Biosensors and Bioelectronics 26 (2011) 37423747

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Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .co

Charac thas the b

Nicholas , GoRavi Gooa Faculty of Agr w Zeab Department oc Universit ded Leibniz Institu , Germe Lincoln Ventuf School of Biol

a r t i c l

Article history:Received 1 DeReceived in reAccepted 9 February 2011Available online 16 February 2011

Keywords:Microbial fuel cellYeastMediated tranMediator-lessSecreted redox

d littly nonre w

transfers electrons to anelectrode through the secretionof a reducedmolecule that is notdetectablewhenwashed cells are rst resuspended but which accumulates rapidly in the extracellular environment. It is asingle molecule that accumulates to a signicant concentration. The occurrence of mediatorless electrontransfer was rst established in a conventional microbial fuel cell and that phenomenon was furtherinvestigated by a number of techniques. Cyclic voltammetry (CV) on a yeast pellet shows a single peak

1. Introdu

Most mias the biocstruction, fuHigson, 200fuel cells thture. Bennefor use in scanodemediacceptorbulications froS. cerevisiaefuel cell as limited in thdifference bblue (0.23

AlthoughS. cerevisea

CorresponE-mail add

0956-5663/$ doi:10.1016/j.sfertransfermolecule

at 450mV, a scan rate study showed that the peak was due to a solution species. CVs of the supernatantconrmeda solution species. It appears that, given its other attributes,A. adeninivorans is a good candidatefor further investigation as a MFC catalyst.

2011 Published by Elsevier B.V.

ction

crobial fuel cell development has focused bacterial cellsatalyst and many reports are available on their con-nction and performance (Bullen et al., 2006; Davis and7; Logan et al., 2006; Logan, 2009). Reports onmicrobialat use eukaryote cells are however rare in the litera-tto (1990) described a Saccharomyces cerevisiae fuel cellhool science. The fuel cell used methylene blue as theator andpotassium ferricyanide as the cathode electrontwasdescribedas sluggish. Signicantly, all otherpub-m Bennetto involve prokaryote cells as the biocatalyst.was also described as a sluggish biocatalyst and the

lethargic by Wilkinson (2000). These fuel cells are alsoat themaximumpotential of the cell is restricted by theetween the E0 values (vs Ag/AgCl at pH 7) of methyleneV) and ferricyanide (0.28V).most early yeast MFC research was performed with

e, it is not an ideal organism. An important prereq-

ding author. Tel.: +64 3 364 2987x7029; fax: +64 3 364 2590.ress: keith.baronian@canterbury.ac.nz (K.H.R. Baronian).

uisite for any MFC is the ability of the organism or consortia oforganisms to completely oxidise the substrate to make availablethe maximum number of electrons. Yeast can have a fully respira-tory or fully fermentative catabolism and in some cases a mixedrespiratory-fermentative catabolism. S. cerevisiae operates a mixedfermentation/respiration mode even in the presence of oxygen,the natural terminal electron acceptor for the electron transportchain. In S. cereviseae the ratio of fermentation to respiration variesbetween strains but is approximately 80:20, respectively. Furtherin some yeast the production of ATP in TCA and electron trans-port/chemiosmosis isnotmaximal, e.g.S. cereviseaeproducesa totalof 14 ATP per glucose molecule in mitochondrial process, whichis well short of the typical net 2830 achieved by most aerobes.Most of the substrate energy thus remains in the end product offermentative pathway. The situation from the MFC perspective isworse when a soluble terminal electron acceptor is not available,for example in anaerobic conditions and fermentation becomes thesole catabolic pathway that produces ATP. Theoretically only about5% of the energy available in glucose is converted to ATP and themajority of the energy and thus the number of electrons availablethrough oxidation of the substrate, remains in the products of fer-mentation. Furthermore the absence of functional mitochondriain anaerobic conditions removes the pathways required for non-

see front matter 2011 Published by Elsevier B.V.bios.2011.02.011terisation of yeast microbial fuel cell wiiocatalyst

D. Hasletta, Frankie J. Rawsonb, Frdric Barrirec

neratnea, Keith H.R. Baronianf,

iculture and Life Sciences, P.O. Box 84 Lincoln University, Lincoln 7647, Canterbury, Nef Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New ZealandRennes 1, CNRS UMR 6226, Sciences Chimiques de Rennes, Equipe MaCSE, Francete of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gaterslebenres Ltd., P.O. Box 133, Lincoln, Christchurch 7640, New Zealandogical Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand

e i n f o

cember 2010vised form 21 January 2011

a b s t r a c t

Yeastmicrobial fuel cells have receivethey are robust, easily handled, mostlcases a broad substrate spectrum. Hem/locate /b ios

the yeast Arxula adeninivorans

tthard Kunzed, Neil Pascoe,

land

any

e attention to date. Yeast should be idealMFC catalyst because-pathogenic organisms with high catabolic rates and in somee show that the non-conventional yeast Arxula adeninvorans

N.D. Haslett et al. / Biosensors and Bioelectronics 26 (2011) 37423747 3743

conventional substrates such as pentoses, sugar alcohols, organicacids, aliphatic alcohols, hydrocarbons and aromatic compounds(Walker, 1998). Lipids are ultimately degraded by glycolysis andvia the glyoxalate cycle, and amino acids are degraded to ammo-nium and gor the TCAwill limit thtotal produimately theis achievedtion.

Yeast fuin general,bacterial fudida melibioand ferricy0.18Wm3

tion of a casignicantlis much hibare carbonthe bare cator (Hubenvarious comsium ferriccerevisiae mof 146.71tial of 383the fuel ceblue was tments. ChiaMFC using(2006) usinreport thatdensity ofcoated grapas the biochad maximrespectively

Halme afuel cell. Thsupernatanshowed thamedium by120Wcm(12h).

Higherand Dunn (visiae MFCto well pericantly less(2008).

The selebe made cabetweenspIn this sturans Kurtphysiologic48 C), pH tance (up torange (Bewhen usingsubstrate.

Potassiuacceptor fowith very h

Under acidic conditions the reduction half-reactions are:

MnO4 + 4H+ + 3e MnO2 + 2H2O E0 = 1.68V (1) + 8H+ 2+ 0

ird h

+ 2H

useore

teria

lls

denibnizlebenC sotaine

ltiva

deniat 3bro

0g Lcf, 8en re4/KHed foC.

ls forve ee of Peparrpm

agen

assiussolvlenedM, ad wa

icrob

fuel1984tinuod theiame18merealco

edbyranethe athodfuelctedincludigitlutamate, which is either catabolised via fermentationcycle. In both cases the inhibition of mitochondriae catabolism of lipids and amino acids. Although thection of electrons per unit time may remain approx-same in both anaerobic and aerobic conditions, itby higher throughput of the substrate in fermenta-

el cells have received renewed attention recently, butyeast MFCs still have a lower power output than

el cells. Hubenova and Mitov (2010) report that a Can-sica mediated MFC with methylene blue in the anodeanide in the cathode gave power densities of up to. In another report they describe how the modica-rbon felt electrode with surface nickel nanostructuresy increased the power output to 720mWm2, whichgher than the 36mWm2 that was achieved with a

felt electrode and the 370mWm2 achieved withrbon felt electrode and methylene blue as a media-ova et al., 2010). Gunawardena et al. (2008) exploredbinations of water (O2), methylene blue and potas-

yanide as mediators and electron acceptors in a S.icrobial fuel cell. It generated a maximum power7.7mWm3 with a maximum open circuit poten-

.61.5mV. The maximum operational efciency ofll was 281.8% which occurred when methylenehe mediator in both anode and cathode compart-o et al. (2006) report 2.3nWcm2 for micro machinedS. cerevisiae as the biocatalyst. Walker and Walkerg S. cerevisiae report

3744 N.D. Haslett et al. / Biosensors and Bioelectronics 26 (2011) 37423747

2.5. Voltammetry

Voltametry was performed using an eDAQ potentiostat andeDAQ power lab 2/20 with eCHEM data acquisition software.

Linea Swcomprisingauxillary elperformedwith the valat +400mVon Lecloth w

Cyclic vobon electroauxiliary eltically withwas wrappelectrode suthe well atthe electrodthe electrod0.1M glucosupernatansample.

The scanfrom250 toperformed

2.6. Retenti

Cellsweor with ferrcose. Analys(LSV). At thin PBS at 4cyanide andconditionsanalysed by

3. Results

3.1. Retenti

Fig. 1 shtor responsstatic reeccatabolism.response toto any decli

3.2. Mediatfuel cell ano

The fuelPBS only inthePBSwas0.5M in PBSon the addithrough thethat is abovin power afby a decay t

3.3. Mediat

There isand A. aden

.1

1

0

100

1000

10987654321

time (days)

ferricyanide

ferricyanide, glucose

ferricyanide, TMPD

ferricyanide, TMPD, glucose

etention of TMPD by A. adeninivorans cells. Cells were incubated for 1h atth TMPDand ferricyanide, orwith ferricyanide alone, bothwith andwithoutand the supernatant was analysed by LSV. At the end of the experiment cellsshed and stored in PBS at 4 C until next use. For the next experiment freshide and glucose were added to the cells to replicate their supply in the dayment, incubated for 1h at 37 C and the supernatant analysed by LSV. Thisre was repeated on days 6 and 9. The difference in the with and withouttrialsthe m

.02

.01

0

.01

.02

.03

.04

.05

.06

.07

.08

.09

50403020100

time (min)

4

etection of mediatorless transfer of electrons from A. adeninivorans to thee. The fuel cell was run for 15min without a cathode electron acceptor.of KMnO4 to the cathode at 15min results in a surge of current followedto a level above that seen in the rst 15min. Control shows a small peak onof KMnO4 followed by a decay to the 015min levels. Electrode geometricrea 0.001018m2.

). Although the maximum growth temperature for S. cere-s usually given as 37 C, catabolism operates as usual up toWalker and Walker, 2006) and its low power output is notthe elevated temperature in the MFC.

0

.005

0.01

.015

0.02

.025

0.03

.035

302520151050

time (min)

A. adeninivorans

S. cerevisiae

Acellular control

ower output in mediator-less S. cerevisiae and A. adeninivorans MFCs. Cell2.5, temp. 37 C, rotated at 80 rpm, external resistance 100. Electrode geo-urface area 0.001018m2. (Error bars represent 1SE of the mean, n=9.)eep Voltammetry (LSV) used a three-electrode systemof a Pt micro-disc electrode (100M diameter), a Pt

ectrode and a Ag/AgCl reference electrode. Scans werefrom +425mV to +100mV at a scan rate of 10mVs1

ues reported as the mean steady state current recordedfrom three LSVs. The Ptworking electrodewas polishedith 0.05m alumina before each measurement.

ltammogramswereperformedusinga3mmglassy car-de, Ag/AgCl reference electrode and a platinum wireectrode. The glassy carbon electrode was mounted ver-the electrode surface uppermost. The electrode body

ed in Sellotape to form a well 2mm deep above therface. The reference and auxiliary electrodes enteredangles from above. Cells were allowed to settle ontoe surface before executing analysis. Total volumeabovee was 100L comprising 75L of cell cream, 10L ofse and 15L of PBS buffer. CVs of YEPD and growtht were the controls with 25L of PBS added to 75L of

rate was 100mVs1 scanning from 0.5V to 0.8V and650mV at a scan rate of 50mVs. Scan rate studieswereat intervals from 2mVs1 to 400mVs1.

on of TMPD

re incubated for1hat37 CwithTMPDand ferricyanide,icyanide alone, both in the presence and absence of glu-is of the supernatant was by linear sweep voltammetrye end of the experiment, cells were washed and storedC until next use. For the next experiment, fresh ferri-glucosewere added to replicate theday1experimental

and samples were incubated for 1h at 37 C and againLSV. This procedure was repeated on days 6 and 9.

on of TMPD by A. adeninivorans cells

ows that there is a rapid decrease in the double media-e while the single mediator responses remain almostting the continuous production of NADH/NADPH byWe thus attribute the decline in the double mediatora loss of the lipophilic mediator from the cells and notne in catabolism.

or-less electron transfer from A. adeninivorans to thede

cell was set up with organisms in PBS in the anode andthe cathode and allowed to run for 20min. At 20minwithdrawn fromthe cathodeand replacedwithKMnO4. A control without cells was also run. Fig. 2 shows thattion of KMnO4 to the cathode there is a surge in powerexternal circuit followed by a decay to a constant level

e the 020min level. The control shows a smaller surgeter the addition of KMnO4 at 20min but it is followedo pre 20min levels.

or-less fuel cell power output

a clear difference in the power output from S. cerevisiaeinivorans fuel cells operating in mediator-less mode

0

1

i (nA

)

Fig. 1. R37 Cwiglucosewere waferricyan1 experiproceduglucose1SD of

-0

-0

0

0

0

0

0

0

0

0

0po

wer

den

sity

(w

m-2

)

Fig. 2. DelectrodAdditionby decayadditionsurface a

(Fig. 3visiae i45 C (due to

0

0

0

0

pow

er d

ensi

ty (

Wm

-2)

Fig. 3. POD600 =metric sconrms the responses are from catabolism. (Error bars representean, n=9.)

KMnO

N.D. Haslett et al. / Biosensors and Bioelectronics 26 (2011) 37423747 3745

0

20

40

60

0

i (nA

)

Fig. 4. Incubatferricyanide,wn=9.) The diffeare from catab

Two expobtained inadeninivorapresence anorganisms ilength of tiglucose dem

The secorans, was peither fromfrom a largecell creamsA. adeninivobut anequivcream.

Fig. 5b shobtained wpeak currenrate indicatcontrol andbe from a so

ThiswasPBS) and on0 i.e. as soonot presentpresent in bthe molecuthat signicdid not occcontinued dsupernatanof the electpeak potenand is show

4. Discussi

We havemediators(Baronian eto our MFCator was disno disadvan

A) CVV to 80mVoot of scan rate. Scan rates were from 2 to 400mVs1.

issue of the use of mediators in continuous ow MFCs hasidely discussed. For example, Schroeder (2007), states inC review that due to a number of severe disadvantages,diated MFC has, with the exception of some fundamentalh, been generally abandoned. Themain problem is the needular addition of the exogenous mediator, which is techno-ly unfeasible and environmentally questionable.maximum power density we have observed with TMPD as

odic mediator and KMnO4 as the cathode reductant in theinivorans MFC is 1.030.06Wm2 which is close to thet yeast MFC output reported by Ganguli and Dunn (2009).724824time (h)

A. adeninivoransA. adeninivorans + gluS. cerevisiaeS. Cerevisiae + glu

ion of S. cerevisiae andA. adeninivoranswith thehydrophilicmediator,ith andwithout glucose (glu). (Error bars represent1SEof themean,rence in the with and without glucose trials conrms the responsesolism.

eriments were undertaken to investigate the resultsSection 3.3. First an incubation of S. cerevisiae and A.

ns with the hydrophilic mediator ferricyanide in thed absence of glucose shows that the response of boths essentially the same and this result persisted over theme a batch MFC may operate (Fig. 4). The response toonstrates that this is a signal generated by catabolism.nd, cyclic voltammetry of S. cerevisiae and A. adeninivo-erformed to search for the presence of redox signals,the cells in PBS or from a small volume of supernatantnumber of cells. Typical voltammograms obtained for

of A. adeninivorans and S. cerevisiae are shown in Fig. 5a.rans Cvs have an irreversible oxidation peak at +0.42Valentpeakwasnot observed inCVsof S. cereviseaeyeast

ows aplot of the faradaic current for thepeak at +0.42Vith A. adeninivorans cell cream at varying scan rates. Thets obtainedwere proportional to the square root of scaning thatmagnitudeof thepeakcurrent isunderdiffusionit can be inferred from this nding that the peak mustlution species.conrmedbyperforming CVs on the cell cream (cells inthe supernatant from the cream. This was done at timen as the cream was produced and at 1h. The peak was

Fig. 5. (500mble at 42square r

Thebeen whis MFthe meresearcfor reglogical

Thethe anA. adenhighesin either the cream or the supernatant at 0h but wasoth the creamand the supernatant at 1h indicating thatle accumulated in the extracellular buffer over time andantdirect electron transfer fromthecell to theelectrodeur. Accumulation of the molecule in the supernatanturing an18h incubation. The peak was larger in the

t than in the cream suggesting that the cells block somerode area in the cream CVs. The 1h supernatant anodictial was 0.45V with an anodic peak current at 4.4An in Fig. 6.

on

used a system comprising lipophilic and hydrophilicto detect large substrate dependant signals in yeastt al., 2002). Initially the system was simply transferredinvestigation but in the process, the hydrophilic medi-carded because it reduced the potential of the MFC andtages were noticed by it absence.

Fig. 6. Cyclic vtrifuged in a m50mVs1.s of cell creams of A. adeninivorans (A) and S. cerevisiae (B) from00mV (vs Ag/AgCl) at 100mVs1. A peak for A. adeninivorans is visi-. (B) A plot of peak currents obtained for Arxula cell cream versus theoltamogram of the supernatant from A. adeninivorans cell cream cen-icrofuge, 13,000 rpm for 10min. The scan was from 250 to 650mV at

3746 N.D. Haslett et al. / Biosensors and Bioelectronics 26 (2011) 37423747

This suggested that an investigation into the retention of medi-ator by the cell should be made because if the lipophilic mediatorremainedassociatedwith the cell, theproblemsassociatedwith theuse of mediators in continuous ow MFCs referred to above, maybe at least pous ow MFcurrent in tsignals fromthe wholeator was racurrent. Ferand the resduring thewhen glucocatabolism.

As stateyeast MFCsmediator-lerates, howeand similarmesophilicprobably du

The resmediator-leelectrode. Bintracellulafrom contathat surrouble methodmolecule.

Recent sregarding mmajority ofthe cell to tthe cell anda dual cathwith the ceseparated fhowever inproduce. Thcells produionised watnot diffusinthe 2nd anferred by da short-livethe impact.transfer froP. anomalaand entrapage of cellsmonolayertrodes gaveenzymes thdrogenase(2010) repoadded medmetabolitesthis type oet al., 2010)

This stuthe mediatocerevisiae wreported foZhang (199ently in the

was investigated by rst checking the magnitude of reduction of ahydrophilic mediator by both species. Fig. 4 shows that the reduc-tion of ferricyanide is essentially the same by both species, bothwith and without glucose, which suggests that the quantity of

ns avande coninivoot innextcanut thcantectaicyathe

ed incan rle wtrifur pealecundenely apresand satanevidxulaidesthealso

en pre tranretiolikelytputFC co

clus

st shave

ionsanyof thoutpns aivoraS. ceto t

inivo

wled

s woew

mmeandch, S3838n, NZfor h.artially negated. We attempted to simulate a continu-C and Fig. 1 shows that there is a dramatic reduction ofhe lipophilic trial over a short period of time while the

the ferricyanide only trials remained constant overexperiment. It seems likely that the lipophilic medi-pidly lost to the environment resulting in the loss ofricyanide accesses the membrane surface tPMETs onlyponses indicate that the cells are functioning normallyexperimental period and the larger signals detectedse was included conrmed that the signals were from

d in Section 1, the power outputs reported for mostare comparatively low. The low power output of thesess yeast MFCs has been attributed to low catabolicver, the high currents seen in the mediated yeast MFC,growth rates suggest the catabolic rates in yeast andbacteria are similar and the very low power outputs aree to the difculty in accessing intracellular electrons.

ult of the TMPD experiment led us to investigatess transfer of electrons from the yeast cell to theecause most eukaryote redox molecules are locatedrly and cell membrane tPMETs are probably shieldedct with an electrode by a 100200nm thick rigid wallnds the cell membrane, it seemed that the only possi-of mediator-less transfer would be via a secreted redox

tudies, however, have arrived at different conclusionsediator-less electron transfer to an electrode. Thereports support the idea that electron transfer from

he electrode is by some kind of direct contact betweenthe electrode. Ducommun et al. (2010) constructed

ode and dual anode MFC. The anode A had contactlls in a glucose water medium whereas anode B wasrom the cells by dialysis membrane. Anode B couldteract with soluble redox molecules that the cells mayey report that the anode that had contact with theced current whereas the anode in contact with de-er did not, indicating that a soluble redox species wasg from the cells through the dialysis membrane toode. They speculate that electrons are either trans-irect contact of the cell with the electrode or thatd electro-active molecule is released at the time ofPrasad et al. (2007) also support the notion of directm the cell surface to the electrode. They immobilizedcells by two different methods; physical adsorptionment of cells on a gold electrode and covalent link-to a gold electrode modied with a self-assembled

of cystamine. They report that CVs with these elec-peaks that corresponded to the potential of redox

at were isolated from the cell membrane (lactate dehy-and ferrireductase). Conversely Hubenova and Mitovrt that Candida melibiosica gave a small current with noiator and concluded that it was probably from secretedacting as electron shuttles and provided evidence for

f transfer in their subsequent publication (Hubenova.dy attempted to resolve the question of the origin ofr-less MFC signal seen with A. adeninvorans (Fig. 3). S.as added as a reference organism because of the resultsr that yeast by Ducommun et al. (2010) and Halme and5). A. adeninivorans and S. cerevisiae performed differ-mediator-lessMFC. The difference in their performance

electrosimilarin thesA. adendoes n

TheA peakscan, bvisiae sany defor ferroxidisedetect

A smolecuby cena largethe moindepepositivit wasducedsupern

Thethat Arit provpeak inan MFC

Givthat ththe secis alsocell ouand M

5. Con

Yeasome hconditand msomepowerelectroadeninthan abe dueA. aden

Ackno

Thiety of NProgranologyResearPROJ-1missioOriezmentsailable to ferricyanide at the cell membrane surface ismay also indicate that catabolic rates of the two speciesditions are similar (Fig. 5a). The higher current seen inrans in the MFC must therefore be from a species thatteract with ferricyanide.checkwas toperformaCVof cell pellets of both species.

be seen at 0.42V vs Ag/AgCl pH 7 in the A. adeninivoransere is no corresponding peak observable in the S. cere-(Fig. 5b). Ganguli and Dunn (2009) also did not reportble peaks in CVs of S. cereviseae. The half wave potentialnide vs Ag/AgCl is 0.28 V at pH 7 and thus it would notA. adeninivorans molecule and it would not have beenthe ferricyanide investigation above.ate study of the yeast cream (Fig. 6) showed that theas soluble. The removal of cells from the yeast creamgation at 13,000 rpm produced a supernatant that hadk, suggesting that removal of cells permitted more of

le to interact with the electrode. The peak potential wast of pH within the range of pH 412 but it did shiftt lower pHs. That it was not detected at time 0 butent at 1h indicates that the molecule was being pro-ecreted over time. Its concentration in the yeast creamt was estimated to be 0.1mM.ence presented above is an unequivocal demonstrationexports an electro-active molecule and it appears thatmost of the current in the Arxula MFC. The absence of aS. cerevisiae scans and the small signals it produces insupports this notion.evious evidence and that presented here, it is possiblesfer of electrons fromyeast cells to the anode is both byn of redox molecules and by direct electron transfer. Itthe contribution of each type of transfer to a yeast fuel

will varywith species and probablywith cell cultivationnditions.

ion

ould make good biocatalysts for MFCs; they are robust,high levels of tolerance to variations in environmentale.g. pH, salinity, temperature, most are non-pathogens,have high growth rates. This work sought to clarifye problems that have been seen in yeast MFCs. Lowut is related to the difculty in accessing catabolicnd not an inherent slow rate of catabolism. An A.ns MFC was shown to have a higher power outputrevisiae MFC and this difference was demonstrated tohe production of an extracellular redox molecule byrans.

gements

rk was funded by: The Marsden Fund (The Royal Soci-Zealand), The Dumont dUrville NZ/France S&T Support(New Zealand Ministry of Research, Science and Tech-French Ministry for Foreign Affairs). The Foundation forcience and TechnologyNZ (DET biotechnologies project,-NMTS-LVL). NDH thanks the Tertiary Education Com-, for a NZ Enterprise PhD scholarship. We thank Vincentis assistance with the ferricyanide reduction experi-

N.D. Haslett et al. / Biosensors and Bioelectronics 26 (2011) 37423747 3747

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Characterisation of yeast microbial fuel cell with the yeast Arxula adeninivorans as the biocatalystIntroductionMaterials and methodsCellsCultivation and preparation of cellsReagentsMicrobial fuel cellVoltammetryRetention of TMPD

ResultsRetention of TMPD by A. adeninivorans cellsMediator-less electron transfer from A. adeninivorans to the fuel cell anodeMediator-less fuel cell power output

DiscussionConclusionAcknowledgementsReferences