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International Biodeterioration & Biodegradation 105 (2015) 21e29

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International Biodeterioration & Biodegradation


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Short communication

Biodegradation of gamma irradiated low density polyethylene and


polypropylene by endophytic fungi
Sana Sheik a, K.R. Chandrashekar a, b, *, K. Swaroop b, H.M. Somashekarappa b
a
Department of Applied Botany, Mangalore University, Mangalagangothri, 574199, Mangalore, Karnataka, India
b
Centre for Application of Radioisotopes and RadiationTechnology (CARRT), Mangalore University, Mangalagangothri, 574199, Karnataka, India

a r t i c l e i n f o a b s t r a c t

Article history: Biodegradation of plastic waste through fungal strains offer a solution to serious issues of pollution as
Received 10 July 2015 fungi are known to release plastic degrading enzymes. In the present study, the isolated endophytic fungi
Received in revised form from two endemic plants, Psychotria flavida and Humboldtia brunonis which produced laccase enzymes
6 August 2015
and grew profusely over hydrophobic surface of plastic films were tested for biodegrading ability. The
Accepted 9 August 2015
Available online xxx
fungi were inoculated on the polythene and polypropylene films irradiated with different doses of ra-
diation, (0e1000 kGy for Low Density Polyethylene and 0e100 kGy for Polypropylene) and incubated for
90 days. The extent of biodegradation pattern of endophytic fungi was measured for the highest dose
Keywords:
Lasiodiplodia theobromae
mainly by analyzing changes using FTIR spectroscopy, DSC, SEM, alteration in viscosity and thereby
LDPE average molecular weight. The decrease in intrinsic viscosity and average molecular weight of gamma
FTIR irradiated LDPE strips inoculated with Aspergillus sp., Paecilomyces lilacinus from H. brunonis and Lasio-
Biofilm diplodia theobromae from Psychotria flavida indicate fungal efficiency in plastic degradation. Only
L. theobromae from P. flavida could degrade irradiated polypropylene film with 0.3 mg on actual weight
loss basis. Further work on employing these endophytic fungi in biodegradation of plastics is warranted.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction Polyethylene is a polymer consisting of long chains of the monomer


ethylene (IUPAC name ethene). Polypropylene (IUPAC name poly-
Much of the nature remains to be explored, particularly mi- propene), are synthesized propene monomers which are typically
crobial environments (Newman et al., 2003). One such unexplored obtained from oil refinery as gaseous products. Nowadays, a wide
and less studied microorganism is the endophytic fungi, those variety of petroleum-based synthetic polymers are produced
microbes that reside in internal tissues of living plants without worldwide to the extent of approximately 140 million tons per year
causing any immediate overt negative effects or external symptoms and remarkable amounts of these polymers are introduced in the
(Aly et al., 2011). The enormous importance of studies on endo- ecosystem as industrial waste products (Shimao, 2001). Biodegra-
phytic system is the ability to produce metabolites and innovation dation is governed by different factors that include polymer char-
of microbial source which are valuable in bioremediation acteristics, type of organism, and nature of pretreatment. The
(Ste˛ pniewska and Kuzniar, 2013). The huge variety of the metabolic polymer characteristics such as its mobility, tacticity, crystallinity,
pathways employed by endophytes makes them valuable tools for molecular weight, the type of functional groups and substituents
bioremediation of pollutants and biotransformation of organic present in its structure, and plasticizers or additives added to the
substances (Gai et al., 2009; Kim et al., 2012). polymer all play an important role in its degradation (Artham and
Semicrystalline low density polyethylene (LDPE) and poly- Doble, 2008; Gu et al., 1998; Gu et al. 2000b, 2011; Gu, 2003a,b).
propylene (PP) of extremely recalcitrant, high hydrophobic nature The prerequisite condition for biodegradation is that the
are major persistant plastics dumped in the environment. microorganism should be able to use the polymer as its sole source
of carbon. LDPE, an extremely high molecular weight large sized
polymer molecule is made up of methylene which microorganism
is unable to transport directly into the cells. . In the current study,
* Corresponding author. Department of Applied Botany, Mangalore University,
Mangalagangothri, 574199, Mangalore, Karnataka, India. inorder to boost biodegradation of plastics, films were pretreated
E-mail address: profkrchandrashekar@gmail.com (K.R. Chandrashekar). with gamma radiation to induce photo oxidation. Photo oxidation

http://dx.doi.org/10.1016/j.ibiod.2015.08.006
0964-8305/© 2015 Elsevier Ltd. All rights reserved.
22 S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29

enhances the rate of biodegradation of polymer that results in the enzyme in the presence of 3 ml acetate buffer. The change in the
generation of large surface area due to its embrittlement and also absorbance of the reaction mixture containing guaiacol was
produces a greater degree of hydrophilicity due to the introduction monitored at 470 nm for 10 min of incubation using Spectropho-
of carbonyl groups (Arkatkar et al., 2009). The biodegradation al- tometer. A control test was conducted in parallel with absence of
ways follow photodegradation and chemical degradation (Shah enzyme source. Enzyme activity is measured in U/ml which is
et al., 2008). The initial breakdown of a polymer can result from a defined as the amount of enzyme catalyzing the production of
variety of physical and biological forces (Swift, 1997). Biodegrad- 1 mmol of colored product per min per ml.
ability of large molecules such as cellulose acetate and electronic
insulation polyimides were studied previously by many workers 2.2. Inoculation of endophytic fungi to gamma irradiated LDPE and
(Gross et al., 1995; Gu et al., 1996a) A comprehensive study of PP films
polyolefin biodegradation has shown that some microorganisms
could utilize polyolefins with low molecular weight (Yamada LDPE (Thickness e 20 mm, density e 0.93 g/cm2, melting point e
Onodera et al., 2001). 114  C) and PP films (Thickness e 20 mm, density e 0.91 g/cm2,
Realizing the capability of microorganisms to produce diverse melting point e 160  C) were purchased from Janani Plastic in-
bioactive molecules and the existence of unexplored microbial di- dustry, a commercial manufacturing unit, Baikampady, Mangalore,
versity, research is underway to isolate and screen microbes of India. The films were cut into strips and irradiated at different doses
diverse habitat and unique environment for discovery of novel of gamma radiation ranging from 0 to 1000 kGy for LDPE. LDPE
metabolites (Mohanta et al., 2007). The unique biological niche of films were not subjected to gamma radiation for more than
endophytes as endosymbionts of tissues rich in complex carbon 1000 kGy as films were slightly brittle and further inoculation with
polymers justifies the investigation of their wider metabolic ca- higher doses would cleave the films resulting in inaccurate weight
pabilities (Russell et al., 2011). Plants that are endemic are also loss measurements. In case of PP, strips were subjected to
more likely to lodge endophytes with active natural products than 0e100 kGy doses as the doses beyond 100 kGy lead to pyrolysis in
other plants (Strobel and Daisy, 2003). The anticipated topic is to these films. Individual pre weighed strips were surface sterilized
unlock the poorly investigated trapped microorganism of novel using70% alcohol and aseptically transferred to the conical flasks
metabolic capabilities for polymer degradation inside two endemic containing 50 ml of Rose Bengal broth medium, to which fungal
plants viz. Humboldtia brunonis Wall. and Psychotria flavida Talbot. strains were inoculated separately. Unirradiated LDPE and PP
of Western Ghats, India. inoculated with/without fungi served as control. The flasks were
maintained for 90 days under aseptic conditions. Subsequently,
2. Materials and methods strips were washed thoroughly in distilled water, shade dried and
weighed for final weight. From the data collected, weight loss of the
2.1. Isolation and selection of endophytic fungi for biodegradation plastic film was calculated.

Healthy, disease free, mature leaf and stem samples of P. flavida


2.3. Characterization and analysis
Talbot and H. brunonis Wall collected from Western Ghats were
transferred to sterile bags and processed within 24 h. Samples were
Samples were characterized for changes in their viscosity,
surface sterilized, excised into small pieces (0.5 cm size), plated in
spectra, morphology and thermal behavior with reference to con-
potato dextrose agar medium for more than 10 days, and examined
trol films.
for endophytic fungal emergence. The emerging fungal mycelium
Average Molecular Weight Determination: Average molecular
was sub cultured in potato dextrose agar for 7 days or till sporu-
weight was determined by viscometric measurements using an
lation. The spores along with the fruiting bodies were used for
Ostwald Viscometer (Gadag and Shetty, 2006). The intrinsic vis-
identification. Few of the fungi were deposited and morphologi-
cosity was determined by dissolving the samples in 20 ml of p-
cally identified by Agharkar Research Institute, Pune. Few of the
xylene for LDPE and 20 ml toluene for PP, followed by heating upto
representative morphotypes and non sporulating fungi of each leaf
80  C. Inorder to find out viscosity, flow time measurements for
and stem samples were identified purely on molecular basis. DNA
various solvents (Xylene for LDPE, Toluene for PP) and polymer
was extracted using CTAB method (HiPurA TMPlant DNA isolation
solvents system were measured using the following equation,
kit-HIMEDIA). PCR was conducted to amplify the internal tran-
scribed spacer (ITS) region of the extracted DNA, using the primers h1 r2 t2
ITS 1 and ITS 4 (White et al., 1990) under the following conditions: h2 ¼
r1 t1
95  C for 3 min followed by 35 cycles of 95  C for 30 s, 50  C for 45 s,
72  C for 90 s, and final extension at 72  C for 10 min. PCR amplicons where, t1 and t2 are the time of flow of the water and xylene/
were electrophorized in 1.2% agarose gel. The amplified PCR toluene and r1 and r2 are the respective densities and h1 is the
products were sequenced by BIOSERVE (Hyderabad) using ABI 3130 coefficient of viscosity of water. The average molecular weight (M)
(48 capillary) or 3730Xl (96 capillary) electrophoresis instrument. A was determined following MarkeHouwink equation
BLAST (Basic Local Alignment Search Tools) was used to search for
closest match sequences in the GenBank database and the se- h ¼ KMa
quences were submitted to gene bank.
Among the consortia of endophytes isolated from two plant where K and a are constants for a given polymer-
species, six fungi were selected on the basis of their ability: i) To esolventetemperature system (K ¼ 0.000218, a ¼ 0.725 for poly-
produce laccase enzyme which was confirmed by the oxidation of propylene e toluene and K ¼ 0.000510, a ¼ 0.725 for LDPE e xylene
colorless 1-naphthol amended medium to bluish violet color system).
(Hankin and Anagnostakis, 1975) ii) To grow profusely forming Spectral analysis: The spectrum was taken from 400 to 4000
biofilm over the control plastic films incubated for a month. wavenumbers cm1 using FTIR spectrophotometer (IR Prestige e
Estimation of Laccase: The Laccase activity was assayed at room 21 SHIMADZU).
temperature using 10 mM Guaiacol in 100 mM sodium acetate Surface Morphology: Determinations of surfacial biofilm for-
buffer (pH 5.0). For this, 1 ml of Guaiacol was mixed with 1 ml mation and degradation characteristics were observed using
S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29 23

Scanning Electron Microscope (ZEISS e SIGMA) after the samples


were metalized with gold particles.
Thermal behavior: The changes in the thermal properties of the
treated films with highest dose of gamma radiation and control
were analyzed through Differential Scanning Calorimetry over
Universal thermal analyzer (SDT Q 600 e WATERS) at temperatures
ranging between 25 and 400  C under nitrogen atmosphere
(100 ml min1) with heating rate of 10  C. The percentage crys-
tallinity was calculated as follows,
Fig. 1. A) L. theobromae attached to Polypropylene film B) Pestalotiopsis sp e Quali-
DHm tative test showing the presence of Laccase enzyme.
%Crystallinity ¼  100
DHm+
bacteria and fungi, has been shown to degrade polyethylene due to
where, DHm ¼ polymer enthalpy of fusion as in the thermogram, in conserved copper binding sites which couple the oxidation of a

Jg1; DHm ¼ enthalpy of fusion of PP, pure crystalline, corre- substrate with the cleavage of dioxygen bonds, leading to the
sponding to 207 Jg1 (Vander et al., 1998). In case of pure crystalline capability to degrade plastics particularly polyethylene. So far,

LDPE value of DHm is equal to 280 Jg1 (Ikhlef et al., 2012). several fungal endophytes as for instance, Colletotrichum gloeo-
sporioides isolated from Piper betle, Monotospora sp., an endophytic
3. Results and discussions fungus in Cynodon dactylon, Phomopsis longicolla of Bixa orellana,
Discosia sp. from Calophyllum inophyllum followed by Chaetomium
3.1. Preliminary selection of endophytic fungi with biodegrading sp from Alpinia calcarata, have been reported to produce laccase
ability enzymes in culture conditions (Wang et al., 2006; Sunitha et al.,
2013; Sidhu et al., 2014).
Among the endophytic fungi isolated from two plant species, six There have been no reports on the biofilm formation of endo-
fungi were selected (Table 1) viz. Cunnighamella echinulata, Pesta- phytic fungi on polyethylene and polypropylene. However there are
lotiopsis sp, Hypoxylon anthochroum, Paecilomyces lilacinus, Asper- reports on biofilm formation on the polyethylene by soil fungi such
gillus sp, Lasiodiplodia theobromae on the basis of their ability to as Aspergillus, Fusarium, Rhizopus arrhizus, Penicillium sp etc
produce both laccase enzyme and profuse biofilm formation (Fig. 1) (Mahalaxmi and Niren, 2012; Raaman et al., 2012; Das and Santosh,
on the plastic films incubated for a month. In the present study, 2014). Biodegradation depends upon the formation of biofilm,
from the plant P. flavida, none of the fungal endophytes except specifically a layer of deposition of the microorganism and their
L. theobromae, were able to produce laccase. The amount of laccase secreted polysaccharides etc on the polymer surface followed by
enzyme produced by these six endophytic fungi is shown in Fig. 2, the breakdown of the polymer to low molecular weight oligomers
where the production level of laccase in L. theobromae was high probably due to the enzymes secreted by the microbes after which
(10.70 ± 1.53 U/ml) and in Hypoxylon anthochroum quite low they are easily assimilated by them (Arkatkar et al., 2009). The
(1.18 ± 0.10 U/ml). Laccase enzyme present in number of different ability of the fungal strains to form a biofilm on polyethylene was

Table 1
Laccase production and biofilm formation by endophytic fungi on plastics.

Plants Endophytic fungal species Accession number Laccase test Adherence on plastic

Humboldtia brunonis Cunnighamella echinulata NFCCI 3251 þ þ


Pestalotiopsis sp. KF913516 þ þ
Hypoxylon anthochroum KF913518 þ þ
Paecilomyces lilacinus NFCCI 3250 þ þ
Curvularia clavata Jain NFCCI 2864 þ e
Curvularia pallescens Boedijn NFCCI 2865 þ e
Fusarium fusaroides NFCCI 2886 þ e
Fusarium oxysporum NFCCI 2885 e e
Lasodiplodia theobromae KF913502 e e
Guignardia sp. KF913507 e e
Aspergillus sp. NFCCI 3249 þ þ
Alternaria alternata KF913536 e e
Phanerochaete sp KF913532 þ e
Debaryomyces hansenii KF913533 e e
Meyerozyma guilliermondii KF913531 e e
Psychotria flavida Lasiodiplodia theobromae KF913500 þ þ
Bipolaris papendorfii KF913497 e e
Curvularia lunata KF913498 e e
Pestalotiopsis clavispora KF913515 e e
Guignardiamangiferae KF913508 e e
Meyerozyma guilliermondii KF913530 e e
Talaromyces flavus KF913527 e e
Cylindrocladium sp KF913525 e e
Phialemonium dimorphosporum KF913521 e e
Phanerochaete chrysosporium KF913520 e e
Meyerozyma caribbica KF913517 e e
Pestalotiopsis clavispora KF913515 e e
Phyllosticta capitalensis KF913508 e e

NFCCI accession code e identified by Agharkar Research Institute and deposited in National Fungal Culture Collection of India, Agharkar Research Institute, Pune, KF
accession code-for gene sequences submitted to NCBI, GenBank.
24 S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29

3.3. Determination of changes in viscosity and average molecular


weight

A discernible decrease in viscosity and molecular weight in


gamma radiation exposed LDPE and PP films were observed after
three months of incubation compared to control (Table 2).
Bonhomme et al. (2003) has reported that with the fungal activity,
polyethylene with a starting molecular weight in the range of 4000
to 28,000 was degraded to units with a lower molecular weight of
500 after three months of liquid cultivation, which indicated the
biodegradation of polyethylene. The mechanisms of degradation of
recalcitrant polymers involve utilization of the polymer and/or
additives by microorganisms as a source of carbon and energy (Gu
et al., 1996b). More specifically, important factors affecting the rate
of biodeterioration include material composition, (Bos et al., 1999;
Busscher et al., 1990; Gu et al., 2000b; Wiencek and Fletcher, 1995),
Fig. 2. Quantification of Laccase enzyme. molecular weights, atomic composition and the chemical bonds in
the structure, the physical and chemical characteristics of the sur-
faces (Becker et al., 1994; Caldwell et al., 1997; Callow and Fletcher,
attributed to the gradual decrease in hydrophobicity of its surface 1994), the indigenous microflora, and environmental conditions.
(Gilan et al., 2004). During degradation, extracellular enzymes from microorgan-
isms breakdown complex polymers yielding short chains or smaller
3.2. Percentage weight loss of LDPE and PP films molecules, e.g., oligomers, dimers, and monomers that are smaller
enough to pass through the semi-permeable membranes. These
The LDPE films inoculated with P. lilacinus, Aspergillus sp and short chain length molecules are then mineralized into end prod-
L. theobromae incubated for 90 days, showed weight loss during the ucts e.g. CO2, H2O, or CH4, which are utilized as carbon and energy
course of incubation period (Fig. 3a). Weight loss was not observed source (Gu, 2003a,b).
in LDPE films irradiated upto 200 kGy. However, percentage of
weight loss in LDPE films increased with increasing doses of radi- 3.4. Analysis of films by Fourier transform infrared spectroscopy
ation (Fig. 3b). In case of polypropylene film, only L. theobromae and plausible mechanism for biodegradation
inoculated film showed weight loss (Fig. 4a). There was a gradual
increase in weight loss from 40 kGy dose onwards (Fig. 4b). There The results of the FTIR analysis of the polymer film displayed a
was no weight loss in the control, inoculated with/without fungi. number of peaks reflecting the complex nature. Control LDPE and
The soil fungi, Aspergillus glaucus and Aspergillus fumigatus were PP films were slightly different than gamma irradiated films (Fig. 5a
reported to cause biodegradation of 7.26% and 4.26% of plastics and b), where carbonyl groups appeared from 200 kGy to 1000 kGy
(Kathiresan, 2003; Singh et al., 2012). However, there are no reports and 60 kGye100 kGy in gamma irradiated LDPE and PP film
on the effect of endophytes in degradation of LDPE and PP films. respectively. Irradiated (1000 kGy) control film (Fig. 6b) showed
Synthetic polymers such as polyolefins are not completely inert several peaks that correspond to addition of oxygen and photo-
towards micro-organisms, but have demonstrated certain, though xidation of film compared to unirradiated control film (Fig. 6a). The
limited long term biodegradability (Albertsson, 1978). The exposure gamma irradiated LDPE (1000 kGy) rendered wavenumber
of polyolefins to gamma radiation increases the hydrophilicity of 1712 cm1 related to carbonyl group formation which was absent in
these films due to photo oxidation enabling microorganisms to control. The area of peak at wavelength (3100e3500) cm1 was
colonize and initiate the process of degradation through production broad and bended in irradiated (1000 kGy) fungal treated samples
of enzymes particularly laccase. The laccase enzymes are known to (Fig. 6c), noticeable for the broad OeH stretching band of hydroxyl
degrade wood, plastic, paint, including jet fuel (Buddolla et al., groups and adsorbed water corresponding to decrease in hydro-
2014). phobicity and attributing to degradation.

a
actual weight loss

0.4
P. lilacinus
0.3
in mg

0.2 Aspergillus sp
0.1 L.theobromae
0
0 100 200 300 400 500 600 700 800 900 1000
Doses KGy

b 4
% Weight loss

2 P. lilacinus
0 Aspergillus sp
0 100 200 300 400 500 600 700 800 900 1000 L.theobromae
Doses KGy

Fig. 3. a: Actual Weight loss in gamma irradiated fungal inoculated LDPE films after 90 days of incubation, b: Percentage Weight loss in gamma irradiated fungal inoculated LDPE
films after 90 of days incubation.
S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29 25

a
0.3

actual weight loss


0.2

in mg
0.1 L.theobromae

0
0 20 40 60 80 100
Doses KGy

b 1.5
% weight loss

0.5 L.theobromae
0
0 20 40 60 80 100
Doses KGy

Fig. 4. aActual Weight loss in gamma irradiated fungal inoculated PP films after 90 days of incubation, b: Percentage Weight loss in gamma irradiated fungal inoculated PP films
after 90 days of incubation.

FTIR spectra revealed remarkable shift in the group frequencies dioxide, water and methane are produced during anaerobic
in terms of wavenumber (cm1) of LDPE in the presence and biodegradation (Gu et al., 2000a). Possible mechanism of breaking
absence of fungi over untreated LDPE as control. The irradiated down of LDPE and PP in the present study is illustrated below (Fig. 7
(1000 kGy) LDPE devoid of fungi compared to fungal treated irra- a, b). Gamma Irradiation breaks the polymer chains, creating free
diated (1000 kGy) film revealed a number of peak shifts that radicals, which may combine with oxygen in air, facilitating
indicate the changes in the macromolecular segments of the LDPE ongoing oxidative degradation of the polymer.
and induced bioactive hydrolysis in polymer film. Moreover, change The primary mechanism for the biodegradation of high molec-
in the fingerprint region of the IR spectrum between 1300 and ular weight polymer is the oxidation or hydrolysis by enzyme to
900 cm1 was observed in fungal treated irradiated (1000 kGy) create functional groups that improves its hydrophilicity. Conse-
film. Peaks of fungi treated films were similar to irradiated control quently, the main chains of polymer are degraded resulting in
but the stretching band of OH frequency was more intense which polymer of low molecular weight and feeble mechanical properties,
shows bioactive hydrolysis. In L. theobromae, peak at wavelength thus, making it more accessible for further microbial assimilation
2368 cm1, was sharper and larger compared to irradiated control. (Albertsson and Karlsson, 1990; Albertsson et al., 1987; Huang et al.,
Peak in sample at wavelength 864 cm1 was broad unlike spectrum 1990).
of the irradiated control. Similarly, small peak appeared in sample
at 1573 cm1, which was not present in control. Weiland et al. 3.5. Scanning electron microscopy
(1995) had observed similar changes at 1575 cm1 where part of
the carbonyl groups formed during thermal oxidation (COOH from SEM image of the untreated films revealed the intactness of the
chain ends and from the hydrolysis of ester groups) are trans- film compared to the films treated with fungi (Fig. 8). The PP film
formed into carboxylate absorbing at 1575 cm1 by fungi. In the exposed to 100 kGy did not show any cracks on the surface (Fig. 8A).
biodegradation of polyethylene, initial abiotic step involves the Inoculated fungi showed profuse colonization (Fig. 8B). The fungus
oxidation of polymer chain leading to the formation of carbonyl colonization induced the occurrence of fissures in the irradiated
groups. These groups eventually form carboxylic groups, which film inoculated with fungi (Fig. 8C). LDPE Control films had an
subsequently undergo b oxidation (Albertsson et al., 1987) and are appearance of smooth surface with no pits, cracks, or any particles
completely degraded via citric acid cycle resulting in the formation attached on the surface (Fig. 8D). The LDPE film exposed to
of CO2 and H2O. During exposure to gamma rays in the presence of 1000 kGy radiation and treated with fungus showed the formation
oxygen, polyolefins undergo photo oxidation and a number of of fungal colony (Fig. 8E) and formation of fungal spores (Fig. 8F).
compounds that contain carbonyl groups are formed (Hasan, 2012). LDPE film was transformed from smoothness to brittleness due to
Microorganisms such as bacteria and fungi are involved in the fungal colonization as compared to control. The well resolved worn
degradation of both natural and synthetic plastics. Carbon dioxide out pressure developed areas, penetration of fungal hyphae in both
and water are produced during aerobic biodegradation and carbon LDPE and PP film surface may be due to biofilm formation,

Table 2
Determination of viscosity and molecular weight.

Plastic sample Viscosity (ή) (pose) Average molecular weight in grams

LDPE Control 0.8349 27,114.40


LDPE gamma irradiated (1000 KGy) Control 0.8088 25,953.74
LDPE gamma irradiated (1000 KGy) þ Aspergillus sp 0.7963 25,398.02
LDPE gamma irradiated (1000 KGy) þ P. lilacinus 0.7884 25,055.32
LDPE gamma irradiated (1000 KGy) þ L. theobromae 0.7700 24,249.34
PP Control 0.6639 63,826.34
PP gamma irradiated Control 0.6280 59,020.00
PP gamma irradiated þ L. theobromae 0.6053 56,195.30
26 S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29

3.6. DSC

The changes in the thermal properties of the treated LDPE and


PP films were analyzed through determination of bulk structural
characteristics with reference to untreated film as control. The
melting temperature or heat of fusion can reveal even slight vari-
ations in the fine structure of irradiated LDPE (Badr et al., 2000).
DSC was used to examine the change in the melting transitions and
variation in the heat of fusion for control with/without fungi and g-
irradiated (highest dose of 1000 kGy for LDPE and 100 kGy for PP)
with/without fungal treated films. The peak temperature melting
(Tm), heat of fusion (DHf) and the onset temperature (To), recorded
from DSC thermograms is given in Table 3.
In the current study, peak temperature melting (Tm) of non
irradiated LDPE vaguely increased compared to irradiated LDPE
film of highest dose (1000 kGy). In case of non irradiated PP, peak
melting temperature distinctly increased than irradiated PP film
with/without fungi of highest dose (100 kGy). Conversely, heat of
fusion (DHf) and percentage of crystallinity decreased compared to
gamma irradiated samples. Increased crystallinity during irradia-
tion has been ascribed to chemicrystallization resulting from the
morphological rearrangement of polymer fragments after chain
scission events (Luongo, 1963; Carisson and Wiles, 1971). The in-
crease in the enthalpy could be attributed to the expected increase
in the degree of crosslinking occurring at higher irradiation doses
(Abou et al., 2000).
There was not much difference for Tm and To in gamma irradi-
ated LDPE films treated with/without fungi, while heat of fusion
and percentage of crystallinity increased in gamma irradiated
fungal treated samples compared to gamma irradiated control. As
chain scissions in the amorphous phase can encourage the crys-
tallization process, an increase in the crystalline content of a
semicrystalline polymer must be considered as degradation sign
Fig. 5. a: Gamma irradiated LDPE films. b: Gamma irradiated PP films. (Ana et al., 2000). Because in semicrystalline polymers degradation
starts in the amorphous phase and the interfacial regions, repre-
sents an increase in the number of the smallest crystals. This in-
indicating strong adhering capabilities and action of inhabitant dicates a high microbial activity on the amorphous phase and on
microbes on film. Some authors have reported the efficient biofilm the smaller crystals. The effect of the microorganisms in semi-
formation on polyethylene surface by Aspergillus niger which crystalline polymers is expected to occur in the amorphous phase
showed surface damage facilitating degradation of thermally rather than in the crystals because the latter are more resistant to
oxidized polyethylene (Garima et al., 2011). The structural changes enzymatic attack (Shogren et al., 1992).
in the form of pits and erosions observed through scanning electron Manzur et al. (1997) have proposed that the amorphous fraction
microscopy indicated surface damage of PE incubated with Fusa- has two components: 1) the one that surrounds the crystalline
rium sp. AF4. which is capable of adhering LDPE and cause surface particles, and 2) the one that defines the limits of crystalline blocks
damage (Shah, 2007). of the crystalline mosaic.

Fig. 6. FTIR of (a) control and (b) Gamma irradiated (1000 kGy) without fungal treated LDPE film; (c) Gamma irradiated (1000 kGy) fungal treated LDPE film.
S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29 27

Fig. 7. Plausible mechanism for degradation a) Gamma radiation b) Further enhancement for degradation by fungus.

Fig. 8. A) PP Gamma irradiated (100 kGy) control, B) Fungal growth forming Biofilm on gamma irradiated PP (100 kGy), C) Fissures formed on Gamma irradiated (100 kGy) PP after
incubation with fungus D) LDPE Gamma irradiated (1000 kGy) control E) Mycelia attached on Gamma irradiated (1000 kGy) LDPE forming biofilm and F) Gamma irradiated
(1000 kGy) LDPE with massive conidia of fungi.

Table 3
Thermal parameters of films.

Sample kGy DH J/g (ToC) Tm Xc

LDPE Control 0 30.73 (104.08) 114.09 10.97


LDPE Control þ Aspergillus sp. 29.21 (102.47) 115.6 10.43
LDPE Control þ P. lilacinus 28.57 (102.31) 115.43 10.20
LDPE Control þ L. theobromae 31.08 (99.45) 113.84 11.11
LDPE gamma irradiated Control 1000 39.23 (103.02) 110.88 14.01
LDPE gamma irradiated þ Aspergillus sp. 47.62 (99.04) 111.22 17.00
LDPE gamma irradiated þ P. lilacinus 40.63 (100.27) 110.49 14.51
LDPE gamma irradiated þ L. theobromae 67.47 (100.27) 110.70 24.09
PP Control 0 23.45 (150.53) 160.32 11.32
PP Control þ L. theobromae 23.19 (150.46) 161.58 11.20
PP gamma irradiated Control 100 31.34 (136.91) 148.68 15.14
PP gamma irradiated þ L. theobromae 37.50 (143.65) 153.35 18.11
28 S. Sheik et al. / International Biodeterioration & Biodegradation 105 (2015) 21e29

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