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Molecular Approaches and MetaGenomic Investigations for optimizing Clean-up of PAH contaminated sites

Molecular Approaches and MetaGenomic Investigations for optimizing Clean-up of PAH contaminated sites

Periodic Report Summary - MAGICPAH (Molecular approaches and metagenomic investigations for optimising clean-up of PAH contaminated sites)

Project context and objectives:

MAGICPAH context

Polyaromatic hydrocarbons (PAH)s are widespread in various ecosystems and are pollutants of great concern due to their potential toxicity, mutagenicity and carcinogenicity. Microbial degradation represents the major mechanism responsible for the ecological recovery of PAH-contaminated sites and a huge body of investigations are available mainly on single isolates from soils capable of mineralising low-molecular-weight PAHs under aerobic conditions. However, less is known about the bacteria capable of utilising PAHs containing five or more rings as a carbon and energy source and knowledge on the metabolism of PAHs under anaerobic conditions is practically absent.

The rapid development of molecular techniques in recent years allows immense insights into the processes on site, including identification of organisms active in target sites, community member interactions and catabolic gene structures. More knowledge on the actual potential of indigenous microbial metabolism towards PAHs, on the processes involved and on the diversity and ecology of the organisms involved would permit us to more precisely understand the long-term fate of such pollutants and to better direct our efforts to sustainable decontamination / detoxification of polluted environments.

PAHs in the environment are to a major degree strongly adsorbed and it is yet not known whether the adsorbed fraction of PAH can be attacked by microbial enzymes, or whether dissolution in aqueous media is required. In that case, the degradation depends critically on the bioavailability, or better the bioaccessibility of PAH. Tools to determine the bioaccessibility and the metabolic activity in different states of bioavailability are thus one goal of the project.

Moreover, on-site biodegradation involves the exposure of a whole mixture of chemical structures to a multispecies metabolic network. Any rational effort to interfere with microbial processes to optimise metabolic performance on site has thus to deal with the enormous complexity present. Fortunately, the last few years have witnessed the emergence of new technological developments and conceptual frameworks which provide fresh approaches to explore complex biological settings, allowing us to move towards a picture of the complete catalytic potential and the metabolic net of the bacterial communities that thrive in polluted sites.

MAGICPAH strategy

MAGICPAH aims to explore, understand and exploit the catalytic activities of microbial communities involved in the degradation of persistent PAHs. It will integrate (meta)genomic studies with in-situ activity assessment based on stable isotope probing particularly in complex matrices of different terrestrial and marine environments. PAH degradation under various conditions of bioavailability will be assessed as to improve rational exploitation of the catalytic properties of bacteria for the treatment and prevention of PAH pollution. We will generate a knowledge base not only on the microbial catabolome for biodegradation of PAHs in various impacted environmental settings based on genome gazing, retrieval and characterisation of specific enzymes but also on systems related bioavailability of contaminant mixtures. MAGICPAH takes into account the tremendous undiscovered metagenomic resources by the direct retrieval from genome/metagenome libraries and characterisation of ensymes through activity screens. These screens will include a high-end functional small-molecule fluorescence screening platform and will allow us to directly access novel metabolic reactions followed by their rational exploitation for biocatalysis and the re-construction of biodegradation networks. Results from (meta)genomic approaches will be correlated with microbial in situ activity assessments, specifically dedicated to identifying key players and key reactions involved in anaerobic PAH metabolism. Key processes for PAH metabolism particularly in marine and composting environments and the kinetics of aerobic degradation of PAH under different conditions of bioavailability will be assessed in model systems, the rational manipulation of which will allow us to deduce correlations between system performance and genomic blueprint. The results will be used to improve treatments of PAH-contaminated sites.

MAGICPAH objectives

The main objectives of MAGICPAH in the context of the above are to:

1. generate a knowledge base of the microbial aerobic catabolome with particular relevance to biodegradation of PAHs in various impacted environmental settings;
2. develop concepts to quantify in situ degradation of PAH employing combined hydrogen and carbon stable isotope analysis;
3. identify key players and key reactions involved in anaerobic PAH metabolism;
4. achieve a detailed understanding on key processes for PAH metabolism in marine and composting environments;
5. develop methods to predict the ultimate fate and the kinetics of aerobic degradation of PAH under different conditions of bioavailability;
6. isolate and sequence novel key players in PAH metabolism to understand the genomic basis of niche specificities that allow microbes to thrive and function in extreme PAH impacted environments;
7. investigate the potential synergistic links between environmental biotechnology and medical biotechnology by assessing novel biocatalysts for their use in new biocatalytic processes;
8. integrate detailed catabolome and reactome information through bioinformatic techniques to re-construct metabolic networks;
9. apply gathered information to improve the treatment performance of PAH contaminated sites.

Project results:

Extensive samplings were performed to obtain materials impacted by industries and anthropogenic activities for subsequent Work package (WP)s. We concentrated on marine sites, including harbours and terrestrial sites, including gas works and woodworks sites. Samples were subjected to basic microbiological analysis, comprising community structure determinations and served as material for (meta)genomic, (meta)transcriptomic and (meta)transcriptomic analyses in subsequent WPs.

Tools to reliably elucidate PAH metabolism in complex communities were developed. To survey microbial communities at high depth and low cost, an Illumina-based barcoded deep sequencing strategy was developed and applied, allowing now a detailed survey of community shifts. To obtain a reliable frame of reactions and enzymes involved in PAH degradation, databases on eight key catabolic enzyme families involved in aerobic and anaerobic degradation were constructed. These carefully curated databases have then been used to design Polymerase chain reaction (PCR) primers and microarray probes to survey the spread and activity of respective genes in contaminated environments and to obtain a reliable annotation of genes observed in metagenomic and metatranscriptomic surveys. Hidden Markov Models were then built, allowing a precise way of extracting, from omics sequence surveys, gene sequences potentially encoding enzymes related to PAH biodegradation steps.

To be able to construct upgraded databases on anaerobic key steps, the initial steps of the anaerobic naphthalene degradation pathway were elucidated. Two previously unknown dearomatising enzymatic ring reductions were identified in vitro as key processes involved in the anaerobic PAH degradation. Specifically naphthoyl-CoA reductase is a highly conserved key enzyme of anaerobic PAH degradation and represent a perfect target for diagnostic tools. The second dearomatising enzyme was identified to be homologous to the Asoarcus-type bensoyl-CoA reductases. An assay that targets such genes has been developed and genes encoding both types of reductase reactions were subsequently identified in the genomes of two distinct naphthalene degrading cultures.

To access and exploit the enormous biodiversity of the microbial world, through the mining for novel activities a total number of 17 metagenome fosmid libraries and 7 lambda SAP libraries from the DNA of microbial communities from different contaminated marine and terrestrial environments were created and 7 different colorimetric or growth-selection methods plus a PCR-based screen successfully designed, optimised and tested. Using the appropriately optimised methods, a total number of 1200 positive clones out of approximately 1.5 million colonies from metagenome libraries have successfully been identified and selected for in depth analysis- Twenty four inserts were fully sequenced and 44 open reading frames coding biodegradation enzymes of interest has been identified. To understand about the abundance and distribution of aromatic degradation networks in complex microbial communities (meta)genomic and shotgun (meta)proteomics were used to deduce previously unrecognised correlations between systems performance and phylogenetic, genomic and proteomic blueprints, applied mainly to an anthropogenic influenced and PAH-contaminated soil plus two naphthalene-enriched communities derived thereof. Overall, enrichment and bio-stimulation were found to select for different microbial communities and complementary arsenal of genes coding biodegradation-related proteins. The results suggest that communities in contaminated biotopes seem to have a wider metabolic plasticity as compared to what earlier predicted.

As a step further in understanding community behaviour when confronted with PAHs, messenger ribonucleic acid (mRNA) sequencing has been successfully applied to obtain a meta-transcriptomic overview of contaminated groundwater samples. Together with the optimisation of methods to obtain mRNA from soil, this will allow now obtaining metatranscriptomic overviews of contaminated soil and sediment samples and the improvement of methods to isolate (meta)proteomes of PAH degrading microbial consortia now allows protein-stable-isotope-probing experiments.

Two special environments have been analysed in micro- and mesocosm studies for identifying key processes and key players. For providing strategies for clean- up of polluted marine sediments we have developed a mesocosm system where biostimulations were carried out and nutrient and temperature impact assessed to mitigate the oil spills and enhance the degradation efficacy. As composting environments are promising to enhance the PAH degradation in contaminated soil a microcosm screening test was conducted to study the pyrene degradation potential. As matured compost turned out to be the most successful soil amendment the respective unknown PAH degrading microorganisms are now aimed to be discovered.

Micro- and mesocosm studies are also used in concert with mathematical modelling to predict the ultimate fate and the kinetics of aerobic degradation of PAH under different conditions of bioavailability. Experiments were done following a newly developed methodology applying dynamic passive dosing. Microbial growth and degradation were described mathematically and a combined model for sorption and bacterial metabolism was set up. The model contains the compartments non-aqueous phase liquids or solids, dissolved compound, adsorbed and sequestered compound, bacterial mass and biotic, as well as abiotic non-extractable residues. The model structure was tested and could be verified by comparison of numerical simulations with analytical solutions (BEST equation). Typical scenarios could be modelled in order to investigate coupled processes and first simulations of experimental results were done. Several scenarios were run to determine the effectiveness of various treatment options. Preliminary results lead to the conclusion that the most successful strategy is to enhance the bioaccessibility of PAH.

Our analysis and enrichments from marine sediments confirmed members of the genus Cycloclasticus as key microorganisms in the aerobic breakdown of PAHs and particularly Cycloclasticus zancles plays a predominant role. Thus, its genome was sequenced revealing a large repertoire of genetic determinants for the uptake of mineral nutrients that are limiting in marine environments, particularly following a sudden input of oil, which leads to severe imbalances in carbon / nitrogen and carbon/phosphorus ratios. The battery of genetic determinants for scavenging functions enables C.zankles 7-ME to efficiently exploit its aromatic catabolic functions in response to a sudden appearance of hydrocarbons. We found that genome of C. zankles contains three large operons with more than fifteen different enzymes belonging to four classes of ring-cleavage dioxygenases. The strong biodegradation potential evidenced by the exceptional multiplicity of key aromatic degradation genes may explain the broad substrate range for utilisation of PAH compounds. To get crucial knowledge on anerobic PAH degradation, we are currently performing sequencing of the only phenanthrene degrading anaerobic enrichment culture available thus far. With the metagenome sequence of this worldwide unique culture available in the near future, initial insights into the unknown degradation pathways of phenanthrene and other three / four-ringed PAHs will be obtained.

In order to use biodegradative bacteria and their functions for novel applications, we performed a detailed characterisation of bacterial capabilities to degrade and transform azzarenes and out of battery of strains those subjected to future gene mining have been decided for. To recruit enzymes for new products, we optimised the protocols for activity assays, cloning, expression, purification and biochemical characterisation and have selected promising activities mined from the metagenome as targets for crystallisation.

Potential impact:

MAGICPAH focuses on the study, understanding and exploiting of the relevant molecular microbial diversity and the molecular biological processes, which play a major role in the removal of PAH contaminants from soils, sediments and wastewaters. PAH within the complex matrices from all types of sources show different behaviour regarding toxicity and bioavailability for degradation, which was not considered in most previous studies of the environmental fate. Therefore, the application of source related matrices in the experiments will provide new insights into the fate and bioavailability of PAH under actual environmental conditions.

The focus on microbes and processes relevant in composting will deliver new insights in a fast turnover of hazardous chemicals of environmental relevance and may offer new testing strategies for chemicals and for the treatment of contaminated environments. The work of will provide a good chance for a challenging comparison of the results from metagenomic approaches with microbial in situ activity assessment based on stable isotope probing in complex environmental systems.

The focus on microbes and processes relevant in marine ecosystems will directly contribute to environmental policies concerning PAHs to protect the resource base upon which marine-related economic and social activities depend.

MAGICPAH will provide knowledge on the distribution and diversity of processes and microbiota involved in metabolism of PAHs in diverse matrices and will provide new tools for assessing the occurrence of such activities.

The results of MAGICPAH will enable:

- a more reliable risk assessment (PAH chemical activity, bioavailability, plant uptake, transport, and mobility) of PAH contaminations in terms of bioavailability;
- a chance for better treatment processes (combined with fertilising effects and plant growth promotion!);
- improved quality control of treatment processes;
- use of biodegradation enzymes for synthesis of valuable production intermediates.

Project website:

Project information

Grant agreement ID: 245226


Closed project

  • Start date

    1 April 2010

  • End date

    31 March 2014

Funded under:


  • Overall budget:

    € 4 515 496

  • EU contribution

    € 2 997 137

Coordinated by: