Rubber particles (RP) were purchased from Trelleborg AB, Germany. For all experiments performed, a particle size between 1 and 2 mm was used. To avoid contamination, rubber particles were washed with 70% ethanol, dried at 80°C for 24 h and sterilised at 121°C for 20 min in separate aluminium envelopes.

Ten different fungal species, namely Coriolus multicolor, Gonnoderma aplanatum, Lentinula edodes, Lenzites trabea, Lenzites betulinus, Pleurotus ostreatus, Pleurotus eryngii, Postia placenta, Stereum hirsutum and Trametes versicolor, were studied. The strains were incubated at 28°C using plates with potato dextrose agar (PDA) and malt extract agar (MEA). After obtaining a fully grown plate, 0.5 g of rubber particles was added to each plate under sterile conditions and incubated for 4 weeks. Each experiment was performed in triplicate.

After the incubation period, the rubber particles were carefully collected from the agar plate under sterile conditions and washed with 70% ethanol and 10% sodium hypochlorite until total removal of the fungi. Subsequently, the solvent was removed, and the rubber particles were dried at 50°C for 72 h. Control samples were treated under the same conditions to prevent the potential oxidative effect of sodium hypochlorite from affecting subsequent analyses.

The mass of the rubber particles was analysed before and after the incubation period with the fungi. The loss of mass was calculated as follows: % weight reduction = ( m i − m f ) m f ⋅ 100 , (1) where m_i is the initial mass of the rubber particles and m_f is the final mass of the rubber particles.

10 mg of dried rubber particles were analysed by attenuated total reflectance (ATR-FTIR). The infrared spectra were obtained using an FTIR spectrometer (Agilent, Cary 630) with the ATR technique. Absorbance was measured in the wavelength range of 400–40,00 cm⁻¹, with a resolution of 4 cm⁻¹. For each sample, 64 scans were performed, and the background was subtracted using the Agilent MicroLab PC software.

An electron microscope TESCAN VEGA 3 with probe EDS BRUKER QUANTAX was used. The method was based on the ASTM E1508 standard “Standard Guide for Quantitative Analysis by Energy-Dispersive Spectroscopy”. Samples were covered by a cathodic spray system with Palladium Gold in the Hummer 6.2 equipment.

Liquid cultures of T. versicolor and P. ostreatus were performed by cutting three 1 × 1 cm² plugs from fully grown PDA plates and poured into 250-ml shake flasks with 50 ml of potato dextrose broth (PDB). Sterile rubber particles were incorporated to the flasks while the corresponding controls did not contain rubber particles. Cultivations were performed at 30 °C, 100 rpm for 5 days. Supernatants were obtained by centrifugation at 10,000 × g for 20 min at 4°C and used for enzyme activity measurements.

Laccase and peroxidase activities were quantified spectrophotometrically (Agilent, Cary 100 Bio) at 415 nm by detecting the oxidation of 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). For laccase activity, 100 μL of sample were mixed with a solution containing 900 μL of 10 mM ABTS and 0.2 M sodium acetate pH 5.0. For peroxidase activity, 100 μL of sample were mixed with a solution containing 800 μL of 10 mM ABTS and 100 M sodium acetate pH 5.0 and 100 μL of 20 mM H₂O₂.

Two Basidiomycetes species were selected in this study as sample sets of the biodegradation of vulcanised rubber. Publicly available genomic assemblies were downloaded from the Joint Genome Institute’s fungal genome portal MycoCosm (Grigoriev et al., 2014), JGI Genome Portal - unified access to all JGI genomic datasets (Mukherjee et al., 2021) and NCBI Genome Assembly (Schoch et al., 2020).

Two specific enzymes (laccase EC 1.10.3.2 and manganese peroxidase EC 1.11.1.13) were searched in sequences deposited in a public database for the 10 fungal species analysed in this study. The database used consisted of identical protein groups of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

For analysis of the similarity at the amino acid level among the sequences of laccases and manganese peroxidase, we used the MEGA-X (Kumar et al., 2018) roand T-COFFEE (https://coffee.crg.cat/apps/tcoffee/) (Notredame et al., 2000) software packages with the default parameters. To generate the protein alignment figures, the ESPript 3.0 software (https://espript.ibcp.fr/) (Robert and Gouet, 2014) was used, highlighting the regions with higher similarity among the sequences: black represents 100% similarity, grey represents 90 to 80% and white similarity below 70%. Each analysis used sequences of laccases and manganese peroxidase separately from Pleurotus ostreatus and Trametes versicolor.

To evaluate the presence of a conserved domain among the sequences of laccases and manganese peroxidase from P. ostreatus and T. versicolor, we used the Conserved Domain Database (CDD) from the NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and PFAM (http://pfam.xfam.org/) programmes (Lu et al., 2020).

The fungal strains were evaluated in terms of radial growth when incubated in PDA and EM media in Petri dishes, and no significant differences were found between the culture media used (data not shown). Figure 1A shows some of the fungi incubated with the vulcanised rubber particles already incorporated. There was a different fungus-rubber interaction depending on the fungal strain used. Three different behaviours stand out, namely total coating, partial coating and zero coating. Of the 10 strains analysed, only P. ostreatus and T. versicolor achieved a total coating of the vulcanised rubber particles, whereas the strains C. multicolor, L. edodes, L. betulinus and P. eryngii showed partial coating. The time required to reach a fully-grown plate was different for each fungus, which was qualitatively analysed by the relative growth rate (Figure 1B). Based on this, P. ostreatus obtained complete growth in the shortest time (between 24 and 30 h), followed by P. eryngii and T. versicolor (from 36 to 42 h). In general, the observations of the level of coating were related to the percentage mass loss of the vulcanised rubber particles at the end of the incubation periods, while the relative growth rate was not always a precise indicator. The highest mass loss was 7.5 ± 0.3%, obtained after cultivation with T. versicolor, followed by 6.1 ± 0.4% obtained with P. ostreatus. Based on these results, vulcanised rubber particles treated with T. versicolor and P. ostreatus strains were selected for surface analysis.

Bredberg et al. (2002) studied the detoxification of vulcanised rubber by different wood-rotting fungi, using the aromatic polymeric dye polyvinylamine sulfonate anthrapyridone (Poly-R478) as a model compound. The authors showed that only three strains were able to biodegrade Poly-R478, namely Pleurotus sajor-caju, T. versicolor and Recinicium bicolor. Subsequently, they used the treated rubber in cultures with Acidithiobacillus ferrooxidans and reached higher growth rates compared to rubber without fungal treatment. It was assumed that the extracellular enzymes from the selected fungi degraded aromatic structures and that the rubber obtained had a reduced toxicity. As in our study, only the cultures with white rot fungi obtained positive results.

Changes in the rubber surface were analysed by FTIR (Figure 2). The functional groups observed may indicate the presence of compounds such as black carbon, aromatic oils, antioxidants, accelerators, among others (Altenhoff et al., 2019). Doublet was observed between 2,926 and 2,853 cm⁻¹ (stretching of CH₂), which was drastically reduced after the treatment, with the greatest reduction in T. versicolor with EM medium and P. ostreatus with EM medium (Gorassini et al., 2016). A loss of a signal was also observed at approximately 1,715 cm⁻¹ in the treatment with T. versicolor on EM and PDA media and for P. ostreatus on EM and PDA media, corresponding to carbonyl groups (C=O), which are typical of the rubber chain (Gorassini et al., 2016). A band at approximately 1,550 cm⁻¹, associated with carboxylate or conjugated ketone, was totally lost after cultivation of both fungal species (Stelescu et al., 2017). After incubation with T. versicolor, a small band close to 1,455 cm⁻¹, assigned to C-H bending of CH₂, was maintained in PDA, but it decreased using EM. However, for P. ostreatus, the band decreased for the assay with PDA and increased significantly in the treatment with EM. Most likely, the sporulation of the fungus was influenced when PDA medium was used, and therefore, the enzymatic metabolism involved in the decrease of the band was influenced by the sporulation process stimulated by PDA (Su et al., 2012). We detected a variation in the polymer structure after treatments corresponding to alkene groups (–C=CH) at a wavelength of 874 cm⁻¹. This band was not present in the control, but it appeared after incubation with T. versicolor and P. ostreatus on both media (Colom et al., 2016). One reason for this may be the exposure of groups related to isoprene and butadiene present in rubber, which, before treatment, were hidden in more interior areas of the material.

These results show that the treatment using T. versicolor with EM allowed a greater transformation of the surface of the rubber material, suggesting that the EM medium favours the degradation in a better way than PDA for T. versicolor. For P. ostreatus, the PDA medium allowed, in general, a greater decrease in bands versus the control. The EM medium could stimulate the generation of secondary metabolites, allowing a more efficient degradation of rubber compared to the type of metabolites stimulated by the PDA medium.

We used SEM analysis to demonstrate the changes in the rubber surface against mechanical, chemical or biological treatments (Li et al., 2012; Aboelkheir et al., 2019). In this study, SEM analysis revealed morphological changes of the analysed rubber surface after biological treatments (P. ostreatus and T. versicolor using PDA medium) in comparison with the control at 500x (Figure 3). In both treatments, particle size was approximately 500 to 1,200 µm and was therefore not influenced by the treatments, in contrast to a previous study (Li et al., 2012). The size reported in this work is compatible with treatments with lower-performance grinding. When comparing the control versus the biological treatments, an increase in the roughness of the material was observed, which was similar for T. versicolor and P. ostreatus. After the treatment, the surface showed cracks, facilitating the breaking of the rubber particles by subsequent treatments, such as mechanical ones, since they can spread rapidly, generating fractures (Kroon, 2011). This result is also related to the elemental analysis of C, O, S, Si, and Zn on the rubber surface (Table 2), where the carbon content decreased in the rubber particles subjected to the treatments. The opposite was observed for O and S; it should be noted that the PDA medium lacks sulphates, and therefore, the increase in S and O is not a result of the culture medium. We therefore recommend that the rubber particles were washed with ethanol and a solution of chlorine before superficial analysis (see Methodology), and therefore, the increases in Si and Zn after the biological treatment are not due to contamination, as both elements are commonly present in tyres (Adathodi et al., 2018; Altenhoff et al., 2019). Zinc is used in the non-accelerated vulcanisation of rubber, whereas silicium is used as a filler (Rattanasom et al., 2007). With P. ostreatus, a higher percentage of Si was detected compared to T. versicolor, whereas for Zn, the percentage was higher with the use of T. versicolor. The changes at the surface can be attributed to oxidation-reduction reactions by the enzymes secreted by the microorganisms, exposing a different proportion of the elements initially arranged on the surface. It is suggested that the observed changes were due to enzymatic processes related to the assimilation of microorganisms towards the components present on the surface of the treated material, which is corroborated by the loss of rubber mass after treatment.

Laccase and peroxidase activities were detected in all the conducted experiments (Figure 4). In cultures of T. versicolor, the presence of rubber particles revealed a 2.8-fold increase of laccase activity and 3.3-fold increase of peroxidase activity compared to the cultures without rubber particles. Likewise in cultures of P. ostreatus, laccase and peroxidase activities increased by double when rubber particles were in the cultivation medium. A maximum laccase activity of 0.128 U ml⁻¹ was calculated for T. versicolor cultures, a 18% more than for P. ostreatus cultures. An enhanced laccase and peroxidase activity can be achieved by the addition of different mediators into the cultivation medium such as xylidine, ferulic acid, veratrylalcohol, pyrogallol and copper (Vranska et el. 2016), enzymes like feruloyl esterase, aryl-alcohol oxidase, quinone reductases, lipases, catechol 2, 3-dioxygenase (Kumar and Chandra 2020) or by culture media optimization (Wang et al., 2014; Hahn Schneider 2018). The increase of the laccase and peroxidase activity in the cultures of T. versicolor and P. ostreatus predicts the affinity of these ligninolytic enzymes for aromatic substrates present in the rubber composition.

The presence of laccase and peroxidase enzymes has been described during the degradation process of natural rubber by the bacteria Bacillus subtilis (Nayanashree and Thippeswamy, 2015). Nevertheless, the enzymatic mechanism is still unknown and rubber oxygenases (Lcp, RoxA, RoxB) have been well characterized for the cleavage of natural rubber (Andler et al., 2018a; Jendrossek and Birke, 2020). The complexity of working with multi-components substrates as rubber is to reveal which of those parts are directly related to the activity of the extracellular enzymes. The current work has shown that the presence of rubber act as an inducer of laccase and peroxidase activities, and provides a more concrete result in the role of these groups of enzymes for vulcanized rubber biodegradation. However, it would be necessary to study the effect of laccases and peroxidases with each of the different components of vulcanized rubber to gain a deeper understanding of the reaction mechanisms.

Obtaining genome data is the initial step in understanding the biology of P. ostreatus and T. versicolor. Fungi present a wide variety of functional proteins that play different roles in the acquisition of nutrients and protection against nearby organisms or unfavourable environmental conditions (Kim et al., 2016).

The genomes of P. ostreatus and T. versicolor have been sequenced, and an analysis of the published fungal genomes, sourced from the public database (Grigoriev et al., 2014; Schoch et al., 2020; Mukherjee et al., 2021), revealed that the P. ostreatus haploid genome contains 11 chromosomes, with a length of 34.3 Mbp and a G + C content of ∼51%. For T. versicolor, there is no information in the public database about the number of chromosomes; however, the total genomic size is 44.8 Mb, with a G + C content of ∼58% (Table 1). Although there is a large variation in the genome size in fungi, the average genome size of fungal species taken during this study was 40.0 Mb. Fungal species harbour small genomes with highly specific genic regions and reduced non-coding regions (Galagan et al., 2003; Martinez et al., 2004). Fungi belonging to the phylum Basidiomycota have an average genome size of 46 Mb (Mohanta and Bae, 2015). In line with this, the sequenced genomes of Pleurotus taxa were assembled to less than 50 M bp but exhibited large numbers of annotated protein-coding genes and few constituent TEs (Table 1). However, as mentioned before, there were genome size variations among the two species, which could have arisen from unequal TE variations. The number of transposable elements (TEs), including retrotransposons and DNA transposons, accounted for approximately 253 of the P. ostreatus genome and 233 of the T. versicolor genome. For P. ostreatus, it was possible to determine notable genomic differences in the regions corresponding to TEs, where 80 identified TE families represented 2.5–6.2% of the genome sizes (Téllez-Téllez and Díaz-Godínez, 2019). Transposable elements are undoubtedly an important source of genetic variation in fungi, as previously found for other fungal species (Labbé et al., 2012).

Considering the coding gene sequences in fungi, on average, the Basidiomycota group encodes for 15,431.51 genes in their genomes. The average numbers of annotated genes are 12,330 for P. ostreatus per genome and 14,572 for the T. versicolor genome (Table 1). For the noncoding gene, we found scaffolds of the genome assembly, with 315 and 234 genes, respectively. There were three and two pseudogenes predicted, corresponding to 0.24% of the genome assembly of P. ostreatus and 0.014% of the genome assembly of T. versicolor.

Comparative analysis of fungal genomes showed that fungi are highly divergent. The P. ostreatus genome sequence assembly was distributed across of 12 and 572 scaffolds. Of these, 56 were smaller than 1 kb (Alfaro et al., 2016; Castanera et al., 2016). The genome of T. versicolor comprised only 283 scaffolds. The average protein coding genes in P. ostreatus were 12,330, with 14,302 for T. versicolor. These coding genes have homologues with known proteins deposited in the NCBI nr, Pfam, SwissProt and TrEMBL databases.

Both fungal species can partially degrade vulcanised rubber, and genome data revealed that both of fungi encode for a large set of enzymes involved in the degradation of rubber materials. The protein coding genes with enzymes for P. ostreatus included 1,677 genes (Table 1). For T. versicolor, such data were not available in the public repositories. However, based on a previous study, T. versicolor has an expansion of the AA2 gene family (26 genes), a feature that is also found in the central polyporoid clade (Miyauchi et al., 2018). The putative peroxidase genes (PoPOD) were obtained from the Joint Genome Institute (JGI) (Mukherjee et al., 2021). The gene density and the mean size of the protein-coding genes in both species of fungi used in this study secrete different enzymes, among which are laccases, manganese peroxidases, versatile peroxidases, glycosylhydrolases, peptidases and fungal esterases/lipases (Ruiz-Dueñas et al., 2011). These enzymes can degrade complex compounds such as lignin as well as certain industrial pollutants contaminating vulcanised rubber. Despite the advances in comparative genomics, the genera Pleurotus and Trametes are under-exploited, and a variety of potential biotechnological applications in different industries can be elucidated based on their genomes.