APPLIED MICROBIAL AND CELL PHYSIOLOGY Isolation of the exoelectrogenic denitrifying bacterium Comamonas denitrificans based on dilution to extinction Defeng Xing & Shaoan Cheng & Bruce E. Logan & John M. Regan Received: 22 May 2009 / Revised: 18 August 2009 / Accepted: 2 September 2009 / Published online: 25 September 2009 # Springer-Verlag 2009 Abstract The anode biofilm in a microbial fuel cell (MFC) is composed of diverse populations of bacteria, many of whose capacities for electricity generation are unknown. To identify functional populations in these exoelectrogenic communities, a culture-dependent approach based on dilu- tion to extinction was combined with culture-independent community analysis. We analyzed the diversity and dy- namics of microbial communities in single-chamber air-cathode MFCs with different anode surfaces using denaturing gradient gel electrophoresis based on the 16S rRNA gene. Phylogenetic analyses showed that the bacteria enriched in all reactors belonged primarily to five phyloge- netic groups: Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria. Dilution-to- extinction experiments further demonstrated that Comamonas denitrificans and Clostridium aminobutyricum were domi- nant members of the community. A pure culture isolated from an anode biofilm after dilution to extinction was identified as C. denitrificans DX-4 based on 16S rRNA sequence and physiological and biochemical characteriza- tions. Strain DX-4 was unable to respire using hydrous Fe (III) oxide but produced 35 mW/m 2 using acetate as the electron donor in an MFC. Power generation by the facultative C. denitrificans depends on oxygen and MFC configuration, suggesting that a switch of metabolic pathway occurs for extracellular electron transfer by this denitrifying bacterium. Keywords Comamonas denitrificans . Exoelectrogen . Denitrifying bacteria . Microbial community . Dilution to extinction . Microbial fuel cell Introduction Microbial fuel cells (MFCs) show great promise as a method for energy production during wastewater treatment (Logan and Regan 2006a; Lovley 2008; Rittmann et al. 2008). The power output of these systems is primarily affected by the system architecture, but the microbial ecology can be important as well (Logan and Regan 2006b; Rabaey et al. 2007; Rittmann 2006; Xing et al. 2008b). In recent years, power production by MFCs has increased by several orders of magnitude through system architecture improvements that have reduced the internal resistance (Logan and Regan 2006a), such as modifying the reactor configuration (He et al. 2007; Liu and Logan 2004; You et al. 2008; Zuo et al. 2007), improving electrodes (Fan et al. 2007a; Logan et al. 2007), increasing solution conductivity (Cheng and Logan 2007; Fan et al. 2007b; He et al. 2008; Torres et al. 2008), providing flow through a porous anode, and reducing electrode spacing (Cheng et al. 2006b). As a result of these improvements, the microbial community or the specific microorganisms on the anode are now becoming factors in the level of power production. Therefore, it is important to understand the physiology of Electronic supplementary material The online version of this article (doi:10.1007/s00253-009-2240-0) contains supplementary material, which is available to authorized users. D. Xing : S. Cheng : B. E. Logan : J. M. Regan (*) Engineering Environmental Institute, The Pennsylvania State University, 212 Sackett Building, University Park, PA 16802, USA e-mail: jregan@engr.psu.edu D. Xing : S. Cheng : B. E. Logan : J. M. Regan Department of Civil and Environmental Engineering, The Pennsylvania State University, 212 Sackett Building, University Park, PA 16802, USA Appl Microbiol Biotechnol (2010) 85:1575–1587 DOI 10.1007/s00253-009-2240-0