La décontamination microbienne est sujet majeur de préoccupation du secteur agroalimentaire. Des nouvelles technologies physiques de décontamination, dites athermiques, sont d’un emploi croissant. La Lumière Pulsée, utilisée pour décontaminer les surfaces et les liquides clairs, en fait partie. Elle utilise des flashes de lumière blanche riches en UV, et délivrés en moins d'une milliseconde. La plupart des traitements par lumière pulsée sont définis dans la littérature par des paramètres spécifiques à l'équipement utilisé. Le but de cette étude a été dans un premier temps de caractériser le traitement par lumière pulsée par les grandeurs physiques appropriées (fluence, tension aux bornes de la lampe, etc…), en reliant une dose de lumière à niveau de décontamination microbienne. L'équipement pilote de la société CLARANOR a révélé des réduction logarithmiques allant jusqu'à plus de 5 unités sur des spores de B. subtilis, et de plusieurs autres espèces de bactéries sporulées, avec des fluences inférieures à 1,5 J/cm², appliquée en un seul flash La mise au point d'une méthode d'inoculation par spray à permis d'évaluer l'efficacité décontaminante de la lumière sur différentes surfaces, y compris des hydrophobes, par pulvérisation des microorganismes en couches formées d’une seule épaisseur de cellules. L'application de la technologie sur des surfaces inertes comme le polystyrène a montré une décontamination notamment sur des spores de B. subtilis, et d'A. niger, supérieures à 4 cycles logarithmiques en utilisant des fluences inférieures à 1 J/cm². L'influence des facteurs liés au système d'éclairage a montré une importance capitale des longueurs d'onde UV, mais ne permettent pas de réduire l'efficacité à la seule action de la dose UV-C. L'efficacité de la technologie a permis de réaliser une étude concernant la décontamination de sirop de sucre dans une optique d'application industrielle. Une réduction supérieure à 3 cycles logarithmiques de spores d'A. acidoterrestris dans du sirop de saccharose a été obtenue en flux continu, sur une épaisseur de 10 mm de liquide / Microbial decontamination is a major concern in the food industry. Non-thermal physical technologies are increasingly used. Pulsed Light used to decontaminate surfaces and clear liquids is one of these new technologies. Pulsed Light uses intense flashes of white light rich in UV, delivered in less than one millisecond. Most of treatments are characterised in the literature using parameters which are specific to the equipment. The aim of this study was firstly to characterise the PL treatment in expressing a log reduction as a function of the dose received by the microorganism. The pulsed light pilot of the CLARANOR company allowed a high decontamination of B. subtilis spores and other sporulating bacterial species, with more than 5 log reductions at fluences lower than 1.5 J/cm², obtained in only one flash. The development of a spray inoculation method was made to evaluate the decontamination efficiency on different surfaces, including hydrophobic surfaces, with a monolayer inoculation. The Pulsed light efficiency on inert surfaces such as polystyrene lead to high decontaminations including B. subtilis and A. niger spores, with more than 4 log reductions using fluences lower than 1 J/cm² in both cases. The influence of the physical factors of the light showed that UV wavelengths are essential for the decontamination, but the efficiency is not totally explained by the action of the UV-C dose. The efficiency of pulsed light allowed to study sugar syrup decontamination, in view of industrial application. Three log reductions of A. acidoterrestris spores were obtained in 10 mm thickness sugar syrup, using a flow-through system

Dans les environnements naturels, industriels ou médicaux, les microorganismes sont majoritairement présents en étant associés aux surfaces dans des communautés hautement organisées appelées biofilms. Ces édifices biologiques constituent une stratégie de survie étonnement efficace témoignant d’une grande capacité de résistance à différent stress environnementaux tels que les traitements de nettoyage et de désinfection. L’impact des biofilms d’un point de vue sanitaire est donc considérable du fait qu’ils permettent la persistance et la transmission de germes pathogènes dans l’environnement. Dans ce contexte, ce travail de thèse avait pour objectif une meilleure compréhension des phénomènes limitant l’efficacité de désinfectants au sein des biofilms en s’appuyant notamment sur des techniques innovantes d’imagerie de fluorescence non-invasive. Le but final étant d’apporter des éléments utiles à l’optimisation des traitements de désinfection. Dans une première partie, une méthode d’investigation structurale à haut-débit par microscopie confocale a été développée et utilisée pour étudier la diversité architecturale des biofilms bactériens formés par un large panel de souches. Cette étude nous a permis d’identifier des souches d’intérêt en termes de structures de biofilms formés pour la suite du travail. Nous avons notamment pu mettre en évidence la capacité de B. subtilis à former des structures importantes et avec une architecture spécifique dans un système immergé. Dans une deuxième partie, les dynamiques d’action spatiotemporelles de désinfectants ont été visualisées dans les biofilms de souches de P. aeruginosa ou B. subtilis par des approches de microscopie confocale de fluorescence en temps réel. L’utilisation de cette technique nous a permis de mettre en évidence les difficultés de pénétration du chlorure de benzalkonium au sein des structures formées par différentes souches de P. aeruginosa. La corrélation des paramètres cinétiques d’inactivation et des données obtenues par la caractérisation biochimique de la matrice suggère un rôle majeur des substances extracellulaires dans la limitation de pénétraton du désinfectant. Nous avons également pu montrer une résistance marquée du biofilm formé par une souche de B. subtilis isolée d’un dispositif médical à l’acide péracétique, à la concentration et au temps d’utilisation du biocide dans le milieu médical. De plus, les structures tridimensionnelles formées par cette souche étaient capables de protéger le pathogène Staphylococcus aureus dans un biofilm mixte vis-à-vis du même traitement soulignant l’importance des interactions multi-espèces dans la résistance des bactéries aux désinfectants et la persistance de pathogènes dans nos environnements. / In natural, industrial or medical environments, microorganisms are present mainly in being associated with surfaces in highly organized communities called biofilms. These biological structures cosntitute a surprisingly effective survival strategy showing a large ability to withstand environmental stresses such as cleaning and disinfection treatments. Therefore, biofilms have a considerable impact on public health because they allow the persistence and transmission of pathogens. In this context, this work aimed to better understand the phenomena limiting the effectiveness of disinfectants in biofilms noticeably by using innovative imaging fluorescence non-invasive techniques. The ultimate goal was to provide data which can help to optimize disinfection treatments. In the first part, a high-throughput structural method based on confocal microscopy was developed and used to study the architectural diversity of bacterial biofilms formed by a wide range of strains. This study allowed us to identify strains of interest in terms of biofilm structure for the second part of the work. In particular, we demonstrated the ability of B. subtilis to form protruding structures with a specific architecture in a submerged system. In the second part, the spatiotemporal dynamic of the action of disinfectants were visualized in the biofilms of P. aeruginosa or B. subtilis strains by a time-lapse fluorescence confocal microscopy method. Using this technique, we showed that benzalkonium chloride encountered problems of penetration in the biofilms formed by P. aeruginosa strains. The correlation of kinetic inactivation parameters and data obtained by the characterization biochemical matrix suggested a key role of extracellular substances in the penetration limitations of the disinfectant. We also observed a pronounced resistance of the biofilm formed by a strain of B. subtilis isolated from a medical device to peracetic acid at the in-use concentration and time of biocide in medical areas. In addition, three-dimensional structures formed by this strains afforded protection to the pathogen Staphylococcus aureus in mixed biofilm against the same treatment This point highlights the importance of multi-species interactions in bacterial resistance to disinfectants and in the persistence of pathogens in our environments.

Bacillus subtilis Stämme vom FZB - FZB24, FZB37 und FZB38 des FZB Biotechnik, Berlin - und ihre Stoffwechselprodukte wurden als Agenzien für eine Resistenzinduktion gegen Schädiger in drei Wirtspflanzen-Erreger-Systemen, Vicia faba - Uromyces appendiculatus, Vicia faba - Aphis fabae und Triticum aestivum - Rhopalosiphum padi untersucht. Prä-inokulativ wurden Blätter und Saatgut der Pflanzen mit den Testsubstanzen behandelt. Zur Testung gelangten Bakterien-Kulturfiltrat (KF), KF-Zenrifugationsüberstände, die vegetativen Zellen und Sporen der B. subtilis Stämme. Nach lokaler Applikation der KF-Überstände von B. subtilis auf die Wirtspflanzenblätter konnte eine erhebliche Breite antibiotischer (entwicklungshemmender) Wirkungen auf A. fabae und R. padi beobachtet werden. Eine Untersuchung der freien Aminosäuren im Phloemsaft von Vicia faba zeigte, daß sich nach Befall von Aphis fabae speziell bei den zuvor mit bakteriellem Kulturfiltrat und KF-Überstand behandelten Pflanzen im Vergleich zu der nur mit Wasser behandelten Kontrolle, die Konzentration von neun Aminosäuren änderte. / In topical and systemic plant treatment, in three host–parasite systems, i.e. Vicia faba - Uromyces appendiculatus, Vicia faba - Aphis fabae and Triticum aestivum - Rhopalosiphum padi the culture filtrate and supernatant of Bacillus subtilis (FZB24, FZB37 and FZB38 from FZB Biotechnik Berlin) was shown to inhibit the development of urediospores produced by Uromyces appendiculatus. The performance of Aphis fabae and Rhopalosiphum padi was evaluated using life table tests where the aphids'' development time (tD), pre-reproduction time (td), relative growth rate (RGR) and intrinsic rate of natural increase (rm) were assessed. A wide range of antibiosis effects in Aphis fabae and Rhopalosiphum padi was observed when the supernatant of Bacillus subtilis was used as foliar topical treatment. The tested aphids presented longer development and pre-reproduction time; conversely a lower relative growth rate and intrinsic rate of natural increase was observed. The investigation of the free amino acids of the phloem sap of V. faba plants, showed the concentration of the amino acid serine remained unchanged in the supernatant induced plants in this study, which was interpreted as the major reason for the observed antibiosis effect on the tested aphids.

Untersuchungen wurden durchgeführt, um die Wirkung von B. subtilis und deren Metaboliten auf den Meloidogyne- und Pratylenchus-Befall und ihre Vermehrung festzustellen sowie um die möglichen Wirkmechanismen zu studieren. Substratbehandlungen mit B. subtilis FZB 24® führten zu einem höherenMeloidogyne-Befall und einer verstärkten Nematodenvermehrung. Trotz verstärktem Befall wurde das Pflanzenwachstum verbessert (induzierte Toleranz). Weiterhin wurden durch "antibiotikafreie" Kulturfiltrate (KF) aus der bakterielen Übergangs- und stationären Phase ähnliche Wirkungen erzielt. Außerdem wurde eine systemische Wirkung von B. subtilis auf den Meloidogyne-Befall an Tomate nachgewiesen. B. subtilis bzw.die KF-behandelte Testpflanzen zeigten stärkere Anlockwirkung auf Meloidogyne-Larven (M. incognita, M. arenaria) als unbehandelte. KF (50, 10, 1%) von B. subtilis zeigten keine nematizide Wirkung auf die Meloidogyne-Larven. KNO3 als Trägersubstanz für das Bakterienpräparat besaß ähnliche Wirkungen auf den Meloidogyne-Befall und die Nematodenvermehrung.Ebenfalls wurde das Pflanzenwachstum durch KNO3-Zufuhr gefördert. Der Einsatz des nematodenfangenden Pilzes Arthrobotrys superba reduzierte den Meloidogyne-Befall (30% Gallenreduktion). Hingegen wurde durch die kombinierte Anwendung von A.superba und B. subtilis FZB 24® der Bekämpfungserfolg von A. superba reduziert. Die exogene Applikation von Phytohormonen bzw. Präkursoren zeigte keine Wirkung auf das Wachstum der Testpflanzen. Die Vermehrung von Meloidogyne wurde durch IAA und die Kombination von IAA und Kinetin gefördert. In den getesteten Konzentrationen der Phytohormone wurde keine Wirkung auf die Mortalität der Wurzelgallenälchen- (Meloidogyne-) Larven beobachtet. Der Gehalt von Enzymen (Chitinase, Glucanase und Peroxidase) aus dem Sproß behandelter Tomatenpflanzen wurde bestimmt. B. subtilis-Isolate (FZB 24® , S18) reduzierten die Population von Wurzelläsionsnematoden, Pratylenchus penetrans (nicht signifikant) [9 bzw. 15-20% pro Wurzelsystem bzw. g Wurzel]. Das Pflanzenwachstum wurde an befallenen Pflanzen durch beide Isolate verbessert (induzierte Resistenz). Es wurden keine Unterschiede zwischen den Bakterien-Isolaten festgestellt. KNO3 führte ebenfalls zu einer Verminderung der Nematodenpopulation. Die Ergebnisse werden hinsichtlich möglicher Wirkmechanismen des RhizobakteriumsB. subtilis und des nematodenfangendes PilzesA. superba zur Regulierung der Nematodenpopulation bei Tomate diskutiert. / Investigations were made to know about the effects of Bacillus subtilis and its metabolites on the infestation of tomato plants with root-knot and lesion nematodes. Further more, experiments were carried out to clear up the mode of actions ofB. subtilis and its culture filtrates on infestation of tomato seedlings and reproduction of root-knot nematodes. Substrate applications ofB. subtilis FZB 24® leaded to an increasement of infestations intensity and reproduction of root-knot nematodes (M. arenaria). Eventhough, bacterized and inoculated plants with root-knot nematodes showed better growth than with bacteria untreated plants (induced tolerance). In addition "antibiotica free" culture filtrates (CF) from transitional and stationary bacterial growth phase also promoted reproduction of root-knot nematodes. These CF elicitized tolerance of tomato plants towardsMeloidogyne too. It was proved that B. subtilis could induce a systemic reaction of tomato plants towards root-knot infestation. Besides that test plants treated with B. subtilis (cells) or CF were more attractive to Meloidogyne-Larvae (M.arenaria & M.incognita) than untreated once. CF in 50, 10 and 1% concentrations did not have a nematicidal effect on the root-knot larvae. KNO3 -the carrier of the bacterial preparate (B. subtilis FZB 24®) - had also the same effects on infestation and reproduction of root-knot nematodes. Plant growth was promoted due to application of KNO3. The use of nematode trapping fungus, Arthrobotrys superba gave some range of nematode (root-knot) control (30% gall reduction). Whereas, with the combination ofA. superba and B. subtilis FZB 24® the effect of the fungus was reduced. The application of exogenic phytohormones and precursores showed no effect on plant growth. Reproduction of Meloidogyne was promoted by IAA and combination of IAA and kinetin (not significant). In the tested concentrations of these phytohormones there was no direct mortality effect on root-knot larvae. Content of some enzymes (chitinase, glucanase and peroxidase) from shoot of treated tomato plants was determind. B. subtilis-Isolates (FZB 24® and S18) reduced the population of lesion nematodes,Pratylenchus penetrans in attacked plants (not significant) [9% per root system and 15-20% per g root]. The treatment improved the predisposition of the plants to lesion nematodes (induced resistance). Plant growth was also improved. There was no difference between the bacterial isolates in their effect. KNO3 reduced also nematode population. In general the results would be explained and discussed towards possible mode of actions of rhizobacterium B.subtilis and nematode trapping fungus A. superba.

Der Einfluss der Rhizosphärenbakterien Bacillus subtilis FZB 24, FZB 37, FZB 42, Bacillus pumilus RK 13 und Streptomyces graminofaciens und des Algenpräparates Goemar Fruton Spezial® (Ascophyllum nodosum) auf Wachstum, Ertrag und Toleranz gegenüber Fusarium spp. bei Spargel wurde untersucht. Es wurden mehrjährige Parzellenfeldversuche und Modellversuche unter kontrollierten Bedingungen mit natürlich Fusarium spp. belasteten Böden und mit speziellen Fusarium Erregern inokulierten Substraten durchgeführt. Die Analyse des verwendeten Nachbaubodens ergab eine wesentlich höhere Kontamination mit Fusarium spp. (56%) in Relation zum Fruchtfolgeboden (14%). F. oxysporum war die dominante Fusariumart. Desweiteren wurde eine ganze Reihe anderer Arten, wie z.B. F. acuminatum, F. culmorum, F. proliferatum und F. culmorum bestimmt, was die Komplexität der Wurzel- und Stängelfäuleerkrankung belegt. Analog zu diesem Ergebnis konnten im Vergleich zur Fruchtfolgevariante von den in dem Nachbauboden kultivierten Spargelpflanzen signifikant mehr Fusarium spp. (80%) isoliert werden. Das Wurzelwachstum war hier extrem reduziert. Unter diesen konduktiven Bedingungen gelang es durch Rhizombakterisierung (107 cfu/ml) mit B. subtilis FZB 42, eine signifikante Förderung des Wurzelwachstums relativ zur unbehandelten Kontrolle und zur B. subtilis FZB 37 Variante zu erzielen. In Pathogenitätstests konnten F. culmorum, F. oxysporum und F. proliferatum als bedeutende Spargelpathogene mit signifikant reduzierter Trieb- und Wurzelmasse nachgewiesen werden, während F. acuminatum nur geringe Symptome verursachte. Eine bakterielle Saatgutbeizung (108 cfu/ml) und zusätzliche präinfektionelle Gießapplikation (107 cfu/ml) konnte eine Infektion mit F. oxysporum f.sp. asparagi nicht verhindern. Dennoch waren die negativen Effekte im Vergleich mit der nicht bakterisierten Kontrolle in den B. subtilis Varianten FZB 24 und vor allem bei FZB 42 deutlich kompensiert. Diese Ergebnisse lassen vermuten, dass eine Resistenz- bzw. Toleranzinduktion ein potentieller Wirkmechanismus der Bakterien ist. In den Feldversuchen erbrachte eine Bakterisierung einjähriger Rhizome (107 cfu/ml) zur Pflanzung und ergänzende Gießbehandlungen (108 cfu/ml/1l/m) in den folgenden 2 Jahren eine tendenzielle Reduzierung der Trieblänge und des Triebdurchmessers, insbesondere in Kombination mit dem Algenpräparat. Einen eindeutigen Einfluss auf das Sortierungsergebnis konnte nicht nachgewiesen werden. Im Gegensatz hierzu resultierte eine Saatgutbakterisierung mit B. subtilis FZB 24 in einem generell geförderten Triebwachstum und einer signifikant gesteigerten Wurzelentwicklung. Auch die anderen Mikroorganismen erbrachten eine tendenzielle Wuchsförderung. Es konnte eine gesicherte Korrelation zwischen Wurzelmasse und Knospenanzahl ermittelt werden. Blattapplikationen mit Goemar Fruton Spezial® während der Hauptvegetationsphase hatten keinen Einfluss auf das Pflanzenwachstum. / The effects of the rhizobacteria Bacillus subtilis, Strain FZB 24, FZB 37and FZB 42, Bacillus pumilus RK 13, Streptomyces graminofaciens N6 and the alga Ascophyllum nodosum (Goemar Fruton Spezial®) on plant growth, yield and Fusarium spp. tolerance of Asparagus officinalis (L.) were investigated. The trials were carried out under field conditions over sev-eral years by sawing and planting and as pot trials in the greenhouse and climate chamber with soil naturally infested with Fusarium spp. and with steamed substrates inoculated with particularly Fusarium species. Asparagus replant soil was considerable higher infested with Fusarium spp. (56 %) than fresh soil without asparagus history (14 %). F. oxysporum was the most determined Fusarium species followed by F. redolens, F. acuminatum, F. culmorum, F. proliferatum, F. solani a.o.. That indicates the complex character of root and crown rot of asparagus. From asparagus plants cultivated in replant soil a significant higher rate of Fusarium isolations (80 %) could be worked out compared to the fresh soil treatment. The root growth in the contaminated soil was extremely reduced. In these conductive situation bacterial treatments with B. subtilis FZB 42 done as rhizom soaking (107cfu/ml) resulted in significantly increased root growth up to 32, 9 % related to the non treated control. B. subtilis FZB 37 was ineffective. F. oxysporum, F. culmorum and F. proliferatum showed a high pathogenicity to asparagus seedlings, manifested in significantly reduced fresh and dry weights of shoots and roots, while F. acuminatum caused only slight symptoms. None of the tested microorganisms applicated as seed coating (108 cfu/ml) and substrate drenching before pathogen inoculation (107 cfu/ml) could prevent seedlings from being infected by F. oxysporum f. sp. asparagi. The negative effects of infection are significantly compensated by Bacillus subtilis FZB 42 and FZB 24. The results support the conclusion, that induced tolerance is a potential mechanism of bacterial mode of action. Under field conditions rhizom bacterization of one year old asparagus plants (107 cfu/ml) before planting and additional soil drenching (108 cfu/ml/1,5l/m) during the following two years reduced shoot length, shoot diameter and yield, especially in combination with Goemar Fruton Spezial®. There was no remarkable influence on spear quality. Seed coating with B. subtilis FZB 24 (108 cfu/ml) and a soil drenching (108 cfu/ml/1l/m) in summer however re-sulted in higher shoots and significantly increased fresh weight and dry substance of roots. But also the other tested microorganisms showed a plant growth promoting trend. There was a significant positive correlation between root fresh weight and number of buds. By spraying the phylloclads four times during the main vegetation season the alga application only gave a slight improvement of plant growth in the field trials.

A citocinese bacteriana é controlada por diversas proteínas que se agrupam em um complexo chamado divisomo. O cerne do divisomo é constituído por FtsZ, uma proteína homóloga à tubulina eucariótica, que se auto-associa formando uma estrutura chamada anel Z. O anel Z serve como arcabouço e recruta diversas outras proteínas componentes do divisomo para o sítio onde o septo será sintetizado na célula. A formação do anel Z é modulada por proteínas que se ligam diretamente a FtsZ e regulam a sua auto-associação, tanto induzindo como inibindo a sua polimerização. Apesar de muitos destes moduladores de FtsZ já serem conhecidos, muito pouco se sabe sobre o mecanismo pelo qual eles controlam a estruturação do anel Z in vivo. O objetivo do presente trabalho foi estudar a interação entre FtsZ e um modulador de divisão, a proteína ZapA, da bactéria gram-positiva Bacillus subtilis. Para isso construímos uma biblioteca de mutantes de ftsZ por \"Error Prone PCR\", com aproximadamente 1 substituição por cópia de ftsZ e contendo um total de 1x105 clones. A partir dessa biblioteca, utilizamos duas triagens genéticas para identificar mutantes incapazes de interagir com ZapA. Na primeira estratégia, selecionamos 12 mutantes de FtsZ resistentes à superexpressão de uma forma tóxica de ZapA, que bloqueia a divisão, causando filamentação e morte das células. Surpreendentemente, apesar destes mutantes serem insensíveis ao efeito de ZapA, ensaios citológicos mostraram que nenhum deles perdeu a interação com ZapA. Como as mutações foram mapeadas nas vizinhanças do sítio catalítico e de polimerização de FtsZ, e como a maioria delas confere resistência cruzada aos efeitos de outros moduladores de FtsZ, suspeitamos que elas afetassem a estabilidade do polímero de FtsZ e, consequentemente, o comportamento do anel Z. Essas suspeitas foram confirmadas em ensaios de FRAP e cálculos de proporção de FtsZ no anel Z, indicando que os mutantes formam um anel Z mais estável que o normal. Como não obtivemos mutantes que perderam a interação com ZapA na primeira triagem, aplicamos a biblioteca em uma segunda estratégia de triagem genética, procurando um mutante de FtsZ que voltasse a interagir com um mutante de ZapA que não se liga mais a FtsZ (ZapAN62A). Esta estratégia de ganho de função identificou um candidato, FtsZE91V , que, tanto por critérios genéticos como citológicos, voltou a interagir com ZapAN62A. Apesar do mutante FtsZE91V mostrar-se capaz de restaurar a interação com ZapAN62A, ele não afetou a interação com ZapA selvagem, segundo nossos ensaios de microscopia de fluorescência e viabilidade. O mutante FtsZE91V, mapeia na hélice H3 de FtsZ. Esta hélice está exposta na superfície de FtsZ (compõe um dos lados da molécula de FtsZ) de uma maneira compatível com a idéia de que ela seria importante para interações laterais entre polímeros de FtsZ. Nossos resultados apontam, portanto, que a hélice H3 deve ser o sítio de interação para ZapA em FtsZ. / The bacterial cytokinesis is ruled by a number of proteins that constitute the divisome complex. FtsZ, a homologue of eukaryotic tubulin, is the main component of the divisome and self-associates in a structure named Z ring. The Z ring works as a scaffold and recruits the other components of divisome, establishing itself where the septum will be synthesized in the cell. Some of these proteins interact directly with FtsZ and control self-association, promoting polymerization or preventing it. Although there have been discovered many of FtsZ modulators, little is known about the mechanisms that control the formation of the Z ring in vivo. The aim of this work was study de interaction between FtsZ e one of its division modulators, ZapA protein, on Bacillus subtilis grampositive bacteria. We created a mutagenized ftsZ plasmid library by error prone PCR, which contained 1,0x105 transformants and exhibited a mutation rate of one substitution per ftsZ copy. The library was transformed into a modified Bacillus subtilis strain and we performed two genetic screenings to select cells with FtsZ mutants incapable of interacting with ZapA. In first strategy, we selected 12 resistant ftsZ mutants for a toxic ZapA overexpression, that blocked division and caused filamentation and cell death. Surprisingly, although these mutants were insensitive to ZapA effect, cytological assays showed that none of them lost interaction with ZapA. As the substitutions were mapped around the catalytic and interaction site of FtsZ structure and showed resistance to other modulators, we suspected that the mutations were affecting the polymer stability of FtsZ and, consequently, the behavior of Z ring. This hypothesis was confirmed by FRAP experiments and by calculations of FtsZ proportions in Z ring, pointing out that the mutants form more stable Z rings. As we didnt\' find mutants that lost their ZapA´s interaction, we applied our library in a second genetic screen, looking for mutants that return to interact with a ZapA mutant (ZapAN62A) that doesn´t bind to FtsZ anymore. This gain of function strategy identified one candidate, FtsZE91V, which returns to interact with ZapAN62A in our genetic and cytological assays. Although the mutant FtsZE91V showed itself capable to interact with ZapAN62A, that didn´t affect the interaction with wild type ZapA by our fluorescent microscopy and viability assays. The substitution E91V was mapped on H3 helix of FtsZ structure. This helix is exposed on FtsZ surfaces (on FtsZ´s lateral side), being compatible with the idea that lateral interaction is important in FtsZ polymers. So, we concluded that helix H3 is the binding site of ZapA in FtsZ.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2019-02-06T18:32:21Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Gislaine Gabardo.pdf: 2546849 bytes, checksum: 3e5114c040fc11f334d987259a7b5968 (MD5) / Made available in DSpace on 2019-02-06T18:32:21Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Gislaine Gabardo.pdf: 2546849 bytes, checksum: 3e5114c040fc11f334d987259a7b5968 (MD5) Previous issue date: 2018-12-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste trabalho foi avaliar a aplicação de produtos alternativos isolados e associados a fungicida em diferentes épocas de semeadura na eficiência no controle de doenças foliares, alterações fisiológicas, desfolha, componentes de rendimento, produtividade e qualidade fisiológica das sementes obtidas na cultura da soja (Glycine max). Foram conduzidos experimentos à campo em Ponta Grossa, PR, Brasil, durante as safras 2015/2016, 2016/2017 e 2017/2018 com as cultivares NA 5909 e TMG 7062. Os tratamentos para os experimentos foram: 1 - testemunha (água) (V4, V6, R1 e R5.1), 2 - Bacillus subtilis linhagem QST (V4, V6, R1 e R5.1), 3 - Bacillus subtilis linhagem QST (V4 e V6) associado a azoxistrobina + benzovindiflupir (R1 e R5.1), 4 - quitosana 1% (V4, V6, R1 e R5.1), 5 - quitosana 1% (V4 e V6) associado a azoxistrobina + benzovindiflupir (R1 e R5.1), 6 - enxofre (V4, V6, R1 e R5.1), 7 - enxofre (V4 e V6) associado a fungicida (R1 e R5.1), 8 - hipoclorito de sódio (V4, V6, R1 e R5.1), 9 - hipoclorito de sódio (V4 e V6) associado a azoxistrobina + benzovindiflupir (R1 e R5.1), 10 - azoxistrobina + benzovindiflupir (R1 e R5.1), 11 - azoxistrobina + benzovindiflupir (V6, R1 e R5.1). Em todos os tratamentos com fungicida foi adicionado o adjuvante Nimbus® (0,5 v/v). Na semeadura em outubro ocorreu a menor severidade de doenças. A área abaixo da curva de progresso da doença (AACPD) da ferrugem asiática foi menor na cultivar TMG 7062, nas duas épocas de semeadura e safras. Em todas as safras e épocas de semeadura, os tratamentos alternativos com enxofre e quitosana (isolados), os tratamentos alternativos associados ao fungicida e o fungicida aplicado duas e três vezes, diminuíram a severidade da ferrugem asiática e do oídio nas duas cultivares. O fungicida elevou os teores de clorofila α, β e total na cultivar TMG 7062. A cultivar TMG 7062 apresentou maior condutância estomática, assimilação de CO2 e transpiração, em relação à NA 5909. A desfolha foi reduzida para as duas cultivares nos tratamentos com enxofre isolado; os produtos alternativos associados ao fungicida e o fungicida aplicado em duas ou três vezes, em todas as safras e épocas de semeadura. Os componentes de rendimento afetados foram o número de vagens por planta e a massa de mil grãos. Houve redução na produtividade quando a cultura foi semeada em dezembro. A cultivar TMG 7062, na primeira época de semeadura não respondeu aos tratamentos para a produtividade. Os tratamentos com fungicida, enxofre, bem como os produtos alternativos associados, evitaram danos a produtividade em todas as épocas de semeadura, safras e cultivares. Destacou-se o tratamento de enxofre associado ao fungicida que foi equivalente ao tratamento com fungicida aplicado três vezes, havendo possibilidade da inserção desta estratégia de manejo pelos produtores da região. Com relação as sementes obtidas, a cultivar NA 5909 apresentou maiores valores de germinação que a TMG 7062. Os produtos alternativos testados não apresentaram efeitos fitotóxicos à germinação das sementes das duas cultivares. Na segunda época de semeadura ocorreu menor incidência de patógenos nas sementes fato que pode justificar a maior germinação nesta época para as duas cultivares. Os tratamentos que reduziram a incidência de patógenos nas sementes foram o fungicida isolado, B. subitilis e o enxofre, isolados e associados ao fungicida nas duas cultivares analisadas. Não houve diferença para a germinação e sanidade das sementes submetidas a duas ou três aplicações foliares do fungicida. / The objective of this work was to evaluate the application of alternative products isolated and associated to fungicide in different sowing dates in the control efficiency of foliar diseases, physiological changes, defoliation, yield components, productivity and physiological quality of the seeds obtained in the soybean crop (Glycine max). Experiments were conducted in the field in Ponta Grossa, PR, Brazil, during the 2015/2016, 2016/2017 and 2017/2018 growing seasons with cultivars NA 5909 and TMG 7062. Treatments for the experiments were: 1-control (water) (V4, V6, R1 and R5.1), 2- Bacillus subtilis strain QST (V4, V6, R1 and R5.1), 3-Bacillus subtilis strain QST (V4 and V6) associated with azoxystrobin + benzovindiflupir (R1 and R5. 1), 4-chitosan 1% (V4, V6, R1 and R5.1), 5-chitosan 1% (V4 and V6) associated with azoxystrobin + benzovindiflupir (R1 and R5.1), 6-sulfur (V4, V6 , R1-R5.1), 7-sulfur (V4 and V6) associated with fungicide (R1 and R5.1), 8-sodium hypochlorite (V4, V6, R1 and R5.1), 9- sodium hypochlorite ( V4 and V6) associated with azoxystrobin + benzovindiflupir (R1 and R5.1), 10-azoxystrobin + benzovindiflupir (R1 and R5.1), 11-azoxystrobin + benzovindiflupir (V6, R1 and R5.1). In all treatments, Nimbus® adjuvant (0.5 v / v) was added. At the sowing in october the lowest disease severity occurred. The Asian rust area under the disease progress curve (AACPD) was lower in the cultivar TMG 7062, in the two sowing dates and growing seasons. In all growing seasons and sowing dates, alternative treatments with sulfur and chitosan (isolated), alternative treatments associated with fungicide and fungicide applied twice and three times, decreased the severity of Asian rust and powdery mildew in both cultivars. The fungicide increased the levels of chlorophyll α, β and total in cultivar TMG 7062. The cultivar TMG 7062 presented greater stomatal conductance, assimilation of CO2 and perspiration, in relation to NA 5909. The defoliation was reduced for both cultivars in the treatments with: sulfur isolated; the alternative products associated to the fungicide and the fungicide applied in two or three times, in all the seasons and sowing dates. The yield components affected were the number of pods per plant and the thousand grains weight. There was a reduction in productivity when the crop was sown in December. The cultivar TMG 7062 in the first sowing date did not respond to treatments for yield. The treatments with fungicide, sulfur, as well as the associated alternative products, prevented damages to productivity in all sowing dates, growing seasons and crops and cultivars. The sulfur treatment associated to the fungicide that was equivalent to the treatment with fungicide applied three times was highlighted, being possible the adaption of this management strategy by the producers of the region. Regarding the seeds obtained, the cultivar NA 5909 presented higher values of germination than the TMG 7062. The alternative products tested did not present phytotoxic effects to the seed germination of the two cultivars. In the second sowing dates there was a lower incidence of seed pathogens, which may justify the higher germination for both cultivars. The treatments that reduced the incidence of pathogens in the seeds were the isolated fungicide, B. subitilis and sulfur, isolated and associated to the fungicide in the two cultivars analyzed. There was no difference for the germination and sanity of the seeds submitted to two or three foliar applications of the fungicide.

La membrane bactérienne a fait l'objet de nombreuses études de localisation de protéines et de phospholipides. Par fusion d’une protéine fluorescente (GFP) aux gènes d’intérêt, il est alors possible d‘observer la localisation des protéines associées par microscopie. La plupart de ces observations ont été réalisées à l’aide de microscopes dits à épifluorescence. Afin d’obtenir une qualité d’image suffisante, il était nécessaire de surexprimer la protéine observée, insérée à un locus ectopique non naturel. Ce travail de thèse a permis d’utiliser une nouvelle technologie acquise dans notre laboratoire, le microscope à fluorescence par réflexion totale interne (TIRFM), plus puissant que le microscope à épifluorescence utilisé précédemment. Cette technologie a permis une caractérisation plus détaillée de la localisation de protéines d’intérêt, placées sous contrôle de leur promoteur naturel. Il a également été possible de caractériser la dynamique des foci observés. Nous avons concentré notre étude sur 3 protéines: (i) SecA pour l’étude de la translocation des protéines du cytoplasme vers la membrane, (ii) YidC pour l’insertion des protéines dans la membrane, (iii) PgsA pour la synthèse des phospholipids. Les foci se déplacent dynamiquement et s’associent de manière transitoire dans la membrane. L’observation sur la durée de ces foci, et l’analyse de leur intensité moyenne au cours des observations, montre que SecA se déplace sur l’ensemble de la membrane de manière uniforme. L’analyse du déplacement des foci montre une relation quadratique entre la distance moyenne parcourue par les foci en fonction du temps. Ce résultat est en accord avec l’hypothèse d’un mouvement brownien des foci. Les foci sont observés dans les différentes phases de croissance des cellules, et le nombre de foci présents dans une cellule de la longueur de celle-ci. SecA-GFP a été testés dans un certain nombre de contextes génétiques. La localisation a été perturbée lors de la déplétion de pgsA. Cependant, comme PgsA est une protéine essentielle, il ne peut être exclu que ce changement de localisation apparaît des cellules qui sont mortes ou mourantes. Dans une souche mutante ΔclsA, on n’observe aucune différence dans la localisation de SecA en phase exponentielle, mais on aperçoit une relocalisation aux poles en phase stationnaire de croissance. La voie Tat est responsable du transport des protéines devant être exportées dans un état structuré, par exemple dans le cas de l’incorporation d’un co-facteur. À ce jour, la régulation du système Tat est peu connues, de même que les interactions entre les différentes sous-unités du système Tat et d'autres protéines dans le cytoplasme, dans la membrane ou dans la paroi cellulaire. Des fusions de les gènes de la voie Tat ont été co-exprimées deux à deux dans des cellules de levure, et leur capacité à interagir in vivo a été testée par la méthode dite du double hybride chez la levure. Nous avons généré un réseau d’interaction autour des cinq composants de système Tat. Pour déterminer les implications fonctionnelles des composants du réseau, nous avons travaillé en collaboration avec le laboratoire de Jan-Maarten van Dijl. Nous avons utilisé une collection de souches mutantes pour lesquels certains composants individuels du réseau ont été retires. Trois a été observe d’etre nécessaires pour la sécrétion Tat-dépendante. Nous avons étudié la localisation des fusions GFP avec ces proteins. On a observé une localisation double de HemAT selon l’état physiologique de la cellule. En phase exponentielle, les cellules de B. subtilis sont généralement présentes sous forme de chaînes dans lesquelles le septum de division a déjà été formé, mais la séparation cellulaire n'a pas encore eu lieu. Une fusion de la GFP à CsbC apparaît de façon homogène dans la membrane. / In the years since the cloning of GFP, the field of bacterial cell biology has characterized a variety of specific protein localization patterns in the bacterial membrane. The vast majority of early subcellular localization studies made use of inducible GFP fusions, which generally required the presence of high concentrations of inducer, and can therefore be considered to be overexpressed. An outstanding question remains over the organization of natively expressed proteins in the membrane. Here, we have investigated the localization of functional GFP fusions to proteins catalyzing important membrane processes; the secretion motor protein SecA, the membrane insertase YidC1, and the essential phospholipid synthase PgsA using total internal reflection fluorescence microscopy (TIRFM). This allowed natively expressed proteins to be localized with temporal resolution that can capture their dynamics. We characterized dynamic complexes dispersed throughout the membrane displaying diffusive movement with no preferred trajectories. Further characterization focused upon identifying conditions in which the localization pattern was disturbed. A polar mislocalization was identified in a cardiolipin mutant strain. The yeast two-hybrid (Y2H) approach is a robust approach to detect binary interactions on a proteome-scale. We performed genome-wide Y2H screens as well as targeted Y2H analyses for specific interactions involving components of the Sec and Tat secretion machineries of B. subtilis, revealing an intricate protein-protein interaction network involving 71 proteins. Furthermore, three proteins identified in the Tat network, WprA, CsbC and HemAT, were shown to be important for effective protein secretion via the B. subtilis Tat system, indicating that our yeast two hybrid assays reveal biologically significant interactions involving membrane proteins. The studies provide a novel proteomic view on the interaction network of the secretion systems of B. subtilis.

AgÃncia Nacional do PetrÃleo / Este trabalho teve como objetivo avaliar a produÃÃo de biossurfactante por cepas de Pseudomona aeruginosa e Bacillus subtilis, utilizando suco de caju, integral e clarificado, como matÃria-prima nÃo convencional. Nos ensaios com P. aeruginosa ATCC 10145 em mesa agitadora, avaliou-se a suplementaÃÃo do suco de caju integral (CAJN) com Ãleo de soja, como fonte de carbono, e com diferentes fontes de nitrogÃnio: peptona, NaNO3 e (NH4)2SO4, sendo estes resultados comparados com os obtidos utilizando caldo nutritivo e com meio CAJN. A maior reduÃÃo na tensÃo superficial (41 %) foi obtida no suco de caju suplementado com peptona (CAJP) apÃs 24 h de cultivo. Neste ensaio, observou-se uma reduÃÃo da tensÃo superficial do meio de 50 para 29,5 dina cm-1. Jà em meio suplementado com NaNO3 e (NH4)2SO4, obteve-se, respectivamente, uma reduÃÃo na TS de 37,14 e 15,85% apÃs 72 horas de cultivo. Estudou-se a suplementaÃÃo do meio CAJP com glicerol e Ãleo de soja. Nestes ensaios, observou-se um alto crescimento celular, obtendo uma densidade Ãptica (a 600nm) de 5,0 com 48 h de cultivo, contudo uma pequena reduÃÃo da tensÃo superficial (16,51 %) ao utilizar glicerol. Com base nos resultados conduzidos em mesa agitadora, os meios CAJP e CAJNaNO3 foram selecionados para estudos em fermentador de bancada. Realizaram-se ensaios utilizando biorreator a 30ÂC, 200 rpm e sem aeraÃÃo, porÃm nÃo se observou o mesmo perfil de produÃÃo de ramnolipÃdeos ocorrido em mesa agitadora. Tal fato pode ter ocorrido devido à falta de oxigÃnio no meio de cultivo. Acompanhou-se a estabilidade tÃrmica, efeito da variaÃÃo de pH e da concentraÃÃo de NaCl, na atividade emulsificante do biossurfactante produzido em CAJP e sua composiÃÃo quÃmica. O biossurfactante produzido por P. aeruginosa demonstrou-se estÃvel a variaÃÃes de temperatura, pH e concentraÃÃes de NaCl, e emulsionou todos os hidrocarbonetos estudados e Ãleo de soja. Em paralelo, diferentes ensaios foram realizados visando otimizar o meio de cultivo para a produÃÃo de surfactina por B. subtilis usando CAJN e suco de caju clarificado (CAJC). Os melhores resultados foram obtidos quando se utilizou meio mineral suplementado com extrato de levedura e formulado com CAJC, de maneira que a concentraÃÃo de glicose fosse de 10 g.L-1. Nestes ensaios, obteve-se uma reduÃÃo de 21,37 % na tensÃo superficial e observou-se a presenÃa de surfactina atravÃs das anÃlises conduzidos em HPLC. No entanto, a mÃnima tensÃo superficial alcanÃada foi superior a 39 dina.cm-1. Portanto, avaliaram-se outras cepas de B. subtilis, doze ao total, quanto à capacidade de produzir surfactina utilizando CAJC. ApÃs 48 horas de cultivo com as cepas BE 08, a tensÃo superficial do meio de cultivo livre de cÃlulas atingiu 28,0  1,0 dina.cm-1, que tambÃm apresentou atividade emulsificante. Os resultados obtidos neste trabalho indicam que o suco de caju à uma matÃria-prima adequada para a produÃÃo de biossurfactantes. / The aim of this work was to investigate the use of natural and clarified cashew apple juice as an alternative raw material for biosurfactant production by Pseudomonas aeruginosa and Bacillus subtilis. In the assays with P. aeruginosa ATCC 10145 on rotary shaker, the influence of medium (CAJN) supplementation with soybean oil, as source carbon, and with different sources of nitrogen: peptone, NaNO3 and (NH4)2SO4, were investigated. Results were compared with the obtained when Nutritive Broth (NB) and CAJN were used as culture medium. Maximum reduction in the Surface Tension (41%)was obtained when P. aeruginosa was grown on CAJP, after 24 h of cultive. In these assays, the surface tension was reduced from 50 to 29.5 dina.cm-1. When P. aeruginosa was grown on CAJN supplemented with NaNO3 or (NH4)2SO4, the reduction in the Surface Tension was of 37.14 and 15.85 %, respectively, after 72 h of cultive. Evaluated CAJP supplemented with glycerol and soybean oil. In these assays, high growth was observed, an optical density of 5,0 at 600 nm with 48h of culture was observed, however small reduction in surface tension (16,51 %) was achieved using glycerol as carbon source. Based on the results in flasks, the mediums CAJP and CAJNaNO3 were selected for further studies in a biorreator. The assays were conduced in biorreator at 30ÂC, 200 rpm and without aeration. Nevertheless, the expected profile of rhamnolipids production was not observed. Such fact may have happened due to the lack of oxygen in the cultivation medium, since the process was conducted without aeration. The stability of biosurfactant produced by P. aeruginosa in CAJP against NaCl, pH and temperature and its chemical structure were evaluated. The biosurfactante produced by P. aeruginosa was stable to temperature and variations, as well as against different NaCl concentrations. Furthermore, it emulsified all the studied hydrocarbons and soybean oil. No protein was detected in the extracted biosurfactant; it however contained carbohydrate. The highest biosurfactant production occurred with 48h,when CAJP was used as culture medium (3.86 g of biosurfactant for 1000 mL de medium) and the poorest in NB. In parallel, different assays were performed to optimize the culture media for surfactin production by Bacillus subtilis using CAJN and clarified cashew apple juice (CAJC). Best results were obtained when mineral medium supplemented with yeast extract (5 g.L-1) was used and formulated with CAJC (glucose concentration - 10 g.L-1). In these assays, a reduction of 21.37 % in the surface tension was obtained and production of surfactin was observed by HPLC. However, best results of surface tension were higher than 39 dina.cm-1. Therefore, twelve strains of Bacillus sp. were evaluated regarding the ability of producing surfactin when grown on CAJC. After 48 hours of cultivation, with strain BE 08, the surface tension of the fermented broth, free of cells, reached 28.0  1.0 dina.cm-1,and it also presented emulsifying activity. The results obtained in this work indicate that the cashew apple juice is an appropriate raw material for biosurfactants production.

Made available in DSpace on 2017-05-12T14:47:01Z (GMT). No. of bitstreams: 1 Larissa_ Kummer.pdf: 3836984 bytes, checksum: 1746a7b3c060c75c722565a4bf452739 (MD5) Previous issue date: 2014-02-21 / High concentrations of heavy metals in the soil can affect the sustainability of ecosystems and the health of humans and animals. The metal availability in the environment is related to the characteristics of each element, historical and source of contamination, as well as the properties of each soil. The presence of more than one element is common in contaminated areas and their interaction can affect their behavior in the environment. Researches have been developed to study the behavior of metals in different types of soils and thus help in cases of remediation. In recent years, the soil washing with biosurfactant has been presented as a promising method of remediation with little or no effect on the physico-chemical and microbiological characteristics of the soil, but the costs of obtaining this biosurfactant are still high, because most manufacturers use artificial means for production. Thus, this study had the objective of evaluating the remediation potential of the biosurfactant obtained from the fermentation of cassava water through the action of the bacteria Bacillus subtilis. This biosurfactant was characterized as surfactin, an anionic lipopeptide. Soils of different origins were used, one of them typical of the southwestern state of Paraná and the other from the northwest. The soils were first evaluated according to their potential for adsorption of the elements copper, zinc and lead in monometallic and multimetalic conditions, representing non-competitive and competitive conditions respectively. This evaluation was carried out by tests of adsorption and application of the matematical models of Langmuir and Freündlich. Soils were characterized chemically, physically and mineralogically. After that, it was performed the process of artificial contamination of these soils for application in the experiments of soil washing with biosurfactant in different conditions, having pH and concentration of biosurfactant solution as the main variables. Furthermore, it was also assessed the adsorption s capacity for metals by biosurfactant in liquid medium. The results showed that metals have different behaviors related to the adsorption and desorption to soil and to the biosurfactant. The soil type is also very important for the efficiency of metal removal. The clay soil showed higher adsorption capacity and therefore lower capacity of metal removal when compared to the sandy soil. In general, the soils showed the following sequence of adsorption capacity: Pb > Cu > Zn. The Pb was the element that less desorved by the washing process. It can also be concluded that, when soils are contaminated by more than one element at the same time, its ability to leach is greater than when the element is alone in the medium. This situation occurs because of differences between the competitive processes that take place in the active sites. The washing experiments showed that the biosurfactant was not able to improve the efficiency of removal of metals. The results obtained by the control treatments (only pure water) had very similar values to those that contained biosurfactant. When the wash solution containing the biosurfactant was in high concentrations, decrease in removel efficiency was found in some of the samples. Analysis of high performance liquid chromatography showed that the biosurfactant was adsorbed to soil samples, which is the consequence of not observing the effectiveness of the extractor in the removal of metals. It is notable, however, that the surfactin obtained has the potential to bind to metals, since the tests of adsorption to metals was confirmed by experiments. According to the results obtained, it can be inferred that the surfactin has greater potential for metal removal in liquid media than in solid medium, because of the lower possibility of adsorption. In soil, the results indicated potential use of this biosurfactant as stabilizing of metals in methods of remediation "in situ". / Concentrações elevadas de metais pesados no solo podem afetar a sustentabilidade dos ecossistemas e também a saúde dos seres humanos e animais. A disponibilidade do metal no ambiente está relacionada às características de cada elemento, histórico e fonte de contaminação, bem como às propriedades de cada solo. A presença de mais de um elemento em áreas contaminadas é comum e a interação entre eles pode afetar o seu comportamento no ambiente. Diante do problema, pesquisas vêm sendo realizadas a fim de estudar o comportamento dos metais em diferentes tipos de solos e assim auxiliar nos procesos de remediação. Nos últimos anos, a lavagem do solo com biossurfactante tem sido apresentada como um método promissor de remediação com pequeno ou nenhum efeito sobre as características físico-químicas e microbiológicas do solo, porém os custos de obtenção deste biossurfactante ainda são altos, pois a maioria dos fabricantes utiliza meios artificiais para sua produção. Neste sentido, este trabalho teve como objetivo geral avaliar o potencial de remediação do biossurfactante obtido a partir do bioprocessamento da manipueira pela ação de bactérias Bacillus subtilis. Este biossurfactante foi caracterizado como surfactina, um lipopeptídeo aniônico. Foram utilizados solos de origens distintas, sendo um deles típico da região sudoeste do estado do Paraná e outro da região noroeste. Os solos utilizados foram primeiramente avaliados de acordo com o seu potencial de adsorção dos elementos cobre, zinco e chumbo em condições monometálicas e multimetálicas, representando condições não-competitivas e competitivas, respectivamente. Esta avaliação foi feita por meio de testes de adsorção e aplicação de modelos matemáticos de Langmuir e Freündlich. Os solos foram caracterizados química, física e mineralogicamente. A partir de então realizou-se o processo de contaminação artificial destes solos para posterior aplicação dos experimentos de lavagem com o biossurfactante em diferentes condições, sendo as variáveis pH e concentração da solução de biossurfactante como as principais. Além disso, também foi avaliada a capacidade de adsorção dos metais pelo próprio biossurfactante, em meio líquido. Os resultados mostraram que os metais apresentam comportamentos distintos quanto a adsorção e dessorção ao solo e ao biossurfactante. O tipo de solo também é muito importante para a avaliação da eficiência de remoção de metais. O solo argiloso apresentou maior capacidade de adsorção e consequentemente menor capacidade de remoção dos metais quando comparado ao solo arenoso. De modo geral, os solos apresentaram a seguinte sequência de capacidade de adsorção: Pb > Cu > Zn. O Pb foi o elemento que menos dessorveu pelos processos de lavagem. Foi possível também concluir que quando os solos estão contaminados por mais de um elemento ao mesmo tempo, a capacidade de lixiviar-se é maior do que quando o elemento está sozinho no meio. Esta situação ocorre em virtude dos processos competitivos existentes entre os sítios ativos. Os experimentos de lavagem mostraram que o biossurfactante não foi capaz de melhorar a eficiência de remoção dos metais. Os resultados obtidos pelos tratamentos controle (somente água pura) tiveram valores muito semelhantes aos que continham biossurfactante. Quando a solução de lavagem continha o biossurfactante em altas concentrações, foi encontrada, em algumas amostras, queda na eficiência de remoção. Análises de cromatografia líquida permitiram concluir que o biossurfactante foi adsorvido às amostras de solo, sendo esta a consequência da não observação da eficácia do extrator na remoção dos metais. Cabe ressaltar, entretanto, que a surfactina obtida apresenta potencial de ligar-se aos metais, uma vez que os testes de adsorção desta aos metais foi confirmado pelos experimentos realizados. De acordo com os resultados encontrados, pode-se inferir que a surfactina tem maior potencial de remoção de metais em meio líquido do que em meio sólido, devido a menor possibilidade de adsorção na matriz sólida. Em solo, os resultados indicaram potencial de utilização deste biossurfactante como agente de estabilização dos metais em métodos de remediação in situ .