Download PDF Winter Bridge on Frontiers of Engineering December 15, 2022 Volume 52 Issue 4 From novel applications of microbes to DEI in engineering to the potential for hydrogen energy, Frontiers of Engineering participants tackle today’s challenging world issues. The winter issue of The Bridge showcases research by early-career engineers as shared at the 2022 US FOE symposium. "Life...Finds a Way": Sustainable Capture and Upcycling of Plastics by Microbes Thursday, December 15, 2022 Author: Ross R. Klauer, Mark A. Blenner, and Kevin V. Solomon Advances in synthetic biology and machine learning enable new engineering approaches to enhance plastic degradation via enzymes. Plastics, widely used for their low cost and durability, pose a grand environmental challenge. Every year, more than 380 million tonnes of plastics are produced globally, using 6 percent of produced petroleum (Zhu et al. 2016). Less than 10 percent of these materials are reused or recycled, leading to significant waste accumulation and environmental pollution; for example, an estimated 8 million tonnes of plastic leak into ocean systems each year (Geyer et al. 2017). Plastic-related environmental damage is estimated to cost at least $75 billion annually. Most of the damage is attributed to waste from consumer packaging, encompassing plastics such as polystyrene (PS), high- and low-density polyethylene (HDPE and LDPE), and polypropylene (PP) (Geyer et al. 2017; MacArthur et al. 2016; UNEP 2014). Lack of Infrastructure for Recycling Plastics A small fraction of total plastics is recycled because of a lack of economical solutions. Many consumer plastics are thermoplastics, which in theory can be infinitely melted and recycled into new plastic products in a process called mechanical recycling. However, these plastics tend to degrade in quality with each round of recycling because of contamination with dyes, labels, and the materials they once contained. These recycling limitations may be mitigated to a degree by proper sorting, cleaning, and/or chemical pretreatment, but these steps are labor-intensive, costly, and don’t work in practice (Li et al. 2022). Chemical recycling, in which plastics are chemically converted to other materials, is energy-intensive and similarly sensitive to contaminants, which can poison the catalysts that mediate the chemical conversion and frequently produces low-value products (Rahimi and García 2017). In light of these challenges, there is a critical need for new technologies that can process or sort mixed plastics waste streams and depolymerize the most abundant plastic wastes (HDPE, LDPE, PP, and PS) into valuable “upcycled” commodity chemicals. Using Biology to Degrade and Upcycle Plastics Biological systems, namely microorganisms, have evolved over millions of years to thrive in their native environment by capturing and converting available carbon, hydrogen, nitrogen, and oxygen, along with other trace elements. More importantly, they do this in environments with a complex mixture of “food,” toxins, and “nonfoods,” effortlessly sorting needed nutrients from other environmental components. Degradation In resource-poor environments, “life finds a way” by evolving novel enzymes or biomolecular protein-based catalysts that can assimilate carbon from even recalcitrant materials such as plastics. Microbes have been reported to degrade HDPE (Devi et al. 2015), LDPE (Sen and Raut 2015), PP (Jeon and Kim 2016), and PS (Ho et al. 2017). These microbial partners, frequently viewed as nuisances or pathogens, offer new hope for the development of sustainable recycling of plastics. The use of microbial systems to deconstruct plastics would mitigate the high energy requirements of chemical recycling with near-ambient processing and could improve economics by eliminating the need for expensive metal catalysts, sorting, and perhaps pretreatment. Upcycling via Synthetic Biology Biology can not only degrade recalcitrant materials such as plastic, it can upcycle or convert them into new products via cellular metabolism. Biological systems leverage complex metabolic pathways, or biochemical reaction networks, to convert inputs like plastic into energy and cellular building blocks that frequently resemble industrial chemicals. These chemical transformations are mediated by enzymes encoded in genes that form a part of an organism’s DNA. Metabolic engineers introduce new enzymes to this network, remove others, or balance the abundance of enzymes to drive carbon through the pathways to specific products. These changes are frequently implemented with tools from the field of synthetic biology (synbio) to build efficient microbial cell factories. For example, other recalcitrant substrates such as lignocellulose found in agricultural residues have been upcycled by microbial systems into value-added products such as biofuels, fine chemicals (e.g., fragrances), and commodity chemicals such as ethyl acetate (Hillman et al. 2021; Ragauskas et al. 2014). Microbes, frequently viewed as nuisances or pathogens, offer new hope for the development of sustainable recycling of plastics. Aside from waste upcycling, synbio is involved in nearly all aspects of modern life, from products such as cold-active laundry detergents, alternative “meat” burgers, thin films used for touchscreens, and cancer treatments (Voigt 2020). Discovering and Engineering Enzymes for Plastic Degradation Utility and Limitations of Plastic-Active Enzymes The discovery of enzymes that can break up carbon-exclusive polymer backbones is paramount, as this first degradative step is the most chemically difficult processing step because of the strength of carbon-carbon bonds that make up plastic polymers. Some progress has been made toward the isolation of efficient enzymes for the degradation of other plastics. For example, PETases that degrade polyethylene terephthalate (PET) were first identified seven years ago from microbes that lived in plastic-contaminated soils (Yoshida et al. 2016). The activity of this enzyme is enhanced at higher temperature, degrading plastic wastes in hours and days rather than in decades when no enzyme is used (Son et al. 2019). But these operating conditions also degrade the enzyme over time, requiring periodic and expensive inputs of fresh enzyme. Recent advances in machine learning and mechanistic insight into how protein structure controls performance have been leveraged to tweak the initial discovery and make supercharged PETases that are almost 40 times more efficient and stable even at elevated temperatures (Lu et al. 2022). Plastic-Eating Insect Larvae Despite the successes of PETases, they are unable to degrade plastics such as HDPE, LDPE, PP, and PS, which account for the majority of all postconsumer wastes. Enzymes for these nonhydrolysable plastics remain elusive, but a handful of biological systems have been reported to degrade these materials. The most promising among them include insect larvae such as the yellow mealworm (the larvae of flour beetles) that can consume plastics such as PS as their primary nutritional source, even when they contained toxic components (Brandon et al. 2020; Yang et al. 2015a,b). The microbes living in the gut of plastic-eating insect larvae are essential for the organism to consume plastic and express enzymes central to plastics degradation (Yang et al. 2015b). To identify these promising new enzymes, the Solomon and Blenner labs at the University of Delaware are isolating and studying microbes from the gut microbiome of the yellow mealworm (Tenebrio molitor larvae). We have assembled a growing library of plastic-degrading microbes that we are studying through integrated systems biology and synbio approaches (figure 1). With data science, we are sorting through these massive datasets to discover novel enzymes that can efficiently tackle the plastics crisis and deploy these enzymes in new microbial factories to sustainably produce needed medicines, fuels, materials, and chemicals. Questions to Be Addressed Although microbial systems offer exciting promise for a sustainable future, many questions remain before bringing this technology to industrial scale, such as: Which microbes can process waste plastics? Which enzymes degrade plastics such as HDPE, LDPE, PP, and PS? What is the chemical fate of plastics that are degraded by microbes? What is the molecular mechanism for enzymatic plastic degradation? How can enzymes be engineered to enhance the rate of degradation? How can microbes be engineered to produce needed materials, medicines, and chemicals? How do microbes collaborate or work together to efficiently degrade plastics? What process equipment is needed to support industrial-level scale-up of plastic handling and upcycling? Conclusion Microbes from diverse environments such as landfills and the digestive tracts of worms have evolved to express enzymes that can break down difficult substrates, like plastic, for sustenance. Advances in data science and molecular sciences such as next-generation sequencing have greatly accelerated enzyme discovery. Parallel advances in synthetic biology and machine learning enable new engineering approaches to enhance plastic degradation via these enzymes. Synthetic biology can extend plastic degradation to upcycling by rewiring an organism to biochemically transform its carbon source (plastic) into a useful industrial compound. Coupling biological tools with engineering principles, this technology can be scaled for large-scale plastics upcycling. Using microbes to degrade plastic, an industrial process can be developed that is self-sorting, with different microbial processes tied to individual plastics. Moreover, leveraging biology provides the potential for an economic plastic waste handling solution by eliminating the need for mechanical sorting, reducing energy costs by operating near ambient conditions, and turning plastics into higher-value materials. 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About the Author:Ross Klauer is a PhD student, Mark Blenner an associate professor, and Kevin Solomon an assistant professor, all in the Chemical & Biomolecular Engineering Department, University of Delaware.