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1 Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto-City, Kumamoto 860-8555, Japan
2 Department of Bioengineering, Yatsushiro National College of Technology, 2627 Hirayama-shinmachi, Yatsushiro, Kumamoto 866-8501, Japan
3 Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto-City, Kumamoto 860-8555, Japan
Correspondence
Toru Shigematsu
shige{at}kumamoto-u.ac.jp
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ABSTRACT |
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-alanine, citrate, D-fructose, glycerol, isobutyrate, isophthalate, D(-)-mannitol, maleate, malonate, phenylacetate, propionate, protocatechuate, terephthalate, D-tryptophan and L-tryptophan. Comparisons of phenotypic and genotypic characteristics with other known species belonging to the genus Delftia suggest that strain T7T represents a novel species, for which the name Delftia tsuruhatensis sp. nov. is proposed; strain T7T is the type strain (=IFO 16741T=ATCC BAA-554T).
Published online ahead of print on 20 June 2003 as DOI 10.1099/ijs.0.02285-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain T7T is AB075017.
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). A large amount of terephthalate is discharged during production of PET bottles and purification of terephthalic acid. Biological degradation of this compound has therefore been of interest in recent years.
Several micro-organisms have been shown to degrade terephthalate under aerobic conditions, including the Gram-positive bacteria Nocardia sp. strain DSM 43251 (Engelhardt et al., 1979), a Bacillus species (Karegoudar & Pujar, 1985) and Dietzia sp. strain GS-1 (Sugimori et al., 2000). Among the Proteobacteria, Bordetella sp. strain K1, Pseudomonas sp. strain C4S (Kimura & Ito, 2001) and two Comamonas testosteroni strains, T-2 (Schläfli et al., 1994) and YZW-D (Wang et al., 1995), have demonstrated the ability to degrade terephthalate.
Most of these micro-organisms have been reported to produce protocatechuate as an intermediate metabolite. The enzymic system responsible for the degradation of terephthalate has been thoroughly studied for C. testosteroni strain T-2. In that strain, terephthalate is degraded to protocatechuate by the terephthalate 1,2-dioxygenase system (TERDOS) (Schläfli et al., 1994) and protocatechuate is then metabolized via the meta cleavage pathway. Another C. testosteroni strain, YZW-D, reportedly uses a similar enzyme system for degradation of terephthalate (Wang et al., 1995).
Recently, a novel terephthalate-assimilating bacterium, designated strain T7T, was isolated from the activated sludge of a domestic wastewater treatment plant. The isolate could utilize terephthalate as sole carbon and energy source. Based on 16S rRNA gene sequences and phylogenetic analyses, strain T7T was placed in the genus Delftia; no members of this genus have previously demonstrated the ability to degrade terephthalate. Based on phenotypic and phylogenetic characterizations, it is suggested that strain T7T constitutes a novel species, for which the name Delftia tsuruhatensis sp. nov. is proposed.
For enrichment of terephthalate-assimilating bacteria, a laboratory-scale activated sludge process was constructed. The aeration tank with a working volume of 3 l was maintained at 30 °C and air was supplied at 3 l min-1. A 2·8 l aliquot of activated sludge, which was provided by Kumamoto Hokubu sewage works (Tsuruhata, Kumamoto, Japan), was used as the initial reactor seed. The mixed liquor suspended solids (MLSS) concentration of the activated sludge, determined according to the Japanese standard method (Japanese Industrial Standards Committee, 1986), was 1620 mg l-1. Concentrations of total organic carbon (TOC) and inorganic carbon in the activated sludge supernatant, obtained after centrifugation at 10 000 r.p.m. for 10 min, were respectively 11·3 and 49·1 mg l-1. The pH of the supernatant was 6·86. A basal salt medium supplemented with sodium terephthalate was fed continuously into the reactor at a rate of 1·2 l day-1. The basal salt medium (pH 7·6) contained the following (l-1): K2HPO4, 3·48 g; KH2PO4, 0·435 g; (NH4)2SO4, 1 g; MgSO4.7H2O, 0·2 g; FeCl3.6H2O, 0·02 g; NaCl, 0·1 g; and CaCl2.2H2O, 0·1 g. Concentrations of sodium terephthalate were increased stepwise as follows: 200 mg l-1 for days 1–12; 500 mg l-1 for days 12–21; and 1000 mg l-1 for days 21–26. After 26 days continuous cultivation, the MLSS concentration of the culture broth in the reactor increased to 2800 mg l-1 and the concentrations of TOC and terephthalate in the reactor supernatant were respectively 15 and <1 mg l-1. After 26 days continuous cultivation, the culture broth was used for isolation of terephthalate-assimilating bacteria with the basal salt medium containing sodium terephthalate at a concentration of 500 mg l-1 supplemented with 1·5 % agar. A 1 ml portion of the culture broth was diluted with sterilized water, inoculated onto agar plates and incubated at 30 °C. Following the first screening using an agar plate, positive colonies were cultivated in the basal salt medium containing sodium terephthalate at a concentration of 1000 mg l-1 at 30 °C with continuous shaking. An isolate with a specific growth rate of 0·65 h-1 was obtained and designated strain T7T.
Strain T7T was cultivated in liquid basal salt medium containing sodium terephthalate at a concentration of 15 mM and the terephthalate in the culture broth was quantified according to Yumihara et al. (2002). The initial terephthalate concentration of 15 mM was degraded completely within 12 h (Fig. 1a). TERDOS activity of the cell lysate of the isolate was determined using a Clarke-type oxygen electrode according to the method of Schläfli et al. (1994) (Fig. 1b). The result suggests that the isolate degrades terephthalate by TERDOS, as described previously for C. testosteroni strain T-2 (Schläfli et al., 1994).
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. With sodium terephthalate concentrations between 0·5 and 4·0 g l-1, the oxygen consumption rate of the isolate was constant, at approximately 1·0 µl min-1. At sodium terephthalate concentrations below 0·2 g l-1 or between 6·0 and 10·0 l-1, a lower oxygen consumption rate of approximately 0·8 µl min-1 was observed. These results indicated that sodium terephthalate concentrations below 4·0 g l-1 had no inhibitory effect on respiration of the isolate.
Colonies of strain T7T on nutrient broth plates were white. The cells did not produce water-soluble pigments or fluorescent pigments on King A or King B media. Cells appeared as slightly curved, short rods, 0·7–1·2x2·4–4·0 µm, and occurred singly or in pairs. They were motile. Gram-staining, preformed according to standard microbiological procedures (Gerhardt et al., 1994), was negative. The isolate accumulated poly--hydroxybutyrate, determined according to Stanier et al. (1966).
Strain T7T grew at 10–40 °C, with optimum growth at 35 °C. No growth was detected after 10 days incubation at 7 or 42 °C. A pH range of 5 to 9 was suitable for growth, with an optimum response at pH 7·0. The doubling time was approximately 1·06 h under optimum growth conditions in liquid basal salt medium containing 1000 mg sodium terephthalate l-1. The isolate could utilize the following compounds as sole energy and carbon sources under aerobic conditions: acetamide, -alanine, citrate, D-fructose, glycerol, isobutyrate, isophthalate, D(-)-mannitol, maleate, malonate, phenylacetate, propionate, protocatechuate, terephthalate, D-tryptophan and L-tryptophan. The isolate could not utilize the following compounds: DL-3-aminobutyrate, D(+)-arabitol, catechol, 2,3-dihydroxybenzoate, D-glucose, lactose, phthalate, L-serine, starch, sucrose, L-valine and D(+)-xylose. Arginine dihydrolase, catalase, oxidase, lipase (Tween 80 hydrolysis) and urease activities, evaluated according to standard microbiological procedures (Gerhardt et al., 1994), were positive. The isolate performed meta cleavage of protocatechuate, determined according to Hugh & Gilardi (1974). It could not denitrify, although the strain reduced nitrate to nitrite, as determined by the method of Stanier et al. (1966). The isolate could not hydrolyse starch. The isolate demonstrated no fermentative response, determined by the O/F reaction (Hugh & Leifson, 1953), using D-glucose, D(-)-fructose or terephthalate as substrates in OF basal medium (Difco).
Whole-cell fatty acids were extracted as methyl esters according to Komagata & Suzuki (1987) and analysed by GC-MS. The most abundant fatty acids in strain T7T were C16 : 0 (33·2 % total fatty acid methyl esters), C16 : 1 (31·1 %) and C18 : 1 (24·6 %). In addition, significant amounts of cyclopropanoic acid (C17 : 0; 3·6 %), 3-OH C10 : 0 (2·6 %), C12 : 0 (2·0 %), C15 : 0 (1·6 %) and C14 : 0 (1·3 %) were detected. 3-OH C8 : 0 was not detected. The DNA G+C content of strain T7T, measured according to Kamagata & Mikami (1991), was 66·2 mol%.
For determination of the 16S rRNA gene sequence of strain T7T, cells were lysed according to Hiraishi (1992). The 16S rDNA fragment was amplified by PCR (Hiraishi et al., 1994) using the following universal primers: forward, 5'-AGAGTTTGATCATGGCTCGA-3' (positions 8–27 of the Escherichia coli 16S rRNA gene); and reverse, 5'-GGCTACCTTGTTACGACTT-3' (positions 1510–1492). The sequence of the amplified 16S rDNA fragment (1456 bp) was aligned with reference sequences using CLUSTAL X, version 1.8 (Thompson et al., 1997), and a phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) with the Kimura two-parameter model (Kimura, 1980) using MEGA version 2.1 (Kumar et al., 2001) (Fig. 2). Sequence similarities between strain T7T and relatives were as follows: Delftia sp. EK3, 99·8 %; Delftia sp. AN3, 99·7 %; Delftia acidovorans MBIC1305, 99·4 %; D. acidovorans MC1, 99·2 %; D. acidovorans BP2, 98·7 %; D. acidovorans ATCC 15668T, 98·6 %. The tree indicated that the isolate is apparently affiliated with the genus Delftia, which was introduced by the relocation of [Comamonas] acidovorans on the basis of phylogenetic and phenotypic analyses (Wen et al., 1999). The sequences of strains belonging to the genus Delftia form two distinct clusters, both with significant bootstrap values (69 and 88). One cluster, with a bootstrap value of 69, includes the sequences of D. acidovorans strains ATCC 15688T, MBIC1305, BP2 and MC1. The sequence of strain T7T and two Delftia strains, AN3 and EK3, were assigned to the other cluster (bootstrap value of 88). Therefore, the sequences of strains belonging to the genus Delftia can be divided in two distinct phylogenetic groups. DNA–DNA hybridization was performed according to Willems et al. (2001) at a hybridization temperature of 52 °C using total DNAs of strain T7T and D. acidovorans ATCC 15668T. Hybridization values were calculated as means of four duplicate hybridizations, with a mean standard deviation of ±5·2 %. As an internal control, reciprocal hybridizations were carried out. Using DNA from either strain T7T or ATCC 15668T as the labelled DNA, hybridization values with DNA from ATCC 15668T or strain T7T were respectively 68·7 and 68·6 %. The DNA–DNA hybridization value between the two strains was thus not significantly below the threshold value of 70 % used for species delineation (Stackebrandt & Goebel, 1994), and therefore does not provide a clear indication of the taxonomic placement of strain T7T within the genus Delftia.
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shows comparative phenotypic properties for strain T7T and D. acidovorans. The isolate could not utilize 3-aminobutyrate, whereas most D. acidovorans strains (20 of 21 strains tested), including the type strain, can utilize this substrate (Willems et al., 1991). 3-OH C8 : 0, which is characteristic of members of the genus Delftia, was not detected among the cellular fatty acids, and the G+C content of the isolate (66·2 mol%) was slightly lower than those of other strains belonging to the genus. On the basis of the phenotypic and phylogenetic analysis above and of the DNA–DNA hybridization value of around 70 %, it is proposed that strain T7T should be classified as a novel species in the genus Delftia, for which the name Delftia tsuruhatensis sp. nov. is proposed.
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Gram-negative, motile, slightly curved, short rods (0·7–1·2x2·4–4·0 µm). Cells occur individually or in pairs. Growth occurs between 10 and 40 °C; optimum growth at 35 °C. Growth occurs at pH 5·0–9·0; optimum growth at pH 7·0. Doubling time is approximately 1·06 h under optimum growth conditions. Non-fermentative. Positive for arginine dihydrolase, catalase, oxidase, lipase (Tween 80 hydrolysis) and urease activities and meta cleavage of protocatechuate. Denitrification ability is not observed, although nitrate reduction is found. Does not hydrolyse starch. The following compounds can be utilized as energy and carbon sources: acetamide, -alanine, citrate, D-fructose, glycerol, isobutyrate, isophthalate, D(-)-mannitol, maleate, malonate, phenylacetate, propionate, protocatechuate, terephthalate, D-tryptophan and L-tryptophan. The following compounds cannot be utilized: DL-3-aminobutyrate, D(+)-arabitol, catechol, 2,3-dihydroxybenzoate, D-glucose, lactose, phthalate, L-serine, starch, sucrose, L-valine and D(+)-xylose. The most abundant fatty acids are C16 : 0, C16 : 1 and C18 : 1. Cyclopropanoic acid (C17 : 0), 3-OH C10 : 0, C12 : 0, C15 : 0 and C14 : 0 are detected in smaller amounts. 3-OH C8 : 0 is not detected. The total DNA G+C content of the type strain is 66·2 mol%.
The type strain, T7T (=IFO 16741T=ATCC BAA-554T), was isolated from activated sludge in Tsuruhata, Kumamoto Prefecture, Japan.
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ACKNOWLEDGEMENTS |
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, terephthalate concentration; 
, OD546). (b) TERDOS activity (Schläfli et al., 1994