Cadmium Accumulation and Tolerance of Two Salix Genotypes Hydroponically Grown in Presence of Cadmium

Abstract

In general, according to previous studies, pioneer species do not require arbuscular mycorrhizal fungi (AMF) to increase their growth and survival in tropical systems. The aim of this study was to determine the dependence response to AMF of Heliocarpus appendiculatus, a pioneer species, at different phosphorus (P) levels. In a greenhouse experiment, H. appendiculatus seedlings were grown in pots with a sterile vermiculite-sand mixture (1:1). Two sets of pots were set up: One set was inoculated (150 spores per pot) with indigenous AMF from a tropical rain forest at “Los Tuxtlas” (Veracruz, Mexico); the other set was not inoculated. To each set, 0, 0.02, 0.2, and 2 g L^?1P was added. All pots were watered with 250 mL of nutrient solution. Mycorrhizal plants showed a higher total dry weight and relative growth rate in 0.02 g L^?1P concentration, while nonmycorrhizal plants responded positively at 0.2 g L^?1P; a decrease in plant responses at higher P levels was observed in both treatments. H. appendiculatus showed to have higher relative dependence at lower P concentration (≈50%). As levels of P increased, mycorrhizal colonization decreased. Successful growth of pioneer species during succession process may be improved if there is AMF content in soils, prior to disturbance.     

Molecular Monitoring of SRB Community Structure and Dynamics in Batch Experiments to Examine the Applicability of in situ Precipitation of Heavy Metals for Groundwater Remediation (15 pp)

Background, Aims and Scope   Sulfate-reducing bacteria (SRB) are known for their capacity to reduce and precipitate heavy metals (HM) as metal sulfides, offering the opportunity to create an in situ reactive zone for the treatment of heavy metal-contaminated groundwater, a process called in situ metal precipitation (ISMP). The applicability of the ISMP technology first has to be investigated at a laboratory scale before going into an on site application. The evaluation and optimization of the ISMP process is facilitated when physical/chemical analysis techniques are combined with molecular tools that specifically monitor the abundance, diversity and dynamics of the indigenous sulfate reducing microbial community. In this study, batch experiments were conducted in order to investigate the feasibility of ISMP as a groundwater remediation strategy for an industrial site contaminated with elevated levels of Zn, Cd, Co and Ni. Methods   The potential of different types of carbon source/ electron donor (lactate, acetate, methanol, ethanol, Hydrogen Release Compound?, molasses) to stimulate the sulfate reduction and metal precipitation activity of the naturally present (or indigenous) SRB community was explored. In addition, the effect of amending vitamin B12 and yeast extract was evaluated. The ISMP process was monitored by combining analytical analyzes of process parameters (SO42-concentration, heavy metal concentrations, pH, Eh) with molecular tools such as SRB subgroup and genus specific PCR, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis of clone sequences, based on either the 16S rRNA or the dsr (dissimilatory sulfite reductase) gene. Results and Discussion   The efficiency of different carbon-sources to stimulate the ISMP process followed the order HRC 〉 molasses 〉 methanol 〉 lactate 〉 ethanol 〉 acetate. Within 10 weeks, the highest sulfate and metal removal efficiencies ranged from 85% to 99%. Addition of yeast extract boosted the ISMP process, whereas vitamin B12 negligibly affected SRB activity. Analysis of the sulfate reducing population by SRB subgroup and genus specific PCR demonstrated that members of the genus Desulfosporosinus dominated in all batch tests, while 16S rDNA DGGE profiles additionally revealed the presence in the microbial communities of non-sulfate reducing bacteria within the family Clostridium and the -proteobacteria. The dsrB-based DGGE profiles allowed us to assess the diversity and dynamics of the sulfate reducing community and added to a better understanding of the effects of different batch conditions on the ISMP process. Remarkably, all dsrB sequences affiliated with the dsrB gene sequence cluster found in Desulfotomaculum, which received their xenologous dsrB gene from the -proteobacteria. Conclusions   The batch experiments, which aimed at stimulating the activities of the indigenous SRB communities, demonstrated that these communities were present and that their activities could be used to obtain efficient in situ precipitation of the contaminating heavy metals. This opens the possibility to test this concept in the future as an on site demonstration as part of the groundwater strategy for the heavy metal contaminated site. Although batch setups are suitable for preliminary feasibility studies for ISMP, they do not reflect the in situ situation where sulfate and heavy metal and metalloid polluted groundwater are supplied continuously. A sulfate reducing strain JG32A was isolated from whose 16S rRNA gene affiliated with the genus Desulfosporosinus, while its dsrB gene sequence clustered with Desulfotomaculum dsrB gene sequences, which received their xenologous dsr genes from -proteobacteria. Therefore we hypothesize that the batch experiments enrich members of the Desulfosporosinus genus that possess a non-orthologous dsrB gene. Recommendation and Perspective   The next step towards an on site pilot test for ISMP will be the setup of a series of column experiments, with process conditions that are selected based on the above mentioned results. This will allow to define optimal ISMP process conditions and to test its long-term efficacy and sustainability before going into an on site bioremediation application. By applying the described molecular tools together with physical-chemical analyzes, it can be investigated whether the same SRB community is enriched and which type of C-source is most effective in promoting and sustaining its growth and sulfate-reduction activity.     

Tolerance of Two Hydroponically Grown Salix Genotypes to Excess Zinc

Woody cuttings from two Salix genotypes (genotype I—clone LUC-31, Salix alba and genotype II- clone STOTT, Salix viminalis) were grown hydroponically for 14 days at increasing concentrations of Zn: control, 50, 100 or 150 μ M Zn. Genotype tolerance to excess zinc (Zn) was evaluated using a root elongation test. The changes in growth, Zn, iron (Fe), copper (Cu) and manganese (Mn) concentrations as well as photosynthetic performance were used as additional evaluation criteria. Photosynthetic pigments concentrations in Zn-exposed cuttings of genotype II decreased as compared with genotype I, which corresponded well with the higher leaf Zn accumulation, decreased Fe concentrations as well as lowered photosynthetic rate. Based on the indicators used, genotype I (Salix alba) was classified as more tolerant to excess Zn than genotype II (Salix viminalis).     

Rehabilitation of the nematode fauna in a phytostabilized,heavily zinc-contaminated,sandy soil

Background Aim and Scope  The Maatheide in Lommel, Belgium, is an extremely metal contaminated, sandy area where vegetation has disappeared over ca. 130 hectares due to the activities of a former pyrometallurgical zinc smelter. To reduce the environmental impact of this area a rehabilitation strategy had to be developed. Therefore, in the centre of this area, an experimental phytostabilization (grass) field of three hectares had been installed in 1990. After a grass cover had been established, the development of the nematode fauna in the phyto-stabilized soil was studied. Nematodes act at various levels in soil ecosystems: herbivorous species extract their food from plant roots, bacterivorous and fungivorous species feed on microbes, predatory species consume other nematodes, and omnivorous species have mixed diets. In a mature soil ecosystem that normally exercises its manifold functions, a diverse nematode fauna occurs, reflecting the intactness of the ecosystem. As such, this fauna is indicative of crop growth, vegetative diversity, organic matter decomposition, microbial activity and diversity, and the maturity of the soil ecosystem. Methods  A metal immobilizing soil amendment (beringite) and municipal waste compost (to improve the nutrient status and water-retaining capacity) were incorporated in the soil and metal tolerant ecotypes of grasses were sown. Soil samples for nematode analyses were taken four times. Results  As a result of the treatment, pH of the soil increased and the water extractable amount of Zn was strongly reduced. Grass growth revitalized the impoverished soil ecosystem, organisms as well as metabolic processes. The nematode fauna of the experimental field in the Maatheide has been studied since 1997 and the recovery of the various feeding guilds and taxa was checked again in 2002. Nematode densities and feeding guilds have normalized, with omnivores and predators as the last guilds to reappear. Up to 27 species with a current diversity were observed in the grass-covered experimental plot, but a number of ubiquitous species, present at a comparable site at some distance, remained absent. Conclusion  It can thus be concluded that rehabilitation of an impoverished soil ecosystem is possible in heavily contaminated soils by means of phytostabilization, but there are some limitations on rehabilitation, since a number of common nematode species remained absent. Further research should indicate if this absence is indicative of a loss of essential processes in the soil. Recommendation and Perspective. Phytostabilization of heavily zinc-contaminated, sandy soil also remediates impoverished soil ecosystems. In particular, the recovery of nematode feeding categories is indicative for the normalization of soil life. The absence/presence of a number of ubiquitous taxa should be checked again after some time to verify if recovery is completed.     

The application of molecular tools to follow up bioremediation

Journal of Soils and Sediments -     

Amendment-Induced Immobilization of Lead in a Lead-Spiked Soil: Evidence from Phytotoxicity Studies

Lead immobilization was evaluated on soils spiked with increasingconcentrations of Pb (as Pb-acetate) using the following soilamendments: bentonite, zeolite, cyclonic ash, compost, lime,steelshot, and hydroxyapatite. The immobilization efficacy of theamendments was evaluated according to the following criteria:Ca(NO₃)₂-extractable Pb as an indicator of Pbphytoavailability, morphological and enzymatic parameters of beanplants (Phaseolus vulgaris) as indicator of phytotoxicity, and Pb concentration in edible tissue of lettuce (Lactuca sativa). The lowest reductions in Ca(NO₃)₂-extractablesoil Pb occurred when bentonite and steelshot were applied. Phytotoxicity from application of steelshot was confounded by toxic amounts of Fe and Mn released from the by-product which killed the lettuce seedlings. Addition of zeolite induced poorplant growth independent of Pb concentration due to its adverseeffect on soil structure. Substantial reductions in Ca(NO₃)₂-extractable Pb were observed when cyclonic ash, lime, compost and hydroxyapatite were applied. In general,these amendments reduced Pb phytotoxicity concomitant with reduced Pb concentration in lettuce tissue. Cyclonic ash, limeand compost further improved plant growth and reduced oxidativestress at low soil Pb concentrations due to soil pH increase mitigating Al or Mn toxicity.     

Phytoremediation: European and American trends successes, obstacles and needs

Phytoremediation is an emerging technology based on the use of green plants to remove, contain, inactivate or destroy harmful environmental pollutants. Recent developments in Europe and the USA show that the approach is somewhat different on both sides of the Atlantic. In Europe, phytoremediation has more basically been research driven and, based on the outcomes, applications have been envisaged. By contrast, the approach in the USA is more application and experience driven. In spite of a growing track record of commercial success, more demonstration projects are needed to prove that phytoremediation is effective in order to rigorously measure its underlying economics, and to expand its applications. More fundamental research is also required to better understand the complex interactions between pollutants, soil, plant roots and micro-organisms at the rhizosphere level, to increase the bioavailability of pollutants, to fully exploit the metabolic diversity of plants and, thus, to successfully implement this new green technology.     

Successes and limitations of phytotechnologies at field scale: outcomes,assessment and outlook from COST Action 859

Purpose  

Many agricultural and brownfield soils are polluted and more have become marginalised due to the introduction of new, risk-based legislation. The European Environment Agency estimates that there are at least 250,000 polluted sites in the member states that require urgent remedial action. There is also significant volumes of wastewaters and dredged polluted sediments. Phytotechnologies potentially offer a cost-effective in situ alternative to conventional technologies for remediation of low to medium-contaminated matrices, e.g. soils, sediments, tailings, solid wastes and waters. For further development, social and commercial acceptance, there is a clear requirement for up-to-date information on successes and failures of these technologies based on evidence from the field. This review reports the outcomes from several integrated experimental attempts to address this at both field and market level in the 29 countries participating in COST Action 859.     

Potential of willow and its genetically engineered associated bacteria to remediate mixed Cd and toluene contamination

Purpose

The purpose of this study was to investigate if bacteria with beneficial properties that were isolated from willow growing on a metal-contaminated site can be further equipped with genes coding for a specific degradation pathway to finally obtain transconjugants that can be inoculated in willow to improve phytoremediation efficiency of mixed contaminations.

Materials and methods

Cultivable rhizosphere bacteria and root endophytes were isolated from willow (cv. Tora) growing on a metal-contaminated soil. All isolated strains were tested for their metal resistance and potential to promote plant growth. The two most promising strains were selected and were equipped with the pTOM plasmid coding for toluene degradation. Both transconjugants were inoculated separately and combined in willow cuttings exposed to mixed Cd–toluene contamination, and their effect on phytotoxicity, Cd uptake, and toluene evapotranspiration was evaluated.

Results and discussion

Many of the isolated strains tested positive for the production of siderophores, organic acids, and indole acetic acid (IAA) and showed increased Cd resistance. The Cd-resistant, siderophore-producing rhizosphere strain Burkholderia sp. HU001 and the Cd-resistant root endophyte Pseudomonas sp. HU002, able to produce siderophores, organic acids, and IAA, were selected as receptors for conjugation with the toluene-degrading Burkholderia vietnamiensis BU61 as a donor of the pTOM-TCE plasmid. Although inoculation with the individual transconjugant strains had no effect on plant growth and negatively affected Cd uptake, their combined inoculation resulted in an increased shoot biomass upon Cd–toluene exposure did not affect Cd uptake and strongly reduced evapotranspiration of toluene to the atmosphere.

Conclusions

In this study, inoculation of willow with a consortium of plant-associated bacteria equipped with the appropriate characteristics resulted in an improved phytoremediation of a mixed Cd–toluene contamination: the degradation of toluene was improved leading to a decreased toxicity and evapotranspiration, while Cd uptake and translocation were not affected.