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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong, Yuseong, Daejeon, Republic of Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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9c, 15 : 0 iso and 17 : 0 iso as the major fatty acids. The G+C content of the genomic DNA was 64.6 mol%. The 16S rRNA gene sequence of strain LCS2T was found to be most similar to that of Rhodanobacter fulvus IAM 15025T (97.4 % similarity). The results of DNA–DNA hybridization and phenotypic analysis showed that strain LCS2T can be distinguished from all known Rhodanobacter species and therefore represents a novel species of the genus, for which the name Rhodanobacter thiooxydans sp. nov. is proposed. The type strain is LCS2T (=DSM 18863T =KCTC 12771T).
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), isolated from a soil sampled from a wood-treatment site and which degrades lindane (
-hexachlorocyclohexane); Rhodanobacter fulvus (Im et al., 2004), isolated from soil mixed with rotten rice straw and which produces 
-galactosidase; and Rhodanobacter spathiphylli (De Clercq et al., 2006), isolated from the rhizospheres of Spathiphyllum plants grown in a compost-amended potting mix. Although the species were first isolated from widely separated geographical locations, all members of the genus Rhodanobacter have emerged from soil environments.
Various denitrification processes to remove nitrate from wastewater have been developed and studied. The autotrophic denitrification process can use sulfur particles as an energy source. This process is cheaper and easier to handle than the heterotrophic denitrification approach, involving organic chemicals (Batchelor & Lawrence, 1978; Claus & Kutzner, 1985; Koenig & Liu, 1996; Zhang & Lampe, 1999), but the exact microbial roles are not yet known.
During a study of the role of the bacterial communities that reside in the biofilms on sulfur particles used in an autotrophic denitrification process, a laboratory-scale upflow anaerobic sludge blanket reactor fed with such sulfur particles was used to enumerate and identify the bacteria involved. A biofilm from sulfur particles was thoroughly suspended in 50 mM phosphate buffer (pH 7.0). The suspension was spread on one-tenth-strength R2A agar (Difco). The agar plates were incubated for 1 month at room temperature under both aerobic and anaerobic conditions, and each single colony was incubated once again using half-strength R2A agar. Consequently, seven strains were isolated under aerobic conditions. They were subjected to identification by means of conventional biochemical tests, analysis of the cellular fatty acids and partial 16S rRNA gene sequencing. Only one strain, LCS2T, was shown to represent a novel species.
The effect of thiosulfate on growth of strain LCS2T was tested in succinate/mineral medium containing (l–1) 0.4 g NH4Cl, 0.8 g K2HPO4, 0.3 g KH2PO4, 2.7 g sodium succinate hexahydrate, 0.1 g yeast extract and 5 ml mineral solution (Frankel et al., 1997). The pH was adjusted to 7.5 with NaOH. The medium was supplemented with various amounts of thiosulfate, resulting in final concentrations ranging from 0 to 20 mM. Growth in liquid medium at 30 °C with vigorous shaking was investigated. Autotrophic growth was tested by omitting succinate from the succinate/mineral medium and replacing the yeast extract with 0.5 ml vitamin solution l–1 (Frankel et al., 1997). The ability to grow anaerobically was determined by supplementing the R2A or succinate/mineral medium with KNO3 (2 g l–1) or Fe4(P2O7)3 (3.72 g l–1) as an alternative electron acceptor and then incubating the inoculated medium in anaerobic jars (Spring et al., 2001). The reduction of nitrate and the oxidation of thiosulfate were monitored by using an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and an anion-exchange column (Metrosep Anion Supp 4; Metrohm).
In our experiments, R. fulvus IAM 15025T and R. spathiphylli LMG 23181T were obtained from culture collection centres, but the type strain of the type species of the genus, R. lindaniclasticus LMG 18385T (the only known strain of this species), could not be included, as the strain no longer exists (Mergaert et al., 2002). All strains were cultivated identically and tested under the same conditions for comparative studies. For the analysis of fatty acids, the strains were cultivated in tryptic soy broth (Difco) at 30 °C for 4 days because of the slow growth of strain LCS2T.
The Gram reaction was performed as described by Gerhardt et al. (1994). Cell morphology and motility were observed under a phase-contrast microscope (Optiphot; Nikon) at 1000x magnification with cells grown for 1–7 days on R2A agar. Oxidase activity was tested using 1 % (w/v) tetramethyl-p-phenylenediamine, and catalase activity was tested using 3 % H2O2. Growth was investigated at temperatures ranging from 5 to 45 °C (using increments of 5 °C), at pH 4–10 (using increments of 1 pH unit) and in different salt concentrations (1, 2, 3 and 5 % NaCl). Hydrolysis of casein and starch was tested on casein agar and starch (Difco). An H2S-production test was performed on triple-sugar–iron agar (BBL). Carbon-source-utilization tests and additional physiological tests were performed using API 20NE, API 32GN, API 50 CH and API ZYM galleries according to the instructions of the manufacturer (bioMérieux).
Fatty acid methyl esters were prepared and analysed as described previously (Klatte et al., 1994) using the standard Microbial Identification System (MIDI) for automated GC analysis (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Isoprenoid quinones were extracted and purified as described previously (Tindall, 1990) and dried preparations were dissolved in 200 µl 2-propanol; 1–10 µl samples were separated by HPLC without further purification.
Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Rainey et al. (1996). The 16S rRNA gene sequence was aligned with published sequences retrieved from EMBL by using CLUSTAL_X (Thompson et al., 1997) and was edited using BioEdit (Hall, 1999). The phylogenetic tree was constructed on the basis of the neighbour-joining method (Saitou & Nei, 1987); distances were estimated using the Kimura two-parameter model (Kimura, 1983) with MEGA version 3.1 (Kumar et al., 2004). The resultant neighbour-joining tree topology was evaluated by using bootstrap analysis (Felsenstein, 1985) based on 1000 resampled datasets. The DNA G+C content was determined by HPLC after hydrolysis, as described by Tamaoka & Komagata (1984), and non-methylated 
DNA (Sigma) was used as a standard. DNA–DNA hybridization to determine genomic relatedness was performed fluorometrically by following the method of Ezaki et al. (1989) using DNA probes labelled with photobiotin (A1935; Sigma) and 96-well microdilution plates (Greiner Bio-One) at 50.8 °C.
The effect of thiosulfate on the growth of strain LCS2T in succinate/mineral medium was quantified by determining the cellular dry weight (Table 1). It is clear that the growth yield was dependent on the amount of added thiosulfate. The optimal thiosulfate concentration was 10 mM and the increase in biomass yield was obtained with the optimal concentration of thiosulfate. This result was similar to that reported for Limnobacter thiooxidans CS-K2T (Spring et al., 2001). Increases in the sulfate concentration, coupled with decreasing thiosulfate concentration, were observed. The concentration of thiosulfate in the medium decreased from 10 to 2 mM and the sulfate concentration reached 12 mM at stationary phase.
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Analysis of the 16S rRNA gene sequence of strain LCS2T, consisting of 1482 nt, indicated its membership of the genus Rhodanobacter (Fig. 1), the intrageneric relatedness of which ranges from 96.0 to 98.5 % among the type strains of species. Strain LCS2T showed 97.4, 96.9 and 96.8 % sequence similarity with respect to the type strains of R. fulvus, R. lindaniclasticus and R. spathiphylli, respectively.
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; Wayne et al., 1987).
Strain LCS2T differed significantly from the type strains of Rhodanobacter species in terms of the acid-production, carbon-source-utilization and substrate-hydrolysis profiles, and also with regard to nitrate reduction. The phenotypic characteristics are summarized in Table 2 and the species description.
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9c (22.0 %), 15 : 0 (21.5 %) and 17 : 0 iso (13.2 %) were predominant; the detailed fatty acid composition is shown in Table 3. Strain LCS2T has a fatty acid profile similar to those of the type strains of Rhodanobacter species, but it has a larger proportion of 17 : 1 iso 9c and a smaller proportion of 11 : 0 iso. Interestingly, the fatty acid profiles were rather different from those described by De Clercq et al. (2006) because of differences in the conditions employed. Strain LCS2T and the two Rhodanobacter type strains were cultured under identical conditions, as mentioned above, for 4 days in order to secure sufficient quantities of fatty acids because of the slow growth of the novel strain.
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Description of Rhodanobacter thiooxydans sp. nov.
Rhodanobacter thiooxydans (thi.o.ox'y.dans. Gr. n. thion sulfur; Gr. adj. oxus sharp, acid; N.L. v. oxydo to make acid, to oxidize; N.L. part. adj. thiooxydans oxidizing sulfur).
Cells are aerobic, non-motile, non-spore-forming rods (0.6–0.8x1.5–3.0 µm). Gram-negative and oxidase- and catalase-positive. Good growth is observed on R2A agar at 25–35 °C and pH 5–8. Growth occurs in the presence of 1 and 2 % NaCl. Colonies are yellowish, convex and circular with entire edges. Uses carboxylic acids and amino acids as energy and carbon sources, but cannot grow autotrophically. Thiosulfate is oxidized to sulfate in the presence of an organic carbon source. Elemental sulfur is not deposited intracellularly or extracellularly during growth on media containing thiosulfate. -Galactosidase activity is present. Nitrate is reduced, but nitrite is not. Indole and H2S are not produced. Aesculin and gelatin are hydrolysed, but casein, starch and urea are not. Acid is produced from aesculin and D-fucose, but not from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -D-xylose, galactose, glucose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. The following compounds are utilized as sole carbon sources: D-glucose, N-acetyl-D-glucosamine, maltose, D-ribose, glycogen and valerate. The following carbon sources are not utilized: L-arabinose, mannose, gluconate, caprate, adipate, D-mannitol, citrate, phenylacetate, L-rhamnose, inositol, sucrose, itaconate, suberate, malonate, acetate, DL-lactate, L-alanine, 5-ketogluconate, 3-hydroxybenzoate, L-serine, salicin, D-melibiose, L-fucose, D-sorbitol, propionate, L-histidine, 2-ketogluconate, 3-hydroxybutyrate, 4-hydroxybenzoate and L-proline. 2-Naphthyl phosphate (pH 8.5), 2-naphthyl butyrate, L-leucyl 2-naphthylamide, 2-naphthyl phosphate (pH 5.4), naphthol-AS-BI-phosphate and 6-bromo-2-naphthyl -D-glucopyranoside are hydrolysed, but 2-naphthyl caprylate, 2-naphthyl myristate, L-valyl 2-naphthylamide, L-cystyl 2-naphthylamide, N-benzoyl-DL-arginine 2-naphthylamide, N-glutaryl-phenylalanine 2-naphthylamide, 6-bromo-2-naphthyl -D-galactopyranoside, 2-naphthyl -D-galactopyranoside, naphthol-AS-BI--D-glucuronide, 2-naphthyl -D-glucopyranoside, 1-naphthyl N-acetyl--D-glucosaminide, 6-bromo-2-naphthyl -D-mannopyranoside and 2-naphthyl -L-fucopyranoside are not hydrolysed. The major quinone is ubiquinone Q-8. The predominant fatty acids are 17 : 1 iso 9c (22.0 %), 15 : 0 iso (21.5 %) and 17 : 0 iso (13.2 %). The G+C content of the DNA of the type strain is 64.6 mol%.
The type strain, LCS2T (=DSM 18863T =KCTC 12771T), was isolated from a biofilm of sulfur particles used in an autotrophic denitrification process.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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