Seleção e caracterização de Papiliotrema flavescens BI281, uma nova levedura oleaginosa isolada no sul do Brasil

O biodiesel de terceira geração, cuja matéria prima é obtida a partir de microrganismos, é a alternativa promissora para substituir grandes cultivos com finalidade combustível. Os organismos oleaginosos mais estudados são microalgas e leveduras, onde a levedura mostra algumas vantagens que conferem viabilidade e potencial para ser aplicada em nível industrial. Porém, o esforço de triagem de leveduras selvagens com capacidade acumuladora de lipídio não é suficiente e novas metodologias devem ser desenvolvidas para acelerar o processo de seleção. Além disso, a biodiversidade de leveduras do Rio Grande do Sul (Brasil) tem sido pouco explorada, apesar dos relatos na literatura do grande potencial de acúmulo de lipídios em leveduras da região. Neste sentido, o objetivo principal desta tese foi avaliar a habilidade de acumulação de lipídios em leveduras de uma coleção, cujas fontes de isolamento foram (1) estruturas de plantas bromélias do parque Itapuã (Viamão, RS), e (2) plantas macrófitas associadas a marismas da lagoa dos Patos, Rio Grande (RS). De 55 leveduras recuperadas e avaliadas, quatro linhagens mostraram capacidade oleaginosa a partir de determinação gravimétrica, observação em microscopia de fluorescência e leituras de cinética do acúmulo de lipídios usando intensidade de fluorescência. Três linhagens foram identificadas como Papiliotrema flavescens (BI281, BI283, BI296) e uma linhagem como Vishniacozyma sp. BI237. Estas espécies são descritas pela primeira vez como oleaginosas. Foi testado o corante fluorescente 2-(1,3-benzothiazol-2-yl)-5-(N,N-dyethylamino)phenol, denominado nessa tese como “Gaúcho Dye (GD)”. Este corante foi comparado com o vermelho de Nilo em duas leveduras oleaginosas e uma levedura não oleaginosa. Os resultados sugerem que o corante GD é lipofílico, marca fluorescência nas gotas lipídicas, e em espectrofluorimetria mostra duas bandas de emissão, o que é relacionado com a sua natureza química. O mecanismo de transferência de protons intramolecular no estado excitado (ESIPT em inglês) pode ser evidenciado de acordo com o solvente empregado e células com acúmulo de lipídios. Os resultados sugeriram a segunda banda de emissão com maior afinididade lipofílica à molécula. Por tudo isso, as propriedades espectroquímicas são vantajosas quando comparadas com o vermelho de Nilo, principalmente pela sua fotoestabilidade. Assim, esta tese envolveu a ampliação da lista de leveduras relatadas como acumuladoras de lipídios, a otimização da produção de lipídios em P. flavescens BI281 e obtenção do seu perfil lipídico. P. flavescens BI281 está sendo proposta como matéria prima para produção de biodiesel. Além disso, um novo corante para marcação de leveduras oleaginosas com propriedades fotoquímicas melhoradas foi avaliado.Third generation biodiesel, where the feedstock are oleaginous microorganisms, is an promissor alternative to replace vegetable crops to produce fuel. The most studied oleaginous organisms are microalgae and yeasts, where yeast showed advantages that offer economic viability and potential to be applied in industry. However, the effort of native yeast screening with lipid-accumulating ability is not enough, and new methodologies should be developed to accelerate the selection process. Moretheless, the yeast biodiversity in Rio Grande do Sul (Brazil) had been little explored besides reports literature of great potential lipid-accumulating ability in yeasts of the region. In this sense, the objective of this thesis was to assess the lipid-accumulating ability in our yeast collection, with focus in two isolation sources: (1) bromeliads plants and structures from Itapuã park (Viamão, RS), and (2) macrophyte plants associated to a marshland in “lagoa dos Patos”, Rio Grande (RS). From 55 recovered and assessed yeasts, four strains showed oleaginous ability using gravimetric determination, observation in fluorescence microscopy and fluorescence kinetics readings. Three strains were identified as Papiliotrema flavescens (BI281, BI283, BI296) and the other one as Vishniacozyma sp. BI237. These species are described for first time as oleaginous in this Thesis. We tested the fluorescent dye 2-(1,3-benzothiazol-2-yl)-5-(N,Ndyethylamino) phenol, and was called “Gaúcho Dye (GD)”. This dye was compared to Nile red in two oleaginous strains and one non-oleaginous strain. Our results suggested that “GD” dye is lipophilic, stain lipid droplets, presents ESIPT property only in presence of oleaginous yeast and showed advantageous characteristics when compared to Nile red., being photostable. Therefore, this thesis involved list expansion of novelty oleaginous species, optimization of lipid production in P. flavescens BI281 and obtained their lipid profile. P. flavescens BI281 is proposed as feedstock for biodiesel. Furthermore, a new fluorescent dye to stain oleaginous yeasts, called GD, with improved photochemical properties, was assessed

Estudio de factibilidad sistema de alimentación por cable en el transporte de pasajeros de la Comuna del Café y su integración al sistema Megabus en la estación Aeropuerto

T 388.042 M76;100 p.En el estudio de factibilidad se buscan datos y analizan elementos que den una referencia al análisis técnico, operativo y financiero previo, de la conección a través de un intercambiador modal que integre el sistema Megabus con la alimentación por cable a la Comuna del Café, en la estación actual Aeropuerto.Universidad Libre de Pereir

Epidemiological aspects and characterization of the resistance profile of Fusarium spp. in patients with invasive fusariosis

Introduction. The remarkable intrinsic resistance of Fusarium species to most antifungal agents results in high mortality rates in the immunocompromised population. Aims. This study aimed to investigate the epidemiology, clinical features and antifungal susceptibility of Fusarium isolates in patients with invasive fusariosis. Methodology. A total of 27 patients admitted to a referral hospital from January 2008 to June 2017 were evaluated. Antifungal susceptibility testing of isolates was performed by broth microdilution according to the Clinical and Laboratory Standards Institute guidelines. Results. Haematological malignancy was the predominant underlying condition, with an incidence of invasive fusariosis of 14.8 cases per 1000 patients with acute lymphoid leukaemia and 13.1 cases per 1000 patients with acute myeloid leukaemia. The Fusarium solani species complex (FSSC) was the most frequent agent group, followed by the Fusarium oxysporum species complex (FOSC). Voriconazole showed the best activity against Fusarium, followed by amphotericin B. Itraconazole showed high minimum inhibitory concentration values, indicating in vitro resistance. Clinical FSSC isolates were significantly (P<0.05) more resistant to amphotericin B and voriconazole than FOSC isolates. Conclusion. The present antifungal susceptibility profiles indicate a high incidence of fusariosis in patients with haematological malignancy. Species- and strain-specific differences in antifungal susceptibility exist within Fusarium in this setting

Determinación de la relación entre el porcentaje del grupo amino y el grado de desacetilación del quitosano

Several methods such as elemental analysis or infrared spectroscopy technique (IR) have been already developed to determine the degree of N-acetylation, for chitin and chitosan. This paper proposed the potentiometric technique to determine the relationship between the amino group proportion and the deacetylation degree of chitosan, using two chitosan samples obtained from different sources: one, by previously established protocols, and a commercial chitosan. The polymers tested were characterized by X-ray Diffraction and Fourier Transform Infrared Spectroscopy to verify the purity of the polymers. The proposed technique allowed us to demonstrate that the molecular weight of the synthesized chitosan are within the range reported for the biopolymer with amino group proportions similar to those obtained for samples of commercial chitosan. Resumen Varios métodos han sido desarrollados para determinar el grado de N-acetilación de la quitina y el quitosano, como el análisis elemental, o la espectroscopía de infrarrojo (IR). En este trabajo se propuso emplear el método potenciométrico para determinar la relación entre el porcentaje del grupo amino y el grado de desacetilación del quitosano, para lo cual se emplearon dos muestras de quitosano obtenido de diferentes fuentes (utilizando protocolos establecidos previamente) y un Espectroscopía de Infrarrojo con Transformada de Fourier para corroborar la pureza de los polímeros. La técnica propuesta permitió demostrar que el peso molecular de los quitosanos sintetizados se encuentra dentro de los márgenes reportados para dicho biopolímero, con una proporción del grupo amino muy similar a la obtenida para muestras de quitosano comercial

Identification by PCR and evaluation of probiotic potential in yeast strains found in kefir samples in the city of Santa Maria, RS, Brazil

Kefir is a product elaborated from the symbiotic fermentation of different microorganisms. The Kluyveromyces and Saccharomyces genera are the major representatives of the yeasts found in kefir microbiota. The only pobiotic yeast commercialized as an oral medication, is the Saccharomyces boulardii. The present work involved the microbiological quality examination of six kefir samples in the city of Santa Maria/RS, the yeasts isolation present in the samples and the identification of them by PCR (Polymerase chain reaction). Then, their probiotic potential was evaluated by in vitro technique. After that, microbiological analysis confirmed that kefir samples were suitable for consumption once the microbiological quality was established. Nineteen yeast strains were isolated from six different kefir samples; it was identified, by PCR analysis, but only three species were identified from these microorganisms in the present article: Saccharomyces cerevisiae, Hanseniospora uvarum and Kazachstania unispora. Nevertheless, by simulating the passage of isolated strains through the gastrointestinal environment, it was observed that they could not be considered probiotics. The results indicate that, in an isolated way, the yeast presents in kefir samples, in the city of Santa Maria, RS, can´t be considered probiotics according to the tests performed

Bactérias e leveduras associadas às etapas de produção de queijo Colonial e avaliação de seu potencial enzimático

fferent types of microorganisms are important in cheese-making because of the contributions their metabolism offers during the process. Few microorganisms present in Colonial cheese are known, in addition to the ones that are introduced to kick-start the processes or the ones that are associated with infections or poisonings. This study aimed to identify, by MALDI-TOF and/or DNA sequencing, the bacteria and yeasts isolated from samples collected in the main stages of Colonial cheese production, i.e., a type of cheese produced in the southern region of Brazil. The lytic capacity of these microorganisms at 5 °C and 30 °C was also evaluated. The 58 bacterial strains were distributed in 10 species among the genera Bacillus, Citrobacter, Klebsiella, Lactococcus, Paenibacillus, Staphylococcus and Raoutella. From the 13 yeasts strains analyzed, three species were identified as following: Candida pararugosa; Meyerozyma guilliermondii; and Rhodotorula mucilaginosa. In three yeasts isolates it was possible to identify only the genus Candida sp. and Trichosporon sp. The species L. lactis (48%) and M. guilliermondii (46%) were, respectively, the predominant bacteria and yeasts species isolated. The highest microbial lytic activity observed was at 30 °C. Lipase activity on isolates was proportionally more observed with yeasts and proteolytic activity with bacteria. Lower caseinase and lipase activity was observed at 5 °C, demonstrating the importance of refrigeration in controlling microbial activity. This research highlighted the cultivation of some microorganisms that are part of the Colonial cheese microbiota as well as that several of them can hydrolyze various compounds present in milk and that could be associated with its maturation or, in uncontrolled circumstances, could be the cause of product deterioration.Durante o processo de produção do queijo, é necessária a presença de diversos microrganismos que contribuem na sua elaboração pela ação de seu metabolismo. Pouco é conhecido sobre os microrganismos presentes no queijo Colonial, além daqueles inseridos para iniciar o processo ou daqueles indesejados, potenciais causadores de infecção ou intoxicação. Este trabalho teve como objetivo principal identificar, através de MALDI-TOF e/ou sequenciamento de DNA, bactérias e leveduras isoladas de amostras coletadas nas principais etapas de produção de queijo Colonial, um tipo de queijo produzido na Região Sul do Brasil. Também foi verificada a capacidade lítica destes microrganismos a 5 °C e 30 °C. Foram analisados 58 isolados de bactérias, sendo identificadas 10 espécies de bactérias distribuídas nos gêneros Bacillus, Lactococcus, Paenibacillus, Staphylococcus, Citrobacter, Klebsiella e Raoutella. Dos 13 isolados de leveduras, foram identificadas três espécies: Candida pararugosa, Meyerozyma guilliermondii e Rhodotorula mucilaginosa. Em três isolados de leveduras, foi possível a identificação somente dos gêneros Candida sp. e Trichosporon sp. As espécies Lactococcus lactis (48%) e M. guilliermondii (46%) foram os isolados predominantes de bactéria e levedura, respectivamente. A maior atividade lítica dos microrganismos identificados foi na temperatura de 30 °C. Proporcionalmente, foi verificado maior número de isolados com atividade de lipase pelas leveduras e maior atividade proteolítica pelas bactérias. Menor atividade de caseínase e lipase foi observada a 5 °C, demonstrando a importância da refrigeração no controle da atividade microbiana. Este trabalho mostrou, através de cultivo, alguns dos microrganismos que fazem parte da microbiota do queijo Colonial e que vários deles possuem capacidade de hidrolisar vários compostos presentes no leite. Mostrou também que podem estar associados com a maturação do mesmo ou, em circunstâncias não controladas, que poderiam ser a causa de deterioração do produto

Chromoblastomycosis caused by Rhinocladiella similis : Case report

We report a case of chromoblastomycosis in lesions on the chest and foot. Itraconazole was chosen as the initial treatment for this patient, who was followed up for 8 months before becoming noncompliant. The pathogenic fungal species was identified as Rhinocladiella similis by ITS region sequencing. In vitro analyses indicate that the fungus was sensitive to posaconazole and itraconazole. This report presents R. similis as a new agent of chromoblastomycosis and raises the hypothesis that this species could be more resistant to some antifungals than R. aquaspersa

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p- nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p <0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0 seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well

The oleaginous yeast Meyerozyma guilliermondii BI281A as a new potential biodiesel feedstock : selection and lipid production optimization

A high throughput screening (HTS) methodology for evaluation of cellular lipid content based on Nile red fluorescence reads using black background 96-wells test plates and a plate reader equipment allowed the rapid intracellular lipid estimation of strains from a Brazilian phylloplane yeast collection. A new oleaginous yeast,Meyerozyma guilliermondii BI281A, was selected, for which the gravimetric determination of total lipids relative to dry weight was 52.38%for glucose or 34.97%for pure glycerol. The lipid production was optimized obtaining 108 mg/L of neutral lipids using pure glycerol as carbon source, and the strain proved capable of accumulating oil using raw glycerol from a biodiesel refinery. The lipid profile showed monounsaturated fatty acids (MUFA) varying between 56 or 74% in pure or raw glycerol, respectively. M. guilliermondii BI281A bears potential as a new biodiesel feedstock

Incorporación de la habilidad de coordinación y del módulo de personalización de sesiones al simulador de otorrinolaringología Wesst-OT

This paper describes the process of incorporating a virtual environment for the practice of the coordination skill to the Web Environment for Surgical Skills in Training in Otolaryngology, WESST - OT. Additionally, a complementary module which allows session customization and the inclusion of anomalies and pathologies in the paranasal region is presented.Este artículo describe el proceso de incorporación de un ambiente virtual en el cual se puede practicar la habilidad de coordinación requerida para la cirugía endoscópica de senos paranasales al simulador Wesst. OT, Web Environment for Surgical Skills Training in Otolaryngology. Además, se presenta un módulo Complementario que permite la personalización de sesiones de práctica a través de la introducción de anomalías y de patologías que se presentan comúnmente en la región paranasal