Orientador: Elias Basile Tambourgi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T17:48:02Z (GMT). No. of bitstreams: 1 Ehrhardt_DanielaDiniz_M.pdf: 1295191 bytes, checksum: fa70dc68bb798428fff1677668957a8a (MD5) Previous issue date: 2015 / Resumo: O crescente comprometimento com questões ambientais, juntamente com as novas legislações vigentes fizeram com que o uso de surfactantes sintéticos se tornasse inviável pela indústria. Assim, o desenvolvimento de novas tecnologias para produção de biossurfactantes aumentou consideravelmente. Biossurfactantes são grupos de compostos químicos produzidos por microrganismos através da biodegradação de matérias-primas renováveis. A utilização de biossurfactantes tornou-se uma alternativa bastante interessante em substituição aos surfactantes sintéticos, pois geram menor impacto ambiental devido à sua biodegrabilidade, diversidade estrutural e baixa toxicidade. Entretanto, os altos custos de produção são fatores limitantes para sua aplicação industrial. Sendo assim, o presente trabalho teve como objetivo a produção de biossurfactante, através da fermentação do resíduo do processamento do abacaxi, por Bacillus subtilis, a 37 ? C. O resíduo do abacaxi como substrato de fonte renovável garante o baixo custo na produção do surfactante microbiológico, uma vez que este fruto é extensivamente cultivado no Brasil. Foram realizadas fermentações com três diferentes concentrações de glicose (1%, 3%, 5%) e, posteriormente, enriquecendo o substrato com glicerol comercial (3%, 5% e 10%). Foram realizados testes de tensão superficial e indíce de emulsão e observou-se melhores resultados com 5% de glicose e sem adiação de glicerol, com redução da tensão em 25% e índices de emulsão de 67%, entretando não foi observado estabilidade do bissurfactante produzido quando expostos à condições extremas de temperatura (100°C e 121°C), variação de pH de 4 a 8 e frente à força iônica com soluções de NaCl em concentrações de 2,5%, 5%, 10% e 20% / Abstract: The growing commitment to environmental issues, with the new current legislation made the use of synthetic surfactants become unviable in industry. Thus, the development of new technologies for producing biossufactantes increased considerably. Biosurfactants are groups of chemical compounds produced by microorganisms by biodegradation renewable raw materials. The use of biosurfactants has become an interesting alternative to replace synthetic surfactants because they generate less environmental impact due to their biodegradability, low toxicity and structural diversity. However, the high production costs are limiting factors for their industrial application. Therefore, this study aimed to produce biosurfactant by Bacillus subtilis at 37 °C, usign the residue from processing of pinneaple as substrate. The pineapple residue as a renewable source substrate ensures low cost production of microbial surfactant, since this fruit is extensively cultivated in Brazil. Fermentations were performed at three different glucose concentrations (1%, 3%, 5%) and then enriching the substrate with commercial glycerol (3%, 5% and 10%). The surface tension and emulsion index analysis showed that the best results was with 5% glucose and without glycerol, with tension reduction of 25% and emulsion index of 67%. However, was observed that the bissurfactant produced wasn't stable when exposed to extreme conditions of temperature (100 ° C and 121 ° C), pH range 4 to 8 and ionic strength of NaCl solutions with concentrations of 2.5%, 5%, 10% and 20% / Mestrado / Engenharia Química / Mestra em Engenharia Química

Made available in DSpace on 2017-06-01T18:20:40Z (GMT). No. of bitstreams: 1 leonila_maria_leandro_acioly.pdf: 728560 bytes, checksum: 908cf4cbcd51d01cea2358383b75e4a1 (MD5) Previous issue date: 2009-01-24 / The biosurfactants biopolymers are used in various industrial sectors, but still, are not economically competitive with synthetic surfactants, due to high production costs, where the substrate is 30% of the final costs. In search of alternative and low cost in the present study, we investigated the sources agribusiness, rice bran (Oryza sativa), potato (Solanum tuberosum) and soybean (Sorghum vulgare), as a liquid medium for biosurfactant production by Bacillus subtilis UCP0999. Assays were performed with and without the addition of (2g / L), peptone, ammonium sulfate and either (0.01 mL / L) of micro-nutrients, which were inoculated with 5% of a culture of B. subtilis (106 CFU / mL), kept under orbital agitation of 200 rpm for 30 ° C for 72 h. At the end of fermentation, the cells collected corresponded to biomass and metabolic cell-free liquid were evaluated pH, surface tension, emulsification index (motor oil, diesel and kerosene) and extracting the biosurfactant produced. The results showed that the bacteria used and the rice bran supplemented with soy peptone, producing a surfactant to reduce surface tension of water to 72 mN / m to 37.80 mN / m and 37.70, respectively. The soy supplemented with micronutrients to reduce the tension 35.60 mN / m. On the other hand, when the used rice bran supplemented with nutrients and micro ammonium sulfate produced a greater quantity of biosurfactant with 8.60 and 6.1 g / L, respectively, showing no significant results regarding the reduction of surface tension . The biosurfactants produced showed high emulsification in motor oil, kerosene and diesel with values from 30 to 100%, with the highest rates with potato broth and soy supplemented with micronutrients and ammonium sulphate. The biosurfactant produced in the different media formulated demonstrated great potential for emulsification of hydrophobic substance and surface tension reduction, allowing for future use in bioremediation processes. / Os biossurfactantes são biopolímeros utilizados em vários setores industriais, porém ainda, não são economicamente competitivos com os surfactantes sintéticos, devido aos elevados custos de produção, onde o substrato representa 30% dos custos finais. Na busca de fontes alternativas e de baixo custo, no presente trabalho foi investigada as fontes agroindustriais, farelo de arroz (Oryza sativa), batata inglesa (Solanum tuberosum) e soja (Sorghum vulgare), como meio líquido para produção de biossurfactante por Bacillus subtilis UCP0999. Os ensaios foram realizados com e sem a adição de (2g/L) de peptona, sulfato de amônia e ou (0,01mL/L) de micro nutrientes, os quais foram inoculados com 5% de uma cultura de B. subtilis (106 UFC/mL), mantidos sob agitação orbital de 200rpm, por 30oC por 72 h. Ao final da fermentação, as células coletadas corresponderam a biomassa e do liquido metabólico livre de células foram avaliados pH, tensão superficial, índice de emulsificação (óleo de motor, diesel e querosene) e extração do biossurfactante produzido. Os resultados demonstraram que a bactéria utilizou o farelo de arroz e soja suplementado com peptona, produzindo um surfactante que reduziu a tensão superficial da água 72 mN/m para 37,80 mN/m e 37,70, respectivamente. A soja suplementada com micro nutrientes reduziu a tensão para 35,60 Mn/m. Por outro lado, quando utilizou o farelo de arroz suplementados com micro nutrientes e sulfato de amônia, produziram uma maior quantidade de biossurfactante, com 8,60 e 6,1 g/L, respectivamente, não apresentando resultados significativos quanto a redução da tensão superficial. Os biossurfactantes produzidos apresentaram altos índices de emulsificação em óleo de motor, querosene e diesel com valores de 30 a 100%, sendo os mais altos índices com caldo de batata e de soja, suplementados com micro nutrientes e sulfato de amônia. O biossurfactante produzido nos diferentes meios formulados demonstrou grande potencial de emulsificação de substancias hidrofóbicas e redução da tensão superficial, possibilitando utilização futura nos processos de biorremediação.

A divisão bacteriana é efetuada por um complexo macromolecular conhecido como divisomo. Um componente central do divisomo é FtsZ, uma proteína homóloga de tubulina que se polimeriza no meio da célula formando uma estrutura em forma de anel (anel Z). O controle da divisão é exercido por proteínas que modulam a habilidade de FtsZ de formar o anel Z. Dois fatores principais estão envolvidos na seleção do correto sitio de divisão. O melhor estudado é o sistema Min, o qual é responsável pelo bloqueio específico de sítios de divisão não desejados nos polos da célula. O componente do sistema Min que inibe a polimerização de FtsZ é a proteína MinC e é sabido que MinC requer MinD para se ativar, mas o mecanismo dessa ativação não está completamente compreendido. No presente trabalho investigamos o papel da associação de MinD à membrana na ativação de MinC. Usando um mutante que não mais se associa à membrana (MinDΔMTS) mostramos que o efeito de MinC em inibir a divisão celular é altamente dependente de seu recrutamento à membrana por MinD. No entanto, ensaios in vitro mostraram que o complexo MinCDΔMTS é mais eficiente em desfazer polímeros de FtsZ que MinC sozinho, indicando que MinD promove a ativação de MinC por outro mecanismo além de recrutamento à membrana. Esta ativação pode resultar de um efeito alostérico ou da criação de um sítio para FtsZ na interface do complexo MinCD, porém resultados preliminares não conseguiram detectar aumento da afinidade de MinC por FtsZ quando na presença de MinD. / Bacterial division is performed by a macromolecular complex known as the divisome. The central component of the divisome is FtsZ, a tubulin protein homolog, which polymerizes at the mid-cell forming a ring-like structure (Z-ring). This division is regulated by proteins that modulate ability of FtsZ to form the Z-ring. Two principal factors are involved in selecting the correct site of division. The best-studied factor is the Min system, which is responsible for the specific blockade of unwanted potential sites in the cell poles. The component of the Min system that inhibits FtsZ polymerization is the MinC protein. MinC requires the MinD protein for activation, but the mechanism of this activation is not completely understood. Here, we investigate the role of the association of MinD to the membrane during MinC activation. Using a mutant that does not interact with the membrane (MinDΔMTS) we show that the effect of MinC in inhibiting cell division is highly dependent on its recruitment to the membrane by MinD. However, in vitro assays show that MinCDΔMTS is more efficient in disrupting FtsZ polymers than MinC alone, indicating that MinD promotes MinC activation by a mechanism other than membrane recruitment. This activation could be due to an allosteric effect or the formation of a site for FtsZ on the MinCD interface; however, preliminary results could not detect any increase in the affinity of FtsZ to MinC in the presence of MinD.

Os manguezais são ambientes ricos em biodiversidade, cuja funcionalidade reside na ciclagem dos nutrientes e seu principal representante vegetal é Rhizophora mangle. Este estudo objetivou conhecer a diversidade bacteriana endoglicolítica e a tolerância à salinidade de rizobactérias associadas à R. mangle. Das amostras de plantas do manguezal de Bertioga (contaminado com petróleo) e Cananéia (não impactado) isolou-se 129 bactérias, das quais 30 apresentaram atividade endoglicolítica, com Bacillus subtilis isolado 39a como melhor produtor. A presença do gene EglA foi confirmada por amplificação com primers específicos. As linhagens testadas para salinidade mostraram-se halotolerantes, com destaque para o 39a, que cresceu em NaCl 20%. A microscopia eletrônica pós-cultivo em diferentes salinidades mostrou produção de biofilme em concentrações altas. Os resultados indicam que a preservação do ecossistema cria um ambiente bacteriano mais diverso e mostra Bacillus spp. como principal produtor de endoglicanase, além de responder ao stress salino formando biofilme. / Mangroves are environments so rich in biodiversity which functionality made by nutrient cycling. The main vegetable specie is Rhizophora mangle. This study objected to know bacterial endoglucolytic diversity and tolerance saline of rhizobacteria associated to R. mangle. Plants from Bertioga (oil contaminated) and Cananéia (not polluted) were sampled. From both sites, 129 bacteria were isolated, which most diversity observed from Cananéia. These isolates, 30 presented endoglucolytic activity and Bacillus subtilis (strain 39a) was characterized as top producer. The presence of EglA gene was confirmed using specific primers. The salinity test showed halotolerance, mainly strain 39a that growth untill about 20% NaCl. The scan electron microscopy of strains allowed biofilm production at elevated salinity that suggest the biofilm as tolerance mechanism to saline environment. The results indicated that ecosystem preservation makes a most diversity bacterial environment and that Bacillus spp. are main endoglucanase producer and response to saline stress producing biofilm.

Chez Bacillus subtilis comme pour toutes les bactéries, le métabolisme central du carbone est essentiel pour la croissance de la cellule. Elle utilise le glucose (source de carbone glycolytique) et le malate (source de carbone gluconéogenique) comme sources de carbone préférentielles. Ces deux sources de carbone sont capables d'induire la répression catabolique au travers de la protéine régulatrice CcpA et ainsi d'établir une hiérarchie dans l'utilisation des sources alternatives de carbone. Au centre du métabolisme du carbone se trouve le pyruvate que B. subtilis est capable d'utiliser comme seule source de carbone, mais son transporteur reste inconnu.Des analyses transcriptomiques ont montré que seul l'opéron ysbAB était spécifiquement induit en présence de pyruvate, et nous avons montré que sa délétion entraînait une perte de croissance presque totale sur pyruvate. En utilisant des protéines étiquetées, nous avons mis en évidence qu'YsbA et YsbB formaient un complexe se localisant à la membrane. Nous avons ensuite montré que ce complexe est le transporteur principal du pyruvate et fonctionne comme un transporteur par diffusion facilitée. A l'aide d'une fusion rapportrice, nous avons démontré que l'opéron lytST situé en amont d'ysbAB, et codant pour un système à deux composants, était responsable de l'induction d'ysbAB. Nous avons également montré qu'en plus d'une répression par CcpA en présence de glucose ou de malate, une régulation dépendante de l'activité enzyme malique de MaeA s'exerce sur ysbAB. Cette régulation est due à l'accumulation de pyruvate dans la cellule qui perturbe l'activation d'ysbAB par LytST.Nous avons aussi montré qu'une régulation indépendante de CcpA s'exerce sur dctP, le gène codant pour le transporteur du succinate et du fumarate en présence de malate, suggérant un mécanisme similaire à celui observé pour ysbAB. Enfin, nous avons montré que le flux métabolique traversant MaeA était également impliqué dans la régulation par CcpA de l'entrée des sources glycolytique par le malate. / In Bacillus subtilis like for all the bacteria, the central carbon metabolism is essential for growth. It uses glucose (a glycolytic carbon source) and malate (a gluconeogenic carbon source) as preferential carbon sources. These two carbon sources are able to induce carbon catabolite repression through the transcription factor CcpA and thus establishing a hierarchy in the use of alternative carbon sources. The pyruvate is in the middle of the carbon metabolism, and can be used by B. subtilis as sole carbon source; however its transporter remains unknown.Transcriptome analyses revealed that the only operon specifically expressed in cells grown on pyruvate is ysbAB, and we showed that its deletion led to a strong growth defect on pyruvate. Using tagged proteins, we highlighted that YsbA and YsbB formed a complex localized at the membrane. We next showed that this complex is the major pyruvate transporter, and operates as a facilitated transporter. Using a reporter fusion, we showed that the operon lytST located upstream of ysbAB, and coding for a two-component system, is responsible for the induction of ysbAB. We also showed that besides the CcpA-mediated repression by both glucose and malate, an additional regulation mechanism through the malic enzyme activity of MaeA is acting on ysbAB. This regulation is due to the accumulation of pyruvate in the cell which hinders the LytST-mediated induction of ysbAB.We also showed that a CcpA-independent repression is exerted on dctP, the gene coding for the succinate and fumarate transporter, in the presence of malate, suggesting a regulation mechanism similar to the one observed for ysbAB. Finally, we showed that the metabolic flux going through MaeA is also involved in the CcpA-dependent repression of the genes coding for glycolytic transporter in presence of malate.

La transcription bactérienne est un processus dans lequel l'information codée dans l'ADN est transféré à l'ARN messager (ARNm). Au cours de la dernière étape de ce procédé, la terminaison de la transcription, l'ARNm est libéré et peut être utilisé pour la synthèse des protéines. Un type de terminaison de la transcription décrit chez les bactéries est la terminaison Rho-dépendante. Le rôle de Rho a été largement étudié dans le modèle à Gram négatif bactérie, Escherichia coli dans laquelle Rho est une protéine essentiel est abondant. En revanche, la connaissance de Rho chez les bactéries qui il ne sont pas essentiels et est présent en faibles quantités: par exemple Gram-positif Bacillus subtilis reste limité..Pour étudier le rôle de Rho dans le contrôle de l'expression des gènes chez B. subtilis plusieurs analyses à grande échelle ont été réalisées, y compris des tests d'interactions physiques et fonctionnelles et une analyse globale des changements observés dans l'expression des gènes en corrélation avec la production de protéines.En effet, un ensemble des Rho-spécifiques interactions physiques et fonctionnelles ont été établies. En outre, de nouveaux phénotypes de mutant dépourvu de rho ont été décrits ce qui élucider le rôle de Rho dans le contrôle des différents aspects de la physiologie cellulaire. / Bacterial transcription is a process in which the information encoded in DNA is transferred to messenger RNA (mRNA). During the final step of this process, transcription termination, mRNA is released and can be used for protein synthesis. One type of transcription termination described in bacteria is Rho-dependent termination. The role of Rho has been widely investigated in model Gram-negative bacterium, Escherichia coli in which Rho is essential an abundant protein. In contrast, the knowledge about Rho inbacteria in which it is not essential and is present in low amounts, i. e. Gram-positive Bacillus subtilis remains limited.To investigate the role of Rho in control of gene expression in B. subtilis several large-scale analysis were performed. In effect, a set of Rho-specific physical and functional interactions were established. Additionally, new phenotypes of rho-null mutant were described unraveling the role of Rho in control of different aspects of cell physiology.

Cell walls of Bacillus subtilis macrofibers have been biochemically analyzed to determine the contribution of various surface polymers in the twist regulation. Helix hand inversion was induced by a variation in either the growth temperature or the nutritional composition of the culture medium. Initial experiments had demonstrated a fivefold difference in the sensitivity of right- and left-handed forms to muramidases indicating modifications of peptidoglycan as a possible mechanism underlaying inversion. An examination of lysozyme susceptibility of purified cell walls and whole cells derived from the two structural forms, however, exhibited no significant difference suggesting loss of the relevant component(s), perhaps biomechanical in nature, during disintegration of macrofibers. The effect of various twist modulators such as trypsin, ammonium sulfate and D-alanine on the development of helical twist in both switchable and "fixed" mutants were studied. The interaction matrices have established D-alanine as the most potent of right-factors. Intestinal alkaline phosphatase is reported as a newly discovered antagonist to the development of leftward twist. Heat inactivation and protein purification experiments strongly indicated that twist modulation was due to the phosphatase activity rather than minor protease contaminants. The chemical composition of cell walls purified from right- and left-handed structures was determined. No twist correlated differences in the overall content of peptidoglycan, teichoic acid and teichuronic acid were detected. Evidence is presented for the absence of correlation between the extent of ester-linked alanine substitution and twist state. These findings suggest that gross changes in wall composition is perhaps not the mechanism for hand inversion. From the profiles of the wall associated proteins, a 200 Kdal band has been identified whose presence is strongly correlated with the development of leftward twist. This polypeptide was found to be highly sensitive to trypsin; a property it shares with a previously proposed left-twist protein. Preliminary evidence for isolation of left-hand specific polyclonal antibodies is also presented. FJ7, a switchable mutant, was successfully transformed with a plasmid containing the Streptococcus transposon Tn917. A small bank of insertional mutants has been constructed for the isolation of mutants impaired in helix hand inversion.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Bacillus subtilis DNA was cloned by ligating restriction endonuclease-generated fragments to plasmid vectors. The plasmid pUB110 was the vehicle in the construction of eight recombinant plasmids, pNM1 through pNM8. Each bears one or more EcoRI fragment(s) of B. subtilis chromosomal DNA. Recovery of the plasmids from host cells demonstrated that recombinant plasmids that bear some homology to the B, subtilis chromosome may be maintained outside of the chromosome in recombination-proficient hosts. The mean size of cloned fragments was 0.78 megadaltons (Mdal). The recombinant plasmid pNM1 interferes with the mechanism that blocks chromosomal recombination in B. subtilis cells that carry the recE4 mutation. Low-level chromosomal recombination at several loci was demonstrated when chromosomal DNA was accompanied by pNM1 in the transformation of recE4 recipient cells. The recombinant plasmid does not appear to code for recE gene products nor does it produce novel proteins when assayed in minicells of B. subtilis. An alternative approach to cloning B. subtilis DNA was successfully accomplished with the vector plasmid pHV33. The vector functions in both B. subtilis and E. coli hosts. B. subtilis chromosomal DNA was digested with Bg1II, then ligated to the unique BamHI site of pHV33. Ligation products were introduced into E. coli by transformation. Plasmid DNAs were isolated from transformants, pooled into several lots, then used to transform auxotrophic B. subtilis recipient cells. The procedure resulted in the construction of two new recombinant plasmids, pNM1055 and pNM1326. B. subtilis cells with the aroD120 mutation restored their ability to synthesize aromatic amino acids when pNM1055 was introduced. The same effect was observed in E. coli recipient cells that had the equivalent mutation. E. coli cells that carried pNM1326 produced granular colonies characteristic of the extraordinary filamentous growth exhibited by individual cells. The pNM1326 plasmid coded for a 16,000 dalton polypeptide produced in abundant quantities in E. coli hosts. A deletion derivative of pNM1326 did not produce the polypeptide, nor was filamentous growth of host cells exhibited. A plasmid-borne fragment of B. subtilis DNA affects cells growth and division of E. coli hosts.

Título original: Avaliação da degradação da mistura diesel/biodiesel por colorimetria / Orientador: Ederio Dino Bidoia / Banca: Adriano Pinto Mariano / Banca: Regina Teresa Rosim Monteiro / Banca: Sandra Imaculada Maintinguer / Banca: Dejanira de Franceschi de Angelis / Resumo: A projetada escassez de combustíveis fósseis, a alta necessidade energética e as pressões ambientais tornaram necessária uma mudança na matriz energética mundial. Nesse sentido, verifica-se a expansão do mercado de combustíveis derivados de fontes renováveis, predominando o etanol para automóveis e o biodiesel adicionado ao diesel para caminhões, ônibus, tratores e transportes marítimos. Além de diversificar a matriz energética, o biodiesel apresenta algumas vantagens do ponto de vista ambiental em relação ao diesel de petróleo como por exemplo, maior biodegradabilidade. Acidentes ambientais com petróleo e derivados causam danos consideráveis ao meio ambiente, gerando uma preocupação pública enorme, que pressiona para soluções rápidas e econômicas. Dentre as alternativas de tratamento para quando ocorre um derramamento de petróleo, a biorremediação emerge como um processo simples e de baixo custo quando comparado a outras alternativas, além se ser menos agressiva e a mais adequada para manutenção do equilíbrio ecológico. O objetivo deste trabalho foi avaliar a biodegradação de misturas de diesel e biodiesel nas proporções de 3, 5, 7, 10, 25, 50 e 80% em solo e em meio aquoso, verificando se a adição de inóculo da bactéria Bacillus subtilis e a adição do surfactante químico Tween® 80 auxilia na biodegradação de tais misturas. Também foi analisada, para as misturas de 5, 25 e 50% de biodiesel em diesel, a eficiência do processo de biorremediação, a fitotoxicidade e a diversidad... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The shortage of fossil fuels, high energy requirements and environmental pressures have made it necessary to change the world energy matrix. In this sense, there is the expansion of the market of fuels derived from renewable sources, predominating ethanol for automobiles and biodiesel for trucks, buses, tractors and maritime transport. In addition to diversifying the energy matrix, biodiesel has some advantages from an environmental point of view in relation to petroleum diesel, such as higher biodegradability. Environmental accidents with oil and by-products cause considerable damage to the environment, generating a huge public concern, pushing for quick and cost-effective solutions. Among the treatment alternatives for when an oil spill occurs, bioremediation emerges as a simple and low-cost process when compared to other alternatives, as well as being less aggressive and more adequate to maintain the ecological balance. The objective of this work was to evaluate the biodegradation of diesel and biodiesel mixtures in the proportions of 3, 5, 7, 10, 25, 50 and 80% of biodiesel in diesel in soil in aqueous medium, checking if the addition of strains of Bacillus subtilis and the addition of the chemical surfactant Tween® 80 assists in the biodegradation of such mixtures. The efficiency of the bioremediation process regarding microbial metabolism, ecotoxicity and diversity of the microbial community was also analyzed for blends of 5, 25 and 50% biodiesel in diesel. With the res... (Complete abstract click electronic access below) / Doutor