Biodegradability of Synthetic Plastics Polystyrene and ...

Biodegradability of Synthetic Plastics

Polystyrene and Styrofoam by Fungal

Isolates

By

Naima Atiq

Department of Microbiology Quaid-i-Azam University

Islamabad

2011

Biodegradability of Synthetic Plastics

Polystyrene and Styrofoam by Fungal

Isolates

A Thesis submitted in the partial fulfillment of the

requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

By

Naima Atiq

Department of Microbiology

Quaid-i-Azam University

Islamabad

2011

IN THE NAME OF ALLAH

THE MOST

COMPASSIONATE

AND

MERCIFUL

DEDICATED TO

AMMI AND AbBu

DECLARATION

The material contained in this thesis is my original work and

I have not presented any part of this thesis/work elsewhere

for any other degree.

Naima Atiq

CERTIFICATE

This thesis by Naima Atiq is accepted in its present form by

the Department of Microbiology, Quaid-i-Azam University,

Islamabad, as fulfilling the thesis requirement for the degree

of Doctor of Philosophy in Microbiology.

Internal examiner

___________________

(Dr. Safia Ahmed)

External examiner

___________________

External examiner

___________________

Chairperson

___________________

Dated: _____________

Table of Contents

Topic Page No.

List of Tables

List of Figures

List of Abbreviations

Acknowledgements

Abstract

Chapter 1 Introduction

Chapter 2 Literature Review

Chapter 3 Material & Methods

Chapter 4 Results

Chapter 5 Discussion

Conclusions

Future prospective

References

Appendices

I

II

IX

XI

XII

1

9

27

43

124

131

132

133

148

i

LIST OF TABLES

Table No. Title Page.

No.

3.1 Composition of Mineral Salt Media 29

3.2 PCR mixture for fungal DNA amplification 31

3.3 PCR mixture for Bacterial 16S ribosomal DNA Amplification 33

3.4 Experimental conditions for bacterial 16S ribosomal DNA

amplification

33

3.5 PCR mixture for amplification of ribosomal DNA for DGGE 35

4.1 Molecular identification of microorganisms isolated from

polystyrene film

50

4.2 CO2 evolved in 8 weeks duration of biodegradation of PS by

R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3

measured gravimetrically by Sturm test (Test; PS as sole

Carbon source, Control; no carbon source)

57

4.3 Gel permeation chromatography analysis of PS films treated

with fungal isolates after 8 weeks incubation at 30ºC, 120rpm

61

4.4 Gel permeation chromatography analysis of EPS films treated

with fungal isolates after 8 weeks incubation at 30ºC, 120rpm

70

4.5 Gel permeation chromatography analysis of EPS beads

treated with fungal isolates after 8 weeks incubation at 30ºC

78

4.6 Gel permeation chromatography analysis of UV pre-treated

PS films biodegraded by fungal isolates after 8 weeks

incubation at 30ºC, 120rpm

85

4.7 Gel permeation chromatography analysis of heat pre-treated

PS films biodegraded by fungal isolates after 8 weeks


93

4.8 Gel permeation chromatography analysis of PS films treated

with fungal isolates with 0.01% glucose after 12 weeks


104

4.9 Gel permeation chromatography analysis of soil buried PS

films biodegraded by fungal isolates for 6 months

120

ii

LIST OF FIGURES

Fig. No. Title Page

No.

1.1 Polymerization of styrene to produce polystyrene. 2

1.2 Types of polystyrene. 2

2.1 Styrene metabolic pathways found in bacteria (dotted line) and

fungi (bold line) composed after Francesc et al.,(2005);

O’Leary et al., (2002); Prenafeta Boldu´ et al., (2001); Weber

et al., (1995); Cox (1995); and Holland et al., (1993). (1)

Phialophora sessilis CBS 238.93; and (2) Clonostachys rosea

CBS 102.94.

21

3.1 Experimental set up of Sturm test for biodegradation studies. 39

4.1 Growth of fungal strains on polystyrene films after soil burial

(8 months) on mineral salts agar medium.

45

4.2 Environmental scanning electron micrographs of Polystyrene

films used for isolation of microorganisms showing fungal

growth 250x (a) and 2000x (b).

46

4.3 Colony morphology of selected fungal strains NA1 (a), NA2

(b) and Na3 (c) on potato dextrose agar medium.

47

4.4 Agarose gel electrophoresis visualisation of PCR Amplified

DNA using 0.8% agarose gel in TAE buffer (Lane 1,

HyperLadderTM 1; Lane 6, PCR product from strain NA1;

Lane7 from NA2 and Lane 8, PCR products from strain NA3).

48

4.5 Denaturing gradient gel electrophoresis (40% - 60%) analysis

of fungal diversity with plastic films buried in soil for 8

months, used for isolation of fungal strains (Lane 5, 6, 7, and

8).

50

4.6 Growth of fungal isolates on PS in MSM agar plates after 8

weeks incubation period at 30ºC, PS film on surface, R. oryzae

NA1 (a), A. terreus NA2 (b), P. chrysosporium NA3 (c),

Polystyrene film partially submerged in media and checked for

growth of R. oryzae NA1 (d), A. terreus NA2 (e) and P.

chrysosporium NA3 (f) on agar plate.

54

iii

4.7 Environmental Scanning Electron Micrographs of PS films

inoculated on MSM agar plates after 8 weeks incubation period

with R. oryzae NA1 (a) A. terreus NA2 and (b) P.

chrysosporium NA3 (c) (1000x).

55

4.8 Environmental scanning electron micrographs of PS films

control (a), treated with R. oryzae NA1 (b), A. terreus NA2 (c)

and P. chrysosporium NA3 (d) in shaker (30°C, 120rpm) for 8

weeks (2000x).

57

4.9 FT-IR spectra of PS treated with R. oryzae NA1 (a), A. terreus

NA2 (b) and P. chrysosporium NA3 (c) in shaker (30ºC,

120rpm) for 4 weeks (w4), 8 weeks (w8) and 12 weeks (w12)

along with untreated control.

58

4.10 FT-IR spectra of PS treated with R. oryzae NA1 (a), A. terreus

NA2 (b) and P. chrysosporium NA3 (c) in static conditions

(30ºC) for 4 weeks ((w4), 8 weeks (w8)) and 12 weeks (w12)


59

4.11 1H-NMR analysis of Polystyrene control (a) treated with R.

oryzae NA1 (b) A. terreus NA2 (c) and P. chrysosporium NA3

(d) in shaker (30ºC, 120rpm) for 8 weeks.

61

4.12 1H-NMR analysis of PS control (a) treated with R. oryzae NA1

(b) A. terreus NA2 (c) and P. chrysosporium NA3 (d) in static

conditions (30ºC) for 8 weeks.

62

4.13 HPLC analysis of biodegradation products of polystyrene

treated with R. oryzae NA1, A. terreus NA2 and P.

chrysosporium NA3 in shaker (30ºC, 120rpm) (a) in static

conditions (30°C) (b).

63

4.14 FT-IR spectra of EPS treated with R. oryzae NA1 (a) A. terreus

NA2 (b) and P. chrysosporium NA3 (c) in shaker (30ºC,

120rpm) for 4 weeks ((w4), 8 weeks (w8)) and 12 weeks (w12)


67

4.15 FT-IR spectra of EPS treated with R. oryzae NA1 (a) A. terreus

NA2 (b) and P. chrysosporium NA3 (c) in static conditions

(30ºC) for 4 weeks (w4), 8 weeks (w8) and 12 weeks (w12).

68

iv

4.16 1H-NMR analysis of EPS film control (a) treated with R. oryzae

NA1 (b) A. terreus NA2 (c) and P. chrysosporium NA3 (d) in

static conditions (30ºC) for 8 weeks.

70

4.17 HPLC analysis of biodegradation products of EPS treated with

R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 in

shaker (30ºC, 120rpm) (a) in static conditions (30ºC) (b).

71

4.18 Growth of fungal isolates on EPS beads in mineral salt media

agar plates after 8 weeks of incubation at 30°C in static

conditions control (no fungi) (a) inoculated with R. oryzae NA1

(b) A. terreus NA2 (c) and P. chrysosporium NA3 (d).

74

4.19 Environmental scanning electron micrographs of EPS beads

control (a) treated with R. oryzae NA1 (b) A. terreus NA2 (c)

and P. chrysosporium NA3 (d) in static conditions (30ºC) for 8

weeks (70x)

75

4.20 Environmental scanning electron micrographs of EPS beads

control (a) treated with R. oryzae NA1 (b) A. terreus NA2 (c)

and P. chrysosporium NA3 (d) in static conditions (30ºC) for 8

weeks (2000x)

76

4.21 1H-NMR analysis of EPS beads control (a), treated with R.

oryzae NA1(b) A. terreus NA2 (c) and P. chrysosporium NA3

(d) in static conditions (30ºC) for 8 weeks

78

4.22 HPLC analysis of EPS beads treated with R. oryzae NA1, A.

terreus NA2 and P. chrysosporium NA3 in shaker (30ºC,

120rpm) (a) in static conditions (30ºC) (b) for the degradation

products 1-phenyl-1,2-ethandiol, 2-phenylethanol,

phenylacetaldehyde and styrene oxide.

79

4.23 FT-IR spectra of UV 1 hour pretreated PS incubated with R.

oryzae NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3

(c) in shaker (30°C, 120rpm) for 4 weeks (w4) and 8 weeks

(w8) along with untreated control.

82

4.24 FT-IR spectra of UV 2hour heat pretreated PS incubated with

R. oryzae NA1 (a), A. terreus NA2 (b) and P. chrysosporium

NA3 (c) in shaker (30ºC, 120rpm) for 4 weeks (w4) and 8

83

v

weeks (w8) along with untreated control.

4.25 1H-NMR analysis of UV 1hour pretreated PS control (a),

treated with R. oryzae NA1 (b) with A. terreus NA2 (c) and

treated with P. chrysosporium NA3 (d) in shaker (30ºC,

120rpm) for 8 weeks.

85

4.26 1H-NMR analysis of UV 2hour pretreated PS control (a),

treated with R. oryzae NA1 (b) with A. terreus NA2 (c) and

treated with P. chrysosporium NA3 (d) in shaker (30ºC,


86

4.27 HPLC analysis for the UV pre-treated Polystyrene

biodegradation products in culture broth of shake flask

experiment (30ºC, 120rpm) with R. oryzae NA1, A. terreus

NA2 and P. chrysosporium NA3 for 8 weeks 1hour UV pre

treated (a) 2hour UV pre treated (a).

87

4.28 FT-IR spectra of 60ºC 1hour heat pretreated PS incubated with


NA3(c) in shaker (30ºC, 120rpm) for 4 weeks (w4) and 8


90

4.29 FT-IR spectra of 80ºC 1hour heat pretreated PS incubated with


NA3 (c) in shaker (30ºC, 120rpm) for 4 weeks (w4) and 8


91

4.30 1H-NMR analysis of the broth of heat (60ºC, 1hour) pre treated

PS control with no fungal treatment (a) treated with R. oryzae

NA1 (b) A. terreus NA2 (c) and P. chrysosporium NA3 (d) in

shaker (30ºC, 120rpm) for 8 weeks.

93

4.31 1H-NMR analysis of heat (80°C, 1hour) pretreated PS control

(a), treated with R. oryzae NA1 (b) A. terreus NA2 (c) and P.

chrysosporium NA3 (d) in shaker (30ºC, 120rpm) for 8 weeks.

94

4.32 HPLC analysis of heat pre-treated PS incubated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 in shaker

(30ºC, 120rpm) for 8 weeks 60ºC 1hour heat treated (a), 80ºC

1hour heat treated (b), in shaker (30ºC, 120rpm).

95

vi


control (a), treated with R. oryzae NA1 (b), A. terreus NA2 (c)

and P. chrysosporium NA3 (d) with 0.01% glucose in media in

shaker (30ºC, 120rpm) for 8 weeks (2000x).

98

4.34 FT-IR spectra of PS incubated with R. oryzae NA1 (a), A.

terreus NA2 (b) and P. chrysosporium NA3 (c) with 0.01%

glucose, in shaker (30ºC, 120rpm) for 4 weeks (w4) and 8


99



glucose in static conditions at 30ºC for 4 weeks (w4) and 8


100

4.36 FT-IR spectra of PS incubated with R. oryzae NA1, A. terreus

NA2 and P. chrysosporium NA3 with 0.1% glucose at 30ºC,

120rpm after 4 weeks.

101



glucose in static conditions at 30ºC for 4 weeks (w4), 8 weeks

(w8) and 12 weeks (w12) along with untreated control.

102

4.38 HPLC analysis of broth of PS films after incubation with R.

oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 with

added 0.01% glucose in shaker (30ºC, 120rpm) (a), in static

conditions (30ºC) (b).

104

4.39 HPLC analysis of broth of PS films incubated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 with added

0.1% glucose in shaker (30°C, 120rpm) for 4 week (b), in static

conditions (30ºC) (a).

105

4.40 FT-IR spectra of EPS incubated with R. oryzae NA1 (a), A.


glucose, in shaker (30ºC, 120rpm) for 4 weeks (w4) and 8


107



108

vii

glucose at 30ºC for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

4.42 FT-IR spectra of EPS incubated with R. oryzae NA1, A. terreus

NA2 and P. chrysosporium NA3 with 0.1% glucose at 30ºC,

120rpm after 4 weeks.

109



glucose in static conditions at 30ºC for 4 weeks (w4), 8 weeks

(w8) and 12 weeks (w12) along with untreated control.

110

4.44 HPLC analysis of broth samples of EPS films treated with R.

oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 in

shaker (30ºC, 120rpm) (a) in static conditions (30ºC) (b) with

added 0.01% glucose.

111

4.45 HPLC analysis of broth samples of EPS films treated with R.

oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 (30ºC,

120rpm) for 4 week (a) in static conditions (30ºC) (b) with

added 0.1% glucose in shaker.

112


buried in soil inoculated with R. oryzae NA1 (a), A. terreus

NA2 (b) and P. chrysosporium NA3(c) at 30ºC for 4 months

115

4.47 Denaturing gradient gel electrophoresis (DGGE) (40% - 60%)

analysis of fungal community attached to polystyrene films

buried in unsterilized soil (3) with fungal isolates NA1 (4),

NA2 (5), NA3 (6) and sterilized soil (10) with fungal isolates

NA1 (11), NA2 (12), NA3 (13).

116

4.48 FT-IR spectra of soil buried PS films incubated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 at 30ºC for 2

months (a) for 4 months (b) for 8 months (c).

117

4.49 FT-IR spectra of soil buried PS films inoculated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 in

unsterilized soil (a) sterilized soil (b) in flower pots for 6

months.

118

4.50 FT-IR spectra of PS starch blend film, incubated with R. oryzae 121

viii

NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) at

30ºC, 120rpm for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

4.51 FT-IR spectra of PS starch blend film, incubated with R. oryzae

NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) at

30ºC for 4 weeks (w4) and 8 weeks (w8) along with untreated

control.

122

4.52 HPLC analysis of PS starch blend films treated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 in shaker

(30ºC, 120rpm) (a), in static conditions (30ºC) (b).

123

1A Phylogenetic tree of R. oryzae NA1 148

2A Phylogenetic tree of A. terreus NA2 149

3A Phylogenetic tree of P. chrysosporium NA3 150

ix

List of Abbreviations

ABS Acrylonitrile-Butadiene Styrene

AFM Atomic Force Microscopy

ATR Attenuated Total Reflection

BLAST Basic Local Alignment Search Tool

CF Consumption Factor

DGGE Denaturing Gradient Gel Elecctrophoresis

DSC Differential Scanning Calorimetry

EH Epoxide Hydrolase

ELISA Enzyme-Linked Immunosorbent Assay

EPDM-g-PS Ethylene-Propylene-Diene-graft-Polystyrene

EPS Expandable Polystyrene

ESEM Environmental Scanning Electron Microscopy

FTIR Fourier Transform Infrared Spectroscopy

FTIR-PAS Fourier Transform Infrared Photoacoustic

Spectroscopy

GPC Gel Permeation Chromatography

GPS General purpose polystyrene

HDPE High-Density Polyethylene

HIPS High-Impact Polystyrene

HPLC High Pressure Liquid Chromatography

LDPE Low-Density Polyethylene

LDH Layered Double Hydroxides

LP Lignin Peroxidases

MALDITOFMS Matrix-Assisted Laser Desorption/Inonization Time-

Of-Flight Mass Spectrometry

MALS Multi-Angle Light Scattering Detection

MFR Mass-Flow Rate

Mn-Ps Manganese Peroxidases

MSW Municipal Solid Waste

x

MSM Mineral Salts Media

NMR Nuclear Magnetic Resonance Spectroscopy

PAA Phenylacetic Acid

PAN Polyacrylonitrile

PB Polybutadiene

PCL Polycaprolactone

PEO Poly(ethylene oxide)

PET Poly(ethylene terephthalate)

PHA Polyhydroxyalkanoate

PMS Poly(p-maleimidostyrene)

PS Polystyrene

SAN Styrene-Acrylonitrile

SBC Styrene Butadiene Copolymer

SBR Styrene-Butadiene Rubber

SEC Size Exclusion Chromatography

SEM Scanning Electron Microscopy

SLS Static Light Scattering

SPMA 3-Sulfopropyl Methacrylate

sPS Syndiotactic Polystyrene

TCA Tricarboxylic Acid

TGA Thermogravimetric Analysis

TMPTA Trimethylol Propane Trimethacrylate

TPS Thermoplastic Starch

UPR Unsaturated Polyester Resin

XRD X-Ray Diffraction

xi

Acknowledgements

In the Name of Allah the most beneficent and merciful. I owe my deepest thanks and praise to

Allah Almighty, Who gave me opportunities and courage to explore the world of

Knowledge. I don’t have words that can completely express my humble gratitude to Allah

who enabled me to complete this research work successfully.

I present Salam to Holy Prophet Hazrat Mohammad (Peace be upon him), who is a

blessing for mankind and enjoined upon his followers (Men and Women) to seek Knowledge

from cradle to grave.

Special thanks and recognition to the efforts of my Knowledgeable and most intellectual

supervisor Dr. Safia Ahmed, Professor and Chairperson , Department of Microbiology,

Quaid-i-Azam University, Islamabad, Whom valuable guidance, permanent motivation and

kind attention made it possible for me to accomplish my research work and thesis write up.

She will be a continuous source of inspiration for me in my life.

I express my sincere and deep regard to the respected Dr. Abdul Hameed Professor and Deen,

Biological Sciences, Quaid-i-Azam University, Islamabad, for his support and cooperation

during the course of my research endeavour.

My deepest thanks are extended to Dr. Fariha Hassan, Assistant Professor, Department of

Microbiology and Dr. Amer Ali Shah, Assistant Professor, Department of Microbiology,

Quaid-i-Azam University, Islamabad, for their support and assistance.

I appreciate very much the nice working environment in the Microbiology Research

Laboratory and wish to express my heartfelt thanks to all my lab fellows and department staff

members for their kind cooperation. I am grateful to Dr. M. Ishtiaq Ali, Saadia Andleeb, Dr.

Imran Javed, Masroor hussain and Bashir Ahmad for the splendid assistance in the laboratory

work, as well as M. Sajjad Shaukat for technical advice.

I appreciate the Higher Education commission, Pakistan for the financial support in all

respects during my study period, without which it was impossible for me to achieve the

objectives of my research work. I highly acknowledge the Indigenous 5000 scholarship

programme of HEC which facilitated the students to take up further studies at postgraduate

level. I also thank Dr. Geoff D. Robson and organisers of International Research Support

Initiative Program (IRSIP) for giving me opportunity to do research work abroad.

I am indebted to express special thanks to my family members whom continuous help,

motivation and prayers gave me strength and encouragement.

I would like to thank all those people who helped me and made this study to complete

successfully and apologise that I couldn’t mention personally one by one.

Naima Atiq

xii

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.

xiii

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.

Chapter1 Introduction

1

1.1 POLYSTYRENE

Polystyrene (PS) is a multipurpose polymer that is used in varied applications in rigid

and foamed form. General purpose polystyrene (GPS) is clear and hard which is used

in packaging, laboratory ware, and electronics. The excellent physical and processing

properties make polystyrene suitable for a lot of applications than any other plastic

(Meenakshi et al., 2002).

Styrofoam is the trade name given to expanded polystyrene (EPS) which is used in

foam form for packaging as well as insulation in various industrial fields in the world

(Kan and Demirboga, 2009). EPS is moulded into sheets for thermoforming into trays

for packaging of fish, meat and cheeses, egg crates, tubs for food and cups. Both

foamed sheets and molded tubs are extensively used in take out restaurants because

they are lightweight, stiff and have excellent thermal insulation capability.

Polystyrene is a vinyl polymer, which make up a large family of polymers that are

made from vinyl monomers containing C=C bonds. Polystyrene molecules possess

long hydrocarbon backbone, with a benzene ring linked to every other carbon atom.

Styrene is used to produce polystyrene by free radical polymerization (Fig.1.1).

On the basis of structure polystyrene can be classified into three forms (Fig.1.2). The

polystyrene containing all of the phenyl groups on one side is termed as isotactic

polystyrene. If the phenyl groups are randomly distributed then it is called atactic

polystyrene. The free radical vinyl polymerization process yields atactic polystyrene.

The polystyrene containing phenyl groups on alternating sides of the chain is

described as syndiotactic polystyrene (sPS), which is highly crystalline. It has the

tendency to crystallize very quickly which gives it the favourable properties of high

melting temperature and chemical resistance. Structurally sPS can have more than one

crystalline form and it shows a complex polymorphic behaviour. Four main

crystalline modifications and several subforms of sPs are known (Saitoh et al., 2003;

Gupper and Kazarian, 2005).

http://www.pslc.ws/mactest/vinyl.htm

http://www.pslc.ws/mactest/radical.htm

http://www.pslc.ws/mactest/crystal.htm


2

Figure 1.1 Polymerization of styrene to produce polystyrene

Figure 1.2 Types of polystyrene


3

1.2 HISTORY OF POLYSTYRENE

Edward Simon accidently discovered polystyrene in 1839 in Germany. From the

resin of Turkish sweetgum tree, Liquidambar orientalis he obtained an oily substance,

which thickened into jelly in air; he described it as styrol oxide. In 1845 John Blyth

and August Wilhelm von Hofmann observed that the same changes occur in styrol in

the absence of oxygen. They named it as metastyrol. Marcelin Berthelot in 1866

identified that it is a polymerization process that change styrol to metastyrol. It was

described in the thesis of Hermann Staudinger (1881-1965) that a chain reaction

occurs in styrol by heating resulting in the formation of macromolecules which was

later called polystyrene. In Germany, I. G. Farben, company in 1931 started producing

polystyrene in Ludwigshafen. The Koppers Company in Pittsburgh, Pennsylvania

produced expanded polystyrene in 1959. Polystyrene with syndiotactic conformation

was synthesized for the first time in the early 1980s.

1.3 SYNTHESIS OF POLYSTYRENE

The polystyrene synthesis begins by heating the natural gas or crude oil in a "cracking

process." The yield of ethylene is dependent on the cracking temperature and is more

than 30% at 850°C. The next step in polystyrene production is alkylation of benzene

with ethylene to form ethyl-benzene. Dehydrogenation of ethylbenzene forms styrene,

which is then polymerized to yield polystyrene.

Polystyrene products are made by injection blow molding, extrusion, injection stretch

blow molding and thermoforming depending upon their applications. Extrusion and

injection molding is mostly used for clear, hard and brittle type of general purpose

polystyrene products. Extruded polystyrene foam is produced by extrusion in the form

of sheets for insulation in construction industry and other insulation purposes.

Expanded polystyrene foam products are mostly produced by thermoforming.

1.3.1 Synthesis of Expanded Polystyrene

Expanded polystyrene (EPS) is produced by using a blowing agent (pentane) to

expand the polymeric chains in order to achieve a low density foamed polystyrene.

The polystyrene (90-95%) with blowing agent (5-10%) is placed in the chamber and

then heated (100-110 ºC) by dry or wet steam, above the glass transition temperature

http://en.wikipedia.org/wiki/Berlin

http://en.wikipedia.org/wiki/Liquidambar

http://en.wikipedia.org/w/index.php?title=John_Blyth&action=edit

http://en.wikipedia.org/wiki/August_Wilhelm_von_Hofmann

http://en.wikipedia.org/wiki/Marcelin_Berthelot

http://en.wikipedia.org/wiki/Hermann_Staudinger

http://en.wikipedia.org/wiki/Macromolecule

http://en.wikipedia.org/wiki/IG_Farben

http://en.wikipedia.org/wiki/Ludwigshafen

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of polystyrene (98 ºC). The blowing agent starts vaporising (iso-pentane at 28 ºC,

normal-pentane at 35 ºC and cyclo-pentane at 49 ºC) and permeates through the

polymer. The rising temperature causes the polymer chains to become softer and the

internal pressure of the blowing agent causes the polymer to expand. The expanded

beads are then cooled for aging purpose. The EPS beads are moulded into sheets or

tubs, cups etc by heating with steam that causes the external surface to become soft

and beads stick to each other.

1.4 OTHER POLYSTYRENE BLENDS AND COPOLYMERS

Styrene polymers have unique properties useful to produce wide range of products

(Meenakshi et al., 2002). To achieve specific properties for a particular application

styrene is mixed with other monomers such as butadiene, acrylonitrile etc. to make

blends, copolymers, graft copolymers. The improvement of the impact properties are

achieved by producing styrenic polymers such as high-impact polystyrene (HIPS) or

ABS (Vishwa-Prasad and Singh, 1997) which are used in electrical and electronic

equipment (Brennan et al., 2002).

1.4.1 High Impact Polystyrene

Poly(styrene-butadiene-styrene), or SBS is a hard rubber used in the manufacture of

tyres and tyre products also contain polystyrene in it. SBS rubber usually called as

high-impact polystyrene, or HIPS is a thermoplastic elastomer. Polybutadiene (PB)

has double bonds in it that cause polymerization with styrene as a graft copolymer.

The HIPS polymer is a multiphase system in which Polybutadiene is dispersed in a

rigid polystyrene (PS) matrix. Thus HIPS has improved fracture resistance, reduced

transparency, modulus and tensile strength (Vishwa-Prasad and Singh, 1997).

1.4.2 Polystyrene- Polyacrylonitrile copolymer (SAN)

Polyacrylonitrile (PAN) and polystyrene have favourable properties for use in

automotive industry, architecture, railway and aerospace. SAN copolymer has better

mechanical properties as compared to the Polyacrylonitrile and polystyrene.

Polystyrene–polyacrylonitrile is generally utilised in the automobile making, home

wiring and other applications (Wang et al., 2008).

http://www.pslc.ws/mactest/sbs.htm

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1.4.3 Acrylonitrile-butadiene-styrene (ABS)

Acrylonitrile-butadiene-styrene (ABS) is a blend that is manufactured by SAN and

polybutadiene rubber (Arnold et al., 2009). ABS has flexibility of composition,

structure and properties on the basis of ratio of monomer for diverse applications.

Styrene component imparts rigidity and easy processibility, acrylonitrile give

chemical and heat stability while, toughness and impact strength are dependet on

butadiene. ABS is used in the electronics for manufacturing parts of electronics and

automobile industry. ABS is also blended with other polymers for different

applications such as ABS/polycarbonate and ABS/polyvinyl chloride.

1.5 USES OF POLYSTYRENE

Polystyrene is used in packaging, electronics, construction, house and medical ware

and disposable food services (Meenakshi et al., 2002). Expanded polystyrene (EPS) is

used for protective packaging in electrical, pharmaceutical and retail industries etc.,

because of light weight, shock resistance, cushioning properties, and flexibility in

design possibilities. Because of thermal insulation properties, EPS is used in cold

rooms, refrigeration and building insulation (Kannan et al., 2009).

End-functionalized polystyrene act as lubricants and polymeric surfactants because

they modify the wetting behaviour of surfaces (Park et al., 2008). Organic crystals

such as polystyrene coated Meta-nitroaniline have their uses in optical devices

(Adhyapak et al., 2008). Polymeric material with a biomolecule is used for the

manufacture of biosensors, bioreactors and in the medical field (Hagiwara et al.,

2008). Polystyrene is commonly used for cell culture (Shim et al., 2008). It is used in

disposable Petri plates and other biomedical containers for its optical transparency,

durability and cost effectiveness, inert nature and nontoxicity.

When a polymer matrix is mixed with inorganic nanoparticles, the thermal (Garcia et

al., 2009), mechanical (Uhl and Wilke, 2002), optical, electrical, magnetic and

flammability properties of such a nanocomposite are much different from the polymer

matrix itself (Manzi-Nshuti et al., 2009).


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1.6 ENVIRONMENTAL EFFECTS OF POLYSTYRENE

Synthetic plastics are used in many fields such as packing, household, agricultural,

marine and architectural. Plastics have replaced natural resources like cotton, wood

and metals because of their light-weight, and durability. The average growth rate of

plastic industry in Pakistan is 15% per annum. The medium sized plastic processing

units are situated in different places in Pakistan (Sabir, 2004).

Polystyrene (PS) is a widely used thermoplastic. Its hardness, hydrophobic nature and

chemical composition cause it to persist in nature without any decomposition for

longer period of time thus cause environmental pollution (Singh and Sharma, 2008).

Floating marine debris include a large proportion of plastics especially Styrofoam that

pose a serious problem to marine life and natural ecosystems (Hinojosa and Thiel,

2009). Polystyrene packaging products are discarded in dumps, landfills or simple

litter after their useful application (Kiatkamjornwong et al., 1999). As the waste

plastic material has become a serious problem, so recycling is taking attention to save

environment and resource recovery (Pantano et al., 2009). Polystyrene waste requires

the transportation of big volume waste, which is costly and make recycling

economically unfeasible. Waste EPS doesn’t decompose in nature and causes

environmental pollutions (Kan and Demirboga, 2009)

1.7 HEALTH EFFECTS

Polystyrene is manufactured from monomer styrene. Styrene is a volatile, colourless,

strong-smelling, oily liquid. Styrene is not harmful in very small amounts in air or

food. Styrene exposure for a short time can result in eye and mucous membrane

irritation and gastrointestinal problems in humans. Styrene and its metabolites are

known to cause serious negative effects on human health (Mooney et al., 2006)

Styrene causes neurological impairment, toxic effect on liver, central nervous system.

Styrene is metabolised by a number of microbes in natural environments. Styrene

biotransformation causes the production of styrene oxide that is more toxic to human

health. Migration of styrene from expanded polystyrene cups into the hot beverages is

reported which is dependent on the fat content, Exposure temperature and time

(Khaksar and Ghazi-Khansari, 2009).


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1.8 DEGRADATION OF POLYSTYRENE

Polymers are weathered due to environmental factors like light and temperature. The

conditions of use play key role in the degradation of plastics. Polystyrene losses its

mechanical and tensile properties due to effect of UV light and heat

(Kiatkamjornwong et al., 1999). UV light induces the production of free radicals by

oxidation. Free radicals cause the chains of polymer to breakdown.

1.8.1 Biodegradation of Polystyrene

Biodegradation is the breakdown of a substance by the activity of living thing or a

product of living thing e.g. enzyme. Biodegradation of plastics involves extracellular

and intracellular enzymes. Extracellular enzymes chop down the long chains of

polymer molecules into simpler water soluble compounds that are easily taken up by

the cell membranes of microorganisms. The intracellular enzymes further convert

these molecules into the forms that can enter into the biochemical and synthetic

pathways of the cellular metabolism. There are few reports of polystyrene

biodegradation in literature mostly carried out by bacterial species i.e. actinomycete

Rhodococcus ruber (Mor and Sivan, 2008), Bacillus, Xanthomonas,

Sphingobacterium (Eisaku et al., 2003), Serratia marcescens, Pseudomonas sp. and

Bacillus sp. (Galgali et al., 2002), Bacillus coagulans (Kiatkamjornwong et al., 1999).

The fungi reported to carry out biodegradation of polystyrene include Curvularia

species (Motta et al., 2009), brown rot Gleophyllum trabeum and white rot

Basidiomycete, P. chrysosporium, Trametes versicolor and Pleurotus ostreatus

(Milstein et al., 1992).

Synthetic polymers are resistance to biodegradation due to high molecular weight

structural complexity and hydrophobic surfaces. These properties make the polymer

inaccessible to the microbial enzymes. For improved biodegradation polymers are

blended with natural polymers like starch, cellulose and lignin that increase the

microbial adherence and attack on the polymer and when the biodegradable part is

consumed the synthetic polymer losses its mechanical properties.

Fungi are the major decomposers in natural ecosystems and are able to colonise a

wide variety of diverse environmental conditions possessing an extremely important

ecological niches. In nature Fungi are the major causative agents of spoilage of food,


8

timber, cotton, paper etc (Pinzari et al., 2006). Fungi are successfully used to degrade

plastics and other xenobiotics (Francesc et al., 2006). Excellent adherence and

colonisation properties give advantage to the fungi for bioremediation. Once

established on a surface the fungi cover the whole area by forming mycellial mat.

Fungi are able to withstand longer periods of stress conditions and due to saprotrophic

nature they are capable of producing a diverse arsenal of enzymes that are able to

degrade the recalcitrant compounds (Gu and Gu, 2005).

AIM AND OBJECTIVES

The aim of the present study was to investigate the biodegradation of polystyrene by

indigenous fungal isolates and methods to accelerate the biodegradation process. The

aim was achieved by the following specific objectives.

• To Isolate and characterise the fungal strains associated with polystyrene

films.

• To study biodegradation of polystyrene in solid and liquid media using

selected fungal isolates.

• To analyse the biodegradation products of polystyrene by HPLC, NMR and

FTIR for possible degradation pathway.

• To Study mineralisation of polystyrene by Sturm test analysis

• To study the effect of UV and thermal pre-treatment on biodegradation

Chapter2 Literature Review

9

Polystyrene has exclusive chemical, physical, mechanical properties and applications.

It is utilised in packaging, automotive industry, telecommunication, electronics,

building insulation etc. However, its durability has caused serious pollution problems

because the polystyrene plastic waste accumulates in the environment. The build up

of discarded plastics has caused a worldwide environmental problem. Nature is unable

to get rid of plastic waste, as the majority of plastics are not decomposed by

microorganisms. Worldwide plastic production is increasing day by day as a result the

amount of plastics wastes is also raised enormously (Mukai and Doi, 1995). As the

available landfill space is decreasing the costs of solid waste disposal is also rising.

Alternatively plastic recycling is quite limited due to low economical value and

incineration is not favourable due to environmental pollution caused by emission of

harmful gases.

2.1 MECHANICAL DEGRADATION OF POLYSTYRENE

Thin films of polystyrene coated on solid surfaces were considerably degraded by

mechanical forces, on a molecular scale. The polymer degraded by non-random

multiple scissions as demonstrated by molecular weight distributions analysed by gel

permeation chromatography. The study described that the high molecular weight

polystyrene will be converted to medium and then to low molecular weight

polystyrene molecules. Polymer chain entanglement was used to explain the mode of

degradation by using computer simulations. The time of degradation, thickness of the

film and the molecular weight of the polymer determine the degradation rate (Nash

and Jacob, 1972).

Styrene-ethylene/butylene-styrene elastomer grafted with maleic anhydride (SEBS-g-

MA) and trimethylol propane trimethacrylate (TMPTA) as compatibilizers were

added to composites of recycled polymers i.e. polypropylene (PP), poly(ethylene

terephthalate) (PET), polystyrene (PS), low-density polyethylene (LDPE) and high-

density polyethylene (HDPE). Compatibilizer addition and electron radiation of the

blends was done to improve their mechanical properties and to study the possibility of

their use for plastic waste recycling (Zenkiewicz and Kurcok, 2008).

Thermo-oxidative ageing and Multiple processing were used as simulation techniques

to study the degradation of high-impact polystyrene (HIPS). Degradation was


10

analysed by melt mass-flow rate (MFR) measurements, differential scanning

calorimetry (DSC), FTIR and tensile testing. Thermo-oxidative ageing and multiple

processing introduces modification in the chemical structure of HIPS causing

oxidative instability and changes the physical properties of the materials. Service life,

processing, and mechanical recycling affects polybutadiene phase. The modifications

introduced during the life cycle of HIPS also determine its recyclability and

performance in market (Vilaplana et al., 2006).

2.2 CHEMICAL AND ELECTROCHEMICAL DEGRADATION OF

POLYSTYRENE

Polystyrene was treated by oxidizing agent alone, oxidizing agent with a transition

metal complex and oxidizing agent with inorganic acid. The FTIR spectroscopy of the

treated samples demonstrated the introduction of carbonyl and hydroxyl groups in the

polymer chains (Motta et al., 2009).

Artificial strong acid rain was used to check surface erosion of atactic polystyrene

insulating material. The surface conductivity of aged material increased due to

changes in physical and chemical structure of the deteriorated surface of PS.

Conductivity, pH and ion concentration of acid rain influence the degradation rate of

polystyrene. The concentration of actual rainwater and normal acid rain was

inadequate to cause noticeable erosion on PS insulation (Wang, 2000).

Polystyrene was subjected to oxidative degradation in solution by peroxides

(Sivalingam et al., 2003). Various concentrations of peroxide, different reaction

times, heating cycles and microwave irradiation were studied. The thermal-assisted

process was less effective then oxidative degradation with microwave.

2.3 PHOTODEGRADATION OF POLYSTYRENE

Kiatkamjornwong et al., (1999) reported that photodegradation of polystyrene in air

causes discoloration (yellowing), cross-linking, and chain scission due to oxidation. A

photodegradation mechanism was also proposed on the basis of the IR spectra of the

photoirradiated film, which indicated the formation of peroxy radical and

hydroperoxide intermediate formation. The photochemical reactions cause the

dissociation of a polystyryl radical by creating an electrochemical excited state. The


11

polystyryl radical is converted to peroxy radicals by reacting with oxygen. The peroxy

radical causes further chain scission and formation of carbonyl compounds.

Kiatkamjornwong et al., (1999) evaluated of the photosensitivity of plastic sheet after

different exposure times (0.5, 5, 10 or 21 hr.). FT-IR analyses of photo irradiated

polystyrene showed increase in the absorption peaks at 1742-1745cm-1

indicating the

presence of ketone, carbonyl groups. For polymers consisting of hydrocarbons,

oxidation must precede biodegradation.

The photo-oxidative degradation (λ ˃ 300 nm, 60°C) of high impact polystyrene

(HIPS) had been reported by Israeli et al., (1994). The photo-oxidized films were

treated with SF4 and NH3, to identify the various hydroperoxides, alcohols and

carbonyl species. Unstable intermediate photoproducts, peroxyl radicals, α, β-

unsaturated ketones and secondary hydroperoxides were produced due to

polybutadiene photooxidation. Photoproduct distribution profile showed that

photodegradation causes cross-linking at the surface of HIPS which make the polymer

impermeable to oxygen and stops the further oxidation process. However longer

wavelength exposure did not show any photoproduct distribution because cross-

linking reactions were not adequate to decrease the oxygen permeability (Israeli et al.,

1994).

Vacuum-ultraviolet irradiation of polystyrene films in the presence of oxygen

produced OH- and C=O-functionalized surfaces and morphological changes. These

changes can be used for secondary functionalization, enhanced aggregation or

printing, and microstructurization. The oxidative fragmentation occurred because of

reactive oxygen species (hydroxyl radicals, atomic oxygen, ozone) leading to

electronic excitation of the polymer causing homolysis of C–C bond and C-centred

radicals. Ozonation of the polystyrene caused oxidative functionalization of the

polymer surface but could not initiate the fragmentation of the polymer backbone

(Gejo et al., 2006).

Polychromatic 254 nm and 365 nm light were used to irradiate the styrene-butadiene

copolymer, which produced carbonyl group. A 254 nm irradiation caused unstable

hydroperoxide concentration, Weak crosslinking and strong discolouration.


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Irradiation resulted predominantly in acetophenone chromophores and carboxylic acid

groups by chain breakage in the aliphatic regions (Allen et al., 2004).

The blends of poly(vinyl methyl ether) and polystyrene were subjected to

photooxidation. The photodegradation products were studied by infrared

spectrometry. The characterisation of the oxidation products has shown that there are

interactions between the two polymers. The modifications of the surface and the

modifications of the chemical structure of the macromolecules induced by irradiation

correlated with each other. Atomic Force Microscopy (AFM) has shown that the

changes of the surface are dependent on the irradiation time (Mailhot et al., 2000).

Nanoclay of Layered double hydroxide (LDH) organo-modified by 3-sulfopropyl

methacrylate (SPMA) was added to polystyrene as filler. The nanoclay was dispersed

into PS by benzoyl peroxide by adding small amount of initiator. The level of

dispersion and degradation due to UV light was evaluated by thermal analysis UV–

visible, FTIR and high resolution 13

C NMR spectroscopy and X-ray diffraction. Filler

only influenced the oxidation rate of the polymer (Leroux et al., 2005).

2.4 THERMAL DEGRADATION OF POLYSTYRENE

Thermal degradation of foamed polystyrene beads was investigated by Mehta et al.,

(1995). Polymer beads collapsed at 110-120°C, melted a 160°C, started to vaporise at

275°C and completely volatilize at 460-500°C. Heat of degradation was not affected

significantly by the density of the polymer or bead size. HIPS films were exposed to

80, 100, and 120°C temperatures in the presence of air to study their thermal

degradation. Thermal oxidation led to a highly crosslinked and oxidized surface of

polybutadiene due to saturation of double bonds. The oxidation of the polystyrene

matrix can be initiated by the radicals produced during thermal degradation of

polybutadiene. That caused the HIPS matrix to become thermally less stable than pure

polystyrene due to the presence of polybutadiene (Israeli et al., 1994).

The degradation kinetics of polystyrene (PS) in supercritical benzene were studied at

various temperatures (300-330 ˚C) at 5.0 MPa. The degradation rate coefficients

obtained in supercritical benzene were higher than the rate coefficients observed for

degradation of PS in subcritical solvents at high pressures and in solvents at normal

pressures (Karmore and Madras, 2000). The Combustion of expanded polystyrene


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with and without flame retardant was studied by Rossi et al., (2001) and analysis of

smoke for the products formation showed that pyrolysis (producing styrene, alpha-

methylstyrene, 1,3-diphenylpropane) dominated over thermal oxidation (producing

phenol and benzaldehyde) during combustion. The fire retardant modified the

composition of the smoke and concentration of the gaseous products.

A new heating medium by a batch system at 190-280°C was used to clarify the

manner in which thermal decomposition of polystyrene is initiated. Thermal

decomposition of polysyrene was analyzed by GC, GPC, IR, 13

C-NMR and GC-MS.

2,4,6-triphenyl-1-hexene (trimer), 2,4-diphenyl-1-butene (dimer) and styrene

(monomer) were found o be the major decomposition products. Ethylbenzene,

propylbenzene, naphthalene, benzaldehyde, biphenyl and 1,3-diphenylpropane were

present in minor quantities. Thermal decomposition of polystyrene started near 190°C

which is used as moulding temperature of PS (Saido et al., 2003).

Allen et al., (2004) reported the thermal and photooxidation of high styrene butadiene

copolymer (SBC) as shown by the presence of aromatic ketones, aldehydes,

lactones/peracids, α,β-unsaturated carbonyl species anhydrides and hydroxyl group in

the matrix were formed. Environmental pollution is caused by waste expanded

polystyrene because it cannot biodegrade in natural environment. Kan and

Demirboga, (2009) developed recycling process for utilizing waste EPS in concrete

technology, after heat treatment at 130 ◦C and 15 min. This technique reduced the

volume about 20 times and increased the compressive strength, thermal conductivity

and density of waste EPS.

Elashmawi et al., 2008 studied the films of polyvinyl acetate and Polystyrene

(PS/PVAc) blends. Significant changes in differential scanning calorimetry (DSC),

XRD and FT-IR analysis were indicative of miscibility and interactions of both the

polymers. The results of DSC gave distinct glass transition temperatures for each

blend supporting that the blend was miscible. Watanabe et al., (2009) reported a

pyrolysis-GC/MS system for rapid evaluation of oxidative, thermal and photo

degradation of polymers in a very small quantity. PS degradation was analysed by the

system. After Exposure to air at 60°C for 1 hour the degradation products with UV

irradiation were benzoic acid, acetophenone, phenol and benzaldehyde and without


14

UV irradiation toluene, styrene and benzaldehyde, acetic acid, formic acid, acetone,

acetaldehyde and formaldehyde by oxidative thermal degradation of PS.

2.5 BIODEGRADATION OF POLYSTYRENE

Since the discovery synthetic plastics, the research was mainly focussed on

developing durable materials or very slowly degrading materials in natural

environment. The scarce landfill space, hazards of waste incineration and increasing

costs of disposing solid wastes have caused scientists to discover new approaches for

waste management. Biodegradation of synthetic polymers is a valuable solution to

this environmental problem. Polymers with higher molecular weights and decreased

solubility in aqueous environments are more resistance to microbial attack then

oligomers dimers and monomers of the same polymer. Microorganisms produce two

groups of enzymes, extracellular and intracellular. Extracellular enzymes tend to

depolymerise the long chains of polymer in order to absorb it through the cell

membrane and assimilate the degradation products further inside the cell by

intracellular enzymes. Hence Molecular weight, crystallinity and physical form are

the most important properties of polymers that determine their biodegradability

(Motta et al., 2009).

Sielicki et al., (1978) reported microbial degradation of 1,3-diphenylbutane (styrene

dimer) and [beta-14

C] polystyrene in liquid enrichment cultures and soil. [14

C]

polystyrene degradation rates in soil was measured by 14

CO2 evolution and found 1.5

to 3.0% after 4 months. Soil microorganism Nocardia, Micrococcus, Pseudomonas

and Bacillus species metabolized 1,3-diphenylbutane in enrichment cultures.

Kaplan, et al., (1979), studied the biodegradation of 14

C-labeled polystyrene phenol

formaldehyde and poly (methyl methacrylate) using five groups of soil invertebrates,

17 fungal species, and mixed microbial communities including sludge, soil, manure,

garbage, decaying plastics. Biodegradation was checked by the evolution of 14

CO2.

Fungi in axenic cultures degraded 0 to 0.24% polystyrene during 35 days. Rate of

decomposition by mixed microbial system remained 0.04 to 0.57% in 5-11 weeks.

Polystyrene is a durable thermoplastic that is generally believed to be non-

biodegradable. Otake et al., (1995) found that a sheet of polystyrene buried in soil for

32 years had no sign of degradation.

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Pseudomonas sp. and Bacillus sp. for styrene degradation, and Xanthomonas sp. and

Sphingobacterium sp. for polystyrene decomposition were isolated and identified by

16S ribosomal DNA analysis. Bacillus sp. STR-Y-O strain decomposed both styrene

and polystyrene 40 % and 56 % of initial concentrations, respectively, in 8 days.

Limonene melted expanded polystyrene; styrene and polystyrene were decomposed

by the isolated microorganisms in this study (Eisaku et al., 2003). Fungal degradation

studies of oxidized polystyrene using Curvularia species were reported by Motta et

al., (2009). The fungus colonized the oxidized polystyrene in sabouraud plates within

9 weeks. Hyphae adhering to and penetrating the polymer’s surface were observed in

microscopic examinations. The fungus utilised PS by co-metabolism.

Raberg and Hafren, (2008) applied the plastics polystyrene and polycaprolactone to

the wood samples and treated them by brown rot (Postia placenta) for 8 weeks in agar

plates. The polystyrene treated wood samples were significantly protected from

decay. The ability of actinomycete Rhodococcus ruber C208 for biodegradation and

biofilm formation on polystyrene was analysed by Mor and Sivan, (2008). The strain

was isolated in a study for biodegradation of polyethylene. The strain produced

colonies on synthetic medium agar plates containing polystyrene powder. 0.8%

weight loss was observed within 8 weeks of treatment. The study reported that R.

ruber C208 was able of partial biodegradation, biofilm formation and colonization of

polystyrene (Mor and Sivan, 2008).

2.6 BIODEGRADATION OF POLYSTYRENE BLENDS

Polymer blends consisting of biodegradable polymer with other polymers are

described as bioblends. Compatibility with other components is the necessity of a

successful bioblend development. Mohamed et al., (2007) reported that

intermolecular interactions were present between biodegradable polycaprolactone

(PCL) and polystyrene (PS) in blended form. Decreased thermal stabilization of

PCL/PS bioblend was observed by thermogravimetric analysis (TGA).

High impact polystyrene with starch was degraded for 12 weeks by concentrated

activated sludge. Concentrated activated sludge was effective in polymer degradation

and starch accelerated the structural changes in that work (Jasso et al., 2004).


16

Styrene (1-phenylethene) graft copolymers of lignin were tested for biodegradation by

brown rot fungus G. trabeum, white rot fungi T. versicolor, P. chrysosporium and P.

ostreatus. Polystyrene pellets could not be degraded. White rot fungi secreted lignin

degrading oxidative enzymes and also degraded polystyrene component of the

copolymer (Milstein et al., 1992). Nuclear magnetic resonance (NMR) and FTIR

spectroscopy of lignin-(1-phenylethylene) graft copolymers (lignin-styrene graft

copolymers) were analyzed to further study the biodegradation by white rot fungi.

Copolymer spectra showed the loss of functional groups after incubation with fungi.

NMR spectra also demonstrated reduced resonances of aromatic region. Scanning

electron microscopy showed degradation of the surface (Milstein et al., 1994).

Kiatkamjornwong et al., (1999) prepared Graft copolymers of Cassava starch and

polystyrene and tested for degradation by UV irradiation, soil burial, outdoor

exposure and the resistance against bacteria (Bacillus coagulans 352). Analysis of

thermal properties, molecular weight, extent of degradation and tensile properties of

the polymer revealed that graft copolymer was easily degraded. Longer time was

taken by the plastics to degrade in soil burial test and no significant degradation

occurred upon indoor exposure. The destroyed areas of starch in composite PS sheets

indicated that bacteria promote the biodegradation of polystyrene plastics.

Galgali et al., (2002), used a novel strategy to increase the rate of degradation of

functionalized polystyrene. They used maleic anhydride (14% by weight) to

functionalize polystyrene and anchored small quantity of different monomeric sugars

like glucose, lactose or sucrose on it. Pure cultures of soil bacteria (Serratia

marcescens, Pseudomonas sp. and Bacillus sp.) were used to study their growth

pattern on these polymers. Infra red (IR) spectroscopy analysis, weight loss data and

Gel Permeation Chromatography data illustrated that polymers were degraded by the

bacteria. Dried waste veins of the leaves and stems of Musa paradiciaca (banana)

were used to prepare cellulose acetate and blended with polystyrene. The films of

cellulose acetate–polystyrene were made to study morphological changes, chemical

and mechanical properties of blend (Meenakshi et al., 2002).

Ward et al., (2006) reported a two step method for the conversion of polystyrene to

polyhydroxyalkanoate (PHA). Polystyrene was converted to styrene oil by pyrolysis

(520 ˚C) and then Pseudomonas putida CA-3 (NCIMB 41162) transformed it to PHA.

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A single pyrolysis and four fermentation steps produced 6.4 g of PHA from 64 g of

polystyrene.

Polystyrene was blended with cassava starch (Manihot esculenta Crantz) and natural

plasticizer Buriti oil from a palm tree (Mauritia flexuosa L.). Plasticizer aided in the

conversion of starch into thermoplastic starch (TPS) and its blending to PS. Presence

of starch made the blend a biodegradable material because the mechanical strength

was lost and polymeric chain breakage occurred when the starch content was digested

by the microorganisms. X-ray diffraction and kinetic studies had shown that thermal

stability and the activation energy of the PS/TPS blends, is much lower compared to

PS (Pimentel et al., 2007).

Polystyrene was modified with hydrophilic monomer and natural polymers by graft

copolymerization. Biodegradation of these polymers was studied by soil burial

method. The copolymers with starch showed 37% biodegradation after soil burial of

160 days (Singh and Sharma, 2008). Schlemmer et al., (2009) reported that blending

polystyrene with thermoplastic starch (TPS) blends and using glycerol and buriti oil

as plasticizers enhanced biodegradation. Soil burial test was performed to check

biodegradation of PS/TPS blends in perforated boxes for 6 months.

2.7 BIODEGRADATION OF STYRENE

The metabolic pathways of bacteria for styrene biodegradation are well established

and studies on genetic and physiological aspects of styrene biodegradation have been

reported (Hartmans et al., 1990; Jindrova et al., 2002; O’Leary et al., 2002; Leoni et

al., 2005).

Reports in literature indicate that in aerobic conditions the majority of organisms

convert styrene to 2-phenylethanol or phenylacetaldehyde, by vinyl side chain

oxidation, followed by further oxidation to give phenylacetic acid (phenylethanoic

acid) (Hartmans et al., 1989; O’Connor et al., 1995; O’Connor et al., 1996; Velasco et

al., 1998). Styrene monooxygenase oxidises styrene to form styrene epoxide

(Hartmans, 1995). Liu et al., (2006) reported bacterial epoxide hydrolase (EH) which

was capable of enantioselective hydrolysis of racemic styrene oxide.


18

styrene oxide isomerase causes the isomerisation of the epoxystyrene to

phenylacetaldehyde (Utkin et al., 1991; Velasco et al., 1998). However, Marconi et

al., (1996) reported that in Escherichia coli, the expression of a 2.3-kb BamHI DNA

fragment from Pseudomonas fluorescens ST converted styrene epoxide to 2-

phenylethanol. However, in Xanthobacter sp. 124X and Corynebacterium sp. ST-10

the phenylacetaldehyde is reduced to 2-phenylethanol by a phenylacetaldehyde

reductase enzyme (Itoh et al., 1997; Jones et al., 1997).

The upper pathway was reported in many bacterial strains, for example Xanthobacter

strain 124X (Hartmans et al., 1989), Xanthobacter strain S5 (Hartmans et al., 1990),

Pseudomonas sp. Strain Y2 (Utkin et al., 1991; Velasco et al., 1998), Pseudomonas

putida CA-3 (O’Connor et al., 1995) P. fluorescens ST (Marconi et al., 1996), and

Pseudomonas sp. strain VLB120 (Panke et al., 1999). Corynebacterium sp. AC-5 uses

a shorter form of upper pathway to metabolize styrene (Itoh et al., 1997). Meta-

cleavage products are also formed during styrene metabolism in various

microorganisms. P. fluorescens strain produced 2-hydroxyphenylacetic acid and

phenylacetic acid during styrene metabolism (Baggi et al., 1983).

Further conversions after phenylacetic acid production are called lower pathway of

styrene metabolism (Ferrandez et al., 1998; Mohamed et al., 2002). In P. putida U

(Olivera et al., 1998) and Escherichia coli W strains (Ferrandez et al., 1998), PAA

was first activated to phenylacetyl-CoA (PACoA) to yield acetyl-CoA. After

conversion to phenylacetyl-CoA it undergoes many enzymatic reactions and finally it

was metabolised by the tricarboxylic acid (TCA) cycle (Alonso et al., 2003; Martinez-

Blanco et al., 1990; Mohamed et al., 2002; O’Leary et al., 2001). The study of a

series of mini- Tn5 mutants of P. putida CA-3 showed that phenylacetyl-CoA is

converted to acetyl-CoA which is used in TCA (O’Leary et al., 2005).

Lee et al., (2006) reported the biodegradation efficiency of styrene by Daldinia

concentrica KFRI 40-1, Trametes versicolor KFRI 20251 and Phanerochaete

chrysosporium KFRI 20742, and reached 99% during one day of incubation. P.

chrysosporium KFRI 20742 produced succinic acid, butanol and 2-phenyl ethanol

during styrene metabolism.


19

A biofilter using white rot fungi for the biodegradation of styrene was reported by

Braun Lullemann et al., (1997). White-rot fungi Phanerochaete chrysosporium,

Bjerkandera adusta, Trametes versicolor and Pleurotus ostreatus (two strains) were

taken in liquid culture and contact surface was increased by using perlite.

Lignosulphonate was found to be the best inducer for T. Versicolor and P. ostreatus

while wood meal resulted in good induction for P. Chrysosoporium and B. adusta.

HPLC analysis detected benzoic acid, 2-phenylethanol and phenyl-1,2-ethanediol and

as degradation products of [14

C]styrene by treatment with P. Ostreatus (Braun

Lullemann et al., 1997).

Styrene utilising fungi Exophiala jeanselmei and Clonostachys rosea were isolated

and enriched during biofiltration of styrene polluted air (Cox et al., 1993). Penicillium

species including P. Minioluteum, P. cf. Miczynskii, P. cf. Janthinellum and P.

fellutanum were also reported to biodegrade styrene (Cox et al., 1997; Paca et al.,

2001).

Rhodococcus rhodochrous NCIMB 13259 utilise styrene involving a cis-glycol

pathway by dioxygenation to give styrene cis-glycol, followed by dehydrogenation to

form 3-vinylcatechol. Ortho cleavage results in 2-Vinylmuconate accumulation in the

growth medium. Meta cleavage formed acetaldehyde and pyruvate as a result of the

action of enzymes (Warhurst et al., 1994) (Fig. 2.1).The yeast-like fungus Exophiala

jeanselmei disintegrate styrene by oxidation (Cox et al., 1996).


20

Figure 2.1 Styrene metabolic pathways found in bacteria (dotted line) and fungi

(bold line) composed after Francesc et al.,(2006); O’Leary et al.,

(2002); Boldu et al., (2001); Weber et al., (1995); Cox, (1995); and

Holland et al., (1993). (1) Phialophora sessilis CBS 238.93; and (2)

Clonostachys rosea CBS 102.94


21

2.8 TECHNIQUES USED IN BIODEGRADABILITY STUDIES

The test methods generally used for assessing polymer biodegradability had been

developed according to the material and applications. The methods include laboratory,

simulation and field tests. The existing methods have limitations in certain aspects

e.g. in literature presence of microorganisms on the polymer surface is used as an

indication of biodegradation. Only a small selection of fungal and bacterial species

are used for simulation testing are not representatives of microbial population of

different geographical areas (Gu and Gu, 2005).

Rate of biodegradation is dependent upon conditions of the surroundings such as

humidity and aeration. The main environmental conditions reported in literature are

classified by Van Der Zee et al., (1994) as aerobic high solids conditions such as

littering and composting, aerobic aquatic environment including sewage treatment,

marine and fresh water, anaerobic high solids environment as land filling and

anaerobic digestion, anaerobic aquatic environment e.g. waste water and sewage

treatment.

Under aerobic conditions, heterotrophic microorganisms carry out biodegradation of

complex materials and produce H2O, CO2 and microbial biomass. Anaerobes in

methanogenic conditions generate H2O, CH4, CO2 and microbial biomass or H2O,

CO2 and H2S in sulfidogenic conditions. Both environmental conditions exist in

nature, but greater population of microorganisms are aerobic as compared to anaerobs

(Gu and Gu, 2005).

2.8.1 Plate Test

Mould test methods are used to study the biodegradability of a test specimen. These

qualitative tests are based on visual quantification of fungal growth on the material

surface. ASTM G21-96 method rates the observed growth on specimens between 0

(no growth) and 4 (heavy growth). Aureobasidium pullulans, Aspergillus niger,

Penicillium purpurogenum, Stachybotrys chartarum, Chaetomium globosum and

other fungal species are used in these tests. A mixed fungal spore suspension

containing a known concentration of spores is spread on the test specimen, which is

placed on an agar-based medium. Specimens are incubated at optimal conditions for

fungal growth for a defined period of time. Many of the mould fungi known to grow


22

on wood have also been isolated from plastics, for example, Aureobasidium,

Aspergillus and Penicillium, so they are important screening organisms for both

plastic and wood (Schirp et al., 2008).

Biodegradation of two poly(3-hydroxyalkanoates) as particles in solid media agar

plates was investigated by Augusta et al., (1993). Distinct circular clear zones were

produced by seven strains of microorganisms isolated from sewage sludge. Strains

achieving high zone growth were able to degrade both poly(3-hydroxybutyrate) and

Poly(3-hydroxybutyrate-co-valerate, indicating that similar enzymatic processes are

involved in biodegradation. The solid ager plat test technique is useful in determining

the degradation abilities of microorganisms and establishing biodegradability of a

material. Agar plate test was utilised by Raberg and Hafren, (2008) to study the

biodegradability of plastic treated wood samples by brown rot Postia Placenta.

2.8.2 Weight Loss Measurement

The determination of weight loss or gravimetric method gives a quantitative

measurement of biodegradation. It is used widely for the biodegradability assessment

of polymeric materials insoluble in water. The drawback of this method is that the

synthetic recalcitrant polymers do not degrade well and there is very little change in

molecular mass of the polymer. The attached biomass and mechanical or hydrolytic

loss of polymer other than biodegradation can also cause misleading results.

2.8.3 Monitoring Metabolic CO2 Production or O2 Consumption

The biodegradability of a polymer is calculated quantitatively with laboratory test

methods studying the metabolism of the polymeric material by monitoring CO2

production or O2 consumption. The Standardised test protocols are prepared so that

following the procedures biodegradability of a given polymer can be validated. The

Sturm test (Sturm, 1973) is used for estimation of CO2 produced due to mineralisation

of polymer materials. The process also has disadvantages such as underestimation

(Muller et al., 1992). The test requires lots of manual work and human error can also

cause misleading results.

Itavaara and Vikman (1995) introduced CO2 measurement by determining changes in

electrical conductivity of a basic solution (0.1 M KOH) in an automated Sturm test.


23

Calmon et al., 2000 described an automated system of CO2 determination by IR

spectroscopy to overcome the disadvantages. ISO 14852 involves the evaluation of

CO2 produced as a result of biodegradation (Mezzanotte et al., 2005).

2.8.4 Soil Burial

Soil burial test is performed to biodegrade the polymeric material by soil

microorganisms. This technique is also used to isolate microorganisms capable of

depolymerisation of a polymer (Kyrikou and Briassoulis, 2007).

Silva et al., (2007) observed that agar or soil involving tests were more useful in

determining deterioration of wood plastic composites under laboratory conditions. It

is desirable to determine the biodegradability of plastics in natural environment where

the wasted plastic is disposed. Soil burial is employed as a field test for plastic

biodegradation because of the similarity to actual conditions of disposal (Orhan et al.,

2004). Soil burial is the most promising method among other methods used in studies

of polyolefins and modified polystyrene biodegradation (Singh and Sharma, 2008).

Biodegradation of pure polystyrene and grafted polystyrene was evaluated by soil

burial. Films were recovered from the soil after six months. Gravimetric method and

FTIR spectra of films were used to determine biodegradation. 37% biodegradation

was found after 160 days in starch polystyrene blend (Singh and Sharma, 2008).

Grima et al., (2000) described a test method to assess polymer biodegradation under

simulated soil conditions. Carbon dioxide (CO2) production, by the test reactors was

used as the determinant of biodegradation.

2.8.5 Composting

Composting is an important waste management strategy. It is carried out in specially

designed compositing facilities and need specific infrastructure. Sludge taken from

different sources consists of microbial flora with variable metabolic capabilities. The

composition of the inoculum for composting strongly influences the process of

biodegradation (Mezzanotte et al., 2005).


24

2.9 ANALYTICAL TECHNIQUES USED IN BIODEGRADATION STUSIES

2.9.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR is useful to elucidate chemical and physical structure, hydrogen bonding, end

group detection, degradation reactions, crosslinking behavior of molecules and

copolymer composition in liquid and solid form of chemicals and polymers. FTIR

technique is employed in the biodegradation studies of polymers to assess the

chemical changes due to microbial activity (Milstein et. al., 1994; Galgali, et. al.,

2002; Mohamed et al.,, 2007; Singh and Sharma, 2008; Elashmawi et al., 2008).The

oxidation products such as aldehydes, ketones, esters and lactones contain carbonyl

groups, visible as peaks at specific wave number in FTIR spectra, are detected in

aerobic biodegradation.

To study the transparent polystyrene in the form of sheet transmission IR is more

useful than ATR (attenuated total reflection) (Wang et al., 2000). Allen et al., (2004)

determined the chemical changes in the styrene-butadiene copolymer due to thermal

and photoxidation by FTIR spectroscopy.

2.9.2 Gel Permeation Chromatography (GPC)

GPC or size exclusion chromatography (SEC) is used for the determination of

molecular mass distribution of polymers to determine changes in molecular weight

after biodegradation (Walter et al., 1995; Peng and Shen, 1999; Kale et al., 2006).

Polystyrene resins of known molecular weight are used to calibrate the instrument.

GPC is a conventional technique that separates molecules according to their

molecular size.

Saito et al.,, (2004) compared molecular weight determination efficiency of matrix-

assisted laser desorption/inonization time-of-flight mass spectrometry

(MALDITOFMS), conventional static light scattering (SLS), 1

H NMR, SEC coupled

with multi-angle light scattering detection (SECMALS) and size-exclusion

chromatography (SEC) using polystyrene. The results showed that SEC calibrated

with polystyrenes was a reliable technique for molecular weight determination.



25

2.9.3 Nuclear Magnetic Resonance Spectroscopy (NMR)

Determination of soluble fraction, tensile strength, molecular weight changes etc. is

indirect evaluation method as they don’t give any idea of chemistry of polymer

changes. NMR spectroscopy (1H and

13C NMR) is the most versatile method that can

be used as analytical tool for biodegradation in many studies (Massardier-Nageotte et

al., 2006; Shah et al., 2008; Schlemmer et al., 2009). Solid state NMR techniques

and pulsed low-resolution 1H NMR can be used to study of solid polymers samples.

NMR technique was used to investigate the gamma irradiation effects on polymers

commonly used in packaging of food. The threshold dose for a significant effect on

acrylonitrile–butadiene–styrene, high impact polystyrene, styrene–acrylonitrile, poly-

butadiene and polystyrene was determined. There was little effect of γ irradiation on

polystyrene in the absence of stabilizers. The results confirmed that polystyrene can

be used for irradiated packaging of food (Pentimalli et al., 2000). Molecular dynamics

can be studied by NMR. Multidimensional NMR method is recently used to

determine ultra-slow exchange in polymers (Qiu and Mirau, 2000).

2.9.4 Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) has diverse applications in studies of polymers.

Surface morphology of the films of polymers can be studied by SEM. The samples

are generally sputter-coated with gold or some metal ions before SEM examination.

The variable pressure SEM technique is helpful in direct observation without any

surface metallization at suitable magnification (Pinzari et al., 2006).

The scanning electron microscopic study of the starch-g-polystyrene graft copolymers

biodegraded by Bacillus coagulans 352 and soil burial test showed the degraded

regions of starch as holes in the sample sheets (Kiatkamjornwong et al., 1999).

Scanning electron microscopic examination was used to evaluate the compatibility of

random and triblock copolymers (Josepha et al., 2005). Surface morphologies of

polystyrene and its graft copolymers with starch and acrylic acid (Singh and Sharma,

2008) and ethylene-propylene-diene-graft-polystyrene (EPDM-g-PS) were studied by

SEM (Pticek et al., 2007).


26

2.9.5 High Pressure Liquid Chromatography (HPLC)

High pressure liquid chromatography (HPLC) is widely used to separate molecules on

the basis of their size. The principle is that the larger molecules will elute earlier as

they pass through the spaces in the column, while smaller ones elute later because

they will take longer to pass through the pores in the gel. HPLC is used to analyze the

metabolic degradation products of xenobiotics.

Styrene, epoxystyrene, phenylacetaldehyde, and 2-phenylethanol biotransformation

products were detected by reverse-phase high pressure liquid chromatography

(Marconi et al., 1996; Beltrametti et al., 1997). Phenylacetic acid concentration in the

broth of P. putida CA-3 culture was analyzed by high performance liquid

chromatography (Ward et al., 2005). Standard styrene solutions of 1, 5, 10, 20, and 30

mg/l were used to get a standard curve for quantitative analysis and study the

efficiency of styrene degradation (Lee et al., 2006).

2.9.6 Thermogravimetric Analysis (TGA)

TGA implies the heat treatment under controlled condition to record mass changes in

the sample due to heating. But it does give any information about the evolved gases

produced during thermal degradation of the sample (Bhandare et al.,, 1997). The

dynamic thermogravimetry can be used to study the temperature at the maximum

decomposition rate, rate of decomposition and the activation energy (Carrasco and

Pages, 1996).

Chapter 3 Materials and Methods

27

3.1 CHEMICALS

The chemical compounds and media were acquired from the BDH laboratory chemical

division (Pool Dorset, England), DIFCO laboratories (Detroit, Michigan, USA), Fluka

(Germany), Sigma Aldrich and Merck (Darmstadt, Germany). Polystyrene (Mol. wt.

100,000 Da) was obtained from Fluka. Expanded polystyrene beads were generously

provided by Styrotech (United Kingdom).

3.2 PREPARATION OF POLYSTYRENE FILMS

Pure polystyrene (Fluka, Mol. wt. 100,000 Da) in the form of granules was dissolved in

chloroform (Fisher Scientific) (2% w/v) and sonicated (Sonicator Heat Systema

Ultrasonics INC cell disrupter model W225R) to get homogeneous solution. The clear

solution was transferred to Petri plates and placed at room temperature to evaporate the

solvent. After 24 hrs the polystyrene film was peeled off. Similar procedure was used to

prepare films from expanded polystyrene.

3.3 MEDIA

Mineral salt media containing inorganic salts and no carbon source was used in the

present study (Kiatkamjornwong et al., 1999; Table 3.1). The pH of media was adjusted

to 7.0 prior to sterilization. The medium was sterilized at 121ºC for 15 min.

3.4 SOIL BURIAL FOR ISOLATION OF MICROBES

Soil from garbage dumping area of Quaid-i-Azam University, Islamabad, Pakistan was

mixed with manure (1: 0.25). Films prepared from expanded polystyrene were buried in

the prepared soil placed in a flower pot. To enhance the microbial activity in the soil, 400

ml glucose solution (2%) was added to the soil. The buried films recovered after 8

months (May- November 2006) were utilized to isolate the microbes that can utilize

polystyrene as a sole carbon source.


28

Table 3.1 Composition of Mineral Salt Media

Contents Quantity (g/l)

K2 HPO4 1.0

KH2PO4 0.2

NaCl 1.0

CaCl2. 2H2O 0.002

H3BO3 0.005

NH4(SO4)2 1.0

MgSO4. 7H2O 0.5

CuSO4. 5H2O 0.001

ZnSO4. H2O 0.001

MnSO4. H2O 0.001

Fe2 (SO4) 3. 6H2O 0.01

Distilled water 1L


29

3.5 ISOLATION OF POLYSTYRENE DEGRADING MICROORGANISMS

Polystyrene and expanded polystyrene films were recovered from soil after eight months

(May-November 2006). The films were cut into pieces, washed with sterilized water and

placed on mineral salt medium (MSM) agar plates (Motta et al., 2009). The plates were

incubated at 30ºC. Mineral salt medium consisted of only inorganic salts, and no carbon

source was added. For isolation of bacterial strains loop full of inoculum was taken from

MSM plates and streaked on nutrient agar plates. Nutrient agar plates were incubated at

30ºC. To isolate fungi small pieces of mycelia were picked up by needle and transferred

to potato dextrose agar plates (FormediumTM

Hunstanton England). Fungal cultures were

incubated at 37ºC. To exclude the growth of fungi 0.5% antifungal agent nystatin (1%

(w/v) stock sol.) was added to nutrient agar media. Streptomycin (0.5% (w/v) stock sol.)

was used as an anti bacterial agent (0.5%) in potato dextrose agar media. Sub-culturing

many times was done to separate mixtures of microorganisms and to get pure cultures.

Serial dilution and plating onto nutrient agar and potato dextrose agar plates were also

used to isolate bacteria and fungi.

3.6 SELECTION AND IDENTIFICATION OF FUNGAL STRAINS

All the isolated microorganisms were subjected to shake flask conditions at 30 ºC, 120

rpm with MSM and Polystyrene as a sole carbon source. Fungal strains for

biodegradation experiments were selected on the basis of the FTIR spectroscopy results

of fungal treated polystyrene films in shake flask conditions (4 and 8 weeks treatment).

The selected fungal strains were characterized on the basis of molecular identification

and morphology. The cultures were maintained on potato dextrose agar plates and slants.

3.7 MOLECULAR IDENTIFICATION OF FUNGAL STRAINS

3.7.1 DNA Extraction

Isolated fungi were grown in potato dextrose broth media at 120 rpm, 30ºC. After the

maximum growth, the mycelia were harvested by filtration. Fungal mycelia were washed

twice with distilled water to remove traces of media. Mycelia were dried by an air pump

by applying negative pressure.


30

Fungal mycelia were converted to fine powdered form in liquid nitrogen by grinding with

the help of pestle and mortar. DNA was extracted according to Qiagen kit mini protocol

(Qiagen Ltd., Crawley, United Kingdom). The quality and quantity of extracted DNA

was determined by nanodrop spectrophotometer (Nanodrop™ 1000). The extracted DNA

was visualized by agarose gel electrophoresis. Agarose gel (0.8% (w/v) in 1x TAE buffer

(20mM Tris acetate, 10mM Sodium Acetate, 0.5mM Na2-EDTA (pH 7.4)) containing

0.1µl ethedium bromide solution was prepared. DNA was run at 80V for 20 minutes.

Gel was observed under UV light to check the DNA bands using UVI tec Gel

Documentation system.

3.7.2 PCR Amplification of Ribosomal DNA

The extracted DNA was subjected to PCR (Bio-Rad i cycler) to amplify the ribosomal

DNA segments. Fungal universal primers ITS-1 and ITS-4 (Invitrogen) were used to

amplify the ITS regions of fungal ribosomal DNA genes (White et al., 1990). DNA

polymerase kit (Bioline BiotaqTM

) and dNTPs (Bioline) were used. Primer sequences

used were ITS-1(5' TCCGTAGGTGAACCTGCGG) and ITS-4 (5'

TCCTCCGCTTATTGATATGC). The PCR reaction mixture 50 µl was prepared (Table

3.2) and run for 35 cycles with 94ºC denaturation temperature for 1 minute, annealing at

56ºC for 1 minute and amplification at 72ºC for 1 minute.

3.8 MOLECULAR IDENTIFICATION PROTOCOL FOR BACTERIA

3.8.1 DNA Extraction

A loop full of bacterial culture was transferred to 100 µl of filter sterilized distilled water,

vortex and boiled for 5 minutes. The sample was centrifuged at13,000 rpm for 10

minutes. Supernatant containing DNA was taken carefully and pellet was discarded. The

extracted DNA was run at 80V and 400mA for 35 minutes on agarose gel (0.8% (w/v) in

TAE buffer 1x containing 0.1µl ethidium bromide solution. Concentration of DNA was

determined by nanodrop spectrophotometer (Nanodrop™ 1000).


31

Table 3.2 PCR mixture for fungal DNA amplification

Contents Quantity

ITS1 5µl

ITS4 5µl

MgCl2 2µl

dNTPs 1µl

Enzyme Taq polymerase 5µl

Buffer 5µl

H2O 13µl

DNA Sample 5µl

Total PCR reaction volume 50µΙ

Dilutions in sterilized water

dNTPs (each 1µl ) 4:96

ITS1 1:49

ITS4 1:49

Enzyme 1:19


32

3.8.2 PCR Amplification of the 16S Ribosomal DNA

The supernatant containing the extracted DNA was used to amplify 16S ribosomal DNA

segments by PCR (Bio-Rad i cycler). Bioline, BiotaqTM

DNA Polymerase kit and dNTPs

set was used. Primer sequences used for bacteria were 6S-27F (5'-

AGAGTTTGATCCTGGCTCAG-3') and 16S-1492R (5'-

TACGGTTACCTTGTTACGACTT-3'). The PCR mixture used as listed in table 3.3 and

experimental conditions used are shown in Table 3.4.

3.9 PCR PRODUCT PURIFICATION

The concentration of amplified DNA was determined by nanodrop spectrophotometer

(Nanodrop™ 1000). QIAquick® PCR Purification Kit (Qiagen Ltd., Crawley, United

Kingdom) was used to purify PCR products. After purification DNA was run on agarose

gel with the Hyperladder1 (Bioline Ltd., London, United Kingdom) to determine the size

of amplified DNA segment.

3.10 DNA SEQUENCING

The purified DNA along with the primer dilutions were sequenced from the sequencing

facility at Faculty of life Sciences, University of Manchester, United Kingdom.

3.11 BLAST SEARCH FOR SEQUENCE HOMOLOGY

The sequencing results were subjected to Blast search at National Centre for

Biotechnology Information (NCBI) nucleotide collection database to identify the fungal

strain and study the closely related species. The Sequence Files were converted to Fast

alignment sequence tool (FASTA) files, annotated in sequin software and submitted to

NCBI Gene Bank to obtain the accession numbers.

3.12 FUNGAL DIVERSITY ASSOCIATED WITH PLASTIC FILM

The microbial population attached to the soil buried PS films was studied by Denaturing

Gradient Gel Electrophoresis (DGGE). DNA extraction kit (Fast Prep-24 MPTM

FastDNA®Kit BIO 101 Systems Q-Biogene) was used to extract DNA from Polystyrene


33

Table 3.3 PCR mixture for bacterial 16S ribosomal DNA amplification

Content Quantity (µl)

Buffer (10x) 5

MgCl2 (50mM) 1.5

16S-27 (50picomol/µl) 0.4

16S-1492 (50picomol/µΙ) 0.4

dNTPs (10mM) 1

Taq polymerase 0.2

H2O 36.5

DNA sample 5

Total PCR reaction volume 50

Table 3.4 Experimental conditions for bacterial 16S ribosomal DNA amplification

Cycle 1 1x 94ºC for 3 min.

Cycle 2 30x

step 1: 94ºC for 1 min.



Cycle 3 1x 72ºC for 10 min.

Cycle 4 1x 4ºC for ∞


34

films. DGGE specific primers were used to amplify ribosomal DNA sequences of

extracted DNA by PCR (Table 3.5). Forward primer (JB206c) had GC clamp (Muyzer et

al., 1993). Forward primer JB206c

(5/CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGAAGTAAAAG

TCGTAACAAGG3/) and reverse primer GM2 (5

/CTGCGTTCTTCTTCATCGAT3

/) was

used.

3.12.1 Denaturing Gradient Gel Electrophoresis (DGGE)

The amplified DNA was run on the DGGE gel. Gel Tank (Bio-Rad DcodeTM

Universal

Mutation Detection System), Power supply (Bio-Rad Power PAC 300). 30% (w/v)

Acrylamide: 0.8% (w/v) Bis-Acrylamide (Protoflow Gel, Flowgen Bioscience) was used

for gel electrophoresis. Denaturing gel was made by adding 6.6ml of 30% acrylamide,

2.1g of urea and 0.4ml of TAE buffer (50x) to make final volume of 20ml. 25%, 55%,

40% and 60% gel contained formamide, 2ml, 4.4ml, 3.2ml and 4.8ml respectively, while

urea was 2.1g, 4.6g, 3.4g and 5gm respectively in the gel solution. 10% Ammonium

persulfate (APS) 150l and N,N,N',N'-Tetramethylethylene diamine (TEMED) 8.5 l

were added to the gel just before pouring. Loading dye for DGGE containing 0.1%

Bromophenol blue sodium salt (BPB), 0.1% Orange G and 60% Glycerol was mixed with

the PCR products (1:1) for loading.

3.12.2 Casting the Gel

The gel was casted immediately after mixing the solutions since it will start to polymerize

after the addition of oxidising agents.

1. Glass plates (large & small), spacers and comb were wiped with 70% ethanol.

Glass plates and spacers were fixed on the casting stand with sandwich clamps.

2. Two 50 ml syringes were fitted with the gel solutions (Low & High) and fixed to

the gradient maker. There should be no air in the syringes and the tubes.

3. Syringe needle were fixed at the middle of the gel chamber and connected with

the syringe tubes.


35

Table 3.5 PCR mixture for amplification of ribosomal DNA for DGGE

Contents Quantity

GM2 2.5µl

JB206 2.5µl

MgCl2 1.5µl

dNTPs 1µl

Enzyme Taq polymerase 0.75µl

Buffer 5µl

H2O 31.75µl

DNA Sample 5µl

Dilutions in sterilized water

dNTPs (each 2.5µl ) 10:90

GM2 1:9

JB206 1:9


36

4. The gel was dispensed by turning the handle of the gradient maker at a constant

speed.

5. The comb was placed into the gel chamber carefully avoiding any air trap.

6. The gel was allowed to solidify for 2 hours.

7. The gel comb was removed after solidification and the wells were washed with

deionised H2O several times.

3.12.3 Running the Gel

The core was placed into the tank ((Bio-Rad DcodeTM

Universal Mutation Detection

System) containing 6 L of 1 x TAE buffer. The circulation was turned on to warm up the

buffer up to 60 °C. As the buffer attained the required temperature, the core was taken out

of the tank. The gel plates were attached to the core. The core was placed with the gel

into the tank and the wells were washed with 1 x TAE buffer. The samples were loaded

into the wells. The gel running conditions used were 42V, 400mA and 999min. for fungi

and 63 V, 400mA and 999 min for bacteria.

3.12.4 Gel staining

Staining solution containing 1 x TAE, 20 ml and SYBR Gold 2l was mixed in 50ml

falcon tube. The gel staining was performed by the following steps.

1. A support was placed in a plastic tray on a level surface.

2. The two glass plates containing the gel were separated. The start site of the loaded

samples was marked and the glass plate with the gel attached to it was placed on

the support in the plastic tray.

3. The staining solution (20-40 ml) was poured onto the gel so that it covered the

entire gel surface. The tray was covered with a box and left for 30 min in the dark

because SYBR Gold dye is light sensitive.

4. The gel was washed with deionised H2O for several times. The gel was soaked in


37

deionised water for 10 min. in the dark.

5. The gel was visualised on gel documentation system (UVI tec Gel Doc).

3.13 POLYSTYRENE BIODEGRADATION EXPERIMENTS

All experiments were carried out in triplicates.

3.13.1 Inoculum Preparation

Spore suspension was prepared of fungal cultures. Fungi were grown in tissue culture

bottles. 20 ml of tween 20 solution (0.05% v/v) was added to each bottle, shake well and

transferred to sterile collection tubes. Concentration of spores/ml was determined by

MOD-FUCH’S ROSETHAL (0.2mm Depth 1/116mm2

WEBER England). The spore

count of Rhizopus oryzae NA1, Aspergillus terreus NA2 and Phanerochaete

chrysosporium NA3 were 5.28 X105, 4.96X10

5, 4.57 X10

6 respectively. Spore suspension

was added to MSM so that final concentration was 10-3

spore per ml. Streptomycin 0.5 %

(0.05g/10 ml stock solution) was used as antibacterial agent.

3.13.2 Fungal Growth on MSM Agar Plates containing Polystyrene

The potential of selected fungal strains to grow on solid MSM with polystyrene as sole

carbon source was analyzed. MSM agar plates (2% w/v agar) were prepared. Polystyrene

films were dipped in 70% ethanol for 15 minutes (Calil et al., 2006) for surface

sterilization. The sterilized films were placed on MSM agar plates in one set of

experiment and embedded in MSM agar media in the second set. 0.5ml of fungal spore

suspension was used as inoculum. The plates were incubated at 30ºC for 8 weeks.

3.13.3 Sturm Test

Sturm test (Sturm, 1973) was used to study the metabolic utilization of polystyrene by

fungal strains (Fig. 3.1). The assembly consisted of ten vessels with 300ml content. First

four contained 3M KOH solution; central two contain MSM with spore suspension spore

suspension and last four contain 1M KOH solution. The central vessel containing the

polystyrene films was the test and the other control without any carbon source.


38

Figure 3.1 Experimental set up of Sturm test for biodegradation studies


39

The assembly was set in a way that air passed through the two vessels of 3M KOH

solution, test and finally through two vessels 1M KOH solution. The same was the case

with control set up. 3 M KOH solutions absorb all the atmospheric CO2 and that air was

supplied to test and control vessels where it picked up metabolic CO2. Finally the

metabolic CO2 was absorbed in 1M KOH solution, which was titrated against BaCl2

solution to produce the BaCO3 precipitates after 30 days incubation period. The

difference in weights of precipitates of test and control gave the concentration of CO2

produced as a result of biodegradation of polystyrene.

3.13.4 Treatment of PS and EPS Films with Fungal Isolates in static and shake flask

Polystyrene and EPS films were dipped in spirit for 15 minutes (Calil et al., 2006) for

surface sterilization and then added in flasks (250ml) containing MSM (100ml). The

flasks were incubated in a shaker at 120 rpm, 30ºC. Similar experiments were also set up

in static conditions at 30ºC. The experiments continued for 8 to 16 weeks.

3.13.5 UV Pre-Treatment of PS Films

Polystyrene films were irradiated by UV light (230V, 50Hz LF-106.L UVI tec limited

England) for 1 hr. and 2hr. UV lamp of 254nm wave length at an exposure distance of 3

cm was used. UV treated PS films were allowed to biodegrade by isolated fungi in MSM

under shaking condition at 120 rpm and 30ºC.

3.13.6 Heat Pre-Treatment of PS Films

Polystyrene films were exposed to heat treated at 60ºC and 80ºC for 1hr in oven (LTE

G215 Oven). Heat treated PS films were used in biodegradation studies by isolated fungi

at 30ºC and 120rpm in shaker.

3.13.7 Preparation of Polystyrene, Starch blend Films

Starch 5% (w/w) (Sigma Chemicals Co. starch soluble ACS Reagent) was added to the

PS solution in chloroform and prepared films of polystyrene, starch blend. These films

were also studied for biodegradation by the isolated fungi.


40

3.13.8 Addition of Carbon Source to Media

Glucose (BDH VWR International Ltd AnalaRc

England) was added to MSM to check

the effect of carbon source on the process of biodegradation of polystyrene and EPS films

in shake flask and static conditions. 0.01% and 0.1% (w/v) concentrations of glucose in

MSM media were used for experiments.

3.13.9 Biodegradation of Expanded Polystyrene Beads

Isolated fungi were allowed to grow with expanded polystyrene (EPS) beads (Styrotech,

England) in MSM to check the biodegradation of EPS in static and shaking conditions at

30°C. The EPS beads were dipped in 70% ethanol for 15 minutes with continuous

shaking and then dried in the Laminar flow hood before placing them in flasks. 0.25gm

of EPS beads were added to 250ml flask (100ml MSM) while 1gm was added to 1l flask

(500ml MSM). The biodegradation was studied using inoculums of all the three fungal

isolates (NA1, NA2 and NA3) in shake flask experiments at 30ºC for 12 weeks.

3.13.10 Soil burial at 30ºC

Garden soil taken from Quaid-i-Azam University, Islamabad campus, was passed through

1mm sieve and placed in 500ml conical flasks (300g soil). The soil was sterilized twice

by autoclaving (121ºC, 15psi). The flasks were inoculated with the isolated fungal strains.

Sterilized water was added to saturation and flasks were kept at 30ºC. Samples of

polystyrene films were taken after 2, 4 and 8 months and analyzed by FTIR.

3.13.11 Soil Burial at Room Temperature

Polystyrene films were buried in sterilized and non-sterilized garden soil collected from

Quaid-i-Azam University, Islamabad campus, at 6 inches depth in flower pots (Plastic)

and inoculated with the spore suspension of isolated fungi. One un-inoculated control of

sterilized and non-sterilized soil was also prepared. The soil burial experiment started in

May 2007 and continued for six months until November 2007. The recovered

Polystyrene films were analysed for biodegradation by FTIR.


41

3.14 ANALYSIS OF BIODEGRADATION

Biodegradation of polystyrene and Expanded polystyrene was studied by the following

analytical techniques.

3.14.1 FTIR Spectroscopy

Polystyrene films were recovered at repeated intervals and analyzed by FT-IR

spectroscopy (Bio-Rad Merlin Excaliber). Untreated PS film was used as control. The

absorbance was taken in the mid IR region of 400-4000 cm-1

wave number. FT-IR

analysis was done for almost all samples of all the experiments except the films used for

isolation and incubated on MSM agar plates.

3.14.2 Environmental Scanning Electron Microscopy

Environmental scanning electron microscopy (ESEM) (FEI Quanta 200) was used to

determine the changes on the surface of polystyrene film and colonization of fungi.

Analysis was carried out using low vacuum 0.68 Torr mode, 30KV and LFD (large field

Detector). The films recovered from all treatments were analysed by ESEM. The EPS

beads incubated with fungi were also observed for surface colonization of fungi by

ESEM.

3.14.3 Gel Permeation Chromatography

The changes in molecular weight distribution were evaluated by Gel permeation

chromatography (Viscotek GPC max VE 2001GPC Solvent/Sample Module, RI Detector

VE 3580). Column set: PL 2MB500A was used for GPC studies.

Tetrahydrofuran (THF) (Fisher Scientific) was used as mobile phase. The flow rate was

adjusted at 1 ml/ min. The injection volume was 100 µl. PS samples were dissolved in

THF 0.01% (w/v) and filtered through 0.45 µm syringe filters (Millex®-HV PVDF

13mm). Column temperature was adjusted at 30ºC and detection temperature at 35 ºC. n-

Dodecane was used as a marker.

GPC studies of PS Films obtained from shake flask experiments, UV pre-treatment, heat


42

pre-treatment and soil burial were carried out. The EPS films and beads treated with

fungi were also analysed by GPC.

3.14.4 Nuclear Magnetic Resonance Spectroscopy (NMR)

1H-NMR spectroscopy was done at 300MHz (BRUKER spect). PS samples were

dissolved in deutrated chloroform 0.02 gm/2ml (w/v). The solution was filled up to 5 cm

in NMR tubes (Wilmad Labglass, Sigma Aldrich, 5mm thin wall, 8// length) and placed

in the rack for spectroscopy.

NMR spectroscopy of PS film samples acquired from shake flask and static incubation

with fungi, UV pre-treatment, heat pre-treatment experiments was accomplished. EPS

films incubated with fungi in static conditions and EPS beads treated by fungi were also

studied by NMR spectroscopy.

3.14.5 High Pressure Liquid Chromatography (HPLC)

The biodegradation products produced during the process of biodegradation were

detected by High Pressure Liquid Chromatography (HPLC) (Shimadzu). The Prominence

Liquid Chromatograph system consisted of SIL-20AC Prominence Autosampler, DGU

20A5 Prominence Degasser, CTO-10AS VP Shimadzu column oven, RF-10AXL

Shimadzu Florescence Detector and Spd-20A Prominence UV/VIS Detector. The

analysis was carried out by LC solution software.

C18 column (Supleco INC LI Chrospher RP18 5µl, 259mm x 4.6mm) was used for

chromatographic analysis. Mobile phase was 70:30 acetonitrile and water respectively.

Flow rate was adjusted at 1ml/min and 10 µl injection volume was used. The UV 210

wave length was used to detect metabolites. Samples were centrifuged and filtered

through 0.2µm filter papers before analysis. The standards used for HPLC analysis of

biodegradation products were 2-phenyl ethanol, 1-phenyl-1, 2-ethanediol,

Phenylacetaldehyde, Styrene oxide and Styrene. Liquid media samples of almost all the

experiments were subjected to HPLC analysis.

Chapter 4 Results

43

4.1 ISOLATION OF POLYSTYRENE DEGRADING MICROORGANISMS

Polystyrene films buried in soil for eight months were used as a source to isolate

microorganisms capable to degrade polystyrene. Within 4 weeks of incubation on

MSM agar the entire surface of the culture medium was covered by the microbial

consortia especially fungal mycellia in all plates (Fig. 4.1). Environmental Electron

Microscopic examination of the films that were used for isolation showed mixed

microflora adhering to the polymeric surface and fungal hyphae forming spores in all

the samples (Fig. 4.2). Six fungal isolates and five bacterial isolates were isolated and

identified on the basis of preliminary studies of biodegradation (Table 4.1).

4.2 SELECTION OF FUNGAL ISOLATES

The isolated bacterial and fungal strains were tested for the ability of adherence,

growth and biodegradation of polystyrene in liquid media in shake flask conditions.

Based on FTIR spectroscopy results three fungal strains NA1, NA2 and NA3 were

selected for further biodegradation experiments (Fig. 4.3).

4.3 IDENTIFICATION OF FUNGAL STRAINS

4.3.1 Morphological Characteristics

The morphology of fungal isolates on potato dextrose agar media was studied to

identify the fungi. The colonies of the strain NA1 were woolly and initially white,

quickly becoming gray and then develop mature sporangia as small black dots in the

mycelium. Growth rate was very rapid filling the Petri plate within 2 to 3 days (Fig.

4.3a). Colonies of NA2 were pale coloured and smooth which turned yellow and then

brown with the appearance of spores (Fig. 4.3b). After sporulation the colonies

assume a powdery appearance. The media beneath the colony became yellow. The

colonies of strain NA3 were slow growing, smooth and flat on the agar surface with

white powdery appearance. The colour of mature mycelium was pale yellow (Fig.

4.3c).

4.3.2 Molecular Characteristics

The selected fungal strains NA1, NA2 and NA3 were identified on the basis of

Chapter 4 Results

44

conserved sequences of 5.8S and 18S ribosomal RNA by molecular identification

techniques. Agarose gel electrophoresis visualization of the PCR products along with

Hyper Ladder I was used to determine the quality of DNA and fragment length (Fig.

4.4). The fragment length of the PCR products was 600 base pairs of partial 18S

complete ITS1, 5.8S, ITS4 and partial 28S ribosomal DNA. The sequences were

subjected to BLAST search in National centre for biotechnology information (NCBI)

database (Appendix A). The strains were identified as Rhizopus oryzae NA1,

Aspergillus terreus NA2 and Phanerochaete chrysosporium NA3. The sequences

were submitted to NCBI Gene bank and accession numbers were obtained (Table

4.1).

4.4 DGGE ANALYSIS OF MICROBIAL COMMUNITY

The diversity of fungi adhered to the plastic films, used for isolation, were studied by

denaturing gradient gel electrophoresis. Several DNA bands were observed along the

increasing gradient of denaturing agent that separates DNA fragments according to

their melting point and GC content. The gel picture observed under UV light showed

that numerous fungi were able to adhere and grow with the PS film (Fig. 4.5).

Chapter 4 Results

45

Figure 4.1 Growth of fungal strains on polystyrene films after soil burial (8

months) on mineral salts agar medium.

Chapter 4 Results

46

Figure 4.2 Environmental scanning electron micrographs of Polystyrene films

used for isolation of microorganisms showing fungal growth 250x (a)

and 2000x (b).

a

b

Chapter 4 Results

47

Figure 4.3 Colony morphology of the selected fungal strains NA1 (a), NA2 (b)

and NA3 (c) on potato dextrose agar medium.

a b

c

Chapter 4 Results

48

Figure 4.4 Agarose gel electrophoresis visualisation of PCR Amplified DNA

using 0.8% agarose gel in TAE buffer (Lane 1, HyperLadderTM

1;

Lane 6, PCR product from strain NA1; Lane7 from NA2 and Lane 8,

PCR products from strain NA3).

1 2 3 4 5 6 7 8

Chapter 4 Results

49

Tab

le 4

.1

Mole

cula

r id

enti

fica

tion o

f m

icro

org

anis

ms

isola

ted f

rom

poly

styre

ne

film

Acc

essi

on

nu

mb

er

FJ6

54

43

0

FJ6

54

43

1

FJ6

54

43

3

FJ6

54

43

2

FJ6

54

43

4

FJ6

54

43

5

FJ6

54

43

6

FJ6

54

43

7

FJ6

54

43

8

FJ6

54

43

9

FJ6

54

44

0

Iden

tifi

cati

on

Rh

izo

pu

s o

ryza

e

Asp

erg

illu

s t

erre

us

Ph

an

ero

cha

ete

chry

sosp

ori

um

Ga

lact

om

yces

geo

tric

um

Asp

erg

illu

s f

lavu

s

Nec

tria

m

au

riti

cola

Mic

rob

acte

riu

m s

p.

Pa

enib

aci

llu

s

uri

na

lis

Sta

ph

ylo

cocc

us

sp.

Bac

illu

s sp

.

Pse

ud

om

on

as

aeru

gin

osa

%

ho

mo

log

y

99

-10

0

97

96

-98

93

-99

99

99

-10

0

97

91

-94

97

97

98

F

val

ue

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Qu

ery

cov

erag

e

99

%

90

%

95

%

70

-95

%

91

%

96

-97

%

97

%

97

%

99

%

99

%

97

%

Sco

re o

f

ho

mo

log

y

10

92

13

20-1

33

5

98

4-1

040

56

1-7

00

13

46-1

34

7

96

7-9

71

17

12-1

71

6

14

58-1

59

1

17

70-8

85

3

17

39

94

1-9

42

Acc

essi

on

nu

mb

ers

of

seq

uen

ces

giv

ing

ho

mo

log

y w

ith

tra

nsc

rip

t

EU

48

427

4.1

, E

U8

62

186

.1

FJ4

62

76

7.1

, F

J03

77

54

.1

AB

36

16

44

.1,

AB

36

16

45

.1

EF

15

91

52

.1, E

F0

879

83

.1

EF

66

15

66

.1, E

F6

615

64

.1

EU

74

783

4.1

, D

Q4

590

04

.1

EU

71

437

7.1

, E

U7

14

354

.1

EF

21

28

93

.1, A

F3

95

03

3.1

AP

00

67

16.1

, L

37

60

0.1

FJ6

41

03

6.1

, F

J64

10

17

.1

FJ7

95

68

7.1

, F

J37

41

25

.1

Iso

late

s

NA

1

NA

2

NA

3

NA

5

NA

9

NA

10

NA

23

NA

26

NA

28

NB

6

NB

26

Fu

ng

i

Bac

teri

a

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=171467195&dopt=GenBank&RID=9CNEYCHC012&log$=nucltop&blast_rank=2



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=217323600&dopt=GenBank&RID=WVBMUBK3012&log$=nucltop&blast_rank=2

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=199599793&dopt=GenBank&RID=WVBMUBK3012&log$=nucltop&blast_rank=3

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=157678733&dopt=GenBank&RID=TMHAPS6D011&log$=nucltop&blast_rank=1



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=119960474&dopt=GenBank&RID=X1AFFFWR012&log$=nucltop&blast_rank=1



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=158144451&dopt=GenBank&RID=X1B6GPAZ014&log$=nucltop&blast_rank=4



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=190351708&dopt=GenBank&RID=X1BZ5RT6012&log$=nucltop&blast_rank=1



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=189483985&dopt=GenBank&RID=X1EBGNSX01R&log$=nucltop&blast_rank=1



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=146261622&dopt=GenBank&RID=X1EUSVDK014&log$=nucltop&blast_rank=1



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=68445725&dopt=GenBank&RID=X1FFYYHA016&log$=nucltop&blast_rank=1

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=576606&dopt=GenBank&RID=X1FFYYHA016&log$=nucltop&blast_rank=2

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=224021839&dopt=GenBank&RID=X1FY7N8X016&log$=nucltop&blast_rank=1



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=225302488&dopt=GenBank&RID=MXUU0GHC016&log$=nucltop&blast_rank=5



Chapter 4 Results

50

Figure 4.5 Denaturing gradient gel electrophoresis (40%-60%) analysis of fungal

diversity with plastic films buried in soil for 8 months, used for

isolation of fungal strains (Lane 5, 6, 7, and 8).

1 2 3 4 5 6 7 8

Chapter 4 Results

51

4.5 BIODEGRADATION STUDIES OF POLYSTYRENE

Biodegradation of polystyrene was evaluated in the static and shake flask

fermentation experiments at 30ºC. Inoculum for experiments was prepared in 0.05%

Tween 20 solution to achieve uniform number of spores in each experiment.

4.5.1 Growth of Fungal Isolates on Polystyrene

The biodegradation ability of the selected fungal strains was individually analyzed by

their growth on PS film in MSM agar plates. Fungal growth was visible to the naked

eye on the surface of media after eight weeks treatment period (Fig. 4.6). ESEM

examination of the films showed mycellial growth and adherence of fungal isolates on

the surface of PS film. NA3 was not found to have profound growth on PS surface

(Fig. 4.7).

4.5.2 Sturm Test Analysis

Carbon dioxide evolution test also known as Sturm test was performed to establish

metabolic utilization of polystyrene by the selected fungal strains. After 4 weeks of

incubation at room temperature it was observed that there was more CO2 produced in

test containing the polymer films. The net metabolic CO2 produced as a result of

utilization of the carbon embedded in PS polymer chains was found to be highest for

P. chrysosporium NA3 (2.93 g/l). R. oryzae NA1 and A. terreus NA2 produced

almost similar amount of CO2 1.65 g/l and 1.42 g/l respectively as a result of PS

biodegradation (Table. 4.2).

4.5.3 Environmental Scanning Electron Microscopic (ESEM) Analysis

The adherence and growth of fungal isolates in shake flask conditions was observed

by environmental scanning electron microscopy (ESEM). The ESEM micrographs

showed profuse growth of fungal mycelia on polystyrene film in 8 weeks treated

samples with the three isolates (Fig. 4.8). The fungal isolates were able to adhere and

grow with PS.

4.5.4 Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The Fourier Transform Infrared Spectroscopy (FTIR) analysis of films recovered in

Chapter 4 Results

52

static and shake flask experiments showed changes in intensities in different regions

of spectra showing some changes in the structure of PS due to biodegradation. FTIR

spectroscopy of the polystyrene film treated under 30ºC, 120rpm conditions by R.

oryzae NA1 showed increase in intensities at 528 cm-1

,702 cm-1

, 1215 cm-1

, 1365 cm-

1, 1735 cm

-1, 2326 cm

-1, 3024 cm

-1(aryl-H stretching vibrations)

and 3402 cm

-1(Fig.

4.9a). The highest intensities in these regions were observed at four and eight week

incubation period.

PS films treated with A. terreus NA2 showed increase in absorbance intensity at 536

cm-1

, 702 cm-1


, 1365 cm-1

, 1450

cm-1

, 1600 cm-1

(C=C stretching vibrations in aromatic ring), 1735 cm-1

, 2916 cm-1

,

3024 cm-1

(aryl-H stretching vibrations) in shake flask experiment (Fig. 4.9b) .

Significant increase was observed in absorption spectra of 4 and 8 week incubation

with A. terreus NA2.

P. chrysosporium NA3 treated PS samples in shake flasks showed changes in 536 cm-

1, 748 cm

-1(mono substituted aromatic compound), 1215 cm

-1, 1365 cm

-1, 1450 cm

-1,

1600 cm-1


, 2357 cm-1

, 3024

cm-1

(aryl-H stretching vibrations), 3313 cm-1

(Fig. 4.9c). There were more obvious

changes observed at 4, 8 and 16 week treatment periods with P. chrysosporium NA3.

When polystyrene films were treated in liquid media in static conditions by the three

fungal isolates R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3, FTIR

absorption spectra showed similar changes of increased intensities around 100-1200

cm-1

, 1735 cm-1

and 3000 cm-1

and 3400 cm-1

regions were observed (Fig. 4.10).

4.5.5 Gel Permeation Chromatography (GPC) Analysis

The changes in molecular weight due to biodegradation of pure polystyrene were

studied by Gel Permeation chromatography (GPC). The weight average molecular

weight (Mw) of the control polystyrene film was 243086 Da, while the Mw after 8

week fungal treatment in shaking conditions were 246252 Da, 302013 Da, 245528 Da

when treated by R. oryzae NA1, A. Terreus NA2 and P. chrysosporium NA3

respectively. The number average molecular weight (Mn) of control was observed as

72318 Da. The Mn of R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3

were 103263 Da, 154057 Da, and 111188 Da respectively. There was a decrease in

Chapter 4 Results

53

polydispersity (Mw/Mn) after treatment. A. terreus NA2 showed lowest

polydespersity (1.96) followed by P. chrysosporium NA3 (2.208) and R. oryzae NA1

(2.385) as compared to untreated control (3.361) (Table 4.3).

4.5.6 Nuclear Magnetic Resonance (NMR) Analysis

Proton nuclear magnetic resonance (1H-NMR) spectra showed aliphatic protons (-CH-

CH2-CH-) of PS in the 1-2 ppm signal region. The aromatic protons of PS gave

signals around 6-7 ppm. The increase in number of peaks was observed in the two

regions. In both shake flask and static condition maximum change in NMR spectra

was observed in the PS film treated by P. chrysosporium NA3 followed by A. terreus

NA2 (Fig. 4.11 and Fig. 4.12).

4.5.7 High Pressure Liquid Chromatography (HPLC) Analysis

High pressure liquid chromatography (HPLC) analysis was employed to detect the

biodegradation products in the extracellular media. 1-phenyl-1,2-ethandiol was

detected in 16 week incubated shake flask media sample of R. oryzae NA1, while it

was present in all the samples (8-16 week) of A. terreus NA2 and P. chrysosporium

NA3 (Fig. 4.13a). The highest concentration (21 ppm) was found in 4 week incubated

media sample of A. terreus NA2. 1-phenyl-1,2-ethandiol was found in all the samples

of static conditions with highest concentration (34.6 ppm) in 4 weeks sample of

biodegradation experiment with P. chrysosporium NA3 (Fig. 4.13b). 2-phenylethanol

(2.5 ppm) was observed in 8 week incubated shake flask media sample and 4 week

incubated static condition sample (2.9 ppm) of P. chrysosporium NA3.

Chapter 4 Results

54

Figure 4.6 Growth of fungal isolates on PS in MSM agar plates after 8 weeks

incubation period at 30ºC, PS film on surface, R. oryzae NA1 (a), A.

terreus NA2 (b), P. chrysosporium NA3 (c), Polystyrene film partially

submerged in media and checked for growth of R. oryzae NA1 (d), A.

terreus NA2 (e) and P. chrysosporium NA3 (f) on agar plate.

(a) (b) (c)

(d) (e) (f)

Chapter 4 Results

55

Figure 4.7 Environmental Scanning Electron Micrographs of PS films inoculated

on MSM agar plates after 8 weeks incubation period with R. oryzae

NA1 (a) A. terreus NA2 and (b) P. chrysosporium NA3 (c) (1000x).

(a)

(b)

(c)

Chapter 4 Results

56

Table 4.2 CO2 evolved in 8 weeks duration of biodegradation of PS by R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 measured

gravimetrically by Sturm test (Test; PS as sole Carbon source, Control;

no carbon source)

Fungal

isolate

CO2 Produced in

TEST (g/l)

CO2 Produced in

CONTROL(g/l)

CO2 Evolved due to

biodegradation (g/l)

NA1 19.81 + 0.94 18.16 + 0.63 1.65 + 0.35

NA2 4.06 + 0.71 2.64 + 0.43 1.42 + 0.27

NA3 6.47 + 0.91 3.50 + 0.78 2.97 + 0.45

Chapter 4 Results

57

Figure 4.8 Environmental scanning electron micrographs of PS films control (a),

treated with R. oryzae NA1 (b), A. terreus NA2 (c) and P.

chrysosporium NA3 (d) in shaker (30ºC, 120rpm) for 8 weeks (2000x).

(b) (a)

(c) (d)

Chapter 4 Results

58

(a)

(b)

(c)

Figure 4.9 FTIR spectra of PS treated with R. oryzae NA1 (a), A. terreus NA2 (b)

and P. chrysosporium NA3 (c) in shaker (30ºC, 120rpm) for 4 weeks

(w4), 8 weeks (w8) and 12 weeks (w12) along with untreated control.

-0.05

-1E-16

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

so

rban

ce

Wavenumber cm-

4w

8w

12w

16w

control

536

12151365

1735

2326

3024

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

so

rban

ce

Wavenumber cm-

w4

w8

w12

w16

control

12151365

1735

2326

3024

536

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

w4

w8

w12

w16

control

1735

13651215

536

2326

2912

3359

Chapter 4 Results

59

(a)

(b)

(c)

Figure 4.10 FTIR spectra of PS treated with R. oryzae NA1 (a), A. terreus NA2 (b)

and P. chrysosporium NA3 (c) in static conditions (30ºC) for 4 weeks

((w4), 8 weeks (w8)) and 12 weeks (w12) along with untreated control.

-0.05

0

0.05

0.1

0.15

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

w12

1029

16272322

3302

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

w12

10261612 3325

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

w12

1026

16083352

Chapter 4 Results

60

Table 4.3 Gel permeation chromatography analysis of PS films treated with

fungal isolates after 8 weeks incubation at 30ºC, 120rpm

Treatment with

Weight average

molecular weight

Mw (Daltons)

Number average

molecular weight

Mn (Daltons)

Polydispersity

(Mw/Mn)

No fungus (PS control) 243086 72318 3.361

R. oryzae NA1 246252 103263 2.385

A. terreus NA2 302013 154057 1.96

P. chrysosporium NA3 245528 111188 2.208

Chapter 4 Results

61

Figure 4.11 1H-NMR analysis of Polystyrene control (a) treated with R. oryzae

NA1 (b) A. terreus NA2 (c) and P. chrysosporium NA3 (d) in shaker (30ºC, 120rpm)

for 8 weeks.

Chapter 4 Results

62

Figure 4.12 1H-NMR analysis of PS control (a) treated with R. oryzae NA1 (b) A.

terreus NA2 (c) and P. chrysosporium NA3 (d) in static conditions

(30ºC) for 8 weeks.

Chapter 4 Results

63

(a)

(b)

Figure 4.13 HPLC analysis of biodegradation products of polystyrene treated with

R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 in shaker

(30ºC, 120rpm) (a) in static conditions (30ºC) (b).

0

5

10

15

20

25

4 8 12 16 4 8 12 16 4 8 12 16

Time (weeks) Time (weeks) Time (weeks)

NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment

1-phenyl 1, 2 ethandiol

2-phenylethanol

0

5

10

15

20

25

30

35

4 8 12 4 8 12 4 8 12


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

Chapter 4 Results

64

4.6 BIODEGRADATION STUDIES OF EXPANDED POLYSTYRENE FILMS

Biodegradation of expanded polystyrene (EPS) in the form of thin films was

evaluated in order to determine whether the fungal isolates are able to grow and

degrade expanded polystyrene.

4.6.1 Fourier Transform Infrared Spectroscopy (FTIR) analysis

The FTIR analysis of films recovered in static and shake flask experiments was

carried out in order to determine the structural changes in EPS films. The FTIR

spectra showed increased absorbance intensities in different regions indicating

changes in the structure of EPS due to biodegradation. The EPS film samples treated

in shake flask conditions with R. oryzae NA1 showed increased absorbance at 1450

cm-1

, 2908 cm-1

after 12 week treatment (Fig. 4.14a), while in static conditions the

changes were observed at 536 cm-1

, 744 cm-1

(mono substituted aromatic compound),

1022 cm-1

, 1450 cm-1

, 1597 cm-1

, 1608 cm-1

(C=C stretching vibrations in aromatic

ring), 3255 cm-1

showing maximum increase after 12 week treatment (Fig. 4.15a).

A. terreus NA2 treated EPS films showed increase in absorbance intensity at 1022 cm-

1, 1450 cm

-1, 2908 cm

-1 after 12 week treatment in shake flask experiment (Fig. 4.14b)

while biodegradation experiment in static conditions the intensities increased at 536

cm-1

, 744 cm-1


, 1450 cm-1

, 1597

cm-1

, 1604 cm-1


(Fig. 4.15b).

P. chrysosporium NA3 treated EPS samples in shake flasks exhibited increase in

absorption peaks at 1026 cm-1

, 1450 cm-1

, 3024 cm-1

(Fig. 4.14c). There was no

significant difference in FTIR spectra of week 4 and 12. In static conditions the EPS

films treated with P. chrysosporium NA3 showed changes in FTIR spectra at 536 cm-

1, 744 cm

-1(mono substituted aromatic compound), 1072 cm

-1, 1450 cm

-1, 1608 cm

-

1(C=C stretching vibrations in aromatic ring), 3224 cm

-1(Fig. 4.15c). The changes in

spectra were more evident in 4 and 8 week treated samples.


The weight average molecular weight (Mw) determined by gel permeation

chromatography (GPC) showed a marked decrease in EPS film samples after 8 weeks

Chapter 4 Results

65

treatment with R. oryzae NA1, 174400 Da; A. terreus NA2, 169226 Da and P.

chrysosporium NA3, 172910 Da as compared to control 179618 Da. The number

average molecular weight (Mn) of control was observed as 86808 Da. The Mn of

treated sample with R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 were

74229 Da, 67499 Da and 64731 Da respectively. There was an increase in

polydispersity (Mw/Mn) in treated sample. P. chrysosporium NA3 treatment showed

highest polydespersity (2.671) followed by polydispersity of A. terreus NA2 treated

EPS (2.507) and R. oryzae NA1 treated sample (2.349) as compared to control (2.069)

(Table 4.4).


1H-NMR spectra of the EPS films treated for 8 weeks by the fungal isolates showed

signals in aliphatic (1-2 ppm) and aromatic regions (6-7 ppm). The number of peaks

decreased in the spectra of EPS film treated by R. oryzae NA1, while increase was

observed in case of EPS film treated by A. terreus NA2. The intensities in the

aliphatic and aromatic region also changed in treated samples as compared to control

(Fig. 4.16).


Expanded polystyrene biodegradation products were analysed by HPLC and it was

observed that 1-phenyl-1,2-ethandiol and 2-phenylethanol were detected in media

when EPS was incubated with the fungal isolates in shake flask experiments (Fig.

4.17a). 1-phenyl-1,2-ethandiol was found in all the samples of the isolates up to 12

weeks except the sample of 4 week A. terreus NA2 treatment experiment. The highest

concentration of 1-phenyl-1,2-ethandiol (24.8 ppm) was detected in 8 week sample of

biodegradation experiment with R. oryzae NA1 while 2-phenylethanol was detected

in 8 weeks sample of R. oryzae NA1 treated (3.6) and A. terreus NA2 treated (3 ppm)

and it was found in all the shake flask samples of P. chrysosporium NA3 (4 week to

12 week) (Fig. 4.17a).

When biodegradation of EPS was tested in static conditions by the three fungal strains

the highest concentration of 1-phenyl-1,2-ethandiol (34 ppm) was found in 8 weeks

sample of A. terreus NA2. 2-phenylethanol was observed in all the samples of A.

terreus NA2 and 8 week incubated sample of P. chrysosporium NA3 (2.5 ppm).

Chapter 4 Results

66

Styrene oxide was detected in 4 week (11 ppm) and 8 week (8.5 ppm) sample of A.

terreus NA2 treated sample in static conditions (Fig. 4.17b).

Chapter 4 Results

67

(a)

(b)

(c)

Figure 4.14 FTIR spectra of EPS treated with R. oryzae NA1 (a) A. terreus NA2

(b) and P. chrysosporium NA3 (c) in shaker (30ºC, 120rpm) for 4

weeks ((w4), 8 weeks (w8)) and 12 weeks (w12) along with untreated

control.

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w12

1450

2908

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w12

1022 2908

1450

-0.03

-0.01

0.01

0.03

0.05

0.07

0.09

0.11

0.13

0.15

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w12

10261450

3224

Chapter 4 Results

68

(a)

(b)

(c)

Figure 4.15 FTIR spectra of EPS treated with R. oryzae NA1 (a) A. terreus NA2

(b) and P. chrysosporium NA3 (c) in static conditions (30ºC) for 4

weeks (w4), 8 weeks (w8) and 12 weeks (w12).

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

w12

536

1022

1450

1597

16083255

744

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w12

536

1022

1450

1597

1604

3378

744

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w12

536

1072

1450

1608

3224

744

Chapter 4 Results

69

Table 4.4 Gel permeation chromatography analysis of EPS films treated with

fungal isolates after 8 weeks incubation at 30ºC, 120rpm

Treatment with

Weight average

molecular weight

Mw (Daltons)

Number average

molecular weight

Mn (Daltons)

Polydispersity

(Mw/Mn)

No fungus (EPS control) 179618 86808 2.069

R. oryzae NA1 174400 74229 2.349

A. terreus NA2 169226 67499 2.507


Chapter 4 Results

70

Figure 4.16 1H-NMR analysis of EPS film control (a) treated with R. oryzae NA1

(b) A. terreus NA2 (c) and P. chrysosporium NA3 (d) in static

conditions (30ºC) for 8 weeks.

Chapter 4 Results

71

(a)

(b)

Figure 4.17 HPLC analysis of biodegradation products of EPS treated with R.

oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 in shaker

(30ºC, 120rpm) (a) in static conditions (30ºC) (b).

0

5

10

15

20

25

4 8 12 4 8 12 4 8 12


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

0

5

10

15

20

25

30

35

4 8 12 4 8 12 4 8 12


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

styrene oxide

Chapter 4 Results

72

4.7 BIODEGRADATION STUDIES OF EXPANDED POLYSTYRENE (EPS)

BEADS

Biodegradability of expanded polystyrene in beads form, was determined in the static

and shake flask conditions at 30ºC.

4.7.1 Growth of Fungal Isolates on EPS Beads

The biodegradation capability of the selected fungal strains was individually analyzed

by their growth on EPS beads in MSM agar Media. Fungal growth was visible to the

naked eye on the surface of EPS beads after eight weeks treatment period (Fig. 4.18).

The colour of the media also changed from pale yellow to dark brown by R. oryzae

NA1, deep yellow by A. terreus NA2 and P. chrysosporium NA3.


The growth of fungal isolates on EPS bead surface in static conditions was observed

by environmental scanning electron microscopy (ESEM) (Fig. 4.19). The ESEM

micrographs showed dense growth of fungal mycelia on EPS beads after 8 weeks

incubation with the fungal isolates (Fig. 4.20). The fungal isolates were able to

colonize EPS in beads form.


The changes at molecular level brought about by biodegradation of EPS beads were

analyzed by Gel Permeation chromatography (GPC). The observed weight average

molecular weight (Mw) of the control EPS bead was 196859 Da, while the Mw after 8

week fungal treatment in static conditions were 208169 Da, 198066 Da and 195050

Da when treated by R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3

respectively. The number average molecular weight (Mn) of control was observed as

92823 Da. The Mn of samples treated with R. oryzae NA1, A. terreus NA2 and P.

chrysosporium NA3 were 101668 Da, 107553 Da and 87588 Da respectively. The

polydispersity (Mw/Mn) of EPS bead treated by A. terreus NA2 was found to be the

lowest (1.842), followed by R. oryzae NA1 (2.048) as compared to control (2.121).

The polydispersity of EPS bead treated by P. chrysosporium NA3 was 2.227 (Table

4.5).

Chapter 4 Results

73


Proton nuclear magnetic resonance (1H-NMR) spectra of EPS beads after fungal

treatment also showed two distinct regions i.e. aliphatic and aromatic. Increase in

number of peaks and height of peak was observed in treated samples as compared to

control. Maximum changes were observed in spectra of EPS bead treated with P.

chrysosporium NA3 (Fig. 4.21).


High pressure liquid chromatography (HPLC) analysis of the broth of EPS beads

degradation experiments by selected fungal isolates showed the presence of 1-phenyl-

1,2-ethandiol in the extracellular media of all the samples (shaking and static) in

varying concentrations. 2-phenylethanol was detected in all the shake flask samples (4

and 8 week) except 8 week sample of EPS beads incubated with R. oryzae NA1.

Styrene oxide (3.6 ppm) was detected in 4 week sample of A. terreus NA2 treatment

in shaking conditions (Fig. 4.22a). In static conditions samples from two sets of

experiments (250ml and 1l volume) were analyzed. The 250 ml flask samples

incubated under static experiment contained1-phenyl-1,2-ethandiol and 2-

phenylethanol in all the samples (4-12 week) except with R. oryzae NA1 that didn’t

contain any biodegradation product at 12 week incubation (Fig. 4.22b).

In 1l flask samples 1-phenyl-1,2-ethandiol was present in all the samples (4-11 week)

with highest concentration of 36.8 ppm in 4 week sample when EPS was treated with

P. chrysosporium NA3 (Fig. 4.22b). The product 2-phenylethanol was present in 4

week treated sample by R. oryzae NA1 and all the samples of treatment by A. terreus

NA2 and P. chrysosporium NA3. Phenylacetaldehyde was detected in 4 week (9.9

ppm), 8 week (11.9 ppm) and 11 week (7.7 ppm) samples of A. terreus NA2. Styrene

oxide was detected in 4 weeks samples of R. oryzae and A. terreus NA2 at

concentration of 2 ppm, 1.7 ppm respectively. Styrene oxide was present in 4 (1.5

ppm) and 8 week (3.7 ppm) samples of P. chrysosporium NA3 treated experiment.

Chapter 4 Results

74

Figure 4.18 Growth of fungal isolates on EPS beads in mineral salt media agar

plates after 8 weeks of incubation at 30ºC in static conditions control

(no fungi) (a) inoculated with R. oryzae NA1 (b) A. terreus NA2 (c)

and P. chrysosporium NA3 (d).

(c) (d)

(a) (b)

Chapter 4 Results

75

Figure 4.19 Environmental scanning electron micrographs of EPS beads control

(a), treated with R. oryzae NA1 (b), A. terreus NA2 (c) and P.

chrysosporium NA3 (d) in static conditions (30ºC) for 8 weeks (70x)

a b

c d

Chapter 4 Results

76

Figure 4.20 Environmental scanning electron micrographs of EPS beads control

(a), treated with R. oryzae NA1 (b), A. terreus NA2 (c) and P.

chrysosporium NA3 (d) in static conditions (30ºC) for 8 weeks (2000x)

b

a

c d

Chapter 4 Results

77

Table 4.5 Gel permeation chromatography analysis of EPS beads treated with

fungal isolates after 8 weeks incubation at 30ºC

Treatment with

Weight average

molecular weight

Mw (Daltons)

Number average

molecular weight

Mn (Daltons)

Polydispersity

(Mw/Mn)

No fungus (EPS control) 196859 92823 2.121

R. oryzae NA1 208169 101668 2.048

A. terreus NA2 198066 107553 1.842


Chapter 4 Results

78

Figure 4.21 1H-NMR analysis of EPS beads control (a), treated with R. oryzae

NA1(b), A. terreus NA2 (c) and P. chrysosporium NA3 (d) in static

conditions (30ºC) for 8 weeks

Chapter 4 Results

79

(a)

(b)

Figure 4.22 HPLC analysis of EPS beads treated with R. oryzae NA1, A. terreus

NA2 and P. chrysosporium NA3 in shaker (30ºC, 120rpm) (a), in static

conditions (30ºC) (b) for the degradation products 1-phenyl-1,2-

ethandiol, 2-phenylethanol, phenylacetaldehyde and styrene oxide.

0

5

10

15

20

25

30

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

styrene oxide

0

5

10

15

20

25

30

35

40

4 8 12 4 8 12 4 8 12 4 8 11 4 8 11 4 8 11

Time (weeks)

Time (weeks)

Time (weeks)

Time (weeks)

Time (weeks)

Time (weeks)

NA1 NA2 NA3 NA1 NA2 NA3

250ml flask 1l flask

Co

nce

ntr

atio

n (p

pm

)

Treatment


2-phenylethanol

phenylacetaldehyde

styrene oxide

Chapter 4 Results

80

4.8 BIODEGRADATION OF POLYSTYRENE FILMS PRETREATED BY

UV RADIATION

On the basis of the hypothesis that a preliminary treatment causing oxidation of the

polymer will enhance the biodegradation process, the PS samples were treated with

UV light for 1 and 2 hours and studied for the biodegradation by the selected fungal

isolates in shake flask conditions.


The Fourier Transform Infrared Spectroscopy (FTIR) analysis of films exhibited

increase in intensities in different regions of spectra demonstrating changes in the

structure of PS due to biodegradation when compared to UV treated control. R. oryzae

NA1 treated UV 1 hr. samples showed increase in intensities at 536 cm-1

, 748 cm-1

,

1026 cm-1

, 1361 cm-1

, 1450 cm-1

(C=C stretching vibration of aromatic compounds),

1735 cm-1

, 3313 cm-1

in 8 weeks sample (Fig.4.23a). In UV 2hr. 8 week treated

sample with R. oryzae NA1 the intensities of absorbance increased at 536 cm-1

, 748

cm-1

, 1450 cm-1

and 2912 cm-1

(Fig.4.24a).

PS UV 1hr. pre-treated films incubateded with A. terreus NA2 showed increase in

absorbance intensity at 536 cm-1

, 744 cm-1

, 1022 cm-1

, 1450 cm-1

, 1597 cm-1

in shake

flask experiment(Fig.4.23b) . The UV 2hr. pretreated demonstrated pronounced

increase around 500- 1600 cm-1

, 2357 cm-1

, 2800- 3000 cm-1

and 3400 cm-

1(Fig.4.24b).

P. chrysosporium NA3 caused the increased absorbance in UV 1hr. pre treated PS

samples in shake flasks at 500-1600 cm-1

, 2357 cm-1

, 2800 - 3000 cm-1

and 3400 cm-1

(Fig.4.23c). There was less increase in peak intensity as compared to other isolates in

UV 2hr. samples (Fig.4.24c).


The changes in molecular weight of the UV pretreated PS due to biodegradation were

studied by Gel Permeation chromatography (GPC). The weight average molecular

weight (Mw) of the UV pretreated control polystyrene film was 245356 Da. The

lowest Mw was found after UV 1hour treatment by A. terreus NA2 (223818 Da). In

Chapter 4 Results

81

UV 2hour treated samples the lowest Mw was found in the sample incubated with A.

terreus NA2 (220418 Da) as compared to control (250833 Da). The number average

molecular weight (Mn) decreased in all the UV 2hour pretreated samples incubated

with the fungal isolates (Table 4.6).


Proton nuclear magnetic resonance (1H-NMR) spectra of UV 1hour irradiated PS

showed increase in number of peaks in aliphatic and aromatic signal region after

treatment with the fungal isolates. Maximum number of peaks in NMR spectra was

found in the PS film treated by P. chrysosporium NA3 followed by R. oryzae NA1

(Fig. 4.25). The UV 2hour pretreated samples showed decrease in number of peaks

after incubation with R. oryzae NA1 and A. terreus NA2 while increase in case of P.

chrysosporium NA3 treatment (Fig. 4.26).


HPLC analysis of the extracellular media of UV 1hour pretreated PS incubated with

the fungal isolates showed the presence of 1-phenyl-1,2-ethandiol and 2-

phenylethanol in all the samples of P. chrysosporium NA3 and 8 week sample of A.

terreus NA2 (Fig. 4.27a). In UV 2hour pretreated samples 1-phenyl-1,2-ethandiol and

2-phenylethanol were detected in the media incubated with P. chrysosporium NA3. 1-

phenyl-1,2-ethandiol was present in 8 week sample of A. terreus NA2 and P.

chrysosporium NA3 treatment. 2-phenylethanol was present in broth of 8 week

sample of PS treated with A. terreus NA2 (Fig. 4.27b).

Chapter 4 Results

82

(a)

(b)

(c)

Figure 4.23 FTIR spectra of UV 1 hour pretreated PS incubated with R. oryzae

NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) in shaker

(30ºC, 120rpm) for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500 4000

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

536

1026

1450 1735

1361

3313

748

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

536

1022

1450

1597

744

-0.1

0

0.1

0.2

0.3

0.4

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

744

14502904

536

Chapter 4 Results

83

(a)

(b)

Figure 4.24 FTIR spectra of UV 2 hour heat pretreated PS incubated with R. oryzae

NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) in shaker


untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

532 748

1450

2912

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

1026

1450

2916

532

1627

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

1022

536

1450

2916

705

1597

Chapter 4 Results

84

Table 4.6 Gel permeation chromatographic analysis of UV pre-treated PS films

biodegraded by fungal isolates after 8 weeks incubation at 30ºC,

120rpm

UV 1hour Pre treated

polystyrene

Weight average

molecular weight

Number average

molecular weight

Polydispersity

Mw (Daltons) Mn (Daltons) (Mw/Mn)

With no fungus (control) 245356 94319 2.601

with R. oryzae NA1 244531 102720 2.381

with A. terreus NA2 223818 64118 3.491

with P. chrysosporium

NA3

226780 80186 2.828

UV 2 hour pre treated

polystyrene

Weight average

molecular weight

Number average

molecular weight

Polydispersity



with R. oryzae NA1 222637 89219 2.495

with A. terreus NA2 220418 86755 2.541


NA3

227830 85399 2.668

Chapter 4 Results

85

Figure 4.25 1H-NMR analysis of UV 1hour pretreated PS control (a), treated with

R. oryzae NA1 (b), with A. terreus NA2 (c) and treated with P.


(a)

(b)

(c)

(d)

Chapter 4 Results

86

Figure 4.26 1H-NMR analysis of UV 2hour pretreated PS control (a), treated with

R. oryzae NA1 (b), with A. terreus NA2 (c) and treated with P.


(a)

(b)

(c)

(d)

Chapter 4 Results

87

(a)

(b)

Figure 4.27 HPLC analysis for the UV pre-treated Polystyrene biodegradation

products in culture broth of shake flask experiment (30ºC, 120rpm)

with R. oryzae NA1, A. terreus NA2 and P. chrysosporium NA3 for 8

weeks 1hour UV pre treated (a) 2hour UV pre treated (a).

0

2

4

6

8

10

12

14

16

18

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment

1-phenyl1, 2 ethandiol2-phenylethanol

0

2

4

6

8

10

12

14

16

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

Chapter 4 Results

88

4.9 HEAT PRE-TREATMENT OF POLYSTYRENE FILMS

Heat causes oxidation of the polymer which enhances the biodegradation process. The

PS samples were heat pre treated at 60ºC and 80ºC for 1 hour and then treated by the

selected fungal isolates in shake flask conditions. The results of degradability of PS

samples are as under.

4.9.1 Fourier Transform Infrared Spectroscopic (FTIR) Analysis

The Fourier Transform Infrared Spectroscopy (FTIR) analysis of heat pretreated PS

films incubated with the fungal isolates depicted increase in absorbance intensities in

500-1600 cm-1

, 2357 cm-1

, 2800 - 3000 cm-1

and 3400 cm-1

region (Fig. 4.28) with

maximum increase in 60ºC pretreated PS incubated with R. oryzae NA1 for 8 weeks.

In 80ºC heat treated samples maximum change was observed in P. chrysosporium

NA3 treated samples. Absorbance at 1083 cm-1

increased up to 1.66 after 8 weeks

treatment as compared to control 0.03 (Fig.4.29).


Gel Permeation chromatography (GPC) of 60ºC heat pretreated PS showed decrease

in polydispersity in A. terreus NA2 (2.45) and P. chrysosporium NA3 (2.87) treated

samples as compared to control (3.564) (Table 4.7). In 80ºC pretreated sample weight

average molecular weight (Mw) decreased in R. oryzae NA1 treated sample (218921

Da) as compared to control (220338 Da) while degradation experiments with A.

terreus NA2 and P. chrysosporium NA3 showed increased Mw. (Table 4.7).


Proton nuclear magnetic resonance (1H-NMR) spectra of heat pretreated PS showed

increase in number of peaks in aliphatic and aromatic signal region after treatment

with the fungal isolates. Maximum number of peaks in NMR spectra was found in the

PS film treated by P. chrysosporium NA3 followed by A. terreus NA2 in 60ºC heat

pre treated samples (Fig. 4.30). The 80ºC heat pretreated samples showed decrease in

number of peaks as compared to control in R. oryzae NA1 and A. terreus NA2 treated

samples (Fig. 4.31).

Chapter 4 Results

89


HPLC analysis of the extracellular media of 60ºC pre-treated PS showed the presence

of 1-phenyl-1,2-ethandiol and 2-phenylethanol in 8 week sample of P. chrysosporium

NA3, R. oryzae NA1 and 4 week sample of A. terreus NA2 (Fig. 4.32a). In 80ºC

pretreated samples 1-phenyl-1,2-ethandiol was detected in all the media samples

except 8 week sample of R. oryzae NA1. The highest concentration of 1-phenyl-1,2-

ethandiol appeared in 8 week sample of P. chrysosporium NA3 (20.6 ppm). 2-

phenylethanol was detected in 4 and 8 week sample of A. terreus NA2.

Phenylacetaldehyde was detected in 4 week sample of P. chrysosporium NA3 (15

ppm) (Fig. 4.32b).

Chapter 4 Results

90

(a)

(b)

(c)

Figure 4.28 FTIR spectra of 60ºC 1hour heat pretreated PS incubated with R.

oryzae NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3(c) in

shaker (30ºC, 120rpm) for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

4w

w8

744

1026

1450

2912

1712

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

694

29081735

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

686

1450

1735

536

Chapter 4 Results

91

(a)

(b)

(c)

Figure 4.29 FTIR spectra of 80ºC 1hour heat pretreated PS incubated with R.

oryzae NA1 (a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) in

shaker (30ºC, 120rpm) for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

744

1026 2912

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

748536

1022 2908

1612

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

536

10831446

1608

2904

Chapter 4 Results

92

Table 4.7 Gel permeation chromatography analysis of heat pre-treated PS films

biodegraded by fungal isolates after 8 weeks incubation at 30ºC,

120rpm

60ºC 1hr pre-treated

Polystyrene

Weight average

molecular

weight

Number average

molecular weight

Polydispersity



with R. oryzae NA1 219191 61707 3.552

with A. terreus NA2 232386 94716 2.454


NA3

236557 82289 2.875

80ºC 1hr pre-treated

Polystyrene

Weight average

molecular

weight

Number average

molecular weight

Polydispersity



with R. oryzae NA1 218921 63909 3.426

with A. terreus NA2 234435 89826 2.61


NA3

232142 73191 2.013

Chapter 4 Results

93

Figure 4.30 1H-NMR analysis of the broth of heat (60ºC, 1hour) pre treated PS

control with no fungal treatment (a) treated with R. oryzae NA1 (b) A.

terreus NA2 (c) and P. chrysosporium NA3 (d) in shaker (30ºC,


(a)

(b)

(c)

(d)

Chapter 4 Results

94

Figure 4.31 1H-NMR analysis of heat (80ºC, 1hour) pretreated PS control (a),

treated with R. oryzae NA1 (b) A. terreus NA2 (c) and P.


(a)

(b)

(c)

(d)

Chapter 4 Results

95

(a)

(b)

Figure 4.32 HPLC analysis of heat pre-treated PS incubated with R. oryzae NA1,

A. terreus NA2 and P. chrysosporium NA3 in shaker (30ºC, 120rpm)

for 8 weeks 60ºC 1hour heat treated (a), 80ºC 1hour heat treated (b), in

shaker (30ºC, 120rpm).

0

2

4

6

8

10

12

14

16

18

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment

1-phenyl1, 2 ethandiol

2-phenylethanol

0

5

10

15

20

25

4 8 4 8 4 8

Time (ppm) Time (ppm) Time (ppm)

NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment

1-phenyl1, 2 ethandiol2-phenylethanol

phenylacetaldehyde

Chapter 4 Results

96

4.10 EFFECT OF GLUCOSE ON POLYSTYRENE BIODEGRADATION

PS films were biodegraded by fungal isolates with 0.01% and 0.1% glucose in media

to study the effect of additional carbon source on biodegradation process. Fungal

growth was more rapid and dense mycelium was visible around the PS films after

eight weeks treatment period.


The growth of fungal isolates on PS film surface in shaking conditions was observed

by environmental scanning electron microscopy (ESEM) after 16 weeks of incubation

period. The ESEM micrographs showed abundant growth of fungal mycelia on PS

films (Fig. 4.33).


The Fourier Transform Infrared Spectroscopy (FTIR) analysis of PS films incubated

with the fungal isolates with added glucose showed increase in absorbance intensities

in 500-1600 cm-1

, 2357 cm-1

, 2800 - 3000 cm-1

and 3400 cm-1

region. There were

more changes in intensities of peaks in spectra of PS film samples of shake flask

experiment with R. oryzae NA1 and A. terreus NA2 with 0.01% glucose as compared

to the PS samples incubated with the same isolates static conditions (Fig. 4.34 and

Fig. 4.35). Similar spectral changes were observed in P. chrysosporium NA3 treated

samples with 0.01% glucose in shake flask and static conditions.

The PS samples incubated with P. chrysosporium NA3 with 0.1% glucose exhibited

more absorbance intensities in 500-1600 cm-1

, 2357 cm-1

, 2800 - 3000 cm-1

and 3400

cm-1

region, in shake flask conditions as compared to the other fungal isolates (Fig.

4.36). There were more pronounced modifications in the spectra of PS treated by A.

terreus NA2 with 0.1% glucose in static conditions at 12 week incubation period. The

region around 3400 cm-1

received more changes in treated samples as compared to

control (Fig. 4.37).

Chapter 4 Results

97


Gel Permeation chromatography (GPC) of PS treated by fungal isolates with 0.01%

glucose showed decrease in molecular weight and polydispersity as compared to

control (Table. 4.8). A. terreus NA2 treated PS film showed the lowest weight

average molecular weight (Mw) (229770 Da) with 0.01% glucose after 12 week

incubation as compared to control (243086 Da).


In 0.01% added glucose conditions 2-phenylethanol was detected in all samples of P.

chrysosporium NA3 and A. terreus NA2 in shake flask experiments by HPLC

analysis (Fig. 4.38a). 1-phenyl-1,2-ethandiol was found in all samples of P.

chrysosporium NA3 and 8 week sample of A. terreus NA2 in shaking conditions with

added 0.01% glucose.

HPLC analysis of the extracellular media of 0.01% added glucose in static condition

showed the presence of 1-phenyl-1,2-ethandiol in all the samples except 8 week

sample of R. oryzae NA1 and 2-phenylethanol in 8 week sample of P. chrysosporium

NA3 (2.5 ppm). The highest concentration of 1-phenyl-1,2-ethandiol (11 ppm) was

observed in 8 week sample of P. chrysosporium NA3 (Fig. 4.38b).

The 0.1% added glucose condition 4 week sample of P. chrysosporium NA3 showed

the presence of 1-phenyl-1,2-ethandiol (19 ppm), 2-phenylethanol (8.3 ppm) and

styrene oxide (1.5 ppm), while A. terreus NA2 treated 4 week sample contained 1-

phenyl-1,2-ethandiol (16.7 ppm), 2-phenylethanol (2.9 ppm) (Fig. 4.39a). In static

conditions 1-phenyl-1,2-ethandiol was present in all the samples, 2-phenylethanol was

also detected in all samples except 4 week sample of R. oryzae NA1 and

Phenylacetaldehyde was present in all samples of A. terreus NA2 (Fig. 4.39b).

Chapter 4 Results

98

Figure 4.33 Environmental scanning electron micrographs of PS films control (a),

treated with R. oryzae NA1 (b), A. terreus NA2 (c) and P.

chrysosporium NA3 (d) with 0.01% glucose in media in shaker (30ºC,

120rpm) for 8 weeks (2000x).

(c) (d)

(a) (b)

Chapter 4 Results

99

(a)

(b)

(c)

Figure 4.34 FTIR spectra of PS incubated with R. oryzae NA1 (a), A. terreus NA2

(b) and P. chrysosporium NA3 (c) with 0.01% glucose, in shaker


untreated control.

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

w4

w8

control

12151365

1735

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

so

rba

nc

e

Wavenumber cm-

w4

w8

control12151361

17352912

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

so

rba

nc

e

Wavenumber cm-

w4

w8

control

748

11991361 1735 2908

Chapter 4 Results

100

(a)

(b)

(c)


(b) and P. chrysosporium NA3 (c) with 0.01% glucose in static

conditions at 30ºC for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

1033

1627

2912

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

748

1450

1616

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

1029

748

1450

1616

2912

Chapter 4 Results

101

Figure 4.36 FTIR spectra of PS incubated with R. oryzae NA1, A. terreus NA2 and

P. chrysosporium NA3 with 0.1% glucose at 30ºC, 120rpm after 4

weeks.

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

NA1

NA2

NA3

1450

1735

1026

748

2912

3024

Chapter 4 Results

102

(a)

(b)

(c)


(b) and P. chrysosporium NA3 (c) with 0.1% glucose in static

conditions at 30ºC for 4 weeks (w4), 8 weeks (w8) and 12 weeks


-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

w12

748

3217102

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w12

690

1002 16203217

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w12

702

3294

1450

1597

Chapter 4 Results

103

Table 4.8 Gel permeation chromatography analysis of PS films treated with

fungal isolates with 0.01% glucose after 12 weeks incubation at 30ºC,

120rpm

Treatment with

Weight average

molecular weight

Mw (Daltons)

Number average

molecular weight

Mn (Daltons)

Polydispersity

(Mw/Mn)

No fungus (control) 243086 72318 3.361

R. oryzae NA1 239024 100607 2.376

A. terreus NA2 229770 81777 2.81


Chapter 4 Results

104

(a)

(b)

Figure 4.38 HPLC analysis of broth of PS films after incubation with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 with added 0.01%

glucose in shaker (30ºC, 120rpm) (a), in static conditions (30ºC) (b).

0

2

4

6

8

10

12

14

16

18

20

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

0

2

4

6

8

10

12

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

Chapter 4 Results

105

(a)

(b)

Figure 4.39 HPLC analysis of broth of PS films incubated with R. oryzae NA1, A.

terreus NA2 and P. chrysosporium NA3 with added 0.1% glucose in

shaker (30ºC, 120rpm) for 4 week (a), in static conditions (30ºC) (b).

0

2

4

6

8

10

12

14

16

18

20

NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Fungal strains


2-phenylethanol

styrene oxide

0

10

20

30

40

50

60

70

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

phenylacetaldehyde

Chapter 4 Results

106

4.11 EFFECT OF GLUCOSE ON EPS BIODEGRADATION

EPS films were biodegraded by fungal isolates with 0.01% and 0.1% glucose in

media.


The Fourier Transform Infrared Spectroscopy (FTIR) analysis of EPS films incubated

with the fungal isolates with glucose showed increase in absorbance intensities in

500-1600 cm-1

, 2357 cm-1

, 2800 - 3000 cm-1

and 3400 cm-1

region. There was more

increase in peak intensities in both static and shake flask conditions with R. oryzae

NA1 and A. terreus NA2 treatment with 0.01% glucose addition to media (Fig. 4.40

and Fig. 4.41). PS samples incubated in shaking conditions with 0.1% glucose showed

maximum increased peak intensities with P. chrysosporium NA3 treatment (Fig.

4.42). While in static conditions R. oryzae NA1 treatment with 0.1% glucose

produced more prominent changes in peak intensities (Fig. 4.43).


In 0.01% added glucose conditions 1-phenyl-1,2-ethandiol was found in all samples

of P. chrysosporium NA3 and A. terreus NA2 treatment of EPS and 2-phenylethanol

was present in P. chrysosporium NA3 treatment in shake flask experiments (Fig.

4.44a). 1-phenyl-1,2-ethandiol was found in all samples of P. chrysosporium NA3

treatment and 8 week sample of A. terreus NA2 and R. oryzae NA1 in static

conditions with added 0.01% glucose (Fig. 4.44b).

HPLC analysis of the extracellular media of 0.1% glucose in shaking conditions

showed the presence of 1-phenyl-1,2-ethandiol and 2-phenylethanol in all the

samples. Styrene oxide was detected in all samples of P. chrysosporium NA3 (11.4

ppm) and A. terreus NA2 (1.6 ppm) (Fig. 4.45a). In samples of static conditions with

0.1% glucose in media 1-phenyl-1,2-ethandiol was present in all samples, 2-

phenylethanol was present in all samples of P. chrysosporium NA3 and A. terreus

NA2 treatment and Phenylacetaldehyde was found in A. terreus NA2 treated samples

(Fig. 4.45b).

Chapter 4 Results

107

(a)

(b)

(c)

Figure 4.40 FTIR spectra of EPS incubated with R. oryzae NA1 (a), A. terreus

NA2 (b) and P. chrysosporium NA3 (c) with 0.01% glucose, in shaker


untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

748

11492912

1527

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

748

11492916

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

748

1145 2916

1450

Chapter 4 Results

108

(a)

(b)

(c)


NA2 (b) and P. chrysosporium NA3 (c) with 0.01% glucose in static

conditions at 30ºC for 4 weeks (w4) and 8 weeks (w8) along with

untreated control.

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

705

1033 2912

1450

1524

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

744

1022

1604 3305

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

7441450

1912

Chapter 4 Results

109

Figure 4.42 FTIR spectra of EPS incubated with R. oryzae NA1, A. terreus NA2

and P. chrysosporium NA3 with 0.1% glucose at 30ºC, 120rpm after 4

weeks.

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

CONTROL

NA1

NA2

NA3

1103

1450

33521627

Chapter 4 Results

110

(a)

(b)

(c)


NA2 (b) and P. chrysosporium NA3 (c) with 0.1% glucose in static

conditions at 30ºC for 4 weeks (w4), 8 weeks (w8) and 12 weeks


-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w12

1026

1627 3359

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w123356

1608

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

w123309

1450

702

Chapter 4 Results

111

(a)

(b)

Figure 4.44 HPLC analysis of broth samples of EPS films treated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 in shaker (30ºC,

120rpm) (a) in static conditions (30ºC) (b) with added 0.01% glucose.

0

2

4

6

8

10

12

14

16

18

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment

1-phenyl 1, 2 ethandiol2-phenylethanol

8

8.5

9

9.5

10

10.5

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


Chapter 4 Results

112

(a)

(b)

Figure 4.45 HPLC analysis of broth samples of EPS films treated with R. oryzae

NA1, A. terreus NA2 and P. chrysosporium NA3 (30ºC, 120rpm) for 4

week (a) in static conditions (30ºC) (b) with added 0.1% glucose in

shaker.

0

5

10

15

20

25

NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Fungal strains


2-phenylethanol

styrene oxide

0

5

10

15

20

25

30

35

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

phenylacetaldehyde

Chapter 4 Results

113

4.12 BIODEGRADATION OF POLYSTYRENE BY SOIL BURIAL

PS films were buried in soil in flasks (controlled conditions) and flower pots (at room

temperature) and evaluated for biodegradation. The selected fungal isolates were also

added to study their biodegradation ability in soil.


The growth of fungal isolates on PS film surface after soil burial for 4 months was

observed by environmental scanning electron microscopy (ESEM). The ESEM

micrographs showed that the fungi were able to colonise the PS in soil (Fig. 4.46).

4.12.2 Denaturant Gradient Gel Electrophoresis (DGGE) Analysis

The fungal community able to adhere and grow with the polystyrene surface along

with the selected fungal isolates was studied by denaturant gradient gel

electrophoresis (DGGE). DGGE separates DNA on the basis of GC content and heat

stability. The gel picture showed more number of bands in unsterile soil indicating

more fungal species adhering to PS as compared to the sterilized soil PS film sample

(Fig. 4.47).


The Fourier Transform Infrared Spectroscopy (FTIR) analysis of PS films buried in

soil under controlled conditions with the fungal isolates show increase in absorbance

intensities in 500-1600 cm-1

region (Fig. 4.48). R. oryzae NA1 showed more

prominent peaks as compared to other isolates after 4 months incubation at 30ºC (Fig.

4.48b).

The soil buried films in natural conditions showed increased absorbance in 750-1300

cm-1

and 3400 cm-1

region (Fig. 4.49).


Gel Permeation chromatography (GPC) of soil buried PS samples demonstrated

decrease in molecular weight and polydispersity as compared to control. In the

unsterile soil buried samples the decrease in weight average molecular weight (Mw)

was almost similar in un-inoculated control (186195 Da), inoculated with R. oryzae

Chapter 4 Results

114

NA1 (186069 Da) and A. terreus NA2 (186557 Da) as compared to control (243086

Da). In sterilized soil conditions the same pattern of Mw decrease was observed

(Table. 4.9).

Chapter 4 Results

115

Figure 4.46 Environmental scanning electron micrographs of PS films buried in

soil inoculated with R. oryzae NA1 (a), A. terreus NA2 (b) and P.

chrysosporium NA3(c) at 30˚C for 4 months

(c)

(a) (b)

Chapter 4 Results

116

Figure 4.47 Denaturing gradient gel electrophoresis (DGGE) (40% - 60%) analysis

of fungal community attached to polystyrene films buried in

unsterilized soil (3) with fungal isolates NA1 (4), NA2 (5), NA3 (6)

and sterilized soil (10) with fungal isolates NA1 (11), NA2 (12), NA3

(13).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Chapter 4 Results

117

(a)

(b)

(c)

Figure 4.48 FTIR spectra of soil buried PS films incubated with R. oryzae NA1, A.

terreus NA2 and P. chrysosporium NA3 at 30ºC for 2 months (a) for 4

months (b) for 8 months (c).

-0.05

0

0.05

0.1

0.15

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

NA1

NA2

NA3

2916

705

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

NA1

NA2

NA3

690

10222912

1450

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

NA1

NA2

NA3

1018

2912

Chapter 4 Results

118

(a)

(b)

Figure 4.49 FTIR spectra of soil buried PS films inoculated with R. oryzae NA1, A.

terreus NA2 and P. chrysosporium NA3 in unsterilized soil (a)

sterilized soil (b) in flower pots for 6 months.

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

500 1000 1500 2000 2500 3000 3500 4000

Ab

sorb

ance

Wavenumber cm-

Control

Uninoculated soil

NA1

NA2

NA3

1026

3400

-0.05

0

0.05

0.1

0.15

0.2

0.25

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

no inoculum

NA1

NA2

NA3

1018

3400

Chapter 4 Results

119

Table 4.9 Gel permeation chromatography analysis of soil buried PS films

biodegraded by fungal isolates for 6 months at room temperature

`

Unsterilized soil burial

Treatment

Weight average

molecular weight

Number average

molecular weight

Polydispersity


Untreated Control PS 243086 72318 3.361

Unsterilized soil

uninoculated control 186195 61457 3.03

R. oryzae NA1 186069 77107 2.413

A. terreus NA2 186557 72337 2.579


Sterilized soil burial

Treatment

Weight average

molecular weight

Number average

molecular weight

Polydispersity


Untreated Control PS 243086 72318 3.361

Sterilized soil uninoculated

control 199217 87213 2.284

R. oryzae NA1 189834 39147 4.849

A. terreus NA2 189260 77152 2.453


Chapter 4 Results

120

4.13 BIODEGRADATION OF POLYSTYRENE STARCH BLEND

PS was blended with starch (5% w/w) and its biodegradation by the selected fungal

isolates was determined in shake flask and static conditions.


The Fourier Transform Infrared Spectroscopy (FTIR) analysis of PS starch blend

films showed increase in peak intensity around 1000-1700 cm-1

and 3400 cm-1

. In

shaking conditions the similar increase was observed in 4 and 8 week samples of the

three fungal isolates (Fig. 4.50). The 8 weeks treated samples showed maximum

absorbance in these regions by the fungal isolates in static conditions, especially the

area around 3400 cm-1

region showed increased absorbance values of 0.149, 0.058,

0.057 after incubation with R. oryzae NA1, A. terreus NA2, P. chrysosporium NA3

respectively as compared to control (Fig. 4.51).

4.13.2 High Pressure Liquid Chromatography Analysis

High pressure liquid chromatography analysis of the media samples showed the

presence of 1-phenyl-1,2-ethandiol and 2-phenylethanol in all the samples of fungal

isolates in shaking conditions, with highest concentration of 1-phenyl-1,2-ethandiol

(17.7 ppm) and 2-phenylethanol (4.5 ppm) in 8 week sample of A. terreus NA2 (Fig.

4.52a). In static conditions 1-phenyl-1,2-ethandiol and 2-phenylethanol were found in

the samples of P. chrysosporium NA3 and 4 week sample of R. oryzae NA1 (Fig.

4.52b).

Chapter 4 Results

121

(a)

(b)

(c)

Figure 4.50 FTIR spectra of PS starch blend film, incubated with R. oryzae NA1

(a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) at 30ºC, 120rpm

for 4 weeks (w4) and 8 weeks (w8) along with untreated control.

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

15971022

3400

-0.05

0

0.05

0.1

0.15

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

1600

3321

1300

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

Control

w4

w8

1311 1600 2916

1712 3400

Chapter 4 Results

122

(a)

(b)

(c)

Figure 4.51 FTIR spectra of PS starch blend film, incubated with R. oryzae NA1

(a), A. terreus NA2 (b) and P. chrysosporium NA3 (c) at 30ºC for 4

weeks (w4) and 8 weeks (w8) along with untreated control.

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

744

34001600

536

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

1026

1635 3400

-0.05

0

0.05

0.1

0.15

0.2

500 1000 1500 2000 2500 3000 3500

Ab

sorb

ance

Wavenumber cm-

control

w4

w8

1022

1604 3400

Chapter 4 Results

123

(a)

(b)

Figure 4.52 HPLC analysis of PS starch blend films treated with R. oryzae NA1, A.

terreus NA2 and P. chrysosporium NA3 in shaker (30ºC, 120rpm) (a),

in static conditions (30ºC) (b).

0

2

4

6

8

10

12

14

16

18

20

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol

0

2

4

6

8

10

12

14

4 8 4 8 4 8


NA1 NA2 NA3

Co

nce

ntr

atio

n (

pp

m)

Treatment


2-phenylethanol


124

Biodegradation is a complex irreversible process causing the scission of polymeric

chains and structural alterations by the activity of enzymes. Most of the synthetic

plastics degrade in the natural environment by a very slow process owing to their

structural complexity, high molecular weight and hydrophobicity (Albertsson and

Karlsson, 1990; Otake et al., 1995; Sudhakar et al., 2007; Mor and Sivan, 2008).

Polystyrene (PS) is a synthetic polymer and is resistant to degradation as a

consequence of its crystallinity, mechanical properties and molecular weight

(Arvanitoyannis and Biliaderis, 1999; Kiatkamjornwong et al., 1999; Schlemmer et

al., 2009). In literature fewer reports describe the microbial utilization of polystyrene

as carbon source (Sielicki et al., 1978; Kaplan, et al., 1979; Otake et al., 1995; Eisaku

et al., 2003; Motta et al., 2009; Mor and Sivan, 2008).

The aim of this study was to investigate the process of polystyrene biodegradation and

effects of various treatments on the biodegradation process. The microorganisms were

isolated from soil, possessing the ability to metabolise the synthetic polymer

polystyrene as a sole carbon source. The potential of the selected isolates to degrade

polystyrene and expanded polystyrene was studied in detail in laboratory. The effect

of pre-treatments i.e. ultra violet light and heat, exposure to natural environment by

soil burial and additional carbon source on biodegrading capability of the selected

fungal isolates was investigated.

The expanded polystyrene thin films buried in soil for eight months were utilized to

isolate microorganisms. Soil burial method is used to determine biodegradability of

polymers because it is very close to natural disposal conditions of plastics (Orhan et

al., 2004; Singh and Sharma, 2008; Silva et al., 2007; Kyrikou and Briassoulis, 2007).

Otake et al., 1995 reported that polystyrene sheet that remained buried in soil for 32

years had no biodegradation signs.

Microorganisms that are known to biodegrade polystyrene include actinomycete

Rhodococcus ruber (Mor and Sivan, 2008), Curvularia species (Motta et al., 2009),

Bacillus, Xanthomonas, Sphingobacterium (Eisaku et al., 2003), Serratia marcescens,

Pseudomonas sp. and Bacillus sp. (Galgali et al., 2002), Bacillus coagulans

(Kiatkamjornwong et al., 1999), brown rot Gleophyllum trabeum, white rot



125

Basidiomycete, Trametes versicolor, Pleurotus ostreatus, and P. chrysosporium

(Milstein et al., 1992).

In the present study three fungal isolates were selected on the basis of preliminary

screening of isolates by FTIR spectroscopy analysis of treated polystyrene films. The

isolates were identified by morphology and ribosomal conserved sequences by

molecular identification. On the basis of morphological relationship obtained by Blast

search in NCBI database the strains were identified as R. oryzae NA1, A. terreus NA2

and P. chrysosporium NA3 and the accession numbers in Genbank are FJ654430,

FJ654431 and FJ654433 respectively. Fungi are successfully used to degrade plastics

and other xenobiotics (Francesc et al., 2006). P. chrysosporium is also reported to

biodegrade polymeric materials (Manzur et al., 1997; Sutherland et al., 1997; Shimao,

2001; Gusse et al., 2006).

Biodegradation studies were carried out in shake flask (120rpm) and static conditions

at 30ºC and at room temperature in Sturm test and soil burial conditions.

Adherence of fungal isolates with the polymer surface was observed by

environmental Scanning Electron Microscopy. The scanning electron micrographs of

PS treated in shake flask, buried in soil and EPS beads showed that the fungal isolates

were able to establish mycelia on the polymer surface (Fig.4.8, 4.19, 4.20, 4.46).

More abundant fungal growth was observed in case of addition of glucose to the

mineral salts media (Fig. 4.33). Growth and establishment of a microorganism on a

polymer is the first step for biodegradation of a polymer surface (Motta et al., 2009).

Colonisation of polystyrene for longer period of time in carbon starvation condition

also indicated that the fungi were able to utilize carbon embedded in polymer chains

as carbon and energy source. Carbon starvation conditions might also encourage the

microbial growth and adherence to the polymer (Mor and Sivan, 2008).

The biodegradation of polystyrene by the fungal isolates was studied by CO2

evolution test also known as Sturm test (Sturm, 1973). A lot of modified forms of

CO2 evolution test are reported in literature but the basic purpose was to study the

complete assimilation of polymeric carbon (Muller et al., 1992; Zee et al., 1994;

Chandra and Rustgi, 1998; Calmon et al., 2000). R. oryzae NA1, A. terreus NA2 and

P. chrysosporium NA3 produced 1.65, 1.42 and 2.93g/l CO2 respectively as a result of


126

PS biodegradation (Table 4.2). Due to the carbon starvation conditions in mineral

salts media used for biodegradation studies very low amount of CO2 is produced

(Shah et al., 2008).

Surface changes of treated films were studied by FTIR spectroscopy. FTIR

spectroscopy enables the study of a polymer at structural level and any change in the

chemical structure can be easily identified. The important absorbance peaks of

polystyrene are CH2 asymmetric and symmetric stretching around 2924 cm-1

and

2852 cm-1

, 3026 cm-1

(aromatic C–H stretches), 756 cm-1

(out-of-plane C–H bending

mode of the aromatic ring), 698 cm-1

(ring-bending vibration) and 1600 cm-1

and 1491

cm-1

(benzene ring) (Allen et al., 2004; Jang and Wilkie, 2005).

The treated samples showed maximum increase in absorbance in the important

regions of FTIR spectra with respect to biodegradation i.e. 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 ring), 1600 cm-1

(C=C stretching vibrations in aromatic ring),

1735 cm-1

, 2916 cm-1

, 3429 cm-1

(Li et al., 2005; Leroux et al., 2005; Albunia et al.,

2006; Elashmawi et al., 2008). The characteristic absorbance bands of vinyl polymers

are 960 cm-1

(transvinylene R–CH=CH–R), 910 cm-1

(terminal vinyl –CH=CH2 and

terminal vinylidene –CR=CH2) and 842 cm-1

(Wang et al., 2000). More changes in

FTIR spectra were observed in shake flask experiments as compared to static

conditions.

Biodegradation is usually initiated by adding oxygen to the substrate. It is established

that oxidation causes the carbonyl (>C=O) as well as hydroxyl (–OH) groups

formation in the substrate. Infrared spectroscopy can be used to find out the existence

of these groups, because quite intense absorbance peaks of >C=O (1740 cm-1

) and –

OH (3400 cm-1

) bonds appear in the infrared spectrum where no other polystyrene

bands are present (Wang et al., 2000). Carboxylic acids, esters and alcohols also

present absorbance peaks in 1000-1200 cm-1

region but polystyrene also has

absorbance in this region but modification in this region with the appearance of other

oxidation peaks at 1740 cm-1

and 3400 cm-1

support the evidence of oxidation process

during biodegradation (Motta et al., 2009). Moreover absorbance peaks at 756 cm-1

,

698 cm-1

indicate the monosubstituted benzene ring in the polymer. In shake flask

experiments maximum changes were observed in PS films in 1000-1700 cm-1

region


127

while in static conditions the increase in absorbance occurred around 3400 cm-1

region. The experiments with glucose added to media also showed increased

absorbance in both 1000-1700 cm-1

and 3400 cm-1

region. The decrease of absorption

peaks after longer time of exposure with the fungus was also observed which

indicated that initial oxidation products were further degraded and utilised by the

fungi.

Molecular weight distribution was characterised by gel permeation chromatography to

study the changes caused by the biodegradation process. The weight average

molecular weight (Mw) as well as number average molecular weight (Mn) increased

in the treated samples of polystyrene films and EPS beads as compared to control

while decreased in case of expanded polystyrene (Table 4.3, 4.4 and 4.5). The

increase in molecular weight may be attributed to chain cleavage and further

crosslinking (Tsuji and Ikada, 1998; Pticek et al., 2007). Changes in molecular weight

indicate the underlying process and rate of degradation of the polymer (Siracusa et al.,

2008). The polydispersity decreased in PS and increased in EPS films (Table 4.3 and

4.4). The Mw decreased in all polystyrene samples pre-treated by UV and increased

in case of heat pre-treatment (Table 4.6 and 4.7). Weight average molecular weight

and polydispersity decreased in incubation condition with added glucose in mineral

salts media and also in all samples of soil buried polystyrene films (Table 4.8, 4.9).

Polydispersity indicate the number and distribution of chain lengths in a polymer

sample. If the polymer has a narrow range of polymer chain lengths the polydispersity

is low while a high value of polydispersity is observed when the chain lengths vary

considerably.

Proton nuclear magnetic resonance (1H-NMR) spectra of the polystyrene samples,

allowed to biodegrade by the fungal isolates were compared with the control to study

the process of biodegradation (Grivet and Delort, 2009). Polystyrene structurally

consists of an aliphatic chain with allied aromatic groups linked to every other carbon

atom. The aliphatic and aromatic protons appeared in 1-2 ppm and 6-7 ppm signal

region respectively in the H1NMR spectra. In almost all samples no new peak

appeared other then the characteristic aliphatic and aromatic protons of polystyrene.

The observation may be attributed to slow biodegradation process, appearance of

unstable products or the biodegradation products may have displaced quickly in to the


128

surrounding media e.g. styrene is a volatile compound. Oxidation by-products may

also partially dissolve in aqueous surroundings. The signal strength and number of

peaks varied between the control and treated samples.

Detection of biodegradation products was accomplished by high pressure liquid

chromatography (HPLC). HPLC standards were selected on the basis of styrene

degradation products reported in literature by the fungi (Cox, 1995; Cox et al., 1996;

Francesc et al., 2006; Lee et al., 2006; Boldu et al. 2001: Weber et al. 1995; O’Leary

et al. 2002). 1-Phenyl-1,2-ethandiol was dominant with highest concentration of 21.3

ppm in media sample of polystyrene incubated with A. terreus NA2 in shake flask and

34.6 ppm in static conditions with P. chrysosporium NA3 (Fig.4.13) (Lee et al.,

2006). 1-Phenyl-1,2-ethandiol, 2-Phenylethanol, Phenyacetaldehyde and styrene

oxide was detected in different concentrations in broth samples of biodegradation

experiments.

The HPLC results when correlated with FTIR spectroscopy results, suggest that

biodegradation process of polystyrene is oxo-biodegradation (Arvanitoyannis and

Biliaderis, 1999), which is not a rapid process (Scott and Wiles, 2001). During oxo-

degradation process, peroxidation results in the formation of biodegradable products

with low molar mass like carboxylic acid, alcohols, aldehydes and ketones (Chiellini

et al., 2007). The microorganisms responsible for the biodegradation process

assimilate the biodegradation products inside the cells (Gu and Gu, 2005; Kyrikou

and Briassoulis, 2007). The peroxidation reaction is mainly initiated by the

oxygenases and peroxidases enzymes released by the microorganisms in the

surrounding media in case of biodegradation (Van den Brink et al., 1998; Francesc et

al., 2006; Kumar and Goswami, 2008; Vatsyayan et al., 2008), UV light in

photodegradation (Sudhakar et al., 2007) and heat in thermal degradation processes

(Mailhot et al., 2000; Contat-Rodrigo and Greus, 2002; Chiellini et al., 2007).

The data obtained by HPLC also back up the assumption that the biodegradation

products were mobile and remained in media that is why they did not appeared in the

NMR analysis of the treated polystyrene and expanded polystyrene films. The fungal

isolates A. terreus NA2 and P. chrysosporium NA3 showed more active

biodegradation in terms of detected biodegradation products as compared to R. oryzae

NA1.


129

The biodegradation studies were not only conducted on thin films but the fungal

isolates were also allowed to grow on expanded polystyrene beads that are used to

make expanded polystyrene packaging products. The fungi grew rapidly with visibly

covering the surface of EPS beads and changing the colour of the media without any

other carbon source. The HPLC results showed that the EPS beads were actively

biodegraded by the fungi especially by the A. terreus NA2.

UV light promotes the oxidation process of polymers by generating free radicals

(Mailhot et. al., 2000; Singh and Sharma, 2008; Ojeda et al., 2009). Thermal

degradation of polystyrene is well documented (Jang and Wilkie, 2005; Singh and

Sharma, 2008). On the assumption that pretreatment with UV light and heat

pretreatment (60 ºC & 80 ºC for 1 hr.) may facilitate the biodegradation process,

experiments were conducted on UV and heat pretreated polystyrene films. After

incubation with the fungi the analysis of the films showed that UV and heat

pretreatment did not have any profound effect on biodegradation process. The

increased exposure time of pretreatment may increase the biodegradation efficiency.

Pre-aging treatments promote the ultimate disintegration of the samples by microbial

attack (Albertsson et al., 1995; Contat-Rodrigo and Greus, 2002; Chiellini et al.,

2007). UV pretreatment of 2 hours resulted in decrease of weight average molecular

weight of polymer. The heat pretreated (80 ºC for 1 hr.) polystyrene films after

biodegradation by P. chrysosporium NA3 resulted in increased absorption intensities

in 1000-1200 cm-1

region.

Effect of additional carbon source on biodegradation was studied by adding glucose in

media and by blending starch with polystyrene. The addition of glucose in media

resulted in visible good fungal growth. There was more biodegradation of polystyrene

and expanded polystyrene with glucose as evident by GPC, FTIR and HPLC results.

The results indicated that the addition of carbon source enhance the biodegradation

process. Polystyrene starch blend showed more changes in FTIR spectra in static

conditions while more biodegradation products were detected in shake flask

conditions. Starch increases the porosity and the surface/content ratio of conventional

synthetic polymers. As the microorganisms assimilate starch, the structural integrity

of the surrounding plastic is lost. The deterioration of the mechanical properties


130

facilitates the degradation and promotes the microbial attack of the polymeric matrix

(Kiatkamjornwong et al., 1999; Zuchowska et al., 1998; Schlemmer et al., 2009).

Biodegradation process of polystyrene was also studied in field conditions by soil

burial method along with the studies in laboratory conditions (Hesselsoe et al., 2008;

Watanabe et al., 2009). Appearance of similar dominant bands in the denaturant

gradient gel electrophoresis analysis of the soil attached to buried polystyrene films

indicated that some soil fungi were the dominant colonizers of the polymer. The

scanning electron micrographs of the soil buried films in the flasks showed the

colonization of the polymer by the fungi. The soil buried films of polystyrene for six

months showed very significant degradation in FTIR and GPC analysis. Significant

decrease in weight average molecular weight in the soil buried samples with no added

fungal inoculum indicated that other soil microbes were also involved in the

biodegradation process of polystyrene.

Conclusions

131

Conclusions

From the present study it is concluded that:

1. The soil contains microorganisms capable of adherence and growth with

polystyrene as a sole carbon source thus bringing about its biodegradation.

2. The isolates Rhizopus oryzae NA1, Aspergillus terreus NA2 and Phanerochaete

chrysosporium NA3 were able to colonize polystyrene film surface for longer

period of time without any other carbon source indicating that the isolates utilised

the polymer as carbon source.

3. The increased carbon dioxide production with polystyrene in Sturm test indicated

that biodegradation process is going on.

4. The techniques SEM, FTIR, GPC and NMR proved to be effective analytical tools

to study plastic materials such as polystyrene and expanded polystyrene films and

beads.

5. The detection of degradation products in the surrounding media of the treated

samples by HPLC confirmed that the fungal isolates were able to not only

depolymerise but biodegrade the polystyrene, expanded polystyrene films and

EPS beads.

6. The UV and heat pre-treatments did not affect biodegradation process but pre-

treatment for longer time could give better results.

7. The addition of glucose showed enhanced biodegradation of polystyrene. More

biomass was produced with additional carbon source resulting in decrease in

molecular weight and more changes in treated samples.

Future Perspectives

132

Future Perspectives

Mechanism of biodegradation of polystyrene can be studied and degradation by-

products could be identified.

Enzymes involved in biodegradation process could be isolated and their

applications could be studied.

Environmental impact of strain involved in biodegradation and the by-products

should be studied for field applications.

Genes involved in biodegradation could be studied in detail.

Strain improvement could be accomplished in order to get desired results for

biodegradation applications.

Field scale trials could be done for bioremediation of plastic waste contaminated

site.

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133

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Appendix

148

Figure 1A Phylogenetic tree of Rhizopus oryzae NA1

R. oryzae CNRMA 03.411 18S ribosomal ...

R. oryzae CNRMA 03.413 18S ribosomal ...

R. oryzae CNRMA 04.48 18S ribosomal R...

R. oryzae CBS 112.07 18S ribosomal RN(2)

R. oryzae NRBC 5384

R. oryzae JCM 12786

R. oryzae CBS 109939

R. oryzae CBS 395.95

R. oryzae NRRL 2710

R. oryzae R-55

R. oryzae R-69

R. oryzae R-612

R. oryzae CTSP F4

R. oryzae IP 4.77 18S ribosomal RNA+i...

R. oryzae DG-B1

R. oryzae CBS 112.07 18S ribosomal RN...

NA1 FJ654430 R.oryzae

R. oryzae SU-B3

7.3963

1.9355

21.1775

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

12.4556

6.2750

14.7682

12.6843

5

Appendix

149

Figure 2A Phylogenetic tree of Aspergillus terreus NA2

A. terreus UOA/HCPF 9927 18S ribosoma...



A. terreus UOA/HCPF 10158-2 18S ribos...

A. terreus UOA/HCPF 10178 18S ribosom...

A. terreus UOA/HCPF 10213A 18S riboso...






A. terreus 18S ribosomal RNA+internal...


E. sp. DC492

A. terreus NBRC 4100 internal transcr...

A. tubingensis NRRL 4851 internal tra...

A. terreus GX7-4A 18S ribosomal RNA+i...

NA2 FJ654431.1| A. terreus0.014769

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.000000

0.002167

0.000000

0.012603

0.000869

0.000836

0.000909

0.002

Appendix

150

Figure 3A Phylogenetic tree of Phanerochaete chrysosporium NA3

u. fungus(3)

u. fungus(13)

u. fungus(2)

u. fungus(11)

u. fungus(4)

u. fungus(7)

u. fungus(5)

u. fungus(12)

P. chrysosporium NA3

P. chrysosporium UD 03/06 18S ribosom...

P. chrysosporium PV1

u. fungus(6)

P. chrysosporium liu

u. fungus

u. fungus(9)

P. chrysosporium TS03

u. fungus(8)

P. chrysosporium IFM 47473 small subu...

P. chrysosporium IFM 47494 small subu...

P. chrysosporium Bm-4

f. endophyte sp. P29-005 P29-005 inte...

P. chrysosporium UD 117/08 18S riboso...

P. chrysosporium FP 102074 18S riboso...

u. fungus(10)

P. chrysosporium KCTC 6728 18S riboso...

P. chrysosporium KCTC 6293 18S riboso...

P. chrysosporium FCL208 18S ribosomal...

12.925

0.155

0.000

0.000

0.008

17.872

0.196

0.006

14.237

9.630

15.317

17.568

12.896

18.674

0.005

7.522

0.000

10.012

12.364

2.753

10.796

0.099

24.553

14.863

0.717

13.031

8.389

5.165

7.209

1.799

4.641

5.731

3.628

1.677

1.611

0.443

0.510

2.307

5.460

9.597

6.612

0.063

5

Publications

Papers Submitted/ Published

Isolation and identification of polystyrene biodegrading bacteria from soil

Atiq, N, S. Ahmed, M. I. Ali, S. Andleeb, B. Ahmed, G. Robson (2010). African

Journal of Microbiology Research. 4(14), 1537 – 1541

An investigation of polystyrene utilization as a carbon source by Rhizopus

oryzae NA1

Naima Atiq, Safia Ahmed, Mohammed Ishtiaq Ali, Saadia Andleeb, Mohammed

Sajjad Shaukat, Geoffrey D. Robson (Submitted for publication in Polymer

Degradation and Stability (PDST-S-09-01105))

Evaluation of Biodegradability of Expanded Polystyrene and its Blends

Naima Atiq, Mariam Asif, Saadia Andleeb, Mohammed Ishtiaq Ali, Mohammed

Sajjad Shaukat, Safia Ahmed (Submitted for publication in Iranian Polymer Journal

(df5120))

Papers Presented in Conferences

Isolation of Polystyrene Degrading Soil Microorganisms

Naima Atiq, Fariha Hassan, Aamer Ali Shah, Safia Ahmed, Abdul Hameed

Sixth International Biennial Conference of Microbiology, March 18-21, 2007

Islamabd, Pakistan, (Poster Presentation).

Fungal Degradation of Polystyrene

Safia Ahmed, Mariam Asif, Fariha Hassan, Naima Atiq, Aamer Ali Shah, Abdul

Hameed

BIOMicroWorld 2007 II International Conference on Environmental, Industrial and

Applied Microbiology, 28th

Nov- 1st Dec 2007, Seville, Spain, (Paper accepted for

presentation).

An investigation of polystyrene utilization by Aspergillus terreus NA2


Sajjad Shaukat, Safia Ahmed

Economic and Social Impact of Fungal Deteriogens, (organized by British

Mycological Society and the International Biodeterioration and Biodegradation

Society), 6th - 7th April 2009, University of Manchester, (Poster presentation).

Colonisation and Biodegradation of Polystyrene by Indigenous Isolated Strain

Na3 Phanerochaete Crysosporium

Naima Atiq, Safia Ahmed, Muhammed, Ishtiaq Ali, Saadia Andleeb, Geoff Robson

Paper accepted for presentation in 3rd Congress of European Microbiologists, FEMS

2009, June 28-July 2, 2009, Gothenburg, Sweden, (Paper accepted for presentation).

Biodegradation Studies of Expanded Polystyrene Blends



9th

International Seminar on Polymer Science and Technology, Iran Polymer and

Petrochemical Institute, 17-21 October, 2009, Tehran, IRAN, (Oral Presentation).

Evaluation of Biodegradability of Expanded Polystyrene by Rhizopus oryzae NA1



9th

International Seminar on Polymer Science and Technology, Iran Polymer and

Petrochemical Institute, 17-21 October, 2009, Tehran, IRAN, (Oral Presentation).

Colonisation and Biodegradation of Expanded polystyrene beads by indigenous

isolated fungal strains

Naima Atiq, Safia Ahmed, Aamer Ali Shah, Mohammed Ishtiaq Ali, Saadia Andleeb,

Geoffrey D. Robson

BioMicroWorld2009- III International Conference on Environmental, Industrial and

Applied Microbiology, 2-4 December, 2009, Lisbon, Portugal, (Virtual Presentation).

Biodegradability of Synthetic Plastics Polystyrene and ...

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