Planning meeting (Chatham, UK)

WG 1 will address problems specific to the treatment of organic pollutants by whole plants, plant cells and tissues, harvested plant biomass.

The first meeting of Working Group 1 was organized by Dr P. J. Harvey, of Greenwich University and held in the Chatham campus of the University of Greenwich on March 4/5th 1999. The meeting was attended by 30 participants from 13 different countries.

It was organized as a general meeting at which participating members were asked to outline the relevant expertise of their research groups and institutions in relation to the overall objectives of the working group. (Abstracts)

A summary of the general trends, from existing research programmes, to emerge from the meeting is given on the page of objectives.

Participants have also appointed Dr Patricia Harvey (University of Greenwich, UK) and Prof. Pantouses Kaltsikes (Agricultural University of Athens, Greece) as WG1 coordinators, and Prof. Paula Castro (Universidade Catolica Portuguesa, Porto, Portugal) as WG1 delegate on the STSM Committee.

Prof. Pantouses Kaltsikes has proposed to organize the next WG1 meeting in Greece (Autumn 1999).

Plant enzymes involved in the metabolism of organic pollutants (Geneva, CH)

The meeting took place in the facilities of the Battelle Research Centre, at Carouge-Geneva, from 9^th to 11^th of December 1999. The local organizers were Dr Augusto Porta (Battelle) and Dr Jean-Paul Schwitzguébel (EPFL).

More than 40 scientists from 15 COST countries attended the meeting, focused on "Plant enzymes involved in the metabolism of organic pollutants". For the first time, one of the Indian scientists associated with COST Action 837, Dr Rishi Shanker, was present. 6 main lectures and 12 short oral presentations were performed, each followed by a discussion (Abstracts).

Saturday morning was devoted to the visit of the cantonal Laboratory for Agronomy and the academic Laboratory of Bioenergetics, at Lullier-Jussy, in the countryside near Geneva. Several research projects and case studies on plant ecophysiology and phytoremediation were presented.

On Friday afternoon, an interesting round table discussion was moderated by Dr P. Harvey and Prof. P. Kaltsikes, WG1 co-ordinators:

Food for Thought

• Many results have been presented, very interesting from a scientific point of view, but they seem far away from the remediation of contaminated sites and wastewaters: how can we link such data to actual pollution problems, or use them from a practical point of view ? To establish links between basic research and applications, contacts with WG4 are needed. On the other hand, how can we generate funds for practical developments, on the basis of research data ?
• Can we use this information to target development of phytotoxicity tests, which could be useful for the agro-food area and the safety of the food chain ?
• The bioavailability and transport into the root systems (uptake) are often bottlenecks in the phytoremediation of organic pollutants: how can we improve the situation ?
• Many single data are available on transport mechanisms and detoxification enzymes: how can we put them together to estimate enzyme activities numbers, flux numbers, flesh on metablic networks and homeostasis, stress or toxicity reactions ?

From a plant physiology point of view, what is the meaning of phytoremediation ? Why plants can support a particular role to extract molecules from the environment (soil, water, air), to transform and accumulate them ? Which metabolic networks are dealing with a particular contaminant ? When is the overload of plants by organic pollutants reached ? What are the limits between homeostasis metabolism, stress reactions and toxicity for different pollutants ?

Potential for increasing plant performance in the phytoremediation of organic pollutants (Grenoble, FR)

1. From identification of plant metabolism and metabolic endpoints to altering metabolism

In the environment, two different pollution situations can occur:

• A non-point source contamination (a lot of plants are able to cope with low concentrations of pollutants)
• A localized pollution, with very high concentrations of contaminants (several plants can adapt to such a situation and will colonize these sites)

Our dream is to remove organic pollutants from soil and water. Alternatively, pollutants should become less bioavailable. Exploiting natural biodiversity and improving on nature are two important possibilities for increasing plant performance in the phytoremediation of organic pollutants. The ultimate goal is to provide constructed wetlands or contaminated sites managers with efficient tools to remove the pollutants.

The xenome can be defined as specific plant genes and enzymes able to act on xenobiotic compounds. The structure and function of many detoxification enzymes is rather well known, but bottleneck is often neither the intensity of metabolism nor enzyme activities, but the penetration of pollutants in the plant (passive versus active transport), which also depends on the properties and bioavailability of the xenobiotic compounds, the size and shape of the root system, or the ratio between root surface and cell volume.  However, if the metabolism is efficient, it creates a favourable concentration gradient.

Should we construct the appropriate enzyme for a particular pollutant, with the right active site? But de-pollution depends also on the evapo-transpiration rate, thus on the functioning of stomata, and uptake is often the most critical point for phytoremediation. Plant roots may excrete not only enzymes like peroxidases, but also small soluble organic molecules (exudates), acting as biosurfactants, thus able to enhance the bioavailability and the uptake of pollutants. Mathematical modelling can be useful, but much more important factors to improve phytoremediation are root biotechnology (using rhizogenic Agrobacterium to induce root proliferation); plant hairy-root technology and rhizosphere biotechnology.

The ploidy of domesticated plants plays probably a role in the level of antioxidants and of detoxification enzymes, but what about wild plants? For example, do early corn varieties have important GST activities?  Many plants are around us, but who will screen them? Exploiting the natural biodiversity is thus an important issue in the choice of appropriate species for phytoremediation among agricultural plants (cultivation well known), hybrid poplars or willows (high water use), or wild plants growing in contaminated areas. Plant taxonomy and phytochemistry should be the first steps in the adequate use of the huge biochemical potential of plant kingdom, with very specific metabolism (plants often produce natural chemicals whose structure is close to xenobiotic compounds).

Plants are very flexible and react very quickly to environmental stress: we have to help them to grow and to concentrate pollutants. The next step would be to harvest plants, then to use them as a possible source of fuel or fibres.

2. Role of the external environment on phytoremediation

The effect of the availability of the xenobiotics and its influences on the phytoremediation were discussed. High available concentration of the xenobiotic as well as the steric configuration of the compound can affect the enzyme activity. The enzyme activity can also be affected by the toxicity of chemicals or a deficient environment. But the big problem is the bioavailability, which determines the rate of degradation. It was shown that ageing decreases the availability, while depressed redox potential and organic material could decrease the persistence of a substance. It was also asked, whether we need to remove unavailable, bound substances. The bioavailability can be increased by artificial surfactants, soluble organic matter, biosurfactants produced by microbes and plants. The artificial ones, however, are normally added in excess, which may leach and be toxic. Bacterial and plant surfactants are continuously produced and non-toxic. In the case of plants, they are localized and coupled with absorption. Increased bioavailability may be risky since it can give a progressive leaching of the pollutant, which can be transferred to the food chain and other species in the surrounding soil may be contaminated. Therefore, a risk assessment should be included.

The Working Groups discussed about what is expected from plant metabolism in order to reduce the different types of soil, water or air contamination, and what we can expect from GMOs. It was stressed that large root absorption area, big root tip mass, high enzyme activity, increase of bioavailability using exudates are all important factors. GMOs could be used where plants have limitations, maybe also to increase the availability. How, nobody could answer on.

3. Poster Session

Five posters were contributed to book of abstracts and during the poster session four of them were presented and discussed.

P. Kanekar presented results about removing of ethylenethiourea (ETU) from Mancozeb wastewater by microbial degradation under aerobic conditions at laboratory scale. A mixed culture of Arthrobacter sulphureus and Microbacterium lacticum was used in this experiment. The two-stage process of biodegradation appears to be efficient in the removing of ETU: 99.6% removed after 72 hours treatment.

C. Loutre has compared N- and O-glucosyltransferase activities participating in herbicide metabolism in maize and soybean. The experiments were set up under in vitro conditions. The enzyme activities were induced 2-3 times by presence of xenobiotic. No glucosyl-S-transferase activity has been detected.

Y. Wang tested different plant species, such as Salix viminalis, Lupinius perennis, Trifolium subterranneum, Triticum aestivum and Brassica napus, for removing of creosote (85% PAH) from the soil. The remediation of contaminated soil was more efficient with plants than without. Therefore the possible effect of plants is either phytostimulation of microorganisms degrading PAH present in the soil or phytodegradation of PAH. The degradation rate of 2-rings PAH was higher than 3-6 rings compounds.

In the context of an Indo-Swiss project, involving 3 Indian and 2 Swiss partner institutes, K. Wenger presented preliminary results on the use of plant and microbial potential to enhance phytoremediation of soils contaminated by hexachlorocyclohexane and atrazine. They have used crop plants like maize, sunflower, and Coriandrum sativum. One Indian cooperating institute (NEERI, Nagpur) screened plant species for the “biomarker” detoxification enzymes e.g. glutathione S-transferase. The effect of rhizosphere will be studied and as come from the discussion the effect of mycorrhiza would be as well as interesting.

4. Concluding workshop

The aim of this workshop was to summarize and conclude the whole meeting. The subject was divided into three headings:

1. What about GMOs?
2. What about the availability of xenobiotics?
3. What about plant intracellular metabolism?

1. GMOs:

Diverse questions related to GMOs were pointed out:

• Environmental concerns: Are GMOs acceptable? What about gene transfer into the environment? Etc.
• Efficiency problems: Increasing an enzyme production does not inevitably lead to an increase of metabolism. (Example: a recent paper related that the increase of a peroxidase in tobacco led to leaf fall)

Moreover, a lack of knowledge about natural plant species was recognised. Most of the plants that were explored in phytoremediation were crops or weeds selected by agronomical practices. Exploration of natural species, especially on contaminated sites, is required. Why should we go for GMOs when natural variability is not yet explored? In conclusion, a database of natural species growing on contaminated sites is needed if we want to improve our knowledge and optimise phytoremediation techniques.

In summary, we recognize that:

• Plants useful for degradation have to be identified
• We are aware of agronomic considerations
• Data on natural species have to be collected

2. What about the availability of pollutants?

Should we increase bioavailability of pollutants in the environment in order to be able to remove them, knowing that it will in the short-term, increase leaching? Or should we concentrate on the release of plant peroxidase in order to form bound residues?

Maximizing root surface is also very important in order to optimise exchange surface between soils and plants. Mycorrhizae may be useful depending on the type of pollutants and plants.

In summary, we recognize that:

• Exudates may enhance or diminish bioavailability
• Root surface is very important
• Mycorrhizae may well play a role

3. What about plant intracellular metabolism?

Do we know metabolic activities useful for phytoremediation? Where are these activities in plants? What are their rates? Is it sufficient for our goal?

In summary, we recognize that:

• We already know a batch of enzymes available for phytoremediation
• Proteomics could help us to find a huge amount of others
• Lots of metabolites are already identified
• Information has to be collected in order to determine pathways taken by the xenobiotics in plants

5. Summary:

Pollutants

·        It is notable that papers were presented on atrazine –inferring at the present time a greater knowledge base on atrazine metabolism than on that for another pollutant

·        Debate focussed on strategies, either for pollutants to be rendered more soluble and mobile for degradation to CO₂ or on whether they should be rendered immobile: no firm conclusions reached since both have their merits

·        Regarding solubility, it is inappropriate to start with the two extreme types of pollutant –water-insoluble and water-soluble: the spectrum in between is of more concern

·        Pollutants should be studied case by case

Species

·        There are both natural populations that represent a rich resource for investigation as well as GMOs that over-express a particular enzyme as starting points for research –both have their merit.

·        We seem to have little idea about whether there is a relationship between plant species and pollutant amelioration: the key point could be agricultural practice –but the data is not available.

·        There is merit in assembling data from the literature on species that have been investigated in relation to a pollutant –for whatever reason.

·        It would be invaluable to have test kits to screen for plants possessing the ability to survive/take-up pollutant.

Pollutant-plant interface

Soil

·        In so far as there are reports of plant exudates that act as natural surfactants to enhance the bioavailability of a given pollutant for metabolism either by microbial consortia or by plants, plants that possess root systems with large surface areas should be sought.

·        There seems to be a discrepancy between root hairs producing exudates for microbial communities and mycchorizal systems that support plant growth –do they contribute to microbial biodegradation of pollutant?

·        The equilibrium between binding to soil humic compounds and their mobilisation will shift in the direction of mobilisation when they are taken up and metabolised

·        Plant transpiration stream

Leaf

·        Air-borne pollution will penetrate leaves but it is not known the extent to which this could be detrimental to plant health

·        Plantibodies to trap pollutants in plants represent another approach to preventing pollutant excretion

Metabolism

·        Papers presented on peroxidases; oxidases; transferases; and more recently ECS synthase indicate the limit of our knowledge. The proteomic approach may well hold surprises in relation to which enzymes may be important –but is expensive and needs good collaborative links to be set up.

·        We have little clear idea of either the regulatory mechanisms –what governs partitioning in one direction compared to another -or what the rate-limiting steps with respect to the current repertoire of enzymes might be.

·        Guesses are valuable for directing screening programmes but transgenic plants to test guesses may prove invaluable.

Would anyone be prepared to buy shares in a company claiming to remediate rapidly? Not yet, but if the time-scale was longer, possibly.

[Home COST 837 | home WG1 ]