BACKGROUND: Chloride salts are major impurities in biodiesel-derived crude glycerol that can impact dihydroxyacetone (DHA) production. Ion exchange was performed to remove these salts. DHA production from crude glycerol was investigated, before and after an ion exchange treatment in shake-flask fermentation and batch fermentation. DHA production from treated crude glycerol was further studied in fed-batch fermentation. RESULTS:In shake-flask fermentation, the DHA production from the treated crude glycerol was 56.1 ± 1.87 g L −1 . This is 16.2 g L −1 (41%) higher than the DHA production from crude glycerol without the ion exchange treatment at 72 h. The DHA production from the treated crude glycerol was 61.9 ± 2.57 g L −1 , with a DHA production yield (DHA moles per glycerol moles) of > 99 ± 4.4% at 138 h in the batch fermentation. The DHA concentration from the treated crude glycerol was 8.1 g L −1 higher than in the crude glycerol fermentation. In fed-batch fermentation, the DHA production was not significantly higher than that in the batch fermentation due to product inhibition when the DHA concentration reaches 65.05 ± 4.52 g L −1 or more, after 156 h.CONCLUSION: This study shows that salt impurities in crude glycerol negatively impact the DHA production by Gluconobacter thailandicus TBRC 3351 cultured in crude glycerol minimal media. Removing chloride salts from crude glycerol can improve the DHA yield, both in the shake-flask and the batch fermentation. Fed-batch fermentation can also increase the DHA production, but to a lesser extent because of the product inhibition mechanism.