(501a) Microbial Upcycling of Post-Consumer Polyethylene Waste into Protein-Based Materials

The overwhelming majority of plastics used today are derived from petrochemical feedstocks and are considered "recalcitrant", meaning they are not readily degraded in most natural environments. Even with ideal application of conventional recycling strategies, these plastics can only be recycled a limited number of times before end-of-life disposal by incineration or dumping in landfills, leading ultimately to greenhouse gas emissions, pollution and disruption of natural ecosystems, and loss of feedstock material. Polyethylene is especially problematic, as it is predominantly used for short lifespan applications such as single use packaging. These challenges have led to a growing interest in processes that convert waste materials into higher value products that may share little to no chemical similarity with the starting material. This process, known as "upcycling", has been widely applied to produce value-added fuels, chemicals, and materials, through methods such as pyrolysis and catalytic conversion reactions. Microbial upcycling is another strategy, which unlike the aforementioned approaches, has the ability to readily produce biodegradable or biologically active materials. Upcycling of plastics using microbial cultures has been used to produce several biodegradable materials, such as polyhydroxyalkanoate, fatty acids, and biosurfactants, but the field is lacking in documentation of upcycled protein-based materials. Within this context, silk proteins are some of the most desirable biomaterials to target for microbial upcycling due to their unmatched combination of properties that includes, but is not limited to, high toughness, biocompatibility, biodegradability, self-assembly capacity, thermal stability, solvent resistance, and ability to act as an optical waveguide. In addition, silk fibroins can be manufactured into a diverse array of constructs, such as coatings, fibers, and hydrogels, facilitating their use in a wide variety of applications. Developing an upcycling process to produce protein-based materials, such as recombinant silk, represents an innovation that can eliminate waste while simultaneously generating biodegradable value added materials.

Our work aimed to establish a microbial process for converting waste polyethylene into biodegradable recombinant silk proteins through use of the Pseudomonas genus. Several Pseudomonas species have demonstrated the ability to utilize the pyrolysis products of post-consumer polyethylene, which is chiefly comprised of alkanes, as a sole carbon source in flask cultures. Our work has uncovered factors that influence the growth of Pseudomonas when alkanes are used as the sole carbon source. These variables include the amount of alkane supplied, the amount and type of nitrogen source used, the addition of exogenous biosurfactant, and the presence of antibiotic selection marker. Results show cell densities as high as 7x109 cfus/ml can be achieved in a minimal media containing only hexadecane as a carbon source, which is comparable to cell densities achieved with common microbial media (such as Luria-broth). There is a substantial variation in the preferred culturing conditions among different Pseudomonas species, including that P. aeruginosa grows optimally in flasks with 1% w/v of alkane while optimal conditions for P. oleovorans include 0.1% w/v of alkane. Moreover, 1.9 g/L of ammonium nitrate is the preferred carbon source in this media for P. aeruginosa while 2.5 g/L of ammonium chloride is necessary for the optimal growth of P. oleovorans. Interestingly, the addition of exogenous biosurfactants to the cultures offers no benefit to the rate or overall amount of cell growth. Our work shows that the use of plasmid vectors can be used in a Pseudomonas host to achieve recombinant production of green fluorescent protein (GFP) and silk protein in Luria-broth. Plasmids have also been used to achieve the production of GFP in an alkane based media with a P. oleovorans host, at levels of 12 mg/L. However, plasmids are poorly maintained in the alkane based media, showing only 1-2% maintenance after 24 hours of culture time. Furthermore, the use of antibiotic selection severely inhibits growth in this media. Therefore, genomic integration of recombinant constructs was undertaken to improve the efficiency of the system and produce upcycled silk proteins, and represents a key strategy for future work in this space. Thus, this work expands the capability of microbial upcycling processes and contributes to future sustainability by demonstrating the first production of heterologous proteins from an alkane feedstock and reports on culturing parameters necessary to facilitate practical levels of cell growth and protein production when alkanes are supplied as a sole carbon source.