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Fungal Degradation of Poly(L-lactide) in Soil and in Compost

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The biodegradability of polymers by microorganisms is generally studied in a real environment that contains a natural mixture of fungi and bacteria. The present research mainly focused on the purely fungal degradation of poly(l-lactide), PLLA, to enclose the part of fungi in a real process of biodegradation and to understand the kinetics of biodegradation. Respirometric tests were realized in soil at 30 °C, and in compost at 30 and 58 °C. Results indicated that temperature is the predominant parameter governing the fungal degradation of PLLA. Moreover, in real compost, the biodegradation kinetics of the PLLA revealed a synergy between bacteria and fungi. The curves of PLLA and cellulose biodegradation were modeled by Hill sigmoïd. Fungal degradation was completed by investigating the physical and the chemical properties of the polymer during the process of degradation using several analytical methods such as matrix assisted laser desorption ionization-time of fly spectroscopy, infrared spectroscopy, size exclusion chromatography, and differential scanning calorimetry. These experiments led to a better understanding of the various stages of fungal degradation of PLLA: hydrolysis as well as mineralization. Furthermore, metabolizing products (by-products) of PLLA was investigated also.
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1 23
Journal of Polymers and the
Environment
formerly: `Journal of Environmental
Polymer Degradation'
ISSN 1566-2543
J Polym Environ
DOI 10.1007/s10924-011-0399-9
Fungal Degradation of Poly(l-lactide) in
Soil and in Compost
Zoubida Saadi, Aurore Rasmont,
Guy Cesar, Hilaire Bewa & Ludovic
Benguigui
Fungal Degradation of Poly(L-lactide) in Soil and in Compost
Zoubida Saadi Aurore Rasmont Guy Cesar
Hilaire Bewa Ludovic Benguigui
ÓSpringer Science+Business Media, LLC 2011
Abstract The biodegradability of polymers by microor-
ganisms is generally studied in a real environment that
contains a natural mixture of fungi and bacteria. The present
research mainly focused on the purely fungal degradation of
poly(L-lactide), PLLA, to enclose the part of fungi in a real
process of biodegradation and to understand the kinetics of
biodegradation. Respirometric tests were realized in soil at
30 °C, and in compost at 30 and 58 °C. Results indicated
that temperature is the predominant parameter governing
the fungal degradation of PLLA. Moreover, in real compost,
the biodegradation kinetics of the PLLA revealed a synergy
between bacteria and fungi. The curves of PLLA and cel-
lulose biodegradation were modeled by Hill sigmoı
¨d.
Fungal degradation was completed by investigating the
physical and the chemical properties of the polymer during
the process of degradation using several analytical methods
such as matrix assisted laser desorption ionization-time of
fly spectroscopy, infrared spectroscopy, size exclusion
chromatography, and differential scanning calorimetry.
These experiments led to a better understanding of the
various stages of fungal degradation of PLLA: hydrolysis as
well as mineralization. Furthermore, metabolizing products
(by-products) of PLLA was investigated also.
Keywords Biodegradation of bio-polymer
Fungi Modified ASTM test MALDI-TOF
Physico-chemical characterization
Introduction
Industrialists and scientists have studied polymer degra-
dation by microorganisms (biodegradation) in order to
obtain a solution for the problems of accumulation of
plastic wastes in the environment. In the case of PLLA,
some studies on this subject were investigated.
Under defined experimental conditions, poly (L-lactide)
is more resistant to microbial attack in the environment
than other microbial and synthetic polymers [1]. Microor-
ganisms degrading PLLA identified in literature are limited
to Actinomycetes Amycolatopsis type [2,4], and a bacte-
rium, Bacillus Brevis (also called brevisbacillus)[5]. The
microorganisms [3,4] such as Amycolatopsis are effective
for the treatment of waste plastics PLLA and evaluation of
degradation of this polymer. The earlier work on the sub-
ject [4] showed that the temperature of the incubation
medium and the content of the culture medium nutrients
(e.g. the percentage of yeast extract) influence the degra-
dation of PLLA by an Actinomycete genus Amycolatopsis.
Another study on fungi degrading PLLA was made in 1996
by Torres et al.[6]. It showed that, among 14 fungi tested,
only two of them: Fusarium Moniliforme and Penicillium
Roqueforti can assimilate DL-lactic acid, and partially sol-
uble racemic oligomers (Mw=1,000 g/mol) derived from
PLLA. They observed that the Moniliforme Species are
Z. Saadi (&)L. Benguigui
UCO2M, Universite
´du Maine, Avenue O. Messiaen,
72085 Le Mans, France
e-mail: zoubidasaadi9@hotmail.com
A. Rasmont
Materia Nova, 1 Rue des Foudriers, 7822 Ghislenghien, Belgium
G. Cesar
SERPBIO, BordaXuri II, Lot 16, Le Stracq Pattarrian,
64240 La Bastide Clairence, France
H. Bewa
ADEME, 20 Avenue du Gre
´sille
´, BP-90 406, 49004 Angers
Cedex 01, France
123
J Polym Environ
DOI 10.1007/s10924-011-0399-9
capable of growing on a film of poly(lactic acid-co-glycolic
acid; Mw=150,000 g/mol) after 2 months of incubation
at 28 °C on a synthetic agar medium. Moreover, Torres
et al. showed that these species have a limited ability to
mineralize the copolymer PLLA/PGA. Jarerat and Tokiwa
[7] studied fungal degradation of PLLA using Tritirachium
album (ATCC 22563) in a liquid culture using basal
medium and PLLA film and no significant film degradation
was observed. However, the addition of 0.1% water-solu-
ble gelatin, about 76% of the PLLA film had been degraded
by the fungus strain after 14 days of cultivation at 30 °C.
Gelatin induces the production of a proteinase leading to
rupture of the PLLA chains. Furthermore, Jarerat and To-
kiwa investigated the degradation activity by the culture
filtrate of the fungus on Different solid substrates such as
PLLA, poly(e-caprolactone), poly(butylene succinate),
poly(b-hydroxybutyrate, silk fibroin, and elastin. The cul-
ture filtrate of T album showed degradation activity against
PLLA, silk fibroin and elastin, but not against poly(b-
hydroxybutyrate), poly(bythylene succinate) and poly(e-
caprolactone). The PLLA-degrading enzyme produced by
this strain was likely to be protease rather than poly(b-
hydroybutyrate)-depolymerase or lipase.
Jarerat et al. [1] confirmed that gelatin increases the
degradation of PLLA in a liquid medium at 30 °C. The
strain used was an Actinomycete Saccharothrix Wayway-
andensis (also called Lentze Waywayandensis) showing a
bioassimilation of L-lactic acid (degradation product).
According to the study of Tomita et al. [8], in the cultivation
at 60 °C in a liquid medium the poly (D-lactic acid; PDLA)
was highly degraded by a soil bacterium, Bacillus Stearo-
thermophilus. For residual PDLA, gel permeation chro-
matograms showed a marked shift of the whole peak to low
molecular weight region, and moreover, degree of crystal-
linity increased, while melting temperature decreased.
Tomita et al. [9] isolated Geobacillus Thermocatenulantus
another soil bacterium degrading PLLA (Mn=47,000 g/mol)
at 60 °C, and which has an optimum growth temperature of
about 60 °C.
The aim of this work was mainly dedicated to fungal
degradation of PLLA in soil and in compost at 30 and
58 °C, respectively, and to real biodegradation (bacteria
and fungi) in compost at 58 °C. Moreover, we investigated
the evolution of physico-chemical properties of PLLA
throughout the degradation with fungi (biotic media) and
without them (abiotic media).
Apart from the research core, the results of this study
suggest several ways to reduce environmental problems
such as to deteriorate a material with specific fungal
strains, to better control the kinetics of plastics biodegra-
dation (mulching films, packaging materials), and to
accelerate a process of biodegradation.
Experimental
Materials and Reagents
Samples, Samples Preparation, and Elemental Analyses
Samples: pure poly(L-lactide; Scheme 1), without any
additives, was obtained from Materia Nova formerly called
NATISS (Nature for Innovative and Sustainable Solutions,
Belgium) Society. The PLLA is a biopolymer, made from
renewable resources. The molecular mass in weight Mw) was
of 175,000 g/mol. The PLLA is a semi-crystalline polymer
with a glass transition temperature of 61 °C, a melting
temperature of 165 °C, and a degree of cristallinity of 32%.
Cellulose material (less than 20 microns) used as posi-
tive control in the biodegradation tests and all chemicals
were purchased from Sigma-Aldrich. All of the reagents
used were analytical grade.
Samples preparation: material in the form of powder
was used to evaluate the percentage of biodegradation
(CO
2
released) in the biodegradation tests as it allows a
uniform mixture of polymer and matrix. To obtain a fine
powder of PLLA, beads of this polymer were frozen into
liquid nitrogen N
2
and then were ground using an ultra-
centrifuge mill. The powder sample was sieved through a
500 microns mesh.
To follow the chemical and physical properties of the
polymer during the biodegradation tests we used the polymer
in form of a film for an easy sampling. A hydraulic press,
Agila, was used to make 0.1 mm thick film samples as so: the
polymer beads were warmed at 120 °C and compressed at
150 bars. The studies were carried out with 1 cm 92cm
pieces of film. The analyses of the thermal properties of
PLLA such as the degree of cristallinity were made on films
rather than on powder in order to take into account the
macromolecular reorganization that can occur in the struc-
ture of polymer during the heat pressing process.
Elemental analyses: the elemental analysis of materials
has been made to determine the percentage of carbon,
hydrogen, oxygen, nitrogen, and metals in PLLA and cel-
lulose materials. The amount of Total Organic Carbon
(C
TOC
) contained in the test material allows us to calculate
the theoretical amount of CO
2
(mCO
2theoretical
; g) that can
be produced by total oxidation of the tested material
(g) using the following equation:
CH C
nCH3
O
O
Scheme 1 Chemical structure
of PLLA
J Polym Environ
123
mCO2theoretical ¼CTOC ð44=12ÞWð1Þ
where W is the weight of tested material sample (g), 44 and
12 are the molar mass of CO
2
and atomic mass of carbon
(g/mol), respectively.
The value of mCO
2theoretical
for the tested material will
be used to calculate its percentage of biodegradation
(%CO
2
) during a biodegradation test.
The elemental analyses were performed in the service of
microanalysis of the CNRS (at Gif sur Yvette, France).
Measurements were made directly on polymer samples
without pre-treatment, by burning. An infrared spectrom-
eter from Perkin–Elmer was connected and used as the
detector. The measurements were performed twice on each
sample, and a third measurement was made if the first and
the second one gave inconsistent results.
As a result of this test, the percentage of carbon in our
PLLA is 50 wt% and in our positive reference (cellulose) is
41 wt%.
Other Reagents
Agricultural soil and mature compost were used for the
biodegradation tests. The soil and compost were respec-
tively sieved on 2 and 5 mm meshes and obvious plant
material, stones, or other inert materials were removed.
The pH of the soil was in the range of 6–8 while that of
compost was between 7 and 9.
All the experiment vessels, mineral media, soil, and
compost were sterilized by autoclaving at 121 °Cata
pressure of 1 bar for 20 min or by decontamination using a
0.5 wt% sodium hypochlorite solution. Tests were prepared
beneath a Holten laminar flow hood. The water used in all
experiments was purified by filtration (MilliQ system,
Millipore, USA).
Note, the compost used for real compost tests (bacteria
and fungi together) did not need to be sterilized as its
natural flora is necessary to carry out a real biodegradation
test.
Microorganisms and Inoculums Preparation
Fungi: the following fungal strains cited in the ISO 846
standard [10] were used in the biodegradation tests:
Aspergillus Niger (DSM 1957), Chaetomium globosum
(DSM 1962), Paecilomyces variotti (DSM 1961), Penicil-
lium pinophilium (DSM 1064) and Trichoderma Viridens
(DSM 1963). These fungal strains were supplied by the
German DSMZ (Deutsche Sammlung von Mikroorganis-
men und Zellkulturen GmbH) in lyophilized form and
distributed separately in sterile glass tanks.
Inoculums preparation: at first, each fungal strain was
rehydrated separately. In a second step, a specific amount
of hydrated fungus (500 lL) was inoculated into a solution
composed of 250 mL of basal liquid medium for fungi and
25 mL of glucose solution to obtain five fungal pre-cul-
tures which were incubated at 30 °C on a rotary shaker
(150 rpm) during 72 h.
The composition of the basal liquid medium for fungi is:
2 g of NaNO
3
; 0.7 of KH
2
PO
4
; 0.3 g of K
2
HPO
4
; 0.5 g of
(MgSO
4
,7H
2
O); 0.5 g of KCl; 0.01 g of (FeSO
4
,7H
2
O);
and 1 L of distilled water. The pH was adjusted to 6–6.5 by
NaOH solution at 0.01 mol/L.
Then, each fungal pre-culture was washed several times
with the basal medium for fungi to remove all source of
carbon and was then diluted with the same solution to get a
defined concentration.
The fungal consortium used to inoculate the soil and
compost (previously sterilized) was prepared by mixing
five similar amounts from each pre-fungal culture. The
final fungal consortium was diluted with the basal medium
for fungi to get a final concentration of 2 910
5
cells/mL
(Beckman Coulter Multisizer Counter).
Description of the Experimental Biodegradation Set-Up
‘Modified ASTM’’ (American Society for Testing and
Materials) test is an adaptation from the ASTM D5988-03
standard [11]. Modifications principally happened in the
type of incubation apparatus, jars instead of desiccators,
and on using a different absorbing solution such as a
hydroxide sodium solution instead of a Barium hydroxide
solution or a potassium hydroxide solution. The purpose of
this test is to determine the degree of aerobic biodegrada-
tion (in presence of oxygen) of organic compounds in
contact with a matrix, soil or compost, as a function of
time.
The percentage of biodegradation of the tested material
(in our case PLLA and cellulose) is determined by com-
paring the amount of CO
2
released during its microbial
degradation with the theoretical amount of CO
2
(mCO
2theoretical
) that could be produced by the tested
material (g) [12], as shown in the equation below:
%CO2¼mCO2test mCO2control
ðÞ100
mCO2theoretical ð2Þ
where mCO
2test
is the amount of CO
2
evolved in each
beaker containing tested material (g), mCO
2control
is the
amount of CO
2
evolved in the blank beaker (g).
For testing the biodegradation of PLLA in the form of
powder, 1-L air-tight jars were used. In soil tests, nine jars
were used: three for soil controls (blank tests); three for the
positive material (cellulose reference), and another three
jars for test material. In compost tests, nine jars were also
used: three for compost controls (blank tests), three for the
positive material (reference sample with cellulose), and
J Polym Environ
123
three jars for test material. In each case, three jars were also
included as technical controls (air control), containing only
an absorbing solution and distilled water.
For fungal degradation in soil and compost, we added
the fungal consortium solution (2 910
5
cells/mL) in the
solid media to bring the moisture content to 80–100% of
the moisture-holding capacity of the soil and compost.
The PLLA biodegradation test in a real environment
(natural flora) at 58 °C was realized in real and mature
compost. Distilled water was added to the solid media to
bring the moisture content to 80–100% of the moisture-
holding capacity of compost.
Each jar test contained three beakers and was prepared
as shown in the Table 1. The beakers #3 that contained
solid media were weighed before the incubation at 30 °C
for a biodegradation test in soil, and at 30 and 58 °C for a
biodegradation test in compost.
The CO
2
produced in each jar reacts with 0.20 N
hydroxide sodium solution (NaOH) to produce Na
2
CO
3
.
The Na
2
CO
3
is precipitated as barium carbonate (BaCO
3
)
by BaCl
2
solution, as shown below.
CO2þ2NaOH !Na2CO3þH2Oð3Þ
Na2CO3þBaCl2!2NaCl þBaCO3ð4Þ
The amount of carbon dioxide produced is determined by
titrating the remaining sodium hydroxide with 0.10 N
hydrochloric acid to thymolphthalein end-point.
NaOH þHCl þthymolphtalein
!NaCl þH2Oþthymolphtalein ð5Þ
The sodium hydroxide traps were removed and titrated
before their capacity was exceeded.
At each removal of the traps, the beakers were
reweighed to check moisture loss and left open to fresh air
before replacing 10 mL of fresh 0.20 N NaOH solution and
resealing the jar. Distilled water could be added periodi-
cally into the medium in order to maintain the initial
weight of the beaker.
Moreover, other jars contained mixture of PLLA film
and compost were prepared to follow the evolution of
the physico-chemical properties of PLLA during a
process of biodegradation or hydrolysis at 58 °C. The
biodegradation tests were performed in a biotic medium
(polymer mixed with compost sterilized and then inoc-
ulated by fungi), and also in an abiotic medium (polymer
mixed with a compost sterilized and not inoculated by
fungi) to investigate the effect of the hydrolysis
degradation.
Chemistry and Physico-Chemical Analyses
Extraction of Small Products (by-Degradation Products)
During the incubation process, some visible film fractions
were periodically isolated from the compost, washed with
purified water, dried for 48 h, and then analyzed by
Infrared Spectroscopy, Size Exclusion Chromatography
(SEC), and Differential Scanning Calorimetry (DSC).
Smaller fragments invisible to the naked eye were collected
by several washes of the compost with chloroform and then
analyzed by Matrix Assisted Laser Desorption Ionization-
Time Of Fly Spectroscopy (MALDI-TOF).
Matrix Assisted Laser Desorption Ionization-Time
of Fly Spectroscopy
MALDI-TOF analyses were carried out on a Bruker Biflex
III equipped with a nitrogen laser (k=337 nm). The
irradiation samples were prepared from THF or CH
2
Cl
2
solutions using dithranol as matrix and ammonium acetate
as dopant.
Infrared Spectroscopy
FTIR analyses were performed on a Perkin Elmer
TM
Instrument equipped with an ATR Golden Gate sampling
accessory. Surfaces were analyzed in the attenuated total
reflection mode (ATR). Spectrum analyses were performed
on Spectrum One software in the 4,000–400 cm
-1
range.
Spectrums are represented in wavelength (cm
-1
) versus
absorbance (% A).
Table 1 Description of beakers and jars used for ASTM test apparatus
Jar for control
(no materials)
Jar for positive material
(cellulose reference)
Jar for test material (PLLA) Jar for technical
control
Beaker #1 10 mL of distilled water Idem Idem Idem
Beaker #2 10 mL of 0.20 N NaOH
absorbing solution
Idem Idem Idem
Beaker #3
(solid media)
25 g of soil (in ASTM test in
soil)
Or, 3 g of compost (in ASTM
test in compost)
50 mg in carbon of cellulose
mixed with 25 g of soil
(ASTM test in soil), or with
3 g of compost (ASTM test
in compost)
50 mg in carbon of PLLA
mixed with 25 g of soil
(ASTM test in soil), or with
3 g of compost (ASTM test
in compost)
Nothing (air control)
J Polym Environ
123
Size Exclusion Chromatography
Molecular weights and polydispersity were determined by
SEC.SEC’s studies were carried out at room temperature
using a HPLC/SEC Waters system consisting of Waters
pumps and a Waters refractive index detector. The Waters
columns were HR column, 5 lm, HT column, 10 lm and
HMW column, 20 lm in particle sizes. Chloroform was
used as the eluent at a flow rate of 1.0 mL/min. Solutions
of polystyrene standards were used for the calibration.
Differential Scanning Calorimetry
The DSC measurements were performed using a Differ-
ential Scanning Calorimetry Q-1000 of TA Instruments.
Sealed aluminum pans were used, and measurements were
done under nitrogen atmosphere using the following
sequence: first heating rate at 10 °C/min up to 190 °C,
cooling rate at 20 °C/min until 0 °C and a second heating
rate at 10 °C/min up to 190 °C. The glass transition tem-
perature (T
g
) and melting temperature (T
m
) were measured
and calculated from the second heating in order to over-
come the thermal history of the sample.
Results and Discussion
Modified ASTM Tests
In Sterilized and Inoculated Soil
A first series of biodegradation tests was performed in an
agricultural soil, sterilized and inoculated with a fungal
consortium, during 220 days at 30 °C. During the bio-
degradation process, the amount of CO
2
released has been
monitored as a function of time for the PLLA and the
reference cellulose (see Fig. 1). For each material, three
replicates were performed and averaged. The amount of
CO
2
released during the biodegradation of cellulose
increases rapidly from day 0 to day 75 and stabilizes finally
at a level of 84%. In the case of PLLA, no release of CO
2
was observed until the 19th day. This lag time corresponds
to a step of hydrolysis of the longs chains of PLLA that is
necessary for later biodegradation. Only a slight amount of
released CO
2
was finally monitored for PLLA, which
explains the low level of biodegradation (9%) reached in
the present test conditions.
In Sterilized and Inoculated Compost
A second series of tests was performed on the same
materials in compost, sterilized and inoculated with the
fungal consortium, at 58 °C for 230 days. During the
incubation, the amount of released CO
2
has been monitored
as a function of time (see Fig. 2). Note, three replicates
were performed and averaged.
The bioassimilation of cellulose and PLLA starts almost
instantly at this temperature. The amount of released CO
2
during cellulose biodegradation increases from day 0 to
day 90 to reach subsequently a plateau corresponding to
90% of biodegradation.
The first phase of bioassimilation observed in the case of
PLLA between days 0 and 6 is related to the biodegrada-
tion of some short PLLA chains available at the beginning
0 50 100 150 200
0
20
40
60
80
100
PLLA
Cellulose
(gCO2/gtheor.CO2)*100
Time (days)
Fig. 1 Biodegradation test of PLLA and cellulose powders by
modified ASTM in sterilized and inoculated soil by fungi at 30 °C.
Biodegradation is expressed as the percentage of carbon dioxide
generated out of the theoretical amount of organic carbon in the
polymer. The solid line shows the degradation curve, which was
calculated with the Hill model
0 50 100 150 200 250
0
20
40
60
80
100
(gCO2/gtheor.CO2)*100
Time (days)
PLLA
Cellulose
Fig. 2 Biodegradation test of PLLA and cellulose powders by
modified ASTM in sterilized and inoculated compost by fungi at
58 °C. Biodegradation is expressed as the percentage of carbon
dioxide generated out of the theoretical amount of organic carbon in
the polymer. The solid line shows the degradation curve, which was
calculated with the Hill model
J Polym Environ
123
of the test. The slight deceleration observed between days 6
and 19 reflects the time required for the hydrolysis of
PLLA chains that precedes the next bioassimilation step.
During it, the amount of CO
2
significantly increases to
reach a plateau corresponding to 90% of biodegradation.
Moreover, the same curve trend has been obtained for the
mineralization of PLLA powder in real compost [13,14].
The high degree of PLLA biodegradation is related to the
temperature of incubation, 58 °C, rather than the use of
compost as matrix. This has been confirmed by another
biodegradation test performed on the same materials in
sterilized and inoculated compost. However, the temperature
of incubation was 30 °C instead of 58 °C, even if 30 °C
doesn’t represent the real temperature of compost. The bio-
degradation curves (see Fig. 3) showed at first a lag time
followed by a very slight bioassimilation corresponding at
5% of biodegradation after 50 days of incubation. First step
is due to hydrolysis phase as explained previously.
The clear effect of temperature on the biodegradability
of PLLA indicates that its structure has to be hydrolysed
before microorganisms can consume it as a nutrient source.
In optimal conditions of temperature and matrix, it is
possible to reach comparable degrees of bioassimilation
between PLLA and cellulose in the presence of fungi.
Comparing the Degradation in Real Compost
and in Inoculated Compost
Respirometric tests in real compost were performed on
PLLA and cellulose to investigate the effect of natural flora
(presence of bacteria and fungi) on their biodegradation
process. The amount of CO
2
released during the biodeg-
radation at 58 °C was monitored as a function of time (see
Fig. 4). The Fig. 4shows that, despite a lag time of
2 weeks, PLLA reaches a very high degree of biodegra-
dation, around 90%, and is degraded almost as well as the
cellulose reference. However, curve trends are different
during the earliest days showing different processes. The
2 weeks of lag time observed in the case of PLLA is
actually the requirement time for hydrolysis reactions
before the bioassimilation by microorganisms.
At 58 °C, PLLA biodegradation is thus more acceler-
ated in real compost than in sterilized and inoculated
compost. As a result, a mixture of fungi and bacteria (real
environment) is more effective (synergistic effect) than a
purely fungal mixture.
Semiempirical Model for Predicting Biodegradation
Profiles of PLLA and Cellulose
In order to better quantify our results on PLLA and cel-
lulose, the biodegradation curves were studied using the
mathematical model of Hill that is suitable to predict bio-
degradation profiles and life times of the materials [15].
All of the curves of biodegradation (amount of CO
2
released as a function of time) were modeled with Hill
model. The obtained fitting curves are shown together with
the experimental data (Figs. 1,2,3,4). The fitting parame-
ters and the calculated half-life times, as well as the
maximal biodegradation rates, are given in the Table 2.
0 20 40 60 80 100 120 140
0
20
40
60
80
100
PLLA
Cellulose
(gCO2/gtheor.CO2)*100
Time (days)
Fig. 3 Biodegradation test of PLLA and cellulose powders by
modified ASTM in sterilized and inoculated compost by fungi at
30 °C. Biodegradation is expressed as the percentage of carbon
dioxide generated out of the theoretical amount of organic carbon in
the polymer. The solid line shows the degradation curve, which was
calculated with the Hill model
0 50 100 150 200
0
20
40
60
80
100
(g CO2 /g theor.CO2)*100
Time (days)
PLLA
Cellulose
Fig. 4 Biodegradation test of PLLA and cellulose powders by
modified ASTM in real compost (bacteria and fungi) at 58 °C.
Biodegradation is expressed as the percentage of carbon dioxide
generated out of the theoretical amount of organic carbon in the
polymer. The solid line shows the degradation curve, which was
calculated with the Hill model
J Polym Environ
123
Where, Y
1, 2
.is the percentage of degradation [%] at
time t in days, a
1, 2
is the percentage of degradation [%] at
infinite time, c
1, 2
is the half-life time in days, R
2
is the
correlation coefficient of the Hill model, d is the lag time,
and b is the curve radius of the sigmoidal function.
In modified ASTM test at 30 °C (both in compost and
soil), the biodegradation half-life times for cellulose (12
and 11 days in soil and compost, respectively) are shorter
than those for the PLLA (30 and 60 days in soil and
compost, respectively) which confirmed that the cellulose
is a fast degrading material.
In modified ASTM test at 58 °C (both in inoculated and
in real compost), the Hill model gives two equations Y
1
and Y
2
because of the lag time (except for the PLLA in real
compost where there is only one equation, Y
1
). The bio-
degradation half-life times of cellulose and PLLA were
compared taking into account only those obtained from the
second equation (Y
2
) as it represents more the curve of
biodegradation.
The biodegradation half-life times, in real compost at
58 °C, for PLLA (39 days) and cellulose (36 days) are
almost comparable; therefore, the PLLA can be considered
among the fast degrading polymers at high temperature of
degradation. The biodegradation half-life time, in inocu-
lated compost at 58 °C, for PLLA (80 days) is longer than
that for cellulose (39 days). Even if the biodegradation of
PLLA is delayed comparing to cellulose its biodegradation
level is still as important as that of cellulose at the end of
the incubation (around 90% as shown previously).
Chemistry and Physico-Chemical Characterization
In order to understand the process of biodegradation and to
identify the products of degradation (by-products), some
Table 2 Hill models (Y1 and Y2) used to predict biodegradation profiles of PLLA and cellulose in the modified ASTM test and fitting parameter
values
Test in inoculated
soil at 30 °C
Test in inoculated
compost at 30 °C
Test in inoculated compost
at 58 °C
Test in real compost
at 58 °C
PLLA material
Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)
a
1
=8.663 a
1
=3.360 a
1
=12.075 a
1
=90.216
b
1
=2.666 b
1
=58.320 b
1
=1.564 b
1
=4.005
c
1
=30.697 c
1
=60.329 c
1
=7.343 c
1
=39.502
R
2
=0.985 R
2
=1.000 R
2
=0.990 R
2
=0.997
0BtB214 0 BtB140 0 BtB13 0 BtB190
Y
2
=d?(a
2
*t^b
2
/(c^ b
2
?t^ b
2
))
a
2
=96.410
b
2
=2.191
c
2
=80.636
d=6.980
R
2
=0.998
13 BtB230
Cellulose material
Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)Y
1
=a
1
*t^ b
1
/(c
1
^b
1
?t^ b
1
)
a
1
=90.232 a
1
=96.881 a
1
=52.595 a
1
=13.101
b
1
=1.452 b
1
=1.374 b
1
=0.907 b
1
=2.605
c
1
=12.151 c
1
=11.284 c
1
=34.512 c
1
=7.101
R
2
=0.989 R
2
=0.992 R
2
=0.997 R
2
=1
0BtB214 0 BtB120 0 BtB15 0 BtB16
Y
2
=d?(a
2
*t^b
2
/(c^ b
2
?t^ b
2
)) Y
2
=d?(a
2
*t^b
2
/(c^ b
2
?t^ b
2
))
a
2
=81.536 a
2
=87.603
b
2
=3.904 b
2
=4.368
c
2
=39.949 c
2
=36.825
d=15.124 d =9.437
R
2
=0.998 R
2
=0.995
15 BtB230 16 BtB120
J Polym Environ
123
complementary analyses were carried out during the bio-
degradation tests in compost at 58 °C.
By-Products Analyzed by MALDI-TOF
The molar weights of the by-products formed after 90 days
of biodegradation of PLLA in inoculated compost at 58 °C
are reported in the Table 3. As MALDI-TOF gives only the
distribution in weights of by-products, their nature was
determined using a theoretical calculus of weights that
allows matching theoretical and experimental data
(Table 3). The theoretical weights were calculated by fix-
ing the number of PLLA unities and by adding the weight
of extremities of chain and the weight of the sodium ion
(Na
?
). The calculated theoretical weights demonstrated
that there are two groups of oligomers as can be shown
below:
Group 1 :theoretical molar weight
¼xMPLLA þMOH þMHþMNaþð6Þ
Group 2 :theoretical molar weight
¼xMPLLA þMOH þ2MNaþð7Þ
The comparison of experimental and theoretical data con-
cludes in the formation of two types of PLLA oligomers:
oligomers with carboxylic acid (COOH) in chain-extremity
(group 1) and others with carboxylate (COO
-
Na
?
)in
chain-extremity (group 2).
The nature of these two groups indicates that the fungal
degradation of PLLA occurs at the chain-ends. Actually,
the microorganism consumes the monomer in the extremity
of chain and consequently involves a process of depoly-
merization of PLLA.
Modification in PLLA Structure After Degradation:
Infrared Analyses
Figure 5shows the FTIR spectra in the attenuated total
reflection mode (ATR) of PLLA at its initial state (t =0)
and after 70 days of degradation in biotic and abiotic
media. These spectra were made in the 450–4,000 cm
-1
range.
Peak positions and assignments for PLLA at its initial
state (unaged) are: m(–CH–) stretch at 2,997 and
2,985 cm
-1
; carbonyle ester at 1,759 cm
-1
,d(–CH–)
deformation at 1,456, 1,383 and 1,368 cm
-1
,m(–C–O–)
stretch at 1,269, 1,186, 1,134, 1,091 and 1,045 cm
-1
, and
stretch m(–C–C–) at 871 cm
-1
.
Spectra comparison before and after degradation
showed a decrease in the band at 1,260 cm
-1
that corre-
sponds to m(–C–O–) stretch and the formation of a new
band at 1,600 cm
-1
that corresponds to carboxylate ions in
the spectrum of the biotic media. The appearance of car-
boxylate ions at the 1,600 cm
-1
position is due to micro-
organisms which consume lactic acid and its oligomers on
the surface and leave carboxylate ions at the chain end.
This result is in accordance with those obtained by
MALDI-TOF analysis. However, the decrease in the band
at 1,260 cm
-1
is due to a hydrolysis of ester linkages.
Molecular Weight and Polydispersity Changes
Table 4shows the changes in the average molecular weight
in number (Mn) and in polydispersity (I
p
) of PLLA in the
biotic (with fungi) and abiotic (sterile) media as a function
of time. In a biotic medium, a process of biodegradation
can occur; in an abiotic one, only chemical hydrolysis can
happen. During the first 38 days of degradation, both Mn
and I
p
decrease in biotic and abiotic medium. After 38 days
Mndecreases more in the biotic media than in the abiotic
media and I
p
increases considerably in the biotic one. The
decrease in I
p
during the first 38 days is due to a cleavage
in polymer chains by chemical hydrolysis of ester bonds
[14,16]. This step of non-random degradation that occurs
in the amorphous region of polymer easily reached induces
a decrease in the molecular weight [17]. The apparent
increase in I
p
after 38 days in the biotic media indicates a
preferential degradation near the chain ends. When the
molecular weight decreases significantly by chemical
hydrolysis, the microorganisms (biotic media) assimilate
the carboxylic end group products and promote the bio-
degradation of PLLA. Thus, the molecular weight varia-
tions measured by SEC are in correlation with the
formation of two chain-ends revealed by MALDI-TOF.
Thermal Behaviour of PLLA During the Biodegradation
Several changes are observed in the thermal properties of
PLLA during the biodegradation process at 58 °C
(Table 5): decreases of T
g
from 60.8 to 43.8 °C and of T
m
from 165 to 142 °C between the beginning of the test and
the day 70.
Table 3 Molar weights of by-products extracted from compost by
chloroform after 90 days of biodegradation of PLLA at 58 °C
m/z =molar weight
Group 1 Group 2
1698 1720
1770 1792
1842 1864
1914 1936
1986 2008
2058 2080
J Polym Environ
123
It appears clearly through our tests that working at a
temperature of degradation close to the T
g
of the polymer
increases the extent of degradation. A previous study [18]
also showed that temperature is a major parameter in PLLA
degradation since an important loss in weight is obtained at
60 °C and no at 40 °Cor50°C. In fact, rubbery state of
PLLA allows a best absorption of water in the polymer
matrix. The soluble oligomers can so be released by
hydrolysis in the medium of degradation and finally be
assimilated by the fungi.
The fact that T
m
decreases can indicate that the deg-
radation occurs not only in the amorphous phase of PLLA
samples but damages also its crystalline regions [9]. In
fact, the occurred damage in the amorphous phase and the
formation of small fragments (which are easily crystalli-
sable) leads to an increase of the degree of cristallinity in
the earliest days of the biodegradation. Then, this degree
of cristallinity starts to decrease which means that the
crystalline regions starts to be damaged [19] because
of the bioassimilation of the small fragments by the
microorganisms.
Conclusion
In the objective to compare purely fungal degradation
and real biodegradation, respirometric tests (‘‘modified’
ASTM) of PLLA were conducted in solid media (soil and
compost) which have been sterilized and then inoculated
by fungi, and in real compost (bacteria and fungi together).
Results showed clearly that the mineralization kinetic for
PLLA is faster in real compost than in inoculated one. It
results from a synergy between bacteria and fungi and
especially from the diversity of enzymes released in
the medium. Nevertheless, the biodegradation process
achieved the same rates at the end of the test in each media.
In sterilized and inoculated soil, at 30°C, the rate of
fungal degradation of PLLA remains very low: 8% after
220 days of incubation. In sterilized and inoculated com-
post, at 58°C, it rises to 90% after 230 days of incubation.
The incubation temperature appears to be the crucial
parameter for degradation. Indeed, at high temperature, the
conformations adopted by the polymer are certainly more
accessible to the enzymes of fungi.
A follow up of the physic-chemical properties of poly-
mers before and during biodegradation has allowed better
understanding of the fungal degradation kinetics of PLLA
4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450,0
cm-1
A
PLLA t = 0
t = 70 days (Abiotic)
t = 70 days (Biotic)
Fig. 5 FTIR spectra of PLLA
before and after 70 days of
degradation in biotic (compost
has been sterilized and
inoculated by fungi) and abiotic
(compost has been sterilized)
media
Table 4 Changes in average molecular weight in number (Mn) and
in polydispersity indice (I
p
) of PLLA during the biodegradation test in
biotic and abiotic media at 58 °C
Biotic media: Mn
(g/mol); (I
p
)
Abiotic media: Mn
(g/mol); (I
p
)
Day 0 116,000; (1.50) 116,000; (1.50)
Day 38 2,100; (1.30) 1,900; (1.45)
Day 70 780; (2.13) 1,600; (1.40)
Table 5 T
g
and T
m
variation during the biodegradation of PLLA in
sterilized and inoculated compost with fungi at 58 °C
T
g
(°C) T
m
(°C)
Day 0 60.8 165
Day 38 54.6 152
Day 70 43.8 142
J Polym Environ
123
and putting in evidence its structural modification. In the
case of PLLA, a hydrolysis of ester bands occurs at the
beginning due to high temperature and humidity, then
the oligomer fragments formed by hydrolysis are bioas-
similated by microorganisms. During these two steps,
hydrolysis and bioassimilation, both molecular weights and
glass transition decrease.
MALDI-TOF has led to locate the site of degradation
and to identify the nature of oligomers formed. For PLLA,
the degradation happens at chain-ends. The produced
oligomers have two kinds of chain-ends: oligomers with
alcohol and carboxylic acid in chain ends, and oligomers
with alcohol and carboxylate in chain-ends.
Acknowledgments The scientific and financial supports received
from the French Environment and Energy Management Agency
(ADEME) and the ‘Region des Pays de la Loire’ are gratefully
acknowledged; Thanks are also extended to Materia Nova Company
formerly called NATISS (Nature for Innovative and Sustainable
Solutions) for their scientific support and to Mr. G. Cesar for his
precious help in biodegradation tests.
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J Polym Environ
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A new automated set-up based on the original Sturm test was developed for determining the biodegradability of proteic mulching films. This method is based on the measurement of released (respiratory metabolism of microorganisms) carbon dioxide to determine film biodegradability. Maintenance of constant aerobic conditions in thermoregulated bioreactors and continuous measurement of released CO2 with an infrared differential gas analyser were the main advantages of this set-up. It was verified with a plasticised proteic film that soil extracts could be used as microbial sources instead of activated sludge that was usually used in laboratory biodegradation tests. To cite this article: S. Lefaux et al., C. R. Chimie 7 (2003).RésuméUn nouveau dispositif de mesure basé sur le principe du test de Sturm a été mis au point et adapté à la quantification de la biodégradation de films protéiques destinés au paillage agricole. Ce montage expérimental permet de déterminer la biodégradabilité potentielle d'un film polymère en mesurant le dioxyde de carbone respiratoire dégagé lors de la dégradation du matériau testé par des micro-organismes. Ce dispositif est équipé d’un analyseur différentiel à infrarouge, qui mesure en continu le CO2 dégagé de six réacteurs thermorégulés. Les expériences réalisées en conditions aérobies sur un film protéique plastifié ont montré que la microflore du sol pouvait être retenue comme inoculum bactérien à la place des boues activées habituellement utilisées dans les tests de laboratoire. Pour citer cet article : S. Lefaux et al., C. R. Chimie 7 (2003).
Article
A thermophile which degrades poly(l-lactic acid) (PLA) film was isolated from 153 soil samples with an enrichment culture technique at 60 °C. Under this temperature condition, the strain grew on PLA and the dissolved total organic carbon (TOC) concentration in the medium changed according to the growth stage, i.e., after the TOC rapidly reached the minimum, it increased gradually and then decreased again thereafter. As for residual PLA, viscosity number decreased rapidly but weight decreased initially slowly. Gel permeation chromatograms of the residual PLA showed a shift of the whole peak to low molecular weight region, which suggested the occurrence of degradation to the inside of the solid sample. The strain has high specificity for PLA, and the culture broth did not show esterase activity. It was identified as a neighboring species to Geobacillus thermocatenulatus, which has an optimum growth temperature of around 60 °C.
Article
The biotic and abiotic degradation of poly (L-lactide) (PLLA) has been studied with pyrolysis gas chromatography mass spectrometry (Py-GC-MS). A mixed culture of compost micro-organisms was used as the biotic medium. Size-exclusion chromatography (SEC), gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) were utilized to monitor the degradation and degradation mechanism. Differences in pH, molecular weight, surface structure, and degradation mechanisms were noted between sample aged in biotic and abiotic medium. Using fractionated Py-GC-MS at 400 and 500°C, acetaldehyde, acrylic acid, lactoyl acrylic acid, two lactide isomers, and cyclic oligomers up to the pentamer were identified as thermal decomposition products of PLA as well as some other not completely identified products. The ratio of meso-lactide to L-lactide was lower in the sample aged in the biotic media than the abiotic media. This is a result of the preference of the micro-organisms for L-form of lactic acid and lactoyl lactic acid rather than the D-form that in turn influences the formation and the amounts of meso and D,L-lactide during the pyrolysis. Based on SEM micrographs, it was shown that degradation in the biotic medium proceeded mainly via a surface erosion mechanism, whereas bulk erosion was the predominant degradation mechanism in the abiotic medium. The SEC and Py-GC-MS data indicate that degradation was faster in the biotic than in the abiotic sample. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 2369–2378, 2000
Article
In order to assess the effects of morphology on the degradation characteristics of high-molecular weight poly(l-lactic acid) (PLA100), specimens of similar sizes were processed by compression moulding and made either amorphous by quenching (PLA100A) or semicrystalline by annealing (PLA100C). PLA100A specimens were allowed to age in iso-osmolar saline and pH 7.4 phosphate buffer at 37 C for periods up to 2 years, whereas PLA100C specimens were studied in the buffer only. Various techniques were used to monitor comparatively the effects of morphology on the mechanism of hydrolytic degradation for these two types of PLA100 specimens: weighing, enzymatic assay, potentiometry, viscoelasticimetry, size-exclusive chromatography, (SEC), X-ray scattering and differential scanning calorimetry. As in the case of non-crystallizable members of the poly(-hydroxy acid) family, degradation was found to proceed more rapidly in the centre than at the surface for both PLA100A and PLA100C specimens. However, the observed multimodal SEC chromatograms have been assigned primarily to differences of degradation rates in amorphous and crystalline domains, regardless of the initial morphology. Indeed, it was found that initially amorphous PLA100A crystallized as degradation proceeded. Furthermore, PLA100A specimens retained mechanical properties for longer than semicrystalline PLA100C specimens, probably because of the sensitivity of the latter to stress and solvent microcracking. When the integrity of the polymer mass was lost, the residual crystalline matter, initially present or formed during degradation, appeared to be very resistant and was still present in a powdered form after 2 years. It is concluded that the morphology is a critical factor for the degradation of PLA100 and that the degradation of bioresorbable devices derived from this polymer should depend very strongly on both the thermal history and the initial crystallinity. The effects of the morphology do not depend significantly on the nature of the ageing medium, provided that the ionic strength is the same.
Article
Out of 25 Amycolatopsis strains, 15 formed clear zones on agar plate emulsified with poly(L-lactide) (PLA), suggesting a large distribution of PLA degraders within this genus. The clear zones were also observed with other polyesters and silk fibroin plates. In liquid cultures of PLA degraders, there were strains with and without ability to assimilate degradation products like L-lactic acid.
Article
Poly(l-lactide) (PLLA) was rapidly (bio)degraded by a mixed culture of compost microorganisms. After 5 weeks in biotic environment, the films had fragmented to fine powder, while the films in corresponding abiotic medium still looked intact. Analysis of the low molecular weight products by GC-MS showed that microorganisms rapidly assimilated lactic acid and lactoyl lactic acid from the films. At the same time, a new degradation product, ethyl ester of lactoyl lactic acid was formed in the biotic environment. This product cannot be formed by abiotic hydrolysis and it was not detected in the abiotic medium. The degradation of the PLLA matrix was monitored by differential scanning calorimetry (DSC), size exclusion chromatography (SEC) and scanning electron microscopy (SEM). A rapid molecular weight decrease and increasing polydispersity was observed in the biotic environment. In the abiotic environment only a slight molecular weight decrease was seen and the polydispersity started decreasing towards 2.0. This indicates different degradation mechanisms, i.e. preferred degradation near the chain ends in the biotic environment and a random hydrolysis of the ester bonds in the abiotic environment. SEM micrographs showed the formation of patterns and cracks on the surface of the films aged in biotic medium, while the surface of the sterile films remained smooth. The SEM micrographs showed a large number of bacteria and mycelium of fungi growing on the surface of the biotically aged films.