The booming of biodiesel industry all over the world has led to generate a large amount (10% v/v) of crude glycerol, created an oversupply problem. Herein, we compared the product concentrations of major metabolic products attained from pure and crude glycerol biotransformation process using an adapted mutant strain Klebsiella variicola SW3. Real-time qPCR and glycerol dehydrogenase (GDH) enzyme activity assay revealed that the overexpression of GDH gene resulted in an increased GDH enzyme activity, led to a markedly boosted 2,3-butanediol (2,3-BD) production. Based on these results, the SW3 strain obtained from wild type strain Klebsiella variicola SRP3 displayed a 139-fold increased 2,3-BD production of 82.5 g/L from 59.3 a, yielding 0.62 g/g using pure glycerol. However, in a batch culture, a final 33.5 g/L of 2,3-BD was accumulated within 96 h from 50 g/L glycerol. Moreover, the strain SW3 withstanding high concentration (200 g/L) of crude glycerol displayed 64.9 and 29.25 g/L 2,3-BD in fed-batch and batch cultures respectively. Therefore, this bioconversion of crude glycerol to 2,3-BD a value-added green product with potential industrial applications as a liquid fuel or fuel additive would represent a remarkable alternative to add value to the biodiesel production helping biodiesel industries development. (C) 2017 Published by Elsevier Ltd.