Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis. and designated as P putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils 'WFOs', vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l(-1)) and rhamnolipids yield (4.1 g l(-1)) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (mu) of 0.272 h(-1), specific product yield (q(p)) of 0.318 g g(-1) h and volumetric productivity (P(v)) of 0.024 g l(-1) h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l(-1) and surface tension as 31.2 mN m(-1). (c) 2006 Elsevier Ltd. All rights reserved.