FEMS Microbiology Letters 223 (2003) 129^134

www.fems-microbiology.org

A novel electrochemically active and Fe(III)-reducing bacterium

phylogenetically related to Aeromonas hydrophila,
isolated from a microbial fuel cell
Cuong Anh Pham a , Sung Je Jung a , Nguyet Thu Phung a , Jiyoung Lee a ,
In Seop Chang a , Byung Hong Kim a; , Hana Yi b , Jongsik Chun b
a
Water Environment and Remediation Research Centre, Korea Institute of Science and Technology, Hawolgok-dong, Sungpook-ku,
Seoul 136-791, South Korea
b
School of Biological Science, Seoul National University, Seoul 151-742, South Korea

Received 28 March 2003; accepted 17 April 2003

First published online 16 May 2003

Abstract

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.
: 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : PA3; Dissimilatory Fe(III) reduction; Cyclic voltammogram; Electrochemically active bacteria; Microbial fuel cell; Aeromonas hydrophila sp.

1. Introduction Electrochemical techniques, including cyclic voltamme-

Various bacteria reduce Fe(III) through their respira-
tory, fermentative or photosynthetic metabolism. Some
of them are able to conserve energy for growth by cou-
pling the oxidation of organic acids, aromatic hydrocar-
bons and H2 to Fe(III) reduction. These include species of
the genera Geobacter [1], Geovibrio [2], Shewanella [3,4], a
sulfate-reducing bacterium, Desulfotomacum reducens [5],
among others. Fermentative bacteria Clostridium butyri-
cum [6] and Clostridium beijerinckii [7] are known to re-
duce Fe(III) as an electron sink during glucose metabo-
lism. In the metabolism of a photosynthetic bacterium,
Rhodobacter capsulatus, Fe(III) acts as an auxiliary oxi-
dant with physiological importance for redox poising
under anaerobic photoheterotrophic growth conditions [8].
* Corresponding author. Tel. : +82 (2) 958 5831;
Fax : +82 (2) 958 5839.
E-mail address : bhkim@kist.re.kr (B.H. Kim).
0378-1097 / 03 / $22.00 : 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00354-9
try, have been used to characterize redox proteins includ-
ing cytochromes [9]. In general, bacterial cells containing
electrochemically active proteins are electrochemically in-
active as their cell wall structures consist of non-conduct-
ing material such as lipid and peptidoglycan. Mediators
were used to facilitate the electron transfer between an
electrode and electrochemically inactive microbial cells
[10]. Alternatively, the bacterial cells can be modi¢ed
with hydrophobic conducting polymers to render the elec-
trochemical activity [11].
An Fe(III)-reducing bacterium, Shewanella putrefaciens,
is known to localize the majority of its membrane-bound
cytochromes on its outer membrane [12]. The outer mem-
brane cytochromes are believed to be involved in the re-
duction of water-insoluble Fe(III). Intact cells of anaero-
bically grown S. putrefaciens were electrochemically active
and the bacterium could grow in a fuel cell-type electro-
chemical cell in the absence of electron acceptors [13].
Similar studies were made using another Fe(III)-reducing
bacterium, Geobacter sulfurreducens [14].

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In this laboratory, electrochemically active microbes
have been enriched in a fuel cell-type electrochemical cell
using di¡erent sources of wastewater as the electron do-
nor. Attempts were made to isolate microbes from the
various electron donor and acceptor combinations of the
enrichment culture, but colony-forming units on conven-
tional 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 elec-
tron 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 charac-
terized by electrochemical methods, including cyclic vol-
tammetry and fuel cell techniques.
2. Materials and methods
2.1. Bacterial strains
The bacterial strain used in the study was isolated from
the anode of a mediator-less microbial fuel cell (MFC) fed
with arti¢cial wastewater containing 5 mM sodium ace-
tate. A piece (1 cm3 ) of anode was transferred to a pres-
sure tube (Bellco Glass, Vineland, NJ, USA) containing
10 ml sterile anaerobic saline solution and shaken vigor-
ously to separate microbial cells from the electrode. The
suspension was serially diluted and plated on phosphate-
bu¡ered basal medium (PBBM) [15] containing 10 mM
sodium acetate and 20 mM ferric pyrophosphate as elec-
tron 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 [16]. The strain designed as
PA3 was one of the colonies with halos. Also used was
Aeromonas hydrophila KCTC 2358, obtained from the Ko-
rean Collection of Type Cultures (Taejon, Korea).
2.2. Culture conditions
PBBM was used to isolate bacterial strains. In certain
experiments strictly anaerobic techniques were employed
[17]. The medium was anaerobically dispensed into pres-
sure tubes or serum vials (160 ml; Wheaton Scienti¢c,
Millville, NJ, USA), which were stoppered with butyl rub-
ber bungs (Bellco Glass) and crimped with aluminum
caps. Solid medium was prepared by adding agar to
PBBM to a ¢nal concentration of 2% (w/w). Acetate was
used as an electron donor at a ¢nal 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 an-
aerobic conditions. Luria^Bertani (LB) medium was used
FEMSLE 11010 2-6-03
for cultivation and maintenance of the isolate under aero-
bic conditions. For the study on the utilization of electron
donors and acceptors, anaerobically sterilized stock solu-
tions of electron donors and acceptors were added to an-
aerobic PBBM to a ¢nal concentration of 20 mM. Tripli-
cate 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 bu¡er
containing 100 mM NaCl to remove nutrients remaining
from the LB broth before being suspended in the same
bu¡er. After 5 days of incubation, the cultures were ana-
lyzed for cellular protein content, remaining electron do-
nors 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 suspen-
sions for cyclic voltammetry and MFC. Cells were washed
and suspended in 50 mM phosphate bu¡er containing 100
mM NaCl. PBBM was used to prepare cell suspension for
fuel cell experiments. The suspensions contained 0.2 N
0.02 g dry cell l31 . All cultures were made at 30‡C.
2.3. Morphological characterization
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 (BioMerieux0 , Marcy
l’Etoile, France) and con¢rmed according to the standard
methods [18]. For scanning electron microscopy, the cells
were collected on a nucleopore ¢lter (pore size 0.2 Wm,
Whatman Scienti¢c, Maidstone, UK) and treated as pre-
viously described [6]. Scanning electron micrographs were
taken using an S-4200 FE-SEM (Hitachi, Tokyo, Japan)
after the specimens were coated with gold [6].
2.4. Physiological and biochemical characterization
Commercial identi¢cation kits (API 20E and API 20
NE, BioMerieux0 ) were used for physiological and bio-
chemical characterization, and the results were analyzed
with APILAB Plus (BioMerieux0 ). A number of addition-
al tests were made. Oxidase activities were determined us-
ing commercial oxidase reagent (BioMerieux0 ) and other
characteristics were determined by standard methods [18].
2.5. 16S rDNA gene sequencing and analysis
Genomic DNA extracted from the isolate was used for
polymerase chain reaction (PCR)-mediated ampli¢cation
of 16S rDNA using 27f and 1492r primer pairs. The am-
plicon was puri¢ed using a puri¢cation kit (GENE-
CLEAN Turbo, Q-BIO gene, Carlsbad, CA, USA) prior
to cloning into Escherichia coli DH5K using the pGEM-
 C.A. Pham et al. / FEMS Microbiology Letters 223 (2003) 129^134 131

T0 easy vector system I (Promega, Madison, WI, USA).

The cloned 16S rDNA was sequenced and analyzed as
described earlier [6]. The 16S rDNA sequence of the iso-
late has been deposited in GenBank under accession num-
ber AF468055.

2.6. Hybridization

Genomic DNA was extracted from isolate PA3 and

A. hydrophila KCTC 2358 to conduct DNA^DNA hybrid-
ization according to the method described by Goodfellow
et al. [19].

2.7. Cyclic voltammetry and MFC

Fig. 1. Scanning electron micrographs of the isolate PA3.

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 bu¡er containing 100 mM NaCl [13]. The cell
suspension contained 0.2 N 0.02 g dry cell l31 . The cell
suspension was used to test electrochemical activities using
cyclic voltammetry and fuel cell techniques as described
earlier [6]. MFCs inoculated with the cell suspension
were fed with PBBM containing yeast extract to give ¢nal
COD values of 250 or 1260 mg l31 . The MFC was con-
nected through a resistance of 500 6 to measure the po-
tential drop between the anode and cathode as current.
2.8. Analyses
Fe(II) was analyzed spectrophotometrically using the
ferrozine method to measure Fe(III) reduction [20]. 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
[21] 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 [22]. Acetate was deter-
mined using a gas chromatograph (Varian 3300) with
£ame ionization detector. Glucose was quanti¢ed using a
PGO-enzymatic glucose kit (Young-dong Pham. Co.,
Seoul, Korea).
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 [1]. Fe(III)-reducing colonies
were picked and transferred to PBBM with acetate and
ferric citrate as the electron donor and acceptor, respec-
tively.
3.2. Morphological and physiological characterization
The isolate PA3 showed a Gram-negative reaction. Cells
were straight rod-shaped, 2.3^2.5 Wm long, and 0.5^0.7 Wm
wide (Fig. 1). On nutrient agar, colonies were bu¡, 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 physiolog-
ical and biochemical tests, including the commercial iden-
ti¢cation kits (API 20E and API 20 NE).

3. Results and discussion

3.1. Isolation of PA3

An isolate, PA3, was selected among the Fe(III)-reduc-
ing colonies on a solid medium containing ferric pyro-
phosphate as the electron acceptor. Fe(III)-reducing colo-
nies were recognized easily on the solid medium as their
Fig. 2. Phylogenetic position of the isolate Aeromonas sp. PA3 with the
related taxa. Similarity and distance matrices were calculated with Clus-
tal W (version 1.8). The phylogenetic tree was constructed based on
available 16S rRNA sequences. The scale bar represents the expected
number of changes per sequence position.

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3.3. Phylogenetic analysis and DNA^DNA hybridization

The sequence similarity of the 16S rRNA gene was com-

pared with those of reference organisms obtained from
GenBank data libraries. The result revealed that the iso-
late 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 identi¢ed as a strain of A. hydro-
phila, designated as A. hydrophila PA3 based on morpho-
logical, biochemical and physiological characteristics as Fig. 3. CVs of an A. hydrophila PA3 cell suspension grown under anaer-
obic conditions with (dotted line) or without (solid line) ferric citrate as
well as 16S rDNA sequence homology.
an electron acceptor.

3.4. Utilization of electron donors and acceptors by

A. hydrophila PA3 bic conditions or under anaerobic conditions without fer-
ric citrate did not show the electrochemical activity (Fig.
A. hydrophila PA3 was cultivated for 5 days using 3). The CV obtained showed di¡erent shapes of oxidation
PBBM containing various electron donor^acceptor combi- and reduction peaks with an apparent redox potential of
nations, and the cellular protein content and the utiliza- around 50 mV against the Ag/AgCl reference electrode.
tion of electron donors and acceptors were measured. This asymmetry shows that the redox reaction of the cell
Cellular protein of 0.36 N 0.03 g l31 was produced from suspension is a quasi-reversible reaction. c-Type cyto-
the cultures made on non-fermentable electron donors chromes were found in A. hydrophila ATCC7966 grown
such as acetate, glycerol and hydrogen without electron under anaerobic conditions with ferric citrate [23]. It is
acceptor. Yeast extract (1 g l31 in PBBM) is believed to plausible that the electrochemical activity observed in
support the bacterial growth. Under similar conditions, this organism is due to the c-type cytochromes observed
glucose and pyruvate produced cellular protein contents in A. hydrophila ATCC7966.
of 1.29 and 0.42 g l31 , respectively. This result shows It is interesting to note that electrochemical activity was
that the bacterium can ferment glucose and pyruvate. observed in the cell suspension of S. putrefaciens grown
Good growth was observed on glucose with any of the under anaerobic conditions without Fe(III) [11], while
electron acceptors used. With pyruvate as the electron A. hydrophila PA3 was electrochemically active only
donor the bacterium reduced Fe(III), nitrate and sulfate when grown under anaerobic conditions with Fe(III). If
with cellular protein contents over 1 g l31 . Acetate sup-
ported 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 pro-
tein 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 ace-
tate with nitrate or oxygen as the electron acceptor, but
not with Fe(III). It is not clear if A. hydrophila PA3 con-
sumes acetate in an MFC environment.

3.5. Electrochemical activity
Washed cell suspensions of A. hydrophila PA3 were used
to determine the electrochemical activity using cyclic vol-
tammetry. Oxidation/reduction peaks were observed in the
cyclic voltammogram (CV) of cells grown under anaerobic
conditions with ferric citrate, but cells grown under aero-
Fig. 4. CVs of anaerobically grown A. hydrophila PA3 cell suspension
treated under di¡erent conditions after 30 min exposure to air. Full
line, aeration for 30 min; dotted line, 20 min N2 gassing after aeration;
small-dash line, incubation for 20 min with 20 mM NaNO3 after aera-
tion; dash-dotted line, incubation for 20 min with 20 mM NaNO3 and
20 mM glycerol after aeration; and long-dash line, incubation for
20 min with 20 mM NaNO3 and 20 mM glycerol in an electrochemical
cell poised at 200 mV after aeration.

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 C.A. Pham et al. / FEMS Microbiology Letters 223 (2003) 129^134 133

the electrochemical activity is related to the localization of

c-type cytochromes to the cell surface [13], these two or-
ganisms might have di¡erent 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 e¡ects of exposure to air
on the electrochemical activity. The cell suspension grad-
ually 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 nitro-
gen, 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, fol- Fig. 6. Current generation by cell suspension of A. hydrophila PA3 in
lowed by incubation with a combination of glycerol and MFCs with di¡erent fuel concentrations. The fuel cell was run with a
resistance of 500 6. Yeast extract with a COD value of 250 mg l31
nitrate, and with nitrate. These results revealed that elec-
(dotted line) or 1260 mg l31 (solid line) was injected as fuel to monitor
trochemical activity is reversibly inactivated when cells are the current generation.
exposed to air.
A similar cell suspension was tested for its ability to
generate current in a fuel cell-type electrochemical cell with acetate as fuel, current was not increased. This result
(Fig. 5). When the fuel cell was inoculated with the cell is consistent with the previous results. The isolate did not
suspension, the current was maintained at a background consume acetate under Fe(III)-reducing conditions,
level of less than 0.1 mA, and increased rapidly to over though acetate is oxidized under aerobic conditions or
1.8 mA when yeast extract as fuel (250 mg l31 as COD) under denitrifying conditions. It is not clear what was
was supplied. After the initial peak, the current decreased the function of this isolate in the MFC fed with acetate.
with a shoulder ¢nally to the background level in around The bacterium might participate in the process converting
7 h. Similar current-generation patterns were observed, fuel into electricity as a member of the microbial consor-
and explained as due to the current generation being lim- tium.
ited not by the biological reaction but by other factors, After MFCs inoculated with the cell suspension were
such as oxygen supply to the cathode and proton perme- run for 5 days, the anode was removed, and observed
ability through the membrane [24,25]. Repeated fuel sup- under a scanning electron microscope (Fig. 7). The elec-
ply gave similar responses. When the current dropped to trode was covered with bacterial cells of short rods with a
the background level, the COD value was less than 5 mg similar shape as shown in Fig. 1. The current reached the
l31 . When fuel was supplied in a higher concentration of maximum immediately after the addition of fuel, and the
1260 mg l31 as COD, the maximum current of 0.3 mA was electron microscopic observation showed that the elec-
generated and the current generation lasted more than 17 h trode was covered with bacterial cells with uniform shape.
(Fig. 6). It is interesting to note the formation of a bio¢lm-like
When the fuel cell containing the cell suspension was fed

Fig. 5. Current generation of single-strain isolate PA3 in an MFC. The

fuel cell was connected through a resistance of 500 6. Yeast extract Fig. 7. Scanning electron micrograph of the anode from the MFC inoc-
with a COD value of 250 mg l31 was injected as fuel at the points indi- ulated by cell suspension of A. hydrophila PA3. The electrode was re-
cated by arrows. moved after 5 days of operation with yeast extract as fuel.

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134 C.A. Pham et al. / FEMS Microbiology Letters 223 (2003) 129^134
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.
Acknowledgements
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.
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