A biomembrana, que é uma membrana de celulose bacteriana (C6H10O5)n formada na superfície do meio de cultivo durante a fermentação acética, foi obtida através do cultivo associado de Gluconacetobacter xylinus (formalmente Acetobacter xylinum) e Saccharomyces cerevisiae em meio de folhas de chá verde, resíduos de frutas (abacaxi, mamão, laranja), resíduos de vegetais (beterraba), vinho e colágeno em condições estáticas a 28 ± 2°C de 7 a 30 dias de cultivo. Foi incorporado à biomembrana, extrato hidroalcoólico de Calendula officinalis, devido as suas propriedades anti-inflamatórias, antioxidantes e cicatrizantes. A espessura, o diâmetro e o peso da biomembrana foram mensurados e foram calculados a produtividade, bem como o fator de conversão de açúcar em celulose. A caracterização da biomembrana foi realizada por Differential Scanning Calorimetric, espectroscopia infravermelho, Brunauer-Emmett-Teller, resistência à tração e alongamento, microscopia eletrônica (Escola Politécnica - USP) e difração de raio-X. Através destas análises verificou-se que a biomembrana obtida nos diferentes meios de cultivo é composta por celulose, o tamanho médio dos poros variou de 517,9 a 1582,0 nm, a resistência à tração variou de 0,76 a 4,32 kN/m e o índice de cristalinidade entre 75% e 91%, a espessura da biomembranas variou de 0,16 a 6,38 mm. Foram realizados 576 experimentos, a maior produtividade (8,23 g de celulose/dia) foi atingida no meio de mamão com suco de laranja (suco de mamão: 50% v/v e suco de laranja: 19% v/v) em 7 dias de cultivo. O maior fator de conversão (2,36 g celulose/g de açúcar) foi obtido no meio de chá verde em 25 dias de cultivo. A adição de 1,5% p/v de colágeno ao meio de chá verde dobrou a massa da biomembrana. A incorporação do extrato de calêndula aumentou a flexibilidade, a cristalinidade e as propriedades mecânicas da biomembrana de chá verde. / The biomembrane, cellulose membrane (C6H10O5)n formed in medium surface, was obtained from an associate culture Gluconacetobacter xylinus (formally Acetobacter xylinum) and Saccharomyces cerevisiae in green tea leaves, fruit residues (pineapple, papaya, orange), vegetables residues (beet), wine and collagen media in static condition , at 28 ± 2 ºC in 7 - 30 days cultivation. The Calendula officinalis hydroalcoholic extract was incorporated in the Biomembrane, due to its anti-inflammatory, anti-oxidants and cicatrizing proprieties. The biomembrane thickness, diameter and weight were measured. The productivity and conversion factor from cellulose to sugar were calculated. The biomembrane caracterization was performed by Differential Scanning Calorimetric, infrared spectroscopy, Brunauer-Emmett-Teller, resistance to tension, elongation, eletrocnic microscopy and raio-X difraction. In these analyses were verified that biomembrane obtained in different media were composed by cellulose, average porous size varied from 517.9 to 1582.0 nm, the resistance to tension varied from 0.76 to 4.32 kN/m and cristalinity index varied from 75% to 91%. The biomembrane thickness varied from 0,16 to 6,38 mm. It was performed 596 tests, the highest bacterial cellulose yield (8.23 ± 0.58 g cellulose/day) was obtained in papaya with orange (papaya juice: 50% v/v and orange juice: 19% v/v) in 7 cultivation days. The highest conversion factor (2,36 g cellulose/g sugar) was obtained in green tea medium in 25 days. The addition of 1.5% w/v collagen to the green tea media increased 2 times the biomembrane weight. The biomembrane absorption capacity for water and Marigold hydroalcoholic extract (1:1), were from 1.73 to 22 and, from 1.75 to 24 times dry weight, respectively. The Marigold extract improved the green tea biomembrane flexibility, cristalinity, and physical proprieties.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-22T16:20:14Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Renata_Almeida_PPGG_041115.pdf: 4144409 bytes, checksum: 709008675e5c4c4efeeb7b0b06e8e4ee (MD5) / Made available in DSpace on 2016-04-22T16:20:14Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Renata_Almeida_PPGG_041115.pdf: 4144409 bytes, checksum: 709008675e5c4c4efeeb7b0b06e8e4ee (MD5) Previous issue date: 2015-07-30 / CAPES / Reuni / CNPq / A cana-de-açúcar tem papel relevante na economia brasileira, apesar de estar sucetível a fatores bióticos e abióticos que influenciam para redução de produtividade agrícola e industrial. Apesar de existirem organismos como agentes causais de doenças que contribuam com danos à cana-de-açúcar, micro-organismos simbiontes interagem com a cultura conferindo benefícios no processo, tais como a fixação de Nitrogênio. Dessa forma, a análise das proteínas envolvidas na resposta a fatores bióticos pode auxiliar no desenvolvimento de novas variedades. No presente trabalho, foi analisado o proteoma da interação da cana-de-açúcar com patógeno (Leifsonia xyli subsp. xyli; Lxx) e/ou com o simbionte (Gluconacetobacter diazotrophicus; Gd) por 2-DE (Eletroforese bidimensional) seguida de MS (Espectrometria de massa). Os resultados da eletroforese bidimensional com a interação cana-de-açúcar/Gd mostraram que 173 spots foram diferencialmente expressos. A análise por espectrometria de massa identificou 65 proteínas. Tais proteínas foram principalmente associadas com produção de energia, componentes de fotossistemas e indução de resposta a estímulos ambientais. Na interação cana-de-açúcar/Lxx, em análise da eletroforese bidimensional, se observou 138 spots com expressão diferenciada. A análise por espectrometria de massa identificou 56 proteínas responsivas a estresses bióticos, defesa e metabolismo de carboidratos. Durante a interação simultânea com Gd e Lxx, o proteoma da cana-de-açúcar a análise da eletroforese bidimensional mostrou que 142 spots apresentaram nível de expressão alterada. A análise por espectrometria de massa identificou 30 proteínas associadas com metabolismo de carboidratos e defesa contra estresses bióticos. Os resultados obtidos compõem um conjunto de proteínas (e genes codificantes) com provável utilidade como marcadores funcionais auxiliares no melhoramento genético, na seleção de variedades com interação benéfica de crescimento/fixação biológica de nitrogênio com Gd, e/ou maior resistência à patogenicidade de Lxx. / The sugarcane has an important role in the Brazilian economy, despite its suceptibility to biotic and abiotic factors that decrease agricultural and industrial productivity. Although there are parasitic organisms that contribute to damage sugarcane, symbiotic microorganisms interact with the culture, providing benefits in the process, such as nitrogen fixation. Thus, the analysis of proteins involved in the response to biotic factors may assist the developing new sugarcane varieties. In this study, we analyzed the proteome of the interaction of sugarcane with pathogen (Leifsonia xyli subsp. xyli; Lxx) and / or symbiont (Gluconacetobacter diazotrophicus; Gd) by 2-DE (Two-dimensional electrophoresis) followed by MS (Mass Spectrometry). The results of two-dimensional electrophoresis with sugarcane / Gd interaction showed that 173 spots showed altered expression level. Analysis by mass spectrometry identified 65 proteins. These differentially expressed proteins were primarily associated with energy production, and induction components photosystems response to environmental stimuli. Since the sugarcane interaction / Lxx the analysis of two-dimensional electrophoresis demonstrated that 138 spots showed altered expression level. The analysis by mass spectrometry identified 56 proteins responsive to biotic stresses, defense and carbohydrate metabolism. During simultaneous interaction with Gd and Lxx, the proteome analysis of sugarcane showed that 142 spots with altered expression level. Analysis by mass spectrometry identified 30 proteins associated with carbohydrate metabolism and protection against biotic stresses. The results obtained in this work comprise a set proteins (and encoding genes) with probable utility as auxiliary functional markers in plant breeding, in the selection of varieties with beneficial interaction for growth/biological nitrogen fixation with Gd, and/or increased resistance to pathogenic Lxx.

Nitrogen (N) is an essential plant nutrient, widely applied as N-fertilizer to improve yields of agriculturally important crops. An alternative to fertilizer use could be the exploitation of plant growth-promoting bacteria, capable of enhancing growth and yield of many plant species. Azospirillum and Gluconacetobacter are root colonizing, free-living, N2-fixing bacteria (diazotrophs) with the potential to transfer fixed N to associated plants. The purpose of this study was to evaluate the agronomic efficiency of two diazotrophs, Azospirillum lipoferum and Gluconacetobacter azotocaptans, inoculated onto wheat. Physiological parameters and yield components were evaluated. The objectives of this study were to: 1) determine the survival of each diazotroph species on wheat seeds over time; 2) determine the survival of A. lipoferum and G. azotocaptans inoculated on wheat seed treated with a fungicide seed treatment, Dividend® XL RTA®; 3) determine if inoculation of wheat with the diazotrophs under controlled conditions causes an increase in dry matter, N2-fixation and N uptake; 4) determine if fertilizer N applied at three levels influences atmospheric N2-fixation by A. lipoferum or G. azotocaptans; 5) determine if inoculation of wheat with A. lipoferum or G. azotocaptans under field conditions causes any increase in dry matter, N2-fixation and N uptake; 6) determine if N-fertilization levels under field conditions influenced N2-fixation by A. lipoferum or G. azotocaptans. In order to meet these objectives lab, growth chamber, and field studies were completed. Laboratory investigations revealed that the decline in recovery of colony forming units (CFU) of G. azotocaptans was not significantly different (P<0.05) for any seed treatment. There was a general decrease in CFU over time regardless of seed treatment. Analysis of the recovered CFU of A. lipoferum over time showed that there was a significant difference (P<0.05) between both the non-sterilized seed and the Dividend® XL RTA® treated seed when compared sterilized seed. Recovery of CFU on sterilized seed declined at a more rapid rate compared to the other two seed treatments. Gluconacetobacter azotocaptans and A. lipoferum were not negatively influenced by the Dividend® XL RTA® seed treatment. Also, both diazotrophs were able to compete with other microorganisms that may have been on the seed coat of unsterilized seeds. Azospirillum lipoferum and G. azotocaptans were able to fix atmospheric N, but, there were no significant (P<0.05) differences between the diazotroph species. Additions of fertilizer N enhanced N2-fixation, in both the growth chamber and field studies. As the amount of fertilizer N increased, so did the %Ndfa and N uptake. In the growth chamber study, inoculated wheat, and fertilized with 12.2 and 24.5 µg N g-1 had the highest %Ndfa of 25.5%, and wheat fertilized with 24 µg N g-1 had the highest N uptake (1.3 g pot-1) at maturity. In the field study, inoculated wheat fertilized with of 80 kg N ha-1 had significantly higher (P<0.05) %Ndfa (10.5%) compared to wheat grown with the other fertilizer levels, which also corresponded to the highest N uptake in wheat plants (47 kg ha-1). The diazotrophs also affected the partitioning of N in the wheat plants differently. Wheat inoculated with A. lipoferum had significantly higher (P<0.05) amounts of N accumulated in heads of plants, and wheat inoculated with G. azotocaptans had significantly higher (P<0.05) amounts of N accumulated in stems of plants. However, this trend was not evident in the field study.

Made available in DSpace on 2014-12-17T15:18:10Z (GMT). No. of bitstreams: 1 CarolinaCSO.pdf: 673645 bytes, checksum: 65edb3dd5d260d66ee98b34da41f4185 (MD5) Previous issue date: 2009-02-27 / Gluconacetobacter diazotrophicus ? uma alfa-proteobact?ria Gram-negativa, tolerante a meios ?cidos, fixadora de nitrog?nio atmosf?rico e foi a primeira bact?ria diazotr?fica endof?tica isolada da cana-de-a??car. Por sua vez, Gluconobacter oxydans, tamb?m alfa-proteobact?ria Gram-negativa, possui a capacidade de oxidar incompletamente alco?is e carboidratos. Ambas de interesse biotecnol?gico e industrial, essas bact?rias tiveram seus genomas seq?enciados completamente em 2007. Desta forma, foi de interesse desse trabalho analisar e comparar os genes de reparo do DNA devido sua import?ncia na manuten??o da integridade gen?mica. Sendo assim, as vias de reparo presentes nos dois organismos foram identificadas, utilizando como base uma terceira alfa-proteobact?ria, a Caulobacter crescentus, cujos genes de reparo foram descritos por um trabalho anterior e tamb?m os genes bem estabelecidos para o reparo do DNA em Escherichia coli. Para esse estudo, um banco de dados contendo ort?logos para os genes de reparo de DNA encontrados nos organismos foi criado e an?lises comparativas por similaridade usando o pacote Blast e o software Clustal foram feitas. Este estudo demonstrou que as principais vias de reparo ao DNA reparos por excis?o, reparo direto, reparo recombinacional e reparo pelo sistema SOS est?o presentes nos organismos analisados, demonstrando, na maioria das vezes, boa similaridade com E. coli. Interessantemente, foram encontradas duplica??es g?nicas nos quais uma das c?pias estava presente no cromossomo e a outra, no plasm?deo, como no caso de UvrD, DnaE e Ssb, possivelmente caracterizando eventos de transfer?ncia lateral. Por fim, uma grande novidade foi a identifica??o de ort?logos para RecB em G. diazotrophicus e G. oxydans e de ort?logos duplicados de RecD em G. diazotrophicus. At? o momento, n?o havia sido relatada a presen?a de membros da via de inicia??o RecBCD do reparo recombinacional em alfaproteobact?rias

β-glucosidases are important components of the cellulase enzyme system in which they not only hydrolyse cellobiose to glucose, but also remove the feedback inhibition effects of cellobiose on exoglucanase and endoglucanase thereby increasing the rate of cellulose degradation to fermentable sugars. A total of 166 proteins were identified as β-glucosidases after manual BLASTp search on the Aspergillus comparative database from eight species. Evidence for Horizontal Gene Transfer (HGT) of bacterial origin of some β-glucosidase genes was provided by their lack of introns, absence of some fungal specific amino acid insertions in their sequences and unusual positions in phylogenetic trees showing similarities to bacterial proteins. A rapid plate assay based on Congo red methods was developed to study the optimum parameters such as pH and temperature for growth of strains and activities of the enzymes produced. Bacterial cellulose (BC) was produced by Gluconacetobacter xylinus. For the first time a fully detailed characterization by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), Differential scanning calorimeter (DSC), Thermogravimetric analysis (TGA) and 13Carbon Solid State Nuclear Magnetic Resonance (SSNMR) of pure BC before and after treatment with a commercially available Aspergillus cellulase enzyme was demonstrated. Two encoding sequences for novel Aspergillus nidulans hydrophobin genes ANID_05290.1 and ANID_07327 that do not fall into either the class I or class II category of hydrophobins were successfully cloned. Two encoding sequences for a novel β-glucosidase gene from an Aspergillus niger strain from Nigeria were amplified and cloned from genomic DNA using PCR. Aspergillus nidulans β-glucosidases (AN2227 and AN1804) expressed in Pichia were purified to homogeneity by using ammonium sulphate precipitation and DEAE-Sephadex A-50 chromatography. Both enzymes had a remarkably broad pH and temperature profile. Further experiments on the development of a technology for lignocellulose degradation based on co-production of β-glucosidase with hydrophobin for biofuel production are suggested.