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-caprolactone) plastic granules as fixed bed, and emended description of the genus Thermomonas
Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, KL Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Jean Swings
jean.swings{at}ugent.be
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-caprolactone); the granules were used as a fixed bed in a denitrification reactor. All the strains showed similar fatty acid profiles. The 16S rRNA gene sequences of five strains were phylogenetically related to Thermomonas spp. Repetitive extragenic palindromic DNA-PCR (REP-PCR) fingerprinting revealed four groups, and DNA hybridizations between representative strains showed that the strains belonged to two new species within the genus Thermomonas, for which the names Thermomonas fusca (type strain LMG 21737T=DSM 15424T) and Thermomonas brevis (type strain LMG 21746T=DSM 15422T) are proposed. Both species are able to grow at low temperatures, but not at 50 °C, and are non-haemolytic. Both species can be differentiated by several other phenotypic features from earlier described species of the genus Thermomonas. Cell extracts contain mainly branched fatty acids, with C15 : 0 iso, C17 : 1 iso 
9c, C11 : 0 iso 3OH and C11 : 0 iso as main constituents. The G+C content of the DNA of the novel species is between 67·6 and 68·7 mol%.
Published online ahead of print on 23 May 2003 as DOI 10.1099/ijs.0.02684-0.
The EMBL accession numbers for the 16S rRNA gene sequences of Thermomonas fusca LMG 21736, LMG 21737T, LMG 21738 and LMG 21739 and Thermomonas brevis LMG 21746T are AJ519985, AJ519986, AJ519987, AJ519988 and AJ519989, respectively.
Present address: Louis Parentstraat 7, B-8301 Knokke-Heist, Belgium. 
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) and the synthetic polymer poly(-caprolactone) (Boley et al., 2000) as sources of carbon for denitrification and electron donor. Both polymers have been shown to be biodegradable by many bacteria (Mergaert & Swings, 1996; Mergaert et al., 2000).
Previously, we characterized the biofilm on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) granules used in a continuous-upflow fixed-bed reactor, by isolation and identification of the dominant microflora (Mergaert et al., 2001b). In a parallel experiment, a reactor with poly(-caprolactone) with a similar set-up was investigated and large numbers of bacteria were isolated from the biofilm on the granules and characterized by fatty acid analysis (Boley et al., 2003). While most organisms were identified as Acidovorax spp., a group of 22 strains with similar fatty acid profiles remained unidentified; representative strains of this group were found to be phylogenetically related to Thermomonas haemolytica (type species) and Thermomonas hydrothermalis, two recently described, slightly thermophilic species isolated from kaolin slurry (Busse et al., 2002) and a hot spring (Alves et al., 2003), respectively. The purpose of the present study was to further characterize these 22 isolates by using a polyphasic taxonomic approach. On the basis of the results presented here, two new species of the genus Thermomonas are proposed.
Strains were isolated from biofilms growing on poly(-caprolactone) granules (Union Carbide), which were used as a fixed bed in a continuous-upflow denitrification reactor, according to methods described previously (Mergaert et al., 2001b). In brief, the granules were taken aseptically from the reactor, the biofilm was washed off, and serial dilutions were plated onto R2A agar (Difco) and incubated aerobically at 20 °C. The 22 strains investigated in this study were all isolated from granules sampled at the anoxic top of the fixed bed in June 1998 and August 1999. The strain designations are listed in Table 1. As reference strains, T. haemolytica LMG 19653T, LMG 19654 and LMG 19656 were included in some experiments. Strains were pre-cultured on R2A agar for 3 days at 28 °C, unless mentioned otherwise.
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). Phylogenetic analysis was performed using the BIONUMERICS software package (Applied Maths; http://www.applied-maths.com/), taking into account the homologous nucleotide positions after discarding all unknown bases and gaps. Using the same software package, a neighbour-joining dendrogram (Saitou & Nei, 1987) was constructed based on global alignment of the sequence similarities (Fig. 1). Strains LMG 21738 and LMG 21739 showed a pairwise sequence similarity of 99·7 %, and the similarity between strains LMG 21736 and LMG 21737T was 99·8 %. Sequence similarity between these two couples was 97·7–97·9 %, and with strain LMG 21746T the sequence similarity was 97·9–98·0 % and 96·2 %, respectively. The latter percentage is a strong indication that the sequenced strains belong to more than one species (Stackebrandt & Goebel, 1994). After a FASTA search, the sequences of the isolates were compared to related sequences available from the EMBL database. In the phylogenetic dendrogram generated, the sequences from the strains grouped with those of T. haemolytica LMG 19653T (95·0–97·2 % pairwise sequence similarity) and T. hydrothermalis SGM-6T (95·0–96·6 %) within the Xanthomonas branch of the 
-Proteobacteria. Strains LMG 21736 and LMG 21737T showed highest sequence similarity (99·6 %) to an unidentified Fe(II)-oxidizing bacterial strain, BRG3, isolated from freshwater sediment after anaerobic chemolithoautotrophic enrichment conditions using Fe(II) as the sole electron donor and nitrate as the electron acceptor (Buchholz-Cleven et al., 1997).
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; Rademaker et al., 2000) and to select representative strains for further study by DNA–DNA hybridization analysis. REP-PCR genomic fingerprints were prepared for all strains listed in Table 1
, using the primers REP1R-I and REP2-I (Versalovic et al., 1991), as described by Rademaker & de Bruijn (1997) and Rademaker et al. (2000). Numerical analysis was carried out using the BIONUMERICS software package, as described by the same authors. On the basis of numerical and visual comparisons, four groups, each consisting of strains with almost identical profiles, could be distinguished (Table 1). Representatives from each REP-PCR cluster were compared by DNA–DNA hybridization analysis and their G+C contents (mol%) were determined (Table 2). DNA was prepared according to the method of Pitcher et al. (1989), with the modifications described by Logan et al. (2000). Alternatively, the method of Marmur (1961) was applied. DNA base content was determined using a HPLC method. DNA was degraded enzymically into nucleosides as described by Mesbah et al. (1989). The obtained nucleoside mixture was then separated by HPLC using a Waters Symmetry Shield C8 column thermoregulated at 37 °C. The solvent was 0·02 M NH4H2PO4 l-1 (pH 4·0) with 1·5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference. DNA from the strains had a G+C content of 67·6–68·7 mol%, which is in the range reported for T. haemolytica (67·1–68·7 mol%; Busse et al., 2002) and slightly higher than that reported for T. hydrothermalis (64·7 mol%; Alves et al., 2003).
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, using a HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 50 °C. Reciprocal experiments were performed for every pair of strains and the results given are the means. The relative DNA binding between the strains showing REP-PCR fingerprint types A, B and C was 82–102 %. This confirms the findings of Stackebrandt & Goebel (1994) that 16S rRNA gene sequence similarities down to 97 % are found between strains showing DNA reassociation values of more than 70 %, a percentage generally accepted as the minimum for an intraspecies genomic relationship (Wayne et al., 1987). These strains showed 49–57 % relative DNA binding with strain LMG 21746T, which represented REP-PCR fingerprint group D. Strain LMG 21737T, representing REP-PCR cluster A, and strain LMG 21746T, representing REP-PCR cluster D, showed 40 and 41 % DNA binding, respectively, to T. haemolytica LMG 19653T. Thus, the 22 strains isolated from a denitrification reactor belong to two new species (Wayne et al., 1987) within the genus Thermomonas. For the 18 strains from REP-PCR clusters A, B and C, the name Thermomonas fusca is proposed; for the four strains showing REP-PCR profile type D, the name Thermomonas brevis is proposed.
Phenotypic characteristics were determined for all strains of T. fusca, T. brevis and T. haemolytica. Most methods, including API 20NE and API ZYM tests, have been described by Mergaert et al. (2002), and the incubation temperature was 28 °C. Growth at 4, 14, 20, 28, 37 and 50 °C was assessed on R2A agar after 5 days incubation. Haemolysis was tested on Columbia agar base (Oxoid) supplemented with 10 % defibrinated horse blood after 2 days incubation at 37 °C. Reduction of nitrate was tested as described by Mergaert et al. (2001b). Cell morphology was investigated by phase-contrast microscopy and flagella staining was performed using the method of Heimbrook et al. (1989).
Cells of T. fusca were straight, filament-forming rods, while cells of T. brevis were non-filamentous. Motile cells from both species showed single polar flagella. The strains grew well on R2A agar and tryptic soy agar (TSA; BBL), between 10 and 37 °C, and under anoxic conditions in nutrient broth supplemented with nitrate. At 50 °C, no growth was detected, and the inocula were killed off, as evidenced by further incubation at 28 °C. At 14 or 20 °C, the colonies of T. fusca were brown in colour with a dark centre, while at 28 °C two strains, LMG 21741 and LMG 21742, failed to produce the brown pigment, but were faint-yellow. At 37 °C, only strains showing REP-PCR profile type C (Table 1) were able to produce brown-pigmented colonies, while the other strains remained non-pigmented. Strains of T. haemolytica and T. brevis were non-pigmented regardless of the incubation temperature.
Biochemical and physiological characteristics of the strains tested are given below. Different reactions were obtained for gelatin liquefaction with strains of T. haemolytica, and for C14-lipase, trypsin and chymotrypsin with strains of T. fusca. The species can be differentiated from each other and from T. hydrothermalis (Alves et al., 2003) by several features, which are listed in Table 3.
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). For all Thermomonas strains tested, cell extracts contained mainly branched fatty acids, with C15 : 0 iso, C17 : 1 iso 9c, C11 : 0 iso 3OH and C11 : 0 iso as main constituents (Table 4). The fatty acid composition of T. haemolytica was similar to those reported by Busse et al. (2002), although the latter authors used different temperatures of incubation for cultivation of cells. Within T. fusca broad ranges of relative amounts were found for the fatty acids C14 : 0 iso and C16 : 0 iso, but there was no clear-cut correlation with genomic data and phylogenetic similarity. The three species differed from each other in their relative amounts of the main fatty acids C15 : 0 iso and C17 : 1 iso 9c. The fatty acid compositions were also similar for those reported for T. hydrothermalis strains (Alves et al., 2003), except for lower amounts of C17 : 0 iso 9c detected in the latter species, most probably as a result of the higher growth temperature (50 °C) used by Alves et al. (2003), a phenomenon demonstrated for T. hydrothermalis and discussed by Busse et al. (2002).
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Cells are rods that form filaments, and are 0·7 µm wide by 3–10 µm long. Motile by means of a single polar monotrichous flagellum or non-motile. Aerobic, but anoxic growth occurs with nitrate as electron acceptor. Growth occurs on complex medium at 4–37 °C, but not at 50 °C; optimum growth occurs at 28–37 °C. On R2A agar, convex, circular, smooth, translucent colonies of about 2 mm diameter and with entire margins are formed after 5 days incubation at 28 °C. At 20 °C, brown colonies with a dark centre are produced, but at higher temperatures some strains remain non-pigmented or are faint-yellow in colour. Nitrate and nitrite are reduced. Non-haemolytic. Positive for the following enzymes: oxidase, catalase, alkaline phosphatase, C4-esterase, C8-esterase, valine arylamidase, cystine arylamidase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and 
-glucosidase. Negative for the following enzymes: arginine dihydrolase, urease, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -mannosidase, N-acetyl-glucosaminidase and -fucosidase. Aesculin is not hydrolysed. Gelatin is liquefied. No growth occurs on glucose, mannose, N-acetylglucosamine, maltose, arabinose, mannitol, gluconate, caprate, adipate, malate, citrate or phenylacetate. Indole is not produced. Other characteristics are as for the genus. The G+C content of the DNA is 67·6–68·7 mol%. Isolated from biofilm grown on poly(-caprolactone) plastic granules that were used as a fixed bed in a denitrification reactor.
The type strain is Thermomonas fusca LMG 21737T (=DSM 15424T).
Description of Thermomonas brevis sp. nov.
Thermomonas brevis (bre'vis. L. fem. adj. brevis short).
Cells are non-filamentous rods that are 0·7 µm wide by 3–10 µm long. Motile by means of a single polar monotrichous flagellum. Aerobic, but anoxic growth occurs with nitrate as electron acceptor. Growth occurs on complex medium at 4–37 °C, but not at 50 °C; optimum growth occurs at 28–37 °C. On R2A agar, convex, circular, smooth, translucent colonies of about 2 mm diameter and with entire margins are formed after 5 days incubation at 28 °C. Regardless of the incubation temperature, non-pigmented colonies are formed. Nitrate and nitrite are reduced. Non-haemolytic. Positive for the following enzymes: oxidase, catalase, alkaline phosphatase, C4-esterase, C8-esterase, valine arylamidase, cystine arylamidase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -glucosidase and N-acetyl-glucosaminidase. Negative for the following enzymes: arginine dihydrolase, urease, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -mannosidase and -fucosidase. Aesculin is hydrolysed. Gelatin is liquefied. Growth occurs on glucose, mannose, N-acetylglucosamine and maltose, but not on arabinose, mannitol, gluconate, caprate, adipate, malate, citrate or phenylacetate. Indole is not produced. Other characteristics are as for the genus. Isolated from biofilm grown on poly(-caprolactone) plastic granules that were used as a fixed bed in a denitrification reactor.
The type strain is Thermomonas brevis LMG 21746T (=DSM 15422T). The G+C content of its DNA is 68·4 mol%.
Emended description of the genus Thermomonas
The inclusion of two additional species (this report) as well as T. hydrothermalis (Alves et al., 2003) in the genus requires an emendation of the description of Thermomonas. The genus is as described by Busse et al. (2002) with the following modifications and emendations. Phylogenetically, the genus belongs to the Xanthomonas branch within the -Proteobacteria. Cells are either rods or filaments. Species differ with regard to maximum and minimum growth temperatures and are thus either mesophilic or slightly thermophilic. The G+C content of the DNA is between 64·7 and 68·7 mol%.
The type species of the genus is Thermomonas haemolytica.
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1·0 % of total). Also in trace amounts of total in some strains: C10 : 0, C10 : 0 3OH, C10 : 0 iso, C10 : 0 anteiso, C12 : 0 iso, C12 : 0 iso 3OH, C13 : 0 anteiso, C14 : 1