(91a) Integration of the Carboxylate Platform with Mcl-PHA Fermentation to Enable a Universal Route from Waste to Bioplastics

Medium chain length-polyhydroxyalkanoates (mcl-PHA) have unique advantages to replace petroleum-based plastics due to their biodegradability, excellent physicochemical properties, and capacity to be produced from organic wastes as raw materials. However, broader application of mcl-PHA is hindered by the high process cost and low yields. Therefore, we have developed efficient P. putida strains to overcome these issues using organic waste as raw material. To this end, we have engineered P. putida based on systems biology and evaluated the metabolic capacity of the strains to utilize organic acids as the main mcl-PHA precursors. More important, we have integrated the carboxylate platform with mcl-PHA fermentation to determine the potential application of this technology using real organic waste as feedstock. The carboxylate platform is a variant of the anaerobic digestion process that can transform organic matter into short chain length organic acids (scl-OA) by suppressing the methanogenic archaea. These scl-OA (C2-C5) can also be converted into more valuable medium chain length organic acids (mcl-OA) containing 6 to 8 carbons by modifying the carboxylate platform conditions to favor chain elongation. This is a technology that our commercial partners have scaled-up to demonstration plants but requires efficient methods for the extraction of organic acids. We therefore propose to bio-upgrade the carboxylate fermentation broth containing unextracted scl-OA and mcl-OA by using the P. putida metabolic versatility for mcl-PHA production, providing an attractive alternative to waste valorization and plastic sustainability. Our results show that our engineered P. putida strains containing overexpression of genes phaC1 and phaJ4 involved in mcl-PHA formation, as well as suppression of gene phaZ required for mcl-PHA depolymerization, are efficient to increase yields and titers during fermentation of model organic acids. Overall, mcl-PHA productions were higher when longer organic acids are present in the medium. More importantly, waste-derived organic acids –obtained as a mixed broth after the carboxylate platform–, reached similar mcl-PHA titers and conversion efficiencies compared to synthetic media containing model organic acids at same concentrations. Bioplastic characterization and further mcl-PHA fermentation improvement was carried out (> 5 g mcl-PHA/L and 70% mcl-PHA content) and will be discussed during the talk. Overall, our work demonstrates for the very first time the potential of these two biochemical platforms to integrate a universal route from waste to elastomer bioplastics. Further development of this technology may also help abating atmospheric CO₂ by sequestration using the Wood–Ljungdahl pathway during a modified carboxylate platform combined with mcl-PHA fermentation.