A rapid UHPLC-HILIC method for algal guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and the potential separation mechanism.

A fast and facile hydrophilic interaction liquid chromatography (HILIC) method was developed and applied to quantify physiologically important ppGpp and its analogues in a tough sample, the astaxanthin-accumulating alga Hameatococcus pluvialis. The method is able to analyze simultaneously seven nucleotides, including ppGpp at the order of pmol g-1 cells within 12 min. Mechanism on the elution order was investigated. It was found that 1) phosphate salt competed for the amide groups on the HILIC column with the phosphate groups of the nucleotides; 2) intramolecular hydrogen bonds might contribute to the elution order by offsetting and reducing the number of free hydrogen acceptor/donor of the nucleotide molecules interacting with the amide groups. This is the first HILIC method for ppGpp, which is feasible and applicable to a wide range of samples, especially tough samples, e.g., algae and plants.

Development of Carbon-Based Solid Acid Catalysts Using a Lipid-Extracted Alga, Dunaliella tertiolecta, for Esterification.

Novel carbon-based solid acid catalysts were synthesized through a sustainable route from lipid-extracted microalgal residue of Dunaliella tertiolecta, for biodiesel production. Two carbon-based solid acid catalysts were prepared by surface modification of bio-char with sulfuric acid (H2SO4) and sulfuryl chloride (SO2Cl2), respectively. The treated catalysts were characterized and their catalytic activities were evaluated by esterification of oleic acid. The esterification catalytic activity of the SO2Cl2-treated bio-char was higher (11.5 mmol Prod.∙h⁻¹âˆ™g Cat. ⁻¹) than that of commercial catalyst silica-supported Nafion SAC-13 (2.3 mmol Prod.∙h⁻¹âˆ™g Cat. ⁻¹) and H2SO4-treated bio-char (5.7 mmol Prod.∙h⁻¹âˆ™g Cat. ⁻¹). Reusability of the catalysts was examined. The catalytic activity of the SO2Cl2-modified catalyst was sustained from the second run after the initial activity dropped after the first run and kept the same activity until the fifth run. It was higher than that of first-used Nafion. These experimental results demonstrate that catalysts from lipid-extracted algae have great potential for the economic and environment-friendly production of biodiesel.

Salinity stress increases lipid, secondary metabolites and enzyme activity in Amphora subtropica and Dunaliella sp. for biodiesel production.

Amphora subtropica and Dunaliella sp. isolated from Tunisian biotopes were retained for their high lipid contents. Respective optimized parameters for rapid growth were: pH 9 and 10, light period 21 and 24h and temperature 31 and 34°C, respectively. After optimization, Amphora subtropica growth rate increased from 0.2 to 0.5day(-1) and Dunaliella sp. growth rate increased from 0.38 to 0.7day(-1). Amphora subtropica biomass production, productivity and lipid content increased from 0.3 to 0.7gL(-1)(dw), 69-100mgL(-1)d(-1)(dw) and 150-190gkg(-1)(dw), respectively, and Dunaliella sp. from 0.5 to 1.4gL(-1)(dw), 124-200mgL(-1)d(-1) (dw) and 190-280gkg(-1)(dw), respectively. Often to overcome trade-off between microalgae rapid growth and high lipid content which are often conflicting and very difficult to obtain at the same time, separation in a growth stage and a lipid accumulation stage is obvious. Salinity stress in a single stage of culture was studied. Compared to the optimal concentration of growth, excess or deficiency of NaCl engendered the same cellular responses by implication of oxidative stress systems and reactivation of defense and storage systems. Indeed, increasing salinity from 1M to 2M for Amphora subtropica or decreasing salinity from 3M to 2M for Dunaliella sp. have both increased lipids content from (220 and 280) to (350 and 430)gkg(-1), carotenoids from (1.8 and 2.4) to (2.3 and 3.7)pgcell(-1), TBARS amount from (10.4 and 5.3) to (12.1 and 10.7)nmolmg(-1) proteins and SOD activity from of (46.6 and 61.8) to (71.6 and 79.4)Umg(-1) proteins, respectively. With further improved fatty acids profile, the microalgae strains could be potent candidates for biofuel production.

Interaction of TGA@CdTe Quantum Dots with an Extracellular Matrix of Haematococcus pluvialis Microalgae Detected Using Surface-Enhanced Raman Spectroscopy (SERS).

The present study reports the localization and interaction of thioglycolic acid (TGA) capped CdTe quantum dots (TGA@CdTe QDs) within the extracellular matrix (ECM) of Haematococcus pluvialis (Chlorophyceae) microalgae (HPM) after an incubation period of 5 min. Changes in the Raman spectrum of HPM induced by the adsorption of the TGA@CdTe QDs are successfully found by using naked gold anisotropic structures as nano-sensors for surface-enhanced Raman scattering (SERS effect). Raman spectroscopy results show that TGA@CdTe QDs interact with the biomolecules present in the ECM. Sample preparation and characterization by complementary techniques such as confocal and electron microscopy are also used to confirm the presence and localization of the nanoparticles in the algae. This research shows new evidence on early accumulation of QDs in plant cells and would further improve our understanding about their environmental impact.

Dissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae.

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600 kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Comparative lipidomic profiling of two Dunaliella tertiolecta strains with different growth temperatures under nitrate-deficient conditions.

The metabolic changes that occur in Dunaliella tertiolecta upon exposure to low temperatures and nitrate deficiency were analyzed by exploring the fatty acid composition and lipid profile of two strains that were acclimated to different temperatures. The results indicate that the levels of linolenic acid (C18:3) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) were significantly higher in the low-temperature (15 °C) strain (SCCAP K-0591) than in a strain grown at 21 °C (UTEX LB999). In addition, DGTS accumulated in LB999 under nitrate-deficient conditions, while the levels of most lipids, including DGTS, remained almost consistent in K-0591. The higher levels of DGTS in K-0591 suggest that DGTS could play a role in adaptation to low temperatures and nitrate deficiency in this organism. The results of this research could be applied to the development of new microalgal strains with tolerance of low temperature and nitrate deficiency by metabolic engineering targeted to DGTS species.

Dunaliella salina exhibits an antileukemic immunity in a mouse model of WEHI-3 leukemia cells.

Dunaliella salina has been shown to have antioxidant property and induce apoptotic cell death of human cancer cells in vitro. However, there is no information available on D. salina showing an antileukemia effect or immunomodulatory activity in vivo. This study applied D. salina to syngeneic leukemia-implanted mice (BALB/c and WEHI-3) to investigate its immunological and antileukemia properties. Oral administration of D. salina (184.5, 369, and 922.5 mg/kg) inhibited spleen metastasis and prolonged the survival in BALB/c mice that had received an intravenous injection of WEHI-3 cells. The results revealed that D. salina had reduced spleen enlargement in murine leukemia. It had also increased the population and proliferation of T-cells (CD3) and B-cells (CD19) following Con A/LPS treatment on flow cytometry and MTT assay, respectively. Furthermore, D. salina increased the phagocytosis of macrophages and enhanced the cytotoxicity of natural killer cells on flow cytometry and LDH assay. Moreover, D. salina enhanced the levels of interferon-γ and interleukin 2 (IL-2) but reduced the levels of IL-4 and IL-10 in leukemic mice. In conclusion, these results demonstrated that the application of D. salina had beneficial effects on WEHI-3 leukemic mice by prolonging survival via modulating the immune responses.

Organization of astaxanthin within oil bodies of Haematococcus pluvialis studied with polarization-dependent harmonic generation microscopy.

Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga, Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.

Antifouling potential of the marine microalga Dunaliella salina.

Marine organisms have usually been viewed as sources of environmentally friendly compounds with antifouling activity. We performed a series of operations to investigate the antifouling potential of the marine microalga Dunaliella salina. For the ethyl acetate crude extract, the antialgal activity was significant, and the EC50 value against Skeletonema costatum was 58.9 µg ml(-1). The isolated purified extract was tested for antifouling activity, the EC 50 value against S. costatum was 21.2 µg ml(-1), and the LC50 against Balanus amphitrite larvae was 18.8 µg ml(-1). Subsequently, both UHR-TOF-MS and GC-MS were used for the structural elucidation of the compounds, and a series of unsaturated and saturated 16- and 18-carbon fatty acids were detected. The data suggested that the fatty acid extracts from D. salina possess high antifouling activity, and could be used as substitutes for potent, toxic antifouling compounds.

Influence of PbS nanoparticle polymer coating on their aggregation behavior and toxicity to the green algae Dunaliella salina.

The potential hazards of nanoparticles (NPs) to the environment and to living organisms need to be considered for a safe development of nanotechnology. In the present study, the potential toxic effects of uncoated and gum Arabic-coated lead sulfide nanoparticles (GA-coated PbS NPs) on the growth, lipid peroxidation, reducing capacity and total carotenoid content of the hypersaline unicellular green algae Dunaliella salina were investigated. Coatings of PbS NPs with GA, as confirmed by Fourier transform infrared spectroscopy, reduced the toxicity of PbS NPs. Uncoated PbS NP toxicity to D. salina was attributed to higher algal cell-NP agglomerate formation, higher lipid peroxidation, lower content of total reducing substances and lower total carotenoid content. Low levels of Pb(2+) in the growth culture media indicate that PbS NP dissolution does not occur in the culture. Also, the addition of 100 µM Pb(2+) to the culture media had no significant (P>0.05) effect on algal growth. The shading of light (shading effect) by PbS NPs, when simulated using activated charcoal, did not contribute to the overall toxic effect of PbS NPs which was evident by insignificant (P>0.05) reduction in the growth and antioxidant capacity of the algae. When PbS NP aggregation in culture media (without algal cells) was followed for 60 min, uncoated form aggregated rapidly reaching aggregate sizes with hydrodynamic diameter of over 2500 nm within 60 min. Effective particle-particle interaction was reduced in the GA-coated NPs. Aggregates of about 440 nm hydrodynamic diameter were formed within 35 min. Afterwards the aggregate size remained constant. It is concluded that PbS NPs have a negative effect on aquatic algae and their transformation by GA capping affects NPs aggregation properties and toxicity.

Lipidomic profiling of snow algae by ESI-MS and silver-LC/APCI-MS.

The main analytical benefit of this study is the development of methods enabling a rapid determination of total lipids of algae by lipidomic analysis and detailed identification and quantification of a complex mixture of natural TAGs by silver-LC/APCI-MS and NARP-LC/APCI-MS. Both types of chromatography can readily identify, both qualitatively and semiquantitatively, triacylglycerols containing 16:3 and 16:4 acids in the molecule. We conclude that the genus Chloromonas is a major producer of C16 PUFAs mostly contained in TAGs. Since more detailed studies in this field have been stymied by the shortage of 16:3 and 16:4 FAs, we decided to study the alga Chloromonas as a potential biotechnological source of C16 PUFAs.

Carotenoid and fatty acid compositions of an indigenous Ettlia texensis isolate (Chlorophyceae) under phototrophic and mixotrophic conditions.

Ettlia oleoabundance (formerly known as Neochloris oleoabundance) is an attractive candidate for biodiesel production because of its high lipid accumulation, and it's taking the majority of the attention among the strains of Ettlia genus; however, potential of the other genus members is unknown. An indigenous strain from Salda Lake (South West Turkey) identified by 18S rDNA sequencing as Ettlia texensis (GenBank accession no: JQ038221), and its fatty acid and carotenoid compositions under phototrophic and mixotrophic conditions was investigated to evaluate the potential of the strain for commercial uses. A threefold increase was observed in total lipid content (total fatty acids; from 13% to 37%) in mixotrophic culture respect to the phototrophic growth conditions. The oleic acid (C18:1) and alpha-linolenic acid (18:3) were the major unsaturated fatty acids accounting for 40% and 13.2% of total fatty acids in mixotrophic culture, respectively. Carotenoid analyses of the mixotrophic culture revealed the metabolite canthaxanthin, a commercially valuable carotenoid used mainly for food coloring, was the major constituent among other pigments. The possible use of E. texensis in biotechnological applications is discussed.

Growth, antioxidant capacity and total carotene of Dunaliella salina DCCBC15 in a low cost enriched natural seawater medium.

Dunaliella is currently drawing worldwide attention as an alternative source of nutraceuticals. Commercially, ß-carotene making up over 10% of Dunaliella biomass is generating the most interest. These compounds, because of their non-toxic properties, have found applications in the food, drug and cosmetic industry. The ß-carotene content of Dunaliella cells, however, depends heavily on the growth conditions and especially on the availability of nutrients, salinity, irradiance and temperature in the growth medium. A chemically well defined medium is usually required, which significantly contributes to the cost of pigment production; hence a desire for low cost marine media. The present study aimed at evaluating the suitability of six different media, especially exploiting local potential resources, for the mass production of Dunaliella salina DCCBC15 as functional food and medicine. The efficacy of a new selected low-cost enriched natural seawater medium (MD4), supplemented with industrial N-P-K fertilizer, was investigated with respect to biomass production, chlorophyll, antioxidant capacity, and total carotene by Dunaliella though culture conditions were not optimized yet. This new medium (MD4) appears extremely promising, since it affords a higher production of Dunaliella biomass and pigments compared with the control, a common artificial medium (MD1), while allowing a substantial reduction in the production costs. The medium is also recommended for culturing other marine algae.

Effects of dietary Dunaliella salina extract and highly unsaturated fatty acids on the fecundity and lipid content of pond-reared Penaeus japonicus brood-stock.

Five basic diets containing fresh squid meat and trash fish were supplemented with different amount of Dunaleilla salina extract (DSE) and highly unsaturated fatty acids (HUFA). Supplemented diets were fed to pond-reared Penaeus japonicus broodstock. Diet A was solely squid and trash fish. Diets B 1 and B2 were supplemented with 400 and 600mg DSE.kg-1 diet, respectively. Diets C1 and C2 were supplemented with HUFA 5 and 10g.kg-1 and 400mg.kg-1DSE, respectively. The results showed that the group fed diet C2 had the best reproductive performance in all experimental groups. It had the highest proportion of spawns (73.5%) and egg production per female (589.0) than all the other experimental groups. The fatty acid composition strongly affected fecundity and stress tolerance of broodstock. The results showed that both HUFA and beta-carotene DSE may play role in stress tolerance and reproductive performance

Production of all trans-beta-carotene by using impinging flow of supercritical carbon dioxide anti-solvent pulverization.

This work investigated column elution chromatography coupled with supercritical anti-solvent precipitation to produce carotenoid rich microsized particulates from microalgal Dunaliella salina species. The extract contained carotenoids ranging from 61.3 mg/g(salina) to 72.5 mg/g(salina) using ultrasonic stirred ethyl ether or tetrahydrofuran (THF) extraction. When 10 L of ethyl alcohol was employed to elute the THF extract, purity of trans-ß-carotene is 823.6 mg/g with a recovery of 86.2%. It was found that the supercritical anti-solvent of THF solution at 160 bar and 318 K produced powdered particulates with a purity of carotenoids above 90%. Subsequently, a central composite response surface design method was used to design supercritical anti-solvent precipitation of carotenoid-rich THF solution. This was accomplished by increasing the pressure from 140 bar to 180 bar and the time from 40 min to 60 min at a feed flow rate of 0.2 mL/min. A CO(2) flow rate of 15 L/min and a temperature of 318 K were also used to determine the effects on purity and recovery of trans-ß-carotene. The combined process produced micronized precipitates with a mean particle size ranging from 3.5 µm to 19 µm and the purity of trans-ß-carotene attained was 926.8 mg/g with a recovery of 54%.

Effects of spray-drying and storage on astaxanthin content of Haematococcus pluvialis biomass.

The main objective of this study was to evaluate the stability of astaxanthin after drying and storage at different conditions during a 9-week period. Recovery of astaxanthin was evaluated by extracting pigments from the dried powders and analysing extracts by HPLC. The powders obtained were stored under different conditions of temperature and oxygen level and the effects on the degradation of astaxanthin were examined. Under the experimental conditions conducted in this study, the drying temperature that yielded the highest content of astaxanthin was 220°C, as the inlet, and 120°C, as the outlet temperature of the drying chamber. The best results were obtained for biomass dried at 180/110°C and stored at -21°C under nitrogen, with astaxanthin degradation lower than 10% after 9 weeks of storage. A reasonable preservation of astaxanthin can be achieved by conditions 180/80°C, -21°C nitrogen, 180/110°C, 21°C nitrogen, and 220/80°C, 21°C vacuum: the ratio of astaxanthin degradation is equal or inferior to 40%. In order to prevent astaxanthin degradation of Haematococcus pluvialis biomass, it is recommended the storage of the spray dried carotenized cells (180/110ºC) under nitrogen and -21°C.

Production, extraction, and quantification of astaxanthin by Xanthophyllomyces dendrorhous or Haematococcus pluvialis: standardized techniques.

For many years, benefits and disadvantages of pigments production either by microalgae or yeasts have been under analysis. In this contribution we shall deal with Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) and Haematococcus pluvialis, which are known as major prominent microorganisms able to synthesize astaxanthin pigment. Then, the usual trend is to look for optimal conditions to conduct astaxanthin synthesis. From one side, pigment production by H. pluvialis is promoted under cellular stress conditions like nutrient deprivation, exposition to high light intensity, aeration. On the other side, X. dendrorhous is able to show significant increase in astaxanthin synthesis when grown in natural carbon sources like coconut milk, grape juice. The main aim of this chapter is to describe optimal environmental conditions for astaxanthin production by X. dendrorhous or H. pluvialis.

Continuous microalgae cultivation in a photobioreactor.

New biomass sources for alternative fuels has become a subject of increasing importance as the nation strives to resolve the economic and strategic impacts of limited fossil fuel resources on our national security, environment, and global climate. Algae are among the most promising non-food-crop-based biomass feedstocks. However, there are currently no commercially viable microalgae-based production systems for biofuel production that have been developed, as limitations include less-than optimal oil content, growth rates, and cultivation techniques. While batch studies are critical for determining basic growth phases and characteristics of the algal species, steady-state studies are necessary to better understand and measure the specific growth parameters. This study evaluated the effects of dilution rate on microalgal biomass productivity, lipid content, and fatty acid profile under steady-state conditions with continuous illumination and carbon dioxide supplemention for two types of algae. Continuous cultures were conducted for more that 3 months. Our results show that the productivity of Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) when the dilution rate varied from 0.08 to 0.64 day(-1). The biomass productivity of C. minutissima reached a maximum value (137 mg/L/day) at a dilution rate of 0.33 day(-1), while the productivity of Dunaliella tertiolecta varied from 46 to 91 mg/L/day at a dilution rate of 0.17 to 0.74 day(-1). The biomass productivity of D. tertiolecta reached a maximum value of 91 mg/L/day at a dilution rate of 0.42 day(-1). Moreover, the lipid content had no significant change with various dilution rates.

Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans.

Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre-log, log, late-log, stationary, and senescent growth phases, and the cells were analyzed by bulk fluorescence emission, flow cytometry, and hyperspectral fluorescence imaging. In addition to intrinsic spectroscopic signatures provided by scatter and endogenous fluorescence, Nile Red staining was employed to monitor relative changes in lipid concentration. Analysis of the fluorescence images and temporal data sets was performed using multivariate curve resolution and fitting to logistic growth models to extract parameters of interest. The spectral components independently isolated from the image and temporal data sets showed close agreement with one another, especially relating to chlorophylls and Nile Red in polar and neutral lipid fractions, respectively. The fastest accumulation and highest total neutral lipid per cell and per chlorophyll were obtained with punctuated nitrate limitation during log phase growth on day 4 of culture. The presence of unbound chlorophyll in the resulting lipid bodies supports a membrane recycling TAG accumulation mechanism mediated by chloropolast-ER lipid exchange. Furthermore, an increase in cell size, indicated by forward scatter, was also found to correlate with increased neutral lipid, providing a size selection mechanism for passive harvest of algal cells at peak lipid enrichment.

An intervention study in obese mice with astaxanthin, a marine carotenoid--effects on insulin signaling and pro-inflammatory cytokines.

Astaxanthin (ASX), a xanthophyll carotenoid from the marine algae Hematococcus pluvialis, has anti-obesity and insulin-sensitivity effects. The specific molecular mechanisms of its actions are not yet established. The present study was designed to investigate the mechanisms underlying the insulin sensitivity effects of ASX in a non-genetic insulin resistant animal model. A group of male Swiss albino mice was divided into two and fed either a starch-based control diet or a high fat-high fructose diet (HFFD). Fifteen days later, mice in each dietary group were divided into two and were treated with either ASX (6 mg kg(-1) per day) in olive oil or olive oil alone. At the end of 60 days, glucose, insulin and pro-inflammatory cytokines in plasma, lipids and oxidative stress markers in skeletal muscle and adipose tissue were assessed. Further, post-receptor insulin signaling events in skeletal muscle were analyzed. Increased body weight, hyperglycemia, hyperinsulinemia and increased plasma levels of tumor necrosis factor-α and interleukin-6 observed in HFFD-fed mice were significantly improved by ASX addition. ASX treatment also reduced lipid levels and oxidative stress in skeletal muscle and adipose tissue. ASX improved insulin signaling by enhancing the autophosphorylation of insulin receptor-ß (IR-ß), IRS-1 associated PI3-kinase step, phospho-Akt/Akt ratio and GLUT-4 translocation in skeletal muscle. This study demonstrates for the first time that chronic ASX administration improves insulin sensitivity by activating the post-receptor insulin signaling and by reducing oxidative stress, lipid accumulation and proinflammatory cytokines in obese mice.