Abstract: Based on the experiment of screening nitrogen sources, mixotrophicKnop mediumwas optimized by using of Plackett-Burman experimental design and the one-at-a-time strategy and the cell density of transgenic Chlorella was improved from 1. 12g/ L to 5. 11g/Lin 250mLflasks. Using the optimized medium, the cell density of the transgenic Chlorella in 250 mL flasks and 5L bioreactorwere 3. 39 and 3. 17 fold respectivelywhat can beobtained in themixotrophic Knop mediumwhile the expression capa-bilities of NP-1 were kept unchanged.