Investigation and Assessment for an effective approach to the reclamation of Polycyclic Aromatic Hydrocarbon (PAHs) contaminated site: SIN Bagnoli, Italy

Native plant species were screened for their remediation potential for the removal of Polycyclic Aromatic Hydrocarbons (PAHs) contaminated soil of Bagnoli brownfield site (Southern Italy). Soils at this site contain all of the PAHs congeners at concentration levels well above the contamination threshold limits established by Italian environmental legislation for residential/recreational land use, which represent the remediation target. The concentration of 13 High Molecular Weight Polycyclic Aromatic Hydrocarbons in soil rhizosphere, plants roots and plants leaves was assessed in order to evaluate native plants suitability for a gentle remediation of the study area. Analysis of soil microorganisms are provides important knowledge about bioremediation approach. Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria are the main phyla of bacteria observed in polluted soil. Functional metagenomics showed changes in dioxygenases, laccase, protocatechuate, and benzoate-degrading enzyme genes. Indolacetic acid production, siderophores release, exopolysaccharides production and ammonia production are the key for the selection of the rhizosphere bacterial population. Our data demonstrated that the natural plant-bacteria partnership is the best strategy for the remediation of a PAHs-contaminated soil.

PAHs in the plants system. The families more present in the Bagnoli brownfield site are Poaceae (20 taxa, 14.4%), Fabaceae (15 taxa, 10.8%), Asteraceae (14 taxa, 10.1%) and Apiaceae (6 taxa, 4.3%). These plants are adapted and able to survive and reproduce under contaminated soil condition 18 . PAHs concentrations in the plant system (root and leaves) were determined for 16 native plant species. Pearson correlation analysis suggested a . The X-axis is scaled in probability (between 0 and 100%) and shows the percentage of the Y variable whose value is less than the data point. The Y-axis displays the range of the data variables.  Figure 6 highlight PAHs accumulation in roots and leaves respectively by analysed plant species. The different profiles of PAHs in plant tissues indicated that the 4-ring and 5-ring PAHs mostly contributed to total concentration of PAHs in both roots and leaves. Higher molecular weight PAHs are generally incapable of translocation to the aboveground plant parts because of their low water solubility, and they are strongly bound to the roots 19 . An organic element enters a plant roots for the presence of the lipid in a plant that, even at small amounts, is usually the principal source for highly water-insoluble contaminants 20 . Lin et al. 21 demonstrated that the storage of PAHs in maize plant (Zea maize L.) was directly congruent to the lipid level in tissues that allows the free diffusion within cells.
In a recent studies 22,23 shown that seeds of Zea mays are ecotoxicity indicator on soils contaminated with petro derivates.
As the PAHs, level in leaves is roughly not significative; our interest must focus on root system. Among the analysed plant species, Pip and Fes show the highest ΣPAHs root concentration, with mean content of 26.4 and 10.2 mg/kg, respectively. However, Dac demonstrates to be the best root bioaccumulator. In addition, Lot and Dit show promising ΣPAHs root phytostabilization rates (Fig. 6). Pip has suitable attributes like as fast growth and high root cover; in addition, it is able to stimulate soil microbial communities 24,25 .
The considerable PAHs content in roots was not only imputable to the contamination by adherent soil particles. However, as suggested by Fismes et al. 26 it is possible that part of the PAHs measured in plants roots can be due to their adsorption on the roots epidermis. "In fact, root peels are mainly made of suberin, a polyester with phenolic and aromatic functions presenting a lipophilic pole 27  www.nature.com/scientificreports www.nature.com/scientificreports/   Fig. 7. In the soils collected directly from three plants (Pip, Lot and Pla), 82% of the bacterial communities were found to be in common. Therefore, the microbial community composition were highly heterogeneous. The analysis of soils showed that within Proteobacteria, Alphaproteobacteria and Gammaproteobacteria were the taxa with higher proportions, both containing known species of PAH degraders and Actinobacteria populations as well. Firmicutes and Deltaproteobacteria were relatively less of all other groups ( Table 2). All three rhizosphere soils investigated showed Gammaproteobacteria as the most abundant class with Pseudomonadaceae family. In literature is known the use of Pseudomonas genera employed in bioremediation study of soil polluted by PAHs 30 reported that the degradation of complex mixtures could be done using a pool of bacteria with different functions and capacity to utilize as source of carbon and energy the hydrocarbons [31][32][33] . Because the endophyte communities is mainly present in phylum Proteobacteria 34 , we hypothesize that our identified bacteria, belonging for 82% to phylum of Proteobacteria, could act as endophytes. In Table 2 there are the main genera of bacteria that behave like endophytes, Pseudomonas, Bacillus, Burkholderia, Rhizobium and Microbacterium [34][35][36][37][38] . A good method to reduce crude oil is a biodegradation process via cometabolism. Gałazka et al. 30 and Doong and Lei 39 reported that in case of cometabolism hydrocarbons could not be a font of carbon and energy but has function as co-substrates, so their degradation is due by the presence of diverse microorganisms (e.g. parathion is cometabolized by Pseudomonas stutzeri to 4-nitrophenol and diethylphosphate, and phenol is then used as a source of carbon and energy by P. aeruginosa); the researcher conclude that cometabolism is one of the most important mechanism in the transformation of PAHs in soil. Pseudomonas aeruginosa make PAH-oxidative enzymes and release rhamnolipids 40 . Bacillus cereus degrade both LMW and HMW PAHs 41,42 . Besides, Cavalcanti et al. 43 emphasizing the adjunct of consortia composed by Pseudomonas aeruginosa and Burkholderia cepacia strains in the removal of phenanthrene and pyrene from a soil contaminated by a lubricating oil mixture containing PAH. In polluted soil the presence of Alphaproteobacteria, family as Bradyrhizobiaceae (slow-growing rhizobia), Rhizobiaceae, Nitrobacter and Sphingobium, known in literature to do nodules on the roots of leguminous plants and fixing nitrogen, is important. Komaniecka et al. 44 called "bacteroids" the endosymbiotic rhizobia, in which nitrogen fixation takes place. All bacterial species isolated in this study are known to be a good PAHs degradation 45 . Different paper reported the import role of bacterial consortium. Vaidya et al. 46 reported the use of Pseudomonas, Burkholderia and Rhodococcus (PBR) was able to degrade 99% of pyrene under microcosm conditions. Other consortiums like Pseudomonas sp. and Bacillus sp. isolated from oil sludge that are able to reduce total petroleum hydrocarbons from 63 to 84% in six weeks 47 ; and the consortium composed of Pseudomonas aeruginosa and Rhodococcus sp. isolated from soil polluted with oily sludge demonstrated 90% degradation of hydrocarbons in 6 weeks 47 . Plant roots release exudates as sugars, organic acids, fatty acids, secondary metabolites, nucleotides and inorganic mixture that perform an important key in establishing and determining the rhizosphere microbial population 48 . These exudates determine and regulate directly or indirectly the activity of biodegradative microorganisms. In this study, the capability of isolates strain to make indole-3-acetic acid, siderophores, exopolysaccharides and ammonia was evaluated ( Table 2). Thirty-nine of the isolates were able to produce Indolacetic acid (IAA). All three-rhizosphere soil had Paenibacillus polymyxa as the best IAA producing. Both in Poaceae and in Plantaginaceae rhizosphere soil Rhizobium leguminosaurum produced highest amount of IAA. Mycobacterium sp. MOTT36Y, instead, produced only in Poaceae family highest amount of IAA. Rhizosphere of Plantaginaceae and Fabaceae had Pseudomonas cichorii as good IAA producer. Plantaginaceae and Fabaceae showed respectively typical strains with high IAA Paenibacillus sp. JDR-Z, Bacillus cereus group, Klesbiella aerogenes for the first and Rhizobium etli bv mimosae for the second one. In this study, the 60% of isolates were found to have ability for siderophores production. Rhodococcus jostii produced in all three-rhizosphere soils high amount of siderophores; only in Plantaginaceae, also Bacillus cereus group showed siderophores activity. The three-rhizosphere soils showed 50% of isolates capable of producing EPS. The high amount of EPSs production was present in Burkholderia cenocepacia, Burkholderia ambifaria AMMD, Bacillus

Beta Proteobacteria Bukholderiales
Alcaligenaceae  In contrast with the heterogeneity of the taxonomy of bacteria structure (82%) in three different rhizosphere soils, there are important alterations in the abundance of the genes encoding enzymes for PAHs degradation (Fig. 8). Our results showed a high gene abundance of several oxidoreductases involved in PAH metabolism, as a test of the PAH degradation potential by native soil microorganisms 11,[49][50][51] . In fact, the dioxygenase family genes (naphthalene 1,2-dioxygenase, extradiol dioxygenase, benzoate 1,2 dioxygenase, protocatechuate 4,5-dioxygenase (alpha and beta chain) and 1,2-dihydroxynaphthalene dioxygenase) are the most abundant in PHAs contaminated soils, particularly in rhizosphere soil of Plantago lanceolata as compared to Piptatherum miliaceum and Lothus corniculatus (Fig. 8). Therefore, from our data, it is possible to hypothesize different ways of degradation of the PAHs, although all could start by an initial oxidation by laccases followed the activity of dioxygenases and decarboxylases, helping the ulterior degradation of PAHs; hypothesizing that natural soil bacterial populations are able to use specific enzymes and common pathways to degrade PAHs.

Conclusions
Our obtained data showed that PAHs contamination at the Bagnoli brownfield site (Southern Italy) is relevant and its origin is mainly due to the industrial activities and processes that had been working for decades. Almost the 44% of the soil samples in the industrial district were contaminated by PAHs with 4, 5, and 6 ring. Media contents of ∑PAHs in the soil were higher than the maximum permitted concentrations for residential/recreational land use, as it represent the remediation objective stated by Italian Government for Bagnoli site recovery. All PAHs congeners overcomed the recommended limits for corresponding soil categories and BghiP, BaP and IP mainly control pollution. Analysis of diagnostic ratios showed that petroleum and coal combustion were the main sources of PAHs in the investigated site. The studied uptake by native plants indicates that PAHs root accumulation is promoted and movement to the aboveground biomass (translocation) is limited. Interesting PAHs roots accumulation rate were observed for several native species. Based on our results, plant PAHs concentration levels mainly depend on rhizosphere soil PAHs content and type of plant species. Maximum PAHs root accumulation was found in monocotyledons Poaceae such as Pip and Fes. These type of plants are known to have a fibrous root system with several moderately branching roots growing from the stem. While, a taproot system is common in dicotyledons. Monocots fibrous root system consisting of a mass of similarly sized roots that maximizes absorption. Boyle and Shann 52 showed that also microbial activity was higher in monocot rhizosphere soils than dicot ones soils and demonstrated that monocot rhizosphere soil degraded many organic contaminants faster than dicot soils. Piptatherum miliaceum (L.) Coss on has suitable attributes such as rapid growth and high root cover and stimulates soil microbial communities 24 . Besides, our data indicated that Rhizoremediation process involve both plants and their associated rhizosphere microbes. Various microbes include as Pseudomonas aeruginosa, Pseudomonas fluoresens, Mycobacterium spp., Rhodococcus spp., Paenibaciluus spp. are involved in PAHs degradation 48,53 . Our bacteria that for about 85% belong to the phylum of Proteobacteria in general have a marked propensity to the endophytic habitat. This is very important both for determining the performance of the plant and for the degrading activity of their rhizosphere 34 . Functional metagenomics showed that the abundance of genes degrading PAHs is present in three rhizosphere soils, confirming the metabolic potential of PAHs-degrading microorganisms present into contaminated soil, and assuming a possible ways of degradation PAHs in contaminated soils 11,12 . The root-exudates help the activity of the microbes, which respond to the exudate, buffet rapidly during the bioremediation of soils. This is in concordance with previous works in which it was markedly noted a strong rhizosphere effect on degradation of PAHs through the stimulation of indole-3-acetic acid,  Table 2. Primary screening of the assessment of potential PGP by bacteria isolates recovered from rhizosphere soil of Piptatherum miliaceum, Lothus corniculatus and Plantago lanceolata. In bold the bacteria isolates typical of Lothus corniculatus, in underline those typical of Plantago lanceolata. The isolates were categorized into three groups according to the produced amount: + low concentrations (< 1 µ g/ml), + + moderate concentrations (1-2.99 µ g/ml) and + + + high concentrations (> 3 µ g/ml). www.nature.com/scientificreports www.nature.com/scientificreports/ siderophores, exopolysaccharides and ammonia. Our data show that PAHs pollution selects a specific rhizosphere microflora that degrades and helps the plant to adapt better and therefore to high performance. Therefore, these results can lead to innovative biotechnological applications in bioremediation processes in the field. In conclusion, our results demonstrated that the microbe-assisted phytoremediation was found to be the most advantageous approach obtaining high rates of degradation of PAHs in relation to other strategies, demonstrating that Piptatherum miliaceum, Lothus corniculatus and Plantago lanceolata association could be an environmentally sound management approach for the treatment of aged PAH-polluted soils.

Materials and Methods
Site description and sampling. The Bagnoli brownfield area (2 km 2 ; situated in correspondence of Phlegrean Fields between 40°49′30″91 and 40°47′30″North, and 14°9′30″ and 14°12′0″ East) falls into the western part of the city of Naples (Campania region), Southern Italy. This industrial district was one of the most important integrated steelworks in Italy during the last century until its closure in the nineties due to economic and environmental reasons. Because of industrial activities, large amount and types of contaminants were generated and dispersed throughout the whole area. The sampling campaign was conducted during 2017 with the identification of 76 areas characterized by high concentrations of PAHs in which the dominant plant species were identified. The most represented species are shown in Fig. S1 (Supplementary Materials). Roots and leaves of each species were gathered and stored in sterile polypropylene tubes. Rhizosphere soil samples of the above plants were also gathered and stored in polypropylene tubes and kept at 4 °C for further analysis. All analysis were performed in triplicate.

Soil and plant (roots and leaves) PAHs analyses. The analysis of PAHs comprised of 13PAHs and
followed the procedure US-EPA method 8270D. The target analytics (Table 3) were extracted by an accelerated solvent extractor, purified using a silica gel column, and detected by a gas chromatograph (7890 A, Agilent, USA) coupled with a mass spectrometer (5977B, Agilent, USA). Deuterated fluorene, Deuterated fenanthrene, Deuterated chrysene and Deuterated perylene were added to the samples prior to extraction, and were considered as internal standards for quantification of the 13 PAHs as described by the USEPA 54   www.nature.com/scientificreports www.nature.com/scientificreports/ Brief as described in Guarino et al. 10 , cultures were incubated in 250 ml of a mineral medium in conical flasks at 28 °C in an orbital shaker (200 rpm) for five days. Then, to isolate the greatest number of strains, 100 ml of serial tenfold dilutions of bacterial cultures were propagated on two different solid media: LB and R2A (Sigma-Aldrich, Milan Italy) for to not overlook slower growing colonies. First colonies were visualized after 4 days of incubation at 28 °C and after other five days, a total count of colonies was performed 10 . For each rhizosphere soil have been obtained and preserved fifty-one colonies per medium and per samples were randomly selected and maintained as pure cultures 10  and subjected to homology comparison (BLAST analysis) at the National Center for Biotechnology Information (NCBI) server (www.ncbi.nlm.nih.gov/blast/Blast.cgi) 10 .

In vitro tests for plant growth promoting (PGP) traits. The bacteria strains were analysed for Plant
Growth Promoting activities (PGP). Indolacetic acid (IAA) production, siderophores release, exopolysaccharides (EPSs) production and ammonia production were determined as described by Guarino et al. 10 .
Library preparation and sequencing PHAs degrading genes. 'Nugen Ovation Ultralow System V2′ kit (Nugen, San Carlos, CA) was used for library preparation. The samples were quantified and quality tested using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Libraries were processed and sequenced on MiSeq (Illumina, San Diego, CA), pair-end with 300 cycles per read. Base calling and demultiplexing were performed on instrument. Analysis were focused on the genes involved Polycyclic Aromatic Hydrocarbons (PAHs) degradation as KEGG elements reported in a recent work 11 . Gene sequences were downloaded from Uniprot, and the short reads obtained from the sequencing experiments were blasted against the above-mentioned genes using BLASTX v2.2.29. All reads resulting in a hit with an e-value lower than 0.1 were retained for analysis.
Statistical data analyses. Univariate statistical analyses were performed to show the single-element distribution. The data below the instrumental detection limit (IDL) were assigned a value corresponding to 50% of the detection limit 59 . The Pearson correlation coefficients between PAHs concentrations in plants and rhizosphere soils were calculated. The statistical significance of the results was verified at the significance level of alpha < 0.05.  Table 3. List of PAHs analysed and their abbreviations, also used in the text and figures. The number of rings and molecular weights are also reported.