International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 4(11), 58-61, November (2015) Int. Res. J. Environment Sci. International Science Congress Association 58 Screening and Isolation of Polyethylene degrading Bacteria from various sources Botre S., Jadhav P., Saraf L., Rau K. and Wagle A.* Guru Nanak Khalsa College, Nathalal Parekh Marg, Matunga, Mumbai-19, Maharashtra, INDIA Guru Nanak Institute of Research and Development, Mumbai, INDIAAvailable online at: www.isca.in, www.isca.me Received 15th September 2015, revised 28th October 2015, accepted 13th November 2015 AbstractPlastics are the most commonly used polymers for routine applications. The accumulation of plastics is a threat to environment as it causes pollution, creating an imbalance in the ecosystem, thus proving to be hazardous. The ways to degrade plastic have not been successful. At the same time natural degradation of plastics is too time consuming. The most eco-friendly approach to resolve this ever growing and persistent issue is the microbial degradation route. The main objective of the present study is to isolate and screen for bacteria having the capability to degrade low density polyethylene (LDPE) which is a major cause of environmental pollution. The bacteria were isolated from various sources after serial dilution on M9 medium incorporated with LDPE. The selected isolates were comparatively screened by an Agar cup method to find strong LDPE degraders. These isolates were further tested for the extent of degradation using a film degradation assay. Total 20 cultures were obtained during the primary screening as probable LDPE degraders. In the agar cup method, 7 high LDPE degraders were selected. The percentage degradation was found out using the film degradation assay and the two most promising isolates were obtained from sewage and marine sources. These isolates were identified using biochemical methods as belonging to the Genus Staphylococcus. Keywords:Plastics, degradation, eco-friendly, ldpe, agar cup etc. Introduction Plastic is the most useful synthetic substance made up of elements extracted from the fossil fuel resources. It has made possible most of the industrial and technological revolutions of the 19th and 20th centuries. LDPE is the most commonly occurring non-biodegradable waste material which constitutes approximately 60% of the total plastic production. The global usage of polyethylene is increasing at a rate of 12% yearly and around 140 million tones of synthetic polymers are produced worldwide each year. Plastic wastes are one of the factors in causing environment pollution, because of their semi-permanent stability in the environment. There are many disadvantages of plastic like combustibility, deformation under load, embrittlement at low temperature etc and the major one is that they are not biodegradable and because of their high molecular weight, cross linkages, high number of aromatic rings and halogen substitution they clog in the environment. The percentage of plastic waste has been increased in the landfills leading to pollution of the environment. A biological process, bioremediation breaks down various pollutants and reduces solid waste. Microbial degradation of plastic and polythene is a major interest in recent years. Biodegradable polymers are polymers which can be degraded naturally by micro-organisms such as fungi, bacteria, algae etc present in the environment. There are various physical, chemical and biological forces which causes initial breakdown of polymer. Microbial degradation of plastics converts polymer into oligomers and monomers. This microbial degradation may be based on aerobic and anaerobic metabolisms. The aim of the present study was to isolate microorganisms from varied natural sources and screen them for potential polyethylene degrading capability. The objective of the work carried out was to identify and characterize the high potential LDPE degraders. The isolation was carried out through serial dilution method of samples from sewage, oil sludge, marine, compost, tar sample and oil contaminated soil. The screening of potential LDPE degraders was carried out using a sequential screening procedure using an agar cup method followed by a film degradation assay. On identification the two selected bacteria were found to be belonging to genus Staphylococcus. Material and Methods Materials: Samples were collected in sterile test tubes, from varied sources like Compost (CC), Marine (M), Oil Sludge (OS), Sewage (S), Tar Sample (TS) and Oil Contaminated Soil (OCS). LDPE (Low Density Poly Ethylene) powder and LDPE films (Provided by Aegis Polymers). M9 agar and M9 broth (Prepared as per Maniatis, Manual for Molecular Biology). LB agar (Obtained from HI-Media, prepared as per manufacturer’s recommendation) Methodology: Primary screening and isolation of polymer degrading microorganisms: In primary screening the samples collected from the above mentioned sources were serially International Research Journal of Environment Sciences Vol. 4(11), 58-61, November (2015) International Science Congress Association diluted up to the range of 10-6 and alternate dilutions inclu the undiluted sample were spread plated on modified M9 agar containing 0.1% LDPE powder as the only source of carbon. The plates were kept for incubation at 37°C up to 72 hours. After incubation, growth was observed and the distinct colonies were chos en for each of the sources. These isolates were preserved on LB slants till further study. Secondary screening for high capacity LDPE degraders Next these probable plastic degrading isolates were screened further to select the ones which could withstand concentration and thereby eliminate the weak LDPE degraders. Hence the LDPE concentration was increased to 1% as the only available source of Carbon. The method adopted for secondary screening was Agar Cup Assay. Wells were bored in the M9 agar and culture suspension with adjusted OD of 0.3(620nm) was added in the respective wells of the selected isolates. Plates were incubated up to 72 hours at 37°C and growth was observed on the plates. The colonies which could utilize the high concentration of LDPE grew around the periphery of the well and were shortlisted for the next study. Film degradation assay: Pre- weighed alcohol sterilized 3*3 cm LDPE films were transferred to 50ml of respective M9 broth inoculated with isolates from secondary screening flask was maintained with the film without any culture suspension. The flasks were put on shaker conditions at 37°C/150rpm for 3 weeks and the OD was checked at 620nm at regular intervals. After 3weeks, the LDPE films were weighed again and the percent plastic degradation by the microbes was determined and compared.Results and Discussion The current study deals with screening, isolation and extent of plastic degradation by the isolates from various sources. Twenty bacterial colonies were isol ated from primary screening using 0.1%LDPE as the sole source of carbon in M9 Agar medium. In primary screening four bacteria were isolated from compost soil, three from marine, four from oil sludge, four from sewage and five from oil contaminated soil. Th e tar sample did not show any significant bacterial growth (figure-Figure-1 Microbial growth of sewage sample during Primary screening Environment Sciences __________________________________ ___________ Association and alternate dilutions inclu ding the undiluted sample were spread plated on modified M9 agar containing 0.1% LDPE powder as the only source of carbon. The plates were kept for incubation at 37°C up to 72 hours. After incubation, growth was observed and the distinct colonies en for each of the sources. These isolates were Secondary screening for high capacity LDPE degraders : Next these probable plastic degrading isolates were screened further to select the ones which could withstand high LDPE concentration and thereby eliminate the weak LDPE degraders. Hence the LDPE concentration was increased to 1% as the only available source of Carbon. The method adopted for secondary screening was Agar Cup Assay. Wells were bored in the M9 and culture suspension with adjusted OD of 0.3(620nm) was added in the respective wells of the selected isolates. Plates were incubated up to 72 hours at 37°C and growth was observed on the plates. The colonies which could utilize the high LDPE grew around the periphery of the well weighed alcohol sterilized 3*3 cm LDPE films were transferred to 50ml of respective M9 broth inoculated with isolates from secondary screening . A control maintained with the film without any culture suspension. The flasks were put on shaker conditions at 37°C/150rpm for 3 weeks and the OD was checked at 620nm at regular intervals. After 3weeks, the LDPE films were weighed percent plastic degradation by the microbes was The current study deals with screening, isolation and extent of plastic degradation by the isolates from various sources. Twenty ated from primary screening using 0.1%LDPE as the sole source of carbon in M9 Agar medium. In primary screening four bacteria were isolated from compost soil, three from marine, four from oil sludge, four from sewage e tar sample did not - 1). Microbial growth of sewage sample during Primary The agar cup secondary screening method, which had 1% LDPE in M9 Agar, showed prominent growth of seven cultures around the periphery of the respective wells. These seven isolates were found to have a greater ability to withstand such high LDPE concentrati on as compared to the other thirteen isolates from primary screening. Thus agar cup secondary screening provided an efficient and quick method for comparative screening of LDPE degraders. The seven cultures named as CC1, OCS3, OCS5, OCS1, OCS4, M1 and SWG film degradation assay (figure-2). Figure - Microbial growth during secondary screening Pre- weighed LDPE films were subjected to degradation with the above mentioned seven isolates. Out of the seven isolates M1 and S4 showed maximum degradation in liquid M9 medium, which confirmed higher plastic degrading capacity These two isolates were further characterized using Gram’s staining and biochemical tests .Gram staining of the two isolates showed Gram positive cocci Figure - Gram positive cocci in Clusters for marine isolate Figure - Gram positive cocci in Clusters for sewage isolate ___________ ISSN 2319–1414 Int. Res. J. Environment Sci. 59 The agar cup secondary screening method, which had 1% LDPE in M9 Agar, showed prominent growth of seven cultures around the periphery of the respective wells. These seven isolates were found to have a greater ability to withstand such high LDPE on as compared to the other thirteen isolates from primary screening. Thus agar cup secondary screening provided an efficient and quick method for comparative screening of LDPE degraders. The seven cultures named as CC1, OCS3, OCS5, OCS1, OCS4, M1 and SWG were selected for further - 2 growth during secondary screening weighed LDPE films were subjected to degradation with the above mentioned seven isolates. Out of the seven isolates M1 maximum degradation in liquid M9 medium, which confirmed higher plastic degrading capacity (table-1). These two isolates were further characterized using Gram’s .Gram staining of the two isolates showed Gram positive cocci in clusters (figure-3 and 4). - 3 Gram positive cocci in Clusters for marine isolate - 4 Gram positive cocci in Clusters for sewage isolate International Research Journal of Environment Sciences _____________________________________________ ISSN 2319–1414Vol. 4(11), 58-61, November (2015) Int. Res. J. Environment Sci. International Science Congress Association 60 Table-1 Result for extent of degradation of LDPE films by the isolatesSource O.D (620nm) after 72 hours Initial weight (g) Final weight (g) % degradation Control 0 0.0225 0.0225 - CC1 0.22 0.0207 0.0195 1.2 OCS3 0.12 0.0214 0.0210 0.4 OCS5 0.12 0.0220 0.0213 0.7 OCS1 0.11 0.0211 0.0207 0.4 OCS4 0.13 0.0222 0.0216 0.8 M1 0.25 0.0209 0.0184 2.5 S4 0.28 0.0237 0.0209 2.8 Table-2 Consolidated result for microscopy and biochemical tests for marine and sewage isolatesTest M1 (Marine) S4 (Sewage) Gram nature Gram positive cocci in clusters Gram positive cocci in clusters Catalase Positive Positive Hemolysis Carbohydrate Utilization Positive Positive Vogues Proskeur’s Negative Negative Citrate Positive Positive Urease Negative Negative Nitrate reduction Positive Positive The cultures were found to be catalase positive and -hemolytic in nature (figure-5, 6 and 7). Thus, both the bacteria were identified belonging to the genus Staphylococcus. On biochemical classification as per Bergey’s Manual (table-2). Figure-5 Catalse test for sewage isolate Figure-6 Catalase test for marine isolate Figure-7 Hemolysis test for sewage and marine isolates ConclusionSome degradability is present in natural conditions, but microbes also exhibit degradation in laboratory condition on culture media. The two strains of bacteria M1 and SWG isolated from marine and sewage sources respectively, were found to have higher ability to degrade LDPE and belonging to Staphylococcus genus. References1.Raaman N et al, Biodegradation of plastic by Aspergillus spp. isolated from polythene polluted sites around Chennai, J. Acad. Indus. Res., 1(6), 313-316 (2012) 2.Ambika devi K. et al, Isolation of polythene degrading bacteria from marine waters of Viskhapatnam, India, Int. International Research Journal of Environment Sciences _____________________________________________ ISSN 2319–1414Vol. 4(11), 58-61, November (2015) Int. Res. J. Environment Sci. International Science Congress Association 61 J. Curr. Microbiol. App. Sci 3(10) 269-283 (2014) 3.Uttiya dey et al, An approach to polymer degradation through microbes, IOSR Journal of Pharmacy,2(3), 385-38 (2012) 4.Katarzyna Leja et al, Polymer Biodegradation and Biodegradable Polymers, Polish J. of Environ. Stud., 19(2), 255-266 (2010) 5.Sharma Prabhat et al, Studies on isolation and identification of active microorganisms during degradation of polythene/starch film, International Research Journal of Environmental Sciences, (2013) 6.Molecular Cloning, A Laboratory Manual 1st ed. Maniatis, 68 (1982) 7.Bergey D.H.1. and Breed R.S., Bergey's manual of determinative bacteriology, American Society for Microbiology, 1, (7th ed) Baltimore, Williams and Wilkins Co, (1957) 8.Albertson AC et al, the mechanism of biodegradation of polyethylene, polymer degradation and stability, 18, 73-87 (1987) 9.Sreedevi S., Solid Waste Generation and its Management: A Case Study, Int. Res. J. Environment Sci., 4(1), 90-93 (201510.Hadad D, Geresh S and Sivan A, Biodegradation of polyethylene by the thermophilic bacterium Brevibacillusborstelensis, J. Appl. Microbiol, 98, 1093-1100 (2005) 11.Chaudhari Y., Bhavana P. and Fulekar M.H., PHA: Production Application and its Bioremediation in Environment, Int. Res. J. Environment Sci.,1(2), 46-52 (2012) 12.Kimi Jain, Isolation of microorganisms carrying biodegradation of plastic, Department of Biotechnology and Environmental Sciences, (2011) 13.KL Hoffmann, J Renickova, K Kozakova, PRuzicka, D Alexy Bakos and L. Precnerova, Polym. Degrad. Stab, 76, 511-519, (2003) 14.Kamble Asmita, Tanwar Shubhamsingh and Shanbhag Tejashree, Int. Res. J. Environment Sci., 4(3), 77-85 (2015) 15.Hamilton J.D., Reinert K.H., Hogan J.V. and Lord W.V., Polymers as solid waste in municipal landfills, J. Air Waste Manage. Assoc., 43, 247-251 (1995) 16.Singh M.K. and Singh Reeta Devi, Int. Res. J. Environment Sci.,2(3), 6-10 (2013)17.Albertsson AC and Karlsson S, The influence of biotic and abiotic environments on the degradation of polyethylene, Prog. Polym. Sci., 15, 177-192 (1990) 18.Pooja Thakur, Screening of Plastic degrading bacteria from dumped soil area, Department of Life Science National Institute of Technology, http://ethesis.nitrkl.ac.in/3141/1/pooja_thesis_1.pdf (2015) 19.Bonhomme S et al, Environmental Biodegradation of polyethylene, Polym Degrad Stab, 81, 854-860 (2003)20.Sonil Nanda et al, Biodegradability of polyethylene by Brevibacillus, Pseudomonas, and Rhodococcusspp, New York Science Journal, (2010)