A facultative anaerobic bacterium was isolated from a mediator-less microbial fuel cell fed with artificial wastewater containing acetate and designated as PA3. The isolate was identified as a strain of Aeromonas hydrophila based on its biochemical, physiological and morphological characteristics as well as 16S rDNA sequence analysis and DNA–DNA hybridization. PA3 used glucose, glycerol, pyruvate and hydrogen to reduce Fe(III), nitrate and sulfate. Cyclic voltammetry showed that PA3 was electrochemically active and was the culture collection strain A. hydrophila KCTC 2358. Electricity was generated from a fuel cell-type reactor, the anode compartment of which was inoculated with cell suspensions of the isolate or A. hydrophila KCTC 2358. The electrochemical activities are novel characteristics of A. hydrophila.
Various bacteria reduce Fe(III) through their respiratory, fermentative or photosynthetic metabolism. Some of them are able to conserve energy for growth by coupling the oxidation of organic acids, aromatic hydrocarbons and H2 to Fe(III) reduction. These include species of the genera Geobacter, Geovibrio, Shewanella[3,4], a sulfate-reducing bacterium, Desulfotomacum reducens, among others. Fermentative bacteria Clostridium butyricum and Clostridium beijerinckii are known to reduce Fe(III) as an electron sink during glucose metabolism. In the metabolism of a photosynthetic bacterium, Rhodobacter capsulatus, Fe(III) acts as an auxiliary oxidant with physiological importance for redox poising under anaerobic photoheterotrophic growth conditions .
Electrochemical techniques, including cyclic voltammetry, have been used to characterize redox proteins including cytochromes . In general, bacterial cells containing electrochemically active proteins are electrochemically inactive as their cell wall structures consist of non-conducting material such as lipid and peptidoglycan. Mediators were used to facilitate the electron transfer between an electrode and electrochemically inactive microbial cells . Alternatively, the bacterial cells can be modified with hydrophobic conducting polymers to render the electrochemical activity .
An Fe(III)-reducing bacterium, Shewanella putrefaciens, is known to localize the majority of its membrane-bound cytochromes on its outer membrane . The outer membrane cytochromes are believed to be involved in the reduction of water-insoluble Fe(III). Intact cells of anaerobically grown S. putrefaciens were electrochemically active and the bacterium could grow in a fuel cell-type electrochemical cell in the absence of electron acceptors . Similar studies were made using another Fe(III)-reducing bacterium, Geobacter sulfurreducens.
In this laboratory, electrochemically active microbes have been enriched in a fuel cell-type electrochemical cell using different sources of wastewater as the electron donor. Attempts were made to isolate microbes from the various electron donor and acceptor combinations of the enrichment culture, but colony-forming units on conventional solid media were less than 0.1% of the number of microbes estimated from DNA and protein analyses of the enriched electrode (paper in preparation). The isolate, PA3, used in this study was one of the colonies formed on a solid medium, with ferric pyrophosphate as the electron acceptor. The isolate was characterized as a strain of the genus Aeromonas, and was able to reduce ferric iron Fe(III), nitrate and sulfate. The isolate was also characterized by electrochemical methods, including cyclic voltammetry and fuel cell techniques.
Materials and methods
The bacterial strain used in the study was isolated from the anode of a mediator-less microbial fuel cell (MFC) fed with artificial wastewater containing 5 mM sodium acetate. A piece (1 cm3) of anode was transferred to a pressure tube (Bellco Glass, Vineland, NJ, USA) containing 10 ml sterile anaerobic saline solution and shaken vigorously to separate microbial cells from the electrode. The suspension was serially diluted and plated on phosphate-buffered basal medium (PBBM)  containing 10 mM sodium acetate and 20 mM ferric pyrophosphate as electron donor and acceptor, respectively, and incubated in an anaerobic glove box (Coy Lab. Products, Grass Lake, MI, USA). Some colonies formed haloes around them on the plate, due to Fe(III) reduction . The strain designed as PA3 was one of the colonies with halos. Also used was Aeromonas hydrophila KCTC 2358, obtained from the Korean Collection of Type Cultures (Taejon, Korea).
PBBM was used to isolate bacterial strains. In certain experiments strictly anaerobic techniques were employed . The medium was anaerobically dispensed into pressure tubes or serum vials (160 ml; Wheaton Scientific, Millville, NJ, USA), which were stoppered with butyl rubber bungs (Bellco Glass) and crimped with aluminum caps. Solid medium was prepared by adding agar to PBBM to a final concentration of 2% (w/w). Acetate was used as an electron donor at a final concentration of 5–20 mM with or without an electron acceptor. Ferric citrate or ferric pyrophosphate were used as a soluble form of Fe(III) to prepare solid medium.
The isolate PA3 could grow either under aerobic or anaerobic conditions. Luria–Bertani (LB) medium was used for cultivation and maintenance of the isolate under aerobic conditions. For the study on the utilization of electron donors and acceptors, anaerobically sterilized stock solutions of electron donors and acceptors were added to anaerobic PBBM to a final concentration of 20 mM. Triplicate cultures were initiated with cell suspensions of isolate PA3 at 5% (v/v). The cell suspension was prepared from aerobically grown overnight cultures using LB broth. Cells were washed three times with 50 mM phosphate buffer containing 100 mM NaCl to remove nutrients remaining from the LB broth before being suspended in the same buffer. After 5 days of incubation, the cultures were analyzed for cellular protein content, remaining electron donors and acceptors [Fe(II) formed in the case of Fe(III) as electron acceptor].
Cultures were made anaerobically using LB medium added with 20 mM ferric citrate to prepared cell suspensions for cyclic voltammetry and MFC. Cells were washed and suspended in 50 mM phosphate buffer containing 100 mM NaCl. PBBM was used to prepare cell suspension for fuel cell experiments. The suspensions contained 0.2±0.02 g dry cell l−1. All cultures were made at 30°C.
A light microscope (Jenalumar, Carl Zeiss Jena, Jena, Germany) was used to determine the Gram reaction. The Gram reaction was determined by using the Difco Gram-stain set. Motility was observed by using the commercial motility medium API M medium (BioMerieux®, Marcy l'Etoile, France) and confirmed according to the standard methods . For scanning electron microscopy, the cells were collected on a nucleopore filter (pore size 0.2 µm, Whatman Scientific, Maidstone, UK) and treated as previously described . Scanning electron micrographs were taken using an S-4200 FE-SEM (Hitachi, Tokyo, Japan) after the specimens were coated with gold .
Physiological and biochemical characterization
Commercial identification kits (API 20E and API 20 NE, BioMerieux®) were used for physiological and biochemical characterization, and the results were analyzed with APILAB Plus (BioMerieux®). A number of additional tests were made. Oxidase activities were determined using commercial oxidase reagent (BioMerieux®) and other characteristics were determined by standard methods .
S rDNA gene sequencing and analysis
Genomic DNA extracted from the isolate was used for polymerase chain reaction (PCR)-mediated amplification of 16S rDNA using 27f and 1492r primer pairs. The amplicon was purified using a purification kit (GENECLEAN Turbo, Q-BIO gene, Carlsbad, CA, USA) prior to cloning into Escherichia coli DH5α using the pGEM-T® easy vector system I (Promega, Madison, WI, USA). The cloned 16S rDNA was sequenced and analyzed as described earlier . The 16S rDNA sequence of the isolate has been deposited in GenBank under accession number AF468055.
Genomic DNA was extracted from isolate PA3 and A. hydrophila KCTC 2358 to conduct DNA–DNA hybridization according to the method described by Goodfellow et al. .
Cyclic voltammetry and MFC
Bacterial cells grown in LB medium containing ferric citrate for 72 h under anaerobic conditions were harvested, washed three times and suspended in 50 mM anaerobic phosphate buffer containing 100 mM NaCl . The cell suspension contained 0.2±0.02 g dry cell l−1. The cell suspension was used to test electrochemical activities using cyclic voltammetry and fuel cell techniques as described earlier . MFCs inoculated with the cell suspension were fed with PBBM containing yeast extract to give final COD values of 250 or 1260 mg l−1. The MFC was connected through a resistance of 500 Ω to measure the potential drop between the anode and cathode as current.
Fe(II) was analyzed spectrophotometrically using the ferrozine method to measure Fe(III) reduction . Fresh medium was used as the control. Liquid chromatography (M930, Young-Lin, Anyang, Korea) was used to quantify sulfate and nitrate with IC-pak Anion HR column (Waters, Milford, MA, USA), and pyruvate with Aminex HPX-87H column (Bio-Rad Laboratories, Richmond, CA, USA). Protein was measured by the Biuret method  with bovine serum albumin as a standard. Hydrogen was measured using a gas chromatograph (Varian 3300, Varian, Sunnyvale, CA, USA) with thermal conductivity detector as described previously . Acetate was determined using a gas chromatograph (Varian 3300) with flame ionization detector. Glucose was quantified using a PGO-enzymatic glucose kit (Young-dong Pham. Co., Seoul, Korea).
Results and discussion
Isolation of PA3
An isolate, PA3, was selected among the Fe(III)-reducing colonies on a solid medium containing ferric pyrophosphate as the electron acceptor. Fe(III)-reducing colonies were recognized easily on the solid medium as their growth resulted in haloes around the colonies in the green-colored ferric pyrophosphate medium. Colonies grown on ferric pyrophosphate medium were typically less than 5 mm in diameter. Colonies were yellow and domed and appeared to be coated with Fe(II) mineral, similar to that observed in Geobacter species . Fe(III)-reducing colonies were picked and transferred to PBBM with acetate and ferric citrate as the electron donor and acceptor, respectively.
Morphological and physiological characterization
The isolate PA3 showed a Gram-negative reaction. Cells were straight rod-shaped, 2.3–2.5 µm long, and 0.5–0.7 µm wide (Fig. 1). On nutrient agar, colonies were buff, circular and convex with an entire margin and a size of 3–4 mm. Similar reactions were observed for the isolate PA3 and the type strain A. hydrophila KCTC 2358 in all physiological and biochemical tests, including the commercial identification kits (API 20E and API 20 NE).
Phylogenetic analysis and DNA–DNA hybridization
The sequence similarity of the 16S rRNA gene was compared with those of reference organisms obtained from GenBank data libraries. The result revealed that the isolate PA3 is a member of the Aeromonas subphylum of Gram-negative bacteria (Fig. 2). A. hydrophila was the nearest neighbor, with a 16S rDNA sequence similarity of 99%. Genomic DNA extracted from the isolate was nearly 100% homologous with that of A. hydrophila KCTC 2358.
The isolate PA3 was identified as a strain of A. hydrophila, designated as A. hydrophila PA3 based on morphological, biochemical and physiological characteristics as well as 16S rDNA sequence homology.
Utilization of electron donors and acceptors by A. hydrophila PA3
A. hydrophila PA3 was cultivated for 5 days using PBBM containing various electron donor–acceptor combinations, and the cellular protein content and the utilization of electron donors and acceptors were measured.
Cellular protein of 0.36±0.03 g l−1 was produced from the cultures made on non-fermentable electron donors such as acetate, glycerol and hydrogen without electron acceptor. Yeast extract (1 g l−1 in PBBM) is believed to support the bacterial growth. Under similar conditions, glucose and pyruvate produced cellular protein contents of 1.29 and 0.42 g l−1, respectively. This result shows that the bacterium can ferment glucose and pyruvate.
Good growth was observed on glucose with any of the electron acceptors used. With pyruvate as the electron donor the bacterium reduced Fe(III), nitrate and sulfate with cellular protein contents over 1 g l−1. Acetate supported good growth of the bacterium with nitrate under aerobic conditions, but the bacterium did not consume acetate with ferric citrate or sulfate as electron acceptors. Hydrogen showed similar results. With glycerol as the electron donor, the bacterium produced more cellular protein with ferric citrate than with nitrate.
A. hydrophila PA3 has been isolated from a mediator-less MFC fed with acetate as sole fuel. Current generation from an MFC has been reported to be related to Fe(III)-reducing activity [13,14,22]. This organism consumed acetate with nitrate or oxygen as the electron acceptor, but not with Fe(III). It is not clear if A. hydrophila PA3 consumes acetate in an MFC environment.
Washed cell suspensions of A. hydrophila PA3 were used to determine the electrochemical activity using cyclic voltammetry. Oxidation/reduction peaks were observed in the cyclic voltammogram (CV) of cells grown under anaerobic conditions with ferric citrate, but cells grown under aerobic conditions or under anaerobic conditions without ferric citrate did not show the electrochemical activity (Fig. 3). The CV obtained showed different shapes of oxidation and reduction peaks with an apparent redox potential of around 50 mV against the Ag/AgCl reference electrode. This asymmetry shows that the redox reaction of the cell suspension is a quasi-reversible reaction. c-Type cytochromes were found in A. hydrophila ATCC7966 grown under anaerobic conditions with ferric citrate . It is plausible that the electrochemical activity observed in this organism is due to the c-type cytochromes observed in A. hydrophila ATCC7966.
It is interesting to note that electrochemical activity was observed in the cell suspension of S. putrefaciens grown under anaerobic conditions without Fe(III) , while A. hydrophila PA3 was electrochemically active only when grown under anaerobic conditions with Fe(III). If the electrochemical activity is related to the localization of c-type cytochromes to the cell surface , these two organisms might have different regulatory mechanisms for the biosynthesis and translocation of c-type cytochromes.
A cell suspension prepared from an anaerobic culture with Fe(III) was used to test the effects of exposure to air on the electrochemical activity. The cell suspension gradually lost its electrochemical activity when exposed to air, with a complete loss within 30 min (Fig. 4). When the air-exposed cell suspension was gassed with oxygen-free nitrogen, its electrochemical activity was restored. The recovery was most extensive under nitrogen gassing when it was poised at 200 mV against the Ag/AgCl reference electrode for 20 min in an electrochemical cell with glycerol, followed by incubation with a combination of glycerol and nitrate, and with nitrate. These results revealed that electrochemical activity is reversibly inactivated when cells are exposed to air.
A similar cell suspension was tested for its ability to generate current in a fuel cell-type electrochemical cell (Fig. 5). When the fuel cell was inoculated with the cell suspension, the current was maintained at a background level of less than 0.1 mA, and increased rapidly to over 1.8 mA when yeast extract as fuel (250 mg l−1 as COD) was supplied. After the initial peak, the current decreased with a shoulder finally to the background level in around 7 h. Similar current-generation patterns were observed, and explained as due to the current generation being limited not by the biological reaction but by other factors, such as oxygen supply to the cathode and proton permeability through the membrane [24,25]. Repeated fuel supply gave similar responses. When the current dropped to the background level, the COD value was less than 5 mg l−1. When fuel was supplied in a higher concentration of 1260 mg l−1 as COD, the maximum current of 0.3 mA was generated and the current generation lasted more than 17 h (Fig. 6).
When the fuel cell containing the cell suspension was fed with acetate as fuel, current was not increased. This result is consistent with the previous results. The isolate did not consume acetate under Fe(III)-reducing conditions, though acetate is oxidized under aerobic conditions or under denitrifying conditions. It is not clear what was the function of this isolate in the MFC fed with acetate. The bacterium might participate in the process converting fuel into electricity as a member of the microbial consortium.
After MFCs inoculated with the cell suspension were run for 5 days, the anode was removed, and observed under a scanning electron microscope (Fig. 7). The electrode was covered with bacterial cells of short rods with a similar shape as shown in Fig. 1. The current reached the maximum immediately after the addition of fuel, and the electron microscopic observation showed that the electrode was covered with bacterial cells with uniform shape. It is interesting to note the formation of a biofilm-like structure on the electrode surface in a short period of time. These results show that the current was generated through the electrochemical activity of A. hydrophila PA3, not through contaminating microorganisms.
To the authors' surprise, the culture collection strains A. hydrophila KCTC 2358 showed similar electrochemical activity in cyclic voltammetry. Electrochemical activity might be a general property of A. hydrophila and related bacteria with the ability of Fe(III) reduction.
The study was supported by a grant from the Korea Institute of Science and Technology and The Ministry of Science and Technology (National Research Laboratory Program) in Korea.