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Microbial networks for PAC cycling in polluted soils

Periodic Reporting for period 2 - NETPAC (Microbial networks for PAC cycling in polluted soils)

Reporting period: 2017-06-16 to 2018-06-15

Polycyclic Aromatic Hydrocarbons (PAHs) are soil pollutants of special concern due to their recalcitrance and (geno)toxicity. Bioremediation, exploiting natural degrading capabilities of soil microorganisms, is a sustainable alternative for the restoration of those soils. However, this biotechnology is still not sufficiently effective, due to i) the limited degradation rates of PAHs; and ii) the presence of other toxicologically relevant polycyclic aromatic compounds (PACs), such as oxygenated PAHs (oxy-PAHs) and nitrogen heterocyclic PACs (N-PACs), whose fate and, in the case of oxy-PAHs, formation are normally overlooked. 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 reliable bioremediation approaches for PAH-impacted soils. Within NETPAC we applied state-of-the-art molecular microbial ecology and analytical chemistry tools in combination with stable isotope tracers to obtain a systems biology insight into the complex metabolic networks dealing with PAC-biodegradation and bioavailability in situ, integrating genomics, transcriptomics and metabolomics data. At this point, major contributions from NETPAC are:
- To provide the first evidence of the direct implication of a bacterial metabolite of pyrene in increased soil genotoxicity after bioremediation. Formation of this heretofore unknown metabolite was associated to the activity of recently described groups of pyrene-degrading bacteria.
- To develop a stable isotope assisted metabolomics approach to understand PAH-metabolism in situ. This method allowed reconstructing compound specific metabolic pathways in soils.
- To identify an unprecedented abundance and diversity of N-PACs in PAH-contaminated soils by the application of nontarget high resolution analytical methods.
This report covers the progress of NETPAC between June 2015 and July 2018. The work was developed at the University of North Carolina (UNC, USA) and the Spanish National Research Council (IRNAS-CSIC, Spain). 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, we applied these methods i) to identify a bacterial metabolite contributing to the increased genotoxicity observed in a PAH-contaminated soil after bioremediation (; ii) to reveal the actual diversity and abundance of N-PACs present in PAH-contaminated environmental samples (; and iii) to reconstruct compound specific metabolic pathways for PAH-biodegradation in contaminated soils by the application of Stable Isotope Assisted Metabolomics ( The library of uniformly labeled 13C-PACs available at UNC was capitalized to identify the microbial key players associated with the assimilation of target contaminants in a creosote-contaminated soil. Considering the concern posed by HMW-PAHs, including recognized carcinogens enriched in the residual fraction of contaminant after bioremediation, we applied DNA-SIP to identify the microbial communities actively assimilating model compounds within this class. This method, in combination with high throughput sequencing, also shed light on the major phylotypes and functions implicated in the cycling of polar PACs co-occurring with PAHs. Considering the unexpected diversity of N-PACs revealed by the nontarget analysis, and their isomer-selective biodegradation, special attention was paid to the microbial processes underlying the biodegradability of these heterocyclic compounds.

Dissemination of NETPAC has already resulted in three publications in international top-ranked scientific journals in the field of environmental sciences (Environmental Science & Technology and Environmental Science & Technology Letters), and a book chapter that has been recently accepted for publication in the third edition of Comprehensive Biotechnology. Additional publications are expected to be delivered soon. Impact of NETPAC on a broader audience, including not only scientists, but also stakeholders and legislators has been achieved through communications in eleven international conferences including ISME conference, the Battelle Bioremediation Symposium, the SETAC Europe and ACS Annual Meetings, the European Bioremediation Conference and AquaConsoil.
The Thematic Strategy for Soil Protection specifies contamination as one of the eight main threats to soil, with 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 regulated PAHs. 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. NETPAC has provided the first direct evidence of microbial activity as contributing to that increased genotoxicity (a bacterial metabolite of pyrene accumulated in bioremediated soil). This metabolite, not previously identified, suggests that previous knowledge on metabolic pathways for PAH biodegradation, gathered from the study of pure cultures, might not be sufficient to understand the reactions actually occurring in situ. The Stable Isotope Assisted Metabolomics method developed within NETPAC might be a decisive step forward towards the understanding of those reactions. Nontarget analysis of PAH-contaminated samples also revealed an unexpected diversity of N-PACs. The limited and isomer-selective biodegradability of higher molecular weight N-PACs, including well-known carcinogens, suggested their potential contribution to risk. Results from NETPAC should raise awareness on policy makers and stakeholders about these classes of co-occurring contaminants overlooked during risk management of PAH-contaminated soils, and contribute to broaden measures of risk beyond the current list of 16 regulated compounds.

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 is the work 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 opens new lines of evidence on how to modulate these processes in the environment. NETPAC provides new tools to achieve a more effective and predictable remediation, with PAC removal below current reference levels, and minimizing the potential risks associated with this biotechnology.