Biodiesel production from non-edible Calophyllum inophyllum lion oil with high levels of Free Fatty Acid (FFA) (acid value -6.732 mg KOH/g of oil) was investigated using whole-cell biocatalysts. Rhizopus oryzae cells immobilized within reticulated polyurethane foams were used as biocatalysts for biodiesel production. The effects of reaction parameters such as methanol-to-oil molar ratio, water content, and temperature for the production of biodiesel through methanolysis in a packed-bed reactor (PBR) were studied. Molar ratio of methanol-to-oil 12:1, water content 15%v/v, cell concentration - 20% and temperature 35 C were found to be the optimum. The yield of biodiesel obtained in batch methanolysis from C inophyllum oil under optimized condition was 92%. Long-term stability of immobilized cells for methanolysis was verified using re-usability studies. (C) 2013 Elsevier Ltd. All rights reserved.