M. Angeles Vargas

Carotenoid content of chlorophycean microalgae : factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta)

Fifteen strains of chlorophycean microalgae have been investigated with regard to their carotenoid profile. Lutein, beta-carotene and violaxanthin were present in virtually all of the strains, lutein, in general, being the most abundant carotenoid, whereas canthaxanthin and astaxanthin were found in some strains only. Chlorella fusca SAG 211-8b, Chlorococcum citriforme SAG 62.80, Muriellopsis sp., Neospongiococcum gelatinosum SAG B 64.80 and Chlorella zofingiensis CCAP 211/14 exhibited high lutein levels, the latter strain containing in addition substantial amounts of astaxanthin. Muriellopsis sp. was further characterized, since besides a high lutein content (up to 35 mg l(-1) culture), it had the highest growth rate (up to 0.17-0.23 h(-1)) and maximal standing cell density (up to 8 x 10(10) cells l(-1) culture). These levels of lutein are in the range of those reported for astaxanthin in Haematococcus and for beta-carotene in Dunaliella, microalgae of recognized interest for the production of these carotenoids. Lutein content of Muriellopsis sp. increased during the exponential phase of growth, with the highest value being recorded in the early stationary phase. Maximum levels of lutein in Muriellopsis sp. cultures were recorded at 20-40 mM NaNO3, 2-100 mM NaCl, 460 micromol photon m(-2) s(-1), pH 6.5 and 28 degrees C, conditions which were, in general, also optimal for cell growth. Growth-limiting conditions, such as pH values of 6 or 9 and a temperature of 33 degrees C, were found to stimulate carotenogenesis in Muriellopsis sp. This strain represents a potential source of lutein, a commercially interesting carotenoid of application in aquaculture and poultry farming, as well as in the prevention of cancer and diseases related to retinal degeneration.

Lutein production by Muriellopsis sp. in an outdoor tubular photobioreactor

The effect of dilution rate, mixing and daily solar cycles on lutein and biomass productivity of the green unicellular alga Muriellopsis sp. has been studied, throughout the year, in an outdoor tubular photobioreactor. Highest productivity values, for both lutein (about 180 mg m(-2) per day) and biomass (about 40 g (dry weight) m(-2) per day) were achieved on May and July. Values for the optimal dilution rate varied, being lower in May (0.06 h(-1)) than in November (0.09 h(-1)). Similar values for photosynthetic efficiency (about 4%) were recorded throughout the year, indicating that optimization of culture conditions was achieved for each experimental period. Along the daily solar cycle, there was a fast increase of lutein content of Muriellopsis sp. in response to irradiance during the early hours of daytime, with maximal lutein content (about 6 mg (g dry weight)(-1)) being recorded at noon, and decreasing slowly, thereafter. An increase in cell growth was observed following the establishment of maximum lutein/chlorophyll ratio, which might indicate a role for lutein in protecting cells from photodamage.

Exopolysaccharide production by the cyanobacterium Anabaena sp. ATCC 33047 in batch and continuous culture

Abstract The halotolerant, filamentous, heterocystous cyanobacterium Anabaena sp. ATCC 33047 released, during the stationary growth phase in batch culture and, at low dilution rate, in continuous culture, large amounts of an exopolysaccharide (EPS) to the culture medium. Different environmental, nutritional and physical parameters affected production and accumulation of the EPS. The presence of either a combined nitrogen source or NaCl at high concentration led to decreased EPS production, without affecting cell growth. In contrast, generation of the EPS was markedly enhanced in response to an increase in either air flow rate, temperature or irradiance. In continuous culture, accumulation of EPS in the medium increased in response to a decrease in the dilution rate, with maximal EPS productivity being reached at a dilution rate of 0.03 h −1 .

Outdoor cultivation of a nitrogen-fixing marine cyanobacterium, Anabaena sp. ATCC 33047.

Optimization of conditions for outdoor production of the nitrogen-fixing cyanobacterium Anabaena sp. ATCC 33047 has been pursued. In open ponds operated under semi-continuous regime biomass productivity values achieved ranged from 9 g (dry weight) m(-2) per day, in winter, to over 20 g m(-2) per day, in summer, provided that key operation parameters, including cell density, were optimized. Under these conditions the efficiency of solar energy conversion by the cells was fairly constant throughout the year, with photosynthetic efficiency values higher than 2%. The cyanobacterial biomass was rich in high-value phycobiliproteins, namely allophycocyanin and phycocyanin, for which open cultures of marine Anabaena represent a most interesting production system. The performance of Anabaena cultures operated under continuous regime in a closed tubular reactor has also been assessed outdoors, in winter. Biomass productivity values similar to those obtained in the ponds have been recorded for the closed system. Additionally, under these conditions, the cells excreted to the medium large amounts of a previously characterized exopolysaccharide, at production rates as high as 35 g m(-2) per day (1.4 g l(-1) per day). Properly operated closed cultures of this strain of Anabaena appear most suitable for outdoor mass production of valuable extracellular polysaccharides.

Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Factors affecting the production of biomass by a nitrogen-fixing blue-green alga in outdoor culture

The effectiveness of an aeration-shaking (air-lift) system for outdoor biomass photoproduction by the N2-fixing filamentous blue-green alga Anabaena variabilis was evaluated and the influence of relevant factors on the productivity of the system was assessed. Air at a flow rate of 60 liters per liter of cell suspension per h was enough, by itself, to promote adequate turbulence and to supply the gaseous nutrients (CO2 and N2) needed for maximal productivity. The addition of either or both, CO2 and combined nitrogen (as KNO3 or NH4Cl), did not result in any increase in productivity. In summer and winter, optimal cell density for a suspension depth of 25 cm was 0·2–0·3 g (dry weight) liter−1 and 0·1–0·2 g (dry weight) liter−1 respectively. Reciprocally, optimal suspension depth for a cell density of 0·2 g (dry weight) liter−1 was 20–25 cm in summer and below 15 cm in winter. Optimal values for pH and temperature were 8·2–8·4 and 30–35°C, respectively. Under optimal conditions, mean productivity values were about 13 g (dry weight) m−2 day−1 in summer and 6 g (dry weight) m−2 day−1 in winter. Net protein content of A. variabilis cells was higher than 50%, and nitrogen accounted for 10% of the dry biomass. From the productivity and nitrogen content data, the N2 fixation rate in outdoor cultures of A. variabilis can be estimated to be higher than 1 g N m−2 day−1, i.e. more than 3 t N per hectare per year when values are extrapolated both in time and area.

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments. Composition and growth performance of ten filamentous heterocystous strains

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growth performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent type being C-phycocyanin, followed by alloppycocyanin, with levels of 17 and 11% d.wt, respectively, in some strains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10% d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains tolerated an irradiance of ca 2000 µmol photon m-2 s-1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited the widest optimum range of both temperature (30–45 and 25–40 °C) and pH (6.5–9.5 and 6.0–9.0) for growth, the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of two phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m-2 d-1 during summer. The growth performance of the allophycocyanin-rich Anabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m-2 d-1.