The indigenous strain Botryococcus braunii TN101 was isolated and acclimatized under laboratory condition. Upstream and downstream process was thoroughly explored for biofuel production. During semi-continuous cultivation, the alga was grown under batch mode for 6 days; thereafter 40% of algal culture was harvested at every three days interval. At semi-continuous system, the indigenous strain grows well and produces high biomass productivity of 33.8 g m(-3) day(-1). A two step combined harvesting process was designed using ferric iron and organic polymer Poly-(D)glucosamine and harvested 99.5% of biomass. Lipid extraction was optimized using different solvents, cyclohexane and methanol at 3:1 ratio supported for maximum extraction of lipids in Botryococcus up to 26.3%. Physicochemical properties of lipid was analyzed and found, saponification values 184, ester values 164, iodine values 92 and the average molecular weight of the lipids are 920 g mol(-1). The lipid contains 9.7% of FFA level, therefore, a simultaneous esterification and transesterification of free fatty acids and triacylglycerides were optimized for biodiesel production and the methyl ester yield was recorded up to 84%. In addition, an optimization study was carried out for the removal of pigments present in the biodiesel; the result revealed that 99% of pigments were removed from the biodiesel using activated charcoal. The biodiesel profile was analyzed by H-1 and C-13 NMR and GC-MS analyzer, methyl palmitate and methyl oleate was the major fatty acid found. Based on the areal and volumetric biomass productivity, it is estimated that the indigenous strain can produce 101 tons ha(-1) year(-1) of biomass. (C) 2014 Elsevier Ltd. All rights reserved.