Plastic has tremendously used due to its excellent physical properties such as versatility, malleability, chemical, and physical resistance, and ease of production compared to other materials. Polyester polyurethane (PUR) is a major plastic material that is widely used in many industrial applications to produce foams, elastomers, coatings, and adhesives (Howard, 2002). The estimated production of PUR is approximately 8 million tons annually (Matsumura et al., 2006). It is mainly produced via the nucleophilic reaction from two monomers (a diol and a highly toxic and reactive diisocyanate) and is derived from the toxic substance phosgene in the presence of some additives. Reprocessing of PUR generates toxic residues, such as aldehydes, ammonia, cyanide, isocyanates, nitrogen oxides, and vinyl chloride, which are hazardous to humans, disrupt ecological processes, and contribute to environmental pollution (Cregut et al., 2013). PUR is also physically stable, making it resistant to biological degradation (Yang et al., 2001). Moreover, the large amount of produced PUR materials has resulted in a massive increase in plastic waste (Miskolczi et al., 2004). Hence, effective methods for the degradation of plastic wastes are needed to solve the problems posed by PUR waste accumulation.

Recently, various bacteria and fungi have been demonstrated to degrade PUR materials. Fungi in some genera have been reported to degrade PUR, including Alternaria, Aspergillus, Phoma, Penicillium, Plectosphaerella, Geomyces, Nectria, and Neonectria (Cosgrove et al., 2007; Schmidt et al., 2017). In addition, Aspergillus niger, Aspergillus flavus, Aspergillus vesicolor, Penicillium funicutosum, Aureobasidium pullulaus, some species of Trichoderma sp., and Chyaetomium glubosum also can degrade PUR reported by Darby and Kaplan (1968). Some bacterial species, including a few species of Corynebacterium genus, Enterobacter agglomerans, and Bacillus subtilis have also been reported to efficiently degrade PUR samples (Rowe and Howard, 2002; Mahajan and Gupta, 2015; Schmidt et al., 2017). However, few organisms have been demonstrated to degrade PUR as the sole carbon source (Shah et al., 2008; Russell et al., 2011).

Several reports have indicated that the active enzymes involved in microbial degradation of PUR are esterases, lipases, proteases, and ureases, suggesting the degradation of the PUR substrate by the cleavage of the ester bond (Tokiwa and Calabia, 2004; Shah et al., 2008; Kale et al., 2015; Danso et al., 2019; Ru et al., 2020). These enzymes were isolated from various microorganisms such as Pseudomonas fluorescens, Comamonas acidovorans, Pseudomonas chlororaphis, Pseusomonas aeruginosa, Acinetobacter gerneri, Bacillus subtilis MZA-75, Comamonas acidovorans TB-35, Aspergillus terreus, and Chaetomium globosum (Allen et al., 1999; Yang et al., 2001; Shah et al., 2008). Although both fungi and bacteria have been reported to degrade PUR, the actual mechanism of the biodegradation process remains unknown. The ability of these microorganisms to degrade PUR offers the possibility of being able to degrade other complex polymers as well (Russell et al., 2011). A massive number of microorganisms could have the degradation ability to colonize the surface of PUR (Glaser, 2019). Therefore, investigating other PUR-degrading microorganisms and their enzymatic activities is essential for efficient PUR waste management (Liu et al., 2021).

This study presents the Impranil biodegradation potential by various fungi. The study also demonstrates their ability to degrade Impranil on a culture plate, in a liquid medium, and with Impranil as the sole carbon source. Analysis of its CO₂ production, enzymatic activities, and degradation products was also performed.

All Impranil-degrading fungi belong to the phylum Ascomycota. Several fungal species in this phylum have been reported to have PUR degradation ability, such as Pestalotiopsis microspore (Russell et al., 2011), Cladosporium herbarum (Álvarez-Barragán et al., 2016), Cladosporium cladosporioides (Álvarez-Barragán et al., 2016), Penicillium griseofulvum (Brunner et al., 2018), Alicycliphilus spp. (Oceguera-Cervantes et al., 2007), and Xepiculopsis graminea (Álvarez-Barragán et al., 2016). Although many fungi have already been reported as PUR degraders, most of the species that are described in this work, as well as their ability to degrade Impranil, have been rarely reported. Impranil was found to employ as a representative of PUR in searching for PUR-degrading microorganisms (Russell et al., 2011; Nakkabi et al., 2015; Hung et al., 2016; Magnin et al., 2019) due to their various useful attributes resulting it ideal for biodegradation assays such as its commercial availability, application as a coating material, stability to pH 4–8, tolerance to high temperature up to 80°C, and rapid rate of dissolution compared to other polyurethanes. However, the formulation of Impranil is proprietary and its colloidal state has ultimately made Impranil a precarious choice for polyurethane-degrading microorganisms (Biffinger et al., 2015). The active fungi showed different Impranil degradation abilities, probably due to differences in the physiology, biochemistry, and genetics among the fungal species (Brunner et al., 2018). The fungus E. clematidis showed the highest Impranil degradation ability among all the active fungi that were tested, based on the formation of a halo in medium plates. It is classified as part of the genus Allophaeosphaeria of the Phaeosphaeriaceae family. However, we found no references related to the degradation of E. clematidis. Thus, this is the first report on the ability of E. clematidis to degrade Impranil. Recently, Phaeosphaeriaceae was evaluated as a big family, consisting of more than 300 species of economically important plant pathogens, endophytes, or saprobes on plants (Phookamsak et al., 2014). The degradation activity suggests that the fungi in this family might be a promising source of biodiversity for testing important bioremediation activities.

We found that E. clematidis in solid and liquid screening assays (with Impranil as the sole carbon source) had the highest rate of Impranil degradation compared to other fungi in this study. This observation indicates that E. clematidis growth in the medium and its metabolism in the medium could be harnessed for Impranil degradation. The growth of E. clematidis was investigated through a modified Sturm test, which is based on the amount of CO₂ released by the fungal culture in the presence and absence of Impranil. The growth of the fungus E. clematidis in the presence of ester hydrolysis products revealed that it utilized the products as a carbon source and mineralized to CO₂ and H₂O. Various studies used CO₂ evolution tests to evaluate the degradation ability of microorganisms and found that several microbes released high amounts of CO₂ during culturing, with polymer as a carbon source (Nakkabi et al., 2015; Bano et al., 2017; Hung et al., 2019). In the present study, the quantity of CO₂ released by E. clematidis in the presence of Impranil as a carbon source was significantly higher than when Impranil is absent, suggesting that Impranil biodegradation was enhanced by fungal activity, as reflected by the corroborative levels of enzyme production and CO₂ liberation.

Chemical composition analysis of the E. clematidis culture was performed using FTIR spectroscopy and GC-MS. Both techniques provide evidence to predict the mechanism of how the fungus attacks the polymer during biodegradation (Mahalakshmi et al., 2012; Shah et al., 2013; Muhonja et al., 2018). As far as the mechanism of degradation of Impranil is concerned, the fungus E. clematidis easily degraded the aliphatic segment to form various alcohol compounds detected by the GC-MS. However, the formulation of Impranil is proprietary (Biffinger et al., 2015). These compounds could be derived from both the polyester and polyisocyanate portions of PUR (Shah et al., 2013). FTIR analysis revealed that E. clematidis degraded the polyester portion of the Impranil. The dramatic variation of the carbonyl signal (1,715 cm^–1) in the FTIR spectra were related to the attack of the ester bonds that are present in the polyol fractions, as well as the attack of the urethane groups (Mahalakshmi et al., 2012; Shah et al., 2013; Álvarez-Barragán et al., 2016; Muhonja et al., 2018). Russell et al. (2011) also reported that a decrease in the peak intensity at 1,715 cm^–1 is consistent with the hydrolysis of the ester bond in the urethane linkage of PUR. Moreover, the decrease in the C-N-H bond peaks at 1,340 cm^–1, and 1,020 cm^–1 indicates that the urethane groups were eliminated. The reduction of the C=O and C-N-H signals confirms the degradation of Impranil by the fungus E. clematidis. The FTIR spectrum measured from the liquid medium without fungi showed a strong peak broadening at 3,270–3,335 cm^–1, indicating the overlap of N-H and O-H- related peaks (Oprea, 2010). FTIR spectroscopy has been applied to monitor the variation in characteristic peaks related to polymer biodegradation.

Evidence of Impranil biodegradation by the fungus E. clematidis was also obtained by GC-MS analysis. New chemical compounds were detected after a 2-week incubation of E. clematidis and A. niger in the medium. The reduction of compounds containing ester bonds at the 15:00–38:00 min-mark increased alcohol compounds, probably due to the hydrolysis of the ester bonds in the polymer chain of PUR (Howard, 2002; Russell et al., 2011; Álvarez-Barragán et al., 2016). Ester compounds, 2E,4E-hexadienol acetate, and 3Z-hexenyl isobutanoate, may be synthesized by the combination of PUR precursors that are degraded by the fungi (Russell et al., 2011; Álvarez-Barragán et al., 2016). This work suggests that the use of fungi to degrade PUR waste presents an alternative, sustainable way of recovering PUR precursors for recycling. However, the compounds generated during the degradation of PUR by fungi might be related to oxidative reactions (Oprea, 2010; Yarahmadi et al., 2017).

FTIR spectroscopy and GC-MS were successfully applied for the analysis of chemical structure changes due to biodegradation by microorganisms. For example, Álvarez-Barragán et al. (2016) studied the biodegradative activities of selected environmental fungi on PUR. FTIR and GC-MS analyses revealed structural changes in PUR. A decrease in carbonyl groups (1,729 cm^–1) and N-H bonds (1,540 and 1,261 cm^–1) was measured by FTIR spectroscopy whereas a decrease in ester compounds and an increase in alcohols and hexane diisocyanate were shown by GC-MS analysis. The results obtained by both techniques indicated the hydrolysis of ester and urethane bonds. Shah et al. (2013) confirmed the degradation of PUR by Pseudomonas aeruginosa strain MZA-85 using FTIR and GC-MS. IR spectra indicated that the peak at 1,725 cm^–1, which corresponds to ester linkage, decreased in the treated PUR samples, showing ester hydrolysis. The peak at 1,685 cm^–1, obtained from the treated PUR sample, represents the overlap of carboxylic acid and amide carboxyl peaks, while the broadened N-H peak at 3,325 cm^–1 represents the presence of free hydroxyl groups in the treated sample. The peak at 1,135 cm^–1, which is attributed to the ester group was absent in the treated sample, confirming ester hydrolysis and the production of free hydroxyl groups after incubation with P. aeruginosa strain MZA-85. In addition, GC-MS analysis demonstrated two compounds, including 1,4-butanediol and adipic acid, which were detected in the treated sample only.

Many studies reported the esterase activities of microorganisms in the PUR degradation (Akutsu et al., 1998; Rowe and Howard, 2002; Mohanan et al., 2020). The enzymatic degradation of PUR occurs in two stages including adsorption of enzymes on the polymer surface in the first stage, followed by hydro-peroxidation/hydrolysis of the bonds in the second stage (Akutsu et al., 1998; Mohanan et al., 2020). Sato (1981) reported the protease, urease, and esterase activities produced by A. niger while culturing with PUR. Similarly, the structural changes due to Impranil degradation by the fungus E. clematidis are also correlated with the same enzymes. However, the rate of polymer biodegradation varied depending on several factors including chemical structures, and molecular weights (Mohanan et al., 2020). Of these three enzymes, esterase appears to be the most responsible for Impranil degradation. It is noted that the enzyme responsible for PUR degradation is extracellular, secreted, and diffusible (Oceguera-Cervantes et al., 2007; Loredo-Treviño et al., 2012; Hung et al., 2016). Extracellular enzymes secreted by microorganisms have played an important role in polymer degradation via depolymerization reaction by breaking down polymers into smaller molecules (Howard, 2002). The polymer chain is degraded by extracellular enzymes and becomes a degradation product. It can then be assimilated into microbial cells and used as carbon sources for producing carboxylic acid and alcohol that ultimately undergo respiration to produce energy (Kay et al., 1991; Nakajima-Kambe et al., 1995; Hung et al., 2016). In this study, extracellular enzymes esterase, lipase, and urease were investigated. E. clematidis was able to produce esterase enzymes which are believed to play a role in Impranil degradation. This result was in agreement with the study of Biffinger et al. (2015) showing the partial degradation of Impranil was significantly achieved by enzyme esterase. They reported that the enzymes (a Pseudomonas sp. esterase showed significant esterase activities and partially cleared Impranil by partial hydrolysis. Esterase enzymes have been isolated and reported by several studies to degrade PUR. Noor et al. (2020) found esterase in the outer membrane of P. aeruginosa, demonstrating a particular affinity to long-chain acyl esters. Purified esterase was also isolated from the outer membrane of PUR-degrading bacteria C. acidovorans strain TB35 and P. aeruginosa PAO1 (Akutsu et al., 1998; Wilhelm et al., 1999). Mukherjee et al. (2011) also reported the ability of esterase enzymes from P. aeruginosa to degrade polyurethane diol. Moreover, esterase was purified from a PUR-degrading fungus, Curvularia senegalensis (Loredo-Treviño et al., 2012). The esterase enzyme attacked the ester bond cleavage in the soft segments of PUR. Yu et al. (2010) reported the role of esterase in the catalysis of the cleavage of ester bonds in the polymer chain in PUR. In addition, 6-hydroxyhexanoate was generated from the hydrolysis of the ester linkages in polycaprolactone polyol-based PUR by an esterase enzyme (Magnin et al., 2019). Shah et al. (2013) also described that esterase that was purified from the strain P. aeruginosa MZA-85 specifically damaged the ester bonds in the aliphatic or soft segment, leading to the depolymerization of the polymer chain to degradation products. These products could be further metabolized through different metabolic pathways (Brzostowicz et al., 2002; Yoshida et al., 2016). However, there is limited information about the mechanism of degradation of Impranil by esterase secreted from E. clematidis. Therefore, the mechanism of how esterase is secreted by E. clematidis should be further studied in detail. Optimal conditions for the fungal activity must also be improved before applying it on a larger scale for commercial use.

This work establishes that various fungi are useful biodiversity sources with potential application in Impranil degradation. It is interesting to note that E. clematidis had the highest capacity for Impranil degrading. FTIR spectroscopy and GC-MS analysis indicate that Impranil was successfully degraded by E. clematidis, with Impranil as the sole carbon source. The fungus E. clematidis degraded the polymer chain of Impranil, constituting the soft segment, and produced degradation products such as 5Z-octenol, and 2E,4E-hexadienol acetate. The ester and urethane groups in the polymer chain of Impranil were attacked via the esterase enzyme. Further studies, which explain the mechanism behind the esterase enzyme production of E. clematidis and its role in PUR degradation, are required for its potential application in polyurethane waste management.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

SK and TS performed experiments on solid- and liquid-medium assays. SK performed FTIR, GC-MS analysis, Sturm test, enzyme activity assay, analyzed data, and wrote the first draft of the manuscript. PP edited, reviewed the manuscript, and supervised the work. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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