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Astaxanthin is a valuable and highly demanded ketocarotenoid pigment, for which the chlorophycean microalga Haematococcus pluvialis is an outstanding natural source. Although information on astaxanthin accumulation in H. pluvialis has substantially advanced in recent years, its underlying molecular bases remain elusive. An integrative metabolic and transcriptomic analysis has been performed for vegetative Haematococcus cells, grown both under N sufficiency (green palmelloid cells) and under moderate N limitation, allowing concurrent active cell growth and astaxanthin synthesis (reddish palmelloid cells). Transcriptional activation was noticeable in reddish cells of key enzymes participating in glycolysis, pentose phosphate cycle and pyruvate metabolism, determining the adequate provision of glyceraldehyde 3 phosphate and pyruvate, precursors of carotenoids and fatty acids. Moreover, for the first time, transcriptional regulators potentially involved in controlling astaxanthin accumulation have been identified, a knowledge enabling optimization of commercial astaxanthin production by Haematococcus through systems metabolic engineering.

A promising approach to CO2 abatement is the use of photosynthetic microorganisms. Different microalgae (Chloroccocum sp., Porphyridium purpureum, Scenedesmus vacuolatus) and cyanobacteria (Anabaena PCC7119, Anabaena PCC7120, Anabaena PCC 7937, Nostoc PCC 9202, Nostoc punctiforme) were cultivated in photobioreactors operated as chemostat, simulating light conditions analogous to those prevailing outdoors and compared on their ability to fix CO2 efficiently. Due to its high biomass productivity and CO2 fixation rate, Scenedesmus vacuolatus was selected and the effect of different culture parameters (i.e. dilution rate, temperature, pH and impinging irradiance on surface's photobioreactor) on its biomass productivity and CO2 fixation was further investigated. In optimal culture conditions, S. vacuolatus rendered 0.63 g biomass L− 1 d− 1, resulting 1.15 gCO2 assimilated L− 1 d− 1. Based on the data obtained in this study, a mathematical model was developed to describe growth and CO2 bio-fixation by S. vacuolatus. Finally, the potential of this microalga for carbon capture was further tested using synthetic flue gases as the source of CO2.

Microalgae are a major natural source for a vast array of valuable compounds, including a diversity of pigments, for which these photosynthetic microorganisms represent an almost exclusive biological resource. Yellow, orange, and red carotenoids have an industrial use in food products and cosmetics as vitamin supplements and health food products and as feed additives for poultry, livestock, fish, and crustaceans. The growing worldwide market value of carotenoids is projected to reach over US$1,000 million by the end of the decade. The nutraceutical boom has also integrated carotenoids mainly on the claim of their proven antioxidant properties. Recently established benefits in human health open new uses for some carotenoids, especially lutein, an effective agent for the prevention and treatment of a variety of degenerative diseases. Consumers' demand for natural products favors development of pigments from biological sources, thus increasing opportunities for microalgae. The biotechnology of microalgae has gained considerable progress and relevance in recent decades, with carotenoid production representing one of its most successful domains. In this paper, we review the most relevant features of microalgal biotechnology related to the production of different carotenoids outdoors, with a main focus on beta-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. We compare the current state of the corresponding production technologies, based on either open-pond systems or closed photobioreactors. The potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae is also discussed.

The nitrogen-fixing cyanobacterium Anabaena sp. ATCC 33047, which generates substantial amounts of exopolysaccharide, was immobilized by entrapment within the reticulate network of polyurethane foam discs. The immobilized Anabaena sp. system has been investigated as a potential biosorbent for the removal of cadmium from aqueous solutions. The results showed that it was a highly fast process, with 80 % of the adsorption taking place in the first 10 min, reaching full equilibrium in about 50 min. Data analysis indicated that the behaviour of the system accurately fits to a monolayer adsorption model (Langmuir isotherm). The maximal biosorption capacity determined for the immobilized Anabaena sp. system was as high as 162 mg Cd (II) per gram dry biomass. The outstanding properties established for immobilized Anabaena sp. in polyurethane foam underline the relevance of such a system as an alternative to current treatments of variety effluents or wastewater contaminated with cadmium.

A basic criterion in the selection of microalgae suitable as source of oil for biodiesel should be their actual capacity to produce lipids or, more properly, the fatty acid yield. Performance assessment of ten preselected microalgae under both batch and continuous culture points to the latter approach as the most adequate for evaluating fatty acid productivity. Differences were patent in continuous culture among strains that otherwise had analogous oil accumulation potential under batch culture. Some promising strains under batch culture (like Muriella aurantiaca and Monoraphidium braunii) exhibited, however, values for actual fatty acid productivity lower than 40 mg L-1d-1 in continuous regime. The analysis performed in photochemostat under continuous culture regime revealed the great potential of Chlorococcum olefaciens, Pseudokirchneriella subcapitata and Scenedesmus almeriensis as oil producing microalgae. Fatty acid productivity levels over 90 mg L-1d-1 were recorded for the latter strains under moderate nitrogen limitation, conditions which led to an enrichment in saturated and monounsaturated fatty acids, a more suitable profile as raw material for biodiesel. The continuous culture methodology employed represents a sound procedure for screening microalgae for biofuel production, providing a reliable evaluation of their fatty acid production capacity, under conditions close to those of outdoor production systems

The optimization of the operation, under the climatic conditions of southern Spain, of an experimental plant for β-carotene production by Dunaliella has been pursued. The effects of mixing, culture depth, cell density and dilution cycles on β-carotene and biomass productivity were studied under a semicontinuous culture regime in open tanks outdoors. Using 3 m2-surface containers, the highest productivity values, for both β-carotene and biomass, were recorded with a flow rate of 0.55 m s−1; 10 cm depth; 0.7 − 0.9 × 106cell ml−1, population density; and dilution cycles of two days. An average annual productivity of 1.65 g (dry wt) m−2 d−1 was estimated for Dunaliella biomass, being that for β-carotene of about 0.1 g m−2 d−1. Under these optimized conditions, experiments have been carried out at the Cadiz Bay with 20 m2-surface tanks during a whole-year cycle. The results obtained have validated this location and the operating conditions established as being most appropriate for efficient mass production of β-carotene rich D. salina.

The extent of biological CO2 fixation was evaluated for outdoor cultures of the cyanobacterium Anabaena sp. ATCC 33047. Culture conditions were optimized indoors in bubble-column photochemostats operating in continuous mode, subjected to irradiance cycles mimicking the light regime outdoors. Highest values achieved for CO2 fixation rate and biomass productivity were 1 and 0.6gL-1day-1, respectively. The comparison among different reactors operating simultaneously - open pond, horizontal tubular reactor and vertical flat-panel - allowed to assess their relative efficiency for the outdoor development of Anabaena cultures. Despite the higher volumetric CO2 fixation capacity (and biomass productivity) exhibited by the tubular photobioreactor, yield of the flat-panel reactor was 50% higher than that of the tubular option on a per area basis, reaching values over 35g CO2 fixed m-2d-1. The flat-panel reactor actually represents a most suitable system for CO2 capture coupled to the generation of valuable biomass by Anabaena cultures

Rotifers, used in aquaculture as feed for early larval stages, are poor in carotenoids compared with copepods, the natural most common prey item of marine fish larvae. A minimal sufficient level of carotenoids in fish larvae may be essential for a correct development. The freshwater microalga Muriellopsis sp. has been identified as a potential source of the carotenoid lutein with possible application in aquaculture. Lutein is a potent antioxidant, with potentially beneficial effects as a carotenoid for fish larvae. This study describes the lutein enrichment of the rotifer Brachionus sp. when incubated with homogeneously dispersed suspensions of freeze-dried, lutein-rich Muriellopsis sp. in seawater. A short enrichment time (2 or 2.5 h) resulted in a higher lutein content of the rotifers than a long enrichment time (13.5 or 24 h). Although the concentration of lutein in enriched rotifers, 0.06–0.16 mg (g dry weight) 1 was low compared with its content in Muriellopsis sp. [about 3 mg (g dry weight) 1 ], it was in the range reported for natural copepod populations. Therefore, we conclude that freeze-dried biomass of Muriellopsis sp. can be used to enrich rotifers with lutein, providing an adequate level of this carotenoid for marine fish larvae.

The effect of operation regime and culture system on carotenoid productivity by the halotolerant alga Dunaliella salina has been analyzed. Operation strategies tested included batch and semi continuous regime, as well as a two-stage approach run simultaneously in both, open tanks and closed reactor. The best results were obtained with the closed tubular photobioreactor. The highest carotenoid production (328.8mgcarotenoidl-1 culture per month) was achieved with this culture system operated following the two-stage strategy. Also, closed tubular photobioreactor provided the highest carotenoid contents (10% of dry weight) in Dunaliella biomass and β-carotene abundance (90% of total carotenoids) as well as the highest 9-cis to all-trans β-carotene isomer ratio (1.5 at sunrise). © 2010 Elsevier B.V., The skilful technical assistance of Dr. R. Rodriguez is very much appreciated. This work was supported by Comisión Interministerial de Ciencia y Tecnología (grant no. IFD 1997-1780 co-financed with FEDER funds from EU), IFAPA (CO3-125) and Plan Andaluz de Investigación (group no. CVI131), Spain.

The influence of nitrate and ammonium assimilation on glycogen metabolism has been determined in intact Anacystis nidulans cell actively fixing CO2. Assimilation of nitrate or ammonium resulted in significant decreases in both the incorporation into glycogen of newly fixed carbon and the accumulation of glycogen by the cells, the magnitude of these effects depending on the light intensity. The depression in glycogen synthesis induced by nitrogen assimilation was more marked at low light intensity, and especially when ammonium was the nitrogen source. Under these conditions, specific radioactivity of the glycogen pool was particularly high, indicating enhanced turnover of glycogen. Thus, in addition to a more general depressing effect of nitrogen assimilation on the carbon flow to glycogen, degradation of glycogen appears to be stimulated by ammonium assimilation at low (but not at high) light intensity.

Summary The effect of free Cd 2+ ions on growth, photosynthesis, photosynthetic pigments (chlorophyll and phycobiliproteins), nitrate and ammonium uptake and divalent cation (Mg 2+ , Ca 2+ ) content in Anabaena PCC 7119 grown in a medium containing nitrate or ammonium was investigated. After 96 h of exposure, the metal causes an inhibition of photosynthesis in cells of Anabaena PCC 7119 growing in NH 4 + and NO 3 - media and a decrease in the photosynthetic pigments. Likewise, Cd 2+ toxicity elicited a reduction in the intracellular concentrations of Mg 2+ and Ca 2+ and an inhibition in ammonium uptake, resulting in changes in the cellular structure. The possible relation between the differential response in each culture medium and pH is discussed.

Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O(2) evolution and in CO(2) fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N(2) fixation is independent of its effects on photosynthesis and reductant supply.

Performance of Dunaliella salina cultures outdoors in a closed tubular photobioreactor has been assessed. Optimization of conditions involved verification of the effect of several determining factors on the yield of both biomass and carotenoids. Maximal biomass productivity (over 2g (dry weight) m(-2) d(-1) or 80 gm(-3) d(-1)) was achieved at 38 cm s(-1), flow rate; 2 x 10(9) cells l(-1), initial population density; 25 degrees C, temperature; semi-continuous regime, keeping a cell density interval between 2 x 10(9) and over 4 x 10(9) cells l(-1). Coverage of the tubular loop with a sunshade screen to avoid light-induced damage of cells was essential to maintain growth performance. The cellular beta-carotene level increased significantly during the light period, as also did that of lutein. The rise in the beta-carotene level could be accounted by the 9-cis-isomer, with all-trans-beta-carotene remaining steady during the light period. By sunset, the ratio between 9-cis- and all-trans-isomers of beta-carotene amounted to 1.5, with over 60% of total beta-carotene corresponding to the 9-cis-isomer. Removal of sunshade enhanced carotenoid accumulation by cells to reach up to 10% of dry biomass. Cultivation of Dunaliella in closed tubular photobioreactor, thus represents a suitable approach for the production of a high-quality microalgal biomass enriched in the valuable 9-cis-isomer of beta-carotene and lutein.

The effect of boron on heterocystous and nonheterocystous dinitrogen fixing Cyanobacteria was examined. The absence of boron in culture media inhibited growth and nitrogenase activity in Nodularia sp., Chlorogloeopsis sp., and Nostoc sp. cultures. Examinations of boron-deficient cultures showed changes in heterocyst morphology. However, cultures of nonheterocystous Cyanobacteria, Gloeothece sp. and Plectonema sp., grown in the absence of boron did not show any alteration in growth or nitrogenase activity. These results suggest a requirement of boron only by heterocystous Cyanobacteria. A possible role for this element in the early evolution of photosynthetic organisms is proposed.

Summary The requirement of sodium for photosynthesis and nitrate assimilation in a mutant of Nostoc muscorum, unable to fix dinitrogen, was examined. After 24 h of culture, sodium deficient cells showed changes in the contents of photosynthetic pigments as well as in photosynthesis and nitrate assimilation. Both CO2 consumption and photosynthetic oxygen evolution were reduced when cells were cultured for 24 h in absence of sodium. Similarly nitrate uptake was inhibited in sodium deficient cells. Nitrate reductase activity however was increased by 37% under these conditions. When assays were performed after 2 h of deficiency only the photosynthetic activities (both photofixation of CO2 and photoevolution of O2) were altered by the absence of sodium. These results suggest that the impairment of nitrate assimilation is a secondary effect of sodium deficiency and that the requirement of sodium for photosynthesis is independent of its effects on nitrate assimilation.

The effect of boron on nitrogenase activity has been studied. When cells were dependent on N(2) fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization.

We describe a simple and efficient non-chromatographic method for the purification of murine IgG3 and IgM monoclonal antibodies (MAbs) which takes advantage of their euglobulin properties. Following filtration, ascitic fluid is dialysed against demineralized water and centrifuged at 22,000 X g for 30 min. The resulting precipitate is dissolved in a high salt buffer (0.1 M Tris-HCl, 1 M NaCl, pH 8). A second cycle of dialysis and centrifugation yields a product of high purity. Nine IgG3 MAbs and eight IgM MAbs were purified by this procedure. Recovery was greater than 90% for seven of nine IgG3 MAbs. It was less reproducible for IgM MAbs and ranged from 40% to greater than 90% depending on antibody and batch. Purity was assessed by SDS-polyacrylamide gel electrophoresis. The purified immunoglobulin was essentially free of albumin, transferrin, and other mouse ascites proteins. No loss of antibody function was observed.