Themoanaerobacterium calidifontis sp. nov., a novel anaerobic, thermophilic, ethanol-producing bacterium from hot springs in China

Shumei Shang 0 1 2 Long Qian 0 1 2 Xu Zhang 0 1 2 Kunzhi Li 0 1 2 Irbis Chagan 0 1 2 0 S. Shang L. Qian X. Zhang K. Li I. Chagan ( 1 S. Shang Faculty of Environmental Science and Engineering, Kunming University of Science and Technology , Chenggong Campus, Kunming 650500, China 2 Communicated by Harald Huber 3 ) Faculty of Life Science and Biotechnology, Kunming University of Science and Technology , Chenggong Campus, Kunming 650500, China A novel thermophilic Gram staining positive strain Rx1 was isolated from hot springs in Baoshan of Yunnan Province, China. The strain was characterized as a hemicellulose-decomposing obligate anaerobe bacterium that is rod-shaped (diameter: 0.5-0.7 m; length: 2.0-6.7 m), spore-forming, and motile. Its growth temperature range is 38-68 C (optimum 50-55 C) and pH range is 4.5-8.0 (optimum 7.0). The maximum tolerance concentration of NaCl was 3 %. Rx1 converted thiosulfate to elemental sulfur and reduced sulfite to hydrogen sulfide. The bacterium grew by utilizing xylan and starch, as well as a wide range of monosaccharide and polysaccharides, including glucose and xylose. The main products of fermentation were ethanol, lactate, acetate, CO2, and H2. The maximum xylanase activity in the culture supernatant after 30 h of incubation at 55 C was 16.2 U/ml. Rx1 DNA G + C content was 36 mol %. 16S rRNA gene sequence analysis indicated that strain Rx1 belonged to the genus Thermoanaerobacterium of the family 'Thermoanaerobacteriaceae' (Firmicutes), with Thermoanaerobacterium aciditolerans 761-119 (99.2 % 16S rRNA gene sequence similarity) being its closest relative. DNADNA hybridization between Rx1 and T. aciditolerans 761119 showed 36 % relatedness. Based on its physiological and biochemical tests and DNA-DNA hybridization analyses, the isolate is considered to represent a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium calidifontis sp. nov. is proposed, with the type strain is Rx1 (=JCM 18270 = CCTCC M 2011109). - The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Rx1 is AB544080. The biotechnological potential and evolutionary significance of thermophiles has led to intensive research focused on anaerobic, saccharolytic, and thermophilic bacteria, which are members of the genera Thermoanaerobacter, Thermoanaerobacterium, and Clostridium (Xue et al. 2001). Within the genus Thermoanaerobacterium, eight species have been isolated, and more recently the taxonomic relationships between some of these species have been better defined (Romano et al. 2010). Most studies of eubacterial thermophilic anaerobes have focused on the saccharolytic bacteria, which form ethanol and lactate and are promising tools for creating alternative fuels from plant biomass (Shaw et al. 2008; Patel et al. 2006). Moreover, their ethanol production capacity involving utilization of glucose and the other hexose transformed from cellulose materials has been the major focus of those studies. Since only a few of the natural anaerobic thermophilic microorganisms identified to date are capable of efficiently fermenting xylose and the other pentose transformed from hemicellulose materials, we aimed to isolate novel thermophilic anaerobic hemicellulose-decomposing bacteria from hot springs sample. The Baoshan region is situated in the southwest of Yunnan Province and encompasses a large number of natural hot springs. Water and sediment samples were collected from the regions hot springs and measured temperature (5070 C) and pH (6.07.5). Using oat spelt xylan as substrate, we isolated a Gram staining positive, obligately anaerobic, thermophilic bacterium (strain Rx1). In this paper, we describe the isolation and characterization of the ethanol-producing strain Rx1. The physiological, biochemical, and phenotypic features of Rx1 were determined, and the results of DNADNA relatedness studies indicated that Rx1 is a new member of the genus Thermoanaerobacterium. The name Themoanaerobacterium calidifontis is proposed. Materials and methods Strains and culture conditions Thermoanaerobacterium aciditolerans DSM 16487T and Thermoanaerobacterium saccharolyticum DSM7060T were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) and were cultured in modified medium 640 at 55 C. All culturing of Rx1 was performed in modified DSMZ medium 640, containing (per liter distilled water) 0.9 g NH4Cl, 0.9 g NaCl, 0.4 g MgCl2 6 H2O, 0.75 g KH2PO4, 1.5 g K2HPO4, 2.5 mg FeCl3 6 H2O, 0.75 g cysteineHCl H2O, 2 g tryptone, 1 g yeast extract, 1 ml trace element solution SL-10 (see medium 320, DSMZ), 0.5 mg resazurin, 5 ml vitamin solution (see medium 141, DSMZ, filter-sterilized), 10.0 g substrate. After adjusting pH to 7.0, the medium was prepared anaerobically under 100 % N2 and dispensed 5 ml portions into 15 ml Hungate tubes. Finally, the medium solutions were autoclaved. Initial enrichment was carried out at 60 C by inoculating the samples of mixed sediment and water into 250 ml anaerobic reagent bottles (50 ml modified medium 640). Oat spelt xylan was added as substrate, and the culture incubated until visible growth was observed. The ethanol-producing cultures were isolated through several serial liquid dilutions and applied to the anaerobic phytagel (1.2 %, w/v) shake-roll tube technique (Ljungdahl and Wiegel 1986) with xylose as substrate. Pure strains were stored as liquid cultures under anaerobic conditions at 4 and 80 C. One of the isolated strains was strain Rx1 for further characterization. Morphological, physiological, and biochemical analysis Gram reaction was determined using a Gram staining kit (Guangdong Huankai Microbial Sci. & Tech Co., Ltd. Guangdong, China). Cell morphology was examined by phase-contrast microscopy (Leica, Germany). Bacteria tolerance of NaCl (05 %, w/v), temperature (from 35 to 70 C), pH (from 3.5 to 8.5), and substrates (complete list in Table 1) was determined by growing on the modified 640 medium for four days and measuring the optical density (OD) at 600 nm using a 4802 UV/VIS double-beam spectrophotometer (Unico, Dayton, NJ, USA). Metabolite products were measured by high-performance liquid chromatography (HPLC) and gas chromatography (GC-SC2), as previously described (Shaw et al. 2008; Romano et al. 2010; Ren et al. 2008). Ethanol tolerance was determined by supplementing the modified 640 medium with ethanol (from 0 to 5.0 %, v/v) after autoclaving. The ability of Rx1 to convert thiosulfate, sulfate, sulfite, and elemental sulfur was assessed by adding thiosulfate (7 g/l), sulfate (2 g/l), sulfite (0.76 g/l), or elemental sulfur (2 g/l), respectively, to the modified medium 640 (Kublanov et al. 2007; Liu et al. 1996). All the tests were performed in triplicate. Phase-contrast microscopy was used to visualize the sulfur globules formation (Lee et al. 2007). The gas from the headspace (10 ml) culture of Rx1 was transfer (...truncated)