Miguel G. Guerrero

Outdoor cultivation of microalgae for carotenoid production: current state and perspectives.

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

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

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 β-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.

Interferon regulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis in three distinct populations

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.

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Background: IRF5 is a transcription factor involved both in the type I interferon and the toll-like receptor signalling pathways. Previously, IRF5 has been found to be associated with systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel diseases. Here we investigated whether polymorphisms in the IRF5 gene would be associated with yet another disease with features of autoimmunity, multiple sclerosis (MS). Methods: We genotyped nine single nucleotide polymorphisms and one insertion-deletion polymorphism in the IRF5 gene in a collection of 2337 patients with MS and 2813 controls from three populations: two case–control cohorts from Spain and Sweden, and a set of MS trio families from Finland. Results: Two single nucleotide polymorphism (SNPs) (rs4728142, rs3807306), and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF5 gene, showed association signals with values of p<0.001 when the data from all cohorts were combined. The predisposing alleles were present on the same common haplotype in all populations. Using electrophoretic mobility shift assays we observed allele specific differences in protein binding for the SNP rs4728142 and the 5 bp indel, and by a proximity ligation assay we demonstrated increased binding of the transcription factor SP1 to the risk allele of the 5 bp indel. Conclusion: These findings add IRF5 to the short list of genes shown to be associated with MS in more than one population. Our study adds to the evidence that there might be genes or pathways that are common in multiple autoimmune diseases, and that the type I interferon system is likely to be involved in the development of these diseases.

Lutein production by Muriellopsis sp. in an outdoor tubular photobioreactor

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.

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

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.

Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans

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 .

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

Abstract The photosynthetic nature of the initial stages of nitrate assimilation, namely, uptake and reduction of nitrate, has been investigated in cells of the cyanobacterium Anacystis nidulans treated with l -methionine dl -sulfoximine to prevent further assimilation of the ammonium resulting from nitrate reduction. The light-driven utilization of nitrate or nitrite by these cells results in ammonium release and is associated with concomitant oxygen evolution. Stoichiometry values of about 2 mol oxygen evolved per mol nitrate reduced to ammonium and 1.5 mol oxygen per mol nitrite have been determined in the presence of CO2, as well as in its absence, with nitrate or nitrite as the only Hill reagent. This indicates that in A. nidulans water photolysis directly provides, without the need for carbon metabolites, the reducing power required for the in vivo reduction of nitrate and nitrite to ammonium, processes which are besides strongly inhibited when the operation of the photosynthetic noncyclic electron flow is blocked. Evidence indicating the participation of concentrative transport system(s) in the uptake of nitrate and nitrite by A. nidulans is also presented. The operation of these energy-requiring systems seems to account for the sensitivity to ATP-synthesis inhibitors exhibited by nitrate and nitrite utilization in l -methionine dl -sulfoximine-treated cells. The utilization of nitrate by A. nidulans cells, concomitant with oxygen evolution, can therefore be considered as a genuinely CO2-independent photosynthetic process that makes direct use of photosynthetically generated assimilatory power.

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

 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.

Regulation of Nitrite Reductase in the Cyanobacterium Anacystis nidulans

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.