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Den globala ekonomin är idag starkt kopplad till utsläpp av växthusgaser samtidigt som det finns en stark enighet bland världens ledande länder att kraftigt minska de globala utsläppen i enlighet med Parisavtalet. Vätgas som produceras från förnyelsebara energikällor anses utgöra en nyckelroll för ett antal olika applikationsområden de kommande decennierna, där bland transportsektorn. Trots att framställningsprocessen bygger på väl utvecklad teknik finns det än idag väldigt få storskaliga produktionsanläggningar av grön vätgas, men teknikutvecklingen inom området är skyndsam. Rederi AB Gotland är idag Sveriges äldsta rederi och således en av de största aktörerna inom Gotlands transportsektorn. Företaget ser idag över möjligheten för att driftsätta Sveriges första storskaliga vätgasdrivna gods- och passagerarfartyg, GotlandHorizon, vilket är en viktig del i företagets miljöarbete. Huvudsakligen avser företaget attvätgasen produceras lokalt på Gotland, vilket föranleder till en rad olika tekniska utmaningarrelaterade till elproduktion, vätgasframställning och distributionssystem. Med bakgrund av detta har en förstudie tillsammans med Uppsala universitet och projektet “Vätgasbaserad färjetrafik” genomförts för att påvisa och kartlägga viktiga aspekter kring ett framtida produktionssystem av grön vätgas samt kartlägga vilka tekniska lösningar som inom tidsramen för projektet är tekniskt genomförbara. Resultatet av förstudien ska kunna användas som grund för utformning av framtida beräkningsmodeller. Av förstudien framgår det att vattenelektrolys i kombination med en utbyggnation av vindkraft teoretiskt kan möta både det efterfrågade elbehovet för elektricitet och således Gotland Horizons vätgasbehov. Det uppskattade elbehovet för framställning av vätgas genom vattenelektrolys motsvarar dock Gotlands idag totala energikonsumtion, vilket såldes utgör en storutmaning. En annan viktig faktor för processen är en tillförlitlig processvattenförsörjning. Gotland har de senaste åren haft en problematisk grundvattensituation samt att dricksvattenproduktionen på Gotland är begränsad. I studien har de viktiga aspekterna kring utformningen av produktionssystemets analyserats. De ekonomiska aspekterna har också redovisats för att ligga till grund för en optimeringsmodell för vidare analys och optimering av produktionssystemet. Av de beräkningsmodeller som genomförts påvisas att både havsbaserad- samt landbaserad vindkraft kan tillgodose behovet av elproduktion för vattenelektrolys, det är snarare en fråga om hur systemet ska optimeras samt vilka synergieffekter som respektive system kan medförasom bestämmer systemets utformning. Solenergi har ansetts vara tekniskt möjligt men till bakgrund av att efterfrågan på elektricitet året runt är hög anses anläggningen bli orealistiskt stor. Också aspekter gällande produktionssystemet utformning, centraliserat eller decentraliserats, har diskuterats. Till bakgrund av de stora ekonomiska storskalsfördelarna som uppskattas för elektrolysörer inom de kommande åren anses ett centraliserat produktionssystem vara det mest tänkbara utifrån ett ekonomiskt perspektiv. Det har också konstaterats att havsbaserade vätgaspipelines kan bli aktuellt vid havsbaserad vätgasproduktion, det för att minimera kapitalkostnaderna för distributionen av energivektor, vilket skulle kunna minska produktionskostnaderna för vätgas från havsbaserad vindkraft. / The global economy today is strongly linked to greenhouse gas emissions while there is a strong consensus among the world's leading countries to significantly reduce global emissions in accordance with the Paris Agreement. Hydrogen produced from renewable energy sources is considered to play a key role within a several different application areas in the coming decades, including the transport sector. Even though the production process is based on welldeveloped technology, there are still very few large-scale production facilities of green hydrogen, but technological development in the field is rapid. Rederi AB Gotland is today Sweden's oldest shipping company and thus one of the largest players in Gotland's transport sector. The company is currently reviewing the possibility of commissioning Sweden's first large-scale hydrogen-powered freight and passenger vessel, Gotland Horizon, which is an important part of the company's environmental work. Mainly, the company intends that the hydrogen is produced locally on Gotland, which leads to a variety of technical challenges related to electricity production, hydrogen production and distribution systems. With this background, a feasibility study together with Uppsala University and the project "Hydrogen-based ferry traffic" has been carried out to demonstrate and map important aspects of a future production system of green hydrogen and to map which technical solutions within the time frame of the project are technically feasible. The results of the feasibility study can be used as a basis for designing future calculation models. The feasibility study shows that water electrolysis in combination with an expansion of wind power can theoretically meet both the demanded electricity demand for electricity and thus Gotland Horizon's hydrogen needs. However, the estimated electricity demand to produce hydrogen through water electrolysis corresponds to Gotland's current total energy consumption, which was sold poses a major challenge. Another important factor for the process is a reliable process water supply. In recent years, Gotland has had a problematic groundwater situation and the drinking water production on Gotland is limited. In the study, the important aspects of the design of the production system have been analyzed. The economic aspects have also been accounted for to form the basis for an optimization model for further analysis and optimization of the production system. From the calculation models carried out, it is shown that both offshore and onshore wind power can meet the need for electricity production for water electrolysis, it is rather a question of how the system should be optimized and what synergies each system can bring that determine the design of the system. Solar energy has been considered technically possible, but given that the demand for electricity all year round is high, the plant is considered to be unrealistically large. Aspects of the design of the production system, centralised or decentralised, have also been discussed. Considering the large economic economies of scale appreciated for electrolysers in the coming years, a centralized production system is considered the most conceivable from an economic perspective. It has also been recognized that offshore hydrogen pipelines may be relevant in offshore hydrogen production, in order to minimize the capital costs of energy vector distribution, which could reduce the production costs of hydrogen from offshore wind.

Nanotechnology is a relatively new research topic that attracts increasing interest from scientists and engineers all over the world, due to its novel applications. The use of nanomaterials has extended to a broad range of applications, for example chemical synthesis, microporous media synthesis and catalytic combustion, contributing to achievement of improved or promising results. Microemulsion (ME) is considered a powerful tool for synthesis of nanomaterials, due to its unique properties. This thesis concentrates on the use of the ME as a catalyst synthesis route for obtaining metal nanoparticles for two challenging concepts: Hydrogen production by a membrane reactor and selective catalytic oxidation (SCO) of ammonia in gasified biomass. Particularly for the scope of the fist concept presented in this thesis, palladium nanoparticles were synthesised from ME in order to be deposited on zeolite composite membranes to improve the H2 / CO2 separation (hydrogen production) ability. The membranes impregnated with Pd nanoparticles were then tested in a metal reactor for the permeance and selectivity towards H2 and CO2. Regarding the second concept, cerium-lanthanum oxide nanoparticles were prepared by conventional methods and from ME in order to be tested for their activity towards SCO of ammonia in gasified biomass. The environmental importance of these two applications under investigation is great, since both are involved in processes contributing to the minimisation of the harmful exhaust gases released to the atmosphere from numerous industrial applications, such as the oil industry and heat-and-power production (for example combustion of natural gas or biomass in a gas turbine cycle). Regarding these applications, separation and capture of CO2 from exhaust gases and oxidation of the fuel-bound ammonia in gasified biomass directly to nitrogen, minimising at the same time NOx formation, are rated as very important technologies. The results obtained from this work and presented analytically in this thesis are considered successful and at the same time promising, since further research on the ME method can even lead to improvement of the current achievements. The first part (Chapter 2) of the thesis gives a general background on the ME method and the applications in the two concepts under investigation. Additionally, it describes how the nanoparticles corresponding to the concepts were synthesised. The second part (Chapter 3) of the thesis describes the different Pd-nanoparticle impregnation methods on the zeolite composite membranes and the results obtained form the permeation tests. In parallel with impregnation methods, various aspects that affect the Pd impregnation efficiency and the membrane performance such as duration, temperature and calcination conditions are discussed thoroughly. The third and final part of the thesis (Chapter 4) concerns the preparation of the cerium-lanthanum oxide catalysts and the activity tests (under simulated gasified biomass fuel conditions) carried out in order to monitor the activity of these catalysts towards the SCO of ammonia. Additionally, a comparison of the activity between identical catalysts prepared by conventional methods and the ME method is discussed. / QC 20101216

The goal of this research was to develop a series of mixed-metal supramolecular complexes with specialized light absorbing units to probe perturbation of excited-state properties by ligand deuteration and long-term complex stability via racemization of initially enantiopure light absorbing subunits. Varying bidentate polypyridyl terminal ligands (TL), bridging ligands (BL), reactive metal center (RM), or number of Ru(II) light absorbers (LA) tunes the electrochemical, spectroscopic, photophysical, and photochemical properties within the supramolecular architecture. Ru(II) monometallics of the design [(bpy)2Ru(prolinate)](PF6) utilize prolinate as a chiral directing ligand to impart chirality to the Ru(II) LAs in the synthesis of more sophisticated supramolecular complexes. Ru(II) monometallics of the design [(TL)2Ru(BL)](PF6)2 (TL = bpy or d8-bpy; BL = dpp or d10-dpp; bpy = 2,2′-bipyridine; dpp = 2,3-bis(2-pyridyl)pyrazine) covalently couple two TLs and one BL to a central Ru(II) metal center forming a LA subunit. Larger bi- and trimetallic complexes are formed by coupling an additional Ru(II), Rh(III), or Pt(II) metal center to an existing Ru(II) LA through a BL. Ru(II),Ru(II), Ru(II),Rh(III), and Ru(II),Pt(II) bimetallics of the design [(TL)2Ru(BL)Ru(TL)2](PF6)4, [(TL)2Ru(BL)RhCl2(TL′)](PF6)3, and [(TL)2Ru(BL)PtCl2](PF6)2 (TL/TL′ = bpy or d8-bpy; BL = dpp or d10-dpp) couple only one Ru(II) LA to a Ru(II), Rh(III), or Pt(II) metal center through the BL. Ru(II),Rh(III),Ru(II) trimetallics of the design [{(TL)2Ru(BL)}2RhCl2](PF6)5 (TL = bpy or d8-bpy; BL = dpp or d10-dpp) covalently couple two Ru(II) LAs to a central Rh(III) RM through polyazine BLs. The complexes discussed herein are synthesized using a building block approach, permitting modification of the supramolecular architecture through multiple synthetic steps. Electrochemical analysis of the mono-, bi-, and trimetallic complexes displays several common features: a Ru-based HOMO and either a bridging ligand or Rh-based LUMO. TL and BL modification by ligand deuteration does not affect the electrochemistry of the Ru(II), Ru(II),Ru(II), Ru(II),Rh(III), or Ru(II),Rh(III),Ru(II) complexes. Likewise, utilizing a single enantiomer of the LA subunit does not modify the redox behavior of Ru(II), Ru(II),Pt(II), or Ru(II),Rh(III),Ru(II) complexes. All of the mono-, bi-, and trimetallic complexes are efficient light absorbers throughout the UV and visible with π→π* intraligand (IL) transitions in the UV and Ru(dπ)→ligand(π*) metal-to-ligand charge transfer (MLCT) transitions in the visible. Ligand deuteration does not affect the light absorbing properties of the complexes, while incorporation of chiral LA subunits imparts a preference for circularly polarized light (CPL) absorbance into supramolecular complexes. Photoexcitation of the Ru(dπ)→dpp(π*) 1MLCT results in near unity population of short-lived, weakly emissive Ru(dπ)→dpp(π*) ³MLCT excited state. In the Ru(II), Ru(II),Ru(II), and Ru(II),Pt(II) complexes, the 3MLCT excited state relaxes to the ground state by emission of a photon or vibrational relaxation processes. In the Ru(II),Rh(III) and Ru(II),Rh(III),Ru(II) complexes, the 3MLCT excited state is efficiently quenched by intramolecular electron transfer to populate a non-emissive Ru(dπ)→'Rh(dσ*) metal-to-metal charge transfer (3MMCT) excited state. Utilizing a deuterated BL, the excited-state lifetimes and quantum yield of emission (Φem) are increased for Ru(II), Ru(II),Ru(II), Ru(II),Rh(III) and Ru(II),Rh(III),Ru(II) complexes. The Ru(II),Rh(III) and Ru(II),Rh(III),Ru(II) complexes have previously been shown to be exceptional photochemical molecular devices (PMD) for photoinitiated electron collection (PEC). The ability of these complexes to undergo multiple redox cycles, efficiently absorb light, populate reactive excited states, and collect electrons at a reactive Rh metal center fulfills the requirements for H2O reduction photocatalysts. Photolysis of the Ru(II),Rh(III) and Ru(II),Rh(III),Ru(II) complexes with 470 nm light in the presence of a sacrificial electron donor and H2O substrate yields photocatalytic H2 production. Varying the BL from dpp to d10-dpp in the bimetallic architecture results in enhanced, although relatively low, catalyst efficiency producing 40 ± 10 μL H2 with dpp and 80 ± 10 μL H2 with d10-dpp in a CH3CN solvent system after 48 h photolysis. The trimetallic architecture showed no enhancement in photocatalytic efficiency and produced 210 ± 20 μL H2 with dpp and 180 ± 20 μL H2 with d10-dpp in a DMF solvent system after 20 h photolysis. The Ru(II),Rh(III) and Ru(II),Rh(III),Ru(II) complexes' behavior differs in that the excited state lifetime is the most important factor for bimetallic catalyst functioning, but intramolecular electron transfer is the most important factor for the trimetallic photocatalysts. Another important property to understand with these catalysts is their long-term stability in solution. In order for these mixed-metal complexes to be industrially useful, they must perform for long periods of time without degradation in the presence of H2O substrate and electron donors in solution. Previous examinations of Ru(II),Rh(III),Ru(II) photocatalysts have found that they can perform for ca. 50 h of photolysis, but are not as effective as the initial few hours. Special care was taken to synthesize enantiopure LA subunits and incorporate them into Ru(II),Pt(II) and Ru(II),Rh(III),Ru(II) architectures to study their photolytic stability by monitoring how long the complexes retained their chirality using electronic circular dichroism (ECD) spectroscopy. After photolyzing for longer than 200 hours with an LED light source, the quantum yield for racemization (Φrac) for the Ru(II),Pt(II) and Ru(II),Rh(III),Ru(II) architectures is 2.6 ⨉ 10⁻⁸ and 0.72 ⨉ 10⁻⁸ respectively. Also, by photolyzing in the presence of free bpy, the bi- and trimetallic complexes racemize via a non-dissociative trigonal twist mechanism. This dissertation reports the detailed analysis of the electrochemical, spectroscopic, photophysical, and photochemical properties of a series of selectively deuterated [(TL)2Ru(BL)](PF6)2, [(TL)2Ru(BL)Ru(TL)2](PF6)4, [(TL)2Ru(BL)RhCl2(TL′)](PF6)3, and [{(TL)2Ru(BL)}2RhCl2](PF6)5 (TL = bpy or d8-bpy; BL = dpp or d10-dpp; bpy = 2,2′-bipyridine; dpp = 2,3-bis(2-pyridyl)pyrazine) supramolecular complexes and a series of [(bpy)2Ru(prolinate)](PF6), [(bpy)2Ru(dpp)](PF6)2, [(bpy)2Ru(dpp)PtCl2](PF6)2, and [{(bpy)2Ru(dpp)}2RhCl2](PF6)5 supramolecular complexes with enantiopure light absorbing subunits. The design of the supramolecular architecture and intrinsic properties of each subunit contribute to the function of these systems. The careful design, synthesis and purification, thorough characterizations, and experimentation have led to deeper understanding of the molecular properties required for efficient H2O reduction. / Ph. D.

La production biologique d'hydrogène (H2) représente une technologie possible pour la production à grande échelle durable de H2 nécessaire pour l'économie future de l'hydrogène. Cependant, l'obstacle majeur à l'élaboration d'un processus pratique a été la faiblesse des rendements qui sont obtenus, généralement autour de 25%, bien en sous des rendements pouvant être atteints pour la production de biocarburants à partir d'autres processus. L'objectif de cette thèse était de tenter d'améliorer la production d'H2 par la manipulation physiologique et le génie métabolique. Une hypothèse qui a été étudiée était que la production d'H2 pourrait être améliorée et rendue plus économique en utilisant un procédé de fermentation microaérobie sombre car cela pourrait fournir la puissance supplémentaire nécessaire pour une conversion plus complète du substrat et donc une production plus grande d'H2 sans l'aide de l'énergie lumineuse. Les concentrations optimales d’O2 pour la production de H2 microaérobie ont été examinées ainsi que l'impact des sources de carbone et d'azote sur le processus. La recherche présentée ici a démontré la capacité de Rhodobacter capsulatus JP91 hup- (un mutant déficient d’absorption-hydrogénase) de produire de l'H2 sous condition microaérobie sombre avec une limitation dans des quantités d’O2 et d'azote fixé. D'autres travaux devraient être entrepris pour augmenter les rendements d'H2 en utilisant cette technologie. De plus, un processus de photofermentation a été créé pour améliorer le rendement d’H2 à partir du glucose à l'aide de R. capsulatus JP91 hup- soit en mode non renouvelé (batch) et / ou en conditions de culture en continu. Certains défis techniques ont été surmontés en mettant en place des conditions adéquates de fonctionnement pour un rendement accru d'H2. Un rendement maximal de 3,3 mols de H2/ mol de glucose a été trouvé pour les cultures en batch tandis que pour les cultures en continu, il était de 10,3 mols H2/ mol de glucose, beaucoup plus élevé que celui rapporté antérieurement et proche de la valeur maximale théorique de 12 mols H2/ mol de glucose. Dans les cultures en batch l'efficacité maximale de conversion d’énergie lumineuse était de 0,7% alors qu'elle était de 1,34% dans les cultures en continu avec un rendement de conversion maximum de la valeur de chauffage du glucose de 91,14%. Diverses autres approches pour l'augmentation des rendements des processus de photofermentation sont proposées. Les résultats globaux indiquent qu'un processus photofermentatif efficace de production d'H2 à partir du glucose en une seule étape avec des cultures en continu dans des photobioréacteurs pourrait être développé ce qui serait un processus beaucoup plus prometteur que les processus en deux étapes ou avec les co-cultures étudiés antérieurément. En outre, l'expression hétérologue d’hydrogenase a été utilisée comme une stratégie d'ingénierie métabolique afin d'améliorer la production d'H2 par fermentation. La capacité d'exprimer une hydrogénase d'une espèce avec des gènes de maturation d'une autre espèce a été examinée. Une stratégie a démontré que la protéine HydA orpheline de R. rubrum est fonctionnelle et active lorsque co-exprimée chez Escherichia coli avec HydE, HydF et HydG provenant d'organisme différent. La co-expression des gènes [FeFe]-hydrogénase structurels et de maturation dans des micro-organismes qui n'ont pas une [FeFe]-hydrogénase indigène peut entraîner le succès dans l'assemblage et la biosynthèse d'hydrogénase active. Toutefois, d'autres facteurs peuvent être nécessaires pour obtenir des rendements considérablement augmentés en protéines ainsi que l'activité spécifique des hydrogénases recombinantes. Une autre stratégie a consisté à surexprimer une [FeFe]-hydrogénase très active dans une souche hôte de E. coli. L'expression d'une hydrogénase qui peut interagir directement avec le NADPH est souhaitable car cela, plutôt que de la ferrédoxine réduite, est naturellement produit par le métabolisme. Toutefois, la maturation de ce type d'hydrogénase chez E. coli n'a pas été rapportée auparavant. L'opéron hnd (hndA, B, C, D) de Desulfovibrio fructosovorans code pour une [FeFe]-hydrogénase NADP-dépendante, a été exprimé dans différentes souches d’E. coli avec les gènes de maturation hydE, hydF et hydG de Clostridium acetobutylicum. L'activité de l'hydrogénase a été détectée in vitro, donc une NADP-dépendante [FeFe]-hydrogénase multimérique active a été exprimée avec succès chez E. coli pour la première fois. Les recherches futures pourraient conduire à l'expression de cette enzyme chez les souches de E. coli qui produisent plus de NADPH, ouvrant la voie à une augmentation des rendements d'hydrogène via la voie des pentoses phosphates. / Biological hydrogen (H2) production represents a possible technology for the large scale sustainable production of H2 needed for a future hydrogen economy. However, the major obstacle to developing a practical process has been the low yields that are obtained, typically around 25%, well below those achievable for the production of other biofuels from the same feedstock. The goal of this thesis was to improve H2 production through physiological manipulation and metabolic engineering. One investigated hypothesis was that H2 production could be improved and made more economical by using a microaerobic dark fermentation process since this could provide the extra reducing power required for driving substrate conversion to completion and hence more H2 production might be obtained without using light energy. The optimal O2 concentrations for microaerobic H2 production were examined as well as the impact of carbon and nitrogen sources on the process. The research reported here proved the capability of Rhodobacter capsulatus JP91 hup- (an uptake-hydrogenase deficient mutant) to produce H2 under microaerobic dark conditions with limiting amounts of O2 and fixed nitrogen. Further work should be undertaken to increase H2 yields using this technology. In addition, a photofermentation process was established to improve H2 yield from glucose using R. capsulatus JP91 hup- strain either in batch and/or continuous culture conditions. Some technical challenges in establishing the proper operational conditions for increased H2 yield were overcome. A maximum yield of 3.3 mols of H2/ mol of glucose was found for batch cultures whereas in continous cultures it was 10.3 mols H2/ mol glucose, much higher than previously reported and close to the theoretical maximum value of 12 mols H2/ mol glucose. In batch cultures the maximum light conversion efficiency was 0.7% whereas it was 1.34% in continuous cultures with a maximum conversion efficiency of the heating value of glucose of 91.14%. Various approaches to further increasing yields in photofermentation processes are proposed. The overall results suggest that an efficient single stage photofermentative H2 production process from glucose using continuous cultures in photobioreactors could be developed which would be a much more promising alternative process to the previously studied two stage photofermentation or co-culture approaches. Furthermore, the heterologous expression of hydrogenases was used as a metabolic engineering strategy to improve fermentative H2 production. The capability of expressing a hydrogenase from one species with the maturation genes from another was examined. One strategy demonstrated that the orphan hydA of R. rubrum is functional and active when co-expressed in E. coli with hydE, hydF and hydG from different organisms. Co-expression of the [FeFe]-hydrogenase structural and maturation genes in microorganisms that lack a native [FeFe]-hydrogenase can successfully result in the assembly and biosynthesis of active hydrogenases. However, other factors may be required for significantly increased protein yields and hence the specific activity of the recombinant hydrogenases. Another strategy was to overexpress one of the highly active [FeFe]-hydrogenases in a suitable E. coli host strain. Expression of a hydrogenase that can directly interact with NADPH is desirable as this, rather than reduced ferredoxin, is naturally produced by its metabolism. However, the successful maturation of this type of hydrogenase in E. coli had not been previously reported. The Desulfovibrio fructosovorans hnd operon (hndA, B, C, and D genes), encoding a NADP-dependent [FeFe]-hydrogenase, was expressed in various E. coli strains with the maturation genes hydE, hydF and hydG of Clostridium acetobutylicum. Hydrogenase activities were detected in vitro, thus a multi-subunit NADP-dependent [FeFe]-active hydrogenase was successfully expressed and matured in E. coli for the first time. Future research could lead to the expression of this hydrogenase in E. coli host strains that overproduce NADPH, setting the stage for increased hydrogen yields via the pentose phosphate pathway.

Les défis conjoints du changement climatique d'origine anthropique et la diminution des réserves de combustibles fossiles sont le moteur de recherche intense pour des sources d'énergie alternatives. Une avenue attrayante est d'utiliser un processus biologique pour produire un biocarburant. Parmi les différentes options en matière de biocarburants, le bio-hydrogène gazeux est un futur vecteur énergétique attrayant en raison de son efficacité potentiellement plus élevé de conversion de puissance utilisable, il est faible en génération inexistante de polluants et de haute densité d'énergie. Cependant, les faibles rendements et taux de production ont été les principaux obstacles à l'application pratique des technologies de bio-hydrogène. Des recherches intensives sur bio-hydrogène sont en cours, et dans les dernières années, plusieurs nouvelles approches ont été proposées et étudiées pour dépasser ces inconvénients. À cette fin, l'objectif principal de cette thèse était d'améliorer le rendement en hydrogène moléculaire avec un accent particulier sur l'ingénierie métabolique et l’utilisation de bioprocédés à variables indépendantes. Une de nos hypothèses était que la production d’hydrogène pourrait être améliorée et rendue plus économiquement viable par ingénierie métabolique de souches d’Escherichia coli producteurs d’hydrogène en utilisant le glucose ainsi que diverses autres sources de carbone, y compris les pentoses. Les effets du pH, de la température et de sources de carbone ont été étudiés. La production maximale d'hydrogène a été obtenue à partir de glucose, à un pH initial de 6.5 et une température de 35°C. Les études de cinétiques de croissance ont montré que la μmax était 0.0495 h-1 avec un Ks de 0.0274 g L-1 lorsque le glucose est la seule source de carbone en milieu minimal M9. .Parmi les nombreux sucres et les dérivés de sucres testés, les rendements les plus élevés d'hydrogène sont avec du fructose, sorbitol et D-glucose; 1.27, 1.46 et 1.51 mol H2 mol-1 de substrat, respectivement. En outre, pour obtenir les interactions entre les variables importantes et pour atteindre une production maximale d'hydrogène, un design 3K factoriel complet Box-Behnken et la méthodologie de réponse de surface (RSM) ont été employées pour la conception expérimentale et l'analyse de la souche d'Escherichia coli DJT135. Le rendement en hydrogène molaire maximale de 1.69 mol H2 mol-1 de glucose a été obtenu dans les conditions optimales de 75 mM de glucose, à 35°C et un pH de 6.5. Ainsi, la RSM avec un design Box-Behken était un outil statistique utile pour atteindre des rendements plus élevés d'hydrogène molaires par des organismes modifiés génétiquement. Ensuite, l'expression hétérologue de l’hydrogénases soluble [Ni-Fe] de Ralstonia eutropha H16 (l'hydrogénase SH) a tenté de démontrer que la mise en place d'une voie capable de dériver l'hydrogène à partir de NADH pourrait surpasser le rendement stoechiométrique en hydrogène.. L’expression a été démontrée par des tests in vitro de l'activité enzymatique. Par ailleurs, l'expression de SH a restaurée la croissance en anaérobie de souches mutantes pour adhE, normalement inhibées en raison de l'incapacité de réoxyder le NADH. La mesure de la production d'hydrogène in vivo a montré que plusieurs souches modifiées métaboliquement sont capables d'utiliser l'hydrogénase SH pour dériver deux moles d’hydrogène par mole de glucose consommé, proche du maximum théorique. Une autre stratégie a montré que le glycérol brut pourrait être converti en hydrogène par photofermentation utilisant Rhodopseudomonas palustris par photofermentation. Les effets de la source d'azote et de différentes concentrations de glycérol brut sur ce processus ont été évalués. À 20 mM de glycérol, 4 mM glutamate, 6.1 mol hydrogène / mole de glycérol brut ont été obtenus dans des conditions optimales, un rendement de 87% de la théorie, et significativement plus élevés que ce qui a été réalisé auparavant. En prolongement de cette étude, l'optimisation des paramètres a également été utilisée. Dans des conditions optimales, une intensité lumineuse de 175 W/m2, 30 mM glycérol et 4.5 mM de glutamate, 6.69 mol hydrogène / mole de glycérol brut ont été obtenus, soit un rendement de 96% de la valeur théorique. La détermination de l'activité de la nitrogénase et ses niveaux d'expression ont montré qu'il y avait relativement peu de variation de la quantité de nitrogénase avec le changement des variables alors que l'activité de la nitrogénase variait considérablement, avec une activité maximale (228 nmol de C2H4/ml/min) au point central optimal. Dans la dernière section, la production d'hydrogène à partir du glucose via la photofermentation en une seule étape a été examinée avec la bactérie photosynthétique Rhodobacter capsulatus JP91 (hup-). La méthodologie de surface de réponse avec Box-Behnken a été utilisée pour optimiser les variables expérimentales de façon indépendante, soit la concentration de glucose, la concentration du glutamate et l'intensité lumineuse, ainsi que d'examiner leurs effets interactifs pour la maximisation du rendement en hydrogène moléculaire. Dans des conditions optimales, avec une intensité lumineuse de 175 W/m2, 35 mM de glucose, et 4.5 mM de glutamate,, un rendement maximal d'hydrogène de 5.5 (± 0.15) mol hydrogène /mol glucose, et un maximum d'activité de la nitrogénase de 246 (± 3.5) nmol C2H4/ml/min ont été obtenus. L'analyse densitométrique de l'expression de la protéine-Fe nitrogenase dans les différentes conditions a montré une variation significative de l'expression protéique avec un maximum au point central optimisé. Même dans des conditions optimales pour la production d'hydrogène, une fraction significative de la protéine Fe a été trouvée dans l'état ADP-ribosylée, suggérant que d'autres améliorations des rendements pourraient être possibles. À cette fin, un mutant amtB dérivé de Rhodobacter capsulatus JP91 (hup-) a été créé en utilisant le vecteur de suicide pSUP202. Les résultats expérimentaux préliminaires montrent que la souche nouvellement conçue métaboliquement, R. capsulatus DG9, produit 8.2 (± 0.06) mol hydrogène / mole de glucose dans des conditions optimales de cultures discontinues (intensité lumineuse, 175 W/m2, 35 mM de glucose et 4.5 mM glutamate). Le statut d'ADP-ribosylation de la nitrogénase-protéine Fe a été obtenu par Western Blot pour la souche R. capsulatus DG9. En bref, la production d'hydrogène est limitée par une barrière métabolique. La principale barrière métabolique est due au manque d'outils moléculaires possibles pour atteindre ou dépasser le rendement stochiométrique en bio-hydrogène depuis les dernières décennies en utilisant les microbes. À cette fin, une nouvelle approche d’ingénierie métabolique semble très prometteuse pour surmonter cette contrainte vers l'industrialisation et s'assurer de la faisabilité de la technologie de la production d'hydrogène. Dans la présente étude, il a été démontré que l’ingénierie métabolique de bactéries anaérobiques facultatives (Escherichia coli) et de bactéries anaérobiques photosynthétiques (Rhodobacter capsulatus et Rhodopseudomonas palustris) peuvent produire de l'hydrogène en tant que produit majeur à travers le mode de fermentation par redirection métabolique vers la production d'énergie potentielle. D'autre part, la méthodologie de surface de réponse utilisée dans cette étude représente un outil potentiel pour optimiser la production d'hydrogène en générant des informations appropriées concernant la corrélation entre les variables et des producteurs de bio-de hydrogène modifiés par ingénierie métabolique. Ainsi, un outil d'optimisation des paramètres représente une nouvelle avenue pour faire un pont entre le laboratoire et la production d'hydrogène à l'échelle industrielle en fournissant un modèle mathématique potentiel pour intensifier la production de bio-hydrogène. Par conséquent, il a été clairement mis en évidence dans ce projet que l'effort combiné de l'ingénierie métabolique et la méthodologie de surface de réponse peut rendre la technologie de production de bio-hydrogène potentiellement possible vers sa commercialisation dans un avenir rapproché. / The joint challenges of anthropogenic climate change and dwindling fossil fuel reserves are driving intense research into alternative energy sources. One attractive avenue is to use a biological process to produce a biofuel. Among the various biofuel options, biohydrogen gas is an attractive future energy carrier due to its potentially higher efficiency of conversion to usable power, low to non-existent generation of pollutants and high energy density. However, low yields and production rates have been major barriers to the practical application of biohydrogen technologies. Intensive research on biohydrogen is underway, and in the last few years several novel approaches have been proposed and studied to surpass these drawbacks. To this end the main aim of this thesis was to improve the molar hydrogen yield with special emphasis of metabolic engineering using the interactive effect with bioprocess independent variable. One investigated hypothesis was that H2 production could be improved and made more economically viable by metabolic engineering on the facultative hydrogen producer Escherichia coli from glucose as well as various other carbon sources, including pentoses. The effects of pH, temperature and carbon source were investigated in batch experiments. Maximal hydrogen production from glucose was obtained at an initial pH of 6.5 and temperature of 35°C. Kinetic growth studies showed that the μmax was 0.0495 h−1 with a Ks of 0.0274 g L−1 when glucose was the sole carbon source in M9 (1X) minimal medium. Among the many sugar and sugar derivatives tested, hydrogen yields were highest with fructose, sorbitol and d-glucose; 1.27, 1.46 and 1.51 mol H2 mol−1 substrate respectively. In addition, to obtain the interactions between the variables important for achieving maximum hydrogen production, a 3K full factorial Box–Behnken design and response surface methodology (RSM) were employed for experimental design and analysis on a metabolically engineered Escherichia coli strain, DJT135. A maximum molar hydrogen yield of 1.69 mol H2 mol−1 glucose was obtained under the optimal conditions of 75 mM glucose, 35°C and pH 6.5. Thus, RSM with Box–Behnken design was a useful statistical tool for achieving higher molar hydrogen yields by metabolically engineered organisms. Furthermore, the heterologous expression of the soluble [Ni-Fe] hydrogenase from Ralstonia eutropha H16 (the SH hydrogenase) was attempted to demonstrate the introduction of a pathway capable of deriving hydrogen from NADH to surpass the stoichiometric molar hydrogen yield. Successful expression was demonstrated by in vitro assay of enzyme activity. Moreover, expression of SH restored anaerobic growth on glucose to adhE strains, normally blocked for growth due to the inability to re-oxidize NADH. Measurement of in vivo hydrogen production showed that several metabolically engineered strains were capable of using the SH hydrogenase to derive 2 mol H2 per mol of glucose consumed, close to the theoretical maximum. Using another strategy, it was shown that crude glycerol could be converted to hydrogen, a possible future clean energy carrier, by photofermentation using Rhodopseudomonas palustris through photofermentation. Here, the effects of nitrogen source and different concentrations of crude glycerol on this process were assessed. At 20 mM glycerol, 4 mM glutamate, 6.1 mol hydrogen/mole of crude glycerol were obtained under optimal conditions, a yield of 87% of the theoretical, and significantly higher than what was achieved previously. As a continuation of this study, multiprocess parameter optimization was also involved. Under optimal conditions, a light intensity of 175 W/m2, 30 mM glycerol, and 4.5 mM glutamate, 6.69 mol hydrogen/mole of crude glycerol were obtained, a yield 96% of theoretical. Determination of nitrogenase activity and expression levels showed that there was relatively little variation in levels of nitrogenase protein with changes in process variables whereas nitrogenase activity varied considerably, with maximal nitrogenase activity (228 nmol of C2H4/ml/min) at the optimal central point. In the final section, hydrogen production from glucose via single-stage photofermentation was examined with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup-). Response surface methodology with Box–Behnken design was used to optimize the independent experimental variables of glucose concentration, glutamate concentration and light intensity, as well as examining their interactive effects for maximization of molar hydrogen yield. Under optimal condition with a light intensity of 175 W/m2, 35 mM glucose, and 4.5 mM glutamate, a maximum hydrogen yield of 5.5 (±0.15) mol H2/mol glucose, and a maximum nitrogenase activity of 246 (±3.5) nmol C2H4/ml/min were obtained. Densitometric analysis of nitrogenase Fe-protein expression under different conditions showed significant variation in Fe-protein expression with a maximum at the optimized central point. Even under optimum conditions for hydrogen production, a significant fraction of the Fe-protein was found in the ADP-ribosylated state, suggesting that further improvement in yields might be possible. To this end an AmtB- derivative of Rhodobacter capsulatus JP91 (hup-) was created by conjugating in amtB::Km using the suicide vector pSUP202. Preliminary experimental results showed that the newly metabolically engineered strain, R. capsulatus DG9, produced 8.2 (±0.06) mol hydrogen/mole of glucose under optimal conditions in batch cultures (light intensity, 175 W/m2; 35 mM glucose, and 4.5 mM glutamate). Western blot analyses of the ADP-ribosylation status of the nitrogenase Fe-protein were investigated on metabolically engineered strain R. capsulatus DG9. In brief, the progress on hydrogen production technology has been limited due to the metabolic barrier. The major metabolic barrier is due to lacking of potential consistent molecular tools to reach or surpass the stochiometric biohydrogen yield since last decades using microbes. To this end a novel approach “metabolic engineering” seems very promising to overcome this constraint towards industrialization to ensure the feasibility of hydrogen production technology. In this present study it has been shown that metabolically engineered facultative (Escherichia coli) anaerobe and photosynthetic bacteria (Rhodobacter capsulatus and Rhodopseudomonas palustris) can produce hydrogen as a major product through fermentative mode by metabolic redirection toward potential energy generation. On the other hand, response surface methodology has depicted in this study as another potential tool to statistically optimize the hydrogen production by generating suitable information concerning interactive correlation between process variables and metabolically engineered biohydrogen producers. Thus, multi process parameter optimization tool has been creating a novel avenue to make a crosslink between lab scale and pilot scale hydrogen production by providing potential mathematical model for scaling up biohydrogen production using metabolically engineered biohydrogen producers. Therefore, it has been clearly revealed in this project that combined effort of metabolic engineering and response surface methodology can make biohydrogen production technology potentially feasible towards its commercialization in near future.

Heterogeneous catalysts form a highly important part of everyday life, ranging from the production of fertiliser enabling the growth of crops that sustain much of the world's population to the production of synthetic fuels. They constitute a key part of the chemical industry and contribute towards substantial economic and environmental benefits. Heterogeneous catalysts are also believed to have an important role to play in a future hydrogen economy, reducing our requirements for fossil fuels. To this end, formic acid has been proposed as a potential hydrogen storage material for small portable devices. Additionally, formic acid has historically been used as a probe molecule to study catalyst materials and recent developments in the knowledge of its decomposition pathways and the preferred sites of these reactions, establish a good foundation for further study. This work explores a range of novel modification techniques that alter the activity of Pd nanoparticles to decompose formic acid to H₂ and CO₂. The methods used are the addition of polymers, attaching various functional groups to the surface of the catalyst support and decoration of nanoparticles with sub-monolayer coverages of another metal. Using a range of characterisation methods including FTIR of an adsorbed CO probe, XRD and XPS coupled with computational modelling, it is found that these methods result in some significant electronic and/or geometric alterations to the Pd nanoparticles. For polymer modification, the nature of the pendent group is highly important in determining the effects of the polymer on the Pd particles, with all the tested polymers resulting in varying degrees of electronic donation to the Pd surface. The geometric modifications caused by the polymers also varied with pendent groups; with amine containing pendent groups found to selectively block low coordinate sites, preventing the undesired dehydration of formic acid which results in poisoning of the Pd catalyst by the resulting CO. Attachment of amine groups to the surface of metal oxide catalyst supports, is demonstrated to result in dramatic electronic promotional effects to the supported Pd nanoparticles, and when an amine polymer is attached to the support surface the geometric modification is again observed. Finally decoration of Pd nanoparticles with a sub-monolayer coverage of a second metal is examined, resulting in some similar electronic and geometric effects on Pd nanoparticle surfaces to those observed with polymer modification with corresponding changes in formic acid decomposition activity. Overall, a number of methods are displayed to tune the catalytic activity and selectivity of Pd nanoparticles for formic acid decomposition, resulting in catalysts with some of the highest reported TOF's at room temperature. These modification methods are believed to be potentially applicable to a wide range of other catalytic reactions that operate under mild conditions.

Frente a um panorama de escassez de combustível convencional fóssil e disponibilidade hidráulica para a produção de energia elétrica, fontes alternativas de energia renovável se tornam cada vez mais urgentes e necessárias. O hidrogênio é o elemento químico mais abundante disponível no planeta, tornando-se um potente candidato para suprir essa escassez de energia, pois, é capaz de conter um grande potencial energético comparável aos combustíveis fósseis, como a gasolina ou carvão. Sendo ele um vetor energético, não se tem a possibilidade de sua \"extração\" direta, porém, técnicas de obtenção do hidrogênio são bastante flexíveis, conhecidas e utilizadas comercialmente. Este trabalho teve como objetivo o estudo e construção de um sistema para produção de energia limpa a partir da produção de hidrogênio gasoso por eletrólise da água em meio alcalino, fazendo uso de um painel fotovoltaico como fonte alternativa de energia elétrica, bem como, a análise da eficiência energética desta produção. Para garantir a obtenção dos gases hidrogênio e oxigênio separadamente foi utilizada uma membrana separadora de gases, o Zirfon® Perl, permitindo a eletrólise pelo eletrolisador projetado e construído especificamente para este estudo, sem a necessidade de um sistema de circulação do eletrólito. Para uma análise comparativa das eficiências, produziu-se hidrogênio por eletrólise alcalina de uma solução aquosa de KOH (1M) com energia fornecida por uma fonte externa DC e por um painel solar ligado diretamente ao eletrolisador. O cálculo dessa eficiência foi feito pela relação entre a energia fornecida à eletrólise pelas fontes (solar e externa) e a energia do hidrogênio produzido com base no seu poder calorífico inferior (LHV). Além disso, curvas de carga de tensão e corrente do painel solar foram levantadas e estudadas para garantir a efetividade da ligação direta com o eletrolisador, bem como ensaios de EIE (Espectroscopia de Impedância Eletroquímica) foram realizados para análise da corrosão do eletrodo anódico e determinação da resistência ôhmica do sistema eletrolisador. Foram realizadas medidas e obtidas a eficiência energética com quatro montagens de sistemas eletrolisadores: 4 células com suprimento de energia por uma fonte externa DC, resultando em uma eficiência de 48,21%; 4 células e painel solar, 44,85%; 1 célula e fonte externa, 23,52 %; 1 célula e painel solar, 23,10%. Também foram calculadas eficiências energéticas da produção do hidrogênio em função do aumento de energia fornecida à eletrólise. Todas as aquisições dos parâmetros da eletrólise foram realizadas com um sistema automático projetado e construído especificamente para este trabalho, de forma que os dados foram obtidos a cada segundo, fornecendo valores em tempo real. Os resultados obtidos, tanto sobre os parâmetros importantes para que uma eletrólise para produção de hidrogênio possa se tornar eficiente energeticamente, como sobre as soluções para o desenvolvimento de um sistema com painel fotovoltaico que alimente diretamente o eletrolisador, serão de vital importância para continuação da pesquisa que envolva a utilização otimizada do hidrogênio em locais onde a energia elétrica proveniente de concessionárias seja escassa ou até mesmo inexistente. / The scenario of conventional fuel shortages (fossil and hydraulic availability for the production of electricity) shows us that alternative sources of renewable energy become more urgent and necessary. Hydrogen is the most abundant chemical element available on the planet, making it a potent candidate to address this energy shortage, since it is able to contain a large energy potential comparable to fossil fuels such as gasoline or coal. Being an energetic vector, one does not have the possibility of its direct \"extraction\", but techniques of obtaining hydrogen are quite flexible, known and used commercially. The objective of this work was the study and construction of a system for the production of clean energy from the production of gaseous hydrogen by electrolysis of water in alkaline medium, making use of a photovoltaic panel and an external source of electric energy, as well as the analysis the energy efficiency of this production. In order to guarantee the hydrogen and oxygen gases separately, a gas separation membrane, Zirfon® Perl, was used allowing electrolysis by the electrolyzer, designed and built specifically for this study, without the need for an electrolyte circulation system. For a comparative analysis of efficiencies, hydrogen was produced by alkaline electrolysis of an aqueous solution of KOH (1M) with energy supplied by an external DC source and by a solar panel connected directly to the electrolyser. The calculation of this efficiency was made by the relation between the energy supplied to the electrolysis by the sources (solar and external) and the energy of the hydrogen produced based on its Lower Heat Value (LHV). In addition, voltage and current load curves of the solar panel were collected and studied to ensure the effectiveness of the direct connection with the electrolyser, as well as IEE (Electrochemical Impedance Spectroscopy) tests were carried out to analyze the corrosion of the anodic electrode and determination of the ohmic resistance of the electrolyser system. Measurements and energy efficiency were obtained with four electrolyser systems assemblies: 4 cells with energy supply by an external DC source, resulting in an efficiency of 48.21%; 4 cells and solar panel, 44.85%; 1 cell and external source, 23,52%; 1 cell and solar panel, 23.10%. Energy efficiencies of hydrogen production were also calculated as a function of the increase in energy supplied to the electrolysis. All the electrolysis parameters were acquired with an automated system designed and built specifically for this work, so that the data were obtained every second, providing real-time values. The results obtained in this work, both on the important parameters so that an electrolysis for hydrogen production can become energy efficient, and on the solutions for the development of a system with photovoltaic panel that directly feed the electrolyser, will be of vital importance for continuation of the research that involves the optimized use of hydrogen in places where the electric energy coming from concessionaires is scarce or even non-existent.