Bacterial Communities on Electron-Beam Pt-Deposited Electrodes in a Mediator-Less Microbial Fuel Cell HO IL PARK, † DAVID SANCHEZ, ‡ SUNG KWON CHO, § AND MINHEE YUN †, * 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, Entero- bacter 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 environ- mental 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 metano- genic anaerobic digestion, ethanol fermentation, and hy- drogen 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 (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 nano- particles 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 non- EAB 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 elec- trodes 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 bio- logical techniques including denaturing gradient gel elec- trophoresis (DGGE), restriction fragment length polymor- phism (RFLP), and 16S rRNA sequencing. Finally, we compared bacterial communities of Aeromonas genus con- sortia 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, 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. Environ. Sci. Technol. 2008, 42, 6243–6249 10.1021/es8006468 CCC: $40.75  2008 American Chemical Society VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 6243 Published on Web 07/03/2008