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Degradation of polyurethane by bacterium isolated from soil and assessment

of polyurethanolytic activity of a Pseudomonas putida strain

Article  in  Environmental Science and Pollution Research · March 2014

DOI: 10.1007/s11356-014-2647-8 · Source: PubMed

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Environ Sci Pollut Res
DOI 10.1007/s11356-014-2647-8
BIODEGRADABILITY ASSESSMENT OF ORGANIC SUBSTANCES AND POLYMERS
Degradation of polyurethane by bacterium isolated from soil
and assessment of polyurethanolytic activity of a Pseudomonas
putida strain
Yu-Huei Peng & Yang-hsin Shih & Yen-Chun Lai &
Yuan-Zan Liu & Ying-Tong Liu & Nai-Chun Lin
Received: 23 November 2013 / Accepted: 10 February 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract The increasing usage and the persistence of polyes-
ter polyurethane (PU) generate significant sources of environ-
mental pollution. The effective and environmental friendly
bioremediation techniques for this refractory waste are in high
demand. In this study, three novel PU degrading bacteria were
isolated from farm soils and activated sludge. Based upon 16S
ribosomal RNA gene sequence blast, their identities were de-
termined. Particularly robust activity was observed in Pseudo-
monas putida; it spent 4 days to degrade 92 % of Impranil
DLNTM for supporting its growth. The optimum temperature
and pH for DLN removal by P. putida were 25 °C and 8.4,
respectively. The degradation and transformation of DLN in-
vestigated by Fourier transformed infrared spectroscopy show
the decrease in ester functional group and the emergence of
amide group. The polyurethanolytic activities were both pre-
sented in the extracellular fraction and in the cytosol. Esterase
activity was detected in the cell lysate. A 45-kDa protein
bearing polyurethanolytic activity was also detected in the
extracellular medium. This study presented high PU degrading
activity of P. putida and demonstrated its responsible enzymes
during the PU degradation process, which could be applied in
the bioremediation and management of plastic wastes.
Keywords Polyurethane . Biodegradation . Pseudomonas
putida . Fourier transformed infrared spectroscopy
Responsible editor: Robert Duran
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-014-2647-8) contains supplementary material,
which is available to authorized users.
Y.<H. Peng : Y.<h. Shih (*) : Y.<C. Lai : Y.<Z. Liu : Y.<T. Liu :
N.<C. Lin
Department of Agricultural Chemistry, National Taiwan University,
No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan, China
e-mail: yhs@ntu.edu.tw
Introduction
Polyurethane (PU) is a class of polymers derived from the
condensation of polyisocyanate and polyol. Depending on
the different types of components, PUs can adopt various
forms ranging from flexible to rigid and from liquid to solid
elastomer. Therefore, PUs are suitable to produce a wide
variety of products. The global consumption of PUs was
about 8 million tons in 2000 and is expected to reach 9.6
million tons in 2016. Their increasing usage and their per-
sistence have raised concerns about huge waste litter and
serious environmental pollution. Recently, the bioremedia-
tion method was proposed because of its low cost and
reducing environmental impact. The effective and meaning-
ful bioremediation techniques for refractory and hazardous
waste need preliminary investigations of novel degraders and
clarifications of the main mechanism for their microbial
degradation.
Biodegradation of PU has been reported in several bacteria
or fungi isolated from soils or plant, such as Pseudomonas
putida, Alicycliphilus sp., or Pestalotiopsis microspora
(ElSayed et al. 1996; Oceguera-Cervantes et al. 2007; Russell
et al. 2011). Few of these degraders took PU as a sole carbon
source. For example, Pseudomonas aeruginosa decomposed
PU within mini-salt medium (Mukherjee et al. 2011; Shah
et al. 2013). The studies on degradation kinetics, optimal
conditions, and the characteristics of responsible enzymes
were only restricted in Pseudomonas fluorescens, Pseudomo-
nas chlororaphis, Comamonas acidovorans, Aspergillus
terreus, and Acinetobacter gerneri (Allen et al. 1999;
Howard and Blake 1998; Howard et al. 2012; Howard et al.
1999; Wales and Sagar 1988). The putative polyurethanases
(PUases) could be secreted in culture medium, presented in
cytosol, or anchored on cell membrane (Howard and Blake
1998; Mukherjee et al. 2011; Nomura et al. 1998). Recombi-
nant esterases, proteases, and ureases could digest PU through

cleaving the ester or peptide bonds (Zachinyaev et al. 2009).
Purified PUases presented either protease or esterase activity
and would be blocked by serine hydrolase inhibitor, soybean
trypsin inhibitor, or bivalent cation chelator (Allen et al. 1999;
Howard et al. 2012). Therefore, the putative PUases did not
restrict to a single type of enzyme. The functions of PUases
were illustrated only when the responsible genes were identi-
fied. So far, only four genes encoding PUases have been
cloned and characterized from environmental microorgan-
isms: PulA from P. fluorescens, PueA and PueB from
P. chlororaphis, and PudA from C. acidovorans (Howard
et al. 2001; Nomura et al. 1998; Stern and Howard 2000;
Vega et al. 1999). Parsimony analysis indicated that these
enzymes were evolved independently instead of a single ori-
gin (Howard et al. 2001).
The objective of this study was to isolate novel PU
degrading microorganisms from environmental samples in
Taiwan. Three active bacteria species were identified. Degra-
dation kinetics was performed with liquid medium so that
microorganisms which utilize PU as sole carbon source were
identified. The optimal conditions for bacterial growth and
removal of PU, transformation of the polymer, the
polyurethanolytic activities, and the correspondent enzyme
for one of the isolate, P. putida, have been characterized. Our
results illustrated the possible and quick biodegradation pro-
cess of PU by indigenous microorganisms. Such information
is necessary when the degraders are applied in PU waste
management and in the process of monomer recycling.
Materials and methods
Isolation of PU degrading bacterial strains
A collection containing about 500 soil microorganisms was
isolated from the farm of the National Taiwan University and
the Taoyuan District Agricultural Research and Extension
Station. To establish this collection, 5 g of soil was resuspend-
ed in 45 mL of physiological saline solution (0.90 %w/v of
NaCl) and serially diluted followed by plating 100 mL each of
the diluted solution on Luria-Bertani (LB) agar plates
(10 g L−1 NaCl, 10 g L−1 tryptone, 5 g L−1 yeast extract, and
15 g L−1 agar). After incubating at 30 °C for 1 to 2 days,
colonies with distinctive morphology were taken and
subcultured until axenic cultures were obtained. These micro-
organisms were propagated onto PU plates that contained 1 %
(v/v) Impranil DLNTM (an anionic aliphatic polyester polyure-
thane dispersion from Bayer MaterialScience) and agar
(17 g L−1) in minimum salt medium (MSM). The composition
of MSM was as follows: 7 g L−1 K2HPO4, 2 g L−1 KH2PO4,
1 g L−1 (NH4)2SO4, 0.1 g L−1 MgSO4⋅7H2O, 0.001 g L−1
ZnSO 4⋅7H2 O, 0.0001 g L−1 CuSO 4⋅5H 2O, 0.01 g L−1
FeSO4⋅7H2O, and 0.002 g L−1 MnSO4⋅6H2O, pH adjusted
Environ Sci Pollut Res
to 7.2. The liquid polymer was added after autoclaving the
medium in order to prevent degradation. Plates were incubat-
ed at 30 °C for 1–2 weeks. Colonies with clear zones were
isolated. Activated sludge was obtained from aerobic tanks at
the wastewater treatment facilities (Bojue Community, New
Taipei City, Taiwan). The sludge was serially diluted by the
diluent solution which contained 42.5 mg L−1 KH2PO4 and
405 mg L−1 MgSO4⋅7H2O (pH 7.2). A 0.2-mL aliquot of the
appropriate dilution was spread onto PU plates and then the
plates were incubated at 30 °C for 1–2 weeks. Colonies
bearing PU degradation activities were isolated and
subcultured with PU plates until axenic cultures were obtain-
ed. The LB was used to amplify each strain under 37 °C
overnight. The isolated culture was mixed with equal volume
of 50 % glycerol and stocked at −80 °C.
Identification of the isolated strains
A 1.5-mL overnight LB culture of each strain was used to
extract genomic DNA by the Tissue & Cell Genomic DNA
Purification Kit (GeneMark, Tainan, Taiwan) according to the
manufacturer’s instructions. For 16S ribosomal RNA (rRNA)
gene analysis, genomic DNA was amplified using primer 27 F
(5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-
TACGGTTACCTTGTTACGACTT-3′). PCR was performed
by Minicycler PTC-150 (Bio-Rad, Hercules, CA, USA), in a
50-μL mixture, which contained 1 U Taq polymerase, 20
pmol of each primer, and 10 nmol of dNTP. The annealing
temperature was 56 °C, and the reaction was carried out for
30 cycles. Sequences of the amplified fragments were identi-
fied by MB Mission Biotech, Inc. (Taipei, Taiwan) using the
Applied Biosystems 3730xl DNA Analyzer (Foster City,
USA). The obtained sequences were deposited into the
GenBank database using Basic Local Alignment Search Tool
(BLAST) of the National Center for Biotechnology Informa-
tion. The most similar sequences and their accession numbers
were acquired. A threshold of 97 % similarity was taken to
define the species. Phylogenetic analysis was performed using
the Vector NTI program, version 7. The phylogram was
constructed according to the neighbor-joining method and
was displayed by the TreeView program.
Measurement of the PU degradation
Degradation kinetics and optimal degradation conditions were
performed in liquid PU medium prepared by the same recipe
of the PU plate while agar was not added. In these experi-
ments, DLN was the sole carbon source for the organisms to
metabolize and grow. The single colony of bacteria was grown
in 3-mL LB medium under 37 °C overnight. The culture was
then diluted to 30-mL LB medium and grown for another 4 h.
The log-phase culture was washed with an equal volume of
MSM twice and then resuspended to an OD600 of ~0.5. In
Environ Sci Pollut Res
order to compare the degradation activities of isolated micro-
organisms within a short period, 12.5 mL of the resuspended
cells were applied in 37.5 mL of liquid PU medium, making
1 % (v/v) as the final concentration of DLN. When different
conditions of temperatures or pH values were compared, the
inoculated cell ratio was less in order to reveal the effect of
different conditions: 5 mL of the resuspended cells were
applied in 45 mL of liquid PU medium. The pH of the medium
was adjusted by the diluted NaOH or HCl solution before
autoclave. Five-milliliter aliquots were seeded into glass tubes
for batch experiments. The noninoculated liquid PU medium
was used as the blank control. The tubes were shaken at
100 rpm in the orbital shaker incubator (Yih Der LM-530R,
Taipei, Taiwan) and kept in the dark. A too high rotation speed
makes DLN aggregate on the wall and precipitate in the
medium. At a specific time point during degradation, a
0.5-mL portion of each tube was transferred to an Eppendorf
tube and centrifuged at 3,300×g for 5 min to pellet bacteria
cell bodies. The absorbance of the supernatant was measured
using an Infinite M200 plate reader (Tecan, Research Triangle
Park, NC), with MSM as a blank. DLN was an aliphatic
compound containing a hydroxyl group based upon the infor-
mation from the supplier. The absorbance values were highest
at 300 nm and lowest at 900 nm within this scanning range,
probably due to the scattering than due to the presence of any
chromophores (Supplemental Fig. S1A). In order to avoid the
interference from other metabolites produced during the deg-
radation process, the detection wavelength of 400 nm was
chosen because the absorbance ratio of experimental settings
to cell-free control was the lowest and the most stable at
around 400 nm (S. Strobel, personal communication, Supple-
mental Fig. S1B). The absorbance ratio of a similar concen-
tration of DLN standard (0.05 %) to cell-free control also
showed the same pattern (Supplemental Fig. S1C). The effect
of temperature and pH was also performed as above under the
selected temperature or pH. Different dilutions of DLN with
MSM were measured to construct a standard curve for
converting the absorbance to the DLN concentration (Supple-
mental Fig. S2). The cell number of bacteria was measured by
the plate count method on LB plates. The growth rate was
calculated based upon the formula: k=[ln (m2 / m1)] / t2 −t1, in
which m stands for the cell number in specific time t. All
experiments were performed in triplicate.
Fourier transformed infrared spectroscopy (FT-IR) analysis
of PU degradation
The organic functional groups of DLN before and after cul-
turing with bacteria were analyzed by FT-IR. At the end of
DLN degradation, a 10-mL portion of the culture medium was
centrifuged at 3,300×g for 5 min. The noninoculated liquid
PU medium was used as a control. The supernatant was then
passed through a 0.22-μm filter to remove bacterial cell body
or relatively large particles. The filtrate was air-dried and the
dried powder was pelletized together with potassium bromide
powder. FT-IR spectra were obtained from a Horiba FT-720
spectrophotometer, working in the range of wavenumber 550–
4,000 cm−1.
Enzyme assay
When DLN was almost degraded, the cells of P. putida were
spun down and resuspended to an OD600 of ~0.5 with 50 mM
phosphate buffer made by K2HPO4 and KH2PO4 (pH 7.0).
Cells were broken by sonication with a Branson Sonifier 450
(output control 3, Duty cycle 20 %) for six times, each
consisting of 10-s pulses with 10-s intervals on ice. After
centrifugation at 17,000×g for 15 min at 4 °C, the supernatant
was then passed through a 0.22-μm filter to remove bacterial
cell body or relatively large particles. Protein concentration
was determined by the Bradford assay (Bio-Rad, Richmond,
CA). In-tube degradation assay was performed by adding
0.1 mg of lysate proteins into 0.6 % of DLN in the same
buffer and shaken at 37 °C. Esterase assay was performed
according to the method of Allen et al. (1999), using p-
nitrophenyl acetate as substrate (0.5 mM) and 0.05 and
0.1 mg of lysate proteins.
Zymogram
The analysis was performed by the method of Chang et al.
(2004) with slight modification. The supernatant of the culture
in which P. putida has degraded almost the DLN was used.
Protein was precipitated by 10 % TCA for 30 min at 4 °C.
After centrifugation, the protein pellet was resuspended by
acetone. Protein was centrifuged again and resuspended in
SDS sample buffer and subjected to electrophoresis on a SDS-
PAGE containing 1 % DLN. After electrophoresis, the gel was
washed twice for 30 min at 4 °C in 100 mM Tris-HCl buffer
(pH 7) containing 25 % isopropanol and then incubated in
Tris-HCl buffer for 30 min at 4 °C one time to remove SDS.
After incubated in Tris-HCl buffer for 16 h at 37 °C to renature
proteins, the gel was soaked in 0.1 % Congo red solution for
30 min at room temperature and destained with 1 M NaCl
until a clear zone appeared. The protein size was referred to
the total protein analyzed by SDS-PAGE and stained by
Coomassie blue solution.
Data analysis and statistics
Statistical analysis was carried out by SPSS 19 (IBM SPSS
Inc, Chicago, IL). Univariate analysis of variance
(UNIANOVA) was applied to the repeated data obtained
under different conditions. The Tukey’s test was used for post
hoc tests of significant differences between means.
 Environ Sci Pollut Res

Results and discussion (A)

Initial PU clearance screen

DLN is a milky suspension and becomes transparent once

(B)
degradation has occurred. A clear zone would present on the
PU plate around the colonies of microorganisms which can 100%
degrade PU. Therefore, PU degraders could be isolated from Blank
A6

DLN degradation (%)

environmental samples directly by vision (Howard and Blake 80%
A12
1998; Russell et al. 2011). A collection of 13 colonies were
60% 4-47
chosen and then grouped into four types of microorganisms
according to their morphologies. All of the clear zones were
with little opacity, indicating incomplete degradation. 40%

According to the sequences of amplified 16S rRNA gene

20%
fragment, the identity of each strain has been identified. Three
of them were 99 % identical to Pseudomonas otitidis (strain
0%
81f, accession number AB698739.1); one isolate is 99 % 0 1 2 3 4
identical to P. putida (strain AS90, accession number Time (day)
AY622320.1); the others were 99 % identical to Burkholderia
gladioli (strain 343-3318, accession number HM231304.1) (C)
(Table 1). The relationship of these three species with their
1.00E+40
closely related species was also assigned by reconstructing a
phylogenetic tree (Supplemental Fig. S2). A6 in the first group A6
CFU/ml

of isolates clustered with the species of P. otitidis. Besides A12

Pseudomonas plecoglossicida, P. putida was the closest to
A12. Strain ID number 4-47 in the third group showed the
highest relationship with B. gladioli. P. putida is able to
degrade various organic compounds with aromatic or aliphatic
functional groups, such as toluene, phenol, and alkanes
(Mordocco et al. 1999; Rojo 2009). It could grow on solid
agar containing PU-painted metal coupons (ElSayed et al.
1996). Because of the wide range of subtracts and the non-
pathogenic nature, P. putida was used for developing biore-
mediation strategies to remove hazardous chemicals. The
other two PU degraders isolated in this study have not yet
been reported before.
PU degradation kinetics
The degradation kinetics of three different species (A6, A12,
and 4-47) was performed at 30 °C (Fig. 1b). Within the initial
2 days, A12 showed the highest degradation efficiency that
45 % of DLN was degraded. A6 degraded 25 % of DLN and
4-47 showed less degradation (16 %). The degradation kept in
the following days; consequently, A6 and A12 degraded 92 %
1.00E+30
4-47
1.00E+20
0 1 2 3 4
Time (day)
Fig. 1 Degradation of DLN by the three isolated bacteria under 30 °C. a
The visual images for the residual amount of DLN at day 3. b Degrada-
tion kinetics of DLN of bacteria during DLN degradation. c The growth
curve of bacteria during DLN degradation. The experiments were per-
formed in triplicate. The error bar indicated the standard deviation
of the DLN and 4-47 degraded 47 % of DLN at day 4.
Compared to the blank control, these three strains showed
significant degradation ability (A12: F1, 28 =17.514, P<0.001;
A6: F1, 28 =11.768, P<0.01; 4-47: F1, 28 =8.981, P<0.001).
Post hoc Tukey’s test revealed that the DLN degradation
percentage by the three degraders was different with the blank
control after day 2; A12 was the fast degrader at the same time
point (P<0.001). Compared to the growth curve during deg-
radation, the cell number of the three isolates increased with
DLN degradation (Fig. 1c). Post hoc Tukey’s test revealed that

Table 1 Identification of three

PU degradation bacteria ID Sequence length (bp) Closest match (BLAST search) Identity (%) Accession no.

A6 1,407 Pseudomonas otitidis 99 AB698739.1

A12 1,402 Pseudomonas putida 99 AY622320.1
4-47 1,396 Burkholderia gladioli 99 HM231304.1
Environ Sci Pollut Res
the cell number of 4-47 was significantly larger than the others
(P<0.001). At the end of degradation (day 3 to day 4), the
growth rates of A6 and A12 decreased from 8.0 to 3.9 and 8.0
to 3.4 day−1, respectively. This might be due to the shortage of
carbon source and energy source. Since all of the degraders
grew during the degradation, the degrading by-products could
be not toxic for the survival and growth of these degraders.
The growth rate for B. gladioli was the highest (9.5 day−1)
while its degradation activity was the lowest. On the contrary,
P. putida showed the highest degradation activity while the
cell number was the least. P. putida was also well known for its
abilities in breaking down various aromatic or aliphatic hy-
drocarbons which were hazardous chemicals caused by burn-
ing fuel, coal, tobacco, and other organic matters (Sohn et al.
2010; Wang et al. 2007). P. otitidis has been reported bearing a
high capacity to decolorize triphenylmethane dyes (Wu et al.
2009). B. gladioli has been reported as a pesticide degrader
(Malghani et al. 2009).
The effect of temperature and pH
The degradation of DLN with P. putida was temperature
dependent. The degradation rate and total removal amount
were highest at 25 °C, followed by 30 °C and then 35 °C
(A)
(B)
100%
DLN degradation (%)
80%
60%
40%
20%
0%
0 2 4 6
Time (day)
Fig. 2 Effect of temperature on the DLN degradation efficiency of
Pseudomonas putida. a The visual images for the residual amount under
different temperatures at day 5. Bk: no inoculation blank control; A12:
(Fig. 2). No DLN was degraded at 40 °C. The degradation
kinetics was significantly different under different tempera-
tures (F3, 56 =5.289, P<0.01). Post hoc Tukey’s test revealed
that at day 2 and day 5, the DLN degradation percentage at 25,
30, and 35 °C was significantly different with that at 40 °C and
with each other (except 30 and 35 °C at day 2) (P<0.001). The
cell numbers were decreased through the increasing of tem-
perature (data not shown). A previous report also mentioned
that the growth of P. putida was quickest at 25 °C and stopped
at 45 °C (Li et al. 2010). In this study, the growth rate
correlated to the degradation efficiency at above temperatures,
except 40 °C. The inhibition of DLN degradation at 40 °C
may be due to the weak enzymatic activity. According to the
results, 25 °C was selected for subsequent experiments and
also indicated that the indigenous P. putida can degrade PU in
the common environment without elevating the temperature.
Figure 3 shows the degradation activity of P. putida under
different initial pH conditions. The original medium pH
values ranged from 5.5 to 8.4. The degradation rate and total
removal amount were the highest under pH 8.4, followed by
pH 7.0 and lowest at pH 5.5. The degradation kinetics was
significantly different under different pH values (F4, 70 =
5.569, P<0.001). Post hoc Tukey’s test revealed that after
day 2, the DLN degradation percentage at pH 8.4, 7.2, 7.0,
Blank P. putida
25 25
30 30
35 35
40 40
8
inoculated with P. putida. b The degradation kinetics under different
temperatures. The experiments were performed in triplicate. The error
bar indicated the standard deviation
 Environ Sci Pollut Res

(A)

(B)
100%

80%
DLN degradation (%)

Blank P. putida

60% pH5.5 pH5.5

pH5.8 pH5.8

40% pH7.0 pH7.0

pH7.2 pH7.2
20% pH8.4 pH8.4

0%
0 2 4 6
Time (Day)
Fig. 3 Effect of initial medium pH value on the DLN degradation control; A12: inoculated with P. putida. b The degradation kinetics under
efficiency of Pseudomonas putida. a The visual images for the residual different pH values. The experiments were performed in triplicate. The
amount under different pH values at day 6. Bk: no inoculation blank error bar indicated the standard deviation

and 5.8 was significantly different with that at pH 5.5 and with
each other (except 7.2 and 7.0 at day 2) (P<0.001). The low
degradation ability below pH 6.0 was consistent with a previ-
ous report (Fortner et al. 2003). DLN was not lysed in acidic
or basic solutions because the concentrations of DLN in blank
controls were stable. The pH values decreased at day 6 in
inoculated samples: pH 5.5 shifted to pH 4.4, pH 7.0 shifted to
pH 6.6, and pH 8.4 shifted to 7.0. The reduction of medium
pH value may have been caused by the secretion of organic
acids into the medium, corresponding to the FT-IR result (see
(asterisk in Fig. 4). It stood the C=O stretching vibration,
which was characterized by the ester carbonyl functional
group of DLN (Gokulakumar and Narayanaswamy 2008).
The absorption peak disappeared in the inoculated medium,
indicating hydrolysis of ester. The absorption peak around
3,230–3,400 cm−1 in DLN and 3,404 cm−1 in the inoculated
80
Bk
70 P. putida
Transmittance (%)

60
below). The growth of P. putida was the fast between pH 7.0
and 7.4 (Hudcova et al. 2011); however, the optimal pH for 50
DLN removal in this study was out of this range, indicating 40
that high enzymatic activity may overcome the low growth 30
rate when the original pH value was 8.4.
20

FT-IR analysis of PU degradation 10

3500 3000 2500 2000 1500 1000
Wavelength (cm-1)
The degradation and transformation of PU were inspected by Fig. 4 IR spectra of DLN before (Bk, gray line) and after (P. putida,
FT-IR spectroscopy. In the blank control without P. putida black line) degradation by Pseudomonas putida. The asterisk indicated
inoculation, an absorption peak at 1,735 cm−1 was shown the ester carbonyl functional group of DLN
Environ Sci Pollut Res

sample was characterized by the hydrogen bond N–H 150%

stretching and O–H stretching. The absorption peaks around
2,780–2,950 cm−1 presented in both samples indicated the C–

H stretching. The peak at 1,560 cm−1 that corresponded with
amide II and the peak at 1,385 cm−1 that corresponded with
amide III or C–H bonding were absent in the control sample
(Khanam et al. 2007). Coupled with the broadened peak
between 3,230 and 3,404 cm−1, these results indicated the
presence of amino acids (Guillén and Cabo 2000; Khanam
et al. 2007) and the possible urethane cleavage. However,
since DLN was the sole carbon source for P. putida during
the degradation process, it offered the materials for cell growth
and metabolism. Therefore, the amide bond detected in the
medium might originate in some metabolites or proteins
which could be secreted in the medium due to the growth of
the organism. This also supported the cell growth during the
degradation process and the dependence of DLN of P. putida.
The degradation of PU by other microorganisms has also
been investigated by FT-IR. PU film pieces degraded by
P. aeruginosa showed the disappearance of ester linkage
(Shah et al. 2013). PU could de degraded by commercialized
esterase, protease, or urease (Howard 2011). Mukherjee et al.
(2011) also presented that the degradation of polyurethane
diol by P. aeruginosa is partly mediated by its esterase activity.
Pestolotiopsis microspora spent 16 days to completely de-
grade its sole carbon source—DLN. The ester stretch of
DLN also disappeared in the IR spectra. However, there were
no other metabolites that have been detected in the literature
(Russell et al. 2011). Since their detection time point was in
the middle of the degradation (day 6 compared to day 16), the
amount of metabolites might be too low to be detected.
esterase activity
100%
50%
0%
Bk low high
Fig. 5 The activity of intracellular esterase of Pseudomonas putida
measured when 0.05 mg (low) or 0.1 mg (high) of lysate proteins was
used. Bk: blank control. The experiments were performed in triplicate.
The error bar indicated the standard deviation. *P<0.001 as compared
with control and low dosage of input protein by Tukey’s test
2011). Therefore, the medium supernatant was used for the
zymogram assay. Five major proteins were presented in the
medium according to SDS-PAGE and Coomassie blue stain-
ing (asterisks in Fig. 6a). A clear band on the zymogram
corresponded to one of the major bands, indicating this protein
was involved in the degradation of DLN (arrow in Fig. 6b). Its
molecular weight was about 45 kDa. Several PUases have
been purified or identified from various bacteria, ranging from
27 to 65 kDa (Howard et al. 2001; Howard et al. 1999; Vega
et al. 1999). Membrane-bound PUase has also been found in
C. acidovorans (Akutsu et al. 1998; NakajimaKambe et al.

Characterization of putative PUase

The polyurethanolytic activity of P. putida was assayed by in-

tube degradation assay and esterase assay. In-tube degradation
assay was performed by incubating 0.1 mg of cell lysate
proteins with 0.6 % DLN in phosphate buffer at 37 °C. After
6 days of incubation, 35 % of DLN was degraded (data not
shown). Esterase activity was detected by the generation of
by-product—p-nitrophenol. When 0.1 mg of cell lysate pro-
teins were added, the absorbance value of the experimental
sample was 36 % more than that of the control (Fig. 5, high).
The difference between the sample and the control was not
obvious when a lower amount of lysate proteins was added
(Fig. 5, low). Post hoc Tukey’s test revealed a significant
increase of esterase activity only when a higher dosage of
lysate protein was loaded (P<0.001). The PU degrading ac-
tivity of P. putida was probably caused by the esterase activity.
The polyurethanolytic activities were detected by cell ly-
Fig. 6 a SDS-PAGE of extracellular proteins of Pseudomonas putida
sate and extracellular medium by radial diffusion assay (data was presented in lane 2. The molecular weight of markers was presented
not shown). The extracellular distribution of PUases was in lane 1 and in the unit of kilodaltons. b Zymogram of the same sample
reported previously (Howard et al. 2012; Russell et al. showed a clear zone which was correspondent to a 45-kDa protein in (a)

1997). Whether P. putida bears more PUases and whether they
are distributed in other cellular fractions would be interesting
questions and waited to be investigated.
Conclusions
Three PU degradation bacteria (13 isolated strains) were iso-
lated from a microbial library containing 500 strains from soils
and activated sludge. They utilized DLN as sole carbon source
for growth. Pseudomonas putida showed the strongest degra-
dation ability when compared to the other two novel de-
graders. The optimum temperature and pH for DLN removal
were 25 °C and 8.4, respectively. By FT-IR observation at the
end of the degradation process, the transformation of DLN
into metabolites was proposed. Both the extracellular medium
and cytosol presented polyurethanolytic activity. The enzy-
matic activity of the cell lysate was assayed by in-tube DLN
degradation assay and esterase assay. Besides, a 45-kDa pro-
tein bearing polyurethanolytic activity was detected on the
zymogram. Our results indicated that P. putida degraded DLN
through the extracellular enzyme. This better understanding
would be helpful in applying PU for waste management and
material recycling.
Acknowledgments The authors thank Bayer Material Science for of-
fering free sample.
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