A two-step statistical experimental design was employed to optimize the medium for vitamin B-12 production from crude glycerol by Propionibacterium freudenreichii sp. shermanii. In the first step, using Plackett-Burman design, five of 13 tested medium components (calcium pantothenate, NaH2PO4 center dot 2H(2)O, casein hydrolysate, glycerol and FeSO4 center dot 7H(2)O) were identified as factors having significant influence on vitamin production. In the second step, a central composite design was used to optimize levels of medium components selected in the first step. Valid statistical models describing the influence of significant factors on vitamin B-12 production were established for each optimization phase. The optimized medium provided a 93% increase in final vitamin concentration compared to the original medium. (C) 2011 Elsevier Ltd. All rights reserved.