Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism.
ABSTRACT A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.
- SourceAvailable from: Feng Zhao[Show abstract] [Hide abstract]
ABSTRACT: Microorganisms capable of generating electricity in microbial fuel cells (MFCs) have gained increasing interest. Here fourteen exoelectrogenic bacterial strains were isolated from the anodic biofilm in an MFC before and after copper (Cu) shock load by Hungate roll-tube technique with solid ferric (III) oxide as an electron acceptor and acetate as an electron donor. Phylogenetic analysis of the 16S rRNA gene sequences revealed that they were all closely related to Enterobacter ludwigii DSM 16688T within the Enterobacteriaceae family, although these isolated bacteria showed slightly different morphology before and after Cu shock load. Two representative strains R2B1 (before Cu shock load) and B4B2 (after Cu shock load) were chosen for further analysis. B4B2 is resistant to 200 mg L-1 of Cu(II) while R2B1 is not, which indicated the potential selection of the Cu shock load. Raman analysis revealed that both R2B1 and B4B2 contained c-type cytochromes. Cyclic voltammetry measurements revealed that strain R2B1 had the capacity to transfer electrons to electrodes. The experimental results demonstrated that strain R2B1 was capable of utilizing a wide range of substrates, including Luria-Bertani (LB) broth, cellulose, acetate, citrate, glucose, sucrose, glycerol and lactose to generate electricity, with the highest current density of 440 mA·m-2 generated from LB-fed MFC. Further experiments indicated that the bacterial cell density had potential correlation with the current density.PLoS ONE 11/2014; 9(11):e113379. · 3.53 Impact Factor
- Materials Research Bulletin 01/2015; 61:76–82. · 1.97 Impact Factor
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ABSTRACT: Dissimilatory metal-reducing bacteria, such as Geobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption of imcH, encoding an inner membrane c-type cytochrome, eliminated the ability of G. sulfurreducens to reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but the imcH mutant retained the ability to reduce Fe(III) oxides with potentials of ≤-0.1 V versus SHE. The imcH mutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to -0.1 V versus SHE triggered exponential growth. At potentials of ≤-0.1 V versus SHE, both the wild type and the imcH mutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not trigger imcH mutant growth. These results demonstrate that G. sulfurreducens possesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments where Geobacter is found. Insoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein in Geobacter sulfurreducens which is required only for electron transfer to high-potential compounds, and we show that G. sulfurreducens has the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell. Copyright © 2014 Levar et al.mBio 10/2014; 5(6). · 6.88 Impact Factor
Vol. 60, No. 10
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1994, p. 3752-3759
Copyright © 1994, American Society for Microbiology
Geobactersulfurreducens sp. nov., a Hydrogen- and Acetate-
Oxidizing Dissimilatory Metal-Reducing Microorganism
FRANK CACCAVO, JR.,1 DEBRA J. LONERGAN,2 DEREK R. LOVLEY,2 MARK DAVIS,3
JOHN F. STOLZ,2 AND MICHAEL J. McINERNEYl*
Department ofBotany and Microbiology, University of Oklahoma, Norman, Oklahoma 730191; Department of
Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 152823; and Water Resources Division,
U.S. Geological Survey, Reston, Virginia 220922
Received 7 March 1994/Accepted 29 July 1994
A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a
hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an
obligately anaerobic, nonfermentative, nonmotile, gram-negative rod. PCA grew in a defined medium with
acetate as an electron donor and ferric PP1, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA,
fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction
of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutryate,
isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA
did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor.
Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were
characteristic ofc-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta
subgroup of the proteobacteria. Its closest known relative is Geobacter metalireducens. The ability to utilize
either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition
to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new
species name, Geobacter su(furreducens, is proposed.
Dissimilatory iron-reducing microorganisms can gain energy
to support growth by coupling the oxidation of organic acids,
alcohols, H2, or aromatic compounds to the reduction of a
variety of metals (26). Recent studies with both pure cultures
and sediments demonstrate that sulfur- and sulfate-reducing
bacteria also have the potential to enzymatically reduce Fe(III)
(11). The marine, sulfur-reducing microorganism Desulfu-
romonas acetoxidans is closely related to Geobacter metallire-
ducens phylogenetically and conserves energy for growth by
coupling the oxidation of organic acids and alcohols to the
reduction of Fe(III) (41). Several sulfate-reducing bacteria can
enzymatically reduce Fe(III) and U(IV) but are unable to grow
with these metals as a terminal electron acceptor (34, 35). A C3
cytochrome purified from Desulfovibrio vulgaris reduces U(VI)
and amorphous iron oxide (30). Lipid analysis of a salt marsh,
siderite, concretion formation found that organisms similar to
G. metallireducens and Shewanella putrefaciens were not abun-
dant in the Fe(III)-reducing zone, while organisms similar to
Desulfovibrio species were enriched in the concretions (11).
Sulfate-reducing bacteria are abundant in Fe(III)-reducing
zones of deep aquifers in the Atlantic coastal plain (27).
Previous studies of freshwater and marine dissimilatory
Fe(III)- and Mn(IV)-reducing bacteria have generated a tro-
phic model that describes the complete mineralization of
complex organic matter with Fe(III) as the sole electron
acceptor (26). In this model, the primary fermentation prod-
ucts are acetate and H2. Acetate is oxidized to CO2 by Fe(III)
reducers with a metabolism like that of G. metallireducens (28,
29), strain 172 (27), or D. acetoxidans (41). The reduction of
Fe(III) can be coupled to H2 or lactate oxidation by organisms
similar to S. putrefaciens (33), Pseudomonas sp. (3), or strain
*Corresponding author. Phone: (405) 325-6050. Fax: (405) 325-
BrY (9). To date, no organism which can couple the oxidation
of both acetate and hydrogen to the reduction of Fe(III) has
This study reports the isolation of a dissimilatory iron-,
cobalt- and sulfur-reducing microorganism that can use both
acetate and hydrogen as electron donors. The unique physiol-
ogy of strain PCA expands our understanding of the trophic
groups present in Fe(III)-reducing environments and further
substantiates the interrelationship between the biogeochemical
cycles of iron and sulfur in both freshwater and saline anaer-
MATERIALS AND METHODS
Source of the organism. Inocula for enrichments were
collected by taking cores of a hydrocarbon-contaminated ditch
near Norman, Okla. The top 3 cm of sediment was extruded
into glass tubes, stoppered, and immediately transported back
to the laboratory, where it was placed in an anaerobic chamber.
Media and cultivation. Standard anaerobic techniques were
used throughout the study (5, 7). All anaerobic media were
boiled and cooled under a constant stream of 80% N2-20%
C02, dispensed into aluminum-sealed culture tubes under the
same gas phase, capped with butyl rubber stoppers, and
sterilized by autoclaving (121°C, 20 min). Additions to sterile
media, inoculation, and sampling were done by using syringes
and needles (5). All incubations were at 35°C in the dark.
The basal medium contained the following (in grams per
liter of deionized H20): NaHCO3, 2.5; NH4Cl, 1.5; KH2PO4,
0.6; KCl, 0.1; vitamins, 10 ml; and trace minerals, 10 ml (29).
For enrichment of iron-reducing bacteria, sodium acetate (10
mM) was added as the electron donor and soluble ferric PP, (3
g/liter) was added as the electron acceptor. The enrichment
was initiated by adding 1.0 g (wet weight) of anaerobic
sediment to sterile tubes containing the acetate-Fe(III) basal
METAL- AND SULFUR-REDUCING MICROORGANISM
medium (10 ml) inside the anaerobic chamber. The head-
spaces of enrichment tubes were evacuated and replaced with
80% N2-20% CO2 immediately after inoculation.
The basal medium was modified to test for the use of
different electron donors or acceptors. The use of different
electron acceptors was tested with sodium acetate (10 to 30
mM) as the sole electron donor. Duplicate determinations
were done for each electron acceptor. The experiment was
repeated if replicates differed from the mean by more than 10
to 15%. Negative controls for the use of alternate electron
acceptors did not contain acetate. Inocula (10%) for electron
acceptor experiments were taken from cultures grown with
basal medium, acetate, and ferric citrate. The use of poorly
crystalline Fe(III) oxide (ca. 100 mM) and Fe(III) citrate (20
mM) as alternative reducible forms of Fe(III) was determined
by monitoring the increase in cell number and Fe(II) over
time. Synthetic MnO2 (30 mM) (29) was used to evaluate the
potential for Mn(IV) reduction, and the production of Mn(II)
was monitored over time. Sodium nitrate, sodium thiosulfate,
sodium sulfite, sodium sulfate, fumaric acid, or malic acid was
added to the basal medium from anaerobic stock solutions to
provide 20 mM. Growth on these acceptors was monitored by
measuring the increase in cell density at anA54. U(VI) was
provided as uranyl chloride (0.5 mM); the loss of visible color
in the medium and the accumulation of a black precipitate of
reduced uranium (uraninite) were monitored over time as
evidence of U(VI) reduction. Elemental sulfur was added
aseptically as sublimed sulfur flowers (1.0 g/liter) to sterile
medium under a stream of sterile 80% N2-20% CO2. The
production of sulfide and an increase in cell numbers were
monitored over time. Co(III)-EDTA medium was prepared as
described previously (18). The decrease of Co(III) and in-
crease in cell numbers over time were monitored as indicators
of reduction and growth, respectively.
The use of different electron donors was tested with the
basal medium without acetate and with ferric citrate as the
electron acceptor. The electron donors were added to the basal
medium from sterile anaerobic stocks to give a final concen-
tration of 10 to 30 mM. Ten milliliters of a 90% H2-10% CO2
mixture was added to the gas phase to test for the use of H2 as
an electron donor. Inocula (10%) for electron donor experi-
ments were from acetate-ferric citrate cultures
transferred and grown in basal medium with ferric citrate so
that any acetate which was transferred was used before inoc-
ulation into experimental tubes. The use of a compound as an
electron donor was determined by comparing the production
of Fe(II) to that by negative controls that lacked the compound
and to positive controls that contained 30 mM sodium acetate.
Duplicate determinations were done for each donor. Stoichi-
ometries for the extent of Fe(III) reduction during acetate or
H2 oxidation were determined by measuring Fe(II) production
when acetate (4 mM) or H2 (15 kPa) was provided as the sole
The optimum temperature for Fe(III) reduction by PCA was
determined by growing PCA in basal medium with acetate and
ferric citrate at different temperatures and by monitoring the
production of Fe(II) over time. Strain PCA's ability to reduce
Fe(III) under saline conditions was determined by growing it
in basal medium with acetate, ferric citrate, and no NaCl or
MgCl2 (FW), with 10 g of NaCl and 5 g of MgCl2 per liter (1/2
SW), or with 20 g ofNaCl and 10 g of MgCl2 per liter (SW) and
by determining the concentration of Fe(II) produced after 2
and 5 days.
16S rRNA gene sequencing. Cells from an actively growing
culture of PCA were harvested by centrifugation and stored at
-70°C. The method devised by Murray and Thompson (37), as
described by Ausubel et al. (2), was used to isolate DNA from
the frozen cell pellet. The DNA was treated with RNase and
diluted 10-fold prior to amplification. The 16S rDNA was
amplified using primer 50F (5'-AACACATGCAAGTCGAA
CG-3') (22) and eubacterial primer 1492R (5'-GGTTACCT
TGTTACGACTT-3') (13, 44). The 16S rDNA PCR product
was resuspended in water after purification with a Wizard PCR
Prep System (Promega Corp., Madison, Ohio). Automated dye
dideoxy terminator sequencing of both strands was performed
on a model 373A DNA sequencing system (Applied Biosys-
tems, Foster City, Calif.) by the Michigan State University
Sequencing Facility. Oligonucleotides complementary to the
conserved regions of the eubacterial 16S rDNA were chosen to
prime the sequencing reactions and were synthesized on either
a model 394 DNA-RNA synthesizer or a model 380B DNA
synthesizer (Applied Biosystems). Sequence alignments were
either performed manually or were obtained from the Ribo-
somal Data Base Project (23). Evolutionary distances were
computed as described previously (21) and were used to
construct a distance tree by the least-squares algorithm de-
scribed by DeSoete (12). The topology of the phylogenetic
analysis was confirmed by the maximum likelihood method
(14, 23, 39). The 16S rRNA sequence of Geobacter sulfurredu-
cens PCA has been deposited in GenBank.
Cytochrome content. Cytochrome analysis was done with
cells grown in basal medium with acetate (30 mM) and Fe(III)
citrate. A dithionite-reduced minus air-oxidized difference
spectrum of whole cells was obtained on a Shimadzu 2101PC
spectrophotometer. Cells (3.51 mg of protein per ml) were
resuspended in 20 mM piperazine-N,N'-bis(2-ethanesulfonic
acid) (PIPES) buffer at pH 7.0.
Electron microscopy. Cells grown in basal medium with
acetate and Fe(III) citrate were initially fixed by the addition of
glutaraldehyde directly into the anaerobic tubes (final concen-
tration, 2.5%). The cells were then transferred to evacuated
centrifuge tubes anoxically and were spun at 4,000xg for 20
min. The spent medium was carefully removed to avoid
precipitation of soluble iron. The cell pellet was then washed
three times in 0.1 M cacodylate buffer and was treated with 1%
osmium tetroxide. After dehydration in an ethanol series and
propylene oxide, the cells were embedded in Spurr's low-
viscosity medium. Thin sections were stained with uranyl
acetate and lead citrate and observed on a Philips 201 trans-
mission electron microscope at 60 kV.
Analytical techniques. Fe(III) reduction was monitored by
measuring the accumulation of Fe(II) over time. The amount
of Fe(II) solubilized after 15 min in 0.5 N HCI was determined
with ferrozine as previously described (29). Co(III)-EDTA
reduction was determined by measuring the loss of Co(III)
over time spectrophotometrically at 535 nm. Sulfide was
measured colorimetrically (42). Cell numbers in cultures grow-
ing with Fe(III) or Co(III) were determined by a modification
of the epifluorescence microscopy technique (20) as previously
described (29). Cell densities in cultures without metals were
monitored spectrophotometrically at 540 nm. Acetate was
quantified by high-pressure liquid chromatography (1). H2 was
measured by gas chromatography (33).
Isolation. The primary enrichment was transferred three
times (10% inoculum) into the basal medium with sodium
acetate and ferric PP1. The Fe(III) in the initial enrichment
tube was completely reduced within 3 days. Fe(III) reduction
was observed as a change in the medium color from yellow to
green to a clearing of the medium and the formation of a white
VOL. 60, 1994
CACCAVO ET AL.
FIG. 1. Transmission electron micrograph of a thin section of
strain PCA. Bar, 0.2 ,.m.
precipitate. After the third transfer, the enrichment was seri-
ally diluted to extinction in this medium. Subsequent transfers
became completely reduced within 1 day. The highest dilution
which was positive for Fe(III) reduction (10-8) was again
serially diluted in this medium. The highest dilution which was
positive for Fe(III) reduction (10-9) completely reduced the
Fe(III) within 7 days and was then streaked for isolation on
anaerobic ferric PP1
addition of a final concentration of 1.5% (wt/vol) purified agar
to the basal medium with acetate and ferricPPi.A pure culture
was obtained by repeatedly restreaking isolated colonies onto
agar slants until an isolate of uniform colony and cell morphol-
ogy was obtained. Colonies formed on agar slants within 7
days. Colonies were pinpoint and turned white along with the
surrounding agar when the Fe(III) in the medium was reduced.
Cells from isolated colonies were single straight rods 2 to 3 p.m
in length by 0.5 p.m in width (Fig. 1). They were nonmotile and
stained gram negative. No spores were observed in wet mounts
by phase-contrast microscopy. The obligately anaerobic micro-
organism was designated strain PCA. The culture was main-
tained on basal medium with acetate and ferric citrate.
Electron donors and acceptors. Strain PCA grew by the
oxidation of acetate coupled to the reduction of Fe(III) PP,
(Fig. 2A), poorly crystalline Fe(III) oxide (Fig. 2B), or Fe(III)
citrate (data not shown). The increase in cell numbers coin-
cided with the production of Fe(II) in the presence of acetate
and each of the electron acceptors. No Fe(III) reduction or cell
growth occurred in the absence of acetate. With Fe(III) citrate
as the electron acceptor, the ratio of Fe(II) produced to
acetate consumed was 6.8 + 0.4 (mean ± standard deviation
for five cultures). Considering that some acetate is likely to
have been incorporated for cell synthesis, these results suggest
agar slants, which were made by the
FIG. 2. Growth of PCA in basal medium with acetate
electron donor and ferricPPi(A) or poorly crystalline ferric oxide (B)
as an electron acceptor.
that strain PCA oxidizes acetate according to the following
equation: CH3COO- +
8Fe(II) + 9H+. Growth of PCA on ferric oxyhydroxide
resulted in the formation of a black, magnetic precipitate
which was presumably magnetite.
Strain PCA also coupled the oxidation of hydrogen to the
reduction of Fe(III) citrate (Fig. 3A and B). The addition of
Casamino Acids (0.25 g/liter) slightly enhanced growth and
Fe(II) production. No growth or Fe(III) reduction occurred
with Casamino Acids alone or in controls which lacked H2. For
each mole of H2 consumed, 1.7 ± 0.2 mol (mean ± standard
deviation for five cultures) of Fe(II) was produced. This
suggests that H2 was oxidized according to the following
equation: H2 + 2Fe(II) -> 2H+ + 2Fe(HI).
Strain PCA did not use sulfur, glucose, lactate, fumarate,
propionate, butyrate, isobutryate, isovalerate, succinate, yeast
extract, phenol, benzoate, ethanol, propanol, or butanol with
Fe(III) as an electron donor (Table 1). The small amounts of
Fe(II) produced in cultures with glucose, lactate, malate,
propanol, methanol, and yeast extract were similar to those
found in cultures without an electron donor and probably
represent the amounts of Fe(II) produced from the small
amounts of acetate in the inocula. No Fe(II) was produced
with many of the electron donors tested, suggesting that these
compounds inhibited the ability of PCA to use the small
amount of acetate in the medium.
Strain PCA coupled the oxidation of acetate to the reduction
of Co(III)-EDTA (Fig. 4). An increase in cell numbers over
time was followed by a concomitant decrease in the concen-
tration of Co(III). Growth and reduction of Co(III) were
electron donor dependent since there was not an increase in
cell numbers or a decrease in Co(III) in the absence of acetate.
Fel) + acetate
Fe(II) no acetate
APPL. ENVIRON. MICROBIOL.
METAL- AND SULFUR-REDUCING MICROORGANISM
FIG. 3. Growth (A) and Fe(II) production
PCA was grown with Fe(III) citrate as an elec
donor (ND), hydrogen (H2), Casamino Acids ((
Casamino Acids (H2 + CA) as an electron dor
Strain PCA did not use Mn(IV) or U(VI
Strain PCA was capable of growth
electron donor and elemental sulfur as
acceptor (Fig. 5). Cell numbers and su
increased over time only when acetate
medium. Strain PCA grew with malate or
TABLE 1. Electron donors used for Fe(III'
FIG. 4. Growth and Co(III)-EDTA reduction by PCA with acetate
as an electron donor.
electron acceptors. No growth occurred in the absence of
acetate. PCA was not capable of growth with oxygen, nitrate,
sulfate, sulfite, or thiosulfate with acetate as an electron donor.
There was no increase in absorbance for any of these acceptors
with or without acetate.
The optimum temperature for Fe(III) reduction was 35°C.
No Fe(II) production was observed at 4 or 50°C. Strain PCA
reduced Fe(III) after 2 days in FW medium. Fe(III) reduction
was observed after 5 days in 1/2 SW medium but not in SW
Cytochromes. The difference spectra of whole cells showed
the presence of c-type cytochromes (Fig. 7). The reduced
cytochromes had absorbance peaks at 552, 522, and 419 nm.
Phylogeny. Comparison of the 16S rRNA sequence of strain
PCA with the 16S rRNA sequences of selected representatives
of the proteobacteria places it in the delta subdivision of the
proteobacteria (Fig. 8). Analysis of the secondary structure
andsignaturenucleotides confirmed thephylogenetic place-
ment. Strain PCA shares a 95% similarity with G. metalliredu-
cens and an 89% similarity with D. acetoxidans.
(B) by PCA with H2.
ctron acceptor and no
CA), or hydrogen plus
as electron accep-
with acetate as an
the sole electron
was present in the
fumarate (Fig. 6) as
) reduction by PCA
after 5 days (mM)
Strain PCA is the first bacterium described for pure culture
that couples the oxidation of acetate or hydrogen to the
reduction of Fe(III). These results suggest that Fe(III)-reduc-
ing bacteria cannot be broadly distinguished phylogenetically
or metabolically as acetate oxidizing (delta proteobacteria
[Geobacter species]) or hydrogen oxidizing (gamma proteobac-
teria [Shewanella species]) as in previous models (26). The use
of either acetate or hydrogen as an electron donor by a single
1 02030 40
FIG. 5. Growth and H2S production by PCA with acetate as an
electron donor and S as an electron acceptor.
VOL. 60, 1994
CACCAVO ET AL.
FIG. 6. Growth of PCA with acetate as
fumarate or malate as an electron acceptor.
species has been previously demonstra
reducing (45) and methanogenic (4) bactc
determined whether, under Fe(III)-reduc
can compete with G. metallireducens
organisms such as Shewanella species for H2. Since these
experiments were done using Fe(III) citrate as the electron
acceptor, it is not known ifPCA can use CO2 as the sole carbon
source and thus grow autotrophically with H2 and Fe(III).
Fe(III) and sulfur reduction. The ability of strain PCA to
couple the oxidation of acetate to the reduction of either
Fe(III) or S° provides further evidence for the interrelation-
ship between the microbial iron and sulfur cycles in anaerobic
environments. Previous studies have shown that the metal-
reducing microorganisms S. putrefaciens (40) and strain BrY
(9) reduce thiosulfate in the absence of a metal. The sulfur-
reducing microorganism D. acetoxidans was recently shown to
grow by dissimilatory reduction of Fe(III) and Mn(IV) (41).
Reduced sulfur compounds can be microbiologically reoxi-
dized using iron oxide or manganese as terminal electron
acceptors, and certain species may be involved in the cycling of
both elements (32, 43). Sulfate-reducing bacteria have been
shown to enzymatically reduce, but not grow with, Fe(III),
U(VI) (34, 35), and Cr(VI) (31) as the terminal electron
acceptor. The abundance of sulfate-reducing bacteria in the
Fe(III)-reducing zone of deep aquifers in the Atlantic coastal
fumarate + acetate
malate + acetate
an electron donor and
Lted in the sulfate-
eria. It remains to be
ing conditions, PCA
for acetate or with
FIG. 7. Difference spectrum of whole cells of G. sulfurreducens. Abs, absorbance.
APPL. ENvIRON. MICROBIOL.
METAL- AND SULFUR-REDUCING MICROORGANISM
FIG. 8. Phylogenetic tree showing the relationship between G.
sulfurreducens and representative delta proteobacteria. The sequence
of Escherichia coli was used as the outgroup. The tree was inferred.
The bar represents a 0.1-nucleotide change per position, i.e., 0.1
evolutionary distance unit.
plain and in salt marsh siderite concretions in which Fe(III)-
reducing bacteria similar to G. metallireducens and S. putrefa-
ciens were not detected suggests an in situ correlation between
microbially mediated iron and sulfur geochemistry. These
observations suggest that metal reduction may be an important
ecological function of sulfur- and sulfate-reducing bacteria and
that microorganisms physiologically defined as dissimilatory
metal-reducing bacteria such as Geobacter species, Shewanella
species, and strain BrY (26) are certainly not the only, and
possibly not even the dominant, metal-reducing bacteria in
some anaerobic environments. The evidence presented above
enables us to expand the Fe(III)-reducing food chain model to
include metal- and sulfur-reducing microorganisms like strain
PCA and D. acetoxidans, as well as sulfate-reducing microor-
ganisms which can enzrmatically reduce metals.
Co(III) reduction. 6Co(III) is a nuclear by-product that is
often codisposed with the synthetic chelator EDTA. The
Co(III)-EDTA complex is extremely stable to chemical reduc-
tion and exchange reactions and is thus highly mobile in
saturated subsurface environments. The dissimilatory reduc-
tion of Co(III)-EDTA by strain PCA represents a novel mode
of microbial metabolism. The fact that growth is coupled to
cobalt reduction suggests that the reduction of cobalt is an
energy-yielding process. Biologically, cobalt is found in vitamin
Bl2 and other corrin ring molecules. The biologically active
forms of these molecules function in hydrogen rearrangement
and methyl transfer reactions (25). Cobalt was not previously
thought to undergo biologically mediated oxidation-reduction
reactions, although such a role for cobalt has been proposed in
the formation of methane (6) and acetate (19). Further studies
on the dissimilatory reduction of Co(III)-EDTA will be pub-
lished elsewhere (18). In addition to finding a new biologically
important role for cobalt, this discovery also has important
biotechnological implications. Microbial reduction of Co(III)-
EDTA may limit the far-field migration of 60Co in contami-
nated subsurface environments. Co(II)-EDTA strongly ad-
sorbs to aluminum oxides in soils and subsoils, while Co(III)-
EDTA does not (17). Microbial reduction of Co(III)-EDTA
will increase the likelihood that 60Co will adsorb to subsurface
materials and prevent the far-field migration of this contami-
Isolation. Previously, Fe(III)-reducing bacteria were en-
riched for and isolated with poorly crystalline Fe(III) oxide
serving as the sole terminal electron acceptor (9, 27, 29).
However, this approach presents several problems. Growth on
Fe(III) oxide is slow, and the enrichment process is lengthy.
Since iron oxide is insoluble, it is difficult to perform physio-
logical studies on isolates in this medium, and nitrate is used as
the electron acceptor to obtain isolates. This technique thus
selects for metal-reducing bacteria which are also capable of
reducing nitrate. The use of Fe(III) PP1 facilitated the enrich-
ment and isolation of the Fe(III)-reducing bacterium PCA.
Fe(III) PP1 is a soluble form of ferric iron that contains both
citrate and phosphate as chelating agents. The soluble Fe(III)
is more quickly reduced than insoluble iron oxide (Fig. 2A and
B) (36). Strain PCA was isolated from sediment in approxi-
mately 40 days, while another organism was concurrently
enriched and isolated on acetate and iron oxide in approxi-
mately 90 days (8). Interestingly, PCAwas isolated with Fe(III)
PPi as the sole electron acceptor and was unable to reduce
nitrate, whereas other Fe(III) reducers, such as G. metallire-
ducens, used nitrate. Although Fe(III) PP1 was used success-
fully in the primary enrichments from which strain PCA was
isolated, similar enrichments with phenol or palmitate as the
sole electron donor yielded fermentative isolates that presum-
ably fermented the citrate chelator and reduced the Fe(III) in
a nondissimilatory manner (8). Fe(III) PP1 would therefore be
most useful as a soluble source of Fe(III) in solid isolation
media or in secondary enrichments.
Taxonomic and phylogenetic relationship to Geobacter spe-
cies and Desulfuromonas species. The physiological character-
istics and phylogeny of strain PCA suggest a relationship to
both Geobacter species and Desulfuromonas species. All three
organisms are members of the delta proteobacteria (28, 46)
and are obligately anaerobic, gram-negative rods which con-
tain c-type cytochromes and couple the oxidation of acetate to
the reduction of Fe(III) (15, 24, 28, 38, 41). It is not yet known
whether PCA oxidizes acetate via the citric acid cycle, as do G.
metallireducens (10) and D. acetoxidans (16). Like G. metalli-
reducens (29), PCA is nonmotile, whereas Desulfuromonas
species are motile. Strain PCA is similar to D. acetoxidans in
that it is capable of using Fe(III), So, fumarate, and malate but
not U(VI) as terminal electron acceptors with acetate as the
electron donor. Desulfuromonas species do not use H2 as an
electron donor, as does PCA, and several Desulfuromonas
species use other organic electron donors, while PCA does not
(41). These phenotypic differences and phylogenetic analyses
preclude the inclusion of strain PCA in the genusDesulfuromo-
Phylogenetic analysis of the 16S rRNA sequence of strain
PCA indicated that it is most closely related to G. metalliredu-
cens. Both species are capable of growth by coupling the
oxidation of acetate to the reduction of Fe(III) and Co(III).
However, the two organisms differ in many respects as well.
PCA differs from all previously described metal-reducing
bacteria in its inability to reduce Mn(IV). PCA is able to use
H2 as an electron donor for Fe(III) reduction. PCA does not
use nitrate as an electron acceptor, while G. metallireducens
does (29). G. metallireducens is able to use several alcohols and
aromatic compounds as electron donors for Fe(III) reduction
(29), while PCA cannot. Strain PCA reduced Fe(III) in
medium which contained up to one-half the NaCl concentra-
tion found in seawater, while G. metallireducens can grow only
in freshwater medium (29). On the basis of phenotypic and
phylogenetic characteristics, we propose that strain PCA be
established as the type strain of a new species, Geobacter
Description of Geobacter sulfurreducens sp. nov. Geobacter
sulfurreducens (sul'fer.re.du'cens. L. n. sulfur, sulfur; L. part.
adj. reducens, converting to a different state; N.L. adj. sulfurre-
ducens, reducing sulfur). Rod-shaped, gram-negative cells 2 to
3 by 0.5 ,um, nonmotile, with no spore formation. Strict
anaerobic chemoorganotroph which oxidizes acetate with
Fe(III), S, Co(III), fumarate, or malate as the electron
acceptor. Hydrogen is also used as an electron donor for
Fe(III) reduction, whereas other carboxylic acids, sugars, alco-
hols, amino acids, yeast extract, phenol, and benzoate are not.
Temperature optimum is 30 to 35°C. Cells contain c-type
VOL. 60, 1994
3758CACCAVO ET AL.
cytochromes. Grows in up to one-half the NaCl concentration
Habitat. G. sulfurreducens was enriched from surface sedi-
ments of a ditch in Norman, Okla., with acetate as the electron
donor and ferricPPias the electron acceptor.
Type strain. The type strain of G. sulfurreducens is PCA.
We thank Tom Schmidt, Harry Jenter, Elizabeth Phillips, and Sue
Lootens for technical assistance.
M.D. was supported by the NASA Planetary Biology Intern Pro-
gram. F.C. and M.J.M. were supported by contract DE-F66589ER-
14003 from the Department of Energy.
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