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Previous Article | Next Article 
Applied and Environmental Microbiology, August 2000, p. 3650-3653, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Thiosulfate Disproportionation by
Desulfotomaculum thermobenzoicum
Bradley E.
Jackson
and
Michael J.
McInerney*
Department of Botany and Microbiology,
University of Oklahoma, Norman, Oklahoma 73109
Received 7 February 2000/Accepted 29 May 2000
 |
ABSTRACT |
Desulfotomaculum thermobenzoicum, but not
Desulfotomaculum nigrificans, Desulfotomaculum
ruminis, or Desulfosporosinus orientis, grew by
disproportionation of thiosulfate, forming stoichiometric amounts of
sulfate and sulfide; sulfite was not disproportionated. The addition of
acetate enhanced growth and thiosulfate disproportionation by D. thermobenzoicum compared to those observed with thiosulfate alone.
| |
TEXT |
Disproportionation is a general term
used to describe a process by which an element or a compound in a state
of intermediate oxidation is converted to substances in higher and
lower oxidation states. Specifically, the biological disproportionation
of thiosulfate occurs via an intramolecular redox change at each of the
sulfur atoms (21) and produces 1 mol of sulfate and 1 mol of
sulfide, with a Gibb's free energy change of approximately 
22 kJ/mol
(equation 1).
|
(1)
|
Although the disproportionation of thiosulfate yields only a small
amount of free energy, microorganisms capable of this metabolism are
abundant in both freshwater and marine environments (1, 6),
and it is a dominant process for thiosulfate transformation in marine
and freshwater sediments and in benthic cyanobacterial mats
(6-9).
To date, the only microorganisms known to metabolize inorganic sulfur
compounds by a disproportionation mechanism are gram-negative, sulfate-reducing bacteria that cluster within the delta subclass of the
Proteobacteria (11). Desulfovibrio
sulfodismutans, Desulfocapsa thiozymogenes,
Desulfocapsa sulfoexigens, and strain NTA3 are the only
known microorganisms capable of growth by thiosulfate disproportionation (1, 2, 4, 5), while other sulfate reducers have been reported to disproportionate but not grow with thiosulfate, sulfur, or sulfite (11). This communication
reports on the growth of a gram-positive, thermophilic sulfate-reducing bacterium by thiosulfate disproportionation.
Organisms and methods of cultivation.
The type strains of
Desulfotomaculum thermobenzoicum (49756),
Desulfotomaculum nigrificans (19998), Desulfotomaculum
ruminis (23193), and Desulfosporosinus orientis (19365)
were obtained from the American Type Culture Collection (Manassas,
Va.). The microorganisms were cultivated in a basal medium containing
(per liter) 0.4 g of MgCl2 · 6H2O,
0.1 g of CaCl2 · 2H2O, 1.0 g
of NaCl, 0.25 g of NH4Cl, 0.1 g of
KH2PO4, 0.1 g of yeast extract (Difco),
2.0 g of NaHCO3, 2.0 g of TES
[N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid],
0.001 g of resazurin, and 0.5 g of dithiothreitol. A trace metal
solution and vitamin solution (12) were added to the medium in amounts of 5.0 and 10.0 ml per liter, respectively. The trace metal
solution contained the following (per liter): 2.0 g of
nitrilotriacetic acid (adjusted to pH 6.0 with 1.0 M KOH), 1.0 g
of MnCl2 · 4H2O, 1.0 g of
FeCl2 · 4H2O, 0.2 g of
CoCl2 · 6H2O, 0.2 g of
ZnCl2, 0.5 g of H3BO3,
0.02 g of CuCl2 · 2H2O, 0.02 g
of NiCl2 · 6H2O, 0.02 g of
Na2MoO4 · 2H2O, 0.02 g
of Na2SeO4, and 0.02 g of
Na2WO4. The medium was adjusted to a pH of 7.4 and boiled under a gas stream of 80% N2-20%
CO2.
All procedures for the anaerobic preparation and use of media and
solutions were essentially those of Balch and Wolfe (3). The
basal medium was boiled and dispensed in 10-ml aliquots into serum
tubes (18 by 150 mm) under a stream of an oxygen-free, 80% N2-20% CO2 gas mixture. The tubes were sealed
with blue rubber stoppers (Bellco Glass, Vineland, N.J.) and crimped
with aluminum seals, and the pressure of each tube was brought to 135 kPa with the above-described gas mixture prior to autoclaving. When
hydrogen was the electron donor, the headspace of each tube was
aseptically exchanged three times by evacuation with vacuum and
repressurization with an 80% H2-20% CO2 (170 kPa) gas mixture. All additions to anaerobic and sterile media were
performed aseptically using syringes and needles flushed with
oxygen-free nitrogen gas. Stock solutions of thiosulfate, sulfate,
sulfite, and acetate were sterilized by filtration (0.2 µm pore
size). The headspace of each solution then was exchanged with a 100%
N2 gas phase, as described above. The initial concentration
of thiosulfate ranged from 5 to 15 mM. For some experiments, acetate
was added as a carbon source at an initial concentration of 3 mM.
Medium volumes varied from 10.5 to 12.5 ml after additions; volume was
constant among treatments within an experiment.
Inocula for experiments were obtained from cultures grown in basal
medium with 5 mM thiosulfate and an 80% H2-20%
CO2 gas phase (180 kPa) to late exponential phase. Each
tube was inoculated with 2 ml of culture, about a 20% (vol/vol)
inoculum size. Inoculations were performed aseptically using syringes
and needles flushed with oxygen-free nitrogen gas. D. ruminis and D. orientis cultures were incubated at
37°C, and D. thermobenzoicum and D. nigrificans cultures were incubated at 62°C. All cultures were incubated without shaking unless hydrogen was the electron donor. Cultures with hydrogen
as the electron donor were shaken at 200 rpm.
To minimize the transfer of sulfur compounds, a washed cell suspension
was used as the inoculum for performing a detailed sulfur balance of
D. thermobenzoicum. All manipulations were done aseptically
and anaerobically using an anaerobic chamber and sterile materials. All
plasticware was placed inside the anaerobic chamber 24 h prior to
use. Cultures of D. thermobenzoicum were grown on hydrogen
and thiosulfate to late exponential phase as described above. The
cultures (20 ml) were transferred to a sterile centrifuge tube inside
the anaerobic chamber. The centrifuge tube was sealed, brought outside
the anaerobic chamber, and then centrifuged (11,500 × g; 15 min; 4°C). The cell pellet was resuspended in 2 ml of anaerobic, sterile basal medium. Two hundred microliters of the cell
suspension was used to inoculate each experimental tube.
Analytical techniques.
Growth of cultures was monitored
spectrophotometrically by measuring the increase in absorbance at 600 nm. Direct cell counts were performed at 400-fold magnification with a
phase-contrast microscope and a Petroff-Hausser counting chamber
(10). A total sample volume of 500 µl was removed to
quantify all sulfur species. Samples were immediately analyzed for
volatile sulfides and total reduced inorganic sulfur. Volatile sulfides
were quantified spectrophotometrically by the methylene blue assay as
described by Tanner (16). Total reduced inorganic sulfur was
determined by using a modified single-extraction chromium reduction
assay as previously reported (21). Elemental sulfur was
determined as previously reported (20). Sulfoxyanions (thiosulfate, sulfate, and sulfite) were quantified by suppressed ion
chromatography using a Dionex Ion Chromatograph DX-500 equipped with a
CD-20 conductivity detector and an AS-11 column. Thiosulfate and
sulfite were also quantified with the ion chromatography system by
measuring the absorbance at 230 nm with an AD-20 absorbance detector.
In either case, the chromatograph was run isocratically with a mobile
phase of 7 mM NaOH at a flow rate of 2.0 ml min
1 for 5 min, and then the mobile phase concentration was linearly increased to
35 mM NaOH for an additional 7 min.
Growth by thiosulfate disproportionation.
A small amount of
growth (a change of 0.06 absorbance units) was observed when D. thermobenzoicum was incubated in basal medium with 4.5 mM
thiosulfate (Fig. 1). Sulfate was
produced concomitant with growth and thiosulfate disappearance. After 6 days, the concentration of sulfide produced was 4.7 mM. Thus, D. thermobenzoicum transformed approximately 4.5 mM thiosulfate to
approximately 4.0 mM sulfate and 4.7 mM sulfide (96.7% sulfur
recovery) after 6 days of incubation. The ratio of sulfate produced to
thiosulfate consumed was about 0.9, which is close to that
theoretically predicted for thiosulfate disproportionation (equation
1). In basal medium without thiosulfate, no increase in absorbance was
observed and sulfate was not produced. Abiotic transformation of
thiosulfate to sulfate or sulfide was not observed. Additionally,
D. thermobenzoicum cultures did not show an increase in
absorbance in basal medium with 5 mM sulfate, indicating that the small
amount of yeast extract in the medium did not support growth.
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FIG. 1.
Growth and thiosulfate disproportionation by D. thermobenzoicum in basal medium with 4.5 mM thiosulfate. Results
are expressed as the means and standard deviations of triplicate
determinations.
|
|
When growth was followed by total cell counts (Fig.
2), the number of D. thermobenzoicum cells increased linearly from approximately 3.5 × 106 to 1.6 × 107 cells/ml
over a period of 10 days in sulfate-free basal medium with 8 mM
thiosulfate. In basal medium without thiosulfate, cell numbers did not
increase. Growth of D. thermobenzoicum in basal medium with
thiosulfate was sustained through at least four successive transfers.
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FIG. 2.
Increase in cell numbers of D. thermobenzoicum in basal medium with 8 mM thiosulfate. Data are
the means and average deviations of duplicate determinations.
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|
In basal medium with 10 mM thiosulfate, growth or thiosulfate
transformation by D. nigrificans, D. ruminis, or
D. orientis was not observed. In addition, thiosulfate
metabolism by these organisms was not stimulated even after 3 mM
acetate was added as a carbon source for biosynthesis. Sulfite (5 mM)
was not transformed by any of the four species tested. However, all
four species grew with hydrogen (170 kPa of an 80%
H2-20% CO2 gas phase) and 10 mM thiosulfate
or 5 mM sulfite as the electron acceptor, indicating that this medium
does support growth of these organisms if a suitable electron donor is
supplied (data not shown).
Stoichiometry and effect of acetate on thiosulfate
disproportionation by D. thermobenzoicum.
D.
thermobenzoicum disproportionated approximately 26 µmol of
thiosulfate to 24 µmol of acid volatile sulfide and 25 µmol of
sulfate when grown in basal medium with 15 mM thiosulfate (Table 1). The ratio of sulfate to sulfide was
1.0, a value that is nearly identical to that for the theoretical
stoichiometry of thiosulfate disproportionation (equation 1). When the
small amount of sulfur (approximately 4 µmol or 7% of the total
sulfur) recovered in the total reduced inorganic sulfur pool is
included, the sulfur recovery is 101%. The latter material may be in
the form of iron sulfides, since elemental sulfur was not detected;
however, this observation was not investigated further.
When 3 mM acetate was added to the medium, the extent of both growth
and thiosulfate disproportionation significantly increased (Table 1).
D. thermobenzoicum is not known to utilize acetate as an
electron donor for sulfate or thiosulfate reduction and presumably uses
acetate only as a carbon source for biosynthesis (17).
Consistent with this, we did not observe any growth of D. thermobenzoicum in the presence of acetate and sulfate (Table 1).
D. thermobenzoicum converted approximately 146 µmol of
thiosulfate to 135 µmol of sulfate, 169 µmol of sulfide, and 10 µmol of sulfite. The sulfur recovery was 107%, and the ratio of
sulfate to sulfide was 0.8, again in agreement with the stoichiometry
of thiosulfate disproportionation (equation 1). Growth was not observed
in tubes that contained acetate alone (Table 1). Thiosulfate has been reported to chemically decompose at elevated temperatures
(13). However, less than 2% of the initial thiosulfate was
abiotically transformed in control tubes amended only with thiosulfate
(without cells) under the experimental conditions used here, i.e.,
62°C and 135 kPa of N2/CO2 (Table 1). In
addition, thiosulfate was not transformed in the presence of
heat-killed cells.
Conclusions.
D. thermobenzoicum is phylogenetically
distinct from previously described thiosulfate-disproportionating
bacteria, clustering with low-G+C-content gram-positive bacteria
(clostridia) within a separate division of the domain
Bacteria. To date, a disproportionation metabolism has been
conclusively documented only within the gram-negative, mesophilic
sulfate-reducing bacterial group, whose members phylogenetically cluster within the delta subclass of the Proteobacteria.
Physiologically, our results are consistent with the fact that the
ability to disproportionate sulfoxyanions seems to be limited to
bacteria that reduce sulfate (11). However, a
disproportionation metabolism is apparently not unique to
gram-negative, sulfate-reducing bacteria but is found among
gram-positive bacteria as well.
The addition of acetate significantly stimulated both growth and
thiosulfate disproportionation. D. thermobenzoicum achieved twice the cell density and disproportionated sixfold more thiosulfate when grown in medium with acetate added as the carbon source than it
did in medium without acetate. The final cell concentration for growth
of D. thermobenzoicum by thiosulfate disproportionation was
very similar to that observed by Bak and Pfennig (2) for D. sulfodismutans. Thiosulfate disproportionation did
support the growth of D. thermobenzoicum through repeated
transfers in medium without acetate added. This medium contained low
levels of yeast extract that could have served as the carbon source
(0.05 g/liter). Alternatively, D. thermobenzoicum may have
used carbon dioxide as a carbon source, since D. thermobenzoicum possesses the enzymatic ability to assimilate
carbon via the carbon monoxide dehydrogenase pathway (18).
To date, only strain NTA 3 has been observed to grow autotrophically by
thiosulfate disproportionation (1).
Disproportionating bacteria are numerically significant and have been
detected in high numbers in freshwater mud and marine sediments
(2, 9). The genus Desulfotomaculum has the
ability, unique among sulfate-reducing bacteria, to form endospores,
indicating that bacteria possessing a disproportionation metabolism can
persist through extreme or unfavorable environmental conditions. The
finding that D. thermobenzoicum effectively
disproportionates thiosulfate suggests that this transformation may
occur in thermophilic as well as freshwater and marine environments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73109-0245. Phone: (405) 325-6050. Fax: (405) 325-7619. E-mail: mcinerney{at}ou.edu.
Present address: Department of Civil Engineering, Northwestern
University, Evanston, IL 60208.
| |
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Applied and Environmental Microbiology, August 2000, p. 3650-3653, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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