R E S EARCH ART I C L E

ENV IRONMENTAL CHEM I STRY

1State Key Laboratory of Agricultural Microbiology College of Science College of FoodScience and Technology Huazhong Agricultural University Wuhan 430070 China 2KeyLaboratory of Urban Pollutant Conversion Institute of Urban Environment Chinese Acad-emy of Sciences Xiamen 361021 ChinaThese authors contributed equally to this workdaggerCorresponding author Email hyhanmailhzaueducn (HH) fzhaoiueaccn (FZ)

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

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101126sciadv1600858

Microbial synthesis of highly dispersed PdAu alloyfor enhanced electrocatalysis

Jiawei Liu1 Yue Zheng2 Zilan Hong1 Kai Cai1 Feng Zhao2dagger Heyou Han1dagger

Biosynthesis based on the reducing capacity of electrochemically active bacteria is frequently used in the reduction ofmetal ions into nanoparticles as an eco-friendly way to recyclemetal resources However those bionanoparticles can-not beuseddirectly as electrocatalysts because of the poor conductivity of cell substrates This problemwas solvedbya hydrothermal reaction which also contributes to the heteroatomdoping and alloying between Pd and AuWith theprotection of graphene the aggregation of nanoparticles was successfully avoided and the porous structure wasmaintained resulting in better electrocatalytic activity and durability than commercial PdC under both alkaline(CH3CH2OH 615-fold ofmass activity) and acidic (HCOOH 658-fold ofmass activity) conditions The strategydevelopedin this work opens up a horizon into designing electrocatalysts through fully utilizing the abundant resources in nature

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INTRODUCTION

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Direct liquid fuel cell is considered a promising green energy device forthe conversion of chemical energy into electricity because of its highoverall electric power efficiency excellent fuel flexibility and envi-ronmental friendliness (1ndash3) Despite its renewable and convenientfeatures the further development of direct liquid fuel cell is hinderedby the low energy conversion efficiency of commercial PtC catalystswith relatively low catalytic activity poor durability and high costTherefore enormous efforts have beenmade to seek alternatives to cur-rent commercial catalysts in the past few decades (4ndash10)

Several strategies have been proposed to enhance the performance ofelectrocatalysts First binary alloy nanocatalysts are fabricated by alloy-ing Pd or Pt with other transition metals such as PtAu PdCu PdNiPdAu and PdPtAg (11ndash15) Moreover several nonmetals have alsobeen used as dopants into noble metals including Pd-P Pd-Ni-Pand Pt-Ni-P (16ndash18) Multialloys are proven to enhance the catalyticactivity through the regulation of the surface electronic structure ofthe catalyticmetal by alloying other elements Another effective strategyto enhance the catalytic activity per unit is seeking and exploiting novelcatalyst substrates (such as carbon substrates) with chemical doping(19ndash24) Among various element dopings nitrogen doping is frequentlyused to enhance the electric conductivity of carbonmaterials and inducen-type semiconductor property (25ndash27) In addition numerous effortshave also beenmade to find an efficient way to anchor nanoparticles onthose supports (21) Traditionalmethods of chemical synthesis can suc-cessfully fabricate alloy catalysts but the addition of toxic surfactantstabilizer and reducing agents makes the synthesis process complicatedand environmentally unfriendly which violates the concept of ldquogreenenergyrdquo Those capping agents around the nanoparticles may severelylimit the activity of catalysts (28)

How to fabricate advanced electrocatalysts by a more facile andgreen method encourages us to develop an alternative protocol froma microbial perspective It has been reported that metal nanoparticlescan be green-synthesized by the metal-respiring bacterium for de-

chlorination of environmental contaminants andHeck coupling reaction(29ndash32) However the nanoparticles synthesized by microorganismshave rarely been used as electrocatalysts because of the poor conductivenature ofmicrobial cells (33) High-temperature carbonization reaction isa commonway to increase the conductivity ofmicrobial cells (33ndash35) butit cannot avoid the aggregation of those nanoparticles at a high loadingamount limiting their catalytic performance (33) Thereforemaintainingthe balance between increasing the conductivity of microbial cells andavoiding the aggregation of nanoparticles still remains a challenge to besolved

To solve the aforementioned problems and develop an efficient elec-trocatalyst for small organicmolecular electrooxidation through a greenprotocol this study in situndashsynthesized bio-PdAu nanoparticles on thesurface of an electrochemically active bacterium without adding toxicsurfactant stabilizer chemical reductant and bonding agent In thisprocess electrochemically active bacterium served as (i) a reducingagent (ii) a supporting material and (iii) doping heteroatom sourcesat the same time The as-prepared coreshell bacteriaPdAu (BPA)was further coated by graphene oxide (GO) to forma three-dimensional(3D) coreshellshell bacteriaPdAuGO hybrid biofilm (BPAGB) Fi-nally the doping process carbonization of bacteria and reduction ofGO were carried out through a facile hydrothermal reaction and theas-prepared hybrid biofilm became a 3D porous heteroatom-dopedbio-PdAureduced GO (rGO) hybrid (DPARH) with clean surfaceand excellent conduction With the protection of graphene the aggre-gation of nanoparticles was successfully avoided under this synergisticcondition The green-synthesized DPARH shows superior electrocata-lytic activity and durability and canmarkedly accelerate the energy con-version on the anode of a direct liquid fuel cell

RESULTS AND DISCUSSION

Shewanella oneidensisMR-1 a typical electrochemically active bacteri-um was used to reduce the metal ions (Pd and Au) and biosynthesizePd-Aunanoparticles located on the surface of the bacteria According toprevious works various kinds of metal nanoparticles can be biosynthe-sized by microorganisms through extracellular electron transfer (36)From the perspective of extracellular electron transfer previous studies

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proposed the following two principal mechanisms of metal respirationFirst electrons could be directly delivered from the bacterium to themetal ions via either membrane cytochromes or electrically conductivebionanowires Second soluble redox molecules such as flavin serve asmediators in an indirect electron transfer from the bacteria to the metalions (31) Here cells of S oneidensisMR-1were collected by washing toremove residual extracellular compounds to avoid the occurrence of in-direct metal reduction Considering the enrichment medium (LB) con-sisting of abundant nutrient elements Shewanella cells are not easily aptto generate bionanowires to achieve electron transfer under nutrient-rich environments (37) On the basis of experimental procedures andour results membrane cytochromes might play a main role in metalreduction for biosynthesis of PdAu nanoparticles Because the complexwith OmcA and MtrC of S oneidensis MR-1 has been proven to bethe terminal reductase that is crucial for extracellular reduction (38)the cells of the mutant (without OmcA and MtrC) were used as acompared experiment Compared with the reducing performanceof the DOmcAMtrC mutant the native S oneidensis MR-1 showeda stronger reduction with Pd and Au ions (fig S1) Therefore theOmcAMtrC complex worked as one of the key outer membrane pro-teins in this process which means that the direct electron transfer ofthe membrane cytochrome would be the key to biosynthesis of PdAunanoparticles

The target material was fabricated in the following three steps(Scheme 1) First inspired by the redox capacity of the electrochemicallyactive bacterium the synthesis process started separately with the in situbioreduction of Pd and Au nanoparticles on the cytomembrane ofS oneidensis MR-1 respectively Second the BPA was further coatedby GO to form a 3D structure which largely enhanced the specific sur-face area as well as stably anchored the PdAu nanoparticles in the mid-dle layer between the bacteria and GO Meanwhile the self-assembledsandwich structure coated by GO was constructed to further maintainthe bionanoparticles from aggregation after a hydrothermal reactionFinally the as-prepared hybrid biofilm was mildly hydrothermally re-acted to improve the conductivity of the cell substrate On the basis ofthese steps DPARH was successfully green-synthesized

The formation process of DPARH electrocatalyst was observed byscanning electron microscopy (SEM) imaging to demonstrate thegrowth mechanism Pd nanoparticles were relatively uniformly distrib-uted on themembrane of S oneidensisMR-1 (Fig 1A) in the first stages

Scheme 1 Formation mechanism for DPARH NPs nanoparticles

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

The nanoparticles were densely distributed after reacting with AuCl4minus

(Fig 1B) The cells with nanoparticles were connected to form 3Dnetwork structures by GO coating (Fig 1C) After the hydrothermal re-action the skeleton of the cells was reduced in size because of the releaseof enchylema under high temperature but the porous structure was stillobviously maintained and PdAu nanoparticles did not aggregate clearlywhich ismuch better thanKOHactivation at 420degC (temperature ramp3degCmin) for 3 hours under argon flow from the same raw materials(Fig 1D and fig S2A) (33)

Considering the attractive nanostructure and properties of DPARHwe investigated the electrocatalytic activity of this catalyst toward smallorganic molecular oxidation including ethanol and formic acid Theas-prepared catalyst showed a much higher electrocatalytic activity anddurability than commercial PdC under both alkaline (CH3CH2OH615-fold mass activity and 58-fold area activity) and acidic (HCOOH658-fold mass activity and 633-fold area activity) conditions (Fig 2)and performed much better than many other catalysts reported in re-cent studies (table S1) What leads to the excellent performance ofelectrooxidation inspired us to further explore the features of this de-signed catalyst fromspace structure and composition to surface electronicstructure

The more detailed structure information of BPAGB and DPARH ispresented in the following transmission electrionmicrosopy (TEM) andhigh-angle annular dark-field scanning TEM (HAADF-STEM) ele-mental mapping images The high-resolution TEM (HRTEM) imagingdemonstrated that BPAGB consists of small primary nanoparticles withan average diameter of 495 nm (Fig 3A2) and the diameter ofDPARHis slightly increased to 661 nm (Fig 3B2) By comparing this with theTEM images of heteroatom-doped bio-PdAu (DPA) (fig S2B) it can beseen that PdAu nanoparticles are easy to aggregate with each other toform a much larger structure without the protection of GO The latticeplanes with an interplanar distance of about 225 and 236 Aring which areconsidered as the (111) plane of face-centered cubic (fcc) metallic Pdand Au are distributed in different nanoparticles of BPAGB indicatingthat Pd and Au nanoparticles are separated from each other (Fig 3A3)However the interplanar distance of 230 and 201 Aring considered as the(111) and (200) planes of fcc metallic PdAu are found in DPARHindicating that PdAu nanoparticles become alloy after hydrothermalreaction To directly prove this phenomenon the microview was fur-ther illustrated by the HAADF-STEM elemental mapping Pd and Au

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nanoparticles were separated on the membrane of cells (Fig 3 B2 andB3) but they fused together to form an alloy after the hydrothermalreaction (Fig 3 D2 and D3)Whenmapping other elements includingP S andNwe found that P and Swere doped into themetal nanoparticlesbecauseheteroatomdopingbenefits the electrocatalytic performanceof cat-alysts as reported in previous studies (16)On the basis of the location ofP and S the P and S elements may be mainly derived from redox pro-teins coated on the cells during the metal reduction of biosynthesisThose membrane proteins participate in the biosynthesis of metal na-noparticles as a reductant and are bound with metal nanoparticles si-multaneously In addition this phenomenonmeant that themembraneprotein not only participates in the reducing and supporting process butalso plays a crucial role in doping heteroatom into our catalyst whichextends the function of bacteria in three ways (i) reducing metal ions(ii) supporting material and (iii) doping heteroatom More accurate

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changes of the structure were further analyzed by powder x-ray diffrac-tion (XRD) pattern

Figure 4 shows the XRD patterns of S oneidensis MR-1 BPAGBand DPARH The Pd and Au reflection peaks are separated in BPAGBfurther proving that they are two separated phases in this stage As toDPARH there is only a series of peaks corresponding to the cubic phaseof PdAu alloy indicating that the nanoparticles in DPARH are a PdAualloy rather than Pd or Au nanoparticles which is in accordance withthe result of HAADF-STEM elemental mapping (39)

The change in the surface electronic structure of Pd and nitrogenbonding configurations in PBABG and DPARH was investigated byx-ray photoelectron spectroscopy (XPS) as shown in Fig 5 Thebinding energies of Pd 3d52 and Pd 3d32 for DPARH are 33548 and34078 eV respectively which are evidently higher than those forPBABG (33518 and 34058 eV respectively) and positively shifted tohigher binding energy after hydrothermal reaction The movement ofbinding energy in Pd indicates the obvious change in the electronicstructure of as-prepared materials because of intra- or interatomiccharge transfer between Pd and Au when they are alloyed in theDPARH which might enhance the activity and durability of electroca-talytic ethanol and formic acid oxidation reaction (40) Moreover thehigh-resolution peak of N1s in BPABG and DPARH could be dividedinto two characteristic peaks corresponding to pyridinic N and pyrrolicN It is well known that pyridinic N pyrrolic N and graphitic (orquaternary) N are the three main types of nitrogen moiety and theformer two are the two common types that can be found in most ofthe N-doped graphene treated by hydrothermal reaction (41ndash46) Withthe addition of urea in the reaction the quaternaryN ismore likely to beobserved in the products (45 46) Because there is no urea in our hy-drothermal reaction there are therefore only two common types of ni-trogen moiety (pyridinic N and pyrrolic N) in our as-prepared catalystAs previously reported the pyridinic N (39914 and 39835 eV) resultedin a p-electron in the graphene layers to form a p-conjugated systemBecause of the contribution of pyridine and pyrrol functionalities thepyrrolic N (39989 and 3996 eV) contributed two p-electrons to the psystem (47) The pyridinic N content was higher in DPARH indicatingthat the nitrogen bonding configurations changed during the hydro-thermal reaction which benefits the electrocatalytic activity of the cat-alyst (48) In addition theXPS test ofDPARH from0 to 600 eVwas alsocarried out to obtain the semiquantitative atomic ratio of Pd Au P Nand S elements which can be calculated by the following equation ninj =(IiSi)(IjSj) where n denotes the concentration of atom I denotes theintensity of photoelectrical peak area and S denotes the relative atomic

Fig 2 General electrocatalytic activities of optimized catalyst Mass activities of DPARH and commercial PdC catalysts in CH3CH2OH and HCOOHelectrooxidation

Fig 1 Morphological features of as-preparedmaterials inbiosynthesisprocess SEM images of (A) coreshell bacteriaPd (BP) (B) BPA (C) BPABGand (D) DPARH

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sensitivity factors The atomic ratio of Pd Au P N and S elements is322810834324709548 (fig S3 and table S2) On the basis of this re-sult the mass ratio of Pd and Au is 6238 which is similar to that testedby inductively coupled plasma atomic emission spectroscopy (6733)

In previous studies the rGO was proven to be better than GO inconduction (49 50) indicating that the reduction of GO can greatly in-

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

fluence the electrocatalytic performance which was supported by Ra-man tests As shown in fig S4 each of the Raman spectra shows twovisible bands TheD band at around 1330 cmminus1 is related to the partiallydisordered structures or the structural defects of graphene whereas theG band at about 1590 cmminus1 corresponds to the ordered sp2 bonded car-bon The DG intensity ratio of DPARH BPAGB GO and rGO are127 103 073 and 126 respectively indicating that in the synthesisprocess of DPARH the GO is partly reduced by bacteria and furtherreduced after the hydrothermal reaction (51 52)

The electrochemically active surface areas of DPARH and commer-cial PdC catalysts were evaluated by calculating CO stripping experi-ment results from cyclic voltammograms (CVs) and tested in 01 MHClO4 (fig S5) By assuming that the oxidation of a monolayer ofCO on Pd corresponds to a charge of 420 mC cmminus2 and by normalizingthe result by Pd loading amount the electrochemically active surfaceareas of DPARH were calculated to be 5566 m2 gminus1 which is similarto those of commercial PdC (5265 m2 gminus1)

Muchhigher electrocatalytic activity and durabilitywere achieved byoptimizing the synthesis conditions such as PdAu ratio GO amounttemperature and time of hydrothermal reaction The DPARH withPdAu ratio of 32 coated with GO (10 ngliter) and synthesized at200degC for 6 hours showed the best electrocatalytic performance (figsS6 and S7 and table S3) The electrocatalytic activity of Pd-based catalystis affected by its composition because of the changes in the surface

Fig 3 Morphological features and element distributionof as-preparedmaterials before and after the hydrothermal reaction TEM (A1) andHRTEM(A2 and A3) imaging and HAADF-STEM elemental mapping (B1 to B6) of BPAGB TEM (C1) and HRTEM (C2 and C3) imaging and HAADF-STEM elementalmapping (D1 to D6) of DPARH after hydrothermal reaction

Fig 4 Structure features XRD pattern of S oneidensis MR-1 BPAGB andDPARH au arbitrary units

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electronic structure of Pd which has been proven inmany previous stu-dies (12 40) Hydrothermal temperature and treatment time mainlycontribute to a different degree of carbonization resulting in a differentelectrical conductivity With increasing hydrothermal temperature andtreatment time the carbonization of bacteria is gradually complete thusincreasing their electrical conductivity and electrocatalytic performanceIt should be noted that with the treatment time increased to 9 hours theelectrocatalytic performance is slightly decreased which may be

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

attributed to the aggregation of nanoparticles In addition the amountof GO also affects the catalytic performance and it can be mainlyattributed to the exposure of surface active sites When there is littleGO in the catalyst it is hard to build the 3D structure and impossibleto avoid the aggregation of nanoparticles just like the case withoutGO However with an excessive amount of GO those PdAu nanopar-ticles would be enclosed by thick GO preventing the interaction be-tween the small organic molecules and the PdAu active site which

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Fig 5 Surface atom features XPS of BPAGB and DPARH

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Fig 6 Detailed electrocatalytic performances of as-prepared catalysts (A) CVs of the DPARH- DPA- DP- and commercial PdC catalystndashmodified electro-des in 1 M KOH + 1 M ethanol at a scan rate of 50 mV sminus1 versus AgAgCl (saturated KCl) (B) Chronoamperometric curves of these catalyst-modifiedelectrodes in 1MKOH+1Methanol atminus03 V for 2000 s (C) CVs of these catalyst-modified electrodes in 05MH2SO4+ 05MHCOOHat a scan rate of 50mV sminus1(D) Chronoamperometric curves of these catalyst-modified electrodes in 05MH2SO4+05MHCOOHat 01 V for 2000 s The Pdmass amounts of all the catalystswere about 1 mg in each electrode

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decreases their catalytic activity To gain a better understanding of theexcellent electrocatalytic performance of DPARH the products suchas DPA and heteroatom-doped bio-Pd (DP) obtained from the processof fabricating DPARH were also investigated As illustrated by the CVin Fig 6 (A andC) the performances of the DP- DPA- and DPARH-modified electrodes were gradually improved under both alkaline andacidic conditions The forward anodic peak current is considered torepresent the activity of catalyst The DP has the lowest activity inour as-prepared catalysts which is similar to that of commercial PdCWith the addition of theAu element the catalytic activity ofDPA showeda 41-fold increase (554 A mgminus1) over that of PdC in ethanol oxidationand a 47-fold (145 Amgminus1) increase in formic acid oxidation indicatingthat alloying with the Au element tremendously enhances the single Pdcatalysts Moreover DPARH shows the highest activity in terms of peakcurrent (830 Amgminus1 in ethanol electrooxidation and 204 in Amgminus1 for-mic aicd electrooxidation) The further enhanced catalytic activity mightbe mainly attributed to the large surface area of the 3D porous structurewith GO coating in DPARH In addition the onset potential in the for-ward scan of ethanol oxidation onDPARH exhibited significantly highernegative shift than that on commercial PdC During the period of etha-nol electrooxidation the dissociative adsorption of ethanol to adsorbedCOads and CHx species usually takes place at lower potentials leadingto the complete oxidation of ethanol to CO2 The negative shift of theonset potential indicates that ethanol is more easily oxidized by DPARHwhich is concordant with a previous study reporting that the functional-ization of N-doping in carbon support could markedly enhance the ki-netics of the ethanol electrooxidation (53 54)

To further test the stability of theDPARHcatalyst long-term chron-oamperometric measurement technique was carried out From the re-sponse curves shown in Fig 6 (B and D) it can be learned that the Auelement can efficiently avoid the current decay and maintain the highelectrocatalytic activities of DPARH and DPA for a much longer timethan that of DP and commercial PdC This is likely to be attributed tothe fact that the gold component can effectively prevent the poisoning ofthe intermediate species (40)Moreover the PdAunanoparticles are fixedon the 3D porous skeleton built by GO which also benefits the durabilityof DPARH Both activity and durability experiment results prove that theDPARH is a promising electrocatalyst for the electrooxidation of ethanoland formic acid

18

CONCLUSION

Here an excellent electrocatalyst was fabricated for small organic mo-lecular electrooxidation under both acid and alkaline conditions usingthe biologically mediated method The electrochemically active bacte-rium replaced the toxic surfactant stabilizer and chemical reducingagent and in situndashsynthesized bio-PdAu nanoparticles on its surfaceextending the function of bacteria in three ways (i) reducing metalions (ii) supportingmaterial and (iii) doping heteroatom The hydro-thermal reaction serves a quadruple purpose in the synthesis processcarbonizing bacterial cells alloying Pd and Au nanoparticles functio-nalizing carbon supports and reducing GO Because of its 3D porousstructures alloy nature carbon support and heteroatom doping theDPARH showed an excellent electrocatalytic activity and durabilitytoward ethanol and formic acid oxidation compared with the com-mercial PdC and many other chemically synthesized catalysts Thisstudy offers a new perspective about the utilization of bionanoparticles

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

to markedly accelerate the energy conversion on the anode of fuelcells

MATERIALS AND METHODS

ChemicalsTetrachloroauric(III) acid hydrate [HAuCl44H2O analytical grade(AR)] palladium(II) chloride (PdCl2 AR) potassium dihydrogenphosphate (KH2PO4 AR) disodium hydrogen phosphate dodeca-hydrate (Na2HPO4 AR) ammonium chloride (NH4Cl AR) magne-sium sulfate (MgSO4 AR) sodium chloride (NaCl AR) ammoniumsulfate [(NH4)2SO4 AR] nitrilotriacetic acid [N(CH2CO2H)3 AR]iron(II) sulfate heptahydrate (FeSO4middot7H2O AR) manganese(II) chlo-ride tetrahydrate (MnCl2middot4H2O AR) cobalt(II) chloride hexahydrate(CoCl2middot6H2O AR) copper(II) sulfate pentahydrate (CuSO4middot5H2OAR) zinc chloride (ZnCl2 AR) nickel(II) chloride hexahydrate(NiCl2middot6H2O AR) boric acid (H3BO3 AR) aluminum potassium sul-fate dodecahydrate [AlK(SO4)2middot12H2OAR] and sodiummolybdate di-hydrate (Na2MoO4middot2H2O AR) were purchased from SinopharmChemical Reagent Co Ltd Sodium selenite (Na2SeO3 98) and sodiumtungstate dihydrate purum (NaWO4middot2H2O 99)were supplied by Sigma-Aldrich In all of the experiments ultrapure water with a conductivity of182 megohmmiddotcm was used to dissolve and dilute chemical reagents

Bacterial cultureS oneidensisMR-1was obtained from the American Type Culture Col-lection and was incubated in LBmedium (pH 70) at 30degCwith shakingat 120 rpm for 24 hours Then S oneidensis MR-1 cells were concen-trated by centrifugation at 6000 rpm for 5 min and washed twice withsterilized buffer [pH 70 KH2PO4 (3 gliter) Na2HPO4 (6 gliter)NH4Cl (1 gliter) NaCl (05 gliter) CaCl2 (0011 gliter) MgSO4

(0012 gliter)] Finally the washed S oneidensisMR-1 cells were resus-pended into 20ml of buffer and purgedwith nitrogen gas to remove thedissolved oxygen

Synthesis of BP and DPSodium lactate [2 ml 60 (ww)] was added into 1 liter of M9 bufferand purged with nitrogen gas to form the reaction solution Cell resus-pension solution (1 ml) was injected into 24 ml of as-prepared reac-tion solution followed by injecting 100 ml of 40 mM H2PdCl4 Aftershaking it for 15 hours the BP was synthesized and washed in 05NaCl solution

The as-prepared BP was resuspended into a Teflon autoclave with20 ml of water and treated at 200degC for 6 hours After cooling down toroom temperature the DP was fabricated and washed in 50 alcoholsolution followed by dispersion into 1 ml of 50 alcohol solution forstorage

Synthesis of BPA and DPAThese BPA and DPA were synthesized as described above for the fab-rication of BP and DP The only difference was that at t = 05 hours inthe synthesis of BP 50 ml of 20 mMHAuCl4 was injected into the reac-tion solution

Synthesis of BPABG and DPARHThese BPABGandDPARHwere synthesized as described above for thepreparation of BPA and DPA except that at t = 1 hour in the synthesis

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of BPA 250 ml of GO (1 mgml) was also injected into the reactionsolution

Morphological characterizationMaterials for SEM analysis were resuspended in 25 (wv) glutar-aldehyde phosphate-buffered solution (100 mM pH 70) for 24 hoursThen the silicon slice coated with Formvar film was added into dis-persed solution [phosphate-buffered solution (100 mM pH 70)] ofmaterials for 2 hours After washing three times with phosphate-bufferedsolution (100mM pH 70) the cells were fixed again in 25 (wv) glu-taraldehyde for 1 hour Next the cells were dehydrated in a gradientethanol series (30 twice and 50 70 90 95 and 100 twice) for 15 mineach After drying at 60degC for 12 hours the cells were placed onto a carbonsubstrate for SEM observation (S-4800 FE-SEM Hitachi)

Samples for TEM analysis were prepared by dripping a single dropof diluted sample dispersion on copper grids A JEM-2100F HRTEMequippedwith an accelerating voltage of 200 kVwas used to record theHRTEM images STEM elemental maps weremade on a FEI TECNAIF30 microscope operated at 300 kV under the HAADF mode

Structure characterizationTheXRDanalysis of all the sampleswas accomplishedwith a BrukerD8Advance x-ray diffractometer equipped with a Cu Ka radiation sourceXPS experiments were measured by a Thermo VGMultilab 2000 spec-trometer equipped with a monochromatic Al Ka radiation source atroom temperature Inductively coupled plasma atomic emission spec-troscopy (ICP-AES) was tested by Optima-7000DV (PerkinElmer)

Electrochemical measurementThe electrochemical measurements of ethanol and formic acidelectrooxidation reaction were performed in a three-electrode cell con-sisting of a glassy carbon (GC 5 mm in diameter) an AgAgCl(saturated KCl) reference electrode and a platinum wire counterelectrode using CHI 660E electrochemical workstation (CHInstruments Inc) The GC electrode was successively polished with10- 03- and 005-mmalumina powder andwashedwith deionizedwa-ter followed by sonication in 8MHNO3 ethanol and deionized waterrespectively before the electrochemical experiments Then theelectrode was dried under nitrogen atmosphere For all the electrocata-lytic tests 4 ml of as-prepared catalysts and commercial PdC catalystethanal aqueous solution [11 (vethanalvwater)] were dropped on the sur-face of each electrode and dried at room temperature Then 5 ml of0028 Nafion (diluted from 5 Nafion by ethanol Sigma-Aldrich)was added and dried before the experiment The Pd mass amounts ofall the tested catalysts on each GC electrode were almost 1 mg whichwere measured by ICP-AES

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at httpadvancessciencemagorgcgicontentfull29e1600858DC1fig S1 The mechanism of biosynthesisfig S2 Morphological feature of different biocatalystsfig S3 Atomic ratio of as-prepared catalystfig S4 Feature of graphenefig S5 CO stripping for the DPARH and commercial PdCfig S6 Ethanol electrooxidation activities of the DPARH synthesized under differentconditionsfig S7 Formic acid electrooxidation activities of the DPARH synthesized under differentconditions

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

table S1 Electrocatalytic mass activities of different catalysts in previous studiestable S2 Atomic ratio of the DPARH from the XPS datatable S3 Electrocatalytic mass activities of DPARH synthesized under different conditionsReferences (55ndash64)

REFERENCES AND NOTES1 M Winter R J Brodd What are batteries fuel cells and supercapacitors Chem Rev 104

4245ndash4269 (2004)2 B C H Steele A Heinzel Materials for fuel-cell technologies Nature 414 345ndash352 (2001)3 A S Aricograve P Bruce B Scrosati J-M Tarascon W van Schalkwijk Nanostructured materials

for advanced energy conversion and storage devices Nat Mater 4 366ndash377 (2005)4 N Tian Z-Y Zhou S-G Sun Y Ding Z L Wang Synthesis of tetrahexahedral platinum

nanocrystals with high-index facets and high electro-oxidation activity Science 316732ndash735 (2007)

5 X Huang S Tang X Mu Y Dai G Chen Z Zhou F Ruan Z Yang N Zheng Freestandingpalladium nanosheets with plasmonic and catalytic properties Nat Nanotechnol 6 28ndash32(2011)

6 C Bianchini P K Shen Palladium-based electrocatalysts for alcohol oxidation in half cellsand in direct alcohol fuel cells Chem Rev 109 4183ndash4206 (2009)

7 H Lee S E Habas G A Somorjai P Yang Localized Pd overgrowth on cubic Pt nanocrys-tals for enhanced electrocatalytic oxidation of formic acid J Am Chem Soc 1305406ndash5407 (2008)

8 X Yu P Pickup Recent advances in direct formic acid fuel cells (DFAFC) J Power Sources182 124ndash132 (2008)

9 Y Tang W Cheng Nanoparticle-modified electrode with size- and shape-dependent elec-trocatalytic activities Langmuir 29 3125ndash3132 (2013)

10 Y Tang W Cheng Key parameters governing metallic nanoparticle electrocatalysis Nanoscale7 16151ndash16164 (2015)

11 C Zhu S Guo S Dong PdM (M = Pt Au) bimetallic alloy nanowires with enhanced elec-trocatalytic activity for electro-oxidation of small molecules Adv Mater 24 2326ndash2331(2012)

12 J Liu Z Huang K Cai H Zhang Z Lu T Li Y Zuo H Han Clean synthesis of an eco-nomical 3d nanochain network of pdcu alloy with enhanced electrocatalytic performancetowards ethanol oxidation Chemistry 21 17779ndash17785 (2015)

13 J Chang L Feng C Liu W Xing X Hu An Effective PdndashNi2PC anode catalyst for directformic acid fuel cells Angew Chem Int Ed Engl 53 122ndash126 (2014)

14 J W Hong Y Kim D H Wi S Lee S-U Lee Y W Lee S-I Choi S W Han Ultrathin free-standing ternary-alloy nanosheets Angew Chem Int Ed Engl 55 2753ndash2758 (2016)

15 Y Yamauchi A Tonegawa M Komatsu H Wang L Wang Y Nemoto N Suzuki K KurodaElectrochemical synthesis of Mesoporous PtndashAu binary alloys with tunable compositionsfor enhancement of electrochemical performance J Am Chem Soc 134 5100ndash5109(2012)

16 L Zhang Y Tang J Bao T Lu C Li A carbon-supported Pd-P catalyst as the anodic cat-alyst in a direct formic acid fuel cell J Power Sources 162 177ndash179 (2006)

17 R Jiang D T Tran J P McClure D Chu A class of (PdndashNindashP) electrocatalysts for the eth-anol oxidation reaction in alkaline media ACS Catal 4 2577ndash2586 (2014)

18 L-X Ding A-L Wang G-R Li Z-Q Liu W-X Zhao C-Y Su Y-X Tong Porous Pt-Ni-Pcomposite nanotube arrays Highly electroactive and durable catalysts for methanolelectrooxidation J Am Chem Soc 134 5730ndash5733 (2012)

19 S Yang L Zhi K Tang X Feng J Maier K Muumlllen Efficient synthesis of heteroatom (N orS)-doped graphene based on ultrathin graphene oxide-porous silica sheets for oxygenreduction reactions Adv Funct Mater 22 3634ndash3640 (2012)

20 D Wei Y Liu Y Wang H Zhang L Huang G Yu Synthesis of N-doped graphene bychemical vapor deposition and its electrical properties Nano Lett 9 1752ndash1758 (2009)

21 C Huang C Li G Shi Graphene based catalysts Energy Environ Sci 5 8848ndash8868 (2012)

22 J Tang J Liu C Li Y Li M O Tade S Dai Y Yamauchi Synthesis of nitrogen-dopedmesoporous carbon spheres with extra-large pores through assembly of diblock co-polymer micelles Angew Chem Int Ed Engl 54 588ndash593 (2015)

23 B Wu D Hu Y Kuang B Liu X Zhang J Chen Functionalization of carbon nanotubes byan ionic-liquid polymer Dispersion of Pt and PtRu nanoparticles on carbon nanotubes andtheir electrocatalytic oxidation of methanol Angew Chem Int Ed Engl 48 4751ndash4754(2009)

24 Y Shao J Liu Y Wang Y Lin Novel catalyst support materials for PEM fuel cells Currentstatus and future prospects J Mater Chem 19 46ndash59 (2009)

25 J Shui M Wang F Du L Dai N-doped carbon nanomaterials are durable catalysts foroxygen reduction reaction in acidic fuel cells Sci Adv 1 e1400129 (2015)

26 J Zhang Z Xia L Dai Carbon-based electrocatalysts for advanced energy conversion andstorage Sci Adv 1 e1500564 (2015)

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27 H B Yang J Miao S-F Hung J Chen H B Tao X Wang L Zhang R Chen J GaoH M Chen L Dai B Liu Identification of catalytic sites for oxygen reduction and oxygenevolution in N-doped graphene materials Development of highly efficient metal-free bi-functional electrocatalyst Sci Adv 2 e1501122 (2016)

28 X Chen G Wu J Chen X Chen Z Xie X Wang Synthesis of ldquocleanrdquo and well-dispersivePd nanoparticles with excellent electrocatalytic property on graphene oxide J Am ChemSoc 133 3693ndash3695 (2011)

29 K B Narayanan N Sakthivel Biological synthesis of metal nanoparticles by microbes AdvColloid Interface Sci 156 1ndash13 (2010)

30 S De Corte T Hennebel J P Fitts T Sabbe V Bliznuk S Verschuere D van der LelieW Verstraete N Boon Biosupported bimetallic PdndashAu nanocatalysts for dechlorination ofenvironmental contaminants Environ Sci Technol 45 8506ndash8513 (2011)

31 X Wu F Zhao N Rahunen J R Varcoe C Avignone-Rossa A E Thumser R C T Slade Arole for microbial palladium nanoparticles in extracellular electron transfer Angew ChemInt Ed Engl 50 427ndash430 (2011)

32 V S Coker J A Bennett N D Telling T Henkel J M Charnock G van der LaanR A D Pattrick C I Pearce R S Cutting I J Shannon J Wood E Arenholz I C LyonJ R Lloyd Microbial engineering of nanoheterostructures Biological synthesis of a mag-netically recoverable palladium nanocatalyst ACS Nano 4 2577ndash2584 (2010)

33 L Xiong J-J Chen Y-X Huang W-W Li J-F Xie H-Q Yu An oxygen reduction catalystderived from a robust Pd-reducing bacterium Nano Energy 12 33ndash42 (2015)

34 H Sun L Cao L Lu Bacteria promoted hierarchical carbon materials for high-performancesupercapacitor Energy Environ Sci 5 6206ndash6213 (2012)

35 Z Guo G Ren C Jiang X Lu Y Zhu L Jiang L Dai High performance heteroatomsquaternary-doped carbon catalysts derived from Shewanella bacteria for oxygen reduc-tion Sci Rep 5 17064 (2015)

36 T J Park K G Lee S Y Lee Advances in microbial biosynthesis of metal nanoparticlesAppl Microbiol Biotechnol 100 521ndash534 (2016)

37 Y A Gorby S Yanina J S McLean K M Rosso D Moyles A Dohnalkova T J BeveridgeI S Chang B H Kim K S Kim D E Culley S B Reed M F Romine D A Saffarini E A HillL Shi D A Elias D W Kennedy G Pinchuk K Watanabe S Ishii B Logan K H NealsonJ K Fredrickson Electrically conductive bacterial nanowires produced by Shewanella onei-densis strain MR-1 and other microorganisms Proc Natl Acad Sci USA 103 11358ndash11363(2009)

38 J M Myers C R Myers Overlapping role of the outer membrane cytochromes of Shewa-nella oneidensis MR-1 in the reduction of manganese(IV) oxide Lett Appl Microbiol 3721ndash25 (2003)

39 A-K Herrmann P Formanek L Borchardt M Klose L Giebeler J Eckert S KaskelN Gaponik A Eychmuumlller Multimetallic aerogels by template-free self-assembly of AuAg Pt and Pd nanoparticles Chem Mater 26 1074ndash1083 (2014)

40 L Y Chen N Chen Y Hou Z C Wang S H Lv T Fujita J H Jiang A Hirata M W ChenGeometrically controlled nanoporous PdAu bimetallic catalysts with tunable PdAu ratiofor direct ethanol fuel cells ACS Catal 3 1220ndash1230 (2013)

41 K Zhang P Han L Gu L Zhang Z Liu Q Kong C Zhang S Dong Z Zhang J Yao H XuG Cui L Chen Synthesis of nitrogen-doped MnOgraphene nanosheets hybrid materialfor lithium ion batteries ACS Appl Mater Interfaces 4 658ndash664 (2012)

42 S-K Park A Jin S-H Yu J Ha B Jang S Bong S Woo Y-E Sung Y Piao In situ hydro-thermal synthesis of Mn3O4 nanoparticles on nitrogen-doped graphene as high-performance anode materials for lithium ion batteries Electrochim Acta 120 452ndash459(2014)

43 R Nie J Shi W Du W Ning Z Hou F-S Xiao A sandwich N-doped grapheneCo3O4

hybrid An efficient catalyst for selective oxidation of olefins and alcohols J Mater ChemA 1 9037ndash9045 (2013)

44 C Nethravathi C R Rajamathi M Rajamathi U K Gautam X Wang D Golberg Y BandoN-doped graphenendashVO2(B) nanosheet-built 3D flower hybrid for lithium ion battery ACSAppl Mater Interfaces 5 2708ndash2714 (2013)

45 L Sun L Wang C Tian T Tan Y Xie K Shi M Li H Fu Nitrogen-doped graphene withhigh nitrogen level via a one-step hydrothermal reaction of graphene oxide with urea forsuperior capacitive energy storage RSC Adv 2 4498ndash4506 (2012)

46 Y-H Lee K-H Chang C-C Hu Differentiate the pseudocapacitance and double-layer ca-pacitance contributions for nitrogen-doped reduced graphene oxide in acidic and alkalineelectrolytes J Power Sources 227 300ndash308 (2013)

47 J R Pels F Kapteijn J A Moulijn Q Zhu K M Thomas Evolution of nitrogen function-alities in carbonaceous materials during pyrolysis Carbon 33 1641ndash1653 (1995)

48 D Guo R Shibuya C Akiba S Saji T Kondo J Nakamura Active sites of nitrogen-dopedcarbon materials for oxygen reduction reaction clarified using model catalysts Science351 361ndash365 (2016)

Liu et al Sci Adv 2016 2 e1600858 30 September 2016

49 C Goacutemez-Navarro R T Weitz A M Bittner M Scolari A Mews M Burghard K KernElectronic transport properties of individual chemically reduced graphene oxide sheetsNano Lett 7 3499ndash3503 (2007)

50 L Li M Chen G Huang N Yang L Zhang H Wang Y Liu W Wang J Gao A greenmethod to prepare PdndashAg nanoparticles supported on reduced graphene oxide and theirelectrochemical catalysis of methanol and ethanol oxidation J Power Sources 263 13ndash21(2014)

51 T Li N Li J Liu K Cai M F Foda X Lei H Han Synthesis of functionalized 3D porousgraphene using both ionic liquid and SiO2 spheres as ldquospacersrdquo for high-performanceapplication in supercapacitors Nanoscale 7 659ndash669 (2015)

52 Y-C Yong Y-Y Yu X Zhang H Song Highly active bidirectional electron transfer by aself-assembled electroactive reduced-graphene-oxide-hybridized biofilm Angew ChemInt Ed Engl 53 4480ndash4483 (2014)

53 P Wu Y Huang L Kang M Wu Y Wang Multisource synergistic electrocatalytic oxidationeffect of strongly coupled PdM (M = Sn Pb)N-doped graphene nanocomposite on smallorganic molecules Sci Rep 5 14173 (2015)

54 H Jin T Xiong Y Li X Xu M Li Y Wang Improved electrocatalytic activity for ethanoloxidation by PdN-doped carbon from biomass Chem Commun 50 12637ndash12640 (2014)

55 S Hu F Munoz J Noborikawa J Haan L Scudiero S Ha Carbon supported Pd-basedbimetallic and trimetallic catalyst for formic acid electrochemical oxidation Appl Catal BEnviron 180 758ndash765 (2016)

56 S Fu C Zhu D Du Y Lin Facile one-step synthesis of three-dimensional PdndashAg bimetallicalloy networks and their electrocatalytic activity toward ethanol oxidation ACS Appl MaterInterfaces 7 13842ndash13848 (2015)

57 S Yao G Li C Liu W Xing Enhanced catalytic performance of carbon supported palla-dium nanoparticles by in-situ synthesis for formic acid electrooxidation J Power Sources284 355ndash360 (2015)

58 Q Wang Y Liao H Zhang J Li W Zhao S Chen One-pot synthesis of carbon-supportedmonodisperse palladium nanoparticles as excellent electrocatalyst for ethanol and formicacid oxidation J Power Sources 292 72ndash77 (2015)

59 N Su X Chen Y Ren B Yue H Wang W Cai H He The facile synthesis of single crys-talline palladium arrow-headed tripods and their application in formic acid electro-oxidationChem Commun 51 7195ndash7198 (2015)

60 B Cai D Wen W Liu A-K Herrmann A Benad A Eychmuumlller Function-led design ofaerogels Self-assembly of alloyed PdNi hollow nanospheres for efficient electrocatalysisAngew Chem Int Ed Engl 54 13101ndash13105 (2015)

61 G-T Fu B-Y Xia R-G Ma Y Chen Y-W Tang J-M Lee Trimetallic PtAgCuPtCu coreshellconcave nanooctahedrons with enhanced activity for formic acid oxidation reaction NanoEnergy 12 824ndash832 (2015)

62 C Bi C Feng T Miao Y Song D Wang H Xia Understanding the effect of ultrathin AuPdalloy shells of irregularly shaped AuAuPd nanoparticles with high-index facets onenhanced performance of ethanol oxidation Nanoscale 7 20105ndash20116 (2015)

63 D Bin B Yang F Ren K Zhang P Yang Y Du Facile synthesis of PdNi nanowire networkssupported on reduced graphene oxide with enhanced catalytic performance for formicacid oxidation J Mater Chem A 3 14001ndash14006 (2015)

64 Y Wang Q He J Guo J Wang Z Luo T D Shen K Ding A Khasanov S Wei Z GuoUltrafine FePd nanoalloys decorated multiwalled carbon nanotubes toward enhanced eth-anol oxidation reaction ACS Appl Mater Interfaces 7 23920ndash23931 (2015)

Acknowledgments We thank Y Jiang (State Key Laboratory of Physical Chemistry of SolidSurfaces Department of Chemistry College of Chemistry and Chemical Engineering XiamenUniversity Xiamen 361005 China) for the help in electrochemical measurement Funding Weacknowledge the financial support from the National Natural Science Foundation of China(21375043 21175051 and 21322703) Author contributions HH and FZ proposed the con-cept designed the experiments and supervised the project JL YZ ZH and KC carried outthe experiments Competing interests The authors declare that they have no competinginterests Data and materials availability All data needed to evaluate the conclusions inthe paper are present in the paper andor the Supplementary Materials Additional data re-lated to this paper may be requested from the authors

Submitted 22 April 2016Accepted 18 August 2016Published 30 September 2016101126sciadv1600858

Citation J Liu Y Zheng Z Hong K Cai F Zhao H Han Microbial synthesis of highly dispersedPdAu alloy for enhanced electrocatalysis Sci Adv 2 e1600858 (2016)

8 of 8

Microbial synthesis of highly dispersed PdAu alloy for enhanced electrocatalysisJiawei Liu Yue Zheng Zilan Hong Kai Cai Feng Zhao and Heyou Han

DOI 101126sciadv1600858 (9) e16008582Sci Adv

ARTICLE TOOLS httpadvancessciencemagorgcontent29e1600858

MATERIALSSUPPLEMENTARY httpadvancessciencemagorgcontentsuppl2016092629e1600858DC1

REFERENCES

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York Avenue NW Washington DC 20005 2017 copy The Authors some rights reserved exclusive licensee American (ISSN 2375-2548) is published by the American Association for the Advancement of Science 1200 NewScience Advances

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