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FEMS Microbiology Letters 71 (1990)123-128 123 Published by Elsevier FEMSLE 04119 Aerobic respiration in sulfate-reducing bacteria * Wahraud Dilling and Heribert Cypionka Faku#iit OirBiologie~ Unwersttiit Konstan=.Konstanz, F R.G. Received 20 April 1990 Revision r~ceived 2 May 1990 Accepted 4 May 1990 Key words: Sulfate reduction; Aerobic respiration in anaerobes; Oxidation of sulfur compounds; Desulfovibrio desulfuri6~ms 1. SUMMARY Cultures of Desulfovibrio desulfuri¢~ms strain CSN (incubated in a sulfide- and sulfate-free medium) reduced up to 5 mM 0 2 with H 2 as electron donor. Aerobic respiration was not cou- pled with growth, but resulted in ATP formation. Washed cells incubated in H ~-saturated phosphate buffer revealed respiration rates of up to 250 nmol 0 2 rain i rag protein- i. The uncoupler earbonyl- cyanide m-chlorophcnylhydrazone (CCCP) stimulated the respiration rate and abolished ATP formation. The terminal oxidase has not yet been identified. Respiration was microaerophilic, insen- sitive to cyanide and azide, but inhibited after heat treatment of the cells (80" C for 10 min). The pH optimum was at pH 6 with less than 5070 activity at pH 4,5 and pH 9. Besides H 2, organic electron donors (formate, ethanol, lactate or pyruvate) and inorganic sulfur compounds (H2S, thiosulfate, sulfite) were used as electron donors for aerobic respiration. Sulfite and thiosulfate were * Dedicated to Prof. Dr. Norbert Pfennig on the oc:asion of his 6Sth birthday. Correspondence to: Heribert Cypionka, Fakult~it fiir Biologic, Llnivets|t[itKonstan:z,P~tfach 5560,D-7750Konstanz, F.R.G. oxidized completely to sulfate. The capability of aerobic respiration was also detected in Oesulfo- vibrio vulgaris. D. sulfodismutans, Desulfobacterium autotrophicum. Desulfobulbus propionicus, and De- sulfococc~s multworans. 2, INTRODUCTION Since their first description in 1895 by Be- ijerinck [1], dissimilatory sulfate-reducing bacteria are known as strict anaerobes. Molecular oxygen blocks dissimilatory sulfate reduction and growth. However, sulfate-reducing bacteria are found near the oxic/anoxic interface in sediments or micro- bial mats [2]. In the absence of H2S and thiols, the cells may survive oxic conditions without loss of viability [3]. Sulfate reducers may even take ad- vantage of the vicinity of O 2, if the (biological or purely chemical) oxidation of H2S leads to the formation of oxidized sulfur compounds which can be used as electron acceptor by them [3]. Here we report on the discovery that several sulfate-re- ducing bacteria are able to utilize molecular oxygen directly as electron acceptor. So far, no aerobic growth was obtained, but the cells revealed respi- ration rates comparable to those of aerobic bacteria. Respiration was coupled to energy con- 0378-IG97/gO/$03.50 © 1990 Federation of European Microbiological Societies

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Page 1: Aerobic respiration in sulfate-reducing bacteria

FEMS Microbiology Letters 71 (1990) 123-128 123 Published by Elsevier

FEMSLE 04119

Aerobic respiration in sulfate-reducing bacteria *

Wahraud Di l l ing and Heriber t Cypionka

Faku#iit Oir Biologie~ Unwersttiit Konstan=. Konstanz, F R. G.

Received 20 April 1990 Revision r~ceived 2 May 1990

Accepted 4 May 1990

Key words: Sulfate reduction; Aerobic respiration in anaerobes; Oxidation of sulfur compounds; Desulfovibrio desulfuri6~ms

1. SUMMARY

Cultures of Desulfovibrio desulfuri¢~ms strain CSN (incubated in a sulfide- and sulfate-free medium) reduced up to 5 mM 0 2 with H 2 as electron donor. Aerobic respiration was not cou- pled with growth, but resulted in ATP formation. Washed cells incubated in H ~-saturated phosphate buffer revealed respiration rates of up to 250 nmol 0 2 rain i rag protein- i. The uncoupler earbonyl- cyanide m-chlorophcnylhydrazone (CCCP) stimulated the respiration rate and abolished ATP formation. The terminal oxidase has not yet been identified. Respiration was microaerophilic, insen- sitive to cyanide and azide, but inhibited after heat treatment of the cells (80" C for 10 min). The pH optimum was at pH 6 with less than 5070 activity at pH 4,5 and pH 9. Besides H 2, organic electron donors (formate, ethanol, lactate or pyruvate) and inorganic sulfur compounds (H2S, thiosulfate, sulfite) were used as electron donors for aerobic respiration. Sulfite and thiosulfate were

* Dedicated to Prof. Dr. Norbert Pfennig on the oc:asion of his 6Sth birthday.

Correspondence to: Heribert Cypionka, Fakult~it fiir Biologic, Llnivets|t[it Konstan:z, P~tfach 5560, D-7750 Konstanz, F.R.G.

oxidized completely to sulfate. The capability of aerobic respiration was also detected in Oesulfo- vibrio vulgaris. D. sulfodismutans, Desulfobacterium autotrophicum. Desulfobulbus propionicus, and De- sulfococc~s multworans.

2, INTRODUCTION

Since their first description in 1895 by Be- ijerinck [1], dissimilatory sulfate-reducing bacteria are known as strict anaerobes. Molecular oxygen blocks dissimilatory sulfate reduction and growth. However, sulfate-reducing bacteria are found near the oxic/anoxic interface in sediments or micro- bial mats [2]. In the absence of H2S and thiols, the cells may survive oxic conditions without loss of viability [3]. Sulfate reducers may even take ad- vantage of the vicinity of O 2, if the (biological or purely chemical) oxidation of H2S leads to the formation of oxidized sulfur compounds which can be used as electron acceptor by them [3]. Here we report on the discovery that several sulfate-re- ducing bacteria are able to utilize molecular oxygen directly as electron acceptor. So far, no aerobic growth was obtained, but the cells revealed respi- ration rates comparable to those of aerobic bacteria. Respiration was coupled to energy con-

0378-IG97/gO/$03.50 © 1990 Federation of European Microbiological Societies

Page 2: Aerobic respiration in sulfate-reducing bacteria

]24

servation, and allowed the complete oxidation of sulfur compounds instead of sulfate reduction. Concerning different modes of respiration, De- sulfovibrio desulfuricans strain CSN appears to be the most versatile organism known today.

3. MATERIALS AND METItODS

3.1. Organisms and cultivation The bacterial strains used (see Table 1) were

grown with H 2 and CO 2 or lactate, with sulfate (10 raM) or nitrate (10 raM) as electron acceptor in the basal medium described previously [4].

3.2. Determination of 02 Oxygen in the gas phase was determined by

means of a gas chromatograph (Carlo Erba, Rodano, Italy, model FTV 4300) using a molecu- lar sieve column (1.8 m, 60/:80 mesh, 80°C) with helium as carrier and a hot wire detector. Respira- tion of washed cell suspensions was measured by means of an oxygen electrode (Yellow Springs Instruments, Model 59). Washed cells were in- cubated at 30°C in N:sa tara ted 50 mM phos- phate buffer, pH 7. O 2 and H 2 were added as 02- or H z .aturated buffer assumed to contain 1.17 mM O 2 or 0.68 mM H 2 at atmospheric pressure and 25°C.

Table I

O 2 reduction in various sulfate-reducing bacteda

Sulfate-reducing bacterium Capability of O z reduction a

Desulfooibrio desulfuricans Strain CSN 141 q- + Desulfooibrio de*ulfurkxms Strain Essex

(DSM 642) + + Desulfovibrio vulgaris Strain Marburg

(DSM 2119) + Desulfovtbrio salexigens (DSM 2638) Desldfol d,r,o ,djodismut,, ,' IDSM 3696) + Desulfouibrio oriemis tNCIB 8382) -- Desulfobacterium autotrophiclmt

(DSM 3382) + Desulfobldbus propionicus tDSM 2032) + Desulfocoecus mutttvorans (DSM 2059) (+)

a Washed cell suspensions were tested in the presence of H~, lactate, H2S and sulfite as deenon donors, Soveral strains could not use all of the electron donors for 02 reduction.

3.3. Determination of ATP ATP was determined after extraction with per-

chloric acid [5] by the hiciferin-luciferase method using a kit from LKB (No. 1243-107) in a LKB 1250 luminometer (LKB-Wallac, Finland).

4. RESULTS AND DISCUSSION

4.1. 0 2 uptake by cultures of Desul[ovibrio de- sulfuricans CSN

In a study on respiration-driven proton translo- cation by Desulfovibrio desulfurieans, we observed the highest H + / H 2 values with O 2, but not with electron acceptors used by the strain during growth (sulfate, sulfite, thiosulfate, nitrate, nitrite) [6]. Therefore, we started an investigation on 02 re- duction by sulfate-reducing bacteria. Since H2S is known to undergo spontaneous autoxidation for- ming toxic compounds [3], a nitrate-reducing= ~.t aia in a sulfate-free medium was used.

D. desulfurican.~ strain CSN [4,7] was incubated in a sulfate- and sulfide-free growth medium (200 ml) under a gas phase (920 ml) containing 80% (v/v) H 2 and 20% CO 2. In intervals, a total of 25 ml (1 mmol) O 2 was added to the headspace. The culture bottles were vigorously shaken. In these experiments, 5 or 10 ml 02 was consumed within one or two days (Fig. 1). There was no aerobic growth in our experiments - - the increase of the optical density in Fig. 1 is due to the reduction of nitrate, which was present in low concentration (1 mM). Because a small concentration of thlosulfate (50 tiM, as sulfur source for biosynthesis) was present in our growth experiments, we were not immediately sure whether 02 consumption oc- curred via formation of H2S from thiosulfate and H2, and subsequent chemical oxidation of H2S by O 2. However, O a reduction was also obtained with washed cells of D. desntfuritxms in phosphate buffer with no sulfur compound present.

4.2. 02 reduction by washed ceils of D. desulfaricans Under an H 2 atmosphere, washed cells of D.

desulfuricans reduced 02 pulses (added as 02- saturated buffer) with rates of up to 250 nmol 02 min- L mg protein- i. These rates are comparable to those of aerobic bacteria such as Escheriehia

Page 3: Aerobic respiration in sulfate-reducing bacteria

1 • 5 0 i i i i i ~ i i i

II

II

II 1 .20 ~ _ 0--2 '~ G r o w t h --

0 . 9 0

0 . 6 0

4 - - - 5 n l 0 2 - ' - - I ~ 1 ~ 4 - - - 5 n l 0 2

" I 'In

0 . 0 0 I I I I I 0 . 0 0 0 0 4 0 8 0 1 2 0 I GO 2 0 0

T L ~ z Q ( h ) Fig. I. Oxygen uptake by a culture of Dezulfovibrio desulfuricans CSN. The cells were vigorously shaken at 30°C in a sulfide-free growth medium (200 ml) under a heedspace (920 ml) containing 80% (v/v) H 2 and 20% CO.,. The mineral medium contained 1 mM nitrate (additional electron acceptor). 2 mM acetate (additional carbon sourCe) and 50 ~M thiosulfate (sulfur source), In intervals, 5

or 10 ml O: was added to the headspac¢. Growth was followed as optical density, and O 2 by means o[ a gas chromatograph.

%

0 • 250

C]

r'1 0 . ~ 0 0

Q

0 . 1 5 0 ~r~

0 . 1 0 0 ~

0 . 0 ~ ) ~

coil Respiration was iaicroaerophilic. Maximum rates were observed below 4% air saturation (10 p.M dissolved 02). Oxygen reduction with lower rates (25-50 nmol 02 min - t mg protein - t ) was obtained if H 2 was added to cells incubated in the presence of 1% 02 saturation (Fig. 2). Organic substrates used for aerobic respiration were for-

t .... 'ore t++ . . . .

I " t~ce

Fig. 2. Aerobic H~ oxidation by Oestdfaoibrio desulfuricons CSN. Washed cells (0.16 mg protein/ml) were incubated at 300(2 in N2-saturated phosphate buffer (50 raM, pH 7.0). The additions arc indicated in nmol. Addition of uncoupler (50 pM

CCCP) led to increased respiration rates.

mate, lactate, pyruvate, and ethanol. The latter three compounds were incompletely oxidized to acetate as typical for D. desulfuricans (consump- tion of 1 O2 per lactate or ethanol, and 0.5 02 per pyruvate).

The oxygen-reactive compound in D. de- sulfuric~ms CSN has not yet been identified. Res- piration was insensitive to the classical inhibitors cyanide and azide (1 mM each), but was in- activated by heat tratment of the cells (17% resid- ual activity after incubation at 800C for 10 min, 0.6% activity after cooking for 15 rain). The pH optimum of H 2 oxidation with 02 was at 6.0; less than 50% activity was obtained at pH values 4.5 and 9. The cells contained cytoehromes (mainly cytochrome c). Catalase activity was not detected.

The capacity of aerobic respiration was present constitutively after growth with sulfate or nitrate. Immediately after changing to anoxic conditions, the cells were able to reduce sulfate to H2S.

Page 4: Aerobic respiration in sulfate-reducing bacteria

126

4.3. Coupling to energy conservation 02 reduction was coupled to energy conserva-

tion. This follows from the experiments on vec- torial proton translocation described above [6]. Furthermore, H~ oxidation with 02 was coupled to A T P formation (Fig. 3). ATP formation could be blocked by an uncoupler (CCCP), which at the same time st imulated the respirat ion rate (Figs. 2 and 3).

4.4. Oxidation of sulfur compounds Besides H z and organic substrates, reduced

sulfur compounds were utilized as substrates for

s.o

4.o 1~ ~ Ha-aatur.teu I~,r ~ r

i 3 . O

O.O ' r ' I J

B • N2-~t ,~aten b~fvQr • 4.0 ~ at 1~. i1~ aaturatLon

i3.O

z.o

o . o , , r i , i i T i i I i • ~o l:m *eo 24o

vi~e ~see)

Fig. 3. ATP formation during Hz oxidation with O z by De- sulfov~brio desulfuricans CSN. Washed cells (1.6 mg protein/ml) were incubated in phosphate buffer at pH 7. (A) ATP forma- tion upon O~ addition to cells incubated in H~-saturated buffer (0). (B) ATP formation upon H~ addition to cells incubated at l~ O 2 saturation (m). Control ¢xporimenls (@. to) were carried oat in the presence of an uncoupler (50 ~M

CCCP).

3o s;~o~" 30 ~z °~3 -

g 6002 ~-~

60 0 2

S rain b

0

Fig. 4. Complete oxidation of Ihio~ulfat¢ by Desulfovibrio desulfuricans CSN. Conditions as described in Fig. 2, except that the call concentration was 0.35 mg protein/ml. Additions

are indicated in nmol.

aerobic respiration. Thiosulfa te (Fig. 4) and sulfite were completely oxidized to sulfate consuming 2.0 and 0 . 5 0 ~ , respectively. While sulfite could also be oxidized by ceil-free extract of D. desulfuricans, thiosulfate was oxidized by whole ceils only. Sulfi te oxidat ion by cell-free extract resulted in A T P for- mat ion (up to 0.5 A T P per sulfite) indieathag the involvement of A T P sulfurylase [7].

HaS was slowly oxidized with Ot ( there was no significant autoxidat ion at mieromolar concentra- tions of H2S and 0 2 ) or, under anoxic condit ions. wi th ni t ra te (detected by means of a sulfide elec- trode). We did not determine the stoichiometry of sulfide oxidat ion in our prel iminary experiments. It appears, however, that D. aesulfurivans CSN can carry out all steps of the sulfur cycle in reductive as well as in oxidat ive direction.

4.5, Aerobic respiration by various sulfate-reducing bacteria

The capabil i ty of aerobic respirat ion appears common among freshwater and mar ine sulfate reducers. Out of 9 strains studied so far, at least 6 reduced oxygen with ei ther H2, lactate, H~S or sulfite (Tab le 1). In most cases, the respirat ion

Page 5: Aerobic respiration in sulfate-reducing bacteria

Table 2 Different Uthotrophic m~le~ of energy conservation in De- sulfovibrio dcsulfurica~¢ CS,~I

Reduclion of inorganic sulfur compounds Sulfate reduction 4H2+SO4 z- + H + ~ H S +4}t20 Thiosulfate reduction 4Hz+S20 ~- ~ 2 H S +3H20 Sulfit~ reduction 3H2+HSO j- ~ H S - + 3 H e O Sulfur reduction H2 + S ° ~ H S - +H +

Ferawntation of inorganic su(fur compounds Thiosulfate disproponionation s~o~- + H ~ O ~ H S - +SOl- +H ~ Sulfite dispmportionation 4HSO~" ~ HS - + 3SO~ - + 3H ÷

Reduction of oxidized nitrogen compounds Nitrate reduction to ammonia 4H2 +NO ~- +2H + ~ N H ~ +3H20 Nitrite reduedon to ammonia 3H 2 +NO 2- +2H + ~ N H 2 +2H20

ReductioR Of oxygen O z reduction with H 2 ( ' KnallgaLs' reaction) 2H2 +O2 ~ 2H20 Oxidation of sulfur compounds SaO ~- +202+HzO~2SO42- +2H + 2HSO 3 + O z ~ 2 S O $- +2H*

rates were lower than wi th D. desulfurieans, an d n o t al l of the subs t ra tes were used. Somet imes , O 2 u p t ak e was on ly observed af ter p re incaba t ion un- d e r H 2, b u t no t unde r N 2 at low O z concent ra t ion .

4. 6. Conclusions Derulfovibrio desulfuricans C S N has the mos t

versat i le respi ra tory me tabo l i sm kno w n so far (Ta- ble 2). Th e re is no o ther o rgan i sm k n o w n that can

use the reduc t ion of 0 2, NO~ ' , NO~ ' , SO~ - , S O ~ - , SzO ~ - or S O for energy conservat ion . All these e lect ron acceptors are used wi th H 2 as electron d o n o r ( l i tho t rophic energy conservat ion) . F u r t l ~ - more , the s t ra in can carry ou t a l i tho t rophic fer- men t a t i o n by d i sp ropor t iona t ion of th iosal fa te or

~27

sulfite to sulfate plus H 2 S [7,8], a n d the comple te oxida t ion of su l fur co mp o unds .

Recent ly , su l fu r -me tabo l i z ing a rchaebac te r ia were descr ibed that can oxidize H 2 S in the pre~- euce of 02 , an d reduce sulfur to H 2 S u n d e r anoxic condi t ions [9]. However , these bacter ia c a n n o t re- duce sulfate, n i t ra te or nitr i te.

A l t h o u g h sul fa te- reducing bacter ia (so far) d id no t grow wi th O2, their capabi l i ty of aerobic respi- ra t ion migh t be of ecological impor tance . U p 1o 50% of the O 2 c o n s u m p t i o n in m a r i n e s ed imen t s is d u e to ox ida t ion of H 2 S [10]. Su l fa te - reduc ing bacter ia r iving near the o x i c / a n o x i c b o u n d a r y in sed iments m a y co mp e t e wi th ae rob ic sulfur-oxidiz- ing bacter ia l ike Thiobacillus o r Beggiatoa.

A C K N O W L E D G E M E N T S

W e t h an k S i m o n e D a n n e n b e r g , Michae l Kroder , Chcis toph Marseha l l and H e n r i k Sass who con t r ibu ted valuable exper iments to this s tudy. T h i s work was suppor t ed by a g ran t of the Deutsche Forschungsgemeinsehaf t .

R E F E R E N C E S

[1] Beijernick, W.M. (1895) Centralbl. Bakteriol. It. Abt. 1, 1-9; 49-59; 104-114.

[21 Jorgensen, B.B. (1977) Marine Biol. 41, 7-17. [3l Cypionka, H., Widde|, F. and Pfennig, N. (1985) FEMS

Microbiol. Ecol. 27.189-193. [41 Cypionka. H. (1989) Arch. MicrobioL 152. 237-243. [5] Blaut, M. and Gouschalk, G. (1984) Eur. J. Biochem. 141,

217-222. [6] Fitz, R. and Cypionka, H. (1989) Arch. Microbiol. 152,

369-376. [7] gariimer, M. and Cypionka, H. (1989) Arch. Mierobiol.

151, 232-237. |8l Bak, F. and Cypionka, H. (1987) Nature 326. 891-892. {9] Stetter, K.O. (1989) in Autotrophic Bacteria (Schleg¢l,

H.G. and Bowie, B., eds.), pp. 167-176, Science Teeh Publ., Madison.

[10l 3orgensen. B.B. (1982) Nature 296, 643-645.