Development of In Situ Solid-Phase Absorption Fermentation in Microbial Production of Terpenoids

Abstract In situ product removal (ISPR), which emerged in the 1990s, enables real-time extraction of metabolites directly from bioreactors. Since then, ISPR has been applied to diverse compounds, including terpenoids, enhancing yield by mitigating product inhibition and degradation. Among different ISPR approaches, solid-phase extraction offers unique advantages such as mechanical solid-liquid separation to remove culture media and cells, thereby further simplifying downstream processing. However, there are currently no side-by-side comparative ISPR fed-batch studies between solid and liquid extractants. This study investigates the use of solid absorptive polymers as in situ extractants for high-titre production of viridiflorol, a terpenoid flavor. In 10 mL mini bioreactors, ∼5% (w/v) solid extractant (Hytrel 3548) loading achieved comparable titre and yield to its liquid counterpart. Additionally, reducing the polymer size from 4 to 1 mm significantly increased extraction efficiency by up to 52%, highlighting the importance of mass transfer considerations for optimal ISPR efficiency. Upon scale-up to a high-yield fed-batch fermentation, the solid-phase system reached a viridiflorol titre of 10.43 g/L, representing ∼53.5% of the yield with the liquid extractant (19.51 g/L). Crucially, the absence of emulsions allowed for a streamlined downstream workflow for solid extractants, achieving 98% purity via simple recrystallization. The ISPR approach was further validated using another flavor product, α-ionone, demonstrating the broad applicability and transferability of solid-phase ISPR in various fermentation processes.