Thermophiles were the first extremophile to be discovered. They are defined as groups of microorganisms which grow at a temperature above 45oC, some of them actively grow at 80oC (Madigan and Mars, 1997). Natural environments for thermophilic microorganisms are widespread on Earth's surface (Bale et al., 1997; Barns et al., 1994). These can be isolated from different environments such as deep ocean-basin cores, shallow marine hot springs, petroleum reservoirs, deep-sea hydrothermal vents and the leachate of a waste pile from a canning factory (Rahman et al., 2004; Bae et al., 2005).
The majority of the thermophilic bacterial species described belong to the genus Bacillus genetic groups 1 and 5 (Rainey et al., 1994). Recently, all the thermophilic Bacillus species previously assigned to group 5 have been transferred to the genus Geobacillus. So far there are 11 validly described Geobacillus species including Geobacillus stearothermophilus, G. caldoxylosilyticus, G. kaustophilus, G. subterraneus, G. thermocatenulatus, G. thermoleovorans, G. thermoglucosidasius, G. thermodenitrificans, G. toebii, G. uralicus and G. uzenensis (Logan & Berkeley, 1984; Claus & Berkeley, 1986; Golovacheva et al., 1975; Suzuki et al., 1983; Priest et al., 1988; White et al., 1993; Zarilla and Perry, 1987; Manachini et al., 2000; Ahmad et al., 2000; Nazina et al., 2001; Fortina et al., 2001; Sung et al., 2002). Members of this genus exhibit high levels of 16 S rRNA sequence similarity (98.5-99.2%).
For several decades, thermophilic bacteria have attracted the interest of many scientists due to their biotechnological potential in addition to scientific curiosity. It is reported that thermophilic strains produce interesting biological molecules including unusual enzymes, antibiotics, anti algal compounds, anti-cancer substances and secreted sugars (Ladenstein and Antranikian, 1998). Thermostable enzymes such as ï¡-amylase, cellulase, ï¡-glucosidase, ï¡-galactosidase, ï¢-glucosidase, ï¢-galactosidase, protease, pullulanase and xylanase, which are stable in the range of 90-105°C, can be obtained from thermophilic organisms (Adams et al., 1995; Adams and Kelly, 1998).
In recent years, the use of 16S rRNA gene has been regarded as the 'gold standard' for identification and phylogenetic analysis of bacteria (Ludwig and Schleifer, 1999). The conserved nature, manageable size (1.5 kb), high information content of rRNA gene and extensive database have resulted in the use of 16S rRNA gene as an ideal molecular chronometer in bacterial phylogeny. The 16S rRNA molecule comprises conserved region and nine variable regions (V1-V9) which provide genetic information to distinguish bacteria up to species and subspecies levels (Woese, 1987).
Tectonic framework of the Middle East and geothermal activity in Pakistan owes its origin to the collision of the Indian Plate with the Eurasian Plate, whereby the Main Mantle Thrust (MMT) and the Main Karakoram Thrust (MKT) have been produced. Geothermal manifestations are concentrated along these features. It may be noted that hot springs occur in the Gilgit, Hunza and Yasin valleys in the Northern areas as well as in Azad Kashmir. The geothermal system here is the result of the collision of the Indian and Eurasian plates. Hot springs are scattered and their temperature ranges up to 91°C (Bakht, 2000).
The numbers of hot springs occurrence are large but the knowledge on hot spring microflora is scanty (Adhikary and Sahu, 1987). Different regions of Pakistan deserve special attention to explore the vast potentialities of endemic and diverse microflora for their commercial usage. So the present study is planned for isolation and characterization of thermophilic microflora from hot springs of Azad Kashmir and their potential in biotechnology.
MATERIALS AND METHODS
Sample collection
Water samples from different distances from the source (where the water oozes out) of the hot spring was collected separately in sterile wide mouthed thermal glass container, which kept the temperature of the samples constant, as well as in the aseptic culture tubes containing 1.0% nutrient broth medium. These tubes were incubated for two hours in the same place into the water and transported to the lab for further processing (Khalil et al., 1998). In situ measurements of temperature, pH and EC was done to create the profile of hot springs.
Isolation of thermophilic bacteria
Thermophilic bacteria were isolated on petri plates containing LB agar medium (1% tryptone, 1% NaCl, 0.5% yeast extract, 1.5% agar) through serial dilution of the samples. One hundred microlitre of the sample was poured over the media and incubated at 60oC while same quantity of sample was inoculated to the flask containing LB medium to isolate extreme thermophiles (Clark et al., 1958; Llarch et al., 1997; Narayan et al., 2008).
Morphological and biochemical characterization
Different morphological and biochemical tests like Gram's staining, colony morphology, motility test, oxidation fermentation test and QTS-20 Tests were performed according to Wiegel and Ljungdahl (1981) and MacFaddin (2000).
Physiological characterization
In order to study the physiological behavior of the pure cultures, growth was determined at various incubation temperatures (20oC- 85oC). The pH range for growth was studied by growing the cultures overnight (16 hrs) at 65±1oC in LB medium adjusted to different pH (4-10) separately (Anton et al., 2002). Turbidity will be monitored at 600 nm on a double beam UV/VIS scanning spectrophotometer (Nair and Surendran, 2004).
Molecular characterization
Chromosomal DNA was isolated following protocol of Kronstad et al. (1983). 16S rDNA was amplified by PCR using two universal primers 9F and 1510R.
9F 5'-GAGTTTGATCCTGGCTCAG-3
1510 ´5- GGTTACCTTGTTACGACTT-3´
PCR amplification was carried out with 1 μl (50ng) DNA as a template, 5 µl of 2.5 mM dNTPs, l µl of 10 µM of each primer, 5 µl of 25 mM magnesium chloride, 5 µl of IX PCR buffer (75 mM Tris-HCl pH 8.8 at 25°C, 20 mM (NH4)2SO4 and 0.01% tween 20) and l µl of 2.5 units of Taq DNA polymerase in a 50 μl reaction mixture. The reaction mixture was incubated at 94°C for 5 min and then subjected to 35 cycles of 94°C for 30 sec, 54°C for 30 sec and 72°C for 1 min; followed by one final cycle of 72°C for 10 min. The PCR product was analysed by 1% agarose gel electrophoresis, stained with ethidium bromide, and visualized with UV light. The PCR product was purified using spin prep gelmelt kit (Novagen). The purified 16S rDNA was then sequenced using ---------------------------------. The purified sequencing reaction mixtures were electrophoresed automatically using --------------------.Highest similarities of the 16S rDNA gene sequence of the new strain to that of other bacteria were searched using the BLAST tool of GenBank (Altschul et al., 1997). The 16S rDNA sequence of strain (-----) was aligned with representative 16S rDNA sequences of related taxa using clustal w software (Thompson et al., 1994). Phylogenetic tree was constructed using neighbor-joining method (Saitou and Nei, 1987) and Jukes and Cantor model (Jukes and Cantor, 1969) employing the software MEGA 4.0 (Tamura et al., 2007)
Biotechnological potential
Isolated thermophilic bacteria were grown on carboxymethylcellulose, starch, xylan and filter paper as sole carbon source to check their biotechnological potential.
RESULTS AND DISCUSSION
Isolation and morphological studies
Few thermophilic bacteria were isolated from the hot spring of Azad Kashmir (PH 7.9, 63-85oC, EC (ms) 1.15). Isolation was performed by sampling water from surface, centre and bottom of hot spring. More than 20 colonies were isolated on LB agar plate. The bacterial culture show good growth in LB medium and on LB agar plates (inoculated plate covered with a layer of 0.8% sterile agar) after incubation at 65oC, indicating the facultative anaerobic nature of the culture. One colony showing the best growth was chosen for further study. The isolate had small, smooth, round and slimy colonies. Morphological observation of the isolate under light microscope showed that it is rod shaped bacteria. Further analysis indicated that the isolate was gram positive and motile (Fig.1). Narayan et al. (2008) isolated Geobacillus stearothermophilus strains from the Savusavu hot springs in Fiji.
Physiological and biochemical studies
Growth was observed at temperature between 20-85oC and pH ranges from 4-10 (Fig. 2&3). No growth was observed below 35oC and above 80oC. The isolate showed good growth between 55-65oC while maximum growth was obtained at 65oC and no growth was obtained at pH below 5.5 and above 8.5 while maximum growth was observed at pH 7.0. The isolate gave positive test for gelatin hydrolysis, acid production from glucose and ortho-nitrophenyl ï¢-D- galactopyranoside while gave negative result for Arginine Dihydrolase, Lysine Decarboxlase, Ornithine Decarboxylases, citrate utilization, H2S Production, Urease, Tryptophan Deaminases, Indole production, Acetoin production, Fermentation/oxidation (Mannitol, Inositol, Sorbitol, Rhamnose, Sucrose, Melibiose, Amygdalin, Arabinose). The results of morphological, physiological and biochemical were compared with the characteristics given in Bergey's manual and indicated that the isolate belong to the Geobacillus stearothermophilus. Similar results were reported by Claus and Berkeley, 1986).
Molecular and phylogenetic studies
Amplified 16S ribosomal RNA fragment of about 1500 bp size was obtained from isolate using 9F and 1510R universal primers (Fig. 4) and was partially sequenced (around --- bp) using an automated DNA sequencer. The sequence was compared with 16S rDNA nucleotide sequences present in GenBank. Nucleotides BLAST search of the 16S rRNA sequences showed that the isolate had ---------- percent identity with Geobacillus stearothermophilus. A phylogenetic tree was constructed by aligning 16S rRNA sequences of ----- type strains taken from GenBank, NCBI and sequence of the isolate from this study (Fig. ---). The phylogenetic tree showed that the isolate was closely related to Geobacillus stearothermophilus.
Biotechnological potential of isolate
The organism showed good growth in the presence of CMC, xylan and starch as sole carbon source indicating that the isolate may has the potential to produce CMcase, xylanase and amylase while no growth was observed when filter paper was used as sole carbon source. Thermophiles are promising sources of heat-stable enzymes. In addition to higher thermostability, proteins from thermophiles often showed higher stability toward organic solvents and higher activity at elevated temperature. In addition, genetic engineering in altering the stability of enzymes is a difficult task and laborious processes. Therefore, efforts have been focused on the screening of microorganisms harboring intrinsically stable biocatalysts (Schmidt-Dannert et al., 1996). Geobacillus stearothermophilus has been a subject of study for its amylases, xylanases, proteases, lipases, CMcase and avicellase (Vihinen et al., 1994; Sinchaikul et al., 2001, Kim et al., 2002; Rehman et al., 2003; Makky, 2009; Zhang et al., 2010). Important industrial enzymes from Geobacillus stearothermophilus have been cloned and expressed successfully (Jorgensen et al., 1991; Ozcan and Ozcan, 2008; Jhang et al., 2009; Asmaa et al., 2011).
The isolated strain is of significant importance in industry and applied science. Geobacillus stearothermophilus spores are included among the materials being autoclaved in industry. Death of the spores indicate that the autoclave is functioning properly and sterilization was successful (Lemieux et al., 2006). Geobacillus stearothermophilus has very high cadmium ion absorption potential and can be used for metal mobilization in drinking water and also to improve the waste treatment of soil (Hetzer et al., 2006).
This study indicated that Geobacillus Stearothermophilus is an important thermophilic strain, present in the hot spring of Azad Kashmir, that can be exploited for its industrial and biotechnological importance.
