To date, the subject of extracellular respiration has been most thoroughly explored through studies concerning the reduction or oxidation of iron and manganese minerals. As several reviews have been published on this topic recently (Croal et al., 2004; Tebo et al., 2005; Weber et al., 2006), here, we restrict our discussion to a few general comments.
Thanks to numerous biochemical and genetic studies over the past two decades in model organisms such as Shewanella oneidensis, Geobacter sulfurreducens, Acidithiobacillus ferroxidans, Bacillus sp. strain SG-1 and Pseudomonas putida, the general scheme for electron transfer in iron/manganese-oxidizing/reducing bacteria is now known. Diverse membrane-bound and -soluble (i.e. periplasmic) electron carriers, such as c-type cytochromes, quinones and multicopper oxidases, can play a role in these systems. Commonly, proteins involved in electron transfer to or from iron/manganese (hydr)oxides are localized to the outer membrane of Gram-negative bacteria or the exosporium of spores from Gram-positive bacteria (Myers and Myers, 1992; Francis et al., 2002; Yarzabal et al., 2002; Mehta et al., 2005; 2006). It is generally assumed that this subcellular location facilitates direct interaction with the metal substrate. In addition to redox-active proteins, extracellular polymeric substances may also affect adherence to mineral surfaces and mediate metal reactivity, as seen in acidophilic iron-oxidizing bacteria (Sand and Gherke, 2006). Biophysical experiments with S. oneidensis have shown that specific attractive forces are induced between the cell and a mineral surface during conditions in which electron transfer from the cell to the mineral is expected (Lower et al., 2001). Although these forces have not yet been shown to be due to any particular protein, recent biochemical experiments with OmcA, an outer membrane c-type cytochrome from S. oneidensis (Myers and Myers, 1998), have indicated that it can bind hematite with high affinity and transfer electrons to it directly (Xiong et al., 2006). Interestingly, however, OmcA did not bind goethite, the mineral used in the Lower et al. study. How different minerals are recognized by the cell is unknown, but may involve a number of subtle effects, as S. oneidensis appears capable of discriminating even between different single crystal faces (Neal et al., 2005). In a separate study, scanning tunnelling microscopy and tunnelling spectroscopy were used to characterize the electron tunnelling properties of OmcA and MtrC (also known as OmcB, another outermembrane c-type cytochrome) immobilized as molecular monolayers on Au(III) surfaces (Wigginton et al., 2007). In this case, modified versions of the native proteins containing a tetracysteine sequence were used, and it remains to be seen whether the conclusions from these in vitro biophysical experiments apply in vivo in the presence of iron oxides. For more details on this topic, see the review by Shi et al. (2007).
In addition to direct electron transfer to minerals, it has also been established that indirect electron transfer can occur via small molecules that act either to chelate metals and deliver them to an intracellular metal oxidoreductase, or by themselves serving as electron shuttles. A large number of research groups have made important contributions to this area; for a thorough review of this topic, we refer the reader to Kappler and Straub (2005). In the case of metal chelators, ligands such as citrate and nitro-tri-acetic acid (NTA) are able to deliver soluble iron to c-type cytochromes inside the cell, although it is clear that some metal chelates are also reduced extracellularly (Beliaev and Saffarini, 1998; Beliaev et al., 2001; Myers and Myers, 2002; Lies et al., 2005). In the latter case, a host of molecules – organic and inorganic, endogenously or exogenously produced – can shuttle electrons back and forth between cells and mineral surfaces. This might be particularly relevant in the context of biofilms, where the majority of cells are not in direct contact with the mineral surface (Hernandez and Newman, 2001). Although the oxidoreductases that act on these molecules and their respective subcellular locations have not been determined for the majority of the known electron shuttles, it was recently shown that at least some of these molecules require the same outer membrane proteins for their reduction that are required for mineral reduction, which blurs the distinction between the ‘direct’ and ‘indirect’ pathways (Lies et al., 2005).
Irrespective of the potential substrate promiscuity of the oxidoreductases involved in these processes (see below), a novel mechanism of extracellular electron transfer has been described recently in studies with G. sulfurreducens (Reguera et al., 2005) and S. oneidensis (Gorby et al., 2006) that invokes electron transfer through microbial ‘nanowires’ (Fig. 2). In the case of G. sulfurreducens, these nanowires appear to comprise a new class of pili (the so-called ‘geopilins’) that are required for Fe(III) oxide reduction. Interestingly, these pili appear to localize to one side of the cell and are induced only under certain conditions. Twitching motility does not appear to occur under these conditions; thus, Reguera et al. (2005) concluded that the geopili do not play an indirect role in Fe(III) oxide reduction by mediating surface motility. Moreover, the geopili are not required for Fe(III) oxides to attach to the cell (i.e. mutants lacking geopili can still bind mineral particles on the cell surface), yet when they are expressed they have a high affinity for Fe(III) oxides. Based on measurements of their electrical conductivity using atomic force microscopy, the geopili were shown to be electrically conductive. From this, it was suggested that geopili serve as direct electron conduits between the cell and the mineral surface. How the amino acids that comprise the pili conduct electrons is not yet understood, nor is it known with which electron-donating proteins they interact inside the cell.
Figure 2. ‘Nanowires’ produced by two different Fe(III)-reducing bacteria. A. Transmission electron micrograph of platinum-shadowed ‘geopili’ produced by Geobacter sulfurreducens (courtesy of Gemma Reguera, Michigan State University). The length of the longer cell is approximately 2 μm. The image has been colorized to enhance contrast. B. Atomic force micrograph of Shewanella oneidensis (courtesy of Pamela Gross, University of Southern California). The large appendage is the flagellum; the shorter pili are electrically conductive nanowires. The length of the cell is approximately 3 μm.
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Geobacter sulfurreducens is not alone in its ability to produce nanowires. Gorby et al. (2006) showed that S. oneidensis produces electrically conductive nanowires in response to electron acceptor limitation. In comparing the electrical conductivity of the nanowires produced by the wild type with that of mutants defective in the production of the outer membrane c-type cytochromes OmcB (also known as MtrC) and OmcA by scanning tunnelling microscopy, evidence was found that suggested the nanowires were poor electrical conductors in the absence of these cytochromes. Whether these cytochromes are physically incorporated into the nanowires themselves, or whether they facilitate electron transfer through the nanowires indirectly, is not known. An important additional finding of the Gorby et al. study was that nanowires can also be produced by other bacterial genera in addition to mineral reducers. They identified electrically conductive nanowires between cells of the photosynthetic cyanobacterium Synechocystis strain PCC 6803 when they were cultivated under conditions of CO2 limitation. In addition, electrically conductive nanowires were also produced by the thermophilic fermentative bacterium Pelotomaculum thermopropionicum when grown in monocultures on fumarate or in co-cultures with Methanothermobacter thermoautotropicus on propionate. It seems possible therefore that nanowire production might serve as a general mechanism for electron transfer both within and between species under certain conditions. Elucidation of what exactly these conditions are, how nanowire production serves the physiological needs of the cell under these conditions, and the mechanisms of their biosynthesis and cellular localization, are priorities for future work.
Electrodes are an important substrate for extracellular respiration in a more applied context. Since the 1970s, understanding how microbes catalyse the conversion of substrates directly into electricity has been relevant to optimizing the performance of MFCs (Rabaey and Verstraete, 2005). Electrodes can serve either as electron donors or as electron acceptors for microorganisms, depending on whether the electrode is functioning as a cathode or anode respectively. Many different types of microorganisms with different physiological capabilities (some fermentative, others respiratory) have been shown to catalyse electricity generation in fuel cells. These organisms have been enriched from diverse environments, ranging from organic wastewaters to aquatic sediments (Bond et al., 2002; Tender et al., 2002; Holmes et al., 2004; Kim et al., 2004; Rabaey et al., 2004; Reimers et al., 2006). The manner in which they generate electricity can vary from catalysing electron transfer directly via outer membrane proteins, to oxidizing/reducing soluble redox mediators that shuttle electrons to/from the electrode, in direct analogy to what occurs for minerals. The potential for artificial mediators to facilitate electron transfer in MFCs has been known for a long time (Roller et al., 1984; Park et al., 2000), but more recently, some MFC bacteria have been shown to produce mediators themselves (Bond and Lovley, 2005; Rabaey et al., 2005). A recent study of electricity production by G. sulfurreducens growing on graphite electrodes demonstrated that the same ‘geopilins’ that play a role in Fe(III) oxide reduction by this organism also contribute to maximum power output when G. sulfurreducens is growing as a multilayered biofilm (Reguera et al., 2006). Interestingly, if G. sulfurreducens grows on electrodes under conditions where cells do not stack on top of each other, the geopili do not appear to affect current production (Holmes et al., 2006). Although there are obvious parallels between electron transfer to/from electrodes and to/from minerals, it appears that the particular proteins needed to catalyse electron transfer reactions in these two contexts differ (Holmes et al., 2006). Moreover, in natural environments, with mixed communities colonizing deployed electrodes, a variety of mechanisms might operate simultaneously, with both organic and inorganic substrates (e.g. sulphur species in the marine environment) potentially participating in electron transfer reactions at both the anode and the cathode (Reimers et al., 2006).