Degradation assessment of low density polythene (LDP) and polythene (PP) by an indigenous isolate of Pseudomonas stutzeri

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1 Journal of Scientific & Industrial Research Vol. 63, March 2004, pp Degradation assessment of low density polythene (LDP) and polythene (PP) by an indigenous isolate of Pseudomonas stutzeri Anjana Sharma* and Amitabh Sharma Bacteriology Laboratory, Department of Bioscience, Rani Durgavati University, Jabalpur Received: 27 August 2003; accepted: 20 November 2003 Tensile strength, elongation, per centage extension, CFU, BOD and turbidity of plastics are assessed to study the extent of degradation of low density polyethylene (LDP) and polypropylene (PP) using Pseudomonas stutzeri under laboratory test conditions. Throughout the investigation both the plastic types are found to undergo qualitative and quantitative changes by bacteria but PP is found to be more biodegradable as compared to LDP. Keywords: Low density polythene, Polythene, Isolate of Pseudomonas stutzeri, Pseudomonas stutzeri IPC: Int Cl. 7 : C 01 C 31/10 Introduction Plastics the synthetic polymers of carbon, hydrogen, and oxygen are derived from petrochemicals. They are versatile family of materials, which are suitable for wide range of applications. Plastics being xenobiotic compounds, resistant to degradation, constitute about 5-8 per cent of dry weight of municipal solid waste. The instrumental effects of these polymers on the environment, range from ozone depletion to the environmental toxicology of agriculture and aquatic ecosystem. There are different methods for disposal of plastics such as, incinerating, recycling 1, landfills 2, and biodegradation. The ability of microorganisms to degrade extracellular polymers depends on the secretion of specific depolymerases that hydrolyze the polymer to water soluble products 3. Although many types of biodegradable plastics are available like, photodegradable and starch linked but bacterial degradable plastics are of great interest 4. Thus the present study was carried out to assess the biodegradation of non-biodegradable plastics by an indigenous isolate of Pseudomonas stutzeri. Material and Methods Plastic and soil samples were collected from the environment that was rich in plastic wastes. Bacterium was isolated from these samples and was maintained on nutrient agar slants after culturing. The bacterium was identified, following differential and selective, morphological, cultural, and biochemical tests 5, using Bergey s manual of systemic bacteriology 6 and probabilistic identification of bacteria computer kit 7. Low density polyethylene (LDP) and polypropylene (PP) used in everyday life were obtained from plastic manufacturing factories. Low density polyethylene was used in the form of films (LDP 1 ) and balls (LDP 2 ). The plastic samples were treated with conc. HNO 3 for 24 h at room temperature and then boiled for 4 h. Degradation of these treated plastic samples was carried out by studying morphological and qualitative alterations after bacterial activity at 15 d interval up to 45 d. (i) Morphological Assessment of Treated Plastic Films

2 294 J SCI IND RES VOL 63 MARCH 2004 Table 1 Identification of bacteria on the basis of morphological and biochemical tests using Bergey s manual of systematic bacteriology and PIB computer kit Test Results Gram stain - Shape Rod Motility + Methyl red - Voges Proskauer - Indole + Catalase + Oxidase + Simmon citrate - Arginine dehydrolase - Hydrogen sulphide - Starch hydrolysis + Gelatin liquification - Glucose fermentation - Gas production - Sucrose fermentation - Lactose fermentation - Hydrogen sulphide - Fermentation - Oxidation + Pseudomonas stutzeri Changes due to bacterial degradation were assessed qualitatively by measuring changes in tensile strength, extension, and elongation of test plastic films (1.5 cm in length and 20 cm in width) using Tensilometer 8. Tensile strength was calculated as: Tensile strength N/cm 2 = Breaking load in N/Cross sectional area in cm 2. Per cent extension was obtained by the following formula: R2 R1 Per cent extension = R1 100, where, R1 is the reading on the extension scale after fixing the specimen and R2 the reading of the specimen when it ruptures. (ii) Qualitative Estimation of Bacterial Degradation Qualitative estimation of bacterial degradation was assessed by considering following parameters: Biochemical oxygen demand (BOD) 7 was analyzed using samples, soil, and water in the ratio of 1:2:12. It *Author for correspondence Ph. No. (0761) ,Fax: (0761) , , anjoo_ 1999@yahoo.com

3 SHARMA & SHARMA: DEGRADATION ASSESSMENT OF LOW DENSITY POLYTHENE 295 was then inoculated and BOD was measured regularly at 15 d interval up to 45 th d of incubation, following Winkler s method 9. Twelve flasks containing about 1200 ml of distilled water and 600 g of sterilized soil, 300 g of carbon source, i.e., LDP, PP and glucose were used. Out of these 12 flasks, in 4 flasks 300 g of LDP was used as a carbon source. Three of them were inoculated with bacterial culture and one was kept as control. Same procedure was opted by taking PP and glucose as carbon source. The DO of the control was taken at zero day and from the same flask 300 ml was incubated for 3 d and BOD was analyzed at regular intervals of 15 d up to 45 d. Colony forming units (CFU/mL) and turbidity were analyzed by using mineral salt solution 10 as the basal medium with glucose plastic samples (1per cent w/v) as the carbon source. Readings for CFU were noted on the zero day and at 15 d interval up to 45 th d of incubation. Turbidity at 620 nm was noted on 5 th d and after every 5 d interval up to 45 d. Results and Discussion The bacterium isolated from the natural environment was identified as Pseudomonas stutzeri. (Table 1). It was used for the assessment of biodegradability of LDP and PP. All experiments were done in triplicates and carried out for 45 d and the results are presented as average of the three. Tensile strength (TS) and extension of treated plastic films with and without the inoculum were studied experimentally at 15 d interval. The changes in per cent extension, tensile strength, and elongation were analyzed. There was reduction in per cent extension, which was found to be maximum in polypropylene, 6.4 per cent more than in low density polyethylene and 13.3 per cent in comparison to control. Tensile strength was found to decrease with increase in incubation period on polypropylene. Steep reduction in elongation was recorded in both the plastic films with increased incubation time with a minimum 2.5 cm reduction observed in polypropylene (Table 2). The minimum per centage change in the tensile strength was in LDP on 15 th d i.e., -18 per cent and maximum was observed in PP on 45 th d, i.e. 66 per cent (Figure 1).

4 296 J SCI IND RES VOL 63 MARCH 2004 Duration Zero day treated with HNO 3 Table 2 Change in tensile strength, elongation and percentage extension of different plastic samples Per cent extension Polypropylene Samples Tensile strength Elongation Per cent N/cm 2 extension Low density polyethylene Tensile strength N/cm 2 Elongation cm cm 15 th d Control cm cm Sample cm cm 30 th d Control cm cm Sample cm cm 45 th d Control cm cm N/cm 2 = Newton/cm 2 Sample cm cm Figure 1 Change in tensile strength per cent of treated plastic samples in the presence of bacteria with increase in incubation Figure 2 Change in turbidity values at 620 nm of the media containing different bacterial treated plastic samples in comparison with glucose There was generally an increase in tensile strength, usually with decrease in extension, in the early stages of degradation. This being followed by a decrease in tensile strength and further reduction in extension, as also reported by Whitney et al 8. This may be due to cleavage of the polymer chain.

5 SHARMA & SHARMA: DEGRADATION ASSESSMENT OF LOW DENSITY POLYTHENE 297 Figure 3 Change in BOD (mg/l) during degradation of plastic samples in comparison with glucose. Figure 4 Total viable count (log CFU/mL) during degradation of different plastic samples (LDP and PP) along with glucose Degradation was therefore considered to have taken place when there was a statistically significant increase or decrease in tensile strength or decrease in extension. The loss of tensile strength after incubation in comparison to control at room temperature, may be purely because of P. stutzeri activity. The maximum turbidity was accounted in glucose on 45 th d (1.99). The growth curve of bacteria in media containing three carbon sources, i.e. LDP, PP and glucose was observed (Figure 2). Higher growth rate was found with glucose in comparison to LDP and PP, but it was observed that the growth proportionally increased in minimal media and it may be predicted that the bacteria was solely dependent on polythene films for its carbon source. The BOD test with slight procedural modification can be used to measure biodegradability of modified plastic films 5. Assuming that only bacteria in the form of organic nutrients used up the components of plastic sheet and soil, virtually the mass loss was interpreted for the BOD increase. Maximum BOD values were recorded with glucose on 45 th d and minimum with LDP on 15 th and 30 th d. Thus, as the incubation period increased, BOD values increased, because the bacteria used the dissolved oxygen and the organic nutrients were consumed (Figure 3).

6 298 J SCI IND RES VOL 63 MARCH 2004 The cfu/ml of the media containing plastic films when observed on specific incubation time intervals, minimum counts 10 1 and maximum 10 3 were recorded for LDP 2 on 15 th d and for glucose on 45 th d of incubation, respectively (Figure 4). The increase in cfu/ml with the increase in incubation period indicates that bacteria might be utilizing plastic as its sole source of carbon, thus may be supporting and enhancing its degradation. Acknowledgement The authors are thankful to Government Engineering College Jabalpur for permitting them to use the tensile testing machine 1.4-f (STD). References 1 Atlas R M, Microbial ecology: fundamentals and applications, Third ed, 1993, pp Fiechter A, Plastics from bacteria & for bacteria: poly (B- hydroxyalkanoates) as natural, biocompatible & biodegradable polyesters (Springer-Verlag, New York) 1990, pp Molitoris H P, Moss S T, Konig de G J M & Jendrossek D, Scanning electron microscopy of polyhydroxyalkanate degradation by bacteria, Appl Microbial Biotech, 46 (1996) Krieg R N& Holt G J, Bergey s manual of systematic bacteriology, Vol 1 (Williams & Wilkins Co., Baltimore) MacFaddin F J, Biochemical tests for identification of medical bacteria, (Williams & Wilkins Co., Baltimore, MD) Krupp R L& Jewell J W, Biodegradability of modified plastic films in controlled biological environments, Environ Sci Technol, 26, (1992), Bryant T N, Probabilistic identification of bacteria, Med Statistics & Computing, (University of Southampton, Southampton, UK) Whitney J P, Swafflied H C & Graffham J A, The environmental degradation of thin plastic films, Int Biodeteriot Biodegrad, 31 (1993) APHA (American Public Health Association), Standard method for examination of water & wastewater analysis, 16 th ed, (APHA- AWWA-WPC1, Washington DC) Starnecker A & Menner M, Assessment of biodegradability of plastics under standard composting conditions in laboratory test system, Int Biodeteriorat Biodegrad, (1996) 85 *Author for correspondence Ph. No. (0761) ,Fax: (0761) , , anjoo_ 1999@yahoo.com

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International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 4 Number 6 (2015) pp. 708-713 http://www.ijcmas.com Original Research Article Enrichment, Isolation and Identification