Effect of incubation temperature on production of amylase in various agroindustrial wastes by alkaliphilic actinobacteria

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1 Bioscience Discovery, 8(3): , July RUT Printer and Publisher Print & Online, Open Access, Research Journal Available on ISSN: (Print); ISSN: X (Online) Research Article Effect of incubation temperature on production of amylase in various agroindustrial wastes by alkaliphilic actinobacteria Vidhale NN, Pundkar VM Shri Shivaji Science College, Amravati, (MS), India vmpundkar@gmail.com Article Info Received: , Revised: , Accepted: Keywords: Amylase, Actinomycetes, Saline soil, Agroindustrial wastes. Abstract Annually 960 million tonnes of wastes are being produced globally and ~350 million tonnes of biomass waste is generated every year from agriculture. These waste if not utilized properly lead to environmental impact including global warming. Hence in present study attempt was made to utilize agroindustrial waste like wheat bran, rice bran, dal mill waste, oil mill waste, vegetable waste, molasses for the production of amylase, an useful enzyme in various industries, by alkaliphilic actinobacteria using solid state fermentation. It has been reported from the study that oil mill waste, rice bran, vegetable waste, wheat bran, dal mill waste and molasses produced remarkable amylase activity at 37 C to 40 C incubation temperature after 120 hrs of incubation period containing 25% moisture. Maximum amylase activity was recorded in oil mill waste (4.25 mg/ml) followed by rice bran (4.00 mg/ml), vegetable waste (3.95 mg/ml), wheat bran (3.00 mg/ml), dal mill waste (2.25 mg/ml) and molasses (1.15 mg/ml) at 37 to 40 C of incubation temperature after 120 hrs of incubation period. INTRODUCTION Globally 960 million tonnes of waste are being produced out of them ~350 million tonnes of biomass waste is generated every year from agriculture. This agricultural waste is a rich source of carbohydrate proteins and if utilized for production of industrially valuable products will minimize environmental and agricultural pollutions (Asokan et al., 2007). Hence the study was undertaken for utilizing agricultural waste like Wheat bran, Rice bran, Oil mill waste, dal mill waste, vegetable waste, Molasses to produce industrially valuable biomolecule the enzyme amylase by Solid State fermentation. Several enzymes hydrolyzed starch. Among those enzymes extracellular enzymes such as - amylase (Endo-1, 4-glucan gluecohydrolase) cleaves 1, 4, - - D glucosidic linkage present in straight linear chain of polysaccharides (Poornima et al., 2005) Fermentation methods such as solid state fermentation shows high volumetric productivity, followed by increase in concentration of products, very less effluent generated and very handful fermentation equipment (Renege et al., 2012). Wheat bran, Rice bran, Oil mill waste, Dal mill waste, Vegetable waste and molasses, waste were used in this study as solid substrate (Nadagouda et al., 2015). Surprisingly saline soil of vidarbha region of Maharashtra in India, having high ph above 8 predominate alkaliphilic actinomycetes (Singh et al., 2010). These actinomycetes have great potential of producing amylase and hence were undertaken for production of amylase from agricultural waste (Ruhi et al., 2014) ISSN: X (Online)

2 MATERIALS METHODS Sample collection from saline sites Soil samples were collected in new sterile zip locked bags using sterile spatula from different saline sites of Vidarbha region of Maharashtra State (Vidhale and Pundkar, 2016). Isolation of alkaliphilic actinomycetes: Actinomycetes isolation agar (Himedia M490) supplemented with glycerol (5ml/L) and nystatin (50 g/ml) as antifungal antibiotics was employed for isolation of actinobacteria. Screening of amylolytic alkaliphilic actinomycetes strain: Production of amylase was detected by flooding plate with grams iodine solution. Efficiency of starch hydrolysis was determined on the basis of diameter of zone of decolouration against the blue colour background (Selvam et al., 2011). Inoculums and Fermentation medium: Agroindustrial waste was grinded to size about 2mm in homogenizer and then sieved through mesh screens to obtain a particle having diameter between 0.4 to 0.85mm. In solid state fermentation 5gm of each agroindustrial waste was weighed and hydrated with 5ml of basal salt solution and adjusted with moisture content from 25%. Initially the 5 gm each agroindustrial waste was hydrated with basalt salt solution and after sterilization in autoclave was inoculated with 7 days old inoculum culture of actinomycetes in conical flask and incubated at different incubation temperature (25 to 50 0 C) for 7 days. Enzymes Assay: Enzyme was assayed by DNS-method (Bernfeld 1955). Amylase activity was measured by Spectrophotometric assay. (Suganthi et al., 2011), (George et al., 1945). RESULTS AND DISCUSSION Alkaliphilic actinomycetes were isolated from soil samples collected from different saline sites. Total 64 strains of alkaliphilic actinomycetes were isolated and identified. Among those 64 isolates, the strain showing maximum zone of clearance against dark blue background (starch agar plate assay) was chosen for production of amylase. The amylase activity shown by alkaliphilic actinomycetes strain at different incubation temperature is shown in table 1 and figures 1 to 6. Vidhale and Pundkar Bioscience Discovery, 8(3): , July In the present investigation it has been noted that in general, incubation temperature 37 C and 40 C the optimum temperature for amylase production from agroindustrial waste containing 25% moisture further was maximum amylase activity was recorded in oil mill waste (4.25 mg/ml) followed by rice bran (4.00 mg/ml), vegetable waste (3.95 mg/ml),wheat bran (3.00 mg/ml), dal mill waste (2.25 mg/ml) and molasses (1.15 mg/ml) at 37 to 40 C incubation temperature after 120 days of incubation period. More or less similar amount of amylase was also recorded after 96hrs of incubation at same incubation temperature. Vijayan et al., (2015) reported optimum temperature 37 C for amylase activity from agroindustrial waste by bacteria isolated from cassava waste (Tamil Nadu, India). Tambekar et al., (2012) studied the physiological and cultural diversity of bacilli characterized from Lonar lake (M.S) India. Alkaliphilic bacilli produce extracellular protease, amylase, and lipase enzyme might have potential value for several biotechnological industries. Similarly, again Tambekar et al., (2017) isolated Bacillus aerophillus from hyper saline environment which produce enzyme at optimum temperature 80 C. Nadagouda et al., (2016) used rice bran as cheap and easily available agricultural waste material which was treated with Tricoderma viride GSG12 under solid state fermentation technique. The findings was at optimum incubation temperature of 32 C influenced the cellulose production. Hence the agroindustrial waste like oil mill waste, rice bran, vegetable waste, wheat bran and dal mill waste can be a good source of amylase if utilized by alkaliphilic actinomycetes under solid state fermentation, which can minimized environmental impact and serve the purpose of waste management. REFERENCES: Asokan P, Saxena M, Asolekar SR, Solid waste generation in India and building materials. Building and Environment, 42(6): Bernfeld P, Amylases α and β. Meth. Enzymol. 1: George L. Peltier and Beckord LD, Sources of amylase-producing Bacteria. J. Bacteriology, 50(6): Karanwal Akansha and Varsha Nigam, Production of Amylase enzyme by isolated microorganisms and its application. Int. J. Pharm. and Bio. Sci, 3(4): ISSN: (Print)

3 Table 1: Effect of incubation temperature on the production of amylase in various agroindustrial wastes by alkaliphilic actinobacteria. Types of waste Temperature Production of amylase (mg/ml) at different incubation period in hours Mean ± SD Value 1.Wheat bran ± ± ± ± ± ± Rice bran ± ± ± ± ± ± Dal mill waste ± ± ± ± ± ± Oil mill waste ± ± ± ± ± ± Vegetable ±0.30 waste ± ± ± ± ± Molasses ± ± ± ± ± ±0.23 Vidhale and Pundkar ISSN: X (Online)

4 Fig 1: Production of amylase by alkaliphilic actinobacteria at different incubation temperature using 25% moisture content in wheat bran by SSF. Fig 2: Production of amylase by alkaliphilic actinobacteria at different incubation temperature using 25% moisture content in Rice bran by SSF. Bioscience Discovery, 8(3): , July ISSN: (Print)

5 Fig 3: Production of amylase by alkaliphilic actinobacteria at different incubation temperature using 25% moisture content in Dal mill waste by SSF. Fig 4: Production of amylase by alkaliphilic actinobacteria at different incubation temperature using 25% moisture content in oil mill waste by SSF. Vidhale and Pundkar ISSN: X (Online)

6 Fig 5: Production of amylase by alkaliphilic actinobacteria at different incubation temperature using 25% moisture content in Vegetable waste by SSF. Fig 6: Production of amylase by alkaliphilic actinobacteria at different incubation temperature using 25% moisture content in Molasses by SSF ISSN: (Print)

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