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Background: Microalgae's low tolerance to high CO2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO2 content—an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO2 conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO2) and at various growth phases. Results: Cells cultivated with 10% CO2 exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO2 conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO2 conditions, resulting in improved CO2 fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO2 conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae. Conclusions: We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future. [ABSTRACT FROM AUTHOR]

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]

• Rapid induction of lipids in Chlorella by mild electric treatment (31 mA and 4 h). • 2.1-fold improvement of triacylglycerol (TAG) content relative to the control. • Promotion of biosynthesis of polyunsaturated linoleic and linolenic acids. • Electric lipid stimulation through transformation and de novo biosynthesis. In this work, a new stress-based method for rapid induction of triacylglycerol (TAG) and total and polyunsaturated fatty acid accumulations in Chlorella sp. by mild electric stimulation is presented. When a cathodic current of 31 mA (voltage: 4 V) was applied to the algal cells for 4 h, the TAG content of the electro-treated cells was sharply increased to a level 2.1 times that of the untreated control. The contents of the polyunsaturated linoleic (C18:2n6) and linolenic (C18:3n3) acids in the electro-treated cells were also 36 and 57% higher than those in the untreated cells, respectively. Cyclic voltammetry and various biochemical analyses indicate that TAG and fatty acid formations are electro-stimulated via de novo fatty acid biosynthesis and metabolic transformation in the Chlorella cells. [ABSTRACT FROM AUTHOR]