrion, Beverly, MA). The probe was placed directly into the serum bottle and
equilibrated for at least 10 min before recording the value. After the incubation, dopant incorporation into the magnetite was monitored by measuring acid-extractable ferrous ion and metal concentrations from the final medium subsamples. Acidextractable Fe(II) concentrations were determined by measuring the absorbance at 510 nm on a UV-spectrophotometer (Hewlett Packard 8453) by the 1,10-phenanthroline method (APHA/ AWWA/WEF, 1992) with 0.5 M HCl solution for extraction and dilution. The other subsamples were diluted into 1% nitric acid solution, then, analyzed for total iron and metal concentrations in the final medium using ICP-MS (Inductively Coupled PlasmaPolyScan Iris Spectrometer, Thermo Jarrell Ash, Franklin, MA). Monitoring increases in microbial biomass was accomplished using the BCA™ protein assay (Pierce, Rockford, IL) using bicinchoninic acid. Subsamples were removed at 0, 1, 2, 3 and 7 days with washing–centrifuging twice using phosphate buffered saline solution containing the ingredients (g/L); 1.18 Na2HPO4, 0.22 NaH2PO4·H2O and 8.5 NaCl and colorimetric detection at 562 nm (UV-spectrophotometer, Hewlett Packard 8453). Quantitation of total protein was standardized to albumin. Solid mineral phases from the reduction experiments were washed by repeated cycles of centrifugation and redispersion in deionized water five times, collected by freeze drying and stored under N2 gas. Magnetic susceptibility was measured for screening magnetization using a magnetic susceptibility meter (SM-30, Terraplus, Inc., CO), which uses a measuring field on the order of 10− 6 SI. The solid slurry was smeared on a glass slide with ethyl alcohol for acquiring the mineralogical composition of the precipitated or synthesized phases using an Xray diffractometer (XRD, PAD V, Scintag, Inc., Sunnyvale, CA) equipped with Cu–Kα radiation at 45 kV/40 mA, with a scan rate of 2–3° 2θ/min. Average crystallite size was derived from XRD result using Scherrer equation (Wilson, 1962). Size distribution was measured by dynamic light scattering (3000 HS Zetasizer, Malvern Instruments, U.K.). The samples were diluted in deionized water and sonicated for 8 min with Darvan 821A, a dispersant (Burke et al., 1998), then filtered through a 0.2 μm syringe filter. Filtration insured that agglomerated particles and bacterial cells remaining after washing would not be counted in the size analysis. A scanning electron microscope (SEM, XL30-FEG, Phillips) with energy dispersive X-ray analysis (EDX) was used for the analysis of morphology, mineralogy and chemistry of the solid mineral phases. Transmission electron microscopy (TEM, FX 2000, JEOL, Japan) was used to study the morphology of the precipitated crystallites of iron minerals (Zhang et al., 1998). 3. Results and discussion 3.1. Synthesis of metal-substituted magnetites Based on the preliminary incubation of a Zn-mixed precursor (ZnxFe1−xOOH, x = 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5), the iron-reducing bacterium TOR-39 reduced Fe(III) in Znmixed akaganeite and produced black Zn-substituted magnetic particles (ZnyFe3−yO4) which could be recognized as distinct from brown non-magnetic akaganeite by observing magnetic attraction
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to a hand-held magnet after 30 day incubation (Fig. 2A). Precipitates at 0.4 and 0.5 CMF remained brownish and nonmagnetic (Fig. 2B). The limit of maximum mixing amounts of mixed-M precursors which could produce magnetites lay between 0.3 and 0.4. This indicated that mixed-M precursors could be microbially synthesized to magnetite with the magnetite structure having about 1/3 of divalent and 2/3 of trivalent cations. The range of CMF was similar for other metals (Mn, Co and Ni) except Cr (0.05–0.1), which is a well known toxic element. All samples except the whole Cu-series and the highly Cr-mixed precursor (Cr0.1Fe0.9OOH) showed magnetic response to a hand magnet, even though there was different magnetic strength according to bacteria species and the type and amount of substituted elements. Fig. 3 illustrates the representative changes in pH and Eh of the final medium after 30 days incubation of TOR-39 and 75 days of PV-4 with various Ni-mixed precursors. The other series of mixed precursors exhibited similar trends. The initial pH and Eh of the medium was 8.0–8.2 and between − 260 and − 70 mV wobbling due to degassing differences for media
Fig. 2. Test for the maximum substitution of metal for microbial transformation of magnetite. (A) Photograph after 30 days incubation of Zn-substituted magnetites. Black precipitates exhibiting magnetic attraction from 0.2 and 0.3 and brownish unreacted Zn-mixed akaganeites from 0.4 and 0.5 CMF. (B) Magnetic susceptibility change of precipitate after 30 days incubation with Zn-mixed precursors and TOR-39.
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Fig. 3. Final pH and Eh of the medium that produced Ni-substituted magnetites from NixFe1−xOOH, where x = 0.02, 0.1, 0.2 and 0.3. Straight lines: pure magnetite as a control.
preparation. All samples after incubation with TOR-39 showed a pH range between 7.1–7.25 except for Cu-mixed precursors (7.7–7.8), while all pH values of PV-4 incubated samples ranged from 7.8 to 8.2. Mixed-M precursors could be synthesized to M-substituted magnetites above the pH of control, pure magnetite irrespective of thermophilic or psychrotolerant species. Although additional 15 mM of MOPS (pH 7.8) was added to TOR-39 media, pH dropped to around 7.1 due to organic acid and CO2 formed by glucose fermentation (Zhang et al., 1996). By contrast, PV-4 kept the pH at around 8.0 without MOPS buffer. The samples containing NixFe1−xOOH at 0.2 and 0.3 CMF exhibited a pH similar to the initial media pH at around 8.2 and this lack of pH change was attributed to the absence of microbial activity by PV-4. The pH change can be associated with the presence of sizable microbial activity of samples containing precursors including low amounts of divalent ions, because this microbial reductive activity leads to produce available reduced ferrous ions to incorporate into the magnetite structure. The pH of the PV-4 media exhibited more variation than TOR-39 media (Fig. 3). It is likely concluded that the limited and constrained incubation condition (65 °C) of TOR-39 media prevented variation in duplicate samples. Even though the Eh of the final medium decreased to between − 370 and − 260 mV for both TOR-39 and PV-4, a comparison between species cannot be made to each control sample due to the gap of the initial Eh values followed by degassing and media preparation. Final concentrations of acid (0.5 M HCl)-extractable ferrous ion in the final medium substantiated microbial reduction according to the amounts of mixed metal (Data not shown). Ferrous ion concentration in the TOR-39 culture (i.e., Co-mixed precursors 0.22–0.49 mM) was more stable than that of PV-4 (i.e., 0.02–3.26 mM). This identical trend among mixed metals
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Fig. 4. Metal concentration in the final medium with TOR-39 at 65 °C for 30 days.
such as Zn, Co, Ni and Mn was likely caused by the fact that TOR-39, which could start to make magnetite within 1–2 days, left a small amount of ferrous ion in the medium after 30 days. In contrast, PV-4, which required up to 3 months for magnetite accumulation, could not use a significant amount of reduced iron in the medium. Similarly, Li et al. (2004) found that the concentration of acid-extractable ferrous ion peaked at the lepidocrocite phase among the sequence of ferrihydrite–intermediate phase–lepidocrocite–magnetite transformation using
GS-15. All the samples except Cu-mixed precursors and Cr0.1 precursor incubated with TOR-39 had lower ferrous ion concentrations than did control samples containing pure precursor. This indirectly indicated that most of the reduced ferrous ions were incorporated into the M-substituted magnetite because the mixed-M precursor would have a lower mole fraction of iron than that of pure akaganeite. This trend was also reflected in the metal concentration in the final medium (Fig. 4). The concentrations of Cr were below the detection limit because of the low CMF of Cr-mixed precursor. The fact that concentrations of Zn were predominantly lower than Mn, Co and Ni was affected by the high free energy of formation of franklinite than any other M-substituted magnetite and even pure magnetite (Moon et al., 2007). The common trend was increasing metal concentration along with the increased CMF in the precursor, however, Mn concentration decreased due to the precipitation of rhodochrosite as a subsidiary mineral phase. Theoretical concentration of metals was 10.8 mM at 0.3 CMF. Only Ni-mixed precursor at 0.2 and 0.3 CMF exceeded 0.3 mM due to the incongruent incorporation into the magnetite structure (Moon et al., 2007). 3.2. Mineralogical investigation of synthesized M-substituted magnetite Fig. 5 shows XRD patterns of the M-substituted magnetites (Mn, Co, Cr and Ni) by metal reducing bacteria TOR-39 and
Fig. 5. X-ray diffraction patterns of the M-substituted magnetites synthesized from mixed-M magnetite precursors at different CMF of (A) MnxFe1−xOOH, x = 0.05, 0.1, 0.2 and 0.3, (B) and (C) CoxFe1−xOOH and NixFe1−xOOH, x = 0.02, 0.1, 0.2 and 0.3, (D) CrxFe1−xOOH, x = 0.02, 0.05 and 0.1. Abbreviations: A, mixed-M akaganeite; M, M-substituted magnetite; R, rhodochrosite; ⁎, not oxalate treated.
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PV-4 with various mixed-M precursors having different mixing levels. Up to x = 0.3 of Mn, Zn, Co and Ni and 0.05 of Cr-mixed precursors formed M-substituted magnetite using TOR 39 and the mixed-M precursor. Rhodochrosite (MnCO3) was precipitated from MnxFe1−xOOH precursors (0.1, 0.2 and 0.3 CMF) irrespective of bacterial species. It was observed that PV-4 could not make M-substituted magnetites with Ni0.2, Ni0.3 and Zn0.3 due to toxicity. XRD analyses of minerals formed by
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thermophilic and psychrotolerant bacteria under a N2 atmosphere showed that M-substituted magnetites (MyFe3−yO4) were predominantly formed during the microbial Fe(III) reduction of mixed-M precursors (MxFe1−xOOH). Metal incorporation into magnetite was ascertained by SEM and EDX analysis (Moon et al., 2007) and the morphology of precipitated single crystallites of M-substituted magnetites was examined by TEM analysis (Fig. 6). Substituted magnetites
Fig. 6. Transmission electron microscopic images and crystallite size analysis of individual magnetite grains using MxFe1−xOOH precursor (where M was Zn, Co, Mn and Ni, respectively) with TOR-39 for 4 weeks at 65 °C.
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Table 1 Comparison of maximum mixing concentration of metals for microbially reductive production from precursor to magnetite Traditional method a
New precursor method b
b1 mM of Cr Using 1.0 mM of K2CrO2− 4 with FeOOH b10 mM of Co Using 10 mM of Co(III)-EDTA with FeOOH N2 mM of Ni Using 2 mM of NiCl2 with FeOOH
N1.8 mM of Cr Using 36 mM of Cr0.05Fe0.95 O(OH) N10.8 mM of Co Using 36 mM of Co0.3Fe0.7 O(OH) N10.8 mM of Ni Using 36 mM of Ni0.3Fe0.7O(OH)
a Minimum concentration suppressing microbial activity (values from Roh et al., 2001). b Maximum concentration in which strain can survive and make magnetite in this study.
with Zn, Co, Mn and Ni exhibited averaged crystallite size of 25, 51, 53 and 75 and median size of 22, 46, 49 and 74 nm, respectively. The Zn- and Mn-substituted magnetites showed a narrow size distributions indicating these elements were congruently incorporated into magnetite crystal structure. Congruent incorporation was supported by comparing the metallic cation mole fraction among mixed-M precursors, M-substituted magnetite and final medium concentrations (Moon et al., 2007). Zn exhibited greater incorporation than Co or Ni. The average size of Co-substituted magnetite was similar to that of Mn-substituted magnetite but it had wide size distribution. The large crystallite size of Ni-substituted magnetite was likely due to incongruent incorporation of Ni into the magnetite structure as reflected by; final medium had higher Ni cation mole fraction than those of mixed precursor and substituted magnetite compared to Zn or Co (Moon et al., 2007) and similar size to pure magnetite (median 85.2 nm by XRD, data not shown). 3.3. Advantages of new method Table 1 shows that the first critical advantage of this new mixed precursor method was to increase the permissible levels
of substituted magnetites with toxic metals. In the new method, mixed precursor, MxFe1−xOOH (where, x N at least 0.3 for Mn, Ni, Co, Zn and 0.05 for Cr) was converted to M-substituted magnetite at higher substitution levels than could be achieved by the previous method (Roh et al., 2001) using pure magnetite precursor as akaganeite phase and soluble metal salts. The microbially induced magnetites incorporate metals in amounts that could be toxic to the bacteria if the metal ions were added in the salt form; Cr (1.8), Co (1.1) and Ni (5.4). The advantage of this method was dramatically improved in the case of toxic metals such as Ni and Cr. Advantage of the mixed precursor method was confirmed by the comparing microbial biomass as measured by total protein amount using BCA protein assay (Fig. 7). Zn-substituted magnetites, irrespective of methods produced were within the tested range of Zn from 1.8 mM to 7.2 mM incubated with TOR-39 at 65 °C. After two day incubations each set had an identical trend; traditional method exhibited almost constant or slight decreases compared to 2 day concentration, while the new mixed method exhibited continuous increase. Therefore the predominant difference between two methods was confirmed by P value 0.0053 for statistical differences after 2 days of incubation. In contrast to Zn, toxic Ni-substituted magnetites were produced throughout the range of 1.8–7.2 mM for new method but only as high as 1 mM for traditional method (Data not shown). Samples of 1 mM of Ni salt exhibited similar biomass to Ni at 1.8 mM (0.02 CMF) for the mixed precursor method and produced magnetite. Though growth occurred with Ni salts at 4 and 7 mM the precipitates remained dark brown with a very weak magnetic response. In contrast, the mixed precursor method produced magnetite at 7.2 mM. Again, the new mixed precursor method was much more effective in the case of toxic metals. Zinc-substituted magnetites transformed from both 36 mM of Zn0.15Fe0.85OOH and FeOOH with about 6 mM of dissolved ZnCl2 were compared under the same conditions. The runproduct using the Zn-mixed precursor collected after 30 hours showed a stronger magnetic signature than the pure akaganeite with zinc chloride solution (Fig. 8A). Microbial production of magnetite, ascertained by semi-quantitative analysis of mineral phases using relative intensity (Chung, 1974), was complete in
Fig. 7. Comparison of microbial activity of TOR-39 incubated at 65 °C represented by total protein using the BCA protein assay. Each value was from triplicates and standardized to an albumin standard.
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Fig. 8. The comparative study with TOR-39 incubated at 65 °C for (A) magnetic susceptibility (10− 6 SI), (B) quantitative analysis between magnetite and precursor, (C) crystallite size, (D) crystallinity with height, area and FWHM and (E) size distribution by zetasizer. Symbols: solid, magnetite from 36 mM Zn0.15Fe0.85OOH and open, magnetite from FeOOH + ZnCl2 6 mM.
one week when using the new mixed precursor method as compared to three weeks when Zn2+ was added as a salt (Fig. 8B). The mass of magnetite among precipitates from the new method increased faster than that of the traditional method. Detailed examination of the XRD pattern for crystallite size using the Scherrer equation and crystallinity by height, area and full width at half maximum (FWHM) indicated fast growth of crystallite and good crystal structure with sharper peaks of Znsubstituted magnetite from Zn-mixed precursor than those produced by the traditional method (Figs. 8C and D). Zn-mixed precursors enabled a faster growth rate of the magnetite with an average size of 40 nm after 2 weeks and 100 nm after 3 weeks incubation in contrast to 22 and 25.7 nm, respectively, using the salt and pure akaganeite method, as determined by the zetasizer analysis. Based on the asymmetry with long tails toward large size in log-scale, large aggregate particles were not dispersed even after the application of a dispersant and sonication.
However, the higher precipitate accumulation rate with the new method was in good agreement with those calculated by the XRD. The traditional method's 10 to 20 nm sized magnetites exhibited limitation in size even after 4 weeks incubation. This new method enabled microbial synthesis of magnetite to substitute larger amounts of dopants such as toxic elements and led to faster phase transformation rates and magnetization with good crystal habit and large crystallite size after short incubation times. Generally, microbial reduction can produce magnetite particles of well-defined size (typically tens of nanometers) and crystallographic morphology. Microbially synthesized Msubstituted magnetite from the new mixed precursor method is another alternative to enhance magnetite properties like size control, capacity for substitution of toxic metals and magnetic response. Finally, the new method can expand the application of microbial induced substituted magnetite according to the flexibility of wide range of substitution amounts.
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