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Microalgae characterization methodologies towards biorefinery routes and unit operation
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O conceito de biorrefinaria, quando aplicado à biomassa de microalgas, depende da exploração total de todos os constituintes bioquímicos disponíveis. Os produtos de microalgas são usados num número significativo de aplicações, como indústria alimentar, alimentação animal, cosmética, produtos farmacêuticos, produção de energia, tratamento de efluentes ou bioplásticos. Uma abordagem de biorrefinaria maximiza os usos potenciais desta poderosa matéria-prima.
Considerando os trabalhos exploratórios anteriores realizados na A4F, uma rota da biorrefinaria de Nannochloropsis sp. foi selecionada e está agora a ser implementada, pelo que uma caracterização extensiva desta microalga ao longo do processo de biorrefinaria (como biomassa de entrada ou produto final) foi o principal objetivo deste projeto.
As células de Nannochloropsis sp. são pequenas, cobertas por uma espessa parede celular que protege os produtos intracelulares localizados principalmente em glóbulos ou ligados às membranas celulares, tornando sua extração um desafio. Com o objetivo de desenvolver e validar metodologias de caracterização de biomassa de Nannochloropsis sp., este trabalho centrou-se em métodos analíticos para realizar tanto o fracionamento e quantificação das classes lipídicas disponíveis como a quantificação do teor de proteínas.
Esta tese descreve a otimização da técnica de extração em fase sólida para permitir a caracterização detalhada das classes de lipídios usando uma pequena coluna de sílica Sep-Pak (690mg) e diferentes sistemas de eluição de solvente. O fracionamento das classes de lipídios em lipídios neutros e lípidos polares foi efetivamente alcançado usando a sequência de eluição que compreende 2 fases móveis, são elas Hexano: Éter dietílico (7:1) e Metanol: Acetona: Hexano (2:2:1) respetivamente, conforme descrito por Sá et al.. GC foi usada para análise composicional das classes de lipídios fracionadas.
O Método BCA foi escolhido para este projeto como o método de quantificação de proteínas mais apropriado. Os resultados mostram a importância do rompimento celular como pré-tratamento considerando o teor máximo de proteína obtido de 52.5%, valor em linha com o intervalo descrito por Hulatt et al. (50-55% da biomassa seca), com um erro médio de aproximadamente 2% entre réplicas.
The biorefinery concept when applied to microalgae biomass relies on the full exploration of all the biochemical constituents available. Microalgae products are used in a significant number of applications such as food industry, animal feed, cosmetics, pharmaceuticals, energy production, effluent treatment, or bioplastics. A biorefinery approach maximizes the potential uses of this powerful raw material. Considering previous exploratory works done at A4F, a Nannochloropsis sp. biorefinery route has been selected and is now being implemented, therefore an extensive characterization of this microalgae throughout the biorefinery process (as input biomass or final products) has been the main objective of this project. Nannochloropsis sp. cells are small, covered with a thick cell wall that protects intracellular products mainly located in globules or bound to cell membranes, making its extraction challenging. Aimed at developing and validating methodologies to characterize the Nannochloropsis sp. biomass, this task focused on analytical methods to carry out both the fractionation and quantification of lipid classes available and the quantification of protein content. This thesis describes optimization of solid phase extraction techniques to enable detailed characterization of lipid classes using a small silica Sep-Pak column (690mg) and different elution solvent systems. The fractionation of lipid classes into neutral lipids and polar lipids was effectively reached by using elution sequence comprising 2 mobile phases being Hexane: Diethyl Ether (7:1) and Methanol: Acetone: Hexane (2:2:1) respectively, as described by Sá et al.. GC was used for compositional analysis of the fractionated lipid classes. The BCA Method was chosen for this task as the most appropriate protein quantification method. Results show the importance of cell disruption as a pre-treatment considering the maximum protein content obtained of 52.5%, value in line with range described by Hulatt et al. (50-55% of dry biomass), with an average error of approximately 2% between replicas.
The biorefinery concept when applied to microalgae biomass relies on the full exploration of all the biochemical constituents available. Microalgae products are used in a significant number of applications such as food industry, animal feed, cosmetics, pharmaceuticals, energy production, effluent treatment, or bioplastics. A biorefinery approach maximizes the potential uses of this powerful raw material. Considering previous exploratory works done at A4F, a Nannochloropsis sp. biorefinery route has been selected and is now being implemented, therefore an extensive characterization of this microalgae throughout the biorefinery process (as input biomass or final products) has been the main objective of this project. Nannochloropsis sp. cells are small, covered with a thick cell wall that protects intracellular products mainly located in globules or bound to cell membranes, making its extraction challenging. Aimed at developing and validating methodologies to characterize the Nannochloropsis sp. biomass, this task focused on analytical methods to carry out both the fractionation and quantification of lipid classes available and the quantification of protein content. This thesis describes optimization of solid phase extraction techniques to enable detailed characterization of lipid classes using a small silica Sep-Pak column (690mg) and different elution solvent systems. The fractionation of lipid classes into neutral lipids and polar lipids was effectively reached by using elution sequence comprising 2 mobile phases being Hexane: Diethyl Ether (7:1) and Methanol: Acetone: Hexane (2:2:1) respectively, as described by Sá et al.. GC was used for compositional analysis of the fractionated lipid classes. The BCA Method was chosen for this task as the most appropriate protein quantification method. Results show the importance of cell disruption as a pre-treatment considering the maximum protein content obtained of 52.5%, value in line with range described by Hulatt et al. (50-55% of dry biomass), with an average error of approximately 2% between replicas.
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Palavras-chave
Biorefinaria Microalgas Nannochloropsis sp Extração fase sólida Fracionamento de lípidos Método BCA Biorefinery Microalgae Solid Phase Extraction Lipid Fractionation BCA Method