Made available in DSpace on 2016-06-02T19:02:43Z (GMT). No. of bitstreams: 1 5706.pdf: 7869131 bytes, checksum: 3a35e736b3418ca357ef4fc2e657c0af (MD5) Previous issue date: 2013-10-16 / Universidade Federal de Minas Gerais / Enzymes are potent catalysts, but operationally fragile, expensive and soluble. Industrial applications of enzymes, often, are possible only using immobilized enzyme. Nowadays, various studies have been performed aiming to immobilize enzymes onto magnetic carriers, which allow the selective recovery of the derivative by applying an external magnetic field even in complex reaction media containing other suspended solids. There are many studies using magnetic carriers in enzymes immobilization procedures, however there are no commercially available enzymes immobilized onto magnetic materials. In these studies usually are used carriers with not ideal characteristics for applications in industrial processes. The present study aimed to develop new magnetic carriers and methods for immobilization of enzymes in these carriers, penicillin G acylase (PGA) and cellulases have been used as model enzymes. The thesis was divided into five parts, in the first part (Chapter 1) the state-of-art is presented. The second part (Chapter 2) describes the synthesis of magnetic carriers robust, cheap and with good characteristics for applications in bioprocesses. For this purpose were tested the synthesis of silica magnetic microparticles (SMMps) in water-in-oil micro-emulsion using sodium silicate as silica source and superparamagnetic iron oxide nanoparticles as magnetic core. Materials with good magnetic properties, high surface area and mesoporous structure were obtained. SMMps structure was characterized, it was possible to control the final structure of the material according to the synthesis conditions. In the third part of this study (Chapter 3) was evaluated a new concept in enzymes immobilization using magnetic materials. Magnetic tags were co-aggregated with PGA and cross-linked with glutaraldehyde, producing magnetic cross-linked enzymes aggregates (M-CLEAs). Several reaction conditions were tested producing M-CLEAs with different characteristics and strong response to external magnetic fields. Derivatives with good recovered activity and increased thermal and methanol 50% (v/v) stabilities were obtained. M-CLEAs presented superior performance, in comparison with the free enzyme, in penicillin G hydrolysis experiments, being reused for three reaction cycles without loss of activity. In the fourth part of this study (Chapter 4) the immobilization of the Trichoderma reesei cellulolytic complex onto 17 carriers using 60 different immobilization conditions was evaluated. Covalent methods to cellulases immobilization resulted in total loss of the enzymatic activity. The immobilization by adsorption allowed preserving a portion of the enzymatic activity, however, the enzyme was desorbed from the carrier with the increase in the ionic strength. The best results were achieved for adsorption in MANAE-agarose followed by cross-linking with glutaraldehyde. Hydrolysis experiments using insoluble substrates showed that it is possible to hydrolyze such substrates even using immobilized enzyme onto porous carriers. The derivative was reused for ten reaction cycles (hydrolysis of filter paper) saving more than 90% of its activity. Finally, in Chapter 5, the T. reesei cellulolytic complex was immobilized by adsorption onto SMMp activated with amino groups followed by glutaraldehyde cross-linking achieving good results in terms of recovered activity. / Enzimas são potentes catalisadores, porém frágeis operacionalmente, caras e solúveis. Aplicações industriais desses catalisadores, muitas vezes, são possíveis apenas com o uso de enzima imobilizada. Estudos indicam que o uso de suportes magnéticos para imobilizar enzimas pode permitir a recuperação seletiva do derivado através da aplicação de um campo magnético externo mesmo em meios complexos contendo outros sólidos em suspensão. Apesar de existirem muitos estudos empregando suportes magnéticos para imobilização de enzimas, não existem enzimas imobilizadas em materiais magnéticos disponíveis comercialmente. Nestes estudos geralmente são utilizados suportes magnéticos com características não ideais para aplicações em bioprocessos. O presente estudo teve como principal objetivo o desenvolvimento de novos suportes magnéticos e métodos para imobilização de enzimas nestes suportes, a enzima penicilina G acilase (PGA) e celulases foram utilizadas como modelo. O estudo foi dividido em cinco partes, no Capítulo 1 é apresentada uma introdução indicando o estado da arte. O Capítulo 2 apresenta o preparo de novos suportes magnéticos robustos, baratos e com características ótimas para aplicações em bioprocessos. Nesta etapa foi testada a síntese de micro-partículas magnéticas de sílica (SMMps) em micro-emulsão água-em-óleo, empregando silicato de sódio como fonte de sílica e nanopartículas superparamagnéticas de óxido de ferro como núcleo magnético. Os materiais obtidos apresentaram excelentes propriedades magnéticas, alta área de superfície e estrutura mesoporosa. A partir da caracterização físico-química e morfológica das SMMps foi possível controlar a estrutura final do material de acordo com as condições de síntese. No Capítulo 3 foi avaliado um novo conceito em imobilização de enzimas empregando materiais magnéticos. Neste estudo etiquetas magnéticas foram co-agregadas com PGA e entrecruzadas com glutaraldeído, gerando agregados enzimáticos entrecruzados com propriedades magnéticas (M-CLEAs). Várias condições reacionais foram testadas rendendo M-CLEAs com diferentes características e com resposta robusta a campos magnéticos externos. Derivados imobilizados com boa atividade recuperada e incremento na estabilidade térmica e frente a metanol 50% (v/v) foram obtidos. M-CLEAs apresentaram desempenho superior ao observado para a enzima livre em experimentos de hidrólise de penicilina G, sendo reutilizados por três ciclos reacionais sem perda de atividade. No Capítulo 4 foi avaliada a imobilização do complexo celulolítico de Trichoderma reesei em 17 suportes, empregando 60 diferentes condições de imobilização. Os experimentos de imobilização realizados empregando técnicas de imobilização por união covalente ocasionaram perda total de atividade enquanto métodos de imobilização por adsorção permitiram conservar boa atividade enzimática, porém a enzima dessorveu do suporte com o aumento na força iônica do meio. Os melhores resultados foram alcançados para adsorção em MANAE-agarose seguido de entrecruzamento com glutaraldeído. Experimentos de hidrólise de substratos insolúveis mostraram que é possível hidrolisar este tipo de substrato mesmo com enzima imobilizada em suportes porosos. O derivado foi reutilizado por dez ciclos (hidrólise de papel filtro) conservando mais de 90% de sua atividade. Por fim, no Capítulo 5, o complexo celulolítico de T. reesei foi imobilizado por adsorção em SMMp ativado com grupos amino seguido de entrecruzamento com glutaraldeído apresentando bons resultados em termos de atividade recuperada.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-21T14:30:40Z No. of bitstreams: 1 DissRASac.pdf: 11251604 bytes, checksum: 6c180d000983f8c0f5a08597c2d53676 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-09-27T20:04:15Z (GMT) No. of bitstreams: 1 DissRASac.pdf: 11251604 bytes, checksum: 6c180d000983f8c0f5a08597c2d53676 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-09-27T20:04:24Z (GMT) No. of bitstreams: 1 DissRASac.pdf: 11251604 bytes, checksum: 6c180d000983f8c0f5a08597c2d53676 (MD5) / Made available in DSpace on 2016-09-27T20:10:13Z (GMT). No. of bitstreams: 1 DissRASac.pdf: 11251604 bytes, checksum: 6c180d000983f8c0f5a08597c2d53676 (MD5) Previous issue date: 2015-03-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Cassava is cultivated worldwide, being Brazil the fourth largest producer. The root industrial processing in the country, aiming to obtain mainly flour and starch, generates carbohydrate-rich residues (e.g., starch, cellulose, and hemicellulose), which could be used to produce value-added products by enzymatic route, mainly using immobilized enzymes that are more operationally stable, allowing to be easily recovered and reused in the process. Thus, this work aimed the biotransformation of residual starch from cassava processing in maltose, using immobilized β-amylase in a Couette–Taylor–Poiseuille vortex flow reactor, which can promote perfect mixture under lower shear stress in the reactional medium compared to the conventional stirred-tank reactor. Cassava bagasse and peel of two starch-processing industries from São Paulo State were physicalchemically characterized and showed about 47% and 55% (dry mass) of residual starch, respectively. The starch was enzymatically extracted from the residues using a α- amylase, followed by maltose production catalyzed by immobilized barley β-amylase. Among the immobilization methods studied in this work, the best one for β-amylase was protein aggregation using bovine serum albumin (BSA) or soybean protein (PS) as protein feeder, followed by cross-linking with glutaraldehyde (CLEA technique). This protocol yielded immobilized β-amylase with 82.67% and 53.26% of recovered activity, respectively. Besides, the CLEAs were highly stables at 40oC, retaining more than 80% of the initial activity after 12 hours. The maltose syrup production from starch was performed using a Couette–Taylor–Poiseuille vortex flow reactor, in order to evaluate the β-amylase CLEAs (in this case CLEA of β-amylase prepared with soybean protein, here named CLEA-β-PS). It was achieved around 70% of maltose conversion in a short reaction time (4 hours), showing that is viable the use of residual starch as raw material for the production of maltose catalyzed by β-amylase CLEA in a Couette–Taylor– Poiseuille vortex flow reactor. / A mandioca é cultivada em todo mundo, sendo o Brasil o quarto maior produtor. O processamento industrial da raiz no país visa principalmente à produção de farinha e fécula, gerando resíduos ricos em carboidratos (amido, celulose, hemicelulose) que poderiam gerar produtos de valor agregado por biocatálise enzimática, particularmente usando enzimas imobilizadas, por serem mais estáveis operacionalmente e poderem ser facilmente recuperadas e reutilizadas no processo. Assim, este trabalho teve como objetivo a biotransformação do amido residual dos resíduos do processamento da mandioca em maltose, usando a enzima β-amilase imobilizada em reator de fluxo em vórtices (RFV) Couette–Taylor–Poiseuille, reator este que pode promover mistura perfeita com menor tensão cisalhante no meio reacional, comparado a um reator de mistura perfeita convencional. Os resíduos bagaço e casca de mandioca de duas fecularias do interior de São Paulo foram caracterizados físico-quimicamente e apresentaram teores de amido por volta de 47% e 55% (b.s.), respectivamente. A extração do amido dos resíduos foi realizada enzimaticamente utilizando uma α-amilase, então, o amido liquefeito foi utilizado na produção de maltose catalisada pela β-amilase de cevada imobilizada. Dentre os métodos de imobilização estudados, o mais satisfatório para a imobilização de β-amilase foi a reticulação de enzimas agregadas (CLEA), utilizando albumina de soro bovino (BSA) ou proteína de soja (PS) como proteínas inertes, retendo 82,67% e 53,26% da atividade oferecida, respectivamente. Os CLEAs apresentaram estabilidades ao pH ligeiramente maiores que a β-amilase livre em seus respectivos valores de pH mais estáveis. Além disso, os CLEAs foram muito estáveis a 40ºC, retendo mais de 80% da atividade inicial após 12 horas de encubação. A conversão do amido em maltose foi realizada em um RFV, com a finalidade de estudar seu comportamento frente aos CLEAs de β-amilase (neste estudo CLEA de β-amilase preparado na presença de proteína de soja, aqui nomeado CLEA-β-PS). A conversão de amido em maltose foi de aproximadamente 70% em curto tempo de reação (4 horas), demonstrando a viabilidade do uso de amido residual como matéria-prima para a produção de maltose catalisada por CLEA de β-amilase em reator de fluxo em vórtices de Couette–Taylor–Poiseuille. Palavras chave: resíduos de mandioca, amido, maltose, beta-amilase de cevada, imobilização enzimática, CLEA, reator de fluxo em vórtices.

Made available in DSpace on 2016-06-02T19:55:23Z (GMT). No. of bitstreams: 1 2455.pdf: 1413452 bytes, checksum: 0d85e80e19b01ab27cd6e9dd30beb3b9 (MD5) Previous issue date: 2009-04-02 / Financiadora de Estudos e Projetos / The industrial production of 6-APA includes: (1) cultivation of Penicillium chrysogenum; (2) extraction with organic solvents, (3) crystallization; (4) penicillin hydrolysis by immobilized penicillin acylase; (5) extraction of phenyl acetic acid (AFA); (6) precipitation of 6-APA at its isoelectric point ( pH ~ 3,6). The scientific community and industry have interest in reducing the number of process steps required for 6-APA production. In this thesis a new method for 6-APA production is presented. In this process, the simultaneous production and hydrolysis of penicillin was carried out. The 6-APA was extracted from culture broth using ionic adsorbent. To demonstrate the technical viability of the process a suitable biocatalysts to perform the hydrolysis of penicillin in the complex media has been developed. The enzymatic extract, containing PGA was partially purified by affinity adsorption on agarose-tryptophan, it was necessary to prepare the biocatalyst. The apparent purification factor obtained was 4,5 and purified PGA was immobilized on agaroseglyoxil by multipoint covalent attachment. The biocatalysts obtained show stability under conditions of sterilization and application in bioreactor. However, their mechanical stability under vigorous conditions of agitation used in stirred tank bioreactors was not satisfactory. Three strategies were used to avoid fragmentation of the biocatalyst. The first strategy was to involve the impellers with a helicoidal structure. In this system the biocatalyst was maintained under agitation in external bulk of the apparatus. In the second strategy, the biocatalyst was introduced into the bioreactor as the biomass density reached a maximum, in this case, the cultivation was carried out under constant agitation speed (300 rpm). An airlift bioreactor was used as third strategy to maintain the pellet structure. These systems were efficient in increasing medium agitation without destroying the pellets. Complete hydrolysis of penicillin (30 g / L) was obtained after five days of cultivation and extraction of 6-APA on ionic exchanger was investigated. The extraction of 6-APA by ionic interaction using chitosan modified with glutaraldehyde and arginine is a good method for recovery it. However, optimization in this method is necessary to achieve the recovery of 6-APA at satisfactory levels for the pharmaceutical industry. The new method for production of 6-APA shows that is possible to eliminate the use of organic solvents and to reduce the number of process steps. / A produção industrial de ácido 6-aminopenicilânico (6-APA) inclui etapas de cultivo de Penicillium chrysogenum, extração com solvente orgânico, cristalização, hidrólise enzimática e precipitação. O interesse industrial e científico em reduzir o número de etapas deste processo tem motivado pesquisadores a buscar processos alternativos para obtenção de 6-APA. Neste trabalho, um novo processo é apresentado para a produção de 6-APA, cujas inovações envolvem a hidrólise de penicilina durante o cultivo de P. chrysogenum, a recirculação de ácido fenilacético (AFA) e extração de 6-APA ao final do cultivo utilizando adsorvente iônico. Para atender aos requerimentos do novo processo, foi desenvolvido um biocatalisador para atuar no complexo meio de cultivo. O preparo deste biocatalisador exigiu o uso de extrato enzimático purificado e uma metodologia para purificação de penicilina G acilase (PGA) foi investigada. Um fator de purificação aparente de 4,5 vezes foi obtido e a enzima foi ligada a agarose utilizando a técnica de imobilização covalente multipontual. O biocatalisador obtido apresentou boa estabilidade química em condições de esterilização e aplicação em biorreator. Entretanto, sua estabilidade mecânica sob condições rigorosas de agitação em biorreatores tipo tanque agitado e aerado não foram satisfatórias. Para solucionar este problema três estratégias foram avaliadas: (1) utilizando-se uma peça em forma de hélice envolvendo os impelidores, (2) adicionando-se o biocatalisador ao biorreator após a concentração de células atingir seu valor máximo e utilizando velocidade de agitação constante de 300 rpm, (3) usando um biorreator tipo air lift . As três estratégias permitiram manter a integridade do biocatallisador. Hidrólise completa de penicilina (30 g/L) foi obtida em 120 h de cultivo e a extração de 6-APA em coluna de troca iônica foi estudada. O método de extração de 6-APA através de interação iônica utilizando quitosana-arginina apresentou resultados promissores, entretanto, um aperfeiçoamento do método ainda faz-se necessário para atingir a recuperação de 6-APA em níveis satisfatórios para a indústria farmacêutica. Os resultados obtidos indicam que é possível eliminar o uso de solventes orgânicos na produção de 6-APA, além disso, a redução no número de etapas torna este processo mais simples e conseqüentemente reduz o tempo de produção e custo do produto final. Portanto, o processo desenvolvido neste trabalho é promissor para a aplicação na indústria farmacêutica.

In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-21T18:21:21Z No. of bitstreams: 1 marcosrodriguesfacchinicerqueira.pdf: 6161892 bytes, checksum: a58d0d5ce0d0b333fd8a3b50155105e4 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-22T12:39:49Z (GMT) No. of bitstreams: 1 marcosrodriguesfacchinicerqueira.pdf: 6161892 bytes, checksum: a58d0d5ce0d0b333fd8a3b50155105e4 (MD5) / Made available in DSpace on 2017-03-22T12:39:49Z (GMT). No. of bitstreams: 1 marcosrodriguesfacchinicerqueira.pdf: 6161892 bytes, checksum: a58d0d5ce0d0b333fd8a3b50155105e4 (MD5) Previous issue date: 2016-09-09 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O foco deste trabalho foi o desenvolvimento e aplicação de microreatores enzimáticos, visando sua aplicação em sistemas de análise por injeção em fluxo. Em cima disso, dois substratos poliméricos foram utilizados para a avaliação de imobilização enzimática: um à base de poli (metil metacrilato) (PMMA) e outro em uma resina do éter diglicídico do bisfenol-A (BADGE). Uma impressora à laser de CO2 foi utilizada para confeccionar os dispositivos nas dimensões desejadas. Para o sucesso da imobilização, os sistemas foram previamente tratados com polietilenoimina (PEI) visando a introdução de grupamentos funcionais reativos na superfície dos materiais de partida. Num primeiro estudo, baseado na modificação de PMMA, a ativação do material foi conseguida após tratamento dos microcanais com PEI em meio de dimetilsulfóxido (DMSO). Já no segundo caso o tratamento com PEI envolveu a simples mistura mecânica dos materiais, objetivando a cura da resina empregada. Após a ativação dos materiais com PEI, as enzimas foram imobilizadas após passagem de uma mistura de glutaraldeído (um agente espaçador) e as enzimas. Dentre as enzimas estudadas estão a glicose oxidase (GOx), a ascorbato-oxidase (AAO), a catalase (CAT), a glutamato dehidrogenase (GDH), além de um sistema híbrido baseado na imobilização simultânea das enzimas glicose oxidase (GOx) e horseradish peroxidase (HPR). A caracterização dos sistemas desenvolvidos foi feita primordialmente por meio da espectroscopia Raman. Além disso, a aplicação de alguns dos sistemas frente a amostras reais e o cálculo de parâmetros cinéticos e operacionais dos microreatores confeccionados foram reralizados. Essas avaliações foram feitas baseadas em sistemas de detecção desenvolvidos no laboratório por técnicas eletroquímicas e por espectroscopia no visível. Como grande benefício dos sistemas desenvolvidos, podem ser destacados a velocidade e a simplicidade de implementação do processo de imobilização e operação. / The focus of this work is the development and application of enzymatic microreactors aiming their application through flow injection analysis systems. On top of that, two polymeric substrates were used for the evaluation of enzyme immobilization: one based on poly (methyl methacrylate) (PMMA) and another baed on a bisphenol-A diglycidyl ether resin (BADGE). A CO2 laser printer was used to fabricate the devices at the desired dimensions. For the success of the immobilization systems have been pretreated with polyethyleneimine (PEI) in order to introduce reactive functional groups on the surface of the starting materials. In a first study, based on PMMA modification, the activation of the material was achieved after treating microchannels with PEI in dimethylsulfoxide (DMSO). In the second case, treatment with PEI involved simply a mechanical mixture of the two materials, in order to cure the resin. After activation of materials with PEI, the enzymes were immobilized after passage of a mixture of glutaraldehyde (a spacer agent) and enzymes. Among the enzymes studied are glucose oxidase (GOx), ascorbate oxidase (AAO), catalase (CAT), dehydrogenase glutamate (GDH), and a hybrid system based on the simultaneous immobilization of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HPR). The characterization of the developed systems was primarily done by Raman spectroscopy. Moreover, application of some of the proposed systems to real samples and calculation of kinetic and operational parameters are presented during the study. These evaluations were made with detection systems based on electrochemical and visible spectroscopy techniques, all developed at the laboratory. One great benefit of the developed systems, are the speed and simplicity of implementation the immobilization process and operation of the devices.

Les matériaux hydroxydes doubles lamellaires (HDL) sont une classe d'argiles anioniques synthétiques dont la structure est basée sur celle du brucite Mg(OH)2 dans lesquelles une partie des cations métalliques divalents sont été remplacés par des ions trivalents donnant ainsi des feuillets chargés positive. Cette charge est équilibrée par l'intercalation d'anions dans la région interlamellaire hydratée. Les identités et les rapports des cations di- et trivalents et l'anion interlamellaire peuvent être varié sur une large gamme, donnant lieu à une large classe de matériaux isostructurales. Le matériau d’origine de cette classe est l’hydrotalcite (HT) et les HDL sont par conséquent également connus comme des matériaux de type hydrotalcite. Bien que les caractéristiques de base de la structure soient bien comprises, des aspects structurels détaillés ont fait l'objet de certaine controverse dans la littérature afin de maîtriser leurs propriétés et leurs applications potentielles. Dans ce travail de thèse nous avons retenu deux types de HDL MgAl et ZnAl qui ont été largement introduits dans diverses applications, tels que la sorption des molécules d'intérêt biologique (enzyme et médicament) et l'élaboration d'électrodes. La spécificité de ce travail repose sur l’immobilisation d’une enzyme modèle, la lactate déshydrogénase dans ces deux matrices ainsi qu’un médicament anti-bactérien, la berbérine, afin d’étudier les interactions entre ces deux biomolécules et la phase HDL introduite et de répondre à leurs exigences d'applications dans le domaine médical. Dans un second temps nous avons tenté d’étudier les deux phases mentionnées de plus en plus fine en termes de structure, morphologie et profil électrochimique en vue de les employer en tant que matériaux d’électrode pour le développement de biopile / DHs are a class of synthetic anionic clays whose structure is based on brucite-like layers Mg(OH)2 inwhich some of the divalent cations have been replaced by trivalent ions giving positively-charged sheets.This charge is balanced by intercalation of anions in the hydrated interlayer regions. The identities andratios of the di- and trivalent cations and the interlayer anion may be varied over a wide range, giving rise toa large class of isostructural materials. The parent material of this class is the naturally occurring mineralhydrotalcite and LDHs are consequently also known as hydrotalcite-like materials. Although the basicfeatures of the structure are well understood, detailed structural aspects have been the subject of somecontroversy in the literature. In this thesis, we have selected two types of LDH, MgAl and ZnAl, which havebeen widely introduced in various applications, such as sorption of molecules of biological interest (enzymeand drug) and the development of electrodes. The specificity of this work lies on the immobilization of amodel enzyme, lactate dehydrogenase in both matrices as well as an anti-bacterial drug, berberine, inorder to study the interactions between these two biomolecules and the introduced LDH phase and tobetter address their challenges of applications in the medical field. Second, we have tried to study the twophases mentioned above more and more accurately in terms of structure, morphology and electrochemicalprofile in order to use them as electrode materials for microbial fuel cell device

Abstract Climate change and its warming effects on vegetation and soils are a widely recognized phenomenon. In addition to warming, the understorey vegetation in northern environments has been subjected to several environmental changes, such as increasing nitrogen (N) and other disturbances. This thesis examines the effects of N-fertilization and disturbances on the vegetation biomass and abundance, plant community composition and plant, soil and microbial N and C pools. Seedling establishment of the most common dwarf shrubs (deciduous Vaccinium myrtillus, evergreens V. vitis-idaea and Empetrum nigrum ssp. hermaphroditum) was investigated after artificial disturbance treatments (vegetation and soil removal). These studies were conducted in the boreal and subarctic ecosystems and in the forest-tundra ecotone in northern Finland. N-fertilization and disturbances enhanced the amount of graminoids in plant communities, and the recovery ability of graminoids was enhanced after N-fertilization, which homogenized the vegetation and resulted in a new stable state in the plant community. The recovery ability of evergreen dwarf shrubs was low after disturbances. Disturbances created habitats for seed germination, but the seedling establishment of dwarf shrubs studied was still limited by seed availability. N-fertilization had no effect on microbial biomass. Instead, microbial biomass decreased with disturbance treatment in the boreal forest. However, the concentration of N increased in above-ground vegetation, both after N-fertilization and disturbance without any indication of N immobilization, suggesting that plant species captured the available N effectively for their recovery. The study shows that the likely outcome of N enrichment, when combined with disturbances, is the enhanced growth of graminoids. The seedling establishment does not compensate for the reduction of the vegetative recovery of evergreen dwarf shrubs, which makes evergreen dwarf shrubs sensitive to environmental changes. As the understorey is more resilient to perturbations in the boreal forest than in the subarctic ecosystem, these results emphasize the sensitivity of the vegetation to simultaneous environmental changes in the northernmost ecosystems. Moreover, microbial properties are more resilient to environmental changes than is above-ground vegetation. / Tiivistelmä Ilmaston muutos ja siitä aiheutuvan lämpenemisen vaikutus kasvillisuuteen ja maaperään on laajasti tunnustettu ilmiö. Lämpenemisen lisäksi pohjoisten alueiden aluskasvillisuuteen kohdistuu useita muutospaineita, kuten lisääntynyt typpipitoisuuden nousu ja kasvillisuutta muokkaavat häiriöt. Tässä tutkimuksessa mitattiin lisääntyneen typpipitoisuuden ja häiriöiden vaikutus kasvillisuuden biomassaan ja runsauteen sekä yhteisörakenteeseen, sekä kasvilajeihin, maaperään ja mikrobibiomassaan sitoutuneen typen ja hiilen määrään. Lisäksi tutkittiin yleisimpien varpukasvien (lehtensä pudottava mustikka, ikivihreät puolukka ja variksenmarja) siemenellistä lisääntymistä kokeellisen häiriön (kasvillisuuden tai maaperän poisto) jälkeen. Tutkimukset tehtiin boreaalisessa ja subarctisessa ekosysteemeissä sekä metsänrajaympäristössä Pohjois-Suomessa. Typpilannoitus ja häiriöt lisäsivät heinien määrää kasviyhteisöissä. Lisäksi typpilannoitus edisti heinien kasvullista palautumiskykyä häiriön jälkeen, joka johti kasvillisuuden homogenisoitumiseen ja kasviyhteisön uuteen tasapainotilaan. Häiriöt heikensivät ikivihreiden varpujen kasvullista palautumista häiriön jälkeen. Häiriö loi sopivia elinympäristöjä siementen itämiselle, mutta tutkittujen lajien siementen määrä rajoitti siemenellistä lisääntymistä. Typpilannoitus ei vaikuttanut mikrobibiomassaan, mutta häiriö vähensi mikrobibiomassaa boreaalisessa ekosysteemissä. Kuitenkin kasvien typen pitoisuudet lisääntyivät sekä lannoituksen että häiriön jälkeen ilman viitteitä typen sitoutumisesta mikrobibiomassaan. Tämä viittaa siihen, että kasvit käyttävät maaperän typen tehokkaasti häiriön jälkeiseen palautumiseen. Väitöskirjan mukaan typen lisääntyminen häiriöiden yhteydessä edistää heinien esiintymistä. Koska ikivihreiden varpujen siemenellinen lisääntyminen ei kompensoi häiriöstä kasvulliselle palautumiselle aiheutuvaa haittaa, ovat ikivihreät varvut erityisen herkkiä häiriöille. Aluskasvillisuus on vastustuskykyisempi ympäristön muutoksille boreaalisessa kuin subarktisessa ekosysteemissä, mikä korostaa pohjoisimpien alueiden herkkyyttä yhtäaikaisille ympäristön muutoksille. Maaperän olosuhteet ovat kasvillisuutta kestävämpiä ympäristön muutoksille.

Ces travaux de thèse pluridisciplinaires à l‘interface entre biocatalyse et nanomatériaux visent la conception de matériaux biohybrides innovants par assemblage dans des conditions douces de matériaux inorganiques de type hydroxydes doubles lamellaires (HDL) avec des enzymes. La première partie de ce mémoire est consacrée à la caractérisation des interactions physico-chimiques entre les HDL et la fructose-6-phosphate aldolase (FSA) catalysant la formation stéréosélective de liaisons C-C pour conduire à des polyols chiraux. Les structures lamellaires HDL permettent un confinement efficace de systèmes enzymatiques grâce à leur structure bidimensionnelle poreuse, leurs propriétés physico-chimiques favorables à l‘échange ionique et leur biocompatibilité. Différentes stratégies d‘immobilisation de la FSA dans des matrices d‘HDL ont été explorées, le taux d‘immobilisation et l‘activité biocatalytique étant fortement dépendant de la méthode d‘assemblage et de la nature des phases HDL. Le taux d‘immobilisation de l‘enzyme obtenu par coprécipitaton est supérieur à celui obtenu par adsorption. Dans une deuxième partie, un bioréacteur a été élaboré par un assemblage hiérarchisé constitué de la FSA, de nanoplaquettes d‘HDL et de billes de polysaccharide, ce dernier jouant le rôle de matrice macrostructurante. De façon notable, le taux d‘encapsulation de l‘enzyme dans la matrice macroscopique est amélioré lorsque le biocatalyseur est pré-encapsulé dans les nanoplaquettes d‘HDL. Ceci est attribué aux interactions électrostatiques favorables entre les chaînes de polysaccharide et les HDL, facilitant une charge de matière plus importante. L‘efficacité catalytique du bioréacteur obtenu et sa recyclabilité ont été démontrés. Dans la troisième partie de cette thèse, nous décrivons pour la première fois la conception de bionanoréacteurs enzymes@HDL par co-immobilisation de systèmes bi- ou tétra-enzymatiques dans les HDL permettant de réaliser des cascades multienzymatiques biomimétiques. L‘immobilisation des différentes enzymes prises séparément a d‘abord été optimisée afin de déterminer les conditions de co-immobilisation et de réaliser les cascades biocatalytiques en phase hétérogène. Ces bionanoréacteurs, dont nous avons démontré la recyclabilité, ont été appliqués pour la synthèse de sucres phosphorylés de série D. Enfin, une cascade multienzymatique a été conçue de novo en solution aqueuse et optimisée pour synthétiser différents sucres phosphorylés rares de série L. / This multidisciplinary thesis at the biocatalysis/nanomaterial interface perfectly aims at designing innovative biohybrid materials by the assembly of inorganic materials the Layered Double Hydroxides (LDH) with enzymes under mild conditions. The first part of this thesis is devoted to the characterization of physico-chemical interactions between the LDH and the fructose-6-phosphate aldolase (FSA) catalyzing the stereoselective C-C bond formation to provide chiral polyols. LDH structures allow the effective confinement of enzymatic systems thanks to their opened two-dimensional structure as well as their chemical surface properties at the nanoscale and their biocompatibility. The FSA immobilization in different LDH matrices by different methods was studied. Biocatalytic activity is highly dependent on the method of assembling, modulating the final amount of FSA. The retaining activity rate of co-precipitated material was higher than that obtained for the adsorbed enzyme. In a second part, a bionanoreactor was developed based on a hierarchized assembly of FSA, LDH nanoplatelets and polysaccharide beads acting as a macrostructuring matrices. Significantly, the encapsulated enzyme rate in the beads was improved when the biocatalyst was pre-encapsulated in LDH nanoplatelets. This is attributed to favorable electrostatic interactions between the polysaccharide chains and LDH, facilitating a higher catalyst loading. The catalytic efficiency of the prepared bioreactor and its recyclability were demonstrated. In the third part of this thesis, we describe for the first time the design of bionanoreactors ―enzymes@LDH‖ by co-immobilisation of two and four enzymes in LDH allowing biomimetic multienzymatic cascades. We first studied the immobilization of the different enzymes taken separately. Then we worked on the optimization of the biocatalytic cascades in heterogeneous phase. These bionanoreactors, for which we have shown the recyclability, have been applied to the synthesis of D-series phosphorylated sugars. Finally, a multienzymatic cascade was de novo designed in aqueous homogeneous solution. It was optimized for the synthesis of rare L-phosphorylated sugars.

Les REFIOM (Résidus d'Epuration des Fumées d'Incinération des Ordures Ménagères) issus de l'incération des déchets ménagers contiennent des métaux lourds comme le plomb ou le cadmium et sont en France uniquement stockés en centre d'enfouissement technique de classe 1 pour dangereux, en étant stabilisés par une vitrification. Afin de trouver des solutions pour le stockage ou la valorisation à long terme des REFIOM sans danger pour l'environnement, nous avons ciblé les vitrocéramiques et les céramiques frittées à hautes températures et hautes pressions. Nous avons utilisé des méthodes de la minéralogie physique par l'intermédiaire de synthèses à hautes températures, de synthèses à hautes températures et à hautes pressions en autoclave à chauffage externe, d'observations en microscopie électronique à balayage, de microanalyses chimiques EDX (Energy Dispersive X-Ray spectrometry), d'analyses en microsondes, de caractérisation structurale par diffraction de rayons X et d'expériences de lixiviation dynamique. Nous avons mis au point des protocoles de synthèses et d'analyses. Par ce biais, nous constatons pour les vitrocéramiques que le plomb ou le cadmium sont incorporés dans des cristallites et dans des nouvelles phases cristallines, eux-mêmes englobés dans une matrice vitreuse. Cette voie dite "double barrière" (cristaux + verre) semble prometteuses pour l'immobilisation du plomb et du cadmium (au regard des analyses EDX et des expériences de lixiviation). Pour les céramiques frittées, comme pour les SYNROC (SYNthetic ROCk) synthétisées pour les déchets nucléaires, de nouvelles phases cristallines incorporant Pb et Cd sont observées et seraient a priori résistantes pour le stockage de ces éléments toxiques. Dans ces deux cas de nouveaux matériaux capables d'incorporer massivement du plomb et du cadmium ont été mis en évidence. Des tests de durabilité permettront d'envisager une valorisation éventuelle de tels matériaux / MSWI 5Municipal Solide Waste Incinerator) fly ashes from the incineration of domestic waste contain heavy metals such as lead or cadmium. In France, these fly ashes are only stored under vitrified forms in class-1 type landfills for hazardous waste. In order to find solutions for long-term storage or valorization of the MSWI fly ashes, we studied glass-ceramics and sintered ceramics at high pressures and/or hight temperature. We used methods of mineral physics to : synthetize at high temperature, synthetize at higt temperature and high pressure using autoclaves with external heating system, observe by electron microcopy, make EDX (Energy Dispersive X-Ray spectrometry) chemical microanalysis, make microprobe analysis, structurally characterize and perform leaching test. We established experimental protocols for the synthesis and analysis of produced materials. For glass-ceramics, we observe that lead and cadmium are incorporated inside expected crystallites and new crystal phases, themselves embedded by a glassy matrix. This so-called "double barrier" (crystals + glass) is a promising way towards a substainable of lead and cadmium (after EDX analysis and leaching experiements). For sintered ceramics, as for the SYNROC (SYNthetic ROCk) with nuclear waste, new crystal phases incorporating Pb and Cd are found and might display a high resistant for the storage of these toxic elements. In both cases, new materials incorporating large amounts of lead and cadmium were formed. Durability tests may give new ways for a valorization of such materials

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 ´ 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.