Chromium has been used extensively in the industry process of metal refinishing and electroplating. It is also a byproduct of the processing of fissionable materials at United States Department of Energy facilities. Chromate (Cr (VI)) is soluble and readily absorbed by cells, while the reduced form of chromium, Cr (III), is insoluble. Thus means of reducing Cr (VI) to Cr (III) in the environment is a potential means of remediation. Desulfovibrio desulfuricans strain 27774 is a sulfate reducing bacterium that can reduce Cr (VI). It also can respire nitrate to ammonia. As some sites of chromium contamination also contain high concentrations of nitrate, an investigation of Cr (VI) reduction under nitrate reducing growth conditions by D. desulfuricans strain 27774 was conducted. A growth medium that was compatible with the colorimetric assay for Cr (VI) and did not itself reduce Cr (VI) was formulated. Cell assays determined that Cr (VI) reduction was primarily in the supernatant, catalyzed by a secreted secondary metabolite. A genomics investigation identified two pathways as possible mechanisms. / Bayer School of Natural and Environmental Sciences / Environmental Science and Management (ESM);= / MS / Thesis

Les procédés actuellement utilisés à bord de la Station Spatiale Internationale permettent un traitement physico-chimique des déchets avec un recyclage partiel des éléments en minimisant le réapprovisionnement en eau et oxygène. Voyager plus loin et sur de plus longues durées n’est possible que si un système de support de vie, permettant d’assurer une autonomie plus importante, est mis en place. Le projet MELiSSA est un système de support de vie biorégénératif ayant pour objectifs de recycler les déchets produits par l’homme, de régénérer l’oxygène et l’eau et d’assurer une production de nourriture. L’utilisation de microorganismes pour assurer ces différentes fonctions, n’est réalisable qu’en ayant la meilleure connaissance possible et surtout la maîtrise des processus et des souches impliqués. L’objectif de cette thèse est de développer un modèle prédictif pour le compartiment nitrificateur de la boucle MELiSSA. Dans un premier temps, les souches Nitrosomonas europaea ATCC® 19718 et Nitrobacter winogradskyi ATCC® 25391 ont été étudiées et ont permis de définir un modèle cinétique de ces deux bactéries en culture immergée. Dans un second temps, ce modèle a été combiné à un modèle N-bacs en série pour composer un modèle de nitrification dans une colonne à lit fixe à biomasse fixée : le modèle NitriSim. Ce modèle a été testé sur une expérience de long-terme menée dans une colonne à lit fixe dont le support est composé de billes Biostyr®. Le dernier chapitre de ce manuscrit décrit l’utilisation d’une souche dégradant l’urée, Cupriavidus pinatubonensis, dans le réacteur à lit fixe. Les différents résultats ont permis de définir un modèle de nitrification dans un réacteur à lit fixe et de présenter des perspectives possibles pour le traitement de l’urine au niveau du compartiment nitrifiant de MELiSSA. / Physico-chemical processes are used on the International Space Station for partial recycling of wastes in order to minimize the resupplies of oxygen and water. Further and longer explorations can only be possible if a life support system is applied to enhance autonomy to the station. MELiSSA is a biological life support system project aiming for waste recycling, water and air regeneration and production of simple food. The only way to assure these functions is the use of microorganisms which is requiring a high knowledge and a perfect control of processes implicated. The thesis objectives consist in developing a predictive model for the MELiSSA nitrifying compartment. First, a kinetic study of Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391 in submerged bioreactors allowed defining a kinetic model of nitrification. Secondly, this model was combined to an N-tanks in series one to compose a nitrification model in fixed-bed biorectors : NitriSim. It was applied on a long-term experiment in a fixed-bed bioreactors where the bed was composed of Biostyr® beads. The last part of this work was about the use of a urea-degradating bacteria, Cupriavidus pinatubonensis, in the fixed-bed bioreactor. All of these results leaded to the development of a nitrification in fixed-bed reactors model and opened up interesting prospects for the urine degradation in the nitrifying compartment of MELiSSA.

Published Article / The development of tolerance to low pH levels and the existence of cross-resistance may promote survival of bacteria in acidic foodstuff and in acidic environments such as the human stomach, in so doing escalating the probability of food poisoning. Similar to antimicrobial resistance developing, there is growing concern that effectiveness of organic acids may decrease as a result of the emergence of acid-tolerant food-borne pathogens. The objectives of this study were to determine the development of acid tolerance in selected food-borne pathogenic bacteria and to explore the activity of organic acids against acid tolerant pathogens. Bacterial strains were screened for acid-tolerance and susceptible strains were induced through exposure to increasing concentrations of an inorganic acid, as well as acidic foodstuffs. Susceptibility to six organic acids at various pH levels was assessed in order to evaluate the possible relationship between altered antimicrobial activity and acid tolerance. Salmonella enterica sv. Enteritidis ATCC 13076 and Escherichia coli ATCC 25922 were found to rapidly develop acid tolerance, while intrinsic acid tolerance was noted in Salmonella enterica sv. Typhimurium ATCC 14028. Pseudomonas aeruginosa ATCC 27853 demonstrated intermediate intrinsic acid tolerance. As expected, pH played a significant role in inhibitory activity of the organic acids as these compounds exhibit optimum antimicrobial activity at a lower pH (pH ≤5). It is, however, necessary to further elucidate the two-way role of pH in foodstuff concomitant to the addition of an organic acid.

O objetivo deste estudo foi avaliar por métodos microbiológicos e microscópia eletrônica de varredura a aderência bacteriana sobre superfície lisa e rugosa (modificada por irradiação a laser) de titânio comercialmente puro (\'Ti\' cp). Os corpos-de-prova eram em forma de discos (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 e Staphylococcus epidermidis ATCC 12228 RP 62A foram às bactérias selecionadas. A topografia das superfícies foram avaliadas por meio de microscópio eletrônico de varredura (MEV). As medidas do ângulo de contato permitiram conhecer a molhabilidade (hidrofobicidade) das superfícies. Os discos de \'Ti\' cp foram incubados em meio de cultura caldo Mueller Hinton inoculado com suspensão bacteriana da ordem de \'10 POT.8\' (UFC)/mL, durante 1, 6, 24, 48 e 72 horas. A cada intervalo de tempo, os discos foram retirados, lavados em solução salina fisiológica e após este procedimento foram colocados em novos tubos contendo 5,0 mL de solução salina fisiológica esterilizada e submetidos ao banho ultrassônico de 40 kHz por oito minutos. A seguir, da suspensão bacteriana resultante foram realizadas diluições seriadas (\'10 POT.-1\'-\'10 POT.-6\'), semeadas em ágar Mueller Hinton e as placas incubadas em estufa bacteriológica a 37 graus Celsius para aguardar o desenvolvimento bacteriano. As colônias crescidas foram contadas, enumeradas e o valor expresso em UFC/mL para cada diluição. Os discos removidos do banho ultrassônico foram preparados para observação por MEV. A medida do ângulo de contato foi realizada em equipamento denominado goniômetro. O resultado da avaliação da viabilidade das células de biofilme de S. mutans sobre superfície lisa em média foi de 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' e sobre superfície rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' a nível 0,05 as médias não são significantemente diferentes. Em média as células viáveis do biofilme de S. epidermidis sobre superfície lisa foram de 6,68 \'+ OU -\' ) 5,83 x \'10 POT.6\' a nível 0,05 as médias não são significativamente diferentes. As células viáveis do biofilme de S. epidermidis, recuperadas em vários intervalos de tempo (1, 6, 24, 48 e 72h) da superfície rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' a nível 0,05 são significantemente diferentes. Em relação à molhabilidade a superfície lisa é hidrofóbica, com um ângulo de 75 graus e a superfície rugosa hidrofílica, com um ângulo < 7 graus. Apesar da superfície lisa ser hidrofóbica e a superfície rugosa hidrofílica ambas as superfícies permitiram a aderência bacteriana e formação de biofilme, fato comprovado por MEV e por cultura. Comparando-se as superfícies lisa e rugosa - modificada por meio físico (aplicação de feixe de laser de alta intensidade \'Nd\':YAG) não foi observada redução significante no número de bactérias aderidas à superfície rugosa, o que permite concluir que a modificação de superfície por laser de alta intensidade cria superfície favorável para aderência de S. mutans e S. epidermidis, sem reduzir a aderência bacteriana, o que pode ser fator de risco para adquirir infecção. / The objective of this study was assessed by microbiological methods and the scanning electron microscope on bacterial adhesion to smooth and rough (modified by laser irradiation) of commercially pure titanium (\'Ti\' cp). The bodies-of-proof was in the form of discs (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 and Staphylococcus epidermidis ATCC 12228 RP62A were selected bacteria. The topography of the areas were evaluated by scanning electron microscope (SEM). Measures the angle of contact allowed to know the wettability (hydrophobicity) of the areas. Of \'Ti\' cp discs were incubated in culture media Mueller Hinton broth inoculated with bacterial suspension of approximately \'10 POT.8\' (CFU)/mL, for 1, 6, 24, 48 and 72 hours. Each time, the discs were removed, washed in saline solution and after this procedure were placed into new tubes containing 5.0 mL of sterile saline solution and submitted to the ultrasonic bath for eight minutes of 40 kHz. Then, the resulting bacterial suspension were serially diluted (\'10 POT.-1\'-\'10 POT.-6\'), grown in Mueller Hinton agar plates and incubated in bacteriological incubator at 37 Celsius degrees to await the development blight. The grown colonies were counted, listed and the value expressed in CFU/mL for each dilution. Discs removed from the ultrasonic bath were prepared for observation by SEM. The measure of the angle of contact was made in equipment called goniometer. The result of evaluating the viability biofilm cells of S. mutans on smooth surface was on average of 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' at the 0,05 and on area rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' at the 0,05 average is not significantly different. On average the cells of the biofilm of S. epidermidis on smooth 6,68 \'+ OU -\' 5,83 x \'10 POT.6\' at the 0,05 average is not significantly surface were different. The cells of the biofilm of S. epidermidis, recovered at various at intervals of time (1, 6, 24, 48 and 72h) of the area rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' 0,05 are significantly different. In relation to the smooth surface wettability is hydrophobic, with an angle of 75 degrees and the hydrophilic surface rough, with an angle < 7 degrees. Despite the smooth surface is rough hydrophobic and hydrophilic surface areas have both the adhesion and formation of bacterial biofilm, a fact evidenced by SEM and by culture. Comparing the smooth and rough surfaces - modified by the physical environment (application of the laser beam of high intensity \'Nd\': YAG) was not observed a significant reduction in the number of bacteria adhered to the surface rough, which indicates that the modification of surface by high-intensity laser creates favorable surface for adhesion of S. mutans and S. epidermidis, without reducing the bacterial adhesion, which may be a risk factor for acquiring infection.

O objetivo deste estudo foi avaliar por métodos microbiológicos e microscópia eletrônica de varredura a aderência bacteriana sobre superfície lisa e rugosa (modificada por irradiação a laser) de titânio comercialmente puro (\'Ti\' cp). Os corpos-de-prova eram em forma de discos (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 e Staphylococcus epidermidis ATCC 12228 RP 62A foram às bactérias selecionadas. A topografia das superfícies foram avaliadas por meio de microscópio eletrônico de varredura (MEV). As medidas do ângulo de contato permitiram conhecer a molhabilidade (hidrofobicidade) das superfícies. Os discos de \'Ti\' cp foram incubados em meio de cultura caldo Mueller Hinton inoculado com suspensão bacteriana da ordem de \'10 POT.8\' (UFC)/mL, durante 1, 6, 24, 48 e 72 horas. A cada intervalo de tempo, os discos foram retirados, lavados em solução salina fisiológica e após este procedimento foram colocados em novos tubos contendo 5,0 mL de solução salina fisiológica esterilizada e submetidos ao banho ultrassônico de 40 kHz por oito minutos. A seguir, da suspensão bacteriana resultante foram realizadas diluições seriadas (\'10 POT.-1\'-\'10 POT.-6\'), semeadas em ágar Mueller Hinton e as placas incubadas em estufa bacteriológica a 37 graus Celsius para aguardar o desenvolvimento bacteriano. As colônias crescidas foram contadas, enumeradas e o valor expresso em UFC/mL para cada diluição. Os discos removidos do banho ultrassônico foram preparados para observação por MEV. A medida do ângulo de contato foi realizada em equipamento denominado goniômetro. O resultado da avaliação da viabilidade das células de biofilme de S. mutans sobre superfície lisa em média foi de 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' e sobre superfície rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' a nível 0,05 as médias não são significantemente diferentes. Em média as células viáveis do biofilme de S. epidermidis sobre superfície lisa foram de 6,68 \'+ OU -\' ) 5,83 x \'10 POT.6\' a nível 0,05 as médias não são significativamente diferentes. As células viáveis do biofilme de S. epidermidis, recuperadas em vários intervalos de tempo (1, 6, 24, 48 e 72h) da superfície rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' a nível 0,05 são significantemente diferentes. Em relação à molhabilidade a superfície lisa é hidrofóbica, com um ângulo de 75 graus e a superfície rugosa hidrofílica, com um ângulo < 7 graus. Apesar da superfície lisa ser hidrofóbica e a superfície rugosa hidrofílica ambas as superfícies permitiram a aderência bacteriana e formação de biofilme, fato comprovado por MEV e por cultura. Comparando-se as superfícies lisa e rugosa - modificada por meio físico (aplicação de feixe de laser de alta intensidade \'Nd\':YAG) não foi observada redução significante no número de bactérias aderidas à superfície rugosa, o que permite concluir que a modificação de superfície por laser de alta intensidade cria superfície favorável para aderência de S. mutans e S. epidermidis, sem reduzir a aderência bacteriana, o que pode ser fator de risco para adquirir infecção. / The objective of this study was assessed by microbiological methods and the scanning electron microscope on bacterial adhesion to smooth and rough (modified by laser irradiation) of commercially pure titanium (\'Ti\' cp). The bodies-of-proof was in the form of discs (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 and Staphylococcus epidermidis ATCC 12228 RP62A were selected bacteria. The topography of the areas were evaluated by scanning electron microscope (SEM). Measures the angle of contact allowed to know the wettability (hydrophobicity) of the areas. Of \'Ti\' cp discs were incubated in culture media Mueller Hinton broth inoculated with bacterial suspension of approximately \'10 POT.8\' (CFU)/mL, for 1, 6, 24, 48 and 72 hours. Each time, the discs were removed, washed in saline solution and after this procedure were placed into new tubes containing 5.0 mL of sterile saline solution and submitted to the ultrasonic bath for eight minutes of 40 kHz. Then, the resulting bacterial suspension were serially diluted (\'10 POT.-1\'-\'10 POT.-6\'), grown in Mueller Hinton agar plates and incubated in bacteriological incubator at 37 Celsius degrees to await the development blight. The grown colonies were counted, listed and the value expressed in CFU/mL for each dilution. Discs removed from the ultrasonic bath were prepared for observation by SEM. The measure of the angle of contact was made in equipment called goniometer. The result of evaluating the viability biofilm cells of S. mutans on smooth surface was on average of 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' at the 0,05 and on area rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' at the 0,05 average is not significantly different. On average the cells of the biofilm of S. epidermidis on smooth 6,68 \'+ OU -\' 5,83 x \'10 POT.6\' at the 0,05 average is not significantly surface were different. The cells of the biofilm of S. epidermidis, recovered at various at intervals of time (1, 6, 24, 48 and 72h) of the area rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' 0,05 are significantly different. In relation to the smooth surface wettability is hydrophobic, with an angle of 75 degrees and the hydrophilic surface rough, with an angle < 7 degrees. Despite the smooth surface is rough hydrophobic and hydrophilic surface areas have both the adhesion and formation of bacterial biofilm, a fact evidenced by SEM and by culture. Comparing the smooth and rough surfaces - modified by the physical environment (application of the laser beam of high intensity \'Nd\': YAG) was not observed a significant reduction in the number of bacteria adhered to the surface rough, which indicates that the modification of surface by high-intensity laser creates favorable surface for adhesion of S. mutans and S. epidermidis, without reducing the bacterial adhesion, which may be a risk factor for acquiring infection.

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C 33H55N6O15) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C20H36N4O10, C31H 55N6O15, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C 33H55N6O15) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C20H36N4O10, C31H 55N6O15, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.

Siderophores are small, iron chelating molecules produced by many bacteria to help meet the iron requirements of the cell. Multiple metabolic functions require iron as it serves as a cofactor in many enzymes and cellular processes. However, in the presence of oxygen and at physiologic pH, iron forms insoluble ferric complexes that cause the nutrient to be unavailable to bacterial cells. Siderophores alleviate this limitation by chelating the ferric iron, rendering it soluble and available for uptake. One group of microorganisms known for their ability to produce siderophores is the rhizobia. These bacteria are characterized both by their formation of symbiotic relationships with leguminous plants and their ability to fix atmospheric nitrogen. Rhizobium leguminosarum ATCC 14479, which infects the red clover Trifolium pratense, was found to produce a trihydroxamate siderophore. Purification and chemical characterization identified this siderophore as Vicibactin that has been found to be produced by other rhizobial strains.

O H2 pode ser obtido por processos biológicos, como a fermentação, conduzidos à temperatura e pressão ambientes. Para tal são utilizadas matérias-primas renováveis, ricas em carboidratos, como as biomassas lignocelulósicas e de algas. Estas biomassas têm estrutura química complexa e requerem uma etapa de pré-tratamento e/ou hidrólise antes da sua utilização na fermentação. Processos de hidrólise podem liberar, tanto monossacarídeos, quanto substâncias potencialmente inibidoras de fermentação. Esse estudo avaliou o efeito de 3 potenciais inibidores de fermentação (5-hidroximetilfurfural -HMF, ácido levulínico AL e ácido fórmico AF), derivados da hidrólise de biomassas. Ensaios cinéticos de fermentação em batelada foram realizados com o microrganismo produtor de H2, Clostridium beijerinckii Br21, utilizando glicose como fonte de carbono e diferentes concentrações de cada inibidor. O efeito do HMF, do AL e do AF foram avaliados nas faixas de concentração de 0,5 a 2,5 g/L, de 1,0 a 4,0 g/L, e de 0,5 a 2,0 g/L, respectivamente. Retiraram-se amostras do gás produzido e do líquido para estimar as velocidades específicas de produção de H2, do crescimento celular e de consumo de glicose, nos ensaios com e sem a presença de inibidor (controle). Foi observada inibição de todos os parâmetros avaliados, comparados ao controle. Houve um aumento do tempo para início da produção e diminuição do rendimento de H2 com o aumento da concentração de todos os inibidores. Os resultados das fermentações permitiram estimar a concentração dos compostos que inibem 50% a produção de H2, o crescimento celular e o consumo do substrato (CI50). Os valores de CI50 obtidos para a produção de H2 pelo HMF, AL e AF foram 0,89, 2,50 e 1,15 g/L, respectivamente. Para o crescimento celular a CI50 do HMF, AL e AF foram 1,42, 2,08 e 1,46 g/L, respectivamente. Para o consumo de substrato a CI50 foi 3,23, 3,79 e 0,43 g/L, para o HMF, AL e AF, respectivamente. As concentrações de CI50 para a produção de H2 foram testados em 2 microrganismos distintos, o C. beijerinckii Br21 e o Clostridium acetobutylicum ATCC 824, para fins comparativos. Assim pode-se verificar a inibição na produção de H2 no C. beijerinckii Br21 de 49,3, 48,7 e 51,3%, enquanto que o C. acetobutylicum ATCC 824 apresentou inibição de 45,5, 61,3 e 59,6%, para o HMF, AL e AF, respectivamente. Foi estimada também a concentração de compostos que inibem 25% a produção de H2, a CI25, a fim de realizar misturas com os inibidores e testá-las em ambos os microrganismos. Os valores obtidos de CI25 para HMF, AL e AF foram 0,66, 2,15 e 0,89 g/L, respectivamente. A partir desses valores foram feitas 4 misturas distintas: HMF+AL, HMF+AF, AL+AF e HMF+AL+AF. A inibição da produção de H2 a partir dessas misturas em C. beijerinckii Br21foram de 58,9, 58,4, 49 e 85,9%, enquanto que para o C. acetobutylicum ATCC 824 obteve-se os valores de 67,6, 66,6, 55,5 e 88,8%, para HMF+AL, HMF+AF, AL+AF e HMF+AL+AF, respectivamente. Portanto, pode-se notar que o C. acetobutylicum ATCC 824 mostrou ser mais sensível aos efeitos causados pelos inibidores, sendo que o HMF parece atuar mais sobre a produção de H2, enquanto que os ácidos têm efeito mais global no metabolismo da bactéria. Esses estudos mostraram os limites destes compostos, quando se deseja utilizar hidrolisados de biomassas como matéria-prima para a produção fermentativa do H2.pelas espécies de Clostridium estudadas. / H2 can be obtained by biological processes, such as fermentation, conducted at ambient temperature and pressure. Renewable raw materials like lignocellulosic and algae biomass, which are rich in carbohydrates, can be used for this purpose. These types of biomass have complex chemical structures and require a pretreatment and/or hydrolysis step before they are used in fermentation. Hydrolysis may release not only monosaccharides but also potentially fermentation-inhibiting substances. This study evaluates how three potential fermentation inhibitors (5-hydroxymethylfurfural (HMF), levulinic acid-(LA), and formic acid (FA) derived from algal biomass hydrolysis affect H2 production. Kinetic batch fermentation assays were performed by using the H2-producing microorganism Clostridium beijerinckii Br21, glucose as carbon source, and different concentrations of each inhibitor. The effect of HMF, LA, and FA on H2 production was evaluated for inhibitor concentrations ranging from 0.5 to 2.5 g/L, 1.0 to 4.0 g/L, and 0.5 to 2.0 g/L, respectively. Samples of the produced gas and liquid were taken to estimate the specific rates of H2 production, cell growth, and glucose consumption in the assays conducted in the presence or in the absence (control) of an inhibitor. Increasing inhibitor concentration delayed the onset of H2 production and diminished the H2 yield. The fermentation results allowed us to estimate the inhibitor concentration that inhibited 50% of the H2 production, cell growth, and substrate consumption rates, designated IC50. Concerning the H2 production rate, IC50 was 0.89, 2.50, and 1.15 g/L for HMF, LA, and FA, respectively. As for the cell growth rate, IC50 was 1.42, 2.08, and 1.46 g/L for HMF, LA, and FA, respectively. Regarding the substrate consumption rate, IC50 was 3.23, 3.79, and 0.43 g/L for HMF, LA, and FA, respectively. IC50 was also tested in the presence of C. beijerinckii Br21 or Clostridium acetobutylicum ATCC 824 and one of the inhibitors. The H2 production rate decreased by 49.3, 48.7, and 51.3% in the presence of C. beijerinckii Br21 and of HMF, AL, or AF, respectively. In the presence of C. acetobutylicum ATCC 824 and of HMF, AL, or AF, the H2 production rate reduced by 45.5, 61.3, and 59.6%, respectively. The inhibitor concentration that inhibited 25% of H2 production, IC25, was also determined so that mixtures of the inhibitors could be prepared and tested in the presence of the microorganisms. HMF, LA, and FA afforded IC25 of 0.66, 2.15, and 0.89 g/L, respectively. On the basis of these values, four different mixtures were prepared: HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA. In the presence of C. beijerinckii Br21, HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA inhibited H2 production by 58.9, 58.4, 49, and 85.9%, respectively, whereas in the presence of C. acetobutylicum ATCC 824, inhibitions were 67.6, 66.6, 55.5, and 88.8% respectively. Therefore, C. acetobutylicum ATCC 824 was more sensitive to the effects caused by inhibitors. HMF seemed to affect the H2 production rate more, whereas acids appeared to act more globally on bacterial metabolism. These results reveal the concentration limits of the tested inhibitors when biomass hydrolysates are employed as raw material for fermentative H2 production.

Ce travail est basé sur l’étude expérimentale, étape par étape, du procédé de lyophilisation, afin de comprendre les impacts des différents paramètres du procédé sur la viabilité d’une souche modèle probiotique de type lactobacillus casei. Nous avons tout d’abord étudié les propriétés thermodynamiques des formulations considérées à base de lactose et de polyvinylpyrrolidone, (PVP) en commençant dans un premier temps par l’obtention du diagramme d’état du système amorphe constitué du binaire eau-PVP, puis le diagramme de fusion du ternaire eau-PVP-lactose afin d’en déduire les paramètres clefs pour l’optimisation des cycles de lyophilisation de ces suspensions bactériennes. Dans la deuxième partie, nous avons caractérisé par microscopie électronique à balayage (MEB) la localisation des bactéries au sein de la phase solide amorphe des lyophilisats poreux. Ensuite, les différentes formulations ont été soumises à différents protocoles de congélation (vitesse de refroidissement ; recuit) afin d’obtenir les meilleurs résultats en termes de taux de survie des bactéries. Avec la formulation sélectionnée précédemment nous nous sommes intéressés à l’influence des paramètres opératoires de sublimation (température étagère et pression totale de sublimation) conduisant aux meilleurs taux de survie des bactéries. Nous avons observé que nos cellules probiotiques, avec ces formulations, pouvaient être lyophilisées, au-dessus de la température limite de collapse, sans impacter la viabilité des cellules présentes ou insérées au sein de la phase matrice poreuse du lyophilisat final, ce dernier présentant de plus, de bonnes propriétés d’usage en termes de stabilité en vue d’une mise en forme galénique ou d’un stockage ultérieur / This work is based on the experimental study, step by step, of the freeze-drying process of a model probiotic strain of lactobacillus caseï type to understand the impact of the numerous factors (formulation; freezing protocol; operating conditions) on the survival rates of these bacteria in the final lyophilisate. Firstly, we investigated the thermo-dynamical and physical properties (vitreous transition and melting temperatures) of formulations based on lactose and polyvinylpyrrolidone (PVP) protectants and their mixture. Thus, we have determined the phase diagrams and the melting diagram of the water+PVP binary system and of the ternary water-PVP-lactose system. Next, we determined the optimal freezing protocol (freezing rates; annealing treatment) with different formulations which led to the best survival rates. Next, in a preliminary study we have characterized by SEM (scanning electron microscopy) the location of the cells inserted inside the solid amorphous phase of the porous matrix of the different lyophilisates. Secondly, with the pre-selected formulation, we experienced the influence of the main operating sublimation parameters (shelf temperature and total gas pressure), leading to highest product quality in terms of bacteria survival ratios of the final lyophilisates. We observed that these probiotics cells, with this formulation, could be freeze-dried above the limit collapse temperature without impacting significantly the viability of the freeze-dried cells and with lyophilisates of high stability attributes