Introduction

Microbes are believed to survive and interact with different ecosystem through various ways for their survival in many environment. Thermophilic bacteria in hot springs survive at moderate to high temperature adopting different characteristics than the normal bacteria. Thermophilic bacilli grow best at temperature between 45⁰ to 75⁰ C and there fore they can survive in hot spring at higher temperature (1). For several decades thermophilic bacteria have been focused for biotechnological and academic interest. In  particular phenotypic and genotypic characterization  of thermophilic bacteria from hot spring has done from different region in world including India.(2,3,4 ).Although there are several hot springs present in Orissa India, few have been studied (3, 4 ) and many are left about the knowledge of microbial communities. Here we report the isolation and characterization of thermo stable bacteria isolated from a hot spring Tarabalu which has not explored earlier in Orissa, India.

Materials and Methods

Water samples of volume 500ml were collected from 5-6 hot springs at Tarabalu present at one location scattered over 100 meter radius in sterile glass bottle and transported to the Microbiology Department, Regional Medical Research centre, Bhubaneswar, India and analyzed within 24hr.Temperature and P^H of the water of the hot spring was measured at the time of collection with thermo-meter and P^H indicator paper respectively. Immediately after reaching the laboratory, 100µl of water sample was streaked on to the Nutrient Agar (Himedia, Mumbai, India) plate which was then sealed in plastic bags to prevent from drying and incubated at 57⁰ C for 7 days. Aliquots of 1ml, 2ml and 3ml of water were added to the Nutrient broth and incubated at 57⁰ C in orbital shaking water bath for 7 days. The broth in flasks were observed daily for turbidity and subculture were made from those flasks showing turbidity on to Nutrient Agar plates and incubated at 57⁰ C sealed in plastic bags to prevent drying. Colonies on Nutrient Agar were purified by serial transfers.

Morphology and motility of the bacterium was observed by Gram’s stain and hanging drop method respectively. The hot spring isolate was subjected for various physiological and biochemical characterization in order to detect the difference if any by following the conventional methods. For different biochemical tests, the test strains was grown on nutrient agar plates and a single colony was picked up for inoculation into specific medium for detection of Cytocrome oxidase, Tryptophanase, Urease, Catalase, Coagulase,  Nitrate reduction and  utilization of  Glucose, Sucrose, Lactose and Citrate. Amylase activate of the bacteria was tested by the starch agar-iodine method (5); Proteases activity was tested on skim milk agar (6); lipases activity was tested using Tween 20 and Tween 80 (Sigma Chemical Company, U. S. A.) as substrates in phosphate buffer (7).

The growth temperature range of strain was examined by measuring optical density (OD) at 405nm of the culture incubated at 57⁰ C -100⁰ C through 75⁰ C in 100ml of Nutrient broth in Orbital shaker. The P^H range for growth was examined by measuring the O.D. at 405nm of the culture incubated at ambient temperature and P^H 6.0-10.0.

Antimicrobial susceptibility was tested by the modified Kirby-Bauer disk diffusion technique (8) with commercially available discs (Himedia, Mumbai, India) ampicillin (10μg), chloramphenicol (30μg), co-trimoxazole (25μg), ciprofloxacin (5μg), furazolidone (100μg), gentamicin (10μg), neomycin (30μg), nalidixicacid (30μg), norfloxacin (10μg), streptomycin (10μg), tetracycline (30μg), rifampicin (5mcg), kanamycin (30mcg), cephotaxime (30mcg), carbenicillin (100mcg), azithromycin (15mcg), piperacillin (100mcg), and doxycyclin (30mcg). Characterization of strains as being susceptible or resistant was based on the size of inhibition zone around each disc according to manufacturer’s instruction, which matched interpretive criteria recommended by the WHO (9).

Results and Discussion

The temperature of the hot spring at Tarabalu was 57⁰ C. The P^H of the spring water was 7.0. The strain formed round, flat, smooth, grayish white colonies with 5-6mm diameter. It was motile gram negative, rod shaped and aerobic. The shape and size of the cell were observed by light microscope.

The growth P^H range of strain was 6. 5 – 9. 5 and the optimum P^H was 7. 0. The growth temperature range was 65⁰ C -100⁰ C and the optimum temperature was 75⁰ C which indicates that it was thermo stable bacteria.

Several biochemical characteristics such as utilization of Glucose, lactose & manitol; reduction of Nitrate; absence of Gas & Hydrogen Sulphide in TSI Agar medium; and absence of Cytochrome Oxidase, Catalase, Coagulase, Urease, Lipase, Protease and Glutamase enzymes were observed. The presence of the enzyme Amylase was detected at 57⁰ C.

The hot spring isolate was susceptible to ampicillin, chloramphenicol, co-trimoxazole, furazolidone, gentamicin, neomycin, nalidixicacid, streptomycin, tetracycline, norfloxacin, ciprofloxacin, rifampicin, kanamycin, azithromycin, piperacillin, doxycyclin and resistant to carbenicillin, and cephotaxime

Phenotypically our hot spring strain is completely different from strain isolated at Atri (3, 4). Detection of Amylase evidenced as a very useful enzyme which has wide industrial application (10). It is used in starch and detergent related industries.  In this study our hot spring   is amylase positive at 57⁰C. To the best of our knowledge this is first report of isolation and characterization thermophilic bacteria from a new hot spring at Tarabalu which has considerable significance. Further genetic analysis is required to find its genus and species position.

Acknowledgements

We thankfully acknowledge “Indian Council of Medical Research ,New Delhis” for it’s funding to conduct the study.

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