Optimization of amylase production from Bacillus subtilis and Pseudomonas aeruginosa using sugarcane bagasses by submerged fermentation

Murugaiah K, Kesavan R., Dhanaraj T.S.*

PG and Research Department of Biochemistry, Enathi Rajappaa College, Enathi, Pattukkottai, Thanjavur Dt., Tamilnadu, India - 614 615

ABSTRACT:

Enzymes are considered as nature’s catalysis most enzymes today (and in future) produced from the microorganisms. In the present study, Bacillus subtilis and Pseudomonas aeruginosa were isolated from the soil samples. The isolated organisms were identified using standard microbiological methods. The inoculums were prepared with waste substrate (sugarcane bagasse). The isolated bacterial strain were inoculated in the medium and incubated at 30˚C for 3 days. The high-level amylase production was observed in Bacillus subtilis inoculated medium so this strain used for the optimization studies. The amylase activity was optimized in various physical parameters such as temperature, pH and chemical parameters like nitrogen and carbon sources. The maximum production of amylase was recorded at 35˚ C and pH 7.0. The amylase production by Bacillus subtilis was recorded maximum in starch-supplemented medium when compared to other carbon sources (maltose and glucose). Among the various nitrogen sources (Ammonium sulphate, ammonium chloride and ammonium nitrate) used, Ammonium sulphate supplemented medium showed the significant results in the production of amylase by Bacillus subtilis.

KEYWORDS:Amylase,sugarcane bagasse, Bacillus subtilis, Pseudomonas aeruginosa and Submerged Fermentation

INTRODUCTION:

Amylases are starch degrading enzymes. They are widely distributed in microbial, plant and animal kingdoms. They degrade starch and related polymers to yield products characteristic of individual amylolytic enzymes. Initially the term amylase was used originally to designate enzymes capable of hydrolyzing a-1,4- glucosidic bonds of amylose, amylopectin, glycogen and their degradation products ^{[5, 7]}. Amylases are important enzymes used in the food industry for hydrolysis of starch to yield glucose syrups amylase –rich flour and proper formation of dextrin during baking. Also they are employed in textile industry for removal of starch sizing. In the production of detergents, amylases are used to dissolve starches from fabrics ^[6]. Amylases constitute a class of industrial enzymes, which alone form approximately 25% of the enzyme market covering many industrial processes such as sugar, textile, paper, brewing, distilling industries and pharmaceuticals^{[11, 12, 13]}. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium commonly found in soil ^[10]. The microbial source of amylase is preferred to other sources because of its plasticity and vast availability. Microbial amylase has almost surpassed the synthetic sources in different industries ^[13]. Nowadays amylase from these sources is vastly used in amylase production under extreme conditions of pH and temperature There are various reports on starch degrading microorganisms from different sources and respective amylase activity ^[2].

Amylases are enzymes, which hydrolyze starch molecules to give diverse products including dextrins, and progressively smaller polymers composed of glucose units^[14].

MATERIALS AND METHODS:

Sample Collection and Isolation of Bacteria

The soil sample was collected from bagasse decomposed area at Thanjavur. The bacteria isolate from the collected soil sample by serial dilution techniques. From the 10^-5 and 10^-7 dilution 0.1 ml of sample taken and spreaded over the nutrient agar medium and the plates were incubated at 37°C for 24 hrs.

Screening of amylase production

The amylase productivity organism was screened by using starch agar. Starch agar medium was prepared and sterilized and poured into sterile petriplates and allowed to solidify. Then test organism was streaked on cooled starch agar medium. After the inoculation the plates were incubated at 37°C for 24 hours. After the incubation period few drops of iodine solution was poured on the medium and observed the clear zone formation.

Identification of amylase producing bacteria

In the screening, test positive results noted in two strains the strains were identified based on the cultural, morphological and biochemical characteristics. The morphological and biochemical tests were done by the following methods as described by Aneja ^[3] in Experiments in Microbiology, Plant pathology and Biotechnology, to identify bacteria.

Fermentation medium and condition

Sugarcane bagasse - 1.5 g; yeast extract - 100 mg; KH₂PO₄- 100 mg; Na₃ , citrate - 250 mg_;MnSo₄, H₂O - 50 mg; Mg SO₄, 7 H₂O - 250 mg; CaCl₂ - 50 mg; Distilled water - 100 ml. After sterilization the flasks were cooled, inoculated with 5% inoculums (Bacillus subtilis and Pseudomonas aeruginosa) and placed on a rotary shaker (250 rpm) at 30˚C.

Crude enzyme preparation and enzyme assay

To obtain crude enzyme 48 hr old cultures were transferred to micro centrifuge tubes and centrifuged at 4000 rpm for 15min. Cells were discarded and the supernatant was used as the enzyme for various assays. Amylase assay was done by using a reaction mixture consisting of 1 ml substrate solution (1.1% soluble starch in 50mM phosphate buffer pH 7.2) and 100 ml of enzyme solution, reaction was stopped by adding 2 ml of dinitrosalicylic acid (DNSA) and cooled for 10min. Optical density of each sample was taken at 540 nm using spectrophotometer (Shimadzu, Japan).

Enzyme activity was expressed in units (1 units/ml = amount of enzyme which releases 1 μ mole glucose under the assay condition).

Optimization for amylase enzyme production

In order to increase the amount of amylase production using various Temperature, pH, nitrogen and carbon sources were added. The fermentation medium was prepared and autoclaved. The flasks were cooled and inoculated with Bacillus subtilis.

Statistical Analysis

The results obtained in the present investigation were subject to statistical analysis like Mean and Standard Deviation (SD) by Zar ^[15].

RESULTS AND DISCUSSION:

The isolation and identification of amylase producing bacterial strains in the starch casein plates were carried out. More number of colonies was observed after incubation period in the plates. In this study starch agar medium was prepared and streak isolated organisms and incubate at 37 °C for 24hrs. In this observation Bacillus subtilis was produced high clear zone around the colonies when compared than Pseudomonas aeruginosa (Plate – I). Asgher et al.,^[4] reported the similar results on time – course studies on α amylase production and cell growth of Bacillus subtilis grown in basal medium supplemented with 1% waste potato as inducers substrate. α - amylase production peaked (44.84 U/ml) at 48 hrs and was found to decline gradually up to 96 hrs (12 - 20 U/ml). It was observed that maximum α -amylase production by Bacillus subtilis occurred when cell production reached the peak. It is also supported by the report on the kinetics of enzyme synthesis was more of the growth associated type. The isolated organisms were identified based on the morphological Cultural and biochemical examinations. The isolated bacterial species compared with Bergey’s manual systemic classification the isolated colonies were identified as Bacillus subtilis and Pseudomonas aeruginosa.

Table-1: Estimation of amylase using waste substrate

S.No

Test organisms

Amylase productivity

(Unit/min/ml)

Bacillus subtilis

Pseudomonas aeruginosa

0.83 ± 0.02

0.64 ± 0.01

Table-2: Amylase productivity of Bacillus subtilis in different pH and Temperature

S.No	Amylase productivity (Unit/min/ml)
S.No	pH		Temp (°C )
1 2 3 4 5	5 6 7 8 9	0.62 ± 0.01 1.04 ± 0.02 1.34 ± 0.02 1.23 ± 0.01 0.94 ± 0.02	25 30 35 40 45	0.85 ± 0.01 0.93 ± 0.01 1.14 ± 0.02 0.74 ± 0.02 0.54 ± 0.02

Table- 3: Amylase production by Bacillus subtilis in different sources

S No.	Microorganism	Enzyme activity (U/min/mg)
		Carbon sources		Nitrogen sources
1.	Bacillus subtilis	Starch	1.23 ± 0.04	Ammonium sulphate	1.12 ± 0.03
		Maltose	0.95 ± 0.03	Ammonium chloride	1.21 ± 0.30
		Glucose	0.83 ± 0.02	Ammonium nitrate	1.05 ± 0.01

Plate – I: Screening of amylase production

The identified organism is able to produce amylase enzyme. The amylase enzyme produced by Bacillus subtilis and Pseudomonas aeruginosa were accessed using waste substrates (bagasse). The results are presented in Table - 1. From the results, we came to know that Bacillus subtilis was most efficient organism to produce maximum amount of amylase from bagasse. Adinarayana Kunammeni et al.,^[1] reported that the maximum amylase production (281U/g) was obtained at a pH of 6.0. The inoculums level was also an important factor for the production of amylase. High inoculums levels are inhibitory in nature. The highest enzyme production (267 U/g) was obtained at an inoculums level of 10% (v/w).

Effect of Physical Factors on Amylase Production

Amylase production was also analyzed using various pHsuch as 5, 6, 7, 8 and 9. The results are presented in the Table - 2. The maximum amylase production was recorded in the pH 7.0 (1.34 ± 0.02 U/mg/min) when compared with other pH. Adinarayana Kunammeni et al.,^[1] reported that the maximum amylase production (281U/g) was obtained at a pH of 6.0. The inoculums level was also an important factor for the production of amylase.

Amylase production was analyzed at different temperatures such as 25, 30, 35, 40 and 45. Among these temperatures, the maximum amylase production was recorded at 35˚C (1.14 ± 0.02 u/mg/min) when compared with other temperatures. The results are presented in the Table - 2. Bacillus sp. was capable of producing amylase in the range of 25– 45°C with maximum production at 35°C (1.14). The related results were observant by Asgher et al.,^[4]. Very recently ^[9]reported that a thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced maximum extra cellular thermo stable α-amylase at 40°c in a medium containing low starch concentration.

Effect of Nutritional Factors on Amylase Production

Amylase production was analyzed using various carbon sources such as starch, maltose and Glucose. The experiment results are presented in Table - 3. Among the various carbon sources used in the present study, the amylase production was recorded maximum in starch-incorporated medium (1.23 ± 0.04 U/mg/min) when compared with all other carbon sources. Optimization of growth condition is a prime steps in using microorganisms in fermentation technology ^[8]. In the present study, we observed 30˚C as the optimum growth temperature for the production of amylase. Amylase production was analyzed using various nitrogen sources such as Ammonium sulphate, Ammonium chloride and Ammonium nitrate. The results are presented in Table - 3. Among the various nitrogen sources used in the present study, the amylase production was recorded maximum in Ammonium chloride incorporated medium (1.21 ± 0.30 U/mg/min) when compared with other nitrogen sources.

CONCLUSION:

Hence, the Bacillus subtilis can be recommended for the commercial production of amylase in sugar cane bagasse substrate. Further experiments will be done to purify the secreted amylase and stability studies will be performed to enhance the application of enzyme to commercial level

ACKNOWLEDGEMENT:

The authors are thankful to Muthaiyah Research Foundation, Thanjavur for offering facilities to carry out this study.

REFERENCES:

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Received on 03.04.2012

Modified on 18.04.2012

Accepted on 22.04.2012

Research J. Science and Tech. 4(2): March-April 2012: 86-89