Thesis (doctoral)--Universität Bremen, 2003. / Includes bibliographical references.

Universiẗat, Diss., 1999--Wuppertal.

Techn. Hochsch., Diss., 2004--Aachen.

In dieser Arbeit wurden PCR-Primer für den Nachweis von Genen des bakteriellen Chloraromaten-Abbaus entwickelt. Als Zielgene wurden hierfür die Gene der Chlorcatechol-1,2-Dioxygenasen und Chlormuconat-Cycloisomerasen des modifizierten ortho-Weges ausgesucht. Die entwickelten Primer wurden an verschiedenen Chloraromaten abbauenden Bakterien getestet. Es gelang dabei erstmals, Fragmente von Chlormuconat-Cycloisomerase-Genen aus alpha-Proteobakterien zu erhalten. Mit den neu entwickelten Primern zum Nachweis der Chlorcatechol-1,2-Dioxygenase-Gene wurden aus den Stämmen Burkholderia sp. 3CB-1 und Rhodococcus opacus 1CP auch Fragmente amplifiziert, die nur relativ geringe Ähnlichkeiten zu bereits bekannten Genen aufwiesen. Um die Sequenzdatenbasis für das Primerdesign zu erweitern, wurden außerdem Chlorcatechol-Gencluster aus zwei Vertretern der alpha-Proteobakterien kloniert und sequenziert. Aus dem Stamm Sphingomonas sp. TFD44 konnten dabei zwei verschiedene Gencluster charakterisiert werden, von denen nur eines einen kompletten Satz der Chlorcatechol-Gene enthielt. Die beiden Gencluster aus dem anderen Stamm, Sphingomonas sp. EML146, wiesen Homologien zu diesem Gencluster auf. Die Konstruktion einer Knockout-Mutante und Proteinanreicherungen ergaben Hinweise auf weitere Chlorcatechol-Abbaugene in Sphingomonas sp. TFD44.

Die Arbeit befasst sich mit der Anwendung der dielektrischen Erwärmung mittels Radiowellen auf unterschiedlichen Gebieten der Umwelttechnik. Im Mittelpunkt stehen die Aufheizung von Schüttbetten aus Adsorbenzien und Katalysatoren sowie die thermisch unterstützte Reinigung kontaminierter Böden und Feststoffe, insbesondere unter dem Gesichtspunkt des Einsatzes bei in-situ-Sanierungsvorhaben. Aus stofflicher Sicht wurde die Erwärmung technisch relevanter Feststoffe wie Aktivkohlen, Molekularsieben (Zeolithen) und anderer Katalysatoren ebenso wie die Behandlung von Modellböden und realen Bodenproben untersucht. Die durchgeführten physikalisch-chemischen Grundlagenuntersuchungen bezogen sich vor allem auf die Fragen, inwiefern sich die Radiowellen-Erwärmung von konventionellen Heizmethoden unterscheidet, ob und unter welchen Bedingungen eine selektive Erwärmung einzelner Feststoffkomponenten (z.B. Katalysatorkomponenten oder Partikel) möglich ist und welchen Einfluss der Wassergehalt und die Temperatur auf den Energieeintrag, den Schadstofftransport sowie den Prozess der Schadstoffdesorption besitzen. Der verfahrenstechnische Aspekt der Arbeit umfasst die Entwicklung der Methode der Radiowellen-Erwärmung vom Labor- bis in den Feldmaßstab, die Entwicklung und den Test eines praxistauglichen und automatisierten Systems zur Feststofferwärmung, die simultane Anwendung mehrerer Frequenzen elektromagnetischer Felder zur optimierten Erwärmung mit größerer Temperaturhomogenität, die Erprobung unterschiedlicher Elektrodengeometrien sowie die Durchführung von Sanierungsvorhaben mit Modellcharakter.

Terrestrial environments are highly complex and dynamic. It consists of various types of soils which are constantly exposed to fluctuating conditions affecting their physical and biological properties. Moreover, soils are delivering several ecosystem services with high relevance for the human well-being such as water purification, nutrient cycling, or biodegradation. For many of those ecosystem services, microorganisms are the main drivers. In consequence, it is important to understand the functional response of microbial ecosystems to disturbances. Thus, identifying key factors for the functional stability of microbial ecosystems in terrestrial environments is of high interest. A powerful tool for analysing dynamics and underlying mechanisms of ecosystems are computational simulation models. Within this doctoral thesis, a spatiotemporally explicit bacterial simulation model was developed for assessing dynamics of biodegradation as a typical microbial ecosystem function under the influence of disturbances. Disturbances were introduced as lethal events for the bacteria within a certain, randomly picked disturbance area. The disturbance characteristics vary in the spatial configuration and frequency of the disturbance events. Functional stability was analysed in terms of the ability to recover the function after a single disturbance event, i.e. functional resilience, and the ability to maintain the function during recurrent disturbance events, i.e. functional resistance. Key factors for functional stability were assessed by systematically varying properties and processes of the microbial ecosystem and characteristics of the disturbance regime. Simulation results show a high influence of the disturbance characteristics, especially its spatial distribution pattern, on the stability of biodegradation. Functional resistance and resilience increase with fragmentation of the spatial pattern of the disturbances. The frequency of recurrent disturbance events proved also essential for the functional resistance: if the disturbances occur too often, the emergence of a functional collapse may not be preventable. However, if the fragmentation of the applied disturbance patterns increases, the function is also maintained under more frequent disturbances without a functional collapse. Ecological processes such as bacterial dispersal and growth are shown to enhance the biodegradation performance, but only under specific disturbance regimes, again depending on frequency and fragmentation of the disturbances. Dispersal networks are shown to increase the functional stability in many scenarios and, thus, may serve as a buffer mechanism against disturbances. Therefore, strategies facilitating these ecological processes, for instance stimulating fungi that act as dispersal networks for bacteria, or modulating the physical soil structure to alter the spatial configuration of disturbances are proposed to increase the functional stability of microbial ecosystems.

Biochar is defined as a carbon-rich, fine-grained, porous substance, which is produced by pyrolysis biomass with little or no oxygen. Biochar is usually produced from crop residues, wood biomass, animal litters, and solid wastes. Recently, biochar is increasingly receiving attention as an environmental-friendly approach, especially as a climate change mitigation strategy. Biochar is especilly demonstrated to remove diesel oil (DO) from soil and water. In this report, 4 biofilm forming bacteria including Klepsiella sp. VTD8, Pseudomonas sp. BQN21, Rhodococcus sp. BN5 and Stenotropomonas sp. QND8 were used to attach to biochar produced from husk to estimate the capacity of their DO removal. As the results, removal efficiency of biofilm formed by each strain VTD8, BQN21, BN5 and QND8 were 67, 73, 75 and 68 % with initial concentration of 39 g/l, respectively. On the other hand, mix species biofilm attached to husk carrier and without carrier degraded 98 and 78 %. Using husk without bacteria as absortion control, the amount of DO removal was 23 %. These results gave hint that using biochar produced from husk as carrier for biofilm forming bacteria to attach may increase efficiency of DO pollution treatment. / Than sinh học (biochar) là một chất xốp có các gốc carbon và có nguồn gốc từ quá trình nhiệt phân sinh khối các loại chất thải, động, thực vật,… dưới điều kiện hạn chế oxy hoặc không có oxy. Hiện nay biochar đã được ứng dụng rộng rãi trong xử lý môi trường. Đặc biệt các biochar còn được chứng minh là có thể xử lý dầu diesel (diesel oil - DO) có trong đất và nước. Trong nghiên cứu này, chúng tôi sử dụng 4 chủng vi khuẩn tạo màng sinh học tốt là Klepsiella sp. VTD8, Pseudomonas sp. BQN21, Rhodococcus sp. BN5 và Stenotropomonas sp. QND8 để gắn lên chất mang là biochar làm từ trấu nhằm đánh giá hiệu quả xử lý DO của chúng. Kết quả cho thấy, sau 7 ngày, các chủng VTD8, BQN21, BN5 và QND8 có khả năng phân hủy 67, 73, 75 và 68 % DO với hàm lượng ban đầu là 39 g/l. Trong khi đó, hiệu suất của màng sinh học tạo thành bởi hỗn hợp các chủng này khi không có chất mang biochar trấu và khi có chất mang biochar trấu lần lượt là 78 và 98 %. Còn sử dụng chất mang biochar trấu không có vi sinh vật làm đối chứng thì thu được hiệu suất hấp phụ DO là 23 %. Như vậy, kết quả này mở ra tiềm năng ứng dụng biochar trấu làm chất mang cho các chủng vi khuẩn tạo màng sinh học để nâng cao hiệu quả xử lý ô nhiễm dầu.

In this study, a well biofilm-forming bacterial strain was isolated from oil contaminated water and sediment samples collected in petroleum storage Duc Giang, Hanoi. It was identified as Paracoccus sp. DG25 and registered in the GenBank database with the accession numbers KJ608354. Several biophysical and bio-chemical conditions for the biofilm formation of the strain were estimated such as pH, temperature, carbon sources and nitrogen sources. As the results the biofilm forming capacity was highest at pH 7, 37 oC, on maltose and supplemented with KNO3. Using these optimal conditions, the formed biofilm degraded 76.07 % of pyrene after 7 day-incubation, with the initial concentration of 300 ppm by high-performance liquid chromatography (HPLC) analysis. To our knowledge, there is rare publication on pyrene degradation by biofilm-forming bacteria. Therefore, the obtained results show that biofilm formed the strain Paracoccus sp. DG25 may considerably increase the degrading efficiency of pyrene and may lead to a new approach to treat polycyclic aromatic hydrocarbons containing in petroleum oil contaminated water in Vietnam. / Trong nghiên cứu này, từ các mẫu đất và nước nhiễm dầu lấy tại kho xăng Đức Giang, Hà Nội, chúng tôi đã phân lập được chủng vi khuẩn có khả năng tạo màng sinh học tốt. Chủng vi khuẩn này đã được phân loại và định tên là Paracoccus sp. DG25 với số đăng ký trên ngân hàng Gen là KJ608354. Chúng tôi cũng đã nghiên cứu một số điều kiện hóa lý ảnh hưởng tới khả năng hình thành màng sinh học như pH, nhiệt độ, nguồn Carbon và nguồn Nitơ. Kết quả cho thấy, chủng DG25 có khả năng tạo màng tốt nhất ở các điều kiện pH 7, 37 oC, nguồn Carbon là maltose và nguồn Nitơ là KNO3. Sử dụng các điều kiện tối ưu này để tạo màng và đánh giá khả năng phân hủy pyrene của màng tạo thành. Bằng phương pháp sắc ký lỏng cao áp, chúng tôi đã đánh giá được hàm lượng pyrene bị phân hủy sau 7 ngày nuôi tĩnh bởi màng sinh học của chủng DG25 lên tới 76,07 % với nồng độ ban đầu là 300 ppm. Cho tới nay, chưa có nhiều công bố về hiệu quả phân hủy pyrene của các chủng vi khuẩn tạo màng sinh học. Do vậy, kết quả đạt được này mở ra khả năng sử dụng màng tạo thành bởi chủng DG25 để nâng cao hiệu quả phân hủy pyren và có thể mở ra phương pháp mới nhằm xử lý các hợp chất hydrocarbon thơm có trong nước ô nhiễm dầu ở Việt Nam.

Mikroorganismen sind in der Lage, zahlreiche Xenobiotika abzubauen. Dazu nutzen sie unter aeroben Bedingungen oft einleitend Oxygenasen. Durch diese kann molekularer Luftsauerstoff aktiviert und auf organische Moleküle übertragen werden. Danach können die Verbindungen in den Metabolismus der Mikroorganismen eingeschleust und teils oder vollständig abgebaut werden. Am Beispiel des Styrols zeigen wir hier eine solche Abbauroute und wie wir diese biotechnologisch nutzen können, um interessante Verbindungen zu synthetisieren. Zielmoleküle der gesamten Enzymkaskade sind dabei diverse Phenylessigsäurederivate.

Die Toluol-Dioxygenase von Pseudomonas putida F1 ist eine Rieske-Dioxygenase und besteht aus Reduktase-, Ferredoxin- und Oxygenase-Komponente. Sie katalysiert den ersten Schritt im aeroben Abbau von Toluol. Ein effizienter Elektronentransfer zur terminalen Oxygenase-Komponente - an der die Sauerstoffaktivierung und Umwandlung von Toluol zum cis-Toluol-Dihydrodiol stattfindet - setzt eine reibungslose Interaktion aller Komponenten voraus. Die Ergebnisse der Stopped-flow-Messungen in der reduktiven Halbreaktion zeigen, dass NADH die Reduktase mittels Hydridtransfer reduziert, wodurch ein stabiler Ladungstransfer-Komplex zwischen NAD+ und FADH- entsteht. In der oxidativen Halbreaktion wird dieser dann durch einen Elektronenakzeptor über das blaue Semichinon zum Chinon oxidiert. Dabei zeigt sich, dass der Ladungstransfer-Komplex die Reaktion der Reduktase mit Sauerstoff unterdrückt. Eine Erklärung hierfür liefert die Kristallstruktur des Ladungstransfer-Komplexes. Die Reaktion mit Sauerstoff wird dadurch unterdrückt, dass das NAD+ koplanar mit dem Isoalloxazinring ist und den reaktiven N5-C4a Teil des FADs schützt und zudem den Isoalloxazinring in eine planare, weniger sauerstoffempfindliche Konformation zwängt. Durch die Bildung des Reduktase-Ferredoxin-Komplexes wird ein effizienter Elektronentransfer folgendermaßen ermöglicht: a) das Ferredoxin bindet an die Reduktase aufgrund elektrostatischer Anziehung entgegengesetzter Oberflächenladungen beider Proteine, b) die hydrophobe Region, die die beiden Redoxzentren umgibt, fungiert als Ein- und Ausgang für Elektronen und c) die geringe Entfernung von 11.7 Å zwischen beiden Kofaktoren erlaubt einen schnellen Elektronentransfer. Die Ergebnisse dieser Arbeit zeigen, dass der Elektronentransfer zwischen Reduktase und Ferredoxin durch die Bildung eines stabilen Ladungstransfer- und Reduktase- Ferredoxin-Komplexes beeinflusst wird und dadurch das Problem einer ungewollten Reaktion mit Sauerstoff umgangen wird. / The toluene dioxygenase from Pseudomonas putida F1 is a three-component Rieske non-heme iron dioxygenase comprising of a reductase, ferredoxin and an oxygenase component. It catalyzes the initial step in the aerobic degradation of toluene to cis-toluene dihydrodiol. A smooth interaction between all three components needs to be ensured to efficiently transfer the electrons derived from NADH oxidation to the terminal oxygenase component where molecular oxygen is activated and used for the hydroxylation of toluene. The results of the kinetic studies of the reductive half reaction of reductase reveal that NADH reduces the reductase, resulting in the formation of a stable charge transfer complex between NAD+ and FADH-. Oxidation of the charge transfer complex by an electron acceptor proceeds via the neutral semiquinone to the quinone state of FAD. It is shown that the charge transfer complex suppresses the reaction of the reductase with dioxygen. An explanation for this change in reactivity can be deduced from the structure of the charge transfer complex. Its slower reaction with dioxygen results from NAD+ lying coplanar with the FAD shielding its reactive N5-C4a locus and the forced planarity of the isoalloxazine ring. The formation of the reductase-ferredoxin complex allows efficient electron transfer from reductase to ferredoxin because a) the oppositely charged interacting surfaces of both proteins facilitate the pre-orientation of the ferredoxin on the reductase, b) a hydrophobic region surrounding the two redox centers in the complex acts as an exit/entrance port for electrons and c) the short edge-to-edge distance between both cofactors of 11.7 Å guarantees a fast electron transfer. The results demonstrate that the electron transfer between reductase and ferredoxin is governed by the formation of a stable charge transfer and of a reductase-ferredoxin complex with which the problem of an unwanted side reaction with dioxygen is obviated.