Periodic Reporting for period 1 - PrOBiEM (Characterization of the anaerobic degradation of phenanthrene by a novel sulfate-reducing enrichment culture for the development of bioremediation methods) Reporting period: 2016-06-02 to 2018-06-01 Summary of the context and overall objectives of the project Polycyclic aromatic hydrocarbons (PAHs) are a group of contaminants mainly derived from natural gas and petroleum. Due to their stable ring structures, poor water solubility and chemical stability, these contaminants are difficult to degrade and persist for long time in the environment, where they pose a hazard to the environment and human health. The enormous concern about PAHs-contamination lies on the fact that high percent of human activities depend on petroleum, not only as fuels for transportation but also for the production of asphalt, plastics, synthetic fibers, lubricants, detergents and fertilizers. Petroleum extraction and refining activities result in PAHs discharge to the environment. Among PAHs-remediation methods is biodegradation of these compounds by the activity of microorganisms. Many bacteria are capable of using PAHs as a source of carbon and energy, as part of a metabolism that also relies in oxygen. However, saturated PAH-contaminated sites rapidly become anoxic, and microorganisms able to use these compounds as a carbon source in the absence of oxygen are crucial for their degradation. So far, information about the responsible organisms or the mechanisms for PAHs degradation in the absence of molecular oxygen are very scarce. The overall aim of the PrOBiEM project is to elucidate the mechanisms for the anaerobic degradation of phenanthrene by a bacterial enrichment culture (TRIP) obtained from a natural asphalt lake. Specifically, to identify enzymes involved in the degradation of this PAH, which is composed by three benzene rings. Phenanthrene is an EPA (United States Environmental Protection Agency) Priority Pollutant and is a major component of the total content of PAH compounds in the environment. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "For characterizing the phenanthrene-degrading TRIP bacterial culture, a metagenomics analysis was performed, including assembling the genomes of the different members of the culture and elucidating the metabolic potential of the dominant organisms. Moreover, the transcriptome and proteome of the TRIP culture was evaluated during growth with phenanthrene as substrate. This allowed identifying genes encoding enzymes potentially involved in the anaerobic degradation of phenanthrene. On the other hand, different strategies were applied aiming at isolating the phenanthrene-degrading bacteria in pure culture, particularly the dilution until extinction and role-tube methods.The results of this project were presented in conferences organized by the German Association for General and Applied Microbiology (VAAM); the Federation of European Microbiological Society (FEMS) and the International Society for Subsurface Microbiology (ISSM), as well as during the Annual meetings of the Marie Curie Alumni Association. Moreover, a manuscript has been prepared for submission to the journal Environmental Microbiology. Finally, scientific outreach activities were performed to communicate the research objectives and results to a brother audience, including an interactive stand during Essen´s ""Day of Movement"", ""Girl´s Day"" at University Duisburg-Essen and assuming the role of Social Media engagement Manager of the Communication Working Group of the Marie Curie Alumni Association." Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This project provided information about the previously unknown metabolic pathway for the anaerobic biodegradation of phenanthrene. Genes were identified in the genome of the dominant organism of the phenanthrene-degrading TRIP culture, encoding enzymes similar to those involved in the degradation of naphthalene. These enzymes were expressed during growth with phenanthrene, verifying that bacterial degradation of three-ring PAHs in the absence of oxygen follows mechanisms similar to the degradation of the model compound naphthalene, composed of two benzene rings.The impact of this project lies on the fact that PAH-contamination is a social and economic concern, as petroleum exploitation is important for many activities of the modern industrial society. Despite the EU environmental protection laws regulating petroleum extraction and refining activities, the high percent of human activities dependent on petroleum products result in PAHs being liberated to the environment, producing noxious effects in human health. The results obtained during this project extended the current state of knowledge on anaerobic degradation of PAHs from naphthalene to higher molecular weight compounds, as well as provided novel research aspects in this subject such as the evaluation of the transcriptome of a PAH-degrading culture. Gaining profound knowledge about the enzymes and genes involved in the degradation of these contaminants provides markers for assessing the self-cleaning potentials of contaminated sites as well as for monitoring bioremediation processes. probiem.jpg
