Universiẗat, Diss., 2002--Giessen.

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-02-23T19:56:49Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO Dayana Montero Rodríguez.pdf: 2077714 bytes, checksum: 9616b20ba65f46313573608ced7b6d62 (MD5) / Made available in DSpace on 2016-02-23T19:56:49Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO Dayana Montero Rodríguez.pdf: 2077714 bytes, checksum: 9616b20ba65f46313573608ced7b6d62 (MD5) Previous issue date: 2015-02-25 / CNPQ / Serratia marcescens UCP/WFCC 1549, isolada do solo do semi-árido do Estado de Pernambuco - Brasil, foi investigada quanto o seu potencial de biodegradação de combustíveis, como também na produção de lipídeos e biossurfactante. A degradação de combustíveis foi avaliada utilizando o meio basal Bushnell Hass (BH), o indicador redox 2,6- diclorofenol – indofenol e a cepa de S. marcescens selvagem e aclimatada em diferentes concentrações do óleo diesel (2, 4, 6, 8, 10, 12 e 15%). Os resultados obtidos demonstraram que a bactéria aclimatada a 15% do óleo diesel apresentou os melhores índices de degradação, com valores de 79,63% para o biodiesel de algodão, 65,57% para o biodiesel de girassol, 60,50% para o diesel, 57,20% para gasolina e 39,26% para querosene. Além disso, S. marcescens demonstrou propriedade de crescer e acumular lipídeos (> 40%) utilizando resíduos agro-industriais (manipueira e óleos vegetais pós-fritura). Os lipídeos produzidos mostraram perfis de ácidos graxos com maior porcentagem em ácidos graxos monoinsaturados, sugerindo uma composição que corresponde às características requeridas para o biodiesel. Ao mesmo tempo, S. marcescens demonstrou habilidade para converter resíduos agroindustriais (manipueira e óleo de milho pós-fritura) em associação com lactose, na produção de biossurfactante, empregando um planejamento fatorial 23. A seleção da melhor condição do planejamento foi avaliada pela variável resposta tensão superficial. O melhor resultado foi obtido no meio constituido por 6% de manipueira e 7,5% de óleo de milho pós-fritura, na ausência de lactose, com uma redução da tensão superficial da água de 72 para 26,2 mN/m. O biossurfactante produzido apresentou propriedade emulsificante (EI24), com valores superiores a 60% de emulsificação utilizando os óleos de soja, diesel, motor e motor queimado. Adicionalmente, o biossurfactante demonstrou estabilidade na redução da tensão superficial frente a diferentes valores de pH, temperatura e NaCl, e mostrou excelente eficiência na remoção de óleo de motor em água, areia de praia e sedimento de mangue (78%, 88,27% e 73,70%, respectivamente). Portanto, S. marcescens UCP/WFCC 1549 demonstrou seu elevado potencial biotecnológico para a produção de biodiesel de boa qualidade, assim como de biossurfactante com aplicação promissora em processos de biorremediação de ecossistemas contaminados com petróleo e seus derivados. / Serratia marcescens UCP/WFCC 1549, isolated from soil of the semi-arid of state of Pernambuco, Brazil, was investigated with regard to their potential to fuel biodegradation as well as for the production of lipids and biosurfactant. The degradation was assessed using Bushnell Hass (BH) medium, the redox indicator 2,6-dichlorophenol – indophenol and S. marcescens wild-type and acclimatized in different concentrations of diesel (2, 4, 6, 8, 10, 12 and 15%). The obtained results showed that strain acclimatized in 15% diesel oil exhibited the best degradation index (79,63% of cotton biodiesel, 65,57% of sunflower biodiesel, 60,50% of diesel, 57,20% of gasoline and 39,26% of kerosene). Also, S. marcescens demonstrated the ability to grow and accumulate lipids (> 40%) using agro-industrial residues (cassava wastewater and waste vegetable oils). The produced lipids exhibited balanced profiles of fatty acids, mainly monounsaturated fatty acids which correspond with biodiesel requirements. In addition, S. marcescens showed ability to produce biosurfactant by bioconversion of agro-industrial residues (cassava wastewater and corn waste oil), in association with lactose, through a 23 factorial design. The best result was obtained in medium containing 6% cassava wastewater and 7,5% corn waste oil, in absence of lactose, with reduction of surface tension of water from 72 to 26,2 mN/m. The biosurfactant had good properties in the emulsification of hydrophobic compounds (EI24 > 60% of soybean oil, diesel oil, engine oil and burned engine oil). Moreover, the biosurfactant demonstrated stability in a wide range of pH, temperature and salinity. Also, it showed excellent efficiency on dispersion of engine oil in water (78%) as well as removing it in beach sand and mangrove sediment (88,27% and 73,70%, respectively). Then, S. marcescens UCP/WFCC 1549 demonstrated their high biotechnological potential for production of good quality biodiesel, as well as biosurfactant with promising application in bioremediation processes.

Orientadores : Tomomasa Yano, Gleize Villela Carbonell / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-02T09:06:04Z (GMT). No. of bitstreams: 1 Escobar_MarceloMartins_M.pdf: 3080727 bytes, checksum: 98512962f37a5bf4b47e1b822cf13f86 (MD5) Previous issue date: 2002 / Mestrado

Orientador: Hélia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T16:00:04Z (GMT). No. of bitstreams: 1 Carvalho_PriscilaHoffmann_D.pdf: 4102242 bytes, checksum: 1496382da70a395d87d5c8536317d42d (MD5) Previous issue date: 2013 / Resumo: A isomaltulose e a trealulose são dissacarídeos isômeros estruturais, que podem ser obtidos a partir da sacarose utilizando-se glicosiltransferase bacteriana. Esses dissacarídeos são considerados açúcares alternativos de grande potencial para uso nas indústrias de alimentos e farmacêutica porque são hidrolisados e absorvidos mais lentamente e apresentam baixo potencial cariogênico comparado com a sacarose. Foi estudada a imobilização de células de Serratia plymuthica ATCC 15928, produtora de glicosiltransferase por gelificação iônica em gel alginato contendo transglutaminase (TG) e também a utilização de células livres para a conversão de sacarose em isomaltulose e trealulose. Utilizando-se células livres de Serratia plymuthica ATCC 15928 foi obtido 70% de conversão em isomaltulose e 8% de trealulose a 25°C por 10 bateladas de 15 minutos, a partir de solução de sacarose 30%. Entre as cinco amostras de alginato de sódio testadas, para a imobilização das células de S. plymuthica ATCC 15928 com e sem adição de TG, foram obtidos melhores resultados (médio de três bateladas) de conversão de sacarose (37,4% de isomaltulose) utilizando o alginato de sódio B, de alta viscosidade (14.000cP Sigma ¿ A 7128) em presença de TG. Nas condições estudadas (1,7% de alginato de sódio, 30% de massa celular úmida, solução de cloreto de sódio 0,2Mol/L, 2% de TG e 35% de sacarose) também houve maior facilidade de formação de grânulos uniformes. A presença de TG como agente de reticulação na matriz de imobilização melhorou a estabilidade de conversão por três bateladas onde observou-se resultado médio 27% maior com relação a matriz com o mesmo tipo de alginato (B) em ausência de TG. A composição da matriz de imobilização com adição de TG foi otimizada por metodologia de planejamento experimental, assim como a adição de gelatina como fonte de proteína adicional para promoção de ligações cruzadas catalisadas pela TG. Os melhores resultados de conversão de sacarose (solução 35%) em isomaltulose (72,66% de isomaltulose e 8% de trealulose em 4 bateladas de 24horas) foram obtidos utilizando-se matriz de polissacarídeo-proteína composto de 1,7% de alginato de sódio 14.000cP (Sigma®-A7128), 0,25mol/L de CaCl2, 0,5% de gelatina, 3,5% de TG e concentração de massa celular úmida superior a 35% (m:v). Verificou-se que a adição de ALMP na matriz de alginato de cálcio-gelatina-TG para imobilização de S. plymuthica, testada por planejamentos experimentais seqüenciais, não aumentou a estabilidade da taxa de conversão de sacarose em isomaltulose quando comparada com as células imobilizadas em matriz de alginato de cálcio-gelatina-TG. Em processo contínuo utilizando-se coluna empacotada com células de S. plymuthica imobilizadas em matriz otimizada e descrita acima, foi obtida taxa de conversão média de 64% de sacarose em isomaltulose durante 200 horas de processo, equivalente a 0,27g de isomaltulose/g de células imobilizadas/hora em coluna a 25°C e fluxo de substrato (35% de sacarose) 0,2mL/min / Abstract: The isomaltulose and trehalulose are disaccharides and structural isomers, which can be obtained from sucrose using bacterial glycosyltransferase. These disaccharide are considered alternative sugars with great potential for use in the food and pharmaceutical industries because they are hydrolyzed and absorbed more slowly and have a low cariogenic potential compared with sucrose. The conversion of sucrose to isomaltulose and trehalulose was estudied using immobilized and free cells of Serratia plymuthica ATCC 15928. The cells were immobilized by ionic gelation in alginate gel containing transglutaminase. Using free cells of Serratia plymuthica ATCC 15928 was obtained 70% isomaltulose conversion and 8% trehalulose conversion at 25° C in 10 batches of 15 minutes from a 30% sucrose solution. Among the five samples of sodium alginate tested for S. plymuthica ATCC 15928 cells immobilization, with or without the addition of TG, the best results (average of three batches) were obtained using sodium alginate B, high viscosity (14.000cP Sigma - A 7128) in the presence of TG, leading to 37.4% isomaltulose conversion from sucrose. In the studied conditions (1.7% sodium alginate, 30% wet cell mass solution of sodium chloride 0.2 Mol/L, 2% TG, 35% sucrose) was also easier to form uniform granules. The presence of TG as a crosslinking agent in the immobilization matrix improved the stability during three batches, resulting in an 27% higher average conversion with respect to a same type of alginate (B) matrix in absence of TG. Immobilization matrix compositions with addition of TG was optimized by experimental design methodology, as well as the addition of gelatin as a protein source for promoting additional crosslinking catalyzed by TG. The best results conversion of sucrose (35% solution) into isomaltulose (72.66% of isomaltulose and 8% of trehalulose in 4 batches of 24 hours) were obtained using proteinpolysaccharide matrix composed of 1.7% alginate 14.000cP sodium (Sigma® A7128), 0.25 Mol/L CaCl2, 0.5% gelatin, 3.5% TG, and wet cell mass concentration of 35% (w:v). It has been found that the addition of ALMP (amidated low methoxyl pectin) into the calcium alginate-gelatin-TG matrix for immobilization of S. plymuthica, tested by sequential experimental design, do not increase the stability of sucrose to isomaltulose conversions rate when compared with cells immobilized in calcium alginate -gelatin-TG matrix. In continuous process using a packed column with S. plymuthica cell's immobilized in the optimized matrix described above, it was obtained an average conversion rate of 64% sucrose to isomaltulose during a 200 hours process, equivalent to 0.27g isomaltulose per gram of immobilized cell per hour, in a column at 25° C and using flow substrate (35% sucrose) of 0.2 mL / min / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

Phytopathogenic Dickeya species inflict large economic losses on a variety of crops. A lack of effective chemical control methods has generated interest in the use of bacteriophages (phages) as a novel tool for biocontrol. In the last decade, six phages have been isolated in Belgium and Poland using Dickeya solani as the host. Previous work in this laboratory has isolated ninety phages capable of infecting D. solani. The majority have been morphologically classified as members of the Ackermannviridae family. In agreement with findings in Salmonella and Klebsiella species, the capsule of D. solani is a likely receptor of Ackermannviridae family phages. Analysis of D. solani strains carrying reporter fusions suggested that the capsule genes are expressed in response to nutritional stress, however disruption of the capsular polysaccharide cluster did not significantly impact virulence. Experiments assessing capsular polysaccharide as a putative receptor for Ackermannviridae family phages in nosocomial pathogen Serratia produced inconclusive results. Phageresistance due to random transposon mutagenesis identified genes encoding transcription factors and regulators, but none directly linked to capsular polysaccharide production. Thirteen phages were capable of infecting a wider host range of Dickeya species. Morphological and genomic analysis showed that six were Podoviridae family members, whilst the other seven were Myoviridae family members. These are part of the recently defined 'hairy Myoviridae', characterised by a distinct morphology. Another member of this grouping was isolated during this study, but is more closely related to phages of Erwinia amylovora. A subset of the Ackermannviridae family phages were shown to be capable of facilitating transduction. This makes them unsuitable for use in the environment due to the risk of deleterious horizontal gene transfer. This is also true for the Myoviridae family members, but not for one of the Podoviridae family members. This phage could therefore be a promising candidate for therapeutic use.

Thesis (Ph. D.)--University of New South Wales, 2004. / Also available online.

Thesis (doctoral)--Universität Hamburg, 1980.

Orientador: Nora Marcela Haun Quiros / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-20T23:09:59Z (GMT). No. of bitstreams: 1 Melo_PatriciadaSilva_M.pdf: 29401299 bytes, checksum: 332e5428465f8888ffa7ea12b727bca7 (MD5) Previous issue date: 1996 / Resumo: Chromobacterium violaceum é um microrganismo de larga distribuição podendo ser encontrado no solo, água e no Rio Negro (Amazônia). Esta bactéria Gram negativa produz um pigmento denominado violaceína, que apresenta alguma atividade tripanocida. Como a violaceína é produzida em pequena quantidade o propósito inicial desse estudo foi investigar cepas diferentes provenientes do Rio Negro que produzissem a violaceína em um maior rendimento. Um sedimento coletado do Rio Negro foi inoculado em caldo simples, 30°C/24 h e posteriormente inoculado através de estrias em ágar nutriente. Somente três tipos de colônias - branca, amarela e vermelha cresceram (todas bactérias Gram negativas). Após dois dias de incubação a colônia branca adquiriu tonalidade violeta indicando a produção de violaceína. Análises espectrais, UV/Vis, IV (Infra Vermelho) e RMN (Ressonância Magnética Nuclear), confirmaram a presença da violaceína. Para determinar as características de C. violaceum e sua variante não pigmentada, testes laboratoriais foram realizados. A coloração pelo Gram, motilidade e estudos bioquímicos e de crescimento indicaram que as colônias branca/violeta e amarela eram C. violaceum. A colônia vermelha era Serratia marcescens (Chromobacterium prodigiosum) a qual produz o pigmento com ação antibiótica chamado prodigiosina. Os dados espectrais (UV/Vis, IV e RMN) reforçam essa conclusão. O pequeno número de bactérias isoladas na amostra confirma a alta atividade antibiótica dos pigmentos produzidos pela C. violaceum (violaceína) e S. marcescens (prodigiosina) na água do Rio Negro. A quimioterapia da doença de Chagas permanece um problema sem solução, e a pesquisa para drogas alternativas está em andamento. A terapia atual dessa doença é insatisfatória e somente o Nifurtimox está em uso, com diversas restrições na administração em pacientes crônicos, devido aos seus efeitos colaterais. Desse modo é de importância fundamental a pesquisa de novas drogas com mecanismos de ação diferentes do Nifurtimox com o objetivo de evitar esses problemas. A violaceína e a prodigiosina extraída da C. violaceum e da S. marcescens, respectivamente apresentam atividade tripanocida, a primeira possui um ID50. de 46 mM e a segunda, menos que 100 mM. A avaliação da titotoxicidade foi realizada através da inibição da síntese de DNA, redução do MTT e captação do Vermelho Neutro (VN), utilizada em células de hamster chinês V-79 (M8). No teste de viabilidade através da redução do MTT o ID50 foi de 6 mM para a prodigiosina, 7mM para a violaceína e 500 mM para o Nifurtimox, no teste do VN o ID50 para a prodigiosina foi de 1,0 mM, 12 mM para a violaceína e 250 mM para o Nifurtimox. A prodigiosina resultou em um valor de ID50 de 20 mM, o Nifurtimox de 100 mM e a violaceína de 5 mM obtidos através da inibição da síntese de DNA / Abstract: Chromobacterium violaceum is a widely distributed microorganism. It is in soil, water and in the Rio Negro (Amazon). This Gram negative rod shaped bacteria produces the pigment violacein, which has shown trypanocide activity. Since violacein is produced in small quantity, the inicial purpose of this study was to investigate differents strains of bacteria from Rio Negro which may produce violacein in highest yield. Sediment was collected from Rio Negro, inoculated in simple broth, 30°C /24 h and striated in simple agar. Only three kinds of colonies - white, yellow, and red grew (rod Gram negative bacteria). After two days the white strain changed to violet indicating violacein production. Spectral analysis, UV/Vis (UV/Visible), IR (Infra Red) and NMR (Nuclear Ressonance Magnetic), confirmed the presence of violacein. In order to determine of C. violaceum characteristics and its non pigmented variants, laboratories tests were undertaken. Gram'stainning, motility, morphological, growth and biochemical studies indicated that the white, yellow and violet colonies were C. Violaceum. The red one was Serratia marcescens (Chromobacterium prodigiosum) which produces the red pigment antibiotic prodigiosin. The spectral data (UV/Vis, IR and NMR) reinforce this conclusion. The low number of microorganisms isolated in the sample confirm the high antibiotic activity of the pigments produced by C. Violaceum (violacein) and Serratia marcescens (prodigiosin) in the Rio Negro water. The chemotherapy of Chagas' disease remains an unsolved problem, and the search for alternative drugs is in course. Current therapy of this disease is unsatisfactory and only Nifurtimox is in general used, with several restricted applicability for chronic patients, as well being deleterious effects. Thus, it is of fundamental importance to search for new drugs with different mechanism of action of Nifurtimox in order to avoid these problems. We have found a potential compounds for the treatment of Chagas' disease, the pigments extracted from S. marcescens and C. violaceum, respectively prodigiosin (ID50 of less 100 mM) and violacein (ID50 of 46 mM). Evaluation ID50 through DNA synthesis inhibition, soluble tetrazolium/formazan (MTT) and Neutral Red (NR) tests on V-79 Chinese hamster (M-8) cells were carried out. Using MTT viability test, ID50 was 6 mM for prodigiosin, 7 mM for violacein and 500 mM for Nifurtimox, and for NR test the ID50 was 1.0 mM, 12 mM and 250 mM for prodigiosin, violacein and Nifurtimox, respectively. Prodigiosin resulted in a ID50 value of 20 mM, for violacein of 5 mM and for Nifurtimox of 300 mM obtained through DNA synthesis inhibition / Mestrado / Bioquimica / Mestre em Ciências Biológicas

Hereditary bacterial symbiosis is a common mechanism by which eukaryotic hosts can acquire traits beneficial for their fitness. Many insects have symbiotic associations with bacteria that trace back millions of years, whose function and evolution are well characterized. Insects can also possess more recently derived symbionts that are closely related to free-living bacteria, and often play a role in host defense. Serratia symbiotica is a recently derived symbiont that infects aphids and provides protection against heat stress, and possibly also plays a nutritional role. Many aspects of the biology of recent symbionts are less well studied, including the diversity of functional roles and evolution among hosts for single lineages of symbionts, the molecular mechanisms that contribute to defense, the early stages of symbiont genome evolution, and interactions with hosts. This dissertation focuses upon S. symbiotica to contribute research addressing each of these themes. Functional studies revealed that S. symbiotica lysis during heat-shock is correlated with protection of the nutritional symbiont Buchnera , and that S. symbiotica has a large effect upon aphid metabolite pools. Despite this large metabolic effect, S. symbiotica does not seem to dramatically influence expression of aphid genes, including those involved in immunity. Analysis of the evolution of S. symbiotica lineages in different aphid hosts revealed this symbiont is common in the aphid subfamily Lachninae, but did not support the obligate nutritional role hypothesized in the literature for this group. Finally, comparison of the S. symbiotica genome to close free-living relatives revealed a genome undergoing massive decay, and provided a rare opportunity to examine the evolution of a recently acquired symbiont.

Serratia entomophila, the causative agent of amber disease, is an endemic bacterium used for the biocontrol of New Zealand grass grub larvae. Although the available biopesticide is effective, its use is limited to areas where sub-surface application is feasible, and is also impacted by soil conditions such as moisture levels and osmolarity. The aim of this study was to elucidate the responses of S. entomophila to osmotic and desiccation stresses in relation to challenges encountered during production, storage and soil application, with the goal of developing a more robust and versatile biocontrol agent. RpoS is a key factor in the stress response of many enteric bacteria. In order to dissociate the effect of RpoS from subsequent cellular stress studies, an rpoS mutant was constructed by site-directed mutagenesis. Assessment of the rpoS mutant showed that RpoS was not implicated in NaC1 or desiccation tolerance of S. entomophila. The rpoS mutant was instead found to have enhanced salt tolerance and could be distinguished from the wild-type by the ability to ferment arabinose, a phenotype that was confirmed through complementation. Complete abolition of the amber disease process was observed using an rpoS strain also missing the Sep virulence genes, suggesting that RpoS is a regulator of the S. entomophila anti-feeding prophage (Afp). These findings indicate a subtle interplay between NaC1 tolerance, virulence and RpoS-mediated regulation of amber disease in S. entomophila. A transposon mutagenesis screen was carried out to identify genes associated with NaC1 tolerance in S. entomophila. Fourteen mutants displaying NaC1 sensitivity were identified, two of which had mutations in genes with potential implications for the formulation of the bacterium as a biocontrol agent. The gene leuO that encodes a LysR-family transcriptional regulator was found to be essential for S. entomophila NaC1 tolerance. The toxicity of increased cellular LeuO from an over-expression vector led to the investigation of the effects of leuO mutation on the proteome. Multiple protein changes observed by two-dimensional gel analysis suggested that LeuO may be a global regulator in S. entomophila, as has been hypothesised for Salmonella species. A second NaC1-sensitive mutant contained an insertion in afp15, the product of which is thought to be involved in assembly of the Afp. As well as being sensitive to NaC1, the afp15 mutant was unable to induce the anti-feeding component of amber disease, again highlighting the link between stress tolerance and virulence in S. entomophila. This study also determined that pre-exposure to NaC1 in conjunction with the provision of exogenous glycine betaine significantly enhanced the survival of S. entomophila either in a desiccated state or after application to soil, regardless of the soil moisture content. The implication of this finding on the future formulation of S. entomophila led to investigation of the underlying genetic mechanisms involved in glycine betaine synthesis and NaC1 tolerance. The genes involved in glycine betaine biosynthesis from choline were identified through genomic comparison, degenerate PCR and primer walking. A 6.5 kb region was sequenced and found to contain four genes with homology and similar chromosomal arrangement to the E. coli bet genes (betTIBA). The S. entomophila betIBA genes comprised an operon, flanked by the divergently-transcribed betT gene whose product is responsible for choline transport. To ascertain the relative transcription levels of components of the bet operon, quantitative RT-PCR was performed. Results of qRT-PCR showed that choline in conjunction with NaC1 induced the greatest levels of bet gene transcription, and that levels of the betA transcript were significantly lower than those of the other bet genes. Examination of the betA 5� non-coding region identified a previously undetected hairpin region, possibly accounting for the observed decrease in betA transcript levels. The findings of this study have significantly advanced our understanding of how S. entomophiia responds to stress, and will contribute to the development of formulation strategies for the production of a robust product capable of application to pasture by a range of teclmiques. In addition, there is significant potential to utilise these findings in the development of other bacterial inocula for a range of biotechnological applications.