MICROBIOLOGY
LETTERS
ELSEVIER
FEMS
Microbiology
Letters
166 (1998)
181-186
A combined pathway of sulfur compound disproportionation
in
Desulfovibrio desulfuricans
Heribert
Institute
for ChrmistrJ
Cypionka
md Biology
*, Andrea
of the Marine
Environment
M. Smock,
(ICBM),
D-261 1I Oldenburg,
Michael
Curl wn Ossietzky
E. Biittcher
Universitiit
’
Oldenburg,
P. 0. Bus 2503
Germcrn~
Received 27 May 1998; revised 22 July 1998; accepted 22 July 1998
Abstract
The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation
by the sulfatereducing bacterium Desulfovibrio
desulfuricans
were followed by isotope mass spectrometry. During disproportionation,
‘(*Sthiosulfate was preferentially metabolized, and the residual thiosulfate became enriched in “S. The sulfate formed was
isotopically heavier than the inner sulfur of the consumed thiosulfate. Vice versa, the sulfide formed was isotopically lighter
than the outer sulfur of the consumed thiosulfate. These results indicate that thiosulfate is cleaved to intermediates that
undergo further disproportionation
to sulfate and sulfide in a second step. These intermediates are probably elemental sulfur
and sulfite. It is concluded that disproportionation
of thiosulfate, sulfite and elemental sulfur includes a combined
pathway.
0 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keyword.~:
Elemental
Thiosulfate
disproportionation;
Sulfate-reducing
bacterium;
Stable isotope;
Sulfur isotope fractionation:
Sulfite;
sulfur
1. Introduction
Thiosulfate
is an important
intermediate
in the
sulfur cycle of marine and limnic sediments. It is
mainly formed during chemical and biological reoxidation of sulfide which originates from the dissimilatory reduction of sulfate. Thiosulfate can undergo
various transformations,
i.e. oxidation, reduction or
disproportionation
[l]. The latter appears to be
* Corresponding author.
Tel.: +49 (441) 9706-360; Fax: +49 (441) 798-3583;
E-mail: [email protected]
’ Present address: Max-Planck-Institute for Marine Microbiology. Celsiusstr. 1~ D-28359 Bremen, Germany.
0378.1097/98/$19,00
Q 1998 Federation
PII: SO378-1097(98)00330-9
of European
Microbiological
quantitatively
most important at the oxic-anoxic interface of sediments [24]. Many sulfate-reducing
bacteria can carry out this process [5]. Besides thiosulfate, sulfite or elemental sulfur can be disproportionated. Especially disproportionation
of elemental
sulfur has been found to be a relevant reaction for
the sulfur cycle in several recent studies [6-91. The
relations between the different sulfur disproportionation processes have, however, not been clarified.
Microbial sulfate and thiosulfate reduction both
discriminate
against sulfate containing
the heavier
(““S) isotope and lead to the formation of sulfide
depleted in 34S [lo, 111. However, solid phase sulfides
found in most sediments which should reflect the
stable isotopic composition of dissolved sulfide [12]
Societies. Published by Elsevier Science B.V. All rights reserved.
are often isotopically lighter than expected from sulfate reduction alone [13]. It has been shown that in
sediments a large part of sulfide is recycled via thiosulfate disproportionation
to sulfide and sulfate, and
it has been suggested that this step may lead to an
additional depletion in ‘r’S compared to the initial
sulfide [4]. In this case the disproportionation
could
have a considerable
impact on the sulfur isotope
composition of solid phase sulfides in natural sediments and thus be important for the interpretation
of stable isotope variations through the geological
record.
I‘;
2. Materials and methods
The freshwater strain Desu@~vibrio rksuljiuicun.~
(DSM 9104) was cultivated
with lactate
(15 mM) and thiosulfate (10 mM) as described by
Smock et al. [1 I]. Washed cells (0.42 mg protein
ml-‘) were incubated at 30°C in an oxygen-free
phosphate buffer (50 mM, pH 7.0). Sulfide was continuously flushed out with Np and trapped in 250 mM
NaOH. The steady-state sulfide concentration
was
generally below 0.4 mM. Thus, further reactions of
sulfide were avoided. Chemical analyses were carried
out as described by Smock et al. [ll]. Sulfate was
separated from thiosulfate-containing
solutions by
precipitation with 10 mM Ba(NO:,)z. Coprecipitation
of BaS20:I was avoided by complexing Ba’+ with
10 mM citric acid (pH 5).
Sulfur isotope ratios (“‘S/:“S) were measured by
means of combustion isotope-ratio-monitoring
mass
spectrometry (C-irmMS) as described by Biittcher et
al. [14]. After conversion to BaSOl or AglS, the
different sulfur species were combusted in a Carlo
Erba EA 1108 elemental analyzer connected to a
Finnigan MAT 252 mass spectrometer via a Finnigan MAT Conflo II split interface. The liberated SO2
was transported
in a continuous
stream of helium
(5.0 grade). ‘r,‘S/“2Sratios are given in the 6 notation
with respect to the Vienna-Canon
Diabolo Troilite
(V-CDT) standard according to:
CSN
Replicate
analyses agreed within
+ 0.2 %O 6:“‘s val-
Residual thiosulfate
Residual outer sulfur
4
:
A-
Sulfate produced
4
*
Outer sulfur consumed
?4 x
Inner sulfur consumed
4
R
I
1
Sulfide produced
I
I
3
5
Incubation time (days)
Fig. I, Sulfur isotope fractionation
during thiosulfate dlsproportionatlon by De.wlfovihrio dcwdfuriccms. A: Concentrations
of
thiosulfate and the products formed. B: Isotope composition of
the sulfur atoms of the residual thiosulfate. C: Isotope composition of the sulfate and sulfide produced compared to the inner
and outer sulfur consumed.
H. Cypionka et al. IFEMS
Microbiology
ues of +20.6%
and -32.3%
were obtained for
IAEA intercomparison
distributes NBS-127 (BaSOd)
and IAEA-S-3 (AgsS), respectively.
The isotopic composition
of the consumed inner
and outer sulfur atoms of thiosulfate was calculated
after the Rayleigh equation for fractionation
in a
closed system [15]. The fractionation
factor for this
calculation was derived from the change in isotopic
composition of inner and outer sulfur of the residual
thiosulfate.
Letters 166 (1998)
in sulfate reduction partially working in reverse direction. Thiosulfate should be reduced to sulfide and
sulfite in a first step (Eq. 3), followed by oxidation of
sulfite to sulfate (Eq. 4), which is necessary to regain
the electrons consumed for thiosulfate reduction.
S&-
+ Z[H]+HsS + SO;-
SO;- + H,O+SO;Summing
The cells disproportionated
thiosulfate over 5 days
at a nearly constant rate of about 0.14 pmol thiosulfate per hour and mg protein (Fig. 1A). The amounts
of sulfate and sulfide formed were equal (in agreement with Eq. 4). The sulfur atoms of the thiosulfate
used had different isotope signatures of the outer
(S34S = +0.6 %,o) and inner (634S = +14.4 %O) sulfur
atoms (Fig. 1B). During disproportionation,
32Sthiosulfate was preferentially
metabolized,
and the
residual thiosulfate
became enriched in 34S (Fig.
1B). This result was mainly due to discrimination
against 34S of the inner sulfur atom. The sulfate
formed was isotopically heavier than the inner sulfur
of the consumed thiosulfate (Fig. 1C). Vice versa, the
sulfide formed was isotopically lighter than the outer
sulfur of the thiosulfate consumed. A parallel experiment at 19°C (not shown) gave similar results. The
overall isotope balances, according to Eq. 2 (with X
indicating the mole fraction, i indicating products
and residual substrate)
634S(S20i-)
= ZXi’6i
were generally
+ 2[H]
(3)
(4)
Eqs. 3 and 4 yields:
SsO;- + H20+HzS
3. Results
183
181-186
A
+ SO;-
(5)
sod*-
1
P/
o=s=o
s-
(
H;S
(2)
near 100% in both cases.
4. Discussion
Desulfovibrio desulfiiricans has a constitutive thiosulfate reductase like most sulfate-reducing
bacteria
and a constitutive capacity of thiosulfate disproportionation [5]. According to a previous study [5] we
expected that disproportionation
is catalyzed by thiosulfate reductase and some of the enzymes involved
Fig. 2. Fates of the sulfur atoms during thiosulfate disproportionation. A: Direct disproportionation
should conserve the isotope
signature of the inner and outer sulfur atoms. B: Thiosulfate disproportionation
via cleavage to sulfite and sulfur, which are disproportionated
in a second step. Part of the outer S is transformed to sulfate and part of the inner S to sulfide. The
numbers indicate the stoichiometries of sulfur and sulfite disproportionation.
The pathway of sulfur disproportionation
might include sulfite as an intermediate, which is shown in Fig. 3.
For the complete disproportionation
(Eq. 5) we
therefore expected to find the isotope signature of
the inner sulfur in the produced sulfate and the signature of the outer sulfur in the produced sulfide
(Fig. 2A).
However. our observations are not in agreement
with a direct transformation
of the inner and outet
sulfur atoms to sulfate and sulfide, respectively. Instead, it must be assumed that thiosulfate is first
transformed
to intermediates
that undergo further
reactions coupled to sulfur isotope fractionation.
The most simple explanation (Fig. 2B) is that thiosulfate is not reduced in the first step, but cleaved to
sulfur and sulfite (Ey. 6). Both of these intermediates
can undergo further disproportionation
(Eqs. 7 and
X):
$0;:
-s” + so-;
I6 I
The sum of Eqs. 6 8 yields Eq. 5. Thuh, disploportionation
of thiosulfaate, sulfite and elemental sulfur would be parts of a combined
pathway.
Although
this pathway looks more complicated
than our preliminary assumption, it is in agreement
with the following observations and might help to
explain some open questions.
The capacities of sulfite and sulfur disproportionation are constitutively
present in Dcxd/o~~ihrio
tlcw~~/u~icwr~v and in many other sulfate reducers
[5-71. Both the disproportionation
of sulfur and SLIIfite have been shown to result in the formation
of ‘“S-depleted
sulfide and “‘S-enriched
sulfate
[16,17].
Physiologically, the cleavage of thiosulfate to WIfur and sulfite might be easier to perform than reduction of the outer sulfur atom to sulfide according
to Fig. 2A. since the standard midpoint potential of
this reduction at pH 7 is -414 mV. Thus, usually an
electronegative electron donor is required. which is
not available under the conditions of thiosulfate disproportionation.
The proposed thiosulfate cleavage
to sulfite and sulfur avoids this critical step. The
enzyme catalyzing this reaction, rhodanese. is widespread and has also been found in sulfate-reducing
bacteria (181. Since the oxidation states of the outer
and inner sulfur in thiosulfate are ~ 1 and +5, rcspectively [l9], already the first step can be regarded
as a disproportionation
that changes the redox states
of the sulfur atoms.
Our model is also confirmed
by earlier unpublished results obtained
with the same strain
using inner or outer ““S-radiolabeled
thiosulfate
(Cypionka and Jorgensen. unpublished
results). In
those experiments
Il43%
of the inner sulfur was
reduced to sulfide, while a similar part of the outet
sulfur was oxidized to sulfate. Whereas
those
results remained unexplained at the time of the experiments. they agree with our present model which
predicts that part of the inner sulfur is reduced to
sulfide. and part of the outer sulfate oxidized to sulfate.
Another feature in favour of the combined disproportionation
pathway is that the disproportionation
of elemental sulfur is also an intermediate step of the
complete oxidation
of sulfide by sulfate-reducing
bacteria. Several sulfate reducers. including the strain
studied here, can oxidize inorganic sulfur compounds
if an electron acceptor such as oxygen or nitrate is
available [20,21]. The complete oxidation of sulfide
takes place via oxidation of sulfide to elemental sulfur (Eq. 9), followed by the disproportionation
of
sulfur to sulfate and sulfide (Eq. IO).
1 H?S + Z O?--1 S” + 1 H&
I S” t 1 HnO-SO;
Summing
+ :I IIpS + %H’
(9)
(10)
these gives:
H-,S r 2 oL’-’ SO_r~ + % II
(II)
In combination
(Eq. 1I). a complete oxidation of
sulfide to sulfate is achieved, although the electron
acceptor is only involved in the oxidation of sulfide
to sulfur.
The two sulfur atoms of thiosulfate underwent
diKerent fractionation.
The residual inner sulfur
atom was much more enriched in ‘“S than the residual outer sulfur atom (Fig. 1). This must be caused
by different metabolic pathways of the two sulfur
atoms. In a study on sulfur isotope fractionation
during reduction of thiosulfate by Desu&wibt+u de.su~furictrrz.s[ 1 I] we also found different fractionation
185
H. Cypionka et al. IFEMS Microbiology Letters 166 11998) 1X1-186
/O\/
-s
-A=o
::
so42-
&3= +I0 %o
6”%(HzS) = 0.25*(6”“S(SO;-)
@“S(S”)
+ Q) + 0.667
+ Q) + O.O83@“‘S(S”) + E~ -t e4)
(12)
63‘ls(so:-)
= o.75+9~4s(so~-)
0.25@“S(~)
+ Es)+
+ E:<+ Q)
(13)
%o
Fig. 3. Fractionation
factors of reactions involved in thiosulfate
disproportionation
by Desulfovibrio desuljiiricans. E indicates the
isotope enrichment factors [15]. For the reduction of the key intermediate sulfite to sulfide an E value of -30%
was assumed
according to [17], which allowed to calculate the other F values.
of the two sulfur atoms. Sulfide produced from the
inner sulfur was depleted in 34S by 15 %O, sulfide
produced from outer sulfur by 5.0%0.
The mode1 in Fig. 2B shows the ultimate fate of
the sulfur atoms but not the intermediate
reaction
steps. From our earlier studies on sulfide oxidation
by sulfate-reducing bacteria [20,21] we know that the
pathway of sulfur disproportionation
includes sulfite
as an intermediate of sulfate formation. Also in the
case of thiosulfate reduction, sulfite is known as an
intermediate. If one assumes that the oxidative part
during elemental sulfur disproportionation
includes
sulfite as an intermediate
(Fig. 3), and that sulfite
disproportionation
results in a 34S depletion
of
-30 %O (~1) in the produced sulfide [17], the remaining three fractionation
factors can be calculated according to the mass balances (Eqs. 12 and 12, for the
indices of E see Fig. 3).
Table 1
Sulfur isotope distributions
Incubation
time (days)
1
3
5
“For the calculations
experimentally [17].
as measured
and expected for thiosulfate
Swso:-
)LLICWlll(.<l
+11.3
+13.1
+14.4
it was assumed
6u4S(SW
This yields a depletion of - 13 %O (~2) in s4S for the
reduction of elemental sulfur to sulfide, which is
close to the value of - 16 %O found during disproportionation
of sulfur by Desulfobulbus propionicus
[161. Correspondingly,
sulfur isotope fractionation
during oxidation
of sulfite to sulfate must be
+IO% (Q), and that of sulfur oxidation to sulfite
+26%0 (~4). The data found in our experiments are
in excellent agreement with these assumptions (Table
1).
Summarizing,
we conclude that the disproportionation of thiosulfate by Desulfovibrio desulfuricans
does not directly lead to the formation of sulfate
and sulfide, but includes intermediate reactions that
allow sulfur isotope fractionation.
The simplest explanation is a common pathway of disproportionation of thiosulfate, sulfite and elemental sulfur. This
is the recently discovered pathway that also catalyzes
the oxidation of sulfide to sulfate in sulfate-reducing
bacteria. Based on field observations,
Thamdrup et
al. suggested [6] that thiosulfate could possibly be an
intermediate
of sulfur disproportionation.
By contrast, our results indicate that elemental sulfur is an
intermediate
of thiosulfate disproportionation.
The
sulfur isotope patterns of sedimentary reduced sulfur
often cannot be explained by microbial sulfate reduction alone [l3]. The combined pathway described
disproportionation
)r,ilnll;*Krl*
+12.0
+13.1
+ 14.4
that sulfide produced
by disproportionation
by the combined
pathway
G”‘S(H~S),,,,,,,,,,.,I
S’“S(HzS)
-20.3
-20.4
~19.8
-20.2
-19.6
-19.0
of sulfite was depleted
<,~cu~ii,<~ti”
in “‘S by -30%
as found
here may contribute to the development
of sulfur
isotope signatures in the geological record.
Acknowledgments
We thank Kirsten Habicht, Bo Thamdrup,
Bo
Barker Jerrgensen and Hans Brumsack for the communication
of unpublished
results and for helpful
discussions during the preparation
of the manuscript. This study was supported by a grant of the
Deutsche Forschungsgemeinschaft
(Cy 116-3).
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