Javier Vigara

Carotenoids as protective response against oxidative damage in Dunaliella bardawil

In the present work the relation between carotenoids production and cell response mechanisms to oxidative damage was studied. High light intensity and nitrogen starvation, both conditions, which may increase the oxidative damage in microalgae, significantly increased total carotenoids content in Dunaliella bardawil, the effect of N-starvation being more noticeable when acting synergetically with light on carotenoid production. S-starvation stimulated carotenoids production as much as N-starvation. The use of norflurazon, inhibitor of phytoene desaturase that blocks formation of epsilon-carotene from phytoene, caused a decrease of carotenoid content down to 5% that of the control cells incubated without the inhibitor. The decrease in the oxygen consumption rate of D. bardawil cells exposed to norflurazon suggests a connection between carotenoids desaturation and chloroplastic oxygen species dissipation processes reported in the literature for other algae. It is an indication of the carotenoids involvement in chloroplastic response mechanisms to oxidative damage.

Microalgae mediated photoproduction of β-carotene in aqueous–organic two phase systems

Improving productivity is a usual requirement for most biotechnological processes, and the utilisation of two-phase aqueous organic systems has proved to be an effective way to improve the productivity of poorly water-soluble or toxic compounds. The high hydrophobicity of beta-carotene, which is highly demanded by the pharma and agrofood industry, makes it a good candidate for aqueous/organic biphasic photoproduction. In the present work we have investigated the viability of a two-phase system for the production of beta-carotene by the marine microalgae Dunaliella salina using decane as organic phase. Decane, with a logP(octanol) value of 5.6, showed no toxicity to Dunaliella cells for more than 72 h, and its ability for beta-carotene extraction is acceptable. Transferring Dunaliella cells from standard to carotenogenic conditions caused inhibition of chlorophyll production and induced a strong synthesis of beta-carotene. The two-phase aqueous/decane system was stable and beta-carotene content of the cells was increasing during 4-days. About 8% of the total carotenoids produced were excreted and extracted into the decane phase.

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.

Organic solvent toxicity in photoautotrophic unicellular microorganisms

Abstract The use of aqueous-organic two-phase systems in microalgae biocatalyzed processes can improve productivity and overcome lack of water solubility and poor extracellular excretion of many natural products produced by microalgae. In the present work the toxic effect of various solvents belonging to two different homologous series on several marine and freshwater microalgae and one cyanobacteria have been studied. Typical sigmoidal plots were obtained when plotting solvent biocompatibilty versus the Hansh parameter “log Poct” (logarithm of the solvent partition coefficient in a standard octanol/water system). Tolerance of microalgae was found to be intermediate between that reported for bacteria and for plant cell suspensions. Anabaena, a blue-green alga with more similarities with bacteria than the others algae studied, was found the most tolerant. Good correlation was found between the aqueous critical solvent concentration and the Hansh parameter for all the microalgae studied. The membrane critical solvent concentration for each microalgae was calculated applying Osborne’s model. The protective effect of cell immobilization by gel entrapment against solvent toxicity has also been investigated, and surprisingly good results were obtained even for long-term experiments.

Cadmium increases the activity levels of glutamate dehydrogenase and cysteine synthase in Chlamydomonas reinhardtii

Abstract The presence of 300 μM Cd2+ in culture medium was found to be toxic to Chlamydomonas reinhardtii, reducing growth and productivity by about 48%. Approximately 30% of the total cadmium in the medium was accumulated by the alga resulting in 0.88% of the algal dried biomass. Elemental analysis indicated a cadmium-dependent decrease in the C (about 3.2%) and N content (about 7.1%) within C. reinhardtii, while the S content increased by approximately 7.5%. In parallel, Cd2+ produced a significant activation of the aminating glutamate dehydrogenase (EC 1.4.1.2) activity and also NAD+- and NADP+-isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) activities upon 24 h of the exposure to 150 μM of the metal. These data are consistent with the key role of the glutamate dehydrogenase/isocitrate dehydrogenase system to supply the glutamate required for the Cd2+-induction of phytochelatin synthesis in the alga. Moreover, the presence of cadmium in the culture medium enhances the sulfate uptake rate and the components of the cysteine synthase complex within the cells such as the serine acetyltransferase (EC 2.3.1.30) and O-acetyl-L-serine (thiol)lyase (EC 4.2.99.8) activities.

Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii

Despite the biotechnological interest of microalgae, no robust and stable methods for genetic transformation of most microalgal strains exist. The scanty and disperse data about the efficiency of heterologous promoters in microalgae and the use of different transformation methods, DNA quantities and reporter genes in the existing studies makes very difficult a real comparison of their efficiency. Using Chlamydomonas reinhardtii as a host, we have evaluated the efficiency of the heterologous promoters of cauliflower mosaic virus 35S (CaMV 35S) and Agrobacterium nopaline synthase (NOS) genes. These promoters were fused to the paromomycin conferring‐resistance aminoglycoside 3′‐phosphotransferase encoding gene (APHVIII), and C. reinhardtii was transformed by the glass beads agitation method. The transformation efficiency and the APHVIII transcript and protein levels were evaluated in a series of transformants for each promoter. The chimeric promoter HSP70A/RBCS2 and the promoter‐less APHVIII marker gene were used for comparison. We found significantly higher transformation efficiencies and higher level of APHVIII expression in those transformants harboring the NOS promoter than in those transformed with CaMV 35S promoter. The NOS promoter, widely used for genetic manipulation of higher plants, has been very rarely used for the transformation of microalgae. The results shown here suggest the possibilities of this heterologous promoter as an efficient system for the genetic manipulation of microalgae.

Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii

The carotenoid biosynthesis pathway catalyses the synthesis of essential pigments that are crucial for light harvesting and photoprotection in photosynthetic organisms. It allows the production of several commercially important compounds and is the target of many herbicides. In the present work we studied the influence of light on the carotenoid composition and the expression of genes encoding the main steps of the pathway in the freshwater microalga Chlamydomonas reinhardtii. We observed that there is an activation of the xanthophyll cycle in response to high light, but also in response to other stress conditions, such as nitrogen starvation, which has not been reported previously. We analysed the expression level of (1) genes encoding the two first enzymes of the pathway, phytoene synthase and phytoene desaturase; (2) the enzymes responsible for the cyclization of lycopene, lycopene β-cyclase and lycopene ε-cyclase; (3) zeaxanthin epoxidase, which catalyses the epoxidation of zeaxanthin; and (4) the three known carotene hydroxylases, directly involved in the synthesis of xanthophylls from α and β-carotene. Measurements of carotenoid content in the presence of inhibitors of protein and carotenoid synthesis suggest that only one of the two possible routes for the synthesis of zeaxanthin upon transference to high light, either the de novo synthesis of carotenoids or the interconversion of violaxanthin and zeaxanthin, is dependent on protein synthesis. The high increase in the transcript levels of the cytochrome-dependent carotene β- and ε-hydroxylases in response to high light suggests an important role of these enzymes in regulation of xanthophyll synthesis upon light stress. These conclusions may be of high interest if efficient engineering of the pathway is to be accomplished.

A new approach to express transgenes in microalgae and its use to increase the flocculation ability of Chlamydomonas reinhardtii

The choice of strong efficient promoters is a critical step in the development of efficient transformation systems for microalgae; however, the physiological and genetic diversity among microalgae groups makes very difficult to develop standard universal plasmids for a wide number of microalgal species as has been achieved for higher plants. Here, we propose a new approach to express transgenes in microalgae: cotransformation with two naked promoterless genes, a selectable antibiotic-resistant gene and a gene of our interest. These genes are randomly inserted into the nuclear genome, where their transcription relies on their adequate insertion in a region adjacent to an endogenous genomic promoter or in frame with a native gene. In a high percentage of the transformants obtained, both genes are, not only adequately incorporated in the nuclear genome, but also efficiently transcribed and translated. This transformation method is validated in the model microalga Chlamydomonas reinhardtii with the bleomycin-resistant gene from Streptoalloteichus hindustanus (ShBLE) as gene of interest, and it is employed to express a flocculin gene from Saccharomyces bayanus (SbFLO5), which is responsible for the flocculation process in yeasts. Chlamydomonas reinhardtii transformants exhibited self-flocculation abilities between 2- and 3.5-fold higher than the control untransformed strain. The successful cotransformation of C. reinhardtii with two promoterless genes opens doors for the establishment of a universal transformation system based on endogenous promoters, applicable to any microalgal species.

Electron microscopic studies on immobilized growing Chlamydomonas reinhardtii cells

Abstract Electron microscopic observations of Chlamydomonas reinhardtii cells immobilized in agar beads revealed that many colonies are assembled over the gel surface and are enlarged during growth. When the microalga reached the stationary phase of growth, the chlorophyll content in the gel beads remained constant, but the gel surface showed microinterstices that allow cell release to the culture medium. The viability was maintained by the cells; thus, this system could be interesting in applied processes operating continuously.

Effect of Selenate on Viability and Selenomethionine Accumulation of Chlorella sorokiniana Grown in Batch Culture

The aim of this work was to study the effect of Se(+VI) on viability, cell morphology, and selenomethionine accumulation of the green alga Chlorella sorokiniana grown in batch cultures. Culture exposed to sublethal Se concentrations of 40 mg·L−1 (212 μM) decreased growth rates for about 25% compared to control. A selenate EC50 value of 45 mg·L−1 (238.2 μM) was determined. Results showed that chlorophyll and carotenoids contents were not affected by Se exposure, while oxygen evolution decreased by half. Ultrastructural studies revealed granular stroma, fingerprint-like appearance of thylakoids which did not compromise cell activity. Unlike control cultures, SDS PAGE electrophoresis of crude extracts from selenate-exposed cell cultures revealed appearance of a protein band identified as 53 kDa Rubisco large subunit of Chlorella sorokiniana, suggesting that selenate affects expression of the corresponding chloroplast gene as this subunit is encoded in the chloroplast DNA. Results revealed that the microalga was able to accumulate up to 140 mg·kg−1 of SeMet in 120 h of cultivation. This paper shows that Chlorella sorokiniana biomass can be enriched in the high value aminoacid SeMet in batch cultures, while keeping photochemical viability and carbon dioxide fixation activity intact, if exposed to suitable sublethal concentrations of Se.