Fungal diversity for bioremediation: Tackling co-contaminations in a decomissioned military site

Military sites, both active and decommissioned, represent a unique situation in which both biodiversity loss and protection may occur, depending on how those sites are managed. Exclusion zones and good practices such those provided in Natura 2000 report for military sites — can maintain or even increase the detected biodiversity. However, potential and identified contaminations in military zones are soil threats of increasing interest, especially when co-contaminations by organic and inorganic pollutants are established. Remediating such complex contaminations through conventional techniques is not only economically unsustainable but can also impact soil biodiversity. Novel methods to tackle co-contaminations may be found in biotechnological applications of the bioresources isolated from those same contaminated sites, even though there is a knowledge gap to be filled regarding the potentialities of autochthonous microbial communities. Therefore, this study aimed at gaining insight on the culturable fungal community of a decommissioned military site in Italy and the potentialities in fungal bioremediation. To reach the aims of the study, soil samples were collected in six sampling plots and the fungal communities were isolated. Furthermore, the fungal community associated with the rhizosphere of a specimen of Plantago lanceolata L., a wild herb largely distributed in the site, was similarly studied for the same purposes. The results showed high differences in species' abundances among samples with Penicillium, Aspergillus and Trichoderma as the most abundant genera. The analysis of alpha diversity and evenness revealed that the samples with the lowest abundance in Colony Forming Units (CFUs) showed the highest values of Shannon’s Diversity Index (H’) and Simpson Diversity Index (D1), pointing to a lack of a dominant species among the isolates from those samples, while in samples with higher CFU values a more varied situation arises. In fact, sample S28 had the lowest diversity indexes but also the second highest CFU abundance, pointing to a dominance of few species, especially Penicillium S28A5, which was the most abundant species isolated from all samples. The analysis of genera alpha diversity revealed a similar situation, except for sample S22, whose diversity of genera showed to be the lowest among all samples, with a clear dominance of the Penicillium genus. Focusing on the rhizosphere sample, the results of alpha revealed a highly diverse community of culturable fungi, in both species and genera. The analysis of beta diversity, using Sørensen’s index, showed that while the samples shared few common species, the isolated genera mostly overlapped. To evaluate the potentialities of the isolated species, a set of screenings were performed on a selected group of 30 species, which included the most abundant species and a species for each genus isolated. The assays performed in this study were the Remazol Brilliant Blue R (RBBR) and Fe-Chromeazurol S (Fe-CAS) decolorization assay, which are reported in literature as proxy test to investigate the ability to degrade complex organic compounds and to produce siderophores in response to metallic and non-metallic elements. The species showing the best performances were further tested to assess their tolerance to zinc (Zn), lead (Pb), Polycyclic Aromatic Hydrocarbons (PAHs) and mixtures of organic and inorganic pollutants (Zn-PAH and Pb-PAH) in in-vitro assays. The results of the screenings pointed to Gliomastix S28RE2 and Westerdykella S28RA1 having high capacities of degrading RBBR, while Acremonium S76A16, Aspergillus S56C4 and the aforementioned Gliomastix species showed to be able to produce high quantities of siderophores. Eleven species were chosen to be tested in the tolerance tests on Zn and PAHs, while four strains isolated from a Pb contaminated sample were exposed to Pb and PAHs. Overall, Penicillium S56C6 showed the best results in both test conditions, retaining more than 70% of its growth rate when compared with control, while Mucor S56E4 showed no change in growth rate, but suffered a loss in mycelium density. To conclude, several strains isolated from this decommissioned military site showed promising potentialities for possible application in bioremediation and further studies are currently underway to develop microbial consortia to enhance their performances