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>
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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 ...

The overall goal of this experimental procedure is to determine the amount of current that can be conducted through a living material in situ as well as the mechanism employed by the living material to transport the current. This method could help answer key questions in the field of microbial electrochemistry such as, How can certain biofilms move electrons over hundreds of microns to support cellular metabolism? The main advantage of this technique is that the interdigitated electrodes give a very high signal-to-noise ratio and can be scaled to fit into many different reactor configurations.Those method can provide insight into multi-cell length extracellular electron transport in biofilms. It can also be applied to other systems, such as electrically conductive polymer films. Researchers new to this method will struggle because it requires a bipotentiostat to set the potentials of both electrodes separately and measure current at each electrode separately.To begin, obtain commercially available IDA electrodes patterned on a non-conductive substrate or synthesize them using standard lithographic methods. Prepare conductive silver epoxy according to the manufacturer's instructions with a mixing rod or pipette tip. Then, place a wire on each gold pad and secure them in place using lab tape.Cover the wire and pad with silver epoxy using a mixing rod or pipette tip. Carefully move the apparatus to an 80 degree Celsius oven for one hour to cure. After the epoxy cures, use a multimeter to ensure electrical conductivity between the end of the wire and the pads.The resistance between the wire and the pads should be less than five ohms. Also, use the multimeter to verify that no conductive epoxy is connecting multiple electrode pads, as this can short the array. If conductive epoxy is connecting multiple leads, use the scribe to isolate the leads.Next, remove the tip of a 15 milliliter conical centrifuge tube to use as a mold for the insulating material. Use a 21 gauge needle to make two small holes in the bottom of the mold for the wires to protrude through. Insert the IDA into the mold and insert the wires through the holes in the bottom of the mold.Next, prepare a thermal electrical and waterproof insulating material. Flame retardant polyurethane resins are often suitable. Pipette the insulator into the mold so that the silver epoxy is completely covered and allow the insulator to dry according to manufacturer's specifications.To set up the electrochemical cell, insert the IDA, counter electrode, and reference electrode into the electrochemical cell. Fill the electrochemical cell with sterile medium suitable for biofilm growth. For Geobacter sulfurreducens, use freshwater medium excluding fumarate.Next, connect the electrodes to a bipotentiostat. Connect one IDA electrode to the working lead one, the other IDA electrode to the working lead two, the reference electrode to the reference lead, and the counter electrode to the counter lead. Perform a control experiment by performing cyclic voltammetry on electrode one and holding electrode two at open circuit to ensure that the electrodes are not connected.To grow the relevant electroactive biofilm, inoculate the electrochemical reactor from a stock culture or enrichment of the desired electrochemically active microorganisms using standard aseptic microbiological techniques. For standard tests, inoculate in a 1 to 20 ratio of inoculum-to-reactor volume. Set the stirring in the reactor to the desired level and set the incubator or water bath to the desired temperature based on the growth conditions of the biofilm of interest.Incubate the system based on specific requirements of the microorganism of interest until the biofilm bridges the gap separating the two electrodes. For stationary Geobacter sulfurreducens biofilm, incubate for seven to ten days at 30 degrees Celsius. Begin setup of the experimental parameters that will be used to determine the current potential dependence for the gating measurements as described in the text protocol.Set up the bipotentiostat software to perform the gating measurements over the selected range at the selected source drain voltage and at the desired scan rate. Additionally, perform a baseline measurement as described in the text protocol. Perform gating measurements using the same conditions under turnover with soluble electron donor or acceptor present in non-turnover without soluble electron donor or acceptor conditions.Obtain a control reactor to ensure that the set point and actual temperature of the medium is the same. Place a thermometer or a thermocouple in a control reactor where the IDA would be. Then, make source strain current measurements over the range of temperatures selected under turnover and non-turnover conditions using the bipotentiostat.To do so, set and hold the IDA at the gate potential that yields maximum conducted current using the bipotentiostat. Record the maximum conducted current using the bipotentiostat while the temperature is cycled between the range of temperatures selected using the onboard controls of the water bath or incubator. Cycle the temperature from one set point to the other and back against using the onboard controls of the incubator or the water bath.This determines the reversibility of the reaction to ensure that the temperature cycling does not harm the biofilm. Finally, reset the temperature back to the normal growth temperature and allow the system to stabilize. Shown here are representative results of an IDA that is functioning properly in freshwater medium.Versus one where the electrodes have been shorted. Representative electrochemical gating measurements performed on a Geobacter sulfurreducens biofilm under non-turnover conditions with a source strain voltage of 10 millivolts exhibit the peak shaped conducted current versus gate potential curve characteristic of redox conductivity. Here, representative raw data shows the dependence of the conducted current on the temperature of the reactor medium.The decrease in conducted current with decreasing temperature is another characteristic of redox conductors. Once mastered, this technique can be done in approximately two days if it is performed properly. While attempting this procedure it is important to remember to make sure that the IDA bands are not shorted prior to inoculating the reactor.The implications of this technique extend towards therapy when applied to study how to disrupt cellular metabolism of biofilm forming pathogens. This procedure can be applied to any living material able to transport electrons in order to address additional questions, such as the correlation between biofilm refology and conductivity.

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