Beruflich Dokumente
Kultur Dokumente
POLYMER
TESTING
Polymer Testing 25 (2006) 597–604
www.elsevier.com/locate/polytest
Material Behavior
Abstract
In this work, we compared the biodegradation of poly(e-caprolactone) (PCL), cellulose acetate (CA) and their blends
using an aerobic biodegradation technique known as the Sturm test, and by their carbon dioxide (CO2) production and
susceptibility to attack by a mixture of fungal strains in solid and liquid media in which samples of each blend were the
only source of carbon. The extent of biodegradation was assessed by the loss of mass. The polymers were also
characterized by thermal analysis using differential scanning calorimetry (DSC). The 40PCL/60CA blend showed faster
biodegradation than the other blends because of its higher CO2 production in aerobic medium (Sturm test). PCL was more
susceptible to attack by a mixture of fungi on solid medium than was CA but showed a lower loss of mass (8.4%) than the
latter polymer; the 60PCL/40CA blend showed the greatest loss of mass during the period of evaluation. In contrast, in
liquid medium, PCL showed a greater loss of mass. Blends of PCL with CA reduced the melting temperature (Tm) of PCL
and increased the Tm of CA, indicating immiscibilty of the polymers. The blends were also less crystalline, which favoured
their biodegradation.
r 2006 Published by Elsevier Ltd.
for the production of inexpensive, rapidly degrad- substances are exposed to an inoculum of compost
able plastics that are easily digested by micro- from municipal solid waste, after which aerobic
organisms. composting occurs in an environment where tem-
Cellulose acetate (CA) is one of the most perature, aeration and humidity are closely mon-
important plant-derived polymers because of its itored and controlled [14]. Another method for
wide variety of applications [3]. Although chemi- evaluating the biodegradation of polymers is by
cally simple, cellulose is a structurally complex measuring the loss of mass when exposed to a
polymer, and several enzymes are required for the mixture of selected fungal strains in culture media
complete degradation of this molecule [4]. The with the plastic sample as sole source of carbon. The
chemical modification of cellulose affects its biode- initial phase of biodegradation involves modifica-
gradability, and CA has been the most studied tion of the substrate to yield a product that is itself
derivative. The high degree of substitution (DS) of an intermediate and is subject to further metabo-
cellulose esters decreases their biodegradability. lism.
Thus, for example, reverse-osmosis membranes In general, the number of microbial cells or the
prepared from CA with a DS of 2.5 quickly lose biomass of the species acting on the chemical of
their semi-permeability because of susceptibility to interest increases as degradation proceeds [19]. Pure
microbial attack [5]. polymers are usually not degraded because of their
Blends of two or more polymeric materials or hydrophobic character, which inhibits the enzy-
copolymers that are not covalently bound [6] matic activity of the microorganisms.
represent a good alternative for reducing the final In this work, the biodegradation of PCL, CA and
cost of industrial products. Various combinations of their blends was assessed by CO2 production in the
such blends have been used to produce conventional Sturm test and by the loss of mass of samples
and biodegradable plastics [7,8]. incubated with a mixture of biodegrading fungal
Biodegradation can be defined as the microbial- strains in solid and liquid media with the samples of
catalyzed conversion of a polymeric substrate into plastic as sole source of carbon. The polymers were
biogas under aerobic ( carbon dioxide [CO2]) and also characterized by thermal analysis using differ-
anaerobic (CH4) conditions in a biologically active ential scanning calorimetry (DSC) to determine the
environment. Mineralization should be distin- extent to which crystallinity influenced the biode-
guished from biodestructability, which is reflected gradation.
in a loss of mass, film or fiber disintegration, or a
loss of physical properties [5]. Hydrolysis is the
principal mechanism by which enzymes degrade 2. Experimental
cellulose-based polymers. The first step in depoly-
merization occurs outside of microbial cells through 2.1. Materials
the action of extracellular enzymes. After cleavage,
the resulting small oligomers can be transported 2.1.1. Polymers
into cells for final mineralization [4]. PCL was supplied in pellet form by Union
Biodegradability tests have been developed to Chemical Carbide Ltd. (P-767) (Cubatão, SP,
quantify the ability of microorganisms to degrade Brazil) and had a melting index of 1.970.3 [20], a
these polymers [9,10]. Several approaches can be density of 1145 g/cm3 and a weight-average mole-
used, including direct incubation in an environment cular weight (Mw) of 50,000 g/mol.
containing an undefined biocinesis and testing in CA type 15-4051 was supplied by Rhodia Acetow
highly defined synthetic media with selected cultures Brazil Ltda. (Santo André, SP, Brazil). The average
of microorganisms [11]. DS was 2.4, the degree of polymerization was
Standard methods for investigating the biodegra- 500–600 and the specific viscosity was 40.295.
dation of plastics under laboratory conditions have Low-density polyethylene (LDPE) was supplied by
been published by ASTM International [12–14], the Union Carbide Quı́mica Ltd. (DUCB 6000 NT) and
International Organization for Standardization had been used in extrusion processes. This LDPE
(ISO) [15–17], and the Deutsche Norm/ European was classified as Type I—Class A—5 (ASTM D
Standard [18]; all of these tests simulate natural 1248), with a melting index of 0.370.1 g/10 min [20],
conditions. The Sturm test is an aerobic biodegra- a density of 0.92070.003 g/cm3 (ASTM D 1505)
dation technique based on CO2 production. The test and an Mw of 36,415 g/mol.
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M.R. Calil et al. / Polymer Testing 25 (2006) 597–604 599
Grade cellulose type 1.02331.0500 for thin layer 10 1C min1. Two heating cycles were used for each
chromatography was supplied by Merck S.A. (São film. The PCL was initially heated to 90 1C to
Paulo, SP, Brazil) and had a particle size of eliminate the thermal history of the sample and then
o20 mm. cooled to room temperature before being immedi-
ately reheated to 250 1C. For CA film, isothermal
2.1.2. Microorganisms treatment (150 1C for 15 min) was followed by
The mixture of fungi consisted of the following reheating to 250 1C. The second scan was done at
species: Aspergillus niger (ATCC 9642), Penicillium the same heating rate. All of the DSC experiments
pinophillum (ATCC 9644), Chaetomium globosum were done in triplicate and the thermograms shown
(ATCC 6205), Aerobasidium pullulans (ATCC refer to the second heating. The crystallinity of PCL
15233) and Gliocadium virens (ATCC 9645). All of was determined using a heat of fusion value (DHo)
these microorganisms were obtained from the of ¼ 139.5 J/kg [21] for 100% crystalline PCL.
culture collection of the Fundac- ão Tropical André
Tosello (Campinas, SP, Brazil), except for G. virens, 2.4. Compost inoculum
which was obtained from the Adolfo Lutz Institute
(São Paulo, SP, Brazil). The compost inoculum consisted of three-month-
old, well-aerated compost derived from the organic
2.2. Blend preparation fraction of municipal solid waste and sieved (screen
mesh, o10 mm) to remove large, inert material. The
Blends of PCL with CA (60/40 and 40/60 wt%) compost produced 75 mg of CO2 per gram of
were prepared by casting. Pure PCL and CA were volatile solids during the first 10 days of the test,
represented by 100/0 and 0/100 wt%, respectively. had a pH of 7.4, a C:N ratio of 43:1, and a total dry
The solutions were prepared by dissolving 15% (w/ solids content of 52%.
w) of each polymer in acetone with stirring at
6573 1C for 6 h. The mixtures were then poured 2.5. Biodegradation tests
into culture dishes and the solvent was allowed to
evaporate in an atmosphere saturated with acetone. 2.5.1. Biodegradation by the Sturm test
For the Sturm test, PCL, CA and their blends In this test, PCL, CA and PCL/CA blends (60/40
were ground using a model TE-625 mill (Tecnal and 40/60 wt%), as well as analytical grade cellulose
Equipamentos para Laboratório Ltda., Piracicaba, for thin layer chromatography (particle size
SP, Brazil) to provide particles with a size o10 o20 mm; positive control) and LDPE (negative
mesh. control), were used. To assess the CO2 production
by the composted organic matter, four identical
2.3. Thermal analysis systems were assembled for each polymer analyzed,
with each system consisting of the polymer, a
Thermal analysis was done using a DSC 204 negative control, a positive control and a blank
TASC 414/3A differential scanning calorimeter containing 600 g of dry solids (inoculum only).
(Netzsch-Gerätebau GmbH, Bavaria, Germany) in Triplicate samples of each polymer were placed in
a nitrogen atmosphere, at a heating rate of recipient B (reactor) (Fig. 1) and immersed in the
Ar O2 CO2
Suction
Reactor
Ba(OH)2 Ba(OH)2
100 mL 100 mL
Sample
Compressor
composted organic matter in the follow proportion: interval and the samples were then incubated at
600 g of humus, 100 g of polymer and 50% of 28 1C for 63 days. At the end of this period, the
distilled water. The reactor was maintained at samples were weighed and the loss of mass was
5872 1C [14], and the system was monitored every calculated as the difference between the first and
24 h for 46 days. The CO2 produced by polymer second weighings.
degradation was collected in receptacle C and the
extent of aerobic biodegradation during the test was 2.5.3. Liquid medium
determined by titration to measure the amount of The loss of mass by pre-weighed samples was
CO2. measured after aerobic incubation in liquid medium
inoculated with the mixed spore suspension. Plastic
2.5.2. Biodegradation in culture medium samples (10 mm 10 mm 1 mm) were disinfected
2.5.2.1. Preparation of culture medium. The liquid for 15 min in 70% ethanol, rinsed twice (15 min
culture medium consisted of the following salts (in each) in sterile distilled water, and incubated at
g/l): KH2PO4 0.7, MgSO4 7H2O 0.7, NH4NO3 1.0, 28 1C for 140 days. Periodically, the samples (in
NaCl 0.005, FeSO4 7H2O 0.002, ZnSO4 7H2O triplicate) were retrieved aseptically, washed with
0.002 and MnSO4 H2O 0.001. The pH was adjusted sterile distilled water, dried, weighed and then
to 6.5 by adding 0.001 N NaOH. The solid returned to the flasks containing the culture
medium contained the salts indicated above and medium.
15 g of agar/l.
3. Results and discussion
2.5.2.2. Inoculum preparation. The fungi were
grown separately on an appropriate medium at 3.1. Thermal analysis
28 1C for two weeks. Spore suspensions were
collected by flooding the culture plates with 10 ml Table 1 shows the melting temperature and
of a 0.5% (w/v) solution of Tween 80 followed by crystallinity values for PCL, CA and PCL/CA
careful removal of the spores with a triangular blends, and Fig. 2 shows the DSC thermograms
Drigalsky rod. The spores were transferred to a for the second heating.
sterile, 125 ml glass-stoppered Erlenmeyer flask The equilibrium melting temperature, i.e., the
containing 45 ml of sterile water and 10–15 glass melting temperature (Tm) corresponding to the
beads 5 mm in diameter, followed by vigorous melting point of an infinitely large lamella, was
shaking. The suspensions were filtered through glass 68.3 1C for PCL. PCL is a highly flexible polyester
wool and centrifuged. The supernatant liquid was that can crystallize during rapid cooling, and its
discarded and the residue resuspended in 50 ml of blending with CA reduced this Tm by 9%–62.3 1C in
sterile water. This washing procedure was repeated the 60PCL/40CA blend, and by 8.5%–62.5 1C in the
three times for the spores from each fungal species. 40PCL/60CA blend. In contrast, the presence of
The spore concentration in the final suspension was PCL slightly increased (by 3%) the Tm of CA in the
counted in a Newbauer chamber (erythrocytomer) 60PCL/40CA and 40PCL/60CA blends, indicating
and adjusted to obtain 1072.1 106 spores/ml. that PCL and CA were immiscible, as previously
reported [22], probably because there were too few
2.5.2.3. Mixed fungal spore suspension. A final
spore suspension for the biodegradation assay was
obtained by mixing equal volumes of spores from Table 1
Melting temperatures of PCL, CA, PCL/CA blends, and
each species prepared as described above.
crystallinity of PCL and PCL/CA blends
2.5.2.4. Solid medium. The polymer films PCL/CA Melting temperature Crystallinity (%)
(10 mm 10 mm 1 mm) were weighed, disinfected formulation (1C)
(wt%)
for 15 min in 70% ethanol, rinsed twice (15 min
PCL CA PCL Blend
each) in sterile distilled water, and placed on a
3–6 mm deep layer of solidified medium in sterile 100/0 68.3 — 54.2 54.2
dishes. The entire surface of the medium was 60/40 62.3 228.8 60.0 36.0
inoculated with the suspension of mixed spores. 40/60 62.5 230.3 55.0 22.0
0/100 — 224.1 — —
Three replicates were prepared for each time
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50 LDPE
aromatic ring and, although this group could act
as a proton donor to form hydrogen bonds with 40
proton acceptor polymers, such an interaction is
30
insufficient to ensure miscibility of the polymers
[24]. El-Shafee et al. [25] reported miscible blends 20
mediated by interaction of the amorphous regions
10
of poly(3-hydroxybutyrate) (PHB) with CA buty-
rate (CAB) based on an analysis of the melting 0
point depression of the crystalline polymer in the
blend. Buchanan et al. [26] observed that when the 0 10 20 30 40 50
Tm of CA was plotted versus the DS, a minimum Time (days)
was observed close to a DS of 2.3, and the resulting Fig. 3. Biodegradation of PCL, CA, and PCL/CA blends based
curve allowed extrapolation to a DS of 2.4, on CO2 production.
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disorder between the polymers in this blend, as so did the mass retention, indicating that CA was
shown by DSC. The resulting decrease in crystal- less biodegradable than CA in liquid medium.
linity and the increase in amorphous regions would
in turn facilitate hydrolysis of the polymers and 3.2.3. Solid medium
attack by microorganisms present in the compost Table 2 shows the loss of mass of PCL, CA and
inoculum. After the necessary induction time, which the 60PCL/40CA and 40PCL/60CA blends in solid
corresponded to aging for about 13 days, the
microorganisms acted more efficiently and pro- Table 2
duced more CO2. Loss of mass (%) of LDPE, PCL, CA and PCL/CA blends
incubated with fungi in petri dishes
3.2.2. Liquid medium Samples Loss of mass (%)a
Fig. 4 shows the changes in mass retention for
PCL, CA and their blends in liquid medium LDPE 0.02
inoculated with a mixture of fungi. PCL showed Pure PCL 8.4
60PCL/40CA 25.8
the highest loss of mass (86.5%) and CA, the 40PCL/60CA 20.6
smallest (2.8%), with the 60PCL/40CA and 40/ Pure CA 14.3
PCL/60CA blends having intermediate values
a
(37.5% and 61.2%, respectively). The values are the average of three determinations.
Although CA showed only limited degradation
compared to PCL and the PCL/CA blends, this low
level of degradation was nevetheless evident when
compared with the corresponding control (LDPE)
values. The CA used here had a DS of 2.4, which
indicated a reduced number of free hydroxyls and
an enhanced interaction with water that would PCL CA
inhibit hydrolysis and, consequently, decrease bio-
degradation. Many microorganisms degrade macro-
molecules through the action of hydrolytic enzymes
secreted into the culture medium [28]. The degrada-
tion of PCL and its blends by fungal enzymes seen
here may have been facilitated by the liquid medium
(presence of water). As the amount of CA increased,
20PCL/80CA 40PCL/60CA
100
80
Mass retetion (%)
60PCL/40CA
60
60PCL/40CA
40
100PCL/0CA
60PCL/40CA
20 40PCL/60CA
0PCL/100CA
LDPE
0
0 20 40 60 80 100 120 140 80PCL/20CA 80PCL/20CA
Time (days)
Fig. 4. Biodegradation of PCL, CA, and PCL/CA blends by Fig. 5. Appearance of polymer samples after exposure to fungi in
fungi (liquid medium). petri dishes.
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