The availability of petroleum sources in the near future is constrained, so nowadays renewable energy resources are largely focused. One of the most paramount renewable energy resources is the biodiesel. During the biodiesel engenderment, excess crude glycerol is engendered which contains methanol, free fatty acids and salt. The biodiesel engenderment results in 10% of glycerol as byproducts. The carbon content in the waste glycerol can be used to serve as a cheap carbon source for the production of high value chemicals, like dihydroxyacetone (DHA), succinic acid, ethanol, citric acid, propanediol, etc. Gluconobacter oxydans is a gram-negative rod or oval-shaped non-motile bacterium belonging to the family Acetobacter, capable of producing DHA. Under optimized condition of 10% (v/v) substrate concentration, 5% (v/v) inoculum at pH 6 and temperature 30°C, maximum yield of 81.64% (v/v) was obtained. Glyceric acid was produced as a byproduct during DHA production was confirmed by qualitative test, FTIR and GCMS analysis. © 2020 Elsevier B.V., All rights reserved.