Dissertations / Theses on the topic 'Hydrocarbonoclastic'

Crude oil spills damage marine ecosystems due to the potentially toxic nature of the petrochemical hydrocarbon constituents and their recalcitrance to degradation. Polycyclic aromatic hydrocarbon components (PAHs) are one group of hydrocarbons in crude oil that are of particular concern due to their genotoxicity and potential to bioaccumulate. Their potential to cause damage in marine environments can be mitigated by the presence and activities of hydrocarbonoclastic bacteria. The phycosphere of marine eukaryotic phytoplankton (micro-algae) has recently been shown as an important biotope where hydrocarbonoclastic bacteria can be found, and the association between these organisms is largely unexplored. This thesis presents new insight into the relationship between these organisms by performing enrichment experiments with crude oil and individual hydrocarbon substrates, as well as wholegenome analysis of an algal-associated hydrocarbon-degrader, and using molecular probes for the in situ visualization (by CARD-FISH) and whole-community analysis (by Flow-FISH) of hydrocarbonoclastic bacteria associated with laboratory cultures and field samples of micro-algae. Results demonstrated variations in the tolerance of different diatom species to PAHs, and that intermediate metabolites formed from the partial biodegradation of PAHs can be more harmful to diatoms compared to that of their parent PAH compounds. Thalassiosira pseudonana presence enhanced phenanthrene dissolution and PAH-degrading bacteria formed cell clusters in EPS aggregates. The genome of an obligate hydrocarbonoclastic bacterial species (Polycyclovorans algicola) found associated with marine micro-algae possessed genes involved in cell communication, horizontal gene transfer and nutrient sharing that may play an important role in the organism’s association with its eukaryotic host cells. Indeed, these interactions are likely to allow these hydrocarbonoclastic bacteria to be supported on the phycosphere of micro-algae in the absence of petrochemical exposure. Microcosm experiments with field samples of micro-algae and bacterial consortia showed that crude oil biodegradation, in particular the PAH fraction, was enhanced compared to that by the free-living bacterial community, and revealed certain groups (e.g. members of the order BD7-3) that had not previously been reported to become enriched in the presence of crude oil. Using Flow-FISH, epibiotic members of the genus Marinobacter were found associated with micro-algae in sea surface field samples, and CARD-FISH was used to show this same group associated with the soft tissues of the coral Lophelia pertusa from polyp samples collected from different coral mounds in the deep North Atlantic.

Doutoramento em Engenharia Química
Actualmente são conhecidas poucas estirpes bacterianas capazes de produzir biossurfactantes (BSFs) em condições de microaerobiose ou anaerobiose. Estas bactérias têm um papel importante não só em processos naturais (ex. formação de biofilmes ou de hidratos de gás), como podem ter diversas aplicações biotecnológicas (ex. estratégias de biorremediação e aplicações industriais). As bactérias produtoras de BSFs em condições de limitação de oxigénio, com capacidade para degradar hidrocarbonetos são de particular interesse para estratégias de biorremediação de locais contaminados com hidrocarbonetos de petróleo (PHs) e na recuperação microbiana de petróleo (MEOR). Neste contexto, o objectivo deste trabalho foi o isolamento, identificação e a caracterização de bactérias anaeróbias ou anaeróbias facultativas produtoras de BSF e degradadoras de hidrocarbonetos (hidrocarbonoclásticas) na perspetiva da sua aplicação biotecnológica em condições de limitação de oxigénio. Foram escolhidos dois ambientes contaminados com PHs como potenciais fontes de bactérias hidrocarbonoclásticas produtoras de BSFs: vulcões de lama (MV) de mar profundo do Golfo de Cádis (Oceano Atlântico) e o sistema estuarino da Ria de Aveiro (Portugal). Foram preparadas culturas de enriquecimento com sedimentos subaquáticos recolhidos nestes dois habitats, como potenciais inóculos de bactérias anaeróbias facultativas. Um design experimental fatorial foi usado para testar o efeito do crude como fonte de carbono, e de nitrato e/ou sulfato, como aceitadores terminais de eletrões. De forma a melhor compreender a estrutura das comunidades bacterianas envolvidas na biodegradação de PHs nos MV do mar profundo procedeu-se à sequenciação do gene 16S rRNA das comunidades bacterianas de culturas de enriquecimento com sedimento de dois MVs, um activo e outro inactivo, e com ou sem adição de crude e/ou nitrato. Detetou-se uma diferenciação entre as comunidades dos dois MVs, independentemente dos suplementos a que as culturas foram expostas, sendo que Alphaproteobacteria e Bacilli predominaram nas culturas com sedimentos de MV activo e inactivo, respectivamente. De uma forma menos acentuada, tanto o nitrato como o crude afetaram a composição das comunidades bacterianas. Géneros de bactérias que só foram detectados nos ensaios com adição de crude (ex. Erythrobacteraceae no MV activo e Acidimicrobiale no MV inactivo) poderão ser usados como indicadores da presença de hidrocarbonetos de petróleo nestes habitats. A biodegradação de PHs nas culturas com crude foi avaliada por cromatografia gasosa acoplada a espectrometria de massa. De uma forma geral, as comunidades de culturas do MV activo foram capazes de degradar n-alcanos de tamanho inferior a C13 e compostos monoaromáticos, enquanto as comunidades do MV inactivo apresentaram a capacidade de metabolizar vários tipos de hidrocarbonetos aromáticos policíclicos. A presença de nitrato apenas afectou positivamente a biodegradação de alcanos, e não teve efeito ou foi mesmo inibitória da biodegradação de outros hidrocarbonetos. A partir de todas as culturas, com todos os tipos de sedimentos, dos MVs do Golfo de Cádis e do estuário da Ria de Aveiro, foi possível isolar-se um total de 13 isolados capazes de sobreviver exclusivamente com crude como fonte de carbono e produzir BSF em condições de aerobiose. Destas, apenas duas não foram capazes de produzir BSFs em anaerobiose. A sequenciação do gene 16S rRNA dos isolados permitiu identifica-los como pertencendo aos géneros Pseudomonas, Bacillus, Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter e Curtobacterium, a maioria dos quais não tinha ainda membros conhecidos como produtores de BSF em anaerobiose. Os resultados obtidos com este trabalho permitiram caracterizar melhor as comunidades envolvidas na degradação de PHs em MVs de mar profundo. Conseguiu-se ainda isolar e identificar estirpes, tanto de mar profundo como de ambiente estuarino, capazes de degradar PHs e produzir BSFs em condições de anaerobiose. Estas estirpes apresentam elevado potencial biotecnológico para aplicações como MEOR e biorremediação em ambientes com escassez de oxigénio.
So far, only few bacterial strains are known to produce biosurfactants (BSFs) under microaerobic or anaerobic conditions. However, these bacteria are not only involved in important natural processes (e.g. biofilm and gas hydrates formations) but can also be used in several biotechnological applications (e.g. bioremediation strategies and industrial applications). Bacteria able to produce BSFs under oxygen-limiting conditions that are also able to degrade hydrocarbons, are of particular interest to bioremediation strategies of sites contaminated with petroleum hydrocarbons (PHs) and microbial enhanced oil recovery (MEOR) strategies. In this context, this work aims at isolating, identifying, and characterizing BSF-producing and hydrocarbon-degrading (hydrocarbonoclastic) bacteria grown under anaerobic conditions, which can be used in biotechnological applications under oxygen limitation. Two environments contaminated with PHs were chosen as potential sources of hydrocarbonoclastic BSF-producing bacteria: deep-sea mud volcanos from the Gulf of Cadiz (Atlantic Ocean), and the estuarine system of Ria de Aveiro (Portugal). Enrichment cultures were prepared using subaquatic sediments from both sites, as potential sources of facultative anaerobic bacteria. A factorial experimental design was used to test the effect of crude oil as carbon source, and nitrate and/or sulfate, as terminal electron acceptors. Aiming at better understanding the structure of bacterial communities involved in PHs biodegradation at deep-sea MVs, sequencing of the 16S rRNA gene was performed for bacterial communities from cultures containing sediments from two MVs, active and inactive, and with or without crude oil and/or nitrate. A distinction between the communities of MVs with different activity, independent of the supplements was observed. Alphaproteobacteria and Bacilli were the predominant classes found in enrichment cultures inoculated with active and inactive MVs sediments, respectively. In a minor scale, nitrate and crude oil additions also affected the composition of bacterial communities. Therefore, genera that only appeared in cultures with crude oil. (e.g. Erythrobacteraceae in active MV cultures and Acidimicrobiale in inactive MV cultures) can be used as biosensors of the presence of PHs in these habitats. Biodegradation of PHs in cultures containing crude oil was assessed by gas chromatography coupled with mass spectrometry. Overall, communities from active MV cultures were able to degrade n-alkanes below C13 and monoaromatic hydrocarbons, while communities from inactive MV cultures presented the ability to metabolize several types of polycyclic aromatic hydrocarbons. The presence of nitrate only had a positive effect on the biodegradation of alkanes, and had no effect or even an inhibitory effect on the biodegradation of other hydrocarbons. A total of 13 isolates able to survive on crude as carbon source and produce BSF under aerobic conditions were obtained from all cultures either from sediments of the Gulf of Cadiz MVs or the estuarine system of Ria de Aveiro. Only two isolates failed to produce BSF under anaerobiosis. Sequencing of 16S rRNA gene was used to establish the identification of isolates as Pseudomonas, Bacillus, Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter and Curtobacterium. Most of these genera had never been described as able to produce BSFs under anaerobic conditions. The results obtained in this work allowed to better characterize the deep-sea communities involved in PHs degradation, as well as, to identify strains from deep-sea and estuarine sediments able to degrade PHs and produce BSFs under anaerobic conditions. These bacteria present high biotechnological potential for applications in oxygen-limiting environments, such as, MEOR and bioremediation of environments contaminated with PHs.

En milieu marin le carbone organique particulaire (POC) représente 25 % du carbone organique total. Sa dégradation est réalisée par des microorganismes hétérotrophes ayant mis en place diverses stratégies pour parvenir à le dissoudre et l’assimiler. Peu d’études se sont intéressées à la dégradation des composés polymériques et/ou hydrophobes, quasiment insolubles dans l’eau constituant le POC. Parmi ces composés, on retrouve les lipides et les hydrocarbures regroupés sous le terme de COH (composés organiques hydrophobes). La dégradation des COH est réalisée par des bactéries dîtes oléolytiques ayant entre autre pour stratégie la formation de biofilms également qualifiés d’oléolytique. Nos connaissances sur la diversité et la fonctionnalité des biofilms oléolytiques se limitent actuellement aux bactéries spécifiquement étudiées pour leur capacité à dégrader les HC. Ainsi la dégradation des lipides est souvent négligée alors que cette famille de molécules représente une part significative du POC.La diversité taxonomique des bactéries formant des biofilms oléolytiques a été déterminée par un criblage de 199 souches marines sur 4 substrats : un alcane (paraffine), un triglycéride (tristéarine), un acide gras (acide palmitique) et une cire (l’hexadécyl palmitate). Cette étude a révélé que les bactéries oléolytiques (formant un biofilm sur au moins 1 des substrats) sont relativement répandues parmi les bactéries marines puisque qu’elles représentent 18.7 % des souches testée. Cette étude montre également que les bactéries capables d’assimiler les alcanes sont également capables d’assimiler au moins un lipide. Les bactéries hydrocarbonoclastes, jusqu’alors décrites comme spécialisées, voir restreintes à l’assimilation des hydrocarbures, présentent donc une gamme de substrats s’étendant aux lipides. La corrélation positive entre la capacité d’assimilation des alcanes et l’assimilation des lipides suggère un lien physiologique entre l’assimilation de ces deux familles de COH. L’activité lipase qui est essentielle à l’assimilation des triglycérides mais pas à l’assimilation des alcanes, a été mesurée dans des cultures de souches oléolytiques poussant sur acétate, triglycéride ou hexadécane. Comme attendu, les cultures sur triglycérides montrent toutes une surexpression de l’activité lipases par rapport aux cultures sur acétate. Les cultures sur hexadécane montraient aussi une surexpression de l’activité lipase renforçant l’idée d’un lien physiologique entre dégradation des alcanes et dégradation des lipides. De plus les souches oléolytiques n’ont pas montré de capacité à former un biofilm sur une surface inerte hydrophobe telle que le polystyrène ou sur une surface hydrophile telle que le verre à la hauteur de celles constaté sur COH. Une étude quantitative de l’adhésion sur COH et substrats inertes réalisée par microscopie montre que l’adhésion (dans les conditions testées) n’est pas un facteur déterminant de la formation de biofilm sur ces mêmes substrats. Cela suggère que la spécificité de formation de biofilm sur les substrats COH, ne réside pas dans l’adhésion mais vraisemblablement dans les étapes de développement du biofilm plus tardives.Enfin, les biofilms oléolytiques mettant en jeux des produits extracellulaires (enzymes et facteurs de solubilisation) qui constituent des biens communs, sont propices à l’établissement de comportements sociaux. Nous avons mis en évidence des comportements synergiques (5/8 des comportements observés) ou compétitifs au sein de biofilm oléolytiques (3/8 des comportements observés)
Particulate organic carbon (POC), in marine environment, accounts for 25% of total organic carbon. POC degradation is carried out by heterotrophic microorganisms which have developed strategies to dissolve and assimilate it. Few studies have investigated the degradation of the polymeric and / or hydrophobic components of POC, which are almost insoluble in the water. Among these compounds, there are lipids and hydrocarbons (HC) grouped under the term of HOCs (hydrophobic organic compounds). The degradation of the HOCs is carried out by oleolytic bacteria which form biofilms at the HOC– water interface. Our knowledge of the diversity and functionality of oleolytic biofilms is mostly limited to HC degrading bacteria, while the degradation of lipids is often neglected although this family of molecules represents a significant part of the POC. A screening of 199 marine strains on 4 substrates: an alkane (paraffin), a triglyceride (tristearin), a fatty acid (palmitic acid) and a wax ester (hexadecyl palmitate) was performed to determine the taxonomic diversity of bacteria able to form oleolytic biofilms. This study revealed that oleolytic bacteria (forming a biofilm on at least 1 substrate) were relatively widespread among marine bacteria since they represented 18.7% of tested strains. This study also showed that bacteria able to assimilate alkanes were also able to assimilate at least one lipid. Hydrocarbonoclastic bacteria, previously described as specialized, or restricted to the assimilation of hydrocarbons, have actually a substrate range spanning from HC to lipids. The positive correlation between the ability to form a biofilm on alkanes and on lipids suggested a physiological link between the assimilation of these two HOC families. The lipase activity, which is essential for triglycerdides assimilation but not for the alkanes assimilation, was measured in oleolytic strains cultures growing on acetate, triglyceride or hexadecane. As expected, overexpression of lipase activity was observed in cultures on triglycerides compared to cultures on acetate. Moreover, overexpression of lipase activity was also observed in cultures on hexadecane reinforcing the idea of a physiological link between alkanes and lipids degradation.Oleolytic strains exhibited a very weak ability to form a biofilm on the inert surfaces (non-nutritive) polystyrene or glass compared to the HOC nutritive surface indicating that oleolytic strains have a specificity for HOC to form a biofilm. A quantitative study of adhesion on HOC and inert substratums carried out by microscopy shows that adhesion (in the tested conditions) is not a determining factor of the biofilm formation on these same substrates. This suggests that the specificity of biofilm formation on HOC substrates does not reside in adhesion but presumably in later biofilm development stages.Lastly, oleolytic biofilms, involving extracellular products (enzymes and solubilization factors) that constitute public goods, are favorable to the establishment of social behaviors. We have demonstrated synergistic behaviors (5/8 of observed behaviors) or competitive behaviors (3/8 of observed behaviors) in oleolytic biofilms

La croissance de Marinobacter hydrocarbonoclasticus SP17 sur alcane à longue chaîne conduit à la formation d’un biofilm à l’interface entre le milieu aqueux et l’alcane. Il a été montré que cette croissance s’accompagne d’un détachement important de cellules issues du biofilm. Le but de ces travaux de recherche consistait, à décrire le développement de ce biofilm, à déterminer les mécanismes moléculaires mis en place lors de sa formation et à caractériser les cellules qui s’en détachent. L’observation du biofilm par microscopie confocale à balayage laser a montré que son développement conduit à la formation d’une structure tridimensionnelle élaborée, résultant d’une croissance sous forme de microcolonie. Une étude protéomique a montré que l’adaptation des cellules à l’utilisation de l’hexadécane et la formation du biofilm qui en découle s’accompagnent de changements importants au niveau de l’expression des gènes. L’identification des protéines surexprimées a mis en évidence deux protéines potentiellement impliquées dans le transport de l’hexadécane et l’induction d’un système de sécrétion de type VI. Cette étude a aussi permis de révéler le phénotype original des cellules nouvellement détachées du biofilm dont le profil protéique se distingue nettement des cellules du biofilm mais aussi de celui de cellules planctoniques cultivées sur substrat soluble. L’étude de différentes caractéristiques physiologiques a permis de confirmer leur singularité, telle que leur capacité accrue à coloniser une interface alcane-milieu, ceci étant probablement lié à la consommation d’inclusions lipidiques accumulées avant leur détachement du biofilm
Growth of Marinobacter hydrocarbonoclasticus SP17 on long chain alkane leads to the formation of a biofilm at the alkane-water interface. It was shown that a huge production of biofilm detached cells occurred during it development. The aim of the present study was to describe the development of this biofilm, to determine molecular mechanisms involved in alkane utilisation and biofilm synthesis and to characterise the biofilm detached cells. The observation of biofilm by confocal laser scanning microscopy showed that development leaded to an elaborated tridimensional structure resulting from the expansion of microcolonies. A proteomic study showed that adaptation to alkane utilisation and to biofilm lifestyle involved a drastic change in gene expression. Identification of proteins modulated revealed two proteins probably involved in alkane transport and the overexpression of a type VI secretion system. This analysis also showed that cells freshly detached from the biofilm, exhibited a protein pattern clearly different from that of biofilm and other planktonic cells grown on soluble substrate. Determination of several physiologic properties corroborated this finding. Detached cells showed also an increased ability to colonize alkane-medium interface most likely linked to the their intracellular lipidic reserves

Bien que les travaux portant sur la biodégradation des hydrocarbures soient bien documentés, très peu d’études ont toutefois été effectuées dans les milieux de vie extrême. A ce jour, la plupart des études portant sur le devenir des hydrocarbures et le rôle joué par les micro-organismes hydrocarbonoclastes dans leur biodégradation ont été conduites dans des milieux presentant principalement des conditions physico-chimiques standards (e.g. pression hydrostatique de 0,1 MPa, salinité comprise entre 0 et 39 g L-1). Or, une partie importante des hydrocarbures déversés dans l’environnement peut aussi contaminer les milieux océaniques profonds (pression hydrostatique supérieure à 10 MPa) ainsi que les milieux hypersalés côtiers (salinité supérieure à 150 g L-1). Par ailleurs, le traitement des rejets hypersalins contaminés par les hydrocarbures qui sont générés par les exploitations pétrolières posent des problèmes sérieux et couteux. Ce travail de recherche porte sur l’étude de l’activité hydrocarbonoclaste de procaryotes présents dans des milieux extrêmes (hyperbares et hypersalés) pour d’une part, comprendre les processus naturels de biodégradation dans ces écosystèmes et d’autre part permettre le développement futur de techniques de réhabilitation spécifiques. Dans ce but, des expérimentations en laboratoire ont été réalisées. Une mise au point de méthodes d’incubation sous pression hydrostatique nous a permis d’obtenir des informations métaboliques et physiologiques importantes sur une souche bactérienne piézotolérante hydrocarbonoclaste (Marinobacter hydrocarbonoclasticus #5) lorsqu’elle se développe sous pression hydrostatique (35 MPa) sur hexadécane comme seule source de carbone et d’énergie. Cela confirme la capacité de certaines bactéries à dégrader les hydrocarbures en conditions hyperbares. Les résultats obtenus soulignent par ailleurs l’importance de la synthèse de cires dans le fonctionnement énergétique des souches hydrocarbonoclastes et soulèvent de nombreuses questions quant à l’augmentation du degré d’insaturation des acides gras cellulaires à pression hydrostatique élevée. D’une manière intéressante, nos résultats montrent aussi que les micro-organismes qui jouent potentiellement un rôle important dans la biodégradation des hydrocarbures dans les milieux océaniques profonds méditerranéens appartiennent aux mêmes genres que ceux isolés des milieux côtiers, notamment Alcanivorax. Notons également que dans les conditions de culture utilisées certaines souches isolées dégradent préférentiellement le n-hexadecane (e.g. Alcanivorax venustensis, Rhodobacter) ou l’heptadecane (e.g. Marinobacter sp.). De même, les archées halophiles extrêmes hydorcarbonoclastes que nous avons isolées appartiennent aux deux genres ubiquistes des environnements hypersalés que sont Haloarcula et Haloferax. En fonction des souches, les archées halophiles isolées montrent un taux important de dégradation de 32 à 95% (0.5 g L-1) du n-heptadécane après 30 jours d’incubation à 40°C dans les milieux sursalés synthétiques (225 g L-1 NaCl). Certaines souches comme Haloferax sp. MSNC 14 sont capables de dégrader à la fois des n-alcanes et un hydrocarbure aromatique polycyclique (HAP), le phénanthrène. Les résultats obtenus au cours de ce travail de thèse constituent une contribution à l’amélioration de nos connaissances sur le devenir des hydrocarbures dans les milieux océaniques profonds et les milieux hypersalins, jusqu’alors peu étudiés. Au regard de ces résultats, il apparaît indispensable de poursuivre l’étude des milieux extrêmes et de la biodiversité microbienne unique qu’ils abritent. Mots clés : hydrocarbures, biodégradation, souches hydrocarbonoclastes, milieux extrêmes, pression hydrostatique, salinité
Limited information is available on the ability of prokaryotes living in the extreme environments to degrade hydrocarbon. To date, most studies on hydrocarbon biodegradation were conducted on microorganisms isolated from environments with so-called “standards” physicochemical properties (e.g. hydrostatic pressure 0,1 MPa and salinity between 0 and 39 g L-1), despite the evident occurrence of hydrocarbons in extreme environments. An important part of hydrocarbon spilled in the environment could contaminate the deep-sea (hydrostatic pressure higher than 10 MPa) as well as hypersalin (salinity higher than 300 g L-1) coastal areas. Moreover, hydorcarbon-contaminated hypersaline waste water produced during oil exploitation require the improvement of the remediation treatments. The present work deals with the hydrocarbon-degrading activity of prokaryotes living in these extreme environments. In order to deepen our knowledge on the hydrocarbon-degrading microorganisms potential role and also to contribute to the development of specific rehabilitation techniques in these type of environments, various experiments were carried out. A bacterial cultivation method under hydrostatic pressure was developped, through which we obtained important metabolical and physiological informations on a hydrocarbon-degrading piezotolerant bacterium (Marinobacter hydrocarbonoclasticus #5) grown using hexadecane as sole source of carbon and energy. Results confirm the capacity of certain bacteria to degrade the hydrocarbon under hyperbaric (35 MPa) conditions. They also highlight the importance of wax production in energetical functionning and raise numerous questions regarding the increase, under elevated hydrostatic pressure, of unsaturated fatty-acid degree that constitute the wax. Interestingly, our results indicated that the same groups of bacteria, in particular those belong to Alcanivorax, were potentially involved in hydrocarbon biodegradation in deep-sea environments as in coastal waters. Moreover, with the cultivation condition used, isolated strains preferred to degrade either n-hexadecane (e.g. Alcanivorax venustensis, Rhodobacter) or n-heptadecane (e.g. Marinobacter sp.). Similar result was obtained from experiments carried out with archaea isolated from a shallow crystallizer pond sample, in that these microorganisms belong to two ubiquistes genus (Haloarcula and Haloferax) in hypersalin environments. Depending on the strain, extremely halophilic archaea isolated degraded 32 to 95% (0.5 g L-1) of n-heptadecane after 30 days of incubation at 40°C in 225 g L-1 NaCl artificial medium. One of the strains (MSNC 14) was also able to degrade phenanthrene. This study provides useful informations on hydrocarbon biodegradation by microorganisms in deep-sea and hypersalin environments, which remains yet to be fully explored. Further studies seems thus indispensable to elucidate the physico-chemical and biological properties involved in these processes, as well as works on the particular microbial biodiversity living in this type of environment. Keywords: alkanes, biodegradation, hydrocarbon-degrading strains, extreme environments, hydrostatic pressure, salinity

Lors de la croissance sur n-alcanes, la bactérie marine Marinobacter hydrocarbonoclasticus SP17 forme un biofilm à l'interface eau-hydrocarbure. La formation de biofilms n'a été observée que sur des substances insolubles et métabolisables, les n-alcanes de C8 à C28 et les alcools gras à 12 et 16 carbones. Par contre, il n'y a pas formation de biofilms à la surface des alcanes non métabolisables pristane, heptamethylnonane et dotriacontane. La formation de biofilms par M. Hydrocarbonoclasticus SP17 serait donc spécifique des interfaces nutritives, c'est-à-dire les interfaces entre la phase aqueuse et un substrat insoluble. La perturbation du développement du biofilm par une forte agitation conduit à une sévère réduction de la croissance et du taux de dégradation de l'hexadécane. La formation du biofilm est donc nécessaire à une assimilation efficace de l'hexadécane. L'absence d'émulsification de l'hexadécane et la production de surfactants associée aux cellules montrent que la capture de l'alcane par les cellules se fait par contact avec l'interface. Le rôle du biofilm pourrait être d'établir et/ou maintenir une interaction entre les cellules et le substrat. Nos travaux montrent que l'adaptation à l'utilisation de l'hexadécane comme source de carbone conduit à la formation de plusieurs types cellulaires au sein de la culture. Des cellules forment le biofilm à l'interface eau-substrat où se fait l'assimilation du substrat et la croissance bactérienne. Les cellules du biofilm accumulent une grande quantité de cires sous forme d'inclusions cytoplasmiques (0,47 mg/mg de protéines). Parallèlement, apparaît une population de cellules planctoniques ne se divisant pas et provenant du détachement du biofilm. Ces cellules contiennent cinq fois moins de cires (0,09 mg/mg de protéines) que les cellules du biofilm. Bien qu'elles contiennent une quantité significative de cires, l'absence de croissance des cellules détachées est due à une carence en source de carbone et d'énergie exogène puisque l'addition d'acétate permet leur croissance. Une étude physicochimique de la première étape du développement du biofilm, l'adhésion à l'hexadécane, a été entreprise en utilisant un tensiomètre dynamique à goutte. Il est montré que l'adsorption des cellules à l'interface eau-hexadécane est limitée par leur diffusion de la phase aqueuse vers l'hexadécane. L'adsorption des cellules s'interrompt avant qu'elles recouvrent la totalité de la surface disponible. Lorsque les cellules atteignent l'interface, elles produisent des composés tensio-actifs qui s'adsorbent et recouvrent l'interface. Un comportement différent des cellules aux interfaces eau-hexadécane et eau-heptaméthylnonane a été mis en évidence, indiquant que M. Hydrocarbonoclasticus SP17 est capable de différencier une interface nutritive (hexadécane) d'une interface non-nutritive (heptamethylnonane)
During growth on n-alkanes, Marinobacter hydrocarbonoclasticus SP17 forms a biofilm at the water/hydrocarbon interface. Biofilm formation was only observed on insoluble and metabolizable substances such as n-alkanes from C8 to C28 and fatty alcohols dodecanol and hexadecanol. These bacteria do not form a biofilm on the non metabolizable pristane, heptamethylnonane and dotriacontane. Biofilm formation seems to be controlled by the presence of nutritives interfaces. The perturbation of biofilm development by vigorous agitation results in a decrease in growth and rate of hexadecane degradation. Biofilm formation is required for efficient hexadecane assimilation. Both the absence of emulsification and the presence of tensioactives substances associated to the cells show that hexadecane uptake is due to interfacial contact. Ours experiments show that adaptation to hexadecane as carbon source leads to different cellular types in the same culture. Biofilm cells, located at the water/hydrocarbon interface, accumulate a large amount of waxes ester (0. 47 mg/mg of proteins). In contrast, planktonic cells originated from biofilm dispersion accumulate five time less of waxes ester (0. 09 mg/mg of proteins). Although they contain significant amount of waxes, cells detached from the biofilm could not grow due to carbon limitation and exogenous energy as demonstrated by their ability to grow after acetate addition. A physico-chemistry study of the bacterial adhesion step to hexadecane by using drop tensiometer shows that adsorption of cells on hydrocarbons is limited by Brownian diffusion. Adsorption of bacteria is stopped before the entire surface is colonized. The interruption of cell adsorption is due to production of surface active compounds by bacteria located at the interface. Different behaviours at water/hexadecane interface and the water/heptamethylnonane interface show that M. Hydrocarbonoclasticus SP17 is able to discriminate hexadecane as nutritive interface and heptamethylnone as non nutritive surface

Les écosystèmes côtiers sont des milieux complexes au sein desquels les communautés microbiennes, jouant un rôle majeur dans leur fonctionnement et leur maintien, s’adaptent et sont tolérantes à des conditions environnementales fluctuantes. En effet, au rythme des marées et de l'activité de la macrofaune, des oscillations oxie/anoxie influencent la composition et la dynamique des communautés microbiennes et par conséquent leur implication métabolique. Afin d’appréhender le devenir du pétrole dans ces écosystèmes, il est donc indispensable d’apporter des connaissances sur l’écologie des microorganismes intervenant dans son élimination, notamment dans des conditions oscillantes anoxie/oxie. Ainsi, ce travail de thèse a eu pour objectif de décrypter l’assemblage de communautés microbiennes hydrocarbonoclastesde sédiments intertidaux soumises à des oscillations anoxie/oxie en présence de pétrole lors d’une expérience en bioréacteurs. Les réponses écologiques des communautés bactériennes globales et de micro-organismes sulfato-réducteurs en conditions oscillantes ont pu être décrites en comparaison avec celles obtenues en conditions d’oxie ou d’anoxie permanentes, par l’analyse des données obtenues par séquençage haut-débit des gènes de l’ARN 16S et dsrB au niveau transcriptionnel. Ces études comparatives ont mis en évidence des profils écologiques en réponseaux conditions oscillantes, pouvant être répandus dans différents environnements marins côtiers. En réponse à ces conditions particulières, de nombreux microorganismes semblent avoir le potentiel à tolérer et/ou s’adapter aux différentes conditions d'oxygénation. Cette capacité d’acclimatation rapide des communautés bactériennes aux conditions oscillantes se sont accompagnées de capacités de dégradation équivalentes ou supérieures dans ces conditions par rapport à la condition d’oxie permanente montrant l’influence des oscillations anoxie/oxie sur le devenir du polluant dans les environnements pollués soumis à ces conditions
Coastal ecosystems are complex environments in which microbial communities, playing a major role in their functioning and maintain, are tolerant and adapt to changing environmental conditions. Indeed, the tides and the macrofauna’s activity generate oxic/anoxic oscillations which influence the composition and dynamics of microbial communities and consequently their metabolic in volvement. To understand the fate of oil in these ecosystems, it is essential to provide knowledge on the ecology of microorganisms involved in these systems, taking into account anoxic/oxicoscillating conditions. Thus, this thesis aimed to decipher the organization of hydrocarbonoclastic microbial communities inhabiting intertidal sediments, when they are subjected to anoxic/oxic oscillations in an experiment in bioreactors with oil addition. Ecological responses of bacterial communities and sulfate-reducing microorganisms in oscillating conditions have been described comparing with those obtained with permanent oxic or anoxic conditions, using high-throughputsequencing analyses of the 16S rRNA and dsrB genes at the transcriptional level. These comparatives studies have highlighted ecological profiles in response to the oscillating conditions, which can be prevalent in different coastal marine environments. In response to these particular conditions, many organisms seem to have the potential to tolerate and / or adapt to the different conditions of oxygenation. This rapid acclimation capacity of bacterial communities tothese changing conditions have been accompanied by equivalent or greater degradation capacity under these conditions compared to the permanent oxic condition, showing the influence of the anoxic/oxic oscillations on the fate of pollutant in environments subjected tothese conditions

Dissertation for the Master’s Degree in Structural and Functional Biochemistry
The majority of bacterial heavy metal resistance systems are regulated by twocomponent signal transduction systems. Stimuli from the environment interact with the histidine kinase, which in turn activates the response regulator by phosphorylation. The effector domain of the response regulator then binds to DNA, eliciting the specific response. Analysis of the Marinobacter hydrocarbonoclasticus genome revealed the presence of genes, copXAB, that code for proteins associated with copper response. The biochemical characterization of the two-component signal transduction system, copSR, is of interest due to the vital role it plays in the regulation of expression of the copXAB operon. The genes that encode for the CopR and CopS_C (cytosolic sensor domain of CopS) proteins were heterologously expressed in E.coli and expression was optimized for the production of soluble protein using LB medium. Due to solubility problems, the genes that code for these proteins were cloned as hexahistidine or glutathione S-transferase fusion proteins. CopR and its domains were optimally expressed at 16°C for 16 and 3 h after induction, respectively, whilst CopS_C was expressed at 37°C during 3 h after induction. Proteins were purified using different chromatographic strategies, most of them using affinity chromatography. The yields of pure protein per liter of growth culture obtained after complete purification from the soluble cellular extract were: 0.14 to 0.23 mg/L for CopR; 0.42 mg/L, CopR_NHis6; for the CopR_CHis6 it was 0.16 mg/L and 4.2 mg/L of CopS_C. The molecular mass of each protein was determined by gel filtration, 31 kDa for CopR, 17.5 kDa for CopR_NHis6, 15.1 kDa for CopR_CHis6 and 38.2 kDa for CopS_C. In the case of CopS_C there is the possibility that a dimer is formed, which should be evaluated. From the evaluation of disulfide bonds, using SDS PAGE and PAGE gels, all proteins or protein domains appeared to be monomers when in the presence of β-MEtOH. Circular dichroism evaluated the state of folding of the CopS_C and CopR proteins, which were shown to be folded in which the α-helix structures predominate. A model structure for CopR was also determined which agrees with this analysis. However, in the case of the CopR domains, the data obtained merely indicate folding, due to the low concentrations of the proteins. Phosphorylation and electrophoresis mobility shift assays of the CopR protein were, for the most part, inconclusive. However, in the absence of BSA, formation of the CopR:DNA complex in a gel filtration column is observed, though requires additional evaluation.

A partir des caracteristiques phenotypiques et de l'analyse de la sequence de l'arn ribosomal 16s, une bacterie marine halotolerante a ete denommee marinobacter hydrocarbonoclasticus nov. Gen. Nov. Sp. (atcc 49840). La croissance sur acetate ou sur eicosane est affectee par la salinite: le temps de latence et le temps de generation sont augmentes, bien que la biomasse ne varie pas significativement. La biodegradation de l'eicosane (80-90%) n'est pas modifiee par la salinite. La souche presente une adherence moderee (40% des cellules adherees a l'hexadecane) et produit un emulsifiant extracellulaire, sans toutefois pseudosolubiliser le substrat insoluble. M. Hydrocarbonoclasticus accumule la glycine betaine a partir du milieu et ne la degrade pas. Une inhibition de la croissance et de la biodegradation par la glycine betaine a ete mise en evidence. Toutefois, la glycine betaine exerce un effet positif sur l'activite specifique de degradation ainsi que sur l'activite emulsifiante a basses osmolarites. C'est la premiere fois qu'un effet negatif de la glycine betaine sur la croissance d'une bacterie est rapporte. La souche synthetise de l'ectoine comme osmolyte principal. M. Hydrocarbonoclasticus est donc capable de s'adapter aux hautes concentrations en nac1 par l'accumulation de solutes compatibles principalement l'ectoine

Le but de cette étude est d'évaluer l'effet qu'exercent les hydrocarbures pétroliers sur les communautés bactériennes sédimentaires marines et de mettre en évidence la validité de l'utilisation des acides gras phospholipidiques (AGPL) comme biomarqueurs en écologie microbienne. La démarche adoptée à consisté à analyser : (1) la biotransformation d'hydrocarbures dans des cultures pures, sur des microcosmes ainsi qu'au niveau d'expérimentations m situ. (2) la composition en AGPL. Nous avons pu mettre en évidence que les communautés microbiennes sédimentaires réagissent et modifient leur composition en AGPL en réponse à une contamination artificielle par des hydrocarbures. L'analyse des AGPL s'est montrée efficace pour mettre en évidence les modifications lipidiques globales au sein des sédiments qui sont la résultante de l'effet des hydrocarbures sur les membranes bactériennes et des modifications structurelles des communautés
Some petroleum hydrocarbons such as n-eicosane, phenanthrene, and a crude oil (BAL 250) were used as model organic contaminants to study the effects of petroleum hydrocarbons on marine sedimentary bacterial compartment. The focus of this study is on the fate of the contaminants, microbial growth, phospholipid ester-linked fatty acid quantification and also their modification as a response to hydrocarbons contamination. Laboratory (in vitro) and fields (in situ) experiments were conducted to obtain information on the effect of petroleum hydrocarbons on the phospholipidic composition of sedimentary marine bacteria. A particular interest was also given to laboratory microcosms. We conclude that microbial communities modify their structure resulting from an artificial contamination by petroleum hydrocarbons. These modifications are related to a high biotransformation activity when contaminant was applied, and are followed by an increase in microbial biomass of hydrocarbonoclastic and non-hydrocarbonoclastic bacteria

Les composés organiques hydrophobes (HOCs), lipides et hydrocarbures, représentent une part significative de la matière organique dans l’environnement marin. Leur faible solubilité dans l’eau exige de la part des bactéries qui les dégradent des adaptations physiologiques permettant de stimuler leur transfert de masse de la phase organique vers la phase aqueuse où ils sont assimilés. La formation de biofilm à l’interface HOC-eau est l’une de ces adaptations. La bactérie marine Marinobacter hydrocarbonoclasticus (Mh), qui est capable d’utiliser un catalogue assez large de HOCs comme les alcanes, les alcools gras et les triglycérides, a été utilisée comme modèle d’étude de la formation de biofilms aux interfaces HOCs-eau. Le but de mes recherches était de : (i) mener la caractérisation fonctionnelle des gènes aupA et aupB, qui sont surexprimés en condition de biofilm sur hexadécane et (ii) dresser, par une étude de transcriptomique, une liste de gènes potentiellement impliqués dans l’adhésion et la formation de biofilm aux interfaces HOCs-eau dans le but d’appréhender les mécanismes moléculaires mis en jeu. L’étude fonctionnelle de aupA et aupB a révélé que ces deux gènes forment un opéron dont l’expression est activée par divers types de HOCs. Il a aussi été démontré qu’ils sont impliqués dans le transport de l’hexadécane et dans la formation de biofilm sur alcanes. La protéine AupA est localisée dans la membrane externe de Mh et AupB, une lipoprotéine présumée, est située dans la membrane interne. AupA appartient à une sous-famille de transporteurs FadL-like, spécifique des bactéries marines hydrocarbonoclastes (HCB). La distribution phylogénétique de l'opéron aupAB limitée aux bactéries marines ayant la capacité de dégrader les alcanes et sa présence en nombreuses copies chez certaines souches d’Alcanivorax sp. suggèrent fortement que les protéines Aup joueraient un rôle primordial dans l’adaptation des HCB à l’utilisation d’alcanes comme sources de carbone et d’énergie. L’analyse transcriptomique des cellules de Mh adhérées (après 15 min ou 3 h de contact) ou formant un biofilm aux interfaces HOCs-eau a révélé une modification importante et précoce de leur transcriptome. De nombreux gènes intervenant dans le métabolisme des HOCs, la production de polysaccharides, la synthèse d’acides aminés et de protéines ribosomales présentent une expression modulée dès 15 min d’adhésion. La surexpression des gènes de flagelle et du chimiotactisme conjointement avec celle de gènes de pili en condition d’adhésion évoquent une possible mobilité des cellules de Mh à l’interface dans les étapes précoces du développement du biofilm. De plus, il semblerait que le facteur de transcription RpoN soit impliqué dans la régulation de la formation de biofilm chez Mh et que les prophages puissent intervenir dans la structure et/ou la dispersion du biofilm. Enfin, le rôle potentiel d’un îlot génomique dans la formation de biofilm sur trioléine a été suggéré
Hydrophobic organic compounds (HOCs), such as lipids and hydrocarbons, represent a significant part of the organic matter in the marine environment. Their low solubility in water requires from bacteria that degrade them physiological adaptations to stimulate their mass transfer from the organic to the aqueous phase where they are assimilated. Biofilm formation at the HOC-water interface is one of those adaptations. The marine bacterium Marinobacter hydrocarbonoclasticus (Mh) which is able to use a broad range of HOCs such as alkanes, fatty alcohols and triglycerides, was used as a model to study the biofilm formation at HOCs-water interfaces. The aim of my research was to (i) conduct the functional characterization of aupA and aupB genes which are overexpressed in biofilm on hexadecane, (ii) draw up a list of genes, through a transcriptomic study, that are potentially involved in adhesion and biofilm formation at HOCs-water interfaces in order to understand the molecular mechanisms involved.Functional study of aupA and aupB revealed that these two genes form an operon whose expression is activated by various types of HOCs. They have also been shown to be involved in the transport of hexadecane and in biofilm formation on alkanes. The AupA protein is localized in the outer membrane and the predicted lipoprotein AupB is located at the inner membrane. AupA belongs to a subfamily of the FadL-like transporters, specific to marine hydrocarbonoclastic bacteria (HCB). The phylogenetic distribution of the aupAB operon restricted to marine bacteria having the ability to degrade alkanes and its presence in multiple copies in somestrains of Alcanivorax sp. strongly suggest that Aup proteins play a key role in the adaptation of HCB to use alkanes as carbon and energy sources. The transcriptomic analysis of Mh cells adhering (after 15 min or 3 h of contact) or forming a biofilm at HOCs-water interfaces revealed significant and early changes in their transcriptome. The expression of many genes involved in the metabolism of HOCs, polysaccharides production, amino acids and ribosomal proteins synthesis is modulated as early as 15 min of adhesion. The overexpression of flagella and chemotaxis genes together with that of pili in adhesion condition suggest a possible motility at the interface during the early stages of biofilm development. In addition, it appears that the transcription factor RpoN is involved in the regulation of biofilm formation in Mh and that prophages could play a role in the structure and/or dispersal of the biofilm. Finally, a potential role of a genomic island in biofilm formation ontriolein was suggested

Les principaux objectifs de ce travail de recherche étaient (i) l'étude de la composition lipidique de quatre bactéries Gram-négatives hydrocarbonoclastes Marinobacter hydrocarbonoclasticus, Marinobacter aquaeolei, Acinetobacter calcoaceticus et Pseudomonas oleovorans en fonction de la source de carbone, (ii) la détermination des différents voies métaboliques de dégradation des hydrocarbures comportant une chaîne alkyle et (iii) mieux connaître la composition en Β-hydroxy acides lipopolysaccharidiques de leurs membranes extérieures. La première partie de ce travail présente une description qualitative et quantitative des lipides "non liés", labiles en milieu basique et labiles en milieu acide de M. hydrocarbonoclasticus en fonction de l'hydrocarbure (alcanes normaux et ramifiés, phényl et cyclohexyl-alcanes, et 1-alcène) utilisé comme source unique de carbone et d'énergie. Ces résultats ont montré que la structure chimique des lipides identifiés suit celle de l'hydrocarbure fourni: apparition de proportions significatives et dans certains cas très importantes d'acides gras issus de la dégradation de ces composés. Les Β-hydroxy acides des LPS sont moins sensibles au changement de la structure de l'hydrocarbure. Le n-Β-12:0 est toujours dominant alors que les Β-hydroxy acides provenant de la dégradation des hydrocarbures peuvent être incorporés aux LPS mais ne constituent que des composés mineurs. La présence d'intermédiaires métaboliques de chaque hydrocarbure dans les lipides "non liés" a permis la détermination des différents voies métaboliques de dégradation. La deuxième partie de ce travail porte sur la composition lipidique des bactéries M. aqueolei, A. calcoaceticus et P. oleovorans en fonction de la source de carbone (acétate ou alcane). Cette partie a montré le degrés de variabilité des acides gras chez les bactéries Gram-négatives M. aquaeolei et A. calcoaceticus: substitution des acides gras normaux pairs par des acides gras de même parité et même squelette que l'alcane utilisé. Chez P. oleovorans l'utilisation d'alcanes courts se manifeste aussi bien sur la distribution des acides gras membranaire que sur la composition en Β-hydroxy acides des LPS. Enfin, les Β-hydroxy acides issus des quatre bactéries étudiées sont essentiellement normaux pairs de C10 à C14 dont le composé prédominant est dépendant de l'espèce bactérienne.

Les phospholipides sont les principaux constituants des membranes bactériennes. Par ailleurs, les croissances in vitro sur des xénobiotiques comme les n-alcanes, les composés aromatiques et les alcools aboutissent généralement à une réponse adaptative des membranes bactériennes. Cette réponse est révélée par une modification majeure de la composition des acides gras phospholipidiques. À propos de ce travail, nous avons étudié différentes souches bactériennes hydrocarbonoclastes cultivées in vitro sur des substrats qui étaient soit solubles, soit hydrophobes, et ce afin de déterminer les effets des xénobiotiques sur les acides gras phospholipidiques mais également sur les phospholipides intacts. Par la suite, nous avons proposé une expérimentation in situ impliquant l'élucidation structurale et l'analyse quantitative des acides gras et des lipides polaires extraits de sédiments marins. Ces sédiments ont été prélevés à proximité d'une raffinerie durant l'hiver et la fin du printemps afin d'estimer les influences respectives des teneurs en hydrocarbures et de la contribution lipidique des macrophytes au cours des saisons
Phospholipids are the major components of bacterial membrane. Furthermore, the growths in vitro on xenobiotics such as n-alkanes, aromatic compounds and alkanols bring about to a bacterial membrane adaptive response. Generally, this response is revealed by major modification of the phospholipid ester-linked fatty acids composition. Concerning this work, we studied different hydrocarbon-degrading bacteria cultured in vitro on either a soluble or hydrophobic substrates so as to determine the effects xenobiotics on both phospholipid ester-linked fatty acids and intact phospholipids. Afterward, we proposed an in situ experiment involving the structural determination and the quantitative analysis of fatty acids and polar lipids extracted from marine sediments. These sediments were collected near to a petroleum refinery and during either the winter or the late spring in order to estimate the influence of hydrocarbon amounts and the seasonal lipid contribution of the macrophytes respectively

Le travail réalisé dans le cadre de cette thèse a pour but de fournir de nouvelles approches méthodologiques pour caractériser les communautés microbiennes en milieu océanique profond et de mesurer leurs activités. Dans le cadre de cette thèse, nous avons tout d'abord amélioré une technique d'hybridation in situ (CARD-FISH) pour dénombrer les groupes phylogénétiques majeurs en milieu marin profond. En Mer Tyrrhénienne, le pourcentage des Bacteria est toujours plus élevé que celui des Crenarchaea et des Euryarchaea. Alors que le pourcentage des Crenarchaea augmente avec la profondeur, celui des Euryarchaea est relativement homogène le long de la colonne d’eau. Dans le cadre d'une collaboration avec le Centre de Physique des Particules de Marseille sur le programme ANTARES (télescope sous-marin à neutrino), nous avons étudié quel pouvait être le rôle des bactéries luminescentes dans le bruit lumineux détecté quelquefois par le télescope. Une première étape a été de développer une approche quantitative par la méthode CARD-FISH sur les ARNm des gènes lux qui n'a été qu'en partie concluante mais donne des résultats préliminaires encourageants. Notre attention s'est portée sur l'identification puis la caractérisation d'une souche isolée à 2200 m de profondeur à proximité du site ANTARES (nommée Photobacterium phosphoreum ANT-2200). Pour mieux comprendre le rôle des bactéries bioluminescentes en milieu profond, nous avons développé un système hyperbare spécifique pour évaluer l’effet de la pression hydrostatique sur cette souche. Alors que cette souche a été caractérisée comme piezotolérante (croissance non sensible à des variations de pression), la lumière produite est plus élevée à la pression de 22 MPa (2200 m simulée) qu'à 0.1 MPa (pression atmosphérique). Enfin, une étude complémentaire m'a conduit à étudier l'effet de la pression hydrostatique sur une souche hydrocarbonoclaste Marinobacter aquaeolei #5. Là encore, nous montrons que la modification de la pression hydrostatique n’influence pas le taux de croissance (souche piezotolérante), mais peut fortement modifier les voies métaboliques, notamment la quantité et la nature des cires produites. Les cires intracellulaires s’accumulent sous formes de corps lipidiques avec une proportion moins importante à 35 MPa alors que le rapport d'insaturation des acides gras membranaires et la quantité de cires di-insaturées augmentent à la pression 35 MPa. Par ces études, nous suggérons que la pression hydrostatique joue un rôle important au niveau de la physiologie des bactéries marines et la distribution des procaryotes dans les écosystèmes marins profonds
The purpose of this work is to provide new methodological approaches to characterize the microbial communities in deep-sea waters and to measure their activities. Within the framework of this thesis, first of all we improved the technique of in situ hybridization (CARD-FISH) to count the major phylogenetic groups in deep-sea zones. At the Tyrrhenian Sea, the percentage of Bacteria was always higher than Crenarchaea and Euryarchaea. Whereas the percentage of Crenarchaea increases with depth, while Euryarchaea is relatively homogeneous along the water column. A second step, within the framework of the ANTARES program in collaboration with the Centre de Physique des Particules de Marseille, we have studied the role of the luminescent bacteria in the luminous background detected sometimes by the telescope. We first developed a quantitative approach using CARD-FISH on mRNA of the Lux genes which was only partly conclusive but gave encouraging preliminary results. Then, we characterized a luminous strain isolated at 2200 m-depth close to the ANTARES site (named Photobacterium phosphoreum ANT-2200). For better understanding the role of the bioluminescent bacteria in deep-sea, we developed a specific hyperbaric system to evaluate the effect of the hydrostatic pressure on this strain. Whereas this strain was characterized as piezotolerante (growth non sensible to variations of pressure), bioluminescence was always higher at 22 MPa (2200 m simulated) than at 0.1 MPa (atmospheric pressure). Finally, a complementary study was done to study the effect of the hydrostatic pressure on a hydrocarbonoclastic bacteria Marinobacter aquaeolei #5. We have showed that the modification of the hydrostatic pressure does not influence growth rate (piezotolerante strain), but can strongly modify the metabolic ways, in particular the quantity and the nature of produced waxes. The intracellular waxes accumulated in the cells were proportionally less important at 35 MPa than at O.1 MPa. At the contrary, the unsaturation ratio of the membrane fatty acids and the quantity of diunsaturated waxes esters increased at 35 MPa. By these studies, we demonstrated that the hydrostatic pressure plays an important role on the physiological level of the marine bacteria and the distribution of the procaryotes in the deep sea ecosystems

La relation entre la structure des communautés bactériennes et le fonctionnement de l’écosystème dans l’environnement marin est encore mal connue. L’étude de la dynamique de ces communautés en présence hydrocarbures est fondamentale pour comprendre l’impact environnemental des polluants et le développement d’outils pour minimiser ses effets (bioremédiation). Les communautés bactériennes marines en mer Méditerranéen ont été caractérisées dans leur totalité (approche gène ribosomale de l’ARNr 16S) et sa fraction active (ARNr 16S) permettant d’établir une relation entre groupes phylogénétiques actives et des paramètres globaux d’activité. Cette caractérisation, avec notamment l’approche parallèle ADN/ARN 16S, a permis également d’étudier l’effet de la température et de la fertilisation dans la dynamique des communautés bactériennes de la mer Méditerranée et des eaux froides de Kerguelen en présence de contamination par du pétrole brut ou du diesel. Finalement l’utilisation des techniques d’isolement de bactéries a permit d’isoler et caractériser une nouvelle souche bactérienne capable de dégrader des hydrocarbures à haut poids moléculaire

La pollution des écosystèmes marins côtiers par les hydrocarbures, en particulier les HAPs, est un problème environnemental majeur. Le même constat est fait pour le littoral Sud de Sfax (Tunisie) dont la pollution presque généralisée menace sérieusement les ressources naturelles existantes dans la région. La caractérisation physico-chimique des échantillons marins prélevés à partir des trois ports (plaisance, commerce et pêche) de la ville de Sfax, prouve une contamination par des micropolluants organiques (hydrocarbures) et inorganiques (métaux) qui sont considérés comme des excellents traceurs de la pollution urbaine et industrielle et ils font partie des composés les plus toxiques étant donné leur faible biodégradation. La méthode d’empreinte moléculaire (PCR-SSCP) montre une dominance du domaine Bacteria suivie des Eucarya et des Archaea au sein des échantillons marins étudiés. Les analyses statistiques par le logiciel R ont montré l’absence de corrélation entre la communauté bactérienne identifiée par PCR-SSCP et les paramètres physico-chimiques étudiés.Dans une autre partie de travail, quatre souches bactériennes marines hydrocarbonoclastes ont été isolées et caractérisées sur les plans phénotypique et phylogénétique, après des enrichissements sur des HAPs et sur le pétrole brut: FLU5 de Bacillus stratosphericus, NAPH6 de Pseudomonas aeruginosa, PYR2 de Bacillus licheniformis isolées sur le fluoranthène, le naphtalène et le pyrène, respectivement, en présence de 30 g/l NaCl; et CO100 de Staphylococcus sp., isolée sur le pétrole brut, en présence de 100 g/l NaCl. Les analyses chromatographiques, GC-MS ou GC-FID, montrent les capacités biodégradatives intéressantes de ces composés récalcitrants par les bactéries isolées. En outre, ces quatre souches bactériennes, sont capables de produire des biosurfactants nommés BS-FLU5, BS-NAPH6, BS-PYR2 et BS-CO100, sur plusieurs sources de carbones, y compris l’huile de friture résiduelle, un substrat bon marché, minimisant ainsi le coût élevé de production de ces tensioactifs. Les analyses MALDI-TOF/MS, des biosurfactants BS-FLU5, BS-PYR2 et BS-CO100 purifiés, montrent qu’il s’agit des lipopeptides, les biosurfactants BS-NAPH6 sont de nature rhamnolipidique, sur la base des analyses FTIR. Ces quatre biosurfactants sont caractérisés par des propriétés tensiactives intéressantes : une faible CMC, une importante réduction de la tension de surface... Ils sont stables vis-à-vis d’une large gamme de pH, de température et de salinité. De plus, ces agents tensioactifs sont doués d’activité de remobilisation des hydrocarbures contenus dans des sols pollués par. Les biosurfactants BS-FLU5, BS-PYR2 et BS-CO100, présentent des activités anti-adhésives et anti-biofilms intéressantes contre des biofilms de certains microorganismes pathogènes. Par ailleurs, une propriété cicatrisante remarquable sur des plaies d’excision chez un modèle expérimental de rats de race Wistar, a été montrée par les quatre biosurfactants pour des concentrations de l’ordre de 5 et 10 mg/ml, en comparaison avec un cicatrisant de référence (CICAFLORA®). A noter que, l’évaluation de la cytotoxicité des biosurfactants étudiés, a montré qu’ils n’ont pas des effets toxiques sur des cellules rénales humaines HEK-239 à des concentrations jusqu’à 1000 µg/ml pour BS-FLU5 et BS-CO100 et jusqu’à 200 µg/ml pour BS-NAPH6 et BS-PYR2. La production des biosurfactants de la souche FLU5 à l’échelle pilote (deux fermenteurs de 20 et 100 litres, volume total), en présence d’un milieu économique, montre une augmentation des quantités des biosurfactants produits par rapport à l’échelle laboratoire (erlenmeyer, 1 litre). L’ensemble de ces résultats prometteurs, montrent que les souches marines isolées FLU5, NAPH6, PYR2 et CO100, ainsi que leurs biosurfactants demeurent d’intérêts biotechnologiques pour divers types d’applications, tels que la bioremédiation, l’agroalimentaire, la cosmétique
Pollution of coastal marine ecosystems by hydrocarbons, in particular polycyclic aromatic hydrocarbons (PAHs), is a major environmental problem. The South coast of Sfax (Tunisia) is an example of a polluted ecosystem subject to both urbanization and industrialization including the outfall of untreated domestic sewage and wastewaters, fishery activities, as well as ship traffic and boat pollution. The physico-chemical characterization of the seawater taken from three harbours (pleasure, commercial and fishing) of the city of Sfax, showed a heavy contamination by organic and inorganic micropollutants. These are excellent tracers of urban and industrial pollution, and they are among the most toxic compounds due to their low biodegradation.The molecular fingerprinting technique (PCR-SSCP) showed the dominance of the Bacteria domain followed by Eucarya and Archaea within the studied marine samples. Statistical analysis using the R software, showed that no correlation was identified between the bacterial community identified by PCR-SSCP and the studied physico-chemical parameters.In another part, four marine, aerobic and hydrocarbonoclastic strains: Bacillus stratosphericus FLU5, Pseudomonas aeruginosa NAPH6, Bacillus licheniformis PYR2, isolated after enrichments on fluoranthene, naphthalene and pyrene, respectively, and in the presence of 30 g/l NaCl. Strain Staphylococcus sp. CO100 was isolated after enrichment on crude oil, in the presence of 100 g/l. Chromatographic analysis (GC-MS or GC-FID), showed the interesting biodegradative capacities of these recalcitrant compounds by the isolated bacteria.Besides, these strains showed their capacity to produce efficient surface active agents BS-FLU5, BS-NAPH6, BS-PYR2 and BS-CO100, on several substrates and in particular the residual frying oil, which is a cheap and renewable carbon source alternative, thus minimizing the high cost of producing surfactants. The MALDI-TOF/MS analysis of the purified BS-FLU5, BS-PYR2 and BS-CO100 biosurfactants revealed that they are belonging to lipopeptide family. FTIR analysis showed the glycolipid nature, more precisely the rhamnolipid type, of biosurfactant BS-NAPH6.These four biosurfactants are characterized by interesting tensioactive properties (low CMC, important surface tension reduction...). Furthermore, these surface active agents showed interest stability against a broad range of pH, temperature and salinity. The application of these biosurfactants, in oil recovery, from hydrocarbons-contaminated soil, showed that they were more effective on the hydrocarbon-remobilization than some tested synthetic surfactants. The biosurfactants BS-FLU5, BS-PYR2 and BS-CO100, were found to have notable anti-adhesif and anti-biofilm activities, being able to prevent and eliminate the biofilm formation by pathogenic microorganisms. Moreover, the four tested biosurfactants showed an interesting healing activity, on the wound site in a rat model. They increased significantly the percentage of wound closure when compared to the untreated and CICAFLORA® (a reference pharmaceutical product) treated groups, using two different concentrations (5 and 10 mg/l). Interestingly, the evaluation of the cytotoxicity of the studied biosurfactants, showed that they have no toxic effects on human HEK-239 cells at concentrations up to 1000 μg/ml for BS-FLU5 and BS-CO100 and up to 200 μg/ml for BS-NAPH6 and BS-PYR2. An attempt to produce biosurfactant produce by strain FLU5 on a pilot-scale (fermentors of 20 and 100 liter, as total volume), using a cost-effective medium, was also performed. Preliminary results showed an increase in the quantities of biosurfactantsBS-FLU5 produced on a pilot-scale compared to the lab-scale (Erlenmeyer of 1 liter).These results highlight the interest for potential use of strains FLU5, NAPH6, PYR2 and CO100, as well as their biosurfactants, in a wide variety of industrial, environmental and biotechnological applications

Compounds from the marine environment exhibit a wide variety of biological activities, and thus hold much promise as potential drugs. The halichondrins, isolated from the Kaikoura sponge Lissodendoryx sp. are no exception to this, demonstrating potent anticancer activity. Novel cytotoxic compounds have also been isolated from the Chatham Rise sponge Lamellomorpha strongylata. Knowledge of the cellular origins of such compounds is desirable, in order to establish if the sponge or associated micro-organisms are producing the compounds of interest. Siderophores are also important molecules, which are produced on demand by bacteria in order to obtain sufficient iron necessary for their growth. Knowledge of the biosynthesis of these compounds has potential for the control of undesirable bacteria, such as the anthrax-causing pathogen Bacillus anthracis. Cell separation studies have been carried out on Lamellomorpha strongylata, locating a swinholide in sponge-associated filamentous bacteria and theonellapeptolides in sponge-associated unicellular bacteria. A microscopic analysis of dissociated cells from Lissodendoryx sp. was also undertaken. The structures of four new halichondrins (3.13 - 3.16), isolated from Lissodendoryx sp., have been determined from spectral data. All of these compounds are very similar to known B series halichondrins, with differences occurring only beyond carbon 44. As biological activity has been shown to be derived from the portion of the molecule between carbons 1 and 35, they all retain good activity in the P388 assay as expected. A new siderophore, petrobactin sulfonate (4.2), was characterised, along with three cyclic imide siderophore derivatives (4.3 - 4.5). Petrobactin sulfonate is the first marine siderophore containing a sulfonated 3,4-dihydroxy aromatic ring. The structures were elucidated from spectral data, resulting in a revision of the NMR assignments of petrobactin.

L'objectif de ce travail consistait a apporter une contribution a l'etude des lipides des communautes microbiennes marines. Cette etude basee sur l'utilisation de methodes chimiques et de techniques d'analyses spectroscopiques concerne plus precisement les micro-organismes marins thermococcus hydrothermalis et pyrococcus abyssi, archaea hyperthermophiles sulfo-oxydantes isolees de sites hydrothermaux marins, et marinobacter hydrocarbonoclasticus, bacterie marine ubiquiste halotolerante extreme. Cette recherche presente une description qualitative et quantitative des lipides de t. Hydrothermalis. Ses lipides neutres correspondent aux ethers de glycerol libres et a un melange de quatre hydrocarbures tetraterpeniques, lycopadienes et lycopatrienes, identifies pour la premiere fois chez une archaea, dans cette etude. Leur decouverte a une implication geochimique fondamentale. Ils pourraient etre en partie a l'origine du lycopane retrouve en quantite trace dans tous les oceans. L'analyse des lipides polaires a conduit principalement a l'identification du biphytanyl glycerol diether et du dibiphytanyl diglycerol tetraether. Un resultat important de ce travail correspond a l'absence de cyclopentane dans les chaines biphytanyles des tetraethers. Ce travail fournit egalement une identification precise des lipides de m. Hydrocarbonoclasticus, selon la source de carbone retenue pour sa culture (acetate ou n-eicosane). Elle a montre sa grande aptitude a ingerer les hydrocarbures presents dans le milieu, meme en tres faible concentration, et a suggere l'idee d'utiliser cette bacterie comme indicateur de pollution. Des acides gras non fonctionnalises et fonctionnalises originaux, chloro et/ou methoxy, ont ete caracterises. Enfin, cette etude a mis en evidence le degre de variabilite de certains des lipides de m. Hydrocarbonoclasticus, selon le substrat choisi, et a permis d'evaluer leur interet effectif comme marqueurs de parametres environnementaux, en milieu marin.

Tese de Doutoramento em Engenharia Química e Biológica.
Bacterial storage lipids are being considered as viable alternative feedstocks for industrial and biotechnological applications, compared to conventional ones. The production of these bacterial compounds can be obtained from different carbon sources, including inexpensive and recalcitrant wastes. This thesis explores the potential of using hydrocarbonoclastic bacteria to obtain lipid reserve substances from hydrocarbon-based wastes, promoting a more economic and environmentally sustainable biotechnological process combining pollution reduction and production of added value compounds. The production of storage lipids by a hydrocarbon degrading bacterium, Rhodococcus opacus B4, from glucose, acetate and hexadecane, was reported for the first time and compared to Rhodococcus opacus PD630, the best known triacylglycerols (TAG) accumulating bacterium. TAG was the main storage compound produced by both strains. R. opacus B4 presented 2 to 3 fold higher TAG volumetric productions than R. opacus PD630 when cultivated in hexadecane. Subsequently, the ability of R. opacus B4 to decontaminate hexadecane-contaminated cork sorbents while producing lipid storage compounds was shown. Two types of cork sorbents, natural and thermally treated cork, were used. R. opacus B4 was able to degrade practically all hexadecane impregnated in both cork sorbents and TAG was the main neutral lipid compound produced. The potential of the obtained lipid-rich biomass for biomethane production was assessed and an efficent conversion to methane was observed. An indigenous microbial community enriched in carbon storage producing hydrocarbon degrading bacteria was developed from a hydrocarbon-based wastewater (containing lubricant and engine oil waste), using feast and famine conditions in the form of alternating periods of presence of the carbon substrate followed by its absence. The most dominant bacteria identified belongs to hydrocarbon degrading Rhodococcus, Acinetobacter and Pseudomonas genera, known for producing mainly triacylglycerols (TAG), wax esters (WE) and polyhydroxyalkanoates (PHA), respectively. The enriched community was able to accumulate small amounts of TAG and PHA, and higher levels of a non-identified lipid as well as to efficiently degrade the wastewater hydrocarbons. The effect of different cultivation parameters on storage compound production by the developed enriched community was evaluated, using central composite circumscribed design based on surface response methodology. The interaction between carbon and nitrogen concentrations positively influenced PHA accumulation whereas interaction between carbon and nitrogen concentrations with cultivation time affected PHA in a negative way. Regarding neutral lipids production, nitrogen concentration and the interaction between carbon and nitrogen concentrations were the significant parameters. Neutral lipids produced were essentially TAG, presenting a highly diversity of chemical structures composed by a narrow range of fatty acids. Finally, a genome-based comparative analysis of genes and metabolic reactions responsible for TAG, WE and polyhydroxybutyrate (PHB) biosynthesis in members of Rhodococcus, Acinetobacter, Alcanivorax and Pseudomonas genera was performed to get deeper knowledge on carbon storage compounds metabolism in hydrocarbonoclastic bacteria. The presence of genes coding for complete metabolic pathways for TAG and PHB biosynthesis was detected in all species. Rhodococcus strains are highly enriched in genes involved in TAG and PHB metabolism, whereas Alcanivorax, Acinetobacter and Pseudomonas have a high number of genes coding for enzymes involved in PHB production.
Os lípidos de reserva bacterianos têm sido considerados como matéria-prima alternativa aos lípidos convencionais para aplicações biotecnológicas e industriais. A produção destes compostos pode ser obtida a partir de diferentes fontes de carbono, incluindo resíduos recalcitrantes e economicamente acessíveis. Esta tese teve como principal objetivo explorar o potencial de bactérias hidrocarbonoclásticas para a produção de lípidos de reserva a partir de resíduos contendo hidrocarbonetos. Esta capacidade pode contribuir para o desenvolvimento de um processo biotecnológico enconomicamente e ambientalmente sustentável, envolvendo a descontaminação biológica de resíduos e a produção de compostos de valor acrescentado. A produção de lipidos de reserva em Rhodococcus opacus B4, uma bactéria degradadora de hidrocarbonetos, a partir de glucose, acetato e hexadecano, foi descrita pela primeira vez e comparada com a bactéria Rhodococcus opacus PD630, considerada como a melhor produtora de triacilgliceróis (TAG). Os triacilgliceróis foram os principais compostos produzidos por ambas as estirpes. R. opacus B4 apresentou uma produção volumétrica de TAG duas a três vezes superior à de R. opacus PD630 quando cultivadas em hexadecano. A capacidade de R. opacus B4 para degradar hexadecano adorvido a cortiça e produzir lípidos de reserva foi avaliada. Foram utilizados dois tipos de adsorventes de cortiça, cortiça natural e cortiça termicamente tratada. R. opacus B4 degradou eficazmente o hexadecano impregnado em ambas as cortiças e os triacilgliceróis foram os principais lípidos de reserva produzidos. O potencial de produção de metano a partir da biomassa rica em lípidos obtida foi analisado, tendo-se observado uma eficiente conversão a metano. O desenvolvimento de uma comunidade microbiana enriquecida em bactérias hidrocarbonoclásticas produtoras de compostos de reserva foi obtido a partir de águas residuais contendo lubrificantes e óleo de motor, aplicando períodos alternados de presença e ausência de fonte de carbono. As bactérias mais dominantes identificadas pertencem a géneros hidrocarbonoclásticos, nomeadamente Rhodococcus, Acinetobacter e Pseudomonas, conhecidas como produtoras de TAG, ésteres de cera (WE) e polihidroxialcanoatos (PHA), respetivamente. A comunidade enriquecida acumulou baixas quantidades de TAG e PHA e quantidades superiores de um lípido desconhecido, tendo degradado eficientemente os hidrocarbonetos presentes na água residual. O efeito de diferentes parâmetros de cultivo na produção de compostos de reserva pela comunidade enriquecida foi avaliado através de um desenho fatorial de experiências baseado na metodologia de superfície de resposta (RSM). A interação entre as concentrações de carbono e azoto influenciou positivamente a acumulação de PHA enquanto que as interações entre o período de cultivo e as concentrações de carbono e azoto e o tempo de cultivo afetaram negativamente a produção de PHA. A produção de lípidos neutros foi significativamente influenciada pela concentração de azoto e pela interação entre as concentrações de carbono e azoto. Os lípidos neutros produzidos foram essencialmente TAG, apresentando uma elevada diversidade de estruturas químicas compostas por uma ampla gama de ácidos gordos. Foi efetuada uma análise comparativa de genes e reações metabólicas responsáveis pela síntese de TAG, WE e polihidroxibutiratos (PHB) em espécies pertencentes aos géneros Rhodococcus, Acinetobacter, Alcanivorax e Pseudomonas. A presença de genes que codificam para enzimas pertencentes a todas as vias metabólicas relativas à produção de TAG e PHB foi detetada em todas as espécies estudadas. As estirpes de Rhodococcus opacus apresentam um número elevado de genes envolvidos no metabolismo de TAG e PHB, enquanto que Alcanivorax borkumensis SK2, Acinetobacter baylyi ADP1 e Pseudomonas putida KT2440 possuem uma grande diversidade de genes que codificam para enzimas envolvidas na produção de PHB.
Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/64500/2009.
Fundação para a Ciência e a Tecnologia (FCT) Strategic Project of UID/BIO/04469/2013 unit.
The project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462).
The Project BioEnv - Biotechnology and Bioengineering for a sustainable world, ref Norte - 07-0124-FEDER-000048, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER.

A actina, um componente central do citoesqueleto eucariótico, é responsáve lpela manutenção da forma celular, entre outras funções.Pensava-se que, em procariotas, este tipo de proteínas não existia, dado que estudos preliminares não haviam detetado elementos do citoesqueleto. No entanto, foram já identificadas homólogas estruturais e funcionais da actina em bactérias, nomeadamente a MreB, uma proteína do citoesqueleto de procariotas com uma estrutura helicoidal associada à membrana celular,envolvida na determinação da forma de células em bastonete. Com base nas características destas proteínas, supõe-se que a função biológica será semelhante à actina. Neste trabalho, realizou-se a clonagem da MreB de Marinobacter hydrocarbonoclasticusno vetor de expressão pET-22b(+), sobre-expressão em meio LB (condições ótimas de crescimento: 37 ºC, 190 rpm, indução a D.O.600 nm= 0,6 com 0,5 mM IPTG)e purificação através de centrifugações diferenciais e cromatografia de troca aniónica e filtração em gel. Determinou-se a massa molecular da proteína por filtração em gel (48kDa) e gel de SDS-PAGE (42,48 kDa) e o coeficiente de extinção molar (ε280 nm= 27 900 M-1cm-1), por espetroscopia de UV-Visível. Os resultados obtidos por dispersão de luz permitem concluir que, à semelhança da MreB de outras bactérias, também a de M.hydrocarbonoclasticustem a capacidade de polimerizar, sendo esta reação dependente da concentração de proteína e de KCl, ocorrendo apenas na presença de nucleótidos, com uma concentração crítica semelhante à MreB de Thermotoga maritima (≈3 nM). Verificou-se, ainda, que a velocidade de polimerização da MreB se ajusta a uma função sigmoidal descrita por -.Através da espetroscopia de RMN, determinou-se que oATP e GTP interagem de maneira semelhante com a proteína, adotando estes uma conformação tal que se ligam através da pentose e do grupo–NH2do anel de pirimidina; já o CTP ligar-se-á apenas através da pentose.

Na economia moderna movida a combustíveis fosseis, todos os produtos petroquímicos são bastante importantes para a sociedade, mas devido ao seu uso excessivo são considerados um dos principais poluentes no Mundo, causando efeitos nocivos e por vezes irreversíveis no meio ambiente. A atuação adequada e atempada em caso de contaminação é essencial e por isso, a procura de soluções que visam remediar o efeito causado por estes compostos quando se encontram indevidamente nas águas e nos solos, continua a ser um assunto pertinente e atual. Entre as diferentes estratégias disponíveis para remoção e tratamento destes contaminantes, a biorremediação através da utilização de organismos vivos, apresenta-se como uma das viáveis, quer pela sua viabilidade económica quer pela abordagem mais sustentável pelo uso de tecnologia verde. Nesta dissertação, primeiramente realizou-se uma pesquisa bibliográfica metódica, que a conduz à submissão de um artigo científico intitulado Bioremediation of soils contaminated with hydrocarbons: one overview. Posteriormente, realizaram-se estudos experimentais, referentes à verificação da existência e caracterização de organismos hidrocarbonoclásticos em amostras de solo obtidas durante o mês de outubro de 2020 na Mata da Machada e na Praia da Alburrica, concelho do Barreiro. Os estudos laboratoriais prosseguiram com a determinação da taxa de biorremediação de hexano, gasolina, tolueno, por culturas simples ou mistas das colónias selecionadas. A determinação da biodegradabilidade dos hidrocarbonetos recorreu à utilização do indicador redox 2,6-diclorofenolindofenol através do método espetrofotométrico utilizando suspensão celular. As colónias foram caracterizadas morfologicamente e bioquimicamente, pela realização do Teste Gram, onde na amostra Mata da Machada identificou-se, através das sete colónias, uma Gram-Negativa, duas Gram-Positivas e quatro que continham estirpes Gram + e Gram - . Na amostra Praia da Alburrica, através das cinco colónias obtidas, identificou-se duas GramPositivas e três com estirpes Gram + e Gram -.O crescimento em meio seletivo de Pseudomonas CN Agar Base, permitiu identificar Pseudomonas aeruginosa na amostra Mata da Machada e na amostra Praia da Alburrica. Os resultados demonstraram que na amostra da Mata da Machada, apesar de ser um local protegido e uma reserva natural apresentam bactérias, incluindo, Pseudomonas aeruginosa, que conseguem degradar os hidrocarbonetos estudados. Nas amostras da Praia da Alburrica, sendo uma zona fluvial, a existência destas bactérias que conseguem degradar hidrocarbonetos era mais provável, tendo acontecido esse desenvolvimento através da utilização dos hidrocarbonetos estudados. A degradação dos hidrocarbonetos ocorreu mais facilmente através das interações positivas entre organismos.
In the modern economy powered by fossil fuels, all petrochemical products are very important to society, but due to their excessive use they are considered one of the main pollutants in the world, causing harmful and sometimes irreversible effects on the environment. Proper and timely action in case of contamination is essential and, therefore, the search for solutions that aim to remedy the effect caused by these compounds when they are unduly found in water and soil, remains a relevant and current issue. Among the different strategies available for the removal and treatment of these contaminants, bioremediation through the use of living organisms is one of the viable ones, both for its economic viability and for a more sustainable approach through the use of green technology. In this dissertation, first, a methodical bibliographic research was carried out, which led to the submission of a scientific article entitled Bioremediation of soils contaminated with hydrocarbons: one overview. This research allowed to select the methodology of the experimental studies. Subsequently, experimental studies were carried out, referring to the verification of the existence and characterization of hydrocarbonoclastic organisms in soil samples obtained during the month of October 2020 in Mata da Machada and Praia da Alburrica, municipality of Barreiro. Laboratory studies continued with the determination of the rate of bioremediation of hexane, gasoline, toluene, by single or mixed cultures of selected colonies. The biodegradability determination of hydrocarbons resorted to the use of the redox indicator 2,6- dichlorophenolindophenol through the spectrophotometric method using cell suspension. Colonies were morphologically and biochemically characterized by performing the Gram Test, where in the Mata da Machada sample, one Gram-Negative, two Gram-Positive and four containing Gram + and Gram - strains were identified through the seven colonies. In the Praia da Alburrica sample, through the five colonies obtained, two Gram-Positive and three with Gram + and Gram - strains were identified.The growth on selective medium of Pseudomonas CN Agar Base allowed the identification of Pseudomonas aeruginosa in the sample Mata da Machada and in the sample Praia da Alburrica. The results showed that in the Mata da Machada sample, despite being a protected site and a natural reserve, there are bacteria, including Pseudomonas aeruginosa, which manage to degrade the studied hydrocarbons. In the samples from Praia da Alburrica, being a river area, the existence of these bacteria that manage to degrade hydrocarbons was more likely, and this development occurred through the use of the studied hydrocarbons. Degradation of hydrocarbons occurred more easily through probably due positive interactions between organisms.

Substantial part of the emissions of nitrous oxide (N2O), a powerful greenhouse gas, to the atmosphere comes from the incomplete denitrification in bacteria. N2O can only be detoxified by nitrous oxide reductase (N2OR), which catalyzes the last step of this pathway. This enzyme contains two distinct centers per monomer: CuA, the electron transfer center and “CuZ”, a tetranuclear copper-sulfide center, which can exists in two forms CuZ(4Cu2S) and CuZ*(4Cu1S). Most of the studies on the denitrification pathway have used soil denitrifying bacteria as models, while marine bacteria are understudied. This thesis presents an analysis of denitrification pathway of Marinobacter hydrocarbonoclasticus a marine bacterium capable of respiring nitrate under oxygen-limiting conditions. Here, the effect of pH (6.5, 7.5 and 8.5) on the denitrification pathway of this organism, as well as on the N2OR isolated from each of those growths, was investigated. These enzymes were characterized through biochemical, spectroscopic and structural studies. The expression profile of genes encoding the enzymes and accessory proteins involved in denitrification was analyzed, together with quantification of the by-products, nitrate and nitrite. These results showed lower levels of nirS expression at pH 6.5, which correlates with the accumulation of nitrite detected. In parallel, whole-cells reduction rates of NO and N2O demonstrated that denitrification is impaired at more acidic conditions, as the whole-cells are not able to reduce external N2O when grown at pH 6.5. The N2OR isolated from each growth exhibits differences at the “CuZ center”. At acidic growth conditions, N2OR has “CuZ center” mainly as CuZ*(4Cu1S), whereas when isolated from growths at 7.5 and 8.5, it is mainly as CuZ(4Cu2S). This was supported by spectroscopic data, sulfide quantification, and inspection of “CuZ center” X-ray structure, demonstrating the presence of an additional sulfur atom in the CuZ(4Cu2S) form. The effect of exogenous ligands on both forms of the “CuZ center” was re-visited and clarified. Direct electrochemistry of N2OR is reported for the first time, with the two signals observed, assigned to CuA and CuZ(4Cu2S) centers, with reduction potentials being in line with the ones determined by potentiometry (272 ± 10 mV and 65 ± 10 mV vs SHE at pH 7.6, respectively). This form of N2OR has lower specific activity (0.004 ± 0.001 U/mg) in the presence of physiological electron donor, cytochrome c552, compared to a N2OR with CuZ*(4Cu1S) (1.25 ± 0.07 U/mg). Fully reduced CuZ*(4Cu1S) is catalytically competent and in the presence of a stoichiometric amount of N2O originates CuZº intermediate. CuZº species can be reduced through intramolecular electron transference (IET) from CuA center, in a reaction 104 faster than IET in the CuZ*(4Cu1S). In the absence of substrate or electrons a novel “CuZ center” intermediate species is formed with a maximum absorption band at 617 nm, and having a [1Cu2+-3Cu1+] oxidation state. These studies shed new lights on the catalytic cycle, which was reassessed and discussed here.