ISSN Original Article Biodegradation of Polyethylene by Bacillus cereus

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Available online at http://www.urpjournals.com Advances in Polymer Science and Technology: An International Journal Universal Research Publications.

Studies and Research in Microbiology, Bioscience Complex, Kuvempu University, Jnanasahyadri, Shankaraghatta-577 451, Shivamogga(Dist.,), Karnataka, INDIA 1 Dept. of P.G.

1 Available online at Advances in Polymer Science and Technology: An International Journal Universal Research Publications. All rights reserved ISSN Original Article Biodegradation of Polyethylene by Bacillus cereus Sowmya, H.V., 1 Ramalingappa., 2 Krishnappa, M. and B. Thippeswamy. Dept. of P.G. Studies and Research in Microbiology, Bioscience Complex, Kuvempu University, Jnanasahyadri, Shankaraghatta , Shivamogga(Dist.,), Karnataka, INDIA 1 Dept. of P.G. Studies and Research in Microbiology, Davanagere University, Shivagangotri, Tholuhunse, Davanagere, Karnataka, INDIA 2 Dept. of P.G. Studies and Research in Applied Botany, Bioscience complex, Jnanasahyadri, Kuvempu University, Shankaraghatta , Shivamogga (Dist.,), INDIA Received 17 April 2014; accepted 06 May 2014 Abstract Bacillus cereus was isolated from a local dumpsite of Shivamogga district for use in the biodegradation of polyethylene. Soil sample of that dumpsite was used as source for isolation of Bacillus cereus. Degradation was carried out using autoclaved, UV treated and surface sterilized polyethylene. Degradation was monitored by observing weight loss and changes in physical structure by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FTIR) Spectroscopy. Bacillus cereus was able to degrade treated polyethylene (14%) more efficiently than autoclaved (7.2%) and surface sterilized polyethylene (2.4%). Enzymes responsible for polyethylene degradation were screened from Bacillus cereus. Enzymes were identified as laccase and manganese peroxidase. These enzymes were produced in large amount, enzyme activity was calculated using spectrophotometric method. By observing these results we can conclude that, Bacillus cereus may act as solution for the problem caused by polyethylene in nature Universal Research Publications. All rights reserved Key Words: Polyethylene; Bacillus cereus; Laccase; Manganese peroxidase; Biodegradation. INTRODUCTION High molecular weight polymers like polyethylene are not susceptible to microbial attack. Polyethylene is one of the most abundant commercially produced synthetic polymers. The annual production is approximately 80 million metric tons. During past three decades, plastic materials are increasingly used in transportation, food clothing, shelter construction, medical and recreation industries. Plastic are advantageous as they are strong, light weight and durable. But, lack of degradability and the closing of landfill sites, as well as growing water and land pollution problems have led to concern about plastics. With the excessive use of plastics and increasing pressure being placed on capacities available for plastic waste disposal, the need for biodegradable plastics and biodegradation of plastic has assumed increasing importance in the last few years. Biodegradation is necessary for water soluble or water immiscible polymers, because they eventually enter water streams which can neither be recycled nor incinerated [18]. The polyethylene is the most commonly found solid waste that has been recently recognized as a major threat to marine life. The polyethylene could sometimes cause blockage in intestine of fish birds and marine mammals [20, 17]. The degradation of polyethylene can occur by different molecular mechanisms such as chemical, thermal, photo and biodegradation [4]. Biodegradability is evaluated by weight loss, tensile strength loss, changes in percent elongation and changes in polyethylene molecular weight distribution. Physicochemical distribution is initiated by treatment with acid at 70⁰C and UV irradiation of the polyethylene film. These pretreatment favors the microbial degradation of polyethylene. The solid waste related problems pose threat to mega cities. So, an attempt has been made in this paper to isolate the potent bacterium that degrades polyethylene from the soil of dumpsite area. MATERIALS AND METHODS 1. Collection of soil sample: Soil sample was collected from a local dumpsite of Shivamogga district and brought to the laboratory, preserved under laboratory conditions for further use. 2. Isolation and identification of bacterium from soil: Enrichment procedure was used for the isolation of bacterium, where polyethylene was used as sole source of carbon. Isolated bacterium was identified based 28

2 on macroscopic appearance, Gram s staining and by performing biochemical tests using standard manuals [16]. The colonies were preserved at 4⁰C in 2% agar slants of medium malt and yeast extract [14]. 3. Screening of bacterium for polyethylene degradation 3.1. Plate assay: The isolated bacterium was inoculated to medium which contained 0.3g of NH 4 NO 3, 0.5g of K 2 HPO g of NaCl, 0.02g of MgSO 4.7H 2 O, 2g of agar, 0.5g of polyethylene and 100ml distilled water [14]. This agar plate test is also a simple semi- quantitative method to know depolymerization of polymer by the organism. After inoculation with bacterium into the medium containing fine particles of polyethylene, the formation of a clear hallow around the colony indicates the first step of bacterial biodegradation [13] Degradation of Polyethylene: The pre-weighed discs of Autoclaved, Surface sterilized and UV treated polyethylene of 1cm diameter prepared from polyethylene bags were aseptically transferred to the conical flask containing 50ml of Mineral Salt Medium. Loop full of organisms was added to medium. Control was maintained with polyethylene discs in the microbe free medium. Triplicates were maintained for each type of polyethylene and left on shaker. After three months of incubation, the plastic discs were collected, washed thoroughly using distilled water, dried in hot air oven at 50⁰C over night and then weighed for final weight [7]. 4. Confirmation of polyethylene degradation. Polyethylene degradation was confirmed by using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FTIR) Spectroscopy. 5. Screening of enzymes responsible for polyethylene degradation Earlier studies revealed that, laccase and manganese peroxidase are responsible for polyethylene degradation. So, we carried out screening, mass production and calculated activity of these enzymes Screening of laccase and manganese peroxidase enzyme The isolated bacterium was screened for the laccase production using laccase screening medium (LSM). Bacterium was inoculated in LSM agar plate and the plate was incubated for 7 days in dark condition. The substrate utilized reddish brown color in screening medium indicated the positive strain for laccase [22]. For manganese peroxidase, H 2 O 2 was used to the same medium Mass production by sub-merged fermentation The mass level production of the enzyme was carried out in mineral salt medium under suitable environmental conditions [21] Enzyme assay 1 ml of the culture supernatant was added with one ml of 2mM guaiacol and 3ml 10mM Sodium acetate buffer (ph 4.6). The reaction mixture was incubated at 30⁰C for 15 mins. The color change was measured using spectroscope at 450 nm. One unit of laccase activity was defined as amount of enzyme required to hydrolyze guaiacol during incubation period. For the enzyme activity calculation of manganese peroxidase same procedure was used but for the reaction mixture 1 ml of H 2 O 2 was added and incubated [15] Protein estimation Protein estimation was done to calculate specific activity of enzymes. The protein concentration was determined by the Lowry s method, as described by Lowry s (1951) using Bovine Serum Albumin (BSA) as a standard. RESULTS 1. Isolation and Identification of Bacterium Bacillus cereus was isolated and identified as Gram positive rod. Biochemical test performed also confirmed organism (Table 1). Bacillus cereus was selected for study because of its common predominant presence in its native region of soil contaminated with waste polyethylene plastic bags. 2. Screening of bacteria for polyethylene degradation 2.1. To check ability of bacterium to grow on medium containing polyethylene Bacillus cereus was able to grow on agar medium containing polyethylene as sole carbon source. This showed its capacity to utilize polyethylene as carbon source and to degrade polyethylene Degradation of autoclaved polyethylene Bacillus cereus was able to degrade autoclaved polyethylene, indicating its capacity to use polyethylene as sole carbon source. The weight loss for autoclaved polyethylene was 7.2% (Table 2) Degradation of UV treated polyethylene Bacillus cereus was able to degrade UV treated polyethylene more efficiently than autoclaved and surface sterilized (Fig. 1). The weight loss for UV treated PE was 14% (Table 3). Degradation was more here because of pretreatment using UV light and acid. UV light is known as initiator of polyethylene oxidation and enhances the bacterial degradation when compared with its corresponding UV untreated control [8]. Both UV and acid treatment causes pro-oxidant and photo-oxidant to produce free radicals on the long chain, causing the material to lose some of its physical properties to become oxidized and more accessible to microbial biodegradation [2]. The bacterial attachment was found on the surface of the plastic and it indicates possible utilization of plastic as carbon source. Fig.1. Biodegradation of different types of polyethylene 2.2. Degradation of surface sterilized polyethylene Bacillus cereus was able to degrade surface sterilized polyethylene. This method confirmed that this organism can utilize polyethylene without any pre- 29

Mannitol Negative 10 Glucose Positive 11 Arabinose Negative 12 Trehalose Positive Table 2. Weight loss of autoclaved polyethylene 1. 0.10 0.0928 0.0072± 0.0002 7.2 ± = Standard Deviation.

3 Table 1. Biochemical tests performed for Bacillus cereus. Sl. No. Test Bacillus cereus 1 Malonate Negative 2 Voges Proskauer s Positive 3 Citrate Positive 4 ONPG Negative 5 Nitrate reduction Negative 6 Catalase Positive 7 Arginine Negative 8 Sucrose Negative 9 Mannitol Negative 10 Glucose Positive 11 Arabinose Negative 12 Trehalose Positive Table 2. Weight loss of autoclaved polyethylene ± ± = Standard Deviation. * = Mean Table 3. Weight loss of UV treated polyethylene ± ± = Standard Deviation, * = Mean Table 4. Weight loss of surface sterilized polyethylene ± ± = Standard Deviation, * = Mean Table 5. Enzyme activity of Laccase and Manganese peroxidase Enzyme/Weeks Laccase Manganese peroxidase treatment like, heat, UV light and acid. The weight loss for surface sterilized polyethylene was 2.4% (Table 4). 3. Confirmation of polyethylene degradation 3.1. Observation of discs using SEM Autoclaved (Fig. 2), UV treated and surface sterilized polyethylene (Fig. 3) showed morphological changes when observed through SEM. Formation of holes, disruption of polyethylene structure confirmed degradation capacity of Bacillus cereus Observation of discs using FTIR FTIR results showed formation of ketone, aldehyde, carboxylic acids, alcohol, phenol, ester and aromatic compounds. Following are the figures showing FTIR spectrum of control, autoclaved (Fig. 4), UV treated and surface sterilized polyethylene (Fig. 5). Fig. 2. SEM photographs of control and autoclaved polyethylene Fig. 4. FTIR spectrum of control and autoclaved polyethylene. Fig. 3. SEM photographs of UV treated and surface sterilized polyethylene Fig. 5. FTIR spectrum of UV treated and surface sterilized polyethylene 30

4 In FTIR spectrum control polyethylene degradation products were not formed. FTIR spectrum of autoclaved, UV treated and surface sterilized polyethylene showed formation of carboxylic acids, aldehydes, alochols, esters, ehers, aromatics, alkene and phenol group formation at different frequencies indicating their degradation by Bacillus cereus. 4. Screening and characterization of polyethylene degrading enzymes Bacillus cereus showed positive result for both laccase and manganese peroxidase enzymes. 4.1 Mass production of enzymes. Laccase and manganese peroxidase enzymes were produced in large amount using submerged fermentation Enzyme assay Activity of manganese peroxidase ( IU/ml) was more compared to laccase activity ( IU/ml) after ninth week of incubation (Table 5) (Fig. 6). Fig.6. Enzyme activity of Laccase and Manganese peroxidase 4.3. Protein estimation Specific activity of manganese peroxidase ( µmol/ml/mg/min) was more compared to laccase ( µmol/ml/mg/min). DISCUSSION Bacillus cereus was isolated and identified as Gram positive rod. Biochemical test performed also confirmed organism. Bacillus cereus was grown on medium containing polyethylene and agar. After the growth of Bacillus cereus on polyethylene containing medium, it was screened for degradation of autoclaved, UV treated and surface sterilized polyethylene. Bacillus cereus was able to degrade UV treated polyethylene (14%) more efficiently than autoclaved (7.2%) and surface sterilized (2.4%). After the treatment of polyethylene with UV light, Bacillus cereus was able to degrade it more efficiently. Carbonyl groups are produced by UV light or oxidizing agents and these groups are the main factors at the beginning of the degradation, being attacked by microorganisms that degrade the shorter segments of polyethylene chains [1]. Degradation of natural and synthetic polyethylene was carried out using 3 species of Pseudomonas. Natural polymer contained 6% of vegetable starch. Degradation was monitored by weight loss. Pseudomonas sp. isolated from sewage sludge sump was found efficient in degradation with 46.2% for natural and 29.1% for synthetic polyethylene. In contrast Pseudomonas sp. from household garbage dump was lowest in degradation with 31.4% of natural and 16.3% of synthetic. Pseudomonas sp. from textile effluent drainage site degraded 39.7% of natural and 19.6% of synthetic polyethylene [11]. Degradation of polyethylene using microorganisms isolated from compost soil was studied. Degradation was studied by inoculating isolated organisms into Mineral salt medium containing 1 gram of polyethylene films as sole carbon source. Degradation was studied using SEM and FTIR. Degraded products were analyzed by Gas Chromatography [10]. SEM and FTIR were also used in our study to evaluate biodegradation. SEM results showed formation of cavities and erosions. Degradation of low - molecular - weight poly- -ethylene (LMWPE) was carried out using thermophilic bacterium Chelatococcus sp. Degradation was studied by using FTIR. The FTIR peaks corresponding to alkenes also were more intense, indicating that dehydrogenations occurred concomitantly with microbial induced oxidation [6]. Our FTIR results also showed formation of alkenes. Pseudomonas sp. was more efficient in degrading polyethylene when compared to Rhodococcus and Brevibacillus. Pseudomonas sp. degraded 40.5% of polyethylene after 3 weeks of incubation [12]. Our isolated species was able to degrade 9.5% of autoclaved polyethylene; it degraded 35% of UV treated and 6.2% of surface sterilized polyethylene after 3 months of incubation. Degradation of high molecular weight polyethylene was carried out with partially purified manganese peroxidase from Phanerochaete chrysosporium. Experiment was carried out under nitrogen limited and carbon limited conditions [19]. Bacillus cereus also showed positive result for manganese peroxidase enzyme, indicating its role in degradation. Degradation of polyethylene in the presence of Tween 80, Mn(II) and Mn(III) chelator was carried out. Results confirmed the role manganese peroxidase as key enzyme in biodegradation of polyethylene [5]. Role of laccase-mediator system was investigated for biodegradation of polyethylene in presence of 1-hydroxybenzotriazole (HBT). Laccase of Trametes versicolor was used. Degradation of polyethylene was confirmed by changes in relative elongation, relative tensile strength and molecular weight distribution. All these results confirmed degradation of polyethylene by laccase mediator system [3]. In our work, Bacillus cereus showed positive result for laccase indicating its possible role in polyethylene degradation. CONCLUSION Degradation of polyethylene was carried out with Bacillus cereus which was isolated from dumpsite soil. This organism was able to degrade polyethylene. Efficiency of Bacillus cereus to use UV treated polyethylene as sole source of carbon was much better than autoclaved and surface sterilized. Degradation was monitored by weight loss, SEM and FTIR studies. Weight loss of UV treated polyethylene (14%) was more followed by autoclaved (7.2%) and surface sterilized (2.4%). FTIR results showed formation of aldehyde, alcohol, carboxylic acid, aromatic, alkene and ether group formation indicating degradation of polyethylene by isolated bacteria. All these results confirmed polyethylene degradation. Enzymes responsible for polyethylene degradation were identified as laccase and manganese peroxidase. Optimum conditions for increased polyethylene degradation can be studied in future. 31

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Int. J. Environ. Res., 9(3):823-830, Summer 2015 ISSN: 1735-6865 Polyethylene Degradation by Fungal Consortium Sowmya, H.V. 1, Ramalingappa, B. 2*, Nayanashree, G. 1,Thippeswamy, B. 1 and Krishnappa, M.