Alkaliphilus halophilus sp. nov., a strictly anaerobic and halophilic bacterium isolated from a saline lake, and emended description of the genus Alkaliphilus

Inland saline and hypersaline lakes harbour diverse communities of halophilic micro-organisms, including many anaerobic species (Ollivier et al., 1994). Most anaerobic, moderately halophilic bacteria described are classified in the order Halanaerobiales, and grow optimally at about 6.0 % NaCl (Oren, 2006). In this paper, we describe a novel, obligately anaerobic and halophilic bacterium with optimum growth at 7.5 % NaCl, isolated from sediments of Xiaokule salt lake (3 ° 35′ N 8 ° 29′ E) in Xinjiang Province, China.

Xiaokule salt lake is located on a plateau at an altitude of about 4480 m above sea level. There is no river supply to the lake, and atmospheric water and groundwater are the only water sources. The low temperature, low nutrient levels and high salt content make it a relatively isolated ecosystem. A mud sample was collected from the bottom of Xiaokule salt lake from a depth of about 0.3 m. About 1 g of this sample was incubated anaerobically in 40 ml enrichment medium (DSMZ medium 210) at 32 °C for 1 week and pure cultures were obtained by the Hungate roll-tube technique (Hungate, 1969; Bryant, 1972) under a gas phase of O₂-free N₂. Three strains were isolated and purified. Two of them stained Gram-negative and were identified as belonging to the genus Halanaerobium. The third, designated strain E2R^T, stained Gram-positive and is characterized in detail herein.

The medium for the cultivation of strain E2R^T had the following composition (per litre distilled water): 2.0 g yeast extract (BD), 2.0 g tryptone (BD), 1.0 g KCl, 0.3 g KH₂PO₄, 0.5 g MgSO₄ . 7H₂O, 75 g NaCl, 10 ml Na₂S . 9H₂O (5 %, w/v), 0.4 g l-cysteine, 0.001 g resazurin and 10 ml trace element solution M144 (see DSMZ medium 144). The medium was adjusted to pH 8.0 with NaOH. To test the effect of salt concentration on growth, NaCl was added at 0, 0.5, 1, 2, 3, 5, 7.5, 10, 12, 15 or 20 % (w/v). To examine the pH range for growth, MES (pH 5.5–6.0), PIPES (pH 6.5–7.0), Tricine (pH 7.5–8.5), CAPSO (pH 9.0–9.5) or CAPS (10.0–11.5) was added at a concentration of 25 mM. The temperature range for growth was determined by incubating at 4, 10, 15, 20, 25, 28, 32, 37, 42, 45, 50, 55 and 60 °C. To test the use of substrates and potential electron acceptors, a basal medium was prepared based on JCM medium 378 without tryptone, crotonic acid or trace vitamins and with 0.1 % yeast extract (BD). For substrate utilization tests, Casamino acids (BBI), yeast extract (BD), tryptone (BD), starch (each at 10 g l⁻¹), alcohols (0.1 %, v/v) and organic acids (20 mM) were added directly to the basal medium; sugars (20 mM) were added from filter-sterilized concentrated solutions. To test for the use of accessory electron acceptors, S⁰, sodium thiosulfate, fumarate or crotonate was added at a concentration of 10 mM. Strain E2R^T and the reference strains listed below were incubated under a gas phase of O₂-free N₂ and utilization was confirmed by growing the strain in the same substrate for two subcultures. All experiments were conducted in duplicate. For comparison of phenotypic and other properties, Alkaliphilus oremlandii DSM 21761^T and Alkaliphilus crotonatoxidans JCM 11672^T were grown under identical conditions. These physiological properties are detailed in Table 1⇓ and in the species description.

Cell morphology and motility were examined by using optical (BX 40; Olympus) and transmission electron (H-600; Hitachi) microscopy. Cells of strain E2R^T were straight to slightly curved rods (0.5–0.9×1.6–6 μm). In the late-exponential and stationary phases of growth, the rods formed terminal endospores (Fig. 1⇓). Strain E2R^T was obligately anaerobic; no growth was observed in the presence of air. The generation time was about 7 h under optimal conditions. No growth occurred in the absence of NaCl; growth was obtained at NaCl concentrations between 0.5 and 15 %, with an optimum at 7.5 %. Growth occurred at 15–42 °C, with an optimum at 32 °C. The pH range for growth was 5.5–9.0 (optimum, pH 8.0).

Fatty acid methyl esters were extracted from cells grown in DSMZ medium 104b PYX without fructose until an OD₆₀₀ of 0.1 was reached, and were analysed by using GC/MS (Kuykendall et al., 1988). The fatty acid profiles of strains E2R^T, A. oremlandii DSM 21761^T and A. crotonatoxidans JCM 11672^T are compared in Table 2⇓. The predominant component of all three strains was iso-C_15 : 0, but a much higher level was found for strain E2R^T. In addition, iso-C_15 : 1 F (double bond position unknown) represented 13.6 % of the total fatty acids in strain E2R^T and 6.3 % in A. oremlandii DSM 21761^T, but only 1.4 % in A. crotonatoxidans JCM 11672^T.

For determination of the G+C content of the genomic DNA and for 16S rRNA gene sequence determination and phylogenetic analysis, DNA was prepared and purified as described by Marmur (1961). The DNA G+C content of strain E2R^T was determined by thermal denaturation (T_m) (Marmur & Doty, 1962) with Escherichia coli K-12 DNA as the calibration standard; the DNA G+C content of strain E2R^T was 28.5 mol%. The 16S rRNA gene was amplified with primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGYTACCTTGTTACGACTT-3′). The sequence between positions 28 and 1491 (E. coli numbering) was compared with all closely related sequences by using the EzTaxon service (Chun et al., 2007). Preliminary 16S rRNA gene sequence comparisons showed that strain E2R^T was related most closely to the type strains of recognized Alkaliphilus species, namely A. oremlandii DSM 21761^T (95.5 % similarity), Alkaliphilus transvaalensis JCM 10712^T (93.2 %), Alkaliphilus peptidifermentans DSM 18978^T (92.9 %) and A. crotonatoxidans JCM 11672^T (91.2 %). The almost-complete 16S rRNA gene sequence (1449 nt) of strain E2R^T was aligned by using clustal x software (version 1.8). Phylogenetic trees were constructed by using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods with the mega4 program package (Tamura et al., 2007) and by using the maximum-likelihood method (Felsenstein, 1981) with the phylip 3.6 program (Felsenstein, 1993). Evolutionary distances were calculated according to the algorithm of Kimura's two-parameter model (Kimura, 1980) for the neighbour-joining method and indicated that the new isolate clustered within the genus Alkaliphilus (Fig. 2⇓). Trees constructed with the other two methods showed similar topologies (Fig. 2⇓ and Supplementary Fig. S1, available in IJSEM Online).

Strain E2R^T could be distinguished from the related alkaliphiles A. transvaalensis JCM 10712^T and A. peptidifermentans DSM 18978^T, both of which grow optimally above pH 9.0 and do not grow below pH 7.5 (Takai et al., 2001; Zhilina et al., 2009). In addition, under a nitrogen gas phase, strain E2R^T utilized not only proteinaceous substrates and some organic acids but also oligosaccharides, which were not metabolized by A. oremlandii DSM 21761^T. Strain E2R^T could be differentiated from A. crotonatoxidans JCM 11672^T based on its ability to utilize tryptone, sucrose and lactate, together with tolerance to NaCl. Based on the phenotypic characteristics described above, phylogenetic analysis, elevated amounts of iso-C_15 : 0 and relatively low DNA G+C content, strain E2R^T is considered to represent a novel species of the genus Alkaliphilus, for which the name Alkaliphilus halophilus sp. nov. is proposed.