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Dissertation (Ph.D.) -- Auburn University, 2005. / Vita. Includes bibliographical references (ℓ. 117-132).

L'un des avantages majeurs des réactions de bioconversion résulte du fait que le milieu réactionnel des biocatalyseurs est l'eau. Ce dit avantage constitue l'une des principales limitations de ces réactions de bioconversion lorsqu'il s'agit de substances lipophiles non solubles dans l'eau connue les stérols. L'efficacité d'un procédé de bioconversion de substances lipophiles solides dépend essentiellement du contact et de 1'interaction entre le biocatalyseur et ce substrat lipophile. Les solutions proposées dans la littérature font appel à des solvants et des produits chimiques de natures souvent toxiques, inflammables et explosives. Ces solutions décrites font perdre à la bioconversion son caractère de biotechnologie blanche. Dans ce travail, nous avons montré qu'en plus de ses avantages connus, l'immobilisation passive de biocatalyseurs au sein d'un support poreux peut favoriser l'interaction cellule-substrat lipophile et augmenter le taux de bioconversion sans utiliser de solvants et de produits chimiques. La réaction modèle étudiée est le clivage de la chaine latérale des stérols par des mycobactéries en vue de l'obtention des androsténones précurseurs naturels des stéroïdes, molécules à forte valeur biologique ajoutée. Le support d'immobilisation le plus performant a été le fruit sec de Luffa cylindrica. Par rapport aux supports organiques classiques tels que les gels de polyacrylamide, les mousses de polyuréthane, la silicone et les plastiques, le fruit sec de Luffa cylindrica présente les avantages suivants : (i) c'est un produit naturel, (ii) biodégradable, (iii) peu onéreux, (iv) non toxique pour les microorganismes, (v) stable du point de vue mécanique et thermique, (vi) et réutilisable. / One of the major advantages of using biocatalysts in organic synthesis is that water constitutes the reaction medium. However, water becomes a serious problem when bioconversion deals with lipophilic compounds, in particular those poorly soluble in water such as sterols. Bioconversion of lipophilic compounds depends on the close contact between the hydrophobic substrate and the biocatalyst. Increasing this contact requires usually the use of huge amounts of chemical which are often toxic, flammable and explosive. In this work, we showed that passive cell immobilization in porous materials can increase the contact between microorganisms and lipophilic substrates without using chemicals. The side chain cleavage of sterols was studied as a model multistep microbial bioconversion of lipophilic compounds. This reaction allows the production of androstenones which are the natural precursors of steroids. Among the studied immobilization carriers, the dried fruit of Luffa cylindrica was the most efficient Compared to other organic support carrier such as alginate beads, polyurethane foams, silicones and plastics, the dried fruit of Luffa cylindrica is advantageous since it is natural, renewable, biodegradable, cheap, mechanically strong, free of toxicity and it doesn't need a chemical pretreatment.

La vanillyl alcool oxydase est un biocatalyseur prometteur d’un point de vue réactionnel en raison de sa faible sélectivité sur les composés phénoliques substitués en position para. Elle catalyse l’oxydation d’une large gamme de dérivés 4-hydroxybenzylique, des réactions de déméthylation oxydative, de désamination, de déshydrogénation et d’hydroxylation ; notamment celle de l’eugénol. L’hydroxylation de ce dernier conduit à la formation d’alcool coniférylique. Dans le but d’utiliser l’eugénol comme substrat de biosytnhèse de dérivés vanilliques, nous avons développé, au cours de ce travail de thèse, deux souches exprimant le biocatalyseur d’intérêt : une souche de levure et une souche bactérienne.La première partie de la thèse a donc consisté à mettre au point un système d’expression pour la production de VAO dans la levure de boulanger et un procédé d’hydroxylation de l’eugénol en fermenteur. Le principal apport réside dans la construction de la souche 93645, qui contient la cassette d’expression VAO et une activité oxydase propre permettant la bioconversion de l’alcool coniférylique formé en acide férulique. En fonction des conditions de fermentation, 20 g/l d’alcool coniférylique ou 27 g/l d’acide férulique sont produits par les cultures de S. cereviaise- VAO 93645. La reproductibilité des procédés, ainsi que leur faisabilité à l’échelle pilote, ont été démontrés.Dans la seconde partie de cette étude, le même travail de clonage a été réalisé dans la souche Amycetales Streptomyces setonii ATCC 39116. La bactérie, reclassifiée en 2009 sous le nom d’Amycolatopsis sp 39116, est connue pour sa capacité à bioconvertir l’acide férulique en vanilline ; d’où l’intérêt d’exprimer l’enzyme VAO dans cette souche. Plusieurs stratégies de clonage ont été expérimentées et une souche recombinante, exprimant une activité VAO active a été obtenue. Les conditions optimales pour l'utilisation de cette dernière dans le cadre de la production de vanilline et d’alcool coniférylique ont été identifiées. Elles conduisent à la biosynthèse de 0,4 g/l vanilline et de 15 g/l d’alcool coniférylique. Les résultats mettent en évidence une activité insuffisante des oxydases de Streptomyces sur l’alcool coniférylique formé. Ce type de production n’a encore jamais été réalisé chez S. setonii et ces premiers résultats demandent encore des mises au point avec, sans doute, le clonage d’oxydases hétérologues permettant la bioconversion de l’alcool coniférylique formé en acide férulique. / Our aim was to develop a process for the biosynthesis of vanillin derivatives from eugenol.Vanillyl alcohol oxidase isolated from Peniciiiium simplissicimum, catalyzes the hydroxylation of eugenolinto coniferyl alcohol. In this study, two strains expressing the biocatalyst were constructed: a yeast,Saccharomyces cerevisiae, and a bacteria; Streptomyces setonii.It has been demonstrated that the wild strain Saccharomyces cerevisiae can bioconvert coniferyl alcohol, most probably due to its dehydrogenase activity. Strain 93645, genetically modified to expressvanillyl-alcohol oxidase, enabled us to optimize an industrial scale process for the production of natural ferulic acid.Streptomyces setonii strain ATCC 39116 was also genetically engineered to over-express VAO. Abioconversion process was developed leading to a coniferyl alcohol concentration of 15 g/1 coniferyl alcohol. The impact of several parameters; such as temperature, substrate addition mode and pH, werealso explored to improve the bioconversion reaction of coniferyl alcohol to vanillin. The amounts of product resulting from bacterial biosynthesis were however too low for implementation of an industrial process.

Xylitol is a high value polyalcohol being used in pharmaceutical, hygiene, and food products due to its functional properties such as anticariogenic, antibacterial as well as low calorie and low glycemic properties. An alternative route for xylitol production is the biotechnological method in which microorganisms or enzymes are involved as catalysts to convert xylose into xylitol under mild conditions of pressure and temperature. This method is unlike the conventional chemical method that requires high pressure and temperature and results in low product yield. The goal of this research is to employ an integrated process using all fractions of an agro-industrial biomass (oat hull) for xylitol bioproduction, preferably in a repeated batch bioconversion process, with C. guilliermondii as the biocatalyst. Processes including hydrolysis, biomass delignification, hydrolysate detoxification using adsorption process, and finally free- and immobilized-cell bioconversions were employed in this study. The kinetics of acid-catalyzed hydrolysis of hemicellulose was investigated under mild conditions (temperature: 110ºC to 130ºC and catalyst (H2SO4) concentrations from 0.1 to 0.55 N) to determine the kinetic mechanism and generation of monosaccharides (xylose, glucose, and arabinose) as well as the microbial inhibitors consisting of acetic acid, furfural, and hydroxymethylfurfural (HMF) in the hydrolysate. A maximum recovery of 80% was attained for xylose as the main monosaccharide and the substrate for xylitol; its generation in the hydrolysate followed a single-phase 2-step kinetic mechanism similar to that of the HMF. However, a single-phase mechanism with no decomposition could describe the formation of arabinose, acetic acid, and furfural. Glucose generation followed a biphasic mechanism (fast and slow releasing) apparently with no decomposition. In the alkaline delignification of the hydrolysis byproduct (solid fraction) and the intact (crude) biomass, kinetic models based on biphasic mechanism consisting of bulk and terminal phases gave the best results and fit to the experimental data. In the bulk phase, where the temperature ranged from 30ºC to 100ºC, the reaction rate constant varied from 0.15 to 0.19 1/min for the intact biomass and from 0.25 to 0.55 1/min for the hydrolysis byproduct. According to the models, accelerated lignin removal with the increased operating temperature could be due to the shift of the process from the terminal phase to the bulk phase. The values obtained for the activation energies herein ( 33 kJ/mol) were less than the values reported in the literature for other lignocellulosic materials. The removal or reduction of the microbial inhibitors in the medium was carried out by activated carbon (adsorptive detoxification). According to the results using the Langmuir model with the activated carbon as the adsorbent, the maximum monolayer capacities of 341, 211, and 46 mg/g were obtained, respectively, for phenol, furfural, and acetic acid. Thermodynamic analyses indicated that the adsorption of the three abovementioned chemicals by the activated carbon was exothermic (enthalpy: H0), spontaneous (free energy: G0), and based on the affinity of the solute toward the adsorbent (entropy: S0). In the concentrated hydrolysate, the removal of phenols, as the main inhibitor, was very successful such that by activated carbon doses of 1.25%, 2.5%, and 5% (w/v) they could be reduced to 34%, 13%, and 3% of the initial concentration (8.7 g/l), respectively. During xylitol bioproduction process in the repeated batch mode using C. guilliermondii, variables of pH control, medium supplementation, and cell recycling proved to be more important than medium detoxification. Processes involving pH-controlled condition combined with nitrogen supplementation and a mild detoxification performed very well with consistent conversion parameters in the successive batches; values of over 0.8 g/g, 0.55 g/l/h, and 53 g/l were obtained respectively for xylitol yield, volumetric productivity, and final concentration. On the other hand, in a single-batch bioconversion, there was no need for supplementing the medium with the nitrogen source. Kinetic modeling of the process showed that substrate (xylose) as well as co-substrate (glucose) consumption, product (xylitol) formation, and cell regeneration could be predicted by a diauxic model. In the aerated free-cell and immobilized-cell systems, aeration rates of 1.25 vvm and 1.25-1.5 vvm were required for free-cell and immobilized-cell systems, respectively, to reach the maximum bioconversion performance. In the immobilized-cell system, cell support also played an important role in this biotransformation. Application of the support based on the delignified hydrolysis byproduct resulted in high and consistent bioconversion parameters in all batches comparable to the ones in the free-cell system. However, bioconversions using the lignin-rich material (hydrolysis byproduct) resulted in a lower efficiency in the first batch which could be partly improved in the second batch and almost fully increased in the third batch to nearly reach performance parameters comparable to the ones obtained in the free-cell system. Overall, the integrated process employed in this investigation helps fill in the knowledge gaps existing on the lignocellulosic biomass application for xylitol bioproduction and biorefinery industries.

Rapid expansion of the biodiesel industry has led to a surplus of crude glycerol. This thesis investigates the ability of fungi to utilize crude glycerol as an alternative to conventional carbon substrates for growth and lipid production. Screening revealed that 40 of the 61 isolates tested had increased biomass yield, compared to glucose, when crude glycerol was utilized; 29 of these isolates possessed the ability to completely metabolize 14 g•L-1 of glycerol after 7-14 days. The top four candidates belonged to the genera Galactomyces and Mucor. Overall, Galactomyces sp. proved to be better suited for lipid production. In addition to producing biomass with a high lipid content (up to 45 % w/w), Galactomyces sp. also exhibited high biomass yields (up to 25 g•L-1). The results obtained in this study compare favourably, and in some cases exceed, other literature reported values for biomass and lipid production using glycerol. / CanAdvance and Agriculture and Agri-Food Canada

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Microbial actions on coal have long been identified as a source of methane in coalbeds. Andrew Scott (1995) was the first to propose imitating the natural process of biogenic gasification, possibly leading to recharging coalbed methane (CBM) reservoirs, or setting up natural gas reservoirs in non-producing coalbeds. This study was aimed at identifying the changes in coal properties that affect gas deliverability in coal-gas reservoirs, when treated with microbial consortia to generate/enhance gas production. The experimental work tested the sorption and diffusion properties for the coal treated and, more importantly, the variation in the relevant parameters with continued bio-conversion since these are the first two phenomena in CBM production. During the first phase, single component sorption-diffusion experiments were carried out using pure methane and CO2 on virgin/baseline coals, retrieved from the Illinois basin. Coals were then treated with nutrient amended microbial consortia for different periods. Gas production was monitored at the end of thirty and sixty days of treatment, after which, sorption-diffusion experiments were repeated on treated coals, thus establishing a trend over the sixty-day period. The sorption data was characterized using Langmuir pressure and volume constants, obtained by fitting it over the Langmuir isotherm. The diffusion coefficient, D, was estimated by establishing the variation trend as a function of pore pressure. The pressure parameter was considered critical since, with continued production of methane, the produced gas diffuses into the coal matrix, where it gets adsorbed with increasing pressure. During production, the pressure decreases and the process is reversed, gas diffusing out of the coal matrix and arriving at the cleat system. The results indicated an increase in the sorption capacity of coal as a result of bioconversion. This was attributed to increased pore surface areas as a result of microbial actions. However, significant hysteresis was observed during desorption of methane and was attributed to preferential desorption from sorption sites in the pathways leading to pore cavities. This is corroborated by the increased rates of diffusion, especially for methane, which exhibited rates higher than that for CO2. This contradicted the results for untreated/baseline coal, which were in agreement with previous studies. Effort was made to explain this anomaly by the non-monotonic dependence of effective diffusion coefficient on the size of the diffusing particles, where in coalbed environments, CO2 has smaller kinetic diameter than methane.

Notre objectif était d’identifier des cytochromes P450 capables d’oxygéner des mono et sesquiterpènes, pour produire des molécules aux propriétés organoleptiques intéressantes labellisés « naturelles » par l’industrie des arômes et du parfum.Nous avons identifié 7 couples P450-substrat catalysant une conversion in vitro supérieure ≥ 45 % et/ou formant un produit attendu. Les quantités de produit obtenu par bioconversion dans la levure restent insuffisantes pour un procédé industriel. Les facteurs limitants ont été identifiés dans le cas du valencène comme : 1) la toxicité induite par les produits, 2) l’accumulation du β-nootkatol dans les membranes, 3) l’inhibition de l’enzyme par les produits réactionnels. Trois cytochromes P450 d’Arabidopsis thaliana impliqués dans le métabolisme indolique oxydent activement le limonène. Ils s’expriment dans les inflorescences et constituent le premier exemple de P450s suceptibles de participer à deux voies métaboliques indépendantes chez les plantes. / Our aim was to identify cytochromes P450 catalyzing hydroxylation of mono-and sesquiterpenes to produce functionalized "natural" compounds with interesting organoleptic properties for the flavor and fragrance industry. We identified 7 P450-substrate pairs showing . 45 % in vitro conversion and/or forming an expected product. The amounts of products resulting from yeast bioconversion were however too low for implementation of an industrial process. Factors limiting the nootkatone production from the P450-dependent bioconversion of valencene were identified : 1) toxicity for yeast of the ƒÀ-nootkatol and nootkatone products, 2) ƒÀ-nootkatol accumulation in endomembranes, 3) products inhibition of valencene hydroxylation. Three previously characterized P450s from Arabidopsis thaliana in indolic metabolism were shown to actively oxidize limonene. They are expressed in inflorescences and may provide the first demonstrated case of multifunctional P450s involved in independent plant pathways.

Dans le contexte de la bioraffinerie végétale pour la production de molécules et d'énergie, des prétraitements sont nécessaires pour augmenter la réactivité de la biomasse ligno-cellulosique. Cette thèse s'insère dans une thématique dont l'objectif général est d'établir les bases d'une raffinerie du végétal par des procédés par voie sèche. Cette étude a pour objectif de développer et comprendre le fractionnement mécanique poussé de la paille de blé jusqu'à des tailles sub-millimétriques et d'évaluer les effets sur des procédés de bioconversions énergétiques. La paille de blé présente une grande hétérogénéité à plusieurs niveaux d'échelle (du cm au µm). Un diagramme de broyage multi-étapes à l'échelle pilote (>1kg) a permis d'obtenir une large gamme de tailles de particules, par 3 modes de sollicitation distincts: i) broyages à grille sélective produisant des tailles du grossier (800 µm) au fin (50 µm), ii) broyage à jet d'air (ultrafin, ~20 µm) et iii) broyage à boulets (ultrafin, ~10 µm). Une méthodologie d'analyse morphologique des particules a été développée par analyse d'images de microscopie optique. La paille soumise aux mécanismes complexes de rupture lors de broyages produit une forte variabilité des formes et compositions des particules. L'analyse multiple de co-inertie a permis d'évaluer de façon globale les morphologies des particules. Globalement, le broyage diminue la taille et les facteurs de forme des particules, avec quelques exceptions dues aux configurations matérielles. La dégradabilité enzymatique (saccharification) des poudres produites a été améliorée par la réduction de la taille des particules. Jusqu'à ~100 µm la solubilisation des polysaccharides augmente puis se stabilise à 36 % des polysaccharides totaux et 40 % de la cellulose. Seuls les échantillons issus du broyage à boulets dépassent cette limite et atteignent 46 % d'hydrolyse des polysaccharides totaux et 72 % de la cellulose. Ceci est lié à une augmentation de l'efficacité enzymatique due à la diminution de la cristallinité de la cellulose (de 22 à 13%). Ces résultats du broyage à boulets sont comparables à ceux de l'explosion à la vapeur, avec une meilleure préservation des hémicelluloses. Cette amélioration d'efficacité enzymatique s'est traduite par des dégradations anaérobies (biogaz) accélérées et légèrement augmentées (cas du broyeur à boulets). La décomposition aérobie dans le sol a été améliorée par le broyage grossier, mais les broyages plus fins n'ont pas entrainé de gain. Les caractéristiques de la paille broyée peuvent varier selon l'intensité et le mode de broyage. Bien que tous les broyages permettent la réduction de la taille, le broyage à grilles et le broyage à jet d'air n'engendrent pas de changements dans la structure fine des polymères pariétaux. Seul le broyage à boulets a engendré des changements de la structure interne des particules notamment en réduisant la cristallinité de la cellulose et en solubilisant partiellement les hémicelluloses. Ces résultats permettent de mettre en évidence que la fragmentation mécanique poussée par voie sèche est une alternative possible aux prétraitements utilisés en raffinerie végétale. / In a context of plant biorefinery for the production of molecules and energy, pretreatments are necessary to increase the reactivity of the lignocellulosic biomass. This thesis is part of a general project aiming to establish the bases for a dry plant refinery. This study aimed to develop and understand advanced mechanical fractionation of wheat straw down to sub-millimeter sizes and to assess its effects on bioconversion processes for bioenergy. Wheat straw exhibited a high heterogeneity at several scale levels (from cm to μm). A multistep diagram of dry grinding at pilot-scale (> 1 kg) produced a wide range of particle sizes by three distinct mode of action: i) sieve-based grinding producing particle sizes from coarse (800 μm) to fine (50 μm), ii) air-jet milling (ultra-fine, ~ 20 μm) and iii) ball milling (ultra-fine, ~ 10 "m). A morphological analysis of particles was developed by image analysis from light microscopy. Subjecting wheat straw to the complex breaking mechanisms during grinding produced particles highly variable in shapes and compositions. A multiple co-inertia analysis allowed the evaluation of the overall particle morphologies. Generally, grinding reduced the size and shape descriptors of particles, with some exceptions due to equipment configurations. The enzymatic degradability (saccharification) of produced powders was improved by reducing their particle size. Until ~ 100 μm the polysaccharides solubilisation was increased and then stabilised at 36% total polysaccharides and 40% cellulose. Only samples from ball milling overcome this limit and attained hydrolysis yields of 46% total polysaccharides and 72% cellulose. This is due to an increase in enzymatic efficiency by the reduction of cellulose crystallinity (from 22 to 13%). These results of ball milling are comparable to those of steam explosion process, with a better preservation of hemicelluloses. This improved enzymatic efficiency resulted in faster and slightly more extensive (ball milling case) anaerobic degradations (biogas). Aerobic decomposition in the soil was improved by coarse grinding, but finer grinding did not result in a further increase. The characteristics of ground straw varied depending on grinding intensity and mode. Although all grindings could reduce the size, sieve-based grinding and air-jet milling did not allow changes in the fine structure of cell wall polymers. Only ball milling led to changethe internal structure of particles especially reducing cellulose crystallinity and partially solubilising hemicelluloses. These results demonstrate that advanced mechanical fragmentation by dry processes is a possible alternative for pretreatments in a plant refinery