Name: characterizing electron transport through living biofilms
Uploaded: 2018-06-01T12:30:00
Description: Watch this Scientific Journal Video about Characterizing Electron Transport through Living Biofilms at JoVE.com

M.D.Y, S.M.G-S., and L.M.T. acknowledge the Office of Naval Research (Award #N0001415WX01038 and N0001415WX00195), the Naval Research Laboratory, and the Naval Research Laboratory Nanosciences Institute; M.Y.E.-N. is supported by the U.S. Department of Energy Grant DE-FG02-13ER16415.

1. Interdigitated microelectrode array (IDA) preparation
<ol>
	<li>Obtain commercially available IDA electrodes patterned on a nonconductive substrate or synthesize them using standard lithographic methods.28 
	NOTE: IDA dimensions and/or materials can be varied based on desired conditions for different experiments. IDAs used here were obtained commercially and consisted of two interdigitated gold microelectrodes patterned on a glass substrate connected to large electrode pads at opposite e...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+dechlorination+of+2-chlorophenol+by+Anaeromyxobacter+dehalogenans+with+an+electrode+serving+as+the+electron+donor.">Reductive dechlorination of 2-chlorophenol by Anaeromyxobacter dehalogenans with an electrode serving as the electron donor.</a> Environ Microbiol Report. 2, 289-294 (2010).</li><li>Yates, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Electrochemical+Energy+Storage+and+Recovery+in+a+Combined+Electrotrophic+and+Electrogenic+Biofilm.">Microbial Electrochemical Energy Storage and Recovery in a Combined Electrotrophic and Electrogenic Biofilm.</a> Environ Sci Technol Lett. 4, 374-379 (2017).</li><li>Tender, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+microbially+generated+power+on+the+seafloor.">Harnessing microbially generated power on the seafloor.</a> Nature Biotechnology. 20, 821-825 (2002).</li><li>Yates, M. D., Siegert, M., Logan, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+evolution+catalyzed+by+viable+and+non-viable+cells+on+biocathodes.">Hydrogen evolution catalyzed by viable and non-viable cells on biocathodes.</a> Int J Hydrogen Energ. 39, 16841-16851 (2014).</li><li>Fokina, O., Eipper, J., Winandy, L., Kerzenmacher, S., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+performance+of+a+biofuel+cell+cathode+with+laccase-containing+culture+supernatant+from+Pycnoporus+sanguineus.">Improving the performance of a biofuel cell cathode with laccase-containing culture supernatant from Pycnoporus sanguineus.</a> Bioresource Technol. 175, 445-453 (2015).</li><li>Yates, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermally+activated+long+range+electron+transport+in+living+biofilms.">Thermally activated long range electron transport in living biofilms.</a> Phys Chem Chem Phys. 17, 32564-32570 (2015).</li><li>Yates, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+conductivity+of+living+Geobacter+sulfurreducens+biofilms.">Measuring conductivity of living Geobacter sulfurreducens biofilms.</a> Nat Nano. 11, 910-913 (2016).</li><li>Snider, R. M., Strycharz-Glaven, S. M., Tsoi, S. D., Erickson, J. S., Tender, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+electron+transport+in+Geobacter+sulfurreducens+biofilms+is+redox+gradient-driven.">Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven.</a> Proc Natl Acad Sci USA. 109, 15467-15472 (2012).</li><li>Strycharz-Glaven, S. M., Snider, R. M., Guiseppi-Elie, A., Tender, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+electrical+conductivity+of+microbial+nanowires+and+biofilms.">On the electrical conductivity of microbial nanowires and biofilms.</a> Energ Environ Sci. 4, 4366-4379 (2011).</li><li>Malvankar, N. S., Tuominen, M. T., Lovley, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+"On+electrical+conductivity+of+microbial+nanowires+and+biofilms"+by+S.+M.+Strycharz-Glaven,+R.+M.+Snider,+A.+Guiseppi-Elie+and+L.+M.+Tender,+Energy+Environ.+Sci.,+2011,+4,+4366.">Comment on "On electrical conductivity of microbial nanowires and biofilms" by S. M. Strycharz-Glaven, R. M. Snider, A. Guiseppi-Elie and L. M. Tender, Energy Environ. Sci., 2011, 4, 4366.</a> Energy Environ. Sci. 5, 6247-6249 (2012).</li><li>Malvankar, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+metallic-like+conductivity+in+microbial+nanowire+networks.">Tunable metallic-like conductivity in microbial nanowire networks.</a> Nat Nanotechnol. 6, 573-579 (2011).</li><li>Strycharz-Glaven, S. M., Tender, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+the+'Comment+on+"On+electrical+conductivity+of+microbial+nanowires+and+biofilms"'+by+N.+S.+Malvankar,+M.+T.+Tuominen+and+D.+R.+Lovley,+Energy+Environ.+Sci.,+2012,+5.">Reply to the 'Comment on "On electrical conductivity of microbial nanowires and biofilms"' by N. S. Malvankar, M. T. Tuominen and D. R. Lovley, Energy Environ. Sci., 2012, 5.</a> Energy Environ. Sci. 5, 6250-6255 (2012).</li><li>Strycharz-Glaven, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Transport+through+Early+Exponential-Phase+Anode-Grown+Geobacter+sulfurreducens+Biofilms.">Electron Transport through Early Exponential-Phase Anode-Grown Geobacter sulfurreducens Biofilms.</a> Chem Electro Chem. 1, 1957-1965 (2014).</li><li>Chidsey, C. E., Feldman, B. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+metallic-like+conductivity+in+microbial+nanowire+networks.">Tunable metallic-like conductivity in microbial nanowire networks.</a> Nat Nano. 6, 573-579 (2011).</li><li>Ing, N. L., Nusca, T. D., Hochbaum, A. 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During the setup of the IDA, it is critical to test that the source and the drain are not shorted together prior to electrochemical gating measurements, as this will alter the ISD vs. EG curve and could lead to erroneous results and interpretations. It is also critical to select VSD and v such that the current is linearly dependent on VSD and independent of v. If this is not the case, then the equations described above cannot be utilized to calculate conductivity.
<p class=...

Extracellular electron transport (EET) is a process that enables certain microorganisms to transport electrons between intracellular metabolic processes and insoluble electron acceptors or donors that reside outside the cell, ranging from natural minerals to electrodes. In some cases, EET enables microorganisms to form electrically conductive multi-cell thick biofilms on electrode surfaces, in which cells not in direct contact with the electrode can still utilize it as a metabolic electron acceptor or donor. There is considerable interest in such biofilms as electrode catalysts for various applications, such as microbial electrosynthesis, contaminant sensing/removal, and remote energy generation and storage,7,8,9,10,11,12,13,14 due to the diversity of metabolic processes performed by microorganisms and the durability of microbial biofilms compared to enzyme-based bioelectrodes.15,16 In addition, EET pathways may potentially be utilized to electrically control or signal changes in naturally occurring or genetically engineered microbial metabolic processes involved, for example, in production of a desired product or detection of a target analyte or stimulus. The electrical conductivity of electrocatalytic biofilms, which sets them apart from other biological materials, is a central aspect of their electrocatalytic properties, yet little is understood about the underlying EET process in the electrode environment, and that which is known is highly contested.17,18,19,20,21,22,23,24
Described here is a 2-electrode method to measure conductivity through living, electrode-grown biofilms using interdigitated electrode arrays (IDAs). IDAs consist of parallel rectangular electrodes patterned on flat glass surface such that every other band is connected at opposite sides of the array resulting in 2 electrodes (the source and drain). Careful examination of an IDA (see for example, Figure 6.12b of ref #1) reveals that that the gaps separating adjacent bands are also connected in such a way as to form a single gap that weaves back and forth across the array separating the two electrodes. The result is a long and narrow gap separating the source and drain electrodes, yielding very high source-drain currents when a conductive material is formed, cast, polymerized, or grown (in the case of the type of biofilms considered here) over the array. In addition, the small size of the electrodes results in small background current due to capacitance charging and to change in oxidation state of the conductive material with change in gate potential, since the amount of material needed to make conductivity measurements using IDAs is so small. The technique of IDA-based electrochemical gating described here, developed to characterize thin film conductive polymers,2,3,4,25 has only recently been applied to living systems.18 Another technique used to measure conductivity of living biofilms utilized a large format split source and drain electrodes and source meters to set the gate potential.26,27 However, concerns over these methods have been detailed previously.18
The protocol below encapsulates our experience with making conductivity measurements of living Geobacter sulfurreducens and biocathode MCL biofilms. G. sulfurreducens is a model electrode reducing organism able to use insoluble materials, including electrodes, as the sole metabolic electron acceptor. Additionally, it forms thick biofilms that are able to transport electrons over multiple cell lengths, making it an ideal model organism to study anodic long-distance extracellular electron transfer. We also include details for the study of biocathode MCL, an aerobic, autotrophic mixed community biofilm isolated from the cathode of a benthic microbial fuel cell. Biocathode MCL (named for the three primary constituents &#8211; Marinobacter, Chromatiaceaea and Labrenzia) is capable of oxidizing an electrode as its sole electron donor and transporting electrons over multiple cell lengths, making it an interesting cathodic system to study. Additionally, biocathode MCL has the highest reported conductivity for a living system to date using these methods. The inclusion of these diverse electroactive biofilms in this protocol is meant to highlight that this technique is applicable to measure the transport of electrons through any living biofilm able to electrically interact with electrodes.

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			<td height="21" style="height:21px;width:259px;">IDAs</td>
			<td style="width:192px;">CH Instruments</td>
			<td style="width:219px;">012125</td>
			<td style="width:337px;">Manufactured by ALS-Japan; sold by CH Instruments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Wire</td>
			<td style="width:192px;">Digikey</td>
			<td style="width:219px;">W7-ND</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Conductive silver epoxy</td>
			<td style="width:192px;">Electron microscopy sciences</td>
			<td style="width:219px;">12670-EE</td>
			<td></td>
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			<td height="21" style="height:21px;width:259px;">Insulating material</td>
			<td style="width:192px;">3M</td>
			<td style="width:219px;">2131-B</td>
			<td>Scotchast flame retardant compound</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">15 mL conical centrifuge tube</td>
			<td style="width:192px;">VWR</td>
			<td style="width:219px;">89004-368</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">21g needle</td>
			<td style="width:192px;">VWR</td>
			<td style="width:219px;">BD-305165</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">5 mL pipette tips</td>
			<td style="width:192px;">VWR</td>
			<td style="width:219px;">82018-842</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">5 mL pipettor</td>
			<td style="width:192px;">VWR</td>
			<td>89079-976</td>
			<td></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Freshwater medium components</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:219px;">All standard laboratory chemicals</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Ammonium chloride</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium phosphate monobasic</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium bicarbonate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Artificial seawater medium components</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:219px;">All standard laboratory chemicals</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium chloride</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Magnesium chloride hexahydrate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Magnesium sulfate heptahydrate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Potassium chloride</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium bicarbonate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Calcium chloride dihydrate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Ammonium chloride</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Potassium phosphate dibasic</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
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			<td style="width:192px;">Basi</td>
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			<td height="21" style="height:21px;width:259px;">Recirculating water bath</td>
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			<td>152-5256</td>
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			<td height="21" style="height:21px;width:259px;">Bipotentiostat</td>
			<td style="width:192px;">Pine Instruments</td>
			<td style="width:219px;">WD-20</td>
			<td>http://www.voltammetry.net/pine/aftermath/user</td>
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			<td style="width:192px;">ATCC</td>
			<td style="width:219px;">51573</td>
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</table>

characterizing electron transport through living biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

IDAs were wired, insulated and tested to ensure that the two electrodes were electrically isolated from each other (Figure 1). Reactors were assembled, inoculated with G. sulfurreducens, and incubated until a biofilm bridged the gap between the electrodes. The G. sulfurreducens biofilm can be visually seen to be covering the array. Other biofilms may require the researcher to do an electrochemical gating measurements to see if the two electr...

Watch this Scientific Journal Video about Characterizing Electron Transport through Living Biofilms at JoVE.com

Characterizing Electron Transport through Living Biofilms

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

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