28 T. Macek et al. Biotechnology Advances 18 2000 23–34
metabolism were studied. The effect of PCBs on the changes of the level of peroxidase activ- ity and the pattern of peroxidase isoenzymes was also followed [36,50,51]. Lee and Fletcher
[52] suggested that cytochrome P450, rather than peroxidases, is involved in the PCB degra- dation pathway. Nevertheless, we found a significant positive correlation between peroxidase
content and PCB disappearance during incubation of cultures in the presence of PCB [51].
8. Plant cells cultivated in vitro as a tool for phytoremediation experiments
Most experiments used to establish phytoremediation techniques were done with normal soil-grown or hydroponically grown plants. Recently, as more and more effort is directed to-
ward research to understand and improve the performance of plants in remediation technolo- gies, the number of results obtained with the help of in vitro plant cell and tissue cultures is
rapidly increasing. The concept is not new—in vitro cultivated plant cells have been used in studies of herbicide resistance and metabolism for many years. Other organic xenobiotics
have also been studied, as in the case of pentachlorophenol, which was shown to be metabo- lized by wheat and soybean cell suspension cultures yielding glucosides and nonextractable
residues as described by Langebartels and Harms [28]. The ability of plant cells to metabo- lize PCBs was demonstrated 10 years ago by Groeger and Fletcher [53]. Transformation of
TNT by a hairy root culture of
Catharanthus roseus was investigated by Hughes et al. [54].
They did not detect mineralization of TNT; instead, products such as 4-amino-2,6-dinitrotol- uene and 2-amino-4,6-dinitrotoluene were identified.
Following transformation of 3,4-dichloraniline by leaves and suspension cultures of soy- bean, it was observed that both systems almost completely metabolize this compound during
48 h. Mostly N-glucosyl and N-malonyl conjugates were analyzed in leaves. These conju- gates were bound to cell wall structures. Cells of suspension cultures produced soluble
N-malonylconjugates, which were excreted into the medium. It was proven that axenic cell cultures are able to metabolize certain compounds by common metabolic pathways. Plant
cell tissue culture can be a useful system with some advantages in comparison with intact plants [55]. These advantages include: 1 the material can be grown under standard labora-
tory conditions, 2 the growth is independent of the weather or climate, and 3 in vitro cul- tures often grow more rapidly. The exploitation of transformed hairy root cultures is espe-
cially rewarding, as discussed by Macková et al. [15,37], Hughes et al. [54], Macek et al. [43,56], and Betts [20]. Plant roots transformed by
Agrobacterium rhizogenes exhibit all fea-
tures of normal plant roots and grow rapidly under defined aseptic conditions in vitro [57], thus allowing the distinction to be made between the plant metabolism itself and the effect of
the complex interaction between plants and microbial communities in the rhizosphere [58]. Metabolism of many other organic compounds has been addressed with the help of plant cell
and tissue cultures, because in addition to the above advantages this model facilitates obtain- ing results with much lower analytical expenses.
9. Practical approaches
Phytoremediation has already been successfully implemented. Trees of the Salicaceae
family willow and poplar have been planted at several locations because of their flood tol-
T. Macek et al. Biotechnology Advances 18 2000 23–34 29
erance and fast growth. Schnoor et al. [5] used the hybrid poplar Imperial Carolina for in- creasing soil suction and decreasing downward migration of pollutants. This system was
used to control agricultural runoff along a small creek in Iowa, and as final cap on a landfill in Oregon. The University of Washington has developed multiple hybrid poplar clones, some
with growth rates of 10–15 feet per year. These trees were found to have remarkable ability to take up and degrade certain halogenated organic solvents. In laboratory conditions the
poplar tissues were used to examine the metabolism of trichloroethylene. Phytotransforma- tion of perchlorate using parrot-feather
Myriophyllum aquaticum was described by Susarla
et al. [59]. This plant has already been successful in the remediation of soils contaminated with TNT as well as other contaminants e.g. TCE, PCP. There are numerous Department of
Defence sites across the USA with explosives contaminated groundwater. The U.S. Army Environmental Centre is developing technologies to effectively clean up groundwater con-
taminated with residues of explosives like TNT, RDX, octahydro-1,3,5,7-tetranitro-1,3,5,7- tetraazocine HMX, and DNT. Current groundwater cleanup technologies, such as granular
activated carbon and advanced oxidation, have proven to be labor-intensive and costly. One potential treatment alternative is phytoremediation using constructed wetlands [60]. The
U.S. EPA National Exposure Research Laboratory identified, in bench scale testing, a plant nitroreductase, which in cooperation with other plant enzymes is able to degrade TNT, RDX,
and HMX. An artificial wetland used to demonstrate the feasibility of using selected plants to clean up explosive-contaminated groundwater was constructed at the Milan Army Ammu-
nition Plant, and experiments were begun in 1995. A pilot-scale plant lagoon system was constructed and operated by the Georgia Institute of Technology. Results indicated that the
TNT removal in plant cells matched laboratory batch study predictions. Removal percent- ages relative to TNT loading for all species ranged from 85.4 to 99.7.
In addition, the U.S. Air Force is trying to clean TCE from groundwater using poplar trees and the U.S. Army is endeavoring to clean 2,4,6-trinitrotoluene and RDX from contaminated
wetlands, using a variety of plants. Pradhan et al. [12] used phytoremediation as a primary remediation technology and as a final polishing step for treatment of soil contaminated with
PAHs. Three plant species, alfalfa Medicago sativa
, switch grass Panicum virgatum
, and little bluestem grass
Schizachyrium scoparium were successfully used. A 57 reduction in
total PAH concentration was observed after 6 months of treatment. Phytoremediation was also tested in pot experiments in soil contaminated with petroleum hydrocarbons during the
Gulf War. Three domestic plants, broad beans Vicia faba
, alfalfa Medicago sativa
, and ryegrass
Lolium perenne were tested. This study also described the rhizospheric effects of
the leguminous plants alfalfa and broad bean, and demonstrated high degradation rates due to plant-microbe interactions. In this study the selection of plants was completely random
and further studies are needed to optimize the technology and to identify key parameters. The determination of the most efficient plant species to degrade a particular compound is the
most important step in this technology [14].
Similar examples were described by Boyajian and Carreira [61]. In pilot sites at numerous industrial facilities for petroleum hydrocarbons and wood preservatives, phytoremediation
using various grasses and their associated bacterial populations has had limited success. Care should be taken, as mentioned previously, to choose the proper plant species. For exam-
ple, at the Naval Air Station Joint Reserve Base, Fort Worth, TX formerly Carswell Air
30 T. Macek et al. Biotechnology Advances 18 2000 23–34
Force Base eastern cottonwood trees were planted to clean up trichloroethylene from a shal- low, thin aerobic aquifer. The organism was chosen, because “they [eastern cottonwoods]
are indigenous to the site, they are members of the same genus as poplar trees and grow at similarly rapid rates” The trees were planted to intercept and reverse the flow gradient and
to determine if cottonwoods can metabolize TCE and its daughter compounds under field conditions [62]. The system decreased the TCE content; however, the reasons leading to the
choice of eastern cottonwood seems to be quite unsatisfactory and lacking in support of re- search results, including the risk of survival problems for plants after long-term exposure to
the contaminants. The above approach cannot generally be considered as optimal. This is of- ten the problem of agencies funding remediation experiments, where time pressure leads
sometimes to support of projects lacking the time-consuming attempts to explain experimen- tally molecular mechanisms involved in the desired activity.
Many of these problems can be addressed by the selection of the proper type of plants. Phytoremediation has a number of inherent technical limitations. The contaminant must be
within, or drawn toward, the root zones of plants usually the top 3–6 feet of soil. This im- plies water, depth, nutrient, atmospheric, and physical and chemical limitations. In addition,
the site must be large enough to make farming techniques appropriate. It must not present an eminent danger to human health or further environmental harm [8]. There may also be a con-
siderable delay in the time needed for obtaining satisfactory cleanup results between phy- toremediation and ‘dig and dump’ techniques. Although plants have upward of 100 million
miles of roots per acre, their root system may not extend deeply enough to eliminate all of the contamination [61].
10. Future prospects
