Isolation and optimization of the growth conditions of thermophilic microorganism from hot springs

Nong Lam University, Ho Chi Minh City 55 Isolation and optimization of the growth conditions of thermophilic microorganism from hot springs Kha M. Tran1, Van T. T. Le1, Duy D. Ngo2, Khanh Q. Hoang2, Phong V. Nguyen3, & Tri H. Nguyen1∗ 1Department of Biology, Nong Lam University, Ho Chi Minh City, Vietnam 2Institute of Tropical Biology, Ho Chi Minh City, Vietnam 3Department of Biotechnology, Nong Lam University, Ho Chi Minh City, Vietnam ARTICLE INFO Research paper Received: April 24, 2018 Revised: May 30 Accepted: June 17, 2018 Keywords Bacillus spp. Hot spring Isolation Spore staining Thermophilic microorganism ∗Corresponding author Nguyen Huu Tri Email: nhtri@hcmuaf.edu.vn ABSTRACT The aim of this study was to isolate and optimize the growth conditions of thermophilic microorganism from hot springs. The isolation was conducted by using the mineral salt basal medium supplemented with 0.6% yeast extract at 500C. Totally, 33 isolates of thermophilic microorganism were isolated from hot springs at Truong Xuan (Khanh Hoa province) and Binh Chau (Ba Ria - Vung Tau province). The effects of temperature (45 - 800C), pH (pH 6 - 9) and carbon sources (malate, pyruvate, acetate, glucose, fructose, or carbon dioxide) on the growth of isolates were exam- ined. In addition, the isolate morphology was also investigated by Gram and spore staining. The isolated thermophilic microor- ganism showed the diversity in colony morphology and color ap- pearance. Most of them were rod shaped, spore-forming and most grew well at 500C and pH 7. The highest growth of all isolates was observed under malate, glucose, or fructose, as an organic car- bon source and unable to use carbon dioxide. Six out of 33 ther- mophilic microorganism isolates (namely BM7, BS5, NS1, NS3, NS4, and NW6) grew rapidly under high temperatures from 50 - 550C and their morphology characteristics showed high similarity to Bacillus sp. The study evidenced the polymorphic diversity of thermophiles in the geothermal hot spring ecosystems. Cited as: Tran, K. M., Le, V. T. T, Ngo, D. D., Hoang, K., Nguyen, P. V., & Nguyen, T. H. (2018). Isolation and optimization of the growth conditions of thermophilic microorganism from hot springs. The Journal of Agriculture and Development 17(3), 55-60. 1. Introduction Hot springs, the emerged water bodies pro- duced by geo-thermally heated groundwater, are scattered all over the globe, in every continent and even under the sea. In Vietnam, there are more than 287 hot springs and water contain- ing dissolved minerals distributed in different re- gions of the country (Cao et al., 1998). There are many previous studies that focus on inves- tigation about geological features of geothermal areas (Rastogi et al., 2010; Tran et al., 2012; Tu- lasi et al., 2013). Besides, the geothermal ecosys- tems such as hot springs and volcanic erup- tion areas are the habitat of thermophilic mi- croorganisms. Based on the range of optimal growth temperature, thermophiles are classified into the following groups: moderate thermophiles (40-600C), extreme thermophiles (60-850C) and hyperthermophiles (>850C) (Tulasi et al., 2013). These thermophilic-derivative products could be applied in biotechnology as industrially valuable compounds. Extremophiles have provided an in- teresting and challenging platform for researchers since they were explorered. Besides growth under the extreme conditions, extremophiles could pro- www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 17(3) 56 Nong Lam University, Ho Chi Minh City duce thermophilic enzymes, biogradable plastic, biofuel, etc. (Tulasi et al., 2013). Thermophilic microorganisms capable of biosynthesis of heat- resistant enzymes are widely used in the industry where production conditions require high temper- atures (Gaughran et al., 1947). During the past few years, the interest in diversity, ecology, and physiology and biochemistry of thermophiles has increased rapidly in Vietnam. The thermophilic bacterium species Geobacillus caldoxylosilyticus was isolated from sedimental sludge of My Lam hot spring in Tuyen Quang province, Vietnam (Tran et al., 2012). Furthermore, this strain be- came promising candidate in industry due to its capability of producing thermostable enzymes such as cellulase and amylase (Tran et al., 2012). The southern of Vietnam is very rich in hot springs. One is Truong Xuan hot spring (M’ Dung village, Ninh Hoa, Khanh Hoa), and another is Binh Chau hot spring (Binh Chau commune, Xuyen Moc district, Ba Ria – Vung Tau) that is very famous in Vietnam. The diversity of micro- bial communities in these hot springs has not yet been fully studied. This study aimed to isolate, optimize, and evaluate the carbon utilization of thermophilic microorganism isolated from these locations. Results from this study were a prelim- inary step to apply thermophilic microorganism and their products in biotechnology. 2. Materials and Methods Soil, muddy, and water samples were col- lected at Truong Xuan hot spring (12031’20”N, 108059’00”E, Ninh Hoa, Khanh Hoa), and Binh Chau hot spring (10036’21”N, 107033’29”E, Xuyen Moc, Vung Tau). Hot water in Truong Xuan hot spring was bubbled from the vein in the rock with temperature ranging from 370C to 670C. The pH was recorded in the range of 7.7- 8.0 indicating alkaline environment. Binh Chau hot spring is the largest hot spring (more than 1 km2) in Vietnam. Water temperature in the veins ranged from 430C to 650C with many bub- bles, and smell hydrogen sulfide (H2S). The pH was recorded in the range of 7.8-9.2 indicating al- kaline environment. The temperature of the sam- pling site is unstable, normally, the temperature at the sampling sites was lower than that at the veins. Samples were randomly collected from differ- ent sites of off flow and stored in 500 mL sterile containers (Hildur et al., 2011). They were imme- diately brought into the laboratory and analyzed within 24 hours. In total, 24 samples were col- lected from Truong Xuan (14 samples) and Binh Chau (10 samples) hot springs. The samples (soil, muddy, and water) were collected separately in the vacuum flask, transported to laboratory and analyzed within 24 hours. The mineral salt basic (MSB) medium used for microorganism growth and trace element solution with the components are shown in Table 1 and Table 2 (Goto et al., 1977). The isolation medium was MSB supplemented with 0.6% yeast (w/v) so-called as MSBY medium, pH 7. Table 1. The components of basic cultivation medium Deionized Water 1.0 L (NH4)2SO4 2.0 g KH2PO4 1.0 g MgSO4.7H2O 0.5 g K2HPO4 2.0 g NaCl 0.5 g FeSO4.7H2O 0.0011 g CaCl2 0.03 g Trace elements solution 0.5 mL Final pH 7.0 Table 2. The components of trace elements solution Deionized Water 1.0 L MoO3 0.004 g ZnSO4.7H2O 0.028 g CuSO4.5H2O 0.002 g H3BO3 0.004 g MnSO4.5H2O 0.004 g CoCl2.6H2O 0.004 g 2.1. Isolation of thermophilic microorganisms from hot springs The thermophilic microorganisms were isolated based on the possible growth at 500C. Fifty Cel- sius degree was chosen to make the initial isola- tion temperature to isolate those microorganisms that were capable of growing by 500C or more. The procedure of enrichment was as follows: 1 gram of soil, sludge or 1 mL of water was di- luted in 5 mL of MSB supplemented with 6 g/L of yeast extract and incubated at 500C for 48 The Journal of Agriculture and Development 17(3) www.jad.hcmuaf.edu.vn Nong Lam University, Ho Chi Minh City 57 hours. The growth of microorganisms was ob- served and recorded via estimation of the envi- ronmental opacity in test tubes. A five tenfold serial dilution was performed, and then spread on MSBA plates (MSB medium supplemented with agar 3% (w/w) and incubated at 500C for 72 hours. Single colonies growing on plates were transferred into freshly prepared MSBA slants and kept at -200C for further study. The isolates were investigated by observation of colony mor- phology, Gram stain, and sporulation (Goto et al., 1977). 2.2. Optimization of the growth condition of the isolated thermophilic microorganisms In order to determine the optimal temperature for the growth of isolated thermophilic microor- ganisms, each isolate was inoculated in 5 mL of MSBY medium (pH 7) in a test tube in range of temperature from 450C to 800C, shaken at 180 rpm for 12 hours. Then, the optimum pH value was examined between 6 and 9 at the optimal temperature. The pH value was adjusted by using 1M NaOH solution. The microorganism growth was determined at 3-hour intervals by measuring the optical density (OD) of the cultures at 540 nm and streaked onto freshly prepared MSBA plate (Goto et al., 1977). The high thermo-tolerance isolates were selected for further experiments. 2.3. Investigation of the potential use of dif- ferent carbon sources of the isolated ther- mophilic microorganism The carbon sources were used in this study including organic substrates [acetate (C2), pyru- vate (C3), malate (C4), glucose (C6), or fructose (C6)] and inorganic substrate (CO2). The concen- tration of carbon in the organic compounds was equivalent to 15 mM. In order to evaluate the use of CO2, the isolates were cultured in MSB medium with the addition of H2: O2: CO2 (80%: 10%: 10%) (Goto et al., 1977). The cultures were incubated in a reciprocating shaker at the optimal temperature and pH. The initial OD value at 540 nm was 0.04-0.06. The microorganism growth in various carbon sources was recorded within 72 hours. The mean value OD540 of triplicates for each experiment was an- alyzed by using Microsoft Excel 2013 software. 3. Results and Discussion 3.1. Isolation of the thermophilic microorgan- isms from hot springs Thirty three isolates that could grow at 500C were isolated from 24 soil, muddy, and water samples from two hot springs in Khanh Hoa (16 isolates) and Ba Ria – Vung Tau (17 isolates) provinces. Of 33 isolates, 11 isolates were ob- tained from soil (33.3%), 8 isolates from muddy (24.3%) and 14 isolates from water (42.4%) sam- ples (Table 3). The colonies were appeared in various of color (beige, white, yellow, or pink) including 7 isolates were beige-colored, 11 were white, 14 were yellow, and 1 was pink on MSBA medium (Figure 1). Figure 1. Diversity of colonial morphology of isolates microorganism from hot springs on MSBA. The Gram-positive isolates were 18 isolates /33 (54.5%) of the collection. Of the 33 isolates, 31 (94%) were rod-shaped, with the size of cells in range of 0.16 – 0.8 ± m, 20 isolates (60.6%) were able to form oval endospores and had the size of the spores in range of 0.10 – 0.41 ± m (Figure 2). This result showed the diversity of the ther- mophilic microorganism communities in geother- mal area. Figure 2. Spore shapes of the isolates under micro- scope observation (magnificent 1000X). A: BS2; B: NW7; C: NW6; D: BM5; E: BS5; F: BM5; G: BS4; H: BM8. www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 17(3) 58 Nong Lam University, Ho Chi Minh City Table 3. Thermophilic microorganism isolated from hot springs Hot spring Sample Isolate Notation Binh Chau (B) (Ba Ria – Vung Tau) Water (W) 3 BW1,2,3 Soil (S) 6 BS 1,2,3,4,5,6 Mud (M) 8 BM 1,2,3,4,5,6,7,8 Truong Xuan (N) (Nha Trang, Khanh Hoa) Water (W) 11 NW1,2,3,4,5,6,7,8,9,10,11 Soil (S) 5 NS1,2,3,4,5 Mud (M) 0 Total 33 3.2. Optimization growth conditions of ther- mophilic isolates In order to optimize the temperature and pH for microorganism growth, the isolates were cul- tivated at temperature range from 450C to 800C and pH range from 6 to 9. The result was shown in details in Table 4. The aim of this study was to isolate the microbes that were capable of growing from 500C, therefore the intended study temper- ature range was 45, 50, 55, 60, 65, 70, 75, 800C. However, at the temperature higher than 550C the growth of microorganisms isolated was very weak. So, we focused on testing from 50 to 550C. At pH 9, the growth of microorganisms could not be observed, then the data were not shown in Ta- ble 4. After 12 hours of incubation, the OD540 val- ues of six isolates including BM7 (0.73 ± 0.06, at 500C), BS5 (0.67±0.02, at 520C), NS1 (0.71 ± 0.03, at 55oC), NS3 (1.04 ± 0.05, at 500C), NS4 (0.93 ± 0.04, at 500C), and NW6 (0.82 ± 0.09, at 550C) were higher than the others. Of these, isolates, BM7, NS3, NS4 grew optimal at 500C with OD540 from 0.73 to 1.04, while growth of isolate BS5 was optimal at 520C with OD540 at 0.67 ± 0.02. Isolates NS1 and NW6 were op- timal at 550C with high OD540 at 0.71 ± 0.03 and 0.82 ± 0.09, respectively. The pH investiga- tion also showed that isolate BS5 grew optimal at pH 6, isolates BM7, NS1, NS3, NS4 grew opti- mal at pH 7 while NW6 was optimized at pH 8. Moreover, the highest OD540 (1.18 ± 0.08) was recorded in isolate NS3 at pH 7. 3.3. Evaluation of the use of different carbon sources Of the 33 isolates, 9 (27.3%) grew on acetate, 15 (45.5%) grew on pyruvate and 24 (72.7%) were able to use malate. All isolates were seen growth well in MSB medium with the supplement of glu- cose, or fructose. Under CO2 condition, microor- ganism were grown in MSB medium (pH 7) with a final gas phase consisting of H2: O2: CO2 (80%: 10%: 10%) at 500C. However, none of isolate was able to grow under autotroph condition. After 7 days of continuous observation, the turbidity environmental change was not found in the test tubes. This result suggested that there is no iso- late could be fixed CO2 or grown autotrophically. Experimental results showed that most isolates developing favorably in the presence of malate, glucose, or fructose. Malate acts as an intermedi- ary helps the microbes produce energy as well as metabolite to produce amino acids via the tricar- boxylic acid cycle, whereas glucose or fructose is easily metabolized via glycolysis pathway (Kim et al., 2008). Hence, the isolates could favorably utilize this organic substrate. It is now very well-known that extreme ther- mophiles are mostly distributed among the gen- era of Bacillus, Clostridium, Thermoanaerobac- ter, Thermus, Thermotoga, Aquifex (Tulasi et al., 2013). In which, Bacillus is a large and diverse genus that is widely distributed in soil and ther- mal water areas (Claus & Berkeley, 1986). During the past few decades, a great diversity of microor- ganisms has been discovered that exist in hot en- vironments. In a previous report of Nguyen et al. (2015), 64 aerobic isolates of thermophilic mi- croorganism were identified from muddy and hot water of Binh Chau hot spring. The percentage of microorganism with cellulase, amylase and pro- tease activities is 19%, 67% and 24% of total 64 microorganism isolates, respectively. Cellular structure and enzyme activities are deeply affected by temperature of habitat. For any microbe to grow at high temperature, their proteins must be able to resist heat. Hence, ther- mophiles have accumulated various thermostable enzymes that are high potential application in biotechnology. The thermophilic strains of Bacil- The Journal of Agriculture and Development 17(3) www.jad.hcmuaf.edu.vn Nong Lam University, Ho Chi Minh City 59 Table 4. OD540 values of 33 isolates at different temperatures and pH No. Isolate Temperature pH 500C 520C 550C 6.0 7.0 8.0 1 BM1 0.40 ± 0.03 0.30 ± 0.02 0.23 ± 0.03 0.10 ± 0.02 0.41 ± 0.04 0.09 ± 0.02 2 BM2 0.59 ± 0.04 0.33 ± 0.02 0.15 ± 0.01 0.16 ± 0.02 0.57 ± 0.06 0.08 ± 0.01 3 BM3 0.44 ± 0.01 0.34 ± 0.03 0.29 ± 0.01 0.25 ± 0.01 0.44 ± 0.02 0.19 ± 0.03 4 BM4 0.36 ± 0.03 0.45 ± 0.02 0.23 ± 0.02 0.02 ± 0.01 0.37 ± 0.03 0.06 ± 0.01 5 BM5 0.24 ± 0.02 0.18 ± 0.04 0.14 ± 0.05 0.26 ± 0.04 0.27 ± 0.02 0.24 ± 0.05 6 BM6 0.40 ± 0.03 0.22 ± 0.01 0.22 ± 0.07 0.15 ± 0.03 0.44 ± 0.04 0.13 ± 0.03 7 BM7 0.73 ± 0.06 0.50 ± 0.02 0.47 ± 0.03 0.54 ± 0.05 0.72 ± 0.04 0.57 ± 0.04 8 BM8 0.45 ± 0.02 0.42 ± 0.03 0.22 ± 0.04 0.02 ± 0.01 0.47 ± 0.03 0.12 ± 0.02 9 BS1 0.41 ± 0.05 0.26 ± 0.02 0.25 ± 0.03 0.14 ± 0.03 0.42 ± 0.02 0.16 ± 0.02 10 BS2 0.43 ± 0.02 0.10 ± 0.03 0.05 ± 0.02 0.23 ± 0.02 0.45 ± 0.03 0.27 ± 0.02 11 BS3 0.53 ± 0.03 0.42 ± 0.07 0.34 ± 0.06 0.15 ± 0.03 0.50 ± 0.03 0.13 ± 0.03 12 BS4 0.35 ± 0.03 0.27 ± 0.03 0.18 ± 0.03 0.17 ± 0.01 0.37 ± 0.01 0.10 ± 0.01 13 BS5 0.46 ± 0.07 0.67 ± 0.02 0.30 ± 0.02 0.71 ± 0.05 0.65 ± 0.06 0.52 ± 0.04 14 BS6 0.35 ± 0.03 0.43 ± 0.05 0.23 ± 0.03 0.14 ± 0.03 0.39 ± 0.02 0.31 ± 0.03 15 BW1 0.40 ± 0.01 0.34 ± 0.02 0.24 ± 0.04 0.50 ± 0.03 0.43 ± 0.03 0.30 ± 0.03 16 BW2 0.42 ± 0.03 0.38 ± 0.03 0.22 ± 0.06 0.18 ± 0.06 0.44 ± 0.02 0.08 ± 0.01 17 BW3 0.60 ± 0.03 0.22 ± 0.01 0.29 ± 0.06 0.11 ± 0.02 0.58 ± 0.05 0.17 ± 0.04 18 NS1 0.45 ± 0.02 0.59 ± 0.01 0.71 ± 0.03 0.32 ± 0.05 0.76 ± 0.03 0.40 ± 0.04 19 NS2 0.62 ± 0.03 0.41 ± 0.03 0.23 ± 0.02 0.34 ± 0.04 0.61 ± 0.05 0.08 ± 0.01 20 NS3 1.04 ± 0.05 0.52 ± 0.05 0.56 ± 0.03 0.83 ± 0.07 1.18 ± 0.08 0.73 ± 0.09 21 NS4 0.93 ± 0.04 0.46 ± 0.01 0.30 ± 0.04 0.92 ± 0.04 0.94 ± 0.10 0.57 ± 0.05 22 NS5 0.43 ± 0.05 0.34 ± 0.06 0.30 ± 0.03 0.50 ± 0.03 0.43 ± 0.01 0.13 ± 0.02 23 NW1 0.51 ± 0.01 0.20 ± 0.02 0.36 ± 0.03 0.14 ± 0.03 0.54 ± 0.03 0.11 ± 0.02 24 NW2 0.26 ± 0.03 0.03 ± 0.01 0.02 ± 0.01 0.04 ± 0.02 0.24 ± 0.01 0.05 ± 0.01 25 NW3 0.30 ± 0.04 0.38 ± 0.02 0.23 ± 0.03 0.23 ± 0.03 0.31 ± 0.03 0.14 ± 0.03 26 NW4 0.55 ± 0.02 0.03 ± 0.01 0.34 ± 0.05 0.19 ± 0.04 0.53 ± 0.06 0.12 ± 0.04 27 NW5 0.42 ± 0.02 0.24 ± 0.02 0.22 ± 0.01 0.15 ± 0.02 0.41 ± 0.05 0.07 ± 0.02 28 NW6 0.68 ± 0.03 0.46 ± 0.06 0.82 ± 0.09 0.48 ± 0.03 0.65 ± 0.06 0.98 ± 0.12 29 NW7 0.48 ± 0.02 0.30 ± 0.04 0.06 ± 0.02 0.16 ± 0.01 0.51 ± 0.04 0.09 ± 0.02 30 NW8 0.42 ± 0.05 0.12 ± 0.03 0.31 ± 0.05 0.26 ± 0.02 0.40 ± 0.01 0.14 ± 0.03 31 NW9 0.61 ± 0.02 0.04 ± 0.01 0.51 ± 0.02 0.12 ± 0.01 0.59 ± 0.03 0.06 ± 0.01 32 NW10 0.60 ± 0.04 0.16 ± 0.03 0.40 ± 0.03 0.13 ± 0.03 0.64 ± 0.04 0.05 ± 0.02 33 NW11 0.53 ± 0.05 0.48 ± 0.04 0.35 ± 0.02 0.19 ± 0.01 0.52 ± 0.05 0.22 ± 0.01 Table 5. Characteristics of six selected thermophilic microorganism isolates Characteristic BM7 BS5 NS1 NS3 NS4 NW6 Shape Rod Rod Rod Rod Rod Rod Color Cream Yellow Orange Cream White White Gram/Spore +/+ +/+ +/+ +/+ +/+ +/+ Optimum growth temperature (0C) 50 52 55 50 50 55 Optimum growth pH 7 6 7 7 7 8 CO2 – – – – – – Acetate – – – – – – Pyruvate – – – – – – Malate + + + + + + Glucose + + + + + + Fructose + + + + + + +: positive; –: negative www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 17(3) 60 Nong Lam University, Ho Chi Minh City lus that synthesized cellulase, amylase and pro- tease have a great significance for many fields of industry (Rastogi et al., 2010). Figure 3. Image profiles of six selected thermophilic microorganism isolates. Table 5 and Figure 3 described the profile of six selected thermophilic microorganism isolates that were selected from thermophilic microorgan- ism collection in this study in details. In order to explore the potential application of six selected thermophilic microorganism isolates, the identifi- cation of microorganism to species as well as en- zyme activity screening is required. Recently, the most effective approach to microorganism taxon- omy may be analysis of 16S rDNA molecules by oligonucleotide sequencing. Detailed information of the molecular identification for six selected mi- croorganism isolates will be announced very soon elsewhere. 4. Conclusions From the sources of samples collected form the geothermal areas, we have successfully con- structed the collection of thermophilic microor- ganism including of 33 isolates that are evaluated in terms of morphology, microscopy, and growth test on different substrates. Six selected iso- lates were Gram-positive, rod-shaped, and spore- forming. These characteristics of six selected iso- lates with the optimum growth temperature from 50-550C were found highly similar to Bacillus species. The achievement in collection of ther- mophiles is the preliminary step in effort to be able to apply the thermophilic microbes into the biotechnology sector. Acknowledgements We appreciate Nong Lam University – Ho Chi Minh City for the financial support under Re- search Project Code. CS-CB16-KH-03. We also thank our colleagues from Institute of Tropi- cal Biology (Vietnam Academic of Sciences and Technology), who provided insight and expertise that greatly assisted the research. References Cao, T. D., Do, T. H., Nguyen, K. N., Chau, V. Q., & Vu, N. T. (1998). Geographic Atlas of Vietnamese. Ha Noi, Vietnam: Education Publishing House. Claus, D., & Berkeley, R. C. W. (1986). The genus Bacil- lus. In Sneath, P. H. A. (Ed.). Bergey’s manual of systematic bacteriology (1105-1139). Baltimore, USA: Williams and Wilkins. Gaughran, E. R. (1947). The thermophilic microor gan- isms. Bacteriological reviews 11(3), 189. Goto, E., Kodama, T., & Minoda, Y. (1977). 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