Presence of Different Bacterial Species in Thermal Sources and Novelty in Their Industrial Enzyme Productions

In this study, one hundred and thirty isolates were isolated from water and sludge samples taken from hot springs located in different regions of Turkey. Among them, eleven isolates were chosen according to conventional (morphological, physiological and biochemical tests) and molecular methods (rep-PCR and 16S rRNA sequencing). These bacteria were then tested for their capability to produce valuable enzymes. As a result; species belonging to Bacillus, Anoxybacillus, Aeribacillus, Enterococcus, Exiguobacterium and Paenibacillus were identified. Test strains were found to have optimum reproductive potential at pH 5.0-9.0 and 15-65°C, usually at a concentration of 1.0-10.0% (w/v) NaCl. In addition, all thermotolerant bacteria were Gram, endospore (except E. profundum), catalase and oxidase (except E. faecium and E. profundum) positive, and rod-shaped (except E. faecium). It was observed that all isolates had a 99% similarity percentage as a result of 16S rRNA sequence analysis. All of the isolates were capable of producing industrially important enzymes moreover, eight of them could produce at least two of these enzymes. Test strains had high potential of industrial enzyme production, and the enzymes from these thermo-tolerant isolates will be widely used in biotechnological processes.

Natural geothermal areas are found in tectonic active zone regions where earth crust movements in the world. Turkey is one of the richest countries in the world in the aspect of geothermal sources. However, the number of scientific studies on the determination of microorganism flora in thermal sources in different geographic regions of our country and determination of species with potential to be used in industry is very limited 41 . Until now, conventional methods based on phenotypic characters have been used in the diagnosis of thermophilic microorganisms in scientific studies conducted both in the world and in our country. The identification of microorganisms by morphological, physiological and biochemical tests has been insufficient to determine the differences between the desired microorganisms. Moreover, the conventional methods need long-term experiments, over labor, experienced and trained researchers, and the results are open to alternative interpretations, other known disadvantages of these methods 36 . Therefore, in recent years, molecular techniques have been developed to be used in the diagnosis of microorganisms. Genetic profiles [16-23S rRNA-PCR, rep-PCR, ERIC-PCR, BOX-PCR and (GTG)5-PCR] are commonly used methods 35 . Rai and Mukherjee 45 defined enzymes as green chemicals, specific biocatalysts that provide the realization and control of all reactions occurring in the living organism under suitable conditions, most of which are of protein structure and naturally produced only by living things. microbial enzymes which have applications in food industry, food and beverage production, cleaning and processing of garments in the textile industry, paper and detergent industries, medicine and medical field diagnosis of diseases continue to change with each passing day especially with biotechnological approaches and can be included in different application areas. The worldwide production volume of industrial enzymes, most of which have extracellular properties, was $ 1 million in 1995; In 2000, approximately 60% of the industrial enzymes used in the world market were produced in Europe and 40% in the USA and Japan 30 . The need for non-toxic and more effective methods has enabled the production of enzymes to scale up and penetrate into different markets thanks to the rapidly growing biotechnology. Thermophilic organisms have a very important place due to their biotechnological potential. Their most important biotechnological features are; to produce enzymes that can catalyze biochemical reactions at much higher temperatures than normal organisms 23 . These enzymes are thermophilic enzymes, and these enzymes are preferred in many industrial areas due to their pH changes and their stability to high temperatures. Thermostable enzymes play a role in the hydrolysis of protein, lipid and polysaccharide substrates in molecular biology 7 . Proteases from thermophilic enzymes are used in food industry, pharmacology, leather and detergent industry in a wide industrial area. enzymes such as lipase, esterase, amylase, cellulase and xylanase; used in detergent, sugar, textile and paper industries 55 .
Thermostable enzymes are more advantageous than other enzymes, and both the industrially important and the thermostable enzyme producer, the introduction of new source microorganisms into the literature, will be able to respond to the needs of industry today and to shed light on possible future studies. The aim of the study was isolation and identification of new and industrially valuable bacterial strains from different geothermal resources of Turkey, presentation of their novel potential to produce industrially important enzymes and multi-enzyme productions.

MATERIAlS AND METHODS Purification of thermo-tolerant isolates
The water and sludge samples from Armutlu (Yalova), Germencik (Ayd n), Yildizburnu (Izmir), Havza (Samsun), Guroymak (Bitlis) and Karakurt (K rsehir) hot springs were collected and the samples were transferred to Nutrient Agar (NA) medium (peptone from meat 5.0 gL -1 ; meat extract 3.0 gL -1 ; agar 12.0 gL -1 ) in order to incubate (Thermo Scientific Heratherm, the USA) at 55°C for 24 h. The growing colonies were spread on NA to obtain pure cultures and were eliminated by morphology differences in plates, as a first step. The pure, single and different colonies were stored in the Nutrient Broth (NB) with 15% glycerol content at -86°C for further studies 3 .

Gene amplification, cloning and genomic fingerprinting of isolates
For DNA extraction of each isolate, a single colony from NA medium was selected and inoculated to NB at 55°C 150 rpm. After the incubation period, genomic DNA isolation was carried out according to Promega WizardR DNA purification kit and purified DNA was stored at +4°C until use 47 . Test isolates were subjected to rep-PCR with the special primers of (GTG) 5 and BOX elements to obtain genomic fingerprinting 4,6,20 . To obtain the PCR products, 50 ng of purified DNA was used as the template in 30 ml reaction mixture. The 16S rRNA of the isolates was amplified by Polymerase Chain Reaction (PCR) with the forward primer 27-F:(5' AGAGTTTGATYMTGGCTCAG3') and the reverse primer 1492-R:(5' GGTTACCTTGTTACGACTT 3') 5 . The amplified fragments were cloned into E. coli JM101 strain with a vector system (pGEM-T, Promega, the UK) and the clones were sequenced (Macrogen, Amsterdam, the Netherlands). The results of 16S rRNA gene sequencing were analyzed using the GenBank (http://blast.ncbi. nlm.nih.gov/blast.cgi) and EzTaxon (http://www. eztaxon.org/) servers 9 . Considering the results of the study, a phylogenetic tree was formed via the neighbor-joining method by using the software package MEGA 4.0 13 .
The PCR products (27 mL) were mixed with 3 mL gel loading buffer (6X) and subjected to agarose (1.5% w/v) gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer at 100 V for 110 min.
After separation of the amplification products by the gel, the fragments were stained with ethidium bromide solution (2 mL Etbr/100 mL 1X TAE buffer). The amplified DNA product was monitored using Gel Documentation System.

Conventional identification
The test isolates were subjected to conventional tests. The pH (Mettler Toledo) and temperature range for bacterial growth were tested between pH 3.0-11.0 and 15-65°C. The NaCl requirement for growth was also tested in NB medium containing 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0% (w/v) NaCl. Cell and colony morphology (Leica ICC50 HD light microscope), Gram and endospore staining, motility and the presence of catalase and oxidase reactions were also investigated by the method of Prescott 44 and Sari 47 .

Preliminary experiments of industrial enzymes Amylase
Amylase assay was performed at 55°C for two days in a modified-medium which contained yeast extract (1 gL -1 ), starch (5 gL -1 ) and agar (15 gL -1 ) 58 . The amylase activity was screened by Lugol's solution. Clear and large zones were evaluated as positive, or as amylase production; however, no zone formation was evaluated as negative, or as no amylase production.

Cellulase
A medium containing carboxymethylcellulose (10 gL -1 ), peptone (5 gL -1 ), yeast (5 gL -1 ), KH 2 PO 4 (1 gL -1 ), MgSO 4 .7H 2 O (0.2 gL -1 ), NaCl (10 gL -1 ) and agar (15 gL -1 ) was used for cellulase assay. Incubation was carried out at 55°C for two days, and after the incubation, the plates were stained by Congo-Red Dye. The presence of clear zone was accepted as positive 47 . lipase Test isolates were inoculated on tributyrin agar medium which contained 1% (w/v) tributyrin (glycerol tributyrate) and incubated at 55°C. Formation of hydrolysis zone around the culture on plate was controlled for two days, and the strains with transparent and the highest zone formation (lipolytic activity) were determined as lipase producers 12 .

Protease
Protease activity was conducted in medium which contained NB (8 gL -1 ), Skimmed Milk (10 gL -1 ) and agar (15 gL -1 ) at 55°C for two days. The plates were evaluated according to the zone formations, and the observation of a halo zone indicated positive protease activity 42 .

Genomic fingerprinting of thermophiles
The first step of this study was the isolation of thermopilic bacteria from water and sludge samples of Armutlu (Yalova), Germencik (Aydn), Yildizburnu (Izmir), Havza (Samsun), Guroymak (Bitlis) and Karakurt (Krsehir) hot springs. One hundred and thirty bacterial isolates were obtained however this number was firstly reduced by eliminating according to colony differences observed on plate surface. As conventional analysis alone was not sufficient for the identification of bacteria, rep-PCR [(GTG) 5 -PCR and BOX-PCR] method was used which clearly showed the differences between microorganisms' genomic fingerprinting. (GTG) 5 -PCR, gave the first information about isolates. It was observed that the isolates usually yielded up to sixteen polymorphic bands between 350-3500 bp and eight polymorphic bands between 700-2500 bp while EA 9 isolate gave no bands (Fig. 1). BOX-PCR, another genomic fingerprint method, was applied and it was determined that the test isolates were given two types of polymorphic bands, usually five bands between 300-1200 bp and two bands between 1200 -4000 bp (Fig. 2). 16S rRNA region, which was evolutionarily conserved, was amplified using universal primers; to identify the isolates at species level. As a result of the analysis, eleven different species were detected, resulted in the fragments of 1500 bp. The sequences of cloned isolates were compared with the sequences of other bacteria in the GenBank and the nucleotides were analyzed using BLAST and EzTaxon ( Table 1). The phylogenetic relationship between thermopilic bacteria was presented by phylogenetic tree (Fig. 3) rep-PCR [(GTG) 5 -PCR and BOX-PCR], which was molecular method used for this purpose, was a highly effective method used to reveal the diversity in the ecosystem, the phylogenetic relationship between the strains and to distinguish genetically close micro-organisms at species and sub-species level 4 . BOX-PCR and (GTG) 5 -PCR methods were found to be very successful in demonstrating the genomic differences between strains in parallel with literature data 3,4,6,58 .
To determine the salt concentration for growth of the isolates, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12% (w/v) NaCl were used (   microbial growth in pH 3.0, 5.0, 7.0, 9.0 and 11.0 were monitored; EA4 and EA11 were detected to develop in pH 6.0-7.0; EA1, EA3, EA5, EA6, EA7 and EA8 were in pH 7.0-9.0; EA2, EA9 and EA10 were in pH 5.0-9.0. The isolates were incubated at 15°C, 25°C, 35°C, 45°C, 55°C and 65°C to determine the temperatures at which they developed and the temperature values of the test strains were determined. Although the temperature ranges of thermophiles varied, EA9 and EA10 were determined that they could develop at 15-55°C, EA5 and EA6 were at 25-55°C, EA3 and EA7 were at 35-65°C, others were 35-55°C. As a result of the morphological analysis of the isolates obtained in parallel with the literature data, all test strains were Gram and endospore (except E. profundum) positive, motile and had bacilli cell morphology except E. faecium (coccus). Growth ranges for pH, temperature and salt concentration were 5-9 pH, 15-65°C and 1-10%(w/v). Finally, all the isolates were catalase and oxidase positive (except E. faecium and E. profundum). Yadav 57 carried out the isolation and identification of 150 thermophilic bacteria from hot springs in Nepal. The 16S rRNA fragments of the isolates were amplified with a size of about 1.5 kbs (1500 bp) and the microorganisms were belonging to the genus Anoxybacillus, Aeribacillus, and Bacillus at a ratio of ≥95. The bacilli isolates were gram positive, endospore forming and their optimum growth temperature were between 55-65°C. However, in this study, the isolates were thermo-tolerant due to large temperate range, 15-65 o C. Aanniz 1 isolated and identified 240 bacteria from different sources in Morocco. All isolates were Gram and endospore positive. When they examined the BOX-PCR and 16S rRNA sequence of the isolates, the dominant species was Bacillus (97.5%); 119 of them were B. licheniformis and 6 of the species belonging to Aerobacillus. Norashirene 38 obtained six thermophilic isolates from Malaysia. The optimum growth temperatures  of these isolates were 55°C and pH was 7.5, and they were aerobic, catalase and oxidase positive. Poli 43 carried out the isolation of thermophilic bacteria by taking water and mud samples from the geothermal areas in Italy. These test strains were aerobic, endospore and Gram positive, motile, rod shaped and their optimum growth was at 65°C and pH 7.2. The salt concentrations in which the test strains could develop were tested at 1-10%(w/v), and the results were generally parallel to the literature data. Tambekar 52 revealed the presence of the strains that could reproduce in 8% (w/v) salt concentration, in parallel with our data. However, B. halodurans (EA6) was found to be able to withstand up to 8% (w/v) salt concentration in contrast to the literature 51 .
It was noticeable that the growth of E. faecium, a lactic acid bacterium at 15-55°C, considering that mesophilic bacteria generally did not show growth above 45°C. However, Orr 39  found some members of the microorganisms contained in Enterococci were thermo-tolerant. Svec 50 reported that, (GTG) 5 -PCR method could be used effectively in the identification of the genus Enterecoccus at species and sub-species level. Abdel-Rahman 2 identified E. faecium with 99% similarity ratio and as unable to utilize oxygen as an electron acceptor. They also concluded that; E. faecium could grow at 45-50°C and pH 9.6. Al-Mariri 8 collected white cheese samples and identified thermophilic streptococci. As a result; E. faecium isolates were gram positive, non-sporulation and could grow at 4-6.5%(w/v) salt concentration while the salt concentration for this study was 2-6%(w/v). In another study; E. faecium was identified as catalase positive 21 however Abdel-Rahman 2 and Jensen 28 identified as catalase negative. According to Schleifer and Kilpper-B‫ה‬lz 48 , some strains of E. faecium were motile. Crapart 15 isolated lactic acid-producing thermo-tolerant bacterium, E. profundum, from hydrothermal vent and the growth conditions were parallel to our study with 12-49°C, 5.5-9.5 pH and 0-11%(w/v) salt concentration. This bacterium was characterized as motile, Gram and catalase positive, oxidase negative while Kasana and Pandey 29 also concluded as non-sporulation.

Enzyme production capacities of thermophiles
Enzyme production abilities of these eleven isolates were tested in enzyme specific solid media. As a result; all of them were producers of at least one enzyme which were industrially valuable (Table 3). EA2, EA3, EA4, EA5, EA7 and EA8 were amylase producers. The only cellulase producer isolate was EA6. Except EA6 and EA11, the rest of the isolates were positive in terms of lipase production. EA6 and EA10 gave negative results in protease growth media however, the other isolates were protease producers. EA2, EA3, EA7 and EA8 were xylanase producers.
A. pallidus, which was isolated from desert soil by Aanniz 1 , could grow at 30-80°C, 0.5-10%(w/v) salt concentration and produce amylase and protease. Koc 31 determined A. pallidus C196 and D642 as potential lipase producers due to use of olive oil as substrate.
The isolation from different locations of Turkey resulted in identification of A. pallidus as amylase producer 12 . With optimum growth conditions between 40-65°C, 0-3% (w/v) NaCl and 5.0-9.5 pH A. geothermalis could hydrolyze casein and starch due to protease and amylase activity, respectively 19 while A. geothermalis in this study could also able to produce lipase and xylanase. Namsaraev 37 determined A. mongoliensis, whose optimum growth was at 35-75°C, 0-5% (w/v) NaCl concentration and 5.5-10.8 pH, as a thermophilic protease and amylase producer due to the hydrolysis of starch, casein and gelatin. A. mongoliensis in this study was also identified as lipase and xylanase producer. Derekova 16 was isolated and identified A. rupiensis, which could survive between 35-67°C, 5.5-8.5 pH, for the first time with ability to degrade xylan, starch and casein. Jardine 27 was also proposed A. rupiensis as amylase, protease and celllulase producer. Yanmis 58 isolated A. rupiensis from geothermal regions of Turkey and it was declared as amylase producer. However, lipase production of A. rupiensis was determined in this study.
The lipolytic activity of B. halodurans was proved by many of the studies 17,46,53 and Vargas 53 determined the optimum ranges at 25-55°C and 2.5-10% (w/v) NaCl. As a potential producer, amylase 25 and xylanase 32 were other enzymes that B. halodurans produced. However, B. haloduransin this study was identified as cellulase producer. Aanniz 1 isolated B. licheniformis, which could survive at 30-75°C, 0.5-10% (w/v) salt content and 7-8.2 pH, had the potential to produce amylase, protease and cellulase. Archana and Satyanarayana 11 isolated cellulase-free xylanase from thermostable B. licheniformis. Baltaci 12 classified two of the isolates as B. licheniformis and they were also amylase, lipase, protease and cellulase producers. B. licheniformis, from thermal regions of Turkey 58 , could also able to produce cellulase and amylase.
Although B. paralicheniformis was isolated from fermented foods 18,40 , there was no record of thermophilic B. paralicheniformis and its enzyme production. B. paralicheniformis in this study could able to produce amylase, lipase, protease and xylanase. E. faecium was firstly isolated by Orr 39 from hospital samples as thermo-tolerant however there was no report about enzyme production of thermo-tolerant E. faecium. However, lipase and protease production ability of E. faecium was determined in this study. Moderately thermophilic E. profundum was defined by Crapart 15 but its industrial enzyme abilities never examined while lipase production of E. profundum discovered in this study. P. dendritiformis, which was included in the genus Bacillus, was first discovered in 1990s and reclassified as a separate genus 49 . However, there is no record about P. dendritiformis from thermophilic sources while protease production was determined in this study.
In addition, the isolation locations of organisms were important during the isolation and identification of thermo-tolerant organisms ( When the multienzyme production capabilities of the isolates were examined, it was observed that all the isolates, except B. halodurans, E. profundum and P. dendritiformis, were able to produce more than one enzyme. In a study, in order to determine the strains which have the potential to produce industrially important enzymes, Yadav 57 observed that 135 of 150 thermophilic isolates have the potential to produce at least one extracellular hydrolytic enzyme.

CONClusiON
As a conclusion, this research contained remarkable points in terms of presenting different thermo-tolerant species in the World and Turkey, microbial flora of thermal regions in Turkey and unknown abilities of these thermo-tolerant species to produce industrially valuable enzymes. The first isolation of B. albus, B. paralicheniformis and P. dendritiformis from thermal regions in the World was carried out. A. geothermalis, A. mongoliensis, B. albus, B. halodurans, B. paralicheniformis, E. faecium, E. profundum and P. dendritiformis were firstly isolated from hot springs of Turkey. A. geothermalis, A. mongoliensis, B. albus, B. halodurans, B. paralicheniformis, E. faecium, E. profundum and P. dendritiformis were identified as producers of some of industrial enzymes, for the first time. Eight different species with the potential to produce multi-enzyme were identified. Since thermostable enzymes play an important role in industrial and biotechnological processes, the enzyme/multi-enzyme production potentials of the isolates identified in this study has been one of the another notable results.