International Journal of Systematic and Evolutionary (2000), 50, 2141–2149 Printed in Great Britain

Thermoanaerobacter subterraneus sp. nov., a novel thermophile isolated from oilfield water

Marie-Laure Fardeau,1 Michel Magot,2 Bharat K. C. Patel,3 Pierre Thomas,1 Jean-Louis Garcia1 and Bernard Ollivier1,3

Author for correspondence: Bernard Ollivier. Tel: j33 4 91 82 85 76. Fax: j33491828570. e-mail: ollivier!esil.univ-mrs.fr

1 Laboratoire de A new thermophilic, anaerobic glucose-fermenting, Gram-positive, rod-shaped Microbiologie IRD, bacterium, designated strain SEBR 7858T, was isolated from an oilfield water Universite! s de Provence et de la Me! diterrane! e, ESIL sample. Under optimal conditions on a glucose-containing medium (3% NaCl, case 925, 163 Avenue de 65 SC and pH 75), the generation time was 25 h. No growth occurred at 35 or Luminy, 13288 Marseille 80 SC, nor at pH 55or90. Strain SEBR 7858T possessed lateral flagella. Spores cedex 9, France were undetected but heat-resistant forms were present. Strain SEBR 7858T 2 Sanofi Recherche, Unite! fermented a range of carbohydrates to acetate, L-alanine, lactate, H and CO . de Microbiologie, 2 2 31676 Labe' ge, The isolate reduced thiosulfate and elemental sulfur, but not sulfate or sulfite France to sulfide. In the presence of thiosulfate, the ratio of acetate produced per 3 School of Biomolecular mole of glucose consumed increased, suggesting a shift in the use of electron and Biomedical Sciences, acceptors during carbohydrate metabolism. The DNA GMC content was 41 Faculty of Science, Griffith mol%. Based on 16S rRNA gene sequence analysis, the strain was almost University, Brisbane, Queensland 4111, equidistantly related to all members of the genus Thermoanaerobacter (mean Australia similarity 92%). Based on phenotypic, genomic and phylogenetic characteristics, strain SEBR 7858T was clearly different from all members of the genus Thermoanaerobacter and was therefore designated as a new species, Thermoanaerobacter subterraneus sp. nov. The type strain is SEBR 7858T (l CNCM I-2383T, DSM 13054T).

Keywords: thiosulfate reduction, Thermoanaerobacter subterraneus sp. nov., thermophile, phylogeny, oil well

INTRODUCTION sugar beet and sugar cane extraction juices, thermal spas, volcanic hot springs, non-volcanic geothermally In the last two decades, intensive research on heated subsurface aquifers, hydrothermal vents and anaerobic, thermophilic, carbohydrate-fermenting oil-producing wells (Cayol et al., 1995; Cook et al., micro-organisms from marine and terrestrial volcanic 1996; Jin et al., 1988; Kozianowski et al., 1997; Larsen hot springs has led to the isolation of several new et al., 1997; Lee et al., 1993; Leigh et al., 1981; genera and species in the domains and Slobodkin et al., 1999; Wiegel, 1986; Wynter et al., Archaea. The major rationale for this research stems 1996). Thermoanaerobacter species originating from from the biotechnological potential and the basic oil-producing wells have only recently been studied evolutionary traits of these microbes. In Bacteria, and have been found to be similar to surface-inhabiting particular attention has been paid to members of the Thermoanaerobacter species (Cayol et al., 1995; Magot order Thermotogales and the family Thermoanaerobi- et al., 2000; Wynter et al., 1996). We report here on the aceae. The latter includes the genera Thermoanaero- characterization of a new member of this genus, bacter and Thermoanaerobacterium, whose members Thermoanaerobacter subterraneus sp. nov., which was reduce thiosulfate or elemental sulfur, respectively, to isolated from a French oilfield. sulfide (Lee et al., 1993; Wiegel & Ljungdahl, 1981). Thermoanaerobacter species are thermophilic, hetero- METHODS trophic, saccharolytic anaerobes found in soil, faeces, Collection site. Strain SEBR 7858T was isolated from the ...... ‘Lacq Supe! rieur’ oilfield located in south-west France. The The GenBank accession number for the 16S rRNA gene sequence of in situ temperature of the geological formation, at a depth of strain SEBR 7858T is AF195797. 645 m, was 52 mC. A water sample was aseptically collected

01423 # 2000 IUMS 2141 M.-L. Fardeau and others at the wellhead, as described previously (Bernard et al., sequencing of the 16S rRNA gene have been described 1992), transported to the laboratory and stored at 4 mC until previously (Andrews & Patel, 1996). The 16S rRNA gene required. sequence was manually aligned with reference sequences of Medium, enrichment and isolation. Enrichment and routine various members of Bacteria using the editor ae2 (Maidak et growth were performed using a culture medium containing al., 1999). Reference sequences were obtained from the −" Ribosomal Database Project (Maidak et al., 1999), EMBL (l ): NH%Cl, 1n0g;K#HPO%,0n3g;KH#PO%,0n3 g; MgCl# ;6H#O, 1n0 g; CaCl#;2H#O, 0n1 g; NaCl, 10n0 g; KCl, 0n2g; and GenBank databases. Positions of sequence and align- Na#S#O$,3n16 g; glucose, 20 mM; sodium thiosulfate, ment uncertainty were omitted from the analysis. The 20 mM; cysteine\HCl, 0n5 g; yeast extract (Difco), 2n0g; pairwise evolutionary distances based on 1224 unambiguous Bio-trypticase (BioMe! rieux), 2n0 g; trace mineral element nucleotides were computed by using the method of Jukes & solution (Balch et al., 1979), 10 ml; resazurin, 1n0 mg. The Cantor (1969) and dendrograms were constructed from pH was adjusted to 7n0 with 10 M KOH and the medium was these distances by using the neighbour-joining method, both boiled under a stream of O#-free N# gas and cooled to room of which were from part of the  suite of programs temperature. Five or 20 ml aliquots were dispensed into (Felsenstein, 1993). A maximum-likelihood approach with Hungate tubes or serum bottles, respectively, under a stream FastDNAml was used as an alternative method for tree of N#\CO# (80:20, v\v) gas and the vessels were autoclaved construction (Olsen et al., 1994). for 45 min at 110 mC. Prior to inoculation, Na#S;9H#O and NaHCO$ were injected from sterile stock solutions to final RESULTS concentrations of 0n04 and 0n2%, respectively. An aliquot of the water sample was inoculated into 20 ml medium and Enrichment and isolation incubated at 70 mC without agitation to initiate an en- richment culture. Several pure cultures were obtained by Enrichment cultures were positive after incubation at picking well isolated colonies that developed in the culture 70 mC for 3 d and H#S was detected, presumably de- medium solidified with 4% (w\v) Phytagel (Sigma; Deming rived from thiosulfate reduction. Microscopic exam- & Baross, 1986) by the repeated use of the Hungate roll- ination revealed the presence of rod-shaped bacteria. tube method (Hungate, 1969). One of these was used for Single, well isolated colonies (3 mm diam.) that de- subsequent studies. veloped in Phytagel roll tubes after 3 d incubation at Characterization studies. pH, temperature and NaCl ranges 70 mC were picked and serially diluted in Phytagel roll for growth were determined using the culture medium. The tubes at least twice before the culture was considered medium in Hungate tubes was adjusted to the desired pH, pure. Several axenic cultures were obtained and the measured at ambient temperature, by injecting 10% (w\v) culture designated strain SEBR 7858T was used for sterile anaerobic stock solutions of NaHCO$ or Na#CO$. further studies. NaCl was weighed directly in the tubes prior to dispensing the culture medium. The strain was subcultured at least once under the same experimental conditions prior to deter- Morphology mination of growth rates. Substrates were tested in culture T medium lacking glucose at a final concentration of 20 mM. Strain SEBR 7858 was a rod-shaped bacterium To test for electron acceptors, sodium thiosulfate, sodium (0n5–0n7i2–8 µm), which occurred singly (Fig. 1a). sulfate, sodium sulfite and elemental sulfur were added to No motility was observed but the cells possessed the culture medium at final concentrations of 20 mM, laterally inserted peritrichous flagella (Fig. 1b). No 20 mM, 2 mM and 2% (w\v), respectively. spores were observed under microscopic examination Light and electron microscopy. Light microscopy was per- (electron and light microscopy), but cultures exposed formed as described by Cayol et al. (1994). Electron at 120 mC for 45 min could be subcultured, suggesting microscopy was performed as described by Fardeau et al. the presence of heat-resistant forms. Electron micro- (1997a). scopy of ultrathin sections revealed a Gram-posi- Analytical techniques. Unless otherwise indicated, duplicate tive cell wall composed of a thin dense surface layer culture tubes were used throughout this study. Growth was separated from a thicker inner layer (Fig. 1c). measured by inserting tubes directly into a model UV-160A spectrophotometer (Shimadzu) and measuring changes in OD&)!. Sulfide was determined photometrically as colloidal Optimum growth conditions CuS by using the method of Cord-Ruwisch (1985). H#,CO#, Strain SEBR 7858 T was strictly anaerobic and did not sugars, alcohols, volatile and non-volatile fatty acids were grow in culture medium containing traces of oxygen, measured as described previously (Fardeau et al., 1996). - Alanine was measured by HPLC as described by Moore et as indicated by the anaerobiosis indicator, resazurin. al. (1958). The strain grew optimally at 65 mC (temperature range between 40 and 75 mC, but not at 80 mC; Fig. 2a) and a Determination of DNA GjC content. The DNA GjC content was determined by the DSMZ (Deutsche Sammlung pH of 7n5 (pH range between 6n0 and 8n5; Fig. 2b). The von Mikroorganismen und Zellkulturen GmbH, Braun- isolate grew in the presence of 0–3% NaCl with an schweig, Germany). DNA was isolated and purified by optimum of 0% at pH 7n0 and 70 mC (Fig. 2c). chromatography on hydroxyapatite and its GjC content was determined by using HPLC as described by Mesbah et Substrates for growth al. (1989). Non-methylated λ DNA (Sigma) was used as the standard. Yeast extract or Bio-trypticase was required for 16S rDNA sequence analysis. The methods for the purifi- growth on carbohydrates. Yeast extract could not be cation and extraction of DNA and the amplification and replaced by a vitamin mixture (Balch et al., 1979).

2142 International Journal of Systematic and Evolutionary Microbiology 50 Thermoanaerobacter subterraneus sp. nov.

0·30 (a) (a) 0·25

0·20

0·15

0·10

0·05 0 30 40 50 60 70 80 90 Temperature (°C)

0·30 (b)

) 0·25 –1 0·20 (b) 0·15

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Growth rate (h 0·05 0 5 678910 pH

0·25 (c) 0·20

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0·05 (c) 0 010203040 NaCl (g l–1)

...... Fig. 2. Effect of (a) temperature (pH 7n5, no NaCl), (b) NaCl (65 mC, pH 7n5) and (c) pH (65 mC, no NaCl) on the growth of strain SEBR 7858T cultivated in basal medium.

fructose, -galactose, -glucose, -lactose, -malt- ose, -mannose, -xylose, -ribose, mannitol, cellobi- ose, pyruvate, melibiose, starch and xylan, but not - xylose, -arabinose, -rhamnose, sorbose or sucrose. Glycerol was used poorly. Acetate, -alanine, lactate,

...... H# and CO# were produced from glucose fermentation. Fig. 1. (a) Phase-contrast micrograph of strain SEBR 7858T. Bar, From 0n6to0n8mol-alanine was produced per mole 10 µm. (b) Electron micrograph of a negatively stained culture glucose consumed (Table 1). of strain SEBR 7858T showing laterally inserted flagella. Bar, 2 µm. (c) Electron micrograph of an ultrathin section of strain T SEBR 7858 showing the cytoplasmic membrane (cm), the inner Effect of electron acceptors layer (il) and the outer layer (ol). Bar, 0n5 µm. Strain SEBR 7858T reduced thiosulfate, elemental sulfur and sulfite, but not sulfate to sulfide. The SEBR 7858T grew on the following substrates (at a presence of thiosulfate altered the concentration of concentration of 20 mM unless otherwise indicated) in metabolites during glucose oxidation, indicating that the presence of thiosulfate as the electron acceptor: - thiosulfate modified the metabolic pathway of strain

International Journal of Systematic and Evolutionary Microbiology 50 2143 M.-L. Fardeau and others

Table 1. End products from glucose metabolism by Thermoanaerobacter subterraneus, in the presence or absence of thiosulfate (20 mM)

Growth conditions Amount of glucose Amount of product (mmol)* consumed (mmol)

Acetate Lactate L-Alanine H2 H2S

Glucose 9n410n61n28n05n70 Glucosejthiosulfate 8n620n30 0 0n221n9 * Data were corrected for non-utilizable glucose present in the medium.

Thermoanaerobacter siderophilus Thermoanaerobacter wiegelii 99 Thermoanaerobacter sulfurophilus 5% Thermoanaerobacter thermohydrosulfuricus 97 Thermoanaerobacter ethanolicus Thermoanaerobacter acetoethylicus

98 Thermoanaerobacter brockii subsp. lactiethylicus Thermoanaerobacter brockii subsp. finnii Thermoanaerobacter brockii subsp. brockii 100 100 Thermoanaerobacter mathranii Thermoanaerobacter thermocopriae Thermoanaerobacter subterraneus 100 Thermoanaerobacterium group

...... Fig. 3. Dendrogram indicating the position of strain SEBR 7858T amongst members of the genus Thermoanaerobacter. Sequences of the following type strains were obtained from the Ribosomal Database Project, version 7.0 (Maidak et al., 1999): Thermoanaerobacter acetoethylicus HTB2/WT (l ATCC 33265T, DSM 2359T), Thermoanaerobacter brockii subsp. brockii HTD4T (l ATCC 33075T, DSM 1457T), Thermoanaerobacter brockii subsp. finnii AKO-1T (l DSM 3389T), Thermoanaerobacter brockii subsp. lactiethylicus SEBR 5268T (l DSM 9801T), Thermoanaerobacter ethanolicus JW 200T (l ATCC 31550T, DSM 2246T), Thermoanaerobacter kivui ATCC 33488T (l DSM 2030T), Thermoanaerobacter mathranii A3T (l DSM 11426T), Thermoanaerobacter siderophilus SR4T (l DSM 12299T), Thermoanaerobacter sulfurophilus L-64T (l DSM 11584T), Thermoanaerobacter thermocopriae (l IAM 13577T), Thermoanaerobacter thermohydrosulfuricus E100- 69T (l DSM 567T), Thermoanaerobacter wiegelii Rt8.B1T (l DSM 10319T). Bootstrap values, expressed as a percentage of 100 replications, are shown at branching points. Only values above 95% were considered significant and reported. Scale bar indicates 5 substitutions per 100 nt.

T SEBR 7858 . In the presence of thiosulfate, -alanine DNA GjC content was not detected, hydrogen production decreased, The G C content of isolate SEBR 7858T was whereas sulfide production increased and acetate was j 41 mol% (as determined by HPLC). the only acid detected (Table 1). DISCUSSION 16S rDNA sequence analysis Strains of various physiological groups of thermophilic A total of 1372 nt of the 16S rDNA of strain SEBR bacteria have been previously isolated from deep 7858T, corresponding to position 20–1470 of the subsurface environments such as oilfields and aquifers. Escherichia coli 16S rDNA, was sequenced. 16S rDNA In addition to methanogens and sulfate-reducing sequence analysis and comparison with species repre- bacteria, heterotrophic micro-organisms, including sentative of Bacteria indicated that strain SEBR members of the order Thermotogales and the family 7858T was a member of the genus Thermoanaerobacter Thermoanaerobiaceae (e.g. the genus Thermoanaero- and formed a deep line of descent almost equidistant to bacter), have been isolated and described (Andrews & the 12 taxonomically validated species and subspecies Patel, 1996; Cayol et al., 1995; Fardeau et al., 1997a; with available sequences, with a mean similarity of Jeanthon et al., 1995; Magot et al., 2000; Ravot et al., 93% (Fig. 3). 1995; Slobodkin et al., 1999; Stetter et al., 1993;

2144 International Journal of Systematic and Evolutionary Microbiology 50 Thermoanaerobacter subterraneus sp. nov.

T Wynter et al., 1996). Strain SEBR 7858 , a Gram- H# and CO#. This constitutes the first experimental positive thermophilic anaerobic bacterium, isolated evidence of -alanine production by a Thermo- from a French oilfield, is phylogenetically related to anaerobacter species. We have detected the presence of Thermoanaerobacter species. The presence of a Ther- -alanine from glucose fermentation in Thermo- moanaerobacter species in oilfields was first reported in anaerobacter brockii and Thermoanaerobacter ethano- 1995. The isolate was found to be a phylogenetic licus, but it is a minor product compared to strain relative of Thermoanaerobacter brockii and Thermo- SEBR 7858T (unpublished data). As for some members anaerobacter finnii (Cayol et al., 1995). It was recog- of Archaea (Kengen & Stams, 1994; Kobayashi et al., nized as a novel subspecies of the terrestrial micro- 1995), -alanine production from glucose metabolism organism Thermoanaerobacter brockii and named has been reported in hyperthermophilic and thermo- Thermoanaerobacter brockii subsp. lactiethylicus. Be- philic members of Bacteria (e.g. Thermotoga, Thermo- cause of the large number of new isolates belonging to sipho and Fervidobacterium, order Thermotogales) and Thermoanaerobacter, the of this genus was has been suggested to be a remnant of an ancestral revised on the basis of 16S rRNA sequence and metabolism (Ravot et al., 1996). -Alanine is also DNA–DNA hybridization and now comprises 11 produced by a Gram-positive anaerobe belonging to validated species, 3 subspecies and 3 non-validated the genus (O$ rlygsson et al., 1995). It is species (Table 2). possible that -alanine production from carbohydrate The isolation of strain SEBR 7858T extends our fermentation is more widespread amongst members of knowledge on the diversity of microbes from sub- Bacteria than previously believed. terrestrial ecosystems and confirms that Thermo- Strain SEBR 7858T differs from its closest phylogenetic anaerobacter species, similar to members of the order relatives, such as Thermoanaerobacter brockii and Thermotogales, may be important components of the Thermoanaerobacter ethanolicus, by fermenting meli- heterotrophic microflora of oilfield reservoirs (Grassia biose and by using sucrose and xylose poorly. In et al., 1996; Magot et al., 2000). Studies on a meth- contrast to Thermoanaerobacter brockii (Zeikus et al., anogenic archaeon, Methanocalculus halotolerans 1979), strain SEBR 7858 T has a much higher DNA T isolated from an African oilfield (Ollivier et al., 1998), GjC content (41% for SEBR 7858 compared to and the results reported by L’Haridon et al. (1995) 30–31% for Thermoanaerobacter brockii). The isolate suggest that indigenous micro-organisms might in- also differs from Thermoanaerobacter ethanolicus habit oilfield reservoirs. However, the presence of (Wiegel & Ljungdahl, 1981) as -alanine is the major Thermoanaerobacter species in oilfield waters as a end product. Phylogenetic, genomic and phenotypic result of anthropogenic contamination rather than as characteristics of strain SEBR 7858T confirm that it an indigenous microflora cannot be ruled out entirely represents a new species of the genus Thermoanaero- and further investigations are needed to understand bacter for which we propose the name Thermo- their distribution. Nevertheless, Thermoanaerobacter anaerobacter subterraneus sp. nov., reflecting its sub- species have a wide nutritional spectrum for growth, terrestrial origin. which includes carbohydrates and amino acids (Fardeau et al., 1997b; Faudon et al., 1995; Magot et al., 2000); oilfields may contain such nutrients, pro- Description of Thermoanaerobacter subterraneus sp. vided by other members of the bacterial community, nov. that will possibly support their growth and\or vi- Thermoanaerobacter subterraneus (sub.ter.rahne.us. L. ability. pref. sub less than; L. n. terra earth; L. adj. subter- Strain SEBR 7858T oxidizes hydrogen in the presence raneus under the earth, describing its site of isolation). of sulfur compounds. This may lead to corrosion of Cells are rods (0n5–0n7i2–8 µm), occur singly or in oilfield installations when these latter compounds are pairs and possess laterally inserted flagella. Spores are available in oilfield waters. The presence of a hydro- not observed under microscopic examination but gen-oxidizing Thermoanaerobacter species was pre- cultures exposed to 120 mC for 45 min could be viously reported from oilfields (Fardeau et al., 1993a, subcultured, suggesting the presence of heat-resistant b). In addition, thiosulfate reduction by Thermo- T forms. Electron microscopic examinations reveal a anaerobacter species, including strain SEBR 7858 , Gram-positive cell wall. Round colonies (3 mm diam.) may be of significance in mineralization processes in develop in Phytagel roll tubes after 3 d incubation at thermal terrestrial or subterrestrial environments, 70 mC. Chemo-organotrophic and obligately anaerobic since this reduction facilitates oxidation of organic member of the domain Bacteria. Optimum tempera- matter as reported previously (Fardeau et al., 1996). ture for growth is 65 mCatpH7n5; temperature range T Strain SEBR 7858 has many features typical of some for growth is 40–75 mC. Optimum pH is 7n5; growth members of the genus Thermoanaerobacter. It is Gram- occurs between pH 6n0 and 8n5. Halotolerant, growing positive, thermophilic (optimal temperature for in the presence of up to 3% NaCl. Yeast extract or growth is 65 mC), anaerobic and contains heat-resistant Bio-trypticase is required for growth on carbo- cells. The isolate reduces thiosulfate, sulfite and el- hydrates. Growth on sugars is highly enhanced by the emental sulfur, but not sulfate to sulfide. In addition, it presence of both yeast extract and Bio-trypticase. ferments carbohydrates to -alanine, acetate, lactate, Yeast extract cannot be replaced by vitamins. Fer-

International Journal of Systematic and Evolutionary Microbiology 50 2145 M.-L. Fardeau and others

Table 2. Characteristics that differentiate members of the genus Thermoanaerobacter ...... Data were taken from the following sources for the following species: 1, Thermoanaerobacter subterraneus, this study; 2, ‘Thermoanaerobacter acetigenus’, Nielsen et al. (1993); 3, Thermoanaerobacter acetoethylicus, Ben-Bassat & Zeikus (1981); 4, Thermoanaerobacter brockii subsp. brockii, Zeikus et al. (1979); 5, Thermoanaerobacter brockii subsp. finnii, Schmid et al. (1986); 6, Thermoanaerobacter brockii subsp. lactiethylicus, Cayol et al. (1995); 7, ‘Thermoanaerobacter cellulolyticus’, Taya et al. (1988); 8, Thermoanaerobacter ethanolicus, Wiegel & Lungdahl (1981); 9, Thermoanaerobacter italicus, Kozianowski et al. (1997); 10,

Character 1 2 3 4 5678

Type strain DSM 13054T DSM 7040 DSM 2359T DSM 1457T DSM 3389T DSM 9801T DSM 8991 DSM 2246T Source Oil well Hot spring Hot spring Hot spring Lake Kivu Oil well Hot spring Hot spring Temperature range (mC) 40–75 50–78 40–80 35–85 40–75 40–75 50–85 37–78 Optimum temperature (mC) 65 65–68 65 65–70 64–66 55–60 75 70 pH range 6–8n55n2–8n65n5–8n55n5–9n5  5n6–8n8 6–9n54n4–9n9 Optimum pH 7n57 7n56n5–6n8  8–8n3 6–8 GjC content (mol%) 41 34n9–36n5 30–32 29–32n4323537n7 37–39 Reduction of Smj kk j   #− Reduction of S#O# jkjjjj  Flagella Peritrichous k Peritrichous k Peritrichous Peritrichous Peritrichous Peritrichous Substrates used: Arabinose kjkk kk Cellobiose jjjjjjjj Fructose jj  jjjj Galactose jj  jjjj Glucose jjjjjjjj Inositol       kk Lactose jjjjjjjj Maltose jjjjjjjj Mannitol jk  jjkk Mannose jjjkjjjj Melezitose       kk Melibiose j     kkk Raffinose  j     jk Rhamnose kk   kkk Ribose jk  jjkj Starch jjjj jjj Sucrose kjjjjjjj Trehalose  j     kk Xylose jjkkjjjj Xylan jj    j  Formate         Lactate  kk k    Pyruvate jkkjjj j Beef extract         Tryptone   jk    Yeast extract  kj  j   Amygdalin       kk Cellulose  k  k  kjk Chitin         Aesculin       kj Glucosamine         Glycerol j     kkk Glycogen       k  Hemicellulose         Inulin       k  Pectin   kk  k  Diagnostic fermentation Ethanol, Ethanol, Ethanol, Ethanol, Ethanol, Ethanol Lactate Ethanol, products from glucose* lactate, isobutyrate isobutyrate, lactate lactate lactate -alanine butyrate, isovalerate, valerate

* All species tested produced acetate, H# and CO# with the exception of Thermoanaerobacter siderophilus which did not produce acetate. † Homoacetogenic species.

2146 International Journal of Systematic and Evolutionary Microbiology 50 Thermoanaerobacter subterraneus sp. nov.

Table 2 (cont.) ...... Thermoanaerobacter kivui, Zeikus et al. (1979); 11, ‘Thermoanaerobacter lactoethylicus’, Kondratieva et al. (1989); 12, Thermoanaerobacter mathranii, Larsen et al. (1997); 13, Thermoanaerobacter siderophilus, Slobodkin et al. (1999); 14, Thermoanaerobacter sulfurophilus, Bonch-Osmolovskaya et al. (1997); 15, Thermoanaerobacter thermocopriae, Jin et al. (1988); 16, Thermoanaerobacter thermohydrosulfuricus, Wiegel et al. (1979); 17, Thermoanaerobacter wiegelii, Cook et al. (1996). , Not determined.

9 1011121314151617

DSM 9252T DSM 2030T DSM 9003 DSM 11426T DSM 12299T DSM 11584T IAM 13577T DSM 567T DSM 10319T Hot spring Lake Kivu Hot spring Hot spring Hydrothermal Cyanobacterial Hot spring Hot spring Geothermal vent mats water 45–78 50–72 42–75 47–78 39–78 44–75 47–74 40–78 38–78 70 66 65 70–75 69–71 55–60 60 68 65–68  5n3–7n3 5–8n54n7–8n84n8–8n24n5–8 6–8 5–9 5n5–7n2 76n476n8–7n86n3–6n56n8–7n26n5–7n36n9–7n56n8 34n43834n6373229n8–30n86n7–37n837n634n4–36n8 k  jjjj kk j   jjj jj kkkPeritrichous Peritrichous Peritrichous  Lateral Peritrichous j  jjkjjjk j kjjjjjjj j jjjjjjjj jkjk  kjj j jjjjjjjj  k    j  kk jkjj jj j j kjjjjjjj  kjj jk j jj j   jjj j   j   k  k j   j   k  k jk j   kjj   jk jkjk  k  j   kjk j   kjjkkj jkj jjkjj j kjjjjkjj jk j   j  j j  jjjj jj    jk k  k  j   kk    k   kj  k  j   jj jk     j         j     j   kj    k   j   j    k  kkkj  j        j j                kj jk kj  kj k   j   j   j      j   j   k   k   Ethanol, Acetate† Ethanol, Ethanol, Ethanol, Ethanol, Ethanol, Ethanol, lactate, Ethanol, lactate, lactate, lactate lactate lactate butyrate, formate, propanol, lactate, succinate propionate, lactate 2-propanol, propionate butyrate, butyrate, isovalerate isovalerate, isocaproate

International Journal of Systematic and Evolutionary Microbiology 50 2147 M.-L. Fardeau and others ments cellobiose, -fructose, -galactose, -glucose, Cord-Ruwisch, R. (1985). A quick method for the determination -lactose, -maltose, -mannose, melibiose, - of dissolved and precipitated sulfides in cultures of sulfate- ribose, starch, -xylose, mannitol, pyruvate and xylan. reducing bacteria. J Microbiol Methods 4, 33–36. Glycerol is poorly utilized and -arabinose, -rham- Deming, J. W. & Baross, J. A. (1986). Solid medium for culturing nose, sorbose, sucrose and -xylose are not used. black smoker bacteria at temperature to 120 mC. Appl Environ Acetate, -alanine, lactate, H# and CO# are produced Microbiol 51, 238–243. during glucose fermentation. Elemental sulfur, thio- Fardeau, M.-L., Cayol, J.-L., Magot, M. & Ollivier, B. (1993a). H# sulfate and sulfite, but not sulfate, are used as electron oxidation in the presence of thiosulfate by a Thermoanaero- acceptor. 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