Development of fermentation process for fungal fucoidanases production

Dissertação de mestrado em Chemical and Biological EngineeringThis thesis is focused in the development of an integral bioprocess for fungal fucoidanases production, in order to develop new strategies of fermentative conditions and parameters. In the recent years, the interest in the research field of fucoidans and fucoidanases has strongly increased due to the high potential in medical exploitation of fucoidans and its degradation products. Fucoidans are highly sulfated polysaccharides of brown algae widely used in fields as food and beverage, pharmacy, health medicine and cosmetics, due to their important biological properties as antitumoral, anticoagulant and antiviral activities. Enzymes with known specificities that catalyze the degradation of fucoidan are important tool for studying the relation between structure and biological role of this class of polysaccharide. Firstly, Aspergillus niger PSH, Mucor sp. 3P, and Penicillium purpurogenum GH2 were selected among several fungal strains, isolated from Northeast Mexican desert, for their fucoidan hydrolyzing ability, by measuring the strains kinetic and morphometric behavior over plate assays containing fucoidan as target polysaccharide and testing different nitrogen sources. Furthermore, submerged fermentations testing the synergy of fucoidan with others sugars for inducing high enzyme titles showed that A. niger PSH synthesized the highest titles of sulfated fucan-degrading enzymes with the culture media of fucoidan-sucrose. Secondly, Fucus vesiculosus algae were collected at Praia Norte, Viana de Castelo, Portugal, for hydrothermal extraction of sulfated polysaccharides (fucoidan) using microwave and autohydrolysis processes. Experimental designs were applied evaluating different conditions of temperature/ pressure, time and alga/water ratio to establish a condition to maximize the extraction results. For microwave assisted extraction at 120 psi, 1 min and 1:25 alga:water rate was the best condition for the fucoidan recovery (18%); whereas the extraction by autohydrolysis showed optimal yield at 180 °C for 20 min (16.5% w/w). The obtained products were characterized and it was verified the presence of fucose as the main constituent of these polysaccharides and a SO3 content higher than 20%. Moreover, as fucoidan is said to possess so many interesting bioactivities, the extracted products were analyzed for antioxidant activity, displaying a strong free radical scavenging effect; this behavior is possibly associated with the high content of sulphate groups. Finally, the solid stated fermentation (SSF) system was assessed for the induction of fucoidanases using the selected fungal strains and the Fucus vesiculosus algae, obtained after both hydrothermal extraction. A rotational drum bioreactor was designed to carry out the culture experiments. The results showed that the maximum fucoidanase activity was obtained with Mucor sp. 3P and algae after autohydrolysis pre-treatment production (3.82 U L-1); also the mixture of the solids showed to influence in the induction of the enzyme production. Moreover, a SSF scale up experiments showed that controlling the moisture content and the addition of inert support on algae substrates increment the enzyme production (9.62 U L-1). These results indicate, that the fucoidan hydrolytic enzyme can be extracellular, induced by a solid substrate rich of fucose and fucoidan. In general, with the established and developed methods of this thesis was possible to determine the solid state fermentation parameters for fucoidan hydrolytic enzyme production using terrestrial fungal strains and also allowed to set up the extraction conditions and physicochemical characteristics for the production of sulfated fucans by environmental friendly process. This opens up new vistas to modify fucoidan and to develop the postulated bioactive potentials.Esta tese foca-se no desenvolvimento de um bioprocesso integral para a produção de fucoidanases fúngicas, a fim de desenvolver novas estratégias de condições fermentativas e parâmetros. Nos últimos anos, o interesse na área de pesquisa de fucoidanos e fucoidanases aumentou significativamente devido ao elevado potencial na exploração médica de fucoidanos e dos seus produtos de degradação. Os fucoidanos são polissacarídeos de algas castanhas, altamente sulfatados, bastante usados em áreas como a dos alimentos e bebidas, farmacêutica, médica e cosmética, devido às suas importantes propriedades biológicas como as suas actividades antitumoral, anticoagulante e antiviral. Enzimas com especificidades conhecidas que catalisam a degradação de fucoidano são uma ferramenta importante para o estudo da relação entre a estrutura e papel biológico desta classe de polissacarídeos. Numa primeira fase, Aspergillus niger PSH, Mucor sp. 3P e Penicillium purpurogenum GH2 foram seleccionadas entre várias estirpes de fungos isoladas do deserto do Nordeste do México, devido à sua capacidade de hidrolisar fucoidano, através da medição do comportamento cinético e morfométrico das estirpes em placas de ensaio contendo fucoidano como polissacarídeo alvo e testando diferentes fontes de azoto. Além disso, as fermentações submersas testando a sinergia de fucoidano com outros açúcares para indução de elevadas actividades enzimáticas evidenciaram que A. niger PSH sintetizou a mais elevada actividade de enzimas que degradam fucanos sulfatados com o meio de cultura de fucoidano-sucrose. Numa segunda fase, algas Fucus vesiculosus foram recolhidas na Praia Norte, Viana do Castelo, Portugal, para extracção hidrotermal dos polissacarídeos sulfatados (fucoidanos) usando processos de microondas e auto-hidrólise. Foram aplicados desenhos experimentais para avaliar diferentes condições de temperatura/pressão, tempo e razão alga/água para estabelecer uma condição para maximizar os resultados de extracção. Na extracção assistida por microondas, 120 psi, 1 min e uma razão de 1:25 água:água, foram as melhores condições para a recuperação de fucoidano (18%); enquanto que a extracção por auto-hidrólise revelou um rendimento óptimo a 180 ºC durante 20 minutos (16.5% w/w). Os produtos obtidos foram caracterizados e verificouse a presença de fucose como o principal constituinte desses polissacarídeos e um conteúdo em SO3 superior a 20%. Além disso, como se diz que o fucoidano possui tantas bioactividades interessantes, os produtos extraídos foram analisados a nível de actividade antioxidante, exibindo um forte efeito na eliminação de radicais livres; este comportamento está possivelmente associado ao elevado conteúdo em grupos sulfato. Finalmente, o sistema de fermentação em estado sólido (FES) foi avaliado para a indução de fucoidanases utilizando as estirpes fúngicas seleccionadas e as algas Fucus vesiculosus, obtidas após extracção hidrotermal. Foi desenhado um bioreactor de tambor rotativo para realizar os ensaios de cultura. Os resultados demonstraram que a actividade máxima de fucoidano foi obtida com Mucor sp. 3P e com algas após produção com pré-tratamento de auto-hidrólise (3.82 U L-1); a mistura dos sólidos também demonstrou a influência na indução da produção da enzima. Além disso, as experiências de aumento de escala de FES mostraram que, controlando o teor de humidade, a adição do suporte inerte em substratos de algas aumenta a produção de enzima (9.62 U L-1). Estes resultados indicam que a enzima que hidrolisa o fucoidano pode ser extracelular, induzida por um substrato sólido rico em fucose e fucoidano. De um modo geral, com os métodos estabelecidos e desenvolvidos nesta tese foi possível determinar os parâmetros de fermentação em estado sólido para produção de enzimas que hidrolisam o fucoidano usando estirpes de fungos terrestres e também foi possível estabelecer as condições de extracção e as características físico-químicas para a produção de fucanos sulfatados por um processo amigo do ambiente. Isto abre novas vistas para modificar o fucoidano e desenvolver os potenciais bioactivos postulados

Enzymatic hydrolysis of hidrothermally pretreated agave bagasse

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Enhancement and modeling of enzymatic hydrolysis on cellulose from Agave bagasse hydrothermally pretreated in a horizontal bioreactor

One of the major challenges in biofuels production from lignocellulosic biomass is the generation of high glucose titers from cellulose in the enzymatic hydrolysis stage of pretreated biomass to guarantee a cost-effective process. Therefore, the enzymatic saccharification on cellulose at high solid loading is an alternative. In this work, the agave bagasse was hydrothermally pretreated and optimized at 194°C/30min, obtaining a pretreated solid rich in cellulose content (>46.46%), and subjected to enzymatic hydrolysis at high solid levels. A horizontal bioreactor was designed for enzyme saccharification at high solid loadings [25% (w/v)]. The bioreactor improved mixing efficiency, with cellulose conversions up to 98% (195.6g/L at 72h). Moreover, mathematical modeling of cellulase deactivation demonstrated that cellulases lose most of their initial activity in the first hours of the reaction. Also, cellulose was characterized by X-ray diffraction, and the pretreated solids were visualized using scanning electron microscopy.This project was funded by the Secretary of Public Education of Mexico - Mexican Science and Technology Council (SEP-CONACYT) with the Basic Science Project-2015-01 (Ref. 254808). Marcela Sofía Pino also thanks the National Council for Science and Technology (CONACYT, Mexico) for her Master Fellowship support (grant number: 611312/452636), and Dr. Michele Michelin thanks the Portuguese Foundation for Science and Technology (FCT) for her postdoctoral fellowship (SFRH/BPD/100786/2014).info:eu-repo/semantics/publishedVersio

Production of mexican brown macroalgae fucoidan and fucosidases under an integral green technology bioproceses by the biorefinery concept

Marine ecosystem can be considered a rather exploited source of natural substances with enormous bioactive potential. In Mexico macro-algae study remain forgotten for research and economic purposes besides the high amount of this resource along the west and east coast. For that reason the Bioferinery Group of the Autonomous University of Coahuila, have been studying the biorefinery concept in order to recover high value byproducts of Mexican brown macro-algae including polysaccharides and enzymes to be applied in food, pharmaceutical and energy industry. Brown macroalgae are an important source of fucoidan, alginate and laminarin which comprise a complex group of macromolecules with a wide range of important biological properties such as anticoagulant, antioxidant, antitumoral and antiviral and also as rich source of fermentable sugars for enzymes production. Additionally, specific enzymes able to degrade algae matrix (fucosidases, sulfatases, aliginases, etc) are important tools to establish structural characteristics and biological functions of these polysaccharides. The aims of the present work were the integral study of bioprocess for macroalgae biomass exploitation by the use of green technologies as hydrothermal extraction and solid state fermentation in order to produce polysaccharides and enzymes (fucoidan and fucoidan hydrolytic enzymes). This work comprises the use of the different bioprocess phases in order to produce high value products with lower time and wastes

Fungal growth on coffee husks and spent ground under solidstatecultivation conditions

The processing of coffee generates significant amounts of agricultural wastes. Coffee husks, comprised of dry outer skin, pulp and parchment, are probably the major residues from the handling and processing of coffee (1). Coffee spent ground is the main coffee industry residue obtained during the processing of raw coffee powder to prepare instant coffee. Coffee husks and spent ground are generated in more than two millions tons yearly (2), and the major problem encountered by the industries is the disposal of these residues, since they contain some amount of caffeine, polyphenols and tannins, which makes them toxic in nature (3). Filamentous fungi are microorganisms able to growth over complex substrates behind minimal conditions, and play an important role in the generation of natural compounds with high commercial interest. Therefore, the aim of the present work was to evaluate the ability of some fungal strains to growth on coffee husks (basically the parchment skin the hull that surrounds the coffee bean), and spent grounds, as an alternative to add value to these toxic residues. Strains from the genus Aspergillus, Penicillium, Mucor and Neurospora were used. Microbial growth was carried out in Petri plates containing 30% of coffee husks or spent ground and 70% of CzapekDox saline media, pH 5.0. The plates were inoculated with a suspension containing 5´106 spores/g dry residue, and maintained at 28ºC for 5 days. The spore suspension was prepared by scrap down the spores from PDA plates with a sterilized solution of 0.2% Tween 80, and counted in a Neubauer chamber. Cultivations were done in duplicate to each fungal in each different substrate. Radial growth rate (Ur, mm/h) was monitored kinetically measuring colony diameters every 12h. All the evaluated fungal strains showed mycelium presence over both residues. For almost all the strains, the invasion capacity was higher in coffee spent ground than in coffee husks. Highest growth rates were obtained with Neurospora crassa, with values of 0.99 and 0.76 mm/h for spent ground and husks, respectively. It was thus concluded that coffee husks and spent grounds can be successfully used as substrate for fungal strains growth. Among the evaluated strains Neurospora crassa gave the best results and could be thus evaluated in solidstate fermentation processes for the obtainment of compounds with commercial interest from these two agroindustrial residues

Identification and evaluation of fungal strains with fucoidan degradation potential

Ten fungal strains isolated of Mexican semi-desert were screened for ability to hydrolyze fucoidan in order to search microorganism capable to produce sulfated fucans-degrading enzymes. Plate assay and liquid fermentation experiments were carried out using Laminaria japonica fucoidan as only carbon source, testing three nitrogen sources. Growth was observed only in Aspergillus niger PSH, Mucor sp., and Penicillum purpurogenum GH2 in fucoidan-urea medium. The activity of fucoidanases was determined by reduced sugars. Aspergillus niger PSH showed the highest activity titles. This research indicate that filamentous fungi, using specific medium, are sources enable to induce active metabolism that act toward this class of polysaccharide

Efecto en las propiedades mecánicas de una resina pinífera modificada biodegradable, al utilizarla como compatibilizante o acoplante en formulaciones elastoméricas sin o con fibra de agave y hule de poli(estireno-butadieno). Un paso hacia la formulación

En este trabajo se valora la eficiencia de una resina de pino modificada biodegradable (A-30), al utilizarla como compatibilizante o acoplante para sustituir al resorcinol (que es tóxico y no es biodegradable), comparando las propiedades mecánicas (contra formulaciones preparadas usando el sistema resorcinol-hexametilentetramina (R-HMT) en: a) polímeros elastoméricos reticulados de estireno-butadieno (SBR), y: b) compósitos de SBR reforzados con fibra de agave de desecho. Para la comparación como compatibilizante, aquí se preparan formulaciones equivalentes con A-30 o R-HMT. Para la comparación de los compósitos equivalentes, se varía el agente de acoplamiento (R-HMT o A-30) y la cantidad de azufre como agente de entrecruzamiento (2,5 partes por cien de elastómero (phr), 7,5 phr o 15 phr). En las pruebas de tracción a temperatura ambiente, y el análisis mecanodinámico con barridos de temperatura (de - 70 °C a 40 °C) y frecuencia (de 0,4 Hz a 40 Hz), se obtuvieron incrementos en módulos (hasta un 74 % en módulo de Young) y en capacidad de deformación (hasta 260 %), para los materiales conteniendo el agente A-30, por encima de las formulaciones en las que se usó el resorcinol. El mejor desempeño como compatibilizante o acoplante del A-30, se reflejó también con claridad morfológicamente mediante microscopía electrónica de barrido, al mostrar mayor compacidad en la matriz formulada y mayor cercanía entre fibra y matriz en muestras fracturadas criogénicamente. Se ha demostrado aquí, que el A-30 puede sustituir al par R-HMT con éxito, ya que funciona mejor como agente compatibilizante o acoplante y es biodegradable. Este tipo de agentes tiene un gran potencial para contribuir al desarrollo de elastómeros y compósitos flexibles amigables con el medio ambiente