Polymer Degradation and Stability 84 (2004) 411e417

www.elsevier.com/locate/polydegstab

Eects of storage of a mature compost

on its potential for biodegradation of plastics
Hea-Sun Yanga, Jin-San Yoonb, Mal-Nam Kima,)
a

Department of Biology, Sangmyung University, 7 Hongji-dong Chongro-gu, Seoul 110-743, South Korea
b
Department of Polymer Science and Engineering, Inha University, Incheon 402-751, South Korea
Received 20 October 2003; received in revised form 15 December 2003; accepted 2 January 2004

Abstract
Biodegradation of plastics was tested in the compost stored at
20 (C, 4 (C and 20 (C for dierent periods. Viable cells in the
compost stored at
20 (C were expected to be fewer than those in the compost stored at 4 (C and 20 (C, because microbes may be
under stress or even be killed due to the formation of ice crystals at the subzero temperature. Mesophilic bacteria and mesophilic
actinomycetes were fewer in number in the compost stored at 20 (C than in the compost stored at the other two lower temperatures
contrary to expectation. In contrast, both thermophilic bacteria and thermophilic actinomycetes were fewest in the compost stored
at
20 (C as was expected, indicating that thermophilic microbes were more susceptible to stress in the freezing conditions than the
mesophilic ones. Activity of the exo-enzymes plausibly excreted by the microbes in the compost decreased as a result of the storage.
Nevertheless, biodegradation of cellulose in the compost was almost independent of the storage time and temperature. In contrast,
biodegradability of both polycaprolactone (PCL) and poly(butylene succinate) (PBS) depended strongly on the storage conditions.
From the point of view that the existing standards for biodegradation tests of plastics in compost accept reproducibility of cellulose
biodegradability as a criterion for the validity of the biodegradation tests, a new standard of the compost preparation should be
provided to guarantee more reliable results on the biodegradability of plastics.
2004 Elsevier Ltd. All rights reserved.
Keywords: Compost; Storage; Biodegradation; Bacteria; Actinomycetes

1. Introduction
Synthetic plastics give rise to water clogging phenomenon in soil burial sites and thus cause rain water overow, provoking serious contamination of nearby rivers
and sea [1]. Moreover, accumulation of synthetic plastics
in cultivation soil reduces crop production. Many animals
and sh are killed due to swallowing of debris of synthetic
plastics, which in turn disrupts the ecosystem.
Recycling of plastics is not economically feasible in
many cases due to deterioration of mechanical properties and excessive cost. Development of biodegradable
plastics is under investigation because it may provide

) Corresponding author. Tel.: C82-2-2287-5150; fax: C82-2-3949585.

E-mail address: mnkim@smu.ac.kr (M.-N. Kim).
0141-3910/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymdegradstab.2004.01.014

a solution to the above-mentioned environmental problems. Food garbage can be composted together with
biodegradable packaging materials, which may contribute to a signicant reduction of municipal solid waste.
Evaluation of biodegradability of plastics is usually
carried out in mature compost, because many kinds of
microbes are present in the compost and the evaluation
may represent eventual fate of the plastics in nature.
Evaluation of biodegradability of plastics appeared
to be satisfactorily reproducible in a compost made from
cow fodder [2]. The compost for the biodegradability
tests was harvested 1e2 weeks after the onset of the
maturation stage. The harvest time was kept constant
because the content of organic matters and the microbial community should be changed with time.
As more than 45 days are required to produce fresh
compost, the biodegradation tests can only be started
with a signicant delay in many cases.

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H.-S. Yang et al. / Polymer Degradation and Stability 84 (2004) 411e417

2. Methods

settle down for 10 min. The supernatant was pipetted

(100 ml) and was dispensed into each of the 20 microcupules. The microcupules were then covered and
incubated at 37 (C for 24 h. After incubation, one drop
each of Reagent A and of Reagent B (API Analytab
Products, New York) was added to all the microcupules.
Enzyme activity was graduated into ve classes, value of
5 corresponding to the highest activity.

2.1. Composting

2.4. Storage of the compost

The animal fodder was purchased from Sam Yang

Co. (Korea). Initial moisture content was adjusted to be
65 wt.% by adding an appropriate amount of sawdust.
A laboratory-scale composting reactor, 50 cm in diameter and 120 cm in depth, was used. Air was supplied
to the composting mass at 1e15 l/min. The temperature
during the composting was controlled not to exceed
58 (C using by-pass air supply as soon as the
temperature became higher than 58 (C.

A mature compost was put into a glass bottle (500 ml)

which was sealed with a cotton plug. Each bottle was
stored at
20 G 2 (C, 4 G 2 (C and 20 G 2 (C for different periods.

In this study, a matured compost was stored at

20 (C, 4 (C and 20 (C for dierent periods. Eects of
the storage conditions on the biodegradability of plastics
in the compost were examined to explore a possibility of
the biodegradation tests without delay.

2.2. Analysis of compost

2.2.1. Physicochemical analysis
Moisture content was determined from the weight
loss after drying at 105 (C for 1 day. The compost
samples (20 g) and distilled water (80 ml) were mixed for
30 min, then the pH of the supernatant was measured
using a Beckman pH meter (pH F34) according to ISO
14855 [3]. C/N ratio was measured using an elemental
analyser (LECO CHNS-932).
2.2.2. Microbial analysis
Bacteria and actinomycetes, both mesophilic and
thermophilic, were enumerated by plate counting methods. Incubation time was 1 day for bacteria and 7 days
for actinomycetes. The isolation media were as follows:
plate count agar (Difco) for bacteria; and actinomycete
isolation agar (Difco) for actinomycetes. Incubation was
at 37 (C for mesophilic bacteria and 27 (C for mesophilic actinomycetes. For thermophilic bacteria and
actinomycetes, the incubation temperature was 55 (C.

2.5. Biodegradation of biodegradable plastics in

compost
2.5.1. Biodegradable plastics
Polycaprolactone (PCL, TONE POLYMER P-787,
Union Carbide (USA), Mn 180; 000, Mw 325; 000)
and poly(butylene succinate) (PBS, G4500, Ire Chemical
Co. (Kor), Mn 41; 000, Mw 106; 000) were powdered in cryogenic conditions and subjected to the biodegradation test. Cellulose (Sigma, sigmacell type 101)
was used as a positive control.
2.5.2. Biodegradation test
Biodegradation test in the compost using the laboratory-scale reactor was conducted according to ASTM
D5338-92 [4]. The air ow rate was controlled at 40 ml/
min. A mixture of mature compost (200 g, wet weight)
and test materials (5%, on dry basis) was introduced and
incubated at 58 (C. CO2 produced from the reactor was
absorbed by 0.4 N potassium hydroxide and 2 N barium
chloride solution, and the amount was determined by
titrating the solution with 0.2 N HCl.

3. Results and discussion

2.3. Compost enzyme activity

3.1. Physicochemical characteristics of the compost

Compost enzyme activity was measured using APIZYM kit (API Analytab products, Plainview NY, USA).
APIZYM is a commercial semi-quantitative micromethod designed for systematic and rapid study of 19
enzymatic reactions. It consists of a series of microcupules containing dehydrated chromogenic substrates
for the 19 dierent enzymes and a control (a microcupule without any substrates for the enzymes).
Enzyme extracts were prepared by mixing 10 g of the
compost sample with 90 ml of sterilized distilled water
for 30 min. The suspension solution was allowed to

Fig. 1 demonstrates temperature prole in the course

of composting of a cow fodder. The high temperature
stage, where the temperature of the compost was kept
approximately at 58 (C, lasted for about 2 weeks. Since
excessively high temperature during the high temperature stage would either completely kill the microbes or at
least reduce their physiological activity, it should be
strictly controlled to get reproducible results of biodegradability.
Storage conditions of the mature compost would also
make changes in physicochemical characteristics of the

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H.-S. Yang et al. / Polymer Degradation and Stability 84 (2004) 411e417

3.2. Microbial community in the compost

70
compost temperature
ambient temperature

60
50
40
30
20
10
0
0

10

20

30

40

Time (Day)
Fig. 1. Temperature prole during the composting process.

compost and community of the microbes and their

physiological activity exerting signicant inuences
upon biodegradability of plastics in the compost [5].
Fresh compost was harvested 1 week after the onset
of the maturation stage of the composting. It was stored
at dierent temperatures and its physicochemical characteristics are presented in Table 1 as a function of
storage time.
Fresh compost contained 65 wt.% of water and its pH
was 8.5. The water content decreased to 59 wt.% as the
storage time increased to 90 days independently of the
storage temperature. Stenberg et al. [6] also reported that
the water content of a cultivating soil after 13 months of
storage at
20 G 2 (C was almost the same as that of the
soil stored at 2 G 2 (C.
pH and total carbon content of the compost decreased
with the storage time, and the decrease was more
signicant at 20 (C than at
20 (C or at 4 (C. This is
because physiological activity of the microbes was higher
at 20 (C to mineralize larger amount of bioassimilable
carbon than at the other two temperatures.

The number of viable mesophilic bacteria is shown in

Fig. 2 as a function of storage conditions. Mesophilic
bacteria in the compost stored for 30 days were
decreased two to three times in number compared to
those in the fresh compost.
It is interesting to observe that viable mesophilic
bacteria in the compost stored not only for 30 days but
also for 60 days and 90 days at 20 (C were smaller in
number than those in the compost stored for the same
period of time at
20 (C or at 4 (C.
Conditioning the stored compost at 17 (C for 2 days
produced negligible eect on the cell number of
mesophilic bacteria, indicating that these bacteria regain
their physiological activity quite rapidly.
Ice crystals were observed in the compost stored at

20 (C. Formation of ice crystals in the compost increases the concentration of salts in the liquid phase and
thus causes osmotic stress to microbes. Intracellular ice
crystals may also damage sensitive microbial cells [6].
Therefore it was expected that viable cells in the
compost stored at 20 (C should be more numerous than
those in the compost stored at
20 (C. However, Figs. 2
and 3 demonstrate that not only mesophilic bacteria but
also mesophilic actinomycetes were fewer in the
compost stored at 20 (C than in the compost stored at
the other two lower temperatures.
Cold-adapted microbes can grow in refrigerated
conditions [7] and microbes were found to continue
respiration in frozen soil [8,9]. Ostroumov et al. [10]
reported that an important factor for long-term survival
of microbes in frozen state was the transfer of unfrozen
liquid water and ions.
In contrast to mesophilic microbes, cells of both
thermophilic bacteria (Fig. 4) and thermophilic actinomycetes (Fig. 5) were fewer in the compost stored at

20 (C than in the compost stored at 20 (C, indicating
that thermophilic microbes are more susceptible to the
stress in refrigerated conditions than are mesophilic ones.
Anderson et al. [11] reported that microbial biomass
in soil depended strongly on the storage conditions. In

Table 1
Physicochemical characteristics of the compost as a function of the storage conditions
Fresh
compost

Moisture contents (wt.%)

pH
Total carbon (wt.%)
Total nitrogen (wt.%)
C/N

65.0
8.5
44.0
3.1
14.1

Storage time
30 days

60 days

90 days

Storage temperature

Storage temperature

Storage temperature

20 (C

4 (C

20 (C

20 (C

4 (C

20 (C

20 (C

4 (C

20 (C

64.2
8.3
43.2
3.1
14.1

65
8.4
43.9
3.12
14.1

62.7
8.3
41.6
3.3
12.6

64.9
8.2
42.7
3.0
14.4

64.3
8.2
44.0
3.1
14.1

62.6
7.8
41.6
4.0
10.4

59.8
8.2
42.1
2.8
14.9

58.5
7.8
43.7
3.1
14.1

59.3
7.7
39.4
3.6
10.9

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H.-S. Yang et al. / Polymer Degradation and Stability 84 (2004) 411e417

109

108

107

fresh compost
-20/30/2
4/30/2
20/30/2
-20/60/2
4/60/2
20/60/2
-20/90/2
4/90/2
20/90/2

106

1010

Viable cell number (cfu ml-1)

Viable cell number (cfu ml-1)

1010

105

109

108

107

fresh compost
-20/30/2
4/30/2
20/30/2
-20/60/2
4/60/2
20/60/2
-20/90/2
4/90/2
20/90/2

106

105

Fig. 2. Eect of storage conditions on mesophilic bacterial population

in an animal fodder compost. The symbol N1/N2/N3 means that the
compost has been stored at N1(C for N2 days, and then conditioned at
17 (C for N3 days.

Fig. 4. Eect of storage conditions on thermophilic bacterial

population in an animal fodder compost. The symbol N1/N2/N3
means that the compost has been stored at N1 (C for N2 days, and
then conditioned at 17 (C for N3 days.

contrast, Blume et al. [12] found that the size of the

microbial biomass in both sandy loam and silt loam soils
in winter was almost the same as that in the corresponding soils collected in summer.

Alkaline phosphatase, acid phosphatase, leucine

arylamidase and N-acetyl-b-glucosaminidase were highly active, while esterase catalysing polyester degradation
and a- and b-glucosidase catalysing cellulose degradation showed a medium activity.
Tiquia et al. [13] found that alkaline phosphatase, acid
phosphatase, leucine amino-peptidase and esterasee
lipase were active in compost made from poultry and
yard trimmings. They also demonstrated that alkaline
phosphatase, acid phosphatase, esterase, esteraseelipase,
leucine amino-peptidase, b-glucosidase and N-acetyl-bglucosaminidase were highly active in the compost from
pig manure and sawdust.
Activity of the enzymes was greater in the fresh
compost than that corresponding to the stored compost.

The substrates corresponding to the enzymes, which

were expected to be secreted by the microbes in the
compost, were added to the supernatant of the solution
suspended with the compost and colour change of the
supernatant solution was monitored to determine the
activity of the enzymes. The enzyme activity was
graduated into 1e5 classes as shown in Table 2 where
higher number corresponds to higher enzymatic activity.
1010

109

108

107

fresh compost
-20/30/2
4/30/2
20/30/2
-20/60/2
4/60/2
20/60/2
-20/90/2
4/90/2
20/90/2

106

105
Fig. 3. Eect of storage conditions on mesophilic actinomycete
population in an animal fodder compost. The symbol N1/N2/N3
means that the compost has been stored at N1 (C for N2 days, and
then conditioned at 17 (C for N3 days.

1010

Viable cell number (cfu ml-1)

3.3. Activity of enzymes in the compost

109

108

107

fresh compost
-20/30/2
4/30/2
20/30/2
-20/60/2
4/60/2
20/60/2
-20/90/2
4/90/2
20/90/2

106

105
Fig. 5. Eect of storage conditions on thermophilic actinomycete
population in an animal fodder compost. The symbol N1/N2/N3 means
that the compost has been stored at N1 (C for N2 days, and then
conditioned at 17 (C for N3 days.

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H.-S. Yang et al. / Polymer Degradation and Stability 84 (2004) 411e417

Table 2
Relative activity of enzymes in the supernatant of the compost suspension
Enzymes

Fresh
compost

Storage conditions of the compost

30 days

60 days

90 days

4 (C

20 (C

4 (C

20 (C

4 (C

20 (C

Phosphatases
Alkaline phosphatase
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase

5
4
3

5
3
3

5
4
2

3
3
2

5
3
2

3
2
2

5
3
2

Esterases
Esterase
Esteraseelipase
Lipase

2
3
3

2
2
2

2
2
3

1
2
2

1
0
2

1
2
1

1
2
1

Arylamidase
Leucine arylamidase
Valine arylamidase
Cystine arylamidase

4
2
1

3
2
1

3
2
0

2
1
0

3
0
0

2
1
0

3
0
0

Proteases
Trypsin
a-Chymotrypsin

1
1

0
0

0
0

0
0

0
0

0
0

0
0

Glycosyl hydrolases
a-Galactosidase
b-Galactosidase
b-Glucuronidase
a-Glucosidase
b-Glucosidase
N-acetyl-b-glucosaminidase
a-Mannosidase
a-Fucosidase

1
1
2
3
3
4
0
0

0
0
2
3
3
4
0
0

0
0
2
1
2
2
0
0

0
0
1
2
2
4
0
0

0
0
1
1
1
2
0
0

0
0
0
1
1
3
0
0

0
0
0
1
0
1
0
0

Low intensity (value of 1); moderate intensity (values of 2e4); high intensity (value of 5).

Activity of phosphatase decreased from class 3e5 to

class 2e5 and that of esterase went down from class 2e3
to class 1e3. Enzymes such as cystine arylamidase,
trypsin, a-chymotrypsin, a-galactosidase and b-galactosidase were slightly active in the fresh compost, while
they became completely inactive in the stored compost.

Cellulose and PCL are both biodegraded fast but

with dierent enzymes. PCL and PBS are polymers with
ester linkages but their biodegradability is signicantly
dierent.
100

80

Figs. 6 and 7 show biodegradation behaviour of

cellulose in the compost. Average of three experiments
was taken. It can be seen that biodegradability of
cellulose in the compost was found to be identical
independently of both storage time and temperature.
In the case of PCL, identical biodegradability was
observed in the compost stored for 30 days independently of storage temperature, while the biodegradability in the compost stored for 90 days depended upon the
storage temperature as can be seen in Figs. 8 and 9.
Biodegradability of PBS changed with the storage
temperature not only in the compost stored for 90 days
but also in the compost stored for 30 days as shown in
Figs. 10 and 11. The results were contrary to the
expectation in that the biodegradability of PBS was
faster in the stored compost than in the fresh compost.

CO2 evolution

3.4. Biodegradation of plastics in the compost

60

40
fresh compost
-20/30/2
4/30/2
20/30/2

20

0
0

10

15

20

25

30

Time (Day)
Fig. 6. Cellulose biodegradation in a compost stored for 30 days at
dierent temperatures. The symbol N1/N2/N3 means that the compost
has been stored at N1 (C for N2 days, and then conditioned at 17 (C
for N3 days.

416

100

100

80

80

CO2 evolution

CO2 evolution

H.-S. Yang et al. / Polymer Degradation and Stability 84 (2004) 411e417

60

40
fresh compost
-20/90/2
4/90/2
20/90/2

20

60

40
fresh compost
-20/90/2
4/90/2
20/90/2

20

0
0

10

15

20

25

30

Time (Day)

10

15

20

25

30

Time (Day)

Fig. 7. Cellulose biodegradation in a compost stored for 90 days at

dierent temperatures. The symbol N1/N2/N3 means that the compost
has been stored at N1 (C for N2 days, and then conditioned at 17 (C
for N3 days.

Fig. 9. PCL biodegradation in a compost stored for 90 days at dierent

temperatures. The symbol N1/N2/N3 means that the compost has been
stored at N1 (C for N2 days, and then conditioned at 17 (C for N3
days.

Biodegradation results in Figs. 6e11, in which rate of

mineralization of plastics into CO2 was compared,
cannot be analysed with the data in Table 2 because
the latter data correspond only to activity of the exoenzymes. Not only the exo-enzymes but also intracellular enzymes and transfer of the products from chain
cleavage of the plastics aect mineralization of plastics.
According to ISO 14855 [3], the quality of the
compost used for biodegradability tests of plastics is

evaluated by its biodegradation ability for cellulose. ISO

14855 proposed a criterion for the validity of the
biodegradation tests, stating that amount of CO2
produced in the three replicates at the end of the
biodegradation tests should be within 20% error range.
The present study clearly reveals that biodegradability of PCL and PBS in the compost depended
signicantly on the storage conditions in spite of the
fact that cellulose biodegradation was hardly aected by

100

100

80

CO2 evolution

80

CO2 evolution

fresh compost
-20/30/2
4/30/2
20/30/2

60

40
fresh compost
-20/30/2
4/30/2
20/30/2

20

60

40

20

0
0

10

15

20

25

30

Time (Day)
Fig. 8. PCL biodegradation in a compost stored for 30 days at dierent
temperatures. The symbol N1/N2/N3 means that the compost has been
stored at N1 (C for N2 days, and then conditioned at 17 (C for N3
days.

10

15

20

25

30

Time (Day)
Fig. 10. PBS biodegradation in a compost stored for 30 days at
dierent temperatures. The symbol N1/N2/N3 means that the compost
has been stored at N1 (C for N2 days, and then conditioned at 17 (C
for N3 days.

H.-S. Yang et al. / Polymer Degradation and Stability 84 (2004) 411e417

100

References
fresh compost
-20/90/2
4/90/2
20/90/2

80

CO2 evolution

417

60

40

20

0
0

10

15

20

25

30

Time (Day)
Fig. 11. PBS biodegradation in a compost stored for 90 days at
dierent temperatures. The symbol N1/N2/N3 means that the compost
has been stored at N1 (C for N2 days, and then conditioned at 17 (C
for N3 days.

the storage conditions. Therefore it can be concluded

that a standard for the compost preparation should be
presented for reliability of biodegradation tests of
plastics in the compost.

Acknowledgements
This work was supported by an Interdisciplinary
Research Program grant R01-2002-000-00146-0 from
the Korea Science and Engineering Foundation.

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