Feasibility Of Labile Zn Phytoextraction Using Enhanced Tobacco And Sunflower: Results Of Five- And One-Year Field-Scale Experiments In Switzerland

Phytoextraction with somaclonal variants of tobacco and sunflower mutant lines (non-GMs) with enhanced metal uptake and tolerance can be a sustainable alternative to conventional destructive decontamination methods, especially for stripping bioavailable zinc excess in topsoil. The overall results of a 5-year time series experiment at field scale in north-eastern Switzerland confirm that the labile Zn pool in soil can be lowered by 45-70%, whereas subplots without phytoextraction treatment maintained labile Zn concentrations. In 2011, the phytoextraction experiment site was enlarged by a factor of 3, and the labile 0.1M NaNO3 extractable Zn concentration in the soil was reduced up to 58% one period after harvest. A Mass Balance Analysis confirmed soil Zn decontamination in line with plant Zn uptake. The plants partially take Zn from the non-labile pool of the total. The sustainability of Zn phytoextraction in subplots that no longer exceed the Swiss trigger value is now assessed over time. In contrary to the phytoextraction of total soil Zn which needs a long cleaning up time, the bioavailable Zn stripping is feasible within a few years period.

Phytoremediation of polluted soils: hype, hope and facts

Jean-Paul Schwitzguebel

Different plant species, ecotypes, varieties or cultivars can be selected, tailored and harnessed for the remediation of polluted soils, sites and brownfields, as well as for the phytotreatment of domestic or industrial wastewater. Several approaches are possible, but phytoremediation does always rely on in situ extraction, detoxification or immobilization of xenobiotic chemicals or trace elements. Phytoremediation has a strong potential as a natural, solar-energy driven approach for soils and sites moderately polluted over large surfaces, if the most appropriate plants have been carefully selected and the adequate agronomic methods are applied to manage the phytoavailability of contaminants. It can already offer a sustainable and less expensive alternative to soils or sites restoration, as compared to traditional physico-chemical techniques, even if the time required reaching the target end-points is often a severe drawback. Depending on the type of soil to be treated, as well as on the pollutants, their concentration and ageing, different approaches can be considered and will be presented with some examples of successful applications, but also highlighting their limitations: • Phytoextraction - uptake of trace elements into roots, then translocation into shoots, followed by the harvest and destruction of the contaminated plants, with possible recycling strategies to recover metals from biomass or ash. Two types of plants can be used: plants able to hyperaccumulate one or several trace elements, but producing low biomass; or plants accumulating moderate amounts of metals or metalloids, but producing high biomass. • Phytostabilisation - based on the immobilization of inorganic contaminants by adsorption to plant roots or soil particles, or precipitation in the root area, thus preventing their migration in soil, groundwater or air, and decreasing erosion, runoff and leaching. For large contaminated areas phytostabilisation is probably the only reasonable option to restore ecosystems. The most effective approach is the use of indigenous plant species, after the addition of appropriate soil amendments. • Phytodegradation - exploiting the huge potential and biodiversity of plant metabolism to detoxify, transform or degrade xenobiotic organic chemicals, like pesticides, explosives or dyes. • Phytostimulation - enhanced microbial metabolism by root exudates; plant/microbial interactions are important for such rhizospheric processes, especially for very hydrophobic compounds. In spite of a growing track record of commercial success, more large-scale trials are needed to definitively prove that phytoremediation is effective and applicable in many different situations, to rigorously assess its economics and to convince stakeholders and local authorities that it is a reality, not a dream. However, to be recognized as a mature and efficient technology, phytoremediation still needs significant progress, even if it is the focus of intensive research and development, from numerous laboratory basic studies and greenhouse experiments to successful demonstration sites. More fundamental research is still required to better understand and control the numerous metabolic processes occurring in the plant and its different organs, as well as the complex interactions between pollutants, soil, plant roots, and the rhizospheric and endophytic microorganisms. However, experimental conditions can modify plant physiology and biochemistry, and it is not obvious if results obtained with young and small plantlets under well-defined laboratory conditions can be extended to real full-scale fields. If the small-scale experimentation in laboratory or in greenhouse is highly valuable to better understand the basic plant molecular and biochemical mechanisms involved in the uptake, translocation, detoxification and sequestration of toxic trace elements and xenobiotic compounds, their direct transposition in the field is somewhat questionable. On the other hand, short-term experimental conditions in small pots used to cultivate plantlets do not allow the normal growth and development of roots and shoots, and thus impose stress which can affect the performance of plants and the efficiency of phytoremediation. In the field the following factors do play a major role, but they cannot be assessed under laboratory conditions: soil heterogeneity, meteorological uncertainty, total concentrations and spatial variability of contaminants, texture and depth of soil layers, nutrient and water availability, problems with the homogeneous input of amendments, differences in rooting depth and density, variability of soil moisture, impacts of pests, pathogens and herbivores. Molecular and biochemical mechanisms of plant adaptation to real stress conditions are still poorly understood and signalling pathways involved remain unclear. However, adaptation to environmental changes is crucial for plants and thus for the efficiency of phytoremediation. Most studies deal either with organic xenobiotics or with trace elements, whereas in the field, a mixed pollution is generally observed. Information on the probable antagonistic or possible synergistic effects of mixed pollutants on their mutual accumulation and detoxification remains however scarce and further studies are urgently needed to better understand the mechanisms involved and improve phytoremediation efficiency. On the other hand, the bioavailable fraction of soil contaminants, but not the total concentration, should be considered as the most important one from an ecological, toxicological and health standpoint, and is determined by the chemical properties of the pollutant, soil characteristics, ageing processes and biota behaviour. Ageing of pollutants usually decreases bioavailability, but root exudates, root-induced rhizosphere changes, mycorrhizal fungi and rhizospheric bacteria play a major role in the dynamics and the ability of pollutants to enter into plants; spreading of pollutants to the surroundings and groundwater can also be affected. The bioavailability of contaminants and their uptake by crops are also essential parameters for establishing risk-based regulatory guidelines and enhancing food safety. Finally, the economics of phytoremediation of organic pollutants is generally favourable, but cost is an acute problem for the treatment of trace elements. Ideally, plants should produce biomass with added value or should be used to recover valuable products. The fate of plant biomass after harvest should thus be considered in the context of biorefineries. Lignocellulosic feedstock can be used for fibers or biofuel production, after extraction of added-value products like oil for lubricants, fragrances or fine chemicals. Incineration of plant material should allow the selective recovery of metals from ash after energy production, thus providing an economically sound process, depending on the type and concentration of metal, as well as on the market price of the trace elements under consideration. With such approaches, marginal contaminated soils or brownfields can be cleaned-up, whereas owners are offered valuable sources of income instead of set-aside scenarios. In such a situation, time is no longer a limiting factor, and the term phytomanagement should be more appropriate than phytoremediation

2014

Analysis of microbial community structures and functions in heavy metal-contaminated soils using molecular methods

Fabienne Gremion

The contamination of agricultural land and groundwater by heavy metals is essentially linked to human activities. A major problem with heavy metals is that they cannot be biodegraded and therefore reside in the environment for long periods of time if they are not removed. Thus, depending of the kind and depth of contamination, different remediation techniques were developed. One of these methods, called "phytoextraction", uses the ability of so-called hyperaccumulating plants to extract high amounts of heavy metals. Accumulation of heavy metals in the environment is a serious concern for animal and human health. At the microscopic scale, heavy metals may have also deleterious effects on bacteria which are the key-players of the different nutrient turnovers in soils. Consequently, ecosystem functioning can be seriously perturbed and the long-term soil fertility may be threatened. The recent development of molecular biology greatly contributed to the discovery of the microbial diversity and its function in the soil. However, to date only a small number of studies used molecular methods to investigate the impacts of heavy metals on the bacterial community. In this thesis, a pot experiment was conducted under controlled conditions with one hyperaccumulating plant (Thlaspi caerulescens) grown in two different soils, a long-term and an artificially heavy metal-contaminated soil. The impact of heavy metals on the microbial community was then investigated with several molecular methods. Moreover, the decrease of the bioavailable heavy metals concentrations in soil due to plant uptake allowed to study the consequences on bacterial community structure and function. Based on the 16S ribosomal RNA and the corresponding gene (16S rDNA), four clone libraries were constructed to retrieve information on the structure of the microbial community and the potentially active part of the microbial community in the rhizosphere of Thlaspi caerulescens grown during three months in a long-term contaminated soil. The data obtained with the two clone libraries (rRNA and rDNA) from the rhizosphere of Thlaspi caerulescens were compared with the bulk soil data to identify any effect of the plant on the soil microbial community structure. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, α-Proteobacteria, β-Proteobacteria, Acidobacteria and the Planctomycetales. The taxa dominating the bacterial community structure in the bulk soil also dominated the rhizosphere community, indicating that the plant did not exert a major influence on the overall bacterial diversity. However, all dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries as compared to the rDNA libraries. On the contrary, sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA. Seventy per cent of these clone sequences were related to two subgroups of the Rubrobacteria, which was an indication that this group of bacteria was probably metabolically active in heavy metal-contaminated soils. Fluorescence in situ hybridization (FISH) was used for the in situ detection and quantification of selected bacterial groups previously detected in the clone libraries from the rhizosphere of Thlaspi caerulescens. By applying the most general probe EUB338, only 20% of the total rhizosphere microbial community could be detected. Based on this result, it was difficult to conclude with certitude which were the most dominant bacteria in the rhizosphere. However, despite this low detection rate, it was possible to detect the major groups present in the rhizosphere clone libraries using group-specific oligonucleotide probes. As part of our sequences were affiliated to two emerging bacterial groups, the Acidobacteria and the Rubrobacteria, two new probes were designed, Acido228 (specific for the subgroup 1 of the Acidobacterium division) and Rubro198 (specific for the all Rubrobacteria subclass), for the detection of these microorganisms. These two probes were first checked for their specificity with pure cultures and finally applied in the rhizosphere soil allowing for the first time the detection of these organisms in situ. Finally, the impact of heavy metals and a subsequent one-year phytoextraction with Thlaspi caerulescens on the soil microbial community was investigated in an artificially heavy metal-contaminated soil. All the different molecular and culture-dependent techniques used, denaturing gradient gel electrophoresis (DGGE), community level physiological profile (CLPP) and potential ammonium-oxidation measurement showed that the heavy metal addition induced drastic changes in the bacterial community. Moreover, the analysis of the different bacterial DGGE patterns (Bacteria, β-Proteobacteria and ammonia-oxidising bacteria) obtained during this experiment showed that one-year phytoremediation was not sufficient to recover the initial community present in the non-contaminated soil. However, with the CLPP analysis, it was possible to detect a stimulating effect of the plant on a part of the microbial community in both contaminated and non-contaminated soils. The most obvious result was obtained in the contaminated soil where the number of substrates metabolised increased significantly in the presence of the plant as compared to the unplanted contaminated samples. The measurement of the potential ammonium-oxidation was used as a criterion for soil quality. Although this test showed that the ammonia-oxidising bacteria were significantly stimulated in the planted non-contaminated soil samples, the positive effect of the plant on these bacteria was not sufficient to overcome the inhibition induced by the presence of the heavy metals in the contaminated soil, even after one year phytoremediation. To conclude, molecular methods in combination with culture-dependent techniques have proven in this study to be very useful for the detection of the changes induced by the heavy metals in the structure and the function of the microbial community. Moreover, the molecular techniques contributed to the identification of bacteria which could be potentially used for the bioremediation of contaminated soils thus offering new perspectives of investigation and technology development.

EPFL2003

Increased tolerance of sunflower mutant seedlings to Cd and Zn in hydroponic culture

Erika Nehnevajova, Jean-Paul Schwitzguebel

Cadmium and zinc show similarities in their chemistry, geochemistry and environmental properties. Whereas Cd is a phytotoxic non-essential element, Zn is an essential trace element for plant growth and development. Zinc plays a fundamental role in several key cellular functions, such as protein metabolism, gene expression, chromatin structure, photosynthetic carbon metabolism and indole acetic acid metabolism (Vallee and Falchuk, 1993; Marschner, 1995; Bonnet et al., 2000; Cakmak and Braun, 2001). Zinc is also an important component of many enzymes and proteins (Broadley et al., 2007). Cadmium can readily inhibit most of the Zn-dependent processes by binding to the membrane and to enzyme active sites, thus inactivating their functions (Aravind and Prasad, 2005). However, increasing Zn concentrations can replace such wrongly bound Cd (Van Assche and Clijsters, 1990; Shaw et al., 2004). Cadmium has been shown to cause many morphological, physiological, biochemical and structural changes in plants, such as growth inhibition, reduction in photosynthesis, transpiration or water imbalance (Sanità di Toppi and Gabbrielli, 1999; Schützendübel et al., 2001; Benavides et al., 2005; Mishra et al., 2006). Cadmium and high zinc concentration affect plant growth and metabolism, but the intensity of their toxic effects depends on plant species and the way and duration of metal exposure. Plants called hyperaccumulators are able to tolerate and accumulate extraordinary levels of trace elements in their above-ground tissue without developing any toxicity symptoms (Baker and Brooks, 1989; Reeves and Baker, 2000). Their high metal accumulation capability makes them interesting for the decontamination technique called phytoextraction, which uses plants for environmental clean-up (Salt et al., 1998). However, their low biomass production strongly limits the real application of this soil decontamination strategy. The ideal plant for phytoextraction should provide both a high biomass and a high tolerance and metal accumulation (Schwitzguébel et al., 2002). High biomass producing plants with improved tolerance to trace elements and with an enhanced metal accumulation capacity should be good candidates for metal removal from contaminated area. Previous studies have shown enhanced tolerance and metal accumulation properties in transgenic tobacco plants (Pomponi et al., 2006; Gorinova et al., 2007; Wojas et al., 2008). However, the main disadvantage of genetic engineering is still public acceptance and free-land application. In vitro breeding techniques have also been successfully used to obtain tobacco lines with a considerably enhanced Cd, Zn and Cu tolerance and metal accumulation (Herzig et al., 1997; Guadagnini et al., 1999; Herzig et al., 2003). In addition, the chemical mutagen ethyl methane sulphonate has been used to develop new sunflower lines with a significantly enhanced biomass and metal uptake on a metal contaminated field (Nehnevajova et al., 2007, 2009). Although these field experiments have revealed the potential of sunflower mutants for metal phytoextraction, their tolerance to trace elements and the effect of Cd and Zn on growth and photosynthetic pigments were not studied within previous free-land experimentation. The aim of the present work was thus to assess the tolerance to Cd and excess Zn of selected sunflower mutant lines with improved biomass and metal uptake capacity, by measuring different physiological parameters. For such a purpose, sunflower seedlings of eight selected mutant lines were cultivated under hydroponic conditions in the presence and in the absence of Cd and Zn to study the effect of these trace elements on (1) growth parameters, such as root elongation, root and shoot dry mass; (2) chlorophyll content; (3) carotenoid content; (4) Cd and Zn accumulation capacity of sunflower mutants; and (5) a possible correlation between Cd and Zn accumulation in sunflower shoots and roots.

2009