This summary focuses on phytoremediation and heavy metals. See also – Rhizosphere and Phytoremediation – organic pollutants.
This section is aimed at compiling information that may be useful in planning phytoremediation experiments and related investigations of rhizosphere processes. Based on the author’s observation of good examples and shortcomings in the literature, some do’s and don’t’s are discussed and reference is made to relevant publications.
THE ROLE OF RHIZOSPHERE PROCESSES IN PHYTOREMEDIATION
Phytoremediation (plant-assisted bioremediation) refers to the use of plants to stabilise / immobilise contaminants in soils or sediments (Phytostabilisation / phytoimmobilisation), to remove organic pollutants via microbial degradation in the plant rhizosphere or by metabolising them after uptake in plant organs, to volatilise some metals and metalloids by the formation of volatile compounds by the action of rhizosphere microorganisms or after uptake in plant organs, or to extract metals / metalloids via uptake in harvestable plant parts, i.e. typically shoots.
Among the various factors that control the success of a particular phytoremediation process, the action of the plant rhizosphere has been pointed out as one of the keys in establishing phytoremediation crops under (typically) harsh conditions, as a means to control pollutant bioavailability and uptake in plants (phytoextraction, phytoimmobilisation), and to enhance the degradation or volatilisation of pollutants (Wenzel 2009). As far as rhizosphere action is concerned, the terms rhizoremediation, rhizodegradation and rhizovolatilisation have been in use as well. The role of rhizosphere action in phytoremediation has been covered in several reviews to which the reader is referred for further details (Anderson et al. 1993; Anderson and Coats 1994; Dzantor 2007; Fitz and Wenzel 2002; Kuiper et al. 2004; McGrath et al. 2001; Meharg and Cairney 2000; Newman and Reynolds 2004; Siciliano and Germida 1998; Wenzel 2009).
PHYTOREMEDIATION EXPERIMENTS – DO’S & DON’TS
It is not possible to provide a general protocol for conducting experiments on phytoremediation and related rhizosphere processes. Basically, the same methods can be used that are available in other plant-soil experiments and generally in rhizosphere research. The reader is referred to the comprehensive COST 631 Handbook of Methods Used in Rhizosphere Research (Luster and Finley 2006), available online at: http://www.rhizo.at/default.asp id=574&lid=2
However, based on my reading of numerous publications related to the topic I suggest it may be useful to consider the following aspects:
- Hydroponic versus soil-based experiments to evaluate the phytoremediation potential: Hydroponic studies are useful to screen and to study the response of potential phytoremediation crops in controlled conditions independent of specific soil properties (e.g. Dos Santos Utmazian et al., 2007a). However, results often deviate substantially from soil experiments and may be particularly misleading if the plants can not be easily grown in hydroponics (e.g. Salix caprea, a Cd and Zn hyperaccumulating willow tested for phytoextraction of these metals) (Dos Santos et al., 2007b). To evaluate the phytoremediation potential, it is highly mandatory to test candidate plant species for phytoremediation also in pot and field studies with real soil. Note that metal concentrations in plant tissues and translocation factors (i.e., the ratio of element concentration in shoot to element concentration in root) can deviate substantially from soil-grown plants. It is not justified to evaluate the phytoremediation potential of a given plant species solely based on hydroponic experiments.
- Choice of experimental soil: Soil-based phytoremediation experiments including several treatments often require a large amount of experimental soil. Careful selection of the experimental soils is a key to obtain useful data.
- Pollution level: The choice of the pollution level depends on the phytoremediation process under study and the pollution problem addressed. Phytoextraction is likely to work within reasonable time only for moderately polluted soil. Therefore, it is recommended to include soils with pollution levels above accepted intervention thresholds (well above background values) e.g. for agricultural soils but well below threshold above which require remediation because they are not acceptable even for non-agricultural or recreational activities (i.e., industrial land use). Phytostabilisation / phytoimmobilisation experiments may require another set of soils, potentially including also extremely high pollution levels. Useful criteria to select experimental soils include – apart from the total pollutant concentration – apparent distribution constants (Kd values) of the pollutants of interest and measurements of the kinetics of metal replenishment as provided by diffusive gradients in thin films (DGT) (Wenzel 2009).
- Other soil properties: The experimental soils should ideally cover the range of soil pH expected to occur in real world conditions. It is particularly important to consider the different behaviour of many pollutants, especially metals, in calcareous versus non-calcareous soils. Ideally, the experimental design should include both, and if possible, moderately acid and acid soil. Carbonate content and pH are not only strong modifiers of metal replenishment, solubility and speciation, but can also change adsorption to root surfaces and thus uptake by the plant. Conclusions on phytoremediation potential are therefore restricted to the (range of properties of the) experimental soil(s) used.
- Source of soil pollution: The source of contaminant of the experimental soil may be long-term pollution in the field (e.g. by mining and smelter activities, air pollution, waste application etc.) or may derive from spiking uncontaminated soils in the laboratory for the purpose of the experiment. Both approaches have their advantages and drawbacks. Field-contaminated soils are close to real world conditions in terms of pollutant bioavailability, i.e., due to gradual pollutant input over longer periods the pollutants are typically becoming partially immobilised and thus less bioavailable by various processes including diffusion in soil minerals and faces of organic matter, co-precipitation and occlusion, and in the case of organic pollutants also by microbial degradation. These phenomena are also known as ageing and are important drivers of natural attenuation. As pollutant bioavailability is a major control of the success of phytoremediation, it is vital to use either gradually polluted soil collected in the field or aged spiked soil material in order to obtain meaningful results.
- Soils gradually polluted in the field: Field-polluted soil does not need much specific preparation other than described in the section on soil preparation (below). The advantage of using such soil relates to the realistic representation of bioavailability. However, if the influence of the pollution level on the phytoremediation potential of a plant species is studied, spiking the pollutant to a uncontaminated soil may be superior as all other soil properties can be held constant.
- Spiked soil material: To obtain meaningful results with spiked soil material, it is vital to carefully mix small portions of spiked soil rather than add the pollutant to the whole batch at once. Smaller batches of spiked soil material can then be mixed with uncontaminated soil to obtain the final soil concentration in step-wise procedure (for details of such procedures see Langer et al., 2009; Unterbrunner et al., 2007). Apart from this, it is crucial to incubate the spiked soil material for at least several weeks or months and recommended to check the progress of contaminant ageing using appropriate extraction procedures (Unterbrunner et al., 2007). For metals, the process can be accelerated by increasing the temperature (compare Barrow, 1998, Langer et al., 2009) and alternating drying and rewetting cycles. Ideally, metal bioavailability, for instance represented by a suitable chemical extraction procedure (usually neutral salt extractions or 1 M NH4NO3) should approach a plateau before the soil can be used in the experiment. Otherwise, as an artefact, pollutant solubility and bioavailability will considerably change (decrease) during the experiment, especially during the initial weeks. As many phytoremediation experiments only last a few weeks or months, using spiked soil without appropriate ageing represents a major drawback.
- Soil preparation: Typically, soils are sieved using screens with mesh sizes in the range of 2-5 mm. Before sieving, soils are typically air-dried. Sieving soils with fine texture (loam and clay soils) may generate problems with water logging during the experiment. In this case, it may be required to mix the soil with quartz sand or perlite in order to achieve reasonable drainage of irrigation water. To minimise differences between replicates, careful homogenisation of the soil material is required by repeated mixing of smaller soil batches. The homogeneity should be confirmed by analysis of several samples taken from the homogenised soil material. After filling the soils in pots or other experimental devices (e.g. rhizoboxes), they should be allowed to re-equilibrate before planting (e.g. Unterbrunner et al. 2007). To this end, the soil should be moistened to obtain the soil moisture content targeted during the experiment (e.g., 80% of the field capacity) and left for at least some days, better weeks under the ambient conditions of the experiment.
- Fertilisation: Soil may be fertilised before or during the experiment to avoid nutrient deficiencies.
- Duration of experiment: Many phytoremediation experiments are rather short-term, i.e. few weeks only. This is a major shortcoming in assessing the phytoremediation potential of a plant species, especially if freshly spiked or insufficiently aged soil is used and if the soil is not allowed to re-equilibrate after re-moistening. Moreover, a short-term experiment does not allow predictions about changes of pollutant availability over extended periods. This is of specific interest in phytoextraction and phytodegradation / rhizodegradation studies as these processes are known to require at least several years before reaching remedial targets. Even more so, if perennial plants such as willows or poplars are used as the annual biomass production of these tree species increases substantially during the initial years after planting with related changes in metal concentrations and contents in plant tissues and removal from soil (e.g. Wieshammer et al., 2007).
- Choice of plant species: In the current literature, it is often claimed that an investigated plant has high potential for phytoextraction. However, the metal concentrations in harvestable plant parts (typically shoots) are often relatively low and in some cases even below that of the soil. When planning a phytoextraction experiment it is important to keep in mind that phytoextraction is going to work only if the bioconcentration factor (i.e., the ratio of metal concentration in harvestable plant tissues to that in the soil, on dry matter basis) is large and at least above 1. These requirements are met only by metal hyperaccumulator and some accumulator plants (see below). It is therefore not reasonable to conduct phytoextraction experiments with “normal” (crop) plants unless the phytoextraction process is substantially accelerated by other means such as application of chelating agents (e.g. EDTA). This also applies if bioaugmentation with rhizobacteria or mycorrhizal fungi is used as microorganisms can enhance phytoextraction only to some extent (perhaps by a factor of 2 (metal concentration) and 5 (metal content); see Lebeau et al. 2008) but not overcome the shortcomings of the plant. Note that the information provided in the review of Lebeau et al. (2008) is to a large extent based on experiments with short duration and soils that had been sterilised before inoculation and may not reflect real world conditions.
- Metal hyperaccumulator and accumulator plants: The literature reflects a continued misconception regarding the use of the term hyperaccumulation. For a plant species to be termed a metal hyperaccumulator it is not sufficient to exceed so called hyperaccumulation thresholds (typically 100 times the metal concentrations considered as normal in plants). The plant must also meet two other requirements: (1) a bioconcentration factor >1 and (2) a translocation factor >1 (see e.g. Baker et al. 2000; McGrath et al. 2001). These characteristics of metal hyperaccumulator plants are also essential for phytoextraction crops to achieve reasonable metal removal rates.
MICROBIAL INOCULATION STUDIES
To be developed
PHYTOREMEDIATION / RHIZOREMEDIATION OF ORGANIC POLLUTANTS
To be developed
MINIMAL DATASET SUGGESTED IN PUBLICATIONS ON PHYTOEXTRACTION
To be developed
Anderson TA, Coats JE (1994) Bioremediation through rhizosphere technology. ACS Symp Ser:563. Am Chem Soc, Washington, DC Anderson TA, Guthrie EA,Walton BT (1993) Bioremediation in the rhizosphere. Plant roots and associated microbes clean contaminated soil. Environ Sci Technol 27:2630-2636
Baker AJM, McGrath SP, Reeves RD, Smith JAC (2000) Metal hyperaccumulator plants: a review of the ecology and physiology of a biochemical resource for phytoremediation of metal-polluted soils. In Phytoremediation of Contaminated Soil and Water. Eds. Terry N, Bañuelos G, Vangronsveld J, pp 85-107. Lewis Publishers, Boca Raton, FL, USA.
Barrow, NJ (1998) Effects of time and temperature on the sorption of cadmium, zinc, cobalt, and nickel by a soil. Austr J Soil Res 36, 941-950.
Lebeau T, Braud A, Jézéquel K (2008) Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: A review. Environ Poll 153:497-522
Dos Santos Utmazian MN, Wieshammer G, Vega R, Wenzel WW (2007a) Hydroponic screening for metal resistance and accumulation of cadmium and zinc in twenty clones of willows and poplars. Environ Poll 148:155-165
Dos Santos Utmazian MN, Wenzel WW (2007b) Cadmium and zinc accumulation in willow and poplar species grown on polluted soils. J Plant Nutr Soil Sci 70:265-272
Dzantor EK (2007) Phytoremediation: the state of rhizosphere “engineering” for accelerated rhizodegradation of xenobiotic contaminants. J Chem Technol Biotechnol 82:228-232
Fitz WJ, Wenzel WW (2002) Arsenic transformations in the soil-rhizosphere-plant system: fundamentals and potential application to phytoremediation. J Biotechnol 99:259-278
Kuiper I, Lagendijk EL, Bloemberg GV, Lugtenberg BJJ (2004) Rhizoremediation: a beneficial plant-microbe interaction. Mol Plant Microbe Interact 17:6-15
Langer I, Krpata D, Fitz WJ, Wenzel WW, Schweiger PF (2009) Zinc accumulation potential and toxicity threshold determined for a metal-accumulating Populus canescens clone in a dose-response study. Environ Poll 157:2871-2877
Luster J, Finlay R (eds) (2006): Handbook of methods used in rhizosphere research. Birmensdorf, Swiss Federal Research Institute WSL. 536 pp
McGrath SP, Zhao FJ, Lombi E (2001) Plant and rhizosphere processes involved in phytoremediation of metalcontaminated soils. Plant Soil 232:207-214
Meharg AA, Cairney JWG (2000) Extomycorrhizas-extending the capabilities of rhizosphere remediation. Soil Biol Biochem 32:1475-1484
Newman LA, Reynolds CM (2004) Phytodegradation of organic compounds. Curr Opin Biotechnol 15:225-230
Siciliano SD, Germida JJ (1998) Mechanisms of phytoremediation: biochemical and ecological interactions between plants and bacteria. Environ Rev 6:65-79
Unterbrunner R, Wieshammer G, Hollender U, Felderer B, Wieshammer-Zivkovic M, Puschenreiter M et al (2007) Plant and fertiliser effects on rhizodegradation of crude oil in two soils with different nutrient status. Plant Soil 300:117-126
Wieshammer G, Unterbrunner R, Bañares García T, Zivkovic MF, Puschenreiter M, Wenzel WW (2007) Phytoextraction of Cd and Zn from agricultural soils by Salix ssp. And intercropping of Salix caprea and Arabidopsis halleri. Plant Soil 298:255-264
Wenzel WW (2009) Rhizosphere processes and management in plant-assisted bioremediation (phytoremediation) of soils. Plant Soil 321:385-408