Scaling up algal cultures to large volumes for commercial production is a challenging task that involves high capital and operating cost. Hence, the feasibility of a laboratory scale culture process is important to be tested, before expanding the production. This study aims to investigate the scaling up of Chlorella vulgaris, FSP-E cultivation by discovering the best culture pathway using life cycle assessment, LCA. The study also emphasize on the techniques that can be used in large-scale algal operations, for photo-bioreactor cleaning and maintenance. Culture mediums plays a significant role in overall costing for microalgae growth processes, and their supply were addressed during the scaling up cultivation systems in terms of flow mixing, pipeline costing. Cradle to gate LCA technique was used in this study considering every phase in the lifecycle, starting from culture medium, carbon dioxide (CO2), air, and water supplies for microalgae growth till end-product dried biomass generation, including storage. Three growth media applications were compared between (1) BG-11, a chemical-based medium; (2) a combination of biscuit waste from a dairy processing facility with BG-11; and (3) re-cycling of Path 2′s residual culture medium after harvesting. The results indicated that recycling of remaining culture water obtained from dried biomass back into the photo-bioreactor was identified to be the most sustainable Chlorella vulgaris production process that can be considered for a commercial facility for bio-fuel production.