Rapid biphasic synthesis of phosphatidylglycerol by phospholipase D in an oscillatory baffled reactor

Phospholipase D (PLD)-catalyzed transphosphatidylation offers a mild route to functional phospholipids, but implementation is limited by poor substrate accessibility, long batch times and poorly controlled liquid–liquid interfaces. Here, we established a formulation-controlled biphasic platform for converting phosphatidylcholine (PC) to phosphatidylglycerol (PG) in a 250 mL oscillatory baffled reactor (OBR). The study integrates formulation screening, operational kinetics, reactor benchmarking and multiscale characterization. A toluene-rich mixed organic solvent enabled high PC loading, while an organic-to-water ratio of 1:3 and a PLD loading of 0.1 wt% performed best among the tested conditions. In operational benchmarking, the OBR achieved the highest PG formation within 120 min and the most uniform dispersion, while its estimated nominal power density was of the same order of magnitude as that of the Applikon reactor. Its resolved-droplet apparent interfacial area density was approximately 11-fold and threefold higher than those of the beaker and Applikon systems, respectively. Under the selected OBR conditions, PG accounted for 96.14% and 99.26% of the quantified PC–PG pool after 60 and 120 min, respectively, while apparent PA formation was 0.34% after 120 min. Microscopy, ATR-FTIR and SAXS/WAXS further revealed phase-ratio-dependent differences in microscale aggregation, local phospholipid environments and lamellar organization. These results demonstrate how organic-phase design and reactor-generated dispersion can be jointly engineered to intensify high-loading biphasic lipid biocatalysis.