Archives of

Arch Mlcrobiol (1990) 153:399-404

Microbiology 9 Springer-Verlag 1990

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov. Hang Min* and S. H. Zinder Department of Mmrobiology, Stocking Hall, Cornell University, Ithaca, NY 14853, USA Received October 5, 1989/Accepted November 29, 1989

Abstract. An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58~ thermophilic anaerobic bioreactor. The cells of strain C A M Z were 0.7 gm by 2 - 5 gm rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The G r a m stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H2/CO2. Lactate and pyruvate were oxidized completely to CO2 with sulfate as electron acceptor. Sulfate was required for growth on Hz/COz, and both acetate and sulfide were produced from Hz/COz-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain C A M Z was 5 5 - 6 0 ~ and the optimum p H value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain C A M Z grown on lactate, H2/CO2, or acetate with sulfate were 7.2, 18.1, and 30.8 gmol methyl viologen reduced rain-1 [mg protein]-1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The m o l % - G + C of strain CAMZ's D N A was 49.7. The new species name Desulfotomaeulum thermoaeetoxidans is proposed for strain CAMZ. Key words: Sulfate reduction - Thermophile - Carbonmonoxide dehydrogenase - Acetate oxidation Acetogen - Desulfotomaculum thermoacetoxidans

diversity of sulfate-reducing bacteria, especially due to the studies of Widdel, Pfennig and collaborators (Pfennig et al. 1981). An important finding was the existence of sulfate reducers which are capable of oxidizing acetate, an important intermediate in anaerobic metabolism. Some sulfate reducers, such as Desulfobacter postgatei and Desulfobacter hydrogenophilus, use variations on the citric acid cycle for acetate oxidation (reviewed by Thauer, 1988), while others, such as Desulfovibrio baarsii, Desulfobacterium autotrophicum, and Desulfotomaculum acetoxidans (Schauder et al. 1986; Spormann and Thauer 1988; Schauder et al. 1989) use a pathway resembling a reversal of the acetogenic pathway (Fuchs 1986; Ljungdahl 1986) in which a key enzyme has carbon monoxide dehydrogenase activity. In the genus Desulfotomaculum, which consists of spore-forming sulfate reducers, D. aeetoxidans and D. sapomandens are the two mesophilic species which can oxidize acetate (Widdel and Pfennig 1981 ; Cord-Ruwisch and Garcia 1985). Recently, a thermophilic species, D. geothermicum, was isolated from saline geothermal groundwater. It was capable of oxidizing fatty acids longer than acetate to CO2, but was unable to grow on acetate. In the present study, we report the isolation and characterization of a thermophilic spore-forming sulfatereducing bacterium, strain CAMZ, which can use acetate, short chain fatty acids, and alcohols as electron donors and can form acetate from H2/CO2 in the presence of sulfate.

Materials and methods

Culture medium and growth conditions In the past decade, there has been a growing appreciation and understanding of physiological and morphological

* Present address. Department of Environmental Science, Zhejiang Agricultural University, Hangzhou, Peoples Republic of China Offprint requests to: S. H. Zinder

The basal culture medium and anaerobic glove-box techniques were those described for culturing a thermophilic Methanothrix by Zinder et al. (1987) except that mercaptoethane sulfonate was omitted as a reducing agent, and other energy sources, such as 10 mM lactate and 10 mM sulfate were added instead of acetate when desired. Solid agar medium contained the same ingredients except that 20 g/1 prepurified agar (Difco, Detroit, MI, USA) was added. Cul-

400 tures were generally incubated at 58~ The final pH of medium was 6.5 at 60~ Cultures were grown in 5 or 10 ml amounts in 27 ml aluminlum crimp tubes with butyl stoppers (Bellco Biotechnol., Vineland, N J, USA), in 50 ml amounts in butyl rubber-stoppered 120 ml serum vials or m 300 ml rubber stoppered side-arm flasks, or in 500 ml amounts in 1 liter rubber-stoppered bottles. Roll tubes contained 7 ml of agar-solidified medium in 27 ml aluminum crimp tubes.

Isolation and determination of purity of isolate The culture was isolated by tenfold serial dilution of a thermophilic methanogenic benzoate enrichment (see Results) into roll tubes containing agar medium with 10 mM each of sodium lactate and sodium sulfate. Isolated colonies formed within a few days and were picked with sterile Pasteur pipettes and were serially diluted and reisolated in roll tubes. The isolated colonies were transferred into liquid medium. The purity of isolate was checked microscopically and by inoculating the culture into medium containing yeast extract or medium with sugars (glucose, sucrose, xylose and cellobiose).

Physiological studies Optical densities of cultures grown in 18 x 150 mm aluminum-crimp tubes were recorded at 600 nm using a Sequoia 340 digital Spectrophotometer (Fisher Scientific, Rochester, NY, USA). Reported results represent the average of duplicate cultures. Cardinal temperatures were determmed by incubating cultures in water baths with different temperatures. The heat-resistance of spores was determined by incubating freshly inoculated cultures at 90~ and 100~ for the indicated times followed by incubahon at 60~ and by measuring the optical density of the cultures eight days later. The effect of medium pH on growth was studied by manipulating the CO2-bicarbonate equilibrium as previously described (Zinder et al. 1987).

Chemical determinations Samples for sulfide analysis taken from both the liquid and the headspace were preserved in zinc acetate and assayed by the methylene blue method as described by Brock et al. (1971). Gas samples were taken using I ml Glaspak syringes (Fisher Scientific) fitted with Mininert valves (Supelco, Inc., Beltefonte, PA, USA) to maintain pressure. Methane was measured using a GowMac 550 thermal conductivity gas chromatograph (Gow-Mac Inst. Co., Bound Brook, N J, USA) using He as a carrier gas as previously described (Zinder and Koch 1984). H2 was determined using a Carle series 100 thermistor detector gas chromatograph with N2 as carrier gas as previously described (Lee and Zinder 1988). Aqueous acetate was determined using high performance liquid chromatography as described by Zinder and Koch (1984).

Carbon monoxide dehydrogenase activity assay Cultures grown in 500 ml medium with either 10 mM lactate and 10 mM sulfate, 20 mM acetate and 10 mM sulfate, or H2/COz and 10 mM SO~-. Cells were harvested inside an anaerobic glove box using a Sorval SS-34 centrifuge operated at 5,000 rpm (3,000 x g) for 15 min and washing the pellet in assay buffer. The cell suspensions were loaded into a French pressure cell (SLM/Aminco, Urbana, IL, USA) and were broken twice at ca. 1,000 atm (100 MPa) using a Carver Laboratory Press (Fred S. Carver Inc., Summit, N J, USA). Disrupted cells were collected in a vial flushed with nitrogen or argon, and cell debris was collected removed by anaerobic centrifugation. Assay tubes (13 x 100 mm) were filled with 3 ml of

25 mM N-2-Hydroxyethylpiperazlne-N1-2-ethanesulfonlc acid (HEPES) pH 7.5 with 5 mM B-mercaptoethanol and were fitted with a rubber stopper in an anaerobic glove box. These tubes were evacuated and flushed with CO gas followed by addition of methyl viologen to a final concentration of 1 mM. The tubes were heated to 60~C, using a block heater (American Scientific Products, McGaw Park, IL, USA). The reaction was initiated with 0.1 ml of an appropriate dilution of crude extract of strain CAMZ cells. The absorbance at 578 nm was measured every 5 min using a Sequoia 340 Digital Spectrophotometer and the results were converted to activity using an extinction coefficient of 9.8 mM - I cm -~ for reduced methyl viologen. Protein was determined by the Coomassie brilliant blue method using reagents purchased from Bio-Rad (Richmond, CA, USA). Bovine serum albumin (BSA) (Sigma Chemical Co., St Lores, MO, USA) was used as a protein standard and the results were corrected for the anomalously high reading for BSA.

DNA G + C ratio DNA was purified by use of hydroxyapatite (Herdman et al. 1979) and its melting point was determined as previously described (Lee and Zinder 1988). The result represents the average of three determinations, and Escherichia coli DNA (51 tool% G + C) was used as a standard.

Chemicals All chemicals used were at least reagent grade.

Results

Isolation, morphology and tool% G + C The culture was derived f r o m a 58 ~ C e n r i c h m e n t converting 2 m M b e n z o a t e to m e t h a n e in which acetate a c c u m u lated as a t r a n s i e n t intermediate. The e n r i c h m e n t was derived from a t h e r m o p h i l i c (58~ a n a e r o b i c digestor c o n v e r t i n g cellulosic waste to m e t h a n e ( Z i n d e r et al. 1984). The three m a j o r m o r p h o t y p e s were a n a u t o fluorescent rod r e s e m b l i n g Methanobacterium thermoautotrophicum, a sheathed rod resembling Methanothrix, a n d a l e m o n - s h a p e d s p o r e - f o r m e r r e s e m b l i n g Desulfotomaculum acetoxidans. A t t e m p t s to isolate a b e n z o a t e oxidizer o n a l a w n of m e t h a n o g e n s in the m a n n e r o f M o u n t f o r t a n d B r y a n t (1982) did n o t succeed. Since the spore-former bore a s t r o n g m o r p h o l o g i c a l r e s e m b l a n c e to Desulfotomaculum, the culture was diluted into anaerobic agar roll tubes with m e d i u m c o n t a i n i n g 10 m M each of lactate a n d sulfate. Colonies o b t a i n e d were transferred, isolated a n d checked for p u r i t y as described in Materials a n d methods. The isolated strain was f o u n d n o t to use b e n z o a t e with sulfate as a n electron acceptor, or in coculture with m e t h a n o g e n s . It was designated by the n a m e of C A M Z ( C h i n a - A m e r i c a - M i n - Z i n d e r ) . Cells of strain C A M Z were straight or slightly curved rods with p o i n t e d ends (Fig. 1 A), 0.7 g m by 2 - 5 g m in size, o c c u r r i n g single or in pairs. Weak motility was observed at r o o m t e m p e r a t u r e , with m o r e vigorous m o tility observed if the slide was heated to near 60 ~C. The f o r m a t i o n of p h a s e - b r i g h t spores was observed, which

401 Table 1. Growth of Strain CAMZ on different electron donors as electron acceptor with 10 mM SO 2- ~ Electron donor (raM)

Maximum optical density (at 600 nm)

Days needed to reach the maximum O.D.

Propionate (5) Pyruvate (5) Pyruvate (5) (without SO~-) Lactate (5) Acetate (5) Formate (5) Malate (5) Butyrate (5) Succinate (5) Valerate (5) Alanine (5) Propanol (10) Butanol (10) Iso-butanol (10) Pentanol (10) H2/CO2 (excess)

0.120 0.212

6 4

0.144 0.171 0.087 0.074 0.296 0.147 0.146 0.126 0.255 0.046 0.304 0.211 0.024 0.098

5 4 10 5 5 17 7 18 5 18 l8 18 18 6

Acetate produced (mM) 0 0 5.7 0.83 1.5 0 0 5.0 0 0.95 0 0.95 6.31 0 0.53 2.51

a Substrates tested but not used by strain CAMZ included: benzoate, glucose, sucrose, celtobiose, xylose, palmitate and ethanol b Residual acetate left in medmm 160

Acetate

15o ~

H2

--

O

o ,~

Fig. 1. A Phase contrast micrograph of vegetative cells of strata CAMZ. B Phase contrast micrograph of a mainly sporulated culture of strain CAMZ. Marker bar = 10 gm

E 3

140

2 v

were spherical a n d c e n t r a l a n d c a u s e d swelling in the cells (Fig. 1 B). G a s vesicles were n o t o b s e r v e d at a n y time. T h e cells s t a i n e d G r a m negative. T h e G + C r a t i o o f the D N A was d e t e r m i n e d to be 49.7 m o l % .

Sulfide

T

1

120" 5

Electron donors used by strain CAMZ E l e c t r o n d o n o r s used for sulfate r e d u c t i o n b y strain C A M Z i n c l u d e d lactate, p y r u v a t e , acetate, f o r m a t e , a n d a v a r i e t y o f s h o r t - c h a i n c a r b o x y l i c acids a n d a l c o h o l s (Table 1). P y r u v a t e c o u l d serve as a g r o w t h s u b s t r a t e in the a b s e n c e o f sulfate while l a c t a t e c o u l d not. T h e d o u b ling times o f strain C A M Z g r o w n o n lactate, acetate, a n d H2-CO2 were 11 h, 32 h, a n d 9 h, respectively. S o m e acetate was d e t e c t e d after g r o w t h o n lactate, b u t y r a t e , valerate, p r o p a n o l , b u t a n o l , or p e n t a n o l . G r o w t h with H2/CO2 r e q u i r e d the presence o f sulfate, a n d a c e t a t e was a l w a y s f o r m e d . A s s h o w n in Fig. 2, sulfide a n d acetate were p r o d u c e d s i m u l t a n e o u s l y w h e n g r o w i n g o n H 2 / C O 2 a n d m o r e a c e t a t e t h a n sulfide was f o r m e d .

<

130

Days

I 10

I 15

0

Fig. 2. Utilization of H 2 and production of sulfide and acetate by a culture of strain CAMZ grown at 58~ on Hz/COz-sutfate Table 2. Growth of strain CAMZ on 10 MM lactate with different electrons acceptors a Electron acceptor (10 mM except S~

Maximum optical density (600 nm)

Time required to reach maximum optical density

SO4 $202 S~

0.27 0.41 0.14

6 6 16

a No growth was detected with 10 mM SO~, NO;-, NO~, betame. or 2 mM SO~ as electron acceptor

402 Table 3. Effect of addition of NaC1 on growth of strain CAMZ, on

03

10 mM lactate + 10 mM sulfate

02

E (D

c5 d

Maximum optical density (600 nm)

Time required to reach maximum optical density days)

0 5 10 15 20 25

0.27 0.19 0.17 0.16 NG a NG a

6 12 15 15

a No growth

0.1

o.o

NaC1 added (g/l)

I 40

,

I 50

,

I 60

I 70

Temperature (~

Fig. 3. Effect of temperature on growth of strain CAMZ, as determined by measuring the optical density of cultures at 600 nm after four days incubation

Electron acceptors used by strain CAMZ Strain CAMZ could use SO4a -, $20 2-, and S ~ as electron acceptors with lactate as electron donor (Table 2). Growth with thiosulfate was always more rapid than with sulfate and higher culture densities were obtained. Sulfide concentrations rarely exceeded 2 mM, indicating sulfide toxicity, similar to most strains of Desulfotomaculum (Klemps et al. 1985). SO 2-, N O ; , NO~- and betaine could not serve as electron acceptors for growth of strain CAMZ. No growth occurred and no methane was formed in sulfate-free medium with lactate present as the electron donor when strain C A M Z was incubated in co-culture with the methanogen Methanobacterium thermoautotrophicum strain T H F (data not shown), which is capable of using either H 2 / C O 2 or formate (Zinder and Koch 1984). No significant methanogenesis was detected in cocultures in which strain C A M Z was grown with pyruvate in the absence of sulfate.

Nutrition The culture grew in a chemically defined medium, with the only organic nutrient additions being a vitamin solution (Balch et al. 1979). Cultures transferred when the vitamin solution was omitted did not grow more than one transfer (data not presented).

Effects of temperature, pH, and salinity on growth The optimum temperature for strain C A M Z was 5 5 60~ (Fig. 3) with little growth below 45~ or above 65~ The heat-resistance of the spores was tested.

Spores of strain C A M Z could survive at 90~ for 30 rain and 100~ for 10 min. Cultures were no longer viable after heating to 100~ for 15 min or longer (data not presented). The optimum pH range for growth of strain CAMZ was 6 . 5 - 7 . 0 with growth occurring from pH 6.0 to 7.5 (data not presented). Some sulfate-reducing bacteria, especially those isolated from saline habitats, require sodium chloride for their growth (Pfennig et al. 1981). In order to determine the salt-tolerance of strain CAMZ, cells were inoculated into the medium with sodium chloride concentrations of between 0 and 25 g/1 (Table 3). Sodium chloride was inhibitory, with no growth above 15 g/1 (0.26 M), clearly showing that strain CAMZ is not marine.

Carbon monoxide dehydrogenase activity The specific activities of carbon monoxide dehydrogenase of strain CAMZ grown on lactate, H2/CO2 and acetate with sulfate were 7.2, 18, and 31 gmol methyl viologen reduced m i n - 1 [mg protein]- 1 respectively.

Discussion

Because of its ability to use sulfate as an electron acceptor and to form spores, strain C A M Z should be placed in the genus Desulfotomaculum. Since it is capable of oxidizing acetate and is thermophilic we propose the name Desulfotomaculum thermoacetoxidans. Table 4 compares the properties of some species of Desulfotomaculum. Strain CAMZ bears considerable morphological and physiological resemblance to the three other Desulfotomaculum species able to use acetate: D. acetoxidans, D. sapomandens and D. geothermicum. Clearly, the resemblence between strain C A M Z and D. geothermicum is the closest, with the two cultures showing similar growth temperatures, substrate spectra, and mol% G + C in their DNA. However, there are also significant differences between them. Attributes present in strain C A M Z but not in D. geothermicum include the ability to grow at 60~ and the ability to grow on acetate. Attributes present in D. geothermicum but not in strain C A M Z include the ability to grow at salinities of 15 g/1 NaC1 and greater, the pres-

403 Table 4. Properties of various Desulfotomaculum species a Characteristic

D. nigrificans

D. orient&

D. acetoxidans

D. sapomandens

D. geothermicum

D. thermoacetoxidans

Optimum growth temperature mol% G + C NaC1 requirement Gas vesicles

55 45-47 . --

3 0 - 37 41.7 . --

37 37.5

38 48

5 5 - 60 49.7

+

+

54 50.4 +b +

Growth with: Acetate + SO2Propionate + SO2Butyrate + SO2Lactate + SO~Ethanol + SO~H2/CO2 + SO~Benzoate + SO4zPyruvate without SO2Lactate without SO2SO~- as e- acceptor $20~- as e- acceptor S~ as e- acceptor Methanogen as e acceptor

+ + + + + + +e

+ + + _ + ND

+ + + + _ ND + + + ND

+ + + + + ND d ND + + ND

+ + + + + + + + --

.

+ + + _ c + + + § f

.

--

a Data from Pfennig et al. 1981; Widdel and Pfennig 1981; Cord-Ruwisch and Garcia 1985; Klemps et al. 1985; Campbell and Singleton 1986; Daumas et al. 1988 b NaC1 not required, but growth was optimal at 35 g/1 NaC1 c Listed as - by Pfennig et al. (1981) and Campbell and Singleton (1986) but unlikely since Klemps et al. (1985) showed growth on lactate without SO2- by D. orient& d Not determined e Lactate as e- donor f Pyruvate as e donor

ence o f gas vesicles, a n d the ability to use sulfite as a n electron acceptor or e t h a n o l as electron donor. These differences are significant e n o u g h to w a r r e n t their being separate species. The presence o f high levels o f c a r b o n m o n o x i d e d e h y d r o g e n a s e indicates t h a t strain C A M Z , like D. acetoxidans ( S c h a u d e r et al. 1986; S p o r m a n n a n d T h a u e r 1988), uses a c a r b o n m o n o x i d e - a c e t y l c o e n z y m e A p a t h w a y for acetate o x i d a t i o n . T h e ability to u s e H 2 - C O 2 with sulfate a n d p r o d u c e acetate resembles D. orientis ( K l e m p s et al. 1985). However, D. orientis c a n grow h o m o acetogenically o n lactate in the absence of sulfate, while strain C A M Z c o u l d n o t a l t h o u g h it did grow o n p y r u v a t e alone. It is possible t h a t acetyl c o e n z y m e A p r o d u c e d f r o m H 2 / C O 2 is the actual g r o w t h substrate d u r i n g g r o w t h o f strain C A M Z o n H2/CO2-sulfate. Both D. nigrificans a n d D. orientis c o u l d grow o n lactate in coculture with h y d r o g e n o t r o p h i c m e t h a n o g e n s ( K l e m p s et al. 1985), while strain C A M Z could not. N o m e t h a n e was p r o d u c e d even w h e n strain C A M Z was g r o w i n g o n p y r u v a t e w i t h o u t sulfate in the presence o f a m e t h a n o g e n i n d i c a t i n g that strain C A M Z has essentially n o capacity to serve as a d o n o r for interspecies electron transfer. The ability s t r a i n C A M Z to p r o d u c e acetate in the presence o f high H2 a n d oxidize acetate in the absence of H 2 is r e m i n i s c e n t o f the acetate oxidizing rod described by Lee a n d Z i n d e r (1988) which c o u l d p r o d u c e acetate f r o m h y d r o g e n a n d CO2 w h e n h y d r o g e n was a b u n d a n t , a n d c o u l d oxidize acetate to CO2 w h e n a h y d r o g e n o t r o p h i c m e t h a n o g e n kept h y d r o g e n below a critical p a r t i a l pressure.

Description o f Desulfotomaculum thermoacetoxidans DesulfotomacuIum thermoacetoxidans sp. nov. thermo. acet. oxi. dans. Gr. Adj, thermos, h o t ; L . n . acetum v i n e g a r ; M . L . n o u n aeidum aceticum acetic acid; M . L . v. oxido m a k e acid, oxidize; M . L . thermoacetoxidans, oxidize acetate u n d e r h o t c o n d i t i o n s .

Morphology straight or slightly curved rods, 0.7 g m by 2 - 5 gin, with p o i n t e d ends. Spores are spherical a n d located in the center o f cells, c a u s i n g swelling. Motile. G r a m stain is negative.

Culture conditions. Strict a n a e r o b e , t e m p e r a t u r e optim u m is 5 5 - 6 0 ~ with r a n g e b e t w e e n 4 5 - 6 5 ~ pH o p t i m u m is 6.5 with range between 6 . 0 - 7 . 5 . N o signific a n t NaC1 r e q u i r e m e n t for growth. V i t a m i n s are required for growth. Growth substrates. E l e c t r o n d o n o r s used with sulfate as acceptor: p r o p i o n a t e , pyruvate, lactate, acetate, formate, malate, b u t y r a t e , succinate, valerate, a l a n i n e , p r o p a n o l , b u t a n o l , i s o - b u t a n o l , p e n t a n o l a n d H2./CO2. O x i d a t i o n is to CO2, a l t h o u g h some acetate m a y accumulate. Benzoate, glucose, cellobiose, xylose, palmitate, a n d e t h a n o l were n o t used as electron d o n o r s . E l e c t r o n acceptors used with lactate as d o n o r : S O l - , $ 2 0 ~ - , or S~ SO 2 - , N O ~, N O y a n d b e t a i n e n o t used. M e t h a n o g e n i c bacteria n o t used as acceptor with lactate or p y r u v a t e as donor. tool% G + C: 49.7

404

Source. T h e r m o p h i l i c (58 ~ C) a n a e r o b i c digestor converting cellulosic wastes to m e t h a n e . Type strain. Strain C A M Z . Acknowledgements. This research was supported by Grant No. DEFG02-85ERI3370 from the US Department of Energy. Hang Min was partially supported by National Natural Science Foundation of China.

References Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43 : 2 6 0 - 296 Brock TD, Brock ML, Bott TL, Edwards MR (1971) Microbial life at 90~ the sulfur bacteria of Boulder Spring. J Bacteriol 107:303-314 Campbell LL, Singleton Jr R (1986) The genus Desulfotomaculum. In: Sheath PHA (ed) Bergey's manual of systematic bacteriology, IX ed, vol 2. Williams and Wilkins, Baltimore, pp 12001202 Cord-Ruwisch R, Garcia JL (1985) Isolation and characterization of an anaerobic benzoate-degrading spore-forming sulfate-reducing bacterium, Desulfotomaculum sapomandens sp. nov. FEMS Microblol Lett 29:325-330 Daumas S, Cord-Ruwisch R, Garcla JL (1988) Desulfotomaeulum geothermieum sp. nov., a thermophilic, fatty acid-degrading, sulfate-reducing bacterium isolated with H2 from geothermal ground water. Antonie van Leeuwenhoek 54:165-178 Fuchs G (1986) CO2 fixation in acetogenic bacteria: variations on a theme. FEMS Microbiol Lett 39:181 - 213 Herdman M, Janvier M, Waterbury JB, Rlppka R, Stanier RY, Mandel M (1979) Deoxyribonucleic acid base composition of Cyanobacteria. J Gen Microbiol 111 : 63 - 71 Klemps R, Cypionka H, Widdel F, Pfennig N (1985) Growth with hydrogen and further physiological characteristics of Desulfotomaculum species. Arch Microbiol 143:203 - 208 Lee M, Zinder SH (1988) Isolation and characterization of a thermophilic bacterium which oxidizes acetate in syntrophic association with a methanogen and which grows acetogenically on H2-CO2. Appl Environ Microbiol 54:124-129

Note added in proof

Desulfotomaculum thermoacetoxidans has been sent to the Deutsche Sammlung von Mikroorganismen for deposition

Ljungdahl LG (1986) The autotrophic pathway of acetate synthesis in acetogenic bacteria. Ann Rev Microbiol 40:415- 450 Mountfort DO, Bryant MP (1982) Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge. Arch Mlcrobiol 133:249-256 Pfennig N, Widdel F, Trfiper HG (1981) The dissimilatory sulfatereducing bacteria. In: Starr MP, Stolp H, Trfiper HGo Balows A, Schtegel HG (eds) The prokaryotes, vol I. Springer, Berlin, pp 9 2 6 - 944 Schauder R, Eikmanns B, Thauer RK, Wlddel E, Fuchs G (1986) Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle. Arch Mlcrobiol 145 : 162-172 Schauder R, Preuss A, Jetten M, Fuchs G (1989) Oxidative and reductlve acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum. 2. Demonstration of the enzymes of the pathway and comparison of CO dehydrogenase. Arch Microbiol 151 : 8 4 - 89 Spormann AM, Thauer RK (1988) Anaerobic acetate oxidation to COz by Desulfotomaculum acetoxidans: demonstration of enzymes required for the operation of an oxidative acetyl-CoA/ carbon monoxide dehydrogenaes pathway. Arch Microbiol 150: 374- 380 Thauer RK (1988) Citric-acid cycle, 50 years on: modifications and an alternative pathway in anaerobic bacteria. Eur J Biochem 176: 4 9 7 - 508 Widdel F, Pfennig N (1977) A new anaerobic, sporing, acetateoxidizing sulfate-reducing bacterium, Desulfotomaculum (emend.) acetoxidans. Arch Macrobiol 112: 119 -- 122 Widdel F, Pfennig N (1981) Sporulation and further nutritional characteristics of Desulfotomaculum acetoxidans. Arch Mlcrobiol 129 :401 - 402 Zinder SH, Anguish T, Lobo AL (1987) Isolation and characterization ofa thermophilic acetotrophic strain of Methanothrix. Arch Microbiol 146: 315- 322 Zinder SH, Cardwell SC, Anguish T, Lee M, Koch M (1984) Methanogenesis in a thermophilic anaerobic digestor: Methanothrtx sp. an important aceticlastic methanogen. Appl Environ Mlcrobio147 : 796- 807 Zinder SH, Koch M (1984) Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture. Arch Microbiol 138:263- 272