Succinic Acid (SA) is an intermediate in the production of fine and commodity chemicals. No commercial SA bioproduction process exists due to process limitations including end product inhibition and high product separation costs, which account for 70% of total production costs. Two-Phase Partitioning Bioreactors (TPPBs) can increase volumetric productivity through in-situ product removal, although SA uptake by polymers requires a pH below the pKA2 of SA (4.2). Sparging CO2 gas into the bioreactor was proposed to temporarily lower the pH of the medium, allowing for SA uptake. At 1atm CO2 sparging lowered the pH of Reverse Osmosis (RO) water to 3.8 but only to 4.75 in medium, requiring the use of H2SO4 and KOH for pH adjustment in subsequent experiments. Polymers were screened for SA uptake and the effect of pH on uptake from 2.2 to 6.2 was also studied. Only Hytrel® 8206 showed non-zero uptake with a partition coefficient for SA of 1.3. Cell cultures of Actinobacillus succinogenes was exposed to pH 4.2 for times from 5 minutes to 4 hours to determine whether cells could grow after low pH exposure. A. succinogenes resumed growth after up to 4 hours of low pH exposure, giving a sufficient time span for SA uptake in the bioreactor. A single-phase run was operated as a benchmark for comparison to the TPPB system which removed SA from the fermentation broth by pH cycling; lowering the pH to 3.8 for uptake, then increasing it to 6.7 to continue bioproduction. Uptake from fermentation broth took 60 minutes, within the time causing no effect on cell growth from low pH exposure. The two-phase run yielded 1.39g/L•h, unchanged compared to the single-phase run which gave 39g/L of SA after 28 hours. Though pH cycling reduced the concentration of SA through polymer uptake, the salts added for pH adjustment hindered further cell growth. The TPPB system demonstrated that SA can be efficiently removed from solution without complex separation methods. Future work will use pressurized vessels to increase the solubility of CO2 and lower the pH of fermentation broth for SA uptake without the need for strong acids. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-04-18 08:07:51.379

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. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-28 17:41:45.458