Isolation , characterization and identification of amylase producing halothermophilic isolates from Howz Soltan Lake , Iran

Halophilic bacteria are a group of salt tolerant microorganisms; the production of enzymes by halophilic bacteria, due to their activities in harsh environments, is considered as a major field of investigation. Hence, the present study was conducted for the isolation of halothermophilic bacteria from Howz Soltan salt lake. For this purpose 40 samples were collected from five regions of the Howz Soltan lake. The samples were diluted and cultivated on Molten haloid agar (MH) with different salt concentrations (5-35%). Then the plates were incubated at 35-70°C in aerobic conditions for 7-10 days. The strains were screened for amylase production based on differences in enzyme production. In total 205 strains were grown on media including 5-15% salt concentrations. Of all, 18 strains were hyperhalophilic and could grow on media including 20-35% salt concentrations at 40-70°С. Three (3) isolates were identified as best amylase producers. In addition, the results indicated that maximum enzyme activity was observed at 48 h (18.145 U/ml). The optimal pH, temperature and NaCl concentration for amylase production were 7.0-7.5, 40°C and 15% (2.5 M), respectively. Taxonomic and phylogenetic analysis using 16SrRNA gene sequencing revealed that they belonged to Bacillus licheniformis and Gracilibacillus while the third isolate was closer to β proteobacterium.


INTRODUCTION
Halophilic bacteria are a group of salt tolerant microorganisms.These bacteria are divided into three groups: Slightly, moderately and extremely halophiles (Larsen, 2001).However, survival of halophilic bacteria in the environment depends on concentration of Na + and K + , but compatible solutes in their cells culminate their survival in ocean, salt lakes and salt fields (Fukushima et al., 2007).Although production of energy in some halophilic bacteria depends on their red pigment (bacteriorhodopsin), non pigmented halophilic bacteria could obtain energy in the environment with high salt concentrations (Dave et al., 2006).Nowadays, production of enzymes such as amylases, proteases and nucleases by halophilic bacteria due to their activities in harsh environments is considered as a major field of investigation (Alqueres et al., 2007).
Therefore, the present study was conducted to isolate and identify halothermophilic bacteria from Howz Soltan Lake in Iran, in order to evaluate their potential for amylase production.

Sample collection
Forty (40) soil and water samples were collected from five different areas of Howz Soltan Lake in Iran (Figure 1).All of the samples were taken from surface and depth of 10-50 cm.The samples were transferred to the laboratory for detection of halophilic microorganisms.

Evaluation of halothermophilic strains for amylase production
Amylolytic activity of the isolates was assessed using MH medium supplemented with 1% (w/v) soluble starch (Gonzalez et al., 1978).
To perform the test, all the isolates were separately cultivated onto the medium and the plates were incubated at 40°C.After 48 h, 0.5%(w/v) I2 and 5% (w/v) KI solution and equal volume of 1 N HCl (0.1 N KI3 reagent) was added onto the plates and the colonies surrounded by clear zone were considered for hydrolysis of starch by bacteria (Zobell, 1941).Then, amylase producing bacteria were identified and characterized based on their morphological, physiological and biochemical tests (Tigue et al., 1995).

Phenotypic identification
The fresh isolates were morphologically and phenotypically characterized using different biochemical tests: Gram reaction, motility, growth at different salt concentrations, catalase and oxidase production, indole and urease production, gelatin liquefaction, hydrolytic activities, ability to use different compounds such as citrate and various carbon sources.

Determination of microbial growth and amylase activity
The growth studies of the halothermophilic strains were performed using overnight cultures (1% v/v containing 1.5 x 10 12 CFU /ml) from the agar plates into 1% (w/v) starch MH broth medium.The effect of pH on microbial growth was assayed at different pH values using sodium acetate buffer (pH 5.0 -6.5), and Tris-HCl buffer (pH 7.0 -9.0).Equally, the effect of temperature on microbial growth was evaluated by growing the strains at different temperatures (35-65°C) at optimum pH value for 96 h.
For the evaluation of enzyme production and activity, the halophilic strains were inoculated into the starch MH medium and the samples were incubated in incubator shaker (150 rpm) for 48 h at 37°C (pH 7.0).Then, the cultures were centrifuged at 10,000 rpm for 20 min at 4°C, and the supernatants were used for the estimation of the amylase activity (Gonzalez et al., 1978;Tigue et al., 1995).In addition, liquefying activity of the enzyme was evaluated in the presence of 0.02 M sodium phosphate buffer (pH7) using 0.1 mg of enzyme supernatant and 0.1 ml of 0.1% (w/v) soluble starch substrate at 40°C.Following 15 min, the reaction was stopped by the addition of KI3 reagent.The starch-iodine complex formation was quantified using the absorbance at 550 nm (Sirimati et al., 2003).Then, hydrolytic activity of enzyme was determined by measuring the decrease in iodine color by Spectrophotometer.One unit of enzyme activity is defined 0.0284 optical density reduction of blue color intensity of starch iodine solution at 40°C.Enzyme production (U/ml) = OD Control -OD sample / 0.0248 and the soluble starch (0.1 % w/v) was used as standard (Ventosa et al., 1989).

Optimum pH, temperature and NaCl concentration for amylase production
The effects of pH on enzyme production were assayed at different pH values from 5.0 to 9.0.The following buffers were used in the cultures: sodium acetate buffer (pH 5.0 -6.5), and Tris-HCl buffer (pH 7.0 -9.0(.The optimum pH for enzyme production was examined at 40°C for a duration of 48 h taking the samples for every 6 h and the amylase activity was determined under standard enzyme assay condition (Anupama and Jayaraman, 2011).Similarly, the temperature required for optimum production of enzyme was studied by culturing the strains at different temperatures, in the range of 25 to 65°C at optimum pH for 48 h.In addition, the effect of NaCl concentration on enzyme production was tested.The role of salt on amylase production was investigated in the NaCl concentration range of 0 M to 6 M (0-35%) at optimum pH and temperature and then amylase activity was determined.The  activity of the enzyme stored at 4°C was used as the control groups.

Confirmation of halothermophilic enzyme producing strains
Identification of halothermophilic enzyme producing strains was verified by sequencing the 16S rRNA genes.This method was carried out as follows: DNA was extracted from the isolates by a standard kit (Roche-Germany).Then amplification of 16S rRNA gene was performed by PCR method and eventually the products were sent to Macrogen in South Korea (http://www.macrogen.com/)for DNA sequencing.

DNA extraction and amplification of 16S rRNA gene
DNA was extracted from the isolated strains using High Pure Template DNA PCR kit (Roche-Germany) according to the supplier's instructions.The purity of the extracted DNA was assessed based on absorbance of the extracted DNA at 260 and 280 nm using a Biophotometer (Eppendrof-Germany) and then the purity was calculated based on absorbance ratio 260/280 nm.The extracted DNA with ratio (260/280nm) 1.9≤ corresponding to 121 μg DNA ml -1 was used for amplification of 16S rRNA by PCR.Amplification of 16S rRNA was performed using universal primers produced by TAG Kopenhagen (Denmark).The sequence of Forward and Reverse primers were 5'-AGGAGGTGATCCAACCGCA-3' and 5'-AACTGGAGGAAGGTGGGGA-3' respectively.Each reaction was performed in a total volume of 25.5 µl containing 14.5 µl of molecular biology-grade water (Sigma Aldrich Company Ltd.), 2.5 µl of 10×PCR buffer (Cinagen-Iran), 1 µl of each forward and reverse PCR primers, 1 µl of a 10 mM dNTPs (Cinagen-Iran), 0.5 µl of Smar Taq polymerase (Cinagen-Iran), 1 µl of 50mM MgCl2 (Cinagen-Iran) and 5 µl of DNA template.
PCR amplification conditions on an Eppendorf thermocycler were as follows: 95°C for 4 min, followed by 35 cycles of 95°C for 40 s, 56°C for 30 s, and 72°C for 40 s, with a final extension at 72°C for 5 min and storage at 4°C.All PCR products obtained were run on a 1.5% (w/v) agarose gel with a 100 bp DNA ladder (Fermentas-Russia).PCR products were electro-phoresed at 75V for 20 min; DNA was visualized using ethidium bromide and photographed by Uvidoc (England).After visualization of pure DNA bands, the PCR products were sent to Macrogen in South Korea (http://www.macrogen.com/)for DNA sequencing.The 16S rRNA sequenced data for bioinformatic applications were subjected to BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) in order to identify each respective 16S rRNA gene amplicon.

Screening of halothermophilic bacterial strains
In total, 205 strains were able of growing on MH media containing 5-15% of salt concentration.The results obtained from microscopic analysis of the isolates indicated that 153 and 25 strains were Gram positive bacilli and cocci, respectively.However, 27 isolates were Gram negative bacilli.Of all the isolates, 34 strains were able of growing on media containing 15-35% of salt, so they can be considered as hyperhalophilic bacteria.Out of that, 18 strains were hyperhalophilic and moderately thermophilic with the ability to grow on media including 20-35% of salt concentration at 35-65°С.

Isolation of halothermophilic strains for amylase production
Totally, 18 halothermophilic bacterial isolates were screened for amylase production on MH medium supplemented with 1% (w/v) soluble starch (Singh et al., 2001).Colonies with highest clear zone on the plates by the addition of KI 3 reagent were selected as potential amylase producing strains.Among the isolated strains, 6 amylase positive strains were selected by exposing the agar plates to KI 3 solution.Based on differences in enzyme production by clear zone on the plates, the highest amylase producers (three strains) were chosen for further investigation.

Microbial growth and amylase production assay
The growth profile shows that these strains have a fast growth rate as it entered log phase within 4 h of lag phase and, the bacterial growth reaches its maximum at 48 h (Figure 2).In addition, growth of the strains occurred over a pH range of 5.0 to 9.0.However, the growth was maximum at pH 7.5 (Figure 3).On the other hand, the strains were able to grow at temperatures between 35-65°C, but optimum temperature for all of the isolates was 40°C (Figure 4 a-c) ,which indicate that, the strains could possibly be presumed as moderately thermophilic (Ventosa et al., 1989).In addition, the results obtained from bacterial growth in the presence of 6-30% NaCl indicated that two of the isolates were able to tolerate NaCl up to 3 M and with increasing the salt concentration, growth decreases (Figure 5).The results from maximum enzyme production indicated that maximum  rate was observed at 48 h of growth when the organisms were in the stationary phase (Figure 6).This indicates that the enzyme production is growth associated and was induced by the presence of substrate.

Effect of pH , temperature and NaCl concentration on amylase production
As seen in Figure 7 the optimum pH for maximum Production of enzyme was 7.5 (specific activity 18.145 U/ml).In addition, the strains were grown on wide range of temperatures ranging from 25 to 65°C.However, the optimum temperature for enzyme production was 40°C (Figure 8).
In order to investigate the salt tolerance, amylase activity was assayed in the presence of different NaCl concentrations (0-6M).As seen in Figure 9 at 1-1.5 M salt concentration specific activity was observed.With increasing the salt concentration up to 3 M specific activity increased and then with increasing the salt concentration the activity decreased which this phenomenon  clearly indicates that the enzyme is halotolerant.

Phenotypic identification
The results obtained from phenotypic identification (Table 1) indicated that enzyme producing halothermophilic isolates were aerobic, Gram positive, rod shaped and spore forming.According to the biochemical results they belong to the genera Gracibacillus, Bacillus licheniformis and unknown.

16SrRNA sequencing
The isolates were identified according to the 16S rRNA genetic analysis.BLAST analysis of the strains revealed that it had a closest match (99% and 96%) with Gracibacillus and Bacillus licheniformis respectively.The sequence is deposited in NCBI Gene Bank with accession numbers HM021766.1 and GQ340506.1.However, the third strain was not matched, but this isolate was the closer to β proteobacterium.

DISCUSSION
In this study, several halophilic bacterial strains were isolated from Howz Soltan Lake, but few strains produced amylase enzyme.According to the 16S rDNA sequences the strains belonged to halothermophilic strains of B. licheniformis, Gracilibacillus and β proteobacterium, which were able to produce amylase with high level.The characterized strains showed optimum growth between pH 7.0 and 7.5 in the presence of various NaCl concentrations.Therefore, according to the salt tolerance (up to 4M) these strains could be described as hyper halophilic microorganisms (Dave et al., 2006).In addition the strains had optimal production of amylase enzyme in pH 7.5 at 40-45°C which is similar to Bacillus aquimaris VITP4 (Anupama and Jayaraman, 2011) and Halobacillus sp.(Coronado et al., 2000;Amoozegar et al., 2003).Mostly, the scientists reported that amylase from halophilic Bacillus sp (Mathabatha, 2010) present an optimum production at 40 to 70°C (Carvalho et al., 2008).In addition Shafiei et al in 2010 showed that moderately halophilic bacterium Nesterenkonia sp.Strain F was able to grow in the pH range 6.0 to 10.0 (Shafiei et al., 2010).On the other hand, The highest mean amylase production (18.145U/ml) of halothermophilic strains amylase enzyme (95%) were observed between 40 -50°C in pH 7.0 -7.5.However, enzyme production were stable at 60°C, but the enzyme stability was better than that of other researcher findings (Tan et al., 2008;Prakash et al., 2009).Therefore, according to the findings it should be noted that the amylase producing halothermophilic strains might be called halophilic because the halophilic enzymes are usually inactive when the NaCl or KCl concentration is less than 2 M (Mader et al., 2000) as Pomares et al. (2003) showed that the optimum salt concentration for amylase production of Haloferax mediterranei was 3 M NaCl.Therefore, this halophilic stability and adaptation of halothermophilic strains with capability to produce amylase (higher than the other halophilic strains than that of other researcher isolates) suggested that, these strains could be a good choice for some biotechnological application.In conclusion, elucidation of the origin of the halophilic bacteria clarifies influence on salt lake ecosystem of the central desert zone of Iran.Above-mentioned considerations invite further empirical investigation.It is thought that the collection of the central desert zone in Iran develops the study.