Aplicaciones biotecnológicas y mediambientales de la microalga Chlorella sorokiniana

Laburpena

The term microalga is comprised of a heterogeneous group of unicellular microorganisms that perform oxygenic photosynthesis with high capacity to fix CO2 and produce O2, their efficiency being between 10 and 50 times greater than higher plants. In recent years, microalgae have awakened interest as potential CO2 fixer organisms and producers of biofuel with a low carbon footprint. Furthermore, the uses of microalgal biomass is increasing in order to obtain high value compounds for cosmetics, pharmaceutic and human or animal nutritional industries. Moreover, some microalgae can use industrial wastewaters as nutrients or tolerate high concentrations of contaminants, such as heavy metals, subsequently creating the potential to be bioremediation systems. These applications are discussed in detail within the introductory chapter (Chapter 1). This thesis also incorporates a chapter where methodology is described (Chapter 2), and a further four chapters which describes the results obtained using the chlorophyte microalga Chlorella sorokiniana, whose class is Trebouxiophyceae. This microalga not only has a high growth rate, but also is a robust species able to grow under adverse conditions. The studies deepen in the biotechnological and environmental applications of this microalga, including its cultivation with industrial wastewaters (Chapter 3) and heavy metals (Chapter 4). Additionally, the optimization of a genetic modification method through electroporation (Chapter 5), and a comparative between Chlorella sorokiniana and other 57 microalgae relating to amino acids profile and nutritional value (Chapter 6) has been performed. The research described in Chapter 3 shows that the microalga Chlorella sorokiniana can grow in mixotrophic cultures using different industrial wastes. In this chapter, a fed-batch 2 L bioreactor using wine waste lees, and the optimal concentration of nutrients previously selected, was developed, and used to get a high productivity in biomass (11.0 g L-1) and neutral lipids (38% DW) terms. In Chapter 4 the effect that heavy metals, such as copper and cadmium, or metalloids, such as arsenic (III and V), produced in Chlorella sorokiniana was evaluated. The parameters studied were growth, nitrogen assimilation and intracellular accumulation that Chlorella sorokiniana has relating to these heavy metals and metalloids, being the microalga a high cadmium accumulator. Moreover, the effect that these heavy metals produce in gene expression and enzymatic activity levels in different antioxidant enzymes, such as ascorbate peroxidase and catalase, or enzymes involved nitrogen and sulphur assimilation, have been tested, showing a general upregulation for most of these enzymes. Finally, a quantitative proteomic approach that compares the expression of Chlorella sorokiniana proteins with or without cadmium in the culture media was performed. The results enable us to conclude that different metabolic pathways, such as photosynthesis, oxidative phosphorylation, glycolysis, TCA cycle or ribosomal proteins biosynthesis are downregulated with cadmium in the culture medium. However, other pathways, such as photorespiration, antioxidant enzymes, gluconeogenesis, nitrogen and sulphur assimilation or biosynthesis of some amino acids are upregulated under the same conditions. In Chapter 5 the optimization of an electroporation method for nuclear transformation in the microalga Chlorella sorokiniana is described. For this method, different heterologous promoters, such as the CaMV 35S promoter of cauliflower mosaic virus, the nopaline synthase promoter, NOS, from Agrobacterium tumefaciens, and the hybrid promoter HSP70S/RBCS2 from Chlamydomonas reinhardtii were tested. These promoters direct the expression of the resistance paromomycin gene APHVIII. The most suitable electroporation parameters were 2.5 kV of electric field strength and 3 electric pulses. The most efficient promoter for Chlorella sorokiniana is the CaMV 35S promoter, showing higher number of transformants and higher gene and protein expression. Additionally, a cryopreservation method, at -80 °C, using ethylene glycol 5% v/v as cryopreservant and able to guarantee cell viability of transformants over 365 days, was developed. Finally, in Chapter 6, the aminoacidic profile of 57 microalgae belonging to 8 phyla and 38 genera, including Chlorella sorokiniana, is compared. This study includes structural and free amino acids. 17 amino acids were determined by GC-MS, being the most common in almost all the species glutamate/glutamine, aspartate/asparragine, alanine, leucine and lysine. Moreover, miozoa, as phylum, and Ochromonas sp. and Chroomonas salina, as species, showed higher essential amino acids content and, consequently, the highest nutritional value. Furthermore, a dependence between free amino acids profile and taxonomic groups was stablished for most of the microalgae.