Bacterial Communities onElectron-Beam Pt-DepositedElectrodes in a Mediator-LessMicrobial Fuel CellH O I L P A R K , † D A V I D S A N C H E Z , ‡

S U N G K W O N C H O , § A N D M I N H E E Y U N † , *

Department of Electrical and Computer Engineering,Department of Civil and Environmental Engineering,Department of Mechanical Engineering and Materials Science,University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received March 4, 2008. Revised manuscript received June2, 2008. Accepted June 4, 2008.

The content of a bacterial consortium found on an electronbeam(e-beam)Pt-depositedelectrodeinamediator-lessmicrobialfuel cell (MFC) using glucose and glutamate as fuel isreported in this paper. The e-beam Pt-deposited electrodeand electrochemically active bacteria (EAB) consortium weredeveloped to improve the mediator-less MFC performance.Denaturing gradient gel electrophoresis (DGGE), restrictionfragment length polymorphism (RFLP), and 16S rRNA sequencingwere used to identify the EAB consortia. Sequencing resultsshowed that clone ASP-31 was predominant and was similar toAeromonas hydrophila, an Fe(III)-reducing and EAB. Thephylogenetic tree analysis disclosed the presence ofγ-proteobacteria groups such as Aeromonas genus, Entero-bacter asburiae, and Klebsiella oxytoca. These resultssuggest that MFC performance of the e-beam Pt-depositedelectrode with Aeromonas genus consortia dominated by A.hydrophila was higher than other MFCs within a short period.With the e-beam Pt-deposited electrode and Aeromonasgenus consortia in the mediator-less MFC, it is possible toincrease the efficiency of electron transfer between the bacteriaand the electrode.

IntroductionMicrobial fuel cells (MFCs) are a promising technology foran alternative energy source. Fossil fuels such as coal andpetroleum produce greenhouse gases that lead to environ-mental pollution and they are limited. Consequently, a newtype of energy source must be developed. Renewable energysources such as hydroelectric, biomass, geothermal, wind,and solar power have been investigated (1). The use ofbiomass as a renewable energy source is environmentallyfriendly and highly valuable. In the past, biomass has beenconverted into bioenergy using processes such as metano-genic anaerobic digestion, ethanol fermentation, and hy-drogen fermentation (2–4). Recently, a novel process usingMFCs to create bioenergy have been proposed (5).

MFCs have typically shown lower levels of efficiency inpower generation when compared to other types of fuel cells

(6). To improve efficiency of the MFCs, many researchershave studied limiting factors such as bacterial metabolism,bacterial electron transfer, performance of the protonexchange membrane, internal and external resistance of theelectrolytes, efficiency of the cathode oxygen diffusion andsupply (7–9), and Pt-deposited electrodes such as Pt nano-particles on carbon nanotube (10). In particular, the electrontransfer from the bacteria to the electrode is very importantin the MFC process (11). To overcome this critical limitingfactor, it is imperative to study a catalyst that effectivelytransfers the electrons from the bacteria to the electrode(12–14) and the corresponding bacterial community in themediator-less MFCs (7, 9). Earlier, we reported the use ofe-beam Pt as a catalyst for the development of high-efficiencyMFCs (15).

Many bacteria cannot produce electricity without amediator present in the MFC. Typically a mediator facilitatesthe electron transfer between the bacteria and the electrodein the system (“mediated MFC”) (16, 17). However, mediatorsare inefficient, expensive, and limited in long-term MFCoperation (7). Other bacteria can transfer electrons withoutthe use of mediators in the MFCs (“mediator-less MFC”).These bacteria, also known as electrochemically activebacteria (EAB), can reduce metals including iron (Fe) andsulfur. Mediator-less MFCs can be operated using EAB suchas Aeromonas hydrophila (18), Clostridium butyricum (19),Desulfobulbus propionicus (20), Enterococcus gallinarum (21),Geobacter sulfurreducens (22), Rhodoferax ferrireducens (23),and Shewanella putrefaciens (24, 25) or EAB-containingconsortia. When EAB were enriched on an anode electrodein mediator-less MFC using anaerobic sludge, non-EAB werealso present in the anode electrode (7). The EAB and non-EAB consortia generated a current that was 6 times higherthan the current generated by the EAB pure culture (11).This suggests that non-EAB play a critical role in generatingelectron donors for the EAB as a result of their metabolism(7). The EAB such as S. putrefaciens and G. sulfurreducensand the electricity generated by these pure enrichments orthese relative consortia are the focus of many studies(21, 23, 25). However, there are not enough bacterialcommunity studies on the Aeromonas genus consortiadominated by A. hydrophila. A few other studies havereported on A. hydrophila strains or consortia. Pham et al.(18) reported that A. hydrophila was an Fe(III)-reducingbacterium and electrochemically active bacterium. Lee et al.(25) showed that the AC-155 clone was a related to theAeromonas genus in the phylogenetic tree.

In this paper, we fabricated e-beam Pt-deposited elec-trodes to increase the efficiency of electron transfer betweenthe bacteria and the electrode in a mediator-less MFC. Wethen monitored the MFC’s performance with enrichedAeromonas genus consortia dominated by A. hydrophila. Thebacterial communities were analyzed using molecular bio-logical techniques including denaturing gradient gel elec-trophoresis (DGGE), restriction fragment length polymor-phism (RFLP), and 16S rRNA sequencing. Finally, wecompared bacterial communities of Aeromonas genus con-sortia dominated by A. hydrophila to other EAB consortia inthe mediator-less MFC.

Experimental SectionThe Mediator-Less Microbial Fuel Cell (MFC) System. Theprinciple of the mediator-less MFC system is shown in Figure1. The anode and cathode compartments are separated bya cation exchange membrane (Nafion-112; Dupont, Wilm-ington, DE) (26). The anode compartment was continuously

* Corresponding author phone: 412-648-8989; fax: 412-648-8003;e-mail: yunmh@engr.pitt.edu.

† Department of Electrical and Computer Engineering.‡ Department of Civil and Environmental Engineering.§ Department of Mechanical Engineering and Material Science.

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Published on Web 07/03/2008

provided with nitrogen gas to maintain anaerobic conditionsand the cathode compartment with air-saturated water. Bothcompartments contained a sheet of electrode paper (1.0 ×4.0 cm). The MFC was electrically loaded with a fixed externalresistor (10 Ω). The potential across the resistor was measuredby a multimeter (model 2701 DMM; Keithley Instruments,Inc., Cleveland, OH) that was connected in parallel, andrecorded using a personal computer through a data acquisi-tion system controlled by the LabVIEW program (NationalInstruments Corporation, Austin, TX) (15).

Plain Toray carbon paper (TGPH-120, E-TEK, Somerset,NJ) was used as the electrode substrate in this study. Wedeposited Pt (1000 Å thickness) on the carbon paper usingan e-beam evaporator (VE-180, Thermoionics Laboratory,Inc., Port Townsend, WA) following a process provided bythe manufacturer (Note: e-beam Pt-deposited electrode), anddeposited Pt (1500 Å thickness) on the carbon paper usingelectrochemical deposition (Note: Pt-Blk electrode) (15).

Scanning Electron Microscopy (SEM). An SEM was usedto capture images of the e-beam Pt-deposited electrodesurface before and after installation in the MFC. Electrodeswere imaged prior to installation in the MFC. EAB were thenenriched on the anode electrode. The e-beam Pt-depositedelectrodes were removed from the MFC reactors and rinsedwith a sterile medium. The electrodes were imaged again bySEM (e-LiNE, Raith GmbH, Dortmund, Germany) set at 10.0kV. To compare adhesion strength of Pt to carbon paperbetween the two deposition methods both Pt-Blk and e-beamPt-deposited electrodes were subjected to 1 h sonification.The electrodes were also imaged before and after by a SEMset at 10.0 kV.

Enrichment of Electrochemically Active Bacteria (EAB).EAB were enriched from anaerobic sludge taken from theFranklin Township Municipal Sanitary Authority (FTMSA)in Pittsburgh, PA. The EAB were inoculated using an artificialwastewater containing 50 mM phosphate buffer (pH 7.0),glucose and glutamate fuel (27), trace mineral solution (28),and salt solution (29).

DNA Extraction and Polymerase Chain Reaction (PCR)Amplification of 16S rRNA Sequencing. A detailed descrip-tion of the DNA extraction and PCR amplification of 16SrRNA sequencing were given in the literature (30). Briefly,total chromosomal DNA was extracted from the anaerobic

sludge and the bacteria on the e-beam Pt-deposited anodeelectrode in the mediator-less MFC using Super Soil Kit (MOBIO Laboratories, Carlsbad, CA). The DNA was purified usingGENECLEAN Turbo (Qbiogene, Irvine, CA) before its use asa template for PCR amplification. For the 16S rRNA genesequence, the purified DNA was amplified using the forwardprimer (27f: 5′-AGA GTT TGA TCM TGG CTC AG-3′) and thereverse primer (1492r: 5′-GGT TAC CTT TGT TAC GAC TT-3′). PCR amplification was performed using the GeneAmpPCR System 9700 (Applied Biosystems, Foster City, CA) withan initial denaturation at 94 °C for 5 min followed by 30cycles of denaturation at 92 °C, annealing at 55 °C for 30 s,and extension at 72 °C for 30 s, before the final extension at72 °C for 7 min. For the bacterial community analysis usingDGGE, the extract was amplified by a nested PCR using theforward primer (GC-341f: 5′-CGC CCG CCG CGC CCC GCGCCC GGC CCG CCG CCC CCG CCC CCC TAC GGG AGG CGACAG-3′) and a reverse primer (534r: 5′-ATT ACC GCG GCTGCT G-3′). Nested PCR amplification was performed twiceaccording to the program. The first PCR amplification wasperformed as follows: Initial denaturation at 94 °C for 9 minfollowed by 30 cycles of denaturation at 94 °C for 30 s,annealing at 55 °C for 30 s, and extension at 72 °C for 30 s,before the final extension at 72 °C for 7 min. The second PCRamplification was performed using the amplicons of the firstPCR as a DNA template according to initial denaturation at94 °C for 5 min followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 55 °C for 45 s, and extension at 72°C for 1 min, before the final extension at 72 °C for 7 min.

Denaturing Gradient Gel Electrophoresis (DGGE) Analy-sis. DGGE is a molecular fingerprinting method that separatesPCR-generated DNA products. The PCR of environmentalDNA can generate templates of differing DNA sequencesthat represent many of the dominant bacteria (31). DGGEwas performed by using a DCode system (Bio-Rad Labora-tories, Hercules, CA) according to the manufacturer’s in-structions. The PCR amplification was loaded onto 10%polyacrylamide gel with a denaturing gradient ranging from30 to 60% consisting of urea and formamide. The gel wasstained using SYBR Gold (Molecular Probes, Carlsbad, CA)for 30 min before DNA bands were observed by a NucleoTechsystem (Bio-Rad Laboratories) under ultraviolet illumination(32, 33).

FIGURE 1. Schematic diagram of a mediator-less MFC. The anode and cathode compartments are separated by a cation exchangemembrane. The fuel is oxidized by microorganisms that produce electrons and protons in the anode compartment. The electrons flowvia wire to the cathode compartment while the protons pass through the membrane.

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16S rRNA Sequencing and Phylogenetic Analysis. PCRproducts amplified using a 27f/1492r primer pair were clonedinto the pGEM-T vector system (Promega, Madison, WI) andtransformed into competent Escherichia coli GC5 competentcells (NextGen Sciences, Ann Arbor, MI). The transformantswere plated on Luria-Bertani (LB) agar medium containingampicillin, 5-bromo-4-chloro-3-indolyl--D-galactopyrano-side (X-gal), and isoprophyl--D-thiogalactopyranoside (IPTG).Ampicillin-resistant and -galactosidase-negative clones weretransferred to a liquid medium of the same composition (34).

Plasmids were then extracted from 80 colonies using theFast Minipreps Kit (Eppendorf North America, Westbury,NY). Replicate representative colonies were sequenced withT7 forward/SP6 reverse primers using an automatic se-quencer system (ABI 3730; Davis Sequencing, Davis, CA).The sequences were compared to those of the National Centerfor Biotechnology Information (NCBI) BLAST GenBanknucleotide sequence algorithm. Parsimony phylogenetic treeswere constructed by the neighbor-joining method using thePHYDIT 3.1 program (Seoul National University, Korea). The16S rRNA gene sequences have been submitted to the NCBIGenBank under accession numbers EF679178 throughEF679197.

Result and DiscussionElectrode Characterization. We fabricated an e-beam Pt-deposited electrode to increase the efficiency of electrontransfer in a mediator-less MFC. We compared electrodesurfaces using a SEM before installing the electrodes intothe mediator-less MFC, and after enriching the EAB on theanode electrodes, as shown in Figure 2. Figure 2a shows amicroscopic view of the carbon paper electrode without anyPt depositions. Numerous straight carbon rods cross overeach other, layer by layer, forming a mesh-like structure.Figure 2b shows the e-beam Pt-deposited electrode. Theentire area of the exposed carbon paper is uniformly coveredwith Pt. A SEM image of the e-beam Pt-deposited electrodesurface after EAB enrichment revealed that the electrodeswere covered with bacteria (Figure 2c).

Improved Efficiency of an Electron Transfer by theE-Beam Pt-Deposited Electrodes and Enrichment of EAB.Two factors, Pt as a catalyst and EAB consortia, are veryimportant to improving the efficiency of electron transfer.We investigated the use of an e-beam Pt-deposited electrodeand EAB consortia, containing EAB and non-EAB, in amediator-less MFC. For the first few days of this study, weenriched the EAB and non-EAB on the anode electrode usingan anaerobic sludge and an artificial wastewater containinga glucose and glutamate fuel. The glucose and glutamatefuel was oxidized by the EAB producing electrons and protonsin the anode compartment. The electrons were transferredto the electrode by EAB and then via an electric wire to thecathode compartment (34). Next, we investigated the MFCperformance using carbon paper, Pt-Blk, and e-beam Pt-deposited electrodes in the anode compartments. As shownin Figure 3, the current density of a carbon paper electrodewithout Pt deposition was around 0.05 A/m2, the currentdensity of the Pt-Blk electrode was around 0.25 A/m2, andthe current density of the e-beam Pt-deposited electrodewas around 0.9 A/m2. However, the e-beam Pt-deposited

FIGURE 2. SEM images of (a) carbon paper electrode, (b) e-beamPt-deposited electrode, and (c) EAB and non-EAB on e-beamPt-deposited electrode in the mediator-less MFC.

FIGURE 3. Current density of the carbon paper without Pt,Pt-Blk electrode and e-beam Pt-deposited electrode with andwithout bacteria on the anode compartments.

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electrode without bacteria showed about 0.0005 A/m2. Thecurrent density of the e-beam Pt-deposited electrode wasabout 18 times higher than the current density of the carbonpaper without Pt deposition. It is important to note thatalthough the e-beam Pt-deposited electrode (1000 Å thick-ness) was thinner than the Pt-Blk electrode (1500 Å thickness),the current density of the e-beam Pt-deposited electrodewas 3.6 times higher than the density of the Pt-Blk electrode.This was a remarkable improvement due mainly to theexcellent coverage and uniformity of Pt using the e-beamdeposition process as opposed to the electrochemicaldeposition process used on the Pt-Blk electrode (15). Theseresults suggest that the e-beam Pt-deposited electrode andEAB consortia in the mediator-less MFC increase theefficiency of electron transfer between the bacteria and theelectrode by the excellent coverage of the e-beam Pt-deposited electrode (15). Additionally a 1 h sonification ofboth the e-beam Pt-deposited and Pt-Blk electrodes con-firmed that e-beam deposition provides a stronger adhesionto the carbon electrode than the Pt-Blk electrode usingBransonic ultrasonic cleaner (5510R-DTH, Branson Ultra-sonic Corporation, U.S.) at 25 °C and an input sonificationpower density of 30 W/gal. Effective sonication power densitywas not measured. Results show that the e-beam electrodesincurred little damage while the Pt-Blk electrodes wereseparated from the underlying carbon paper (Figure 4).

Profiling Bacterial Communities Using DGGE. EABconsortia of the EAB and non-EAB present on the anodeelectrode play an important role in the mediator-less MFCprocess; however, selection and classification of EAB aredifficult using traditional microbiological techniques, such

FIGURE 4. SEM images of (A) Pt-Blk electrode (electrochemical deposi-ted) and (B) e-beam Pt-deposited electrode before and after sonification and(C) Pt-Blk electrode and (D) e-beam Pt-deposited electrode for 1 h.

FIGURE 5. Comparison of the bacterial community in theanaerobic sludge and on the e-beam Pt-deposited anode electrodeby using DGGE (denaturing gradient: 30-60%; S: anaerobic sludge;A: e-beam Pt-deposited anode electrode).

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as microscopy and cultivation. The molecular biologicaltechniques such as DGGE, RFLP, and 16S rRNA sequencinghave allowed the study of bacterial communities at a differentlevelsthe genetic level (36). They represent the most powerfulapproach to explore bacterial communities in natural samples(32). In particular, DGGE is effective for separating envi-ronmental DNA samples. DGGE can separate many bandsof the PCR products based on sequence differences indifferential denaturing characteristics of the DNA (37). Asshown in Figure 5, DGGE bands of the anaerobic sludge (S)based on the e-beam Pt-deposited anode electrode (A) wererevealed in between 12 and 15 detectable bands. DGGE bandsof each sample indicate that several bands were shown inthe same position in this case. Six bands of the anaerobicsludge were dominant. S1 and S2 bands were more prevalentthan other bands in the anaerobic sludge. Two bands of thee-beam Pt-deposited anode electrode (A), A1 and A2, weremost dominant. The A1 band was found in both the anaerobicsludge and the anode electrode. When comparing the results

of the 16S rRNA sequencing, the A1 band seems to play animportant role in the mediator-less MFC. The A2 bandshowed weakly in the anaerobic sludge, but showed clearlyon the anode electrode. The A2 band could potentially beresponsible in part for the generated electricity in themediator-less MFC.

Bacterial Classification by 16S rRNA Sequencing. Toidentify bacterial classification, E. coli GC5 colonies contain-ing the PCR products of 16S rRNA were sequenced with T7forward/SP6 reverse primers using an automatic sequencingsystem, and were compared to those of the NCBI BLASTGenBank nucleotide sequence algorithm. As a result, 11different groups were identified in the mediator-less MFC(Table 1). The most abundant clone was ASP-31 (25%sequence abundance) in the bacterial community of ourmediator-less MFC. A sequence type of clone ASP-31,represented in the clone library, showed 98% similarity to A.hydrophila. Clone ASP-31 seems to be the predominantbacterium in the anaerobic sludge (S1) and on the anode

TABLE 1. Bacterial Clones Retrieved from a Mediator-Less MFC Enriched with a Glucose and Glutamate Fuel

clones (access no.) similar relatives (accession no.) homology (%) class

ASP-31 (EF679181) Aeromonas hydrophila (AF468055) 98 γ-proteobacteriaASP-32 (EF679181) Aeromonas media (X60410) 98 γ-proteobacteriaASP-33 (EF679181) Enterobacter cloacae subsp. Dissolvens (DQ988523) 98 γ-proteobacteriaASP-35 (EF679181) Enterobacter asburiae (AJ506159) 98 γ-proteobacteriaASP-37 (EF679181) Enterobacter sp. Px6-4 (EF175731) 95 γ-proteobacteriaASP-38 (EF679181) Klebsiella sp. F51-1-2 (DQ277701) 99 γ-proteobacteriaASP-39 (EF679181) Klebsiella oxytoca (AF543296) 98 γ-proteobacteriaASP-40 (EF679181) Aeromonas sp. ydcc-5-1 (DQ837027) 98 γ-proteobacteriaASP-41 (EF679181) Aeromonas molluscorum (AY532691) 99 γ-proteobacteriaASP-42 (EF679181) Citrobacter freundii (AF025365) 99 γ-proteobacteriaASP-43 (EF679181) Enterobacter cloacae (DQ202394) 98 γ-proteobacteria

FIGURE 6. Phylogenetic analysis of the bacterial community of the e-beam Pt-deposited anode electrode in the mediator-less MFCwith glucose and glutamate fuel.

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electrode (A1) (Figure 5). The second-most abundant clone,ASP-32 (20% sequence abundance), showed 98% similarityto Aeromonas media. The sequence types of some cloneswere similar to the Aeromonas genus such as Aeromonasmolluscorum (98% similarity) and Aeromonas sp. ydcc-5-1(98% similarity). Some clones were similar to Klebsiellaoxytoca (98% similarity) which is also an Fe(III)-reducingbacterium. Enterobacter such as K. oxytoca ferments ferriccitrate as a carbon and energy source (38).

Sequences of 16S rRNA clones were obtained from themediator-less MFC and compared with those sequences inthe NCBI algorithm. Representatives were used to constructa phylogenetic tree (Figure 6). In our mediator-less MFC, allsequences obtained from the 16S rRNA clone library of theanode electrode sample, were γ-proteobacteria. Thesebacteria in our mediator-less MFC with a glucose andglutamate fuel belong to the γ-proteobacteria group but inparticular our consortia was dominated by A. hydrophila.This is a remarkable phenomenon because these types ofstudies have never been reported in previous literature. Loganet al. reported that over 97% of the detected sequencesbelonged to the γ-proteobacteria group, but predominantbacteria in the MFC with the mineral salts medium wereShewanella spp. (39). Choo et al. reported that 33.6% of thepredominant γ-proteobacteria such as Acinetobacter waspresent in the mediator-less MFC with a glucose andglutamate fuel (40). It is possible for the Aeromonas genusdominated by A. hydrophila to produce electricity withinthree days. The consortium was easily enriched in the MFCusing a copiotrophic glucose and glutamate fuel. Therefore,the MFC with Aeromonas genus consortia dominated by A.hydrophila can produce electricity more quickly than otherEAB (12, 41) as well as a higher power density of our MFCsthan others (15).

In summary, our results indicate that the current densityfrom MFCs using e-beam Pt-deposited electrodes were higherthan the MFCs using Pt-Blk electrodes. A. hydrophila clonesASP-31 were predominant on the e-beam Pt-depositedelectrodes. Relatively better coverage and stronger adhesionof e-beam Pt-deposited electrodes were one of the contribu-tions for producing higher current density than any otherelectrodes. However, further investigation is required in orderto fully understanding the relationship between e-beam Ptelectrode and the bacteria.

AcknowledgmentsThis work was supported by the Mascaro SustainabilityInitiative (MSI) at the University of Pittsburgh. We thank C.Brucker with the Wastewater Treatment Plant of the FranklinTownship Municipal Sanitation Authority (FTMSA) andNational Energy Technology Laboratory (NETL) in Pittsburgh,PA, for the facilities and indirect financial assistance.

Supporting Information AvailableInformation regarding the Restriction fragment length poly-morphism analysis can be found in the manuscript supple-ment. This material is available free of charge via the Internetat http://pubs.acs.org.

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