REDUCTIVE DEHALOGENATION OF GAS-PHASE TRICHLOROETHYLENE USING HETEROGENEOUS CATALYTIC AND ELECTROCHEMICAL METHODSXiumin Ju, Ph.D.The University of Arizona, 2005Director: Dr. Robert G. ArnoldThe first part of this work investigates catalytic hydrodechlorination (HDC) of gas-phase trichloroethylene (TCE) using 0.5 wt.% Pt/g-Al2O3 and 0.0025 wt.% Pt/SiO2 in packed-bed reactors. TCE was efficiently transformed on the platinum surface using H2 as reducing agent. The main products of the reaction were ethane and chloroethane. In the case of Pt/Al2O3, more than 94% TCE conversion efficiency was maintained for over 700 hours of operation at 100ºC at a residence time of 0.37 seconds. At 22ºC, severe catalyst deactivation was observed. Catalyst deactivation was attributed to coking and chlorine poisoning. A series of treatments including (i) hydrogen gas addition at high temperature (oxygen free) to remove chlorine and (ii) oxygen addition at 500ºC to remove coke were attempted to regenerate the deactivated catalyst. Only hydrogen treatment partially restored catalyst activity. When using Pt/SiO2, catalyst deactivation was severe even at 100ºC, probably due to low surface area of Pt and the silica support. Adding KOH to the packed Pt/SiO2 catalyst during (otherwise) normal operation slowed catalyst deactivation. Adding O2 to the influent improved catalyst activity and slowed deactivation.The second part of this research involves the destruction of gas-phase TCE using an electrochemical reactor similar in design of a polymer electrolyte membrane (PEM) fuel cell. With a proton-conducting membrane in the middle, the anode and cathode comprised of carbon cloth and carbon-black-supported Pt were hotpressed together to form a membrane electrode assembly (MEA). TCE contaminated gas streams were fed to the cathode side of the fuel cell, where TCE was reduced to ethane and hydrochloric acid. The results suggest that TCE reduction occurs via a catalytic reaction with atomic hydrogen that is reformed on the cathode's surface rather than an electrochemical reduction via direct electron transfer. Substantial conversion of TCE was obtained, even in the presence of molecular oxygen in the cathode chamber. The process was modeled successfully by conceptualizing the cathode chamber as a plug flow reactor with a continuous source of H2(g) emanating from the boundary.

Perfluorooctane sulfonate (PFOS) and related perfluoroalkyl surfactants (PFAS) are ubiquitous contaminants of increasing public concern due to their environmental persistence, toxicity, and bioaccumulation. These perfluorinated compounds have been used for more than half a century in a wide variety of industrial and consumer products ranging from stain repellents such as Teflon® to aqueous fire-fighting foams and to grease-proof food packing. The public health and environmental risks posed by PFAS have driven environmental agencies and the industry to restrict their use to specific applications where they cannot be replaced by other chemicals. The sources and pathways of PFOS and its derivatives in the environment are not well understood. Analysis of environmental samples is critical to understand the fate, transport and persistence of these emerging contaminants. Techniques based on fluorine nuclear magnetic resonance (¹⁹F NMR) spectroscopy and high performance liquid chromatography (HPLC) with suppressed conductivity detection were successfully developed to monitor the presence of PFAS in water samples. Chromatographic separation of C₄ to C₈ PFAS surfactants was achieved using a C₁₈ reversed-phase column and a mobile phase consisting of a mixture of boric acid and acetonitrile at mixing ratios ranging from 75:25 to 45:55 (v/v). The combination of these two techniques was very effective for characterization and routine quantification of PFOS and related chemicals. Analytical methods based on ¹⁹F NMR, HPLC-suppressed conductivity detection, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were employed to characterize commercial PFOS samples. Linear and branched PFOS isomers in a percentage ratio of 75:25 were identified. Municipal wastewater treatment systems are one of the major sources of PFAS emissions into the environment. The presence of PFAS in sewage sludge from two wastewater treatment plants in Tucson, Arizona, was investigated. Sludge samples were washed with acetic acid and extracted with a mixture of acetic acid and methanol. The extract was cleaned and concentrated by means of solid phase extraction. LC-MS/MS operating in the selective ion monitoring (SIM) mode was employed to assess the presence of perfluorosulfonates, perfluorosulfonamides, and perfluorocarboxylates in sewage sludge samples. PFOS was the only perfluoroalkyl chemical detected in municipal sludge samples at a concentration of 77 ± 5 g kg⁻¹ sludge dry weight. Cost-effective treatment techniques for removing PFAS from industrial effluents are needed to minimize discharges of these pollutants. Reductive dehalogenation is widely applied to the degradation of highly chlorinated compounds. Hence, the susceptibility of PFOS and related compounds to biological and chemical reductive dehalogenation was evaluated in batch assays. PFAS were not reductively dehalogenated by different microbial consortia even after periods of incubation exceeding 2 y, confirming the high resistance of these compounds to microbial degradation. The anaerobic biodegradability of PFOS and perfluorobutane sulfonate (PFBS) samples exposed to electrochemical pretreatment with boron-doped diamond film electrodes was also investigated. The oxidation decreased the concentration of PFAS and dissolved organic carbon in solution, confirming the destruction of these compounds. However, the oxidative treatment did not enhance the susceptibility of PFAS to microbial degradation even after extended periods of incubation (> 1 y). In contrast, PFOS was reductively dehalogenated with a biomimetic system based on vitamin B12 as the catalyst and Ti(III) citrate as the reducing agent. The optimal treatments conditions of the reaction were 260 μM vitamin B₁₂, 36 mM Ti(III) citrate, 70°C and solution pH 9.0. Interestingly, branched PFOS isomers were more prone to degradation by vitamin B₁₂ catalysis compared to the linear isomer. Removal of 3 mol Fper mol of technical PFOS and 12 mol F- per mol of branched PFOS isomers was achieved. Defluorination of PFOS was also observed at environmental relevant conditions of 30°C and pH 7.0, albeit at lower degradation rates. Fluoride and carbon dioxide were identified as the major products of the chemical defluorination. Traces of partially fluorinated volatile compounds were also detected in the headspace. The feasibility of removing PFAS compounds from aqueous streams by sorption onto granular activated carbon (GAC), zeolite, and wastewater treatment sludge was examined in batch isotherm experiments. The fluorocarbon chain and the functional group influenced sorption of the anionic surfactants, PFOS adsorbed more strongly to GAC than perfluorooctanoic acid (PFOA) and PFBS. Activated carbon showed the highest affinity for PFOS (Freundlich K(F) values of 36.7 to 60.9) followed by the hydrophobic, high-silica zeolite NaY (Si/Al 80, K(F) of 31.8) and lastly anaerobic sludge (K(F) of 0.95 to 1.85). GAC sorption is a suitable treatment for the removal of anionic perfluoroalkyl surfactants when present at low concentrations. Fluoride has been identified as the major product of the reductive dehalogenation of PFOS and derivatives. Thus, the toxicity of inorganic fluoride towards the main microbial populations responsible for the removal of organic constituents and nutrients in wastewater treatment processes was also studied. Fluoride concentrations ranging from 18 to 43 mg L⁻¹ caused 50% inhibition (IC₅₀) of the activity of propionate- and butyratedegrading microorganisms and of acetate-utilization by methanogens evaluated under mesophilic and thermophilic conditions. All other microbial populations evaluated in this study, i.e., glucose fermenters, aerobic glucose-degrading heterotrophs, denitrifying bacteria, and H₂-utilizing methanogens tolerated fluoride at very high concentrations (> 500 mg L⁻¹). In the same manner, H₂-utilizing methanogens also tolerated PFOS and PFBS at concentrations as high as 200 and 500 mg L⁻¹, respectively.

This work focused on using a microbubble dispersion to deliver hydrogen and carbon dioxide to anaerobic consortia to stimulate their ability to reductively dehalogenate tetrachloroethylene all the way to ethene and ethane. A continuous flow system, consisting of six anaerobic soil column bioreactors, inoculated with sediments from Virginia Tech's Duck Pond, was used for this study. Two columns received microbubbles containing hydrogen and carbon dioxide, two received sodium propionate, and two were not fed a substrate. A 30 micromolar PCE solution was delivered to the consortia at 3 ml/min. Microbubbles containing a mixture of 90% hydrogen and 10% carbon dioxide were effectively produced in a closed spinning disk generator, and were acceptable for delivering the gases to the columns. After the biodegradation study was completed, the microbubbles were found to have a pH of 4.4, due to the carbon dioxide. Microbubbles amended with NaOH to 0.01 molar yielded pH neutral microbubbles with improved stability. Methane was measured in all six columns throughout the experiment, verifying that methanogens were present. Methane levels were highest in the propionate columns, showing the the methanogens there were more active. Methane levels in the microbubble columns were similar to those in the control columns. Propionate and acetate were not detected in the columns where propionate was fed, showing that proton reducers and acetoclastic methanogens were both active. Recovery of PCE and the degradation products was almost 90% in the microbubble and control columns where most of the PCE was recovered in the effluent. The predominant product in both systems was TCE, although some ethene was detected in all four columns. The control consortia produced TCE averaging about five micromolar while the microbubble columns averaged about two micromolar TCE. One of the components of the microbubbles probably caused the lowered amounts of PCE reduction. That some ethene was seen in the microbubble columns suggests different conditions can be found to stimulate the further reduction of PCE with hydrogen and carbon dioxide microbubbles. The product recovery in the propionate columns was about 64%. Over half of the injected PCE was dechlorinated to ethene and ethane. / Ph. D.

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

Foi estudada a degradação do pentaclorofenol (PCP) em reator aneróbio horizontal de leito fixo (RAHLF) de volume de 2000 mL. O reator foi inoculado com microrganismos oriundos de reatores aneróbios não previamente adaptados a PCP. Atividade microbiana foi monitorada através de técnicas clássicas na presença do organoclorado na faixa de 2,0 a 13 mg/L de PCP. O reator apresentou eficiência de 97% na remoção de DQO e completo desaparecimento do composto de PCP em todas as concentrações testadas. A fração orgânica foi consumida totalmente na primeira terça parte do reator de acordo com os valores determinados de ácidos voláteis, DQD e PCP. Não foi verificada inibição da atividade de culturas microbianas. Os exames microscópicos, fluorescência e varredura, permitiram verificar o predomínio de microrganismos pertencentes ao Domínio Archea. As técnicas moleculares PCR, ARDRA e hibridação slot-blot confirmaram o predomínio do Domínio Archaea e possibilitaram a verificação de alterações na diversidade das populações após adição de 2 mg PCP/L. Conclui-se que o reator sem prévia adaptação do inóculo foi eficiente para o tratamento do PCP, e os microrganismos relacionados às Archaea metanogênicas acetocláticas podem estar envolvidas na degradação deste composto. / The degradation of pentachlorophenol (PCP) was studied in a 2000 mL. Horizontal Anaerobic Immobilized System (HAIS). The reactor was inoculated with microorganisms obtained from an anaerobic reactor without previous adaptation to the PCP. The microbial activity was evaluated by using classic techniques in order to . monitor its behavior during the HAIS fed with a range of PCP between 2.0 to 13 mg/L. The reactor presented 97% of efficiency in the removal of COD and complete decrease of PCP in alI concentrations tested. The total consumption of organic fraction took place mainly in the first third part of the reactor according the values of volatile fatty acids, COD and PCP obtained. Microbial inhibition was not verified in during HAIS operation. Microscopic examinations allowed certifying the Archaea Domain predominance according the morphologies observed. The molecular techniques polimerase chain reaction (PCR), ARDRA and slot-blot hibridation confirmed the predominance of Archaea Domain and alIowed verifying some changes in the population\'s diversity under additions of 2mg PCP/L. The efficiency of PCP decreased in the anaerobic reactor was related to the presence of Archaea Domain, especially the acetoclastic methanogens, whose where probably involved with the organochlorine compound degradation.

Foi estudada a degradação do pentaclorofenol (PCP) em reator aneróbio horizontal de leito fixo (RAHLF) de volume de 2000 mL. O reator foi inoculado com microrganismos oriundos de reatores aneróbios não previamente adaptados a PCP. Atividade microbiana foi monitorada através de técnicas clássicas na presença do organoclorado na faixa de 2,0 a 13 mg/L de PCP. O reator apresentou eficiência de 97% na remoção de DQO e completo desaparecimento do composto de PCP em todas as concentrações testadas. A fração orgânica foi consumida totalmente na primeira terça parte do reator de acordo com os valores determinados de ácidos voláteis, DQD e PCP. Não foi verificada inibição da atividade de culturas microbianas. Os exames microscópicos, fluorescência e varredura, permitiram verificar o predomínio de microrganismos pertencentes ao Domínio Archea. As técnicas moleculares PCR, ARDRA e hibridação slot-blot confirmaram o predomínio do Domínio Archaea e possibilitaram a verificação de alterações na diversidade das populações após adição de 2 mg PCP/L. Conclui-se que o reator sem prévia adaptação do inóculo foi eficiente para o tratamento do PCP, e os microrganismos relacionados às Archaea metanogênicas acetocláticas podem estar envolvidas na degradação deste composto. / The degradation of pentachlorophenol (PCP) was studied in a 2000 mL. Horizontal Anaerobic Immobilized System (HAIS). The reactor was inoculated with microorganisms obtained from an anaerobic reactor without previous adaptation to the PCP. The microbial activity was evaluated by using classic techniques in order to . monitor its behavior during the HAIS fed with a range of PCP between 2.0 to 13 mg/L. The reactor presented 97% of efficiency in the removal of COD and complete decrease of PCP in alI concentrations tested. The total consumption of organic fraction took place mainly in the first third part of the reactor according the values of volatile fatty acids, COD and PCP obtained. Microbial inhibition was not verified in during HAIS operation. Microscopic examinations allowed certifying the Archaea Domain predominance according the morphologies observed. The molecular techniques polimerase chain reaction (PCR), ARDRA and slot-blot hibridation confirmed the predominance of Archaea Domain and alIowed verifying some changes in the population\'s diversity under additions of 2mg PCP/L. The efficiency of PCP decreased in the anaerobic reactor was related to the presence of Archaea Domain, especially the acetoclastic methanogens, whose where probably involved with the organochlorine compound degradation.

This master thesis is focused on groundwater remediation of chlorinated ethylenes and methanes in the area of chemical factory Spolchemie in the Czech Republic, Ústí nad Labem city. For these purposes nano zero valent iron particles were used. For the remediation two separate applications of different types of particles together in suspension with tracer (lithium chloride), were carried out. The results from the first application were evaluated by the supervisor of this thesis and are briefly summarized and discussed together with the second application which was evaluated by the author of this thesis. Second application of particles was carried out in October 2015 and observed for 424 days. Observation consisted of monitoring of groundwater level, physical-chemical parameters and collection of water samples for the analysis of concentrations of chlorinated hydrocarbons, their degradation products and several chosen ions. Nanoiron particles had the assumed effect onto the physical-chemical parameters and reduction of contamination. The application of tracer had proven the expected groundwater flow and made a possibility to distinguish between the process of reductive dehalogenation and dilution caused by the injected water, the dilution did not exceeded 5 % in most monitored points. Other...

O estudo que ora se apresenta integrou o conjunto de pesquisas do sub-projeto - Diversidade de Bactérias Associadas à Degradação de Compostos Recalcitrantes, do projeto temático BIOTA FAPESP - Ecologia Molecular e Taxonomia Polifásica de Bactérias de Interesse Ambiental e Agro-Industrial. Apresenta caráter inovador, na medida em que procurou avaliar o potencial de aplicação biotecnológica de microrganismos anaeróbios de uma área severamente contaminada no Brasil, o estuário de Santos-São Vicente, em degradar o pentaclorofenol (PCP). A pergunta fundamental a ser respondida pelos resultados experimentais realizados foi: são os microrganismos autóctones do estuário capazes de servirem de inóculo para degradar o pentaclorofenol em biorreatores sob condições metanogênicas? Dois grupos de amostras foram avaliados, o primeiro, uma parcela composta por vários sedimentos coletados no estuário e, o segundo, sedimentos coletados na região do Largo de Canéus e na frente da Companhia Siderúrgica Paulista (COSIPA). O estabelecimento da determinação cromatográfica do PCP e congêneres menos clorados para o monitoramento experimental mostrou que na análise da presença dos clorofenóis nos sedimentos, o método de extração por ultrassom com posterior metilação dos analitos foi adequado para concentração mínima de 200 'mü'g clorofenóis/Kg sedimento para 2,3; 2,6 diclorofenóis; 2,4,6 e 2,3,6 triclorofenóis. Contudo, não foi adequado para a determinação do PCP e 2,3,4 triclorofenol. Para o meio de cultivo, o método de extração do PCP por agitação em vórtex e acetilação dos analitos mostrou-se adequado para todos os clorofenóis e com limite de quantificação de 0,1 mg/L. A avaliação do potencial metanogênico foi realizada com as amostras compostas do estuário enriquecidas sob condições halofílicas. O valor estável de metano no biogás de 50% foi obtido nos primeiros 20 dias de incubação. O sedimento nessas condições foi utilizado como inóculo para fins de isolamento de culturas metanogênicas, redutoras do íon sulfato e degradadoras de PCP. Não foram obtidas culturas desalogenadoras; porém foram isoladas arquéias metanogênicas, cultivadas em metanol, acetato e formiato de sódio, bem como bactérias cultivadas em lactato de sódio na presença e ausência de sulfato de sódio. Ensaios fisiológicos aliados aos métodos moleculares FISH e DGGE permitiram identificar arquéias metanogênicas do gênero Methanosarcina sp. e microrganismos do domínio Bacteria. Os sedimentos individualmente estudados foram coletados com maior controle de anaerobiose empregando-se amostrador do tipo corer. O enriquecimento destes sedimentos, inicialmente sob concentração de 2,5 mg PCP/g STV e com adições periódicas de 50% da concentração inicial do clorofenol e 1,25 a 2,5 g/L de glicose por 13 meses a 30 graus Celsius, resultou na obtenção de culturas degradadoras do PCP sob anaerobiose estrita. Nos reatores controles sem PCP, já primeiros 20 dias de incubação 70% de metano foi determinado no biogás. Nos reatores com PCP, a produção do metano (20%) iniciou após 100 dias. A adsorção foi o principal mecanismo de remoção de 50% do PCP nas primeiras 12 horas de incubação dos enriquecimentos. Posteriormente, a redução do PCP no meio de 77% para a amostra de Canéus e 70% para a da COSIPA foi relacionada a mecanismos de biodegradação anaeróbia, como a desalogenação redutiva. Exames microscópicos mostraram a seleção de microrganismos na presença de PCP, com predomínio de bacilos formadores de esporos semelhantes ao gênero Clostridium sp., filamentos diversos e filamentos semelhantes as arquéias metanogênicas acetoclásticas relacionadas ao gênero Methanosaeta sp. O enriquecimento foi realizado sob condições halofílicas, segunda a salinidade de cada amostra estudada no estuário. Não houve perda do clorofenol por volatilização. Os resultados obtidos no enriquecimento anterior viabilizaram o estudo seguro do potencial anaeróbio dos microrganismos oriundos do sedimento da COSIPA em metabolizar o PCP em reator contínuo do tipo RAHLF. Assim, sedimento enriquecido sob condições metanogênicas e halofílicas foi inoculado no RAHLF, controlando-se rigorosamente a anaerobiose. O reator preenchido com cubos de espuma de poliuretana foi operado por 126 dias e tempo de detenção hidráulica de 18 horas, com meio de cultura salino contendo glicose (1 g/L) como principal fonte de carbono e PCP-Na nas concentrações de 5, 13, 15 e 21 mg/L. O desempenho do RAHLF foi estável com boa eficiência durante toda a operação. A redução dos níveis de matéria orgânica medida em DQO variou de 70 a 100% e a de PCP foi de 99%, com detecção de intermediários menos clorados e sob teores de metano no biogás de 20%. Da massa de 1111,73 mg de PCP aplicada no reator, 286,9 mg ficou retida no sistema pelo processo de adsorção nas biopartículas e 824,83 mg foi biodegradada. As análises morfológicas dos tipos celulares, em conjunto com as técnicas moleculares DGGE e FISH, revelaram a presença de grupos microbianos do domínio Archaea pertencente à família Methanosarcinacea e grupos do domínio Bacteria. A participação de grupos de domínio Bacteria, cuja estrutura dos tipos microbianos na comunidade variou ao longo do RAHLF e das concentrações de PCP, e dos organismos metanogênicos da família Methanosarcinacea, possibilitou responder a questão inicialmente formulada, uma vez que se pode afirmar que os microrganismos autóctones foram capazes de degradar o PCP sob condições metanogênicas e halofílicas, com eficiência adequada. A prática para a seleção dos microrganismos retirados do ambiente estuarino, de interesse para biotecnologia anaeróbia aplicada ao saneamento ambiental, que empregou o reator do tipo RAHLF, parece promissora para avanços da engenharia na remediação biológica de uma área cuja relevância é indiscutível para o estado de São Paulo / This study has an innovative character, once aimed to evaluate the biotechnological application of anaerobic microorganisms degrading pentachlorophenol (PCP) at a severely polluted area in Brazil, estuary of Santos-São Vicente, São Paulo state. This study integrated a group of researches of the sub-project - Diversity of Bacteria Associated to the Degradation of Recalcitrant Compounds, supported by major project BIOTA FAPESP which theme is Molecular Ecology and Polyphasic Taxonomy of Bacteria of Environmental and Agriculture-Industrial Interest. The fundamental question to be answered by the accomplished experimental results was: are the autochthonus microorganisms of the estuary capable of serving as inoculum to degrade the PCP in bioreactors under methanogenic conditions? In order to evaluate this hypothesis, two groups of samples were collected: sediments taken from several sites of the estuary and sediments taken from two sites, one severely contaminated with organochlorine compounds, in front of the São Paulo Metallurgical Company (COSIPA) and other, less contaminated, in the area of Canéus. The establishment of protocol for the PCP and less chlorinated compounds chromatographic determination showed that the extraction method by ultrosonication followed by metilation of the chlorinated compounds was appropriated to evaluate concentrations of 200 ug chlorophenol/Kg of sediment, 2,3; 2,6 dichlorophenols; 2,4,6 and 2,3,6 trichlorophenols. However, it was not appropriated for evaluation of PCP and 2,3,4 trichlorophenol. For the culture medium, the extraction method by vortex agitation followed by acetilation was appropriated for all of the chlorophenols compounds. The quantification limit was 0,1 mg/L. The evaluation of the methanogenic potential and PCP biodegradation was accomplished with the samples of the estuary enriched under halophylic conditions. In the experiments without PCP was possible to obtain cultures of Methanosarcina sp, identified by FISH technique and cells of domain Bacteria, identified by DGGE. In the experiment with PCP dehalogenated cultures were not obtained. To evaluate PCP anaerobic biodegradation by sediments of COSIPA and Caneus sites, sediments collected under anaerobic conditions by a corer device were enriched in a halophylic brine medium with glucose and PCP at concentration of 2.5 mg PCP/g VTS. Periodic additions of 50% of the initial concentration of PCP and 1.25 to 2.5 g/L of glucose were done. With this strategy was possible to obtain PCP dehalogenated cultures. The adsorption was the main mechanism of 50% of PCP removal in the first 12 hours of incubation. The PCP reduction of 77% for Caneus reactor and 70% for COSIPA reactor was related to anaerobic biodegradation. Microscopic exams showed selection of microorganisms, with predominance of cells related to Clostridium sp., and filaments related to methanogenic acetoclastic archaea Methanosaeta sp. chlorophenols volatilization was not observed. The biotechnological application of HAIB reactor in PCP biodegradation was evaluated using sediment of COSIPA site previously enriched with glucose. The reactor was operated by 126 days and hydraulic detention time of 18 hours, with saline brine medium containing glucose (1 g/L) and NaPCP in concentrations of 5, 13, 15 and 21 mg/L. PCP amendments did not affect the overall performance and functional stability of the process. COD and PCP reduction was close to 80% and 100%, respectively with detection of trichlorophenols and dichlorophenols. Percentage of methane in the biogas closed to 30%. Adsorption analyses demonstrated that 287 mg of PCP was removed by adsorption in the biofilm and 825 mg was removed by biodegradation. Biofilm DGGE-profiling showed the presence of specific bands of Bacteria and Archaea domains when PCP was amended. The appearance of new bands of Bacteria showed that this organisms had a direct influence at PCP dehalogentation. Archaea organisms of Methanosarcinaceae family had an indirect influence in this metabolism. This thesis demonstrated that HAIB reactors, using autochthonous microorganisms under halophylic and methanogenic conditions, are a potential alternative for organochlorines bioremediation

O estudo que ora se apresenta integrou o conjunto de pesquisas do sub-projeto - Diversidade de Bactérias Associadas à Degradação de Compostos Recalcitrantes, do projeto temático BIOTA FAPESP - Ecologia Molecular e Taxonomia Polifásica de Bactérias de Interesse Ambiental e Agro-Industrial. Apresenta caráter inovador, na medida em que procurou avaliar o potencial de aplicação biotecnológica de microrganismos anaeróbios de uma área severamente contaminada no Brasil, o estuário de Santos-São Vicente, em degradar o pentaclorofenol (PCP). A pergunta fundamental a ser respondida pelos resultados experimentais realizados foi: são os microrganismos autóctones do estuário capazes de servirem de inóculo para degradar o pentaclorofenol em biorreatores sob condições metanogênicas? Dois grupos de amostras foram avaliados, o primeiro, uma parcela composta por vários sedimentos coletados no estuário e, o segundo, sedimentos coletados na região do Largo de Canéus e na frente da Companhia Siderúrgica Paulista (COSIPA). O estabelecimento da determinação cromatográfica do PCP e congêneres menos clorados para o monitoramento experimental mostrou que na análise da presença dos clorofenóis nos sedimentos, o método de extração por ultrassom com posterior metilação dos analitos foi adequado para concentração mínima de 200 'mü'g clorofenóis/Kg sedimento para 2,3; 2,6 diclorofenóis; 2,4,6 e 2,3,6 triclorofenóis. Contudo, não foi adequado para a determinação do PCP e 2,3,4 triclorofenol. Para o meio de cultivo, o método de extração do PCP por agitação em vórtex e acetilação dos analitos mostrou-se adequado para todos os clorofenóis e com limite de quantificação de 0,1 mg/L. A avaliação do potencial metanogênico foi realizada com as amostras compostas do estuário enriquecidas sob condições halofílicas. O valor estável de metano no biogás de 50% foi obtido nos primeiros 20 dias de incubação. O sedimento nessas condições foi utilizado como inóculo para fins de isolamento de culturas metanogênicas, redutoras do íon sulfato e degradadoras de PCP. Não foram obtidas culturas desalogenadoras; porém foram isoladas arquéias metanogênicas, cultivadas em metanol, acetato e formiato de sódio, bem como bactérias cultivadas em lactato de sódio na presença e ausência de sulfato de sódio. Ensaios fisiológicos aliados aos métodos moleculares FISH e DGGE permitiram identificar arquéias metanogênicas do gênero Methanosarcina sp. e microrganismos do domínio Bacteria. Os sedimentos individualmente estudados foram coletados com maior controle de anaerobiose empregando-se amostrador do tipo corer. O enriquecimento destes sedimentos, inicialmente sob concentração de 2,5 mg PCP/g STV e com adições periódicas de 50% da concentração inicial do clorofenol e 1,25 a 2,5 g/L de glicose por 13 meses a 30 graus Celsius, resultou na obtenção de culturas degradadoras do PCP sob anaerobiose estrita. Nos reatores controles sem PCP, já primeiros 20 dias de incubação 70% de metano foi determinado no biogás. Nos reatores com PCP, a produção do metano (20%) iniciou após 100 dias. A adsorção foi o principal mecanismo de remoção de 50% do PCP nas primeiras 12 horas de incubação dos enriquecimentos. Posteriormente, a redução do PCP no meio de 77% para a amostra de Canéus e 70% para a da COSIPA foi relacionada a mecanismos de biodegradação anaeróbia, como a desalogenação redutiva. Exames microscópicos mostraram a seleção de microrganismos na presença de PCP, com predomínio de bacilos formadores de esporos semelhantes ao gênero Clostridium sp., filamentos diversos e filamentos semelhantes as arquéias metanogênicas acetoclásticas relacionadas ao gênero Methanosaeta sp. O enriquecimento foi realizado sob condições halofílicas, segunda a salinidade de cada amostra estudada no estuário. Não houve perda do clorofenol por volatilização. Os resultados obtidos no enriquecimento anterior viabilizaram o estudo seguro do potencial anaeróbio dos microrganismos oriundos do sedimento da COSIPA em metabolizar o PCP em reator contínuo do tipo RAHLF. Assim, sedimento enriquecido sob condições metanogênicas e halofílicas foi inoculado no RAHLF, controlando-se rigorosamente a anaerobiose. O reator preenchido com cubos de espuma de poliuretana foi operado por 126 dias e tempo de detenção hidráulica de 18 horas, com meio de cultura salino contendo glicose (1 g/L) como principal fonte de carbono e PCP-Na nas concentrações de 5, 13, 15 e 21 mg/L. O desempenho do RAHLF foi estável com boa eficiência durante toda a operação. A redução dos níveis de matéria orgânica medida em DQO variou de 70 a 100% e a de PCP foi de 99%, com detecção de intermediários menos clorados e sob teores de metano no biogás de 20%. Da massa de 1111,73 mg de PCP aplicada no reator, 286,9 mg ficou retida no sistema pelo processo de adsorção nas biopartículas e 824,83 mg foi biodegradada. As análises morfológicas dos tipos celulares, em conjunto com as técnicas moleculares DGGE e FISH, revelaram a presença de grupos microbianos do domínio Archaea pertencente à família Methanosarcinacea e grupos do domínio Bacteria. A participação de grupos de domínio Bacteria, cuja estrutura dos tipos microbianos na comunidade variou ao longo do RAHLF e das concentrações de PCP, e dos organismos metanogênicos da família Methanosarcinacea, possibilitou responder a questão inicialmente formulada, uma vez que se pode afirmar que os microrganismos autóctones foram capazes de degradar o PCP sob condições metanogênicas e halofílicas, com eficiência adequada. A prática para a seleção dos microrganismos retirados do ambiente estuarino, de interesse para biotecnologia anaeróbia aplicada ao saneamento ambiental, que empregou o reator do tipo RAHLF, parece promissora para avanços da engenharia na remediação biológica de uma área cuja relevância é indiscutível para o estado de São Paulo / This study has an innovative character, once aimed to evaluate the biotechnological application of anaerobic microorganisms degrading pentachlorophenol (PCP) at a severely polluted area in Brazil, estuary of Santos-São Vicente, São Paulo state. This study integrated a group of researches of the sub-project - Diversity of Bacteria Associated to the Degradation of Recalcitrant Compounds, supported by major project BIOTA FAPESP which theme is Molecular Ecology and Polyphasic Taxonomy of Bacteria of Environmental and Agriculture-Industrial Interest. The fundamental question to be answered by the accomplished experimental results was: are the autochthonus microorganisms of the estuary capable of serving as inoculum to degrade the PCP in bioreactors under methanogenic conditions? In order to evaluate this hypothesis, two groups of samples were collected: sediments taken from several sites of the estuary and sediments taken from two sites, one severely contaminated with organochlorine compounds, in front of the São Paulo Metallurgical Company (COSIPA) and other, less contaminated, in the area of Canéus. The establishment of protocol for the PCP and less chlorinated compounds chromatographic determination showed that the extraction method by ultrosonication followed by metilation of the chlorinated compounds was appropriated to evaluate concentrations of 200 ug chlorophenol/Kg of sediment, 2,3; 2,6 dichlorophenols; 2,4,6 and 2,3,6 trichlorophenols. However, it was not appropriated for evaluation of PCP and 2,3,4 trichlorophenol. For the culture medium, the extraction method by vortex agitation followed by acetilation was appropriated for all of the chlorophenols compounds. The quantification limit was 0,1 mg/L. The evaluation of the methanogenic potential and PCP biodegradation was accomplished with the samples of the estuary enriched under halophylic conditions. In the experiments without PCP was possible to obtain cultures of Methanosarcina sp, identified by FISH technique and cells of domain Bacteria, identified by DGGE. In the experiment with PCP dehalogenated cultures were not obtained. To evaluate PCP anaerobic biodegradation by sediments of COSIPA and Caneus sites, sediments collected under anaerobic conditions by a corer device were enriched in a halophylic brine medium with glucose and PCP at concentration of 2.5 mg PCP/g VTS. Periodic additions of 50% of the initial concentration of PCP and 1.25 to 2.5 g/L of glucose were done. With this strategy was possible to obtain PCP dehalogenated cultures. The adsorption was the main mechanism of 50% of PCP removal in the first 12 hours of incubation. The PCP reduction of 77% for Caneus reactor and 70% for COSIPA reactor was related to anaerobic biodegradation. Microscopic exams showed selection of microorganisms, with predominance of cells related to Clostridium sp., and filaments related to methanogenic acetoclastic archaea Methanosaeta sp. chlorophenols volatilization was not observed. The biotechnological application of HAIB reactor in PCP biodegradation was evaluated using sediment of COSIPA site previously enriched with glucose. The reactor was operated by 126 days and hydraulic detention time of 18 hours, with saline brine medium containing glucose (1 g/L) and NaPCP in concentrations of 5, 13, 15 and 21 mg/L. PCP amendments did not affect the overall performance and functional stability of the process. COD and PCP reduction was close to 80% and 100%, respectively with detection of trichlorophenols and dichlorophenols. Percentage of methane in the biogas closed to 30%. Adsorption analyses demonstrated that 287 mg of PCP was removed by adsorption in the biofilm and 825 mg was removed by biodegradation. Biofilm DGGE-profiling showed the presence of specific bands of Bacteria and Archaea domains when PCP was amended. The appearance of new bands of Bacteria showed that this organisms had a direct influence at PCP dehalogentation. Archaea organisms of Methanosarcinaceae family had an indirect influence in this metabolism. This thesis demonstrated that HAIB reactors, using autochthonous microorganisms under halophylic and methanogenic conditions, are a potential alternative for organochlorines bioremediation

A pesquisa que ora se apresenta visou estabelecer as condições nutricionais adequadas para o uso do sedimento do estuário de Santos - São Vicente do Estado de São Paulo, como inóculo no reator anaeróbio horizontal de leito fixo (RAHLF) no processo de degradação anaeróbia do pentaclorofenol (PCP) em busca da aplicação da tecnologia em escala real, assim como identificar grupos microbianos envolvidos no processo. Para tanto, sedimento do estuário de Santos-São Vicente, com características metanogênicas foi utilizado. Os microrganismos provenientes do sedimento estuarino foram enriquecidos sob condições metanogênicas e halofílicas, visando a utilização do sedimento como inoculo nos ensaios nutricionais e na operação dos reatores do tipo RAHLF. O meio de cultivo salino Biota, suplementado com glicose e formiato, foi utilizado para o desenvolvimento da comunidade microbiana metanogênica halofílica. Testes de degradação do PCP foram realizados previamente sob diferentes concentrações de nitrogênio e fósforo, com vistas a uma melhor compreensão da relação N:P adequada para o processo anaeróbio. Os resultados provenientes do acompanhamento da diversidade microbiana do domínio Bacteria nas diferentes relações testadas indicaram a seleção de distintas comunidades microbianas, resultando em diferentes velocidades de degradação do PCP. A relação N:P de 10:1 foi a que apresentou melhores resultados, pois além da rápida degradação do PCP quando comparada com as outras relações, apresentou a maior diversidade de microrganismos. Posteriormente, o sistema RAHLF foi operado com vazão média afluente de aproximadamente 44 mL/hora, com meio mineral salino Biota (DQO:N:P de 1000:130:45) para R1 e com a alteração para relação DQO:N:P de 1000:10:1 para R2. Duas diferentes estratégias foram adotadas para partida dos reatores. Para R1, optou-se por acrescentar PCP na concentração inicial de 10,0 mg/L, durante 110 dias causando desestabilização da metanogênese e acúmulo de PCP, requerendo intervenção para recuperação do reator pelo período de 90 dias. Na partida do RAHLF 2, optou-se pelo aumento gradual de concentração do PCP de 0,5 mg/L a 12,0 mg/L durante 52 dias. Após estabelecimento da metanogêsenese, R1 foi alimentado durante 270 dias com 5,0 mg PCP/L, durante 41 dias com 8,0 mg/L e 59 dias com 12 mg/L. O balanço de massa no reator RAHLF 1 demonstrou que 0,52% do PCP adicionado saiu no efluente e que não ocorreu adsorção no sistema. 22,34 mg de 2,4,6 TCP, intermediário da degradação do PCP, ficaram adsorvidos na biopartícula. Os resultados das análises de diversidade microbiana apontaram para mudança da comunidade microbiana do domínio Bacteria ao longo do período operacional e morfologias de bacilos fluorescentes semelhantes a Methanobacterium sp estiveram presentes no reator. No RAHLF 2, a degradação do PCP foi de 100%, até a concentração de 10,0 mg/L. No final da fase com 12,0 mg PCP/L, a concentração no efluente foi de 1,4 mg PCP/L, com eficiência média de remoção de 93,2 \'+ ou -\' 5,5%. 2,4,6 TCP foi o intermediário principal no efluente do reator. 4,06% do PCP adicionado ao sistema foram encontradas no efluente e 15,94% ficaram adsorvidas nas biopartículas do reator. Portanto, considera-se que 80% do PCP adicionado sofreu degradação anaeróbia microbiana. A presença dos microrganismos Methanocalcullus e Methanosaeta na fase final de operação do RAHLF 2 e determinadas no sedimento coletado foi considerada fundamental para manter estabilidade do reator. Essa descoberta contribui com informações sobre a real diversidade microbiana de ecossistemas tropicais, sobretudo em habitats anaeróbios, bem como sobre as condições nutricionais e os procedimentos necessários para confiná-la em reatores e usá-la em processos de biorremediação. / The research presented here aimed to determine the optimal nutritional conditions for the use of sediment from the Santos-São Vicente estuary in the state of São Paulo, Brazil, as an inoculum for a horizontal-flow anaerobic immobilized biomass reactor (HAIB) applied to the anaerobic degradation of pentachlorophenol (PCP), seeking to apply the technology on the real scale and to identify the microbial groups involved in the process. To this end, sediment with methanogenic characteristics from the Santos-São Vicente estuary was used. The microorganisms from the estuarine sediment were enriched under methanogenic and halophilic conditions, aiming to use the sediment as an inoculum in nutritional assays and in the operation of HAIB reactors. Biota saline culture medium supplemented with glucose and formiate was used to develop the halophilic methanogenic microbial community. PCP degradation tests were carried out previously under different concentrations of nitrogen and phosphorus in order to gain a better understanding of the optimal N:P ratio for the anaerobic process. The findings on the microbial diversity of the domain Bacteria at the various ratios tested here indicated the selection of distinct microbial communities, resulting in different PCP degradation velocities. The N:P ratio utilized was 10:1 since it presented the best results not only in terms of faster PCP degradation than the other ratios but also highest diversity of microorganisms. The HAIB reactor was then operated with a mean inflow of approximately 44 mL/hour, using the biota saline mineral medium with a COD:N:P ratio of 1000:130:45 in R1 (reactor 1) and a COD:N:P ratio of 1000:10:1 in R2. Two distinct strategies were adopted to start up the reactors. In R1 PCP was added at an initial concentration of 10.0 mg/L for 100 days, causing destabilization of the methanogenesis and accumulation of PCP, requiring a 90-day intervention for the reactor\'s recovery. To start up R2, the PCP concentration was increased gradually from 0.5 mg/L to 12.0 mg/L for 52 days. After methanogenesis was established, R1 was fed for 270 days with 5.0 mg of PCP/L, followed by 41 days with 8.0 mg/L and 59 days with 12 mg/L. The mass balance in R1 indicated that 0.52% of the added PCP exited through the reactor\'s outflow and that adsorption of the system did not occur. 22.34 mg of 2,4,6 TCP, an intermediary of PCP degradation, was adsorbed in the bioparticles. The results of the analysis of microbial diversity indicated a change in the microbial community of the domain Bacteria along the operational period, with fluorescent bacilli morphologies resembling Methanobacterium sp present in the reactor. PCP degradation in R2 was 100% up to a concentration of 10.0 mg/L. At the end of the phase with 12.0 mg PCP/L, the effluent concentration was 1.4 mg PCP/L, with a mean removal efficiency of 93.2 \'+ or -\' 5,5%. 2,4,6 TCP was the main intermediary in the reactor\'s effluent. 4.06% of the PCP added to the system was found in the effluent and 15.94% was absorbed in the bioparticles of the reactor. Therefore, it was concluded that 80% of the added PCP underwent microbial anaerobic degradation. The presence of Methanocalcullus and Methanosaeta microorganisms in the final operating phase of R2, which was determined in the collected sediment, was considered fundamental for maintaining the reactor\'s stability. This discovery contributes to the body of information about the real microbial diversity of tropical ecosystems, above all in anaerobic habitats, and about the nutritional conditions and procedures involved in confining these microorganisms in reactors and using them in bioremediation processes.