Plastics in the Anthropocene

In today’s world, there are no longer any environments that have not been affected by plastic pollution. Plastics have even been found in extremely remote areas such as deep-sea sediments, submarine canyons, and Arctic Sea ice (Horton et al., 2017; MacLeod et al., 2021). This worldwide phenomenon manifests in a way as diverse as the ecosystem it occupies. Plastic pollution varies from massive rocky plastiglomerates, formed from a mixture of plastic debris, volcanic rocks, sediment, and organic materials to the microscopic microplastics that pollute waterways and accumulate in the base of aquatic food chains (Horton et al., 2017; MacLeod et al., 2021).

Environmental sustainability is a particularly pressing issue as Canada is famous for its natural beauty and large sectors of the economy are based around ecotourism, fisheries or natural resource production.

In 2018, the Canadian Council of Ministers of the Environment announced their vision and strategy toward achieving zero plastic waste. The initiative is an effort to guide Canada toward the circular economy: an economic system in which products and materials are retained, recycled, and reused to reduce and conserve natural resources (Government of Canada, 2023). This strategy relies on the three pillars of preventing plastic waste, collecting all plastics, and recovering the value of plastics (Government of Canada, 2023).

In accordance with these measures, the Canadian government set a Zero Plastic Waste Target for 2030 with multiple steps aiming to extend product usage and reduce 75% of federal plastic waste. Some of the steps already in effect include the phasing out of certain plastic materials. Other phases focus on recycling plastics already in circulation to reduce plastic waste, which is a concern across the provinces. In 2023, the Canadian government proposed further changes and bans on manufacturing and importing single-use plastic (Government of Canada, 2023). While the majority of Canadians agree that drastic measures need to be taken to reduce plastic pollution, the practical steps for achieving this goal are still unclear.

In North America, efforts to reduce plastic waste and increase recycling face many challenges such as contamination of organic waste, mixing of different types of plastic, and unfavourable economic incentives (Ackerman & Levin, 2023). Despite having recognized the issue, Canadians still discard over 3 million tons of plastic annually, of which only 9% undergoes recycling, leaving the remaining plastic doomed for landfills (Government of Canada, 2023). This year UManitoba iGEM wanted to help bridge the gap between good intentions and actionable solutions.

The Road to PLAnet Zero

We began by looking into our local province of Manitoba. The province of Manitoba is known for its wide expanses and extensive waterways. The natural world is central to the lives of all Manitobans and is both a source of employment through natural resources as well as our personal well-being. Recent research found a high influx of approximately 400 million pieces of microplastics annually into Lake Winnipeg, the largest freshwater lake in Manitoba (Warrack et al., 2017). Therefore, our first idea was to degrade microplastics in Lake Winnipeg. This idea was eliminated in the Design phase. Our research indicated that the primary issue with microplastic pollution in Manitoba waters was the volume of the body of water involved, and the need to filter the water for microplastic removal (Warrack et al., 2017). Altering algae to filter plastic out of the water and degrade it was considered but deemed unrealistic for the time being.

The second idea was to develop a bioreactor system for the degradation of the most common plastic PET (polyethylene terephthalate). At this point, we had already begun to consult with Dr. Levin, a plastic degradation expert, who has experience with the use of microbes to degrade various types of plastic. The first key insight was that the province of Manitoba is currently making large efforts to reduce the production of PET plastics (Government of Canada, 2023). In fact, many distributors of single-use plastics such as restaurants, sporting events and concerts are now required to use bioplastics. We were thrilled to hear that the province was already making a substantial commitment to the circular economy.

We learned through conversations with the Multi-Material Stewardship of Manitoba, that high-tech bioreactors to deal with mixed waste like we had been envisioning, were not a feasible solution in our province. The shift to PLA bioplastics across Canada provides an invaluable opportunity to ensure that an adequate solution is implemented before it becomes just another plastic accumulating in our landfills. As stated, PLA has lots of promise. It can be made from renewable feedstock, has mechanical properties that allow conventional manufacturing techniques, and breaks down into a bio-available monomer. These outstanding properties make PLA a leader in bioplastics accounting for 24% of bio-based polymers produced worldwide (Zaaba & Jaafar, 2020). However, to our surprise biodegradability can have some significant fine print.

PLA is estimated to degrade 20 times faster than high-density polyethylene on land under appropriate conditions (Chams et al., 2020). However, PLA is only degradable at high temperatures of approximately 60˚C in the presence of O2 and moisture (Karamanlioglu & Robson, 2013; Nampoothiri et al., 2010). These temperatures are reached in commercial composting facilities, but only for a short period of time in each composting cycle. The unfortunate result is that compost with significant PLA content needs to be processed several times. Our final design is aimed at the improvement of PLA plastic degradation while working alongside current government initiatives and existing infrastructure to improve PLA composting.

PLAnet Zero

The idea behind PLAnet Zero is to develop biological catalysts that are compatible with the composting system used in our hometown of Winnipeg. We began with four candidate enzymes for use in PLA breakdown. Est119, GEN0105, MGS0156, RPA1511 and Thermoanaerobacter thermohydrosulfuricus lipase (TTL). Prototyping and benchmarking were done with purified enzymes. This helps us understand how much activity our constructs have under ideal conditions.

Of course, purification would present an unacceptable cost for application in widespread composting. The first prototype was to simply add all cell contents to the compost as a lysate. This was good, but we wanted our system to be as user-friendly as possible. The prototype for PLAnet Zero has the chassis organism expressing PLA breakdown proteins on its cell membrane. Our whole cell catalysts can easily be produced on-site in bulk in nutrient broth. Once the proteins are expressed, the cells and growth solution are simply added to the compost along with the water, which is added for moisture.

PLAnet Zero is intended to be added directly to any commercial or large-scale composting mixture. These compost products are always sterilized before use to ensure the containment of the biological agents and the elimination of any pathogens, a common practice already being followed by Canadian composting facilities.

Going further, we began searching for ways to improve on the prototype. Inspired by the use of the non-canonical amino acid norleucine to improve PET breakdown, we substituted norleucine into our PLA breakdown catalysts. With the help of computer-aided design, we managed to increase the binding affinity of our catalysts for PLA analogs 4-nitrophenyl butyrate and 4-nitrophenyl octanoate.

What’s next for PLAnet Zero?

Prototype field tests

We are thrilled to be able to share PLAnet Zero at iGEM 2023, but we are not done yet! Due to time constraints, we were not able to field-test our prototypes. However, we do plan to test PLAnet Zero in composting systems. The first stage of field tests would be in collaboration with the Sustainability in Action Facility, at our university and would test the efficacy of our device using a series of small-scale composting assays. The next stage of tests would be in a larger composting system, Such as the rapid bio-digestion machines at EnviroClean. EnviroClean is a composting research facility that allows for tight control over composting conditions.

More enzyme engineering & characterization

After initial testing, optimization, and surface display, there was only time for the first round of improvement to the enzymes themselves. In the future, we will attempt further non-canonical amino acids, look into mutagenesis libraries, and investigate the possibility of site-specific residue incorporation for more targeted non-canonical amino acid replacement. More enzymatic characterization is required to determine the optimal working temperature for each enzyme. Ideally, we need a collection of different enzymes that can work at different temperatures or that each enzyme is robust enough for different temperatures. Due to the wide fluctuation in temperatures in our hometown Winnipeg (-30˚C in the winter to 30˚C in the summer), maintaining one working temperature for the composting setting can be challenging. Therefore, having different working temperatures enables flexibility across current composting infrastructure, and would permit composting throughout the year.