Grantee Research Project Results

Final Report: A Synthetic Biology Approach to Recycling Waste Plastic into Biodegradable Plastic

EPA Contract Number: 68HERC21C0026
Title: A Synthetic Biology Approach to Recycling Waste Plastic into Biodegradable Plastic
Investigators: Brewer, Alec V
Small Business: Transfoam LLC
EPA Contact: Richards, April
Phase: I
Project Period: March 31, 2021 through September 29, 2021
Project Amount: $100,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2021) RFA Text |  Recipients Lists
Research Category: Small Business Innovation Research (SBIR)

Description:

The environmental havoc wreaked by petroleum-based plastics is well recognized. Drilling, fracking and refining processes cause unmeasured physical and chemical damage to local ecosystems, as do the 8 million tons of petroplastics that pollute our oceans each year. In addition to the environmental effects of raw material harvesting and plastic pollution, it is estimated that humans consume upwards of 50,000 microplastic particles daily - or approximately a credit card’s worth of plastic each week. Much of this is a result of direct consumption during use in foodservice goods, and the remainder is attributable to accumulation in the environment. Lack of incentive to collect, sort, and recycle plastics has caused widespread contamination of the environment with materials fragmenting into the microplastics threatening human and wildlife health.

Polyhydroxybutyrate (PHB) is a fully bio-based and biodegradable polymer with properties suited to replace Polypropylene (PP) and Polyethylene (PE). Unlike petroplastics, which fragment into microplastics, PHB offers a nontoxic end of life. Unlike many bioplastics, PHB does not require industrial composting; rather, PHB is metabolized by microbes found throughout terrestrial and marine ecosystems. The major bottlenecks of widespread use of PHB are raw material acquisition and associated costs, which currently drives PHB prices to over two to five times that of bio-based alternatives.

Transfoam is a biomanufacturing platform that uses a highly-engineered microorganism to turn waste-based feedstocks into PHB. Our biological design distinctly combines the genetic capabilities of 3 microorganisms into one continuous process to create a more efficient and modular system. Our current approach aims to optimize the yield and cycle time of turning treated waste plastic into PHB, lending itself to a more sustainable and economical use of recycled plastic waste. By decoupling “bio-based” from meaning “plant-based,” our bioprocess contributes to increased recycled content in PHB, and overcomes issues associated with the overutilization of farmland and nearby bodies of water required by plant-based raw materials. Transfoam’s upcycling of waste polystyrene to produce PHB is the first of its kind to offer a beginning and end of life solution to global plastic pollution, emphasizing three environmental and financial benefits:

1. Incentivizing proper collection of new and existing polystyrene waste,

2. Reducing footprint of PHB feedstock acquisition and its overall lifecycle,

3. Increasing accessibility to PHB with synthetic biology to help meet unmet demand.

Summary/Accomplishments (Outputs/Outcomes):

During Phase I, Transfoam successfully demonstrated a proof of concept for producing polyhydroxybutyrate (PHB) from styrene in engineered E. coli TG1 strains. Denovo AI Software analysis and modeling of the secondary structure of ribonucleic acids informed synthetic design of plasmid to drive high translation rates of each homologous protein. Expression of all 8 heterologous proteins required for styrene conversion into PHB was demonstrated via quantitative immunoblotting. Plasmid libraries with ranging expression values were synthesized and performance of each operon variant was screened for performance in styrene degradation and PHB production through hightroughput functional analysis. Indigo formation was used as the functional assay to analyze genetic variants of the styrene degradation pathway and demonstrate functionality of heterologous enzymes. Nile Red staining and gas chromatography-mass spectroscopy (GC-MS) were used to demonstrate the production of PHB from styrene, glucose, and LB media. Finally, PHB was recovered using an aqueous-based extraction method and additional processing techniques were developed to further increase the purity of  PHB.

Conclusions:

Technical:

●      Models suggest consistent yields of around 60% (g PHB/g styrene) and productivity of 1-2 g PHB/L/hr should be met to achieve price competitiveness with current manufacturers. Advancements in synthetic biology can be employed to achieve these metrics, including a number of optimizations made to the genetic constructs introduced and their method of introduction, as well as the genome of the organism itself through selective pressure and targeted genome engineering.

●      Tests of this kind will continue to be performed in micro-scale experiments to minimize fermentation costs while performing extensive genomic engineering, before moving into benchtop bioreactor fermentation (2-10L).

●      As we continue to iterate upon our genetic design and implementation at this scale, we will use the several grams of PHB generated each week for material characterization and standardization. These standards are established by organizations, such as the American Society for Testing and Materials (ASTM) and the International Organization for Standardization (ISO), and define protocols for a number of material properties in isolation, blends and in specific product applications.

●      Upon nearing the metrics set forth in (models), we can begin scaling up our bioprocess, achieving kilogram scale production by late 2022/early 2023 and scaling iteratively by 1.5-10x (varied by scale) to achieve pilot, semi-works, and full-scale production, separated each by 2-2.5 years.

Commercial:

●     PHBs have a variety of uses, and can most commonly be found in foodservice and cosmetic goods and packaging, agricultural films and fertilizers, textiles and a variety of medical applications. We will work with customers to develop PHB formulations suited for their intended product applications.

●     We will first target our marketing towards public benefit corps in the consumer food-service goods segment due to the alignment of our PHB’s distinctly-low footprint with their commitment to delivering the healthiest and most sustainable goods to market.

●      Initially, we will purchase chemically recycled polystyrene to feed into our bioprocess, but will later license and/or co-develop infrastructure from a polystyrene recycler to more actively contribute to its collection and remediation and ensure a more constant supply of the large industrial volumes of waste polystyrene.

●      Transfoam consists of 5 full-time members and is currently pre-revenue, but expects to be able to both expand our team and begin selling PHB by late 2022/early 2023.