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Biovalorization of lignocellulosic wastes for sugar alcohols production by the yeast Komagataella pastoris
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Xilitol e arabitol são álcoois de açúcar naturais usados como alternativas à sucrose e adoçantes artificiais, que pertencem à lista de compostos de valor acrescentado a serem produzidos a partir de biomassa de baixo custo. As suas ótimas propriedades e benefícios para a saúde têm atraído a atenção das indústrias alimentares e farmacêuticas, mas as suas aplicações continuam limitadas pelo preço e baixa disponibilidade.
O objetivo principal desta tese foi avaliar, pela primeira vez, a capacidade de levedura Komagataella pastoris DSM 70877 para produzir xilitol e arabitol utilizando resíduos lignocelulósicos como substrato, particularmente, cascas de banana, dreche, carolos de milho, bagaço e engaço de uva e serradura. Uma via biotecnológica promissora e alternativa à produção química em larga escala.
A primeira parte do trabalho focou-se na obtenção de licores ricos em açúcares e fermentáveis, a partir dos diferentes resíduos lignocelulósicos, submetendo-os a uma hidrólise ácida diluída. Na dreche, engaço de uva e serradura revelou-se uma maior recuperação de açúcares, originando hidrolisados com um teor total de monossacarídeos entre 12.9 e 21.5 g/L e diferentes rácios de glucose, xilose e arabinose. Os inevitáveis subprodutos tóxicos do processo foram também detetados nos hidrolisados, nomeadamente, furfural, 5-HMF e ácido acético, e, portanto, na tentativa de os reduzir/eliminar recorreu-se a tratamentos com carvão ativado.
Na segunda parte deste trabalho, os hidrolisados foram testados como substratos para o cultivo de K. pastoris e produção de álcoois de açúcar. Entre os ensaios em Erlenmeyer, o hidrolisado da dreche levou à maior produção de xilitol, 3.97 g/L, com um rendimento de xilitol/xilose de 0.47 g/g. Ensaios em bioreator também foram realizados, em batch e fed-batch, testando diferentes substratos e condições de arejamento. Os substratos selecionados foram dreche, cascas de banana, mistura de dreche (ou engaço de uva) com alimentação de serradura e ainda, uma mistura de três substratos: dreche (ou engaço de uva) e cascas de banana com alimentação de serradura. A levedura alcançou uma produção máxima de xilitol de 1.33 g/L (rendimento 0.18 gxilitol/gxilose) num ensaio em batch, com uma taxa de fluxo de ar de 0.5 L/min e usando como única fonte de carbono o hidrolisado de dreche concentrado 2 vezes e destoxificado.
Apesar das baixas produções alcançadas, neste trabalho provou-se o potencial da levedura para produzir xilitol a partir de hidrolisados hemicelulósicos. Assim, este estudo terá de ser validado e aprofundado para desenvolver e otimizar o processo.
Curiosamente, percebeu-se que a cultura estava a canalizar parte do carbono fornecido pelos hidrolisados hemicelulósicos para outras vias metabólicas e/ou produção de outros compostos, como detetado pela análise de HPLC. Abre-se assim a possibilidade da levedura K. pastoris ser capaz de produzir compostos de grande interesse e valor comercial.
Xylitol and arabitol are natural sugar alcohols used as alternatives to sucrose and artificial sweeteners, that belong to the list of value-added compounds to be produced from low-cost biomass. Their great properties and health benefits have attracted the attention of food and pharmaceutical industries, but their applications are still limited by cost and lack of availability. The main goal of this thesis was to evaluate, for the first time, the ability of the yeast Komagataella pastoris DSM 70877 to produce xylitol and arabitol by using lignocellulosic waste materials as feedstocks, namely, banana peels, brewers’ spent grains (BSG), corncobs, grape pomace, grape stalks and sawdust, a promising biotechnological route as an alternative to the chemical large-scale production. The first part of this work was focused on obtaining sugar-rich and fermentable liquors from different lignocellulosic waste materials, by subjecting them to dilute acid hydrolysis. The higher sugar recoveries were achieved for BSG, grape stalks and sawdust, leading to hydrolysates with total monosaccharides’ contents between 12.9 and 21.5g/L, with different ratios of glucose, xylose and arabinose. The inevitable toxic by-products generated were also detected in the hydrolysates, furfural, 5-HMF and acetic acid, and attempts were done to reduce/eliminate them by treatment with activated charcoal. In the second part of this work, the hydrolysates were tested as substrate for cultivation of K. pastoris and sugar alcohols production. Within shake flask assays, BSG hydrolysate attained the highest xylitol production of 3.97 g/L, with a xylitol/xylose yield of 0.47 g/g. Bioreactor cultivations were also performed in batch and fed-batch modes, testing different feedstocks and aeration conditions. The selected feedstocks were BSG, banana peels, a mixture of BSG (or grape stalks) with feeding of sawdust and triple mixture of BSG (or grape stalks) and banana peels with feeding of sawdust. The yeast achieved a maximum xylitol production of 1.33 g/L (yield of 0.18 gxylitol/gxylose) in a batch cultivation with an airflow rate of 0.5 L/min, using twofold concentrated and detoxified BSG hydrolysate as the sole carbon source. Despite the low productions achieved, this work proved the yeast potential to produce xylitol from hemicellulosic hydrolysates. Thus, further research is required in order to develop and optimize the process. Interestingly, it was noticed that the culture was channeling part of the carbon provided by the hemicellulosic hydrolysates into other metabolic pathways and/or different products, as detected by HPLC analysis. This opens the possibility of K. pastoris to be able to produce other compounds of great interest and commercial value.
Xylitol and arabitol are natural sugar alcohols used as alternatives to sucrose and artificial sweeteners, that belong to the list of value-added compounds to be produced from low-cost biomass. Their great properties and health benefits have attracted the attention of food and pharmaceutical industries, but their applications are still limited by cost and lack of availability. The main goal of this thesis was to evaluate, for the first time, the ability of the yeast Komagataella pastoris DSM 70877 to produce xylitol and arabitol by using lignocellulosic waste materials as feedstocks, namely, banana peels, brewers’ spent grains (BSG), corncobs, grape pomace, grape stalks and sawdust, a promising biotechnological route as an alternative to the chemical large-scale production. The first part of this work was focused on obtaining sugar-rich and fermentable liquors from different lignocellulosic waste materials, by subjecting them to dilute acid hydrolysis. The higher sugar recoveries were achieved for BSG, grape stalks and sawdust, leading to hydrolysates with total monosaccharides’ contents between 12.9 and 21.5g/L, with different ratios of glucose, xylose and arabinose. The inevitable toxic by-products generated were also detected in the hydrolysates, furfural, 5-HMF and acetic acid, and attempts were done to reduce/eliminate them by treatment with activated charcoal. In the second part of this work, the hydrolysates were tested as substrate for cultivation of K. pastoris and sugar alcohols production. Within shake flask assays, BSG hydrolysate attained the highest xylitol production of 3.97 g/L, with a xylitol/xylose yield of 0.47 g/g. Bioreactor cultivations were also performed in batch and fed-batch modes, testing different feedstocks and aeration conditions. The selected feedstocks were BSG, banana peels, a mixture of BSG (or grape stalks) with feeding of sawdust and triple mixture of BSG (or grape stalks) and banana peels with feeding of sawdust. The yeast achieved a maximum xylitol production of 1.33 g/L (yield of 0.18 gxylitol/gxylose) in a batch cultivation with an airflow rate of 0.5 L/min, using twofold concentrated and detoxified BSG hydrolysate as the sole carbon source. Despite the low productions achieved, this work proved the yeast potential to produce xylitol from hemicellulosic hydrolysates. Thus, further research is required in order to develop and optimize the process. Interestingly, it was noticed that the culture was channeling part of the carbon provided by the hemicellulosic hydrolysates into other metabolic pathways and/or different products, as detected by HPLC analysis. This opens the possibility of K. pastoris to be able to produce other compounds of great interest and commercial value.
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Palavras-chave
Lignocellulosic wastes hemicellulosic hydrolysates sugar alcohols xylitol K. pastoris