Olimpio Montero

Nannochloropsis (Eustigmatophyceae) as source of commercially valuable pigments

Pigment composition and its variation with culture agewere analyzed in six strains of Nannochloropsis(Eustigmatophyceae). The capacity for accumulationof the ketocarotenoids astaxanthin and canthaxanthinwas higher in N. salina and N. gaditanathan in the other strains studied here. Theinfluence of salinity (15 to 100 practical units) onpigment production was studied in N. gaditana,where a defined pattern of variation could not befound apart from a notable increase in zeaxanthin at100‰. In cultures grown in a photobioreactor and athigh cell densities of about 109 cells mL-1,pigment production reached: 350 mg L-1 forchlorophyll a, 50 mg L-1 for violaxanthin,5 mg L-1 for canthaxanthin, 3 mg L-1 forastaxanthin. The highest contents of canthaxanthin andastaxanthin obtained in experiments with N.gaditana were 19.4 and 14.6 ng pigment (106cells)-1, respectively, which accounts for 0.7%dry weight. By means of xanthophyll cycle inductionthrough exposure of cells to high irradiance and at40 °C, conversion of violaxanthin intozeaxanthin may attain up to 70% of the violaxanthincontent, which corresponds to 0.6% dry weight. Theresults indicate that interest in Nannochloropsis as a source of valuable pigments isnot related to its capacity for single pigmentaccumulation, but the availability of a range ofpigments such as chlorophyll a, zeaxanthin,canthaxanthin and astaxanthin, each with highproduction levels.

Extraction of carotenoids and chlorophyll from microalgae with supercritical carbon dioxide and ethanol as cosolvent

The extraction of carotenoids and chlorophylls using carbon dioxide modified with ethanol as a cosolvent is an alternative to solvent extraction because it provides a high-speed extraction process. In the study described here, carotenoid and chlorophyll extraction with supercritical CO(2 )+ ethanol was explored using freeze-dried powders of three microalgae (Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina) as the raw materials. The operation conditions were as follows: pressures of 200, 300, 400 and 500 bar, temperatures of 40, 50 and 60 degrees C. Analysis of the extracts was performed by measuring the absorbance and by using empirical correlations. The results demonstrate that it is necessary to work at a temperature of 50-60 degrees C and a pressure range of 300-500 bar, depending on the type of microalgae, in order to obtain the highest yield of pigments. The best carotenoid/chlorophyll ratios were obtained by using supercritical fluid extraction + cosolvent instead of using conventional extraction. The higher selectivity of the former process should facilitate the separation and purification of the two extracted pigments.

UV-B radiation increases cell permeability and damages nitrogen incorporation mechanisms in Nannochloropsis gaditana

Abstract.This study shows the response of Nannochloropsis gaditana, a marine nannoplanktonic species, exposed to UV radiation for 7 days. PAR, UV-A and UV-B ratios used were within the range likely to be observed in nature, a photoperiod of 12L:12D was maintained, and light irradiances were modified daily to promote cell acclimation. Growth, pigment content, internal nitrogen and carbon content, and photochemical efficiency using PAM fluorometry were assessed in nutrient replete cultures. Cell size, autofluorescence and cell permeability were analysed by flow cytometry. Results showed a cessation of growth after day 3 and a progressive decrease was observed in Fv/Fm values in cultures exposed to UV-B (plus UV-A and PAR). Flow cytometry analysis also demonstrated an increase in membrane permeability caused by UV-B damage. Cells that showed an increase in membrane permeability also exhibited a proportional decrease in cellular nitrogen content. The results support the conclusion that UV-B radiation can affect N. gaditana nitrogen incorporation mechanisms by direct damage or indirectly by damage to membrane structure and to the photosynthetic apparatus with resulting effects on energy and reductant demand. In contrast, the presence of UV-A radiation was beneficial to cells exposed to PAR plus UV-A when compared to those exposed to only-PAR from day 4. This response resulted in cells with a higher nitrogen content and without changes in membrane permeability.

Effects of dissolved inorganic carbon availability on growth, nutrient uptake and chlorophyll fluorescence of two species of marine microalgae

Growth of two species of marine microalgae, namely Nannochloropsis gaditana Lubian (Eustigmatophyceae) and Nannochloris maculata Butcher (Chlorophyceae), was investigated in cultures submitted to three different concentrations of dissolved inorganic carbon (DIC). Cultures of N. gaditana grown in the absence of DIC in the medium and aerated with less than 0.0001% (v/v) CO2 in air (low DIC conditions) showed a reduction in final cell biomass of approximately 56% as compared with the biomass obtained in cultures grown under control conditions (2 mM DIC in the medium and aerated with air-equilibrated levels of CO2, i.e. 0.03% (v/v) CO2). Growth was not observed in N. maculata cultured under low DIC conditions. A concentration of 1% (v/v) CO2 in air (high DIC conditions) did not modify growth of N. gaditana in relation to that in the control-culture but enhaced growth of N. maculata. Nutrient (NO3− and PO43−) uptake was also analyzed under the different growth conditions. The uptake of NO3− and PO43− by N. maculata was dependent on the inorganic carbon level; thus, whereas no nutrient absorption was observed in the low DIC-culture, growth at the highest inorganic carbon concentration caused an accelaration of the uptake. Capacity to use nitrate was restricted in N. gaditana cells under low DIC conditions, but nutrient uptake was similar in cultures adapted to air levels of CO2 and to CO2-enriched air. Chlorophyll fluorescence measurements were used to determine the photochemical efficiency of photosystem II and the non-photochemical quenching. A similar pattern of evolution of the actual quantum yield of photosystem II (φPSII) was observed in all cultures of N. gaditana over the growth period, without development of non-photochemical quenching. In contrast, changes in φPSII of N. maculata differed between treatments and were concurrent with carbon and nutrient availabilities. Non-photochemical quenching rose in this alga when carbon or phosphate limitation constrained proton dissipation from the lumen. Results are discussed in relation to the particular carbon uptake mechanism of each alga.

Markers of Monocyte Activation Revealed by Lipidomic Profiling of Arachidonic Acid-Containing Phospholipids

Stimulated human monocytes undergo an intense trafficking of arachidonic acid (AA) among glycerophospholipidclasses. Using HPLC coupled to electrospray ionization mass spectrometry, we have characterized changes in the levels of AA-containing phospholipid species in human monocytes. In resting cells, AA was found esterified into various molecular species of phosphatidylinositol (PI), choline glycerophospholipids (PCs), and ethanolamine glycerophospholipids (PEs). All major AA-containing PC and PI molecular species decreased in zymosan-stimulated cells; however, no PE molecular species was found to decrease. In contrast, the levels of three AA-containing species increased in zymosan-activated cells compared with resting cells: 1,2-diarachidonyl-glycero-3-phosphoinositol [PI(20:4/20:4)]; 1,2-diarachidonyl-glycero-3-phosphocholine [PC(20:4/20:4)]; and 1-palmitoleoyl-2-arachidonyl-glycero-3-phosphoethanolamine [PE(16:1/20:4)]. PI(20:4/20:4) and PC(20:4/20:4), but not PE(16:1/20:4), also significantly increased when platelet-activating factor or PMA were used instead of zymosan to stimulate the monocytes. Analysis of the pathways involved in the synthesis of these three lipids suggest that PI(20:4/20:4) and PC(20:4/20:4) were produced in a deacylation/reacylation pathway via acyl-CoA synthetase–dependent reactions, whereas PE(16:1/20:4) was generated via a CoA-independent transacylation reaction. Collectively, our results define the increases in PI(20:4/20:4) and PC(20:4/20:4) as lipid metabolic markers of human monocyte activation and establish lipidomics as a powerful tool for cell typing under various experimental conditions.

Effects of high irradiance and temperature on photosynthesis and photoinhibition in Nannochloropsis gaditana Lubián (Eustigmatophyceae)

Photosynthesis and photoinhibition of the marine microalga Nannochloropsis gaditana Lubian (Eustigmatophyceae) have been determined under different irradiance and temperature regimes by using O 2 evolution and pulse amplitude modulated fluorescence techniques. The photochemical efficiency (F v /F m ) decreased to a similar extent (65 % of the initial) at both 25 and 35 °C at high irradiance of white light (1500 μmol m -2 s -1 ) as well as at low irradiance (150 μmol m -2 s -1 ) (75 % of the initial); however, photoinhibition was less pronounced at low irradiance after 4 h of exposure. Recovery of photochemical efficiency at 25 and 35 °C treatments occurred during the first 2 h when transferred to darkness at 25 °C. O 2 evolution showed a similar variation at 25 and 35 °C. Recovery of photosynthesis at 25 and 35 °C treatments was slower than that of photochemical efficiency, taking 24 h to return to the initial values. At 40 °C, fluorescence parameters as well as O 2 evolution dropped after 15 min of exposure at both quanta fluence rates and did not return during the recovery period in darkness. At this temperature, damage to the photosynthetic apparatus was found even at low light, possibly through an effect of temperature on the water-splitting complex. Violaxanthin decreased and antheraxanthin and zeaxanthin increased under high irradiance at all temperatures. This result indicates the activity of the xanthophyll cycle acting as a photoprotection system. According to the data, three processes are proposed to be involved in the acclimation of the photosynthetic apparatus to the irradiance-temperature stress: 1) PSII reaction center turnover accompanied by changes in open PSII reaction center efficiency; these pathways have been found to fluctuate complementary to each other, 2) zeaxanthin-associated dissipation of excess absorbed energy and 3) reduction of the effective cross-section.

Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: Cross talk of secondary metabolite pathways

The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H(+) antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence.

Calcium-independent phospholipase A2-mediated formation of 1,2-diarachidonoyl-glycerophosphoinositol in monocytes.

Phagocytic cells exposed to exogenous arachidonic acid (AA) incorporate large quantities of this fatty acid into choline and ethanolamine glycerophospholipids, and into phosphatidylinositol (PtdIns). Utilizing liquid chromatography coupled to MS, we have characterized the incorporation of exogenous deuterated AA ([2H]AA) into specific PtdIns molecular species in human monocyte cells. A PtdIns species containing two exogenous [2H]AA molecules (1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol) was readily detected when human U937 monocyte‐like cells and peripheral blood monocytes were exposed to [2H]AA concentrations as low as 160 nm to 1 μm. Bromoenol lactone, an inhibitor of Ca2+‐independent phospholipase A2 (iPLA2), diminished lyso‐PtdIns levels, and almost completely inhibited the appearance of 1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol, suggesting the involvement of deacylation reactions in the synthesis of this phospholipid. De novo synthesis did not appear to be involved, as no other diarachidonoyl phospholipid or neutral lipid was detected under these conditions. Measurement of the metabolic fate of 1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol after pulse‐labeling of the cells with [2H]AA showed a time‐dependent, exponential decrease in the level of this phospholipid. These results identify 1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol as a novel, short‐lived species for the initial incorporation of AA into the PtdIns class of cellular phospholipids in human monocytes.

Comparative Sensitivity of Seven Marine Microalgae to Cumulative Exposure to Ultraviolet-B Radiation with Daily Increasing Doses

Abstract A comparative study of the relative sensitivity to ultraviolet radiation (UVR) among seven marine microalgae from the classes Cyanophyceae (Synechococcus sp.), Rhodophyceae (Porphyridium cruentum), Cryptophyceae (Rhodomonas salina), Prymnesiophyceae (Isochrysis galbana), Bacillariophyceae (Chaetoceros sp.), Chlorophyceae (Dunaliella salina), and Prasinophyceae (Tetraselmis suecica), was carried out. Daily exposure for 4 h over a 4-day period with daily increasing doses of UVR (mainly ultraviolet-B, UV-B) led to a continuous decrease in the effective quantum yield (ϕPSII) in Rhodomonas salina, Chaetoceros sp. and Isochrysis galbana. In contrast, Tetraselmis suecica did not show a significant inhibition over the 4-day period of exposure. Synechococcus sp., Dunaliella salina and Porphyridium cruentum were able to restore, at least partially, the values of ϕPSII during the recovery period (white light only, 20 h), even though a significant decrease in ϕPSII was also measured in these algae after each exposure. Relative inhibition, defined here as the decrease in ϕPSII after exposure as compared to the value before exposure and normalised to the latter value (RI = ∆PSII/ϕPSII), was fitted against UV-B irradiance according to an equation of the type y = 1 − (a x)c e−bx, where y is RI, x is the UV-B irradiance (W m−2), and a, b and c are constants determined from the fit, using unweighted and weighted irradiances. From the fit, the UV-B irradiance for 50% relative inhibition of ϕPSII (RI = 0.5, E50 [W m−2]), was calculated for each alga. Rhodomonas salina and Isochrysis galbana exhibited the lowest values of E50 (0.21 and 0.25, respectively, for unweighted irradiance), being the most sensitive algae to UV-B, while the highest value was calculated for Dunaliella salina (0.47 for unweighted irradiance). The E50 could not be calculated for Tetraselmis suecica since this alga showed an RI maximum of 0.1, and was therefore considered as a species which was highly tolerant of UV-B. A rise in optical density of each algal cell suspension at 680 and 750 nm, as well as in chlorophyll a content determined from methanolic extracts, was evident over the experimental period in all of the species except for Isochrysis galbana and Rhodomonas salina. In these two algae, in contrast to the other species, there was a notable increase in the carotenoid/chlorophyll a ratio, although this had negligible effects for the protection against deleterious UV-B. The phycobilin/chlorophyll a ratio showed a gradual decrease with time in the three phycobilin-containing algae, but it was particularly evident in R. salina after the third day of exposure.

Relationship between bio-optical characteristics and photoinhibition of phytoplankton

The relationship between the bio-optical properties of different microalgae and photoinhibition after short-term exposure (15 and 30 min) to solar radiation was analyzed. Photoinhibition was determined as the decrease in oxygen production and in the in vivo-induced chlorophyll fluorescence. Microalgae with different chlorophyll concentrations, cell size and volume were used. Both photoinhibition and recovery of oxygen production and quantum yield were higher after 30 than after 15 min exposure to solar radiation. Photoinhibition was reduced when UV-A and UV-B radiations were eliminated from the solar radiation. The decrease of effective quantum yield and oxygen production was not dependent on cell size, biovolume or chlorophyll concentration in the algal cultures. It was, however, related to the bio-optical property of the cultures. manifested as the specific attenuation coefficient (Kc). As a general response, the inhibition of