Biodegradation of polyurethane - a review

Biodegradation of polyurethane - a review

(Parte 1 de 4)

Biodegradation of polyurethane: a review

GaryT. Howard∗ Department of Biological Sciences, Southeastern Louisiana University, SLU 10736, Hammond, LA70402, USA

Abstract

Lack of degradabilityand the closing of land)l sites as wel as growing water and land polution problems have led to concern about plastics. Increasingly, raw materials such as crude oil are in short supply for the synthesis of plastics, and the recycling of waste plastics is becoming more important. As the importance of recycling increases, so do studies on elucidation of the biodegradability of polyurethanes. Polyurethanes are an important and versatile class of man-made polymers used in a wide variety of products in the medical, automotive and industrial )elds. Polyurethane is a general term used for a class of polymers derived from the condensation of polyisocyanates and polyalcohols. Despite its xenobiotic origins, polyurethane has been found to be susceptible to biodegradation by naturallyoccurring microorganisms. Microbial degradation of polyurethanes is dependent on the manyproperties of the polymer such as molecular orientation, crystallinity, cross-linking and chemical groups present in the molecular chains which determine the accessibility to degrading-enzyme systems. Esterase activity (both membrane-bound and extracellular) has been noted in microbes which allow them to utilize polyurethane. Microbial degradation of polyester polyurethane is hypothosized to be mainly due to the hydrolysis of ester bonds bythese esterase enzymes. ? 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Polyurethane; Degradation; Polyurethanase; Esterase

1. Introduction

Polyurethanes (PU) are present in many aspects of modern life. Theyrepresent a class of polymers that have found a widespread use in the medical, automotive and industrial )elds. Polyurethanes can be found in products such as furniture, coatings, adhesives, constructional materials, )bers, paddings, paints, elastomers and synthetic skins.

Polyurethanes are replacing older polymers for various reasons. The United States government is phasing out chlorinated rubber in marine and aircraft and coatings because they contain environmentallyhazardous volatile organic compounds (Hegedus et al., 1989; Reisch, 1990). Auto manufacturers are replacing latex rubber in car seats and interior padding with PU foam because of lower densityand greater 9exibility(Ulrich, 1983).

Other advantages of PUs are that theyhave increased tensile strength and melting points making them more durable (Bayer, 1947). Their resistance to degradation by water, oils, and solvents make them excellent for the replacement of plastics (Saunders and Frisch, 1964). As coatings, they exhibit excellent adhesion to manysubstances, abrasion

∗ Tel.: +1-504-549-3501; fax: +1-504-549-3851. E-mail address: ghoward@selu.edu (G.T. Howard).

resistance, electrical properties and weather resistance for industrial purposes (Saunders and Frisch, 1964; Urbanski et al., 1977; Fried, 1995).

2. Physical and chemical properties of polyurethanes

Polyurethanes were )rst produced and investigated by

Dr. Otto Bayer in 1937. Polyurethane is a polymer in which the repeating unit contains a urethane moiety. Urethanes are derivatives of carbamic acids which exist onlyin the form of their esters (Dombrow, 1957). This structure can be represented bythe following, generalized amide-ester of carbonic acid:

Variations in the R group and substitutions of the amide hydrogen produce multiple urethanes. Although all PUs contain repeating urethane groups, other moieties such as urea, ester, ether and aromatic maybe included (Saunders and Frisch, 1964). The addition of these functional groups mayresult in fewer urethane moieties in the polymer than functional groups.

0964-8305/02/$-see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S0964-8305(02)00051-3

The urethane linkage results most readilythrough the reaction of an isocyanate, –N!C!O, with an alcohol, –OH (Dombrow, 1957; Kaplan et al., 1968). The hydrogen atom of the hydroxyl group is transferred to the nitrogen atom of the isocynanate (Bayer, 1947). The major advantage of PU is that the chain is not composed exclusivelyof carbon atoms but rather of heteroatoms, oxygen, carbon and nitrogen (Bayer, 1947). The simplest formula for PU is linear and represented by

(–R–O–C–NH–R2–NH–C–O–)n where n is the number of repetitions and R2 is a hydrocarbon chain. R represents a hydrocarbon containing the OH group. Diisocyanates are employed in PU production reactions because theywill react with anycompound containing an active hydrogen (Dombrow, 1957).

For industrial applications, a polyhydroxyl compound can be used. Similarly, polyfunctional nitrogen compounds can be used at the amide linkages. Bychanging and varying the polyhydroxyl and polyfunctional nitrogen compounds, different PUs can be synthesized (Dombrow, 1957). Polyester or polyether resins containing hydroxyl groups are used to produce polyester- or polyether-PU, respectively (Urbanski et al., 1977).

Variations in the number of substitutions and the spacing between and within branch chains produce PUs ranging from linear to branched and 9exible to rigid. Linear PUs are used for the manufacture of )bers and molding (Urbanski et al., 1977). Flexible PUs are used in the production of binding agents and coatings (Saunders and Frisch, 1964). Flexible and rigid foamed plastics, which make up the majorityof PUs produced, can be found in various forms in industry(Fried, 1995). Using low molecular mass prepolymers, various block copolymers can be produced. The terminal hydroxyl group allows for alternating blocks, called segments, to be inserted into the PU chain. Variation in these segments results in varying degrees of tensile strength and elasticity. Blocks providing rigid crystalline phase and containing the chain extender are referred to as hard segments (Fried, 1995). Those yielding an amorphous rubbery phase and containing the polyester=polyether are called soft segments. Commercially, these block polymers are known as segmented PUs (Young and Lovell, 1994).

3. Polyurethane degradation

After years of production of PUs, manufacturers found them susceptible to degradation. Variations in the degradation patterns of diHerent samples of PUs were attributed to the manyproperties of PUs such as topologyand chemical composition (Pathirana and Seal, 1983). The regularityin synthetic polymers allows the polymer chain to pack easily, resulting in the formation of crystalline regions. This limits accessibilityof the polymer chains to degradative agents.

4. Decreasing polyurethane degradation

Research was initiated to elucidate whether additives to the chemical structure of PUs could decrease biodegradation. Kanavel et al. (1966) observed that sulfur-cured polyester and polyether PUs had some fungal inertness. However, theynoted that even with fungicides added to the sulfur- and peroxide-cured PUs, fungal growth still occurred on the polyester PUs and most fungicides had adverse eHects on the formulations. Kanavel et al. (1966) also recognized the need for physical testing of the PUs after extended exposure to the activityof fungi.

Santerre et al. (1994) varied the amount of degradation products released by varying the physical makeup of the polyester PUs, as coatings on glass tubes or as )lms. This implied that while urethane and urea groups are susceptible to hydrolysis, they are not always accessible to the enzyme and degradation maynever proceed past the polymer surface. Although the polyether PUs showed no signi)cant degradation, theyconsistentlyshowed higher radiolabel products release from soft-segment-labeled, enzyme-incubated samples than controls. The authors attributed these results to the shielding of ester sites bysecondarystructures and hydrogen bonding within the hard segment.

Santerre and Labrow (1997) tested the eHect of hard segment size on the stabilityof PUs against cleavage. Analysis was performed with polyether PUs and their susceptibility to cholesterol esterase. Three polyether PUs were synthesized with varying molar ratios of [14C]-diisocyanate to chain extender and constant polyether makeup. A ten-fold increase in enzyme concentration of cholesterol esterase previously used (Santerre et al., 1994) was utilized to approach plateau values for polyether PU hydrolysis. Upon treatment with cholesterol esterase, Santerre and Labrow (1997) observed that radiolabel release was signi)cantlydependent on the amount of hard segment contained within the polymer. In the polymer with the lowest concentration of hard segment, higher numbers of carbonyl groups are exposed to the surface. With increased hard segment size, a greater number of carbonyl groups are integrated into secondary hard segment structures through hydrogen bonding. The investigators also concluded that an increase in hard segment size does lead to restrictions in polymer chain mobility.

In the medical )eld PUs show resistance to macromolecular oxidation, hydrolysis and calci)cation (Marchant, 1992). Polyurethane elastomers are being used in place of other elastomers due to higher elasticityand toughness, and resistance to tear, oxidation and humidity(Dombrow, 1957; Saunders and Frisch, 1964; Ulrich, 1983). In addition, polyether derivatives are inexpensive to produce as prepolymers, which can lower the overall cost of polymer production.

Huang and Roby(1986) tested the biodegradabilityof polyamide-urethanes for medical purposes. They synthesized PUs with long repeating units and alternating amide and urethane groups from 2-aminoethanol. The resulting partial crystalline )bers were observed to undergo hydrolysis bysubtilisin less readilythan polyamideesters with degradation proceeding in a selective manner. The amorphous regions on the PU were being degraded prior to the crystalline regions. These )bers showed promise as absorbable sutures and implants where in vivo degradation is needed. The investigators also noted that PUs with long repeating units and hydrophilic groups would less likely to pack into high crystalline regions as normal PUs, and these polymers were more accessible to biodegradation.

Tang et al. (1997) added surface-modifying macromolecules (SMM) containing 9uorinated end groups to the base PU to reduce the material’s susceptibilityto hydrolysis by lysosomal enzymes. Synthesized polyester urea-urethanes were radiolabled with [14C] and coated onto small hollow tubes. Biodegradation experiments were carried out using methods previouslyestablished bySanterre et al. (1994). Results indicated that degradation was inhibited bythe SMM surface. DiHerent SMM formulations provided varying degrees of enzyme resistance. It was noted that some SMM formulations were incompatible with the PU and led to increased biodeterioration. The mechanism of inhibition was not deduced and will be the subject of further study.

In an attempt to increase biocompatibilityand reduce bacterial adhesion on PU surfaces, Baumgartner et al. (1997) synthesized phosphonated PUs. They used glycerophosphorylcholine (GPC) as the chain extender, which incorporated phosphorylcholine head groups into the PU backbone. This gave the PU surface some characteristics of a red blood cell surface. Physical tests on the PU showed a small decrease in tensile strength and transition temperature with increasing GPC concentration. Water absorption bythe PU was increased with increased GPC content. To test bacterial adhesion to the PU, Baumgartner et al. (1997) used a radial 9ow chamber. Theypassed a culture of Staphylococcus aureus across phosphonated and unphosphonated PU at a rate of 8 ml min−1. The phosphonated PU showed a decrease in bacterial adhesion with increased GPC content.

5. Increasing polyurethane degradation

Lack of degradabilityand increasing depletion of land- )l sites as wel as growing water and land problems have led to concern about plastics (Kawai, 1995). As more and more raw materials (e.g. crude oil) become in short supply for the synthesis of plastics, recycling of waste plastics is becoming important (Schnabel, 1981). Degradability problems promoted researchers to investigate modi)cation or productions that led to either chemicallydegradable or biodegradable PUs.

Huang et al. (1981) derived polyester PUs from polycaprolactonediols in an eHort to produce biodegradable PUs for use in the medical )eld. Several diHerent PUs were made containing polyester subunits of various lengths. The polymers were subjected to degradation bythe enzyme axion and two species of fungi. The enzyme and fungi degraded each PU. In addition, it was also noted that there was an increase in the biodegradabilityof the polyester PUs with increase in the chain length of the polyesters.

In a later study, Phua et al. (1987) observed that two proteolytic enzymes, papain and urease degraded a medical polyester PU. The PU they tested was BiomerJ, segmented, cross-linked polyester PU. Although cross linking was previouslydescribed as a wayof inhibiting degradation (Kaplan et al., 1968), papain (molecular weight 20:7 kDa) had little diMcultyin diHusing into the )lm and causing breaks in the structural integrity. Urease activity, because of its size (molecular weight 473 kDa), was limited to the PU surface and therefore was not signi)cant. Phua et al. (1987) also proposed that papain degraded the polymer by hydrolyzing the urethane and urea linkages producing free amine and hydroxyl groups. The eHect of papain on polyether PU was assessed byMarchant et al. (1987). Comparison of papain activity to aqueous hydrolysis resulted in both releasing degradation products. Ether linkages were non-enzymatically hydrolyzed by water while degradation of the urethane groups was dependent on the presence of the proteolytic enzyme.

Labrow et al. (1996) treated polyester PU and polyether

PU with human neutrophil elastase and porcine pancreatic elastase. The polyester PU was readilydegraded byporcine pancreatic elastase at a rate 10 times higher than byhuman neutrophil elastase. The rate of polyester PU degradation by porcine pancreatic elastase was also 10 times higher than its activityagainst the polyether PU. Human neutrophil elastase had no signi)cant activityagainst the polyether PU. These results indicate a distinct similarityto the degradation of PUs bycholesterol esterase (Santerre et al., 1993, 1994; Santerre and Labrow, 1997). Inhibition of porcine pancreatic elastase was achieved with the elastase speci)c inhibitor NMSAAPVCMK.

6. Fungal biodegradation

After years of production of PUs, manufacturer’s found them susceptible to degradation. Variations in the degradation patterns of diHerent samples of PUs were attributed to the manyproperties of PUs such as molecular orientation, crystallinity, cross-linking, and chemical groups presented in the molecular chains which determine the accessibilityto degrading-enzyme systems (Pathirana and Seal, 1983). The regularity in synthetic polymers allows the polymer chains to pack easily, resulting in the formation of crystalline regions. This limits accessibilityof the polymer chains to degradation whereas, amorphous regions on the PU can degrade more readily. Huang and Roby (1986) observed PU degradation proceeded in a selective manner, with the amorphous regions being degraded prior to the crystalline regions. Also, it was observed that PUs with long repeating units and hydrolytic groups would be less likely to pack into high crystalline regions as normal polyurethanes, and these polymers were more accessible to biodegradation. Several investigators have suggested microbial attack on PUs could be through enzymatic action of hydrolases such as ureases, proteases and esterases (Evans and Levisohn, 1968; Hole, 1972; Flilip, 1978; GriMn, 1980).

Several reports have appeared in the literature on the susceptibilityof PUs to fungal attack (Darbyand Kaplan, 1968; Kaplan et al., 1968; Ossefort and Testroet, 1966). These studies revealed that polyester-type PUs are more susceptible to fungal attack than other forms. In addition, polyether PUs were noted to be moderatelytoo highlyresistant. Boubendir (1993) isolated enzymes with esterase and urethane hydrolase activities from the fungi Chaetomium globosum and Aspergillus terreus. These organisms did not grow solelyon PU and the enzymes had to be induced. Induction of the enzymes was accomplished by addition of liquid polyester PU to the growth media. Activity of the enzymes was determined by assays based on ethyl carbamate (urethane) as arti)cial substrate.

Four species of fungi, Curvularia senegalensis, Fusarium solani, Aureobasidium pullulans and Cladosporium sp. were isolated based on their abilityto utilize a colloidal polyester PU (Impranil DLNTM) as the sole carbon and energysource (Crabbe et al., 1994). Curvularia senegalensis was observed to have a higher PU-degrading activityand therefore subsequent analysis of this fungal isolate was carried out. An extracellular polyurethanase (PUase) displaying esterase activitywas puri)ed from this organism. The protein has a molecular mass of 28 kDa, is heat stable at 100 C for 10 min and inhibited by phenylmethylsulphonyl9uoride (PMSF).

Wales and Sagar (1988) proposed a mechanism for the degradation of polyester PUs by extracellular esterases. Polyurethane degradation is the result of synergistic activity between endopolyurethanases and exopolyurethanases. Endoenzymes hydrolyze the PU molecule at random locations throughout the polymer chain leading to loss of tensile strength. Exoenzymes remove successive monomer units from the chain ends however, show little loss of tensile strength.

7. Bacterial biodegradation

In a large-scale test of bacterial activityagainst PUs, Kay et al. (1991) investigated the abilityof 16 bacterial isolates to degrade polyester-PU. Seven of the isolates tested degraded PU when the media was supplemented with yeast extract. Two isolates, Corynebacterium sp. and Pseudomonas aeruginosa, could degrade PU in the presence of basal media. However, none of the isolates grew on PU alone. Physical tests of the degraded polyester PU revealed different but signi)cant decreases in tensile strength and elongation for each isolate. In a further study, (Kay et al., 1993), tested the chemical and physical changes in degraded polyester PU. Polyurethanes taken from Corynebacterium sp. cultures had signi)cant reductions in both tensile strength and elongation after three days of incubation. Infra-red spectrophotometer analysis revealed the ester segment of the polymer to be the main site of attack. The investigators noted that supplementing the media with glucose inhibited esterase production. However, addition of PU did not increase esterase activity.

Halim El-Sayed et al. (1996) tested the growth of several species of bacteria on PU militaryaircraft paint. The investigators isolated Acinetobacter calcoaceticus, two Pseudomonas sp., Pseudomonas cepacia, and Arthrobacter globiformis. In addition, the U.S. Navysupplied two strains of A. calcoaceticus, Pseudomonas aeruginosa and Pseudomonas putida. All species were capable of utilizing the polyurethane paint as a sole carbon and energy source with the exception of P. cepacia. Using 9uorescein diacetate as an esterase substrate, the remaining species showed esterase activityin the absence of PU. This data indicated that the PUases were constitutivelyexpressed.

In an additional study, Comamonas acidovorans strain

TB-35 was isolated from soil samples byits abilityto degrade polyester PU (Nakajima-Kambe et al., 1995). Solid cubes of polyester PU were synthesized with various polyester segments. The cubes were completely degraded after 7 days incubation when they were supplied as the sole carbon source and degraded 48% when theywere the sole carbon and nitrogen source. Analysis of the breakdown products of the PU revealed that the main metabolites were from the polyester segment of the polymer. Further analysis of strain TB-35 revealed that the degradation products from the polyester PU were produced by an esterase activity(Nakajima-Kambe et al., 1997). Strain TB-35 possess two esterase enzymes, a soluble, extracellular and one membrane-bound. The membrane-bound enzyme was found to catalyze the majority of the polyester PU degradation. The membrane-bound PUase enzyme was puri)ed and characterized (Akutsu et al., 1998). The protein has a molecular mass of 62 kDa, heat stable up to 65 C and inhibited byPMSF. The structural gene, pudA, for the PU esterase was cloned in Escherichia coli. Upon nucleotide sequencing of the open reading frame (ORF), the predicted amino acid sequence contained a Gly-X-Ser-X-Gly motif characteristic of serine hydrolases. The highest degree of homologywas detected with the Torpedo californica acetylcholinesterase (T AchE), possessing the Ser–His–Glu catalytic triad, with the glutamate residue replacing the usual aspartate residue. Similarityin the number and positions of cysteine and salt bonds was very apparent between PudA and T AchE, as were also identities of sequences and their positions in the -helix and -strand regions between

Table 1 Characteristics of puri)ed PUase isolated byour laboratory

Bacterial Molecular Enzyme Inhibition Heat isolate mass (kDa) speci)citystable a

P. 7uorescens 29 Protease PMSF + P. 7uorescens 48bc Esterase PMSF − P. chlororaphis 63bc Esterase=protease PMSF + P. chlororaphis 31 Esterase PMSF + C. acidovorans 42 Esterase=protease PMSF=TI + aEnzyme activity (100%) after 10 min at 100 C. bEnzyme has been cloned and expressed in E. coli. cGene has been sequenced.

the two. In the neighborhood of the glutamate residue of the Ser199–His433– Glu324 catalytic domain of PudA, there were three hydrophobic domains, one of which constituted the surface-binding domain, which occurred in the C-terminus of most bacterial poly(hydroxyalkanoate)(PHA) depolymerases.

(Parte 1 de 4)

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