Mechanistic Aspects of Polyurethane Biodeterioration

D.S. Wales & B.F. Sagar

Shirley Institute, Wilmslow Road, Didsbury, Manchester M20 8RX, UK

ABSTRACT

Polyester-based polyurethanes are particularly susceptible to bio-

degradation by a wide range of micro-organisms. Many of them
produce extracellular esterases, which have been used to study the
degradation of model polyurethanes. The polymer containing only
urethane links, and the polymers containing ester and urethane links
were degraded, indicating that both are susceptible to enzymatic
hydrolysis. A polyester-based polyurethane containing an effective
biocide was still degraded by the isolated enzyme preparations. Partial
characterisation of the crude esterase preparations from four micro-
fungi by gel permeation chromatography has indicated possible
differences in the mechanism ofpolyurethane degradation by different
micro-organisms. Analysis of the MW distributions of a polyester-
based polyurethane before and after microbial degradation showed
that bulk MW; although reduced, is not unduly affected by the action
of micro-organisms, but polydispersity of the polymer increases.

INTRODUCTION

Although all organic polymers contain a potential carbon source for

microbial growth, most synthetic polymers are resistant to biodegradation
(potts et al., 1973). Polyester-based polyurethanes, however, are particularly
susceptible to biodegradation of their polymer structure by a wide range of

351
352 D.S. Wales, B.F. Sagar

micro-organisms (Darby & Kaplan, 1968; Kaplan et al., 1968); the

mechanism is believed to be mediated through hydrolytic enzymes that
attack the polyester component of the polyurethanes (Pathirana & Seal,
1985; Wales & Sagar, 1985). However, since polyether-based polyurethanes
also exhibit limited susceptibility to microbial degradation (Kaplan et al.,
1968), it is likely that the urethane links of the polyurethane structure are
also hydrolysed enzymatically.

MATERIALS AND METHODS

Production of crude esterase preparations

Aspergillus terreus, Chaetomium globosum, Cladosporium cladosporioides,

Rhizopus stolonifer and Trichoderma viride were grown in batch culture in
5 litres stirred fermenters at 30°C in PL medium, and crude esterase
preparations isolated by acetone precipitation (Wales & Sagar, 1985).

Commercial enzymes

The following commercial enzymes were obtained: Palatase (lipase) and

Alcalase (protease)-(Novo Industri NS); Lipase A (John & E. Sturge
Ltd); Papain (protease) and Takamine fungal protease (Miles Kali-
Chemie GmbH).

Estimation of esterase activity

Esterase activity was estimated by the spectrophotometric method

described by Wales & Sagar (1985).

Synthesis of polyurethanes

Four polyurethanes have been studied PUI (derived from butane-l,4-diol

and isophorone diisocyanate) and PU2 (from poly(ethylene adipate) and
toluene diisocyanate) were synthesised as described previously (Wales &
Sagar, 1985). PU3 was synthesised similarly from poly(ethylene adipate),
butane-l,4-diol and toluene diisocyanate; a biocide, Actifresh PDlll6
(British Sanitised), was added to a portion of this polymer at a
concentration of O· 5% by weight of solids before the films were cast PU4
was a commercial polyester-based polyurethane.
 Mechanistic aspects of polyurethane biodeterioration 353

Degradation of polyurethanes on agar plates

Dumb-bell shaped specimens (length 12 cm, gauge width, 0·6 cm) of

PU3 (with and without biocide) or PU4 were placed on two types of agar
plate, one a mineral salts agar (MSA) as described in BS6085 (Anon.,
1981), the other a minimal nutrient agar (MNA) containing the same
salts as MSA but with added mycological peptone (1% w/v) and malt
extract (1 % w/v). The plates containing PU3 were inoculated with single
spore preparations of Chaetomium globosum, Cladosporium cladosporioides
or Trichoderma viride, prepared as described in BS6085 (Anon., 1981). The
PU4 specimens were inoculated with a mixed spore preparation of
Aspergillus amstelodami, A. niger, Aureobasidium pullulans, Penicillium
funiculosum, P. ochro-chloron, Rhizopus stolonifer, Stachybotris atra and
Ulocladium oudomansi. After incubation at 28 ± 1°C for 4 weeks, the
dumb-bells were removed from the plates, washed, and their tensile
strength determined (Anon., 1976).

Enzyme treatment of polyurethanes

Dumb-bell shaped specimens of polyurethanes were exposed to enzyme

solutions (0·4 units/ml) as described by Wales & Sagar (1985).

Gel permeation chromatography of crude enzyme preparations

The crude enzyme preparations fromA. te"eus, C. globosum, R. stolonifer

and T. viride were subject to gel permeation chromatography (Wales &
Sagar, 1985).

Gel permeation chromatography of PU4

Molecular weight distribution measurements of PU4 before and after

biodegradation were made by gel permeation chromatography of the
polymer, dissolved in tetrahydrofuran (THF) on a 60 cm mixed pore size
column ofPL-GEC (styrene based) with a mobile phase (THF) flow rate
of 1 mllmin; the eluate was passed through a UVIKON ultraviolet
detector (280 mm) and the resulting signal analysed on a BBC 128K
microcomputer. Molecular weights quoted from this analysis should not
be taken as absolute values; they have been calculated using the Mark-
Houwink constants for polystyrene (the calibration standard), since
constants for PU4 were not available, but comparisons between
undegraded and degraded samples are valid.
354 D.S. Wales, B.F Sagar
RESULTS AND DISCUSSION

Enzymatic degradation of polyurethanes

The esterase elaborated by A. terreus and the commercial protease

Alcalase were most effective in degrading PU1; significant reductions in
tensile strength were also effected by the esterase produced by C.
globosum and Lipase A (Table 1). The breaking extension ofthis polymer
was not significantly affected, however, by treatment with any of the
enzyme preparations except for papain. These results demonstrate the
susceptibility of the urethane groups to enzymolysis, since they are the
only hydrolysable bonds in PUl. Whether both esterases and proteases
are implicated remains uncertain since the protease Alcalase possesses
4-nitrophenyl acetate hydrolase activity, presumably due to a con-
taminating esterase, and the possibility that both the crude laboratory
esterase preparations from A. terreus and C. globosum contain protease
activity has not been eliminated.
Only the esterase from R. stolonifer and Takamine fungal protease
failed to reduce significantly the tensile strength ofPU2 (Table 1). Thus
TABLE 1
Tensile Properties of PUI and PU2 after Enzyme Treatment

Enzyme PUl PU2

Tensile Loss over Breaking Tensile Loss over Breaking

strength control extension strength control extension
(MN/m2) (%) (%) (MN/m2) (%) (%)
Buffer 14'1 5'5 35'8 513
A. te"eus 8·8 37·6° 8'2 28·8 19'6b 447
esterase
C. globosum 11'3 19'8° 8'5 28'4 20·6° 495
esterase
R. stolonifer 15'2 +7·8 2'5 29'9 16'5 519
esterase
T. viride 13·1 7'1 4·8 32'4 9'5° 508
esterase
Lipase A 11-4 19·2° 2'2 31-8 l1'2 C 511
Palatase 11'9 15'6 6'7 24'3 32'2 c 446
Alcalase 8'6 39'0< 1'7 29·5 17·6c 482
Papain 13·1 7·1 0·3 29'6 17·3 c 489
Takamine 14·1 0'0 2-3 35'0 2'2 512
fungal protease

°Significantly different from the buffer control at the 95% confidence level.
bSignificantly different from the buffer control at the 98% confidence level.
CSignificantly different from the buffer control at the 99% confidence level.
 Mechanistic aspects of polyurethane biodeterioration 355

there is ample evidence from these results and the results of Pathirana &
Seal (1985) that the ester links in polyester-based polyurethanes are
susceptible to enzymatic hydrolysis.

Biodegradation of a polyester-based polyurethane containing a biocide

PU3, without the inclusion of a biocide, was degraded by T. viride, C.

globosum and C. cladosporioides on MSA and MNA plates, and by aseptic
treatment with their crude esterase preparations (Table 2). No significant
degradation of PU3 containing Actifresh PD 1116 was observed on the
agar plates but aseptic treatment with the crude esterase preparations
isolated from these three micro fungi again caused substantial reductions
in the tensile strength (Table 2). These results have important implications
concerning the degradation of polyurethane coated fabrics in the field;
growth of micro-organisms on surface contamination may produce
extracellular esterase, which can migrate to the polyurethane surface,
and cause degradation despite the polymer being protected with a biocide.

Gel permeation chromatography

Esterase enzymes
Separation of the crude laboratory esterase preparations from C.
globosum, R. stolonifer and T. viride (Fig. 1) produced only single peaks of
esterase activity, coinciding with major protein peaks (MWs 135000,
150000 and 160 000 daltons respectively). Separation of the crude
esterase fromA. te"eus, however, resulted in two peaks of esterase activity
with MWs of 104 000 and 160000 daltons; protein peaks were associated
with both of these esterase peaks, and a third protein peak (MW 40 000
daltons) was also observed. These results indicate that the complexity of
enzymatic breakdown of polyurethane varies from one micro-organism
to another. The two peaks of esterase activity from the separation of the
crude enzyme preparation from A. te"eus may indicate the presence of
both endo- and exo-activity. Endo-acting esterase would have a marked
effect on the degree of polymerisation by cleaving bonds randomly along
the polymer chain, resulting in appreciable loss in tensile strength but
relatively little concomitant weight loss, in contrast to the action of an
exo-esterase, which removes successive monomer units from the chain
ends, resulting in a disproportionate weight loss relative to the effect on
tensile strength. Work carried out previously by the authors has
indicated that biodeterioration of polyester-polyurethanes by C. globosum
or T. viride produces significant losses in tensile strength with little or no
weight loss, whereas A. te"eus caused appreciable losses in both tensile
strength and weight.
 \J.)
VI
0\

TABLE 2
Biodeterioration of PU3 with and without Biocide

Micro-organism Treatment No additive Biocide treated

Tensile Loss over Breaking Tensile Loss over Breaking

strength control extension strength control extension
(MN/rrr) (96) (96) (MN/rrr) (96) (96)
Control (0·5% MSA 14·8 652 7·3 848
MNA 12·8 660 7·2 764 !::1
sodium azide) !'l
Buffer 12-1 668 6·5 923
~
T. viride MSA 12·9 12.80 550 7·4 +1·3 804 s.o~
MNA 7·5 41.46 568 7·2 0·0 900 ~
Esterase 6·9 43.06 800 2-1 67.76 1120 !"!l
15·1 +2·0 604 7·0 4·1 816 ~
C. globosum MSA ~
MNA 6·5 49.2 6 520 7·9 +9·7 763 ....
Esterase 7·5 38.06 650 3·6 44.66 900
C. cladosporioides MSA 9·5 35.86 532 7·8 +6·8 846
MNA 11·0 14.1 0 620 7·9 +9-7 867
Esterase 7·8 35.56 834 2·3 64.66 1076

°Significantly different from the control at the 95% confidence level.

6Significantly different from the control at the 99% confidence level.
 Yj"" 0·03 300 0·08
R. stolonifer
E 0·07
Yj"" 0·06
0·02 200
:fiE
"oJ E 0·05
Yjc 0·04 ~
g..
~ 100 ,... 'e 0.03 §
ILl 0·01 "i ~.
<I 0 0 .02 I i / \12001 ;:;.
E \rJVI 100 CI
~ CI ~ 0'01
E E ~
:~ 01. Yo ok , I ?z.rt10 W o I-Y. , ,V 0"- 0 Q ....orn--IO ~
u
. <I 1.:l"
111
2330 40 50 60 70 80 90100 .5 23 30 40 50 60 70 80 90 100 ·i
0·12 r J\ 600 ! 0-08 600 () ~
tI -~ r ]
CII C. globosum 1 f 'g
111 0.10 500 Q. ~ 0-07 t T. viride 500 d:
~
'- 111 ;:::
tI 0·06
400 tI ~
1ii 0·08 CII .400 So
ILl /I
0·05
'-
§
tI 0·04 ~300 'I>
~
til
..
ILl 0·03
1\ ~.
!}
0-02 ~ \ ~
I. ro ~.
O-O~ A. "I ; ...,,:00 i:!
::~lrL~~
23 30 40 50 60 70 80 90 100 2330 40
tic 50 60 70 80 90 100
g.
;:,:
Elution volume (ml)

Fig. 1. Esterase activity (0-0) and protein (e-e) after separation of crude enzyme preparations from Aspergil/us terreus, Trichoderma viride,
Rhizopus stolonifer and Chaetomium globosum on a Sephadex G-IOO column. ...,
~
358 D.S. Wales, B.F. Sagar

Molecular weight distribution of biodegraded PU4

GPC analysis ofthe MW distribution ofPU4, after exposure to the mixed

inocula on MSA and MNA plates, indicated (Table 3) a greater reduction
in MW and increase in polydispersity on MSA than on MNA; the
polymer on MNA was hardly affected in terms of bulk MW but showed
an increase in polydispersity. These results, taken in conjunction with
the changes in tensile strength (Table 3), show that microbiological
degradation need not unduly affect the bulk of the polymer. In this
experiment it seems likely that the extensive reduction in strength results
from the initiation of superficial cracks, which propagate through the
film under tension. The negligible losses in weight vis-iI-vis the large
reductions in tensile strength of the microbiologically-degraded polymer
(Table 3) suggest that much of the degradation is mediated by endo-
acting enzymes. The pattern of reduction in molecular weight could also
be expected from attack by endo-acting enzymes.

TABLE 3
Molecular Weights of PU4 by GPC and Comparison with Tensile Strength and Weight
Loss

Treatment Relative MW Polydispersity Relative Relative

MW MN MW/MN tensile weight
strength loss

Control 1 1 1'58 1 1
Mixed spore MSA 0'86 0'72 1'89 0·30 1
Mixed spore MNA 0-98 0'90 1'73 0'34 I

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