Submitted by GUILHERME DEOMEDESSE MINARI null (gdminari@gmail.com) on 2016-03-02T21:34:04Z No. of bitstreams: 1 Dissertação Guilherme D. Minari.pdf: 1275061 bytes, checksum: b7c17ca0d2c40870526f019d55c9b421 (MD5) / Rejected by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão do arquivo submetida é considerada a versão final do trabalho, não sendo possível realizar alterações posteriormente. Por favor verifique as páginas que contém a ficha catalográfica e o certificado de aprovação e realize uma nova submissão certificando-se de que o arquivo PDF contém a versão final do trabalho. Agradecemos a compreensão. on 2016-03-04T13:14:40Z (GMT) / Submitted by GUILHERME DEOMEDESSE MINARI null (gdminari@gmail.com) on 2016-03-04T18:31:39Z No. of bitstreams: 2 Dissertação Guilherme D. Minari.pdf: 1275061 bytes, checksum: b7c17ca0d2c40870526f019d55c9b421 (MD5) Dissertação_Guilherme_Deomedesse_Minari.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-04T19:56:27Z (GMT) No. of bitstreams: 1 minari_gd_me_jabo.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) / Made available in DSpace on 2016-03-04T19:56:27Z (GMT). No. of bitstreams: 1 minari_gd_me_jabo.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) Previous issue date: 2016-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A contaminação do solo pode ser decorrente do descarte inadequado de substâncias com potencial poluente ou do uso agrícola prolongado com aplicação de materiais corretivos de acidez, fertilizantes e defensivos. O objetivo deste estudo foi avaliar a distribuição espacial de cádmio (Cd), cromo (Cr) e níquel (Ni) em Latossolo sob plantio direto, convencional e convencional com aplicação de lodo de esgoto, em áreas com exploração agrícola há mais de 80 anos. As concentrações dos metais em 422 amostras de solo foram mensuradas por espectroscopia de absorção atômica com chama ar-acetileno, sendo os extratos obtidos por extração com HNO3, H2O2 e HCl em sistema aberto (USEPA-3050B). As variáveis pH e matéria orgânica também foram determinadas. Os mapas de distribuição espacial foram elaborados a partir do software Spring v. 5.2.7 e auxiliaram na visualização da dispersão dos metais na área de estudo, indicando níveis de contaminação. A análise estatística multivariada por componentes principais diferenciou as amostras de solo de mata nativa das que estão sob atividade agrícola, apontando a uma mesma fonte de contaminação para Cr e Ni, como também para a correlação da matéria orgânica com Cd. Conclui-se que a área estudada apresenta contaminação, em larga escala, por Cd e Cr, e contaminações pontuais por Ni, estando alguns valores dentro da faixa de intervenção. / Soil contamination may result from inappropriate use of substances with pollution potential or from the prolonged agricultural use with application of acidity corrective materials, fertilizers and pesticides. The aim of this work was to evaluate the spatial distribution of cadmium (Cd), chromium (Cr) and nickel (Ni) in latosol under no tillage, conventional tillage and conventional with sewage sludge application, in agricultural exploration areas for over 80 years. Metals concentration in 422 soil samples were measured by atomic absorption spectroscopy with acetylene-air flame, obtaining the extracts by extraction with HNO3, H2O2 e HCl in open system (USEPA-3050B). The variables pH and organic matter were obtained. Maps of spacial distribution were elaborated by software Spring v. 5.2.7 and assisted in the visualization of the metals dispersion in the study area, indicating contamination levels. The statistical multivariate by main components differentiated the native forest soil samples from those under agricultural activity, pointing to a same contamination source to Cr and Ni, and to the organic matter correlation to Cd as well. It can be concluded that the studied area presents contamination in large scale by Cd and Cr and pontual contamination by Ni, being some of the values into the intervention group.

This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.

The aim of this work is to study the possibility of microbial production of polyhydroxyalkanoates (PHA). PHA can be used as biodegradable materials. Bacterial strain Cupriavidus necator was used for laboratory production of PHA. This bacterium was cultivated in medium with various precursors to produce copolymers of 3HB with 3HV or 4HB. Another part of the work was aimed at cultivation of C. necator on different waste substrates, especially oils, with the aim to achieve the highest production of polymer. Another large part of the thesis is dedicated to isolation strategies of PHA using enzymes. Commercially used proteases – alcalase and pancreatin – can be used with advantages for digestion of bacterial cells. A number of optimization experiments showed that application of proteases leads to enhancement of PHA purity to about 13%. Purity increase up to 90 % was achieved by adding a surfactant, which promotes the solubility of non-PHA forming polymer. This surfactant increases the purity of 20 % when compared to control. The last part of presented work deals with the use of enzyme solution isolated from Bacillus subtilis medium. Its application to C. necator culture led to the yield of polymer at a purity exceeding 95 %. These results could represent the basis for new isolation strategies, which can lead to more efficient yield of PHA.

Předložená disertační práce se zabývá studiem produkce a degradace polymerních materiálů s využitím mikroorganismů. Hlavní pozornost je upřena ke studiu produkce polyesterů bakteriálního původu - polyhydroxyalkanoátů. Tyto materiály jsou akumulovány celou řadou bakterií jako zásobní zdroj uhlíku, energie a redukční síly. Díky svým mechanickým vlastnostem, kterými silně připomínají tradiční syntetické polymery jako jsou polyetylén nebo polypropylén, a také díky své snadné odbouratelnosti v přírodním prostředí, jsou polyhydroxyalkanoáty považovány za ekologickou alternativu k tradičním plastům vyráběným z ropy. Polyhydroxyalkanoáty mají potenciál najít řadu aplikací v průmyslu, zemědělství ale také v medicíně. Významná část předložené práce je zaměřena na produkci polyhydroxyalkanoátů z odpadních substrátů pocházejících především z potravinářských výrob. Testována byla odpadní syrovátka nebo odpadní oleje z různých zdrojů. Právě využití levných odpadních substrátů je strategií, která by mohla přispět ke snížení ceny polyhydroxyalkanoátů a tím usnadnit jejich masové rozšíření. Podle výsledků dosažených v této práci jsou právě odpadní olejové substráty velice perspektivní cestou k ekonomicky rentabilní biotechnologické produkci polyhydroxyalkanoátů. Další část předložené práce se zabývá studiu spojení metabolické role polyhydroxyalkanoátů a stresové odpovědi bakterií. V této práci bylo zjištěno, že expozice bakteriální kultury řízené dávce etanolu nebo peroxidu vodíku významně navýší dosažené výtěžky a to o přibližně 30 %. Po aplikaci výše zmíněných stresových faktorů došlo k aktivaci metabolických drah vedoucí k odbourání stresového faktoru z média. Výsledkem bylo navýšení poměru NAD(P)H/NAD(P)+, což vedlo k částečné inhibici Krebsova cyklu a naopak aktivaci biosyntetické dráhy polyhydroxyalkanoátů. Mimoto došlo k významnému navýšení molekulové hmotnosti výsledných materiálů. Podle těchto výsledků se regulovaná aplikace vhodně zvolených stresových podmínek zdá být zajímavou strategií, která vede nejen k navýšení celkových výtěžků, ale také významnému zlepšení vlastností polymeru. Poslední část disertační práce se zabývala studiem procesu biodegradace polyuretanových materiálů. Polyuretanové eleastomery byly modifikovány rozličnými biopolymery za účelem navýšení jejich biodegradability. Tyto materiály byly posléze vystaveny působení směsné termofilní kultury jako modelového systému, který simuluje přirozené konsorcium bakterií. Přítomnost testovaných materiálů v kultivačním médiu vedla k neobvyklým růstovým charakteristikám bakteriální kultury. V průběhu prvních několika dní byl růst kultury silně inhibován, nicméně po překonání této neobvykle dlouhé lag-fáze došlo k intenzivnímu nárůstu kultury. Hlavní podíl na hmotnostním úbytku testovaných materiálů během experimentů měl samovolný rozpad materiálů, nicméně byl pozorován i vliv bakteriální kultury, kdy míra biotické degradace závisela na použitém modifikačním činidle. Nejvyšší míra biotické degradace byla pozorována u polyuretanového materiálu modifikovaného acetylovanou celulózou. Lag-fáze byla způsobena uvolněním nezreagovaného katalyzátoru (dibutylcínlaurát) a polyolu do kultivačního média. Bakteriální kultura se však po čase dokázala na přítomnost toxických látek v médiu adaptovat nebo je dokázala eliminovat.

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS). / Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

CnrX est un senseur périplasmique, ancré à la membrane, appartenant au complexe CnrYXH qui contribue à réguler l'expression des gènes impliqués dans la résistance au nickel et au cobalt chez Cupriavidus metallidurans CH34. La résistance est induite par la libération de CnrH, un facteur sigma de type ECF (Extracytoplasmic Function), par le complexe CnrYX en réponse à Ni et Co. Nous avons cherché à comprendre la manière dont CnrXs, le domaine senseur de CnrX, détecte les ions métalliques, les stratégies utilisées pour sélectionner spécifiquement Ni ou Co ainsi que la nature du signal engendré par cette interaction. Les techniques spectroscopiques et biophysiques telles que l'UV-visible, la RPE, le XAS et l'ITC ont permis d'étudier les sites métalliques en solution. Le dimère de CnrXs possède quatre sites de liaison au cobalt. Deux des sites (sites F) sont retrouvés dans la protéine entière dont nous avons maintenant un excellent modèle avec le mutant CnrXs-H32A. Les deux autres sites (sites E) ont un signal spectroscopique atypique probablement dû à la formation d'un complexe binucléaire de cobalt. Nous présentons également des structures à haute résolution de CnrXs dans ses formes apo et métallées par le nickel, cobalt ou zinc. Nous avons établi que la forme zinc est la forme inactive de la protéine et que le mécanisme de détection est engendrée par la substitution du zinc par le nickel et le cobalt dans le site F, conduisant à une modification majeure du site de liaison au métal. Tandis que le zinc est pentacoordiné dans une sphère 3N2O, Ni et Co recrutent le soufre de la seule méthionine (Met123) comme sixième ligand pour former un site octaédrique. Nous suggérons que Met123 soit l'interrupteur moléculaire dont la liaison avec le métal fait évoluer la structure de la protéine vers une conformation active. A notre connaissance, ces résultats constituent la première étude structurale et spectroscopique d'un senseur de métal périplasmique impliqué dans un système de transduction du signal dépendant d'un facteur sigma de type ECF.

Made available in DSpace on 2017-07-10T18:08:19Z (GMT). No. of bitstreams: 1 Manoela E Boff Zortea.pdf: 4020211 bytes, checksum: 0f828faa5dff1470fa28c16252974f2d (MD5) Previous issue date: 2012-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The diversity of use of polymeric materials causes concern because the use of non-renewable source for its production, as well as the environmental problems caused by the accumulation of the volume of solid waste generated. Seeking to minimize this problem were sought alternative products similar to plastics of petrochemical origin, one of them is the biodegradable polymer. The polyhydroxyalkanoates (PHAs) are polyesters synthesized by microorganisms from natural substances such as carbon and energy reserves, and accumulated by microbial cell in the form of intracellular granules, which may represent up to 80 % of the dry cell. The most studied polymer of this class is the polyhydroxybutyrate (P(3HB)) a biodegradable polymer that has similar features to the plastics from petroleum. This study aimed to develop for growth of bacteria Cupriavidus necator for the production of poly (3-hydroxybutyrate) using medium supplemented with cassava bagasse, whey and soybean oil. The first crops were carried out using a 2³ factorial design, to obtain the best condition at this stage of growth with the supplements tested. Other experiments used medium limited in nitrogen and higher amounts of supplements. The results obtained from the experimental design showed that the highest biomass production during growth was obtained in experiments which showed through the three supplements in their central levels, followed by testing supplements containing cheese whey and soybean oil combined or the presence of only one of these two components. Four experiments with increasing concentrations of supplement showed that waste and cheese whey hydrolyzed starch presented as good culture media for growth of C. necator, and the increased production of cell biomass (5,8 g.L-1) at the time of the nutrient nitrogen limitation occurred in the culture containing 100 % hydrolyzed starch conversion factor and the best substrate was obtained in cells in medium containing the highest amount supplement cheese whey (50 %). / A diversidade de utilização de materiais poliméricos acarreta preocupação devido a utilização de fonte não renovável para a sua produção, bem como os problemas ambientais causados pelo acúmulo do volume de lixo sólido gerado. Procurando minimizar este problema buscaram-se alternativas de produtos semelhantes aos plásticos de origem petroquímica, uma delas é o polímero biodegradável. Os polihidroxialcanoatos (PHAs) são poliésteres sintetizados por micro-organismos como substâncias naturais de reserva de carbono e energia, sendo acumulados pela célula microbiana na forma de grânulos intracelulares, podendo representar até 80% da massa seca celular. O polímero mais estudado desta classe é o polihidroxibutirato (P(3HB)) um polímero biodegradável que possui características semelhantes às dos plásticos provenientes de petróleo. O presente trabalho teve como objetivo desenvolver estratégias de crescimento da bactéria Cupriavidus necator para produção de poli (3-hidroxibutirato), utilizando meio suplementado com bagaço de mandioca, soro de queijo e óleo de soja. Os primeiros cultivos foram realizados seguindo um planejamento experimental 2³, visando obter nesta etapa a melhor condição de crescimento com os suplementos testados. Os demais experimentos utilizaram meio limitado em nitrogênio e maior quantidade de suplementos. Os resultados obtidos através do planejamento experimental mostraram que as maiores produções de biomassa na fase de crescimento foram obtidas nos experimentos cujo meio apresentava os três suplementos em seus níveis centrais, seguido pelos ensaios que continham os suplementos soro de queijo e óleo de soja combinados ou somente a presença de um destes dois componentes. Quatro experimentos com aumento nas concentrações de suplemento mostraram que os resíduos soro de queijo e hidrolisado amiláceo apresentaram-se como bons meios de cultivo para o crescimento de C. necator, sendo que a maior produção de biomassa celular (5,8 g.L-1) no momento da limitação do nutriente nitrogênio ocorreu no cultivo contendo 100 % de hidrolisado amiláceo e o melhor fator de conversão de substrato em células foi obtido no meio contendo maior quantidade do suplemento soro de queijo (50 %).

This diploma thesis deals with the application of evolutionary engineering to PHA producing bacterial strains. The aim of the thesis is to prepare strains adapted to levulinic acid, a selected stress factor, by methods of evolutionary engineering, and then to characterize these strains. The theoretical part deals with evolutionary engineering and polyhydroxyalkanoates predominantly. The bacterial strain Cupriavidus necator H16 was used for evolutionary experiments. Levulinic acid and levulinic acid in the presence of the MMS mutagen were applied to prepare adapted strains. Selection of mutants was evaluated on the basis of growth potential and PHA content in biomass. Polymers produced by five obtained PHA-producing mutants and control were characterized using GC-FID, SEC-MALS, DSC and FT-IR. It was found that a higher content of 3HV in the copolymer led to a lower crystallinity and hence to a lower melting point, nevertheless, only the copolymer of the M0151 strain did not fit this trend. In addition to the characteristics of the polymers, the strains themselves were evaluated from the biochemical point of view by determining the activities of selected enzymes of the citrate, glyoxalate and 2-methylcitrate cycle, selected enzymes generating NADPH, levulic acid catabolism enzyme and PHA biosynthesis enzymes. On the basis of the obtained data, the possible adaptation strategies were discussed, when the E0575 strain was most differentiated from original culture. Values of specific enzyme activities were subjected to AHC and PCA statistical analysis methods.

This diploma thesis deals with the utilization of physico-chemical analysis in study on microbial stress–response. The main content of this work was to propose and optimize techniques and methods which are generally used in different industries. Two bacterial strains, Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4 producing polyhydroxyalkanoates (PHA) under certain conditions, were used as model microorganisms. Initially, microscopic teques such as transmission electron microscopy (TEM), cryo scanning electron microscopy (cryo SEM) and atomic force microscopy (AFM) were proposed and tested for the characteriozation of morphological differences of bacteria. Furthermore, viscoelastic properties of bacteria were determined and compared by the oscillatory tests of the rheology method considering another type of samples. Thermal analysis methods, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) in particular, focused on water transport and behavior of intracellular water influenced by presence of the granules of PHB in bacteria. The last technique, utilized in the study, was dynamic and electrophoretic light dispersion during pH changes for characterization of the surface properties of bacteria such as size, zeta potential and the isoelectric point.

First artificial PHB granules were prepared under the terms of this thesis. The effect of used PHB solvent, ultrasonic bath temperature and time, solvent evaporation temperature and stabilizing agent nature was investigated using dynamic and electrophoretic light scattering. The most proper parameters were demonstrated at samples which were prepared by dissolving of PHB in chloroform, stabilizing with CTAB or lecithin, ultrasonifying at 35 °C followed by chloroform evaporating at 60 °C. Based on ATR-FTIR and Raman spectroscopy results, it was found out that PHB within the artificial granules were in crystalline form. The native PHB granules were isolated from Cupriavidus necator using either lysosyme, deoxyribonuclease and cell disruption by ultrasonification or digestion with alcalase, SDS and EDTA. Granules obtained by both isolation procedures were characterized by ATR-FTIR, Raman spectroscopy, light scattering techniques and DSC. According to the analyses results, the second mentioned procedure turned out to be more effective for obtaining the polymer in amorphous state. Furthermore, the polymer within granules recovered by using this procedure was thermally more stable. Last but not least, the native PHB granules samples were exposed to effect of acetone, lipase and sodium hypochlorite, assuming that polymer crystallinity should be increased by these chemicals in varying degrees. The highest degree of crystallinity was achieved after their treatment with lipase.