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Biodegradation of low-density polyethylene (LDPE) by isolated fungi

in solid waste medium

Sahebnazar Zahra, Shojaosadati Seyed Abbas
, Mohammad-Taheri Mahsa, Nosrati Mohsen
Biotechnology Group, Chemical Engineering Department, Faculty of Engineering, Tarbiat Modares University, Tehran, P.O. Box 14115-143, Iran
a r t i c l e i n f o
Article history:
Accepted 21 September 2009
Available online 17 November 2009
a b s t r a c t
In this study, biodegradation of low-density polyethylene (LDPE) by isolated landll-source fungi was
evaluated in a controlled solid waste medium. The fungi, including Aspergillus fumigatus, Aspergillus ter-
reus and Fusarium solani, were isolated from samples taken from an aerobic aged municipal landll in
Tehran. These fungi could degrade LDPE via the formation of a biolm in a submerged medium. In the
sterilized solid waste medium, LPDE lms were buried for 100 days in a 1-L ask containing 400 g sterile
solid waste raw materials at 28 C. Each fungus was added to a separate ask. The moisture content and
pH of the media were maintained at the optimal levels for each fungus. Photo-oxidation (25 days under
UV-irradiation) was used as a pretreatment of the LDPE samples. The progress of the process was mon-
itored by measurement of total organic carbon (TOC), pH, temperature and moisture. The results obtained
from monitoring the process using isolated fungi under sterile conditions indicate that these fungi are
able to grow in solid waste medium. The results of FT-IR and SEM analyses show that A. terreus and A.
fumigatus, despite the availability of other organic carbon of materials, could utilize LDPE as carbon
source. While there has been much research in the eld of LDPE biodegradation under solid conditions,
this is the rst report of degradation of LDPE by A. fumigatus.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Currently, more than 150 million tons of petroleum-based syn-
thetic polymers are produced each year (Shimao, 2001). Some
polymers, such as low-density polyethylene (LDPE), are used in
many applications due to their useful physical and chemical prop-
erties. LDPE is durable, light-weight, easily processed and charac-
teristically inert, and these properties make it appropriate for
many industrial uses (Mark, 1999). However, LDPE is hardly de-
graded after disposal, which pollutes the environment and disturbs
the ecosystem (Bastioli, 2005). The increasing levels of LDPE waste,
decreasing landll capacity and very slow rate of LDPE degradation
in the environment have caused the research tendency to decrease
the amount of waste LDPE buried in the nature. Current methods
for addressing the problem include recycling, chemical recovery,
pyrolysis and hydrolysis, incineration (with energy recovery) and
biodegradation (Hamid, 2000).
Recently, efforts have focused on the biodegradation of LDPE
wastes due to the disadvantages of other methods such as cost
and pollution. Microorganisms play a signicant role in biological
decomposition of materials (Shah et al., 2008), however, a major
obstacle to biodegradation is the resistance of LDPE to biological
attack because of its hydrophobicity, high molecular weight and
its lack of functional groups recognized by microbial enzymatic
systems (Hamid, 2000). Biodegradation of LDPE could be acceler-
ated either by polymer pretreatments such as photo-oxidation,
thermo-oxidation and chemical-oxidation to create additional
functional groups (C@O bonds). The C@O bonds ready the polymer
surface for microorganisms attack. In other word, basically the rst
step in polymer biodegradation is oxidation (Albertson et al., 1998;
Brown et al., 1974; Chiellini et al., 2003; Cornell et al., 1984; Pan-
dey and Singh, 2001), or by inoculating soil or compost with micro-
organisms (bacteria and fungi) that are able to biodegrade LDPE
(Gilan et al., 2004; Shah, 2007). In most studies, fungi were consid-
ered for the degradation of LDPE due to their ability to form hydro-
phobic proteins that can attach to the polymer surface
(Seneviratne et al., 2006; Kershaw and Talbot, 1998), their genera-
tion of degrading enzymes that are well-matched to the insoluble
LDPE (Shah et al., 2008), the faster growth of fungal biomass in soil
compared to bacteria (Kim and Rhee, 2003), and the growth exten-
sion and penetration into other locations through the distribution
of hyphae. Also, fungi survive environments with low nutrient
availability, low pH and low moisture well.
However, there are still many understudied areas of research in
the degradation of LDPE by fungi. Characterization of LDPE degra-
dation by solid waste-source fungi is among these attractive topics
because of their compatibility with a waste-rich environment
(such as landll and composting) that contains a variety of dis-
carded polymers.
0956-053X/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +98 21 82883341; fax: +98 21 82883381.
E-mail addresses:, (S.S. Abbas).
Waste Management 30 (2010) 396401
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The aim of this study was to evaluate the ability of fungi that
could be found naturally in landll to degrade LDPE. Aspergillus
fumigatus, Aspergillus terreus and Fusarium solani were isolated
from aerobic area in the typical aged landll because of their dem-
onstrated ability to degrade LDPE. The performances of these three
fungi in solid waste medium were investigated in this work.
2. Materials and methods
2.1. Materials
Commercial granules of LDPE were provided from one of the
stocks of Irans National Petrochemical Commercial Company
(INPCC). LDPE lms with thickness of 15 lm were made from this
material using a blowing lm extruder. LDPE lms were irradiated
for 25 days with UV-irradiation in a laminar ow cabinet and then
cut into pieces of about 1 1 cm.
2.2. Isolation of fungi
A random sample of landll soil (1 g) and fragmented LDPE that
had been buried in soil for 10 years were suspended into 100 ml of
mediumA (0.3 g NH
, 0.5 g K
, 0.1 g NaCl, 0.02 g MgSO
0.01 g yeast extract, 1 lg biotin, 20 lg riboavin, 1 lg folic acid,
20 lg nicotinic acid in 100 mL). Fragmented and irradiated LDPE
(1 g) was also added to 100 ml of medium A as a carbon source.
The culture was incubated with shaking for 1 month at 180 rpm
and 28 C. Then, 1 ml of the suspension was put into 4 ml of fresh
medium A and incubated for 1 week. A 5 ll sample of this culture
was spread onto medium B (0.3 g NH
, 0.5 g K
, 0.1 g
NaCl, 0.02 g MgSO
O, 0.5 g irradiated polyethylene powder,
2 g agar, 1 lg biotin, 1 lg folic acid, 20 lg nicotinic acid in
100 mL), and the plates were incubated at 28 C for another
3 weeks. To conrm LDPE utilization by the isolated fungi, various
experiments were carried out culturing fungi in medium B plus
LDPE, medium B plus LDPE without vitamins, medium B without
LDPE, medium B without LDPE and vitamins at 28 C for 25 days.
2.3. Identication of isolated fungi
Fungi grew on medium B plus LDPE and medium B plus LDPE
without vitamins. Colonies were then identied morphologically.
In addition, the genomic DNA of one of the species was extracted
and ITS region PCR-amplied and sequenced (Naderi, 2008). From
the bioinformatic analysis of the sequencing data, the isolated fun-
gal strains were identied as A. fumigatus, A. terreus and F. solani.
2.4. Preparation of spores from the isolated fungi
Isolated fungi were cultured on potato dextrose agar (PDA)
plates at 28 C. After completion of sporulation, spores of fungi
on the plate surface were washed with physiological serum includ-
ing peptone (0.1% v/v) and Tween 80 (0.05% v/v), then transferred
to glycerol solution (20% v/v) at a concentration of 5 10
spores/mL before storage at 70 C.
2.5. Evaluation of LDPE degradation in liquid culture
Spores of each fungus were incubated on PDA medium at 28 C
for 32 h, and then hyphae were harvested to inoculate PDB med-
ium. Cultivation was done at 30 C on a rotary shaker at 180 rpm
for 2 days. A 10-ml sample of the suspension described above
was also used to inoculate medium C (0.3 g NH
, 0.5 g
, 0.1 g NaCl, 0.02 g MgSO
O, 0.1 g yeast extract in
100 mL). Cultures were incubated at 28 C on a rotary shaker at
180 rpm for 2 weeks. A 10-ml sample of this suspension was again
added to 100 ml of medium D (0.3 g NH
, 0.5 g K
, 0.1 g
NaCl, 0.02 g MgSO
O, 0.5 g fragmented and irradiated LDPE as
the sole carbon source in 100 mL) in a 250 ml shaking ask. Shak-
ing was carried out at 180 rpm and 28 C. In order to evaluate LDPE
degradation, after 3 months, a small amount of ethanol was added
to asks, and the asks were placed in an oven at 80 C for 24 h. To
separate the remaining LDPE pieces, the mixture was ltered and
the LDPE on the lter paper was added to an Erlenmeyer ask con-
taining water and Tween 80 and shaken for 12 h. The mixture was
ltered, and the LDPE on the lter paper was washed with ethanol
and then dried at 25 C. Scanning electron microscopy was used to
check for changes in the surface of LDPE samples.
2.6. Detection of LDPE degradation in solid waste medium
2.6.1. Fermentation process
The composition of raw materials used for the process was as
follows (dry weight): 36% vegetable waste, 20% rice, 2% paper
waste, 29% mature compost and 13% sawdust as a bulking agent.
The C/N ratio of the initial material was approximately 29:1. Fungi
were inoculated separately in the solid waste medium. The initial
moisture content and pH were adjusted to optimal values for each
of the fungi by addition of distilled water and HCl. The optimized
moisture content and pH for A. fumigatus, A. terreus and F. solani
were as follows: 50% and 7, 40% and 7, 70% and 5, respectively
(Sahebnazar, 2008). Approximately 400 g of raw materials were
mixed and autoclaved at 121 C and 14 psi for 15 min, loaded into
a 1000-ml Erlenmeyer ask, and then LDPE pieces (1 1 cm) were
mixed with the materials. Three Erlenmeyer asks were prepared
and each of them was inoculated with 5 10
spores/g (dry mate-
rial). Erlenmeyer asks were covered with glass wool to reduce
heat loss to the surrounding environment (Fig. 1). Aeration was
performed by slowly mixing the contents of the ask daily. Mois-
ture content was controlled by adding an appropriate amount of
sawdust. Sampling was performed weekly and samples were com-
pletely homogenized for analyses. After 100 days, the process was
terminated, and LDPE pieces were washed in ethanol and distilled
water and then dried. Samples of LDPE were analyzed for
Fig. 1. Schematic of the compost system.
S. Zahra et al. / Waste Management 30 (2010) 396401 397
2.6.2. Measuring the chemical and physicochemical parameters
The moisture content of materials was determined by drying
the samples at 105 C for 24 h (Tiquia and Tam, 1998). The TOC
was measured using potassium dichromate and sulfuric acid
(Baruah and Barthakur, 1997). The pH value of an aqueous extract
was measured using a digital pH meter. The extract was obtained
by mechanically shaking the samples for 2 min and collecting the
supernatant after centrifugation at 5300 rpm for 15 min at a
solidwater ratio of 1:10 (wet weight/volume) for 1 h (Zhu, 2007).
2.6.3. Analysis of LDPE degradation
Scanning electron microscopy (SEM) analysis was used to
examine changes in the surface of LDPE samples during their deg-
radation. LDPE lms were coated with gold by BAL-TEC-SCDOOS,
and the surface was investigated using a Philips-X LP30 scanning
electron microscope.
Structural changes in the LDPE surface was investigated using
the EQUINOX 55 FT-IR spectrometer. A spectrum was taken from
400 to 4000 wavenumbers cm
for each LDPE lm.
Molecular weight changes of polyethylene were measured by
high-temperature gel-permeation chromatography (HT-GPC) with
a thermostat operating at 140 C and tetrachlorobenzene (TCB) as
the liquid phase.
3. Results and discussion
3.1. Monitoring of the process
The temperature increased due to the accumulation of fungal
metabolic heat and biodegradation of organic compounds, and
reached 36 C, 35 C and 31.5 C for A. terreus, F. solani and A. fumig-
atus, respectively. Afterwards, the temperature of the asks fell to
the environmental temperature. The decrease of temperature re-
sulted from a depletion of organic matter (Grima et al., 2002)
(Fig. 2).
The pH value is a key factor for the survival and activity of fungi.
However, environmental pH varies according to the metabolites
present. Generally, the pHshould be between 7 and 9. In this study,
the pH increased early in the process and leveled off near 9 for all
fungi. This is due to ammonication of nitrogen components
(Fig. 3).
The moisture content varied over time as shown in Fig. 4. It was
observed that in all experiments, moisture increased along with
the process that resulted from the production of water during
the fungi growth in aerobic condition. In other hand, generation
of the carbon dioxide (in an aerobic metabolism) results in de-
crease in the amount of the total organic carbon (TOC).
Previous investigators mentioned that moisture content is an
important factor affecting microbial activity in the process and
ultimately the rate of degradation (Davis, 2005; Liang et al.,
2003). On one hand, a lack of moisture leads to a decrease of the
fungal metabolic and the developmental rates. Conversely, excess
moisture causes the packing down of substrate and anaerobiosis
(Grima et al., 2002). The best fungi growth is happened in moisture
content range of 5070%. That is why the moisture content must be
controlled during a successful fungi growth in process. In this
study, sawdust was added when the moisture content surplused
TOC decreased signicantly during the process. The initial burst
might be causedbydecompositionof easilydegradedTOC. However,
difcult-to-degrade TOC, suchas lignin, wouldbe decomposedgrad-
ually in curing phase (Solano et al., 2001). In this study, the largest
decrease of the TOC occurred from the beginning to the 28th
day as TOC concentration decreased from its initial amount of
Fig. 2. Temperature prole for the composting process with each fungus.
Fig. 3. pH prole for the composting process with each fungus.
Fig. 4. Moisture prole for the composting process with each fungus.
Fig. 5. TOC prole for the composting process with each fungus.
398 S. Zahra et al. / Waste Management 30 (2010) 396401
3926.6%, 25% and 24.6%by the individual growth of different fungi
A. terreus, A. fumigatus and F. solani, respectively. However, after the
curing phase more TOCdecrease was happenedtoo. At the endof the
process, the TOC concentrations were 22.5%, 18.7% and 22.6% for A.
terreus, A. fumigatus and F. solani, respectively (Fig. 5).
3.2. Scanning electron microscopy analysis
Scanning electron microscopy (SEM) was used to monitor
changes inthe surface of LDPEsamples during the process. SEMpho-
tomicrographs (Fig. 6) of samples before process had a smooth sur-
face with no defects (Fig. 6a). However, after process with A.
terreus and A. fumigatus, the samples possessed pitted and eroded
surfaces (Fig. 6c and e). The pits were observed on the surface, sug-
gesting that the fungi penetrate into the LDPE matrix during degra-
dation. Figs. 6d and f indicate penetration of fungal hyphae into the
LDPE matrix. The surface of the polymer after biological attack was
physically weak and readily disintegrated under mild pressure. For-
mation of biolms of A. terreus and A. fumigatus was evident on the
surface and was considered to be a result of the surface moistness.
Water couldspreadsmoothlyonthesurfaceof UVexposedLDPElm
because the surface had been modied to be hydrophilic. Therefore,
microorganisms could also expand their colonies over the surface
and form a fungal biolm. LDPE degradation by A. terreus is consis-
tent with results obtained previously (Shah et al., 2008). Fig. 6b
shows that F. solani did not degrade the surface of LDPE signicantly.
This nding is not consistent with previous results (Otake et al.,
1995; Shah et al., 2008). This is may be due to the conditions used
for the growth of the fungus in this experiment, and further investi-
gation and optimization may be necessary.
Fig. 6. SEM micrographs of LDPE lms before and after incubation with each fungus for 100 days. (a) Blank (no UV-irradiation, no incubation); (b) after UV-irradiation,
incubation with Fusarium solani; (c) after UV-irradiation, incubation with Aspergillus terreus; (d) after UV-irradiation, incubation with Aspergillus terreus (penetration of
hyphae into the LDPE matrix); (e) after UV-irradiation, incubation with Aspergillus fumigatus; (f) after UV-irradiation, incubation with Aspergillus fumigatus (penetration of
hyphae into the LDPE matrix).
S. Zahra et al. / Waste Management 30 (2010) 396401 399
Fig. 7. FT-IR spectra of LDPE lms before and after UV-irradiation and incubation with each fungus for 100 days from 400 to 4000 wavenumbers cm
Fig. 8. FT-IR spectra of UV-irradiated LDPE lms before and after incubation with each fungus for 100 days. (a) Blank (no UV-irradiation, no incubation); (b) after UV-
irradiation, no incubation; (c) after UV-irradiation, incubation with Aspergillus terreus; (d) after UV-irradiation, incubation with Aspergillus fumigatus; (e) after UV-irradiation,
incubation with Fusarium solani.
Fig. 9. GPC results of UV-irradiated LDPE lms. (a) Before incubation with mixture of fungi; (b) after incubation with mixture of fungi.
400 S. Zahra et al. / Waste Management 30 (2010) 396401
3.3. FT-IR analysis
The degradation of LDPE and the structural changes induced by
degradation were analyzed by FT-IR analysis using characteristic
bonds ranging from 1700 to 1760 cm
. Fig. 7 shows FT-IR spectra
of LDPE lms before and after UV-irradiation and incubation with
each fungus for 100 days from 400 to 4000 wavenumbers cm
Changes of the C@O bond are magnied in Fig. 8.
One main characteristic bond was observed for the irradiated
LDPE that was not observed in the original samples. This bond
was observed in the vicinity of 1720 cm
and assigned to the
C@O group that was formed by UV-irradiation. The intensity of
the C@O bond at 17001760 cm
was signicantly decreased dur-
ing the process with fungi due to utilization of carbonyl group by
fungi for LDPE degradation. These nding support Hasans results
(Hasan et al., 2007). The surface area under curves was measured
using Upos software, and the results were as follows: 0.00608,
0.10409, 0.08914, 0.07233 and 0.09527 for samples a, b, c, d and
e, respectively (Fig. 8). According to these results, A. fumigatus
was the most effective fungus for LDPE degradation.
3.4. HT-GPC analysis
Molecular weight changes of polyethylene were shown in Fig. 9.
Molecular weight the irradiated LDPE was decreased from its ini-
tial amount of 6673954686 KD during incubation with a mixture
of fungi. The results of present study are in agreement with the
most reports on polyethylene biodegradation (Mueller, 2006;
Albertsson and Karlsson, 1990).
4. Conclusion
The results obtained from observing the process using isolated
fungi under sterile conditions indicate that these fungi are able
to grow in solid waste medium. The results from FT-IR and SEM
analyses show that A. terreus and A. fumigatus, despite the avail-
ability of organic carbon from other materials, could utilize LDPE
as a carbon source. These fungi are able to degraded LDPE without
any additives. UV-irradiation facilitated the biodegradation of
LDPE. Regarding other work in the eld of LDPE biodegradation
in solid waste environments such as Shah, Otake, Albertsson, Ohta-
ki, Jitendra and Hasans studies, this is the rst report and investi-
gation of LDPE degradation by A. fumigatus (Albertsson, 1980;
Hasan et al., 2007; Ohtaki et al., 1998; Otake et al., 1995; Shah,
2007). Results of this research show that isolated fungi have great
potential for LDPE biodegradation in the composting process. There
is great potential for the development of a process for degrading
LDPE in a composting environment using fungi in the near future.
The authors would like to express their sincere thanks to the Hi-
tech Industries Center of IRAN and Dr. Vosoughi and Dr.
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