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And FC 2004 Were Isolated from Sediment Samples Collected from Mae Fang Hot Spring, Northern Thailand, Using 480G Medium

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And FC 2004 Were Isolated from Sediment Samples Collected from Mae Fang Hot Spring, Northern Thailand, Using 480G Medium CHARACTERIZATION AND IDENTIFICATION OF TWO HYPERTHERMOPHILIC P1 BACTERIA ISOLATED FROM A HOT SPRING IN THAILAND: A THERMOTOGA SP. STRAIN FC 1002 AND A FERVIDOBACTERIUM SP. STRAIN FC 2004 Wirojne Kanoksilapathama, Porranee Keawrama, Patlada Pasomsupa, Maria C. Portillob and Juan M. Gonzálezb a Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom, 73000, Thailand bIRNAS-CSIC, Avda. Reina Mercedes 10, 41012 Sevilla, Spain SEPTEMBER 10-13, 2012 E-mail: [email protected] INTRODUCTION Hyperthermophilic and thermophilic, anaerobic bacteria belonging to order Thermotogales, thrive within extreme environments including, marine hydrothermal systems, petroleum reservoirs and continental springs. Nine official genera (Fervidobacterium, Geotoga, Kosmotoga, Marinitoga, Petrotoga, Thermococcoides, Thermosipho, Thermotoga and Oceanotoga) have been reported. Cells of these genera are usually surrounded by a characteristic toga, a sheath-like structure ballooning over the end(s). They are capable of utilizing 2- 2- proteins and carbohydrates for heterotrophic growths. Many of them can gain energy from anaerobic respiration using S2O3 , SO4 and So as terminal electron acceptors. The aims of this study were to characterize growth kinetics and metabolisms, and classify taxonomic positions of two new fresh water anaerobic hyperthermophilic isolates. RESULTS Strain FC 2004 MATERIALS & METHODS Strain FC 1002 MORPHOLOGY ORGANISMS Strains FC 1002 and FC 2004 were isolated from sediment samples collected from Mae Fang Hot Spring, Northern Thailand, using 480G medium. MEDIA 480G, pH 7.5 (per liter) SEM and phase contrast micrographs of strain NaCl (0.5 g), NH4Cl (0.33 g), CaCl2.2H2O (0.15 g), MgCl2.6H2O (0.35 g) KCl (0.3 g), KH2PO4 (0.3 g), pancreatic FC 2004 reveal ensheathed rods sized of digestion of casein (1 g), yeast extract (0.5 g), A5 solution 0.6x1.2-2 µm with a balloon at an end. (1 ml), 0.2% Resazurin solution (0.5 ml). SEM and phase contrast micrographs of Filamentous cells sized up to 30-40 µm long Carbohydrate test medium (CTM) strain FC 1002 reveal rods sized of were occasionally detected. This medium contains similar compositions to the 480G, 0.5x2.5-5 µm with a toga at the ends. except that 0.1 g of pancreatic digestion of casein and 0.05 16S rDNAs & PHYLOGENETIC ANALYSES g of yeast extract were used. This basal medium containing GROWTH CONDITIONS 0.1% carbohydrate was employed for fermentation testing. Phylogenetic analyses on 16S rDNAs of Optimal conditions for growths of strains strains FC 1002 (JF339227) and FC 2004 CARBOHYDRATE FERMENTATION FC 1002 (85oC, pH7.5, 0.05-0.2 %NaCl) (JF339224 and JF339225) versus reference o Glucose concentration (0.005-0.1%) was and FC 2004 (78-85 C, pH7.5-8, 0-0.1 sequences belonging to order Thermotogales optimized using CTM, the basal medium. %NaCl) were determined. revealed that strains FC 1002 and FC 2004 Strain FC 2004 (approx. 105 cells/ml) was are members of Thermotoga and Fervidobact- inoculated and incubated at 80 oC for 48 h. erium, respectively. Kosmotoga olearia NR 044583 77 100 Marinitoga spp. 100 99 99 Geotoga spp. DISCUSSION & CONCLUSION 100 56 Petrotoga spp. Thermopallium natronophilum X91822 Summary is shown in Table 1. Based on 100 Fervidobacterium islandicum AW-1 |AF434670 99 85 Fervidobacterium changbaicum CBS-1 |NR 043248 morphology, physiology, habitats, G+C 58 62 Fervidobacterium pennavorans |EF565822 96 Fervidobacterium gondwanense AB39 |NR 036997 96 content of genomes and phylogenetic Fervidobacterium nodosum |CP000771|:c814384-813004 75 Strain FC2004 |JF339224+JF339225 ************** this study 100 analyses, strains FC 1002 and FC 2004 Thermosipho spp. 78 Thermotoga thermarum DSM 5069 |NR 024751 were identified as Thermotoga sp. FC 79 Thermotoga hypogea SEBR 7054 |NR 029205 100 Thermotoga subterranea SL1 |NR 025969 1002 and Fervidobacterium sp. FC 2004. 99 Thermotoga elfii SM-2 |EU276416 Thermotoga lettingae TMO |NR 027542 Compared with T. maritima and T. 94 Thermotoga maritima |M21774 Increased cell densities were substantially 98 Thermotoga neapolitana DSM 4359 |NR 027530 petrophila, strain FC 1002 requires the 98 Thermotoga petrophila RKU10 |NR 042374 detected, and decreasing concentrations of Strain FC 1002 |JF339227 ********************************************this study residual sugar (%) were obtained. The CTM lowest NaCl for optimal growth, grows containing 0.1% of a test carbohydrate was optimally at the highest temperature and 0.05 Strain FC 1002 was placed at the deepest chosen for further studies. has the lowest G+C content (41.3 mol%). position of a subgroup that was composed of o Strains FC 1002 and FC 2004 remarkably Both strain FC 2004 (85 C) and F. chang- Thermotoga neapolitana, T. maritima and T. o utilized glucose, maltose, sucrose, starch, baicum (80 C) are hyperthermophiles. petrophila (bootstrap score of 98). The 16S cellobiose and carboxymethyl cellulose However, FC 2004 is unable to utilize rDNA sequence of strain FC 2004 shares high (CMC). Strain FC 1002 utilized lactose, but lactose and has approx. 12 mol% G+C similarities to Fervidobacterium pennavorans, not strain FC 2004. content higher (44 vs 31.9 mol%). F. islandicum and F. changbaicum (96%); F. Strain FC 2004 is able to degrade duck Moreover, the deepest positions observed nodosum (94%) and F. gondwanense (92%), feather, but not strain FC 1002. in the branches suggest that both hyper- respectively. Moreover, this strain was thermophilic bacteria (strains FC 1002 positioned at the earliest point of the branch ACKNOWLEDGEMENTS and FC 2004) might be novel species. (bootstrap score of 96). This research was financed by the Scientific Promotion and Development Fund, Faculty of Science, Silpakorn University (RGI 2553-06) and Silpakorn University Research and Development Institute (SURDI 54/01/18 and SURDI 55/01/05). JMG and MCP acknowledge Table 1. Summary & comparisons. CMC=carboxymethyl cellulose, NR=not report, H=hyperthermophile, T=thermophile support from a CSIC movility program, PA1001993 and PA1002058, and the Andalusian Government Bio288 which included FEDER funds. REFERENCES Cai J, Wang Y, Liu D, Zeng Y, Xue Y, Ma Y & Feng Y (2007). Fervidobacterium changbaicum sp. nov., a novel thermophilic anaerobic bacterium isolated from a hot spring of the Changbai Mountains, China Int. J. Syst. Evol. Microbiol. 57, 2333 – 36 Friedrich AB & Antranikian G (1996). Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Appl. Environ. Microbiol. 62, 2875 – 82 Huber R, Langworthy TA, Kölnig H, Thomm M, Woese CR, Sleytr UB & Stetter KO (1986). Thermotoga maritima sp. nov. represents a new genus of uniquely extremely thermophilic eubacteria growing up to 90°C. Arch. Microbiol. 144, 324 – 33 Huber R, Woese CR, Langworthy TA, Kristjansson JK & Stetter KO (1990). Fervidobacterium islandicum sp. nov., a new extremely thermophilic eubacterium belonging to the “Thermotogales.” Arch. Microbiol. 154(2), 105 – 11 Takahata Y, Nishijima M, Hoaki T, Maruyama T. (2001). Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan. Int. J. Syst. Evol. Microbiol. 51(Pt 5), 1901 – 9 .
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