Isolation of Thermophilic Bacteria from Bora Hot Springs in Central Sulawesi

Thermophilic bacteria can survive at high temperature, in which hot spring is one of its habitats. Indonesia has many hot springs with potential as a habitat for thermophilic bacteria. The purpose of this study was to obtain isolates thermophilic bacteria from Bora hotspring located in Central Sulawesi. This study applied a descriptive-observational study design, characteristics of bacterial properties identified using conventional methods according to the Bergey’s Manual of Determinative Bacteriology. The study was conducted in 3 stages. The first stage was bacteria cultivation on the appropriate media, followed by stage of isolated and the last stage by identified characteristics of thermophilic bacteria which included microscopic and macroscopic morphology, Physiological and biochemistry test. The results of the isolates indicted 4 representative isolated of thermophilic bacteria from Bora Hot Spring namely TM022, TM023, TM024, TM026. The bacteria isolates obtained were bacillus, coccus and Gram negative and positive, while the physiological test of all isolates were able to grow and showed changes in the medium. This study is useful in providing characteristic of indigenous thermophilic bacteria isolates that produces thermostable enzymes.

microorganisms conducted in one region (Mahmudah et al, 2016).This study used conventional exploration method through macroscopic observation, microscopic, physiology test and biochemical test.
The following study determines genus identification and optimization of bacterial growth.Determination of genus of bacterial isolate in this study was in accordance with Bergey's Manual of Determinative Bacteriology with microscopic observation through several tests such as morphological test.Some of these bacterial identification include colony morphology (size, shape, color) and macroscopic morphology as well as breeding.
The geothermal springs located in the Bora Sigi Biromaru have an average temperature of 45-70 o C. Currently the area is used as tourist destination by surrounding resident Knowing the role of thermophilic microorganisms as a thermostable enzyme producer is necessary to explore the potential of thermophilic bacteria by by isolating, characterizing and identifying.Study on the isolation, characterization and identification of thermophilic bacteria had never been done and was considered as novelty in this study.The study provides benefits by giving information on the thermophilic bacteria diversity of thermostable enzyme producers from the hot springs.

INTRODUCTION
Indonesia is a country with much geothermal activity and high biodiversity.Indonesia also has the largest geothermal resources in the world, with as many as 252 locations spread over 26 Provinces.The large number of hydrothermal areas, waste composting, petroleum drilling wells and natural gas shows potentials that can be empowered and applied in the development of science.Even studies in the last two decades revealed that 99% of the bacteria present in the environment have not been yet explored for biotechnology applications (Panda et al., 2013;Wijayati et al., 2014).
Thermophilic bacteria are microorganisms that live in extreme condition up to a temperature of 121 o C. Groups of thermophilic microorganisms also produce the most enzymes compared to animals and plants.
This enzyme is widely used because of its intrinsic-thermostability and its resistance to changes in physical and chemical factors.Currently, there are only 2 types of thermophilic bacteria enzyme most widely used as a biocatalyst which are derived from the group Thermusaquaticus and alkaliphiles is sellulase 103 (Khalil, 2011).In addition, protease enzymes derived from thermophilic bacteria are also widely used for bio-industrial food, pharmaceutical and various biological applications because of its stable properties such as are resistant to various solvents and acidic -alkaline pH (Mohammad et al, 2017).
Research regarding thermophilic bacteria is getting wider attention.Some local thermophilic bacteria isolates have been successfully isolated from a number of places.Isolated microorganisms in Indonesia including thermophilic bacteria from Pacet Hot Spring (8 genus including Thermussp, Acetogeniumsp, Bacillussp, Thermotrixsp, Thermodesulfo bacterium sp, Thermomicobriumsp, Pseudomonassp and Sulfobacillussp) (Asnawi, 2006).A study conducted by Maria and Surya, (2012) succeeded in identifying 2 genera namely Bacillussp and Vibrio sp.
A study related to thermophilic bacteria from Bora Hot Springs has previously been conducted by Khoridah, (2015) and Hardi et al. (2016), which successfully isolated B1211 bacteria which has potential use for the production of chitinolytic and chitinases enzymes.The highest activity of chitinase enzyme from B1211 bacteria isolate, is 0.75 U/mL obtained with colloidal chitin 1% concentration and temperature 60 o C.
Isolation, characterization and identification are the first steps of exploration of indigenous nal method; all data obtained according to the phenotype of representative thermophilic bacterial isolates during macroscopic and microscopic observations and were used for conventional identification by using Bergey's Manual of Determinative Bacteriology identification key book.The data collected in this study consisted of primary data and secondary data.

Isolation and Selection of Thermophilic Bacteria
Isolation of bacteria was performed using spread method.The water sample contained in the bottle was homogenized, then taken 1 ml using pipette into petri dish containing selective medium Organic Gaus Agar (OGA) and Thermus Medium Modified (TMM).The medium allowed to be transported at the temperature of 50 ° C for 48 hours.Therefore, it would resulted to representative bacteria.The growing bacteria were inoculated back into sterile petridish containing OGA and TMM mediums using quadrant method, then incubated at 50 ° C for 24-48 hours.

Characterization of Thermophilic Bacteria
Characterization of thermophilic bacteria was done through macroscopic observation to the colony formed, colony edge, elevation, texture and pigmentation.Microscopic observation to the shape of bacterial cells was performed by Gram staining.
Physiological tests were performed by growing isolates based on observed growth of various media, temperature, pH and NaCl.Carbohydrate fermentation biochemical test.Isolate bacteria taken 1 ose and put into medium carbohydrate broth of glucose, sucrose, lactose.Media that contained isolate, incubated for 2 days.Any changes in color were observed.Red indicates the absence of acid while yellow indicates the presence of acid.The formation of gas and color changes in the medium was caused by the addition of PP (Phenolphthalein) indicator.The fermentation activity could be observed with acid and gas production during the growth phase.

RESULTS AND DISCUSSION
All isolates of thermophilic bacteria obtained were the result of selection of microbial colonies grown on selective OGA (Organic Gaus Agar) and TMM (Thermus Medium Modified) medium.Acquired isolates were identified based on macroscopic, microscopic, physiological and biochemical descriptions.The isolation found 4 isolates of thermophilic bacteria from representative Bora Hot Spring is TM022, TM023, TM024 and TM026.

Characterization Macroscopic and Microscopic Thermophilic Bacterial
Bacterial characterization was performed on four isolates of thermophilic bacteria is TM022, TM023, TM024, TM026 isolates which had different colony form.
Differences in the macroscopic characteristics of thermophilic bacteria were due to the effect of the medium used so as to exhibit different phenotypic characteristics (Sianturi, 2008).The bacterial phenotype characteristics are not static so it is possible that the same bacteria may exhibit different morphological characteristics (Ochman et al., 2005).
In colony color observation, all isolates had the same basic white color (figure 1).This result was similar to the study conducted by Dirnawan et al., (2000) on the exploration of thermophilic bacteria from Mount Pancar, which also showed coloration dominated by white.A study conducted by Fitri and Yasmin (2011) also showed that most of the bacterial colonies were round-shaped, the color of the base colonies was beage -white.The colony shape and the size also varied greatly depending on the species.
Differences in the morphologic appearance of microorganism colonies on the media were one of the efforts of bacteria to survive in conditions of stress or toxic environment.At the beginning of growth, the bacteria used the carbon source present in the medium, so the colonies grew slighly.When the carbon source in the medium began to decrease, the bacteria tried to break the carbon bond, so it would be easy to use.Such activity usually caused bacterial colonies to become more rough and somewhat wrinkled (Alwi, 2007).
The microscopic observations of thermophilic bacterial isolates were generally Grampositive, coccus shaped, whereas the TM022 isolate was Gram-negative and bacillus shaped (Table 2).
The Study carried out by Akanbhiet al., (2010) on the identification of thermophilic bacteria from aerobic sources generally resulted the bacteria obtained were Gram positive.Gram positive bacteria have cell walls with low lipid concentrations, which will dissolve by 96% aklohol and form small pores, so the main dye (crystal violet) is difficult to remove (Jumawita et al., 2014).In addition Gram positive bacterial groups generally have aerobic and facultative anaerobic properties and have the ability to live on physiological conditions such as temperature, pH , and and salt concentration.
The discovery of Gram negative and positive isolates in Bora Hot Spring can be linked to environmental conditions.According to Mahmudah et al., (2016) Gram-negative bacteria require relatively simpler nutrients compared to Grampositive bacteria.This means the ability of this group of bacteria to grow in a wider environment than Gram positive bacteria

Physiological Test
The diversity of thermophilic bacteria found in Bora Hot Spring is influenced by the environmental conditions that support bacterial life such as biotic and abiotic factors.As the environ-ment has high temperatures, hot water becomes one environment for microorganisms.The temperature of the environment suitable for microba varies and some are resistant to high temperature.Microbial sustainability at high temperatures is caused because thermophilic bacteria have different protein structures compared to mesophyll microbes so that they are able to survive at extreme temperatures.The existence of biotic components is capable of supporting the growth of thermophilic microorganisms, such as deciduous leaves, branches, grains, pollen, and insecticides found around hot springs as organic substances that can be utilized by living microorganisms in hot springs (Dirnawan et al., 2000 ).
One of the abiotic factors that affect bacterial life is temperature and pH.Microbes that inhabit aquatic environments have characteristics that vary in regards to physiochemical parameters including temperature, salinity, pH and nutrient load (Pitri et al., 2015;El Gayar et al., 2017).Groups of extreme microorganisms generally thrive in extreme ecosystems.The environment is strongly influenced by very high or low pH, high temperature, high salinity, high pressure and various these combinations (Aanniz et al., 2015).
All bacterial isolates grew on 50oC (Table 3).Madigan et al., (2000) states that microorganisms that grow optimally at temperatures above 45 o C are thermophilic bacteria, whereas according to Rudiger et al., (1994), that the optimum growth temperature of thermophilic microorganisms can be distinguished in general Observation of pH parameter of all isolate of thermophilic bacteria indicated that the optimum pH was in range of 6-8.5. Brock (1997), explains that thermophilic bacteria can survive in extreme conditions with an optimum pH of about 6-8.5 and 4.5 -8.7 pH in hot alkaline water (Pitri et al., 2015).Sari (2012) states that an alkaline hot spring has a high mineral content that allows thermophilic microorganisms to survive and can affect the growth and diversity of thermophilic microorganisms.
Regions that have a degree of acidity (pH), gas and mineral composition, various oxidation/ reduction and nutrients can lead to the presence of genetic diversity and metabolism of every living microorganism in it (Pitri et al., 2015).Testing of NaCl concentration showed that all isolates were able to grow at 2% NaCl concentration, whereas at 6-10% NaCl concentration were not able to grow.These results were consistent with the study of Syafriani et al. (2013) that bacterial isolates were capable of growing and developing at 3% NaCl content compared 5% concentration, while the 7% NaCl concentration, bacteria cannot live.At 3% NaCl concentration the color of the media turned to cloudy, while the 7% NaCl concentration the media looked clear.According to Prasetyo (2006), the more cloudy the media the higher the optical density (OD) and the more population increase of cells in the media.

Biochemical Test
In the carbohydrate test of glucose, lactose and sucrose, it was found that all isolates of thermophilic bacteria were able to ferment carbohydrates which indicated by change of medium color from green to yellow (Table 4).The result of fermentation test of glucose, lactose and maltose carbohydrates all isolates showed positive result (Table 4).This was indicated by the color change of fermentation medium of glucose and maltose from green to yellow, while lactose test indicated no color change in during incubation for 24 hours.
According to Harley (2002), the yellow color of the media was caused by the phenol solution that forms acid in the carbohydrate media.The resulting lactose fermentation did not change color, because bacteria are able to ferment lactose and cause variation to medium color.
The color change was caused by isolates capability to use glucose, lactose and maltose as the carbon source.In addition, carbohydrate fermentation could occur aerobically on the agar surface and the anaerobically at the base of the medium.On the surface of the medium, glucose catabolism was done through the Embden-Meyerhof path resulted in pyruvic acid which degraded completely in cycle citric acid to CO 2 , H 2 O, and energy (Haryani et al., 2012).

CONCLUSION
Based on the result of study, there were 4 isolate thermophilic bacteria, TM022, TM023, TM024, TM026.Characterization of thermophilic bacterial isolates macroscopically showed different phenotypic properties, whereas microscopic observation showed bacillus and coccus shaped bacterial cells as well as Gram positive and negative bacteria.Observation of physiological test of all isolates grew at 50 o C, pH 6.8-8.6 and 2% NaCl concentration.
This study contributes to the development of conservation strategies and provides information on the characteristic diversity of indigenous thermophilic bacteria as a potentialthermostable enzyme producer from Bora Hot Spring.The novelty of this research is the determination of characterization and identification of thermophilic bacteria isolate based on macroscopic observation, microscopic, physiological test, biochemical test and optimization of its growth in various media.

Figure 1 .
Figure 1.Macroscopic characterization of representative thermophilic bacteria from Bora Hot Spring A : Morphologic Observation of Macroscopic Isolate TM022 B : Morphologic Observation of Macroscopic Isolate TM023 C : Morphologic Observation of Macroscopic Isolate TM024 D : Morphologic Observation of Macroscopic Isolate TM026

Table 1 .
Macroscopic characterization of representative thermophilic bacterial isolates from Bora Hot Spring

Table 2 .
Microscopic characterization of representative thermophilic bacterial isolates from Bora C, mesophile 13-45 o C, thermophile 45-65 o C, extreme thermophilic temperature 65-85 o C and hypertermofil at temperatures above 85 o C. o

Table 3 .
Observation of Physiological tests representative thermophilic bacterial isolates from Bora Hot Springs

Table 4 .
Observation of biochemical tests of representative thermophilic bacterial isolates from Bora Hot Springs