Periodic Report Summary 2 - MAGICPAH (Molecular Approaches and MetaGenomic Investigations for optimizing Clean-up of PAH contaminated sites)
Project Context and Objectives:
MAGIC PAH context
Polyaromatic hydrocarbons (PAHs) 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 mineralizing low-molecular-weight PAHs under aerobic conditions. However, less is known about the bacteria capable of utilizing 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 optimize 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 Objectives
The main objectives of MAGICPAH in the context of the above are:
i) to generate a knowledge base of the microbial aerobic catabolome with particular relevance to biodegradation of PAHs in various impacted environmental settings
ii) to develop concepts to quantify in situ degradation of PAH employing combined hydrogen and carbon stable isotope analysis
iii) to identify key players and key reactions involved in anaerobic PAH metabolism
iv) to achieve a detailed understanding on key processes for PAH metabolism in marine and composting environments
v) to develop methods to predict the ultimate fate and the kinetics of aerobic degradation of PAH under different conditions of bioavailability
vi) to 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
vii) to investigate the potential synergistic links between environmental biotechnology and medical biotechnology by assessing novel biocatalysts for their use in new biocatalytic processes.
viii) to integrate detailed catabolome and reactome information through bioinformatic techniques to re-construct metabolic networks
ix) to 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 and tools to reliably elucidate PAH metabolism in complex communities developed. To survey microbial communities at high depth and low cost, an Illumina barcoded deep sequencing strategy was developed. Curated databases on key catabolic enzyme families were used to survey the spread of respective genes in contaminated environments and to obtain a reliable annotation of genes observed in metagenomic/transcriptomic surveys. Assays that target previously unknown de-aromatizing reductions in naphthalene degradation were developed and genes encoding both types of reductases identified in distinct naphthalene degrading cultures. Sequencing and global proteome analyses indicates Deltaproteobacteria affiliated to ‘strain N47’ as key microorganisms.
To access and exploit the enormous biodiversity, metagenome fosmid and lambda ZAP libraries were created. Different methods were designed and used to select a total of 1200 clones related to PAH degradation. More than 300 clones were sequenced evidencing mainly proteins only distantly related to previously described ones. We then generated a pipeline for high throughput selection, cloning, expression, purification, characterization and crystallization of a large number of gene products showing that MAGICPAH metagenomes contain enzymes with unprecedented activities towards multiple substrates and others of previously unknown catabolic diversity.
To understand about the abundance and distribution of aromatic degradation networks in complex communities we have made significant progress in providing an affinity-based enrichment methodology to isolate proteins that might bind/act upon the attached substrates, and in generating multi-omic datasets of a number of communities and pure cultures. As a step further mRNA sequencing has now been successfully applied to contaminated soil samples.
Marine and composting environments have been analyzed in micro- and mesocosm studies for identifying key processes and key players and enhance degradation. Experiments in compost imply a ´super-degrading community` with intense metabolic interactions rather than a single key player in composting environments.
Analysis of marine sediments confirmed Cycloclasticus as key microorganism in the aerobic breakdown of PAHs. Genome sequencing revealed a large repertoire of genetic determinants for the uptake of mineral nutrients which enables Cycloclasticus to efficiently exploit its aromatic catabolic functions in response to a sudden appearance of hydrocarbons. The strong biodegradation potential evidenced by the exceptional multiplicity of key aromatic degradation genes may explain the broad substrate range for utilization of PAHs.
To get crucial knowledge on anerobic PAH degradation, the only phenanthrene degrading anaerobic enrichment culture available thus far was sequenced. Initial insights into the unknown degradation pathways of phenanthrene were obtained, indicating the degradation to occur via initial carboxylation.
Micro- and mesocosm studies are also used in concert with mathematical modeling to predict the ultimate fate and the kinetics of aerobic PAH degradation under different conditions of bioavailability. The developed combined model for sorption and bacterial metabolism is applied to the experimental studies but also to the simulation of remediation options with the goal to evaluate the best remediation strategy for PAH contaminated soils and sediments. The addition of PAH-sorbing amendments to contaminated soil reduces their freely dissolved concentrations which limits their bioavailability and uptake by organisms. This can lead to reduced toxicity, but can also decrease biodegradation. Even though the addition of mature compost in the short term slightly decreased desorption, a positive effect on PAHs degradation was proven. This effect varies depending on compost composition.
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 fertilizing effects and plant growth promotion!)
- improved quality control of treatment processes
- use of biodegradation enzymes for synthesis of valuable production intermediates
List of Websites:
magicpah.org
MAGIC PAH context
Polyaromatic hydrocarbons (PAHs) 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 mineralizing low-molecular-weight PAHs under aerobic conditions. However, less is known about the bacteria capable of utilizing 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 optimize 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 Objectives
The main objectives of MAGICPAH in the context of the above are:
i) to generate a knowledge base of the microbial aerobic catabolome with particular relevance to biodegradation of PAHs in various impacted environmental settings
ii) to develop concepts to quantify in situ degradation of PAH employing combined hydrogen and carbon stable isotope analysis
iii) to identify key players and key reactions involved in anaerobic PAH metabolism
iv) to achieve a detailed understanding on key processes for PAH metabolism in marine and composting environments
v) to develop methods to predict the ultimate fate and the kinetics of aerobic degradation of PAH under different conditions of bioavailability
vi) to 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
vii) to investigate the potential synergistic links between environmental biotechnology and medical biotechnology by assessing novel biocatalysts for their use in new biocatalytic processes.
viii) to integrate detailed catabolome and reactome information through bioinformatic techniques to re-construct metabolic networks
ix) to 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 and tools to reliably elucidate PAH metabolism in complex communities developed. To survey microbial communities at high depth and low cost, an Illumina barcoded deep sequencing strategy was developed. Curated databases on key catabolic enzyme families were used to survey the spread of respective genes in contaminated environments and to obtain a reliable annotation of genes observed in metagenomic/transcriptomic surveys. Assays that target previously unknown de-aromatizing reductions in naphthalene degradation were developed and genes encoding both types of reductases identified in distinct naphthalene degrading cultures. Sequencing and global proteome analyses indicates Deltaproteobacteria affiliated to ‘strain N47’ as key microorganisms.
To access and exploit the enormous biodiversity, metagenome fosmid and lambda ZAP libraries were created. Different methods were designed and used to select a total of 1200 clones related to PAH degradation. More than 300 clones were sequenced evidencing mainly proteins only distantly related to previously described ones. We then generated a pipeline for high throughput selection, cloning, expression, purification, characterization and crystallization of a large number of gene products showing that MAGICPAH metagenomes contain enzymes with unprecedented activities towards multiple substrates and others of previously unknown catabolic diversity.
To understand about the abundance and distribution of aromatic degradation networks in complex communities we have made significant progress in providing an affinity-based enrichment methodology to isolate proteins that might bind/act upon the attached substrates, and in generating multi-omic datasets of a number of communities and pure cultures. As a step further mRNA sequencing has now been successfully applied to contaminated soil samples.
Marine and composting environments have been analyzed in micro- and mesocosm studies for identifying key processes and key players and enhance degradation. Experiments in compost imply a ´super-degrading community` with intense metabolic interactions rather than a single key player in composting environments.
Analysis of marine sediments confirmed Cycloclasticus as key microorganism in the aerobic breakdown of PAHs. Genome sequencing revealed a large repertoire of genetic determinants for the uptake of mineral nutrients which enables Cycloclasticus to efficiently exploit its aromatic catabolic functions in response to a sudden appearance of hydrocarbons. The strong biodegradation potential evidenced by the exceptional multiplicity of key aromatic degradation genes may explain the broad substrate range for utilization of PAHs.
To get crucial knowledge on anerobic PAH degradation, the only phenanthrene degrading anaerobic enrichment culture available thus far was sequenced. Initial insights into the unknown degradation pathways of phenanthrene were obtained, indicating the degradation to occur via initial carboxylation.
Micro- and mesocosm studies are also used in concert with mathematical modeling to predict the ultimate fate and the kinetics of aerobic PAH degradation under different conditions of bioavailability. The developed combined model for sorption and bacterial metabolism is applied to the experimental studies but also to the simulation of remediation options with the goal to evaluate the best remediation strategy for PAH contaminated soils and sediments. The addition of PAH-sorbing amendments to contaminated soil reduces their freely dissolved concentrations which limits their bioavailability and uptake by organisms. This can lead to reduced toxicity, but can also decrease biodegradation. Even though the addition of mature compost in the short term slightly decreased desorption, a positive effect on PAHs degradation was proven. This effect varies depending on compost composition.
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 fertilizing effects and plant growth promotion!)
- improved quality control of treatment processes
- use of biodegradation enzymes for synthesis of valuable production intermediates
List of Websites:
magicpah.org
