Polymer Degradation and Stability 85 (2004) 855e863

www.elsevier.com/locate/polydegstab

Soil burial and enzymatic degradation in

solution of aliphatic co-polyesters
Paola Rizzarellia,), Concetto Puglisia, Giorgio Montaudob,)
a
Istituto di Chimica e Tecnologia dei Polimeri, Consiglio Nazionale delle Ricerche; Viale A. Doria 6, 95125 Catania, Italy
b
Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 Catania, Italy
Received 19 January 2004; received in revised form 22 March 2004; accepted 31 March 2004

Abstract

A series of high molar mass aliphatic homo- and co-polyesters was obtained from 1,4-butandiol and methyl esters of succinic,
adipic, sebacic acids, and these materials were characterised by 1H NMR, SEC, DSC, X-ray and viscosity. Good filmability was
achieved for all the polymers. The biodegradability of poly(butylene succinate-co-butylene sebacate), P(BSu-co-BSe), and
poly(butylene succinate-co-butylene adipate), P(BSu-co-BAd), samples, with different composition, was investigated under
controlled soil burial conditions. Film samples were also assayed to enzymatic attack by lipase from Mucor miehei or from
Rhizopus arrhizus. The biodegradation was evaluated as weight loss and the relative normalised weight loss rates were compared.
The influence of crystallinity, molar mass, chemical structure and melting temperature upon biodegradation was studied. The weight
loss of poly(3-hydroxy butyrate), P(HB), of poly(3-hydroxy butyrate-co-3-hydroxy valerate) 76/24, P(HB-co-HV) 76/24, and of two
commercial Bionolle samples, was also investigated under soil burial conditions. The results allow a direct comparison of the soil
burial degradability of polyesters having different structures.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Aliphatic co-polyesters; Soil burial degradation; Enzymatic degradation; Crystallinity

1. Introduction loss under controlled soil burial conditions. Poly(3-

Aliphatic polyesters are among the most interesting
candidates for biodegradable materials [1] in agricul-
tural and sanitary fields as well as in packaging ap-
plications. The first products introduced commercially
were poly(3-hydroxy butyrate), P(HB), and poly(3-
hydroxy butyrate-co-3-hydroxy valerate) co-polymers,
P(HB/HV) [2], and more recently poly(butylene succi-
nate), P(BSu), and poly(butylene succinate-co-butylene
adipate), P(BSu-co-BAd), have appeared on the market
under the trade name of Bionolle [3].
We have performed the synthesis and the structural
characterisation of a set of co-polyesters, obtained from
1,4-butandiol and methyl esters of succinic, adipic, seba-
cic acids (Table 1), and have investigated their weight
) Corresponding authors. Fax: C39-095-221541.
E-mail addresses: prizzarelli@dipchi.unict.it (P. Rizzarelli), gmontaudo@
dipchi.unict.it (G. Montaudo).
hydroxy butyrate), P(HB), poly(3-hydroxy butyrate-
co-3-hydroxy valerate) 76/24, P(HB-co-HV) 76/24, and
two Bionolle commercial samples, were also investigated
for comparison. The lipase-induced hydrolysis of poly
(butylene succinate-co-butylene sebacate), P(BSu-co-
BSe), and poly(butylene succinate-co-butylene adipate),
P(BSu-co-BAd), has been also performed here us-
ing two different lipase strains, which actually showed
different hydrolytic activity towards the same co-
polyester.
The rates of biodegradation of polymers are influ-
enced by several factors including molar mass [6],
chemical structure [7e10], co-polymer composition
[11], stereochemistry [12e14], hydrophilic/hydrophobic
balance [15] and chain mobility [16]. Lately, crystallinity
has been singled out as the factor that affects mostly
enzymatic degradation of polymers [8,10,17,18]. It is
long known [19,20] that the degradation of aliphatic
co-polyesters is faster with respect to the single homo-
polyesters, and that co-polyesters made from moieties

0141-3910/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymdegradstab.2004.03.022
856 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863

Table 1
Properties of the co-polyesters analysed
No. Samplea Method Composition 1
H NMR hsp/cc SEC DSC WAXD
b (dL/g)
Composition Mnd Mw d
D Tm ((C) Tg ((C) Crystallinitye
(%)
1 P(BSu) I 0.56 28 000 49 700 1.78 114 e 34
2 P(BSu) II 1.60 95 400 188 800 1.98 116 ÿ33 32
3 P(BSe) I 0.43 17 800 31 500 1.77 65 e 56
4 P(BSe) II 0.74 33 600 61 400 1.83 65 ÿ55 40
5 P(BAd) I 0.30 10 500 23 500 2.24 63 e e
6 P(BAd) II 1.08 36 150 101 500 2.81 64 ÿ53 38
7 P(BSu-co-BSe) I 90/10 90/10 0.50 32 600 55 800 1.71 106 ÿ42 35
8 P(BSu-co-BSe) II 70/30 70/30 0.95 53 400 104 000 1.95 83 ÿ53 30
9 P(BSu-co-BSe) I 50/50 48/52 0.42 21 900 37 000 1.69 48 ÿ51 26
10 P(BSu-co-BSe) II 50/50 47/53 0.70 41 290 72 150 1.75 49 e 29
11 P(BSu-co-BSe) II 30/70 29/71 0.85 48 300 89 500 1.85 56 ÿ53 33
12 P(BSu-co-BSe) I 10/90 10/90 0.39 24 000 39 500 1.65 63 e e
13 P(BSu-co-BSe) II 10/90 e 1.29 51 700 108 800 2.10 64 ÿ52 35
14 P(BSu-co-BAd) II 80/20 79/21 2.03 85 000 210 000 2.47 88 ÿ32 26
15 P(BSu-co-BAd) II 70/30 69/31 0.91 47 800 107 000 2.24 80 ÿ45 23
16 P(BSu-co-BAd) II 50/50 48/52 1.04 51 800 150 800 2.91 58 ÿ52 11
17 P(BSu-co-BAd) II 30/70 29/71 1.00 56 700 120 000 2.12 39 ÿ52 23
18 P(BSu-co-BAd) II 20/80 17/83 0.77 39 700 79 900 2.01 48 ÿ53 35
19 Bionolle 3001 1.69 52 000 112 300 2.16 93 e 29
20 Bionolle 1001 1.79 59 950 126 100 2.10 114 e 28
21 P(HB)f e e e e 180h e e
22 P(HB-co-HV)g 76/24 e e e e e e e e
a
P(BAd) = poly(butylene adipate); P(BSu) = poly(butylene succinate); P(BSe) = poly(butylene sebacate); P(BSu-co-BSe) = poly(butylene
succinate-co-butylene sebacate); P(BSu-co-BAd) = poly(butylene succinate-co-butylene adipate).
b
Carried out in CDCl3.
c
Determined in CHCl3, 30 (C.
d
Number-average and weight-average molar masses determined using the calibration curve obtained with PS standards in CHCl3.
e
Determined using the Vonk’s method [4].
f
SigmaeAldrich.
g
Marlborough Biopolymers Ltd.
h
Ref. [5].

containing six carbon atoms are more rapidly hydro-
lysed, whereas increasing or decreasing the spacing be-
tween ester groups made the polyesters less susceptible
to enzymatic degradation. Preferential hydrolytic cleav-
age induced by lipases has been observed [21]. In partic-
ular, it was observed that these enzymes prefer cleaving
sebacic ester bonds in P(BSu-co-BSe) co-polymers,
whereas succinic ester bonds appear to be hydrolysed
faster than adipic ester bonds in P(BSu-co-BAd) co-
polyesters [21].
It has long been known that extra-cellular hydrolytic
enzymes, such as lipases, are able to digest films of
aliphatic polyesters [22]. Lipases are water-soluble en-
zymes that act on insoluble substrates and consist of
a large family showing the same overall fold structure [23].
The activity is maximal only when the enzyme is adsorbed
at an oilewater interface. This unique property is known
as interfacial activation. X-ray crystallographic studies
[24,25] have shown that lipases contain buried catalytic
sites and that activation occurs as a result of a conforma-
tional change induced upon binding to a lipid interface.
In aqueous media an a-helical lid covers the active site of
the enzyme and blocks the access to the substrate. When
in contact with a hydrophobic surface, the a-helical lid
rolls back upon the body of the molecule, whereby the
active site becomes fully accessible, substantially enhanc-
ing the hydrophobicity around the active site, and the
enzyme assumes the active conformation. This two-stage
model implies that the closed conformation is stable in an
aqueous medium rendering the active centres inaccessible
to water-soluble substrates [24,25]. However, the primary
products generated by lipase hydrolysis of polyester films
undergo further degradation at longer reaction times. In
fact, the LC/ESI-MS analysis at various times provided
evidence that lipase catalysis is active also in water
solution, a hydrophobic effect induced by the aliphatic
units of these polyesters [21].
Degradation experiments using a single lipase strain
are more specific with respect to soil burial conditions
(where a mixture of enzymes may be present), however,
to measure the weight loss in controlled soil burial
conditions provides a realistic test of the biodegradabil-
ity of polymeric materials, since mineralization rates
cannot be determined with enzyme assays.
 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863
2. Experimental section
2.1. Materials
Dimethyl succinate, dimethyl adipate, dimethyl seba-
cate and titanium (IV) butoxide were purchased from
SigmaeAldrich (MI, Italy) whereas 1,4-butanediol was
purchased from Janssen Chimica. The reagents were
purified by vacuum distillation before use. Showa
Denko (Germany) provided two commercial polyesters,
having the trade name Bionolle 1001 and 3001. Com-
mercial P(3HB) (Mw 800 000) was purchased from
SigmaeAldrich and P(3HB-co-3HV) 76/24 was ob-
tained from Marlborough Biopolymers Ltd.
2.2. Polyester synthesis
Homo- and co-polyesters were synthesised by melt
polymerisation starting from stoichiometric amounts
(method I) of dimethyl esters and 1,4-butandiol, or
using an excess of 10% (method II) of 1,4-butanediol,
in the presence of titanium (IV) butoxide as trans-
esterification catalyst [10]. The polymeric materials
obtained were characterised by viscometry, size-exclu-
sion chromatography (SEC), 1H NMR spectroscopy,
differential scanning calorimetry (DSC) and wide angle
X-ray diffraction (WAXD).
2.3. Viscometry
The reduced viscosity, hsp/c, was measured by an
Ubbelohde viscometer at a concentration of 0.5 g/dL in
chloroform at 30 G 01 (C.
2.4. 1H NMR analysis
The 200 MHz 1H NMR spectra were recorded at
room temperature on a Bruker A-CF 200 spectrometer
using deuterated chloroform as solvent. Spectra were
processed by WINNMR (Bruker), using tetramethylsi-
lane as standard.
2.5. DSC
Differential scanning calorimetry was performed us-
ing 5 mg samples under nitrogen flow with a Mettler
DSC 20 thermal analysis instrument, in the temperature
range 30e160 (C at a heating rate of 10 (C/min. The
melting temperatures (Tm) were taken as the peak
temperature of the melting endotherm.
The determination of the glass transition temperatures
was performed on 25 mg samples under nitrogen flow
with a Mettler DSC 30 thermal analysis instrument, in
the temperature range ÿ100 to 25 (C at a heating rate of
10 (C/min. The glass transition temperatures (Tg) were
calculated as the midpoint of the heat capacity change.
857
2.6. X-ray analysis
The crystalline content of hot-pressed films was de-
termined by wide angle X-ray diffraction (WAXD) using
a Cu Ka radiation (l = 1.5406 Å) with a Bruker D5005
operating at 40 kV and 30 mA. The degree of crystal-
linity was evaluated by Vonk’s method [4].
2.7. SEC analysis and molar mass estimates
The SEC analyses were performed in CHCl3 with
a Waters 6000A apparatus equipped with five ultra-
styragel columns (in the order 105, 104, 103, 500, 100 Å
pore size) connected in series, using a Waters R401
differential refractometer. A polymer solution (100 mL,
5 mg/mL) was injected and eluted at a flow rate of 1 mL/
min. The average molar masses of the polyesters were
calculated by the calibration curve obtained using a set
of primary polystyrene standards using a software pur-
chased from Polymer Labs.
2.8. Films
Co-polyesters films (thickness 0.150e0.250 mm G
0.01 mm) were obtained by hot pressing the polymer
powder for 1 min under a pressure of 200e250 kg/cm2
(Carver C27 Laboratory Press) between two Teflon
plates, containing a spacer, at 25e30 (C above the
melting temperature. The hot-pressed films were stored
at room temperature for at least three weeks before use
in order to reach equilibrium crystallinity.
Films of P(3HB) (2 ! 2 cm; initial weight 15e20 mg;
thickness 0.040e0.070 mm G 0.01 mm) were prepared
by solvent-casting.
2.9. Soil burial degradation test
Tests were carried out at 30 G 0.1 (C, under mois-
ture controlled conditions. Triplicate specimens of each
polyester film were placed in a series of darkened vessels
containing a multi-layer substrate (Scheme 1). The
polymer films (2 ! 2 cm; initial weight 45e78 mg) were
sandwiched between two layers of a mixture of milled
perlite (100 g) and of soil (200 g), moistened with
100 mL of distilled water. The bottom and top layers
were filled with 60 g of perlite moistened with 120 mL of
distilled water. Perlite was added to increase aeration
of the soil and the amount of water retained. A flow of
moistened air was supplied from the bottom of each
vessel every 24 h for 15 min.
The films were removed after 15 days, brushed softly,
washed with distilled water several times and dried
under vacuum in the presence of P2O5 at room temper-
ature, to constant weight. The degree of degradation
was evaluated as the weight loss normalised with respect
to the initial surface area [26]. The surface area exposed
858 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863
PERLITE (wet)
MIX: MIX:
milledmilled
dry perlite
dry + soil + dist. water
60 g perlite +
Polymer film
120 mL dist. water
MIX: milled dry perlite + soil + dist. water
PERLITE (wet)
Net
Grate
AIR Water
Scheme 1. Representation of the soil burial degradation test apparatus.
to burial degradation was considered as the sum of the
two sides of the films (8 cm2).
2.10. Enzymatic degradation in water
Polyester films (2 ! 0.75 cm; initial weight 20e30 mg)
were incubated, in duplicate, at 37 G 0.1 (C, in vials
containing 1.5 mL of 0.1 M potassium phosphate buffer
(pH = 7.4), in the presence of lipase (100 mg/mL). The
lipases used were extracted from the micro-organisms
Mucor miehei or Rhizopus arrhizus. The films were re-
moved from the enzymatic solution after 20 h, washed
with distilled water several times, and dried under
vacuum in the presence of P2O5 at room temperature,
to constant weight (Mettler M3 microbalance; reproduc-
ibility 1 mg).
The activity of the lipase from Mucor miehei was
1.6 U/mg. One unit is defined as the amount of enzyme
that catalyses the release of 1 mmol of oleic acid (triolein
as substrate) per minute at pH 8 and 40 (C.
The activity of the lipase from Rhizopus arrhizus was
1.9 U/g. One unit is defined as the amount of enzyme
that catalyses the release of 1 mmol of butyric acid
(tributyrin as substrate) per minute at pH 8 and 40 (C.
Polyester films were previously immersed in 1.5 mL
of potassium phosphate buffer solution, without enzyme
addition, to estimate the weight loss caused by chemical
hydrolysis. After 20 h, films were washed with distilled
water and dried under vacuum, over P2O5 at room
temperature, to constant weight. The procedure was
repeated until there was no weight loss in two successive
immersions.
The degree of biodegradation was evaluated as weight
loss normalised by initial surface area [26]. The surface
area exposed to enzymatic solution was considered as the
sum of the two sides of the film samples (3 cm2).
3. Results and discussion
The structure and properties of the homo- and co-
polyesters synthesised are listed in Table 1. These
samples contain butylene succinate (BSu), butylene
adipate (BAd) and butylene sebacate (BSe) units. The
structure and properties of commercial biodegradable
polyesters with the trade name of Bionolle 1001 and
200 g mixed soil +
100 g milled perlite + 3001 were also investigated.
100 mL dist. water The co-polyesters composition was inferred from the
relative intensity of the 1H NMR signals of the BSu
(2.628 ppm) and BAd (2.332 ppm) or BSu (2.628 ppm)
and BSe (2.294 ppm) units, and it was found to be
almost equal to the feed ratio used in the synthesis
(Table 1). The commercial samples Bionolle 1001 and
Bionolle 3001 were identified as a P(BSu) homo-polymer
and as a P(BSu-co-BAd) 80/20 co-polymer, respectively
(Table 1).
The biodegradability of the samples listed in Table 1
was investigated under controlled soil burial conditions.
Film samples were also assayed to enzymatic attack in
water. Two lipases were used: from Mucor miehei or
Rhizopus arrhizus. The biodegradation was evaluated by
monitoring the normalised weight loss and the influence
of chemical composition, chemical structure, molar
mass, crystallinity and melting temperature was investi-
gated.
Fig. 1aee shows the results concerning P(BSu-co-
BAd) co-polyesters. In Fig. 1aec the normalised weight
loss is plotted versus the BSu content for the three
biodegradation tests. If the biodegradation degree were
influenced only by the co-polyesters composition, the
dependence of the normalised weight loss versus BSu
content should be linear and this is not the case here
(Fig. 1aec). In Fig. 1d and e the crystallinity index and
the melting temperature, respectively, are plotted versus
BSu content. The crystallinity index shows a minimum
value at 50% whereas the melting temperature at 30%
of butylene succinate units. The random sequence of the
co-monomeric units destroys, as expected, the crystal-
line lattice of the homo-polymers and less ordered,
amorphous, areas are formed.
If the crystallinity index or the melting temperature
were the predominant factor influencing the biodegra-
dation of P(BSu-co-BAd) films, the minimum value of
these parameters should correspond to the maximum
value of the normalised weight loss. The inspection of
the data in Fig. 1a and c reveals that the normalised
weight loss curves in the soil burial test and the enzy-
matic attack by lipase from Mucor miehei, respectively,
present a maximum biodegradation degree at 50 mol%
of BSu units. On the other hand the enzymatic bio-
degradation curve by lipase from Rhizopus arrhizus
shows a maximum at 30% of butylene succinate units
(Fig. 1b). It should be deduced that crystallinity affects
soil burial degradation and enzymatic hydrolysis by
lipase from Mucor miehei while melting temperature
influences biodegradation by lipase from Rhizopus
arrhizus. However, it is evident that the enzymatic de-
gradation curves are not symmetric and that P(BSu)
 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863 859

Normalised weight loss (mg/cm2)

1.6 Soil burial test
(a)

1.2

0.8

0.4

0.0
Normalised weight loss (mg/cm2)

3 Lipase from Rhizopus arrhizus

(b)

5
Normalised weight loss (mg/cm2)

Lipase from Mucor miehei

4 (c)

50

(d)
Crystallinity index ( )

40

30

20

10

120
110
Melting temperature (°C)

100
(e)
90
80
70
60
50
40
30
100 90 80 70 60 50 40 30 20 10 0
BSu content (mol )

Fig. 1. Normalised weight loss versus BSu content in P(BSu-co-BAd) films in: (a) soil burial degradation tests (15 days); (b) enzymatic degradation
assays (20 h) by lipase from Rhizopus arrhizus; (c) enzymatic degradation assays (20 h) by lipase from Mucor miehei; (d) crystallinity index versus BSu
content in P(BSu-co-BAd) films; (e) melting temperature versus BSu content in P(BSu-co-BAd) films.
860 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863
homo-polymer, the sample with the highest melting
temperature, is not susceptible to biodegradation both
enzymatic and microbial. Besides, P(BSu-co-BAd) 20/80
has a higher crystallinity content (35%) than P(BSu-co-
BAd) 80/20 or 70/30, but it is degraded faster. This
result appears to be due both to the lower melting
temperature and to the higher content of BAd units.
P(BSu-co-BAd) 50/50 and P(BAd) have similar melting
temperature, different crystallinity index and compara-
ble normalised weight loss by lipase from Rhizopus
arrhizus. The biodegradation degree concerning the soil
burial tests appears to be less influenced by melting
temperature and crystallinity with respect to enzymatic
degradation.
In Fig. 2aee are shown the data concerning P(BSu-
co-BSe) co-polyesters. Both crystallinity index and melt-
ing temperature plotted, respectively, in Fig. 1d and in
Fig. 1e versus BSu content, show a minimum value at
50% of butylene succinate units. In the meantime, the
normalised weight loss due to the enzymatic attack by
lipase from Rhizopus arrhizus (Fig. 2b) shows a maxi-
mum biodegradation degree at 50 mol% of BSu units.
The enzymatic degradation curve is not symmetric:
P(BSu-co-BSe) 30/70 is degraded faster than P(BSu-co-
BSe) 70/30 and it should be correlated to the different
melting temperature as well as to their chemical com-
position (Fig. 2b). In fact a preferential hydrolytic cleav-
age of sebacic ester bonds in P(BSu-co-BSe) co-polymers
has been recently reported by the analysis of water-
soluble monomers and co-oligomers by liquid chroma-
tography/electrospray ionization mass spectrometry
(LC/ESI-MS) [21].
The enzymatic biodegradation curve by lipase from
Mucor miehei shows a different trend (Fig. 2c): the nor-
malised weight loss increases with BSe content reaching
a maximum at 30% of butylene succinate units. The
P(BSu-co-BSe) 10/90 and P(BSe) homo-polymer, have
a higher crystallinity index and a lower melting temper-
ature than P(BSu-co-BSe) 70/30 but comparable nor-
malised weight loss, indicative of a higher degree of
biodegradability induced by sebacate units in these co-
polyesters. In contrast, the normalised weight loss curve
(Fig. 2a) concerning the soil burial test shows the highest
normalised weight loss at 70% of BSu units. The con-
tribution of different factors produces a less selective
biodegradation and the influence of chemical structure,
crystallinity and melting temperature is less evident.
Furthermore to check the effect of molar mass on the
enzymatic degradation, we have compared the normal-
ised weight loss of P(BSu-co-BSe) 50/50 with different
molar masses (samples 9 and 10, Table 1). The change of
molar mass does not affect sensibly the crystallinity
index, the melting temperature and the biodegradation
level for both lipases used. The independence of nor-
malised weight loss from the molar mass of samples
should indicate that the lipases used are endo-type
enzymes that randomly cleave ester bonds of the poly-
mer chain.
In Fig. 3a the normalised weight losses by lipase from
Mucor miehei are compared for both series of synthetic
co-polyesters. Although P(BAd) and P(BSe) have simi-
lar crystallinity index and comparable melting temper-
atures, P(BAd) is hydrolysed faster. However, the
P(BSu-co-BSe) 30/70 sample shows the highest biodeg-
radation rate and it could be correlated to the higher
susceptibility of butylene sebacate bonds to enzymatic
hydrolysis as well as to the higher hydrophilic/hydro-
phobic ratio, that should induce a faster interfacial
activation of the lipase.
In Fig. 3b the normalised weight losses by lipase from
Rhizopus arrhizus are compared for the P(BSu-co-BAd)
and the P(BSu-co-BSe) co-polyesters. The activity of
lipase from Rhizopus arrhizus is lower than that of lipase
from Mucor miehei and the co-polyesters with a higher
BAd content are hydrolysed faster. P(BAd) is the unique
homo-polymer susceptible to enzymatic attack by lipase
from Rhizopus arrhizus.
In Fig. 4 the normalised weight loss values obtained
in the controlled soil burial degradation tests of the
polyesters investigated are compared, including poly(3-
hydroxy butyrate), P(HB), poly(3-hydroxy butyrate-co-
3-hydroxy valerate) 76/24, P(HB-co-HV) 76/24 and two
Bionolle commercial samples. P(HB) and P(HB-co-HV)
76/24 show a higher biodegradation rate than Bionolle
samples but lower than some P(BSu-co-BSe)s and
P(BSu-co-BAd)s. Among the homo-polyesters, P(BAd)
appears more susceptible to biodegradation. More spe-
cifically, P(BAd) and P(BSe) have similar melting tem-
peratures and comparable crystallinity, but again the
former biodegrades twice as fast as the latter. This
should indicate that adipate bonds are hydrolysed faster
than sebacate bonds. Nevertheless P(BSu-co-BSe) 70/30
shows the highest biodegradation rate, although it has
a higher crystallinity index than P(BSu-co-BAd) 70/30
(Table 1).
4. Conclusions
P(HB) and P(HB-co-HV) 76/24 show excellent soil
burial biodegradation. The low biodegradation of the
two Bionolle samples might represent the result of a
compromise between biodegradation properties and
physical properties (melting point). The co-polyesters
synthesised and analysed here show quite satisfactory
biodegradation levels. Butylene adipate units are more
susceptible to the attack by lipase from Rhizopus ar-
rhizus, whereas butylene sebacate units are more suscep-
tible to the attack by lipase from Mucor miehei. Butylene
succinate units appear to be the less susceptible.
Even if crystallinity has been singled out as the factor
that most affects enzymatic degradation of polymers
 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863 861

2.0

Normalised weight loss (mg/cm2)

Soil burial test
1.6 (a)

1.2

0.8

0.4

0.0
Normalised weight loss (mg/cm2)

1.2
Lipase from Rhizopus arrhizus
1.0
(b)
0.8

0.6

0.4

0.2

0.0
Normalised weight loss (mg/cm2)

7
6 Lipase from Mucor miehei
(c)
5
4
3
2
1
0

46
Crystallinity index ( )

42 (d)
38

34

30

26

120
110
Melting temperature (°C)

100
(e)
90
80
70
60
50
40
100 90 80 70 60 50 40 30 20 10 0
BSu content (mol )

Fig. 2. Normalised weight loss versus BSu content in P(BSu-co-BSe) films in: (a) soil burial degradation tests (15 days); (b) enzymatic degradation
assays (20 h) by lipase from Rhizopus arrhizus; (c) enzymatic degradation assays (20 h) by lipase from Mucor miehei; (d) crystallinity index versus BSu
content in P(BSu-co-BSe) films; (e) melting temperature versus BSu content in P(BSu-co-BSe) films.
862 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863

Lipase from Mucor miehei Lipase from Rhizopus arrhizus

P(BSu)
(a) (b)
P(BSu-co-BSe) 90/10

P(BSu-co-BSe) 70/30

P(BSu-co-BSe) 50/50

P(BSu-co-BSe) 30/70

P(BSu-co-BSe) 10/90

P(BSe)

P(BSu-co-BAd) 80/20

P(BSu-co-BAd) 70/30

P(BSu-co-BAd) 50/50

P(BSu-co-BAd) 20/80

P(BAd)

0 1 2 3 4 5 6 7 0.0 1.0 2.0 3.0

Normalised weight loss (mg/cm2) Normalised weight loss (mg/cm2)

Fig. 3. Normalised weight losses for P(BSu-co-BAd) and P(BSu-co-BSe) films in enzymatic degradation tests (20 h) (a) by lipase from Mucor miehei
and (b) by lipase from Rhizopus arrhizus.

[8,10,17,18], a combined effect of chemical structure, Biodegradation in soil burial tests is less selective
melting temperature and crystallinity appears to influ- than that in solution, mediated by a single enzyme.
ence the activity of the lipase from Mucor miehei and However, mineralization rates cannot be determined
from Rhizopus arrhizus. with enzyme assays.

4.0
P(BSu-co-BSe) 70/30
P(BSu-co-BAd) 50/50

P(BSu-co-BSe) 50/50

3.5
P(BSu-co-BAd) 30/70

P(BSu-co-BSe) 30/70

3.0
Normalised weight loss (mg/cm2)

P(BSu-co-BAd) 20/80
P(BSu-co-BAd) 70/30

P(HB-co-HV) 76/ 24

2.5
P(HB)*

2.0
P(BSu-co-BSe) 10/90
P(BAd)
P(BSu-co-BAd) 80/20

1.5
Bionolle 3001

1.0
Bionolle 1001
P(BSe)
P(BSu)

0.5

0.0

Fig. 4. Normalised weight losses for P(BSu-co-BAd), P(BSu-co-BSe), Bionolle, P(HB) and P(HB-co-HV) 76/24 film samples in soil burial tests. *Films
of P(HB) were prepared by solvent-casting.
 P. Rizzarelli et al. / Polymer Degradation and Stability 85 (2004) 855e863
Acknowledgements
Financial support from the National Council of Re-
search (CNR, Rome) is gratefully acknowledged.
The authors wish to thank Mr. Gaetano Pastorelli
from the Istituto di Chimica e Tecnologia dei Polimeri,
Section of Catania, for setting up the soil burial test
apparatus and for continuous and skilled technical
support. We wish to thank Prof. G. Malandrino, Uni-
versity of Catania, for her help in the use of WAXD
instrument and Prof. G. Spoto, University of Catania, for
his help concerning the computational determination of
crystallinity index.
References
[1] Gross RA, Kalra B. Biodegradable polymers for the environment.
Science 2002;297(5582):803e7.
[2] Holmes PA, Wright LF, Collins SH. (Imperial Chemical In-
dustries PLC, UK). 3-Hydroxy butyrate polymers. E Patent
69497; 1983.
[3] Fujimaki T. Processability and properties of aliphatic polyesters,
‘BIONOLLE’, synthesized by polycondensation reaction. Polym
Degrad Stab 1998;59(1e3):209e14.
[4] Vonk CG. Computerization of Ruland’s X-ray method for
determination of the crystallinity in polymers. J Appl Crystallogr
1973;6:148e52.
[5] Ballistreri A, Montaudo G, Garozzo D, Giuffrida M, Montaudo
SM. Microstructure of bacterial poly(hydroxy butyrate-co-
hydroxy valerate) by fast atom bombardment mass spectrometry
analysis of the partial pyrolysis products. Macromolecules 1991;
24(6):1231e6.
[6] Tokiwa Y, Suzuki T, Takeda K. Two types of lipases in hydrolysis
of polyester. Agric Biol Chem 1988;52(8):1937e43.
[7] Nagata M, Kiyotsukuri T, Minami S, Tsutsumi N, Sakai W. Enzy-
matic degradation of poly(ethylene terephthalate) co-polymers
with aliphatic dicarboxylic acids and/or poly(ethylene glycol). Eur
Polym J 1997;33(10e12):1701e5.
[8] Nagata M, Machida T, Sakai W, Tsutsumi N. Synthesis, charac-
terization, and enzymatic degradation studies on novel network
aliphatic polyesters. Macromolecules 1998;31:6450e4.
[9] Abe H, Doi Y. Structural effects on enzymatic degradabilities for
poly[(R)-3-hydroxybutyric acid] and its co-polymers. Int J Biol
Macromol 1999;25(1e3):185e92.
[10] Montaudo G, Rizzarelli P. Synthesis and enzymatic degradation
of aliphatic copolyesters. Polym Degrad Stab 2000;70(2):305e14.
863
[11] Nikolic MS, Djonlagic J. Synthesis and characterization of
biodegradable poly(butylene succinate-co-butylene adipate)s.
Polym Degrad Stab 2001;74(2):263e70.
[12] Zhang Y, Gross RA, Lenz RW. Stereochemistry of the ring-
opening polymerization of (S )-b-butyrolactone. Macromolecules
1990;23(13):3206e12.
[13] Kemnitzer JE, McCarthy SP, Stephen P, Gross RA. Poly(b-
hydroxybutyrate) stereoisomers: a model study of the effects of
stereochemical and morphological variables on polymer biological
degradability. Macromolecules 1992;25(22):5927e34.
[14] Abe H, Matsubara I, Doi Y, Hori Y, Yamaguchi A. Physical
properties and enzymatic degradability of poly(3-hydroxybuty-
rate) stereoisomers with different stereoregularities. Macromole-
cules 1994;27:6018e25.
[15] Mukai K, Doi Y, Sema Y, Tomita K. Substrate specificities in
hydrolysis of polyhydroxyalkanoates by microbial esterases.
Biotech Lett 1993;15(6):601e4.
[16] Cao A, Okamura T, Ishiguro C, Nakayama K, Inoue Y, Masuda
T. Studies on syntheses and physical characterization of bio-
degradable aliphatic poly(butylene succinate-co-3-caprolactone)s.
Polymer 2002;43(3):671e9.
[17] Mochizuki M, Mukai K, Yamada K, Ichise N, Murase S,
Iwaya Y. Structural effects upon enzymatic hydrolysis of
poly(butylene succinate-co-ethylene succinate)s. Macromolecules
1997;30:7403e7.
[18] Gan Z, Abe H, Doi Y. Crystallization, melting, and enzymatic
degradation of biodegradable poly(butylene succinate-co-14 mol
ethylene succinate) copolyester. Biomacromolecules 2001;2(1):
313e21.
[19] Fields R, Rodriguez F, Finn RK. Microbial degradation of
polyesters: polycaprolactone degraded by P. pullulans. J Appl
Polym Sci 1974;18:3571e9.
[20] Fields R, Rodriguez F. Proceedings of the Third International
Biodegradation Symposium. In: Sharpley JM, Kaplan AM,
editors. Barking, England: Applied Science; 1976. p. 775.
[21] Rizzarelli P, Impallomeni G, Montaudo G. Evidence for selective
hydrolysis of aliphatic copolyesters induced by lipase catalysis.
Biomacromolecules 2004;5:433e44.
[22] Tokiwa Y, Suzuki T. Nature 1977;270:76e8.
[23] van Tilbeurgh H, Egloff MP, Martinez C, Rugani N, Verger R,
Cambillau C. Interfacial activation of the lipaseeprocolipase
complex by mixed micelles revealed by X-ray crystallography.
Nature 1993;362:814e20.
[24] Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M,
Misset O. Bacterial lipases. FEMS Microbiol Rev 1994;15(1):
29e63.
[25] Derewenda U, Brzozowski AM, Lawson DM, Derewenda ZS.
Catalysis ate the interface: the anatomy of a conformational
change in a triglyceride lipase. Biochemistry 1992;31(5):1532e41.
[26] Scandola M, Focarete ML, Frisoni G. Simple kinetic model
for the heterogeneous enzymatic hydrolysis of natural poly(3-
hydroxybutyrate). Macromolecules 1998;31(12):3846e51.