Rhamnolipids are surface-active molecules produced by Pseudornonas aeruginosa as congener mixtures. They are considered "green" alternatives to synthetic surfactants used in industrial, remediation and pharmaceutical applications. Optimizing yield as well as controlling congener distribution are necessary steps for successful commercialization of rhamnolipids. This study used a mixture of glucose and fatty acids of different chain length (C-12-C-22) and saturation (C-18:1 and C-18:2) to produce monorhamnolipids and determine the effect of fatty acid substrates on rhamnolipid yield, percent carbon conversion and congener distribution. Results show that 1% glucose + 0.25% stearic acid (C-18) produced the greatest yield (2.1 g L-1) compared to other glucose-fatty acid combinations (0.8-1.8 g L-1)(.) Various glucose + C-18 ratios were then tested to optimize yield and percent substrate carbon conversion to monorhamnolipid. Results revealed a positive linear correlation between the mass percent of C-18 used and the percent carbon conversion. A mass percent of 67% C-18 was optimal resulting in a 44% carbon conversion and a yield of 13.7 gL(-1) monorhamnolipid. For all fatty acid substrates tested, the RhaCioCio was the most abundant and RhaC(10)C(12:1) was the least abundant of the four major congeners produced. However, the relative amount of RhaC(10)C(8) and RhaC(10)C(12) congeners was dependent on several factors: in general, fatty acid substrates with relatively short chain length (C-12 and C-14), unsaturated fatty acid substrate (c(18:2)), and longer cultivation time resulted in a higher RhaC(10)C(8)/RhaC(10)C(12) ratio. These findings will assist in mass production of monorhamnolipids and controlling the specific congeners produced. (C) 2014 Elsevier Ltd. All rights reserved.