Soil amendment and rhizobacterial inoculation improved Cu phytostabilization, plant growth and microbial activity in a bench-scale experiment.

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 organic amendments, tolerant plants, and plant growth promoting rhizobacteria to reduce environmental risk and improve fertility of soils impacted by mining.

Beneficial traits of root endophytes and rhizobacteria associated with plants growing in phytomanaged soils with mixed trace metal-polycyclic aromatic hydrocarbon contamination.

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.

Effect of plant root exudates on the desorption of hexachlorocyclohexane isomers from contaminated soils.

Plants and their associated microbiota can have a significant impact on the behaviour of soil contaminants. Particularly, root exudation is one of the most important plant-associated processes in this respect, as it may have a substantial effect on the bioavailability of soil contaminants, specially of hydrophobic contaminants strongly sorbed by soil. The aim of the present study was to evaluate the effect of root exudates (natural and artificial) on the desorption of α-, ß-, δ- and γ-isomers of hexachlorocyclohexane (HCH) from contaminated soil, using batch experiments. Natural root exudates were obtained from Holcus lanatus plants growing in the same (contaminated) area. Fifteen compounds (mainly organic acids and phenolic compounds) usually found in root exudates were also tested, individually or as mixtures (1 and 10 mM). Both natural and artificial exudates favoured the mobilization of sorbed HCH in soil. The effect was highly significant for α-, ß- and γ-HCH isomers, for which the desorption rates increased by 23.0, 26.8 and 15.5% in the presence of natural root exudates and by 40.1, 25.9 and 25.6% in the presence of the artificial mixture (at 10 mM). The δ-HCH desorption rates increased by less than 10%. The effect of individual exudate components was very variable, but increased with the carbon content, reflecting the significance of hydrophobic interactions between the exudates and HCH molecules in the desorption of these last from soil. These findings indicate that plants may significantly influence the bioavailability of persistent contaminants, with major implications for improving phyto- and bioremediation procedures.

Modelling the ex situ bioremediation of diesel-contaminated soil in a slurry bioreactor using a hydrocarbon-degrading inoculant.

Bioremediation is a soil clean-up technique which exploits the metabolic capacity of microorganisms to degrade soil contaminants. A model was developed to simulate the ex situ bioremediation of a diesel-contaminated soil in a bio-slurry reactor inoculated with a diesel-degrading bacterial strain. Mass transfer processes involving desorption of diesel from soil to water and volatilization of diesel from water, and biodegradation by the bacterial inoculant were included in the model by using Weibull sigmoid kinetics and logistic/Monod kinetics respectively. Model parameters were estimated in batch-based abiotic and biodegradation experiments. Sensitivity analysis revealed the importance of maintaining a high bacterial density in the system for maximum bioremediation efficiency. The model was validated using a pilot bioreactor monitored for 528 h, which removed almost 90% of the diesel present in the system. The results revealed the capacity of the model to predict the bioremediation efficiency under different scenarios by adapting the input parameters to each system.

Improving the growth of Ni-hyperaccumulating plants in serpentine quarry tailings.

Phytomining techniques based on metal-hyperaccumulating plants can be implemented in serpentine quarry wastes for Ni recovery. However, strategies must be developed to overcome the unfavourable plant growth conditions that these substrates present and to optimize Ni yields. In this study, the Ni hyperaccumulators Alyssum serpyllifolium, Alyssum inflatum, and Alyssum bracteatum were evaluated for their Ni phytoextraction efficiency from quarry tailings. Effects of two organic amendments, composted municipal sewage sludge and cow manure, on plant growth and physiological status and Ni removal were determined. Organic amendments were incorporated at two addition rates (5% and 20% w/w). The best-performing hyperaccumulators were A. inflatum and A. serpyllifolium. Organic amendments improved plant biomass production, photosynthetic efficiency and nutrition, but reduced shoot Ni concentrations. However, the stimulation in biomass resulted in significantly enhanced Ni yields. The most promising results were found using low addition rates and after manure incorporation.

Assessing the agromining potential of Mediterranean nickel-hyperaccumulating plant species at field-scale in ultramafic soils under humid-temperate climate.

Nickel (Ni) agromining of ultramafic soils has been proposed as an eco-friendly option for metal recovery, which can also improve the fertility and quality of these low productive soils. The selection of adequate plant species and the analysis of their performance under the different climatic conditions are of interest for optimising the process and evaluating its full viability. A one-year field experiment was carried out to evaluate the viability of the two Ni-hyperaccumulating Mediterranean species, Alyssum murale and Leptoplax emarginata, for agromining purposes in ultramafic soils under a humid-temperate climate. Field plots of 50 m2 were established and the soil was fertilised with gypsum and inorganic NPK fertilisers prior to cropping. Alyssum murale produced a slightly higher Ni yield than L. emarginata, but Ni bioaccumulation was dependent on the plant phenological stage for both species, being maximal at mid-flowering (4.2 and 3.0 kg Ni ha-1, respectively). In both species, Ni was mainly stored in the leaves, especially in leaves of vegetative stems, but also in flowers and fruits in the case of L. emarginata. The main contributors to Ni yield of A. murale were flowering stems and their leaves, while for L. emarginata they were flowering stems and fruits. Implementing the agromining system increased soil nutrient availability, and modified microbial community structure and metabolic activity (due to fertilisation and plant root activity). The soil bacterial communities were dominated by Proteobacteria, Actinobacteria, Acidobacteria and Chloroflexi, and the agromining crops modified the relative abundance of some phyla (increasing Proteobacteria, Bacteroidetes and Nitrospirae and reducing Acidobacteria and Planctomycetes). Cultivating A. murale increased the densities of total culturable bacteria, while L. emarginata selected Ni-tolerant bacteria in its rhizosphere. In summary, both species showed great potential for their use in Ni agromining systems, although optimising soil and crop management practices could improve the phytoextraction efficiency.

Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation.

Bioremediation of polluted soils is a promising technique with low environmental impact, which uses soil organisms to degrade soil contaminants. In this study, 19 bacterial strains isolated from a diesel-contaminated soil were screened for their diesel-degrading potential, biosurfactant (BS) production, and biofilm formation abilities, all desirable characteristics when selecting strains for re-inoculation into hydrocarbon-contaminated soils. Diesel-degradation rates were determined in vitro in minimal medium with diesel as the sole carbon source. The capacity to degrade diesel range organics (DROs) of strains SPG23 (Arthobacter sp.) and PF1 (Acinetobacter oleivorans) reached 17-26% of total DROs after 10 days, and 90% for strain GK2 (Acinetobacter calcoaceticus). The amount and rate of alkane degradation decreased significantly with increasing carbon number for strains SPG23 and PF1. Strain GK2, which produced BSs and biofilms, exhibited a greater extent, and faster rate of alkane degradation compared to SPG23 and PF1. Based on the outcomes of degradation experiments, in addition to BS production, biofilm formation capacities, and previous genome characterizations, strain GK2 is a promising candidate for microbial-assisted phytoremediation of diesel-contaminated soils. These results are of particular interest to select suitable strains for bioremediation, not only presenting high diesel-degradation rates, but also other characteristics which could improve rhizosphere colonization.

Assessing phytotoxicity of trace element-contaminated soils phytomanaged with gentle remediation options at ten European field trials.

Gentle remediation options (GRO), i.e. in situ stabilisation, (aided) phytoextraction and (aided) phytostabilisation, were implemented at ten European sites contaminated with trace elements (TE) from various anthropogenic sources: mining, atmospheric fallout, landfill leachates, wood preservatives, dredged-sediments, and dumped wastes. To assess the performance of the GRO options, topsoil was collected from each field trial, potted, and cultivated with lettuce (Lactuca sativa L.) for 48days. Shoot dry weight (DW) yield, photosynthesis efficiency and major element and TE concentrations in the soil pore water and lettuce shoots were measured. GRO implementation had a limited effect on TE concentrations in the soil pore water, although use of multivariate Co-inertia Analysis revealed a clear amelioration effect in phytomanaged soils. Phytomanagement increased shoot DW yield at all industrial and mine sites, whereas in agricultural soils improvements were produced in one out of five sites. Photosynthesis efficiency was less sensitive than changes in shoot biomass and did not discriminate changes in soil conditions. Based on lettuce shoot DW yield, compost amendment followed by phytoextraction yielded better results than phytostabilisation; moreover shoot ionome data proved that, depending on initial soil conditions, recurrent compost application may be required to maintain crop production with common shoot nutrient concentrations.

Use of plant growth promoting bacterial strains to improve Cytisus striatus and Lupinus luteus development for potential application in phytoremediation.

Plant growth promoting (PGP) bacterial strains possess different mechanisms to improve plant development under common environmental stresses, and are therefore often used as inoculants in soil phytoremediation processes. The aims of the present work were to study the effects of a collection of plant growth promoting bacterial strains on plant development, antioxidant enzyme activities and nutritional status of Cytisus striatus and/or Lupinus luteus plants a) growing in perlite under non-stress conditions and b) growing in diesel-contaminated soil. For this, two greenhouse experiments were designed. Firstly, C. striatus and L. luteus plants were grown from seeds in perlite, and periodically inoculated with 6 PGP strains, either individually or in pairs. Secondly, L. luteus seedlings were grown in soil samples of the A and B horizons of a Cambisol contaminated with 1.25% (w/w) of diesel and inoculated with best PGP inoculant selected from the first experiment. The results indicated that the PGP strains tested in perlite significantly improved plant growth. Combination treatments provoked better growth of L. luteus than the respective individual strains, while individual inoculation treatments were more effective for C. striatus. L. luteus growth in diesel-contaminated soil was significantly improved in the presence of PGP strains, presenting a 2-fold or higher increase in plant biomass. Inoculants did not provoke significant changes in plant nutritional status, with the exception of a subset of siderophore-producing and P-solubilising bacterial strains that resulted in significantly modification of Fe or P concentrations in leaf tissues. Inoculants did not cause significant changes in enzyme activities in perlite experiments, however they significantly reduced oxidative stress in contaminated soils suggesting an improvement in plant tolerance to diesel. Some strains were applied to non-host plants, indicating a non-specific performance of their plant growth promotion. The use of PGP strains in phytoremediation may help plants to overcome contaminant and other soil stresses, increasing phytoremediation efficiency.

Enhanced Degradation of Diesel in the Rhizosphere of after Inoculation with Diesel-Degrading and Plant Growth-Promoting Bacterial Strains.

The association of plants and rhizospheric bacteria provides a successful strategy to clean up contaminated soils. The purpose of this work was to enhance diesel degradation in rhizosphere by inoculation with selected bacterial strains: a diesel degrader (D), plant growth-promoting (PGP) strains, or a combination (D+PGP). Plants were set up in pots with the A or B horizon of an umbric Cambisol (A and B) spiked with diesel (1.25%, w/w). After 1 mo, the dissipation of diesel range organics (DRO) with respect to = 0 (i.e., 1 wk after preparing the pots with the seedlings) concentration was significantly higher in inoculated than in noninoculated (NI) pots: The highest DRO losses were found in A D+PGP pots (close to 15-20% higher than NI) and in B D pots (close to 10% higher). The water-extractable DRO fraction was significantly higher at = 30 d (15-25%) compared with = 0 (<5%), probably due to the effects of plant root exudates and biosurfactants produced by the degrader strain. The results of this experiment reflect the importance of the partnerships between plants and bacterial inoculants and demonstrate the relevance of the effect of bacterial biosurfactants and plant root exudates on contaminant bioavailability, a key factor for enhancing diesel rhizodegradation. The association of lupine with D and PGP strains resulted in a promising combination for application in the rhizoremediation of soils with moderate diesel contamination.

Biochar and compost amendments enhance copper immobilisation and support plant growth in contaminated soils.

Contamination of soil with trace elements, such as Cu, is an important risk management issue. A pot experiment was conducted to determine the effects of three biochars and compost on plant growth and the immobilisation of Cu in a contaminated soil from a site formerly used for wood preservation. To assess Cu mobility, amended soils were analysed using leaching tests pre- and post-incubation, and post-growth. Amended and unamended soils were planted with sunflower, and the resulting plant material was assessed for yield and Cu concentration. All amendments significantly reduced leachable Cu compared to the unamended soil, however, the greatest reductions in leachable Cu were associated with the higher biochar application rate. The greatest improvements in plant yields were obtained with the higher application rate of biochar in combination with compost. The results suggest joint biochar and compost amendment reduces Cu mobility and can support biomass production on Cu-contaminated soils.

Leachability of volatile fuel compounds from contaminated soils and the effect of plant exudates: A comparison of column and batch leaching tests.

Volatile fuel compounds such as fuel oxygenates (FO) (MTBE and ETBE) and BTEX (benzene, toluene, ethylbenzene and xylene) are some of the most soluble components of fuel. Characterizing the leaching potential of these compounds is essential for predicting their mobility through the soil profile and assessing the risk of groundwater contamination. Plant root exudates can play an important role in the modification of contaminant mobility in soil-plant systems, and such effects should also be considered in leaching studies. Artificially spiked samples of A and B horizons from an alumi-umbric Cambisol were leached in packed-columns and batch experiments using Milli-Q water and plant root exudates as leaching agents. The leaching potential and rate were strongly influenced by soil-contaminant interactions and by the presence of root exudates. Organic matter in A horizon preferably sorbed the most non-polar contaminants, lowering their leaching potential, and this effect was enhanced by the presence of root exudates. On the other hand, the inorganic components of the B horizon, showed a greater affinity for polar molecules, and the presence of root exudates enhanced the desorption of the contaminants. Column experiments resulted in a more realistic protocol than batch tests for predicting the leaching potential of volatile organic compounds in dissimilar soils.

Influence of plant root exudates on the mobility of fuel volatile compounds in contaminated soils.

Vegetation and its associated microorganisms play an important role in the behaviour of soil contaminants. One of the most important elements is root exudation, since it can affect the mobility, and therefore, the bioavailability of soil contaminants. In this study, we evaluated the influence of root exudates on the mobility of fuel derived compounds in contaminated soils. Samples of humic acid, montmorillonite, and an A horizon from an alumi-umbric Cambisol were contaminated with volatile contaminants present in fuel: oxygenates (MTBE and ETBE) and monoaromatic compounds (benzene, toluene, ethylbenzene and xylene). Natural root exudates obtained from Holcus lanatus and Cytisus striatus and ten artificial exudates (components frequently found in natural exudates) were added to the samples, individually and as a mixture, to evaluate their effects on contaminant mobility. Fuel compounds were analyzed by headspace-gas chromatography-mass spectrometry. In general, the addition of natural and artificial exudates increased the mobility of all contaminants in humic acid. In A horizon and montmorillonite, natural or artificial exudates (as a mixture) decreased the contaminant mobility. However, artificial exudates individually had different effects: carboxylic components increased and phenolic components decreased the contaminant mobility. These results established a base for developing and improving phytoremediation processes of fuel-contaminated soils.

Phytoremediation of hexachlorocyclohexane (HCH)-contaminated soils using Cytisus striatus and bacterial inoculants in soils with distinct organic matter content.

The performance of Cytisus striatus in association with different microbial inoculant treatments on the dissipation of the insecticide hexachlorocyclohexane (HCH) was studied. Two soils with different organic matter (A and B soil) content were spiked with 0 or 65 mg HCH kg(-1). Plants were either not inoculated (NI), or inoculated with the endophyte Rhodococcus erythropolis ET54b and the HCH-degrader Sphingomonas sp. D4 separately or in combination (ET, D4 and ETD4). Unplanted pots were also established. HCH phytotoxicity was more pronounced in the B soil. Soil HCH concentrations in unplanted pots were similar to initial concentrations, whereas concentrations were reduced after plant growth: by 20% and 8% in A and B soil, respectively. Microbial inoculants also modified HCH dissipation, although effects were soil-dependent. Inoculation with the combination of strains (ETD4) led to a significant enhancement in HCH dissipation: up to 53% in the A soil and 43% in the B soil.

Identification of metalliferous ecotypes of Cistus ladanifer L. using RAPD markers.

The genetic diversity of Cistus ladanifer ssp. ladanifer (Cistaceae) growing on ultramafic and non-ultramafic (basic and schists) soils in the NE of Portugal was studied in order to identify molecular markers that could distinguish the metal-tolerant ecotypes of this species. Random Amplified Polymorphic DNA (RAPD) markers were used in order to estimate genetic variation and differences between populations. The RAPD dataset was analysed by means of a cluster analysis and an analysis of molecular variance (AMOVA). Our results indicate a significant partitioning of molecular variance between ultramafic and non-ultramafic populations of Cistus ladanifer, although the highest percentage of this variance was found at the intra-population level. Mantel's test showed no relationship between inter-population genetic and geographic distances. A series of RAPD bands that could be related to heavy metal tolerance were observed. The identification of such markers will enable the use of Cistus ladanifer in phytoremediation procedures.