The genomes of Vischeria oleaginous microalgae shed light on the molecular basis of hyper-accumulation of lipids.

BACKGROUND: With the urgent need to reduce carbon emissions, and the dwindling reserves of easily exploitable fossil fuel, microalgae-based biofuels that can be used for transport systems and CO2 abatement have attracted great attention worldwide in recent years. One useful characteristic of microalgae is their ability to accumulate high levels of lipid content, in particular under conditions of nitrogen deprivation, with numerous species identified so far. However, a trade-off between levels of lipid accumulation and biomass productivity hinders the commercial applicability of lipids from microalgae. Here, we sequenced the genomes of Vischeria sp. CAUP H4302 and Vischeria stellata SAG 33.83, which can accumulate high content of lipids rich in nutraceutical fatty acids and with excellent biomass yield in nitrogen-limiting culture. RESULTS: A whole-genome duplication (WGD) event was revealed in V. sp. CAUP H4302, which is a rare event in unicellular microalgae. Comparative genomic analyses showed that a battery of genes encoding pivotal enzymes involved in fatty acids and triacylglycerol biosynthesis, storage polysaccharide hydrolysis, and nitrogen and amino acid-related metabolisms are expanded in the genus Vischeria or only in V. sp. CAUP H4302. The most highlighted is the expansion of cyanate lyase genes in the genus Vischeria, which may enhance their detoxification ability against the toxic cyanate by decomposing cyanate to NH3 and CO2, especially under nitrogen-limiting conditions, resulting in better growth performance and sustained accumulation of biomass under the aforementioned stress conditions. CONCLUSIONS: This study presents a WGD event in microalgae, providing new insights into the genetic and regulatory mechanism underpinning hyper-accumulation of lipids and offering potentially valuable targets for future improvements in oleaginous microalgae by metabolic engineering.

Evaluation and Transcriptome Analysis of the Novel Oleaginous Microalga Lobosphaera bisecta (Trebouxiophyceae, Chlorophyta) for Arachidonic Acid Production.

Arachidonic acid (AA) is an omega-6 long-chain polyunsaturated fatty acid and is important for human health. The coccoid green microalga Lobosphaera bisecta has been reported to be able to accumulate high AA content under certain conditions. Nutrient management and light intensity had significant effects on the biomass and accumulation of lipids and AA in L. bisecta SAG2043. Both a high nitrogen concentration (18 mM) and high light intensity (bilateral light-300 µmol m-2 s-1) were beneficial to the growth of L. bisecta, and the replacement of culture medium further enhanced the biomass, which reached 8.9 g L-1. Low nitrogen concentration (3.6 mM) and high light significantly promoted the accumulation of lipids and AA. The highest lipid and AA content reached 54.0% and 10.8% of dry weight, respectively. Lipid compositions analysis showed that 88.2% of AA was distributed within the neutral lipids. We then reconstructed the lipid metabolic pathways of L. bisecta for the first time, and demonstrated that the upregulation of a key desaturase and elongase in the Δ6 pathway was conducive to the accumulation of fatty acids toward AA synthesis. L. bisecta SAG2043 exhibits high biomass, lipid and AA production. It may be a potential candidate for AA production.

Optimum Production Conditions, Purification, Identification, and Antioxidant Activity of Violaxanthin from Microalga Eustigmatos cf. polyphem (Eustigmatophyceae).

Violaxanthin is a major xanthophyll pigment in the microalga Eustigmatos cf. polyphem, but the amount produced after propagation can vary depending upon culture conditions. In this study, the effects of cultivation time, nitrogen concentration, light intensity, and culture mode on violaxanthin production were investigated. The results showed that this microalga vigorously grew and maintained a high level of violaxanthin in the fed-batch culture, and the highest violaxanthin productivity of 1.10 ± 0.03 mg L-1 d-1 was obtained under low light illumination with 18 mM of initial nitrogen supply for ten days. Additionally, violaxanthin was purified from E. cf. polyphem by silica gel chromatography and preparative high-performance liquid chromatography (PHPLC), and identified with high-resolution mass spectrometry (HRMS). The antioxidant activity of the purified violaxanthin was evaluated by three tests in vitro: reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical assays. The strongest inhibition of purified violaxanthin occurred during the scavenging of ABTS⁺ radicals, with EC50 of 15.25 µg mL-1. In conclusion, this is the first report to investigate the effects of different culture conditions on violaxanthin accumulation in E. cf. polyphem and provide a novel source for the production of violaxanthin that can be used for food and pharmaceutical applications.

[Effects of different nitrogen sources and concentrations on starch and lipid biosynthesis by Desmodesmus insignis].

Objective: In order to improve biofuel production by Desmodesmus insignis, we studied the effect of different nitrogen sources and concentrations on the growth, total lipids, carbohydrate and starch accumulation of Desmodesmus insignis. Methods: D. insignis was cultivated in basic general-11 medium containing 5 different initial nitrogen concentrations (3, 6, 9, 12 and 18 mmol/L) supplied in the form of sodium nitrate, ammonium bicarbonate and urea. Biomass was determined by dry weight, total lipids by gravimeter, and carbohydrates and starch by phenolsulfuric acid method. Results: NaNO3, NH4HCO3 and CO(NH2)2 were all suitable for the growth of D. Insignis. When 3 mmol/L NaNO3 was used, the peak lipid content reached to 32.61% (d.w). The maximum content and productivity of carbohydrate and starch were 56.54% (d·w), 55.33% (d·w) and 0.24 g/(L·d), 0.23 g/(L·d) respectively, when 18 mmol/L NH4HCO3 was used. Urea could also obtain relative high content of biomass, total lipids, carbohydrates and starch. Conclusion: Integrating these results with production costs, we could suggest ammonium bicarbonate and urea as nitrogen source at the concentration of 18 mmol/L.

Preliminary characterization, antioxidant properties and production of chrysolaminarin from marine diatom Odontella aurita.

A new chrysolaminarin, named CL2, with a molecular mass of 7.75 kDa, was purified from the marine diatom, Odontella aurita, using DEAE-52 cellulose anion-exchange chromatography and Sephadex G-200 gel-filtration chromatography. The monosaccharide and structural analysis revealed that CL2 was a glucan mainly composed of glucose, which was linked by the ß-d-(1→3) (main chain) and ß-d-(1→6) (side chain) glycosidic bond, demonstrated by infrared spectroscopy (IR) and nuclear magnetic resonance (NMR). The antioxidant activity tests revealed that the CL2 presented stronger hydroxyl radical scavenging activity with increasing concentrations, but less was effective on reducing power analysis and scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The influences of nitrogen concentration and light intensity on chrysolaminarin production of O. aurita were further investigated in a glass column photobioreactor, and a record high chrysolaminarin productivity of 306 mg L-1 day-1 was achieved. In conclusion, the chrysolaminarin CL2 from O. aurita may be explored as a natural antioxidant agent for application in aquaculture, food and pharmaceutical areas.

A novel sulfur supply strategy for maximizing lipid production in Tribonema minus (Xanthophyceae).

Tribonema minus, a promising filamentous oleaginous microalga, was cultured under different nutrient concentrations and different culture modes (fed-batch culture, two-step culture) to study the method of rapid regulation of its lipid metabolism. In contrast to many other oleaginous microalgae, T. minus did not show that nitrogen stress promoted lipid accumulation; however, sulfur deficiency promoted rapid lipid accumulation with a maximum lipid content of 54% of dry weight. Increasing the MgSO4 concentration significantly increased nitrogen uptake and biomass (10.09 g/L). Lipid productivity was significantly increased by the two-step culture using a medium with a high concentration of MgSO4 in the first step and a sulfur-free medium in the second step. In addition, it was found that the lipid content of T. minus was negatively correlated with the intracellular sulfur content when the intracellular sulfur content was below 0.6%. This study provides a new approach for industrial lipid production in T. minus.

The growth, lipid accumulation and adaptation mechanism in response to variation of temperature and nitrogen supply in psychrotrophic filamentous microalga Xanthonema hormidioides (Xanthophyceae).

BACKGROUND: Microalgae are promising feedstocks for production of renewable biofuels and value-added bioproducts. Temperature and nitrogen supply are important environmental and nutritional factors affecting the growth and metabolism of microalgae, respectively. In this study, the growth and lipid accumulation of filamentous microalgae Xanthonema hormidioides under different temperatures (5, 7, 10, 15, 20, 25, 27 and 30 °C) and initial nitrogen concentrations (3, 9, 18 mM) were investigated, and its adaptive mechanisms of tolerance to low temperature and nitrogen stress were analysis by proteomics. RESULTS: The optimum temperature range for the growth of X. hormidioides was between 15 and 20 °C, and the algal cells had slow growth rate at 5 °C and could not survive at 30 °C. The maximum biomass concentration was 11.73 g L-1 under the temperature of 20 °C, and the highest total lipid content was 56.63% of dry weight. Low temperature did not change the fatty acids profiles but promoted the accumulation of unsaturated fatty acids of X. hormidioides. The maximum contents of palmitoleic acid, eicosapentaenoic acid and total fatty acid were 23.64%, 2.49% and 41.14% of dry weight, respectively. Proteomics was performed under three temperature (7, 15, 25 °C), two nitrogen concentrations (3 and 18 mM) and two cultivation times (day 3 and 12). A total of 6503 proteins were identified. In the low temperature, photosynthesis-related proteins were down-regulated to protect the photosynthetic apparatus. The up-regulation of key enzymes DGAT and PDAT demonstrated the accumulation of TAGs under low nitrogen treatment. The proteins related to ribosome, phosphatidylinositol signaling system, antioxidant system and cold shock proteins (CSPs) in X. hormidioides were co-upregulated under the treatment of low temperature, which can alleviate the damages induced by temperature stress and maintain the normal growth and metabolism of algal cells. CONCLUSIONS: X. hormidioides is a psychrotolerant microalga. It is an oleaginous filamentous microalga containing hyper palmitoleic acid and a certain amount of eicosapentaenoic acid with great potential for biofuel development, as well as for applications in nutritional health products and other industries.

Maximizing fucoxanthin production in Odontella aurita by optimizing the ratio of red and blue light-emitting diodes in an auto-controlled internally illuminated photobioreactor.

Fucoxanthin has multiple beneficial effects on human health. However, an efficient cultivation strategy for hyper-production of microalgae-based fucoxanthin has been seldom achieved. Here, an auto-controlled photobioreactor (PBR) installed internal light-emitting diodes illumination with adjustable spectra ratio was firstly used to culture Odontella aurita. The results showed that red light (RL) was more suitable for cell growth and fucoxanthin accumulation than blue light (BL) and white light. The biomass and fucoxanthin production were further promoted by optimizing the ratios of RL and BL, with 8:2 giving the highest productivities of 570 and 9.41 mg L-1 d-1, respectively, which is the highest fucoxanthin productivity ever reported under photoautotrophic cultivation. Pilot-scale cultivation demonstrated its promising feasibility in commercial fucoxanthin production. Our study represents a pioneering work of harnessing the PBR with internal illumination for hyper-production of microalgae-based fucoxanthin, and provides feasible strategies for high-efficient production of other value-added products in related species/strains.

Beneficial Changes in Growth Performance, Antioxidant Capacity, Immune Response, Hepatic Health, and Flesh Quality of Trachinotus ovatus Fed With Oedocladium carolinianum.

The purpose of this study is to assess the feasibility of astaxanthin-rich Oedocladium carolinianum as an immunostimulant in the diet for Trachinotus ovatus. Three experimental diets containing 0% (OC0), 1% (OC1), and 5% (OC5) O. carolinianum powder were formulated for 6-week feeding trials. The results indicated that the OC5 diet boosted the growth performance through decreasing the feed conversion ratio and increasing digestive enzyme activities and intestinal villus length. Meanwhile, fish fed with the OC5 diet promoted antioxidant ability via stimulating the Nrf2-ARE signal pathway and enhancing antioxidant enzyme activities. Furthermore, the OC5 diet exerted hepatoprotective effects by suppressing the lipid deposition and inflammation response and enhancing the transport capacity of cholesterol. Besides, the OC5 diet improved the non-specific immunity by activating the lysozyme and complement system and increasing the nitric oxide content and total nitric oxide synthase activity. Dietary O. carolinianum supplementation promoted the deposition of astaxanthin in the whole body. Therefore, a diet supplemented with 5% O. carolinianum is recommended to boost the growth, antioxidant capacity, immune response, and flesh quality of T. ovatus.

Effects of Barranca yajiagengensis Powder in the Diet of Trachinotus ovatus on the Growth Performance, Antioxidant Capacity, Immunity and Morphology of the Liver and Intestine.

Barranca yajiagengensis, a novel filamentous microalga, can accumulate lutein under high-light and low-nitrogen conditions. It is well known that lutein has antioxidant, anti-inflammatory and immune-modulating properties. The purpose of this study is to evaluate the effects of including lutein-rich B. yajiagengensis powder in the diet of Trachinotus ovatus on the growth performance, antioxidant capacity, immunity, liver, and intestinal morphology. For this aim, three experimental diets containing 0% (BY0), 1% (BY1), and 5% (BY5) B. yajiagengensis powder were formulated for six-week feeding trials. The results indicated that growth performance, feed utilization, and intestinal morphology were not affected by different diet treatments. Fish fed with the BY5 diet promoted antioxidant ability by activating the Nrf2-ARE signal pathway and enhancing antioxidant enzymes activities. Furthermore, the BY5 diet improved non-specific immunity and antibacterial ability by activating lysozymes and the complement system and increasing the nitric oxide (NO) content and total nitric oxide synthase activity. Dietary B. yajiagengensis supplementation improved the liver morphology and exerted hepatoprotective effects. Therefore, as a natural source of lutein, B. yajiagengensis has the potential as a safe and non-toxic immunostimulant for T. ovatus. A diet supplemented with 5% B. yajiagengensis is recommended to improve the growth, antioxidant capacity, immune response, and liver health of T. ovatus.

Physicochemical Characteristics of Cellulose Nanocrystals Derived from the Residue of Filamentous Microalga Tribonema utriculosum.

Tribonema biomass is considered promising biorefinery feedstock for the co-production of biodiesel and valuable bioproducts; however, the extraction of these useful compounds produces large amounts of algal residues, which produce increased environmental concerns. Herein, cellulose was extracted from the waste residue of T. utriculosum via alkalization and bleaching, followed by the production of high-value-added cellulose nanocrystals (CNCs) via acid hydrolysis. The hydrolysis was performed with 60% (wt%) H2SO4 at a yield of 13.31%, resulting in the generation of rod-shaped nanoparticles averaging 39.5 nm in diameter and 239.2 nm in length. The structural characterization analysis revealed that the prepared CNCs had high crystallinity (73.0%) due to the removal of non-cellulose components and amorphous regions by chemical treatment, as well as possessing good aqueous suspension stability (zeta potential = - 40.1 mV). Although the CNCs showed lower thermal stability than extracted cellulose, they spanned a broader temperature range due to two-stage degradation behaviour, with higher residue weight (16.7%). This work represents the first report on the preparation of a high-value-added industrial product, CNCs, from the filamentous microalga T. utriculosum, aiming to maximize benefits from waste algal residue reutilization.

Transcriptomic analysis unravels the modulating mechanisms of the biomass and value-added bioproducts accumulation by light spectrum in Eustigmatos cf. Polyphem (Eustigmatophyceae).

Light spectrum can influence microalgal growth and metabolites accumulation significantly. However, the related mechanism has not been fully elucidated. Here, an oleaginous microalga Eustigmatos cf. polyphem, which also featured with high content of palmitoleic acid (POA) and ß-carotene, was cultured with LEDs-based red light (RL) and blue light (BL). The results showed that the biomass, total lipid content and POA content were much higher under RL than these under BL, regardless of nitrogen concentration. However, the ß-carotene content under RL was significantly lower than that under BL. Transcriptomic analysis revealed that photosynthesis, central carbon metabolism, fatty acid and glycerolipid biosynthesis were elevated, supporting the fast cell growth and high lipid content with POA under RL. In contrast, upregulation of key enzymes in carotenoids biosynthesis and suppression of ß-carotene conversion promoted ß-carotene accumulation under BL. These findings provide a feasible strategy for promoting lipids, POA and ß-carotene in E. cf. polyphem.

Biomass, lipid accumulation kinetics, and the transcriptome of heterotrophic oleaginous microalga Tetradesmus bernardii under different carbon and nitrogen sources.

BACKGROUND: Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass, and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO3 and CO(NH2)2). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. RESULTS: T. bernardii can accumulate high biomass concentrations in heterotrophic batch culture where the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass exceeded 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism-related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely come from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time. CONCLUSION: T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid.

Resourceful treatment of cane sugar industry wastewater by Tribonema minus towards the production of valuable biomass.

Tribonema minus was cultivated in different concentrations of sugarcane wastewater (SW) diluted with mBG-11 medium to produce biomass for biodiesel, bioproduct, and biomaterial production. The results showed that T. minus grew mixotrophically in 50%SW, with the highest biomass accumulation (7.86 g/L) and nutrient removal efficiency (84.85% of nitrogen, 62.57% of phosphorus, and 44.72% of COD). Excluding 100%SW, the chrysolaminarin and cellulose contents increased with increasing SW concentration; the highest contents of 8.11% and 25.69% dry weight were reached in 75%SW, respectively. Although fewer lipids and palmitoleic acid accumulated at higher SW concentrations, their productivities were significantly higher than those in the control due to the higher contribution of biomass. Moreover, the fatty acid profiles produced at the tested concentrations showed superior biodiesel properties. These findings suggested that the addition of mBG-11 medium to SW might be an effective strategy for valuable biomass production in T. minus and SW bioremediation.

Comparative transcriptome analysis of a long-time span two-step culture process reveals a potential mechanism for astaxanthin and biomass hyper-accumulation in Haematococcus pluvialis JNU35.

BACKGROUND: Among all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process. RESULTS: In the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine-urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass. CONCLUSIONS: To the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, ß-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.

Integrated biorefinery strategy for tofu wastewater biotransformation and biomass valorization with the filamentous microalga Tribonema minus.

This study focused on the feasibility of using different concentrations of tofu wastewater (TW) as alternative media for Tribonema minus cultures to produce valuable biorefinery feedstock. T. minus grew mixotrophically in 100% TW with larger carbohydrate (30.99% of dry weight (DW)), protein (15.56% of DW) and chrysolaminarin (6.93% of DW) accumulations than that of in mBG-11 medium. The highest biomass concentration, 7.77 g/L, was achieved in 100% TW, and nutrient removal efficiencies of T. minus at this concentration ranged from 60.49% to 93.60%. Although smaller neutral lipid and palmitoleic acid amounts were detected in 100% TW, their productivities reached 133.77 and 67.19 mg/L/d, respectively, due to the largest biomass yield contribution, which were comparable to those in mBG-11 medium. These findings demonstrated that TW is a promising alternative medium, and an integrated TW biotransformation and biomass valorization process is proposed to achieve better economic performance and environmental sustainability.

Production of fucoxanthin, chrysolaminarin, and eicosapentaenoic acid by Odontella aurita under different nitrogen supply regimes.

Microalgae are recognized as promising producers of many bioactive products, but their utility is limited due to high production costs. We subjected the marine diatom Odontella aurita to three nitrogen supply regimes [initial low nitrogen (ILN), initial high nitrogen (IHN), and initial high nitrogen plus supplementary nitrogen (SN)] to investigate the accumulation of three high-value bioactive components: fucoxanthin, chrysolaminarin and eicosapentaenoic acid (EPA). We found that SN conditions maximized fucoxanthin accumulation: a maximum productivity of 6.01 mg L-1 d-1 was obtained, a 4.32-fold and 1.42-fold increase over production in the ILN and IHN groups, respectively. After nitrogen was depleted in the growth medium, chrysolaminarin became the dominant energy storage compound. Chrysolaminarin content rose to 60.33% of dry weight (DW) in the ILN group, and 46.27% of DW in the IHN group. Variations in fatty acid composition across the different nitrogen supply regimes indicated that EPA primarily accumulated in the glycolipids, especially when nitrogen supply was sufficient. The maximum productivity of chrysolaminarin (161.55 mg L-1 d-1) and EPA (9.37 mg L-1 d-1) was observed in the IHN group. However, IHN conditions did not maximize overall content of either compound. Our results demonstrated that O. aurita is potentially useful as a producer of a variety of bioactive products; the compounds produced by this species can be controlled by altering the nitrogen supply.

Evaluation of oleaginous eustigmatophycean microalgae as potential biorefinery feedstock for the production of palmitoleic acid and biodiesel.

This study aimed to evaluate the potential of six oleaginous eustigmatophytes for use as biorefinery feedstock for the co-production of palmitoleic acid (PA) and biodiesel under different initial nitrogen concentrations (INCs). Six eustigmatophytes were studied, the nitrogen deficiency strategy significantly stimulated the simultaneous hyper-accumulation of PA and lipids, and led to a desirable fatty acid profile (FAP), except in Vacuoliviride sp. and Nannochloropsis oculata. Particularly, Eustigmatos cf. polyphem exhibited great potential when supplied with 1 mM INC and yielded the highest PA (29.71% of dry weight (DW)) and lipid (72.01% of DW) contents, as their productivities increased to 96.26 and 232.79 mg/L/d, respectively. Furthermore, neutral lipids accounted for 91.82% of the total lipids and were rich in PA, and the favourable FAPs of C16-C18 (87.95%) and monounsaturated FAs (70.10%) ensured good biodiesel properties including the cetane number (55.69) and iodine value (92.81 gI2/100 g), and all met the standard requirements.

Effective flocculation of target microalgae with self-flocculating microalgae induced by pH decrease.

A flocculation method was developed to harvest target microalgae with self-flocculating microalgae induced by decreasing pH to just below isoelectric point. The flocculation efficiencies of target microalgae were much higher than those flocculated only via pH decrease. The mechanism could be that negatively charged self-flocculating microalgal cells became positively charged during pH decrease, subsequently attracted negatively charged target microalgae cells to form flocs and settled down due to gravity. Microalgal biomass concentration and released polysaccharide (RPS) from target microalgae influenced flocculation efficiencies, while multivalent metal ions in growth medium could not. Furthermore, neutralizing pH and then supplementing nutrients allowed flocculated medium to be recycled for cultivation. Finally, Spearman's Rank Correlation Coefficients (Rs) between flocculation efficiency and key factors were also investigated. These results suggest that this method is effective, simple to operate and allows the reuse of flocculated medium, thereby contributing to the economic production from microalgae to biodiesel.