Process may have advantages over traditional cleanup methods

Researching the phytoremediation of plutonium

Phytoremediation-using plants to clean up the environment-relies on the fact that plants are essentially solar-driven pumps, transpiring water from their roots through their leaves. In the process, they can degrade organic chemicals and stabilize contaminants in their root zone, or even take up contaminants into their leaves, thus allowing them to be removed from the soil.

Phytoremediation has distinct advantages over traditional cleanup methods: it is safe, relatively inexpensive, usually simple to implement with existing agricultural practices, and has a positive public image. Phytoremediation of metals has been particularly successful. A few techniques are being explored commercially, including phytostabilization and phytoextraction.

Phytostabilization relies on the plants to decrease contaminant migration by decreasing wind and water erosion of soils, while the plants pull soluble species into their root zone where metals can be sorbed onto roots and stabilized.

Phytoextraction takes the process a step further; it relies on metal-accumulating plants to actually take up metals into their aboveground parts, thereby removing toxic metals from soils and water. Metal-loaded plants are harvested, dried, ashed, composted, or stored. The volume of resulting waste is generally a fraction of waste produced by many current, more invasive remediation technologies, and associated costs are much less.

Most research on metal phytoextraction has focused on finding naturally occurring plants that can take up a large amount of metal compared to their biomass (called hyperaccumulators), and then developing these plant species through genetic alteration for improved field performance, and for a wider range of metal uptake.

Elise Deladurataye, a student in the Structural Inorganic Chemistry Group (C-SIC), checks to see if barley plants have enough nutrient solution to grow for one more day before being harvested to determine their uranium uptake levels. The barley seeds are first sprouted on a small water-soaked mat and then transferred to a nutrient-rich growth solution, where they grew hydroponically in a controlled-atmosphere growth chamber in a Chemistry Division laboratory. The bottles containing the plant solutions are equipped with an air bubbler inlet to make sure the growth solution doesnÕt stagnate and a HEPA-filtered outlet. The plants grow in the nutrient solution with either no iron, normal iron levels, or high iron levels (well-fertilized). After adjusting to the iron levels for one week, a solution of uranium is added to the nutrient solution and the plants grow in (and take up) the uranium. The plants are harvested by clipping off the stems and the roots and letting them completely dry in small vials. The dry plant parts are digested in acid and then analyzed for uranium content using ICP (Inductively-coupled Plasma atomic emission) spectroscopy and compared to uranium concentration standards.

Work with hyperaccumulators has focused on commercially produced "heavy" metal contaminants-such as lead, mercury, chromium, cadmium, zinc, copper, arsenic, and nickel-rather than radionuclides such as plutonium, thorium, and uranium. These groups of metals are chemically very different, so it is unlikely that any known hyperaccumulators will be able to hyperaccumulate plutonium or other actinides. Researchers have even speculated that no hyperaccumulator will ever be found for the actinides.

Graminaceous plants produce and release phytosiderophores, a family of nonprotein amino acid that enhances solubility and uptake of iron and possibly other metals from the environment. Phytosiderophores are derivatives of mugineic acid. Shown here is the mugineic acid family of phytosiderophores, nicotianamine (a phytosiderophore biosynthetic precursor), EDTA, and citric acid.

Fortunately for actinide phytoremediation, true hyperaccumulators are not absolutely necessary. A plant with high biomass but a lower percent of metal uptake will remove the same amount of metal from the soil as a plant that has low biomass (such as most hyperaccumulators) but a higher percent of metal uptake.

Maximum efficiency is still achieved by increasing the percent of metal uptake into the plant. This can be done by increasing the metal solubility, increasing the ability of the metal to cross the root membrane, and increasing the amount of metal translocated into the higher plant parts.

Increasing metal solubility alone has been shown to dramatically improve phytoextraction efficiency. Chelators such as EDTA or citrate are added to soils to improve metal solubility and increase plant uptake, a process called chelate-enhanced, or induced, phytoextraction. For example, researchers at Phytotech Inc., a metal phytoremediation company, published work in "Environmental Science and Technology," [1998, 32(13), 2004] showing that added citrate can increase plant uptake of uranium more than 1,000-fold.

Unfortunately, adding such chelators can backfire. Even though they increase metal solubility, the rate of metal translocation into the plant roots may still be limited. This can lead to large amounts of solubilized metals that are not able to get into the plant, leading to increased soil migration and leaching to ground waters. Luckily, there may be a better way to "trick" plants into taking up plutonium, or at least trapping it in the plant root zone.

Plutonium behaves similarly to iron, a necessary nutrient in biological systems. Both iron(III) and plutonium(IV) readily hydrolyze, have rich redox chemistry, have high coordination numbers, have similar charge-to-radius ratio, and often have predictably similar binding affinities for the same chelating ligands. Both have extremely low solubility in the environment. Successful development of many plutonium complexation and decontamination agents has relied on this iron-plutonium similarity by exploiting iron chelating ligands.

We hope to be able to use plant iron-uptake systems to trick plants to take up large amounts of plutonium, or at least trap it in the plant root zone.

Graminaceous plants (grasses such as barley, oat, and wheat) produce and release phytosiderophores, a family of nonprotein amino acid that enhances solubility and uptake of iron and possibly other metals from the environment (see figure at left). Phytosiderophores, all derivatives of mugineic acid, form stable complexes with numerous metals.

Researchers have used ultraviolet-visible (UV-Vis) spectroscopy to examine the binding of plutonium(IV) to mugineic acid (MA) or EDTA over a large pH range.

Phytosiderophores have been shown to enhance soil mobility of copper, iron, manganese, and zinc as much as microbial siderophores and more than some synthetic chelators. Some researchers have shown that phytosiderophores are up to 100 times more efficient than anthropogenic or bacterial iron chelators for iron uptake into graminaceous plants, presumably because they are the chelate form recognized by the plant roots. They are notably similar to EDTA in structure and metal-binding chemistry. Both have multi-carboxylate chelating groups and form extremely strong complexes with iron and other transition metal ions.

We have examined the binding of plutonium(IV) to mugineic acid over a large pH range using ultraviolet-visible (UV-Vis) spectroscopy (see figure above). Mugineic acid prevents hydrolysis and precipitation of plutonium(IV) to a pH greater than 10; spectra of plutonium(IV)-EDTA and plutonium(IV)-mugineic acid are nearly identical at all but the highest pH. In this pH range, plutonium(IV)- EDTA is believed to be a hydroxo rather than aquo complex, as it is under acidic pH.

The plutonium-mugineic acid complex may not undergo this change, or it may occur at an even higher pH. Possible structures of the plutonium(IV)EDTA and plutonium(IV)-mugineic acid complex are shown in the figure at right. These data suggest that mugineic acid will be as powerful a ligand as EDTA for keeping plutonium species soluble in the environment. We are currently directly testing the rate siderophore complexes or whether they are trapped in the plant root zone. Alternatively, we need to know if phytosiderophores create soluble species that aren't taken up or broken down, and therefore contribute to plutonium migration.

Pictured are proposed structures of the plutonium-EDTA complex as the pH increases, and possible analogous structures for the plutonium-mugineic acid complex. Data suggest that mugineic acid will be as powerful a ligand as EDTA for keeping plutonium species soluble in the environment.

To test if phytosiderophores can aid in actinide uptake, we are examining the effects of plant iron status. Iron-starved plants produce more phytosiderophores than iron-saturated ones, and have dramatically higher uptakes of zinc, nickel, manganese, copper, and even cadmium (under some growth conditions), than do high-iron nutritional plants.

We are growing both a phytosiderophore-producing plant (barley) and a non-phytosiderophore-producing "hyperaccumulator" species (Indian mustard) under variable iron conditions, and with actinides added as various chelated forms (phytosiderophore-chelated, citric acid-chelated, and unchelated) to see how this affects uptake into the plant.

Our initial tests with uranium show some interesting results. Barley plants grown in hydroponic solutions with 1,000 parts per million uranium with or without citrate have low uranium uptake into plant shoots (typically 0.2Ð3.5 milligrams per gram plant dry weight), with no consistent response to plant iron status, presence of citrate, or pH (pH 5 or 6), although on average, uptake with citrate was slightly higher (see figure above). This was surprising because we expected citrate and decreased pH to cause significantly higher shoot uptake because of their ability to keep uranium soluble. We did not expect the iron status of barley plants to have a major effect on uranium uptake into shoots because, although the phytosiderophore could contribute to uranium solubility, it is unlikely the iron(III)-phytosiderophore uptake system in plants would recognize and take up a uranium(VI) phytosiderophore complex.

Initial tests with uranium show some interesting results. Barley plants were grown in hydroponic solutions with 1,000 parts per million uranium with or without citrate; without added iron, with 7.5 micromoles added iron (ÒnormalÓ), and with 100 micromoles of added iron (ÒhighÓ); and at a pH of 5 and 6. Error bars show one standard deviation among the three to four plants grown at each condition.

Barley plant roots have very high sorption of uranium from solution, as has been observed with other plant species. We observed values from 4 milligrams uranium to more than 600 milligrams uranium per gram dry root weightÑone to two orders of magnitude more than in shoots (see figure on page 30). Interestingly, we did observe a significant difference in root uranium binding based on growth conditions.

Barley roots have significantly more uranium bound without citrate present than with citrate, regardless of pH and iron status. This could simply be due to increased precipitation of uranium onto the roots without citrate. However, without citrate present, roots bound significantly more uranium at pH 5 than at pH 6. This may be caused by such rapid precipitation of uranium from solution at pH 6 that it is unavailable for interaction with the plant roots. And at pH 5, roots of plants with excess iron bound more uranium than roots of plants grown without iron. This could have many causes. There could be increased microorganism presence on the Ò'igh-iron" roots increasing sorption of metals, or there could be increased solubilization of uranium on the roots by chelators, such as phytosiderophores, when plants are iron starved.

We will soon test plutonium uptake into plants under these same conditions, where we expect iron status and phytosiderophores to make significant differences. We now know phytosiderophores are good chelators for plutonium(IV), and have a good chance of being recognized by the iron-uptake system of the plant because of the similarities between iron and plutonium.

If plutonium-phytosiderophore complexes are recognized and translocated by the phytosiderophore uptake system in plants, plutonium solubilization may be balanced with uptake, eliminating a problem of chelate-induced phytoextraction. Phytosiderophores may also improve translocation into higher plant parts. Their biosynthetic precursor, nicotianamine, is ubiquitous in plants and is believed to affect cation (copper, iron, zinc, and manganese) mobilization and distribution in plants, presumably having a direct influence on plant metal uptake and translocation.

Even if efficient uptake into the higher parts of the plant cannot be achieved, we will determine how phytosiderophores will affect phytostabilization. Phytosiderophores will chelate actinides. These chelated actinides could be fluxed to the root zone of the plant, as are other metal-phytosiderophore complexes, and could have increased precipitation onto the plant roots. Concentrating contaminants and fixing them onto the plant roots may help prevent migration and improve the efficiency of phytostabilization. Alternately, phytosiderophores could chelate actinides and prevent precipitation onto plant roots, thereby increasing the risk of contaminant migration.

We think this research holds enormous potential. Grasses grow anywhere and everywhere, from arid and semiarid sites, like the Nevada Test Site and the Rocky Flats Environmental Technology Site, to wet and subtropical sites like Oak Ridge National Laboratory and the Savannah River Site.

This article was contributed by Christy Ruggiero, Elise Deladurantaye, Brandy Duran, and Stephen Stout of Structural Inorganic Chemistry (C-SIC); and Scott Twary of the Bioscience Division Szilard Resource (B-3)

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