Int J Pharm Pharm Sci, Vol 8, Issue 5, 379-386Original Article
EXTRACELLULAR ALKALINE PROTEASE PRODUCING HALO-ALKALITOLERANT BACTERIA ISOLATED FROM MARINE COASTS OF ODISHA
ABHAS KUMAR MAHARANAa*, ISHITA SINHAa, PRAKASH CHANDRA BEHERAb, PRATIMA RAYa
aDepartment of Microbiology, College of Basic Science and Humanities, Orissa University of Agriculture and Technology, Surya Nagar, Bhubaneswar, Odisha 751003, India, bDepartment of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Surya Nagar, Bhubaneswar, Odisha 751003, India
Email: abhas_maharana@yahoo.com
Received: 27 Feb 2016 Revised and Accepted: 08 Apr 2016
ABSTRACT
Objective: The objective of the present study was on the isolation of alkali-tolerant bacteria from sediment samples of different coasts of Odisha, having potentiality to produce alkaline protease.
Methods: About 25 sediment samples were collected and analyzed for pH and moisture contents. Then isolation of alkali-tolerants was done using Horikoshi media at 10.3 pH. Isolates were analyzed for producing alkaline protease by plate assay method both at pH 6 and 10. Effects of temperature on protease production were also determined. Besides a new method of quantification of enzymes were adapted. Along this the isolates were partially characterized and identification was done using PIBWin software.
Results: About 80 isolates were initially isolated, and 11 isolates were considered based on maximal zones of clearances at alkaline pH. Maximum solubilisation index (SI) was found to be 30 mm by 3 isolates viz. AP2, AP8 and AP13 while maximum hydrolytic run percentage (HR%) was found to be 65.39% by AP3. About 45.46% isolates had capability for protease production at 37 °C and 18.18% at 57 °C while 81.82% isolates showed production at 17 °C. AP8 was the good producers of alkaline protease having SI 39 mm at pH 10 while incubating at 47 °C. Isolates were characterized partially by cultural, morphological, biochemical and physiological tests, which were belonged to the genera of Bacillus, Virgibacillus and Micrococcus. The isolated bacteria showed growth at pH ranges from 4-12 and can tolerate 12% NaCl concentrations for their growth.
Conclusion: Due to the above unique features and capability to produce alkaline proteases by the marine isolates, can be used significantly in various industries.
Keywords: Alkali-tolerant, Alkaline protease, Halo-tolerant, Hydrolytic run, Odisha-coast
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
‘Extreme’ or ‘normal’, are those two words that differentiate the extremophiles from other microorganisms. Alkaliphiles are those microorganisms, which can thrive optimally at pH above 9 and showing little or no growth at near neutral pH. Nevertheless, alkalitolerants are capable of growing over a wider range of pH, with pH optima at approaching neutral pH [1]. Alkaliphilic microorganisms are widely distributed in nature and can be found in almost all environments without the restriction of alkalinity. Even so, a few of the naturally-occurring alkaline environments, namely soda soils, lakes, and deserts, harbor a wide range of these types [2]. The dilute alkaline springs, desert soils and oils containing decaying proteins or forest soil also act as the habitat for them, where the pH values of these environments are commonly around 10 and above [3-6]. There are many physiological differences in the adaptive nature of alkaliphiles than the neutrophiles. The major difference is in the cell wall; alkaliphiles have cell wall containing acidic polymers such as galacturonic acid, gluconic acid, glutamic acid, aspartic acid, and phosphoric acid [7]. However, they also have an excess of hexoamines and amino-acids in peptidoglycan layer. Alkaliphiles usually require at least sodium ions for growth as it is essential for effective solute transport [8].
Proteases or exopeptidases (EC 3.4.11-18) are a complex group of hydrolytic enzymes capable of hydrolyzing the peptide bonds in a protein molecule to amino acids and smaller peptides [9]. Proteases are classified into 4 major groups, i.e. serine-, thiol-, metallo- and acid proteases [10]. Further, it was sub-grouped assuming the side chain specificity of the enzyme and properties of their active centers [11]. The protease types depend on the composition of the production medium. Therefore, one microorganism can produce various types of proteases.
Microbial proteases have immense applications as compared to other hydrolytic enzymes in various spheres of science and industries and there is a high demand of alkaline protease in detergent industries, which make it as a potent enzyme. Besides, utilizing in detergent industries alkaline proteases can be used in bioremediation, food and dairy industries, leather processing and bio-film degradation [12].
Proteases can be available from plants, animals and microorganisms. However, proteases from microbial source are preferred due to diversified uses and compatibility. There are many reports of getting alkaline proteases from bacteria and fungi. The dominant genera of bacteria producing alkaline proteases are Bacillus and Pseudomonas due to their survival at extreme alkalinity. About 15 alkaliphilic aerobic bacteria from soil and water samples of Egyptian Soda Lake reported, producing alkaline protease (61 U/ml) at 48 h of incubation [13]. About 20 alkaliphilic were isolated bacteria from industrial effluents, Kott Lakh-Patt, Lahore, Pakistan and belonged to the genera of Escherichia, Alcaligenes, Aeromonas, Pseudomonas, Marinococcus, Neisseria, Natronobacterium, Micrococcus, Sporosarcina, Pleisomonas and Cupriavidus [14]. Dodia and co-workers isolated 8 moderately halophilic and alkaliphilic bacteria from saline habitat of coastal Gujarat, India [15].
There are very few reports on alkaline protease producing bacteria from the coastal region of Odisha. Therefore, the present study focuses on the isolation of alkali-tolerant bacteria having the potentiality to produce alkaline protease and quantifying the enzyme activity by a new method i.e. determining hydrolytic run percentage.
MATERIALS AND METHODS
Sample collection and analysis
A total of 25 sediment samples were collected from 5 sites of coastal regions of Odisha i.e. Chandipur (21.47 ° N, 87.02 ° E), Chilika (19.7167 ° N, 85.318 ° E), Gopalpur (19.27 ° N, 84.92 ° E), Puri (19.81 ° N, 85.83 ° E) and Paradip (20.2654 ° N, 86.68 ° E). All the sediments were collected in sterile plastic polythene and were brought to the Department of Microbiology, O. U. A. T., BBSR (Odisha) for further study. After collection of samples, pH and moisture content were calculated accordingly.
Sediment pH was determined by taking about 10 g of samples, weighed into a 50 ml size beaker and 20 ml of 0.01 M CaCl2 (1.47 g CaCl2.2H2O in 1000 ml deionize water) was added to the sample [16]. The preparation was allowed to stand for 30 min with occasional stirring before determination of pH of the supernatant by digital pH meter 335 (Systronics, India).
For moisture content, about 10 g of sediment from each sample was taken and dried at 60±1 °C for 24-30 h in hot air oven and percentage of moisture content was determined [17]:
Isolation of alkali-tolerant bacteria
For the isolation of alkaliphilic or alkali-tolerant bacteria, Horikoshi medium was used, which comprises (g/l: glucose, 10.0 g; peptone, 5.0 g; yeast extract, 5.0 g; KH2PO4, 1.0 g; MgSO4.7H2O, 0.2 g; Na2CO3, 10.0 g; NaCl, 40.0 g) having 10.3 pH [18]. About 1 g sediment sample was inoculated in the above 100 ml medium. After 96 h, serial dilution was carried out in Ringer’s solution and 0.1 ml of diluents was spread on the sterilized pre-cooled nutrient agar plate (pH 8) with Fluconazole (0.015% w/v). Then the plates were incubated at 37±1 °C for 24 h. The CFU/g of each sediment sample was calculated. The strains were maintained on nutrient agar slants (pH 8) at 4 °C for further study. Bacterial isolates were revived in an interval of 2-3 w.
Screening for alkaline protease production
For the production of alkaline protease, all the isolated bacteria were screened on skim milk agar (Hi-media) maintained at pH 6 and 10, to differentiate the nature of enzyme. Isolates were spot inoculated on the above agar plate incubated at 37±1 °C for 96 h. The zone of clearance around the colony was regarded as positive for enzyme production. Besides, another direct method was applied for isolation and screening of alkaline protease producing bacteria. For that the serially diluted sediment samples were directly applied to skim milk agar (pH 10) and the isolates showing zone of clearances at 37±1 °C were selected for further study.
Qualitative optimization of alkaline protease
Qualitative optimization was done in skim milk agar plates by using potent isolates. It was based on the solubilisation index (SI), which was calculated by subtracting the colony diameter from the total diameter. The optimizing parameters investigated were pH (4, 6, 8, 10 and 12) and temperatures (17, 27, 37, 47 and 57 °C).
Quantitative screening of alkaline protease
In addition to the plate assay method another new method was created for the quantitative determination of alkaline protease by “Hydrolytic Run (HR)” method. This method was conducted in tubes. Skim milk agar (Hi-media) was prepared having 10 pH and distributed equally to test tubes having 5 ml each. After autoclave the height of the stab was measured in each case and aseptically 0.1 ml bacterial isolates (prepared in nutrient broth having pH 8 and 24 h old) were applied and incubated at different temperatures of 27, 37 and 47 °C for 3 d. After every 24 h, HR% was calculated by the following:
Where “R” denotes, the actual height of the assay stab medium and “r” denotes, the height left un-hydrolyzed. By this new method the potentiality of isolates were measured. The optimizing parameter investigated by using the above method was incubation temperatures (27, 37 and 47 °C).
Partial characterization of bacterial isolates
Afterward the screening of alkaline protease producers, isolates were subjected for partial characterization by cultural (colour, texture, margin, elevation, density and size), morphological (Gram variability), standard biochemical and physiological tests (pH and temperature). The results were analyzed in PIBWin Software for probabilistic identification of bacteria [19].
Statistical analysis
The data recorded during the investigation was subjected to Pearson’s correlation coefficient determination (r), Student’s T-test and Analysis of Variance (ANOVA), and the conclusions were drawn accordingly.
Table 1: Physico-chemical parameters and alkaliphilic bacterial load of different sediment samples
Sampling sites |
S. No. |
pH |
Moisture content (%) |
Bacterial load (CFU/g)× 103 |
Chandipur |
1 |
7.17±0.21 |
26.7476±1.07 |
0.267±0.033 |
2 |
7.71±0.11 |
16.9576±2.1 |
1.133±0.767 |
|
3 |
7.31±0. 1 |
21.6344±1.11 |
0.4±0.2 |
|
4 |
7.73±0.13 |
30.0343±2.41 |
1.533±0.567 |
|
5 |
8.12±0.12 |
27.4632±1.77 |
3.267±1.133 |
|
Chilika |
6 |
7.72±0.1 |
55.0202±0.57 |
25.7±4.2 |
7 |
6.75±0.04 |
21.0175±0.59 |
0.4±0.1 |
|
8 |
7.21±0.02 |
47.6811±1.12 |
5.2±2.5 |
|
9 |
7.53±0.2 |
46.7435±1.67 |
2.767±1.633 |
|
10 |
8.11±0.08 |
45.5714±1.1 |
9.667±1.433 |
|
Gopalpur |
11 |
7.37±0.2 |
13.482±2.13 |
0.367±0.233 |
12 |
7.45±0.12 |
15.4483±1.87 |
0.867±0.033 |
|
13 |
7.11±0.11 |
14.0412±1.25 |
0.3±0.1 |
|
14 |
7.31±0.1 |
19.6983±2.01 |
0.567±0.233 |
|
15 |
7.19±0.2 |
20.5549±2.88 |
0.333±0.067 |
|
Puri |
16 |
7.13±0.22 |
26.1396±1.09 |
0.267±0.233 |
17 |
7.21±0.16 |
17.812±2.04 |
0.533±0.266 |
|
18 |
7.16±0. 1 |
21.6088±1.71 |
0.333±0.166 |
|
19 |
7.33±0.3 |
27.7011±0.88 |
0.5±0.2 |
|
20 |
7.48±0.1 |
27.4922±1.7 |
0.733±0.166 |
|
Paradip |
21 |
7.55±0.14 |
10.6704±1.92 |
0.967±0.333 |
22 |
7.47±0.11 |
17.4273±1.11 |
0.633±0.166 |
|
23 |
7.64±0.06 |
18.0421±2.19 |
1.067±0.233 |
|
24 |
7.16±0.1 |
37.3013±1.01 |
0.233±0.166 |
|
25 |
7.51±0.04 |
36.4901±2.1 |
1±0.5 |
All values are mean±SD, sample size, n=3
Table 2: Pearson correlation coefficient and T-test for significant study
Parameters |
Moisture content (%) |
pH |
pH |
r = 0.288 t = 7.966** |
|
Bacterial load (CFU/g) |
r = 0.678* t = 2.209** |
r = 0.415* t = 2.228** |
*correlation is significant (p<0.05), **population mean is significant (p<0.05, 2-tailed)
RESULTS AND DISCUSSION
Sample characterization
A total of 25 sediment samples were investigated for physicochemical properties like pH and moisture content percentage. The pH values of sediment samples were near neutral to slight alkaline ranged from 6.75±0.04 to 8.12±0.12 and moisture content varied with sampling sites, ranged from 10.6704±1.92 to 55.0202±0.57% (table 1). Microbiological analysis revealed 25.7±4.2 × 103 CFU/g bacterial load at pH 10.3 from Chilika sediments. The minimum (0.233±0.166 × 103 CFU/g) bacterial load was found from Paradip sediments. About 80 isolates from five sites were selected on the basis of their macro-morphology on agar medium and stored for further screening.
From statistical analysis, it is observed that there is a positive and significant correlation between the bacterial load with both moisture content and pH (p<0.05). The t-test revealed that there is a significant variation between all the population means of all variables (table 2). There is a non-significant correlation found among pH and moisture content (p>0.05), which corroborates the results of [20]. Alkaline bacteria from coastal regions of Odisha showed a significant correlation between pH and moisture content [21]. The similar significant correlations among the bacterial load with pH were also reported [21-22].
Screening for alkaline protease
All the isolates were screened for alkaline protease production in skim milk agar plates at 10 pH. From this, it was found that only 22 of 80 isolates (27.5%) were positive for alkaline protease production and designated as AP1-AP22. Further investigation revealed that, 11 isolates (50%) of 22 showed a maximal zone of clearances at pH 6 than respective plates of 10 pH. Therefore, for further analysis rest 11 isolates were taken. Table 3 depicts the effect of different pH of skim milk agar for the production of protease enzymes by the above 11 isolates. All the isolates showed a zone of clearance at pH 8–12, while 63.64% and 9.09% isolates were positive for protease hydrolysis at pH 6 and 4 respectively. Fig. 1 depicts the zone of clearances by isolates at pH 8. Optimum zone of clearance was found at alkaline pH, which denotes the alkalinity nature of the protease produced by the isolates.
Table 3: Effect of pH for protease production by isolates
Isolates code |
Solubilization indexes at different pH on 48 h of incubation |
||||
4 |
6 |
8 |
10 |
12 |
|
AP1 |
0 |
0 |
8±2 |
10±2 |
12±2 |
AP2 |
0 |
0 |
15±1 |
17±1 |
11±1 |
AP3 |
1±0 |
9±1 |
10±2 |
14±2 |
17±3 |
AP5 |
0 |
0 |
9±1 |
17±1 |
5±1 |
AP7 |
0 |
6±2 |
9±1 |
10±2 |
9±1 |
AP8 |
0 |
0 |
9±1 |
14±2 |
11±3 |
AP9 |
0 |
2±0 |
17±1 |
15±3 |
14±2 |
AP10 |
0 |
15±1 |
15±1 |
16±2 |
9±1 |
AP12 |
0 |
9±1 |
10±2 |
11±1 |
8±2 |
AP13 |
0 |
7±1 |
15±1 |
16±2 |
11±1 |
AP21 |
0 |
12±2 |
12±2 |
12±2 |
10±2 |
All values are mean±SD, ANOVA revealed that there is a significant differences found among the SI with respect to various pH investigated (p<0.05), sample size, n=3
Table 4: Qualitative and quantitative analysis of alkaline protease at pH 10 by isolates at 37 °C
Isolates code |
Inoculum (600 nm) |
HR% at different incubation period (h)** |
SI at different incubation period (h)* |
|||||
24 |
48 |
24 |
48 |
72 |
96 |
120 |
||
AP1 |
1.386±0.026 |
42.308±3.846 |
61.539±7.143 |
2±0 |
7±1 |
9±1 |
12±2 |
20±2 |
AP2 |
1.546±0.007 |
12.5±7.692 |
16.667±3.704 |
13±3 |
16±2 |
21±1 |
26±2 |
30±2 |
AP3 |
0.856±0.026 |
34.615±4.167 |
65.385±15.3846 |
6±2 |
14±2 |
16±2 |
18±3 |
20±2 |
AP5 |
1.451±0.002 |
34.615±3.704 |
50±3.571 |
7±3 |
17±1 |
19±1 |
21±3 |
22±2 |
AP7 |
1.548±0.013 |
17.857±3.846 |
28.571±3.846 |
7±1 |
9±1 |
11±1 |
14±4 |
16±2 |
AP8 |
0.831±0.026 |
37.037±4.167 |
48.148±3.571 |
4±2 |
9±1 |
15±1 |
20±0 |
30±2 |
AP9 |
1.726±0.007 |
32±4.167 |
60±4.167 |
12±2 |
15±3 |
17±3 |
26±0 |
26±2 |
AP10 |
1.420±0.026 |
15.385±7.143 |
34.615±3.571 |
11±1 |
13±1 |
16±2 |
25±1 |
28±2 |
AP12 |
1.707±0.013 |
21.429±3.571 |
32.143±4.167 |
6±0 |
8±2 |
12±2 |
18±2 |
23±1 |
AP13 |
1.779±0.026 |
39.286±3.571 |
53.571±4.167 |
10±2 |
12±2 |
14±2 |
28±0 |
30±2 |
AP21 |
1.531±0.026 |
35.714±3.571 |
64.286±7.143 |
9±1 |
11±1 |
16±2 |
21±1 |
26±2 |
All values are mean±SD, *ANOVA revealed that there is a high significant difference found in SI along the incubation period (p<0.001), **ANOVA revealed that there is a significant difference found in HR% of all isolates along the incubation period (p<0.05), Sample size, n=3
Fig. 1: Alkali-tolerant isolates showing zone of clearances at pH 8.0 at 37 °C
Quantitative analysis of alkaline protease
Table 4 revealed that from 11 isolates maximum zone of clearance at 10 pH was shown by 3 isolates (SI = 30±2) by AP2, AP8 and AP13, while the minimum by AP7 (SI = 16±2) after 5 d of incubation.
Nevertheless, the quantification experiment result was somehow different from the above qualitative test, which is depicted in table 4. The maximum HR% was seen by isolate AP3, i.e. 65.385±15.3846% while the minimum by the isolate AP2 (16.667±3.704%). From qualitative experiment, the potent producers were AP2, AP8 and AP13 while by quantitative experiment, the potent producer was AP3. There is no report on the hydrolytic run of any isolates as it is a new methodology that gives information regarding the "isolates" for vertical hydrolysis of substrates.
Effects of temperature for alkaline protease
All the isolates were screened for protease by taking skim milk agar maintaining at different incubation temperatures (table 5 and 6). Among all the isolates about 18.18% were competent to produce alkaline protease at 57 °C at pH 10, whereas 81.82% could capable of showing zone of clearances at 17 °C while incubating for 8 d. If maximum solubilisation index was considered about 45.46% isolates at 27 °C, 45.46% at 37 °C and 9.09% at 47 °C showed more than 20 SI. Isolate AP8 showed maximum solubilisation index of 39±1 at pH 10 while incubating at 47 °C.
Table 6 revealed the hydrolytic run percentage of the isolates at different temperatures. Maximum HR% was found by AP1 while incubating at 27 °C (65.517±3.846%) and AP3 at 37 °C (65.385±7.143%) and at 47 °C, AP5 showed the maximum (53.846±0%).
Partial characterization of alkaline protease producers
All the protease producing isolates again characterized partially to know the behavior and identification to their genus level. The biochemical results were depicted in table 7. Among all the isolates only 9.1% were positive for motility, 54.6% for citrate utilization, 90.9% for catalase test whereas about 18.2% were negative for esculin hydrolysis and nitrate reduction test. All isolates except one (9.1%) were positive for anaerobic growth. Table 8 represents about 20 sugar tests, where all the isolates were negative for lactose, 18.18% were positive for inulin and sorbitol. The micro-and macromorphological characteristics were given in table 9. All were bacilli except AP7, which is a Gram-positive coccus. Identification was done by putting appropriate results in the matrix available in PIBWin software, where only the identification is probabilistic as denoted in scores in the parenthesis (table 9). The isolated alkali-tolerant bacteria belonged to the genera of Bacillus, Virgibacillus and Micrococcus. There are many reports of getting alkaline proteases from bacterial origins like Bacillus cereus [23-24], Bacillus smithii [25], Virgibacillus [26] and Micrococcus luteus [27].
Table 5: Effect of incubation temperature for alkaline protease production by isolates
Isolates code |
Solubilization indexes at varied incubation temperature ( °C) at pH 10 |
||||
17 (8 d) |
27 (4 d) |
37 (4 d) |
47 (4 d) |
57 (4 d) |
|
AP1 |
0 |
13±1 |
7±3 |
16±2 |
0 |
AP2 |
2±0 |
25±3 |
12±2 |
1±0 |
0 |
AP3 |
0 |
14±2 |
10±2 |
16±2 |
0 |
AP5 |
6±2 |
12±2 |
15±1 |
9±1 |
7±1 |
AP7 |
2±0 |
17±1 |
17±1 |
9±1 |
0 |
AP8 |
13±1 |
20±2 |
22±2 |
39±1 |
11±3 |
AP9 |
9±1 |
23±1 |
22±2 |
12±2 |
0 |
AP10 |
9±1 |
19±1 |
23±1 |
11±1 |
0 |
AP12 |
11±1 |
18±0 |
13±1 |
7±1 |
0 |
AP13 |
3±1 |
24±0 |
30±2 |
10±2 |
0 |
AP21 |
2±0 |
21±1 |
26±2 |
6±0 |
0 |
All values are mean±SD; ANOVA revealed high significant differences between the SI of isolates at varied temperatures (p<0.001), Sample size, n=3
Table 6: Effect of incubation temperature on HR% at pH 10 by isolates
Isolates code |
Hydrolytic Run (%) at different temperatures for varied incubation period (h) |
||||||||
47 °C |
37 °C |
27 °C |
|||||||
24 |
48 |
72 |
24 |
48 |
72 |
24 |
48 |
72 |
|
AP1 |
0 |
20±3.846 |
28±7.143 |
0 |
42.308±0 |
61.538±3.704 |
0 |
34.483±4.167 |
65.517±3.846 |
AP2 |
0 |
0 |
0 |
0 |
12.5±7.143 |
16.667±7.143 |
0 |
24.138±0 |
37.931±0 |
AP3 |
19.231±0 |
30.769±3.846 |
34.615±3.846 |
0 |
34.615±3.846 |
65.385±7.143 |
0 |
37.931±3.846 |
58.621±3.846 |
AP5 |
15.385±0 |
26.923±7.143 |
53.846±0 |
0 |
34.615±4.167 |
50±3.846 |
0 |
33.333±0 |
43.333±3.704 |
AP7 |
4±0 |
28±3.846 |
48±0 |
0 |
17.857±4.167 |
28.571±4.167 |
0 |
21.429±3.846 |
35.714±3.846 |
AP8 |
0 |
0 |
52±3.846 |
0 |
37.037±0 |
48.148±3.571 |
0 |
27.586±7.143 |
31.034±4.167 |
AP9 |
0 |
26.923±7.143 |
42.308±4.167 |
0 |
32±7.143 |
60±0 |
0 |
32.143±0 |
50±0 |
AP10 |
0 |
0 |
0 |
0 |
15.385±3.704 |
34.615±3.704 |
0 |
25.926±7.143 |
40.741±0 |
AP12 |
0 |
0 |
0 |
0 |
21.429±3.846 |
32.143±3.846 |
0 |
29.63±3.846 |
40.741±7.143 |
AP13 |
0 |
0 |
0 |
0 |
39.286±7.143 |
53.571±3.846 |
0 |
17.857±0 |
42.857±0 |
AP21 |
0 |
0 |
0 |
0 |
35.714±4.167 |
64.286±3.704 |
0 |
25.926±3.846 |
40.741±7.143 |
All values are mean±SD; ANOVA revealed high significant differences between the HR% of isolates at different temperatures for varied incubation period (p<0.001), Sample size, n=3
Table 7: Biochemical characterization of alkaline protease producing isolates
Isolates code |
Mo |
MR |
VP |
In |
Ci |
Ca |
Ox |
Es |
Ur |
PS |
NR |
AN |
AP1 |
- |
- |
+ |
- |
- |
+ |
+ |
+ |
+ |
- |
+ |
+ |
AP2 |
- |
- |
- |
- |
- |
+ |
- |
- |
+ |
- |
- |
+ |
AP3 |
+ |
- |
+ |
- |
- |
+ |
+ |
+ |
+ |
- |
+ |
+ |
AP5 |
- |
+ |
- |
- |
+ |
+ |
+ |
+ |
+ |
- |
+ |
+ |
AP7 |
- |
- |
- |
- |
- |
+ |
+ |
- |
+ |
- |
- |
+ |
AP8 |
- |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
+ |
AP9 |
- |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
+ |
AP10 |
- |
- |
+ |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
+ |
AP12 |
- |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
+ |
AP13 |
- |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
+ |
AP21 |
- |
- |
- |
- |
- |
- |
+ |
+ |
- |
- |
+ |
- |
Mo: Motility test; MR: Methyl red test; VP: Voges-Proskauer test; In: Indole production; Ci: Citrate Utilization; Ca: Catalase; Oxi: Oxidase; Es: Esculin Hydrolysis; Ur: Urease test; PS: Phosphate solubilization; NR: Nitrate reductase; AR: Anaerobic growth; (+): Positive; (-): Negative.
Table 8: Sugar utilization tests of alkaline protease producing isolates
Isolates code |
Sugars investigated |
|||||||||||||||||||
Mn |
Ce |
Ma |
Gl |
Su |
Me |
La |
Fr |
Rh |
Is |
Ga |
Xy |
Mo |
Ar |
Du |
De |
Tre |
In |
So |
Sa |
|
AP1 |
+ |
+ |
- |
+ |
+ |
- |
- |
- |
- |
+ |
- |
- |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
AP2 |
+ |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
- |
- |
- |
- |
- |
+ |
AP3 |
+ |
+ |
- |
+ |
+ |
- |
- |
- |
- |
- |
+ |
- |
+ |
- |
+ |
+ |
+ |
+ |
+ |
+ |
AP5 |
+ |
+ |
+ |
+ |
+ |
- |
- |
- |
- |
+ |
+ |
- |
+ |
- |
+ |
+ |
+ |
- |
- |
+ |
AP7 |
- |
- |
- |
- |
- |
+ |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
- |
- |
- |
- |
- |
- |
AP8 |
+ |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
- |
- |
- |
- |
- |
- |
- |
AP9 |
- |
+ |
+ |
+ |
- |
+ |
- |
+ |
- |
- |
+ |
- |
- |
+ |
+ |
- |
- |
- |
- |
- |
AP10 |
- |
+ |
- |
+ |
- |
- |
- |
+ |
- |
- |
- |
+ |
- |
+ |
+ |
+ |
- |
- |
- |
- |
AP12 |
+ |
- |
+ |
- |
+ |
+ |
- |
+ |
- |
+ |
- |
- |
+ |
+ |
- |
+ |
- |
- |
- |
- |
AP13 |
+ |
- |
- |
- |
- |
+ |
- |
+ |
- |
- |
- |
- |
- |
+ |
- |
- |
- |
- |
- |
+ |
AP21 |
- |
- |
- |
- |
- |
- |
- |
+ |
- |
- |
+ |
+ |
- |
+ |
- |
+ |
- |
- |
+ |
- |
Mn: Mannitol; Ce: Cellobiose; Ma: Maltose; Gl: Glucose; Su: Sucrose; Me: Melibiose; La: Lactose; Fr: Fructose; Rh: Rhamnose; Is: Inositol; Ga: Galactose; Xy: Xylose; Mo: Mannose; Ar: Arabinose; Du: Dulcitol; De: Dextrose; Tre: Trehalose; In: Inulin; So: Sorbitol; Sa: Salicin; (+): Positive; (-): Negative.
Table 9: Micro-and macro-morphological study and probabilistic identification of isolates
Isolates code |
Micro-morphological characterization |
Macro-morphological characterization on NA at pH 8 |
Bacteria identified (PIBWin Score) |
AP1 |
Gram+ve, small rods, central spore |
Big colony, irregular margin, creamy, flat surface, opaque, dry and hyper growth at pH 8.0 within one-day |
Bacillus cereus (0.99993) |
AP2 |
Gram+ve, small rods, short chain, central spore |
Big, round, creamy, convex, raised, entire, opaque and aggregation at middle |
Bacillus cereus (0.99732) |
AP3 |
Gram+ve, tiny rods to coccobacilli |
Big, irregular, rhizoidal, creamy to white, flat, opaque, sticky, hyper growth at pH 8.0 within one day and somehow same as AP1 |
Bacillus cereus (0.89744) |
AP5 |
Gram+ve, thin rods |
Medium, round, regular, creamy, opaque, grooved at middle and raised at the border. |
Bacillus cereus (0.89336) |
AP7 |
Gram+ve, cocci |
Tiny, round, regular, lemon yellow, convex, and opaque |
Micrococcus luteus (0.90346) |
AP8 |
Gram+ve, small rods, streptobacilli, central spore |
Big, round to oval, irregular, creamy, flat, opaque and wheel like colony |
Virgibacillus (0.81499) |
AP9 |
Gram+ve, tiny, coccobacilli |
Big, round to oval, flat, regular, creamy, smooth, opaque and lambda like marks on colony |
Bacillus carotarum (0.95797) |
AP10 |
Gram+ve, tiny, coccobacilli, in short chain |
Medium, round, regular, yellowish white, convex, opaque and circular rings on colony |
Bacillus carotarum (0.91842) |
AP12 |
Gram+ve, tiny, coccobacilli |
Medium, round, irregular, creamy, rough, flat, opaque and some clumps at middle of colony |
Bacillus smithii (0.71708) |
AP13 |
Gram+ve, long rods, short chain, central spore |
Medium to big, round, regular, creamy, raised, flat to convex and not sticky as AP21 |
Bacillus cereus (0.99826) |
AP21 |
Gram+ve, long chain, streptobacilli, central spore |
By some means same as AP13, but slightly change in furrow patterns, and sticky, c reamy, round, regular, raised, distinct furrows like triangular shape and opaque |
Virgibacillus (0.99425) |
Physiological characterization of isolates
For physiological tests, isolates were investigated for its tolerance to various pH and salt concentrations, which are depicted in fig. 2 and fig. 3, respectively. All the bacterial isolates investigated were alkali tolerant having pH optimum near to alkaline. However, among the isolates AP5, AP7 and AP21 have optimum growth at alkaline pH.
Johnvesly and Naik reported an alkaline and thermostable protease from Bacillus sp. JB-99, which showed maximum production at pH 9 and the purified product, was stable at pH 9 and more active in 11 pH [28]. The similar findings were observed while working on alkaline and thermostable protease by Bacillus stearothermophilus F1 [29]. Virgibacillus pantothenticus reported an extracellular thermostable serine protease having pH optima at 10 and temperature at 50 °C [26]. Similarly, there was a report about protease from a halophilic Virgibacillus sp. EMB13 [30]. Production of alkaline protease by Bacillus cereus was reported in new industrial culture mediums, which had pH optima at 7 and temperature for optimum production was at 60 °C [23]. Protease production capabilities of Micrococcus luteus and Bacillus sp. isolated from abattoir environment were reported [27]. They reported the optimum temperature and pH for Micrococcus luteus was 37 °C and pH 7 while in case of Bacillus sp. it was 47 °C and 9 pH. Whatever it may be the above bacterial spp. showed the maximum alkaline protease production at near to 7 while could tolerate 12 pH.
From the salt tolerance test, it was observed that all the isolates needed additional salts for their optimal growth, which was confirmed by analyzing the control lacking of extra salts from growth medium (fig. 3). All isolates have optimum growth at 2% (w/v) NaCl except AP5, which in turn has been optimum at 4%. However, all showed growth at 12% NaCl, which confirms them as halo-tolerants. It is apparent because all the isolates were from marine sources.
Fig. 2: Effect of initial pH on growth of isolates, and values are mean±SD for three distinctive experiments, n=3, ANOVA test revealed that there is a high significant difference in growth among the isolates along varied pH of the medium (p<0.001)
Fig. 3: Effect of salt concentrations (% w/v) on growth of isolates, and values are mean±SD for three distinctive experiments, n=3, ANOVA test revealed that there is a high significant difference in growth among the isolates along varied concentrations of NaCl (p<0.001)
CONCLUSION
From the above study, it can be concluded that marine sources are the reservoir of various alkali-tolerant bacteria, which are the good sources of alkaline protease. In our study, isolates found from marine sources were mostly Gram positives. The isolates were not only active in alkaline pH but also can tolerate 12% NaCl, which makes them as halo-alkali tolerant bacteria. The alkaline proteases produced from the above isolates have immense applications in various fields of sciences and industries. The new methodology adapted here for quantification of alkaline protease can strengthen the results found from qualitative and shows the real potentiality of the isolates.
ACKNOWLEDGEMENT
Authors would like to thank the DST, Govt. of Odisha for the financial support in the project having F. No. ST-11-(SC)-34/2014-2371/ST and also grateful to HOD, Department of Microbiology, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India for providing laboratory support.
CONFLICTS OF INTERESTS
All authors have none to declare
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