Sequential fill-and-draw fermentation strategy provides an approach to increase the productivity by replenishing nutrients and minimizing the toxic effects of by-products. In the present work, the same strategy was adopted using lignocellulosic industrial rice-straw C-6 hydrolysate stream to produce rhamnolipids from Achromobacter sp. (PS1) in a 6 L bioreactor with a working-volume of 2 L. The production results showed overall rhamnolipid production of 22.03 g/L in 15 days observed at par with 19.35 g/L obtained under shake flask conditions in 18 days. At each sequential feed (2 % sugars), a rise in dissolved oxygen (D.O) concentration was observed in the range between 60-530/0 which declined to 47-39 % with consecutive depletion in sugar concentration under no D.O control. For maximum extraction of rhamnolipids from culture broth, the synergistic effect of sweep floccoagulation using FeCl3 at 0.4 % (w/v) followed by its acidification and solvent extraction was adopted which resulted in maximum recovery of 97.5 % compared to 89.05 % recovery obtained in simply acidification followed by solvent extraction. The characterization of partially purified biosurfactant using tandem-MS revealed six-congeners, Rha-C-10-C-10 and Rha-Rha-C-10-C-10 being the most abundant. Oil recovery of 92.21 % from motoroil impregnated sand using crude rhamnolipid further added the value to the biosurfactant.