Die Nutzung von Kraftstoffen auf der Basis von Biodiesel erlaubt es, konventionelle, etablierte Antriebstechniken mit Nachhaltigkeit zu kombinieren. Daher kann in den nächsten Jahren mit einer weiteren weltweiten Steigerung der Biodieselproduktion gerechnet werden. Damit verbunden ist jedoch auch ein Anstieg der anfallenden Menge an Glycerol, das bei der Herstellung von Biodiesel als Koppelprodukt entsteht. Da traditionelle Märkte für Glycerol kaum mehr Wachstum zeigen, müssen neue Wege zur effizienten Verwertung von Glycerol gefunden werden. Die vorliegende Arbeit beschäftigt sich mit der heterogen-katalysierten Umsetzung von Glycerol zu Acrolein. Unter Verwendung von ZSM-5-Katalysatorproben mit unterschiedlichem Si/Al-Verhältnis und phosphorsäure-modifizierten Katalysatorproben konnte die Existenz von Synergieeffekten zwischen den texturellen, strukturellen und aciden Eigenschaften in der heterogen-katalysierten Glyceroldehydratisierung zu Gunsten der Acroleinselektivität eindeutig nachgewiesen werden. Eine selektive Umsetzung von Glycerol zu Acrolein wird daher an solchen Katalysatorproben ermöglicht, die ausgewogene texturelle, strukturelle und acide Eigenschaften aufweisen.

[ES] La presente tesis doctoral aborda el desarrollo de nuevos procesos catalíticos centrados en la valorización del glicerol, subproducto principal en la síntesis de biodiesel. El objetivo principal del trabajo consiste en utilizarlo como fuente de carbono para la producción de heterociclos nitrogenados de interés industrial, en concreto, para la producción de 2-metilpiperazina y 2-metilpirazina. Debido a la baja reactividad del glicerol y las drásticas condiciones de reacción que serían necesarias para llevar a cabo las transformaciones a estos heterociclos, se ha planteado como paso previo el estudio de la optimización y el entendimiento del proceso de deshidratación selectiva de glicerol a hidroxiacetona (o acetol). A través de la obtención de este compuesto intermedio, se han podido desarrollar procesos de producción de los heterociclos nitrogenados eficientes y selectivos, en condiciones de reacción moderadas. A este respecto, los precursores de hidrotalcitas del tipo Cu-Mg-Al dan lugar a una familia de materiales basados en óxidos mixtos Cu-Mg-Al capaces de llevar a cabo la deshidratación selectiva de glicerol a acetol en continuo con rendimientos del ¿40%. Además, estos catalizadores son estables durante más de 8 horas, mostrando también excelente capacidad de regeneración y reusabilidad. Del mismo modo, la combinación de centros ácido-base y redox exhibida por estos materiales ha permitido, a través de la combinación de estudios catalíticos y de caracterización, avanzar en el estado del arte en lo que respecta a la comprensión de esta reacción de deshidratación catalítica de glicerol. De esta forma, se ha podido comprobar el papel fundamental de las especies de Cu y, en concreto de las especies Cu(I) presentes en los catalizadores, en la generación de gliceraldehido como intermedio clave para la producción de acetol. Del mismo modo, los centros ácidos del catalizador facilitan la primera adsorción del glicerol, acelerando así la reacción. No obstante, la necesidad de alcanzar productividades de acetol más elevadas para asegurar el éxito de la estrategia global motivó el desarrollo y estudio, en este proceso de deshidratación selectiva de glicerol en continuo, de una segunda familia de catalizadores basados en óxido de cobre soportado sobre diferentes óxidos metálicos (SiO2, Al2O3 y ZrO2), combinando centros ácidos de Lewis y una alta exposición del Cu. La adecuada selección y optimización de estos materiales lleva a lograr, con varios de ellos, rendimientos del 60% a acetol con concentraciones de glicerol en la alimentación mucho más elevadas. Una vez establecidos varios sistemas catalíticos para la producción de acetol, se abordó la producción de los heterociclos nitrogenados de interés a partir de la combinación de esta molécula con etilendiamina. En concreto, el catalizador Pd/TiO2-Al2O3 tiene alta actividad específica hacia la formación de 2-metilpiperazina (80% de rendimiento), gracias a su elevada exposición de centros de Pd insaturados, crítica para activar el doble enlace C=N y así proceder a la hidrogenación de las iminas intermedias. En segundo lugar, el catalizador CuO/Al2O3-npw, muestra rendimientos del 50% a la 2-metilpirazina, siendo posible alcanzar valores cercanos al 60% cuando se usa el método de precipitación-deposición por micelas en fase reversa para incorporar las nanopartículas de CuO. Finalmente, se llevó a cabo una prueba de concepto para la síntesis de 2-metilpirazina a partir de glicerol en un reactor multi-lecho especialmente diseñado para realizar las dos etapas del proceso en "one-pot" con el mismo catalizador basado en CuO-soportado, obteniéndose resultados prometedores. En definitiva, se han establecido dos nuevas rutas catalíticas para la producción de heterociclos nitrogenados con glicerol como la principal fuente de carbono a través de su derivado acetol, abriéndose así nuevas perspectivas en el campo de la valorización sostenible de moléculas derivadas de biomasa. / [CA] La present tesi doctoral aborda el desenvolupament de nous processos catalítics centrats en la valorització del glicerol, subproducte principal en la síntesi de biodièsel, utilitzant-lo com a font de carboni per a la producció d'heterocicles nitrogenats d'interès industrial. En concret, per a la producció de 2-metilpiperazina i 2-metilpirazina. A causa de la baixa reactivitat del glicerol i les dràstiques condicions de reacció que serien necessàries per a dur a terme les transformacions a aquests heterocicles, s'ha plantejat com a pas previ un estudi detallat escometent l'optimització i l'enteniment del procés de deshidratació selectiva de glicerol a hidroxiacetona (o acetol). A través de l'obtenció d'aquest compost intermedi, s'han desenvolupat processos de producció dels heterocicles nitrogenats eficients i selectius, en condicions de reacció moderades. Referent a això, els precursors hidrotalcítics Cu-Mg-Al donen com a resultat una família de materials basats en òxids mixtos Cu-Mg-Al capaços de dur a terme la deshidratació selectiva de glicerol a acetol en continu amb rendiments del 40%. Així mateix, aquests catalitzadors són estables durant més de 8 hores, mostrant a més una excel·lent regenerabilitat i reusabilitat. De la mateixa manera, la combinació de centres àcid-base i redox exhibida per aquests materials ha permès, a través de la combinació d'estudis catalítics i de caracterització, avançar significativament en l'estat de l'art pel que fa a la comprensió d'aquesta reacció catalítica. D'aquesta manera, s'ha pogut comprovar el paper fonamental del Cu i, en concret del Cu(I), en la generació de gliceraldehid com a intermedi de reacció clau. Per altra banda, els centres àcids del catalitzador faciliten la primera adsorció del reactiu, accelerant així la reacció. No obstant això, la necessitat d'aconseguir productivitats de acetol més elevades per a assegurar l'èxit de l'estratègia global va motivar l'ús, en aquest procés de deshidratació selectiva de glicerol en continu, d'una segona família de catalitzadors basats en òxid de coure suportat sobre diferents òxids inorgànics d'alta àrea (SiO2, Al2O3 i ZrO2), combinant centres àcids de Lewis i una alta exposició del Cu. L'adequada selecció i optimització d'aquests materials aconsegueix, amb alguns d'ells, rendiments del 60% a acetol amb concentracions de glicerol en l'alimentació molt més elevades. Una vegada establits diversos sistemes catalítics per a la producció d'acetol, es va abordar la producció dels heterocicles nitrogenats d'interès a partir de la combinació d'aquesta molècula amb etilendiamina. En concret, el catalitzador Pd/TiO2-Al2O3 té una alta activitat específica cap a la formació de 2-metilpiperazina (80% de rendiment), gràcies a la seua elevada exposició de centres de Pd insaturats, crítica per a activar el doble enllaç C=N i així procedir a la hidrogenació de les imines intermèdies. En segon lloc, s'ha pogut comprovar que, en presència d'un catalitzador principalment àcid contenint Cu, és possible realitzar la dehidrociclació d'acetol amb etilendiamina per a obtindre 2-metilpirazina i altres alquilpirazinas. Concretament, el catalitzador CuO/Al2O3-npw, mostra rendiments del 50% a la 2-metilpirazina, sent possible aconseguir valors pròxims al 60% quan s'utilitza el mètode de precipitació-deposició per micel·les en fase revessa per a incorporar les nanopartícules de CuO. Finalment, es va dur a terme una prova de concepte per a la síntesi de 2-metilpirazina a partir de glicerol en un reactor multi-llit especialment dissenyat per realitzar les dues etapes del procés en "one-pot" amb el mateix catalitzador basat en CuO-suportat, amb resultats prometedors. D'aquesta manera, s'han establit dues noves rutes catalítiques per a la producció d'heterocicles nitrogenats amb glicerol com a la principal font de carboni, a través del seu derivat acetol obrint així noves perspectives en l'àmbit de la valorització sostenible de les molècules derivades de la biomassa. / [EN] This doctoral thesis addresses the development of new catalytic processes centered on glycerol valorization, which is the main by-product of biodiesel synthesis. In this sense, the main aim focused on using it as a carbon source to generate nitrogen heterocycles of industrial interest, specifically, to produce 2-methylpiperazine and 2-methylpyrazine. Due to the low reactivity of glycerol and the severe reaction conditions necessary to carry out the transformations towards these N-heterocycles, previous detailed research to optimize and understand the selective dehydration process of glycerol to hydroxyacetone (or acetol) was undertaken. Through obtaining this intermediate compound, it has been possible to develop efficient and selective nitrogen heterocycles production processes, under moderate reaction conditions. In this regard, Cu-Mg-Al hydrotalcite precursors give rise to a family of materials based on Cu-Mg-Al mixed oxides capable of carrying out the selective dehydration of glycerol to acetol continuously with yields of 40%. In addition, these catalysts are stable for more than 8 hours under operational conditions, showing excellent regeneration capacity and reusability. In the same way, through the combination of catalytic and characterization studies, the interesting mix of acid-base and redox centers exhibited by these materials has allowed for advancing significantly in the state of the art regarding understanding this glycerol catalytic dehydration reaction. Hence, it has been possible to verify the fundamental role of Cu species and, specifically, Cu(I) species present in the catalysts, in the generation of glyceraldehyde as a critical reaction intermediate for acetol production. Similarly, the acid centers of the catalyst facilitate the first adsorption of glycerol, thus accelerating the reaction. However, the need to achieve higher acetol productivities from glycerol to stand a chance to succeed in the overall strategy motivated the development and study of a second family of catalysts based on copper oxide supported on different metal oxides (SiO2, Al2O3 and ZrO2) combining Lewis acid centers and high Cu exposure. The proper selection and optimization of these materials lead to reaching, with several of them, yields of 60% to acetol with much higher glycerol concentrations in the starting feed. Once several catalytic systems had been established to produce acetol, the generation of the nitrogen heterocycles of interest from the combination of this molecule with ethylenediamine was investigated. Specifically, the Pd/TiO2-Al2O3 catalyst presents high specific activity when forming 2-methylpiperazine (80% yield). These excellent results could be attributed to the enhanced exposure of unsaturated Pd centers observed in this material, critical for activating the C=N double bond and thus proceeding to the hydrogenation of the intermediate imines. Secondly, the CuO/Al2O3-npw catalyst yields 50% to 2-methylpyrazine, reaching values close to 60% when the precipitation-deposition method by micelles in reverse phase is used to incorporate the CuO nanoparticles. Finally, a proof of concept of 2-methylpyrazine synthesis starting from glycerol by using a specially designed multi-bed catalytic reactor to perform the two-steps process in one-pot with the same CuO-supported catalyst was assayed, with promising results. In summary, two new catalytic routes have been established to produce nitrogen heterocycles with glycerol as the main carbon source through its derivative hydroxyacetone, thus opening new perspectives in the field of sustainable valorization of biomass-derived molecules. / Mazarío Santa-Pau, J. (2021). Catalytic transformations of glycerol via hydroxyacetone into nitrogen heterocycles of industrial interest [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/179915 / TESIS

Vid produktion av biodiesel produceras även råglycerol (10 vikt%) som biprodukt och utbudet av råglycerol har ökat över de senaste åren till följd av att biodieselproduktionen ökat. Marknadsvärdet för råglycerol är lågt, men det är en utmärkt föregångare till att producera mervärdesprodukter för exempelvis läkemedelsindustrin, kemikalieindustrin eller kosmetika. Genom att reformera glycerol till mervärdesprodukter ökar konkurrenskraftigheten för biodiesel och värdekedjans ekonomiska hållbarhet. Elektrokemisk reformering av glycerol är en lovande metod för att producera dess mervärdesprodukter, eftersom processen är justerbar och miljövänlig. En ytterligare fördel är att vätgas kan samproduceras med mervärdesprodukterna genom denna metod och energikonsumtionen för att producera vätgas genom denna metod är ungefär hälften av vad som krävs vid vattenelektrolys. Denna studie utvärderar hur masstransport påverkar selektiviteten och prestandan för elektrokemisk oxidation av glycerol, genom att utföra elektrokemiska experiment, karaktärisera processens katalysator och analysera produkter som produceras.  Den experimentella uppställningen innefattar en roterande diskelektrod med ett elektrodepositerat lager av Pd, på ett Ni-substrat, i varierande tjocklek. Elektroden undersöks i elektrolyter bestående av varierande koncentration av NaOH (1, 0.5 och 0.25 M) och 0.5 M glycerol vid 25 oC. Systemet konstrueras även som modell i Comsol Multiphysics 6.0 för att simulera experiment. Resultaten visar att masstransport av glycerol begränsar systemet genom att orsaka deaktiveringen av katalysatorn. Vid de lägre NaOH koncentrationerna begränsar även masstransport av OH- reaktionshastigheten genom att miljön omgärdande elektroden blir mindre oxidativ. Slutligen visas att reaktionsmekanismen vid alla tjocklekar av katalysatorn utgörs av två parallella reaktionsvägar. En ökande tjocklek hos katalysatorn ökar selektiviteten mot den ena reaktionsvägen framför den andra, samt ökar antal oxidationssteg som åstadkoms vid reaktionen. / Biodiesel production gives rise to 10 wt% crude glycerol as a byproduct, which is becoming increasingly available on the market owing to the increased biodiesel production over the last few years. Crude glycerol has a low market value, but it is an excellent precursor to producing value added products for the pharmaceutical industry, chemical industry or cosmetics to mention a few. By producing value added products with biodiesel derived glycerol, the ability of biodiesel to compete with fossil fuels is improved and its economical sustainability is promoted. One of the most promising methods to convert glycerol into its value added products is electrochemical reforming, as it poses high tunability and is an environmentally friendly process. Additionally, the process produces hydrogen concurrently and halves the energy consumption compared to producing hydrogen from water electrolysis. This study evaluates the impact of mass transport in the glycerol electrooxidation reaction (GEOR), as a parameter that can be used to finetune product selectivity and optimise the system performance. To do so, the study employs electrochemical experiments, catalyst characterisation and product analysis. The experimental setup consists of a rotating disk electrode (RDE) cell with electrodeposited Pd on Ni substrate at varying thicknesses, operating in 1, 0.5, 0.25 M NaOH and 0.5 M glycerol electrolyte at 25 oC. Additionally, the system is modelled in Comsol Multiphysics 6.0 which is used to perform simulations. It is found that mass transport of glycerol is limiting the system by causing the deactivation of the catalyst. At lower NaOH bulk concentrations for the thicker electrodes, the mass transport of OH- also limits the reaction rate by causing the formation of a locally less oxidative environment. Finally, it is found that all electrodes pose dual reaction mechanisms. Increasing the electrode thickness promotes one reaction mechanism over the other as well as it increases the number of oxidation steps achieved in the reaction.

The two main requirements for ceria-zirconia hydrodeoxygenation (HDO) catalysts are the presence of defect sites to bind oxygenates and the ability to adsorb and dissociate hydrogen. Two types of sites were identified for exchange of hydrogen and deuterium. The activation energy for one type of site was associated with H2-D2 exchange through oxygen defect sites. The activation energy for the second type of site was associated with H2-D2 exchange through hydroxyl groups and correlated with crystallite size. Ceria-zirconia can convert guaiacol, a model pyrolysis oil compound, with a high selectivity to phenol, an HDO product. Ceria-zirconia catalysts had a higher conversion of guaiacol to deoxygenated products as well as a higher selectivity towards phenol than pure ceria. They did not deactivate over the course of 72 hours on stream, whereas coking or the presence of water in the feed can cause serious decay of common HDO catalysts HDO. Therefore, ceria-zirconia catalysts are promising HDO catalysts for the first step of deoxygenation. The stability of supported Ru on ZrO2 in acidic or basic environments at reaction temperature is examined. In this study, the ruthenium dispersion is greatly increased by hydrothermal treatment in acidic and basic pH without alterations to the surface area, pore volume, pore size or crystal structure. An increase in Ru dispersion showed an increase in the selectivity to propylene glycol relative to ethylene glycol. A decrease in total Lewis acid site concentration was correlated with a decrease in the ethylene glycol yield. The conclusions of this study indicate that stability of catalysts in realistic industrial environments is crucial to the design of catalysts for a reaction.

Master of Science / Department of Grain Science and Industry / Leland McKinney / Three experiments were conducted to evaluate value added uses for dried distillers grains with solubles (DDGS) and crude glycerol in swine and poultry diets. In Exp.1, crude glycerol from multiple biodiesel production facilities was evaluated for storage and variability characteristics. Part one reviewed the storage capabilities of crude glycerol at room temperature and excessive heat conditions on ferrous and stainless steel metal. There was no notable corrosion during the two-month experiment for either metal type under each condition. There was a slight discoloration observed on the ferrous metal stored under excessive heat conditions, but no pitting or sign of corrosion was noted. No changes were observed in the stainless steel under either environment condition. Part two evaluated the variability of crude glycerol from multiple biodiesel production facilities. Representative samples of the parent feedstock and resultant glycerol was collected an analyzed. There was considerable variation between samples, particularly when comparing glycerol from the different feedstocks (vegetable vs. animal). Exp. 2 and 3 took place in the Feed Processing Research Center in the Department of Grain Science at Kansas State University. Pellet quality and electrical energy consumption was evaluated by reviewing production rate, conditioning and hot pellet temperatures, motor load, and pellet durability index (PDI) as testing parameters. In Exp. 2, diets containing varying levels of crude glycerol were evaluated on pellet quality and pellet mill performance in a pilot mill and in a commercial facility. For part one, a corn-soy based swine grower diet was formulated to contain 0, 3, 6, and 9% crude glycerol. Each diet was steam conditioned to 150, 170 and 190 °F in an atmospheric conditioner and pelleted. An interaction existed between glycerol and conditioning temperature. For all diets containing glycerol, roll skid occurred and the pellet mill plugged as conditioning temperature approached 190°F. Pellet quality increased linearly (P < 0.01) with increasing levels of glycerol. Part two took place at Don’s Farm Supply in Newell, IA. A corn-soy based turkey grower diet was formulated to contain 3% glycerol and pelleted. Results from part two were consistent with part one, in that there was an interaction between conditioning temperature and the addition of glycerol. Exp. 3 evaluated DDGS on pellet quality and electrical consumption. A poultry diet was formulated to contain 0, 15, or 30% DDGS and steam conditioned to 140, 160, and 180°F. As conditioning temperature increased, pellet quality in diets containing DDGS significantly improved (P>.001). Electrical consumption in diets containing DDGS compared to the control showed no significance in reduction in energy usage (P>.001). In conclusion, the production of pelleted diets containing crude glycerol is a feasible option; however, conditioning temperatures should be kept minimal and storage and variation of the material should be considered. In addition, improved pellet quality in pelleted diets containing DDGS is in part by conditioning at higher temperatures, respectively. The data suggests that the addition of crude glycerol and DDGS in animal diets can serve as a beneficial feed additive.

Master of Science / Department of Animal Sciences and Industry / Terry A. Houser / The objectives of this study were to determine the impact of 0 and 20% dried distillers grains with soluble (DDGS) and increasing levels of glycerol (0, 2.5 and 5%) in grow-finishing rations on bacon quality and to determine the relationship between belly firmness and slicing yield for commercially produced bacon. A total of 84 barrows (PIC, initially 31.03 kg) were fed corn-soybean meal-based diets organized in a 2 x 3 factorial with primary effects of DDGS (0 or 20%) and glycerol (0, 2.5, or 5%) as fed. Belly length was measured from flank end to blade end. Belly thickness was measured at eight locations evenly spaced around the perimeter of the belly. Belly firmness was measured by centering bellies perpendicularly (skin side up and skin side down) over a stainless steel smokestick and measuring the flex between the edges on the ventral and dorsal edges of the belly. Bellies were injected at 12% of the skinned belly weight resulting in a final concentration of 1.74% salt, 0.5% sugar, 0.3% sodium phosphate, 120 ppm sodium nitrite, and 500 ppm sodium erythorbate in the bellies. Bellies were cooked to an internal temperature of 53oC, chilled, pressed and sliced for evaluation. Belly slice yield was calculated by determining the yield of #1 type bacon slices. Proximate analysis and fatty acid analysis were evaluated by taking every 10th bacon slice beginning from the caudal end to make a composite sample for each belly. Iodine value was calculated using the resulting fatty acid content results. Twenty bacon slices were removed from the belly one-third the length of the belly from the cranial end for sensory analysis and cooking yields. Sensory characteristics were evaluated on an 8-point scale for brittleness, bacon flavor intensity, saltiness and off-flavor. There were no significant DDGS x glycerol interactions on any parameters measured (P > 0.08). Inclusion of 20% DDGS in pig diets decreased belly firmness (P < 0.04) as measured by the belly flop fat side down method. Twenty percent DDGS decreased the percentage of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, vaccenic acid, total saturated fatty acids, and total monounsaturated fatty acids (P < 0.01). In contrast, 20% DDGS increased the percentage of linoleic acid, α-linolenic acid, eicosadienoic acid, total polyunsaturated fatty acids and decreased unsaturated: saturated fatty acid ratios, polyunsaturated: saturated fatty acid ratios, and iodine values (P < 0.01). Statistical correlation analysis of belly processing characteristics showed that by increasing belly weight there will be an increase in smokehouse yields (R = 0.81), increasing smokehouse yields will increase slice yield (R = 0.71), increasing belly thickness results in firmer bellies (R = 0.94) and increasing belly firmness will increase slice yields (R = 0.60). Fatty acid content did not correlate with any belly processing characteristic (R < 0.50). Iodine values were highly correlated with Total MUFA (R = 0.83) Total PUFA (R = 0.79), Total TFA (R = 0.75), and UFA: SFA ratio, and PUFA: SFA ratios (R = 0.83). The inclusion of 0, 2.5 and 5% glycerol in swine diets did not affect any measured parameters in this study. In conclusion, feeding DDGS at a level of 20% decreased belly firmness and changed the fatty acid profile; however, it did not affect belly processing or sensory characteristics. Glycerol fed at 2.5 or 5.0% did not affect belly quality, fatty acid profile, or sensory characteristics of bacon.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Development of analytical methods to characterise biodiesel has become central to the overall success of the marketing of biodiesel fuel. In this regard, different bodies including the American Society for Testing and Materials (ASTM) and the European normalization (EN) have come up with various methods to determine important biodiesel parameters such as total glycerol, methanol and the fatty acid methyl esters (FAMEs), etc. Various studies have been conducted on the parameters mentioned above using a variety of instrumentation and sample preparations. The best methods reported are those that have been adopted by both the ASTM and EN standards. The purpose of this study was to develop alternative analytical methods to both the recommended ASTM and EN methods and, in some cases, to make modifications to both standards (ASTM D 6571 and EN 14214) and methods to determine total and bound glycerol, the ester content and also methanol content in biodiesel. Moreover, water washing after transesterification and the effect this practice has on biodiesel cold flow properties such as kinematic viscosity, cloud and pour point and density were evaluated. The possibility of using the iodine value to predict the feedstock source of an unknown biodiesel was also investigated. Six different vegetable oil samples were transesterified with methanol and used for this study. The six samples used were palm, crown, sunflower, waste vegetable oil (wvo), peanut and rapeseed biodiesel. Quantitative results indicated that the use of programmable temperature volatilisation (PTV) for total glycerol did not produce the required repeatability of between 1-4% relative standard deviation(RSD) for total glycerol analyses in biodiesel with precision of 25%, 86%, 25% and 56% for free glycerol (FG), monoglycerides (MG), diglycerides (DG), and triglycerides (TG) respectively. The standard requires a relative standard of between 1-4% As an alternative to the method using gas chromatography, normal phase high performance chromatography (HPLC) with binary gradient elution was used to determine the bound glycerol content. This method proved accurate and repeatable with RSD % of 0.33, 1.12, and 1.2 for TG, DG and MG respectively. Following the EN14103 protocol (European standard ester determination), the Zebron ZBWAX column which is comparable to the specification recommended by EN14103 but afforded the determination of ester content from the esters of myristic acid (C14:0) to behenic acid (C22:0) with reproducibility with RSD % of 6.81, 1.91, 7.27, 0.64, 1.18, 1.55, 6.03, 1.96, and 5.21 for methyl esters of myristic, palmitic, stearic, oleic, linoleic, linolenic, arachidoic, gadoleic and behenic acid respectively. Solid phase micro extraction (SPME) using GC-MS was developed as an alternative to both the EN14110 and ASTM D93 protocols for determining the methanol content in biodiesel. For this method, polyethylene glycol fibre (PEG) was used together with a deuterated methanol internal standard and a DB-FFAP (60m×0.25um×0.25um) column. Less volume of sample was required as compared to the EN14214 method. This method was found to be sensitive, accurate and repeatable with a RSD % of 4.82. The Iodine number of biodiesel decrease compared to their corresponding feed stock and therefore predicting the feed stock of an unknown biodiesel was going to be difficult .Results from this study indicated that it is not possible to predict the feed stock source of an unknown biodiesel from its iodine value. The effect of water washing after phase separation on biodiesel cold flow properties such as kinematic viscosity, density, cloud and pour point depended on the type of biodiesel produced. We observed that water washing after transesterification caused an increase in all the cold flow properties of sunflower biodiesel, whereas only the densities and kinematic viscosities increased in the case of palm and waste vegetable oil biodiesel. The cloud and pour point of the latter two diesel samples remained unchanged after water washing. Thus, the effect of water washing on biodiesel cold flow depended on the type of biodiesel. Blending a highly saturated biodiesel (fewer numbers of double bonds) with a less saturated biodiesel (higher number of double bonds) resulted in an improvement of both the pour and cloud points of the resultant biodiesel blend. / AFRIKAANSE OPSOMMING: Die ontwikkeling van analitiese metodes om biodiesel te karakteriseer word tans as ‘n kernmaatstaf gesien om biodiesel suksesvol te bemark. Hiervoor het verskeie liggame wat die Amerikaanse Vereniging vir Toetsing van Materiale (AVTM) en die Europese Normalisering (EN) insluit met verskeie standaard analitiese metodes vorendag gekom om belangrike biodiesel parameters soos bv. totale gliserol, metanol en vetsuur metielesters te meet. Om hierdie parameters te bepaal is van ‘n wye verskeidenheid toetse met verskillende instrumente en monsterbereidings gebruik gemaak. Die beste metodes is deur beide die AVTM en EN aanvaar. Die doel van hierdie studie was om metodes te ontwikkel wat as alternatiewe kan dien tot die wat deur die AVTM en EN voorsgeskryf is. In sommige gevalle is aanpassings tot beide die standaarde (AVTM en EN) en metodes aangebring om die totale en gebonde gliserol-, esteren metanolinhoud te bepaal. Verder is die effek van ‘n water wasstap na transesterifikasie op biodiesel se kouevloei eienskappe gevalueer wat eienskappe soos kinematiese viskositeit, vertroebelingspunt, gietingspunt en digtheid insluit. Die moontlike gebruik van die Jodiumpunt om die bron van die voerstof van ‘n onbekende diesel te bepaal is ook ondersoek. In hierdie studie is ses verskillende oliemonsters van plantaardige oorsprong gebruik wat d.m.v. metanol getransesterifiseer is. Hierdie monsters het palm-, kroon-, sonneblom-, afvalplant-, grondboontjie- en raapsaadolie ingelsuit. Tydens die studie is programmeerbare temperatuur vervlugtiging (PTV) vergelyk met inkolom inspuiting soos deur AVTM D6584/EN14214 vir totale gliserol analise voorgeskryf. Kwantitatiewe resultate het getoon dat die PTV metode nie die verlangde akkuraatheid van ‘n relatiewe standaardafwyking (RS) van 1-4% vir beide vrye en gebonde gliserol kon handhaaf nie. Die akkuraatheid was in die omgewing van 25%, 86%, 25% en 56% vir vrye gliserol (VG), monogliseriede (MG), digliseriede (DG) en trigliseriede (TG), onderskeidelik. Normale fase hoë werkverrigting vloeistofchromatografie met ‘n binêre elueeringsgradiënt is as alternatief tot gaschromatografie (GC) ondersoek om die gebonde gliserolinhoud te bepaal. Al was die GC metode meer sensitief, het die vloeistofchromatografie metode ‘n hoë graad van akuraatheid en herhaalbaarheid getoon met RS% waardes van 0.33, 1.12 en 1.2 wat vir TG, DG en MG, onderskeidelik, verkry is. ‘n Zebron ZB-WAX kolom is vir die EN14103 protokol gebruik. Behalwe vir ‘n groter lengte kon hierdie kolom met spesifikasies soos deur EN14103 voorgeskryf vergelyk word. Met die gebruik van hierdie kolom kon die esterinhoud van miristiensuur (C14:0) tot behensuur (C14:0) bepaal word. ‘n Hoë graad van herhaalbaarheid met RS% waardes van 6.81, 1.91, 7.27, 0.64, 1.18, 1.55, 6.03, 1.96 en 5.21 vir die metielesters van miristien-, palmitien-, stearien-, oleïn-, linoleïn-, linoleen-, aragidoon-, gadoleïen- en behensuur is onderskeidelik verkry. Om die metanolinhoud van die biodiesel te bepaal is soliede fase mikroekstraksie (SFME) m.b.v. gaschromatografie-massaspektrometrie (GC-MS) as alternatiewe tot EN14110 en AVTM D93 ontwikkel. In hierdie metode is daar van poliëtileenglikolvesels (PEG) en gedeutereerde metanol saam met ‘n DB-FFAP kolom (60 mm x 0.25 mm x 0.25 mm) gebruik gemaak. Hierdie metode het ‘n kleiner monstervolume as die EN14214 metode benodig en was sensitief, akkuraat en hehaalbaar wat tot ‘n RS% waarde van 4.82 gelei het. Op grond van die Jodiumwaarde van biodiesel en hul ooreenstemmende voerstowwe het hierdie studie bevind dat die Jodiumwaarde nie gebruik kan word om die voerstof van ‘n onbekende diesel kan voorspel nie. Die effek van ‘n water wasstap na faseskeiding op verskeie kouevloei eienskappe soos kinematiese viskositeit, vertroebelingspunt, gietingspunt en digtheid het van die tipe diesel afgehang. Dit is bevind dat ‘n water wasstap na transesterifikasie ‘n toename in al die kouevloeieienskappe van sonneblomdiesel tot gevolg gehad het. In teenstelling hiermee het slegs die kinematiese viskositeit en digtheid van palm- en afvalplantdiesel vermeerder terwyl hul vertroebelings- en gietingspunte onveranderd gebly het. Die hipotese dat ‘n water wasstap na transesterifikasie tot swak kouevloei eienskappe lei is dus as onwaar bevind aangesien hierdie eienskappe deur die tipe biodiesel bepaal word. Deur ‘n hoogs versadigde biodiesel (lae aantal dubbelbindings) met ‘n minder versadigde biodiesel (hoë aantal dubbelbindings) te vermeng het tot ‘n verbetering van beide die vertroebelings- en gietingspunte gelei. / Centre for Renewable and Sustainable Energy Studies

Oleic acid (18:1), a monounsaturated fatty acid (FA), is synthesized upon desaturation of stearic acid (18:0) and this reaction is catalyzed by the plastidal enzyme stearoyl-acyl carrier protein-desaturase (SACPD). A mutation in the SSI2/FAB2 encoded SACPD lowers 18:1 levels, which correlates with induction of various resistance (R) genes and increased resistance to pathogens. Genetic and molecular studies have identified several suppressors of ssi2 which restore altered defense signaling either by normalizing 18:1 levels or by affecting function(s) of a downstream component. Characterization of one such ssi2 suppressor mutant showed that it is required downstream of low 18:1-mediated constitutive signaling and partially restores altered defense signaling in the ssi2 mutant. Molecular and genetic studies showed that the second site mutation was in the Nitric Oxide Associated (NOA) 1 gene, which is thought to participate in NO biosynthesis. Consistent with this result, ssi2 plants accumulated high levels of NO and showed an altered transcriptional profile of NO-responsive genes. Interestingly, the partial defense phenotypes observed in ssi2 noa1 plants were completely restored by an additional mutation in either of the two nitrate reductases NIA1 or NIA2. This suggested that NOA1 and NIA proteins participated in NO biosynthesis in an additive manner. Biochemical studies showed that 18:1 physically bound NOA1, in turn leading to its degradation in a protease-dependent manner. In concurrence, overexpression of NOA1 did not promote NO-derived defense signaling in wild-type plants unless 18:1 levels were lowered. Subcellular localization showed that NOA1 and the 18:1-synthesizing SSI2 were present in close proximity within the nucleoids of chloroplasts. Indeed, pathogen- or low 18:1- induced accumulation of NO was primarily detected in the chloroplasts and their nucleoids. Together, these data suggested that 18:1 levels regulate NO synthesis and thereby NO-mediated retrograde signaling between the nucleoids and the nucleus. Since cellular pools of glycerol-3-phosphate (G3P) regulate 18:1 levels, I next analyzed the relationship between G3P and 18:1. Interestingly, unlike 18:1, an increased G3P pool was associated with enhanced systemic immunity in Arabidopsis. This was consistent with G3P-mediated transcriptional reprogramming in the distal tissues. To determine mechanism(s) underlying G3P-conferred systemic immunity, I analyzed the interaction between G3P and a lipid transfer protein (LTP), DIR1. In addition, I monitored localization of DIR1 in both Arabidopsis as well as tobacco. Contrary to its predicted apoplastic localization, DIR1 localized to endoplasmic reticulum and plasmodesmata. The symplastic localization of DIR1 was confirmed using several different assays, including co-localization with plasmodesmatal-localizing protein, plasmolysis and protoplast-based assays. Translocation assays showed that G3P increased DIR1 levels and translocated DIR1 to distal tissues. Together, these results showed that G3P and DIR1 are present in the symplast and their coordinated transport into distal tissues is likely essential for systemic immunity. In conclusion, this work showed that low 18:1-mediated signaling is mediated via NO, synthesis of which is likely initiated in the plastidal nucleoids. In addition, my work shows that G3P functions as an independent signal during systemic signaling by mediating translocation of the lipid transfer protein, DIR1.

The conserved metabolites, oleic acid (18:1), a major monounsaturated fatty acid (FA), and glycerol-3-phosphate (G3P) are obligatory precursors of glycerolipid biosynthesis in plants. In Arabidopsis, the SSI2-encoded SACPD is the major isoform that contributes to 18:1 biosynthesis. Signaling induced upon reduction in oleic acid (18:1) levels not only upregulates salicylic acid (SA)-mediated responses but also inhibits jasmonic acid (JA)- inducible defenses. I examined the transcription profile of ssi2 plants and identified two transcription factors, WRKY50 and WRKY51. Although the ssi2 wrky50 and ssi2 wrky51 plants were constitutively upregulated in SA-derived signaling, they were restored in JAdependent defense signaling. Not only did these plants show JA-inducible PDF1.2 expression, but they were also restored for basal resistance to the necrotrophic pathogen, Botrytis cinerea. Overall, my results show that the WRKY50 and WRKY51 proteins mediate both SA- and low 18:1-dependent repression of JA signaling in Arabidopsis plants. My studies also show that cellular G3P levels are important for plant defense to necrotrophic pathogens. I showed that G3P levels are induced in Arabidopsis in response to the necrotrophic fungal pathogen B. cinerea. G3P-dependant induction of basal defense is not via the activities of other defense-related hormones such as SA, JA or the phytoalexin camalexin. Arabidopsis mutants unable to accumulate G3P (gly1, gli1) showed enhanced susceptibility to B. cinerea. Previous studies in our lab identified acyl-carrier protein 4 (ACP4), a component of FA and lipid biosynthesis, as an important regulator of plant systemic immunity. ACP4 mutant plants were defective in systemic acquired resistance (SAR) because they contained a defective cuticle. I further investigated the role of the plant cuticle in SAR by studying the involvement of long-chain acyl-CoA synthetases (LACS), a gene family involved in long-chain FA and cuticle biosynthesis, in SAR. In all, eight lacs mutants (lacs1, lacs2, lacs3, lacs4, lacs6, lacs7, lacs8, lacs9) were isolated and characterized. Six mutants were compromised in SAR. Together, my studies show that the various LACS isoforms contribute differentially to both cuticle formation and systemic immunity in Arabidopsis.

Le biodiesel est un mélange d’esters mono-alkyl utilisé comme carburant alternatif au diesel. Il est obtenu par transestérification des huiles végétales ou de graisses animales par un alcool léger.L’objectif de ce travail consiste en la recherche de catalyseurs solides acides ou basiques capables de promouvoir la réaction de transestérification de l’huile de colza par l’éthanol en conditions aussi douces que celles utilisées en catalyse homogène basique. Pour cela, deux séries de solides acides et basiques ont été préparées et caractérisées par différentes techniques (ATG-ATD, DRX, Isotherme N2). Les propriétés acides et basiques de ces solides ont également été étudiées par calorimétrie et spectroscopie Infra Rouge. Des corrélations entre les activités initiales et les propriétés acides ou basiques de ces catalyseurs ont été recherchées. L’influence des principaux paramètres réactionnels a été examinée sur le catalyseur acide et le catalyseur basique, les plus actifs. Puis une étude cinétique comparative a été réalisée. Enfin la valorisation in situ du glycérol, produit secondaire de la réaction, a été considérée.Nous avons pu montrer que le sel de césium de l’acide 12-tungstophosphorique est le catalyseur acide le plus actif, dans la gamme de solides acides étudiés. Ceci s’explique par la présence de sites acides de BrØnsted forts aptes à promouvoir la réaction en conditions douces de température. Par ailleurs, il est capable de réaliser l’éthérification in situ du glycérol par l’éthanol. En catalyse basique, le nouveau catalyseur préparé, la zircone échangée au césium, est le catalyseur le plus actif parmi les bases solides étudiées. Ce catalyseur est stable dans le milieu réactionnel après élimination par lavage à chaud des espèces Cs labiles. La comparaison des deux catalyseurs montre que la catalyse basique est nettement plus rapide que la catalyse acide. L’étude cinétique permet de proposer des différences mécanistiques entre ces deux types de catalyseurs. / Biodiesel is a mixture of mono-alkyl esters used as alternative fuel. It can be obtained by transesterification of vegetable oils or animal fats with light alcohols.The objective of this work is the research of acid and basic solid catalysts able to promote the transesterification reaction of rapeseed oil with ethanol in conditions as mild as those used in homogeneous base catalysis. For this, two series of solid acids and bases were prepared and characterized by various characterization techniques (TGA-DTA, XRD, Isothermal N2). Calorimetry and Infrared Spectroscopy have also been used to characterize acid and basic properties. Correlations between initial activity and the acidic or basic properties of these catalysts have been investigated. The influence of the main reaction parameters was examined on the most active acid and basic solid. A comparative kinetic study then allowed us to propose reaction mechanisms. Finally the in situ valorisation of glycerol, a by product of the reaction, was carried out.We have shown that the cesium salt of 12-tungstophosphoric acid catalyst is the most active among the solid acids studied. This is explained by the presence of strong BrØnsted acid sites that are able to promote the reaction under mild temperature conditions. This catalyst is capable of achieving in situ etherification of glycerol with ethanol. In base catalysis, the newly prepared catalyst, zirconia exchanged with Cesium, is the most active catalyst among the solid bases studied. This catalyst is stable in the reaction medium after removal of the labile Cs species by hot washing. Comparison of the two catalysts shows that the base catalysis is much faster than acid catalysis. A kinetic comparative study allows us to propose mechanistic differences between these two types of catalysts.