ISOLATION AND IDENTIFICATION OF THERMOPHILIC BACTERIA SPECIES FROM HOT SPRINGS

1. ISOLATION AND IDENTIFICATION OF THERMOPHILIC BACTERIA SPECIES FROM HOT SPRINGS Ercan Akaya, S., Kivanc, M., Çakır, E. Anadolu University, Eskişehir, Turkey ABSTRACT In this study thermophilic bacteria, growing optimally at 55 oC and 65 oC, were isolated from Gazlıgöl, Gecek, Hüdai, Sarıcakaya, Gediz, Eynal and Kızıldere hot springs. Then these isolates were identified according to nutritional characteristics, total protein profiles and cellular fatty acid profiles. Their enzyme activities were also investigated. Bacterial diversity of seven different hot springs were investigated and these isolates were identified as Geobacillus genus. Keywords: Geobacillus, Thermophile, Hot spring Fatty acid compositions The fatty acid compositions of the hot springs isolates and B. stearothermophilus were determined. Major fatty acid were iso fatty acid (Table 2). Table .1. Biochemical characteristics of isolates: (+: positive result %90 ; -:negative result %10; d: positive result %11-89; w: weak growth; Isolates were named useing name of hot spring : Gazlıgöl Gg, Gecek Gc, Ömerli Ö, Hüdai H, Eynal E, Gediz Gd, Sarıcakaya S, Kızıldere K. Table 2: Fatty acid composition of hot springs isolates. INTRODUCTION Figure 1 Phylogenetic tree (Wosse et al., 1990). Nowadays all organisms are assigned into one of three domains, each described by a different type of cells: Archaebacteria, Eubacteria and Eucarya and, all eucaryotes are placed in the domain Eucarya whereas, procaryotes represent a branch which has never evolved past the microbial stage (Figure 1). The procaryotic cell types are placed in the domains Archaea and Eubacteria. These two domains of procaryotes differ in many aspect of their translation apparatus as well as many other important ways and are thought to have diverged early in the history of life on earth. Archaebacteria share some molecular features with eucaryotes. They show atypical cell structure and live in extreme enviroments (high salt and temperature). Eubacteria includes cell with typical procaryotic cell structure (Madigan et al., 2000; Beldüz et. al., 2000; Prescott at al., 1999). It has so many different genus of bacteria. The genus Bacillus is large and diverse collection of aerobic and facultatively anaerobic, rod-shaped, Gram-positive (to Gram-variable), endospore-forming organisms with growth temperature optima in the range >45oC to >70oC have been isolated for over 100 years from wide range of mesophilic and thermophilic environments (Nazina et. al., 2001; Beldüz et.al., 2000; Touzel et. al., 2000). Currently Thermophilic aerobic spore-forming bacils are classified into the genera Bacillus, Alicyclobacillus, Brevibacillus, Aneuribacillus, Sulfobacillus, Thermoactinomyces and Thermobacillus (Nazina et. al., 2001; Touzel et. al., 2000; Heyndrickx et. al., 1997;Dufresne et. al., 1996). Thermophilic species of Bacillus have been isolated from a wide range of environments but only few species were isolated from solfatara field and Hot Springs such as B.caldotenax, B.coldovelox, B.coldolitycus, B.thaemochatenulatus, B.flavothermus, B.tusciae and B.acidocaldarius (Beldüz etal., 2000; Canganella and Trovatelli, 1995). The majority of thermophilic species described belong to the genus Bacillus genetic groups 1 and 5, as judged from their 16S rRNA sequences (Nazina et al., 2001; Rainey et al., 1994). We studied high-temperature hot springs from seven different geographical area and, active and diverse microbial community was found in these thermal ecosystems. Several pure cultures of dominant aerobic, thermophilic, hydrocarbon-oxidizing bacteria were isolated. A preliminary characterization of these bacteria based on phenotypical features indicated that they belong to the genus Bacillus. SDS-PAGE protein profiles Gel electrophoresis of total proteins revealed that the seven hot springs isolates tested had some difference in their protein profiles (Figure.6 ) We showed analyse of the protein patterns of E.coli, B.cereus, B. stearothermophilis, B.flovothermus and our isolates and, figure 6 is a gel photograph of SDS-PAGE analysis. Enzyme activity:Enzyme activity of isolates were showed Table 3. Our isolates have a high thermophilic esterase(C4), esterase lipase (C8) Naftol AS-BI phosphohydrolase, α-glucosidase. MATERİAL AND METHOD Isolations of strains:Seven Gram-positive rods were isolated from water samples of different hot springs. The water temperature of hot springs investigated were around 56-212 oC and pH is 8-9.5. After collection,water samples were immediately transferred to laboratory and inoculated in nutrient broth (NB) and, incubated at 60-65 oC. One day old enrichment cultures were streaked on agar plates in order to obtain single colonies. Purity of the isolates was assessed by colony morphology and microscopy. Nutritional characteristics: The utilisation of organic compounds as carbone source was tested in solide media (10ml) suplemented with 0.5%(w/v) of the following compounds which had been seperately sterilized in stock solutions; glucose, xylose, sucrose, mannitole, sorbitole, arabinose, maltose, fructose. Strains were incubated at 65oC. NaCl tolerance: Nutrient broth was prepared containing 1, 2 and 3 % NaCl, respectively. The growth of isolates at different salt concentrations were tested. Gel electrophoresis of proteins: Extract from cells growing actively on NB medium were obtained according to the methods of Ertan and Arda (1999). Protein concentration in the extracts was measured according to the method of Bradford (1976). Electrophoresis on 12% SDS-PAGE was carried out as described by Ertan and Arda (1999). Proteins were stained in a solution that contained comassie blue R-250 (0.125%), methanol (5%), acetic acid (10%), for 2-4 hours, and visualised by destaining. Molecular mass (10-200 kDa #SM0661) standarts were purchased from Fermentas. Analysis of cellular fatty acids and quinones: Whole-cell fatty acids and the main type of isoprenoid quinones were determined using biomass grown on nutrient agar at 60 oC for 24 h. Fatty acids were extracted according to Pires et.al.(2005). Reaction mixture was analysed with Agilent Midi System (GC Chromotography). Enzyme activity: Cultivation of bacteria was prepared at 60 oC for 24 h. Enzyme activities were determined at 60 oC by API-ZYM. Table 3: Detection of enzymes (+++: intensive positive result; +positive result). H20 Ec. Bc. Bs. K01 Gg03 S02 Ö05 B.flv Gc02 E02 Gd10 M Figure 6. Comassie brillant blue-stained SDS-PAGE showing proteins of total cell extracts of thermophilic bacteria related to the genus Bacillus isolated eight different hot springs and from strain Escherichia coli (Ec.), Bacillus cereus (Bc), Bacillus stearothermophilus (Bs), Bacillus flavothermus (Bf). Moleculer mass marker on the far right lane. RESULTS Isolation of strains and nutritional characteristics Sixty five strains have been isolated from eight hot springs by enrichments and purification and, the seven representative isolates used in this study were selected from 65 strains (Figure 2., 3., 4., 5.). On the surface of nutrient agar, all strains formed round, muscous, small, colourless colonies with a diameter of about 0.5-2.5 mm. Many sporulating cell could be observed in old cultures and, endospores were terminal or subterminal. Isolates were moderately thermophilic organisms able to utilize aerobically a large variety of sugars (Table 1). All strains could grow anaerobically, reducing nitrat to N2; strains Gc02 and H21 could reduced nitrate. All strains produced acid, fructose and maltose. No acid was formed sorbitole and arabinose. The substrates used by all strains as energy and carbon sources tryptone, nutrient broth, yeast extract, acetate. All strains were catalase-positive. Indole and Voges-Proskauer reaction were negative. The strains grew optimally at pH 7.0-8.0, 60 oC and with 1 % (w/v) NaCl in medium. Besides all isolates were tested by VITEC analysis sistems (Table 4). DISCUSSION There is an increasing interest in thermophilic environments, particularly in regard to ecology, evolution as well as general and applied microbiology. Among all known thermophilic species, thermophilic Bacillus spp. and Clostridium spp. are of special interest since they can not only grow but also survive over very extend periods due to sporilation, and they can produce thermostabile enzymes (Beldüz et al., 2000; Sharp et al., 1991). Hot springs with temperature of 56-212 oC and pH 8-9.5 are unique ecological niche for thermophilic bacteria. We have studied the microbiological characteristics of range of high-temperature hot springs in Afyon, Kütahya, Denizli, Eskişehir and isolated eight strains. All seven isolates were moderately thermophilic, neutrophilic, aerobic or facultatively anaerobic, spore-forming rods that utilize a range of carbohydrates. Table 4: Results are compared of biochemical tests, VITEC analises and Fatty acids analysis. The isolates could be distinguished from bacilli group by a number of morphological and biochemical features, nutritional traits (utilization of arabinose, glucose, lactose, saccarose, xylose, mannitole, sorbitole, maltose, fructose), production of gas from nitrate and temperature, pH and NaCl growth ranges and, gram stain. They were negative for the following reactions: indole and Voges-Proskauer test (acettylmethlcarbinol production). No strains utilized sorbitole and growth 28 oC, 37 oC, 80oC, pH 4, %3 NaCl. The genus Bacillus has been extensively studied with respect to fatty acid profiles and B stearothermophillus was assigned to a seperate group in all studies (Nazin et. al., 2001; Manachini, et. al., 2000; Kampfer, 1994). According to our data and data available in the literature, our isolates and all other representatives of group 5, B.stearothermophilus, B. thermoglukosidasius, B. thermoleovarans, B. catenulatus, B. caldolyticus, B. caldotenax and B. caldovelox, contain iso-branched saturated acids (Anderso et al., 1995; Kampfer, 1994). Thus, the fatty acid profile is a useful characteristic that distinguishes this group of bacilli clearly from other mesophiles of the genera Bacillus, Alicyclobacillus, Brevibacillus, Aneuribacillus, Sulfobacillus and Thermobacillus (Nazin et al., 2001). All these data of physiological characterisations, protein profiles, fatty acid analysis and VITEC tests show that our isolates shoud be classified as Geobacillus sp., and here we also report high esterase(C4), esterase lipase (C8) Naphtol AS-BI phosphohydrolase, α-glucosidase. Figure2 Gg03 Ge.stearothermophillus Figure3 Gc02 Ge.stearothermophillus (VITEC) (X100) (VITEC) (X100) REFERENCES BELDÜZ. A.O., DÜLGER. 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