Micro-organismos oriundos de ecossistemas restritivos e pouco explorados como o continente Antártico têm despertado grande interesse na comunidade científica, visto que pesquisas biotecnológicas podem resultar em produtos de alto valor agregado. Dentre estes produtos, enzimas de psicrófilos ou psicrotolerantes, que apresentam alta atividade catalítica aliada à flexibilidade, estimulam a busca de condições que favoreçam a sua produção. Assim, as proteases se destacam neste universo, visto que respondem por cerca de 60% do mercado mundial de enzimas. Neste contexto, o objetivo do presente trabalho foi o de selecionar, dentre os isolados de diferentes amostras do continente Antártico, leveduras produtoras destas enzimas, bem como de estabelecer condições de cultivo laboratorial para o favorecimento da atividade proteolítica. Foram utilizadas 99 leveduras previamente isoladas na Divisão de Recursos Microbianos da Coleção Brasileira de Micro-organismos de Ambiente e Indústria da UNICAMP, das quais 14,14% (n= 14) foram selecionadas como produtoras de proteases por meio de ensaios em placas contendo ágar Sabouraud com leite desnatado (10%). O perfil da atividade proteolítica extracelular, o crescimento e o consumo de açúcar das leveduras selecionadas foram avaliados a partir do cultivo das mesmas em caldo Sabouraud, pH 5,5, a 150rpm, a 25°C por 120h. Todos os isolados selecionados mostraram alguma atividade proteolítica em meio líquido. A levedura Rhodotorula mucilaginosa L07 foi então selecionada para a etapa seguinte, em função da máxima atividade proteolítica (33,36 U.mL-1) observada em meio líquido. Para avaliar o impacto da suplementação do meio de cultivo e da variação de parâmetros físico-químicos sobre a atividade proteolítica desta levedura, e ainda visando o favorecimento da produção de proteases, foi utilizado o planejamento fatorial fracionado 26-2, seguido de planejamento fatorial completo (DCCR 23). Os resultados obtidos permitiram estabelecer um modelo matemático que foi validado, elevando a atividade proteolítica inicial em 45,53%. A caracterização da protease produzida por R. mucilaginosa L07 revelou seu caráter ácido, cuja máxima atividade (71,14 U.ml-1) foi obtida em pH 5,0 a 50°C. Verificou-se ainda que 70% da atividade proteolítica foi conservada quando os ensaios foram realizados a 37°C e 80% em 45 e 60°C. Esta enzima mostrou-se altamente estável após incubação por 1h em temperaturas até 50°C, verificando-se a manutenção da atividade proteolítica. Estas características combinadas com a atividade proteolítica sugerem que esta protease exibe potencial para ser utilizada em processos biotecnológicos. / Microorganisms from restrictive and unexplored ecosystems as the Antarctic continent have been of great interest in the scientific community, because they can synthesize biotechnological products with high added value. Among these products, psychrotolerants or psychrophilic enzymes, which present high activity coupled with flexibility, stimulates the studies regarding to optimize conditions in order to favor their production. Proteases are highlighted in this universe, because that account for about 60 % of the enzymes world market. In this context, the aim of this work was to select among microorganisms strains isolated from Antarctica, specifically protease-producing yeast, as well as establishing its growth conditions at laboratory scale focusing on its proteolytic activity. Ninety-nine previously isolated yeasts (DRM CPQBA / UNICAMP), from different samples of the Antarctic continent were evaluated and 14,14% (n = 14) were selected as protease producers in Sabouraud Agar containing skim milk (10%). The profile of the extracellular proteolytic activity, growth conditions and sugar consumption by selected yeast strains was reported in Sabouraud broth at pH 5.5, agitation rate of 150 rpm, 25 ° C for 120h. The results showed that all selected strains presented proteolytic activity. The maximum activity values was observed with Rhodotorula mucilaginosa L07 (33,36 U.mL-1), which was selected to continue the experiments. In the next step it was studied the effect of nutrients supplementation of the culture medium and the variation of physicochemical parameters on the proteolytic activity of R. mucilaginosa L07 by using an experimental design consisting of 26-2 fractional factorial followed by a full factorial design (CCRD 23). The results obtained allowed to stablish a mathematical model, which was validated, allowing the increase of the initial proteolytic activity in 45.53%. Characterization of protease produced by R. mucilaginosa L07 showed an acid character, and a maximum activity of 71.14 U.ml-1 was obtained at pH 5.0 at 50°C. It was also found that 70% of the proteolytic activity was preserved when the assays were performed at 37°C and 80% at 45 and 60°C. This enzyme was highly stable after incubation at temperatures <= 50°C/ 1 h, maintaining the proteolytic activity. These characteristics combined with enzymatic activity, suggests that this protease exhibits potential use in biotechnological processes.

La grande majorité des colorants alimentaires naturels, utilisés dans la formulation des aliments et des boissons, proviennent des pigments extraits de matières premières végétales. Plusieurs couleurs dérivées de plantes peuvent entraîner des problèmes de formulation. Des facteurs, comme par exemple, la région, le climat, l'environnement, la variété cultivée, ont un effet de nuances de couleurs, de résistance et surtout de stabilité dans le produit final. Par ailleurs, les champignons filamenteux du genre Monascus, Penicillium et Talaromyces sont connus comme d'excellents producteurs de pigments rouges. Ces pigments intéressent de ce fait les industries car ils sont stables, non-toxiques et peuvent être utilisés comme colorants alimentaires.La recherche présentée dans le cadre de cette thèse de doctorat concerne la description des propriétés du pigment rouge que produit la souche de Talaromyces albobiverticillius isolée du milieu marin tropical autour de l'île de La Réunion. Les plans d’expérience (DOE) et la méthodologie des surfaces de réponses (RSM) ont été utilisés pour optimiser les conditions de culture et la formulation du milieu de fermentation, dans le but d'accroître les teneurs en polykétides colorés. Douze structures différentes ont été identifiées dans des extraits intracellulaires et extracellulaires des cultures fongiques, à l'aide de séparations et d'analyses spectroscopiques (HPLC-PDA-ESI/MS et RMN). Les pigments N-thréonine-monascorubramine, N-glutaryl-rubropunctamine et PP-O figurent ainsi parmi les 12 composants.Avec la demande croissante de composés colorés naturels dans le secteur industriel, les champignons isolés du milieu marin semblent présenter de nombreux intérêts. Des essais ont ainsi été menés afin d'étudier 1) l'amélioration des conditions de fermentation en fioles agitées ou en fermenteur de 2 litres; 2) les effets de la teneur en sel marin sur la synthèse des pigments; 3) des méthodes d'extraction respectueuses de l'environnement. Globalement, ces résultats font ressortir le grand potentiel des champignons marins produisant ce colorant rouge et la possibilité d'obtenir les colorants alimentaires adaptés. / It is well known that the vast majority of food colorants used in food and beverage applications comes from the pigments synthesized by plant materials. Besides, stability of many plant-derived colors can create formulation problems. Factors such as the region, the climate, the environment, the cultivar all impact colors shade, strength and overall stability in the final product. As an alternate, fungi of the genus Monascus, Penicillium and Talaromyces are known as excellent producers of red pigments. These red pigments are of industrial interest as they are stable and non-toxic and can be used as food colorants.This present research deals with the selection of high throughput red pigment producing Talaromyces albobiverticillius as a source of polyketide based natural food colorants. Design of Experiments (DoE) and Response Surface Methodology (RSM) have been used to optimize culture conditions and media formulation of fermentation process. Using Box Behnken Design (BBD), the influence of different physical factors on pigment and biomass production was studied using potato dextrose broth as culture media. The best optimal conditions were found to be with initial pH of 6.4, temperature of 24 °C, agitation speed of 164 rpm and fermentation time of 149 h gave 47.93 ± 0.58 mg /L of orange pigment, 196.28 ± 0.76 mg / L of red pigment and 12.58 ± 0.41 g /L of dry biomass. With the application of Plackett- Burman Design (PBD), 16 different media formulations were optimized using various carbon and nitrogen sources. When Sucrose and Yeast extract was used as a basal medium at 24° C, high pigment yield was observed: 695.93 ± 0.29 mg /L of orange pigment, 738.28 ± 0.51 mg / L of red pigment and 6.80 ± 0.37 g /L of dry biomass.Twelve different compounds were detected from the HPLC-PDA-ESI/MS analysis of intracellular and extracellular pigmented extracts. In particular, N-threonine-monascorubramine, N-glutaryl-rubropunctamine and PP-O were tentatively identified among these twelve compounds; further, this work reports for the first time on the PDA, MS and NMR characterization of the here named as N-GABA-monascorubramine derivative (6-[(Z)-2-Carboxyvinyl]-N-GABA-monascorubramine) pigment bearing a cis configuration at the C10-C11 double bond, in Talaromyces albobiverticillius 30548. Attempts were made to study the effects of sea salts on pigment synthesis; sustainable green extraction methods for pigments; upscaling of fermentation from shake flasks to laboratory fermenter. All these experiments with their results were discussed briefly as individual chapters. Overall, these findings bring out the potential of marine-derived red pigment producing fungi and its possibility of obtaining tailor made food colorants.

L'association mycorhizienne implique nombre de plantes et de champignons, étant sans doute la symbiose mutualiste la plus importante et la plus répandue au sein des écosystèmes terrestres. Étant donné que la plupart des arbres forment des mycorhizes arbusculaires ou des ectomycorhizes qui se distinguent par leur écophysiologie, il est judicieux de caractériser les forêts en fonction de leur dominance mycorhizienne afin d'en mesurer les impacts sur les processus écologiques. Ainsi, l'objectif de cette thèse est de quantifier les influences de la dominance mycorhizienne en forêt sur les propriétés abiotiques et biotiques du sol ayant un impact à l'échelle locale sur deux processus associés : la décomposition de la matière organique et la régulation de la diversité végétale. Les forêts étudiées, de dominance mycorhizienne très contrastée, présentent des propriétés physico-chimiques et des communautés microbiennes distinctes au niveau du sol, mais des patrons de distribution verticale des microorganismes du sol d'une similarité inattendue. Dans ces forêts nordiques décidues, la décomposition de la matière organique est favorisée dans les couches supérieures du sol, notamment grâce à la présence du réseau fongique et d'autant plus lorsque les ectomycorhizes prédominent, ce qui prouve l'aspect déterminant du contexte local. L'établissement d'arbres mycorhiziens arbusculaires peut être limité par la combinaison des conditions abiotiques et biotiques édaphiques de la forêt boréale, qui est dominée par les ectomycorhizes, contrairement aux forêts à dominance partagée entre mycorhize arbusculaire et ectomycorhize, où la diversité est favorisée à l'échelle de la communauté. Cette thèse démontre le rôle déterminant, au niveau local, exercé par la dominance mycorhizienne sur les processus écologiques, et soulève l'importance de l'hétérogénéité biotique et abiotique du sol pour mieux saisir le fonctionnement des écosystèmes terrestres. / Mycorrhizas, which involve plants and fungi, are probably the most important and widespread mutual symbioses in terrestrial ecosystems. Since most trees form arbuscular mycorrhizas or ectomycorrhizas that are ecophysiologically distinct from each other, it is useful to characterize forests according to their mycorrhizal dominance in order to measure their respective impacts on ecological processes. The objective of this thesis is to quantify the impacts of forest mycorrhizal dominance on the abiotic and biotic properties of the soil, which influence at the local scale two associated processes: the decomposition of organic matter and the maintenance of plant diversity. The forests studied have opposite mycorrhizal dominance exhibit distinct soil physico-chemical properties and microbial communities, but more similar vertical distribution patterns of microorganisms than expected. Decomposition is favored by organic matter in the upper soil layers, but also by the presence of the fungal network, especially when ectomycorrhizas predominate, illustrating the importance of the local environmental context. Establishment of arbuscular mycorrhizal tree may be limited by the combination of abiotic and biotic edaphic factors of the boreal forest, which is ectomycorrhizal-dominated, in contrast to forests with shared dominance between arbuscular mycorrhizas and ectomycorrhizas, where tree species diversity is favored at the community level. This thesis demonstrates the decisive role, at the local scale, played by mycorrhizal dominance on ecological processes, and raises the importance of soil biotic and abiotic heterogeneity to better understand the functioning of terrestrial ecosystems.