Name: characterizing electron transport through living biofilms
Uploaded: 2018-06-01T12:30:00
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M.D.Y og S.M.G-S. LMT erkjenner den Office of Naval Research (Award #N0001415WX01038 og N0001415WX00195), den Naval Research Laboratory og den Naval Research Laboratory Nanosciences Institute; M.Y.E.-N. støttes av den amerikanske avdelingen av energi Grant DE-FG02-13ER16415.

1. interdigitated microelectrode matrise (IDA) forberedelse
<ol><li>Få kommersielt tilgjengelig IDA elektroder mønstret på et nonconductive substrat eller syntetisere dem ved hjelp av standard litografisk metoder. 28 Merk: IDA dimensjoner og/eller materialer kan varieres basert på ønskede vilkår for forskjellige eksperimenter. IDAs brukes her ble innhentet kommersielt og besto av to interdigitated gull microelectrodes mønster fra en barometer substrate koblet til store elektrode pads p...

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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. 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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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Under installasjonen av IDA er det viktig å teste at kilden og avløpet ikke er kortsluttet sammen før elektrokjemiske gating målinger, da dette vil endre ISD vs EG kurve og kan føre til feilaktige resultater og tolkninger. Det er også viktig å velge VSD og v slik at gjeldende lineært avhengig av VSD og uavhengig av v. Hvis dette ikke er tilfelle, kan ikke deretter ligningene beskrevet ovenfor brukes til å beregne ledningsevne.
Minst to bakgr...

Ekstracellulære elektronet transport (lunsj) er en prosess som gjør at visse mikroorganismer å transportere elektroner mellom intracellulær metabolske prosesser og uløselig elektron acceptors eller givere som bor utenfor cellen fra naturlige mineraler til elektroder. I noen tilfeller kan EET mikroorganismer til elektrisk ledende flercellede tykk biofilm på elektroden overflater, der cellene ikke i direkte kontakt med elektroden kan fortsatt utnytte det som en metabolsk elektron acceptor eller donor. Det er betydelig interesse slik biofilm som elektrode katalysatorer for forskjellige programmer, for eksempel mikrobiell electrosynthesis, forurensning sensing/fjerning, og eksterne energi og lagring,7,8,9 ,10,11,12,13,14 på grunn av mangfoldet av metabolske prosesser av mikroorganismer og holdbarheten av mikrobielle biofilm forhold til enzym-baserte bioelectrodes. 15 , 16 i tillegg EET trasé kan potensielt utnyttes til elektrisk kontroll eller signal endringer i naturlig forekommende eller genmodifisert mikrobiell metabolske prosesser involvert, for eksempel i produksjon av en ønsket produkt eller deteksjon for en målet analytt eller stimulans. Elektrisk ledningsevne electrocatalytic biofilm, som skiller dem fra andre biologiske materialer, er et sentralt aspekt av electrocatalytic egenskaper, men lite forstått om underliggende EET prosessen i elektrode-miljøet og det som er kjent er svært omstridt. 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24
Beskrevet her er en 2-elektrode metode for å måle ledningsevne gjennom levende, elektrode dyrket biofilm bruker interdigitated elektrode matriser (IDAs). IDAs består av parallelle rektangulære elektroder mønstret på flat glassoverflaten slik at alle andre band er koblet på motsatte sider av array resulterer i 2 elektroder (kilde og avløp). Nøye undersøkelse av en IDA avslører (se for eksempel figur 6.12b av ref #1) at hullene mellom tilstøtende band er også koblet på en slik måte at skjemaet en eneste gap som vever frem og tilbake over array skille de to elektrodene. Resultatet er en lang og smal avstand skiller source og elektrodene, gir svært høy kilde strømforbruk strøm når en ledende materiale er dannet, kastet, polymerized eller vokst (ved av biofilm anses her) matrisen. Dessuten, resulterer den lille størrelsen på elektrodene i liten bakgrunn gjeldende kapasitans lading og endre oksidasjonstallet for det ledende materialet med endring i gate potensial, siden mengden av materiale for å gjøre ledningsevne mål med IDAs er så liten. Teknikken av IDA-baserte elektrokjemiske gating beskrevet her, utviklet for å karakterisere tynnfilm ledende polymerer,2,3,4,25 har nylig brukt på levende systemer. 18 en annen teknikk som brukes til å måle ledningsevne levende biofilm benyttes et stort format source og elektroder og kilde meter å sette porten potensielle. 26 , 27 men bekymringer over disse metodene har blitt beskrevet tidligere. 18
Protokollen nedenfor sammenfatter vår erfaring med å lage ledningsevne målinger av levende Geobacter sulfurreducens og biocathode MCL biofilm. G. sulfurreducens er en modell elektrode redusere organisme bruke uløselig materialer, inkludert elektroder, som den eneste metabolske elektron acceptor. I tillegg danner tykke biofilm som kan transportere elektroner over flere celle lengder, noe som gjør det til en ideell modell organisme til å studere anodic langdistanseløpet ekstracellulære elektron overføring. Vi har også detaljer for studier av biocathode MCL, en aerobic, autotrophic blandet samfunnet biofilm isolert fra katoden en bunnlevende mikrobiell brenselcelle. Biocathode MCL (oppkalt etter tre viktigste bestanddelene- Marinobacter, Chromatiaceaea og Labrenzia) er i stand til oksiderende en elektrode som sin eneste elektron donor og transport elektroner over flere celle lengder, gjør det en interessant Katodisk system å studere. I tillegg har biocathode MCL høyest rapporterte ledningsevne for et levende system ennå med disse metodene. Inkludering av disse ulike electroactive biofilm i denne protokollen er ment å markere at denne teknikken er anvendelig å måle transport av elektroner gjennom noen levende biofilm elektrisk samhandler med elektroder.

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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</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="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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Ag/AgCl reference electrode</td>
			<td style="width:192px;">Basi</td>
			<td style="width:219px;">MF-2079</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Graphite rod counter electrode</td>
			<td style="width:192px;">Electron microscopy sciences</td>
			<td>70230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Recirculating water bath</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td>152-5256</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Stir bars</td>
			<td style="width:192px;">VWR</td>
			<td>58947-114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">G. sulfurreducens culture</td>
			<td style="width:192px;">ATCC</td>
			<td style="width:219px;">51573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Jacketed reactor</td>
			<td style="width:192px;">Pine Instruments</td>
			<td>RRPG085</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing electron transport through living biofilms

Her viser vi metoden elektrokjemiske gating benyttes for å karakterisere elektrisk ledningsevne av elektroden dyrket mikrobiell biofilm fysiologisk relevante vilkår. 1 disse målingene utføres på levende biofilm i vandig medium bruker kilde og avløp elektroder mønstret på en glassoverflate i en spesialisert konfigurasjon som tabelldata interdigitated elektrode (IDA). En biofilm er dyrket som strekker seg over gapet koble kilden og avløp. Potensialer brukes elektrodene (ES og ED) genererer en kilde strømforbruk gjeldende (ISD) gjennom biofilm mellom elektrodene. Avhengighet av elektrisk ledningsevne på gate potensial (gjennomsnittet av source og potensial, EG = [ED + ES] / 2) bestemmes av systematisk endre porten potensielle og måle den resulterende kilde strømforbruk gjeldende. Avhengighet av konduktivitet på gate potensielle gir mekanistisk informasjon om ekstracellulære elektronet transport prosessen underliggende elektrisk ledningsevne den bestemte biofilm under etterforskning. Elektrokjemiske gating måling metoden beskrevet her er basert på som brukes av M. S. Wrighton2,3 og kolleger og R. W. Murray4,5,6 og kolleger i 1980 er for å undersøke tynnfilm ledende polymerer.

IDAs var kablet, isolert og testet for å sikre at de to elektrodene var elektrisk isolert fra hverandre (figur 1). Reaktorer var samlet, inokulert med G. sulfurreducens, og inkubert til en biofilm Brokoblet gapet mellom elektrodene. G. sulfurreducens biofilm sees visuelt skal dekke matrisen. Andre biofilm kan kreve forskeren å gjøre en elektrokjemisk gating mål å se om de to elektrodene har elektrisk koblet. Mikroskopi bør også brukes...

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

Characterizing Electron Transport through Living Biofilms

En protokoll for å måle elektrisk ledningsevne av levende mikrobiell biofilm fysiologisk relevante vilkår er presentert.

Karakterisere elektronet Transport gjennom levende biofilm

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

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