Toward a Circular Bioeconomy: Designing Microbes and Polymers for Biodegradation

Abstract

Polymer production is rapidly increasing, but there are no large-scale technologies available to effectively mitigate the massive accumulation of these recalcitrant materials. One potential solution is the development of a carbon-neutral polymer life cycle, where microorganisms convert plant biomass to chemicals, which are used to synthesize biodegradable materials that ultimately contribute to the growth of new plants. Realizing a circular carbon life cycle requires the integration of knowledge across microbiology, bioengineering, materials science, and organic chemistry, which itself has hindered large-scale industrial advances. This review addresses the biodegradation status of common synthetic polymers, identifying novel microbes and enzymes capable of metabolizing these recalcitrant materials and engineering approaches to enhance their biodegradation pathways. Design considerations for the next generation of biodegradable polymers are also reviewed, and finally, opportunities to apply findings from lignocellulosic biodegradation to the design and biodegradation of similarly recalcitrant synthetic polymers are discussed.

Introduction

Recent reports estimate annual global plastic production at 460 million tons, of which less than 10% is recycled.¹ The remaining waste is sent to landfills (50%), is incinerated (19%), or becomes environmental litter (22%).^1,2 Furthermore, the 70% of the plastics entering the environment through landfills or litter remain pervasive for hundreds of years after being discarded³⁻⁵ (Table 1). Both abiotic and biotic processes drive fragmentation of these plastics in the environment; this can lead to the formation of microplastics, which can be transported far from the source of the initial plastic via wind and rain.⁶ Microplastics are also able to enter the food chain, which poses a risk to the health of ecosystems, animals, and humans.⁷ Marine plastics are estimated to be responsible for an annual loss of $500–$2500 billion to marine ecosystem services, based on 2011 data, with the cost predicted to be even higher now.⁸

Table 1. Estimated Degradation Half-Lives of Plastics Buried in Land⁵.

Global market share (%)	Plastic-based item	Made of	Degradation half-life (years)
24a	Bags, bottles	Low-density polyethylene	5
16	Food containers, medical devices	Polypropylene	800
5	Bottles, clothes	Poly(ethylene terephthalate)	>2500b
5	Insulating packaging	Polystyrene	>2500b
11	Pipes	Poly(vinyl chloride)	>2500b
24a	Pipes	High-density polyethylene	5000

^a24% represents a combined fraction of HDPE, LDPE, and LLDPE.

^bRelevant studies detected no measurable degradation; the value of 2500 years was obtained by calculating a specific surface degradation rate based on the sensitivity of the balance used in the study

The impervious nature of synthetic polymers is a necessary quality for certain applications, such as aerospace materials⁹ or medical devices;¹⁰ however, this recalcitrance is excessive and unnecessary for many single-use polymers. To reduce the amount of polymer waste entering the environment, new designs for more degradable polymeric materials must be developed. Additionally, much more can be learned from the enzymatic machinery of microbial communities that can break down these materials. The relationship between the design and microbiological breakdown of polymeric materials is not yet fully understood, which has hindered the development of intrinsically circular, carbon-neutral biopolymers.¹¹

Polymeric materials have become essential to our everyday lives. Removing them from our livelihoods altogether is therefore untenable, but approaches can be taken to mitigate the rapid buildup of synthetic polymers. There are two key goals: (i) altering the polymer design and (ii) enhancing polymer biodegradation. These goals will enable the replacement of materials that do not require such long lifespans (e.g., polyethylene (PE), poly(ethylene terephthalate) (PET), polyurethane (PUR), polystyrene (PS)) with next-generation biodegradable polymers. Approaches to the design of these polymers should draw inspiration from polymers that have known degradation mechanisms in nature or by incorporating some “trigger” into the polymer backbone to enable microbial degradation and catabolism. Additionally, there is a need to improve current recycling techniques by improving chemical catalysis (not discussed in this review) or by discovering and designing new microbes and microbial consortia with the potential for polymer metabolism.¹²⁻¹⁷ These microbes and their enzymes can then be engineered to degrade recalcitrant polymers even more efficiently.¹⁸⁻²¹

This proposed polymer carbon life cycle requires careful selection and tailoring of the carbon feedstock and the materials synthesized from it (Figure 1). This review discusses recent breakthroughs in (i) the discovery and engineering of synthetic polymer-degrading microbes and enzymes, (ii) how future polymers can be designed to further enhance their biodegradation, and (iii) how the biodegradation of lignocellulose can provide valuable insight into understanding and facilitating the degradation of recalcitrant synthetic materials.

Figure 1.

A vision to achieve a circular carbon life cycle. Microbes convert plant biomass to value-added biopolymers, which can be converted back to polymer precursors or broken down and metabolized by more microbes at the end of their lives, releasing metabolites and carbon dioxide to contribute to the growth of new plants.

Current Status of Synthetic Polymer Biodegradation

Changes in mass, molecular weight, functional groups, contact angle, mechanical properties, thermal properties, or surface characteristics have previously been used to measure the biodeterioration of a polymer.²²⁻²⁴ However, these factors alone are not sufficient evidence of polymer biodegradability.^24,25 Standardized protocols, such as those from the American Society for Testing and Materials (ASTM) and International Organization for Standardization (ISO), have been developed to measure the conversion of polymer carbon into carbon dioxide, which better quantifies how much of a polymer remains in the test environment. This also allows for more consistent comparisons of the polymer biodegradability between conditions. These tests, however, do not account for the polymer carbon that is converted to microbial biomass. The use of isotope-labeled polymers to measure the conversion of the polymer carbon into carbon dioxide and microbial biomass is therefore the most effective method of assessing polymer biodegradability, although it is more expensive and difficult than the ASTM and ISO methods.²⁴

Conventional petroleum-derived plastics such as PE, polypropylene (PP), and poly(vinyl chloride) (PVC) currently represent 51% of the global plastic market (Table 1) and are predominantly considered non-biodegradable (Table 2). These polymers possess entirely aliphatic backbones, producing densely packed molecules with high crystallinity levels (Table 2), rendering them less susceptible to biodegradation.²⁶ Bio-based polymers, or bioplastics (BPs), are derived from renewable biomass sources. BPs that have the same structure as petroleum-based non-biodegradable polymers, such as Bio-PE, are still non-biodegradable. However, BPs that have more labile bonds along their backbones, such as poly(lactic acid) (PLA), cellulose acetate (CA), and thermoplastic starch (TPS), and the petroleum-based poly(butylene adipate terephthalate) (PBAT), are biodegradable under specific conditions.²⁶ TPS is primarily used for food packaging and foam production and is often used in a blend with PLA, resulting in TPS–PLA plastics.²⁷ CA is found in different consumer products, including textiles, plastic films, and cigarette filters²⁸ (structures of some common biodegradable polymers are shown in Figure 2). The incorporation of biodegradable motifs that disrupt the polymer’s crystallinity can enhance the biodegradability of petroleum-based polymers as well; integrating cellulose, hemicellulose, or other well-described biodegradable components has been explored to help microbes overcome the crystallinity of polymers and promote their biodegradation.²⁷⁻³³

Table 2. Most Abundant Plastics, Their Uses, and Known Biodegradation Paths,,,−.

^aPolyethylene, polypropylene, and polystyrene have been reported to be degraded by certain insects (e.g. waxworm larvae), but there is no commercial approach to biodegrade these materials yet, so for this review they will still be considered as non-biodegradable.

Figure 2.

Structures of some common biodegradable plastics. PHA, starch, cellulose acetate, and PLA are bioplastics, and PBAT is petroleum-based.

Economic viability is a crucial consideration for developing industrially relevant polymers. For example, a biodegradable poly(acrylic acid) was developed by installing ester bonds along the backbone, which facilitate environmental hydrolysis and subsequent biodegradation.³⁴ A significant improvement was observed, with 27.5% biodegradation achieved in 28 days, contrasting with traditional poly(acrylic acid), which does not biodegrade at all.³⁴ However, the economic viability of this synthesis is hampered by the prohibitively expensive starting monomer, 2-methylene-1,3-dioxepane (USD $380 for 1 g from Advanced ChemBlocks Inc., as of May 2024). It is important to find the balance between developing novel polymers and keeping the synthesis costs manageable for the polymer to be appropriately scaled up. Studying how nature synthesizes and breaks down recalcitrant materials, the microbes, and enzymes involved in the degradation mechanisms will guide future innovations to develop a polymer carbon life cycle.

Revealing Polymer-Associating Microbes through Omics Technologies

As synthetic polymers accumulate in different environments, they encounter and influence microbial communities. Omics tools, such as 16S rRNA/ITS gene sequencing, shotgun metagenomics, (meta)transcriptomics, (meta)proteomics, and metabolomics, can be used to probe the different levels of microbial response to polymer exposure (Table 3). Researchers can use this information to better understand which microbes associate with specific types of plastics and even to design new polymers that degrade based on expected enzymatic machinery in a given environment. When omics tools are combined with materials characterization or biodegradation efficiency of the polymer of interest, patterns between the polymer design and microbial response can be established. Moreover, discoveries from omics can further inform the development of genetic engineering tools that can be utilized to enhance microbial capacities for polymer degradation.

Table 3. Omics Tools Used to Investigate Microbial Response to Polymers.

Omics tool	Use
16S/ITS sequencing	Identification of bacteria/fungi composition in a microbial community and changes in their relative abundances in response to different conditions
Metagenomics	Identification of all taxonomic composition in a microbial community, changes in their relative abundances in response to different conditions, and their metabolic profiles
(Meta)transcriptomics	Identification of differential gene expression in a microorganism (or microbial communities) in response to different environmental conditions
Meta(proteomics)	Identification of proteins produced by a microorganism (or microbial community) in response to different environmental conditions
Metabolomics	Identification of metabolites produced by a microorganism (or microbial community) in response to different environmental conditions

16S rRNA gene amplicon sequencing and metagenomics enable the identification of polymer-degrading microbes in an environmental sample.³⁹⁻⁴¹ When a microbial community is exposed to a polymer, increases in the relative abundance of specific taxa can indicate their capability to utilize the polymer as a carbon source. These omics tools were employed to study the PET-degrading capabilities of microbial communities isolated from marine plastic debris in Wales, UK,⁴² and to investigate the degradation of biodegradable polymers by microbiomes isolated from the Tsurumi River in Japan.⁴³ In the former case, the microbial community was incubated for 6 weeks with PET products as a sole carbon source.⁴² 16S sequencing was then used to track the dynamics of the community in response to PET and determined that Alcanivoraceae became the most dominant microbe. Moreover, two other novel marine isolates capable of PET degradation, Thioclava sp. BHET1 and Bacillus sp. BHET2 were also identified.⁴² In the latter study, 16S sequencing was employed to investigate the degradation of five biodegradable polymers: polycaprolactone (PCL), poly(butylene succinate-co-adipate) (PBSA), poly(butylene succinate) (PBS), PBAT, and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH). This led to the identification of putative plastic-degrading bacteria as well.⁴³ Another study assembled a metagenomic library from DNA isolated from soil exposed to polyurethanes and identified three new urethanases.³⁵ These are the first reported enzymes that directly hydrolyze urethane bonds efficiently.³⁵

In comparison to 16S or ITS sequencing, which only sequence selected organisms or genes of bacteria or fungi, respectively, shotgun metagenomics is the untargeted sequencing of all microbial genomes present in a sample and can be utilized to identify genes and metabolic pathways involved in polymer degradation.⁴¹ Shotgun metagenomics was used to identify the microbial communities growing on PET and PHA biofilms in coastal marine environments⁴⁴ and to study the plastic degrading potential of the microbiome colonizing solid municipal waste.⁴⁵ The PHA film was significantly enriched in sulfate-reducing microbes, and analysis of the gene pool revealed enrichment of depolymerase (20-fold increase) and esterase (2.5-fold increase) genes.⁴⁴ Metagenomic reconstruction of genomes revealed three novel species, Desulfovibrio, Desulfobacteraceae, and Desulfobulbaceae, all possessing genes integral for BP biodegradation and sulfate reduction.⁴⁴ The microbial community grown on a PET biofilm had an insignificant difference from the ceramic film negative control.⁴⁴ To study the communities growing on solid municipal waste, six samples were taken from soil, three from leachate, and one from compost, and it was identified that communities were dominated by bacteria, specifically Proteobacteria, Firmicutes, and Actinobacteria.⁴⁵ The functional potential of the microbes was analyzed for biodegradation of PE, PET, and PS; multiple enzymes that have been documented for degrading these materials were identified within the solid municipal waste microbiome.⁴⁵ Enzymes for oxidizing PE (e.g., laccases and monooxygenases), PET hydrolases, and enzymes for degrading PS (e.g., phenylacetaldehyde dehydrogenase) were identified, but no growth tests were performed, so it is unknown whether these species can adapt to metabolize these plastics as a sole carbon source.⁴⁵

These omics tools can also be used in studies aimed at isolating polymer-degrading microorganisms from their community. Metagenomics, metatranscriptomics, and metaproteomics were utilized to identify the key enzymes and microbial interactions that drove an enriched marine consortium’s ability to biodegrade a PBAT-based copolyester.⁴⁶ The use of several omics tools in that study enabled the analysis of gene expression and protein biosynthesis patterns involved in this biodegradation process, including those of PETase-like and MHETase-like enzymes.⁴⁶ In another study, a bacterium isolated from deep sea also showed potential to degrade waterborne PUR.⁴⁷ Using transcriptomics, the researchers found that Bacillus velezensis GUIA produces proteases, lipases, and oxidoreductases which were all upregulated in the presence of PUR, and the oxidoreductase Oxr-1 was determined to play the key role in the degradation of PUR.⁴⁷ Researchers in Switzerland isolated 34 microbial strains from plastics buried in alpine or arctic soils; five of these strains (three fungal and two bacterial) had a sequence similarity considerably below the 98.65% taxonomic threshold,⁴⁸ suggesting that Neodevriesia sp., Mortierella sp., Lachnellula sp., Streptomyces sp., and Amycolatopsis sp. are newly isolated species with potential to break down plastics.⁴⁹ Of the 34 strains isolated, none were able to degrade PE; however, 19 strains were able to degrade PUR. Neodevriesia sp. and Lachnellula sp. were able to degrade all the biodegradable plastics tested.⁴⁹ These results suggest that the alpine and arctic soils likely possess a unique microbiome with useful biotechnological applications.^49,50

Other organisms, including worms and fungi, have also displayed remarkable proficiency for breaking down synthetic polymers.^14,51−56 Mealworms (the larvae of Tenebrio molitor) fully digested Styrofoam in under 24 h, converting 47.7% into CO₂; 49.2% was egested as fecula, and 0.5% was incorporated into biomass.^51,52 Over the course of 1 month, mealworms fed exclusively a diet of Styrofoam thrived as well as those fed a normal diet.^51,52 These worms were also able to grow on PE, achieving similar degradation rates as PS.⁵⁵ Microbiome analysis revealed that two species, Citrobacter sp. and Kosakonia sp, were primarily responsible for PE and PS biodegradation in the worm guts.⁵⁵ Another species of worm, Zophobas atratus, was also able to thrive on an exclusive diet of Styrofoam, converting 36.7% of the ingested Styrofoam into CO₂ within 16 days.⁵⁴ In another study, Z. atratus was fed HDPE, PP, and PS to increase the abundance of plastic-degrading bacteria in their gut microbiome, aiming to establish a microbial community capable of thriving on plastics.⁵³ The primary bottleneck preventing PE biodegradation is the introduction of oxygen into the polymer chain, fragmenting the polymer into smaller, digestible pieces.⁵⁷ Typically, this oxidation step requires abiotic pretreatment. However, Galleria mellonella can oxidize unpretreated PE within 1 h,⁵⁷ making it the most efficient organism discovered so far for degrading PE. It was found that the gut microbiome was surprisingly not responsible for the degradation of PE. Instead, enzymes from the saliva of G. mellonella were identified as the key agents, including two enzymes named Demetra and Cibeles that self-associate into a heterocomplex (which could oxidize and break down PE) and two hexamerins, Ceres (only able to oxidize PE) and Cora (able to degrade PE).^57,58

Fungi have also shown the ability to degrade polyethylene microplastics,⁵⁹ polypropylene,⁶⁰ and other recalcitrant materials.⁶¹⁻⁶³Aspergillus terreus achieved 25.29% degradation when grown on a UV-pretreated PP metallized film, while Engyodontium album achieved 27.08% degradation on a metallized PP film pretreated with heat over a period of 90 days.⁶⁰ However, these studies failed to investigate the mechanisms by which these fungi degrade recalcitrant materials. Gaining insight into how these microbes break down such materials could significantly contribute to the design of new materials more prone to biodegradation. Additionally, understanding which enzymes and microbes should be targeted for genetic engineering could enhance the microbial metabolism of synthetic polymers.

In general, polymer biodegradation studies that collect various types of biological information can address more specific questions about microbes and degradation mechanisms within the microbial community. The application of these omics tools will continue to aid in the discovery of new microbes and enzymes capable of degrading polymers. This information is an essential precursor to developing strategies for improving the polymer-degrading activity of such microbes.

Toward Enhanced Bioplastic Biodegradation

PLA, which has ester bonds along its backbone, is often considered the model circular bioplastic because its starting material, lactic acid, is from fermented corn rather than fossil fuels.^64,65 The ester bonds of PLA can be readily hydrolyzed, converting the polymer back to lactic acid (Figure 3). This lactic acid can either then be fully metabolized by environmental microbes or be used to resynthesize PLA, thus eliminating its presence in the environment. However, its biodegradability strongly depends on the test environment.⁶⁶ A study found that PLA achieved 90% biodegradation during a period of 200 days under industrial composting conditions but only 4% biodegradation when buried in soil.⁶⁷ This trend can be found among other bioplastics as well; cigarette butts are one of the most notorious littered items and are made of CA, yet CA is not as biodegradable as it is often assumed to be.^26,33 The biodegradation of CA largely depends on the degree of acetyl substitution (DS) and the degradation environment.^26,33 Over 55 days in a controlled compost environment, cellulose acetate powder with a DS of 1.5 and 2.5 achieved 45–50% mineralization, however powder with a DS of 3.0 achieved 9% mineralization, and cellulose acetate granules with a DS of 2.5 only reached 15% mineralization.³³ Starch blends follow similar patterns to CA and PLA.^32,66,68 Wheat-starch-derived plastics buried in soil for 660 days only achieved 19.7% biodegradation,⁶⁸ and plastarch (a polypropylene–starch blend) achieved 55% biodegradation after composting for 120 days.⁶⁸ These limitations are important to consider in the design of next-generation bioplastics and waste collection strategies.

Figure 3.

Ester bonds along the PLA backbone are susceptible to enzymatic and chemical degradation, yielding its starting material, lactic acid.

A multitude of bioplastics have been developed, but to replace traditional petroleum-based plastics, further studies are needed to develop new bioplastics, improve current bioplastics, and characterize the biodegradation of these materials. It is well-documented that lower molecular weight facilitates easier microbial metabolism of a substrate.⁶⁹⁻⁷³ Consequently, researchers are developing strategies to integrate built-in degradability “triggers” into polymer structures to render the residual polymer fragments more easily digested. Researchers at Colorado State University and the National Renewable Energy Laboratory have been drawing attention to the idea of “intrinsically circular polymers” (iCPs).⁷⁴ These iCPs are kinetically trapped and are inherently and selectively depolymerizable to their monomer state when subjected to thermal/light energy, catalysis, or some other external stimulus. An example of how this can be incorporated into polymers was shown by researchers who nanoscopically dispersed enzymes into polyesters. Burkholderia cepacia lipase (BC-lipase) and Candida antarctica lipase (CA-lipase) were embedded in PCL, and proteinase K was embedded in PLA along with a previously developed four-monomer random heteropolymer to disperse the enzymes.²¹ Incorporating these enzymes into the polymers resulted in a less than 10% change in elastic modulus and tensile strength, while near-complete degradation was achieved within just a few days in a standard compost setup.²¹

Recent research has also established biochar soil amendments as an effective strategy to mitigate greenhouse gas emissions and improve the biodegradation of soil contaminants.^75,76 Biochar is a carbon-rich solid material obtained from the pyrolysis of organic waste or biomass.⁷⁶ Adding biochar to soil has been shown to significantly enhance polycyclic aromatic hydrocarbon (PAH) removal^77,78 and reduce the bioavailability of heavy metals such as chromium and lead.⁷⁹ Biochar can also enhance the biodegradation of bioplastics during composting. When biochar from livestock manure was added to compost containing polyhydroxyalkanoate (PHA) microplastics, it was found to increase the degradation of PHAs by 20% over 60 days.⁸⁰ The addition of biochar also enriched the compost with microbes that were more efficient at degrading PHAs.⁸⁰ More recently, research has been looking into making biochar composite polymers to enhance the biodegradability of plastics and bioplastics.⁸¹⁻⁸⁴ Biochar from postconsumer food waste was added as a filler material to PLA and HDPE, and it was found to decrease the thermal degradation and melt viscosity of PLA and made no difference in the thermal degradation and increased the melt viscosity for HDPE.⁸⁴ Under composting conditions, though, the PLA–biochar composite had double the mass loss compared to neat PLA over 40 days.⁸⁴ Another study investigated the effect biochar additions had on recycled plastics and found that biochar additions increased the tensile strength, stiffness, and flexural storage modulus of recycled HDPE but resulted in a more brittle material.⁸² Altogether, the studies presented above underscore the potential of biochar in enhancing degradable composite materials. However, further research is needed to enhance the mechanical performance of these materials and to understand the microbial response to them.

Additives have become a common approach to grant materials desirable characteristics; however, the effects additives have on polymers are not always clear. Common additives include fillers (e.g., clays, silica, alumina), which reinforce structure and reduce synthesis cost; plasticizers (e.g., phthalates), which adjust polymer flexibility; flame retardants (e.g., poly(bromo diphenyl ethers)), which prevent ignition; colorants (e.g., cadmium, chromium, cobalt), which determine polymer color; and others.⁸⁵⁻⁸⁷ The effect these additives have on the biodegradation of plastics is still poorly understood, and results vary drastically depending on the additive used. Some additives may reduce biodegradability by being toxic to microbes, altering the hydrophobicity or hydrophilicity of the polymer surface, or adding antimicrobial properties. Some additives claim to enhance biodegradation by incorporating biodegradable components that disrupt the polymer network and reduce crystallinity.⁸⁷⁻⁸⁹ However, conflicting reports have found that additives claiming to promote polymer biodegradation may be unwarranted.⁹⁰ Five additives that claim to increase biodegradation of PET and PE were tested under composting, anaerobic digestor, and soil burial conditions, and none of the additives significantly increased biodegradation.⁹⁰ Lignin has also been investigated as an additive for many biopolymers, such as starch, protein, cellulose, or PLA, and it was shown to improve the flexibility, mobility, and workability of polymers.⁹¹⁻⁹⁴ The absence of clear research in this field and the conflicting reports on biodegradable additives suggest that further work should elucidate the effects these additives have on polymer biodegradation and the impact on the microbiome.⁸⁹

Genetic Tools to Engineer Degradation Activities of Polymer-Degrading Microbes

Genetic Engineering Techniques to Promote Enzymatic and Microbial Biodegradation of Synthetic Polymers

As researchers continue to identify plastic-degrading microbes and enzymes, advances in genetic engineering tools enable their enhancement. Ideonella sakaiensis’ key enzymes, PETase and MHETase, have been engineered through different methods to improve their degradation abilities.^18,95−100 A study developed a high-throughput directed evolution platform to engineer PETases.⁹⁷ Twenty-one mutations were introduced into the new enzyme, HotPETase, leading to a notable enhancement in both thermostability and activity.⁹⁷ Determining the structure of proteins can be important in engineering enzymes. A 0.92 Å X-ray structure of IsPETase revealed key insights into the structure of PETase, highlighting features of the enzyme that could be changed to improve its activity.¹⁰⁰ Based on the crystal structure of PETase, rational protein engineering was used to increase its thermostability and efficiency.⁹⁶ Several limitations of PETase were identified based on its crystal structure, and variants were developed to overcome these limitations. One such limitation that was addressed was the interruption of the central β-sheet in IsPETase caused by an abnormality in one of the surrounding β-strands.⁹⁶ This interruption prevented formation of hydrogen bonds between proline and leucine.⁹⁶ The variant, IsPETase^P181A, addressed this and increased its melting temperature, but overall, there was not a significant improvement over wild-type PETase.⁹⁶ A few more iterations of rational design based on the crystal structures of the wild-type enzyme and the variants led to IsPETase^{S121E/D186H/R280A}, which exhibited significantly increased thermal stability and degradation activity over wild-type PETase.⁹⁶ Another study employed a 3D self-supervised convolutional neural network (MutCompute) to suggest five mutations to the wild-type PETase. This led to the creation of the mutant FAST-PETase, which enhanced PET hydrolytic activity across a range of pH levels and temperatures between 30 and 50 °C.¹⁸ The recovered monomers from this degradation study were used to resynthesize PET, demonstrating the potential circularity of this platform.¹⁸

The discovery of I. sakaiensis led to the rapid discovery of multiple other organisms capable of breaking down PET. A study testing five different enzymes reported to possess PET-degrading abilities (including IsPETase) found leaf-branch compost cutinase (LCC) to have a depolymerization rate 33 times more efficient than any of the other enzymes and the highest thermostability.¹⁹ Eleven of LCC’s amino acids were identified for site-specific mutagenesis to improve the catalytic activity, resulting in an enzyme (LCC^ICCG) capable of achieving 90% conversion of PET in 10 h.¹⁹ Furthermore, the terephthalic acid degradation products were recycled back into PET and processed into plastic bottles with the same properties as those derived from petrochemical sources.¹⁹ A comparison of four of the most efficient PET degraders developed so far (LCC^ICCG,¹⁹ FAST-PETase,¹⁸ HotPETase,⁹⁷ and a polyester hydrolase [PES-H1^L92F/Q94Y]¹⁰²) found that LCC^ICCG’s degradation rate outperformed the other enzymes to convert PET into its monomers, ethylene glycol and terephthalic acid.¹⁰³ Based on these results, LCC^ICCG emerges as the prime candidate for industrial-scale reactions, aligning precisely with Carbios’ plans.¹⁰⁴

While most of the above genetic engineering strategies have involved enhancing the biodegradation activities of organisms that are already capable of degrading plastics, strategies to take these enzymes and express them in model organisms represent another strategy. For example, Escherichia coli is a very well-studied organism, making it much easier to scale up and use as a cell factory to produce large quantities of a desired enzyme. E. coli was engineered to secrete PETases to breakdown PET into terephthalic acid and ethylene glycol using sec-dependent signal peptides.^105,106 It was found that by fusing signal peptides from E. coli to PETase and transforming PETase into E. coli, PETases could be produced and transported out of the cells for PET degradation,¹⁰⁵ thus highlighting the potential for E. coli to be used as a cell factory to scale up the production of these enzymes. PelB, the signal peptide responsible for transporting the proteins to the periplasm, was subjected to random mutagenesis, and the resulting variants improved the secretion efficiency 1.7-fold.¹⁰⁶ Similar methods were used for extracellular PETase secretion with Bacillus subtilis,¹⁰⁷Phaeodactylum tricornutum,¹⁰⁸ and Chlamydomonas reinhardtii,¹⁰⁹ and an alternative approach to express PETases on the cell surface was used to turnPichia pastoris GS115¹¹⁰ andVibrio natriegens¹¹¹ into whole-cell biocatalysts.

E. coli was also engineered to upcycle PET to vanillin¹¹² and adipic acid,¹¹³ a molecule with applications toward food and cosmetics and a nylon precursor, respectively. E. coli MG1655 RARE (reduced aromatic aldehyde reduction) was used as the host strain, and the cells were transformed with plasmids containing the enzymes terephthalate 1,2-dioxygenase (TPADO), dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid dehydrogenase (DCDDH), carboxylic acid reductase (CAR), and catechol O-methyltransferase (COMT) to convert terephthalic acid to vanillin in 79% yield.¹¹² They also showed that the engineered E. coli could convert postconsumer PET waste into vanillin by pretreating the PET with LCC enzymes.¹¹² Similar techniques were used to transform E. coli with the genes necessary to convert PET into adipic acid.¹¹³

Engineering enzymes and microbes remains a time-intensive process, which has inhibited the rate of discovery and improvement of these polymer-degrading systems. Employing high-throughput instruments and machine learning significantly lowers the time and energy barriers that have prevented further improvements of these mechanisms.

Machine Learning Can Assist Plastic Biodegradation

Machine learning platforms that are capable of rapidly predicting and screening information from microbes, enzymes, and genomic databases will greatly improve the discovery and engineering of novel enzymes with the ability to break down and convert recalcitrant polymers to value-added materials (Figure 4). As the volume of biological information collected for databases like Carbohydrate-Active Enzymes (CAZy),¹¹⁴ Plastics-Active Enzymes (PAZy),¹¹⁵ and the Joint Genome Institute (JGI) Genome Portal¹¹⁶ continuously increases, computational algorithms will have a larger pool of data to draw conclusions from.¹¹⁵

Figure 4.

Machine learning is a powerful tool to facilitate novel enzyme discovery and engineering and is already responsible for major advances in plastic biodegradation.

Recently, machine learning was implemented to develop the plastic enzymatic degradation (PED) framework.¹¹⁷ This PED framework can predict the ability of an enzyme to degrade plastics by analyzing protein sequences to identify specific patterns. The framework outperformed many other sequence-based classification models and identified that hydrophobicity, heat capacity, frequency of occurrence of alanine in the sequence, and the enzyme’s molecular weight are the most important structural motifs for an enzyme to degrade plastics.¹¹⁷ This PED framework works by inputting an enzyme sequence and plastic type, and the framework outputs “degradable” or “nondegradable” with an accuracy of 90.2% (tested with 230 enzyme–plastic pairs from 129 enzyme sequences and 11 types of plastics).¹¹⁷

Hidden Markov models (HMMs) provide probabilistic models to sort large linear sequences, are powerful tools in computational biology, and are often used for protein structure prediction and large-scale genome sequence analysis.¹¹⁸ Recently, a deep-sea Candidatus Bathyarchaeota archaeon, known for its ability for methane metabolism and nitrate reduction,^119,120 was found to express an esterase capable of degrading PET comparable to wild-type IsPETase and LCC.¹²¹ This discovery marked the first reported case of an archaeal PETase and was facilitated by employing an HMM to analyze global genome and metagenome databases.¹²¹ In this example, specifically an HMM was used to parse the National Center for Biotechnology Information (NCBI) genome and metagenome databases, pinpointing potential PETases.¹²¹

Another computational tool with major impacts on biology is AlphaFold.^122−124 AlphaFold v1.0 leverages physical and biological protein structure information along with multisequence alignments into its machine learning platform to predict the structure of proteins with exceptional accuracy.¹²² AlphaFold granted scientists unprecedented access to structural models for virtually any protein based simply on the amino acid sequence.¹²² AlphaFold v2.0 expands the known protein–sequence space even further and was released with a public database that provides extensive access to highly accurate protein structure models.¹²³ Now AlphaFold v3.0 has been released and offers significant improvements in prediction accuracy and has been expanded beyond proteins to include predictions of the structures and interactions of ligands, ions, nucleic acids, and modified residues as well as complexes of these.¹²⁴ AlphaFold v3.0 was also released with a user-friendly server, letting scientists perform their own predictions for the structures and interactions of proteins, DNA, RNA, and some other small molecules.¹²⁴

While AlphaFold has garnered significant attention in areas such as drug discovery, proteomics, and the understanding of diseases, its capabilities provide important insights to understand the enzymes and mechanisms responsible for the biodegradation of complex polymers as well. An HMM approach was used to identify 74 thermotolerant PET hydrolases, which were then subjected to experimental screening and AlphaFold for structural characterization.¹²⁵ Thirty-seven of these enzymes were active for PET hydrolysis, 23 of which had not been previously reported.¹²⁵ AlphaFold revealed diverse enzyme architectures but found that the most active PET degraders had a highly conserved core domain with a nine-stranded β-sheet flanked by eight or nine α-helices.¹²⁵ Another study developed a new PET variant, TurboPETase, which achieves near-complete biodegradation of PET in 8 h at industrially relevant loadings and outperforms other well-known PET hydrolases as well.¹²⁶ AlphaFold was used to analyze the structure of TurboPETase, and it was suggested that the improved performance could result from a more flexible substrate binding cleft, improved charge–charge interactions on the PETase surface, and the introduction of a disulfide bond, which contributes to improved thermostability.¹²⁶ AlphaFold has also provided some structural insights into polyurethane biodegradation by the enzyme polyurethane esterase A (PueA) from Pseudomonas chlororaphis and represents a good starting point to develop improved iterations of this polyurethane degrading enzyme.¹²⁷

Recent years have seen a surge in discoveries of novel polymer-degrading enzymes and engineering strategies to improve these systems. This surge can largely be attributed to machine learning and high-throughput instruments significantly decreasing the time and energy necessary to pursue these innovations. Where previously scientists were limited by the number of reactions that they were capable of running in a given time, high-throughput platforms are quick and relentless, while machine learning enables more targeted experimentation, granting far more results in the same time frame than were previously possible.

Leveraging Lignocellulose to Design Next-Generation Renewable Polymers

Lignocellulose provides the foundation for plant cell walls. It is a renewable and abundant material that is responsible for endowing plants with structure and rigidity, making it a promising carbon source to replace petroleum.^128,129 Lignocellulose can be used to create new plastic materials through various means. First, studying the degradation of lignocellulose can provide valuable insights and inform research efforts aimed at understanding and improving the biodegradation of bioplastics. Second, lignocellulose and its fractions offer a renewable and sustainable source of raw materials to produce new plastic materials, contributing to the development of environmentally friendly alternatives to traditional plastics.

From Lignocellulose Degradation to Enhanced Bioplastics Degradation

Due to similarities in composition, understanding how microorganisms degrade and catabolize lignocellulose can offer insights into the breakdown of bioplastics. The enzymatic mechanisms in lignocellulolytic organisms can also serve as starting techniques that can be engineered for targeted degradation of breakout products. Furthermore, both lignocellulose and bioplastics undergo degradation under diverse environmental conditions.^{22,32,66,129−132} By examining factors such as temperature, moisture, and microbial activity that influence lignocellulose degradation, researchers can gain a deeper understanding of how these factors impact bioplastics degradation.

Lignocellulose is composed of cellulose, hemicellulose, and lignin, three of the four most abundant polymers found in nature.^15,128,133 Cellulose is a polysaccharide comprising many d-glucose units, and hemicellulose is made of many sugars, such as xylose, arabinose, mannose, and galactose. Both cellulose and hemicellulose are abundant, durable, and easily biodegraded via enzymatic hydrolysis into simple sugars.¹³⁴ Lignin, in contrast, has a much more complex structure; p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol undergo dehydrogenative polymerization to form guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units, linked together by C–O and C–C bonds (Figure 5)¹³⁵

Figure 5.

Structural examples of lignocellulose. (A) Cellulose is composed of repeating glucose units. (B) Hemicellulose is composed of many sugars such as xylose, arabinose, mannose, and galactose. (C) Coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol are the primary monomers which polymerize to form lignin, with H, G, and S units that are linked together by various key C–O and C–C bonds (colored). (D) Example arrangement of how monomers form lignin. Biological lignin can take on many forms, which are not all captured here.

Lignin’s structure makes it much more difficult for microbes to break down and catabolize as a carbon source;^128,132,134 however, certain microorganisms have demonstrated the ability to break down lignin. The aerobic bacterium Thermobifida fusca and the anaerobic bacterium Clostridium thermocellum are examples of lignolytic bacteria that effectively solubilize carbohydrates and lignin within lignocellulose simultaneously.¹³⁶ These species use ligninolytic enzymes, such as laccases, lignin peroxidases (LiPs), versatile peroxidases (VPs), and manganese peroxidases (MnPs). These enzymes are the most widely characterized enzymes responsible for the breakdown of lignin in both bacteria and aerobic fungi.^{131,133,137,138} Furthermore, carbohydrate-active enzymes (CAZymes) produced by certain fungal and bacterial species have proven to be quite effective at overcoming lignin’s recalcitrance.^15,139,140 NMR studies identified that termites can efficiently break down both C–C and C–O ether bonds.^141,142Coptotermes formosanus were fed softwood, hardwood, and grass lignocellulose diets and were found to target β-5 or β–β bonds over β-O-4 bonds¹⁴¹ (see Figure 5). Another study fed ¹³C-labeled Populus x canadensis stems to the higher termite Nasutitermes sp. and found that after passage through the gut, there was significant depletion in all primary lignin bonds, β-O-4, 4-O-β, β–β, β-5, and 5–5.¹⁴² Anaerobic gut fungi (AGF) have also emerged as specialists in the breakdown and utilization of lignocellulose via their CAZymes largely employed through cellulosomes.^{13,15,143−146} Mechanisms for AGF lignin degradation are yet uncharacterized, but recent work with 2D-HSQC NMR has demonstrated their ability to break down and reorganize un-pretreated, naturally occurring lignin under anaerobic conditions to access the more nutrient-rich cellulose and hemicellulose components of the plant biomass.¹⁵ These anaerobic fungi preferentially removed syringyl units by remodeling β-aryl ether units and phenylcoumarans (β-O-4 and β-5 bonds, respectively).¹⁵

Insights from the Microbial Metabolism of Lignocellulose May Enhance the Biodegradation of Synthetic Polymers

Microbes responsible for breaking down plant biomass provide a starting point to speed up the evolution necessary to breakdown synthetic polymers and can provide clues for designing next-generation biodegradable polymers. The three components of lignocellulose are made of many repeating C–C and C–O bonds (Figure 5), with microbes typically choosing to target ester bonds; structural similarities between lignin and certain aromatic-rich synthetic polymers suggest that enzymes capable of degrading lignin also may be strong candidates to degrade other synthetic polymers.^{130,147−149}

Lignocellulose provides plants with protection and rigidity, and it is naturally recalcitrant toward biodegradation. However, over time, certain organisms have evolved to degrade and extract nutrition from these plants. These organisms developed the tools to overcome the complex aromaticity and hydrophobicity of lignin and the crystallinity of cellulose microfibrils.¹⁵⁰ Different species employ different mechanisms to degrade lignocellulose; cellulose and hemicellulose can be broken down via enzymatic hydrolysis using different variations of secreted extracellular enzymes.^130,150,151 The biodegradation of cellulose liberates glucose and cellobiose as primary products, and the biodegradation of hemicellulose yields pentoses (xylose, arabinose) and hexoses (mannose, glucose, galactose).¹⁵⁰ Lignin is typically broken down through the actions of various enzymes (e.g., laccases and peroxidases¹³³) or chemical reactions (Fenton chemistry^130,151). While the glycosidic bonds connecting the various sugar units in cellulose and hemicellulose are susceptible to hydrolysis, they are not found in synthetic plastics; however, the ester bonds (β-O-4 and 4-O-5 in Figure 4D) and C–C bonds (5–5, β–β, and β-5 in Figure 4D) found in lignin are more reminiscent of bonds found in synthetic plastics. Figure 6 highlights some of the similarities between products that can be obtained from the biodegradation of lignin (Figure 6A) and some example polymers with bonds and characteristics similar to those of lignin (Figure 6B). The chemicals shown in Figure 6A are common starting materials to synthesize various chemicals, polymers, or pharmaceuticals. Studying how microbes break down the ester and carbon–carbon bonds in lignin can provide valuable insight into understanding how to breakdown similar synthetic polymers and to designing next-generation biodegradable biopolymers to replace current petroleum-based plastics.

Figure 6.

Biodegradation of lignin can provide insight into how microbes break down natural, recalcitrant materials, while providing access to (A) lignin-derived polymer precursors to renewably synthesize (B) various commodity chemicals and polymers.

Large-scale screening of microbes from lignin-rich environments may provide an interesting starting point to identify novel microbes to improve the biodegradation of lignin and to break down synthetic polymers. The digestive tracts of herbivores provide such an opportunity. The cow rumen has a suite of enzymes that are tailored to breakdown polyesters to derive nutrition from plant biomass, and recent evidence shows that rumen fluid can also hydrolyze certain synthetic polyesters, including PET, PBAT, and poly(ethylene furanoate) (PEF).¹⁵² Plant-biomass-degrading enzymes such as cutinases and lipases are known to target ester bonds in plastics and bioplastics,^153,154 and peroxidases and laccases have also been shown to target synthetic plastics.¹³⁰ A lignin peroxidase from Phanerochaete chyrsosporium grown on PVC decreased the weight by 31% over 4 weeks,¹⁵⁵ and these enzymes play an important role in the fungal biodegradation of lignin¹³³ and highlight some of the impacts lignocellulose-degrading enzymes may have on synthetic plastic biodegradation. Furthermore, the bacteria Rhodococcus ruber was identified to grow on PE, secreting a laccase that achieved 2.5% biodegradation of PE over 30 days.¹⁵⁶ Microbes from the guts of worms have also displayed a propensity to degrade synthetic plastics.^{14,51−54,57,58}

Certain organisms and enzymes have displayed the ability to utilize both lignocellulose and synthetic plastics, but there are still many obstacles that must be overcome before these lignocellulose-degrading systems can efficiently be translated to plastic biodegradation. The general lack of hydrolyzable bonds in petroleum-based plastics (PE, PS, PP, etc.) render these materials far more recalcitrant and crystalline than lignocellulose. Understanding and emulating how nature has designed polymers will provide key insights to develop an arsenal of next-generation renewable polymers to satisfy humanity’s needs while remaining susceptible to microbial degradation to prevent further accumulation of these toxic materials in our environments.

Conclusions

Microorganisms with remarkable abilities for biotechnological applications continue to be discovered.^{14,16,35,47,49,157} Having the tools to both discover and improve these novel microbes’ enzymatic machinery and ability to process recalcitrant materials will streamline their translation to industrial applications. After their discovery, several polymer-degrading enzymes have been improved considerably through genetic engineering techniques such as mutagenesis,^18,19 directed evolution,⁹⁷ and rational design.¹⁰⁰ Machine learning and high-throughput techniques have already made significant contributions to discovering new polymer-degrading enzymes^121,125 and identifying genetic modifications to improve enzyme activity^18,126,127 and will continue to increase the rate of innovation by decreasing the time and energy scientists must input to obtain results.¹²⁴

Developing this renewable carbon life cycle for polymers can be significantly improved by drawing insights from how nature breaks down lignocellulose. Cellulose and hemicellulose can be degraded efficiently by enzymatic hydrolysis, but the full utilization of this feedstock has been hindered by lignin.¹³⁴ Improved understanding of heterogeneous bacterial and fungal consortia,^13,158 studies characterizing lignin degradation mechanisms,^15,141,159 and the discoveries of anaerobic fungi and bacteria’s ability to localize their suite of lignocellulolytic enzymes with cellulosomes^15,160 will forge the way to improved bioprocessing of lignin. A deeper understanding of these processes will translate to enhanced biodegradation of synthetic polymers and biopolymers. As more research elucidates the mechanisms via which microbes break down recalcitrant polymers, this information should be used to design new, biodegradable polymers and biopolymers. Not every polymer can be replaced with a biodegradable alternative, but many can and should be. Incorporating labile bonds along polymer backbones that are traditionally exclusively carbon provides a handle to break down a polymer chemically^34,161 or via enzymatic digestion.^21,73 When such bonds are broken, the resulting fragments are more compatible for microbial digestion than the original polymer.⁶⁹⁻⁷²

Considering the large volume of waste that ends up discarded in the natural environment, the next generation of bioplastics must confront these limitations. Designing and synthesizing petroleum-based, non-biodegradable plastics might seem cost-effective in the short term, but the long-term consequences of plastic waste pollution will be far more costly and energy-intensive than designing polymers that do not become recalcitrant waste.^8,25,74,152 As microbial machinery and polymer design become optimized, their interactive roles will establish a circular carbon cycle and thus contribute to ameliorating Earth’s environmental crisis.

Author Contributions

V.M., C.B.A., M.C., and M.A.O. wrote and edited the manuscript. M.C. and M.A.O. provided funding support. M.A.O. coordinated project management.

The authors declare no competing financial interest.