Sécuriser un approvisionnement fiable de micro-algues est récemment devenu un enjeu industriel. Pour assurer la croissance de micro-algues dans des photobioréacteurs clos, un transfert de masse optimum de l'oxygène et du dioxyde de carbone doit être assuré. Dans cette thèse, une étude du transfert de masse gaz-liquide dans les conduites horizontales a été menée. Dans les trois premiers chapitres, un modèle unidimensionnel de transfert de masse dans le photobioréacteur a été développé. Tout d'abord, le transfert de masse entre une bulle de gaz allongée et un écoulement liquide turbulent a été 'étudié expérimentalement. En considérant l'interface comme étant plane, les coefficients de transfert de masse mesurés sont proches d'une corrélation de Lamourelle (1972). Le modèle de Taitel pour les écoulements stratifiés a été comparé à des modèles plus complets pour la prédiction de l'interface des bulles allongées. Une approche analytique basée sur un bilan de masse et utilisant les modèles testés a ensuite été développée et adaptée à un photobioréacteur afin de prédire l'efficacité de la conversion du CO2 en biomasse en fonction des paramètres d'exploitation. Les deux derniers chapitres visent à développer une simulation numérique du transfert de masse gaz-liquide. Une mesure de la concentration en CO2 dans le sillage d'une bulle de gaz ascendante a été effectuée à l'aide d'une méthode améliorée de traitement des données de Fluorescence Induite par Plan Laser (FIPL). Enfin, une simulation numérique a été réalisée sous COMSOL / Securing a reliable supply of microalgae has recently become an industrial stake. To ensure successful growing of microalgae in enclosed, tubular photobioreactor systems as in Microphyt, an optimum mass transfer of oxygen and carbon dioxide should be secured. In this thesis an investigation of the gas-liquid mass transfer in horizontal pipes has been conducted. In the first three chapters, a one dimensional mass transfer model in horizontal gas-liquid flows will be developed and applied to horizontal photobioreactors. Firstly, a study of mass transfer between an elongated gas bubble under a turbulent liquid flow immobilized in a duct has shown that under the hypothesis considering the interface as a flat plane estimated, the measured mass transfer coefficients appear to be well fitted by a correlation from Lamourelle (1972). The interface prediction for stratified flows have been compared to more complete unit-cell models for intermittent flow interface and found to be a good first estimate. The photobioreactor’s conversion efficiency of CO2 into biomass as a function of operating parameters is investigated using an analytical approach to complete the mass balance and classical two-phase flow approach from Taitel (1976). The last two chapters aim at developing a numerical simulation of gas-liquid mass transfer. A measurement of CO2 wake structures behind free rising bubbles have realized using an improved data treatment method for Planar laser-induced fluorescence (PLIF) and pH sensitivity of fluorescein sodium. Finally, an implementation of the experimental measurements under COMSOL has been realised

Le métabolisme central de la souche E. coli MG1655 Δpgi ΔudhA Δedd Δqor a été rationnellement modifié afin de produire deux moles de NADPH et deux moles de NADH lors de l’oxydation du glucose en acétyl-CoA, alors qu’une souche sauvage produit quatre moles de NADH. La conséquence de cette modification est une forte diminution de son taux de croissance sur milieu minimum et glucose. Afin d’évaluer les aptitudes de cette souche à s’adapter à un tel stress métabolique, son évolution in vivo a été forcée lors de cultures par repiquages successifs sur glucose. Ainsi, après quatre cultures d’évolution un clone pur a été réisolé et caractérisé : un taux de croissance multiplié par six par rapport à la souche non évoluée a été mesuré. L’analyse par CGS (Séquençage par comparaison de génomes) a permis de corréler l’augmentation du taux de croissance à l’apparition d’une mutation, NuoF*(E183A), dans la sous-unité NuoF du complexe respiratoire I, complexe NADH-dépendant. Des études biochimiques et physiologiques de l’impact de cette mutation ont permis de démontrer que le complexe I évolué peut oxyder à la fois le NADPH et le NADH, créant ainsi une nouvelle voie d’oxydation du NADPH dans la cellule. L’évolution in vivo a ensuite été poursuivie au cours de onze repiquages et un nouveau clone pur a été réisolé et caractérisé : un taux de croissance proche de la souche sauvage et onze fois supérieur à celui de la souche non évoluée a alors été mesuré. L’analyse par CGS a permis cette fois de corréler l’augmentation du taux de croissance à l’apparition de deux mutations : NuoF*(E183A) et d’une deuxième dans la sous-unité α de l’ARN polymérase, rpoA*. Enfin, une deuxième souche E. coli MG1655 ΔpfkAB ΔudhA Δedd Δqor a été construite afin de détourner son métabolisme pour produire cette fois trois moles de NADPH et une mole de NADH lors de l’oxydation du glucose en acétyl-CoA. Cette souche étant incapable de se développer en milieu liquide et glucose, une étape de criblage en milieu solide et glucose a permis de sélectionner des clones capables de croître sur glucose. Tous ces clones possédaient soit la mutation NuoF*(E183A), soit une nouvelle mutation NuoF*(E183G), dont la caractérisation biochimique a montré que les deux enzymes évoluées permettent l’oxydation du NADPH par la chaîne respiratoire. Le phénomène d’évolution in vivo a conduit à la création d’une nouvelle fonction pour le NADPH qui n’est plus seulement impliqué dans les réactions de synthèse anabolique mais qui peut être utilisé pour produire directement de l’énergie catabolique. La compréhension globale du phénomène d’évolution a finalement permis la conception de nouvelles souches adaptées pour la production NADPH-dépendante de composés chimiques d’intérêt / Bacterial metabolism is characterized by robustness and plasticity that allow it to adjust too many metabolic perturbations. This present work demonstrates Escherichia coli abilities of evolution and adaptation under stress of NADPH accumulation. We constructed the E. coli MG1655 Δpgi::FRT ΔudhA::FRT Δedd::FRT Δqor::FRT strain where central metabolism has been rationally engineered to produce two mol of NADPH and two mol of NADH during the oxidation of glucose to acetyl-CoA, while a wild-type strain produces 4 mol of NADH per mole of glucose. Consequently, this strain presents a weak growth on glucose mineral medium. So as to evaluate bacterial abilities to overcome such metabolic stress, in vivo evolution of this strain has been forced in laboratory by serial transfer subcultures. After four evolution subcultures, an individual clone has been characterized by a six fold increased growth rate compared to non-evolved strain. CGS (Comparative Genome Sequencing) analysis allowed us to correlate growth improvement with one mutation apparition in respiratory complex: NuoF*(E183A) in NuoF subunit from the NADH dependant complex I. Further biochemical and physiological studies demonstrated that the evolved respiratory complex is able to oxidize both NADH and NADPH, resulting in a new NADPH reoxydation pathway in the cell. In vivo evolution experiments were then continued until eleven subcultures, where a new individual clone has been characterized by an eleven fold increased growth rate compared to non-evolved strain. Additional CGS analysis allowed us to correlate growth improvement with apparition of two mutations: NuoF*(E183A) and another mutation within the RNA polymerase alpha subunit, rpoA*. Thus, a second E. coli MG1655 ΔpfKA::FRT ΔpfKB::FRT ΔudhA::FRT Δedd::FRT Δqor::FRT strain has been rationally engineered to produce three mol of NADPH and one mole of NADH per mole of glucose oxidized to acetyl-coA. As this train was unable to growth in liquid glucose mineral medium, we performed a solid-state screening on glucose mineral medium that led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed an E183G mutation, both having NADH and NADPH oxidizing ability. This result highlights need of this new NADPH reoxydation pathway for NADPH accumulating cells. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy. Finally, global understanding of evolution process allowed conception of new engineered strains, well designed for NADPH dependant production of chemicals of interest

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Hydrocarbons provide excellent feed stocks for bioconversion processes to produce value added products using various micro-organisms. However, hydrocarbon-based aerobic bioprocesses may exhibit transport problems where the bioconversion is limited by oxygen supply rather than reaction kinetics. Consequently, the overall volumetric oxygen transfer coefficient (KLa) becomes critical in designing, operating and scaling up of these processes. In view of KLa importance in hydrocarbon-based processes, this work evaluated KLa measurement methodologies as well as quantification of KLa behavior in aerated agitated alkane-solid-aqueous dispersions. A widely used KLa measurement methodology, the gassing out procedure (GOP) was improved. This improvement was done to account for the dissolved oxygen (DO) transfer resistances associated with probe. These resistances result in a lag in DO response during KLa measurement. The DO probe response lag time, was incorporated into the GOP resulting in the GOP (lag) methodology. The GOP (lag) compared well with the pressure step procedure (PSP), as documented in literature, which also incorporated the probe response lag time. Using the GOP (lag), KLa was quantified in alkane-solid-aqueous dispersions, using either inert compounds (corn flour and CaCO3) or inactive yeast cells as solids to represent the micro-organisms in a hydrocarbon bioprocess. Influences of agitation, alkane concentration, solids loading and solids particle sizes and their interactions on KLa behavior in these systems were quantified. In the application of an accurate KLa measurement methodology, the DO probe response lag time was investigated. Factors affecting the lag, which included process conditions such as agitation (600-1200rpm), alkane concentration (2.5-20% (v/v), alkane chain length (n-C10-13 and n-C14-20), inert solids loading (1-10g/L) and solids particle sizes (3-14μm) as well as probe characteristics such as membrane age and electrolyte age (5 day usage), were investigated. Kp, the oxygen transfer coefficient of the probe, was determined experimentally as the inverse of the time taken for the DO to reach 63.2% of saturation after a step change in DO concentration. Kp dependence on these factors was defined using 22 factorial design experiments. Kp decreased on increased membrane age with an effect double that of Kp decrease due to electrolyte age. Additionally, increased alkane concentration decreased Kp with an effect 7 times higher compared to that of Kp decrease due to increased alkane chain length. This was in accordance to Pareto charts quantification. KLa was then calculated, using the GOP (lag), according to equation [1] which incorporates the influence of Kp. Equation 1 is derived from the simultaneous solution of the models which describe the response of the system and of the probe to a step change in DO. 1 1 * L p p p K at K t L p p La C K e K ae C K K = -  - - -  -   [1] The KLa values documented in literature from the PSP and KLa calculated by the GOP (lag) showed only a 1.6% difference. However KLa values calculated by the GOP (lag) were more accurate than KLa calculated by the GOP, with up to >40% error observed in the latter according to t-tests analyses. These results demonstrated that incorporating Kp markedly improved KLa accuracy. Consequently, the GOP (lag) was chosen as the preferred KLa measurement methodology. KLa was determined in n-C14-20-inert solid-aqueous dispersions. Experiments were conducted in a stirred tank reactor with a 5L working volume at constant aeration of 0.8vvm, 22ºC and 101.3kPa. KLa behavior across a range of agitations (600- 1200rpm), alkane concentrations (2.5-20% (v/v)), inert solids loadings (1-10g/L) and solids particle sizes (3-14μm) was defined using a 24 factorial design experiment. In these dispersions, KLa increased significantly on increased agitation with an effect 5 times higher compared to that of KLa increase due to interaction of increased alkane concentration and inert solids loading. Additionally, KLa decreased significantly on increased alkane concentration with an effect 4 times higher compared to both that of increased solids particle sizes and the interaction of increased agitation and solids particle size. In n-C14-20-yeast-aqueous dispersions, KLa was determined under narrowed process conditions better representing typical bioprocess conditions. KLa behavior across a range of agitations (600-900rpm), alkane concentrations (2.5-11.25% (v/v)) and yeast loadings (1-5.5g/L) using a 5μm-yeast cell was defined using a 23 factorial design experiment. In these dispersions, KLa increased significantly on increased agitation. Additionally, KLa decreased significantly on increased yeast loading with an effect 1.2 times higher compared to that of KLa decrease due to interaction of increased alkane concentration and yeast loading. In this study, the importance of Kp for accurate KLa measurement in alkane based systems has been quantified and an accurate and less complex methodology for its measurement applied. Further, KLa behavior in aerated alkane-solid-aqueous dispersions was quantified, demonstrating KLa enhancement on increased agitation and KLa depression on increased alkane concentration, solids loading and solids particle sizes. / AFRIKAANSE OPSOMMING: Koolwaterstowwe dien as uitstekende voervoorraad vir ´n verskeidenheid van mikroorganismes wat aangewend word in biologiese omsettingsprosesse ter vervaardiging van waardetoevoegende produkte. Hierdie biologiese omsettingsprosesse word egter vertraag weens die gebrek aan suurstoftoevoer onder aerobiese toestande. Die tempo van omsetting word dus beheer deur die volumetriese suurstofoordragkoeffisiënt (KLa) eerder as die toepaslike reaksiekinetika. Die bepaling van ´n akkurate KLa word dus krities tydens die ontwerp en opskalering van hierdie prosesse. Met dit in gedagte het hierdie studie die huidige metodes om KLa te bepaal geëvalueer en die gedrag van KLa in goed vermengde en belugde waterige alkaanmengsels met inerte vastestowwe, soos gisselle, in suspensie ondersoek. ´n Deesdae populêre metode om KLa te bepaal, die sogenaamde gasvrylatingsprosedure (GOP) is in hierdie studie verbeter. Die verbetering berus op die ontwikkeling van ´n prosedure om die suurstofoordragsweerstand van die pobe wat die hoeveelheid opgeloste suurstof (DO) meet, in berekening te bring. Hierdie weerstand veroorsaak ´n vertragin in the responstyd van die probe. Die verbeterde metode, GOP (lag), vergelyk goed met die gepubliseerde resultate van die drukstaptegniek (PSP) wat ook die responstyd in ag neem. GOP (lag) is ingespan om KLa te gekwantifiseer vir waterige alkaan-vastestof suspensies. Inerte componente soos mieliemeel, kalsiumkarbonaat en onaktiewe gisselle het gedien as die vastestof in suspensie verteenwoordigend van die mikroörganismes in ´n koolwaterstof bio-proses. Die invloed van vermengingstempo, alkaan konsentrasie, vastestof konsentrasie en partikelgrootte asook die interaksie van al die bogenoemde op KLa is kwatitatief bepaal in hierdie studie. Faktore wat die responstyd van die DO probe beïnvloed is ondersoek. Hierdie faktore is onder meer vermengingstempo (600-1200opm), alkaankonsentrasie (2.5-20% (v/v)), alkaankettinglengte (n-C10-13 en n-C14-20), vastestofkonsentrasie (1-10g/L) en partikelgrootte (3-14 μm). Faktore wat die eienskappe van die probe beïnvoed, naamlik membraan-en elektrolietouderdom (5 dae verbruik), is ook ondersoek. Kp, die suurstofoordragskoeffisiënt, is bepaal deur ´n inkrementele verandering in die suurstofkonsentrasie van die mengsel te maak en die tyd vir 63.2% versadiging van die probelesing te noteer. Die genoteerde tyd is die response tyd van die probe en Kp, die inverse van hierdie tyd. Die afhanklikheid van Kp op die bogenoemde faktore is ondersoek in ´n 22 faktorieël ontwerpte reeks eksperimente. Kp toon ´n afname met ´n toename in membraanouderdom. Hierdie afname is dubbel in grootte as dit vergelyk word met die afname relatief tot die toename in elektrolietouderdom. Verder toon Kp ´n afname met ´n toename in alkaankonsentrasie. Hierdie afname is 7 keer groter relatief tot die afname gesien met die toename in alkaan kettinglengte. Hierdie is in goeie ooreenstemming met Pareto kaarte as kwantifiseringsmetode. KLa is bereken met die inagname van Kp volgens vergelyking [1]: 1 1 * L p p p K at K t L p p La C K e K ae C K K = -  - - -  -   [1] Vergelyking [1] is afgelei vanaf die gelyktydige oplossing van die bestaande modelle wat die responstyd van die pobe vir ´n stapverandering in DO bereken. Die KLa waardes van die PSP metode uit literatuur verskil in die orde van 1.6% van dié bereken deur vergelyking [2]. Hierdie verskil is weglaatbaar. Die KLa waardes verkry uit die GOP metode wat nie Kp in berekening bring nie, verskil met meer as 40% van die huidige, verbeterde metode volgens die statistiese t-test analiese. Dit bewys dat die inagname van Kp ´n merkwaardige verbetering in die akuraatheid van KLa teweeg bring. GOP (lag) kry dus voorkeur vir die berekening van KLa verder aan in hierdie studie. KLa is bereken vir n-C14-20-water mengsels met inerte vastestofsuspensies. Die eksperimente is uitgevoer in ´n 5L geroerde reaktor met ´n konstante belugting van 0.8vvm (volume lug per volume supensie per minuut), 22ºC en 101.3kPa. Die gedrag van KLa met betrekking tot vermengingstempo (600-1200opm), alkaankonsentrasie (2.5-20% (v/v)), vastestofkonsentrasie (1-10g/L) en partikelgrootte (3-14μm) is ondersoek in ´n 24 faktorieël ontwerpte reeks eksperimente. Verder is die invloed van vloeistofviskositeit en oppervlakspanning op KLa ondersoek in ´n 23 faktorieël ontwerpte reeks eksperimente. KLa het ´n beduidende toename getoon met ´n toename in vermengingstempo. Hierdie toename was 5 keer groter as die toename relatief tot die interaksie van alkaan-en vastestofkonsentrasie. KLa het ook beduidend afgeneem met ´n toename in alkaankonsentrasie. Die toename was 4 keer groter as die toename relatief tot die toename in partikelgrootte en die interaksie van vermengingstempo en partikelgrootte. In n-C14-20-water mengsels met gisselsuspensies is KLa bepaal onder kondisies verteenwoordigend van tipiesie biologiese omsettingsprosesse. Die gedrag van KLa met betrekking tot vermengingstempo (600-900opm), alkaankonsentrasie (2.5-11.25% (v/v)) en giskonsentrasie (1-5.5g/L) met ´n partikelgroote van 5μm is ondersoek in ´n 23 faktorieël ontwerpte reeks eksperimente. Hierdie eksperimente het ´n beduidende toename in KLa met ´n toename in vermengingstempo getoon sowel as ´n beduidende afname met ´n toename in giskonsentrasie. Hierdie afname is in die orde van 1.2 keer groter in vergelyking met die interaksie van alkeen- en giskonsentrasie. Hierdie studie bring die kritieke rol wat Kp speel in die akkurate bepaling van KLa in waterige alkaansisteme met inerte vastestofsuspensies na vore. Dit stel verder ´n metodiek voor vir die akurate meting van en kwantifisering van beide Kp en KLa onder aerobiese toestande met betrekking tot vermengingstempo, alkaankonsentrasie, vastestofkonsentrasie en partikelgrootte.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009. / The abundance of alkane by-products formed in South Africa presents a feedstock opportunity for the production of a wide range of commercially important products, such as long-chain dioic acids and alcohols. These compounds are formed as intermediates through the biological conversion of alkanes, a route which is particularly attractive when compared with chemical conversion due to its operation under milder process conditions. Furthermore, advances in genetic manipulation, which enable the accumulation of a range of metabolic intermediates, make the biological route remarkably flexible. From the literature review Yarrowia lipolytica was identified as a promising organism for use in studying alkane bioconversion because of its ability to produce large quantities of fatty acids when grown on n-paraffins as a sole carbon source. The bioconversion of alkanes will not only depend on the genetic modification but also on the process conditions to maximise growth and bioconversion. The overall objective of this project was therefore to investigate the potential of Y. lipolytica for alkane bioconversion by defining the conditions that maximise both cell growth and bioconversion. The Y. lipolytica strains supplied (TVN348, TVN493 and WT), however, were not yet modified to the extent that accumulation of metabolic intermediates was possible. Use was therefore made of a model system in which the alkane substrate was substituted with an even chain alkylbenzene. Since Y. lipolytica is unable to metabolise the benzene ring, the alkylbenzene is converted to the metabolic intermediate, phenyl acetic acid (PAA), and the potential for bioconversion assessed through measuring the accumulation of PAA. The specific objectives of the project were therefore 1) to define and quantify the parameters for the establishment of an effective model system in shake flasks with respect to trace elements, buffering, added nitrogen, oxygen supply, glucose concentration, alkylbenzene substrate and inducer requirements 2) to use the defined model system to identify the most promising strain of Y. lipolytica TVN348, TVN493 and WT 3) to use the defined model system and selected strain for evaluation of the influence of time of substrate addition and glucose concentration on cell growth and bioconversion of Y. lipolytica under controlled conditions in an instrumented bioreactor Furthermore, since poor reproducibility in cell growth and bioconversion had been prevalent in previous studies, it was also aimed to identify and statistically quantify the reproducibility between duplicate or triplicate samples in each experiment and between sets of different experiments with respect to PAA formation and cell concentrations. Studies were conducted in shake flask cultures to define and quantify the parameters for the model system. The parameters assessed included trace elements, buffering, nitrogen concentration, oxygen supply, glucose concentration, alkylbenzene substrate type and possible inducer requirements. Trace elements, phosphate buffering and added nitrogen did not significantly affect the cell growth of Y. lipolytica TVN348. The cell concentration of Y. lipolytica TVN348 and TVN493 was increased by 65% and 43% respectively for an increase in oxygen supply by decreasing the working volume from 150ml to 50ml, while the cell concentration of Y. lipolytica WT was increased by 41% when oxygen supply was increased by switching from non-baffled to baffled flasks in 50ml cultures. Bioconversion was also increased for an increase in oxygen supply: 2.4mM to 29.0mM PAA (Y. lipolytica TVN348) and 1.2mM to 21.7mM PAA (Y. lipolytica TVN493) for a decrease in working volume; 10.5mM to 46.6mM PAA (Y. lipolytica WT) when switching from non-baffled to baffled flasks. These results indicated that adequate oxygen supply is crucial to both growth and bioconversion, and that further study should be conducted in 50ml working volumes. Cell concentrations obtained in 1.6% (wt/v) and 3.2% (wt/v) glucose cultures (3.95x108cells/ml and 4.03x108cells/ml respectively) indicated that cell growth was neither enhanced nor inhibited by 3.2% (wt/v) glucose. Of the range of substrates examined (propylbenzene, butylbenzene, sec-butylbenzene, hexylbenzene, ethyltoluene and tert-butyltoluene for Y. lipolytica TVN348 and TVN493; octylbenzene and decylbenzene for Y. lipolytica WT), hexylbenzene was regarded as the best substrate for bioconversion (14.7mM and 14.1mM PAA for TVN348 and TVN493 respectively; 42.6mM PAA for WT). Lastly, the absence of a requirement for an additional inducer such as ethanol or oleic acid was confirmed when PAA was formed from hexylbenzene in the culture containing additional glucose (25.0mM). This suggested that when using hexylbenzene as substrate, bioconversion was induced provided sufficient glucose was available for cell maintenance. Results from duplicate or triplicate flasks in each individual shake flask experiment were reproducible and conclusions were based solely on results which showed 95% confidence intervals. However, reproducibility problems were experienced with results between different sets of experiments carried out under the same conditions. The model system was therefore defined by: 1) no addition of trace elements, additional buffering or added nitrogen, 2) cultures grown in 50ml volumes to supply an adequate amount of oxygen crucial for growth and bioconversion, 3) 3.2% (wt/v) glucose and 4) addition of 1% (v/v) hexylbenzene at 24h with no inducer requirements. Use of the model system in shake flask cultures to identify the most promising of the three strains of Y. lipolytica supplied demonstrated that there was no significant difference in cell growth or bioconversion between these strains. Y. lipolytica WT (which has no genetic modifications) was therefore used for further investigation until an appropriate strain could be substituted when it became available. The growth and bioconversion of Y. lipolytica WT was further investigated under controlled conditions in a bioreactor. The influence of time of substrate addition (11h, 24h, 48h) and glucose concentration (3.2% and 6.4% (wt/v)) on growth and bioconversion was examined. When hexylbenzene was added at 48h, cell growth was increased (8.90x108cells/ml) when compared to two of the triplicate cultures with hexylbenzene addition at 24h (4.74x108cells/ml and 3.92x108cells/ml) and the culture with hexylbenzene addition at 11h (2.82x108cells/ml). The third of the triplicate cultures with hexylbenzene addition at 24h, on the other hand, exhibited the strongest growth (2.23x109cells/ml). The poor reproducibility between the triplicate cultures with hexylbenzene addition as 24h made it difficult to determine whether hexylbenzene addition at 24h or 48h maximised cell growth. Furthermore, the cell growth was not significantly improved when the glucose concentration was increased from 3.2% (wt/v) to 6.4% (wt/v) (7.47x108cells/ml for 6.4% glucose culture), however it was also not inhibited. The highest amount of specific PAA formed by Y. lipolytica WT was found when hexylbenzene was added at 11h (7.4x10-11mmol PAA/cell), however the highest accumulated PAA was produced in the culture that exhibited the strongest growth with hexylbenzene addition at 24h (41.4mM). This suggested that the bioconversion of hexylbenzene was maximised when it was added during the active growth phase. It is therefore recommended to conduct fed-batch experiments in future to maintain the active growth phase. Accumulated PAA was increased in 6.4% (wt/v) glucose culture (15.2mM PAA) when compared with two of the 3.2% (wt/v) glucose cultures (5.4mM and 4.3mM PAA). These results indicated that the increased glucose concentration did not inhibit the bioconversion. Furthermore, PAA was formed when 5% (wt/v) residual glucose was observed in the culture, suggesting that the bioconversion of hexylbenzene was not inhibited at glucose concentrations as high as 5.0% (wt/v). If future work were to be conducted in bioreactor culture where glucose is added in fed-batch operation, glucose concentrations in cultures of up to 5% (wt/v) could be considered for initial studies. During bioconversion by Y. lipolytica, the PAA measured after hexylbenzene exhaustion did not, however, correspond to 100% conversion. Further, poor reproducibility was found in the bioreactor cultures. The disappearance of hexylbenzene without a corresponding accumulation of PAA and poor reproducibility was investigated by determining whether PAA was further degraded or alternatively, whether other metabolic intermediates were being formed and accumulated from the hexylbenzene. However, substitution of the hexylbenzene with PAA as substrate confirmed that PAA could not be metabolised. Further, NMR analyses of both the aqueous and organic phases of the culture did not identify any additional metabolic intermediates. It is recommended that additional analyses be conducted on the aqueous and organic phases to further assess the possible accumulation of intermediates. The development of the model system in shake flask cultures demonstrated the importance of adequate oxygen supply for both cell growth and bioconversion. It was also shown that no inducer was needed because hexylbenzene acted as its own substrate inducer. Furthermore, comparison of Y. lipolytica strains TVN348, TVN493 and WT under the defined conditions of the model system revealed that the genetically modified strains (TVN348, TVN493) did not exhibit enhanced bioconversion. Bioreactor cultures using the model system under controlled conditions further showed that bioconversion was not inhibited at a 5% (wt/v) residual glucose concentration and suggested that bioconversion was maximised when hexylbenzene was added during active growth phase. This informs on future work, suggesting fed-batch operation in order to extend the active growth phase, where glucose concentrations in the bioreactor of up to 5% (wt/v) can be considered.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2007. / Adequate provision of oxygen to aerobic bioprocesses is essential for the optimisation of process kinetics. In bioprocesses in which the feedstock is an alkane, the supply of sufficient oxygen is of particular concern because the alkane molecular structure is deficient in oxygen. As a result, the oxygen demand has to be met solely by transfer of oxygen to the culture, necessitating a proportionately higher requirement for oxygen transfer. Maximisation of the rate of oxygen transfer is therefore of key importance in optimising the potential for alkane bioconversion, with respect to both operation and scale up. Nevertheless, the oxygen transfer rate (OTR), and its dependence on the overall volumetric mass transfer coefficient (KLa) in alkane-aqueous dispersions is not yet well understood. In view of the importance of an adequate OTR in the optimisation of alkane bioconversion, this study has focused on the identification and elucidation of the factors which underpin the behaviour of KLa in an alkane-aqueous dispersion. KLa behaviour was quantified in terms of the pressures imposed by turbulence and alkane fluid properties, through their influence on the Sauter mean diameter (D32), gas hold up, gas-liquid interface rigidity and gas-liquid interfacial area per unit volume. These properties were correlated with KLa over a wide range of agitation rates and alkane concentrations in alkane-aqueous dispersions. Experiments were conducted in a 5 litre aerated and agitated bioreactor at agitation rates of 600, 800, 1000 and 1200 rpm and alkane (n-C10-C13 cut) concentrations of 0, 2.5, 5, 10, and 20% (v/v). KLa determination was executed using both the gassing out and pressure step methods. The accuracy and reliability of these methods were compared under the full range of agitation rates and alkane concentrations. The pressure step method was conclusively shown to be superior provided that probe response was taken into account, and was therefore used in the correlations. The interfacial areas corresponding to the KLa values were calculated from the combined effects of D32 and gas hold up. D32 was determined from the measurement of the dispersed air bubble diameters by means of a photographic technique and image analysis. Image analysis was performed by a program that was developed in Matlab® using image acquisition and image processing techniques. This program used these techniques to extract information of the gas bubbles in the image. The gas hold up was determined using the dispersion height technique. The behaviour of KLa was shown to be dependent on both agitation and alkane concentration. Increasing agitation from 600 to 1200 rpm increased KLa for each of the alkane concentrations. The influence of agitation on the interfacial area was evaluated over the same range of agitation rates and the relationship between the corresponding KLa values and interfacial areas assessed. Increasing agitation rate similarly enhanced the interfacial area available for transfer for each of the alkane concentrations, resulting in the concomitant increase in KLa. This increase in interfacial area was related directly to a shear-induced decease in D32 and indirectly to an increased gas holdup as a result of the lower rise velocity of the smaller bubbles. In addition to the agitation, the presence of alkane markedly influenced KLa behaviour, but in different ways, depending on the alkane concentration. Alkane concentration between 2.5 and 5% (v/v) reduced D32 at constant agitation of 800, 1000 and 1200 rpm, a likely consequence of decreased surface tension and retarded coalescence conferred by the alkane. The smaller D32 and the consequential enhanced gas hold up served to amplify KLa through increased interfacial area. However, as alkane concentration was increased above 5% (v/v), the gas hold up decreased despite a continued decrease in D32, resulting in a corresponding decrease in both the interfacial area and KLa. This suggests that at the higher alkane concentrations, the influence of viscosity predominated, exerting multiple negative influences on the interfacial area and oxygen transfer coefficient. The trends were however, not observed at the low agitation of 600 rpm, where turbulence was significantly reduced and KLa was repressed for all alkane concentrations. The pressures imposed by turbulence and alkane properties on the interfacial area defined locales of KLa behaviour and three distinct KLa behavioural trends were identified, depending on the agitation rate and alkane concentration. Regime 1 was constrained between 2.5 and 5% (v/v) for agitation rates of 800 rpm and above. Here KLa enhancement was directly associated with increased interfacial area which was the major factor defining KLa in this regime. Regime 2 was constrained by alkane concentrations higher than 5% (v/v) for agitation rates of 800 rpm and above. In this regime, the KLa depression was observed with increasing alkane concentration suggesting a predominant influence of viscosity which would be likely to exert multiple negative influences on KLa, through both the interfacial area and KL. The interfacial area in this regime decreased mainly due to the negative effect of viscosity on gas holdup. Regime 3, characterised by a decline in KLa irrespective of the alkane concentration, occurred at agitation rates smaller than 800 rpm. It is likely that at low agitation rates, the contribution of turbulence was insufficient to exert a positive influence on the interfacial area In this regime, the interfacial decreased through the combined negative effect of increased D32 and decreased gas holdup. The resultant variation in OTR depended directly on the relative magnitudes of the KLa and oxygen solubility and indirectly on the process conditions which defined these magnitudes. Under conditions of enhanced KLa, OTR benefited from the combined increases in KLa and oxygen solubility. However, under conditions of KLa depression, the elevated oxygen solubility did not invariably outweigh the influence of KLa depression on OTR. Consequently, despite the considerably increased solubility of oxygen in alkane-based bioprocesses a potential decrease in OTR through depressed KLa underlines the critical importance of the quantification of this parameter in alkane-aqueous dispersions and the necessity for a definition of the locales of optimal KLa. Through the identification of the parameters which underpin the behaviour of KLa in alkane-aqueous dispersions and the quantification of the effect of process conditions on these parameters, a fundamental understanding of the KLa and OTR in alkane-aqueous dispersions has been developed. This provides a knowledge base for the prediction of optimal KLa in these systems and has wide application across all alkane-based bioprocesses.

Plastics are highly useful economically because of their resistance to diverse types of environmental and chemical agents and their ability to be molded into many types of products. Globally, plastic production is greater than 20 million metric tons per year. However, their widespread use and often their disposable nature results in significant plastic accumulation in the environment. Plastics are made of hydrocarbons, materials that are biodegradable depending on their molecular structure and size. It is hypothesized that pre-treatment of plastic materials could enhance their bioavailability, facilitating their microbial biodegradation. In this dissertation, a process was developed to treat nylon 6,6 polymers by acid hydrolysis to produce a microbial growth medium. The chemical composition of the medium was determined by low pressure liquid chromatography-spectrophotometry and electrospray ionization mass spectrometry and found that the medium was a mixture of molecules with molecular weight > 800 m/z and with similar chemical characteristics to polyamines. There was steady growth of Pseudomonas putida KT2440 in the medium with concomitant substrate biodegradation. Notably, the yeast Yarowia lipolytica grew well in the medium when supplemented with yeast extract. A similar medium derived from nylon 6,6 containing nylon-derived particles supported the growth of Beijerinckia sp. and Streptomyces sp. BAS1. Confocal laser scanning microscopy and flame ionization gas chromatography were used to identify and quantify the production of polyhydroxybutyrate, a type of “bioplastic”. The aforementioned microorganisms were cultivated in a bench-scale bioreactor that was developed as part of this dissertation. The bioreactor had a novel impeller design resulting in enhanced mixing and rotation and also a modular format allowing for diverse configurations. The bioreactor was notable for its durability and low cost. A detailed description of its design is included in the appendices. In summary, plastic materials can potentially be processed into growth media for microorganisms and can be used for production of value-added products. The media described herein can be used in bioconversion processes using a bioreactor.

Le glutamate (Glu) est le principal neurotransmetteur excitateur du système nerveux central. Ses analogues peuvent se comporter comme des ligands sélectifs et sont intéressants pour élucider le rôle spécifique des différents types de transporteurs ou de récepteurs du système glutamatergique. Ce travail avait pour objectif de synthétiser de nouveaux analogues fonctionnalisés du Glu en utilisant les aminotransférases en tant que biocatalyseurs de façon à convertir les analogues de l'acide 2-oxoglutarique (KG) en analogues du Glu. La plupart des analogues du KG se sont avéré substrats de l'Aspartate Aminotransférase d'E. coli (AspAT) permettant la préparation stéréosélective des analogues du Glu correspondants. Une étude de modélisation moléculaire a été réalisée afin d'expliquer la spécificité de substrat de l'AspAt. Quatre analogues se sont comportés comme des ligands sélectifs et offrent des perspectives pour le développement de nouveaux analogues actifs dans le système glutamatergique

Maximizing enzymatic hydrolysis performance can be achieved through the combination of experimental work and modeling. The present work utilizes an enzymatic hydrolysis model based on reaction kinetics, Langmuir adsorption isotherms, and product inhibition of enzymes (β-glucosidase, cellobiohydrolase, and endoglucanase). The model was developed from a 10% w/w corn stover system. Glucose yield sensitivity to changes in parameter values was assessed and linked to biomass and enzyme characteristics. A commercial enzyme cocktail (CEC) was subsequently characterized by FPLC and gel electrophoresis to identify key enzymes/activities, and the CEC was used in the enzymatic hydrolysis of 20% w/w steam-exploded hardwood. The model was applied to experimental data from the enzymatic hydrolysis of the steam-exploded hardwood, which provided characteristic reaction rate and inhibition parameters consistent with cellulose and xylan hydrolysis. These model-based analyses enhanced understanding of hydrolysis at commercially relevant solids loadings, while identifying pathways to improve enzyme cocktails and enhance biomass conversion.

O presente trabalho se propôs a avaliar o efeito de uma etapa de tratamento alcalino no bagaço de malte visando à extração de acetato e de compostos fenólicos, e como consequência diminuir a concentração destes no hidrolisado hemicelulósico. Foi ainda objetivo deste trabalho, estudar o efeito do tratamento alcalino na bioconversão das frações celulósica e hemicelulósica do bagaço de malte, por meio de fermentação com as leveduras Kluyveromyces marxianus e Pichia stipitis, respectivamente. Os resultados mostraram que para todas as condições de tratamento alcalino avaliadas, ocorreu solubilização de acetato, compostos fenólicos e furanos, não sendo observada solubilização de açúcares. A capacidade de solubilização destes compostos aumentou com o aumento da concentração de hidróxido de sódio empregada até o tempo de 40 min, sendo observada uma concentração máxima de 0,86 g/L, 13,0 g/L e 0,58 g/L empregando 1%%, 2% e 2% de NaOH, respectivamente. Para todas as condições avaliadas, o tratamento alcalino do bagaço de malte reduziu a concentração de ácido acético no hidrolisado hemicelulósico. A menor concentração (0,026 g/L) foi obtida após 20 minutos de tratamento com 2% de NaOH. Com relação à concentração xilose, o menor teor desta pentose (8,3 g/L) foi obtido após o tratamento do bagaço com 0,25 % de NaOH por 2 minutos e o maior (22,0 g/L) com 0,5% de NaOH por 60 minutos. Visando estabelecer as condições de tratamento alcalino que permitissem uma redução na concentração de ácido acético, concomitantemente ao favorecimento da solubilização de xilose no hidrolisado hemicelulósico e de glicose no hidrolisado celulósico, as condições de temperatura e concentração de NaOH do tratamento alcalino do bagaço de malte foram otimizadas através de um planejamento estatístico. Nas condições otimizadas (concentração de NaOH de 0,63%, temperatura de 67,6°C e tempo de 20 min), o tratamento alcalino do bagaço de malte promoveu uma redução de 90% na concentração de ácido acético, concomitante ao aumento de 4,5% e 15% na concentração de xilose no hidrolisado hemicelulósico e concentração de glicose no hidrolisado celulósico, respectivamente. Os resultados referentes à bioconversão da fração celulósica demonstraram que para ambas as configurações de processo avaliadas (SHF e SSF) a fermentação da celulignina do material tratado (CLT) forneceu maior concentração de etanol, e essa diferença foi mais pronunciada na SSF. Além disso, na SSF, a produtividade volumétrica total em etanol, a qual considera o tempo de sacarificação e fermentação no processo foi aumentada em 2,2 vezes em relação à SHF. Com relação a bioconversão da hemicelulose, os resultados mostraram que tanto para o hidrolisado do bagaço referência não tratado (HHR) quanto para o previamente tratado com álcali (HHT), a concentração máxima de etanol foi obtida após 89 horas de fermentação, porém na fermentação do (HHT) a produção de etanol foi cerca de 3 vezes superior a obtida no (HHR), passando de 5,8 para 17,4 g/L. Com base nos resultados obtidos, pode-se concluir que a etapa de tratamento alcalino do bagaço de malte, provavelmente promoveu mudanças estruturais na celulignina aumentando a liberação de glicose no hidrolisado e ainda foi capaz de remover substâncias inibidoras do metabolismo microbiano, especialmente o ácido acético, favorecendo o processo fermentativo da fração hemicelulósica. / This study aimed to evaluate the effect of alkaline treatment in brewer\'s spent grain on extraction of acetate and phenolic compounds, and consequently to decrease their levels on hemicellulosic hydrolyzate. In addition, the effect of the alkali treatment on the bioconversion of cellulose and hemicellulose hydrolyzates employing Kluyveromyces marxianus and Pichia stipitis, respectively, was also evaluated. The results showed that for all conditions of treatment studied occurred solubilization of acetate, phenolics and furans, but not sugars. The solubilizing capacity of these compounds increased with increasing concentration of sodium hydroxide employed until the time of 40 min, and the maximum concentration observed was 0.86 g / L 13.0 g / L and 0.58 g / L from 1%, 2% NaOH and 2%, respectively. For all conditions evaluated, alkali treatment of brewer\'s spent grain reduced the acetic acid concentration in hemicellulosic hydrolyzate. The lowest concentration (0.026 g / L) was obtained after 20 minutes with 2% NaOH. With respect to xylose concentration, the lower content of this pentose (8.3 g / L) was obtained after the treatment with 0.25% NaOH for 2 minutes and the highest (22.0 g / L) with 0.5 % NaOH for 60 minutes. To establish optimal conditions of temperature and NaOH concentration in the alkaline treatment aiming to decrease the concentration of acetic acid and, at the same time, increase the solubilization of xylose in the hemicellulose hydrolyzate and glucose in the cellulosic hydrolyzed, a statistical design was used. Under optimized conditions (NaOH concentration of 0.63%, a temperature of 67.6 °C and time of 20 min) alkaline treatment of the brewer\'s spent grain promoted a decrease of 90% in acetic acid concentration, concomitant to an increase of 4.5% and 15% on xylose concentration in the hemicellulose hydrolyzate and on glucose concentration in the cellulosic hydrolyzate, respectively. The results concerning the bioconversion of cellulosic fraction showed that for both configurations of the process evaluated (SHF and SSF) the treated material cellulignin (CLT) provided higher ethanol concentration, and this difference was more pronounced in SSF. In addition, in SSF, the total ethanol volumetric productivity, which considers time saccharification and fermentation process was increased by 2.2 times compared to SHF. In relation to the hemicellulose bioconversion, the results showed that for both hydrolyzate employed, i.e untreated (HHR) and previously treated with alkali (HHT), the maximum ethanol concentration was achieved after 89 hours of fermentation, however the ethanol production in HHT was about 3 times higher obtained in HHR (from 5.8 to 17.4 g/L). Based on these results, we conclude that the alkali treatment, probably induced structural changes in the fraction of cellulignin regarding glucose released in the hydrolyzate and still was able to remove substances inhibitory to microbial metabolism, especially acetic acid, favoring the fermentation of hemicellulose fraction.

O presente trabalho se propôs a avaliar o efeito de uma etapa de tratamento alcalino no bagaço de malte visando à extração de acetato e de compostos fenólicos, e como consequência diminuir a concentração destes no hidrolisado hemicelulósico. Foi ainda objetivo deste trabalho, estudar o efeito do tratamento alcalino na bioconversão das frações celulósica e hemicelulósica do bagaço de malte, por meio de fermentação com as leveduras Kluyveromyces marxianus e Pichia stipitis, respectivamente. Os resultados mostraram que para todas as condições de tratamento alcalino avaliadas, ocorreu solubilização de acetato, compostos fenólicos e furanos, não sendo observada solubilização de açúcares. A capacidade de solubilização destes compostos aumentou com o aumento da concentração de hidróxido de sódio empregada até o tempo de 40 min, sendo observada uma concentração máxima de 0,86 g/L, 13,0 g/L e 0,58 g/L empregando 1%%, 2% e 2% de NaOH, respectivamente. Para todas as condições avaliadas, o tratamento alcalino do bagaço de malte reduziu a concentração de ácido acético no hidrolisado hemicelulósico. A menor concentração (0,026 g/L) foi obtida após 20 minutos de tratamento com 2% de NaOH. Com relação à concentração xilose, o menor teor desta pentose (8,3 g/L) foi obtido após o tratamento do bagaço com 0,25 % de NaOH por 2 minutos e o maior (22,0 g/L) com 0,5% de NaOH por 60 minutos. Visando estabelecer as condições de tratamento alcalino que permitissem uma redução na concentração de ácido acético, concomitantemente ao favorecimento da solubilização de xilose no hidrolisado hemicelulósico e de glicose no hidrolisado celulósico, as condições de temperatura e concentração de NaOH do tratamento alcalino do bagaço de malte foram otimizadas através de um planejamento estatístico. Nas condições otimizadas (concentração de NaOH de 0,63%, temperatura de 67,6°C e tempo de 20 min), o tratamento alcalino do bagaço de malte promoveu uma redução de 90% na concentração de ácido acético, concomitante ao aumento de 4,5% e 15% na concentração de xilose no hidrolisado hemicelulósico e concentração de glicose no hidrolisado celulósico, respectivamente. Os resultados referentes à bioconversão da fração celulósica demonstraram que para ambas as configurações de processo avaliadas (SHF e SSF) a fermentação da celulignina do material tratado (CLT) forneceu maior concentração de etanol, e essa diferença foi mais pronunciada na SSF. Além disso, na SSF, a produtividade volumétrica total em etanol, a qual considera o tempo de sacarificação e fermentação no processo foi aumentada em 2,2 vezes em relação à SHF. Com relação a bioconversão da hemicelulose, os resultados mostraram que tanto para o hidrolisado do bagaço referência não tratado (HHR) quanto para o previamente tratado com álcali (HHT), a concentração máxima de etanol foi obtida após 89 horas de fermentação, porém na fermentação do (HHT) a produção de etanol foi cerca de 3 vezes superior a obtida no (HHR), passando de 5,8 para 17,4 g/L. Com base nos resultados obtidos, pode-se concluir que a etapa de tratamento alcalino do bagaço de malte, provavelmente promoveu mudanças estruturais na celulignina aumentando a liberação de glicose no hidrolisado e ainda foi capaz de remover substâncias inibidoras do metabolismo microbiano, especialmente o ácido acético, favorecendo o processo fermentativo da fração hemicelulósica. / This study aimed to evaluate the effect of alkaline treatment in brewer\'s spent grain on extraction of acetate and phenolic compounds, and consequently to decrease their levels on hemicellulosic hydrolyzate. In addition, the effect of the alkali treatment on the bioconversion of cellulose and hemicellulose hydrolyzates employing Kluyveromyces marxianus and Pichia stipitis, respectively, was also evaluated. The results showed that for all conditions of treatment studied occurred solubilization of acetate, phenolics and furans, but not sugars. The solubilizing capacity of these compounds increased with increasing concentration of sodium hydroxide employed until the time of 40 min, and the maximum concentration observed was 0.86 g / L 13.0 g / L and 0.58 g / L from 1%, 2% NaOH and 2%, respectively. For all conditions evaluated, alkali treatment of brewer\'s spent grain reduced the acetic acid concentration in hemicellulosic hydrolyzate. The lowest concentration (0.026 g / L) was obtained after 20 minutes with 2% NaOH. With respect to xylose concentration, the lower content of this pentose (8.3 g / L) was obtained after the treatment with 0.25% NaOH for 2 minutes and the highest (22.0 g / L) with 0.5 % NaOH for 60 minutes. To establish optimal conditions of temperature and NaOH concentration in the alkaline treatment aiming to decrease the concentration of acetic acid and, at the same time, increase the solubilization of xylose in the hemicellulose hydrolyzate and glucose in the cellulosic hydrolyzed, a statistical design was used. Under optimized conditions (NaOH concentration of 0.63%, a temperature of 67.6 °C and time of 20 min) alkaline treatment of the brewer\'s spent grain promoted a decrease of 90% in acetic acid concentration, concomitant to an increase of 4.5% and 15% on xylose concentration in the hemicellulose hydrolyzate and on glucose concentration in the cellulosic hydrolyzate, respectively. The results concerning the bioconversion of cellulosic fraction showed that for both configurations of the process evaluated (SHF and SSF) the treated material cellulignin (CLT) provided higher ethanol concentration, and this difference was more pronounced in SSF. In addition, in SSF, the total ethanol volumetric productivity, which considers time saccharification and fermentation process was increased by 2.2 times compared to SHF. In relation to the hemicellulose bioconversion, the results showed that for both hydrolyzate employed, i.e untreated (HHR) and previously treated with alkali (HHT), the maximum ethanol concentration was achieved after 89 hours of fermentation, however the ethanol production in HHT was about 3 times higher obtained in HHR (from 5.8 to 17.4 g/L). Based on these results, we conclude that the alkali treatment, probably induced structural changes in the fraction of cellulignin regarding glucose released in the hydrolyzate and still was able to remove substances inhibitory to microbial metabolism, especially acetic acid, favoring the fermentation of hemicellulose fraction.