Strategies for Enhanced Bioproduction of Benzaldehyde Using Pichia Pastoris in a Solid-Liquid Partitioning Bioreactor and Integrated Product Removal by In Situ Pervaporation
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Benzaldehyde (BZA), a biologically derived high-value molecule used in the flavour and fragrance industry for its characteristic almond-like aroma, has also found use in nutraceutical, pharmaceutical, cosmetics, agrochemical, and dye applications. Although, nature-identical BZA is most commonly produced by chemical synthesis, biologically derived BZA, whether by plant material extraction or via microbial biocatalysts, commands much higher prices. The bioproduction of high value molecules has often been characterized by low titers as results of substrate and product inhibition. The current work examined a variety of process strategies and the implementation of a solid-liquid bioreactor partitioning system with continuous integrated pervaporation to enhance the bioproduction of BZA using Pichia pastoris. Previous work on two-phase partitioning bioreactors (TPPBs) for the biotransformation of BZA using Pichia pastoris has had limitations due to long fermentation times and unutilized substrate in the immiscible polymer phase, contributing to complications for product purification. To reduce fermentation times, a mixed methanol/glycerol feeding strategy was employed and reduced the time required for high-density fermentation by 3.5 fold over previous studies. Additionally, because BZA and not the substrate benzyl alcohol (BA) had been found to be significantly inhibitory to the biotransformation reaction, a polymer selection strategy based on the ratio of partition coefficients (PCs) for the two target molecules was implemented. Using the polymer Kraton D1102K, with a PC ratio of 14.9 (BZA:BA), generated a 3.4 fold increase in BZA produced (14.4 g vs. 4.2 g) relative to single phase operation at more than double the volumetric productivity (97 mg L-1 h-1 vs. 41 mg L-1 h-1). This work also confirmed that the solute(s) of interest were taken up by polymers via absorption, not adsorption. BZA and BA cell growth inhibition experiments showed that these compounds are toxic to cells and it was their accumulation rather than low enzyme levels or energy (ATP) depletion that caused a reduction in the biotransformation rate. For this reason, the final strategy employed to enhance the bioproduction of benzaldehyde involved in situ product removal by pervaporation using polymer (Hytrel 3078) fabricated into tubing by DuPont, Canada. This aspect was initiated by first characterizing the custom-fabricated tubing in terms BZA and BA fluxes. The tubing was then integrated into an in situ pervaporation biotransformation and was shown to be effective at continuous product separation, using 87.4% less polymer by mass in comparison to polymer beads in conventional TPPB operation, and improved overall volumetric productivity by 214% (245.9 mg L-1 h-1 vs. 115.0 mg L-1 h-1) over previous work producing BZA.