Bacterial Communities on Electron-Beam Pt-Deposited Electrodes in a

Jul 3, 2008 - Denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), and 16S rRNA sequencing were used to ...
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Environ. Sci. Technol. 2008, 42, 6243–6249

Bacterial Communities on Electron-Beam Pt-Deposited Electrodes in a Mediator-Less Microbial Fuel Cell HO IL PARK,† DAVID SANCHEZ,‡ 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 June 2, 2008. Accepted June 4, 2008.

The content of a bacterial consortium found on an electron beam (e-beam) Pt-deposited electrode in a mediator-less microbial fuel cell (MFC) using glucose and glutamate as fuel is reported in this paper. The e-beam Pt-deposited electrode and electrochemically active bacteria (EAB) consortium were developed to improve the mediator-less MFC performance. Denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), and 16S rRNA sequencing were used to identify the EAB consortia. Sequencing results showed that clone ASP-31 was predominant and was similar to Aeromonas hydrophila, an Fe(III)-reducing and EAB. The phylogenetic tree analysis disclosed the presence of γ-proteobacteria groups such as Aeromonas genus, Enterobacter asburiae, and Klebsiella oxytoca. These results suggest that MFC performance of the e-beam Pt-deposited electrode 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 Aeromonas genus consortia in the mediator-less MFC, it is possible to increase the efficiency of electron transfer between the bacteria and the electrode.

Introduction Microbial fuel cells (MFCs) are a promising technology for an alternative energy source. Fossil fuels such as coal and petroleum produce greenhouse gases that lead to environmental pollution and they are limited. Consequently, a new type of energy source must be developed. Renewable energy sources such as hydroelectric, biomass, geothermal, wind, and solar power have been investigated (1). The use of biomass as a renewable energy source is environmentally friendly and highly valuable. In the past, biomass has been converted into bioenergy using processes such as metanogenic anaerobic digestion, ethanol fermentation, and hydrogen fermentation (2–4). Recently, a novel process using MFCs to create bioenergy have been proposed (5). MFCs have typically shown lower levels of efficiency in power generation when compared to other types of fuel cells * Corresponding author phone: 412-648-8989; fax: 412-648-8003; e-mail: [email protected]. † Department of Electrical and Computer Engineering. ‡ Department of Civil and Environmental Engineering. § Department of Mechanical Engineering and Material Science. 10.1021/es8006468 CCC: $40.75

Published on Web 07/03/2008

 2008 American Chemical Society

(6). To improve efficiency of the MFCs, many researchers have studied limiting factors such as bacterial metabolism, bacterial electron transfer, performance of the proton exchange membrane, internal and external resistance of the electrolytes, efficiency of the cathode oxygen diffusion and supply (7–9), and Pt-deposited electrodes such as Pt nanoparticles on carbon nanotube (10). In particular, the electron transfer from the bacteria to the electrode is very important in the MFC process (11). To overcome this critical limiting factor, it is imperative to study a catalyst that effectively transfers the electrons from the bacteria to the electrode (12–14) and the corresponding bacterial community in the mediator-less MFCs (7, 9). Earlier, we reported the use of e-beam Pt as a catalyst for the development of high-efficiency MFCs (15). Many bacteria cannot produce electricity without a mediator present in the MFC. Typically a mediator facilitates the electron transfer between the bacteria and the electrode in the system (“mediated MFC”) (16, 17). However, mediators are inefficient, expensive, and limited in long-term MFC operation (7). Other bacteria can transfer electrons without the use of mediators in the MFCs (“mediator-less MFC”). These bacteria, also known as electrochemically active bacteria (EAB), can reduce metals including iron (Fe) and sulfur. Mediator-less MFCs can be operated using EAB such as 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-containing consortia. When EAB were enriched on an anode electrode in mediator-less MFC using anaerobic sludge, non-EAB were also present in the anode electrode (7). The EAB and nonEAB consortia generated a current that was 6 times higher than the current generated by the EAB pure culture (11). This suggests that non-EAB play a critical role in generating electron donors for the EAB as a result of their metabolism (7). The EAB such as S. putrefaciens and G. sulfurreducens and the electricity generated by these pure enrichments or these relative consortia are the focus of many studies (21, 23, 25). However, there are not enough bacterial community studies on the Aeromonas genus consortia dominated by A. hydrophila. A few other studies have reported on A. hydrophila strains or consortia. Pham et al. (18) reported that A. hydrophila was an Fe(III)-reducing bacterium and electrochemically active bacterium. Lee et al. (25) showed that the AC-155 clone was a related to the Aeromonas genus in the phylogenetic tree. In this paper, we fabricated e-beam Pt-deposited electrodes to increase the efficiency of electron transfer between the bacteria and the electrode in a mediator-less MFC. We then monitored the MFC’s performance with enriched Aeromonas genus consortia dominated by A. hydrophila. The bacterial communities were analyzed using molecular biological techniques including denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), and 16S rRNA sequencing. Finally, we compared bacterial communities of Aeromonas genus consortia dominated by A. hydrophila to other EAB consortia in the mediator-less MFC.

Experimental Section The Mediator-Less Microbial Fuel Cell (MFC) System. The principle of the mediator-less MFC system is shown in Figure 1. The anode and cathode compartments are separated by a cation exchange membrane (Nafion-112; Dupont, Wilmington, DE) (26). The anode compartment was continuously VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Schematic diagram of a mediator-less MFC. The anode and cathode compartments are separated by a cation exchange membrane. The fuel is oxidized by microorganisms that produce electrons and protons in the anode compartment. The electrons flow via wire to the cathode compartment while the protons pass through the membrane. provided with nitrogen gas to maintain anaerobic conditions and the cathode compartment with air-saturated water. Both compartments contained a sheet of electrode paper (1.0 × 4.0 cm). The MFC was electrically loaded with a fixed external resistor (10 Ω). The potential across the resistor was measured by a multimeter (model 2701 DMM; Keithley Instruments, Inc., Cleveland, OH) that was connected in parallel, and recorded using a personal computer through a data acquisition system controlled by the LabVIEW program (National Instruments Corporation, Austin, TX) (15). Plain Toray carbon paper (TGPH-120, E-TEK, Somerset, NJ) was used as the electrode substrate in this study. We deposited Pt (1000 Å thickness) on the carbon paper using an e-beam evaporator (VE-180, Thermoionics Laboratory, Inc., Port Townsend, WA) following a process provided by the manufacturer (Note: e-beam Pt-deposited electrode), and deposited Pt (1500 Å thickness) on the carbon paper using electrochemical deposition (Note: Pt-Blk electrode) (15). Scanning Electron Microscopy (SEM). An SEM was used to capture images of the e-beam Pt-deposited electrode surface before and after installation in the MFC. Electrodes were imaged prior to installation in the MFC. EAB were then enriched on the anode electrode. The e-beam Pt-deposited electrodes were removed from the MFC reactors and rinsed with a sterile medium. The electrodes were imaged again by SEM (e-LiNE, Raith GmbH, Dortmund, Germany) set at 10.0 kV. To compare adhesion strength of Pt to carbon paper between the two deposition methods both Pt-Blk and e-beam Pt-deposited electrodes were subjected to 1 h sonification. The electrodes were also imaged before and after by a SEM set at 10.0 kV. Enrichment of Electrochemically Active Bacteria (EAB). EAB were enriched from anaerobic sludge taken from the Franklin Township Municipal Sanitary Authority (FTMSA) in Pittsburgh, PA. The EAB were inoculated using an artificial wastewater 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 description of the DNA extraction and PCR amplification of 16S rRNA sequencing were given in the literature (30). Briefly, total chromosomal DNA was extracted from the anaerobic 6244

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sludge and the bacteria on the e-beam Pt-deposited anode electrode in the mediator-less MFC using Super Soil Kit (MO BIO Laboratories, Carlsbad, CA). The DNA was purified using GENECLEAN Turbo (Qbiogene, Irvine, CA) before its use as a template for PCR amplification. For the 16S rRNA gene sequence, the purified DNA was amplified using the forward primer (27f: 5′-AGA GTT TGA TCM TGG CTC AG-3′) and the reverse primer (1492r: 5′-GGT TAC CTT TGT TAC GAC TT3′). PCR amplification was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) with an initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 92 °C, 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. For the bacterial community analysis using DGGE, the extract was amplified by a nested PCR using the forward primer (GC-341f: 5′-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC CCC TAC GGG AGG CGA CAG-3′) and a reverse primer (534r: 5′-ATT ACC GCG GCT GCT G-3′). Nested PCR amplification was performed twice according to the program. The first PCR amplification was performed as follows: Initial denaturation at 94 °C for 9 min followed 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 PCR amplification was performed using the amplicons of the first PCR as a DNA template according to initial denaturation at 94 °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) Analysis. DGGE is a molecular fingerprinting method that separates PCR-generated DNA products. The PCR of environmental DNA can generate templates of differing DNA sequences that represent many of the dominant bacteria (31). DGGE was performed by using a DCode system (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. The PCR amplification was loaded onto 10% polyacrylamide gel with a denaturing gradient ranging from 30 to 60% consisting of urea and formamide. The gel was stained using SYBR Gold (Molecular Probes, Carlsbad, CA) for 30 min before DNA bands were observed by a NucleoTech system (Bio-Rad Laboratories) under ultraviolet illumination (32, 33).

FIGURE 3. Current density of the carbon paper without Pt, Pt-Blk electrode and e-beam Pt-deposited electrode with and without bacteria on the anode compartments. Plasmids were then extracted from 80 colonies using the Fast Minipreps Kit (Eppendorf North America, Westbury, NY). Replicate representative colonies were sequenced with T7 forward/SP6 reverse primers using an automatic sequencer system (ABI 3730; Davis Sequencing, Davis, CA). The sequences were compared to those of the National Center for Biotechnology Information (NCBI) BLAST GenBank nucleotide sequence algorithm. Parsimony phylogenetic trees were constructed by the neighbor-joining method using the PHYDIT 3.1 program (Seoul National University, Korea). The 16S rRNA gene sequences have been submitted to the NCBI GenBank under accession numbers EF679178 through EF679197.

Result and Discussion

FIGURE 2. SEM images of (a) carbon paper electrode, (b) e-beam Pt-deposited electrode, and (c) EAB and non-EAB on e-beam Pt-deposited electrode in the mediator-less MFC. 16S rRNA Sequencing and Phylogenetic Analysis. PCR products amplified using a 27f/1492r primer pair were cloned into the pGEM-T vector system (Promega, Madison, WI) and transformed into competent Escherichia coli GC5 competent cells (NextGen Sciences, Ann Arbor, MI). The transformants were plated on Luria-Bertani (LB) agar medium containing ampicillin, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), and isoprophyl-β-D-thiogalactopyranoside (IPTG). Ampicillin-resistant and β-galactosidase-negative clones were transferred to a liquid medium of the same composition (34).

Electrode Characterization. We fabricated an e-beam Ptdeposited electrode to increase the efficiency of electron transfer in a mediator-less MFC. We compared electrode surfaces using a SEM before installing the electrodes into the mediator-less MFC, and after enriching the EAB on the anode electrodes, as shown in Figure 2. Figure 2a shows a microscopic view of the carbon paper electrode without any Pt depositions. Numerous straight carbon rods cross over each other, layer by layer, forming a mesh-like structure. Figure 2b shows the e-beam Pt-deposited electrode. The entire area of the exposed carbon paper is uniformly covered with Pt. A SEM image of the e-beam Pt-deposited electrode surface after EAB enrichment revealed that the electrodes were covered with bacteria (Figure 2c). Improved Efficiency of an Electron Transfer by the E-Beam Pt-Deposited Electrodes and Enrichment of EAB. Two factors, Pt as a catalyst and EAB consortia, are very important to improving the efficiency of electron transfer. We investigated the use of an e-beam Pt-deposited electrode and EAB consortia, containing EAB and non-EAB, in a mediator-less MFC. For the first few days of this study, we enriched the EAB and non-EAB on the anode electrode using an anaerobic sludge and an artificial wastewater containing a glucose and glutamate fuel. The glucose and glutamate fuel was oxidized by the EAB producing electrons and protons in the anode compartment. The electrons were transferred to the electrode by EAB and then via an electric wire to the cathode compartment (34). Next, we investigated the MFC performance using carbon paper, Pt-Blk, and e-beam Ptdeposited electrodes in the anode compartments. As shown in Figure 3, the current density of a carbon paper electrode without Pt deposition was around 0.05 A/m2, the current density of the Pt-Blk electrode was around 0.25 A/m2, and the current density of the e-beam Pt-deposited electrode was around 0.9 A/m2. However, the e-beam Pt-deposited VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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

FIGURE 4. SEM images of (A) Pt-Blk electrode (electrochemical deposited) 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. 6246

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electrode without bacteria showed about 0.0005 A/m2. The current density of the e-beam Pt-deposited electrode was about 18 times higher than the current density of the carbon paper without Pt deposition. It is important to note that although the e-beam Pt-deposited electrode (1000 Å thickness) was thinner than the Pt-Blk electrode (1500 Å thickness), the current density of the e-beam Pt-deposited electrode was 3.6 times higher than the density of the Pt-Blk electrode. This was a remarkable improvement due mainly to the excellent coverage and uniformity of Pt using the e-beam deposition process as opposed to the electrochemical deposition process used on the Pt-Blk electrode (15). These results suggest that the e-beam Pt-deposited electrode and EAB consortia in the mediator-less MFC increase the efficiency of electron transfer between the bacteria and the electrode by the excellent coverage of the e-beam Ptdeposited electrode (15). Additionally a 1 h sonification of both the e-beam Pt-deposited and Pt-Blk electrodes confirmed that e-beam deposition provides a stronger adhesion to the carbon electrode than the Pt-Blk electrode using Bransonic ultrasonic cleaner (5510R-DTH, Branson Ultrasonic Corporation, U.S.) at 25 °C and an input sonification power density of 30 W/gal. Effective sonication power density was not measured. Results show that the e-beam electrodes incurred little damage while the Pt-Blk electrodes were separated from the underlying carbon paper (Figure 4). Profiling Bacterial Communities Using DGGE. EAB consortia of the EAB and non-EAB present on the anode electrode play an important role in the mediator-less MFC process; however, selection and classification of EAB are difficult using traditional microbiological techniques, such

TABLE 1. Bacterial Clones Retrieved from a Mediator-Less MFC Enriched with a Glucose and Glutamate Fuel clones (access no.) ASP-31 ASP-32 ASP-33 ASP-35 ASP-37 ASP-38 ASP-39 ASP-40 ASP-41 ASP-42 ASP-43

(EF679181) (EF679181) (EF679181) (EF679181) (EF679181) (EF679181) (EF679181) (EF679181) (EF679181) (EF679181) (EF679181)

similar relatives (accession no.)

homology (%)

class

Aeromonas hydrophila (AF468055) Aeromonas media (X60410) Enterobacter cloacae subsp. Dissolvens (DQ988523) Enterobacter asburiae (AJ506159) Enterobacter sp. Px6-4 (EF175731) Klebsiella sp. F51-1-2 (DQ277701) Klebsiella oxytoca (AF543296) Aeromonas sp. ydcc-5-1 (DQ837027) Aeromonas molluscorum (AY532691) Citrobacter freundii (AF025365) Enterobacter cloacae (DQ202394)

98 98 98 98 95 99 98 98 99 99 98

γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria γ-proteobacteria

as microscopy and cultivation. The molecular biological techniques such as DGGE, RFLP, and 16S rRNA sequencing have allowed the study of bacterial communities at a different levelsthe genetic level (36). They represent the most powerful approach to explore bacterial communities in natural samples (32). In particular, DGGE is effective for separating environmental DNA samples. DGGE can separate many bands of the PCR products based on sequence differences in differential denaturing characteristics of the DNA (37). As shown in Figure 5, DGGE bands of the anaerobic sludge (S) based on the e-beam Pt-deposited anode electrode (A) were revealed in between 12 and 15 detectable bands. DGGE bands of each sample indicate that several bands were shown in the same position in this case. Six bands of the anaerobic sludge were dominant. S1 and S2 bands were more prevalent than other bands in the anaerobic sludge. Two bands of the e-beam Pt-deposited anode electrode (A), A1 and A2, were most dominant. The A1 band was found in both the anaerobic sludge and the anode electrode. When comparing the results

of the 16S rRNA sequencing, the A1 band seems to play an important role in the mediator-less MFC. The A2 band showed weakly in the anaerobic sludge, but showed clearly on the anode electrode. The A2 band could potentially be responsible in part for the generated electricity in the mediator-less MFC. Bacterial Classification by 16S rRNA Sequencing. To identify bacterial classification, E. coli GC5 colonies containing the PCR products of 16S rRNA were sequenced with T7 forward/SP6 reverse primers using an automatic sequencing system, and were compared to those of the NCBI BLAST GenBank nucleotide sequence algorithm. As a result, 11 different 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 our mediator-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 predominant bacterium in the anaerobic sludge (S1) and on the anode

FIGURE 6. Phylogenetic analysis of the bacterial community of the e-beam Pt-deposited anode electrode in the mediator-less MFC with glucose and glutamate fuel. VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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electrode (A1) (Figure 5). The second-most abundant clone, ASP-32 (20% sequence abundance), showed 98% similarity to Aeromonas media. The sequence types of some clones were similar to the Aeromonas genus such as Aeromonas molluscorum (98% similarity) and Aeromonas sp. ydcc-5-1 (98% similarity). Some clones were similar to Klebsiella oxytoca (98% similarity) which is also an Fe(III)-reducing bacterium. Enterobacter such as K. oxytoca ferments ferric citrate as a carbon and energy source (38). Sequences of 16S rRNA clones were obtained from the mediator-less MFC and compared with those sequences in the NCBI algorithm. Representatives were used to construct a phylogenetic tree (Figure 6). In our mediator-less MFC, all sequences obtained from the 16S rRNA clone library of the anode electrode sample, were γ-proteobacteria. These bacteria in our mediator-less MFC with a glucose and glutamate fuel belong to the γ-proteobacteria group but in particular our consortia was dominated by A. hydrophila. This is a remarkable phenomenon because these types of studies have never been reported in previous literature. Logan et al. reported that over 97% of the detected sequences belonged to the γ-proteobacteria group, but predominant bacteria in the MFC with the mineral salts medium were Shewanella spp. (39). Choo et al. reported that 33.6% of the predominant γ-proteobacteria such as Acinetobacter was present in the mediator-less MFC with a glucose and glutamate fuel (40). It is possible for the Aeromonas genus dominated by A. hydrophila to produce electricity within three days. The consortium was easily enriched in the MFC using a copiotrophic glucose and glutamate fuel. Therefore, the MFC with Aeromonas genus consortia dominated by A. hydrophila can produce electricity more quickly than other EAB (12, 41) as well as a higher power density of our MFCs than others (15). In summary, our results indicate that the current density from MFCs using e-beam Pt-deposited electrodes were higher than the MFCs using Pt-Blk electrodes. A. hydrophila clones ASP-31 were predominant on the e-beam Pt-deposited electrodes. Relatively better coverage and stronger adhesion of e-beam Pt-deposited electrodes were one of the contributions for producing higher current density than any other electrodes. However, further investigation is required in order to fully understanding the relationship between e-beam Pt electrode and the bacteria.

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Acknowledgments This work was supported by the Mascaro Sustainability Initiative (MSI) at the University of Pittsburgh. We thank C. Brucker with the Wastewater Treatment Plant of the Franklin Township Municipal Sanitation Authority (FTMSA) and National Energy Technology Laboratory (NETL) in Pittsburgh, PA, for the facilities and indirect financial assistance.

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Supporting Information Available

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Information regarding the Restriction fragment length polymorphism analysis can be found in the manuscript supplement. This material is available free of charge via the Internet at http://pubs.acs.org.

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