In recent years, several industrial yeasts, owing to their robust growth and certain other characteristics, have been developed as recombinant host systems for commercial production of heterologous proteins. One such yeast Pichia pastoris has proven to be an excellent host for production of secreted and intracellular proteins (Cereghino and Cregg. 2000). The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of Pichia pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology;(2) the ability of Pichia pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; and (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing. The expression level for a given recombinant protein produced by Pichia pastoris seems to be determined largely by its inherent properties such as amino acid sequence, the tertiary structure and the site for expression (Sreekrishna et al.,1997). The attempts on increasing the protein expression levels by far are focused on genetic manipulations to enhance the gene expression and protein stability. Although this is crucial, there is ample scope to improve the productivity of Pichia pastoris fermentations by undertaking a systematic program of optimizing the entire fermentation process. This work aims at undertaking such a program by focusing on strategy to identify and to characterize trends in the behavior of the system. It can be expected that by addressing the process as a whole, rather than narrowly focusing on the protein expression alone, the methodology proposed here can simplify process scale-up and can be applied to several products made by the same host. Pichia pastoris is methylotrophic yeast. In the Pichia pastoris fermentation, the limiting carbon source is glycerol, method or mixture of both. It can grow on methanol as a sole carbon and energy source. It possesses a highly inducible methanol utilization pathway. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by alcohol oxidase (AOX). AOX, the first enzyme of the pathway, accounts for up to 35% of the total protein in cells grown on limited amounts of methanol. The enzymes undetectable in cells grown on glucose, ethanol or glycerol. There are two genes in Pichia pastoris that code for AOX: AOXI. The AOXI gene product accounts for the majority of alcohol oxidase activity in the cell. This highly inducible and stringently regulated AOXI promoter has been used to construct expression vectors for the production of heterologous proteins in Pichia pastoris. Although some foreign proteins have expressed well in shake-flask cultures, expression levels are typically low compared to fomenter cultures. There are several key aspects of Pichia pastoris fermentations: 1. Fed-batch operation – Controlled addition of glycerol, methanol or mixture thereof. In general, strains are grown initially in a defined medium containing glycerol as its carbon source (growth phase). During this phase, biomass accumulates but heterogonous gene expression is fully repressed. Upon depletion of glycerol, a transition phase is initiated in which additional glycerol is fed to the culture at a growth-limiting rate. Finally, method a mixture of glycerol and methanol is fed to the culture to induce expression (induction phase). The duration of individual substrate feeds, the amount and mode of feeding are critical to optimal fermentation performance. 2.Online measurement and control-One of the most important key parameters in Pichia pastor is expression system is the methanol concentration. Monitoring and controlling this variable are important because high levels of this inductor substrate can be toxic to the cells and low levels of methanol may not be enough to initiate the AOX transcription (Cereghino and Cregg, 2000) This research work aims at investigation the above mentioned aspects by conduction an in depth engineering analysis of the Pichia Pastoris fermentations.

<p>The increased concentration of greenhouse gases in theatmosphere, in particular CO₂, is changing the Earth’s climate. Accordingto the Kyoto protocol, where the international community agreedon binding emission targets, developed countries are committedto reduce their greenhouse gas emissions. The increased use ofbiomass in energy systems is an important strategy to reduce CO₂emissions. The purpose of this thesis has been toanalyse the opportunities for Sweden to further reduce CO₂emissions in the energy system, by rationallyutilising woody biomass energy. The characteristics of currentcommercially operating biofuel-based CHP plants in Sweden aresurveyed and systematically presented. A consistent andtransparent comprehensive reference base for system comparisonsis given. Furthermore, the fuel effectiveness and contributionto CO₂reduction is calculated. The governmentalsubsidies of the CHP plants’investment, expressed as costof specific CO₂reduction, appears to be low.</p><p>The competitiveness of biomass-fuelled energy production inrelation to fossil-based production with carbon capture isanalysed, showing that the biomass-fuelled systems provide acompetitive option, in terms of cost of electricity andefficiencies. The remaining Swedish woody biofuel potential ofat least 100 PJ/yr is principally available in regions with abiomass surplus. Transportation is therefore required to enableits utilisation in a further national and international market.Refining the biofuel feedstock to pellets, or even furtherrefining to motor fuels (DME, methanol or ethanol) or power,could facilitate this transport. Different options for fuelrefining are studied and compared. The entire fuel chain, fromfuel feedstock to end users, is considered and CO₂emissions are quantified. Substituting fuelpellets for coal appears to be the most costeffectivealternative and shows the largest CO₂reduction per energy unit biofuel. Motor fuelsappear more costly and give about half the CO₂reduction. Transportation of the upgraded biofuelpellets is highly feasible from CO₂emissions point of view and does not constitute ahindrance for further utilisation, i.e. the pellets can betransported over long distances efficiently with only limitedemissions of CO₂.</p><p>Bioenergy utilisation has additional features forenvironmental improvement, apart from the CO₂aspect. Waste heat from biofuel-based CHP can becost-effectively used in conjunction with sewage treatment. Theincoming sewage water to the nitrification process can bepreheated with the waste heat, and thereby substantiallyenhance the nitrification and the reduction of ammoniumnitrogen during the winter season.</p><p><b>Keywords:</b>CO₂reduction, energy system, biofuel, CHP, refining,fuel pellets, ethanol, methanol, DME, fuel substitution, sewagewater, nitrification.</p>

In der vorliegenden Arbeit wurde die heterogen-katalysierte, partielle Gasphasenhydrierung von Benzol zu Cyclohexen untersucht. Dazu wurden Träger- und Schalenkatalysatoren für Schüttgutreaktoren sowie Mikrostruktur-Reaktoren mit katalytisch aktivierten Wafern hergestellt und eingesetzt. Die Bildung des thermodynamisch und kinetisch nicht begünstigten Zielproduktes Cyclohexen konnte durch den Reaktionsmodifikator Methanol bewirkt werden. Dieser war in der Lage, die Verhältnisse der Geschwindigkeiten der Teilreaktionen sowie der Sorptionsprozesse an der Katalysatoroberfläche so zu beeinflussen, daß Cyclohexen im Abgasstrom der Reaktion nachgewiesen werden konnte. Dabei gelang es außerdem, die Reaktion unter quasi-stationären Bedingungen an unterschiedlichen Katalysatoren reproduzierbar durchzuführen und mit Hilfe eines einfachen Verfahrens die Katalysatoren zu reaktivieren. Der Einfluß des Trägermaterials von Ruthenium-Katalysatoren auf Umsatzgrad, Selektivität sowie das Desaktivierungsverhalten während der Gasphasenhydrierung wurde ermittelt. Darüber hinaus wurde eine Reihe von unpromotierten und promotierten Ru/Al2O3-Trägerkatalysatoren mit Hilfe des Sol-Gel-Verfahrens präpariert und in der Gasphasenhydrierung von Benzol in Gegenwart des Reaktionsmodifikators Methanol untersucht. Dabei konnte gezeigt werden, daß die zusätzliche Promotierung mit Zinkoxid zu einer weiteren Verbesserung der Cyclohexen-Bildung führte. Die Mikrostruktur-Reaktoren für die partielle Gasphasenhydrierung von Benzol wurden zum einen aus mikrostrukturierten Aluminium-Wafern und zum anderen aus mikrostrukturierten Edelstahl-Wafern gebildet. Beide Wafertypen wurden mit unterschiedlichen Methoden katalytisch aktiviert. Die Aluminium-Wafer wurden durch anodische Oxidation und anschließende Imprägnierung aktiviert, für die Aktivierung der Edelstahl-Wafer wurde ein Sol-Gel-Tauchbeschichtungs- Verfahren mit Imprägnierung angewendet. Bei der Untersuchung der partiellen Gasphasenhydrierung von Benzol an den Mikrostruktur-Reaktoren mit den jeweiligen Waferkatalysatoren zeigte sich, daß die Mikrostruktur-Reaktoren mit Aluminium-Waferkatalysatoren bezüglich der Bildung von Cyclohexen besser geeignet waren, als die Mikrostruktur-Reaktoren mit Edelstahl-Waferkatalysatoren. Es wurde gezeigt, daß ein Mikrostruktur-Reaktor in der partiellen Gasphasenhydrierung von Benzol das gleiche Selektivitäts-Umsatz-Verhalten aufweist, wie ein Schüttgutreaktor mit vergleichbarem Schalenkatalysator.

Soil contamination with petroleum hydrocarbons is a common problem. Toxic compounds such as BTEXs are present in gasoline derivatives. They can move through the soil and contaminate the groundwater, especially if methanol is present. This problem is critical in permeable soil. Although leaching of BTEX compounds from soil to the water is almost temperature dependent, movement of methanol through the soil is not. Methanol can move through the porous soil and reach the groundwater in a short time. It can also dissolve and carry BTEX compounds through the porous soil. Therefore, fast cleanup of the permeable soil which is contaminated with BTEX and methanol is crucial. Chlorine dioxide is an oxidizer, which is easy to use and safe to transport; and may be considered as a treatment technique for soil cleanup. Keywords: Groundwater, soil contamination, BTEX compounds, methanol, chlorine dioxide, soil cleanup

Le milieu interstellaire (MIS) où se forment les étoiles est constitué de gaz très dilué dominé par l'hydrogène moléculaire, et de grains de poussière de taille submicrométrique. Ces poussières jouent un rôle crucial en atténuant la lumière des étoiles lointaines, protégeant ainsi les molécules du gaz des rayonnements ultra-violets, et en servant de catalyseurs à une chimie hétérogène à très basse température. Outre la synthèse de l'hydrogène moléculaire, la surface des grains permet de former des molécules organiques dites complexes comme le méthanol (CH3OH) à partir de l'hydrogénation (et la deutération) du monoxyde de carbone (CO). Les glaces ainsi formées participent à la complexification moléculaire du MIS et seront à terme intégrées au sein de disques de poussières, berceaux des astéroïdes, comètes et exo-planètes. L'objectif de cette thèse est l'étude des mécanismes d'échanges hydrogène-deuterium sur certains groupements fonctionnels de molécules organiques simples, méthanol par exemple, présentes à la surface ou dans les manteaux des grains interstellaires. La thèse est centrée sur une exploration expérimentale de ces processus en phase condensée, à l'aide d'une expérience de cryogénie synthétisant des glaces à très basse température (15K) couplée à un spectromètre infrarouge. Nous montrons que ces échanges se produisent avant la sublimation du manteau de glace sur des groupes fonctionnels capables d'établir des liaisons hydrogènes avec les molécules d'eau voisines. Le processus catalysant est vraisemblablement la cristallisation de la glace d'eau. Des études cinétiques nous permettent d'évaluer les énergies d'activation du transfert H/D (6745K) et de la transition amorphe-cristalline (8100K), et de déterminer la constante de vitesse d'échange dans le domaine de température 120-140~K. Cette constante de vitesse est, de plus, comparée à des calculs semi-classiques basés sur un traitement ab initio. En marge de ces expériences, des observations millimétriques de la molécule de méthanol en direction de proto-étoiles confirment une variabilité des abondances relatives des isotopologues simplement deutérés de cette molécule en fonction de la masse de la protoétoile.

In the production of biodiesel via the transesterification of vegetable oils, purification to international standards is challenging. A key measure of biodiesel quality is the level of free glycerol in the biodiesel. In order to remove glycerol from fatty acid methyl ester (FAME or biodiesel), a membrane separation setup was tested. The main objective of this thesis was to develop a membrane process for the separation of free glycerol dispersed in FAME after completion of the transesterification reaction and to investigate the effect of different factors on glycerol removal. These factors included membrane pore size, pressure, temperature, and methanol, soap and water content. First, a study of the effect of different materials present in the transesterification reaction, such as water, soap, and methanol, on the final free glycerol separation was performed using a modified polyacrylonitrile (PAN) membrane, with 100 kD (ultrafiltration) molecular weight cut off for all runs at 25°C. Results showed low concentrations of water had a considerable effect in removing glycerol from the FAME. The mechanism of separation of free glycerol from FAME was due to the removal of an ultrafine dispersed glycerol-rich phase present in the untreated (or raw) FAME. The size of the droplets and the free glycerol separation both increased with increasing water content of the FAME. Next, three types of polymeric membranes in the ultrafiltration range with different molecular weight cut off, were tested at three fixed operating pressures and three operating temperatures (0, 5 and 25oC) to remove the free glycerol from a biodiesel reactor effluent. The ASTM standard for free glycerol concentration was met for the experiments performed at 25°C. The results of this study indicate that glycerol could be separated from raw FAME to meet ASTM and EN standards at methanol feed concentrations of up to 3 mass%. The process was demonstrated to rely on the formation of a dynamic polar layer on the membrane surface. Ceramic membranes of different pore sizes (0.05 µm (ultrafiltration (UF) range) and 0.2 µm (microfiltration (MF) range)) were used to treat raw FAME directly using the membrane separation set up at temperatures of 0, 5 and 25°C. The results were encouraging for the 0.05 µm pore size membrane at the highest temperature (25°C). The effect of temperature on glycerol removal was evident from its relation with the concentration factor (CF). Higher temperatures promoted the achievement of the appropriate CF value sooner for faster separation. Membrane pore size was also found to affect separation performance. A subsequent study revealed the effect of different variables on the size of the glycerol droplets using dynamic light scattering (DLS). A key parameter in the use of membrane separation technology is the size of the glycerol droplets and the influence of other components such as water, methanol and soaps on that droplet size. The effect of water, methanol, soap and glycerol on the size of suspended glycerol droplets in FAME was studied using a 3-level Box-Behnken experimental design technique. Standard statistical analysis techniques revealed the significant effect of water and glycerol on increasing droplet size while methanol and soap served to reduce the droplet size. Finally, a study on the effect of trans-membrane pressure (TMP) at different water concentrations in the FAME phase on glycerol removal using UF (0.03 µm pore size, polyethersulfone (PES)) and MF (0.1 and 0.22 µm pore sizes, PES) membranes at 25, 40 and 60°C was performed. Results showed that running at 25°C for the two membrane types produced the best results for glycerol removal and exceeded the ASTM and EN standards. An enhancement of glycerol removal was found by adding small amounts of water up to the maximum solubility limit in biodiesel. An increase in temperature resulted in an increase in the solubility of water in the FAME and less effective glycerol removal. Application of cake filtration theory and a gel layer model showed that the gel layer on the membrane surface is not compressible and the specific cake resistance and gel layer concentration decrease with increasing temperature. An approximate value for the limiting (steady-state) flux was reported and it was found that the highest fluxes were obtained at the lowest initial water concentrations at fixed temperatures. In conclusion, dispersed glycerol can be successfully removed from raw FAME (untreated FAME) using a membrane separation system to meet the ASTM biodiesel fuel standards. The addition of water close to the solubility limit to the FAME mixture enables the formation of larger glycerol droplets and makes the separation of these droplets straightforward.

No description available.

No description available.

No description available.

Made available in DSpace on 2014-06-11T19:23:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-30Bitstream added on 2014-06-13T19:09:17Z : No. of bitstreams: 1 perez_l_me_bauru.pdf: 2493638 bytes, checksum: 26329bfa30a60d575a769d58ce9d9493 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A oxidação do metanol apresenta problemas acentuadamente complexos que ainda não foram satisfatoriamente solucionados. Possui um mecanismo duplo caminho, ou seja, diretamente a CO2 ou pelo caminho alternativo via intermediários. Também como intermediário/produto da reação ocorre a formação de CO que é usualmente identificado como o agente bloqueador da superfície eletródica devido à sua adsorção irreversível. Baseado em um estudo recente da reação de oxidação do metanol sobre Pt por espectroscopia de impedância eletroquímica (EIE), este trabalho teve por objetivo aplicar a técnica de EIE no estudo da eletrocatálise da reação de oxidação do metanol sobre fases intermetálicas ordenadas de PtMn, PtSb e PtSn em meio ácido, visto que os metais Mn, Sb e Sn por apresentarem característica oxifílica, podem formar mais facilmente espécies OH que promovem a oxidação de intermediários fortemente adsorvidos nos sítios ativos da superfície eletródica. Os intermetálicos também apresentam maior distância entre os sítios da platina o que pode favorecer uma configuração de adsorção vertical da molécula de CO que é mais fácil de oxidar quando comparado a uma configuração em ponte. Uma análise prévia da atividade catalítica desses materiais para a reação de oxidação do metanol foi realizada empregando-se as técnicas de voltametria cíclica cronoamperometria. Os resultados obtidos mostraram que o processo de oxidação do metanal utilizando os intermetálicos apresentou um deslocamento para valores menos positivos do potencial de início de oxidação, necessitando de uma menor demanda energética para que o processo de oxidação ocorra sobre a superfície destes intermetálicos. A oxidação do metanol apresentou densidade de corrente de corrente de pico superior para esses materiais quando comparado... / Methanol oxidation reaction congregates very complex constraints that were not conveniently solved so far. This reaction usually follows a dual pathway, i.e. direct oxidation to CO2 or through an alternative path via stable intermediates. CO is the most commom identified intermediate of the reaction and surface blocking agent due to its irreversible adsorption characteristic. The here in research is based on recent study performed with the methanol oxidation reaction on Platinum by employing the Electrochemical Impedance Spectroscopy (EIS) technique. The aim of the research was to investigate the methanol oxidation reaction on PtMn, PtSb and PtSn ordered intermetallic surfaces, in acid medium, by means of the EIS technique. These materials were selected to the study since they have oxophilic metals (Mn, Sb and Sn) that could provide OH species on the electrode surface at electrode potentials less positive than polycrystalline Platinum under the same experimental conditions. Moreover, these surfaces also exhibits a larger Pt-Pt distance in comparison to polycrystalline Platinum that inhibits the stable bridge configuration adsorption of CO. The electrochemical data obtained have demonstrated that ehe methanol oxidation reaction on the studied surfaces presented a less positive onset potential as compared to Pt. The materials also have exhibited a higher maxima current densities and lower susceptibility for CO blocking than Pt. EIS spectra obtained fot the reaction taking place on Pt, PtSb and PtSn have presented an inductive component that is characteristic of stable intermediate adsorption process. Steady state measurements have pointed to a change in the mechanism of the reaction probably due to the action of surface oxygenated species. Furthemore, the EIS technique has been proved o be a powerful tool to investigate... (Complete abstract click electronic access below)