Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2015. / Made available in DSpace on 2016-10-19T12:48:21Z (GMT). No. of bitstreams: 1 338958.pdf: 3638990 bytes, checksum: 23eca8bb60257059ff1c08d56f7f4c1e (MD5) Previous issue date: 2015 / A fermentação ABE (acetona-butanol-etanol) da Clostridium acetobutyli-cum ATCC 824, muito usada industrialmente até 1940, é um dos mais importantes bioprocessos industriais. Recentemente, o interesse nesse processo foi retomado devido à necessidade de substituir fontes combus-tíveis fósseis por vetores energéticos renováveis, principalmente biohi-drogênio e biobutanol. O objetivo desta tese é contribuir para uma melhor compreensão dos problemas associados com a detecção precoce e moni-toramento das mudanças fisiológicas da C. acetobutylicum ATCC 824 em fermentações tipo batelada. Pretende-se também refinar o entendimento desse comportamento variável utilizando a informação genômica dispo-nível desta bactéria e assim contribuir para mostrar como algumas ferra-mentas da Engenharia Genômica podem incorporar novas informações aos métodos tradicionais de monitoramento de bioprocessos. É proposta a incorporação em três diferentes níveis, a saber: (i) nível genômico, (ii) nível metabólico e (iii) nível fenotípico. No nível genômico, a presente pesquisa detectou que existem seis regiões de alta similaridade, nas quais foram identificados 15 genes já mapeados. O gene rpoB foi identificado como um forte candidato a marcador molecular de variabilidade fenotípi-ca. Foi proposta uma relação entre a regulação da transcrição do gene rpoB e o pH do meio de cultura. No nível metabólico, dois modelos meta-bólicos foram desenvolvidos, um modelo simplificado de 20 reações e um modelo genômico completo de 502 reações. Foi detectado que o modelo simplificado era mais adequado para detectar as mudanças fenotípicas da bactéria. Foi criado um modelo simplificado protonado que integra infor-mações do pH do meio de cultura. No nível fenotípico, a técnica de colora-ção de Gram mostrou-se eficiente na detecção precoce das mudanças fisiológicas da C. acetobutylicum. Foi demonstrada uma correlação entre os dados de fermentação e mudanças fisiológicas da parede celular da bactéria. Sugere-se, portanto, um novo método de detecção precoce e monitoramento do estado fisiológico da C. acetobutylicum ATCC 824 (e outras espécies de Clostridium) utilizando técnicas de coloração de Gram para determinar o estado fisiológico no qual a bactéria se encontra, e quais os principais produtos metabólicos da sua fermentação.<br> / Abstract : The problem of renewable and sustainable energy is challenging and requires innovative approaches. The production of biohydrogen, and biofuels in general, particularly biobutanol in the so-called ABE fermenta-tion by Clostridium acetobutylicum, discovered in 1861 by Louis Pasteur, although extensively studied, still lacks some fundamental understanding to facilitate monitoring changes in the physiological state and optimiza-tion. Here we propose a genomic engineering approach, divided in three levels: i) genomic, ii) metabolic, iii) phenotypic. At the genomic level, a comparative whole-genome study was performed comparing C. acetobu-tylicum with six other chosen microorganisms and a in silico Gram type experiment; The whole-genome analysis detected one major gene, the rpoB gene, implied in acquired antibiotic resistance, and possibly impli-cated in phenotypic variation during ABE fermentation. A possible link between the pH regulation and rpoB expression is suggested. The 16S rRNA analysis consistently showed Gram-positive classification for the C. acetobutylicum strain used, incapable of detecting the existent phenotypic variations.at the metabolic level, two metabolic network models were developed: a simplified, core model, with 20 reactions, and a genome-wide model, comprising 502 reactions; to translate the molecular and biochemical information to a useful engineering level, a phenotypic rela-tion between the Gram stain response and pH developed during batch fermentation was analyzed. The simplified core metabolic network suc-cessfully captured the observed strain metabolic profile. Additional regu-latory mechanism was added to the simplified metabolic model with the integration of pH regulation. Using the Gram staining method, we demon-strated a strong correlation between bacterial growth and morphological changes of the cell wall that is useful in monitoring early detection of the metabolic stages of C. acetobutylicum ABE fermentation. The Gram stain-ing technique is proposed as an efficient, low cost method to identify the physiological state of the bacteria culture and its connection with the fermentation state.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-04T13:16:48Z No. of bitstreams: 2 Dissertação Carlos Eduardo de Oliveira Lacerda .pdf: 1440207 bytes, checksum: 45ba5069ce1d39e72db4b57677b1c684 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-04T13:16:48Z (GMT). No. of bitstreams: 2 Dissertação Carlos Eduardo de Oliveira Lacerda .pdf: 1440207 bytes, checksum: 45ba5069ce1d39e72db4b57677b1c684 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / REUNI; CAPES / A glicerina bruta, subproduto da produção de biodiesel, vem sendo investigada como fonte de carbono em processos microbianos para a obtenção de bioprodutos de alto valor agregado. A utilização da glicerina na produção de 1,3-propanodiol por via microbiológica é uma forma bastante viável de utilização dessa substância. O processo microbiano ocorre em anaerobiose, onde bactérias (Klebsiella, Citrobacter, Enterobacter e Clostridium) convertem a glicerina bruta em 1,3-propanodiol e vários outros produtos, como: ácido acético, etanol, ácido succínico, ácido lático, ácido cítrico, ácido fórmico, CO2 e H2, de acordo com as vias metabólicas do seu processo fermentativo. Dentre os micro-organismos estudados, no entanto, destacam-se as bactérias Clostridium butyricum e Klebsiella pneumoniae como as de maior utilização e provavelmente as melhores produtoras deste composto, devido a sua tolerância ao substrato, seu rendimento e produtividade. O presente trabalho consistiu em avaliar o comportamento do Clostridium acetobutylicum ATCC 4259 através de fermentações em batelada utilizando glicerol PA como fonte de carbono. A fermentação foi realizada em meio de cultura próprio para obtenção de 1,3-propanodiol, importante monômero utilizado na síntese de polímeros. Foi realizado um planejamento fatorial 2² em estrela com ponto central para avaliar a influência de duas variáveis (glicerol PA e extrato de levedura) no crescimento do micro-organismo. Foram realizados 10 ensaios no planejamento, sendo 9 ensaios em pontos distintos e uma repetição no ponto central. Também foi realizado um experimento de cinética microbiana para estudar em detalhes o crescimento do micro-organismo. Em ambos os casos a técnica de filtração com peso seco foi utilizada para calcular a concentração celular presente no meio. A cinética microbiana mostrou que, para as condições experimentais da pesquisa, a fase exponencial de crescimento ocorreu até o tempo de 14 horas, e uma produtividade celular (P) de 0,0405 g/L.h, uma velocidade máxima de crescimento (μmáx) de 0,0403 h-1 e um tempo de geração (G) de 15,7 h foram alcançados. As melhores respostas obtidas no planejamento fatorial foram 1,42 g/L e 1,40 g/L (30 g/L e glicerol e 2 g/L de extrato de levedura no meio de cultura).

An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.

O H2 pode ser obtido por processos biológicos, como a fermentação, conduzidos à temperatura e pressão ambientes. Para tal são utilizadas matérias-primas renováveis, ricas em carboidratos, como as biomassas lignocelulósicas e de algas. Estas biomassas têm estrutura química complexa e requerem uma etapa de pré-tratamento e/ou hidrólise antes da sua utilização na fermentação. Processos de hidrólise podem liberar, tanto monossacarídeos, quanto substâncias potencialmente inibidoras de fermentação. Esse estudo avaliou o efeito de 3 potenciais inibidores de fermentação (5-hidroximetilfurfural -HMF, ácido levulínico AL e ácido fórmico AF), derivados da hidrólise de biomassas. Ensaios cinéticos de fermentação em batelada foram realizados com o microrganismo produtor de H2, Clostridium beijerinckii Br21, utilizando glicose como fonte de carbono e diferentes concentrações de cada inibidor. O efeito do HMF, do AL e do AF foram avaliados nas faixas de concentração de 0,5 a 2,5 g/L, de 1,0 a 4,0 g/L, e de 0,5 a 2,0 g/L, respectivamente. Retiraram-se amostras do gás produzido e do líquido para estimar as velocidades específicas de produção de H2, do crescimento celular e de consumo de glicose, nos ensaios com e sem a presença de inibidor (controle). Foi observada inibição de todos os parâmetros avaliados, comparados ao controle. Houve um aumento do tempo para início da produção e diminuição do rendimento de H2 com o aumento da concentração de todos os inibidores. Os resultados das fermentações permitiram estimar a concentração dos compostos que inibem 50% a produção de H2, o crescimento celular e o consumo do substrato (CI50). Os valores de CI50 obtidos para a produção de H2 pelo HMF, AL e AF foram 0,89, 2,50 e 1,15 g/L, respectivamente. Para o crescimento celular a CI50 do HMF, AL e AF foram 1,42, 2,08 e 1,46 g/L, respectivamente. Para o consumo de substrato a CI50 foi 3,23, 3,79 e 0,43 g/L, para o HMF, AL e AF, respectivamente. As concentrações de CI50 para a produção de H2 foram testados em 2 microrganismos distintos, o C. beijerinckii Br21 e o Clostridium acetobutylicum ATCC 824, para fins comparativos. Assim pode-se verificar a inibição na produção de H2 no C. beijerinckii Br21 de 49,3, 48,7 e 51,3%, enquanto que o C. acetobutylicum ATCC 824 apresentou inibição de 45,5, 61,3 e 59,6%, para o HMF, AL e AF, respectivamente. Foi estimada também a concentração de compostos que inibem 25% a produção de H2, a CI25, a fim de realizar misturas com os inibidores e testá-las em ambos os microrganismos. Os valores obtidos de CI25 para HMF, AL e AF foram 0,66, 2,15 e 0,89 g/L, respectivamente. A partir desses valores foram feitas 4 misturas distintas: HMF+AL, HMF+AF, AL+AF e HMF+AL+AF. A inibição da produção de H2 a partir dessas misturas em C. beijerinckii Br21foram de 58,9, 58,4, 49 e 85,9%, enquanto que para o C. acetobutylicum ATCC 824 obteve-se os valores de 67,6, 66,6, 55,5 e 88,8%, para HMF+AL, HMF+AF, AL+AF e HMF+AL+AF, respectivamente. Portanto, pode-se notar que o C. acetobutylicum ATCC 824 mostrou ser mais sensível aos efeitos causados pelos inibidores, sendo que o HMF parece atuar mais sobre a produção de H2, enquanto que os ácidos têm efeito mais global no metabolismo da bactéria. Esses estudos mostraram os limites destes compostos, quando se deseja utilizar hidrolisados de biomassas como matéria-prima para a produção fermentativa do H2.pelas espécies de Clostridium estudadas. / H2 can be obtained by biological processes, such as fermentation, conducted at ambient temperature and pressure. Renewable raw materials like lignocellulosic and algae biomass, which are rich in carbohydrates, can be used for this purpose. These types of biomass have complex chemical structures and require a pretreatment and/or hydrolysis step before they are used in fermentation. Hydrolysis may release not only monosaccharides but also potentially fermentation-inhibiting substances. This study evaluates how three potential fermentation inhibitors (5-hydroxymethylfurfural (HMF), levulinic acid-(LA), and formic acid (FA) derived from algal biomass hydrolysis affect H2 production. Kinetic batch fermentation assays were performed by using the H2-producing microorganism Clostridium beijerinckii Br21, glucose as carbon source, and different concentrations of each inhibitor. The effect of HMF, LA, and FA on H2 production was evaluated for inhibitor concentrations ranging from 0.5 to 2.5 g/L, 1.0 to 4.0 g/L, and 0.5 to 2.0 g/L, respectively. Samples of the produced gas and liquid were taken to estimate the specific rates of H2 production, cell growth, and glucose consumption in the assays conducted in the presence or in the absence (control) of an inhibitor. Increasing inhibitor concentration delayed the onset of H2 production and diminished the H2 yield. The fermentation results allowed us to estimate the inhibitor concentration that inhibited 50% of the H2 production, cell growth, and substrate consumption rates, designated IC50. Concerning the H2 production rate, IC50 was 0.89, 2.50, and 1.15 g/L for HMF, LA, and FA, respectively. As for the cell growth rate, IC50 was 1.42, 2.08, and 1.46 g/L for HMF, LA, and FA, respectively. Regarding the substrate consumption rate, IC50 was 3.23, 3.79, and 0.43 g/L for HMF, LA, and FA, respectively. IC50 was also tested in the presence of C. beijerinckii Br21 or Clostridium acetobutylicum ATCC 824 and one of the inhibitors. The H2 production rate decreased by 49.3, 48.7, and 51.3% in the presence of C. beijerinckii Br21 and of HMF, AL, or AF, respectively. In the presence of C. acetobutylicum ATCC 824 and of HMF, AL, or AF, the H2 production rate reduced by 45.5, 61.3, and 59.6%, respectively. The inhibitor concentration that inhibited 25% of H2 production, IC25, was also determined so that mixtures of the inhibitors could be prepared and tested in the presence of the microorganisms. HMF, LA, and FA afforded IC25 of 0.66, 2.15, and 0.89 g/L, respectively. On the basis of these values, four different mixtures were prepared: HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA. In the presence of C. beijerinckii Br21, HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA inhibited H2 production by 58.9, 58.4, 49, and 85.9%, respectively, whereas in the presence of C. acetobutylicum ATCC 824, inhibitions were 67.6, 66.6, 55.5, and 88.8% respectively. Therefore, C. acetobutylicum ATCC 824 was more sensitive to the effects caused by inhibitors. HMF seemed to affect the H2 production rate more, whereas acids appeared to act more globally on bacterial metabolism. These results reveal the concentration limits of the tested inhibitors when biomass hydrolysates are employed as raw material for fermentative H2 production.

O H2 pode ser obtido por processos biológicos, como a fermentação, conduzidos à temperatura e pressão ambientes. Para tal são utilizadas matérias-primas renováveis, ricas em carboidratos, como as biomassas lignocelulósicas e de algas. Estas biomassas têm estrutura química complexa e requerem uma etapa de pré-tratamento e/ou hidrólise antes da sua utilização na fermentação. Processos de hidrólise podem liberar, tanto monossacarídeos, quanto substâncias potencialmente inibidoras de fermentação. Esse estudo avaliou o efeito de 3 potenciais inibidores de fermentação (5-hidroximetilfurfural -HMF, ácido levulínico AL e ácido fórmico AF), derivados da hidrólise de biomassas. Ensaios cinéticos de fermentação em batelada foram realizados com o microrganismo produtor de H2, Clostridium beijerinckii Br21, utilizando glicose como fonte de carbono e diferentes concentrações de cada inibidor. O efeito do HMF, do AL e do AF foram avaliados nas faixas de concentração de 0,5 a 2,5 g/L, de 1,0 a 4,0 g/L, e de 0,5 a 2,0 g/L, respectivamente. Retiraram-se amostras do gás produzido e do líquido para estimar as velocidades específicas de produção de H2, do crescimento celular e de consumo de glicose, nos ensaios com e sem a presença de inibidor (controle). Foi observada inibição de todos os parâmetros avaliados, comparados ao controle. Houve um aumento do tempo para início da produção e diminuição do rendimento de H2 com o aumento da concentração de todos os inibidores. Os resultados das fermentações permitiram estimar a concentração dos compostos que inibem 50% a produção de H2, o crescimento celular e o consumo do substrato (CI50). Os valores de CI50 obtidos para a produção de H2 pelo HMF, AL e AF foram 0,89, 2,50 e 1,15 g/L, respectivamente. Para o crescimento celular a CI50 do HMF, AL e AF foram 1,42, 2,08 e 1,46 g/L, respectivamente. Para o consumo de substrato a CI50 foi 3,23, 3,79 e 0,43 g/L, para o HMF, AL e AF, respectivamente. As concentrações de CI50 para a produção de H2 foram testados em 2 microrganismos distintos, o C. beijerinckii Br21 e o Clostridium acetobutylicum ATCC 824, para fins comparativos. Assim pode-se verificar a inibição na produção de H2 no C. beijerinckii Br21 de 49,3, 48,7 e 51,3%, enquanto que o C. acetobutylicum ATCC 824 apresentou inibição de 45,5, 61,3 e 59,6%, para o HMF, AL e AF, respectivamente. Foi estimada também a concentração de compostos que inibem 25% a produção de H2, a CI25, a fim de realizar misturas com os inibidores e testá-las em ambos os microrganismos. Os valores obtidos de CI25 para HMF, AL e AF foram 0,66, 2,15 e 0,89 g/L, respectivamente. A partir desses valores foram feitas 4 misturas distintas: HMF+AL, HMF+AF, AL+AF e HMF+AL+AF. A inibição da produção de H2 a partir dessas misturas em C. beijerinckii Br21foram de 58,9, 58,4, 49 e 85,9%, enquanto que para o C. acetobutylicum ATCC 824 obteve-se os valores de 67,6, 66,6, 55,5 e 88,8%, para HMF+AL, HMF+AF, AL+AF e HMF+AL+AF, respectivamente. Portanto, pode-se notar que o C. acetobutylicum ATCC 824 mostrou ser mais sensível aos efeitos causados pelos inibidores, sendo que o HMF parece atuar mais sobre a produção de H2, enquanto que os ácidos têm efeito mais global no metabolismo da bactéria. Esses estudos mostraram os limites destes compostos, quando se deseja utilizar hidrolisados de biomassas como matéria-prima para a produção fermentativa do H2.pelas espécies de Clostridium estudadas. / H2 can be obtained by biological processes, such as fermentation, conducted at ambient temperature and pressure. Renewable raw materials like lignocellulosic and algae biomass, which are rich in carbohydrates, can be used for this purpose. These types of biomass have complex chemical structures and require a pretreatment and/or hydrolysis step before they are used in fermentation. Hydrolysis may release not only monosaccharides but also potentially fermentation-inhibiting substances. This study evaluates how three potential fermentation inhibitors (5-hydroxymethylfurfural (HMF), levulinic acid-(LA), and formic acid (FA) derived from algal biomass hydrolysis affect H2 production. Kinetic batch fermentation assays were performed by using the H2-producing microorganism Clostridium beijerinckii Br21, glucose as carbon source, and different concentrations of each inhibitor. The effect of HMF, LA, and FA on H2 production was evaluated for inhibitor concentrations ranging from 0.5 to 2.5 g/L, 1.0 to 4.0 g/L, and 0.5 to 2.0 g/L, respectively. Samples of the produced gas and liquid were taken to estimate the specific rates of H2 production, cell growth, and glucose consumption in the assays conducted in the presence or in the absence (control) of an inhibitor. Increasing inhibitor concentration delayed the onset of H2 production and diminished the H2 yield. The fermentation results allowed us to estimate the inhibitor concentration that inhibited 50% of the H2 production, cell growth, and substrate consumption rates, designated IC50. Concerning the H2 production rate, IC50 was 0.89, 2.50, and 1.15 g/L for HMF, LA, and FA, respectively. As for the cell growth rate, IC50 was 1.42, 2.08, and 1.46 g/L for HMF, LA, and FA, respectively. Regarding the substrate consumption rate, IC50 was 3.23, 3.79, and 0.43 g/L for HMF, LA, and FA, respectively. IC50 was also tested in the presence of C. beijerinckii Br21 or Clostridium acetobutylicum ATCC 824 and one of the inhibitors. The H2 production rate decreased by 49.3, 48.7, and 51.3% in the presence of C. beijerinckii Br21 and of HMF, AL, or AF, respectively. In the presence of C. acetobutylicum ATCC 824 and of HMF, AL, or AF, the H2 production rate reduced by 45.5, 61.3, and 59.6%, respectively. The inhibitor concentration that inhibited 25% of H2 production, IC25, was also determined so that mixtures of the inhibitors could be prepared and tested in the presence of the microorganisms. HMF, LA, and FA afforded IC25 of 0.66, 2.15, and 0.89 g/L, respectively. On the basis of these values, four different mixtures were prepared: HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA. In the presence of C. beijerinckii Br21, HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA inhibited H2 production by 58.9, 58.4, 49, and 85.9%, respectively, whereas in the presence of C. acetobutylicum ATCC 824, inhibitions were 67.6, 66.6, 55.5, and 88.8% respectively. Therefore, C. acetobutylicum ATCC 824 was more sensitive to the effects caused by inhibitors. HMF seemed to affect the H2 production rate more, whereas acids appeared to act more globally on bacterial metabolism. These results reveal the concentration limits of the tested inhibitors when biomass hydrolysates are employed as raw material for fermentative H2 production.

Source d'énergie renouvelable et non polluante, le biohydrogène connaît un intérêt croissant. En culture continue sur glucose, la productivité d'hydrogène la plus élevée est réalisée par la bactérie anaérobie Clostridium acetobutylicum avec 2,4 litres d'hydrogène produit l-1 h-1. Cette production d'hydrogène est catalysée par la [FeFe]-hydrogénase HydA. Pour comprendre et améliorer les capacités de ce micro-organisme pour la production d'hydrogène, nous avons voulu caractériser les relations structure-fonctions de HydA avec ses partenaires d'oxydo-réduction. Par homologie avec l'hydrogénase à fer I de Clostridium pasteurianum, les centres [4Fe-4S] FS4C et [2Fe-2S] FS2, situés en surface de la protéine, pourraient être impliqués dans le transfert inter-moléculaire d'électrons entre HydA et ses partenaires d'oxydoréduction. Notre objectif a alors été de déterminer l'implication de FS4C et FS2 dans ce transfert. Pour cela, des mutations par substitutions d'acides aminés et par délétions de domaines ont été effectuées au niveau de FS4C et FS2. Pour palier des problèmes d'instabilité des hydrogénases à fer native et modifiées, le protocole de purification a été amélioré. L'hydrogénase native a nettement été stabilisée, mais l'instabilité persistante des hydrogénases modifiées est restée une limitation importante pour leur caractérisation catalytique. La perte d'activité des hydrogénases modifiées a pu être corrélée à une perte importante de fonctionnalité de leur site actif et à un nombre d'atomes de fer incorporés inférieur à la valeur théorique. Les partenaires physiologiques d'oxydo-réduction de HydA chez C. acetobutylicum, la ferrédoxine CAC0303 et la flavodoxine, ont été purifiés. Le profil catalytique complet et les paramètres cinétiques des activités de consommation et de production d'hydrogène de HydA native avec différents partenaires d'oxydo-réduction ont été déterminés. L'amélioration du protocole de purification a permis d'augmenter significativement les activités de consommation et production d'hydrogène. Nous avons confirmé la préférence in vitro de HydA à catalyser la consommation de l'hydrogène par rapport à la production. Une valeur très élevée de kcat a été obtenue avec le substrat artificiel, méthyl viologène, en consommation d'hydrogène. Cela semble indiquer que le méthyl viologène pourrait interagir plus ou moins directement avec le site actif de l'enzyme, en évitant le transfert intramoléculaire d'électrons. Des efficacités catalytiques élevées de consommation et de production de l'hydrogène ont été obtenues avec le méthyl viologène (sauf sous sa forme réduite), la ferrédoxine et la flavodoxine. Ce résultat reflète le haut potentiel de HydA pour les réactions liées à l'hydrogène, potentiel conservé aussi bien pour ses partenaires redox physiologiques qu'artificiel. Ainsi, en condition de croissance en carence en fer, la substitution de la ferrédoxine par la flavodoxine ne serait pas une limitation pour l'activité hydrogénase in vivo.

Application of bioengineering technologies for enhanced biological hydrogen production is a promising approach that may play a vital role in sustainable energy. Due to the ability of several naturally occurring microorganisms to generate hydrogen through varying metabolic processes, biological hydrogen has become an attractive alternative energy and fuel source. One area of particular interest is the production of biological hydrogen in organically-rich engineered systems, such as those associated with waste treatment. Despite the potential for high energy yields, hydrogen yields generated by bacteria in waste systems are often limited due to a focus on microbial utilization of organic material towards cellular growth rather than production of biogas. To address this concern and to improve upon current technological applications, metabolic engineering approaches may be applied to known hydrogen producing organisms. However, to successfully modify metabolic pathways, full understanding of metabolic networks involved in expression of microbial traits in hydrogen producing organisms is necessary. Because microbial communities associated with hydrogen production are capable of exhibiting a number of phenotypes, attempts to apply metabolic engineering concepts have been restricted due to limited information regarding complex metabolic processes and regulatory networks involved in expression of microbial traits associated with biohydrogen production. To bridge this gap, this dissertation focuses on identification of phenotype-related biochemical processes within sets of phenotype-expressing organisms. Specifically, through co-development and application of evolutionary genome-scale phenotype-centric comparative network analysis tools, metabolic and cellular components related to three phenotypes (i.e., dark fermentative, hydrogen production and acid tolerance) were identified. The computational tools employed for the systematic elucidation of key phenotype-related genes and subsystems consisted of two complementary methods. The first method, the Network Instance-Based Biased Subgraph Search (NIBBS) algorithm, identified phenotype-related metabolic genes and subsystems through comparative analysis of multiple genome-scale metabolic networks. The second method was the multiple alignments of metabolic pathways for identification of conserved metabolic sub-systems in small sets of phenotype-expressing microorganisms. For both methodologies, key metabolic genes and sub-systems that are likely to be related to hydrogen production and acid-tolerance were identified and hypotheses regarding their role in phenotype expression were generated. In addition, analysis of hydrogen producing enzymes generated by NIBBS revealed the potential interplay, or cross-talk, between metabolic pathways. To identify phenotype-related subnetworks, three complementary approaches were applied to individual, and sets of phenotype-expressing microorganisms. In the first method, the Dense ENriched Subgraph Enumeration (DENSE) algorithm, partial "prior knowledge" about the proteins involved in phenotype-related processes are utilized to identify dense, enriched sets of known phenotype-related proteins in Clostridium acetobutylicum. The second approach utilized a bi-clustering algorithm to identify phenotype-related functional association modules associated with metabolic controls of phenotype-related pathways. Last, through comparison of hundreds of genome-scale networks of functionally associated proteins, the á, â-motifs approach, was applied to identify phenotype-related subsystems. Application of methodologies for identification of subnetworks resulted in detection of regulatory proteins, transporters, and signaling proteins predicted to be related to phenotype-expression. Through analysis of protein interactions, clues to the functional roles and associations of previously uncharacterized proteins were identified (DENSE) and hypotheses regarding potentially important acid-tolerant mechanisms were generated (á, â-motifs). Similar to the NIBBS algorithm, analysis of functional modules predicted by the bi-clustering algorithm suggest cross-talk is occurring between pathways associated with hydrogen production. The ability of these phenotype-centric comparative network analysis tools to identify both known and potentially new biochemical process is important for providing further understanding and insights into metabolic networks and system controls involved in the expression of microbial traits. In particular, identification of phenotype-related metabolic components through a systems approach provides the underlying foundation for the development of improved bioengineering technologies and experimental design for enhanced biological hydrogen production.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2015. / Made available in DSpace on 2016-05-24T17:27:10Z (GMT). No. of bitstreams: 1 338958.pdf: 3638990 bytes, checksum: 23eca8bb60257059ff1c08d56f7f4c1e (MD5) Previous issue date: 2015 / A fermentação ABE (acetona-butanol-etanol) da Clostridium acetobutyli-cum ATCC 824, muito usada industrialmente até 1940, é um dos mais importantes bioprocessos industriais. Recentemente, o interesse nesse processo foi retomado devido à necessidade de substituir fontes combus-tíveis fósseis por vetores energéticos renováveis, principalmente biohi-drogênio e biobutanol. O objetivo desta tese é contribuir para uma melhor compreensão dos problemas associados com a detecção precoce e moni-toramento das mudanças fisiológicas da C. acetobutylicum ATCC 824 em fermentações tipo batelada. Pretende-se também refinar o entendimento desse comportamento variável utilizando a informação genômica dispo-nível desta bactéria e assim contribuir para mostrar como algumas ferra-mentas da Engenharia Genômica podem incorporar novas informações aos métodos tradicionais de monitoramento de bioprocessos. à proposta a incorporação em três diferentes níveis, a saber: (i) nível genômico, (ii) nível metabólico e (iii) nível fenotípico. No nível genômico, a presente pesquisa detectou que existem seis regiões de alta similaridade, nas quais foram identificados 15 genes já mapeados. O gene rpoB foi identificado como um forte candidato a marcador molecular de variabilidade fenotípi-ca. Foi proposta uma relação entre a regulação da transcrição do gene rpoB e o pH do meio de cultura. No nível metabólico, dois modelos meta-bólicos foram desenvolvidos, um modelo simplificado de 20 reações e um modelo genômico completo de 502 reações. Foi detectado que o modelo simplificado era mais adequado para detectar as mudanças fenotípicas da bactéria. Foi criado um modelo simplificado protonado que integra infor-mações do pH do meio de cultura. No nível fenotípico, a técnica de colora-ção de Gram mostrou-se eficiente na detecção precoce das mudanças fisiológicas da C. acetobutylicum. Foi demonstrada uma correlação entre os dados de fermentação e mudanças fisiológicas da parede celular da bactéria. Sugere-se, portanto, um novo método de detecção precoce e monitoramento do estado fisiológico da C. acetobutylicum ATCC 824 (e outras espécies de Clostridium) utilizando técnicas de coloração de Gram para determinar o estado fisiológico no qual a bactéria se encontra, e quais os principais produtos metabólicos da sua fermentação.<br> / Abstract : The problem of renewable and sustainable energy is challenging and requires innovative approaches. The production of biohydrogen, and biofuels in general, particularly biobutanol in the so-called ABE fermenta-tion by Clostridium acetobutylicum, discovered in 1861 by Louis Pasteur, although extensively studied, still lacks some fundamental understanding to facilitate monitoring changes in the physiological state and optimiza-tion. Here we propose a genomic engineering approach, divided in three levels: i) genomic, ii) metabolic, iii) phenotypic. At the genomic level, a comparative whole-genome study was performed comparing C. acetobu-tylicum with six other chosen microorganisms and a in silico Gram type experiment; The whole-genome analysis detected one major gene, the rpoB gene, implied in acquired antibiotic resistance, and possibly impli-cated in phenotypic variation during ABE fermentation. A possible link between the pH regulation and rpoB expression is suggested. The 16S rRNA analysis consistently showed Gram-positive classification for the C. acetobutylicum strain used, incapable of detecting the existent phenotypic variations.at the metabolic level, two metabolic network models were developed: a simplified, core model, with 20 reactions, and a genome-wide model, comprising 502 reactions; to translate the molecular and biochemical information to a useful engineering level, a phenotypic rela-tion between the Gram stain response and pH developed during batch fermentation was analyzed. The simplified core metabolic network suc-cessfully captured the observed strain metabolic profile. Additional regu-latory mechanism was added to the simplified metabolic model with the integration of pH regulation. Using the Gram staining method, we demon-strated a strong correlation between bacterial growth and morphological changes of the cell wall that is useful in monitoring early detection of the metabolic stages of C. acetobutylicum ABE fermentation. The Gram stain-ing technique is proposed as an efficient, low cost method to identify the physiological state of the bacteria culture and its connection with the fermentation state.

: Chez la bactérie Clostridium acetobutylicum, la production de dihydrogène est catalysée par des hydrogénases, enzymes impliquées dans l’oxydation de la ferrédoxine réduite, qui permet la réduction des protons et la formation du gaz. Toutes les hydrogénases à fer partagent un domaine protéique très conservé (le Domaine H), hébergeant le site catalytique inorganique (le Cluster H). L’enzyme CaHydA de C. acetobutylicum, possède également le Domaine F contenant en tous quatre centres fer-soufre dits accessoires. Au cours de ces travaux, l’implication des centres fer-soufre du Domaine F sur les capacités catalytiques de l’enzyme a été étudiée, ainsi que les mécanismes de transfert d’électrons entre l’enzyme et son partenaire physiologique d’oxydoréduction principal : la ferrédoxine 2[4Fe-4S]. Différents variants ciblés de l’hydrogénase ont été créés, produits puis purifiés afin de les caractériser par une combinaison de méthodes biochimiques, électrochimiques, spectroscopiques et de modélisation moléculaire. Ceci a permis de mettre en évidence pour la première fois l’implication des centres fer-soufre accessoires du Domaine F dans les capacités catalytiques de l’enzyme. Enfin, il a été démontré que le centre [2Fe-2S] de surface FS2 de l’enzyme était le point d’entrée des électrons provenant de la ferrédoxine réduite. / The hydrogenase of Clostridium acetobutylicum catalyses the oxydation of reduced ferredoxin, leading to reduction of protons and dihydrogen formation. Among the three different classes of hydrogenases, the [Fe-Fe] Hydrogenases harbor a very conserved domain (H-Domain) containing the inorganic catalytic site (Cluster H). CaHydA from C. acetobutylicum possesses, in addition, the F-Domain containing four accessory iron-sulfur clusters. The involvement of accessory iron-sulfur cluster of F-Domain on the catalytic capacities of the enzyme has never been assessed. Moreover, which of the two surface iron-sulfur cluster of the F-Domain who interacts with the physiological redox partner ferredoxin is unknown. Different CaHydA mutants enzymes, modified in the Fe-S cluster composition of the F-Domain have been purified, and spectroscopically, biochemically and electrochemically characterized. These mutants enzymes, impaired in their catalytic activity both in solution and wired to an electrode, suggested, for the first time that the Fe-S clusters of the F-Domain have a long-range thermodynamic effect on the H-cluster and modulate enzyme’s functions. Moreover, it has been shown, and confirmed by molecular modelling, that the [2Fe-2S] surface cluster FS2 of the enzyme is the entry point for the electrons coming from the reduced ferredoxin.

Genome-scale modeling and new strategies for constraining these models were applied in this research to find new insights into cellular metabolism and identify potential metabolic engineering strategies. A newly updated genome-scale model for Clostridium acetobutylicum, iMM864, was constructed, largely based on the previously published iRS552 model. The new model was built using a newly developed genome-scale model database, and updates were derived from new insights into clostridial metabolism. Novel methods of proton-balancing and setting flux (defined as reaction rate (mmol/g biomass/hr)) ratio constraints were applied to create simulations made with the iMM864 model approximate observed experimental results. It was determined that the following constraints must be applied to properly model C. acetobutylicum metabolism: (1) proton-balancing, (2) constraining the specific proton flux (SPF), and (3) installing proper flux ratio constraints. Simulations indicate that the metabolic shift into solventogenesis is not due to optimizing growth at different pH conditions. However, they provide evidence that C. acetobutylicum has developed strictly genetically regulated solventogenic metabolic pathways for the purpose of increasing its surrounding pH to decrease the toxic effects of high proton concentrations. Applying a ratio constraint for the P/O ratio (a measure of aerobic respiratory efficiency) to the iAF1260 genome-scale model of E. coli K12 MG1655 was explored. Relationships were found between: (1) the P/O ratio, (2) the SPF, (3) the growth rate, and (4) the production of acetate. As was expected, higher acetate production correlates with lower P/O ratios, while higher growth correlates with higher P/O ratios. For the first time, a genome-scale model was able to quantify this relationship and targeting both the P/O ratio and the SFP is required to produce an E. coli K12 strain with either (i) maximized growth rate (and minimized acetate production) or (ii) maximized acetate production (at the expense of cell growth). A gene knockout mutant, Î ndh, was created with E. coli BL-21 to study the effects of forcibly higher P/O ratios on growth. The results suggest that a metabolic bottleneck lies with the NADH-1 complex, the NADH dehydrogenase that contributes to the generation of a proton motive force. / Master of Science