Selection of a recyclable in situ liquid-liquid extraction solvent for foam-free synthesis of rhamnolipids in a two-phase fermentation

Excessive foaming causes instabilities in fermentation processes, particularly when producing biosurfactants, which can be overcome by intensifying the fermentation via insitu product recovery. A reductive, multi-step approach for selecting organic solvents for an in situ liquid-liquid extraction of rhamnolipids produced by recombinant Pseudomonas putida KT2440 was developed. (1) A database consisting of physicochemical parameters for 183 solvents was composed, allowing a pre-selection by setting respective thresholds. (2) The number of solvents was reduced by evaluating their extraction efficiencies regarding rhamnolipids in cell-free cultivation broth and their impact on the growth of P. putida KT2440. (3) The most promising solvent was characterized regarding phase separation, pH-dependency of the extraction, and applicability of back-extraction for product recovery and solvent regeneration. The overall performance was assessed in two-phase (fed-)batch fermentations in lab-scale stirred-tank reactors. The solvent selection approach revealed ethyl decanoate to be a highly suitable and sustainable solvent for the in situ liquid-liquid extraction of rhamnolipids. During the final two-phase fed-batch fermentation, 30 g L-1 of produced rhamnolipids accumulated in the organic phase. Integrating extraction and increasing the partition coefficient by moderately lowering the pH prevented foaming during fermentation, thus resolving the initial process instability. Rapid phase separation and back-extractability allowed product recovery and solvent recycling. The here presented reductive, multi-step solvent selection approach was successfully applied to establish a two-phase fermentation producing rhamnolipids by engineered P. putida KT2440, resolving the foaming challenge. The approach can serve as a blueprint for selecting solvents for in situ liquid-liquid extractions in bioprocesses.