Plant testing with hemp and miscanthus to assess phytomanagement options including biostimulants and mycorrhizae on a metal-contaminated soil to provide biomass for sustainable biofuel production.

The need of biofuels from biomass, including sustainable aviation fuel, without using agricultural land dedicated to food crops, is in constant demand. Strategies to intensify biomass production using mycorrhizal fungi, biostimulants and their combinations could be solutions for improving the cultivation of lignocellulosic plants but still lack well-established validation on metal-contaminated soils. This study aimed to assess the yield of Miscanthus x giganteus J.M. Greef & Deuter and Cannabis sativa L. grown on a metal-contaminated agricultural soil (11 mg Cd, 536 mg Pb and 955 mg Zn kg-1) amended with biostimulants and/or arbuscular mycorrhizal fungi, and the shoot Cd, Pb and Zn uptake. A pot trial was carried out with soil collected from a field near a former Pb/Zn smelter in France and six treatments: control (C), protein hydrolysate (a mixture of peptides and amino acids, PH), humic/fulvic acids (HFA), arbuscular mycorrhizae fungi (AMF), PH combined with AMF (PHxAMF), and HFA combined with AMF (HFAxAMF). Metal concentrations in the soil pore water (SPW), pH and electrical conductivity were measured over time. Miscanthus and hemp shoots were harvested on day 90. Both PH and PHxAMF treatments increased SPW Cd, Pb, and Zn concentrations (e.g. by 26, 1.9, and 22.9 times for miscanthus and 9.7, 4.7, and 19.3 times for hemp in the PH and PHxAMF treatments as compared to the control one, respectively). This led to phytotoxicity and reduced shoot yield for miscanthus. Conversely, HFA and HFAxAMF treatments decreased SPW Cd and Zn concentrations, increasing shoot yields for hemp and miscanthus. Shoot Cd, Pb, and Zn uptakes peaked for PH and PHxAMF hemp plants (in µg plant-1, Cd: 310-334, Pb: 34-38, and Zn: 232-309 for PHxAMF and PH, respectively), while lowest values occurred for PH miscanthus plants mainly due to low shoot yield. Overall, this study suggested that humic/fulvic acids can be an effective biostimulant for increasing shoot biomass production in a metal-contaminated soil. These results warrant further investigations of the HFAxAMF in field trials.

Field assessment of organic amendments and spring barley to phytomanage a Cu/PAH-contaminated soil.

The INTENSE project, supported by the EU Era-Net Facce Surplus, aimed at increasing crop production on marginal land, including those with contaminated soils. A field trial was set up at a former wood preservation site to phytomanage a Cu/PAH-contaminated sandy soil. The novelty was to assess the influence of five organic amendments differing in their composition and production process, i.e. solid fractions before and after biodigestion of pig manure, compost and compost pellets (produced from spent mushroom substrate, biogas digestate and straw), and greenwaste compost, on Cu availability, soil properties, nutrient supply, and plant growth. Organic amendments were incorporated into the soil at 2.3% and 5% soil w/w. Total soil Cu varied from 179 to 1520 mg kg-1, and 1 M NH4NO3-extractable soil Cu ranged from 4.7 to 104 mg kg-1 across the 25 plots. Spring barley (Hordeum vulgare cv. Ella) was cultivated in plots. Changes in physico-chemical soil properties, shoot DW yield, shoot ionome, and shoot Cu uptake depending on extractable soil Cu and the soil treatments are reported. Shoot Cu concentration varied from 45 ± 24 to 140 ± 193 mg kg DW-1 and generally increased with extractable soil Cu. Shoot DW yield, shoot Cu concentration, and shoot Cu uptake of barley plants did not significantly differ across the soil treatments in year 1. Based on soil and plant parameters, the effects of the compost and pig manure treatments were globally discriminated from those of the untreated, greenwaste compost and digested pig manure treatments. Compost and its pellets at the 5% addition rate promoted soil functions related to primary production, water purification, and soil fertility, and the soil quality index.

Potential ability of tobacco (Nicotiana tabacum L.) to phytomanage an urban brownfield soil.

The ability of tobacco (Nicotiana tabacum L. cv. Badischer Geudertheimer) for phytomanaging and remediating soil ecological functions at a contaminated site was assessed with a potted soil series made by fading an uncontaminated sandy soil with a contaminated sandy soil from the Borifer brownfield site, Bordeaux, SW France, at the 0%, 25%, 50%, 75%, and 100% addition rates. Activities of sandblasting and painting with metal-based paints occurred for decades at this urban brownfield, polluting the soil with metal(loid)s and organic contaminants, e.g., polycyclic aromatic hydrocarbons, in addition to past backfilling. Total topsoil metal(loid)s (e.g., 54,700 mg Zn and 5060 mg Cu kg-1) exceeded by seven- to tenfold the background values for French sandy soils, but the soil pH was 7.9, and overall, the 1M NH4NO3 extractable soil fractions of metals were relatively low. Leaf area, water content of shoots, and total chlorophyll (Chl) progressively decreased with the soil contamination, but the Chl fluorescence remained constant near its optimum value. Foliar Cu and Zn concentrations varied from 17.8 ± 4.2 (0%) to 27 ± 5 mg Cu kg-1 (100%) and from 60 ± 15 (0%) to 454 ± 53 mg Zn kg-1 (100%), respectively. Foliar Cd concentration peaked up to 1.74 ± 0.09 mg Cd kg-1, and its bioconcentration factor had the highest value (0.2) among those of the metal(loid)s. Few nutrient concentrations in the aboveground plant parts decreased with the soil contamination, e.g., foliar P concentration from 5972 ± 1026 (0%) to 2861 ± 334 mg kg-1 (100%). Vulnerability to drought-induced embolism (P50) did not differ for the tobacco stems across the soil series, whereas their hydraulic efficiency (Ks) declined significantly with increasing soil contamination. Overall, this tobacco cultivar grew relatively well even in the Borifer soil (100%), keeping its photosynthetic system healthy under stress, and contaminant exposure did not increase the vulnerability of the vascular system to drought. This tobacco had a relevant potential to annually phytoextract a part of the bioavailable soil Zn and Cd, i.e., shoot removals representing here 8.8% for Zn and 43.3% for Cd of their 1M NH4NO3 extractable amount in the potted Borifer soil.

Long-term phytomanagement with compost and a sunflower - Tobacco rotation influences the structural microbial diversity of a Cu-contaminated soil.

At a former wood preservation site contaminated with Cu, various phytomanagement options have been assessed in the last decade through physicochemical, ecotoxicological and biological assays. In a field trial at this site, phytomanagement with a crop rotation based on tobacco and sunflower, combined with the incorporation of compost and dolomitic limestone, has proved to be efficient in Cu-associated risk mitigation, ecological soil functions recovery and net gain of economic and social benefits. To demonstrate the long-term effectiveness and sustainability of phytomanagement, we assessed here the influence of this remediation option on the diversity, composition and structure of microbial communities over time, through a metabarcoding approach. After 9 years of phytomanagement, no overall effect was identified on microbial diversity; the soil amendments, notably the repeated compost application, led to shifts in soil microbial populations. This phytomanagement option induced changes in the composition of soil microbial communities, promoting the growth of microbial groups belonging to Alphaproteobacteria, many being involved in N cycling. Populations of Nitrososphaeria, which are crucial in nitrification, as well as taxa from phyla Planctomycetacia, Chloroflexi and Gemmatimonadetes, which are tolerant to metal contamination and adapted to oligotrophic soil conditions, decreased in amended phytomanaged plots. Our study provides an insight into population dynamics within soil microbial communities under long-term phytomanagement, in line with the assessment of soil ecological functions and their recovery.

From phytoremediation of soil contaminants to phytomanagement of ecosystem services in metal contaminated sites.

Since the emergence of phytoremediation, much research has focused on its development for (i) the removal of metals from soil and/or (ii) the reduction of metal bioavailability, mobility, and ecotoxicity in soil. Here, we review the lights and shades of the two main strategies (i.e., phytoextraction and phytostabilization) currently used for the phytoremediation of metal contaminated soils, irrespective of the level of such contamination. Both strategies face limitations to become successful at commercial scale and, then, often generate skepticism regarding their usefulness. Recent innovative approaches and paradigms are gradually establishing these phytoremediation strategies as suitable options for the management of metal contaminated soils. The combination of these phytotechnologies with a sustainable and profitable site use (a strategy called phytomanagement) grants value to the many benefits that can be obtained during the phytoremediation of metal contaminated sites, such as, for instance, the restoration of important ecosystem services, e.g. nutrient cycling, carbon storage, water flow regulation, erosion control, water purification, fertility maintenance, etc.

Ecosystem services and plant physiological status during endophyte-assisted phytoremediation of metal contaminated soil.

Mining sites shelter a characteristic biodiversity with large potential for the phytoremediation of metal contaminated soils. Endophytic plant growth-promoting bacteria were isolated from two metal-(hyper)accumulator plant species growing in a metal contaminated mine soil. After characterizing their plant growth-promoting traits, consortia of putative endophytes were used to carry out an endophyte-assisted phytoextraction experiment using Noccaea caerulescens and Rumex acetosa (singly and in combination) under controlled conditions. We evaluated the influence of endophyte-inoculated plants on soil physicochemical and microbial properties, as well as plant physiological parameters and metal concentrations. Data interpretation through the grouping of soil properties within a set of ecosystem services was also carried out. When grown together, we observed a 41 and 16% increase in the growth of N. caerulescens and R. acetosa plants, respectively, as well as higher values of Zn phytoextraction and soil microbial biomass and functional diversity. Inoculation of the consortia of putative endophytes did not lead to higher values of plant metal uptake, but it improved the plants' physiological status, by increasing the content of chlorophylls and carotenoids by up to 28 and 36%, respectively, indicating a reduction in the stress level of plants. Endophyte-inoculation also stimulated soil microbial communities: higher values of acid phosphatase activity (related to the phosphate solubilising traits of the endophytes), bacterial and fungal abundance, and structural diversity. The positive effects of plant growth and endophyte inoculation on soil properties were reflected in an enhancement of some ecosystem services (biodiversity, nutrient cycling, water flow regulation, water purification and contamination control).

Enhancement of ecosystem services during endophyte-assisted aided phytostabilization of metal contaminated mine soil.

Endophytic plant growth-promoting bacteria (endophytes) were isolated from a variety of (pseudo)metallophytes growing in an abandoned Zn/Pb mine and then characterized according to their plant growth-promoting traits (i.e. ACC deaminase activity, IAA production, siderophore production, phosphate solubilising capacity, metal and salt tolerance and phenotypic characterization). Initially, under growth chamber conditions, an endophyte-assisted aided phytostabilization study was carried out with Festuca rubra plants (native vs. commercial variety) inoculated with a Pseudomonas sp. isolate and cow slurry as organic amendment. The effect of treatments on soil physicochemical and microbial indicators of soil quality, as well as plant physiological parameters and metal concentrations, was assessed. We performed a complementary interpretation of our data through their grouping within a set of ecosystem services. Although the application of cow slurry had the most pronounced effects on soil quality indicators and ecosystem services, the growth of native F. rubra plants reduced soil bioavailability of Cd and Zn by 19 and 22%, respectively, and enhanced several soil microbial parameters. On the other hand, endophyte (Pseudomonas sp.) inoculation improved the physiological status of F. rubra plants by increasing the content of carotenoids, chlorophylls and Fv/Fm by 69, 65 and 37%, respectively, while also increasing the values of several soil microbial parameters. Finally, a consortium of five endophyte isolates was used for an endophyte-assisted aided phytostabilization field experiment, where lower metal concentrations in native excluder plants were found. Nonetheless, the field inoculation of the endophyte consortium had no effect on the biomass of native plants.

Impact of repeated single-metal and multi-metal pollution events on soil quality.

Most frequently, soil metal pollution results from the occurrence of repeated single-metal and, above all, multi-metal pollution events, with concomitant adverse consequences for soil quality. Therefore, in this study, we evaluated the impact of repeated single-metal and multi-metal (Cd, Pb, Cu, Zn) pollution events on soil quality, as reflected by the values of a variety of soil microbial parameters with potential as bioindicators of soil functioning. Specifically, parameters of microbial activity (potentially mineralizable nitrogen, ß-glucosidase and acid phosphatase activity) and biomass (fungal and bacterial gene abundance by RT-qPCR) were determined, in the artificially metal-polluted soil samples, at regular intervals over a period of 26 weeks. Similarly, we studied the evolution over time of CaCl2-extractable metal fractions, in order to estimate metal bioavailability in soil. Different metals showed different values of bioavailability and relative bioavailability ([metal]bio/[metal]tot) in soil throughout the experiment, under both repeated single-metal and multi-metal pollution events. Both repeated Zn-pollution and multi-metal pollution events led to a significant reduction in the values of acid phosphatase activity, and bacterial and fungal gene abundance, reflecting the negative impact of these repeated events on soil microbial activity and biomass, and, hence, soil quality.