Polymer Degradation and Stability 72 (2001) 323±327
www.elsevier.nl/locate/polydegstab
Degradation of polyethylene by a fungus,
Penicillium simplicissimum YK
Keiko Yamada-Onodera a,*, Hiroshi Mukumoto a, Yuhji Katsuyaya b,
Atsushi Saiganji b, Yoshiki Tani a
a
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
b
Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 11 October 2000; accepted 6 November 2000
Abstract
We isolated a strain of Penicillium simplicissimum, YK, for use in the biodegradation of polyethylene, characterizing the fungus
and examining how to treat the polyethylene before cultivation to make degradation more complete. Degradation was monitored
by high-temperature gel-permeation chromatography of the molecular weight distribution of polyethylene before and after the
fungus was cultivated with it. Polyethylene with starting molecular weights of 4000 to 28,000 had lower molecular weights after 3
months of liquid cultivation with hyphae of the fungus. UV irradiation of polyethylene or its incubation with nitric acid at 80 C for
6 days before cultivation caused functional groups to be inserted into the polyethylene. The strain grew better on a solid medium
with 0.5% polyethylene when it was irradiated for 500 h than when it was not irradiated. Polyethylene with a molecular weight of
100,000 or higher after nitric acid treatment had lower molecular weight after 3 months of liquid cultivation with hyphae of the
fungus. The eciency of polyethylene degradation depended on the growth phase in pure cultivation of the fungus. Functional
groups inserted into polyethylene aided biodegradation. Bioremediation of polyethylene may become possible. # 2001 Elsevier
Science Ltd. All rights reserved.
Keywords: Polyethylene degradation; Bioremediation; Penicillium simplicissimum
1. Introduction
Many polymers have been synthesized chemically in
this century because of the convenience and economy of
the process and because of the stability of the products.
As a result, a large amount of waste polymers has been
produced. Now, most waste polymers are burned for
disposal, but they produce toxic gases. Some polymers
are recycled, but the cost is high. Biodegradable plastics
that contain natural compounds such as polyhydroxy
butyric acid [1] have been developed. New forms of
plastic must retain all of the physical properties needed
by the consumer and must ful®ll safety standards both
when it is being used and after it has been discarded.
Until then, polymers that are not biodegradable will
continue to be used. A new way to degrade non-bio
degradable polymers is needed.
* Corresponding author. Tel.: +81-743-72-5422; fax: +81-743-725429.
E-mail address: [email protected] nara.ac.jp (K. Yamada-Onodera).
Synthetic polymers are made as inert materials with
resistance to micro-organisms. However, polyethylene
was realized to be biodegradable when it was reported
that fungal growth can occur on n-alkane [2], of which
polyethylene is an analogue. Potts [3] and Tsuchii et al.
[4] found that micro-organisms utilize polyethylene with
a molecular weight below 500 and 800, respectively.
Hueck pointed out that polyethylene needs to undergo
some non-biotic degradation before microbial attack
because of its hydrophobicity and its large molecular
dimensions [5]. Albertsson et al. concluded that UV light
or oxidizing agents such as a UV sensitizer are needed at
the beginning of biodegradation of inert materials such
as polyethylene [6] and that biodegradation without
them takes more than 10 years [7]. Polyethylene containing starch as a carbon source to help micro-organisms
grow also was used in experiments about biodegradation
[8,9]. Polyethylene without helpful additives should be
investigated, because it is causing problems now.
Degradation of polyethylene has been monitored in
terms of the growth of micro-organisms [3,5,10],
0141-3910/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0141-3910(01)00027-1
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K. Yamada-Onodera et al. / Polymer Degradation and Stability 72 (2001) 323±327
biochemical oxygen demand of microbes growing on it
[4], 14CO2 generation from polyethylene [6,7,11], weight
loss, tensile strength, and changes during degradation in
percentage elongation [8,9,12], and changes in average
molecular weight [8,9,11]. How micro-organisms utilize
polyethylene of high molecular weights is important.
Many experiments on bioremediation have been done
with soil and sludge [3,5±8,10±12]. Use of a single strain
rather than a mixture makes it easier to manipulate the
culture conditions and to use gene technology.
We found a micro-organism that degraded polyethylene without needing compounds to be added for
easier degradation, and found a way to treat the polyethylene before cultivation for better microbial growth
or to make degradation more complete. The degradation is described here, with the results of high-temperature gel-permeation chromatography (HT-GPC) and
Fourier transform infrared (FT-IR) spectra.
2. Materials and methods
2.1. Chemicals
Polyethylene irradiated for 500 h with UV light or not
irradiated was supplied by Asahi Chemical Industry Co.
as high-density polyethylene. The numbers, n, of functional groups were calculated by the following formulae:
n=0.24A1713/[(density; g/cm3)(thickness of the sample;
mm)] for-COOH groups, n=0.55A1721/(densitythickness) for-CˆO groups, n=0.76A1733/(densitythickness)
for±CHO groups, and n=0.45A1742/(densitythickness)
for±COOC (ester) groups, where A is the absorbance,
measured with a UV tester (SUV-F1, Iwasaki Electronics, Tokyo, Japan). The polyethylene was dissolved in
xylene and recrystallized before being used. Recrystallized polyethylene was treated for 6 days with nitric
acid at 80 C before being used as the sole carbon source
in liquid culture. Other chemicals were obtained commercially and used without further puri®cation.
2.2. Isolation of the micro-organism
Cultivation in 5 ml of a medium in a 16.5165-mm or
21200-mm test tube was at 28 C with reciprocal
shaking at 240 rpm. Shaking was at 120 rpm for 100 ml
of medium in a 500-ml shaking ¯ask. Samples of soil
and leaves were suspended in 100 ml of medium A (0.1 g
of recrystallized polyethylene, 0.3 g of NH4NO3, 0.5 g
of K2HPO4, 0.1 g of NaCl, 0.02 g of MgSO4.7H2O, 0.01
g of yeast extract, 10 mg of thiamin.HCl, 20 mg of ribo¯avin, 20 mg of nicotinic acid, 20 mg of Ca-pantothenate,
20 mg of pyridoxine.HCl, 1 mg of biotin, 10 mg of paminobenzoic acid, 1 mg of folic acid, and distilled
water; pH 6). The cultures were incubated with shaking
for 1 month. Then 1 ml of the suspension was put into 4
ml of fresh medium. After 1 week of shaking, 5 ml of the
culture was spread on a 2% agar plate of medium A and
the plate was incubated for several days. The colonies
were preserved at 4 C in 2% agar slants of medium B
(5% malt extract, 0.3% yeast extract, and distilled
water; pH 5±6).
2.3. Growth assays
Medium C contained (per 100 ml) 0.5 g of polyethylene irradiated or not irradiated (or none; see
below), 0.3 g of NH4NO3, 0.5 g of K2HPO4, 0.1 g of
NaCl, 0.02 g of MgSO4.7H2O, 0.1 g of Triton X-100 (or
none), and distilled water. Spores of a strain from medium B were used for inoculation. For con®rmation of
polyethylene utilization for growth, medium C without
polyethylene, medium C without polyethylene or Triton
X-100, and medium C with polyethylene and Triton X100 were prepared. For another check of polyethylene
utilization, spores were spread on three kinds of plates:
medium C without polyethylene, medium C with 0.1%
polyethylene, and medium C with 0.5% polyethylene.
For identi®cation of the e€ect of the irradiation of
polyethylene on growth, medium C containing polyethylene irradiated for 500 h or not irradiated was used.
Cultivation was at 28 C for 1 week.
2.4. Detection of polyethylene degradation in liquid
culture
Spores of a strain from medium B were incubated in
100 ml of 2% agar medium D (2 g of malt extract, 0.1 g
of peptone, 2 g of glucose, and distilled water) in a 500ml Erlenmeyer ¯ask. After 1 week of incubation of the
¯ask at 30 C, 50 ml of 0.05 M sodium phosphate bu€er
(pH 7) containing 0.3% Tween 80 was added. The mixture was stirred vigorously. Next, 10 ml of this spore
suspension was used to inoculate 100 ml of medium D
in a 500-ml Erlenmeyer ¯ask, which was shaken for 1
week. Hyphae were harvested, homogenized (Labo
Milser, Osaka Chemical Co., Osaka, Japan), and used
to inoculate 100 ml of medium C that contained 0.5 g of
polyethylene treated with nitric acid or not. Ten milliliters of the spore suspension described above also was
used to inoculate 100 ml of medium C that contained
0.5 g of untreated polyethylene. Cultivation was at 30 C
on a rotary shaker at 150 rpm. One or three months
later, the culture medium was ®ltered and suspended in
200 ml of distilled water. For removal of cell constituents, the suspension was sonicated (model 250
Soni®er, Branson, Danbury, CT) at 4 A for 15 min at
room temperature and then about 8 g of NaOH was
added. The mixture was boiled at 95 C for 10 min,
cooled, and ®ltered. The polyethylene on the ®lter paper
was washed with distilled water several times and dried
in a pyrostat at 70 C for 20 h. The distribution of
K. Yamada-Onodera et al. / Polymer Degradation and Stability 72 (2001) 323±327
molecular weights of the polyethylene was examined by
HT-GPC. FT-IR analysis was used for detection of
functional groups in the polyethylene.
HT-GPC was done with a Model 150-CV apparatus
(Waters, Tokyo, Japan) with two m-Styragel HT linear
columns (Waters), the elution speed of 1 ml/min, and odichlorobenzene as the solvent. Temperatures were
135 C for the injector, column, and detector and 50 C
for the pump.
A Herschel FT-IR-500 apparatus (Jasco, Tokyo)
controlled by Jasco software (FT for Windows) was
used for detection of changes in functional groups in
polyethylene. Powdery polyethylene was mixed with
KBr and made into a tablet, which was ®xed to the FTIR sample plate. A spectrum was taken at 400 to 4000
wavenumbers cm 1 for each sample.
3. Results and discussion
A strain that grew on polyethylene was isolated and
identi®ed as P. simplicissimum by the National Collections of Industrial and Marine Bacteria (Aberdeen,
Scotland). We named this strain P. simplicissimum YK.
Imai reported that the growth of wax-inhabiting
organisms on paran was favorably a€ected by the
addition of surface-active substances to the culture
medium, and that a fungus could grow with a surfaceactive substance as the sole carbon source [2]. P. simplicissimum YK likewise grew better in the medium containing a surface-active substance, Triton X-100, than
that without the addition (data not shown), but the fungus did not utilize Triton X-100 as the sole carbon source
(Fig. 1). P. simplicissimum YK grew better on plates with
0.5% polyethylene than on plates with 0.1% polyethylene, and the plate without polyethylene showed little
if any growth (Fig. 2). These results showed that P. simplicissimum YK could utilize polyethylene for growth.
Albertsson et al. [6] concluded that carbonyl groups
are produced by UV light or oxidizing agents and that
these groups are the main factors at the beginning of the
degradation, being attacked by micro-organisms that
325
degrade the shorter segments of polyethylene chains.
Ohtake et al. [8] also observed biodegradation of polyethylene buried in soil for 32±37 years, which was promoted by UV irradiation. Polyethylene without
irradiation or the nitric acid treatment described in
Materials and Methods had no functional groups.
Recrystallization did not cause the addition of functional groups. Polyethylene irradiated for 500 h had, per
1000 carbons, 0.018±COOH group, 0.025±CˆO group,
0.025±CHO group, and 0.011±COOC±(ester) group. P.
simplicissimum YK grew better on the agar plate containing irradiated polyethylene than that with polyethylene not irradiated (Fig. 3). Our results supported
the conclusions of Albertsson and Ohtake et al.
Cornell et al. [10] concluded that photo-oxidative
degradation of polymers does not always facilitate progressive attack by micro-organisms, because the oligomer fractions produced during photo-oxidation may
support microbial growth, but polymers with a high
molecular weight resulted in little or no growth. They
suggested that these observations could explain some of
the contradictions in the literature about microbial
degradation of polymers. Albertsson and Karlsson [7]
concluded that the biodegradation of inert material
such as polyethylene takes more than 10 years and that
that of degradable material containing a UV sensitizer
takes 2 years or less. We observed little if any reduction
of untreated polyethylene during 6 months of cultivation with spores of the fungus (data not shown), but
polyethylene with a molecular weight of 1000 to 10,000
cultivated for 1 month (Fig. 4) or 4000 to 28,000
cultivated for 3 months (Fig. 5) with hyphae of the
Fig. 2. Growth of P. simplicissimum YK on plates with di€erent concentrations of polyethylene as the sole carbon source.
Fig. 1. Growth of P. simplicissimum YK on plates with or without
polyethylene.
Fig. 3. Growth of P. simplicissimum YK on plates with polyethylene
irradiated or not as the sole carbon source.
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K. Yamada-Onodera et al. / Polymer Degradation and Stability 72 (2001) 323±327
Fig. 4. Results of HT-GPC of untreated polyethylene in 1-month
liquid culture with hyphae of P. simplicissimum YK. (a) Before incubation; (b) after 1 month of incubation.
Fig. 6. HT-GPC of polyethylene treated with nitric acid, in 3-month
liquid culture of hyphae of P. simplicissimum YK. (a) Before incubation; (b) after 3 months of incubation.
Fig. 5. Results of HT-GPC of untreated polyethylene in 3-month
liquid culture with hyphae of P. simplicissimum YK. (a) Before incubation; (b) after 3 months of incubation.
Fig. 7. FT-IR analysis of polyethylene. (a) Before nitric acid treatment; (b) after treatment and before incubation; (c) after treatment
and incubation for 3 months in a liquid culture of hyphae of P. simplicissimum YK.
fungus was reduced. As the sole carbon source, polyethylene with a molecular weight higher than 100,000
treated with hot nitric acid was degraded to lower
molecular weights during cultivation with hyphae of the
fungus (Fig. 6). At the same time, in FT-IR analysis of
polyethylene, absorbance at 1620±1640 cm 1 and 840±
880 cm 1 (corresponding to±CˆC±), which appeared
after the nitric acid treatment, decreased during cultivation with this strain (Fig. 7). The results of HT-GPC
and FT-IR analysis showed that some of the double
carbon bonds of polyethylene might be cut by P. simplicissimum YK. Albertsson et al. [6] suggested that
after carbonyl groups are formed in polyethylene, the
polyethylene chain is degraded to shorter pieces through
b-oxidation. The by-products in our experiments are
being investigated. Pometto et al. [9] reported that an
extracellular enzyme(s) from a Streptomyces sp. degrade
starch-polyethylene-prooxidant plastic in 3 weeks, as
seen by changes in the FT-IR spectra. Extracellular
enzymes of hyphae might be helpful for the rapid
degradation of polyethylene.
4. Conclusions
P. simplicissimum YK utilizes intact polyethylene as
the carbon source, but did not utilize Triton X-100. P.
simplicissimum YK grew better on agar plates containing irradiated polyethylene (which had carbonyl groups)
than intact polyethylene (which had no carbonyl
groups). Hyphae of the fungus were more e€ective for
degradation of intact polyethylene than the spores.
When polyethylene treated with hot nitric acid was the
sole carbon source, polyethylene with a molecular weight
higher than 100,000 was degraded to lower molecular
weights with hyphae of the fungus. A decrease in the
K. Yamada-Onodera et al. / Polymer Degradation and Stability 72 (2001) 323±327
absorbance corresponding to ±CˆC± suggested that
some of the double carbon bonds of polyethylene were
cut by P. simplicissimum YK. The time needed for polyethylene degradation depended on the growth phase in
pure cultivation of the fungus. Functional groups inserted into polyethylene aided biodegradation.
Acknowledgements
We thank Mr. S. Tanji, Asahi Chemical Industry Co.,
for the gift of polyethylene and for helpful advice.
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