In this study, heterotrophic growth conditions for Micractinium sp. ME05 cells were investigated for the improvement of biomass production. Plackett Burman (PB) method was used to screen process variables, namely, pH, carbon source and yeast extract concentrations, temperature and inoculum ratio, that affect the biomass production. The Box-Behnken (BB) design of response surface methodology (RSM) was applied to evaluate the interaction effect of process variables and to optimize them. The biomass obtained from PB design was 1.07 g/L and pH, temperature and carbon source concentration were selected based on their positive effect on biomass production. Applying response optimizer tool of RSM, the highest biomass obtained was 2.08 g/L. The results revealed that a 1.9-fold increase in biomass concentration was achieved by manipulating cultivation conditions which would be valuable for large scale cost efficient industrial applications of biomass production.