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

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

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

Solid-state fermentation: a strategy for biological detoxification of coffee industry residues

Coffee is the second largest traded commodity in the world, after petroleum, and therefore, the coffee industry is responsible for the generation of large amount of residues. Among these residues, coffee silverskin (CS) and spent coffee grounds (SCG) are generated in significant amounts and merit special attention. Despite the large generation, most of these residues are unutilized, being discharged to the environment or burned for elimination, which are not environmentally friendly techniques. The discharge to the environment cause severe contamination and environmental pollution problems due to their toxic nature (presence of polyphenols, caffeine, and tannins), and burning results in the production of carbon dioxide, the green house gas. If the toxic constituents present in these materials could be removed, or, at least degraded to a reasonably low level, it would open new opportunities for the utilization of these residues. Therefore, the development of methods to decrease their toxicity or to utilize them as raw material for the production of value added compounds is of great relevance. Solid-state fermentation (SSF) can be defined as the growth of microorganisms on moistened solid substrate, in which enough moisture is present to maintain microbial growth and metabolism, but there is no free-moving water. In recent years, SSF has received more interest from researchers since several studies have demonstrated that this process may lead to higher yields and productivities or better product characteristics than submerged fermentation systems. Based on the above mentioned aspects, the present study consisted in evaluating the ability of seven different fungal strains from the genus Aspergillus, Mucor, Penicillium, and Neurospora, to grow and release phenolic compounds from CS and SCG under solid-state cultivation conditions, as an alternative for biological detoxification of these residues. The biomass production and content of phenolic compounds released from the substrates were monitored during the cultivations. According to the results, Penicillium purpurogenum, Neurospora crassa and Mucor released the highest amount of phenolic compounds from the materials, contributing thus for their detoxification, since phenolic compounds are closely related to the material toxicity. Biological detoxification of CS and SCG provides environmental benefits for the disposal of these residues, as well as economical benefits for the conversion of them to value added products that can be industrially applied

Microwave-assisted extraction of sulfated fucans from brown seaweed and evaluation of fungal strains for enzymesactive production toward this class of polysaccharide

Sulphated polysaccharides from brown seaweedss comprise a complex group of macromolecules with a wide range of important biological properties such as anticoagulant, antioxidant, antiproliferative, antitumoral, anticomplementary, anti-inflammatory, antiviral, antipeptic and antiadhesive activities. Fucoidan is one of the main sulphated fucan, mostly interesting for their biological activities specially the potential to inhibit HIV reverse transcriptase and the possible application as active compound in antiretroviral drugs. However, algae remain largely unexploited and seaweeds can be found in sufficient amount for the commercial exploitation. Usually, most of the processes to recover sulfated polysaccharides from natural sources consist in acid extractions during long reaction times. Specific enzymes able to degrade fucoidan matrix (fucoidanases) are important tools to establish structural characteristics and biological functions of this polysaccharide. Such enzymes, have been only isolated from marine organisms. Reports of fungal microorganisms with enzymatic activity over this sulfated-polysaccharide are scarce. The aims of the present work were: 1) to recovery of sulphated polysaccharides (fucoidan) by microwave-assisted extraction under different operational conditions and 2) the identification of fungal strains able to growth over fucoidan-based media and to produce active fucoidanases

Extraction of sulfated polysaccharides by autohydrolysis of brown seaweed Fucus vesiculosus

The extraction of sulfated polysaccharides (fucoidan) by autohydrolysis (AH) of brown seaweed Fucus vesiculosus was studied. Experimental assays were performed under different conditions of temperature (160 to 200°C) and reaction time (10 to 30 min) according to a 22 central composite design, and the conditions able to maximize the fucoidan yield were selected. The alga degradation and the total sugar yield in the liquor after AH were also determined to each experimental condition. The highest fucoidan yield (∼16.5% w/w) was obtained when the AH process was performed at 180°C for 20 min. This product was characterized by high-performance liquid chromatography, infrared analysis spectroscopy, and thermal gravimetric analyses, which verified the presence of fucose and galactose as main components (70:30% mol ratio, in average) and an SO3 content higher than 20%. AH process under optimum reaction conditions was an effective method to recover fucoidan from F. vesiculosus. The use of this technology brings also important advantages from economical and environmental viewpoints since it does not require the use of chemical solvent and generates less waste when compared to conventional extraction procedures.R.M. Rodriguez-Jasso would like to thank the Mexican Science and Technology Council (CONACYT, Mexico) for Ph.D. fellowship support (grant number 213592/308679). The authors also thank Professor Isabel Sousa Pinto, Rita Araujo PhD from CII-MAR, University of Porto, and Bartolomeu de Souza PhD for the collaboration during the selection and collection of the seaweed used in this study

Kinetic and morphometric evaluation of fucoidan‐degrading fungal strains

Fucoidan is a sulfated fucose hetero‐polysaccharide found in brown algae. This compound has a wide variety of biological activities including anticoagulant, antithrombotic, antitumoral and antiviral (Alexeeva et al. 2002; Ellouali et al. 1993; McClure et al. 1992). Specific enzymes able to degrade fucoidan matrix are important tools to establish structural characteristics and biological functions of this polysaccharide. Such enzymes, called fucoidanases, have been only isolated from marine organisms (Sakai et al. 2004; Giordano et al. 2006). Reports of fungal microorganisms with enzymatic activity over this sulfated‐polysaccharide are scarce. Mycelial growth and morphology of filamentous fungi can be mathematically described by kinetic models, through the estimation of specific growth rate of molds on plates containing target polysaccharide as sole carbon source, using image processing techniques (Loera and Viniegra 1998). In this sense, the aim of this work was to identify fungal strains able to growth over fucoidan media as sources of active fucoidanases, by quantification of kinetic and morphology features, to establish the influence of media composition on growth patterns. Aspergillus niger PSH, Penicillium purpurogenum GH2 and Mucor sp. 3P were the screened strains. Different culture media with and without mineral salts were tested for microbial growth. Fucoidan of Laminaria japonica and urea were used as carbon and nitrogen source. Radial growth rate (Ur) was kinetically monitored measuring colony diameters. Hyphal length (Lav) and diameter (Dh) were quantified by image analyses measurements. All the evaluated strains were able to growth on different fucoidan‐urea media, and their plate invasion capacity and radial growth rate were directly proportional to measured morphometric parameters. The three fungi strains synthesize acting metabolites toward fucoidan matrix, and are important tools for the synthesis of sulfated fucan‐degrading enzymes. These results are, until now, the first report of enzymes able to growth and degrade fucoidan obtained by terrestrial fungus

Hydrothermal processing, as an alternative for upgrading agriculture residues and marine biomass according to the biorefinery concept : a review

The concept of a biorefinery that integrates processes and technologies for biomass conversion demands efficient utilization of all components. Hydrothermal processing is a potential clean technology to convert raw materials such as lignocellulosic materials and aquatic biomass into bioenergy and high added-value chemicals. In this technology, water at high temperatures and pressures is applied for hydrolysis, extraction and structural modification of materials. This review is focused on providing an updated overview on the fundamentals, modelling, separation and applications of the main components of lignocellulosic materials and conversion of aquatic biomass (macro- and micro- algae) into value-added products.The authors Hector A. Ruiz and Bruno D. Fernandes thank to the Portuguese Foundation for Science and Technology (FCT, Portugal) for their fellowships (grant number: SFRH/BPD/77361/2011 and SFRH/BD/44724/2008, respectively) and Rosa M. Rodriguez-Jasso thanks to MexicanScience and Technology Council (CONACYT, Mexico) for PhD fellowship support (grant number: 206607/230415)