Engineering Wild Wastewater Microbes to Degrade PET Plastics

Microplastics are an emerging contaminant of concern that pose risks to aquatic ecosystems and human health. Wastewater treatment plants are a major pathway for microplastics to enter natural water bodies and soil, and there are currently no unit operations designed to explicitly handle microplastic separation or degradation in wastewater treatment plants. With a focus on PET plastics, we propose a bold solution to removing microplastics in wastewater: engineering wastewater microbiomes to biodegrade PET plastics. In this project, we sought to engineer bacteria from a wastewater sample to produce FAST-PETase, an engineered PET hydrolase that can fully depolymerize PET plastics within approximately one week at mesophilic conditions. We developed a broad-host-range plasmid carrying the FAST-PETase gene and a fluorescent reporter, and we successfully transferred the plasmid to endemic wastewater bacteria using bacterial conjugation. We screened for isolates producing FAST-PETase by purifying the 6X-His-tagged enzyme from the supernatant of concentrated cultures, and then confirmed presence of the enzyme using SDS-PAGE. For each isolate, we screened for activity of the produced FAST-PETase by incubating the enzyme with post-consumer PET plastics for five days. We then characterized PET degradation gravimetrically and confirmed significant mass loss. After confirming expression of FAST-PETase in functional form, we assessed the in situ PET biodegradation rates that each isolate could achieve. Collectively, our work has broad implications regarding the use of engineered microbes for addressing the global plastic pollution problem, and we speculate that our findings may have potential applications in other settings like plastic upcycling and bioremediation.