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Microbial networks for PAC cycling in polluted soils

Periodic Reporting for period 1 - NETPAC (Microbial networks for PAC cycling in polluted soils)

Reporting period: 2015-06-16 to 2017-06-15

Polycyclic Aromatic Hydrocarbons (PAHs) are soil pollutants causing special concern due to their high recalcitrance and (geno)toxicity. Despite the extensive knowledge gathered on microbial PAH degradation, soil biorestoration technologies are still not sufficiently effective. A main constraint of environmental PAH biodegradation is their low bioavailability to microbial communities, with the concomitant limited degradation rates. In addition, at PAH-polluted sites, other toxicologically relevant polycyclic aromatic compounds (PAC), such as oxygenated PAHs (oxy-PAHs) and nitrogen heterocyclic PACs (N-PACs), are generally present, their fate and, in the case of oxy-PAHs, formation being normally overlooked. These polar biotransformation products have been implicated as causative of soil genotoxicity after bioremediation, however no specific product has been identified as potential source so far. As a result, the success of bioremediation and its ecotoxicological assessment are often limited. NETPAC aims to identify the microbial communities and functions relevant for PAC biodegradation, and their adaptations to low bioavailability conditions, to further exploit them in novel and more sustainable approaches for biological attenuation of PAH-impacted soils. Molecular microbial ecology and analytical chemistry tools in combination with stable isotope tracers will allow a systems biology insight into the complex metabolic networks dealing with PAH-biodegradation and bioavailability in situ, integrating genomics, transcriptomics and metabolomics data. Diagnostic tools will be developed and applied to monitor a green remediation approach based on enhanced natural attenuation, and to identify the natural microbial adaptations to promote the degradation of the expected low bioavailability residue. Understanding these processes will provide us with tools to assess biodegradation occurrence and, as a final outcome, predict the success of bioremediation thus reducing its uncertainties, one of the main drawbacks of this technology.
This report covers the work developed by Dr. Vila at the laboratory of Prof. Aitken (University of North Carolina, UNC), between June 2015 and June 2017. In this period, we applied metabolomics and microbial ecology tools to analyze the presence, abundance and potential formation of oxy-PAHs and N-PACs in PAH contaminated soils, and identify microbial key players involved in the natural cycling processes not only of PAHs but also of these generally overlooked co-contaminants.

Nontarget analysis combined with tools of metabolomics has been seldom applied in the field of environmental sciences, and to contaminated soils in particular. Within NETPAC, Dr. Vila has been involved in the application of these methods i) to identify the accumulation of a bacterial metabolite contributing to the genotoxicity observed in a PAH-contaminated soil submitted to bioremediation; ii) to reveal an unprecedented diversity of N-PACs present in PAH-contaminated samples collected from four independent sites; and iii) to reconstruct compound specific metabolic pathways for PAH biodegradation in contaminated soils. The availability of a unique library of uniformly labeled 13C-PACs at UNC has been capitalized to identify the microbial key players associated with the assimilation of target contaminants in a creosote-contaminated soil from Spain. Considering the major concern posed by HMW-PAHs, including recognized carcinogens enriched in the residual fraction of contaminant after bioremediation, DNA-SIP has been applied to identify the microbial communities actively contributing to the biodegradation of model compounds within this class. This method in combination with Next Generation Sequencing, has contributed to shed light on the major phylotypes and functions implicated in the cycling of polar aromatic contaminants co-occurring with PAHs. Considering the relevant results obtained within NETPAC regarding the discovery of an unexpected diversity of N-PACs in PAH-contaminated soils, major efforts have been applied to understand the microbial processes underlying the biodegradability of these heterocyclic compounds. The results achieved so far, have been integrated to assess the occurrence of active microbial processes associated with PAC removal during the bioremediation of a real contaminated soil sample under nutrient stimulated conditions.
The Thematic Strategy for Soil Protection specifies contamination as one of the eight main threats to soil, with ca. 3.5 million sites potentially contaminated in Europe, and 0.5 million currently needing remediation. About 13% of those sites are contaminated with PACs in complex mixtures such as coal tar or creosote. Human exposure to PACs in soil or sediment is well known, but risk management at PAC-contaminated sites has barely changed since the 1970s, and is exclusively based on concentration levels of 16 priority-pollutant PAHs. So far, progress on NETPAC highlights the need for a more accurate risk assessment during bioremediation of PAH-contaminated sites. Previous reports revealed that, despite of effective PAH removal, bioremediation might have limited effect on or even increase the genotoxicity of the contaminated soil. Those works pointed to the formation of oxy-PAHs as a potential factor of risk. The identification of a novel bacterial PAH-metabolite as implicated in the genotoxicity observed in bioremediated soil, represents the first direct evidence of microbial activity as contributing to that increased genotoxicity. This metabolite, not previously identified, suggests that previous knowledge on metabolic pathways for PAH biodegradation, gathered from the study of pure bacterial cultures, might not be sufficient to understand the reactions actually occurring in situ. Therefore, the work currently ongoing within NETPAC, aiming to reconstruct the in situ PAH-degradation pathways in soils, might be a decisive step forward towards the understanding of those reactions. Nontarget analysis of PAH-contaminated samples has also revealed and unexpected diversity of nitrogen containing heterocyclic aromatic compounds. The limited biodegradability of higher molecular weight N-PACs, including well-known carcinogens, suggested their potential contribution to risk. NETPAC aims to raise awareness on policy makers and stakeholders about these classes of co-occurring contaminants overlooked during risk management in PAH-contaminated soils, with the final aim to broaden current measures of risk beyond the current list of 16 target compounds.

At this point, NETPAC has identified microbial key players involved in the degradation of model PACs of concern in contaminated soils (HMW-PAHs, oxy-PAHs and N-PACs), and assessed their occurrence during active bioremediation processes. Of special relevance and novelty will be the work soon to be delivered on oxy-PAHs and N-PACs. The identification of the microbial communities and metabolic pathways involved in the in situ cycling, and eventual formation (for oxy-PAHs), of these three classes of contaminants may open new lines of evidence on how to modulate these processes in the environment. The final aim is to provide new tools to achieve a more effective and predictable remediation, with PAC removals below current reference levels, and minimizing the potential risks associated with this biotechnology.
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