Polymer Degradation and Stability 116 (2015) 36e44

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Polymer Degradation and Stability

journal homepage: www.elsevier.com/locate/polydegstab

Biodegradation behavior of three-layer sheets based on gelatin and

poly (lactic acid) buried under indoor soil conditions
Josefa F. Martucci, Roxana A. Ruseckaite*
Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA), Av. Juan B. Justo 4302, 7600 Mar del Plata, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: The biodegradation of three-layer sheets composed by glycerol-plasticized bovine gelatin film as the
Received 3 December 2014 inner layer and coated with two outer layers of poly(lactic acid) (PLA) was examined under indoor soil
Received in revised form burial conditions during 120 days, using the natural soil microflora as degrading medium. The residual
24 February 2015
degraded samples of the multilayer and individual components were taken regularly from the soil to
Accepted 6 March 2015
determine water uptake, weight loss, variations in thermal properties and morphology. The multilayer
Available online 13 March 2015
sheet exhibited reduced water uptake as compared to the gelatin counterpart, which was associated with
the protection of the middle gelatin layer against water by the hydrophobic PLA layer at both sides. The
Keywords:
Biopolymers
gelatin layer almost disappeared after 25 days, while the pure PLA layer suffered marginal weight loss on
Proteins day 120 in soil. On the contrary, the multilayer sheet was degraded to a much greater extent, leading to
Multilayer an overall mass reduction of approximately 20% at the end of the experiment. The presence of gelatin in
Indoor soil degradation the multilayer seems to enhance water availability around the three-layer sheet soil micro-environment,
inducing gelatin hydrolysis (chemical and enzymatic) which favors the abiotic hydrolysis of PLA, and, in
turn, possibly stimulates the action of more active soil microorganisms against PLA. This was experi-
mentally confirmed by the presence of filamentous (actinomycete and fungi) microbes on the surface of
the multilayer. The glass transition temperature of the degraded multilayer samples slightly increased
while the degree of crystallinity augmented significantly up to 78% on day 120, evidencing the crys-
tallization of the amorphous PLA induced by bio/degradation in soil.
© 2015 Published by Elsevier Ltd.

1. Introduction of multilayer structures since their biodegradability and environ-

Multilayer films have gained relevance in many industrial ap-
plications, especially in the fresh food packaging industry, for
extending shelf-life [1]. Lamination is widely used to improve
polymeric films performance by combining the properties of
several types of layers into one film or sheet [2e5]. However, while
the combination of various layers is required for good food pres-
ervation, the recyclability or biodegradability of the multilayer
packaging could become compromised. The impact of packaging
waste on the environment can be minimized by prudently selecting
materials and reviewing packaging expectations in terms of envi-
ronmental impact [6e8].
Polymers derived from natural products, such as carbohydrates
and proteins, offer the greatest opportunities as components
* Corresponding author. Tel.: þ54 223 481 66 00x249; fax: þ54 223 481 00 46.
E-mail addresses: roxana@fi.mdp.edu.ar, rxane888@gmail.com (R.A. Ruseckaite).
mental compatibility are assured. Protein films are excellent oxygen
and aroma barriers at low relative humidity with potential to
replace synthetic oxygen-barrier components in multilayer for-
mulations. Nevertheless, such property fails in high moisture en-
vironments due to the hydrophilic character of most proteins [4,9].
In an attempt to overcome this issue, numerous studies have
focused on the use of bio-polyesters as protective layers of protein
films, including three-layer films from a soybean protein isolate
(SPI) laminated with poly(lactic acid) (PLA) by means of a solvent-
casting procedure [3,10]; bi-layer PLA-SPI films obtained by casting
[7], polyhydroxyalkanoates (PHAs)- ultrathin zein fibers [5] and bi-
layer Bioflex™-isolate whey protein films (IWP) [8]. Most of these
materials have proven to be biodegraded under the stimulus of an
environmental trigger after use [7,8].
In a previous work [4], the authors described the design and
performance of a biodegradable, high barrier, self-adhesive, three-
layer sheet based on glycerol plasticized bovine gelatin layer acting
as the oxygen barrier component and PLA as the outer layers

http://dx.doi.org/10.1016/j.polymdegradstab.2015.03.005
0141-3910/© 2015 Published by Elsevier Ltd.
 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44
produced by compression molding, as a potential thermoforming
packaging solution. The favorable combination into a multilayer
material enhanced the oxygen barrier of the pure PLA component
(a limiting factor for PLA packaging applications), improved mois-
ture resistance, and enhanced the mechanical and impact proper-
ties of its plasticized gelatin counterpart [4]. To consider such a
multilayer sheet as a viable packaging material, it must retain the
physical properties during the in-use period and ensure that
lamination does not compromise the inherent biodegradability of
the individual components under specific conditions, such as in
indoor soil burial experiments.
The susceptibility of gelatin to the action of enzymes (pro-
teases) present in a variety of micro-organisms has been well
documented [11]. Several studies have centered on the effect of
chemical modifications on the rate and extension of gelatin films
biodegradation [12,13]. Conversely, the role that soil microor-
ganisms play in PLA degradation is unclear and remains contro-
versial [14]. PLA degradation is generally believed to follow a two-
step mechanism involving first an abiotic process, which com-
prises the chemical hydrolysis of PLA, followed by the biotic
assimilation of polymer break-down products by the micro-
organisms, generating carbon dioxide, water and biomass under
aerobic conditions [14,15]. As asserted by Tokiwa and Calabia [16],
PLA-degrading microorganisms are not widely distributed in the
natural environment and so, PLA is less susceptible to microbial
attack in the natural biotic medium than other microbial and
aliphatic polyesters. Other studies have suggested that some mi-
crobial enzymes are capable of directly degrading high molecular
weight PLA [17,18]. Reports on the biodegradability of PLA in soil
where temperature does not exceed 30  C remain scarce. Ho and
Pometto [19] found that about 20% of the PLA film was mineralized
to CO2 after 182 days in soil in a laboratory respirometer at 28  C.
In South Finland, the degradation of PLA in soil under real con-
ditions was followed by the evaluation of the appearance of lactic
acid and lactoyl lactic acid with time [20]. After 20 months, lactic
acid and lactoyl lactic acid appeared as a result of hydrolysis, but
the amount of lactoyl lactic acid decreased with time due to the
biotic attack [20]. The long-term degradation of PLA films and fi-
bers in natural Mediterranean soil (where air and soil temperature
never exceeded 40  C and 21  C, respectively) and in simulated soil
burial experiments was followed for 11 months by Rudnik &
Briassoulis [21]. The slow degradation rate of PLA films exposed to
real field conditions and simulated soil burial, suggested that
materials need much more time and/or higher temperatures to
achieve higher degradation levels, most likely because the
degrading temperature is below PLA glass transition temperature
(Tg) [21]. Karamanlioglu and Robson [14] examined the degrada-
tion of PLA coupons in a range of temperature (25e55  C) in soil
and compost. Findings support the direct role of temperature in
PLA biodegradation, being more relevant at temperatures above
PLA Tg. This study suggests that microbes can directly enhance
degradation of high molecular weight PLA over and above
chemical hydrolysis at elevated temperatures and that microor-
ganisms in compost are more active in this process as compared to
soil.
Based on the considerations outlined above and the potential
industrial applications of the three e layer sheets based on PLA
outer layers and plasticized gelatin inner layer, the main objective
of the present study was to analyze the biodegradation of the
multilayer sheet under indoor soil burial experiments. Biodegra-
dation patterns in characterized soil were evaluated through
weight loss determination and water sensitivity was assessed by
measuring the water uptake at selected exposure times. Thermal
properties, crystallinity, visual aspect, as well as morphology
changes were followed on residual materials.
37
2. Experimental
2.1. Materials
Bovine hide gelatin (Ge) type B was kindly supplied by Rousselot
(Argentina), Bloom 150, isoionic point (Ip) 5.3. Poly (lactic acid)
(CML PLA) was purchased from Hycail Finland Oy (Turku, Finland)
and used as received. Glycerol analytical grade (Gly, 98%) was
supplied by DEM Chemicals (Mar del Plata, Argentina).
2.1.1. Sheets formation
Laminate sheets were prepared through a two-step process
described in the previous work by the authors [4]. In the first step,
the individual layers were separately obtained by thermo-
compression. Additionally, the outer PLA layers and the glycerol
(Gly) plasticized gelatin inner layer, Ge-30Gly, were stacked
together and, subsequently, heat-compressed to the desired
thickness. Before processing, PLA pellets were oven dried under
vacuum at 60  C for 4 h. Then, 4e5 g samples of PLA were placed
between two stainless steel plates coated with a Teflon film and
molded into sheets (30 cm2) at 180  C in a hot press (EMS, Buenos
Aires, Argentina) using an aluminum frame to regulate the target
thickness (0.150 mm). The material was kept between plates at
atmospheric pressure for 2 min until melting and then it was
submitted to the following pressure cycle: 3 MPa for 1 min, 5 MPa
for 1 min and 10 MPa for 3 min. Finally samples were quenched
under pressure (10 MPa) up to room temperature (25 ± 2  C) and
stored in a desiccator. Regarding the plasticized-gelatin layer, it was
obtained under conditions similar to those previously described
[13]. Gelatin powder and glycerol (30 wt. % on gelatin dry basis)
were mixed together by using a kitchen mixer (M.B.Z., San Justo,
Buenos Aires, Argentina) at low speed (150 rpm) for 24 h and at
ambient temperature (25 ± 2  C). The Ge-30Gly blends were
thermo-molded into sheets (30 cm2) at 120  C in the same hot press
above mentioned and under the following press cycle: 5 min at
atmospheric pressure and 10 min at 5 MPa. Subsequently, samples
were quenched up to room temperature (25 ± 2  C) at atmospheric
pressure and kept in a desiccator.
In the second stage, PLA and Ge-30Gly individual layers were
piled together and thermo-compressed with no need to add any
adhesive between layers [4]. An aluminum frame was used to
control de final thickness of the multilayer (0.45 mm). Processing
temperature was set at 100  C, slightly above both gelatin and PLA
glass transition temperatures [4,22]. Compression was achieved by
applying 10 MPa for 10 min, followed by a cooling step up to room
temperature at atmospheric pressure. PLA/Ge-30Gly/PLA sheets
(thickness ¼ 0.465 ± 0.046 mm), were stored in an environmental
chamber under controlled temperature (25 ± 2  C) and relative
humidity (65 ± 2% RH) before testing.
Control PLA and Ge-30Gly sheets were obtained under similar
conditions than those described for the individual components of
the multilayer. For comparison reasons, thickness values were fixed
at the same level of that of the multilayer, being 0.0416 ± 0.028 mm
and 0.428 ± 0.018 mm for PLA and Ge respectively. Control samples
were kept under similar storage conditions than multilayer sheet
(25 ± 2  C and 65 ± 2% RH) before testing.
2.2. Indoor soil burial
Biodegradation was studied by means of indoor soil burial ex-
periments [6,13,23]. This strategy is a good approach to test
biodegradability under real soil environment when biodegradation
cannot be measured and quantified in the way proposed by the
standard respirometric testing methods. The aerobic degradation
of individual layers and multilayer sheets was carried out in a
38 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44
series of plastic boxes (dimensions: 80 cm  15 cm  10 cm)
containing commercial Pinocha type soil (typical Argentinean
Argiudol þ pine litter); and the natural microflora present was
used as the biodegrading medium [6]. Pinocha soil characteristics
are as follows: pH 6, organic matter content (OMC), 7.2%; nitrogen,
0.19%; NO 3 , 18 ppm; P, 10.35 ppm; Ca, 12.35 meq/100 g; 2.1 meq/
100 g; Na, 0.3 meq/100 g; K, 1.5 meq/100 g. Studies were performed
at room temperature during 120 days. Specimens were cut in a
rectangular shape (2 cm  3 cm) and then dried at 105  C in a
convection oven until constant weight to remove the moisture
before testing. Specimens' weight was gravimetrically determined
on an analytical balance (accuracy ± 0.0001) and such determi-
nation was taken as the initial weight (m0). Test specimens were
put into stainless-steel mesh envelops and buried in the condi-
tioned soil 8-cm deep. The moisture content of the soil was
maintained at around 40%, and constancy was determined by
weighing approximately 5 g of moist soil in a labeled aluminum
weighing pan and drying at 103e105  C for 18e24 h. Moisture was
adjusted by irrigating with distilled water on a periodic basis.
Samples were carefully removed from soil at appropriate time in-
tervals, depending on the evaluation of the results, and analyzed
following the experimental protocol. Soil temperature and hu-
midity was periodically assessed.
2.3. Methods of analysis and testing
2.3.1. Thickness
Thickness was measured at five random points along the spec-
imens by using a digital micrometer (0-25 ± 0.01 mm, Vernier,
China).
2.3.2. Determination of water uptake (WU)
The average water uptake (WU) of the materials under study
was gravimetrically measured throughout the experiment. Samples
were carefully recovered from the soil at predetermined exposure
times (t) by pulling the holders out, cleaned with distilled water to
remove soil debris and attached biomass, and superficially dried
with a tissue paper and re-weighed (mh). The percentage of water
uptake was quantified with the following equation:
mh  mt
%WU ¼ *100
m0
where m0 is the initial mass, mt is the remaining mass after expo-
sure time ¼ t after drying and mh is the humid mass. The values
reported are the average of three measurements.
2.3.3. Determination of weight loss (WL)
After WU determination, each sample was washed with distilled
water and gently cleaned with a tissue prior to evaluation, and then
dried under vacuum at room temperature to constant weight.
Samples were kept in a dry desiccator until analysis. Average
weight loss (WL) was estimated from:
mt  m0
%WL ¼ *100
m0
where m0 and mt were the initial and residual mass (after dried) at a
given time t. The main difficulties faced by this method were loss of
sample fragments as a result of the thorough cleaning process
undergone and the increase in sample weight due to leftover soil
debris. Thus, an average value of three measurements was calcu-
lated to obtain the reported results.
2.3.4. Scanning electron microscopy (SEM)
The evolution with exposure time of the cross-section mor-
phologies of the control and multilayer sheets was conducted by
observing the cryo-fractured surfaces with a scanning electron
microscope (SEM, JEOL, model JSM-6460 LV) using 10 kV as oper-
ating voltage. The exposed surfaces of all the specimens were
previously sputter-coated with gold. Collected images of degraded
samples were compared with those recorded for the original un-
treated ones.
2.3.5. Differential scanning calorimetry (DSC)
Thermal analysis was performed using a Perkin Elmer Pyris DSC
6 differential scanning calorimeter. The sample (z5 mg) was
heated at a rate of 10  C min1 from 30  C up to 180  C, then
quenched to room temperature and re-heated again up to 180  C,
under a nitrogen atmosphere. Melting temperature (Tm), enthalpy
(DHm) and relaxation enthalpy (DHr) were taken from the first
heating scan. Glass transition temperature (Tg), crystallization
temperature (Tc), and enthalpy (DHc) were determined from the
second scan.
2.3.6. X-ray diffraction (XRD)
Analysis was conducted in a PANalytical X'Pert Prodiffrac-
tometer equipped with Cu Ka radiation source (l ¼ 1.546 Å),
operating at 45 kV and 30 mA as applied voltage and current,
respectively. Diffraction patterns were recorded between 2 and 30
by using a scanning rate of 1 min1.
2.3.7. Statistical analysis
Analysis of variance (one way ANOVA) and Tukey's HSD (hon-
estly significant difference) test were used to evaluate the statistical
significance of any change in weight and water absorption over
time with the significance threshold set at P value <0.05.
3. Results and discussion
The susceptibility of the three-layer sheet and the control in-
dividual components to aerobic biodegradation under indoor soil
burial was measured by using natural microbial consortium pre-
sent in soil as degrading medium. Such mixed undefined microbial
population (including bacteria, actinomycetes, fungi and protozoa,
among others) may act synergistically during degradation and
(1) reproduce under naturally occurring conditions. This situation was
considered as a realistic approach to the biodegradation process in
actual natural environments such as aerobic landfilling [6,23].
3.1. Water uptake
Given the strong influence that water exerts on soil processes,
including microbial growth, soil water availability (water potential)
was kept constant (around 40%) in order to restrict changes in
moisture regime and composition of the active soil microbial
community [6,23]. Soil microbes secrete a variety of enzymes into
the soil water, and said enzymes then begin polymer breakdown. In
addition, abiotic hydrolysis has been reported to act as the initial
step of the environmental degradation of several biopolymers
polymers including PLA [15,21] and gelatin [11,25].
(2)
The progressive percentage of weight gain due to water uptake
during soil burial (Fig. 1) clearly indicates the different water ab-
sorption capacities of the investigated materials. The control Ge-
30Gly sheet exhibited very rapid water uptake reaching a
maximum of about 429% of its initial weight within the first 3 days
of soil burial owing to the hydrophilic character of gelatin and
glycerol [6,13]. After this period of time, WU decreased abruptly
since water sorption promoted the instability of secondary bonds
 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44 39

appearance, as revealed by visual inspection (Fig. 2). At the

Fig. 1. Water uptake (WU) curves of individual components and three-layer sheet
against exposure time.
and the hydrolysis of primary peptide bonds in the gelatin matrix
causing disintegration and facilitating microbial attack [6].
Conversely, neat PLA behaved more hydrophobic, revealing a
limited WU extent not exceeding 1.8% after 120 days of soil burial.
Similar results have been previously reported for a commercial 50
e mm-thick PLA film under Mediterranean soil conditions [21] and
injection molded amorphous and crystalline PLA under controlled
composting [26].
In the case of the multilayer film, WU was initially more rapid
than in the control PLA, and its curve superimposed that of gelatin
up to day 5, reaching a maximum of 42% after 10 days. At this time,
the multilayer film suffered increased weight gain due to the water
absorption of the hydrophilic inner gelatin layer promoted by the
exposed and unprotected edges of the multilayer film specimen [6].
From this point, there is a reduction in WU due to the weight loss
resulting from the gradual hydrolysis of the gelatin layer and the
migration of glycerol. Similar results were observed for SPI/PLA
multilayer films [3], three-layer gelatin films [6], and PLA-
laminated agar/k-carrageenan/clay bio-nano composite [27]. It is
worth noting that WU reduction compared with Ge control sheet in
the three-layer sheet was due to the protection of the middle
gelatin layer against water by the hydrophobic PLA layer at both
sides. At day 40, up to 11% and kept constant until the end of the
experiment. Interestingly, the percentage of water uptake of the
residual multilayer was significantly higher than that of neat PLA
for the same period of time maybe linked to the presence of gelatin
in the multilayer which is known to promote the enzyme capable of
degrading PLA [17,18].
beginning of the incubation period, i.e., after 3 days, the soil-
degraded gelatin sheet reduced 18% its initial weigh. This was
mostly attributed to the leaching out of low-molar-mass com-
pounds, such as glycerol and, to a lower extent, oligopeptides, as it
was previously observed on gelatineglycerol films, soy proteine-
glycerol polymers, and gelatin multilayer films [6,9,13]. Glycerol
would eventually be absorbed by soil or passed through the cell
membrane, being metabolized by microbes [28]. Visually, the
gelatin sample exhibited a wrinkle aspect, which was ascribed to
the loss of plasticizer (inset a, Fig. 2). During the same time frame,
PLA suffered a decrease in weight loss not statistically marked
(p > 0.05) being of nearly 0.12%, which is in accordance with the
hydrophobic character of as-molded PLA sheets. Conversely, the
multilayer sheet exhibited small but significant mass decrease (ca.
3% of its initial mass), chiefly explained by the diffusion of low-
molar-mass compounds into the environment. Multilayer and
PLA sheets samples changed their color and became more opaque
after 7 days (inset c and f, Fig. 2).
At day 14, when most of the control gelatin layer was degraded
(Fig. 2), the maximum WL achieved (see the slope of each curve in
Fig. 2) indicated the following tendency PLA < multilayer << Ge-
30Gly. These results were in accordance with the water uptake
ability of the studied materials (Fig. 1), since water availability is
necessary for chemical hydrolysis of the studied materials, micro-
bial growth and potential assimilation of the degradation product.
The loss of weight of Ge-30Gly sheet was almost complete (about
90%, p < 0.05) after 2 weeks. The highest degradation rate of control
Ge-30Gly was due to the fact that sheet components (c.a. gelatin
and glycerol) are hydrophilic, thus increasing water activity and
promoting soil-inhabiting microbial communities growth in the
range of 20e25  C. Degradation products of gelatin and glycerol
would eventually be adsorbed by soil or passed through the cell
membrane, being metabolized by microbes [11,12,28]. The clear
visual deterioration of the control gelatin layer over 14 days (inset
bin Fig. 2) was a direct evidence of the susceptibility of such ma-
terial to natural soil environment, as previously accounted for [6].
This might be associated with the abiotic hydrolysis and the variety
of gelatin degrading microorganisms present in soil, as reported in
the literature [6,13,25].
The PLA sheet suffered restricted weight changes over the
experiment duration (about 0.8% at day 120, Fig. 2). This could be
connected to the low soil humidity and temperature (average soil

3.2. Weight loss and physical appearance

Average weight loss of specimens exposed to soil environment

was considered as an indicator of degradation. It is worthy of
mention that the average values representing the weight loss of the
films buried in soil were probably underestimated as soil debris and
occluded biomass are difficult to remove without damaging the
samples [13,23]. Despite these potential errors, weight loss data
were used herein as a qualitative tool to evaluate the effect of soil
environment (biotic and abiotic) on the degrading behavior of the
materials under study. Fig. 2. Weight loss (WL) curves of Ge-30Gly, neat PLA and three-layer sheet against
Weight loss of control and multilayer sheets over the soil burial exposure time. Variations of visual aspect are also included: Ge-30Gly (inset a, b), PLA
period was accompanied by macroscopic changes of their physical (inset c, d, e) and three-layer sheet (inset f, g, h).
40 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44

temperature 20 ± 2  C, below Tg PLA ¼ 58  C) necessary to initiate used. The presence of starch was found to facilitate the biodegra-
the hydrolytic degradation of this polyester before the action of dation of the polylactic component, especially in liquid media. Even
microorganisms commences [14,15] and sample degradation is though more experimental work should be carried out, the findings
slowed down [14,21]. Additionally, Tokiwa and Calabia [16] reported herein demonstrate the beneficial effect of gelatin on PLA
explained that PLA-degrading microorganisms are not widely degradation in soil. Similar to PLA sheet, the multilayer sheet
distributed in the natural environment and thus, PLA is less sus- became increasingly brittle and partially opaque over the incuba-
ceptible to microbial attack in the natural biotic medium than other tion time (See inset c-e, Fig. 2). The multilayer lack of flexibility is an
microbial and aliphatic polyesters. Therefore the small weight loss indirect evidence of the loss of the most flexible component, the
of control PLA observed in the present study could be ascribed to plasticized-gelatin layer. The presence of cracks due to leakage of
the low water potential of soil limiting abiotic hydrolysis and to the degradation products as well as the crystallization induced by
scarcity of PLA-degrading microorganisms in the Pinocha soil used biodegradation could be interpreted as the most important factor
in this study. The small increment in WL observed at later stages inducing changes in the refractive index of the degraded samples of
could be attributed to chemical hydrolysis contribution occurring the multilayer.
preferentially in the amorphous regions of the polymer matrix and
the leaching of oligomers which can be totally assimilated by soil
microorganisms such as fungi or bacteria [15,29,30]. It is also 3.3. XRD and DSC of the residual material
possible that some biotic activity may have contributed to PLA
degradation at later stages of soil burial since the relative compo- Variations in crystallinity and thermal properties of all materials
sition of the soil microbial community can change owing to varia- during soil burial were followed by DSC (Fig. 3). The thermal pa-
tions in the soil temperature affecting bacterial and fungal growth rameters are listed in Table 1. Based on the representative ther-
rates. Despite the low extent of weight loss, PLA exhibited signifi- mograms shown in Fig. 3, it can be easily inferred that all the
cant macroscopic changes such as increased brittleness and opacity materials under study suffered physical ageing during soil burial, as
(inset g and h, Fig 2). Increased opacity is directly tied to light evidenced by the enthalpy relaxation (DHr) observed as a sub-Tg
diffusion through the material. Such diffusion can be altered by
various phenomena occurring all along the degradation process
such as the formation of holes in the bulk of the specimen during
degradation, changes in the refraction index of the sample as a
consequence of products formation by the hydrolytic process
[29,31] or the evolution of the polymer matrix crystallinity. It is
known that during degradation in wet soil, the water sorbed allows
crystallization by either enhancing mobility or inducing the
formation of small chains with the ability to crystallize
[21,24,26,31,32].
Unlike the control PLA sheet, the multilayer sheet appeared to
be degraded to a greater extent, leading to an overall mass reduc-
tion of approximately 18% over 14 days. This result could be asso-
ciated with the preferential degradation of the gelatin inner layer
due to water entrance, resulting from the insufficient protection of
the multilayer edges by PLA (idem WU). Visually, the inner layer
edges of the laminate sheet exhibited irregular profiles that could
result from a non-uniform distribution of microorganisms in con-
tact with the sample (inset c, Fig. 2). The gradual degradation of the
inner gelatin layer was clearly visible until it almost disappeared on
day 25 (inset d, Fig. 2). This is likely to be due to the hydrophobic
chains of PLA preventing hydrophilic enzymes from accessing the
gelatin inner layer contained within the multilayer. Thereafter the
WL rate of the multilayer slowed down and leveled off reaching a
final value of 20% at day 120. The increased susceptibility of the
multilayer sheet to soil environment as compared to control PLA
could be linked to the presence of Ge-30Gly in the multilayer. Some
authors have postulated that PLA degradation rate is sharply
increased by adding 0.1% (w/v) gelatin in the basal medium, which
indicates that gelatin induces the enzyme capable of degrading PLA
[17,18]. Reports on PLA film degradation by Saccharothrix way-
wayandensis [17] and by Kibdelosporangiumaridum [18] substanti-
ated that the degradation of PLA is markedly increased by the
addition of gelatin in the culture medium. Moreover, Tokiwa and
Jarerat [33] accounted for an actinomycete strain able to degrade
high-molar-mass PLA. Since actinomycetes are very active in
degrading gelatin in soil [6], it has been suggested that gelatin could
promote a more active microbial soil composition against PLA.
Gattin et al. [34] observed similar behavior in a co-extruded starch/
PLA polymeric film. The authors reported an improved percentage
of mineralization than the required 60% value for the definition of a Fig. 3. Dynamic thermograms of (a) neat PLA and (b) multilayer sheet at various soil
biodegradable material, irrespective of the degrading medium burial periods. (Heating rate: 10  C/min, nitrogen atmosphere).
 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44 41

Table 1 compounds, i.e., glycerol and degradation products. The crystalli-

Glass transition temperature (Tg), melting temperature (Tm) and enthalpy (DHm), zation area started to decrease and enlarge with time, rendering it
crystallization temperature (Tc) and enthalpy (DHc) of individual and multilayer
films obtained by Differential Scanning Calorimetry at 10  C/min under N2
difficult to determine the associated enthalpy. On the contrary, the
atmosphere. area of the melting peak increased, suggesting that the multilayer
sheet was able to crystallize under soil burial conditions (Table 1).
Tg DHr Tm DHm Tc DHc
( C) (J/g) ( C) (J/g) ( C) (J/g)
Interestingly, a double melting peak appeared from the first week
(Fig. 3) with melting temperatures at 144 and 150  C. The multi-
Ge-30Gly 44 13.9 e e e e
layer sheet became increasing crystalline with time, which could be
PLA 58 11.2 147 1.4 113 1.5 related to the presence of two distinct crystals formed during
7 59 4.8 148 2.3 112 3.1
degradation in soil burial [35].
40 58.5 5.4 149 2.0 114 4.2
120 59 5.3 148 1.9 114 4.9
The XRD analysis performed on samples collected after different
exposure times revealed a clear evolution of the morphology of the
Multilayer 58 7.8 148 3.6 nd nd
studied materials upon soil burial (Fig. 4). The absence of any
7 58 5.9 145 and 150 6.0 nd nd
40 59 4.6 145 and 150 7.2 nd nd diffraction peak confirmed the amorphous nature of the neat PLA
120 60 3.7 144 and 150 8.5 nd nd sheet before burying in soil (Fig. 4a). Such situation remained un-
*nd: not determined. changed up to day 120 when a peak at 2 theta 16.5 visibly emerged
from the amorphous halo corresponding to 18% crystallinity. This
was ascribed to the (110)/(200) reflection of an alpha-crystal. Our
endothermic peak. The sub-Tg endotherm is normally detected observations are consistent with reports in the literature indicating
when a polymeric sample is aged at a temperature well below Tg for that the hydrolytic degradation of amorphous PLA in biotic and
a short period of time. If restoration of mobility occurs in the vi- abiotic media induces crystallization, and that yield similar XRD
cinity of Tg, then this endotherm is superposed on the glass tran- peaks after different exposure times [24,27,35,36]. Particularly, Park
sition. Therefore, the area below this endothermic peak at the Tg
can be used to quantify the extent of the enthalpy relaxation.
Before soil burial, the gelatin sheet exhibited a Tg value of nearly
44  C with an associated relaxation enthalpy of about 13.9 J/g due to
physical ageing. With the glass transition temperature at 44  C, the
physical aging of the gelatin sheet is, therefore, anticipated even at
the soil temperature, i.e., about 20  C. As degradation proceeded, Tg
shifted to values below room temperature and could not be
detected, giving indirect evidence of the occurrence of random
chain scissions through abiotic and biotic hydrolysis [6]. On the
other hand, physical ageing was also evident during neat PLA sheet
degradation, as it can be concluded from the endothermic peak at
Tg (Fig. 3a). The area of this peak was reduced after a week of
storage and the value remained almost constant throughout the
experiment (Table 1). This means that, initially, the rate of physical
ageing was very rapid and decreased abruptly with time. The Tg
value of the control PLA sheet remained almost invariable
throughout the experiment at about 58  C, which suggested that
PLA did not suffer important physical degradation. A small melting
peak can be clearly observed at 147  C in the neat PLA sheet before
exposing to soil, associated with the increased chain mobility due
to the moisture absorbed upon conditioning. However, the material
remains amorphous as revealed by the fact that the cold crystalli-
zation exotherm (DHc) and the melting endotherm (DHm) in the
DSC curves yielded very similar heat content (i.e., DHc ¼ 1.5 J/g and
DHm ¼ 1.4 J/g). Along degradation, the amount of crystallizable
material increased upon heating owing to the reorganization of PLA
structure involving molecular rearrangements, but the material
remained almost amorphous (difference between melting and
crystallization enthalpies near zero, Table 1) until day 120 when
crystallization appeared induced by degradation [24,32,35].
Melting temperature hardly varied with time (i.e., from 147 to
148  C before and after day 120 of soil burial, respectively). This
means that the small increase in crystallinity scarcely affected this
parameter. In line with our results, Gallet et al. [20] reported that
thermal properties of PLA (Tg and Tm) were only affected during the
second year of soil burial in south Finland, where the soil temper-
ature is below the glass transition temperature of PLA, as it is the
case of the present work.
As regards the three-layer sheet, the relaxation enthalpy was
reduced with time while Tg values shifted toward slightly higher
ones (i.e., from 58 to 60  C for 0 and 120 days of soil burial, Fig. 4. X-ray patterns of (a) neat PLA and (b) multilayer sheet at various exposure
respectively) due to the loss of soluble low-molar-mass times to soil burial.
42 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44

and Xanthos [37] observed the same behavior during indoor soil
degradation of PLA thick films.
Surprisingly, the multilayer sheet exhibited considerable initial
crystallinity before soil burial (about 16.8%) (Fig. 4b). It was hy-
pothesized that, during compression molding, glycerol might
migrate from the inner layer toward the outer ones enhancing PLAs
chain mobility, and subsequently, favoring the rearrangement of
the amorphous phase into crystalline forms. As biodegradation
proceeded, the peak at 16.5 increased accompanied by some re-
flections at 2 theta 12 and 22 which were indexed as (100)/(010)/
(110) and (110)/(120)/(210) of stereo-complex crystals, similar to
those observed after degradation of low molecular weight PLA [24].
The degree of crystallinity increased until reaching about 78% over
the 120 day period. This result suggests that crystalline structures
were formed during degradation in soil as a consequence of the
crystallization of degraded polymer chains, as previously accounted
for [24].
3.4. Morphology evolution
Fig. 5 depicts SEM photographs of specimens degraded at
different times. Gelatin films can be rapidly degraded by soil
microflora as previously reported [6,13]. Filamentous microorgan-
isms rapidly colonized Ge-30Gly sheet surface from the beginning
of the experiment. It appears that the biodegradation process was
caused by fungi and actinomycete, being particularly active during
the early stages of the process. The presence of filaments of such
microorganisms on the film surface agrees well with the fact that
fungi and actinomycetes are ubiquitous and extremely effective in
gelatin biodegradation even at different moisture regimes [38] and
extreme temperatures [11]. Filamentous fungi (i.e., Aspergilius)
were found to be active at degrading gelatin even at temperatures
as low as 4  C. As biodegradation proceeded, soil bacteria (i.e.,
genus Bacillus and Pseudomonas), as well as yeast like Aureobasi-
dium sp., also become effective in degrading gelatin [11]. Even after

Fig. 5. Evolution of the morphology of individual components and the three-layer sheet with soil burial time.
 J.F. Martucci, R.A. Ruseckaite / Polymer Degradation and Stability 116 (2015) 36e44
3 days, the surface of the sample was significantly eroded and after
14 days, the sample pieces were too small to be recovered. These
observations may suggest that this material could be bio-
assimilated by mixed soil consortia which may act in a synergetic
way promoting its degradation.
In the case of PLA layer, minor morphological changes were
observed with time, i.e., increased roughness at day 120. No mi-
croorganisms were detected on the specimen surface, at least
during the time of the study, supporting the reported scarcity of
PLA-degrading microorganisms in natural soil [16,39]. Indeed, it
has been widely accepted that PLA degrades via simple chemical
hydrolysis in biotic environment and it is only at the last stages of
such process that oligomeric degradation products become small
enough to be processed by microorganisms and indirectly by ani-
mals such as earthworms [14,21,40]. As far as the multilayer sheet is
concerned, morphological differences appeared on the second
week of soil burial. The surface become rougher and some cracks
were visible. As degradation progressed, the extended morpho-
logical changes intensified and even the presence of microorgan-
isms' filaments was verified. Since no microbial attack was
observed on PLA surface, the colonization of the multilayer sheet
surface by filamentous microbes should be related to the attractive
presence of the gelatin layer, giving indirect support to delamina-
tion upon soil burial, which enhanced moisture uptake and
consequently the biodegradability of multilayer as compared to
neat PLA sheet. This observation allows us to conclude that gelatin
sheet is the most bio-susceptible material and that it plays a pivotal
part in the biodegradation of PLA/Ge/PLA multilayer in soil.
4. Conclusions
The soil burial tests show that the degradation of PLA/Ge/PLA
multilayer in Pinocha type soil environment is slow. The multilayer
sheet reached 20% of weight loss upon 120 days of indoor soil
burial, while control PLA was only affected by a marginal variation
during the same time period. The gelatin inner layer enhances
water availability around the three-layer sheet soil micro-
environment, inducing gelatin hydrolysis (chemical and enzy-
matic), which favors the entrance of water and consequently the
abiotic hydrolysis of PLA. The products of the abiotic PLA hydrolysis
possibly stimulate the action of more active soil microorganisms
against PLA. The low extent attained by control PLA was more likely
due to the natural soil microbial flora in the degrading medium and
to the soil temperature which, in general, is lower than PLA Tg.
Results reported here clearly indicate that PLA and multilayer sheet
need much longer time for complete biodegradation under realistic
indoor soil conditions, where the average temperature never
exceeded 20 ± 2  C and soil humidity content was kept at 40%.
Funding
This research was financed by the Consejo Nacional de Inves-

cnicas (CONICET, grant number PIP 112-
tigaciones Científicas y Te
201101-00637) and the Agencia Nacional de Promocio
n Científica y
Tecnolo
gica (ANPCyT, grant number PICT 2010-1791) of Argentina.
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