Continuous extraction and concentration of secreted metabolites from engineered microbes using membrane technology

Abstract

Microalgal cultivation in photobioreactors and membrane separations are both considered sustainable processes. Here we explore their synergistic combination to extract and concentrate a heterologous sesquiterpenoid produced by engineered green algal cells. A hydrophobic hollow-fiber membrane contactor was used to allow interaction of culture broth and cells with a dodecane solvent phase to accumulate algal produced patchoulol. Subsequent continuous membrane extraction of patchoulol from dodecane enabled product concentration in a methanol stream as well as dodecane recovery for its reuse. A structure-based prediction using machine learning was used to model a process whereby 100% patchoulol recovery from dodecane could be achieved with solvent-resistant nanofiltration membranes. Solvent consumption, E-factor, and economic sustainability were assessed and compared with existing patchoulol production processes. Our extraction and product purification process offers six- and two-orders of magnitude lower solvent consumption compared to synthetic production and thermal-based separation, respectively. Our proposed methodology is transferable to other microbial systems for the isolation of high-value isoprenoid and hydrocarbon products.

Biomass generation and heterologous isoprenoid milking from engineered microalgae grown in anaerobic membrane bioreactor effluent

“…UVM4 has previously been genetically and metabolically modified to produce the heterologous plant sesquiterpenoid patchouli alcohol, “patchoulol” (Abdallah et al, 2022; Lauersen et al, 2016). Heterologous terpenoid products can be collected during cultivation of microbial cultures using a two-phase culture-solvent system (Beekwilder et al, 2014; Lauersen, 2019; Overmans et al, 2022). The hydrophobic terpenoid products accumulate in biocompatible solvents as the products are more favored to partition into the solvents than into the cells or culture medium (Gruchattka and Kayser, 2015).…”

“…The milking of a terpenoid co-product during nutrient removal could be considered a highly promising way to increase the economics of lost resources in WWs. Solvent milking can be cyclic, wherein the extraction of the patchoulol from the solvent can be performed and the solvent returned to culture in low OPEX bio-processes (Overmans et al, 2022). Here, we chose the sesquiterpenoid patchoulol as an exemplary product of the engineered algae due to the availability of strains.…”

“…Engineering efforts have been facilitated by the domestication of this alga through several rounds of mutation and transformation (Neupert et al, 2020, 2009) while advances in synthetic transgene design have enabled robust expression of heterologous pathways in this alga (Baier et al, 2020, 2018). The value of engineered algae is enhanced when the recombinant products can be harvested separately from the algal biomass using solvent-culture two-phase systems in a process called ‘microbial milking’ (Overmans et al, 2022; Overmans and Lauersen, 2022).…”