The use of microorganisms for the formation of metal nanoparticles and their application
Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. The development of reliable experimental protocols for the synthesis of nanomaterials over a range of chemical compositions, sizes, and high monodispersity is one of the challenging issues in current nanotechnology. In the context of the current drive to develop green technologies in material synthesis, this aspect of nanotechnology is of considerable importance. Biological systems, masters of ambient condition chemistry, synthesize inorganic materials that are hierarchically organized from the nano- to the macroscale. Recent studies on the use of microorganisms in the synthesis of nanoparticles are a relatively new and exciting area of research with considerable potential for development. This review describes a brief overview of the current research worldwide on the use of microorganisms in the biosynthesis of metal nanoparticles and their applications.
There is an enormous interest in the synthesis of nanomaterials due to their unusual optical (Krolikowska et al. 2003), chemical (Kumar et al. 2003), photoelectrochemical (Chandrasekharan and Kamat 2000), and electronic (Peto et al. 2002) properties. Impressive advances have been observed in various aspects such as the synthesis of nanoscale matter and understanding/utilizing their exotic physicochemical and optoelectronic properties. Recent developments in the organization of nanoscale structures into predefined superstructures ensure that nanotechnology will play an increasingly crucial role in many key technologies of the new millennium. It is gaining importance in areas such as catalysis, optics, biomedical sciences, mechanics, magnetics, and energy science. The synthesis of nanomaterials over a range of chemical composition and high monodispersity is still challenging in material science. Several manufacturing techniques that usually employ atomistic, molecular, and particulate processing in a vacuum or in a liquid medium are in use (Daniel and Astruc 2004). Most of the techniques are capital intensive, as well as inefficient in materials and energy use. Hence, there is an ever-growing need to develop clean, nontoxic, and environmentally benign synthesis procedures. Consequently, researchers in nanoparticle synthesis have turned to biological systems for inspiration.
It is well known that many organisms can provide inorganic materials either intra- or extracellularly (Simkiss and Wilbur 1989; Mann 1996). For example, unicellular organisms such as magnetotactic bacteria produce magnetite nanoparticles (Lovley et al. 1987; Spring and Schleifer 1995; Dickson 1999), and diatoms synthesize siliceous materials (Mann 1993; Oliver et al. 1995; Kroger et al. 1999). Multicellular organisms produce hard inorganic–organic composite materials such as bones, shells, and spicules using inorganic materials to build a complex structure (Lowenstam 1981). These biominerals are composite materials and consist of an inorganic component and a special organic matrix (proteins, lipids, or polysaccharides) that controls the morphology of the inorganic compound. The surface layer bacteria produce gypsum and calcium carbonate layers (Pum and Sleytr 1999; Sleytr et al. 1999). Even though many biotechnological applications such as the remediation of toxic metals employ microorganisms such as bacteria (Stephen and Macnaughton 1999) and fungi (Mehra and Winge 1991), such microorganisms are recently found as possible eco-friendly nanofactories (Southam and Beveridge 1994; Beveridge and Murray 1980). Processes devised by nature for the synthesis of inorganic materials on nano- and microlength scales have contributed to the development of a relatively new and largely unexplored area of research based on the use of microbes in the biosynthesis of nanomaterials (Sastry et al. 2004).
In this article, we provide a brief overview of the current research worldwide on the use of microorganisms such as bacteria and actinomycetes (both prokaryotes), as well as algae, yeast, and fungi (eukaryotes) in the biosynthesis of metal nanoparticles and their applications.
Bacteria in nanoparticle synthesis
Among the microorganisms, prokaryotic bacteria have received the most attention in the area of biosynthesis of nanoparticles. Early studies reveal that Bacillus subtilis 168 is able to reduce Au3+ ions to produce octahedral gold particles of nanoscale dimensions (5–25 nm) within bacterial cells by incubation of the cells with gold chloride (Beveridge and Murray 1980; Southam and Beveridge 1994; Fortin and Beveridge 2000) under ambient temperature and pressure conditions. Organic phosphate compounds play a role in the in vitro development of octahedral Au (Southam and Beveridge 1996), possibly as bacteria–Au-complexing agents. Fe(III)-reducing bacteria Shewanella algae (Konishi et al. 2004) can reduce Au(III) ions in anaerobic environments. In the presence of S. algae and hydrogen gas, the Au ions are completely reduced, which results in the formation of 10- to 20-nm gold nanoparticles.
It is already established that silver is highly toxic to most microbial cells. Nonetheless, several bacterial strains are reported as silver resistant (Silver 2003) and may even accumulate silver at the cell wall to as much as 25% of the dry weight biomass, thus suggesting their use for the industrial recovery of silver from ore material. The silver-resistant bacterial strain Pseudomonas stutzeri AG259 accumulates silver nanoparticles, along with some silver sulfide, in the cell where particle size ranges from 35 to 46 nm (Slawson et al. 1992). Larger particles are formed when P. stutzeri AG259, isolated from a silver mine, is placed in a concentrated aqueous solution of silver nitrate (50 mM; Klaus et al. 1999). Nanoparticles of well-defined size, ranging from a few to 200 nm or more, and distinct morphology are deposited within the periplasmic space of the bacteria. Cell growth and metal incubation conditions may be the reasons for the formation of different particle sizes. The exact reaction mechanisms leading to the formation of silver nanoparticles by this species of silver-resistant bacteria is yet to be elucidated. The ability of microorganisms to grow in the presence of high metal concentrations might result from specific mechanisms of resistance. Such mechanisms include the following: efflux systems, alteration of solubility and toxicity by changes in the redox state of the metal ions, extracellular complexation or precipitation of metals, and the lack of specific metal transport systems (Silver 1996; Beveridge et al. 1997).
Biocomposites of nanocrystalline silver and the bacteria may be thermally treated to yield a carboneous (cermet) nanomaterial with interesting optical properties for potential application in functional thin-film coating (Joerger et al. 2000; Klaus-Joerger et al. 2001). These properties can be modified by varying the silver loading factor. This type of carboneous material is composed primarily of graphite carbon and up to 5% by weight (of the dry biomass) of silver.
Bacteria not normally exposed to large concentrations of metal ions may also be used to grow nanoparticles. The exposure of Lactobacillus strains (Nair and Pradeep 2002), which are present in buttermilk, to silver and gold ions resulted to the large-scale production of metal nanoparticles within the bacterial cells. Moreover, the exposure of lactic acid bacteria present in the whey of buttermilk to mixtures of gold and silver ions can be used to grow alloy nanoparticles of gold and silver.
In addition to gold and silver nanoparticles, there is much attention in the development of protocols for the synthesis of semiconductors (the so-called quantum dots) such as CdS, ZnS, and PbS. These luminescent quantum dots are emerging as a new class of materials for biological detection and cell imaging (Chan et al. 2002), based on the conjugation of semiconducting quantum dots and biorecognition molecules. Clostridium thermoaceticum (Cunningham and Lundie 1993) precipitates CdS at the cell surface as well as in the medium from CdCl2 in the presence of cysteine hydrochloride in the growth medium. Most probably, cysteine acts as the source of sulfide. When Klebsiella aerogenes is exposed to Cd2+ ions in the growth medium, 20- to 200-nm CdS formed on the cell surface. The formation of CdS is confirmed by quantitative energy dispersive x-ray analysis. The buffer composition of the growth medium plays an important role in forming the cadmium sulfide crystallites. Intracellular CdS nanocrystals, composed of a wurtzite crystal phase, are formed when E. coli is incubated with cadmium chloride and sodium sulfide (Sweeney et al. 2004). Nanocrystal formation varies dramatically depending on the growth phase of the cells and increases about 20-fold in E. coli grown in the stationary phase as compared with that grown in the late logarithmic phase. In a remarkable investigation, Labrenz et al. (2000) has shown that spherical aggregates of 2- to 5-nm-diameter sphalerite (ZnS) particles are formed within natural biofilms dominated by sulfate-reducing bacteria of the family Desulfobacteriaceae. A combination of geochemical and microbial processes leads to ZnS biomineralization in a complex natural system. It is appropriate to mention that the concentration of Zn can be significantly reduced to below-acceptable levels for drinking water with the use of this method.
Magnetic Fe sulfide nanoparticles are synthesized by using sulfate-reducing bacteria where particles having a size of a few nanometers are formed on the surface (Watson et al. 1999), and the magnetic mineral is separated from the solution by a high-gradient magnetic field of 1 T. Bacterially produced iron sulfide is an adsorbent for a wide range of heavy metals and some anions. High adsorption of radioactive ions occurs due to high surface area (400–500 m2/g). It may provide a suitable matrix for the long-term safe storage of a number of ions vital to the nuclear industry, particularly the pertechnetate ion (TcO4−; Watson et al. 2001).
All magnetotactic bacteria contain magnetosomes, which are intracellular structures comprising magnetic iron mineral crystals enveloped by a membrane vesicle. The magnetosome membrane (MM) is most likely a structural entity that anchors the crystal at particular locations in the cell, as well as the locus of biological control over the nucleation and growth of the magnetosome crystals. Knowledge of biochemical and genetic controls on magnetic production is essential to understanding how the magnetotactic bacteria produce magnetosomes and organize them in chains. The biomineralization of magnetosome particles is achieved by a complex mechanism that involves the uptake and accumulation of iron and the deposition of the mineral particle with a specific size and morphology within a specific section provided by the MM (Schuler 1999). Since the MM is thought to be of paramount importance in magnetosome formation, researchers have focused on the role of MM proteins, which occur in the MM but not in the soluble (periplasmic or cytoplasmic) fraction or in the cytoplasmic or outer membranes, in magnetosome synthesis. The mam (MM) genes (Grunberg et al. 2001) appear to be conserved in a large gene cluster within several magnetotactic bacteria (Magnetospirillium species and strain MC-1) and may be involved in magnetic biomineralization.
Synthesis of nanoparticles by different microorganisms
Type of nanoparticle
Gold, silver, Au–Ag alloy
Gold, silver, Au–Ag alloy, cadmium sulfide, zirconia
Yeast in nanoparticle synthesis
It has long been recognized that among the eukaryotes, yeasts are explored mostly in the biosynthesis of the semiconductor nanoparticles. Exposure of Candida glabrata (Reese and Winge 1988; Dameron et al. 1989) to Cd2+ ions leads to the intracellular formation of CdS quantum dots. The synthesis of PC is activated in the presence of Cd. The structure of PC involves a repeating sequence of γ-glutamyl–cysteine pairs to give polypeptides the general formula (γ-Glu-Cys)n Gly, with n values commonly ranging from 2 to 6. They bind Cd ions immediately, forming Cd–PC complexes that are transported into the vacuole. Then, the complex is degraded and the nanoparticles are formed. Torulopsis sp., which was found in an extensive screening program (Kowshik et al. 2002a,b), is capable of synthesizing PbS nanocrystals intracellularly when challenged with Pb2+. Crystallites, which are extracted from the biomass by freeze thawing, exhibit a sharp absorption maximum at ∼330 nm and are 2–5 nm in size.
Intensive research activity is currently in progress to fabricate electronic devices using organic materials due to their flexibility, easy processing, and large quantum efficiency for light emission. To improve their stability, efficiency, and color tunability for diverse applications, composites of organic materials with nanoparticles, porous silicon, etc., have been explored. So far, chemically synthesized nanoparticles have been used for device applications, but biogenic nanoparticles are yet to be explored for the same applications. Addressing this important issue, Kowshik et al. (2002a,b) have shown that CdS quantum dots synthesized intracellularly in Schizosaccharomyces pombe yeast cells exhibit ideal diode characteristics. Biogenic CdS nanoparticles in the size range 1–1.5 nm have been used in the fabrication of a heterojunction with poly(p-phenylenevinylene). Such a diode exhibits an approximately 75-mA/cm2 current in the forward bias mode at 10 V, while breakdown occurred at ∼15 V in the reverse direction.
Though yeast has been used to synthesize intracellular nanoparticles for several years, very recently, silver nanoparticles have been synthesized extracellularly by a silver-tolerant yeast strain, MKY3 (Kowshik et al. 2003). Particles with a 2- to 5-nm size range are formed when challenged with Ag+ ions in the log phase of growth.
Fungi in nanoparticle synthesis
The use of fungi in the synthesis of nanoparticles is a relatively recent addition to the list of microorganisms. The use of fungi is potentially exciting since they secrete large amounts of enzymes and are simpler to deal with in the laboratory. However, the genetic manipulation of eukaryotic organisms as a means of overexpressing specific enzymes identified in nanomaterial synthesis would be much more difficult than that in prokaryotes.
The exposure of Verticillium sp. to silver ions resulted to a similar intracellular growth of silver nanoparticles (Mukherjee et al. 2001b). The exact mechanism leading to the intracellular formation of gold and silver nanoparticles by Verticillium is not fully understood at the moment. Since the nanoparticles are formed on the surface of the mycelia and not in the solution, it is thought that the first step involves the trapping of the metal ions on the surface of the fungal cells possibly via electrostatic interaction between the ions and the negatively charged carboxylate groups in the enzymes present in the cell wall of the mycelia. Thereafter the ions are reduced by enzymes present in the cell wall leading to the formation of the nuclei, which subsequently grow through the further reduction of metal ions and accumulation of these nuclei. The ability of Verticillium cells to multiply after exposing to metal ions proves the capability of using microorganisms in the synthesis of nanomaterials (Mukherjee et al. 2001b).
Summary and outlook
In summary, a brief overview of the use of microorganisms such as bacteria, yeasts, algae, fungi, and actinomycetes in the biosynthesis of metal nanoparticles has been depicted. This is an interdisciplinary field “bionanotechnology” that requires the collaboration between physicists, chemists, biologists, and engineers. A case for the serious investigation of microorganisms has been made as a possible alternative to the more popular physical and chemical methods that are currently prevalent. A number of issues need to be addressed from the nanotechnology and microbiology points of view before such biosynthetic procedures can compete with the traditional protocols. The elucidation of biochemical pathways leading to metal ion reduction in the different classes of microbes is necessary to develop a rational microbial nanoparticle synthesis procedure. The surface chemistry of biogenic nanoparticles should be properly recognized. Genetic engineering techniques can potentially be used to improve the particle properties and to control their composition. The shift from bacteria to fungi as a means of developing natural “nanofactories” has the added advantage that downstream processing and handling of the biomass would be much simpler. At present, microbial methods in the synthesis of nanomaterials of varying composition are extreme limited and confined to metal, some metal sulfide, and very few oxides. An extension of the procedures to enable reliable synthesis of nanocrystals of other oxides (TiO2, ZrO2, etc.), nitrides, carbides, etc., could make microbial synthesis a commercially feasible proposition.
We would like to thank S. Senapati, Dr. A. Ahmad, Dr. M.I. Khan, Dr. M. Sastry, and Dr. R. Kumar for their enthusiastic contributions to the experimental work and for their helpful discussions. We also thank Prof. A. Steinbuchel for supporting this review.
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