-
fd
o
,
r
liAbstract
Biodegradation of plastics was tested in the compost stored at
20 (C, 4 (C and 20 (C for dierent periods. Viable cells in
thecompost stored at 20 (C were expected to be fewer than those in
the compost stored at 4 (C and 20 (C, because microbes may beunder
stress or even be killed due to the formation of ice crystals at
the subzero temperature. Mesophilic bacteria and
mesophilicactinomycetes were fewer in number in the compost stored
at 20 (C than in the compost stored at the other two lower
temperaturescontrary to expectation. In contrast, both thermophilic
bacteria and thermophilic actinomycetes were fewest in the compost
storedat 20 (C as was expected, indicating that thermophilic
microbes were more susceptible to stress in the freezing conditions
than themesophilic 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 cellulosebiodegradability 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 phenom-enon in
soil burial sites and thus cause rain water over-ow, provoking
serious contamination of nearby riversand sea [1]. Moreover,
accumulation of synthetic plasticsin cultivation soil reduces crop
production.Many animalsand sh are killed due to swallowing of
debris of syntheticplastics, which in turn disrupts the
ecosystem.
Recycling of plastics is not economically feasible inmany cases
due to deterioration of mechanical proper-
a solution to the above-mentioned environmental pro-blems. Food
garbage can be composted together withbiodegradable packaging
materials, which may contrib-ute to a signicant reduction of
municipal solid waste.
Evaluation of biodegradability of plastics is usuallycarried out
in mature compost, because many kinds ofmicrobes are present in the
compost and the evaluationmay represent eventual fate of the
plastics in nature.
Evaluation of biodegradability of plastics appearedto be
satisfactorily reproducible in a compost made fromcow fodder [2].
The compost for the biodegradabilityEects of storage oon its
potential for bio
Hea-Sun Yanga, Jin-San Y
aDepartment of Biology, Sangmyung University, 7 HbDepartment of
Polymer Science and Engineering
Received 20 October 2003; received in revised fo
Polymer Degradation and Stabities and excessive cost.
Development of biodegradableplastics is under investigation because
it may provide
) Corresponding author. Tel.: C82-2-2287-5150; fax:
C82-2-394-9585.
E-mail address: [email protected] (M.-N. Kim).
0141-3910/$ - see front matter 2004 Elsevier Ltd. All rights
reserved.doi:10.1016/j.polymdegradstab.2004.01.014a mature
compostegradation of plastics
oonb, Mal-Nam Kima,)
ngji-dong Chongro-gu, Seoul 110-743, South Korea
Inha University, Incheon 402-751, South Korea
m 15 December 2003; accepted 2 January 2004
ty 84 (2004) 411e417
www.elsevier.com/locate/polydegstabtests was harvested 1e2 weeks
after the onset of thematuration stage. The harvest time was kept
constantbecause the content of organic matters and the micro-bial
community should be changed with time.
As more than 45 days are required to produce freshcompost, the
biodegradation tests can only be startedwith a signicant delay in
many cases.
-
tiIn this study, a matured compost was stored at20 (C, 4 (C and
20 (C for dierent periods. Eects ofthe storage conditions on the
biodegradability of plasticsin the compost were examined to explore
a possibility ofthe biodegradation tests without delay.
2. Methods
2.1. Composting
The animal fodder was purchased from Sam YangCo. (Korea).
Initial moisture content was adjusted to be65 wt.% by adding an
appropriate amount of sawdust.A laboratory-scale composting
reactor, 50 cm in di-ameter and 120 cm in depth, was used. Air was
suppliedto the composting mass at 1e15 l/min. The temperatureduring
the composting was controlled not to exceed58 (C using by-pass air
supply as soon as thetemperature became higher than 58 (C.
2.2. Analysis of compost
2.2.1. Physicochemical analysisMoisture content was determined
from the weight
loss after drying at 105 (C for 1 day. The compostsamples (20 g)
and distilled water (80 ml) were mixed for30 min, then the pH of
the supernatant was measuredusing a Beckman pH meter (pH F34)
according to ISO14855 [3]. C/N ratio was measured using an
elementalanalyser (LECO CHNS-932).
2.2.2. Microbial analysisBacteria and actinomycetes, both
mesophilic and
thermophilic, were enumerated by plate counting meth-ods.
Incubation time was 1 day for bacteria and 7 daysfor actinomycetes.
The isolation media were as follows:plate count agar (Difco) for
bacteria; and actinomyceteisolation agar (Difco) for actinomycetes.
Incubation wasat 37 (C for mesophilic bacteria and 27 (C for
meso-philic actinomycetes. For thermophilic bacteria
andactinomycetes, the incubation temperature was 55 (C.
2.3. Compost enzyme activity
Compost enzyme activity was measured using API-ZYM kit (API
Analytab products, Plainview NY, USA).APIZYM is a commercial
semi-quantitative micro-method designed for systematic and rapid
study of 19enzymatic reactions. It consists of a series of
micro-cupules containing dehydrated chromogenic substratesfor the
19 dierent enzymes and a control (a micro-cupule without any
substrates for the enzymes).
Enzyme extracts were prepared by mixing 10 g of thecompost
sample with 90 ml of sterilized distilled water
412 H.-S. Yang et al. / Polymer Degradafor 30 min. The
suspension solution was allowed tosettle down for 10 min. The
supernatant was pipetted(100 ml) and was dispensed into each of the
20 micro-cupules. The microcupules were then covered andincubated
at 37 (C for 24 h. After incubation, one dropeach of Reagent A and
of Reagent B (API AnalytabProducts, New York) was added to all the
microcupules.Enzyme activity was graduated into ve classes, value
of5 corresponding to the highest activity.
2.4. Storage of the compost
Amature compost was put into a glass bottle (500 ml)which was
sealed with a cotton plug. Each bottle wasstored at 20G 2 (C, 4G 2
(C and 20G 2 (C for dif-ferent periods.
2.5. Biodegradation of biodegradable plastics incompost
2.5.1. Biodegradable plasticsPolycaprolactone (PCL, TONE POLYMER
P-787,
Union Carbide (USA), Mn 180; 000, Mw 325; 000)and poly(butylene
succinate) (PBS, G4500, Ire ChemicalCo. (Kor), Mn 41; 000, Mw 106;
000) were pow-dered in cryogenic conditions and subjected to the
bio-degradation test. Cellulose (Sigma, sigmacell type 101)was used
as a positive control.
2.5.2. Biodegradation testBiodegradation test in the compost
using the labora-
tory-scale reactor was conducted according to ASTMD5338-92 [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 andincubated at 58 (C. CO2 produced from the reactor
wasabsorbed by 0.4 N potassium hydroxide and 2 N bariumchloride
solution, and the amount was determined bytitrating the solution
with 0.2 N HCl.
3. Results and discussion
3.1. Physicochemical characteristics of the compost
Fig. 1 demonstrates temperature prole in the courseof composting
of a cow fodder. The high temperaturestage, where the temperature
of the compost was keptapproximately at 58 (C, lasted for about 2
weeks. Sinceexcessively high temperature during the high
tempera-ture stage would either completely kill the microbes or
atleast reduce their physiological activity, it should bestrictly
controlled to get reproducible results of bio-degradability.
Storage conditions of the mature compost would also
on and Stability 84 (2004) 411e417make changes in
physicochemical characteristics of the
-
period of time at 20 (C or at 4 (C.
rag
(
.7
.3
.6
.3compost and community of the microbes and theirphysiological
activity exerting signicant inuencesupon biodegradability of
plastics in the compost [5].
Fresh compost was harvested 1 week after the onsetof the
maturation stage of the composting. It was storedat dierent
temperatures and its physicochemical char-acteristics are presented
in Table 1 as a function ofstorage time.
Fresh compost contained 65 wt.% of water and its pHwas 8.5. The
water content decreased to 59 wt.% as thestorage time increased to
90 days independently of thestorage temperature. Stenberg et al.
[6] also reported thatthe water content of a cultivating soil after
13 months ofstorage at20G 2 (Cwas almost the same as that of
thesoil stored at 2G 2 (C.
pH and total carbon content of the compost decreasedwith the
storage time, and the decrease was moresignicant at 20 (C than at
20 (C or at 4 (C. This isbecause physiological activity of the
microbes was higherat 20 (C to mineralize larger amount of
bioassimilablecarbon than at the other two temperatures.
Time (Day)0 10 20 30 40
0
10
Fig. 1. Temperature prole during the composting process.
Table 1
Physicochemical characteristics of the compost as a function of
the sto
Fresh
compost
Storage time
30 days
Storage temperature
20 (C 4 (C 20Moisture contents (wt.%) 65.0 64.2 65 62
pH 8.5 8.3 8.4 8
Total carbon (wt.%) 44.0 43.2 43.9 41
Total nitrogen (wt.%) 3.1 3.1 3.12 3C/N 14.1 14.1 14.1
12.6Conditioning the stored compost at 17 (C for 2 daysproduced
negligible eect on the cell number ofmesophilic bacteria,
indicating that these bacteria regaintheir physiological activity
quite rapidly.
Ice crystals were observed in the compost stored at20 (C.
Formation of ice crystals in the compost in-creases the
concentration of salts in the liquid phase andthus causes osmotic
stress to microbes. Intracellular icecrystals may also damage
sensitive microbial cells [6].
Therefore it was expected that viable cells in thecompost stored
at 20 (C should be more numerous thanthose in the compost stored at
20 (C. However, Figs. 2and 3 demonstrate that not only mesophilic
bacteria butalso mesophilic actinomycetes were fewer in thecompost
stored at 20 (C than in the compost stored atthe other two lower
temperatures.
Cold-adapted microbes can grow in refrigeratedconditions [7] and
microbes were found to continuerespiration in frozen soil [8,9].
Ostroumov et al. [10]reported that an important factor for
long-term survivalof microbes in frozen state was the transfer of
unfrozenliquid water and ions.
In contrast to mesophilic microbes, cells of boththermophilic
bacteria (Fig. 4) and thermophilic actino-mycetes (Fig. 5) were
fewer in the compost stored at20 (C than in the compost stored at
20 (C, indicatingthat thermophilic microbes are more susceptible to
thestress in refrigerated conditions than are mesophilic ones.
Anderson et al. [11] reported that microbial biomassin soil
depended strongly on the storage conditions. In
e conditions
60 days 90 days
Storage temperature Storage temperature
C 20 (C 4 (C 20 (C 20 (C 4 (C 20 (C64.9 64.3 62.6 59.8 58.5
59.3
8.2 8.2 7.8 8.2 7.8 7.7
42.7 44.0 41.6 42.1 43.7 39.4
3.0 3.1 4.0 2.8 3.1 3.6
14.4 14.1 10.4 14.9 14.1 10.93.2. Microbial community in the
compost
The number of viable mesophilic bacteria is shown inFig. 2 as a
function of storage conditions. Mesophilicbacteria in the compost
stored for 30 days weredecreased two to three times in number
compared tothose in the fresh compost.
It is interesting to observe that viable mesophilicbacteria in
the compost stored not only for 30 days butalso for 60 days and 90
days at 20 (C were smaller innumber than those in the compost
stored for the same20
30
40
50
60
70compost temperatureambient temperature
413H.-S. Yang et al. / Polymer Degradation and Stability 84
(2004) 411e417
-
iocontrast, Blume et al. [12] found that the size of
themicrobial biomass in both sandy loam and silt loam soilsin
winter was almost the same as that in the cor-responding soils
collected in summer.
3.3. Activity of enzymes in the compost
The substrates corresponding to the enzymes, whichwere expected
to be secreted by the microbes in thecompost, were added to the
supernatant of the solutionsuspended with the compost and colour
change of thesupernatant solution was monitored to determine
theactivity of the enzymes. The enzyme activity wasgraduated into
1e5 classes as shown in Table 2 wherehigher number corresponds to
higher enzymatic activity.
fresh
compost-20/30/24/30/220/30/2-20/60/24/60/220/60/2-20/90/24/90/220/90/2
1010
108
107
106
105
109
Fig. 3. Eect of storage conditions on mesophilic
actinomycete
population in an animal fodder compost. The symbol N1/N2/N3means
that the compost has been stored at N1 (C for N2 days, and
Via
ble
cell
num
ber
(cfu m
l-1)
1010
109
108
107
106
105
fresh
compost-20/30/24/30/220/30/2-20/60/24/60/220/60/2-20/90/24/90/220/90/2
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 at17 (C for N3 days.
414 H.-S. Yang et al. / Polymer Degradatthen conditioned at 17
(C for N3 days.Alkaline phosphatase, acid phosphatase,
leucinearylamidase and N-acetyl-b-glucosaminidase were high-ly
active, while esterase catalysing polyester degradationand a- and
b-glucosidase catalysing cellulose degrada-tion showed a medium
activity.
Tiquia et al. [13] found that alkaline phosphatase,
acidphosphatase, leucine amino-peptidase and esteraseelipase were
active in compost made from poultry andyard trimmings. They also
demonstrated that alkalinephosphatase, acid phosphatase, esterase,
esteraseelipase,leucine amino-peptidase, b-glucosidase and
N-acetyl-b-glucosaminidase were highly active in the compost
frompig manure and sawdust.
Activity of the enzymes was greater in the freshcompost than
that corresponding to the stored compost.
fresh
compost-20/30/24/30/220/30/2-20/60/24/60/220/60/2-20/90/24/90/220/90/2
Via
ble
cell
num
ber
(cfu m
l-1)
1010
108
107
106
105
109
Fig. 4. Eect of storage conditions on thermophilic bacterial
population in an animal fodder compost. The symbol N1/N2/N3means
that the compost has been stored at N1 (C for N2 days, andthen
conditioned at 17 (C for N3 days.
fresh
compost-20/30/24/30/220/30/2-20/60/24/60/220/60/2-20/90/24/90/220/90/2
Via
ble c
ell n
umbe
r (cfu
ml-1
)
1010
108
107
106
105
109
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
n and Stability 84 (2004) 411e417conditioned at 17 (C for N3
days.
-
oc
N-acetyl-b-glucosaminidase 4 4 2 4 2 3 1
tiActivity of phosphatase decreased from class 3e5 toclass 2e5
and that of esterase went down from class 2e3to class 1e3. Enzymes
such as cystine arylamidase,trypsin, a-chymotrypsin,
a-galactosidase and b-galacto-sidase were slightly active in the
fresh compost, whilethey became completely inactive in the stored
compost.
3.4. Biodegradation of plastics in the compost
Figs. 6 and 7 show biodegradation behaviour ofcellulose in the
compost. Average of three experimentswas taken. It can be seen that
biodegradability ofcellulose in the compost was found to be
identicalindependently of both storage time and temperature.
In the case of PCL, identical biodegradability wasobserved in
the compost stored for 30 days indepen-dently of storage
temperature, while the biodegradabil-ity in the compost stored for
90 days depended upon thestorage temperature as can be seen in
Figs. 8 and 9.
Biodegradability of PBS changed with the storagetemperature not
only in the compost stored for 90 daysbut also in the compost
stored for 30 days as shown inFigs. 10 and 11. The results were
contrary to theexpectation in that the biodegradability of PBS
was
Cellulose and PCL are both biodegraded fast butwith dierent
enzymes. PCL and PBS are polymers withester linkages but their
biodegradability is signicantlydierent.
a-Mannosidase 0 0 0 0 0 0 0
a-Fucosidase 0 0 0 0 0 0 0
Low intensity (value of 1); moderate intensity (values of 2e4);
high intensity (value of 5).
Time (Day)0 5 10 15 20 25 30
CO
2 ev
olu
tion
0
20
40
60
80
100
fresh compost-20/30/24/30/220/30/2
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
(CTable 2
Relative activity of enzymes in the supernatant of the compost
suspensi
Enzymes Fresh
compost
Storage
30 days
4 (C
Phosphatases
Alkaline phosphatase 5 5
Acid phosphatase 4 3
Naphthol-AS-BI-phosphohydrolase 3 3
Esterases
Esterase 2 2
Esteraseelipase 3 2
Lipase 3 2
Arylamidase
Leucine arylamidase 4 3
Valine arylamidase 2 2
Cystine arylamidase 1 1
Proteases
Trypsin 1 0
a-Chymotrypsin 1 0
Glycosyl hydrolases
a-Galactosidase 1 0
b-Galactosidase 1 0
b-Glucuronidase 2 2
a-Glucosidase 3 3
b-Glucosidase 3 3
H.-S. Yang et al. / Polymer Degradafaster in the stored compost
than in the fresh compost.n
onditions of the compost
60 days 90 days
20 (C 4 (C 20 (C 4 (C 20 (C
5 3 5 3 5
4 3 3 2 3
2 2 2 2 2
2 1 1 1 1
2 2 0 2 2
3 2 2 1 1
3 2 3 2 3
2 1 0 1 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
2 1 1 0 0
1 2 1 1 1
2 2 1 1 0
415on and Stability 84 (2004) 411e417for N3 days.
-
tiBiodegradation results in Figs. 6e11, in which rate
ofmineralization of plastics into CO2 was compared,cannot be
analysed with the data in Table 2 becausethe latter data correspond
only to activity of the exo-enzymes. Not only the exo-enzymes but
also intracellu-lar enzymes and transfer of the products from
chaincleavage of the plastics aect mineralization of plastics.
According to ISO 14855 [3], the quality of thecompost used for
biodegradability tests of plastics is
fresh compost-20/90/24/90/220/90/2
Time (Day)0 5 10 15 20 25 30
CO
2 ev
olu
tion
0
20
40
60
80
100
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
(Cfor N3 days.
fresh compost-20/30/24/30/220/30/2
Time (Day)0 5 10 15 20 25 30
CO
2 ev
olu
tion
0
20
40
60
80
100
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
416 H.-S. Yang et al. / Polymer Degradadays.evaluated by its
biodegradation ability for cellulose. ISO14855 proposed a criterion
for the validity of thebiodegradation tests, stating that amount of
CO2produced in the three replicates at the end of thebiodegradation
tests should be within 20% error range.
The present study clearly reveals that biodegradabil-ity of PCL
and PBS in the compost dependedsignicantly on the storage
conditions in spite of thefact that cellulose biodegradation was
hardly aected by
fresh compost-20/90/24/90/220/90/2
Time (Day)0 5 10 15 20 25 30
CO
2 ev
olu
tion
0
20
40
60
80
100
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
N3days.
fresh compost-20/30/24/30/220/30/2
Time (Day)0 5 10 15 20 25 30
CO
2 ev
olu
tion
0
20
40
60
80
100
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
on and Stability 84 (2004) 411e417for N3 days.
-
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Acknowledgements
This work was supported by an InterdisciplinaryResearch Program
grant R01-2002-000-00146-0 fromthe Korea Science and Engineering
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Effects of storage of a mature compost on its potential for
biodegradation of plasticsIntroductionMethodsCompostingAnalysis of
compostPhysicochemical analysisMicrobial analysis
Compost enzyme activityStorage of the compostBiodegradation of
biodegradable plastics in compostBiodegradable
plasticsBiodegradation test
Results and discussionPhysicochemical characteristics of the
compostMicrobial community in the compostActivity of enzymes in the
compostBiodegradation of plastics in the compost
AcknowledgementsReferences
