Dissertations / Theses on the topic 'Bacteria accumulation'

Les aminoglycosides (AG) sont une famille d’antibiotiques qui ciblent le ribosome bactérien. À titre d’exemple il s’agit de la néomycine, la gentamicine et la streptomycine. Quand ces antibiotiques se fixent au ribosome, ils provoquent des erreurs de lectures ou inhibent la synthèse des protéines, ce qui conduit à la mort cellulaire. Même s’ils ont été découverts il y a plus d’un demi-siècle, de nombreux aspects de leur mode d’action restent inconnus. L’accumulation des AG dans les bactéries se passe en trois étapes. La première consiste en une interaction électrostatique avec la membrane. La deuxième est une phase I énergie-dépendante (EDPI). Les antibiotiques rentrent dans le cytoplasme, atteignent les ribosomes, ce qui cause des erreurs de lecture et donc la production de protéines mal repliées. EDPI dépend du niveau énergétique de la cellule et le mécanisme d’entrée à travers les membranes reste inconnu. La troisième étape est la deuxième phase énergie-dépendante (EDPII), où l’antibiotique pénètre dans le cytoplasme en grande quantité par des membranes endommagées lors de la phase I. Le but de cette thèse était de créer de nouveaux outils afin d’étudier l’interaction des AG avec les bactéries et d’appliquer la méthodologie à des bactéries en phase rapide de croissance ou bien en état de persistance. Nous avons synthétisé des conjugués fluorescents des AG aux propriétés bactéricides. Avec ces conjugués nous avons analysé l’interaction des AG avec les bactéries à l’échelle de la cellule unique par microscopie de fluorescence. Nous avons combiné cette technique avec la cytométrie de flux (FACS) pour évaluer la cinétique d’accumulation. Cette étude démontre qu’il y a deux types d’accumulation : une à la périphérie avec interaction à la membrane et une deuxième où l’antibiotique est localisé dans le cytoplasme. Notre analyse démontre aussi que de faibles niveaux d’antibiotiques dans le cytoplasme sont tolérés et n’inhibent pas la croissance cellulaire. En utilisant un inhibiteur des phases EDPI et EDPII nous démontrons que cette technique permet de distinguer les différentes étapes de l’accumulation. Au cours d’ajustements du protocole, nous avons découvert que les AG peuvent entrer dans le cytoplasme par mechano-sensation et activation de canaux mécanosensibles (MS). Ces canaux sont connus pour avoir une affinité pour les AG. Ici pour la première fois nous montrons qu’une manipulation mécanique ouvre les canaux et stimule une entrée massive d’antibiotiques. Ce résultat inattendu pourrait permettre de mieux comprendre le mécanisme d’entrée des AG dans le cytoplasme. Après avoir étudié l’accumulation des AG dans les cellules en croissance nous avons étudié la tolérance aux AG pour les bactéries en phase de dormance : les cellules persistantes. Elles forment une sous-population parmi une population sensible. Elles sont en dormance et tolèrent de fortes doses d’antibiotiques. En absence d’antibiotique elles sortent de l’état de dormance pour reformer une population sensible à l’antibiotique. Par microscopie de fluorescence, nous montrons que les cellules persistantes ont une accumulation périphérique d’AG. Grâce à notre méthodologie, nous avons un outil performant pour identifier les différents états d’accumulation des AG. Avant cette étude il était seulement possible de connaître les niveaux d’accumulation mais la localisation de l’antibiotique demeurait inaccessible. Nous avons avec cette méthode étudié deux mutants d’E. coli, qui sont moins tolérants aux AG et identifié leurs caractéristiques d’accumulation. Enfin, nous avons développé un système de microfluidique adapté à l’étude de nos conjugués fluorescents pour étudier en temps réel l’accumulation par les cellules persistantes
Aminoglycoside (AG) is a family of antibiotic which target bacterial ribosome. Few examples of this family are neomycin, gentamicin and streptomycin. When these antibiotics bind to ribosomes, they cause miscoding or inhibit protein synthesis which consequently leads to cell death. Although discovery of these antibiotics was more than half a century ago, there are many facts about AGs’ action mechanism which remain unknown. AG accumulation in the bacterial cells happens in three steps. First step is cell membrane attachment. This step is driven by an electrostatic interaction with the cationic AGs. Second step is an energy dependent phase I (EDPI). In EDPI, the antibiotic enters into the cytoplasm and reaches ribosomes, causing miscoding and production of misfolded proteins. EDPI depends on cellular energy level, however to date the mechanism by which AGs pass through membranes and enter cytoplasm is unknown. The third step is energy dependent phase II (EDPII) in which the antibiotic enters into the cytoplasm in larger amount due to damages in the membrane that resulted from EDPI. The aim of this PhD was to create new tools to study the interaction of AGs with bacteria and apply the methodology to study fast growing bacteria as well as persister cells. We have made fluorescently-tagged AGs with preserved bactericidal properties. We used these conjugates to track down the interaction of AG at single cell level by fluorescence microscopy. We combined fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis to measure AGs accumulation in the cells at different time points to capture the kinetics of antibiotic penetration. This study showed that there are two accumulations patterns for the drug in cells: in the first step there is a peripheral accumulation, which corresponds to specific binging to cell membrane. Next there is a cytoplasmic accumulation in which the antibiotic in entering into the cytoplasm. According to microscopy time laps study, low levels of cytoplasmic accumulation is tolerated by cells and did not cause cell death. Using FACS analysis, we used an inhibitor of EDPI and EDPII and proved that with this technique we can distinguish different steps of AGs accumulation. During protocol adjustment steps we found that AGs can enter into the cytoplasm as a result of mechanosensation and activation of mechanosensitive (MS) channels. These channels have already been shown to have affinity to AG and here this is a first time that we observed that mechanical manipulation of cells lead to opening of MS channel causing massive cytoplasmic accumulation. This unpredictable result may lead us to a better understanding of the mechanism of AG entrance into the cytoplasm. After studying AG accumulation in fast growing cells, we studied AG tolerance for non-growing cells, which are called persisters. Persisters are antibiotic tolerant sub-population among susceptible bacterial cell population. Persisters are non-growing, dormant cells which tolerate high concentrations of antibiotic. In the absence of antibiotic, they exit this dormant state and grow into an antibiotic susceptible population. By fluorescence microscopy we showed that persister cells have peripheral accumulation of AG. Thanks to our methodology, we have a powerful tool by which we can determine the patterns of AG accumulation. Prior to this study, it was only possible to know the levels of accumulation and not the corresponding patterns. We applied the method to investigate AG accumulation in two mutants of E. coli, which are less tolerant to AG and defined their pattern of accumulation. Finally, we developed a coated microfluidic system, which is adapted to our antibiotics for studying in real time drug accumulation by persister cells

Le transport des micro-organismes, comme par exemple les bactéries, par un fluide se retrouve au centre de thématiques de recherche dans des domaines aussi variés que de la biologie, l’écologie, l’ingénierie et la médecine.Ce manuscrit résume mon étude expérimentale du couplage entre le mouvement microscopique de la nage des bactéries et le mouvement advectif de l’écoulement.La première partie du manuscrit porte sur la rhéologie des suspensions d’E. coli sous faible taux de cisaillement. Pour cette condition, j’ai montré que les perturbations hydrodynamiques induites par la nage réduisent fortement la viscosité. Cet effet peut-être si important pour qu’il soit suffisant pour compenser entièrement la perte visqueuse due au cisaillement.La seconde partie traite des expériences d’écoulement réalisées dans un canal capillaire. Pour cette géométrie, j’ai examiné le couplage pour des écoulements caractérisés par un plus fort taux de cisaillement. Le suivi des trajectoires et le dénombrement des bactéries m’ont permis de mettre en évidence l’existence d’une composante de vitesse normal à la direction de l’écoulement. Cette dernière montre que les bactéries suivent des trajectoires hélicoïdales qui s’enroulent autour du centre du capillaire d’une façon antihoraires. Cette nouvelle composante est corrélée à la migration préférentielle des bactéries dans une couche de localisation proche de la paroi du canal.Les couplages rhéotactiques bactéries/fluide que j’ai étudiés doivent avoir des conséquences potentielles sur le transport en géométries plus complexes qui mériteraient une étude particulière
The question of transfer and spreading of living microorganisms, such as motile bacteria, is of interest in biology and ecology, but also in engineering and medicine.The way in which the background flow affects the behavior of these bacteria and how it impacts the bacterial transport through complex systems and on the macroscopic properties of the fluid remains unclear and little studied.In this thesis, I present an experimental investigation of the coupling between the local bacteria-driven motion and the fluid advection.In a first part, I investigate the rheological response of E. coli suspensions when subjected to weak flows (low shear rates). I show that, in particular conditions, the microscopic perturbations caused by the bacteria highly impact on the macroscopic viscosity of the suspension, leading to a striking viscosity decrease and eventually overcoming the dissipative effects due to viscous loss. I also identify the relevant time scales defining this viscosity decrease.In a second part, I perform experiments in a capillary channel and analyze the coupling for stronger flows (higher shear rates), at which bacteria were found not to impact on the macroscopic viscosity. Instead, by analyzing the bacterial trajectories under flow, I evidence a breakage of the symmetry of this trajectories which, characterized by a preferential migration, causes the localization of the bacteria in a layer that extends over a significant distance from the surface, and thus potentially influencing the bacterial transport in complex systems

Betaine, the predominant compatible solute of bacterial cells, is transported by the ProP and ProU transport systems of E.coli and S.typhimurium under conditions of osmotic stress. Transport via these systems is modulated both at the level of gene expression and the level of activity by osmotic pressure of the medium. The primary aim of this study was to elucidate the mechanism(s) of regulation of betaine accumulation by osmotic pressure. The second objective was to establish a quantitative model of osmoadaptation in enteric bacteria with a view to predicting the survival and growth of the organisms under conditions of osmotic stress. Growth simulation by betaine strongly correlated with intracellular betaine concentration over a limited range. There was an upper limit of growth simulation achieved by the accumulation of betaine. Generally, the ProP and ProU transport systems could substitute for each other in effecting betaine accumulation. A betaine concentration as low as 100nM was able to confer osmoprotection but the growth stimulation was dependent upon the activity of the ProU system. Analysis of steady-state betaine accumulation in S.typhimurium indicated that betaine accumulation is a stress-regulated response. However, this regulation did not lie in the uptake system since they were not feedback regulated. Physiological evidence obtained from experiments using sulphydryl reagents indicated the presence of a novel betaine efflux system, which was proposed to be the site of regulation. Genetic evidence for the existence of this efflux system was sought by the isolation of betaine efflux mutants. A screening procedure was developed to detect the leak of betaine and two mutants were isolated. Analysis of the mutation in one of the mutants, however, indicated that it is located in the proP gene. Future strategies for the isolation of betaine efflux mutants are discussed.

Fusarium head blight (FHB) is a widely occurring plant disease, which is caused by fungi in the genus Fusarium. FHB leads to mycotoxin accumulation on grain, which causes food safety risk and economic loss. In addition to chemical treatments, biological strategies, like application of lactic acid bacteria (LAB), could be useful in preventing and/or eradicating mycotoxigenic Fusarium growth and mycotoxin production.After comparision of the anti-Fusarium activities by a microdilution assay against Fusarium graminearum 08/RG/BF/51, Lactobacillus rhamnosus VT1 was found to have the highest anti-Fusarium activity. Response surface methodology (RSM) was employed to optimize the incubation conditions for the production of cell-free Lactobacillus culture supernatant (CFLCS) from the strain. The best combination included 34??C, 55 hours, and shaking at 170 rpm for production of CFLCS from L. rhamnosus VT1. Under these incubation conditions, a 10% cell-free culture of Lactobacillus rhamnosus VT1 inhibited 83.7% of the Fusarium growth on microplate. MIC value of the CFLCS with a 104 conidia /well inoculum concentration is 18%.To identify the mechanisms of anti-Fusarium activity, a stepwise regression, with ?? to enter = 0.15 and ?? to remove = 0.15, was performed to analyze the data of the RSM design. It was indicated that pH, total acidity, and 3-phenyllactic acid were the most important factors and could be used to explain 39.2% variation of the anti-Fusarium activity. In addition, proteinaceous compounds might be important due to the possible synergistic effect in the CFLCS. CFLCS applied directly to grain not only prevented Fusarium growth, but also changed mycotoxin accumulation. Fusarium growth was inhibited completely by a 50% concentration (V/V) of the CFLCS applied on rice media after 14 days incubation, and almost no mycotoxins were detected. Concentrations of 15%, 30% and 50% of CFLCS as steeping water inhibited Fusarium growth and mycotoxin accumulation on barley in the malting process. Almost no mycotoxins were detected in the samples treated by 50% CFLCS. However, the germination ability of the barley samples was inhibited. In general, the CFLCS showed potential effective anti-Fusarium activity. However, the strategies of application of the CFLCS on grain should be further investigated.

Le sol possède un capital naturel qui lui confère la capacité à produire des services écosystémiques aussi bien culturel que de régulation ou d’approvisionnement, il est indispensable à la Vie telle que nous la connaissons et au développement des activités humaines. Cependant les activités anthropiques et les pollutions, notamment par les éléments traces métalliques (ETMs) tel que le mercure (Hg), perturbent les sols et modifient en profondeur l’organisation des écosystèmes. Face à ces enjeux, des projets de remédiation et de gestion des sites et sols pollués se sont multipliés durant les dernières décennies en vue de futures ré-exploitations de ces sols. Cette thèse s’inscrit dans le cadre des projets ANR-BIOFILTREE et EC2CO FREIDI-Hg gérés par le laboratoire Chrono-Environnement. Mes travaux ont permis l’exploration de la diversité des communautés de microorganismes associées à une plantation de peuplier sur un site contaminé par le Hg et géré par phytomanagement, via les approches combinées de séquençage à très haut débit et par l’approche culture dépendante. Ces méthodes combinées ont permis de révéler i) la diversité des communautés bactériennes et fongiques de la peupleraie ; ii) les groupes de microorganismes particulièrement résistant au Hg (Trichoderma et Pseudomonas) ; et iii) des bactéries promotrices de croissance des plantes (PGPB). Par ailleurs, la compréhension des mécanismes cellulaires liés à l’accumulation de Hg par les microorganismes a été un de mes sujets d’étude en partenariat avec le LIEC (Université de Lorraine). Les modèles eucaryotes Saccharomyces cerevisiae et Podospora anserina ont été utilisés pour tester le rôle potentiel de certains transporteurs d’ions dans l’entrée du Hg dans les cellules fongiques. Les résultats ont montré que le transporteur de magnésium Alr1 situé sur la membrane plasmique pourrait participer au transport du Hg. En outre, une approche de transcriptomique chez Saccharomyces cerevisiae après une courte exposition au Hg des souches mutantes et sauvages a été mise en œuvre. Pour conclure, ce travail de thèse ambitionne d’être un travail de référence pour les futurs projets de phytomanagement en milieux contaminé par le Hg, qui met en avant les communautés de microorganismes et leurs rôles fondamentaux
Soil has a natural capital that gives it the capacity to produce ecosystem services, cultural as well as regulation or supply, it is essential to the Life as we know it and the development of human activities. However, anthropogenic activities and pollution, in particular by trace elements (ETs) such as mercury (Hg), disrupt the soil and modify in depth the organization of ecosystems. Facing these challenges, remediation and management projects for polluted sites and soils have emerged during the last decades with a view to future re-exploitation of these soils. This thesis is part of the ANR-BIOFILTREE and EC2CO FREIDI-Hg projects managed by the Chrono-Environnement laboratory. My Ph-D work explored the diversity of microorganism communities associated with a poplar plantation at a Hg-contaminated site managed by phytomanagement, combining approaches such as very high-throughput sequencing and conventional culture-based techniques. These combined methods revealed i) the diversity of the bacterial and fungal communities of the poplar plantation; ii) the groups of microorganisms particularly resistant to Hg (Trichoderma and Pseudomonas); and iii) plant growth promoting bacteria (PGPB). In addition, understanding the cellular mechanisms related to the accumulation of Hg by microorganisms was one of my objectives carried out in collaboration with the LIEC (University of Lorraine). The eukaryotic models Saccharomyces cerevisiae and Podospora anserina were used to test the potential role of some ion transporters in the entry of Hg into fungal cells. The results showed that the magnesium transporter Alr1 located on the plasma membrane could participate in the transport of Hg. In addition, a transcriptomic approach in Saccharomyces cerevisiae after a short exposure to Hg of mutant and wild strains has been implemented. To conclude, this work aims to be a reference work for future phytomanagement projects in Hg-contaminated environments, which highlights micro-organism communities and their fundamental roles

Heavy metal pollutants, discharged into the ecosystem as waste by anthropogenic activities, contaminate drinking water for millions of people and animals in many regions of the world. Long term exposure to these metals, leads to several lethal diseases like cancer, keratosis, gangrene, diabetes, cardio- vascular disorders, etc. Therefore, removal of these pollutants from soil, water and environment is of great importance for human welfare. One of the possible eco-friendly solutions to this problem is the use of microorganisms that can accumulate the heavy metals from the contaminated sources, hence reducing the pollutant contents to a safe level. In this thesis an arsenic resistant bacterium Lysinibacillus sphaericus B1-CDA, a chromium resistant bacterium Enterobacter cloacae B2-DHA and a nickel resistant bacterium Lysinibacillus sp. BA2 were isolated and studied. The minimum inhibitory concentration values of these isolates are 500 mM sodium arsenate, 5.5 mM potassium chromate and 9 mM nickel chloride, respectively. The time of flight-secondary ion mass spectrometry and inductively coupled plasma-mass spectroscopy analyses revealed that after 120 h of exposure, the intracellular accumulation of arsenic in B1-CDA and chromium in B2-DHA were 5.0 mg/g dwt and 320 μg/g dwt of cell biomass, respectively. However, the arsenic and chromium contents in the liquid medium were reduced to 50% and 81%, respectively. The adsorption values of BA2 when exposed to nickel for 6 h were 238.04 mg of Ni(II) per gram of dead biomass indicating BA2 can reduce nickel content in the solution to 53.89%. Scanning electron micrograph depicted the effect of these metals on cellular morphology of the isolates. The genetic composition of B1-CDA and B2-DHA were studied in detail by sequencing of whole genomes. All genes of B1-CDA and B2-DHA predicted to be associated with resistance to heavy metals were annotated. The findings in this study accentuate the significance of these bacteria in removing toxic metals from the contaminated sources. The genetic mechanisms of these isolates in absorbing and thus removing toxic metals could be used as vehicles to cope with metal toxicity of the contaminated effluents discharged to the nature by industries and other human activities.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible biopolymers. PHAs emerge as a possible solution as substitutes of petroleum based plastics, being produced under the Biorefinery concept, in which wastes and by-products of numerous industries may be used as carbon source. This project aimed the isolation and characterization of organisms able to store PHAs from Hardwood Sulphite Spent Liquor (HSSL), a by-product of the pulp and paper industry. Isolation was performed from a Mixed Microbial Culture (MMC) selected under feast and famine conditions, using some components present in HSSL as substrates, such as acetic acid and xylose. Five pure isolates able to produce PHAs resulted from the successive streaking in solid medium containing HSSL. The purity of the isolates was evaluated through Gram staining and FISH analysis and the PHAs accumulation by Nile Blue staining. Two strains were identified as Rhohococcus spp. and three as Pseudomonas spp.. One isolate of each genus was selected and further studied in terms of growth and PHAs accumulation capability from three distinct carbon sources (HSSL, acetic acid and xylose). Both isolates, Rhodococcus spp. and Pseudomonas spp., were able to grow and use the three carbon sources as well as to produce PHAs. However, both strains showed a higher maximum specific growth rate (μmax) when HSSL was used as carbon source, 0.212 ± 0.0219 h-1 and 0.251 ± 0.0526 h-1, respectively. A qualitative evaluation of the PHAs accumulation through Nile Blue staining exhibited a higher accumulation when acetic acid was used as sole carbon source. In an attempt to identify some of the species responsible for PHAs accumulation of the selected MMC, belonging to the dominant class, Alphaproteobacteria, a 16S rDNA clone library was constructed. It was possible to identity Novosphingobium spp., Sphingobium spp. and Pleomorphomonas spp.
Polihidroxialcanoatos (PHAs) são biopolímeros biodegradáveis e biocompatíveis. Os PHAs são considerados uma solução possível como substitutos dos plásticos derivados do petróleo, podendo ser produzidos no âmbito do conceito de Biorefinaria utilizando resíduos como fonte de carbono. Este trabalho teve como objectivo o isolamento e a caracterização de bactérias produtoras de PHAs a partir de licor de cozimento ao sulfito ácido (HSSL), um sub-produto da indústria papeleira. Os isolamentos foram realizados partindo de uma cultura mista seleccionada para a acumulação de PHAs por imposição de ciclos de fome e fartura, utilizando alguns dos componentes do HSSL como substrato, nomeadamente a xilose e o ácido acético. Após repicagens sucessivas em meio sólido contendo HSSL, foi possível obter cinco isolados puros capazes de acumular PHAs. A pureza dos isolados foi avaliada através de coloração de Gram e análise FISH e a capacidade de acumulação de PHAs por coloração de Azul do Nilo. Duas estirpes foram identificadas como Rhohococcus spp. e três como Pseudomonas spp.. Um isolado de cada género foi seleccionado e estudado em termos de crescimento e capacidade de acumulação de PHAs, a partir de três fontes de carbono distintas (HSSL, ácido acético e xilose). Verificou-se que ambos os isolados, Rhodococcus spp. e Pseudomonas spp., foram capzes de crescer nos três meios e produziram PHAs. Contudo, ambas as estirpe apresentaram uma taxa específica de crescimento (μmax) superior com HSSL como fonte de carbono, 0.212 ± 0.0219h-1 e 0.251 ± 0.0526h-1 respectivamente. Uma avaliação qualitativa da acumulação de PHAs utilizando coloração Azul do Nilo mostrou uma acumulação maior nos ensaios em que o ácido acético era a única fonte de carbono. Numa tentativa de identificar algumas das espécies responsáveis pela acumulação de PHAs da cultura mista seleccionada pertencentes à classe dominante, Alfaproteobactéria, recorreu-se à construção de uma biblioteca de clones 16S rDNA. Foram identificadas as espécies Novosphingobium spp., Sphingobium spp e Pleomorphomonas spp.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Inocular sementes de trigo com a bactéria Azospirillum brasilense pode propiciar a fixação biológica de nitrogênio (FBN), porém tem sido observado efeito mais pronunciado desta inoculação no crescimento inicial de plantas, assim, com o sistema radicular mais desenvolvido, a absorção de nutrientes e água pode ser maior. Com isso, o crescimento, produtividade e exportação de nutrientes da cultura do trigo podem ser maiores, porém a resposta a esta inoculacao pode variar conforme a afinidade da bacteria com os cultivares de trigo. Dessa forma, objetivou-se avaliar o acúmulo de matéria seca e extração de nutrientes em estádios fenológicos, exportação de nutrientes pelos grãos, componentes de produção e produtividade de grãos de cultivares de trigo inoculados ou não com Azospirillum brasilense. O experimento foi desenvolvido em Selvíria - MS, em um Latossolo Vermelho Distroférrico de textura argilosa, em sistema plantio direto. O delineamento experimental foi em blocos ao acaso com quatro repetições, com os tratamentos dispostos em esquema fatorial 3 x 2, sendo três cultivares de trigo (CD 116, IPR CATUARA TM e IAC 385), com ou sem inoculação de sementes por Azospirillum brasilense (300 ml ha-1 do produto, com estirpes Abv5 e Abv6 (garantia de 2x108 UFC mL-1). Procedeu-se também a análise de regressão em esquema de parcela subdividida com quatro repetições, em que as parcelas foram constituídas pelos seis tratamentos descritos acima, e as subparcelas no tempo, por sete épocas de coletas de plantas (antes do afilhamento, afilhamento e antes da adubação nitrogenada de cobertura, folha bandeira, emborrachamento, florescimento, grão pastoso e maturação fisiológica), para o acúmulo de matéria seca e extração de nutrientes. A inoculação com Azospirillum brasilense propiciou maior índice de clorofila foliar (ICF) e acúmulo de matéria seca de raízes, independentemente do cultivar de trigo. Os cultivares de trigo extraíram quantidades totais de nutrientes semelhantes, porém a extração por tonelada de grãos produzida foi diferenciada para K, Mg, S, B, Cu, Mn e Zn. A extração de nutrientes total ou por tonelada de grãos produzida em ordem decrescente foi de N>K>P>S>Ca>Mg>Fe>Mn>Zn≥B>Cu, com ou sem a inoculação com Azospirillum brasilense. As maiores exportações totais de K, Ca, Mg, S e Mn foram obtidas sem a inoculação com Azospirillum brasilense, entretanto, esta bactéria diazotrófica proporcionou maiores exportações de N, P, K, Ca e Mg por tonelada de grãos produzida e de Zn total. As maiores exportações relativas (acima de 50% do que é extraído) foram de Zn, Cu, P, N e B e de Mg apenas com a inoculação. O cultivar CD 116 foi o mais produtivo e que propiciou as maiores exportações totais de nutrientes, apesar da menor absorção de K, Mg, S, Cu, Mn e Zn por grãos produzido em relação aos demais cultivares. A inoculação com Azospirillum brasilense interfere de forma diferenciada nos cultivares de trigo, sendo que o cultivar CD 116 proporcionou a maior produtividade de grãos quando inoculado.
Wheat seeds inoculated with Azospirillum brasilense bacteria can provide biological nitrogen fixation (BNF), but has been observed more pronounced effect of this inoculation in the initial plant growth (phytohormonal effect), thereby, with the further development of root system, the nutrients and water uptake may be greater. Thus, the growth, productivity and nutrients removal from the wheat crop may be higher, but the response to this inoculation may vary according to the affinity of the bacterium with the wheat cultivars. The aim of this study was to evaluate the dry matter accumulation the accumulation of dry matter and nutrients uptake at different growth stages, nutrients export by grains, yield components and grains yield of wheat cultivars inoculated or not with Azospirillum brasilense. The experiment was conducted in Selvíria - MS, in a distroferric Oxisol clayey in no-till system. The experimental design was a randomized block with four replications, with the treatments arranged in a factorial 3 x 2, with three wheat cultivars (CD 116, IPR CATUARA TM and IAC 385) with or without seed inoculation by Azospirillum brasilense (300 ml ha-1 product with strains Abv5 Abv6 (2x108 guarantee UFC mL-1). Also proceeded regression analysis in a split plot scheme with four replications, where the plots were the six treatments described above, and the subplots, seven plants collections seasons (before of tillering, tillering and before nitrogen topdressing, leaf flag, booting, flowering, doughy grain and physiological maturity), for the accumulation of dry matter, extraction and export of nutrients. Azospirillum brasilense inoculation provided greater leaf chlorophyll index (LCI) and accumulation of roots dry matter, regardless of the wheat cultivar. The wheat cultivars uptake similar total amounts of nutrients, but the uptake per ton of grain produced was differentiated for K, Mg, S, B, Cu, Mn and Zn. Total nutrient extraction or per ton of grain produced in descending order was N>K>P>S>Ca>Mg>Fe>Mn>Zn>B>Cu, with or without inoculation with Azospirillum brasilense. The highest total removal of K, Ca, Mg, S and Mn were obtained without inoculation with Azospirillum brasilense; however, this diazotrophic bacterium provided higher removal of N, P, K, Ca and Mg per ton of grain produced and Zn total. The highest relative removal (above 50% that is uptake) was the Zn, Cu, P, N and B and Mg only with inoculation. The CD 116 cultivar was the most productive and provided the highest total nutrients removal, despite the lower uptake of K, Mg, S, Cu, Mn and Zn by grains produced in relation to the other cultivars. The inoculation with Azospirillum brasilense interferes differently in wheat cultivars, and cultivar CD 116 obtained the highest grain yield when inoculated.

Orientador: Marcelo Carvalho Minhoto Teixeira Filho
Resumo: Inocular sementes de trigo com a bactéria Azospirillum brasilense pode propiciar a fixação biológica de nitrogênio (FBN), porém tem sido observado efeito mais pronunciado desta inoculação no crescimento inicial de plantas, assim, com o sistema radicular mais desenvolvido, a absorção de nutrientes e água pode ser maior. Com isso, o crescimento, produtividade e exportação de nutrientes da cultura do trigo podem ser maiores, porém a resposta a esta inoculacao pode variar conforme a afinidade da bacteria com os cultivares de trigo. Dessa forma, objetivou-se avaliar o acúmulo de matéria seca e extração de nutrientes em estádios fenológicos, exportação de nutrientes pelos grãos, componentes de produção e produtividade de grãos de cultivares de trigo inoculados ou não com Azospirillum brasilense. O experimento foi desenvolvido em Selvíria - MS, em um Latossolo Vermelho Distroférrico de textura argilosa, em sistema plantio direto. O delineamento experimental foi em blocos ao acaso com quatro repetições, com os tratamentos dispostos em esquema fatorial 3 x 2, sendo três cultivares de trigo (CD 116, IPR CATUARA TM e IAC 385), com ou sem inoculação de sementes por Azospirillum brasilense (300 ml ha-1 do produto, com estirpes Abv5 e Abv6 (garantia de 2x108 UFC mL-1). Procedeu-se também a análise de regressão em esquema de parcela subdividida com quatro repetições, em que as parcelas foram constituídas pelos seis tratamentos descritos acima, e as subparcelas no tempo, por sete épocas de c... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

The contamination of fresh water by phosphates in poultry litter results in substantial eutrophication of fresh water causing fish kills and other types of environmental damage. The poultry indus try in Kentucky is expanding rapidly. The number of broilers is increasing as more poultry farms are established in the state producing waste that needs disposal. Investigations were made to study the possibility of using microorganisms normally found in poultry litter to sequester phosphate, thereby delaying phosphate runoff after litter is applied to croplands. Little is known, however, about the microflora of poultry litter. Terminal restriction fragment length polymorphism of 16S rDNA from bacteria was used to investigate the bacterial diversity of poultry litter. Poultry litter was collected from a local producer. DNA was isolated using commercial kits and amplified using the polymerase chain reaction with primers specific for bacterial 16S rDNA. The amplified fragments were digested using HhaI restriction endonuclease and the DNA fragment lengths were determined. To determine the sensitivity of this method, known quantities of Escherichia coli cells were spiked into litter prior to DNA extraction. Successful amplification of the bacterial rDNA was highly variable but could be improved by passing the purified DNA through two purification columns in lieu of only one column. The detection threshold for E. coli was 10 cells, however, the results also varied widely. Bacteria capable of hyper-accumulating intracellular phosphate were isolated from poultry litter as possible tools for phosphate remediation in poultry litter. Five strains of phosphate accumulating bacteria were successfully isolated from poultry litter. Poultry litter was suspended in sterile nanopure water and 100μl was plated on BHI plates containing an addtional 750mM K2HPO4. Isolated colonies were screened for intracellular metachromatic granules using the Nile blue stain, a presumptive test for polyphosphate. Positive colonies were cultured in BHI and BHI with supplementation of K2HPO4 and free intracellular phosphate concentrations were determined in cell extracts. Total phosphates were measured in cell extracts subjected to hydrolysis by addition of 12N HCl and heating at 100°C for 60 min. Polyphosphate was determined by subtraction of free phosphates from total phosphates. Results showed five isolates of gram-positive bacteria were obtained from poultry litter. All isolates were cocci arranged in chains or clusters and were catalase positive. All isolates showed considerable levels of intracellular phosphate accumulation, which were comparable to Microlunatus phosphovorus, a bacterium known to hyper-accumulate phosphate. Biolo g analysis indicated four of the five strains isolated were Staphylococcus sp. and one strain was unidentified.

碩士
高苑科技大學
化工與生化工程研究所
100
Polyhydroxyalkanoates (PHAs), one of the biodegradable biosynthetic polymers were function as carbon and energy reservoirs in numerous bacteria. They were known to accumulate inside the bacteria usually when carbon source is excess and one else essential growth nutrient is limited. Shift-down, a temporary unbalanced bacteria culture method established by simple dilution culture procedure can equivalently induce the PHAs accumulation. A temporary unbalanced bacteria growth phenomenon established by shift-down culture procedure was applied to screening PHAs accumulating environment bacteria isolates. The culture method was applied to test PHAs accumulating in standard and environment bacteria isolates. Despite the typical limiting nutrient or C/N ratio, PHAs accumulation seems to be common in bacteria applied of the culture method. This result shown that the shift-down culture method is simple and broad spectrum in screening environment bacteria to find new industry available PHAs produced candidates. The products were characterized as PHB in recovery of Pseudomonas sp. and Bacillus sp.0.03 g/L and 0.07g/L respectively.

129I is of major concern because of its biophilic nature, excessive inventory, long half-life (~16 million yrs), and high mobility in the natural environment that depends on its chemical speciation. Iodide (I-) has the highest mobility than iodate (IO3-) and is the predominant species in the terrestrial environment due to prevailing pH and Eh conditions. In order to transform I- to less mobile organo-iodine (OI), strong oxidants are necessary to activate the first electron transfer step from I- to reactive intermediates. The aim of this study was to determine the influence of naturally occurring aerobic bacteria isolated from an 129I contaminated aquifer (F-area of the Savannah River Site, SC) on I- oxidation and OI formation. It was demonstrated that 3 of 136 strains accumulated I- (0.2~2%) in the presence of H2O2, when incubated in the presence of an environmentally relevant concentration of I- (0.1 microM). The accumulation was likely through electrophilic substitution resulting in the iodination of cellular constituents. The results indicated that culturable I--accumulating bacteria are not directly responsible for the high fraction of oxidized iodine species (IO3- and OI, >50% of total I) present in the SRS F-area. Several bacterial strains were found to be capable of stimulating I- oxidation through excretion of oxidants and enzymes. Organic acids in spent liquid medium from 27 of 84 aerobic bacterial cultures enhanced H2O2-dependent I- oxidation 2-10 fold. Organic acids enhanced I- oxidation by (1) lowering the pH of the spent medium and (2) reacting with H2O2 to form peroxy carboxylic acids, which are strong oxidizing agents. In the absence of H2O2, spent medium from 44 of 84 bacteria cultures showed I- oxidizing capacities. One I- oxidizing bacterium was studied to characterize its extracellular I- oxidizing component(s). The I- oxidizing capability from the spent medium was inactive by treatments with heat and H2O2 and absent under anaerobic conditions. Conversely, NADH, NADPH and FMN additions stimulated I- oxidation in the spend medium. These results indicate an oxidase(s) catalyzed I- oxidation. Understanding the bacterial activities involved with I- oxidation and OI formation is expected to help reduce 129I mobility in water-soil systems.

Bacterial histamine formation in mackerel and albacore was studied by inducing histamine in the muscles under controlled storage conditions. The optimum temperature for histamine formation was 25°C. The highest level of histamine detected was 283 mg/100 g in the 2-day stored mackerel; and 67.1 mg/100 g in the 6-day stored albacore. To identify the bacteria crucial to histamine formation, histamine formers were isolated using the conventional culture method. Enteric bacteria were most frequently isolated from the fish. Weak histamine formers were found in the gill and skin of fresh fish, and they required the enrichment step. Prolific histamine formers were mostly isolated from the decomposed muscles during storage at 25°C. Morganella morganii was the most prolific histamine former, producing >3,000 ppm in culture broth. M. morganii was the most prevalent histamine former in mackerel. In albacore, however, the most prevalent species was Hafnia alvei, a weak histamine former, resulting in less histamine accumulation than mackerel. Weak histamine formers, identified as natural bacteria in the marine environment, were found in mackerel during storage at 4°C after fish became unsuitable for human consumption. At 0°C, neither histamine-forming bacteria nor histamine was detected in fish. M. morganii formed significant amounts of histamine (>200 mg/100 g) in artificially contaminated fresh and frozen mackerel, albacore, and mahi-mahi when the fish were improperly stored at ambient temperatures (25°C). Growth of M. morganii was controlled by storage of fish at 4°C or below, but histamine formation was controlled only during frozen storage. For rapid detection of M. morganii, a PCR assay was developed by designing 16S rDNA targeted primers. Unique primers found for M. morganii were: the forward primer, 5'-CTCGCACCATCAGATGAACCCATAT-3'; and the reverse primer, 5'-CAAAGCATCTCTGCTAAGTTCTCTGGATG-3'. Nine CFU/ml of M. morganii inoculated in albacore homogenate were detected with a 6 h-enrichment of samples in TSB at 37°C. It would be necessary to monitor the presence of M. morganii in fish during handling and storage due to its high histamine-producing capability and prevent its contamination and proliferation after capture. The PCR assay developed in this study would be helpful to routinely monitor its presence in fish.
Graduation date: 2002

碩士
國立高雄海洋科技大學
海洋生物技術研究所
100
The marine bacterium, Paracoccus stylophorae KTW-16, was isolated from the coral Stylophora pistillata, collected from the Kenting sea waters southern Taiwan. In the marine broth (MB) without extra carbon source, P. stylophorae KTW-16 was capable of synthesizing 80 mol% 3-hydroxybutyrate (3-HB) and 20 mol% 3-hydroxyvalerate (3-HV) of PHA copolymer. At 25oC, P. stylophorae KTW-16 accumulated 30% PHA with 75 mol% 3-HB and 25 mol% 3-HV. The 3-HV monomer composition of PHA was decreased with the increase of tryptone in MB. According to this result, the effect of amino acid supplemented to the MB on PHA accumulation was tested. The addition of threonine, valine, and isoleucine significantly enhanced the 3-HV monomer composition. In addition, under the presence of β-oxidation pathway inhibitor acrylic acid in the medium, P. stylophorae KTW-16 synthesized 3-HB, 3-HV and 3-hydroxyhexanoate (3-HHx) terpolymer. The polymer structure was by nuclear magnetic resonance spectroscopy (13C-NMR) analyzed. It confirmed that three monomers were randomly copolymerized. This study also constructed a genomic library of P. stylophorae KTW-16. The PHA synthase gene was cloned. The in vivo substrate specificity of PHA synthase of P. stylophorae KTW-16 was analyzed in the PHA mutant Pseudomonas putida GPp104 PHA-. The PHA synthase of P. stylophorae KTW-16 biosynthesis 80 mol% 3-HB, 18 mol% 3-HHx and 2 mol% 3-hydroxyoctanoate (3-HO) of PHA from 0.4% octanoate as the carbon source, compared with that of Ralstonia eutropha H16 (PhaCH16) synthesizing 96 mol% 3-HB, 3 mol% 3-HHx and 1 mol% 3-HO of PHA. The above results strongly suggested that the PHA synthase of P. stylophorae KTW-16 was of broader substrate specificity than PhaCH16.

Dissertação de Mestrado em Ecologia apresentada à Faculdade de Ciências e Tecnologia
Diversas actividades antropogénicas libertam metais no meio ambiente, o que se tem vindo a tornar um problema grave. Microorganismos modificados podem ser usados como ferramentas promissoras de biorremediação para limpar áreas contaminadas por metais. O tungstênio (W) é um elemento de transição, com alta densidade que é usado em várias indústrias em todo mundo. Alguns microorganismos têm a capacidade de utilizar esse elemento como cofator para enzimas. Eles são capazes de transportar W para dentro das células usando o transportador de tungstênio altamente específico tupABC, que é constituído pela TupA (proteína de ligação W), TupB (proteína formadora de poros transmembranares) e TupC (ATPase periplasmática). Neste estudo, o grupo de genes tupABC da estirpe Sulfitobacter dubius NA4 foi usado para realizar várias construções genéticas na estirpe Escherichia coli DH5α. Foram construídos cinco clones diferentes, tupA_1 (gene completo tupA), tupA_2 (tupA sem sequência de endereçamento), tupA_3 (tupA com sequência de endereçamento do gene ompA), tupBC e tupBCA. Todos os clones foram testados quanto à capacidade de absorção de tungstênio, molibdênio (Mo) e crómio, utilizando diferentes métodos de quantificação de metais. A técnica de ICP-MS foi utilizada como abordagem padrão para a quantificação de W e Mo e os métodos DPC adaptado e ácido tânico foram utilizados como métodos espectrofotométricos para quantificação de W e Mo, respectivamente. O método DPC padrão foi usado para quantificação de cromato. Neste trabalho, concluímos que o clone tupBCA apresentou a maior capacidade de absorção W e Mo quando comparado com os outros clones. Embora a sua capacidade fosse mais relevante para W do que para o Mo. Em conclusão, estes resultados confirmaram que o sistema tupABC é o principal mecanismo de transporte de W para as células e a presença da proteína de ligação ao W é essencial para melhorar a absorção de W pelas células bacterianas. Em relação às técnicas alternativas para quantificação de W e Mo, ambos os métodos espectrofotométricos foram úteis na quantificação de metais, embora tenham mostrado algumas limitações, como a baixa sensibilidade à concentração de metais.
Several anthropogenic activities have released metals in the environment, which has become a serious issue. Modified microorganisms can be used as promising bioremediation tools to clean metal contaminated areas. Tungsten (W) is a transition element, with a high density that is used in several industries around the world. Some microorganisms have the capacity to use this element as cofactor in their enzimes. They are able to transport W into the cells using the highly specific tungsten transporter tupABC, which is constituted by the TupA (W binding protein), TupB (transmembrane pore forming protein) and TupC (periplasmatic ATPase). In this study the tupABC gene cluster of strain Sulfitobacter dubius NA4 was used to perform several genetic constructions in the strain Escherichia coli DH5α. Five different clones were constructed, tupA_1 (tupA full gene), tupA_2 (tupA without addressing sequence), tupA_3 (tupA with ompA gene addressing sequence), tupBC and tupBCA. All the clones were tested to tungsten, molybdenum (Mo) and chromium uptake capability using different metal quantification methods. ICP-MS was used as the standard approach for W and Mo quantification and the adapted DPC and the tannic acid methods were used as spectrophotometric methods for W and Mo quantification, respectively. The standard DPC method was used for chromate quantification. In this work, we concluded that clone tupBCA showed the highest ability to accumulate W and Mo when compared with the other clones. Though its capability was more relevant for W than Mo. In conclusion, these results confirmed that the tupABC system is the main W-transport mechanism to the cells and the presence of the W-binding protein is essential to improve the W uptake by bacterial cells. Moreover, in regard of the alternative techniques for W and Mo quantification, both spectrophotometric methods were useful in metal quantification, although they had shown some limitations, such as their low metal concentration sensitivity.

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology in the Department of Biotechnology, Technikon Natal, 1999.
General removal of phosphorus (P) from wastewater was introduced in Scandanavia in the late 1960's. At that time it was believed that P alone was limiting to algal growth and that the sole removal of P would solve the problem of eutrophication. However, we now know that both P and nitrogen (N) contribute to this deleterious effect and as such, much research has been conducted concerned with both the biological and chemical removal of these nutrients from sewage effluents. Enhanced biological phosphorus removal (EBPR), which is basically the biological accumulation of soluble P (as polyphosphate or poly-P) from the bulk liquid in excess of normal metabolic requirements, still tends to be sensitive to many external parameters and, as such, is subject to fluctuations. This makes it extremely difficult for wastewater treatment installations to achieve and maintain full compliance with strict discharge regulations. A more comprehensive understanding of the microbial community within the mixed liquor of a wastewater treatment system is therefore required which will ultimately assist in improving system design and performance. Chemical and civil engineers, when designing biological wastewater treatment systems, consider only the processes (biological or chemical) taking place within the reactor/s with little or no regard for the individual microbial species or the entire microbial community involved. Process design appears to be tackled empirically from a 'black box' approach; biological reactions or processes occurring within a system such as wastewater treatment are all lumped together and attributed to a single surrogate organism ie., the response of the surrogate to certain stimuli accounts for the total system response. This is similar to an analogy which Professor George Ekama (Dept of Civil Engineering, UCT), a leading scientist in wastewater treatment and process design, refers to where engineers, if, for example, are confronted with modelling the dynamics of carbon dioxide utilisation ofa forest, would recognise the accumulative system response and not give cognisance to each individual tree's contribution. It is true that if one had to consider every microbial species present in a highly organised community such as activated sludge, process models, designed to make quantitative and qualitative predictions as to the expected effluent quality from a particular design, would become increasingly complex and superfluous. It is evident from the countless accomplishments that engineers have succeeded, to a certain degree, in modelling wastewater treatment systems. One only has to consider the tremendous success of biological P (bio-P) removal and nitrification/denitrification processes at full-scale. However, there are limitations to this empirical approach and EBPR processes occasionally deteriorate in phosphate removal efficiency. In order to further optimise biological processes, whether they be organics oxidation, bio-P removal, nitrification or denitrification, biological community analyses will have to play a more significant role in design. The better microbial community structure and function is understood, the better the control and management of the system. With the advent of improved microbial identification and enumeration (to a certain extent) techniques (in situ), it was considered significant to investigate the mechanism ofbio-P removal and to elucidate which bacteria are actively responsible for this process. To this end, experimental work was conducted in two phases: \xAE laboratory, where samples of mixed liquor were obtained from a full-scale wastewater treatment facility exhibiting biological nutrient removal (BNR) characteristics and @ pilot plant, where an enhanced culture ofpolyphosphate accumulating organisms (PAO's) was developed and probed using molecular identification and enumeration techniques (as well as a cultivation-dependent approach). During phase \xAE of experimentat
M

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
O fósforo é um elemento essencial, que está presente em todos os seres vivos. Paradoxalmente, este elemento é ao mesmo tempo responsável por um tipo de poluição aquática devido a causas antropogénicas, e está em risco de escassez no futuro. As lamas ativadas, usadas em estações de tratamento de águas residuais, são ricas em fósforo e alguns países Europeus têm vindo a implementar legislações que fazem com que seja obrigatória a recuperação de fósforo nas estações de tratamento de águas.O objetivo deste trabalho é estudar a eficácia de bioaumentação de lamas ativadas numa estação de tratamento de águas residuais de escala laboratorial, para o melhoramento do processo de remoção de fósforo utilizando estirpes de bactérias nativas ou geneticamente modificadas, capazes de remover fósforo de águas residuais, nomeadamente as estipes Acinetobacter johnsonii 5bvlmeb2 e Escherichia coli BL21_pET30a_ppk1, respetivamente.Nas experiências de bioaumentação, realizada numa estação de tratamento de águas a escala laboratorial, a quantificação diária do fósforo presente no efluente, durante 5 dias, mostrou que a concentração média de fósforo na água, comparada com o controlo, foi reduzida em mais de metade quando se bioaumentou com E. coli BL21_pET30a_ppk1 ao mesmo tempo que a acumulação de polifosfato nas células aumentou substancialmente. Estes resultados indicam que a bioaumentação de lamas ativadas com esta estirpe modificada poderá potencialmente melhorar o desempenho da obtenção biológica de fósforo a partir de águas residuais no futuro e, por isso, mais estudos deverão ser realizados com a estirpe.
Phosphorus is an essential element that is found in every living entity. Paradoxically, not only is it responsible for aquatic eutrophication, due to anthropogenic causes, but is also at risk of shortage in the future. The activated sludge, produced, during wastewater treatment, is rich in this element and European countries have been implementing legislations making nutrient recovery, i.e. phosphorus, from wastewater facilities mandatory.The focus of this thesis is on studying native and genetically modified bacterial strains, such as Acinetobacter johnsonii 5bvlmeb2 and Escherichia coli BL21_pET30a_ppk1, respectively, which were show to be capable of removing phosphorus from wastewater. These will be used to bioaugment activated sludge in a laboratory-scale wastewater treatment plant. for the purpose of understanding if the bioaugmentation is efficient or not in improving the phosphorus removal process.Daily phosphorus quantification of the effluent water from the 5-day bioaugmentation experiments, performed in a laboratory-scale wastewater treatment, showed that the average residual phosphorus concentration was reduced by more than half in comparison to the control when using E. coli BL21_pET30a_ppk1. It was also shown that the polyphosphate uptake increased substantially. These results indicate that activated sludge bioaugmentation using this modified strain could potentially improve biological phosphorus removal in the future and, for that, more studies should be conducted with more depth.
Outro - Bolsa do Programa Erasmus+, durante 3 meses, financiada pela Comissão Europeia

Most chemotherapeutics fail to treat solid tumors because they cannot reach beyond regions proximal to blood vessels and are ineffective against quiescent tumor cells. Motile Salmonella typhimurium, being able to penetrate tumor tissue and chemotax towards distant tumor regions, provides an attractive drug delivery system that could break these therapeutic barriers. This dissertation focuses on two studies using genetic engineering and treatment supplement to upgrade Salmonella tumor-targeting for better therapeutic efficacy. It has previously been shown in tumor cylindroids that Salmonella lacking ribose chemoreceptors (Trg) primarily localized in tumor quiescence. To evaluate this tumor targeting specificity in vivo, a Trg-deficient Salmonella was created from the attenuated strain VNP20009 by deleting its trg gene. Both VNP20009 trg- and VNP20009 were intravenously administered into the 4T1 murine tumor model. At 12 hours, VNP20009 trg- formed twice as many colonies in quiescent tumor regions as VNP20009, with average colony size 60% bigger. It showed improved abilities to target tumor quiescence and penetrate tumor tissue. Evidence suggests that individual Salmonella bacteria may alternatively penetrate tumor tissue, proliferate or stay dormant. These findings have enriched the understanding of Salmonella tumor targeting, and suggest that controlling the origin of Salmonella intratumoral behavioral heterogeneity will benefit the quality and reliability of bacterial anticancer therapy. VNP20009 has been shown to be safe in humans, but accumulates at considerably lower densities in tumors than wild-type Salmonella. Recently it was observed that this may be associated with its inability to synthesize functional lipid A, a pro-inflammatory molecule critical to wild-type Salmonella tumor-invasion. To compensate it, three doses of diphosphoryl lipid A were injected with VNP20009 into the 4T1 murine tumor model. The dose of 2μg lipid A per mouse has raised bacterial count by 4 fold and reduced VNP20009's tumor-targeting variability by 50%. Mathematical simulation of a bacterially produced anticancer peptide diffusing out of VNP20009 colonies was created to give intratumoral peptide concentration and tumor cell killing predictions. Results suggest that injecting VNP20009 with lipid A will enable safe and robust delivery of anticancer agents otherwise unattainable with the bacteria alone.

碩士
國立臺灣大學
環境工程與科學系
86
The object of this research is to investigate how the different C/NH4-N/P(wt/wt/wt) feeding ratios influence the biochemical mechanisms of denitrification and phosphorous removal of the activated sludge in an anaerobic-aerobic alternated reactor. 　　Experimental results indicated that when the feeding ratio is 500/50/10, the polyphosphorus-accumulating bacteria (PAB) become the dominant bacteria in the SBR bioreactor and total-P removal efficiency reaches to 100%. Howerver, while changing the feeding ratio to 500/50/25 and to 500/50/50, the relative increase of P source to NH4-N source results in other bacteria instead of PAB to be the dominant bacteria. By analyzing the quantity of PHB and glycogen contained in the cell of the PAB, we verified that: the decreasing trend of glycogen during the anaerobic stage (ORP≦-200mv) can illustrate that the glycolysis provides the reducing equivalent NADH2 or NAD(P)H to form PHB, however, during the initial anaerobic stage (while the ORP is still ≧-200mv which is due to the influence of aerobic state at the last batch), acetate will form the accumulation of glucose by Glyoxylate cycle, and the Glyoxylate cycle also provides the reducing equivalent to form the PHB. 　　Also by STS modified method, this research discovered that the control of low-molecular-weight polyphosphate(LWP) is rather relative to the mechanism of phosphorous-release during anaerobic stage and the mechanism of phosphorous-uptake during aerobic stage.

碩士
國立臺灣海洋大學
水產養殖學系
100
This study evaluated the efficacies of nanosilver solution (2000 ppm) and nanosilver non-woven fabric applied in aquaculture. The MIC value of nanosilver against Vibrio alginolyticus was 8 ppm; the MBC value was 16 ppm. Nanosilver of 2000 ppm could produce bacteriostatic circles against Vibrio alginolyticus, Vibrio parahaemolyticus, Aeromonas hydrophila and Edwardsiella tarda growth in TSA, with diameters of 14.0 ± 2.0, 14.0 ± 1.5, 7.5 ± 2.5 and 14.5 ± 2.0 mm, respectively. When V. alginolyticus or V. parahaemolyticus reached a bacterial count of 103 CFU/mL in seawater, treatment with 8 ppb nanosilver for 2 hours or 1/2 hour had a complete bacteriostatic effect; when the bacterial count increased to 105 CFU/mL, treatment time with the same concentration increased to 4 and 1 hour. When A. hydrophila or E. tarda reached a bacterial count of 103 CFU/mL in freshwater, treatment with 1 ppb nanosilver for 1/2 hour or 2 hours had a bacteriostatic effect; when the bacterial count increased to 105 CFU/mL, treatment with 2 ppb for 1/2 hour was effective. In the experiment to find the bacteroistatic effect of nanosilver in different salinities of seawater, 15 and 25 ppt seawater which contained 103 CFU/mL V. alginolyticus were treated with 8 ppb nanosilver, and the bacteriostatic effects could last for 2 days; when the salinity was increased to 35 ppt, the effect could only last for 1 day. When the nanosilver concentration was increased to 16 ppb, the effect could last for 4 days in 35 and 25 ppt seawater, and for 5 days in 15 ppt seawater. This experiment also confirmed that nanosilver, less than 16 ppb, would not affect the ammonia-nitrogen, nitrite, dissolved oxygen, pH or redox potential in aquaculture water. In the disinfectant experiment on live feeds, treatment of 16 ppb nanosilver on Brachionus plicatilis for 15 minutes could maintain their vitality and has a good bacteriostatic effect; however, nanosilver was unsuitable to be used for disinfection of Isochrysis galbana. Feeding diets supplemented with 800 ppb nanosilver to Epinephelus coioides for 8 weeks would not affect their growth or survival; nanosilver would not remain in the muscle tissues, but would remain in the liver, intestines, brain, and head kidney. Also, treatment with 25 ppb nanosilver on Acanthopagrus sivicolus fertilized eggs for 10 minutes could result in bacteriostasis, and their hatchability would not be affected. 15 grams of nanosilver non-woven fabric was used as a filter material to treat seawater contained 103 CFU/mL V. alginolyticus, at a water flow rate of 5 ± 0.5 mL/sec. After one filtration process, 50% of the bacteria could be diminished; after four processes, 75% could be diminished. This study assessed that nanosilver and nanosilver non-woven fabric were usable in aquaculture as a bacteriostatic supplement and as a filter material.