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Int J Syst Evol Microbiol 56 (2006), 369-372; DOI  10.1099/ijs.0.63694-0
© 2006 International Union of Microbiological Societies

Tepidimicrobium ferriphilum gen. nov., sp. nov., a novel moderately thermophilic, Fe(III)-reducing bacterium of the order Clostridiales

A. I. Slobodkin1, T. P. Tourova1, N. A. Kostrikina1, A. M. Lysenko1, K. E. German2, E. A. Bonch-Osmolovskaya1 and N.-K. Birkeland3

1 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, 117 312 Moscow, Russia
2 Institute of Physical Chemistry, Russian Academy of Sciences, Leninskiy prospect 31, 119 991 Moscow, Russia
3 Department of Biology, University of Bergen, PO Box 7800, N-5020 Bergen, Norway

Correspondence
A. I. Slobodkin
aslobodkin{at}hotmail.com


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A moderately thermophilic, anaerobic bacterium (strain SB91T) was isolated from a freshwater hot spring at Barguzin Valley, Buryatiya, Russia. Cells of strain SB91T were straight to slightly curved rods, 0·5–0·6 µm in diameter and 3·0–7·0 µm in length. Formation of endospores was not observed. The temperature range for growth was 26–62 °C, with an optimum at 50 °C. The pH range for growth was 5·5–9·5, with an optimum at pH 7·5–8·0. The substrates utilized by strain SB91T in the presence of 9,10-anthraquinone 2,6-disulfonate included peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, alanine plus glycine, alanine plus proline, L-valine and n-propanol. Carbohydrates were not utilized. Strain SB91T reduced amorphous Fe(III) oxide, Fe(III) citrate, Fe(III) EDTA or Fe(III) nitrilotriacetate with peptone, L-valine or n-propanol as an electron donor. Strain SB91T reduced 9,10-anthraquinone 2,6-disulfonate, thiosulfate, elemental sulfur, fumarate and selenite. Strain SB91T survived after exposure to gamma-radiation at a dose of 5·4 kGy. The G+C content of the DNA of strain SB91T was 33 mol%. Analysis of the 16S rRNA gene sequence revealed that the isolated organism belonged to cluster XII of the clostridia. On the basis of its physiological properties and the results of phylogenetic analyses, it is proposed that strain SB91T represents the sole species of a novel genus, Tepidimicrobium; the name Tepidimicrobium ferriphilum gen. nov., sp. nov. is proposed, with strain SB91T (=DSM 16624T=VKM B-2348T) as the type strain.

Abbreviations: AQDS, 9,10-anthraquinone 2,6-disulfonate

Published online ahead of print on 30 September 2005 as DOI 10.1099/ijs.0.63694-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SB91T is AY656718.


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Microbial processes play an essential role in controlling the solubility and mobility of radionuclides in environments contaminated by nuclear waste. The activities of dissimilatory Fe(III)-reducing micro-organisms are of particular importance because they can alter the solubility of radionuclides via direct enzymic reduction or by indirect mechanisms catalysed via a range of electron-shuttling compounds (Lloyd et al., 2002Go; Anderson et al., 2003Go). There is a need for bioremediating micro-organisms that are resistant to both radiation and high temperatures because of the existence of thermally insulated contaminated environments where temperatures are elevated by the decay of long-lived radionuclides (Brim et al., 2003Go). The use of irradiation as a treatment method for the isolation of micro-organisms from thermal environments led to the isolation of hyperthermophilic archaea of the genus Thermococcus (Jolivet et al., 2003Go) and aerobic bacteria of the genera Rubrobacter and Deinococcus (Yoshinaka et al., 1973Go; Suzuki et al., 1988Go; Ferreira et al., 1997Go). In this article, we describe an anaerobic, moderately thermophilic, Fe(III)-reducing micro-organism, strain SB91T, isolated from a continental hot spring after gamma-irradiation of the samples and enrichments, that belongs to a novel genus within the order Clostridiales.

Strain SB91T was isolated from a sample of sediment collected under a cyanobacterial mat developing in a freshwater hot spring in the Alla River (Barguzin Valley, Buryatiya, Russia). The temperature at the sampling site was 50 °C and the pH was close to 8·0. After transportation to the laboratory, an aliquot of the sample was irradiated at 1·5 kGy with a gamma-ray source (60Co) at a rate of 120 Gy min–1 (Institute of Physical Chemistry RAS, Moscow, Russia). An enrichment culture was initiated by inoculating 10 % (w/v) of the irradiated sample into anaerobically prepared, bicarbonate-buffered, sterile (135 °C, 1 h) liquid medium with peptone (10 g l–1) as an electron donor and amorphous Fe(III) oxide (90 mM) as an electron acceptor. Medium composition and preparation techniques were as described previously (Slobodkin et al., 1999Go). The pH of the autoclaved medium was adjusted to 7·5–8·0 (at 25 °C) with 10 % (w/v) NaOH. After three subsequent 5 % (v/v) transfers of the initial enrichment, it was irradiated for the second time at 5·4 kGy under the same conditions. A pure culture of strain SB91T was obtained from this irradiated outgrown enrichment by serial dilution in a medium in which amorphous Fe(III) oxide was replaced by 20 mM 9,10-anthraquinone 2,6-disulfonate (AQDS); this was followed by the selection of well-isolated colonies that had developed in agar shake tubes (1·5 % agar in growth medium). Physiological studies on substrate utilization and on temperature, pH and salinity ranges for growth were carried out in the medium containing AQDS unless noted otherwise. In electron-acceptor utilization experiments, AQDS was omitted. Light and electron microscopy, analytical techniques, DNA extraction and determination of the G+C content were performed as described previously (Slobodkin et al., 1999Go). 16S rRNA gene amplification, sequencing and sequence analysis were done as described previously (Zavarzina et al., 2002Go).

In agar-shake cultures, brown spherical colonies (1·0–1·5 mm in diameter) of strain SB91T appeared after incubation at 50 °C for 18–24 h. Cells of strain SB91T were straight to slightly curved rods, 0·5–0·6 µm in diameter and 3·0–7·0 µm in length (Fig. 1Goa). The cells occurred singly or in short chains, were peritrichously flagellated and exhibited slight tumbling motility. Formation of endospores was not observed. Ultrathin sectioning of strain SB91T revealed a distinct thick peptidoglycan layer in its cell wall (Fig. 1bGo). The outermost layer consisted of small subunits lying outside the peptidoglycan layer.


Figure 1
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  		Fig. 1. Cell morphology of strain SB91T grown in medium with peptone-Fe(III) EDTA. (a) Electron micrograph showing a dividing cell (ultrathin section). (b) Ultrathin sections showing cell wall layers. The outermost wall layer consisted of small subunits (see arrows). Bars, 0·5 µm (a) and 0·1 µm (b).

 
The temperature range for growth of strain SB91T was 26–62 °C, with an optimum at 50 °C. No growth was detected at 64 °C or at temperatures up to 25 °C after incubation for 3 weeks. The pH range for growth was 5·5–9·5, with an optimum between pH 7·5 and 8·0. No growth was detected at pH 5·0 or 10·0. Growth of strain SB91T was observed at NaCl concentrations ranging from 0 to 3·5 % (w/v), but no growth was evident at 4·0 % (w/v). The substrates utilized by strain SB91T included peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate (each at 10 g l–1), L-valine (20 mM), DL-alanine plus glycine (both at 20 mM), DL-alanine (20 mm) plus L-proline (10 mM), and n-propanol (20 mM). Glucose, mannose, lactose, galactose, sucrose, fructose, maltose, L-arabinose, rhamnose, xylose, cellobiose (each at 25 mM), pyruvate, glycerol, formate, acetate, propionate, butyrate, lactate, malate, fumarate, methanol, ethanol, isopropanol, n-butanol, glycine, DL-alanine, L-arginine (each at 20 mM), L-proline (10 mM), benzoate (5 mM), betaine (5 mM), casein, olive oil, starch, xylan, carboxymethyl cellulose, filter paper, chitin (each at 10 g l–1) and H2/CO2 (80 : 20, v/v) were not utilized. Strain SB91T reduced AQDS (20 mM) to 9,10-anthrahydroquinone 2,6-disulfonate with all electron donors utilized. Strain SB91T reduced amorphous Fe(III) oxide (90 mM), Fe(III) citrate (20 mM), Fe(III) EDTA (10 mM) and Fe(III) nitrilotriacetate (10 mM) with peptone (10 g l–1), L-valine (20 mM) or n-propanol (20 mM) as an electron donor. No growth was evident on L-valine or n-propanol in the absence of Fe(III). L-Valine was incompletely oxidized to isobutyrate and n-propanol was oxidized to propionate with Fe(III) as an electron acceptor. Strain SB91T reduced thiosulfate (20 mM) and elemental sulfur (150 mM) to hydrogen sulfide, fumarate (20 mM) to succinate, and selenite (20 mM) to elemental selenium with peptone (10 g l–1) as an electron donor. Reduction of Fe(III), AQDS, thiosulfate, S0 and fumarate enhanced final cell yields and growth rates of strain SB91T two- to threefold in comparison with growth in pre-reduced medium (0·5 g Na2S.9H2O l–1) without an electron acceptor; reduction of selenite did not stimulate growth. Strain SB91T did not use nitrate (20 mM), sulfate (20 mM), selenate (20 mM) or oxygen (0·5, 4·0 or 20 %, v/v, in the gas phase) as electron acceptors with peptone (10 g l–1) as the electron donor.

Cultures of strain SB91T remained viable after gamma-irradiation at a dose of 5·4 kGy, but no growth was obtained from cultures irradiated at 10·0 kGy. While direct comparison of radiation-resistance data obtained for various micro-organisms is not always possible, because of differences in the irradiation conditions used, the level of radiation resistance of strain SB91T could be considered as moderate. Thermophilic members of the genera Deinococcus and Rubrobacter withstand doses of gamma-radiation above 12 and 18 kGy, respectively (Ferreira et al., 1997Go; Chen et al., 2004Go), while the lethal dose for Thermococcus species exceeds 30 kGy (Jolivet et al., 2003Go, 2004Go). The mechanism of radiation resistance of strain SB91T is not connected with the formation of endospores. To date, ionizing-radiation resistance of vegetative cells of members of the Clostridiales has not been reported.

The G+C content of the genomic DNA of strain SB91T was 33 mol% (Tm). Analysis using BLAST indicated that the highest levels of 16S rRNA gene sequence similarity were found with species of the genus Clostridium within the low-G+C-content Gram-positive subgroup of the Bacteria. A comparison of 1493 nt of the 16S rRNA gene sequence of strain SB91T with the sequences of neighbouring reference bacterial strains and some representatives of thermophilic anaerobic bacteria showed that strain SB91T belonged to cluster XII of the clostridia (nomenclature of Collins et al., 1994Go) (Fig. 2Go); it formed a single phylogenetic cluster with Clostridium ultunense, the closest relative (92 % similarity). The levels of 16S rRNA gene sequence similarity with other members of phylogenetic cluster XII ranged between 88 and 91 %. Trees constructed by maximum likelihood and by maximum parsimony had the same topology (data not shown). Transversion analysis (Woese et al., 1991Go) did not affect the phylogenetic position of strain SB91T.


Figure 2
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  		Fig. 2. Phylogenetic tree showing the position of strain SB91T within cluster XII of the clostridia. Bootstrap values (from 100 replications) are shown at branch points; values greater than 80 were considered significant. Bar, 5 substitutions per 100 nt.

 
On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain SB91T is C. ultunense (Schnurer et al., 1996Go). In addition to the fact that there is a significant phylogenetic distance between C. ultunense and strain SB91T, the former is a mesophilic micro-organism with an optimum growth temperature of about 37 °C. It also differs from strain SB91T by its ability to ferment glucose and to form endospores. Cluster XII of the clostridia comprises mesophilic (Tissierella or Soehngenia) as well as thermophilic (Thermohalobacter and Caloranaerobacter) genera, but the low level of 16S rRNA sequence similarity (89 %) does not permit placement of strain SB91T within any of these genera. All thermophilic species of cluster XII require marine (or even higher) levels of salinity for growth, whereas strain SB91T grows optimally in freshwater medium. Dissimilatory Fe(III) reduction has not been reported previously for micro-organisms from this phylogenetic cluster. On the basis of the physiological properties and the results of phylogenetic analyses, we propose that strain SB91T represents the sole species of a novel genus, Tepidimicrobium ferriphilum gen. nov., sp. nov.

Description of Tepidimicrobium gen. nov.
Tepidimicrobium (Te.pi.di.mi.cro'bi.um. L. adj. tepidus moderately warm; N.L. neut. n. microbium microbe; N.L. neut. n. Tepidimicrobium a microbe from a hot spring).

Rod-shaped bacteria. Member of cluster XII of the clostridia (nomenclature of Collins et al., 1994Go). Anaerobic and moderately thermophilic. Neutrophilic. Gram-positive-type cell wall. Grow organotrophically on a number of proteinaceous substrates. Reduce Fe(III), AQDS, thiosulfate, elemental sulfur, fumarate and selenite. Resistant to gamma-radiation at a dose of 5–10 kGy. The type species is Tepidimicrobium ferriphilum.

Description of Tepidimicrobium ferriphilum sp. nov.
Tepidimicrobium ferriphilum (fer.ri.phi'lum. L. n. ferrum iron; Gr. adj. philos loving; N.L. neut. adj. ferriphilum iron-loving).

Shows the following properties in addition to those given in the genus description. Cells are straight to slightly curved rods, 0·5–0·6 µm in diameter and 3·0–7·0 µm in length. Cells occur singly or in short chains and exhibit tumbling motility due to peritrichous flagellation. The temperature range for growth is 26–62 °C, with an optimum at 50 °C. The pH range for growth is 5·5–9·5, with an optimum at pH 7·5–8·0. Growth occurs at NaCl concentrations in the range 0–3·5 % (w/v). Substrates utilized include peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, valine, alanine plus glycine, alanine plus proline, and n-propanol. Glucose, mannose, lactose, galactose, sucrose, fructose, maltose, arabinose, rhamnose, xylose, cellobiose, pyruvate, glycerol, formate, acetate, propionate, butyrate, lactate, malate, fumarate, benzoate, methanol, ethanol, isopropanol, n-butanol, glycine, alanine, arginine, proline, betaine, casein, olive oil, starch, xylan, carboxymethyl cellulose, filter paper, chitin and H2/CO2 are not utilized. Reduces amorphous Fe(III) oxide, Fe(III) citrate, Fe(III) EDTA, Fe(III) nitrilotriacetate, thiosulfate, elemental sulfur, fumarate and selenite. Does not use nitrate, sulfate, selenate or oxygen as electron acceptors. Survives exposure to gamma-radiation at a dose of 5·4 kGy. The G+C content of the genomic DNA is 33 mol% (Tm).

The type strain is SB91T (=DSM 16624T=VKM B-2348T), isolated from a freshwater hot spring at Barguzin Valley, Buryatiya, Russia.


   ACKNOWLEDGEMENTS
 
This work was supported by INTAS grant 01-151, CRDF grant RB2-2379-MO-02, NATO grant LST.CLG.97.8868, grant 04-03-22000-CNRS from Russian Foundation for Basic Research and by the Program ‘Molecular and Cell Biology’ of the Russian Academy of Sciences.


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