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Soil Science and Plant Nutrition
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Effects of soil temperature and anaerobiosis on
degradation of biodegradable plastics in soil and their
degrading microorganisms
Hiroyo Nishide
a

b
, Koki Toyota
a
& Makoto Kimura
a
a
Computer Room, National Institute for Basic Biology, Okazaki, 444-8585, Japan
b
Nagoya University, Laboratory of Soil Biology and Chemistry, Graduate School of
Bioagricultural Sciences, Nagoya, 464-8601, Japan
Published online: 04 Jan 2012.
To cite this article: Hiroyo Nishide , Koki Toyota & Makoto Kimura (1999): Effects of soil temperature and anaerobiosis on
degradation of biodegradable plastics in soil and their degrading microorganisms, Soil Science and Plant Nutrition, 45:4,
963-972
To link to this article: http://dx.doi.org/10.1080/00380768.1999.10414346
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Soil Sci. Plant Nutr., 45 (4), 963-972, 1999
Effects of Soil Temperature and Anaerobiosis on
Degradation of Biodegradable Plastics in Soil
and Their Degrading Microorganisms
Hiroyo Nishidel, Koki Toyota, and Makoto Kimura
Laboratory of Soil Biology and Chemistry, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, 464-8601 Japan
Received July 9, 1999; accepted in revised form October 7, 1999
The bioplastic PHB/HV (copolymer of 3-hydroxybutyrate and 3-hydroxyvaler-
ate) underwent a faster degradation at 30"C than at 52C in soil under aerobic
conditions, while there was no remarkable difference between 30C and 52C in
the degradation rate of PCL PBSA (polybutylene
succinate and agipate), and PBS (polybutylene succinate). PHB showed the
fastest degree of degradation among the four plastics at 30C and PBSA the
fastest at 52C. Degradation of all the four plastics was nor observed both at
30C and 52C under anaerobic conditions for 50 d. Microorganisms on the
degrading plastics appeared to be diverse at 30"C, including bacteria and fungi.
However, among the several to ca. 10 kinds of bacterial and fungal strains
isolated from the degradation sites of each plastic at 30"C, only one or two
fungal strains were able to degrade the respective plastics in vitro. The de-
graders were identified as Mucor sp. (PHB), Paecilomyces sp. (PCL), Aspergil-
lus sp. (PBSA), and Cunninghamella sp. (PBSA). In contrast, only a single
type of fungus was observed at the degradation sites of PCL and PBSA at 52C.
The fungus isolated from PCL and PBSA was identified as Thermomyces sp.
This study demonstrated that soil temperature and anaerobiosis exerted signifi-
cant effects on the degradation of the plastics, and that fungi were mainly
responsible for the degradation of the plastics in soil.
Key Words: biodegradable plastics, degrading microorganisms, fungi, soil
conditions.
963
Synthetic plastics have been used for various purposes, and the production of plastics
has been one of the key industries. However, synthetic plastics with high performance and
stability cause serious problems in waste management (Hrabak 1992; Lee 1995). Recently,
biodegradable plastics have become a cause for public concern. A number of biodegradable
plastics derived from natural products or polymerized from naturally occurring monomers
(Brandl and Puchner 1992; Takiyama and Fujimaki 1994) are commercially available. PI-IB
[poly(3-hydroxybutyrate)] is typical of the former group, and several plastics such as
poly(vinyl alcohol) (PV A), poly(methyl-glutamate) (PGA), polY(E-caprolactone) (peL),
polybutylene succinate (PBS), polybutylene succinate and agipate copolymer (PBS A)
belong to the latter group.
1 Present address: Computer Room, National Institute for Basic Biology, Okazaki, 444-8585 Japan.
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964 H. NISHIDE, K. TOYOTA, and M. KIMURA
It was reported that a variety of microbial strains, including bacteria and fungi,
degraded polyhydroxyalkanoates (PHAs) (Brandl et al. 1995; Mergaert and Swings 1996).
Most of the plastic-degrading microorganisms were screened for their potential to degrade
them in vitro (Delafield et al. 1965; Fields and Finn 1974; Tanaka et al. 1976; Matavulj and
Molitoris 1992; lendrossek et al. 1993; Oda et al. 1995; Pranamuda et al. 1995; Mergaert and
Swings 1996), and in several studies it was shown that plastic-degrading microorganisms
were isolated from the plastics that were degraded in the environment (Mergaert et al. 1992,
1993). Much less attention has been paid to determine whether isolates from the degraded
plastics actually degrade the respective plastics in situ.
Degradation of PHAs was mainly performed by bacteria (e.g., Matavuli and Molitoris
1992; Mergaert and Swings 1996). It was also found that fungi, rather than bacteria, were
mainly responsible for the degradation of PHB in soil (Kimura et al. 1994; Oda et al. 1995).
PHA-degrading microorganisms have been screened for their in vitro ability to degrade in
most of the studies. It is, therefore, necessary to isolate degrading microorganisms from
degraded plastics in the environment in order to determine what kinds of microorganisms
are mainly responsible for the in situ degradation. We successfully isolated several new
plastic-degrading microorganisms from soil under different conditions. Here, we report the
effect of soil conditions on the degradation of biodegradable plastics (PHB/HV, PCL,
PBSA, and PBS), and the microorganisms that degraded the plastics in soil.
MATERIALS AND METHODS
Soil and biodegradable plastics. The soil used (Hapludults; total C = 1.2%, total
N=O.ll%, pH(H
z
O)=6.2) was collected from the Nagoya University Farm, and passed
through a 2-mm mesh screen. Farmyard manure (FYM) was also obtained from the Nagoya
University Farm, and passed through a 4-mm mesh screen. The soil was mixed with 2% (w/
w) of FYM prior to use. The plastic film samples used were as follows: PHB/HV (poly-3-
hydroxybutyrate, (10%) 3-hydroxyvalerate, Zeneca Co.), PCL (poly-caprolactone, Daicel
Chemical Industries), PBSA (poly-butylene succinate and agipate copolymer, Showa
Highpolymer Co.), and PBS (poly-butylene succinate, Showa Highpolymer Co.).
Effect of soil conditions on degradation of biodegradable plastics. Four kilo-
grams of soil that was adjusted to 40% of maximum water holding capacity was placed in
10 L of an air-tight plastic bag. Films (1 cmX 1 cm, 10 to 15 mg in weight) of the plastic
samples were buried in soil, and incubated at 30"C or 52"C for 30 to 50 d. Degradation of
the plastics under anaerobic conditions was also examined as follows. A piece of film was
buried in 20 g of soil placed in a 50 mL flask with an air tight cap and then the air was
replaced with nitrogen. The films were taken out periodically, washed gently in water to
remove adhering soil particles, and the weight of the recovered films was determined. Four
pieces of films were used for each plastic on each sampling day.
Isolation and identification of degrading microorganisms. Microorganisms were
isolated from degradation sites (holes) with a sterile toothpick and purified on nutrient agar
(NA: Eiken Chemical Co.). To ensure their ability to degrade plastics, isolated microorgan-
isms were inoculated into a minimum medium (0.1% NH
4
N0
3
, 0.1% KH
z
P0
4
, 0.05% MgS0
4

7H
z
O, 0.02% KCl) or autoclaved (12l"c for 15 min) soil with respective plastic films as a sole
carbon source and incubated either at 30"C or 52"C. Plastic degradation was estimated based
on microbial growth in the medium and holes formed on the films.
Identification of the fungal strains was performed by the Japan Food Research Lab.,
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Degradation of Biodegradable Plastics in Soil
965
a) 30"C, aerobic conditions b) 52"C, aerobic conditions
100
100

80

80


CI Ci
&:: &::
"2 "2
iii
60
pel iii
60
E E
G/ G/
... ..
.... ....
.c .c
CI CI
PBS
Qi 40 Gi 40
3: 3:
20
HH
20
t
LSD (P < 0,05) PBSA
LSD (P < 0,05)
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Incubation days
Incubation days
Fig, 1. Degradation of the plastics PHB/HV, PCL, PBS, and PBS A in soil under aerobic conditions at
30T (a) and S2C (b). Each symbol is the mean of four replications and bars indicate the least significance
difference (LSD: p < 0.05) for each plastic.
a) 30C, anaerobic conditions
120
100

-;;; 80
,5
I:
'iii
E
60

-fr- PHB/HV
E
CI
'iii ---pel
3: 40

20
-----ts--- P BSA

o 1 0 20 30 40 50
Incubation days



CI
&::
'c
iii
E
G/
...
E
CI
'iii
:s:
b) 52"C, anaerobic conditions
120
80
60
-fr- PHB/HV
---pel
40

20
-----ts--- P BSA
01 1
0 10 20 30 40 50
Incubation days
Fig, 2, Degradation of the plastics PHB/HV, PCL, PBS, and PBSA in soil under anaerobic conditions at
30C (a) and 52C (b). Each symbol is the mean of four replications.
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966 H. NISHIDE, K. TOYOTA, and M. KIMURA
Fig. 3. Microscopic obser-
vation (X 6.3 to 40) of the
plastics buried in soil under
aerobic conditions at 30'C. A:
PHB/HV. B: peL. C: PBSA.
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Degradation of Biodegradable Plastics in Soil 967
Fig. 4. Microscopic obser-
vation (x 6.3 to 40) of the
plastics buried in soil under
aerobic conditions at 52'C. A:
PHB/HV.B: PCL. C: PBSA.
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968 H. NISHIDE, K. TOYOTA, and M. KIMURA
Fig. 5. Degradation of the
plastics by isolated micro-
organisms under aerobic con-
ditions at 30"C. A: PHB/HV,
by Mucor sp. B: peL, by
Paecilomyces sp. C: PBSA, by
Cunninghamella sp.
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Degradation of Biodegradable Plastics in Soil 969
mainly based on morphology.
RESULTS AND DISCUSSION
Effect of soil conditions on degradation of PBB/BV, peL, PBS, and PBSA
Degradation was estimated from the weight loss of the plastics. PHB underwent the
fastest degradation among the four plastics at 30C under aerobic conditions and PBS the
slowest: the estimated half-life (the day when the weight of the recovered films was reduced
to half) was ca. 10 d for PHB and over 25 d for PBS (Fig. 1). At 52C, PBSA underwent the
fastest degradation with a half-life of ca. 7 d, and PHB, PCL, and PBS showed a similar
degradation rate under aerobic conditions. PHB/HV underwent a faster degradation at 30C
than at 52T under aerobic conditions, while PCL, PBS, and PBS A showed a faster
degradation at 52C (Fig. 1).
The plastics were not degraded under anaerobic conditions at both 30C and 52C [Fig.
2; the weight of PCL after 50 d of incubation at 52C was larger than that at a-time, because
of the adhesion of soil particles due to partial melting of PCL (the melting point of PCL
is 5YC)]. These results suggested that the degradation ofPHB, PCL, PBS, and PBSA at 52T
was not physical, but biological. Most of the reports on the biodegradation of PHB in
different environments have been conducted under aerobic conditions. Only a few studies
were reported on the anaerobic degradation of PHB in an estuarine sediment (Janssen and
Harfoot 1990) and sludges (Budwill et al. 1992; Mergaert and Swings 1996). There has been,
hitherto, no report on the anaerobic degradation of PHB in soil.
Isolation of plastic-degrading microorganisms
Two types of degradation of the plastics were observed. In one case, various kinds of
fungi were observed under a microscope (X40) on/around the degradation sites, suggesting
that these fungi may be responsible for the degradation of the plastics. In the other case, no
mycelium was observed around the degradation sites, suggesting that degradation was due
to bacteria, rather than fungi. Under aerobic conditions at 30C, different types of fungi,
based on the color of spores, were observed on the plastics, irrespective of the kinds of
plastics, and they appeared to be the organisms that degraded the plastics (Fig. 3). In
contrast, fungal mycelia were hardly observed on the degradation sites of PHB at 52C (Fig.
4A), indicating that bacteria may be the major microorganisms that degraded PHB at 5TC.
In the cases of PCL and PBSA, only a single type of fungus was observed on the degraded
Table 1. Relative abundance of fungi on degraded plastics and identificaiton of the
plastic-degrading microorganisms.
Plastics Temperature Microscopic observation Identified microorganisms as plastic degraders
PHB/HV 30"C Fungi + + + * Mucor sp.
52"C Fungi + Talaromyces sp., an actinomycete strain
PCL 30"C Fungi + + + Paecilomyces sp.
5TC Fungi + + + Thermomyces sp.
PBSA 30"C Fungi + + + Cunninghamella sp., Aspergillus sp.
5TC Fungi + + + Thermomyces sp.
PBS 30"C Fungi + + + not isolated
52"C Fungi + + + not isolated
* Relative amount of fungal mycelia on the films. +: present, + + +: abundant.
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970 H. NISHIDE, K. TOYOTA, and M. KIMURA
Fig. 6. Degradation of the
plastics by isolated micro-
organisms under aerobic con-
ditions at 52T. A: PHB/HV,
by Talaromyces sp. B: peL,
by Thermomyces sp. C:
PBSA, by Thermomyces sp.
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Degradation of Biodegradable Plastics in Soil 971
plastics at 52e with a marked proliferation around the degradation sites (Fig. 4B, e).
Several to ca. 10 kinds of bacterial and fungal strains were isolated from the degradation
sites of each plastic at 30C. However, only several strains, exclusively fungi, were able to
degrade the respective plastics in inoculation experiments. They showed a similar pattern of
degradation to that observed in soil (Fig. 5), and were considered to be the microorganisms
responsible for the degradation of the plastics in soil. Mucor sp. and Paecilomyces sp. were
identified as PHB/HV- and PeL-degrading microorganisms, respectively (Table 1). Asper-
gillus sp. and Cunninghamella sp. were identified as PBS A-degrading microorganisms.
Unlike the degradation at 30
o
e, a single type of fungus, based on the colony morphology,
was isolated from the degradation sites of peL and PBSA at 52T, and the fungus also
showed a similar way of degradation to that observed in soil (Fig. 6B, e). Both fungi that
degraded either peL or PBSA were identified as Thermomyces sp. Five bacterial and one
fungal strains were isolated from the degradation sites of PHB/HV at 52T, but only one
actinomycete and one fungal strain were able to degrade PHB/HV at 52e in the inoculation
experiments. The fungus was identified as Talaromyces sp.
Although the degradation of PHAs was mainly performed by bacteria, fungal degrada-
tion was also reported (e.g., Matavulj and Molitoris 1992; Mergaert et al. 1993; Oda et al.
1995; Mergaert and Swings 1996). Degradation of PHB/HV, peL, and PBSA in soil at 30
0
e
was remarkably suppressed by the addition of a fungicide Daconil (chlorothalonil,
2,4,5,6-tetrachloroisophthalonitrile), while the addition of streptomycin did not affect
appreciably the degradation of the plastics (PHB/HV, Kimura et al. 1994; peL and PBSA,
data not shown). These results suggest that fungi might be mainly responsible for the
biodegradation of the plastics in soil.
Reports on the microbial degradation of biodegradable plastics other than PH As are
relatively limited compared with those of PHAs. Paecilomyces sp. (Oda et al. 1995),
Penicillium sp. (Tokiwa et al. 1976), and actinomycetes (Mergaert and Swings 1996) were
reported to be PeL-degrading microorganisms. Paecilomyces sp., Acremonium sp., Aspergil-
lus sp. (Nishioka et al. 1994), Paecilomyces sp., and Sphingobacterium sp. (Imada et al. 1998)
and actinomycetes (Mergaert and Swings 1996) were reported to be PBSA-degrading
microorganisms. Talaromyces sp., Thermomyces sp., and Cunninghamella sp. were for the
first time reported to be plastic-degrading fungi in this study.
In conclusion, this study suggested that fungi were mainly responsible for the degrada-
tion of the plastics PHB/HV, peL, PBS, and PBSA in soil, and that microorganisms
responsible for the degradation were different depending on the kinds of plastics and soil
conditions.
Acknowledgments. This study was partly funded by the Biodegradable Plastics Society. We thank the
chairman Mr. A. Hoshino for his encouragement in the study, and Zeneca Co., Daicel Chemical Industries,
and Showa Highpolymer Co. for supplying the plastics.
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972 H. NISHIDE, K. TOYOTA, and M. KIMURA
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