Significance and Impact of the Study: This study highlights the significance of an effective fed-batch strategy for rhamnolipid production in a submerged fermentation using a water-immiscible substrate, based on maximum substrate uptake rate. The impact of this strategy ensured that the substrate was supplied at the rate matching the maximum substrate utilization by the cells without excess feeding, leading to increased rhamnolipid production, yield and productivity. AbstractA fed-batch strategy was established based on the maximum substrate uptake rate (MSUR) of Pseudomonas aeruginosa USM-AR2 grown in diesel to produce rhamnolipid. This strategy matches the substrate feed rates with the substrate demand based on the real-time measurements of dissolved oxygen (DO). The MSUR was estimated by determining the time required for consumption of a known amount of diesel. The MSUR trend paralleled the biomass profile of Ps. aeruginosa USM-AR2, where the MSUR increased throughout the exponential phase indicating active substrate utilization and then decreased when cells entered stationary phase. Rhamnolipid yield on diesel was enhanced from 0Á047 (g/g) in batch to 0Á110 (g/g) in pulse-pause fed-batch and 0Á123 (g/g) in MSUR fed-batch. Rhamnolipid yield on biomass was also improved from 0Á421 (g/g) in batch, 3Á098 (g/g) in pulse-pause fed-batch to 3Á471 (g/g) using MSUR-based strategy. Volumetric productivity increased from 0Á029 g l À1 h À1 in batch, 0Á054 g l À1 h À1 in pulse-pause fed-batch to 0Á076 g l À1 h À1 in MSUR fed-batch.

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