Gordonia paraffinivorans sp. nov., a hydrocarbon-degrading actinomycete isolated from an oil-producing well

The genus Gordonia belongs phylogenetically to the suborder Corynebacterineae, the mycolic acid group within the order Actinomycetales (Stackebrandt et al., 1997). The species of Gordonia show considerable metabolic diversity and are capable of degrading toxic environment-contaminating compounds, as exemplified by the isolation of strains possessing hydrocarbon-oxidizing, rubber-degrading, aromatic-desulphurizing and 3-ethylpyridine-degrading pathways (Kummer et al., 1999; Linos et al., 2002; Kim et al., 1999; Gilbert et al., 1998; Rhee et al., 1998; Yoon et al., 2000). From the point of view of bioremediation, the genus Gordonia is a very important taxon. Some species have been used for decontamination of polluted soils and waters (Bell et al., 1998). The members of the genus Gordonia are widely distributed in various environments such as soil, activated sludge, biofilm and the mangrove rhizosphere (Tsukamura, 1971; Lechevalier & Lechevalier, 1974; Bendinger et al., 1995; Takeuchi & Hatano, 1998; Linos et al., 1999; Kummer et al., 1999). There are no reports of Gordonia species in the producing-well water of oilfields, which has low levels of nutrients, consisting mainly of straight-chain and aromatic hydrocarbons.

In the course of an investigation of the microflora in the Daqing oilfield formation, we isolated several hydrocarbon-degrading strains from well-bore water samples (Nazina et al., 2000). A hydrocarbon-degrading bacterium, designated strain HD321^T, appeared to represent a novel taxon based on preliminary investigations of its phenotypic and phylogenetic characteristics. Here, we describe the physiological, chemotaxonomic and phylogenetic characteristics of strain HD321^T and propose a novel species of the genus Gordonia, Gordonia paraffinivorans sp. nov., for this strain.

Strain HD321^T was isolated from a water sample collected in late May 1996 from a producing well of Daqing oilfield, China. The water sample was inoculated into 100 ml minimal salts medium containing (l⁻¹) 1·0 g NH₄Cl, 0·5 g KH₂PO₄, 0·75 g Na₂HPO₄.12H₂O, 0·2 g MgSO₂.7H₂O, 0·02 g CaCl₂ and 10 ml trace-element solution (Lee et al., 1991), supplemented with 5 ml liquid paraffin. This medium was incubated at 37 °C on a rotary shaker at 250 r.p.m. After the liquid paraffin was emulsified by growth of micro-organisms, a portion of the cultivation medium was diluted and plated on nutrient agar containing (l⁻¹) 10 g tryptone, 3 g beef extract, 5 g NaCl and 15 g agar. Strain HD321^T was isolated on nutrient agar and showed orange-red colonies on glucose potato agar (GPA).

Cell morphology was determined by phase-contrast and electron microscopy. Mobility was determined by the hanging-drop method. The optimal conditions for growth were determined in nutrient broth with 0–14 % NaCl, pH 5–10 and at 4–60 °C. Carbon source utilization was tested in the medium described previously (Kämpfer et al., 1990). Controls were grown without substrate addition. The ability to grow in the presence of 0·1 % oleic acid and 0·001 % zinc chloride was tested as described previously (Kim et al., 1999). The methods used for biochemical tests were described previously (Smibert & Krieg, 1981). The isomeric form of diaminopimelic acid in the cell wall was analysed using TLC according to the method of Komagata & Suzuki (1987). The sugars of the cell wall were analysed as described previously (Saddler et al., 1991). Menaquinones were extracted and purified from freeze-dried cells using the method of Collins (1985) and determined by reversed-phase HPLC. Fatty acid analysis was performed using standard methods and compared to the database of fatty acids in the MIDI Sherlock Microbial Identification System (Microbial ID). Mycolic acid was determined by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). Genomic DNA of the strain was prepared by the method of Marmur (1961) and the purity was checked spectrometrically. The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962). The methods used for PCR amplification of 16S rRNA gene, sequencing of the PCR products and determination of the phylogenetic position were described previously (Zhang et al., 2002). The reference strains used in sequence comparison are shown in Fig. 1⇓.

The novel strain HD321^T had morphological and biochemical properties that were consistent with its classification in the genus Gordonia (Stackebrandt et al., 1988). The strain was a Gram-positive, non-acid-fast, non-motile, rod-shaped and strictly aerobic bacterium. Cells (0·3–0·5×1–2 μm) often occurred singly or in a typical coryneform V-shape. Catalase and urease were positive. Oxidase was negative. Gelatin and cellulose were not hydrolysed. Strain HD321^T grew well at 30–37 °C and very slowly at 20 and 45 °C. Growth occurred in medium containing 0·5–7 % (w/v) NaCl and at pH 5·5–9·5. Some other biochemical properties useful for identifying and differentiating strain HD321^T and type strains of other species of the genus Gordonia are represented in Table 1⇓ and in the description of the species.

The chemotaxonomic properties of strain HD321^T were consistent with the chemotaxonomic markers of the genus Gordonia (Chun et al., 1997; Takeuchi & Hatano, 1998; Stackebrandt et al., 1988). The cell wall of strain HD 321^T contained meso-diaminopimelic acid as the only diamino acid and arabinose and galactose as the major cell-wall sugars, indicating that the wall chemotype was type IV (Lechevalier & Lechevalier, 1970). The predominant menaquinone was MK-9(H₂). The fatty acid profile of strain HD321^T was composed of C_16 : 0 (25·8 %), tuberculostearic acid (10-methyl-C_18 : 0) (22·4 %), C_18 : 1ω9c (20·5 %), summed feature 4 (C_16 : 1ω7c and/or i-C_15 : 0 2-OH) (11·5 %), C_18 : 0 (9·3 %), C_14 : 0 (2·7 %), C_17 : 0 (2·2 %), C_17 : 1ω8c (1·6 %) and C_20 : 0 (1·2 %). Mycolic acids ranged from 52 to 62 carbon atoms (2·8 % C₅₂, 6·7 % C₅₄, 25·2 % C₅₆, 2·5 % C₅₇, 41 % C₅₈, 1 % C₅₉, 19·1 % C₆₀, 1·7 % C₆₂), with C₅₆ and C₅₈ being the principal mycolic acids. The G+C content of the DNA was 66·0 mol%. Fine qualitative and quantitative differences in fatty acid patterns and mycolic acid chain lengths could distinguish strain HD321^T from type strains of other species of the genus Gordonia.

The 16S rDNA of strain HD321^T was amplified by PCR and an almost-complete nucleotide sequence (1472 bp) was determined by direct sequencing. The phylogenetic tree showed that strain HD321^T was in the cluster comprising members of the genus Gordonia and formed a monophyletic clade (Fig. 1⇑). The 16S rDNA sequence similarities between strain HD321^T and the type strains of other Gordonia species with validly published names were 95·3–97·6 %. The highest level of sequence similarity (97·6 %) was to Gordonia amicalis and Gordonia rubripertincta. These values and the results shown in the phylogenetic tree indicate that strain HD321^T represents a novel member of the genus Gordonia (Stackebrandt & Goebel, 1994). Because of the ability of the strain to use paraffin as a carbon source, the name Gordonia paraffinivorans sp. nov. is proposed.