Jana Boulet

Phytoremediation of contaminated soils and groundwater: lessons from the field.

Background, aim, and scopeThe use of plants and associated microorganisms to remove, contain, inactivate, or degrade harmful environmental contaminants (generally termed phytoremediation) and to revitalize contaminated sites is gaining more and more attention. In this review, prerequisites for a successful remediation will be discussed. The performance of phytoremediation as an environmental remediation technology indeed depends on several factors including the extent of soil contamination, the availability and accessibility of contaminants for rhizosphere microorganisms and uptake into roots (bioavailability), and the ability of the plant and its associated microorganisms to intercept, absorb, accumulate, and/or degrade the contaminants. The main aim is to provide an overview of existing field experience in Europe concerning the use of plants and their associated microorganisms whether or not combined with amendments for the revitalization or remediation of contaminated soils and undeep groundwater. Contaminations with trace elements (except radionuclides) and organics will be considered. Because remediation with transgenic organisms is largely untested in the field, this topic is not covered in this review. Brief attention will be paid to the economical aspects, use, and processing of the biomass.Conclusions and perspectivesIt is clear that in spite of a growing public and commercial interest and the success of several pilot studies and field scale applications more fundamental research still is needed to better exploit the metabolic diversity of the plants themselves, but also to better understand the complex interactions between contaminants, soil, plant roots, and microorganisms (bacteria and mycorrhiza) in the rhizosphere. Further, more data are still needed to quantify the underlying economics, as a support for public acceptance and last but not least to convince policy makers and stakeholders (who are not very familiar with such techniques).

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.

Short Rotation Coppice Culture of Willows and Poplars as Energy Crops on Metal Contaminated Agricultural Soils

Phytoremediation, more precisely phytoextraction, has been placed forward as an environmental friendly remediation technique, that can gradually reduce increased soil metal concentrations, in particular the bioavailable fractions. The aim of this study was to investigate the possibilities of growing willows and poplars under short rotation coppice (SRC) on an acid, poor, sandy metal contaminated soil, to combine in this way soil remediation by phytoextraction on one hand, and production of biomass for energy purposes on the other. Above ground biomass productivities were low for poplars to moderate for willows, which was not surprising, taking into account the soil conditions that are not very favorable for growth of these trees. Calculated phytoextraction efficiency was much longer for poplars than these for willows. We calculated that for phytoextraction in this particular case it would take at least 36 years to reach the legal threshold values for cadmium, but in combination with production of feedstock for bioenergy processes, this type of land use can offer an alternative income for local farmers. Based on the data of the first growing cycle, for this particular case, SRC of willows should be recommended.

Bacteria associated with oak and ash on a TCE-contaminated site: characterization of isolates with potential to avoid evapotranspiration of TCE

Background, aim, and scopeAlong transects under a mixed woodland of English Oak (Quercus robur) and Common Ash (Fraxinus excelsior) growing on a trichloroethylene (TCE)-contaminated groundwater plume, sharp decreases in TCE concentrations were observed, while transects outside the planted area did not show this remarkable decrease. This suggested a possibly active role of the trees and their associated bacteria in the remediation process. Therefore, the cultivable bacterial communities associated with both tree species growing on this TCE-contaminated groundwater plume were investigated in order to assess the possibilities and practical aspects of using these common native tree species and their associated bacteria for phytoremediation. In this study, only the cultivable bacteria were characterized because the final aim was to isolate TCE-degrading, heavy metal resistant bacteria that might be used as traceable inocula to enhance bioremediation.Materials and methodsCultivable bacteria isolated from bulk soil, rhizosphere, root, stem, and leaf were genotypically characterized by amplified rDNA restriction analysis (ARDRA) of their 16S rRNA gene and identified by 16S rRNA gene sequencing. Bacteria that displayed distinct ARDRA patterns were screened for heavy metal resistance, as well as TCE tolerance and degradation, as preparation for possible future in situ inoculation experiments. Furthermore, in situ evapotranspiration measurements were performed to investigate if the degradation capacity of the associated bacteria is enough to prevent TCE evapotranspiration to the air.Results and discussionBetween both tree species, the associated populations of cultivable bacteria clearly differed in composition. In English Oak, more species-specific, most likely obligate endophytes were found. The majority of the isolated bacteria showed increased tolerance to TCE, and TCE degradation capacity was observed in some of the strains. However, in situ evapotranspiration measurements revealed that a significant amount of TCE and its metabolites was evaporating through the leaves to the atmosphere.Conclusions and perspectivesThe characterization of the isolates obtained in this study shows that the bacterial community associated with Oak and Ash on a TCE-contaminated site, was strongly enriched with TCE-tolerant strains. However, this was not sufficient to degrade all TCE before it reaches the leaves. A possible strategy to overcome this evapotranspiration to the atmosphere is to enrich the plant-associated TCE-degrading bacteria by in situ inoculation with endophytic strains capable of degrading TCE.

Endophytes and their Potential to Deal with Co-contamination of Organic Contaminants (Toluene) and Toxic Metals (Nickel) during Phytoremediation

The aim was to investigate if engineered endophytes that are capable of degrading organic contaminants, and deal with or ideally improve uptake and translocation of toxic metals, can improve phytoremediation of mixed organic-metal pollution. As a model system, yellow lupine was inoculated with the endophyte Burkholderia cepacia VM1468 possessing (a) the pTOM-Bu61 plasmid, coding for constitutive toluene/TCE degradation, and (b) the chromosomally inserted ncc-nre Ni resistance/sequestration system. As controls, plants were inoculated with B. vietnamiensis BU61 (pTOM-Bu61) and B. cepacia BU72 (containing the ncc-nre Ni resistance/sequestration system). Plants were exposed to mixes of toluene and Ni. Only inoculation with B. cepacia VM1468 resulted in decreased Ni and toluene phytotoxicity, as measured by a protective effect on plant growth and decreased activities of enzymes involved in antioxidative defence (catalase, guaiacol peroxidase, superoxide dismutase) in the roots. Besides, plants inoculated with B. cepacia VM1468 and B. vietnamiensis BU61 released less toluene through the leaves than non-inoculated plants and those inoculated with B. cepacia BU72. Ni-uptake in roots was slightly increased for B. cepacia BU72 inoculated plants. These results indicate that engineered endophytes have the potential to assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation.

Contrasting colonization and plant growth promoting capacity between wild type and a gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar:

This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.

Bacteria associated with yellow lupine grown on a metal-contaminated soil: in vitro screening and in vivo evaluation for their potential to enhance Cd phytoextraction.

In order to stimulate selection for plant-associated bacteria with the potential to improve Cd phytoextraction, yellow lupine plants were grown on a metal-contaminated field soil. It was hypothesised that growing these plants on this contaminated soil, which is a source of bacteria possessing different traits to cope with Cd, could enhance colonisation of lupine with potential plant-associated bacteria that could then be inoculated in Cd-exposed plants to reduce Cd phytotoxicity and enhance Cd uptake. All cultivable bacteria from rhizosphere, root and stem were isolated and genotypically and phenotypically characterised. Many of the rhizobacteria and root endophytes produce siderophores, organic acids, indole-3-acetic acid (IAA) and aminocyclopropane-1-carboxylate (ACC) deaminase, as well as being resistant to Cd and Zn. Most of the stem endophytes could produce organic acids (73.8%) and IAA (74.3%), however, only a minor fraction (up to 0.7%) were Cd or Zn resistant or could produce siderophores or ACC deaminase. A siderophore- and ACC deaminase-producing, highly Cd-resistant Rhizobium sp. from the rhizosphere, a siderophore-, organic acid-, IAA- and ACC deaminase-producing highly Cd-resistant Pseudomonas sp. colonising the roots, a highly Cd- and Zn-resistant organic acid and IAA-producing Clavibacter sp. present in the stem, and a consortium composed of these three strains were inoculated into non-exposed and Cd-exposed yellow lupine plants. Although all selected strains possessed promising in vitro characteristics to improve Cd phytoextraction, inoculation of none of the strains (i) reduced Cd phytotoxicity nor (ii) strongly affected plant Cd uptake. This work highlights that in vitro characterisation of bacteria is not sufficient to predict the in vivo behaviour of bacteria in interaction with their host plants.