**1. Introduction**

The overture of remediation of polluted soils for sustainable innovation is one of the critical steps in addressing current global environmental issues.

Nowadays, several physicochemical and biological solutions have been developed for the recovery of contaminated water [1–3] and soils [4,5], making the optimal selection a problematic ye<sup>t</sup> crucial step for the success of the reclamation [6,7].

However, remediation activities also have an environmental impact since they often use chemical products or processes, with consequent consumption of raw materials and energy that could compromise the sustainability of the approach or even invalidate its beneficial aspects [8].

In the light of this, environmentally unsound technologies for the remediation of heavy metal contaminated soils (chemical extraction, chemical oxidation, stabilization/solidification, solvent extraction, etc.) should be replaced by green and sustainable technologies. The aim of the latter is not only to eliminate or reduce contamination but also to minimize the environmental impact (reduction of air emissions, minimization of energy use, decrease of waste, etc.) and create synergies between different sectors and activities (ecosystems protection, circular economy, climate change and resilience). Concerning this, sustainable phytomanagement can make a significant contribution to supporting this transition towards sustainable remediation.

**Citation:** Pietrini, I.; Grifoni, M.; Franchi, E.; Cardaci, A.; Pedron, F.; Barbafieri, M.; Petruzzelli, G.; Vocciante, M. Enhanced Lead Phytoextraction by Endophytes from Indigenous Plants. *Soil Syst.* **2021**, *5*, 55. https://doi.org/10.3390/ soilsystems5030055

Academic Editors: Matteo Spagnuolo, Paola Adamo and Giovanni Garau

Received: 9 July 2021 Accepted: 2 September 2021 Published: 3 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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Remediation technologies based on the new natural-based solution (NBS) approach enable the achievement of the goals established by current environmental policies to protect natural resources [9]. Among the NBS remediation measures, a growing focus is on phytoremediation [10–12], which is the set of remediation technologies with plants as the main actors to remediate organic and inorganic contaminants in soil and other environmental matrices (sediments, water). The interest in these phytotechnologies has increased over time, given some significant advantages in terms of low cost, simplicity of operation and environmental benefits, as highlighted by some recent LCA-based studies [13,14]. In addition, the combined use with other solutions to further increase the overall sustainability is under investigation [15].

Among the different phytoremediation technologies, phytoextraction is considered a non-invasive technique to remove heavy metals from contaminated soil in an environmentally friendly and economical way [16,17]. In phytoextraction, metals are absorbed by roots from the soil solution and then transferred and accumulated in various tissues of the plant. Traditional phytoextraction requires hyperaccumulating species able to accumulate high amounts of metal without suffering physiological damage. However, these species exhibit low biomass production and slow growth rate, making phytoextraction a slow process with long implementation times [17,18].

Several studies have focused on improving phytoremediation efficiency using fastgrowing, high biomass tolerant species, and the aid of chelating agents for increased metal uptake from the soil. The main chelating agents include ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetate (DTPA), ethylene glycol tetraacetic acid (EGTA), hydroxyethyliminodiacetic acid (HEIDA), and ethylenediamine disuccinic acid (EDDS). These compounds can accelerate the release of heavy metals bound to soil particles into the soil solution, thus increasing the phytoavailable metal fraction [19]. Indeed, the only metals that plants can absorb are those in bioavailable form, i.e., present in soluble forms in the soil solution [20,21].

Among the various assisted-phytoextraction approaches to maximize the technique's effectiveness, an exciting alternative is the use of plant growth-promoting rhizobacteria (PGPR) [22]. This strategy involves rhizobacteria that can stimulate plant growth by both facilitating the bioavailability of soil nutrients and modulating the production of phytohormones (including auxins, cytokines, gibberellic acid, 1-aminocyclopropane-1-carboxylate deaminase—ACCD) and modulating plant hormone levels [22,23]. In addition, through microbial processes active in the rhizosphere, PGPR can also promote the mobility and bioavailability of metals in the soil, increasing their uptake by plants [23,24].

This work aimed to investigate the single and synergistic effect of PGPR and EDTA on the growth and Pb uptake in two tolerant species, *Brassica juncea* L. (Indian mustard) and *Helianthus annuus* L. (sunflower), to evaluate the phytoremediation potential of a Pb-contaminated site.

The selection of bacterial strains capable of improving the efficiency of phytoremediation is a fundamental step. In this study, the addition of a microbial consortium with indigenous endophytic bacteria allowed detectable levels of phytoextraction to be obtained even without the addition of mobilizing chemical agents.

#### **2. Materials and Methods**

#### *2.1. Site Description and Soil Sampling*

The contaminated site under investigation is located in an area of 40,000 m<sup>2</sup> near an urban area in Italy, previously affected for many years by a deposit for the storage of industrial wastes, active until the beginning of the 90s.

Being adjacent to a pond and at a distance of about 1 km from the sea, the site is characterized by sediments of marine and alluvial origin (silts, clayey silts, fluvio-lacustrine and marshy clays, arenaceous and conglomeratic, as well as alluvial deposits) and by a superficial layer (4–5 m depth) mainly composed of homogeneous artificial backfill soils of clayey, silty sand nature.

Previous chemical analyses performed on the soil of the area under examination revealed a relevant Pb contamination, which exceeded the contamination threshold concentrations (CSC) established by the Italian regulation (D.Lgs 152/2006) for sites intended for industrial use (1000 mg kg−1) [25]. The highest Pb concentration found in the area was approximately 2200 mg kg−1.

For the soil sampling to be addressed for the laboratory activities, 6 sampling points (SP1 to SP6) were chosen within the site. The selection was based on the geological and morphological characteristics of the soils and favoring the points where previous analytical campaigns had already detected the presence of Pb.

Approximately 50 kg of soil was collected from each sampling point with an excavator to a maximum depth of 2.5 m.

The soil samples were sieved at 2 cm on-site and then transported in special containers to the laboratories for the soil characterization analysis. Based on the results of characterization, which showed that the soil samples were homogeneous, a single soil sample was prepared for the phytoextraction tests, obtained by mixing the soil aliquots (~20 kg) from each soil sample. On this soil (Pb-soil), the determination of the total and bioavailable lead content was performed again.

#### *2.2. Soil Characterization and Pb Analysis*

The soil samples were air-dried, ground, and sieved (0–2 mm) before analysis. The physicochemical characteristics of the soil were determined following the procedures reported in Methods of soil analysis [26]. Soil pH and electrical conductivity (EC) were determined by glass electrodes with a soil/water ratio of 1:2.5 and 1:2, respectively. The cation exchange capacity (CEC) was measured by exchange with barium acetate (pH 8.1) and titration with EDTA (0.05 N). Particle size distribution (sand, silt, and clay) was evaluated by the pipette method [27].

For the determination of total Pb content, soil samples were digested in a mixture of HNO3 (65%, *v*/*v*) and H2O2 (30%, *v*/*v*) using a microwave oven (FKV-ETHOS 900), according to EPA method 3051-A [28].

Potentially bioavailable concentrations of Pb in soil were determined according to an extraction procedure that sequentially involves the use of H2O (to extract soluble Pb), KNO3 1 M (to extract exchangeable Pb), and EDTA 1% (to extract Pb retained with bonds also of covalent nature) with a soil/extractant ratio of 1:5 and extraction time5h[29,30].

EDTA was selected because it is one of the most effective mobilizing agents for Pb and can be applied effectively in various soil types [31–33].

A single extraction with EDTA was also performed. The concentration selected was 2 mM, frequently used in phytoextraction tests [15,31,34]. Higher concentrations of EDTA would mobilize excessive quantities of metal, which could give rise to leaching phenomena towards the aquifer [18,19,35].

The extraction was carried out by shaking the soil and extractant (ratio of 1:5) for 2 h. All extracts were centrifuged at 15,000 rpm for 15 min and filtered.

All analyses were performed in triplicate, and the mean value was recorded.

#### *2.3. Microcosm Experimental Design*

To study the role of bacterial inocula on phytoremediation processes, alone or in combination with the EDTA mobilizing agent, a microcosm experimental campaign was performed.

Each microcosm was prepared by adding 300 g of Pb-soil and sowing *B. juncea* var. Scala or *H. annuus* var. Paola, at doses of 0.5 g and 5 seeds per pot, respectively.

The experimentation lasted about 30 days and was conducted in a growth chamber (CCL300BH-AS S.p.A., Perugia, Italy). The growth conditions were the following: photoperiod of 14 h light at 25 ◦C and 10 h dark at 19 ◦C, photosynthetic photon flux density (PPFD) of 200 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1, and relative humidity 65%.

The experiment consisted of the following treatments: Pb-soil treated with 10<sup>8</sup> CFU (Colony-Forming Unit) per gram of soil of bacterial consortium SG\_1 (PGPR) prepared as described in Section 2.6, Pb-soil treated with PGPR and EDTA 2 mM (PGPR + EDTA), and Pb-soil without any treatment (CT, control).

The study design consisted of a completely random design with three replicates per treatment. Bacterial inocula were added approximately 9 days after planting. EDTA addition was carried out after approximately 15 days, splitting the total dose over 5 days to avoid possible Pb toxic effects on plant growth resulting from rapid release and high metal mobilization [36].

Pots were thoroughly watered with tap water at field capacity, maintaining this condition throughout the experiment without fertilizer addition.

Plants were harvested 15 days after the addition of EDTA. Plant organs (roots and shoots) were washed with deionized water and then dried at 40 ◦C to constant weight to determine their Pb content. Roots were further treated in an ultrasonic bath (Branson Sonifier 250 ultrasonic processor, Branson Ultrasonic Corporation, Brookfield, CT, USA) to remove any residual soil.

Dried plant tissues were weighed, dry weight (DW) recorded, and powdered. Total Pb content was determined in the dried vegetal samples after overnight acid pre-digestion with HNO3 (65%, *v*/*v*) and H2O2 (30%, *v*/*v*) according to US-EPA 3052 [37]. Quantification of Pb in soil, extract, and vegetal samples was performed by inductively coupled plasma optical emission spectroscopy (ICP-OES) Liberty AX, Varian.

## *2.4. Test of Phytotoxicity*

Soil toxicity was assessed by a phytotoxicity screening test based on germination inhibition and root extension.

The assay was performed in a Petri dish (10 cm diameter) filled with 10 g of Pb-soil moistened with deionized water to saturation, over which was placed a Whatman #1 filter and 10 seeds of *Lepidium sativum* L. [38]. *L. sativum* is a biological indicator of soil toxicity screening highly sensitive to phytotoxic compounds.

Hydrated quartz sand was used as a negative control. Five replicates were performed for each soil (Pb-soil and negative control). The closed Petri dishes were placed in a germination chamber in the dark at 25 ± 1 ◦C. After 72 h, the number of germinated seeds was counted, and the root length was measured. The germination index (*GI*%) and the radical extension inhibition (*Inh*%) were estimated by combining the values of seed germination and root elongation:

$$GI^{\diamondsuit} = \frac{G\_{\sf s} \ast L\_{\sf s}}{G\_{\sf c} \ast L\_{\sf c}} \ast 100\tag{1}$$

$$Inh\% = \frac{L\_{\varepsilon} - L\_{\varepsilon}}{L\_{\varepsilon}} \ast 100\tag{2}$$

*Gs* and *Gc* are the average numbers of seeds germinated in the contaminated soil samples and in the negative control, respectively. *Ls* and *Lc* are the radical lengths (mm) for the contaminated soil sample and negative control, respectively.
