J. Gen. Appl. Microbiol., 59, 261‒266 (2013) Full Paper Hydrogenophaga electricum sp. nov., isolated from anodic biofilms of an acetate-fed microbial fuel cell Zen-ichiro Kimura and Satoshi Okabe* Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060‒8628, Japan (Received October 25, 2012; Accepted April 2, 2013) A Gram-negative, non-spore-forming, rod-shaped bacterial strain, AR20T, was isolated from an- odic biofilms of an acetate-fed microbial fuel cell in Japan and subjected to a polyphasic taxo- nomic study. Strain AR20T grew optimally at pH 7.0‒8.0 and 25°C. It contained Q-8 as the pre- dominant ubiquinone and C16:0, summed feature 3 (C16:1ω7c and/or iso-C15:02OH), and C18:1ω7c as the major fatty acids. The DNA G+C content was 67.1 mol%. A neighbor-joining phylogenetic tree revealed that strain AR20T clustered with three type strains of the genus Hydrogenophaga (H. flava, H. bisanensis and H. pseudoflava). Strain AR20T exhibited 16S rRNA gene sequence similarity values of 95.8‒97.7% to the type strains of the genus Hydrogenophaga. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain AR20T is considered a novel species of the genus Hydrogenophaga, for which the name Hydrogenophaga electricum sp. nov. is pro- posed. The type strain is AR20T (= KCTC 32195T = NBRC 109341T). Key Words—Hydrogenophaga electricum; hydrogenotrophic exoelectrogen; microbial fuel cell Introduction the MFC was analyzed. Results showed that bacteria belonging to the genera Geobacter and Hydrogenoph- Microbial fuel cells (MFCs) are devices that are able aga were abundantly present in the anodic biofilm to directly convert the chemical energy of organic community (Kimura and Okabe, 2013). We isolated a compounds into electric energy with microorganisms bacterium closely related to the genus Hydrogenopha- (Bullen et al., 2006; Davis and Higson, 2007; Kim et al., ga (H. electricum strain AR20T). 1999). Electricity generation in a mediator-less MFC is The genus Hydrogenophaga was proposed by Wil- linked to the ability of certain bacteria, called exoelec- lems et al. (1989) with the reclassification of Pseudo- trogens, to transfer electrons outside the cell to the monas species as Hydrogenophaga flava, H. pallero- anode electrode in the MFC (Logan, 2006). In our pre- nii, H. pseudoflava and H. taeniospiralis. Subsequently, vious study, a MFC fed with acetate was constructed five other species, Hydrogenophaga intermedia (Cont- with activated sludge as a source of microorganisms, zen et al., 2000), H. atypical, H. defluvii (Kämpfer et al., and the microbial community of the anodic biofilm in 2005), H. caeni (Chung et al., 2007) and H. bisanensis (Yoon et al., 2008) have been described. Phylogenetic analysis based on 16S rRNA gene sequences showed * Corresponding author: Satoshi Okabe, Division of Environ- that the genus Hydrogenophaga falls within the family mental Engineering, Faculty of Engineering, Hokkaido Univer- sity, North-13, West-8, Kita-ku, Sapporo, Hokkaido 060‒8628, Comamonadaceae, class Betaproteobacteria (Anzai et Japan. al., 2000; Kämpfer et al., 2005). In this paper, the re- Tel & Fax: +81‒(0)11‒706‒6266 sults of an examination of the phenotypic characteris- E-mail: [email protected] tics of strain AR20T are described, along with the phy- 262 KIMURA and OKABE Vol. 59 logenetic placement of the strain. The name Hydro- uid medium (Chung and Okabe, 2009a) under the genophaga electricum sp. nov. is proposed with strain conditions described by Malik and Schlegel (1981). AR20T as the type strain. Utilization of various substrates, enzyme activities and other physiological and biochemical properties were Materials and Methods tested by using the API 20NE and API 50CH (bioMéri- eux, Marcy l’Etoile, France); utilization of various sub- Test strain and cultivation. Strain AR20T was iso- strates was determined by inoculating the API 50CH lated by means of the standard dilution plating tech- strip with cells suspended in AUX medium (bioMéri- nique on R2A agar (Difco, Japan BD, Tokyo, Japan) at eux). Utilization of various substrates was also tested 25°C. The morphological, physiological and biochemi- as described by Kämpfer et al. (1991). cal characteristics of strain AR20T were investigated by Minimum inhibitory concentrations (MIC) were de- using standard cultivation techniques at 25°C. Cell termined in R2A broth containing the following antibi- morphology was examined by transmission electron otics: chloramphenicol, ampicillin, tetracycline, kana- microscopy (Beveridge et al., 1999). Cells were fixed mycin and neomycin. with glutaraldehyde (2.5%, w/v), negatively stained Chemotaxonomic analyses. Total cellular fatty with phosphotungstic acid (0.2%, w/v), and visualized acid analysis of cells grown on YPG agar (Japan BD, using a JEM 1400 transmission electron microscope Tokyo, Japan) for 48 h at 30°C was performed by (JEOL, Tokyo, Japan). Gas Liquid Chromatography (GLC) according to the Growth at various temperatures (4‒50°C) was mea- instructions of the Microbial Identification System sured. The pH range for growth was determined in (MIDI) Sherlock version 6.0 (Sasser, 1990) with the R2A broth that was adjusted to various pH values RTSBA6 MIDI database (MIDI Inc., Newark, DE, USA). (pH 4.5‒10.5 at intervals of 0.5 pH units) by the addi- Quinone was extracted with a chloroform-methanol tion of HCl and Na2CO3. Growth in the various NaCl mixture, purified by thin layer chromatography and concentrations (0‒5.0% (w/v) at intervals of 1.0%) was analyzed by reversed-phase HPLC (L-7000, Hitachi, investigated by using R2A medium. Tokyo, Japan) as described previously (Hiraishi, 1988). Growth under anaerobic conditions was determined 16S rRNA gene sequencing and phylogenetic analy- after incubation in an anaerobic chamber in R2A broth sis. The 16S rRNA gene of strain AR20T was PCR supplemented with potassium nitrate and nitrite (0.1%, amplified, purified and sequenced directly with an au- w/v), both of which had been prepared anaerobically tomated DNA sequencer (ABI PRISM 3100-Avant Ge- under a nitrogen atmosphere. Nitrate and nitrite reduc- netic Analyzer; Life Technologies, Grand Island, NY, tion were studied as described by Lanyi (1988). Elec- USA). Sequence data were compiled with the BioEdit trochemical activity of the AR20T was evaluated using program. Multiple alignment of sequence data, calcu- H-type MFCs that consisted of 500 ml anode and cath- lation of the corrected evolutionary distance (Kimura, ode glass chambers separated by a proton exchange 1980), and construction of neighbor-joining phyloge- membrane (PEM, NafionTM 117, Dupont Co., Fayette- netic tree (Saitou and Nei, 1987) were performed us- ville, NC, USA) (Chung et al., 2011). The anode cham- ing the CLUSTAL W program ver. 1.83 (Thompson et ber was filled with 500 ml of a sterilized mineral medi- al., 1994). The topology of the tree was evaluated by um (Chung and Okabe, 2009b) containing H2 gas as bootstrapping with 1,000 resamplings (Felsenstein, the sole electron donor and a carbon electrode as the 1985). sole electron acceptor. The cathode chamber was DNA base composition. Genomic DNA was ex- filled with 500 ml of phosphate buffer (80 mM and pH tracted and purified according to the method of Mar- 7.1) containing 50 mM ferricyanide. The H-type MFCs mur (1961). The guanine plus cytosine (G+C) ratio of were operated at 25 ± 2°C. Utilization of thiosulfate genomic DNA was determined by the HPLC/PDA was tested in R2A broth supplemented with 10 mM method with external nucleotide standards as de- Na2S2O3･ 5H2O as described by Spring et al. (2004). scribed by Mesbah (1989). DNA-DNA hybridization The concentrations of nitrate, nitrite and thiosulfate in were performed using the quantitative dot-blot hybrid- R2A broth were measured by ion chromatograph (ICS; ization with the Alkphos Direct Labelling and Detection Dionex, Osaka, Japan). Chemolithoautotrophic growth System with CDP-star (GE Healthcare Japan, Tokyo, of strain AR20T with hydrogen gas was tested on a liq- Japan) as described previously (Kubota et al., 2005). 2013 Hydrogenophaga electricum sp. nov. 263 Results and Discussion few reports on the presence and ability of hydrogeno- trophic exoelectrogens (Chung et al., 2009b; Pham et Morphology and cultural characteristics al., 2003), strain AR20T has the highest ability to gener- Morphological, cultural, physiological and biochem- ate current to date. The cultural, physiological and bio- ical characteristics of strain AR20T are shown in Fig. 1, chemical characteristics of strain AR20T are apparently Fig. 2 and Table 1. The cells were Gram-negative, non- different from all other nine Hydrogenophaga species spore-forming, motile, and rod-shaped organisms. and Xenophilus azovorans strain KF46FT, for example Strain AR20T grew under anoxic conditions in R2A broth with nitrate or nitrite as a terminal electron ac- ceptor. This strain grew well in a temperature range from 20 to 30°C, but did not grow at 4°C on R2A agar or YPG agar (Difco) (Table 1). Strain AR20T could gen- erate an electrical current via oxidation of hydrogen in a pure culture MFC (Fig. 2). Although there are only a Fig. 2. Characterization of the electrochemical activity of strain AR20T in a two-chamber MFC. Change in power density was shown when hydrogen was used as the sole electron donor without any other dissolved electron acceptors. Hydrogen gas (100% H2) was bubbled di- rectly into the anode culture fluid for 10 min as indicated by ar- Fig.