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Electrically conductive pili: Biological function and potential applications in electronics
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Review Article Electrically conductive pili: Biological function and potential applications in electronics Derek R. Lovley Electrically conductive pili (e-pili) enable long-range electron exchange between microorganisms and their extracellular environment and show promise as a ‘green’ electronic material. Most studies have focused on the e-pili of G. sulfurreducens, which play a role in Fe(III) oxide reduction and interspecies electron transfer, and confer conductivity to current-producing biofilms. However, a diversity of other microorganisms have recently been found to possess e-pili, including e-pili with unique structures. Aromatic amino acids are key elements in electron transport along the length of e-pili and the conductivity of synthetic e-pili can be tuned by manipulating aromatic content. e-Pili are attractive as an electronic material because they can be produced from renewable feedstocks and are biodegradable, yet they are also highly robust for device fabrication. Basic information on the structure of e-pili, mechanisms for electron transport, and other electronic properties is required in order to better understand their biological function and to guide the design of synthetic e-pili for applications as electronic components. Address Department of Microbiology, University of Massachusetts, Amherst, MA, USA Corresponding author: Lovley, Derek R. ([email protected])
Current Opinion in Electrochemistry 2017, XX:XX–XX This review comes from a themed issue on Physical & NanoElectrochemsitry 2017 Edited by Feng Zhao and Jens Ulstrup For a complete overview see the Issue and the Editorial Available online XX XXXX 2017 http://dx.doi.org/10.1016/j.coelec.2017.08.015 2451-9103/© 2017 Elsevier B.V. All rights reserved.
Introduction Electrically conductive pili (e-pili) enable electron transport over unprecedented distances for a biological protein and confer unique properties to microorganisms of biogeochemical and practical significance [1]. Furthermore, e-pili are a sustainable ‘green’ electronic material with diverse potential applications [2]. Following the initial discovery of e-pili in Geobacter sulfurreducens [3], highly divergent hypotheses were put forth on e-pili function www.sciencedirect.com
within microbial communities and the mechanisms of electron transport along e-pili [1]. However, recent experimental evidence has culled some of these early concepts and has provided a foundation for further hypothesis development. The purpose of this review is to summarize these recent studies, focusing on publications within the last two years, with the exception that key earlier papers are mentioned when required for context. Current concepts on mechanisms for electron transfer along e-pili are discussed, as well as the possible biological functions of e-pili, and emerging technologies based on e-pili (Figure 1).
What is an e-pilus and who has them? An e-pilus is any pilus that is sufficiently electrically conductive along its length to promote long-range electron exchange between the microbe expressing e-pili and the external environment. Conductivity data for e-pili under physiologically relevant conditions is limited (Box 1). The specific threshold conductivity required to satisfy the definition of an e-pilus is dependent on its function. Rough guidelines for the e-pili conductivity necessary for electron transport to extracellular electron acceptors, such as Fe(III) oxides, other cells, and electrodes, are available from studies with G. sulfurreducens. The native e-pili with a conductivity at pH 7 of 51 mS/cm [4● ] effectively supported growth under conditions that required long-range electron transport, consistent with theoretical calculations [5]. Heterologous expression of pili with higher conductivity (277 S/cm at pH 7) did not further improve growth dependent on extracellular electron transfer [6]. Pili with conductivities of 30–40 μS/cm [4● ,5] were not sufficiently conductive to support extracellular electron transfer [5,7]. However, it is conceivable that there may be other, as yet unverified, functions of e-pili, such as electrical communication to the cell that a pilus has contacted a specific surface. Conductivities in the μS/cm range may be adequate for these potential alternative functions. The e-pili of G. sulfurreducens are homologous to the type IV pili that are common in bacteria and archaea [8●● ,9]. The pilin genes of a number of Geobacter species and closely related microorganisms are highly similar to the pilin gene of G. sulfurreducens [10,11]. Similar pilin monomer genes are also found in a few phylogenetically diverse microorganisms [10]. Several of these genes have yielded e-pili when expressed in G. sulfurreducens [6,12●● ]. Some pilin monomers not closely related to the G. sulfurreducens pilin can also assemble into e-pili, Current Opinion in Electrochemistry 2017, 000:1–9
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Figure 1
Examples of biological function and materials application of electrically conductive pili.
demonstrating the possibility of broad distribution of e-pili in the microbial world [12●● ]. Furthermore, there is a growing list of microorganisms with other pilus-like filaments that are electrically conductive, at least in vitro [13●● ,14]. A wide diversity of microorganisms are known to be electroactive and many more are likely to be discovered [15]. Like much of the microbial world, many of these microorganisms may be difficult to culture, especially in sufficient quantities to study the properties of their pili. Heterologous expression in G. sulfurreducens of pilin monomer genes (Figure 2) from the (meta)genomes of difficult-toculture microbes is an option to rapidly screen whether pilin genes have the potential to yield e-pili [12●● ]. Determining the composition of conductive filaments is key. For example, it was initially thought that Shewanella oneidensis possessed conductive pili, but subsequent investigation demonstrated that the conductive filaments were artifacts that form when membrane extensions are dried and chemically fixed [16,17]. Cryoelectron microscopy revealed that the cytochrome-based electron transport along these filaments that was previCurrent Opinion in Electrochemistry 2017, 000:1–9
ously proposed [16] is unlikely in vivo because the cytochromes within the membrane extensions are spaced to far apart for cytochrome-to-cytochrome electron transfer [18●● ]. It is also important to document the conductivity of any filaments that are proposed to be conductive. For example, it was speculated that the pili of a sulfate-reducing microorganism were the conduit for interspecies electron transfer during syntrophic anaerobic methane oxidation [19]. However, when the pilin gene from the sulfate reducer was expressed in G. sulfurreducens the resultant pili were poorly conductive, suggesting that there must be another route for the interspecies electron exchange [12●● ].
Mechanisms for electron transport along e-pili Electron transport mechanisms for e-pili have primarily been investigated with e-pili from G. sulfurreducens and related proteins. Individual e-pili that are free of other proteins or metals have substantial conductivities (50 mS/cm–1 kS/cm) [4● –6,20,21●● ]. Propagation of charge along cytochrome-free regions of G. sulfurreducens e-pili was also documented with electrostatic force microscopy www.sciencedirect.com
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Filament type
Solvents
Fixation
Dried (D)/ Hydrated (H)
Two Probe or Four Probe
Conductivity
Reference
Rhodopseudomonas palustris G. sulfurreducens e-pili
+ −
+ −
D H
Two-probe Both
[14] [4]
G. sulfurreducens e-pili Synthetic e-pili in G. sulfurreducens
+ −
− −
D H
Two-probe Two-probe
G. metallireducens pilin in G. sulfurreducens
−
−
H
Two-probe
35–72 µS/cm 51 mS/cm (pH 7) 187 mS/cm (pH 2) 40 µS/cm (pH 10.5) 1.4 - 4.3 S/cm 10 S/cm (pH 7) 393 S/cm (pH 2) 300 mS/cm (pH 10.5) 277 S/cm (pH 7)
1
For a comprehensive review of additional filaments in which conductivity has been measured across the filament width, typically after chemical fixation, see reference (13).
[20] [21]
[6]
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Examples of types of conductivity measurements along the length of e-Pili and other conductive filaments.1
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Taking Measure of e-Pili There has been little consistency between laboratories in the methods by which the conductivity of e-pili and other microbial filaments have been evaluated. In many studies the filaments have been treated rather harshly for a biological protein, with extraction in solvents and/or fixation with glutaraldehyde, which cross-links peptides. These treatments could significantly change the filament structure from that found in vivo. Measuring conductivity across the diameter of filaments with the assumption that this reflects corresponding conduction along the length of the filament is common. Another strategy is to measure the conductivity of e-pili networks. Network conductivity can provide evidence for long-range electron transport through e-pili, but the conductivities of e-pili networks are much lower than the conductivities of individual e-pili, presumably due to resistances in pili-to-pili electron transfer. Conductivity along the length of e-pili has been measured with two-probe and four-probe methods. Although more technically difficult, four-probe measurements are preferable because they eliminate errors in conductivity estimates due to contact resistance between the e-pili and the electrodes. Very few measurements along the length of individual e-pili at physiologically relevant conditions (hydrated; no chemical fixation; pH 7) are available due to the technical difficulty of such measurements. Of course when e-pili are being processed as an electronic material, the relevant concern is the conductivity and other properties of the e-pili under the processing conditions required for material/device fabrication and concerns about biological relevance are eliminated.
Current Opinion in Electrochemistry 2017, 000:1–9
Please cite this article as: Lovley, Electrically conductive pili: Biological function and potential applications in electronics, Current Opinion in Electrochemistry (2017), http://dx.doi.org/10.1016/j.coelec.2017.08.015
Box 1
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Figure 2
Method for a rapid screen of pilin genes with heterologous expression in Geobacter sulfurreducens to determine if the pilins have the potential to assemble into electrically conductive pili.
[22]. These direct experimental measurements negate multiple previous theoretical models which suggested that cytochromes were essential for e-pili conductivity (for a detailed review see Refs. [1,17]). Aromatic amino acids are important for e-pili conductivity. Substituting alanine for key aromatic amino acids in e-pili reduces conductivity [4● ,7,20]. Introducing more rings by substituting a tryptophan for the carboxyl terminal phenylalanine and tyrosine in the G. sulfurreducens pilin monomer increased e-pili conductivity [21●● ], as did expressing the more aromatic-rich pilin monomer of G. metallireducens [6]. The conductivity of pili that assemble from larger pilins is also dependent on aromatic amino acid content [12●● ]. One potential explanation for these observations is that overlapping π -π orbitals of aromatic amino acids confer a metallic-like conductivity, similar to that in synthetic organic conducting polymers [1,17]. Data supporting this hypothesis for G. sulfurreducens e-pili includes structural evidence for π -π stacking and a correlation between the degree of π -π stacking and e-pili conductivity [4● ,22–24]. Conductivity response to changes in temperature and pH are consistent with metallic-like conductivity [4● ,23], as is the charge propagation along e-pili observed with electrostatic force microscopy [22]. An alternative model is that the aromatic amino acids are important constituents in paths for electron hopping along the e-pili [20,25]. The primary experimental eviCurrent Opinion in Electrochemistry 2017, 000:1–9
dence presented to support the electron hopping model was analysis of e-pili conductivity at two temperatures [20]. The study had major limitations. Conductivity was not analyzed within the temperature range in which evidence for metallic-like conductivity was previously observed [23] and in the temperature studies conductivity was examined across the diameter of the e-pili rather than along their length [20]. Furthermore, a subsequent detailed analysis of the conductivity along the length of G. sulfurreducens e-pili networks has confirmed a temperature response consistent with metallic-like conductivity [26]. Different theoretical models for G. sulfurreducens e-pili structure have been invoked to support both proposed conductivity mechanisms [20,24,25,27,28], but the limitations of such modeling efforts should be recognized. For example, a number of theoretical models for G. sulfurreducens e-pili concluded that aromatic amino acids could not be positioned close enough for π -π stacking, yet there is clear experimental evidence for this aromatic amino acid arrangement [24]. Experimental data trumps model predictions. The experimental challenge for determining the structure of G. sulfurreducens e-pili is their thin diameter (ca. 3 nm). However, the larger pilins recently found to also yield e-pili [12●● ] should assemble into pili that are closer in diameter to the type IV pili and related filaments whose structure can readily be solved with data from www.sciencedirect.com
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cryo-electron microscopy [29,30●● ]. Initially, structurefunction analysis of conductive mechanisms in e-pili may be more readily addressed by studying these larger e-pili.
Natural roles for e-Pili: direct interspecies electron transfer and Fe(III) reduction A full understanding of the mechanisms for e-pili conductivity is not necessary to appreciate the remarkable biological capabilities that long-range electron transport along e-pili can provide to microorganisms. e-Pili permit cells to make direct electrical connections with insoluble electron acceptors multiple cell lengths from the cell surface [1,31–33]. For example, direct interspecies electron transfer (DIET) with Geobacter species as the electron donating partner has been shown to be important in anaerobic digesters converting brewery wastes to methane [34,35] and in rice paddy soils, an important source of atmospheric methane [36]. Metatranscriptomic results suggest that genera other than Geobacter species may also be actively participating in DIET via e-pili in terrestrial wetland systems [36]. e-Pili have the potential to provide a more direct and potentially faster method of electron exchange between species than relying on diffusive interspecies electron carriers such as hydrogen [32,33]. This understanding has led to an important practical application: electrically conductive materials that can substitute for e-pili can be added to anaerobic digesters to promote DIET and accelerate and stabilize the conversion of wastes to methane [33,37]. G. sulfurreducens also requires e-pili for Fe(III) oxide reduction [5,7,38]. It seems likely that as the distribution of e-pili in the microbial world is better understood it will be found that e-pili play an important role in DIET and Fe(III) oxide reduction in diverse microorganisms.
Role of e-Pili in conductive biofilms The evolution of some Geobacter species to participate in DIET is a likely explanation for their effectiveness in growing thick conductive biofilms on the anodes of bioelectrochemical devices [39]. This conclusion is in accordance with the studies which first suggested that e-pili networks conferred conductivity to G. sulfurreducens biofilms, permitting cells at distance from the anode to contribute to current production by transferring electrons to the conductive e-pili network [23,40]. Many subsequent studies proposed an alternative model in which cytochrome-to-cytochrome electron transfer was thought to be the mechanism for long-range electron transport through the biofilm matrix [41–46]. However, physical evidence does not support the cytochrome-to-cytochrome electron transfer model [47]. Furthermore, the analysis of electrochemical data put forth to support the cytochrometo-cytochrome electron transfer model is flawed because the data was fit to models developed for simple, well-defined homogeneous systems, which are much www.sciencedirect.com
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different than physically and chemically complex heterogeneous biofilms [48●● ]. The key assumption in developing the cytochrome-to cytochrome electron transfer model was that cytochromes in the extracellular matrix are positioned close enough for direct cytochrometo-cytochrome electron exchange [49]. There are just not enough extracellular cytochromes for this to happen [1,50]. Most of the cytochromes are bound to cells (Figure 3). The majority of cytochromes are intracellular [1,51]. Although the cytochromes can be oxidized and reduced in electrochemical analyses, they cannot contribute to cytochrome-to-cytochrome electron transfer through the extracellular matrix [52]. Even if all of the cytochromes in G. sulfurreducens biofilms were freely extracellular (they clearly are not), the abundance of cytochromes would be too low to position cytochromes close enough for longrange electron transfer throughout the biofilms [1,50]. It is only near the biofilm/anode interface that an abundance of extracellular cytochromes has been documented [53]. The lack of sufficient cytochromes in the bulk of the extracellular matrix of G. sulfurreducens biofilms may explain why it has been necessary to assume physically unrealistic parameters to make the electrochemical data fit the cytochrome-to-cytochrome biofilm conductivity model [45,50]. A protein-based experimental system that mimicked the model for cytochrome-to-cytochrome conductivity in biofilms with hemin as the electron carrier [54●● ] demonstrated that it was necessary to add hemin concentrations that were orders of magnitude higher than the extracellular heme content of G. sulfurreducens biofilms [50] in order to achieve conductivities comparable to G. sulfurreducens biofilms. Determining the spacing of cytochromes within a extracellular matrix is readily feasible [18●● ]. Proponents of the cytochrome-to-cytochrome model for electron transport through Geobacter biofilms should directly experimentally test their hypothesis with such approaches rather than making unwarranted conclusions from indirect electrochemical analysis. As previously reviewed [1,55,56], simple, direct experiments, which do not require a mathematical model with physically unrealistic assumptions, have ruled out the cytochrome-to-cytochrome conductivity hypothesis and are consistent with the alternative hypothesis that e-pili enable long-range electron transfer through G. sulfurreducens biofilms. The e-pili model has led to novel strain designs to increase biofilm conductivity and current output, whereas the cytochrome-based model has not [1,55]. In multiple studies it has been demonstrated that genetically modified strains of G. sulfurreducens that produce poorly conductive pili generate low current densities even though they maintain high densities of outersurface cytochromes [5,7,12●● ,38,57]. As recent evidence has confirmed [58,59], the importance of cytochromes in current production has never been in doubt [31], but as Current Opinion in Electrochemistry 2017, 000:1–9
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Figure 3
Model for electron transport to electrodes in Geobacter sulfurreducens biofilms based on [31]. Recent studies [47,57] have further confirmed the roles of cytochromes in short-range electron exchange and e-pili in long-range electron transfer. Note that most of the cytochromes are within or bound to the cells and unavailable to freely exchange electrons within the extracellular matrix.
reemphasized in recent models [47,57], e-pili are important conduits for electron long-range electron transport as part of a complex electron transport system (Figure 3).
e-Pili as sustainable electronic materials As the biological role of e-pili has become clearer, some research focus has shifted to investigating the possibility that e-pili might serve as a renewable electronic material. e-Pili have many attractive material properties [2]. For example, e-pili can readily be mass produced from renewable feedstocks, contain no toxic components, and can be recycled as a degradable organic material. They are thinner than many other nanowire materials of similar conductivity [21●● ]. e-Pili properties such as conductivity and diameter can be tuned through genetic modifications of e-pilin amino acid composition [4● ,7,21●● ] and e-pili transistor properties [60] might also be optimized in a similar fashion. The ability to express pili with diverse structures and properties in G. sulfurreducens [5,6,12●● ,21●● ,38] suggests that it will possible to make additional modifications to functionalize e-pili with various amino acid sequences to enable diverse sensing functions and linking e-pili with other materials to produce composites [2]. Although one of the best features of e-pili is that they can be fabricated and function in water, the robustness of the e-pili allows them to retain conductivity when processed in organic solvents, under vacuum, or at high temperatures [2]. These Current Opinion in Electrochemistry 2017, 000:1–9
properties make e-pili amendable for more traditional materials fabrication approaches. The first applications for e-pili as electronic materials are likely to be in nanowire sensing devices. e-Pili intrinsically have dynamic sensitivity to pH [4● ], an important parameter of biomedical and environmental significance. Furthermore pH change is an output in many enzyme-based sensors. e-Pili may naturally respond to other environmental parameters. Furthermore, e-pili are more amenable than other nanowire materials such as silicon or carbon nanotubes to structural modifications to add linkers that can directly interact with analytes of interest, or to bind enzymes or antibodies [2]. The biocompatibility of e-pili and the potential for e-pili to make electrical connections between cells, or between cells and traditional electronics, suggest broad possibilities for bioelectronic/biomedical applications.
Conclusions Significant advances in e-pili research can be expected in the near future. Elucidating the structure of an e-pilus will be a major breakthrough, providing a better understanding of the mechanisms for electron transport along e-pili and serving as a guide for the design of synthetic e-pili with novel properties. Continued prospecting through the microbial world for additional e-pili www.sciencedirect.com
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[12●● ,13●● ] will improve understanding of their biological role and provide a broader range of electronic materials that may be useful as components in materials and devices. The archaea appear to be a particularly rich repository of diverse pili [9] worthy of further exploration. Fantastic improvements in the ability to visualize the organization of pili and associated proteins within cells [61● ,62] will aid in understanding how e-pili interact with other electron transport proteins to facilitate extracellular electron exchange. Nanowires have increasingly diverse applications, especially as biomedical sensing devices [63● ]. It seems likely that such applications will be a major focus of future e-pili research.
Acknowledgment Research in my laboratory on e-pili is supported by supported by Office of Naval Research Grants N000141310549 and N000141612526.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: •
Paper of special interest. Paper of outstanding interest.
••
1.
Malvankar NS, Lovley DR: Microbial nanowires for bioenergy applications. Curr Opin Biotechnol 2014, 27:88–95.
2.
Lovley DR: e-Biologics: fabrication of sustainable electronics with ‘green’ biological materials. mBio 2017, 8:e00695-17.
3.
Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR: Extracellular electron transfer via microbial nanowires. Nature 2005, 435:1098–1101.
Adhikari RY, Malvankar NS, Tuominen MT, Lovley DR: Conductivity of individual Geobacter pili. RSC Adv 2016, 6:8354–8357. First report on the conductivity of individual e-pili. Dmonstrated that conductivity is high enough to support extracellular electron transfer and the impact of pH on conductivity. 4. ●
5.
Tan Y, Adhikari RY, Malvankar NS, Ward JE, Nevin KP, Woodard TL, Smith JA, Snoeyenbos-West OL, Franks AE, Tuominen MT, et al.: The low conductivity of Geobacter uraniireducens pili suggests a diversity of extracellular electron transfer mechanisms in the genus Geobacter. Front Microbiol 2016, 7:980.
6.
Tan Y, Adhikari RY, Malvankar NS, Ward JE, Woodard TL, Nevin KP, Lovley DR: Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens yields pili with exceptional conductivity. mBio 2017, 8:e02203-16.
7.
Vargas M, Malvankar NS, Tremblay P-L, Leang C, Smith JA, Patel P, Synoeyenbos-West O, Nevin KP, Lovley DR: Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. mBio 2013, 4:e00105-13.
8. Berry J-L, Pelicic V: Exceptionally widespread nanomachines ●● composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol Rev 2015, 39:134–154. Excellent review of type IV pili. 9.
Makarova KS, Koonin EV, Albers S-V: Diversity and evolution of type IV pili systems in Archaea. Front Microbiol 2016, 7:667.
10. Holmes DE, Dang Y, Walker DJF, Lovley DR: The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microbial Genomics, vol 2 2016. http://dx.doi.org/10.1099/mgen.1090.000072. 11. Shu C, Xiao K, Yan Q, Sun X: Comparative analysis of type IV pilin in Desulfuromonadales. Front Microbiol 2016, 7:2080. www.sciencedirect.com
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12. Walker DJF, Adhikari RY, Holmes DE, Ward JE, Woodard TL, ●● Nevin KP, Lovley DR: Electrically conductive pili from genes of phylogenetically diverse microorganisms. ISME J, vol 11 2017. http://dx.doi.org/10.1038/ismej.2017.141. Demonstrated that microorganisms other than Geobacter species possess e-pili and that long (>100 amino acids) pilins can yield e-pili. 13. Sure S, Ackland ML, Torriero AJ, Adholeya A, Kochar M: Microbial ●● nanowires: an electrifying tale. Microbiology 2016, 162:2017–2028. Excellent review of the diversity of electrically conductive filaments in microorganisms. 14. Venkidusamy K, Megharah M, Schroder U, Karouta F, Mohan SV, Naidu R: Electron transport through electrically conductive nanofilaments in Rhodopseudomonas palustris strain RP2. RSC Advances 2015, 5:100790–100798. 15. Koch C, Harnisch F: Is there a specific ecological niche for ● electroactive microorganisms? ChemElectroChem 2016, 3:1282–1295. Excellent review of the diversity of microorganisms that have the capacity for extracellular electron exchange. 16. Pirbadian S, Barchinger SE, Leung KM, Byun HS, Jangir Y, Bouhenni RA, Reed SB, Romine MF, Saffarini DA, Shi L, et al.: Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components. Proc Natl Acad Sci USA 2014, 111:12883–12888. 17. Lovley DR, Malvankar NS: Seeing is believing: novel imaging techniques help clarify microbial nanowire structure and function. Environ Microbiol 2015, 7:2209–2215. 18. Subramanian P, Pribadian S, El-Naggar MY, Jensen GJ: The ●● ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryo-tomography. bioRxiv 2017. doi: http://dx.doi.org/10.1101/103242 . Demonstrates that cytochrome spacing in Shewanella membrane extensions is too great for conduction along filaments by cytochrome-to-cytochrome electron transfer. 19. Wegener G, Krukenberg V, Riedel D, Tegetmeyer HE, Boetius A: Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria. Nature 2015, 526:587–590. 20. Lampa-Pastirk S, Veazey JP, Walsh KA, Feliciano GT, Steidl RJ, Tessmer S, Reguera G: Thermally activated charge transport in microbial protein nanowires. Sci Rep 2016, 6:23517. 21. Tan Y, Adhikari RY, Malvankar NS, Pi S, Ward JE, Woodard TL, ●● Nevin KP, Xia Q, Tuominen MT, Lovley DR: Synthetic biological protein nanowires with high conductivity. Small 2016, 12:4481–4485. Demonstrates the potential to increase e-pili conductivity and decrease diameter with changes in the amino acid sequence of the pilin monomer. 22. Malvankar NS, Yalcin SE, Tuominen MT, Lovley DR: Visualization of charge propagation along individual pili proteins using ambient electrostatic force microscopy. Nat Nanotechnol 2014, 9:1012–1017. 23. Malvankar NS, Vargas M, Nevin KP, Franks AE, Leang C, Kim B-C, Inoue K, Mester T, Covalla SF, Johnson JP, et al.: Tunable metallic-like conductivity in nanostructured biofilms comprised of microbial nanowires. Nat Nanotechnol 2011, 6:573–579. 24. Malvankar NS, Vargas M, Nevin KP, Tremblay P-L, Evans-Lutterodt K, Nykypanchuk D, Martz E, Tuominen MT, Lovley DR: Structural basis for metallic-like conductivity in microbial nanowires. mBio 2015, 6:e00084-15. 25. Feliciano GT, Steidl RJ, Reguera G: Structural and functional insights into the conductive pili of Geobacter sulfurreducens revealed in molecular dynamics simulations. Phys Chem Chem Phys 2015, 17:22217–22226. 26. Ing NL, Nusca TD, Hochbaum AI: Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution. PCCP 2017 in press. http://dx.doi.org/10.1039/C1037CP03651E. Current Opinion in Electrochemistry 2017, 000:1–9
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27. Reardon PN, Mueller KT: Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J Biol Chem 2013, 288:29260–29266. 28. Xiao K, Malvankar NS, Shu C, Martz E, Lovley DR, Sun X: Low energy atomic models suggesting a pilus structure that could account for electrical conductivity along the length of Geobacter sulfurreducens pili. Sci Rep 2016, 6:23385. 29. Kolappan S, Coureuil M, Yu X, Nassif X, Egelman EH, Craig L: Structure of the Neisseria meningitidis Type IV pilus. Nat. Commun. 2016, 7:13015. 30. Poweleit N, Ge P, Nguyen HN, Loo RRO, Gunsalus RP, Zhou ZH: ●● CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pilus. Nat. Microbiol. 2016, 2:16222. Important insights into the structure of filaments related to type IV pili that are prevalent in archaea. 31. Lovley DR: Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination. Energy Environ Sci 2011, 4:4896–4906. 32. Lovley DR: Happy together: microbial communities that hook up to swap electrons. ISME J 2017, 11:327–336. 33. Lovley DR: Syntrophy goes electric: direct interspecies electron transfer. Ann Rev Microbiol 2017, 71:643–664. 34. Rotaru A-E, Shrestha PM, Liu F, Shrestha M, Shrestha D, Embree M, Zengler K, Wardman C, Nevin KP, Lovley DR: A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energy Environ Sci 2014, 7:408–415. 35. Shrestha PM, Malvankar NS, Werner JJ, Franks AE, Rotaru A-E, Shrestha M, Liu F, Nevin KP, Angenent LT, Lovley DR: Correlation between microbial community and granule conductivity in anaerobic bioreactors for brewery wastewater treatment. Bioresource Tech 2014, 174:306–310. 36. Holmes DE, Shrestha PM, Walker DJF, Dang Y, Nevin KP, Woodard TL, Lovley DR: Metatranscriptomic evidence for direct interspecies electron transfer between Geobacter and Methanothrix species in rice paddy soils. Appl Environ Microbiol 2017, 83:e00223-17. 37. Cheng Q, Call D: Hardwiring microbes via direct interspecies electron transfer: mechanisms and applications. Environ Sci Process Impacts 2016, 18:968–980. 38. Liu X, Tremblay P-L, Malvankar NS, Nevin KP, Lovley DR, Vargas M: A Geobacter sulfurreducens strain expressing Pseudomonas aeruginosa type IV pili localizes OmcS on pili but Is deficient in Fe(III) oxide reduction and current production. Appl Environ Microbiol 2014, 80:1219–1224. 39. Rotaru A-E, Woodard TL, Nevin KP, Lovley DR: Link between capacity for current production and syntrophic growth in Geobacter species. Front Microbiol 2015, 6:744. 40. Reguera G, Nevin KP, Nicoll JS, Covalla SF, Woodard TL, Lovley DR: Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells. Appl Environ Microbiol 2006, 72:7345–7348.
44. Phan H, Yates MD, Kirchofer ND, Bazan GC, Tender LM: Nguyen T-Q: Biofilm as a redox conductor: a systematic study of the moisture and temperature dependence of its electrical properties. PCCP Commun 2016, 18:17815. 45. Zhang X, Philips J, Roume H, Guo K, Rabaey K, Prevoteau A: Rapid and quantitative assessment of redox conduction across electroactive biofilms by using double potential step chronoamperometry. ChemElectroChem 2017, 4:1026–1036. 46. Virdis B, Millo D, Donose BC, Lu Y, Batstone DJ, Kromer JO: Analysis of electron transfer dynamics in mixed community electroactive microbial biofilms. RSC Adv 2016, 6:3650. 47. Ordonez MV, Schrott GD, Massazza DA, Busalmen JP: The relay network of Geobacter biofilms. Energy Environ Sci 2016, 9:2677–2681. 48. Babauta JT, Beyenal H: Biofilm electrochemistry: Biofilms in ●● Bioelectrochemical Systems: From Laboratory Practice to Data Interpretation. Edited by Beyenal H, Babauta JT. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2015:121–175. Excellent overview of the electrochemical analysis of biofilms. 49. Boyd DA, Snider RM, Erickson JS, Roy JN, Strycharz-Glaven SM, Tender LM: Theory of redox conduction and the measurement of electron transport rates through electrochemically active biofilms: Biofilms in Bioelectrochemical Systems: From Laboratory Practice to Data Interpretation. Edited by Beyenal H, Babauta JT. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2015:177–210. 50. Malvankar NS, Tuominen MT, Lovley DR: Lack of involvement of c-type cytochromes in long-range electron transport in microbial biofilms and nanowires. Energy Environ Sci 2012, 5:8651–8659. 51. Bonanni PS, Schrott GD, Robuschi L, Busalmen JP: Charge accumulation and electron transfer kinetics in Geobacter sulfurreducens biofilms. Energy Environ Sci 2012, 5:6188–6195. 52. Korth B, Rosa LFM, Harnisch F, Picioreanu C: A framework for modeling electroactive microbial biofilms performing direct electron transfer. Bioelectrochemistry 2015, 106:194–206. 53. Inoue K, Leang C, Franks AE, Woodard TL, Nevin KP, Lovley DR: Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens. Environ Microbiol Rep 2010, 3:211–217. 54. Amdursky N, Wang X, Meredith P, Riley DJ, Payne DJ, ●● Bradley DDC, Stevens MM: Electron hopping across hemin-doped serum albumin mats on centimeter-length scales. Adv Mater 2017. http://dx.doi.org/10.1002/adma.201700810. Important experimental investigation of the potential for long-range conduction in a protein-based system via electron hopping. 55. Malvankar NS, Rotello VM, Tuominen MT, Lovley DR: Reply to ‘Measuring conductivity of living Geobacter sulfurreducens biofilms’. Nat Nanotechnol 2016, 11:913–914. 56. Malvankar NS, Lovley DR: Electronic conductivity in living biofilms: physical meaning, mechanisms, and measurement methods: Biofilms in Bioelectrochemical Systems: From Laboratory Practice to Data Interpretation. Edited by Beyenal H, Babauta JT. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2015:211–248.
41. Lebedev N, Strycharz-Glaven SM, Tender LM: Spatially resolved confocal resonant raman microscopic analysis of anode-grown Geobacter sulfurreducens biofilms. ChemPhysChem 2014, 15:320–327.
57. Steidl RJ, Lampa-Pastirk S, Reguera G: Mechanistic stratification in electroactive biofilms of Geobacter sulfurreducens mediated by pilus nanowires. Nat. Commun. 2016, 7:12217.
42. Yates MD, Golden JP, Roy J, Strycharz-Glaven SM, Tsoi S, Erickson JS, El-Naggar MY, Barton SC, Tender LM: Thermally activated long range electron transport in living biofilms. Phys Chem Chem Phys 2015, 17:32564–32570.
58. Esteve-Canales M, Kuzume A, Borjas Z, Füeg M, Lovley D, Wanlowski T, Esteve-Nunez A: A severe reduction in the cytochrome C content of Geobacter sulfurreducens eliminates its capacity for extracellular electron transfer. Environ Microbiol Rep 2015, 7:219–226.
43. Yates MD, Strycharz-Glaven SM, Golden JP, Roy J, Tsoi S, Erickson JS, El-Naggar MY, Barton SC, Tender LM: Measuring conductivity of living Geobacter sulfurreducens biofilms. Nat Nanotechnol 2016, 11:910–913.
59. Peng L, Yang Y: Cytochrome OmcZ is essential for the current generation by Geobacter sulfurreducens under low electrode potential. Electrochim Acta 2017, 228:447–452.
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60. Adhikari R: Study of Charge Transport Mechanism in Microbial Nanowires. University of Massachusetts; 2016. 61. Chang Y-W, Rettberg LA, Treuner-Lange A, Iwasa J, ● Søgaard-Andersen L, Jensen GJ: Architecture of the type IVa pilus machine. Science 2016:351. Amazing visulalization of how type IV pili and associated proteins are oragnized in the cell.
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62. Hospenthal MK, Costa TRD, Waksman G: A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nature Reviews Microbiology 2017, 15:365–379. 63. Zhou W, Dai X, Lieber CM: Advances in nanowire bioelectronics. ● Rep Prog Phys, vol 80 2017 016701. Excellent overview of the potential applications of nanowires as bioelectronic devices.
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Review Article Electrically conductive pili: Biological function and potential applications in electronics Derek R. Lovley Electrically conductive pili (e-pili) enable long-range electron exchange between microorganisms and their extracellular environment and show promise as a ‘green’ electronic material. Most studies have focused on the e-pili of G. sulfurreducens, which play a role in Fe(III) oxide reduction and interspecies electron transfer, and confer conductivity to current-producing biofilms. However, a diversity of other microorganisms have recently been found to possess e-pili, including e-pili with unique structures. Aromatic amino acids are key elements in electron transport along the length of e-pili and the conductivity of synthetic e-pili can be tuned by manipulating aromatic content. e-Pili are attractive as an electronic material because they can be produced from renewable feedstocks and are biodegradable, yet they are also highly robust for device fabrication. Basic information on the structure of e-pili, mechanisms for electron transport, and other electronic properties is required in order to better understand their biological function and to guide the design of synthetic e-pili for applications as electronic components. Address Department of Microbiology, University of Massachusetts, Amherst, MA, USA Corresponding author: Lovley, Derek R. ([email protected])
Current Opinion in Electrochemistry 2017, XX:XX–XX This review comes from a themed issue on Physical & NanoElectrochemsitry 2017 Edited by Feng Zhao and Jens Ulstrup For a complete overview see the Issue and the Editorial Available online XX XXXX 2017 http://dx.doi.org/10.1016/j.coelec.2017.08.015 2451-9103/© 2017 Elsevier B.V. All rights reserved.
Introduction Electrically conductive pili (e-pili) enable electron transport over unprecedented distances for a biological protein and confer unique properties to microorganisms of biogeochemical and practical significance [1]. Furthermore, e-pili are a sustainable ‘green’ electronic material with diverse potential applications [2]. Following the initial discovery of e-pili in Geobacter sulfurreducens [3], highly divergent hypotheses were put forth on e-pili function www.sciencedirect.com
within microbial communities and the mechanisms of electron transport along e-pili [1]. However, recent experimental evidence has culled some of these early concepts and has provided a foundation for further hypothesis development. The purpose of this review is to summarize these recent studies, focusing on publications within the last two years, with the exception that key earlier papers are mentioned when required for context. Current concepts on mechanisms for electron transfer along e-pili are discussed, as well as the possible biological functions of e-pili, and emerging technologies based on e-pili (Figure 1).
What is an e-pilus and who has them? An e-pilus is any pilus that is sufficiently electrically conductive along its length to promote long-range electron exchange between the microbe expressing e-pili and the external environment. Conductivity data for e-pili under physiologically relevant conditions is limited (Box 1). The specific threshold conductivity required to satisfy the definition of an e-pilus is dependent on its function. Rough guidelines for the e-pili conductivity necessary for electron transport to extracellular electron acceptors, such as Fe(III) oxides, other cells, and electrodes, are available from studies with G. sulfurreducens. The native e-pili with a conductivity at pH 7 of 51 mS/cm [4● ] effectively supported growth under conditions that required long-range electron transport, consistent with theoretical calculations [5]. Heterologous expression of pili with higher conductivity (277 S/cm at pH 7) did not further improve growth dependent on extracellular electron transfer [6]. Pili with conductivities of 30–40 μS/cm [4● ,5] were not sufficiently conductive to support extracellular electron transfer [5,7]. However, it is conceivable that there may be other, as yet unverified, functions of e-pili, such as electrical communication to the cell that a pilus has contacted a specific surface. Conductivities in the μS/cm range may be adequate for these potential alternative functions. The e-pili of G. sulfurreducens are homologous to the type IV pili that are common in bacteria and archaea [8●● ,9]. The pilin genes of a number of Geobacter species and closely related microorganisms are highly similar to the pilin gene of G. sulfurreducens [10,11]. Similar pilin monomer genes are also found in a few phylogenetically diverse microorganisms [10]. Several of these genes have yielded e-pili when expressed in G. sulfurreducens [6,12●● ]. Some pilin monomers not closely related to the G. sulfurreducens pilin can also assemble into e-pili, Current Opinion in Electrochemistry 2017, 000:1–9
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Figure 1
Examples of biological function and materials application of electrically conductive pili.
demonstrating the possibility of broad distribution of e-pili in the microbial world [12●● ]. Furthermore, there is a growing list of microorganisms with other pilus-like filaments that are electrically conductive, at least in vitro [13●● ,14]. A wide diversity of microorganisms are known to be electroactive and many more are likely to be discovered [15]. Like much of the microbial world, many of these microorganisms may be difficult to culture, especially in sufficient quantities to study the properties of their pili. Heterologous expression in G. sulfurreducens of pilin monomer genes (Figure 2) from the (meta)genomes of difficult-toculture microbes is an option to rapidly screen whether pilin genes have the potential to yield e-pili [12●● ]. Determining the composition of conductive filaments is key. For example, it was initially thought that Shewanella oneidensis possessed conductive pili, but subsequent investigation demonstrated that the conductive filaments were artifacts that form when membrane extensions are dried and chemically fixed [16,17]. Cryoelectron microscopy revealed that the cytochrome-based electron transport along these filaments that was previCurrent Opinion in Electrochemistry 2017, 000:1–9
ously proposed [16] is unlikely in vivo because the cytochromes within the membrane extensions are spaced to far apart for cytochrome-to-cytochrome electron transfer [18●● ]. It is also important to document the conductivity of any filaments that are proposed to be conductive. For example, it was speculated that the pili of a sulfate-reducing microorganism were the conduit for interspecies electron transfer during syntrophic anaerobic methane oxidation [19]. However, when the pilin gene from the sulfate reducer was expressed in G. sulfurreducens the resultant pili were poorly conductive, suggesting that there must be another route for the interspecies electron exchange [12●● ].
Mechanisms for electron transport along e-pili Electron transport mechanisms for e-pili have primarily been investigated with e-pili from G. sulfurreducens and related proteins. Individual e-pili that are free of other proteins or metals have substantial conductivities (50 mS/cm–1 kS/cm) [4● –6,20,21●● ]. Propagation of charge along cytochrome-free regions of G. sulfurreducens e-pili was also documented with electrostatic force microscopy www.sciencedirect.com
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Filament type
Solvents
Fixation
Dried (D)/ Hydrated (H)
Two Probe or Four Probe
Conductivity
Reference
Rhodopseudomonas palustris G. sulfurreducens e-pili
+ −
+ −
D H
Two-probe Both
[14] [4]
G. sulfurreducens e-pili Synthetic e-pili in G. sulfurreducens
+ −
− −
D H
Two-probe Two-probe
G. metallireducens pilin in G. sulfurreducens
−
−
H
Two-probe
35–72 µS/cm 51 mS/cm (pH 7) 187 mS/cm (pH 2) 40 µS/cm (pH 10.5) 1.4 - 4.3 S/cm 10 S/cm (pH 7) 393 S/cm (pH 2) 300 mS/cm (pH 10.5) 277 S/cm (pH 7)
1
For a comprehensive review of additional filaments in which conductivity has been measured across the filament width, typically after chemical fixation, see reference (13).
[20] [21]
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Examples of types of conductivity measurements along the length of e-Pili and other conductive filaments.1
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Taking Measure of e-Pili There has been little consistency between laboratories in the methods by which the conductivity of e-pili and other microbial filaments have been evaluated. In many studies the filaments have been treated rather harshly for a biological protein, with extraction in solvents and/or fixation with glutaraldehyde, which cross-links peptides. These treatments could significantly change the filament structure from that found in vivo. Measuring conductivity across the diameter of filaments with the assumption that this reflects corresponding conduction along the length of the filament is common. Another strategy is to measure the conductivity of e-pili networks. Network conductivity can provide evidence for long-range electron transport through e-pili, but the conductivities of e-pili networks are much lower than the conductivities of individual e-pili, presumably due to resistances in pili-to-pili electron transfer. Conductivity along the length of e-pili has been measured with two-probe and four-probe methods. Although more technically difficult, four-probe measurements are preferable because they eliminate errors in conductivity estimates due to contact resistance between the e-pili and the electrodes. Very few measurements along the length of individual e-pili at physiologically relevant conditions (hydrated; no chemical fixation; pH 7) are available due to the technical difficulty of such measurements. Of course when e-pili are being processed as an electronic material, the relevant concern is the conductivity and other properties of the e-pili under the processing conditions required for material/device fabrication and concerns about biological relevance are eliminated.
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Box 1
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Figure 2
Method for a rapid screen of pilin genes with heterologous expression in Geobacter sulfurreducens to determine if the pilins have the potential to assemble into electrically conductive pili.
[22]. These direct experimental measurements negate multiple previous theoretical models which suggested that cytochromes were essential for e-pili conductivity (for a detailed review see Refs. [1,17]). Aromatic amino acids are important for e-pili conductivity. Substituting alanine for key aromatic amino acids in e-pili reduces conductivity [4● ,7,20]. Introducing more rings by substituting a tryptophan for the carboxyl terminal phenylalanine and tyrosine in the G. sulfurreducens pilin monomer increased e-pili conductivity [21●● ], as did expressing the more aromatic-rich pilin monomer of G. metallireducens [6]. The conductivity of pili that assemble from larger pilins is also dependent on aromatic amino acid content [12●● ]. One potential explanation for these observations is that overlapping π -π orbitals of aromatic amino acids confer a metallic-like conductivity, similar to that in synthetic organic conducting polymers [1,17]. Data supporting this hypothesis for G. sulfurreducens e-pili includes structural evidence for π -π stacking and a correlation between the degree of π -π stacking and e-pili conductivity [4● ,22–24]. Conductivity response to changes in temperature and pH are consistent with metallic-like conductivity [4● ,23], as is the charge propagation along e-pili observed with electrostatic force microscopy [22]. An alternative model is that the aromatic amino acids are important constituents in paths for electron hopping along the e-pili [20,25]. The primary experimental eviCurrent Opinion in Electrochemistry 2017, 000:1–9
dence presented to support the electron hopping model was analysis of e-pili conductivity at two temperatures [20]. The study had major limitations. Conductivity was not analyzed within the temperature range in which evidence for metallic-like conductivity was previously observed [23] and in the temperature studies conductivity was examined across the diameter of the e-pili rather than along their length [20]. Furthermore, a subsequent detailed analysis of the conductivity along the length of G. sulfurreducens e-pili networks has confirmed a temperature response consistent with metallic-like conductivity [26]. Different theoretical models for G. sulfurreducens e-pili structure have been invoked to support both proposed conductivity mechanisms [20,24,25,27,28], but the limitations of such modeling efforts should be recognized. For example, a number of theoretical models for G. sulfurreducens e-pili concluded that aromatic amino acids could not be positioned close enough for π -π stacking, yet there is clear experimental evidence for this aromatic amino acid arrangement [24]. Experimental data trumps model predictions. The experimental challenge for determining the structure of G. sulfurreducens e-pili is their thin diameter (ca. 3 nm). However, the larger pilins recently found to also yield e-pili [12●● ] should assemble into pili that are closer in diameter to the type IV pili and related filaments whose structure can readily be solved with data from www.sciencedirect.com
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cryo-electron microscopy [29,30●● ]. Initially, structurefunction analysis of conductive mechanisms in e-pili may be more readily addressed by studying these larger e-pili.
Natural roles for e-Pili: direct interspecies electron transfer and Fe(III) reduction A full understanding of the mechanisms for e-pili conductivity is not necessary to appreciate the remarkable biological capabilities that long-range electron transport along e-pili can provide to microorganisms. e-Pili permit cells to make direct electrical connections with insoluble electron acceptors multiple cell lengths from the cell surface [1,31–33]. For example, direct interspecies electron transfer (DIET) with Geobacter species as the electron donating partner has been shown to be important in anaerobic digesters converting brewery wastes to methane [34,35] and in rice paddy soils, an important source of atmospheric methane [36]. Metatranscriptomic results suggest that genera other than Geobacter species may also be actively participating in DIET via e-pili in terrestrial wetland systems [36]. e-Pili have the potential to provide a more direct and potentially faster method of electron exchange between species than relying on diffusive interspecies electron carriers such as hydrogen [32,33]. This understanding has led to an important practical application: electrically conductive materials that can substitute for e-pili can be added to anaerobic digesters to promote DIET and accelerate and stabilize the conversion of wastes to methane [33,37]. G. sulfurreducens also requires e-pili for Fe(III) oxide reduction [5,7,38]. It seems likely that as the distribution of e-pili in the microbial world is better understood it will be found that e-pili play an important role in DIET and Fe(III) oxide reduction in diverse microorganisms.
Role of e-Pili in conductive biofilms The evolution of some Geobacter species to participate in DIET is a likely explanation for their effectiveness in growing thick conductive biofilms on the anodes of bioelectrochemical devices [39]. This conclusion is in accordance with the studies which first suggested that e-pili networks conferred conductivity to G. sulfurreducens biofilms, permitting cells at distance from the anode to contribute to current production by transferring electrons to the conductive e-pili network [23,40]. Many subsequent studies proposed an alternative model in which cytochrome-to-cytochrome electron transfer was thought to be the mechanism for long-range electron transport through the biofilm matrix [41–46]. However, physical evidence does not support the cytochrome-to-cytochrome electron transfer model [47]. Furthermore, the analysis of electrochemical data put forth to support the cytochrometo-cytochrome electron transfer model is flawed because the data was fit to models developed for simple, well-defined homogeneous systems, which are much www.sciencedirect.com
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different than physically and chemically complex heterogeneous biofilms [48●● ]. The key assumption in developing the cytochrome-to cytochrome electron transfer model was that cytochromes in the extracellular matrix are positioned close enough for direct cytochrometo-cytochrome electron exchange [49]. There are just not enough extracellular cytochromes for this to happen [1,50]. Most of the cytochromes are bound to cells (Figure 3). The majority of cytochromes are intracellular [1,51]. Although the cytochromes can be oxidized and reduced in electrochemical analyses, they cannot contribute to cytochrome-to-cytochrome electron transfer through the extracellular matrix [52]. Even if all of the cytochromes in G. sulfurreducens biofilms were freely extracellular (they clearly are not), the abundance of cytochromes would be too low to position cytochromes close enough for longrange electron transfer throughout the biofilms [1,50]. It is only near the biofilm/anode interface that an abundance of extracellular cytochromes has been documented [53]. The lack of sufficient cytochromes in the bulk of the extracellular matrix of G. sulfurreducens biofilms may explain why it has been necessary to assume physically unrealistic parameters to make the electrochemical data fit the cytochrome-to-cytochrome biofilm conductivity model [45,50]. A protein-based experimental system that mimicked the model for cytochrome-to-cytochrome conductivity in biofilms with hemin as the electron carrier [54●● ] demonstrated that it was necessary to add hemin concentrations that were orders of magnitude higher than the extracellular heme content of G. sulfurreducens biofilms [50] in order to achieve conductivities comparable to G. sulfurreducens biofilms. Determining the spacing of cytochromes within a extracellular matrix is readily feasible [18●● ]. Proponents of the cytochrome-to-cytochrome model for electron transport through Geobacter biofilms should directly experimentally test their hypothesis with such approaches rather than making unwarranted conclusions from indirect electrochemical analysis. As previously reviewed [1,55,56], simple, direct experiments, which do not require a mathematical model with physically unrealistic assumptions, have ruled out the cytochrome-to-cytochrome conductivity hypothesis and are consistent with the alternative hypothesis that e-pili enable long-range electron transfer through G. sulfurreducens biofilms. The e-pili model has led to novel strain designs to increase biofilm conductivity and current output, whereas the cytochrome-based model has not [1,55]. In multiple studies it has been demonstrated that genetically modified strains of G. sulfurreducens that produce poorly conductive pili generate low current densities even though they maintain high densities of outersurface cytochromes [5,7,12●● ,38,57]. As recent evidence has confirmed [58,59], the importance of cytochromes in current production has never been in doubt [31], but as Current Opinion in Electrochemistry 2017, 000:1–9
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Figure 3
Model for electron transport to electrodes in Geobacter sulfurreducens biofilms based on [31]. Recent studies [47,57] have further confirmed the roles of cytochromes in short-range electron exchange and e-pili in long-range electron transfer. Note that most of the cytochromes are within or bound to the cells and unavailable to freely exchange electrons within the extracellular matrix.
reemphasized in recent models [47,57], e-pili are important conduits for electron long-range electron transport as part of a complex electron transport system (Figure 3).
e-Pili as sustainable electronic materials As the biological role of e-pili has become clearer, some research focus has shifted to investigating the possibility that e-pili might serve as a renewable electronic material. e-Pili have many attractive material properties [2]. For example, e-pili can readily be mass produced from renewable feedstocks, contain no toxic components, and can be recycled as a degradable organic material. They are thinner than many other nanowire materials of similar conductivity [21●● ]. e-Pili properties such as conductivity and diameter can be tuned through genetic modifications of e-pilin amino acid composition [4● ,7,21●● ] and e-pili transistor properties [60] might also be optimized in a similar fashion. The ability to express pili with diverse structures and properties in G. sulfurreducens [5,6,12●● ,21●● ,38] suggests that it will possible to make additional modifications to functionalize e-pili with various amino acid sequences to enable diverse sensing functions and linking e-pili with other materials to produce composites [2]. Although one of the best features of e-pili is that they can be fabricated and function in water, the robustness of the e-pili allows them to retain conductivity when processed in organic solvents, under vacuum, or at high temperatures [2]. These Current Opinion in Electrochemistry 2017, 000:1–9
properties make e-pili amendable for more traditional materials fabrication approaches. The first applications for e-pili as electronic materials are likely to be in nanowire sensing devices. e-Pili intrinsically have dynamic sensitivity to pH [4● ], an important parameter of biomedical and environmental significance. Furthermore pH change is an output in many enzyme-based sensors. e-Pili may naturally respond to other environmental parameters. Furthermore, e-pili are more amenable than other nanowire materials such as silicon or carbon nanotubes to structural modifications to add linkers that can directly interact with analytes of interest, or to bind enzymes or antibodies [2]. The biocompatibility of e-pili and the potential for e-pili to make electrical connections between cells, or between cells and traditional electronics, suggest broad possibilities for bioelectronic/biomedical applications.
Conclusions Significant advances in e-pili research can be expected in the near future. Elucidating the structure of an e-pilus will be a major breakthrough, providing a better understanding of the mechanisms for electron transport along e-pili and serving as a guide for the design of synthetic e-pili with novel properties. Continued prospecting through the microbial world for additional e-pili www.sciencedirect.com
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[12●● ,13●● ] will improve understanding of their biological role and provide a broader range of electronic materials that may be useful as components in materials and devices. The archaea appear to be a particularly rich repository of diverse pili [9] worthy of further exploration. Fantastic improvements in the ability to visualize the organization of pili and associated proteins within cells [61● ,62] will aid in understanding how e-pili interact with other electron transport proteins to facilitate extracellular electron exchange. Nanowires have increasingly diverse applications, especially as biomedical sensing devices [63● ]. It seems likely that such applications will be a major focus of future e-pili research.
Acknowledgment Research in my laboratory on e-pili is supported by supported by Office of Naval Research Grants N000141310549 and N000141612526.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: •
Paper of special interest. Paper of outstanding interest.
••
1.
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7.
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Electrically conductive pili: Biological function and potential applications in electronics Lovley
60. Adhikari R: Study of Charge Transport Mechanism in Microbial Nanowires. University of Massachusetts; 2016. 61. Chang Y-W, Rettberg LA, Treuner-Lange A, Iwasa J, ● Søgaard-Andersen L, Jensen GJ: Architecture of the type IVa pilus machine. Science 2016:351. Amazing visulalization of how type IV pili and associated proteins are oragnized in the cell.
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62. Hospenthal MK, Costa TRD, Waksman G: A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nature Reviews Microbiology 2017, 15:365–379. 63. Zhou W, Dai X, Lieber CM: Advances in nanowire bioelectronics. ● Rep Prog Phys, vol 80 2017 016701. Excellent overview of the potential applications of nanowires as bioelectronic devices.
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Please cite this article as: Lovley, Electrically conductive pili: Biological function and potential applications in electronics, Current Opinion in Electrochemistry (2017), http://dx.doi.org/10.1016/j.coelec.2017.08.015