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Mine driven trace elements' pollution entails environmental risks and causes soil infertility. In the last decades, in situ techniques such as phytostabilization have become increasingly important as ways to tackle these negative impacts. The aim of this study was to test the individual and combined effects of different aided phytostabilization techniques using substrate from barren tailings of a Cu mine, characterized by extreme infertility (high acidity and deficiency of organic matter and nutrients). The experiment analyzed the growth of Populus nigra L. planted alone (P) or in co-cropping with Trifolium repens L. (PT), in pots containing mine soil amended with compost (1, 10, compost, soil, w/w) non inoculated (NI) or inoculated with plant growth promoting rhizobacteria (PGP), mycorrhizae (MYC) or a combination of bacterial and fungal inocula (PGPMYC). Non-amended, non-planted and non-inoculated reference ports were also prepared. Plants were harvested after 110 days of plant development and several biometric and phytopathological parameters (stem height, aerial biomass, root biomass, wilting, chlorosis, pest and death) and macro and micronutrient composition were determined. The growth substrate was analyzed for several physicochemical (pH, CECe, and exchangeable cations, total C and N, P Olsen and availability of trace elements) and microbiological (community level physiological profiles: activity, richness and diversity) parameters. The use of the amendment, P. nigra plantation, and inoculation with rhizobacteria were the best techniques to reduce toxicity and improve soil fertility, as well as to increase the plant survival and growth. Soil bacterial functional diversity was markedly influenced by the presence of plants and the inoculation with bacteria, which suggests that the presence of plant regulated the configuration of a microbial community in which the inoculated bacteria thrive comparatively better. The results of this study support the use of organ

Most worldwide policy frameworks, including the United Nations Sustainable Development Goals, highlight soil as a key non-renewable natural resource which should be rigorously preserved to achieve long-term global sustainability. Although some soil is naturally enriched with heavy metals (HMs), a series of anthropogenic activities are known to contribute to their redistribution, which may entail potentially harmful environmental and/or human health effects if certain concentrations are exceeded. If this occurs, the implementation of rehabilitation strategies is highly recommended. Although there are many publications dealing with the elimination of HMs using different methodologies, most of those works have been done in laboratories and there are not many comprehensive reviews about the results obtained under field conditions. Throughout this review, we examine the different methodologies that have been used in real scenarios and, based on representative case studies, we present the evolution and outcomes of the remediation strategies applied in real soil-contamination events where legacies of past metal mining activities or mine spills have posed a serious threat for soil conservation. So far, the best efficiencies at field-scale have been reported when using combined strategies such as physical containment and assisted-phytoremediation. We have also introduced the emerging problem of the heavy metal contamination of agricultural soils and the different strategies implemented to tackle this problem. Although remediation techniques used in real scenarios have not changed much in the last decades, there are also encouraging facts for the advances in this field. Thus, a growing number of mining companies publicise in their webpages their soil remediation strategies and efforts; moreover, the number of scientific publications about innovative highly-efficient and environmental-friendly methods is also increasing. In any case, better cooperation between scientists and o

Farmyard manure, an amendment traditionally used for improving the fertility of sandy soils in arid climates, is becoming scarce and expensive. Its shortage makes it necessary to evaluate, at field scale, the suitability and medium-term performance of other cheap, highly available substitutes. A field trial was established to analyze the effects of a single application of three organic residues on barley yield and nutrient uptake and selected soil properties after two consecutive harvests. Municipal solid waste compost (MSWC), sewage sludge compost (SSC) and farmyard manure (FYM) were tested at rates of 0, 20, 40 and 60 t ha−1. Adding all three organic amendments increased organic matter, cation exchange capacity and available P, Ca, Mg and K in the soil, the grain yield (up to 51%), and the barley plants’ nutrient contents. After the second harvest, a positive residual effect of the amendment was observed in plant yield (up to 77%) and nutrient contents. MSWC and SSC induced slight increases on the extractable fractions (BCR protocol) of Co, Cu and Ni, relative to the unamended soil. The results demonstrate the positive immediate and residual effect of the amendments evaluated as fertilizers for agricultural purposes.

Lindane and other 1,2,3,4,5,6-hexachlorocyclohexane (HCH) isomers are persistent organic pollutants highly hydrophobic, which hampers their availability and biodegradation. This work aimed at (i) investigating genes encoding enzymes involved in HCH degradation in the bacterium Sphingobium sp. D4, (ii) selecting strains, from a collection of environmental isolates, able to mobilize HCHs from contaminated soil, and (iii) analysing the biodegradation of HCHs by strain D4 in co-culture with HCH-mobilizing strains or when cultivated with root exudates. Fragments of the same size and similar sequence to linA and linB genes were successfully amplified. Two isolates, Streptomyces sp. M7 and Rhodococcus erythropolis ET54b able to produce emulsifiers and to mobilize HCH isomers from soil were selected. Biodegradation of HCH isomers by strain D4 was enhanced when co-inoculated with HCH mobilizing strains or when cultivated with root exudates. The degrader strain D4 was able to decompose very efficiently HCHs isomers, reducing their concentration in soil slurries by more than 95% (from an average initial amount of 50 ± 8 mg HCH kg soil) in 9 days. The combination of HCH-degrading and HCH-mobilizing strains can be considered a promising inoculum for future soil bioremediation studies using bioaugmentation techniques or in combination with plants in rhizodegradation assays.

Purpose: Rhizosphere processes are known to modify uptake of elements from soil, but limited information is available for hyperaccumulators. We investigated labile Ni fractions and their kinetics of replenishment in the rhizospheres of the Ni-hyperaccumulator Odontarrhena serpyliffolia, the Ni-excluder Holcus lanatus and in bulk soils collected at the same serpentine outcrop. Methods: Labile Ni fractions in rhizosphere and bulk soil were evaluated using conventional extractions and also by the Diffusive Gradients in Thin Films (DGT) technique. DGT data were used to predict the kinetics of Ni resupply using DIFS modelling. Chemical imaging of Ni distribution along roots using DGT coupled with laser-ablation was conducted. Results: Labile Ni forms were higher in both rhizosphere than in bulk soils, together with an increase in dissolved organic C, cation exchange capacity and the Ca/Mg ratio. Ni fractionation indicated a shift towards less stable Ni fractions in the rhizosphere, particularly in the hyperaccumulator. DIFS modelling showed that the rhizosphere of the excluder was able to sustain the initially lower soluble Ni concentration through replenishment from the solid phase, while Ni resupply in the rhizosphere of the hyperaccumulator was not sufficient to maintain the initially high concentrations of soluble Ni. However, the amount of DGT-labile Ni was higher in the rhizosphere of the hyperaccumulator compared to the excluder in all deployment times. Conclusion: Our data suggest that compounds derived from root activity, in particular DOC, are important controls of Ni availability to plants growing on serpentine soil.

Initial experiments using Mediterranean Ni-hyperaccumulator plants for the purpose of phytomining were carried out in the 1990s. In order to meet commercial phytoextraction requirements, a technology has been developed using hyperaccumulator species with adapted intensive agronomic practices on naturally Ni-rich soils. Ultramafic soils in the Balkans and other parts of Europe display a great variability in Ni concentrations and available Ni levels. In Albania, Vertisols are currently being used for low-productivity agriculture (pasture or arable land) on which phytomining could be included in cropping practices. In northwestern Greece (Pindus and Vourinos mountain regions), agricultural soils may occur on ultramafic Cambisols. In Spain and Austria, these soils are much more erratically distributed and are seldom used for crops and pastures. In the Balkans, Odontarrhena chalcidica (synonym Alyssum murale) occurs widely on these ultramafic soils and is a spontaneous weed that grows among other crops. Field studies across Europe have now been carried out outside Mediterranean areas and have evaluated the Ni-hyperaccumulator O. chalcidica, as well as two other species native to northwestern Greece (Bornmuellera emarginata and B. tymphaea). At each site, local hyperaccumulator plants were tested for comparison (Noccaea goesingense in Austria and Odontarrhena serpyllifolia s.l. in Spain), in the context of two recent EUfunded projects (by Agronickel and LIFEAgromine). Soil and crop management practices are being developed in order to optimize the Ni agromining process. Field studies have evaluated the potential benefits of fertilization regimes, crop selection, and cropping patterns (introducing agroecological practices), and bioaugmentation using plant-associated microorganisms.

The diversity of cultivable bacteria associated with plants from phytomanaged soils with mixed trace metal (TM) and polycyclic aromatic hydrocarbon (PAH) contamination in Pierrelaye (France) was evaluated. The emphasis was on the cultivable bacterial community since the overall objective is to obtain inoculants to improve the remediation of this type of contaminated site. Root endophytic and rhizosphere soil bacterial counts were determined, and isolates were pooled by amplified rDNA restriction analysis and identified by 16S rDNA sequencing. Isolates were further characterized for the production of plant growth-promoting (PGP) substances, and resistance to TM. The selected strains were evaluated for their ability to degrade PAHs. The potential of cell-free microbial supernatant to increase the mobilisation of PAHs from the polluted soil of Pierrelaye was also evaluated. Proteobacteria and Actinobacteria dominated the collection of isolates, and differences in taxonomic diversity were observed between plant species (Populus or Zea mays) and depending on the remediation treatment (Populus inoculation with mycorrhizae or Populus intercropping with Alnus). The majority of isolates exhibited at least one of the tested PGP traits, as well as resistance to more than one TM. Several rhizosphere, endophyte and even one bulk soil isolate showed high rates of fluoranthene and pyrene reduction. The endophyte Rhizobium strain MR28 isolated from maize and degrading pyrene produced bioemulsifying molecules capable of improving the availability of PAHs from the soil of Pierrelaye. A selection of the most interesting strains was made for further re-inoculation experiments in order to assess their potential in rhizoremediation processes.

Soils from arid and semi-arid ecosystems are generally very low in organic matter content, poor in nutrients and typically with sandy texture. The application of different organic amendments has been proposed as an adequate approach to improve the quality of these soils for their use in agriculture. The use of organic wastes of different origins (agricultural, industrial, urban, etc.) as soil amendments has a dual goal: i) improving soil fertility and quality, ii) reducing the environmental problem that poses the disposal of these residues. However, despite of the beneficial effects of these residues, undesirable changes may also occur in agricultural soils after their addition. For example, the presence of various pollutants of anthropogenic origin in organic wastes may cause adverse effects on soil microbiota. Generally, the arid and semi-arid soils of Tunisia are well characterised. However, the use of organic amendments to improve the quality of these soils has been scarcely investigated. Soil biochemical properties, and specifically soil enzyme activities, have been often used to investigate the impact of different amendments on soil quality, because they are highly sensitive to human or environmental perturbations. In this work, the results of a field trial established for investigating the effectiveness of three organic residues (composted municipal solid waste, composted sewage sludge and farmyard manure) to improve the quality of one agricultural soil from Tunisia are reported. The soil had a sandy texture, alkaline pH (pH 8.3) and was very poor in organic matter (0.21 and 0.03% of total C and N, respectively). Each of the organic residues was applied in triplicate at three different doses in nine sub-plots randomly distributed; three untreated sub-plots were also established for comparison. One, 6 and 18 months after the soil amendments, surface (0-20 cm) soil samples were collected from all the treated and untreated subplots. The soil samples were analyse

The thesis focuses on the (1) characterisation and comparison of the bacterial communities associated with Ni-hyperaccumulating and metal-excluding plants growing in ultramafic soils naturally enriched in TE and (2) analysis of bacterial communities in soils contaminated with TE and treated with phytoremediation strategies. The work planned aimed to obtain information about bacteria potentially involved in the mechanisms of TE (hyper)accumulation and/or exclusion and to study the relationships between the bacterial communities and the changes induced in soil properties by phytomanagement.

This thesis aims to compare phytoextraction and phytostabilisation capacity of different plant species and to study the changes that they or their associated microorganisms produce in the bioavailability of metals in the rhizosphere. The soil-plant-bacteria system of the two subspecies of the Ni-hyperaccumulator Alyssum serpyllifolium, endemic to the Iberian Peninsula were studied with the aim to characterise its associated bacterial community. Rhizospheric processes involved in Ni solubilisation were also studied and experiments for the improvement of phytomining efficiency using soil organic amendment were carried out. On the other hand, greenhouse and field experiments were carried out on Zn, Cd and Pb or Cu-contaminated soils; the objective being the evaluation of phytoremeditiation potential of metal-(hyper)accumulators or metal-excluding plant species growing in metal contaminated soils. Moreover, in the same soils studies were carried out to improve phytoremediation efficiency through the use of soil fertilisation, organic amendments or bacterial inoculation.

The metal hyperaccumulating species play an important role in soil phytoremediation techniques. The Thesis Project focuses on the development of various methods to improve the ability of nickel phytoextraction in hyperaccumulating species. There are a variety of techniques used for this purpose. This study focuses primarily on the use of agricultural practices to increase the bioavailability of metals in soil, inoculation of plants with microorganisms involved in the process of phytoextraction and external application of growth promoting substances in the plant. Through the development of these techniques is possible to obtain plants with a greater potential for phytoextraction of heavy metals and therefore most likely to use in soil remediation. In this study is also included the study of five different populations of the nickel hyperaccumulator Alyssum serpyllifolium (ssp. lusitanicum and ssp. malacitanum) of the Iberian Peninsula (also known as A. pintodasilvae and A. malacitanum). Different populations included in this study represent the main serpentinitic areas of the Iberian Peninsula: the region of Tras-os-Montes (Morais and Samil) in the northeastern Portugal, Melide in the province of A Coruña, and Sierra de Aguas and Sierra Bermeja, located in the province of Malaga. The main conclusions of this Thesis can be summarised as: - The application of plant growth regulators (PGRs) or phytohormones may increase biomass production of Ni hyperaccumulating species such as Alyssum and Noccaea and, consequently, their metal phytoextraction capacity. The most effective PGR for enhancing Ni removal was the IAA-based product. - Bioagumentation using PGPR can be considered as a potential tool to optimised Ni phytoextraction (or phytomining) technologies, but it remains to be seen whether or not plant-associated microorganisms can further improve the shoot Ni removal on a field scale. This study has identified candidate strains which hence, could be useful for future fiel

[Background and aims] Microbe-assisted phytoremediation is particularly effective for organic pollutants. The leguminous shrub Cytisus striatus (Hill) Rothm. has been proposed as a candidate species for the rhizoremediation of hexachlorocyclohexane (HCH)-contaminated sites. The aim of this study was to improve the performance of this species using microbial inoculants., [Methods] C. striatus was grown in substrates contaminated with 0, 10 and 35 mg HCH kg−1 for 8 weeks. Plants were either not inoculated (NI), or inoculated with the endophyte Rhodococcus erythropolis ET54b and the HCH-degrader Sphingomonas sp. D4 (isolated from a HCH-contaminated soil) on their own or in combination (ET, D4 and ETD4)., [Results] Inoculation with both bacterial strains (ETD4) resulted in decreased HCH phytotoxicity and improved plant growth. HCH-exposed plants inoculated with ETD4 presented a 120–160 % increase in root, and 140–160 % increase in shoot biomass, and led to a decrease in the activities of enzymes involved in anti-oxidative defence. APOD activity was reduced by up to 37 % in shoot tissues and 25 % in root tissues, and corresponding activities of SOD were reduced by up to 35 % and 30 %. HCH dissipation was enhanced in the presence of C. striatus but no significant effect of microbial inoculants was observed., [Conclusions] Inoculating C. striatus with this combination of bacterial strains is a promising approach for the remediation of HCH-contaminated sites.

Strains of Fusarium verticillioides produce mycotoxins such as fumonisins, which are common contaminants of maize and maize-based products worldwide and considered to be hazardous for human and animal health, due to their nephrotoxic, hepatotoxic, immunosuppressive and carcinogenic activity (references cited in Butron et al. 2006, Dalie et al. 2012). Biological control of Fusarium infection is therefore of great interest for food safety and could contribute towards reducing the use of toxic chemicals with fungicide activity. Many plant-associated bacteria, both epiphytes and endophytes, are widely recognized as agents of biological control against plant diseases and infections (Compant et al. 2010). Numerous phylogenetically diverse bacterial strains showing inhibitory activity against different fungi have been isolated (Bacon & Hinton 2002, Compant et al. 2005, Dalie et al. 2010, McSpadden Gardener 2007, Yoshida et al. 2012).

Strains of Fusarium verticillioides produce mycotoxins such as fumonisins, common contaminants of maize and maize-based products worldwide and considered to be hazardous for human and animal health. Biological control of Fusarium infection is of great interest for food safety The study describes the isolation and identification of a bacterial endophyte from kernels of the corn line A509 resistant to Fusarium. Initial studies on the inhibition of a fumonisin- producing Fusarium verticillioides by the isolated bacterial endophyte are also presented. The analysis include the growth of the bacterial strain and fungus in different media as well as the effect of bacterial metabolites on fungal growth.

Bromoxynil, frequently applied as bromoxynil octanoate, is a post-emergence herbicide, widely used for control of broad-leaved weeds on a variety of crops, particularly since the banning of atrazine in most countries. This study analysed the degradation of bromoxynil octanoate by microorganisms from non-organically managed agricultural soils developed over granite (G) and schist (S) as well as from an organically managed agricultural soil developed over granite (Go). As a means of analysing the role of the most abundant members of the soil microbial community, degradation of bromoxynil octanoate was evaluated in microcosms incubated with distinct amounts of soils. Furthermore, the effect of the herbicide on soil microbial communities (community level physiological profiles, CLPP) was analysed. Bromoxynil octanoate was rapidly degraded by microorganisms from the 3 soils and was no longer detectable three days after initiating the experiment. At this time, about half of the herbicide applied was present as the active compound, bromoxynil. Thereafter, bromoxynil was degraded more rapidly in G and S than in Go, in which the herbicide was still detectable 2 weeks after its application. In the short-medium term (3-15 days), the metabolites 3,5-dibromo-4-hidroxybenzamide (DBHB amide) and/or 3,5-dibromo-4- hidroxybenzoic acid were detected in all three soils. The most abundant members of the soil microbial communities were also able to degrade bromoxynil and its ester, though this degradation was slower, or incomplete, and bromoxymil and/or DBHB amide were detected 6 weeks after application. The herbicide had little effect on the functionality of the microbial communities in the soils.

The plant-microorganism-soil system of three pseudometallophytes (Betula celtiberica, Cytisus scoparius and Festuca rubra) growing in a Pb/Zn mine was characterised. Plant metal accumulation, soil metal fractions (rhizosphere and non-vegetated) and bacterial densities were determined. Total Cd, Pb and Zn in non-vegetated soils was up to 50, 3000 and 20,000 mg kg−1 dry weight, respectively. The residual fraction dominated non-vegetated soils, whereas plant-available fractions became important in rhizosphere soils. All plant species effectively excluded metals from the shoot. F. rubra presented a shoot:root transport factor of ≤0.2 and this population could be useful in future phytostabilisation trials. Culturable bacterial densities and diversity were low (predominantly Actinobacteria). Rhizosphere soils hosted higher total and metal-tolerant bacterial densities. Seventy-four metal-tolerant rhizobacteria were isolated, and characterised genotypically (BOX-PCR, 16S rDNA) and phenotypically [Cd/Zn tolerance, biosurfactant production and plant growth promoting (PGP) traits]. Several isolates resisted high concentrations of Cd and Zn, and only a few presented PGP traits. Fourteen isolates were evaluated for promoting plant growth of two species (Salix viminalis and Festuca pratensis). Thirteen inoculants enhanced growth of F. pratensis, while only three enhanced growth of S. viminalis. Growth enhancement could not always be related to isolate PGP traits. In conclusion, some isolates show potential application in phytostabilisation or phytoextraction techniques.

Bacterial strains were isolated from the rhizosphere of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum (A. pintodasilvae; M, S, and L), one population of Ni-hyperaccumulator A. serpyllifolium subsp. malacitanum (A. malacitanum; SB), and one population of the non-hyperaccumulator A. serpyllifolium subsp. serpyllifolium (A. serpyllifolium; SN). Isolates were characterized genotypically by BOX-PCR genomic DNA fingerprinting and comparative sequence analysis of partial 16S rRNA gene, and phenotypically by their Ni tolerance (0–10 mM), presence of plant growth promoting traits (indoleacetic acid (IAA)-, siderophore-, or organic acid-production, and phosphate solubilization) or capacity to produce biosurfactants. Among the collection of rhizobacteria, 84 strains were selected (according to their BOX-PCR profiles and phenotypic characteristics) to assess their ability to modify Ni extractability from Ni-rich (serpentine) soils. Metabolites produced by 13 of the isolates mobilized soil Ni (originating from the rhizosphere of both Ni-hyperaccumulators and non-hyperaccumulator). In contrast, Ni extraction using culture medium filtrates which had supported the growth of 29 strains was significantly reduced. The remaining strains had no effect on Ni mobility. Bacterial induced Ni mobilization was not related to Ni resistance or the phenotypic traits tested. Isolates with potential use in phytoremediation techniques will be further studied in a plant-microorganism-soil system.

Inoculation of plants with their associated microorganisms is a promising strategy for improving phytoremediation of organic contaminants. Isolation and characterisation of these organisms from plants growing in contaminated sites will permit the identification of candidate strains for re-inoculation studies. The diversity of culturable endophytic and rhizoplane bacteria found in association with Cytisus striatus plants growing at a hexachlorocyclohexane (HCH)-contaminated site was studied. A total of 97 strains of endophytic bacteria were isolated from the root, stem and leaf tissues, and 49 from the rhizoplane. They were further characterised genotypically (BOX-PCR, 16S rDNA sequencing, presence of linA and linB genes) and phenotypically (trace metal tolerance, capacity to produce biosurfactants and plant growth promoting (PGP) traits). Proteobacteria and Actinobacteria dominated the isolate collection, and taxonomic diversity was strongly tissue-specific. The linA and linB genes were not detected in the isolate collection. The majority of isolates had at least one of the PGP traits tested, whereas biosurfactant-producing strains were less frequent. Resistance to more than one trace metal was generally restricted to endophytes isolated from shoot tissues. The PGP characteristics found in an important number of the bacterial isolates obtained in this study could be particularly useful for exploiting the phytoremediation potential of C. striatus.

In this study we determine culturable microbial densities (total heterotrophs, ammonifiers, amylolytics and cellulolytics) and bacterial resistance to Co, Cr, and Ni in bulk and rhizosphere soils of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum and the excluder Dactylis glomerata from ultramafic sites (two populations in Northeast (NE) Portugal (Samil (S), Morais (M)) and one population in Northwest (NW) Spain (Melide (L)). The relationship between bioavailable metal concentrations (H2O-soluble) and microbial densities were analysed. Significant differences in microbial densities and metal-resistance were observed between the two species and their three populations. The hyperaccumulator showed higher microbial densities (except cellulolytics) and a greater rhizosphere effect, but this was only observed in S and M populations. These populations of A. serpyllifolium also showed selective enrichment of Ni-tolerant bacteria at the rhizosphere where Ni solubility was enhanced (densities of Ni-resistant bacteria were positively correlated with H2O-soluble Ni). These rhizobacteria could solubilise Ni in the soil and potentially improve phytoextraction strategies.