Chlorella sp. HS2, which previously showed excellent performance in phototrophic cultivation and has tolerance for wide ranges of salinity, pH, and temperature, was cultivated heterotrophically. However, this conventional medium has been newly optimized based on a composition analysis using elemental analysis and ICP-OES. In addition, in order to maintain a favorable dissolved oxygen level, stepwise elevation of revolutions per minute was adopted. These optimizations led to 40 and 13% increases in the biomass and lipid productivity, respectively (7.0 and 2.25 g l-1 d-1). On this basis, heat shock was successfully adopted in novel Chlorella sp. HS2 cultivation as a lipid inducer for the first time. Considering its fast and cost-effective characteristics, heat shock will enhance the overall microalgal biofuel production process.