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International Biodeterioration & Biodegradation 60 (2007) 245–249

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Biodegradation of automotive waste polyester polyurethane foam using

Pseudomonas chlororaphis ATCC55729
R. Gautama, A.S. Bassia,, E.K. Yanfulb, E. Cullenb
a
Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ont., Canada N6A 5B9
b
Department of Civil and Environmental Engineering, The University of Western Ontario, London, Ont., Canada N6A 5B9
Received 16 January 2007; received in revised form 19 February 2007; accepted 16 March 2007
Available online 11 May 2007

Abstract

Automotive waste polyester polyurethane (PUR) foams represent a major solid waste management problem. In the
present investigation, we examined the capacity of Pseudomonas chlororaphis ATCC 55729 to biodegrade waste polyester
PUR foam obtained from an automotive industry in shake cultures. Ammonia nitrogen, pH and diethylene glycol (DEG) concentrations
were found to increase steadily over a period of 12 days. Furthermore, scanning electron photomicrographs of foam pieces also
showed evidence of biodegradation. This shows that waste PUR foams can be successfully biodegraded under controlled laboratory
environment.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Polyester polyurethane foam; Foam biodegradation; Polyurethane esterase enzyme; Degradation by-products

1. Introduction Studies related to non-foamed PUR materials have

Polyurethane (PUR) foams are synthetic polymers
widely used throughout the world for diverse applications.
Enormous quantities of used PUR foams are disposed of in
landfills and incinerators, which cause resource depletion
and litter problems (Zheng et al., 2005). Biodegradation
may provide an innovative solution to these problems.
Biodegradation of both foamed (Hedrick and Crum,
1968; Filip, 1978; Martens and Domsch, 1981; Bentham
et al., 1987; Kay et al., 1991) and non-foamed (Montgomery
et al., 1995; Howard et al., 1999; Nakajima-Kambe et al.,
1999) PUR materials have been previously examined.
These investigations have provided evidence that PUR
materials are biodegradable.
One possible PUR metabolic pathway is shown in Fig. 1
(Nakajima-Kambe et al., 1997; Gautam et al., 2007). However,
there is a lack of information on the formation of degradation
by-products such as diethylene glycol (DEG) and ammonia.
Corresponding author. Tel.: +1 519 661 2111x88324;
fax: +1 519 661 3498.
E-mail address: abassi@uwo.ca (A.S. Bassi).
shown promising results. However, it is noted that non-
foamed PUR materials possess physical and chemical
properties that are distinct from those of foamed PUR
materials (Gautam et al., 2007). This difference in proper-
ties may affect the biodegradation process parameters such
as rates of biodegradation, microbial growth and forma-
tion of biodegradation byproducts. Therefore, more studies
are needed to understand the waste PUR foams biode-
gradation process if the goal is to design bioreactors or
bioprocesses for practical applications.
In this paper, we describe the biodegradation of
waste polyester PUR foam obtained from an automotive
industry using Pseudomonas chlororaphis ATCC 55729.
The type of foam chosen in this study is particularly
used as seat cushions in automobiles. P. chlororaphis was
chosen because several previous studies have successfully
used this bacterium to biodegrade PUR products. Our
study also presents, for the first time, the kinetics of
generation of ammonia nitrogen and DEG during the
biodegradation experiments. This information should be
helpful in designing waste PUR foam biodegradation
systems.

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doi:10.1016/j.ibiod.2007.03.009
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246 R. Gautam et al. / International Biodeterioration & Biodegradation 60 (2007) 245–249

CH3
O O O H
H

N C O (CH2)4 C O C N

Urethane bond Ester bond

m
n
Typical aromatic Typical polyester
isocyanate group polyol group

(Typical polyester urethane polymer)

H2O
Hydrolysis

O O

HO C (CH2)4 C OH HO (CH2)2 O (CH2)2 OH

(Adipic acid) (Diethylene glycol)

CH2OH O

CH3CH2 C CH2OH N C O

CH2OH H
(Trimethylol propane) (Unknown compound)

Fig. 1. (Adapted from Gautam et al., 2007.)

2. Materials and methods 2.2. Total ammonia nitrogen, pH and DEG measurement

2.1. Bacterial strain and cultivation
All reagents were purchased from Sigma-Aldrich (St. Louis, Mo) unless
otherwise noted. Freeze-dried culture of P. chlororaphis (ATCC 55729)
were grown overnight in soy trypticase broth (VWR International,
Mississauga, Ont., Canada) (2971 1C, 125 rpm) and used as an inoculum.
Liquid culture medium for foam biodegradation experiments was
prepared according to Crabbe et al. (1994): Solution A: K2HPO4
(50 g l 1), KH2PO4 (25 g l 1); solution B: MgSO4
7H2O (50 g l 1); solution
C: MnCl2
4H2O (2 g l 1), CuCl2
2H2O (28 mg l 1), ZnCl2 (22 mg l 1),
CaCl2
6H2O (40 mg l 1), FeCl3
6H2O (150 mg l 1); solution D: gelatin
(4 g l 1), NH4Cl (0.4 g l 1), yeast extract (20 mg l 1), Na2MoO4 (20 mg l 1)
were autoclaved separately at 121 1C for 20 min and while they were still
hot (E70 1C), 20 ml of solution A, 10 ml of solution B and 1 ml of solution
C were aseptically added to 970 ml of solution D to make a total of 1 l
solution.
The serial dilution method was used to measure the colony-forming
units (i.e. cfu ml 1) in the inoculum and in the culture broth containing
foam pieces. Soy trypticase agar plates were used to perform the cfu
counts.
Large pieces (50  50  3 cm) of waste polyester PUR open cell
foams were obtained from a local automotive industry. They
were cut to 0.5  0.5  0.5 cm dimensions, washed twice with
distilled water and oven dried at 90 1C to constant weight. One gram of
the dried foam pieces was autoclaved and aseptically introduced into a
500 ml Erlenmeyer flask containing 200 ml of sterile medium. Four
identical flasks were used; three of them were inoculated with 0.1 ml of
P. chlororaphis inoculum prepared above. One uninoculated flask was
used as control. All flasks were incubated by shaking at 150 rpm and
2971 1C.
A total volume of 5.5 ml of the liquid phase was collected periodically
from each culture for cfus, total ammonia nitrogen (NH3 N), pH and
DEG analyses. NH3 N was determined using a HachTM DR/2000
spectrophotometer and Hach ammonia–nitrogen measurement reagent kit
(ClearTech Industries Inc., Mississauga, Ont., Canada). For the final
weight measurement, foam pieces were collected periodically, they were
vigorously washed with distilled water three times and oven dried at 90 1C
to constant weight.
DEG was extracted with ethyl acetate according to the protocol
described by Nakajima-Kambe et al. (1997). Five milliliters of liquid
culture was centrifuged (10,000  g), filtered (0.45 mm) and acidified to pH
1 by adding 15 ml of concentrated HCl. The solution was mixed with an
equal volume of ethyl acetate and extracted twice. The solvent phases were
combined, dehydrated over anhydrous Na2SO4 and concentrated to 1 ml
in a vacuum desiccator. DEG was analyzed by gas chromatography (GC)
using a Varian Chrompack GC (model CP 3800) equipped with a CP-SIL
5CB (10 m  0.53 mm ID) column and a flame ionization detector. The
oven temperature program was as follows: 80 1C initial, 1 min holding
time, 16 1C min 1 increment to a final 250 1C. The carrier gas was nitrogen.
A calibration curve in the range of 2–40 mg l 1 of DEG in ethyl acetate
was prepared to quantify the DEG concentration in unknown samples.
2.3. Assay to measure PUR esterase activity
P. chlororaphis (ATCC 55729) produces an extra-cellular PUR esterase
(PURE), which is associated to PUR biodegradation (Allen et al., 1999;
Howard et al., 1999). In this study, we measured PURE activity produced
in the culture supernatant at the 12th day of incubation. The culture
supernatant was centrifuged at 10,000  g and filtered (0.45 mm). Total
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protein concentration was measured using the Bradford method
(Bradford, 1976) with bovine serum albumin (BSA) as a standard.
PURE produces p-nitrophenol (PNP) and acetic acid from p-
nitrophenyl acetate (PNPA). PNP has a characteristic yellow color with
maximum wavelength of absorption at 405 nm. For the determination of
enzymatic activity, the 8 mM PNPA solution was prepared according to
Kay et al. (1993). PURE activity of culture supernatants was measured in
1.5 ml cuvet containing 0.8 ml of 0.1 M phosphate buffer, 0.066 ml of the
culture supernatant and 0.134 ml of the 8 mM PNPA solution. PNP was
monitored at 405 nm using a Varian Cary 50 UV–vis spectrophotometer
and an extinction coefficient value of 18.5 ml mmol 1 cm 1 (Akutsu et al.,
1998).
2.4. Scanning electron photomicrograph
Foam pieces were collected periodically from liquid culture medium.
They were washed vigorously with distilled water and then dried in an
oven at 90 1C to constant weight. Dried foam pieces surfaces were
examined by scanning electron microscope (SEM) to determine evidence
of biodegradation.
3. Results and discussion
3.1. Bacterial growth, total ammonia nitrogen, pH and foam
weight
The viable cell count of the inoculum was approximately
2  109 cfu ml 1. Under the growth conditions used, the
growth curve for P. chlororaphis reached a stationary phase
in approximately 8 to 10 h after inoculation. The viable cell
concentration remained constant at 5  107 cfu ml 1 from
then until the end of the experiment. The specific growth
rate was calculated to be 1.03 h 1. In the control flask,
microorganisms were not detected.
As shown in Fig. 2, NH3 N concentration in the
inoculated flasks remained constant for the first 2 days
and then increased steadily to reach a maximum of
approximately 40 mg l 1 on the 12th day. The initial
8.8 50
8.6
8.4 40
8.2
NH3 N (mg l-1)
30
8.0
pH
7.8
20
7.6
7.4 10
7.2
7.0 0 0
0 2 4 6 8 10 12
Time (day)
Fig. 2. Time course change in ammonia nitrogen concentration and pH.
&, J: pH and total ammonia nitrogen in inoculated flasks, respectively.
X, n: pH and total ammonia nitrogen in control flasks, respectively.
247
concentration of approximately 2 mg l 1 is attributed
to the presence of ammonium salt in the liquid
medium. NH3 N concentration in the control flask
remained constant throughout the experiment. The
soluble substrates available in the culture media were most
likely exhausted rapidly during initial bacterial growth.
Bacteria might have then started targeting PUR foam for
energy. Therefore, the generation of ammonia is attributed
to the biodegradation of PUR foam in the inoculated
flasks.
Similarly, as shown in Fig. 2, the pH in the inoculated
flasks steadily increased and reached a value of 8.6. It is
noted that for the initial 2 days, there was no change in pH
in the inoculated flasks. This result is consistent with the
NH3 N concentration results. In the control flask, the pH
remained constant.
The weight measurement data did not show any
significant weight loss in the foam samples. However, the
12-day incubation period was most likely too short to
obtain precise estimations of weight loss.
3.2. Diethylene glycol
Consistent with above observation about NH3 N, as
shown in Fig. 3, DEG concentration increased steadily,
starting at the second day, at a rate of approximately
0.9 mg l 1 day 1 in the inoculated flasks. No DEG was
detected in the control flask. It is noteworthy that DEG
production rate was almost linear, which may reflect mass
transfer limitations during the biodegradation of the solid
plastic foams. A similar observation was reported by
Nakajima-Kambe et al. (1997) in an investigation involving
solid polyester PUR pieces by Comamonas acidovorans TB-
35. However, to our knowledge DEG production during
PUR foam biodegradation has never been reported
previously.
10
8
Diethylene glycol (mg l-1)
6
4
2
0 2 4 6 8 10 12
Time (day)
Fig. 3. Time course change in diethylene glycol concentration. K, m:
diethylene glycol concentrations in inoculated and control flasks,
respectively.
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3.3. Total protein concentration and activity assay 3.4. Scanning electron photomicrograph

The total protein concentration and the PURE specific
activity of the culture supernatant at the end of the
experiment were measured to be 0.02 mg ml 1 and
0.069 U mg 1, respectively. No PURE activity was detected
in the uninoculated control. PURE activity from culture
supernatant was not further purified. However, a comparison
of specific activities of crude culture supernatants reported in
different research studies is presented in Table 1. This table
shows that the specific activity of the culture supernatant in
this study is within the range reported in other works.
SEM photomicrographs of undegraded, sixth day
and 12th day foam samples are presented in Fig. 4
(A, B and C). As shown in this figure, the foam
matrices in the sixth and 12th day samples reveal increased
number of holes. The presence of holes in the foam
matrix provides a visual evidence of the biodegradation of
foam caused by P. chlororaphis. Akutsu et al. (1998)
obtained similar SEM photographs; however, in their
experiment, they used purified PURE to degrade solid
PUR pieces.

Table 1
Comparison of specific activities of crude culture supernatants

PUR source (substrate) Bacteria Protein conc (mg ml 1) Specific activity (U mg 1)a Reference

Automotive PUR foam waste P. chlororaphis 0.02 0.069 Present study

5 mm PUR cubes C.acidovorans 0.93 0.030 Akutsu et al. (1999)
ImpranilTM E. coli expressing P. fluorescens genes 2.19 0.097 Vega et al. (1999)
5 mm PUR cubes C.acidovorans 3.00 2.200 Akutsu et al. (1998)b
ImpranilTM C.acidovorans 0.04 3.375 Allen et al. (1999)
a
Values obtained using p-nitrophenyl acetate as substrate (in all studies, 1 U is defined as the amount of enzyme required to liberate 1 mmol of p-
nitrophenol per minute).
b
Intracellular enzyme as opposed to extra cellular enzyme in other studies.

Fig. 4. Scanning electron micrographs of foam samples with 100x magnification: (A) undegraded foam, (B) and (C) foam samples subjected to
biodegradation for six and 12 days, respectively. Appearance of holes is evident in the foam matrices after subjecting them for biodegradation.
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4. Conclusions
Automotive waste polyester PUR foams were subjected
to biodegradation using P. chlororaphis in a defined liquid
medium. The growth of bacteria and the production of
ammonia nitrogen and DEG were monitored for a period
of 12 days. SEM photomicrographs of foam samples were
also taken to visually identify surface changes occurring in
the foam matrix. The liquid medium showed increased
concentration of ammonia nitrogen, DEG and pH. In the
landfills where waste foams are usually disposed of, they
may generate ammonia and other toxic chemicals such as
DEG, which end up in landfill leachate. This study shows
that it is important to treat such waste foams separately.
The rate of generation of DEG was calculated to be
approximately 0.9 mg l 1 day 1 and the rate was found to
be linear, reflecting mass transfer limitations during
biodegradation of solid plastic foams. PURE specific
activity of the culture supernatant was 0.069 U mg 1.
Scanning electron photomicrographs of foam samples also
revealed visual evidence of biodegradation.
Acknowledgments
The authors would like to thank the Natural Sciences
and Engineering Council of Canada (NSERC) Discovery
Grants to A. Bassi and E. Yanful for providing support to
the ongoing PUR foam biodegradation research at the
University of Western Ontario. R. Gautam is especially
thankful to Ivan Malek and Ross and Jean Clark scholar-
ships.
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