Reuterin (3 hydroxypropionaldehyde), secreted from L. reuteri strains, is a potentially valuable chemical and broad spectrum antimicrobial substance. The patented bacteria strain, L. reuteri DPC16, is supported by Drapac Ltd, and is successfully used in commerce. However, the low yield of reuterin greatly restricts its commercial use. To improve the conversion of glycerol to reuterin, this project studied the two step process of reuterin production from glycerol to reuterin by L. reuteri DPC16. The influence of initial glycerol concentration, biomass concentration, pH value, culture age, conversion time and temperature on the production of reuterin were investigated. The results showed that maximum reuterin production was achieved by fermenting 350mmol/L (initial concentration) of glycerol for 2h at 25 and pH 6.8 using 25g/L of 20h old resting DPC16 cells.