Reflexions, le site de vulgarisation de l'Universit de Lige

Soil remediation: appearances can be deceiving

8/21/13
Using trees to extract heavy metals from polluted soils. What a great idea! The scientific community has been
successfully working on this idea for more than twenty years. Among the woody species studied, the willow
and its many species and sub-species offer promising prospects. However, the health of these trees that can
successfully concentrate heavy metals in their biomass while still producing wood in abundance might actually
be under threat. This knowledge is important for the managers of polluted sites
Today, we have a much more detailed knowledge of those plants that are capable of accumulating-sometimes
at very high concentrations-heavy metals such as zinc, cadmium, copper, lead and nickel. This is a veritable
godsend because most of these metals (described by scientists as MTEs or Metal Trace Elements ) are
toxic to the ecosystem and, ultimately, to humans. Thus the idea was born: why not use these plants, and
therefore shrubs and trees equipped with the same properties for the remediation of soils polluted by industrial
or domestic activity? Even better: by using woody species we could kill two birds with one stone. That is to say,
we could remove the pollution from soils and produce biomass, wood that could be used as fuel, for example.
In 1983, the term "phytoremediation" appeared in scientific literature for the first time. It describes all the
techniques involved in the use of plants in the fight against the risks associated with pollutants. These
techniques are based on tolerance mechanisms in the sense that, despite the stresses caused by high levels
of metals, some plant species clearly succeed in surviving and reproducing without the slightest hitch. "For a
quarter of a century, research into phytoremediation has mainly been concentrated on phytoextraction. This
involves the tolerance mechanisms by which the plant succeeds in extracting pollutants from the soil and
concentrating them in the harvestable parts, the twigs and leaves. Yet other plants, on the other hand, can
conserve the metals in the roots and the rhizosphere which is the part of the soil that is influenced by the
roots and their associated microorganisms. These plants are to be prioritized for phytostabilisation", comments
Aricia Evlard, of the Laboratory of Environmental Toxicology of Gembloux Agro-Bio Tech. A distinction
needs to be made between these two concepts.
The willow tree to the rescue of our watercourses
In order to properly understand the doctoral thesis defended last May by Aricia Evlard, it would be necessary
to give a brief history of the context in which she got the idea for her work. During 2009, the young researcher
was immersed in the ECOLIRIMED project. Spread over the period between 2008 and 2012, this European
initiative, placed under the banner of Interreg IV and coordinated by the Walloon Centre for Agronomic
Research (CRA-W), aims to identify the best local woody species to consolidate and remediate riverbanks in
the North of France, Belgium and Luxembourg.

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After consulting the volume of information that has

been collected over the last few years on the willow (a species reputed for its quick growth and its faculties of
adaptation to diverse ecosystems), Aricia Evlard asks the following questions. Could it be that we are on the
wrong track? Is it possible that we are persisting in a vain search for the "miracle tree", a sort of idealized "great
photovoltaic pump" which would be capable both of depolluting the environment by accumulating metals and
producing great quantities of biomass over a period of many years? "If we were to consider phytoremediation
alone as a means to clean up polluted soil, the species of trees offered to the managers of polluted sites
would require a period of 30 to 600 years to finish the job. Yet management officials have to answer shortterm objectives: depollution without delay! Likewise, studies carried out on cleaning by woody plants do not
always include the "health" of the tree in question. As trees can live a very long time, those that are used to
accumulate metals could prove to be intolerant to pollution and, after a few years could weaken and die. What
would happen if we scientists advised managers to use these metal-accumulating but intolerant trees in the
long term? It is therefore crucial in the general context of phytoremediation, to include a monitoring program
to assess the tolerance of these woody species to metals".
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Instantaneous measurement of fluorescence

Aricia Evlard subjected 41 willow clones from the

cloning park of the CRA-W located at Grand-Manil (Gembloux), to a substrate contaminated with heavy metals.
Instead of being satisfied with mere empirical observation (phenotypical) of their growth, she investigated the
tolerance of the young trees to these pollutants (1). For two years she used physiological methods which
consist in measuring chlorophyll fluorescence, to measure the sugars and pigments present in the leaves and to
identify the expression of proteins (known as a "proteomic" approach). "The more the plant becomes stressed
by the metals the more it emits fluorescence. This fluorescence can be measured by a fluorometer, a technique
which has the advantage of being non-destructive and delivering immediate results. Pigments and sugars
also constitute parameters of stress which are interesting to study. During metal stress, the concentration
in sugars can increase for example. This is linked to the roles sugars play in the protection of proteins and
the maintenance of cellular activities". Finally, the identification of certain proteins can indicate the presence
of reactive oxygen species (ROS), oxygen free radicals that are more toxic than oxygen. "Under normal
circumstances, there is always a minimum of ROS produced in the cells. The cell has defense mechanisms
that can make them disappear before they can do any damage. These antioxidant defense mechanisms can
be enzymatic (again, proteins) or non-enzymatic. However, when the plant is stressed, the ROS are formed
more rapidly than they are eliminated. The result of this is that the ROS interact with the macromolecules
(DNA, lipids and proteins) to the point of deactivating the antioxidant defense mechanisms. Indirectly, the
metals cause various disorders of the cellular metabolism (loss of membrane integrity, altered photosynthesis,
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accelerated senescence, etc.)" The vital functions of the plant are therefore reduced or inhibited. Finally the
plant in question dies.
After having subjected the 41 clones to these four examinations, it clearly emerged that one of them stands
out from the rest. Here it is not important for the purposes of the demonstration which species or sub-species
is identified. "The main thing is that this clone was not only part of those which produced the most biomass in
the presence of metals, but also stood out from the others by having the highest levels of metals in its twigs.
In reality, during a supplementary study, it became clear that it was the least tolerant to metal stress from
a physiological and proteomics viewpoint. This study was also able to demonstrate that the good individual
"phytoextractor" or the most tolerant individual is not neccessarily that which produces the most biomass".
Accumulation and good health are not synonymous!
The main lesson learned from this research: a tree that "holds its own" against the metals, and which produces
a lot of biomass, is not neccessarily in good health due to its weak tolerance to MTEs. For this reason, it is
not necessarily a good phytoextractor in the long term. This fact is even more interesting when we consider
that the duration of a short rotation coppice-the type of crop generally used for energy purposes-is at least
twenty years.
Another observation made during this thesis: while the clones of willow tested at Gembloux seem less
interesting from the point of view of remediating a polluted site within a reasonable timescale, they appear,
on the other hand, to be more interesting to use in the context of phytostabilisation. "In fact they seem to be
tolerant to the metals but slow down the transfer of these to the upper parts of the plant and concentrate them
around the area of the rhizosphere. In other words, when this type of individual is planted, the metals are
better "handled" (this has yet to be studied in situ) than in the absence of planting: the metals do not travel
(or at least very rarely) towards the water tables. Also, because the transfer of pollutants to the twigs and
leaves is reduced, the contamination of the biomass is also reduced as well as the restitution of the metals
due to leaf-fall".
A necessary precaution must be taken, however: because part of the metals remain likely to move towards
the biomass, this woody material must be treated in controlled combustion units, that is to say, equipped with
specific filters. This is the recommendation of Aricia Evlard with regard to current legislation overseas because
here in Belgium there are no regional regulations governing this subject.
The endophytes show themselves
Subsequently, in order to increase knowledge about the phytoremediation potential of the willow, Aricia Evlard
concentrated on the mushrooms present around the roots. What role do these play in the fixation of metals?
How exactly do the fungal cells act? Does their presence slow down the flow of metals to the plant (acting as a
barrier) or, on the contrary, does their presence facilitate the flow of metals to the above-ground plant surfaces?
With her team, the doctoral student concentrated on the job of perfecting an in vitro protocol aimed at
assessing the sensitivity of the fungal strains associated with the roots of alder and willow trees. First she
made a collection of 91 fungal strains taken from the banks of the non-navigable watercourses of Wallonia.
The objective: to test the tolerance of these strains in vitro by subjecting them to various concentrations of
cadmium (1, 5, 10, 25 or 50 mg per liter of culture medium) this heavy metal was chosen due to its high level
of toxicity. The first surprise: against all expectations, the majority of the strains showed themselves to be
perfectly tolerant to the metal, even for a maximal dose of 50 mg. "I was expecting lethal effects. Yet this was
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certainly not the case". Second surprise: "among the most resistant strains, I discovered endophytes, more
particularly 'Dark Septate Endophytes', that is to say, mushrooms belonging to the sub-branch of ascomycetes
and which live inside roots ; they are not parasites nor are they really symbiotic (2).

It seems that tolerance to these high doses of cadmium can be explained by a phenomenon of absorption of
melanin present in the walls of the mushroom. "We can reasonably suppose that this absorption phenomenon
can also be observed for other metals, but this remains to be established. We have already reproduced this
procedure with Zinc: the results are in the process of being interpreted. In any event, we can, at this stage,
posit the theory that the role of this microflora in the control of metals could be important in the willow and
alder trees. It would be interesting to test the most highly resistant strains by inoculating them with woody
species used in phytoremediation".
Finally, in the light of this work, what hopes can we place in phytoextraction and phytostabilisation? "It must
be remembered that these two approaches are slow and are spread out over several years. They are only
useful when used as a complement to traditional physico-chemical techniques when there is persistent residual
pollution. In addition, these more radical techniques very often disturb the structure of the soil and leave it very
fragile. Phytoremediation projects by means of the willow tree can help to compensate for the loss of natural
resources and the damage caused by traditional techniques. It is in this sense that phytoremediation can
be considered as a complementary and/or compensatory measure, described in the 'soil' legislation recently
passed in Wallonia.

(1) For this purpose she is the beneficiary of assistance from the Gabriel Lippmann Public Research
Centre (Luxembourg).
(2) At the present stage of knowledge.
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