El estrés abiótico en el metabolismo del nitrógeno y en el sistema antioxidante de la microlga acidófilaCoccomyxa onubensis

Resumo

Microalgae are unicellular eukaryotic microorganisms comprising an incredibly diverse group of multiple shapes and sizes and also, being capable of performing oxygenic photosynthesis. Due to its simple cellular structure, chemical energy obtained from the photosynthetic process is preferentially invested to generate new cells. In this sense, photosynthetic microorganisms constitute the basis of every food chain and, therefore, sustaining life on the planet. Among these organisms, some groups can be found under very restrictive natural conditions whose specific physicochemical properties are sometimes considered extreme for life. Accordingly, the most important variables affecting the functions and structures of cellular components and therefore, establishing an environment as extreme are: temperature, pH, pressure, oxygen availability, salinity and exposure to radiation. The natural properties of the ecosystem clearly affect the physiology of these microorganisms, as it occurs to the microalga object of study of the present Thesis. Coccomyxa onubensis Is an acidophilous microalgae isolated from the rio Tinto mining basin (Huelva). In its natural environment, it is capable of growing at acidic pH (approximately 2.5) and at high concentrations of metals. Nowadays, acidophilic microorganisms research presents as an interesting and useful tool with many biotechnological application; for example, mineral processing with microbial intervention (biomineria) and bioremediation of metal-polluted environments. These processes mediated by microorganisms are considered "clean technologies", as they have lower energy requirements, lower costs and, fundamentally, less environmental impact than traditional non-biological processes. In microalgae, the presence of heavy metals significantly affects the cellular metabolism generating oxidative stress and affecting different metabolic pathways. Therefore, Chapter 1 of this Doctoral Thesis has focused on the study of the behavior of C. onubensis cells under different nitrogen sources (nitrate, ammonium and ammonium nitrate) at neutral pH (7) and acid pH (2.5). The effect of the consumption of these nitrogen sources on the pH of the culture medium has also been evaluated. In addition, it has been tested the role of different metals (copper, cadmium and mercury) and metalloids (arsenite and arsenate ) on the nitrate and ammonium employment by the microalga. It has been found that the pH of the medium has no effect on the consumption of nitrogen, that the consumption of nitrate and ammonium basifies and acidifies the pH of the culture medium respectively. Indeed, nitrate consumption is more sensitive than ammonium to metallic stress. On the other hand, microalgae use the synthesis of phytochelatins to minimize the effects caused by exposure to metals, taking into account the demand for glutamate necessary for the synthesis of phytochelatins. In Chapter 2, evaluation of three enzymes, nitrite reductase, glutamine synthetase and glutamate dehydrogenase, which are involved either directly or indirectly in the Nitrogen biosynthesis pathway, has been carried out. The indicated enzymes have been kinetically and molecularly characterized, and the effect of diverse abiotic stress has been tested. Results have shown inhibition of nitrite reductase and glutamine synthetase, and activation of the dehydrogenated glutamate. Additionally, the presence of heavy metals also causes an increase in the levels of reactive oxygen species (ROS), with hydrogen peroxide (H202) acting as the most determinant compound to signal the stressful changes undergone due to their high stability and lasting half-life. In this context, the role of three enzymes, catalase, ascorbate peroxidase and glutathione reductase, known as ROS scavengers, which participate directly or indirectly in the elimination of H202 has been studied in Chapter 3. The enzymes have been characterized kinetically and molecularly, generally displaying a common increase, especially of the catalase. Finally, in Chapter 4, research on the effects of abiotic stress caused by the presence of heavy metals and metalloids on the cellular growth of C. onubensis has been performed. To conclude, the microalga capacity of bioaccumulation when exposed to copper, cadmium, zinc or arsenic has also been studied resulting on arsenic hiperaccumulation on the C. onubensis microalga, specially under acidic culture conditions.