Bioprocess Monitoring and Control

Process monitoring and control are fundamental to all processes; this holds especially for bioprocesses, due to their complex nature. Usually, bioprocesses deal with living cells, which have their own regulatory systems. It helps to adjust the cell to its environmental condition. This must not be the optimal condition that the cell needs to produce whatever is desired. Therefore, a close monitoring of the cell and its environment is essential to provide optimal conditions for production. Without measurement, no information of the current process state is obtained. In this book, methods and techniques are provided for the monitoring and control of bioprocesses. From new developments for sensors, the application of spectroscopy and modelling approaches, the estimation and observer implementation for ethanol production and the development and scale-up of various bioprocesses and their closed loop control information are presented. The processes discussed here are very diverse. The major applications are cultivation processes, where microorganisms were grown, but also an incubation process of bird’s eggs, as well as an indoor climate control for humans, will be discussed. Altogether, in 12 chapters, nine original research papers and three reviews are presented

Photophysical characterization of dynamically linked polymers for self-healing applications

The thesis at hand deals with the spectroscopic and photophysical characterization of dynamically linked polymers, so called dynamers. Dynamers have already found extensive use in the fields of sensor systems or self-healing materials, but the combination of their dynamic chemistry with optical properties such as absorption or emission is still an open field of research. The present thesis is divided in three main parts. First, dynamers that rely on the Diels-Alder functionality were investigated by steady-state and time-resolved, i.e. transient absorption and time-resolved emission, spectroscopies. The chromophores embedded in the polymer scaffold belonged to the class of oligo(arylene ethynylene)s, which are known for their pronounced emission properties. In particular, the influence of the dynamer structure on their ground- and excited state properties both in solution as well as in thin films was studied. Additionally, energy transfer experiments in different polymer compositions were conducted. Second, an imine based polymer was probed with regards to its photostability. Utilizing different excitation energies and solvent properties, different photochemical deactivation pathways were found. Last, an imine-based polymer system was investigated that could partially self-heal its absorption properties after photodamage. Different polymer sructures which affected polymer mobility in films were tested and general rules for the design of optically active self-healing polymers were derived. Keywords: dynamic polymers, dynamic chemistry, self-healing, time-resolved spectroscopy, transient absorption spectroscopy, energy transfer, arylene ethynylene, phenylene ethynylene, photoisomerization, photooxidation

APPLIED PHOTOPROPERTIES OF PHENYLENE ETHYNYLENES

Light-absorbing molecules can be used as powerful tools to perturb and understand biological systems by fluorescence, sensitization, or photochemical reactions. A thorough understanding of the delivery of dyes to specific biochemical targets and the processes that control the fate of excited-state energy is needed to engineer useful technology out of organic photochemistry. This thesis presents four projects investigating different aspects of pathogen destruction and biochemical sensing in a variety of systems, using the properties of p-phenylene ethynylenes (PEs), an especially flexible and well-studied class of conjugated molecules. Of particular relevance, some PEs are found to be effective dyes for amyloid protein aggregates both in solution and in mouse and human brain tissue. As well, control of the solvent microenvironment can be used to tune accessibility of the triplet state, which has implications for targeted photodynamic inactivation of both pathogens and cancer cells

ANTIMICROBIAL ACTIVITY AND MECHANISTIC STUDY FOR THE POLY(PHENYLENE ETHYNYLENE) (PPE)-BASED CATIONIC CONJUGATED POLYELECTROLYTES AND OLIGO-PHENYLENE ETHYNYLENES

My dissertation work focuses on recent progress made in elucidating the intermolecular interactions between a novel class of synthetic phenylene ethynylene (PPE)-based conjugated polyelectrolyte polymers (CPEs) and oligomers (OPEs) and multiscale cellular targets that give rise to their remarkable broad spectrum biocidal activity. We studied the interactions and self-assembly behaviors of the CPEs and OPEs with a set of vital biomolecules, including lipids, proteins and nucleic acids, that reveal the potential pathways by which the synthetic biocidal agents could exert toxicity. Then, we explored the antimicrobial effects and mechanisms of the CPEs and OPEs on multiple clinically relevant pathogens, with an emphasis on the morphological damages induced by the biocidal compounds towards the pathogens. The discussion about the cytotoxicity of these materials against mammalian cells and human tissues to can help us to explore the potential applications of the CPEs and OPEs as antiseptics. We also pose some unanswered questions about their antimicrobial mechanisms, which provide directions for the future study

The effect of fluorine substituents on the physical and structural properties of conjugated molecular materials

A series of selectively fluorinated tolans of the general formulae C(_6)F(_5)-C=C-C(_6)H(_4)X and C(_6)H(_5)-C=C-C(_6)F(_4)X (where X = I, Br, CI) have been synthesized via homogeneous palladium-catalysed Sonogashira cross-coupling and organolithium chemistry. Several of their crystal structures have been solved from X-ray diffraction data, and their molecular packing is described in terms of arene-perfluoroarene and halogen-halogen interactions. Diffraction-quality crystals of a number of binary arene-perfluoroarene complexes of hexafluorobenzene and octafluoronaphthalene with several mismatched polyaromatic hydrocarbons have been obtained and their crystal structures solved from X-ray diffraction data. All of the structures have been shown to consist of infinite stacks of alternating components. The individual structures are compared and contrasted in detail, and those of the HFB complexes are found to closely resemble those predicted from ab initio DFT calculations, which implies that the interactions are over 90 % electrostatic in nature, in contrast with previous calculations on related complexes. A number of selectively fluorinated 4,4'-bis(phenylethynyl)tolan (BPET) derivatives containing fluorinated and non-fluorinated phenyl rings, have been synthesized from palladium-catalysed Sonogashira cross-coupling of various tolan-based precursors. They are observed to strongly absorb in the UV range 336 - 342nm, which are directly comparable to the absorptions for similarly fluorinated 1,4-bis(phenylethynyl)benzene derivatives which suggests that an effective conjugation length (ECL) of 3-4 repeat units is applicable for these phenylene ethynylene systems. They are observed to fluoresce very strongly in the range 372-410 nm. The diethynylbenzene derivatives 1,4-diethynyltetrafluorobenzene and 1,4- diethynyl-2,5-difluorobenzene have been synthesized from the hydrodesilation of their trimethylsilylated precursors. Their crystal structures have been solved from X-ray diffraction data, and are described in terms of C=C-H F and C=C-H π(C=C) interactions

Antifungal Activity of Cationic Conjugated Polyelectrolytes and Oligomers against Candida albicans

Candida albicans is a human fungal commensal and an opportunistic pathogen. It is a polymorphic fungus that is able to invade host cells and disseminate through tissues. C. albicans also form biofilms on medical devices and mucosal surfaces, which aid in the development of drug resistance, tissue colonization and evasion of host immunity. Disseminated candidiasis is associated with high mortality rates, especially in immunocompromised hosts. Concern about resistance to current antifungal therapies has led to a push to develop novel, alternative treatments. In this study, we investigated the antifungal activities of two agents: PPE-DABCO, a poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolyte (CPE), and EO-OPE-1 (DABCO), an oligo-phenylene ethynylene (OPE). Both compounds generate singlet oxygen upon UV-irradiation. The effect of both compounds against C. albicans was tested in the dark and with photoactivation using light of the appropriate wavelength for each agent. We demonstrated that both compounds showed enhanced antifungal activity when irradiated with UV-light, reducing the viability of yeast in suspension. Activation of PPE-DABCO with the 405nm confocal laser was also able to significantly reduce the viability of C. albicans yeast and hyphal cells. Furthermore, by limiting the time of exposure to the 405nm laser to 20 minutes, we were able to isolate the effects of photoactivation of PPE-DABCO from effects due to phototoxicity, by assessing the subsequent hyphal growth. Neither dark nor light treatment with either compound was able to prevent the biofilm formation of surviving cells. This study provides evidence that PPE-DABCO and EO-DABCO act as potent antifungal agents upon photoactivation, and photoactivation is necessary to achieve full antifungal efficacy to reduce the viability of yeast and hyphae and prevent morphological transitioning

Carbohydrate functionalized oligo (phenylene ethynyene)s (OPEs): synthesis, photophysical and biological properties

Tesis doctoral inédita cotutelada por la Università degli studi di Messina, Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali y la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Orgánica. Fecha de lectura: 21 de marzo de 201

Synthesis and Characterization of Sequence-Defined Stiff Oligomers Using the Sonogashira Reaction

Konjugierte Stäbchenmoleküle sind interessante Verbindungen für elektronische Anwendungen. Mehrere iterative Syntheseverfahren zu monodispersen Phenylalkin-Oligomeren wurden publiziert, aber nur ein Beispiel zu derartigen sequenzdefinierten Oligomeren existiert. Dabei könnten sequenzdefinierte, konjugierte Oligomere einen wichtigen Beitrag zur Erforschung von Struktur-Eigenschafts-Beziehungen liefern. In dieser Arbeit wird ein lineares Syntheseverfahren zu sequenzdefinierten Phenylalkin-Oligomeren in Lösung vorgestellt. Dafür wurden verschiedene Bausteinmoleküle mit einem Halogen und einer geschützten Dreifachbindung synthetisiert. Verschiedene Dialkyloxysubstituenten zur besseren Löslichkeit weisen zusätzlich elektronenschiebende Eigenschaften auf. Des Weiteren wurden Elektronenakzeptor-Bausteine hergestellt. Die Stäbchenmoleküle wurden über Zyklen von Sonogashira Kreuzkupplung und anschließender Entschützung der Dreifachbindung realisiert. Zunächst wurde Phenylacetylen als Starteinheit mit einem Bausteinmolekül umgesetzt und das entsprechende Monomer erhalten. Mit dem vorgestellten Syntheseverfahren konnten ein monodisperses und ein sequenzdefiniertes Pentamer in jeweils zehn Reaktionsschritten erhalten werden. Die Ausbeute für das monodisperse Pentamer belief sich dabei auf 18% und 116 Milligramm. Für das sequenzdefinierte Pentamer wurde eine Ausbeute von 3.2% und 73.6 Milligramm erhalten. Die finalen Produkte sowie die entsprechenden Intermediate wurden vollständig mit 1H und 13C-NMR, IR-Spektroskopie und Massenspektrometrie charakterisiert. Zusätzlich wurde SEC und DSC durchgeführt und die photophysikalischen Eigenschaften mit UV/Vis-Spektroskopie untersucht. Ein direkter Oligomerisierungsversuch zu monodispersen Stäbchenmolekülen mit einem Kettenstopper führte zu einem Gemisch aus vielen Oligomeren und rechtfertigt die iterative Reaktionsführung. In dieser Arbeit wurden außerdem Stäbchenmoleküle mit Farbstoffen, die eine thermisch aktivierte verzögerte Fluoreszenz aufweisen, mittels Sonogashira Reaktion verknüpft und vollständig charakterisiert. Mit diesen Verbindungen könnten optoelektronische Vorgänge in organischen Solarzellen untersucht werden

Organic Photochemistry: Remote Delivery of Reactive Oxygen Intermediates via a Heterogeneous Approach

Photosensitized oxidation reactions produce a number of intermediates species, which are generated in varying amounts over time. This complexity presents major challenges in the study of oxidation processes. Mechanistic efforts to separate and deliver reactive oxygen intermediates enable their controlled use in processes such as bacterial inactivation. This thesis describes a heterogeneous reaction approach taken to control the generation and delivery of reactive oxygen intermediates. The mechanistic details of photosensitized reactions were elucidated via synthetic, materials, and physical organic techniques to optimize the delivery of reactive oxygen intermediates. This thesis contains six chapters as described below. Chapter 1 gives a short background on molecular organic photochemistry, to provide a sense of the current state of photochemistry research, as well as an outline of the thesis. Chapter 2 describes a physical-organic study on the photodecomposition of dicumyl peroxide co-adsorbed with sensitizers 4,4¢-dimethylbenzil or chlorin e6 on dry silica. Dicumyl peroxide was decomposed by heterogeneous photosensitization under UV and white lamp irradiation and monitored by the desorption of products acetophenone, 2-phenylpropan-2-ol, and α-methylstyrene using 1H NMR spectroscopy and GC/MS. Dicumyl peroxide and sensitizer were co-adsorbed on silica in 1:4 up to 200:1 ratios, a high peroxide destabilization occurring in a ratio of about 10:1. This increased photodecomposition corresponds to sensitizer–peroxide distances of up to 6–9 Å on silica. Furthermore, a higher photostability of dicumyl peroxide was observed on silica than in a homogeneous acetonitrile solution, where the surface attenuated the diffusion of alkoxy radical geminate pairs apart from each other. A mechanism is proposed that explains how the sensitizer and peroxide separation distance, and geminate recombination of alkoxy radical pairs lead to higher and lower peroxide O–O bond homolysis efficiencies on silica, respectively. This biphasic system can thus serve both to destabilize and stabilize a peroxide; this may be of practical use in a surface used for the delivery of alkoxy radicals for bacterial disinfection. Chapter 3 describes the study of a new series of alkyl chain pterin conjugates using photochemical and photophysical methods, as well as theoretical DFT and solubility calculations. Reactivity patterns for the alkylation of pterin were examined both experimentally and theoretically. The theoretical calculations were carried out using density functional theory (DFT) methods. 2D NMR spectroscopy was used to characterize the pterin derivatives, clearly indicating that the decyl chains were coupled to either the O4 or N3 site on the pterin. At a temperature of 70 °C, the pterin alkylation regioselectively favored the O4 alkylation over the N3 alkylation. The O4 alkylation was also favored when using solvents in which the reactants had increased solubility, e.g., N,N-dimethylformamide and N,N-dimethylacetamide, rather than solvents in which the reactants had a very low solubility, e.g., tetrahydrofuran and dichloromethane. Two additional adducts were also obtained from an N-amine condensation of DMF solvent molecule as byproducts. In comparison to the natural product pterin, the alkyl chain pterins have reduced fluorescence quantum yields (ΦF) and enhanced singlet oxygen (1O2)quantum yields (Φ∆). The DMF-condensed pterins were found to be more photostable compared with the alkylated pterins bearing a free amine group. The alkyl chain pterins efficiently intercalate in large unilamellar vesicles; this is a good indicator of their potential use as photosensitizers in biomembranes. Our study serves as a starting point where the synthesis can be expanded to produce a wider series of lipophilic, fluorophilic, and photooxidatively active pterins. Chapter 4 describes the synthesis of new chlorin e6 silica conjugates and interfacial photooxidation studies. Porous silica and nonporous fumed silica were used as solid supports to evaluate the effect of solid supports on 1O2 production. Chlorin e6 conjugated silica was embedded on to superhydrophobic surfaces to generate bi- and triphasic photocatalytic systems. Finally, photooxidation efficiencies of interfacial systems were evaluated for applications in bacteria inactivation. Chapter 5 describes a photooxidation study on prenylsurfactants [(CH3)2C=CH(CH2)nSO3- Na+ (n = 7, 9, 11)] to probe the “ene” reaction mechanism of 1O2 at an air–water interface. Increasing the number of carbon atoms in the hydrophobic chain increased the regioselectivity for a secondary rather than a tertiary surfactant hydroperoxide, arguing for an orthogonal alkene on water. The prenylsurfactants and a photoreactor technique enabled a certain degree of interfacial control of the hydroperoxidation reaction on a liquid support, where the oxidant (airborne 1O2) is delivered as a gas. Chapter 6 is a review of literature techniques developed so far to understand the delivery of 1O2. This chapter strives to push the idea of 1O2 delivery further by examining two types of delivery: First, the transport of 1O2 in the presence of physical and chemical quenchers is described. Second, the transport of 1O2 by carrier compounds is described. Singlet oxygenation examples include endoperoxides and hydroperoxides

An Evaluation of p-Phenylene Ethynylenes and their Interactions with Pathogenic Microbes

Antibiotics are critical to the welfare of humans domestically and abroad, and their importance cannot be overlooked. However, over the past century, the rise of antibiotic-resistant bacteria and fungi has warranted the investigation of alternative antimicrobials and decontamination strategies. Recently, a new class of antimicrobial has come into view and appears to offer promise in our ongoing defense against pathogenic microbes. These compounds, known as p-phenylene ethynylenes, fundamentally differ from traditional antibiotics in that they are light-activated and deal broad-spectrum damage in a detergent-like manner. Despite the promise of these compounds, there was up until recently good reason to believe that they were simply not suitable for use outside of the laboratory. A series of studies, described herein, was conducted to address these legitimate concerns. First, the compatibility of oligomers with surfactant was investigated, and found to confer numerous biocidal benefits. Similar oligomers were found to be sporicidal and fungicidal. Polymers were also investigated, and were found to be well-suited for attachment onto glass surfaces. The culmination of these studies foreshadows the use of these compounds as anti-biowarfare agents, and at the point-of-care