J. Anal. Appl. Pyrolysis 85 (2009) 480–486
Contents lists available at ScienceDirect
Journal of Analytical and Applied Pyrolysis
journal homepage: www.elsevier.com/locate/jaap
Microbial deterioration of Mowilith DMC 2, Mowilith DM5 and Conrayt
poly(vinyl acetate) emulsions used as binding media of paintings by
pyrolysis-silylation-gas chromatography–mass spectrometry
Marı́a Teresa Doménech-Carbó a,*, Giovanna Bitossi a, Juana de la Cruz-Cañizares a,
Fernando Bolı́var-Galiano b, Marı́a del Mar López-Miras b, Julio Romero-Noguera b,
Inés Martı́n-Sánchez c
a
Instituto Universitario de Restauración del Patrimonio de la Universitat Politécnica de València, Laboratorio de Análisis Fı´sico-Quı´mico y
Control Medioambiental de Obras de Arte, Camino de Vera s/n, 46022 Valencia, Spain
b
Departamento de Pintura, Facultad de Bellas Artes, Universidad de Granada, Avda. Andalucı´a s/n, 18071-Granada, Spain
c
Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva, 18071-Granada, Spain
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 30 June 2008
Accepted 18 October 2008
Available online 28 October 2008
Evaluation of the deterioration produced by microbiological attack on Mowilith DM5, Mowilith DMC2
and Conrayt poly(vinyl acetate) (PVA) emulsions has been carried out using pyrolysis-gas
chromatography/mass spectrometry (Py-GC/MS). The proposed method includes the on-line derivatization of PVA emulsions using hexamethyldisilazane during pyrolysis. Specimens consisting of thin films
formed on glass slides from dried PVA emulsions have been used. Py-GC–MS analyses performed on the
specimens where the fungi Aspergillus niger, Penicillium chrysogenum, Trichoderma pseudokoningii,
Cladosporium cladosporioides, Chaetomium globosum, Rhizopus oryzae, Aureobasidium pullulans, and the
bacteria Streptomyces cellulofans, Bacillus amyloliquefaciens, Arthrobacter oxydans and Burkholderia cepacia
were inoculated and allowed to grow, enable an evaluation of the effect of these microorganisms on the
composition of the PVA emulsion. Decrease in the relative content of external plasticiser of phthalate type
used in these PVA emulsions has been the main effect observed. Moreover, a different behavior was
observed depending on the plasticiser present in every commercial PVA emulsion studied. Diisobutyl
phthalate, used in Conrayt emulsion slightly varied its content in specimens inoculated with bacteria
whereas dibutyl phtalate used in Mowilith DMC2 emulsion noticeably decreased its content in the
specimens inoculated with fungi, thus suggesting that the effects of the metabolic processes associated to
the latter microorganisms on the studied PVA emulsions are more significant than those from bacteria.
ß 2008 Elsevier B.V. All rights reserved.
Keywords:
Py-GC/MS
Poly(vinyl acetate) emulsion
Binding media
Hexamethyldisilazane
Biodeterioration
Bacteria
Fungi
Diisobutyl phthalate
Butyl phthalate
External plasticiser
1. Introduction
Since the introduction of the synthetic polymers in the art
and art conservation field a few years after the invention of
cellulose nitrate by Schönbein in 1846, these materials have
become widespread, and nowadays they are found not only as
materials forming the art object but also as adhesives,
consolidants, inpainting varnishes or fillers of missing parts
used in restoration works [1]. Despite the better properties that,
in general, these materials exhibit if they are compared to the
traditional binding media and varnishes, they also are affected,
* Corresponding author. Tel.: +34 9638 79312; fax: +34 9638 77319.
E-mail address: tdomenec@crbc.upv.es (M.T. Doménech-Carbó).
0165-2370/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jaap.2008.10.010
in more or less extent, by deterioration or biodeterioration
processes [2]. In particular, paintings are sometimes subjected
to microbial attack [3–5], which results in the lowering of the
mechanical strength of the support, fading and detachment of
the paint layer or spots produced by coloring metabolic byproducts [6]. Thus, study of the biodeterioration of synthetic
binders and varnishes has attracted the attention of the
conservation profession given their demonstrated ability for
undergoing morphological and chemical changes on microbial
attack. Thus, different methods have been reported for assessing
the biodeterioration of synthetic resins commonly present in
paintings, sculptures and other contemporary art objects
including instrumental techniques such as optical and scanning
electron microscopy (SEM) [7–9], spectrophotometry [8,9],
determination of the weight loss of specimens inoculated with
M.T. Doménech-Carbó et al. / J. Anal. Appl. Pyrolysis 85 (2009) 480–486
selected microorganisms [10] or characterization of structural
changes by means of FTIR, and Raman spectroscopy [9,11] and
FTIR-PAS spectroscopy [9]. In particular, the ‘‘Standard Practice
for Determining Resistance of Synthetic Polymeric Materials to
Fungi’’ (ASTM G21-96(2002)) and the ‘‘Standard Practice for
Determining Algal Resistance of Plastic Films’’ (ASTM G2996(2002)) [2,9] – methods extensively used for this purpose –
are based on measurement of the visual appearance, optical and
electronic microscopic observation, as well as on the measurement of mechanical and electrical properties.
Poly(vinyl acetate) (PVA) emulsions have been widely used in
the 20th century for preparing pictorial binding media and
protective coatings of paintings in Spain. A number of additives
are included in these commercial emulsions, which improve
chemical and mechanical properties of the paint film or coating.
Among them it can be mentioned external plasticisers such as
dibutyl phthalate, isobutyl phthalate or ethyl phthalate [12].
Internal plasticisation obtained by copolymerization of vinyl
acetate with softer monomers such as acrylates or vinyl versatates
(commercial mixtures of highly branched C9 and C10 vinyl esters),
among others, has been reported [13].
Some works can be found in literature specifically dedicated to
the study of biodeterioration of PVA resins used in art and art
conservation [2,8,14]. Interestingly, Heyn et al. [11] have reported
significant microbial growth in PVA emulsions Mowilith DM5 and
Mowilith 20. Measurement of pH and analysis of organic acid
formation, mainly citric and ethanoic acid, was considered by
HPLC. The microbiological agents were, in this case, Aspergillus
versicolor, Aspergillus niger, Cladosporium herbarum, Cladosporium
sphaerospermum, Engyodontium album, Penicillium aurantiogriseum, Penicillium chrysogenum, Trichoderma longibrachiatum,
Debaryomyces hansenii, Rhodotorula mucilaginosa, Bacillus subtilis,
Bacillus licheniformis and Rhodococcus fascians. It should be noted
that no works have been found reporting the study of biodeterioration of PVA resins due to bacteria attack.
On the other hand, Cappitelli et al. [2] have extended the
discussion, stressing the significant role of the additives included
in resin-based products as main carbon source of the attacking
microorganisms, suggesting that the polymer, more resistant than
additive compounds, could be initially unaltered—but the biomass
produced could generate unspecific enzymes (under so-called ‘‘cometabolic conditions’’) which result in resin attack in a second
step.
In a prior paper [15], the authors have shown a biodeterioration study performed on Mowilith 50, a PVA resin exempt of
additives. The aim of this work was to evaluate the role of the pure
PVA resin as potential carbon source of microorganisms. The
results obtained suggested that the culture media was not
completely consumed during incubation of the microorganisms,
so that the PVA resin was used to a low extent as carbon source. At
the same time, the suitability of pyrolysis-gas chromatography–
mass spectrometry (Py-GC–MS) as a complementary technique
for evaluating biodeterioration processes was considered. For this
purpose, a method based on the on-line derivatization of samples
with hexamethyldisilazane (HMDS) was applied [16]. This
method is based on the formation of trimethylsilyl ethers and
esters, and has been proposed as an alternative to other commonly
employed derivatization reagents such as tetramethyl ammonium hydroxide, to improve the detection of hydroxylated
compounds and to avoid isomerization and unwanted alkylation
reactions.
In this paper is describe the biodeterioration study performed
on three PVA emulsions frequently used as binding media and/or
consolidants, namely, Mowilith DMC2, Mowilith DM5 and
Conrayt, in order to evaluate the role of the plasticisers used in
481
the PVA emulsions as potential carbon source of microorganisms.
In the experiments performed a series of selected microorganisms
have been induced to grow under drastic conditions, so that they
have completely consumed the nourishment provided by the
culture medium and thus, impelled to use the substances
composing the PVA emulsion as carbon source. For this purpose,
on-line derivatization of samples with HMDS has been applied
with Py-GC–MS.
2. Experimental
2.1. Solvents, reagents and culture media
2.1.1. Analytical reagents and reference materials
The following reagents were used to treat the samples:
hexamethyldisilazane (HMDS) (purity 99%) and acetone (Sigma–
Aldrich, Steinheim, Germany), Tween 80 (Sigma–Aldrich, Steinheim, Germany).
Mowilith DMC 2 emulsion (co-polymer of vinyl acetate and
dibutyl maleate (35%)) (Hoechst) and Mowilith DM5 emulsion (copolymer of vinyl acetate and n-butyl acrylate (35%)) (Hoechst) and
Conrayt emulsion (Rayt SA) have been the materials used in this
study. It should be noted that suppliers of these products do not
provide information concerning the external plasticisers of
phthalate type identified in the commercial products studied
(vide infra).
2.1.2. Culture media
Tryptone soy broth (TSB) medium (Scharlau Chemie, Barcelona,
Spain) was used for bacteria cultures and complete medium (CM)
composed by yeast extract 0.5%, malt extract 0.5% and glucose 1%
was used for fungal cultures.
2.2. Instrumentation
2.2.1. Pyrolysis-gas chromatography–mass spectrometry
Pyrolysis-gas chromatography–mass spectrometry was carried
out with an integrated system composed of a CDS Pyroprobe 1000
heated filament pyrolyser (Analytical Inc. New York, USA), and a
Gas Chromatograph Agilent 6890N (Agilent Technologies, Palo
Alto, CA, USA) coupled with an Agilent 5973N mass spectrometer
(Agilent Technologies) and equipped with a pyrolysis injection
system. A capillary column HP-5MS ((5%-phenyl)-methylpolysiloxane; 30 m, 0.25 mm i.d., 0.25 mm) was used. Pyrolysis was
performed at 700 8C for 10 s, using a precalibrated Pt coil type
pyrolyser (CDS pyprobe). The pyrolyser interface and the inlet
were set at 250 8C. Samples were injected in split mode (split ratio
1:40). The GC column temperature conditions were as follows:
initial temperature 50 8C, held for 2 min and then increased at
5 8C min1 up to 100 8C, increased at 15 8C min1 to 295 8C, and
held for 10 min. Helium gas flow was set to 1.2 mL min1. The
electronic pressure control was set to the constant flow mode with
vacuum compensation.
Ions were generated by electron ionization (70 eV) in the
ionization chamber of the mass spectrometer. The mass spectrometer was scanned from m/z 20 to m/z 800, with a cycle time of 1 s.
Agilent Chemstation software G1701CA MSD was used for GC–MS
control, peak integration and mass spectra evaluation. EI mass
spectra were acquired in the total ion monitoring mode, and the
peak area (TIC) data were used for obtaining values of peak area
percentage. The temperatures of the interface and the source were
280 8C and 150 8C, respectively. NIST and Wiley Library of Mass
Spectra were used for identifying compounds.
The CLIMAS culture chamber (Génesis Instrumentación,
Madrid, Spain) was used for incubation trials.
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M.T. Doménech-Carbó et al. / J. Anal. Appl. Pyrolysis 85 (2009) 480–486
2.3. Preparation of test specimens
A series of test specimens were prepared by brushing the PVA
emulsions in three successive thin layers on glass slides of standard
size (24 mm 80 mm). Then, the specimens were dried at room
temperature during 2 months.
2.4. Microorganism inoculation and incubation
The microorganisms studied were recognized biodeterioration agents, selected from an extensive review of the literature
[8,11,17–20], among fungi and bacteria species susceptible to
originate processes of biodeterioration in synthetic resins. All
species studied are ubiquitous saprophytes, abundantly distributed in the atmosphere, and came from collection stocks of
the Spanish Collection of Type Cultures (CECT, Colección
Española de Cultivos Tipo). The microorganisms used were
described below.
2.4.1. Fungi
Aspergillus niger (An) (CECT 2088, ATCC 9029), Penicillium
chrysogenum (Pc) (CECT 2306, ATCC 8537), Trichoderma pseudokoningii (Tp) (CECT 2937), Cladosporium cladosporioides (Cc) (CECT
2110, ATCC 16022), Chaetomium globosum (Cg) (CECT 2701, ATCC
6205), Rhizopus oryzae (Ro) (CECT 2339, ATCC 11145), Aureobasidium pullulans (Ap) (CECT 2703, ATCC 9348).
2.4.2. Bacteria
Streptomyces cellulofans (Sc) (CECT 3242, ATCC 29806), Bacillus
amyloliquefaciens (Ba) (CECT 493, ATCC 23842), Arthrobacter
oxydans (Ao) (CECT 386, ATCC 14358), Burkholderia cepacia (Bc)
(CECT 322, ATCC 17759).
In order to obtain fungal spores, lyophilized collection stocks
were hydrated in CM broth and incubated for 1 week (28 8C, 75%
RH). Afterwards, cultures were spread on solid CM medium and
incubated for 15 days. Sporulated cultures were suspended in 2 mL
of Tween 80, 0.1%. After centrifugation, pellets were washed and
resuspended in 2 mL of distilled water. The suspensions were
filtered through glass wool to eliminate any remains of mycelia.
After a count in a Neubauer chamber, concentration was adjusted
to 106 spores mL1. In a similar way, bacterial suspensions (107–
108 cells mL1) were obtained after centrifugation and washing
with distilled water to eliminate any possible remain of culture
media.
Finally, a single species of the selected fungi and bacteria was
inoculated on each previously prepared test specimen containing a
single dried PVA emulsion. 20 mL of the fungal or bacterial
suspensions described above were applied on each test specimen,
which covered an area of at ca. 20 mm2 of the dried polymeric film.
A total of three replicates were prepared for each type of test
specimen containing a single PVA emulsion and a single species of
fungus or bacterium. Previous experiments helped us to establish
the optimal conditions for incubating the model varnish specimens. The specimens were incubated for 15 days in darkness at
28 8C and 85% RH, water activity (aw) = 0.85. After this, the biomass
of microorganisms formed on the inoculated are was completely
removed.
2.5. Preparation of samples for Py-GC–MS analysis
A sample was taken scraping about 1 mg of the polymeric film
from the inoculated area exposed to the microbial attack in each
PVA specimen with the help of a scalpel. The same procedure was
carried out to take a blank sample from the parts of the specimen
which were not inoculated. A total of three replicates for each
sample of blank and each sample of inoculated area were analyzed
for every type of specimen.
2.6. Derivatization of samples
Samples scraped from the specimens were placed in a
microquartz pyrolysis tube and then two small portions of quartz
wool were introduced in both sides of the quartz tube for avoiding
undesirable displacements of the sample. Afterwards, 5 mL of
HMDS were added.
3. Results and discussion
Ethanoic acid together with benzene are the main products
formed as consequence of the breaking down of the PVA
polymer chains during pyrolysis via a side group elimination
mechanism [21]. Thus, these compounds have been suggested as
a marker of PVA emulsions for analytical purposes by this author
[22]. Characterization of PVA emulsions used in art and art
conservation has usually been performed by direct Py-GC/MS
[13,21,22]. Recently, Doménech-Carbó et al. have proposed a
new method based on the on-line derivatization of the PVA
pyrolysates with HMDS [15,16]. This method has as major
advantage the ability to form the trimethylsilyl ester of
carboxylic acids, in particular, of ethanoic acid. The proposed
method improves the efficiency in the formation of this
pyrolysis product and avoids the appearance of a fronting peak
for ethanoic acid as usually occurs with strong polar organic
compounds such as carboxylic acids. In turns, the benzene peak
is overlapped by the more intense peak from the derivatization
reagent, hindering its identification and quantification in some
cases. Additionally, a series of peaks can be recognized at higher
retention times, namely, 1,4-dihydronaphthalene (12.77 min),
and 1,2-dihydronaphthalene (13.09 min) and naphthalene
(13.55 min).
Table 1 summarizes the declared composition and the
compounds identified in the analysis of the set of samples excised
from the un-inoculated area of the specimens prepared with the
three studied PVA emulsions, which were used as blanks in the
series of experiments performed. Different peaks corresponding to
the external plasticiser can be observed in the pyrogram of the
commercial PVA emulsions studied. Thus, peak at 20.43 min
ascribed to diisobutyl phthalate is found in Conrayt samples.
Similarly, peak at 18.03 min is ascribed to dibutyl ester of
butanedioic acid and peak at 18.23 min is ascribed to dibutyl
ester of 2-butenedioic acid, the two latter are associated with the
dibutyl maleate present in Mowilith DMC2. Additionally, peak at
21.09 min ascribed to the external plasticiser dibutyl phthalate is
identified in this PVA film. Finally, peak appearing in the specimens
of Mowilith DM5 at 5.75 min is ascribed to n-butyl acrylate and
peak at 18.37 min, associated to diethyl phthalate, is also found in
this PVA emulsion in a low amount. It is interesting to note that, no
indication about use external plasticisers is made in the declared
composition provided by the suppliers and reported in literature
[1].
On the basis of the identified compounds, a comparative study
has been carried out in an attempt to recognize the changes in
composition due to the microbial attack. The methodology
proposed is based on the calculation of the parameter R defined
as the ratio of the peak area of the external plasticiser i (i represents
the external phthalate plasticisers found in the specimens studied,
namely, ethyl phthalate, dibutyl phthalate and isobutyl phthalate
and also the three PVA emulsions studied, namely, Mowilith DMC 2
Mowilith DM5 and Conrayt as a different plasticiser is present in
each PVA emulsion as shown in Table 1) recognized in the samples
M.T. Doménech-Carbó et al. / J. Anal. Appl. Pyrolysis 85 (2009) 480–486
483
Table 1
Commercial art and conservation PVA emulsions studied, declared composition and main compounds identified in the analysis carried out by means on-line silylationpyrolysis GC–MS.
Compounds identified
Benzene
Ethanoic acid, TMS ester
Styrene
n-Butyl acrylate
2-Trimethylsilyloxypropanoic
acid, TMS ester
1,4-Dihydronaphthalene
1,2-Dihydronaphthalene
Naphthalene
Benzoic acid, TMS ester
2-Methylbenzoic acid, TMS ester
Benzoic acid, butyl ester
Butanedioic acid, dibutyl ester
2-Butenedioic acid, dibutyl ester
Diethyl phthalate
Diisobutyl phtalate
Dibutyl phthalate
a
b
Peak
tr (min)
Mw
PVA art and conservation commercial emulsions
Mowilith DMC 2
Mowilith DM5
Conrayt
Hoechst (supplier)
Hoechst (supplier)
Rayt España (supplier)
Co-polymer of vinyl acetate and
di-butyl maleate (35%)
(declared compositiona)
Co-polymer of vinyl acetate and
butyl acrylate (35%) (declared
compositiona)
Polyvinyl acetate
(declared compositiona)
Blank
Inoculatedb
Blank
Inoculatedb
Blank
Inoculatedb
1
2
3
4
5
2.20
2.67
5.65
5.75
10.48
78
132
104
128
234
H
H
H
–
–
H
H
H
–
H
H
H
–
H
–
H
H
–
–
H
H
H
–
–
–
H
H
–
–
H
6
7
8
9
10
11
12
13
14
15
16
12.77
13.09
13.55
14.60
15.96
16.27
18.03
18.23
18.37
20.43
21.09
130
130
128
194
208
298
230
228
222
278
278
H
H
H
H
H
H
H
H
H
–
H
H
H
H
H
H
H
H
H
–
–
H
–
–
–
H
H
H
–
–
–
–
–
–
–
H
–
–
–
–
–
–
–
–
–
–
H
–
–
–
–
–
–
H
–
–
–
H
–
–
–
–
–
–
H
–
Presence of ethyl, dibutyl and isobutyl phtalates was not indicated in the declared composition of the studied PVA emulsions.
Compounds identified in the PVA emulsion specimens inoculated with the microorganisms listed in Section 2.4.
analyzed to the value of the peak area of the trimethylsilyl ester of
ethanoic acid:
Ri ¼
Ai
;
Aa
where Ai is the value of the peak area of the external plasticiser i
and Aa is the value of peak area of the trimethylsilyl ester of
ethanoic acid obtained in the pyrogram of a specimen.
Thermal degradation mechanisms of addition polymers and
copolymers of vinyl acetate have been described in literature. In
particular, thermal degradation of PVA has been extensively
studied by McNeill et al. [23,24] concluding that, breaking down of
the chains of PVA polymer occurs between 250 8C and 400 8C. This
process as been described in terms of a side group elimination
reaction which results in the quantitative formation of ethanoic
acid and benzene molecules as a simultaneous process. Therefore,
it can be assumed that this process is quantitative in the
experimental pyrolysis conditions applied here (700 8C). On the
other hand, the Ri blank value found in the blank sample from each
one of the three studied PVA emulsions can be considered a
reference value, as it is correlated straightforward to the relative
content of plasticiser in the PVA emulsion specimen i not subjected
to microbial attack but subjected to the incubation conditions.
Comparison of the Ri blank value and the Ri inoculated value found in
the specimen i inoculated with a specific microorganism can be
used for assessing the changes in the relative content of plasticiser
which takes place in the studied PVA emulsions after microbial
attack. A decrease in the R value from the blank sample to the
sample of the inoculated area of the PVA specimen (DRi < 0, where
DRi = Ri inoculated Ri blank) indicates that this biodeteriorated
specimen experienced a loss of plasticiser, which is ascribed to
the activity of the inoculated microorganisms.
Likewise, a negative value for DRi can also be ascribed to an
increase in the content of free ethanoic acid in the biodeteriorated
sample as consequence of the metabolic activity of the microorganisms, which results in the breakdown of the polymer chains.
Fig. 1. Pyrogram obtained from the specimen of Mowilith DMC 2 inoculated with the fungus Chaetomium globosum. (1) Benzene, (2) ethanoic acid, TMS ester, (3) Styrene, (5)
2-trimethylsilyloxypropanoic acid, TMS ester, (6) 1,4-dihydronaphthalene, (7) 1,2-dihydronaphthalene, (8) naphthalene, (9) benzoic acid, TMS ester, (10) 2-methylbenzoic
acid, TMS ester, (11) benzoic acid, butyl ester, (12) butanedioic acid, dibutyl ester, (13) 2-butenedioic acid, dibutyl ester, (16) dibutyl phthalate.
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M.T. Doménech-Carbó et al. / J. Anal. Appl. Pyrolysis 85 (2009) 480–486
Fig. 2. Pyrogram obtained from the specimen of Mowilith DMC 2 inoculated with the bacterium Bacillus amyloliquefaciens. (1) Benzene, (2) ethanoic acid, TMS ester, (3)
styrene, (5) 2-trimethylsilyloxypropanoic acid, TMS ester, (6) 1,4-dihydronaphthalene, (7) 1,2-dihydronaphthalene, (8) naphthalene, (9) benzoic acid, TMS ester, (10) 2methylbenzoic acid, TMS ester, (11) benzoic acid, butyl ester, (12) butanedioic acid, dibutyl ester, (13) 2-butenedioic acid, dibutyl ester.
Fig. 3. Bar chart illustrating the shift of R values for specimens of Mowilith DMC 2
inoculated with bacteria and fungi. Rblank: peak area of dibutyl phthalate/peak area
of trimethylsilyl ester of ethanoic acid found in the blank sample and Rinoculated:
peak area of dibutyl phthalate/peak area of trimethylsilyl ester of ethanoic acid
found in the sample from an area of the specimen inoculated with a specific
microorganism. Fungi: Aspergillus niger (An), Penicillium chrysogenum (Pc),
Trichoderma pseudokoningii (Tp), Cladosporium cladosporioides (Cc), Chaetomium
globosum (Cg), Rhizopus oryzae (Ro), Aureobasidium pullulans (Ap). Bacteria:
Streptomyces cellulofans (Sc), Bacillus amyloliquefaciens (Ba), Arthrobacter oxydans
(Ao), Burkholderia cepacia (Bc).
This second effect and the previously described of loss of plasticiser
are undistinguished from our approach and thus, the experimental
DRi value obtained in each inoculated PVA emulsion specimen is
the result of both processes that, presumably, take place during the
microbial attack. Therefore, this parameter can be considered an
indicator of the extent of the deterioration processes that affect the
PVA emulsion subjected to the attack of a specific microorganism.
Figs. 1 and 2 show the pyrograms corresponding to specimens
of Mowilith DMC 2 inoculated with the fungus C. globosum and the
bacterium B. amyloliquefaciens. Both pyrograms are dominated by
the peak corresponding to the trimethylsilyl ester of ethanoic acid.
Interestingly, while a peak of dibutyl phtalate can be seen in the
pyrogram corresponding to the specimen with B. amyloliquefaciens,
significant reduction of this peak is observed in the pyrogram from
the specimen inoculated with C. globosum.
Rdibutyl phthalate values obtained in the series of specimens
prepared with Mowilith DMC2 are shown in Fig. 3. In general,
acceptable repeatability is obtained under the experimental
conditions applied for performing the analyses as suggested by
the values of relative standard deviation from the three replicates
analyzed for each type of specimen containing a single microorganism and a single PVA emulsion, which are in the range 2–4%.
As it is shown in Fig. 3, fungi, in general, have significantly
modified the composition of the PVA films resulting in a decrease
of the plasticiser/ethanoic acid ratio apart from specimens
inoculated with A. pullulans, whereas specimens inoculated with
bacteria do not exhibit remarkable change, apart from S. cellulofans.
These results suggest that, in general, specimens have been
specially sensitive to the fungi attack.
Figs. 4 and 5 show the pyrograms corresponding to specimens
of Conrayt inoculated with the fungus A. niger and the bacterium A.
oxydans. The pyrograms are also dominated by the peak
corresponding to the trimethylsilyl ester of ethanoic acid and
peak corresponding to the diisobutyl phthalate plasticiser appears
in both pyrograms. Fig. 6 shows that the effect of the microbial
attack in this PVA emulsion is notably lower than in Mowilith
DMC2 and only specimens inoculated with the fungus A. niger and
the set of bacteria, specially, A. oxydans and B. amyloliquefaciens
exhibited a slight decrease in the Rdibutyl phthalate value.
Fig. 7 shows the pyrogram found in the specimen prepared with
Mowilith DM5 and inoculated with P. chrysogenum. Interestingly,
Fig. 4. Pyrogram obtained from the specimen of Conrayt inoculated with the fungus Aspergillus niger. (1) Benzene, (2) ethanoic acid, TMS ester, (5) 2trimethylsilyloxypropanoic acid, TMS ester, (8) naphthalene, (15) diisobutyl phthalate.
M.T. Doménech-Carbó et al. / J. Anal. Appl. Pyrolysis 85 (2009) 480–486
485
Fig. 5. Pyrogram obtained from the specimen of Conrayt inoculated with the bacterium Arthrobacter oxydans. (1) Benzene, (2) ethanoic acid, TMS ester, (5) 2trimethylsilyloxypropanoic acid, TMS ester, (8) naphthalene, (15) diisobutyl phthalate.
Fig. 6. Bar chart illustrating the shift of R values for specimens of Conrayt inoculated with bacteria and fungi. Rblank: peak area of dibutyl phthalate/peak area of trimethylsilyl
ester of ethanoic acid found in the blank sample and Rinoculated: peak area of dibutyl phthalate/peak area of trimethylsilyl ester of ethanoic acid found in the sample from an
area of the specimen inoculated with a specific microorganism. Fungi: Aspergillus niger (An), Penicillium chrysogenum (Pc), Trichoderma pseudokoningii (Tp), Cladosporium
cladosporioides (Cc), Chaetomium globosum (Cg), Rhizopus oryzae (Ro), Aureobasidium pullulans (Ap). Bacteria: Streptomyces cellulofans (Sc), Bacillus amyloliquefaciens (Ba),
Arthrobacter oxydans (Ao), Burkholderia cepacia (Bc).
Fig. 7. Pyrogram obtained from the specimen of Mowilith DM5 inoculated with the fungus Penicillium chrysogenum. (1) Benzene, (2) ethanoic acid, TMS ester, (5) 2trimethylsilyloxypropanoic acid, TMS ester, (8) naphthalene.
peak corresponding to diethyl phthalate is not present in the
pyrogram of both blank and inoculated samples suggesting that
elimination of this external plasticiser was mainly associated to
the incubation conditions.
Finally, it is interesting to note that according with the results
obtained by Heyn et al. [11], the metabolic activity of most of the
fungi tested in this study results in the formation of hydroxycarboxylic acids, in particular 2-hydroxypropanoic acid.
4. Conclusions
The proposed method based on the on-line HMDS derivatization with Py-GC–MS has proven to be successful for recognizing
the changes occurring after microbial attack with the studied
bacteria and fungi. These changes mainly involve the decrease in
the relative content of phthalate plasticiser to ethanoic acid in the
studied specimens of PVA emulsions.
Different behavior has been found in the three studied
commercial PVA emulsions depending on the type of plasticiser
present. Diethyl phthalate is sensitive to the environmental
conditions during the incubation so that its relative content is
drastically decreased in both the inoculated and the un-inoculated
parts of the specimens. Conrayt specimens containing diisobutyl
phthalate slightly vary their plasticiser content, in particular in
specimens inoculated with bacteria. In contrast, the molecules of
dibutyl phthalate maintain their relative content in specimens
486
M.T. Doménech-Carbó et al. / J. Anal. Appl. Pyrolysis 85 (2009) 480–486
inoculated with bacteria whereas significantly reduce their
content when fungi are present.
Finally, the appearance of a peak ascribed to 2-hydroxypropanoic
acid in the specimens inoculated with fungi has been correlated to
the metabolic activity of microorganisms in good agreement with
prior works found in literature [11] where occurrence of hydroxylated acids was reported.
Acknowledgements
Financial support is gratefully acknowledged from the Spanish
‘‘I+D+I MEC’’ projects CTQ2005-09339-CO3-01 and 03, and the
Generalitat Valenciana ‘‘I+D+I’’ project ACOMP/2007/138, which
are also supported by ERDEF funds as well as the SB2005-0031
project ascribed to the program of postdoctoral stages of novel
researchers in Spanish Universities and research centers from the
Ministerio de Educación y Ciencia (MEC).
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