Biodegradation of low density polyethylene (LDPE) by fungi … · 2018-12-15 · K.Vijaya Ramesh et...

Journal of Microbiology and Biotechnology Research

Scholars Research Library

J. Microbiol. Biotech. Res., 2011, 1 (4):131-136

(http://scholarsresearchlibrary.com/archive.html)

ISSN : 2231 –3168

CODEN (USA) : JMBRB4

131 Available online at www.scholarsresearchlibrary.com

Biodegradation of low density polyethylene (LDPE) by fungi isolated from municipal landfill area

R. Pramila and K.Vijaya Ramesh*

Dept. of Plant Biology & Plant Biotechnology, Quaid-e-Millath Govt. College for Women, Chennai

______________________________________________________________________________

ABSTRACT The present article reveals the biodegradation of low density polyethylene by two fungal strains isolated from municipal landfill area. The degrading ability of the two fungal strains was evaluated by performing colonization studies, SEM and Sturm test analysis. Colonization studies on LDPE film was performed over a period of one month by measuring the fresh weight of the fungus. LDPE films colonized by fungus were analysed by scanning electron microscope (SEM) for any structural changes caused in the LDPE films. LDPE degradation by the fungal strains was further evaluated by measuring the CO2 evolved which was calculated gravimetrically and volumetrically by Sturm test. The isolated fungi showed a promising degradation pattern as analyzed by the above mentioned three methods. Fungi were identified as Mucor circinilloides and Aspergillus flavus. Keywords: Biodegradation, LDPE, Scanning electron microscopy, Sturm test, Mucor, Aspergillus. ______________________________________________________________________________

INTRODUCTION

Plastics are synthetic substances produced by chemical reactions. Plastics are polymers, that consist of monomers linked together by chemical bonds.Among polymers, polyethylene is a thermoplastic polymer produced by monomers of ethylene.Polyethylene contains the chemical elements carbon and hydrogen.Polyethylene is classified into several different categories such as HDPE.LLDPE,LDPE. Low density polyethylene(LDPE) is a thermoplastic made from petroleum.LDPE materials are strong, light-weight and durable thus are widely used for manufacturing various containers, dispensing bottles, plastic bags and various molded laboratory wares. Plastic bags brings a lot of convenient to peoples life but at the same time it also brings long term harms.The waste materials collected are of all types including plastic materials such as polyethylene bags,plastic cups,etc,when they burnt produce toxic gases posing a health

K.Vijaya Ramesh et al J. Microbiol. Biotech. Res., 2011, 1 (4):131-136 ______________________________________________________________________________


hazard.Inhaling of such gases causes lung diseases and cancer.The decomposition of plastic bags takes about 1000years. Previous work done on biodegradation of LDPE revealed that microorganisms such as bacteria, fungi and actinomycetes are involved in the degradation of both natural and synthetic plastics [1]. Biodegradation of polyethylene has been studied extensively earlier [2-5] but the results were based on polyethylene blended with starch. There are few reports that deal with microbial degradation of polyethylene materials,[6-10]. Fungi survive environments with low nutrient availability, low pH and low moisture as well. The aim of this study was to investigate the ability of fungi isolated from municipal landfill area to degrade LDPE.

MATERIALS AND METHODS : Preparation of LDPE powder: [9]LDPE sheets were cut into bits and immersed in xylene. It was boiled for 15 min as xylene dissolves the LDPE film and the residue was crushed while it was warm by using band gloves. The LDPE powder so obtained was washed with ethanol to remove residual xylene and allowed to evaporate to remove ethanol. The powder was dried in hot air oven at 60◦C over night. Isolation of fungal strain: Soil sample was collected from the municipal solid waste landfill area,Pallikaranai,Chennai.5gm of soil sample was inoculated in sterilized synthetic medium(SM).SM contains the following constitutions in 1000ml distilled water(K2HPO4, 1 g; KH2PO4, 0.2 g; NaCl, 1 g; CaCl2.2H2O, 0.002 g; (NH4)2SO, 1 g; MgSO4.7H2O, 0.5 g; CuSO4.5H2O, 0.001 g; ZnSO4.7H2O,0.001 g; MnSO4.H2O, 0.001 g and FeSO4.7H2O, 0.01 g.100mg of LDPE powder was added.Incubated at room temperature for 1week. Colonization study: The colonizing capacity of the fungi on LDPE film was studied by growing the fungi in petriplates. SM was aseptically poured into petriplates. LDPE sheets were cut into small pieces 2cm×2cm of similar weight, disinfected with 70% ethanol for 30 min and transferred to sterile distilled water for 20 min. Five LDPE sheets of same weight were placed in petriplates containing the Synthetic Medium without yeast extract and they were inoculated with 5 similar sized colonies of fungi using the cork borer. The petriplates were incubated at room temperature and results were observed after 1 week to 28 days. Scanning electron microscope analysis: LDPE films colonized by fungal strains for period of 1month were analyzed by scanning electron micrographs. CO2 evolution test– Modified Sturm Test [11]: CO2 evolved as a result of LDPE biodegradation was determined gravimetrically and volumetrically by Sturm test. Gravimetric analysis: Test and control bottles containing SM supplemented with LDPE powder was prepared. Fungal strain was added in the test bottle. Sterile air was allowed to flow through 1M KOH solution containing bottles (air pretreatment bottles). The CO2 free air was passed to the test bottles. The CO2 free air was utilized by the inoculum that released CO2 (after metabolizing LDPE) in the absorption bottle. The test was performed at room temperature for 1week.After 1week the amount of CO2 produced in absorption bottle was calculated by adding 0.1 M Bacl2 which forms a precipitate of barium carbonate. CO2 released was gravimetrically



calculated by measuring the weight of the precipitate formed (Barium carbonate). Difference in the values obtained in the control and test was noted. The dissolved carbon dioxide present in the medium was also measured volumetrically using titration method. Briefly, sample (medium- 25ml) was taken in a conical flask and 0.05ml of 0.1N Thiosulphate solution was added. After the addition of 2 drops of methyl orange indicator, this solution was titrated against 0.02M Sodium Hydroxide solution. End point was the change in color from orange red to yellow. Following this, two drops of phenolphthalein indicator was added and titration continued till a pink color developed. Volumes of the titrant used were noted and the amount of CO2 evolved was calculated using the formula:

A X B X 50 X 1000 V where A= mL of NaOH titrant B= normality of NaOH

V= mL of the sample

RESULTS

From the colonization studies, there was an increase in the fresh weight of the fungal isolates observed after 7 to 28days of the experiments(Table 1) SEM analysis: Scanning electron micrographs of LDPE films treated with Aspergillus flavus and Mucor circinelloides showed structural changes such as cracks,formation of pits, sporangia and spores grown through LDPE film were observed. CO2 evolution test (Sturm test):The total amount of CO2 evolved as calculated gravimetrically and volumetrically for the isolate Mucor circinelloides was found to be 5.9g/L and Aspergillus flavus evolved around 4.4g/L of CO2 (Table 2) Identification of fungal strains: Fungal strain was identified by slide culture technique as Aspergillus flavus and Mucor circinelloides

DISCUSSION

The present study deals with the isolation of polyethylene degrading micro organisms from the municipal landfill soil. Low density polyethylene films were used in this study.The micro organisms with the ability to degrade LDPE were isolated in synthetic medium supplemented with LDPE powder and these organisms were used for degradation study. Several methods were employed to monitor the biodegradation of the polyethylene. Colonization study with the fungi showed a result of visible increase in the fresh weight of fungus after 7th to 28th days . Fungal strains found colonized on the surface of LDPE causing some physical changes that was evident in our study by Scanning electron microscope results. There are previous report for the result of SEM analysis after 2months of incubation with fungal strains[9,12-14]..



Sturm test was commonly employed for evaluation of the biodegradability of polymer materials.Modified sturmtest have been used for the measurement of CO2 in gaseous form in SM.

A

B

C

Fig:1 Scanning electron micrographs of LDPE A)control; B)Treated with Aspergillus flavus; C) Treated with Mucor cicinelloides

Table 1 Fresh weight of the fungus

Name of the isolate Fresh weight of the fungus After 7 After 14 After 21 After 28 days days days days Aspergillus flavus 0.015 0.018 0.021 0.34 Mucor cicinelloides 0.007 0.009 0.021 0.029



Table 2 Quantification of carbon dioxide evolution after degradation of LDPE (1 week reading) TheCO2 evolution test gave a valid data about the degradation rate. We obtained very good results from the CO2 evolution test done for a period of 1 week (Sturm test). There has been a report on biodegradation of LDPE with a fungal strain [12,14]with a Sturm test value of 10g/L and 1.85g/L for 30days period. Sturm test was commonly employed for evaluation of the biodegradability of polymer materials.Modified sturm test have been used for the measurement of CO2 both in gaseous and dissolved form in SM. TheCO2 evolution test gave a valid data about the degradation rate. We have done some preliminary experiments to prove that LDPE can be degradaded if the microbial strain is isolated. From the present study it can be concluded that fungal isolates are able to grow on minimal medium with LDPE as a sole carbon source. Hydrophobic nature of LDPE film acts as substratum for microorganisms which colonize the surface of the LDPE films. Production of CO2 during Sturm test indicates positive degradability test for the polyethylene and this test has given fulfillment to the objective of this study.

Acknowledgements The authors are grateful to the University Grants Commission for providing financial assistance for the completion of this work.

REFERENCES

[1] Gu,J.D.,T.E.Ford,and R. Mitchell.2000a.Microbial corrosion of metals. In :Review.(Ed),The Uhlig Corrosion Handbook,2nd Edition. Wiley ,New York, pp. 915-927.

[2] Albertsson, A. C. 1980. Europ. Polym. J. 16: 623-630. [3] Breslin, V.T. 1993. J. Environ. Polym. Degrad. 1: 127-141. [4] Breslin, V.T., R. L. Swanson, 1993. J. Air Waste Management Association. 43: 325-335. [5] Imam, S.H., Gould, J.M., 1990. Applied and Environmental Microbiology 56: 872–876. [6] Lee, B., A.L. Pometto III, A. Fratzke, and T.B. Bailiey Jr. (1991). Appl. Environ. Microbiol., 57: 678-685. [7] El-Shafei, H.A., N.H. El-Nasser,. A. L. Kansoh.,.and A.M. Ali. (1998) Polym. Degrad. Stab. 62: 361-365. [8] Kathiresan, K. (2003). Rev. Biol. Trop. 51: 629-633. [9] Shah, A. A.,(2007). Role of microorganisms in biodegradation of plastics. Ph. D. Thesis. Quaid-i-Azam University, Islamabad, Pakistan.

[10] Shah, A. A., F. Hassan., A. Hameed., and S. Ahmed. (2008). Biotech. Adv. 26: 246-265. [11] Muller, R. J., J. Augusta, and M. Pantke. 1992. Material and Organismen. 27: 179-189 [12] Aamer Ali Shah, H. Fariha, H. Abdul, and A. Javed Iqbal.( 2009). Afri. J. Microb. Res. 3: 658-663.

Name of the isolate Total CO2 evolved (g/L) Aspergillus flavus 4.4507 Mucor cicinelloides 5.9328



[13] S. Zahra., S. A. Shojaosadati., M. Mohammad-Taheri., and M. Nosrati. (2010). Waste Management. 30: 396-401. [14] Muhammad Ishtiaq Ali, P. Qaiser, A. Bashir, J. Imran, R. Raja, A. Saadia, A. Naima, G. Pir Bux, A. Safia, and H. Abdul. (2009). Int. J. Agri. Biol. 11: 577-580.

Biodegradation of low density polyethylene (LDPE) by fungi … · 2018-12-15 · K.Vijaya Ramesh et...

Documents

Transcript of Biodegradation of low density polyethylene (LDPE) by fungi … · 2018-12-15 · K.Vijaya Ramesh et...