Bioconversion of biodiesel-derived waste glycerol into high-value products is proposed as a solution to improve economic viability of biorefineries. Several microorganisms are able to metabolize glycerol into 2,3-butanediol (2,3-BD), a promising bulk chemical with wide applications: solvent, fuel additive, and feedstock for synthetic rubber production, among them. In the present work, a wide screening of microorganisms present both into the wastewater treatment system in a biodiesel industry and in culture collections was carried out in order to evaluate their potential as new 2,3-BD producer biocatalysts. Two microorganisms for 2,3-BD production from glycerol as sole carbon source have been selected, namely Raoultella planticola CECT 843 and Raoultella terrigena CECT 4519. Raoultella strains belong to the nonpathogenic bacteria class (biosafety level 1). This genus has not been previously described as biocatalyst for the studied process. The influence of operational temperature, organic acid addition, and yeast extract concentration on 2,3-BD yield and productivity have been studied through Taguchi design methodology as well as initial glycerol concentration. Based on these results, the feasibility of the process employing pure glycerol and different samples of raw glycerol has been demonstrated. (C) 2016 Elsevier Ltd. All rights reserved.