The use of high pressure carbon dioxide for in situ product recovery of butyric acid in a two phase partitioning bioreactor

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Peterson, Eric Charles
in situ product recovery , Two-phase partitioning bioreactor , butyric acid , Clostridium tyrobutyricum
The production of organic acids in solid-liquid two-phase partitioning bioreactors (TPPBs) is challenging, as acids absorb into amorphous polymers only in their undissociated form. Thus, at near-neutral fermentation pH values, where dissociated conjugate base species are dominant, partitioning is restricted. This thesis utilizes high pressure CO2 to achieve temporary pH reductions during the production of butyric acid (pKa=4.8) by Clostridium tyrobutyricum, permitting acid absorption and in situ product recovery (ISPR), reducing end-product inhibition and improving fermentative performance. A growth medium with minimized buffering was developed which did not interfere with pH reductions, and using CO2 sparging a pH of 4.8 was achieved, compared to 5.3 in the original medium, with no difference in cell growth. Buffering from the accumulation of butyric acid during fermentation was observed, however, ISPR of butyric acid was demonstrated using CO2 at atmospheric pressure, yet this did not achieve improvements in reactor performance, and it was concluded that high pressure CO2 may overcome buffering to improve recovery. To determine what pH would need to be reached with CO2, a first-principles study of pH-dependent partitioning was performed, identifying partition coefficient (PC), polymer fraction (F) and pH as variables for determining organic acid recovery. Through polymer screening based on thermodynamic affinity for butyric acid, an absorptive polymer was selected (Pebax®2533), and partitioning tests for both butyric acid (PC=4.2) and benzoic acid (PC=70, pKa=4.2) validated pH-dependent partitioning models. 60 bar pCO2 was shown to achieve up to 40% recovery of butyric acid, yielding a distribution coefficient (D) of 1.8, and 90% recovery of benzoic acid (D=24), demonstrating clear improvement over atmospheric pressures. Finally, exposure of cell populations to 60 bar pCO2 showed no adverse biological effect, and at this pressure medium buffering effects were substantially overcome. During fed-batch production of butyric acid the use of high pCO2 provided the necessary pH reductions to achieve substantial acid recovery during fed-batch production of butyric acid, as overall yields, titres, and volumetric productivities were increased by 35%, 60%, and 96%, respectively. High pCO2 represents a novel method for achieving ISPR of butyric acid, which could be extended to the production of other organic acids.
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