Herminia Rodríguez

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.

BIOCHEMICAL COMPOSITION AND FATTY ACID CONTENT OF FILAMENTOUS NITROGEN-FIXING CYANOBACTERIA

 The biochemical composition and fatty acid content of twelve strains of filamentous, heterocystous, nitrogen‐fixing cyanobacteria have been determined. When grown under diazotrophic conditions, protein, carbohydrate, lipid, and nucleic acids comprised 37–52%, 16–38%, 8–13%, and 8–11% of the dry weight, respectively. The presence of a combined nitrogen source resulted in an increase in the protein content of the cells and a decrease in the levels of lipids and carbohydrates, although biomass productivity was not affected significantly. Biochemical composition also changed during culture growth, with the highest levels of proteins and lipids occurring as the culture entered stationary phase, whereas the highest levels of carbohydrate and nucleic acids were found during the exponential phase. Total fatty acid levels in the strains assayed ranged between 3 and 5.7% of the dry weight. With regard to fatty acid composition, all strains showed high levels of polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SAFAs), with values of 24–45% and 31–52% of total fatty acids, respectively, whereas the levels of monounsaturated fatty acids (MUFAs) were in general lower (11– 32%). Palmitic acid (16:0) was the most prevalent SAFA, whereas palmitoleic (16:1n‐ 7) and oleic acid (18:1n‐9) were the most abundant MUFAs in all the strains. Among PUFAs, γ‐linolenic acid (GLA, 18:3n‐6) was present at high levels (18% of total fatty acids) in Nostoc sp. (Chile) and at lower levels (3.6% of total fatty acids) in Anabaenopsis sp. The presence of GLA has not been previously reported in these genera of cyanobacteria. The rest of the strains exhibited high levels (12–35% of total fatty acids) of α‐linolenic acid (ALA, 18:3n‐3). Linoleic acid (18:2n‐6) was also present at a substantial level in most of the strains. Eicosapentaenoic acid (EPA, 20:5n‐3) was also detected in Nostoc sp. (Albufera). Some filamentous nitrogen‐fixing cyanobacteria therefore represent potential sources of commercially interesting fatty acids.

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 Lutein Production in Chlorella sorokiniana (Chorophyta) by Improvement of Culture Conditions and Random Mutagenesis

Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

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.

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.

Overexpression of an exogenous phytoene synthase gene in the unicellular alga Chlamydomonas reinhardtii leads to an increase in the content of carotenoids.

Phytoene synthase (PSY) catalyses the first step in the production of carotenoids, which has been described as a key regulatory step in the carotenoids biosynthetic pathway. PSY gene from Dunaliella salina was constitutively expressed in Chlamydomonas reinhardtii under the control of the RBCS2 and HSP70A promoters and targeted to the chloroplast by the RBCS2 transit peptide. DsPSY overexpression resulted in a stable increase in the corresponding PSY transcript level and in the content of carotenoids such as violaxanthin, lutein, and β‐carotene, reaching between 125 and 260% the levels in control untransformed cells.

ISOLATION AND CHARACTERIZATION OF A LYCOPENE β‐CYCLASE GENE FROM THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

The isolation, characterization, and regulation by light and nitrogen of the lycopene β‐cyclase gene from Chlorella zofingiensis Dönz (CzlcyB), involved in the biosynthesis of astaxanthin and lutein, have been performed in this work. These carotenoids are of high commercial value as dyes in food and as nutraceuticals. The open reading frame (ORF) of CzlcyB encoded a polypeptide of 546 amino acids. A single copy of CzlcyB has been found in C. zofingiensis. The chararacteristic Rossmann or dinucleotide binding fold, present in most lycopene cyclases, has been also identified in the LCYb of C. zofingiensis (CzLCYb). Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to cycle both lycopene and δ‐carotene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the lycopene β‐cyclases (LCYb) of the chlorophycean microalgae studied, being very closely related to LCYb of plants. Transcript levels of CzlcyB were increased under nitrogen deprivation, but no increase was observed under high‐light conditions. However, high irradiance triggered astaxanthin synthesis, while nitrogen deprivation by itself could not induce it. The combination of high irradiance and nitrogen deprivation led to a significant enhancement of the astaxathin accumulation.

Seasonal Differences in Photochemical Efficiency and Chlorophyll and Carotenoid Contents in Six Mediterranean Shrub Species under Field Conditions

The effects of summer and winter stress on the chlorophyll and carotenoid contents and photosystem 2 efficiency were examined in six Mediterranean scrub species. These six species belong to two different plant functional types: drought semi-deciduous (Halimium halimifolium L., Rosmarinus officinalis L., Erica scoparia L.) and evergreen sclerophylls (Juniperus phoenicea L., Pistacia lentiscus L., Myrtus communis L.). Two sites with different water availability were chosen. In the xerophytic site, despite they belong to two different functional types, R. officinalis and J. phoenicea showed a similar response. These were the most affected species in summer. H. halimifolium showed optimal values of Fv/Fm and non-significant seasonal changes in xanthophyll content. In the mesic site, E. scoparia and M. communis were apparently the most affected species by winter climatic conditions. P. lentiscus presented a pattern similar to H. halimifolium, except for elevated F0 values. In all the studied species, lutein plus zeaxanthin content was negatively correlated with Fv/Fm in summer and with leaf water potential, thus indicating that the thermal dissipation of energy was a general pattern for all species. Under stress, plant response is more species-specific than dependent on its functional type.