Journal of Polymers and the Environment, Vol. 8, No.

4, October 2000 (䉷 2002)

Aerobic Biodegradation of Polymers in Solid-State

Conditions: A Review of Environmental and
Physicochemical Parameter Settings in Laboratory
Simulations

Sophie Grima,1,2,3 Véronique Bellon-Maurel,1 Pierre Feuilloley,1 and Françoise Silvestre2

During the last few years, biodegradable polymers have been developed to replace petrochemical
polymers. Until now, research devoted to these polymers essentially focused on their biodegradability.
There is now a need to bear out their nontoxicity. To verify this, the biodegradation must be carried
out in accelerated laboratory tests which allow the metabolites and residues to be recovered. To
reproduce the natural conditions (compost, field) as closely as possible, degradation experiments
must be run on solid-state substrates. We review studies of aerobic degradation in solid-state
substrates. This article focuses in particular on the environmental, physical, and chemical parameters
(such as substrate nature, moisture, temperature, C/N ratio, and pH) that influence biodegradation
kinetics. This study also aims at finding the solid substrate most adapted to residues and metabolite
recovery. The most significant parameters would appear to be the substrate type, moisture content,
and temperature. Inert substrates such as vermiculite are well suited to residue extraction. This
review also opens the field to new research aimed at optimizing conditions for aerobic solid-state
biodegradation and at recovering the metabolites and residues of this degradation process.

KEY WORDS: Solid-state biodegradation; environmental parameters; inert substrate; moisture; pH control.

INTRODUCTION oped, and on the other hand, their nontoxicity for the
environment must be borne out.
The gradual replacement of petrochemical plastics
The first point has already been addressed by numer-
by biodegradable materials has become a major issue.
Consequently, two key points have to be examined ous researchers. Methods to assess polymer biodegrada-
closely: on the one hand, rapid methods to assess biodeg- tion have been extensively studied [1–3] and have led to
radation of these biodegradable polymers must be devel- several standards (ASTM, ECN, OECD [4–7]).
Thus, our aim is to develop a method to study the
1
degradation mechanisms in detail, and in particular, those
Cemagref, 361 rue JF Breton, BP 5095, 34033 Montpellier Cedex
01, France.
likely to produce toxic agents. The first step is to set up a
2
Laboratoire de Chimie Agro-industrielle UMR 1010 INRA/INPT- protocol to study biodegradation in laboratory conditions.
ENSIACET, Ecole Nationale Supérieure des Ingénieurs en Arts Chim- Biodegradation must be carried out in a medium that, on
iques et Technologiques, 118 route de Narbonne, 31077 Toulouse
one hand, is close to the natural medium (such as soil or
cedex, France.
3
To whom all correspondence should be addressed. E-mail: compost), and on the other hand, allows the researcher
sgrima@ensct.fr to recover the metabolites and residues.

183
1566-2543/00/1000-0183/0 䉷 2002 Plenum Publishing Corporation
184 Grima, Bellon-Maurel, Feuilloley, and Silvestre

In an aqueous medium biodegradability tests are of the definitions found in different standards are
generally easier to set up and more reproducible. More- reviewed by Van der Zee [10].
over, this medium is more conducive to the recovery of In summary, the term biodegradation means frag-
the metabolites produced during biodegradation. How- mentation with loss of mechanical properties or chemical
ever, poor representation of real conditions is a major modifications through the actions of microorganisms
drawback. Erroneous conclusions could easily be drawn [10].
as biodegradation mechanisms differ among substrates First, the distinction between degradation and bio-
[8, 9]. degradation must be made. Degradation is an irreversible
The medium closest to the natural condition is a solid change in the chemical structure of a polymer involving
medium. Biodegradation studies on soils and compost a deleterious change in properties [10].
are relatively well described in the literature. However, The polymers can be degraded by five interacting
recovering residues in such a complex medium is a diffi- mechanisms [1]:
cult task. Therefore, laboratory biodegradation on inert
● photodegradation by natural daylight;
solid media is also considered.
● oxidation by chemical additives;
The first step in this research program is to review
● thermal degradation by heat;
the studies on solid-state biodegradation processes in field
● mechanical degradation by mechanical effects;
and laboratory conditions, and in various media such
and
as compost, soil, or inert material. This review aims at
● biodegradation by microorganisms.
analyzing the environmental parameters that influence
the degradation kinetics in solid-state media, at describing Biodegradation is a degradation catalyzed by micro-
the measurement methods employed to assess biodegrad- organisms, ultimately leading to the formation of carbon
ability in these media, and at studying the potentiality of dioxide, water, and new biomass.
using inert media. It points the way to new research areas The chemical process can be summarized by the
related to the simulation of biodegradation processes in following Eqs. (1) and (2):
inert media, which allows metabolites and residues to be Aerobic conditions (C ⫽ carbon):
easily extracted. Cpolymer ⫹ O2 → CO2 ⫹ H2O ⫹ Cresidue
In this paper are successively presented the external (1)
parameters that influence biodegradation kinetics: the ⫹ Cbiomass ⫹ salts
material concentration in the solid medium, the environ-
Anaerobic conditions:
mental conditions (temperature, pH, moisture, oxygen
availability, composition, and concentration of inorganic Cpolymer → CO2 ⫹ CH4 ⫹ H2O ⫹ Cresidue
nutrients of the solid medium), the microbial population (2)
⫹ Cbiomass ⫹ salts
(concentration, nature, and interactions), the presence or
the absence of other degradable substances, and the condi- Complete (or total) biodegradation (or mineraliza-
tions and properties of the test system (volume and shape tion) occurs when no residue remains, i.e., when the
of the vessels). original product is completely converted into gaseous
The measurement procedures currently used in labo- products and salts.
ratory solid-state fermentation are studied. The specific Recently, the concept of an “environmentally accept-
problem of the easy recovery of residues is also addressed. able biodegradable polymer” has been introduced by
Swift [11]. Consequently, the biodegradation definition
must include not only the degree of biodegradation, but
BIODEGRADATION: DEFINITIONS AND also the impact of the polymer by-products on the envi-
MECHANISMS ronment.
Definitions Biodegradation can occur in two different environ-
ments: aerobic (oxygen available) and anaerobic (no oxy-
The definitions of biodegradation are not clear. They gen present). These two environments are subdivided
depend on the application range of the polymers (biomed- into aquatic and solid environments. Thus, there are four
ical area or natural environment). In this article, we dis- different environments. We will focus on just one of them:
cuss only biodegradation in the natural environment. In solid-state biodegradation with aerobic conditions.
addition, many different definitions have officially been There are three key elements indispensable for bio-
adopted, depending on the background of the defining degradation. If any one of these elements is lacking, the
standard organizations and their particular interests. All entire process will not happen [10, 12]:
Aerobic Biodegradation of Polymers 185

1. Organisms: The basis for any biodegradation ● procaryotes which have no nucleus and include
process is the existence of microorganisms with eubacteria and archeobacteria; and
the appropriate metabolic pathways to synthesize ● eucaryotes whose DNA is contained in a nucleus
enzymes specific for the target polymer to initiate delimited by a double membrane and which
the depolymerization process and to mineralize include protozoa, unicellular algae, and fungi.
the monomers and oligomers formed by this
Fungi and bacteria have evolved during millions of
process.
years and have developed the ability to degrade every
2. Environment: Some factors are indispensable to
kind of organic compound after an adaptation period.
the biodegradation process. These include tem-
perature, moisture, salts, and oxygen. The most
significant element is the moisture factor. Enzymes
3. Substrate: The structure of the polymer influ-
ences the biodegradation process. These struc- Enzymes are biological catalysts that can induce
tural factors include chemical bonds, degree and enormous increases in reaction rates in an environment
type of branching, degree of polymerization, otherwise unfavorable for biochemical reactions. They
degree of hydrophobicity, stereochemistry, are produced by cells (animal, plant, or microbial).
molecular weight distribution, crystallinity, and From a structural point of view, all enzymes are
other aspects of morphology. specialized proteins, i.e., polypeptides with a complex
three-dimensional structure ranging in molecular weight
from 103 to 106 Da.
Different Steps of Biodegradation The enzyme activity is closely related to the confor-
Biodegradation takes place in two stages: the depo- mational structure, which creates certain regions at the
lymerization of the plastic, and the mineralization. surface forming an active site. With the loss of this confor-
The first stage is a chain cleavage. First occurs the mation, the enzyme activity disappears. The interaction
fragmentation. During this phase the contact area between between enzyme and substrate takes place in this active
the polymer and the microorganisms increases. Then, the site and leads to the chemical reaction. The active sites
decomposition of the macromolecules into shorter chains are specific for a given substrate or a series of substrates.
takes place. The enzymes are classified in six groups according
This step normally occurs outside the organism due to their activities: hydrolases, esterases, isomerases,
to the size of the polymer chain and the insoluble nature reductases, lyases, and ligases [13]. The enzymes have
of many polymers. Extracellular enzymes are responsible different mechanisms of catalysis. In polymer biodegra-
for the polymeric chain cleavage. The enzymes involved dation, the mechanisms are typically biological hydrol-
in this step are either endo-enzymes (random cleavage ysis and biological oxidation.
on the internal linkages of the polymer chains) or exo-
enzymes (sequential cleavage on the terminal monomer
Biodegradation Mechanisms
units in the main chain) [10, 12].
The second step corresponds to the mineralization. The main degradation mechanisms are oxidation and
Once sufficiently small-size oligomeric fragments are hydrolysis. They can be initialized by chemical or biologi-
formed, they are transported into cells where they are cal compounds.
bioassimilated by the microorganisms and then mineral-
ized. From this mineralization process are produced gases
Chemical Hydrolysis
(CO2, CH4, N2, H2), water, salts, minerals, and new bio-
mass. The chemical hydrolysis reaction is catalyzed by
acid or basic compounds.
The Different Organisms Involved in Biological For instance, for polyesters, the reaction is [14]
Degradation RCOOR⬘ ⫹ H2O ⇔ RCOOH ⫹ R⬘OH (3)
Microorganisms
The byproduct, RCOOH, is an acid and is able to
The term microorganism covers a heterogeneous accelerate the hydrolysis by autocatalysis. From a macro-
group of living beings which have a microscopic size scopic point of view, this hydrolysis occurs in two steps.
and are, for the most part, unicellular. The microorgan- The first step is a random hydrolysis leading to a decreas-
isms belong to two major groups: ing molar mass. During the second step, the molecular
186 Grima, Bellon-Maurel, Feuilloley, and Silvestre

fragments are solubilized and the matter disappears. This (synthetic polyesters composed by different ␣,␻-aliphatic
degradation occurs preferentially in the amorphous area diols and dicarboxylic acids) in different kinds of soil
of the polymer. (volcanic ashes, gley, red-yellow soil, and sand). They
showed that the biodegradation rate depends on the family
and ratio of the microorganism populations in the soil
Biological Hydrolysis and thus, of the soil type.
Unlike the chemical hydrolysis, the biological Other studies confirm these findings [20, 23]. Differ-
hydrolysis reaction is catalyzed by enzymes. A large num- ences up to 7-fold are noticed in the material weight
ber of different enzymes are involved, depending of the loss measured after burial in different types of soil [23].
type of bond to be hydrolyzed. In general, they are called Scanning electron microscopy (SEM) measurements con-
depolymerases. Glycosidic bonds, peptide bonds, and firm this soil influence. Furthermore, these researchers
ester bonds are affected by this kind of reaction. think that the use of one particular soil for the biodegrada-
The reaction products of an enzymatic hydrolysis tion test is not suitable. A mixture of several soils would
or a chemical hydrolysis are the same. The only difference increase the microbial diversity.
is the catalyst involved in the reaction. However, these results are in contradiction with the
As a result of the enzyme size, this reaction occurs first results obtained by Goheen et al. [24]. Goheen et
on the surface of the polymer. Thus, with an enzymatic al. [24] studied the influence of the soil nature, as the
hydrolysis, the polymer weight decreases and the molar organic matter content, on the biodegradability rate.
mass barely changes, unlike in chemical hydrolysis. Sieved silt, loam, and sand were tested. The test materials
were a blend of cornstarch and polyethylene (PE), the
latter in major quantity. The soil nature did not seem to
SOLID MEDIUM NATURE AND INFLUENCE influence the biodegradability. However, conclusions by
ON THE INOCULUM Goheen et al. [24] are due to the fact that the polymers are
principally polyethylene-based and degrade very slowly.
For solid media, biodegradation can be carried out in These materials are, now, no longer considered to be bio-
three different substrates: soil, compost, and vermiculite. degradable.
To obtain a better reproducibility and to more easily
compare the results, a standard soil can be used. It is
Soil
nevertheless difficult to define a standard soil because its
Generally, for laboratory experiments, natural soils composition is not always the same. Södergard et al. [25]
of different textures are collected. Soil is usually com- and Witt et al. [26] follow the indications of the German
posed of sand (majority), clay, and silt. The organic matter norm DIN 53739D for the soil burial test. Södergard et
content of the soil is less than 5% [15, 16]. The soil al. [25] use the ED 73 soil, but the composition is not
particle size is a major factor. The soil must have small given in the article. Witt et al. [26] use a soil sold by a
enough particles to allow components to be well distrib- German company and made up of 25% clay, 60% peat,
uted, and for water and air to go through easily. However, and 15% bark humus.
soil particles should be large enough to avoid soil packing Standardizing the soil composition would be an
and, consequently, anaerobiosis. Thus, the soil is sieved improvement. But this measure is not simple to imple-
before use in order to keep particle size smaller than 2 ment at a national scale. For the moment, the inoculum
mm. Plant material, stones, and others inert materials are is only defined in terms of origin and pretreatment.
also removed through sieving [17].
The soil, collected in fields or forests, comes from
the upper layer called rhizosphere. There, the soil is influ- Compost
enced by the roots and microorganisms which are numer-
ous and diverse [18]. But, the drawbacks of this layer Compost is an organic soil conditioner obtained by
are temperature, pH, moisture and oxygen supply changes the biodegradation of a mixture principally made up of
due to the diurnal and seasonal cycles. These conditions various vegetable residues, occasionally with other
are much less stable that the ones encountered in the sub- organic material, and having a limited mineral content.
surfacic soils. The composts used for the biodegradation and disin-
In real conditions, the test materials are buried at a tegration tests are mature, i.e., a 4-month-old compost,
depth between 10 and 20 cm [19–21]. Nishioka et al. with a Rottegrad grade of V (temperature between 20⬚
[22] studied the degradation of different kinds of Bionolle and 30⬚C).
Aerobic Biodegradation of Polymers 187

Gu et al. [27] and Tosin et al. [28] studied the posed a synthetic compost made up of the following
influence of compost composition on the biodegradation mixture (in weight percentage): 45% shredded leaves,
rate. According to Gu et al. [27], the differences between 19.8% shredded news and computer paper (1:1 ratio),
the rates of film weight losses are not due to the different 2.5% saw dust, 11.2% meat waste, and 21.5% food waste.
C/N ratios, but to the differences in the physical form This mixture has a C/N ratio of 30–40. In the studies of
and nutritional contents of the compost. The biodegrada- Gu et al. [27] and Yue et al. [32], synthetic composts are
tion rate depends on the compost formulation. Changes described with similar components but with different per-
in the formulation may, indeed, influence mixture poros- centages.
ity and compost pH, which are major biodegradation Yue et al. [32] and Day et al. [33] studied the micro-
factors. bial activity during composting. The compost has a high
Tosin et al. [28] compared the weight losses of sam- composting activity during the first 28 days. Compost
ples buried in mature compost to those placed in fresh samples taken after 5 weeks have significantly reduced
synthetic waste. The materials tested were Mater-Bi activity. After 49 days, the composting process has ceased
ZI01U (starch, polycaprolactone, and natural plasticizers) and the compost can be used as a mature compost.
and paper. At the beginning of the test (⬍7 days), the In the works of Yue et al. [32], the samples are
weight loss of paper in mature compost was significantly submitted to a simulated municipal solid waste (SMSW)
higher than that in synthetic waste. At day 14, the rate at a temperature of 55⬚C, a constant moisture content of
of biodegradation in synthetic waste was slightly higher 54% of the total water holding capacity, and in two differ-
than that in mature compost. A total degradation was ent ways. Group 1 is made up of samples that are continu-
observed in both systems after 25 days. ously exposed to the biomass; measurements are taken
The same situation was observed for Mater-Bi. Sam- every 3 days of exposure. Group 2 is made up of samples
ples were totally degraded after 25 days in fresh synthetic that are exposed to the biomass for only 3 days, and then
waste, but only after 45 days in mature compost. measured, and different samples are successively exposed
These results indicate that the type of compost influ- all along the composting cycle.
ences the biodegradation rate of Mater-Bi. Nutrient avail- In group 2, the weight losses increase between days
ability, which is a crucial condition for degradation, seems 10 and 15. The number of microorganisms initially
to be higher in fresh synthetic waste. Furthermore, nitro- increases, remains constant during a stationary phase,
gen supply is better and more evenly distributed. On and decreases during a decline phase where the nutrients
the other hand, the synthetic waste cannot be used for are consumed.
respirometric measurements. The substrate releases too The weight losses of group 1 are compared with the
much CO2 and thus masks the amount of CO2 released cumulative weight losses from the individual subgroups
by the test material. of group 2. Group 1 samples, which are continuously
Kim et al. [29] confirm this last remark. They sug- exposed, exhibit an important increase in weight losses.
gest the use of a predigested compost to avoid an exces- The attachment of the microorganisms on the material
sive background noise. The compost is thus incubated at surface plays a major role, especially at the end of the
50⬚C during 45 days. test when the microbial population decreases. It is prefera-
The European Committee for Normalisation (ECN) ble to have a continuous process to authorize a longer
standard draft [30] recommends certain mature compost attachment time at the material surface. Moreover, the
conditions in order to standardize the tests: microbial activity does not remain constant during the
composting; there are increasing and decreasing phases.
● compost made up of municipal solid organic
Thus, on the one hand the biodegradation changes
waste;
dependently on the compost nature, and on the other
● compost between 2 and 4 months old;
hand, for a given composition, it is not constant but linked
● compost pH value between 7.0 and 9.0;
up with the microbial activity.
● total dry solid content between 50% and 55% of
The commercial composts, i.e., prepared at an indus-
the wet solid content;
trial scale, are generally made up of vegetable waste
● volatile solids more than 30% of the dry solids;
[33–35]. These composts are regularly turned over in
and
order to homogenize the decomposing material and to
● a specific average voluminal mass (400 kg/m3).
avoid anaerobiosis.
The reproducibility is improved when synthetic These facilities have various sizes. For instance, 2
compost is used (International Organization for Standard- m high, 3 m wide, and 30 m long [34], or 80 m long and
ization [ISO] standard draft [31]). Kim et al. [29] pro- 2 m2 cross section [33].
188 Grima, Bellon-Maurel, Feuilloley, and Silvestre

To verify that the composting process is correctly reproducibility. The priming effect causes a over-
occurring, pH, temperature, moisture, and concentrations estimation of the biodegradability: glucose and
of O2 and CO2 are monitored. After 1 week, the pH glucose polymers contained in the compost can
increases from the initial value of 5 up to 8. After the stimulate the mineralization of organic matrix
thermophilic step, it slowly decreases down to a plateau [38].
at 6. The temperature rapidly increases from 20 to 60⬚C,
However, vermiculite’s drawback is due to its ionic
then decreases gradually to 20⬚C. The temperature and
nature. Its surface is rich with negative charges that can
pH curves are very typical for natural composting [35].
interact with positively charged degradation products
These temperature variations are parallel to the O2 and
through ionic bonds. Extraction with high ionic strength
CO2 evolutions [33].
solutions could reduce this potential problem.
On the other hand, moisture content decreases.
To conclude, most articles dealing with solid-state
biodegradation describe compost experiments. Experi-
Vermiculite [9, 36–39] ments on soil are generally carried out in the field. The
problem with soil perhaps lies in the correct inoculation
Vermiculite is a clay mineral (Mg,Fe,Al)3(Al,Si)4- and the difficult survival of exogenous microorganisms.
O10(OH)2, 4H2O, used in flakes, i.e., in an expanded Vermiculite offers an appealing alternative and inocula-
shape. This medium is known to be particularly suitable tion by compost inoculum has proven to be successful.
as a microbial carrier, allowing survival and full activity No article was found about vermiculite inoculation by
of microbes [40]. It can be employed to study material soil extract, and this should be studied.
degradation and metabolite formation in solid-state fer-
mentation (SSF). Degli-Innocenti’s team studied the
replacement of mature compost by an activated vermicu- BIODEGRADABILITY EVALUATION
lite layer in solid-state respirometric tests [9, 36–39]. Current Measurements
Before use, vermiculite must be inoculated by a
suitable microbial population, through a prefermentation Biodegradability assessment methods are similar in
process. Vermiculite is first washed several times, dried soil or compost media. They mainly involve weight loss
in an oven, and ground with a mill endowed with an measurements (disintegration tests), consumed O2
8-mm sieve. Then, it is blended with mineral salts and (BOD), dissolved organic carbon (DOC), or exhausted
organic nutrients and inoculated with a compost extract. CO2 quantification (biodegradability tests). The follow-
Microorganisms are allowed to grow by incubation at ing additional analyses can be carried out: infrared (IR),
50⬚C during 3 or 4 days. The inoculum does not depend ultraviolet (UV)-visible, and nuclear magnetic resonance
on the substrate, which is inert, but on the compost. (NMR) spectrometry, gel permeation chromatography (to
Biodegradation rates of Mater-Bi and cellulose obtained determine molecular mass distribution), mechanical prop-
on this medium are practically identical to that measured erties, and surface analysis by electron microscopy.
in mature compost. The biodegradation rate of the cellu- In biodegradation tests, the biodegradation value can
lose is even higher in the vermiculite than in compost [38]. vary according to the measurement method [41]. For
Vermiculite shows the following advantages: instance, DOC measurement cannot determine the differ-
ence between carbon eliminated by biodegradation and
● Because it does not contain substantially available carbon eliminated by other processes such as adsorption
organic carbon, it produces less CO2 than the on the biomass or stripping into the air. Thus, it is
mature compost. Thus, the background noise is important to refer to the test method. CO2 measurements
lower in biodegradation tests [38]. are generally preferred. They are more precise and sim-
● Vermiculite allows us to easily collect the degra- pler to implement than other test methods.
dation products and polymer residues at the end When radiolabeled compounds are used, CO2 emit-
of the experiment, by a simple centrifugation. ted by the material can be distinguished from the one
This is useful to perform the carbon balance, to exhausted; for instance, from the solid medium [42].
detect any toxic metabolite, or to study the differ- However, because radiolabeled material is difficult to
ent steps of the biodegradation. This would be obtain, this technique is still costly and difficult to per-
impossible with a mature compost, because its form.
structure is heterogeneous and complex [9]. In real conditions (field or industrial compost), it is
● Vermiculite avoids any priming effect, which can impossible to measure CO2, DOC, or BOD. But, weight
be found in controlled compost, leading to a better losses [43], gel permeation [19], and IR analysis [24],
Aerobic Biodegradation of Polymers 189

although more difficult to carry out than in laboratory Validity Criteria

conditions, have been reported. For disintegration tests,
In compost degradation, three validity criteria
Decriaud-Calmon [44] presented a new method based on
exist [30]:
image analysis, where the surface eliminated by biodegra-
dation is quantified. However, this method is difficult to ● The percentage of mineralization for the reference
apply to burial in compost because the samples are dis- material must be higher than 70% after 45 days.
torted due to high temperature levels. ● The deviation of the percentage of biodegradation
for the reference substance in the different vessels
must be lower than 20% at the end of the test.
The Special Case of Vermiculite Tests ● The compost inoculum in the blank control must
produce after 10 days of incubation more than 50
In the special case of vermiculite, in addition to the and less than 150 mg of carbon dioxide per gram
respirometric measurement, the products retrieved at the of volatile solids.
end of the reaction can be analysed. Tosin et al. [9]
studied this point and in particular the extraction methods. In vermiculite degradation, the same validity criteria
Two materials were degraded: are met, except for the CO2 emission of the blank control,
which has no limit [39].
● Mater-Bi ZI01U: blend of starch, polycaprolac- In soil degradation, criteria are less strict. Only one
tone (PCL) and minor amounts of natural plasti- condition is required: the CO2 emission of the reference
cizers bioreactors (i.e., those containing only the soil) must be
● Mater-BI 2030/489: similar in composition to ZI less than 20% of the mean value of the CO2 emission [17].
01U, 18% being substituted by an equal amount of Therefore, the validity criteria for degradation on
Estane. Estane 54351 is a 4,4⬘ diphenyl methane inert substrate is that the reference vessel produce high
diisocyanate (MDI) thermoplastic polycaprolac- enough (70% of mineralization) and reproducible enough
tone-based polyurethane. The 4,4⬘ diamino diphe- (deviation between vessels less than 20%) amounts of
nyl methane (MDA), an hazardous by-product, is CO2.
created during its biodegradation.

C/N RATIO
(–CO–NH–␸–CH2 –␸–NH–CO–): group MDI
This ratio is highly dependant on the mineral
(–NH2 –␸–CH2 –␸–NH2 –): group MDA medium nature. The composition of the latter is not
defined, contrary to that in liquid medium tests. Soil or
These two materials degrade at a different rate but compost samples are sometimes used with additional
completely disappear after 30 days. Metabolites are only compounds.
detected in the 2030/489 extracts. Water extraction C/N ratio is slightly variable and must be found
enables the retrieval of MDA, identified by high-per- between 10:1 and 40:1 [17, 30, 45]. If this ratio is higher
formance liquid chromatography (HPLC). Dimethyl sulf- than 40:1, it can be lowered by adding ammonium chlo-
oxide (DMSO) extraction (Estane solvent) enables the ride or urea. Indeed, lack of nitrogen can be a limiting
recovery of Estane residues. Sulfuric acid precipitation factor for the growth of microorganisms, which need it
of this extract gives an Estane residue in which the PCL for proteic synthesis.
fraction has been partially degraded. A relative enrich- As an exception, Yabannavar et al. [15, 46] use a
ment of the –NH– groups (MDI) in comparison with the 100:1 ratio, by adding (NH4)2HPO4 as fertilizer.
–CH2– groups (PCL) due to the degradation has been Thus, C/N will be set between 10:1 and 40:1.
noted as a result of this extractive process.
To conclude, in solid-state biodegradation, the respi-
rometric methods are used provided that the large amount MOISTURE CONTENT CONTROL AND
of CO2 produced by the matrix does not hinder the meas- INFLUENCE
urement of CO2 produced by the sample degradation. In
that case, only physical measurements such as weight Water is the main transportation and distribution
loss are available. Vermiculite has the advantage of medium for microorganisms and nutrients. Consequently,
allowing not only respirometric measurements, but also water is absolutely required for the biodegradation proc-
chemical extraction. esses. The noticeable degradation differences between
190 Grima, Bellon-Maurel, Feuilloley, and Silvestre

aqueous media and solid-state media are essentially due capacity have been studied. An increase of the degrada-
to the low water availability in the latter [41]. Thus, the tion rate with increasing humidity has been observed.
water availability is a critical factor for the microbial Degradation with incubation at constant moisture (50%),
development and metabolic activity. The water availabil- and degradation with several drying and rewetting cycles
ity must reach an intermediate level to maintain the above (from 20 to 90% and reciprocally) have been compared.
processes. A lack of moisture leads to a decrease of the This second method shows a lower degradation rate than
metabolism and the microorganism development rates. the first method. Consequently, this study recommends
Conversely, a moisture excess causes substrate packing use of a constant and high humidity rate in the medium.
down and anaerobiosis.
Two different ways of controlling the substrate mois-
ture amount are available, one by monitoring the relative Compost
humidity of air over the substrate, and another by monitor- The influence of moisture content on the biodegrada-
ing the water content of the substrate itself. tion process has been studied by Gu et al. [27]. The
First, the gas water content over the substrate is required duration time for total disappearance of cellulose
fixed. The optimal value is between 60 and 70% and acetate films (substitution degree: 1.7) with moisture con-
can be obtained using saturated solutions of ammonium tents of 60, 50, and 40% was, respectively, 6, 16, and 30
nitrate [47]. days. Obviously, the weight loss rate is strongly correlated
Second, the total substrate water holding capacity to the compost humidity, and increased humidity
corresponds to the residual water content at the end of improves the degradation. However, Suler et al. [52]
the drainage, after a massive addition of water. This is show that the upper level should not exceed 70%.
determined by sample drying during 24 h at 110⬚C.
Three different ways to set the desired water amount
are possible: TEMPERATURE CONTROL AND INFLUENCE
1. Humidified air is generated by bubbling air
through erlenmeyer flasks containing distilled Temperature monitoring is similar in compost and
water. This method allows a constant amount of in soil. The bioreactor temperature is controlled by means
moisture [8, 22, 32, 33, 45, 48–50]. of thermostated aeration [22, 30, 46], or by hot water
2. To keep constant the soil moisture content after bath [8, 45, 49, 50]. In contrast, optimal degradation
adding an amount of water determined by values are not comparable: temperature settings for opti-
weighing [51], the soil sample is placed in a mal degradation are different in the two media due to
desiccator vessel with water at the bottom. This different ecotypes.
method keeps soil from drying during the incuba-
tion period.
Soil
3. Other authors [8, 15, 22, 28, 46] use another
method for setting the soil moisture content. To Current and suitable conditions for the development
control this level of water, the vessels are weighed of soil microorganisms are required. Therefore, constant
once or twice a week. Then, distilled water is temperatures between 25⬚ and 27⬚C are necessary. With
added to offset the weight losses. However, this respect to soils under temperate climate, temperature
method does not allow a constant moisture level, ranges from 10⬚ to 25⬚C are appropriate and indicative
since the weight loss is not exclusively due to of such natural conditions.
the dehydration. But, this procedure is able to
maintain a high water amount in the test medium.
This way is presently used for the vermiculite tests. Compost
For this medium, the initial moisture value is 70% of the The optimal temperature is higher in a compost than
total activated vermiculite holding capacity. in a soil because the compost microorganisms are thermo-
philic. A temperature of 58⬚C is recommended by the
Soil ECN standard draft [30]. Lower temperatures, near 53⬚C,
have been studied by other authors [27, 32, 48, 53].
The moisture content influence on the biodegrada- Nevertheless, temperatures lower than 65⬚C are strongly
tion process has been studied by Garcia-Valcarcel et al. recommended; beyond this limit, several microbial spe-
[51]. Soils with 20, 50, and 90% of total water holding cies die and limit the degradation rate. Protein, fat, and
Aerobic Biodegradation of Polymers 191

complex sugar (cellulose and hemicellulose) cleavages method, however, is not universal: the amount of water
are accelerated by these high temperatures. to add depends on the moisture content of the substrate.
Optimal conditions for poly lactic acid (PLA) degra-
dation in compost have been investigated by Agarwal et
al. [54]. Temperature influence has been tested at 40⬚, Soil
50⬚, and 60⬚C. In this study, a temperature increase is Soil pH is situated between 5.5 and 6.5. It can be
correlated with an increase of polymer chain degradation adjusted to 7.5 by adding CaCO3 [15], in order to obtain
and with a decrease of molecular weights. For the PLA, a pH favorable to the hydrocarbon degradation [42].
temperatures higher than the glass transition temperature
are compulsory for the above degradation. An external
temperature of 35⬚C and monitoring of the bioreactor Compost
with three thermocouples have been stipulated by Day
During the composting process, pH increases.
et al. [33]. A typical temperature curve is observed: an
Because pH is a key parameter of biodegradation, it is
initial rapid self-heating to 50⬚C within few hours, fol-
necessary to monitor its value. At the beginning of the
lowed by a cooling period (down to 46⬚C). Afterward,
process, pH decreases weakly due to the carboxylic acid
the material self-heats and remains above 60⬚C for several
formation. Later, if the pH value is less than 7.0, biodegra-
days. At last, the temperature decreases slowly.
dation can be inhibited by compost acidification and con-
To set up a simulation as close as possible to the
sequently, the disappearance of microorganisms.
conditions of a commercial compost, several authors have
In this case, the volatile fatty acids should be quanti-
imposed a temperature profile similar to the one described
fied. If the amount is higher than 2 g/kg of dry solid, the
above [8, 45, 49]: during 1 day, the temperature is kept
test is nonvalid.
at 35⬚C, then it is raised to 58⬚C during 4 days, then
reduced to 50⬚C for optimum composting conditions until
day 28, and finally at 35⬚C until the end of the test. Hot Conclusion
water baths are used.
These two methods have been compared by Pagga The pH value is a key factor for the survival and
et al. [8]. At the end of the test on paper, the biodegradabil- activity of microorganisms. However, pH varies ac-
ity level is the same. However, the time necessary to cording to the metabolites. Maintaining the pH neutral
reach it is shorter when the temperature is kept constant or weakly basic is therefore a challenge.
at 58⬚C. After 30 days, the biodegradability is 93% for
a constant temperature and 88% for a temperature profile.
CONCENTRATION AND SHAPE OF THE
POLYMER IN THE SUBSTRATE
Conclusion
Whatever the substrate, the film degradation is a
In compost, if the aim is to accelerate the test, a
surface erosion mechanism. Thus, the area/volume ratio
constant temperature is preferred. If the aim is to be as
must be maximum [20, 55, 56]. Samples in powder will
close as possible to the real conditions, a profile tempera-
therefore degrade more easily. The optimal concentration
ture such as the one described above is recommended.
of test material depends on the substrate.
In soil, no similar study has been carried out: simulating
the degradation in soil at various temperatures from 10⬚
to 30⬚C could be useful to find a way to accelerate the Soil
process.
The quantity of test material input must be large
enough to avoid the CO2 production to be masked by the
pH INFLUENCE AND MONITORING one of the substrates. Sharabi et al. [42] showed that with
a ratio lower than 0.5 mg of sample per gram of soil (i.e.,
For these solid substrates, pH must be neutral or a 0.5:1000 ratio), the CO2 release is hard to distinguish
weakly basic [8, 28, 30, 32, 33, 45, 48, 49]. To measure from the background noise.
its pH, a sample of 10 g of substrate is drawn, added to The ISO standard draft [17] recommends a ratio of
500 mL of distilled water, mixed for 5 min with a mag- 1:1000, i.e., 200 mg of material for 200 g of soil. This
netic stirrer, and allowed to decant. When the sediments ratio will be increased or decreased, if the materials are
are settled, pH is measured in the supernatant [33]. This known to degrade slowly or rapidly, respectively [15, 22].
192 Grima, Bellon-Maurel, Feuilloley, and Silvestre

The material/soil ratio was studied by Keursten et For real-life tests, it is not possible to set environ-
al. [16] on rubber. Different rubber/soil ratios were inves- mental parameters at precise values. Duration can then
tigated. CO2 release, Y, is modeled by a first-order kinet- be much longer: samples are collected at 3, 6, 9, 12, 24,
ics: Y ⫽ Ymax ⫻ [1 ⫺ exp(⫺␣T )] where Ymax is the and 36 months [20, 44].
maximal biodegradation level, ␣ is the biodegradation
parameter, and T is the temperature. Ymax increases and
Compost and Vermiculite
␣ decreases when the amount of rubber input increases.
Keursten et al. [16] deduced an effect of the degree of Material degradation is faster in compost than in
contact between the rubber and the soil on this degrada- soil. The duration to achieve the maximal degradation
tion parameter. At low content (10%), the contact between rate varies according to the experimental conditions and
rubber particles and soil was found to be optimal, giving the test materials (inherent material characteristics and
the highest possible value for ␣ and the lowest value for particle size). A 45-day duration is recommended by the
the half-life time. The half-life time is the time after ECN project and ASTM standard [30, 45]. The duration
which half of the polymer weight is degraded. may be extended, but never longer than 6 months.

Compost AERATION AND AIR FLOW

Degli-Innocenti et al. [57] studied the starch degra- Because the biodegradation process must be aerobic,
dation in a compost according to the starch amount. They aeration is required. This is achieved using air where CO2
showed that the higher the material concentration, the and other substances toxic for microorganisms which
more precise the measurement. They did not study the could hinder the biodegradation process have been
degradation time as did Keursten [16]. removed. Aeration, however, tends to dry the medium.
In the ECN TC261 standard draft [58], 600 g of The aeration level must be finely tuned: if too low,
compost and 100 g of test material (in dry weight) have fermentation slows down; if too high, drying and mass
to be introduced in bioreactors with a minimal volume cooling may occur. The air flow range for compost varies
of 3 L. The material/compost ratio is then huge: 1:6. usually from 100 to 150 mL/min for vessel volume
between 3.3 and 4.7 L [8, 27, 32, 33, 48, 53]. For a soil,
the air flow is a little weaker. O2 outflow level is an
Vermiculite excellent indicator of good aeration [45]: it must be fre-
In these tests, the material/vermiculite ratio is even quently measured to ensure it is higher than 6%. O2
higher. The ECN TC249 standard draft [39] advocates concentration levels are determined by means of dedi-
to use a 1:3 ratio for the material/vermiculite ratio (in cated sensors (e.g., paramagnetic analyzer) or by gas
dry weight). chromatography.
To ensure homogenous aeration conditions, bioreac-
tors must be regularly shaken. Compost porosity can be
Conclusion improve by adding inert materials [8].
According to Spence et al. [50], aerating the medium
The concentration of the test sample has an influence
through the vessel bottom is more effective than through
on the degradation duration and on the precision. A high
the top (head space). This method reduces the drying of
concentration leads to a better precision (the background
the medium. Head space aeration is recommended in the
noise is lower), but a low concentration accelerates the
current ISO standard draft [17]. However, results obtained
process.
by this standard raise questions of reliability: short circuits
and gas concentration gradient could affect the biodegra-
TEST DURATION dation tests. It could be useful to modify this point in the
future standard draft version.
Soil
The test duration is very variable: it can extend OTHER CONDITIONS
from 45 days [23] to 6 months [19]. In short-term tests,
measurements are made weekly, whereas samples are To facilitate sample recovery, samples can be placed
collected every month when disintegration tests are to in nylon or polyethylene nets [44] or in cups [24]. Incuba-
be performed. tion could occur in the dark [22] or under diffuse light
Aerobic Biodegradation of Polymers 193

[15]. Light does not seem to be a crucial biodegradabil- released by the substrate itself. Moreover, the degradation
ity parameter. can be stimulated by the presence of glucose or its poly-
In soil, few studies on other environmental condi- mers, which increases the CO2 release of the solid
tions have been investigated. Polyethylene films have medium [42, 57].
been exposed to UV light before burial [46]. This irradia- Consequently, according to its nature, the material
tion has no significant influence on the CO2 release. better degrades in compost or in soil. Thus, it can be
Akahori et al. [19] did a similar experiment and exposed preferable to simulate one environment rather than
samples to solar radiation for 2 weeks. These authors another to be representative of what will happen in
did not notice any significant physical property changes real conditions.
after exposure.
Regarding disintegration tests in compost, the biore-
CONCLUSION AND PROSPECTS
actor size is also normalized: the recommended bioreactor
is a 30 ⫻ 19.5 ⫻ 9.5 cm box of an approximate volume
This review has mainly found articles dealing with
of 5–6 L. It is pierced by two holes to make the gaseous
laboratory simulation of compost processes. The last
exchange easier [28]. However, numerous tests are per-
research related to compost, and in particular, to material
formed in others types of reactors.
residues after composting, investigates inert media.
For soil, articles are much fewer: some articles have
COMPARISON BETWEEN SOIL AND been found on soil simulation in the laboratory, but none
COMPOST DEGRADATION was discovered about inert media aimed at replacing soils.
However, soil simulations would be highly necessary
To better understand plastic film behavior when sub-
for agricultural applications, for instance to predict the
jected to microorganisms, several authors [23, 26, 29, 54,
evolution of degradation residues after years of agricul-
59] compare degradation rates in soil and compost. The
tural film use.
degradation is always faster in compost. Even if a com-
post test seems more reliable and more favorable, one
must be careful. Two types of mistakes can be encoun- Description of a Test Method on Vermiculite
tered in biodegradation in compost.
The analysis of these articles allows us to describe
First, the degradation process in compost can be a
the main guidelines for a simulation of soil in laboratory:
chemical hydrolysis and not a biodegradation. Because
temperatures in compost are higher than those in soil, a ● Substrates: The substrate has to be close to the
chemical acid hydrolysis may complete the biological real-life substrate. The concern with soil perhaps
degradation. This hydrolysis is catalyzed by substances lies in the difficult survival of exogenous microor-
already present in the compost or by degradation products ganisms. Vermiculite offers an appealing alterna-
such as acids coming from polyesters. tive, and inoculation by compost inoculum has
This chemical hydrolysis balances the problems of proven to be successful. No article was found
oligomer solubility. Witt et al. [26, 59] showed that poly- about vermiculite inoculation by soil extract, and
mers with a polymerization degree of 5–6 could be this should be studied.
degraded in compost, whereas in soil, dimers and mono- ● The duration of the experiment varies from 45 to
mers only were assimilated by microorganisms. Conse- 180 days.
quently, Witt et al. [26, 59] propose degradation ● The preferred measurement system for biodegrad-
mechanisms for aromatic and aliphatic oligoesters: (a) ability assessment is a respirometric method (CO2
slow hydrolysis of longer polymers followed by (b) measurement), provided that the large amount of
mono- and dimers dissolution and metabolization by CO2 produced by the matrix does not hinder the
microorganisms. measurement of CO2 produced by the sample deg-
Agarwal et al. [54] studied the degradation of PLA radation. A destructive technique (measurement
in compost and in an abiotic system. They did not find of sample weight loss) would lead to a cumber-
any difference when the films were in contact, or not, some experiment. Vermiculite has the advantage
with the microorganisms. Torres de Dominiguez [14] of allowing not only respirometric measurements,
showed the same process with PLA: a chemical hydrol- but also chemical extraction.
ysis only was involved in the degradation. ● The C/N ratio must be set between 10:1 and 40:1.
Second, measuring CO2 production in compost does ● Moisture must be controlled in order to be set
not assuredly mean a degradation, because CO2 can be between 40 and 60% of the water holding capac-
194 Grima, Bellon-Maurel, Feuilloley, and Silvestre

ity. This control is basically provided by bubbling that pH remain above 7, without need of a finer
the aeration air into water, and by a weekly pH control. Conversely, in soil, pH is a combina-
weighing and rehydrating of the reactors. tion of the effects of its geological origin and of
● The temperature must be close to the natural one. the vegetation, and thus, pH is stable. Microor-
In compost, the influence of the temperature and ganisms extracted from soils are very sensitive
temperature profile on the reaction rate has been to soil pH variation. Therefore, the pH must be
evaluated. In soil, no similar study has been car- accurately maintained. Urea is one of the nutri-
ried out. Simulating the degradation in soil at ments recommended for prefermentation before
various temperature from 10⬚ to 30⬚C could be vermiculite inoculation [39]. Urea degradation
useful to find a way to accelerate the process. leads to pH increase, which can inhibit microor-
● The pH must be neutral or weakly basic. ganisms. One investigation would be to replace
● The concentration of material (over substrate, dry urea by another nitrogen source that would not
weights) varies from 1:1000 to 1:6 according to release OH⫺ ions.
the substrate. This parameter should be carefully 3. The inoculum concentration advocated for the
studied in the future. prefermentation phase in compost simulation
● Grinding the film accelerates the process. studies is very high (108 CFU), due to the micro-
● The aeration is required with a flow rate of 30 organism density of compost. In soil, the concen-
mL/min per liter of vessel. tration after extraction is much lower (typically
105 to 106 CFU). Therefore, the concentration
dealt with in the standard ECN TC 249 WG 9
New Fields of Research cannot be reached. Moreover, due to a lower
temperature (24⬚ instead of 58⬚C), kinetics are
Although these guidelines, synthesized from the lit-
slower in soil simulation. To develop a standard
erature survey, are useful, they are not yet precise enough.
for soil simulation, the duration of the prefer-
Transposing the methods of mineral bed (developed for
mentation phase should be addressed with regard
compost) to soil is not straightforward and readers should
to these points.
be encouraged to consider the following points as key
4. Another consequence of the lesser activity of soil
issues, in particular in the frame of standard development.
microorganisms is that the amount of material to
Therefore, this synthesis opens new fields of research:
be degraded should be lowered. The concentra-
1. The inert substrate used for compost simulation tion of material is definitely an issue to address.
is exclusively vermiculite. The reasons for that
More generally, the studies described in this article
choice have been described above: no additional
defined the values of the key parameters taken individu-
CO2 production, no priming effect, and easy
ally. A more complete study should also consider the
chemical extraction. Because of its high water interaction of these parameters. Experimental design
holding capacity (300%), vermiculite perfectly
could be carried out for this purpose. Because of the
fits compost simulation (110% of water holding duration of the experiments, automation of solid-state
capacity for compost to 290% for Reed peat) fermentation benches could also be studied to increase
[60], whereas the soil water holding capacity is
the number of fermentation experiments run in parallel.
much lower (typically ranking from 28 to 45%
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