Desenvolvimento e aplicação de métodos analíticos para caracterização de biomassas microalgais

Abstract

Microalgal biomass has compounds of commercial importance, lipids, proteins, carbohydrates, pigments and toxins are the most researched and applied. However, synthesis intermediates can also be studied to help understand the metabolic pathways performed by these microorganisms. The objective of this work was to evaluate microalgal metabolites and to characterize biomass obtained from cultivations with different external conditions. Analyzes were carried out with integrated sample preparation methods, and for pigments, specifically, a new extraction method was developed. Except for pigments that are thermally unstable and determined by spectrophotometry or liquid chromatography (LC), gas chromatography (GC) presents itself as a suitable analytical tool for characterizing metabolites. This technique has the advantage of separating and detecting gases, volatile organic compounds, polar (organic acids, amino acids, sugars and sugar alcohols) and non-polar (fatty acids and sterols) metabolites, in addition to high resolution and selectivity. GC was used in the study of the Scenedesmus obliquus metabolomics, allowing the identification and quantification of organic acids, free amino acids and fatty acids. In addition to the precursors, proteins, lipids and total chlorophylls were determined. The synthesis of precursor metabolites was affected by the external cultivation conditions, different photoperiods, 24:0 and 12:12 (hour:hour; light:dark), and also by the phases of cell growth, exponential, stationary and declining. At the end of the cultivations, the highest concentrations of biomass (4020 mg L-1) and protein (47.30%) were obtained in the cultivation with constant lighting. In the 12:12 photoperiod (light:dark) culture, S. obliquus triggered a higher production of lipids (23.00%) and chlorophylls (26.40 mg g-1). The major pigments present in microalgae are chlorophylls and carotenoids, and there are still limitations in the extraction methods applied in these biomasses, such as high solvent consumption and time. For the method development of simultaneous extraction of chlorophylls and carotenoids from the microalgae Spirulina sp. and S. obliquus, different mechanical homogenization times and extracting solvents were evaluated. Satisfactory recovery values, from 101.11-124.18% for most pigments, in relation to the maceration method (MM) used as a reference, were obtained with 30 min of homogenization and extraction with chloroform followed by ethanol (PM30E). In general, the PM30E pigment profiles showed fewer isomers and possible degradation compounds, in addition to a reduction of about five times in the volume of organic solvents and is four times faster than MM. Subsequently, the characterization of S. obliquus biomasses cultivated with 3, 5, 10, 15, 20 and 25% of CO2 was carried out. Cultures with 3, 5, and 10% CO2 showed greater synthesis of amino acids and proteins (greater than 60% of dry biomass). The highest concentrations of chlorophylls were found in cultures with 15, 20 and 25% CO2, 24.23, 23.06 and 30.79 mg g-1. The total carotenoids were three times bigger cultures with 3, 5 and 10% of CO2 and all-trans-β-carotene was the main compound. The synthesis of lipids was intensified with the increase of CO2 percentages, as well as the accumulation of polyunsaturated fatty acids.