A Review on the Role of Insects and Their Gut Microbiota in Plastic Biodegradation

A review on the role of insects and their gut microbiota in plastic biodegradation

Carlos Nahuel Regalsky Mallar

Leopold-Franzens-Universität Innsbruck Faculty for Biology

Master Thesis in Environmental Management of Mountain Areas

Main Supervisor (UIBK): Univ.-Prof. Dr.-Ing. Anke Bockreis Leopold-Franzens-Universität Innsbruck

Second Supervisor (UNIBZ): Assist. Prof. Dr. Sergio Angeli Freie Universität Bozen

Co-Supervisor: Dr. Sabine Robra Leopold-Franzens-Universität Innsbruck

∙ October 2020 ∙

Abstract

Plastics, synthetic organic polymers manufactured mainly from petrochemicals, have become a fundamental part of modern society. Since large-scale production of plastic started around 1950 it has increased dramatically reaching around 380 million metric tons per year in 2015 with an all-time total of 8,300 Mt estimated to have been produced across the globe by 2018. Some of the characteristics that make plastic an appealing material, such as low-cost fabrication, high durability, lightweight and hydrophobic nature, make it also a problematic waste, especially since most of the production is conceived for single-use packaging. Moreover, global plastic waste management has not been able to keep the pace with its ever-increasing production, resulting in more than 4,900 million tons of plastic waste estimated to have accumulated in landfills and natural environments by 2017 and 11,000 Mt projected to accumulate in the environment by 2025. Uncontrolled littering and leaching of plastic waste to the environment has made it a ubiquitous pollutant across the globe, raising concerns about harmful effects on ecological systems. Efforts on prevention, reduction and mitigation of the urgent plastic waste problem have promoted research of different fields, ranging from adequate policymaking to the development of bio-based polymers. A viable solution to the “plasticene” problem will presumably involve a collective action of multiple fields. Microbial strains capable of using plastic as a sole carbon resource have been found in a diversity of places, from natural marine and terrestrial environments to city landfills and wastewaters. Additionally, some invertebrates have been observed to chew and ingest plastic. Further screening of their gut microbiome has revealed new strains capable of colonizing and feeding on plastic. Nevertheless, although multiple reports of microbial plastic degradation have been published to this date, reviews on the subject point out the inconclusive nature of some experimental results and argue that complete biodegradation has not been proved yet.

In particular, this review article focuses on existing research addressing plastic biodegrading using insects and related microbial strains and enzymes isolated from their gastrointestinal tract. By conducting a narrative review, using relevant keywords across multiple search engines and carrying out backward and forward reference searches on relevant publications, the present work aims to explore the role of insects and their gut microbiome in plastic biodegradation, ultimately discussing their potential as a biotechnology for plastic waste management. Finally, the limitations, knowledge gaps, and recommendations for future research are delineated.

Abstract (Deutsch)

Kunststoffe, synthetische organische Polymere, die hauptsächlich aus Erdölderivaten hergestellt werden, sind zu einem grundlegenden Bestandteil der modernen Gesellschaft geworden. Seit Beginn der Massenproduktion von Kunststoffen um 1950 herum ist deren Herstellungsmenge dramatisch angestiegen: Allein im Jahr 2015 errichte sie 380 Millionen Tonnen und insgesamt wurden weltweit schätzungsweise 8,300 Mio. t bis zum Jahre 2017 produziert. Eigenschaften wie die kostengünstige Herstellung, die hohe Haltbarkeit, das geringe Gewicht und die Hydrophobie machen Kunststoff zu einem attraktiven Material. Ebenso machen diese ihn aber auch zu einem problematischen Abfallstoff, zumal bei der Produktion größtenteils Einwegverpackungen hergestellt werden. Die globale Kunststoff- Abfallwirtschaft konnte mit der stetig steigenden Produktion nicht Schritt halten, was dazu führte, dass bis 2017 schätzungsweise mehr als 4,900 Mio. t Kunststoffabfälle auf Deponien und in der Umwelt angehäuft wurden und bis 2025 voraussichtlich 11,000 Mio. t in der Umwelt anfallen werden. Unkontrollierte Vermüllung und Auswaschung von Kunststoffabfällen in die Umwelt haben Plastik zu einem allgegenwärtigen Schadstoff auf der ganzen Welt gemacht, wodurch die Sorgen bezüglich dessen schädlicher Auswirkungen auf die Ökosysteme zunehmend steigen. Die Bemühungen um die Vermeidung, Verringerung und Eindämmung des dringenden Kunststoffabfallproblems involvieren verschiedene Forschungsbereiche, die von einer angemessenen Politikgestaltung bis zur Entwicklung biobasierter Polymere reichen. Eine tragfähige Lösung des "plasticene"-Problems wird vermutlich die Zusammenarbeit mehrerer Bereiche erfordern. Mikrobenstämme, die in der Lage sind, Plastik als einzige Kohlenstoffquelle zu nutzen, wurden an einer Vielzahl von Orten gefunden, von natürlichen Meeres- und Landumgebungen bis hin zu städtischen Mülldeponien und Abwässern. Zudem konnte beobachtet werden, dass einige wirbellose Tiere Plastik kauen und aufnehmen können. Ein Screening ihres Darmmikrobioms zeigte neue Stämme, die in der Lage sind, Plastik zu besiedeln und sich davon zu ernähren. Obwohl bisher bereits mehrere Berichte über den mikrobiellen Kunststoffabbau veröffentlicht worden sind, weisen Reviews zu diesem

iii

Thema aber auf die nicht schlüssige Natur einiger experimenteller Ergebnisse hin; Ein vollständiger biologischer Abbau sei noch nicht erreicht worden. Dieses Review konzentriert sich auf bestehende Forschungsarbeiten, bezüglich des biologischen Abbaus von Kunststoff anhand von Insekten und aus deren Gastrointestinaltrakt isolierten mikrobiellen Stämmen und Enzymen. Durch das Verfassen einer narrativen Übersicht, unter der Verwendung relevanter Schlüsselwörter in verschiedenen Suchmaschinen und der Durchführung von Rückwärts- und Vorwärts- Referenzrecherchen in relevanten Publikationen, soll die vorliegende Arbeit die Rolle von Insekten und ihrem Darmmikrobiom beim biologischen Abbau von Kunststoffen untersuchen und schließlich ihr Potenzial als Biotechnologie für die Kunststoffabfallentsorgung diskutieren. Abschließend werden die aktuellen Grenzen sowie Wissenslücken diskutiert und Empfehlungen für zukünftige Forschung beschrieben.

Acknowledgments

First and outmost I want to thank my family for their unconditional love and support, always encouraging and enabling me to grow and improve myself.

I wish to thank my supervisors, particularly Dr. Sabine Robra and Dr. Anke Bockreis for their constant and readily assistance. Their reassuring words and guidance were key to finishing this work. Thanks to my co-supervisor Dr. Sergio Angeli for remaining involved, even after the Corona pandemic forced us to abandon our original project. Thanks to Dr. Sara Bortolini for voluntarily granting me her time, availability and feedback.

Thank you to all the lovely people and friends that have given me their hands, time, thoughts and advices, both in and outside the academic world. Special thanks to my girlfriend Lea Gibitz and her family, for making me feel like home and bear with me through thick and thin.

Thank you to the universities of Bolzano, Innsbruck and all the EMMA professors involved, for I’ve learned so much more than I hoped for.

vii

Contents

Abstract ...... i Abstract (Deutsch) ...... iii Acknowledgments ...... vi List of Figures ...... ix List of Abbreviations ...... ix 1 INTRODUCTION ...... 1 1.1 Plastic material ...... 1 1.2 Plastic society ...... 2 1.3 Plastic world ...... 3 1.4 Plastic waste management ...... 4 1.5 Plastic biodegradation ...... 6 1.6 A word about insects ...... 8 2 METHODOLOGY ...... 10 3 RESULTS ...... 12 3.1 Summary of the search results ...... 12 3.2 Coleoptera ...... 18 3.2.1 Overview ...... 18 3.2.2 Tenebrionidae ...... 20 3.2.3 Lucanidae ...... 31 3.3 Lepidoptera ...... 31 3.3.1 Overview ...... 31 3.3.2 Pyralidae ...... 34 3.4 Diptera ...... 41 3.4.1 Culicidae ...... 41 3.4.2 Stratiomyidae ...... 41 4 DISCUSSION ...... 43 4.1 Plastics and Insects ...... 45 4.2 Role of insects in plastic waste biodegradation ...... 47 5 FINAL REMARKS ...... 51 6 REFERENCES ...... 53 7 FINAL APPENDIX ...... 67

viii

List of Figures

Figure 1. Plastic biodegradation process ...... 7 Figure 2. Methodology used in the present review ...... 11

List of Abbreviations

1H MNR: Hydrogen (proton) nuclear magnetic resonance AFM: Atomic force microscopy ATP: Adenosine triphosphate BP: Bioplastic BSF: Black soldier fly BSFL: Black soldier fly larvae CE: Circular economy CH4: Methane (Carbon tetrahydride) CO2: Carbon dioxide CYP: Cytochromes P450 DNA: Deoxyribonucleic acid EPS: Expanded polystyrene ER: Excreta residue FT-IR: Fourier-transform infrared spectroscopy GC-MS: Gas chromatography – mass spectrometry GPC: Gel permeation chromatography H2O: Water (dihydrogen oxide) HBCD: Hexabromocyclododecane HDPE: High-density polyethylene HPLC–MS: High-performance liquid chromatography coupled with mass spectrometry HT-GPC: High-temperature gel permeation chromatography LC-MS/MS: Liquid chromatography with tandem mass spectrometry LDPE: Low-density polyethylene LLDPE: Linear low-density polyethylene LMCO: Laccase-like multicopper oxidase Mn: Number average molecular weight MP: Microplastic mtDNA: Mitochondrial deoxyribonucleic acid Mw: Weight average molecular weight NO2: Nitrogen dioxide OTU: Operational taxonomic unit PBS & PBSA: Polybutylene succinate & Polybutylene succinate adipate PCL: Polycaprolactone PE: Polyethylene PET: Polyethylene terephthalate PLLA & PDLLA: Poly (L-lactic acid) & Poly (DL-lactic acid)

PP: Polypropylene PS: Polystyrene PUR: Polyurethane PVC: Polyvinyl chloride rRNA: Ribosomal ribonucleic acid RT-PCR: Reverse transcription polymerase chain reaction SBR: Styrene-butadiene SEM: Scanning electron microscopy SEM-EDS: Scanning electron microscopy coupled with energy dispersive X-Ray spectroscopy SR: Survival rate TGA: Thermal gravimetric analysis THF: Tetrahydrofuran TOC: Total organic carbon WC: Wax Comb XPS: Extruded polystyrene XRD: X-ray powder diffraction

INTRODUCTION

1.1 Plastic material

Plastic – from the Greek πλαστικός/plastikos: fit for molding or shaping1,2 - is a term used for a wide range of malleable materials commonly made from synthetic organic polymers of high molecular mass3. During manufacture, synthetic polymer resins are mixed with different additives, resulting in a product with the characteristic features that plastic consumers all over the world are familiar with: a lightweight, flexible, versatile material that is also resistant and durable4–6. The most common polymer resins globally used are polyethylene (PE, 36% of global plastic production by weight), polypropylene (PP, 21%), polyvinyl chloride (PVC, 12%), polyethylene terephthalate (PET, <10%), polyurethane (PUR, <10%), and polystyrene (PS, <10%). In total, these 6 types of polymer resins account for about 92% of all plastic ever produced. Packaging production, dominated by PE, PP, and PET resins, accounts for roughly 42% of the global resins used7. The synthetic polymer resins that makeup plastics are complex substances made of long- chain molecules (polymers) composed of repeating units of small carbon-based molecules (monomers), connected by strong chemical bonds. Depending on the polymers' composition and arrangements, plus the additives mixed during manufacture, different desirable properties can be obtained, making plastic an intrinsically multipurpose material3,5,8,9. In Europe, plastic demand is mostly dominated by packaging (40%), building and construction (20%), automotive (10%), and electrical and electronic (6%). The remaining sectors, including medical and leisure, compose the remaining quarter (24%) of total plastic use10. Plastic materials used for packaging have a short in-use phase (less than a year) and are commonly discarded after a single-use, making them the most frequent plastic waste found in landfills and as microplastics (particles with diameters between 1mm and 1µm11) in the environment7,12. Additives, chemical compounds of diverse nature, are used during manufacture to improve the performance, functionality, and aging properties of plastics. They are mainly

divided into functional additives (e.g. stabilizers, plasticizers, flame retardants), colorants (e.g. pigments), fillers (e.g. clay, barium sulfate), and reinforcements (e.g. glass fibers)6. Geyer et al. (2017) estimated that non-fiber plastics (examples of synthetic fibers are nylon, rayon, and some types of polyester13) contain, on average, 93% polymer resin and 7% additives by mass. In other cases, additives such as plasticizers, reinforcements, and fillers may comprise up to 70% in weight of any given plastic product, especially those used for construction6. Of all additives used during plastic manufacture in the last decade, the most common ones are plasticizers (35%), fillers (28%), and flame retardants (13%)7. However, without adequate plastic waste management, discarded plastic products may eventually reach the environment, where the benefits of using additives during plastic production are offset by their widely documented potential to contaminate soil, air, water, and food6.

1.2 Plastic society

The outstanding characteristics of plastic mentioned in the section above, coupled with the inexpensive production thereof, especially when it comes to packaging applications, have made it an extremely popular material of high demand9,14. Plastic has become a ubiquitous material of our modern society, extensively used in several sectors of the economy15–17. Since mass production started around 1950, the amount of plastic produced has increased annually from an initial 2 million metric tons (Mt) to 388 Mt of plastic produced in 20157,18. Geyer et al. (2017) estimated that, in total, 8300 Mt of plastic were produced until 2015, more than half of which was produced after 20057. Almost 99% of plastics are produced from petrochemicals derived from fossil fuels, which are also used to power the whole refinement and production processes. Because of this, conventional plastic production imposes significant negative externalities upon society,

10,19,20 such as CO2 emissions to the environment . Plastic production currently utilizes about 8% of the total fossil fuel extracted globally. By 2050, under a “business as usual” scenario where production trends continue unchanged, projections estimate that global annual plastic production may quadruple and account for 20% of the global fossil fuel

extraction and 15% of the global annual carbon budget (from the budget calculated to remain below a 2 °C increase in global warming)14,21–25. Large investments betting on the future development of petrochemicals and plastics in particular have stick to the projections mentioned before, however, it seems that, under the current global trend to decouple from fossil-fuels and the disruptive effects of the COVID pandemic on the world economy, the peak demand for fossil-based plastic is most likely to occur in the next years or might even already have occurred in 2019, painting a complicated and grim picture for the future plastics economy 20.

1.3 Plastic world

By 2015, out of the 8,300 Mt of primary plastic produced globally, 500 Mt (≈6%) were recycled, 700 Mt (≈9%) were incinerated, 2,500 Mt (≈30%) were still in use and 4600 Mt (≈55%) were estimated to have been discarded, ultimately accumulating in landfills and natural environments7. Currently, environmental plastic pollution is a widely recognized worldwide problem whose ominous presence influences every environment analyzed so far, from deposition in the deepest marine ecosystems to regional and global transport over wind and rain26,11,27–29. Deposits of plastic material across the earth’s surface make up a distinctive stratal component that has recently been proposed as a key indicator of the so-called Anthropocene, a geological time unit stating the deep human impact on earth’s systems30. High molecular weights, strong chemical bonds between carbons in the polymer chains, extremely hydrophobic surfaces, and recalcitrant additives frequently used during manufacture are some of the features that make current plastic products remarkably resistant to degradation and therefore a very persistent type of waste found in landfills and the environment31,32. Authors have pointed out that, despite the massive scale and urgency of environmental plastic pollution, the literature on plastic degradation rates and resulting byproducts under environmental conditions remain very limited17,33. At present, the best-known route of environmental plastic depletion starts with exposure to abiotic factors, such as UV light and mechanical wearing caused by waves and winds

grinding plastic on rocks and sediments, eventually breaking larger plastics into smaller pieces. After this so-called “weathering”, the resulting smaller particles have a much larger surface area which makes them amenable to further biodegradation31,34. The term biodegradation refers to a complex and stepwise process of polymer (both synthetic and natural) degradation predominantly carried out by biological activities17,34. More details about this process are presented below in the section of “plastic biodegradation”. The small plastic particles, named microplastics, that result from weathering and fragmentation of macroplastic or that have been purposely manufactured in micro scales, remained largely overlooked by the scientific community until 2011, when the number of publications started increasing exponentially, almost doubling in number with each passing year12. Hartmann et al. (2019) argue that the fast development and relatively young age of the plastic pollution research field have led to a lack of consensus on the definition of some concepts and categorization of different plastic debris. The inconsistent and sometimes ambiguous terminology used in different reports likely leading to confusion and miscommunication, further hampering progress in plastic pollution research and related mitigation measures. While the complexities of elaborating a common framework for plastic debris categorization are discussed by the above- mentioned authors, this review adheres to the recommendations, criteria, and categories proposed therein. Briefly, size categories used in the present review are: macroplastics for particles that have 1 cm or larger diameters; mesoplastics for particles with diameters between 1 cm and 1 mm; microplastics, for particles between 1 mm and 1 µm; and nanoplastics, for particles between 1 µm and 1 nm11. It is important to note that, despite constant efforts and advances, the miniature size of nanoplastics makes them inherently elusive to direct detection in environmental matrices, suggesting that environmental plastic pollution might be more severe than what has been quantified so far35–37.

1.4 Plastic waste management

Research suggests that plastic waste constitutes, on a rough average, about 10% in weight of the annual solid waste collected worldwide38–41. In 2015, 58% of global plastic

waste was landfilled, and only 18% recycled7. In a business-as-usual scenario where the production, waste generation, and waste mismanagement of plastics keep their current trends, the mass of mismanaged plastic waste - waste that is left in uncontrolled landfills and is more likely to leach to the environment than being properly processed - is estimated to double by 2050 reaching 12,000 Mt7,42,43. To successfully tackle this problem, experts emphasize the need for integrated efforts from several different sectors, from policymaking to increasing recycling rates, with actions addressing local, regional, and global issues44,45. However, projections of various scenarios designed to reduce plastic pollution between 2016 and 2040 estimate that substantial amounts of plastic waste will remain mismanaged worldwide, even under a “system change” scenario where all the currently available interventions are immediately implemented43,46. Estimations show that in 2017, 32% of the 25 Mt of plastic waste produced in Europe were collected for recycling47. A significant amount of the collected waste was then exported to other non-EU countries48, where different environmental standards for plastic waste management are applied47,49. For the past quarter of the century, China was the world’s largest importer of plastic waste, handling almost half of the globally produced plastic waste meant for recycling. However, starting in 2018, the Chinese government enacted a new policy called “The National Sword”, banning the import of most of this plastic waste into the country. The ban has posed an important challenge to the global plastic waste management, disrupting the global trade network. None of the other large economies involved in the network is able to take on the role of China in the short-term, increasing the risk of illegal dumping and incinerating of plastic waste and redirecting the flow of waste towards other countries, which are usually developing countries not well prepared to manage their own plastic waste stream50,51. The challenge posed by the Chinese ban can also prove to be an opportunity, particularly low prices of plastic waste pushing investment and innovation towards a more robust and complete recycling and recovery system regionally developed. In the case of Europe, keeping waste plastics within the EU could reduce their net global environmental impact by securing a more scrutinized management, under EU standards set towards a circular economy (CE) of plastics52. Since 2012 the circular plastic economy concept has

increasingly gained the attention of policy-making decisions for its goal of minimizing the number of materials and products to become waste, based on the recovery and recycling of products that reach their intended end-of-use stage47,53. Circular plastic economy legislations are reported to have stimulated better plastic waste management practices, like the case of countries with landfill bans (i.e. Austria, Belgium, Denmark, Germany, Luxembourg, the Netherlands, Norway, Sweden, and Switzerland) where on average less than 5% of the collected plastic waste is reported to be dumped into landfills54,55. However, because recycling alternatives currently remain relatively expensive, the major improvements in plastic waste management trends all across Europe are mainly driven by an increase in incineration rates, where materials (plastic waste feedstock) leave the

40,56,57 value chain as emissions of GHGs (e.g. CO2) and hazardous atmospheric pollutants . In a thorough review, Ilyas et al. (2018) identified five fundamental strategies currently used to handle plastic waste: recycling, depositing in landfills, incineration, conversion into useful materials (e.g. fuel), and microbial degradation (biodegradation). Each of these strategies presents specific advantages but serious limitations, causing mismanaged accumulation of plastic material and its eventual leakage to natural environments18,57.

1.5 Plastic biodegradation

Plastic biodegradation is considered by some researchers as a form of feedstock or tertiary recycling, where long synthetic polymer chains are converted to smaller molecules by chemical reactions, much like the degradation of naturally occurring polymers (e.g. starch, cellulose, lignin)32,34,58. This process is carried out by organisms (mainly microorganisms) that degrade and assimilate materials59, thus the particularity of this form of recycling is that it potentially conserves the intrinsic value of the materials used and incorporates them into biological cycles58. Furthermore, the enzymes produced by the organisms to degrade the material can act under mild conditions without the need for additional energy or expensive machinery59.

Plastic biodegradation can be fundamentally separated into the following steps (Figure 1.): Fragmentation and deterioration, where biotic (e.g. microbial communities, insects and other decomposer organisms) and abiotic (e.g. sunlight, wind, waves) agents fragment plastic materials into tiny fractions and alter the physical and chemical properties of the constituent polymers; depolymerization, where enzymes (mainly depolymerases) and other catalytic agents secreted by organisms interacting with the plastic surfaces (e.g. microbial communities imbedded in biofilms that are attached to the plastic surface) cut polymers into smaller and simpler components (e.g. oligomers, dimers) progressively reducing their molecular weight; the resulting molecules that are small enough may be recognized by cellular receptors and enter the cell cytoplasm by crossing the plasmic membrane. Once in the cytoplasm, they are integrated into the cellular metabolism (e.g. cyclic acid cycle) producing energy, biomass, and various primary and secondary metabolites in a step called assimilation; finally, simple (e.g. CO2,

N2, CH4, H2O) and complex (e.g. organic acids, antibiotics, terpenes) metabolites from the previous step are excreted to extracellular surroundings in a process called mineralization34,60–63.

Figure 1. Plastic biodegradation process

Design and production of intentionally biodegradable plastics under certain conditions is a specially interesting option for replacing traditional recalcitrant plastics in nondurable applications such as packaging and agricultural films60,64. However, complete biodegradation is yet to be conclusively proven and biodegradation rates greatly vary according to environmental conditions (e.g. temperature, pH, humidity), meaning that these new-generation plastics do not translate to a “continue to throw away” solution, but rather contribute to improving end-of-life management options such as tertiary recycling and biodegradation60,65–67. Biodegradation of plastic waste is perceived by many authors as the most attractive management alternative, providing a cost-effective and environmentally friendly option to deal with collected plastic waste and remove accumulated plastic debris from natural environments while potentially retaining the value of plastic for subsequent use in a circular economy. However, the application of this strategy is not yet scalable nor cost- effective, prompting for further research31,57,59,68.

1.6 A word about insects

The incredible diversity of insects has proven to be a valuable biotechnological resource for multiple applications in sectors such as agriculture, medicine, and industry69. Insect biotechnology, also called yellow biotechnology due to the color of insect hemolymph (analogous to blood in vertebrates), has historically provided humanity with useful materials such as honey, silk, and wax. Armed by a long and successful evolutionary history, insects have developed a huge arsenal of active compounds that help them defend themselves against enemies and diseases or to explore novel food sources70. The diversification and evolutionary success of insects are deeply connected to the myriad relationships they have established with beneficial microorganisms, which are known to upgrade nutrient-poor diets; aid digestion of recalcitrant food components; neutralize toxic substances; and many other functions. These beneficial microorganisms are predominantly found in the digestive tract where they are mediators of the varied diets and lifestyles of their insect hosts71,72.

Insects provide important ecosystem services in many of the earth’s environments73. Termites and their gut symbionts, for example, have a very important place in the environmental carbon cycle by breaking down plant litter. Thanks to synergic activities and a set of specific cellulolytic enzymes produced by both the termites and their microbial symbionts, they can efficiently digest lignocellulose (i.e. dried plant matter), a natural polymer that is highly recalcitrant to enzymatic attack, and convert it into insect biomass 73–75. Black soldier fly (Hermetia illucens L., Diptera: Stratiomyidae) and its microbial symbionts, on the other hand, can aptly digest a vast array of organic substrates (e.g. manure from diverse animals, decaying organic matter, agricultural waste) converting them into valuable insect protein and fat, additionally transforming the initial substrate into ready-to-use quality compost41,76–81. These and other sophisticated bioconversion systems performed by insects are valuable resources for further biotechnological applications in medical, agricultural, and industrial fields69,72,73,77,78,80,82–87. In the present review, the focus is set on research involving insect biodegradation of plastic as a novel source of microbial strains and enzymes of biotechnological value, capable of degrading the most common synthetic polymers found in waste streams and the natural environment.

METHODOLOGY

The present work constitutes neither a systematic nor a comprehensive review. Rather, the following review on the potential role of insects in plastic biodegradation is elaborated in the form of a narrative literature review (NLR); the methodology thereof is explained in the subsequent paragraphs. A limited number of articles were included following the below-described selection criteria. First, to determine the relevance of a review on the subject, already existing reviews dealing with the same or similar subjects were searched for88 in different academic search engines: Web of Science, Microsoft academic, and Google Scholar89 using a specific/exact search of the keywords plastic and polymer biodegradation, in combination with keywords review and literature (e.g. “plastic biodegradation” review). An initial screening of the results by publication date (last 10 years) and title relevance was undertaken. The resulting collection of reviews was manually filtered by answering the following question: does the review make any mention of primary research on plastic degradation by a specimen - or the gut-microbiota thereof – belonging to the taxonomic groups of arthropods or insects? All resources that affirmatively answer the question were included for further examination. Back and forward reference searches were carried out (citation mining90) to identify original research and additional biodegradation reviews that may not have been previously included. Next, additional articles not included in the previous step were searched for, including keywords extracted from the reviews and articles examined so far. Using the same academic search engines as before, specific/exact keywords plastic and polymer biodegradation were combined with the keywords: impacts, processing, polyethylene, polystyrene, polyester, polypropylene, polyvinyl, polylactide, polyurethane, LLDPE, LDPE, HDPE, PET, PVC, PUR, PVA, insect, invertebrate, larva, worm, moth, Coleoptera, Lepidoptera, Diptera, Arthropoda, gut microbiome (e.g. “plastic biodegradation” worm). The search results were manually filtered by analyzing title and abstract content, subjecting them to the following inclusion criteria: - Is the experiment relevant to plastic degradation?

- Is it a publication dealing with primary research or empirical data? - Do the methods include the specimen and type of plastic analyzed? - Does the research involve at any point a specimen belonging to the phylum Arthropoda? Primary research detected during citation mining was also filtered out following the same inclusion criteria. An additional crosscheck was carried out to verify that all the collected resources were published in scientific peer-review outlets. Duplicates were eliminated and the final collection of primary empirical research was organized into a matrix table. Supplementary information and updates were checked and reviewed when provided in the publication or made easily available. To better delineate the scope of the present review, the focus is set on a waste management perspective, granting priority to (a) arthropods that may have already been involved with waste management practices or with a natural history suggesting potential waste processing capabilities, (b) arthropods documented to have been reared in the past, that are currently mass-produced or that show potential for mass-rearing and (c) model organisms that have been widely studied. Furthermore, marine arthropods (e.g. copepods, amphipods) and some soil engineers (e.g. ants, termites, collembola) are not included in the matrix table but are considered to broaden the overall analysis.

Figure 2. Methodology used in the present review

RESULTS

3.1 Summary of the search results

A small number of reviews discussing the effect of microplastics on ecosystem services provided by insects73 and mentioning the abilities of insects to perform synthetic polymer degradation83,84,91 have been identified, the latter mainly referencing ground-breaking research on polyethylene biodegradation using waxworms, conducted by Yang et al. (2014). The first reports - more than 50 years ago - of insects damaging or consuming plastic- packaging92 and describing their plastic-eating behaviour93 focused primarily on preventing losses of the packaged commodities by some common house-pests rather than inspecting the insects' biodegradation capabilities. Although pest control continues to be thoroughly researched94,95, increasing awareness of widespread plastic-packaging pollution9 and scientific serendipity96 have fostered a new area of research related to the potential of insects as an emerging biotechnological resource for plastic biodegradation83,84,91,97 and entomoremediation86,98. The first mention of insect plastic-biodegrading potential comes from Suzuki et al. in 2012 while isolating an enzyme capable of degrading several bioplastics films made of poly(butylene succinate) (PBS). Two years later, Yang et al. (2014) published the first report involving insects - and their gut microbiome - degrading polyethylene (PE), an oil- derived plastic widely considered as non-biodegradable. The laboratory hosting the latter research (the Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education (Beijing, China)) is also involved with several other reports of plastic biodegradation using insects99,100. To the date when the literature search was realized for the present review, thirty-five (35) peer-reviewed publications concerning the role of insects in plastic biodegradation have been found (see Table 1), further delineating the effect of plastic exposure on insects and the biodegradation rates (or the lack thereof) of plastic commonly found in waste streams.

The publications in Table 1 are compiled chronologically, according to the insect specie(s) and plastic-type(s) used during the multiple experiments. Ten (10) different types of synthetic polymers have been reviewed for insect biodegradability. While some experiments evaluate more than one type of plastic, nineteen (19) articles include polystyrene (PS) for experimentation, eighteen (18) include polyethylene (PE), and other plastics and bioplastics (SBR, PVC, PUR, PBSA, PBS, PCL, PLLA & PDLLA) are included in only one (1) publication each. Research employing eleven (11) species of the Insecta class, belonging to three (3) orders (Coleoptera; Lepidoptera; Diptera) and distributed in five (5) different families (Tenebrionidae; Pyralidae; Noctuidae; Culicidae; Stratiomyidae) explore the organisms’ capability for chewing, ingesting and/or partially degrading synthetic polymers; additionally, two (2) yeast strains isolated from the guts of a beetle species (Aegus laevicollis, Lucanidae: Coleoptera) are reported to produce enzymes able to degrade several biodegradable plastics, most of which are produced from renewable resources.

; ; -

- ; ;

–

ቃ

; ; 7.

mical

PUR PUR

Enzyme Enzyme

푝푙푎푠푡푖푐

generation generation

풊풏풅풊풗풊풅풖풂풍풔

풏

e, e, viability of 표푓

∗

; ; 11.

푚푔

Chromatographic

푑푎푦푠

ቂ

eating behavior and behavior eating

ase chain reaction (RT reaction chain ase

locate the articles in the in articles the locate

chemical chemical composition of

Polyvinyl chloride;

–

polymer

sis (XRD)); sis

PVC PVC

Plastic(e.g.change surface scanning

rate expressed rate as expressed

code) to bette to code)

; 3. ;

Analyze insects' plastic insects' Analyze

ity, bioaccumulation of xenobiotic substances, xenobiotic of bioaccumulation ity,

Ray diffraction analy diffraction Ray

pupal pupal and pupation period), pupation rat

Reference number ( number Reference

Polystyrene Polystyrene (polystyrene foam, Styrofoam®, expanded PS,

–

experimentation

ence ence analysis, activity analysis, API ZYM, API 20 NE, ABTS assays,

; PS

ic substrate weight reduction rate (ingestion or consumption rate), inputrate), consumption or (ingestion rate reduction weight substrate ic

density; low density; high density);

H NMR), turbidity assay, X assay, turbidity NMR), H

estimation estimation of the specific plastic ingestion

microbiome influence & microbial strain isolation (e.g. selective media culture, totalculture, media selective (e.g. isolation strain microbial & influence microbiome

; ; r

; 8. ;

DNA DNA and mitochondrial DNA (mtDNA) analysis (e.g. global mtDNA methylation level (HPLC

products (insect frass) analysis as commodity value (e.g. viability of insect frass as agriculturalfrassinsectasof (e.g. viability commodity value as analysis (insect frass) products

bioplastics

Thermogravimetric Thermogravimetric analysis (e.g. thermal gravimetric analysis (TGA), melt flow index (MFI))

Polyethylene Polyethylene (linear low

Hologenome Hologenome functional analysis

; ; 13.

6. Other

–

PE PE

e the insects gut microbiome (e.g. microbial community composition, strain isolation, antibiotics screening) antibiotics isolation, strain composition, community microbial (e.g. microbiome gut insects the e

; ; 14.

MS);

produced), fractionproduced), of frass, extractable polymer intoxins in frass)

Analyz

IR), nuclear magnetic resonance ( resonance magnetic nuclear IR),

For further information refer to theinformationoriginal to publicatio referfurther For

Functional metagenomics (e.g. PICRUSt, KEGG pathway, QIIME analysis, CLC Microbial Genomics)

Insect biomass and by and Insectbiomass

MS/MS, GC

- -

Analyze Analyze the plastic substrate (e.g. change in average molecular weight, carbon mass balance, fate of ingested carbon,

; ** ;

; 12. ;

; ; 10.

butadiene butadiene rubber;

ommercial ommercial feed in fisheries)

mass loss of the initial weight as a percentage (%); Ref. No No Ref. (%); percentage a as weight initial the of mass loss

throughput screening (HTS), operational taxonomic units (OTU), TaqMan assay, Reverse transcription Reverse assay, TaqMan (OTU), units taxonomic operational (HTS), screening throughput

Styrene

Gene sequencing (e.g. Illumina sequencing of 16S rDNA (bacteria), 18S rRNA (fungi), shotgun metagenomics, next

Plastic carbon mass balance and gravimetric analysis (e.g. plast (e.g. analysis gravimetric and balance mass carbon Plastic

SEM), atomic force microscopy (AFM) fluorescence microscope, surface chemical components, water contact angle, number of hole

–

forming forming units (CFU) assays, antibiotic screening & suppression treatment assay, morphological characterization (ESEM), bioche

GPC, GPC, HPLC, GPC, LC

; 2. ; -

;

SBR SBR

PBSA, PBSA, PBS, PCL, PLLA, PHB, PDLLA

Recommended Recommended publication

Reviewed articles ordered by type of insect and type of plastic used for used plastic of type and insect of type by ordered articles Reviewed

. .

; * ;

generation generation development, bioaccumulation of toxins (body burden analysis), effect of insect biomass as feedstock, relative bio

Insect population characteristics (e.g. survival rate, growth rate (larval weight change; larval, pre

Carbon isotope tracers (e.g. isotope ratio mass spectrometry) mass ratio isotope (e.g. tracers isotope Carbon

(FT infrared (e.g. analysis Spectroscopic

- -

medium, medium, insect biomass as c Table fertil periods, pupation rate, survival growth, insect on impact consumption, plastic affecting factors (e.g. biology changes in mitochondrial DNA); additives); plastic toxic potentially of fate 1. 2 biomass) insects’ outputmass respirometry vs. balance, test(CO electron microscopy ( 4. analysis (e.g. HT number of colony characterization) high (NGS), sequencing PCR), PacBio RS II sequencing) characterization and enzymatic activity (e.g. enzyme purification, partial amino acid sequ proteome analysis) gland salivary MS/MS), reactive oxygen species detection (ROS)) extruded PS); Polyurethane; as expressed rate depletion article 13

3.2 Coleoptera

3.2.1 Overview Tenebrio molitor has been reported to consume PS, PE, PVC, two types of rubber SBR and PUR without significant impact on their survival rate (SR) compared to control groups fed standard diets100–109 completing their life cycle (reach adult phase and reproduce)101,102,104,105 although in one experiment with PS, SR decreased to 20% over a period of 45 days and then remained stable for 2 months, after which some larvae started to pupate110. Results suggest T. molitor is able to degrade different types of plastics (and microplastics108) commonly found in waste streams107 and that the ability to consume some plastic types is ubiquitous in mealworms from different geographic regions103. Mealworms consuming only synthetic polymers have been documented to maintain their weight100,102,104 and even increase it106, however, in some cases, they have also been documented to shrink and lose weight108,110. Supplementing a plastic diet with a standard co-feed (bran, oat, corn, yeast, protein, etc.) has been shown to increase plastic consumption, improve pupation rates and promote larval weight gain102,103,105,107,111 in different members of the darkling beetle family (Tenebrionidae). In the case of mealworms, for example, optimal removal of PS from feed has been reported at 25°C when larvae are fed with bran containing 6 to 11% (weight) PS102. HBCD, a plastic additive that has been banned from production for posing a threat to the environment, does not accumulate in mealworms nor does it affect the SR and PS ingestion of the insect105. Recently, it has been proved that exposure to plastic (PUR) leads to epigenetic changes in mealworms, increasing methylation rates of mitochondrial DNA, and registering lower ATP levels in comparison to control groups109. Mass balance and carbon tracing analysis have revealed that some darkling beetles can convert a considerable fraction of the carbon from the ingested plastic (e.g. PS) into

107,112,113 100 CO2 , while a marginal amount is mineralized and stored as fatty acid . This plastic conversion ability has been reported to improve over the experimentation period, with more production of CO2 and less amount (weight) of residual polymers being excreted by the insects107,113.

Compared to the original plastic substrate fed to larvae of the Tenebrionidae family, spectrophotometric, thermogravimetric, and chromatographic analyses of the residual plastic (e.g. partially degraded polymers) recovered from insect excreta (frass) reveal novel substances100,102,103,106–108,111,113,114 (e.g. products of Styrofoam oxidation like phenyl derivatives100,110 and release of sulfur from the breaking down of cross-linking bonds in thermosetting plastics106 with lower molecular weight100,102,103,106–108,111,113,114 and a different thermodegrading profile100,106,108,113,114, indicating changes in the chemical structure of the original plastic substrate. When treated with antibiotics (e.g. gentamicin) dark, yellow and giant mealworms were unable to reduce the molecular weight of the

103,111–113 112 ingested polymers or produce CO2 , suggesting a fundamental role of gut microbiota in plastic degradation. Metagenomic analyses show strong evidence of shifts in the gut microbial communities of plastic fed mealworms and other darkling beetles101,103,107,111,114,115 (e.g. Citrobacter sp. and Kosakonia sp. were strongly associated to PE and PS diets, suggesting plastic degrading capabilities107) towards operational taxonomic units (OTUs) with functional profiles associated to the degradation of synthetic polymers (e.g. functional profiles linked to the degradation of aromatic compounds and hydrocarbons101,114). Multiple microbial strains involved in plastic degradation have been identified in guts of darkling beetles (e.g. Serratia marcescens, Klebsiella oxytoca and Pseudomonas aeruginosa116; Bacillus spp. and Stenotrhophomonas sp.110; Parabacteriodes spp. and Clostridium sp.115) and pure isolated strains have been proven to grow on (TM1 and ZM1117) and partially degrade (Exiguobacterium sp. strain YT2112, Acinetobacter sp. AnTc-1114) plastic, although some experiments fail to identify the presence of plastic degrading microorganisms reported in previous publications110,116. Researchers have also been able to isolate yeast strains with plastic degrading abilities (BPD1A and BPD2A) from other beetles (Lucanidae family), further applying novel purification methods to isolate a cutinase-like enzyme (CmCut1) with bioplastic degrading activities produced by one of the strains (BPD1A)118.

3.2.2 Tenebrionidae 3.2.2.1 Tenebrio molitor Yang et al. (2015a) published the first academic report on expanded PS (polystyrene - commercially known as Styrofoam) biodegradation using yellow mealworm (a common name for the larvae of Tenebrio molitor)100. The experiment explored the larvae biology and population dynamics, while also examining the fate of the PS-substrate consumed. Over time, survival rates of the treatment group with mealworms fed with PS as their sole carbon source were not significantly different compared to a control group fed with conventional wheat bran. A battery of widely used techniques to determine polymer degradation34,68 (chromatography, thermal characterization, and spectrophotometry analysis) were used to compare the egested fecula (frass) of PS-fed mealworms against the original (virgin) PS substrate. Decreases in the molecular weights - and thus the molecular weight distributions - of the samples revealed changes in the chemical structure and composition of the Styrofoam substrate, with the appearance of novel compounds (attributed to phenyl derivatives, possible decomposition products of PS119) and relative depletion of the original high-density, long-chain PS polymers. Carbon mass balance analysis using closed-system incubators showed that the mealworms’ PS-digesting activity increased with time, starting with an estimated 20% of the carbon ingested by larvae converted to

CO2 after the first 4 days, reaching up to almost half of the consumed PS-carbon

13 transformed into CO2 after 16 days of treatment. Moreover, C isotope tracer experiments showed that a fraction (only about 0.5%) of the ingested carbon was mineralized and stored as fatty acids in the mealworms body tissue. PS-fed mealworms did not show any significant change in their biomass during the treatment, as opposed to bran-fed mealworms which increased their biomass by more than 30%. On the other hand, a starving group of mealworms (deprived from any feed) reduced their biomass by about 25%, leading the researchers to state that PS substrate offers a marginal benefit to the mealworms consuming it. A companion article, Yang et al. (2015b)112, builds upon the role of the mealworms’ microbiome on PS biodegradation. In this experiment, a treatment group of mealworms

was fed with a suitable antibiotic (gentamicin) to inhibit any synergic effect of their gut microbiota on PS digestion. Compared to an untreated group of mealworms (control), the gentamicin-treated group was reported to be unable to mineralize carbon from the

Styrofoam, manifested by their inability to produce CO2 or decrease the molecular mass (both weight-average and number-average) of the ingested substrate (analysis was carried out with a 13C-Carbon labeled test and a chromatography test, correspondingly). Furthermore, various strains of potential PS-degrading microorganisms were isolated from a group of untreated mealworms (same procedure as in the previous work with waxworm99), identifying Exiguobacterium sp. strain YT2 as a PS-degrading bacteria. Besides a weight loss of almost 8% of its original dry-weight, a prepared PS-medium inoculated with YT2 showed reduced hydrophobicity, changes in surface chemical components, and lower molecular mass (both Mw and Mn) as showed by the results from water contact-angle test, X-ray photoelectron spectroscopy and gel permeation chromatography, respectively. Correspondingly, the sequence of the isolated YT2 strain was deposited in the GenBank making it available for further study. Chen et al. (2017)101 further investigated changes in the gut microbiota of mealworms fed only with polystyrene, compared to a control group fed only with paper sheets. During a 90-day experiment, both groups showed similar survival rates with some individuals in both groups undergoing metamorphosis. Analysis of high-throughput sequencing of the guts of both groups revealed 179 different OTUs in total, 51% of which were shared between the two groups. However, when OTUs with a relative abundance of less than 0.5% were removed, only 4 OTUs were common to both groups. Furthermore, PICRUSt analysis of the 16S rRNA in the PS group identified in 328 functional profiles (predictions), 17 of which were documented as metabolic pathways related to the degradation of aromatic compounds and alkanes. In comparison to the control group, where these pathways were absent, this result indicated that the guts of PS fed mealworms were enriched with bacteria associated with PS breakdown. Yang et al. (2018a)102 analyzed, in a comprehensive study, the effect of nutrition on PS foam biodegradation using T. molitor larvae by subjecting 3 groups of mealworms to distinct diets simulating food wastes (PS only, PS+Bran, and PS+Protein), additionally

testing the effect of different co-diet mixtures (Bran:PS ratios of 1.3:1, 2.7:1, 8:1, 16:1, and 24:1) under three different temperatures (20, 25 and 30°C) for a total of over 26 tests each with duplicates, including unfed and Bran-only control groups. Over a 32-day test, survival rates between mealworms fed with PS-only, PS + protein, PS + Bran, and Bran-only were not significantly different, as opposed to a control group with no feed at all that showed much lower survival rates. PS-only fed larvae did not show weight gain or loss, while unfed larvae lost an average of 2,6% of their average mass and Bran-only fed larvae gained 32%. Optimal PS removal was reported at 25°C using a bran feed that had 6% to 11% (w/w) PS content (PS removal was nearly double as much as when fed PS alone). Furthermore, six commercially available PS foams that are typically found in urban trash waste were additionally studied, ultimately establishing the main characteristics of the PS-product that influence the degradation rate (mainly the density and molecular weight of the material). Analysis of the insects’ frass revealed partial depolymerization and biodegradation of the original PS-feedstock as evidenced by chromatography (GPC) and spectrography (FT-IR and 1H NMR spectra) analysis, which showed lower molecular weights and new spectrometric peaks (generation of novel compounds), respectively. Additionally, carbon mass balance analysis revealed a reduced extractable fraction of PS present in the frass. Complementary, the impact of a PS + Bran diet on the development of the mealworm’s life cycle stages (larvae, pupae, beetles, egg) was tested, consequently culminating in a second generation of mealworms that showed better capabilities for PS degradation while in turn completing their life cycle (adult beetles) on a PS + bran diet. The ubiquity of PS digestion and partial biodegradation by T. molitor was suggested by Yang et al. (2018b)103 in a publication where academic researchers and “citizen scientists” from 22 different countries reported locally acquired mealworms (thus presumed to represent 22 different strains) surviving on diets of PS-only and PS+Bran. In-depth experiments in 12 of these countries, where chromatography and spectrophotometry analysis were carried out, additionally revealed partial depolymerization and biodegradation of PS foams after ingestion by mealworms. Moreover, the importance of gut microbiota was established in 5 of these sources by treating a group of mealworms

with gentamycin (a bactericidal antibiotic) before feeding them with PS feedstock and comparing the egested frass with the original PS, using an antibiotic-untreated control group to support the results. Tetrahydrofuran (THF) is a widely used dissolvent in biodegradation analysis suited to extract the hydrophobic fraction (presumed to be mainly degraded or partially degraded PS) from the insect frass so it can be analyzed in chromatographic and spectrometric analyzes. The extracted fraction from the antibiotic- treated larvae frass showed no significant difference with the original PS-feedstock, while the residual PS extracted from the untreated group frass showed a reduction in the original molecular weights and molecular weight distribution, suggesting oxidation and ring cleavage of the original polymers in the sample. Ultimately, the microbial communities' composition of the PS-fed mealworms was compared to control groups (unfed and bran-only feedstock), detecting significant changes in the relative compositions. This shift was attributed to the establishment of a microbial community with improved PS degrading capabilities. Aiming to determine if PS could be used as an economical feed alternative for mealworm rearing, Nukmal et al. (2018)104 compared the effects of three different diets using yeast as the standard control feed and two different commercially available PS products - expanded polystyrene (EPS) and extruded polystyrene (XPS) – on the survival rate, larval weight, prepupal periods, pupation periods, pupal weight, imago weight and number of eggs laid. The results obtained support the premise that PS foam is eatable and non- lethal to the larvae of T. molitor. However, PS feedstock does not equate, moreover surpass, productivity effects of standard diets such as yeast in mealworms and thus does not represent an economical alternative for mealworm rearing. On the other hand, since PS foam feed allowed worms to complete their life cycle without significantly affecting their mortality, the use of mealworms in PS waste degradation is worth further investigation. For their experiment, Brandon et al. (2019)105 investigated the fate of hexabromocyclododecane (HBCD – the historically most common flame-retardant additive in plastic manufacture, currently being phased-out and banned after its potentially toxic nature was reported by several sources) in PS-degrading mealworms,

also evaluating HBCD bioaccumulation effects at a secondary trophic level by feeding PS- fed mealworms to Litopenaeus vannamei (pacific whiteleg shrimp), a model aquaculture organism. Experiments using PS products with high and low HBCD concentrations that were still commercially available (proof of the lack of regulation for many plastic products) showed that most of the HBCD consumed by mealworms was egested after 24h and almost of all it after 48 h, with only 0,27% of the ingested HBCD remaining in the mealworms body tissue with little or no signs of bioaccumulation. Moreover, mealworm survival rates, plastic mass ingested and number of pupae were not affected by the different concentrations of HBCD ingested, whereas the addition of bran as a supplement to the PS diet increased plastic mass ingested and number of pupae. Finally, no evidence of HBCD transfer to higher trophic levels (L. vannamei) was observed. The researchers call for further studies of the potential environmental effects of the egested particles since their toxicity remains unknown While assessing the microbiome diversity of PS-fed mealworms, Urbanek et al. (2020)116 reported results that conflict with those documented so far involving the mealworms - and their gut microbiome – PS-biodegrading capabilities. In their experiment, Urbanek et al. observed no significant difference in the weight changes and biochemical composition between different types of PS-fed mealworms and starved mealworms, in contrast to a control group of mealworms fed with a standard bran diet. Moreover, mealworm-gut microorganisms reported in earlier studies to be involved in PS-biodegradation (e.g. Exiguobacterium sp. strain YT2) were not found in any of the PS-treatment groups. Interestingly, bacterial strains (Serratia marcescens, Klebsiella oxytoca, and Pseudomonas aeruginosa) capable of degrading so-called bioplastics120 (PBSA, PBS, and PCL) were found in gut extracts from PS fed mealworms. Similarly, Peña-Pascagaza et al. (2020)110 reported PS-eating mealworms losing weight and shrinking during their 4-month experiment, with a steep decrease in their survival rate after 45 days (keeping a steady 20% survival rate after 105 days until the end of the experiment), moreover the beforementioned gut microbe strains documented as being capable of PS degradation were absent in the mealworms’ guts. Adult mealworms (post metamorphosis) did not ingest any PS foam at all, attributed to changes in jaw-structure

and gut microbiome121,122. After isolation, 9 strains were put to test for PS-degradation capabilities. Three Bacillus strains, identified via 16S rRNA, were documented to modify the PS surface (tunnels, cracks, and cellular aggregates on the PS sheets under SEM microscopy), suggesting their capability of PS degradation. Pathways of PS-degradation via oxidation of styrene to phenylacetate, via the citric acid cycle, is common among different microorganisms (Ho et al., 2018). Bacteria of genus Bacillus stand out for their capacity to transform styrene to phenylacetate. Authors conclude that PS is not nutritious enough to feed mealworms without them resourcing to cannibalism. Aboelkheir et al. (2019)106 studied T. molitor capability to biodegrade vulcanized rubber and tire crumb, both thermosetting plastics with a high degree of cross-linking structure. In a 3-week experiment, they observed no significant difference in the survival rate of the 3 test groups (vulcanized rubber, tire crumb and standard bran (control) fed groups). By the end of the experiment, only the tire crumb group was recorded to lose weight (10.54%) while the rubber and bran fed groups gained weight (9.31% and 22.23%, respectively). Spectrophotometric and thermogravimetric analysis indicated changes in the chemical composition of the original substrate, and surface microscopy revealed the release of sulfur (a constituent of the cross-linking bonds sulfur-sulfur and sulfur-carbon present in thermosetting plastics that undergo vulcanization), suggesting biodegradation of the plastic feedstock by cleavage and breaking down of cross-linking bonds (labeled as bio-devulcanization or bio-desulphurization by the authors of the study). Although these cross-linking bonds provide the thermosetting plastics higher stability (and therefore reduce their biodegradability) they are also the weakest bonds present in the polymer matrix, so that the products of the initial bio-desulphurization may be considerably more recalcitrant to enzymatic attack and thus less prone to biodegradation by mealworms. To determine whether plastics may be broadly susceptible to biodegradation within mealworms, Brandon et al. (2018)107 investigated the fate of PE and mixtures of PE + PS when fed to T. molitor. Over the course of a 32-day experiment, researchers observed mealworms readily consuming and degrading PE and PS. Findings suggested that PE biodegrades at comparable rates to PS, supported by mass balance analysis showing the

conversion of up to 49% of the ingested PE into CO2, calculated as a putative gas fraction. No significant difference has been registered between a group fed only PE and a group fed only PS. PE and PS biodegradation was confirmed by FTIR, 1H NMR and HT-GPC analysis of the samples extracted from frass of the different treatment groups compared to the original plastic substrate, showing that chemical modifications occurred within the mealworms’ guts (consistent with partial degradation and oxidation of the polymers). Next-generation sequencing of the mealworms intestinal tracts identified two OTUs (Citrobacter sp. and Kosakonia sp.) strongly associated with both PE- and PS-only diets, and other OTUs that were specific to each diet (mixed PE + PS, PE + Bran, PS + Bran, Bran only), suggesting that diets influence the composition of gut microbial communities and that these changes potentially enable the insect to degrade dissimilar plastics. On the other hand, consumption and degradation of both PE and PS in the mixed-plastic group suggest that some degradation pathways may be nonspecific. Przemieniecki et al. (2020)115 examined the effect of 5 different feeding regimes (including PE and PS) on the gastrointestinal microbial communities of mealworms, additionally exploring their enzymatic activity and identifying bacterial groups potentially involved with plastic waste degradation. After feeding larvae with cellulose (cardboard), oat, 2 types of PE (regranulate and oxo-degradable) and PS for 2 months, the degrading efficiencies where OAT-10.56%, CEL-5.2%, PE-reg-5.92%, PE-oxo-6.22% and PS- 7.88%. A total sum of 972 microbial species were identified across all treatments, the highest number of individuals and species was noted in the digestive tracts of mealworm larvae fed with cardboard followed by the oxo-biodegradable plastic group. PE regranulate recorded the highest diversity (Shannon’s diversity index) and evenness (Pielou’s evenness index). Significant differences in the composition and structure of microorganisms at phylum, class, and genus levels are detailed in the publication. Here, in the interest of brevity, we note that larvae fed oatmeal and cellulose were colonized by similar bacterial communities, and some similarities were also identified between both PE variants, while the microbiota identified in larvae fed with PS differed most considerably from the remaining bacterial groups. The overall results of enzymatic activity revealed a wide range of enzymes secreted at higher concentrations by digestive tract

homogenates (21 pmol on average) than those secreted by bacteria alone (13 pmol on average). An analysis of the potential role of the evaluated microbiota (information about the microbiome composition and its enzymatic activity) revealed that in the PS variant, the digestive tract of T. molitor contained an additional source of carbon as well as atmospheric nitrogen fixed by bacteria of the orders Rhizobiales, Nitrospirales and Nitrosomonadales. In this way, nitrification and anammox reactions can take place in the digestive tracts of mealworms fed diets deficient in nitrogen compounds. Enzymatic activity and next-generation sequencing supported the identification of enzymes and bacterial groups that participate in waste degradation. A common trait of every analyzed diet was the dominant presence of Parabacteriodes spp. and Clostridium sp., anaerobic bacteria reported to decompose hemicellulose and polyethylene under natural conditions61. Concerned about the excessive amount of microplastics entering the world's natural environments and the threat they pose to ecosystem function, Wu et al. (2018)108 investigated the effect of three common microplastics found in the environment on the feeding and metabolism of three different strains of T. molitor. After 1 month of experimentation, the survival rate of mealworms fed exclusively with LDPE, PS and PVC microplastics (MP) was not significantly different from the control group fed with bran only. On the other hand, the average weight of the mealworms from the three MP treatment groups (and the starving group) decreased during the study, while the bran fed group increased their weight significantly. When comparing feeding habits of mealworms in relation to the different MP, PS was the most consumed, followed by PVC and lastly LDPE. Further comparisons of the crystalline structure, molecular structure, chemical composition and molecular weight were made between untreated MP and MP extracted from insect frass from all treatments. Results indicated that all strains of mealworms were able to change the crystalline structure, molecular structure, chemical composition and average molecular mass (showed by XRD, FTIR, TGA, and GPC analysis, respectively) of all MP types, except for a strain of mealworms (originating from the Guangzhou province in China) where no changes were reported for either PVC and LDPE MP in comparison to the untreated substrate.

Guo et al. (2019)109 investigated epigenetic changes in mitochondrial DNA of T. molitor fed with polyurethane (PUR), while also controlling ATP levels in comparison to a control group fed with bran only. Methylation of mtDNA was reported as a rare event in the control group mealworms, however, methylation content and patterns proved to be affected by exposure of mealworms to PUR. Moreover, recorded ATP levels revealed an inverse correlation with methylation events in mealworms fed with plastic. The ATP and mtDNA gene regulation (by epigenetic methylation) are thought to be stress responses to environmental pollutants.

3.2.2.2 Tenebrio obscurus To further investigate the ubiquity of consumption and biodegradation of synthetic polymers by members of the Tenebrio genus, Peng et al. (2019)111 experimented with T. obscurus (commonly known as dark mealworm) from 4 different geographical regions, additionally comparing the PS biodegrading efficiency of one strain of T. obscurus with that of a T. molitor strain. At the end of the 31-day experiment, results revealed that dark mealworms had a higher PS consumption rate (0,32 mg larvae-1 day-1) than yellow mealworms (0,24 mg larvae-1 day-1) of similar size. The decrease of the number-average molecular weight (Mn) of partially degraded polymers extracted from insects’ excreta was also significantly stronger in T. obscurus (26%) compared to T. molitor (12%). Further FTIR and TGA indicated chemical modification of the original substrate and the formation of functional groups identified as degradation intermediates of PS. When corn flour was added as a co-diet, PS consumption increased for both species. Moreover, the importance of the gut microbiome in PS depolymerization was established with an antibiotic (gentamicin) test, where the treated larvae almost completely lost the ability to degrade PS. Finally, high-throughput sequencing analysis revealed shifts in the gut microbial community of individuals fed with a PS diet, with changes in three predominant families (Enterobacteriaceae, Spiroplasmataceae, and Enterococcaceae).

3.2.2.3 Zophobas morio Tang et al. (2017)117 reported PS biodegradation of microbial strains isolated from T. molitor and Zophobas morio (also known as superworm) via a rather minimalistic set-up,

using Petri plates with PS-basal medium to isolate microbial strains and inoculating them into PS-emulsions to test for turbidity changes (attributed to microbial degradation of the PS substrate). In the experiment, insects of both species were fed PS for 30 days before extracting a suspension of their gut content; the suspension was then plated and incubated in a basal agar medium, later transferred to a PS medium and using streak methods until obtaining pure microbial colonies. Microbial strains from both mealworm and superworm were eventually isolated (strains TM1 and ZM1, respectively) for further experimentation. PS biodegradation was inspected by spectroscopic analysis (turbidity assay) inoculating the isolated strains into of PS-emulsions with and without additional yeast extract. After incubation, the PS-emulsions mixed with yeast extract exhibited changes in turbidity, while PS-only emulsions remained unchanged. Both strains were characterized and sequenced as part of the experiment, but their genetic sequences have not been submitted to the GenBank database yet.

3.2.2.4 Zophobas atratus Zophobas atratus was reported as capable of chewing, ingesting, degrading and mineralizing Styrofoam by Yang et al. (2019)113. Moreover, Z. atratus (common names: superworms or giant mealworms) could consume 4 times more Styrofoam compared to experiments with T. molitor in a previous study by Yang et al. (2015). Larvae were then capable of completing their life cycle (although fertility was not tested). Standard spectrometry, chromatography and thermogravimetry test showed that, after 28 days, the egested products of superworm digestion were different from the original plastic substrate regarding depletion of aromatic rings, appearance of new carbonyl groups (spectrometry), decline in average -number and weight- molecular weights (chromatography and thermogravimetry) which suggested depolymerization and appearance of novel lower molecular weight degradation products. Additionally, mineralization of ingested Styrofoam was tested using sealed glass incubators measuring

CO2 production, revealing that PS fed larvae produced more CO2 than starving larvae

(latter larvae only produce CO2 from basal endogenous respiration). Further carbon mass balance experiments showed that the conversion efficiency of ingested Styrofoam to CO2

increased with time, while carbon egested in frass decreased with time. Finally, gut microbiota was proven to play an essential role in plastic biodegradation after superworms with suppressed gut microbiota (treated with antibiotics) showed impaired PS-degrading ability compared to a control group of untreated superworms. The average molecular weights of polymers extracted from frass were significantly heavier in the antibiotic-treatment group compared to the untreated group, suggesting that gut microbiota is involved in plastic depolymerization.

3.2.2.5 Tribolium castaneum Wang et al. (2018)114 realized a metagenomic analysis of the gut tissues of the Red Flour Beetle Tribolium castaneum fed with PS, later comparing it to standard fed T. castaneum. Results showed that the number of bacterial genes related to the metabolism of aromatic compounds, hydrocarbons, saturated and unsaturated fatty acids was significantly higher in the PS fed larvae compared to the control group, indicating that bacteria related to PS degradation were enriched by the progress of PS-feeding. A bacterial strain identified as Acinetobacter sp. (strain AnTc-1) with strong PS degradation activity was isolated from gut tissues for further studies. After 60 days of incubation of PS powder with AnTc-1, mass loss up to 12% of the starting weight was registered. The presence of biofilms, cavities, and pits was observed on the treated samples using SEM, whereas the control did not show any changes. Furthermore, 1H NMR spectroscopy revealed changes in the chemical structure of the treated samples and TGA analysis showed that the thermal stability of PS was remarkably reduced after incubation with AnTc-1. To verify whether depolymerization of the long-chain structure of the treated PS samples occurred, researchers used GPC to characterize the molecular weight distribution, weight-average molecular mass (Mw), and number-average molecular mass (Mn). Mw and Mn of the treated samples were reduced 13% and 25%, respectively, indicating a shift in the molecular weight distribution towards the shorter polymer chain side.

3.2.3 Lucanidae 3.2.3.1 Aegus laevicollis In 2012, Suzuki et al. characterized a biodegradable plastic (BP)-degrading enzyme, purified from a yeast strain (closely related to Cryptococcus magnus) isolated from the larval midgut of a stag beetle (Aegus laevicollis)118. This is likely the first published academic report to link an insect (concretely its gut microbiome) with the biodegradation of synthetic polymers; in this case a set of biodegradable plastics. Researchers used selective medium and streaking techniques to isolate pure microbial strains from the guts of stag beetles. Two isolated yeast strains (labeled as BPD1A and BPD2A) were pointed out for their BP degrading abilities. A novel method developed by the researchers for affinity purification of relevant enzymes, based on the binding action of the enzyme to an emulsified PBSA substrate, was tested on the BPD1A strain. The resulting purified enzyme - CmCut1 - displayed degradation activities on several BP cast films (PBSA, PBS, PCL, PLLA, and PDLLA), detected by tests measuring water-soluble total organic carbon (TOC) released from the BP cast films after being inoculated with the enzyme, although no degradation rate was reported. Analysis of partial amino acid sequences from the purified enzyme suggests a close relation to the family of cutinase-like enzymes (thus its name CmCut1).

3.3 Lepidoptera

3.3.1 Overview The economic importance of beehive-pests such as Galleria mellonella (greater waxmoth) and Achroia grisella (lesser waxmoth) has led to numerous studies on their ecology, molecular biology, physiology, and more. Besides their apicultural importance, G. mellonella larvae are well-accepted as model organisms for studies on pathogens and insect physiology123,124, and its nutraceutical potential is highlighted in human and animal diets125. On the other hand, Plodia interpunctella (Indian meal moth) is one of the most common and serious pests of stored products, thus also extensively investigated for its economic importance126. There is a broad range of published research techniques

covering the study of these different moths – especially great wax moths – representing a valuable resource to foster further studies127. After Tenebrionidae, the second most studied insect family used in plastic degradation research is the Pyralidae family. Notably, Galleria mellonella has received a lot of attention in the past few years for its plastic degrading abilities. Studies have shown that, compared to a standard diet, G. mellonella larval survival rate and average weight does not vary significantly when fed with different common PE waste materials128 or LDPE pretreated with UV-radiation129. Furthermore, feeding regimes combining plastic substrates and nutrient-rich supplements have been observed to significantly increase the starting average weight130. Gravimetric analyses showed a significant mass reduction in plastic substrates exposed to G. mellonella129,131,132 and fungal strains (Aspergillus flavus) isolated from wax moth’s gut133, supported by microscopy analysis showing physical damages on the plastic substrates129,131,134,135. Additionally, spectroscopic and chromatographic analyses show changes in the chemical structure129,131,132,136 and reduction in the molecular weight131,129 of residual plastic excreted by wax moths and of plastic samples incubated with isolated microbial strains from the insects’ digestive tracks132–134. However, chromatographic analysis in one experiment showed that the molecular weight distribution of residual plastic increased136, a feat that was attributed to the depletion of low molecular weight polymers (smaller molecules) that are readily digested before the degradation of more complex heavier polymers (bigger molecules). The experimental analysis identified ethylene glycol131,132; alcohols, esters, and carbonic acids134; and carbonyl groups133 as some of the products of PE biodegradation via G. mellonella. Based on G. mellonella’s natural history parasitizing beehives and feeding on beeswax (natural polymer with a similar structure to synthetic polymers such as PE), Kong et al. (2019) and Peydaei et al. (2020) suggested and proved that G. mellonella possesses genes encoding for enzymes that enable the insect to degrade PE independently of its gut microbiome. Nevertheless, microbial strains capable of growing on (Acinetobacter sp. ACT126132) and degrading (Enterobacter sp. D1134 and Aspergillus flavus strain PEDX3133)

PE substrates have been isolated from the insects’ gut, suggesting a possible complementary function of the gut microbiome in long-chain polymer degradation. Achroia grisella, another beehive pest known as the lesser wax moth, has also been observed to chew and consume PE films137,138. When fed PE only, A. grisella PE- consumption rates are comparable to those reported for G. mellonella131 and the rate can be significantly increased by supplementing wax-comb to the plastic diet. Adding wax- comb has also been shown to significantly increase A. grisella larval weight and survival rate. Spectroscopic analysis of the excreta from insects fed with PE reveals an increase in the concentration of unsaturated hydrocarbons and the presence of new carbonyl and alcoholic groups compared to the initial substrate, indicating that depolymerization of PE occurs during the digestion process. Yang et al. (2014)99 isolated two PE-degrading bacterial strains, identified as Bacillus sp. YP1 and Enterobacter absuriae YT1, from the guts of Plodia interpunctella (Indian meal moth) larvae fed with LLDPE bags filled with millet grains. Both bacterial strains were observed to grow biofilms and colonies on the surface of inoculated PE films. Further analysis revealed changes in the physicochemical properties of the PE-films’ surface (such as the reduction of the materials hydrophobicity, tensile strength, weight loss, molecular weight decrease, and appearance of new carbonyl groups and water-soluble daughter products), widely attributed to PE degradation. Ultimately, the genomic sequences of both strains were deposited in the GenBank database for further studies. The following year, as a resource to facilitate the study of the enzymes involved with PE biodegradation, a companion article139 was published with the complete genome sequence of Bacillus sp. strain YP1. The analysis of potential metabolic networks revealed a total of 182 genes (out of 4238) involved in known pathways of biodegradation and metabolism of PE. Another member of the Pyralidae family, Corcyra cephalonica (known as common rice moth) has also recently been tested for its polyethylene biodegrading potential. In their experiment, Suresh Kesti et al. (2019)140 aimed at studying the role of the rice moth larvae gut-microbiome in PE degradation by comparing a group of untreated rice moths with a group of gut-microbiome suppressed rice moths pretreated with antibiotics. Before feeding the two groups exclusively with a commercially available PE film, inhibition of gut

bacteria in the antibiotic group was confirmed by the absence of both bacterial colony growth on agar plates and bacterial DNA in an electrophoresis test. Regardless of the pretreatment, both groups of rice moths were able to chew and consume similar quantities of PE after 20 days (20% and 25% of the initial substrate for the control and antibiotic group, respectively), which led the researchers to conclude that C. cephalonica gut-microbiome may not play a central role in PE consumption.

3.3.2 Pyralidae 3.3.2.1 Plodia interpunctella After exposing Indianmeal worms (a common name for the larvae of Plodia interpunctella) to linear low-density polyethylene (LLDPE) plastic bags filled with millet grains, Yang et al. (2014)99 isolated eight strains of potential plastic-degrading microorganisms extracted from the enriched gut content of the larvae. These strains were then screened for their PE degradation activity resulting in two strains (Bacillus sp. YP1 and Enterobacter asburiae YT1) reported as being independently able to biodegrade virgin polyethylene. This assertion stems from observing the formation of biofilms and colonies on the surface of inoculated PE films, consecutively measuring several physicochemical changes on the surface properties of said films such as the reduction of the materials hydrophobicity, tensile strength, weight loss, molecular weight decrease, and appearance of new carbonyl groups and water-soluble daughter products, attributed to polyethylene degradation. The two strains were characterized and identified, ultimately depositing the genomic sequences in the GenBank database for further studies. As a resource to facilitate the study of the enzymes involved with PE biodegradation, a companion article was published a year later139, with the complete genome sequence of Bacillus sp. strain YP1. Following an analysis of metabolic networks, Yang et al. (2015a) reported a total of 182 genes (out of 4238) involved in the pathway of biodegradation and metabolism of the xenobiotic (i.e. synthetic substances found but not produced by the organisms or the environment)141– 143.

3.3.2.2 Galleria mellonella In 2017, Bombelli et al.131 reported fast biodegradation of PE by the larvae of Galleria mellonella (commonly known as greater wax moth), identifying ethylene glycol as a degradation product. The gravimetric analysis revealed that 100 larvae exposed to a commercial PE-plastic bag had consumed 92 mg of the synthetic polymer after 12 hours. Similarly, PE samples treated with wax moth homogenate showed a significant 13% mass loss after 14 hours. Further analysis (AF microscopy and FTIR spectroscopy) showed modifications in the surface integrity and chemical composition compared to a control sample of untreated PE. Further characterization of the treated samples using high- performance liquid chromatography coupled with mass spectrometry (HPLC–MS), indicated the presence of lighter fractions absent in the control sample. Based on previous reports of microbial plastic biodegradation, the authors pointed out that this degradation product might be ethylene glycol. Some experiments have capitalized on the fact that beeswax presents a chemical structure similar to that of PE144. For example, Ren et al. (2019)134 sampled wax moth larvae from bee farms, where natural beeswax constitutes their main food source, screening for PE-degrading microorganisms present in the larvae’s gut. Intestinal homogenates extracted from G. mellonella were incubated in special mediums with PE as the only carbon source, so that only the microorganisms able to take apart PE polymer chains and use the resulting smaller carbon molecules as an energy source, would grow on the medium. After 31 days, a strain identified as Enterobacter sp. D1 was isolated and tested for its PE-biodegrading capabilities. The presence of a biofilm was observed on the surface of the treated PE samples, while changes in the morphology and chemical structure of the treated PE-films (observed with SEM, SEM-EDS, AFM, and FT-IR) indicated that plastic was deteriorated and oxidation reactions had occurred on the surface, as opposed to the control medium (without D1 strain inoculation) were no changes were detected compared to virgin PE. Additional analysis of water-soluble products using liquid chromatography-mass spectrometry (LC-MS) showed that organic compounds (including alcohols, esters, and carbonic acids) were significantly increased

in the treated samples compared to the control ones, with several new compounds detected only in the samples inoculated with the D1 strain. Kundungal et al. (2019)129 compared the biodegrading efficiency of G. mellonella on low- density polyethylene pretreated with solar radiation (PTLDPE) with untreated PE (UTLDPE) samples and natural wax comb (WC). The three substrates and the post degradation products present in the excreta residue (ER) produced by insects of the three groups were characterized using AFM, FT-IR, 1H NMR, and GCMS techniques. Results showed the presence of new carbonyl and alcoholic groups with an increase in unsaturated hydrocarbon in the compounds extracted from the ER of the PTLDPE group, indicating enhanced mineralization of LDPE compared to the UTLDPE group. The survivability of the UTLDPE group was significantly lower than the PTLDPE group, the latter one remaining equal to the WC control group. Gravimetric analysis of post degradation of WC, UTLDPE, and PTLDPE showed 92.03 ± 2.1%, 55.8 ± 1.2%, and 18.57 ± 1.8% weight loss respectively. Vasileva et al. (2019)128 compared the ability of G. mellonella to biodamage (i.e. total weight loss, number of perforations, and total area of perforations) different PE materials commonly found in Russian urban waste streams. No significant difference in survival rates, mobility, weight gain, and melanization between PE fed groups and a control group fed with beeswax was reported, indicating no negative effects on the health of G. mellonella larvae when fed polyethylene waste. In 2019, Zhang et al.133 isolated Aspergillus flavus strain PEDX3, capable of PE- biodegradation, from the guts of wax moth G. mellonella. To do so, wax moths were fed exclusively with PE for two weeks after which their gut contents were extracted and incubated for 30 days in acclimatization media, and pure colonies were progressively isolated. Consequently, isolated strains were then screened for PE-degrading activity, inoculating them on mediums where PE was the only carbon source. Out of 25 isolated strains, the one with the most prominent growth, identified as Aspergillus flavus strain PEDX3 by its morphological and phylogenetic characteristics, was singled out for further experiments. A spectrometry analysis (FT-IR) of the post degradation effect of PEDX3 on PE-films revealed changes within the chemical structure of the medium inoculated with

PEDX3 compared to the control samples, the new absorption peaks detected in the spectrum of the treated samples corresponding to ether and carbonyl groups. Chromatography analysis (HT-GPC) also established that, in comparison to control samples, the samples exposed to strain PEDX3 presented significantly Mw and Mn (values describing the molecular mass of polymers), shifting the distribution curve of the molecular mass composition towards lighter and smaller molecules, indicating depolymerization of PE long-chain structure and formation of lower molecular weight fragments. Gravimetry analysis determined that samples inoculated with Aspergillus flavus PEDX3 had a mass loss percentage of 3.9 ± 1.18% after 28 days of incubation. Finally, gene sequencing analysis (RT-PCR) identified two laccase-like multicopper oxidases (LMCOs) genes, AFLA_006190 and AFLA_053930, displaying up-regulated expression during the experiments, nominating them as candidate enzymes potentially involved in PE degradation. Similarly, Kong et al. (2019)130 found, in a thoroughly extensive genetic and biochemical analysis, an increase in the activity of genes encoding for esterase, lipase, and cytochrome P450 (CYP) enzymes in G. mellonella fed with beeswax. These enzymes are related to the oxidation of long-chain hydrocarbon compounds, which make up around 14% of wax (weight) and are the main constituents of PE135,144. In the subsequent phase of the experiment, chromatographic/spectrometric analysis (GC-MS) revealed no significant difference of post-degradation PE samples taken from wax moths fed with PE + antibiotic treatment and wax moths fed PE + no-antibiotic treatment, indicating that G. mellonella was able to break down PE and beeswax independently of the intestinal microbiota. This was supported by gravimetric results showing that PE-fed wax moths, regardless of being treated or not with antibiotics, maintained a relatively constant weight over the course of 15 days. In comparison, the average weight significantly decreased in a starved group while larvae fed with a PE + nutrition-rich diet (mixed in 3 different proportions) showed a significant increase in their starting weight. In a supplementary study of G. mellonella’s innate ability to degrade PE, Peydaei et al. (2020)135 examined changes in the protein expression of salivary glands in larvae fed with PE compared to larvae fed a normal diet123. Researchers observed changes on the surface

properties (SEM) and chemical structure (FT-IR) of samples incubated separately with homogenates made from the wax moths’ salivary glands and gut tissues for 20 days. Analysis of the protein composition (differential proteomic analysis using LC-MS/MS) of the salivary glands from wax moths fed with and without PE identified 481 proteins present in both groups, PE-consumption having a significant effect on the expression of 13 proteins. Further analysis of the specific functions affiliated to these 13 proteins revealed that PE exposure increased the abundance (upregulation) of ten proteins associated to fatty acid beta-oxidation, cell physiology regulation, and induced secretion of macromolecules, among other functions related to biochemical changes in cells. On the other hand, three proteins were significantly downregulated (decreased abundance) during exposure to PE, the most notable of which was the Juvenile Hormone Esterase, indicating that larvae may extend the length of their last larval instar, associated to a general decline in the metabolism and a reduced energy level in the glands of wax moths consuming PE. Cassone et al. (2020)132 further investigated the contribution of G. mellonella’s intestinal microbiome by examining differences in PE degradation between intact larvae and larvae with a suppressed gut-microbiome (treated with antibiotics); changes in the composition of the wax moths’ gut microbial communities fed with different diets; and isolating PE- degrading bacteria from the larvae gut. After observing that the excreta of larvae fed with PE turned liquid (larvae fed with honeycomb produced solid excreta), researchers tested the role of gut microbiota by feeding the larvae with a broad spectrum of antibiotics. Consumption of PE in both the treated and control group both remained similar, but excreta analysis showed that in the group of antibiotic-treated larvae, where measures of gut bacterial abundance confirmed a 65% reduction after 24 h, only 22% of the individuals continued to produce liquid excreta (as opposed to 62% in the control group) generating, as a group, 40% less liquid excreta (weight) and significantly less ethylene glycol (spectrophotometric analysis) compared to larvae with no antibiotic treatment. Next, the feeding regime was proved to have a significant effect on the abundance of gut microorganisms after 24 h, with PE fed larvae having 7 and 1,7-fold higher abundance than starved and honeycomb-fed larvae, respectively. Subsequent

metagenomic analysis (gene sequencing) further uncovered characteristics of the structure, composition, and diversity of microbial communities in the context of the different feeding regimes. Statistical analysis revealed that only a handful of microbial genera had a significant increase in their abundance when fed with PE in comparison to honeycomb fed groups. Finally, Acinetobacter sp. ACT126 was isolated and incubated for more than 60 weeks on a medium with PE as the sole carbon source, confirming the ability of this strain to use PE as an energy source. Lou et al. (2020)136 further explored the effect of supplementing beeswax or wheat bran as a co-diet on larval survival and plastic degradation in G. mellonella fed with synthetic polymers. Moreover, this is the first experiment to test the degradation abilities of wax moths on a type of plastic other than PE (polystyrene -PS- was also tested). Every 3 days, survival rates of larvae and plastic mass loss were registered and, after 21 days, thermogravimetric, spectrophotometric, and chromatographic analyses were done to examine the chemical changes, depolymerization, and degradation products of plastic fed to wax moths (using TGA, FT-IR, GPC, and GC-MS analysis, respectively). Additionally, differences in the gut microbiome of all 9 feeding regimes tested (starved, beeswax, wheat bran, PE, PS, PE + beeswax, PE + wheat bran, PS + beeswax, and PS + wheat bran) were explored with Illumina Hiseq sequencing of the 16S rRNA gene. Although there was a decrease in plastic consumption in the co-diet treatments (PE + beeswax, PE + wheat bran, PS + beeswax, and PS + wheat bran) compared to feeding regimes with plastic only (PE or PS), beeswax and wheat bran significantly increased the survival rates of G. mellonella when added to their feed. Characterization of plastic degradation indicated that changes had occurred to the different substrates after being excreted by larvae. TGA and FTIR analysis, using samples directly collected from frass (as opposed to GPC and GC-MS where samples needed to be extracted with suitable solvents for their subsequent analysis) revealed that new compounds, different from the ones found in the control (initial substrate), were present in the insects' excreta, indicating oxidation of both PE and PS in the larval gut. GPC, on the other hand, revealed results that might not be intuitively connected to depolymerization and oxidation of plastic. The analysis indicated that the molecular weight distribution of the treated samples increased in comparison to

the control samples, this feat was attributed to the depletion of low molecular weight polymers (smaller molecules) that would be readily digested before the degradation of more complex heavier polymers (bigger molecules). Finally, GC-MS analysis revealed the intermediaries of PE and PS degradation, indicating decreases of complex long-chain carboxylic acid esters in PE and compounds with a benzene ring structure in PS, suggesting that G. mellonella metabolism attacks these types of compounds.

3.3.2.3 Achroia grisella Lesser wax moths, a common name for A. grisella, from different geographic regions (Argentina and India) were reported to chew and consume polyethylene films137,138. Additionally, Kundugal et al. (2019)138 found that lesser wax moths fed exclusively with a PE diet gained weight while consuming, on average per day per larvae, 1.83 mg of PE. Plastic fed wax moths were able to complete their life cycle and produce a 2nd generation with similar PE consuming capabilities as the 1st generation. Nevertheless, the 2nd generation presented strong initial mortality, the young larvae being unable to properly establish themselves on a PE-only diet after hatching from the eggs. Although the larval biomass weight gain, substrate reduction, and survival rate of the PE fed group was significantly lower than the WC control group, this review found that A. grisella presents, together with G. mellonella131, the highest PE consumption rate. Further FT-IR and 1H MNR spectrophotometric analysis revealed changes in the physicochemical properties of the egested insect frass compared to the original PE substrate, with the presence of new carbonyl and alcoholic groups and an increase in unsaturated hydrocarbon concentration. These results suggest that the initial chemical composition and structure of PE were modified. A co-diet of plastic plus conventional feed (in this case PE+WC) allowed for a significantly larger increase in larval weight, PE consumption rate, and higher survival rate compared to a group of lesser wax moths fed solely with PE.

3.3.2.4 Corcyra cephalonica Reports have identified C. cephalonica (commonly known as rice moth) as a worldwide distributed stored grain pest, promoting research to control its economic impact on stored products145. Recently, the rice moth has also been tested for its polyethylene

biodegrading potential. In their experiment, Suresh Kesti et al. (2019)140 aimed at studying the role of the rice moth larvae gut-microbiome in PE degradation by comparing a group of untreated rice moth with a group of gut-microbiome suppressed rice moths pretreated with antibiotics. Before feeding the two groups exclusively with a commercially available PE film, inhibition of gut bacteria in the antibiotic group was confirmed by the absence of both bacterial colony growth on agar plates and bacterial DNA in an electrophoresis test. Regardless of the pretreatment, both groups of rice moths were able to chew and consume similar quantities of PE after 20 days (20% and 25% of the initial substrate for the control and antibiotic group, respectively), which led the researchers to conclude that C. cephalonica gut-microbiome may not play a main/major role in PE consumption.

3.4 Diptera

3.4.1 Culicidae 3.4.1.1 Culex pipiens Al-Jaibachi et al. (2018)146 examined the fate of polystyrene microplastic (MP) particles ingested by larvae (feeding stage) of common household mosquito once they developed into pupae and terrestrial adults (non-feeding stages). Using an epi-fluorescent microscope to track the different sized fluorescent MP particles fed to larvae, the researchers were able to detect 2 µm plastic particles in the Malpighian tubules of adult individuals of the treatment group, proving that ontogenical transfer of MP particles can occur through the development stages in insects.

3.4.2 Stratiomyidae 3.4.2.1 Hermetia illucens Commonly known as Black soldier fly (BSF), H. illucens is a widely studied insect appreciated by its potential to process multiple types of organic waste streams, additionally converting the waste into valuable protein and fats that can be used as feedstock for different animals (poultry, aquaculture, household pet feed and so on)77,79– 81.

Cho et al. (2020)147 speculated that recalcitrant compounds present in BSF substrate may have a negative effect on its growth and survival, likely affecting the efficiency at which they decompose organic waste as well. In their experiment, BSF larvae were fed with PE and PS microparticles (400 to 500 µm) mixed with common food waste at different concentrations (5, 10, and 20% w/w). The effect of salt (NaCl) was additionally tested on the treatment group fed with 20% plastic concentration. Researchers found that the final weight of the larvae in the treatment groups was not significantly lower than the control group (fed with untreated food waste). On the contrary, after 20 days larvae from all 3 PS treatment groups had significantly larger mean larval weight than the control group (up to 17% heavier in the 20% added PS group), while the PE treatment groups showed no significant difference. Moreover, only the treatment group with 5% added PS showed a significantly reduced survival rate at the end of the experiment. On the other hand, adding from 1 to 3% salt concentration (w/w) to food waste mixed with microplastics (20% w/w) proved to have a significant negative effect (incrementing with increased salt concentration) on the mean larval weight and performance traits (survival rate, pupation ratio, and substrate reduction) of the treatment group, compared to a control group without added salt.

DISCUSSION

The massive manufacture, indiscriminate use, and inadequate disposal of plastic products have quickly turned plastic in one of the most conspicuous anthropogenic materials affecting environments worldwide, turning the attention of the scientific community towards the threat of plastic as a potential planetary boundary. This means that, if left unmanaged, humanity’s fixation with plastic might exceed the theorical “safe limits for human development”, breaching the hypothetical thresholds at which vital Earth system processes are capable of adjusting and regulate disrupting perturbations (i.e. plastic pollution), resulting in deleterious and even catastrophic environmental changes that can reach planetary scales148–151. Awareness of environmental plastic pollution dates back to 1970152,153. Since then, continuous and ever-increasing efforts have been undertaken to close knowledge gaps regarding the origin, extend, distribution, toxicity, fate, and other parameters of environmental plastic pollution, but the problem remains largely unsolved18,154,155. Plastic biodegradation is regarded as a particularly promising waste management strategy 60,65,156–158, especially for single-use and short-in-use phase products used for agricultural purposes and packaging, which are widely recognized as the main drivers of global plastic waste mismanagement and environmental pollution7,48. Biodegradation of various synthetic polymers and plastic products has been studied under different environmental conditions. Numerous microbial strains and enzymes isolated from diverse environments (e.g. soil, compost, landfills, seawater) were reported to show degrading capabilities of synthetic polymers that commonly make up plastic packaging products31,32,56,82,156,159–161. The degradation processes and pathways of these plastic products have been thoroughly reviewed, pointing out the methodological challenges and knowledge gaps that are still to be resolved in order to attain conclusive evidence of complete plastic biodegradation5,16,16,17,31–33,56,67,68,156,160,162–168. Recent experiments have revealed that some insects and their microbial symbionts possess plastic biodegradation capabilities, raising interest in their untapped potential as a bioresource for new plastic waste management alternatives83,84,91,169. Actually,

interactions between insects and plastic, particularly plastic packaging, have been described as early as 195093. The large majority of reports aimed at testing and improving the resistance of plastic packaging to the attack of insect “pests”, ensuring the integrity of the packaged product (i.e. food). This reflects on one of the major purposes and benefits of using and developing plastic packaging technologies: reduce food waste170. On the other hand, Schweitzer et al. (2018)25 point out that the historic trends reveal increasing quantities of both food waste and plastic packaging waste in developed economies like Europe, therefore arguing that plastic packaging fails to address important underlying drivers of food waste. Moreover, many environmental impact analyses and life cycle assessments that advocate for the use of plastic packaging may heavily rely on assumptions that ignore or oversimplify the complex reality of current plastic packaging waste management25,154,155,171,172. The development of plastics intentionally manufactured to be readily biodegradable under certain conditions (i.e. biodegradable plastics) and plastics made out of renewable resources instead of petrochemicals (i.e. bioplastics or bio-based plastics) are part of a growing trend of emerging technologies aiming at reducing the burden of plastic packaging waste on the environment. However, sustainable production, use, and disposal of these materials are not exempt of challenges, starting from establishing a standard and international biodegradation criteria to certify and accurately label the so-called “biodegradable” products60,173,174. Just like with conventional non-biodegradable fossil- based plastics produced so far, degradation rates of novel biodegradable and bio-based plastics strongly depend on the environmental factors (e.g. temperature, humidity, pH, oxygen concentration, presence of microorganisms) and the chemical nature of additives and polymers used to make the final product. This means that specific and controlled waste management practices (e.g. composting within defined conditions) are needed once these materials reach their end-of-life phase. Furthermore, if littered or mismanaged, they may potentially release ecotoxic xenobiotics (e.g. plasticizers, microplastics, nanoplastics) to the environment. In this sense, the lack of consistent data at the end-of-use stage in life-cycle assessments of bio-based and biodegradable plastics may strongly influence the conclusions made about the ecotoxic, carbon, and energy

footprints of bioplastics and biodegradable plastics. Ultimately, these types of plastics appear to have many of the same challenges that conventional plastics currently face21,23,33,45,64,66,120,166–168,171,175–191.

4.1 Plastics and Insects

The experiments reviewed in the present work, presented in Table 1, are mainly focused on conventional synthetic fossil-based types of polymers largely used for plastic packaging (e.g. PE and PS), additionally including polymers like PUR, PVC, and SBR which have been detected in the form of microplastics in the environment106,108. Plastic degradation rates are included in the Table 1, whenever recorded in the reviewed papers. However, these rates must be considered carefully, since the plastic substrates used in the different experiments may be similar in composition and even have the same polymer backbone, but are in no way identical. For example, HDPE and LDPE both have a PE backbone, however, because of different degrees of crystallinity and secondary polymer branching, they have quite different physical and chemical characteristics. Density, crystallinity, molecular weight, additives, and other features of the plastic substrates used to feed insects and their isolated gut-microorganisms vary from one experiment to the other. Furthermore, experimental designs (e.g. number of insect larvae used in the experiment, type and amount of co-substrate, pretreatment of plastic substrates), and laboratory conditions (e.g. temperature, humidity) also differ among experiments. All of the above-mentioned factors have been reported to affect degradation rates of the different polymer types21,33,34,68,102,192–194, making comparisons between rates hard to establish. Therefore, degradation rates should best be regarded as an illustration of the factual biodegradation of the different synthetic polymers, eventually being useful as a reference in future experiments that seek to replicate the reported methodologies and outcomes. Depending on their hypotheses and objectives, plastic biodegradation experiments are focused on one or several of the following: the initial plastic substrate; the reaction products of the degradation processes, and the organisms carrying out the degradation

processes. Four common approaches are used to carry out the analyses: monitoring changes in substrate chemical and physical properties; monitoring the depletion of the initial substrates; monitoring reaction products that result from biodegradation, and monitoring accumulation or loss of biomass in the organisms195. Usually, multiple techniques that monitor optical, physical and chemical changes of the initial substrates are combined to corroborate signs of degradation in the plastic substrates and additionally identify the reaction products. For example, gravimetric analyses measuring weight loss often deal with insignificant changes, so, in order to make the analysis more robust, they are combined with other tests like respiratory tests measuring O2 consumption or CO2 production, thermogravimetric analyses which provide more information about changes in the physical and chemical profiles of the samples, spectroscopic analyses that evaluate chemical modifications (e.g. emergence of novel molecules and depletion of original molecules), or chromatography analyses that reveal changes in the molecular weight distribution of the diverse molecules that make up a sample34,168,196–198. On the other hand, direct methods like isotope carbon tracing and compound-specific isotope analysis, aided by fluorescent microscopy, may be cogent enough to independently provide compelling evidence about the pathways and fate of the carbon molecules that compose synthetic polymers and plastics, further elucidating plastic biodegradation processes (e.g. assimilation, mineralization)146,199–203. The numerous techniques used in the reviewed experiments draw from methodologies widely recognized and used in microbial and enzymatic plastic degradation experiments in different countries, with an additional technique for enzyme affinity-purification developed by Suzuki et al. (2012). However, there is currently no encompassing international standard protocol to determine and compare the extent of plastic biodegradation. The methods and techniques used tend to be applied in the countries where they originated and are yet to be equated at an international level5,34,68,168,195,204,205.

4.2 Role of insects in plastic waste biodegradation

Except for Culex pipiens, all of the insect species reviewed (see Table 1) were reported to display plastic degrading abilities, further identifying genes encoding for polymer- degrading enzymes130,135 and isolating symbiotic microorganisms capable of growing and degrading on synthetic polymer substrates103,116,110,115,117,114,118,134,132,99,112. Predominant evidence indicates that survival rates between larvae fed with plastic and standard diets were not significantly different and that insects on a strict plastic diet were able to maintain a constant weight. Although synthetic polymers, in general, show a high caloric value206, supplementing strict plastic diets with standard co-diets (e.g. oatmeal, yeast, protein) has been shown to increase survival rates, weight gain, and plastic consumption in the vast majority of experiments that included this type of test. These co-diets likely provide the additional nutrients needed for improved development. Insects could thus be suitable candidates to process mixed waste streams that are problematic to recycle, such as organic waste streams contaminated with plastic or post-consumer plastic packaging waste with food and organic residues, the latter being otherwise likely managed by incineration207–210. In the present review, the experiments that analyze gut microbial communities present evidence that insects fed with plastic diets undergo changes in the structure and composition of their microbial gut communities, shifting towards functional profiles that are better suited for synthetic polymer biodegradation101,103,111,114,115. However, there is relatively little consistency in the composition of microbial communities between experiments, and plastic degrading microorganisms isolated in one experiment are not identified in others116,110,130. Apart from social insects and a few other exceptions, insects appear to be colonized by microorganisms picked up from the surrounding environments rather than by specialized residents that are transmitted or inherited71,72. In this sense, the environmental habitat and diet are thought to predominantly determine the bacterial diversity in insects’ gut, which is further delineated by their developmental stage and phylogeny211. Often the fortuitous acquisition of a microbial symbiont has been the key step in colonizing specialized niches that otherwise would have remained unavailable or

nutritionally inadequate212. Therefore, plastic-eating insects are likely colonized by microorganisms that linger on the plastic substrate, some of them possessing rudimentary plastic-degrading tools (i.e. promiscuous enzymes that are capable of catalyzing secondary reactions using synthetic polymers as substrates32,213,214) that may enable insects to occupy and utilize plastic polluted niches. Low degradation rates under natural environmental conditions is often mentioned as a main limitation in microbial plastic degradation5,17,31–33,56,67,68,160,162–165. The guts of insects may provide an environment with more suitable conditions (e.g. temperature, oxygen concentration, humidity, pH, additional enzymes and nutrients) for microbial activity. Furthermore, insect digestion has been proved to be efficiently compartmentalized, allowing a sequential and effective breakdown of ingested material under different conditions and microbial communities215. The decomposition of plastic or any other complex polymeric material occurs due to the presence of several simultaneous and synergistic factors and runs in several stages61,163,216. Therefore, compartmentalization of digestion in insects may prove particularly convenient for plastic biodegradation, offering various physicochemical conditions71 that may suit the optimal growing conditions of different microorganisms97, facilitating a cooperative effort to successfully degrade the complex synthetic polymers158. Furthermore, the different feeding habits of insects can also be combined in a stepwise process to efficiently degrade complex polymers. Wang et al. (2017) demonstrated that a two-step biorefinery combining an initial stage with Tenebrio molitor and a second stage with Hermetia illucens (both species reported to show plastic biodegradation abilities) was able to process lignocellulosic crop waste, with satisfactory conversion rate and biodiesel production yield217. The process did not require any additional energy, chemical reagents, or material pretreatments, making it a sustainable recycling technology that does not pose an additional burden to the environment 6,59. Besides biodiesel and animal feed77, H. illucens biomass can be refined into bioplastics218, which in future scenarios could represent an insect-based biorefinery that operates in a complete circular bioplastic scheme: H. illucens biofactories use collected bioplastic waste as feed; during larval digestion, without additional energy or material inputs (eco-friendly), specific enzymatic

reactions convert bioplastic waste into compounds that can be directly extracted from the insect's frass or that become insect biomass; insect biomass and extracted compounds from insect frass are further processed and recycled into new bioplastics, re-entering the value chain. It seems that research into plastic biodegradation using insects and the associated gut microbiome is in the first stage of exploration, where potential candidates are screened for signs of polymer biodegradation. For example, Yang et al. (2016)199 challenged the groundbreaking report of complete PET microbial biodegradation by Yoshida et al. (2016a)219, arguing that the PET test substrate used during the experiments (of significantly lower crystallinity than the common PET bottle commercially used) does not equate to any material currently found in the market or waste streams. Yoshida et al. (2016b)200 replied that the main objective of their experiment was to isolate candidate microorganisms whose enzymes show PET biodegradation activities. Indeed, research on plastic biodegradation so far has mainly dealt with substrates, conditions, and organisms in controlled laboratory settings, gathering vast amounts of data but yet to be scaled and transposed to real-world scenarios. Parallelly, the increasing amount of data from novel microorganisms, enzymes, and genes identified to be involved in polymer degradation constitute the fundamental resource for a new generation of biotechnological tools. These rapidly developing tools from different fields (e.g. environmental biotechnology, synthetic and systems biology) may play a key role in attaining feats like the one described in the previous paragraph, and further applications in bioremediation of environmental plastic polluted sites159,216,220. The recent computational and systems biology developments help to refine the design of superior biocatalysts for biodegradation and biotransformation of desired chemicals, assisting in the analysis of degradation end-products and the prediction of probable mechanisms and metabolic pathways implemented by the microbial community during biodegradation205,221. For example, MetaRouter222 provides the possibility, exploiting data-mining applications, of locating biodegradative pathways for defined chemical compounds according to a given set of constraints and requirements. Integrating this information with the corresponding protein (enzyme) and genome data can further

provide a suitable framework for studying the biological network involved in a complete biodegradation of any type of plastic, given that enough information is available.

FINAL REMARKS

This review does not intend to point out insect biodegradation of plastic as the silver- bullet capable of solving the global plastic pollution problem. We are convinced that, for such a complex issue, no such silver-bullet exists. Instead, there is a need for concrete actions from all sectors involved, spanning from product design to international policies, aiming towards a change in the plastic economy from a linear scheme (extract, produce, consume and throw away) towards a circular one (reduce depletion of natural resources, keep materials in the economy and avoid products to become waste). Instead, this review intends to expand on the potential of insects as a rich and versatile biotechnological resource, with possible applications in waste management. In particular, we hope to encourage further research on the unrealized potential of insects to biodegrade problematic waste, such as post-consumer plastic packaging waste or organic waste contaminated with plastic. For example, insects such as mealworms (Tenebrio molitor) and black soldier fly larvae (Hermetia illucens) have been found to tolerate and process synthetic polymers without reporting serious negative impacts on their biology. Both these insects are currently used in various organic waste processing operations, where the value of different waste streams (e.g. food waste, biomass from agricultural waste) is reclaimed in the form of insect biomass and frass, both of which can be further refined into valuable products with global demand (e.g. biodiesel, bioplastic, feedstock for animal breeding, nutraceutical products, and soil fertilizer, to name a few). Post- consumer plastic packaging waste streams are costly and complicated to recycle under current recycling procedures, mainly because of the highly diverse mixture of synthetic polymers that compose the plastic packages and the organic impurities (e.g. food residues) found in the stream. Similarly, installations where organic waste streams are processed (e.g. composting facilities) might receive waste streams contaminated with plastic, reducing the efficiency and end-product value of the installation. We foresee that insects, such as mealworms and black soldier fly larvae, might find a place in a circular economy scheme, helping in the processing of waste streams like the ones mentioned before.

Another potential field of application for plastic-degrading insects may be bioremediation of plastic polluted sites, where insects may be capable of reducing the mass and volume of plastic material, transforming it into insect biomass, CO2, and secondary products released in insect excreta (frass) that might be quicker to continue biodegrading in situ via microbial activity in the environment (for further reference see ecotoxicogenomics223,224 and bioentomoremediation86,98,220,225). In general, accumulating more information and closing important knowledge gaps about plastic biodegradation, ranging from a thorough characterization of novel microorganisms, genes, enzymes and pathways involved in plastic degradation, to the identification of primary and secondary degradation products and recognition of their effects on the environment, are fundamental to enable fast-developing computational tools to design complete and environmentally friendly degradation schemes for problematic synthetic materials like plastic, that could eventually be materialized in circular economy schemes thanks to parallelly fast-developing advances in synthetic biology.

REFERENCES

1. Henry George Liddell, Robert Scott, A Greek-English Lexicon, πλαστι^κ-ός. http://www.perseus.tufts.edu/hopper/text?doc=Perseus%3Atext%3A1999.04.0057 %3Aentry%3D%2383506&redirect=true. 2. Fried, J. R. Polymer science and technology. (Prentice Hall, 2014). 3. Massy, J. A Little Book about BIG Chemistry. (Springer International Publishing, 2017). doi:10.1007/978-3-319-54831-9. 4. Clayden, J., Greeves, N. & Warren, S. G. Organic chemistry. (Oxford University Press, 2012). 5. Glaser, J. A. Biological Degradation of Polymers in the Environment. Plastics in the Environment (2019) doi:10.5772/intechopen.85124. 6. Hahladakis, J. N., Velis, C. A., Weber, R., Iacovidou, E. & Purnell, P. An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. J. Hazard. Mater. 344, 179–199 (2018). 7. Geyer, R., Jambeck, J. R. & Law, K. L. Production, use, and fate of all plastics ever made. Sci. Adv. 3, e1700782 (2017). 8. Plastics and the environment. (Wiley-Interscience, 2003). 9. Thompson, R. C., Swan, S. H., Moore, C. J. & vom Saal, F. S. Our plastic age. Philosophical Transactions of the Royal Society B: Biological Sciences 364, 1973– 1976 (2009). 10. PEMRG. Plastics - the Facts 2019. An analysis of European latest plastics production, demand and waste data. https://www.plasticseurope.org/application/files/9715/7129/9584/FINAL_web_versi on_Plastics_the_facts2019_14102019.pdf (2020). 11. Hartmann, N. B. et al. Are We Speaking the Same Language? Recommendations for a Definition and Categorization Framework for Plastic Debris. Environ. Sci. Technol. 53, 1039–1047 (2019). 12. Zhang, Y., Pu, S., Lv, X., Gao, Y. & Ge, L. Global trends and prospects in microplastics research: A bibliometric analysis. Journal of Hazardous Materials 400, 123110 (2020). 13. Zubris, K. A. V. & Richards, B. K. Synthetic fibers as an indicator of land application of sludge. Environmental Pollution 138, 201–211 (2005). 14. World Economic Forum, Ellen MacArthur Foundation and McKinsey & Company. The New Plastics Economy: Rethinking the future of plastics. https://www.ellenmacarthurfoundation.org/publications/the-new-plastics-economy- rethinking-the-future-of-plastics (2016). 15. Defruyt, S. Towards a New Plastics Economy. Field Actions Science Reports. The journal of field actions 78–81 (2019). 16. Ahmed, T. et al. Biodegradation of plastics: current scenario and future prospects for environmental safety. Environ Sci Pollut Res 25, 7287–7298 (2018).

17. Raddadi, N. & Fava, F. Biodegradation of oil-based plastics in the environment: Existing knowledge and needs of research and innovation. Science of The Total Environment 679, 148–158 (2019). 18. Ryberg, M., Laurent, A. & Hauschild, M. Z. Mapping of global plastic value chain and plastic losses to the environment: with a particular focus on marine environment. (2018). 19. CIEL. Fueling Plastics. https://www.ciel.org/wp-content/uploads/2017/09/Fueling- Plastics-Fossils-Plastics-Petrochemical-Feedstocks.pdf (2017). 20. Bond, K., Benham, H., Vaughan, E. & Chau, L. The Future’s Not in Plastics: Why plastics demand won’t rescue the oil sector. Carbon Tracker Initiative https://carbontracker.org/reports/the-futures-not-in-plastics/ (2020). 21. Koltzenburg, S., Maskos, M. & Nuyken, O. Polymers and the Environment. in Polymer Chemistry 533–549 (Springer Berlin Heidelberg, 2017). doi:10.1007/978- 3-662-49279-6_21. 22. Hopewell, J., Dvorak, R. & Kosior, E. Plastics recycling: challenges and opportunities. Phil. Trans. R. Soc. B 364, 2115–2126 (2009). 23. Zheng, J. & Suh, S. Strategies to reduce the global carbon footprint of plastics. Nature Climate Change 9, 374–378 (2019). 24. Barra, R., Sunday A Leonard, Whaley, C. & Bierbaum, R. Plastics and the Circular Economy - A STAP Document. (2018) doi:10.13140/RG.2.2.11515.57128. 25. Schweitzer, J.-P. et al. Unwrapped: How throwaway plastic is failing to solve Europe’s food waste problem (and what we need to do instead). A study by Zero Waste Europe and Friends of the Earth Europe for the Rethink Plastic Alliance. Institute for European Environmental Policy (IEEP), Brussels. 28 (2018). 26. Allen, S. et al. Atmospheric transport and deposition of microplastics in a remote mountain catchment. Nat. Geosci. 12, 339–344 (2019). 27. Jamieson, A. J. et al. Microplastics and synthetic particles ingested by deep-sea amphipods in six of the deepest marine ecosystems on Earth. R. Soc. open sci. 6, 180667 (2019). 28. Brahney, J., Hallerud, M., Heim, E., Hahnenberger, M. & Sukumaran, S. Plastic rain in protected areas of the United States. Science 368, 1257–1260 (2020). 29. Bucci, K., Tulio, M. & Rochman, C. M. What is known and unknown about the effects of plastic pollution: A meta‐analysis and systematic review. Ecol Appl 30, (2020). 30. Zalasiewicz, J. et al. The geological cycle of plastics and their use as a stratigraphic indicator of the Anthropocene. Anthropocene 13, 4–17 (2016). 31. Danso, D., Chow, J. & Streit, W. R. Plastics: Environmental and Biotechnological Perspectives on Microbial Degradation. Appl Environ Microbiol 85, e01095-19, /aem/85/19/AEM.01095-19.atom (2019). 32. Wei, R. & Zimmermann, W. Microbial enzymes for the recycling of recalcitrant petroleum-based plastics: how far are we? Microbial Biotechnology 10, 1308–1322 (2017). 33. Chamas, A. et al. Degradation Rates of Plastics in the Environment. ACS Sustainable Chem. Eng. 8, 3494–3511 (2020).

34. Lucas, N. et al. Polymer biodegradation: Mechanisms and estimation techniques – A review. Chemosphere 73, 429–442 (2008). 35. Marine Anthropogenic Litter. (Springer International Publishing : Imprint: Springer, 2015). doi:10.1007/978-3-319-16510-3. 36. Ferreira, I., Venâncio, C., Lopes, I. & Oliveira, M. Nanoplastics and marine organisms: What has been studied? Environmental Toxicology and Pharmacology 67, 1–7 (2019). 37. Schwaferts, C., Niessner, R., Elsner, M. & Ivleva, N. P. Methods for the analysis of submicrometer- and nanoplastic particles in the environment. TrAC Trends in Analytical Chemistry 112, 52–65 (2019). 38. Barnes, D. K. A., Galgani, F., Thompson, R. C. & Barlaz, M. Accumulation and fragmentation of plastic debris in global environments. Phil. Trans. R. Soc. B 364, 1985–1998 (2009). 39. Ross, D. E. & Rogoff, M. J. ‘What a waste…’ the World Bank’s call for action. Waste Manag Res 30, 755–757 (2012). 40. Wong, S. L., Ngadi, N., Abdullah, T. A. T. & Inuwa, I. M. Current state and future prospects of plastic waste as source of fuel: A review. Renewable and Sustainable Energy Reviews 50, 1167–1180 (2015). 41. Abdel-Shafy, H. I. & Mansour, M. S. M. Solid waste issue: Sources, composition, disposal, recycling, and valorization. Egyptian Journal of Petroleum 27, 1275–1290 (2018). 42. Lebreton, L. & Andrady, A. Future scenarios of global plastic waste generation and disposal. Palgrave Communications 5, 1–11 (2019). 43. Lau, W. W. Y. et al. Evaluating scenarios toward zero plastic pollution. Science eaba9475 (2020) doi:10.1126/science.aba9475. 44. Agamuthu, P., Mehran, S., Norkhairah, A. & Norkhairiyah, A. Marine debris: A review of impacts and global initiatives. Waste Manag Res 37, 987–1002 (2019). 45. Prata, J. C. et al. Solutions and Integrated Strategies for the Control and Mitigation of Plastic and Microplastic Pollution. Int J Environ Res Public Health 16, (2019). 46. Jambeck, J. R. et al. Plastic waste inputs from land into the ocean. Science 347, 768–771 (2015). 47. Elliott, T., Gillie, H. & Thomson, A. European Union’s plastic strategy and an impact assessment of the proposed directive on tackling single-use plastics items. in Plastic Waste and Recycling 601–633 (Elsevier, 2020). doi:10.1016/B978-0-12- 817880-5.00024-4. 48. Andrady, A. L. Managing Plastic Waste. in Plastics and Environmental Sustainability 255–293 (John Wiley & Sons, Inc, 2015). doi:10.1002/9781119009405.ch9. 49. European Environment Agency. The plastic waste trade in the circular economy — European Environment Agency. https://www.eea.europa.eu/themes/waste/resource-efficiency/the-plastic-waste- trade-in (2016). 50. Huang, Q. et al. Modelling the global impact of China’s ban on plastic waste imports. Resources, Conservation and Recycling 154, 104607 (2020).

51. Katz, C. Piling Up: How China’s Ban on Importing Waste Has Stalled Global Recycling. Yale School of Forestry & Environmental Studies https://e360.yale.edu/features/piling-up-how-chinas-ban-on-importing-waste-has- stalled-global-recycling (2019). 52. D’Amato, A., Paleari, S., Pohjakallio, M., Vanderreydt, I. & Zoboli, R. Plastic waste trade and the environment. https://www.eionet.europa.eu/etcs/etc- wmge/products/etc-reports/plastics-waste-trade-and-the-environment (2019). 53. Gall, M., Wiener, M., Chagas de Oliveira, C., Lang, R. W. & Hansen, E. G. Building a circular plastics economy with informal waste pickers: Recyclate quality, business model, and societal impacts. Resources, Conservation and Recycling 156, 104685 (2020). 54. Van Eygen, E., Feketitsch, J., Laner, D., Rechberger, H. & Fellner, J. Comprehensive analysis and quantification of national plastic flows: The case of Austria. Resources, Conservation and Recycling 117, 183–194 (2017). 55. Van Eygen, E., Laner, D. & Fellner, J. Circular economy of plastic packaging: Current practice and perspectives in Austria. Waste Manag 72, 55–64 (2018). 56. Wierckx, N. et al. Plastic Biodegradation: Challenges and Opportunities. in Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Biodegradation and Bioremediation (ed. Steffan, R. J.) 333–361 (Springer International Publishing, 2019). doi:10.1007/978-3-319-50433-9_23. 57. Ilyas, M. et al. Plastic waste as a significant threat to environment – a systematic literature review. Reviews on Environmental Health 33, 383–406 (2018). 58. Ignatyev, I. A., Thielemans, W. & Vander Beke, B. Recycling of Polymers: A Review. ChemSusChem 7, 1579–1593 (2014). 59. Lee, A. & Liew, M. S. Ecologically derived waste management of conventional plastics. J Mater Cycles Waste Manag 22, 1–10 (2019). 60. Haider, T. P., Völker, C., Kramm, J., Landfester, K. & Wurm, F. R. Plastics of the Future? The Impact of Biodegradable Polymers on the Environment and on Society. Angewandte Chemie - International Edition 58, 50–62 (2019). 61. Pathak, V. M. & Navneet. Review on the current status of polymer degradation: a microbial approach. Bioresour. Bioprocess. 4, 15 (2017). 62. Singh, B. & Sharma, N. Mechanistic implications of plastic degradation. Polymer Degradation and Stability 93, 561–584 (2008). 63. Ghosh, S., Qureshi, A. & Purohit, H. J. Microbial degradation of plastics: Biofilms and degradation pathways. in Contaminants in Agriculture and Environment: Health Risks and Remediation 184–199 (Agro Environ Media - Agriculture and Ennvironmental Science Academy, Haridwar, India, 2019). doi:10.26832/AESA- 2019-CAE-0153-014. 64. Narancic, T. et al. Biodegradable Plastic Blends Create New Possibilities for End-of- Life Management of Plastics but They Are Not a Panacea for Plastic Pollution. Environ. Sci. Technol. 52, 10441–10452 (2018). 65. Narancic, T. & O’Connor, K. E. Plastic waste as a global challenge: are biodegradable plastics the answer to the plastic waste problem? Microbiology, 165, 129–137 (2019).

66. Leja, K. & Lewandowicz, G. Polymer Biodegradation and Biodegradable Polymers – a Review. 12 (2009). 67. Quecholac-Piña, X., Hernández-Berriel, M. del C., Mañón-Salas, M. del C., Espinosa-Valdemar, R. M. & Vázquez-Morillas, A. Degradation of Plastics under Anaerobic Conditions: A Short Review. Polymers 12, 109 (2020). 68. Kalita, N. K., Kalamdhad, A. & Katiyar, V. Recent Trends and Advances in the Biodegradation of Conventional Plastics. in Advances in Sustainable Polymers (eds. Katiyar, V., Kumar, A. & Mulchandani, N.) 389–404 (Springer Singapore, 2020). doi:10.1007/978-981-15-1251-3_17. 69. Ejiofor, A. O. Insect Biotechnology. in Short Views on Insect Genomics and Proteomics (eds. Raman, C., Goldsmith, M. R. & Agunbiade, T. A.) vol. 4 185–210 (Springer International Publishing, 2016). 70. Hoffmann, K. H. Insect biotechnology – a major challenge in the 21st century. Zeitschrift für Naturforschung C 72, 335–336 (2017). 71. Engel, P. & Moran, N. A. The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev 37, 699–735 (2013). 72. Jang, S. & Kikuchi, Y. Impact of the insect gut microbiota on ecology, evolution, and industry. Current Opinion in Insect Science 41, 33–39 (2020). 73. Oliveira, M., Ameixa, O. M. C. C. & Soares, A. M. V. M. Are ecosystem services provided by insects “bugged” by micro (nano)plastics? TrAC Trends in Analytical Chemistry 113, 317–320 (2019). 74. Watanabe, H. & Tokuda, G. Cellulolytic Systems in Insects. Annu. Rev. Entomol. 55, 609–632 (2010). 75. Brune, A. Symbiotic digestion of lignocellulose in termite guts. Nat Rev Microbiol 12, 168–180 (2014). 76. Parra Paz, A. S., Carrejo, N. S. & Gómez Rodríguez, C. H. Effects of Larval Density and Feeding Rates on the Bioconversion of Vegetable Waste Using Black Soldier Fly Larvae Hermetia illucens (L.), (Diptera: Stratiomyidae). Waste Biomass Valor 6, 1059–1065 (2015). 77. Nguyen, T. T. X., Tomberlin, J. K. & Vanlaerhoven, S. Ability of Black Soldier Fly (Diptera: Stratiomyidae) Larvae to Recycle Food Waste. Environmental Entomology 44, 406–410 (2015). 78. Čičková, H., Newton, G. L., Lacy, R. C. & Kozánek, M. The use of fly larvae for organic waste treatment. Waste Management 35, 68–80 (2015). 79. Müller, A., Wolf, D. & Gutzeit, H. O. The black soldier fly, Hermetia illucens – a promising source for sustainable production of proteins, lipids and bioactive substances. Zeitschrift für Naturforschung C 72, 351–363 (2017). 80. Smet, J. D., Wynants, E., Cos, P. & Campenhout, L. V. Microbial Community Dynamics during Rearing of Black Soldier Fly Larvae (Hermetia illucens) and Impact on Exploitation Potential. Appl. Environ. Microbiol. 84, (2018). 81. Liu, C., Wang, C. & Yao, H. Comprehensive Resource Utilization of Waste Using the Black Soldier Fly (Hermetia illucens (L.)) (Diptera: Stratiomyidae). Animals 9, 349 (2019).

82. Xie, S., Lan, Y., Sun, C. & Shao, Y. Insect microbial symbionts as a novel source for biotechnology. World J Microbiol Biotechnol 35, 25 (2019). 83. Rumpold, B. A., Klocke, M. & Schlüter, O. Insect biodiversity: underutilized bioresource for sustainable applications in life sciences. Reg Environ Change 17, 1445–1454 (2017). 84. Berasategui, A., Shukla, S., Salem, H. & Kaltenpoth, M. Potential applications of insect symbionts in biotechnology. Appl Microbiol Biotechnol 100, 1567–1577 (2016). 85. Gold, M., Tomberlin, J. K., Diener, S., Zurbrügg, C. & Mathys, A. Decomposition of biowaste macronutrients, microbes, and chemicals in black soldier fly larval treatment: A review. Waste Management 82, 302–318 (2018). 86. Bulak, P. et al. Hermetia illucens as a new and promising species for use in entomoremediation. Science of The Total Environment 633, 912–919 (2018). 87. Win, S. S. et al. Anaerobic digestion of black solider fly larvae (BSFL) biomass as part of an integrated biorefinery. Renew. Energy 127, 705–712 (2018). 88. Byrne, J. A. Improving the peer review of narrative literature reviews. Res Integr Peer Rev 1, 12 (2016). 89. Ortega, J. L. Final remarks. in Academic Search Engines 179–181 (Elsevier, 2014). doi:10.1533/9781780634722.179. 90. Atkinson, K. M., Koenka, A. C., Sanchez, C. E., Moshontz, H. & Cooper, H. Reporting standards for literature searches and report inclusion criteria: making research syntheses more transparent and easy to replicate: Reporting Standards for Literature Searches. Res. Syn. Meth. 6, 87–95 (2015). 91. Kesti, S. S. & C.T., S. The Role of Insects and Microorganisms in Plastic Biodegradation A Comprehensive Review. IJSRBS 5, 75–79 (2019). 92. Singh, P. & Jerram, E. M. Plastic Damage by Insects. New Zealand Entomologist 6, 188–188 (1976). 93. Gerhardt, P. D. & Lindgren, D. L. Penetration of packaging films: Film materials used for food packaging tested for resistance to some common stored-product insects. Calif. Agric. 8, 3–4 (1954). 94. Riudavets, J., Salas, I. & Pons, M. J. Damage characteristics produced by insect pests in packaging film. Journal of Stored Products Research 43, 564–570 (2007). 95. Hassan, M. W. et al. Evaluation of Standard Loose Plastic Packaging for the Management of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebriondiae). J Insect Sci 16, (2016). 96. This caterpillar can digest plastic. Nature 545, 8–8 (2017). 97. Sharma, B., Rawat, H., ja, P. & Sharma, R. Bioremediation - A Progressive Approach toward Reducing Plastic Wastes. Int.J.Curr.Microbiol.App.Sci 6, 1116– 1131 (2017). 98. Ewuim, S. C. Entomoremediation - A Novel In-Situ Bioremediation Approach. Animal Research International 10, 1681–1684 (2013).

99. Yang, J., Yang, Y., Wu, W.-M., Zhao, J. & Jiang, L. Evidence of Polyethylene Biodegradation by Bacterial Strains from the Guts of Plastic-Eating Waxworms. Environ. Sci. Technol. 48, 13776–13784 (2014). 100. Yang, Y. et al. Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms: Part 1. Chemical and Physical Characterization and Isotopic Tests. Environ. Sci. Technol. 49, 12080–12086 (2015). 101. Chen, G.-Z. et al. Gut microbiota of polystyrene-eating mealworms analyzed by high-throughput sequencing. /paper/Gut-microbiota-of-polystyrene-eating- mealworms-by-Guan-Zhou-Bai-lu/3838f9cdf29551542c6788ec5c8b1469e118b787 (2017). 102. Yang, S.-S. et al. Biodegradation of polystyrene wastes in yellow mealworms (larvae of Tenebrio molitor Linnaeus): Factors affecting biodegradation rates and the ability of polystyrene-fed larvae to complete their life cycle. Chemosphere 191, 979–989 (2018). 103. Yang, S.-S. et al. Ubiquity of polystyrene digestion and biodegradation within yellow mealworms, larvae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). Author (2018). 104. Nukmal, N., Umar, S., Amanda, S. P. & Kanedi, M. Effect of Styrofoam Waste Feeds on the Growth, Development and Fecundity of Mealworms (Tenebrio molitor). OnLine Journal of Biological Sciences 18, 24–28 (2018). 105. Brandon, A. M. et al. Fate of Hexabromocyclododecane (HBCD), A Common Flame Retardant, In Polystyrene-Degrading Mealworms: Elevated HBCD Levels in Egested Polymer but No Bioaccumulation. Environ. Sci. Technol. acs.est.9b06501 (2019) doi:10.1021/acs.est.9b06501. 106. Aboelkheir, M. G., Visconte, L. Y., Oliveira, G. E., Toledo Filho, R. D. & Souza, F. G. The biodegradative effect of Tenebrio molitor Linnaeus larvae on vulcanized SBR and tire crumb. Science of The Total Environment 649, 1075–1082 (2019). 107. Brandon, A. M. et al. Biodegradation of Polyethylene and Plastic Mixtures in Mealworms (Larvae of Tenebrio molitor ) and Effects on the Gut Microbiome. Environ. Sci. Technol. 52, 6526–6533 (2018). 108. Wu, Q., Tao, H. & Wong, M. H. Feeding and metabolism effects of three common microplastics on Tenebrio molitor L. Environ Geochem Health 41, 17–26 (2019). 109. Guo, B., Yin, J., Hao, W. & Jiao, M. Polyurethane foam induces epigenetic modification of mitochondrial DNA during different metamorphic stages of Tenebrio molitor. Ecotoxicology and Environmental Safety 183, 109461 (2019). 110. Peña-Pascagaza, P. M., López-Ramírez, N. A. & Ballen-Segura, M. A. Tenebrio molitor and its gut bacteria growth in polystyrene (PS) presence as the sole source carbon. Universitas Scientiarum 25, 37–53 (2020). 111. Peng, B.-Y. et al. Biodegradation of Polystyrene by Dark ( Tenebrio obscurus ) and Yellow ( Tenebrio molitor ) Mealworms (Coleoptera: Tenebrionidae). Environ. Sci. Technol. 53, 5256–5265 (2019). 112. Yang, Y. et al. Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms: Part 2. Role of Gut Microorganisms. Environ. Sci. Technol. 49, 12087– 12093 (2015).

113. Yang, Y., Wang, J. & Xia, M. Biodegradation and mineralization of polystyrene by plastic-eating superworms Zophobas atratus. Science of The Total Environment 708, 135233 (2019). 114. Wang, Z., Xin, X., Shi, X. & Zhang, Y. A polystyrene-degrading Acinetobacter bacterium isolated from the larvae of Tribolium castaneum. Science of The Total Environment 726, 138564 (2020). 115. Przemieniecki, S. W., Kosewska, A., Ciesielski, S. & Kosewska, O. Changes in the gut microbiome and enzymatic profile of Tenebrio molitor larvae biodegrading cellulose, polyethylene and polystyrene waste. Environmental Pollution 256, 113265 (2020). 116. Urbanek, A. K., Rybak, J., Wróbel, M., Leluk, K. & Mirończuk, A. M. A comprehensive assessment of microbiome diversity in Tenebrio molitor fed with polystyrene waste. Environmental Pollution 262, 114281 (2020). 117. Tang, Z.-L., Kuo, T.-A. & Liu, H.-H. The Study of the Microbes Degraded Polystyrene. Advances in Technology Innovation 2, 13–17 (2017). 118. Suzuki, K. et al. Affinity purification and characterization of a biodegradable plastic- degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis. Appl Microbiol Biotechnol 97, 7679–7688 (2012). 119. Yang, S. S. et al. Progresses in Polystyrene Biodegradation and Prospects for Solutions to Plastic Waste Pollution. IOP Conf. Ser.: Earth Environ. Sci. 150, 012005 (2018). 120. Thakur, S. et al. Sustainability of bioplastics: Opportunities and challenges. Current Opinion in Green and Sustainable Chemistry 13, 68–75 (2018). 121. Wilson, M. C. L. The morphology and mechanism of the pupal gin-traps of Tenebrio molitor L. (Coleoptera, Tenebrionidae). Journal of Stored Products Research 7, 21–30 (1971). 122. Wynants, E. et al. Effect of post-harvest starvation and rinsing on the microbial numbers and the bacterial community composition of mealworm larvae ( Tenebrio molitor ). Innovative Food Science & Emerging Technologies 42, 8–15 (2017). 123. Jorjão, A. L. et al. From moths to caterpillars: Ideal conditions for Galleria mellonella rearing for in vivo microbiological studies. Virulence 9, 383–389 (2018). 124. Maguire, R., Kelly, S. & Kavanagh, K. Lepidoptera as Models for Studying Fungal Disease. in Reference Module in Life Sciences B9780128096338120000 (Elsevier, 2017). doi:10.1016/B978-0-12-809633-8.12068-0. 125. Francardi, V. et al. Galleria mellonella (Lepidoptera Pyralidae): an edible insect of nutraceutical interest. 100, 6 (2017). 126. Johnson, J. Pest control in postharvest nuts. in Improving the Safety and Quality of Nuts 56–87 (Elsevier, 2013). doi:10.1533/9780857097484.1.56. 127. Ellis, J. D., Graham, J. R. & Mortensen, A. Standard methods for wax moth research. Journal of Apicultural Research 52, 1–17 (2013). 128. Vasileva, A. V. et al. Comparative Analysis of Biodamage of Various Polyethylene Types by Galleria mellonella (Insecta, Lepidoptera, Pyralidae) Larvae. Povolžskij èkologičeskij žurnal 17–27 (2019) doi:10.35885/1684-7318-2019-1-17-27.

129. Kundungal, H., Gangarapu, M., Sarangapani, S., Patchaiyappan, A. & Devipriya, S. P. Role of pretreatment and evidence for the enhanced biodegradation and mineralization of low-density polyethylene films by greater waxworm. Environmental Technology 1–14 (2019) doi:10.1080/09593330.2019.1643925. 130. Kong, H. G. et al. The Galleria mellonella Hologenome Supports Microbiota- Independent Metabolism of Long-Chain Hydrocarbon Beeswax. Cell Reports 26, 2451-2464.e5 (2019). 131. Bombelli, P., Howe, C. J. & Bertocchini, F. Polyethylene bio-degradation by caterpillars of the wax moth Galleria mellonella. Current Biology 27, R292–R293 (2017). 132. Cassone, B. J., Grove, H. C., Elebute, O., Villanueva, S. M. P. & LeMoine, C. M. R. Role of the intestinal microbiome in low-density polyethylene degradation by caterpillar larvae of the greater wax moth, Galleria mellonella. Proc. R. Soc. B. 287, 20200112 (2020). 133. Zhang, J. et al. Biodegradation of polyethylene microplastic particles by the fungus Aspergillus flavus from the guts of wax moth Galleria mellonella. Science of The Total Environment 704, 135931 (2019). 134. Ren, L. et al. Biodegradation of Polyethylene by Enterobacter sp. D1 from the Guts of Wax Moth Galleria mellonella. IJERPH 16, 1941 (2019). 135. Peydaei, A., Bagheri, H., Gurevich, L., de Jonge, N. & Nielsen, J. L. Impact of polyethylene on salivary glands proteome in Galleria melonella. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 34, 100678 (2020). 136. Lou, Y. et al. Biodegradation of Polyethylene and Polystyrene by Greater Wax Moth Larvae (Galleria mellonella L.) and the Effect of Co-diet Supplementation on the Core Gut Microbiome. Environ. Sci. Technol. 54, 2821–2831 (2020). 137. Chalup, A. et al. First report of the lesser wax moth Achroia grisella F. (Lepidoptera: Pyralidae) consuming polyethylene (silo-bag) in northwestern Argentina. Journal of Apicultural Research 57, 569–571 (2018). 138. Kundungal, H., Gangarapu, M., Sarangapani, S., Patchaiyappan, A. & Devipriya, S. P. Efficient biodegradation of polyethylene (HDPE) waste by the plastic-eating lesser waxworm (Achroia grisella). Environ Sci Pollut Res 26, 18509–18519 (2019). 139. Yang, Y., Chen, J., Wu, W.-M., Zhao, J. & Yang, J. Complete genome sequence of Bacillus sp. YP1, a polyethylene-degrading bacterium from waxworm’s gut. Journal of Biotechnology 200, 77–78 (2015). 140. Suresh Kesti, S. & Chandrabanda Thimmappa, S. First report on biodegradation of low density polyethyleneby rice moth larvae, Corcyra cephalonica (stainton). Holist. approach environ. 9, 79–83 (2019). 141. Kawai, F. The Biochemistry and Molecular Biology of Xenobiotic Polymer Degradation by Microorganisms. Bioscience, Biotechnology, and Biochemistry 74, 1743–1759 (2010). 142. Varsha, Y. M., Deepthi CH, N. & Chenna, S. An Emphasis on Xenobiotic Degradation in Environmental Clean up. J Bioremed Biodegrad 02, (2011). 143. Embrandiri, A., Kiyasudeen, S. K., Rupani, P. F. & Ibrahim, M. H. Environmental Xenobiotics and Its Effects on Natural Ecosystem. in Plant Responses to

Xenobiotics (eds. Singh, A., Prasad, S. M. & Singh, R. P.) 1–18 (Springer Singapore, 2016). doi:10.1007/978-981-10-2860-1_1. 144. Tinto, W. F., Elufioye, T. O. & Roach, J. Waxes. in Pharmacognosy 443–455 (Elsevier, 2017). doi:10.1016/B978-0-12-802104-0.00022-6. 145. Allotey, J. & Azalekor, W. Some aspects of the biology and control using botanicals of the rice moth, Corcyra cephalonica (Stainton), on some pulses. Journal of Stored Products Research 36, 235–243 (2000). 146. Al-Jaibachi, R., Cuthbert, R. N. & Callaghan, A. Up and away: ontogenic transference as a pathway for aerial dispersal of microplastics. Biol Lett 14, (2018). 147. Cho, S., Kim, C.-H., Kim, M.-J. & Chung, H. Effects of microplastics and salinity on food waste processing by black soldier fly (Hermetia illucens) larvae. j ecology environ 44, 7 (2020). 148. Rockström, J. et al. Planetary Boundaries: Exploring the Safe Operating Space for Humanity. E&S 14, art32 (2009). 149. Villarrubia-Gómez, P., Cornell, S. E. & Fabres, J. Marine plastic pollution as a planetary boundary threat – The drifting piece in the sustainability puzzle. Marine Policy 96, 213–220 (2018). 150. Jahnke, A. et al. Reducing Uncertainty and Confronting Ignorance about the Possible Impacts of Weathering Plastic in the Marine Environment. Environ. Sci. Technol. Lett. 4, 85–90 (2017). 151. Lithner, D. Environmental and health hazards of chemicals in plastic polymers and products. (Department of Plant and Environmental Science, Faculty of Science, University of Gothenburg, 2011). 152. Marine Anthropogenic Litter. (Springer International Publishing : Imprint: Springer, 2015). doi:10.1007/978-3-319-16510-3. 153. Akbari, M., Khodayari, M., Danesh, M., Davari, A. & Padash, H. A bibliometric study of sustainable technology research. Cogent Business & Management 7, (2020). 154. Vegter, A. et al. Global research priorities to mitigate plastic pollution impacts on marine wildlife. Endang. Species. Res. 25, 225–247 (2014). 155. Rochman, C. M. et al. Classify plastic waste as hazardous. Nature 494, 169–171 (2013). 156. Shahnawaz, Mohd., Sangale, M. K. & Ade, A. B. Case Studies and Recent Update of Plastic Waste Degradation. in Bioremediation Technology for Plastic Waste 31–43 (Springer Singapore, 2019). doi:10.1007/978-981-13-7492-0_4. 157. Bioremediation of Industrial Waste for Environmental Safety: Volume I: Industrial Waste and Its Management. (Springer Singapore : Imprint: Springer, 2020). doi:10.1007/978-981-13-1891-7. 158. Yogalakshmi, K. N. & Singh, S. Plastic Waste: Environmental Hazards, Its Biodegradation, and Challenges. in Bioremediation of Industrial Waste for Environmental Safety (eds. Saxena, G. & Bharagava, R. N.) 99–133 (Springer Singapore, 2020). doi:10.1007/978-981-13-1891-7_6. 159. Ru, J., Huo, Y. & Yang, Y. Microbial Degradation and Valorization of Plastic Wastes. Front. Microbiol. 11, 442 (2020).

160. Montazer, Z., Habibi Najafi, M. B. & Levin, D. B. Challenges with Verifying Microbial Degradation of Polyethylene. Polymers 12, 123 (2020). 161. Wierckx, N. et al. Plastic waste as a novel substrate for industrial biotechnology. Microbial Biotechnology 8, 900–903 (2015). 162. Ho, B. T., Roberts, T. K. & Lucas, S. An overview on biodegradation of polystyrene and modified polystyrene: the microbial approach. Critical Reviews in Biotechnology 38, 308–320 (2018). 163. Mierzwa-Hersztek, M., Gondek, K. & Kopeć, M. Degradation of Polyethylene and Biocomponent-Derived Polymer Materials: An Overview. J Polym Environ 27, 600– 611 (2019). 164. Emadian, S. M., Onay, T. T. & Demirel, B. Biodegradation of bioplastics in natural environments. Waste Management 59, 526–536 (2017). 165. Restrepo-Flórez, J.-M., Bassi, A. & Thompson, M. R. Microbial degradation and deterioration of polyethylene – A review. International Biodeterioration & Biodegradation 88, 83–90 (2014). 166. Lambert, S. & Wagner, M. Environmental performance of bio-based and biodegradable plastics: the road ahead. Chem. Soc. Rev. 46, 6855–6871 (2017). 167. Handbook of biodegradable polymers: synthesis, characterization, and applications. (Wiley-VCH, 2011). 168. Shah, A. A., Hasan, F., Hameed, A. & Ahmed, S. Biological degradation of plastics: A comprehensive review. Biotechnology Advances 26, 246–265 (2008). 169. Brandon, A. M. & Criddle, C. S. Can biotechnology turn the tide on plastics? Current Opinion in Biotechnology 57, 160–166 (2019). 170. Licciardello, F. Packaging, blessing in disguise. Review on its diverse contribution to food sustainability. Trends in Food Science & Technology 65, 32–39 (2017). 171. Hermann, B. G., Debeer, L., De Wilde, B., Blok, K. & Patel, M. K. To compost or not to compost: Carbon and energy footprints of biodegradable materials’ waste treatment. Polymer Degradation and Stability 96, 1159–1171 (2011). 172. Sintim, H. Y. et al. Release of micro- and nanoparticles from biodegradable plastic during in situ composting. Science of The Total Environment 675, 686–693 (2019). 173. Amos, C., Allred, A. & Zhang, L. Do Biodegradable Labels Lead to an Eco-safety Halo Effect? J Consum Policy 40, 279–298 (2017). 174. Viera, J. S. C., Marques, M. R. C., Nazareth, M. C., Jimenez, P. C. & Castro, Í. B. On replacing single-use plastic with so-called biodegradable ones: The case with straws. Environmental Science & Policy 106, 177–181 (2020). 175. Wojnowska-Baryła, I., Kulikowska, D. & Bernat, K. Effect of Bio-Based Products on Waste Management. Sustainability 12, 2088 (2020). 176. Bátori, V., Åkesson, D., Zamani, A., Taherzadeh, M. J. & Sárvári Horváth, I. Anaerobic degradation of bioplastics: A review. Waste Management 80, 406–413 (2018). 177. Nemat, B., Razzaghi, M., Bolton, K. & Rousta, K. The Potential of Food Packaging Attributes to Influence Consumers’ Decisions to Sort Waste. Sustainability 12, 2234 (2020).

178. Nielsen, T. D., Hasselbalch, J., Holmberg, K. & Stripple, J. Politics and the plastic crisis: A review throughout the plastic life cycle. WIREs Energy and Environment 9, e360 (2020). 179. Changwichan, K., Silalertruksa, T. & Gheewala, S. Eco-Efficiency Assessment of Bioplastics Production Systems and End-of-Life Options. Sustainability 10, 952 (2018). 180. Tokiwa, Y., Calabia, B. P., Ugwu, C. U. & Aiba, S. Biodegradability of Plastics. Int J Mol Sci 10, 3722–3742 (2009). 181. Koch Daniel & Mihalyi Bettina. Assessing the change in environmental impact categories when replacing conventional plastic with bioplastic in chosen application fields. Chemical Engineering Transactions 70, 853–858 (2018). 182. Gironi, F. & Piemonte, V. Bioplastics and Petroleum-based Plastics: Strengths and Weaknesses. Energy Sources, Part A: Recovery, Utilization, and Environmental Effects 33, 1949–1959 (2011). 183. Jabeen, N., Majid, I. & Nayik, G. A. Bioplastics and food packaging: A review. Cogent Food & Agriculture 1, (2015). 184. Rossi, V. et al. Life cycle assessment of end-of-life options for two biodegradable packaging materials: sound application of the European waste hierarchy. Journal of Cleaner Production 86, 132–145 (2015). 185. Scott, J. L. & Johns, M. A. Designing and Synthesizing Materials with Appropriate Lifetimes. in Encyclopedia of Sustainability Science and Technology (ed. Meyers, R. A.) 1–29 (Springer New York, 2018). doi:10.1007/978-1-4939-2493-6_1016-1. 186. Razza, F. & Innocenti, F. D. Bioplastics from renewable resources: the benefits of biodegradability: BIODEGRADABLE BIOPLASTICS. Asia-Pac. J. Chem. Eng. 7, S301–S309 (2012). 187. Ren, X. Biodegradable plastics: a solution or a challenge? Journal of Cleaner Production 11, 27–40 (2003). 188. Markowicz, F. & Szymańska-Pulikowska, A. Analysis of the Possibility of Environmental Pollution by Composted Biodegradable and Oxo-Biodegradable Plastics. Geosciences 9, 460 (2019). 189. Johnson, K. E., Pometto, A. L., Somasundaram, L. & Coats, J. Microtox assay to determine the toxicity of degradation products from degradable plastics. J Environ Polym Degr 1, 111–116 (1993). 190. Kubowicz, S. & Booth, A. M. Biodegradability of Plastics: Challenges and Misconceptions. Environ. Sci. Technol. 51, 12058–12060 (2017). 191. Piemonte, V. & Gironi, F. Bioplastics and GHGs Saving: The Land Use Change (LUC) Emissions Issue. Energy Sources, Part A: Recovery, Utilization, and Environmental Effects 34, 1995–2003 (2012). 192. Ojeda, T. Polymers and the Environment. in Polymer Science (ed. Ylmaz, F.) (InTech, 2013). doi:10.5772/51057. 193. Pischedda, A., Tosin, M. & Degli-Innocenti, F. Biodegradation of plastics in soil: The effect of temperature. Polymer Degradation and Stability 170, 109017 (2019).

194. Canopoli, L., Coulon, F. & Wagland, S. T. Degradation of excavated polyethylene and polypropylene waste from landfill. Science of The Total Environment 698, 134125 (2020). 195. van der Zee, M. Analytical Methods for Monitoring Biodegradation Processes of Environmentally Degradable Polymers. in Handbook of Biodegradable Polymers (eds. Lendlein, A. & Sisson, A.) 263–281 (Wiley-VCH Verlag GmbH & Co. KGaA, 2011). doi:10.1002/9783527635818.ch11. 196. Ihms, E. C. & Brinkman, D. W. Thermogravimetric Analysis as a Polymer Identification Technique in Forensic Applications. J. Forensic Sci. 49, 1–6 (2004). 197. Calmon-Decriaud, A., Bellon-Maurel, V. & Silvestre, F. Standard Methods for Testing the Aerobic Biodegradation of Polymeric Materials. Review and Perspectives. in Blockcopolymers - Polyelectrolytes - Biodegradation (eds. Bellon- Maurel, V. et al.) vol. 135 207–226 (Springer Berlin Heidelberg, 1998). 198. Niaounakis, M. Definitions and Assessment of (Bio)degradation. in Biopolymers Reuse, Recycling, and Disposal 77–94 (Elsevier, 2013). doi:10.1016/B978-1-4557- 3145-9.00002-6. 199. Yang, Y., Yang, J. & Jiang, L. Comment on “A bacterium that degrades and assimilates poly(ethylene terephthalate)”. Science 353, 759–759 (2016). 200. Yoshida, S. et al. Response to Comment on “A bacterium that degrades and assimilates poly(ethylene terephthalate)”. Science 353, 759–759 (2016). 201. Jang, C., Chen, L. & Rabinowitz, J. D. Metabolomics and Isotope Tracing. Cell 173, 822–837 (2018). 202. Taipale, S. J. et al. Tracing the fate of microplastic carbon in the aquatic food web by compound-specific isotope analysis. Sci Rep 9, 19894 (2019). 203. Chaves, M. et al. A Practical Fluorescence-Based Screening Protocol for Polyethylene Terephthalate Degrading Microorganisms. J. Braz. Chem. Soc. (2017) doi:10.21577/0103-5053.20170224. 204. Chrzanowski, W. & Dehghani, F. 9 - Standardised chemical analysis and testing of biomaterials. in Standardisation in Cell and Tissue Engineering (ed. Salih, V.) 166– 197a (Woodhead Publishing, 2013). doi:10.1533/9780857098726.2.166. 205. Skariyachan, S., Manjunath, M., Shankar, A., Bachappanavar, N. & Patil, A. A. Application of Novel Microbial Consortia for Environmental Site Remediation and Hazardous Waste Management Toward Low- and High-Density Polyethylene and Prioritizing the Cost-Effective, Eco-friendly, and Sustainable Biotechnological Intervention. in Handbook of Environmental Materials Management (ed. Hussain, C. M.) 431–478 (Springer International Publishing, 2019). doi:10.1007/978-3-319- 73645-7_9. 206. Philp, J. OECD Policies for Bioplastics in the Context of a Bioeconomy, 2013. Industrial Biotechnology 10, 19–21 (2014). 207. Juergen Bertling, Hamann, L. & Bertling, R. Kunststoffe in der Umwelt. (2018) doi:10.24406/UMSICHT-N-497117. 208. Degli Innocenti, F. & Breton, T. Intrinsic Biodegradability of Plastics and Ecological Risk in the Case of Leakage. ACS Sustainable Chem. Eng. 8, 9239–9249 (2020).

209. Wohner, B., Schwarzinger, N., Gürlich, U., Heinrich, V. & Tacker, M. Technical emptiability of dairy product packaging and its environmental implications in Austria. PeerJ 7, e7578 (2019). 210. Tallentire, C. W. & Steubing, B. The environmental benefits of improving packaging waste collection in Europe. Waste Manag 103, 426–436 (2020). 211. Yun, J.-H. et al. Insect Gut Bacterial Diversity Determined by Environmental Habitat, Diet, Developmental Stage, and Phylogeny of Host. Appl. Environ. Microbiol. 80, 5254–5264 (2014). 212. Moran, N. A. Symbiosis. Current Biology 16, R866–R871 (2006). 213. Copley, S. D. Shining a light on enzyme promiscuity. Current Opinion in Structural Biology 47, 167–175 (2017). 214. Newton, M. S., Arcus, V. L., Gerth, M. L. & Patrick, W. M. Enzyme evolution: innovation is easy, optimization is complicated. Current Opinion in Structural Biology 48, 110–116 (2018). 215. Terra, W. R., Ferreira, C. & Baker, J. E. Compartmentalization of digestion. in Biology of the Insect Midgut (eds. Lehane, M. J. & Billingsley, P. F.) 206–235 (Springer Netherlands, 1996). doi:10.1007/978-94-009-1519-0_8. 216. Sheth, M. U. et al. Bioengineering a Future Free of Marine Plastic Waste. Front. Mar. Sci. 6, (2019). 217. Wang, H. et al. Insect biorefinery: a green approach for conversion of crop residues into biodiesel and protein. Biotechnology for Biofuels 10, 304 (2017). 218. Barbi, S., Spinelli, R., Ferrari, A. M. & Montorsi, M. Design and environmental assessment of bioplastics from hermetia illucens prepupae proteins. Environmental Engineering and Management Journal 18, 2123–2131 (2019). 219. Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016). 220. Mohanta, T. K., Mohanta, Y. K. & Mohanta, N. Role of biotechnology in bioremediation. in Handbook of Research on Uncovering New Methods for Ecosystem Management Through Bioremediation 399–432 (2015). doi:10.4018/978-1-4666-8682-3.ch016. 221. de Lorenzo, V. Systems biology approaches to bioremediation. Current Opinion in Biotechnology 19, 579–589 (2008). 222. Pazos, F., Guijas, D., Valencia, A. & De Lorenzo, V. MetaRouter: Bioinformatics for bioremediation. Nucleic Acids Research 33, D588–D592 (2005). 223. Straalen, N. M. V. Peer Reviewed: Ecotoxicology Becomes Stress Ecology. Environ. Sci. Technol. 37, 324A-330A (2003). 224. Zhang, X. Environmental DNA Shaping a New Era of Ecotoxicological Research. Environ. Sci. Technol. 53, 5605–5612 (2019). 225. Shahnawaz, M., Sangale, M. K. & Ade, A. B. Bioremediation Technology for Plastic Waste. (Springer, 2019).

FINAL APPENDIX

Statutory declaration:

I hereby declare on oath, as evidenced by my handwritten signature, that I have written the present thesis independently using nothing other than the indicated sources and aids. All content that was taken literally or in content from other sources is specified as such within the thesis. The present thesis has not yet been submitted in the same or similar form as a Magister- /Master-/Diplomarbeit/Dissertation.

29/10/20 Date Signature