Sustainable in situ extraction of microalgae-derived terpenoids using functionalized silica microparticles

Abstract

Metabolic engineering of microbes for heterologous isoprenoid production has developed into an established technology. While this green approach for producing high-value chemicals holds tremendous potential, current in situ extraction methods employing biocompatible-solvents such as alkanes or perfluorinated compounds, present technical challenges in large-scale bioreactors. Here, we develop a low-cost solvent-free approach based on silanized silica particles as an alternative to solvent incubation on live microbial cultures. We determine the feasibility and specificity of three differently coated (C11, C16, C18) silica particles to extract nine heterologous terpenoid metabolites from engineered Chlamydomonas reinhardtii algae cultures during cultivation. Extraction efficiencies for all compounds except cedrene were reasonably high with C11- and C16-functionalized particles (C11: 6–228 %; C16: 6–223 % compared to dodecane), but particularly C18-coated particles demonstrated potential as alternatives to traditional two-phase extractions (12–235 %). While the extraction efficiencies of microparticles were oftentimes lower compared to alkane-solvents, the particles can be directly implemented in larger-scale cultivations where the use of alkanes poses concerns of flammability and emulsion formation. The present study additionally identified the optimum concentration of particles in culture (2 % of total volume) and that four elution steps with ethanol were sufficient to achieve >99 % product recovery, and proves the feasibility of an upscaled extraction of the sesquiterpenoid patchoulol as a representative metabolite in 5 L hanging-bag algae photobioreactors. This study paves the way for a circular bioprocess for terpene harvest from engineered microbes, while allowing for straightforward product recovery (solid–liquid separation) and reuse of functionalized particles over numerous cycles.