Lipase-displaying whole cells appear to be efficient biocatalysts because of their low preparation costs and simple recycling procedure. The combined utilization of Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML), separately displayed on Pichia pastoris whole cells, to produce biodiesel in co-solvent media was investigated. A response surface methodology incorporating a D-optimal design was employed to obtain the optimum reaction conditions for methyl ester (ME) synthesis. The synergistic effect of the two displayed lipases and the use of tert-butanol and isooctane as the co-solvent media were found to significantly improve the transesterification reaction. Scaled-up reactions using various types of feedstock were carried out in a 0.5-1 stirred reactor under optimum conditions, affording ME yields over 90% in 12 h. Moreover, the ME yields remained above 85% after 20 repeated batch cycles. In conclusion, this biocatalyst affords a promising route to efficient biodiesel production. (C) 2012 Elsevier Ltd. All rights reserved.