Decoupling Growth and Conversion in Bioprocesses: A Sustainable Strategy for trans-Cinnamic Acid Production

Growing concerns over global greenhouse gas emissions, rising petroleum costs, water scarcity, and the increasing demand for environmentally friendly products have driven interest in developing new bioprocess routes using whole-cell biocatalysts for the production of sustainable chemical-based products. However, a major challenge in bioprocesses competing with traditional chemical processes or petroleum-based synthesis is the need to improve key performance metrics (titer, rate and yield; TRY) because of the inherent trade-off between biomass production and product formation. These cannot be optimized simultaneously, especially high productivity, which is a key performance indicator in several biotechnological processes. To address this, production processes using decoupled bioprocesses and employing whole-cell biocatalysts/supernatants recycling systems to fully utilize the cell's high substrate-to-product conversion capability, increase water use efficiency, and reduce wastewater generation represent alternative routes for sustainable chemical production.

This thesis aims to develop a decoupled bioprocess integrated with in-situ product recovery and whole-cell biocatalysts/supernatants recycling system to enhance performance metrics of sustainable trans-cinnamic acid (tCA) production, a model target product used in several industrial sectors, using a whole-cell biocatalyst of recombinant Pseudomonas putida KT2440 expressing phenylalanine ammonia-lyase (PAL) to convert L-Phenylalanine (L-phe) to tCA. Four different PAL genes were cloned into the pPS4 plasmid. Among these, RmXAL showed the highest titer and production rate under optimal conditions of pH 8.5 and a temperature of 37oC. Whole-cell biocatalysts were produced between 10 and 50 gDCW L^-1 using fed-batch fermentation during the first stage, and the process was switched to the tCA production stage. The optimal performance metrics were achieved with a biocatalyst concentration of 30 gDCW L^-1, resulting in a titer of 29.88 g L^-1, overall rate of 1.30 g L^-1 h^-1, yield on glucose of 0.27 g tCA g glucose^-1, and yield on L-phe of 0.75 g tCA g L-phe^-1. Moreover, mathematical models were constructed to describe cell growth, glucose consumption, and tCA production. These models remained effective even when the process was scaled up to a bioreactor 6-fold larger.

To maximize the cell's biotransformation capability and reduce water usage in the production process, the decoupled bioprocess was combined with a product removal technique, enabling the reuse of whole-cell biocatalysts and supernatants in subsequent production cycles. Two methods for removing tCA from the supernatant were compared: a precipitation-based approach involving pH adjustment from alkaline to acidic conditions and a liquid-liquid extraction using various biocompatible solvents. Among these, the decoupled bioprocess integrated with tCA precipitation-based technique, utilizing whole-cell biocatalyst concentrations of 40 gDCW L^-1 and L-phe feed concentration of 35 g_L-phe L^-1, demonstrated the best performance. This process resulted in a titer, rate, and yield that were approximately 4.79-fold, 1.73-fold, and 5.60-fold higher, respectively, compared to the production process without the recycling system. In addition, this approach significantly reduced water consumption and wastewater generation by 85% compared to standard production processes. By aligning with the principles of Sustainable Development Goal 6 (SDG) “Clean water and sanitation” and SDG 12 “Responsible production and consumption”, these strategies support the development of an industrial bioproduction platform to achieve better environmental and economic sustainability.