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                                      Int. J. Hyg. Environ. Health 212 (2009) 61–66

Utilization of chemically oxidized polystyrene as co-substrate
by filamentous fungi
Oriana Mottaa,Ã, Antonio Protob, Francesco De Carlob, Francesco De Caroa,
Emanuela Santoroa, Luigi Brunettia, Mario Capunzoa
Department of Educational Science, Chair of Hygiene, University of Salerno, via Ponte don Melillo, 84084 Fisciano (SA), Italy
Department of Chemistry, University of Salerno, via Ponte don Melillo, 84084 Fisciano (SA), Italy

Received 21 May 2007; received in revised form 31 August 2007; accepted 25 September 2007

   Atactic polystyrene, one of the most widely used chemical products, was subjected to novel chemically oxidative
treatments able to trigger a great variety of physical and chemical changes in the polymer’s chains. The oxidized
polystyrene samples, when analyzed with Fourier transform infrared spectroscopy (FTIR) clearly showed the
formation of carbonyl groups and hydroxyl groups, which increased with the increase in the strength of chemically
oxidative treatments.
   In fungal degradation tests deploying Curvularia species, the fungus colonized the oxidized samples within 9 weeks.
Colonization was confirmed by microscopic examination, which showed that the hyphae had adhered to and
penetrated the polymer’s structure in all the treated samples. Such colonization and adhesion by microorganisms are a
fundamental prerequisite for biodegradation of polymers.
r 2007 Elsevier GmbH. All rights reserved.

Keywords: Polystyrene; Oxidation; Degradation; Fungi; Curvularia

Introduction                                                                   Both natural and synthetic polymers containing
                                                                            specific functional groups, as all materials of organic
   Polymers have unique chemical composition, physical                      origin, are potential substrates for heterotrophic micro-
forms, mechanical properties and applications. Because of                   organisms including bacteria and fungi. Aerobic biode-
their structural versatility, polymers are widely used in                   gradation of natural polymers has been well studied and
product packaging, insulation, structural components,                       many polymer-degrading microorganisms have been
protective coatings, medical implants, drug delivery                        isolated and identified (Gu, 2003), whereas study of
carriers, slow-release capsules, electronic insulation, tele-               biological degradation of synthetic polymers is yet at a
communication, aviation and space industries, sporting                      developing stage, probably because concern about
and recreational equipment and as building consolidants,                    degradation of the environment brought about by large
etc.                                                                        amounts of discarded polymeric materials at the end of
                                                                            their useful life is relatively recent. In fact, for many
                                                                            years research was directed mainly toward developing
    ÃCorresponding author. Tel./fax: +39 089 963083.                        indestructible materials of possibly infinite life or those
     E-mail address: (O. Motta).                            that degrade very slowly in natural environments. Only

1438-4639/$ - see front matter r 2007 Elsevier GmbH. All rights reserved.
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in the last decade or two, because of the increasing costs          Experimental
of disposing solid wastes and scarce landfill space as well
as because of the potential hazards of waste incinera-              Materials and methods
tion, biodegradation of synthetic polymers has emerged
as the most effective potential solution to this environ-              The polymers used were atactic polystyrene 143 E
mental problem.                                                     (BASF) and an aromatic–aliphatic co-polyester, namely
   The biodegradability of any polymer depends on                   Ecoflex (BASF). Ecoflex was used as supplied, whereas
its molecular weight, crystallinity and physical forms              polystyrene 143 E was chemically transformed into more
(Gu et al., 2000). Generally, higher molecular weight               oxidized forms as follows: one treatment involved the
results in greater resistance to degradation by micro-              oxidizing agent alone (sample 1), another involved
organisms, whereas monomers, dimers and oligomers                   adding a transition metal complex to the oxidizing
of a polymer’s repeating units are degraded and                     agent (sample 2), and the third involved adding an
mineralized more easily. High molecular weights lead                inorganic acid to the oxidizing agent (sample 3). All the
to sharply decreased solubility, making the polymer                 treatments involved a 2 h exposure at room temperature
resistant to microbial attack because microorganisms                to the oxidizing agent (with or without the additives).
need to assimilate the substrate through their cellular                All the polymers were sterilized with UV radiation
membrane and then degrade the substrate further by                  before incubation with fungi. Untreated atactic poly-
means of intracellular enzymes. At least two categories             styrene was used as reference.
of enzymes, namely extracellular and intracellular
depolymerizers, are actively involved in biodegradation
of polymers (Doi, 1990; Gu et al., 2000).
   It is commonly recognized that the closer a polymer’s            Fungal growth and culture conditions
structure to that of a natural molecule, the more
easily it is degraded and mineralized (Gu and Gu,                      Microbial degradation of the polymers was studied
2005). Polymers such as cellulose, chitin and poly                  usingCurvularia as the fungal species of choice. The
b-hydroxybutyrate (PHB) are all biologically synthe-                choice was based on environmental sampling carried out
sized and can be completely and rapidly biodegraded                 for an earlier piece of research and on the proven ability
by heterotrophic microorganisms in a wide range                     of the species to degrade Ecoflex. Five other fungi were
of natural environments (Berenger et al., 1985; Byrom,              also considered: Aspergillus niger, Aspergillus flavus,
1991; Frazer, 1994; Gamerith et al., 1992; Gujer                    Mucor sp., Monilia sp., and Penicillium sp. Each was
and Zehnder, 1983; Gunjala and Sulflita, 1993;                       grown on sabouraud plates separately and repeatedly to
Hamilton et al., 1995; Hespell and O’Bryan-Shah,                    obtain pure cultures and identified by microscopic
1988).                                                              examination.
   It is also known that polymers with carbonated linear               Ecoflex samples, each 150 mm thick and measuring
chains, such as polyolefins, are unlikely to be attacked             5 cm by 5 cm, were embedded within the sabouraud agar
by microorganisms. However some research has demon-                 in the plates that had been seeded with the different
strated that ligninolytic and cellulolytic fungi can                fungal species. The plates were incubated at 25 1C for 9
degrade oxidized polymeric chains (Lee et al., 1991;                weeks and examined weekly.
Manzur et al., 1997; Weiland et al., 1994).                            Within 10 days, the entire surface of the culture
   Several attempts have been made to induce carbon-to-             medium excluding the embedded piece of the polymer
oxygen bonds into the polymeric chain, most of them                 was covered by the fungal colony in every plate
involving photo- and thermo-oxidative treatment of                  irrespective of the species. However, after 4 weeks, only
polyolefins, principally polyethylene (PE); a few of the             Curvularia sp. had completely colonized the surface of
attempts report the degradation of the polymer after a              the polymer. After 9 weeks, the Ecoflex samples were
long period of incubation with selected fungi or in                 degraded by Curvularia sp. to such an extent that it was
mature compost (Chiellini and Corti, 2003; Chiellini                impossible to recover the samples from the culture
et al., 2003; Manzur et al., 2004; Pandey and Singh,                plates. Therefore, Curvularia sp. was selected as the test
2001; Volke-Sepulveda et al., 2002).                                fungus, and samples other than Ecoflex were subjected
   Against this background, we sought to find out                    to the same regimen.
whether microorganisms can attack and/or fully degrade                 After 9 weeks, the polymer samples were removed
synthetic polyolefinic materials that have been subjected            from the plates, washed for 3 min with a solution of
beforehand to novel chemically oxidative treatments.                sodium hypochlorite, and rinsed with double-distilled
The results related to structural changes in the polymeric          water before microscopic examination, which was
chain, observed by Fourier transform infrared spectro-              conducted using an optical microscope.
scopy (FTIR), and the behavior of fungi on polymer                     The photographs shown in Fig. 3(c) are of the
surface are reported.                                               specimens stained with lactophenol blue.
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Fourier transform infrared spectroscopy                             sample within less than 2 weeks and degrading it almost
                                                                    totally in less than nine. Most strains of Curvularia sp.
  Infrared spectra in the middle range (4000–400 cmÀ1)              are capable of degrading soil, plants, and cereal grains in
were acquired at a resolution of 2.0 cmÀ1 and a scanning            tropical or subtropical areas, and a few of them are also
number of 32 with a Vector 22 FTIR spectrometer from                found in temperate zones.
Bruker equipped with a deuterated triglycine sulfate                   It is worth noting that the aromatic–aliphatic co-
(DTGS) detector. All the spectra were recorded under                polyester Ecoflex was chosen because of the presence
an N2 atmosphere. The analyses were carried out on a                along its synthetic chain of both aromatic and aliphatic
pressed mixture prepared by using about 5 mg of                     esters and of the respective carbonylic acids as end
powdered polymeric material in 30 mg of dried KBr.                  groups. The formation of such chemical moieties along
                                                                    the polyolefinic chain represents the key factor influen-
                                                                    cing the biodegradability of the treated polymers, which
                                                                    makes Ecoflex an ideal material to study fungal
Results and discussion                                              biodegradation of oxidized polymers.
                                                                       We chose atactic polystyrene, one of the most widely
   In nature, fungi are among the major decomposers,                used synthetic polymers, for our study because its high
particularly in cases of such natural polymers as cellulose         recalcitrance to biodegradation is well known. To
and lignin. However, most biodegradation studies use                induce changes in its structure and thus facilitate the
bacteria, both in the laboratory and in the field. One               mechanism through which microorganisms can assim-
reason is that classical microbiological enrichment tech-           ilate the carbon contained in the polymer, polystyrene
niques favor bacteria since fungi grow more slowly and              was first subjected to novel chemically oxidative
often require co-metabolic substrates for growth; how-              treatments able to transform its polymeric chains into
ever, when fungi are isolated, the mycelial growth allows           more oxidized compounds of presumably lower mole-
rapid colonization of substrates. Several studies have              cular weight. These treatments trigger a great variety of
reported that bacteria are incapable of degrading such              physical and chemical changes, leading to the formation
recalcitrant polymers as polyimides, whereas fungi are              of carbonyl and hydroxyl groups, as is clearly seen by
more effective (Gu et al., 1996, 1997a, b).                         the analysis of the FTIR spectra shown in Fig. 1.
   The biodegradation of a polymer can occur by means                  It is well established that carbonyl (CQO) as well as
of enzymes capable of attacking it and breaking it down             hydroxyl (O–H) groups are the main products in
into chemical compounds small enough to be trans-                   oxidation treatments. Infrared spectroscopy is the best
ported inside a microbial cell and metabolized further.             technique to determine the presence of these groups,
Microbes that can degrade natural polymers may act by               because the absorbance of CQO and O–H bonds is
depolymerizing the compound and/or utilizing the low                quite intense and falls in a region of the infrared
molecular weight intermediates generated in the degra-
dation process (Gu, 2003).
   Generally, microbes cannot degrade synthetic poly-                                     3400 cm-1                   1740 cm-1
mers, principally the polyolefins, which are made up of
only carbon and hydrogen atoms, considered to be
resistant to biodegradation. This is probably due to a
total lack in the polymer’s backbone of sites involving
carbon-to-oxygen bonds (CQO, C–OR, C–OH), which                                                                                                d

are the real target of microbial enzymes.
   To evaluate the effect of different treatments on the                                                                                       c
response of the treated samples to microbial degrada-
tion, we used the samples as co-substrates for the growth
of a filamentous fungal species. Although growth on a                                                                                           b
polymeric surface is not a sufficient condition to prove
that fungus assimilates the carbon contained in the                                                                                            a
polymer, thereby implying its biodegradation, such
growth can be considered both a necessary condition                                4000   3500   3000   2500    2000      1500    1000   500       0
for biodegradation and an easy, fast and clear test to                                                         cm-1
assess the response of macromolecular material to                   Fig. 1. FTIR spectra of polystyrene with and without chemical
biodegradation.                                                     oxidative treatment (from bottom): (a) untreated atactic
   Curvularia sp., with its reported ability to degrade             polystyrene, (b) sample 1, (c) sample 2, and (d) sample 3.
complex natural fibers (Ohkawa et al., 2000), proved to              The hydroxyl (3400 cmÀ1) and carbonyl (1740 cmÀ1) bands are
be the best decomposer of Ecoflex, colonizing the                    indicated.
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spectrum in which no other polystyrene bands are                          All the signals present in the spectra were normalized
present. Fig. 1 presents the infrared spectra of untreated             with respect to the signal at 1601 cmÀ1 due to the in
polystyrene (a) and those of chemically oxidized samples               plane stretching of the C–C bonds of phenylic groups in
(b)–(d). The appearance of the carbonyl band                           polystyrene. This band was used as reference since it
(1740 cmÀ1) was observed in all the treated samples,                   does not change during the degradation of polystyrene.
whereas this band was absent in the untreated poly-                       In fungal degradation tests deploying Curvularia sp.,
styrene; the broadness of this band suggests that the                  the fungus began to colonize the surface of the treated
treatment led to the formation of different carbonylic                 samples after 4 weeks, while the atactic polystyrene
compounds with different chemical environments. In                     sample was completely surrounded. In subsequent
samples 2 and 3, the appearance of the hydroxyl band                   weeks, a slow colonization of the treated samples was
(3400 cmÀ1) was clearly visible and could be related both              observed, whereas atactic polystyrene remained free of
to carboxylic acids and alcoholic compounds. Car-                      fungal colonization, as expected.
boxylic acids, esters and alcohols show an additional                     After 9 weeks, the test was discontinued. Curvularia
absorption band in the region between 1000 and                         had completely invaded the surface of sample 3, partly
1200 cmÀ1 due to characteristic vibrational stretching                 colonized sample 2 and attacked the surface of sample 1
of the C–O bond. Although atactic polystyrene presents                 only at a few points, as shown in Fig. 2 (b)–(d), but
characteristic absorption bands in this region of the                  entirely failed to colonize the surface of atactic
spectrum, as can be seen in Fig. 1(a), in the treated                  polystyrene (Fig. 2(a)), demonstrating the resistance of
samples there was an evident modification of this region,               polystyrene to microbial attack without a preliminary
and the appearance in sample 3 of a broad band at                      chemical oxidative treatment that changes its structure.
1180 cmÀ1 (Fig. 1(d)) confirmed the formation of                           Fig. 3 (a)–(c) shows the results of microscopic
compounds involving C–O bonds.                                         examination of samples 1–3.
   As to the degree of oxidation of the samples, it should                The images clearly show the septate, brown hyphae
be noted that the intensity of absorbance of carbonyl                  penetrating the polymer and forming a well-defined
and hydroxyl groups increased with the strength of                     network on its surface. It has to be emphasized that 10
chemically oxidative treatments of atactic polystyrene.                sections were drawn randomly from each sample for the

Fig. 2. Photographs taken after 9 weeks of incubation with Curvularia species: (a) untreated polystyrene, (b) sample 1, (c) sample 2,
and (d) sample 3.
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Fig. 3. Microscopic observation (400 Â ) of (a) sample 1, (b) sample 2 and (c) sample 3 showing hyphae adhering to and penetrating
the sample as well as conidia growing on the polymer.

microscopic examination to represent the whole surface of             surface of the sample that had been oxidized the most,
the sample in the examination. Microscopic observation on             attacked only a few points on the surface of the least
samples 1 and 2 revealed the hyphae attacking only the                oxidized sample and could not colonize untreated
polymeric surface in the sections colonized by Curvularia             polystyrene at all.
sp., whereas all the analyzed sections of sample 3 revealed              Microscopic examination showed hyphae adhering to
that the hyphae had penetrated the sample beyond its                  and penetrating the polymeric surface and forming
surface. Moreover, the growth of brown, multiseptate                  spores in all the treated samples. Although fungal
conidia, characteristic of Curvularia sp. (8–14 Â 21–35 mm),          growth on a polymeric surface is not a sufficient
was observed on the polymer’s surface.                                condition to prove the assimilation of carbon contained
                                                                      in the polymer, thereby implying its biodegradation,
                                                                      colonization of and adhesion to a polymeric surface by
Conclusions                                                           microorganisms is a fundamental prerequisite to biode-
   Atactic polystyrene was subjected to novel chemically
oxidative treatments to induce chemical changes in its
polymeric chains. CQO, C–O and O–H groups were                        Acknowledgment
detected by FTIR analysis.
   At the end of 9 weeks, when the degradation test was                  The authors acknowledge the financial support from
terminated, Curvularia had completely invaded the                     Italian Ministry of Research MURST – CNR 60%.
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