A bactéria Zymomonas mobilis atraiu considerável interesse nas últimas décadas devido ao seu metabolismo único e eficientes características fermentativas na produção de etanol a partir de açúcares simples. No entanto, apesar das aparentes vantagens na conversão e taxas específicas quando comparada com as leveduras, estas ainda dominam o mercado produtivo de etanol. Dentre os diversos modelos encontrados na literatura para representar o processo fermentativo de glicose através de Z. mobilis, o modelo proposto por Jöbses et al. (1986) aparenta ser bem estruturado e ideal para a criação de estratégias de controle e otimização de processos fermentativos com este microrganismo, viabilizando sua utilização em escala industrial. Fermentações de Z. mobilis em regime contínuo apresentam comportamento oscilatório para baixas taxas de diluição, fenômeno este comprovado experimentalmente. Experimentos laboratoriais foram utilizados para a estimação de parâmetros do modelo de Jöbses, através da técnica de minimização da derivada do erro da função objetivo. O novo modelo ajustado apresenta dinâmica equivalente ao modelo original de Jöbses, fato este comprovado através da construção dos diagramas de bifurcação. Os diagramas de fase dos modelos apresentam algumas diferenças estruturais entre si, entretanto, a ocorrência de multiplicidade de estados estacionários em baixos valores de taxa de diluição está presente em todos. Em bioprocessos, a obtenção de informações sobre o sistema é um tanto complexa, devido a estes meios serem muito suscetíveis a distúrbios, contaminações, além de que medidas de concentrações são relativamente dispendiosas, associadas com um alto tempo morto, o que impossibilita, muitas vezes, a implementação de sistemas de controle eficientes. Para contribuir com a resolução deste problema foi desenvolvida uma nova inferência das variáveis de estado através da informação de consumo de hidróxido de sódio, utilizado no controle de pH do meio. Inferidores de variáveis de estado que utilizam a variação no consumo de hidróxido de sódio foram ajustados e apresentaram resultados promissores, mostrando a viabilidade do desenvolvimento de metodologias para este tipo de análise, tornando a caracterização de sistemas fermentativos mais rápida e acessível, não apenas em nível acadêmico, que a desenvolvida atualmente, principalmente devido ao baixo custo associado, e a dinâmica rápida deste tipo de sensor. / The bacterium Zymomonas mobilis has attracted considerable interest in recent decades due to their unique metabolism and efficient characteristics in the production of ethanol by simple sugars. However, despite the apparent advantages in the conversion and specific rates, when compared with yeast, there is no industrial-scale fermentations with these bacteria. Among the various models found in literature to represent glucose fermentation with Z. mobilis, the model proposed by Jöbses et al. (1986) appears to be well structured and ideal for the creation of control strategies and optimization methods for increase productions. Fermentation in continuous mode with this organism exhibit oscillatory behavior at low dilution rates, phenomenon showed or demonstrated by experiments performed on laboratory scale. A laboratorial experiment also was used for estimation of new parameters group of Jöbses models, using the technique of minimization of error derivative for objective function. The new adjusted model set presents dynamics similar to the original model, fact confirmed by bifurcation analyses of both models. The diagram for these models show structural differences, but both presents steady states multiplicity and Hopf bifurcations. The obtained of fermentation characteristics is very complex, due to these reactive means being very susceptible to disturbances, contamination, among others, and concentrations measures are relatively expensive and whit a high dead time associated, which often prevents the implementation of control tools. The proposed inference of state variables by consumption rate of hydroxide sodium, used for maintenance pH medium, was tested and confirmed. Virtual sensors using the information of consumption rate of hydroxide were adjusted and have shown promising results, demonstrating the feasibility of developing methods of analysis based on this methodology, making the characterization of fermentative systems faster and cheaper them currently developed, mainly due to low cost associated, and the dynamics of this fast type of sensor.

A bactéria Zymomonas mobilis atraiu considerável interesse nas últimas décadas devido ao seu metabolismo único e eficientes características fermentativas na produção de etanol a partir de açúcares simples. No entanto, apesar das aparentes vantagens na conversão e taxas específicas quando comparada com as leveduras, estas ainda dominam o mercado produtivo de etanol. Dentre os diversos modelos encontrados na literatura para representar o processo fermentativo de glicose através de Z. mobilis, o modelo proposto por Jöbses et al. (1986) aparenta ser bem estruturado e ideal para a criação de estratégias de controle e otimização de processos fermentativos com este microrganismo, viabilizando sua utilização em escala industrial. Fermentações de Z. mobilis em regime contínuo apresentam comportamento oscilatório para baixas taxas de diluição, fenômeno este comprovado experimentalmente. Experimentos laboratoriais foram utilizados para a estimação de parâmetros do modelo de Jöbses, através da técnica de minimização da derivada do erro da função objetivo. O novo modelo ajustado apresenta dinâmica equivalente ao modelo original de Jöbses, fato este comprovado através da construção dos diagramas de bifurcação. Os diagramas de fase dos modelos apresentam algumas diferenças estruturais entre si, entretanto, a ocorrência de multiplicidade de estados estacionários em baixos valores de taxa de diluição está presente em todos. Em bioprocessos, a obtenção de informações sobre o sistema é um tanto complexa, devido a estes meios serem muito suscetíveis a distúrbios, contaminações, além de que medidas de concentrações são relativamente dispendiosas, associadas com um alto tempo morto, o que impossibilita, muitas vezes, a implementação de sistemas de controle eficientes. Para contribuir com a resolução deste problema foi desenvolvida uma nova inferência das variáveis de estado através da informação de consumo de hidróxido de sódio, utilizado no controle de pH do meio. Inferidores de variáveis de estado que utilizam a variação no consumo de hidróxido de sódio foram ajustados e apresentaram resultados promissores, mostrando a viabilidade do desenvolvimento de metodologias para este tipo de análise, tornando a caracterização de sistemas fermentativos mais rápida e acessível, não apenas em nível acadêmico, que a desenvolvida atualmente, principalmente devido ao baixo custo associado, e a dinâmica rápida deste tipo de sensor. / The bacterium Zymomonas mobilis has attracted considerable interest in recent decades due to their unique metabolism and efficient characteristics in the production of ethanol by simple sugars. However, despite the apparent advantages in the conversion and specific rates, when compared with yeast, there is no industrial-scale fermentations with these bacteria. Among the various models found in literature to represent glucose fermentation with Z. mobilis, the model proposed by Jöbses et al. (1986) appears to be well structured and ideal for the creation of control strategies and optimization methods for increase productions. Fermentation in continuous mode with this organism exhibit oscillatory behavior at low dilution rates, phenomenon showed or demonstrated by experiments performed on laboratory scale. A laboratorial experiment also was used for estimation of new parameters group of Jöbses models, using the technique of minimization of error derivative for objective function. The new adjusted model set presents dynamics similar to the original model, fact confirmed by bifurcation analyses of both models. The diagram for these models show structural differences, but both presents steady states multiplicity and Hopf bifurcations. The obtained of fermentation characteristics is very complex, due to these reactive means being very susceptible to disturbances, contamination, among others, and concentrations measures are relatively expensive and whit a high dead time associated, which often prevents the implementation of control tools. The proposed inference of state variables by consumption rate of hydroxide sodium, used for maintenance pH medium, was tested and confirmed. Virtual sensors using the information of consumption rate of hydroxide were adjusted and have shown promising results, demonstrating the feasibility of developing methods of analysis based on this methodology, making the characterization of fermentative systems faster and cheaper them currently developed, mainly due to low cost associated, and the dynamics of this fast type of sensor.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Fermentation in Zymomonas mobilis has been described as a catabolic highway, with 50 % of soluble protein comprising glycolytic and fermentative enzymes. In conjunction with one of the fastest observed fermentations, the conversion of glucose to ethanol forms one of the least efficient energy extractions found in nature. The low energy yield of fermentation in Z. mobilis is a result of the usage of the Entner-Doudoroff glycolytic pathway, which has half the energy yield per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway. The work presented in this thesis forms part of a larger project to compare glycolytic regulation in different micro-organisms (i.e., Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus lactis). These organisms were chosen based on their usage of different glycolytic mechanisms. By using supply-demand analysis for quantifying glycolytic regulation as well as similar experimental conditions (e.g. using non-growing cell cultures), we can compare the regulatory behaviour of mechanistically distinct freeenergy supplies. The aim of this thesis was to quantify the importance of anaerobic free-energy generation for the regulation of the Entner-Doudoroff glycolytic pathway in Z. mobilis. We used metabolic control analysis (MCA) and supply-demand analysis to realize this goal. The central message of MCA is that when a metabolic parameter (e.g., a conserved metabolic moiety) is deemed important for affecting a particular steady-state variable (i.e., fermentation flux), its effect on the steady state variable should be tested. An extension to MCA, supply-demand analysis, provides a quantitative framework for analyzing the regulatory importance of cellular commodities such as anaerobic free-energy. This is done through comparing the elasticities of anaerobic free-energy supply and demand, which yields the degree to which the respective reaction blocks control the flux through anaerobic free-energy metabolism, as well as determine the cellular free-energy state (ATP/ADP ratio). The regulation of anaerobic free-energy metabolism in Z. mobilis was investigated with an experimental approach. The key features of our experimental setup were the use of NMR spectroscopy for detecting metabolites, as well as employing non-growing conditions for supply-demand experiments. With NMR spectroscopy metabolites could be detected in real time without using invasive sampling techniques; the use of nongrowing conditions further simplified the analysis by enabling us to correlate fermentative behaviour exclusively with the anaerobic free-energy state. Fermentation of glucose was investigated in the wild type Z. mobilis, a recombinant containing a non-expressing plasmid, or expressing plasmids for over-expressing the glucose facilitator (TCDB 2.A.1.1.4) or glucose-6-phosphate dehydrogenase (EC 1.1.1.49). In addition, ATP demand in the non-expressing recombinant and wild type was perturbed by titrating with the uncoupler acetic acid. Our results show that the anaerobic free-energy demand, the glucose facilitator and glucose-6-phospate dehydrogenase all control the flux of ethanol production in Z. mobilis. The Entner-Doudoroff glycolytic supply activity was found to be sensitive to changes in the ratios of ATP/ADP (elasticity varied between –0.31 and –0.49) and NTP/NDP (elasticity varied between –0.31 and – 0.50). / AFRIKAANSE OPSOMMING: Fermentasie in Zymomonas mobilis word beskryf as ‘n kataboliese snelweg, waar glikolitiese en fermentatiewe ensieme 50% van totale oplosbare proteïene in die sel uitmaak. Hoewel dié fermentasie een van die vinnigstes is wat tot op hede waargeneem is, is die omskakeling van glukose na etanol een van die mees ondoeltreffende energieekstraksies in die natuur. Dié lae energie-opbrengs, soos waarneembaar in fermentasie in Zymomonas mobilis, kan toegeskryf word aan die Entner-Doudoroff metaboliese pad. Hierdie metaboliese pad lewer slegs die helfte van die energie-opbrengs per mol substraat vergeleke met die meer bekende Embden-Meyerhof-Parnas glikolitiese pad. Die navorsing in hierdie tesis is deel van ‘n omvattende projek wat poog om die regulering van glikolise in verskillende mikro-organismes (Z. mobilis, Escherichia coli, Saccharomyces cerevisiae en Lactococcus lactis) te vergelyk. Dié organismes is gekies op grond van die uiteenlopende glikolitiese meganismes waarvan hulle gebruik maak. Ten einde die reguleringsgedrag van meganisties verskillende vry-energie produksieweë m.b.v. vraag-aanbod analise te vergelyk, moet glikolitiese regulering eers onder eenderse eksperimentele kondisies (b.v. nie-groeiende selkulture) gekwantifiseer kan word. Die hoofdoel van hierdie tesis was om die belang van anaerobiese vry-energie produksie vir die regulering van die Entner-Doudoroff glikolitiese pad in Z. mobilis te kwantifiseer. Hiervoor is van Metaboliese kontrole-analise (MKA) en vraag-aanbodanalise (‘n uitbreiding van MKA) gebruik gemaak. MKA is ‘n tegniek waarmee die effek wat ‘n metaboliese parameter (soos metaboliese deel-konservering) op ‘n spesifieke bestendige toestand-veranderlike (soos fermentasiefluksie) het, gekwantifiseer kan word. Vraagaanbodanalise daarenteen, bied ‘n kwantitatiewe raamwerk waardeur die regulatoriese belang van sellulêre kommoditeite (byvoorbeeld anaerobiese vry-energie) geanaliseer kan word. Tydens laasgenoemde proses word die elastisiteit van die anaerobiese vry-energie aanbod en die elastisiteit van die vraag vergelyk. Op hierdie manier kan die mate van beheer wat die onderskeie reaksieblokkie oor die fluksie deur anaerobiese vry-energie metaboliese paaie, sowel as oor die sellulêre vry-energie toestand (ATP/ADP verhouding), bepaal word. In hierdie werk is die regulering van anaerobiese vry-energie metabolisme in Z. mobilis ondersoek deur van ‘n eksperimentele benadering gebruik te maak. Die sleuteleienskappe van dié benadering was om kernmagnetiese-resonansiespektroskopie (KMR spektroskopie) te gebruik om metabolietkonsentrasies te meet, en om van niegroeiende kondisies gebruik te maak vir die vraag-aanbod eksperimente. Metabolietkonsenstrasies kon aaneenlopend bepaal word sonder die gebruik van monsternemingstegnieke wat die reaksie sou kon beïnvloed. Eksterne invloede op die fermentasiegedrag kon ook uitgesluit word deur van nie-groeiende kondisies gebruik te maak, sodat die waargenome fermentasiegedrag uitsluitelik aan die anaerobiese vryenergie toestand toegeskryf kan word. Glukose fermentasie was ondersoek in wilde tipe Z. mobilis, en in drie rekombinante wat onderskeidelik ‘n glukose fasiliteerder ooruitdrukkingsplasmied (TCDB 2.A.1.1.4), ‘n glukose-6-fosfaat dehidrogenase ooruitdrukkingsplasmied (EC 1.1.1.49), en ‘n nieuitdrukkingsplasmied bevat het. Die ATP vraag in die wilde tipe en die nieuitdrukkingsrekombinant is geperturbeer deur titrasies met asynsuur as ontkoppelaar. Die resultate toon dan die anaerobiese vry-energievraag, sowel as die glukose fasiliteerder en glukose-6-fosfaat dehidrogenase, die fluksie van etanolproduksie in Z. mobilis beheer. Die Entner-Doudoroff glikolitiese produksie-aktiwiteit was sensitief vir veranderinge in die ATP/ADP verhouding (elastisiteite was tussen -0.31 en -0.49) en die NTP/NDP verhouding (elastisiteite was tussen -0.31 en -0.50).

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Metabolic networks of cellular systems are complex, in that there are numerous components with multiple non-linear interactions. To understand how these networks work they are often split into manageable pieces and studied individually. However, an individual part is unable to account for the complex properties of systems. In order to study these interactions the eld of systems biology has developed. Systems biology makes use of computers to construct models as a method to describe aspects of living systems. Using cellular pathways, kinetic models of metabolic pathways can be constructed and used as a tool to study the biological systems and provide a quantitative description. This thesis describes the quantitative analysis of a bacterium using a systems biology approach. Zymomonas mobilis is a rod shaped, Gram-negative, non-mobile facultative anaerobe and has one of the fastest observed fermentations, yet least energy e cient extractions found in nature. Furthermore it is the only known micro-organism to use the Entner-Doudoro (2-keto-3-deoxy-6- phosphogluconate) pathway anaerobically. The low energy yield of fermentation in Z. mobilis is a result of the usage of the Entner-Doudoro glycolytic pathway, which has half the energy yield per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway. The work presented in this thesis forms part of a larger project to compare glycolytic regulation in di erent micro-organisms Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus lactis. These organisms were chosen based on their usage of di erent glycolytic mechanisms. Kinetic models are suitable tools to draw a comparison between these organisms. The emphasis here is on the construction of a kinetic model of the Entner-Doudoroff glycolytic pathway as it occurs in Z. mobilis. The aim of this thesis was to characterise as many of the Entner-Doudoro pathway enzymes as possible, under standard conditions. This was done using enzyme assays, to obtain the kinetic parameters of each of the enzymes. Microtitre plate assays were used to characterise most of the enzymes of the Entner-Doudoro pathway. However, not all characterisations could be done using plate assay methods, as some intermediates were not commercially available to perform coupled assays. Nuclear magnetic resonance (NMR) spectroscopy was used to characterise these enzymes. These experimentally obtained parameters were then incorporated in a mathematical framework. Time simulations on the initial model were unable to reach a steady-state, with a build up of metabolic intermediates. A secondary model was constructed (using calculated maximal activities) which allowed us to identify discrepancies in the initial model. This showed that the experimentally determined maximal activities of three enzymes in lower glycolysis were unrealistically low, which might be due to protein denaturation by sonication. A nal model was constructed which incorporated a correction factor for these three enzymes. The models' predicted output (steady-state concentrations and ux) was compared to that of either literature or experimentally determined values, as a method to validate the model. The model output compared well to literature values. The constructed and partially validated kinetic model was then used as an analytical tool to identify points of control and regulation of glycolysis in Z. mobilis. The model presented in this work was also compared to published models. Our model relies much less on literature obtained values, and uses kinetic parameters experimentally determined under the same conditions. The parameters of the published models were obtained from the literature and in many instances, the assay conditions for these parameters were set-up to yield the maximum activity under non-physiological conditions. Furthermore, the number of excluded or assumed parameters is much less in our model. However, introduction of a milder, more predictable extraction technique for preparing cell lysates, should be considered for future work, to obtain the parameters that was not determined during this study. The published models do include reactions not included in our model (e.g ATP metabolism), which should be considered for inclusion, as we strive to construct a detailed kinetic model of glycolysis in Z. mobilis in the future. / AFRIKAANSE OPSOMMING: Sellul^ere metaboliese netwerke is komplekse stelsels, omdat hulle bestaan uit talle komponente met verskeie nie-lineêre interaksies. Om die funksionering van hierdie netwerke te verstaan, word hulle dikwels in hanteerbare stukke verdeel en individueel bestudeer. 'n Enkele komponent is egter nie in staat om die komplekse eienskappe van sulke stelsels te verklaar nie. Die veld van sisteembiologie het ontwikkel met die doel om sulke stelsels te bestudeer. Sisteembiologie maak gebruik van rekenaarmodelle as 'n metode om aspekte van lewende sisteme te beskryf. Kinetiese modelle van metaboliese paaie word gebou en gebruik as gereedskap om die biologiese stelsels te bestudeer en 'n kwantitatiewe beskrywing te bekom. Hierdie tesis beskryf die kwantitatiewe ontleding van 'n bakterie deur middel van 'n sisteembiologiese benadering. Zymomonas Mobilis is 'n staafvormige, Gram-negatiewe, nie-mobiele fakultatiewe ana erobe, en het een van die vinnigste waargenome fermentasies, maar met die minste energie-doeltre ende ekstraksie wat in die natuur aangetref word. Verder is dit die enigste bekende mikro-organisme wat die Entner-Doudoro (2-keto-3-dioksi-6-fosfoglukonaat) pad ana erobies gebruik. Die lae-energieopbrengs van fermentasie in Z. mobilis is 'n gevolg van die gebruik van die Entner-Doudoro metaboliese pad, wat die helfte van die energie-opbrengs per mol substraat lewer, in vergelyking met die bekende Embden-Meyerhof-Parnas pad. Die werk wat in hierdie tesis aangebied word, vorm deel van 'n groter projek om glikolitiese regulering in verskillende mikro-organismes te vergelyk, naamlik Z. mobilis, Escherichia coli, Sac- charomyces en Lactococcus lactis. Hierdie organismes is gekies op grond van hul gebruik van verskillende glikolitiese meganismes. Kinetiese modellering is 'n handige metode om 'n vergelyking tussen hierdie organismes te trek. Hierdie werk fokus op die bou van 'n kinetiese model van die Entner-Doudoro glikolitiese metaboliese pad soos dit in Z. mobilis voorkom. Die doel van hierdie tesis was om so veel moontlik van die Entner-Doudoro ensieme onder standaard-toestande te karakteriseer. Die kinetiese parameters van elk van die ensieme is met behulp van ensimatiese essai's bepaal. Vir die meeste essai's is 96-put mikrotiterplate gebruik, maar nie al die karakteriserings kon met behulp van hierdie metode gedoen word nie, omdat sommige intermediate nie kommersieel beskikbaar was om gekoppelde essai's mee uit te voer nie. Kernmagnetiese resonansie (KMR) spektroskopie is gebruik om hierdie ensieme te karakteriseer. Die eksperimenteel bepaalde parameters is opgeneem in 'n wiskundige raamwerk. Tydsimulasies op die aanvanklike model was nie in staat om 'n bestendige toestand te bereik nie, omdat metaboliete opgebou het. 'n Sekond^ere model is gebou (met behulp van berekende maksimale aktiwiteite) wat ons toegelaat om teenstrydighede in die aanvanklike model te identi seer. Dit het getoon dat die eksperimenteel bepaalde maksimale aktiwiteite van drie ensieme in die laer gedeelte van glikolise te laag was, waarskynlik as gevolg van prote en denaturering tydens die ultrasoniese disintegrasieproses. 'n Finale model is gebou waarin 'n korreksiefaktor vir hierdie drie ensieme opgeneem is. Die modelle se voorspelde uitset (bestendige toestand konsentrasies en uksie) is vergelyk met waardes uit die literatuur of wat ons self bepaal het, as 'n metode om die model te valideer. Die model uitset was in goeie ooreenstemming met hierdie waardes. Die gedeeltelik gevalideerde kinetiese model is voorts gebruik as 'n analitiese instrument om beheer en regulering van glikolise in Z. mobilis te ondersoek. Die model wat in hierdie werk ontwikkel is, is ook vergelyk met die vorige gepubliseerde modelle. Ons model berus baie minder op waardes uit die wetenskaplike literatuur, en maak gebruik van parameters wat eksperimenteel bepaal is, onder identiese toestande. Die parameters van die gepubliseerde modelle is meesal verkry uit die literatuur, en in baie gevalle was die eksperimentele kondisies vir hierdie analises opgestel om die maksimale aktiwiteit te lewer onder nie- siologiese toestande. Verder bevat ons model minder parameters wat of uitgesluit is of wie se waardes aangeneem moes word. In toekomstige werk sal daar egter klem gel^e moet word op 'n minder wisselvallige ekstraksietegniek vir die verkryging van selekstrakte, om sodoende parameters te identi seer wat nie in hierdie werk bepaal kon word nie. Die gepubliseerde modelle sluit ook reaksies in wat nie ingesluit is in ons model nie (bv. ATP metabolisme). Hierdie sou in ag geneem moet word vir insluiting in 'n toekomstige uitgebreide model, om daarna te streef om 'n gedetailleerde kinetiese model van glikolise in Z. mobilis te bou.

Orientador: Crispin Humberto Garcia Cruz / Banca: Vanildo Luiz Del Bianchi / Banca: Marcos Lucca Jr. / Resumo: A produção de etanol combustível a partir de resíduos lignocelulósicos, como tecnologia emergente, conhecida como bioetanol de segunda geração, está sendo bastante pesquisada em virtude de uma preocupação com o desenvolvimento de fontes energéticas renováveis e mais limpas. Neste contexto as indústrias vinícolas têm destaque, já que o subproduto gerado por elas, bagaço e engaço, por exemplo, é de lenta decomposição. Ao mesmo tempo em que há buscas por novos substratos, há também por micro-organismos capazes de fermentar monossacarídeos presentes nestes resíduos para produzir bioetanol, e também que estes consigam fermentar em consórcio sem que haja competição entre eles para o melhor aproveitamento destas fontes de carbono. Neste trabalho foram realizadas fermentações pela bactéria Zymomonas mobilis CCT 4494 e pela levedura Candida tropicalis em hidrolisados ácidos dos resíduos de uvas das espécies Isabel e Bordô, para isto, foi utilizada a concentração de ácido sulfúrico de 1,5% com aquecimento em autoclave a 121 ºC e 1 kgf/cm2. Foram realizadas também, fermentações utilizando meios semi-sintéticos com glicose e xilose (50 g/L) como substratos para a Z. mobilis e C. tropicalis, respectivamente. Nas fermentações realizadas pela bactéria Z. mobilis, utilizando tanto a mistura (50% bagaço e 50% engaço) dos resíduos quanto apenas o engaço não houve detecção de etanol por cromatografia gasosa. Com o meio semi-sintético, em 8 horas de fermentação, foram produzidos 17,0 g/L de etanol. Durante as fermentações efetuadas pela levedura C. tropicalis, houve produção de etanol de 1,04 g/L e 5,89 g/L em 6 horas de fermentação utilizando a mistura dos resíduos e o engaço, respectivamente e, 18,56 g/L em 8 horas em meio semi-sintético. No consórcio dos micro-organismos, a produção de etanol, utilizando primeiramente a mistura e depois o engaço foi de 0,72 g/L e 1,15 g/L, ambas em 8 horas... / Abstract: The production of fuel ethanol from lignocellulosic residues, as an emerging technology, known as second-generation bioethanol is being extensive research because of a concern with the development of renewable and cleaner energy sources. In this context, the wine industry has highlighted, since the by-product generated by them, bagasse and stems, for example, is the slow decomposition. While there search for new substrates, there are also micro-organisms capable of fermenting monosaccharides present in these residues to produce bioethanol, and also that they are able to ferment in a consortium without competition between them for better use of these carbon sources . In this work were carried out fermentations by Zymomonas mobilis CCT 4494 bacteria and the yeast Candida tropicalis in hydrolyzed acid waste grape species Isabel and Bordô, for this, we used the sulfuric ... / Mestre

Orientador: Crispin Humberto Garcia Cruz / Banca: Vanildo Luiz Del Bianchi / Banca: Mauricio Boscolo / Banca: Sandra Regina Ceccato Antonini / Banca: Harvey Alexander Villa Vélez / Resumo: As crises no fornecimento de petróleo, a possibilidade de sua escassez, instabilidade dos preços e, principalmente, os efeitos negativos ao meio ambiente, aumentaram, nos últimos anos, o interesse pela procura de fontes alternativas para produção de energia. A possibilidade de produzir etanol a partir de resíduos lignocelulósicos é um grande atrativo para os pesquisadores, pois reduziria a necessidade em aumentar as áreas plantadas para incremento da produção, a competição direta com a produção de alimentos e o custo de matéria-prima. Diante do exposto, o objetivo deste trabalho foi estudar a produção de etanol por culturas individuais e co-culturas de Zymomonas mobilis e Pachysolen tannophilus usando cascas de banana, maracujá e coco verde hidrolisadas. O pré-tratamento ácido, seguido de hidrólise enzimática das cascas de banana, maracujá e coco, promoveram a liberação de 74,1, 87,2 e 13,4 g L-1 de açúcares totais, respectivamente. Foram avaliadas três concentrações de substrato (5, 10 e 15%) e três pH iniciais dos meios de fermentação (4,5; 5,0 e 5,5). Nas fermentações com a bactéria Z. mobilis, com os três resíduos, foi verificado que o substrato mais concentrado não contribuiu para um aumento significativo da produção de etanol. Além disso, a maior concentração do produto foi de 2,9 g L-1, obtido nas fermentações de cascas de banana hidrolisadas com concentração de 5%. Já nas fermentações com a levedura P. tannophilus, as maiores produções de etanol foram determinadas... / Abstract: Crises in oil supply, the possibility of their scarcity, price instability and, especially, the negative effects to the environment have increased the interest in alternative sources for energy production, in recent years. The possibility of producing ethanol from lignocellulosic residues is a great attractive to the researchers, because it would reduce the need for increasing the planted areas to increase production, the direct competition with food production and the high costs of raw materials. In this context, the objective of this work was to study the ethanol production by individual cultures and co-cultures of Zymomonas mobilis and Pachysolen tannophilus using banana, passion fruit and coconut peels hydrolysate. The acid pretreatment, followed by enzymatic hydrolysis of banana, passion fruit and coconut peels promoted the liberation of 74.1, 87.2 and 13.4 g L-1 of total sugars, respectively. Three substrate concentrations (5, 10 and 15%) and three initial pH of fermentation media (4.5; 5.0 and 5.5) were evaluated. In the fermentations with Z. mobilis bacteria, using the three residues, it was found that the most concentrated substrate did not contribute to a significant increase in ethanol production. In addition, the highest product concentration was 2.9 g L-1, obtained in hydrolysed banana peels fermentations at 5% concentration. In the fermentations with yeast P. tannophilus, the highest ethanol yields were determined using passion fruit peel hydrolysates at 10% solids concentration, providing the ethanol production of 10 g L-1 . And the association of Z. mobilis bacteria and P. tannophilus yeast, in the co-culture process, showed potential for the lignocellulosic hydrolysates fermentation. The inoculation system that showed the best results was the one in which the bacterium was inoculated at the beginning of the process, and after 7 h of fermentation, the yeast ... / Doutor

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-03Bitstream added on 2014-06-13T20:21:44Z : No. of bitstreams: 1 ernandes_fmpg_dr_sjrp.pdf: 1963066 bytes, checksum: 42dcae2ecfe9dfb358788c6490a13653 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O principal produto da fermentação de açúcares por Zymomonas mobilis é o etanol quando glicose e frutose são utilizadas como fontes de carbono. Entretanto, quando sacarose é empregada na fermentação, o rendimento do etanol diminui devido à formação de subprodutos como levana, sorbitol, acetaldeído, ácido acético, pequenas quantidades de alguns álcoois superiores e fenol. A utilização de produtos agroindustriais, como o caldo e melaço de cana-deaçúcar, é uma alternativa para reduzir o custo final dos produtos de fermentação devido à disponibilidade de aquisição e composição química desses substratos. Este trabalho teve como objetivo utilizar substratos alternativos e otimizar as condições de fermentação para a produção de etanol, levana e sorbitol por Zymomonas mobilis CCT 4494. Também foi considerado o efeito da variação do substrato e de sais minerais adicionados nos meios de produção (sintético, caldo e melaços de cana-de-açúcar). Para a obtenção dos produtos de fermentação, foi aplicada a metodologia de superfície de resposta, seguindo um planejamento fatorial do tipo 27-2, de acordo com o modelo proposto por Box e Hunter, onde as variáveis independentes estudadas foram: pH inicial do meio de cultivo, temperatura de incubação, concentração do substrato e efeito da adição de KCl, K2SO4, MgSO4, CaCl2. Durante a realização das fermentações foi observado que a bactéria Zymomonas mobilis CCT 4494 se adaptou nos meios de fermentação contendo altas concentrações de sacarose e suportou a variação do pH e da temperatura de fermentação. O aumento da concentração da fonte de carbono favoreceu a formação dos produtos levana e etanol, entretanto, não houve produção de sorbitol. O meio sintético proporcionou maior rendimento de levana e etanol, enquanto que, os meios alternativos caldo e melaços de cana-de-açúcar... / The main product from fermentation of sugars by Zymomonas mobilis is ethanol when glucose and fructose are used as carbon sources. However, when sucrose is used in the fermentation medium, ethanol yield decreases due to the formation of by-products such as levan, sorbitol, acetaldehyde, acetic acid, small amounts of some superior alcohols and phenol. The use of agro industrial by products, such as sugarcane juice and molasses, is an alternative to reduce the final cost of fermentation products due to the constant availability and to the chemical composition of these by substrates. This study had the aim of using alternative substrates and of optimizing fermentation conditions for the production of ethanol, levan and sorbitol by Zymomonas mobilis CCT 4494. The effect of variation of substrate and mineral salts added to the production media (synthetic, sugarcane juice and molasses) was also considered. To obtain the fermentation products, response surface methodology was employed, following a 27-2 factorial planning, according to the model proposed by Box and Hunter, where the independent variables studied were: initial medium pH, incubation temperature, substrate concentration and effect of the addition of KCl, K2SO4, MgSO4, CaCl2. During the fermentations, it was noted that the bacteria Zymomonas mobilis CCT 4494 well adapted in the media containing high concentrations of sucrose and tolerated pH and temperature variations. The increase of carbon source concentration favored the formation of levan and ethanol, however, there was no sorbitol production. The synthetic medium offered higher levan and ethanol yield, whereas alternative media sugarcane juice and molasses, favored cellular growth. Among the independent variables analyzed with the best medium (synthetic) for biosynthesis of the biopolymer and ethanol, the ones that significantly (p<0.05) affected were KCl, K2SO4, CaCl2... (Complete abstract click electronic access below)

Supercritical fluids have unique properties which may make them ideal as reaction media for biotransformation or extractive solvents. Supercritical fluids are ideal for reducing diffusivity limitations over conventional fluids. Depending on the polarity of the fluid, a supercritical fluid can be similar to conventional organic solvents, but with few of the environmental drawbacks. The use of supercritical fluids in enzymatic research has the advantage of removing mass transport limitations so that they can act as a suitable solvent. In this study, four permeabilization techniques were compared: control, toluene, supercritical carbon dioxide, and freeze/thaw cycles. The model cell systems studied were Z. mobilis and E. coli. The cells were analyzed for lipid profiles, recovery of proteins and enzymatic activity. The use of supercritical carbon dioxide may not be the most effective of the treatments based on total protein or enzyme recovery since the greatest protein and enzyme recovery was with the freeze/thaw treatment. However, it can be selective in removing cofactors from Z. mobilis enabling sorbitol production and minimizing side reactions. In this application, supercritical carbon dioxide does show an advantage over the freeze/thaw treatment. Aspects of the mechanism of permeabilization were investigated based on the lipid profiles of the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM and AFM show changes of the cell surface morphology which indicate that the treatments affect the cellular surface. The use of supercritical carbon dioxide as a reaction medium was investigated. Minute quantities of sorbitol were produced when Z. mobilis and sugars were placed in a supercritical carbon dioxide environment over a period of 24 hours.

This study proposes simulations which present optimized methods for producing fatty acids, fatty alcohols and alkanes using Zymomonas mobilis bacterium by the energy efficient β-oxidation reversal pathway, an eco-friendly alternative to the present petroleum based processes. Zymomonas has advantages of higher carbon intake, higher ethanol tolerance and higher ethanol production efficiency than other organisms. I have improved an earlier Zymomonas genome scale model and used Constraint Based Reconstruction and Analysis (COBRA), a linear optimization based computational tool in Matlab, and to perform flux balance analysis (FBA) based simulations. FBA accounts for formation, consumption, accumulation and removal rate or flux of each metabolite. The results present solution spaces of cell growth rate and product formation rate, which trend with products and their carbon chain length. I have analyzed these solution space trends gaining insight into the Zymomonas’ metabolism, enabling efficient product formation and opening a way for future improvement.

Supercritical fluids have unique properties which may make them ideal as reaction media for biotransformation or extractive solvents. Supercritical fluids are ideal for reducing diffusivity limitations over conventional fluids. Depending on the polarity of the fluid, a supercritical fluid can be similar to conventional organic solvents, but with few of the environmental drawbacks. The use of supercritical fluids in enzymatic research has the advantage of removing mass transport limitations so that they can act as a suitable solvent. In this study, four permeabilization techniques were compared: control, toluene, supercritical carbon dioxide, and freeze/thaw cycles. The model cell systems studied were Z. mobilis and E. coli. The cells were analyzed for lipid profiles, recovery of proteins and enzymatic activity. The use of supercritical carbon dioxide may not be the most effective of the treatments based on total protein or enzyme recovery since the greatest protein and enzyme recovery was with the freeze/thaw treatment. However, it can be selective in removing cofactors from Z. mobilis enabling sorbitol production and minimizing side reactions. In this application, supercritical carbon dioxide does show an advantage over the freeze/thaw treatment. Aspects of the mechanism of permeabilization were investigated based on the lipid profiles of the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM and AFM show changes of the cell surface morphology which indicate that the treatments affect the cellular surface. The use of supercritical carbon dioxide as a reaction medium was investigated. Minute quantities of sorbitol were produced when Z. mobilis and sugars were placed in a supercritical carbon dioxide environment over a period of 24 hours.