Microbial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individ- ual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.

Liquid biofuels produced from biomass have the potential to partly replace gasoline. One of the most promising biofuels is butanol which is produced in acetone-butanol-ethanol (ABE) fermentation. The ABE fermentation is characterized by its low butanol concentration in the final fermentation broth. In this research, the simulation of three in situ recovery methods, namely, vacuum fermentation, gas stripping and pervaporation, were performed in order to increase the efficiency of the continuous ABE fermentation by decreasing the effect of butanol toxicity. The non-integrated and integrated butanol production systems were simulated and optimized based on a number of objectives such as maximizing the butanol productivity, butanol concentration, and butanol yield. In the optimization of complex industrial processes, where objectives are often conflicting, there exist numerous potentially-optimal solutions which are best obtained using multi-objective optimization (MOO). In this investigation, MOO was used to generate a set of alternative solutions, known as the Pareto domain. The Pareto domain allows to view very clearly the trade-offs existing between the various objective functions. In general, an increase in the butanol productivity resulted in a decrease of butanol yield and sugar conversion. To find the best solution within the Pareto domain, a ranking algorithm (Net Flow Method) was used to rank the solutions based on a set of relative weights and three preference thresholds. Comparing the best optimal solutions in each case study, it was clearly shown that integrating a recovery method with the ABE fermentation significantly increases the overall butanol concentration, butanol productivity, and sugar conversion, whereas butanol yield being microorganism-dependent, remains relatively constant.

Th. 3e cycle--Microbiol., biotechnol.--Toulouse--I.N.S.A., 1982. N°: 22.

Made available in DSpace on 2014-10-09T12:23:12Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:05Z (GMT). No. of bitstreams: 1 00868.pdf: 8105372 bytes, checksum: 760345cfee59eb2c2b451f97a789a035 (MD5) / Tese (Doutoramento) / IEA/T / Instituto de Fisica, Universidade de Sao Paulo - IF/USP

abstract: Emergent environmental issues, ever-shrinking petroleum reserves, and rising fossil fuel costs continue to spur interest in the development of sustainable biofuels from renewable feed-stocks. Meanwhile, however, the development and viability of biofuel fermentations remain limited by numerous factors such as feedback inhibition and inefficient and generally energy intensive product recovery processes. To circumvent both feedback inhibition and recovery issues, researchers have turned their attention to incorporating energy efficient separation techniques such as adsorption in in situ product recovery (ISPR) approaches. This thesis focused on the characterization of two novel adsorbents for the recovery of alcohol biofuels from model aqueous solutions. First, a hydrophobic silica aerogel was evaluated as a biofuel adsorbent through characterization of equilibrium behavior for conventional second generation biofuels (e.g., ethanol and n-butanol). Longer chain and accordingly more hydrophobic alcohols (i.e., n-butanol and 2-pentanol) were more effectively adsorbed than shorter chain alcohols (i.e., ethanol and i-propanol), suggesting a mechanism of hydrophobic adsorption. Still, the adsorbed alcohol capacity at biologically relevant conditions were low relative to other `model' biofuel adsorbents as a result of poor interfacial contact between the aqueous and sorbent. However, sorbent wettability and adsorption is greatly enhanced at high concentrations of alcohol in the aqueous. Consequently, the sorbent exhibits Type IV adsorption isotherms for all biofuels studied, which results from significant multilayer adsorption at elevated alcohol concentrations in the aqueous. Additionally, sorbent wettability significantly affects the dynamic binding efficiency within a packed adsorption column. Second, mesoporous carbons were evaluated as biofuel adsorbents through characterization of equilibrium and kinetic behavior. Variations in synthetic conditions enabled tuning of specific surface area and pore morphology of adsorbents. The adsorbed alcohol capacity increased with elevated specific surface area of the adsorbents. While their adsorption capacity is comparable to polymeric adsorbents of similar surface area, pore morphology and structure of mesoporous carbons greatly influenced adsorption rates. Multiple cycles of adsorbent regeneration rendered no impact on adsorption equilibrium or kinetics. The high chemical and thermal stability of mesoporous carbons provide potential significant advantages over other commonly examined biofuel adsorbents. Correspondingly, mesoporous carbons should be further studied for biofuel ISPR applications. / Dissertation/Thesis / M.S. Chemical Engineering 2011

Um den CO2-Ausstoß von Kraftfahrzeugen zu reduzieren, ist die Beimischung von biogenen Kraftstoffen zum fossilen Ottokraftstoff interessant. Der Einsatz dieser führt zum veränderten Rußpartikelausstoß, welcher aufgrund der zunehmend verschärften gesetzlichen Regulierungen im Automobilsektor hinsichtlich Partikelmasse und -anzahl begrenzt ist. In der Arbeit werden Flammenstruktur und Rußbildungsprozess bei der Anwendung flüssiger Brennstoffe mit biogenem Anteil in laminaren, vorgemischten Modellflammen charakterisiert. Durch die Verwendung verschiedenster Messtechniken werden experimentelle Referenzdaten ohne den Einfluss von komplexen Wechselwirkungen, hervorgerufen durch den motorischen Betrieb, erhoben. Anhand der gemessenen Partikelgrößenverteilungen wird der chemisch hemmende Einfluss der biogenen Komponenten Ethanol und Butanol auf die Rußbildung gezeigt. Diese wird verlangsamt, sodass die Partikeldurchmesser abnehmen und die Rußmenge deutlich minimiert wird, wobei bzgl. der Gesamtpartikelanzahl kein eindeutiger Einfluss erkennbar ist.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciencias Fisicas e Matematicas / Made available in DSpace on 2012-10-17T03:10:46Z (GMT). No. of bitstreams: 0Bitstream added on 2016-01-08T21:44:38Z : No. of bitstreams: 1 108797.pdf: 2401340 bytes, checksum: ff9434a0ed4c94373cf186fbc1fc1555 (MD5) / Os efeitos do n-butanol sobre propriedades micelares, estruturais e catalíticas do dodecilsulfato de sódio (SDS) e dodecanoato de sódio (SDOD) foram investigados e comparados com respeito à influência do grupo cabeça dos surfactantes. Foram determinados os valores de concentração micelar crítica (CMC), grau de ionização (a), pH aparente (pHap) e polaridade destes detergentes, em soluções aquosas na ausência e na presença de n-butanol. O número de agregação micelar médio () do SDOD e o de micelas mistas SDOD/n-butanol foram determinados usando o método de Turro. Os efeitos da adição de n-butanol nas propriedades catalíticas de soluções aquosas 0,10 M de SDS (pH 9,15) e 0,10 M de SDOD (pH 9,05), a 25,0°C foram estudados através de medidas de constante de velocidade observada (Kobs) para a reação de hidrólise de anidrido benzóico.

The increased demand for an alternative form of fuel has raised a great interest towards exploring various metabolic pathways and enzymes in several microbial species for hydrocarbon production. In recent years, cyanobacteria have emerged as an attractive microbial host and cyanobacterial metabolic pathways were targeted for engineering to produce "drop in" fuels such as propane and butane. Whilst appealing, practicalities for producing biofuels in cyanobacteria remain challenging, requiring the identification and engineering of natural biocatalysts and their integration into metabolic processes. Cyanobacterial hydrocarbon biosynthesis arises from fatty acid metabolism involving a potential enzyme, aldehyde deformylating oxygenase (ADO), which catalyses the decarbonylation of long-chain fatty aldehydes to alkanes, mainly in the conversion of octadecanal (C17H35CHO) to heptadecane (C17H36) and formate. The substrate specificity and preferences for long-chain aldehyde by ADO necessitates a detailed kinetic and structural characterisation in order to optimise/engineer this enzyme for future biotechnological applications. Thus, the main objective was to identify a potential ADO enzyme that can be optimised for shorter chain alkane production. By studying the substrate specificity and reaction kinetics of different ADO enzymes, it was found that ADO from Prochlorococcus marinus MIT 9313 (PmADO) is a potential target for short chain alkane production. The crystal structural of PmADO was solved and further GC-MS analysis was carried out to identify the chemical origin of a mixture of long-chain fatty acid in the active site, originated from E. coli cells during recombinant over-expression and purification. It was suggested that the structure-guided protein engineering for short-chain alkane production should be carried out along with the removal of this adventitious ligand from the active site in order to increase the alkane production. Four important residues present at the entrance of the ligand-binding cavity were targeted and saturated mutagenesis was performed on PmADO to identify variants that excluded the long fatty acid ligands from the active site but have specificity and higher conversion rates for shorter chain aldehydes. This identified two variants, V41Y and A134F, with the A134F variant that not only exhibiting an improved activity and turnover value of PmADO by four-fold but also improved binding affinity for butyraldehyde by 2 times. Finally the improved variants were incorporated in a host organism (E. coli) and the possibilities for the development of a microbial platform for renewable propane synthesis based on a fermentative clostridial butanol pathway were explored. Four pathways were designed namely atoB-adhE2, atoB-TPC7, nphT7-adhE2 and nphT7-TPC7 routes, which utilise CoA intermediates selected to incorporate ADO as the terminal enzyme. When PmADO was co-expressed with these pathways, the engineered E. coli host produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed A134F variant ofPmADO with an enhanced specificity towards short-chain substrates, and (iii) including a ferredoxin-based electron supply system, the propane titre was increased up to 3.40 ± 0.19 mg/L. It was also shown that the best propane producing pathways are scalable in a 250 mL flask and in a large-scale (up to 30 L) fermentor setup. This thesis focuses on the detailed kinetics and structure-guided characterisation and engineering studies on the ADO enzyme for the development of a renewable microbial biofuel platform.

Orientador: Maria Alvina Krahenbuhl / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-12T02:24:53Z (GMT). No. of bitstreams: 1 Ultchak_FlavianaSalles_M.pdf: 2164181 bytes, checksum: f1468bdd0cb3489326bd20f27ce9101c (MD5) Previous issue date: 2000 / Resumo: Dados de equilíbrio de fases são úteis no projeto e otimização de processos que envolvem separação de fases, como destilação e extração. Malonatos são substâncias orgânicas que atuam como importantes intermediários em várias reações, como na produção de tintas, fármacos, pesticidas e aromas. Mesmo assim, as propriedades físicas dos malonatos são escassas na literatura. Este trabalho tem como objetivo obter as curvas de equilíbrio líquido-vapor isobáricas para três sistemas: dimetilmalonato/n-butanol a 100 e 20OmmHg, dietilmalonato/n-butanol a 200mmHg. Foi utilizado um ebuliômetro de recirculação de fases, da marca comercial "Normag". As concentrações das fases líquidas e vapor foram obtidas por cromatografia gasosa. Observou-se que ocorria uma reação química durante os experimentos, mas que não impediu o desenvolvimento do trabalho. A consistência termodinâmica foi testada pelo método de van Ness-Fredenslund (1977) e os parâmetros de interação binária do coeficiente de atividade para os modelos Wilson, UNIQUAC e NRTL foram estimados pelo método da máxima verossimilhança de acordo com Stragevitch (1997). Apesar da reação química, os dados obtidos são consistentes de acordo com os critérios adotados, mas observou-se que com o aumento da ftação molar do malonato presente, a partir de 0,5, a qualidade dos dados foi prejudicada pela reação química / Abstract: Phase equilibrium data are useful in the project and optimization of process that inc1udes phases separation, such as distillation and extraction. Malonates are organic substances that participate as an important intennediate in production reactions, like as ink, pharmaceuticals, pesticides and ftagrances. However their properties are rare in scientific literature. In this work the isobaric curves ofvapor-liquid equilibrium were measured for three systems: (I) dimethyl malonate+l-butanol at 100 mmHg, (2) dimethyl malonate+l-butanol at 200 mmHg and (3) diethyl malonate+l-butanol at 200 mmHg. Each system was measured by means of an ebuliometer with phase circulation (NORMAG). The liquid and vapor phase concentration was obtained by gaseous chromatography. A chemical reaction was verified during the experiments, but it didn't impede the work development. The thermodynamic consistency of the data was tested using the van Ness-Fredenslund method (1977) and the binary interaction parameters ofthe activity coefficient for the WILSON, UNIQUAC and NRTL models were calculated by the Maximum Likelihood Method in agreement with Stragevitch (1997). However a reaction inside of the equipment occurred during the experimental measurements, the results were considered consistent in agreement with the adopted rules in this work, but was verified that the data quality was impaired by chemical reaction with the increase ofmolar ftaction ofthe malonate, after 0,5 / Mestrado / Desenvolvimento de Processos Químicos / Mestre em Engenharia Química

Fossil based energy resources are dominating the world’s primary energy consumption for the last century. However, with decreasing crude oil reservoirs and the role they play in global warming by emitting greenhouse gases, the focus has been turned towards improved utilization of renewable resources and the need for new, sustainable fuels and chemicals is more urgent than ever. Biomass is a carbon neutral resource that can be used to produce biofuels and other useful chemicals. One such chemical is 1-butanol (or simply butanol), which has great potential as a gasoline substitute because of its favorable fuel properties. Butanol can be produced from acetone, butanol and ethanol (ABE) fermentation using e.g. Clostridium acetobutylicum. However, the concentration of butanol in fermentation in the resulting broth is limited to ca. 20 g/L due to its toxicity for microorganisms. Butyric acid is a precursor to butanol, which is produced prior to butanol in ABE fermentation. Butyric acid is an important industrial chemical, which can be further converted into a number of commercial compounds e.g. acetate butyrate, butyl acetate and butanol. Arginine is a semi-essential amino acid that has vast applications in the field of pharmaceutical and food industry. In addition, arginine can replace inorganic nitrogen as nitrogen source in fertilizers. It can be produced via fermentation of sugars using engineered microorganism like E. Coli, but like butanol its concentration is restricted to approximately 12 g/L. Due to low concentration of these useful chemicals in the resulting fermentation broths recovery of these chemicals remain challenging with today’s options and therefore  novel recovery process should be developed. In this study, zeolite adsorbents were used to recover butanol, butyric acid and arginine from model and real fermentation broths. Zeolite MFI adsorbent efficiently adsorbed butanol from model solutions with a saturation loading of 0.11 g/g- zeolite. On the other hand, adsorption of butyric acid was found to be strongly pH dependent, with high adsorption below and little adsorption above the pKa value of the acid. A structured adsorbent in the form of steel monolith coated with a silicalite-1 film was also used and performance was evaluated by performing breakthrough experiments at room temperature using model ABE fermentation broths and the results were compared with those obtained using traditional adsorbent sin the form of beads. Desorption studies showed that a high quality butanol product with purity up to 95.2% for butanol-water system and 88.5% for the ABE system can be recovered with the structured silicalite-1 adsorbent. Further, zeolite X adsorbents in the form of powder and extrudates was used to recover arginine from a real fermentation broth and also from aqueous model solutions. To the best of our knowledge, this is the first time recovery of arginine from real fermentation broths using any type of adsorbent is reported. Arginine loading of 0.15 g/g was observed at pH 11 using zeolite X powder. The selectivity for arginine over ammonia and alanine from the fermentation broth at pH 11 was 1.9 and 8.3, respectively, for powder and 1.0 and 4.1, respectively, for extrudates. Synthesis gas (CO + H2) can be produced e.g.by gasification of lignocellulose biomass. This synthesis gas can be used to produce methanol, which subsequently may be converted into gasoline using zeolite ZSM-5 catalyst. However, during Methanol to Gasoline (MTG) process, undesirable carbon residue (coke) is formed that gradually reduces the activity of catalyst. It was hypothesized that intracrystalline defects in the zeolite formed during conventional synthesis may accelerate the deactivation rate by coke formation. In this work, a novel ZSM-5 zeolite catalyst essentially free of intracrystalline defects was synthesized and evaluated in the  MTG reaction,. The novel catalyst showed significantly higher resistance towards deactivation by coke formation as compared to a reference catalyst containing defects.