Tunable metallic-like conductivity in microbial nanowire ... · a, Decrease in impedance between split electrodes as biofilms grew over time. b, Circuit models for control systems

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2011.119

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

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Tunable metallic-like conductivity in microbial nanowire networks

Nikhil S. Malvankar, Madeline Vargas, Kelly P. Nevin, Ashley E. Franks, Ching Leang,

Byoung-Chan Kim, Kengo Inoue, Tünde Mester, Sean F. Covalla, Jessica P. Johnson, Vincent

M. Rotello, Mark T. Tuominen*, and Derek R. Lovley*

*Email: M.T.T.([email protected]) or D.R.L. ([email protected])

Supplementary Information

Page 2. Fig.S1| Confocal laser microscopy image of control split-electrodes without biofilm

Page 2. Fig.S2| Confocal laser microscopy image of biofilm not bridging the gap

Page 3. Fig.S3| AC impedance spectroscopy – data, model and fitting results

Page 4. Fig.S4| Acetate removal experiment

Page 5. Fig.S5| Four-probe measurements

Page 6. Fig.S6| Confocal laser microscopy images of aerobically grown biofilms

Page 6. Fig.S7| Comparison of conductance of aerobically grown and G.sulfurreducens biofilms

Page 7. Fig.S8| Confocal laser microscopy image of fumarate-grown biofilm

Page 7. Fig.S9| Confocal images, current and conductance of biofilms of different strains.

Page 8. Fig.S10| Cytochrome denaturing experiments

Page 9. Fig.S11| Control experiments with metallic nanowires and latex nanoparticles.

Page 10. Fig.S12| Conductivity of pili filaments as a function of pH

Page 10. Fig.S13| X-ray diffraction patterns of purified pili filaments

Page 11. Supplementary methods – AC impedance spectroscopy measurements

Page 12. Supplementary references

© 2011 Macmillan Publishers Limited. All rights reserved.

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Supplementary figures

Supplementary Fig. S1| Top view 3-D projection of fluorescent confocal laser microscopy images of split electrodes illustrating a lack of biofilm on the control electrodes not connected to cathode. Scale bar 100 µm. Setup for control electrodes is shown in Fig. 1a. Supplementary Fig. S2| Top view 3-D projection of fluorescent confocal laser microscopy images of split electrodes when only one of the two electrodes was connected to the cathode. A biofilm did not grow on the side not connected to cathode and there was no biofilm bridging the non-conductive gap. Biofilm thickness 50 µm. Scale bar 100 µm.


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Supplementary Fig.S3| AC impedance spectroscopy – data, model and fitting results. a, Decrease in impedance between split electrodes as biofilms grew over time. b, Circuit models for control systems without gap-spanning biofilms and for systems with gap-spanning biofilms. c, Modelling of representative impedance for control electrodes not bridged by a biofilm. d, Modelling of impedance for corresponding electrodes on the same day that were bridged by a biofilm. Inset tables show model parameters. Error bars indicate s.d. (n=5)


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Supplementary Fig. S4| Acetate removal experiment. a, Effect of acetate removal and addition on microbial current production and corresponding acetate concentration. b, Conductivity measurements when acetate was removed and added back to the growth medium. Error bars indicate s.d.


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Supplementary Fig. S5| Four-probe measurements. a, Schematic of microbial fuel cell with four gold electrodes, each separated by 50 µm non-conductive gap, serving as an anode. b, Schematic of conductivity measurements using four electrodes. Current is injected through outer two electrodes and voltage is measured across inner two electrodes.


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Supplementary Fig. S6| Confocal laser microscopy images of aerobically grown biofilms. Cross-sectional and top view 3-D projection of fluorescent confocal scanning laser microscopy images of biofilms of aerobically grown a, Pseudomonas aeruginosa and b, Escherichia coli bridging non-conductive gap. Scale bar 50µm. Pseudomonas biofilm had height 12 ± 1.5 μm. E-coli biofilm height was 38 ± 2.2 μm. Gap is designated by arrows.

Supplementary Fig. S7| Comparison of conductivity of aerobically grown and G.sulfurreducens biofilm. Conductivity of biofilm and control for Pseudomonas aeruginosa, Escherichia coli, and G.sulfurreducens under various conditions. Error bars indicate s.d. for individual measurements for several different biofilms.


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Supplementary Fig. S8| Confocal laser microscopy image of fumarate-grown biofilm. Top view 3-D projection of fluorescent confocal laser microscopy images of the fumarate-grown biofilm of strain DL-1 covering the split electrodes. Biofilm thickness 130 µm. Scale bar 100µm.

Supplementary Fig. S9| Confocal images, current and conductance of biofilms of different strains. Top view 3-D projection of fluorescent confocal laser microscopy images: a, strain KN400 biofilm. Scale bar 100µm. Biofilm height was 40 μm. b, strain BEST biofilm. Scale bar 75 µm. Biofilm height was 60 μm. Gap is designated by arrows. c, Microbial current generation and d, conductance. Error bars show s.d. of individual measurements of several biofilms. (n=5 KN400 and DL-1, n=4 BEST)


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Supplementary Fig. S10| Cytochrome denaturing experiments. a, Conductivity of filaments of wild-type KN400 strain before and after treating with 320 mM cytochrome denaturing reagent β-mercaptoethanol (ME). Bars represent mean ± s.d. (n=3 biological replicates.) b, Haem staining gel of filaments of wild-type KN400 strain before and after treating with cytochrome denaturing reagent ME. Haem containing protein bands were absent after ME treatment, confirming cytochrome denaturation.

a

b


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Supplementary Fig. S11| Control experiments with metallic nanowires and latex nanoparticles. a, Scanning electron microscope image of metallic nanowires. Scale bar, 5 µm. b, Atomic force microscopy image of non-conductive latex nanoparticles. Scale bar, 500 nm. c, Current-voltage response of metallic nanowires showing high conductivity and negligible conductivity for latex nanoparticles and also for buffer solution.


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Supplementary Fig. S12| Conductivity of pili filaments as a function of pH. Conductivity of pili measured using 4-probe approach increased over two orders of magnitude with decreasing pH. Control experiments with media buffer did not show significant change in conductivity with change in pH. Error bars represent s.d. (n=3 biological replicates).

Supplementary Fig. S13| X-ray diffraction patterns of purified pili filaments. The peak at 25o indicates the overlap of π orbitals. Control measurements with buffer media and filaments of PilA mutant strain did not show any peaks. Data is a representative of several biological replicates of filaments of KN400 strains (n=5 for wild-type, n=3 for PilA mutant).


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Supplementary Methods AC impedance spectroscopy measurements

AC impedance spectroscopy was employed to provide an independent probe for conductance measurements between the electrodesS1-S3, to avoid potential contributions of redox charge transfer and ion diffusion to measured dc conductanceS4,S5 and to separate electronic and ionic conductivityS6.

Impedance was measured by sweeping the frequency between 100 mHz and 300 KHz with a 0.1 V amplitude ac voltage excitation using a Solartron 1252/ 1287 impedance analyzer. Data fitting was performed with ZView software (Scribner Inc.) which used LEVM algorithm developed by J. Ross Macdonald. Details of equivalent circuit modelling are described elsewhereS5,S7. Anode and cathode potentials were continuously monitored using Ag/AgCl reference electrode in the anode and cathode chamber respectively. For all impedance measurements, open circuit potential was monitored until it reached a constant value before over imposing ac signal.

The low-frequency impedance of the biofilm decreased as the biofilm grew (Supplementary Fig. S3a). In contrast, the impedance between the control electrodes, which did not have a biofilm, or in which only one electrode had a biofilm, did not change substantially over time.

The ac impedance between the control electrodes without gap-spanning biofilms could be accurately modelled as a series RC circuit (Supplementary Fig.S3b), accounting for solution resistance (R) and interface capacitance (C), with an electrode capacitance in parallelS7. A low frequency cut-off of 100 mHz was used to eliminate any possible contributions from electrochemical reactions occurring at very low frequencyS1,S3,S4. The interface capacitance was calculated asS5:

( ) 1

)1( ppsInterface TRC −−=

Where T and P are parameters associated with the electrolytic double layer and Rs is the solution resistance, representing ionic conductivity. These parameters were obtained by fitting the circuit model (Supplementary Fig. S3b) to the impedance data (Supplementary Fig.S3a). The value of Celectrode was extracted by fitting the model of the solution resistance and the electrode capacitance in parallel at high frequencies and then the value of electrode capacitance was fixed for further fittingS7. The double layer and electrode capacitance values obtained with this approach (Supplementary Fig.S3c) agreed very well with results from the literatureS4-S7, indicating that this modelling approach was valid.


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However, in order to fit the data accurately in the instances in which biofilms spanned the gap it was necessary to add an extra resistor, representing an additional conductive pathway, in parallel to the RC model that described the controls without biofilms (Supplementary Fig. S3b dotted line, Supplementary Fig. S3d). This equivalent circuit method has been previously employed to measure conductivity of nanostructured filmsS8-S10, and serves to separate electronic and ionic conductivity in mixed conductorsS6. The need to include this extra conductance in the circuit model demonstrates that the biofilm is electronically conductive.

The capacitance of the biofilm also increased as the thickness of the biofilm increased. The larger capacitance masked the biofilm conductance evaluated with comparatively high frequency (0.1Hz-300KHz) ac impedance measurements, but with dc measurements the biofilm conductance could still be measured at lower frequencies (10mHz), suppressing the capacitive effect. There was good correspondence between the estimates of conductance obtained with the dc and ac methods, further validating the measurement methodology (Fig.2c).

Supplementary references

S1. Katz, E. & Willner, I. Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: Routes to impedimetric immunosensors, DNA-Sensors, and enzyme biosensors. Electroanalysis 15, 913-947 (2003).

S2. K'Owino, I.O. & Sadik, O.A. Impedance spectroscopy: A powerful tool for rapid biomolecular screening and cell culture monitoring. Electroanalysis 17, 2101-2113 (2005).

S3. Wu, J., Ben, Y.X., Battigelli, D. & Chang, H.C. Long-range AC electroosmotic trapping and detection of bioparticles. Industrial & Engineering Chemistry Research 44, 2815-2822 (2005).

S4. Bard, A.J. & Faulkner, L.R. Electrochemical Methods: Fundamentals and Applications, 2nd ed. (John Wiley & Sons, New York; 2001).

S5. Barsoukov, E. & Macdonald, J.R. Impedance Spectroscopy: Theory, Experiment and Applications. 2nd ed. (John Wiley and Sons Inc., New Jersey; 2005).

S6. Thangadurai, V., Huggins, R.A. & Weppner, W. Use of simple ac technique to determine the ionic and electronic conductivities in pure and Fe-substituted SrSnO3 perovskites. Journal of Power Sources 108, 64-69 (2002).

S7. Hong, J. et al. AC frequency characteristics of coplanar impedance sensors as design parameters. Lab Chip 5, 270-279 (2005).

S8. Bisquert, J. Physical electrochemistry of nanostructured devices. Physical Chemistry Chemical Physics 10, 49-72 (2008).

S9. Meulenkamp, E.A. Electron transport in nanoparticulate ZnO films. J. Phys. Chem. B 103, 7831-7838 (1999).

S10. Kankare, J. & Kupila, E.L. In-situ conductance measurement during electropolymerization. J.Electroanal.Chem. 322, 167-181 (1992).


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Tunable metallic-like conductivity in microbial nanowire ... · a, Decrease in impedance between split electrodes as biofilms grew over time. b, Circuit models for control systems