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(Plastic Cup) Using Micrococcus Luteus and Masoniella Sp Sivasankari

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(Plastic Cup) Using Micrococcus Luteus and Masoniella Sp Sivasankari Scholars Academic Journal of Biosciences (SAJB) ISSN 2321-6883 (Online) Sch. Acad. J. Biosci., 2014; 2(2): 85-89 ISSN 2347-9515 (Print) ©Scholars Academic and Scientific Publisher (An International Publisher for Academic and Scientific Resources) www.saspublisher.com Research Article In Vitro Degradation of Plastics (Plastic Cup) Using Micrococcus Luteus and Masoniella Sp Sivasankari. S* , Vinotha. T Department Of Microbiology, Shri Mathi Indira Gandhi College, Tirchirappalli, Tamil Nadu -620002, India *Corresponding author S. Sivasankari Email: Abstract: Plastic is a broad name given to different polymers with high molecular weight, which can be degraded by various processes. However, considering their abundance in the environment and their specificity in attacking plastics, biodegradation of plastics by microorganisms and enzymes seems to be the most effective process. When plastics are used as substrates for microorganisms, evaluation of their biodegradability should not only be based on their chemical structure, but also on their physical properties (melting point, glass transition temperature, crystallinity, storage modulus etc.). Present paper investigates the possibility of plastic degradation by microbes isolated from forest soil. The invitro degradation was studied by litter bag experiment by taking 1 g of each plastic and buried under forest soil at a depth of 15 cm from the surface during the month of September to February, 2010. An in-vitro experiment was started after collecting the plastic samples from the litter bag experiment and the microbes were isolated from the surface of the plastic. Then the isolated microbes inoculated in the nutrient agar. Result showed that no variety of plastic comfortable degraded under burial condition during 45 days. In the present study, plastics are highly resistant to degradation of plastics by using microorganisms is a great challenge. Hence an attempt has been made to determine the plastics degrading ablity of Micrococcus luteus and Masoniella sp. Keywords: Poly ethylene, in vitro, biodegradation, enzymatic degradation, bio-based plastics, microbial degradation INTRODUCTION environmental conditions or in municipal and Plastics are defined as the polymer (Solid industrial biological waste treatment facilities [3]. A materials) which became mobile on heating and number of biodegradable polyesters, namely thus can be cast into moulds. Plastics are non-metallic Polyhydroxyalkonoates (PHA), polylactides, aliphatic moldable compounds and the materials made from polyester, polysaccharides and copolymer or blends of them, can be pushed into almost any desired shape and the have been developed Successfully to meet retain that shape [1]. They are durable and degrade Specific demands in various fields and industries [4]. very slowly These are highly to biodegradation, leading to pollution and harmful to the natural MATERIALS AND METHODS environment. Collection of sample Polystyrene Collection Plastic materials have been increasingly used Plastics cup were collected from waste disposal in in Food clothing shelter, transportation, Construction, and around Thanjavur. medical and recreation industries. Plastics are advantageous as they are strong, light weighted and Soil Collection: durable. However, they are disadvantageous as they are The samples are collected from the site dumped with resistant to biodegradation leading to pollution and large amount of plastic waste disposal in the area of harmful to the natural environment. In the past 10 years, Thanjavur. several biodegradable plastics have been introduced in the market. However, none of them is efficiently Methodology biodegradable in landfills. For this reason, none of the Inoculum Preparation products has gained widespread use [2]. Nutrient Broth Beef extract - 3 gm Biodegradable plastics opened the way for Peptone - 5 gm new consideration of waste management strategies Sodium chloride - 5 gm since these materials are designed to degrade under Distilled water - 10000 ml 85 Sivasankari S et al., Sch. Acad. J. Biosci., 2014; 2(2):85-89 Potato Dextrose Broth Lactophenol cotton blue is a stain commonly used Potato (peeled) - 200 gm for making semi permanent microscope preparation of Dextrose - 20 gm fungi. It stains the fungal cytoplasm and provides a light Distilled water - 1000 ml blue back ground against which the walls of hyphae can readily be seen. It contains four constituent’s namely Processing of Soil Sample: phenol- which serves cotton blue) and a cover glass was The soil samples were serially diluted as per the placed over the preparation which was then ready for method of Aneja [5]. The suspension from 10-5 and 10-6 microscopic examination. dilutions were inoculated on Nutrient agar plate for the isolation of bacteria. The suspension from 10-2 and 10-3 Requirements: dilutions were inoculated on potato dextrose agar A Young culture of Masoniella sp, medium for the isolation of fungi. Both the plates were Loctophenol cotton blue in a dropper bottle mounted incubated for 24 hrs and respectively. needles, Glass slides, Cover slips, Bunsen burner, 2% alcohol, Nail polish,and Microscope. Identification of Bacterial Isolates Gram Staining Procedure: The gram staining of bacteria was done as per the A drop of lactophenol cotton blue was placed on a procedure given by Aneja [5]. The colonies were clean glass slide. stained by staining method, in order to identify the A small test of fungus, probably with spores and morphology and gram's reaction of the bacterium. A spore bearing structures was transferred into the thin smear was prepared on a clean slide using the drop, using a flamed, coolded needle. isolated individual colony. The smear was heat fixed Then the material was gently teased using the two and dried. The dried smear was than flooded, with the mounted needles. primary stain, crystal violet solution and allowed to Then the stain was gently mixed with the mold stand for one minute. Then it was washed with water structure. and flooded with Gram's lodine solution and allowed to A cover slip was placed over the preparation by stand for minute. Then it was washed with water and avoiding air bubbles. flooded with Gram's lodine solution and allowed to Finally, the slide was observed under the stand for 1 minute. The slide was again washed with microscope. water and decolorized with 95% ethanol for few seconds and washed with running tap water. Then the Microbial Degradation of Plastics: slide was flooded with a counter stain, safranin for 1 Surface Sterilization of Polystyrene minute. After drying the stained smear was observed The degradation of plastics by microorganisms were under microscope to identify the organisms. studied by following the method of Kathirasan [6]. The collected plastics cups were cut into small pieces. The Motility Test: (Hanging Drop method). pieces were cleaned with tap water (to remove dust The motility bacteria were done as per the procedure particles), Surface sterilized with ethanol and again described by Aneja [5]. The motility was studied by washed with distilled water. They were weighed about employing hanging drop method. A drop of culture both 1g one and used for both test and control. was placed on the centre of the cover slip. A pinch of Vaseline was applied over each corner of the cover slip. Media Preparation: Then a cavity slide was placed in an inverted position 1000ml of Nutrient broth Potato dextrose were on the cover slip and the slide was observed under the prepared and autoclaved at 1210C for 15 min. 250ml of microscope. cooled. Nutrient broth and potato dextrose broth was poured into four 500ml conical flasks. The sterile pre Biochemical Tests: weighed plastic pieces were aseptically transformed The biochemical tests for the bacterial isolates were into respective medium. A loopful of Micrococcus done as per the procedure described by Aneja[5]. The Luteus a Masoniella was inoculated into respective isolated organisms were subjected to biochemical test medium. A 250 ml of flask containing only the plastics for identification. The biochemical tests includes - were maintained as control. These flasks were Indole test, Methyl red Test, Voges - proskauer test, incubated at 370C for 35, 45, and 55 days in shaking Citrate Utilization test, Catalase test, Oxidase test and incubators. Triple sugar iron test . Recovers: Identification of Fungal isolates: The Plastic pieces were carefully removed from the The fungal isolates were identified as per the culture (by using forceps) after days of incubation. The procedure described by Aneja [5]. The fungal isolate collected pieces were washed thorough with tap water, that were obtained in potato dextrose agar plates were ethanol and then with distilled water. The pieces were identified by lactophenol cotton blue staining. shade diced and weighed for final weight. The data 86 Sivasankari S et al., Sch. Acad. J. Biosci., 2014; 2(2):85-89 were recorded. The same procedure was also repeated for 45 and 55 days of incubation. Degradation of plastics by Micrococcus Luteus: The results of degradation of plastics by Micrococcus Determination of Degradation of Plastics: Luteus were presented in Table .3. The results clearly The percentage of degradation of plastic pieces by showed that Micrococcus luteus were able to degrade Micrococcus Luteus and Masoniella SP were plastics. determined by calculated the percentage of weight loss of plastics. The percentage of weight loss was Degradation of plastics by Masoniella sp: calculated by the following formula.
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