Enhanced bioproduction of carvone in a two‐liquid‐phase partitioning bioreactor with a highly hydrophobic biocatalyst

Abstract

The microbial biotransformation of (-)-trans-carveol to the flavor and fragrance compound (R)-(-)-carvone by Rhodococcus erythropolis DCL14 was carried out in a 3 L two phase partitioning bioreactor with an immiscible liquid second phase in an effort to improve upon the reactor performance achieved in a single aqueous phase system. The purpose of employing the liquid second phase is to minimize biotransformation rate inhibition due to the accumulation of the toxic substrate (cis-carveol) and product (carvone) in the aqueous phase. 1-Dodecene was chosen as the solvent for this application because it is biocompatible, non-biodegradable and has a superior affinity for the target product (carvone) relative to the other solvents tested. However, when 1-dodecene was used in the biotransformation, the extremely hydrophobic R. erythropolis DCL14 created an emulsion with the organic solvent with significant sequestering of the cells into the organic phase and negligible substrate conversion. To overcome these operational difficulties, silicone oil, which is considered a liquid polymer, was used with the aim of preventing emulsification and sequestration of cells in the non-aqueous phase. Although some emulsification of the water-silicone oil was again created by the cells, operability was improved and, in fed-batch mode, the system was able to convert approximately 2(1/2) times more carveol than a benchmark single aqueous phase system before substrate/product toxicity caused the biotransformation to stop. This study has demonstrated enhancement of a microbial biotransformation for the production of a high value nutraceutical compound via the use of a second partitioning phase, along with operational challenges arising from the use of a highly hydrophobic organism in such systems.

The use of CO2 for reversible pH shifting, and the removal of succinic acid in a polymer‐based two‐phase partitioning bioreactor

“…12 -14 Although immiscible organic solvents were originally used as a sequestering phase for in situ product removal, low-cost commercial polymers have been shown to be equally effective in this capacity, while also possessing numerous advantages in terms of reduced cost, enhanced safety, and improved process operability. 12 One complication of SA separation using immiscible organic solvents or amorphous polymers, is that uptake requires the pH of the solution Carbon dioxide gas dissolves in water forming carbonic acid, which dissociates and lowers the pH, and in theory could be used to lower the pH without the addition of strong acid solutions. This reduction in pH using CO 2 can be reversed by sparging nitrogen through the liquid, driving out the CO 2 and restoring the pH to its original value.…”

Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli

“…Different approaches have been described to overcome uptake limitations in whole-cell biotransformations. The two-liquid-phase concept applied in stirred-tank reactors can increase mass transfer by ensuring maximal substrate availability and typically allows in situ product extraction (13,20,33,37,40,47,53,72). Next to that, the addition of rhamnolipids, synthetic surfactants, and cosolvents has been described as enhancing biotransformation rates.…”

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

“…Activity could be regained by the use of a different organic phase with a higher log P Oct value [80] as a reservoir for α-pinene, "masking" its detrimental effects. Searching for a suitable organic phase for the biotransformation of carveol to carvone with Rhodococcus erythropolis DCL14, Morrish and colleagues determined the critical log P Oct of R. erythropolis DCL14 by exposing the cells to a selection of 12 organic solvents with log P Oct ranging from 1.25 to 9.04 [56]. The critical log P Oct of R. erythropolis DCL14 was found to be 5 in shake flask experiments.…”

“…Surface-active components, often produced by microorganisms themselves as a protective mechanism or to improve substrate availability, impede phase separation and promote the formation of stable emulsions. Phenomena associated with stable emulsions in isoprene-producing bioprocesses were described as difficulties in determination of cell, substrate, or product concentration [56]; formation of a third phase partitioning of cell mass in the organic phase [26]; or problems in recovery of organic solvent and product [96]. The tendency for emulsion formation can change with phase volume ratio.…”

“…Usually a number of organic solvents with appropriate log P Oct values are compiled and experiments conducted to determine biocompatibility, partition coefficients for substrate, and/or product and biodegradability [6,22,37,56,80]. To test for biocompatibility an activity profile can be created by plotting an organic solvent log P Oct against biocatalyst activity.…”