Tackling co-contaminations: potentialities of soil fungi isolated from a decommissioned military site.

Approximately 250 000 sites in Europe are thought to be contaminated and in need of remediation, mostly due to industries and waste processing, with a rising concern over the underestimation of current and past military activities 1 . A recognized common trend is the soil co-contamination by both organic and inorganic contaminants caused by the production and use of ammunitions and weaponry and vehicle maintenance 2–4 . Subsequently, restoring military sites should be a primary objective, aiming to preserve the untouched habitats found within exclusion zones 5,6 and return decommissioned sites to local communities. Unfortunately, the remediation of such contaminations, especially through conventional methods, comes with many drawbacks, both economically and environmentally. Hence, in recent years the potential of biotechnological applications of biological resources, such as fungi, bacteria and plants, has garnered rising interest. Particularly with a focus on organisms isolated from contaminated environments, who may possess useful traits, such as increased tolerance and ability to degrade/detoxify pollutants 7,8 . Therefore, in this study, the microbial community of a decommissioned military site, previously identified as co-contaminated, has been isolated. Consequently, a preliminary screening has been implemented in order to evaluate the potentialities of fungal isolates in the bioremediation of co-contaminated soils. Furthermore, this study also focused on evaluating the potentialities of the rhizospherical fungal community of a specimen of Plantago lanceolata L., a wild herb widely distributed in the site. This study’s results highlighted a high variability in Colony Forming Unit (CFU) abundance among the isolates of the 6 samples collected from the site. The most abundant genera were found to be Penicillium, Aspergillus and Trichoderma, with the strain labelled Penicillium S28A5 being the most represented among the samples. To study the potentialities of the isolates, a representative subset of 30 strains, including the most represented strains and at least a strain for each identified genera, were tested in a two-phase screening. The first phase included the Remazol Brilliant Blue R (RBBR) and the Fe-Chromeazurol S (Fe-CAS) decolorization assays to pinpoint the best candidates for the subsequent in-vitro tolerance tests. The selected strains were tested for their tolerance against Zn (an inorganic contaminant detected in all samples) and a mixture of polycyclic aromatic hydrocarbons (PAHs), in single contaminant and co-presence conditions. The decolorization assays showed that Gliomastix S28RE2 and Westerdykella S28RA1 showed large decolorization halos in the RBBR assay, pointing to a strong capacity of degrading complex organic compounds. Meanwhile, in the Fe-CAS test, Gliomastix S28RE2 as well as Acremonium S76A16 and Aspergillus S56C4 showed the ability to produce high amounts of siderophores, similar to a strong chelating agent used as positive control. Based on the results of the first phase of screenings 11 strains were chosen for the subsequent tolerance test on Zn and PAHs. Based on the analysis performed on the soil samples, a second tolerance test, replacing Zn with Pb, were performed involving 4 strains, isolated from a Pb-contaminated area. These tests revealed that Penicillium S56C6 tolerated both the Zn-PAH and the Pb-PAH mixture, with a tolerance index of more than 70%, while Mucor S56E4 showed to be unaffected by co-presence of Pb and PAH in the growth medium, although showing a less dense mycelium. Overall, several strains isolated from this contaminated site showed promising abilities for application as bioresources in the bioremediation of co-contaminated soils. Further studies are currently ongoing to evaluate the application of those strains in consortia. 1) Contamination from local sources — EEA. https://www.eea.europa.eu/themes/soil/soil-threats. 2) P. Pereira, Barceló, D. & Panagos, P. (2020) Environ. Res. 186, 109501. 3) Stolte, J. et al. EUR 27607. (2016) JRC Scientific and Technical Reports. 4) Siles, J. A. & Margesin, R. (2018) Appl. Microbiol. Biotechnol. 102, 4409–4421. 5) Ellwanger, G., Müller, C., Ssymank, A., Vischer-Leopold, M. & Paulsch, C. (2016). Naturschutz und Biologische Viefalt, 152 6) European Commission (2005) Natura 2000 and the military. http://europa.eu.int/comm/environment/life/home.htm. 7) Ye, S. et al. (2017) Crit. Rev. Biotechnol. 37, 1062–1076. 8) Harms, H., Schlosser, D. & Wick, L. Y. (2011) Nat. Rev. Microbiol. 9, 177–192.