Microbiology (2014), 160, 123–129
DOI 10.1099/mic.0.069930-0
Sulfur oxidation to sulfate coupled with
electron transfer to electrodes by Desulfuromonas
strain TZ1
Tian Zhang, Timothy S. Bain, Melissa A. Barlett, Shabir A. Dar,
Oona L. Snoeyenbos-West, Kelly P. Nevin and Derek R. Lovley
Correspondence
Department of Microbiology, University of Massachusetts, Amherst, MA, USA
Tian Zhang
[email protected]
Received 26 May 2013
Accepted 28 October 2013
Microbial oxidation of elemental sulfur with an electrode serving as the electron acceptor is of
interest because this may play an important role in the recovery of electrons from sulfidic wastes
and for current production in marine benthic microbial fuel cells. Enrichments initiated with a
marine sediment inoculum, with elemental sulfur as the electron donor and a positively poised
(+300 mV versus Ag/AgCl) anode as the electron acceptor, yielded an anode biofilm with a
diversity of micro-organisms, including Thiobacillus, Sulfurimonas, Pseudomonas, Clostridium
and Desulfuromonas species. Further enrichment of the anode biofilm inoculum in medium with
elemental sulfur as the electron donor and Fe(III) oxide as the electron acceptor, followed by
isolation in solidified sulfur/Fe(III) medium yielded a strain of Desulfuromonas, designated strain
TZ1. Strain TZ1 effectively oxidized elemental sulfur to sulfate with an anode serving as the
sole electron acceptor, at rates faster than Desulfobulbus propionicus, the only other organism
in pure culture previously shown to oxidize S6 with current production. The abundance of
Desulfuromonas species enriched on the anodes of marine benthic fuel cells has previously been
interpreted as acetate oxidation driving current production, but the results presented here suggest
that sulfur-driven current production is a likely alternative.
INTRODUCTION
Microbial oxidation of elemental sulfur coupled to electron
transfer to electrodes may be an important process
contributing to the current production of marine sediment
microbial fuel cells (Holmes et al., 2004a, 2004b; Nielsen
et al., 2009; Reimers et al., 2007; Tender et al., 2002) and is
a key reaction in bioelectrical harvesting of electrons from
sulfide wastes (Sun et al., 2009, 2010). Effective conversion
of sulfide to electricity requires microbial catalysis (Lovley,
2006). This is because although sulfide can abiotically
donate electrons to electrodes (Rabaey et al., 2006; Reimers
et al., 2001; Sun et al., 2009), the abiotic reaction typically
only yields two electrons per sulfide, with Su as the primary
product (Lovley, 2006). The extraction of the remaining
three-quarters of electrons potentially available for current
production requires micro-organisms that can oxidize Su to
sulfate with an electrode as an electron donor.
Desulfobulbus propionicus was previously shown to oxidize
Su to sulfate coupled with electron transfer to an electrode
(Holmes et al., 2004a). This organism was chosen for initial
studies on this reaction because micro-organisms closely
related to D. propionicus were abundant on anodes
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
sequence of Desulfuromonas strain TZ1 is JX258673.
069930 G 2014 SGM
harvesting electrical current from marine sediments with
high sulfide content (Holmes et al., 2004b; Tender et al.,
2002) and because D. propionicus was previously shown to
oxidize Su to sulfate with the reduction of Mn(IV) oxide,
another insoluble electron acceptor (Lovley & Phillips,
1994). However, other micro-organisms are often abundant on anodes harvesting electricity in sulfide-containing
environments (Holmes et al., 2004b; Ryckelynck et al., 2005;
Sun et al., 2010; Tender et al., 2002) and their potential role
in current production is not completely understood. For
example, Desulfuromonas species are often the most abundant
micro-organisms colonizing anodes harvesting current from
marine sediments and their presence has been attributed to
their ability to oxidize acetate with current production
(Bond et al., 2002; Holmes et al., 2004b). Here, we report the
discovery of a pure culture of Desulfuromonas that is capable
of elemental sulfur oxidation with current generation.
METHODS
Organism and sediment sources. Desulfobulbus propionicus
(DSM 2032) was obtained from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen and was anaerobically grown at
37 uC in a slightly modified NB basal medium with propionate
(1.5 g l21) as the electron donor and sulfate (3 g l21) as the electron
acceptor as previously described (Holmes et al., 2004a).
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123
T. Zhang and others
(a) 5
(c)
3
Sediment slurry inoculum
ers
Oth
2
Medium exchange
1
3
2
γ -P
160
Time (h)
240
320
400
380
Electron transferred to anode
Sulfate production
414
515
ε-Proteobacteria
2%
Sulfurospirillum
2%
β-Proteobacteria
7%
325
ε-P
248
Sulfurimonas
22 %
Desulfotalea
3%
te
ro
200
74
ria
71
116
te
74
100
ac
ob
mmol /m2 . day
(b) 600
Thiobacillus
24 %
Arcobacter
3 % δ-Proteobacteria
3%
Desulfovibrio
2%
Desulfuromonodales
3%
0
Sediments
1
2
3
cteria
80
teoba
0
Pseudomonas
7%
β-Pro
1
0
Alistipes Clostridium Thalassospira Methylophilus
2%
2%
3%
Sphingobacterium 2 %
2%
Methyloversatilis
Unclassified
2%
9%
rote
oba
cte
ria
mA
4
α-Proteobacteria
4
δ-P
(0–142 h) (142–202 h)(202–245 h)(245–291 h)(291–320 h)
a
teri
bac
o
rote
4
Fig. 1. Enrichment studies with S6 as the electron donor and a graphite anode poised at +300 mV (vs Ag/AgCl) as the sole
electron acceptor. (a) Current production. The initial inoculum was marine sediments diluted with sea water and, where
designated by arrows, the liquid contents of the anode chamber were exchanged with fresh culture medium with additional S6.
(b) Rates of electron transfer to the anode and sulfate production prior to medium exchange. The data shown are representative
of triplicate incubations. The mean standard errors of the current and sulfate measurements were 23 % and 10 %, respectively.
(c) Percentage of 16S rRNA gene sequences in clone libraries of microbial community growth on graphite anode with sulfur
serving as the sole electron donor.
Marine sediments were collected from Boston Harbor, MA and stored
as previously described (Zhang et al., 2010). Seawater was collected
from the same site and stored at 4 uC in the dark. Freshwater medium
(Lovley & Phillips, 1988), which was amended with additional salts
(18 g l21 of NaCl, 5.4 g l21 of MgCl2 and 0.27 g l21 of CaCl2), was
used for enrichment and isolation studies.
Studies with a graphite electrode as the electron acceptor. For
electrode studies, anodes and cathodes were comprised of graphite
stick (65 cm2; Mersen) and suspended in three-electrode, dualchambered microbial fuel cells as previously described (Zhang et al.,
2010). The anode was poised with a potentiostat (ECM8, Gamry
Instruments) at +0.3 V versus Ag/AgCl. In the initial sediment
studies the anode chamber was filled with 200 g of sediment slurry
(sediment/seawater, 1:4 v/v) and the cathode chamber was filled
with 200 ml of seawater. The anode and cathode chambers were
filled with 200 ml of medium for studies with enrichment and pure
culture studies. N2/CO2 (80 : 20) was the gas phase for all studies
and the cathode chamber was continuously flushed with the gas
mixture.
DNA extraction, PCR amplification of 16S rRNA and phylogenetic analysis. DNA was extracted using the FastDNA spin kit (Bio
101). The 16S rRNA genes were amplified by PCR using primers 8F
124
(Eden et al., 1991) and 519R (Lane et al., 1985) for the mixed
microbial community and primers 8F (Eden et al., 1991) and 1492R
(Amann et al., 1990) for Desulfuromonas strain TZ1 as previously
described (Holmes et al., 2004c; Nevin et al., 2005). PCR products
were then purified with a gel extraction kit (Qiagen), and clone
libraries were constructed with a TOPO TA cloning kit (Invitrogen)
according to the manufacturer’s instructions. For each clone library,
96 clones were sequenced with the M13F primer at the University of
Massachusetts Sequencing Facility. Completed gene sequences were
aligned to the greengenes database and classified using the NCBI
database and BLAST search matches for percentage identity. Nucleotide
sequences were aligned using CLC Sequence Viewer (CLCBio) and
phylogenetic trees were inferred using SeaView (Gouy et al., 2010).
Branching order and distances were determined using parsimony and
neighbour-joining algorithms. Bootstrap values were calculated for
100 replicates.
Electron microscopy. The Gram type was determined using Gram
staining and bright-field microscopy as previously described (Nevin
et al., 2005). For transmission electron microscopy, strain TZ1
cultures were placed on 300-mesh carbon-coated copper grids,
incubated for 15 min and then stained with 2 % aqueous uranyl
acetate. Cells were observed using a JEOL 100S transmission electron
microscope at an accelerating voltage of 80 kV. Images were taken
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Microbiology 160
Sulfur oxidation in Desulfuromonas
(a)
(b)
76
97
Pelobacter venetianus (U41562)
Pelobacter acetylenicus (X70955)
Pelobacter carbinolicus (U23141)
Desulfuromonas chloroethenica (U49748)
Desulfuromonas michiganensis (AF357914)
Desulfuromusa bakii (X79412)
Desulfuromusa kysingii (X79414)
Desulfuromonas acetoxidans (AY187305)
88
1000 nm
61
76
95
99
80
91
Desulfuromonas thiophila (Y11560)
Desulfuromonas palmitatis (U28172)
Desulfuromonas strain TZ1T (JX258673)
Desulfuromonas alkaliphilus (DQ309326)
Geobacter hydrogenophilus (U46860)
Geobacter metallireducens (L07834)
Geobacter sulfurreducens (U13928)
Pelobacter propionicus (X70954)
Desulfovibrio desulfuricans (AF098671)
Desulfovibrio vulgaris (EU882409)
Shewanella putrefaciens (X82133)
0.10
Fig. 2. Morphology and phylogenetic analysis of Desulfuromonas strain TZ1. (a) Transmission electron micrograph of strain TZ1
grown on medium with ferric citrate (55 mM) provided as the electron acceptor and acetate (10 mM) as the electron donor as
previously described (Nevin et al., 2005). (b) Neighbour-joining phylogenetic tree of the 16S rRNA sequences of strain TZ1 and
closest relatives supported by bootstrap analysis. The same topology was obtained from parsimony and maximum-likelihood
analyses. The GenBank accession number for the 16S rRNA of Desulfuromonas strain TZ1 is JX258673.
digitally using the MaxIm-DL software and analysed using Image J
(http://rsbweb.nih.giv/ij/index.html).
RESULTS AND DISCUSSION
Microbial communities associated with anaerobic
S‡ oxidation coupled to electrode reduction
In order to search for organisms capable of oxidizing Su with
an electrode as the electron acceptor, marine sediments
collected from Boston Harbor were slurried with seawater
under anaerobic conditions and added to the anode
chambers of bioelectrical systems, comparable to those
previously described with a graphite anode poised at
+300 mV (vs Ag/AgCl) as the sole electron acceptor
(Zhang et al., 2010). In sulfide-based microbial fuel cells,
the abiotic electrocatalysis of H2S into oxyanions of sulfur
might allow the organisms to immediately reuse these to
produce more sulfide by electron abstraction from organic
substrate as a cyclic process within the electrode biofilms.
However, by setting the anode potential at +300 mV vs Ag/
AgCl, the abstraction of electrons from sulfur by sulfuroxidizing species forms sulfate that cannot be reduced again
by the micro-organisms of the anode biofilm. The sediment
slurries were amended with 0.32 g l21 of colloidal sulfur as
an electron donor. Current was generated, accompanied
with concurrent production of sulfate (Fig. 1a, b). When
current production declined, the medium in the anode
chamber was successively replaced with Su-containing
http://mic.sgmjournals.org
medium which was the previously described (Lovley &
Phillips, 1988) freshwater medium amended with extra salts.
Current production continued. With successive enrichment
the stoichiometry approached the production of 1 mol of
sulfate produced per 6 moles of electrons harvested for Su
oxidation to sulfate with an electrode serving as the sole
electron acceptor (Fig. 1), but was always less, presumably
because some electrons from Su oxidation were required to
fix carbon dioxide for cell growth.
After the fourth medium exchange, the biofilm on the anode
was scrapped off and 16S rRNA gene sequences analysed as
previously described (Holmes et al., 2004b). The sequences
recovered were predominantly Proteobacteria with bProteobacteria and e-Proteobacteria each accounting for
more than 25 % of the total sequences (Fig. 1c). Within
the b-Proteobacteria, the majority of the clones were most
similar to Thiobacillus (Fig. 1c). Thiobacillus species are well
known as chemolithotrophs that can anaerobically oxidize
sulfur with the reduction of Fe(III) or nitrate (Friedrich
et al., 2005; Garcia-de-Lomas et al., 2007; Pronk et al., 1992;
Schedel & Trüper, 1980; Sugio et al., 1985; Waksman &
Joffe, 1922). Within the e-Proteobacteria, the majority of
the clones were most similar to Sulfurimonas (Fig. 1c).
Sulfurimonas species have been shown to anaerobically
oxidize sulfur with nitrate as the electron acceptor (Sievert
et al., 2007, 2008; Takai et al., 2006; Zhang et al., 2009).
Pseudomonas (Friedrich et al., 2001; Sun et al., 2009, 2010)
and Clostridia (Michaelidou et al., 2011; Sun et al., 2009,
2010; Zhao et al., 2008, 2009), which have previously
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T. Zhang and others
(b) 100
(a) 2.0
1
2
3
Electron transferred to anode
80
4
mmol /m2 . day
1.5
mA
Desulfuromonas strain TZ1
1.0
0.5
0
Sulfate production
64
57
60
40
20
0
20
40
14
0
60
1
(0–16 h)
Time (h)
(c) 1.0
61
54
9
7
2
(16–31 h)
3
(31–42 h)
7
4
(42–54 h)
(d) 60
1
0.8
Electron transferred to anode
48
Sulfate production
Desulfobulbus propionicus
2
3
4
mA
0.6
mmol /m2 . day
44
0.4
40
36
36
20
0.2
4,5
4
0
0
20
40
60
Time (h)
0
1
(0–15 h)
2
(15–27 h)
4,6
3
(27–41 h)
4,4
4
(41–56 h)
(e)
25 µm
Fig. 3. Sulfur oxidation by Desulfuromonas strain TZ1 and D. propionicus with a graphite anode poised at +300 mV (vs Ag/
AgCl) as the sole electron acceptor. Current production (a, c) and rate of electron transfer to anode and sulfate generation rate
(b, d). (e) Confocal laser scanning microscopy of Desulfuromonas strain TZ1 grown on the graphite anode surface. The cells
were stained with a LIVE/DEAD BacLight Viability kit and imaged with confocal laser microscopy as previously described
(Reguera et al., 2006). The arrows indicate when medium was replaced and additional sulfur was provided. The mean standard
errors of the current and sulfate measurements were 14 % and 6 % for Desulfuromonas strain TZ1 and 18 % and 4 % for D.
propionicus, respectively.
been suggested to be responsible for the oxidation of
sulfur in microbial fuel cells, were also detected (Fig. 1c).
d-Proteobacteria including close relatives to known
126
sulfate-, Fe(III)- and Su-reducing bacteria were in low
abundance (Fig. 1c). Three per cent of the total sequences
found in the anode enrichment were members of the
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Sulfur oxidation in Desulfuromonas
(b) 400
4
1
mA
3
Desulfuromonas strain TZ1
4
2
3
mmol /m2 . day
(a)
2
1
Electron transferred to anode
Sulfate production
300
200
206
186
100
20
0
40
0
60
Time (h)
(c) 2.0
(d)
Desulfobulbus propionicus
1
(0–16 h)
mmol /m2 . day
mA
150
1.0
0.5
4
(43–55 h)
112
141
129
124
100
50
Control current without cells
0
3
(32–43 h)
Electron transferred to anode
Sulfate production
4
3
2
7
200
1
1.5
2
(16–32 h)
56
43
32
28
Control current without cells
0
278
242
0
20
40
60
Time (h)
80
12
10
100
0
1
(0–24 h)
2
(24–52 h)
17
3
(52–73 h)
20
4
(73–93 h)
Fig. 4. Current production (a, c) and rate of electrons transfer to anode and sulfate generation rate (b, d) with/without strain TZ1
and D. propionicus, with addition of a carbon source (100 mM acetate or propionate for strain TZ1 and D. propionicus,
respectively). The arrows indicate when medium was replaced and additional sulfur was provided. The mean standard errors of
the current and sulfate measurements were 16 % and 5 % for Desulfuromonas strain TZ1 and 12 % and 6 % for D. propionicus,
respectively.
Desulfuromonodales order, which includes the genus
Desulfuromonas.
solidified medium (Nevin et al., 2005). A colony from this
extended purification, designated strain TZ1, was selected
for further study.
Enrichment and isolation of a S‡-oxidizing
electrode reducer
Analysis of 96 sequences of a clone library of 16S rRNA
gene sequences indicated that the culture was pure, with
a sequence most closely related (94.8 % similarity) to
Desulfuromonas palmitatis (Fig. 2b), a marine isolate
capable of oxidizing a wide range of fatty acids with
Fe(III) or Su as the electron acceptor (Coates et al., 1995).
Cells of strain TZ1, were Gram-negative rods, 1–3 mm6
0.5–0.8 mm (Fig. 2a).
In an attempt to further enrich for a Su-oxidizing organism,
an inoculum of the anode biofilm was added to the defined
medium with 0.16 g l21 of colloidal sulfur as the sole
electron donor and 100 mM poorly crystalline Fe(III)
oxide (Lovley & Phillips, 1988) as the electron acceptor.
Enrichments were incubated at 30 uC under anoxic
conditions as previously described (Lovley & Phillips,
1988). When Fe(II) sufficient to account for oxidation of
the added colloidal sulfur accumulated, the culture was
transferred (10 %) to fresh medium. After five successive
transfers in liquid medium, the culture was inoculated
(10 %, v/v) into an anaerobic dilution series of the same
medium amended with 2 % noble agar to form roll tubes
(Nevin et al., 2005; Zhang et al., 2012). A single isolated
colony was selected and further purified by repeating serial
dilution in roll tubes and restreaking of single colonies on
http://mic.sgmjournals.org
Anaerobic oxidation of S‡ coupled to electron
transfer to an electrode by Desulfuromonas strain
TZ1 compared to D. propionicus
When strain TZ1 was inoculated into an anode chamber
with Su as the electron donor, current was produced with
concurrent production of sulfate (Fig. 3a, b). Confocal
scanning laser microscopy combined with live/dead staining revealed a thin layer of metabolically active cells on the
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T. Zhang and others
anode surface (Fig. 3e). Current and sulfate production
rates were consistently higher for strain TZ1 than D.
propionicus (Fig. 3), the only other organism in pure
culture previously shown to oxidize Su to sulfate coupled
with electron transfer to an electrode (Holmes et al.,
2004a). Addition of a carbon source (100 mM acetate or
propionate for strain TZ1 and D. propionicus, respectively)
significantly increased rates of current and sulfate
generation for both species (Fig. 4), probably due to the
fact that these two organisms are not effective in fixing
carbon dioxide.
Bond, D. R., Holmes, D. E., Tender, L. M. & Lovley, D. R. (2002).
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by bacteria: emergence of a common mechanism? Appl Environ
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The finding that a Desulfuromonas species is capable of Su
oxidation coupled to electron transfer to electrodes not
only doubles the number of pure cultures known to carry
out this reaction, but also has important implications for
the interpretation of the function of microbial communities colonizing anodes harvesting electricity from marine
sediments. Desulfuromonas species are often the most
abundant species in the biofilms of marine sediment fuel
cells (Bond et al., 2002; Holmes et al., 2004b; Lovley, 2006;
Tender et al., 2002). It was previously assumed that the
primary role of these organisms was the oxidation of
acetate coupled to current production, because acetate is a
key intermediate in anaerobic oxidation of organic matter
and Desulfuromonas and closely related Geobacter species
are highly effective in acetate oxidation coupled to current
production (Lovley et al., 2011). However, sulfide may be
a more abundant electron donor for current production
in marine sediment fuel cells than acetate. This is because
acetate is a transient metabolic intermediate, maintained
at low (approximately 1 mM) concentrations, whereas
sulfide is an end product that builds up in sediments.
Therefore, sulfide produced at substantial (.40 mm)
distances from anodes may contribute to current production (Reimers et al., 2001, 2006). Desulfuromonas species
that can utilize both acetate produced from neighbouring
fermentative micro-organisms, as well as the sulfur
accumulating on anodes from the abiotic oxidation of
sulfide, are likely to have a competitive advantage over
other micro-organisms attempting to colonize marine
sediment fuel cell anodes.
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ACKNOWLEDGEMENTS
This research was supported by the Office of Naval Research (grant
N00014-13-1-0550) and by the Advanced Research Projects AgencyEnergy (ARPA-E), US Department of Energy, under award numbers
DE-AR0000087 and DE-AR0000159.
Lovley, D. R., Ueki, T., Zhang, T., Malvankar, N. S., Shrestha, P. M.,
Flanagan, K. A., Aklujkar, M., Butler, J. E., Giloteaux, L. & other
authors (2011). Geobacter: the microbe electric’s physiology, ecology,
and practical applications. Adv Microb Physiol 59, 1–100.
Michaelidou, U., ter Heijne, A., Euverink, G. J., Hamelers, H. V.,
Stams, A. J. & Geelhoed, J. S. (2011). Microbial communities and
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