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Original title:
Life in pitch - Bacteria in a natural oil emitting lake help to understand anaerobic biodegradation of polycyclic aromatic hydrocarbons 
Translated title:
Leben im Teer - Bakterien eines natürlichen Teersees helfen die Abbauprozesse von polyzyklischen aromatischen Kohlenwasserstoffen zu verstehen 
Document type:
Fakultät Wissenschaftszentrum Weihenstephan 
Lueders, Tillmann (Priv.-Doz. Dr.) 
Lueders, Tillmann (Priv.-Doz. Dr.); Liebl, Wolfgang (Prof. Dr.); Meckenstock, Rainer U. (Prof. Dr.) 
Subject group:
BIO Biowissenschaften 
Anaerobic, anoxic, PAH, phenanthrene, TRIP1, biodegradation, degradation pathway 
Translated keywords:
PAK, Phenanthren, Anreichungskultur TRIP1 
TUM classification:
BIO 250d 
The research of anaerobic degradation of non-substituted polycyclic aromatic hydrocarbons is still in its infancy and most processes therein only poorly understood. Due to the poor bacterial degradation capabilities of PAHs, only few cultures exist that can be used to explore the underlying mechanisms. Their growth times are considerably longer than those of compounds with a smaller molecular weight and the production of biomass is substantially lower as shown by strain-specific FISH analyses and flow cytometry. Elucidation of pathways started in enrichment cultures growing on naphthalene as sole carbon and electron source and is at a point where individual steps are fairly well characterized through years of research. The next logical step is to look at compounds with a higher molecular weight to find similarities or dissimilarities in the pathways. The compound of interest in this thesis is phenanthrene, a three-ringed PAH without known degradation steps apart from a carboxylation as initial reaction (Zhang and Young 1997; Davidova et al. 2007). Parallels to anaerobic naphthalene degradation are expected due to similarities in the aromatic ring structure. In this thesis I wanted to find insights into the anaerobic degradation of phenanthrene as the next sized PAH beyond the initial carboxylation reaction. Therefore, we isolated bacteria able to degrade hydrocarbons under anoxic conditions from a naturally contaminated ecosystem, the pitch lake in Trinidad, Trinidad & Tobago. An enrichment culture growing anaerobically under sulfate reducing conditions was set up from sediments from the pitch lake. This culture was able to degrade phenanthrene as sole carbon and energy source. It was used to elucidate the anaerobic phenanthrene degradation pathway. Beyond the carboxylation reaction in the C2 position, which could be shown indirectly within the framework of this thesis by metabolite analysis and biochemical enzyme assays, I was able to show the ligation reaction from 2-phenanthroic acid to 2-phenanthroyl-CoA within this culture. This step is therefore similar to the ligation in anaerobic naphthalene degradation from 2-napthoic acid to 2-naphthoyl-CoA. Further downstream reductions steps could not be shown with enzyme assays yet. Nevertheless, metabolite analysis was able to indicate a stepwise ring reduction, which again would be in accordance with the naphthalene reduction steps (Eberlein, Estelmann, et al. 2013; Eberlein, Johannes, et al. 2013). The main dominating bacterium within the culture belongs to the Desulfobacteraceae family and made up for 60% of the culture as confirmed by flow cytometry and genome-resolved metagenomics. It has a 93% similarity to the known naphthalene degrading, sulfate reducing strain NaphS2, indicating that degradation pathways of both molecules were the result of similar gene clusters or mechanisms. While looking for life in the naturally forming asphalt, minuscule water droplets have been discovered, that contain living bacteria (Meckenstock et al. 2014). As an extreme and seemingly uninhabitable habitat within the naturally formed asphalt, important questions towards the origin of the bacteria within the pitch, their way of coping with the lack of oxygen and their access to nutrients remain to be answered. In this thesis, sequencing of DNA extracted from both pitch and water droplets gave further insights into the community composition in compartments and their possible interactions. The diversity in the pitch was higher than in the water droplets, indicating a more specialized microbiota in the water droplets. Diversity within the droplets might change compared to the original diversity in the source water during upward movement due to a local selection and evolution on the “micro-scale”. The diversity in both compartments was very low compared to other oil sources, with more than 30% of the contained bacteria without any known relatives. In other samples like marine oil sources, there are only 10% of the bacteria without known relatives. The dominant bacterium in all sequenced droplets was Tepidiphilus sp., it belongs to the family Hydrogenophilaceae within the β-Proteobacteria and is able to degrade organic acids as sole carbon source under nitrate reducing conditions. It is therefore a plausible candidate to live in this extreme environment, but it still is not characterized well yet. In summary this thesis advances our understanding of anaerobic phenanthrene degradation and I was able to discover the similarities between naphthalene and phenanthrene degradation on an enzymatic basis. This also allows us to assume similarities for PAHs of an even higher molecular weight than that of phenanthrene. The diversity of the degrader community just opened a small window into the life in oil and the background metagenome from the pitch serves as a template for a deeper look into this extreme habitat. 
Translated abstract:
Die anaeroben Abbauwege polyzyklischer aromatischer Kohlenwasserstoffe (PAKs) sind bisher weitestgehend unerforscht geblieben. Aufgrund des langsamen Wachstums und geringer Biomassebildung existieren nur wenige Kulturen, anhand derer die Abbauwege aufgeklärt werden können. Des Weiteren sind deutliche Unterschiede der am Abbau beteiligten bekannten Gene zwischen verschiedenen Spezies aufgedeckt worden, was überdies die Aufklärung durch Sequenzierungsdaten verhindert. Der einzig bislang untersuc...    »
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