Magnetite: Structure, Properties and Applications R.U. Onyenwoke 1, and J. Wiegel2 ABSTRACT Magnetic particles are of interest because they: occupy a unique position at the interface of biology and chemistry, and hold high technological potential, i.e., they are easily manipulated with only an external magnetic field. Many magnetic particles are largely produced as a result of microbial iron metabolism, e.g., the low temperature Banded Iron Formations – formed as a result of microbial activity in contrast to the similar high temperature formations. The microbial origins of the Banded Iron Formations are a fact that has only recently (in the last 30 years) been realized by mainstream science. Particles of the mineral magnetite are among the most magnetic that occur in nature. Magnetite is one of the most common products of microbial iron metabolism, and it is, therefore, also a highly abundant mineral. In addition to being a common end-product of microbial metabolism, magnetite is also known to be incorporated into specialized organelles termed “magnetosomes.” These small magnets are used for direction sensing by certain species of bacteria, i.e., magnetotactic bacteria. The presence of magnetite in a Martian meteorite (ALH84001) was even touted as evidence for life on Mars. Yet magnetite particles are also of interest in biotechnology for everything from cell selection, e.g., stem cell tracking, differentiation and biosensing, to a magnetic resonance imaging (MRI) contrast agent to drug screening. Microbially-produced, nano-sized magnetite crystals are highly ordered minerals consistent in size and morphology – characteristics that lend these particles well to nano- and biotechnological applications. In this review, we examine various forms of magnetite from its unique structures and properties to its many points of relevance to biology and biotechnology. Neuroscience Center, University of North Carolina-Chapel Hill, UNC School of Medicine, Chapel Hill, North Carolina1 Department of Microbiology, University of Georgia, Athens, Georgia2 INTRODUCTION Over the past two decades there has been a marked increase in interest in biologically mediated formation of iron (Fe) containing compounds and processes leading to the biomineralization of iron minerals [1; 2; 3; 4]. This interest is in part due to the fact that iron is a highly abundant element (the 2nd most abundant metal after aluminum and the fourth most abundant element in the Earth’s crust) and also because many believe that Fe(III) was the first external electron acceptor of global significance in microbial respiration [5; 6; 7]. However, the role of iron in our planet’s natural history is far from the only scientific appeal of iron and it mineral derivatives. From a biotechnological perspective, magnetic iron oxide nanoparticles with appropriate surface chemistries are ideal for use in numerous in vivo applications, such as: MRI contrast enhancement,

tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery, and cell separation [8; 9]. One of the iron minerals of chief interest among all of the entities composing the field of iron research is the naturally occurring iron oxide magnetite. Magnetite is, as might be expected, 1) highly abundant, 2) a common by-product/“secondary mineral” of metal metabolism/respiration, 3) utilized intracellularly by a surprisingly large number of organisms, and 4) proposed for an ever-increasingly large role in a number of biotechnological applications. The scope of this chapter is to give a brief overview on various aspects of magnetite, especially its biologically-mediated formation, and its applications. What is Magnetite?

Iron (Fe), the first transition metal in group VIIIA of the periodic table, is one of the most abundant elements on earth and is a major part (~4.7%) of the earth’s crust. Most iron-containing minerals which form anaerobically contain iron in the Fe2+ oxidation state (“ferro”). However, upon hydrolysis oxygen rapidly oxidizes the iron from its Fe2+ state to Fe3+ (“ferric”) [3; 10]. Pure carbon-free metallic iron (the enantiotropic α-iron from) is ferromagnetic, however, above 768oC it becomes paramagnetic and above 907oC it converts to the enantiotropic γ-iron form [10; 11]. Carbon containing metallic iron can also be made permanently magnetic, similar to steel. This form of magnetic iron is different from magnetite, the focus of this chapter, and contains iron in both of its oxidation states, i.e., Fe2+ and Fe3+ (which will from here be referred to as Fe(II) and Fe(III), respectively). Iron can form a variety of oxide and oxyhydroxide phases leading to varying, highly abundant mineral formations in terrestrial, aerobic habitats [12]. These iron oxides exist in the form of minerals, some of the more common are: ferrihydrite, lepidocrocite, maghemite, magnetite, hematite, and goethite [3; 13; 14; 15]. Although iron can also exist in an amorphous iron oxyhydride form without a defined stoichiometry of Fe(II)/Fe(III). Magnetite is composed of Fe(II) and Fe(III) oxide typically denoted by ‘Fe3O4’ but more appropriately expressed by its full notation ‘Fe(II)Fe(III)2O4’. Magnetite is one of the most commonly occurring, ferromagnetic minerals on Earth. It is a member of the class of minerals known as the ‘spinels’. These minerals have the general formulation ‘A2+B2

3+O42-‘, A and B can be divalent, trivalent, or quadrivalent cations, including

magnesium, zinc, iron, manganese, aluminum, chromium, titanium, and silicon. The anion is normally oxide, and A and B can also be the same metal under different charges, such as the case of magnetite ‘Fe(II)Fe(III)2O4’ (described above). Spinels form in the isometric/cubic crystal system [16]. Magnetite is typically found in natural terrestrial environments ranging from igneous and metamorphic rocks to all varieties of sedimentary environments [17]. Magnetite is also found intracellularly in multiple lineages of life: the Bacteria, the Protozoa, and the Animals [18; 19; 20; 21]. Additionally, magnetite has also been found in materials believed to be of extraterrestrial origin, e.g., the Martian meteorite ALH84001 [17].

Figure 1. A Model for the Proposed Evolution of Magnetite Biomineralization. Geologic time is shown along the vertical axis in billions of years along with some of the major events in history that may have influenced magnetite biomineralization. These include (a) the origin of the Earth at ~ 4.5 billion years ago (bya); (b) the oldest sedimentary rocks from the Isua complex of Greenland at 3.8 bya; (c) the major deposition of the banded iron formations at 2.5-2.0 bya, which presumably corresponds to the time in which the bulk of the deep oceanic water mass changed from anaerobic to more aerobic conditions; (d) the endosymbiotic origin of eukaryotic cells at about 1.6 bya; (e) the diversification of metazoan phyla at ~0.9 bya; and (f) the widespread appearance of diverse biomineralization at the Precambrian-Cambrian boundary at ~0.6 bya (see Chang and Kirschvink [22]). Structure of Magnetite Magnetite is the most strongly magnetic mineral occurring in nature. Small particles of magnetite in single-domain (SD) or pseudo-SD magnetization states (sizes in the range of 30–70 nm and 70 nm to 20 µm, respectively) are the dominant carriers of magnetization in sediments and rocks. Larger, multidomain particles are less likely to maintain strong and stable natural magnetization over geological time as compared to SD particles [23]. The structure of magnetite is often revealed using X-ray scattering and/or electron diffraction studies [24; 25; 26]. However electron diffraction studies alone are not

accurate enough to unambiguously identify a mineral of interest, for example magnetite and maghemite only present slight differences in their electron diffraction parameters. For the differentiation of magnetite/maghemite for example, 57Fe Mössbauer spectroscopy would be used [27]. Another technique that can convincingly differentiate minerals such as magnetite from maghemite without requiring large amounts of the mineral of interest is electron energy-loss spectroscopy [28]. This technique is able to unravel the oxidation state of iron in a sample analyzed by transmission electron microscopy [28]. Origins of Magnetite Biotic Magnetite Production Magnetite biomineralization is known to occur in multiple lineages of organisms: the Bacteria, the Archaea, the Protozoa, and the Animals (Fig. 1; [18; 19; 20; 21; 22]). Some of the identified functions of biogenic, intracellular magnetite and its mineral precursors include the hardening of chiton (marine mollusk) teeth [29], geomagnetic navigation in bacteria [30; 31], and iron storage in a number of organisms, e.g., fungi, plants, humans, etc. [18; 32; 33; 34; 35]. Prokaryotes (both archaea and bacteria) biomineralize iron oxides, including magnetite, through two methods that differ mechanistically: biologically-induced mineralization (BIM) and biologically-controlled mineralization (BCM). Magnetite nanocrystals produced by BIM are 1) known to be synthesized by dissimilatory iron-reducing microorganisms (DIRM), 2) are deposited external to the cell, and 3) are generally physically indistinguishable from magnetite particles formed inorganically [24; 36; 37]. In the past, BCM has been referred to as “organic matrix-mediated mineralization” [32], and “boundary-organized biomineralization” -- implying membranes play an important role in the biomineralization process [38]. BCM magnetite is synthesized by the magnetotactic bacteria and is precipitated intracellularly as membrane-bound structures called ‘magnetosomes’. Magnetosome magnetite appears to have a crystal morphology and narrow size range unique to the bacterium producing it [26; 39; 40]. The magnetotactic bacteria will be discussed after the description of DIRM. Dissimilatory Iron-Reducing Microorganisms. Dissimilatory iron reduction is the use of Fe(III) as the terminal electron acceptor in electron transport and may be the most important chemical change that takes place in anaerobic soils and sediments [3; 41; 42]. Facultatively and strictly anaerobic microorganisms respire using Fe(III) as terminal electron acceptor, producing Fe(II), under anaerobic conditions. Until relatively recently, the reduction of Fe(III) to Fe(II) had been regarded as a primarily abiotic (chemical) process [43]. A perspective echoed by the belief that the Banded Iron Formations were of abiotic origin, however this notion has been modified more recently (see “Banded Iron Formations” below).

When iron is reduced by DIRM, the typical transformation is from an insoluble oxide/hydroxide form to soluble divalent cations. Poorly crystalline Fe(III) oxides appear to be the primary source of Fe(III) in the Fe(III) reduction zone of soils and sediments. Either amorphous Fe(OH)3 or an ill-defined Fe(III) oxide with a stable intermediate

between amorphous Fe(OH)3 and α-FeOOH (goethite) is likely the Fe(III) form that is reduced in these environments [44]. Secondary minerals, depending on the component of the medium, can then form. These secondary minerals include sulfides, carbonates, phosphates, and oxides [45; 46]. In the presence of 1) Fe(II) ions formed by microbial reduction and 2) Fe(III) ions as a substrate, magnetite will form chemically, as long as the pH is not far below pH 5.0. Thus the ”biological” formation of magnetite is actually a mixed reaction, i.e., a biological reaction (reduction of Fe(III) ions to Fe(II) ions) followed by a chemical reaction between these two oxidation states of iron ions [47]. Until a more recent report by Glasauer et al. [48] showing the intracellular accumulation of iron oxides after reduction by a bacterium, secondary minerals were thought to always accumulate on the exterior of cells, except for the magnetotactic bacteria (see “Magnetotactic Organisms”). It is interesting to note the microbially-mediated reduction of metals is a phenomenon that was first explored decades ago [49; 50; 51; 52; 53]. However, it had been widely assumed the formation of many iron minerals/compounds was the result of abiotic (chemical) processes. A good example of this assertion is the Precambrian era Banded Iron Formations (BIF), alternating layers of iron-rich and amorphous silica-rich layers which contain hematite [Fe(III)2O3] and magnetite as the dominant minerals in the iron-rich layers [54; 55]. Today it is clear that only the high temperature BIFs are the result of abiotic iron precipitation, and that DIRM played a greater than expected role in the co-precipitation of secondary iron minerals in the low temperature BIFs [5; 6; 54]. Presently DIRM have been isolated from a relatively wide number of unique and ubiquitous habitats (Table 1), including mesophilic and thermophilic (with isolates able to grow above the boiling point of water [56; 57; 58]); anaerobic and aerobic soils and sediments, soda lakes, thermal springs, etc. [2; 3; 59; 60; 61; 62; 63; 64; 65]. Magnetite is produced by a number of iron-reducing microorganisms, including: several species of Shewanella [64; 66; 67; 68] and Geobacter [37; 66; 69; 70], Ferribacterium limneticum [61], Geoalkalibacter ferrihydriticus [59], Alkaliphilus metalliredigens [60], and Thermolithobacter ferrireducens [2]. Unlike the BCM magnetite produced by the magnetotactic bacteria, there does not appear to be any functional use that can be ascribed to BIM particles.

Of particular interest are thermophilic, iron-reducing, chemolithoautotrophs, i.e., microorganisms capable of growth utilizing only CO2, H2, and amorphous iron oxides alone [2; 36; 71; 72; 73]. These DIRM are likely to have been involved in the precipitation of the Precambrian era, low temperature BIF [54; 55]. Thermophilic, autotrophic, DIRM may also be much more prevalent than currently reported, and may exist in biosphere pockets deep within the Earth (and possibly other planets, see “Possible Extraterrestrial Origins? below”) [4; 74]. A number of groups [71; 72; 73; 75; 76; 77] have focused on the isolation and characterization of these chemolithotrophic metal-reducing (specifically iron-reducing) strains from deep subsurface environments over the past decade. From these studies and others, a number of novel microorganisms have been isolated, characterized, and studied.

Table 1. Dissimilatory Iron-Reducing Microorganims which Biomineralize Magnetite. *Several species, and strains of species, of Shewanella and Geobacter are known to produce magnetite. NR = Not reported.

Magnetotactic Organisms. Magnetotactic bacteria synthesize intracellular magnetic particles composed of iron oxides and/or iron sulfides, the most common examples include magnetite (Fe3O4) and greigite (Fe3S4), respectively [1; 78]. The nano-sized particles are actually highly specialized, membrane-enveloped organelles [79; 80]. Unlike the magnetite nanoparticles synthesized by the dissimilatory iron-reducing microorganisms through BIM, the magnetite produced by the magnetotactic bacteria is biomineralized intracellularly in vesicles which originate from invaginations of the cell membrane [81; 82]. After the first report of magnetotactic bacteria by [30], X-ray and electron diffraction, 57Fe Mössbauer spectroscopy, and direct lattice imaging studies showed that the intracellular iron particles of these bacteria are invariably made of magnetic iron-containing particles, i.e., magnetite, despite a wide range of biologically unique crystal morphologies and sizes [1; 25; 26]. These magnetic organelles, or magnetosomes as they are often referred, are aligned in chains within a bacterium and are postulated to function as biological compasses that enable the bacterium to migrate along oxygen gradients in aquatic environments under the influence of the Earth’s magnetic field [1; 26; 30; 80; 83]. Not surprisingly, the features of BCM magnetite differ greatly from those produced by the DIRM. BCM magnetite has both high chemical purity

and structural perfection coupled with magnetic properties that differ from those produced through BIM [84; 85; 86; 87; 88]. It is interesting to note that in sediment obtained from the Southern Hemisphere, the majority of the magnetotactic bacteria swim to the South Pole [89]. Whereas in samples obtained from the Northern Hemisphere, they swim in the direction of the North Pole [30; 31]. However, exceptions to this North Pole vs. South Pole swimming bias are known (for an example see [90]). The synthesis of magnetosomes is interesting in and of itself alone as these magnetotactic bacteria must have evolved the appropriate mechanisms to achieve a high degree of control over the biomineralization of perfectly shaped and sized magnetic crystals [26; 40]. The assembly of these crystals into highly ordered chain structures only adds to the complexity of producing these orienting compasses (Fig. 2; [26]). Yet each assembly of magnetosomes appears to be unique for any given magnetotactic bacterium – possibly to best serve the bacterium in its ecological niche [26; 39]. In the bacteria, three distinctive particle morphologies have been reported: sub-rounded cubes and rectangles [80]; hexagonal prisms with flat ends [26]; and "teardrop," "bullet," or irregular shapes [31]. However, it is clear to most efficiently function as magnetic sensors, the magnetosomes must be arranged within the cell to maximize its magnetic dipole. The commonly seen chain arrangement of the magnetosomes best meets this magnetic dipole maximization criterium (though exceptions exist [91]). The unique characteristics of magnetosomes and the biomineralization process involved in their production have attracted a broad interdisciplinary interest. Magnetosomes can easily disperse in aqueous solutions because they are enveloped by an organic membrane, consists mainly of phospholipids and proteins [79; 92], and an individual magnetosome contains a single magnetic domain or magnetite with superior magnetic properties [85]. The biomineralization of magnetosomes and their assembly into chains is also of great interest for the generation of bioinspired magnetic materials and has even been suggested as a biomarker to detect extraterrestrial life [93]. Because of these unique properties, magnetosomes might someday be exploited for a variety of applications in diverse disciplines: microbiology, cell biology, and geobiology to biotechnology [94]. Abiotic Magnetite Production Numerous chemical methods can be used to synthesize magnetic nanoparticles such as magnetite. These methods include: microemulsions, sol-gel syntheses, sonochemical reactions, hydrothermal reactions, hydrolysis and thermolysis of precursors, flow injection syntheses, and electrospray syntheses [8]. A very common method for the production of magnetite nanoparticles is the simple chemical coprecipitation of iron salts. It can also be formed as a secondary mineral phase by thermal decomposition of an Fe-bearing carbonate and recrystallization of the ferrihydrite, green rust and/or ferric oxyhydroxides. But a number of techniques are known to produce magnetite as either a primary or secondary mineral [40]. Some of the best-developed techniques for the synthesis of inorganic magnetite involve its synthesis as a primary mineral phase. The precipitation of magnetite from a bulk

solution is highly effective and large quantities of the mineral can be produced. A number of different methods accomplish the precipitation of magnetite from bulk solutions. These techniques differ mainly in how the Fe(II) is introduced into the solution. But all of these methods are dependent on controlling the pH and alkalinity/concentration of CO2, i.e., conditions required for the stability of magnetite, in the solution [8].

Figure 2. Electron micrographs of various magnetotactic bacteria and their magnetosome chains. The images represent some of the diversity seen in the cell morphology of the magnetosomes, and of the arrangement of the magnetosomes in the bacteria: (a) spirillum with a single chain of cubooctahedral magnetosomes, (b) coccus with two double chains of slightly elongated prismatic magnetosomes, (c) coccus with clustered, elongated magnetosomes, (d) vibrio with elongated prismatic or cubooctahedral magnetosomes arranged in a single chain, (e) vibrio with two chains, and (f) rod-shaped bacterium with bullet-shaped magnetosomes arranged in a multitude of chains. Scale bars in (a-f) represent 1 μm. Part g shows a chain from a similar type of MTB as in part a, and part h shows one from a bacterium as in part b. Scale bars in parts g-h represent 100 nm. Images are reprinted from Faivre and Schuler [26] with permission from the American Chemical Society. For bulk solution “co-precipitation”, Fe(II) and Fe(III) mixtures are introduced in a starting solution under anaerobic conditions. In order to maintain the appropriate alkaline pH for the stability of the magnetite, NH3 [95; 96; 97], NaOH [95; 98; 99], NH4OH [100], or N(CH3)4OH [95] are employed. Interestingly when employing this

technique it is not uncommon for magnetite crystals of several different morphologies (dependent on the exact conditions employed) to form; including cubic, rounded, octahedral and/or irregular [95]. When more uniform crystals of magnetite are required, modifications of the bulk “co-precipitation” technique can be applied. These modifications rely upon limiting the space available for crystal growth by performing the precipitation of magnetite in microemulsions, vesicles, polymer solutions, or gels [101; 102; 103; 104].

Other methods for the inorganic precipitation of magnetite as a primary mineral are: “reduction-precipitation”, the use of constant voltage (the “electrochemical” method), and high temperature precipitation. The “reduction-precipitation” technique involves the precipitation of magnetite by the addition of iron as an Fe(III) solution, i.e., FeCl3. The ferric ions are reduced to ferrous ions, e.g., by Na2SO3, followed by an increase in pH under strictly anaerobic conditions [105; 106; 107], or a constant voltage can applied to reduce Fe(III) ions and to obtain the necessary conditions to produce magnetite [108]. Finally, magnetite can be precipitated at 90ºC using an Fe(II) solution (such as FeSO4) and the addition of KNO3 or N2H4 [106; 109].

Magnetite formation as a secondary mineral phase, as might be assumed, involves the transformation of iron-bearing, primary mineral phases to magnetite at high or low temperatures. Low temperature (20- 30°C) magnetite forms by the transformation of ferrihydrite and/or green rust [66]. Magnetite formation through reactions between soluble iron hydroxides and amorphous iron oxyhyroxide has also been reported [110]. In addition, the thermal decomposition of siderite or other Fe-rich carbonate phases at highly elevated temperatures (>400°C) has been shown to result in the formation of magnetite [111; 112; 113]. This latter mechanism of magnetite formation has been invoked in connection with the controversy over the origin of magnetite crystals contained within the Martian meteorite ALH84001 [93].

Possible Extraterrestrial Origins? The reported similarity of the nanometer-sized magnetite crystals of the Martian meteorite ALH84001 [93; 114; 115; 116] to the biogenic magnetite incorporated into the magnetosomes of magnetotactic bacteria has raised significant scientific debate. At the center of this debate is whether 1) these Martian meteorite magnetite crystals are evidence for the presence of life on ancient Mars, and 2) what criteria can and will be used to distinguish between the biological and inorganic origins of magnetite crystals. The use of six criteria, first proposed by [17], are used by many (but not all) researchers for the determination of whether certain magnetite crystals possibly constitute a biomarker for life. Collectively these criteria are referred to as the magnetite assay for biogenicity (MAB) and include 1) a narrow crystal size range, 2) consistent width/length ratios, 3) chemical purity, 4) crystallographic perfection, 5) unusual crystal morphology, and 6) directional crystal elongation [17; 103; 117; 118]. Thomas-Keprta et al. [17] further suggest that these criteria comprise a rather restrictive and robust biosignature since >30% of magnetites produced by a magnetotactic bacterial strain would not display all six of these properties, i.e., would not be confirmed as biogenic precipitates. Thomas-Keprta et al. propose that up to 25% of the magnetite crystals in the globular carbonate globules of ALH84001 display all six of the properties with the remaining 75%

of the magnetites lacking sufficient characteristics to constrain their origin as either biogenic and/or inorganic [17; 119]. However, there is an abiotic hypothesis, which has been developed by a number groups, for the formation of the magnetite crystals within ALH84001 [112; 120; 121]. The abiotic hypothesis is based on the thermal decomposition of Fe-bearing carbonate to produce magnetite [112; 120; 121]. The implication is that in the case of ALH84001 such an event possibly occurred through impact shock heating. Evidence for this process comes from the observation that nano-dimensional periclase (MgO) crystals are also associated with the carbonate globules in ALH84001, particularly the Mg-rich carbonate. Both magnetite and periclase crystals are frequently associated with voids in the carbonate. This fact suggests a mineralization process in which CO2 is released. Some faceted magnetite and periclase crystals in carbonate are crystallographically oriented with respect to the carbonate crystal lattice [121; 122]. This is a powerful line of evidence that these magnetites formed in situ abiogenically. Golden et al. has demonstrated that thermal decomposition of pure siderite (FeCO3) above 450°C results in the formation of magnetite crystals with a size-range and shape similar to those that have been found in ALH84001 [112; 119]. More recently, Thomas-Keprta et al. [113] sought to resolve the debate between the abiotic, i.e., thermal decomposition, and biotic origin of the ALH84001 magnetite crystals. These authors performed a detailed characterization of the compositional and structural relationships of the carbonate disks and associated magnetite crystals of ALH84001. They then compared their observations with experimental thermal decomposition studies [123; 124] under a range of plausible geological heating scenarios. From their experiments, Thomas-Keprta et al. [113] concluded that the vast majority of the nanocrystal magnetites present in the carbonate disks could not have formed by any of the currently proposed thermal decomposition scenarios. They go on to indicate their evidence supports an alternative allochthonous origin for this magnetite unrelated to any shock or thermal processing of carbonates. Evidence contrary to the biogenic origin of the ALH84001 magnetite has also been produced that does not intrinsically invoke the abiotic, thermal decomposition origin as an alternative. Weiss et al. [125] suggested that the ALH84001 magnetite fulfills only two of the six criteria described by Thomas-Keprta et al. [17; 115]: high chemical purity and an unusually fine-grained (single-domain to superparamagnetic) size distribution/range. Weiss et al. [125] went on to indicate the magnetic measurements they made show that no more than ~10% of the magnetite in ALH84001 can be in isolated chains, one of the most distinctive properties of magnetosomes (BCM magnetite) [126]. Therefore the results of Weiss et al. [125] suggest a difficulty in definitively proving that ALH84001 magnetites are magnetosome in origin. However, even these authors confess that in terrestrial sediments more than half of the magnetosome chain structures present could have been disrupted [127]. The claim made by Thomas-Keptra et al. [17] that ~27% of the ALH84001 magnetite crystals are biogenic can not yet be ruled out. Thus, there are credible arguments and supporters for both the biogenic and abiogenic

hypotheses for the origin of the crystals of magnetite in the ALH84001 meteorite. A more definitive answer to the origin of these magnetite crystals will involve further examination of the ALH84001 meteorite and other ancient Martian meteorites as well as further study of BCM magnetite/magnetosomes. . Applications of Magnetite Magnetic iron oxide particles, such as magnetite or maghemite (γ-Fe2O3), are widely used in the development of medical and diagnostic applications such as magnetic resonance imaging [128], cell separation [129], drug delivery [130] and hyperthermia [131]. Molecular imaging is currently envisioned as one of the most promising applications for the targeted use of iron oxide nanoparticles. Different antibodies, or fragments of antibodies, are coupled to iron oxide nanoparticles and directed to several types and sub-types of receptors either in vitro or in vivo [8] . A modified cellular enzyme-linked immunosorbent assay (ELISA), or cellular magnetic-linked immunosorbent assay (C-MALISA), is an example of a recent application; having the potential for in vitro clinical diagnoses using MRI techniques [132]. An important application of magnetite nanoparticles is the functionalization of techniques for in vitro protein or cell separation. Magnetic separation techniques are relatively simple and all steps of the purification can take place within a single test tube [133; 134; 135; 136]. Magnetic drug targeting is being developed to employ nanoparticles as drug carriers. This technique is thought to be particularly promising in the area of cancer treatment, whereby the side effects of conventional chemotherapies such as radiotherapy and chemotherapy might be avoided. In these studies, magnetic nanoparticles are used as drug carriers and injected into circulating blood, i.e., the thee.,tingrs is administered. The iron oxide magnetic nanoparticles are actually covered by starch derivatives which make the ferrofluid tolerable to the body. The nanoparticles are retained in the region of the tumor by a strong inhomogeneous magnetic field [137]. In one study, the drug mitoxantrone was bound to the phosphate groups of the starch derivatives [137]. Electron microscope investigations have actually validated that so-called ‘ferrofluids’ are thereby enriched at the site of the tumor and in the tumor cells [137]. This method of drug targeting offers the opportunity to treat malignant tumours loco-regionally without systemic toxicity, and these magnetic nanoparticles have the potential for use as a carrier system for a variety of anticancer agents, e.g. radionuclides, cancer-specific antibodies and genes. Ferrofluids have additional therapeutic purposes including hyperthermia treatment. Jordan et al. [138] was the first to show experimentally the high efficiency of a superparamagnetic crystal suspension to absorb the energy of an oscillating magnetic field and convert it into heat. Tumor cells are more sensitive to an increase in temperature than healthy ones [139; 140]. So hyperthermia treatment can be employed

in vivo to increase the temperature of tumor tissue and to potentially destroy the pathological cells. Conclusion

In the last two decades, there has been an increased focus on the study of magnetite synthesis by prokaryotes, including both the dissimilatory iron-reducing microorganisms (DIRM) and the magnetotactic bacteria. The magnetite crystals biomineralized by these two classes of microorganisms, i.e., the DIRM: biologically-induced mineralization (BIM) magnetite, and the magnetotactic bacteria: biologically-controlled mineralization (BCM) magnetite, differ in a great number of ways, e.g., BCM magnetite biomineralization is tightly regulated, in contrast to BIM magnetite biomineralization, to produce crystals with a uniform crystal morphology and narrow size range. Surprisingly magnetite crystals have even been found in the higher organisms, from the Protozoa to even humans. The possible use of nanometer-sized crystals of magnetite as magnetofossils, biosignatures for the past presence of life, is intriguing and led to vigorous debates. Through all of these findings and studies, much has been learned about how organisms biomineralize magnetite and how magnetite is formed inorganically. However much remains to be discovered. Many parameters must affect the chemical composition, crystal morphology and mineral structure of magnetite. The studies of these parameters will lead to increased applications for magnetite in industry and technology, a better understanding of how, and if, magnetite nanocrystals can be studied as magnetofossils, and the full scope of iron metabolism by dissimilatory iron-reducing microorganisms in the origin of life, e.g., the use of Fe(III) as a primordial and relatively efficient external electron acceptor before the advent of the more efficient electron acceptor oxygen at sufficient concentrations.

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