Orientador: Lara Durães Sette / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T06:41:16Z (GMT). No. of bitstreams: 1 Passarini_MichelRodrigoZambrano_D.pdf: 6997453 bytes, checksum: 0a905c32bd8d912eb875c88cff54de8e (MD5) Previous issue date: 2012 / Resumo: O oceano representa um habitat promissor na busca por novos micro-organismos, os quais podem apresentar capacidade de produzir enzimas de interesse industrial diferentes das produzidas por seus parceiros terrestres. Neste contexto, duas amostras da esponja marinha Dragmacidon reticulatum foram coletadas no litoral Norte do Estado de São Paulo, objetivando a caracterização da diversidade fúngica por métodos dependentes e independentes de cultivo, bem como a avaliação da produção, expressão da enzima e caracterização do gene da lacase. Com relação à parcela cultivada das amostras, 108 fungos filamentosos foram isolados. Destes, 64 ribotipos distintos foram submetidos aos experimentos de taxonomia polifásica e aos relacionados com a lacase. Análises macro- e microscópicas, moleculares (genes ribossomais ITS/28S) e pela técnica de MALDI TOF ICMS, resultaram na caracterização de 38 isolados distribuídos em 23 gêneros pertencentes ao Filo Ascomycota e um ao Filo Zygomycota. Este foram posteriormente depositados na Coleção Brasileira de Micro-organismos de Ambiente e Indústria (CBMAI). Dentre os isolados obtidos, uma potencial espécie nova de Penicillium foi identificada. Os resultados da triagem enzimática permitiram a seleção de dois isolados identificados como Nigrospora sp. CBMAI 1328 (0,30 U L-1) e Arthorpyrenia sp. CBMAI 1330 (0,40 U L-1), os quais foram submetidos à uma nova avaliação da atividade e expressão (RT-PCR) com indução de íons cobre e caracterização do genes da lacase. A adição de íons cobre na concentração de 5 mM, proporcionou aumento das atividades enzimáticas dos isolados CBMAI 1328 e CBMAI 1330 em 3,9X (25,2 U L-1) e 1,2X (9,0 U L-1) após 120 h, respectivamente. Os resultados da expressão dos genes da lacase para o isolado CBMAI 1328 foram os mesmos encontrados pela indução com cobre (maior expressão em 5 mM após 120 h), entretanto para o isolado CBMAI 1330, a maior expressão foi após 96 h sem adição de cobre. Os resultados da caracterização dos genes da lacase revelaram a possivel existência de 3 novos putativos genes de lacases marinhas. Com relação à parcela não cultivada, foi possível a identificação de 7 gêneros e de um fungo não cultivado (uncultured fungi) do Filo Ascomycota bem como 3 gêneros e um fungo não cultivado (uncultured fungi) do Filo Basidiomycota a partir da técnica de DGGE e do sequenciamento direto do gene RNAr ITS e do gene 18S. Em ambas as abordagens utilizadas, a maior diversidade foi encontrada na amostra DR9. Os dados derivados do presente trabalho, ressaltam a importância em se utilizar a taxonomia polifásica, bem como empregar metodologias dependente e independente de cultivo (em paralelo) para um melhor e maior conhecimento da real diversidade em amostras ambientais. Estes resultados ampliam o conhecimento dos fungos filamentosos recuperados de esponjas marinhas e demonstram o potencial biotecnológico destes micro-organismos / Abstract: The ocean represents a promissing habitat in the search for new microorganisms, which may have the ability to produce enzymes of industrial interests different from that produced by their terrestrial counterparts. In this context, two samples of the marine sponge Dragmacidon reticulatum were collected on northern coast of São Paulo State, aiming at the characterization of fungal diversity by cultureddependent and -independent approaches as well as the evaluation of the production, characterization and expression of laccase enzyme gene. Regarding the cultivated portion of the samples, around 108 filamentous fungi were isolated, belonging to 64 different taxonomic groups (ribotypes), which were subjected to enzymatic activity assays. Polyphasic taxonomy approaches (macro- and microscopic, molecular - ITS/28S ribosomal genes - and MALDI TOF ICMS analyses) resulted in the characterization of 38 isolates distributed in 23 genera belonging to the phylum Ascomycota and one to the phylum Zygomycota, which were deposited in the Brazilian Collection of Micro-organisms from Environment and Industry (CBMAI). Among the isolates recovered one possible new species of Penicillium was identified. Enzymatic screening allowed the selection of two isolates identified as Nigrospora sp. CBMAI 1328 (0,30 U L-1) and Arthorpyrenia sp. CBMAI 1330 (0,40 U L-1), which were subjected to a new activity evaluation and expression (RT-PCR) with the induction of copper ions and the laccase genes characterization. The addition of copper ions in a concentration of 5 mM resulted in an increase in the enzymatic activities of CBMAI 1328 and CBMAI 1330 strains in 3,9X (25,2 U L-1) and 1,2X (9,0 U L-1) after 120h, respectively. The results of the expression of the laccase genes for CBMAI 1328 strain were the same found by induction with copper (expression increased in 5 mM after 120 h), however for CBMAI 1330 the higher expression was after 96 h without addition of copper. Results of laccase genes characterization revealed the existence of three possible putative new marine laccase genes. Regarding to the uncultured portion of the samples, from the DGGE and direct sequencing of the ITS rRNA gene and 18S gene approaches, was possible to identify seven species of fungi and one uncultured fungus from phylum Ascomycota and three species and one uncultured fungus from phylum Basidiomycota. For both approaches used the greatest diversity was achieved in the DR9 sample. Data derived from the present work highlight the importance of using polyphasic taxonomy as well as applying culturedependent and culture-independent methodologies (in parallel) in order to have a better and greater knowledge of the real diversity in environmental sample. These results expand the knowledge of fungi recovery from marine sponges and demonstrate the biotechnological potential of these micro-organisms / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Establishing a commercial, lignocellulose-based, second-generation ethanol process has received several decades of attention by both researchers and industry. However, a fully economically viable process still remains a long-term goal. The main bottleneck to this achievement is the recalcitrance of lignocellulosic feedstocks, although there are several other factors, such as the huge investment required for second-generation ethanol facilities. An intelligent alternative solution discussed in this thesis is an integrated approach using firstgeneration ethanol plants for second-generation processes. Wheat is the major feedstock for first-generation ethanol in Europe; therefore, wheat-based lignocellulose waste, such as wheat straw, bran, and whole stillage fiber (a waste stream from first-generation wheat-based ethanol plants) was the primary focus of the integration model in this thesis. Since the major share of first-generation ethanol plant economics focuses on the animal feed DDGS (Distillers’ dried gains with solubles), the integration of lignocellulose should be designed in order to maintain DDGS quality. An ethanol-producing edible filamentous fungus, Neurospora intermedia, a potential protein source in DDGS, was considered for use as the fermenting microbe. The morphological and physiological aspects of this fungus were studied in the thesis, leading to the first report of fungal pellet development. An alternative approach of using dilute phosphoric acid to pretreat lignocellulose, as it does not negatively affect fungal growth or DDGS quality, was demonstrated in both the laboratory and on a 1m3 pilot scale. Furthermore, the process of hydrolysis of pretreated lignocelluloses and subsequent N. intermedia fermentation on lignocellulose hydrolysate was also optimized in the laboratory and scaled up to 1 m3 using an in-house pilot-scale airlift bioreactor. Fungal fermentation on acid-pretreated and enzyme-hydrolyzed wheat bran, straw and whole stillage fiber resulted in a final ethanol yield of 95%, 94% and 91% of the theoretical maximum based on the glucan content of the substrate, respectively. Integrating the first- and second-generation processes using thin stillage (a waste stream from first-generation wheat-based ethanol plants) enhanced the fungal growth on straw hydrolysate, avoiding the need for supplementing with extra nutrients. Based on the results obtained from this thesis work, a new model for integrated first- and second-generation ethanol using edible filamentous fungi processes that also adds value to animal feed (DDGS) was developed.

L’avènement de la spectrométrie de masse de type MALDI-TOF a révolutionné la microbiologie en permettant l’identification précise des bactéries et des levures en seulement quelques minutes. En 2010, la MALDI-TOF SM n’était pas applicable à l’identification des champignons filamenteux. Un protocole d’identification générant des spectres interprétables de champignons filamenteux fut d’abord mis au point, suivi par le développement d’une architecture de banque originale. Enfin une banque de références solides, intégrant des références pour la majorité des espèces de moisissures impliquées en pathologie humaine, fut créée en collaboration avec le BCCM/IHEM. Les qualités d’identification de cette banque ont été démontrées non seulement à l’échelle localemais également à l’échelle nationale . Dans un second temps, nous sommes intéressés à l’identification des levures. Nous avons déterminé qu’une extraction complète est le meilleur protocole d’identification des levures en utilisant la banque de référence Bruker ™. Afin d’améliorer le diagnostic clinique,nous avons testé un protocole d’identification sur un séries de 6192 levures isolées de prélèvements cliniques reçus au laboratoire pendant un an. Enfin nous avons pu démontrer en utilisant nos « systèmes » d’identification par MALDI-TOF MS la sous-estimation de la diversité des espèces de moisissures et de levures isolées des prélèvements cliniques, lorsqu’on ne disposait que de techniques conventionnelles d’identification. La mise à disposition d’un tel outil ouvre de grandes perspectives dans le domaine de la mycologie, tant d’un point de vue épidémiologique que clinique. / During the last decade, MALDI-TOF MS has revolutionized microbiology by enabling the accurate identification of bacteria and yeast in only few minutes1. At the beginning of this work in 2010, MALDI-TOF MS was not yet optimized for mold identification. To meet this need, we established an identification protocol to generate interpretable mold spectra. The structure of the reference database was developed taking intoaccount the heterogeneity of molds in culture. Finally, a comprehensive reference database,including references for the majority of molds encountered in human pathology, was created in collaboration with the BCCM/IHEM. Identification performance of this database was tested and validated at both a local scale and an international scale . We also determined the best-adapted pre-treatment protocol to identify yeasts in a routine setting. The protocol was tested on a panel of 6192 yeast isolates recovered from clinical samples submitted to our laboratory over the course of one year. Using our fungal identification system, we were able to identify morphologically similar species and highlight the underestimation of fungal pathogen diversity. The development of our MALDI-TOF MS-based fungal identification system presents numerous opportunities in the field of mycological, from both an epidemiological and clinical point of view. In subsequent studies, defining the clinical meaning of emerging species identified via MALDI-TOF MS will profoundly modify our perspective of fungal diseases.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-15T12:57:52Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Mestrado, Rocha,APS.pdf: 1946617 bytes, checksum: b6dc0fb29dac8660c791c1a05c3de947 (MD5) / Made available in DSpace on 2016-04-15T12:57:52Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Mestrado, Rocha,APS.pdf: 1946617 bytes, checksum: b6dc0fb29dac8660c791c1a05c3de947 (MD5) Previous issue date: 2015-02-20 / CAPEs / Piedra preta é uma micose superficial assintomática, caracterizada pela formação de nódulos rígidos e enegrecidos localizados ao longo do fio capilar. É uma micose considerada rara, inócua, podendo acometer tanto o gênero feminino quanto o masculino em qualquer faixa etária. Piedraia hortae, agente etiológico desta micose, é um fungo filamentoso, demáceo que produz o pigmento melanina, caracterizando uma coloração enegrecida na parede celular e nas estruturas fúngicas. Este pigmento, por sua vez, é caracterizado como um potente fator de virulência, favorecendo, desse modo, seu potencial em causar parasitismo no cabelo. Na literatura, não há referências citando que outros fungos demáceos, que formam ascostroma, ascos e ascosporos, semelhantes ao P. hortae, possam ser agente etiológico dessa feohifomicose. Assim, o objetivo desse trabalho foi avaliar a capacidade de fungos demáceos de formarem nódulos na porção extrafolicular de fios de cabelo humano, semelhantes aos da piedra preta. Foram obtidos onze isolados de fungos demáceos, sendo dez da Coleção de Cultura Micoteca URM, UFPE, os quais estavam preservados sob óleo mineral e posteriormente semeados em meio Àgar Batata Dextrose para estimulação do crescimento; e um isolado proveniente do Laboratório de Micologia Médica. Foram preparadas suspensões dos isolados dos fungos demáceos em 2,0 ml de água destilada esterilizada, ajustada para concentração final de 106 células/mL. Em seguida, vertidos separadamente 0,5 mL de cada suspensão sobre os fios de cabelo contidos nas placas de Petri previamente esterilizada e mantidas a temperatura de 28°C e 37°C. O experimento foi conduzido através de observações macroscópicas e microscópicas dos cabelos durante 40 dias e acompanhado em intervalo de 5 dias. Dois isolados (970 e 3334) mostraram um elevado potencial capaz de causar infecção no fio capilar. Entretanto, oito isolados apresentaram apenas uma colonização. Exophiala dermatitidis e Cladosporium tenuissimo foram capazes de formar nódulos semelhantes ao da piedra preta, porém estruturas de reprodução como ascostroma, ascos e ascosporos, não foram vizualizadas.Todavia, podemos inferir que esses fungos apresentam um potencial capaz de parasitar os fios de cabelo, degradando e destruindo a queratina e os componentes cutiulares, sendo o potente agente de tricomicose. / Black piedra is a superficial mycosis asymptomatic, characterized by the formation of hard lumps and blackened located along the capillary yarn. It is a mycosis considered rare, innocuous and can affect both the female and the male at any age. Piedraia hortae, the etiologic agent of this mycosis, is a filamentous fungus, dematiaceous that making the pigment melanin, featuring a blackish color in the cell wall and the fungal structures. This pigment, in turn, is characterized as a potent virulence factor, facilitating there by the potential to cause parasitic hair. In the literature, there are no references citing that other demáceos fungi that form ascostroma, asci and ascospores, similar to P. hortae, may be etiologic agent of this phaeohyphomycosis. The objective of this paper is to demáceos fungal ability to form nodules in extrafollicular portion of human hair, similar to those of black piedra. Eleven isolates of fungi demáceos, obtained of the Culture Collection URM, UFPE, which were preserved under mineral oil and then plated on potato dextrose agar medium for growth promotion; and one isolated from the Medical Mycology Laboratory. Fungal isolates demáceos suspensions were prepared in 2.0 ml of sterile distilled water, adjusted to a final concentration of 106cells / ml. Then separately poured into 0.5 mL of each suspension over the strands of hair contained in the previously sterile Petri dishes and kept at 28 °C and 37 °C. The experiment was conducted by macroscopic and microscopic observations of the hair for 40 days and monitoring in the range of 5 days. Two isolates (970 and 3334) showed a high potential capable of causing infection in the capillary. However, eight isolates showed only colonization. Exophiala dermatitidis and Cladosporium tenuissimo was capable of forming nodules were similar to those of black piedra; however ascostroma structures such as playback, asci and ascosporos were not displayed. However, we can infer that these fungi have the potential able to parasitize the hairs, degrading and destroying the keratin and cuticulares components, the powerful Trichomycosis agent.

Made available in DSpace on 2017-06-01T18:20:44Z (GMT). No. of bitstreams: 1 katarina_botelho_melo_nascimento.pdf: 1178274 bytes, checksum: a0a26b1671afacf6454fb9b3859e8013 (MD5) Previous issue date: 2013-12-11 / The use of agro-industrial waste in the production of various bioproducts has emerged as a viable alternative for use in the formation of by-products through bioconversion of agro-industrial waste that are increasingly used. The waste material is characterized as highly heterogenous, and they serve both as a source of carbon and energy, as for microbial growth thus reducing the production costs of several microbial enzymes and minimizing the environmental impact that these materials would provoke to be disposed in the environment. Filamentous fungi has a high potential for biotechnological production of enzymes of microbial origin. The tanases are extracellular enzymes that catalyze the cleavage of hydrolyzable tannins, inducible produced by various microorganisms, mainly by filamentous fungi , for ferment solid, liquid or submerged in a wide application in various segments of origin industrial and commercial. This work presents a proposal to formulate considered economical means for tannase production using agroindustrial waste (coffee, grape and orange) by the filamentous fungus Aspergillus isolated from the caatinga of Pernambuco state. The first stage of the research consisted in the selection of the fungus, where he was active degrading tannic acid, but the strain that showed the best activity was the SIS 4. This strain was subsequently tested in culture media by adding 10 g/L of the agroindustrial waste salt solution containing 10 g /L tannic acid. The best results were obtained with the residue of coffee to the line previously selected. / A utilização de resíduos agroindustriais na produção de diversos bioprodutos tem surgido como uma alternativa viável, para o aproveitamento na formação de subprodutos através da bioconversão de resíduos agroindustriais que estão cada vez mais utilizados. Os resíduos se caracterizam como materiais extremamente heterogêneos, e que servem tanto como fonte de carbono e energia, quanto para o crescimento microbiano, reduzindo assim os custos de produção de diversas enzimas microbianas e minimizando o impacto ambiental que esses materiais ocasionariam ao serem descartados no meio ambiente. Os fungos filamentosos apresentam um elevado potencial biotecnológico para produção de enzimas de origem microbiana. As tanases são enzimas extracelulares que catalisam a quebra de taninos hidrolisáveis, induzíveis, produzidas por diversos micro-organismos, principalmente por fungos filamentosos, por fermentação sólida, líquida ou submersa, com vasta aplicação em diversos seguimentos de origem industrial e comercial. Este trabalho apresenta uma proposta de formular meios considerados econômicos para produção de tanases utilizando resíduos agroindustriais (café, uva e laranja) pelo fungo filamentoso Aspergillus isolado da caatinga do estado de Pernambuco. A primeira etapa da pesquisa consistiu na seleção do fungo, onde ele apresentou atividade degradadora do ácido tânico, porém a linhagem que apresentou melhor atividade foi o SIS 4. Essa linhagem, posteriormente, foi testada em meios de cultura adicionando 10 g/L de resíduos agroindustriais a uma solução de sais contendo 10 g/L de ácido tânico. Os melhores resultados foram obtidos com o resíduo de café para a linhagem previamente selecionada.

Vegetable oil is more difficult to degrade by microorganisms in comparison to carbohydrates and protein. Thus, it creates serious environmental and health concerns if oil-derived waste streams produced by restaurants and industries remain untreated. In this study, a strategy has been developed to grow filamentous fungi in pure olive oil so that it can be used as a bench-mark for growth in olive oil mill sidestreams. The growth of different strains (Aspergillus oryzae, Neurospora intermedia and Rhizopus oryzae) was tested in pure olive oil. A pre-germination stage using glucose as carbon and energy source, or the addition of yeast extract, were found necessary for successful fungal growth in olive oil. Here, A. oryzae showed a superior performance in comparison to N. intermedia and R. oryzae. Medium pH did not impact A. oryzae growth in olive oil, whereas a concentration higher than 40 g/L of the latter impaired the growth of the ascomycete. Obtained biomasses from A. oryzae and N. intermedia cultivations in olive oil were analyzed and compared for protein, fat, ash, and alkali-insoluble material (cell wall content), where the presence of olive oil had a steering effect. The fungal biomass of A. oryzae, obtained from cultivation in the absence of olive oil, contained 0.33% fat and 48% protein, whereas the respective values in the presence of olive oil were 31% and 14%. Similar trends on fat and protein contents were observed for the biomass of N. intermedia. Sudan black staining was also performed on fresh biomass which clearly indicated the presence of oil globules inside the fungal cells. This research can be a fundamental step towards treatment of oil-based waste streams, which entails high-energy and costs if treated, or environmental impacts during informal discharges. Moreover, the fact that the composition of fungal biomass can be steered through addition of olive oil increases the versatility of the originated biomass for various applications, namely in feed, food and biofuel production.

Palm oil empty fruit bunches (EFB) is an abundant and cheap lignocellulose material in South East Asia. Its use as the sole medium for producing lignocellulose-hydrolyzing enzymes would increase its commercial value. A newly isolated Aspergillus niger DSM 26641 was investigated for its capability of producing hemicellulases in EFB hydrolysate obtained by treatment with pressurized hot water (1-20%, w/v) at 120-180◦C in a 1 L Parr reactor for 10-60 min. The optimal hydrolysate for the fungal growth and endoxylanase production was obtained when 10% (w/v) of empty fruit bunch was treated at 120◦C or 150◦C for 10 min, giving an endoxylanase activity of 24.5 mU ml-1 on RBB-Xylan and a saccharification activity of 5 U ml-1 on xylan (DNS assay). When the hydrolysates were produced at higher temperatures, longer treatment times or higher biomass contents, only less than 20% of the above maximal endoxylanase activity was detected, possibly due to the higher carbohydrate concentrations in the medium. Transcriptome analysis showed that 3 endoxylanases (expression levels 59-100%, the highest level was set as 100%), 2 b-xylosidases (4%), 4 side chain-cleaving arabinofuranosidases (1-95%), 1 acetyl xylan esterase (9%) and 2 ferulic acid esterases (0.3-9%) were produced together.

For complete degradation of hemicellulose into its monomers from lignocellulose biomass, the synergistic action of a broad range of hydrolytic enzymes is needed. Therefore, production of enzymes from their natural producer is desirable. To obtain a powerful b-1,4-endoxylanase producing fungus, 304 environmental samples were collected from various locations in Singapore, leading to 603 isolates. Among them, 71 exhibiting b-1,4-endoxylanase activity were identified belonging mainly to the genera of Aspergillus, Penicillium, and Trichoderma. Further analysis revealed Aspergillus niger DSM 26641 as a potential and stable b-1,4-endoxylanase producer, being able to grow in hydrothermal lignocellulose hydrolysate exhibiting its maximal b-1,4-endoxylanase activity at pH 4 and 60◦C. This strain is thought to be very suitable for lactic acid production in a simultaneous saccharification and fermentation at pH values below 5.

in English β-N-acetylhexosaminidase (EC 3.2.1.52) belongs to exoglycosidase, and is one of the most abundant enzymes found in organisms from bacteria to human. The fungal β-N-acetylhexosaminidase from Aspergillus oryzae is composed of propeptide and catalytic domain. The propeptide is noncovalently associated with the catalytic domain of the enzyme. Propeptide is essential for the enzyme activity. While the structure of the catalytic domain was desidned by homology modeling, the structure of the propeptide has not been resolved yet. In this study, the position where the propeptide is associated with the catalytic domain, was uncovered. Presented work consists of two parts. First part deals with optimization of production conditions, purification and crystallization of β-N-acetylhexosaminidase from the filamentous fungi Aspergillus oryzae. Second part is devoted to the study of interaction between propeptide and catalytic domain, which was characterized by chemical cross-linking and high-resolution mass spectrometry. It was found that the structural changes of the catalytic domain depend on the presence of the propeptide molecule. Moreover the region of propeptide-catalytic domain interaction was revealed.

Natural strains of the Saccharomyces cerevisiae growing on solid médium form structured, biofilm -like colonies. This ability is depended on the surface adhesin Flo11p. The expression of the FLO11 gene is upregulated by the RNA-binding protein Whi3p, which is likely to have a negative effect on the level of the transcription factor Yap6p. The aim of this study was to determine whether Yap6p affects colony morphology and FLO11 expression. Analysis of FLO11 expression using the fluorescent proteins pFlo11-GFP and Flo11p-DsRed in WHI3-deletion strains demonstrated a negative effect of Yap6p on FLO11 expression and confirmed changes in the effect of Yap6p on FLO11 expression in the presence of NaCl. In the strain overexpressing YAP6, the fluorescence values of pFlo11-GFP and Flo11p-DsRed were lower than in the strain with deletion of the YAP6 and in the presence of NaCl there was observed the largest increase in fluorescence. Although Yap6 protein is thought to have a negative effect on FLO11 expression under standard culture conditions, it seems to be responsible for a significant increase in FLO11 expression in the presence of mild osmotic stress. In WHI3-deletion strains, there was observed a significant increase in structuredness of colonies growing in the presence of NaCl. Analysis of structured...