The fermentation of the hydantoinase-active Pseudomonasputida RU-KM3s was investigated. The hydantoinase activity was found to be reduced in complex media, while the use of high sugar to nitrogen ratios in the growth medium resulted in the formation of extracellular polymers, low biomass, and low enzyme activity. By replacing the primary carbon source from simple carbohydrate to a fatty acid, using an ammonia-deficient medium (designated PP2 medium), a maximum growth rate of 0.29 (+/- 0.005) h(-1) was attained with a biomass of 7.3 (+/- 0.18) g L-1 at a yield of 0.73 g g(-1) and a productivity of 0.61 g g(-1)h(-1). The maximum hydantoinase activity obtained was 187 (+/-19.4) nmol min(-1) mg(-1) (DCW), while that of the N-carbamoylase was 22 (+/- 3.1) nmol min(-1) mg(-1) (DCW). The fermentation was scaled up 50-fold from shake flask to 15 L batch. (c) 2007 Elsevier Inc. All rights reserved.