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The determination of ethanol in fermented substrates is an important parameter for monitoring the production of distilled beverage samples. The correct measurement of its content has a direct impact on the profitability of the process. In this work, a diffusive micro-distillation device (DMDD) is proposed that allows the determination of ethanol directly in the fermented or distilled beverages samples. The DMDD consists of a 5 mL plastic test tube containing a reagent solution of potassium dichromate and sulfuric acid, inserted into another 50 mL polyethylene tube containing the sample. This set is heated in a water bath for 15 min at 80 °C, providing the ethanol diffusion, which reacts with the receptor solution contained in the test tube. The chromium (III) produced by the oxidation reaction, is spectrophotometrically quantified at 589 nm. The proposed procedure has a linear range between 1 and 12% (v/v) with R2 = 0.999 and RSD = 3.8% and results in agreement with those obtained by the distillation-densitometry official method.

Made available in DSpace on 2019-10-04T12:31:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01. Added 1 bitstream(s) on 2021-07-15T15:22:57Z : No. of bitstreams: 1 S1517-83822018000400840.pdf: 1193433 bytes, checksum: 4021607d35f01d91b6816b9a44e1d4ca (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 mu mol mL(-1) respectively. Captan and difenocona-zole were included as positive controls (MIC 16.63 and 0.02 mu mol mL(-1), respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease. (C) 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. Univ Sao Paulo, Dept Ciencias Exatas, Escola Super Agr Luiz de Queiroz, Piracicaba, SP, Brazil Univ Sao Paulo, Dept Genet, Escola Super Agr Luiz de Queiroz, Piracicaba, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Bioquim & Microbiol, Rio Claro, SP, Brazil Univ Fed Sao Carlos, Dept Quim, Sao Carlos, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Bioquim & Microbiol, Rio Claro, SP, Brazil FAPESP: 2014/15760-3 FAPESP: 2013/50228-8 CNPq: 142079/2016-2 : FAPESP: 2012/02000-5 : FAPESP: 2014/05940-4

Introduction Urban pests pose enormous risks to human health. Control initiatives are carried out in regions of high infestation and incidence of accidents caused by scorpions Objective In this study, we aimed to analyze the anti-scorpionic activity of fungal isolates obtained from a cemetery in Brazil. Materials and methods A total of thirteen fungi were subjected to a bioassay test against Tityus serrulatus, and the two isolates with the highest scorpionicidal activity were selected for molecular identification through sequencing of the ITS DNA hypervariable region and large-scale cultivation on liquid medium for secondary metabolite extraction. The crude extracts were partitioned by solid-phase extraction, and the resulting purified extracts were tested for anti-scorpionic activity. The extracts from one of the isolates presented better results and were submitted to UPLC-MS/MS. The metabolomics data were submitted to GNPS website for Molecular Networking and MASST searches. We also performed a MolNetEnhancer analysis to identify the chemical classes of the molecules found in the samples. Results The most promising fungal isolate was identified as Paecilomyces sp. CMAA1686 which has 98% of similarity to Paecilomyces formosus. The sub-fractions C and D had the best activity against the scorpions (54 and 32% mortality, respectively). Molecular Networking and MolNetEnhancer revealed a range of molecular classes in our extracts that are known to include bioactive metabolites from Paecilomyces species. Conclusions The scorpionicidal activity of Paecilomyces sp. CMAA1686 and its secondary metabolites may provide new alternative compounds for biological and chemical control of scorpions from the species T. serrulatus. Paecilomyces sp. CMAA1686 is an isolate that has great potential for isolation of secondary metabolites.

Several plants use secondary metabolites against insects and pathogens attack. Among such metabolites known glycoalkaloids are the most studied. Plants from Solanaceae Family are the most abundant on those alkaloids. Despite alfa-tomatine is a known glycoalkaloid, its specific action against Moniliophthora perniciosa, fungi responsible for witches broom disease in cocoa plantations was obtained in this work. Alfa-tomatine was infusion-extracted from Solanum lycopersicum leaves, and its action against the fungi growth was shown on the bioassays. In these tests, M. perniciosa growth was totally inhibited but other fungus tested, the inhibition was not totally, or did not present effect on growth, showing in this way, the specificity of alfa-tomatine to M. perniciosa. The advantages of work with a natural molecule for the fungi control is the stability of the molecule, its resistance to high temperature and pressure and for its use, the simple method of extraction used. The confirmation of the alfa-tomatine molecule was done by High Performance Liquid Chromatography and Mass Spectrometry in comparison to the Sigma standard.

RESUMO: Este trabalho constou de uma revisão de literatura corn o tema Cronobiologia, que teve como objetivo compilar textos que servissem de subsidios para a compreensão dos pressupostos desta ciência. Visando o entendimento das relações existentes entre os 3 eixos que norteiam os conteúdos de ciências para as escolas públicas do Estado do Parana, a partir de 1990. A Cronobiologia busca explicitar as relações existentes entre fatores geofísicos, como o movirnento de rotação e translação do planeta, suas influências sobre a disponibilidade de energia no ecossistema e suas consequências para as seres vivos.

Many oomycete species are plant pathogens and are responsible for causing significant losses in agriculture. Currently, plant pathogen control is carried out by chemical, biological and physical methods. However, due to the development of resistance to these methods by some pathogens, it is imperative that alternative methods are developed. Brazilian biodiversity is well-known for its species richness and is considered a promising source of natural products. Among the vascular plants, the family Solanaceae A. Juss. (Solanaceae) is considered one of the largest, with distributions across all tropical and temperate regions of the world. The Solanaceae family presents a high diversity of species of economic importance as sources of food, medicinal and ornamental properties. Plants of this family are sources of secondary metabolites of various chemical classes that possess potential diverse applications. Therefore, chemical and biological investigations of these compounds are extremely important as they present alternatives for their potential use in the control of plant pathogens. Here, we report for the first time, the biological activity of 7beta-acetoxywithanolide D, a compound isolated from Acnistus arborescens, against the oomycete Phytophthora cinnamomi. With these results, we emphasize the importance of such studies on plant secondary metabolites, which may present coadjuvant options in the control of plant pathogens.

The basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as "Defence genes, Virulence, and Cell response" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen's own survival in the hostile environment.

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (∼2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

One sorghum commercial genotype (MASSA 03) and nine ICRISAT high-lysine genotypes from India were analyzed for storage protein content, distribution profile, and soluble amino acid concentrations. Storage proteins fraction were extracted and separated by SDS-PAGE. Soluble amino acids contents were determined by HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the sorghum genotypes suggesting genetic variability. Amino acid profile also indicated large variations in the amino acid concentrations. The high lysine and threonine soluble concentrations observed in the seeds of the sorghum genotypes encouraged the use of these genotypes as potential food source due to the better balanced amino acids profile.

O uso de biotécnicas para aprimoramento da reprodução em bovinos pode afetar o desempenho da técnica em si quando utiliza hormônios de origem diferente do animal em questão. No caso da superovulação, o uso de FSP de origem porcina pode levar a resposta imunológica dos animais superovulados. Neste trabalho, anticorpos policlonais contra FSHp foram produzidos em coelhas e testados em fêmeas bovinas para quantificação de anticorpos produzidos após esquemas de superovulação. Embora as vacas apresentassem anticorpos contra FSHp, esta resposta imunológica não interferiu na produção de corpos lúteos e produção de embriões viáveis.

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract-peptone-fructose. In fermentations with a medium containing sucrose (180.3 g L(-1)) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L(-1)) and ethanol production (until 42.3 g L(-1)) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.

The load of pathogenic bacteria, their fate and their dangerousness in the Tiete River were assessed along 100 km starting from the city of Sao Paulo, Brazil. The data were compared with those from two German rivers. High loads of pathogens were found in the Tiete River near the city of Sao Paulo (Escherichia coli 0157:H7, Shigella flexneri, and Shigella boydii), which were absent 30 km downstream of Sao Paulo. The antibiotic resistances observed in the Tiete river were rather low and decreased after the major input in Sao Paulo to significantly lower levels about 30 km downstream. While the Brazilian isolates were more susceptible for ampicillin than the German ones, the reverse was observed for gentamycin. For optimal control of infections in humans critical areas where these bacteria survived longer and their elimination mechanisms should be identified as well as the extent and the origin of antibiotic resistance should be determined.

The goal of this study was to develop conditional flocculant yeasts for use in the alcohol fermentation industry. Promoters were constructed to completely repress gene transcription in the presence of sugar and to support strong expression after the exhaustion of this compound. A fusion of regulatory regions of the ADH2 promoter with the FLO5 core promoter was constructed to regulate the FLO5 gene. This construct was inserted into multicopy plasmids and transformed into laboratory strains of Saccharomyces cerevisiae, whereby the transformed cells were selected by sedimentation from the bulk medium after sugar exhaustion, without decreasing ethanol production. The ADH2-FLO5 region was converted into an integrative cassette to disrupt the CAN1 gene in industrial yeast strains. Transformed cells became resistant to canavanine and demonstrated conditional flocculation. Although ethanol production was significantly decreased in the industrial transformants, this development reveals a promising technology for the substitution of centrifugation in industrial ethanol production.

The aim of the present research was to evaluate the protein polymorphism degrees among forty-eight C. albicans isolates from fourteen anatomical sites of clinical patients by polyacrylamide gel electrophoresis (SDS-PAGE) and numerical analyzes, in order to identify subspecies and their similarities in some infectious niches. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed in cold saline solution. The whole-cell proteins were extracted by cell disruption using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrixes and dendrograms were generated by application of similarity coefficient of simple matching and UPGMA algorithm, respectively. The results obtained showed several C. albicans subtypes and their similarity degrees (80% to 100%). Such data showed that same patients may be infected by two or more C. albicans subtypes in certain anatomical sites (i.e. only in oral cavity of immunocompromised patients, blood, or tracheal secretion), or yet, two or more patients can be infected in identical anatomical sites (i.e. bronchial washing, urine, oral cavity, tracheal secretion, vaginal secretion, and healthy saliva) with a same C. albicans subtype. However, two or more patients also can show infections in corresponding sites (i.e. oral cavity of immunocompromised patients, blood, oropharyngeal secretion, oral cavity, tracheal secretion, vaginal secretion, and healthy saliva) by different C. albicans subtypes. Besides, two or more patients also can be infected with identical or different C. albicans subtypes in different anatomical sites (i.e.1. identical subtypes in vaginal secretion, tracheal secretion, and urine; abdominal secretion and spittle; drainage and oral cavity; catheter and healthy saliva - i.e.2. subtypes different in bronchial washing, oropharyngeal secretion, pulmonary secretion, oral cavity of immunocompromised patients, and blood). Complementary studies involving C. albicans sample isolated from several anatomical sites of immunocompetent or immunocompromised patients (before, during and after specifics therapies) and their families or hospital workers must be done in order to establish the sources of C. albicans colonization. The whole-cell proteins profile performed by SDS-PAGE associated with computer-assisted numerical analysis may provide preliminary criteria for taxonomic and epidemiological studies of such microorganisms.O objetivo da presente pesquisa foi analisar os graus de polimorfismos protéicos entre isolados de C. albicans provenientes de diversos sítios anatômicos de quarenta e dois pacientes clínicos, através do emprego da eletroforese em gel de poliacrilamida (SDS-PAGE) e análise numérica, a fim de se identificar subespécies e suas similaridades nos diversos nichos infecciosos. Culturas celulares foram desenvolvidas em meio YEPD, coletadas por centrifugação e lavadas com solução salina gelada. As proteínas celulares totais, foram extraídas por rompimento celular, usando pérolas de vidro e submetidas à técnica de SDS-PAGE. Após a eletroforese, as bandas de proteínas foram coradas com coomassie-blue e analisadas pelo conjunto de programas estatístico NTSYS-pc versão 1,70. Matrizes de similaridade e dendrogramas foram gerados pela aplicação do coeficiente de similaridade simple-matching e do algoritmo UPGMA, respectivamente. Os resultados obtidos revelaram vários subtipos de C. albicans e seus graus de similaridade (80% a 100%). Tais dados permitiram demonstrar que, certos pacientes podem estar infectados com dois ou mais subtipos de C. albicans em determinados sítios anatômicos (i.e. apenas na cavidade oral de pacientes imunocomprometidos, sangue ou secreção traqueal), ou ainda, dois ou mais pacientes podem estar infectados em sítios anatômicos idênticos (i.e. apenas em lavagem brônquica, urina, cavidade oral, secreção traqueal, secreção vaginal ou saliva saudável) com um mesmo subtipo de C. albicans. No entanto, dois ou mais pacientes também podem apresentar infecções em sítios correspondentes (i.e. apenas na cavidade oral de pacientes imunocomprometidos, sangue, secreção orofaríngea, cavidade oral, secreção traqueal, secreção vaginal e saliva saudável) por diferentes subtipos de C. albicans. Além disso, dois ou mais pacientes também podem estar infectados com subtipos idênticos ou não de C. albicans em diferentes sítios anatômicos (i.e.1. idênticos subtipos na secreção vaginal, secreção traqueal e urina; secreção abdominal e escarro; drenagem e cavidade oral; cateter e saliva saudável - i.e.2. diferentes subtipos em lavagem brônquica, secreção orofaríngea, secreção pulmonar, cavidade oral de pacientes imunocomprometidos e sangue). Dados complementares envolvendo amostras de C. albicans isoladas de vários sítios anatômicos de pacientes imunocompetentes ou imunocomprometidos (antes, durante e após terapias específicas) e seus familiares ou trabalhadores hospitalares, deverão ser obtidos a fim de se estabelecer as possíveis fontes de colonização por esses microrganismos. De modo geral, os perfis de proteínas totais obtidos por SDS-PAGE associados com análise numérica computadorizada, permitem a obtenção de critérios adicionais para os estudos epidemiológicos e taxonômicos de C. albicans.

The assay aimed to evaluate the cell viability of commercial yeast strains for bakery purpose in comparison to a selected strain (Yea-Sacc® 1026 , Alltech®) indicated as a probiotic to be added to animal feed, promising yield gains. The objective of this first study was to check the number of viable cells, once the indicated product to be classified as probiotic needs to provide live cells. We tested the viability of one commercial probiotic strain (Yea-Sacc® 1026 ) in comparison to four bakery commercial yeast. Our results showed a low viability of the probiotic Saccharomyces cerevisiae , which was 7.7 % and the bakery yeasts around 35 % of viable cells. The sustainability calls for alternative sources for animal feeding and the use of microorganisms is very welcome in the livestock chain, provided that the product presented has the characteristics inherent to it.

Caracteres moleculares do morango foram avaliados para conhecer cultivares que estão sendo introduzidas no Brasil. Utilizou-se o método do polimorfismo de DNA amplificado ao acaso (RAPD). Os caracteres moleculares com maior poder de discriminação foram os "marcadores" gerados pelos "primers" Operon B8, Operon B19 e Operon G5, que foram eficientes para discriminar as vinte e seis cultivares estudadas. Os dados foram interpretados com o auxílio de dendograma, mapa de bandas, quadro de identificação e chave dicotômica. Foi possível distinguir seis grupos de similaridade, dois deles com cultivares selecionadas no Brasil, sendo um com 'Campinas', 'Agf 80', 'Piedade', 'Jundiaí' e 'Monte Alegre' e o outro com 'Obaira' e 'Mantiqueira'; três grupos com cultivares introduzidas, sendo o primeiro com 'Lassen', 'Reiko', 'Chandler', 'Pajaro', 'Blackmore' e 'Seascape', o segundo com 'Fern' e 'Oso Grande' e o terceiro com 'Florida Belle' e 'Selva' O último grupo reuniu as cultivares 'Dover' e 'Dabreak' junto com 'Princesa Isabel'. Strawberry cultivars introduced in Brazil were identified through molecular study. The method of Random Amplified Polymorphic DNA (RAPD) was used. Molecular characters with larger discrimination power were produced by the primers Operon B8, Operon B19 and Operon G5. These were efficient to discriminate the twenty six studied cultivars. Data were analised through dendogram, bandmap, picture and dicotomic key. Six similarity groups were distinguished: Two with cultivars selected in Brazil, one with 'Campinas', 'Agf 80', 'Piedade', 'Jundiaí' and 'Monte Alegre' and the other with 'Obaira' and 'Mantiqueira'; three with introduced cultivars the first of which 'Lassen', 'Reiko', 'Chandler', 'Pajaro', 'Blackmore' and 'Seascape', the second with 'Fern' and 'Oso Grande' and the third with 'Florida Belle' and 'Selva'. The last group united the cultivars 'Dover' and 'Dabreak' with 'Princesa Isabel'.

Caracteres moleculares do morango foram avaliados para conhecer cultivares que estão sendo introduzidas no Brasil. Utilizou-se o método do polimorfismo de DNA amplificado ao acaso (RAPD). Os caracteres moleculares com maior poder de discriminação foram os "marcadores" gerados pelos "primers" Operon B8, Operon B19 e Operon G5, que foram eficientes para discriminar as vinte e seis cultivares estudadas. Os dados foram interpretados com o auxílio de dendograma, mapa de bandas, quadro de identificação e chave dicotômica. Foi possível distinguir seis grupos de similaridade, dois deles com cultivares selecionadas no Brasil, sendo um com 'Campinas', 'Agf 80', 'Piedade', 'Jundiaí' e 'Monte Alegre' e o outro com 'Obaira' e 'Mantiqueira'; três grupos com cultivares introduzidas, sendo o primeiro com 'Lassen', 'Reiko', 'Chandler', 'Pajaro', 'Blackmore' e 'Seascape', o segundo com 'Fern' e 'Oso Grande' e o terceiro com 'Florida Belle' e 'Selva' O último grupo reuniu as cultivares 'Dover' e 'Dabreak' junto com 'Princesa Isabel'.Strawberry cultivars introduced in Brazil were identified through molecular study. The method of Random Amplified Polymorphic DNA (RAPD) was used. Molecular characters with larger discrimination power were produced by the primers Operon B8, Operon B19 and Operon G5. These were efficient to discriminate the twenty six studied cultivars. Data were analised through dendogram, bandmap, picture and dicotomic key. Six similarity groups were distinguished: Two with cultivars selected in Brazil, one with 'Campinas', 'Agf 80', 'Piedade', 'Jundiaí' and 'Monte Alegre' and the other with 'Obaira' and 'Mantiqueira'; three with introduced cultivars the first of which 'Lassen', 'Reiko', 'Chandler', 'Pajaro', 'Blackmore' and 'Seascape', the second with 'Fern' and 'Oso Grande' and the third with 'Florida Belle' and 'Selva'. The last group united the cultivars 'Dover' and 'Dabreak' with 'Princesa Isabel'.

A.P.L. D ELAMARE, S . E CHEVERRIGARAY, K .R. D UARTE, L .H. G OMES A ND S .O.P. C OSTA. 2002. Aims: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces. Methods and Results: A monoclonal antibody with strong specificity against Aer. hydrophila was obtained by the fusion of myeloma cells and splenocytes of a mouse immunized with vegetative cells of Aer. hydrophila ATCC 7966, followed by a two-step selection against other species of the genera. ELISA analyses revealed that MAb 5F3 strongly reacts with all the Aer. hydrophila strains evaluated, showing a just basal reactivity against other species of the genera, especially Aer. sobria and Aer. caviae. Conclusions: MAb 5F3 was characterized as an IgG that recognized a polypeptide of approximately 110 kDa. Significance and Impact of the Study: This MAb could be used to detect Aer. hydrophila in human stool samples.

This work compared the efficiency of four methods for the identification of industrial yeast strains and the establishment of a pattern for yeast characterization to be used during industrial fermentation processes, allowing the detection of yeast contaminants. Five strains of yeast currently used in the Brazilian fuel alcohol industry (about 99% of the yeast used for this purpose), and yeast strains isolated from the five major beer industries that represent 95% of the Brazilian beer market were evaluated for their growth and absorption of dyes on differential culture media, their total protein electrophoretic patterns (SDS–PAGE), CHEF chromosome separation patterns, and RAPD profiles. For the identification of brewing yeast, all tested methods were efficient, allowing the identification of at least two different species, one of which was Saccharomyces cerevisiae . The strains used for the fuel alcohol industries were best characterized by SDS–PAGE and RAPD analysis. Those strains share high level of genetic similarity and they are all known as S. cerevisiae strains.

Este trabalho estudou a imobilização da inulinase de Kluyveromyces marxianus em diferentes suportes, com a finalidade de promover a bioconversão da inulina de Helianthus tuberosus. A inulina de H. tuberosus foi extraída dos tubérculos, desproteinizada e concentrada a 25% de açúcares redutores totais (ART). A inulinase de K. marxianus foi concentrada em evaporador rotativo e imobilizada em quitina (com e sem glutaraldeído), alginato sódico (concentrações de 2 e 4%), pectina, membrana de diálise e sílica de porosidade controlada (SPC). Em quitina obteve-se taxas de imobilização de 73Unidades/g com glutaraldeído e 48 U/g sem glutaraldeído, mas a hidrólise foi baixa em ambos os tratamentos, o equivalente a 2,4% por hora. Em gel de alginato sódico, nas concentrações de 2 e 4%, converteram-se, respectivamente, 12% e 26%, em 1h. A imobilização em pectina foi impossibilitada devido à presença de pectinase no extrato enzimático. A contenção da enzima com o substrato em membrana de diálise proporcionou uma recuperação de 50% do ART em 6h. A SPC apresentou taxa de imobilização de 43 U/g sílica, proporcionando a hidrólise de 43% em 1h, entretanto sua atividade foi se exaurindo rapidamente durante o processo devido à inativação natural da enzima e a conformação dos poros da SPC.

Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV) isolated in Brazil. One antibody (8G7G2) isotyped as IgG2b (k light chain) showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV). It can be used in identification of tomato mosaic virus (ToMV).

A simple DNA isolation method was developed with routine chemicals that yields high quality and integrity preparations when compared to some of the most well known protocols. The method described does not require the use of lysing enzymes, water bath and the DNA was obtained within 40 minutes The amount of nucleic acid extracted (measured in terms of absorbancy at 260 nm) from strains of Xanthomonas spp., Pseudomonas spp. and Erwinia spp. was two to five times higher than that of the most commonly used method.

The herbicide Metolachlor (α-chloroacetamide group) inhibits the growth of Saccharomyces cerevisiae on complete, minimal, and non-fermentative media. Spontaneous and induced resistant mutants showed monogenic segregation patterns. Among the resistant clones, 70% were recessives, 16.4% were partially dominants and 13.4% were dominants. The spontaneous partially dominant mutation Mtc1 was mapped on linkage group XV at 33.3 cM from ade2 and 31.7 cM from his3, in a region that is characterized by the presence of several resistant genes. The recessive mutation mtc2 was located on chromosome IV. Although all the mutants had the ability to grow in the presence of the herbicide, they remained affected in their respiration efficiency, indicating two different mechanism of action of Metholachor on yeast cells.

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

Made available in DSpace on 2022-04-28T18:55:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-01-01 The aim of the present research was to evaluate the protein polymorphism degrees among forty-eight C. albicans isolates from fourteen anatomical sites of clinical patients by polyacrylamide gel electrophoresis (SDS-PAGE) and numerical analyzes, in order to identify subspecies and their similarities in some infectious niches. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed in cold saline solution. The whole-cell proteins were extracted by cell disruption using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrixes and dendrograms were generated by application of similarity coefficient of simple matching and UPGMA algorithm, respectively. The results obtained showed several C. albicans subtypes and their similarity degrees (80% to 100%). Such data showed that same patients may be infected by two or more C. albicans subtypes in certain anatomical sites (i.e. only in oral cavity of immunocompromised patients, blood, or tracheal secretion), or yet, two or more patients can be infected in identical anatomical sites (i.e. bronchial washing, urine, oral cavity, tracheal secretion, vaginal secretion, and healthy saliva) with a same C. albicans subtype. However, two or more patients also can show infections in corresponding sites (i.e. oral cavity of immunocompromised patients, blood, oropharyngeal secretion, oral cavity, tracheal secretion, vaginal secretion, and healthy saliva) by different C. albicans subtypes. Besides, two or more patients also can be infected with identical or different C. albicans subtypes in different anatomical sites (i.e.1. identical subtypes in vaginal secretion, tracheal secretion, and urine; abdominal secretion and spittle; drainage and oral cavity; catheter and healthy saliva - i.e.2. subtypes different in bronchial washing, oropharyngeal secretion, pulmonary secretion, oral cavity of immunocompromised patients, and blood). Complementary studies involving C. albicans sample isolated from several anatomical sites of immunocompetent or immunocompromised patients (before, during and after specifics therapies) and their families or hospital workers must be done in order to establish the sources of C. albicans colonization. The whole-cell proteins profile performed by SDS-PAGE associated with computer-assisted numerical analysis may provide preliminary criteria for taxonomic and epidemiological studies of such microorganisms. Departamento de Diagnóstico Oral Laboratório de Microbiologia e Imunologia Universidade Estadual de Campinas, Piracicaba, SP Departamento de Genética Laboratório de Genética e Bioquímica de Plantas Universidade Estadual de São Paulo, Piracicaba, SP Departamento de Genética Laboratório de Genética de Leveduras Universidade Estadual de São Paulo, Piracicaba, SP Departamento de Genética Laboratório de Genética e Bioquímica de Plantas Universidade Estadual de São Paulo, Piracicaba, SP Departamento de Genética Laboratório de Genética de Leveduras Universidade Estadual de São Paulo, Piracicaba, SP

In the last years, the fruit fly Zapronius indianus became the most important plague of Brazilian fig production. A fermentation process is associated with infection of the fruit by this fly. A single yeast species, Candida tropicalis, was identified in all the infected figs. The presence of one species and the low genetic variability (RAPD) of the isolates indicates an uncommonly strict interaction between C. tropicalis and Z. indianus. Nos últimos anos, a mosca-africana-do-figo Zaprionus indianus tem se tornado a praga mais importante desta cultura no Brasil. Um processo fermentativo está associado com a infecção dos frutos por esta mosca. Uma única espécie de levedura, Candida tropicalis, foi identificada nos figos infectados. A presença de uma única espécie de levedura e a baixa variabilidade genética (RAPD) dos isolados indica uma relação muito estreita entre C. tropicalis e Z. indianus.

O tomateiro é uma olerícola de grande importância econômica e uma das mais suscetíveis a viroses, dentre as quais, a causada pelo vírus do mosaico do tomateiro (ToMV), gênero Tobamovirus, que tem como sintomas mosaico verde claro-escuro nas folhas, afilamento dos folíolos e diminuição da produção, entre outros sintomas. Visando a identificação do ToMV, foram produzidos anticorpos monoclonais (MAbs), testados através de PTA- ELISA ("plate trapped antigen- enzyme linked immunoassay"). O MAb (10.H1) foi utilizado para avaliar a capacidade de identificação do ToMV em testes no campo em plantas de tomate infectadas. O MAb não apresentou reação cruzada com TMV (tobamovirus do mosaico do tabaco) nem com extrato de plantas sadias. O ToMV das amostras foi isolado, purificado e re-inoculado em plantas de tomateiro e de tabaco, para confirmação dos sintomas. Em "immunobloting" o MAb 10.H1 reconheceu somente a proteína referente à capa protéica do ToMV (de 17,5 kDa). A especificidade do MAb 10.H1 pode permitir o diagnóstico precoce desta doença na fase de plântulas, ainda em casa de vegetação, evitando assim a disseminação desta virose no campo.

Diversos métodos tem sido propostos para a identificação de leveduras contaminantes em processos fermentativos que utilizam leveduras da espécie Saccharomyces cerevisiae, mas nenhum deles oferece resultados absolutamente satisfatórios com a exatidão e precisão necessárias. Considerando que a identificação poderia ser facilitada com o uso de genes mercadores, foi construído um vetor plasmidial pYGFP3 com o gene da GFP (“green fluorescent protein”) extraída da Aequorea victoria, sob o controle do promotor de ADH2 (álcool desidrogenase II) de Saccharomyces cerevisiae o qual apresenta repressão por glicose. O vetor pYGFP3 foi utilizado para transformação de leveduras S. cerevisiae HD 93.15 D (MAT a, his 3, leu2, trpl, ura3) e X2904-3C (MATa, met1, trp1, ura3), que expressam o gene GFP3 somente em condições de baixo teor de glicose, não interferindo com outras funções metabólicas ou dispêndio de energia durante o processo fermentativo. Repicações sucessívas demonstraram a estabilidade do gene marcador nos transformantes, indicando que o método é viável para identificação simples e segura desta linhagem, podendo-se constituir em recurso adicional de identificação da levedura frente a outras contaminantes do processo fermentativo Several methods have been proposed for identify spoilage yeast during fermentation processes, that uses Saccharomyces cerevisiae, therefore none of them offers absolutely acurancy and precision needed. Considering that the identification could be facilitate with the use of gene markers, a plasmid vector was constructed carrying the GFP gene (green fluorescent protein) from Aequorea victoria, under the control of ADH2 (alcohol dehydrogenase II) promoter from Saccharomyces cerevisiae, which is repressed by glucose. The p YGFP3 vector was used to transform yeast Saccharomyces cerevisiae HD 93.15D (MAT a, his 3, leu2, trpl, ura3) and X2904-3C (MATa, metl, trp 1, ura3) and the GFP3 was expressed only under low levels of glucose, not interfering in other metabolic functions of the cell neither spending energy during the fermentative process. Successive replications shows gene stability into the transformed cells, indicating its viability for simple and secure of this strain, being considered an additional tool to identify spoilage yeast in fermentative processes.

Atualmente a demanda mundial por cacau é superior à sua oferta, impulsionando a pesquisa e desenvolvimento de substitutos. Pesquisa conduzida recentemente evidenciou um potencial substituto ao submeter sementes de jaca dura a processos de fermentação, secagem e torração, resultando na produção de compostos voláteis similares aos do cacau. No entanto, o processo fermentativo foi realizado com a microbiota natural, o que impossibilita o direcionamento da produção de compostos de interesse. Desse modo, o objetivo do presente trabalho foi utilizar cultura starter de dois microrganismos isolados da fermentação da semente do cacau (Kluyveromyces marxianus e Saccharomyces cerevisiae) no processo fermentativo da semente de jaca dura, acompanhar as características da massa fermentativa, caracterizar microbiologicamente e avaliar os compostos voláteis produzidos. No primeiro capítulo são apresentados os dados referentes à primeira etapa do projeto, que envolveu a realização das fermentações em três lotes, contendo um tratamento sem inoculação de leveduras (controle) e dois tratamentos inoculados, sendo o KM, com K. marxianus, e o mix, com K. marxianus e S. cerevisiae. Neste capítulo também foram avaliadas as características da massa fermentativa ao longo do processo fermentativo (pH, exsudação, sólidos solúveis, acidez titulável e temperatura), o consumo de nutrientes (açúcares e proteínas), a produção de etanol e ácidos orgânicos (ácido acético e ácido lático) e a caracterização do conteúdo mineral. Observaram-se diferenças nos parâmetros para os tratamentos, principalmente devido à aceleração da fermentação no tratamento mix, que influenciaram na produção de compostos voláteis. Também foi evidenciada a presença de leveduras, bactérias láticas e bactérias acéticas ao longo do processo. No capítulo 2 estão apresentados os resultados obtidos na segunda etapa do projeto, que envolveu a análise dos compostos voláteis produzidos com perfil aromático semelhante ao do cacau, evidenciando a maximização da produção de alguns compostos voláteis que conferem aroma de chocolate, na presença de mix de culturas starters, indicando o potencial uso das mesmas na fermentação de jaca dura. Currently the world demand of cocoa exceeds it\'s supply, leading the research and development of substitutes. Study recently conducted evidenced a potential substitute by subjecting hard jackfruit seeds to fermentation, drying and roasting processes, resulting in the production of volatile compounds to those of cocoa. However, the fermentative process was carried out with the natural microbiota, which makes it impossible to direct the production of interests compounds. Thus, the aim of the present work was the application of starter cultures of two microrganisms isolated from the cocoa beans fermentation (Kluyveromyces marxianus e Saccharomyces cerevisiae) in the fermentative process of hard jackfruit seeds, the monitoring of the fermentative mass characteristics, the microbiological characterization, and evaluation of volatile compounds produced. In the first chapter are presented the data refering to the first stage of the project, which involved the fermentation in three lots, containing a treatment without yeast inoculation (control) and two inoculated treatments, KM with K.marxianus, and MIX with K.marxianus and S. cerevisiae. In this chapter it was also evaluated the fermentative mass characteristics throughout the fermentation process (pH, exsudation, soluble solids, titratable acidity and temperature), the nutrients consumption (sugars and proteins), the production of ethanol and organic acids (acetic acid and lactic acid) and the characterization of mineral content. Differences were observed in the parameters for the treatments, mainly due to the acceleration of the fermentation in the MIX treatment, which influenced in the volatile compounds production. Also it was evidenced the presence of yeasts, lactic acid bacteria and acetic acid bacteria throughout the process. In chapter two are presented the results obtained in the second stage of the project, which involved the analysis of volatile compounds production and the aromatic profile similar to cocoa, evidencing the maximization of some volatile compounds production that confer chocolate aroma, in the presence of MIX of starters cultures, indicating their potential use in the hard jackfruit fermentation.

Devido à grande expansão das cidades e seu acelerado crescimento e industrialização, a contaminação d\'águas vem aumentando rapidamente, resultado de despejos não controlados em rios e solos, oferecendo um grande risco à saúde. Sendo assim, neste trabalho foi realizada a medição da atividade das desidrogenases (envolvidas na respiração e/ou fermentação), por meio da conversão do 2,3,5-trifenil cloreto de tetrazólio (TTC) em 1,3,5-trifenil formazan (TPF), como parâmetro em teste de toxicidade. Para isto, foram utilizadas leveduras da espécie Saccharomyces cerevisiae, liofilizadas de panificação, como bioindicadoras da presença de elementos possivelmente tóxicos na água, através da medida da atividade metabólica pela conversão do TTC em TPF, sendo denominado de YTOX. No estudo da atividade das desidrogenases, os diferentes fermentos liofilizados de panificação não apresentaram distinção entre si, como também, não demonstraram diferença em relação a atividade das desidrogenases na presença de cádmio, sendo constatada a capacidade de recuperação metabólica da levedura após a exposição ao metal. Além disso, foi utilizada levedura PE-2 fresca para comprovar que o comportamento enzimático é similar, tanto em leveduras frescas como em liofilizadas. Acrescentando que o tempo de recuperação metabólica das leveduras é proporcional às concentrações de metais no meio. Os íons livres dos metais Al, Pb, Zn, Cr, Ba e o semimetal As, causam inibição da atividade das desidrogenases em suas respectivas concentrações, sendo este o primeiro relato da utilização de Ba em estudos com leveduras. No estudo de complexação, fica comprovado que metais complexados causam baixíssima ou nenhuma toxicidade à atividade das desidrogenases da levedura. Também foram testados solventes, como álcool butílico, ácido acético, isopropanol, acetona, cloreto de metila, clorofórmio, acetato de etila, hexano, álcool metílico, benzeno e peróxido de hidrogênio, apresentaram toxicidade por meio da inibição da atividade das desidrogenases em suas concentrações correspondentes. O teste comparativo foi a germinação de sementes de pepino (Cucumis sativus) em soluções de metais com as mesmas concentrações que o YTOX, o que possibilitando comprovar que leveduras são excelentes bioindicadoras de contaminação, por apresentarem um alto grau de homologia com eucariotos superiores, permitindo assim, avaliar aspectos relevantes em relação aos efeitos tóxicos dos compostos avaliados com a biologia humana, além de ser de fácil manutenção e resistente às pequenas variações ambientais. Este trabalho apresenta contribuição no conhecimento sobre atividade das desidrogenases em relação à toxicidade de metais, semimetal e solventes estudados. Due to the great expansion of cities and their accelerated growth and industrialization, water contamination has been increasing rapidly, resulting from uncontrolled dumping in rivers and soils, posing a great risk to health. Thus, in this work the measurement of the activity of dehydrogenases (involved in respiration and / or fermentation) was carried out, by means of the conversion of 2,3,5-triphenyl chloride tetrazolium (TTC) to 1,3,5-triphenyl formazan (TPF) as a parameter in the toxicity test. Saccharomyces cerevisiae, freeze-dried baking yeast were used as bioindicators of the presence of possibly toxic elements in the water, through the measurement of metabolic activity by the conversion of TTC into TPF, being called YTOX. In the study of the activity of the dehydrogenases, the different lyophilized baking fermentations did not distinguish between themselves, nor did they show any difference in relation to the activity of the dehydrogenase in the presence of cadmium, being verified the metabolic recovery capacity of the yeast after exposure to the metal . In addition, fresh PE-2 yeast was used to demonstrate that the enzymatic behavior is similar in both fresh and freeze-dried yeast. Adding that the metabolic recovery time of the yeasts is proportional to the concentrations of metals in the medium. The free ions of metals Al, Pb, Zn, Cr, Ba and the semimetal As, cause inhibition of the activity of the dehydrogenases in their respective concentrations, being this the first report of the use of Ba in studies with yeasts. In the complexation study, it is proven that complexed metals cause very low or no toxicity to the activity of yeast dehydrogenase. Solvents such as butyl alcohol, acetic acid, isopropanol, acetone, methyl chloride, chloroform, ethyl acetate, hexane, methyl alcohol, benzene and hydrogen peroxide were also tested for toxicity by inhibiting the activity of the dehydrogenases in their concentrations correspondents. The comparative test was the germination of cucumber seeds (Cucumis sativus) in solutions of metals with the same concentrations as YTOX, which allows to prove that yeasts are excellent contamination bioindicators, because they have a high degree of homology with superior eukaryotes, allowing thus, to evaluate relevant aspects regarding the toxic effects of the compounds evaluated with human biology, besides being easy to maintain and resistant to small environmental variations. This work presents a contribution in the knowledge about the activity of the dehydrogenases in relation to the toxicity of metals, semimetal and studied solvents.

O aumento da participação dos biocombustíveis na matriz energética mundial pode ajudar a prolongar a existência das reservas de petróleo, mitigar as ameaças representadas pela mudança climática e permitir melhor segurança do fornecimento de energia em uma escala global. Neste cenário, o processo brasileiro da produção de etanol a partir da cana-de-açúcar tem ganhado papel de destaque, pelo alto rendimento e baixo custo da produção. Linhagens de S. cerevisiae são amplamente empregadas nas fermentações industriais e, embora sejam consideradas mais tolerantes em relação a outras, o processo brasileiro impõe uma variedade de fatores estressantes sob a mesma, afetando o seu metabolismo e crescimento. A fermentação com alto teor alcoólico, realizada a partir da utilização de mostos contendo altas concentrações de açúcares, é uma das maneiras mais eficientes de se obter elevados níveis de etanol. No entanto, tal tecnologia procede ocasionando efeitos deletérios adicionais à levedura. Neste contexto, aumentar a tolerância da levedura é de fundamental importância para alcançar um desempenho fermentativo satisfatório. Neste estudo foram avaliadas linhagens de S. cerevisiae, isogênicas a linhagem industrial CAT-1, com a sobre-expressão dos genes TRP1 e MSN2, envolvidos na biossíntese de triptofano e na resposta geral ao estresse, respectivamente. Tais linhagens foram avaliadas quanto ao seu potencial para realizar fermentações com alto teor alcoólico, simulando as condições industriais brasileiras. Os resultados revelaram que o gene MSN2, na versão truncada, favoreceu a linhagem principalmente com relação ao estresse osmótico, aumentando a velocidade de fermentação e o consumo de açúcares. O gene TRP1 promoveu maior crescimento da linhagem em meio YEPD com 8% de etanol, contudo, tornou a linhagem menos viável em concentrações acima deste nível. No presente trabalho também foi avaliado o efeito da suplementação de aminoácidos na fisiologia da linhagem CAT-1 em meio YNB e em mostos de melaço e xarope de cana-de-açúcar. A suplementação com histidida promoveu maior crescimento e viabilidade celular nos diferentes meios testados. Além de histidina, os aminoácidos lisina e alanina aumentaram o crescimento da CAT-1 em mosto de melaço. A suplementação de triptofano e asparagina também promoveu aumento da viabilidade celular em mosto de xarope. Por outro lado, nos testes em microplacas a suplementação com cisteína depreciou o crescimento da linhagem em meio YNB com 10 e 12% de etanol e em mosto de melaço com 20% de ART. Os resultados obtidos indicam que tanto a engenharia genética, quanto a suplementação de aminoácidos podem ser alternativas viáveis para aumentar a tolerância de S. cerevisiae, para suportar condições de múltiplo estresse, encontradas em destilarias brasileiras. The expansion biofuels participation in the world energy matrix would help to extend the existence of fossil fuel reservoirs, mitigate the threats of climate change, and enable a better security of energy supply. The Brazilian process of ethanol production from sugarcane has gained an important role in the global energy scenario, for the high yield and low production cost. S. cerevisiae species is widely used in industrial fermentations for being resistant, but the Brazilian process imposes a variety of stressing factors to the yeast, affecting its metabolism and growth. The Very High Gravity Fermentation is performed by the utilization of musts with high sugar concentration and is one of the most efficient ways for obtaining high ethanol levels. However, this technology causes additional deleterious effects to the yeast. In this context, increasing yeast tolerance is of fundamental importance for a satisfactory fermentative performance. In this study we assessed S. cerevisiae strains - isogenic to the industrial strain CAT-1 - with over expression of TRP1 and MSN2 genes involved to tryptophan biosynthesis and in general stress response, respectively. These strains were evaluated for their potential to perform fermentations with high ethanol content, simulating the conditions of Brazilian distilleries. The results showed that the MSN2 gene in the truncated version improved strain mainly to respond to the osmotic stress, increasing in fermentation velocity and the consumption of sugars. The TRP1 gene overexpression promoted higher growth in YEPD medium with 8% ethanol, however, decreased viability at concentrations above this level. The present work also evaluated the effect of amino acid supplementation on the physiology of the CAT-1 strain in YNB medium and in molasses and syrup of sugarcane. Histidide supplementation increased the growth and cell viability in the different media tested. In addition to histidine, the amino acids lysine and alanine increased the growth of CAT-1 in molasses. Supplementation of tryptophan and asparagine also promoted increased cell viability in sugarcane syrup. On the other hand, in microplate assays, cysteine supplementation decreased growth in YNB medium with 10 and 12% ethanol, and in molasses with 20% ART. The results indicate that both genetic engineering and amino acid supplementation may be viable alternatives to increase tolerance of S. cerevisiae to supporting multiple stress conditions typical in Brazilian distilleries.

O etanol se consagrou como o principal biocombustível e o Brasil detém o processo mais econômico e ambientalmente adequando. O processo industrial brasileiro é único, pois utiliza o reciclo de células de leveduras na fermentação permitindo uma ótima velocidade de fermentação, porém o mesmo ato potencializa os efeitos tóxicos presentes no processo. O substrato de fermentação é composto pela mistura de caldo e melaço de cana-de-açúcar, porém a vantagem econômica do açúcar sobre o etanol faz com que o caldo seja priorizado para a produção de açúcar e o seu subproduto seja mais utilizado na fermentação, nesse caso o melaço. O melaço apresenta diversos compostos inibitórios como sulfitos, excesso de sais, fenóis, furfurais e metais. Devido à larga escala do processo, o substrato não pode ser esterilizado antes da fermentação, logo a presença de microrganismos contaminantes se torna um problema sempre presente. As bactérias contaminantes consomem o substrato e produzem substâncias tóxicas como ácidos orgânicos, fatos que prejudicam o rendimento fermentativo do processo. Quando se considera o longo período de reciclos (aproximadamente 250 dias por safra e pelo menos 2 ciclos por dia) a maioria das linhagens de leveduras empregadas não são capazes de perdurar no processo, seja pela toxicidade do processo e ou pela competição com contaminantes. Portanto há um esforço contínuo no sentido de se buscar novas linhagens mais aptas ao processo industrial. Para tal é imperiosa a disponibilidade de uma metodologia para simular o processo industrial de modo a permitir uma avaliação de pretensas linhagens quanto ao seu desempenho no processo industrial com reciclo de células. No presente trabalho é proposto um protocolo, simulando as várias condições estressantes de uma fermentação industrial com reutilização de células, para a avaliação de linhagens de levedura com diferentes capacidades de colonizarem as dornas de fermentação. Os resultados obtidos mostraram que o protocolo empregado foi capaz de reproduzir o comportamento fisiológico e tecnológico das linhagens quando submetidas no processo industrial, se constituindo numa ferramenta útil para a seleção de leveduras. The ethanol became entrenched as the main biofuel and Brazil owns the most economic and environmentally appropriate process. The Brazilian industrial process is unique, since it utilizes the yeast cell recycle, what allows a great fermentation velocity, but the same process enhances the toxics effects presents on the process. The fermentation subtract is composed by the mixture of sugar cane juice and molasses, however the economic advantages of the sugar over the ethanol makes that the juice get prioritized to the production of sugar and it\'s by-product, the molasses, go into the fermentation process. The molasses contain several toxic compounds, like sulfites, salts excess, phenols, furfural and metals. Due to the large scale of the process, the substrate cannot be sterilized before the fermentation and this cause the presence of contaminant microorganisms an always present problem. The contaminant bacteria consumes the substrate and produce toxics products like organic acids, facts that can hinder the yield of the fermentation process. When the long recycle period (approximately 250 days per season, with at least 2 cycles per day) is take in consideration, most of the yeast strains aren\'t capable to endure the process because of the toxicity of it and or by the competition with the contaminants. Therefore there is a continuous effort in the search of new strains best suited for the industrial process. To do so, it`s imperative the availability of a methodology that can simulate the industrial process and the alleged strains can be evaluated in terms of their performance in the industrial process with cell recycle. It`s proposed a protocol on this work, that simulates several stressful conditions of an industrial fermentation with cell recycle, to evaluate the yeast strains with different capabilities in colonize the fermentation tanks. The results indicate that the protocol utilized was capable of reproduce the physiological and technological behavior of the strains when they were submitted to the industrial process, making it a useful tool for yeast selection.

A crescente demanda por cervejas especiais tem levado o setor a buscar inovações. No âmbito da fermentação, as leveduras constituem o ponto crucial, tanto no que se refere à tolerância aos estresses do processo quanto no que tange à produção dos compostos aromáticos da bebida. Processos cervejeiros com mosto de alta densidade (high-gravity (HG)) impõem condições mais estressantes às leveduras devido à maior pressão osmótica no início da fermentação e maior teor alcoólico ao final da mesma. Leveduras isoladas de processos de bioetanol poderiam ser oportunas à produção de cervejas com mosto HG, podendo contribuir com atributos fisológicos relevantes e também para a obtenção de um produto diferenciado, com peculiaridades de sabor e aroma. Objetivou-se com este trabalho avaliar o potencial fisiológico e tecnológico dessas leveduras quanto à fermentação cervejeira com mosto HG visando à produção de cerveja especial. Para tanto, inicialmente 24 linhagens de bioetanol e três cepas cervejeiras (controle) foram avaliadas quanto ao crescimento em meio contendo maltose e em mosto cervejeiro HG. Sete cepas foram incapazes de se desenvolver satisfatoriamente, sendo inapropriadas para o processo cervejeiro e, por isso, excluídas da seleção. As linhagens selecionadas foram analisadas quanto a parâmetros fisiológicos em fermentações de mosto HG e a outros atributos relevantes ao processo. Cinco linhagens foram selecionadas para a produção das cervejas. As análises físico-químicas e sensorial mostraram que as cepas de bioetanol agregaram características organolépticas de interesse em cervejas. O ambiente de processos produtivos de bioetanol se mostrou como uma fonte oportuna de biodiversidade, até então não explorada, para os processos cervejeiros, destacando linhagens com potencial fisiológico e tecnológico para a elaboração de cervejas especiais diferenciadas, com peculiaridades de sabor e aroma. The increasingly demand for specialty beers has led production sector to search for innovations. In the fermentation scope, yeasts are a crucial point, both because of process stress tolerance as well as beer aromatic compounds production. Brewing process which use high-gravity (HG) worts impose higher stressful conditions to the yeast due to increased osmotic pressure in the beginning, and higher ethanol concentration at the end of fermentation. Yeasts isolated from bioethanol process could be opportune to beer production with HG wort, contributing to both relevant physiological traits and also to obtain differentiated specialty beer, with flavor and aroma particularities. In this work, the physiological and technological potential of bioethanol yeast strains have been evaluated for HG brewery wort fermentation for production of specialty beer. Initially, 24 bioethanol yeast strains and 3 commercial brewing yeasts (controls) were evaluated for growth in maltose medium and in HG brewery wort. Seven bioethanol yeast strains were not able to grow efficiently on maltose and HG wort, and were therefore unsuitable for brewing, being excluded from selection. Selected strains were evaluated for physiological traits in fermentation assessments of HG wort and for other relevant brewing traits. Five strains were selected for beer production. The physicochemical and sensorial analyses demonstrated that the bioethanol strains contributed to desirable organoleptic traits to the beers. The bioethanol process environment presented a valuable source of biodiversity, so far unexploited, to brewing process, highlighting strains with physiological and technological potential to produce differentiated specialty beers, with flavor and aroma particularities.

É crescente a busca por fontes de energia renováveis em substituição a o uso dos combustíveis fósseis, devido a grande preocupação mundial com o aquecimento global e as mudanças climáticas. O Brasil é considerado o detentor do processo de produção de etanol mais economicamente viável. Estimativas apontam para o fato de que a produção de etanol de primeira geração não será suficiente para atender a futura demanda global pelo biocombustível. Diante disto, a produção de etanol a partir da biomassa lignocelulósica se mostra como uma potencial solução. No entanto, durante o pré-tratamento e a hidrólise da biomassa, há formação de vários compostos tóxicos tais como o furfural, HMF, ácido fracos, e compostos fenólicos, os quais exercem efeitos inibitórios sobre as leveduras, tendo como consequência queda no rendimento fermentativo. Além das vantagens tecnológicas que o processo industrial brasileiro apresenta quanto à incorporação da produção de etanol 2G nas plantas já existentes, soma-se a abundância de matéria prima proveniente da própria indústria sucroalcooleira. No entanto, é de extrema necessidade o desenvolvimento de leveduras capazes de resistir as diversas condições inibitórias provenientes do novo substrato, as quais são potencializadas pelo reciclo celular. Neste sentido, o presente trabalho objetivou o desenvolvimento de novas linhagens de leveduras através das técnicas de hibridação e evolução adaptativa/seleção. Para isto, foi realizado o cruzamento massal envolvendo 5 linhagens de Saccharomyces cerevisiae, previamente selecionadas por demonstrar alta tolerância em fermentações em mosto misto a base de hidrolisado lignocelulósico e melaço de cana-de-açúcar. Inicialmente estudos foram realizados com a intenção de se obter altas taxas de esporulação, a fim de se propiciar uma grande quantidade de cruzamentos aleatórios para consequente geração de uma ampla biodiversidade, aumentando assim a possibilidade de se obter indivíduos com fenótipos melhorados. A cultura resultante do cruzamento massal foi seguida de evolução adaptativa/seleção, buscando, após cerca de 51 gerações, um enriquecimento da cultura com as linhagens mais tolerantes. Por meio de avaliação de crescimento em microplacas (DO 600nm), foram selecionadas 10 isolados evoluídos, os quais foram submetidos a ensaio de fermentação em bancada, simulando tanto quanto possível as condições industriais. Ao final, foi possível destacar uma linhagem por apresentar teor de reserva de trealose significativamente maior que as demais linhagens avaliadas, demonstrando assim a geração de um fenótipo melhorado. Searching for renewable energy sources to substitute the fossil fuels use is growing, due to a great concern worldwide for global warming and climate change. Brazil is considered the holder of the most economically viable process of ethanol production. Estimates indicate that ethanol production of first generation will not be enough to supply future global demand for biofuel. Therefore, an ethanol production from the lignocellulosic biomass show up as a potential solution; however, during biomass pretreatment and hydrolysis, several toxic compounds such as furfural, HMF, weak acid, and phenolic compounds are formed, which exert inhibitory effects on yeasts, resulting in a fermentative yield decrease. Besides the technological advantages, presents in Brazilian industrial processes to incorporation of 2G ethanol production in existing factories, add up the abundance of feedstock comes from the own sugar and alcohol industry. However, the development of yeasts strains, resisting to inhibitory conditions from the new substrate which are potentiated by the cellular recycle, is extremely necessary. In this sense, the present work aimed the development of new yeasts strains by hybridization and adaptive evolution techniques. Mass mating was carried out involving 5 strains of Saccharomyces cerevisiae, previously selected by demonstrating high tolerance to fermentation from mixed-must composed by lignocellulosic hydrolyzate and sugarcane molasses. Previous studies were carried out to get high rates of sporulation that promote random crosses and broad biodiversity, in order to obtain individuals with improved phenotypes. The culture resulting from the mass mating was followed by an adaptation/selection, during 51 generations, generating enrichment of more tolerant strains. By means of microplate growth evaluation (DO 600nm), 10 evoluted isolates were selected, which were submitted to lab scale fermentation, simulating as much as possible as industrial conditions. At the end, it was possible to highlight a lineage demonstrating significantly higher trehalose reserve content than the other lineages evaluated, thus demonstrating a generation of an improved phenotype.

A fermentação alcoólica é susceptível à presença de contaminantes e controlar este problema é um desafio para a indústria produtora de etanol, sejam os contaminantes bacterianos ou leveduras selvagens. As leveduras selvagens ocorrem naturalmente no ambiente e nos substratos utilizados na fermentação e são mais difíceis de controlar do que contaminações por bactérias; podem causar excesso de produção de espuma, levando a perda do teor alcoólico e perda de eficiência fermentativa. Características morfológicas particulares de leveduras Saccharomyces cerevisiae podem exacerbar estes traços indesejáveis para a fermentação. Métodos de isolamento e identificação de leveduras selvagens podem consistir de testes de resistência à temperatura, avaliação por microscopia e a utilização de meios diferenciais sintéticos, que podem ser utilizados no isolamento de leveduras selvagens dos mais diversos substratos e identificação de características morfofisiológicas. O emprego de técnicas moleculares como PCR (Polymerase Chain Reaction) e DNA-fingerprinting também são empregadas, sendo capazes de detectar contaminações em pequenas quantidades, além de permitir a diferenciação a nível intra-específico. Foram avaliados 14 isolados de levedura proveniente de ambiente industrial, utilizando as leveduras CAT-1 e PE-2 como linhagens de referência, sendo avaliadas as características morfofisiológicas através de plaqueamento em meio diferenciais como WLN, BiGGY e Nagai, em conjunto com a análise microscópica das células. Os isolados foram também avaliados através de um teste de resistência de temperatura, testes de capacidade fermentativa e crescimento em microplacas em diversos meios. Por fim, foi extraído o DNA das amostras e este foi avaliado através de reação de PCR e sequenciamento a nível intra-específico. Os resultados mostraram grande variação de padrões morfológicos, presença de células deficientes respiratórias petite e crescimento consistente em temperaturas elevadas, característica esta geralmente presente em leveduras selvagens. Quanto à capacidade fermentativa, 12 dos 14 isolados apresentaram esta característica nos meios utilizados. No crescimento em microplaca, os isolados apresentaram comportamentos distintos em diferentes meios, quando comparados com as linhagens referência. Por fim, a análise molecular das amostras apresentou diferentes padrões de banda entre si e em comparação com as linhagens referência, e o sequenciamento a nível intra-específico mostrou que todos os isolados pertencem a espécie Saccharomyces cerevisiae. Alcoholic fermentation is susceptible to the presence of contaminants and controlling this problem is a challenge for the ethanol industry, whether it\'s bacterial contaminants or wild yeasts. Wild yeast naturally occur in the environment and on substrates used in fermentation and are more difficult to control than contamination by bacteria. They may cause excessive foaming, leading to loss of the alcoholic yield and decrease of fermentative efficiency. Particular morphological traits of Saccharomyces cerevisiae yeasts may exacerbate these undesirable traits for fermentation. Methods of isolation and identification of wild yeast may consist of temperature resistance tests, evaluation by microscopy and also the use of synthetic differential media, which can be used to isolate wild yeasts from the most diverse substrates and to identify their morphophysiological characteristics. The use of molecular techniques such as PCR (Polymerase Chain Reaction) and DNA-fingerprinting are also employed, being able to detect contaminations in small quantities, besides allowing intra-specific differentiation. A total of 14 yeast isolates from an industrial environment were evaluated, using yeasts CAT-1 and PE-2 as reference strains. Morphophysiological characteristics were evaluated by plating in differential media such as WLN, BiGGY and Nagai, alongside with microscopic analysis of the yeast cells. The isolates were also evaluated according a temperature resistance test, fermentation capacity tests and a microplate growth assay in several media. Finally, the DNA was extracted from the samples and was evaluated through PCR reaction and sequencing at intra-specific level. The results showed a great variation of morphological patterns, presence of petite respiratory deficient cells and consistent growth at high temperatures, a characteristic that is generally present in wild yeasts. Regarding the fermentative capacity, 12 of the 14 isolates showed this trait in the media used. In the microplate growth assay, the isolates presented different behaviors in different media when compared with the reference strains. Finally, the molecular analysis of the samples presented different band patterns among themselves and in comparison with the reference lines, and the intra-specific sequencing showed that all the isolates belong to the species Saccharomyces cerevisiae.

As principais matérias-primas do chocolate, obtidas a partir das sementes secas do fruto do cacaueiro (Theobroma cacao), são a manteiga e o líquor de cacau. Para se obter matérias-primas de alta qualidade é necessário que o processo que antecede a industrialização, no caso a fermentação, seja padronizado para que sejam formados nas sementes os precursores de aroma, sabor e cor característica do chocolate. No interior do fruto do cacaueiro são encontradas as sementes envoltas por uma mucilagem composta por: água, pectina, sacarose, glicose, frutose, proteínas, ácidos e sais. O processo fermentativo do cacau ocorre sem qualquer tipo de inóculo ou padronização. Devido a este fato, os padrões de qualidade das sementes obtidas são as mais adversas e muitas vezes a presença de compostos interferentes e não desejáveis são formados ao longo desse caminho. Visando a otimização do processo fermentativo este trabalho teve por objetivo selecionar leveduras de ocorrência espontânea presentes na fermentação do cacau, reinoculá-las no processo natural in locu e comparar com o processo de ocorrência espontânea, avaliando assim o potencial do coquetel de leveduras a ser utilizado futuramente para padronizar o processo. Para tanto, foram isoladas 367 linhagens de leveduras de ocorrência espontânea em duas fazendas no sul da Bahia. As linhagens passaram por uma seleção onde foi implementado um programa de seleção composto por três ensaios: ensaio de crescimento em pectina; análise de Açúcar Redutor Total livre (ART); e avaliação de atividade enzimática. Foi possível selecionar três linhagens de leveduras promissoras com potencial pectinolítico as quais foram testadas in locu no município de Itabuna-BA. O processo de isolamento, seleção e reintrodução das linhagens selecionadas no processo fermentativo do cacau se mostrou uma prática altamente eficaz. Os resultados obtidos com a inoculação inicial de leveduras selecionadas, antecipou os eventos como produção de etanol, ácido acético, drenagem do mel e elevação da temperatura em 24 horas em relação ao controle. The fundamental raw material to produce chocolate, obtained from dried seeds of cocoa fruit (Theobroma cacao), are butter and cocoa liquor. In order to obtain high quality of raw materials, it is necessary standardize the procedure before industrialization, known as fermentation, so that the aroma, taste and color precursors of chocolate must be formed in the seeds. Inside the fruits exists a white mucilaginous pulp, which covers the beans, it contains water, pectin, sucrose, glucose, fructose, proteins, acids and salts. The fermentation of cocoa seeds occurs in wooden boxes or piles on the ground without any control or standardization. Due to this fact, the quality of the seeds are the most adverse, the presence are often of interfering compounds and undesirable products could be formed along the way. To optimize the fermentation process this study aimed to select pectinolytic yeasts of spontaneous occurrence from cocoa fermentation, re-inoculate them in the natural process and compare with the spontaneously occurring process. Consequently evaluate the yeast cocktail potential as a standard inoculum. Therefore, we isolated 367 yeast strains from spontaneous cocoa fermentation in two different farms in southern Bahia - Brazil. The strains were analyze to a selection-screening program, which consists of three tests: ability to grow in pectin medium; Total free Reducing Sugar Analysis (ARTL); and evaluation of enzyme activity. It was possible to select three yeast strains with promising pectinolitic potential. Those strains were tested in locu in Itabuna-BA, Brazil. The results of that program, selection and re-introduction in the fermentation process proved to be a highly effective practice. The results obtained with the initial inoculation of selected yeasts, could anticipate the fermentation events in 24 hours, such as the production of ethanol, acetic acid, sweating drainage and temperature rise when compared with the control.

A busca por soluções sustentáveis, levando a um processo energético mais eficiente induziu a novas tecnologias e esforços estão sendo realizados para viabilizar o etanol de segunda geração, com o aproveitamento da biomassa celulolítica como substrato para a fermentação alcoólica. Contudo, durante a hidrólise do bagaço, muitos compostos tóxicos à levedura são formados como o furfural, hidroximetilfurfural, ácido acético, e compostos fenólicos com efeitos depressivos sobre a fermentação. A adição de melaço no hidrolisado de bagaço poderia permitir uma fermentação com maior teor alcoólico, contribuindo para um balanço energético favorável da destilação, além de propiciar nutrientes minerais e orgânicos para a levedura. Tais nutrientes poderiam permitir um processo fermentativo com reciclo de células de leveduras aproveitando assim uma estrutura e conhecimentos já existentes na destilaria de etanol de primeira geração. O reciclo de células permitiria fermentações rápidas, porém impõe repetidas condições estressantes, o que torna um grande desafio a obtenção de linhagens com o perfil de tolerância desejado. Assim este trabalho se propôs a selecionar linhagens de Saccharomyces cerevisiae com múltiplas tolerâncias em relação aos inibidores tanto presentes no hidrolisado como no melaço. Para tal, foram impostas condições estressantes sobre culturas da linhagem SA-1 e de leveduras isoladas de destilarias brasileiras durante cerca de 62 gerações, forçando uma evolução adaptativa ou mesmo um enriquecimento/seleção de indivíduos mais tolerantes. Paralelamente a biodiversidade de linhagens isoladas de destilarias foi avaliada quanto aos atributos de tolerância aos compostos tóxicos presentes no hidrolisado do bagaço. As linhagens com maiores desempenhos foram avaliadas em fermentações com reuso de células empregando-se substrato constituído de hidrolisado e melaço, sendo que 4 linhagens se mostraram superiores às linhagens referenciais. Destas, dois isolados (242 e 408) foram esporulados e os conjuntos dos haploides foram empregados em cruzamentos massais. Simultaneamente, 273 haploides isolados das linhagens 242 e 408 foram avaliados quanto ao crescimento (DO600nm) em substrato constituído por hidrolisado e melaço, sendo que 32 foram selecionados. Após a tipificação segundo o \"mating type\" os mesmos foram utilizados em cruzamentos direcionados mediante micromanipulação, resultando em 35 cruzamentos. Cinco híbridos de cada cruzamento direcionado foram resgatados (155 isolados), que juntamente com 80 isolados oriundos do cruzamento massal, foram novamente avaliados quanto ao crescimento (DO600nm) e a seguir em fermentações com reciclo de células. Cinco linhagens se destacaram como superiores aos parentais, demonstrando que mediante o protocolo empregado foi possível incrementar o perfil de tolerância de Saccharomyces cerevisiae para suportar os estresses impostos por um substrato para produção do etanol de segunda geração. The search for sustainable solutions to improve process efficiency has promoted the development of new technologies, and the use of cellulolytic biomass as the substrate for fermentation has emerged as a promising second-generation ethanol production strategy. However, the hydrolysis of this material results in the formation of toxic compounds to yeast such as furfural, hydroxymethylfurfural, acetic acid and phenolic compounds, with deleterious effects on fermentation. Addition of molasses in the bagasse hydrolysate could allow fermentation with higher alcohol content contributing to a favorable energy balance in the distillation, as well as providing minerals and organic nutrients for the yeast. These nutrients could allow a fermentative process with yeast cell recycle, utilizing the structure and knowledge already existing in first generation process. The cell recycle enables a rapid fermentation, but imposes repeated stress conditions, making it challenging to obtain strains with the desired tolerance profile. The purpose of this study was to select Saccharomyces cerevisiae strains with multiple tolerances to inhibitors present in the hydrolysate and molasses. Stressful conditions were imposed on cultures of SA-1 strain and indigenous strains from Brazilian distilleries for around 62 generations, forcing an adaptive evolution or even an enrichment / selection of more tolerant individuals. In parallel, the biodiversity of the strains from Brazilian distilleries were evaluated with respect to their tolerance to the toxic compounds present in bagasse hydrolysate. The strains that showed higher performance were assessed in fermentations with cell reuse employing substrate composed by hydrolyzate and molasses. Four of the analyzed strains exhibited better performance than the reference strain. Of these, two isolates (242 and 408) were sporulated and the haploids were subjected to mass mating. Simultaneously, 273 haploids rescued from the strains 242 and 408 were evaluated for growth (OD 600 nm) in the substrate consisting of hydrolysate and molasses, and among them 32 were selected. After the characterization according to the \"mating type\", the haploids were utilized in direct mating induced by micromanipulation, totaling 35 crossings. Five hybrids from each direct mating were rescued (totaling 155 isolates), which together with 80 isolated from the mass mating, were evaluated for growth (OD 600 nm) and then in fermentation with cell recycle. 5 strains have excelled as superiors to the reference strain showing that by the protocol employed was possible to increase profile of tolerance of Saccharomyces cerevisiae to resist pressures imposed by a substrate for second-generation ethanol production.

O etanol contribui significativamente para que a matriz energética do país se apresente extremamente favorável quanto à participação da energia renovável. A demanda por este biocombustível é crescente e tecnologias que permitam a sua produção de forma sustentável é de suma importância, como a fermentação com alto teor de etanol, já empregada em alguns países. Linhagens de leveduras com tolerância a múltiplos estresses muito contribuíram para a implantação de tal tecnologia no Brasil. Neste contexto se insere o presente trabalho, o qual busca linhagens de leveduras capazes de suportar os estresses impostos por uma fermentação com alto teor alcoólico. Para tal, 3 entre as melhores linhagens industriais de Saccharomyces cerevisiae atualmente disponíveis (CAT-1, PE-2 e SA- 1), foram utilizadas num programa para a seleção de híbridos para tolerância múltipla aos estresses etanólico, osmótico, ácido, além de outros, inerentes à fermentação com alto teor de etanol. Estas linhagens foram esporuladas e dissecadas para obtenção de células haplóides, as quais foram submetidas a cruzamentos entre- e intra-linhagens. Foram conduzidos cruzamentos massais (aleatórios) cujos produtos foram submetidos à evolução adaptativa em meios com os fatores estressantes em intensidades crescentes no transcorrer de 80 gerações. Ao final da evolução buscou-se variantes prevalentes na população de híbridos, os quais foram submetidos a novos procedimentos seletivos com imposições de várias condições estressantes. Igualmente foram conduzidos cruzamentos direcionados entre haplóides mediante micromanipulação, sendo tais híbridos submetidos ao mesmo procedimento seletivo nos meios estressantes. Assim, a partir de 230 haplóides das 3 linhagens, 174 isolados (120 oriundos dos cruzamentos massais e 54 dos cruzamentos direcionados) foram pré-selecionados pela maior tolerância aos meios seletivos, tendo os seus cariótipos estabelecidos mediante a cariotipagem eletroforética. Os isolados com maior tolerância (27) foram novamente avaliados em fermentações com reciclo de células e sob condições de elevado teor alcoólico (até 14,5% v/v) em mosto de melaço e água. Em todas as etapas da seleção os isolados foram comparados com as linhagens parentais (CAT-1, PE-2 e SA-1), sendo que ao final do processo seletivo destacou-se a linhagem 35B (híbrido entre CAT-1 e PE-2) com atributos fermentativos superiores aos exibidos pelos parentais. Tais atributos fermentativos contemplaram parâmetros bioquímicos, fisiológicos e tecnológicos (rendimento em etanol, viabilidade celular, crescimento em biomassa, formação de glicerol e teores celulares de carboidratos de reserva glicogênio e trealose). Os resultados permitem sugerir que devido às características fermentativas desejáveis do híbrido 35B, o mesmo possa ser empregado no processo industrial para ser avaliado como uma promissora linhagem a conduzir a fermentação com alto teor alcoólico. Ethanol contributes significantly to the energy country matrix, which presents itself as extremely favorable to the share of renewable energy. The demand for this biofuel is increasing and technologies for its production in a sustainable way is of paramount importance such as fermentation with very high gravity, already used in some countries. Yeast strains tolerant to multiple stresses greatly contributed to the deployment of such technology in Brazil. In this context the present work is inserted, which seeks yeast strains capable of withstanding the stresses imposed by high ethanol content fermentation. In order that, three of the best industrial strains of Saccharomyces cerevisiae currently available (CAT-1, PE-2 and SA-1), were used in a program to select hybrids with tolerance towards multiples stresses: ethanolic, osmotic and acid, besides other factors involved in a high ethanol content fermentation. These strains were sporulated and dissected to obtain haploid cells, which were submitted to inter- and intra-strains crossings. Hybrids from polycrossings (random crossings) were subjected to an adaptative evolution in media with increasing stressing action over the course of 80 generations. At the end of evolution, prevalent variants were sought in the hybrids population, and submitted to new selective procedures with several stressing conditions. In the same way, directed crossings (between identified haplois) were performed by micromanipulation, and the resulting hybrids were subjected to the same selective procedure. Therefore, from 230 haploid from the 3 parent strains, 174 were isolated (120 from polycrossings and 54 from directed crossings) and pre-selected for higher tolerance in selective media; moreover their karyotypes were established by electrophoretic karyotyping. Strains showing greater tolerance (27) were again evaluated during cell recycling fermentations with high ethanol content (up to 14.5% v/v) using must formulated with water and molasses. At all stages, the isolates were compared with the parental strains (CAT-1, PE-2 and SA-1), and at the end of the selection process, the strain 35B (hybrid between CAT-1 and PE-2) standed out with fermentative attributes higher than the parentals. The fermentative performance was assessed by biochemical, physiological and technological parameters (ethanol efficiency, cell viability, biomass gain, glycerol formation and cellular levels of reserve carbohydrates - glycogen and trehalose). The results suggest that due to desirable fermentation traits, the hybrid 35B, could be used as starter in industrial fermentation process with high ethanol content.

O presente trabalho teve por objetivo avaliar a atividade celulolítica de fungos isolados de bagaço de cana-de-açúcar e serapilheira, atuantes sobre bagaço de cana-de-açúcar e farelo de arroz como substratos. Dez isolados fúngicos de amostras de bagaço de cana-de-açúcar e material vegetal em decomposição foram avaliados quanto à atividade celulolítica, tendo como referências os fungos Trichoderma reesei QM9414 e T. reesei RUTC30. A atividade celulolítica foi estimada pela capacidade hidrolítica do extrato enzimático dos fungos crescidos em bagaço de cana-deaçúcar, com e sem pré-tratamento térmico e com diferentes proporções de farelo de arroz. A atividade celulolítica foi estimada tanto em papel de filtro, para celulase total, como a carboximetilcelulose sódica, para endoglucanase. Os isolados identificados por análise molecular através da região 26S rDNA foram: Aspergillus giganteus (S1), Aspergillus fumigatus (S2 e F4), Trichoderma viride/Trichoderma hamatum (S3 e S6), Trichoderma koningiopsis (TPB), Paecilomyces variotti (F1), Moniliophthora perniciosa (F2), Acremonium cellulyticus/Penicillium verruculosum (G3) e Trichoderma sp RA305 (MAD). O Trichoderma reesei QM9414 apresentou a maior atividade tanto para celulase total como para endoglucanase. O isolado F1 não diferiu estatisticamente para atividade de endoglucanase em relação à cepa QM9414 em substrato contento somente bagaço. O isolado MAD não diferiu estatisticamente para atividade de celulase total e endoglucanse em relação à cepa QM9414 em substrato contendo 10% de farelo de arroz. Os isolados TPB e S2 apresentaram as maiores atividades para endoglucanase em bagaço de cana-de-açúcar tratado. Tais resultados mostram que a biodiversidade em nichos, como o bagaço de cana-de-açúcar e materiais vegetais em decomposição, podem fornecer linhagens de fungos celulolíticos com grande potencial biotecnológico. The objective of this work was to evaluate the cellulolytic activity of fungi isolated from sugarcane bagasse and decaying plant material, growing on sugarcane bagasse and rice bran as substrate. Ten isolates from sugarcane bagasse and decayed plant material were evaluated for their cellulolytic activity as compared to the fungi Trichoderma reesei QM9414 and T. reesei RUTC30. The cellulolytic activity was estimated by the hydrolytic capacity of the enzymatic extract of fungi grown on sugarcane bagasse, with and without pre-heat treatment and with different proportions of rice bran. It was used as substrates both filter paper, for total cellulolytic activity, and sodium carboxymethyl cellulose, for endoglucanase activity. The isolates identified by molecular analysis using 26S rDNA region were: Aspergillus giganteus (S1), Aspergillus fumigatus (S2 and F4), Trichoderma viride/Trichoderma hamatum (S3 and S6), Trichoderma koningiopsis (TPB), Paecilomyces variotti (F1), Moniliophthora perniciosa (F2), Acremonium cellulyticus/Penicillium verruculosum (G3) and Trichoderma sp RA305 (MAD). Trichoderma reesei QM9414 showed the highest total cellulolytic activity and endoglucanase activity among all isolates. Isolate F1 was not statistically different for endoglucanase activity in relation to strain QM9414 using bagasse as substrate. Isolate MAD was not statistically different for total cellulose activity and endoglucanase activity in relation to strain QM9414 using substrate containing 10% of rice bran. Isolates TPB and S2 had the highest endoglucanase activities using sugarcane bagasse treated as substrate. These results show that biodiversity in niches, such as bagasse and decayed plant material, can provide strains of cellulolytic fungi with great biotechnological potential.