Title of Thesis

Biodegradability of Synthetic Plastics Polystyrene and Styrofoam by Fungal Isolates

Author(s)

Naima Atiq

Abstract
Polystyrene is a rigid plastic that is commonly used in crystalline and foamed form. Biodegradation of polystyrene is very slow in natural environment and it persists for longer period of time as solid waste. The aim of the study was to investigate the biodegradation process of polystyrene and explore the ways to enhance the biodegradation process. Soil burial method was used to isolate microorganisms. The plastic films recovered from soil after 8 months were incubated on mineral salts media (MSM) agar plates for 3 months to get the growth of only those microorganisms that were able to grow with polystyrene for longer time. Six fungal and five bacterial stains were isolated and identified. Three fungal isolates were selected on the basis of biodegradability of polystyrene films in shake flask transformation experiments analysed by Fourier transform Infrared (FTIR) spectroscopy.
The selected fungal strains were characterized taxonomically on the basis of sequence homology of conserved regions of 18S rRNA and were identified as Rhizopus oryzae NA1, Aspergillus terreus NA2 and Phanerochaete chrysosporium NA3. The 18S rRNA sequences were deposited in NCBI database with accession numbers in Genbank FJ654430, FJ654431 and FJ654433 for strain NA1, NA2, NA3 respectively. The biodegradation of polystyrene was studied by CO2 evolution test (Sturm test) all the isolated showed higher CO2 levels in the test as compared to control showing effective mineralization of polystyrene.
Biodegradation studies in liquid media with polystyrene films, expanded polystyrene (EPS) films and beads were conducted in the static and shake flask (120rpm) fermentation experiments at 30 ºC. Scanning electron microscopic (SEM) analysis showed that the fungal isolates were able to establish mycelia on the polymer surface and maximum growth was observed in glucose added mineral salts media. FTIR spectra of the treated films showed increase in absorption spectra around 536 cm-1, 748 cm-1(mono substituted aromatic compound), 1026 cm-1, 1450 cm-1, 1492 cm-1(C=C stretching vibration of aromatic compounds), 2916 cm-1, 3400 cm-1(aryl-H stretching vibrations). Major changes were observed in 1000-1700 cm-1 and 3400 cm-1 region which indicated depolymerisation and degradation into monomers.
Molecular weight distribution was studied by gel permeation chromatography (GPC).The weight average molecular weight and number average molecular weight increased in the samples of polystyrene films and EPS beads treated with the fungal isolates as compared to control while decreased in case of expanded polystyrene. The polydispersity decreased in polystyrene and increased in EPS films. In proton nuclear magnetic resonance (1H-NMR) spectra of polystyrene and expanded polystyrene intensities of the signals were increased in treated samples as compared to control but treated samples did not show any significant change in the spectra.
The degradation products of the polystyrene and expanded polystyrene were analysed by HPLC. 1-phenyl-1,2-ethandiol, 2-phenylethanol and phenyacetaldehyde and styrene oxide, which were oxidation degradation products of monomer styrene, were detected in most of the cases. 1-phenyl-1,2-ethandiol was detected with highest concentration of 21.3 ppm in media sample of polystyrene incubated with A. terreus NA2 in shake flask and 34.7 ppm with P. chrysosporium NA3 in static conditions.
Polystyrene films were given pretreatment of UV irradiation (1-2 hr. at λ 254 nm) and heat (60˚C and 80˚C for 1 hour) and then biodegradation was studied. UV pretreatment of 2 hours showed enhancing effect on biodegradation by fungal isolates indicated a decrease of weight average molecular weight in the treated samples. Heat pretreatments did not show enhancing effect on biodegradation except P. chrysosporium NA3 treatment of heat pretreated polystyrene films. Enhancing effect of glucose on biodegradation of polystyrene films was observed in FTIR spectral analysis, when glucose was used as additional carbon source in mineral salts media,
The soil buried films of polystyrene for six months showed very significant degradation in FTIR and GPC analysis. The scanning electron micrographs of the treated films from all the samples also confirmed the biodegradation process by showing some changes in structure and colonization of fungi on the films. The selected fungal strains are capable of utilising polystyrene as a sole carbon source and have potential to be used for polystyrene biodegradation in the environment.

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