The objective of this work is to develop catalysts that are active in simultaneous conversion of triacylglycerides and fatty acids into fatty acid methyl esters. Three Sn(IV) complexes, named butyl stannoic acid (BTA), di-n-butyl-oxo-stannane (DBTO) and dibutyltin dilaurate (DBTDL), were initially tested as catalysts for esterification reaction of fatty acids in the presence of methanol as alcoholysis agent. Parameters like reaction time, temperature, and catalyst amount were systematically evaluated in this work. All complexes were active at relative high reaction temperatures, but BTA displayed the highest activity. Former studies have already demonstrated that these same complexes display good catalytic activity in methanolysis of triacylglycerides (TAGs). These results prompted us to test BTA catalyst also in simultaneous esterification/transesterification reactions from a mixture of free fatty acids (FFAs) and TAGs in the presence of methanol. The BTA was able to convert mixtures of fatty acid methyl esters (FAMEs) with excellent yields of up to 96% during 4 hours with simple isolation procedures. The BTA was also tested in the hydrolysis of TAG and conversions were quite satisfactory, reaching approximately 98% at 160 ° C. From the results obtained it was possible to determine that, in comparison to hydroesterification reactions, the simultaneous esterification/transesterification is the best route to obtain FAMEs using this type of catalyst. The complexes exhibiting general formula R4-xSn(C11H20O2)x, wherein R = C4H9 or CH3 and x= 1 ou 2, were tested in order to evaluate the influence of the nature of different ligands bearing the metal center, on the esterification conversion. The results obtained in this study may suggest the following catalytic activity order: dimetildiundecenoate tin > dibutildiundec-10-enoate tin > tributilundecenoate tin, supporting a significant influence of the stereo effect in esterification reaction. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste trabalho foi testar e desenvolver catalisadores que sejam ativos tanto em reações de esterificação quanto transesterificação. Compostos de Sn(IV), denominados n-butil hidroxioxi de estanho (BTA), óxido de dibutil estanho (DBTO) e dibutildilaurato de estanho (DBTDL), foram inicialmente testados como catalisadores para a reação de esterificação de ácidos graxos (AG) na presença de metanol como agente de alcoólise. Parâmetros como o tempo de reação, temperatura e quantidade de catalisador, foram sistematicamente avaliadas. Todos os complexos foram ativos a temperaturas de reação entre 80 e 160 ºC, sendo que o BTA apresentou maior atividade com conversões de aproximadamente 96 % em ésteres metílicos de ácidos graxos (% EMAGs). Estudos anteriores já demonstraram que estes mesmos complexos exibem boa atividade catalítica na metanólise de triacilglicerídeos (TAG). Esse conjunto de resultados nos levou a testar o catalisador BTA também em reações simultâneas de esterificação/transesterificação de uma mistura de AG e TAG, na presença de metanol. O BTA foi capaz de converter as misturas em ésteres metílicos de ácidos graxos (EMAGs) com excelentes rendimentos, de até 96% durante 4 horas, com processos de purificação simples. O BTA também foi testado na hidrólise de TAG e as conversões em AG foram bastante satisfatórias, chegando aproximadamente 100 % a 160 ºC durante 10 horas. A partir dos resultados obtidos pôde-se determinar que, comparativamente à hidroesterificação, a reação de esterificação/transesterificação simultânea é a melhor rota a ser empregada na obtenção de EMAGs em presença de matérias primas de baixo custo, utilizando este tipo de catalisador. Foram sintetizados e testados novos complexos, a fim de avaliar a influência da natureza de diferentes ligantes em torno do centro metálico sobre a conversão em reações de esterificação. Com os resultados obtidos neste estudo pode-se sugerir uma ordem de atividade catalítica: dimetil diundecenoato de estanho > dibutildiundecenoato de estanho > tributilundecenoato de estanho, evidenciando uma influência significativa de efeitos estéreos na reação de esterificação.

Os ácidos linoléicos conjugados (CLAs) consistem de um grupo de isômeros geométricos e posicionais do ácido linoléico aos quais tem sido atribuídos efeitos anticarcinogênico, antidiabético, antiaterogênico e modulador da composição corporal. As principais fontes naturais de CLA são o leite e a carne de ruminantes, uma vez que microorganismos ruminais são capazes de formar CLAs e seus precursores como intermediários da biohidrogenação. Para sua identificação e quantificação por cromatografia gasosa (CG) deve se tomar cuidado ao decidir um procedimento apropriado de metilação, a fim de evitar a isomerização dos CLAs. Dessa forma, o objetivo do presente trabalho foi de avaliar criticamente metodologias analíticas para caracterizar os isômeros conjugados c-9, t-11 e t-10, c-12 do ácido linoléico, identificando o melhor método para a esterificação e a melhor programação por cromatografia gasosa para a separação, identificação e quantificação dos CLAs através de sua análise em suplementos, cortes bovinos e laticínios. Para tanto, foram empregadas as técnicas de esterificação ácida com trifluoreto de boro metanólico, esterificação alcalina com metóxido de sódio metanólico e uma esterificação mista usando a combinação dos dois catalisadores na metilação de uma amostra comercial de suplemento de ácido linoléico conjugado na forma de triacilglicerol, o Tonalin® CLA TG 80, da Cognis. Os ésteres metílicos foram identificados sob três condições cromatográficas CLA01, CLA02 e CLA03. Também foram realizadas quantificações dos isômeros c-9, t-11 e t-10, c-12 CLA no Tonalin® CLA TG 80 e FFA 80 através de ressonância magnética nuclear do carbono 13 (13C-RMN). Os resultados obtidos permitiram decidir pelo emprego da técnica de esterificação alcalina, seguido da análise cromatográfica pelo método CLA03 para análise de cinco cortes bovinos fracionados em músculo e capa de gordura, creme de leite, iogurte integral e três tipos de queijo. O método de esterificação alcalina, seguido de separação por CG pelo método CLA03 mostrou-se o mais adequado para uma quantificação segura do perfil de ácidos graxos de alimentos que contenham os isômeros conjugados do ácido linoléico, uma vez que os resultados obtidos concordam com a literatura atual. / Conjugated linoleic acids (CLAs) are a group of geometric and positional isomers of linoleic acid which have been attributed to present anticarcinogenic, antidiabetic, antiaterogenic and body composition modulation effects. Major natural sources of CLA are ruminants milk and meat, since rumen microorganisms are able to produce CLAs and their precursors as intermediates of biohydrogenation. Before identification and quantification by gas chromatography (GC), the methylation procedure should be carefully decided to avoid isomerization of CLAs. Thus, the objective of this study was to critically evaluate analytical methods for characterizing conjugated linoleic acid isomers c-9, t-11 and t-10, c-12, by identifying the best method for esterification and the best gas chromatography programming for separation, identification and quantification of CLAs through quantification of CLAs in supplements, beef and dairy products. In order to accomplish this objetive, the techniques of acid esterification with boron trifluoride methanol, alkaline esterification with methanolic sodium methoxide and mixed esterification using a combination of the two catalysts were employed in the methylation of a commercial sample of conjugated linoleic acid supplement in triacylglycerol form, the CLA Tonalin® TG 80, from Cognis. The methyl esters were identified in 3 chromatographic conditions CLA01, CLA02 and CLA03. CLA isomers c-9, t-11 and t-10, c-12 were also quantified in CLA Tonalin® TG 80 and FFA 80 by carbon 13 nuclear magnetic resonance (13C-NMR). The results indicated the alkaline esterification technique followed by chromatographic analysis by CLA03 method as the best method for analyzing five cuts of beef fractionated in muscle and fat layer, cream, full yoghurt and three types of cheese. The alkaline esterification method followed by GC separation method CLA03 proved to be the most suitable for safe quantification of fatty acid profile in foods that contain conjugated linoleic acid isomers, once the results agreed with the data available in the current literature.

De nouvelles architectures macromoléculaires ont été synthétisées à partir de lignine afin de développer des matériaux pour des applications dans le bâtiment. Trois voies différentes de modifications chimiques ont été retenues afin d’apporter de nouvelles propriétés à la lignine pour répondre à un cahier des charges précis. Dans un premier temps, des chaines de poly(caprolactone) ont été greffées sur la lignine par polymérisation par ouverture de cycle conférant ainsi à la lignine des propriétés particulières en fonction de la longueur des chaines de PCL greffées. La valorisation de la lignine pour la synthèse de polymères fonctionnels repose ainsi sur sa modification chimique. Notre démarche scientifique s’est par la suite axée sur la fonctionnalisation de la lignine afin d’en faire un synthon principal pour la synthèse de polyuréthanes (PU). Cette stratégie a été poursuivi avec l’addition d’acide oléique sur la lignine. Une réaction d’estérification a été mise au point par un procédé non solvanté en l’absence de catalyseur. Cet intermédiaire a été fonctionnalisé en vue d’obtenir un macropolyol réactif pour la synthèse de PU. Les propriétés des polymères ont été investiguées en modulant certains paramètres réactionnels. Les performances des matériaux obtenus ont montré l’intérêt de cette stratégie globale pour les applications finales visées. Enfin, dans une troisième et dernière approche, la réaction d’oxypropylation a été étudiée. Des chaines de polypropylène glycol ont été greffées sur la lignine aboutissant à l’obtention de quatre macropolyols différents avec des architectures macromoléculaires variées. Des PU ont été synthétisés à partir de ces macropolyols et les paramètres réactionnels ajustés en vue de moduler les propriétés finales des matériaux. Tous les PU réalisés ont démontré une séparation de phase attestant d’une organisation spécifique en segments souples/rigides et conférant des propriétés élastomères aux matériaux finaux. Cette étude a permis d’intégrer jusqu’à 89 % de matériaux biosourcés et renouvelables dans les polymères finaux et a montré le fort potentiel de la lignine pour l’élaboration de matériaux performants. / New macromolecular architectures have been performed by using lignin to develop new advanced materials for building applications. Three synthetic pathways have been carried out in order to impart specific properties to lignin-based materials. A Ring-Opening Polymerization has first been performed with ε-caprolactone to graft polymer chains onto lignin. Branching efficiency was confirmed by analysis and allows giving advanced properties to lignin materials. This first modification highlights the importance of lignin functionalization as building block for polymer synthesis. Then, we pursue the investigations by esterifying lignin with oleic acid by a solvent and catalyst-free method. Doubles bond were functionalized to obtain a lignin/fatty acid-based macropolyol that was then used for polyurethane synthesis (PU). Mechanical and thermal properties have been modulated by varying NCO:OH molar ratio and prepolymer molecular weight. Finally, oxypropylation was performed onto lignin by using various reaction parameters to obtain different macromolecular architectures for the resulting polyols. PUs have then been synthesized with the resulting macropolyols. The structure of macropolyols impacts greatly the thermal and mechanical properties of polymers, the influence of NCO:OH molar ratio was also studied.All of the PU reported in this work presented a micro-phase separation resulting in a specific organization (rigid/soft segments) into the polymer matrix. The lignin-based material revealed advanced thermal and mechanical properties with a high bio-based content up to 89%. This work allows emphasizing the potential of lignin as a starting material in the synthesis of sustainable material for building applications.

No description available.

Lignocellulosic biomass is a promising alternative to non-renewable fossil fuel resources for the sustainable production of fuels and chemicals. Levulinic acid (LA) is considered to be a versatile platform chemical which can be derived from biomass and further upgraded to high-value products like levulinate esters. In this work, the direct chemical conversion of lignocellulosic biomass to ethyl levulinate (EL) through LA was studied in collaboration with Gascon Biomass Research Inc. in an effort to valorize their surplus of lignocellulosic feedstock obtained from biomass recycling activities. A novel one-pot, biphasic, acid-catalyzed hydrolysis-esterification process developed by Prof. Tom Baker's lab (uOttawa Chemistry) was used. Kinetic analyses of the overall reaction pathway and major reaction steps were performed to investigate the effect of reaction conditions such as time, temperature, alcohol concentration, and catalyst concentration and evaluate the kinetic and thermodynamic parameters. Reactions were well modeled by power law rate equations with applicability of the Arrhenius expression and exhibited nearly first-order dependence with respect to each of the corresponding reactants and catalyst, as expected. Kinetic studies of LA esterification to various levulinate esters showed that the reaction is endothermic, endergonic, non-spontaneous, and non-rate-limiting. Increased alcohol alkyl chain length and branching reduced the thermodynamic favourability of LA esterification, likely due to steric effects. Process considerations such as reactor pressure, LA and EL partitioning, solvent:feedstock ratio, solvent recycling, and alternative feedstocks were investigated through various lab-scale studies and simulation case studies with UniSim® Design. The results of experimental studies informed the development of a preliminary potential process flow diagram consisting of eleven batch slurry reactors under agitation in parallel. The product streams feed into a continuous separation process composed of flash vessels and distillation columns to separate and purify the various products, intermediates, unconsumed reactants, and recoverable catalyst and solvent. This work ultimately informs decisions regarding future pilot-scale experiments, process design, and optimization for the envisioned large-scale biorefinery to produce EL.

[ES] En la presente tesis doctoral el principal objetivo ha sido desarrollar una nueva metodología para producir monoésteres, diésteres y polioles derivados de ácidos grasos de forma continua. Una producción en continuo incrementa la producción y la productividad por tiempo de catalizador. Además, esta metodología es mucho más sostenible para el medioambiente, ya que hace decrecer el factor E (Factor E = kg totales de sustancias involucradas en el proceso/Kg de producto) y se reducen también los residuos generados, si los comparamos a un proceso discontinuo. Así mismo, el proceso en continuo se desarrollará mediante catálisis heterogénea y mediante sólidos poliméricos de nueva síntesis. Cabe destacar que actualmente no hay en la literatura ninguna referencia de producción en continuo para la esterificación de ácidos grasos lineales de cadena larga con alcoholes. En primer lugar, se realizó un "screening" de diferentes catalizadores a nivel de laboratorio para la reacción de esterificación tanto en "batch" como en un reactor de lecho fijo. De entre los catalizadores probados se vio como catalizadores como el PAFR eran altamente activos a la conversión de ácidos, pero su limitada disponibilidad a nivel comercial limitó su posterior uso. Otra tipología de catalizadores probados fueron los carbones activos funcionalizados con azufre, entre ellos el catalizador que mejor funcionó fue el Norit RBHG. Sin embargo, debido al lixiviado durante la reacción, nos impide utilizarlos a nivel de planta piloto. Por tanto, el catalizador más adecuado para ser probado en planta piloto es la resina Amberlyst-16, ya que ha presentado el mejor rendimiento y es estable a la lixiviación. Con los resultados obtenidos en el laboratorio se diseñó y se fabricó, juntamente con una empresa de ingeniería, una planta piloto para la fabricación de ésteres en un reactor de lecho fijo. En ella se realizaron estudios de viabilidad técnica y económica para la reacción de esterificación de ácido láurico. Los resultados obtenidos indicaron que es posible fabricar 2-EHL a nivel de planta piloto en conversiones cercanas al 98% utilizando un catalizador ácido como es la resina Amberlyst-16. Se llevo a cabo un estudio de viabilidad económica con el que se pudo comprobar cómo con la planta piloto no es rentable fabricar, pero si se decidiera invertir en una planta industrial en continuo para la fabricación de ésteres de ácidos grasos, el retorno de inversión sería prácticamente inmediato, ya que la eficiencia de fabricación es mucho mayor que la que se tiene en una planta de esterificación en batch. Por último, se realizaron pruebas de uso del 2-EHL fabricado en la planta piloto para determinar si era posible utilizar en 2-EHL con la conversión del 98% en formulaciones de tintas digitales cerámicas en base solvente, los resultados obtenidos fueron excelentes, ya que no había variación en las propiedades de la tinta al sustituir el 2-EHL comercial por el 2-EHL fabricado en la planta piloto, dando por concluido el estudio. / [CAT] En aquesta tesi doctoral el principal objectiu ha estat desenvolupar una nova metodologia per produir monoèsters, dièsters i poliols derivats d'àcids grassos de forma contínua. Una producció en continu incrementa la producció i la productivitat per temps de catalitzador. A més, aquesta metodologia és molt més sostenible per al medi ambient, ja que fa decréixer el factor E (Factor E = kg totals de substàncies involucrades en el procés/Kg de producte) i es redueixen també els residus generats, si els comparem a un procés en discontinu. Així mateix, el procés en continu s'ha desenvolupat mitjançant catàlisi heterogènia i mitjançant sòlids polimèrics de nova síntesi. Cal destacar que actualment no hi ha a la literatura cap referència de producció en continu per a l'esterificació d'àcids grassos lineals de cadena llarga amb alcohols. En primer lloc, es va realitzar un "screening" de diferents catalitzadors a nivell de laboratori per a la reacció d'esterificació tant a "batch" com a un reactor de llit fix. Entre els catalitzadors provats es va veure com catalitzadors com el PAFR eren altament actius a la conversió d'àcids, però la seva limitada disponibilitat a nivell comercial va limitar el seu posterior ús. Una altra tipologia de catalitzadors provats van ser els carbons actius funcionalitzats amb sofre, entre ells el catalitzador que millor va funcionar va ser el Norit RBHG. Tot i això, a causa del lixiviat durant la reacción, esta ens impedeix utilitzar-los a nivell de planta pilot. Per tant, el catalitzador més adequat per ser provat a planta pilot és la resina Amberlyst-16, ja que ha presentat el millor rendiment i és estable a la lixiviació. Amb els resultats obtinguts al laboratori es va dissenyar i fabricar, juntament amb una empresa d'enginyeria, una planta pilot per a la fabricació d'èsters en un reactor de llit fix. S'hi van fer estudis de viabilitat tècnica i econòmica per a la reacció d'esterificació d'àcid làuric. Els resultats obtinguts van indicar que és possible fabricar 2-EHL a nivell de planta pilot en conversions properes al 98% utilitzant un catalitzador àcid com és la resina Amberlyst-16. Es va dur a terme un estudi de viabilitat econòmica amb què es va poder comprovar com amb la planta pilot no és rendible fabricar, però si es decidís invertir en una planta industrial en continu per a la fabricació d'èsters d'àcids grassos, el retorn d'inversió seria pràcticament immediat ja que l'eficiència de fabricació és molt més gran que la que es té en una planta d'esterificació en batch. Finalment, es van realitzar proves d'ús del 2-EHL fabricat a la planta pilot per determinar si era possible utilitzar a 2-EHL amb la conversió del 98% en formulacions de tintes digitals ceràmiques en base solvent, els resultats obtinguts van ser excel·lents ja que no hi havia variació en les propietats de la tinta en substituir el 2-EHL comercial pel 2-EHL fabricat a la planta pilot, donant per conclòs l'estudi. / [EN] In the present doctoral thesis, the main objective has been to develop a new methodology to produce mono-esters, di-esters and polyols derived from fatty acids, continuously. Continuous production increases production and productivity per catalyst time. In addition, this methodology is much more sustainable for the environment, since it decreases the factor E (Factor E = total kg of substances involved in the process/Kg of product) and the waste generated is also reduced, if we compare to a discontinuous process. Likewise, the continuous process will be developed through heterogeneous catalysis and through newly synthesized polymeric solids. It should be noted that there is not any current continuous production reference in the literature for the esterification of long-chain linear fatty acids with alcohols. In the first place, a screening of different catalysts was carried out at the laboratory level for the esterification reaction both in batch and in a fixed-bed reactor. Among the catalysts tested, catalysts such as PAFR were found to be highly active in acid conversion, but their limited commercial availability prevented their further use. Another type of catalyst tested was sulfur-functionalized active carbon, and the catalyst that worked best was Norit RBHG. However, due to leaching during the reaction, it prevents us from using them at the pilot plant level. Therefore, the most suitable catalyst to be tested in a pilot plant was the Amberlyst-16 resin, since it presented the best performance and is stable to leaching. With the results obtained in the laboratory, a pilot plant for the manufacture of esters in a fixed-bed reactor was designed and manufactured, together with an engineering company. Technical and economic feasibility studies were carried out for the lauric acid esterification reaction. The results obtained indicated that it is possible to manufacture 2-EHL at a pilot plant level with conversions close to 98%, using an acid catalyst such as Amberlyst-16 resin. An economic feasibility study was carried out which verifies that the pilot plant it is not profitable to manufacture, but if it were decided to invest in a continuous industrial plant for the manufacture of fatty acid esters, the return on investment would be practically immediately, since the manufacturing efficiency is much higher than that of a batch esterification plant. Finally, use tests of the 2-EHL manufactured in the pilot plant were carried out to determine if it was possible to use 2-EHL with a conversion of 98% in solvent-based ceramic digital ink formulations. The results obtained were excellent since there was not any variation in the properties of the ink when substituting the commercial 2-EHL, thus ending the study so far. / Martínez Castelló, A. (2022). Esterificación de ácidos grasos en lecho fijo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191533

Batch distillation with chemical reaction when takes place in the same unit is referred to as batch reactive distillation process. The combination reduces the capital and operating costs considerably. Among many different types of batch reactive distillation column configurations, (a) conventional (b) inverted (c) semi-batch columns are considered here. Three reaction schemes such as (a) esterification of methanol (b) esterification of ethanol (c) hydrolysis of methyl lactate are studied here. Four different types of dynamic optimisation problems such as (a) maximum conversion (b) maximum productivity (c) maximum profit and (d) minimum time are formulated in this work. Optimal design and or operation policies are obtained for all the reaction schemes. A detailed rigorous dynamic model consisting of mass, energy balances, chemical reaction and thermodynamic properties is considered for the process. The model was incorporated within the dynamic optimisation problems. Control Vector Parameterisation (CVP) technique was used to convert the dynamic optimisation problem into a nonlinear programming problem which was solved using efficient SQP (Successive Quadratic Programming) method available within the gPROMS (general PROcess Modelling System) software. It is observed that multi-reflux ratio or linear reflux operation always led to better performance in terms of conversion, productivity for all reaction schemes compared to that obtained using single reflux operation. Feed dilution (in the case of ethanol esterification) led to more profit even though productivity was found to be lower. This was due to reduction in feed price because of feed dilution. Semi-batch reactive distillation opertation (for ethanol esterification) led to better conversion compared to conventional batch distillation, however, the total amount of acetic acid (reactant) was greater in semi-batch operation. Optimisation of design and operation (for ethanol esterification) clearly showed that a single cloumn will not lead to profitable operation for all possible product demand profile. Also change in feed and /or product price may lead to adjust the production target to maximise the profitability. In batch distillation, total reflux operation is recommended or observed at the begining of the operation (as is the case for methnaol or ethanol esterification). However, in the case of hydrolysis, total reflux operation was obseved at the end of the operation. This was due to lactic acid (being the heaviest) was withrawn as the final bottom product.

This study focuses on the development of novel two-stage esterification-transesterification synthesis of biodiesel from used cooking oil (UCO) using novel heterogeneous catalysts. The esterification of the UCO was investigated using three types of ion exchange resins catalysts including Purolite D5081, Purolite D5082 and Amberlyst 15. Of all the catalysts investigated, Purolite D5081 resin showed the best catalytic performance and was selected for further optimisation studies. From the optimisation study, it was found that the external and internal mass transfer resistance has negligible effect on the esterification reaction. At the optimum reaction conditions, Purolite D5081 achieved 92% conversion of FFA. During reusability study, the conversion of FFA dropped by 10% after each cycle and it was found that progressive pore blockage and sulphur leaching were dominant factors that decreased the catalytic performance of the Purolite D5081 catalyst. A kinetic modelling for FFA esterification was carried out using Purolite D5081 as a catalyst. Three types of kinetic models were investigated i.e. pseudo homogeneous (PH), Eley-Rideal (ER) and Langmuir-Hinshelwood-Hougen-Watson (LHHW). Experimental data obtained from the batch kinetic studies was successfully represented by the PH model and a good agreement between experimental and calculated values was obtained. The activation energy for esterification and hydrolysis reaction was found to be 53 and 107 kJ/moL. The transesterification of pre-treated cooking oil (P-UCO) was investigated using various types of heterogeneous catalysts including Purolite CT-122, Purolite CT-169, Purolite CT-175, Purolite CT-275, Purolite D5081, Diaion PA306s and Cs-supported heteropolyacids catalysts. Of all the catalysts investigated, Diaion PA306s catalyst showed the highest conversion of triglycerides and was selected for further optimisation studies. At the optimum reaction conditions, Diaion PA306s achieved ca. 75% of triglycerides conversion. During the reusability study, Diaion PA306s catalyst gave a similar conversion of triglycerides after being reused once. Therefore, it was concluded that the resin can be used several times without losing catalytic activity. Several purification methods have been investigated and dry washing method was chosen as the best alternative for biodiesel purification.

L’induction de la synthèse d’une acide férulique estérase (AFE) a été étudiée chez Streptomyces ambofaciens ATCC 23877. L’activité la plus élevée a été détectée en présence de son de blé désamidonné ou de xylane d’avoine (0,22, 0,21 mU/mg protéine, respectivement). Des productions d’AFE en bioréacteur ont également été réalisées en utilisant 1% (p/v) de son de blé comme inducteur. Le niveau de production de l’AFE a été trois fois plus important en bioréacteur qu’en fiole d’Erlenmeyer. L’AFE de Streptomyces ambofaciens ATCC 23877 et celle de Humicola sp., présente dans un mélange enzymatique commercial (DepolTM 740L), ont été partiellement purifiées et caractérisées. A l’issue de la purification, l’activité AFE de Streptomyces ambofaciens ATCC 23877 a été trop faible pour pouvoir être utilisée ultérieurement en synthèse. Par contre, le potentiel de l’AFE de Humicola sp., concentrée par précipitation à l’acétone, pour la synthèse de différents esters phénoliques a été testé. Les meilleurs rendements de conversion ont été observés lors de l’absence de substitutions sur le cycle aromatique de l’acide phénolique ou en présence de groupements hydroxyles. Les synthèses en milieu non aqueux (M2B2) se sont montrées infructueuses en raison, peut-être, d’un effet néfaste du solvant sur l’enzyme / The induction of the ferulic acid esterase (FAE) synthesis was studied with Streptomyces ambofaciens ATCC 23877. The highest activity was detected in the presence of either destarched wheat bran or oat spelt xylan (0,22, 0,21 mU/mg protein, respectively). FAE productions in bioreactor were also carried out using 1% (w/v) of wheat bran as inducer. The FAE production level was three times higher in bioreactor than in Erlenmeyer flask. FAE of Streptomyces ambofaciens ATCC 23877 and that of Humicola sp., present in an enzymatic commercial mixture (DepolTM 740L), were partially purified and characterised. At the end of the purification, FAE activity of Streptomyces ambofaciens ATCC 23877 was too weak to be used later in synthesis. However, the FAE potential of Humicola sp., concentrated by acetone precipitation, for the synthesis of various phenolic esters was tested. The best conversion yields were observed in the absence of substitution on the phenolic acid aromatic cycle or in the presence of hydroxyl groups. The synthesis in non-aqueous medium (M2B2) were unsuccessful maybe because of an harmful effect of the solvent on the enzyme

Manguezais são ecossistemas localizados na confluência de terra e mar, característicos de áreas tropicais e subtropicais, cobrindo cerca de 18,1 milhões de hectares do planeta. A grande biodiversidade encontrada nestes ambientes ressalta a importância da busca por conhecimentos à seu respeito, como o estudo sobre novos princípios ativos derivados de microrganismos endofíticos presentes nas plantas de manguezais. Desta forma, o propósito do presente trabalho foi determinar a diversidade genética da comunidade de fungos endofíticos presentes em folhas e ramos das principais espécies arbóreas de manguezais de Cananéia e Bertioga (situados no estado de São Paulo, Brasil), e avaliar o potencial biotecnológico destes fungos em relação à produção de antibióticos contra os patógenos humanos Staphylococcus aureus e Escherichia coli, e contra o fitopatógeno Xanthomonas axonopodis citri . Os resultados da primeira etapa do trabalho, que envolveu o isolamento e a caracterização de fungos endofíticos filamentosos, mostraram que a comunidade fúngica associada às plantas de manguezais é formada por pelo menos 35 gêneros diferentes, sendo que os gêneros mais frequentes foram Diaporthe, Fusarium, Trichoderma, Colletotrichum e Xylaria. Grande parte dos gêneros encontrados neste trabalho é de fungos de solo, indicando que eles estão adaptados às condições adversas dos manguezais. Os resultados mostraram que, dentre as linhagens produtoras de antibiótico, 29,41% pertencem ao gênero Diaporthe, o qual apresentou maior frequência na comunidade fúngica estudada. Após a avaliação de 344 fungos quanto ao potencial de atividade antimicrobiana, foi selecionada a linhagem 41.1(1) de D. phaseolorum, um endófito de folha de Laguncularia racemosa, para elucidação da estrutura química do seu antibiótico purificado. Por meio das técnicas de Ressonância Magnética Nuclear e de Espectrometria de Massas o antibiótico foi identificado como ácido 3-hidroxipropiônico o qual apresentou atividade frente aos patógenos humanos Staphylococcus aureus e Salmonella tiphy. A estrutura química deste antibiótico foi modificada por meio de reação química de esterificação de Fischer-Speier para avaliar a relação da estrutura química e atividade biológica deste composto. O produto final da reação química de esterificação do antibiótico ácido 3-hidroxipropiônico não apresentou atividade antimicrobiana, indicando que o grupo hidroxila removido na reação é importante na atividade farmacológica desse composto. Além disso, a linhagem 41.1(1) de D. phaseolorum foi transformada geneticamente pelo sistema Agrobacterium tumefaciens, visando a obtenção de transformantes deficientes para produção de antibiótico e, com isso, a identificação de genes relacionados com a via de biossíntese do antibiótico ácido 3-hidroxipropiônico. A análise das sequências que flanqueiam o T-DNA, obtidas por TAIL-PCR, mostraram que os genes interrompidos nos transformantes estão relacionados com proteínas de domínios conservados envolvidos com diferentes funções como: translação de proteínas, homeostase do íon orgânico Mg2+, transporte intracelular, migração, adesão e proliferação celular e outras funções celulares. A caracterização da biblioteca de agrotransformantes constitui uma ferramenta importante para o estudo da biologia molecular de fungos que produzem compostos bioativos por meio do seu metabolismo secundário. / Mangroves are ecosystems situated beyond land and sea. They are more frequently found in tropical and subtropical areas englobing around 18.1 millions of hectares in the planet. The great biodiversity found in these ecosystems shows the importance of researching them, including studies regarding new compounds derived from endophytic fungi that inhabit these ecosystems. Therefore, the goal of this study was to determine the genetic diversity of the fungal endophytic community found in leaves and branches of the main arboreal species from mangrove of Cananéia and Bertioga (situated in São Paulo state, Brazil), and to evaluate the biotechnological potential of these fungi concerning the production of antibiotics against the human pathogens Staphylococcus aureus and Escherichia coli, and against the phytopathogen Xanthomonas axonopodis citri . The results of the first part of this work, including the isolation and characterization of the filamentous endophytic fungi, showed that the mangrove fungal community is made up of at least 35 different genera, from which the most frequent are Diaporthe, Fusarium, Trichoderma, Colletotrichum and Xylaria. Most of the fungal genera found in this study come from soil, which suggests that they are adapted to the adverse conditions of mangroves. The results show that among the antibiotic-produncing strains, 29.41% belong to the genus Diaporthe, which was the most frequently found in the studied fungal community. After the analysis of 344 fungi regarding the antibiotic activity potential, a strain of D. phaseolorum (a leaf endophyte of Laguncularia racemosa) was selected to unveil the chemical structure of their purified antibiotic. The nuclear magnetic resonance and the mass spectrometry techniques allowed the identification of the antibiotic as 3-hidroxypropionic acid, which displayed activity against the pathogens Staphylococcus aureus and Salmonella tiphy. The chemical structure of this antibiotic was modifyed by the chemical reaction of Fischer-Speier sterification in order to evaluate the chemical structure and biological activity of this compound. The final product of the chemical reaction of 3-hidroxipropionic acid sterification had no antibiotic activity, which suggests that the hydroxil group removed from the reaction is important to the pharmachological activity of this compound. Additionally, the strain 41.1(1) of D. phaseolorum was genetically transformed by the Agrobacterium tumefaciens system, in order to generate antibioticdeficient transformants, which would help to identify genes related to the biosynthesis pathway of the 3- hidroxypropionic acid antibiotic. The TAIL-PCR analysis revealed that the interrupted genes in the tranformants are related to proteins from conserved domains involved in different functions such as protein translation, Mg2+ ion homeostasis, intracellular transport, migration, adhesion and cellular proliferation and other cellular functions. The characterization of the agrotransformants library is an important tool to unveiling the molecular biology of fungi that produce bioactive compounds by the secondary metabolism.