D-Glyceric　acid　(D-GA)　is　a　promising　chemical　for　minimizing　the　toxic　effect　of　acetaldehyde.　It　has　been　successfully　produced　by　bioprocesses　using　glycerol　as　a　substrate.　However,　high　concentrations　of　glycerol　are　not　beneficial　for　cell　growth.　A　two-step　culture　strategy　was　employed　to　deal　with　the　ambivalent　culture　conditions　between　the　growth　of　Acetobacter　tropicalis　and　the　biosynthesis　of　D-GA.　The　first　stage　focuses　on　biomass　accumulation　with　low　initial　glycerol　concentration;　the　second　stage　provides　D-GA　accumulation　through　whole-cell　biocatalysis.　Approximately　2-fold　of　D-GA　yield　and　4.5-fold　of　D-GA　productivity　were　gained　by　the　two-step　culture　strategy　compared　to　the　traditional　fermentation　with　150　g　L-1　of　glycerol.　Our　results　also　showed　that　A.　tropicalis　was　able　to　catalyze　the　conversion　of　glycerol　to　D-GA　in　the　presence　of　up　to　10　%　methanol.　These　findings　have　important　implications　to　enhance　D-GA　yield　by　strategy　optimization　and　reduce　its　cost.　©　2018　Assoc.　of　Chemists　and　Chemical　Engineers　of　Croatia.　All　rights　reserved.