The purpose of this study was to determine the effect of substrate levels and different types of substrates on population changes of mixed cultures of Serratia marcescens and Saccharomyces cerevisiae, as compared to pure cultures.

Microorganisms are of great importance for the large scale elemental cycles and overallfunctioning of most natural ecosystems, and this also includes the ecology and maintenance offreshwater resources. Anthropogenic actions as well as climate change has greatly affectedfreshwaters and it is therefore important to understand how microorganisms react to suchenvironmental changes. I investigated how one such pressure, increased nutrient levels,influenced freshwater microbial communities and their potential to degrade the globallyabundant biopolymer chitin. To assess the effects of changed nutrient levels on functionalsubcommunities within the natural microbiota, I established a collection of mixed culturesoriginating from Lake Erken and two mesocosms from the same lake subjected to either highor low nutrient amendments. I observed that higher nutrient addition greatly increasedbacterial cell numbers in the source community. However, for the emerging mixed culturesgrowing on chitin as a substrate, those originating from the “Low” nutrient amendmentmesocosm treatment featured higher cell growth potential compared to cultures originatingfrom the “High” ones or inoculated with the natural lake water. Moreover, mixed culturesfrom the mesocosms presented higher chitinase extracellular enzymatic activity compared tothe lake cultures. Interestingly, “High” and “Low” mesocosm cultures were quite constrainedin bacterial growth response (low variance for the respective treatment) while the growthpotential in cultures from the lake were much more diverse, indicating a higher degree ofpatchiness and subcommunities with variable ability to profit from chitin as a substrate.Ongoing work will assess how individual microbial lineages react to variable nutrient levelsand how the composition of less diverse but fully functional subcommunities profiting fromchitin will change under such conditions.

Cette étude vise à étudier l'effet de la composition de substrats organiques solides sur les performances de production d'hydrogène, les voies métaboliques associées et les changements des communautés microbiennes dans un réacteur discontinu (sCSTR). L'hydrogène est un vecteur énergétique idéal qui a gagné en intérêt scientifique au cours de la dernière décennie. L'H2 produit par voie biologique, ou biohydrogène, peut être produit par des procédés de fermentation sombre où les déchets organiques sont traités et avec la production de molécules à haute valeur ajoutée. Cependant, l'effet de la composition des déchets organiques solides sur la production de biohydrogène dans la fermentation sombre n'a pas encore été clairement élucidé. Au cours de cette étude, une revue bibliographique a été réalisée sur la production d'hydrogène à partir de déchets agricoles. Cette revue montre qu'une large gamme de performances en hydrogène peut être observée principalement en raison de la variabilité dans les compositions en même type de substrats et des conditions expérimentales appliquées. Après avoir optimisé un protocole de test de potentiel biohydrogène (BHP), une grande variété de substrats organiques solides visant à couvrir un grand panel de déchets a été testée pour fournir des données comparables à analyser. Les résultats d'une régression PLS ont montré que seuls les sucres solubles ou facilement disponibles éteint corrélaient avec la production d'hydrogène. En outre, les rendements d'hydrogène corrélaient aussi bien avec l'accumulation de butyrate, principale voie productrice de bioH2. Un modèle prédictif du rendement en hydrogène en fonction de la teneur en sucres a été proposé. Ensuite, des expériences ont été menées en réacteur semi-continu (sCSTR) avec le topinambour comme substrat solide. Il a été montré qu'une faible charge organique favorisait une production continue d'hydrogène tandis que l'accroissement de la charge organique introduisait la présence de voies concurrentes à la production d'hydrogène. De plus, les profils des empreintes moléculaires basées sur l'ADNr 16s ont montré que l'augmentation de la charge organique avait un impact significatif sur la diversité microbienne en favorisant l'implantation de microorganismes ne produisant pas d'hydrogène tels que des bactéries lactiques. / This study aims to investigate the effect of solid substrates composition on hydrogen production performances, metabolic pathways and microbial community changes in batch reactor and their dynamics in semi continuous reactors (sCSTR). Hydrogen is an ideal energy carrier which has gained scientific interest over the past decade. Biological H2, so-called biohydrogen, can especially be produced by dark fermentation processes concomitantly with value-added molecules (i.e. metabolic end-products), while organic waste is treated. However, the effect of solid organic waste composition on biohydrogen production in dark fermentation has not yet been clearly elucidated. In this study, a bibliographic review was made on hydrogen production from agricultural waste. This survey on literature showed that diverse performances were reported on hydrogen production due to the variability in substrate compositions and experimental conditions. After having optimized a protocol of biohydrogen potential test (BHP), a wide variety of organic solid substrates aiming to covering a large range of solid waste was tested to provide a comparable data analysis. The results of a PLS regression showed that only soluble carbohydrates or easily available carbohydrates correlated with hydrogen production. Furthermore, hydrogen yields correlated as well with butyrate H2-producing pathway which is consistent with the literature knowledge. A predictive model of hydrogen yield according to carbohydrate content was proposed. Then, experiments were carried out in sCSTR with Jerusalem artichoke tubers as a case study. It was shown that low organic loading rate favored continuous hydrogen production while higher organic loading introduced hydrogen competition pathways and decreased the overall hydrogen yields. Moereover, 16S rRNA gene based CE-SSCP profiles showed that increasing OLR had a significant effect on the microbial diversity by favoring the implementation of microorganisms not producing hydrogen, i.e. lactic acid bacteria.

L'hydrogène, qui constitue une solution alternative et durable à l’usage d’énergies fossiles, est produit essentiellement par reformage de combustibles fossiles (95%). Des filières de production plus soucieuses de l'environnement sont envisagées. Deux familles de technologies sont explorées: 1) par décomposition thermochimique ou électrochimique de l'eau et 2) à partir de différentes sources de biomasse. Parmi celles-ci, les cellules d'électrolyse microbienne ou «Microbial electrolysis cell (MEC)» permettent de produire de l'hydrogène par électrolyse de la matière organique. Une MEC consiste en une cathode classique qui assure la production d'hydrogène par la réduction électrochimique de l'eau, associée à une bioanode qui oxyde des substrats organiques en dioxyde de carbone. Ce processus d'oxydation n'est possible que grâce au développement sur l'anode d'un biofilm microbien électroactif qui joue le rôle d'électro-catalyseur. Par rapport aux procédés courants d'électrolyse de l'eau, une MEC requière un apport énergétique 5 à 10 fois plus faibles. En outre, les procédés « classiques » de production de bio-hydrogène par voie fermentaire en cultures mixtes convertissent des sucres avec des rendements limités à 2-3 moles d'hydrogène par mole d'hexose tout en coproduisant des acides organiques. Alimenté par de l'acétate, une MEC produit au maximum 3 moles d'hydrogène/mole d'acétate. Le couplage de la fermentation à un procédé d'électrolyse microbienne pourrait donc produire de 8 à 9 moles d'hydrogène/mole d'hexose, soit un grand pas vers la limite théorique de 12 moles d'hydrogène/mole d'hexose. L'objectif de cette thèse est d'analyser les liens entre la structure des communautés microbiennes dans les biofilms électroactifs et en fermentation, les individus qui les composent et les fonctions macroscopiques (électroactivité du biofilm, production d'hydrogène) qui leur sont associées dans des conditions permettant de réaliser le couplage des deux procédés. L'originalité de cette étude a été de travailler en milieu salin (30-35 gNaCl/L), favorable au transport de charges dans l'électrolyte de la MEC. Dans un premier temps, la faisabilité de la fermentation en conditions salines (3-75 gNaCl/L) a été démontrée en lien avec l'inhibition de la consommation de l'hydrogène produit et une forte prédominance d'une nouvelle souche de Vibrionaceae à des concentrations en sel supérieures à 58 gNaCl/L. D'autre part, la mise en œuvre de biofilms électroactifs dans des conditions compatibles avec la fermentation sombre a permis la sélection d'espèces dominantes dans les biofilms anodiques et présentant des propriétés électroactives très prometteuses (Geoalkalibacter subterraneus et Desulfuromonas acetoxidans) jusqu'à 8,5 A/m². En parallèle, la sélection microbienne opérée lors d'une méthode d'enrichissement utilisée pour sélectionner ces espèces à partir d'une source d'inoculum naturelle sur leur capacité à transférer leurs électrons à des oxydes de Fer(III) a été étudiée. Une baisse des performances électroactives du biofilm liée à une divergence de sélection microbienne dans ces deux techniques de sélection mène à limiter le nombre de cycle d'enrichissement sur Fer(III). Cependant, l'enrichissement sur Fer(III) reste une alternative efficace de pré-selection d'espèces électroactives qui permet une augmentation de rendement faradique de 30±4% à 99±8% par rapport au biofilm obtenu avec un inoculum non pré-acclimaté. Enfin, l'ajout d'espèces exogènes issues de la fermentation sombre sur le biofilm électroactif a révélé une baisse de l'électroactivité du biofilm se traduisant par une diminution de la densité de courant maximale produite. Cette baisse pourrait s'expliquer par à une diminution de la vitesse de transfert du substrat due à un épaississement apparent du biofilm. Cependant, un maintien de sa composition microbienne et de la quantité de biomasse laisse supposer une production d'exopolymères (EPS) dans le biofilm en situation de couplage. / Nowadays, alternative and sustainable solutions are proposed to avoid the use of fossil fuel. Hydrogen, which constitutes a promising energy vector, is essentially produced by fossil fuel reforming (95%). Environmentally friendly production systems have to be studied. Two main families of technologies are explored to produce hydrogen: 1) by thermochemical and electrochemical decomposition of water and 2) from different biomass sources. Among those last ones, microbial electrolysis cells (MEC) allow to produce hydrogen by electrolysis of organic matter. A MEC consists in a classical cathode, which provides hydrogen production by electrochemical reduction of water, associated to a bio-anode that oxidizes organic substrates into carbon dioxide. This process is only possible because of the anodic development of an electroactive microbial biofilm which constitutes an electrocatalyst. In comparison to classical water electrolysis process, a MEC requires 5 to 10 times less electrical energy and therefore reduces the energetic cost of produced hydrogen. Furthermore, classical process of dark fermentation in mixed cultures converts sugars (saccharose, glucose) to hydrogen with a limited yield of 2-3 moles of hydrogen per mole of hexose because of the coproduction of organic acids (mainly acetic and butyric acids). Fed with acetate, a MEC can produce up-to 3 moles of hydrogen per mole of acetate. Therefore, the association of these two processes could permit to produce 8 to 9 moles of hydrogen per mole of hexose, which represents a major step toward the theoretical limit of 12 moles of hydrogen per mole of hexose.Therefore, this work aims at analyzing the relationship between microbial community structures and compositions and the associated macroscopic functions (biofilm electroactive properties, hydrogen production potential) in electroactive biofilms and in dark fermentation in conditions allowing the coupling of the two processes. The originality of this study is to work in saline conditions (30-35 gNaCl/L), which favors the charges transfer in the MEC electrolyte.First of all, feasibility of dark fermentation in saline conditions (3-75 gNaCl/L) has been shown. This was linked to an inhibition of produced hydrogen consumption and the predominance of a new Vibrionaceae species at salt concentrations higher than 58 gNaCl/L. Secondly, electroactive biofilm growth in conditions compatibles to dark fermentation (pH 5.5-7 and fed with different organic acids) allowed to select dominant microbial species in anodic biofilms that present promising electroactive properties (Geoalkalibacter subterraneus and Desulfuromonas acetoxidans) with maximum current densities up to 8.5 A/m². In parallel, the microbial selection occurring during iron-reducing enrichment method used to select species from a natural inoculum source and based on their capacity to transfer electrons to iron oxydes (Fe(III)) has been studied. A decrease of electroactive performances of the biofilm linked to the divergence of microbial selection led to a limitation of the number of iron-enrichment steps. However, enrichment on Fe(III) presents an efficient alternative to pre-select electroactive species with an increase of coulombic efficiency from 30±4% to 99±8% in comparison with a biofilm obtained with a non-acclimated inoculum. Finally, the addition of exogenous bacteria from a dark fermenter on the electroactive biofilm revealed a decrease of electroactivity with a decrease of maximum current density produced. This diminution could be explained by a lower substrate transfer due to an apparent thickening of the biofilm. Nevertheless, the stability of microbial composition and of bacterial quantity on the anode suggests that a production of exopolymers (EPS) occurred.

La production de biopolymères de type polyhydroxyalkanoates (PHA) est une alternative attractive pour remplacer, en partie, les plastiques produits à partir de ressources fossiles. Les contraintes techniques imposées par les cultures pures (substrat purifié, stérilité…) impliquent un coût de production qui rend la production de ces bioplastiques difficilement compétitive par rapport à celle des plastiques conventionnels. L’utilisation de cultures non axéniques permettrait de palier les contraintes des cultures pures mais nécessite une étape de sélection des microorganismes producteurs naturels de PHA. A partir d’un inoculum issu de boues d’épuration et de substrats de types AGV (acide butyrique et acétique), une stratégie de limitation de la croissance par le phosphore pour accumuler du PHB a été mise en place. Nous avons étudié, avec les modes de culture fed-batch et continu, le potentiel de sélection de souches productrices et de production de PHA en fonction des paramètres opératoires (taux de dilution) et environnementaux (degré de limitation en phosphore). L’objectif scientifique a consisté à améliorer les connaissances sur le rôle d’une limitation en phosphore selon les conditions opératoires du procédé, tout d’abord sur la nature des souches sélectionnées, et ensuite sur la croissance et l’accumulation de PHB. Pour cela, une démarche associant l’identification des micro-organismes en dynamique par une technique de pyroséquençage, une caractérisation cinétique des micro-organismes sélectionnés, une analyse procédé et le développement d’une modélisation cinétique a été effectué. L’objectif final du travail visait l’optimisation des procédés de production de PHB en culture non axénique : productivité, rendement, titre final en PHB mais aussi fiabilité et robustesse, en vue de définir une stratégie de production optimale de PHA. Les performances atteintes lors des cultures en fed-batch se situent parmi les meilleures de la littérature (70% de PHA) en cultures mixtes sans étape d’enrichissement préalable en microorganismes producteurs. Les résultats ont montré le rôle de la limitation phosphore sur le déclenchement de la production de PHB. En chémostat, l’analyse des paramètres macro-cinétiques, à partir des sélections microbiennes, a révélé des cinétiques de conversion du substrat carboné en PHB, biomasse catalytique et CO2 dépendantes du degré de limitation en phosphore et du taux de croissance. Le taux de phosphore intracellulaire (dépendant du taux de croissance et du degré de limitation phosphore), est le paramètre gouvernant la conversion du carbone. De plus, ce rôle a été observé pour toutes les populations sélectionnées sous limitation phosphore, démontrant un comportement universel de ces populations face à une limitation phosphore. En parallèle, des études dynamiques en batch à partir de ces populations ont permis de caractériser les paramètres cinétiques des souches, montrant une vitesse maximale de production de PHB de 0,6 et 1,2 Cmol/Cmol.h avec acide acétique et butyrique respectivement. Ces hypothèses réalisées à partir des observations expérimentales ont permis l’établissement d’un nouveau modèle cinétique basé sur le rôle du phosphore intracellulaire sur la conversion du carbone. La confrontation de ce modèle aux résultats expérimentaux a conforté et amélioré la compréhension des processus de dilution intracellulaire du phosphore et de stockage de PHB. Ce modèle a également permis d’explorer une large gamme de conditions environnementales et de prédire les comportements microbiens d’organismes producteurs et non producteurs. A partir des résultats observés et du modèle cinétique établi, les performances de différentes configurations de procédés de production de PHA ont pu être discutées : chémostat simple ou double étage, fed-batch, chémostat et batch... Les performances en termes de productivités, taux de PHB intracellulaires, degré de sélection de producteurs et robustesse du procédé sont comparées. / The production of polyhydroxyalkanoates (PHA) is an attractive alternative for plastics produced from fossil resources. The technical constraints imposed by pure cultures (purified substrate, sterilization ...) involve a high production cost of PHA production, and the production of these bioplastics is hardly competitive. The use of non-axenic cultures would avoid the constraints of pure cultures but requires a selection step of PHA producers. From a microbial inoculum (activated sludge) and AGV (butyric and acetic acid), a strategy for limiting the growth by phosphorus to accumulate PHB was established. From fed-batch and continuous culture, we studied the selection of PHA producers and the production of PHA based on operating parameters (dilution rate) and environmental (degree of phosphorus limitation). The scientific objective was to improve knowledge on the role of phosphorus limitation according to the operating conditions of the process, first about the nature of selected strains, and then about the cellular growth and PHB accumulation. For this, an approach involving identification of microorganisms by pyrosequencing method, a kinetic characterization of selected microorganisms, a process analysis and development of a kinetic modeling were performed. The ultimate goal of the work was the optimization of PHB production processes in non-axenic culture: productivity, yield, final PHB concentration but also reliability and robustness, to define an optimal production strategy of PHA. The performance achieved during the fed-batch cultures are among the best in the literature (70% PHA) in mixed cultures without enrichment step of PHA producers. The results showed the role of phosphorus limitation on the PHB production. Thus, it has been demonstrated the importance of degree of phosphorus limitation to maintain cell growth allowing enrichment in PHA producers explaining the high content of PHA obtained. From microbial selections in chemostat culture, the analysis of macro-kinetic parameters revealed conversion kinetics of the carbon substrate in PHB, catalytic biomass and CO2, dependent on the degree of phosphorus limitation and growth rate. The limits on the degree of plasticity of the intracellular phosphorus (ranging from 3.8% to 0.045%) were identified as a function of the specific growth rate. This intracellular phosphorus content (depending on the growth rate and degree of phosphorus limitation), is the parameter governing carbon conversion. Furthermore, this role of the intracellular phosphorus was observed for all populations selected under phosphorus limitation in this study, demonstrating a universal behavior of these populations face to phosphorus limitation. In parallel, dynamic studies in batch reactor from these selected populations were used to characterize the kinetic parameters of the strains, showing a maximum PHB production rate of 0.6 and 1.2 Cmol/Cmol.h with acetic acid and butyric respectively. These hypotheses made from experimental observations allowed the establishment of a new kinetic model based on the role of intracellular phosphorus on carbon conversion. The comparison of this model with experimental results has strengthened and improved the understanding of the mechanisms of intracellular phosphorus dilution and storage PHB. This model was also used to explore a wide range of environmental conditions and predict microbial behavior of PHA producers and non-producing organisms according to the operating conditions in continuous or batch reactor. From the results observed and the established kinetic model, the performance of PHA production processes of different configurations was discussed: chemostat single or two-stage, fed-batch, chemostat plus batch... The productivities, intracellular PHB content, performances of selection and the reliability of the process are compared.

O estudo das interações planta-microrganismos tem sido de grande interesse ao longo dos últimos anos. Atualmente, as interações que ocorrem entre microrganismos que vivem em estreita relação também vêm merecendo grande atenção, pois forças competitivas e mutualísticas podem induzir a produção de novos metabólitos bioativos. Portanto, estudar interações existentes entre os microrganismos endofíticos que colonizam uma mesma planta parece ser uma estratégia promissora para a obtenção de substâncias quimicamente diferentes, eventualmente bioativas. Através da utilização de culturas mistas de microrganismos, o presente trabalho contribuiu para o conhecimento da relação existente entre os fungos endofíticos SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) e SS84 (Phoma betae), isolados da planta medicinal Smallanthus sonchifolius (yacon), e sua implicação no aumento da diversidade química de produtos naturais microbianos, com o intuito de se identificar metabólitos secundários anticancerígenos. Para isso, os fungos foram cultivados em culturas singles e mistas em meios de cultivo líquidos e semi-sólidos. Foram utilizados diferentes meios de cultura, diferentes maneiras de se estabelecer o cultivo misto e diferentes formas de extração. Os extratos foram analisados química e biologicamente. Após fracionamento, foram isoladas cinco substâncias: afidicolina (I), 3-desóxi-afidicolina (II), estenfiperilenol (III), alterperilenol (IV) e alternariol monometil éter (V), sendo as duas primeiras de origem terpênica e as outras de origem policetídica. As substâncias I e II foram produzidas pelo fungo SS67, sendo que a produção de I aparentemente aumentou nas culturas mistas líquidas com SS13 e SS84, diminuindo consideravelmente na cultura mista com SS77. Devido à afidicolina ser um composto altamente citotóxico, os cultivos do fungo SS67 originaram extratos muito ativos frente aos ensaios de citotoxicidade em células cancerígenas. A substância III, primeiramente, só foi detectada por CLAE-DAD na cultura mista dos fungos SS67 e SS77, e a produção da substância IV foi maior nessa cultura mista que na cultura simples do fungo SS77 (meio fermentativo de extrato de malte). Provavelmente esses compostos foram produzidos por SS77 em resposta à presença de SS67. O extrato obtido durante esse cultivo misto foi o que apresentou maior atividade citotóxica frente à linhagem celular MDAMB-435 (câncer de mama). Posteriormente, a substância III foi também isolada da cultura simples do fungo SS77 cultivado em outras condições (meio fermentativo PDB), juntamente com a substância V. Os experimentos de antagonismo em placa de Petri envolvendo esses dois fungos revelaram, ainda, a presença de vários outros compostos na zona de inibição, que não correspondem às substâncias previamente isoladas de meio líquido, e podem ser responsáveis pelo efeito antagônico observado em meio semi-sólido. Os experimentos de atividade antagônica dos metabólitos produzidos pelos fungos evidenciaram que muitos compostos ativos, provavelmente, são produzidos em quantidades ínfimas, o que impossibilita a detecção por CLAE-DAD. Além disso, verificou-se que a substância I não possui atividade antifúngica significativa contra os fungos SS13, SS50 e SS77 e que a inibição de SS67 por SS77 ocorre devido à produção de compostos difusíveis em meio semi-sólido, e ainda, muito provavelmente, pela produção da substância III e IV em meio líquido, além de outros policetídeos. A produção de metabólitos secundários por fungos endofíticos deve ocorrer em consequência do papel ecológico que desempenham na natureza. Assim, a utilização de culturas mistas desses microrganismos deve induzir a produção de compostos que não seriam produzidos em condições não naturais. / The study of plant-microbe interactions has been of great interest over the past years. Currently, the interactions that occur among organisms that live in close relationship also have been receiving great attention because mutualistic and competitive forces may induce the production of new bioactive metabolites. Therefore, studying the interactions between endophytic microorganisms that colonize the same plant seems to be a promising strategy for obtaining chemically different substances that might also be bioactive. Through the utilization of mixed microbial cultures, this study contributed to knowledge of the relationship among the endophytic fungi SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) and SS84 (Phoma betae), isolated from the medicinal plant Smallanthus sonchifolius (yacon), and its role in the increase of the chemical diversity of microbial natural products, in order to identify anticancer secondary metabolites. For this aim, the fungi were grown in single and mixed cultures, both in liquid and semi-solid media. Different media, different approaches to establish the mixed cultures and different extraction methods were used. The extracts were analyzed chemically and biologically. After the fractionation, five compounds were isolated: aphidicolin (I), 3-deoxy-aphidicolin (II), stemphyperylenol (III), alterperylenol (IV) and alternariol monomethyl ether (V). Compounds I and II are terpene derivatives, while III, IV and V are polyketide derivatives. The substances I and II were produced by the fungus SS67, and the production of I apparently increased in liquid mixed cultures with SS13 and SS84, decreasing considerably in mixed culture with SS77. Due to the high cytotoxic activity of aphidicolin (I), the cultures of the fungus SS67 originated extracts highly active in the cytotoxicity assays in cancer cells. Substance III was only detected by HPLC-DAD in the mixed culture between the fungi SS67 and SS77, and the production of compound IV was higher in this mixed culture when compared to the single culture of the fungus SS77 (malt extract medium). Probably these compounds were produced by SS77 in response to the presence of SS67. The extract obtained during this mixed culture showed the highest cytotoxic activity against the cell line MDAMB-435 (breast cancer). In a subsequent fermentation in PDB medium, compound III was also isolated from the single culture of the fungus SS77 grown, along with compound V. Additionally, antagonism experiments in Petri dishes with these two fungi revealed the presence of several other compounds in the inhibition zone, which does not correspond to the substances previously isolated from the liquid medium, and may be responsible for the antagonistic effect observed in semi-solid medium. The experiments of antagonistic activity of metabolites produced by fungi showed that many active compounds are probably produced in very small quantities, making it impossible to detect by HPLC-DAD. Moreover, it was found that aphidicolin (I) did not have significant antifungal activity against the fungi SS13, SS50 and SS77 and that the SS67 inhibition by SS77 is due to the production of diffusible compounds in semi-solid medium, and likely, due to the production of compound III and IV in liquid medium, and other polyketides. The production of secondary metabolites by endophytic fungi probably occurs as a result of the ecological role they play in nature. Thus, the use of mixed cultures of these microorganisms may induce the production of compounds that would not be produced under unnatural conditions.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.

O estudo das interações planta-microrganismos tem sido de grande interesse ao longo dos últimos anos. Atualmente, as interações que ocorrem entre microrganismos que vivem em estreita relação também vêm merecendo grande atenção, pois forças competitivas e mutualísticas podem induzir a produção de novos metabólitos bioativos. Portanto, estudar interações existentes entre os microrganismos endofíticos que colonizam uma mesma planta parece ser uma estratégia promissora para a obtenção de substâncias quimicamente diferentes, eventualmente bioativas. Através da utilização de culturas mistas de microrganismos, o presente trabalho contribuiu para o conhecimento da relação existente entre os fungos endofíticos SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) e SS84 (Phoma betae), isolados da planta medicinal Smallanthus sonchifolius (yacon), e sua implicação no aumento da diversidade química de produtos naturais microbianos, com o intuito de se identificar metabólitos secundários anticancerígenos. Para isso, os fungos foram cultivados em culturas singles e mistas em meios de cultivo líquidos e semi-sólidos. Foram utilizados diferentes meios de cultura, diferentes maneiras de se estabelecer o cultivo misto e diferentes formas de extração. Os extratos foram analisados química e biologicamente. Após fracionamento, foram isoladas cinco substâncias: afidicolina (I), 3-desóxi-afidicolina (II), estenfiperilenol (III), alterperilenol (IV) e alternariol monometil éter (V), sendo as duas primeiras de origem terpênica e as outras de origem policetídica. As substâncias I e II foram produzidas pelo fungo SS67, sendo que a produção de I aparentemente aumentou nas culturas mistas líquidas com SS13 e SS84, diminuindo consideravelmente na cultura mista com SS77. Devido à afidicolina ser um composto altamente citotóxico, os cultivos do fungo SS67 originaram extratos muito ativos frente aos ensaios de citotoxicidade em células cancerígenas. A substância III, primeiramente, só foi detectada por CLAE-DAD na cultura mista dos fungos SS67 e SS77, e a produção da substância IV foi maior nessa cultura mista que na cultura simples do fungo SS77 (meio fermentativo de extrato de malte). Provavelmente esses compostos foram produzidos por SS77 em resposta à presença de SS67. O extrato obtido durante esse cultivo misto foi o que apresentou maior atividade citotóxica frente à linhagem celular MDAMB-435 (câncer de mama). Posteriormente, a substância III foi também isolada da cultura simples do fungo SS77 cultivado em outras condições (meio fermentativo PDB), juntamente com a substância V. Os experimentos de antagonismo em placa de Petri envolvendo esses dois fungos revelaram, ainda, a presença de vários outros compostos na zona de inibição, que não correspondem às substâncias previamente isoladas de meio líquido, e podem ser responsáveis pelo efeito antagônico observado em meio semi-sólido. Os experimentos de atividade antagônica dos metabólitos produzidos pelos fungos evidenciaram que muitos compostos ativos, provavelmente, são produzidos em quantidades ínfimas, o que impossibilita a detecção por CLAE-DAD. Além disso, verificou-se que a substância I não possui atividade antifúngica significativa contra os fungos SS13, SS50 e SS77 e que a inibição de SS67 por SS77 ocorre devido à produção de compostos difusíveis em meio semi-sólido, e ainda, muito provavelmente, pela produção da substância III e IV em meio líquido, além de outros policetídeos. A produção de metabólitos secundários por fungos endofíticos deve ocorrer em consequência do papel ecológico que desempenham na natureza. Assim, a utilização de culturas mistas desses microrganismos deve induzir a produção de compostos que não seriam produzidos em condições não naturais. / The study of plant-microbe interactions has been of great interest over the past years. Currently, the interactions that occur among organisms that live in close relationship also have been receiving great attention because mutualistic and competitive forces may induce the production of new bioactive metabolites. Therefore, studying the interactions between endophytic microorganisms that colonize the same plant seems to be a promising strategy for obtaining chemically different substances that might also be bioactive. Through the utilization of mixed microbial cultures, this study contributed to knowledge of the relationship among the endophytic fungi SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) and SS84 (Phoma betae), isolated from the medicinal plant Smallanthus sonchifolius (yacon), and its role in the increase of the chemical diversity of microbial natural products, in order to identify anticancer secondary metabolites. For this aim, the fungi were grown in single and mixed cultures, both in liquid and semi-solid media. Different media, different approaches to establish the mixed cultures and different extraction methods were used. The extracts were analyzed chemically and biologically. After the fractionation, five compounds were isolated: aphidicolin (I), 3-deoxy-aphidicolin (II), stemphyperylenol (III), alterperylenol (IV) and alternariol monomethyl ether (V). Compounds I and II are terpene derivatives, while III, IV and V are polyketide derivatives. The substances I and II were produced by the fungus SS67, and the production of I apparently increased in liquid mixed cultures with SS13 and SS84, decreasing considerably in mixed culture with SS77. Due to the high cytotoxic activity of aphidicolin (I), the cultures of the fungus SS67 originated extracts highly active in the cytotoxicity assays in cancer cells. Substance III was only detected by HPLC-DAD in the mixed culture between the fungi SS67 and SS77, and the production of compound IV was higher in this mixed culture when compared to the single culture of the fungus SS77 (malt extract medium). Probably these compounds were produced by SS77 in response to the presence of SS67. The extract obtained during this mixed culture showed the highest cytotoxic activity against the cell line MDAMB-435 (breast cancer). In a subsequent fermentation in PDB medium, compound III was also isolated from the single culture of the fungus SS77 grown, along with compound V. Additionally, antagonism experiments in Petri dishes with these two fungi revealed the presence of several other compounds in the inhibition zone, which does not correspond to the substances previously isolated from the liquid medium, and may be responsible for the antagonistic effect observed in semi-solid medium. The experiments of antagonistic activity of metabolites produced by fungi showed that many active compounds are probably produced in very small quantities, making it impossible to detect by HPLC-DAD. Moreover, it was found that aphidicolin (I) did not have significant antifungal activity against the fungi SS13, SS50 and SS77 and that the SS67 inhibition by SS77 is due to the production of diffusible compounds in semi-solid medium, and likely, due to the production of compound III and IV in liquid medium, and other polyketides. The production of secondary metabolites by endophytic fungi probably occurs as a result of the ecological role they play in nature. Thus, the use of mixed cultures of these microorganisms may induce the production of compounds that would not be produced under unnatural conditions.