Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine. / Ph. D.

Wastewater treatment has developed as a mature technology over time. However, conventional wastewater treatment is a very energy-intensive process. Bioelectrochemical system (BES) is an emerging technology that can treat wastewater and also recover resources such as energy in the form of electricity/hydrogen gas and nutrients such as nitrogen and phosphorus compounds. Microbial electrolysis cell (MEC) is a type of BES that, in the presence of an additional voltage, can treat wastewater and generate hydrogen gas. This is a promising approach for wastewater treatment and value-added product generation, though it may not be sustainable in the long run, as it relies on fossil fuels to provide that additional energy. Thus, it is important to explore alternative renewable resources that can provide energy to power MEC. Waste heat is one such resource that has not been researched extensively, particularly at the low-temperature spectrum. This was utilized as a renewable resource by converting waste heat to electricity using a device called thermoelectric generator (TEG). TEG converted simulated waste heat from an anaerobic digester to power an MEC. The feasibility of TEG to act as a power source for an MEC was investigated and its performance compared to the external power source. Various cold sources were analyzed to characterize TEG performance. To explore this integrated TEG-MEC system further, a hydraulic connection was added between the two systems. Wastewater was used as a cold source for TEG and it was recirculated to the anode of the MEC. This system showed improved performance with both systems mutually benefitting each other. The operational parameters were analyzed for the optimization of the system. The integrated system could generate hydrogen at a rate of 0.36 ± 0.05 m3 m-3 d-1 for synthetic domestic wastewater treatment. For the practical application, it is necessary to estimate the cost and narrow the focus on the functions of the system. Techno-economic analysis was performed for MEC with cost estimation and net present value model to understand the economic viability of the technology. The application niche of the BES was described and directions for addressing the challenges towards a full-scale operation were discussed. The present system provides a sustainable method for wastewater treatment and resource recovery which can play an important role in human health, social and economic development and a strong ecosystem. / Doctor of Philosophy / An average person produces about 50-75 gallons of wastewater every day. In addition to the households, wastewater is generated from industries and agricultural practices. As the population increases, the quantity of wastewater production will inevitably increase. To keep our rivers and oceans clean and safe, it is essential to treat the wastewater before it is discharged to the water bodies. However, the conventional wastewater treatment is a very energy (and thus cost) intensive process. For low-income and developing parts of the world, it is difficult to adapt the technology everywhere in its present form. Furthermore, as the energy is provided mostly by fossil fuels, their limited reserves and harmful environmental effects make it critical to find alternative methods that can treat the wastewater at a much lower energy input. For a circular and sustainable economy, it is important to realize wastewater as a resource which can provide us energy, nutrients, and water, rather than discard it as a waste. Bioelectrochemical systems (BES) is an emerging technology that can simultaneously treat wastewater and recover resources in the form of electricity/hydrogen gas, and nitrogen and phosphorus compounds. Microbial electrolysis cell (MEC) is a type of BES that is used to treat wastewater and generate hydrogen gas. An additional voltage is supplied to the MEC for producing hydrogen. In the long run, this may not be sustainable as it relies on fossil fuels to provide that additional energy. Thus, it is important to explore alternative renewable resources that can provide energy to power MEC. Waste heat is a byproduct of many industrial processes and widely available. This was utilized as a renewable resource by converting waste heat to electricity using a device called thermoelectric generator (TEG). TEG converted simulated waste heat from an anaerobic digester to power an MEC. The mutual benefit for MEC and TEG was also explored by connecting the system electrically and hydraulically. Cost-estimation of the system was performed to understand the economic viability and functions of the system were developed. The present system provides a sustainable method for wastewater treatment and resource recovery which can play an important role in human health, social and economic development and a strong ecosystem.

Le fer est l'un des rares métaux qui ne soit pas produit industriellement par électrolyse. Pour aider au développement d'un tel procédé pour l'acier, l'électrolyse des oxydes de fer en milieu sodique est examinée, selon deux approches. La première démarche consiste en l'étude expérimentale du mécanisme réactionnel. L'électrochimie des ions indique qu'il est possible de produire du métal par électrodéposition en milieu alcalin. Parallèlement, l'étude de la réduction d'une particule d'oxyde hématite révèle qu'elle subit, lors de sa conversion en fer métallique, une transformation macroscopique en phase solide. Les analyses démontrent la formation de magnétite comme intermédiaire réactionnel. La seconde démarche est dédiée à la production du fer métallique, par électrolyse d'une suspension de particules d'oxyde dans diverses configurations de cellules. L'incidence des paramètres de procédé a été établie et permet de proposer des éléments de conception d'une cellule industrielle / Iron is one of the few metals which is not industrially produced by electrolysis. The electrowinning of iron metal from its oxides in alkaline solution has been studied to develop such an ironmaking route. Two approaches have been adopted. The first one concerns the evaluation of the reaction mechanism. The study of iron ions electrochemistry in alkaline media shows that the electrodeposition of iron metal is possible. The study of a single iron oxide particle reduction reveals that a reaction of the hematite solid phase is possible. The analysis of a partially converted particle proves that magnetite is formed as an intermediate. The second field of study is dedicated to the production of iron metal in various electrochemical cells, using a suspension electrolysis process. The influence of the key operating parameters is established to assess the possible scale-up. All these elements are gathered to propose the main features of an industrial cell dedicated to the reaction

Ausgangspunkt der Untersuchungen ist eine signifikante Veränderung des elektrochemischen Abscheidungsverhaltens von Silber bei Wechsel des Elektrolytsystems vom technisch eingesetzten salpetersauren System zu einem methansulfonsauren System. Im letztgenannten wird eine für Silber unübliche Kompaktabscheidung an einer Kathode möglich. Die Grundlagenuntersuchungen konnten zeigen, dass eine Erhöhung der Abscheidungspolarisation die Veränderung hervorruft. Ursächlich für diese erhöhte Abscheidungspolarisation ist nach Auswertung der vorhandenen Messdaten eine Veränderung der Struktur der elektrochemischen Doppelschicht. Die Veränderung der Abscheidungsform des Silbers ließ sich erfolgreich zur Durchführung von Laborelektrolysen nutzen. Dabei konnten bei Stromdichten bis zu 900 A/m^2 kompakte Silberniederschläge erhalten werden. Die Gehalte kritischer Verunreinigungen der Silberraffinationselektrolyse, Blei, Kupfer und Palladium, konnten in den meisten Fällen unterhalb der geltenden technischen Spezifikationen gehalten werden. Eine technische Umsetzung ist demnach ausgehend von den Untersuchungen möglich.:1 Einleitung 1 2 Grundlagen zur elektrolytischen Metallabscheidung 2.1 Elektrochemische Kinetik 2.2 Phasengrenze Kathode-Elektrolyt 2.3 Polarisation 2.3.1 Durchtrittspolarisation 2.3.2 Diffusionspolarisation 2.3.3 Reaktionspolarisation 2.3.4 Kristallisationspolarisation 2.4 Stofftransport 2.4.1 Diffusion 2.4.2 Migration 2.4.3 Konvektion 2.5 Durchtritt an der Phasengrenze 2.6 Metallkristallisation 2.6.1 Keimbildung 2.6.2 Keimwachstum 2.6.3 Inhibition 2.6.4 Elektrokristallisation bei gleichzeitiger Inhibition 3 Elektrolytische Raffination von Silber 3.1 Grundlagen zur Silberraffination 3.2 Verunreinigungen von Feinsilber durch Kupfer, Palladium und Blei 3.3 Technische Umsetzung durch die Möbius-Elektrolyse 3.4 Einsatz von Additiven zur Verbesserung der Kathodenqualität 4 Präzisierung der Aufgabenstellung 33 5 Experimentelle Charakterisierung des Systems 5.1 Beschreibung der Silberabscheidung aus Silbermethansulfonat 5.1.1 Angewandte Messmethoden 5.1.2 Experimentelle Vorgehensweise 5.1.3 Charakterisierung der Abscheidungskinetik 5.1.4 Messung des Diffusionskoeffizienten für Ag+ 5.1.5 Charakterisierung der Kristallisation 5.1.6 Ableitung der 5.2 Applikationsbeispiel 5.2.1 Versuchsdurchführung 5.2.2 Optimierung der kathodischen Abscheidungsqualität durch Veränderung der Elektrolytzusammensetzung5.2.3 Verbesserung der Qualität des kathodischen Niederschlags durch Variation der Elektrolysebedingungen 5.2.4 Charakterisierung des Verhaltens typischer Verunreinigungen während der Silberraffination 6 Zusammenfassung Literaturverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Anhang / The investigations started observing a significant change in the electrochemical deposition behaviour of silver when the electrolyte system is changed from the technically used nitric acid system to a methanesulfonic acid system. In the latter system a compact deposition on a cathode, unusual for silver, becomes possible. The basic investigations could show that an increase in deposition polarization causes the change. The reason for this increased deposition polarization is, according to evaluation of the existing measurement data, a change in the structure of the electrochemical double layer. The change in the deposition form of the silver could be successfully used for laboratory electrolysis. Thereby, compact silver deposits could be obtained at current densities up to 900 A/m^2. The contents of critical impurities of the silver refining electrolysis, lead, copper and palladium, could in most cases be kept below the applicable technical specifications. A technical implementation is therefore possible based on the investigations. Translated with www.DeepL.com/Translator (free version):1 Einleitung 1 2 Grundlagen zur elektrolytischen Metallabscheidung 2.1 Elektrochemische Kinetik 2.2 Phasengrenze Kathode-Elektrolyt 2.3 Polarisation 2.3.1 Durchtrittspolarisation 2.3.2 Diffusionspolarisation 2.3.3 Reaktionspolarisation 2.3.4 Kristallisationspolarisation 2.4 Stofftransport 2.4.1 Diffusion 2.4.2 Migration 2.4.3 Konvektion 2.5 Durchtritt an der Phasengrenze 2.6 Metallkristallisation 2.6.1 Keimbildung 2.6.2 Keimwachstum 2.6.3 Inhibition 2.6.4 Elektrokristallisation bei gleichzeitiger Inhibition 3 Elektrolytische Raffination von Silber 3.1 Grundlagen zur Silberraffination 3.2 Verunreinigungen von Feinsilber durch Kupfer, Palladium und Blei 3.3 Technische Umsetzung durch die Möbius-Elektrolyse 3.4 Einsatz von Additiven zur Verbesserung der Kathodenqualität 4 Präzisierung der Aufgabenstellung 33 5 Experimentelle Charakterisierung des Systems 5.1 Beschreibung der Silberabscheidung aus Silbermethansulfonat 5.1.1 Angewandte Messmethoden 5.1.2 Experimentelle Vorgehensweise 5.1.3 Charakterisierung der Abscheidungskinetik 5.1.4 Messung des Diffusionskoeffizienten für Ag+ 5.1.5 Charakterisierung der Kristallisation 5.1.6 Ableitung der 5.2 Applikationsbeispiel 5.2.1 Versuchsdurchführung 5.2.2 Optimierung der kathodischen Abscheidungsqualität durch Veränderung der Elektrolytzusammensetzung5.2.3 Verbesserung der Qualität des kathodischen Niederschlags durch Variation der Elektrolysebedingungen 5.2.4 Charakterisierung des Verhaltens typischer Verunreinigungen während der Silberraffination 6 Zusammenfassung Literaturverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Anhang

In order to keep the Paris Agreement's goal of limiting global warming to well below 2°C, greenhouse gas emissions should be reduced. However, larger measures need to be implemented as it has been established that today's measures will not be enough. The Port of Gävle has plans to install a water electrolyser for hydrogen production of either Proton Exchange Membrane (PEM) or Alkaline Water Electrolysis(AWE). The size of the electrolyser will be approximately 10 MW and will have the capacity to produce 2,000 tons of fossil-free hydrogen per year that might supply 100 heavy trucks. However, it is currently cheaper with fossil hydrogen production. Therefore, an article review is conducted containing a calculation part where the purpose is to investigate the amount of by-products produced and whether they can be sold in other areas of use to make renewable hydrogen more economically competitive. Information for the study has been retrieved from databases, search engines, companies, authorities and individuals deemed relevant to the study. The by-products from the 10 MW electrolyser in the Port of Gävle have been compared with 1,5 MW and 17 MW electrolysers, then a sensitivity analysis has also beenperformed on the 10 MW electrolysers. The potentially generated heat depends on the type of electrolyser where AWE generates 77 MWh of residual heat per day and PEM potentially generates 67 MWh of residual heat per day. Furthermore, AWE needs 64 kWh of electricity to produce 1 kg of hydrogen while PEM needs 66,5 kWh of electricity per kg of hydrogen produced. Revenues from residual heat sales for AWE were estimated annually to approximately 7 million SEK and for PEM approximately 6 million SEK. For electrolysis-produced oxygen to compete with cryogenic oxygen, the price should not exceed 108 SEK/tonne. For the 10 MW electrolyser, oxygen sales are estimated to generate approximately 1,1 million SEK annually for both AWE and PEM. Total income for AWE will annually be just over 8,1 million SEK and 7.1million SEK annually for PEM. The AWE process is then preferable as it is more economically sustainable as the income from the by-products is 12% higher than PEM due to higher production of oxygen and greater generation of residual heat. / För att hålla Parisavtalets mål att begränsa den globala uppvärmningen till väl under 2°C bör utsläppen av växthusgaser minska. Däremot behöver större åtgärder genomföras då det har konstaterats att dagens åtgärder inte kommer att räcka. Gävle hamn har planer på att installera en vattenelektrolysör för vätgasproduktion av antingen Protonutbytesmembran (PEM) eller Alkalisk vattenelektrolys (AWE). Storleken på elektrolysören kommer vara ungefär 10 MW och har kapaciteten att producera 2000 ton fossilfri vätgas per år som kan försörja 100 tunga lastbilar. Dock är det i dagsläget billigare med fossil vätgasproduktion. Därför genomförs en litteraturstudie innehållande en beräkningsdel. Där syftet är att undersöka mängden biprodukter som produceras samt om de kan säljas inom andra områden för att göra förnyelsebar vätgas mer ekonomiskt konkurrenskraftig. Information för studien har hämtats från databaser, sökmotorer, företag, myndigheter och enskilda personer som ansetts relevanta för studien. Biprodukterna från 10 MW elektrolysören i Gävle hamn har jämförts med 1,5 MW och 17 MW elektrolysörer, sedan har även en känslighetsanalys utförts på elektrolysörerna. Potentialen att generera värme beror på typen av elektrolysör där AWE genererar 77 MWh restvärme per dygn och PEM genererar potentiellt 67 MWh restvärme per dygn. Vidare behöver AWE 64 kWh el för att producera 1 kg vätgas medan PEM behöver 66,5 kWh el per producerat kg vätgas. Intäkterna från restvärmeförsäljningen för AWE beräknades årligen till ungefär 7mnSEK och för PEM ungefär 6 mnSEK. För att elektrolysframställd syrgas ska kunna konkurrera med kryogent framställd syrgas bör inte priset övergå 108 SEK/ton. För 10 MW elektrolysören beräknas syrgasförsäljningen kunna inbringa omkring 1,1 mnSEK årligen både för AWE och PEM. Totala inkomsten för AWE blir drygt 8,1 mnSEK/år och 7,1 mnSEK/år för PEM. AWE processen är att föredra då den är mer ekonomiskt hållbar då inkomsten från biprodukterna är 12% högre än PEM på grund av högre produktion av syrgas samt större generering av restvärme.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for obtaining structural information about chemical centres with unpaired electrons. In complex biological systems, EPR spectroscopy can be used to probe these paramagnetic centres and the long-range interactions between them. This thesis investigates two important types of enzymes, and in particular the role of the iron-sulfur electron-transfer centres they contain, with a variety of EPR techniques. Complex I (NADH:Ubiquinone Oxidoreductase) plays a key role in the electron transfer chain essential to the formation of ATP, and its malfunction has been related to numerous human diseases. It is a giant enzyme that contains the longest relay of iron-sulfur clusters known. EPR experiments conducted on complex I from bovine mitochondria yield crucial insight into the mechanism of efficient long-range electron transfer and bring us a step closer to understanding the functioning of this important complex. Hydrogenases are produced by micro-organisms and catalyse the reversible oxidation of H2. Most hydrogenases, including Hyd-2 from Escherichia coli, are very air-sensitive, but some, including E. coli Hyd-1 and Salmonella Hyd-5, are able to function in the presence of atmospheric levels of O2. Understanding the origins of this 'O2-tolerance' is of paramount importance if hydrogenases are to be exploited in future energy technologies. In this thesis, native E. coli Hyd-1 and Hyd-2, Salmonella Hyd-5, as well as O2-tolerant and O2-sensitive variants of E. coli Hyd-1 are characterised using EPR. The EPR investigations elucidate properties of the active site and the electron-transfer relay and, in conjunction with other techniques, reveal structural and mechanistic details of how a highly unusual iron-sulfur cluster in the electron-transfer chain enables some hydrogenases to sustain catalytic activity in the presence of O2.

The covalent chemistry of individual reactants bound within a protein nanopore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. An efficient method to incorporate unnatural amino acid is semisynthesis, which allows site-specific modification with a chemically-defined functional group. However, relatively little work has been done on engineered membrane proteins. This deficiency stems from attributes inherent to proteins that interact with lipid bilayer, notably the poor solubility in aqueous buffer. In the present work, four different derivatives α-hemolysin (αHL) monomer were obtained either by two- or three-way native chemical ligation. The semisynthetic αHL monomers were successfully refolded to heptameric pores and used as nanoreactors to study single-molecule chemistry. The semisynthetic pores show similar biophysical properties to native αHL pores obtained from an in vitro transcription and translation technique. Interestingly, when αHL pores with one semisynthetic subunit containing a terminal alkyne group were used to study Cu(I)-catalyzed azide-alkyne cycloaddition, a long-lived intermediate in the reaction was directly observed.

SPM, optical microscopy, and electrochemical techniques are used to study a range of electrochemical deposition processes on carbon electrodes, particularly those associated with diffusion-controlled multiple nucleation. Anodic stripping voltammetry for analytical measurements using solid electrodes is addressed in the light of limitations arising from electrode heterogeneity, electrode morphology, inhibited electrodeposition, and incomplete stripping of deposited metal. It is shown, using direct imaging of electrode surfaces, that each of the preceding factors may produce significant deviations from ideal electrode behaviour. The electrochemical nucleation of silver on BDD is examined. Data are obtained for the nucleation rate by interpretation of the deposition voltammetry, and by inspection of in-situ optical microscopic images. The particle distributions are analyzed and a stochastic model of nucleation developed. A model for the potentiostatic nucleation and three dimensional growth of deposits on an electrode surface under hydrodynamic conditions is examined. A wall-tube and stirred cell are used to generate conditions in which the diffusion layer thickness is in the range 10 – 40 μm. It is shown that the model provides excellent fits to the experimental data. A previously unrecognised correlation between the morphology of the PbO2 deposits and their electrocatalytic activity is established. The morphology of the films are observed as a function of time and potential using in-situ AFM. Nanotrench arrays are fabricated on HOPG surfaces. Cyclic voltammetry in simple redox couples is used to provide experimental evidence that the voltammetric response of a graphite electrode is solely due to the edge plane sites, with the basal plane sites having no measurable contribution. Nanotrenches are used as templates in a simple method for generating random assemblies of metal nanobands. This method is shown to be effective for generating gold, silver and copper nanowires. The electrochemical properties of the array are investigated via cyclic voltammetry.

In this work, the use of modified diamond as an electrode material with superlative physical and electrochemical properties was investigated in a number of electrochemical applications. The surface chemistry of three differing forms of diamond, namely boron-doped microcrystalline diamond, boron-doped diamond powder and detonation nanodiamond powder was modified utilising such strategies as hydrogen plasma treatment, reactive ion plasma etching along with various chemical treatments. The surface and functional properties of the modified diamond electrodes were studied using a wide spectrum of techniques. The electrochemical activity of these materials was concomitantly investigated in order to expand the knowledge of diamond electrochemistry and to establish an understanding of how the surface chemistry of these materials impacts their electrochemical performance. In the first study, the nanostructuring strategies of boron-doped diamond surface with platinum nanoparticles were developed. In particular, two types of diamond nanostructures were produced: one consisting of platinum particles located on the top of diamond nanorods, the other with platinum particles located in the bottom of diamond nanopits. For the first time, the experimental evidence proving the mechanism of the diamond nanostructuring process was reported. The electrochemical activity of these nanostructured diamond electrodes with regard to the electrochemical oxidation of glucose and methanol was investigated. In the second study, the relationship between the surface chemistry of three differing forms of diamond, including microcrystalline boron-doped diamond, boron-doped diamond powder as well as detonation nanodiamond powder, and the electrode fouling in the result of the adsorption processes in methyl viologen and anthraquinonedisulfonate solutions was investigated. The influence of two dissimilar surface terminations: hydrophobic H-terminated and hydrophilic O-terminated on the electrode performance was studied in detail. This work provides a useful insight on the likely reasons for the undesirable adsorption occurrence which may be experienced in many electroanalytical applications that utilise solid and powdered forms of diamond. The third project extends the discussion on the study of the diamond electrodes, modified with detonation nanodiamond and boron-doped diamond powders and investigates the electrochemical behaviour of these materials. In this work, charge transport within the diamond powder films, partition coefficients of different redox mediators along with heterogeneous electron transfer constants were identified. The chemical modification of these electrodes with platinum nanoparticles along with the mechanism of nucleation and growth of the latter were studied. The enhanced electrode performance with regard to methanol electrooxidation reaction was demonstrated. The fourth study investigates the preparation of nickel modified boron-doped diamond electrodes and ascertains the relationship between the surface chemistry of the modified diamond and the associated electrocatalytic performance of nickel nanoparticles in hydrogen peroxide and glucose electrooxidation. The fifth study reports on the development of a novel surface functionalization strategy, based on porphyrin and amide coupling chemistry, which allows the creation of hybrid biomimetic diamond interface that was used as the artificial β-alanine receptor.

The use of H2 as an energy carrier has in recent years been identified as a promising future solution to the current energy crisis. Hydrogenases are metalloenzymes found in many microorganisms and are used to catalyse the reversible inter-conversion of protons and H2. These enzymes and their synthetic analogues have been recognised as a way to facilitate the use of H2 as a fuel. A major challenge to the future use of these catalysts is their reactions with small molecule inhibitors, such as oxygen and carbon monoxide. Detailed understanding of the structure and catalytic mechanism of these highly efficient catalysts is vital for the design of bio-inspired functional analogues for use in technological applications. In this thesis electrochemical studies of three [FeFe]-hydrogenases are presented, performed using the technique of protein film electrochemistry. The strong potential dependence of the reaction of these hydrogenases with carbon monoxide and formaldehyde is characterised and rationalised. These studies provide compelling evidence for the formation of a previously ambiguous super-reduced state of [FeFe]-hydrogenase at low potential. This state is shown to be active and stable, and it is proposed that this state is involved in catalytic H2 production. This super-reduced state is shown to have a high affinity for the reversible binding of formaldehyde, but a very low affinity for both carbon monoxide and oxygen. Activation of carbon monoxide inhibited [FeFe]-hydrogenase can be very rapidly induced by the application of a sufficiently reducing potential. These enzymes, considered to be oxygen sensitive, are shown to be extremely tolerant to irreversible oxygen damage at very reducing potentials where the super-reduced state is formed.