Research

Research Accomplishments and Significant findings
(** indicates projects done completely through the University of Washington)

Uptake, Metabolism, Environmental Impacts and Applications of Phytoremediation of Chlorinated Solvents

Laboratory

While previous work done as part of the University of Washington research team revealed three metabolites of trichloroethylene expected from mammalian degradation pathways, these compounds do not accumulate. We have been working to determine other metabolites of TCE in plants outside the expected mammalian metabolites. We have shown that monochloroacetic acid is produced in plants following TCE exposure, but not after exposure to related compounds perchloroethylene or carbon tetrachloride. We also confirmed the degradation of carbon tetrachloride to trichloroacetic acid. 

The use of hybrid technologies such as pump-and-irrigate, increases the effectiveness of phytoremediation on sites with physical limitations. However, this system can lead to exposure to TCE of organism that would normally be protected by the soil column. Also, insects feeding on plants used for remediation purposes could also have increased exposure risk. We have done studies on this subject and found that insect feeding on plants exposed to 50ppm of TCE showed no signs of toxicity, and that soil organisms were protected by the enhanced uptake and degradation in planted soils.  (student:  Sandy Benson)

The use of genetically engineered plants or microbes for remediation purposes can lead to discussions about the ‘escape’ of genes or organisms from the selected areas. One way to deal with these issues is the use of bacterial strains that exist almost exclusively as endophytes within the plant vascular tissue. Researcher at Brookhaven National Laboratory have created a strain of bacteria transformed with the plasmid that degrades both aromatic solvents and TCE and related compounds. We are starting studies with them to look at changes in the metabolite profile and transpiration rates of TCE in colonized plants.  (student:  Lucas Odom) 

**While plant metabolites of TCE seem to follow mammalian degradation pathways, not much is known about the plant genes involved. When at the University of Washington, work was done as part of the team that put mammalian genes known to be involved in TCE degradation into plants. This resulted in an increase in certain metabolites within the plants to levels over 600 time higher than that seen in non-transformed plants. 

At USC, we have worked to find plant genes involved in TCE degradation. This could lead to the development of engineered plants with much greater social acceptance than the use of mammalian genes. Also, knowledge of those plant genes involved in TCE metabolism could lead to the development of screening tools to help select plants suitable to clean-up applications. We have identified four genes with strong protein sequence homology to the conserved amino acids in the active pocket of CYP2E1, and at least one of these has shown to be involved in TCE metabolism via insertional mutations in A. thaliana. We have also transformed these genes into tobacco with strong constitutive promoters and are screening them for changes in metabolic profiles.  (student:  Sarah) 

We are working on a study to look at ways to change the levels of enzymes within plant systems without using genetically engineered plants or bacteria. Certain agricultural chemicals are known to increase the levels of various enzymes in plants, and we are looking at how the application of these supposedly ‘inert’ compounds could decrease plant toxicity and increase metabolism of solvents within the plants.  (student:  Brian Hann) 

Greenhouse
**Hybid poplars are used on most groundwater phytoremediation sites, but there has been speculation as to which clonal lines are best suited for remediation of solvents, or if there is a difference. At study done at the University of Washington has shown that the major difference between the clonal lines is in the amount of water take up by the plants. All clonal lines tested had similar toxicity to trichloroethylene, and all were capable of degrading TCE. Those lines that had the best remediation potential were those that had the fastest growth rates and took up the highest levels of water, which in the field would lead to the greatest level of contaminant uptake.  (student:  Marietta Sharp) 

While the use of such standard plants such as hybrid poplar or willow have been used on the majority of groundwater remediation projects, there has been an increasing interest in the use of ‘native’ vegetation for remediation work. This would allow for the combined remediation and restoration of impacted sites. This can be critically important in areas where there are limitations on the introduction of non-native species, as well as on large scale projects such as the restoration of decommissioned military bases. While at UW, work was done with the plant Leuceana leucocephala to remediate ethylene dibromide in Hawaii. More recently work has been done to look at native and naturalized plants of the Southeast. A study of deciduous trees has shown that sweetgum and sycamore have excellent potential for remediation purposes. In a study of conifers, southern Long Leaf Pine, itself considered vital to revegatation efforts, is capable to uptake and degradation of TCE.  (student:  Sarah)

In two related studies, we have looked at grasses that grow either in freshwater riparian systems, Arundinaria gigantica ssp. Tecta, swithcane, or in brackish water systems, Arundo donax. Both  plants are capable to taking up trichloroethylene and metabolizing it within the plant. Switchcane is a prime candidate plant for restoration purposes in impacted riparian zones. Arundo is not native, but has been proposed as a bioenergy crop.  (students:  Neshia Mohammed and Gillian Connolly)

While it would be nice to assume that contaminated sites contained one chemical and we could study how the plants respond to that chemical, in truth, most sites contain a variety of chemicals. At the Savannah River Site, chlorinated groundwater plumes can run into areas where there is heavy metal or radionuclide contamination. We have done a study to determine what happens within the plant systems when a TCE plume moves into an area already contaminated by nickel. The study looked at changes in TCE metabolism brought on by nickel uptake by the plants and saw that changes in nickel levels within the plant altered the levels of TCE metabolites in the plants. We are currently looking at how the migration of TCE into the area might affect nickel uptake by the plants by either changing bioavailabilty or root porosity.  (technician:  Tom Cirauolo) 

Before a remediation action can start, it is critical to know the extent of the contamination on the site. Typically, this is done by drilling multiple wells on the site until the plume can be accurately modeled. This is both time consuming and costly. NASA has proposed using hyperspectral imaging of pre-existing vegetation to determine if this method can be used to determine the size and boundaries of the plume. We are doing greenhouse studies to determine if this type of imaging can distinguish between exposed and unexposed plants, and to learn if this distinction can be seen at TCE levels low enough to make this a viable tool to use for site characterization.  (technician:  Tom Cirauolo) 

Working with NASA again with hyperspectral imaging, we are collaborating with the USFS to determine if this technology can also lead to the development of a tool that can help to map root  systems underground, and to track changes in root development over time. By screening root/soil systems, we are working with the NASA group for the development of software that can reduce the operator time for monitoring root development, fungal interactions and the presence of root foraging soil organism.  (technician:  Tom Cirauolo) 

Field
When phytoremediation of groundwater was first being proposed, most stakeholders were reluctant to gamble on the new technology. Occidental Chemical, which funded much of the early research, built a one-of-a-kind test treatment facility outside Fife, WA where we could test a variety of trees and chemical in contained, simulated aquifer systems. Over the years, we looked at three different groundwater contaminants, and ten varieties of trees to determine their relative toxicity, uptake rates, metabolism, transpiration and impact on soil pore space contamination.

Phytoremediation of groundwater remediation projects have been install around the country, but there is still reluctance by state regulators to accept a technology that they themselves have not worked with. At the Southern Sector A&M area site on the Savannah River Site, we installed a two acre demonstration plot, with one acre being naturalized plants within a pine plantation, and one acre of planted hybrid poplar. Extensive monitoring was done monthly on TCE concentrations in the irrigation water, the air, the trees and the soil pore water. It was found that the site could treat approximately 1.5 million gallons of contaminated water per growing season without breakthrough beyond the root zone of the plants.  (technician:  Robert Kim) 

With the extensive work we have done showing that most plants have some levels of ability to degrade solvents like TCE, selection of plants for a given site becomes more an issue of which trees will grow well in a given area. While hybrid poplar is still the most common tree used, most clonal lines were created either in the Pacific northwest, or the upper Midwest regions of the country. Thus, determining which lines have the best suitability for the southeast region can be critical. With the USFS, we did a study looking at survival, growth, resistance to disease and insects. We found that there were several lines that did significantly better than others, and that these would probably be the best clonal lines to use for remediation projects in the southeast.

While install tree plantations is highly effective in dealing with contaminated groundwaters, many site managers and owners are now looking more at monitored natural attenuation or MNA as an even more cost effective way to deal with contamination. However, there is still debate among the regulatory community as to what conditions have the potential to lead to a successful MNA treatment site. We did a study at the Chemical, Metal and Pesticide disposal area on the  Savannah River Site to determine if MNA was taking place on the site, and if so, what the temporal and regional conditions were that lead to MNA taking place.  (student:  Neshia Mohammed) 

Site Application
**In 1997, we worked with the Oregon Department of Environmental Quality to install the first ‘pump and irrigate’ system for the treatment of groundwater contamination. The site was an orphan site outside Medford, OR, where an accident had spilled a tanker truck of 1,1,1-trichloroethane. After laboratory study to show that plants could take up the compound, a 7/8-acre test plot was installed. After three years of growth, it was found that the plot of trees could take up over one million gallons of contaminated water per growing season without breakthrough beyond the root zone.

In 1998, the Undersea Naval Warfare Center at Keyport decided to install a phytoremediation system on top of a landfill to control leachate containing TCE and other chemicals that was threatening a protected wetland. Community involvement was very high, and the ceremonial Earth Day planting in 1999 included community members, the base commander and the base chaplin who blessed the trees as we planted. Monitoring on the site is ongoing to determine the extent of the impact of the trees. We have deployed some of the most up-to-date groundwater and plant metabolic instrumentation on the site to deal with problems such as tidal influences on the groundwater.  (student:  Marietta Sharp) 

Production of Plant Crops for Biomass and Biodiesel in the Southeast using Biosolids

Field
The foreign oil situation is only going to get worse, and the development of renewable bio-based fuels will need to be one of the ways that the country deals with this problem. Biomass can be utilized in three major ways for energy production: burning the mass for electricity or steam production, chemical transformation of the biomass for ethanol production, and modification of seed oil for biodiesel. In many instances, these types of energy are economical only if produced locally. We are working on a project using biosolids as a fertilizer regime to grow plants suitable for production level growth in the Southeast such as sunflower, caster bean and soy bean, for both seed oil production, as well as high biomass for ethanol or steam production. This allows the use of waste product to produce two forms of energy from the same crop.  (student:  Brad Temple)  

Conditions Enhancing Carbon Sequestration in Soil Fungal Biomass

Field
Research into carbon sequestration is increasing as the need develops to find a way to reduce the levels of this greenhouse gas in the atmosphere. A lot of work has gone into developing ways to utilize the plants ability to conduct photosynthesis and utilize carbon dioxide, but this is considered short term storage. However, soil fungal biomass has the ability to produce extremely recalcitrant compounds such as glomalin, which will tie up the carbon for much longer periods of time. We are working with the US Forest Service on their short-rotation woody crop plantations to work out which growing regimes will produce the most below ground fungal biomass and also induce the highest level of glomalin production. We are also doing greenhouse studies to determine which plant species will best foster fungal growth and glomalin production under more controlled conditions.  (student:  Rebecca Maska and Jaclin Durant)  

Phyto and Photodegradation of Energetics

Laboratory
Nitroamine Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX) is widely used by the military as an explosive, rocket propellant, and as a key ingredient in C-4, a plastic explosive. When test firing shells during practice maneuvers, incomplete detonation results in widespread areas of varying concentration of RDX. Due to the fact that these ranges must remain open, a remediation option that is low maintenance and still allows use is imperative to find. Our lab is working on using plants with variations in pigmentation to enhance photodegradation of RDX, as well as diverse secondary metabolic pathways that may enhance the degradation within the plant tissue.  (student:  Carrie Beth Hadden) 

Phyto and Enhanced Microbial Degradation of Petroleum Additives and PAH’s

Lab
The US Environmental Protection Agency mandated the addition of methyl-t-butyl ether to gasoline to reduce air pollution and to boost octane ratings. However, when MTBE leaks from underground storage tanks, its high water solubility results in plumes that can be over a mile long and often have no defined source. Greenhouse work has shown that plant can effectively take up MTBE, but metabolism by plants has long been debated. Work in our lab has shown that hydroponic cultures of Arabidopsis are capable of metabolizing MTBE to its primary metabolite, t-butyl ether.

Greenhouse
The dredging of waterways results in large volumes of sediments that are often too contaminated to be placed in open water systems. Thus, much of the sediment in placed in confined placement facilities, while the Navy and Corps of Engineers try to decide how to treat them. We looked using various wetland and wet tolerant plants to determine those that were able to most efficiently dewater the sediments, as well as promote the increase in microbial communities able to degrade contaminants such as PAH’s. We found that swamp pea, annual rye, and barnyard grass are all capable of increasing the numbers of pyrene degraders in the sediments.  (student:  Paul Biery)

**Based on laboratory studies of plant uptake of MTBE, we did a study at the Port Hueneme Naval Facility, and looked at the effect of trees on the site of reducing MTBE concentrations in groundwater. We found the a single mature tree on the base were able to reduce concentrations of MTBE by 50% after passage through the root zone.

Heavy Metal and Mercury Sequestration, Availability and Processing in the Environment

Lab
Due to the proposed plan in build over one hundred new coal burning power plants, there is a real need to have increased knowledge about the fate of mercury in the environment. While the use of higher grade coal and scrubbers will reduce the mercury emission, it will not eliminate them. Much work has been done with brackish and salt water system, but there is much less knowledge about freshwater systems. Our work looked at the effectiveness of bench scale processes to predict what was happening in a field scale application.  (student:  Michele Harmon) 

**There is still a strong debate as to who plants handle toxic metals at a cellular level; with various theories being put forward, and then being altered or dropped as new information becomes available. We proposed to bypass the natural plant systems, and to utilize the work of Dr. Loeb in selected mutagenesis experiments to design peptide systems that would require minimal cellular energy yet bind high levels of heavy metals. cDNA libraries were constructed, inserted in E.coli or yeast and screened for the ability of to impart resistance to metal toxicity. Promising candidate genes could then be transformed into plants for both decreased sensitivity and enhanced remediation activities.   

Field
Construction of artificial wetlands to deal with environmental contaminants is becoming more common, but proper design of the systems can be problematic. Minimizing one contaminant while increasing bioavailability of another is not uncommon. At the Savannah River Site, the construction of a wetland to reduce copper levels was successful, however, the conditions in the wetland favored methyl mercury production. We looked at alternative ways to design these types of systems to reduce metal contamination to below regulatory levels while not producing other more harmful contaminants.  (student:  Michele Harmon) 

The water flow in the Winyah Bay area has been hypothesized to limit water movement in certain areas of the bay, and thus potentially lead to retention of contaminants in the system. In our study, we looked area plants and sediments both upgradient and downstream of Georgetown Steel Corporation to determine if plant activities were increasing metal concentrations in the bay. Sediments were sampled both from the surface and down to three feet, to determine if historical contamination was still present. What we found was that our highest levels of contamination were not near the metal plant, but in our supposed control area, immediately down stream from a bridge. High levels of lead and zinc were found in all sediments in that area, as well as higher levels of zinc in the plants. 

On the Savannah River Site, the use of coal for energy production has resulted in both nuclear and coal impacts on the site. At the D-area ash basins, the dumping of high sulfur coal has resulted in an area with pH typically less than 1, leaching of toxic heavy metals from the ash, and limited revegetation. We worked on two projects on the ash basin, the first was using different types of surface and subsurface amendments to increase survival of plants placed on the site, and to document those plants that were started to colonize the site, determine their uptake of heavy metals, and determine conditions in the ash that allowed them to survive.  (student:  Jaclin Durrant) 

Uptake, Degradation and Stabilization of Pesticides. Preservatives and Agricultural Chemicals

Greenhouse
Dichloropropane was used as an agricultural pesticide for many years until the toxicity to humans was determined. Storage of the chemical on many agricultural chemical depot sites resulted in high levels of localized contamination. We did a study that determined that DCP could be taken up by hybrid poplars and broken down into metabolites similar to those found in humans.  (student:  Mary Loftfield) 

**Pentachlorophenol was long used as a wood preservative, especially in aquatic environments. We did a study to determine if a wetland plants would be capable of either taking up PCP and metabolizing it within the plant tissue, or of enhancing the microbial breakdown of the PCP within the soil. The eventual application of this information was the construction of a bioswale to handle contaminated surface water run-off. 

Site Applications
The Saginaw Mill site in Aberdeen, WA, was the site of a saw mill that produced cedar shingles. In an attempt to preserve the shingles and make them more fire-resistant, the company preserved the shingles with formaldehyde, which was sprayed on in the open. Years of operation resulted in contaminated groundwaters, which are moving toward the Chehalis River, which is a spawning salmon route. Poplars were first determined in laboratory studies to be able to take up the formaldehyde. A site application plan was developed and 5 acres of hybrid poplars planted on the site. Site monitoring is continuing to determine the efficiency of the trees in stopping the movement of the contaminated plume. 

Decades of agricultural support activities in eastern Oregon town of Vale lead to the contamination of ground water with leaked ammonium nitrate, and soil contamination with various pesticides. When the nitrate levels reach a point where it was above regulatory limits for the town, the agricultural firm on site elected to do a clean-up using hybrid poplars. The soil levels of ammonia so high it was toxic to plants, and it required respirators when working with the soil in the greenhouse. Extensive amendments needed to be added to the site to deal with this as well as herbicide contamination in the soils. However, the poplars were established on the site, and are continuing to grow and are monitored for affects on groundwater concentrations.  (student:  Mary Loftfield) 

**The use of compounds such as ethylene dibromide for the control of soil nematodes was widespread. In Hawaii, the Delmonte Corporation had an tanker truck spill, resulting in contamination of the groundwater supplying drinking water for the workers. Preliminary greenhouse studies showed that naturalize plants were able to take up the EDB, and between plant uptake and enhancement of soil microbial activity, a treatment system was designed to pump up the contaminated water and run it through a contained plant system. Delmonte installed the system and proved that the system could remove contaminants to below detection limits.