Nitrogen management in the optimization of microbial plastic waste upcycling

Bioprocesses grant us with broad opportunities to build a circular carbon economy. Using microbes allows us to replace non-renewable resources and change waste management strategies, including recycling and upcycling. However, research still lags on strategies to enable economic viability of these types of bioprocesses relative to the traditional production methods. Tuning the composition of the microbial growth medium is one way to address these issues. This allows us to gain insight into the interactions of nutrients and the focal organisms in order to formulate appropriate media recipes to support the metabolic needs. This step, which is often overlooked in proof-of-concept research, can substantially improve process metrics. After carbon, nitrogen is the most important and costly nutrient and should be given thorough consideration. In this study, we utilized nitrogen sourcing to tackle the biological upcycling of thermally oxo-degraded plastic waste using a previously described non-conventional yeast with the ability to utilize hydrophobic substrates, Candida maltosa. We compare the use of algae extract and casamino acids as organic, amino acid-based nitrogen sources to inorganic ammonium sulfate with the goal of increasing growth metric and biomass production. Our findings show that the use of algae extract and casamino acids promotes 2X-25X higher growth in model compounds and up to 2X on TOD products. Significant changes were observed in elemental composition of the cells in response to the change in nitrogen and carbon source. These changes align with the ~ 3X increase in internal protein content in the case of cells grown on casamino acids and TOD. Membrane properties and fatty acid content of the cells are also largely impacted, with a significant decrease in saturated and unsaturated fatty acid content, resulting in a reduced average lipid length in the case of casamino acid and TOD grown cells. Three proteins, namely the nonspecific lipid transfer protein POX18, the glycine zipper 2 transmembrane domain-containing protein, and the histidine triad nucleotide binding protein HNT1 were expressed in cells grown on TOD. The results from this study highlight the importance of nutrient management in non-model organisms and the biological challenges associated with the utilization of these compounds which can be insightful in future genetic engineering efforts for improved performance.