MICROBIOLOGY
LETTERS
ELSEVIER
FEMS
Microbiology
Letters
144 (1996) 129-134
A common pathway of sulfide oxidation by
sulfate-reducing bacteria
Knut Fuseler, Daniel Krekeler, Ulrike Sydow, Heribert Cypionka *
Institut fiir Chemie und Biologie des Mews.
UniversitiitOldenburg, Postfach 2503, D-26111 Oldenburg, Germany
Received 1 July 1996; revised 14 August
1996; accepted
16 August
1996
Abstract
The pathway of sulfide oxidation with oxygen as electron acceptor was studied with five strains of freshwater, marine and
alkaliphilic sulfate-reducing bacteria. Electrode measurements with washed cells indicated that all strains oxidized sulfide to
elemental sulfur. In a second step, the elemental sulfur formed was disproportionated
to sulfate and sulfide. During this phase,
most of the disappeared sulfide was formed back. Since oxygen could be replaced by nitrate or nitrite as electron acceptor, the
described biphasic reaction was independent of molecular oxygen. With Desuljobulbuspropionicus
and the alkaliphilic strain Z7935, sulfide back-formation
started after oxygen was consumed completely. By contrast, with the freshwater strains
Desufivibrio
desulfuricuns CSN (DSM 9104) and Essex 6 (DSM 2032) and the marine strain PlB, sulfide back-formation
already started before oxygen was consumed. The addition of hydrogen as electron donor increased simultaneously the rate of
aerobic respiration and sulfide back-formation.
Both reactions stopped when the oxygen was consumed, indicating that the
electron transport to oxygen and sulfur was coupled. Sulfide-oxidizing activity (84 nmol Oa mm-’ (mg protein)-l) was found
in the periplasmic fraction prepared by osmotic shock treatment of suspensions of D. desulfuricans CSN. This fraction oxidized
sulfide with oxygen to elemental sulfur. It is concluded that in different sulfate-reducing bacteria sulfide oxidation proceeds via
a common pathway with the formation of elemental sulfur as intermediate and its disproportionation
to sulfate and sulfide.
The process is independent of molecular oxygen.
Keywords:
Sulfate-reducing
bacteria;
Sulfide oxidation;
Elemental
sulfur disproportionation;
Nitrate;
Nitrite;
Periplasmic
space;
Glutathione
1. Introduction
Several sulfate-reducing
bacteria are able to oxidize sulfide, sulfite, thiosulfate and elemental sulfur
with oxygen, nitrate or nitrite as electron acceptor
[l]. Furthermore,
many sulfate reducers can carry
* Corresponding author.
E-mail: [email protected]
0378-1097 /96/$12.00 Copyright
PIISO378-1097(96)00337-O
a disproportionation
of sulfite, thiosulfate
[2]
or elemental
sulfur
[3]. The disproportionation
reactions were suggested to be important
for the
sulfur cycle in sediments [4]. Recently, we reported
that the oxidation of sulfide by Desulfobulbus
proout
0 1996 Federation
of European
pionicus
also
includes
disproportionation
sulfide
to elemental
strain
oxidizes
oxygen
as electron
mental
sulfate.
sulfur
Microbiological
acceptor.
In a second
is disproportionated
Societies. Published
[5]. This
sulfur
with
to
step,
sulfide
by Elsevier Science B.V.
eleand
130
K. Fu.rrkr
et ul I ITEMS Microbiology
Lettws
144 i 1996) IZY- 134
Fig. I. (ax) Sulfide oxidation with oxygen by washed cells of different strains of sulfate-reducing
bacteria. (a) Alkaliphilic strain Z-7935
(0.36 mg protein ml-‘. pH 9.5). (b) Marine strain PlB (1.56 mg protein ml-‘, pH 7.5). (c) Freshwater strain Desulfovihrio desulfiricans
CSN (0.48 mg protein ml-‘, pH 7.0). All tests were carried out in N2-saturated
salt solutions at 30°C. Numbers indicate additions in
nmol to 3 ml assays.
In the present study, we have compared sulfide
oxidation by freshwater, marine and alkaliphilic sulfate-reducing bacteria. We investigated whether the
pathway depends on the presence of molecular oxygen and tried to localize the sulfide-oxidizing activity
in the cell.
2. Materials and methods
2.1. Organisms and cultivation
The freshwater
strains
Desuljhibrio
desu&wicans
CSN (DSM 9104) Essex 6 (DSM 642) and Db. pro-
2.2.
Meusurement
of oxygen, suede
and pH
Freshly harvested and washed cells were studied as
described previously [5] at 30°C in a multi-electrode
chamber (3 ml) which allows the simultaneous measurement of oxygen, sulfide and pH. The freshwater
strains D. desulfuricans CSN and Essex 6, and Db.
propionicus were studied in Nz-saturated
100 mM
KC1 titrated to pH 7.0, the marine strain PlB was
incubated
in a salt solution of 850 mM NaCl,
13 mM KC1 (pH 7.5) and the alkaliphilic strain
Z-7935 in a salt solution
of 320 mM NaCl,
80 mM KCl, 10 mM MgClz and 2 mM KzCOa
(pH 9.5).
pionicus (DSM 2032) were cultivated
in a chemostat
[6] with Hz, CO? and limiting concentrations
of sulfate or nitrate. The alkaliphilic strain Z-7935 was a
kind gift from Dr. Tatjana Zhilina and Dr. Georgy
Zavarzin, Russian Academy of Sciences, Moscow.
This strain was grown in a chemostat at pH 9.5 as
described by Sydow and Cypionka (in preparation).
Strain PlB was isolated by Pave1 Sigalevich and Dr.
Yehuda Cohen in Eilat, Israel, from a hypersaline
pond inoculated with Solar Lake water. This strain
was cultivated in a chemostat [6] with marine salt
concentration.
2.3. Preparution of periplasmic fractions
The periplasmic fraction of D. desu@ricans CSN
was prepared as described by Badziong and Thauer
[7]. Freshly harvested cells were resuspended in Nzsaturated
Tris-HCl buffer (30 mM Tris, 1 mM
EDTA, 20% saccharose, pH 8.0) for 15 min, centrifuged at 10 OOOXg for 10 min, resuspended in 0.1
mM MgClP and centrifuged again at 10 OOOXg for
10 min [8]. For separation of the periplasm, the osmotically shocked cells were suspended in Tris-HCI
t
K. Fuseler et al. I FEMS
Nitrate
50
90
80
Sulfide
J
+
50
30
20
I
10
Fig. 2. Sulfide oxidation
ml-‘).
Microbiology
50
Sulfide
1
min
with nitrate by Desulfobulbus propionicus
(0.45 mg protein
buffer (50 mM Tris, 50 mM EDTA,
170 mM
Na2C0s, pH 9.0). The suspensions were stirred for
30 min at 35°C under Hz and then centrifuged at
10 000 X g for 20 min. The supernatant was adjusted
to pH 7.0 with 1 M HCl.
3. Results
3.1. St&de
Letters 144 (1996)
131
129-134
firmed by the consumption
of protons during the
first phase. The formation of other oxidized sulfur
compounds would have been coupled to acidification. In the absence of oxygen, nearly 75% of the
elemental sulfur formed was transformed to sulfide
due to sulfur disproportionation
[5] according to
4s’ + 4&o
+ 3HS- + 5H+ + So;-
(2)
This phase was accompanied by Ht production.
By contrast, with D. desulfuricans CSN or Essex 6,
or strain PlB sulfide back-formation
started during
oxygen reduction (Fig. lb). Both the rate of aerobic
respiration
and that of sulfide back-formation
stopped when oxygen was consumed. The addition
of hydrogen increased both rates (Fig. lc). Thus, elemental sulfur and oxygen were reduced at the same
time.
The amount of transiently consumed sulfide in the
first step depended on the sulfide-to-oxygen
ratio.
The more sulfide was present, the greater the amount
which disappeared during the first phase.
3.2. SulJde oxidation with nitrate and nitrite
When oxygen was replaced by nitrate or nitrite as
electron acceptor Db. propionicus oxidized sulfide in
a biphasic reaction as found with 02 [5]. Up to 4
sulfide molecules per added nitrate disappeared transiently (Fig. 2) confirming that elemental sulfur was
oxidation with oxygen
When oxygen (13-33 PM) was added to washed
cells in the presence of excess sulfide (45-120 PM),
all of the five strains tested oxidized sulfide in a biphasic reaction. Sulfide was consumed quickly after
the addition of oxygen. In a second phase, part of
the sulfide consumed was restored.
With strain Z-7935 sulfide back-formation
started
when the added oxygen was consumed (Fig. la) as
described for Db. propionicus [5]. The stoichiometry
of sulfide consumption
during the first step and the
pH effect indicated the intermediary
formation
of
elemental sulfur according to
2HS- + O2 + 2H+ --) 2s’ + 2H20
That elemental
sulfur was the intermediate
(1)
was con-
42.5
t
25
L
5
Q,
win
Fig. 3. Oxygen reduction with sulfide by the periplasmic fraction
(0.13 mg protein ml-‘) of Desulfovibrio demlfuricans CSN.
132
K. Fuseler et ul. IFEMS
+HS-
i- .._._
-+
,e,
\-__
Hz0 V-(4+)
e:.q;.T
,_------*’
\\
/G’
so
’ kj
.---’
L
,,’
Microbiology Letters 144 (1996) 129-134
\_
0,
(NO,-, NO,-)
,I
,’
_,’
so42-
Fig. 4. Model of the common pathway of sulfide oxidation in
sulfate-reducing
bacteria. First, sulfide is oxidized to elemental
sulfur with oxygen (or nitrate or nitrite) as electron acceptor (A).
In a second step, elemental sulfur is disproportionated
(B).
Strains, starting sulfide back-formation
after oxygen reduction,
reduce oxygen only with electrons released during sulfide oxidation to elemental sulfur. Strains, which start sulfide back-formation during oxygen reduction, reduce oxygen also with the electrons released during elemental sulfur oxidation to sulfate (C).
the intermediate
according
to
2HS- + 2HsS + NO, + 4H+ + 4s” + NH; + 3&O
oxygen-containing
assays 0.5 oxygen per added sulfide were consumed indicating sulfur formation (Fig.
3). The oxygen was consumed at a rate of 84 nmol
02 mine1 (mg protein))‘. This result was surprising,
because sulfide oxidation with whole cells of D. desulfuricans CSN was not observed under these assay
conditions [l]. Control experiments in the absence of
protein indicated that the chemical sulfide oxidation
was very slow (51 nmol 0s min-l).
Oxidation of elemental sulfur by the periplasmic
fraction was not found. In the presence of reduced
glutathione
(GSH) and elemental sulfur chemical
oxygen consumption
took place, while the agents
alone did not consume oxygen. When elemental sulfur was added to assays of the periplasmic fraction
containing 30 uM oxygen and 1.7 mM GSH biologically catalyzed oxygen consumption
was observed
at a rate of 95 nmol 02 min-’ (mg protein))’
(the
rate of the chemical reaction (6 nmol 02 mm’) was
subtracted). During the phase of oxygen consumption low sulfide production was observed, however,
it is still uncertain whether this kind of sulfide production was catalyzed biologically.
(3)
The stoichiometry
of sulfide consumption
at the
end of the reaction indicated that sulfide was oxidized to sulfate with nitrate as electron acceptor
(Fig. 2) as described by
HS
+ HsS + 2N0,
+ 2HaO + H+ + 2SO,;- + 2NH;
(4)
While the ability of nitrite reduction to ammonia
was constitutively present, that of nitrate reduction
had to be induced by growing the cells in nitratecontaining medium as described earlier [6]. Chemical
oxidation of sulfide with nitrate was not observed.
3.3. Suljide oxidation b-v the periplasmic fraction
D. desulfuricans CSN
of
The capacity of sulfide oxidation with oxygen was
detected in the periplasmic fraction of D. desulfuricans CSN. When oxygen was added to sulfide-containing assays, sulfide consumption
and oxygen reduction
were observed,
while no sulfide backformation took place. When sulfide was added to
4. Discussion
From the present study we conclude that a common mechanism of sulfide oxidation is present in
different sulfate-reducing bacteria. Like most colourless sulfur-oxidizing
and anoxygenic phototrophic
bacteria the strains tested oxidized sulfide via elemental sulfur [9-l 11. The combination of sulfide oxidation to sulfur and sulfur disproportionation
allows
a complete oxidation to sulfate as recently shown for
Db. propionicus [5]. While enzyme preparations
of
sulfite reductases from Chromatium vinosum and D.
desuljiiricans Essex 6 catalyzed the oxidation of sulfide to sulfite [12,13], our data gave no indication for
the involvement of a sulfite reductase operating in
the oxidative direction. Thus, sulfide oxidation is
not the direct reversal of sulfate reduction (Fig. 4).
Differences between the strains were observed concerning the beginning of sulfide back-formation
by
sulfur disproportionation.
While in some strains sulfide back-formation
started after the consumption of
oxygen, in others sulfide back-formation
occurred
during oxygen consumption.
These differences ap-
K. Fuseler et al. I FEMS Microbiology Letters 144 (1996) 129-134
pear to depend on the rate of sulfide oxidation.
When sulfide is oxidized slower than it is formed
back by sulfur disproportionation,
sulfide back-formation occurs in the presence of oxygen. In these
cases, it became obvious that the reduction of oxygen and sulfur was coupled. The rates of aerobic
respiration
and sulfide back-formation
stopped at
the same time when the oxygen was consumed, and
both rates could be increased by the addition of hydrogen as electron donor. While D. desulfuricans reduced oxygen prior to nitrate, sulfate, sulfite or thiosulfate, possibly because of the more favourable
redox potential [6], the cells did not differentiate between elemental sulfur and oxygen. The electrons
released during the oxidative part of elemental sulfur
disproportionation
were consumed
simultaneously
by the reduction of elemental sulfur and oxygen
(Fig. 4).
Since the same biphasic pathway of sulfide oxidation was observed when molecular oxygen was replaced by nitrate or nitrite as electron acceptor, the
oxidation of elemental sulfur and sulfide was independent of molecular oxygen as electron acceptor
and is not catalyzed by an oxygenase as reported
for Thiobacillus thiooxidans [14].
It has been demonstrated that sulfide-oxidizing activity is located in the periplasmic fraction of D.
desulfuricans CSN. The transformation
of elemental
sulfur depended on the presence of GSH as described
for T. thiooxidans and T. ferrooxidans indicating the
involvement of thiol compounds [15,16]. Along the
same lines, the inhibition of sulfur-oxidizing
activity
by sulfbydryl-binding
agents like N-ethylmaleimide
(NEM) for T. ferrooxidans [9] and Db. propionicus
[5] was found.
A feasible hypothesis to explain several of our observations would assume the involvement of a periplasmic cytochrome c, which was already described
as being capable of reducing So [ 171 and is known to
undergo autoxidation
by 02 [18].
Acknowledgments
We thank Dr. Tatjana Zhilina, Dr. Georgy Zavarzin, Pave1 Sigalevich and Dr. Yehuda Cohen for providing bacterial cultures. This work was supported
by the Deutsche Forschungsgemeinschaft.
133
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