Advances in Bioscience and Biotechnology, 2011, 2, 52-58 ABB
doi:10.4236/abb.2011.22009 Published Online April 2011 (http://www.SciRP.org/journal/abb/).
Published Online April 2011 in SciRes. http://www.scirp.org/journal/ABB
Polyurethane foam as substrate for fungal strains
Araceli Loredo-Treviño1, Gilberto García2, Abraham Velasco-Téllez2, Raúl Rodríguez-Herrera1,
Cristóbal N. Aguilar1*
1Group of Bioprocesses. School of Chemistry. University of Coahuila. Saltillo, México;
2Research and Development Division. Nemak SA, García, N.L., México;
3Biotechnology Department. School of Chemistry. University of Coahuila. Saltillo, México.
Received 30 January 2011; revised 11 February 2011; accepted 24 February 2011.
ABSTRACT
Polyurethane is a versatile plastic with several indus-
trial applications in the modern life, but it is consid-
ered as a very recalcitrant material. Biodegradation
of this plastic has been poorly explored, and most of
the studies that have been published focus on bacte-
rial enzymes. In this work, some fungi with the ca-
pacity of growing with polyurethane foam as nutrient
source were isolated from sands contaminated with
this plastic and from DIA/UAdeC collection, testing
their ability to grow on polyurethane as sole carbon
and nitrogen sources and their enzymatic activities
were determined in specific media as well as their
invasion capacity on polyurethane agar plates. 22
fungal strains demonstrated their capacity of grow-
ing on polyurethane. Among the enzymatic activities
evaluate, the most common was the urease activity
(95% of the strains).Protease, esterase and laccase
activities were present in 86%, 50% and 36% respec-
tively. The great ability of the isolated fungal strains
to use polyurethane foam as nutrient opens an im-
portant opportunity to study at detail the biodegra-
dation of this plastic, with clear implications in cell
biology and environmental technology.
Keywords: Polyurethane Biodegradation; Fungal
Enzymes; In-Plate Screening
1. INTRODUCTION
Plastics are synthetic long polymer chains with resis-
tance towards microbial attack. Due to their short time
on Earth, nature has not been able to design new enzy-
matic structures that can attack these synthetic polymers.
This has brought concern about how to degrade them.
There are some mechanisms like photodegradation,
thermal degradation, environment erosion and biodeg-
radation and biodegradation is a process in which or-
ganic substances are degraded by living organisms. This
could occur under different conditions depending on the
environment. Plastics are potential substrates for hetero-
trophic microorganisms [1] and polyurethanes are a type
of plastics widely used in various industries. These plas-
tics are synthesized from polyols and polyisocyantes and
classified in polyesther or polyether polyurethanes de-
pending on what substrates are used and the biological
attack towards them is determined by the type of sub-
strates used in the polymer synthesis despite its xenobi-
otic origin [2].
Polyurethane (PU) biodegradation by microorganisms
had become an important issue in the study of degrada-
tion mechanisms and PU aging. PU biodegradation
could be due to utilization of this material as carbon or
nitrogen source by the microorganisms or to fortuitous
biodegradation in presence of other nutrients and sub-
strates [3].
Both fungi and bacteria have been isolated from the
surface of buried PU and showed degradation capacity
[4]. The diversity of mycromycetes and the great amount
of metabolites they secrete allow them to survive in lim-
ited environments [5]. Among fungi there are Gliocla-
dium roseum, Aspergillus, Emericella, Fusarium, Peni-
cillium, Trichoderma, Gliocladium pannorum, Nectria
gliocladiodes and Penicillium ochrochloron [6].
Aureobasidium pullulans is a primary colonizer of poly-
vinyl chloride [7].
The attack towards PU could be due because of the
enzymes produced by microorganisms [8]. Nomura et al
[9] reported that Comamonas acidovorans TB35 has two
types of extracellular esterases. Meanwhile, Howard et
al [10] found that the supernatant of a liquid culture of
Pseudomonas chlororaphis had three different types of
PU activity. A culture of Alicycliphilus sp showed es-
terase activity when degrading PU [11].
The most important contributions have been made us-
ing bacterial strains and the information of fungal bio-
degradation of PU is scarce and old. For this reason, in

A. Loredo-Treviño et al. / Advances in Bioscience and Biotechnology 2 (2011) 52-58 53
this study, we report the results obtained of a study of
isolation and characterization of fungal strains capable to
use PU as sole carbon or nitrogen source, describing the
associated enzymes to PU biodegradation.
2. MATERIALS AND METHODS
2.1. Samples and Chemicals
Sand samples contaminated with PU were provided by
Nemak corporation. These samples were identified as
M1, M2, M3 and M4. M1 corresponded to sand mixed
with soil and aluminum filings: M2 corresponded to
sand that was discarded in a dumpling and contained
aluminum fillings, also some plant growth was present:
M3 was sand after a process that involved milling and
combustion to eliminate the PU: M4, was sand contami-
nated with PU before the cleaning process. All reagents
were analytical grade and all analysis was performed by
triplicate. PU, diisocyante and diol were provided by
Nemak corporation and these PU was used for every test
unless indicated otherwise. PU used in agar plates radial
growth tests was Sayer Lack Mexicana, S.A. de C.V.
(T-0028/A; batch: 6289513). Physicochemical charac-
terization included humidity content, pH, content of
phenols [12], protein [13] and content of molds and
yeasts according to AOAC manual and Mexican Official
Norms (NOM). All assays were made by triplicate.
2.2. Selection of Microorganisms Capable of Growing
Using PU A s Nutrie nt So urce
Culture media using PU as sole carbon or nitrogen
source were prepared (3 g/L). Also the diisocyanate and
the diol used to synthesize the PU were tested (3 g/L of
each component). Composition of mineral medium used,
either with the PU, the diisocyante and the diol, is de-
scribed as follows. Quantity of every component is in
g/L: K2HPO4, 1; KH2PO4, 0.5; MgSO4·7 H2O, 0.5;
MnCl2·4 H2O, 0.001; CuCl2·2 H2O, 1.4 x 10-5; ZnCl2, 1.1
x 10-5; CoCl2·6 H2O, 2 x 10-5; Na2MoO4·2 H2O, 1.3x10-5;
FeCl3·6 H2O, 7.5 x 10-5 [11]. Culture conditions were
30°C and pH 4.5. Fungal strains were isolated from the
sands (coded NK strains) and Centro Internacional de
Servicios Fitosanitarios (CISEF, S.A. de C.V.) provided
some strains (coded CS strains) and the rest of tested
fungi were from the DIA/UAdeC collection. Inocula
were either spores or mycelium. Fungal growth was ob-
served and the strains capable of growing in the PU me-
dia were selected to continue with the research.
2.3. Fungal Growth in PU Plates
As a parameter of the PU degradation capacity of the
fungi, invasion of agar plates containing PU was evalu-
ated. The culture media used was mineral medium and 3
g/L of PU [15]. Also 2 g/L of glucose were added to
avoid stress and accomplish goo measurements. It is
important to mention that no additional nitrogen source
was added so the microorganism was forced to use PU
as sole nitrogen source, in order for us to be able to re-
late the fungal growth to the PU utilization as a nutrient
source. PU used was a PU coat instead of Nemak PU
because it didn’t show good properties to be used as a
support for this test (data not shown). 1x106 spores/mL
of 10µL of mycelia suspension for non-sporulating fungi,
were inoculated in the center of the plate and allowed to
dry. Measurements were made every 24 hours in two
axes during 96 hours. All tests were done by triplicate.
2.4. Estimation of PU Degradation and Related
Enzymatic Activites
To evaluate if polyurethane is better as a carbon or a
nitrogen source, an experiment was performed using
Trichoderma DIA-T sp and a 1:1 ratio of polyurethane
and additional nutrient. Glucose was used as additional
carbon source and ammonium sulfate as additional ni-
trogen source. The culture was carried out in 7 mL cul-
ture tubes using the mineral medium previously de-
scribed. Also an experiment using PU as sole carbon and
nitrogen source was performed to compare expression of
protein in presence or absence of additional nutrients.
The amount of spores used was 5x105 spores/mL and the
agitation was oscillatory for 9.5 days. Mixing was oscil-
latory and the time of culture was 9.5 days and sampling
was made by sacrificing one tube every 12 hours.
Analyses performed were protein [16], total phenol con-
tent [12], urease activity [17] and esterase activity [18].
2.5. Detection of Associated Enzymes
To establish if the selected molds have the enzymes re-
lated to PU degradation, four specific culture media
were used for the detection of those enzymatic activities.
YES medium to determine protease activity, Tween 80
medium for esterase activity, Christensen urea medium
for urease activity and PDA-ABTS (2,2’-azino-bis
(3-ethylbenzhiazoline-6-sulphhonic acid) medium [14]
for laccase activity. Media composition is described as
follows. Quantity of every component is given in g/L:
YES medium: K2HPO4, 1; KH2PO4, 0.5; MgSO4·7 H2O,
0.5; MnCl2·4 H2O, 0.001; CuCl2·2 H2O, 1.4 x 10-5;
ZnCl2, 1.1 x 10-5; CoCl2·6 H2O, 2 x 10-5; Na2MoO4·2
H2O, 1.3x10-5; FeCl3·6 H2O, 7.5 x 10-5, gelatin/peptone,
0.02. Christensen’s urea medium: Urea, 20, NaCl, 5;
K2HPO4, 2; glucose, 1; phenol red, 0.12. The urea is
sterilized by filtration and then added to the rest of the
medium. Tween 80 medium: peptone, 10; NaCl, 5;
CaCl2, 0.1; Tween 80, 10 mL [11]. PDA-ABTS medium:
Potato-dextrose agar 49; ABTS, 0.1M. To carry out the
experiments, 5 mL were disposed in tubes and inocu-

A. Loredo-Treviño et al. / Advances in Bioscience and Biotechnology 2 (2011) 52-58 54
lated with the fungal strains and incubated 8 days at 30
°C, except for PDA-ABTS medium were the incubation
was carried out in plate. Fungal growth was observed in
YES medium, and this indicated a positive protease ac-
tivity; urease activity was determined by the turn in
color from yellow to pink; esterase activity was evi-
denced by the apparition of a white precipitate in the
medium and laccase activity was evidenced by the appa-
rition of a blue-green halo round the fungal colony.
3. RESULTS AND DIS CUSSION
From 32 fungal strains, 22 grew using PU as nutrient
source. The other 10 grew using either the diol or the
diisocyanate. These 22 strains were isolated from the
sand samples, the group collection and provided by
CISEF. Among the selected genera of fungi were As-
pergillus, Trichoderma, Paecelomyces, Penicillium, Al-
ternaria, and Fusarium. These results are similar to re-
sults reported by Lagauskas et al [5]. They isolated a
great amount of fungal species from several types of
plastics including PU and species of Trich oderma, Peni-
cillium, Paecellomyces and Alternaria. Cosgrove et al [4]
also isolated and identified some Penicillia and Alter-
naria among others from buried PU coupons and 3 also
from buried samples of PU, isolated species of Nectria,
Penicillium and Geomyces. These fungal genera were
isolated from plasticized polyvinyl chloride (PVC) [7].
The recurrent presence of these mycromycetes in soil
buried plastics may be due to the great amount of
exoenzymes that fungi secrete and these enzymes allow
the fungi to degrade plastics and probably use them as
nutrient source [5]. Also, these reports used standardized
soil or soil from template regions. Food Research De-
partment and NK strains were isolated from a Mexican
semidesertic region of extreme environmental conditions
(where temperatures and solar radiation are elevated and
water content very low).
It was important to know if the selected strains had the
enzymatic activities related to PU biodegradation so the
enzymatic characterization was performed. These results
are shown in Table 2.
All the fungi grew on the media, but not every mold
showed all the enzymatic activities. In table 1 it can be
observed that the majority of the fungi presented urease
activity (95.45%). The second more common activity
was protease activity (86.36%), then esterase activity
(50%) and the less common activity detected was lac-
case activity (36.26%). These enzymes, as stated previ-
ously, are related to PU degradation, as reported by Bar-
rat, et al [6]. In recent years the research about PU bio-
degradation has focused in bacteria and their enzymes.
The major part of the enzymes reported as PU degrading
enzymes showed esterase activity (19; 20; 10; 11; 21)
and some showed also protease activity (20; 10) but
none of these reports mention urease activity. The diver-
sity of PUs due to the different components used for
their synthesis could be the reason of the difference be-
tween the enzymes produced by the microorganisms. In
this work the PU used was a polyether PU (information
provided by personal of the automotive company. The
main components used to synthesize the PU are
4,4'-Methylenebis(phenyl isocyanate) and phenol for-
maldehyde) and the fungi capable of using it as nutrient
source must be able to produce enzymes that hydrolyze
the plastic and since there are not ester bonds in the
molecule, the fungi that were isolated must have the ca-
pacity to secrete enzymes other than esterases to attack
the PU molecule and use it degradation products as nu-
trient source. In this experiment we determined the
presence of this type of enzymes since they are the main
group reported responsible of polyurethane degradation.
The invasive capacity of the fungi was evaluated on
agar plates with mineral medium and PU. Results are
shown in Figure 1. It can be seen that the fungi have
different growth rate but Trichoderma DIA-T and PSS
showed the fastest growth velocity (0.7917 mm/h and
0.7813 mm/h respectively).
Invasive capacity of fungi has been used to indicate
Table 1. Enzymatic characterization of the fungi capable of using polyurethane as nutrient source.
Fungi esterase protease urease laccase Fungi esterase protease urease laccase
DIA-T - + + + CS3 + + +
FP310 + + + - CS4 - - + -
PSH + + + - CS6 - - + -
EH2 + + + + CS7 - - + -
EH3T1 + + + + NK2 - + + -
PSS - + - - NK4 + + + -
NH4 + + + + NK6 + + -
GS + + + - NK8 + + + +
GH1 - + + + NK10 - + + +
CS1 - + + - NK12 - + + -
CS2 - + + + NK23 - + + -

A. Loredo-Treviño et al. / Advances in Bioscience and Biotechnology 2 (2011) 52-58 55
Figure 1. Invasive capacity of fungi in polyurethane agar plates.
the susceptibility of a plastic to be degraded by these
microorganisms [22] or the microorganism capacity to
grow using the plastic [7] using visual evaluation and a
rating scale. Here we measured the growth rate on agar
plates containing PU and glucose as a parameter of the
mold capacity to use PU as nutrient source. It is impor-
tant to remark that the fungi provided by the
DIA/UAdeC collection are used in biotransformations of
antimicrobial molecules (polyphenols) and the enzy-
matic battery of these strains is very adapted to recalci-
trant substrates [23]. In other works that used bacteria
capable of degrading PU in an agar plate they measured
the diameter of a clear halo around the colony or around
a well filled with bacterial enzymatic extract (6; 20; 10;
15; 24). Fungi tend to invade the medium where they
grow and it could be a good criterion to find molds ca-
pable of adapt to PU as nutrient source.
Even thought NK12 did not show the best growth rate
(0.1597 mm/h) the mycelium and the spores were very
compact and looked very similar to the strain when it
was cultured in PDA medium and since this strain was
isolated from the contaminated sands probably this mold
is adapted to the PU and was selected as well as Tricho-
derma DIA-T and PSS to further research. Macroscopic
images of every mold in polyurethane agar are presented
in Figure 2.
Polyurethane itself has carbon and nitrogen in its
structure and it was necessary to determine if this mole-
cule is a better carbon or nitrogen source to fungi and the
production of the enzymes capable of hydrolyze the PU.
In Figure 3 the amount of protein from the culture
with Trichoderma DIA-T. as model fungus is shown. It
can be seen that, in a general way, the protein production
produced followed the same behavior in all media.
Around day 1.5 the production begins and decreases in
Figure 2. Macroscopic images of fungi in poly-
urethane agar plates.
day 7.5. In figure 4 the results for esterase production are
shown. It is evident in Figure 3 and 4, esterase activity
can’t be related to protein production, so it can be in-
ferred that this enzymatic activity may not be the re-
sponsible for the hydrolysis of the polyurethane mole

A. Loredo-Treviño et al. / Advances in Bioscience and Biotechnology 2 (2011) 52-58 56
Figure 3 Extracellular production of Trichoderma DIA-T under dif-
ferent culture conditions. Polyurethane as sole carbon and nitrogen
source (); ammonium sulfate as additional nitrogen source ();
glucose as additional carbon source ().
Figure 4. Esterase activity of Trichoderma DIA-T. under different
cultura conditions. Polyurethane as sole carbon and nitrogen source
(); ammonium sulfate as additional nitrogen source (); glucose
as additional carbon source ().
Figure 5. Release of phenolic compounds to the cultura media by
Trichoderma DIA-T under different cultura conditions. Polyurethane
as sole carbon and nitrogen source (); ammonium sulfate as addi-
tional nitrogen source (); glucose as additional carbon source ().
cule and/or its modification. In 2005, Santerre et al, re-
ported that cholesterol esterase could degrade ether type
polyurethanes although there was no presence of ester
bonds in the molecule [25] so, it is possible for a fungal
esterase to hydrolyze this type of PU in later stages of
the culture process.
For urease activity (Figure 5), tendency of the fungi is
to produce this enzyme in the first stages of the culture
and this, related with phenolic compounds released to
the culture media (Figure 5, lines). The enzyme could
modify the polyurethane structure so, from day 5 the
phenolic compounds begin to increase. The measure-
ment of phenolic compounds released to the culture me-
dia could be a parameter to estimate the polyurethane
degradation due to the phenolic molecules contained in
its estructure.
Maximum U/L of urease activity reported in this in-
vestigation (4985 U/L) is higher than reported for bacte-
ria by other authors like Shigeno-Akutsu et al [26] that
report a maximum of 3300 U but for esterase activity.
It’s important to highlight that the majority of the articles
adjudge to esterases the hydrolysis of the polyurethanes
that were tested. In 2009, Ibrahim et al, reported degra-
dation of an ester type polyurethane by Alternaria sp.
and found that the strain produced protease, esterase and
urease enzymes when tested in specific media, but when
they look for the enzymes in the polyurethane culture
filtrate they did not found any urease activity [27]. The
present work would be the first report to our knowledge
to attribute the polyurethane degradation to an urease
enzyme.
4. CONCLUSIONS
From two collections and from sand contaminated with
PU, 22 fungal strains were capable of growing using PU
as carbon source, and all of these strains showed at
least one of the enzymatic activities related with PU
biodegradation being the protease activity the most
common one. Two fungal strains showed the fastest
growth on PU plates. Three strains were selected for
further research on degrading PU based on their invasive
capacity, their morphology on PU plates and their enzy-
matic activities and one of them was used to evaluate the
PU degradation in culture media using different addi-
tional nutrients. Probably, urease is the main enzyme
responsible of the fungal attack towards PU as the re-
sults from different culture media using Trichoderma
DIA-T showed.
5. ACKNOWLEDGEMENTS
The authors thank Nemak corporation for materials and samples pro-
vided; CISEF for fungal strains and Consejo Nacional de Ciencia y
Tecnología (CONACYT) for the student grant (258623/221603).

A. Loredo-Treviño et al. / Advances in Bioscience and Biotechnology 2 (2011) 52-58 57
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