Degradation Of Polyester Polyurethane Research Articles

Colonization and degradation of polyurethane coatings by Pseudomonas protegens biofilms is promoted by PueA and PueB hydrolases

The ability of bacteria and fungi to degrade polymeric substrates is often assessed in liquid-based assays conducted by exposing polymers to planktonic cells, or cell-free supernatants or enzymes generated from them. These assessments miss the opportunity to specifically examine the role of biofilm formation and physiology in degradative processes. In a previous study, we examined the ability of cell-free supernatants from wildtype and hydrolase-deficient Pseudomonas protegens Pf-5 strains to degrade a commercial polyester polyurethane (PU). In this study, we developed the methodology to conduct spatial-temporal analysis of both biofilm colonization and degradation of polyester PU coatings using the same strains. Wild-type Pf-5 grew as confluent biofilms that produced tendrils containing viable cells; in contrast, Pf-5ΔpueAB, a mutant lacking PueA and PueB hydrolases, colonized the PU only by growing as a confluent biofilm but lacked tendril expansion. Degradation of PU by the wild-type hydrolases may alter the substrata, facilitating colonization by the biofilm. Chemical analysis by in situ Fourier transform infrared (FTIR) and Raman spectroscopies revealed the polyester block of the PU was preferentially degraded to varying degrees by the wild-type Pf-5 and mutant biofilms, and showed degradation due to PueB that was undetectable in liquid-based assays. Raman spectroscopy analysis of biofilms of Pf-5ΔpueAB grown on PU revealed a detectable, but the lowest, level of PU degradation relative to other strains. Degradation of PU by this mutant had previously been undetectable and this study revealed the existence of biofilm-associated hydrolytic mechanisms other than those driven by PueA and PueB. Taken together, our data suggest that both PueA and PueB hydrolases play a role in the colonization and degradation of a model polyester PU by P. protegens biofilms. This study demonstrates the importance of using biofilm-based assays to identify mechanisms of microbiologically-influenced degradation.

Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases.

Polyurethanes (PU) are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC), TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU) Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

Degradation of Polyester Polyurethane by Aspergillus sp. Strain S45 Isolated from Soil

A polyurethane (PU)-degrading fungus designated as strain S45 was isolated from a solid waste-dumping site in Islamabad, Pakistan. Strain S45 was identified as Aspergillus sp. through microscopic, morphologic as well as 18S rRNA sequencing (99% similarity with Aspergillus fumigatus). The degradation ability of strain S45, was analyzed through measurement of weight loss and evolution of CO2 as a result of breakdown of PU film. The degradation was confirmed through scanning electron microscopy, fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Surface changes like pits, cracks and holes were observed in scanning electron micrographs upon degradation of PU film by strain S45. FTIR spectra were carefully examined, changes were observed in various peaks at different wavelength ranges that also correspond to degradation of PU film. Differential scanning calorimetric technique was used to measure the melting temperature of PU film pieces before and after incubation with strain S45. DSC curve indicated an increase in melting temperature of PU from 191 to 196 °C that might be due to increase in crystallinity of the exposed PU after degradation of amorphous soft segment. A gradual increase in specific activity of esterase was observed up to the maximum of 15th day of incubation with PU film in mineral salt medium. Enzyme activity was further confirmed by tween 20 agar plates, demonstrated by formation of calcium complex. It is concluded from the results that Aspergillus sp. strain S45 might be applied for the treatment of plastics-contaminated environments.

Degradation of polyester polyurethane by an indigenously developed consortium of Pseudomonas and Bacillus species isolated from soil

This work investigated the degradation efficiency of bacterial co-culture against polyester polyurethane in comparison to the individual strains. For this purpose, a consortium of two already reported polyester polyurethane (PU) degrading bacterial strains Bacillus subtilis MZA-75 and Pseudomonas aeruginosa MZA-85 was developed. The degradation was analyzed through measurement of weight loss, scanning electron microscopy (SEM), fourier transformed infra-red (FT-IR) spectroscopy and Sturm test (CO2 evolution test). Polyester PU degradation was augmented in the presence of co-culture, as indicated by maximum weight reduction in Polyester PU film pieces within 20 days. SEM micrographs showed surface changes in the polyester PU films in the form of pits or holes that resides bacterial cells. Besides degradation of soft segment, a new peak at 2250 cm−1 appeared in FTIR spectrum, which corresponds to free isocyanates due to breakdown of urethane linkages. The quantity of carbon dioxide evolved in Sturm test was much higher due to high degradation rate in co-culture vessel as compared to control as well as the separate cultures. A sharp increase in extracellular esterase activity of co-culture against polyester PU was observed. Butanediol (BD) and Adipic Acid (AA) were detected as degradation products in relatively higher intensity than those observed in pure culture, through gas chromatography-mass spectrometry (GC-MS). Hence, it is concluded that consortium might be better than pure cultures in polyester PU waste management and used as a tool for recycling purified monomers (biochemical depolymerisation/monomerization).

Degradation of polyurethane by bacterium isolated from soil and assessment of polyurethanolytic activity of a Pseudomonas putida strain

The increasing usage and the persistence of polyester polyurethane (PU) generate significant sources of environmental 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 determined. Particularly robust activity was observed in Pseudomonas putida; it spent 4 days to degrade 92% of Impranil DLN(TM) 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 investigated by Fourier transformed infrared spectroscopy show the decrease in ester functional group and the emergence of amide group. The polyurethanolytic activities were both presented 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.

Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75

A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

Degradation of polyester polyurethane by newly isolated Pseudomonas aeruginosa strain MZA-85 and analysis of degradation products by GC–MS

In this report, a polyester polyurethane (PU) degrading bacterium, designated as strain MZA-85, was isolated from soil through enrichment. The bacterium was identified through 16S rRNA gene sequencing; it was completely matched with Pseudomonas aeruginosa type strain. The degradation of PU film pieces by P. aeruginosa strain MZA-85 was investigated by scanning electron microscopy (SEM), Fourier transformed infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM micrographs of PU film pieces, treated with strain MZA-85, revealed changes in the surface morphology. FTIR spectrum showed increase in organic acid functionality and corresponding decrease in ester functional group. GPC results revealed increase in polydispersity, which shows that long chains of polyurethane polymer are cleaved into shorter chains by microbial action. The bacterium was found to produce cell associated esterases based on p-Nitrophenyl acetate (pNPA) hydrolysis assay. 1,4-Butanediol and adipic acid monomers were detected by gas chromatography–mass spectrometry (GC–MS), which were produced as a result of hydrolysis of ester linkages in PU by cell bound esterases. Strain MZA-85 not only depolymerized PU but also mineralized it into CO2 and H2O, as indicated by increase in cells growth in the presence of degradation products as well as detection of CO2 evolution through Sturm test. From the results presented above, it is finally concluded that P. aeruginosa strain MZA-85, as well as its enzymes, can be applied in the process of biochemical monomerization for the pure monomers recycling.

Biodegradation of Polyester Polyurethane by Endophytic Fungi

Bioremediation is an important approach to waste reduction that relies on biological processes to break down a variety of pollutants. This is made possible by the vast metabolic diversity of the microbial world. To explore this diversity for the breakdown of plastic, we screened several dozen endophytic fungi for their ability to degrade the synthetic polymer polyester polyurethane (PUR). Several organisms demonstrated the ability to efficiently degrade PUR in both solid and liquid suspensions. Particularly robust activity was observed among several isolates in the genus Pestalotiopsis, although it was not a universal feature of this genus. Two Pestalotiopsis microspora isolates were uniquely able to grow on PUR as the sole carbon source under both aerobic and anaerobic conditions. Molecular characterization of this activity suggests that a serine hydrolase is responsible for degradation of PUR. The broad distribution of activity observed and the unprecedented case of anaerobic growth using PUR as the sole carbon source suggest that endophytes are a promising source of biodiversity from which to screen for metabolic properties useful for bioremediation.

Fungal Communities Associated with Degradation of Polyester Polyurethane in Soil

Soil fungal communities involved in the biodegradation of polyester polyurethane (PU) were investigated. PU coupons were buried in two sandy loam soils with different levels of organic carbon: one was acidic (pH 5.5), and the other was more neutral (pH 6.7). After 5 months of burial, the fungal communities on the surface of the PU were compared with the native soil communities using culture-based and molecular techniques. Putative PU-degrading fungi were common in both soils, as <45% of the fungal colonies cleared the colloidal PU dispersion Impranil on solid medium. Denaturing gradient gel electrophoresis showed that fungal communities on the PU were less diverse than in the soil, and only a few species in the PU communities were detectable in the soil, indicating that only a small subset of the soil fungal communities colonized the PU. Soil type influenced the composition of the PU fungal communities. Geomyces pannorum and a Phoma sp. were the dominant species recovered by culturing from the PU buried in the acidic and neutral soils, respectively. Both fungi degraded Impranil and represented >80% of cultivable colonies from each plastic. However, PU was highly susceptible to degradation in both soils, losing up to 95% of its tensile strength. Therefore, different fungi are associated with PU degradation in different soils but the physical process is independent of soil type.

Fungi are the predominant micro-organisms responsible for degradation of soil-buried polyester polyurethane over a range of soil water holding capacities.

To investigate the relationship between soil water holding capacity (WHC) and biodegradation of polyester polyurethane (PU) and to quantify and identify the predominant degrading micro-organisms in the biofilms on plastic buried in soil. High numbers of both fungi and bacteria were recovered from biofilms on soil-buried dumb-bell-shaped pieces of polyester PU after 44 days at 15-100% WHC. The tensile strength of the polyester PU was reduced by up to 60% over 20-80% soil WHC, but no reduction occurred at 15, 90 or 100% soil WHC. A PU agar clearance assay indicated that fungi, but not bacteria were, the major degrading organisms in the biofilms on polyester PU and 10-30% of all the isolated fungi were able to degrade polyester PU in this assay. A 5.8S rDNA sequencing identified 13 strains of fungi representing the three major colony morphology types responsible for PU degradation. Sequence homology matches identified these strains as Nectria gliocladioides (five strains), Penicillium ochrochloron (one strain) and Geomyces pannorum (seven strains). Geomyces pannorum was the predominant organism in the biofilms comprising 22-100% of the viable polyester PU degrading fungi. Polyester PU degradation was optimum under a wide range of soil WHC and the predominant degrading organisms were fungi. By identifying the predominant degrading fungi in soil and studying the optimum WHC conditions for degradation of PU it allows us to better understand how plastics are broken down in the environment such as in landfill sites.

Characterization of polyether and polyester polyurethane soft blocks using MALDI mass spectrometry

Selective degradation reactions combined with MALDI analysis have been applied for molecular weight (MW) determination of polyether and polyester polyurethane (PUR) soft blocks. Selective degradation allows recovery of the polyols, and direct observation of the soft block oligomer distribution is possible for the first time by using MALDI. Ethanolamine is applied for polyether PUR degradation. MALDI analysis indicates that the recovered polytetrahydrofuran (pTHF) MW distribution is nearly identical to the unreacted pTHF material. Reduction in the ethanolamine reaction time allows observation of oligomer ions containing the diisocyanate linkage, which provide identification of the diisocyanate. Ethanolamine is not used for polyester PUR's degradation because the ester bonds will be cleaved. Therefore, phenylisocyanate is applied for polyester PUR degradation. Polybutylene adipate (pBA) oligomers were directly observed in the MALDI spectra of the degraded pBA-PUR samples. Comparison of the degraded pBA-PUR oligomer distribution with the unreacted pBA material indicates that low-mass oligomers are less abundant in the degraded pBA-PURs. Oligomer ions containing the diisocyanate linkage are also observed in the spectrum, providing a means for identifying the diisocyanate used for PUR syntheses. Size-exclusion chromatography (SEC) was combined with MALDI to provide accurate MW determination. Narrow MW fractions of the degraded and unreacted polyols were collected and analyzed by MALDI. This method allows precise calibration of the SEC chromatogram. The SEC-MALDI results provide significantly larger Mw and PD values than MALDI alone. Using SEC-MALDI, it was determined that the PD indexes of the pTHF and pBA samples are larger than the assumed values, which are based on the polyol synthesis reactions. The combination of selective degradation with SEC-MALDI, using either ethanolamine or phenylisocyanate, is a viable method for polyurethane polyol characterization.

Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2% N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45 degrees C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity when p-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.

Determination of the polyester polyurethane breakdown products and distribution of the polyurethane degrading enzyme of Comamonas acidovorans strain TB-35

Metabolites produced by the degradation of polyester polyurethane (PUR) by Comamonas acidovorans strain TB-35 were investigated. GC-MS analysis revealed that they were diethylene glycol, trimethylolpropane and adipic acid. These metabolites were considered to be derived from polyester segments of the PUR as a result of hydrolytic cleavage of ester bonds. In addition, when the culture broth was alkaline treated, a previously undetected product was detected by GC analysis. This product was identified as 2,4-diaminotoluene by GC-MS analysis. This indicates that an additional metabolite exists in the culture broth. This metabolite was considered to have been derived from polyisocyanate segments of the PUR. A preliminary study on a PUR degrading enzyme was also performed. Strain TB-35 produced two different esterases, one which is secreted to the culture broth and one which is bound to the cell surface. Between them, only cell-surface-bound esterase catalyzes the degradation of the polyester PUR.