3-Hydroxypropionaldehyde (3-HPA) produced by Lactobacillus reuteri is a broad-spectrum antimicrobial substance of glycerol conversion. The aim of the present work was to optimize 3-HPA production by Lb. reuteri ATCC 53608 using a two-step process. The first step was the production of Lb. reuteri cells in optimal conditions. Cells were then harvested by centrifugation and suspended in glycerol solution, which the resting cells bioconverted to 3-HPA. The effect of biomass concentration, temperature, glycerol concentration, anaerobic/micro-aerophilic conditions, and incubation time was studied for high 3-HPA production. 3-HPA accumulation was limited by the death of cells in contact with high concentrations of 3-HPA. However, a very high 3-HPA concentration of 235 +/- 3 mM was obtained after 45 min of incubation at 30 degrees C in 400 mM glycerol for an initial free-cell concentration of 1.6 0.3 x 10(10) viable cells/ml. A high viability was maintained at low temperatures in the range 5-15 degrees C, but with a slightly lower yield of 3-HPA at 5 degrees C compared with higher temperatures, up to 37 degrees C. Successive 1-h incubations of Lb. reuteri cells in 200 mM glycerol at 15 degrees C to tentatively reuse the cells resulted in decreasing 3-HPA concentrations at the end of each cycle, with two successful production cycles yielding high 3-HPA concentrations of 147 +/- 1 mM and 128 +/- 2 mM.