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VIII. Current Phytoremediation Research and Development
S. D. CUNNINGSIAM ET AL.
rally, they are not common constituents of most soils and may be resistant to
biodegradation primarily because of their insolubility, molecular size, toxicity,
and/or inherent chemical bond energies and configuration. Although use of these
compounds as pesticides or industrial chemicals has dramatically decreased or
ceased in many countries, their persistence has contributed to their continued
detection in the environment and occurrence at hazardous waste sites. Despite this
persistence, the degradation of some chlorinated hydrocarbons has been shown to
be enhanced in the rhizosphere or by microorganisms from rhizosphere soil
compared to nonvegetated soils.
Pentuchlorophenof (PCP) is a widespread chlorinated hydrocarbon contaminant, especially in soil around wood treatment plants. At high concentrations,
PCP is often quite persistent due to its toxicity to a variety of soil organisms. One
approach to remediation of PCP-contaminated soil has been inoculation with
microorganisms capable of metabolizing PCP (Crawford and Mohn, 1985);however, survivability of inoculated organisms is often limiting. Ferro and coworkers ( 1994) explored the possibility of using rhizosphere microorganisms to
accelerate PCP degradation. Crested wheatgrass (Agropyron desertorurn) was
tested for the ability to enhance mineralization of 14C-PCP in a flow-through
soil-plant system in the laboratory. Mineralization of 1%-PCP in the vegetated
system was 22% of the applied 14C after 155 days, whereas only 6% of the 14CPCP was mineralized in the nonvegetated system. A significant portion of the
I4C-PCP and/or metabolites (36%)was taken up by the plants. Leaching of 14CPCP and/or metabolites was greater in the nonvegetated system. Overall, the
vegetated soil had lower levels of PCP-derived material than the nonvegetated
soils at the conclusion of the experiment. Thus, vegetation was beneficial in
increasing mineralization of PCP as well as reducing leaching of PCP and its
Trichloroethylene ( I , I ,2-trichloroethylene) (TCE) is one of the most common
substances found at hazardous wastes sites and in contaminated groundwater.
Walton and Anderson (1990) previously observed accelerated degradation of
TCE in slurries of rhizosphere soil collected from four plant species at a former
solvent disposal site and enhanced mineralization of I4C-TCE in rhizosphere soil
samples. However, the authors speculated that the absence of a living plant may
have lead to conservative estimates of degradation for TCE, and in subsequent
experiments utilizing soil-plant systems composed of soil and vegetation from
the same contaminated field site showed significant mineralization of 14C-TCE.
In this study, specially designed Erlenmeyer flasks were used to monitor the fate
of “T-TCE in the soil-plant systems. In addition to the vegetated samples,
nonvegetated and sterile soil samples were included as separate treatments for
each experiment. The flasks were sealed by coating a nonreactive silicone rubber
sealant between the plant stem and the flask. At 24-hr intervals, headspace within
the flasks was flushed through a series of traps for removing IT-volatile com-
PHYTOREMEDIATION OF SOILS
pounds and W02.Analysis of the I4CO2traps from the experiments showed
that I4CO2production in the vegetated soils was elevated compared with both the
nonvegetated soil and the sterile control soil. In the experiments with soils
containing Lespedeza cuneatu and Pinus tuedu, I4CO2production at the conclusion of the experiment was significantly greater than I4CO, production in nonvegetated and sterile (autoclaved) control soils (Anderson and Walton, 1992,
1995). Most of the 14C02produced from 14C-TCE in the vegetated soils evolved
during the initial 3 days of the experiment. This is in agreement with earlier
observations on the initially rapid disappearance of TCE from the headspace
above aqueous slurries of rhizosphere soil (Walton and Anderson, 1990). In
addition, comparisons of the percentage of I4C-TCE mineralized in the whole
plant systems with previous data on “T-TCE mineralization in L . cuneata rhizosphere soil appeared to confirm the hypothesis that the mineralization rates based
on rhizosphere soil samples gave conservative estimates of mineralization that
would occur in soil containing a living plant.
Polychlorinated biphenyls (PCBs)provide another research story of particular
interest in the relationships between rhizosphere microorganisms and plants.
While the use of microorganisms for remediation of PCB-contaminated environments has been the focus of research for the last 10 years, recently the use of
vegetation for enhancing microbial degradation has also been explored (Donnelly
et a / ., 1994; Donnelly and Fletcher, 1995; Brazil et a / ., 1995).
The use of ectomycorrhizal fungi for bioremediation is a potential technology
for overcoming some of the survival limitations of soil inoculation. The symbiotic relationship of plant-ectomycorrhizal systems may give the fungus a better
chance to compete against indigenous soil microflora and subsequently increase
PCB metabolism in contaminated soils. Donnelly and Fletcher (1995) explored
the metabolism of a PCB stock solution containing 10 or 11 different PCB
congeners (2-6 C1) by ectomycorrhizal fungi in culture flasks over 5 days. Of the
21 fungal species tested, 14 were capable of some PCB metabolism. The number
of congeners metabolized ranged from 0 to 7 and varied among the fungal
species. As expected, the lower-chlorinated congeners were more easily metabolized. Radiigera atrogleba and Cautieria crispa metabolized the most PCB congeners. An interesting note familiar to many in the bioremediation area is that
there was no correlation between taxonomically related species and metabolism
of structurally similar congeners.
Donnelly and co-workers (1994) have also been exploring the use of PCBdegrading bacteria in combination with plants. Previous research from the group
at General Electric (Bedard et a / . , 1986) identified several aerobic bacteria
capable of PCB metabolism. However, success with these organisms in the field
has been limited by the requirement of biphenyl as a cosubstrate. The need to
find other compounds which stimulate the growth of PCB-degrading bacteria led
Donnelly and co-workers to hypothesize that naturally occurring compounds
S. D.CUNNINGHAM ET AL.
produced by plants may be useful. The growth of three bacterial strains shown to
degrade PCB was tested using several known plant compounds including flavonoids and coumarins. Growth on biphenyl served as a control. Several of the
plant compounds supported growrh of the bacterial strains as well as or better
than biphenyl. In addition, bacterial strains grown on the plant compounds
retained the ability to metabolize certain PCB congeners. Results of these studies
suggest that certain plant species or stages of plant growth might be valuable for
enhancing PCB degradation in soil.
Plants can select for different rhizosphere microbial communities. It remains
unproven, however, whether this selection affects degradative rates in the field.
Whether this selection potential translates into differences in the rates of microbial degradation of organic compounds remains to be proven. It is on this premise, however, that recent research by Fletcher et al. (1995) has screened and
selected plants for specific exudate patterns and has examined the rate and timing
of the release of these compounds into the rhizosphere environment.
2. Remediation in Plantu
As mentioned above, research on pesticides has shown that plants have four
known types of reactions which result in the breakage of the C-Cl bound. These
reactions include: (1) monoxygenases, (2) glutathione (or homoglutathoine)
S-transferases, (3) (anti)auxin cell receptor binding (that converts a C-Cl to a CS protein), and (4) a nonenzymatically catalyzed replacement of a C1 to an OH
(in certain aromatic configurations, e.g., triazines). Other mechanisms of C-Cl
bond breakage undoubtedly exist; however, they have not been actively researched.
The capacity of plants to metabolize PCBs has been studied by Groeger and
Fletcher (1988). The extensive screening on whole plant showed a number of
differences in metabolic capacity between plants. To ascertain whether this was
plant metabolism or a microbial-plant community, Groeger and Fletcher went
further and produced and screened plant tissue culture (free from microbial
associations) for PCB metabolism. Cell cultures of rose (Rosa sp., cv. Paul’s
Scarlet) were found to have among the best ability to metabolize PCBs, but the
completeness of metabolism was dependent upon specific PCB congeners.
Recent work on trichloroethylene in hybrid poplar trees has shown that the
plant is relatively insensitive to the presence of the compound and shows no ill
growth effects at 100 mg/liter TCE (many times higher than the level deemed
safe in drinking water). The TCE is taken into the plant and has two detectable
fates: it is either metabolized or bound. The metabolic process produces 2,2,2trichloromethanol and di- and trichloroacetic acid compounds, which would
suggest the presence of an active TCE metabolizing P450 enzyme (Strand et al.,
1995). In addition, a large fraction of the TCE taken up by the plant roots
PHYTOREMEDIATION OF SOILS
becomes bound and unavailable to chemical solvent extraction. More work on
this fraction is also needed from a mechanistic as well as an ecotoxicological
perspective. Based on previous results, this bound fraction may also be unavailable to animals, microbes, and other environmental receptors. Binding pollutants
into the woody tissue of long-lived trees may prove to be an acceptable phytostabilization strategy. A field test of these poplar trees has recently been installed by these same researchers.
The widespread use of pesticides during the last 40 years has facilitated the
growth of retail agrochemical dealerships. Unfortunately, many of these dealerships have experienced soil and water contamination problems from normal
operating procedures and accidents. At pesticide-contaminated sites, a potential
limitation to using vegetation exists because of the presence of mixtures of
herbicide contaminants. Nonetheless, herbicide-resistant plants exist at these
sites, and rhizosphere soils from these plants have previously shown the ability to
degrade mixtures of herbicides (Anderson et u l . , 1994). In addition, previous
studies on the herbicide-degradative capability of rhizosphere soils of other plant
species (Sandmann and Loos, 1984; Lappin et a[., 1985) help support the use of
vegetation in remediating pesticide-contaminated sites. The use of plants in the
remediation process for these materials is also a logical extension of ongoing
research in the landfarming (surface tilling and fertilization) of these same materials (Felsot and Shelton, 1993).
1. Rhizosphere Degradation
Anderson and co-workers have conducted studies utilizing soils and plants
from pesticide-contaminated sites. Preliminary studies indicated increased degradation of atrazine, trifluralin, and metolachlor in rhizosphere soil from Kochia
scoparia. a herbicide-resistant plant, compared with nonvegetated soil (Anderson et a l . , 1994). Subsequent experiments indicated that mineralization of 1%atrazine in soil treated with a mixture of atrazine and metolachlor at concentrations typical of point-source contamination (50 x g/g each) was significantly
greater in rhizosphere soil from Kochia scoparia than in nonvegetated and control soils (Perkovich et a l . , 1995). Soils were collected from an agrochemical
dealership contaminated with several herbicides, including atrazine, metolachlor, trifluralin, and pendimethalin at concentrations well exceeding the field
application rates. Mineralization rates of ring-labeled atrazine in both rhizosphere and nonvegetated soils were quite high (>47% of the initial 14C applied
after 36 days) compared to literature values. Based on the relatively rapid miner-
S . D. CUNNINGHAM ET AL.
alization half-lives of atrazine in both soils, it does not appear that the presence
of metolachlor at 50 pg/g had a negative influence on the degradation. This
research supports the use of rhizosphere microorganisms associated with
herbicide-resistant plants to enhance microbial degradation of atrazine in soil at
contaminated sites. Naturally occurring plants, such as K . scoparia, appear to
have the capacity to be used as in sifu agents of bioremediation by facilitating the
proliferation of microorganisms in surface soil with the ability to mineralize high
concentrations of atrazine.
Anderson and Coats (1995) have also screened other rhizosphere soils from
waste areas for their ability to degrade atrazine and metolachlor. Several soil
samples exhibited the ability to mineralize high concentrations of I4C-atrazine.
These included rhizosphere soils from lambsquarters (Chenopodium berlundieri), foxtail barley (Hordeumjubatum), witchgrass (Panicum capillare), catnip
(Nepeta cataria), and musk thistle (Carduus nutans). Of the 14 species (eight
families) tested, the greatest mineralization of 14C-atrazine was observed in
rhizosphere soils from musk thistle (33.1-1.7%) and catnip (24.1-1.2%). However, none of the 14 rhizosphere samples tested exhibited a positive response for
2. Remediation in Plan@
Actually, modern pesticides, and in particular herbicides, are an ideal target
for phytoremediation. The materials are designed for application on soil and
plant systems. Herbicides which are designed to be applied to the bare soil at
planting, or prior to planting, are specifically designed to move through the soil
to the plant root, be taken up by the plant roots, and be metabolized by the
tolerant species. In one sense, phytodecontarnination occurs in many farmers’
fields, as an overapplication of a pesticide may require more than one cropping
cycle to reduce its bioactivity down to a level where sensitive species can be
grown. The phenomena of “carry-over” is often considered good by farmers who
grow successive monocultures of the same crop (as it reduces their need for
reapplication), but bad by farmers who try to replant with sensitive rotational
Many farmers will relate stories of altering crop management schemes to
accommodate a spill or miss-application (due to incorrect dilution procedures or
malfunctioning equipment). One farmer, who had planted corn into a triazine
spill a decade earlier, thought the Ph.D.’s “concept of phytoremediation of pesticides” was “about half a bucket of common sense” and then went on to inquire
what we did for a living. The mechanism of triazine uptake, degradation, and
characterization is exhaustively studied in the literature, and also apparently
practiced with some skill in the field. It is not, however, a soil decontamination
method approved by most regulatory authorities.
PHYTOREMEDIATION OF SOILS
Over the last decade, the transformation and regeneration of microbes and
plants has advanced greatly. Genes are now commonly moved between microbial
genera, and genes cloned from a wide variety of viral, microbial, plant, and
animal sources are now routinely expressed in some plants. Genetic engineering
for enhanced phytoremediation is in its infancy, yet progress is being made.
Progress has been made in finding and cloning potentially useful genes, transforming and regenerating appropriate species, altering plant characteristic
morphology, changing plant metabolism, and adding degradative capacity to the
1. Degradative Genes
A wide variety of microbial genes that can metabolize xenobiotics have now
been cloned. A small portion of these have been expressed in plant tissue. The
primary purpose behind most of this work to date has been to increase the
tolerance of a crop plant to a particular herbicide. Such altered plants may
increase the sales of the associated herbicides. Examples of this include: ( 1 )
Alzodef tolerance conferred on tobacco by the introduction of the cah gene from
the fungus Myrothecium verrucaricr (Maier-Greiner et ul., 199l ) , ( 2 ) Glofosinate
tolerance in tobacco and potato with the bar gene from Strepromyces hyet al., 1989), and ( 3 ) a glyphosate metabolism system that
g r o s c o p i c ~(DeGreef
may be used in conjunction with the altered tolerance genes (again a microbial
source) in “Round-up ready” germplasm developed by Monsanto. In addition to
these, P450s have been cloned from microbial, plant, and mammalian tissues.
Some of these, including mammalian genes, also have been expressed in plants
(Saito et al., 1991). Many molecular labs are now in the process of isolating the
genes responsible for a specific metabolic activity in microbial, plant, or animal
systems. Insect populations resistant to specific insecticides have even provided a
source of novel glutathione S-transferase activity (Thompson er a l . , 1994). These
genes are being cloned and eventually expressed in plant tissues. In some cases,
the molecular strategy has needed some adjustment (e.g., whole-plant, constitutive expression of a gene coding for the metabolism of a PCB is unlikely to
succeed as PCBs are a lipophilic contaminant that, if available, are tightly bound
into the outer layers of the plant root). Although the molecular biology may be of
intrinsic interest, justifying the project on the basis of phytoremediation potential
is difficult. The best use of molecular biological tools is as part of an integrated
phytoremediation team. To a large degree, the limiting factors in phytoremediation are unknown and hence difficult to target with molecular biological skills.
Relatively few recombinant plants have been made specifically for the purpose
of phytoremediation, however the microbial genes chlorocatechol 1,2-dioxyge-
S. D. CUNNINGHAM ET AL.
nase, catechol 2,3-dioxygenase (Gordon et al., 1990), and chlorophenol hydroxylase (Stomp el al., 1994) have all been engineered into plants for that
2. Other Targets
Much is known about the plant metabolic pathways that produce bioactive
compounds. Of particular interest is the regulation of pathways that determine
the plant’s production of microbial signal molecules, antibiotics, pigments, and
xenobiotic pollutant analogs. The biosynthesis of many of these molecules is
increasingly well understood (Kubasek ef al., 1992), and in some cases genes
(e.g., PAL pathway genes) have been isolated and cloned into vectors appropriate for phytoremediation work. Work at this level would be more likely to
produce research tools than to actually produce field plants at the moment;
however, the prospects are intriguing. Producing paired plants differing in only
one trait (e.g., 100-fold difference in tannin or phenolic exudation) would provide phytoremediation with much needed tools. Other targets of biotechnology
might include altered lignin production or incorporation into cell walls (for
increased phytostabilization), increased lipid concentration or quality in the roots
(for all forms of phytoremediation), altered root permeability, and altered infectivity by mycorrhizae.
3. Plant Transformation
Unfortunately, not all species of plants are equally amenable to transformation
and regeneration. After nearly a decade’s worth of work, monocots and many
trees are still proving difficult to transform and regenerate on a routine basis.
Since many of the target plants for phytoremediation include these types of
plants, we believe continued effort in plant transformation will eventually prove
useful to phytoremediation. In one species, now in trials, altered rooting
morphology was obtained by Agrobucferium rhizogenes transformation (Han et
af., 1993). The resulting trees had greatly increased root mass, surface area, and
soil/root contact. This shows that some tree species are indeed amenable to
molecular techniques, and schemes for their improvement in phytoremediation
have been proposed (Stomp ef al., 1994).
4. Microbial Biotechnology
Not all biotech efforts in phytoremediation are directed to the plant component. Efforts at creating “biased rhizospheres” where microbes have additional
degradative capacities are ongoing in a number of labs working with biocontrol
of plant pests, nitrogen fixation, plant-growth promotion, and myconhizae.
PHYTOREMEDIATION OF SOILS
Few workers have attempted to combine molecular biology, microbial competition in the rhizosphere, and microbial degradation of xenobiotics. One exception is research by Brazil and co-workers (1995). They have inserted the genes
(bph) encoding the biphenyl degradative pathway into the chromosome of two
rhizosphere pseudomonads. Results of tests on the genetically engineered organism demonstrate that growth rate, bph gene expression and stability, and colonization potential of the rhizosphere were not seriously affected. It may therefore
be possible to genetically engineer rhizosphere competent pseudomonads without compromising their competence. Importantly, expression of the bph genes
was detected in rhizosphere soil microcosms. The authors suggest that expanding
the degradative capabilities of rhizosphere-competent microorganisms might be a
good method for generating useful strains for bioremediation applications.
Phytoremediation is an exciting nascent technology. Its development represents an opportunity for cross-discipline research teams to produce a new, and
much needed, technology to remediate environmental contamination. Research
at all levels is needed. We lack many of the fundamental understandings on the
interactions between the system components (plants, their microbial communities, contaminants, and soil, water, and the atmosphere) and field and engineering installation and equipment development. The ability to use natural ecosystems to remediate the environmental damage done by industrial and urban
activites has generated excitement among technologists, owners of contaminated
sites, regulators, and the popular press. Delivering a widely applicable technology, acceptable to the scientific, regulatory, and political communities, is the
current challenge before the research community.
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