Physiology and Biotechnology of the Hydrogen Production with the Green Microalga Chlamydomonas reinhardtii

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga.

Design and development of new green catalytic methodologies for the synthesis of enantiomerically enriched molecules

The following Ph.D work was mainly focused on catalysis, as a key technology, to achieve the objectives of sustainable (green) chemistry. After introducing the concepts of sustainable (green) chemistry and an assessment of new sustainable chemical technologies, the relationship between catalysis and sustainable (green) chemistry was briefly discussed and illustrated via an analysis of some selected and relevant examples. Afterwards, as a continuation of the ongoing interest in Dr. Marco Bandini’s group on organometallic and organocatalytic processes, I addressed my efforts to the design and development of novel catalytic green methodologies for the synthesis of enantiomerically enriched molecules. In the first two projects the attention was focused on the employment of solid supports to carry out reactions that still remain a prerogative of omogeneous catalysis. Firstly, particular emphasis was addressed to the discovery of catalytic enantioselective variants of nitroaldol condensation (commonly termed Henry reaction), using a complex consisting in a polyethylene supported diamino thiopene (DATx) ligands and copper as active species. In the second project, a new class of electrochemically modified surfaces with DATx palladium complexes was presented. The DATx-graphite system proved to be efficient in promoting the Suzuki reaction. Moreover, in collaboration with Prof. Wolf at the University of British Columbia (Vancouver), cyclic voltammetry studies were reported. This study disclosed new opportunities for carbon–carbon forming processes by using heterogeneous, electrodeposited catalyst films. A straightforward metal-free catalysis allowed the exploration around the world of organocatalysis. In fact, three different and novel methodologies, using Cinchona, Guanidine and Phosphine derivatives, were envisioned in the three following projects. An interesting variant of nitroaldol condensation with simple trifluoromethyl ketones and also their application in a non-conventional activation of indolyl cores by Friedel-Crafts-functionalization, led to two novel synthetic protocols. These approaches allowed the preparation of synthetically useful trifluoromethyl derivatives bearing quaternary stereocenters. Lastly, in the sixth project the first γ-alkylation of allenoates with conjugated carbonyl compounds was envisioned. In the last part of this Ph.D thesis bases on an extra-ordinary collaboration with Prof. Balzani and Prof. Gigli, I was involved in the synthesis and characterization of a new type of heteroleptic cyclometaled-Ir(III) complexes, bearing bis-oxazolines (BOXs) as ancillary ligands. The new heteroleptic complexes were fully characterized and in order to examine the electroluminescent properties of FIrBOX(CH2), an Organic Light Emitting Device was realized.

Sulfanyl Radical Addition to Alkynes: Revisiting an Old Reaction to Enter the Novel Realms of Green Chemistry, Bioconjugation, and Material Chemistry

In the last decade considerable attention has been devoted to the rewarding use of Green Chemistry in various synthetic processes and applications. Green Chemistry is of special interest in the synthesis of expensive pharmaceutical products, where suitable adoption of “green” reagents and conditions is highly desirable. Our project especially focused in a search for new green radical processes which might also find useful applications in the industry. In particular, we have explored the possible adoption of green solvents in radical Thiol-Ene and Thiol-Yne coupling reactions, which to date have been normally performed in “ordinary” organic solvents such as benzene and toluene, with the primary aim of applying those coupling reactions to the construction of biological substrates. We have additionally tuned adequate reaction conditions which might enable achievement of highly functionalised materials and/or complex bioconjugation via homo/heterosequence. Furthermore, we have performed suitable theoretical studies to gain useful chemical information concerning mechanistic implications of the use of green solvents in the radical Thiol-Yne coupling reactions.

Sustainable processes using heterogeneous acid catalysts. Some examples of industrial interest.

In recent years the need for the design of more sustainable processes and the development of alternative reaction routes to reduce the environmental impact of the chemical industry has gained vital importance. Main objectives especially regard the use of renewable raw materials, the exploitation of alternative energy sources, the design of inherently safe processes and of integrated reaction/separation technologies (e.g. microreactors and membranes), the process intensification, the reduction of waste and the development of new catalytic pathways. The present PhD thesis reports results derived during a three years research period at the School of Chemical Sciences of Alma Mater Studiorum-University of Bologna, Dept. of Industrial Chemistry and Materials (now Dept. of Industrial Chemistry “Toso Montanari”), under the supervision of Prof. Fabrizio Cavani (Catalytic Processes Development Group). Three research projects in the field of heterogeneous acid catalysis focused on potential industrial applications were carried out. The main project, regarding the conversion of lignocellulosic materials to produce monosaccharides (important intermediates for production of biofuels and bioplatform molecules) was financed and carried out in collaboration with the Italian oil company eni S.p.A. (Istituto eni Donegani-Research Center for non-Conventional Energies, Novara, Italy) The second and third academic projects dealt with the development of green chemical processes for fine chemicals manufacturing. In particular, (a) the condensation reaction between acetone and ammonia to give triacetoneamine (TAA), and (b) the Friedel-Crafts acylation of phenol with benzoic acid were investigated.

Assessment and optimization of chemical industrial processes from a life cycle perspective

During the PhD program in chemistry, curriculum in environmental chemistry, at the University of Bologna the sustainability of industry was investigated through the application of the LCA methodology. The efforts were focused on the chemical sector in order to investigate reactions dealing with the Green Chemistry and Green Engineering principles, evaluating their sustainability in comparison with traditional pathways by a life cycle perspective. The environmental benefits associated with a reduction in the synthesis steps and the use of renewable feedstock were assessed through a holistic approach selecting two case studies with high relevance from an industrial point of view: the synthesis of acrylonitrile and the production of acrolein. The current approach wants to represent a standardized application of LCA methodology to the chemical sector, which could be extended to several case studies, and also an improvement of the current databases, since the lack of data to fill the inventories of the chemical productions represent a huge limitation, difficult to overcome and that can affects negatively the results of the studies. Results emerged from the analyses confirms that the sustainability in the chemical sector should be evaluated from a cradle-to-gate approach, considering all the stages and flows involved in each pathways in order to avoid shifting the environmental burdens from a steps to another. Moreover, if possible, LCA should be supported by other tools able to investigate the other two dimensions of sustainability represented by the social and economic issues.

Physiological studies to optimize algal biomass production in phytoremediation processes

Nowadays microalgae are studied, and a number of species already mass-cultivated, for their application in many fields: food and feed, chemicals, pharmaceutical, phytoremediation and renewable energy. Phytoremediation, in particular, can become a valid integrated process in many algae biomass production systems. This thesis is focused on the physiological and biochemical effects of different environmental factors, mainly macronutrients, lights and temperature on microalgae. Microalgal species have been selected on the basis of their potential in biotechnologies, and nitrogen occurs in all chapters due to its importance in physiological and applicative fields. There are 5 chapters, ready or in preparation to be submitted, with different specific matters: (i) to measure the kinetic parameters and the nutrient removal efficiencies for a selected and local strain of microalgae; (ii) to study the biochemical pathways of the microalga D. communis in presence of nitrate and ammonium; (iii) to improve the growth and the removal efficiency of a specific green microalga in mixotrophic conditions; (iv) to optimize the productivity of some microalgae with low growth-rate conditions through phytohormones and other biostimulants; and (v) to apply the phyto-removal of ammonium in an effluent from anaerobic digestion. From the results it is possible to understand how a physiological point of view is necessary to provide and optimize already existing biotechnologies and applications with microalgae.

Optimization of molecular and crystalline forms of drugs, agrochemicals, pesticides in relation to activity, bioavailability, patentability and to the fabrication of polymorphs, solvates, co-crystals with green chemistry methods

This doctorate was funded by the Regione Emilia Romagna, within a Spinner PhD project coordinated by the University of Parma, and involving the universities of Bologna, Ferrara and Modena. The aim of the project was: - Production of polymorphs, solvates, hydrates and co-crystals of active pharmaceutical ingredients (APIs) and agrochemicals with green chemistry methods; - Optimization of molecular and crystalline forms of APIs and pesticides in relation to activity, bioavailability and patentability. In the last decades, a growing interest in the solid-state properties of drugs in addition to their solution chemistry has blossomed. The achievement of the desired and/or the more stable polymorph during the production process can be a challenge for the industry. The study of crystalline forms could be a valuable step to produce new polymorphs and/or co-crystals with better physical-chemical properties such as solubility, permeability, thermal stability, habit, bulk density, compressibility, friability, hygroscopicity and dissolution rate in order to have potential industrial applications. Selected APIs (active pharmaceutical ingredients) were studied and their relationship between crystal structure and properties investigated, both in the solid state and in solution. Polymorph screening and synthesis of solvates and molecular/ionic co-crystals were performed according to green chemistry principles. Part of this project was developed in collaboration with chemical/pharmaceutical companies such as BASF (Germany) and UCB (Belgium). We focused on on the optimization of conditions and parameters of crystallization processes (additives, concentration, temperature), and on the synthesis and characterization of ionic co-crystals. Moreover, during a four-months research period in the laboratories of Professor Nair Rodriguez-Hormedo (University of Michigan), the stability in aqueous solution at the equilibrium of ionic co-crystals (ICCs) of the API piracetam was investigated, to understand the relationship between their solid-state and solution properties, in view of future design of new crystalline drugs with predefined solid and solution properties.

New and More Sustainable Processes for the Synthesis of Phenolics: 2-phenoxyethanol and hydroxytyrosol

The research work has dealt with the study of new catalytic processes for the synthesis of fine chemicals belonging to the class of phenolics, namely 2-phenoxyethanol and hydroxytyrosol. The two synthetic procedures investigated have the advantages of being much closer to the Green Chemistry principles than those currently used industrially. In both cases, the challenge was that of finding catalysts and methods which led to the production of less waste, and used less hazardous chemicals, safer solvents, and reusable heterogeneous catalysts. In the case of 2-phenoxyethanol, the process investigated involves the use of ethylene carbonate (EC) as the reactant for phenol O-hydroxyethylation, in place of ethylene oxide. Besides being a safer reactant, the major advantage of using EC in the new synthesis is the better selectivity to the desired product achieved. Moreover, the solid catalyst based on Na-mordenite was fully recyclable. The reaction mechanism and the effect of the Si/Al ratio in the mordenite were investigated. In the case of hydroxytyrosol, which is one of the most powerful natural antioxidants, a new synthetic procedure was investigated; in fact, the method currently employed, the hydrolysis of oleuropein, an ester extracted from the waste water processing of the olive, makes use of large amounts of organic solvents (hexane, ethyl acetate), and involves several expensive steps of purification. The synthesis procedure set up involves first the reaction between catechol and 2,2-dimethoxyacetaldehyde, followed by the one-pot reduction of the intermediate to give the desired product. Both steps were optimized, in terms of catalyst used, and of reaction conditions, that allowed to reach ca 70% yield in each step. The reaction mechanism was investigated and elucidated. During a 3-month period spent at the University of Valencia (with Prof. A. Corma’s group), a process for the production of diesel additives (2,5-bis(propoxymethyl)furan) from fructose has been investigated.

Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

Microsedimentological characterization as indicators of sedimentary processes and climate changes during Lateglacial at Laguna Potrok Aike, Santa Cruz, Argentina

Palaeoclimatic and paleoenvironmental high latitude records in the Southern Hemisphere are scarce compared to the northern counterpart. However, understanding global evolution of environmental systems during sudden climate changes is inseparable from an equivalent knowledge of both Hemispheres. In this context, a high-resolution study of lacustrine sediments from Laguna Potrok Aike, Santa Cruz province, Patagonia, Argentina, was conducted for the Lateglacial period using concurrent X-Ray Fluorescence (XRF) and Scanning electron microscope analyses. Peaks of Ca/Si and Mn, and occurrences of the green alga Phacotus lenticularis have been interpreted as variations in ventilation of the water column from 13.6 to 11.1 ka cal. BP. During this interval, mild climate conditions during the Younger Dryas are characterized by relatively weak westerlies favouring the formation of a stratified water body as indicated by preserved manganese and Ca/Si peaks and high Total Organic Carbon (TOC) values. In this environment, water in the epilimnion can reach sufficiently high temperature to allow P. lenticularis to grow. Colder conditions are marked by peaks in Ca without P. lenticularis and occur during the Antarctic Cold Reversal (ACR). In this Lateglacial interval, micropumices were also detected in large amount. Image analysis of thin sections allowed the counting and size measurement of detrital particles and micropumices separately. Micropumices significantly influence the iron and titanium content, hence preventing to use them as proxies of detrital input in this interval.

Ocean acidification alters the calcareous microstructure of the green macro-alga Halimeda opuntia

Decreases in seawater pH and carbonate saturation state (Omega) following the continuous increase in atmospheric CO2 represent a process termed ocean acidification, which is predicted to become a main threat to marine calcifiers in the near future. Segmented, tropical, marine green macro-algae of the genus Halimeda form a calcareous skeleton that involves biotically initiated and induced calcification processes influenced by cell physiology. As Halimeda is an important habitat provider and major carbonate sediment producer in tropical shallow areas, alterations of these processes due to ocean acidification may cause changes in the skeletal microstructure that have major consequences for the alga and its environment, but related knowledge is scarce. This study used scanning electron microscopy to examine changes of the CaCO3 segment microstructure of Halimedaopuntia specimens that had been exposed to artificially elevated seawater pCO2 of 650 µatm for 45 d. In spite of elevated seawater pCO2, the calcification of needles, located at the former utricle walls, was not reduced as frequent initiation of new needle-shaped crystals was observed. Abundance of the needles was 22 %/µm**2 higher and needle crystal dimensions 14 % longer. However, those needles were 42 % thinner compared with the control treatment. Moreover, lifetime cementation of the segments decreased under elevated seawater pCO2 due to a loss in micro-anhedral carbonate as indicated by significantly thinner calcified rims of central utricles (35-173 % compared with the control treatment). Decreased micro-anhedral carbonate suggests that seawater within the inter-utricular space becomes CaCO3 undersaturated (Omega < 1) during nighttime under conditions of elevated seawater pCO2, thereby favoring CaCO3 dissolution over micro-anhedral carbonate accretion. Less-cemented segments of H. opuntia may impair the environmental success of the alga, its carbonate sediment contribution, and the temporal storage of atmospheric CO2 within Halimeda-derived sediments.

Seawater carbonate chemistry and Strongylocentrotus droebachiensis larval and juvenile survival and reporductive processes, 2012

Anthropogenic CO2 emissions are acidifying the world's oceans. A growing body of evidence demonstrates that ocean acidification can impact survival, growth, development and physiology of marine invertebrates. Here we tested the impact of long term (up to 16 months) and trans life-cycle (adult, embryo/larvae and juvenile) exposure to elevated pCO2 (1200 µatm, compared to control 400 µatm) on the green sea urchin Strongylocentrotus droebachiensis. Female fecundity was decreased 4.5 fold when acclimated to elevated pCO2 for 4 months during reproductive conditioning while no difference was observed in females acclimated for 16 months. Moreover, adult pre-exposure for 4 months to elevated pCO2, had a direct negative impact on subsequent larval settlement success. Five to nine times fewer offspring reached the juvenile stage in cultures using gametes collected from adults previously acclimated to high pCO2 for 4 months. However, no difference in larval survival was observed when adults were pre-exposed for 16 months to elevated pCO2. pCO2 had no direct negative impact on juvenile survival except when both larvae and juveniles were raised in elevated pCO2. These negative effects on settlement success and juvenile survival can be attributed to carry-over effects from adults to larvae and from larvae to juveniles. Our results support the contention that adult sea urchins can acclimate to moderately elevated pCO2 in a matter of a few months and that carry-over effects can exacerbate the negative impact of ocean acidification on larvae and juveniles.

Forest Hydrology, Soil Conservation and Green Barriers in Canary Islands

In volcanic islands, the rainfall regime and its torrential nature, together with the steep slopes and the soil types present are considered to be some of the main factors affecting forest hydrology and soil conservation. In such environments, rain regime is generally irregular and characterized by short and intense rainfalls, which could cause destructive flows at times, followed by long periods of rain absence. The volcanic nature of these islands have as a direct resultant steep slopes which influences the runoff volume and speed, as well as the amount of topsoil susceptible to be detached and transported downstream. The soil type also affects the susceptibility to erosion processes. Andisols are the most typical soil on volcanic islands. Their particularities derive their mineral constituents, called short-range-order products, which provide these soils with an increased structural stability, which in turn reduces their susceptibility to erosion. However, the land use changes and the environmental factors such as rain regime and steep slopes may be determinant factor in destabilizing these soils and ultimately a cause for soil erosion and runoffs, which become a threat to the population downstream. Green barriers have been traditionally used to prevent or reduce these processes, also to enhance the dew effect and the fog water collection, and as a firebreak which acts as a barrier to slow or stop the progress of a wildfire. Wooded species present and subsequently their performance have a major influence on their effectiveness. The use of this natural erosion and fire control methods on volcanic islands is discussed in this paper.

Utilizing the Internet of Things to promote energy awareness and efficiency at discrete production processes: Practices and methodology

El actual contexto de fabricación, con incrementos en los precios de la energía, una creciente preocupación medioambiental y cambios continuos en los comportamientos de los consumidores, fomenta que los responsables prioricen la fabricación respetuosa con el medioambiente. El paradigma del Internet de las Cosas (IoT) promete incrementar la visibilidad y la atención prestada al consumo de energía gracias tanto a sensores como a medidores inteligentes en los niveles de máquina y de línea de producción. En consecuencia es posible y sencillo obtener datos de consumo de energía en tiempo real proveniente de los procesos de fabricación, pero además es posible analizarlos para incrementar su importancia en la toma de decisiones. Esta tesis pretende investigar cómo utilizar la adopción del Internet de las Cosas en el nivel de planta de producción, en procesos discretos, para incrementar la capacidad de uso de la información proveniente tanto de la energía como de la eficiencia energética. Para alcanzar este objetivo general, la investigación se ha dividido en cuatro sub-objetivos y la misma se ha desarrollado a lo largo de cuatro fases principales (en adelante estudios). El primer estudio de esta tesis, que se apoya sobre una revisión bibliográfica comprehensiva y sobre las aportaciones de expertos, define prácticas de gestión de la producción que son energéticamente eficientes y que se apoyan de un modo preeminente en la tecnología IoT. Este primer estudio también detalla los beneficios esperables al adoptar estas prácticas de gestión. Además, propugna un marco de referencia para permitir la integración de los datos que sobre el consumo energético se obtienen en el marco de las plataformas y sistemas de información de la compañía. Esto se lleva a cabo con el objetivo último de remarcar cómo estos datos pueden ser utilizados para apalancar decisiones en los niveles de procesos tanto tácticos como operativos. Segundo, considerando los precios de la energía como variables en el mercado intradiario y la disponibilidad de información detallada sobre el estado de las máquinas desde el punto de vista de consumo energético, el segundo estudio propone un modelo matemático para minimizar los costes del consumo de energía para la programación de asignaciones de una única máquina que deba atender a varios procesos de producción. Este modelo permite la toma de decisiones en el nivel de máquina para determinar los instantes de lanzamiento de cada trabajo de producción, los tiempos muertos, cuándo la máquina debe ser puesta en un estado de apagada, el momento adecuado para rearrancar, y para pararse, etc. Así, este modelo habilita al responsable de producción de implementar el esquema de producción menos costoso para cada turno de producción. En el tercer estudio esta investigación proporciona una metodología para ayudar a los responsables a implementar IoT en el nivel de los sistemas productivos. Se incluye un análisis del estado en que se encuentran los sistemas de gestión de energía y de producción en la factoría, así como también se proporcionan recomendaciones sobre procedimientos para implementar IoT para capturar y analizar los datos de consumo. Esta metodología ha sido validada en un estudio piloto, donde algunos indicadores clave de rendimiento (KPIs) han sido empleados para determinar la eficiencia energética. En el cuarto estudio el objetivo es introducir una vía para obtener visibilidad y relevancia a diferentes niveles de la energía consumida en los procesos de producción. El método propuesto permite que las factorías con procesos de producción discretos puedan determinar la energía consumida, el CO2 emitido o el coste de la energía consumida ya sea en cualquiera de los niveles: operación, producto o la orden de fabricación completa, siempre considerando las diferentes fuentes de energía y las fluctuaciones en los precios de la misma. Los resultados muestran que decisiones y prácticas de gestión para conseguir sistemas de producción energéticamente eficientes son posibles en virtud del Internet de las Cosas. También, con los resultados de esta tesis los responsables de la gestión energética en las compañías pueden plantearse una aproximación a la utilización del IoT desde un punto de vista de la obtención de beneficios, abordando aquellas prácticas de gestión energética que se encuentran más próximas al nivel de madurez de la factoría, a sus objetivos, al tipo de producción que desarrolla, etc. Así mismo esta tesis muestra que es posible obtener reducciones significativas de coste simplemente evitando los períodos de pico diario en el precio de la misma. Además la tesis permite identificar cómo el nivel de monitorización del consumo energético (es decir al nivel de máquina), el intervalo temporal, y el nivel del análisis de los datos son factores determinantes a la hora de localizar oportunidades para mejorar la eficiencia energética. Adicionalmente, la integración de datos de consumo energético en tiempo real con datos de producción (cuando existen altos niveles de estandarización en los procesos productivos y sus datos) es esencial para permitir que las factorías detallen la energía efectivamente consumida, su coste y CO2 emitido durante la producción de un producto o componente. Esto permite obtener una valiosa información a los gestores en el nivel decisor de la factoría así como a los consumidores y reguladores. ABSTRACT In today‘s manufacturing scenario, rising energy prices, increasing ecological awareness, and changing consumer behaviors are driving decision makers to prioritize green manufacturing. The Internet of Things (IoT) paradigm promises to increase the visibility and awareness of energy consumption, thanks to smart sensors and smart meters at the machine and production line level. Consequently, real-time energy consumption data from the manufacturing processes can be easily collected and then analyzed, to improve energy-aware decision-making. This thesis aims to investigate how to utilize the adoption of the Internet of Things at shop floor level to increase energy–awareness and the energy efficiency of discrete production processes. In order to achieve the main research goal, the research is divided into four sub-objectives, and is accomplished during four main phases (i.e., studies). In the first study, by relying on a comprehensive literature review and on experts‘ insights, the thesis defines energy-efficient production management practices that are enhanced and enabled by IoT technology. The first study also explains the benefits that can be obtained by adopting such management practices. Furthermore, it presents a framework to support the integration of gathered energy data into a company‘s information technology tools and platforms, which is done with the ultimate goal of highlighting how operational and tactical decision-making processes could leverage such data in order to improve energy efficiency. Considering the variable energy prices in one day, along with the availability of detailed machine status energy data, the second study proposes a mathematical model to minimize energy consumption costs for single machine production scheduling during production processes. This model works by making decisions at the machine level to determine the launch times for job processing, idle time, when the machine must be shut down, ―turning on‖ time, and ―turning off‖ time. This model enables the operations manager to implement the least expensive production schedule during a production shift. In the third study, the research provides a methodology to help managers implement the IoT at the production system level; it includes an analysis of current energy management and production systems at the factory, and recommends procedures for implementing the IoT to collect and analyze energy data. The methodology has been validated by a pilot study, where energy KPIs have been used to evaluate energy efficiency. In the fourth study, the goal is to introduce a way to achieve multi-level awareness of the energy consumed during production processes. The proposed method enables discrete factories to specify energy consumption, CO2 emissions, and the cost of the energy consumed at operation, production and order levels, while considering energy sources and fluctuations in energy prices. The results show that energy-efficient production management practices and decisions can be enhanced and enabled by the IoT. With the outcomes of the thesis, energy managers can approach the IoT adoption in a benefit-driven way, by addressing energy management practices that are close to the maturity level of the factory, target, production type, etc. The thesis also shows that significant reductions in energy costs can be achieved by avoiding high-energy price periods in a day. Furthermore, the thesis determines the level of monitoring energy consumption (i.e., machine level), the interval time, and the level of energy data analysis, which are all important factors involved in finding opportunities to improve energy efficiency. Eventually, integrating real-time energy data with production data (when there are high levels of production process standardization data) is essential to enable factories to specify the amount and cost of energy consumed, as well as the CO2 emitted while producing a product, providing valuable information to decision makers at the factory level as well as to consumers and regulators.

PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.