Neste trabalho, formularam-se diferentes meios de cultivo para a produção econômica de biomassa de Pleurotus sajor-caju PS-2001 em processo submerso, visando o emprego do micélio na alimentação humana. Foram testadas glicose, frutose e sacarose como fontes de carbono, assim como diferentes concentrações de glicose, óleo de soja, solução de sais, extrato de levedura Prodex®, proteína de soja e sulfato de amônio. Estudos realizados em frascos agitados mostraram que 10g.L-1 de glicose promoveram a maior biomassa micelial (5,98g.L-1) e a melhor produtividade (0,048g.L-1.h-1). A adição de 1mL.L-1 de óleo de soja ao meio de cultivo mostrou ser favorável ao crescimento fúngico, também resultando na presença do aroma típico de corpos de frutificação. Os resultados de otimização do meio de cultivo mostraram que proteína de soja (2,3g.L-1), extrato de levedura (1,86g.L-1) e sulfato de amônio (1,57g.L-1) também favoreceram o crescimento. Estudos realizados em biorreator de bancada, utilizando o meio otimizado e nas condições testadas (temperatura de 28ºC, freqüência do agitador de 100rpm, taxa de aeração de 0,5vvm, O2 dissolvido acima de 30% da saturação), quando comparados aos resultados obtidos em frascos agitados com o mesmo meio, mostraram valores de biomassa, rendimento e produtividade superiores (8,18g.L-1, 0,82g.g-1 e 0,08g.L-1.h-1, respectivamente). Ao comparar os principais componentes do micélio seco de P. sajor-caju PS-2001, obtidos no presente trabalho, com o do corpo de frutificação da mesma linhagem, cultivado em serragem de eucalipto, observaram-se semelhanças na composição química com relação ao percentual de carboidratos totais, proteína bruta e minerais; no entanto, os conteúdos de lipídios brutos e de calorias mostraram-se maiores no micélio. O consumo total de sacarose na presença de óleo de soja foi evidenciado quando o cultivo foi realizado em biorreator, o que não ocorreu em frascos agitados. Ao comparar os resultados dos cultivos variando as fontes de carbono (glicose e sacarose) em biorreator, observaram-se maiores valores de biomassa, rendimento e produtividade com glicose. A determinação dos valores de exopolissacarídeos (EPS) correspondeu a 1,18 e 1,58g de matéria seca.L-1 após 120h e 144h, para glicose e sacarose, respectivamente. Os resultados mostraram ausência de atividade antioxidante in vitro dos EPS e do extrato bruto do micélio, avaliadas através da capacidade de redução do radical livre 1,1-difenil-2-picril-hidrazil (DPPH°). Verificou-se que, para produzir 1kg de micélio seco, utiliza-se 1,69kg de sacarose, a um custo de R$1,63, sendo o valor de kw.h-1 consumido de R$1,26, totalizando R$2,89, não computados os custos de mão de obra e de secagem; entretanto, o valor de mercado para 1kg de cogumelo seco é de R$200,00. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-14T17:08:22Z No. of bitstreams: 1 Dissertacao Fernanda G Confortin.pdf: 667228 bytes, checksum: dd5aee415908da35b7dd385fcbfcba0c (MD5) / Made available in DSpace on 2014-05-14T17:08:22Z (GMT). No. of bitstreams: 1 Dissertacao Fernanda G Confortin.pdf: 667228 bytes, checksum: dd5aee415908da35b7dd385fcbfcba0c (MD5) / In this study, different growth media for the economic production of biomass of Pleurotus sajor-caju PS-2001 in submerged process were formulated, aiming the use of mycelium in human feeding. Glucose, fructose and sucrose as carbon sources, as well as different concentrations of glucose, soy oil, salt solution, Prodex® yeast extract, soy protein and ammonium sulphate were tested. Studies that were carried out in shaken flasks showed that 10g.L-1 of glucose led to the larger mycelium biomass (5.98g.L-1) and the best productivity (0.048g.L-1.h-1). The addition of 1mL.L-1 of soy oil to the medium was favorable to fungal growth, also resulting in the presence of the typical aroma of the fruiting body. The results achieved in the optimization of the growing medium showed that soy protein (2.3g.L-1), yeast extract (1.86g.L-1) and ammonium sulphate (1.57g.L-1) enhanced the mycelium growth. Studies carried out in laboratory-scale bioreactor, using the optimized medium, under standard conditions (temperature of 28ºC, initial impeller speed of 100rpm, initial aeration rate of 0.5vvm, and dissolved oxygen over 30% of saturation), showed superior biomass values, yield, and productivity (8.18g.L-1, 0.82g.g-1 and 0.08g.L-1.h-1, respectively) when compared to the results obtained in shaken flasks with the same medium. By comparing the composition of the dry mycelium of P. sajor-caju PS-2001 obtained in this study with the fruiting body of the same strain, grown on eucalyptus sawdust, similarities in the chemical composition regarding the total carbohydrate percentual, crude protein and minerals were observed. However, the contents of crude lipids and calories were higher in the mycelium. The total consumption of sucrose in the presence of soy oil was evidenced when growth was carried out in bioreactor, which did not occurred in shaken flasks. When comparing the results of cultivation on media containing glucose and sucrose in bioreactor, superior values of biomass concentration, yield and productivity with glucose were observed. With media containing glucose and sucrose, exopolyssaccharide (EPS) values of 1.18 and 1.58g of dry material.L-1, after 120h and 144h, respectively, were observed. No in vitro antioxidant activity of both EPS and mycelium crude extract was detected when these materials were evaluated with respect to their capacity of reducing the free radical 1,1-difenil-2-picrilhidrazil (DPPH°). It was calculated that, to produce 1kg of dry mycelium, it is used 1.69kg of sucrose, costing R$1,63, being the value of kw.h-1 consumed of R$1,26, toting up R$2,89, not being computed the labor and drying costs; however, the market value for 1kg of dry mushroom is R$200,00.

Neste trabalho, formularam-se diferentes meios de cultivo para a produção econômica de biomassa de Pleurotus sajor-caju PS-2001 em processo submerso, visando o emprego do micélio na alimentação humana. Foram testadas glicose, frutose e sacarose como fontes de carbono, assim como diferentes concentrações de glicose, óleo de soja, solução de sais, extrato de levedura Prodex®, proteína de soja e sulfato de amônio. Estudos realizados em frascos agitados mostraram que 10g.L-1 de glicose promoveram a maior biomassa micelial (5,98g.L-1) e a melhor produtividade (0,048g.L-1.h-1). A adição de 1mL.L-1 de óleo de soja ao meio de cultivo mostrou ser favorável ao crescimento fúngico, também resultando na presença do aroma típico de corpos de frutificação. Os resultados de otimização do meio de cultivo mostraram que proteína de soja (2,3g.L-1), extrato de levedura (1,86g.L-1) e sulfato de amônio (1,57g.L-1) também favoreceram o crescimento. Estudos realizados em biorreator de bancada, utilizando o meio otimizado e nas condições testadas (temperatura de 28ºC, freqüência do agitador de 100rpm, taxa de aeração de 0,5vvm, O2 dissolvido acima de 30% da saturação), quando comparados aos resultados obtidos em frascos agitados com o mesmo meio, mostraram valores de biomassa, rendimento e produtividade superiores (8,18g.L-1, 0,82g.g-1 e 0,08g.L-1.h-1, respectivamente). Ao comparar os principais componentes do micélio seco de P. sajor-caju PS-2001, obtidos no presente trabalho, com o do corpo de frutificação da mesma linhagem, cultivado em serragem de eucalipto, observaram-se semelhanças na composição química com relação ao percentual de carboidratos totais, proteína bruta e minerais; no entanto, os conteúdos de lipídios brutos e de calorias mostraram-se maiores no micélio. O consumo total de sacarose na presença de óleo de soja foi evidenciado quando o cultivo foi realizado em biorreator, o que não ocorreu em frascos agitados. Ao comparar os resultados dos cultivos variando as fontes de carbono (glicose e sacarose) em biorreator, observaram-se maiores valores de biomassa, rendimento e produtividade com glicose. A determinação dos valores de exopolissacarídeos (EPS) correspondeu a 1,18 e 1,58g de matéria seca.L-1 após 120h e 144h, para glicose e sacarose, respectivamente. Os resultados mostraram ausência de atividade antioxidante in vitro dos EPS e do extrato bruto do micélio, avaliadas através da capacidade de redução do radical livre 1,1-difenil-2-picril-hidrazil (DPPH°). Verificou-se que, para produzir 1kg de micélio seco, utiliza-se 1,69kg de sacarose, a um custo de R$1,63, sendo o valor de kw.h-1 consumido de R$1,26, totalizando R$2,89, não computados os custos de mão de obra e de secagem; entretanto, o valor de mercado para 1kg de cogumelo seco é de R$200,00. / In this study, different growth media for the economic production of biomass of Pleurotus sajor-caju PS-2001 in submerged process were formulated, aiming the use of mycelium in human feeding. Glucose, fructose and sucrose as carbon sources, as well as different concentrations of glucose, soy oil, salt solution, Prodex® yeast extract, soy protein and ammonium sulphate were tested. Studies that were carried out in shaken flasks showed that 10g.L-1 of glucose led to the larger mycelium biomass (5.98g.L-1) and the best productivity (0.048g.L-1.h-1). The addition of 1mL.L-1 of soy oil to the medium was favorable to fungal growth, also resulting in the presence of the typical aroma of the fruiting body. The results achieved in the optimization of the growing medium showed that soy protein (2.3g.L-1), yeast extract (1.86g.L-1) and ammonium sulphate (1.57g.L-1) enhanced the mycelium growth. Studies carried out in laboratory-scale bioreactor, using the optimized medium, under standard conditions (temperature of 28ºC, initial impeller speed of 100rpm, initial aeration rate of 0.5vvm, and dissolved oxygen over 30% of saturation), showed superior biomass values, yield, and productivity (8.18g.L-1, 0.82g.g-1 and 0.08g.L-1.h-1, respectively) when compared to the results obtained in shaken flasks with the same medium. By comparing the composition of the dry mycelium of P. sajor-caju PS-2001 obtained in this study with the fruiting body of the same strain, grown on eucalyptus sawdust, similarities in the chemical composition regarding the total carbohydrate percentual, crude protein and minerals were observed. However, the contents of crude lipids and calories were higher in the mycelium. The total consumption of sucrose in the presence of soy oil was evidenced when growth was carried out in bioreactor, which did not occurred in shaken flasks. When comparing the results of cultivation on media containing glucose and sucrose in bioreactor, superior values of biomass concentration, yield and productivity with glucose were observed. With media containing glucose and sucrose, exopolyssaccharide (EPS) values of 1.18 and 1.58g of dry material.L-1, after 120h and 144h, respectively, were observed. No in vitro antioxidant activity of both EPS and mycelium crude extract was detected when these materials were evaluated with respect to their capacity of reducing the free radical 1,1-difenil-2-picrilhidrazil (DPPH°). It was calculated that, to produce 1kg of dry mycelium, it is used 1.69kg of sucrose, costing R$1,63, being the value of kw.h-1 consumed of R$1,26, toting up R$2,89, not being computed the labor and drying costs; however, the market value for 1kg of dry mushroom is R$200,00.

Polychlorinated biphenyls (PCB) represent relevant persistent organopollutants of the environment and the estimated amount of PCB released into the environment is 750000 metric tons. White-rot fungi have been studied for long time due to their degradative potential toward various aromatic pollutants and it is known that these fungi are able to decompose PCB in vivo. Biodegradation of PCB by the fungus Pleurotus ostreatus was studied in the frame of this work. A high degradative efficiency of P. ostreatus was observed in the first set of experiments, even in the presence of relative high amount of added PCB. Fungus was able to transform 780±50 µg out of the intial amount 1000 µg in 20 ml of a cultivation media within 42 days. A decrease in toxicity was recorded during the degradation that suggests the suitability of this organism for a practical use in decontamination. In vitro experiments with purified laccase induced with Cu2+ from this fungus did not prove any participation of the enzyme in the first step of PCB transformation. The enzyme did not show an ability to degrade PCB even after purification from cultivation media containing PCB. It was found that the first step of PCB transformation can be performed by an intracellular process with microsomal fraction. A degradation of 44-67% was observed for...

A synthetic estrogen 17α-ethinylestradiol (EE2) is the main active component of the hormonal contraceptive pills. The rise of consumption of hormonal contraceptives has increased the risk of the back negative effects of EE2 to aquatic organisms. EE2 belongs to the endocrine disruptive compounds known for mimicking natural hormones. A more detailed examination of the transformation of this compound in vivo and in vitro can contribute to a better understanding of its negative effects. This master thesis is therefore devoted to the study of the metabolism of EE2 in two selected model organisms. The ligninolytic fungus Pleurotus ostreatus is the type of fungi with promising biodegradation ability to a lot of pollutants. These properties have led to numerous studies of the degradation potential of P. ostreatus towards EE2, with the possibility of removing this compound from the environment. EE2 has been degraded by the fungus P. ostreatus in vivo resulting in one hydroxylated metabolite, which estrogenic activity is in need for further study. In vitro studies were carried out with a microsomal fraction isolated from the mycelium of this fungus. The conversion of EE2 in vitro via CYPs dependent on NADPH has not been demonstrated, however using KHP as a cofactor, there was one metabolite of EE2 found,...

Ligninolytic fungi are well known for their ability to degrade a wide range of xenobiotics contaminating the environment, including synthetic industrial dyes. In this work Pleurotus ostreatus was used for decolorization of a synthetic textile dye Remazol Brilliant Blue R (RBBR). To set up a model fungal "fixed-bed" bioreactor the fungus was immobilized on a polyurethane foam and artificially contaminated with a model bacterium Rhodococcus erythropolis. The development of bacterial contamination can be expected during a real application of fungal bio filters in wastewater treatment. The main aim of the work was to study interspecies interactions in the model bioreactors during the dye decolorization. Ligninolytic enzyme activities were followed in the bioreactor cultures as markers of fungal biodegradation ability. In contrast to the controls, no bacterial growth was observed in the P. ostreatus bioreactor culture liquid. The results showed that fungal laccase, pH of the culture liquid, and glucose consumption by the fungus had no effect on the bacterial growth. However, 4*105 - 1,3*106 CFU/ml of R. erythropolis was detected to be associated with the fungal solid support. The presence of these bacteria had no effect on the decolorization performance of the bioreactors. Dye decolorization efficiency...

The current anthropogenic practices of generating single-use waste streams in agriculture, forestry and manufacturing industries have created a host of environmental health problems. Humankind's reliance on non-renewable resources for the production of food and materials, and its current approach to product design and development, are clearly unsustainable. One mitigation strategy to reducing industrial and municipal solid waste, as well as environmental pollution, can be found in using white rot fungi to valorize our planet's most abundant and regenerative natural resource – plant biomass containing lignocellulose. From residual dry plant matter, white rot fungi can be employed through a solid-state fermentation process to produce a variety of edible, nutrient-dense saprotrophic mushrooms in addition to biologically augmented composite materials. Under the framework of the circular economy, agricultural and forestry byproducts with fibers containing lignin, cellulose and hemicellulose may be used as a feedstock for the production of both food and biomaterials – keeping plant biomass revolving through multiple cycles of use and reuse for a variety of product outputs that are biodegradable and help to sequester carbon. In this study, mushrooms were grown on a variety of lignocellulosic substrates derived from agricultural and forestry residues. Hemp-based substrates performed the best of the feedstocks with regard to mushroom yield and mycelium colonization time. Additionally, a number of mycelium composite products were designed and fabricated in this study using residual lignocellulosic plant biomass, including: insulation bricks, acoustical panels, and biodegradable planter pots. In particular, spent mushroom substrate containing hemp hurd and other agricultural and forestry residues showed significant potential in upcycling lignocellulosic plant biomass for the production of both mushrooms and mycelium materials. Regenerative design practices demonstrated how food and materials can be generated from the same lignocellulosic feedstock; therefore, reducing waste, circulating products and materials, and ultimately regenerating nature. / Master of Science / Environmental pollution and natural resource scarcity have encouraged exploration into using biologically based materials for the production of more ecologically friendly products. By valorizing the Earth's most abundant, renewable natural resource for the production of food and materials– dry plant matter containing lignocellulose – waste is reduced, carbon is stored, and materials can remain upcycled through multiple generations of production. Lignocellulosic residues – natural fibers containing the biopolymers lignin, cellulose and hemicellulose – have recently been given increased attention due to their ability to be aggregated and grown into low-cost, lightweight materials using white rot fungi. Mushroom farming has historically valorized lignocellulosic agricultural and forestry residues to grow an edible, nutrient-dense food crop. This thesis investigates the potential of various agricultural and forestry residues for the production of mushrooms and mycelium-based lignocellulosic composites. Furthermore, this study explores the utilization of spent mushroom substrate for the production of several mycelium-based composite products within the framework of the circular economy. Hemp-based substrates demonstrated significant potential in both mushroom production and mycelium composite fabrication, outperforming other agricultural residues in this study with regard to mushroom yield and speed of mycelial growth of Pleurotus ostreatus. More research into the tunable lignocellulosic substrate compositions will continue to help advance mushroom production and mycelium-based composite generation as environmentally friendly materials and production practices continue to gain interest.

Le nombre important de sites contaminés au Québec (Canada) et partout dans le monde est une problématique de santé publique majeure en raison des risques toxicologiques qu’ils présentent pour la santé humaine et environnementale. Dans la municipalité de Varennes (Québec, Canada), située sur la rive sud de l'Île de Montréal, les activités d’une ancienne usine pétrochimique (Pétromont Inc.) ont conduit à l’accumulation de concentrations modérées à élevées d’éléments traces métalliques (ETMs), de biphényles polychlorés (BPCs), d’hydrocarbures pétroliers aliphatiques (C10-C50) et d’hydrocarbures aromatiques polycycliques (HAPs) sur les terrains de la compagnie. En 2010, une culture intensive de saule sur courtes rotations (CICR) a été établie sur le site, afin d’y conduire une expérience de phytoremédiation à grande échelle. Bien que cette plantation de Salix miyabeana ait été implantée dans une optique d'assainissement, aucun effet significatif n'a été signalé sur la concentration des contaminants du sol au cours des premières années de croissance. Les processus d'assainissement basés sur l’utilisation de végétaux peuvent être difficiles à prévoir en milieux naturels et nécessitent des améliorations afin d'en augmenter leur efficacité. La fertilisation des sols avec des amendements organiques, ainsi que la manipulation du microbiome végétal, sont deux techniques agronomiques couramment utilisées pour la gestion des cultures traditionnelles, afin d’augmenter la production de biomasse et améliorer la santé générale des végétaux. Ces approches peuvent également influencer la mobilité et la biodisponibilité de certains composés du sol. Puisque de telles modifications sont connues pour avoir le potentiel d’améliorer considérablement l’efficacité des végétaux à éliminer ou à transformer certains contaminants du sol, ces deux techniques agronomiques présentent un intérêt grandissant dans le domaine de la phytoremédiation. Cette recherche doctorale vise donc à améliorer les connaissances scientifiques dans le domaine de la phytoremédiation appliquée à grande échelle en abordant certains aspects qui touchent à ces deux approches agronomiques. En utilisant la plantation de saules déjà établie, une première étude a été réalisée afin d’évaluer l’impact d’un amendement de sol organique sur l’efficacité phytoremédiatrice des deux cultivars de saules (‘SX61’ et ‘SX64’). À l’intérieur de cette plantation, le sol de certaines parcelles expérimentales a été recouvert de bois raméal fragmenté (BRF) de saules, combiné, ou non, avec du substrat de champignons épuisé (SCE) de Pleurotus ostreatus. Après trois saisons de croissance, les résultats ont montré que l’ajout de SCE au BRF n’avait eu aucun effet sur la croissance des saules, ainsi que sur leur efficacité à extraire ou à réduire la concentration des contaminants présents sur le site. Les résultats suggèrent néanmoins que le BRF contribue à immobiliser certains HAPs dans le sol, en plus d’augmenter l’efficacité des saules à phytoextraire le Zn. La présence de saules semble avoir réduit de façon significative l’atténuation naturelle des C10-C50 sur le site. De plus, les concentrations de BPCs, de Cd, de Ni et de dix HAPs, ont montré des oscillations saisonnières, ce qui suggère que l’évapotranspiration qui a lieu à l’intérieur de la plantation de saules provoque un important flux d'eau et de contaminants solubles en direction des racines. Ainsi, la concentration de certains contaminants pourrait avoir tendance à augmenter à l’intérieur d’une dense plantation de saules au fil du temps. Une deuxième étude a été réalisée à l’intérieur de cette même plantation, afin de vérifier si les augmentations de concentration observées précédemment pouvaient être liées à l’évapotranspiration qui a lieu à l’intérieur d’une plantation de saules. Dans l’optique d’éliminer l’effet de transpiration, des coupes de saules ont été effectuées dans certaines parcelles de la plantation, puis les concentrations des contaminants organiques et inorganiques ont été suivies au fil du temps (24 mois), et comparées avec celles observées dans les parcelles non coupées. Les résultats obtenus ont montré que l'élimination des saules avait bel et bien limité l'accumulation de certains contaminants à la surface du sol, tels qu’observé dans les parcelles non coupées. Ces résultats suggèrent donc encore une fois que la culture intensive de saules à courte rotation peut entrainer une migration de certains contaminants en direction des racines et ainsi augmenter leurs concentrations à la surface du sol près des zones racinaires. Très peu d’études ont rapporté des résultats qui semblent contredire les multiples avantages de purification qui sont habituellement mis de l’avant en phytoremédiation. Toutefois, de tels effets sur la mobilisation des contaminants pourraient être pertinents et souhaitables dans un contexte de gestion du risque. La troisième et dernière étude présentée dans cette thèse explore la diversité des communautés microbiennes associées aux racines des deux cultivars de saules établis sur le site expérimental depuis plusieurs années (six années). Des études antérieures ont permis d’en apprendre davantage sur la composition du microbiome racinaire et rhizosphérique du saule poussant en milieux contaminés, mais la plupart de celles-ci ont été menées sur des individus relativement jeunes. Par conséquent, peu d’information existe concernant les associations microbiennes des individus plus âgés qui ont été établis en milieux contaminés. La caractérisation des communautés fongiques, bactériennes et archéennes a permis de montrer des différences de composition entre les deux cultivars de saules, ainsi qu’entre leurs compartiments (i.e. racines et rhizosphère). Certains groupes taxonomiques, appartenant à chacun des trois domaines, se sont démarqués, de par leur abondance, ou par leurs fonctions écologiques déjà connues et potentiellement bénéfiques pour la survie des végétaux, ou pour augmenter la dégradation et l'extraction de divers contaminants. Cette étude fournit donc de précieuses informations qui pourront servir à l’amélioration de certaines approches d'ingénierie du microbiome favorisant l'établissement, la survie, la croissance et les performances d’assainissement de Salix spp. établis en milieux contaminés. L’ensemble des résultats présentés dans cette thèse ont permis d’alimenter différentes réflexions sur l’intérêt d’utiliser certains amendements organiques et de caractériser le microbiome racinaire et rhizosphérique des saules afin d’améliorer les pratiques et la mise en oeuvre de la phytoremédiation par des saules. Cette thèse met également en lumière un phénomène de migration des contaminants, influencé par la présence de plantes à croissance rapide, qui représente un obstacle pour l’évaluation des performances d’assainissement par des approches de phytoremédiation notamment par des saules. / The large number of contaminated sites in Quebec (Canada) and all around the world is a major public problem because of the toxicological risks they present for human and environmental health. In the municipality of Varennes (Quebec, Canada), located on the south shore of the Island of Montreal, the activities of a former petrochemical plant (Pétromont Inc.) have led to the accumulation of moderate to high concentrations of traces elements (TEs), polychlorinated biphenyls (PCBs), aliphatic petroleum hydrocarbons (C10-C50) and polycyclic aromatic hydrocarbons (PAHs) on the land. In 2010, a short rotation intensive culture (SRIC) of willow has been established on the site, in order to conduct a field-scale phytoremediation experiment. Although this plantation of Salix miyabeana was established with a remediation view, no significant effect was reported on the concentration of soil contaminants during the first years of growth. Plant-based remediation processes can be difficult to predict in the fiel and require improvement in order to increase their effectiveness. Fertilization with organic amendments, as well as manipulating the plant microbiome, are two agronomic techniques commonly employed in traditional crop management, in order to increase biomass production and improve overall plant health. These approaches can also influence the mobility and bioavailability of some compounds in the soil. Since such modifications are known to have the potential to significantly improve the efficiency of plants in removing or transforming soil contaminants, these two agronomic techniques are of growing interest in the field of phytoremediation. This doctoral research aims to improve scientific knowledge in the field-scale phytoremediation application by addressing some aspects that affect these two agronomic approaches. Inside the already established willow plantation, a first study was carried out to assess the impact of soil organic amendment on the phytoremediation efficacy of the two willow cultivars (‘SX61’ and ‘SX64’). The soil of some experimental plots was covered with ramial chipped wood (RCW) combined or not with spent mushroom substrate (SMS) of Pleurotus ostreatus. After three growing seasons, the results showed that the addition of SMS to the RCW had no effect on the growth of the willows, as well as on their effectiveness in removing or reducing the concentration of contaminants on the site. The results nevertheless suggest that RCW helps immobilize some PAHs in the soil, in addition to increasing the efficiency of willows to phytoextract Zn. The presence of willows appears to have significantly reduced the natural attenuation of C10-C50 on the site. In addition, the concentrations of PCBs, Cd, Ni and ten PAHs, showed seasonal oscillations, which suggests that the evapotranspiration inside the willow plantation mobilized some contaminants towards the rooting zones. Thus, the concentration of certain contaminants may tend to increase within a dense willow plantation over time. A second study was carried out inside the same plantation, in order to verify if the increases in concentration observed previously could be linked to the evapotranspiration that takes place inside a willow plantation. In order to eradicate the effect of plant transpiration, willows were harvested in certain plots of the plantation. The concentrations of organic and inorganic contaminants were followed over time (24 months) and compared with those observed in the unharvested plots. The results obtained showed that the removal of the willows limited the accumulation of certain contaminants on the soil surface, as observed in the uncut plots. These results suggested once again that the short rotation intensive culture of willows can lead to the migration of certain contaminants towards the roots and thus increase their concentrations on the soil surface near the root zones. Very few studies have reported results that seem to contradict the multiple purification benefits that are usually put forward in phytoremediation. However, such effects on contaminant mobilization could be relevant and suitable in a risk management context. The third and final study presented in this thesis explores the microbial communities associated with the roots of the two willow cultivars established on the experimental site for several years (six years). Root and rhizosphere microbial communities of Salix spp. have been studied in contaminated environments, but most of studies have been carried out on relatively young hosts. Therefore, little information exists regarding the microbial communities associated with older willows established in contaminated environments. The characterization of fungal, bacterial and Archean communities has shown differences in composition between the two willow cultivars, as well as between their compartments (i.e., roots and rhizosphere). Some taxonomic groups, belonging to each of the three domains, caught our attention, either by their abundance, or by their ecological functions already known to be potentially beneficial for the plant survival, or for increasing the degradation and extraction of various contaminants. This study therefore provides valuable information that can be used to improve certain microbiome engineering approaches that promote the establishment, survival, growth and phytoremediation performance of Salix spp. in contaminated environments. All the results presented in this thesis have fueled various reflections on the interest of using soil organic amendments and characterizing the root and rhizosphere microbiome of willows in order to improve the practices and implementation of phytoremediation with willows. This thesis also highlights a phenomenon of contaminant migration, influenced by the presence of fast-growing woody plants, which represents an obstacle for the evaluation of phytoremediation performance approaches with willows.

El objetivo principal de la presente investigación es la generación de la información necesaria para el diseño de un biosistema integrado que utilice macrófitas para el postratamiento de las aguas mieles del café, buscando que sus efluentes generen el menor impacto negativo posible sobre el ecosistema acuático cafetero, presentando alternativas viables, desde el punto de vista técnico, económico, ambiental y social, para la adecuada disposición de la biomasa generada durante el proceso de depuración. En un primer ensayo, que constó de 18 tratamientos, se determinó el efecto de la concentración, en términos de DQO, de las aguas mieles del café tratadas mediante digestión anaerobia sobre el desempeño de sistemas acuáticos de tratamiento, a escala de mesocosmos, sembrados con las especies flotantes Eichhornia crassipes, Pistia stratiotes, Salvinia auriculata y la especie emergente Typha angustifolia, evaluando la remoción de los parámetros físico-químicos DQO, DBO5, ST, SST, NT, PT, K, S y del grupo de bacterias Coliformes. En un segundo ensayo, que constó de 24 tratamientos, se determinó el efecto de la carga orgánica de los efluentes de los sistemas anaerobios de tratamiento de las aguas mieles del café sobre el desempeño de sistemas acuáticos de tratamiento, sembrados con las mismas especies de plantas y determinando los mismos parámetros de remoción evaluados en el ensayo 1. Considerando el desempeño de las 4 especies acuáticas en la remoción de los parámetros DBO5, SST, NT, PT y K (medida como mg/m2-d) en los 2 ensayos evaluados, se determinó que la mejor especie para el postratamiento de las aguas mieles del café fue E. crassipes, seguida de P. stratiotes, T. angustifolia y S. auriculata. En un tercer ensayo, se evaluó el efecto del monocultivo vs la combinación de especies, en sistemas acuáticos para el postratamiento de las aguas mieles del café. / Rodríguez Valencia, N. (2009). Estudio de un biosistema integrado para el postratamiento de las aguas residuales del café utilizando macrófitas acuáticas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/4342

Tesis por compendio / [ES] La población mundial está en constante crecimiento, por lo que la soberanía alimentaria se ha convertido en un desafío crucial. Se estima que la demanda de alimentos de origen animal aumentará en un 68% para el año 2050, lo cual resulta insostenible a nivel medioambiental. Así, impulsar un mayor consumo de proteína vegetal se plantea como una de las estrategias dirigidas a promover la sostenibilidad ambiental, asegurando la disponibilidad de proteína dietética para toda la población. Si bien ciertas las legumbres y pseudocereales son una excelente fuente de nutrientes y en particular de proteína, también contienen ciertos antinutrientes que pueden limitar su digestibilidad. Este aspecto es especialmente relevante en aquellos grupos poblaciones con alteraciones gastrointestinales, como las que pueden aparecer con la edad en población sénior. En este contexto, el objetivo general de esta tesis doctoral es aplicar la fermentación en estado sólido (FES) como bioestrategia para la obtención de harinas de lenteja y quinoa con digestibilidad y bioaccesibilidad mejoradas. Para alcanzar este objetivo, se llevó a cabo la fermentación con el hongo Pleurotus ostreatus, en dos variedades de lentejas y quinoa, y se estabilizaron posteriormente mediante secado por aire caliente a diferentes temperaturas, además de la liofilización como método de referencia. Posteriormente, las harinas fermentadas fueron digeridas in vitro simulando el proceso digestivo de un adulto sano (estándar de referencia), así como en condiciones alteradas del adulto mayor. Los resultados obtenidos evidenciaron un incremento de proteína total, así como de la actividad inhibidora de la enzima convertidora de angiotensina (ECA), conjuntamente con una disminución del contenido en ácido fítico, todo ello como resultado de la actividad metabólica del hongo sobre el sustrato. A pesar de que la FES también ocasionó una reducción de la actividad antioxidante, el posterior secado por aire caliente, especialmente a 70 °C incrementó este parámetro. Asimismo, la FES y el secado por aire caliente promovieron cambios en el perfil fenólico, disminuyendo algunos compuestos e incrementando otros como el ácido gálico hasta 5 veces su contenido inicial. En cuanto al perfil volátil de las harinas fermentadas, este se caracterizó por un aroma dulce, afrutado y con matices a cacao, acompañado de notas de setas y sustratos cocidos, debido a las concentraciones de benzaldehído, hexanal, nonanal, furfural y 1-octen-3-ol que se generaron durante la fermentación. Por otro lado, con respecto a la digestibilidad de las harinas fermentadas en condiciones estándar de adulto sano, la FES y el secado a 70 °C incrementó la hidrólisis de las proteínas, así como la liberación de aminoácidos hidrófobos y aminoácidos cargados negativamente. La FES también disminuyó la actividad inhibidora de la ECA en los digeridos, sin embargo, ésta aumentó después del secado a 70 °C debido a las melanoidinas generadas durante el secado. Además, las propiedades antioxidantes y la bioaccesibilidad de minerales también se vieron incrementados con la FES y el posterior secado a 70 °C. Finalmente, la simulación de las alteraciones gastrointestinales que comúnmente se dan en el adulto mayor, indicaron que estas impactaban negativamente en la mayoría de los parámetros evaluados, a excepción de la bioaccesibilidad del magnesio, hierro y calcio en comparación con el modelo estándar. En conclusión, se ha logrado mejorar el perfil nutricional y funcional de las nuevas harinas obtenidas por fermentación con el hongo P. ostreatus, y posterior secado por aire caliente en comparación con las harinas obtenidas a partir de sustrato no fermentado, conduciendo esto a una mejora significativa en la digestibilidad y la bioaccesibilidad de los nutrientes, lo que puede ser especialmente relevante para el diseño de alimentos orientados a grupos de población con alta demanda de proteína de fácil digestión. / [CA] La població mundial està en constant creixement, per la qual cosa la sobirania alimentària ha esdevingut un desafiament crucial. S'estima que la demanda d'aliments d'origen animal augmentarà un 68% per a l'any 2050, cosa que resulta insostenible a nivell mediambiental. Així, impulsar un consum més gran de proteïna vegetal es planteja com una de les estratègies dirigides a promoure la sostenibilitat ambiental, assegurant la disponibilitat de proteïna dietètica per a tota la població. Si bé certs els llegums i pseudocereals són una excel·lent font de nutrients i en particular de proteïna, també contenen certs antinutrients que poden limitar-ne la digestibilitat. Aquest aspecte és especialment rellevant en aquells grups de poblacions amb alteracions gastrointestinals, com les que poden aparèixer amb l'edat en població sènior. En aquest context, l'objectiu general d'aquesta tesi doctoral és aplicar la fermentació en estat sòlid (FES) com a bioestratègia per obtenir farines de llentia i quinoa amb digestibilitat i bioaccessibilitat millorades. Per assolir aquest objectiu, es va dur a terme la fermentació amb el fong Pleurotus ostreatus, en dues varietats de llenties i quinoa, i es van estabilitzar posteriorment mitjançant assecat per aire calent a diferents temperatures, a més de la liofilització com a mètode de referència. Posteriorment, les farines fermentades van ser digerides in vitro simulant el procés digestiu d'un adult sa (estàndard de referència), així com en condicions alterades de l'adult més gran. Els resultats obtinguts van evidenciar un increment de proteïna total, així com de l'activitat inhibidora de l'enzim convertidor d'angiotensina (ECA), conjuntament amb una disminució del contingut en àcid fític, com a resultat de l'activitat metabòlica del fong sobre el substrat. Tot i que la FES també va ocasionar una reducció de l'activitat antioxidant, el posterior assecat per aire calent, especialment a 70 °C va incrementar aquest paràmetre. Així mateix, la FES i l'assecatge per aire calent van promoure canvis en el perfil fenòlic, disminuint alguns compostos i incrementant-ne d'altres com l'àcid gàlic fins a 5 vegades el contingut inicial. Quant al perfil volàtil de les farines fermentades, aquest es va caracteritzar per una aroma dolça, afruitada i amb matisos a cacau, acompanyat de notes de bolets i substrats cuits, a causa de les concentracions de benzaldehid, hexanal, nonanal, furfural i 1-octen -3-ol que es van generar durant la fermentació. D'altra banda, pel que fa a la digestibilitat de les farines fermentades en condicions estàndard d'adult sa, la FES i l'assecatge a 70 °C va incrementar la hidròlisi de les proteïnes, així com l'alliberament d'aminoàcids hidròfobs i aminoàcids carregats negativament. La FES també va disminuir l'activitat inhibidora de l'ACA en els digerits, però aquesta va augmentar després de l'assecat a 70 °C a causa de les melanoïdines generades durant l'assecat. A més, les propietats antioxidants i la bioaccessibilitat de minerals també es van veure incrementats amb la FES i el posterior assecat a 70 °C. Finalment, la simulació de les alteracions gastrointestinals que comunament es donen a l'adult major, van indicar que aquestes impactaven negativament a la majoria dels paràmetres avaluats, a excepció de la bioaccessibilitat del magnesi, ferro i calci en comparació del model estàndard. En conclusió, s'ha aconseguit millorar el perfil nutricional i funcional de les noves farines obtingudes per fermentació amb el fong P. ostreatus, i posterior assecat per aire calent en comparació amb les farines obtingudes a partir de substrat no fermentat, conduint-ho a una millora significativa en la digestibilitat i la bioaccessibilitat dels nutrients, cosa que pot ser especialment rellevant per al disseny d'aliments orientats a grups de població amb alta demanda de proteïna de fàcil digestió. / [EN] The world's population is constantly growing, making food sovereignty a crucial challenge. It is estimated that the demand for animal-based food will increase by 68% by 2050, which is environmentally unsustainable. Thus, encouraging greater consumption of plant protein is one of the strategies aimed to promote environmental sustainability by ensuring the availability of dietary protein for the entire population. Although certain legumes and pseudocereals are an excellent source of nutrients and in particular protein, they also contain certain anti-nutrients that can limit their digestibility. This aspect is especially relevant in those population groups with gastrointestinal disorders, such as those that may appear with age in the elderly population. In this context, the general objective of this doctoral thesis is to apply solid-state fermentation (SSF) as a biostrategy to obtain lentil and quinoa flours with improved digestibility and bioaccessibility. To achieve this objective, fermentation with the fungus Pleurotus ostreatus was carried out on two varieties of lentils and quinoa, and subsequently stabilised by hot air drying at different temperatures, in addition by freeze-drying as a reference method. Subsequently, the fermented flours were digested in vitro simulating the digestive process of a healthy adult (reference standard), as well as under altered conditions of the elderly. The results obtained evidenced an increase in total protein, as well as in angiotensin-converting enzyme (ACE) inhibitory activity, together with a decrease in phytic acid content, all as a result of the metabolic activity of the fungus on the substrate. Although SSF also caused a reduction in antioxidant activity, subsequent hot air drying, especially at 70 °C, increased this parameter. Similarly, SSF and hot air-drying promoted changes in the phenolic profile, decreasing some compounds and increasing others such as gallic acid up to 5 times its initial content. The volatile profile of the fermented flours was characterised by a sweet, fruity aroma with hints of cocoa, accompanied by notes of mushrooms and cooked substrates, due to the concentrations of benzaldehyde, hexanal, nonanal, furfural and 1-octen-3-ol that were generated during fermentation. On the other hand, regarding the digestibility of the fermented flours under standard healthy adult conditions, SSF and drying at 70 °C increased the hydrolysis of proteins, as well as the release of hydrophobic and negatively charged amino acids. SSF also decreased the ACE inhibitory activity of the digests, however, it increased after drying at 70 °C due to melanoidins generated during drying. Furthermore, antioxidant properties and mineral bioaccessibility were also increased with SSF and subsequent drying at 70 °C. Finally, simulation of gastrointestinal disturbances commonly found in the older adult indicated that these impacted negatively on most of the parameters evaluated, with the exception of the bioaccessibility of magnesium, iron and calcium compared to the standard model. In conclusion, the nutritional and functional profile of the new flours obtained by fermentation with the fungus P. ostreatus, and subsequent hot air drying has been improved compared to flours obtained from unfermented substrate, leading to a significant improvement in the digestibility and bioaccessibility of nutrients, which may be particularly relevant for the design of foods oriented to population groups with a high demand of easily digestible protein. / Sánchez García, J. (2023). Desarrollo y caracterización de nuevas harinas de lenteja y quinoa fermentadas con Pleurotus ostreatus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202014 / Compendio

Tesis por compendio / [ES] El objetivo de esta tesis doctoral ha sido proporcionar conocimientos fundamentales y prácticos relacionados con la valorización de la biomasa de setas, para comprender la relación estructura-funcionalidad de extractos acuosos ricos en ß-glucanos, en términos de capacidad inmunorreguladora, antioxidante y antiviral, así como las propiedades tecnológicas (gelificantes y emulsionantes). El objetivo de esta tesis doctoral también ha sido explorar la viabilidad de dicha biomasa en el desarrollo de materiales compostables para el envasado de alimentos. Inicialmente, se aplicó un proceso de extracción secuencial, que implicaba varios tratamientos consecutivos tanto acuosos con y sin temperatura como alcalinos, aplicados a setas de gran consumo (P. ostreatus, L. edodes y G. frondosa), con la finalidad de comprender cómo las diferencias iniciales en la composición y la arquitectura de la pared celular afectaban a la extracción de ß-glucano. A partir de los resultados obtenidos, se analizó en profundidad la aplicación potencial de extractos acuosos, ricos en ß-glucanos, del género Pleurotus, explorando cómo la composición de los extractos y la complejidad estructural de ß-glucanos afectaban a su capacidad inmunorreguladora. Los resultados evidenciaron que tanto Pleurotus ostreatus como sus estipes mostraron los mejores resultados, con una actividad inmunoestimulante mucho mayor que las otras especies de Pleurotus exploradas. En la segunda parte de esta tesis, se evaluaron las propiedades funcionales (antivirales y antioxidantes) y tecnológicas (gelificantes y emulsionantes) de los extractos acuosos de ß-glucano, purificados y sin purificar, de Pleurotus ostreatus y sus estipes. El proceso de purificación, como era de esperar, incrementó el porcentaje en carbohidratos (con un mayor aumento en los estipes), lo que se tradujo en una mayor viscosidad y capacidad gelificante. Además, el extracto obtenido de los estipes mostró una fuerte actividad antiviral frente a norovirus murino, probablemente atribuida a la mayor complejidad estructural de sus ß-glucanos. Aunque la presencia de proteínas en los extractos acuosos de ß-glucanos potenció sus propiedades emulsionantes, esta propiedad fue dependiente de la accesibilidad de la proteína para adsorberse en la interfase O/W, lo que también afectó a la viscosidad de las emulsiones resultantes. La quitina y los ß-glucanos son dos de los principales carbohidratos de las setas, y tienen un gran potencial en la formación de materiales de envasado. En la última parte de esta tesis, se investigó la viabilidad de la biomasa de residuos de setas para desarrollar materiales de envasado de alimentos biodegradables y compostables. Los resultados mostraron que la composición de la biomasa de champiñón y las temperaturas de procesado tuvieron un impacto en las propiedades fisicoquímicas de los films desarrollados y, todas fueron biodesintegrables en condiciones de compostaje según la norma ISO 20200. Por lo tanto, esta tesis doctoral representa un importante avance en la valorización de la biomasa de setas (seta entera y estipes) y pone de relieve su idoneidad para desarrollar nuevos ingredientes funcionales y materiales de envasado para aplicaciones alimentarias y de envasado de alimentos. / [CA] L'objectiu d'aquesta tesi ha sigut proporcionar els coneixements fonamentals i pràctics relacionats amb la valorització de la biomassa de bolets, per a comprendre la relació estructura-funcionalitat d'extractes aquosos rics en ß-glucans, en termes de capacitat immunoreguladora, antioxidant i antiviral, així com de les propietats tecnològiques (gelificants i emulsionants). L'objectiu d'aquesta tesi ha estat també l'exploració de la viabilitat d'aquesta biomassa pel desenvolupament de materials compostables per a l'envasat d'aliments. Inicialment, es va aplicar un procés d'extracció seqüencial, que implicava diversos tractaments consecutius, tant aquosos amb temperatura i sense, com alcalins, aplicats a bolets de gran consum (P. ostreatus, L. edodes i G. frondosa), per tal de comprendre com les diferències inicials en la composició i l'arquitectura de la paret cel·lular afectaven l'extracció de ß-glucà. A partir dels resultats obtinguts, es va analitzar en profunditat la potencial aplicació d'extractes aquosos, rics en ß-glucans, del gènere Pleurotus, explorant així com la composició dels extractes i la complexitat structural dels ß-glucans afectaven a la seua capacitat immunoreguladora. Els resultats van evidenciar que tant Pleurotus ostreatus com les seues estipes mostraren els millors resultats, amb una activitat immunoestimulant molt més gran que la obtinguda per a les altres espècies de Pleurotus explorades. A la segona part d'aquesta tesi, es van avaluar les propietats funcionals (antivirals i antioxidants) i tecnològiques (gelificants i emulsionants) dels extractes aquosos de ß-glucà, purificats i sense purificar, de Pleurotus ostreatus i les seues estipes. El procés de purificació, com calia esperar, va incrementar el percentatge en carbohidrats (amb un major augment en els estipes), cosa que es va traduir en una major viscositat i capacitat gelificant. A més, l'extracte obtingut dels estipes va mostrar una forta activitat antiviral contra norovirus murí, probablement atribuïda a la complexitat estructural més gran dels seus ß-glucans. Tot i que la presència de proteïnes en els extractes aquosos de ß-glucans va potenciar les seues propietats emulsionants, aquesta propietat va ser dependent de l'accessibilitat de la proteïna per adsorbir-se a la interfase O/W, cosa que també va afectar la viscositat de les emulsions resultants. La quitina i els ß-glucans són dos dels principals carbohidrats dels bolets, i tenen un gran potencial en la formació de materials d'envasament. Així, a la darrera part d'aquesta tesi, es va investigar la viabilitat de la biomassa de residus de bolets per desenvolupar materials biodegradables i compostables d'envasat d'aliments. Els resultats van mostrar que la composició de la biomassa de bolets i les temperatures de processament van tenir un impacte en les propietats fisicoquímiques de les pel·lícules desenvolupades i, totes elles van ser biodesintegrables en condicions de compostatge segons la norma ISO 20200. Per tant, aquesta tesi doctoral representa un important pas endavant en la valorització de la biomassa de bolets (sencers i estipes) i posa en relleu la seua idoneïtat per desenvolupar nous ingredients funcionals i materials d'envasament per a aplicacions alimentàries i d'envasat d'aliments. / [EN] This doctoral thesis was aimed at providing fundamental and practical knowledge related to the valorisation of underexploited mushroom biomass, in order to understand the structure-function relationship of ß-glucan aqueous extracts in terms of immunoregulatory, antioxidant and antiviral capacity as well as technological (gelling and emulsifying) properties. It was also the aim of this PhD thesis to explore the feasibility of mushroom biomass to develop compostable food packaging materials. Initially, a sequential fractionation process, involving several consecutive cold or hot aqueous and alkaline treatments, was applied to widely consumed mushrooms (P. ostreatus, L. edodes and G. frondosa), in order to understand how the initial differences in composition and cell wall architecture affected ß-glucan extraction. Based on the results, the potential application of ¿-glucan aqueous extracts from Pleurotus genus was deeply analysed, thus exploring how the composition of the extracts and structural complexity of ¿-glucans affected their immunoregulatory capacity. The results evidenced that both Pleurotus ostreatus and its stipes showed the best results, with a much higher immunostimulant activity than the other explored Pleurotus species. In the second part of the thesis, functional (antiviral and antioxidant) and technological (gelling and emulsifying) properties of purified and unpurified¿¿-glucan aqueous extracts from Pleurotus ostreatus and its stipes were evaluated. The purification process, as expected, increased the carbohydrates content (greater in those obtained from the stipes), which resulted in a greater viscosity and gelling capacity. Furthermore, the extract obtained from the stipes showed a strong antiviral activity against murine norovirus, probably ascribed to the higher structural complexity of ß-glucans. Although the presence of proteins in the ¿-glucan aqueous extracts enhanced their emulsifying properties, it depended on the accessibility of the protein to adsorb at the O/W interphase, which also affected the viscosity of the resulting emulsions. Chitin and ß-glucans are two of the major carbohydrates of mushrooms, having a great potential in the formation of packaging materials. Thus, in the last part of this thesis, the feasibility of mushroom waste biomass to develop biodegradable and compostable food packaging materials was investigated. The results showed that the composition of mushroom biomass and the processing temperatures had an impact on the physicochemical properties of the developed films and, all of them were biodisintegrated under composting conditions according to ISO 20200. Therefore, this PhD thesis represents a significant step forward in the understanding of the discarded mushroom biomass (whole biomass and stipes) for valorisation purposes and highlights its suitability to develop new cost-efficient functional ingredients and packaging materials for food and food packaging applications. / This work was performed with the financial support of the CIEN project BIOPRO from “Centro para el Desarrollo Tecnológico Industrial” (CDTI), Ministry of Science and Innovation, Government of Spain. The Accreditation as Center of Excellence Severo Ochoa CEX2021-001189-S funded by MCIN/AEI/10.13039/501100011033 is also fully acknowledged. his study forms part of the AGROALNEXT programme and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17. I1) and by Generalitat Valenciana. Synchrotron experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (proposal 2,022,025,569). IF was supported by a postdoctoral contract grant for the requalification of the Spanish university system from the Ministry of Universities of the Government of Spain, financed by the European Union (NextGeneration EU) (MS21-006). / Pérez Bassart, Z. (2024). Fungal Biomass Valorization for Obtaining Functional Food-Related Materials [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203841 / Compendio