Дисертації з теми "Organohalides"

A serious impediment to the application of fundamental toxicology to the protection of human and environmental health is that most organisms are exposed to mixtures of chemical agents while the majority of toxicology research elucidates the toxic actions of individual agents. Our current state of knowledge is insufficient for predicting the effects of a combination of agents based on the dose-response characteristics of the agents administered singly. Even when dose-response data from mixtures and their individual components are available, no clear consensus exists as to which means are appropriate for determining if an interaction such as synergy or antagonism is indicated by those data. Sound mathematical analysis of toxic interaction is an essential ingredient in this pursuit. Experimental designs and means of data analysis permitting precarious conclusions remain in common use, and impede the characterization and elucidation of xenobiotic interactions. This thesis critiques some of the approaches used to address xenobiotic interaction, and offers specific and novel techniques and guidelines for improved approaches. Increasingly large numbers of toxicants exceed our current ability to assess toxicity. The development of in vitro methods offers an increased ability to examine larger numbers of toxicants and their combinations than conventional in vivo approaches given the finite resources available. This thesis presents evidence supporting the validation of precision-cut liver slice culture as an in vitro model for investigating hepatotoxic interactions of defined binary mixtures. Toxic interactions observed in vivo were demonstrated in the in vitro liver slice culture in two strains of rat. No intrinsic bias was detected by challenging this approach with a sham interaction (one compound combined with itself). Structure-activity based predictions of toxic interaction were demonstrated in liver slice culture. Two separate means of data analysis arrived at the same interpretations of the data for all of the experimental results described above. No toxic interactions were found in a limited but rigorous test of a bacterial toxicity assay, suggesting that interactive toxic responses are sensitive to the choice of biological model. Preliminary experiments were conducted for assessing the effect of mechanistic probes (metabolic manipulations) on established toxic interactions.

Les halogènes présentent des propriétés très intéressantes en chimie organique. L’incorporation de ceux-ci sur des molécules organiques permet de modifier leurs propriétés et les rend indispensables dans plusieurs domaines tels que la chimie pharmaceutique, l’agrochimie et la science des matériaux. En plus de cela, ils sont de très bons intermédiaires de synthèse pour l’obtention de produits hautement fonctionnalisés. Cette thèse portera donc sur le développement de nouvelles méthodes pour l’incorporation d’halogènes à partir de produits de départ simples. Dans le premier projet, une réaction d’hydrofluoration a été développée. Cela permet d’incorporer un atome de fluor sur diverses molécules, à partir d’alcènes qui sont facilement accessibles. Les conditions réactionnelles sont une combinaison entre un acide fort, l’acide méthanesulfonique et une source de fluorure, Et3N·3HF. Les conditions ont pu être appliquées à des alcènes comportant une vaste gamme de groupements fonctionnels. La réaction n’est toutefois compatible qu’avec les alcènes 1,1-disubstitués et trisubstitués. Les rendements obtenus sont généralement bons et comparables à ceux obtenus avec les autres méthodes de la littérature. Dans le deuxième projet, une modification des conditions réactionnelles du premier projet a été réalisée pour permettre l’hydrochloration, l’hydrobromation et l’hydroiodation d’alcènes. Les composés halogénés sont obtenus dans d’excellents rendements, généralement sans purification. Des études mécanistiques ont été effectuées et montre que la réaction est favorisée par la formation d’un intermédiaire acétate, qui provient de la réaction entre le substrat et le solvant, l’acide acétique. Enfin, un exemple de deutérochloration a également été montré. Pour ce qui est du troisième projet, nous nous sommes intéressés à la déoxyfluoration d’alcools. L’objectif était de complémenter les méthodes présentes dans la littérature qui permettent la transformation sur des alcools primaires et secondaires, mais qui sont souvent problématiques pour les alcools tertiaires. En modifiant les conditions réactionnelles du premier projet, nous avons été en mesure de développer une réaction de déoxyfluoration d’alcools tertiaires qui donne d’excellents rendements. La réaction est compatible avec une vaste gamme de groupements fonctionnels, et les produits fluorés correspondants sont obtenus généralement sans purification. Une extension de la méthode pour d’autres types de liaison C–O a aussi été effectuée. Enfin, des études mécanistiques ont été réalisées et ont permis de déterminer que la réaction se déroule via une séquence d’élimination puis d’hydrofluoration. Une modification des conditions réactionnelles de déoxyfluoration a également été effectuée pour les rendre compatibles avec les méthodes de radiofluoration
Halides have very interesting properties in organic chemistry. Organic molecules that bear halides have modified properties which make them interesting in many fields of chemistry such as pharmaceuticals, agrochemicals, and material science. Moreover, they are useful intermediate for the synthesis of a vast array of highly functionalized molecules. This thesis will therefore report on the development of new reaction for the incorporation of halides starting from abundant molecules such as alkenes and alcohols. The first project focused on the development of an hydrofluorination reaction. This allowed for the easy incorporation of a fluorine atom on various molecules starting from easily available alkenes. The reaction uses a combination of a strong acid, methanesulfonic acid, and a fluoride source, triethylamine trihydrofluoride. These conditions were compatible with a wide range of functional groups. However, the reaction was limited to 1,1-disubstituted and trisubstituted alkenes. The yields are generally good and similar to those of other reported methods. For the second project, a modification of the reaction conditions from the first project was performed to allow for the hydrochlorination, hydrobromination and hydroiodination of alkenes. The corresponding halides are obtained in excellent yields, usually without purification. Mechanistic studies have shown that the solvent, acetic acid, plays a role in the stabilization of the carbocation. Finally, an example of deuteriochlorination has been reported using deuterated acetic acid. In the third project, we focused on the transformation of alcohols into fluorides. The main objective of this project was to complement the existing methods of deoxyfluorination which work generally well on primary and secondary alcohols, but not so much on tertiary alcohols. By modifying the conditions from the first project, we were able to develop a deoxyfluorination reaction that gives tertiary fluorides in excellent yields. The reaction is compatible with a vast array of functional groups and the products are usually obtained without purification. The reaction has been extended for the fluorination of other C–O bonds such as ethers and esters. Mechanistic studies have been performed and show that the reaction proceeds in two steps via an elimination/hydrofluorination pathway. Finally, a modification of these conditions has been done to allow for an adaptation of this reaction in radiofluorination of alcohols

Solar cells based on organohalide perovskites (PSCs) have made rapid progress in recent years and are a promising emerging technology. An important next evolutionary step for PSCs is their up-scaling to commercially relevant dimensions. The main challenges in scaling PSCs to be compatible with current c-Si cells are related to the limited conductivity of the transparent electrode, and the processing of a uniform and defect-free organohalide perovskite layer over large areas. In this work we present a generic and simple approach to realizing efficient solution-processed, monolithic solar cells based on methylammonium lead iodide (CH₃NH₃PbI₃). Our devices have an aperture area of 25 cm² without relying on an interconnected strip design, therefore reducing the complexity of the fabrication process and enhancing compatibility with the c-Si cell geometry. We utilize simple aluminum grid lines to increase the conductivity of the transparent electrode. These grid lines were exposed to an UV-ozone plasma to grow a thin aluminum oxide layer. This dramatically improves the wetting and film forming of the organohalide perovskite junction on top of the lines, reducing the probability of short circuits between the grid and the top electrode. The best devices employing these modified grids achieved power conversion efficiencies of up to 6.8%.

La freqüent contaminació d’aigua subterrània per compostos organohalogenats és un greu problema degut als riscs humans i ecològics que se’n deriven. La bioremediació és un tècnica sostenible que permet superar algunes de les limitacions que presenten els tractaments fisicoquímics. En aquest estudi ens proposem obtenir i caracteritzar cultius que contenen bacteris anaerobis capaços de degradar compostos organohalogenats ambientalment perillosos i que es puguin aplicar per a la bioremediació d’aqüífers in situ. En treballs previs realitzats al nostre laboratori es va obtenir un cultiu enriquit que contenia un bacteri dehalorespirador del gènere Dehalogenimonas a partir de sediments de la desembocadura del riu Besòs (Barcelona) que degrada alcans amb halògens situats en carbons adjacents. En aquesta tesis, s’ha identificat la dehalogenasa reductora (RDasa) d’aquesta Dehalogenimonas implicada en la conversió de dibromur d’etilè (EDB) al compost innocu etilè combinant tècniques de proteòmica basades en gels d’electroforesis, tests enzimàtics i nano-cromatografia líquida acoblada a espectrometria de masses (nLC-MS/MS). Aquesta RDasa es va designar com a EdbA. EdbA és la primera RDasa identificada entre les espècies d’aquest gènere bacterià que catalitza una reacció de debromació. A més, és la primera RDasa que s’ha demostrat funcional i que no té cap subunitat B de fixació a la membrana citoplasmàtica codificada de forma adjacent en el seu genoma. Addicionalment, s’ha detectat un enzim ortolog a l’enzim responsable de la degradació de 1,2-diclorpropà a propé (DcpA) com a única RDasa en cultius que transformen 1,2,3-triclorpropà a clorur d’alil mitjançant la combinació de tècniques d’ultracentrifugació, gels d’electroforesis i nLC-MS/MS. Aquesta DcpA es va detectar en la fracció de la membrana tal i com predeien les eines bioinformàtiques emprades. El mecanisme pel qual aquestes dues RDases identificades es fixen a les membranes és encara desconegut. En aquesta treball s’ha obtingut un segon consorci bacterià estable provinent de llots d’una planta de tractament d’aigües residuals industrials i aplicant estratègies de d’enriquiment del cultiu i tècniques de dilució fins a l’extinció. Aquest cultiu fermenta diclorometà (DCM) i dibromometà (DBM) en acetat i format. S’ha demostrat que el bacteri responsable de la fermentació d’aquests dihalometans és un Dehalobacterium i s’ha procedit al seu aïllament. Tanmateix, les interaccions sinèrgiques entre les espècies del consorci han impedit el seu aïllament. Mitjançant la selecció de colònies en cultius semi sòlids, canvis en la composició del medi i l’ús de antibiòtics, s’ha assolit un cultiu on l’abundància de Dehalobacterium és del 67%. L´acompanyen bacteris dels gèneres Acetobacterium i Desulfovibrio, tal i com revelen els anàlisis de genoteques. El fraccionament dels isòtops de carboni durant la fermentació de DCM per aquest cultiu s’ha determinat mitjançant l’anàlisi d’isòtops estables de compostos específics (CSIA). El valor obtingut de -27 ± 2‰ difereix del prèviament publicat per una soca de Dehalobacter (-15.5 ± 1.5‰) que també fermentava DCM. Aquests valors són significativament diferent dels obtinguts per bacteris metilotròfics degradadors de DCM (que varien de -45 a -61‰) i podria permetre la distinció entre vies de degradació de DCM en treballs de bioremediació in situ. Finalment, s’ha demostrat que la presència de co-contaminants que es detecten freqüentment amb DCM, tals com tricloroetilè (TCE), 1,2-dicloroetà (1,2-DCA), cis-dicloroetilè (cis-DCE), 1,1,2-tricloroetà (1,1,2-TCA), àcid perfluorooctanoic (PFOA) i 3,4-dicloroanilina (3,4-DCA) no provoca una inhibició significativa en la degradació de DCM pel cultiu amb Dehalobacterium a les concentracions testades. La concentració de cloroform de 100 mg/L provoca una total inhibició. De manera similar, la presència de 200 mg/L d’àcid perfluorooctanosulfonic (PFOS) i ≥ 25 mg/L de diuron provoquen una inhibició severa, impedint la degradació completa de DCM. Tanmateix, l’activitat degradadora de DCM es recupera quan els cultius inhibits es transfereixen a medi fresc sense co-contaminants.
La frecuente contaminación de las aguas subterráneas por compuestos organohalogenados es un grave problema ambiental debido a los riesgos ecológicos y para la salud humana de ella derivados. La bioremediación es una tecnología sostenible que evita algunos inconvenientes que presentan los tratamientos físico-químicos. En este estudio nos proponemos obtener y caracterizar cultivos que contengan bacterias anaerobias que degraden compuestos organohalogenados ambientalmente peligrosos con potencial para la bioremediación in situ de aguas subterráneas. En trabajos previos de nuestro grupo de investigación, se obtuvo un cultivo enriquecido en bacterias del género Dehalogenimonas procedente de sedimentos del estuario del río Besós (Barcelona) que degrada alcanos con halógenos situados en carbonos adyacentes. En esta tesis se ha identificado la dehalogenasa reductora (RDasa) de esta cepa de Dehalogenimonas implicada en la conversión del dibromuro de etileno (EDB) al compuesto inocuo eteno combinando técnicas de proteómica basadas en geles de electroforesis, ensayos enzimáticos y nano-cromatografía líquida de alta resolución (nLC-MS/MS). Esta RDasa es designada EdbA, y constituye la primera RDasa identificada en este género bacteriano que cataliza una reacción de debromación. Además, es también la primera RDasa en ser demostrada funcional sin una subunidad B de anclaje a la membrana codificada de forma adyacente en el genoma. Adicionalmente, se ha detectado una única RDasa en cultivos que transforman 1,2,3-tricloropropano a cloruro de alilo combinando técnicas de ultracentrifugación, geles de electroforesis y nLC-MS/MS. Esta enzima ortóloga a DcpA, la responsable de la degradación de 1,2-dicloropropano a propeno, ha sido detectada en la fracción proteica de membrana, lo cual concuerta con las predicciones realizadas mediante herramientas bioinformáticas. El mecanismo por el cual EdbA y esta DcpA se anclan a la membrana citoplasmática es desconocido, atribuyéndose a proteínas todavía no descritas. En este trabajo se ha obtenido un segundo consorcio bacteriano estable a partir de lodos de una planta de tratamiento de aguas residuales industriales aplicando técnicas de cultivo de enriquecimiento y dilución por extinción. Este cultivo fermenta diclorometano (DCM) y dibromometano (DBM) a acetato y formato. Se ha demostrado que la bacteria responsable de la fermentación pertenece al género Dehalobacterium, y se ha procedido a su aislamiento. Sin embargo, las interacciones sinérgicas existentes entre las especies del consorcio han impedido obtener un cultivo puro. Seleccionando colonias en medio de cultivo semisólido, aplicando antibióticos y cambios en la composición del medio, se ha obtenido una abundancia relativa de Dehalobacterium del 67%. Le acompañan bacterias de los géneros Acetobacterium y Desulfovibrio, tal y como se detectó mediante análisis de genotecas. El fraccionamiento isotópico del carbono durante la fermentación del DCM por este cultivo fue determinado mediante análisis de isótopos estables de compuestos específicos (CSIA). El valor obtenido, -27 ± 2‰, difiere del publicado previamente para una cepa de Dehalobacter que también fermenta el DCM (-15.5 ± 1.5‰). Estos valores son significativamente diferentes de los obtenidos con bacterias metilotróficas degradadoras de DCM (-45 a -61‰), y podrían permitir diferenciar vías de degradación de DCM en trabajos de bioremediación in situ. Finalmente, se ha demostrado que la presencia de co-contaminantes que se detectan frecuentemente con el DCM, como el tricloroetileno (TCE), 1,2-dicloroetano (1,2-DCA), cis-dicloroetileno (cis-DCE), 1,1,2-tricloroetano (1,1,2-TCA), ácido perfluorooctanoico (PFOA) y 3,4-dicloroanilina (3,4-DCA) no provocan una inhibición significativa en la degradación de DCM por parte del cultivo de Dehalobacterium, a las concentraciones estudiadas. Una concentración de cloroformo de 100 mg/L provoca una inhibición total. De manera similar, 200 mg/L de sulfonato de perfluoroctano (PFOS), y ≥ 25 mg/L de diuron provocan una inhibición severa, impidiendo la degradación completa del DCM. Sin embargo, la actividad degradadora de DCM se recupera cuando los cultivos inhibidos se transfieren a medio libre de co-contaminantes.
The widespread groundwater contamination by organohalide compounds is of a major concern due to the human and ecological risks derived from it. Bioremediation is a sustainable technology that overcomes some limitations of the physical-chemical remediation techniques on these water bodies. In this study, we aimed to obtain and characterize cultures containing anaerobic bacteria capable of degrading organohalide compounds of environmental concern with potential for in situ groundwater bioremediation. In previous work carried out in our laboratory a highly enriched culture containing organohalide-respiring bacteria from the genus Dehalogenimonas degrading vicinally halogenated alkanes was obtained from sediments of the river Besós estuary (Barcelona). In this thesis, the reductive dehalogenase (RDase) from this Dehalogenimonas strain responsible for the catalysis of ethylene dibromide (EDB) to the innocuous ethene was identified combining gel-based proteomic techniques, specific enzymatic tests and nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS). This RDase is therefore designated as EdbA, for ethylene dibromide RDase subunit A. EdbA is the first RDase identified for debrominating catalytic activity among species of this genus. Moreover, it is the first RDase shown to be functional for respiration without an adjacent membrane-anchoring subunit B encoded on the genome. Additionally, combining ultracentrifugation, gel electrophoresis and nLC-MS/MS, an orthologous enzyme of the dichloropropane-to-propene RDase (DcpA) was the only RDase detected in 1,2,3-trichloropropane-to-allyl chloride dehalogenating cultures. This DcpA was detected in the membrane fraction of the crude protein extract, in accordance to its predicted subcellular localization by bioinformatics tools and it is also not co-localised with an rdhB gene. The membrane-anchoring mechanisms of these RDases remains not known and may rely in yet-unidentified proteins. A second stable bacterial consortium was obtained in the present work from slurry samples of an industrial wastewater treatment plant with a combination of enrichment culture strategies and the dilution-to-extinction technique. This culture was demonstrated to ferment dichloromethane (DCM) and dibromomethane (DBM) into acetate and formate. The Dehalobacterium sp. present in this culture was shown to be the responsible for the dihalomethanes fermentation, and the isolation of this strain was attempted. However, the synergic interactions existing among the different accompanying species present in the bacterial consortia impeded the isolation. Despite a pure culture was not achieved via picking up colonies from semisolid agar cultures, changes in the medium composition, and the application of selected antibiotics, a final relative abundance of Dehalobacterium sp. of 67 % was attained. As determined by clone library analysis, bacteria from the genera Acetobacterium and Desulfovibrio remained present in the culture. The carbon isotope fractionation during DCM fermentation by this culture was determined by compound-specific stable isotope analysis (CSIA). The value obtained was -27 ± 2‰ and differs from the previously published value of -15.5 ± 1.5‰ of a Dehalobacter sp. performing also DCM fermentation. These values are yet significantly different from those reported for facultative methylotrophic bacteria degrading DCM (ranging from -45 to -61‰), and this would allow for further differentiation of these degradation pathways during in situ bioremediation works. Finally, the potential inhibitory effect of selected frequent groundwater co-contaminants over DCM degradation by the Dehalobacterium-containing culture was assessed for further in situ bioremediation applications. Trichloroethylene (TCE), 1,2-dichloroethane (1,2-DCA), cis-dichloroethylene (cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), perfluorooctanoic acid (PFOA), and 3,4-dichloroaniline (3,4-DCA) did not show significant inhibitory effects at the concentrations tested. Differently, a total inhibition was caused with a chloroform concentration of 100 mg/L. Also, the presence of 200 mg/L of perfluorooctanesulfonic acid (PFOS), as well as concentrations higher than 25 mg/L of the pesticide diuron caused a severe inhibitory effect, preventing the full depletion of DCM. Nevertheless, DCM degrading activity was recovered when inhibited cultures were transferred to co-contaminant free medium.

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.

Microbial respiration can be highly diverse and adaptable, with many bacteria able to respond to changes in their environment promptly and efficiently. The regulation of respiratory enzymes by highly responsive and precise transcriptional regulators confers distinct advantage for survival in sometimes harsh and extreme conditions. The organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2 is able to utilise a wide range of electron acceptors and respiratory processes through tight regulation of respiratory machinery. An example of this tight regulation of respiratory machinery can been seen by biochemical analysis of the CRP-FNR-type transcriptional regulator family CprK, of which five are present in the strain. CprK1 is able to sense the presence of the physiological ligand, 3-chloro-4-hydroxyphenylacetic acid (CHPA), of reductive dehalogenase CprA1 with nM affinity. In this work we demonstrate that CprK1 is able to distinguish between the chlorinated CprA1 substrate CHPA and the non-chlorinated product 4-hydroxyphenylacetic acid (HPA) by ‘pKa interrogation’ of the 4-hydroxy moiety and by the atomic radius of the ortho-moiety. Through the use of in vitro biophysical and in vivo transcriptional response assays, we show that CprK1 is able to sense a number of halogenated phenols, including phenylacetic acids and nitrophenols. We also demonstrate that a 4-hydroxyl group is essential for CprK1 activation. In Chapter 4, an attempt to modify the effector sensitivity of CprK1 is performed by site-specific and random mutagenesis, and mutant selection assays are developed. We show that CprK1 is highly resistant to effector specificity modifications, with seemingly minor or conservative amino acid changes removing CprK1’s ability to initiate transcription. In Chapter 5, the CprK1 paralogue, CprK4 from D. hafniense DCB-2 is characterised by in vitro biophysical and in vivo transcriptional response assays in order to assess its potential as a biosensor. We show that CprK4 is able to bind cis-regulatory DNA elements dehaloboxes 7 and 10 in the absence of effector by Surface Plasmon Resonance (SPR) protein array; however, we were unable to identify its effectors reliably. Due to the unknown nature of CprK4’s effector, it is still unclear whether CprK4 could be a valuable biosensor.

The evolution of microorganisms over millions of years has led to an impressive adaptability regarding the utilisation of different environmental conditions. The identification of bacterial species with the fascinating features to use cobalamin-dependent metalloenzymes to (i) extract energy from halogenated organic compounds (organohalides) and (ii) transfer methyl groups from lignin breakdowns into central carbon pathways, are examples for this adaptability. The biochemical study of these two cobalamin-dependent enyzmes is the topic of this PhD project. For the extraction of growth energy, organohalides serve as terminal electron acceptorsand are reductively dehalogenated in a respiratory manner termed organohalide respiration. Reductive dehalogenases, the key enzymes in organohalide respiration, catalyse the chemical cleavage between the halogen substituent and the carbon moiety. They use cobalamin and two Fe-S clusters as cofactors and constitute a new and distinct class of cobalamin-dependent enzymes. Their three-dimensional structure and the mechanism of catalysis are unknown, because their hydrophobicity and oxygen sensitivity have hampered their biochemical investigation. Here, a novel purification technology in Escherichia coli for the reductive dehalogenase PceA from Dehalobacter restrictus has been developed, accompanied by methods that allow the in vitro reconstitution of PceA with both cofactors, cobalamin and Fe-S clusters. It has been demonstrated that the soluble expression of PceA is dependent on the covalent fusion of the enzyme to a trigger factor chaperone. Based on these findings, the PceA specific trigger factor PceT has been studied biochemically, resulting in its successful crystallisation. The established protocols for PceA and PceT are transferable to other members of their respective families, which will therefore allow detailed studies of reductive dehalogenases and their associated chaperones in the future. In addition to reductive dehalogenases, organohalide respiring bacteria contain anothercobalamin-dependent enzyme system, termed O-demethylase, which is involved in the carbon metabolism of different anaerobic bacteria. O-demethylases are three-component enzyme systems that transfer methyl groups from aromatic methyl ethers totetrahydrofolate via methylcobalamin intermediates. The different cofactors (substrate,cobalamin and tetrahydrofolate), bind to either of the three individual proteins involvedin O-demethylation. It has been speculated that the same or similar halogenated aromatic molecules are substrates for both organohalide respiration and O-demethylation in the same bacteria. In order to test this proposal, a O-demethylase from Desulfitobacterium hafniense DCB-2 has been studied using X-ray crystallography and biochemistry. As a result, the first crystal structures of the cobalamin-binding protein in complex with cobalamin, and of the methyl acceptor protein in complex with substrate (tetrahydrofolate) and product (methyltetrahydrofolate) from a O-demethylase have been solved toresolutions of 1.5 A, 1.8 A and 1.6 A, respectively. The crystal structures, in combinationwith spectroscopic and biophysical analyses, have led to a proposed mechanism forthe catalysed methyl transfer reaction from methylcobalamin to tetrahydrofolate.

This dissertation is devoted to the development of novel devices for optoelectronic and photovoltaic applications using the promise of inkjet printing with two-dimensional (2D) materials. A systematic approach toward the characterization of the liquid exfoliated 2D inks comprising of graphene, molybdenum disulfide (MoS2), tungsten diselenide (WSe2), and 2D perovskites is discussed at depth. In the first study, the biocompatibility of 2D materials -- graphene and MoS2 -- that were drop cast onto flexible PET and polyimide substrates using mouse embryonic fibroblast (STO) and human esophageal fibroblast (HEF) cell lines, was explored. The polyimide samples for both STO and HEF showed high biocompatibility with a cell survival rate of up to ~ 98% and a confluence rate of 70-98%. An inkjet printed, biocompatible, heterostructure photodetector was constructed using inks of photo-active MoS2 and electrically conducting graphene, which facilitated charge collection of the photocarriers. The importance of such devices stems from their potential utility in age-related-macular degeneration (AMD), which is a condition where the photosensitive retinal tissue degrades with aging, eventually compromising vision. The biocompatible inkjet printed 2D heterojunction devices were photoresponsive to broadband incoming radiation in the visible regime, and the photocurrent scaled proportionally with the incident light intensity, exhibiting a photoresponsivity R ~ 0.30 A/W. Strain-dependent measurements were also conducted with bending, that showed Iph ~ 1.16 µA with strain levels for curvature up to ~ 0.262 cm-1, indicating the feasibility of such devices for large format arrays printed on flexible substrates. Alongside the optoelectronic measurements, temperature-dependent (~ 80 K to 573 K) frequency shifts of the Raman-active E12g and A1g modes of multilayer MoS2 exhibited a red-shift with increasing temperature, where the temperature coefficients for the E12g and A1g modes were determined to be ~ - 0.016 cm-1/K and ~ - 0.014 cm-1/K, respectively. The phonon lifetime τ was determined to be in the picosecond range for the E12g and A1g modes, respectively, for the liquid exfoliated multilayer MoS2. Secondly, an all inkjet printed WSe2-graphene hetero-structure photodetector on flexible polyimide substrates is also studied, where the device performance was found to be superior compared to the MoS2-graphene photodetector. The printed photodetector was photo responsive to broadband incoming radiation in the visible regime, where the photo responsivity R ~ 0.7 A/W and conductivity σ ~ 2.3 × 10-1 S/m were achieved at room temperature. Thirdly, the synthesis of solution-processed 2D layered organo-halide (CH3(CH2)3NH3)2(CH3NH3)n-1PbnI3n+1 (n = 2, 3, and 4) perovskites is presented here, where inkjet printing was used to fabricate heterostructure flexible photodetector devices on polyimide substrates. The ON/OFF ratio was determined to be high, ~ 2.3 × 103 while the photoresponse time on the rising and falling edges was measured to be rise ~ 24 ms and fall ~ 65 ms, respectively. The strain-dependent measurements, conducted here for the first time for inkjet printed perovskite photodetectors, revealed the Ip decreased by only ~ 27% with bending (radius of curvature of ~ 0.262 cm-1). This work demonstrates the tremendous potential of the inkjet printed, composition tunable, organo-halide 2D perovskite heterostructures for high-performance photodetectors, where the techniques are readily translatable toward flexible solar cell platforms as well. Fourthly, metal contacts and carrier transport in 2D (CH3(CH2)3NH3)2(CH3NH3)n-1PbnI3n+1 (n = 4) perovskites is a critical topic, where the use of silver (Ag) and graphene (Gr) inks as metallic contacts to 2D perovskites was investigated. The all inkjet printed Gr-perovskite and Ag-perovskite photodetectors were found to be photo-responsive to broadband incoming radiation where measurements were conducted from λ ~ 400 nm to 2300 nm. The photoresponsivity R and detectivity D were compared between the Gr-perovskite and Ag-perovskite photodetectors, which revealed the higher performance for the Ag-perovskite photodetector. The superior performance of the Ag-perovskite photodetector was also justified with the Schottky barrier analysis using the thermionic emission model through temperature-dependent transport measurements. Finally, this dissertation ends with the description of the first steps for using solution-processed, inkjet printed perovskites for solar cells. The preliminary investigations include the discussion of the chemical formulations for the carrier separation layers, dispersion route, and the variation of solar cell figures of merit with processing.

Organohalide pollution of freshwater and marine sediments threatens human and environmental well-being. In freshwater and marine sediments a natural anaerobic microbiological process called reductive dehalogenation (RD) is carried out by organohalide respiring bacteria (OHRB) and reduces toxicity and improves biodegradability of organohalide pollutants. The reaction is catalyzed by enzymes called reductive dehalogenases (rdh). The marine sediment is the final sink for such dangerous and persistent contaminants, which make the very environment noxious, bioaccumulate in living beings and reach human foodstocks. This work explores reductive dehalogenation, OHRB and their rds’s in the marine environment of two sites of the Adriatic Sea: the Venice Lagoon (VL) and Ravenna Harbor (RH). In a microcosm study, primary sediment from the two sites and from an OHRB-enriched PCB-dechlorinating slurry culture were spiked with different organochloride compounds: hexachlorobenzene (HCBe), 1,2,3,5-tetrachlorobenzene (TeCBe), pentachlorophenol (PCP), 2,3,5-trichlorophenol (TCP) and trichloroethylene (TCE), 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) and a commercial mixture of PCBs (Aroclor© 1254). All compounds were dechlorinated to a certain extent in OHRB-enriched cultures, while primary sediments showed MRD of HCBe, TeCBe, TCP, TCE but not of PCP, and TeCDD, while PCBs dechlorination in VL sediments was not observed in RH but only in VL in previous studies. Microbial community analysis revealed the enrichment of bacteria from the Dehalococcoidia class where dechlorination was taking place in most primary cultures. In enriched cultures the increase of previously identified phylotypes from the same taxon, VLD-1 and VLD-2, correlated with dechlorination. A sediment free TCE-dechlorinating consortium was established in a defined mineral medium. PCR degenerate primer pairs screens and Next Generation Sequencing of amplicons revealed 81 novel rdh homologous gene sequences. Gene expression and proteomic mass spectrometry studies on the TCE-dechlorinating cultures revealed the overexpression of a cluster of three rdh genes hinting at a TCE-dechlorinating activity of one or more of them.

Els compostos orgànics halogenats (organohalogenats) són recalcitrants i presenten efectes tòxics per la salut humana i l’ecosistema. A partir d’uns sediments de la desembocadura del riu Besòs (Barcelona, Espanya) es va establir un cultiu anaerobi estable i no metanogènic que exclusivament dehalogenava cloro- i bromoalcans que tenen els àtoms d’halògens en carbonis adjacents via dihaloeliminació. L’aplicació de cebadors específics derivats a partir del gen 16rRNA de diferents bacteris dehalorespiradors junt amb les observacions fisiològiques del cultiu declorador indicaren que una soca de Dehalogenimonas era la responsable de la degradació dels alcans clorats. L’augment de còpies del gen 16S rRNA de Dehalogenimonas determinades per qPCR demostraren que la decloració de 1,2-dicloropropà (1,2-DCP) es produïa en paral·lel al creixement de Dehalogenimonas. L’anàlisi de l’isòtop de carboni durant la degradació de 1,2-DCP per Dehalogenimonas presenta un fraccionament isotòpic () de -15.0 ± 0.7 que és significativament diferent al d’altres soques de Dehalococcoides encara quetenen la mateixa dehalogenasa reductiva funcional (DcpA) implicada en la degradació de 1,2-DCP a propè. La correlació de fraccionament isotòpic obtingut per a la parella C-Cl (Ʌ = 1.89 ± 0.02) durant la dicloroeliminació de 1,2-DCA per Dehalogenimonas era significativament diferent tant dels obtinguts prèviament per Dehalococcoides catalitzant una reacció similar de dehaloeliminació com dels de l’oxidació aeròbia de 1,2-DCA. Aquests resultats serveixen per il·lustrar la potencialitat de l’ús del fraccionament isotòpic simultani del C-Cl per a diferenciar entre diferents mecanismes de reacció (oxidació, dehalogenació hidrolítica i dehaloeliminació) Desprès d’assolir el repte d’obtenir un cultiu estable, l’aïllament de Dehalogenimonas va ser el següent objectiu. Fins avui, només 3 especies del gènere Dehalogenimonas han estat aïllades i aquesta és la primera que es cultiva a Europa. Per abordar l’aïllament s’ha fet servir el mètode de dilució fins a l’extinció i l’addició d’antibiòtics selectius. Desprès de 13 transferències seqüencials d’aquest cultiu de partida alimentat amb 1,2-DCP i dues dilucions consecutives 1:107 seguides per l’addició d’estreptomicina durant 5 transferències, es va fer una genoteca que va demostrar que el cultiu estava format predominantment per Dehalogenimonas sp. (87%), però també hi havia Desulfovibrio sp. (12%) i una Vellonellaceac no classificada (1%). Actualment s’està continuant el treball en el nostre laboratori per assolir l’aïllament de Dehalogenimonas sp. En aquest estudi s’ha intentat fer una llista preliminar de dehalogenases reductives (RDase) candidates a estar involucrades en la transformació de 1,1,2-tricloroetà (1,1,2-TCA) i 1,2-dibromoetà (EDB) en Dehalogenimonas fent servir la tècnica de shotgun proteomics (LTQ-Orbitrap). A més a més, es van fer electrofòresis amb gel de poliacrilamida blau natiu (BN-PAGE) combinat amb assajos d’activitat decloradora per identificar la RDase responsable de la dicloroeliminació de 1,1,2-TCA. Encara que es va detectar activitat decloradora en una banda del gel, no es va identificar la RDase ni per cromatografia líquida acoblat amb un espectròmetre de masses (LC-MS/MS) ni per electroforesis en gel de poliacrilamida amb dodecil sulfat de sodi. L’absència de RDAse pot ser degut a vàries raons, entre elles la baixa concentració de proteïna i l’ús d’una base de dades que es va construir a partir d’anotacions dels genomes d’altres Dehalogenimonas, perquè la nostra soca no està encara seqüenciada. Finalment, es va construir un co-cultiu sintròfic de Dehalogenimonas i Dehalococcoides mccartyi per tal d’assolir la completa detoxificació de 1,1,2-TCA fins a etè. En aquest co-cultiu, Dehalogenimonas transforma 1,1,2-TCA via dihaloeliminació a clorur de vinil, alhora que Dehalococcoides redueix el clorur de vinil via hidrogenòlisis a etè. S’ha fet un canvi d’escala a un reactor anaerobi de 5 L operant en semicontinu. En aquest estudi es demostra, a partir d’una combinació sintètica de bacteris, la capacitat de detoxificar 1,1,2-TCA quan aquests microorganismes presenten capacitats metabòliques complementàries.
Halogenated compounds (organohalides) are recalcitrant organic compounds, exhibiting toxic effects on human health and the ecosystem. In this study, we aimed to obtain an enrichment culture containing organohalide-respiring bacteria able to transform halogenated alkanes. A stable nonmethanogenic enriched anaerobic culture that exclusively dehalogenates vicinally chlorinated and brominated alkanes via dihaloelimination was established from Besòs River estuary sediments (Barcelona, Spain). Application of genus specific primers targeting 16S rRNA gene sequences together with the observation of physiological characteristics of the dechlorinating culture indicated that a Dehalogenimonas strain was the responsible for chlorinated alkane degradation in the microcosms. The increase of Dehalogenimonas 16S rRNA gene copies using quantitative PCR (qPCR) revealed that 1,2-dichloropropane (1,2-DCP) dechlorination was coupled to Dehalogenimonas growth in this culture. Carbon and dual carbon-chlorine (C-Cl) isotope fractionation during anaerobic biodegradation of 1,2-DCP and 1,2-dichloroethane (1,2-DCA), respectively, by Dehalogenimonas-containing enrichment culture were determined in this study. Compound specific isotope analysis revealed that the Dehalogenimonas-catalyzed carbon isotopic fractionation () of the 1,2-DCP-to-propene reaction was −15.0 ± 0.7‰ and differs significantly from other Dehalococcoides strains eventhough they harbored the same functional reductive dehalogenase (DcpA) for 1,2-DCP-to-propene dechlorination. The dual element C-Cl isotope correlation obtained (Ʌ = 1.89 ± 0.02) during 1,2-DCA-to-ethene dichloroelimination by Dehalogenimonas was significantly discernible from those reported for Dehalococcoides catalyzing a similar dihaloelimination reaction and aerobic oxidation. This illustrates the potential use of dual C-Cl isotope approach to distinguish between different degradation pathways (oxidation, hydrolytic dehalogenation and dihaloelimination). After overcoming the challenges in developing a stable culture, isolation of Dehalogenimonas became the next objective. To date, only three species belonging to Dehalogenimonas genus have been isolated and this study constituted the first evidence of a Dehalogenimonas culture enriched in Europe. The isolation approach consisted of the dilution to extinction method and the addition of selected antibiotics. After thirteen sequential transfer of this culture fed with 1,2-DCP-to-propene and two consecutive 10-7 dilutions followed by the addition of streptomycin for five transfers, a clone library of bacterial amplicons revealed that Dehalogenimonas sp. constituted 87 % of the predominant bacteria, followed by Desulfovibrio sp. (12 %) and unclassified Veillonellaceae (1.2%). Further work is currently underway in our lab to isolate the Dehalogenimonas sp. In this study, a preliminary list of reductive dehalogenase (RDase) candidates involved in the transformation of 1,1,2-trichloroethane (1,1,2-TCA) and 1,2-dibromoethane (EDB) by this Dehalogenimonas culture was attempted using shotgun proteomics analysis (LTQ-Orbitrap). In addition, blue native polyacrylamide gel electrophoresis (BN-PAGE) approach combined with dechlorination activity assays were performed to identify the RDase responsible for 1,1,2-TCA dichloroelimination. Eventhough dechlorination activity was detected in a gel slice of the BN-PAGE of the culture growing with 1,1,2-TCA, no RDase was identified by neither liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis nor sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The absence of RDase can be due to several reasons including low protein content and the use of a database constructed from the published genomic annotations of other isolated Dehalogenimonas because our strain was not sequenced yet. Finally, a syntrophic co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains to achieve complete detoxification of 1,1,2-TCA to ethene was also constructed. In this co-culture, Dehalogenimonas transformed 1,1,2-TCA via dihaloelimination to vinyl chloride, whereas Dehalococcoides reduced vinyl chloride via hydrogenolysis to ethene. Scale up of the cultivation to a 5-L bioreactor operating in fed-batch mode and the synthetic combination of these bacteria with known complementary metabolic capabilities was demonstrated.

Creating new carbon-carbon bonds is one of the most important and challenging reactions in organic synthesis. Metal-catalyzed cross-coupling reactions have emerged as one of the preferred methods of producing new carbon-carbon bonds, and this work led to the 2010 Nobel Prize in Chemistry. This thesis was aimed at expanding the current research in the area of metal-catalyzed cross-coupling reactions to include new applications with dithiolane and dithiane protecting groups. 1,3-Dithiolane and 1,3-dithiane derivatives are particularly interesting molecules in that they can be deprotonated by a strong base to form anions, which can then be used for carbon-carbon bond synthesis. This thesis describes the investigation into the use of dithiolanes and dithianes in metal-catalyzed cross-coupling reactions, as well as some of the challenges faced in performing this sulfur-based chemistry.

This study is focused on one particular group of the halogenated molecules called polychlorinated biphenyls (PCBs) - synthetic, organic compounds derived from biphenyl with bound chlorine atoms. Depending on the position and number of the chlorine atoms, there are theoretically 209 individual PCB congeners. Although PCBs production was brought to a halt thirty years ago, recalcitrance to degradation makes them a major environmental pollutant at a global scale. Large amounts of PCBs were produced in several countries, and former Czechoslovakia belonged to the ten major world producers. Despite the PCB congener resistance to chemical modification, bacterial process of reductive dechlorination, named organohalide respiration (OHR), was shown to be efficient in the dechlorination of extensively chlorinated PCB congeners and a prerequisite step towards their subsequent complete mineralization by aerobic bacteria. In our study, reductive dechlorination of polychlorinated biphenyls (PCBs) was assessed using long term anaerobic microcosms. The microcosms were inoculated with highly contaminated with weathered Delor sediments, sampled from the efflux channel of the former PCB manufacturer Chemko Strazske. After one year of cultivation the chemical analysis showed a degradation of up to 36 % of the highly...

Organohalides have been abundantly utilized as pesticides and in industrial processes for the past 100 years, with over 30 000 sites in Europe still being contaminated today. Because of their recalcitrance, large quantities have accumulated in soils, sediments, and groundwater. Many organohalides can cause multiple adverse health effects, including neurological damage, congenital malformations, and a variety of human cancers. Fortunately, bacterial genera from a diverse range of phyla are capable of detoxifying these organohalides via anaerobic respiration, i.e., by using them as their terminal electron acceptor. These metabolic pathways involve a reductive dehalogenation reaction, during which a chlorine atom dissociates and thereby either immediately reduces the toxicity of the organohalide, or enables it to be further degraded by a broader range of organisms. Thus, organohalide-respiring bacteria can be used for the bioremediation of contaminated environments. To be able to support this application, fundamental research on these reactions and the metabolism of organohalide-respiring bacteria is a prerequisite. Many aspects of the physiology of organohalide-respiring bacteria are unresolved. Organohalide-respiring bacteria harbor up to 38 reductive dehalogenase homologous genes, which putatively encode the key enzymes of reductive dehalogenation. However, the regulation, protein-coding ability, the function of these enzymes as well as their interactions with other proteins has yet to be elucidated. Organohalide-respiring bacteria are difficult to study due to their slow growth, low biomass yields, oxygen sensitivity and genetic inaccessibility. The aim of this thesis was to circumvent these obstacles by introducing new methods for studying organohalide respiration and thereby enabling the formulation of informed predictions about the functions of reductive dehalogenases and the identity of their regulators. For this, obligate and facultative organohalide-respiring bacteria were assessed. To form a basis of the current research in the field, all available genomic, transcriptomic and proteomic literature on organohalide-respiring bacteria were reviewed and compared. Through combining quantitative expression data of hundreds of orthologs and subjecting them to statistical analyses, many new aspects of the metabolism of organohalide-respiring bacteria were uncovered. Especially notable were the unclear expression patterns of reductive dehalogenases and their accessory proteins. An important conclusion from this review was that shotgun proteomics is essential to reveal how many reductive dehalogenase proteins are produced in parallel, but this approach alone cannot clarify the function of these enzymes nor their underlying regulation processes. Therefore, the next chapter of this thesis aimed to extend and refine the standard proteomics approaches. First, proteomics conducted via mass spectrometry requires optimization of sample processing and analysis. Utilizing harsher conditions for protein extraction and digestion substantially improved proteome coverage compared to previous studies, especially of membrane proteins. The combination of this approach with a highly stringent statistical filtering procedure allowed a more detailed, reliable and thus more valid view of the proteome to be obtained from the model organism Sulfurospirillum halorespirans. The quantification of the putative protein histidine kinase provided the first evidence of its involvement in controlling organohalide respiration together with the putative response regulator, forming a complete two-component regulatory system. The quantification of the putative quinol dehydrogenase membrane subunit also supported its involvement in the organohalide respiratory chain of this genus. We observed that S. halorespirans undergoes the same type of peculiar memory-effect as Sulfurospirillum multivorans, that is, continuing to produce its complete dehalogenating machinery even after prolonged cultivation on a non-halogenated electron acceptor. To reveal the underlying mechanism, protein lysine acetylation was additionally measured, which is an important post-translational modification involved in many regulatory processes across all living organisms. Lysine acetylations are, e.g., known to alter the binding properties of DNA-interacting proteins like transcription factors or response regulators but have a range of other regulatory effects. In the first ‘acetylome’ study of an organohalide-respiring bacterium and an Epsilonproteobacterium, one-third of all S. halorespirans proteins were found to be acetylated at one point over the course of a long-term cultivation experiment. Interestingly, the putative response regulator of the two-component regulatory system described earlier was acetylated during the metabolic transition phase, after short-term adaptation to a non-halogenated electron acceptor. Another advancement of shotgun proteomics was its combination with thermal proteome profiling to elucidate substrate specificities of reductive dehalogenases and their regulators. The underlying principle behind thermal proteome profiling is to identify the interaction of a protein with a binding ligand through its impact on the thermal stability of the protein. The thermal stability of hundreds of proteins can be measured in parallel by a proteomics approach. Aliquots of protein extract are first incubated at different temperatures, and the non-denatured fraction of each protein is then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), thus allowing the composition of melting curves of each protein to be determined. With this unbiased approach, unknown protein-ligand interactions can also be identified. In a proof-of-concept study on S. multivorans, we adapted the method to anaerobic conditions and showed that this technique is suitable for the detection of interactions between enzymes and their specific substrates. For example, a melting curve shift was detected when the tetrachloroethene reductive dehalogenase, PceA, bound to its known substrate, trichloroethene. Furthermore, the melting curve shift of the putative response regulator in the two-component regulatory system indicated at least an indirect interaction between it and trichloroethene, providing the first biochemical evidence of its role in organohalide respiration besides mere expression data. In conclusion, this work not only includes the first systematic analysis of all omics-based studies conducted to date but substantially advanced the methods for assessing organohalide-respiring bacteria by providing a more detailed picture of their physiology. Besides methodological advances, it was demonstrated that the two-component regulatory system interacts with halogenated compounds and that its post-translational modification might impact long-term downregulation of the organohalide respiratory apparatus in Sulfurospirillum spp. The insights into the involvement of the two-component regulatory system in the organohalide respiration of Sulfurospirillum spp. would not have been uncovered by using less complex standard shotgun proteomics measurements. In the future, our findings will help to further elucidate regulators and functioning of reductive dehalogenases also in other organohalide-respiring bacteria.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128
Während der letzten einhundert Jahre wurden halogenierte organische Verbindungen großflächig in Industrie und Landwirtschaft eingesetzt, wodurch heute mehr als 30 000 Flächen in Europa kontaminiert sind. Aufgrund ihrer eingeschränkten Abbaubarkeit konnten sich riesige Mengen in Böden, Sedimenten und Grundwasser ausbreiten. Viele halogenierte organische Verbindungen können erhebliche nachteilige Auswirkungen auf die Gesundheit des Menschen haben, u.a. neurologische Schäden, Fehlbildungen und eine Vielzahl von Krebserkrankungen. Glücklicherweise sind bestimmte Bakterientypen unterschiedlicher Phyla in der Lage, diese Stoffe mittels anaerober Atmung, d.h. über deren Nutzung als terminalen Elektronenakzeptor, umzuwandeln. Diese reduktive Dehalogenierung, bei der ein Chlor-Rest abgespalten wird, vermindert die Toxizität der meisten Organohalide bzw. macht sie zugänglich für den Abbau durch ein breiteres Organismenspektrum. Demgemäß können Organohalid-atmende Bakterien für die Bioremediation kontaminierter Flächen genutzt werden. Voraussetzung für deren Einsatz ist jedoch das Verständnis der zugrundeliegenden biochemischen Reaktionen und des Metabolismus der Organohalid-Atmer. Viele Aspekte der Physiologie Organohalid-atmender Bakterien sind noch ungeklärt. Die Organismen besitzen bis zu 38 unterschiedliche Gene, die reduktive Dehalogenasen, die Schlüsselenzyme der Organohalid-Atmung, kodieren. Allerdings sind deren Regulation, Proteinkodierung, die Funktion der einzelnen Enzyme sowie deren Interaktionen mit anderen Proteinen noch unbekannt. Die Forschung an Organohalid-atmenden Bakterien wird durch deren langsames Wachstum, die geringen Zelldichten, die hohe Sensitivität gegenüber Sauerstoff und fehlende gentechnische Methoden erschwert. Ziel dieser Arbeit war es, die genannten Hindernisse mittels neuartiger Methoden an Organohalid-Atmern zu umgehen und damit Regulatoren und Funktionsweise der reduktiven Dehalogenasen zu bestimmen. Hierfür wurden sowohl obligate als auch fakultative Organohalid-atmende Bakterien herangezogen. Als Grundlage führte ich zunächst alle bisher durchgeführten Genomik-, Transkriptomik- und Proteomikstudien zu Organohalid-atmenden Bakterien zusammen. Hunderte zu Orthologen kombinierte und statistisch analysierte quantitative Expressionsdaten lieferten dabei ein umfassendes Bild vom Metabolismus der Organohalid-Atmer. Insbesondere die unklaren Expressionsmuster der reduktiven Dehalogenasen und ihrer akzessorischen Proteine wurden offenbar. Eine wichtige Erkenntnis des Review-Prozesses war, dass Standard-Proteomikansätze zwar unerlässlich sind, um beispielsweise die gleichzeitige Produktion mehrerer reduktiver Dehalogenasen offenzulegen, aber weder deren Funktionen noch Regulation aufklären können. Aus diesem Grund sollten im weiteren Verlauf dieser Arbeit die bisher genutzten Shotgun-Proteomikmethoden weiterentwickelt werden. Für eine umfassende Proteinanalyse mittels Massenspektrometrie müssen zunächst Probenaufarbeitung und Analyse optimiert werden. Durch die Verwendung harscherer Bedingungen bei Proteinextraktion und -verdau konnten wir die Proteomabdeckung, insbesondere unter Membranproteinen, im Vergleich zu früheren Studien erheblich verbessern. In Kombination mit einem sehr stringenten statistischen Filterprozess erlaubte dies einen detaillierten und validen Blick auf das Proteom des Modellorganismus Sulfurospirillum halorespirans. Die Quantifizierung der mutmaßlichen Protein-Histidinkinase ist der erste Beleg dafür, dass diese zusammen mit dem Regulationsprotein im Zweikomponentensystem an der Kontrolle der Organohalid-Atmung in Sulfurospirillum spp. beteiligt ist. Die quantifizierte Membranuntereinheit der Quinoldehydrogenase stützt die Annahme zu deren Beteiligung an der Atmungskette dieses Organismus. Wir konnten weiterhin zeigen, dass in S. halorespirans die gleiche außergewöhnliche Langzeitregulation wie in Sulfurospirillum multivorans wirksam ist, sodass auch nach langanhaltender Kultivierung auf nicht-halogenierten Substraten der komplette Organohalid-Atmungsapparat synthetisiert wird. Zur Aufklärung der zugrundeliegenden Regulation erweiterten wir unsere Analyse um Protein-Lysin-Acetylierungen, wichtige posttranslationale Modifikationen, die an verschiedensten regulatorischen Prozessen in allen Lebewesen beteiligt sind. Protein-Lysin-Acetylierungen beeinflussen z.B. die Wechselwirkungen zwischen Transkriptionsfaktoren oder Regulationsproteinen und der DNA, aber haben noch viele weitere regulatorische Effekte. In dieser ersten „Acetylom“-Studie an einem Organohalid-atmenden Bakterium bzw. einem Epsilonproteobacterium, konnten wir zeigen, dass ein Drittel aller S. halorespirans-Proteine im Verlauf der Langzeitkultivierung mindestens einmal acetyliert wurden. Interessanterweise war auch das mutmaßliche Regulatorprotein des oben erwähnten Zweikomponentensystems während der metabolischen Umstellungsphase, d.h. nach Kurzzeitanpassung an den nicht-halogenierten Elektronenakzeptor, acetyliert. Eine zusätzliche Weiterentwicklung der klassischen proteomischen Messungen war deren Kombination mit Thermal Proteome Profiling, um Substratspezifitäten und Regulatoren von reduktiven Dehalogenasen zu bestimmen. Zugrundeliegendes Prinzip des Thermal Proteome Profiling ist die Identifikation eines Proteinbindungspartners über dessen Einfluss auf die Thermostabilität der Faltung eines Proteins. Die Thermostabilität tausender Proteine kann mit Hilfe eines Proteomikansatzes bestimmt werden. Hierfür werden extrahierte Proteine zunächst aufgeteilt und unterschiedlichen Temperaturen ausgesetzt. Die nicht-denaturierte Fraktion jedes Proteins kann mittels Flüssigchromatographie mit Tandemmassenspektrometrie-Kopplung (LC-MS/MS) quantifiziert und zu Schmelzkurven zusammengesetzt werden. Mit dieser Methode können auch unbekannte Protein-Liganden-Interaktionen identifiziert werden. In unserer Machbarkeitsstudie an S. multivorans konnten wir zeigen, dass die von uns modifizierte Technik auch zur Aufklärung von Enzym-Substrat-Interaktionen und sogar unter anaeroben Bedigungen eingesetzt werden kann. So konnte nachgewiesen werden, dass die Schmelzkurve der reduktiven Tetrachlorethen-Dehalogenase PceA durch Bindung ihres bekannten Substrates Trichlorethen signifikant verschoben wurde. Außerdem deutet die Verschiebung der Schmelzkurve des mutmaßlichen Regulatorproteins des Zweikomponentensystems zumindest auf eine indirekte Interaktion mit Trichlorethen hin und ist damit, abgesehen von bloßen Expressionsdaten, der erste biochemische Beleg für dessen Rolle bei der Organohalid-Atmung. Zusammenfassend beinhaltet diese Arbeit nicht nur die erste systematische Analyse und Kombination aller bisher verfügbaren „Omics“-Studien, sondern auch deren Weiterenwiclung für die Untersuchung organohalid-atmender Bakterien, wodurch ein detailliertes Bild von deren Physiologie geschaffen werden konnte. Neben den technischen Neuerungen konnte gezeigt werden, dass das Zweikomponentensystem von Sulfurospirillum sp. mit halogenierten organischen Verbindungen interagiert und dass dessen posttranslationale Modifikation die Langzeitreulation des Organohalid-Atmungsapparates beeinflussen könnte. Die Einblicke in die Beteiligung des Zweikomponentensystems an der Organohalidatmung in Sulfurospirillum sp. wären durch Nutzung von weniger komplexen Standard-Proteomikmethoden unentdeckt geblieben. In Zukunft können uns diese neu entwickelten Methoden dabei unterstützen, Funktionalität und Regulation von reduktiven Dehalogenasen in anderen Organohalid-Atmern aufzuklären.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128

abstract: Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene.
Dissertation/Thesis
Ph.D. Microbiology 2013