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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]

[Display omitted] • Chlorella sp. ABC-001 showed the best growth and lipid yield with flue gas 10% CO 2. • Chlorella sp. ABC-001 was unaffected by NO but SO 2 severely inhibited cell growth. • Galdieria sulphuraria was adopted to remove SO 2 and enhance CO 2 and NO removal. • SO 2 removal is attributed to ionic interactions as well as assimilation by cells. • The dual-strain system showed stable growth under simulated flue gas supply. This study aims to propose a biological system that allows for direct utilization of flue gas for carbon dioxide capture and utilization by microalgae. The strain Chlorella sp. ABC-001 is employed for its high growth rate as well as lipid and carbohydrate content. Toxicity tests showed that cell growth was unaffected by NO, but the presence of SO 2 showed critical damage on cell growth. Hence, an extremophile alga, Galdieria sulphuraria 5587.1 was applied to build a dual-strain cultivation system to mitigate the effect of SO 2 toxicity and increase CO 2 capture efficiency. All SO 2 was removed by Galdieria culture and the system exhibited stable growth from a simulated flue gas stream containing CO 2 , NO and SO 2. Combined CO 2 biofixation rate of 793 mg/L/d and lipid productivity of 113 mg/L/d was achieved. The results showed that this new cultivation system is a promising alternative for reducing CO 2 emissions from power plants. [ABSTRACT FROM AUTHOR]

The microalgae-based biorefinement process has attracted much attention from academic and industrial researchers attracted to its biofuel, food and nutraceutical applications. In this paper, recent developments in cell-disruption and lipid-extraction methods, focusing on four biotechnologically important microalgal species (namely, Chlamydomonas , Haematococcus , Chlorella , and Nannochloropsis spp.), are reviewed. The structural diversity and rigidity of microalgal cell walls complicate the development of efficient downstream processing methods for cell-disruption and subsequent recovery of intracellular lipid and pigment components. Various mechanical, chemical and biological cell-disruption methods are discussed in detail and compared based on microalgal species and status (wet/dried), scale, energy consumption, efficiency, solvent extraction, and synergistic combinations. The challenges and prospects of the downstream processes for the future development of eco-friendly and economical microalgal biorefineries also are outlined herein. [ABSTRACT FROM AUTHOR]

• Hydrolyzed H. tuberosus replaced fructose in heterotrophic Ettlia sp. cultivation. • Hydrolysates of algal residue replaced yeast extract as N source. • Sequential hydrolysis was developed to utilize C and N sources simultaneously. • Lipid productivity was enhanced with sequential hydrolysates medium. Heterotrophic cultivation of microalgae results in substantial productivity but requires significant nutrient input. Alternative sources of major nutrients such as C and N should be carefully selected to make heterotrophic production of microalgal biomass feasible. In this study, Helianthus tuberosus and lipid-extracted algae (LEA) were hydrolyzed with sulfuric acid to grow Ettlia sp. YC001. Hydrolysis of H. tuberosus with 0.3 N of sulfuric acid resulted in fructose yield of 0.51 g g−1 and glucose yield of 0.14 g g−1 at 80 °C in 1 h. Proteolysis of algal residue with 2 N of sulfuric acid resulted in amino acid yield of 0.38 g g−1 at 110 °C in 24 h. To simplify the production of hydrolysates, the hydrolysis conditions were combined to establish a tailored sequential hydrolysis process. Then, simultaneous neutralization and salt removal of the hydrolysates were done with barium hydroxide. Consequently, cultivation of Ettlia sp. with the diluted sequential hydrolysate of H. tuberosus and LEA successfully achieved 5.90 g L−1 d−1 of biomass productivity and 1.42 g L−1 d−1 of lipid productivity with Ettlia sp. [ABSTRACT FROM AUTHOR]

Microalgae accumulate abundant lipids and are a promising source for biodiesel. However, carbohydrates account for 40% of microalgal biomass, an important consideration when using them for the economically feasible production of biodiesel. In this study, different acid hydrolysis and post-treatment processing of Chlorella sp. ABC-001 was performed, and the effect of these different hydrolysates on bioethanol yield by Saccharomyces cerevisiae KL17 was evaluated. For hydrolysis using H2SO4, the neutralization using Ca(OH)2 led to a higher yield (0.43 g ethanol/g sugars) than NaOH (0.27 g ethanol/g sugars). Application of electrodialysis to the H2SO4 + NaOH hydrolysate increased the yield to 0.35 g ethanol/g sugars, and K+ supplementation further enhanced the yield to 0.41 g ethanol/g sugars. Hydrolysis using HNO3 led to the generation of reactive species. Neutralization using only NaOH yielded 0.02 g ethanol/g sugars, and electrodialysis provided only a slight enhancement (0.06 g ethanol/g sugars). However, lowering the levels of reactive species further increased the yield to 0.25 g ethanol/g sugars, and K+ supplementation increased the yield to 0.35 g ethanol/g sugars. Overall, hydrolysis using H2SO4 + Ca(OH)2 provided the highest ethanol yield, and the yield was almost same as from conventional medium. This research emphasizes the importance of post-treatment processing that is modified for the species or strains used for bioethanol fermentation. [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]