A comparison of solvents for their suitability as solvent-culture two phase systems for living extraction of microbial isoprenoids using the green microalga Chlamydomonas reinhardtii as a model organism

Biotechnology and metabolic engineering of microbes has become a mature technology in many host microorganisms. A key method for the capture and quantification of heterologous and natural microbially produced isoprenoid products is to incubate growing cultures with a bio-compatible solvent. This is a culture-solvent two phase system that allows the continuous extraction, or ‘milking’, of hydrophobic products produced from microbial hosts. Solvent milking systems are highly valuable because they allow capture and subsequent analytics to be performed in a relatively straightforward manner. However, the currently used solvents are petroleum-derived, or can be challenging to work with at scale. Therefore, this thesis sought to investigate the biocompatibility of alternative solvents, including some examples of “green solvents” derived from biomass sources, using a model microalga as a study organism that had been previously engineered to produce the heterologous sesquiterpenoid patchoulol. In total 22 solvents were investigated, their 50% lethal concentration (LC50) determined, and their abilities to accumulate patchoulol from the algal culture compared. Solvents that were not-immediately lethal to algal cells and accumulated patchoulol, were also tested for their capacities for further isolation of patchoulol out of the solvent, either in ethanol, methanol, or through solid phase extraction. This work provides a broad foundation of information which is important for future efforts at microbial product milking and solvent choices for bio-process designs, especially with engineered microalgae.