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)
7 Nadagouda MG, Lingappa K, Malipatil SG and Bheemareddy VS, Screening, characterization and biosynthesis of cellulase from Trichoderma viride on rice bran under solid state fermentation. Science Research Reporter, 5(2): Nadagouda MG, Lingappa K, Bheemareddy VS and Malipatil SG, Optimization of solid fermentation conditions for the production of cellulase by using Trichoderma viride GSG12. Bioscience Discovery, 7(1): Oyeleke SB, Egwi EC, Oyewole, OA, John EE, Production of cellulase and protease from microorganisms isolated from gut of Archachatina marginata (giant African Snail) Science and Technology, 2(1): Poornima R, Kumar SM, Sivakumar K and Pushjpavalli V, Optimization of α-amylase Production by Actinomycete strain AE-19 isolated from shrimp pond. Trend Appl. Sci. Res, 3(1): Renge VC, Khedkar SV and Nikita R. Nandurkar, Enzyme synthesis by fermentation method. A review. Sci. revs. Chem.. commun, 2(4): Ruhi R, Kamble LH, Kadam AS, A report on extracellular enzyme production potential of actinomycetes isolated from sediments of river Godavari, India. Bioscience Discovery, 5(2): Singh SP, Thakur JT, Gohel SD, and Purohit MK, Molecular diversity and enzymatic Bioscience Discovery, 8(3): , July potential of salt-tolerant alkaliphilic actinomycetes. Formatex.info; microbiology 2 : Selvam KB, Vishnupriya and Bose VS, Screening and Quantification marine actinomycetes producing industrial enzymes Amylase, Cellulose and Lipase from south coast of India.. Int. J. Pharmaceutical and biological archives, 2(5): Suganthi R, Benazir JF, Santhi R, Ramesh KV, Anjana H, Nitya M, Nidhiya KA, Kavitha G and Lakshimi R, Amylase Production by Aspergillus niger under solid state fermentation using Agroindustrial wastes. Int. J. Eng. Sci. Technol, 3: Tambekar DH and Dhudale VR, Studies on physiological and cultural diversity of Bacilli characterized from Lonar lake (MS) India. Bioscience Discovery, 3(1): Tambekar DH, Tambeker SD, Jadhav AS amd Bhule PB, Extracellular thermoalkalophilic protiolytic activities of the Bacillus aerophilus : Detection and Priliminary characterization. Bioscience Discovery, 8(2): Vidhale NN and Pundkar VM, Exploration of alkaliphilic actinomycetes for amylase production from agroindustrial waste. Int. J. of life sciences, 4(3); Vijyan M, Jothinathan MK, Murugesan SK, Rangasamy N, SankaranG, Sathasivam V, Prasanth M, Shanthini T, Microbial production of amylase from Cassava waste. Research in Pharmacy, 5: How to Cite this Article: Vidhale NN, Pundkar VM, Effect of incubation temperature on production of amylase in various agroindustrial wastes by alkaliphilic actinobacteria. Bioscience Discovery, 8(3): ISSN: X (Online)
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