Luis M. Lubián

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.

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO2 transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H13CO3− caused a rapid initial drop, followed by a slower decline in the extracellular CO2 concentration. Addition of bovine CA to the medium raised the CO2 concentration by restoring the HCO3−–CO2 equilibrium, indicating that cells were taking up CO2 and were maintaining the CO2 concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO2 concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of 18O from 13C18O2 into water. Exchange of 18O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO2 uptake by active transport for use as a carbon source for photosynthesis.

UPTAKE, EFFLUX, AND PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON BY THE MARINE EUSTIGMATOPHYTE NANNOCHLOROPSIS SP.1

Uptake, efflux and utilization of inorganic carbon were investigated in the marine eustigmatophyte Nannochloropsis sp. grown under an air level of CO2. Maximal photosynthetic rate was hardly affected by raising the pH porn 5.0 to 9.0. The apparent photosynthetic affinity for dissolved inorganic carbon (DIC) was 35 μM DIC between pH 6.5 to 9.0, but increased approximately threefold at pH 5.0 suggesting that HCO3‐ was the main DIC species used from the medium. No external carbonic anhydrase (CA) activity could be detected by the pH drift method. However, application of ethoxyzolamide (an inhibitor of CA) resulted an a significant inhibition of photosynthetic O2 evolution and carbon utilization, suggesting involvement of internal CA or CA‐like activity in DIC utilization. Under high light conditions, the rate of HCO3− uptake and its internal conversion to CO2 apparently exceeded the rate of carbon fixation, resulting in a large leak of CO2 from the cells to the external medium. When the cells were exposed to low DIC concentrations, the ratio of internal to external DIC concentration was about eight. On the other hand, in the presence of 2 mM DIC, conditions prevailing in the marine environment, the internal concentration of DIC was only 50% higher than the external one.

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.

Interaction of UV Radiation and Inorganic Carbon Supply in the Inhibition of Photosynthesis: Spectral and Temporal Responses of Two Marine Picoplankters¶

The effect of ultraviolet radiation (UVR) on inhibition of photosynthesis was studied in two species of marine picoplankton with different carbon concentration mechanisms: Nannochloropsis gaditana Lubián possesses a bicarbonate uptake system and Nannochloris atomus Butcher a CO2 active transport system. Biological weighting functions (BWFs) for inhibition of photosynthesis by UVR and photosynthesis vs irradiance (PI) curves for photosynthetically active radiation (PAR) were estimated for both species grown with an enriched CO2 supply (high dissolved inorganic carbon [DIC]: 1% CO2 in air) and in atmospheric CO2 levels (low DIC: 0.03% CO2). The response to UVR and PAR exposures was different in each species depending on the DIC treatment. Under PAR exposure, rates of maximum photosynthesis were similar between treatments in N. gaditana. However, the cultures growing in high DIC had lower sensitivity to UVR than the low DIC cultures. In contrast, N. atomus had higher rates of photosynthesis under PAR exposure with high DIC, but the BWFs were not significantly different between treatments. The results suggest that one or more processes in N. gaditana associated with HCO3− transport are target(s) for UV photodamage because there was relatively less UV inhibition of the high DIC‐grown cultures in which inorganic carbon fixation is supplied by passive CO2 diffusion. Time courses of photochemical efficiency in PAR, during UV exposure and during subsequent recovery in PAR, were determined using a pulse amplitude modulated fluorometer. The results were consistent with the BWFs. In all time courses, a steady state was obtained after an initial decrease, consistent with a dynamic balance between damage and repair as found for other phytoplankton. However, the relationship of response to exposure showed a steep decline in activity that is consistent with a constant rate of repair. A novel feature of a model developed from a constant repair rate is an explicit threshold for photosynthetic response to UV.

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.

Concentrating cultured marine microalgae with chitosan

Chitosan flocculation of 11 marine species of microalgae belonging to six algal classes, of interest for aquaculture purposes, has been studied. Likewise the effect of pH control, prior and further addition chitosan on flocculation efficiency have been tested. When pH was adjusted to 8·0 after chitosan addition results showed flocculation efficiencies equal or superior to 75%, for chitosan concentrations of 40–80 mg litre−1, pH preadjustment of cultures to 6·5 improved flocculation at smaller chitosan concentrations (10 mg litre−1) in several species. Isochrysis galbana has been the only species which did not respond positively to any treatment. No clear relation between flocculation response and algae taxonomic group has been found. It seems that other factors such as cellular volume and cell wall composition play a more important role in flocculation. The results confirm how to obtain optimum microalgae flocculation in large-scale cultures. An adequate pH control is the best alternative to salinity changes, addition of oxidant compounds, etc.

Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae).

A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC(50) values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC(50) values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media.

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.