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Microalgae are new and sustainable sources of starch with higher productivity and flexible production modes than conventional terrestrial crops, but the downstream processes need further development. Here, ultrasonication (with power of 200 W or 300 W and duration of 10, 15, 20, or 25 min) was applied to simultaneously extract and modify starch from a marine microalga Tetraselmis subcordiformis for reducing the digestibility, and an aqueous two-phase system (ATPS) of ethanol/NaH 2 PO 4 was then used to isolate the starches with varied properties. Increasing ultrasonic duration facilitated the partition of starch into the bottom pellet, while enhancing the ultrasonic power was conducive to the allocation in the interphase of the ATPS. The overall starch recovery yield reached 73 ∼ 87 % and showed no significant difference among the ultrasonic conditions tested. The sequential ultrasonication-ATPS process successfully enriched the starch with purities up to 65 % ∼ 88 %, which was among the top levels reported in microalgal starch isolated. Ultrasonication produced more amylose which was mainly fractionated into the interface of the ATPS. The digestibility of the starch was altered under different ultrasonic conditions and varied from different ATPS phases as well, with the one under the ultrasonic power of 200 W for 15 min at the bottom pellet having the highest resistant starch content (RS, 39.7 %). The structural and compositional analysis evidenced that the ultrasonication-ATPS process could exert impacts on the digestibility through altering the surface roughness and fissures of the starch granules, modulating the impurity compositions (protein and lipid) that could interact with starch, and modifying the long- and short-range ordered structures. The developed ultrasonication-ATPS process provided novel insights into the mechanism and strategy for efficient production of functional starch from microalgae with a potential in industrial application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chrysolaminarin, a kind of water-soluble bioactive β-glucan produced by certain microalgae, is a potential candidate for food/pharmaceutical applications. This study identified a marine microalga Isochrysis zhangjiangensis , in which chrysolaminarin production was investigated via nutrient (nitrogen, phosphorus, or sulfur) deprivations (-N, -P, or -S conditions) along with an increase in light intensity. A characterization of the antioxidant activities of the chrysolaminarin produced under each condition was also conducted. The results showed that nutrient deprivation caused a significant increase in chrysolaminarin accumulation, though this was accompanied by diminished biomass production and photosynthetic activity. -S was the best strategy to induce chrysolaminarin accumulation. An increase in light intensity from 80 (LL) to 150 (HL) µE·m -2 ·s -1 further enhanced chrysolaminarin production. Compared with -N, -S caused more suitable stress and reduced carbon allocation toward neutral lipid production, which enabled a higher chrysolaminarin accumulation capacity. The highest chrysolaminarin content and concentration reached 41.7% of dry weight (%DW) and 632.2 mg/L, respectively, under HL-S, with a corresponding productivity of 155.1 mg/L/day achieved, which exceeds most of the photoautotrophic microalgae previously reported. The chrysolaminarin produced under HL-N (Iz-N) had a relatively competitive hydroxyl radical scavenging activity at low concentrations, while the chrysolaminarin produced under HL-S (Iz-S) exhibited an overall better activity, comparable to the commercial yeast β-glucan, demonstrating I. zhangjiangensis as a promising bioactive chrysolaminarin producer from CO 2 .

Industrial wastewaters usually possess a wide range of nitrate strength. Microalgae-based nitrate-rich wastewater treatment could realize nitrate recovery along with CO 2 sequestration for sustainable biomass production, but the low tolerance of the microalgal strains to high-strength nitrate restricted the treatment process. The present study comprehensively evaluated a euryhaline marine microalga Tetraselmis subcordiformis for photosynthetic nitrate removal and biomass production in synthetic wastewater with a broad range of nitrate strength (0.24-7.0 g NO 3 - -N/L). This alga could acclimate to high nitrate strength up to 3.5 g NO 3 - -N/L (HN) without compromising biomass production. Nitrate could be completely removed within four days when low nitrate (0.24 g NO 3 -N/L (SHN) caused an increased light energy dissipation while decreased the density of photosystem II active reaction center, which partially protect the cells from photodamage and contributed to their acclimation to SHN. The algae also enhanced amino acid/fatty acid proportions essential for maintaining intracellular redox states to cope with the stress caused by LN or SHN. HN and SHN was in favor of protein accumulation and maintenance with enhanced proportion of essential amino acids, which entitled the algal biomass to be of high quality for animal feed applied in livestock graziery and aquaculture. LN facilitated productive starch and lipid accumulation with good quality for biofuels production. The nitrate removal rate and biomass productivity exceeded most of the microalgae reported in literature under similar conditions, which highlighted Tetraselmis subcordiformis as a potent strain for flexible nitrate-rich wastewater remediation coupled with fast CO - -N/L, LN) was loaded. The maximum nitrate removal rate of 331 mg N/L/day and specific nitrate removal rate of 360 mg N/day/g cell was obtained under medium nitrate condition (1.8 g NO 3 - -N/L, MN). High-nitrate stress under 7.0 g NO 3 - -N/L (SHN) caused an increased light energy dissipation while decreased the density of photosystem II active reaction center, which partially protect the cells from photodamage and contributed to their acclimation to SHN. The algae also enhanced amino acid/fatty acid proportions essential for maintaining intracellular redox states to cope with the stress caused by LN or SHN. HN and SHN was in favor of protein accumulation and maintenance with enhanced proportion of essential amino acids, which entitled the algal biomass to be of high quality for animal feed applied in livestock graziery and aquaculture. LN facilitated productive starch and lipid accumulation with good quality for biofuels production. The nitrate removal rate and biomass productivity exceeded most of the microalgae reported in literature under similar conditions, which highlighted Tetraselmis subcordiformis as a potent strain for flexible nitrate-rich wastewater remediation coupled with fast CO 2 bio-mitigation and high-quality biomass production for sustainable algal biorefinery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anaerobic digester effluent containing high levels of ammonia poses a threat to the environment. To hinder this issue, a modern and promising treatment method incorporates both microalgae and their bioconversion potential. When culturing Chlorella vulgaris at a 1:7 digestate supernatant dilution ratio, biomass concentration was 1.33 g L -1 and 66% of ammonia nitrogen was removed. Furthermore, a prior nitrogen-starved seed method, namely over-compensation strategy, was applied to improve both biomass production and nutrient removal. By using nitrogen-starved seeds after a 48 h nitrogen-free stimulation, biomass yield increased by 1.7-times to 2.56 g L -1 . Simultaneously, ammonia nitrogen and total phosphorus removal efficiencies reached 99% and 97% respectively. The enhanced production corresponds to higher chlorophyll fluorescence in the middle and late stages of the culture. In addition, the bioproduct contained 39% carbohydrates, and the proportion of polyunsaturated fatty acids in lipids was 66%. These findings demonstrated that the over-compensation strategy contributed to greater nitrogen removal and high-value bioproduct production in the microalgae-digestate treatment system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Microalgae accumulate starch and lipid as storage metabolites under nutrient depletion, which can be used as sustainable feedstock for biorefinery. Omics analysis coupled with enzymatic and genetic verifications uncovered a partial picture of pathways and important enzymes or regulators related to starch and lipid biosynthesis as well as the carbon partitioning between them under nutrient depletion conditions. Depletion of macronutrients (N, P, and S) resulted in considerable enhancement of starch and/or lipid content in microalgae, but the accompanying declined photosynthesis hampered the achievements of high concentrations. This review summarized the current knowledge on the pathways and the committed steps as well as their carbon allocation involved in starch and lipid biosynthesis, and focused on the manipulation of different nutrients and the alleviation of oxidative stress for enhanced storage metabolites production. The biological and engineering approaches to cope with the conflict between biomass production and storage metabolites accumulation are proposed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microalgae are potential starch producers as alternatives to agricultural crops. This study disclosed the effects and mechanism of phosphorus availability exerted on storage starch production in a starch-producing microalga Tetraselmis subcordiformis in nitrogen starvation conditions. Excessive phosphorus supply facilitated starch production, which differed from the conventional cognition that phosphorus would inhibit transitory starch biosynthesis in plants. Phosphorus enhanced energy utilization efficiency for biomass and storage starch production. ADP-glucose pyrophosphorylase (AGPase), conventionally known to be critical for starch biosynthesis, was negatively correlated to storage starch biosynthesis. Excessive phosphorus supply maintained large cell volumes, enhanced activities of starch phosphorylases (SPs) along with branching enzymes and isoamylases, and increased phosphoenolpyruvate and trehalose-6-phosphate levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. This work highlighted the importance of phosphorus in the production of microalgal starch and provided further evidence for the SP-based storage starch biosynthesis pathway.

In a marine green starch-producing microalga Tetraselmis subcordiformis, the role of starch phosphorylase (SP) in the starch biosynthesis was disclosed by characterizing the enzyme properties and activity variations during the starch accumulation process. TsSP4, a SP isoform accounting for the major SP activity in T. subcordiformis, was unique to be active in a monomer form with a molecular weight of approximately 110kDa. It resembled one of the chloroplast-located SPs (PhoA) in Chlamydomonas reinhardtii with a similarity of 63.3% in sequence, though it possessed the typical L78/80 domain found in the plastidial SPs (Pho1) of higher plants that was absent in PhoA. TsSP4 exhibited moderate sensitivity to ADP-Glc inhibition and had a high activity for longer-chain linear maltooligosacchride (MOS) and amylopectin against highly branched glycogen as the substrates. TsSP4 had 2-fold higher affinity for Glc-1-P in the synthetic direction than for Pi in the phosphorolytic direction, and the catalytic constant k cat for Glc-1-P was 2-fold of that for Pi. Collectively, TsSP4 preferred synthetic rather than phosphorolytic direction. TsSP4 could elongate MOSs even initially with Pi alone in the absence of Glc-1-P, which further supported its synthetic role in the starch biosynthesis. TsSP4 displayed increased activities in the developing and mature stage of starch biosynthesis under nitrogen-starvation conditions, indicating its possible contribution to the amylopectin amplification., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

In the context of sustainable cultivation of microalgae, the present study focused on the use of nitrogen from the hot-water extracted biomass residue of Arthrospira platensis by hydrothermal carbonization (HTC) and the sequential cultivation of the same alga with the HTC aqueous phase (AP). Nearly 90% of the nitrogen recovered from HTC into AP was in the organic form. Under nitrogen-limited condition with HTCAP as nitrogen source the yield and content of carbohydrate were enhanced by 21% and 15% respectively compared with that under the same nitrogen level provided by NaNO3, which entitled HTCAP for the substitution of conventional nitrate. In the same way pilot-scale cultivation of A. platensis in raceway ponds outdoors demonstrated that carbohydrate content of 43.8% DW and productivity of 10.3g/m(2)/d was achieved. Notably 54% of organic nitrogen in the HTCAP could be recycled by cultivation of pre-nitrogen starved A. platensis as seeds under nitrogen limitation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lipids and carbohydrates are main energy storage compounds (ESC) of microalgae under stressed conditions and they are potential feedstock for biofuel production. Yet, the sustainable and commercially successful production of ESC in microalgae needs to consider nitrogen utilization efficiency. Here the impact of different initial cell densities (ICDs) on ESC accumulation in Isochrysis zhangjiangensis under two nitrogen supply modes (an initially equal concentration of nitrogen per-cell in the medium (N1) and an equal total concentration of nitrogen in the culture system (N2)) were investigated. The results demonstrated that the highest ESC yield (1.36gL(-1)) at N1, which included a maximal nitrogen supply in the cultivation system, and the highest ESC content (66.5%) and ESC productivity per mass of nitrogen (3.28gg(-1) (N) day(-1)) at N2, were all obtained under a high ICD of 8.0×10(6)cellsmL(-1). Therefore I. zhangjiangensis qualifies for ESC-enriched biomass production with economized nitrogen utilization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The marine microalga Tetraselmis subcordiformis could photoproduce hydrogen under the regulation of carbonyl cyanide m-chlorophenylhydrazone (CCCP), and a hydrogen production process kinetic analysis was characterized by two peaks, suggesting that two distinct mechanisms might exist in this alga. Therefore, 2D nanoliquid chromatography-tandem mass spectrometry (LC-MS/MS) was introduced to analyze the proteome of samples from different time points. A total of 912 proteins were identified, providing a global view of the cellular responses at the proteomic level. These proteins can be divided into multiple functional groups including stress responses, energy metabolism and redox homeostasis. The quantitative proteomic data provided more details on the electron donors for hydrogen production. During the first stage, photosystem II produced electrons for hydrogen production; during the second stage, metabolites were the major electron donors via nonphotochemical plastoquinone reduction by NADH dehydrogenase.

Fourier transform infrared spectroscopy (FT-IR) was applied in algal strain screening and monitoring cell composition dynamics in a marine microalga Isochrysis zhangjiangensis during algal cultivation. The content of lipid, carbohydrate and protein of samples determined by traditional methods had validated the accuracy of FT-IR method. For algal screening, the band absorption ratios of lipid/amide I and carbo/amide I from FT-IR measurements allowed for the selection of Isochrysis sp. and Tetraselmis subcordiformis as the most potential lipid and carbohydrate producers, respectively. The cell composition dynamics of I. zhangjiangensis measured by FT-IR revealed the diversion of carbon allocation from protein to carbohydrate and neutral lipid when nitrogen-replete cells were subjected to nitrogen limitation. The carbo/amide I band absorption ratio had also been demonstrated to depict physiological status under nutrient stress in T. subcordiformis. FT-IR serves as a tool for the simultaneous measurement of lipid, carbohydrate, and protein content in cell., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Microalgal starch is a potential feedstock for biofuel production. The effects of KNO(3) and MgSO(4) concentrations and light intensity on biomass and starch production by the marine microalga, Tetraselmis subcordiformis, were investigated. Under 200 μmol m(-2) s(-1) irradiance and sulfur-deprived conditions, a starch productivity of 0.62 g L(-1) d(-1) and a starch content of 62.1% based on dry weight (DW) was achieved. A starch content of 54.3% was achieved under low irradiance and nitrogen starvation, which was 6.5% higher than that under nutrient- and light-sufficient conditions. Photosynthetic activity was indispensable for starch accumulation. It is difficult to reach high starch productivity and starch concentration simultaneously. Proper nutrient concentrations are necessary to achieve high starch productivity or starch concentration based on the target. The high starch productivity and starch content suggest that T. subcordiformis is a promising microalgal starch producer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A system coupling ethanol fermentation with microalgae culture was developed, in which CO2 produced during ethanol fermentation was used as carbon source for the growth of Tetraselmis subcordiformis, a microalgae accumulating starch intracellularly. The biomass concentration about 2.0 g DCW/L was achieved within the photobioreactor for the batch culture of 7 days, and intracellular starch accumulation was about 45%. Furthermore, ultrasonic pretreatment and enzymatic hydrolysis were applied to the microalgae biomass, and 71.1% of the intracellular starch was converted into glucose that was fermented sequentially to ethanol by Saccharomyces cerevisiae with an ethanol yield of 87.6% of the theoretical value, indicating that the microalgae biomass could be an alternative feedstock for ethanol production to save grain consumption, and in the meantime mitigate the CO2 emission.

The performance of microalgae-based wastewater treatment processes for ammonium-N (NH4+-N) removal depends on the maintenance of a favorable pH that is critical for minimizing nitrogen escape in the form of free ammonia (NH3) and preventing high-NH3 or extreme-pH stress. This study developed a CO2-inorganic carbon (CO2-IC) buffering system that automatically stabilized pH with the supply of a carbon source for efficient photosynthetic reclamation of NH4+-N by a euryhaline microalga Tetraselmis subcordiformis. The soluble (NaHCO3) and insoluble (CaCO3 and MgCO3) ICs were compared for this purpose. The pH was well controlled in the range of 6.5~8.5 in the CO2-IC system, which was suitable for the photosynthetic growth of T. subcordiformis. The NH4+-N (100 mg/L) was almost completely removed in three days, with the maximum removal rate of 60.13 mg N/L/day and minimal N escape of 19.65% obtained in the CO2-NaHCO3 system. The CO2-IC system also restricted the release of extracellular organic matter by preventing stress conditions. The CO2-NaHCO3 system enabled the highest "normal" starch production suitable for fermentation, while the CO2-CaCO3/MgCO3 system facilitated high-amylose starch accumulation that was conducive to producing bio-based materials and health-promoting ingredients. The proteins accumulated in T. subcordiformis were of good quality for animal feeds. [ABSTRACT FROM AUTHOR]

Thermogravimetric (TG) techniques are widely used in thermal characteristic studies of microalgae combustion and pyrolysis. Ash content is an important parameter for evaluating the cultivation efficiency of photosynthetic microalgae. In this study, microalgal ash content was determined using an optimized protocol that included a heating rate of 20 °C/min from room temperature to 600 °C; a temperature of 600 °C was then maintained for 30 min under an air atmosphere for TG analysis. At temperatures over 600 °C, the ash content was reduced due to the loss of relatively volatile minerals. Four species of microalgae with selectively enriched carbohydrate, protein or lipid contents under different culture conditions were identified via derivative thermogravimetric (DTG) graph analysis, for which characteristic DTG fitting peaks for carbohydrates, protein and lipids were used as reference peaks. Therefore, a qualitative or semi-quantitative analysis of the carbohydrates, proteins and lipids in microalgae based on TG analysis, which concomitantly determines ash content, can be easily performed using only 5–10 mg of sample. The proposed method is rapid and easy, providing composition information for strain screening and physiological studies during microalgae cultivation. [ABSTRACT FROM AUTHOR]

Background: Triacylglycerol (TAG) from photosynthetic microalgae is a sustainable feedstock for biodiesel production. Physiological stress triggers microalgal TAG accumulation. However excessive physiological stress will impair the photosynthesis system seriously thus decreasing TAG productivity because of the low biomass production. Hence, it is critical to quantitatively and timely monitor the degree of the stress while the microalgal cells growing so that the optimal TAG productivity can be obtained. Results: The lack of an on-line monitored indicator has limited our ability to gain knowledge of cellular "health status" information regarding high TAG productivity. Therefore, to monitor the degree of nitrogen stress of the cells, we investigated the correlation between the photosynthetic system II (PS II) quantum yield and the degree of stress based on the high relevancy between photosynthetic reduction and nitrogen stress-induced TAG accumulation in microalgal cells. ΔF/Fm′, which is the chlorophyll fluorescence parameter that reflects the effective capability of PS II, was identified to be a critical factor to indicate the degree of stress of the cells. In addition, the concept of a nitrogen stress index has been defined to quantify the degree of stress. Based on this index and by monitoring ΔF/Fm′ and guiding the supply of nitrogen in culture medium to maintain a stable degree of stress, a stable and efficient semi-continuous process for TAG production has been established. Conclusion: The results indicate that the semi-continuous cultivation process with a controlled degree of stress by monitoring the ΔF/Fm′ indicator will have a significant impact on microalgal TAG production, especially for the outdoor controllable cultivation of microalgae on a large scale. [ABSTRACT FROM AUTHOR]

It's the first comparative transcriptome study of Tetraselmis subcordiformis' H 2 production with carbonyl cyanide m-chlorophenylhydrazone (CCCP) by de novo RNA-seq. The global analysis of the annotated genes and pathways unveiled it a good candidate for biofuels. Focusing on H 2 production, the genes coding active center subunit (HydA) and assembly subunits (HydEF and HydG) of hydrogenase proportionally expressed during the induction but stopped after illuminated; on contrast, the gene of the active centre stabilization subunit (Hyd3) highly expressed during the H 2 evolution, indicating its key role in the prolonged H 2 production. The results also showed genes of HCP4, ppGpp phosphohydrolase and RpoD relating to the hydrogenase expression regulation. On the energy aspect, genes of CF-ATPases and ferredoxin-NADP reductase in chloroplast down-regulated while Mito-ATPase in mitochondria and pyruvate metabolism pathway up-regulated during the H 2 production, consisting with our previous reports on physiological level. [ABSTRACT FROM AUTHOR]