Rozprawy doktorskie na temat „Microbial metabolism”

Nitrogen fixation (catalyzed by the enzyme nitrogenase), cellular respiration (completed through the Tricarboxylic Acid (TCA) cycle) and mercury detoxification (through mercury methylation) are three metabolic processes used by a wide variety of microorganisms, but that also have far reaching impacts on nutrient cycling in the environment. Roseiflexus castenholzii has been found to have a unique nitrogenase gene cluster encoding several nitrogenase homologs, including the structural proteins NifH and NifDK and the radical SAM protein, NifB, necessary for cofactor biosynthesis. However, the genome of R. castenholzii lacks the suite of nitrogenase accessory proteins necessary for nitrogen fixation. To investigate the metabolic role of these nitrogenase homologs, expression and purification protocols were developed that aid in the biochemical characterization of these proteins. Synechococcus sp. PCC 7002 encodes three novel TCA proteins, contrary to previous studies that indicated these phototrophs have incomplete TCA cycles. Expression, purification and preliminary crystallization trials were completed on the three novel TCA proteins in order to gain insight into the structure of the proteins which will elucidate the mechanism of each novel enzyme and provide evidence into the novel TCA cycle utilized by these cyanobacteria. The third project presented examines the role of microorganisms in metabolizing mercury, producing methylmercury and providing an entry point for methylmercury into the food chain in Yellowstone National Park (YNP). In this project, environmental samples were enriched for a sulfate reducing organism and a culture containing three sulfate reducing bacteria (SRB) has been established. The SRB that are present and active in the enrichment samples are known to reduce sulfate and may be responsible for the presence of methyl mercury in algal mats that bioaccumulates through the food chain in YNP. The enrichment of SRB in this culture will enable the identification and characterization of the organisms that are capable of methylating mercury in hydrothermal systems. Collectively, the results presented herein increase the knowledge base of three metabolic processes used by microorganisms: nitrogen fixation, cellular respiration through the TCA cycle and mercury detoxification; these results will contribute to a broader understanding of how these processes have evolved and their impacts on the environment.

Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed November 14, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Orientador: Maria Helena Andrade Santana
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
Made available in DSpace on 2018-08-14T16:51:23Z (GMT). No. of bitstreams: 1 Pires_AlineMaraBarbosa_D.pdf: 5667585 bytes, checksum: 99be814ed0f4b52809cad017f91235eb (MD5) Previous issue date: 2009
Resumo: Neste trabalho, estudou-se a otimização da produção de ácido hialurônico (HA) por cultivo de Streptococcus zooepidemicus em batelada, com base nas alterações metabólicas ao longo do cultivo. Ás condições ambientais estudadas foram a concentração inicial de glicose, controle do pH, íons minerais e fonte de nitrogênio orgânico. Nos cultivos em frascos, a concentração inicial de glicose não alterou nem o crescimento celular nem a produção de HA. Entretanto, no cultivo em biorreator sem o controle do pH, ambos foram fortemente dependentes da concentração inicial de glicose, com maior produção de HA (1,21 g.L-1) no cultivo realizado em meio com 25 g.L-1 glicose. Tal condição nutricional foi a única que apresentou maior conversão de glicose em HA (YHA/S) do que conversão de glicose em massa celular (YX/S). O controle do pH ao longo do cultivo com 25 g.L-1 glicose resultou em maior produtividade de células (0,21 g.L-1.h-1) e de HA (0,10 g.L-1.h-1). apesar dos menores rendimentos em relação à glicose. A combinação desses resultados relaciona o maior direcionamento da fonte de carbono para HA do que para células a uma resposta do microrganismo ao stress ácido ocorrido no cultivo sem controle do pH. Uma análise da distribuição dos fluxos metabólicos nas condições ambientais estudadas demonstrou que as alterações na via de produção de HA foram mais relacionadas à distribuição dos fluxos para os açúcares precursores da síntese do polímero que à disponibilidade de energia (ATP) ou potencial redutor (NADH/NAD+) das células. A total suplementação do meio de cultura com íons minerais (K+, Mg++, Na+, Fe++, Ca++, Mn++, Zn++ e Cu++) foi benéfica para o crescimento celular, porém não alterou a produção de HA de forma significativa. O estudo demonstrou ainda que a qualidade do polímero produzido pode ser modulada pela suplementação do meio com íons minerais. As propriedades reológicas do HA com baixo teor de proteína (0.44 g.g-1) e massa molar média de 4.0 x 106 Da demonstraram elevada densidade de emaranhamento das cadeias devido à alta dependência do módulo elástico com a concentração e desvios da viscosidade complexa com relação à regra de Cox-Merz. O estudo de meios alternativos contendo derivados agroindustriais demonstrou maiores concentrações de HA em meios contendo extrato de levedura como fonte de nitrogênio. Este conjunto de resultados contribui para a otimização da produção de HA, assim como para um melhor entendimento do metabolismo do Streptococcus zooepidemicus.
Abstract: In this work, h was studied the optimization of HA production by hatch culture of Streptococcus zooepidemicus, with focus on the metabolic changes along cultivation. The environmental conditions studied were the initial glucose concentration, pH control, mineral ions and organic nitrogen source. In flask cultivations, the initial glucose concentration had no influence on the amounts of either the biomass or the MA produced. However, in bioreactor cultivations, at non-controlled pH. both were strongly dependent on the initial glucose concentration. The highest HA concentration (1.21 g.L-1) was obtained from 25 g.L-1 glucose, which was the only cultivation where the conversion of glucose to HA (YHA/S) was higher than the one of glucose to biomass (YX/S). Not only did the pH control along cultivation result in higher cell productivity (0.21 g.L-1.h-1), but also in the HA productivity (0.10 g.L-1.h-1), However, the HA and cell yields from glucose were lower. The combination of these results relates the higher direction of the carbon source to the HA synthesis at the expenses of the cell growth to a microbial response to the acid stress observed in non-controlled pH. An analysis of the metabolic flux distribution in the environmental conditions studied shows that the changes in the HA production pathway were more related to die distributions of duxes 10 the precursors of HA synthesis than to the energy availability (ATP) or redox slate (NADH/NAD+) of the cells. The total supplementation of the culture medium with ions was beneficial to die cell growth. However, if did not have any influence on the HA production. Moreover, the results showed that the HA quality may be modulated through the mineral ion supplementation. The rheological properties of HA with low protein content (0.44 g.g-1) and average molecular weight of 4.0 x 106 Da showed the high entanglements density of the HA chains due to the high storage modulus concentration dependence as well as to the complex viscosity deviations with respect to the Cox - Merz rule. Alternative media containing agricultural resources derivates were studied. The higher HA concentrations were produced in media whose organic nitrogen source was yeast extract. This set of results contributes not only to the optimization of the HA production, but also to a better understanding of the Streptococcus zooepidemicus metabolism.
Doutorado
Desenvolvimento de Processos Biotecnologicos
Doutor em Engenharia Química

Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Key players in the marine carbon cycle are the ocean-dwelling microbes that fix, remineralize, and transform organic matter. Many of the small organic molecules in the marine carbon pool have not been well characterized and their roles in microbial physiology, ecological interactions, and carbon cycling remain largely unknown. In this dissertation metabolomics techniques were developed and used to profile and quantify a suite of metabolites in the field and in laboratory experiments. Experiments were run to study the way a specific metabolite can influence microbial metabolite output and potentially processing of organic matter. Specifically, the metabolic response of the heterotrophic marine bacterium, Ruegeria pomeroyi, to the algal metabolite dimethylsulfoniopropionate (DMSP) was analyzed using targeted and untargeted metabolomics. The manner in which DMSP causes R. pomeroyi to modify its biochemical pathways suggests anticipation by R. pomeroyi of phytoplankton-derived nutrients and higher microbial density. Targeted metabolomics was used to characterize the latitudinal and vertical distributions of particulate and dissolved metabolites in samples gathered along a transect in the Western Atlantic Ocean. The assembled dataset indicates that, while many metabolite distributions co-vary with biomass abundance, other metabolites show distributions that suggest abiotic, species specific, or metabolic controls on their variability. On sinking particles in the South Atlantic portion of the transect, metabolites possibly derived from degradation of organic matter increase and phytoplankton-derived metabolites decrease. This work highlights the role DMSP plays in the metabolic response of a bacterium to the environment and reveals unexpected ways metabolite abundances vary between ocean regions and are transformed on sinking particles. Further metabolomics studies of the global distributions and interactions of marine biomolecules promise to provide new insights into microbial processes and metabolite cycling.
by Winifred M. Johnson.
Ph. D.

Methylated amines, such as trimethylamine (TMA) and trimethylamine N-oxide, are nitrogenous compounds that are thought to be ubiquitous in the marine environment. TMA is a product of the anaerobic degradation of quaternary amines, such as glycine betaine and choline. Through a set of complex chemical and biological interactions, methylated amines play a role in regulating the planet’s climate. Microbial degradation of methylated amines is thought to be a sink for these compounds in the marine environment, however some of the key genes and enzymes responsible for the degradation of methylated amines are unknown. Using Ruegeria pomeroyi DSS-3 as the model organism, the key enzymes for the uptake and catabolism of trimethylamine N-oxide were identified and it was discovered that these genes and enzymes are highly expressed in the seawater, as revealed by the re-analysis of a number of recent metatranscriptomic and metaproteomic datasets. Again using R. pomeroyi as the model organism, it was shown that trimethylamine and trimethylamine N-oxide can be oxidised to CO2 to generate reducing equivalents and ATP. The generation of this reducing power results in a number of physiological benefits which are further discussed in detail. It was determined that bacteria possessing trimethylamine monooxygenase, the key enzyme required for the oxidation of TMA could also oxidise the reduced sulfur compound, dimethylsulfide, when supplemented with methylated amines. The ecology of methylated amine-utilising bacteria was investigated using a newly designed primer set targeting the trimethylamine N-oxide demethylase. The results are presented in detail within. The key genes and enzymes essential for the catabolism of the quaternary amine, choline were also discovered, again using R. pomeroyi as the model organism. The occurrence of genes required for the catabolism of choline are widespread among certain groups of marine bacteria known to interaction with eukaryotic biota, suggesting that this compound may be an essential nutrient for these organisms.

Marine organisms are known to produce secondary metabolites which have novel structures and are often biologically active. Chemical studies of biologically active metabolites from three different marine organisms led to the discovery of six new compounds and six previously known compounds. The brown alga Dictyota binghamiae is fairly abundant in British Columbia coastal waters. A chemical study of this alga yielded ten diterpenoids of which four are new compounds. All the new compounds, dictyoxide A (66), dictyol G acetate (68), dictyotriol A diacetate (69), and epidictyol B acetate (70) contain a perhydroazulene carbon skeleton first encountered in the algal metabolite pachydictyol A (29). Dictyoxide A (66) appears to be an artifact of isolation. The acetates 68, 69, 70 were found to be antibacterial and antifungal. Six previously known compounds pachydictyol A (29), dictyol C (32), dictyoxide (35), acetyldictyolal (49) and the acetals 61a and 61b were also isolated from this alga. Chemical studies on an Agelas sp. of sponge collected in Sri Lanka yielded the antimicrobial metabolite desbromooroidin (79). An interesting interaction between the starfish Dermasterias imbricata and the sea anemone Stompia coccinea was observed a long time ago. When contacted by the starfish, the anemone displays an unusual "swimming" response which was, according to other subsequent studies, caused by a single chemical substance in the starfish. A study conducted to elucidate the structure of this starfish metabolite led to the isolation of imbricatine (91), a unique benzyltetrahydroisoquinoline alkaloid. Imbricatine (91) induced S. coccinea swimming response at a very low concentration and also exhibited antitumor activity. Structures of all the new metabolites were determined by spectral analysis, and chemical degradations and chemical interconversions.
Science, Faculty of
Chemistry, Department of
Graduate

Made available in DSpace on 2015-04-11T13:55:41Z (GMT). No. of bitstreams: 1 Cassiane Minelli.pdf: 1637307 bytes, checksum: b5227c9445b1a0b3f8dcc8fcef9bcfd3 (MD5) Previous issue date: 2013-06-24
Conselho Nacional de Desenvolvimento Científico e Tecnológico
One of the biggest successes of Brazilian agriculture is the Pro-Alcohol, whose base of support is the culture of sugar cane, which leaves a lapse of activity in the offseason. In view of this, several alchool industries have been investing in other cultures, where the starch is the primary source for the alcohol production. The pursuit of amylase in thermotolerant Bacillus sp. And rhizobia from Amazonian soils is a strategy to be used for the bio-industries to reduce or stop importing them. Mutation was made from two Bacillus sp. containing amylases by exposure to ultraviolet light. Also tested were 40 strains of rhizobia for the presence of thermophilic amylases. Enzyme assays were performed to check for amylase activity at different temperatures, times, pHs, thermal shock and thermal stability. The Bacillus sp. ITAAM 023M mutant showed higher amylolyse than mutant ITAAM 010M. The amylases of the two Bacillus sp mutants and rhizobias proved quite diverse as their characteristics, indicating they are different from each other. The temperature with higher amylase activity of the mutants ITAAM 010M and ITAAM 023M was 100 ˚C. Amylase from the mutant ITAAM 010M showed greater activity in pH 4.5, and from the ITAAM 023M at pH 7.5. Their amylases showed high sensitivity to thermal shock. Amylase from mutant ITAAM 010M showed high thermostability at temperature of 90 ˚C. The amylase from ITAAM 023M, at a temperature of 80˚C. The highest activity shown by ITAAM 010M amylase was 1.6 U / ml and the ITAAM 023M, 1.5 U / mL. From the 40 rhizobia, 17 grew well in culture medium containing starch, 19 produced amylolytic halo, and 11 proved to be thermophiles. Rhizobia INPA INPA R001 and INPA R020 showed the highest rates of amylolyse. Their amylase showed greater activity at 90˚C. Amylase of INPA R001 showed higher activity at 100˚C, and from INPA R020, 30˚C. Amylase from INPA R001 showed greater activity at pH 4.5 and from INPA R020, at pH 7.5. Their amylases showed high sensitivity to thermal shock. Amylase from INPA R020 showed higher thermostability at 90 °C and from INPA R001 at 100˚C. The higher activity shown by amylase of rhizobia INPA R001 was 1.6 U/mL, and from INPA R020, 7.5 U/mL
Um dos maiores sucessos da agricultura brasileira é o Pró-Álcool, cuja base de sustentação é a cultura da cana-de-açúcar, que deixa um lapso de atividade na entressafra. Em vista disso, diversas usinas vêm investindo em outras culturas, onde o amido é a fonte primária para a produção do álcool. A busca de amilases termotolerantes em Bacillus sp. e em rizóbios de solos da Amazônia é uma estratégia a ser usada para que as bioindústrias reduzam ou parem de importá-las. Foi feita a mutação de dois Bacillus sp. contendo amilases com exposição à luz ultravioleta. Foram testadas também, 40 estirpes de rizóbio quanto à presença de amilases termotolerantes. Foram feitos ensaios enzimáticos para a verificação de atividade amilolítica em diferentes temperaturas, tempos, pHs, choque térmico e termoestabilidade. O mutante de Bacillus sp. ITAAM 023M mostrou maior índice de amilolise do que o ITAAM 010M. As amilases dos dois mutantes de Bacillus sp e dos rizóbios mostraram-se bem diversificadas quanto às suas características, indicando serem diferentes umas das outras. A temperatura com maior atividade das amilases dos mutantes ITAAM 010M e ITAAM 023M foi 100˚C. A amilase do mutante ITAAM 010M mostrou maior atividade em 4,5, e a do ITAAM 023M em pH 7,5. Suas amilases mostraram alta sensibilidade ao choque térmico. A amilase do mutante ITAAM 010M mostrou maior termoestabilidade na temperatura de 90˚C. A do ITAAM 023M, na temperatura de 80˚C. A maior atividade mostrada pela amilase do ITAAM 010M foi de 1,6 U/mL e a do ITAAM 023M, 1,5 U/mL. Dos 40 rizóbios, 17 cresceram bem em meio de cultura contendo amido, 19 produziram halo amilolítico e 11 se mostraram termofílicos. Os rizóbios INPA R001 e INPA R020 mostraram os maiores índices de amilolise. Suas amilases mostraram maior atividade a 90˚C. A amilase do INPA R001 mostrou maior atividade a 100˚C, e a do INPA R020 a 30˚C. A amilase do INPA R001 mostrou maior atividade no pH 4,5 e a do INPA R020 no pH 7,5. Suas amilases mostraram alta sensibilidade ao choque térmico. A amilase do INPA R020 mostrou maior termoestabilidade a 90ºC e a do INPA R001 a 100˚C. A maior atividade amilolítica mostrada pela amilase do rizóbio INPA R001 foi de 1,6 U/mL e a do rizóbio INPA R020, 7,5 U/mL

There is increasing evidence demonstrating a determinant role of gut microbiota in host health, and one underlying mechanism is via gut-microbial metabolites-host interaction in modulating host's cellular functions. SCFAs, main fermentation products of dietary fibres by gut microbiota in GI tract, are considered to be generally beneficial to the host. It has been shown that SCFAs act as substrates for energy metabolism, receptor agonists, and as histone deacetylase (HDAC) inhibitors. However, there are still gaps in the knowledge of their biological effects. It has been hypothesised that SCFAs may act as substrate for energy metabolism as well as ligand for several GPCRs, thus play roles in many cellular functions. Combining 1H nuclear magnetic resonance (NMR) spectroscopy with multivariate statistical analysis of the effect of SCFAs on the hepatic cancer metabolic network identified a metabolic signature associated dose- and time- related SCFA exposure. TCA cycle intermediate, α-keto-β-methylvalerate was strongly correlated with the treatment of SCFAs suggesting the preferential of the cells to SCFAs for energy production. This signature further confirms that SCFAs play direct role in host energy metabolism. Having implemented a harmonised pharmacological assessment of a comprehensive SCFA panel on FFAR2, FFAR3, and GPR109A, this study is the first to reveal that isobutyrate and isovalerate are novel partial agonists for GPR109A. Furthermore, niacin, a classical agonist for GPR109A also has been shown to activate FFAR2 and FFAR3 but with much lower affinity. In another study, the effect of SCFAs on 3T3-L1 adipogenesis and adipocytes lipolysis has been characterised. Dosing the 3T3-L1 cells with isobutyrate, valerate, and isovalerate significantly induce the adipogenesis of the cells. In contrast, other SCFAs have no effect of the 3T3-L1 cell differentiation. Subsequent study focused on the effect of SCFAs on basal adipocyte lipolysis. Mature adipocytes were treated with 100 μM of SCFAs for 3hr and glycerol release was measured. This study revealed the anti-lipolytic property of propionate, butyrate, and valerate by significantly inhibits lipolysis in mature 3T3-L1 adipocytes. Thus, the role of SCFAs in regulating adipocytes functions may be particular important and beneficial in regulating plasma lipid profile and possibly aspects of metabolic syndrome. Together, these data enhance our understanding on the role of SCFAs on important metabolic tissues, which are hepatocyte and adipocyte.

El principal objectiu d'aquest treball ha estat estudiar la producció de metabòlits amb activitat antibiòtica per soques de l'espècie Pseudomonas fluorescens de la col·lecció EPS, i també avaluar la seva potencialitat com a agents de biocontrol. Es va disposar també de diverses soques de P. fluorescens, cedides per altres investigadors, que van utilitzar-se com a referència perquè algunes són actives en control biològic i produeixen metabòlits secundaris d'interès en el biocontrol de malalties de plantes.
La present memòria s'estructura en cinc capítols, que són, introducció al control biològic, descripció de l'etapa de selecció de soques i cerca dels metabòlits produïts, estudi de la producció d'HCN per la soca EPS288, estudi de la producció de l'antibiòtic 2,4-diacetilfloroglucinol (DAPG), i finalment, el darrer capítol, on s'ha estudiat la producció de DAPG sobre material vegetal i la capacitat de colonitzar arrels per diverses soques d'interès.
En l'etapa de prospecció, va demostrar-se que un 37% del total de les soques de la col·lecció EPS produïen HCN, totes de l'espècie P. fluorescens, i un 90% d'aquestes provenien de les arrels de plantes. Es va confirmar la producció dels metabòlits secundaris 2,4-diacetilfloroglucinol, àcid fenazina-1-carboxílic, i pirrolnitrina, per diverses soques de la col·lecció EPS seleccionades mitjançant tècniques moleculars. Així, de la col·lecció EPS, les soques EPS317 i EPS808 produeixen DAPG, la EPS263 àcid fenazina-1-carboxílic i pirrolnitrina i, EPS894, EPS895, EPS945 produeixen àcid fenazina-1-carboxílic.
La producció d'HCN es va estudiar més exhaustivament en la soca EPS288, seleccionada per la seva activitat antifúngica i candidata a agent de biocontrol contra Stemphylium vesicarium, causant de la estemfiliosi de la perera, i contra Penicillium expansum, causant de la podridura blava en conservació de fruita a postcollita. Per aquest estudi, es va dissenyar i validar un sistema per recollir l'HCN a partir de cultius en medi líquid. S'ha demostrat que la temperatura d'incubació, la concentració cel·lular de sembra i la composició del medi de cultiu afectaven a la producció d'HCN. Els medis complexos i la glicina n'afavorien la síntesi i la font de carboni no afectava. La soca EPS288 va produir més HCN que P. fluorescens CHA0, soca de referència productora d'HCN i descrita com a activa en processos de biocontrol de fongs fitopatògens.
En l'estudi de la producció de DAPG, les soques de la col·lecció EPS i de referència, es van comparar en diversos medis de cultiu estudiant l'efecte de la complexitat i consistència del medi, i l'addició de ferro o de glucosa. Va demostrar-se que la producció de DAPG depèn principalment de la soca i de les característiques del medi de cultiu. La glucosa estimula la producció, mentre que el ferro pràcticament no afecta, i en general, el medi sòlid i complex estimula la producció de DAPG. Tanmateix, aquests efectes varien en alguna de les soques assajades donant lloc a comportaments singulars.
En el seguiment del creixement amb un sistema automàtic es va comprovar que la velocitat específica de creixement i la concentració cel·lular assolida al final del cultiu, estaven condicionades per la composició del medi de cultiu.
En les proves d'antagonisme vers fitopatògens que foren seleccionats com a indicadors, va observar-se que tant l'antagonisme in vitro com la inhibició d'infeccions sobre material vegetal estaven parcialment relacionades amb la producció dels metabòlits secundaris estudiats. La promoció del creixement de portaempelts per aquestes soques depenia de la soca i de l'hoste, però no es pogué establir una relació causa-efecte amb el metabòlits produïts. També es va comprovar que algunes de les soques podien sobreviure en ferides de pomes i de peres, on produïren DAPG.
Mutants resistents a rifampicina de diverses soques de la col·lecció EPS i de referència es van inocular en llavors de pomera i de tomatera que es van sembrar i incubar en condicions controlades. Es va fer el seguiment de la població bacteriana total i resistent a rifampicina present a les arrels durant 72 dies. Totes les soques van colonitzar les arrels de les plantes, mantenint una elevada població durant 37 dies, cap d'elles va estimular el creixement ni mostrar efectes fitotòxics, no afectant tampoc signicativament a la població bacteriana espontània de les arrels.
La soca EPS808, una de les seleccionades pel treball, va aconseguir uns nivells de producció de DAPG, una velocitat de creixement i una supervivència relativa a les arrels similar a altres soques de referència descrites com a bons agents de biocontrol. En conseqüència, se la considera una candidata a agent de biocontrol que hauria de ser objecte de futurs estudis d'eficàcia.
El principal objetivo de este trabajo ha sido estudiar la producción de metabolitos con actividad antibiótica por cepas de la especie Pseudomonas fluorescens de la colección EPS, y también evaluar su potencialidad como agentes de biocontrol. Se dispuso de varias cepas de P. fluorescens, cedidas por otros investigadores, que se utilizaron como referencia ya que algunas de estas son activas en el control biológico, y producen metabolitos secundarios de interés para el biocontrol de enfermedades de plantas.
La presente memoria se estructura en cinco capítulos, revisión bibliográfica del control biológico, descripción de la etapa de selección de cepas y de los metabolitos producidos, estudio de la producción de HCN por la cepa EPS288, estudio de la producción de 2,4-diacetilfloroglucinol(DAPG), y finalmente, el último capítulo, donde se ha estudiado la producción de DAPG sobre material vegetal y la capacidad de colonizar las raíces de diversas plantas.
En la etapa de prospección se demostró que el 37% del total de las cepas de la colección EPS producían HCN, todas ellas de la especie P. fluorescens, de las cuales el 90% había sido aislada de las raíces de plantas. Se confirmó la producción de los metabolitos secundarios 2,4-diacetilfloroglucinol, ácido fenazina-1-carboxílico y pirrolnitrina, por diversas cepas de la colección EPS seleccionadas mediante técnicas moleculares. De la colección EPS las cepas EPS317 y EPS808 producen DAPG, la EPS263 ácido fenazina-1-carboxílico y pirrolnitrina y las cepas EPS894, EPS895 y EPS945 producen ácido fenazina-1-carboxílico.
La producción de HCN se estudió en detalle por la cepa EPS288, esta había sido seleccionada por su actividad antifúngica y es candidata a agente de biocontrol contra Stemphylium vesicarium, causante de la estemfiliosis del peral, y contra Penicillium expansum, causante de la enfermedad del moho azul en la conservación de la fruta en poscosecha. Para este estudio se diseñó y se validó un sistema para la recogida del HCN que se desprende de cultivos en medio líquido. La temperatura de incubación, la concentración en la siembra y la composición del medio de cultivo afectaban a la producción de HCN. Los medios de cultivo complejos y la glicina estimulaban su síntesis, mientras que la fuente de carbono no afectaba. La cepa EPS288 produjo más HCN que la P. fluorescens CHA0, cepa de referencia productora de HCN y descrita como activa en procesos de control biológico de hongos fitopatógenos.
En el estudio de la producción de DAPG, las cepas de la colección EPS y de referencia, se compararon en diferentes medios de cultivo estudiando el efecto de la complejidad y la consistencia del medio de cultivo, y de la adición de hierro o de glucosa. Se demostró que la producción de DAPG depende principalmente de la cepa y de la composición del medio de cultivo. La glucosa estimula la producción, mientras que el hierro casi no tiene efecto. Sin embargo, estos efectos varían en alguna de las cepas ensayadas dando lugar a comportamientos singulares.
El seguimiento del crecimiento mediante un sistema automático permitió verificar que la velocidad específica de crecimiento y la concentración celular en la fase estacionaria estaban condicionadas por la composición del medio de cultivo.
En las pruebas de antagonismo frente a hongos fitopatógenos seleccionados como indicadores, se observó que el antagonismo in vitro y la inhibición de infecciones sobre material vegetal correlacionaban parcialmente con la producción de los metabolitos secundarios estudiados. La promoción del crecimiento de portainjertos con esas cepas era función del huésped y de la cepa. Se comprobó que algunas de las cepas sobrevivían en heridas de manzanas y de peras dónde sintetizaban DAPG.
Mutantes resistentes a rifampicina de diversas cepas de la colección EPS y de referencia se inocularon en semillas de manzano y de tomate las cuales se sembraron e incubaron en ambiente controlado. La población bacteriana en las raíces de las plantas se monitorizó durante un periodo de 72 días. Todas las cepas colonizaron las raíces de las plantas manteniendo una elevada población durante, al menos, 37 días. Ninguna de la cepas estimuló el crecimiento ni mostró fitotoxicidad, y no afectaron significativamente a la población bacteriana espontánea de las raíces.
La cepa EPS808, una de las seleccionadas para el presente trabajo, demostró una producción de DAPG, velocidad específica de crecimiento y una supervivencia relativa en las raíces, similares a otras cepas de referencia descritas como buenos agentes de biocontrol. Por lo tanto, se considera a esta cepa como una candidata a agente de biocontrol, que tendría que ser objeto de futuros estudios de eficacia.
The main objective of this work is to study the production of biologically active secondary metabolites produced by Pseudomonas fluorescens strains from EPS collection. Moreover, their potential as biocontrol agents was also evaluated. We used P. fluorescens strains, provided by foreign scientists, that were utilised as a reference because these strains are active as biocontrol agents and produce secondary metabolites involved in biological control.
The present work is divided into five chapters which deal with an introduction to biological control, the selection of strains, the detection and identification of metabolites produced by these bacteria, the study of HCN production by EPS288 strain, the study of 2,4-diacetylphloroglucinol (DAPG) production, and finally, the DAPG production based on fruits and plants, and root colonisation by selected strains.
It was shown that 37% of isolates in EPS collection produced cyanide, all were P. fluorescens, and 90% of them were isolated at roots of different plants. The secondary metabolites 2,4-diacetilphloroglucinol (DAPG), phenazine-1-carboxilic acid (PCA) and pyrrolnitrin were identified and its production by some EPS collection strains, selected with molecular techniques, was demonstrated. The strains EPS317 and EPS808 produce DAPG, EPS263 produces phenazine-1-carboxilic acid and pyrrolnitrin, and the strains EPS894, EPS895, EPS945 produce phenazine-1-carboxilic acid.
The production of cyanide acid by P. fluorescens EPS288 was studied more exhaustively. This strain was selected as a biocontrol agent due to its antifungal activity against Stemphylium vesicarium, that causes brown spot on pear, and Penicillium expansum, that causes the blue mould of fruits during postharvest. A device to collect cyanide produced by bacteria from broth cultures was set up and validated. The incubation temperature, the initial cell density and the composition of growth media clearly affected HCN production by EPS288 strain. Complex media and glycine stimulated HCN production, however, it was not affected by carbon source in defined growth media. The EPS288 strain produced more cyanide in Castric growth media than P. fluorescens CHA0, a reference strain employed in biological control of fungal pathogens.
The reference strains and those from EPS collection which produced DAPG were compared considering the factors complexity and consistence of growth media. The effect of iron and glucose addition were also evaluated. The strain and growth media characteristics were the predominant factors in the DAPG production. Glucose increased DAPG production, while iron addition had no considerable effect, and as general rule, solid and complex media stimulated DAPG production. However these effects are variable depending on the strain.
The monitoring of bacterial growth and DAPG production with an automated absorbance reader showed that the growth rate and cellular yield were affected by growth media.
In antagonism tests against selected fungal pathogens, the in vitro inhibition and inhibition of infections over organic material were, in part, correlated with secondary metabolite production. The rootstock growth promotion caused by some of these strains was dependent on the strain and the host, but a cause-effect relationship with antibiotics produced could not be clearly established. Some strains were able to colonise and produce DAPG in wounds on apples and pears.
Rifampicine resistant spontaneous mutants of the reference and of EPS collection strains were isolated and inoculated on apple and tomato seeds, which were sown and grown under a controlled conditions. The evolution of inoculated strains and the whole bacterial population at the roots were monitored during 72 days. All selected strains efficiently colonised plant roots at high levels for at least 37 days, and nor phytotoxic either plant growth promotion was observed by any strain. Moreover, they did not significantly affected the spontaneous bacterial population at roots.
The EPS808 strain exhibited a 2,4-diacetilphloroglucinol production level, a specific growth rate and a relative root population as high as the reference strains that are referenced to be good biocontrol agents. Thus we conclude that this strain can be considered as a candidate for biological control agent, but further efficacy studies are required.

Five strains (9L, 642A, 714, 5688T and 3384Y) of Histophilus ovis were investigated with respect to iron acquisition. All strains used ovine, bovine and goat, but not porcine or human, transferrins (Tfs) as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine Tf, total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine and goat) were bound by the same receptor(s). An affinity isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. A gene encoding a Pasteurella multocida TbpA homologue was shown to be present in each of two representative strains (9L and 3384Y); these genes were sequenced and determined to be the structural genes encoding the 78-kDa Tf-binding polypeptides. The identification of a fur homologue and a Fur box within the promoter region of tbpA in both strains indicated that Fur (and iron) is responsible for the iron-repressible nature of Tf-binding activity. Although tbpA transcripts were detected by reverse transcription (RT)-PCR with RNA isolated from strains 9L and 3384Y grown under iron-restricted conditions, with strain 3384Y, and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was isolated from cells grown under conditions of iron-restriction in the presence of Tf. The presence of an additional G in the tbpA gene of strain 3384Y grown under iron-replete conditions, compared to organisms grown under iron-restricted conditions plus bovine Tf, is

There is an increasing need to produce fuels and chemical commodities from renewable resources. Current efforts have been mainly focused on liberating sugars from plant-derived lignocellulosic feedstocks. However, lignocellulosic materials have naturally evolved to resist their microbial and enzymatic degradation and this poses a major problem. Due to these difficulties, alternative feedstocks derived from biomass gasification have recently become a major focus of research. The gasification process generates mixtures of hydrogen, carbon monoxide and carbon dioxide, known as syngas, which can be utilised by some autotrophic bacteria as the sole sources of carbon and energy. Cupriavidus necator strain H16 was chosen as a chassis organism for the current investigation as it can grow to high cell densities on CO2/H2 and, under nutrient limiting conditions, stockpiles huge amounts poly[R-(–)-3-hydroxybutyrate] (PHB). The long term aim of the study was to employ metabolic engineering approaches to re-direct carbon flux so that desirable chemicals are produced instead of PHB. Following the establishment of defined growth media, genetic tools, and DNA delivery methods, the natural resistance of the bacterium to a range of desirable target chemicals was tested and 3-hydroxypropanoic acid (3-HP) identified as a suitable target. However, it was noted that C. necator was able to utilise this compound as the sole source of carbon and energy. Hence, several genes involved in the degradation of 3-HP were identified and inactivated through ORF deletion, resulting in strain CNCA13 unable to grow on this compound. However, this strain was still able to co metabolise 3-HP alongside other carbon sources such as fructose or gluconate, necessitating further investigation, including the introduction of additional gene deletions. Some of these deletions belonged to genes or pathways involved in a reductive route for the assimilation of the compound. The inactivation of one of these candidates over the strain CNCA13 led to prevent the co-assimilation of 3-HP alongside fructose. Following strain development, a heterologous pathway designed to produce 3-HP from actyl-CoA in two enzymatic steps was introduced into the organism. The first committed step in this pathway is the carboxylation of acetyl-CoA to malonyl-CoA, catalysed by the enzyme acetyl-CoA carboxylase (ACC). The second step is the reduction of malonyl-CoA to 3-HP, a conversion catalysed by the bifunctional enzyme malonyl-CoA reductase (MCR) or, in some archaea, by the combination of two monofunctional reductases. Genes encoding ACC subunits and MCRs from different bacteria and archaea were codon-optimised, assembled into functional operons and screened for efficient expression in C. necator H16. All genes were found to be expressed, but production of 3-HP could not be observed, even in strains lacking the ability to produce PHB or to consume 3-HP as the sole source of carbon. Thus, further work is needed to efficiently redirect carbon flux through the generated pathway.

Experiments were conducted to examine the cause of lag-phase growth during phenol degradation by mixed microbial cultures that had been acclimated to one of four substrates. Four aerated Imhoff cones were inoculated with wastewater sludge and fed one of four substrates: acetate, egg albumin, vegetable oil, or phenol. Inocula from these cones were injected into batch reactors containing phenol. Time-dependent growth was measured by two methods: most probable number (MPN) and epifluorescence microscopy (EM). The MPN technique was used to distinguish two cell concentrations: total cells and a phenol-degrading community within the total; EM was also used to count total cells. The results indicated that a lag in phenol utilization for all cultures, except the phenol-acclimated cultures, was a result of growth of a phenol-degrading subpopulation, and not due to enzyme induction of the existing population. Similar experiments were conducted using 2,4-dichlorophenol (2,4-DCP), which resulted in no growth and no degradation of 2,4-DCP.

A aplicação de biossólidos em solos agrícolas e/ou florestais é uma prática aceitável, tanto como forma de disposição destes materiais, quanto de melhorar as características físicas, químicas e biológicas do solo. No entanto, pouco se sabe dos impactos decorrentes desta utilização e dos seus efeitos sobre a estrutura das comunidades microbianas dos solos e sua capacidade metabólica. Assim, o objetivo deste trabalho foi determinar alterações quanti-qualitativas na diversidade metabólica dos microrganismos do solo após a adição de biossólidos e identificar possíveis indicadores de alteração de qualidade do solo. O delineamento experimental constituiu-se de 4 doses de biossólidos da ETE-Franca, equivalentes à 6, 12, 24 e 48 Mg ha-1, mantendo-se um controle sem adição de biossólido. Foram utilizadas 4 repetições por tratamento, compreendendo 20 sub-parcelas. Estas sub-parcelas foram incubadas por 1, 2, 4, 8 e 16 semanas, perfazendo um total de 120 parcelas. Após o período de incubação, amostras de solo foram coletadas para as determinações de pH, C-biomassa, Respiração Basal (RB), Respiração Induzida pelo Substrato (RIS), Quociente Metabólico (qCO2) e Diversidade Metabólica. Houve um aumento transiente do pH nos tratamentos com as maiores doses de biossólido, em relação ao controle. Após 16 semanas de incubação os solos com biossólidos apresentaram pH mais baixo do que o controle sem biossólido. O C-biomassa apresentou comportamento semelhante, com os valores decrescendo e tendendo a estabilização, em relação ao controle sem biossólido. A atividade metabólica com base na RB, RIS e MHM indicou uma indução transiente no metabolismo da microbiota do solo nos tratamentos com as maiores doses de biossólido, tanto no início quanto no final do período de incubação. Os maiores valores de qCO2 nos solos com as maiores quantidades de biossólidos sugerem a ocorrência de condições estressantes para a microbiota e sucessão de populações microbianas. A utilização de substratos de C pela comunidade microbiana foi substancialmente influenciada pela quantidade de biossólido adicionado ao solo e pelo período de incubação. Os maiores valores de Riqueza de Substratos (S) e Diversidade Metabólica (H) observados nos tratamentos que receberam as maiores quantidades de biossólido, no início do período de incubação, são indicativos de um estímulo da microbiota nativa e da contribuição de novas células microbianas decorrentes da adição do biossólido. As similaridades entre os perfis de utilização de substratos C diminuíram com o aumento da concentração de biossólido no solo, em relação ao controle sem biossólido, e aumentaram em função do período de incubação, sugerindo uma indução transiente na diversidade metabólica do solo em função da aplicação de biossólido da ETE-Franca. Solos que receberam altas doses de biossólidos apresentaram atividades metabólicas não observadas no solo sem biossólidos, no início do período de incubação, e perderam algumas atividades no final do período de incubação, em relação ao controle sem biossólido. No geral, os resultados sugerem que a aplicação de biossólidos da ETE-Franca no solo argiloso utilizado provoca um distúrbio na fisiologia da microbiota do solo e que aplicações sucessivas de altas doses de biossólido podem ocasionar alterações significativas na capacidade metabólica dos solos.
The use of biosolids in agricultural and/or forestry soils has been an acceptable practice to dispose as well as to improve physical, chemical and biological soil properties. However, the impacts of biosolids application on microbial community structures and metabolic diversity in agricultural soils are relatively unknown. The aim of this work was to determine quantitative and qualitative alterations in the metabolic diversity of a clay soil upon treatment with biosolids. A clay soil was amended with the equivalent to 6, 12, 24 and 48 Mg ha-1 of biosolids from the City of Franca (SP) Sewage Treatment Plant. A control without biosolids was also used. Four repetitions per treatment were used, totalizing 20 sub-parcels. Sub-parcels were incubated for 1, 2, 4, 8 and 16 weeks, after incorporating the biosolids, totaling 120 microcosms. After the incubation period, soil samples were collected to determine pH, Basal Respiration (BR), Substrate Induced Respiration (SIR), C-biomass, Metabolic Quotient (qCO2) and Metabolic Diversity. A transient increase in soil pH was observed in the treatments with the higher concentrations of biosolids, compared to the control without biosolids. After 16 weeks of incubation, the pH of soils with biosolids was lower than in the control without biosolids. The C-biomass also showed a transient increase in soils with biosolids at early time points, and did not statistically differ from the control at late time points. Transient increases in metabolic activities, based on BR, SIR and AWCD (Average Well Color Development), were observed in soils with the higher concentrations of biosolids at early time points. Higher values of qCO2 in soils with higher concentrations of biosolids at the earliest and latest time points suggests stressing conditions to the microbial community and alterations in microbial community structure. The utilization of C-substrates by the soil microbial community was greatly affected by the concentration of biosolids added and by the incubation time. Higher values of Substrate Richness (S) and Metabolic Diversity (H) in the soils with the higher concentrations of biosolids at early time points indicate that biosolids stimulate the activity of soil microorganisms and contribute with new activities. The similarities between the C-sources utilization profiles in the soil amended with biosolids and the control were lower in soils with the higher concentrations of biosolids. The similarities of C-source utilization profiles in the soils with biosolids and the control increased during the incubation time. Soils amended with the higher concentrations of biosolids showed metabolic activities not observed in the control soil at the early time points. The ability to use several C-sources was lost at the latest time point. In general, the results suggest that the incorporation of biosolids to the clay soil disturbs soil microbial physiology and that the successive application of biosolids to the soil may significantly alter the soil metabolic activities.

Biofilm communities were cultivated in rotating annular bioreactors using water from the South Saskatchewan River. The impacts of seasonal variations, nutrients, pollutants and dissolved oxygen on the activity and composition of the biofilms were assessed by using a combination of microcosm assays and molecular biology techniques.
The seasonal pattern in nitrification, denitrification and hexadecane mineralization, and in the occurrence of nirK in the South Saskatchewan River biofilms was: fall greater than winter, which was equivalent to spring. Hexadecane mineralization was higher in fall 1999 than in fall 2001, denitrification was similar in these two years, and no seasonal pattern of nitrification was observed.
The addition of combined nutrients (C, N, and P) resulted in significant increases in the measured bacterial activities and in the predominance of alkB, nirS and nirK in all seasons and years. The addition of individual nutrients did not stimulate hexadecane mineralization, denitrification, and the PCR amplification of nirS and nirK. In fall 1999, CNP and, to a lesser extent P, stimulated nitrification, whereas in fall 2001, no pattern was observed. The results showed that nutrients, especially P, were limiting for bacterial activities, and that the biofilm activities and composition varied with nutrient availability and time of year.
At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification to similar extents in both years, had a negative impact on nitrification and hexadecane mineralization in fall 1999, and a positive impact on these two latter activities in fall 2001. Nickel (0.5 mg liter-1 ) negatively affected denitrification but had no effect on hexadecane mineralization. The alkB and nirS genes were less predominant and absent, respectively, in biofilms grown in the presence of nickel. DGGE analyses indicated that nickel reduced the biofilm bacterial diversity.
The results presented herein provide much needed information on the microbial ecology of river biofilms, and on the impact and interactive effects of pollutant and nutrient inputs on these biofilms. These results and the techniques used in this project can be applied to monitor environmental effects of anthropogenic activities on aquatic biofilms, and can contribute to establish or revise environmental regulations.

Polymicrobial communities often show complex patterns of metabolic and ecological interactions, yet our understanding of how the properties of communities emerge from the metabolic rules of species interactions is still limited. A central feature of metabolic interactions within microbial communities is ‘cross-feeding’, where one species or lineage consumes the metabolic by-products of another. Cross-feeding bacteria excrete and consume a wide range of metabolites and this sets the stage for diverse intra- and inter-specific metabolic interactions. In this thesis, I use ecological and evolutionary theory to address a number of critical questions posed by cross-feeding bacteria, with a particular focus on the role played by microbial metabolism in driving the emergence and dynamics of microbial interactions. First, I explore the conditions that favour the emergence and maintenance of cooperative cross-feeding and show that the evolutionary outcome depends strongly on the shape of the trade-off curves between the costs and benefits of cooperation. Second, I investigate the origins of cross-feeding interactions via single lineage diversification and derive new predictions on the physiological mechanisms that may explain the stable coexistence of a cross-feeding polymorphism that evolved from a single clone. Third, I investigate what are the ecological consequences of cross-feeding metabolic interactions and demonstrate theoretically that a simple mechanism of trade can generate a diverse array of ecological relationships. Furthermore, I show the importance of the metabolic by-product properties in determining the ecological outcome. Fourth, I investigate how metabolic constraints of individual species shape the emergent functional and structural relationships among species. I show that strong metabolic interdependence drives the emergence of mutualism, robust interspecific mixing, and increased community productivity. Furthermore, I show that these emergent community properties are driven by demographic feedbacks. In general, these findings support the idea that bridging microbial ecology and metabolism is a critical step toward a better understanding of the factors governing the emergence and dynamics of polymicrobial interactions.

Ovine (strains 9L and 3384Y) and bovine (strains 649, 2336 and 8025) isolates of Histophilus somni were investigated for their ability to acquire iron from haemoglobin (Hb). Bovine isolates were capable of utilizing bovine, but not ovine, porcine or human Hb as a source of iron. Ovine isolates could not obtain iron from Hb. Bovine isolates bound bovine, ovine, and human Hbs by means of the same iron-repressible receptor(s) and produced a ~120-kDa iron-repressible, outer membrane protein. Using PCR approaches, an iron-regulated operon containing hugX and hugZ homologues and a gene (hgbA) that encodes a TonB-dependent, Hb-binding proteins were identified in strains 649, 9L and 3384Y. In strains 9L and 3384Y, HgbA is truncated offering a possible explanation for their lack of utilization of Hb as an iron source. In strains 2336 and 8025, expression of HgbA was also subject to a form of phase variation.

Zymomonas mobilis is a non-pathogenic, facultatively-anaerobic Gram-negative bacterium, which has historically been used for the fermentation of alcoholic beverages in many tropical/sub-tropical countries. Due to its excellent ethanol-producing capabilities, significant effort has been undertaken over recent years to utilize it for industrial ‘bioethanol’ production. Its physiological and metabolic properties indicate that it may also be an excellent organism for the bio-production of many different types of organic molecules. Consequently, the aim of my thesis was to develop new molecular methodologies that would enable Z. mobilis to be ‘engineered’ for use in future ‘bioproduction’ endeavours. In the first part of my study, I analyzed the native (cryptic) plasmids present within a variety of Z. mobilis strains, including two poorly-studied Z. mobilis strains: NCIMB 11163 and NCIMB 8227. Several plasmid libraries containing restriction-digested fragments of Z. mobilis cryptic plasmid DNA were prepared, and their inserts were sequenced. This enabled the complete DNA sequences of three small (non-integrating, double-stranded DNA) cryptic plasmids to be determined: pZMO1A and pZMO7 from NCIMB 11163, and pZMO1B from NCIMB 8227. Their DNA sequences were analyzed using bioinformatic approaches, to identify open reading frames, and regions of DNA that were putatively involved in transcription or DNA replication. In the second part of this thesis, the minimally-replicating region from plasmid pZMO7 was used to construct a series of Escherichia coli-Z. mobilis shuttle vectors. These vectors were found to be stable within several Z. mobilis strains for over 60 generations without antibiotic selective pressure. A reliable and reproducible method based on quantitative real time PCR (Q-RT-PCR) was developed to accurately determine the copy number of cryptic plasmids and shuttle-vectors present in Z. mobilis cultures. The pZMO7-based shuttle vectors exhibited good compatibility with cryptic plasmids as well as the widely-used pZM2-based shuttle vectors. Genes encoding glutathione S-transferase (GST) as well as green and red fluorescent protein (GFP and RFP) reporters were cloned into various shuttle vector constructs; placing them under the control of endogenous (Ppdc) or exogenous (Plac and Ptac) promoters. Promoter strength was evaluated by quantifying the reporter gene expression. The plasmid-based expression of GFP and RFP was visualized within planktonic and biofilm cultures using confocal laser scanning microscopy (CLSM). Shuttle vector-based GST pull-down experiments were used to study intracellular protein-protein binding interactions. In the third part of my thesis, I explored the potential use of Z. mobilis for the bioproduction of isoprenoid (terpenoid) compounds. Five predicted sesquiterpene synthases (terpene cyclases) of unknown function from the dimorphic fungus Penicillium marneffei, and several terpene cyclases from several other bacteria, fungi and plants were initially functionally-analyzed in E. coli. Several cyclase genes were cloned into E. coli-Z. mobilis shuttle vectors for expression trials within Z. mobilis cells. In summary, this thesis describes the development of a variety of novel methodologies and genetic ‘tools’ that may be used to express heterologous genes within Z. mobilis cells. These will be invaluable for future studies concerned with exploring the biology and industrial applications of this ‘microbial cell factory’.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

Recently the property of some bacteria to exchange electrons with non-soluble electron acceptors, such as minerals, has been discovered. In addition, some of these bacteria can use electrodes as the final electron acceptor. This phenomenon is called Extracellular Electron Transfer (EET) and it can be done through several mechanisms, especially through conductive bacterial nanowires. The main objective of this thesis is the investigation of the polarization properties of such electrochemically active bacteria and their appendages as the polarization plays a key role in EET. The curiosity also stems out from the increasing interest in using such bacteria as a biosensing platform. Specifically, I have studied two types of bacteria, Shewanella oneidensis MR-1 and cable bacteria, from the Desulfobulbaceae family. With this purpose, I have used the Electrostatic Force Microscopy (EFM) technique, which measures the electrostatic force using a nanometric probe, combined with finite element simulations to obtain the polarization properties of bacterial nanowires. The electrostatic force depends mainly on the geometry and dielectric constant of the probe-sample system. In order to do that, first, I have developed a way to obtain the dimensions of objects without damaging them, avoiding any physical contact, by measuring the electrostatic force. I have tested this technique on silver nanowires and bacterial flagella. In this way, I have been able to optimize the EFM technique to nanowire-like biological samples at the nanoscale. Secondly, I have analyzed one of the S. oneidensis appendages, the flagella, and I have compared their properties with the flagella of a non-electrochemically active bacteria, the Pseudomonas aeruginosa. I have obtained a dielectric constant of εSo = 4.3 ± 0.6 for S. oneidensis and εPa = 4.5 ± 0.7 for P. aeruginosa, similar results for both bacteria. In addition, this value corresponds to the dielectric constant of proteins (εr ~ 4) measured with the same technique, in agreement with the fact that flagella are composed of flagellin protein monomers. Later, I have studied the electrical properties of another S. oneidensis appendages, the outer membrane extensions (OMEs), responsible for the extracellular electron transfer. In this analysis, I have obtained a relatively low value of the dielectric constant (εOME = 3.7 ± 0.7) corresponding to a combination of lipids (εr ~ 2) and proteins (εr ~ 4). However, considering that the conduction mechanism of such OMEs is through electron hopping, and electrons are localized, these results do not contradict the literature. I have also studied the electrical properties of the cable bacteria, especially the fibers that are along this filamentous bacterium. The dielectric constant of the fibers was εr = 7 ± 1. However, this result is not compatible with the conductivity reported in the literature. Therefore, a core-shell model was proposed with a conductive core of h ~ 10 – 20 nm. Subsequently, I have finished the nanoscale analysis performing qualitative EFM measurements in liquid over living S. oneidensis bacteria and rehydrated bacteria. Finally, I have connected these nanoscale measurements in dry conditions with macroscale measurements in living S. oneidensis using a microfluidic device that I designed, fabricated and characterized at the Denmark Technical University (DTU) in Copenhagen. The microfluidic device was used to perform two-electrode impedance measurements. In these measurements, the impedance experiences an abrupt change for f ~ 102 – 103 Hz when bacteria were in anaerobiosis. However, further experiments are needed to explain this phenomenon.
Algunas bacterias pueden intercambiar electrones con aceptores de electrones no solubles, como los minerales. Este fenómeno se llama Transferencia de Electrones Extracelulares (EET) y se puede hacer a través de varios mecanismos, especialmente a través de nanocables bacterianos conductores. El objetivo principal de esta tesis es la investigación de las propiedades de polarización de bacterias electroquímicamente activas y sus apéndices. Específicamente, he estudiado dos tipos, Shewanella oneidensis MR- 1 y cable bacteria. Para ello, he utilizado la microscopía de fuerza electrostática (EFM), que mide la fuerza electrostática utilizando una sonda nanométrica, combinada con simulaciones por elementos finitos. La fuerza electrostática depende principalmente de la geometría y la constante dieléctrica del sistema sonda-muestra. Para hacerlo, he desarrollado una técnica para obtener las dimensiones de los objetos evitando el contacto físico con la muestra, midiendo la fuerza electrostática. He probado esta técnica en nanohilos de plata y flagelos bacterianos, optimizándola para muestras biológicas filamentosas en la nanoescala. Después, he analizado los flagelos de S. oneidensis y he comparado sus propiedades con los flagelos de Pseudomonas aeruginosa, una bacteria no electroquímicamente activa, obteniendo similares resultados (εr~4.5). También he estudiado las extensiones de membrana externa (OMEs) de S. oneidensis, responsables del EET, obteniendo un valor bajo de la constante dieléctrica (εOME=3.7±0.7). Sin embargo, considerando que el mecanismo de conducción de tales OMEs es a través de salto de electrones, donde los electrones están localizados, estos resultados no contradicen la literatura. También he estudiado la cable bacteria, especialmente las fibras que se encuentran a lo largo de esta bacteria filamentosa. He obtenido un modelo núcleo-caparazón para las fibras con un núcleo conductor de h~10–20nm compatible con los resultados experimentales. Posteriormente, he realizado mediciones cualitativas de EFM en líquido sobre S. oneidensis viva y rehidratada. Finalmente, he realizado mediciones en la macroescala en S. oneidensis vivo usando un dispositivo microfluídico que diseñé, fabriqué y caractericé en la Universidad Técnica de Dinamarca (DTU), Copenhague. El dispositivo se ha utilizado para realizar mediciones de impedancia de dos electrodos. En estas mediciones, la impedancia experimenta un cambio abrupto para f~102–103Hz cuando las bacterias estaban en anaerobiosis.

The scope of this work is to identify the relation that exists between the alterations of the bacterial metabolism and the exposition to an ultrasonic field (O'Leary et al. 1997, Piyasena et al. 2003, Brujan2004). To made this relation quantifiable they will be used repeatable and reproducible metrological methods. To obtain this result the research will be centred on 3 specific goal: 1. To realise a measurement of the ultrasonic field, generated by an ultrasonic bath, using an hydrophone calibrated specifically with the wavelengths applied (Canney et al. 2008). 2. To choose a method, through uncertainty evaluation, to expose microbes to the ultrasonic field without an excessive alteration of the generated acoustic pressure. 3. To find a method to measure bacterial viability, represented by the planktonic growth and the biofilm development, after exposure to the ultrasounds. Studying the relationship between the bacteria metabolism and ultrasonic exposure it will be possible to identify novels and more precise methods to treat bacterial colonizations. This result will be in fact useful in various fields where the bacterial presence, and in particular biofilm development, is today an unresolved problem. At the same time this research will put the basis for many further studies that will be realized applying the same methods with different bacterial species, ultrasounds exposition conditions or ultrasonic devices.

Characterizing dissolved organic matter (DOM) composition remains a major unresolved problem in aquatic ecology. "Tryptophan-like" dissolved organic matter fluorescence (FDOM) was found to be a much better predictor of heterotrophic bacterial metabolism in 28 Quebec lakes than dissolved organic carbon (DOC), describing 52, 44, 51 and 55% of the variability in bacterial production (BP), bacterioplankton respiration (BR), total bacterial carbon consumption (TBCC), and total plankton community respiration (CR), respectively. In addition, the study provides indirect support for the view that FDOM represents a product of bacterial activity, rather than a bioavailable substrate. This is the first field study to show that fluorescence spectroscopy can be used to characterize an aspect of DOM composition that is related to bacterial metabolism, and provides results that encourage further exploration of the potential uses of DOM fluorescence spectroscopy as a predictive tool.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Scientists and biochemical engineers alike are very interested in the control and regulation of free-energy metabolism in micro-organisms, whether the findings purely satisfy scientific curiosity or translate into the meeting of biotechnology company deadlines. We used a rather fundamental approach to investigate experimentally the control and regulation of yeast free-energy metabolism in anaerobic chemostat cultures using supply-demand analysis. This conceptually simple, quantitative framework, however, may lead to insight into the control properties of various metabolic pathways to be used in biotechnological applications. Supply-demand analysis is based on the theoretical framework of metabolic control analysis (MCA).Sections (of arbitrary size) of a metabolic pathway are grouped together around a linking metabolite. Those steps that produce the intermediate are combined into the supply block while the reactions that remove/consume the intermediate are grouped together as the demand. The elasticity coefficients of the supply and demand blocks (with regard to the linking metabolite concentration) can be used to determine the flux and concentration control coefficients by using the traditional MCAsummation and connectivity theorems. Supply and demand rate characteristics are a powerful visual approach to determine and display the control structure of the pathway under consideration and sets supply-demand analysis apart from traditional top-down analysis. Our first tool of analysis was a structured kinetic model of yeast growing in a chemos tat, constructed by using methods developed in our research group for modelling systems with variable volumes. Independent perturbations of the linking metabolite concentration resulted in a control profile where the control resided mainly in the demand (flux control coefficient of 0.92), as a result of a large negative supply elasticity. This elasticity, however, varied greatly under different conditions, leading to increased flux control by the supply in some cases. We extended our research to an experimental setup of Saccharomyces cerevisiae growing in a glucose-limited chemos tat supplemented with yeast extract as a source of carbon intermediates. This allowed glucose to act solely as the freeenergy source, as confirmed by balancing the glucose flux with the fluxes towards the fermentation products, ethanol and carbon dioxide. We obtained the supply rate characteristic by perturbing the ATPdemand through the addition of benzoate, which uncouples the proton gradient across the cell membrane. The demand rate characteristic was obtained by perturbing the ATP supply through changes in the dilution rate and thus the residual glucose concentration in the fermentor. The concentrations of ATPand ADPwere measured using a luciferase bioluminescence assay, while the fermentation products were measured with HPLCand C02 with an acoustic off-gas analyser. For our experimental conditions the flux-control of energy metabolism resided predominantly in the supply with respect to the linking metabolite [ATP]/[ADP](chosen as an indication of the free-energy state of the cell), i.e. a flux control coefficient of 0.90. Further, the [ATP]/[ADP]was under strong homeostatic control, as evidenced by the low [ATP]/[ADP]control coefficients of ± 0.12. We adjusted the structured kinetic model by varying strategic parameters, so that the results resembled the experimental observations more closely. However, the kinetics of our core model seem to be too simplistic to capture fully the extent of regulation displayed by the experimental system. The model did, however, reveal the regulatory importance of glucose transport into the cell. We conclude that the control and regulation of free energy metabolism in yeast strongly depend on the culturing conditions and on the steady state being analysed.
AFRIKAANSE OPSOMMING: Wetenskaplikes sowel as biochemiese ingenieurs is dikwels geïnteresseerd in die beheer en regulering van vry-energie metabolisme in mikro-organismes, hetsy die bevindinge suiwer wetenskaplike nuuskierigheid bevredig of die haalbaarheid van biotegnologie-maatskappy-mikpunte beteken. Ons het 'n redelik fundamentele benadering gevolg om die beheer en regulering van vry-energie metabolisme in gis eksperimenteel te bepaal in anaerobiese chemostaatkulture met behulp van aanbod- aanvraag analise. Dit is 'n konseptueel eenvoudige, kwantitatiewe raamwerk met die potensiaal om insig te gee in die beheereienskappe van verskeie metaboliese paaie wat nuttig kan wees in biotegnologiese toepassings. Aanbod-aanvraag analise is gebaseer op die teoretiese onderbou van metaboliese kontrole-analise (MKA).Dele (van arbitrêre grootte) van 'n metaboliese pad word gegroepeer rondom 'n verbindingsmetaboliet. Die stappe wat die intermediaat produseer word gekombineer as die aanbod terwyl die reaksies wat die intermediaat verbruik, saamgegroepeer word as die aanvraag. Die elastisiteitskoëffisiënte van die aanbod en aanvraag blokke (met betrekking tot die verbindingsmetabolietkonsentrasie) kan gebruik word om die fluksie en konsentrasie kontrolekoëffisiënte te bereken met behulp van die sommasie en konnektwiteit teoremas van MKA.Aanbod en aanvraag snelheidskenmerkgrafieke is 'n treffende visuele benadering om die kontroleprofiel van die betrokke metaboliese pad te bepaal en te vertoon. Hierdie kenmerk onderskei aanbod-aanvraag analise van bo-na-onder analise. Die eerste deel van ons ondersoek het behels 'n gestruktureerde kinetiese model (van gis wat groei in 'n chemostaat) met behulp van metodes wat in ons groep ontwikkel is om sisteme met variërende volumes te modelleer. Onafhanklike perturbasies van die verbindingsmetaboliet konsentrasie het gelei tot 'n kontroleprofiel waar die kontrole hoofsaaklik in die aanvraag gesetel was (fluksie kontrolekoëffisiënt van 0.92), as gevolg van 'n groot negatiewe aanbod-elastisiteit. Hierdie elastisiteit kan egter grootliks varieer tydens verskillende kondisies, wat lei tot 'n toenemende fluksle-beheer deur die aanbod in sommige gevalle. Ons het ons navorsing uitgebrei na 'n eksperimentele opstelling van Saccharomyces cerevisiae wat groei in 'n glukose-gelimiteerde chemostaat, aangevul met gisekstrak as 'n bron van koolstof-Intermediate. Dit bring mee dat glukose slegs as energiebron dien; dit is wel bevestig deur balanse op te stel van die koolstoffluksie vanaf glukose na koolstofdioksied en etanol as die fermentasieprodukte. Die aanbod snelheidskenmerkgrafiek is gegenereer deur die aanvraag van ATP te manipuleer deur middel van toevoeging van bensoaat, wat die protongradiënt oor die selmembraan ontkoppel. Die snelheidskenmerkgrafiek Vir die aanvraag is gegenereer deur die aanbod van ATP te manipuleer deur middel van 'n variasie in die verdunningstempo en sodoende die residuele glukose konsentrasie in die fermentor. Die konsentrasies van ATPen ADPis bepaal deur middel van 'n lusiferase bioluminessensie-essai, terwyl die fermentasieprodukte met 'n HPLCen CO2 met 'n akoestiese aflaatgasanaliseerder gemeet is. Vir die betrokke eksperimentele toestande was die flukste-kontrole van energiemetabolisme oorwegend in die aanbod met betrekking tot die verbindingsmetaboliet, [ATP]/[ADP](gekies as aanduiding van die vrye-energiestatus van die sel), naamlik 'n fluksie kontrolekoëffisiënt van 0.90. Verder was die [ATP]/[ADP]onder sterk homeostatiese beheer soos duidelik blyk uit die lae [ATP]j[ADP] kontrolekoëffisiënte van ± 0.12. Ons het die gestruktureerde kinetiese model aangepas deur strategiese parameters te verander om sodoende die eksperimentele gedrag te probeer naboots. Die kinetika van ons kernmodel blyk egter te simplisties te wees om die volle omvang van die regulering van die eksperimentele sisteem te vertoon. Die model het egter die belang van glukose transport oor die selmembraan aan die lig gebring. Ons kom tot die gevolgtrekking dat die beheer en regulering van vrye-energie metabolisme in gis sterk afhang van die groeitoestande sowel as die spesifieke bestendige toestand wat ondersoek word.