ISOLATION OF AMYLOLYTIC BACTERIA FROM A BIOFERTILIZER GENERATED FROM MUNICIPAL SOLID WASTE

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1 NSave Nature to Survive 7 (3&4): , 2013 ISOLATION OF AMYLOLYTIC BACTERIA FROM A BIOFERTILIZER GENERATED FROM MUNICIPAL SOLID WASTE AYONA JAYADEV AND S. S. NAVAMI* Department of Environmental Sciences, All Saints College, Thiruvananthapuram, Kerala , INDIA sureshayona@gmail.com ABSTRACT INTRODUCTION Microorganisms produce a vast variety of biochemical molecules which can be used for various purposes by humans. They are one of the most important sources of enzyme production. The advantages of microbial enzymes comprise lower production costs, possibility of large-scale production in industrial fermentors, wide range of physical and chemical characteristics, possibility of genetic manipulation, absence of effects brought about by seasonality, rapid culture development and the use of non-burdensome methods. Currently, microbial enzymes are considered to be increasingly important for sustainable technology and green chemistry (Kiro Mojsov, 2012). The above characteristics make microbial enzymes suitable biocatalysts for various industrial applications (Hasan et al., 2006). Amylase is one of the most important enzymes used in the field of Biotechnology. It is a hydrolytic enzyme and in recent years, interest in its microbial production has increased dramatically due to its wide spread use in food, textile, baking and detergent industries (Verma et al, 2012). Amylase is one of the most important enzymes used in the field of Biotechnology. The advantages of using microorganisms for production of amylases are their ability to produce in bulk and the ease at which it can be manipulated for desired products (Ramesh and Lonsane, 1990). They are used in the starch processing industries for the hydrolysis of polysaccharides such as starch into simple sugar constituents (Omemu et al., 2005). Ikram- Ul-Haq et al., 2009, explained that amylase activity was affected by cultivation methods, substrates (medium) ingredient or toxic accumulation in culture medium. Municipal Solid Waste is one of the most serious problems in the world, currently. One of the efficient methods by which this can be managed is to digest it to produce an end product which can be used as a resource. Several products are reported to be produced from Municipal Solid Waste. One among them is biofertilizer. There are several reports that Municipal Solid Waste can be converted to efficient multi-functional biofertilizer. Biofertilizer generated from Municipal Solid Wastes will be rich in microorganisms with various capacities as these products are the results of microbial action on various substrates like starch, proteins, cellulose etc. Composting is a way of transforming the organic waste into fertilizer, minimizing the use of inorganic compounds that may contaminate the environment. This transformation is the result of the microorganism action, converting complex carbon sources into energy. Enzymes that are produced by the microorganisms to the surrounding environment mediate this process (Pascon et al., 2011). The present study focuses on the screening of biofertilizer generated from Municipal Solid Waste for amylase producing microorganisms. Amylase The present study focuses on the screening of a biofertilizer generated from the degradation of Municipal Solid Waste (MSW) for amylase producing microorganisms. Culturable subset of the microorganisms present in the biofertilizer was enriched after serial dilution. The bacterial isolates were then screened for amylase production in starch agar plates incubating overnight. The plates were flooded with iodine solution to identify amylase producers. Those microbes which showed a halo (clearance zone) on flooding with iodine was selected for further assay. The assay of amylase was done by using starch as the substrate. Amylase activity was measured by quantifying the glucose content in inoculated medium. A total of 18 colonies were enriched from the sample of the biofertilizer. Of these, 11 colonies showed positive amylolytic activity in plate assay. To find the most potent amylase producing microbe, the supernatant of liquid cultures (supplemented with starch) of the selected isolates were tested for the quantity of glucose produced using DNS method. On screening, culture 16 showed highest amylolytic activity (0.37µg/mL glucose produced on amylase activity). KEY WORDS Amylase Municipal Solid Wastes Bio-fertilizer Starch agar, Lugol s iodine solution Received : Revised : Accepted : *Corresponding author 109

2 AYONA JAYADEV AND S. S. NAVAMI producing microbes were then compared to find the comparative efficiency. Finally, the microbes were characterized up to genus level. MATERIALS AND METHODS The sample of the digested Municipal Solid Waste (biofertilizer) was collected from a waste disposal site, Brahmapuram, Cochin, South India. Dried biofertilizer was collected in sterile plastic bags avoiding possibilities of microbial contamination. The samples were brought to the laboratory, refrigerated and were subjected to microbial isolation procedures at the earliest as per standard isolation techniques following the procedures of Aneja, Isolation and Enumeration of Bacteria The bacteria in the biofertilizer were isolated by serial dilution and spread plate method. The microbes enriched were enumerated. The isolated bacterial colonies were subjected to identification of the colony characteristics as per standard chart. Morphologically distinct colonies were selected to further studies after doing gram staining. Detection of Amylase Activity: Starch Agar Plate Assay Method The initial screening of amylase production by bacteria was done by following the procedures of Kaur et al., The isolated colonies were placed on Nutrient Agar medium supplemented with 2% starch. The plates were incubated at 37ºC for 24h. After incubation, plates were flooded with Lugol s iodine solution. Assay of Enzyme Activity For comparing the activity of the isolated microorganisms, the cultures were grown in liquid media (broth) containing starch. After incubation for some time, supernatants of the culture were drawn and it was analyzed to find the concentration of glucose liberated. The concentration of glucose was estimated using DNS method. (Al-Quadan et al., 2009). The absorbance was measured at 560 nm using an UV-VIS spectrophotometer (Systronics, India). Table 1: Morphological characteristic of the bacterial isolates Culture Form Size Surface Texture Color Elevation Margin C1 Circular Medium Rough Dry Opaque Convex Entire C2 Circular Medium Dull Moist Cloudy Raised Entire C3 Rhizoid Large Veined Dry Transparent Flat Curled C4 Irregular Medium Wrinkled Moist Opaque Umbonate Lobate C5 Circular Large Dull Viscous Translucent Raised Entire C6 Oval Medium Dull Moist Cloudy Pulvinate Entire C7 Circular Medium Dull Moist Transparent Raised Entire C8 Rhizoid Medium Wrinkled Dry Cloudy Raised Filiform C9 Oval Punctiform Rough Dry Opaque Pulvinate Entire C10 Irregular Medium Dull Buttery Cloudy Pulvinate Undulate C11 Circular Medium Rough Dry Opaque Convex Entire C12 Circular Punctifom Glistening Viscous Translucent Umbonate Entire C13 Circular Large Rough Moist Cloudy Pulvinate Entire C14 Irregular Medium Dull Dry Opaque Umbonate Undulate C15 Irregular Punctiform Wrinkled Mucoid Translucent Crateriform Lobate C16 Irregular Large Glistening Dry Cloudy Umbonate Entire C17 Irregular Large Rough Dry Cloudy Raised Undulate C18 Ovoidal Medium Rough Dry Cloudy Pulvinate Entire Table 2: Gram staining results of the bacterial isolates Sl. No. Culture Gram (+/-) Shape Forms 1 C1 - ve Minute cocci Clusters 2 C2 - ve Minute cocci Chain 3 C3 + ve Small cocci Chain 4 C4 - ve Cocci Clusters 5 C5 - ve Rod shaped Clusters 6 C6 - ve Small cocci Clusters 7 C7 - ve Small cocci Clusters 8 C8 - ve Rod shaped Clusters 9 C9 + ve Small cocci Chain 10 C10 - ve Minute cocci Clusters 11 C11 + ve Small cocci Cluster 12 C12 - ve Cocci Chain 13 C13 + ve Cocci Cluster 14 C14 - ve Cocci Chain 15 C15 + ve Cocci Ring 16 C16 - ve Rod shaped Clusters 17 C17 + ve Cocci Chain 18 C18 - ve Cocci Cluster Identification of most potent isolate The bacterial isolate which showed the maximum amylase production based on the conversion of starch to glucose was subjected to morphological, cultural and biochemical characteristics as per Bergy s Manuel of Determinative Microbiology. RESULTS AND DISCUSSION Enumeration of Colony Forming Units (CFU) showed that there were 217 x 10-3 CFU/mg of the biofertilizer. Microbes are present everywhere in nature and in biofertilizers derived from Municipal Solid Waste; their number would be very high as the biofertilizer itself is formed by the activity of microbes on wastes. Gram Staining and Morphological Analysis Of the isolated microbes, 18 bacterial colonies were selected on the basis of colony morphology and they were selected for 110

3 ISOLATION OF AMYLOLYTIC BACTERIA Table 3: Activity table of Amylase producing bacteria Trial Volume Volume of Volum of Volume Volume of Volume of OD at 540 Mean OD of enzyme acetate buffer D.H 2 O of DNS NaK tartarate D. H 2 O C1R C1R C3R C3R C4R C4R C7R C7R C8R C8R C11R ml 1.5 ml 1 ml 1 ml 7 ml C11R C12R C12R C13R C13R C14R C14R C15R C15R C16R C16R Incubate in boiling water bath for 5 minutes Cooled the test tube at room temperature Table 4: Concentration of glucose Culture Glucose (µg/ml) C C C C C C C C C C C further studies. The morphological characteristics of the isolated microbial colonies are given in table 1. They were subjected to gram staining and the results are represented as Table 2. Screening for Amylase Activity Amylase activity was detected by the formation of a characteristic clearance zone around the bacterial colonies which was evident when the plates were flooded with 70% Lugol s Iodine solution. It was seen that 11 isolated cultures (C1, C3, C4, C7, C8, C11, C12, C13, C14, C15 and C16) out of the total 18 cultures showed amylolytic activity. Of all these cultures, the culture C 16 showed the best activity by having the largest clearance area. The result of the plate assay for amylase activity expressed as size of clearance zone is shown in Fig. 2. All types of life produce amylase which is a starch degrading enzyme. Of these the most potent are the microbes. Bacterial amylases are having selective advantage over amylases from other sources because of some unique characteristics they possess (Tanyildizi et al., 2005; Gupta et al., 2003; Vihinen and Mantasala 1989 and Pandey et al., 2000). The bacterial 111 isolates producing extracellular amylase will use the starch in the starch-agar medium and will convert it into component glucose molecules (Abe et al., 1988). When iodine solution is added, it gives a violet coloration in areas of presence of starch. In those areas which does not contain starch will remain decolorized. Assay of Enzyme Activity To find the most potent among the cultures showing amylase activity, the cultures were inoculated into a starch containing broth medium and the quantity of glucose formed were analyzed. C 16 was identified as the most potent amylase producer. It produced 37 µg/ml of glucose after inoculation with the culture supernatant. The second maximum activity was 20 µg/ml shown by culture, C 11. The result is compiled as Table 3. The microorganisms are grown in liquid media supplemented with starch. Hence they will use starch as their carbon source (Abe et al., 1988). For metabolizing starch they produce amylase. Amylase breaks starch into glucose. Depending on the amount of amylase produced, the quantity of starch broken down and hence the quantity of glucose formed will increase. Thus the quantity of glucose produced which can be determined spectrophotometrically using DNS method will reflect the quantity of amylase produced in each culture. Some microorganisms can be exceptionally good amylase producers. Sapto et al., 2010 isolated a thermostable á-amylase from Sonai Hot Spring, South East Sulawesi. Different concentrations of glucose were given as substrate to optimize the activity of the potent enzyme. Various reports are available on microorganisms degrading starch from different sources and their respective amylase activity (Aiba et al., 1983). Identification of most potent isolate Based on the colony characteristics, cultural characteristics, gram staining and biochemical characterization, the most potent of amylase producing bacterial isolate was identified to

4 AYONA JAYADEV AND S. S. NAVAMI specific to the type of the initial substrate. The composition and concentration of media greatly affect the growth and production of extracellular amylase producing bacteria (Srivastava and Baruah, 1986). Microorganisms usually degrade carbohydrate first then they will degrade the other composition of nutrition (Mohney, 2006). Mishra and Behera, 2008 reported that increase in substrate concentration, resulted more enzyme activity. The properties of the bacterial isolates of present study can be further optimized to make it suitable for application in starch processing and other food industries. The use of these bacteria that yield enzymes of different qualities may be of value in the modern day biotechnology and industries. In order to attain large scale production of amylase the molecular and functional relationship of amylase producing bacteria have to be studied in detail. ACKNOWLEDGEMENT Figure 1: Screening bacterial isolates for amylase activity Clearance distance (cm) Bacterial cultivars Figure 2: Amylase activity: Clearance zone in plate assay belong to Bacillus sp. Reports are available to show Bacillus sp. is one of the most active amylase producers (Sohail et al, 2005). Baishya and Hridip, 2010 isolated amylolytic bacteria from the gut of some fish species and the most potent was identified to be Bacillus megaterium. Swain et al., (2006) reported that bacteria belonging to Bacillus sp. have the ability to produce large quantities of extracellular amylase. The number of colony forming units is calculated to be 217x10-3 CFU in the present study. A total number of 18 colonies were identified. 12 colonies were found to be of gram negative and 6 were gram positive. 11 colonies showed amylolytic activity. Culture 16 showed highest amylolytic activity with highest production of glucose (0.37µg/mL). Starch is identified to be an important starting material in a variety of industrial processes. Many of these include the digestion of starch using amylase enzymes. Hence several workers have concentrated on the identification and optimization of different categories of amylase enzymes which can be efficiently used in industrial applications (Sohail et al., 2005). In this work, a biofertiliser generated from Municipal Solid Waste was screened for amylase producing bacteria with an idea that biofertilisers produced from this kind of wastes will be rich sources of starch degrading microorganism as it contains mostly starchy substrate (Mishra and Behera, 2008). The work shows that biofertilizers which are generated from Municipal Solid Waste carries microorganisms with activities The authors acknowledge the financial assistance as student project from the Kerala State Council for Science, Technology and Environment. REFERENCES Abe, J., Bergman, F. W., Obata, K. and Hik uri, S Production of raw starch digesting amylase by Aspergillus K-27. Applied Microbiology and Biotechnology. 27: Aiba, S. Kitai, K. and Imanaka, T Cloning and Expression of Thermostable Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis. Appl. Environ. Microbiol. 46: Al-Quadan, F., Akel, H. and Natshi, R Characteristics of a novel highly thermostable and extremely thermophilic alkalitolerant amylase from hyperthermophilic bacillus strain HUTBS71. OnLine J. Biol. Sci. 9: DOI: /ojbsci Aneja, K. A Experiments in Microbiology Plant Pathology and Biotechnology, Fourth Edition, New Age International (P) Ltd., Publishers, New Delhi. p Baishya Debabrat and Hridip Kumar, S Isolation of Amylolytic Bacteria from Viscera of Labeo rohita (Ham.) and Optimization of Alpha Amylase Extraction American J. Microbiology. 1(2): pp Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., Chauhan, B., Microbial á-amylases: a biotechnological perspective. Process Biochem. 38: Hasan, F., Shah, A. A. and Hameed, A Industrial application of microbial lipase. Enzyme and Microbial technology. 39(2): Ikram-Ul-Haq, S. Ali, A. S. and Javed, M. M Mutagenesis of Bacillus licheniformis through ethyl methane sulfonate for alpha amylase production. Pak. J. Bot. 41: Kaur, A., Kaur1, M., Samyal, M. L. and Ahmed, Z Isolation, characterization and identification of bacterial strain producing amylase., J. Microbiol. Biotech. Res. 2(4): Kiro, M Microbial Á-Amylases And Their Industrial Applications: A Review. International J. Management, IT and Engineering. 2(10) Mohney, K Synchronization of Carbohydrate and Protein Metabolism by Ruminal Microbes in Continuous Culture. Theses of Animal Sciences Departmen, Kansas State University s College. Omemu, A. M., Akpan, I., Bankole, M. O. and Teniola, O. D Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil. African J. Biotechnology. (4(1): ISSN: ). 112

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