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Lipid production in microalgae under nitrogen (N) starved condition can be enhanced by excess phosphorus (P) supply in the second stage of two-stage cultivation strategy. However, implementing two-stage cultivation is difficult in large-scale cultivation system as it requires high energy of transferring large algal biomass from first stage to second stage. To address this problem, we have optimized a continuous two-stage (CTS) cultivation strategy using Chlorella sp. HS2, where nitrogen in the growth environment is depleted naturally via consumption. To enhance both biomass and lipid productivity this strategy explored supplementation of additional P from 50% to 2500% of the initial concentration at the start of N-limited second stage of growth. The results of the optimization study in photobioreactor (PBR) showed that supplementing 500% of initial P and 100% of initial other nutrients (O) (N 0 –P 500 –O 100) on 5th day showed the maximum biomass productivity of 774.4 mg L−1 d−1. It was observed that Chlorella sp. HS2 grown in PBR yielded higher biomass (3.8 times), lipid (6.1 times) and carbohydrate (5.5 times) productivity in comparison to the open raceway ponds (ORP) study, under optimum nutrient and carbon supply condition. The maximum lipid (289.6 mg L−1 d−1) and carbohydrate (219.2 mg L−1 d−1) productivities were obtained in TPBR-3, which were 1.9 and 1.3 times higher than that of TPBR-2 (+ve control) and 9.6 and 3.7 times higher than that of TPBR-1 (-ve control), respectively. Fatty acid mainly composed of C 16 /C 18 (84.5%–85.7%), which makes the microalgal oil suitable for biofuel production. This study concluded that feeding excess amount of P is an effective and scalable strategy to improve the biomass and lipid productivity of CTS cultivation. Image 1 • Chlorella sp. HS2 was grown in a CTS cultivation process with optimum carbon supply. • Biomass growth was maximum with 500% excess P supply (N 0 –P 500 –O 100) in 2nd stage. • Both TPBR-3 and TORP-3 achieved the enhanced lipid and carbohydrate productivity. • Provide a strategy for high P utilization and maximizing the biofuel production. [ABSTRACT FROM AUTHOR]

[Display omitted] • Whole-cell biomass utilization was completed with newly integrated biorefinery. • Hydrolysis yielded 89 % of total carbohydrate within 4 min using optimized process. • Volume reduction decreased solvent usage by 75 % and recovered all oil within 20 min. • Bioethanol yield of sole hydrolysate was comparable with conventional YPD medium. • Newly developed process decreased OPEX and CAPEX by 82.7 % and 70.1 %, respectively. Microalgae are a promising source of biomass for the production of biofuels, but their use is currently infeasible because of changes in the biofuel market and the lack of an optimized refinery process. The biomass of oleaginous microalgae consists of up to 50% lipids and 50% carbohydrates and proteins, so the biorefinery process must be able to use whole cell biomass. In this work, an alternative process to the standard biorefinery approach of lipid extraction and treatment of lipid-extracted residue was developed and tested using oleaginous Chlorella sp. ABC-001. First, a hydrolysis process was optimized to achieve cell lysis. At 170 °C and 0.1 N H 2 SO 4 , 89.0% of total carbohydrates were hydrolyzed to monosaccharides within 4 min. Then, a volume reduction process (brief and mild centrifugation) was applied to reduce the extraction time and the use of organic solvent. By reducing the aqueous phase from the hydrolysate, all lipids were extracted by vortexing with just a quarter of the original volume (10 mL) within 20 min. Finally, after extraction the aqueous phase was utilized as the sole growth medium for ethanol production by Saccharomyces cerevisiae KL17. The resulting ethanol yield was 0.39 g ethanol/g sugar, comparable to that achieved using conventional yeast-peptone-dextrose (YPD) medium (0.41 g ethanol/g sugar). Furthermore, relative to the conventional process, techno-economic analysis confirmed that the newly developed process decreased operational expenditures by 82.7% and decreased capital expenditures by 70.1%. [ABSTRACT FROM AUTHOR]