Endophyte-enhanced phytoremediation of DDE-contaminated using Cucurbita pepo: A field trial.

Although the use of the pesticide 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) was banned from the mid-1970s, its most abundant and recalcitrant degradation product, 2,2-bis(p-chlorophenyl)-1,1-dichloro-ethylene (DDE), is still present in terrestrial and aquatic ecosystems worldwide. Zucchini (Cucurbita pepo ssp. pepo) has been shown to accumulate high concentrations of DDE and was proposed for phytoremediation of contaminated soils. We performed a field trial covering a full plant life cycle. C. pepo plants inoculated with the plant growth-promoting endophytic strains Sphingomonas taxi UH1, Methylobacterium radiotolerans UH1, Enterobacter aerogenes UH1, or a consortium combining these 3 strains were grown on a DDE-contaminated field for 100 days. The effects of these inoculations were examined at both the plant level, by evaluating plant weight and plant DDE-content, and at the level of the cultivable and total endophytic communities. Inoculating plants with S. taxi UH1, M. radiotolerans UH1, and the consortium increased plant weight. No significant effects of the inoculations were observed on DDE-concentrations in plant tissues. However, the amount of DDE accumulated by C. pepo plants per growing season was significantly higher for plants that were inoculated with the consortium of the 3 strains. Therefore, inoculation of C. pepo with DDE-degrading endophytes might be promising for phytoremediation applications.

Brownfields to green fields: Realising wider benefits from practical contaminant phytomanagement strategies.

Gentle remediation options (GROs) are risk management strategies or technologies involving plant (phyto-), fungi (myco-), and/or bacteria-based methods that result in a net gain (or at least no gross reduction) in soil function as well as effective risk management. GRO strategies can be customised along contaminant linkages, and can generate a range of wider economic, environmental and societal benefits in contaminated land management (and in brownfields management more widely). The application of GROs as practical on-site remedial solutions is still limited however, particularly in Europe and at trace element (typically metal and metalloid) contaminated sites. This paper discusses challenges to the practical adoption of GROs in contaminated land management, and outlines the decision support tools and best practice guidance developed in the European Commission FP7-funded GREENLAND project aimed at overcoming these challenges. The GREENLAND guidance promotes a refocus from phytoremediation to wider GROs- or phyto-management based approaches which place realisation of wider benefits at the core of site design, and where gentle remediation technologies can be applied as part of integrated, mixed, site risk management solutions or as part of "holding strategies" for vacant sites. The combination of GROs with renewables, both in terms of biomass generation but also with green technologies such as wind and solar power, can provide a range of economic and other benefits and can potentially support the return of low-level contaminated sites to productive usage, while combining GROs with urban design and landscape architecture, and integrating GRO strategies with sustainable urban drainage systems and community gardens/parkland (particularly for health and leisure benefits), has large potential for triggering GRO application and in realising wider benefits in urban and suburban systems. Quantifying these wider benefits and value (above standard economic returns) will be important in leveraging funding for GRO application and soft site end-use more widely at vacant or underutilized sites.

Cadmium-induced and trans-generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana.

Trans-generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well-known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd-exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd-exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.

Exposure of Cucurbita pepo to DDE-contamination alters the endophytic community: A cultivation dependent vs a cultivation independent approach.

2,2-bis(p-chlorophenyl)-1,1-dichloro-ethylene (DDE) is the most abundant and persistent degradation product of the pesticide 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) and is encountered in contaminated soils worldwide. Both DDE and DDT are classified as Persistent Organic Pollutants (POPs) due to their high hydrophobicity and potential for bioaccumulation and biomagnification in the food chain. Zucchini (Cucurbita pepo ssp. pepo) has been shown to accumulate high concentrations of DDE and other POPs and has been proposed as a phytoremediation tool for contaminated soils. The endophytic bacteria associated with this plant may play an important role in the remedial process. Therefore, this research focuses on changes in endophytic bacterial communities caused by the exposure of C. pepo to DDE. The total bacterial community was investigated using cultivation-independent 454 pyrosequencing, while the cultivable community was identified using cultivation-dependent isolation procedures. For both procedures, increasing numbers of endophytic bacteria, as well as higher diversities of genera were observed when plants were exposed to DDE. Several bacterial genera such as Stenotrophomonas sp. and Sphingomonas sp. showed higher abundance when DDE was present, while, for example Pseudomonas sp. showed a significantly lower abundance in the presence of DDE. These findings suggest tolerance of different bacterial strains to DDE, which might be incorporated in further investigations to optimize phytoremediation with the possible use of DDE-degrading endophytes.

Comparison between cultivated and total bacterial communities associated with Cucurbita pepo using cultivation-dependent techniques and 454 pyrosequencing.

Endophytic bacteria often have beneficial effects on their host plants that can be exploited for bioremediation applications but, according to the literature, only 0.001-1% of all endophytic microbes should be cultivable. This study compared the cultivated endophytic communities of the roots and shoots of Cucurbita pepo with the total endophytic communities as determined by cultivation-dependent techniques and 454 pyrosequencing. The ten most abundant taxa of the total communities aligned well with the cultivated taxa; however, the abundance of these taxa in the two communities differed greatly. Enterobacter showed very low presence in the total communities, whereas they were dominantly present in the cultivated communities. Although Rhizobium dominated in total root and shoot communities, it was poorly cultivable and even then only in growth media containing plant extract. Since endophytes likely contribute to plant-growth promotion, cultivated bacterial strains were tested for their plant-growth promoting capabilities, and the results were correlated with their abundance in the total community. Bacillus and Pseudomonas showed promising results when considering cultivability, abundance in the total community and plant-growth promoting capability. This study demonstrated that, although a limited number of bacterial genera were cultivable, current cultivation-dependent techniques may be sufficient for further isolation and inoculation experiments that aim to improve phytoremediation efficiency.

Optimization of isolation and cultivation of bacterial endophytes through addition of plant extract to nutrient media.

Many endophytes have beneficial effects on plants and can be exploited in biotechnological applications. Studies hypothesize that only 0.001-1% of all plant-associated bacteria are cultivable. Moreover, even after successful isolations, many endophytic bacteria often show reduced regrowth capacity. This research aimed to optimize isolation processes and culturing these bacteria afterwards. We compared several minimal and complex media in a screening. Beside the media themselves, two gelling agents and adding plant extract to media were investigated to enhance the number and diversity of endophytes as well as the growth capacity when regrown after isolation. In this work, 869 medium delivered the highest numbers of cultivable bacteria, as well as the highest diversity. When comparing gelling agents, no differences were observed in the numbers of bacteria. Adding plant extract to the media lead to a slight increase in diversity. However, when adding plant extract to improve the regrowth capacity, sharp increases of viable bacteria occurred in both rich and minimal media.

The effect of long-term Cd and Ni exposure on seed endophytes of Agrostis capillaris and their potential application in phytoremediation of metal-contaminated soils.

We examined whether long-term Cd exposure leads to beneficial changes in the cultivable endophytic bacteria present in the seeds of Agrostis capillaris. Therefore the cultivable seed endophytes of Agrostis capillaris growing on a long-term Cd/Ni-contaminated plot (Cd/Ni seeds) were compared with those originating from a non-contaminated plot (control seeds). We observed plant- and contaminant-dependent effects on the population composition between control and Cd/Ni seeds. Also differences in phenotypic characteristics were found: endophytes from Cd/Ni seeds exhibited more ACC deaminase activity and production of siderophores and IAA, while endophytes from control seeds, very surprisingly, showed more metal tolerance. Finally, the 3 most promising seed endophytes were selected based on their metal tolerance and plant growth promoting potential, and inoculated in Agrostis capillaris seedlings. In case of non-exposed plants, inoculation resulted in a significantly improved plant growth; after inoculation of Cd-exposed plants an increased Cd uptake was achieved without affecting plant growth. This indicates that inoculation of Agrostis with its seed endophytes might be beneficial for its establishment during phytoextraction and phytostabilisation of Cd-contaminated soils.

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.

Bacterial communities associated with Brassica napus L. grown on trace element-contaminated and non-contaminated fields: a genotypic and phenotypic comparison.

Cultivable bacterial strains associated with field-grown Brassica napus L. (soil, rhizosphere and roots) from a trace elements (Cd, Zn and Pb) contaminated field and a non-contaminated control field were characterized genotypically and phenotypically. Correspondence analysis of the genotypic data revealed a correlation between soil and rhizosphere communities isolated from the same field, indicating that local conditions play a more important role in influencing the composition of (rhizosphere) soil bacterial communities than root exudates. In contrast, endophytic communities of roots showed a correlation between fields, suggesting that plants on the two fields contain similar obligate endophytes derived from a common seed endophytic community and/or can select bacteria from the rhizosphere. The latter seemed not very likely since, despite the presence of several potential endophytic taxa in the rhizosphere, no significant correlation was found between root and rhizosphere communities. The majority of Cd/Zn tolerant strains capable of phosphorus solubilization, nitrogen fixation, indole-3-acetic acid production and showing 1-aminocyclopropane-1-carboxylate deaminase capacity were found in the rhizosphere and roots of plants growing on the contaminated field.

Changes in the population of seed bacteria of transgenerationally Cd-exposed Arabidopsis thaliana.

Plant-associated bacteria can have beneficial effects on the growth and health of their host. Nevertheless, the role of endophytic bacteria present in seeds has not been investigated in depth. In this study, the cultivable endophytic population of seeds from Arabidopsis thaliana exposed to 2 µm cadmium for several generations (Cd seeds) was compared with a population isolated from seeds of plants that were never exposed to Cd (control seeds). We observed obvious differences between the two types of seed concerning genera present and phenotypic characteristics of the different isolates. Sinorhizobium sp. and Micrococcus sp. were only found in control seeds, while Pseudomonas sp., Bosea sp. and Paenibacillus sp. were only found in Cd seeds. Sphingomonas sp., Rhizobium sp., Acidovorax sp., Variovorax sp., Methylobacterium sp., Bacillus sp. and Staphylococcus sp. occurred in varying numbers in both types of seed. Metal tolerance and 1-aminocyclopropane-1-carboxylate deaminase activity were predominantly found in strains isolated from Cd seeds, while the production of siderophores, indole-3-acetic acid and organic acids was more prevalent in endophytes isolated from control seeds. These data support the hypothesis that certain endophytes are selected for transfer to the next generation and that their presence might be important for subsequent germination and early seedling development.

Groundwater remediation and the cost effectiveness of phytoremediation.

In 1999, phytoremediation was applied at the site of a Belgian car factory to contain two BTEX plumes. This case study evaluates the cost effectiveness of phytoremediation compared to other remediation options, applying a tailored approach for economic evaluation. Generally, when phytoremediation is addressed as being cost effective, the cost effectiveness is only determined on an average basis. This study however, demonstrates that an incremental analysis may provide a more nuanced conclusion. When the cost effectiveness is calculated on an average basis, in this particular case, the no containment strategy (natural attenuation) has the lowest cost per unit mass removed and hence, should be preferred. However, when the cost effectiveness is determined incrementally, no containment should only be preferred if the value of removing an extra gram of contaminant mass is lower than 320 euros. Otherwise, a permeable reactive barrier should be adopted. A similar analysis is provided for the effect determined on the basis of remediation time. Phytoremediation is preferred compared to 'no containment' if reaching the objective one year earlier is worth 7 000 euros.

Phytoextraction of toxic metals: a central role for glutathione.

Phytoextraction has a promising potential as an environmentally friendly clean-up method for soils contaminated with toxic metals. To improve the development of efficient phytoextraction strategies, better knowledge regarding metal uptake, translocation and detoxification in planta is a prerequisite. This review highlights our current understanding on these mechanisms, and their impact on plant growth and health. Special attention is paid to the central role of glutathione (GSH) in this process. Because of the high affinity of metals to thiols and as a precursor for phytochelatins (PCs), GSH is an essential metal chelator. Being an important antioxidant, a direct link between metal detoxification and the oxidative challenge in plants growing on contaminated soils is observed, where GSH could be a key player. In addition, as redox couple, oxidized and reduced GSH transmits specific information, in this way tuning cellular signalling pathways under environmental stress conditions. Possible improvements of phytoextraction could be achieved by using transgenic plants or plant-associated microorganisms. Joined efforts should be made to cope with the challenges faced with phytoextraction in order to successfully implement this technique in the field.