The ever-increasing accumulation of post-consumer plastic in the environment has received much attention in recent years and inspired industry and academia to intensify efforts to achieve a circular polymer economy. Alongside a sustainable waste hierarchy, including waste prevention and minimization, reuse and repair, followed by recycling and other forms of recovery (e.g., incineration with energy capture), alternatives to the conventional, virtually non-degradable synthetic plastic polymers play a key role in the transition to a sustainable and more circular polymer economy. Biodegradable plastics sourced from renewable materials, such as plant oils, are a promising step toward achieving this goal. However, despite their importance to the circular economy, existing bioplastics have so far failed to emerge beyond a minor market share, calling for further innovation in this field (Abe et al., 2021; Abrha et al., 2022; Boey et al., 2022; Nanda et al., 2022; Rosenboom et al., 2022; González-López et al., 2023; Negrete-Bolagay and Guerrero, 2024; Serrano-Aguirre and Prieto, 2024).

In this context, materials such as long-chain aliphatic polyesters (LCAP) with the potential to replace conventional polyolefins such as high-density polyethylene (HDPE) and polypropylene (HDPP) have evolved. LCAP are comprised of ester-linked long-chain dialcohol (e.g., 1,18-octadecanediol) and dicarboxylic acid (e.g., 1,18-octadecanedioic acid) monomers (thus, polyester-C₁₈, C₁₈; abbreviated PE-18,18), which can be sourced from plant oils (Häußler et al., 2021). They possess material properties comparable to HDPE, while also exhibiting high-yield and low-energy chemical recyclability (Häußler et al., 2021). Additionally, the biodegradability of LCAP bioplastics was demonstrated under controlled, thermophilic composting conditions according to ISO 14855-1, a standardized test method for the determination of the ultimate aerobic biodegradability of plastics (Eck et al., 2023). Compost samples that derived from this test and that were used for microbial community analysis, are the subject of this communication. Particularly the PE-2,18 variant of LCAP reached a degradation of 96 ± 2% (n = 3) within two months at 58 °C, achieving near-complete stoichiometric conversion to CO₂ despite its polyethylene-like material properties (Eck et al., 2023). This fulfills regulatory requirements for biodegradability (e.g., according to EN 13432), and demonstrates the high potential of LCAP for achieving a circular plastic economy.

Biodegradation of synthetic polymers is influenced by various factors, particularly the chemical structure of the polymer, including the presence or absence of functional groups susceptible to hydrolysis. As such, a meaningful degradation has only been demonstrated for polymers with hydrolysable bonds present in their backbone structure, for example polyethylene terephthalate (PET), polylactic acid (PLA), polycaprolactone (PCL) and polybutylene adipate terephthalate (PBAT), among others (Yoshida et al., 2016; Meyer-Cifuentes et al., 2020; Ru et al., 2020; Teixeira et al., 2021; Buchholz et al., 2022; Gambarini et al., 2022; Lear et al., 2022; Jendrossek, 2024). In addition to the chemical structure of the polymer chain and other factors such as crystallinity of the plastic material, a diverse and adaptable microbial community is key for successful biodegradation, which may differ strongly between habitats (Shah et al., 2008).

The compost matrix contains a vast diversity of naturally occurring microorganisms which are particularly specialized on the utilization of complex plant polymers such as cellulose, lignin and cutin. Notably, enzymes identified to depolymerize synthetic polyesters often share a close homology to those involved in the degradation of natural polyesters, such as the plant hetero-polyester cutin (Chen et al., 2020; Tournier et al., 2020; Sonnendecker et al., 2022; Arnal et al., 2023; Burgin et al., 2024; Ma et al., 2024). Additionally, the high temperatures of up to 70 °C during industrial composting is close to the glass transition temperature (Tg) of some polymers. These temperatures cause polymer chains in amorphous regions to become more flexible and accessible, enabling increased enzymatic accessibility (Karamanlioglu and Robson, 2013; James-Pearson et al., 2023; Shalem et al., 2024). Therefore, complex soil environments, and thermophilic compost in particular, provide ideal conditions for facilitating the biodegradation of synthetic polymers. This is also evidenced by the frequent isolation of plastic-degrading microorganisms and enzymes from these environments (Buchholz et al., 2022; Gambarini et al., 2022). Particularly thermophilic genera belonging to the Actinomycetota, such as Thermobifida, Streptomyces, and Saccharomonospora have been repeatedly isolated from composting systems and implicated in plastic degradation (Kleeberg et al., 1998; Hu et al., 2010). Other thermophilic actinomycetes, including Pseudonocardia and Thermomonospora, have been associated with PLA degradation through screening of culture collections or sequencing-based studies, highlighting the potential ecological relevance of this phylum for synthetic polymer degradation (Sangwan and Wu, 2008; Apinya et al., 2015; Butbunchu and Pathom-Aree, 2019). In addition, homology-based genome mining revealed that Thermomonospora curvata DSM43183 encodes two putative polyester hydrolases, Tcur1278 and Tcur0390, with high sequence homology to Thermobifida fusca polyesterases and demonstrated potential to hydrolyze the plastic materials PET and PCL in vitro (Wei et al., 2014). While the ecological relevance of this genotype in polyester degradation has not been demonstrated, other Thermomonospora strains isolated from soil, compost, manure, and plant material were reported to degrade natural polymers such as cellulose and rubber latex, indicating their ecological relevance in natural polymer degradation (Henssen, 1957; Ibrahim et al., 2006; Chertkov et al., 2011).

Nevertheless, synthetic-polymer biodegradation studies employing compost are commonly operated as a microbial ‘black-box’. While the standardized test employs standardized incubation conditions (e.g., 58 °C, aeration, moisture control, as for test ISO 14855-1; see below), the individual studies are being conducted with different compost matrices that may contain vastly different microbial communities. Therefore, advancing our ecological understanding of plastic biodegradation in these systems is important not only for validating biodegradation claims, but also for informing material design and developing improved strategies for sustainable waste processing.

To date, few studies have attempted to directly identify the microorganisms involved in the degradation process during thermophilic composting, with varying results. While one study identified clear microbial community shifts in response to biodegradable plastic treatments, other studies also reported significant shifts in HDPE treatments in absence of observable degradation (Esan et al., 2019; Sun et al., 2021; Ruggero et al., 2023). These results demonstrate the challenge of assessing whether plastic-dependent community shifts are in fact associated with polymer degradation, or whether taxa simply make use of an additional colonizable surface (the ‘plastisphere’), especially with respect to plastics widely regarded as non-degradable (Wallbank et al., 2022). Therefore, inferring an organism’s involvement in degradation processes from an increased occurrence in the plastisphere or throughout the overall community might result in erroneous assignment of ecological relevance. As such, studies often lack a link between substrate-specific community shifts in response to plastic exposure and the catabolic potential of candidate taxa to facilitate the observed degradation. Consequently, still relatively little is known about organisms actually catalyzing the in-situ degradation of synthetic polymers, particularly in the context of controlled composting.

In the present study, we report the first microbial community analysis of an LCAP-degrading environment, i.e., thermophilic compost. We aimed not only to identify taxa likely involved in LCAP degradation based on treatment-specific abundance increases, but also to provide functional evidence of their capacity to facilitate the observed degradation process. To this end, we compared the microbial communities across end-point samples of compost material used in the previously reported study of LCAP biodegradation. The composting experiment, as described by Eck et al. (2023), included the LCAP bioplastic materials PE-18,18 (polyester of octadecane-1,18-diol and 1,18-octadecanedioic acid) and PE-12,12 (polyester of dodecane-1,12-diol and 1,12-dodecanedioic acid) and PE-2,18 (polyester of ethane-1,2-diol [ethylene glycol] and 1,18-octadecanedioic acid), and an untreated compost sample as control, each in triplicate incubations.

The microbial community analysis revealed a significant increase of Pseudonocardia and Thermomonospora ASVs in response to LCAP treatment. We provide additional evidence of the functional capacity of Thermomonospora phylotypes to facilitate LCAP depolymerization, by detection of gene homologs of the Thermomonospora curvata polyester hydrolase Tcur1278 (Wei et al., 2014) in the LCAP-degrading reactors by PCR. Additionally, in-vitro testing of this enzyme demonstrated its ability to depolymerize LCAP. Hence, the taxonomic, genetic, and functional evidence combined appoints Thermomonospora as a likely in-situ degrader of LCAP in the investigated environment.

A sequencing-based analysis of compost samples derived from the ISO14855-compliant LCAP degradation study (Eck et al., 2023) revealed treatment-specific shifts in the bacterial community composition, with significantly increased relative abundance of the actinomycetes Pseudonocardia and Thermomonospora in all LCAP-treated samples. The analysis of the fungal community, in contrast, revealed no significant treatment-specific community shifts, indicating a more pronounced effect of polymer treatment on the bacterial community. While these results do not explicitly exclude fungi from the observed degradation process, the data are less likely to reveal the potentially involved organisms.

The observed abundances of Pseudonocardia and Thermomonospora in the LCAP-treated compost were relatively low despite their significant increase compared to the controls (0.3–2.6%), while the bacterial communities overall exhibited remarkably similar core communities, regardless of the applied treatment (Figure 2). This suggests that spiking of the compost with the polymers did not fundamentally alter the compost microbiome, even at the comparatively high loading of the compost with the test substrates. The low relative abundance of ecologically relevant taxa is often part of the difficulty when studying processes within highly active and complex environmental microbial communities (Wellington et al., 2003; Uhlik et al., 2009). Particularly soil environments, and more so thermophilic composting conditions, offer a plethora of alternative nutrients and niches, resulting in a highly diverse community, both phylogenetically and functionally. It can be expected that provision of a novel growth substrate into compost will create an additional niche to be occupied by specialized community members capable of utilizing the substrate for their growth (Goldfarb et al., 2011). However, the recalcitrant nature of LCAP, paired with availability of alternative carbon sources, is unlikely to provide a fitness advantage large enough to fundamentally alter core community dynamics. The provision of such slow-degrading substrates is more likely to result in a low steady-state abundance of community members capable of utilizing that carbon source, particularly those that are able to catalyze the initial, rate-limiting step of the mineralization process.

It has to be considered, however, that this study was based on only a single timepoint and was therefore not able follow community dynamics, which would further inform on the substrate-induced shifts, especially with regard to potential degraders and in reference to the degradation dynamics. At the time of sampling, all incubations had reached a plateau in their CO₂ production, suggesting that active degradation of the substrates had ended and that degrader populations might have been in decline at that point, potentially resulting in low abundances or even loss of degraders (Eck et al., 2023).

According to the associated ASVs, the relative abundance of Thermomonospora increased significantly across all LCAP-treated composts, while Pseudonocardia also increased in cellulose-treated reactors. Both Thermomonospora and Pseudonocardia have previously been implicated in the degradation of PLA, and Thermomonospora species such as T. curvata are known for their ability to degrade cellulose and lignin (Stutzenberger, 1972; McCarthy, 1987; Butbunchu and Pathom-Aree, 2019). Similar functional capabilities have recently been attributed to a Pseudonocardia sp. strain (Sharapova, 2023). These functional associations are in line with a potential involvement in the degradation of cellulose and LCAP in the studied compost samples.

The cellulose-treated compost exhibited further taxa with significantly increased relative abundance, including ASVs associated with order R7C24 and family A4b of the phylum Verrucomicrobiota, and genus Longispora of the phylum Actinomycetota. While not much is known about order-R7C24 and family-A4b bacteria, Verrucomicrobiota include thermoacidophilic members, as well as cellulose degrading strains (Schlesner et al., 2006; Dunfield et al., 2007; Pol et al., 2007; Islam et al., 2008; Bergmann et al., 2011). While cellulose degradation has not yet been demonstrated for members of the genus Longispora, apart from a sequencing-based association of this genus with cellulose degradation in a composting setting, members of the same family (Micromonospora) are known to degrade cellulose (Matsumoto et al., 2003; de Menezes et al., 2008; Shiratori-Takano et al., 2011; Piao et al., 2017; Duan et al., 2022).

These results suggest that, apart from Pseudonocardia, various other potential cellulose-degrading taxa were present in the cellulose treatment. Interestingly, despite their reported capacity to degrade cellulose, Thermomonospora did not establish or maintain a competitive population in the cellulose treatments. While it is possible that Thermomonospora did not contribute to cellulose degradation under the conditions used, an initial population might also have declined below the detection limit following the depletion of cellulose, considering that samples were taken at the end of the degradation experiment. In contrast, Thermomonospora-related phylotypes were consistently enriched in the LCAP treatments, where their relative abundance exceeded that of Pseudonocardia by approximately fourfold. This suggests a stronger association of Thermomonospora with LCAP degradation, while Pseudonocardia may have utilized both cellulose and LCAP substrates, either directly or via secondary degradation products.

The close phylogentic relationship of the detected Thermomonospora ASVs to T. curvata DSM43183, reported to encode the thermostable polyester hydrolase Tcur1278 (Wei et al., 2014), prompted further analysis for potential functional evidence of their involvement in the observed LCAP degradation. For the identified Pseudonocardia ASVs, however, no close association with plastic-degrading functions could be identified.

Qualitative PCR with specific primers confirmed the presence of tcur1278 in the LCAP-treated composts, with the exception of the PE-18,18 treatment. The lack of amplification might be of a technical nature, or suggests a different genotype in these samples, despite the same ASVs being detected as in the other LCAP-treatments. A further functional link was established by demonstrating Tcur1278 activity on PE-2,18 as a representive substrate for LCAP. Results confirmed that Tcur1278 is able to depolymerize PE-2,18, albeit at a lower rate than that observed for the monomer production from PCL as positive control. The repeat units of LCAP are significantly longer than those of PCL, resulting in fewer hydrolysable ester bonds along the polymer chains and, consequently fewer potential cleavage sites for a hydrolase enzyme. This reduced ester-bond frequency might also affect the binding and orientation of the substrate in the active site, and thereby the degradation efficiency. Additionally, the molecular weight of PCL is relatively low with ~14,000 g mol⁻¹ compared to the ~80,000 g mol⁻¹ of PE-2,18, likely increasing its enzymatic depolymerization by offering more end-groups and a less dense polymer-chain arrangement, increasing enzymatic accessibility (Im et al., 2023).

PE-2,18 was chosen to provide proof of concept for LCAP depolymerization due to its higher ester bond frequency compared to the other two LCAP materials, but also due to the facile detection of the depolymerization product ethylene glycol, which is readily soluble in water. In contrast, the long-chain dialcohol and diacid monomers that are released from PE-12,12, PE-18,18 and also PE-2,18, are poorly water-soluble and require solvent extraction and LC–MS-based detection (Schwab et al., 2024). While the activity of Tcur1278 was not tested on PE-12,12 and PE-18,18, their structural similarity to PE-2,18 suggests that depolymerization is likely, potentially at significantly lower efficiency due to the reduced frequency of ester bonds and possibility of increased steric hindrance from longer repeat units. This was previously observed for the commercially available cutinase enzyme of the fungus H. insolens, for which a significantly reduced depolymerization rate was observed with PE-18,18 as substrate compared to PE-2,18 (Eck et al., 2023). It has also been suggested that particularly the length of the diol monomer controls depolymerization rates, at least with regard to the H. insolens cutinase, where increased diol-length resulted in reduced depolymerization rates for LCAP (Schwab et al., 2024).

For all LCAP variants, the respective diacids and diols are non-toxic and expected to be further metabolized through established bacterial pathways. The diacids can be activated and degraded via fatty-acid β-oxidation, while ethylene glycol in funneled into the TCA cycle. The long-chain diols are likely oxidized to the corresponding diacids before entering fatty-acid β-oxidation. Although these pathways are well established in bacteria, future work using isolates or transcriptomics could provide direct evidence for their involvement in LCAP degradation.

Despite the functional link of Tcur1278-encoding Thermomonospora and LCAP degradation, our cultivation attempts using the reference strain T. curvata DSM43183 with LCAP powder as the sole or additional carbon and energy were unsuccessful. However, also cultivation with cellulose powder, a verified substrate, was unsuccessful under the conditions we used. Most likely we were not able to generate the cultivation conditions for inducing the expression of the required depolymerase enzymes. Various factors may influence the successful cultivation including, but are not limited to, the medium composition and the stability of individual components, the availability of specific growth factors, the dependence on other microorganisms for growth on complex polymers, or repression of depolymerase gene expression due to carbon catabolite repression or the absence of inducers. For future studies, we recommend using microcosms containing ‘live’ (non-autoclaved) compost rather than soil, to better simulate the original degradation environment and retain a functionally active microbial community. For axenic cultivation attempts, we also suggest systematic testing and optimization of individual medium components to ensure that all essential nutrients are available and that depolymerase expression is not inadvertently suppressed.

While isolation attempts were not part of this study, various thermophilic filamentous actinomycetes were previously isolated from compost as polyester plastic degraders such as Streptomyces, Thermobifida (formerly Thermomonospora), Saccharomonospora, and Thermoactinomyces, further supporting a high relevance of this taxon for facilitating plastic degradation in these systems (Kleeberg et al., 1998; Hu et al., 2010). Additionally, bioinformatic mining of (meta)genomes has led to the identification of various biochemically confirmed polyester degrading enzymes phylogenetically linked to actinomycetes, including Tcur1278 (Wei et al., 2014; Buchholz et al., 2022). Other than these preceding studies, however, we were able to link Tcur1278-encoding phylotypes of Thermomonospora to the degradation of polyester-based plastics in situ, rather than through enrichment-based isolation or homology-based enzyme mining, providing a rare insight into the ecology of plastic degradation in the context of thermophilic composting.

Thermophilic compost environments play a key role in waste management within a circular economy, as such, an improved understanding of the organisms influencing plastic degradation within these systems is highly relevant to improve this process. Accelerating the biodegradation of biodegradable plastics in compost is of particular interest, given these compounds are expected to be diverted from general waste streams to composting facilities. A better understanding of the degrader communities and functions within these systems can lead to development of optimized plastic-degrading microbial communities to accommodate increases in biodegradable plastic waste. As such, the identification of T. curvata-like phylotypes as active participants in LCAP degradation under thermophilic composting conditions has both ecological and practical implications. As these organisms appear to occupy a distinct niche associated with recalcitrant polymer breakdown, they may contribute to the poorly characterized, but frequently observed, plastic-degradation capacity of thermophilic composting environments. Additionally, the broad substrate spectrum of Tcur1278 including PET, PCL, and now LCAP, suggests a high potential for the depolymerization of various ester-linked plastics. Therefore, targeted enrichment or introduction of such organisms through bioaugmentation strategies could enhance degradation performance.

Overall, our study provides new insight into the thermophilic composting process often used as a microbial ‘black-box’ system for regulatory biodegradation studies, by combining community profiling, differential abundance analysis, and functional gene evidence with enzyme-level validation. Our study strongly suggests Thermomonospora species closely related to T. curvata as a key phylotype for the mineralization of LCAP under thermophilic composting conditions. Not only was this phylotype detectable with increased relative abundance exclusively in LCAP-mineralizing compost reactors, but the presence of tcur1278 in these samples was confirmed, along with the enzyme’s capacity to depolymerize LCAP in vitro. While previous work demonstrated enzymatic activity of Tcur1278 on PET and PCL, its environmental relevance and activity on LCAP had not been investigated. Therefore, our findings not only expand the known substrate range of this enzyme but also demonstrate its presence and likely functional role in an industrial composting context. However, it is important to note that despite our comprehensive analysis, definitive evidence of the attributed role of Thermomonospora and Tcur1278 would require further analysis. Additionally, while we focused on Thermomonospora in this study, Pseudonocardia phylotypes should also be further examined for involvement in LCAP degradation. Future work employing correlative network-based analyses could provide deeper insight into the potential role of microbial interactions in LCAP degradation (Kumar et al., 2024). In addition, isolation attempts or cultivation independent multi-omics approaches that directly link identity and function, could help to unequivocally identify the primary LCAP degraders (Wright et al., 2021; Malik et al., 2023). Dedicated labeling approaches such as nucleic-acid stable isotope probing (DNA/RNA-SIP) in combination with sequencing analyses may provide further confidence in the ecological relevance of candidate taxa by providing direct proof of substrate-carbon assimilation, as previously done with less complex and/or difficult to obtain isotopically-labeled substrates (Luo et al., 2009; Xie et al., 2011; Uhlik et al., 2012; Vogt et al., 2016; Lerner et al., 2020), and recently, for the first time, with a plastic substrate (Odobel et al., 2025).