Rozprawy doktorskie na temat „Production engineering”

This thesis addresses the need for environmentally and socially responsible sources of energy. Biofuels, made from organic matter, have recently become a viable alternative to petroleum-based fossil fuel. Sugar and starch make up the majority of feedstock used in biofuel production as it is easily digested. However, the use of these feedstocks is problematic as they consume resources with negative implications. By using a bacterium able to utilise five and six carbon sugars, such as the thermophile Geobacillus thermoglucosidans, organic lignocellulosic waste material can be used as a feedstock. The aim of this project was to investigate and utilise key genetic regulators of fermentation in G. thermoglucosidans and to construct genetic engineering tools that enable strain development for second generation biofuel production. We have focused on the redox-sensing transcriptional regulator Rex, widespread in Grampositive bacteria, which controls the major fermentation pathways in response to changes in cellular NAD+/NADH ratio. Following the identification of several members of the Rex regulon via bioinformatics analysis, ChIP-seq and qRT-PCR experiments were performed to locate genome-wide binding sites and controlled genes in G. thermoglucosidans. Initial electromobility shift assay experiments were performed to demonstrate the potential for use of Rex from Clostridium thermocellum as an orthogonal regulator. To further this research, novel in vivo synthetic regulatory switches were designed and tested with the aim of controlling gene expression in response to changes in cellular redox state. In addition, new tools for the efficient genetic engineering of G. thermoglucosidans were produced and optimised, including an E. coli-G. thermoglucosidans conjugation method for plasmid transfer and gene disruption.

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Addressing a number of critical challenges caused by centralised production and large scale distribution infrastructures, local production systems designed in a synergistic manner could offer a possible pathway towards sustainability. The thesis focuses on the technical design of local production systems integrating local heterogeneous processes to satisfy local demands through efficient use of locally available renewable resources within technical and ecological constraints. A conceptual and quantitative multi-level framework, based on the Cumulative Exergy Resource Accounting methodology, was first developed for a better understanding of a local production system by considering the production and consumption of products or services as well as ecological processes. A general design framework comprising an optional preliminary design stage followed by a simultaneous design stage based on mathematical optimisation was then developed for solving the design problem towards minimum overall resource consumption. The preliminary design stage considers each supply subsystem individually and allows insights into the potential interactions between them. The simultaneous design stage has the capacity to include all design integration possibilities. A second, insight-based approach was further developed, which offers a new hierarchical and iterative decision and analysis procedure and incorporates design principles and ability to examine design decisions. The multilevel resource accounting framework was demonstrated on ethanol production from cane and successfully revealed how decisions at one level would affect other levels of the system. Both design approaches were illustrated on a case study for the design of local food-energy-water nexus. It showed the advantages of an integrated design of a system which makes use of local resources to meet its demands over a system relying on centralised supplies and over a design without considering integration opportunities between subsystems. The insight-based approach was also found to produce a comparable design to the simultaneous design approach while offering more valuable insights for decision makers.

Biobutanol represents a second generation biofuel, which can be producedfrom renewable resources by microorganisms. A Saccharomyces cerevisiae strainbearing the five butanol synthetic genes (hbd, adhe2, crt, ccr and ERG10) wasconstructed, where the hbd, adhe2, crt and ccr genes are derived from Clostridiumbeijerinckii, while ERG10 is a yeast gene. The genes were transformed individually onsingle cassettes, which integrated into specific chromosomal sites. The single integrantstrains were back‐crossed to create a strain bearing all five butanol synthetic genes. The butanol synthetic enzymes appeared to be highly expressed in the cytosol,however, very little butanol was obtained (< 10 ppm). Therefore, additional geneticmanipulations were made with a view to restoring any redox imbalance channellingthe carbon flux toward the butanol pathway. Deletion of the ADH1 gene in strains withthe butanol pathway improved production to ~250 ppm (203 mg/L) butanol. Furtherimprovement to 360 ppm (292 mg/L) was gained by overexpressing the ALD6 and ACS2genes, that are involved in synthesis of acetyl‐CoA; the precursor for butanolbiosynthesis. However, the replacement of ALD6 with ALD2, which produces NADHinstead of NADPH, didn’t improve butanol yields. In addition, no significantimprovement of butanol yield was obtained when dehydrogenase enzymes from theglycerol biosynthetic pathway were deleted. An initial assessment of the bestconditions for butanol production were semi‐anaerobic growth at 30°C in 2% glucosewith a starting OD600 of 0.1.In this project, another key question was addressed: does the sensitivity of cellsto short chain alcohols like butanol affect butanol production? Previous work in theAshe lab has identified specific point mutations in the translation initiation factor,eIF2B, which generate resistance or sensitive phenotypes to exogenously addedbutanol. Here a comparison of butanol production in sensitive and resistantbackgrounds showed that the butanol yield was 1.5‐2 fold higher in a butanol resistantstrain compared to the sensitive mutant. Generating a ‘super’ butanol resistant strainbearing a GCD2‐S131A mutation in eIF2B promoted a higher butanol yield per cell. However, another consequence of this mutation was reduced growth. So thecombination of these effects meant that the overall butanol concentration in mediawas similar to the control. Overall this work highlights that S. cerevisiae can producebutanol but that further optimisation both at the level of the strain and processengineering would be necessary before this would be of interest to the commercialsector.

The use of microalgae culture to convert CO2 from power plant flue gases into biomass that are readily converted into biofuels offers a new frame of opportunities to enhance, compliment or replace fossil-fuel-use. Apart from being renewable, microalgae also have the capacity to utilise materials from a variety of wastewater and the ability to yield both liquid and gaseous biofuels. However, the processes of cultivation, incorporation of a production system for power plant waste flue gas use, algae harvesting, and oil extraction from the biomass have many challenges. Using SimaPro software, Life cycle Assessment (LCA) of the challenges limiting the microalgae (Chlorella vulgaris) biofuel production process was performed to study algae-based pathway for producing biofuels. Attention was paid to material use, energy consumed and the environmental burdens associated with the production processes. The goal was to determine the weak spots within the production system and identify changes in particular data-set that can lead to and lower material use, energy consumption and lower environmental impacts than the baseline microalgae biofuel production system. The analysis considered a hypothetical transesterification and Anaerobic Digestion (AD) transformation of algae-to- biofuel process. Life cycle Inventory (LCI) characterisation results of the baseline biodiesel (BD) transesterification scenario indicates that heating to get the biomass to 90% DWB accounts for 64% of the total input energy, while electrical energy and fertilizer obligations represents 19% and 16% respectively. Also, Life Cycle Impact Assessment (LCIA) results of the baseline BD production scenario show high proportional contribution of electricity and heat energy obligations for most impact categories considered relative to other resources. This is attributed to the concentration/drying requirement of algae biomass in order to ease downstream processes of lipid extraction and subsequent transesterification of extracted lipids into BD. Thus, four prospective alternative production scenarios were successfully characterised to evaluate the extent of their impact scenarios on the production system with regards to lowering material use, lower energy consumption and lower environmental burdens than the standard algae biofuel production system. A 55.3% reduction in mineral use obligation was evaluated as the most significant impact reduction due to the integration of 100% recycling of production harvest water for the AD production system. Recycling also saw water demand reduced from 3726 kg (freshwater).kgBD- 1 to 591kg (freshwater).kgBD- 1 after accounting for evaporative losses/biomass drying for the BD transesterification production process. Also, the use of wastewater/sea water as alternative growth media for the BD production system, indicated potential savings of: 4.2 MJ (11.8%) in electricity/heat obligation, 10.7% reductions for climate change impact, and 87% offset in mineral use requirement relative to the baseline production system. Likewise, LCIA characterisation comparison results comparing the baseline production scenarios with that of a set-up with co-product economic allocation consideration show very interesting outcomes. Indicating -12 MJ surplus (-33%) reductions for fossil fuels resource use impact category, 52.7% impact reductions for mineral use impact and 56.6% reductions for land use impact categories relative to the baseline BD production process model. These results show the importance of allocation consideration to LCA as a decision support tool. Overall, process improvements that are needed to optimise economic viability also improve the life cycle environmental impacts or sustainability of the production systems. Results obtained have been observed to agree reasonably with Monte Carlo sensitivity analysis, with the production scenario proposing the exploitation of wastewater/sea water to culture algae biomass offering the best result outcome. This study may have implications for additional resources such as production facility and its construction process, feedstock processing logistics and transport infrastructure which are excluded. Future LCA study will require extensive consideration of these additional resources such as: facility size and its construction, better engineering data for water transfer, combined heat and power plant efficiency estimates and the fate of long-term emissions such as organic nitrogen in the AD digestate. Conclusions were drawn and suggestions proffered for further study.

The acquisition of spider silk is a complex and costly process that restricts its availability. Increasing applications stemming from the biomedical and pharmaceutical sectors is driving the demand higher, necessitating the need for efficient large-scale production. This thesis investigates 1) recombinant protein expression systems, 2) major ampullate gland cell culture techniques for natural silk production, and 3) process optimization of recombinant silk protein expression. Using a process engineering analysis, the current E.coli system expression system was found to be a cost-effective and efficient technique for silk production. While a Box-Behnken predictive model was developed to optimize expression conditions based on small-scale E.coli expression data, it failed to translate to a larger-scale. Alternatively, the protein secreting cells that line the major ampullate silk gland were isolated and grown in conditions mimicking the native microenvironment, demonstrating a clear impact on growth of the cells and a potential new source of silk.

Mestrado em Biotecnologia
Bioplastics have been the focus of interest as a sustainable alternative to conventional plastics. Among those, polyhydroxyalkanoates (PHA) can be highlighted, not only for their biocompatibility and biodegradability, but also because they can be produced by mixed microbial cultures (MMC) from agro-industrial wastes. This allows to substantially reduce the production costs and valorize alternative substrates. PHA have a wide range of characteristics according to their composition, which allows them to be used in many applications. The polymers characteristics can be manipulated through the control of several operational parameters during the production process. Production of PHA by MMC in this work was based in a three-stage process: acidification of a by-product of the paper industry, hardwood spent sulphite liquor (HSSL), selection of a PHA accumulating microbial culture and PHA production. The selection step occurred in a sequencing batch reactor (SBR), operated for 180 days, and whose conditions were changed in order to select for a PHA-accumulating culture and with good PHA volumetric production. Three pseudo-stationary states (PSS) were achieved after successive increases in the selective pressure, a clear indication that the MMC was able to adapt to the substrate and to the imposed conditions. In the last step of this work several accumulation assays were performed that allowed for the validation of the use of HSSL acidified under different conditions and Condensate (another byproduct of the paper industry) for PHA production. The best test performed achieved a maximum accumulation of 74.7% cdw and a volumetric productivity of 0.27 gPHA/L.h. This work allowed to show the potential of the use of PHA producing MMC as a way of valorization of agroindustrial byproducts and residues.
Os bioplásticos têm sido foco de interesse como alternativa sustentável aos plásticos convencionais. Entre os vários biopolímeros destacam-se os polihidroxialcanoatos (PHA), não só pela sua biocompatibilidade e biodegradabilidade, mas também porque podem ser produzidos por culturas microbianas mistas (MMC) a partir de resíduos agroindustriais. Desta forma é possível reduzir substancialmente o preço de produção destes polímeros e valorizar substratos alternativos. Os PHA apresentam características muito variadas de acordo com a sua composição, o que permite que sejam utilizados em diversas aplicações. As características do polímero podem ser manipuladas através do controlo de vários parâmetros operacionais durante o processo de produção. A produção de PHA por MMC neste trabalho foi feita com recurso a um processo em três fases: acidificação de um subproduto da indústria papeleira, o licor de cozimento ao sulfito ácido acidificado (HSSL), seleção de uma cultura microbiana acumuladora de PHA e produção de PHA. A seleção ocorreu num reator descontínuo sequencial (SBR), operado durante 180 dias, e cujas condições foram alteradas de forma a selecionar uma cultura acumuladora de PHA e com boa produtividade volumétrica de PHA. Três estados pseudo-estacionários (PSS) foram atingidos após sucessivos aumentos na pressão seletiva, uma indicação clara de que a MMC foi capaz de se adaptar ao substrato e às condições impostas. No último passo do trabalho foram realizados vários testes de acumulação que permitiram validar a utilização de HSSL acidificado em condições diferentes e Condensado (outro subproduto da índustria papeleira) como substratos para a produção de PHA. O melhor teste realizado apresentou uma acumulação máxima de 74.4% cdw e uma produtividade volumétrica de 0.27 gPHA/L.h. Este trabalho permitiu mostrar a potencialidade do uso de MMC produtoras de PHA como forma de valorização de subprodutos e resíduos agroindustriais.

System integration is a key enabler to maximize information value in an engineering context. The valuable information is normally represented by information models which play a decisive role in the implementation of system integration. The information models are designed to efficiently and effectively capture, process and communicate information among different functional and business units. However, use of the information models in implementing system integration is challenged by insufficient support from current settings of modeling architectures. This situation calls for new strategies to ease the use of information models. To address this challenge, this study presents a new twofold solution: Model driven system integration. It includes 1) a modeling architecture to guide the development of information models and 2) an integrated implementation process to guide the use of information models. Thus, this work improves practical applicability of an information model in its entire modeling lifecycle. The results contribute not only to the performance of modeling practices but also to improved understanding of information modeling in system integration. Implementation contexts and implementation models are introduced to develop an implementation-oriented modeling architecture. Further, the potential of information models as a knowledge base to sup-port implementation practices is identified. To concretely discuss behaviors and structures of information models, this study adopts ISO 10303 and the related standards as major references of existing information models. Case studies on model driven system integration validate this research in scenarios concerning kinematic modeling, kinematic error modeling, cutting tools classification and product catalogue modeling. Model driven system integration exhibits high efficiency in implementation, enhanced interoperability and increased value of information models.

QC 20170519

MPQP - Model driven process and quality planning
FBOP - Feature Based Process Planning
DFBB - Digital factory building blocks

The work described was carried out as part of a collaborative Alvey software engineering project (project number SE057). The project collaborators were the Inter-Disciplinary Higher Degrees Scheme of the University of Aston in Birmingham, BIS Applied Systems Ltd. (BIS) and the British Steel Corporation. The aim of the project was to investigate the potential application of knowledge-based systems (KBSs) to the design of commercial data processing (DP) systems. The work was primarily concerned with BIS's Structured Systems Design (SSD) methodology for DP systems development and how users of this methodology could be supported using KBS tools. The problems encountered by users of SSD are discussed and potential forms of computer-based support for inexpert designers are identified. The architecture for a support environment for SSD is proposed based on the integration of KBS and non-KBS tools for individual design tasks within SSD - The Intellipse system. The Intellipse system has two modes of operation - Advisor and Designer. The design, implementation and user-evaluation of Advisor are discussed. The results of a Designer feasibility study, the aim of which was to analyse major design tasks in SSD to assess their suitability for KBS support, are reported. The potential role of KBS tools in the domain of database design is discussed. The project involved extensive knowledge engineering sessions with expert DP systems designers. Some practical lessons in relation to KBS development are derived from this experience. The nature of the expertise possessed by expert designers is discussed. The need for operational KBSs to be built to the same standards as other commercial and industrial software is identified. A comparison between current KBS and conventional DP systems development is made. On the basis of this analysis, a structured development method for KBSs in proposed - the POLITE model. Some initial results of applying this method to KBS development are discussed. Several areas for further research and development are identified.

This final thesis was performed at Ecole Nationale Supériure D’Arts et Métiers (ENSAM) in Lille, France. The aim of the thesis has been to use two different simulation tools to analyse an existing production cell with focus on industrial engineering. The possible use and the usability of the simulation tools are also studied. The models built for simulation have been used to gather data about the cell. After analyse and discussion about the data we came to the following conclusions. The bottleneck in the cell is the Stäubli. A cheap and simple way to improve the cell is by adding new decision points (sensors) to it. The most efficient location of the new decision points is before and after the Stäubli. The production rate reaches its maximum rate for both settings with eight pallets. If the improvements are implemented then there is no reason to change the speed of the conveyor. With basic settings the speed can be increased for better productivity. The most efficient production type for short setup times is single, for both settings. Delmia is useful for visual representation. It’s also useful for measurements of time and distance since the accuracy is very high. Delmia is useful as common platform when to discuss and explain thoughts and ideas about a project. Flow simulation in Quest provides a great understanding of the production and the behaviour of the cell. It is very easy to get data in and out of the program and to compare results and impacts of changes.

Due to technological advancements, the role of the musician has changed dramatically in the 20th and 21st centuries. For the composer or songwriter especially, it is becoming increasingly expected for them to have some familiarity with music production and engineering, so that they are able to provide a finished product to employers, clients, or listeners. One goal of a successful production or engineered recording is to most effectively portray the recorded material. Music theory, and specifically analysis, has the ability to reveal important or expressive characteristics in a musical work. The relationship between musical analysis and production is explored to discover how music analysis can provide a more effective and informed musical production or recording and how a consideration of music production elements, notably timbre and instrumentation, can help to better inform a musical analysis. Two supplemental MP3 files are included with this thesis to demonstrate proposed mixing guidelines derived from the analysis.

Microalgal biotechnology has attracted considerable interest owing to it is potential to provide renewable energy and its capacity to produce molecules such as pigments, fatty acids and other high value compounds, which can be used in the biomaterials, cosmetics and pharmaceutical industries. One class of compounds are the terpenoids: a diverse group of molecules derived from C5 isoprene units that are exploited for their aromatic and bioactive properties. Terpenoid production in microalgae offers an alternative to extraction from plant species or chemical synthesis. However, metabolic engineering technology for microalgae is still in its infancy and far from economic viability. Thus, the aim of this study was to develop engineering tools for the industrial algal species Nannochloropsis gaditana, with the goal of manipulating the main terpenoid pathway located in the chloroplast. In parallel, the effects of such manipulation was studied using the laboratory species Chlamydomonas reinhardtii, for which chloroplast genetic engineering is already established. N. gaditana is a robust marine species well suited to industrial scale cultivation. The availability of a draft genomic sequence, nuclear transformation methodology and a high lipid productivity have positioned N. gaditana as a promising oleaginous alga for metabolic engineering. However, to develop it as an industrially relevant platform, further molecular tools are needed; in particular a reliable chloroplast transformation method. Thus, the aim of the first project was to develop chloroplast transformation for the alga. This involved optimizing the cultivation conditions for N. gaditana, evaluating its sensitivity to herbicides and chloroplast specific compounds in order to identify suitable selectable markers, and to construct chloroplast transformation vectors. In addition, the temporary increase in cell size by inhibition of cytokinesis was investigated in order to facilitate the delivery of DNA into the small chloroplast. C. reinhardtii is the most developed algal model with well-established tools for genetic manipulation, and can be used to study the effect of chloroplast metabolic engineering in other species such as Nannochloropsis. Thus, the second project focused on the manipulation of the terpenoid biosynthetic pathway: specifically, the chloroplast-localized methylerythritol phosphate pathway by over-expressing the rate limiting enzyme; 1-deoxy-D-xylulose-5-phosphate synthase (DXS). An additional dxs gene from the cyanobacterium Synechocystis 6803 was introduced into the chloroplast genome in the hope of improving the productivity of downstream terpenoid metabolites. A number of transgenic lines were obtained and the successful integration was confirmed by molecular analysis. The effects of up-regulating DXS enzyme activity on overall algal growth and terpenoid profile are studied.

Cette thèse a été réalisée en collaboration avec ENNOLYS, une société de biotechnologies spécialisée dans la production de biomasse, d'enzymes et de molécules aromatiques naturelles. Dans ce contexte, l'objectif principal de ce projet de thèse est l'ingénierie métabolique d'actinobactéries, dont celles du genre Amycolatopsis, pour le développement et l'amélioration de procédés de bioproduction de molécules aromatiques naturelles, dont la vanilline. Pour ce faire, nous avons séquencé le génome de la souche industrielle Amycolatopsis ZYL926 afin de mieux connaitre ses potentialités dans la production de métabolites spécialises ainsi que dans la biosynthèse de la vanilline. De plus, par comparaison de séquence nous avons pu identifier des gènes impliqués dans la dégradation de la vanilline (ou de ces intermédiaires de biosynthèse). Ensuite, nous avons identifié des outils (vecteurs et promoteurs) pour l'insertion et l'expression efficace de gènes hétérologues chez Amycolatopsis. En plus, nous avons développé des outils pour la délétion propre (sans marqueur de résistance) de gènes ou de grandes régions génomiques chez ces bactéries. Ces outils génétiques sont polyvalents et susceptibles d'être utilisés chez de nombreuses espèces du genre Amycolatopsis. Enfin, dans le but d'implémenter une voie de biosynthèse de la vanilline à partir d'un substrat peu coûteux, nous avons choisi et étudié quelques enzymes candidates pour chaque nouvelle étape de biosynthèse. Ces études nous ont permis d'identifier parmi les enzymes candidates celles qui sont actives chez Amycolatopsis dans les conditions de bioconversion utilisées industriellement. De plus, nous avons pu étudier une étape limitante et suggérer des moyens pour améliorer l'efficacité de cette voie de biosynthèse
This thesis was carried out in collaboration with ENNOLYS, a French biotechnology company specialised in the production of biomass, enzymes, and natural aromatic molecules. In this context, the main objective of this thesis project is the metabolic engineering of actinobacteria, including those of the genus Amycolatopsis, for the development and improvement of bioproduction processes for natural aromatic molecules, including vanillin, the most used flavouring agent in the world. To this end, we sequenced the genome of the industrial strain Amycolatopsis ZYL926 in order to better understand the potential of this bacterium in the production of specialised metabolites and in the biosynthesis of vanillin. Furthermore, we were able to identify genes involved in the degradation of vanillin (or of its biosynthetic intermediates) by sequence comparison analysis. Secondly, we have identified genetic tools (including vectors and promotors) for the stable insertion and efficient expression of heterologous genes in Amycolatopsis. In addition, we have developed tools for marker-free deletion of genes or large genomic regions in these bacteria. These genetic tools are versatile and could be used in many species of the genus Amycolatopsis. Finally, in order to implement a vanillin biosynthetic pathway from a low-cost substrate, we selected and studied a few candidate enzymes for each new biosynthetic step. These studies enabled us to identify among the candidate enzymes those which are active in Amycolatopsis under the bioconversion conditions used industrially. In addition, we were able to study a limiting step and suggest ways to improve the efficiency of the studied biosynthetic pathway

Computational biology enabled us to manage vast amount of experimental data and make inferences on observations that we had not made. Among the many methods, predicting metabolic functions with genome-scale models had shown promising results in the recent years. Using sophisticated algorithms, such as flux balance analysis, OptKnock, and OptForce, we can predict flux distributions and design metabolic engineering strategies at a greater efficiency. The caveat of these current methods is the accuracy of the predictions. We proposed using flux balance analysis with flux ratios as a possible solution to improving the accuracy of the conventional methods. To examine the accuracy of our approach, we implemented flux balance analyses with flux ratios in five publicly available genome-scale models of five different organisms, including Arabidopsis thaliana, yeast, cyanobacteria, Escherichia coli, and Clostridium acetobutylicum, using published metabolic engineering strategies for improving product yields in these organisms. We examined the limitations of the published strategies, searched for possible improvements, and evaluated the impact of these strategies on growth and product yields. The flux balance analysis with flux ratio method requires a prior knowledge on the critical regions of the metabolic network where altering flux ratios can have significant impact on flux redistribution. Thus, we further developed the reverse flux balance analysis with flux ratio algorithm as a possible solution to automatically identify these critical regions and suggest metabolic engineering strategies. We examined the accuracy of this algorithm using an Arabidopsis genome-scale model and found consistency in the prediction with our experimental data.
Master of Science

Although replication is considered an indispensable part of the scientific method in software engineering, few replication studies are published each year. The rate of replication, however, is not surprising given that replication theory in software engineering is immature. Not only are replication taxonomies varied and difficult to reconcile, but opinions on the role of replication contradict. In general, we have no clear sense of how to build knowledge via replication, particularly given the practical realities of our research field. Consequently, most replications in software engineering yield little useful information. In particular, the vast majority of external replications (i.e., replications performed by researchers unaffiliated with the original study) not only fail to reproduce the original results, but defy explanation. The net effect is that, as a research field, we consistently fail to produce usable (i.e., transferable) knowledge, and thus, our research results have little if any impact on industry. In this dissertation, we dissect the problem of replication into four primary concerns: 1) rate and explicitness of replication; 2) theoretical foundations of replication; 3) tractability of methods for context analysis; and 4) effectiveness of inter-study communication. We address each of the four concerns via a two-part research strategy involving both a theoretical and a practical component. The theoretical component consists of a grounded theory study in which we integrate and then apply external replication theory to problems of replication in empirical software engineering. The theoretical component makes three key contributions to the literature: first, it clarifies the role of replication with respect to the overall process of science; second, it presents a flexible framework for reconciling disparate replication terminology; and third, it informs a broad range of practical replication concerns. The practical component involves a series of replication studies, through which we explore a variety of replication concepts and empirical methods, ultimately culminating in the development of a tractable method for context analysis (TCA). TCA enables the quantitative evaluation of context variables in greater detail, with greater statistical power, and via considerably smaller datasets than previously possible. As we show (via a complex, real-world example), the method ultimately enables the empirically and statistically-grounded reconciliation and generalization of otherwise contradictory results across dissimilar replications—which problem has previously remained unsolved in software engineering.

This PhD thesis focused on nanomaterial (NM) engineering for occupational health and safety, in the frame of the EU project “Safe Nano Worker Exposure Scenarios (SANOWORK)”. Following a safety by design approach, surface engineering (surface coating, purification process, colloidal force control, wet milling, film coating deposition and granulation) were proposed as risk remediation strategies (RRS) to decrease toxicity and emission potential of NMs within real processing lines. In the first case investigated, the PlasmaChem ZrO2 manufacturing, the colloidal force control applied to the washing of synthesis rector, allowed to reduce ZrO2 contamination in wastewater, performing an efficient recycling procedure of ZrO2 recovered. Furthermore, ZrO2 NM was investigated in the ceramic process owned by CNR-ISTEC and GEA-Niro; the spray drying and freeze drying techniques were employed decreasing NM emissivity, but maintaining a reactive surface in dried NM. Considering the handling operation of nanofibers (NFs) obtained through Elmarco electrospinning procedure, the film coating deposition was applied on polyamide non-woven to avoid free fiber release. For TiO2 NF the wet milling was applied to reduce and homogenize the aspect ratio, leading to a significant mitigation of fiber toxicity. In the Colorobbia spray coating line, Ag and TiO2 nanosols, employed to transfer respectively antibacterial or depolluting properties to different substrates, were investigated. Ag was subjected to surface coating and purification, decreasing NM toxicity. TiO2 was modified by surface coating, spray drying and blending with colloidal SiO2, improving its technological performance. In the extrusion of polymeric matrix charged with carbon nanotube (CNTs) owned by Leitat, the CNTs used as filler were granulated by spray drying and freeze spray drying techniques, allowing to reduce their exposure potential. Engineered NMs tested by biologists were further investigated in relevant biological conditions, to improve the knowledge of structure/toxicity mechanisms and obtain new insights for the design of safest NMs.

This PhD thesis focused on nanomaterial (NM) engineering for occupational health and safety, in the frame of the EU project “Safe Nano Worker Exposure Scenarios (SANOWORK)”. Following a safety by design approach, surface engineering (surface coating, purification process, colloidal force control, wet milling, film coating deposition and granulation) were proposed as risk remediation strategies (RRS) to decrease toxicity and emission potential of NMs within real processing lines. In the first case investigated, the PlasmaChem ZrO2 manufacturing, the colloidal force control applied to the washing of synthesis rector, allowed to reduce ZrO2 contamination in wastewater, performing an efficient recycling procedure of ZrO2 recovered. Furthermore, ZrO2 NM was investigated in the ceramic process owned by CNR-ISTEC and GEA-Niro; the spray drying and freeze drying techniques were employed decreasing NM emissivity, but maintaining a reactive surface in dried NM. Considering the handling operation of nanofibers (NFs) obtained through Elmarco electrospinning procedure, the film coating deposition was applied on polyamide non-woven to avoid free fiber release. For TiO2 NF the wet milling was applied to reduce and homogenize the aspect ratio, leading to a significant mitigation of fiber toxicity. In the Colorobbia spray coating line, Ag and TiO2 nanosols, employed to transfer respectively antibacterial or depolluting properties to different substrates, were investigated. Ag was subjected to surface coating and purification, decreasing NM toxicity. TiO2 was modified by surface coating, spray drying and blending with colloidal SiO2, improving its technological performance. In the extrusion of polymeric matrix charged with carbon nanotube (CNTs) owned by Leitat, the CNTs used as filler were granulated by spray drying and freeze spray drying techniques, allowing to reduce their exposure potential. Engineered NMs tested by biologists were further investigated in relevant biological conditions, to improve the knowledge of structure/toxicity mechanisms and obtain new insights for the design of safest NMs.

1. Introduction Biodiesel (BD) is a liquid biofuel that is defined as a fatty acid methyl ester fulfilling standards such as the ones set by European (EN 14214) and the American (ASTM 6751) regulations. BD is obtained by the transesterification (Scheme 1.1) or alcoholysis of natural triglycerides contained in vegetable oils, animal fats, waste fats and greases, waste cooking oils (WCO) or side-stream products of refined edible oil production with short-chain alcohols, usually methanol or ethanol and using an alkaline homogeneous catalyst (Perego and Ricci, 2012). Scheme 1.1. Transesterification reaction. BD presents several advantages over petroleum-based diesel such as: biodegradability, lower particulate and common air pollutants (CO, SOx emissions, unburned hydrocarbons) emissions, absence of aromatics and a closed CO2 cycle. Refined, low acidity oilseeds (e.g. those derived from sunflower, soy, rapeseed, etc.) may be easily converted into BD, but their exploitation significantly raises the production costs, resulting in a biofuel that is uncompetitive with the petroleum-based diesel (Santori et al., 2012; Lotero et al., 2005). Moreover, the use of the aforementioned oils generated a hot debate about a possible food vs. fuel conflict, i.e. about the risk of diverting farmland or crops at the expense of food supply. It is so highly desirable to produce BD from crops specifically selected for their high productivity and low water requirements (Bianchi et al., 2011; Pirola et al., 2011), or from low-cost feedstock such as used frying oils (Boffito et al., 2012a) and animal fats (Bianchi et al., 2010). The value of these second generation biofuels, i.e. produced from crop and forest residues and from non-food energy crops, is acknowledged by the European Community, which states in its RED directive (European Union, RED Directive 2009/28/EC): ‘‘For the purposes of demonstrating compliance with national renewable energy obligations […], the contribution made by biofuels produced from wastes, residues, non-food cellulosic material, and ligno-cellulosic material shall be considered to be twice that made by other biofuels’’. However, the presence of free fatty acids in the feedstock, occurring in particular in the case of not refined oils, causes the formation of soaps as a consequence of the reaction with the alkaline catalyst. This hinders the contact between reagents and the catalyst and makes difficult the products separation. Many methods have been proposed to eliminate FFA during or prior to transesterification (Pirola et al., 2011; Santori et al., 2012). Among these the FFA pre-esterification method is a very interesting approach to lower the acidity since it allows to lower the acid value as well as to obtain methyl esters already in this preliminary step (Boffito et al., 2012a, 2012b; 2012c Bianchi et al., 2010, 2011; Pirola et al., 2010, 2011). Aims of the work The aims of this work are framed in the context of the entire biodiesel production chain, ranging from the choice of the raw material, through its standardization to the actual biodiesel production. The objectives can be therefore summarized as follows: Assessing the potential of some vegetable or waste oils for biodiesel production by their characterization, deacidification and final transformation into biodiesel; To test different ion exchange resins and sulphated inorganic systems as catalysts in the FFA esterification; To assess the use of ultrasound to assist the sol-gel synthesis of inorganic sulphated oxides to be used as catalysts in the FFA esterification reaction; To assess the use of sonochemical techniques such as ultrasound and microwave to promote both the FFA esterification and transesterification reaction. 2. Experimental details 2.1 Catalysts In this work, three kinds of acid ion exchange resins were used as catalysts for the FFA esterification: Amberlyst®15 (A15), Amberlyst®46 (A46) (Dow Chemical) and Purolite®D5081 (D5081). Their characteristic features are given in Tab. 2.1. Various sulphated inorganic catalysts, namely sulphated zirconia, sulphated zirconia+titania and sulphated tin oxide were synthesized using different techniques. Further details will be given as the results inherent to these catalysts will be presented. Catalyst A15 A46 D5081 Physical form opaque beads Type Macroreticular Matrix Styrene-DVB Cross-linking degree medium medium high Functional group -SO3H Functionalization internal external external external Form dry wet wet Surface area (m2 g-1) 53 75 514a Ave. Dp (Ǻ) 300 235 37a Total Vp (ccg-1) 0.40 0.15 0.47 Declared Acidity (meq H+g-1) 4.7 0.43 0.90-1.1 Measured acidity (meq H+g-1) 4.2 0.60 1.0 Moisture content (%wt) 1.6 26-36 55-59 Shipping weight (g l-1) 610 600 1310a Max. operating temp (K) 393 393 403 Tab. 2.1. Features of the ion exchange resins used as catalysts. The acidity of all the catalysts was determined by ion exchange followed by pH determination as described elsewhere (López et al., 2007; Boffito et al., 2012a; 2012b). Specific surface areas were determined by BET (Brunauer, Emmett and Teller, 1938) and pores sizes distribution with BJH method (Barrett, Joyner and Halenda, 1951). XRD, XPS SEM-EDX and HR-TEM analyses were performed in the case of catalysts obtained with the use of ultrasound (Boffito et al. 2012a). Qualitative analyses of Lewis and Brønsted acid sites by absorption of a basic probe followed by FTIR analyses was also carried out for this class of catalysts (Boffito et al, 2012a). 2.2 Characterization of the oils Oils were characterized for what concerns acidity (by acid-base titrations) as reported by Boffito et al. (2012a, 2012b; 2012c), iodine value (Hannus method (EN 14111:2003)), saponification value (ASTM D5558), peroxide value and composition by GC analyses of the methyl ester yielded by the esterification and transesterification. Cetane number and theoretical values of the same properties were determined using equations already reported elsewhere (Winayanuwattikun et al., 2008). 2.3 Esterification and transesterification reactions In Tab. 2.2, the conditions adopted in both the conventional and sonochemically-assisted esterification are reported. For all these experiments a temperature of 336 K was adopted. Vials were used to test the sulphated inorganic oxides, while Carberry reactor (confined catalyst) (Boffito et al., 201c) was used just for the FFA esterification of cooking oil. Rector oil (+ FFA) (g) MeOH (g) catalyst amount vial 21 3.4 5%wt/gFFA sulphated inorganic catalysts slurry 100 16 - 10 g ion exchange resins - 5%wt/gF FA sulphated inorganic catalysts Carberry 300 48 10 g (5 g in each basket) Tab. 2.2. Free fatty acids esterification reaction conditions for conventional and sonochemically-assisted experiments. All the sonochemically-assisted experiments were performed in a slurry reactor. FFA conversions were determined by acid-base titrations of oil samples withdrawn from the reactors at pre-established times and calculated as follows: "FFA conversion (%)=" (〖"FFA" 〗_"t=0" "-" 〖"FFA" 〗_"t" )/〖"FFA" 〗_"t=0" " x 100" In Tab. 2.3, the conditions of both the conventional and ultrasound (US)-assisted transesterification are reported. KOH and CH3ONa were used for conventional experiments, while just KOH for the US-assisted experiments. The BD yield was determined by GC (FID) analysis of the methyl esters. Method Reactor Step gMeOH/100 goil gKOH/100 goil Temp. (K) Time (min) traditional batch step 1 20 1.0 333 90 step 2 5.0 0.50 60 US-assisted batch step 1 20 1.0 313, 333 30 US-assisted continuos step 1 20 1.0 338 30 Tab. 2.3. Transesterification reaction conditions. 3. Results and Discussion 3.1 Characterization and deacidification of different oils by ion exchange resins: assessment of the potential for biodiesel production In Tab. 3.1 the results of the characterization of the oils utilized in this work are displayed. The value in parentheses indicate the theoretical value of the properties, calculated basing on the acidic composition. The acidity of all the oils exceeds 0.5%wt (~0.5 mgKOH/g), i.e. the acidity limit recommended by both the European normative (EN 14214) and American standard ASTM 6751 on biodiesel (BD). The iodine value (IV) is regulated by the EN 14214, which poses an upper limit of 120 gI2/100 g. The number of saturated fatty chains in the fuel determines its behaviour at low temperatures, influencing parameters such as the cloud point, the CFPP (cold filter plugging point) and the freezing point (Winayanuwattikun et al., 2008). The IV are in most of the cases similar to the ones calculated theoretically. When the experimental IV differs from the theoretical one, it is in most of the cases underestimated. This can be explained considering the peroxide numbers (PN), which indicates the concentration of O2 bound to the fatty alkyl chains and is therefore an index of the conservation state of oil. Oils with high IV usually have a high concentration of peroxides, whereas fats with low IV have a relatively low concentration of peroxides at the start of rancidity (King et al., 1933). Moreover, although PN is not specified in the current BD fuel standards, it may affect cetane number (CN), a parameter that is regulated by the standards concerning BD fuel. Increasing PN increases CN, altering the ignition delay time. Saponification number (SN) is an index of the number of the fatty alkyl chains that can be saponified. The long chain fatty acids have a low SN because they have a relatively fewer number of carboxylic functional groups per mass unit of fat compared to short chain fatty acids. In most of the cases the experimental SN are lower than the ones calculated theoretically. This can be explained always considering the PN, indicating a high concentration of oxygen bound to the fatty alkyl chains. Oil Acidity (%wt) IV1 (gI2/ 100 g) PN2 (meqO2 /kg) SN3 (mg KOH/g) CN4 Fatty acids composition (%wt) animal fat (lard)* 5.87 51 2.3 199 62.3 n.d. soybean* 5.24 138 3.8 201 42.4 n.d. tobacco1 1.68 143 (149) 21.9 199 (202) 41.6 (39.8) C14:0 (2.0) C16:0 (8.3) C18:0 (1.5) C18:1 (12.0) C18:2 (75.3) C18:3 (0.6) C20:0 (0.1) C22:0 (0.2) sunflower* 3.79 126 3.7 199 45.4 n.d. WSO5 0.50 118 (129) 71.3 187 (200) 48.9 (44.6) C16:0 (6.9) C18:0 (0.9) C18:1 (40.1) C18:2 (50.9) C18:3 (0,3) C20:0 (0.1) C20:1 (0.4) C22:0 (0.4) palm 2.71 54.0 (53.0) 12.3 201 (208) 61.3 (60.6) 16:0 (43.9) 18:0 (5.6) 18:1 (40.5) 18:2 (8.6) WCO6 2.10 53.9 (50.7) 11.0 212 (196) 59.9 (62.7) C16:0 (38.8) C18:0 (4.1) C18:1 (47.9) C18:2 (4.2) WCO:CRO =3:1 2.12 69.0 (75.5) 30.1 200 (212) 58.1 (55.1) C16:0 (30.1) C18:0 (3.1) C18:1 (51.9) C18:2 (12.0) C18:3 (2.%) C20:0 (0.2) C22:0 (0.1) WCO:CRO =1:1 2.19 76.8 (90.7) 51.3 188 (203) 58.1 (52.8) C16:0 (21.5) C18:0 (2.1) C18:1 (55.8) C18:2 (14.7) C18:3 (5.1) C20:0 (0.8) C22:0 (0.1) WCO:CRO =1:3 2.24 84.5 (104) 62.4 177 (202) 58.1 (49.9) 14:0 (0.1) 16:0 (14.7) 16:1 (0.7) 18:0 (6.85) 18:1 (40.0) 18:2 (37.0) 18:3 (0.25) 20:0 (0.25) 22:0 (0.15) rapeseed (CRO7) 2.20 118 (123) 71.6 165 (200) 52.8 (45.9) C16:0 (4.1) C18:0 (0.1) C18:1 (63.7) C18:2 (20.2) C18:3 (10.2) C20:0 (1.5) C22:0 (0.2) rapeseed* 4.17-5.12 108 (107) 3.5 203 (200) 48.9 (49.5) C16:0 (7.6) C18:0 (1.3) C18:1 (64.5) C18:2 (23.7) C18:3 (2.4) C20:0 (0.5) Brassica juncea 0.74 109 (110) 178 (185) 52.4 (51.1) C16:0 (2.4) C18:0 (1.1) C18:1 (19.9) C18:2 (19.2) C18:3 (10.9) C20:0 (7.2) C20:1 (1.7) C22:0 (0.9) C22:1 (34.8) 24:0 (1.9) safflower 1.75 139 48.9 170 47.1 n.d. WCO: tobacco2 =1:1 4.34 119 (112) 56.0 191 (203) 48.1 (48.0) C16:0 (22.5) C18:0 (3.2) C18:1 (32.0) C18:2 (42.1) C18:3 (0.2) tobacco2 6.17 141 (151) 33.4 183 (201) 44.4 (39.5) C16:0 (8.7) C18:0 (1.6) C18:1 (12.8) C18:2 (76.0) C18:3 (0.7) C20:0 (0.1) C22:0 (0.1) 1Iodine value; 2Peroxide number; 3Saponification number; 4Cetane number; 5Winterized sunflower oil, 6Waste cooking oil; 7Crude rapeseed oil; * refined, commercial oils acidified with pure oleic acid up to the indicated value. Tab. 3.1. Results of the characterization of the oils. The results of the FFA esterification performed on the different oils are given in Fig. 3.1. Fig. 3.1. Results of the FFA esterification reaction on different oils. The dotted line represents a FFA concentration equal to 0.5%wt, i.e. the limit required by both the European and American directives on BD fuel and to perform the transesterification reaction avoiding excessive soaps formation. The FFA esterification method is able to lower the acidity of most of the oils using the ion exchange resins A46 and D5081 as catalysts in the adopted reaction conditions. High conversion was obtained with A15 at the first use of the catalyst, but then its catalytic activity drastically drops after each cycle. The total loss of activity was estimated to be around 30% within the 5 cycles (results not shown for the sake of brevity). A possible explanation concerning this loss of activity may be related to the adsorption of the H2O yielded by the esterification on the internal active sites, which makes them unavailable for catalysis. When H2O molecules are formed inside the pores, they are unable to give internal retro-diffusion due to their strong interaction with H+ sites and form an aqueous phase inside the pores. The formation of this phase prevents FFA from reaching internal active sites due to repulsive effects. What appears to influence the FFA conversion is the refinement degree of the oil. WCO is in fact harder to process in comparison to refined oils (Bianchi et al., 2010; Boffito et al., 2012c), probably due to its higher viscosity which results in limitations to the mass transfer of the reagents towards the catalyst. Indeed, the required acidity limit is not achieved within 6 hours of reaction. A FFA concentration lower than 0.5%wt is not achieved also in the case of WCO mixture 3:1 with CRO and 1:1 with tobacco oil and in the case of the second stock of tobacco oil (tobacco2). This is attributable to the very low quality of these feedstocks due to the waste nature of the oil itself, in the case of WCO, or to the poor conservation conditions in the case of tobacco oilseed. In this latter case, the low FFA conversion was also ascribed to the presence of phospholipids, responsible for the deactivation of the catalyst. BD yields ranging from 90.0 to 95.0 and from 95.0 to 99.9% were obtained from deacidified raw oils using KOH and NaOCH3 as a catalyst, respectively. In Fig. 3.2, the comparison between A46 and D5081 at different temperatures and in absence of drying pretreatment (wet catalyst) is displayed. As expected, D5081 performs better than A46 in all the adopted conditions. Nevertheless, the maximum conversion within a reaction time of 6 hours is not achieved by any of the catalysts both operating at 318 K and in the absence of drying pretreatment. A more detailed study on the FFA esterification of WCO and its blends with rapeseed oil and gasoline was carried out. In Tab. 3.2 a list of all the experiments performed with WCO is reported together with the FFA conversion achieved in each case, while in Fig. 3.3 the influence of the viscosity of the blends of WCO is shown. Fig. 3.2. Comparison between the catalysts. D5081 and A46 at a) different catalysts amounts and b) temperatures and treatments. The results show that Carberry reactor is unsuitable for FFA esterification since a good contact between reagents and catalyst is not achieved due to its confinement. A15 deactivated very rapidly, while A46 and D5081 maintained their excellent performance during all the cycles of use due to the reasons already highlighted previously. The blends of WCO and CRO show an increase of the reaction rate proportional to the content of the CRO, that is attributable to the decreases viscosity (Fig. 3.3), being all the blend characterized by the same initial acidity. Also the use of diesel as a solvent resulted in a beneficial effect for the FFA esterification reaction, contributing to the higher reaction rate. Feedstock %wtFFAt=0 Reactor Cat. gcat/100 goil gcat/100 g feedstock Number of cat. re-uses FFA conv. (%), 1st use, 6 hr 1 WCO 2.10 Carberry A15 3.3 3.3 6 15.4 2 WCO 2.10 slurry A15 10 10 6 71.7 3 WCO 2.10 Carberry A46 3.3 3.3 6 7.7 4 WCO 2.10 slurry A46 10 10 6 62.0 5 WCO 2.10 slurry D5081 10 10 6 63.7 6 CRO 2.20 slurry A46 10 10 10 95.9 7 CRO 2.20 slurry D5081 10 10 10 93.7 8 WCO 2.10 slurry A46 10 10 0 62.0 9 WCO 75 CRO 25 2.12 7.5 71.3 10 WCO 50 CRO 50 2.19 5.0 79.9 11 WCO 25 CRO 75 2.24 2.5 86.1 12 CRO 2.20 10 95.9 13 WCO 75 DIESEL 25 1.74 7.5 76.8 14 WCO 50 DIESEL 50 1.17 5.0 58.7 15 WCO 25 DIESEL 75 0.65 2.5 40.4 16 WCO 25 DIESEL 75 (higher FFA input) 2.44 2.5 63.5 Tab. 3.2. Experiments performed with waste cooking oil. . Fig. 3.3. FFA conversions and viscosities of the blend of WCO with rapeseed oil. 3.2. Sulphated inorganic oxides as catalysts for the free fatty acid esterification: conventional and ultrasound assisted synthesis Conventional syntheses In Tab. 3.3, the list of all the catalyst synthesized with conventional techniques is reported together with the results of the characterization. Catalyst Composition Prep. method precursors T calc. SSA (m2g-1) Vp (cm3g-1) meq H+g-1 1 SZ1 SO42-/ZrO2 one-pot sol-gel ZTNP1, (NH4)2SO4 893 K O2 107 0.09 0.90 2a SZ2a SO42-/ZrO2 two-pots sol-gel ZTNP, H2SO4 893 K 102 0.10 0.11 2b SZ2b SO42-/ZrO2 two-pots sol-gel ZTNP, H2SO4 653 K 110 0.10 0.12 3 SZ3 SO42-/ZrO2 Physical mixing ZrOCl2.8H2O (NH4)2SO4 873 K 81 0.11 1.3 4 SZ4 Zr(SO4)2/SiO2 Impregnation Zr(SO4)2.4H2O SiO2 873 K 331 0.08 1.4 5 SZ5 Zr(SO4)2/Al2O3 Impregnation Zr(SO4)2.4H2O Al2O3 873 K 151 0.09 0.67 6 ZS Zr(SO4)2.4H2O (commercial) - - - 13 0.12 9.6 7 STTO_0 SO42-/SnO2 Physical mixing + impregnation SnO2 TiO2 P25 H2SO4 773 K 16.8 0.10 3.15 8 STTO_5 SO42-/95%SnO2-5%TiO2 773 K 15.9 0.11 3.43 9 STTO_10 SO42-/ 90%SnO2-10%TiO2 773 K 16.5 0.09 5.07 10 STTO_15 SO42-/ 85%SnO2-15%TiO2 773 K 14.9 0.11 7.13 11 STTO_20 SO42-/ 80%SnO2-20%TiO2 773 K 16.9 0.09 7.33 Tab. 3.3. Sulphated inorganic catalysts synthesized with conventional techniques. The FFA conversions of the sulphated Zr-based systems are provided in Fig. 3.4a and show that Zr-based sulphated systems do not provide a satisfactory performance in the FFA esterification, probably due to their low acid sites concentration related to their high SSA. Even if catalysts such as SZ3 and SZ4 exhibit higher acidity compared to other catalysts, it is essential that this acidity is located mainly on the catalyst surface to be effectively reached by the FFA molecules, as in the case of ZS. In Figure 3.4b, the results of the FFA esterification tests of the sulphated Sn-Ti systems are shown. Other conditions being equal, these catalysts perform better than the sulphated Zr-based systems just described. This is more likely due to the higher acidity along with a lower surface area. With increasing the TiO2 content, the acidity increases as well. This might be ascribable to the charge imbalance resulting from the heteroatoms linkage for the generation of acid centres, (Kataota and Dumesic, 1988). As a consequence, the activity increases with the TiO2 content along with the acidity of the samples. For the sake of clarity, in Fig. 3.4c the FFA esterification conversion is represented as a function of the number of active sites per unit of surface area of the samples. Ultrasound- assisted synthesis In Tab. 3.4, the list of all the catalyst synthesized with conventional techniques is reported together with the results of the characterization. Samples SZ and SZT refer to catalysts obtained with traditional sol-gel method, while samples termed USZT refer to US-obtained sulphated 80%ZrO2-20%TiO2. The name is followed by the US power, by the length of US pulses and by the molar ratio of water over precursors. For example, USZT_40_0.1_30 indicates a sample obtained with 40% of the maximum US power, on for 0.1 seconds (pulse length) and off for 0.9 seconds, using a water/ZTNP+TTIP molar ratio equal to 30. SZT was also calcined at 773 K for 6 hours, employing the same heating rate. This sample is reported as SZT_773_6h in entry 2a. Further details about the preparation can be found in a recent study (Boffito et al., 2012b). Entry Catalyst Acid capacity (meq H+/g) SSA (m2g-1) Vp (cm3g-1) Ave. BJH Dp (nm) Zr:Ti weight ratio S/(Zr+Ti) atomic ratio 1 SZ 0.30 107 0.20 6.0 100 0.090 2 SZT 0.79 152 0.19 5.0 79:21 0.085 2a SZT_773_6h 0.21 131 0.20 5.0 n.d.1 n.d 3 USZT_20_1_30 0.92 41.7 0.12 12.5 80:20 0.095 4 USZT_40_0.1_30 1.03 47.9 0.11 9.5 81:19 0.067 5 USZT_40_0.3_30 1.99 232 0.27 4.5 81:19 0.11 6 USZT_40_0.5_7.5 1.70 210 0.20 5.0 78:22 0.086 7 USZT_40_0.5_15 2.02 220 0.20 5.0 80:20 0.13 8 USZT_40_0.5_30 2.17 153 0.20 5.0 78:22 0.12 9 USZT_40_0.5_60 0.36 28.1 0.10 10 79:21 0.092 10 USZT_40_0.7_30 1.86 151 0.16 5.0 78:22 0.11 11 USZT_40_1_15 3.06 211 0.09 7.0 80:20 0.15 12 USZT_40_1_30 1.56 44.1 0.09 7.0 80:20 0.17 Tab. 3.4. Sulphated inorganic Zr-Ti systems synthesized with ultrasound-assisted sol-gel technique. Some of the results of the characterizations are displayed in Tab. 3.4. The results of the catalytic tests are shown in Fig. 3.5 a, b and c. In Fig. 3.5a and 3.5b the FFA conversions are reported for the samples synthesized using the same or different H2O/precursors ratio, respectively. Fig. 3.5. FFA conversions of sulphated inorganic Zr-Ti systems synthesized with ultrasound-assisted sol-gel for a) the same amount of H2O, b) different amount of H2O used in the sol-gel synthesis, c) in function of the meq of H+/g of catalyst Both the addition of TiO2 and the use of US during the synthesis are able to improve the properties of the catalysts and therefore the catalytic performance in the FFA esterification. The addition of TiO2 is able to increase the Brønsted acidity and, as a consequence, the catalytic activity (compare entries 1 and 2 in Tab. 3.4). The improvement in the properties of the catalysts due the use of US is probably caused by the effects generated by acoustic cavitation. Acoustic cavitation is the growth of bubble nuclei followed by the implosive collapse of bubbles in solution as a consequence of the applied sound field. This collapse generates transient hot-spots with local temperatures and pressures of several thousand K and hundreds of atmospheres, respectively (Sehgal et al., 1979). Very high speed jets (up to 100 m/s) are also formed. As documented by Suslick and Doktycz (Suslick and Doktycz, 1990), in the presence of an extended surface, such as the surface of a catalyst, the formation of the bubbles occurs at the liquid-solid interface and, as a consequence of their implosion, the high speed jets are directed towards the surface. The use of sonication in the synthesis of catalysts can therefore improve the nucleation production rate (i.e. sol-gel reaction production rate) and the production of surface defects and deformations with the formation of brittle powders (Suslick and Doktycz, 1990). For the samples obtained with the US pulses with on/off ratio from 0.3/0.7 on, the conversion does not increase much more compared to the one achieved with the sample obtained via traditional sol-gel synthesis. Their conversion is in fact comparable (see samples USZ_40_0.3_30, USZ_40_0.5_30, USZ_40_0.7_30 and SZT in Fig. 3.5a. The similarity in the catalytic performance of these catalysts may be ascribable to the fact that they are characterized by comparable values of SSA (entries 2, 5, 8, 10 in Tab. 3.4) and, in the case of the catalysts obtained with pulses, also by comparable acidities (entries 5, 8, 10 in Tab. 3.4). A high SSA may in fact be disadvantageous for the catalysis of the reaction here studied for the reasons already highlighted in the previous sections. The best catalytic performance is reached by the sample USZT_40_1_30, i.e. the one obtained using continuous US at higher power. This catalyst results in fact in a doubled catalytic activity with respect to the samples prepared either with the traditional synthesis or with the use of pulsed US. In spite the acidity of this catalyst is lower than that of the samples obtained with the US pulses, it is characterized by a rather low surface area (entry 12 in Tab. 3.4) that can be associated with a localization of the active sites mainly on its outer surface. As evidenced by the FTIR measurements (not reported for the sake of brevity), it is also important to highlight, that only in the case of the USZT_40_1_30 sample, a not negligible number of medium-strong Lewis acid sites is present at the surface, together with a high number of strong Brønsted acid centres. The XRD patterns of the samples were typical of amorphous systems, due to the low calcination temperatures. Samples calcined for a long time (SZT_773_6h) exhibit almost no catalytic activity (results not reported for the sake of brevity). This catalytic behaviour might be ascribable to the loss of part of the sulphates occurred during the calcinations step that result also in a very low acid capacity (see Tab. 3.4). For the sake of clarity, in Fig. 3.5c the FFA conversions as a function of the concentration of the acid sites normalized to the surface area are reported for the most significant samples. For what concerns how the water/precursors ratio affects the catalysts acidity, some general observations can be made: increasing it up to a certain amount increases the H+ concentration (compare entries from 6 to 9 and 11 to 12 in Tab. 3.4) because the rate of the hydrolysis and the number of H2O molecules that can be chemically bounded increases. Nevertheless, increasing the water/precursor ratio over a certain amount (30 for pulsed and 15 for continuous US, entries 8 and 11 in Tab. 3.4, respectively), seems to have a negative effect on the acidity concentration. In fact, the risk of the extraction of acid groups by the excess of water increases as well and the US power density decreases. 3.3 Sonochemically-assisted esterification and transesterification Esterification In Tab. 3.5 a list of the sonochemically-assisted esterification experiments is displayed together with the final acidities achieved after 4 hours of reaction. The reactor used for these experiments, provided with both an US horn (20 kHz) and a MW emitter (2450 MHz) is described elsewhere in detail (Ragaini et al., 2012). Standard calorimetric measurements were carried out to measure the actual emitted power (Suslick and Lorimer, 1989). Considering entries from 1 to 6 (rapeseed oil with high acidity), a final acidity lower than 0.5%wt is achieved within 4 hours operating at the conventional temperature of 336 K with all the methods, while this does not happen operating at lower temperatures. In particular, the lowest acidity is achieved at 336 K with MW. Considering entries from 7 to 12, inherent to the raw tobacco oilseed, final acidities lower than 0.5%wt are achieved only with the use of US. It is remarkable that at the temperature of 293 K the FFA esterification reaction rate results 6X faster than the conventional process at the same temperature. In the case of the rapeseed oil with low acidity (entries from 13 to 20), the use of MW increases the FFA conversion at 293 K and 313 K but not at 336 K. Moreover, the higher the applied power, the higher the FFA conversion. Oil Initial acidity (%wt) Cat. Technique Temp. (K) Emitted power (W) Tthermostat (K) Final acidity (%wt), 4 hr 1 Rapeseed oil (5)* 4.2-5.0 A46 conventional 313 - 315 1.18 2 336 338 0.50 3 ultrasound 313 38.5 293 0.55 4 336 313 0.48 5 microwaves 313 61.4 293 0.69 6 336 313 0.32 7 Tobacco 1.17 A46 conventional 293 - 293 0.97 8 313 315 0.55 9 336 338 0.45 10 ultrasound 293 38.5 277 0.48 11 313 293 0.46 12 336 313 0.30 13 Rapeseed oil (2)* 2.0-2.3 D5081 conventional 293 - 277 0.82 14 313 315 0.44 15 336 338 0.25 16 microwaves 293 31.7 277 0.73 17 313 31.7 293 0.34 18 61.4 293 0.37 19 336 31.7 313 0.29 20 61.4 313 0.25 Tab. 3.5. Sonochemically-assisted esterification experiments. The positive effects of acoustic-cavitation in liquid-solid systems are ascribable to the asymmetric collapse of the bubbles in the vicinity of the solid surface. When a cavitation bubble collapses violently near a solid surface, liquid jets are produced and high-speed jets of liquid are driven into the surface of a particle. These jets and shock waves improve both the liquid–solid and liquid-liquid mass transfer (Mason and Lorimer, 1988). MW is considered as a non-conventional heating system: when MW pass through a material with a dipole moment, the molecules composing the material try to align with the electric field (Mingos et al., 1997). Polar molecules have stronger interactions with the electric field. Polar ends of the molecules tend in fact to align themselves and oscillate in step with the oscillating electric field. Collisions and friction between the moving molecules results in heating (Toukoniitty et al., 2005). The increase of the FFA conversion as the power increases may be attributed to the fact that more power is delivered to the system and, therefore, the enhanced temperature effects caused by electromagnetic irradiation are increased with respect to lower powers. Differently the reason why a too high power was detrimental at the temperature of 336 K could be accounted for by two factors: i) the acoustic cavitation is enhanced at lower temperatures due to the higher amount of gas dissolved; ii) possible generation of too high temperatures inside the reaction medium that could have caused the removal of methanol from the system through constant evaporation or pyrolysis. Transesterification Transesterification experiments were performed on rapeseed oil both in batch and continuous mode. For the batch experiments two kinds of reactors were used: a traditional reaction vessel and a Rosett cell reactor, both with two ultrasound horns with different tip diameters (13 and 20 mm), and operating powers. A Rosett cell is a reactor designed to promote hydrodynamic cavitation through its typical loops placed at the bottom of vessel. Sonicators used in this work were provided by Synetude Company (Chambery, France). In Fig. 3.6, results from the conventional and the US-assisted batch experiments are compared. The US methods allows to attain very high yields in much shorter times than the traditional method and using less reagents (see Tab. 2.3) in just one step. The beneficial effects given by the US are attributable to the generation of acoustic cavitation inside the reaction medium leading to the phenomena already described in the case of esterification reaction. In particular, with the use of the Rosett cell reactor, BD yields of 96.5% (dotted lined) are achieved after 10 minutes of reaction. This is likely due to the combined approach exploiting acoustic cavitation along with hydrodynamic cavitation, which is able to provide a very efficient mixing inside the system. The use of the Rosett cell reactor provided transesterification reaction rates up to 15X faster than the conventional process. Continuous experiments were performed using two tubular reactors with different volumes (0.070 L at 35 KHz and 0.700 L at 20 kHz) and different US powers (19.3 and 68.3 W, respectively). The volume of the treated reagents was varied to obtain the same power density in both the reactors. Results are presented in Fig. 3.7. BD yields higher than 96.5% were obtained in the case of the small reactor within a reaction time of ~5 minutes. It is remarkable that BD yields higher than 90% were obtained using pulsed US (2 seconds on, 2 seconds off) after only 18 seconds, corresponding to just one passage in the reactor. In this case the transesterification reaction rate was 300X faster than the conventional process. The beneficial effects of pulses for the reactivity of the transesterification have been extensively reported (Chand et al., 2010; Kumar et al., 2010). In particular, as reported by Chand, when pulses are adopted, excessive heating of the reaction medium is not promoted, so preventing the loss of the gases dissolved in the system that are necessary for the acoustic cavitation to occur. Moreover, excessive heating during the transesterification reaction might lead to evaporation followed by pyrolysis of methanol and its subsequent removal from the reaction environment. 4. Conclusions As a conclusion to this work, some final remarks can be claimed: Feedstocks with a high potential for biodiesel (BD) production are Brassica juncea oilseed, which can be used as feedstock for BD100, Carthamus tinctorus, tobacco, animal fat and waste cooking oil to be used in BD blends with other oils or in diesel blends. However, blending different oils among them or with diesel already during the free fatty acids (FFA) esterification reaction may increase the reaction rate due to the lowered viscosity. Free fatty acids esterification over acid ion exchange resins in slurry reactors remains the preferred method of oils deacidification due to the optimal contact between the reagents and the catalyst and the good durability over time. The final high BD yields obtained for the oils de-acidified with the pre-esterification method over sulphonic ion exchange resins demonstrate its effectiveness in lowering the acidity and the possibility of obtaining high quality biodiesel from the selected feedstocks. Surface acidity and specific surface area of sulphated inorganic systems can be increased by both adding TiO2 and using ultrasound (US) in precise experimental conditions to assist the sol-gel synthesis of the catalysts. Changing the experimental conditions of US during the sol-gel synthesis makes also possible to tune the properties of the catalysts. In spite of not satisfying FFA conversions were obtained, US-assisted sol-gel synthesis turns out to be an extremely interesting method to obtain catalysts with high acidity and surface area. Both US and microwaves (MW) enhanced the FFA esterification reaction rate at temperatures lower than the one used conventionally (336 K). The positive effects of US are attributable to the phenomena generated inside the reaction medium by the acoustic cavitation, while MW are able to generate temperature effects localized in the proximity of the catalyst surface and to increase MeOH-oil solubility. US-assisted transesterification reaction is much faster than conventional transesterification: BD yields higher than 96.5% were achieved in most of the cases within 10 minutes of reaction, whereas the conventional method requires 150 minutes, besides higher reagents amount and higher temperatures. In particular, BD yields higher than 90% were obtained using a continuous reactor and pulsed US within 18 seconds, corresponding to just one passage in the reactor. In this case the transesterification reaction rate resulted to be 300X faster than the conventional process. Suggestions for the continuations of the work concern the further study of the synthesis of sulphated inorganic systems such as SO42-/ZrO2 or SnO2 or TiO2 with US and MW. Future work should also be devoted to the optimization of the experimental variables related to the use of MW and US to promote both FFA esterification and transesterification reactions. References Barrett E.P., Joyner L.G., Halenda P.P., “The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms”, J. Am. Chem. Soc. 1951, 73, 373. Bianchi C.L., Boffito D.C., Pirola C., Ragaini V., “Low temperature de-acidification process of animal fat as a pre-step to biodiesel production”, Catal. Lett., 2010, 134, 179. Bianchi C.L., Pirola C., Boffito D.C., Di Fronzo A., Carvoli G., Barnabè D., A. Rispoli, R. Bucchi, “Non edible oils: raw materials for sustainable biodiesel”, in Stoytcheva M., Montero G. (Eds.): Biodiesel Feedstocks and Processing Technologies, Intech, 2011, pp. 3-22. Boffito D.C., Pirola C., Galli F., Di Michele A., Bianchi C.L., “Free Fatty Acids Esterification of Waste Cooking Oil and its mixtures with Rapeseed Oil and Diesel”, Fuel, 2012a, accepted on 19th October 2012, DOI: 10.1016/j.fuel.2012.10.069. Boffito D.C., Crocellà V., Pirola C., Neppolian B., Cerrato G., Ashokkumar M., Bianchi C.L., “Ultrasonic enhancement of the acidity, surface area and free fatty acids esterification catalytic activity of sulphated ZrO2-TiO2 systems”, J. Catal., 2012b, http://dx.doi.org/10.1016/j.jcat.2012.09.013 Boffito D.C., Pirola C., Bianchi C.L., “Heterogeneous catalysis for free fatty acids esterification rea.ction as a first step towards biodiesel production”, Chem, Today, 2012c, 30, 14. Brunauer S., Hemmett P., Teller E., “Adsorption of Gases in Multimolecular Layers”, J. Am. Chem. Soc. 1938, 60, 309. López D. E., Suwannakarn K., Bruce D. A., Goodwin JG. “Esterification and transesterification on tungstated zirconia: Effect of calcination temperature”, J Catal 2007, 247, 43. Mason T.J., Lorimer J.P., “Sonochemistry, Theory, Applications and Uses of Ultrasound in Chemistry“, Efford, J. Wiley, New York, 1988. Mingos D.M.P.,Baghurst D.R., “Applications of Microwave Dielectric Heating Effects to Synthetic Problems in Chemistry“, Microwave-Enhanced Chemistry, American Chemical Society,Washington, DC, USA, 1997. Perego C., Ricci, M., “Diesel fuel from biomass”, Catal. Sci. Technol., 2012, 1, 1776. Pirola C., Boffito D.C., Carvoli G., Di Fronzo A., Ragaini V., Bianchi C.L., “Soybean oil deacidification as a first step towards biodiesel production”, in D. Krezhova (Ed.): Recent Trends for Enhancing the Diversity and Quality of Soybean Products, Intech, 2011, pp. 321-44. Pirola C., Bianchi C.L., Boffito D.C., Carvoli G., Ragaini V., “Vegetable oil deacidification by Amberlyst : study of catalyst lifetime and a suitable reactor configuration”, Ind. Eng. Chem. Res., 2010, 49, 4601. Ragaini V., Pirola C., Borrelli S., Ferrari C., Longo I., “Simultaneous ultrasound and microwave new reactor: Detailed description and energetic considerations”, Ultrasonics Sonochemistry 2012, 19, 872 Sehgal C., Steer R.P., Sutherland R.G., Verrall R.E., “Sonoluminescence of argon saturated alkali metal salt solutions as a probe of acoustic cavitation”, J. Chem. Phys., 1979, 70, 2242. Suslick K. S., Doktycz, S. J., "The Effects of Ultrasound on Solids" in Mason, T.J.: Advances in Sonochemistry, JAI Press: New York, 1990, vol.1, pp. 197-230. Toukoniitty B., Mikkola J.P., Murzin D.Yu., Salmi T., “Utilization of electromagnetic and acoustic irradiation in enhancing heterogeneous catalytic reactions”, Appl. Catal. A 2005, 279, 1 Winayanuwattikun P., Kaewpiboon C., Piriyakananon K., Tantong S., Thakernkarnkit W., Chulalaksananukul W. et al. “Potential plant oil feedstock for lipase-catalyzed biodiesel production in Thailand”, Biomass. and Bioen. 2008, 32, 1279.

This research involves a study of sane of the methods available for covering material substrates with a metallic coating. The materials studied were metal and ceramic powders, and the research attempted to produce composite powders, which could be used in the powder metallurgy industries as cheaper replacements for existing expensive powders. For example, iron powder particles coated with copper/tin could replace expensive bronze powders in machine bearing production.

This thesis explores how the study of realistic mixes can expand current knowledge about multitrack music mixing. An essential component of music production, mixing remains an esoteric matter with few established best practices. Research on the topic is challenged by a lack of suitable datasets, and consists primarily of controlled studies focusing on a single type of signal processing. However, considering one of these processes in isolation neglects the multidimensional nature of mixing. For this reason, this work presents an analysis and evaluation of real-life mixes, demonstrating that it is a viable and even necessary approach to learn more about how mixes are created and perceived. Addressing the need for appropriate data, a database of 600 multitrack audio recordings is introduced, and mixes are produced by skilled engineers for a selection of songs. This corpus is subjectively evaluated by 33 expert listeners, using a new framework tailored to the requirements of comparison of musical signal processing. By studying the relationship between these assessments and objective audio features, previous results are confirmed or revised, new rules are unearthed, and descriptive terms can be defined. In particular, it is shown that examples of inadequate processing, combined with subjective evaluation, are essential in revealing the impact of mix processes on perception. As a case study, the percept `reverberation amount' is ex-pressed as a function of two objective measures, and a range of acceptable values can be delineated. To establish the generality of these findings, the experiments are repeated with an expanded set of 180 mixes, assessed by 150 subjects with varying levels of experience from seven different locations in five countries. This largely confirms initial findings, showing few distinguishable trends between groups. Increasing experience of the listener results in a larger proportion of critical and specific statements, and agreement with other experts.

El llevat metilotròfic Pichia pastoris (Komagataella sp.) és un dels sistemes d’expressió més atractius per a la producció de proteïna recombinant, mercat contínuament en expansió. El fort promotor del gen de l’alcohol oxidasa 1 (PAOX1), induït per metanol però reprimit per glucosa, glicerol o etanol, és un dels més emprats per aquest propòsit. No obstant, existeixen encara diversos colls d’ampolla fisiològics que limitant el procés. En aquest context, diferents estratègies han estat proposades i provades per tal de millorar la producció heteròloga de molts tipus diferents de proteïna. Les aproximacions més habituals inclouen increment en el nombre de còpies de gen heteròleg, enginyeria de promotors i modificació dels mecanismes de plegament i secreció. L’objectiu d’aquesta tesi ha estat el desenvolupament de noves estratègies per incrementar la producció de proteïna recombinant, emprant la lipasa de Rhizopus oryzae (Rol) com a proteïna model en el sistema d’expressió basat en el PAOX1. Primerament, els gens del factors de transcripció de PAOX1, MXR1 i MIT1, es van sobreexpressar constitutivament per tal de millorar la transcripció de ROL. Això es va confirmar degut a una millora en la capacitat assimilatòria de metanol i un increment en els nivells relatius de mRNA de ROL i varis gens relacionats amb el metabolisme del metanol, i.e. revertint l’efecte de titulació causat per la transcripció de múltiples cassettes d’expressió de ROL. Tot i aquestes millores, els nivells extracel·lulars d’activitat lipàsica no van augmentar de forma significativa en cultius en quimiòstat, apuntant a colls d’ampolla addicionals limitant la producció de Rol. En segon lloc, es van explorar possibles dianes d’enginyeria metabòlica en el metabolisme cel·lular de P. pastoris emprant el model metabòlic a escola genoma (GEM) consens iMT1026 v3.0. Aquest pas in silico va proporcionar diversos knock-outs prometedors que serien experimentalment testats fent servir el sistema d’edició genòmica CRISPR/Cas9. Les simulacions apuntaven a la disponibilitat de NADPH i una limitada aportació de determinats aminoàcids (serina i cisteïna) com a potencials factors limitants de la producció de Rol. Una reducció en el fitnes cel·lular que afecta a la viabilitat de les soques que es buscaven obtenir va impedir la verificació de la majoria dels knock-outs proposats. Finalment, donat que les nostres anàlisis i estudis prèviament publicats identificaven el NADPH com un cofactor important limitant la producció de proteïna recombinant, els nostres esforços es van dirigir a incrementar la seva disponibilitat a través d’estratègies de knock-in de gens. Específicament, vam sobreexpressar dos gens que codificaven per enzims redox, una NADH quinasa i una NADH oxidasa, amb l’objectiu de pertorbar directament l’equilibri redox de la cèl·lula. A més, es va comprovar l’efecte fisiològic d’aquests enzims fent servir diferents mescles co-substrat/metanol com a font de carboni. En resum, vam observar un increment en la producció de proteïna recombinant amb diferents graus de millora depenent de la font de carboni provada. També vam realitzar anàlisis transcriptòmiques i una avaluació in silico dels nostres resultats per tal de presentar una interpretació millor de l’estat fisiològic de la cèl·lula. Dins del nostre coneixement, aquest és el primer estudi dirigit a incrementar la generació de NADPH en un sistema d’expressió basat en PAOX1, en condicions de creixement en metanol. A grans trets, noves estratègies d’enginyeria de soques han estat proposades i provades durant l’execució d’aquest estudi. A més a més, s’han aplicat GEMs i aproximacions relacionades amb biologia de sistemes, demostrant que són eines potents i prometedores per al disseny racional d’organismes industrials.
The methylotrophic yeast Pichia pastoris (Komagataella sp.) is one of the most attractive expression systems for heterologous protein production, which constitutes a continuously expanding market. The strong alcohol oxidase gene 1 promoter (PAOX1), induced by methanol but repressed by glucose, glycerol or ethanol, is one of the most used for this purpose. Nevertheless, there still exist several physiological bottlenecks limiting the process. In this context, several strategies have been proposed and tested in order to improve the heterologous production of many different types of proteins. Common approaches include increasing heterologous gene copy number, promoter engineering and modification of the folding and secretory mechanisms. The aim of this thesis has been the development of new strategies to increase recombinant protein yields, using the Rhizopus oryzae lipase (Rol) as model protein in a PAOX1-based expression system. Firstly, the PAOX1 transcription factor genes MXR1 and MIT1 were constitutively overexpressed aiming at improving ROL transcription. This was confirmed by an improved methanol assimilation capacity and an increase in relative mRNA levels of ROL and several genes related with methanol metabolism, i.e. reverting the titration effect caused by the transcription of multiple ROL expression cassettes. Despite such improvements, extracellular lipase activity levels did not increase significantly in chemostat cultures, pointing out to additional bottlenecks limiting Rol production. Second, possible metabolic engineering targets in P. pastoris’ cell metabolism were explored using the consensus genome-scale metabolic model (GEM) iMT1026 v3.0. This in silico step provided several promising knock-outs which were going to be experimentally tested using the CRISPR/Cas9 genome editing system. The simulations pointed to NADPH availability and limited supply of some amino acids (serine and cysteine) as potential Rol production limiting factors. A reduction in cell fitness affecting the viability of the obtained strains impeded to verify most of the proposed knock-outs. Finally, since our in silico analyses and previously published studies identified NADPH as an important limiting cofactor in recombinant protein production, our efforts were geared towards increasing its availability through gene knock-in strategies. Specifically, we overexpressed two genes encoding redox enzymes, a NADH kinase and a NADH oxidase, with the aim to directly perturb the cell’s redox balance. Further, we tested the physiological effect of these enzymes using different co-substrate/methanol mixtures as carbon source. In short, we observed an increase in recombinant protein production with different degrees of improvement depending on the carbon source(s) tested. We also performed a transcriptomic analysis and an in silico evaluation of our results in order to provide a better interpretation of the cell physiological state. To our knowledge, this is the first study aiming to increase NADPH generation in the PAOX1-based expression system, under methanol growth conditions. Overall, novel strain engineering strategies have been proposed and tested during the execution of this study. Furthermore, GEMs and related systems biology approaches were applied, proving to be promising powerful tools for rational engineering of industrial microorganisms.

Philosophiae Doctor - PhD (Biotechnology)
Parageobacillus thermoglucosidans is a promising “platform” organism to use in the production ofa range of useful metabolites with demonstrated ability to produce ethanol, isobutanol and polylactic acid for bio-degradable plastics. Extensive work has been done in engineering the organism for enhanced ethanol production. However, an often used and highly effective alternative pathway (pyruvate decarboxylase mediated) for ethanol production has not yet been demonstrated in P. thermoglucosidans. We first characterize two novel bacterial pyruvate decarboxylase enzymes (PDC’s) then attempt to express the more thermostable of these enzymes from Gluconobacter oxydans in P. thermoglucosidans to improve ethanol yields. Initial expression was unsuccessful. Analysis of the codon usage pattern for the gene revealed that the codon usage was suboptimal in the heterologous host P. thermoglucosidans. After codon harmonization, we could demonstrate successful expression of the enzyme at 45°C, however not at the bacterium’s optimum growth temperature of 60°C. This was concomitant with enhanced ethanol production close to the theoretical yield possible (0.5g/l).

Articular cartilage disorders are a leading cause of human disability in many countries around the world. In this work, new techniques and strategies were developed to improve the quality of cartilage produced in vitro by methods of tissue engineering. Chondrocytes were isolated from the hip and knee joints of aborted human foetuses. The cells were expanded and seeded into scaffolds and the seeded scaffolds were cultured in perfusion bioreactors. The quality of the final cartilage constructs was assessed biochemically by measuring their content of glycosaminoglycan (GAG), total collagen and collagen type II and histologically by staining cross-sections of the constructs for GAG, collagen type I and collagen type II. The amount of proteoglycan released in the culture medium was also measured at regular intervals. Proteoglycans from tissue-engineered cartilage and spent culture medium were compared and analysed for degradation and capability of aggregation. During monolayer expansion, the chondrocyte differentiation indices decreased, the cell size increased and the percentage of cells present in G2/S??M phase decreased with the greatest changes occurring during the first passage. Expanding chondrocytes in PGA or PGA??alginate scaffolds produced cells with a higher level of differentiation than monolayer-expanded cells. However, PGA and PGA??alginate could not be justified as suitable systems for the routine expansion of chondrocytes mainly because of the relatively low cell proliferation obtained. Two new methods for seeding of cells into scaffolds were investigated using PGA and PGA??alginate as scaffold materials. Both methods produced high seeding efficiencies and homogeneous distribution of cells. When seeded PGA??alginate scaffolds were cultured in perfusion bioreactors, they produced good quality constructs with higher concentrations of extracellular matrix (ECM) components compared with previously described methods. However, when seeded PGA scaffolds were cultured in perfusion bioreactors, they produced small constructs of poor quality. Investigation of the effect of medium flow rate on the PGA scaffolds showed that a low flow rate was needed at the beginning of the culture to enable the cells to form a framework onto which other synthesised elements could deposit. Applying a gradual increase in medium flow rate to PGA scaffolds cultured in perfusion bioreactors solved the shrinkage problem and produced constructs with quality similar to those produced using PGA??alginate scaffolds. A novel compression bioreactor that mimicked the physiological stimulation of cartilage by joint movement was constructed. Using this bioreactor, compressed constructs showed significantly higher wet weight and higher concentrations of GAG, total collagen and collagen type II compared with non-compressed constructs.

The chemical diversity and biological activities of terpene and terpenoids have served in the development of new flavors, fragrances, medicines and pesticides. While terpenes are made predominantly by plants and microbes in small amounts and as components of complex mixtures, chemical synthesis of terpenes remains technically challenging, costly and inefficient. In this dissertation, methods to create new yeast lines possessing a dispensable mevalonate biosynthetic pathway wherein carbon flux can be diverted to build any chemical class of terpene product are described. The ability of this line to generate diterpenes was next investigated. Using a 5.5 L fed bath fermentation system, about 569 mg/L kaurene and approximately 207 mg/L abietadiene plus 136 mg/L additional isomers were achieved. To engineer more highly modified diterpenes might have greater industrial, agricultural or medicinal applications, kaurenoic acid production reached 514 mg/L with byproduct kaurene and kaurenal at 71.7mg/L and 20.1mg/L, respectively, in fed batch fermentation conditions. Furthermore, ZXM lines for engineer monoterpene and ZXB lines for engineer triterpene were generated by additional specific genomic modification, 84.76 ±13.2 mg/L linalool, 20.54±3.8 mg/L nerolidol and 297.7mg/L squalene were accumulate in ZXM144 line ana ZXB line, respectively, in shake flask conditions.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The past few years have introduced a flurry of interest over renewable energy sources. Biofuels have gained attention as renewable alternatives to liquid transportation fuels. Microbial platforms for biofuel production have become an attractive option for this purpose, mitigating numerous challenges found in crop-based production. Towards this end, metabolic engineering has established itself as an enabling technology for biofuels development. In this work we investigate the strategies of metabolic engineering for developing a biodiesel production platform, utilizing the oleaginous yeast Yarrowia lipolytica as the host organism. We establish new genetic tools for engineering Y. lipolytica beginning with an expression vector utilizing the genetic features from translation elongation factor 1-a (TEF). Additionally, a complementary plasmid was developed allowing for multiple plasmid integration. Bioinformatics analysis of intronic genes in hemiascomycetous yeast also identified relationships between functional pathways and intron enrichment, chronicling the evolutionary journey of yeast species. Next gene targets were examined within the lipid synthesis pathway: acetyl-coA carboxylase (ACC), delta9-desaturase (D9), ATP citrate lyase (ACL), and diacylglycerol acyltransferase (DGA). A combinatorial investigation revealed the order of contribution to lipid overproduction (from strongest to weakest): DGA, ACC, D9, ACL. Scale-up batch fermentation of selected strains revealed exceptionally high lipid accumulation and yield. These results demonstrate the balance between cellular growth and lipid production which is being modified through these genetic manipulations. We next explored utilization of alternative substrates to expand the capabilities and utility of Y. lipolytica. For xylose, a prevalent substrate in cellulosic feedstocks, expression of the redox pathway from Scheffersomyces stipitis and adaptation led to successful substrate utilization. Through the use of cofermentation, growth and productivity on xylose was improved dramatically with xylose-to-lipids conversion successfully demonstrated. For acetate, a potentially useful substrate for electrofuel production, lipid production using our strongest performing strain resulted in high lipid accumulation and yield. From this study, metabolic engineering of Y. lipolytica was successfully used to achieve exceptional lipid overproduction from a variety of substrates. Our genetic tools and recombinant strains establish a strong platform for the study and development of microbial processes for the production of biofuels.
by Mitchell Tai.
Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2001.
Includes bibliographical references (leaves 183-210).
A central goal in metabolic engineering is the design of more productive biological systems by genetically modifying metabolic pathways. In this thesis we report such an optimization in the bacterial strain Corynebacterium glutamicum that is employed for the fermentative production of various amino acids such as lysine. The main goal of the research presented here was the application of metabolic and genetic engineering tools in order to investigate the role of the pyruvate node in cellular physiology. This was achieved by integrating the tools of bioinformatics, recombinant DNA technology, enzymology and classical bioengineering in the context of control and genetically engineered strains of C. glutamicum. First, the main anaplerotic pathway responsible for replenishing oxaloacetate, namely pyruvate carboxylase was targeted. After fruitless attempts to establish an in vitro enzymatic activity for this enzyme, our efforts were directed towards its gene identification. This was achieved by designing PCR primers corresponding to homologous regions among pyruvate carboxylases from other organisms. Utilizing these primers, a PCR fragment was isolated corresponding to part of the gene of the C. glutamicum pyruvate carboxylase. The sequence of the complete gene was finally obtained by screening a C. glutamicum cosmid library. In order to investigate the physiological effect that this enzyme has on lysine production, recombinant strains and deletion mutants were generated. The presence of the gene of pyruvate carboxylase in a multicopy plasmid is not sufficient to yield a significant overexpresssion of this enzyme in C. glutamicum. Contrary to our expectations, overexpression of pyruvate carboxylase has a negative effect on lysine production but improves significantly the growth properties of C. glutamicum. A metabolic model was developed according to which pyruvate carboxylase overexpression increases the carbon flux that enters the TCA cycle, thus the higher growth. However due to the presence of a rate-limiting step in the lysine biosynthesis pathway this increased carbon flux does not translate into higher lysine production. The role of aspartokinase, the first step in lysine biosynthesis, was explored as such a potential bottleneck. Its overexpression proves to increase the amount of lysine produced, however it leads to a lower growth and finally a lower productivity. Since pyruvate carboxylase and aspartokinase have opposite effects on cell physiology, the combination of the overexpression of these two enzymes was finally studied. By this simultaneous overexpression, we achieved to create a C. glutamicum recombinant strain with similar growth as that of the control but higher lysine production and productivity. In the context of exploring the physiological role of pyruvate carboxylase, a biotinylated enzyme, two other enzyme that utilize biotin were also investigated namely acetyl-CoA-carboxylase and biotin ligase. The first enzyme was purified to completion and its N-terminal as well internal amino acid sequences were obtained. A cosmid from the C. glutamicum cosmid library was identified that most likely contains the gene of the latter enzyme. In summary, in the present work we have achieved to prove unequivocally the presence of pyruvate carboxylase in C. glutamicum. We have also achieved to characterize the second biotinylated enzyme in this organism, namely acetyl-CoAcarboxylase. The physiological effect of both pyruvate carboxylase and aspartokinase was established and a metabolic model was developed based on these experimental results. This model finally led us to the construction of a new recombinant strain with improved lysine productivity. As such, this work stands as one of the few examples of a primary metabolite production improvement using metabolic engineering techniques.
by Mattheos A.G. Kofas.
Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015.
Page 11 out of sequence; inserted between page 4 and page 5. Page 209 blank. Cataloged from PDF version of thesis.
Includes bibliographical references.
Although petroleum and other fossil fuels have traditionally been used to fulfill our energy needs, rising concerns over energy security and the climate-changing effects of our continual greenhouse gas emissions have led to great interest in developing a domestic source of renewable fuel with low net carbon emissions. Biodiesel is an attractive option for replacing petroleum-based fuels used in the transportation sector due to its compatibility with existing infrastructure. Single cell oils from heterotrophic oleaginous microorganisms as a source of bio diesel allow for high productivity from a wide array of potential feedstocks, including agroindustrial and municipal waste streams. The goal of this work is to use the tools of rational metabolic engineering to improve lipid production in the non-conventional oleaginous yeast Yarrowia lipoytica on two representative carbon sources, glucose and acetate. Previous work in this area achieved considerable success with the simultaneous overexpression of the native acetyl-CoA carboxylase (ACC 1) and diacylglycerol acyltransferase (DGA2) genes; the resulting strain was used as a benchmark to evaluate our own efforts. We began with the compilation of a set of 44 genes and evaluated the effects of the individual overexpression of each gene on the ability of the resulting strain to produce lipids in fermentations of glucose and acetate. The genes tested here represent many different functions potentially important to lipid production, including the Kennedy pathway, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport. Our results demonstrate that a diverse subset of gene overexpressions led to significant improvements in lipid production on at least one substrate. The largest improvements unsurprisingly came from overexpressing genes directly related to triacylglycerol synthesis, such as diacylglycerol acyltransferase DGAI, which on glucose increased the lipid titer, content and yield by 236%. 165%, and 246%, respectively, over our wild-type control strain, and the acylglycerolphosphate acyltransferase SLC1 gene, which increased titer/content/yield on glucose by 86%/73%/87% and on acetate by 99%/91%/151%. Significant improvements were also detected from genes that more indirectly effect lipogenesis, such as glycerol-3-phosphate dehydrogenase GPD (which produces head groups for triacylglycerol molecules) and the 6-phosphogluconolactoase SOL3 (catalyzing the middle step of the NADPH-producing oxidative pentose phosphate pathway). We next chose the aforementioned SLCl, GPD, and SOL3 genes for use in continued rational engineering of our benchmark strain due to the significance of their effects and the lack of redundancy in their likely mechanism of improving lipogenesis when overexpressed along with ACC I and DGA2. The results of this investigation indicate that the strain overexpressing ACC 1, DGA2,'and SLC 1 may be superior to our benchmark strain, increasing lipid content and yield by 24% and 20%, respectively, with a statistically equivalent titer on acetate. This strain produces the highest reported overall lipid yield of an oleaginous yeast on acetate, at 0.207 g lipids/g acetate.
by Andrew Michael Silverman.
Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.
Includes bibliographical references (p. 165-174).
Forward metabolic engineering (FME) is a rational approach to cellular engineering, relying on an understanding of the entire metabolic network to direct perturbations for phenotype improvement. Conversely, inverse metabolic engineering (IME) uses a global, combinatorial approach to identify genetic loci that are important for a given phenotype. These two approaches complement each other in a strain improvement program. FME and IME approaches were applied to poly-3-hydroxybutyrate (PHB)production in Synechocystis PCC6803 [IME] and recombinant E. coli [FME] in this thesis.IME was appropriate for Synechocystis, where metabolic regulation of the native PHB pathway was not well understood. A high throughput screening method was established by developing a staining protocol that quantitatively related nile red fluorescence to PHB content, while maintaining cell viability for both organisms. This was combined with fluorescence activated cell sorting (FACS) to screen for high PHB mutants. A Synechocystis insertion mutagenesis library was screened to identify gene disruptions that increased PHB. Two gene disruptions in proline biosynthesis and an unknown function were identified and characterized.An analogous IME study in E. coli did not find increased PHB mutants, but suggested an FME approach on the PHB pathway. Systematic overexpression of the pathway revealed phaB, acetoacetyl-CoA reductase, limited PHB flux. Beyond this, whole operon overexpression led to even higher PHB fluxes.In a nitrogen-limited chemostat, PHB flux did not change with dilution rate. Unlike prior pleiotropic perturbations, these systematic experiments could clearly conclude that the flux control is in the PHB pathway. At high PHB flux, growth rate was extremely hindered and was accompanied by PHB plasmid genetic instability and rapid PHB productivity loss.
(cont.) Tandem gene duplication (TGD) was developed to slow productivity loss caused by "allele segregation," a fast process that propagates a DNA mutation to all copies of a plasmid. By placing the many copies in tandem, rather than on individual plasmids, allele segregation could be avoided, increasing stability significantly.These methods and results should support PHB engineering in higher photosynthetic organisms and better E. coli PHB production in batch or continuous culture.TGD is a broadly applicable technique for high level recombinant expression.
by Keith E. J. Tyo.
Ph.D.

Due to technological advancements, the role of the musician has changed dramatically in the 20^th and 21^st centuries. For the composer or songwriter especially, it is becoming increasingly expected for them to have some familiarity with music production and engineering, so that they are able to provide a finished product to employers, clients, or listeners. One goal of a successful production or engineered recording is to most effectively portray the recorded material. Music theory, and specifically analysis, has the ability to reveal important or expressive characteristics in a musical work. The relationship between musical analysis and production is explored to discover how music analysis can provide a more effective and informed musical production or recording and how a consideration of music production elements, notably timbre and instrumentation, can help to better inform a musical analysis. Two supplemental MP3 files are included with this thesis to demonstrate proposed mixing guidelines derived from the analysis.

The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n­-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance. In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days. In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold. In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis. Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor. Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.

In the Paris Agreement from 2015, nations agreed to limit the effects of global warming to well below 2°C. To be able to reach those goals, cheap, abundant and carbon neutral energy alternatives needs to be developed. The microorganisms that several billion years ago oxygenated the atmosphere; cyanobacteria, might hold the key for creating those energy technologies. Due to their capacity for photosynthesis, metabolic engineering of cyanobacteria can reroute the carbon dioxide they fix from the atmosphere into valuable products, thereby converting them into solar powered cell factories. Of the many products bacteria can be engineered to make, the production of terpenoids has gained increasing attention for their attractive properties as fuels, pharmaceuticals, fragrances and food additives. In this thesis, I detail the work I have done on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 for terpenoid production. By deleting an enzyme that converts squalene into hopanoids, we could create a strain that accumulates squalene, a molecule with uses as a fuel or chemical feedstock. In another study, we integrated two terpene synthases from the traditional medical plant Coleus forskohlii, into the genome of Synechocystis. Expression of those genes led to the formation of manoyl oxide, a precursor to the pharmaceutically active compound forskolin. Production of manoyl oxide in Synechocystis was further enhanced by engineering in two additional genes from C. forskohlii that boosted the flux to the product. To learn how to increase the production of squalene, manoyl oxide or any other terpenoid, we conducted a detailed investigation of each step in the MEP biosynthesis pathway, which creates the two common building blocks for all terpenoids. Each enzymatic step in the pathway was overexpressed, and increased flux was assayed by using isoprene as a reporter and several potential targets for overexpression were identified. The final part of this thesis details the characterization of native, inducible promoters and ribosomal binding sites in Synechocystis.