Microalgae-derived docosahexaenoic acid (DHA) production has been considered an alternative technology to overcome several disadvantages of fish oil-based DHA production. Despite its potential, efficient production methods for DHA have been rarely reported. Accordingly, in this study we investigated the potential application of Jerusalem artichoke and lipid-extracted algae (LEA) hydrolysates as inexpensive sources of carbon and other nutrients for DHA production usingAurantiochytriumsp. KRS101. Response surface methodology (RSM) with central composite design (CCD) was used to determine the maximum point of DHA productivity in the presence of 51.9 g L(-1)fructose and 4.9 g L(-1)yeast extract. Hydrolysis of 76.6 g L(-1)Jerusalem artichoke powder with 0.25 N sulfuric acid at 70 degrees C yielded the desired concentration of fructose for use as the medium. Cultivation of the microalgae using the Jerusalem artichoke hydrolysate (JAH) and JAH + LEA (JLH-1) resulted in 27.8% and 31.3% improvements in biomass productivity, respectively, compared with the basal medium control. In addition, simultaneous one-step hydrolysis of Jerusalem artichoke and LEA (JLH-2) resulted in 7.2% higher biomass and 47% higher DHA yields than that obtained from hydrolyzing the two components separately (JLH-1). These results suggested that a strategy using JAH combined with LEA could replace expensive nutrient sources and produce efficient and eco-friendly DHA fromAurantiochytriumsp. KRS101.