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This diploma thesis deals with the microbial production of biosurfactants of selected bacterial strains. In order to test the biosurfactant production ability, screening methods were chosen to be able to review the potential of the selected strains to produce biosurfactants. With the scope of the work, 11 bacterial strains, which are used as polyhydroxyalkanoate (PHA) producers, have been tested. The ability to produce biosurfactants was tested in all strains both in complex inoculation and mineral production media. The presence of biosurfactants in Pseudomonas putida was detected on the basis of the results obtained after cultivation in inoculation and production media. The bacteria Pseudomonas fulva was put under more deep study to support their production by cultivation in different types of production media supplemented by different sources of carbon and nitrogen, and the effect of cultivation time was tested as well. Biosurfactants produced by these bacteria were subsequently identified by Fourier transform infrared spectroscopy (FTIR) on the basis of which the substances were identified as rhamnolipids. According to thin-layer chromatography result (TLC), Pseudomonas putida produces a mixture of mono- and dirhamnolipids, with monorhamnolipids being more dominant in our samples.

This diploma thesis focuses on a research of the foam stability of the beer. We specifically observed the influence of ethanol and surface tension on the foam stability, and influence of ethanol and surface tension on the height of the foam. It was observed the influence of the hop acids in non-alcoholic beers. Furthermore, different biosurfactants were added into to the beer to observe better stability of the foam. The theoretical part of this work describes the foam from the physical aspect. There are described fourth key events involved in foam formation. Then it focuses on the beer foam – its structure, substances that influence its behavior and measurement of the foam stability. In this part there are described biosurfactants as well – their characteristics, qualities, distribution and applications. The experimental part is dedicated to the influence of ethanol, surface tension, the amount of iso- acids on foam stability in beer. There is observed the influence of biosurfactants of the foam stability and surface tension, which are added into to the beer. For the measurement of the foam stability was used the Rudin method. Liquid chromatography with DAD detector was used for the measurement the amount of the hop acids. The surface tension was determined by tensiometer. According to the results addition of ethanol changed the characteristics of the foam stability, but it’s not easy to find correlation between the increasing amount of ethanol and its stability. It was found out, that there is connection between the amount of ethanol and surface tension. On the other hand, there is not much connection between the surface tension and foam height. Within the framework of addition of biosurfactants was observed, that these foaming agents interacted the surface tension of the beer (lowering), however these agents accelerated the fall of the beer foam.

Pseudomonas aeruginosa (PA) est une bactérie Gram négatif, pathogène opportuniste, impliquée dans un grand nombre d’infections nosocomiales. Cette bactérie est aussi le principal micro-organisme responsable des surinfections broncho-pulmonaires chez les patients atteints de mucoviscidose. Cette prééminence est due en partie à la capacité de PA à former des biofilms, ce qui lui confèrent une résistance exceptionnelle aux antimicrobiens. Au sein de notre laboratoire, une analyse protéomique différentielle a permis de démontrer, en 2004, l’existence d’un protéome spécifique lorsque la bactérie se développe en mode biofilm. Parmi les protéines spécifiquement exprimées en mode biofilm, la protéine hypothétique PA3731 a été plus particulièrement étudiée. Cette protéine est impliquée dans la formation de biofilm, la production de rhamnolipides, la résistance à la tobramycine et la mobilité de type « swarming ». Des recherches bioinformatiques ont montré que le gène pA3731 appartient à un cluster de 4 gènes allant de pA3729 à pA3732 (système BAC), qui pourraient être impliqués dans l’élaboration et/ou la régulation d’un même système protéique. Cette hypothèse a constitué le point de départ de ce travail de thèse. La présente étude a permis de confirmer l’implication du système BAC dans la formation du biofilm, la résistance aux antibiotiques et la production de rhamnolipides chez PA. Les études protéomiques ont mis en évidence l’implication de ce système dans l’expression de la pompe MexEF-OprN, de la porine OprD, et dans la régulation du Quorum Sensing. Des études intéractomiques, menées en parallèle, ont montré une forte interaction entre la protéine PA3731 et PA3732. Ces études ont également permis de valider une forte interaction entre ces protéines et les rhamnolipides. L’ensemble de ces résultats nous permettent d’avancer une hypothèse quant à l’implication du système BAC dans le transport des rhamnolipides vers le milieu extracellulaire. / Pseudomonas aeruginosa (PA) is a Gram-negative bacterium, opportunistic pathogen, involved in a large number of nosocomial infections. This microorganism is also the main infectious agent involved in bronchopulmonary infections in cystic fibrosis patients. This pre-eminence is partly due to the ability of PA to form biofilms, which confers to the bacterial cells an increased resistance to antibiotics. In our laboratory, a differential proteomic analysis allowed to demonstrate in 2004, the existence of a specific proteome when the bacterium grows in the biofilm mode. This study allowed identifying about 40 proteins, specifically accumulated when bacteria adhere to a surface. Among these proteins, the hypothetical protein PA3731 has been particularly investigated. This protein is involved in the biofilm formation, the rhamnolipids production, the resistance to tobramycin and the swarming mobility. Bioinformatic research showed that the pA3731 gene belongs to a cluster of 4 genes ranging from pA3729 to pA3732 (BAC system), which could be involved in the development and / or regulation of the same protein system. This hypothesis was the starting point of this thesis work. The present study confirmed the involvement of the BAC system in the biofilm formation, the antibiotic resistance and the rhamnolipid production in PA. Proteomic studies highlighted the implication of this system in the expression of the MexEF-OprN pump and that of the OprD porin, and in the regulation of Quorum Sensing. Interactomic investigations, conducted in parallel, showed a strong interaction between PA3731 and PA3732 proteins. These studies have also pointed out a strong interaction between these proteins and rhamnolipids. All these results suggest that the BAC system could play a major role in the transfer of rhamnolipids to the extracellular environment.

Dans leur environnement, les plantes sont fréquemment soumises à des attaques de microorganismes pathogènes. Pour leur faire face, elles mettent en place des mécanismes de défense activés suite à la détection du microorganisme via des motifs moléculaires ou IPs (Invasion Patterns). Les rhamnolipides (RLs) sont des molécules glycolipidiques amphiphiles produites par des bactéries des genres Pseudomonas et Burkholderia. Ces molécules sont capables d’induire, au niveau foliaire chez différentes plantes, une résistance locale contre des microorganismes phytopathogènes. L’acide 3-hydroxydécanoïque (3-OH-C10:0), le constituant de base de la partie lipidique des RLs, active aussi une réponse immune dans la partie aérienne de la plante Arabidopsis thaliana. Cette réponse immune est déclenchée suite à sa perception par le récepteur kinase S-lectine LORE. Les travaux menés au cours ce projet de thèse ont permis de mettre en évidence que le 3-OH-C10:0 est perçu au niveau racinaire par LORE, conduisant à l’activation d’une réponse immune innée et à la mise en place d’une résistance systémique (ISR) efficace contre le champignon nécrotrophe Botrytis cinerea. D’autre part, ces travaux ont révélés que les RLs sont aussi perçus au niveau racinaire et activent une ISR contre B. cinerea n’impliquant pas le récepteur LORE. L’ensemble de ces résultats montrent que les RLs ainsi que le 3-OH-C10:0, sont deux IPs reconnus par A. thaliana au niveau racinaire via des mécanismes indépendants et tous deux conduisant à l’activation d’une résistance systémique. / In their environment, plants are frequently challenged by pathogenic microorganisms. Plants are able to trigger an innate immune response to fight against the infection. This immune response is activated after perception of the microorganisms through Invasion Patterns (IPs). Rhamnolipids (RLs) are amphiphilic glycolipidics molecules produced by some bacterial species including Pseudomonas and Burkholderia. RLs are able to induce an immune response in the aerial part of several plant which is effective against phytopathogens. 3-hydroxydecanoic acid (3-OH-C10:0), a lipid building block from RLs, is known to trigger Arabidopsis thaliana immune responses in leaves after its perception by the bulb-type lectin receptor kinase LORE. In the present work, we showed that the 3-OH-C10:0 is also sensed by roots through LORE, triggering local immune responses and a systemic induced resistance (ISR) effective against the necrotrophic fungus Botrytis cinerea. In addition, this work revealed that RLs are also recognized by root cells, activating a LORE-independent ISR against B. cinerea. This work shows that RLs and 3-OH-C10:0 are different IPs independently recognized by A. thaliana roots but both inducing a systemic resistance in plants.

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