Research ArticleMagnet-Facilitated Selection of ElectrogenicBacteria from Marine Sediment

Larisa Kiseleva1 Justina Briliute1 Irina V Khilyas12 David J W Simpson1

Viacheslav Fedorovich1 M Cohen1 and Igor Goryanin13

1Biological Systems Unit Okinawa Institute of Science and Technology 1919-1 Tancha Onna-son Okinawa 904-045 Japan2Institute of Fundamental Medicine and Biology Kazan (Volga Region) Federal University Ulitsa Kremlyovskaya 18 KazanRepublic of Tatarstan 420008 Russia3School of Informatics University of Edinburgh 10 Crichton Street Edinburgh EH8 9AB UK

Correspondence should be addressed to Igor Goryanin goryaninoistjp

Received 8 April 2015 Revised 3 July 2015 Accepted 13 July 2015

Academic Editor Yu Zhang

Copyright copy 2015 Larisa Kiseleva et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Some bacteria can carry out anaerobic respiration by depositing electrons on external materials such as electrodes therebycreating an electrical current Into the anode chamber of microbial fuel cells (MFCs) having abiotic air-cathodes we inoculatedmicroorganisms cultured from amagnetic particle-enriched portion of a marine tidal sediment reasoning that since some externalelectron acceptors are ferromagnetic electrogenic bacteria should be found in their vicinity Two MFCs one inoculated with amixed bacterial culture and the other with an axenic culture of a helical bacterium isolated from the magnetic particle enrichmenttermed strain HJ were operated for 65 d Both MFCs produced power with production from the mixed culture MFC exceedingthat of strain HJ Strain HJ was identified as aThalassospira sp by transmission electron microscopic analysis and 16S rRNA genecomparisons An MFC inoculated with strain HJ and operated in open circuit produced 47 and 57 of the maximal powerproduced from MFCs inoculated with the known electrogen Geobacter daltonii and the magnetotactic bacterium Desulfamplusmagnetomortis respectively Further investigation will be needed to determine whether bacterial populations associated withmagnetic particles within marine sediments are enriched for electrogens

1 Introduction

As a group bacteria obtain electrons for respiratorymetabolism from a vast range of sources and likewise deliverthese electrons to an equally impressive array of acceptormolecules A growing list of bacteria have been found to beelectrogenic that is capable of reducing external solid elec-tron acceptors From a biotechnological standpoint electro-genic bacteria are of interest because they can efficiently oxi-dize compounds within the anaerobic environment of micro-bial fuel cells (MFCs) while creating an electrical current [1]

Among the electrogenic bacteria a variety of mechanismsare employed for reducing external electron acceptors [2]Two types of ldquonanowiresrdquo have been identified by whichcells can deliver electrons from cell membrane respira-tory proteins type IV pili found in Geobacter spp andcytochrome-containing outermembrane extensions found in

Shewanella spp [3 4] Bacteria may stably associate with asolid electron acceptor surface as attached biofilms [5] ortransiently by electrokinesis a dynamic process in whichbacteria cycle between depositing electrons and swimmingin the vicinity of the acceptor [6] Some bacteria are alsocapable of transferring electrons to external solid electronacceptors via diffusible electron carrying shuttle moleculessome of which can be exchanged between species [7]Other unidentifiedmechanisms for external electron deliveryalmost certainly exist Development of means to screen forand isolate electrogens would aid in the discovery of suchmechanisms

Conductive minerals (eg magnetite Fe3O4) confer

centimeter-long conductivity to anaerobic marine sediments[8] promoting the activity of electrogens in these envi-ronments and enabling applications such as powering ofremote devices and reductive dechlorination of contaminants

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 582471 7 pageshttpdxdoiorg1011552015582471

2 BioMed Research International

[9] Magnetite can facilitate electron transfer from bacteriato external receptors [10] including transfer to nitrate-reducing bacteria [11] and under acidic conditionsmagnetiteit can be an external electron acceptor [12] Recently it wasdemonstrated that magnetite owing to its mixed valency canbehave as a battery being oxidized by phototrophic bacteriain the light and reduced by electrogenic bacteria in the dark[13] We reasoned therefore that electrogenic bacteria maybe found in preferential associationwithmagnetic particles ofmarine sediments Here we describe the isolation and partialcharacterization of an electrogenic bacterium Thalassospirasp strain HJ from a magnetic particle-enriched portion of amarine tidal sediment

2 Materials and Methods

21 Sediment Sampling and Preparation Sandy sedimentfrom Kaichu-Doro Beach (26∘ 191015840 56110158401015840N 127∘ 541015840 010158401015840 EOctober 2013) Okinawa Japan was sampled to 25 cmdepth and placed into 500mL bottles to approximately halfcapacity filling the remainder of the bottle with seawaterAt the laboratory the sample was vigorously mixed andallowed to settle with a magnet pressed against the outsideof the container positioned above the height of the sedimentsurface before mixing After the sediment had settled 4mLof liquid and adherent magnetic particles from the regionclosest to the magnet was sampled with a sterile Pasteurpipette The ldquocapillary racetrack methodrdquo was then usedto enrich for magnet-associated bacteria from the sample[14] Fluid from the capillary was inoculated into a test tubecontaining 10mL culture medium (Difco Marine Broth2216) supplemented with 50mg Lminus1 FeCl

3(Marine-Fe broth)

and grown overnight at 23∘C without shaking A samplewas streaked to Marine-Fe Agar incubated for 2 d and aresulting colony of a spiral bacterium (termed strain HJ) wassubcultured intoMarine-Fe broth 50mL subcultures of boththe original capillary racetrack-derived mixed communityand strain HJ were poured into separate MFCs that weretopped off with sim125mL 01M sodiumpotassium phosphatebuffer (pH 59) and 1mL 2 sodium acetate

22 Operation of Sediment-Derived Culture-InoculatedMicro-bial Fuel Cells Two single-chamber air-cathode MFCs (11986710 cm 119871 12 cm 119882 10 cm) were prepared to have internalworking volume of 175mL The internal MFC chambercontained two anodes (approximately 6 times 8 cm) suspended2-3mm off the bottom of the chamber composed of a layerof 04mm proprietary conductive carbon cloth to which2mmaverage size activated carbon granules were boundwithconducted glue to provide more surface area The granuleshad been prepared from birch precursor and pretreated witha neutral red catalyst to facilitate electron transfer The twocathodes (6 times 8 cm) were graphite plates (3mm thick 60porosity) sprayed on the liquid-facing side with an aqueous5 Fumion membrane polymer (FuMA-Tech Bietigheim-BissingenGermany)while activated carbon granules (treatedwith iron(II) phthalocyanine) were mechanically pressedto the air-facing side using netting frame Unit cathode

Capillary racetrack

S N

MFC

MFC

Figure 1 A flow scheme indicating the procedure for enrichment ofmagnetic particles from sediment and inoculation of marine brothmedium and microbial fuel cells

section (including membrane) was cleaned by immersionin concentrated HCl before module assembly for 20minto remove any organics and was then soaked in steriledistilled water with water changes every 1 h until the pH wasneutralized The reactor cell PVC surfaces were cleaned bywashing with soap and water drying and then wiping downwith acetone solvent Anode and internal reactor areas weresprayedwith 70 ethanol solution just prior to final assemblyThe cathode extended into a bath containing an electrolytesolutionmaintained at pH 2with regular additions of 1 NHClto provide a source of protons for the abiotic reduction ofoxygen to water The MFCs were maintained on a 24 h opencircuit24 h closed circuit cycle using an external resistanceof 40Ω The anode and cathode electrodes were connectedwith a multichannel logger (Graphtec midi LOGGERGL820Japan) for daily voltage measurements The correspondingelectric current was calculated using Ohmrsquos law (119881 = 119868119877)Power density was obtained according to the equation 119875 =119868119881119860 where 119868 is the current 119881 is the voltage and 119860 is theprojected surface area of the cathode

Once weekly after removal of 1mL mixed contents forchemical oxygen demand (COD) analysis the MFCs werefed with approximately 5mL phosphate buffer containing1 g Lminus1 sodiumacetatewhenCODanalysis indicated substratedepletionThe extra volume of the feed was needed to replacelosses due to evaporation through the cathode membraneUpon completion of the monitoring period on the 65th dayof operation due to declining power production the MFCswere disassembled Anodematerial was sampled and bacteriawere isolated from the strain HJ-inoculated MFC to confirmthe presence of strain HJ

23 Bacterial Isolation and Initial Characterization DNAfrom strain HJ was isolated and subject to genomic sequenc-ing [15] Phylogenetic analyses of the 16S rRNA gene (Gen-Bank accession number KP704219) were performed usingthe Phylogenyfr platform [16] Minimum and maximumtemperatures for growth were determined by culturing onMarine Agar plates Catalase and oxidase activities weretested and Gram staining was carried out using standardmicrobiological methods

24 Transmission Electron Microscopy Transmission elec-tron microscope (TEM) imaging was performed on a JEOLJEM-1230R ElectronMicroscope at an accelerating voltage of

BioMed Research International 3

HJ (R)HJ (L)

Mixed (R)Mixed (L)

0 1 2 3 4 5 6 7 8 9

Time (weeks)

HJ (R)HJ (L)

Mixed (R)Mixed (L)

0 1 2 3 4 5 6 7 8 9

Time (weeks)

0

100

200

300

400

500

600

700Vo

ltage

(mV

)

Curr

ent (

mA

)

0

5

10

15

20

25

Figure 2 Electrical output frommicrobial fuel cells inoculated with cultures derived from tidal beach sediment obtained from Kaichu-DoroBeach Okinawa Japan Total anode surface area 151 cm2 (755 cm2 per anode) 175mL volume Plotted lines indicate activity from the mixedculture-inoculated MFC and the strain HJ-inoculated MFC R right and L left anode-cathode couple

100KV Strain HJ cells were prepared from cultures grownat room temperature (23∘C) in 50mL of Marine-Fe brothto an OD

600of 10 The pelleted cells were fixed with 1

osmium tetroxide in 01M cacodylate buffer (pH = 72) for30min and washed with water three times for 5min A dropof fixed bacterial cells was deposited onto a carbon-coatedHF34 200mesh copper grid washed oncewith distilledwaterand stained for 3 to 5 sec with 1 uranyl acetate

25 Inoculation and Monitoring of Microbial Fuel Cells Inoc-ulated with Axenic Bacterial Cultures A micro-MFC arraywas developed from two parts of Plexiglas with three 4 cmmicrochambers to test for current generation by three axenicbacterial cultures Each chamber consisted of an anode anda cathode compartment (8mm deep) separated by a cation-exchange membrane Nafion 117 (196 cm2 DuPont CoDelaware USA) The anode and cathode electrodes (3mmthick graphite plates 45ndash50 porosity Xinghe County MuziCarbon Co Ltd China) were connected with a multichannellogger (Graphtec midi LOGGER GL820 Japan) for dailyvoltage measurements 1mm thick rubber gaskets were usedfor sealing between the anode and cathode compartmentsA titanium screw held the electrode against the membraneand acted as electrical connector Each micro-MFC cham-ber was equipped with a 25 ID mm polyurethane inlettube (FESTO Germany) on the bottom for medium andelectrolyte feeding and one on the top for biogas outputand replacement of exhausted electrolyte For disinfectionthe plexiglas microchambers were soaked in a 10 bleachsolution rinsed with deionized water and then exposed to

UV light for 12 h Graphite plates were washed with 100isopropanol in an ultrasonic bath and then heated in an ovenfor 2 h at 200C Micro-MFCs were operated in the opencircuit mode and current measurements were performedevery 24 hours starting from time of inoculation 50mMiron(II) phthalocyanine withoutN

2sparging was used as the

cathode electrolyte solutionAxenic bacterial cultures (Thalassospira spHJ the known

electrogen Geobacter daltonii and the magnetotactic Desul-famplus magnetomortis) were pregrown with shaking at 37∘Cfor 12 h in 5mL of modified PBS medium containing (perliter) 458 g Na

2HPO4 245 g NaH

2PO4sdotH2O 031 g NH

4Cl

013 g KCl 5 g glucose 5 g yeast extract and 5 g peptoneBacterial cells were harvested washed with 16mMphosphatebuffer (pH 70) and resuspended into 50mL fresh modifiedPBS medium The initial cell concentration was adjustedto an optical density at 600 nm of 03 as measured bya Spectronic GENESYS 5 spectrophotometer (Milton RoyCompany Rochester NY USA) with filtered cell-free culturemedium as reference

The micro-MFCs were inoculated initially with 15mLmedium containing bacterial cells (exceeding the sim10mLvolume of the anode chamber since the porous graphiteabsorbed some liquid) Bacteria-free modified PBS mediumcontaining 01 NaN

3was used for control experiments

All experiments were set up in two technical replicationsand two biological replications The axenicity of the anolytesfor the strain HJ-containing MFCs was determined atthe end of the experiments by plating serial dilutions toMarine Agar medium Experiments were performed at roomtemperature

4 BioMed Research International

(a) (b)

(c) (d)

Figure 3 Transmission electron micrographs of Thalassospira sp strain HJ showing various features (a) A single polar flagellum (b)intracellular electron-dense regions (c) symmetric cell division and (d) asymmetric cell division Representative images are shown

3 Results and Discussion

To obtain magnetic particle-associated bacteria some ofwhich should be electrogenic [12] we subjected tidal beachsediment to the enrichment procedure depicted in Figure 1One MFC was inoculated with a mixed culture derived fromthe magnetic particle-enriched sediment and the other withan axenic culture of a helical bacterium strain HJ isolatedfrom the enriched sediment Both MFCs were found toproduce power with the current and voltage generated fromthe mixed culture MFC being substantially higher (Figure 2)The comparatively low power density observed of strain HJis typical of axenic bacterial cultures relative to that of thecommunities from which they are derived [5] At the endof the experiment strain HJ was reisolated from the MFC toconfirm its retention

Strain HJ was identified by means of microscopic anal-ysis (Figure 3) and 16S rRNA gene sequence comparisons(Figure 4) to be of the genusThalassospira TEM images showcells of 05ndash07120583mwidth and 15ndash57120583m length having single

polar flagella typical of the genus Thalassospira [17] withevidence of both symmetric and asymmetric cell division(Figure 3) One or more electron-dense staining regions canbe seenwithinmost cells (Figure 3) Cells weremotile stainedGram negative and tested positive for catalase and oxidaseactivity Growth occurred on Marine Agar at temperaturesranging from 12∘C to 39∘C and pH values ranging from 55to 105 The closest matching 16S rRNA gene sequence toa named species within the GenBank database was that ofThalassospira profundimaris strainWP0211 (14491453 997identity) which notably differs from strain HJ in its lack offlagella [18]

Micro-MFC chambers inoculated with axenic cultures ofThalassospira sp strain HJ and for comparison Geobacterdaltonii and Desulfamplus magnetomortis showed electricalcurrent production immediately after inoculation but dif-fered in the intensity and shape of the subsequent productioncurve (Figure 5) Thalassospira sp strain HJ and D magne-tomortis displayed gradual increases in current after initia-tion whereas current production from G daltonii decreased

BioMed Research International 5

Thalassospira alkalitolerans (AB786710)Thalassospira xiamenensis M-5 (CP004388)

Thalassospira lucentensis QMT2 (AF358664)

Magnetospira thiophila MMS1 (EU861390)Magnetovibrio blakemorei MV1 (L06455)

01

Rhodospirillum oryzae JA318T (AM901295)Roseospira marina CE2105 (AJ298879)

Azospirillum brasilense DSM1690 (GU256438)Magnetospirillum magneticum AMB-1 (D17514)

Sphingomonas azotifigens NBRC15497 (AB217471)Rhodobacter blasticus ATCC33485 (D16429)

Caulobacter vibrioides FMC1 (JX968034)

Thalassospira profundimaris WP0211 (AY186195)

Thalassospira mesophila (AB786711)

Thalassospira sp HJ (KP704219)

Figure 4 Phylogenetic tree of strain HJ and selected alphaproteobacteria based on 16S rRNA gene sequences The tree was constructedfrom sequences aligned by MUSCLE using maximum-likelihood method of PhyML 30 [16 21] The scale bar reflects evolutionary distancemeasured in units of substitution per nucleotide site

0

5

10

15

20

25

30

35

0 24 48 72 96 120 144 172

Curr

ent (

mA

)

Time (h)0 24 48 72 96 120 144 172

Time (h)

000

005

010

015

020

025

030

035

040

045

Volta

ge (V

)

Strain HJD magnetomortis

G daltoniiControl

Strain HJD magnetomortis

G daltoniiControl

Figure 5 Voltage and current readings (plusmnrange 119899 = 2) from microbial fuel cells inoculated with axenic cultures of Thalassospira sp strainHJ Geobacter daltonii and Desulfamplus magnetomortis or medium only with 1 g Lminus1 sodium azide (control) Anode surface area 196 cm210mL volume

with time After 172 h the current for all axenic culturesdecreased to 5ndash8mA which was attributed with C-sourcedepletion Voltage drop was observed after 144 h from thestrain HJ-inoculated micro-MFC whereas G daltonii andD magnetomortis MFCs demonstrated voltage loss after48 h As a control to determine the contribution of growthmedium components to current and voltage production amicro-MFC was filled with sterile medium Although therewas no observable current generation background voltagewas observed (Figure 5) The maximal single-value powerreadings from the micro-MFCs were of 26Wmminus2 (at 96 h)

for Thalassospira sp strain HJ 55Wmminus2 (at 144 h) for Dmagnetomortis and 46Wmminus2 (at 24 h) for G daltonii

Geobacter is a well-characterized genus of electrogenicbacteria and although we are not aware of prior reportsof electrogenicity by the magnetotactic bacterium D mag-netomortis members of sulfate-reducing members of theDeltaproteobacteria to which D magnetomortis belongswere found to display transcriptomic responses to changes inMFC electrode potential indicative of electrogenic behavior[19]We hypothesize thatmagnetosomes could confer a selec-tive benefit by enabling bacteria to hone in on ferromagnetic

6 BioMed Research International

external electron acceptors A wider survey of magnetotacticbacteria for electrogenicity would be warranted

Recently two marine sediment-derived Thalassospira spisolates were reported to exhibit electrotrophic behavioraccepting electrons from insoluble sulfur [20] but the capacityof these strains to transfer electrons to an anode as we havefound here of Thalassospira sp strain HJ was not examinedExamination of the strain HJ genome does not reveal anyhomologs for the pili or outer membrane extension-typeexternal electron transfer systems found in Geobacter or She-wanella species [15] andmicroscopic imaging of anodes fromstrain HJ-colonized MFCs implies that biofilm formationis not necessary for electrogenicity (data not shown) Thuswe hypothesize that external electron transfer occurs via anelectron shuttle-type mechanism

4 Conclusions

A microbial community derived from magnetic particle-enriched marine sediment displayed electrogenic behaviorand yielded an electrogenic bacterium identified to be aThalassospira species This is the first report of electrogenicbehavior within the genus Thalassospira A more extensivestudy would be needed to determine whether the propor-tion of electrogenic bacteria obtained from this magneticparticle enrichment procedure exceeds that found in theenvironment With their diurnal patterns of flooding anddiversity of mineral components tidal sediments should berich environments to bioprospect for electrogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding was provided by the Okinawa Institute of Scienceand Technology I V Khilyas was supported by RussianGovernment Program of Competitive Growth of KazanFederal University The authors thank M Power World Ltdfor consultations and provision of equipment and T Sasakifor conducting transmission electron microscopy

References

[1] V Gude B Kokabian and V Gadhamshetty ldquoBeneficial bio-electrochemical systems for energy water and biomass pro-ductionrdquo Journal of Microbial amp Biochemical Technology vol 6article 005 p 2 2013

[2] S A Patil C Hagerhall and L Gorton ldquoElectron transfermechanisms between microorganisms and electrodes in bio-electrochemical systemsrdquo Bioanalytical Reviews vol 4 no 2ndash4pp 159ndash192 2012

[3] D R Lovley and N S Malvankar ldquoSeeing is believing Novelimaging techniques help clarify microbial nanowire structureand functionrdquo Environmental Microbiology vol 17 no 7 pp2209ndash2215 2015

[4] S Pirbadian S E Barchinger K M Leung et al ldquoShe-wanella oneidensis MR-1 nanowires are outer membrane andperiplasmic extensions of the extracellular electron transportcomponentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 111 no 35 pp 12883ndash128882014

[5] B E Logan ldquoExoelectrogenic bacteria that powermicrobial fuelcellsrdquo Nature Reviews Microbiology vol 7 no 5 pp 375ndash3812009

[6] H W Harrisa M Y El-Naggarb O Bretschgerc et al ldquoElec-trokinesis is a microbial behavior that requires extracellularelectron transportrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 1 pp 326ndash331 2010

[7] K Rabaey N Boon M Hofte and W Verstraete ldquoMicrobialphenazine production enhances electron transfer in biofuelcellsrdquo Environmental Science amp Technology vol 39 no 9 pp3401ndash3408 2005

[8] N S Malvankar G M King and D R Lovley ldquoCentimeter-long electron transport in marine sediments via conductivemineralsrdquoThe ISME Journal vol 9 no 2 pp 527ndash531 2015

[9] D R Lovley and K P Nevin ldquoA shift in the current new appli-cations and concepts for microbe-electrode electron exchangerdquoCurrent Opinion in Biotechnology vol 22 no 3 pp 441ndash4482011

[10] F Liu A Rotaru P M Shrestha N S Malvankar K P Nevinand D R Lovley ldquoMagnetite compensates for the lack of apilin-associated c-type cytochrome in extracellular electronexchangerdquo Environmental Microbiology vol 17 no 3 pp 648ndash655 2015

[11] S Kato K Hashimoto and K Watanabe ldquoMicrobial inter-species electron transfer via electric currents through conduc-tive mineralsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 25 pp 10042ndash100462012

[12] J E Kostka and K H Nealson ldquoDissolution and reduction ofmagnetite by bacteriardquo Environmental Science amp Technologyvol 29 no 10 pp 2535ndash2540 1995

[13] J M Byrne N Klueglein C Pearce K M Rosso E Appel andA Kappler ldquoRedox cycling of Fe(II) and Fe(III) in magnetite byFe-metabolizing bacteriardquo Science vol 347 no 6229 pp 1473ndash1476 2015

[14] R SWolfe R KThauer andN Pfennig ldquoA lsquocapillary racetrackrsquomethod for isolation of magnetotactic bacteriardquo FEMSMicrobi-ology Letters vol 45 no 1 pp 31ndash35 1987

[15] L Kiseleva S K Garushyants J Briliute D J SimpsonM F Cohen and I Goryanin ldquoGenome sequence of theelectrogenic petroleum-degrading Thalassospira sp strain HJrdquoGenome Announcements vol 3 no 3 Article ID e00483-152015

[16] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 supplement 2 pp W465ndashW469 2008

[17] J I Baldani S S Videira J I Baldani et al ldquoThe family Rho-dospirillaceaerdquo in The Prokaryotes E Rosenberg E DeLongS Lory E Stackebrandt and F Thompson Eds pp 533ndash618Springer Berlin Germany 2014

[18] C Liu Y Wu L Li M Yingfei and Z Shao ldquoThalassospiraxiamenensis sp nov and Thalassospira profundimaris sp novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 2 pp 316ndash320 2007

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

Zoology

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International Journal of

Microbiology

2 BioMed Research International

[9] Magnetite can facilitate electron transfer from bacteriato external receptors [10] including transfer to nitrate-reducing bacteria [11] and under acidic conditionsmagnetiteit can be an external electron acceptor [12] Recently it wasdemonstrated that magnetite owing to its mixed valency canbehave as a battery being oxidized by phototrophic bacteriain the light and reduced by electrogenic bacteria in the dark[13] We reasoned therefore that electrogenic bacteria maybe found in preferential associationwithmagnetic particles ofmarine sediments Here we describe the isolation and partialcharacterization of an electrogenic bacterium Thalassospirasp strain HJ from a magnetic particle-enriched portion of amarine tidal sediment

2 Materials and Methods

21 Sediment Sampling and Preparation Sandy sedimentfrom Kaichu-Doro Beach (26∘ 191015840 56110158401015840N 127∘ 541015840 010158401015840 EOctober 2013) Okinawa Japan was sampled to 25 cmdepth and placed into 500mL bottles to approximately halfcapacity filling the remainder of the bottle with seawaterAt the laboratory the sample was vigorously mixed andallowed to settle with a magnet pressed against the outsideof the container positioned above the height of the sedimentsurface before mixing After the sediment had settled 4mLof liquid and adherent magnetic particles from the regionclosest to the magnet was sampled with a sterile Pasteurpipette The ldquocapillary racetrack methodrdquo was then usedto enrich for magnet-associated bacteria from the sample[14] Fluid from the capillary was inoculated into a test tubecontaining 10mL culture medium (Difco Marine Broth2216) supplemented with 50mg Lminus1 FeCl

3(Marine-Fe broth)

and grown overnight at 23∘C without shaking A samplewas streaked to Marine-Fe Agar incubated for 2 d and aresulting colony of a spiral bacterium (termed strain HJ) wassubcultured intoMarine-Fe broth 50mL subcultures of boththe original capillary racetrack-derived mixed communityand strain HJ were poured into separate MFCs that weretopped off with sim125mL 01M sodiumpotassium phosphatebuffer (pH 59) and 1mL 2 sodium acetate

22 Operation of Sediment-Derived Culture-InoculatedMicro-bial Fuel Cells Two single-chamber air-cathode MFCs (11986710 cm 119871 12 cm 119882 10 cm) were prepared to have internalworking volume of 175mL The internal MFC chambercontained two anodes (approximately 6 times 8 cm) suspended2-3mm off the bottom of the chamber composed of a layerof 04mm proprietary conductive carbon cloth to which2mmaverage size activated carbon granules were boundwithconducted glue to provide more surface area The granuleshad been prepared from birch precursor and pretreated witha neutral red catalyst to facilitate electron transfer The twocathodes (6 times 8 cm) were graphite plates (3mm thick 60porosity) sprayed on the liquid-facing side with an aqueous5 Fumion membrane polymer (FuMA-Tech Bietigheim-BissingenGermany)while activated carbon granules (treatedwith iron(II) phthalocyanine) were mechanically pressedto the air-facing side using netting frame Unit cathode

Capillary racetrack

S N

MFC

MFC

Figure 1 A flow scheme indicating the procedure for enrichment ofmagnetic particles from sediment and inoculation of marine brothmedium and microbial fuel cells

section (including membrane) was cleaned by immersionin concentrated HCl before module assembly for 20minto remove any organics and was then soaked in steriledistilled water with water changes every 1 h until the pH wasneutralized The reactor cell PVC surfaces were cleaned bywashing with soap and water drying and then wiping downwith acetone solvent Anode and internal reactor areas weresprayedwith 70 ethanol solution just prior to final assemblyThe cathode extended into a bath containing an electrolytesolutionmaintained at pH 2with regular additions of 1 NHClto provide a source of protons for the abiotic reduction ofoxygen to water The MFCs were maintained on a 24 h opencircuit24 h closed circuit cycle using an external resistanceof 40Ω The anode and cathode electrodes were connectedwith a multichannel logger (Graphtec midi LOGGERGL820Japan) for daily voltage measurements The correspondingelectric current was calculated using Ohmrsquos law (119881 = 119868119877)Power density was obtained according to the equation 119875 =119868119881119860 where 119868 is the current 119881 is the voltage and 119860 is theprojected surface area of the cathode

Once weekly after removal of 1mL mixed contents forchemical oxygen demand (COD) analysis the MFCs werefed with approximately 5mL phosphate buffer containing1 g Lminus1 sodiumacetatewhenCODanalysis indicated substratedepletionThe extra volume of the feed was needed to replacelosses due to evaporation through the cathode membraneUpon completion of the monitoring period on the 65th dayof operation due to declining power production the MFCswere disassembled Anodematerial was sampled and bacteriawere isolated from the strain HJ-inoculated MFC to confirmthe presence of strain HJ

23 Bacterial Isolation and Initial Characterization DNAfrom strain HJ was isolated and subject to genomic sequenc-ing [15] Phylogenetic analyses of the 16S rRNA gene (Gen-Bank accession number KP704219) were performed usingthe Phylogenyfr platform [16] Minimum and maximumtemperatures for growth were determined by culturing onMarine Agar plates Catalase and oxidase activities weretested and Gram staining was carried out using standardmicrobiological methods

24 Transmission Electron Microscopy Transmission elec-tron microscope (TEM) imaging was performed on a JEOLJEM-1230R ElectronMicroscope at an accelerating voltage of

BioMed Research International 3

HJ (R)HJ (L)

Mixed (R)Mixed (L)

0 1 2 3 4 5 6 7 8 9

Time (weeks)

HJ (R)HJ (L)

Mixed (R)Mixed (L)

0 1 2 3 4 5 6 7 8 9

Time (weeks)

0

100

200

300

400

500

600

700Vo

ltage

(mV

)

Curr

ent (

mA

)

0

5

10

15

20

25

Figure 2 Electrical output frommicrobial fuel cells inoculated with cultures derived from tidal beach sediment obtained from Kaichu-DoroBeach Okinawa Japan Total anode surface area 151 cm2 (755 cm2 per anode) 175mL volume Plotted lines indicate activity from the mixedculture-inoculated MFC and the strain HJ-inoculated MFC R right and L left anode-cathode couple

100KV Strain HJ cells were prepared from cultures grownat room temperature (23∘C) in 50mL of Marine-Fe brothto an OD

600of 10 The pelleted cells were fixed with 1

osmium tetroxide in 01M cacodylate buffer (pH = 72) for30min and washed with water three times for 5min A dropof fixed bacterial cells was deposited onto a carbon-coatedHF34 200mesh copper grid washed oncewith distilledwaterand stained for 3 to 5 sec with 1 uranyl acetate

25 Inoculation and Monitoring of Microbial Fuel Cells Inoc-ulated with Axenic Bacterial Cultures A micro-MFC arraywas developed from two parts of Plexiglas with three 4 cmmicrochambers to test for current generation by three axenicbacterial cultures Each chamber consisted of an anode anda cathode compartment (8mm deep) separated by a cation-exchange membrane Nafion 117 (196 cm2 DuPont CoDelaware USA) The anode and cathode electrodes (3mmthick graphite plates 45ndash50 porosity Xinghe County MuziCarbon Co Ltd China) were connected with a multichannellogger (Graphtec midi LOGGER GL820 Japan) for dailyvoltage measurements 1mm thick rubber gaskets were usedfor sealing between the anode and cathode compartmentsA titanium screw held the electrode against the membraneand acted as electrical connector Each micro-MFC cham-ber was equipped with a 25 ID mm polyurethane inlettube (FESTO Germany) on the bottom for medium andelectrolyte feeding and one on the top for biogas outputand replacement of exhausted electrolyte For disinfectionthe plexiglas microchambers were soaked in a 10 bleachsolution rinsed with deionized water and then exposed to

UV light for 12 h Graphite plates were washed with 100isopropanol in an ultrasonic bath and then heated in an ovenfor 2 h at 200C Micro-MFCs were operated in the opencircuit mode and current measurements were performedevery 24 hours starting from time of inoculation 50mMiron(II) phthalocyanine withoutN

2sparging was used as the

cathode electrolyte solutionAxenic bacterial cultures (Thalassospira spHJ the known

electrogen Geobacter daltonii and the magnetotactic Desul-famplus magnetomortis) were pregrown with shaking at 37∘Cfor 12 h in 5mL of modified PBS medium containing (perliter) 458 g Na

2HPO4 245 g NaH

2PO4sdotH2O 031 g NH

4Cl

013 g KCl 5 g glucose 5 g yeast extract and 5 g peptoneBacterial cells were harvested washed with 16mMphosphatebuffer (pH 70) and resuspended into 50mL fresh modifiedPBS medium The initial cell concentration was adjustedto an optical density at 600 nm of 03 as measured bya Spectronic GENESYS 5 spectrophotometer (Milton RoyCompany Rochester NY USA) with filtered cell-free culturemedium as reference

The micro-MFCs were inoculated initially with 15mLmedium containing bacterial cells (exceeding the sim10mLvolume of the anode chamber since the porous graphiteabsorbed some liquid) Bacteria-free modified PBS mediumcontaining 01 NaN

3was used for control experiments

All experiments were set up in two technical replicationsand two biological replications The axenicity of the anolytesfor the strain HJ-containing MFCs was determined atthe end of the experiments by plating serial dilutions toMarine Agar medium Experiments were performed at roomtemperature

4 BioMed Research International

(a) (b)

(c) (d)

Figure 3 Transmission electron micrographs of Thalassospira sp strain HJ showing various features (a) A single polar flagellum (b)intracellular electron-dense regions (c) symmetric cell division and (d) asymmetric cell division Representative images are shown

3 Results and Discussion

To obtain magnetic particle-associated bacteria some ofwhich should be electrogenic [12] we subjected tidal beachsediment to the enrichment procedure depicted in Figure 1One MFC was inoculated with a mixed culture derived fromthe magnetic particle-enriched sediment and the other withan axenic culture of a helical bacterium strain HJ isolatedfrom the enriched sediment Both MFCs were found toproduce power with the current and voltage generated fromthe mixed culture MFC being substantially higher (Figure 2)The comparatively low power density observed of strain HJis typical of axenic bacterial cultures relative to that of thecommunities from which they are derived [5] At the endof the experiment strain HJ was reisolated from the MFC toconfirm its retention

Strain HJ was identified by means of microscopic anal-ysis (Figure 3) and 16S rRNA gene sequence comparisons(Figure 4) to be of the genusThalassospira TEM images showcells of 05ndash07120583mwidth and 15ndash57120583m length having single

polar flagella typical of the genus Thalassospira [17] withevidence of both symmetric and asymmetric cell division(Figure 3) One or more electron-dense staining regions canbe seenwithinmost cells (Figure 3) Cells weremotile stainedGram negative and tested positive for catalase and oxidaseactivity Growth occurred on Marine Agar at temperaturesranging from 12∘C to 39∘C and pH values ranging from 55to 105 The closest matching 16S rRNA gene sequence toa named species within the GenBank database was that ofThalassospira profundimaris strainWP0211 (14491453 997identity) which notably differs from strain HJ in its lack offlagella [18]

Micro-MFC chambers inoculated with axenic cultures ofThalassospira sp strain HJ and for comparison Geobacterdaltonii and Desulfamplus magnetomortis showed electricalcurrent production immediately after inoculation but dif-fered in the intensity and shape of the subsequent productioncurve (Figure 5) Thalassospira sp strain HJ and D magne-tomortis displayed gradual increases in current after initia-tion whereas current production from G daltonii decreased

BioMed Research International 5

Thalassospira alkalitolerans (AB786710)Thalassospira xiamenensis M-5 (CP004388)

Thalassospira lucentensis QMT2 (AF358664)

Magnetospira thiophila MMS1 (EU861390)Magnetovibrio blakemorei MV1 (L06455)

01

Rhodospirillum oryzae JA318T (AM901295)Roseospira marina CE2105 (AJ298879)

Azospirillum brasilense DSM1690 (GU256438)Magnetospirillum magneticum AMB-1 (D17514)

Sphingomonas azotifigens NBRC15497 (AB217471)Rhodobacter blasticus ATCC33485 (D16429)

Caulobacter vibrioides FMC1 (JX968034)

Thalassospira profundimaris WP0211 (AY186195)

Thalassospira mesophila (AB786711)

Thalassospira sp HJ (KP704219)

Figure 4 Phylogenetic tree of strain HJ and selected alphaproteobacteria based on 16S rRNA gene sequences The tree was constructedfrom sequences aligned by MUSCLE using maximum-likelihood method of PhyML 30 [16 21] The scale bar reflects evolutionary distancemeasured in units of substitution per nucleotide site

0

5

10

15

20

25

30

35

0 24 48 72 96 120 144 172

Curr

ent (

mA

)

Time (h)0 24 48 72 96 120 144 172

Time (h)

000

005

010

015

020

025

030

035

040

045

Volta

ge (V

)

Strain HJD magnetomortis

G daltoniiControl

Strain HJD magnetomortis

G daltoniiControl

Figure 5 Voltage and current readings (plusmnrange 119899 = 2) from microbial fuel cells inoculated with axenic cultures of Thalassospira sp strainHJ Geobacter daltonii and Desulfamplus magnetomortis or medium only with 1 g Lminus1 sodium azide (control) Anode surface area 196 cm210mL volume

with time After 172 h the current for all axenic culturesdecreased to 5ndash8mA which was attributed with C-sourcedepletion Voltage drop was observed after 144 h from thestrain HJ-inoculated micro-MFC whereas G daltonii andD magnetomortis MFCs demonstrated voltage loss after48 h As a control to determine the contribution of growthmedium components to current and voltage production amicro-MFC was filled with sterile medium Although therewas no observable current generation background voltagewas observed (Figure 5) The maximal single-value powerreadings from the micro-MFCs were of 26Wmminus2 (at 96 h)

for Thalassospira sp strain HJ 55Wmminus2 (at 144 h) for Dmagnetomortis and 46Wmminus2 (at 24 h) for G daltonii

Geobacter is a well-characterized genus of electrogenicbacteria and although we are not aware of prior reportsof electrogenicity by the magnetotactic bacterium D mag-netomortis members of sulfate-reducing members of theDeltaproteobacteria to which D magnetomortis belongswere found to display transcriptomic responses to changes inMFC electrode potential indicative of electrogenic behavior[19]We hypothesize thatmagnetosomes could confer a selec-tive benefit by enabling bacteria to hone in on ferromagnetic

6 BioMed Research International

external electron acceptors A wider survey of magnetotacticbacteria for electrogenicity would be warranted

Recently two marine sediment-derived Thalassospira spisolates were reported to exhibit electrotrophic behavioraccepting electrons from insoluble sulfur [20] but the capacityof these strains to transfer electrons to an anode as we havefound here of Thalassospira sp strain HJ was not examinedExamination of the strain HJ genome does not reveal anyhomologs for the pili or outer membrane extension-typeexternal electron transfer systems found in Geobacter or She-wanella species [15] andmicroscopic imaging of anodes fromstrain HJ-colonized MFCs implies that biofilm formationis not necessary for electrogenicity (data not shown) Thuswe hypothesize that external electron transfer occurs via anelectron shuttle-type mechanism

4 Conclusions

A microbial community derived from magnetic particle-enriched marine sediment displayed electrogenic behaviorand yielded an electrogenic bacterium identified to be aThalassospira species This is the first report of electrogenicbehavior within the genus Thalassospira A more extensivestudy would be needed to determine whether the propor-tion of electrogenic bacteria obtained from this magneticparticle enrichment procedure exceeds that found in theenvironment With their diurnal patterns of flooding anddiversity of mineral components tidal sediments should berich environments to bioprospect for electrogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding was provided by the Okinawa Institute of Scienceand Technology I V Khilyas was supported by RussianGovernment Program of Competitive Growth of KazanFederal University The authors thank M Power World Ltdfor consultations and provision of equipment and T Sasakifor conducting transmission electron microscopy

References

[1] V Gude B Kokabian and V Gadhamshetty ldquoBeneficial bio-electrochemical systems for energy water and biomass pro-ductionrdquo Journal of Microbial amp Biochemical Technology vol 6article 005 p 2 2013

[2] S A Patil C Hagerhall and L Gorton ldquoElectron transfermechanisms between microorganisms and electrodes in bio-electrochemical systemsrdquo Bioanalytical Reviews vol 4 no 2ndash4pp 159ndash192 2012

[3] D R Lovley and N S Malvankar ldquoSeeing is believing Novelimaging techniques help clarify microbial nanowire structureand functionrdquo Environmental Microbiology vol 17 no 7 pp2209ndash2215 2015

[4] S Pirbadian S E Barchinger K M Leung et al ldquoShe-wanella oneidensis MR-1 nanowires are outer membrane andperiplasmic extensions of the extracellular electron transportcomponentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 111 no 35 pp 12883ndash128882014

[5] B E Logan ldquoExoelectrogenic bacteria that powermicrobial fuelcellsrdquo Nature Reviews Microbiology vol 7 no 5 pp 375ndash3812009

[6] H W Harrisa M Y El-Naggarb O Bretschgerc et al ldquoElec-trokinesis is a microbial behavior that requires extracellularelectron transportrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 1 pp 326ndash331 2010

[7] K Rabaey N Boon M Hofte and W Verstraete ldquoMicrobialphenazine production enhances electron transfer in biofuelcellsrdquo Environmental Science amp Technology vol 39 no 9 pp3401ndash3408 2005

[8] N S Malvankar G M King and D R Lovley ldquoCentimeter-long electron transport in marine sediments via conductivemineralsrdquoThe ISME Journal vol 9 no 2 pp 527ndash531 2015

[9] D R Lovley and K P Nevin ldquoA shift in the current new appli-cations and concepts for microbe-electrode electron exchangerdquoCurrent Opinion in Biotechnology vol 22 no 3 pp 441ndash4482011

[10] F Liu A Rotaru P M Shrestha N S Malvankar K P Nevinand D R Lovley ldquoMagnetite compensates for the lack of apilin-associated c-type cytochrome in extracellular electronexchangerdquo Environmental Microbiology vol 17 no 3 pp 648ndash655 2015

[11] S Kato K Hashimoto and K Watanabe ldquoMicrobial inter-species electron transfer via electric currents through conduc-tive mineralsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 25 pp 10042ndash100462012

[12] J E Kostka and K H Nealson ldquoDissolution and reduction ofmagnetite by bacteriardquo Environmental Science amp Technologyvol 29 no 10 pp 2535ndash2540 1995

[13] J M Byrne N Klueglein C Pearce K M Rosso E Appel andA Kappler ldquoRedox cycling of Fe(II) and Fe(III) in magnetite byFe-metabolizing bacteriardquo Science vol 347 no 6229 pp 1473ndash1476 2015

[14] R SWolfe R KThauer andN Pfennig ldquoA lsquocapillary racetrackrsquomethod for isolation of magnetotactic bacteriardquo FEMSMicrobi-ology Letters vol 45 no 1 pp 31ndash35 1987

[15] L Kiseleva S K Garushyants J Briliute D J SimpsonM F Cohen and I Goryanin ldquoGenome sequence of theelectrogenic petroleum-degrading Thalassospira sp strain HJrdquoGenome Announcements vol 3 no 3 Article ID e00483-152015

[16] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 supplement 2 pp W465ndashW469 2008

[17] J I Baldani S S Videira J I Baldani et al ldquoThe family Rho-dospirillaceaerdquo in The Prokaryotes E Rosenberg E DeLongS Lory E Stackebrandt and F Thompson Eds pp 533ndash618Springer Berlin Germany 2014

[18] C Liu Y Wu L Li M Yingfei and Z Shao ldquoThalassospiraxiamenensis sp nov and Thalassospira profundimaris sp novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 2 pp 316ndash320 2007

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

HJ (R)HJ (L)

Mixed (R)Mixed (L)

0 1 2 3 4 5 6 7 8 9

Time (weeks)

HJ (R)HJ (L)

Mixed (R)Mixed (L)

0 1 2 3 4 5 6 7 8 9

Time (weeks)

0

100

200

300

400

500

600

700Vo

ltage

(mV

)

Curr

ent (

mA

)

0

5

10

15

20

25

Figure 2 Electrical output frommicrobial fuel cells inoculated with cultures derived from tidal beach sediment obtained from Kaichu-DoroBeach Okinawa Japan Total anode surface area 151 cm2 (755 cm2 per anode) 175mL volume Plotted lines indicate activity from the mixedculture-inoculated MFC and the strain HJ-inoculated MFC R right and L left anode-cathode couple

100KV Strain HJ cells were prepared from cultures grownat room temperature (23∘C) in 50mL of Marine-Fe brothto an OD

600of 10 The pelleted cells were fixed with 1

osmium tetroxide in 01M cacodylate buffer (pH = 72) for30min and washed with water three times for 5min A dropof fixed bacterial cells was deposited onto a carbon-coatedHF34 200mesh copper grid washed oncewith distilledwaterand stained for 3 to 5 sec with 1 uranyl acetate

25 Inoculation and Monitoring of Microbial Fuel Cells Inoc-ulated with Axenic Bacterial Cultures A micro-MFC arraywas developed from two parts of Plexiglas with three 4 cmmicrochambers to test for current generation by three axenicbacterial cultures Each chamber consisted of an anode anda cathode compartment (8mm deep) separated by a cation-exchange membrane Nafion 117 (196 cm2 DuPont CoDelaware USA) The anode and cathode electrodes (3mmthick graphite plates 45ndash50 porosity Xinghe County MuziCarbon Co Ltd China) were connected with a multichannellogger (Graphtec midi LOGGER GL820 Japan) for dailyvoltage measurements 1mm thick rubber gaskets were usedfor sealing between the anode and cathode compartmentsA titanium screw held the electrode against the membraneand acted as electrical connector Each micro-MFC cham-ber was equipped with a 25 ID mm polyurethane inlettube (FESTO Germany) on the bottom for medium andelectrolyte feeding and one on the top for biogas outputand replacement of exhausted electrolyte For disinfectionthe plexiglas microchambers were soaked in a 10 bleachsolution rinsed with deionized water and then exposed to

UV light for 12 h Graphite plates were washed with 100isopropanol in an ultrasonic bath and then heated in an ovenfor 2 h at 200C Micro-MFCs were operated in the opencircuit mode and current measurements were performedevery 24 hours starting from time of inoculation 50mMiron(II) phthalocyanine withoutN

2sparging was used as the

cathode electrolyte solutionAxenic bacterial cultures (Thalassospira spHJ the known

electrogen Geobacter daltonii and the magnetotactic Desul-famplus magnetomortis) were pregrown with shaking at 37∘Cfor 12 h in 5mL of modified PBS medium containing (perliter) 458 g Na

2HPO4 245 g NaH

2PO4sdotH2O 031 g NH

4Cl

013 g KCl 5 g glucose 5 g yeast extract and 5 g peptoneBacterial cells were harvested washed with 16mMphosphatebuffer (pH 70) and resuspended into 50mL fresh modifiedPBS medium The initial cell concentration was adjustedto an optical density at 600 nm of 03 as measured bya Spectronic GENESYS 5 spectrophotometer (Milton RoyCompany Rochester NY USA) with filtered cell-free culturemedium as reference

The micro-MFCs were inoculated initially with 15mLmedium containing bacterial cells (exceeding the sim10mLvolume of the anode chamber since the porous graphiteabsorbed some liquid) Bacteria-free modified PBS mediumcontaining 01 NaN

3was used for control experiments

All experiments were set up in two technical replicationsand two biological replications The axenicity of the anolytesfor the strain HJ-containing MFCs was determined atthe end of the experiments by plating serial dilutions toMarine Agar medium Experiments were performed at roomtemperature

4 BioMed Research International

(a) (b)

(c) (d)

Figure 3 Transmission electron micrographs of Thalassospira sp strain HJ showing various features (a) A single polar flagellum (b)intracellular electron-dense regions (c) symmetric cell division and (d) asymmetric cell division Representative images are shown

3 Results and Discussion

To obtain magnetic particle-associated bacteria some ofwhich should be electrogenic [12] we subjected tidal beachsediment to the enrichment procedure depicted in Figure 1One MFC was inoculated with a mixed culture derived fromthe magnetic particle-enriched sediment and the other withan axenic culture of a helical bacterium strain HJ isolatedfrom the enriched sediment Both MFCs were found toproduce power with the current and voltage generated fromthe mixed culture MFC being substantially higher (Figure 2)The comparatively low power density observed of strain HJis typical of axenic bacterial cultures relative to that of thecommunities from which they are derived [5] At the endof the experiment strain HJ was reisolated from the MFC toconfirm its retention

Strain HJ was identified by means of microscopic anal-ysis (Figure 3) and 16S rRNA gene sequence comparisons(Figure 4) to be of the genusThalassospira TEM images showcells of 05ndash07120583mwidth and 15ndash57120583m length having single

polar flagella typical of the genus Thalassospira [17] withevidence of both symmetric and asymmetric cell division(Figure 3) One or more electron-dense staining regions canbe seenwithinmost cells (Figure 3) Cells weremotile stainedGram negative and tested positive for catalase and oxidaseactivity Growth occurred on Marine Agar at temperaturesranging from 12∘C to 39∘C and pH values ranging from 55to 105 The closest matching 16S rRNA gene sequence toa named species within the GenBank database was that ofThalassospira profundimaris strainWP0211 (14491453 997identity) which notably differs from strain HJ in its lack offlagella [18]

Micro-MFC chambers inoculated with axenic cultures ofThalassospira sp strain HJ and for comparison Geobacterdaltonii and Desulfamplus magnetomortis showed electricalcurrent production immediately after inoculation but dif-fered in the intensity and shape of the subsequent productioncurve (Figure 5) Thalassospira sp strain HJ and D magne-tomortis displayed gradual increases in current after initia-tion whereas current production from G daltonii decreased

BioMed Research International 5

Thalassospira alkalitolerans (AB786710)Thalassospira xiamenensis M-5 (CP004388)

Thalassospira lucentensis QMT2 (AF358664)

Magnetospira thiophila MMS1 (EU861390)Magnetovibrio blakemorei MV1 (L06455)

01

Rhodospirillum oryzae JA318T (AM901295)Roseospira marina CE2105 (AJ298879)

Azospirillum brasilense DSM1690 (GU256438)Magnetospirillum magneticum AMB-1 (D17514)

Sphingomonas azotifigens NBRC15497 (AB217471)Rhodobacter blasticus ATCC33485 (D16429)

Caulobacter vibrioides FMC1 (JX968034)

Thalassospira profundimaris WP0211 (AY186195)

Thalassospira mesophila (AB786711)

Thalassospira sp HJ (KP704219)

Figure 4 Phylogenetic tree of strain HJ and selected alphaproteobacteria based on 16S rRNA gene sequences The tree was constructedfrom sequences aligned by MUSCLE using maximum-likelihood method of PhyML 30 [16 21] The scale bar reflects evolutionary distancemeasured in units of substitution per nucleotide site

0

5

10

15

20

25

30

35

0 24 48 72 96 120 144 172

Curr

ent (

mA

)

Time (h)0 24 48 72 96 120 144 172

Time (h)

000

005

010

015

020

025

030

035

040

045

Volta

ge (V

)

Strain HJD magnetomortis

G daltoniiControl

Strain HJD magnetomortis

G daltoniiControl

Figure 5 Voltage and current readings (plusmnrange 119899 = 2) from microbial fuel cells inoculated with axenic cultures of Thalassospira sp strainHJ Geobacter daltonii and Desulfamplus magnetomortis or medium only with 1 g Lminus1 sodium azide (control) Anode surface area 196 cm210mL volume

with time After 172 h the current for all axenic culturesdecreased to 5ndash8mA which was attributed with C-sourcedepletion Voltage drop was observed after 144 h from thestrain HJ-inoculated micro-MFC whereas G daltonii andD magnetomortis MFCs demonstrated voltage loss after48 h As a control to determine the contribution of growthmedium components to current and voltage production amicro-MFC was filled with sterile medium Although therewas no observable current generation background voltagewas observed (Figure 5) The maximal single-value powerreadings from the micro-MFCs were of 26Wmminus2 (at 96 h)

for Thalassospira sp strain HJ 55Wmminus2 (at 144 h) for Dmagnetomortis and 46Wmminus2 (at 24 h) for G daltonii

Geobacter is a well-characterized genus of electrogenicbacteria and although we are not aware of prior reportsof electrogenicity by the magnetotactic bacterium D mag-netomortis members of sulfate-reducing members of theDeltaproteobacteria to which D magnetomortis belongswere found to display transcriptomic responses to changes inMFC electrode potential indicative of electrogenic behavior[19]We hypothesize thatmagnetosomes could confer a selec-tive benefit by enabling bacteria to hone in on ferromagnetic

6 BioMed Research International

external electron acceptors A wider survey of magnetotacticbacteria for electrogenicity would be warranted

Recently two marine sediment-derived Thalassospira spisolates were reported to exhibit electrotrophic behavioraccepting electrons from insoluble sulfur [20] but the capacityof these strains to transfer electrons to an anode as we havefound here of Thalassospira sp strain HJ was not examinedExamination of the strain HJ genome does not reveal anyhomologs for the pili or outer membrane extension-typeexternal electron transfer systems found in Geobacter or She-wanella species [15] andmicroscopic imaging of anodes fromstrain HJ-colonized MFCs implies that biofilm formationis not necessary for electrogenicity (data not shown) Thuswe hypothesize that external electron transfer occurs via anelectron shuttle-type mechanism

4 Conclusions

A microbial community derived from magnetic particle-enriched marine sediment displayed electrogenic behaviorand yielded an electrogenic bacterium identified to be aThalassospira species This is the first report of electrogenicbehavior within the genus Thalassospira A more extensivestudy would be needed to determine whether the propor-tion of electrogenic bacteria obtained from this magneticparticle enrichment procedure exceeds that found in theenvironment With their diurnal patterns of flooding anddiversity of mineral components tidal sediments should berich environments to bioprospect for electrogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding was provided by the Okinawa Institute of Scienceand Technology I V Khilyas was supported by RussianGovernment Program of Competitive Growth of KazanFederal University The authors thank M Power World Ltdfor consultations and provision of equipment and T Sasakifor conducting transmission electron microscopy

References

[1] V Gude B Kokabian and V Gadhamshetty ldquoBeneficial bio-electrochemical systems for energy water and biomass pro-ductionrdquo Journal of Microbial amp Biochemical Technology vol 6article 005 p 2 2013

[2] S A Patil C Hagerhall and L Gorton ldquoElectron transfermechanisms between microorganisms and electrodes in bio-electrochemical systemsrdquo Bioanalytical Reviews vol 4 no 2ndash4pp 159ndash192 2012

[3] D R Lovley and N S Malvankar ldquoSeeing is believing Novelimaging techniques help clarify microbial nanowire structureand functionrdquo Environmental Microbiology vol 17 no 7 pp2209ndash2215 2015

[4] S Pirbadian S E Barchinger K M Leung et al ldquoShe-wanella oneidensis MR-1 nanowires are outer membrane andperiplasmic extensions of the extracellular electron transportcomponentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 111 no 35 pp 12883ndash128882014

[5] B E Logan ldquoExoelectrogenic bacteria that powermicrobial fuelcellsrdquo Nature Reviews Microbiology vol 7 no 5 pp 375ndash3812009

[6] H W Harrisa M Y El-Naggarb O Bretschgerc et al ldquoElec-trokinesis is a microbial behavior that requires extracellularelectron transportrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 1 pp 326ndash331 2010

[7] K Rabaey N Boon M Hofte and W Verstraete ldquoMicrobialphenazine production enhances electron transfer in biofuelcellsrdquo Environmental Science amp Technology vol 39 no 9 pp3401ndash3408 2005

[8] N S Malvankar G M King and D R Lovley ldquoCentimeter-long electron transport in marine sediments via conductivemineralsrdquoThe ISME Journal vol 9 no 2 pp 527ndash531 2015

[9] D R Lovley and K P Nevin ldquoA shift in the current new appli-cations and concepts for microbe-electrode electron exchangerdquoCurrent Opinion in Biotechnology vol 22 no 3 pp 441ndash4482011

[10] F Liu A Rotaru P M Shrestha N S Malvankar K P Nevinand D R Lovley ldquoMagnetite compensates for the lack of apilin-associated c-type cytochrome in extracellular electronexchangerdquo Environmental Microbiology vol 17 no 3 pp 648ndash655 2015

[11] S Kato K Hashimoto and K Watanabe ldquoMicrobial inter-species electron transfer via electric currents through conduc-tive mineralsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 25 pp 10042ndash100462012

[12] J E Kostka and K H Nealson ldquoDissolution and reduction ofmagnetite by bacteriardquo Environmental Science amp Technologyvol 29 no 10 pp 2535ndash2540 1995

[13] J M Byrne N Klueglein C Pearce K M Rosso E Appel andA Kappler ldquoRedox cycling of Fe(II) and Fe(III) in magnetite byFe-metabolizing bacteriardquo Science vol 347 no 6229 pp 1473ndash1476 2015

[14] R SWolfe R KThauer andN Pfennig ldquoA lsquocapillary racetrackrsquomethod for isolation of magnetotactic bacteriardquo FEMSMicrobi-ology Letters vol 45 no 1 pp 31ndash35 1987

[15] L Kiseleva S K Garushyants J Briliute D J SimpsonM F Cohen and I Goryanin ldquoGenome sequence of theelectrogenic petroleum-degrading Thalassospira sp strain HJrdquoGenome Announcements vol 3 no 3 Article ID e00483-152015

[16] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 supplement 2 pp W465ndashW469 2008

[17] J I Baldani S S Videira J I Baldani et al ldquoThe family Rho-dospirillaceaerdquo in The Prokaryotes E Rosenberg E DeLongS Lory E Stackebrandt and F Thompson Eds pp 533ndash618Springer Berlin Germany 2014

[18] C Liu Y Wu L Li M Yingfei and Z Shao ldquoThalassospiraxiamenensis sp nov and Thalassospira profundimaris sp novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 2 pp 316ndash320 2007

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

(a) (b)

(c) (d)

Figure 3 Transmission electron micrographs of Thalassospira sp strain HJ showing various features (a) A single polar flagellum (b)intracellular electron-dense regions (c) symmetric cell division and (d) asymmetric cell division Representative images are shown

3 Results and Discussion

To obtain magnetic particle-associated bacteria some ofwhich should be electrogenic [12] we subjected tidal beachsediment to the enrichment procedure depicted in Figure 1One MFC was inoculated with a mixed culture derived fromthe magnetic particle-enriched sediment and the other withan axenic culture of a helical bacterium strain HJ isolatedfrom the enriched sediment Both MFCs were found toproduce power with the current and voltage generated fromthe mixed culture MFC being substantially higher (Figure 2)The comparatively low power density observed of strain HJis typical of axenic bacterial cultures relative to that of thecommunities from which they are derived [5] At the endof the experiment strain HJ was reisolated from the MFC toconfirm its retention

Strain HJ was identified by means of microscopic anal-ysis (Figure 3) and 16S rRNA gene sequence comparisons(Figure 4) to be of the genusThalassospira TEM images showcells of 05ndash07120583mwidth and 15ndash57120583m length having single

polar flagella typical of the genus Thalassospira [17] withevidence of both symmetric and asymmetric cell division(Figure 3) One or more electron-dense staining regions canbe seenwithinmost cells (Figure 3) Cells weremotile stainedGram negative and tested positive for catalase and oxidaseactivity Growth occurred on Marine Agar at temperaturesranging from 12∘C to 39∘C and pH values ranging from 55to 105 The closest matching 16S rRNA gene sequence toa named species within the GenBank database was that ofThalassospira profundimaris strainWP0211 (14491453 997identity) which notably differs from strain HJ in its lack offlagella [18]

Micro-MFC chambers inoculated with axenic cultures ofThalassospira sp strain HJ and for comparison Geobacterdaltonii and Desulfamplus magnetomortis showed electricalcurrent production immediately after inoculation but dif-fered in the intensity and shape of the subsequent productioncurve (Figure 5) Thalassospira sp strain HJ and D magne-tomortis displayed gradual increases in current after initia-tion whereas current production from G daltonii decreased

BioMed Research International 5

Thalassospira alkalitolerans (AB786710)Thalassospira xiamenensis M-5 (CP004388)

Thalassospira lucentensis QMT2 (AF358664)

Magnetospira thiophila MMS1 (EU861390)Magnetovibrio blakemorei MV1 (L06455)

01

Rhodospirillum oryzae JA318T (AM901295)Roseospira marina CE2105 (AJ298879)

Azospirillum brasilense DSM1690 (GU256438)Magnetospirillum magneticum AMB-1 (D17514)

Sphingomonas azotifigens NBRC15497 (AB217471)Rhodobacter blasticus ATCC33485 (D16429)

Caulobacter vibrioides FMC1 (JX968034)

Thalassospira profundimaris WP0211 (AY186195)

Thalassospira mesophila (AB786711)

Thalassospira sp HJ (KP704219)

Figure 4 Phylogenetic tree of strain HJ and selected alphaproteobacteria based on 16S rRNA gene sequences The tree was constructedfrom sequences aligned by MUSCLE using maximum-likelihood method of PhyML 30 [16 21] The scale bar reflects evolutionary distancemeasured in units of substitution per nucleotide site

0

5

10

15

20

25

30

35

0 24 48 72 96 120 144 172

Curr

ent (

mA

)

Time (h)0 24 48 72 96 120 144 172

Time (h)

000

005

010

015

020

025

030

035

040

045

Volta

ge (V

)

Strain HJD magnetomortis

G daltoniiControl

Strain HJD magnetomortis

G daltoniiControl

Figure 5 Voltage and current readings (plusmnrange 119899 = 2) from microbial fuel cells inoculated with axenic cultures of Thalassospira sp strainHJ Geobacter daltonii and Desulfamplus magnetomortis or medium only with 1 g Lminus1 sodium azide (control) Anode surface area 196 cm210mL volume

with time After 172 h the current for all axenic culturesdecreased to 5ndash8mA which was attributed with C-sourcedepletion Voltage drop was observed after 144 h from thestrain HJ-inoculated micro-MFC whereas G daltonii andD magnetomortis MFCs demonstrated voltage loss after48 h As a control to determine the contribution of growthmedium components to current and voltage production amicro-MFC was filled with sterile medium Although therewas no observable current generation background voltagewas observed (Figure 5) The maximal single-value powerreadings from the micro-MFCs were of 26Wmminus2 (at 96 h)

for Thalassospira sp strain HJ 55Wmminus2 (at 144 h) for Dmagnetomortis and 46Wmminus2 (at 24 h) for G daltonii

Geobacter is a well-characterized genus of electrogenicbacteria and although we are not aware of prior reportsof electrogenicity by the magnetotactic bacterium D mag-netomortis members of sulfate-reducing members of theDeltaproteobacteria to which D magnetomortis belongswere found to display transcriptomic responses to changes inMFC electrode potential indicative of electrogenic behavior[19]We hypothesize thatmagnetosomes could confer a selec-tive benefit by enabling bacteria to hone in on ferromagnetic

6 BioMed Research International

external electron acceptors A wider survey of magnetotacticbacteria for electrogenicity would be warranted

Recently two marine sediment-derived Thalassospira spisolates were reported to exhibit electrotrophic behavioraccepting electrons from insoluble sulfur [20] but the capacityof these strains to transfer electrons to an anode as we havefound here of Thalassospira sp strain HJ was not examinedExamination of the strain HJ genome does not reveal anyhomologs for the pili or outer membrane extension-typeexternal electron transfer systems found in Geobacter or She-wanella species [15] andmicroscopic imaging of anodes fromstrain HJ-colonized MFCs implies that biofilm formationis not necessary for electrogenicity (data not shown) Thuswe hypothesize that external electron transfer occurs via anelectron shuttle-type mechanism

4 Conclusions

A microbial community derived from magnetic particle-enriched marine sediment displayed electrogenic behaviorand yielded an electrogenic bacterium identified to be aThalassospira species This is the first report of electrogenicbehavior within the genus Thalassospira A more extensivestudy would be needed to determine whether the propor-tion of electrogenic bacteria obtained from this magneticparticle enrichment procedure exceeds that found in theenvironment With their diurnal patterns of flooding anddiversity of mineral components tidal sediments should berich environments to bioprospect for electrogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding was provided by the Okinawa Institute of Scienceand Technology I V Khilyas was supported by RussianGovernment Program of Competitive Growth of KazanFederal University The authors thank M Power World Ltdfor consultations and provision of equipment and T Sasakifor conducting transmission electron microscopy

References

[1] V Gude B Kokabian and V Gadhamshetty ldquoBeneficial bio-electrochemical systems for energy water and biomass pro-ductionrdquo Journal of Microbial amp Biochemical Technology vol 6article 005 p 2 2013

[2] S A Patil C Hagerhall and L Gorton ldquoElectron transfermechanisms between microorganisms and electrodes in bio-electrochemical systemsrdquo Bioanalytical Reviews vol 4 no 2ndash4pp 159ndash192 2012

[3] D R Lovley and N S Malvankar ldquoSeeing is believing Novelimaging techniques help clarify microbial nanowire structureand functionrdquo Environmental Microbiology vol 17 no 7 pp2209ndash2215 2015

[4] S Pirbadian S E Barchinger K M Leung et al ldquoShe-wanella oneidensis MR-1 nanowires are outer membrane andperiplasmic extensions of the extracellular electron transportcomponentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 111 no 35 pp 12883ndash128882014

[5] B E Logan ldquoExoelectrogenic bacteria that powermicrobial fuelcellsrdquo Nature Reviews Microbiology vol 7 no 5 pp 375ndash3812009

[6] H W Harrisa M Y El-Naggarb O Bretschgerc et al ldquoElec-trokinesis is a microbial behavior that requires extracellularelectron transportrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 1 pp 326ndash331 2010

[7] K Rabaey N Boon M Hofte and W Verstraete ldquoMicrobialphenazine production enhances electron transfer in biofuelcellsrdquo Environmental Science amp Technology vol 39 no 9 pp3401ndash3408 2005

[8] N S Malvankar G M King and D R Lovley ldquoCentimeter-long electron transport in marine sediments via conductivemineralsrdquoThe ISME Journal vol 9 no 2 pp 527ndash531 2015

[9] D R Lovley and K P Nevin ldquoA shift in the current new appli-cations and concepts for microbe-electrode electron exchangerdquoCurrent Opinion in Biotechnology vol 22 no 3 pp 441ndash4482011

[10] F Liu A Rotaru P M Shrestha N S Malvankar K P Nevinand D R Lovley ldquoMagnetite compensates for the lack of apilin-associated c-type cytochrome in extracellular electronexchangerdquo Environmental Microbiology vol 17 no 3 pp 648ndash655 2015

[11] S Kato K Hashimoto and K Watanabe ldquoMicrobial inter-species electron transfer via electric currents through conduc-tive mineralsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 25 pp 10042ndash100462012

[12] J E Kostka and K H Nealson ldquoDissolution and reduction ofmagnetite by bacteriardquo Environmental Science amp Technologyvol 29 no 10 pp 2535ndash2540 1995

[13] J M Byrne N Klueglein C Pearce K M Rosso E Appel andA Kappler ldquoRedox cycling of Fe(II) and Fe(III) in magnetite byFe-metabolizing bacteriardquo Science vol 347 no 6229 pp 1473ndash1476 2015

[14] R SWolfe R KThauer andN Pfennig ldquoA lsquocapillary racetrackrsquomethod for isolation of magnetotactic bacteriardquo FEMSMicrobi-ology Letters vol 45 no 1 pp 31ndash35 1987

[15] L Kiseleva S K Garushyants J Briliute D J SimpsonM F Cohen and I Goryanin ldquoGenome sequence of theelectrogenic petroleum-degrading Thalassospira sp strain HJrdquoGenome Announcements vol 3 no 3 Article ID e00483-152015

[16] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 supplement 2 pp W465ndashW469 2008

[17] J I Baldani S S Videira J I Baldani et al ldquoThe family Rho-dospirillaceaerdquo in The Prokaryotes E Rosenberg E DeLongS Lory E Stackebrandt and F Thompson Eds pp 533ndash618Springer Berlin Germany 2014

[18] C Liu Y Wu L Li M Yingfei and Z Shao ldquoThalassospiraxiamenensis sp nov and Thalassospira profundimaris sp novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 2 pp 316ndash320 2007

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Thalassospira alkalitolerans (AB786710)Thalassospira xiamenensis M-5 (CP004388)

Thalassospira lucentensis QMT2 (AF358664)

Magnetospira thiophila MMS1 (EU861390)Magnetovibrio blakemorei MV1 (L06455)

01

Rhodospirillum oryzae JA318T (AM901295)Roseospira marina CE2105 (AJ298879)

Azospirillum brasilense DSM1690 (GU256438)Magnetospirillum magneticum AMB-1 (D17514)

Sphingomonas azotifigens NBRC15497 (AB217471)Rhodobacter blasticus ATCC33485 (D16429)

Caulobacter vibrioides FMC1 (JX968034)

Thalassospira profundimaris WP0211 (AY186195)

Thalassospira mesophila (AB786711)

Thalassospira sp HJ (KP704219)

Figure 4 Phylogenetic tree of strain HJ and selected alphaproteobacteria based on 16S rRNA gene sequences The tree was constructedfrom sequences aligned by MUSCLE using maximum-likelihood method of PhyML 30 [16 21] The scale bar reflects evolutionary distancemeasured in units of substitution per nucleotide site

0

5

10

15

20

25

30

35

0 24 48 72 96 120 144 172

Curr

ent (

mA

)

Time (h)0 24 48 72 96 120 144 172

Time (h)

000

005

010

015

020

025

030

035

040

045

Volta

ge (V

)

Strain HJD magnetomortis

G daltoniiControl

Strain HJD magnetomortis

G daltoniiControl

Figure 5 Voltage and current readings (plusmnrange 119899 = 2) from microbial fuel cells inoculated with axenic cultures of Thalassospira sp strainHJ Geobacter daltonii and Desulfamplus magnetomortis or medium only with 1 g Lminus1 sodium azide (control) Anode surface area 196 cm210mL volume

with time After 172 h the current for all axenic culturesdecreased to 5ndash8mA which was attributed with C-sourcedepletion Voltage drop was observed after 144 h from thestrain HJ-inoculated micro-MFC whereas G daltonii andD magnetomortis MFCs demonstrated voltage loss after48 h As a control to determine the contribution of growthmedium components to current and voltage production amicro-MFC was filled with sterile medium Although therewas no observable current generation background voltagewas observed (Figure 5) The maximal single-value powerreadings from the micro-MFCs were of 26Wmminus2 (at 96 h)

for Thalassospira sp strain HJ 55Wmminus2 (at 144 h) for Dmagnetomortis and 46Wmminus2 (at 24 h) for G daltonii

Geobacter is a well-characterized genus of electrogenicbacteria and although we are not aware of prior reportsof electrogenicity by the magnetotactic bacterium D mag-netomortis members of sulfate-reducing members of theDeltaproteobacteria to which D magnetomortis belongswere found to display transcriptomic responses to changes inMFC electrode potential indicative of electrogenic behavior[19]We hypothesize thatmagnetosomes could confer a selec-tive benefit by enabling bacteria to hone in on ferromagnetic

6 BioMed Research International

external electron acceptors A wider survey of magnetotacticbacteria for electrogenicity would be warranted

Recently two marine sediment-derived Thalassospira spisolates were reported to exhibit electrotrophic behavioraccepting electrons from insoluble sulfur [20] but the capacityof these strains to transfer electrons to an anode as we havefound here of Thalassospira sp strain HJ was not examinedExamination of the strain HJ genome does not reveal anyhomologs for the pili or outer membrane extension-typeexternal electron transfer systems found in Geobacter or She-wanella species [15] andmicroscopic imaging of anodes fromstrain HJ-colonized MFCs implies that biofilm formationis not necessary for electrogenicity (data not shown) Thuswe hypothesize that external electron transfer occurs via anelectron shuttle-type mechanism

4 Conclusions

A microbial community derived from magnetic particle-enriched marine sediment displayed electrogenic behaviorand yielded an electrogenic bacterium identified to be aThalassospira species This is the first report of electrogenicbehavior within the genus Thalassospira A more extensivestudy would be needed to determine whether the propor-tion of electrogenic bacteria obtained from this magneticparticle enrichment procedure exceeds that found in theenvironment With their diurnal patterns of flooding anddiversity of mineral components tidal sediments should berich environments to bioprospect for electrogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding was provided by the Okinawa Institute of Scienceand Technology I V Khilyas was supported by RussianGovernment Program of Competitive Growth of KazanFederal University The authors thank M Power World Ltdfor consultations and provision of equipment and T Sasakifor conducting transmission electron microscopy

References

[1] V Gude B Kokabian and V Gadhamshetty ldquoBeneficial bio-electrochemical systems for energy water and biomass pro-ductionrdquo Journal of Microbial amp Biochemical Technology vol 6article 005 p 2 2013

[2] S A Patil C Hagerhall and L Gorton ldquoElectron transfermechanisms between microorganisms and electrodes in bio-electrochemical systemsrdquo Bioanalytical Reviews vol 4 no 2ndash4pp 159ndash192 2012

[3] D R Lovley and N S Malvankar ldquoSeeing is believing Novelimaging techniques help clarify microbial nanowire structureand functionrdquo Environmental Microbiology vol 17 no 7 pp2209ndash2215 2015

[4] S Pirbadian S E Barchinger K M Leung et al ldquoShe-wanella oneidensis MR-1 nanowires are outer membrane andperiplasmic extensions of the extracellular electron transportcomponentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 111 no 35 pp 12883ndash128882014

[5] B E Logan ldquoExoelectrogenic bacteria that powermicrobial fuelcellsrdquo Nature Reviews Microbiology vol 7 no 5 pp 375ndash3812009

[6] H W Harrisa M Y El-Naggarb O Bretschgerc et al ldquoElec-trokinesis is a microbial behavior that requires extracellularelectron transportrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 1 pp 326ndash331 2010

[7] K Rabaey N Boon M Hofte and W Verstraete ldquoMicrobialphenazine production enhances electron transfer in biofuelcellsrdquo Environmental Science amp Technology vol 39 no 9 pp3401ndash3408 2005

[8] N S Malvankar G M King and D R Lovley ldquoCentimeter-long electron transport in marine sediments via conductivemineralsrdquoThe ISME Journal vol 9 no 2 pp 527ndash531 2015

[9] D R Lovley and K P Nevin ldquoA shift in the current new appli-cations and concepts for microbe-electrode electron exchangerdquoCurrent Opinion in Biotechnology vol 22 no 3 pp 441ndash4482011

[10] F Liu A Rotaru P M Shrestha N S Malvankar K P Nevinand D R Lovley ldquoMagnetite compensates for the lack of apilin-associated c-type cytochrome in extracellular electronexchangerdquo Environmental Microbiology vol 17 no 3 pp 648ndash655 2015

[11] S Kato K Hashimoto and K Watanabe ldquoMicrobial inter-species electron transfer via electric currents through conduc-tive mineralsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 25 pp 10042ndash100462012

[12] J E Kostka and K H Nealson ldquoDissolution and reduction ofmagnetite by bacteriardquo Environmental Science amp Technologyvol 29 no 10 pp 2535ndash2540 1995

[13] J M Byrne N Klueglein C Pearce K M Rosso E Appel andA Kappler ldquoRedox cycling of Fe(II) and Fe(III) in magnetite byFe-metabolizing bacteriardquo Science vol 347 no 6229 pp 1473ndash1476 2015

[14] R SWolfe R KThauer andN Pfennig ldquoA lsquocapillary racetrackrsquomethod for isolation of magnetotactic bacteriardquo FEMSMicrobi-ology Letters vol 45 no 1 pp 31ndash35 1987

[15] L Kiseleva S K Garushyants J Briliute D J SimpsonM F Cohen and I Goryanin ldquoGenome sequence of theelectrogenic petroleum-degrading Thalassospira sp strain HJrdquoGenome Announcements vol 3 no 3 Article ID e00483-152015

[16] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 supplement 2 pp W465ndashW469 2008

[17] J I Baldani S S Videira J I Baldani et al ldquoThe family Rho-dospirillaceaerdquo in The Prokaryotes E Rosenberg E DeLongS Lory E Stackebrandt and F Thompson Eds pp 533ndash618Springer Berlin Germany 2014

[18] C Liu Y Wu L Li M Yingfei and Z Shao ldquoThalassospiraxiamenensis sp nov and Thalassospira profundimaris sp novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 2 pp 316ndash320 2007

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

external electron acceptors A wider survey of magnetotacticbacteria for electrogenicity would be warranted

Recently two marine sediment-derived Thalassospira spisolates were reported to exhibit electrotrophic behavioraccepting electrons from insoluble sulfur [20] but the capacityof these strains to transfer electrons to an anode as we havefound here of Thalassospira sp strain HJ was not examinedExamination of the strain HJ genome does not reveal anyhomologs for the pili or outer membrane extension-typeexternal electron transfer systems found in Geobacter or She-wanella species [15] andmicroscopic imaging of anodes fromstrain HJ-colonized MFCs implies that biofilm formationis not necessary for electrogenicity (data not shown) Thuswe hypothesize that external electron transfer occurs via anelectron shuttle-type mechanism

4 Conclusions

A microbial community derived from magnetic particle-enriched marine sediment displayed electrogenic behaviorand yielded an electrogenic bacterium identified to be aThalassospira species This is the first report of electrogenicbehavior within the genus Thalassospira A more extensivestudy would be needed to determine whether the propor-tion of electrogenic bacteria obtained from this magneticparticle enrichment procedure exceeds that found in theenvironment With their diurnal patterns of flooding anddiversity of mineral components tidal sediments should berich environments to bioprospect for electrogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding was provided by the Okinawa Institute of Scienceand Technology I V Khilyas was supported by RussianGovernment Program of Competitive Growth of KazanFederal University The authors thank M Power World Ltdfor consultations and provision of equipment and T Sasakifor conducting transmission electron microscopy

References

[1] V Gude B Kokabian and V Gadhamshetty ldquoBeneficial bio-electrochemical systems for energy water and biomass pro-ductionrdquo Journal of Microbial amp Biochemical Technology vol 6article 005 p 2 2013

[2] S A Patil C Hagerhall and L Gorton ldquoElectron transfermechanisms between microorganisms and electrodes in bio-electrochemical systemsrdquo Bioanalytical Reviews vol 4 no 2ndash4pp 159ndash192 2012

[3] D R Lovley and N S Malvankar ldquoSeeing is believing Novelimaging techniques help clarify microbial nanowire structureand functionrdquo Environmental Microbiology vol 17 no 7 pp2209ndash2215 2015

[4] S Pirbadian S E Barchinger K M Leung et al ldquoShe-wanella oneidensis MR-1 nanowires are outer membrane andperiplasmic extensions of the extracellular electron transportcomponentsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 111 no 35 pp 12883ndash128882014

[5] B E Logan ldquoExoelectrogenic bacteria that powermicrobial fuelcellsrdquo Nature Reviews Microbiology vol 7 no 5 pp 375ndash3812009

[6] H W Harrisa M Y El-Naggarb O Bretschgerc et al ldquoElec-trokinesis is a microbial behavior that requires extracellularelectron transportrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 1 pp 326ndash331 2010

[7] K Rabaey N Boon M Hofte and W Verstraete ldquoMicrobialphenazine production enhances electron transfer in biofuelcellsrdquo Environmental Science amp Technology vol 39 no 9 pp3401ndash3408 2005

[8] N S Malvankar G M King and D R Lovley ldquoCentimeter-long electron transport in marine sediments via conductivemineralsrdquoThe ISME Journal vol 9 no 2 pp 527ndash531 2015

[9] D R Lovley and K P Nevin ldquoA shift in the current new appli-cations and concepts for microbe-electrode electron exchangerdquoCurrent Opinion in Biotechnology vol 22 no 3 pp 441ndash4482011

[10] F Liu A Rotaru P M Shrestha N S Malvankar K P Nevinand D R Lovley ldquoMagnetite compensates for the lack of apilin-associated c-type cytochrome in extracellular electronexchangerdquo Environmental Microbiology vol 17 no 3 pp 648ndash655 2015

[11] S Kato K Hashimoto and K Watanabe ldquoMicrobial inter-species electron transfer via electric currents through conduc-tive mineralsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 25 pp 10042ndash100462012

[12] J E Kostka and K H Nealson ldquoDissolution and reduction ofmagnetite by bacteriardquo Environmental Science amp Technologyvol 29 no 10 pp 2535ndash2540 1995

[13] J M Byrne N Klueglein C Pearce K M Rosso E Appel andA Kappler ldquoRedox cycling of Fe(II) and Fe(III) in magnetite byFe-metabolizing bacteriardquo Science vol 347 no 6229 pp 1473ndash1476 2015

[14] R SWolfe R KThauer andN Pfennig ldquoA lsquocapillary racetrackrsquomethod for isolation of magnetotactic bacteriardquo FEMSMicrobi-ology Letters vol 45 no 1 pp 31ndash35 1987

[15] L Kiseleva S K Garushyants J Briliute D J SimpsonM F Cohen and I Goryanin ldquoGenome sequence of theelectrogenic petroleum-degrading Thalassospira sp strain HJrdquoGenome Announcements vol 3 no 3 Article ID e00483-152015

[16] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 supplement 2 pp W465ndashW469 2008

[17] J I Baldani S S Videira J I Baldani et al ldquoThe family Rho-dospirillaceaerdquo in The Prokaryotes E Rosenberg E DeLongS Lory E Stackebrandt and F Thompson Eds pp 533ndash618Springer Berlin Germany 2014

[18] C Liu Y Wu L Li M Yingfei and Z Shao ldquoThalassospiraxiamenensis sp nov and Thalassospira profundimaris sp novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 2 pp 316ndash320 2007

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

[19] S Ishii S Suzuki T M Norden-Krichmar et al ldquoA novel meta-transcriptomic approach to identify gene expression dynamicsduring extracellular electron transferrdquoNature Communicationsvol 4 article 1601 2013

[20] A R Rowe P Chellamuthu B Lam A Okamoto and KH Nealson ldquoMarine sediments microbes capable of electrodeoxidation as a surrogate for lithotrophic insoluble substratemetabolismrdquo Frontiers in Microbiology vol 5 article 784 2015

[21] SGuindon J-FDufayardV LefortMAnisimovaWHordijkand O Gascuel ldquoNew algorithms and methods to estimatemaximum-likelihood phylogenies assessing the performanceof PhyML 30rdquo Systematic Biology vol 59 no 3 pp 307ndash3212010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology