International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1853–1862 Printed in Great Britain Hydrogenothermus marinus gen. nov., sp. nov., a novel thermophilic hydrogen-oxidizing bacterium, recognition of Calderobacterium hydrogenophilum as a member of the genus Hydrogenobacter and proposal of the reclassification of Hydrogenobacter acidophilus as Hydrogenobaculum acidophilum gen. nov., comb. nov., in the phylum ‘Hydrogenobacter/Aquifex’ 1 Institut fu$ r Allgemeine Ru$ diger Sto$ hr,1† Arne Waberski,1 Horst Vo$ lker,1 Brian J. Tindall2 Mikrobiologie, Am 1 Botanischen Garten 1–9, and Michael Thomm 24118 Kiel, Germany 2 DSMZ Braunschweig, Author for correspondence: Michael Thomm. Tel: j49 431 880 4330. Fax: j49 431 880 2194. Mascheroder Weg 1b, e-mail: mthomm!ifam.uni-kiel.de 38124 Braunschweig, Germany A novel thermophilic, hydrogen-oxidizing bacterium, VM1T, has been isolated from a marine hydrothermal area of Vulcano Island, Italy. Cells of the strain were Gram-negative rods, 2–4 µm long and 1–15 µm wide with four to seven monopolarly inserted flagella. Cells grew chemolithoautotrophically under an atmosphere of H2/CO2 (80:20) in the presence of low concentrations of O2 (optimum 1–2%). Carbohydrates and peptide substrates were not utilized, neither for energy generation nor as a source of cellular carbon. Growth of VM1T occurred between 45 and 80 SC with an optimum at 65 SC. Growth was observed between pH 5 and 7. NaCl stimulated growth in the range 05–6% with an optimum at 2–3%. Hydrogen could not be replaced by elemental sulfur or thiosulfate as electron donors. Nitrate and sulfate were not used as electron acceptors. The major respiratory lipoquinone was a new menathioquinone. T Analysis of the fatty acids of VM1 revealed straight-chain saturated C18:0 and the unsaturated C18:1ω9c and C20:1ω9c as major components. The GMC content of the total DNA was 43 mol%. Phylogenetic analysis placed strain VM1T near the members of the genera Hydrogenobacter, Thermocrinis and Aquifex on a separate deep-branching phylogenetic lineage. Therefore, it is proposed that strain VM1T (l DSM 12046T l JCM 10974T) represents a novel species within a new genus, for which the name Hydrogenothermus marinus gen. nov., sp. nov., is proposed. In addition, it is shown that Calderobacterium hydrogenophilum should be transferred to the genus Hydrogenobacter; the name Hydrogenobacter hydrogenophilus comb. nov. (DSM 2913T l JCM 8158T)is proposed for this organism. Furthermore, on the basis of 16S rRNA sequence analysis, Hydrogenobacter acidophilus is only distantly related to Hydrogenobacter species. Owing to this finding and its growth at low pH, the name Hydrogenobaculum acidophilum gen. nov., comb. nov., is proposed for Hydrogenobacter acidophilus. The type strain is JCM 8795T (l DSM 11251T). Keywords: hydrogen oxidation, thermophilic bacteria, Aquifex, Hydrogenobacter ................................................................................................................................................................................................................................................................................................................. † Present address: Institut fu$ r Meereskunde, Du$ sternbrooker Weg 20, 24105 Kiel, Germany. The GenBank accession number for the 16S rDNA sequence of strain VM1T is AJ292525. 01805 # 2001 IUMS 1853 R. Sto$ hr and others INTRODUCTION added to the headspace of the serum bottles by use of a sterile filter after autoclaving the medium. Mass cultures of When Aquifex pyrophilus was described (Huber et al., VM1T were grown in a 10 l titanium fermenter (Braun 1992; Burggraf et al., 1992), a new phylum of the Biotech). The fermenter was gassed with 120 ml H#,30ml −" Bacteria was found that represented the deepest CO# and 7n5 ml air min . Working with mixtures of branching of the bacterial kingdom. Aquifex pyrophilus hydrogen and oxygen can lead to highly explosive gas was characterized by its hyperthermophilic and chemo- mixtures when the hydrogen atmosphere contains more lithoautotrophic metabolism, yielding energy from the than 25% air (Aragno & Schlegel, 1992). Under normal oxidation of molecular hydrogen. Other thermophilic, conditions of fermentation, this explosive atmosphere was hydrogen-oxidizing bacteria were found to be related. not formed. To prevent hydrogen entering the room at- mospheere, the fermenter was equipped with a direct exhaust Hydrogenobacter thermophilus (Kawasumi et al., 1984) pipe out of the building and gas-tight bearings. and Calderobacterium hydrogenophilum (Kryukov et al., 1983) also could be grouped together within the In order to analyse carbon source utilization by VM1T,3g order ‘Aquificales’ (Huber et al., 1998). PIPES buffer, adjusted to pH 6n0, was added to 1 l medium as a buffer and NaHCO$ was omitted from the medium. The While Aquifex pyrophilus was isolated from a marine carbon sources meat peptone, tryptone, meat extract, yeast geothermally heated area of the Kolbeinsey Ridge, extract, lactose, -galactose, α--glucose, -ribose, -fruc- members of the genera Hydrogenobacter, Caldero- tose, sucrose, citric acid, α--maltose hydrate, starch, - bacterium and Thermocrinis (Huber et al., 1998) were xylose, -alanine, -proline, -histidine hydrochloride, isolated from freshwater habitats. The only marine glycine, methanol, ethanol, acetic acid, pyruvate, disodium species of the genus Hydrogenobacter reported so far is fumarate, -malate and ammonium formate were added ‘Hydrogenobacter halophilus’, which was isolated from individually at concentrations of 0n1%. The gas atmosphere was 99% H# (300 kPa) and 1% O#. To analyse growth of the a marine hot spring (Nishihara et al., 1990). With strain in the presence of organic carbon sources and in the respect to the optimal temperature of growth, members absence of hydrogen, the strain was cultivated under a of the genera Aquifex and Thermocrinis are hyper- N#\CO# atmosphere (80:20; 300 kPa) in the presence of 1% thermophiles, showing optimal growth at 85 mC. O#. Growth on the carbon sources yeast extract, glucose, Representatives of the genera Hydrogenobacter and starch, peptone, tryptone and maltose, added individually at Calderobacterium show optimal growth around 70 mC. final concentrations of 0n1%, was assayed under a hydrogen- They do not grow at 85 mC. Here, we describe a new free atmosphere. strain of marine origin with an even lower optimal The gas atmosphere was changed to N#\CO# (80:20) to test growth temperature, of 65 mC, representing a new for nitrogen fixation and the ability to use thiosulfate and phylogenetic lineage within the phylum ‘Hydro- elemental sulfur as electron donators. Oxygen was omitted genobacter\Aquifex’. when testing sulfate and nitrate (0n1% KNO$,w\v) as electron acceptors. METHODS Isolation procedure. Pure cultures were obtained by repeated transfers of serial dilution cultures. The cultures were Origin of samples. Strain VM1T was isolated from a marine checked for contamination using a phase-contrast light water sample that also contained sediment. The sample was microscope (Zeiss). The purity of the cultures was confirmed taken from a geothermally heated shallow area at Vulcano by repeated partial sequence analysis of the gene encoding beach, 3–4 m from the shore. The temperature of the sample 16S rRNA. was 83 C. The sample was collected with a 20 ml syringe m Gram staining. Gram staining was performed by using the and was transferred to a 20 ml tube containing a drop of Bacto 3-step Gram-stain procedure (Difco). resazurin solution (0n1%). The tube was sealed with a rubber stopper and reduced by adding a spatula-tip amount of Measurement of growth. Growth experiments were set up in dithionite to protect the sample from oxygen, as we planned 120 ml serum bottles that were incubated in a reciprocally initially to isolate anaerobes. shaking water bath (100 r.p.m.). Growth curves were de- termined by direct counting using a Thoma Blau Brand Culture media. Modified marine medium described by T chamber (Omnilab-Laborzentrum) with a depth of 0n02 mm ZoBell (1941) was used for the isolation of VM1 . This −" under a phase-contrast microscope (Zeiss standard 16). The medium contained (g l ): Bacto yeast extract, 1n0; Bacto doubling times were calculated from the slopes of growth peptone, 5n0; NaCl, 19n4; MgCl#.6H#O, 12n6; NaHCO$, curves of three replicates. When the pH optimum was 0n16; Na#SO%,3n24; KCl, 0n56; elemental sulfur, 2n0; determined, the pH was adjusted 1 d prior to the experiment resazurin (0n1%), 1n0 ml; trace minerals (10i) 10 ml (Balch and readjusted immediately before inoculation using uni- et al., 1979). For large-scale fermentation and subsequent versal pH paper (duotest; Macherey-Nagel). cultivation of the isolate, Bacto peptone and Bacto yeast extract were replaced by 0n3g NH%Cl, and 2n38 g Electron microscopy. A cell suspension of a well-grown CaCl#.2H#O; the amount of elemental sulfur was reduced to culture was applied to Pioloform-covered 300 mesh Cu grid, −" 0n5gl . The pH was adjusted to 7n0 with H#SO% (25%). The washed once with glass-distilled water and sputtered after medium was mixed with an UltraTurrax for 1 min, deoxy- drying with Pt\C at an angle of 40m. A culture was fixed genated under a stream of N# for 20 min and dispensed in overnight at 4 mC with 2% glutaraldehyde and 0n05% 20 ml portions in 120 ml type III borosilicate bottles ruthenium red prior to thin sectioning. After centrifugation (Pharmapack; Stute) under a N# atmosphere. Prior to (10000 g,