Thermococcoides shengliensis gen. nov., sp. nov., a new member of the order Thermotogales isolated from oil-production fluid

The order Thermotogales represents, along with the order Aquificales, the deepest phylogenetic branch in the domain Bacteria (Winker & Woese, 1991). The family Thermotogaceae, the sole family within the Thermotogales, is currently represented by six genera: Fervidobacterium, Thermotoga, Geotoga, Petrotoga, Marinitoga and Thermosipho (Urios et al., 2004). Within the last three decades, it has been recognized that the members of the Thermotogales are frequently isolated from shallow and deep submarine hydrothermal vents, continental hot springs and oil reservoirs (Haridon et al., 2001). Currently, the family Thermotogaceae contains extremely thermophilic bacteria growing up to 90 °C, such as Thermotoga maritima (Huber et al., 1986), as well as thermophiles and moderate thermophiles such as Thermosipho melanesiensis (Antoine et al., 1997) and Geotoga subterranea (Davey et al., 1993). Since the isolation of Thermotoga subterranea (Jeanthon et al., 1995) and Thermotoga elfii (Ravot et al., 1995a) from petroleum reservoirs, several other anaerobic bacterial strains have been obtained. The most recently described was the fermentative bacterium Petrotoga halophila obtained from a Congolese offshore oil well (Miranda-Tello et al., 2007), which grows at an optimum temperature of 60 °C.

In the present paper, we describe a novel thermophilic bacterium, strain 2SM-2^T, isolated from production fluid from the Shengli oil reservoir.

Strain 2SM-2^T was obtained from oil-production water of block L801 in the Shengli oilfield, in which seawater injection was being used to enhance oil recovery. The in situ temperature of the reservoir ranged between 75 and 80 °C, and the in situ pressure was 9.24 MPa. At the sampling points, the total mineralized degree was 9794 mg l⁻¹. The production water from Shengli oilfield was collected in sterile glass bottles, which were closed tightly with rubber stoppers (Cheng et al., 2007). The samples were directly transported to the laboratory and stored at 4 °C before use as a source of inoculum. Enrichments under anaerobic conditions were prepared using a modified Hungate technique (Ollivier et al., 1997). For enrichment, the following medium was used (l⁻¹ distilled water): 23 g NaCl, 1.3 g KCl, 1 g MgCl₂.6H₂O, 0.08 g CaCl₂, 0.5 g NH₄Cl, 0.3 g KH₂PO₄.3H₂O, 1.42 g Na₂SO₄, 5 ml trace element solution and 1 ml vitamin solution (Balch et al., 1979). The pH was adjusted to 7.0 by using 5 M KOH before autoclaving and the medium was dispensed into serum bottles with N₂/CO₂ (4 : 1, 150 kPa) in the headspace. Na₂S.9H₂O and NaHCO₃ were injected from sterile stock solutions to final concentrations of 0.03 % and 0.5 % before inoculation. After incubation without shaking at 65 °C for 2 weeks, fresh culture (5 ml) was then transferred anaerobically into a new bottle of sterile M141 medium (DSMZ, 1993) with methanol (120 mmol l⁻¹) under a stream of N₂. Single colonies were picked and purified by repeated use of the Hungate roll-tube method (Hungate, 1969). The purity of each culture was checked by microscopic examination.

A phase-contrast microscope (Eclipse 80i; Nikon) equipped with a digital camera (DXM-1200C; Nikon) was used for routine observation and to obtain photomicrographs of organisms. Gram reaction and motility were studied according to standard procedures (Doetsch, 1981). Transmission electron microscopy was performed with a Hitachi H7000 transmission electron microscope (Hattori et al., 2000). Cells of isolate 2SM-2^T appeared as cocci and stained Gram-negative. They were non-motile and no spore formation was detected. The cells had a surrounding membrane (Fig. 1a⇓), which is characteristic for members of the order Thermotogales. This outer membrane was easily visible under phase-contrast microscopy during growth. Cells were 0.7–0.9 μm in diameter. Typically, growth in formations of two to six cells enclosed within a large sheath (about 20–25 μm in width) (Fig. 1b⇓) could be seen. Strain 2SM-2^T remained coccus-shaped in all phases of growth. Thin sections revealed the Gram-negative structure of the cell wall, with an outer membrane (Fig. 1c⇓). Most of the outer sheath lysed in lysozyme (1 mg ml⁻¹) after 30 min at 37 °C in the mid-exponential phase of growth.

Growth was monitored by measuring the increase of the OD₆₀₀ with a UV/Visible Beckman DU 800 spectrophotometer. Effects of pH and temperature on strain 2SM-2^T were determined in triplicate in basal medium with glucose (120 mM). The pH range for growth was determined in the culture medium with various buffers at a concentration of 10 mM (Takai et al., 2002). For studies of NaCl requirements, NaCl was weighed directly in the tubes before the medium was dispensed. Strain 2SM-2^T was not able to grow in oxygenated media (oxidation was indicated by the pink colour of the resazurin). The strain grew between pH 6.0 and 8.0, optimum growth occurring at pH 7.0; however, no growth was observed at pH 5.5 or 8.5. The temperature range for growth was between 45 and 75 °C, optimum growth being at 65 °C; growth was not observed at 40 or 80 °C. Strain 2SM-2^T required 0–40 g NaCl l⁻¹ for growth; optimum growth occurred at 15 g NaCl l⁻¹. Under optimal growth conditions (temperature, pH and NaCl), the doubling time of strain 2SM-2^T was approximately 105 min.

To investigate the sensitivity of strain 2SM-2^T to antibiotics, kanamycin, streptomycin, ampicillin, chloramphenicol and erythromycin (all at 100 μg ml⁻¹) were added to sterile basal medium with glucose (120 mM) at 65 °C. Growth of strain 2SM-2^T was inhibited completely by chloramphenicol and erythromycin; kanamycin caused partial inhibition of growth. The novel isolate was resistant to streptomycin and ampicillin.

Strain 2SM-2^T was a strictly anaerobic, fermentative organism. Growth was observed on complex substrates such as beef extract, peptone and trypticase. The utilization of single substrates was studied using basal medium supplemented with 0.01 % yeast extract and the test carbon source. Yeast extract was required for growth with carbohydrates. The basic medium was supplemented with vitamin and trace mineral solutions (5 ml l⁻¹ each) (Balch et al., 1979). To confirm growth on any of the substrates, the novel isolate was subcultured twice in the same medium. The compounds that strain 2SM-2^T was able to use for growth were glucose (5 mM), acetate (10 mM), methanol (10 mM), galactose (5 mM), fructose (5 mM), xylose (5 mM), sucrose (2 mM), maltose (2 mM), sorbitol (2 mM), lactose (2 mM), xylan (2 mM), arabinose (2 mM), formate (10 mM), rhamnose (2 mM), glycerol (1 g l⁻¹), pyruvate (10 mM), starch (2 g l⁻¹), lactate (10 mM) and n-propyl alcohol (10 mM). No growth occurred on H₂/CO₂ (4/1, v/v), mannose (5 mM), tartrate (10 mM), α-ketoglutarate (10 mM), glutamate (10 mM), malate (10 mM), citric acid (10 mM), fumarate (10 mM), raffinose (2 mM), succinate (10 mM), melezitose (2 mM), salicin (2 mM) or mannitol (10 mM). The glucose fermentation products of isolate 2SM-2^T were analysed after incubation of a cell suspension at 65 °C. The fermentation end products from glucose (50 mM) were determined by ion chromatography. Acetate and lactate were measured with an 850 Professional ion chromatograph (Metrohm) with a Metrosep Organic Acids IonPac column. l-Alanine was quantified by 882 Compact ion chromatography (Metrohm) with a Metrosep C 4-150 IonPac column. Acetate (17.5 mM), lactate (1.7 mM), l-alanine (1.7 mM), CO₂, and traces of H₂ were produced in the presence of elemental sulfur. The end products increased in response to growth and glucose consumption. The production of l-alanine as an end product of glucose fermentation had been reported as a special characteristic for Thermotoga species, and it was hypothesized that this is an ancestral metabolism (Ravot et al., 1996). Production of H₂S during glucose metabolism was investigated by the addition of 500 μl 5 mM CuSO₄/50 mM HCl to 0.2 ml of the culture (Alain et al., 2002). A brown precipitate demonstrated the presence of H₂S. Thiosulfate (20 mM), sulfate (20 mM) and elemental sulfur (2 %) (Ravot et al., 1995b) were tested as potential electron acceptors on basic medium. Strain 2SM-2^T reduced thiosulfate, sulfate and elemental sulfur to sulfide. The addition of elemental sulfur increased the growth density and enhanced the utilization of glucose in contrast with thiosulfate and sulfate (data not shown). The presence of elemental sulfur as an electron acceptor might lower the hydrogen partial pressure of the medium during glucose fermentation (Antoine et al., 1997).

Genomic DNA was extracted and purified by using the modified procedure of Charbonnier & Forterre (1994), and its G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962), using a Beckman DU 800 spectrophotometer. Escherichia coli K-12 DNA was used as a reference. The G+C content of the genomic DNA of strain 2SM-2^T was 36.4 mol%, which was lower than that of members of the genus Thermotoga (Balk et al., 2002), but higher than that of members of the genus Thermosipho (Urios et al., 2004).

DNA was extracted with a Soil DNA isolation kit (BioTeke). The 16S rRNA gene was selectively amplified with a TaKaRa Thermocycler Dice TP600, using a TaKaRa 16S rDNA Bacterial Identification PCR kit (Cheng et al., 2007). The universal primer set 27f/1492r (Karita et al., 2003) was used for 16S rRNA gene amplification. PCR products were purified with a TaKaRa Agarose Gel DNA Purification kit version 2.0 and sequenced with an ABI PRISM BigDye Terminator v3.1 Cycle Sequencing kit and an ABI PRISM 3730XL DNA sequencer. The partial 16S rRNA gene sequences (1448 bp) were compared with reference published sequences in the GenBank database by using the blast program (Altschul et al., 1990), and then aligned with closely related sequences in the order Thermotogales by using clustal_x software (Thompson et al., 1997). Phylogenetic trees based on three algorithms were constructed, which included neighbour-joining (Kumar et al., 2004), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Lake, 1987). Bootstrap values were calculated after 1000 replications (Felsenstein, 1985). Strain 2SM-2^T was phylogenetically affiliated to the strain Thermosipho melanesiensis (Antoine et al., 1997) and Thermotoga maritima (Huber et al., 1986) with respective 16S rRNA gene sequence similarities of 86.8 and 86.7 %.

Phylogenetic analysis using 16S rRNA gene sequences revealed that strain 2SM-2^T fell within the cluster of the order Thermotogales and formed a clade distinct from members of the genera Geotoga, Petrotoga and Marinitoga (Fig. 2⇓). The phylogenetic tree constructed by the neighbour-joining method revealed that strain 2SM-2^T branched deeply within the family Thermotogaceae and belonged to no previously known genera in this family. The tree constructed by the maximum-likelihood and maximum-parsimony methods also supported this result (data not shown). The novel isolate was related to the moderately thermophilic strain Thermosipho melanesiensis and hyperthermophilic strain Thermotoga maritima (Fig. 2⇓), which were isolated from marine hydrothermal fields. However, strain 2SM-2^T can be clearly distinguished from Thermotoga maritima by a 10 mol% lower G+C content of its DNA (36 mol%) and by a 15 °C lower optimal growth temperature (Table 1⇓). The rod cells of Thermotoga maritima became coccoid only in stationary phase, but strain 2SM-2^T kept a coccus shape in all phases of growth. The main fermentation products of strain Thermotoga maritima were acetate and lactate. For strain 2SM-2^T, the main products from glucose fermentation were acetate, lactate and l-alanine in the presence of elemental sulfur. In comparison with Thermosipho melanesiensis, the temperature optimum for strain 2SM-2^T was lower (65 versus 70 °C) and the optimum NaCl concentration for growth was also lower (15 versus 30 g l⁻¹) (Table 1⇓). Strain 2SM-2^T differed from Thermosipho melanesiensis by its ability to reduce sulfate and thiosulfate. Moreover, strain 2SM-2^T had a broader range of substrate utilization. It could obtain energy from formate, acetate and xylose, which are not used by Thermosipho melanesiensis.

The different isolation sources reflect the restricted ecological habitats from which the organisms were isolated. Characterized by high temperature, pressure and salinity, oil reservoirs are extreme environments with particular varieties of micro-organisms present. Up to now, the order Thermotogales has been represented by six genera. Strain 2SM-2^T can be differentiated from the other members of the Thermotogales by its unique cell morphology. Strain 2SM-2^T differs from its closest phylogenetic relatives, Thermosipho melanesiensis and Thermotoga maritima, by the optimum growth parameters, the substrates utilized, the electron acceptors reduced and the main products from glucose fermentation (Table 1⇑).

From the above morphological and physiological characteristics and phylogenetic analysis, we propose that strain 2SM-2^T represents a novel species in a new genus, Thermococcoides shengliensis gen. nov., sp. nov.