Acetic acid bacteria (AAB) isolated from natural resources and fermented plant beverages were screened to produce 1,3-dihydroxyacetone (DHA) from non-detoxified crude glycerol. Among them, the isolate NKC115 was identified as Gluconobacter frateurii and produced the highest amounts of DHA. Subsequently, the effects of growth-medium conditions (initial pH, crude glycerol concentration and nitrogen sources) on growth and DHA-production capability were examined. The results showed that the crude glycerol concentration increase to above 100 g/L suppressed growth and DHA production. The highest amount of DHA obtained was 27.50 g/L, from an initial crude glycerol concentration of 100 g/L. Meanwhile, an initial pH of 5.5-7.5 in the YPGc medium did not significantly affect the bacterial growth and DHA production. The optimal nitrogen source was peptone, with DHA production at 34.70 g/L. Furthermore, overexpression of the nhaK2 gene encoding for the Na+(K+)/H+ antiporter from Acetobactor tropicalis SKU1100 in G. frateurii NKC115 improved growth and increased the accumulation of DHA (37.25 g/L) from an initial crude glycerol concentration of 20%. These results indicated that the expression of this antiporter might maintain an optimal intracellular pH and concentration of Na+ or K+, leading to the cells’ ability to tolerate high concentrations of crude glycerol.