Grantee Research Project Results
Final Report: Transgenic Citrate-Producing Plants for Lead Phytoremediation
EPA Contract Number: 68D03043Title: Transgenic Citrate-Producing Plants for Lead Phytoremediation
Investigators: Elless, Mark P.
Small Business: Edenspace Systems Corporation
EPA Contact: Richards, April
Phase: II
Project Period: May 1, 2003 through April 30, 2005
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2002) Recipients Lists
Research Category: Hazardous Waste/Remediation , SBIR - Waste , Small Business Innovation Research (SBIR)
Description:
In 1991, the Secretary of the U.S. Department of Health and Human Services called lead “the number one environmental threat to the health of children in the United States.” Decades of peeling exterior paint, emissions from leaded gasoline car exhaust along streets and highways, and pollution from lead smelters and other industries have deposited lead in the yards around homes. Home improvement projects, such as remodeling and renovation activities, create dust and debris that contribute to further dispersion and elevated soil lead levels. The U.S. Environmental Protection Agency (EPA) estimates that 12 million homes have lead in their yards at levels exceeding the new 400 ppm standard for play areas, while 4.7 million homes exceed the new 1,200 ppm standard for the rest of the yard. Soil lead at small arms firing ranges, manufacturing plants, and other government and industrial sites poses a similar environmental challenge.
Paving, laying sod or gravel, or other techniques can reduce the exposure
hazards presented by soil lead. The only effective technique currently used
for eliminating the hazard, however, is costly excavation of the contaminated
soil and replacement with clean soil. An alternative to soil excavation and
replacement that also can remove the hazard is phytoremediation, or extraction
of lead from the soil using living plants. Phytoremediation has been used to
remove lead from soil at firing ranges, industrial sites, and residential yards.
Because the few plant species that naturally accumulate lead are unsuitable
for cultivation, phytoremediation of lead relies on use of crop species, including
turf grasses, with a combination of soil and foliar-applied amendments that
induce enhanced plant availability of lead and higher plant uptake rates.
Although phytoremediation clearly shows potential as a low-cost, in situ lead remediation technology, two factors tend to increase its cost at many sites. The first is the need to add chelating agents to the soil to make lead available for plant uptake. The cost of purchasing and applying such chelating agents, in the amounts typically applied by Edenspace Systems Corporation, can range up to $20,000 per acre per growing season, or about $12 per ton of treated soil. The second major cost factor is the need to use a water-impermeable liner at some sites (such as with sandy, well-drained soil over shallow groundwater) to prevent slowly degrading chelating agents such as ethylenediamine tetraacetic acid (EDTA) from leaching metals into groundwater. Costs associated with installing and maintaining a liner can more than double the total cost of phytoextraction and the need for a liner may render phytoremediation impractical for many sites, including residential sites. Although rapidly degradable chelating agents such as citric acid are increasingly being used at sites where leaching is a concern, to ensure adequate metal availability to plant roots even these chelating agents must be applied in amounts exceeding plant extraction rates, thereby increasing costs. Matching chelating agent application to plant uptake capabilities would minimize costs and leaching concerns.
Certain plants have developed the ability to acquire plant nutrients such as iron through the production of root exudates (organic acids and siderophores) that chelate iron in the rhizosphere. The genetic manipulation of plants to enhance the production of specific chelates or organic acids would provide a means to enhance the natural uptake of heavy metals and minimize or eliminate the need for soil applied chelating agents.
Together with Professor Neal Stewart of the University of Tennessee, Edenspace investigated the use of transgenic technology to provide plants with the ability to exude significant amounts of citric acid from their roots, seeking to enable cost savings of more than 70 percent in the phytoextraction of lead, uranium, and other metals from contaminated soil. Plant production of rapidly biodegradable citric acid, precisely at the root/soil interface where metal uptake occurs, could eliminate both the need to apply expensive chelating agents and the need for a liner. Attainment of the project's goals offers the possibility of reducing the substantial public health hazard of soil lead by realizing phytoremediation's low-cost potential.
Specific Phase II objectives were as follows:
- Using homozygous transgenic plants of transgenic tobacco lines that strongly express citrate synthase (CS) in the Northern blot analysis, demonstrate the ability of these transgenic plants to hyperaccumulate heavy metals such as lead and uranium in harvestable leaves and stems (bioconcentration factor of greater than 1, whereby bioconcentration factor is the ratio of the metal concentration in the plant to that in the soil).
- Conduct a field demonstration to validate operational performance.
- Develop methods of concentrating lead recovered by the plants to facilitate cost-efficient disposal or recycling, as well as other field techniques that maximize performance and minimize cost.
- Create transgenic lines of a proven lead-accumulating plant species, Brassica juncea, to investigate possible additional improvements in lead extraction efficiency.
Summary/Accomplishments (Outputs/Outcomes):
Soil Characterization. Approximately 60 kg of composite soil from two sites (Fort Meade, MD, and Las Cruces, NM) were collected and brought to Edenspace to include in the laboratory/growth chamber study. These soils were air-dried and sieved to smaller than 2 mm in diameter prior to use. The sieved soils then were analyzed for total lead, following EPA Method 3050 for digestion and EPA Method 6010 for analysis by inductively coupled plasma (ICP) spectroscopy. Total lead content of the Fort Meade soil was high (approximately 6,300 mg/kg), whereas the Las Cruces firing range soil contained a lower lead concentration (about 1,700 mg/kg). Total lead in both soils is at levels that should not hinder phytoextraction performance.
The degree of lead extractability by citric acid was considered to be a major factor for identifying soils having the greatest potential for citric acid-induced phytoextraction of lead. This extractability was assessed by extracting 2.5 g of soil with 25 mL of 2 mM citric acid for 2 hours on an end-to-end shaker, followed by centrifugation and filtration prior to analysis by ICP spectroscopy. Results indicated that the Las Cruces soil (330 mg/kg) and Fort Meade soil (830 mg/kg) were readily extracted with citric acid.
Transgenic Plant Characterization. Northern blot analysis in Phase I demonstrated that the CS transgene was expressed in two lines of transgenic tobacco, 35S-CS and Ubi7-CS. Second-generation T2 plants from these lines were grown in Phase II and tested for citrate production and exudation. Results indicated that citric acid content in shoots of both T2 transgenic tobacco lines was independent of pH and was similar to citric acid content in non-transgenic (Xanthi) tobacco plants. Citrate content of roots was higher in control plants compared to transgenic plants. Citrate exudation also was higher in wild-type plants than in transgenic plants.
Lead Uptake. Results from the growth chamber studies indicate that uptake of lead by both transgenic tobacco lines grown in a firing range soil was not significantly different than that of the control tobacco, with levels less than 200 mg/kg accumulated in all tobacco lines tested. The testing of the foliar amendment designed to stimulate uptake of soluble lead also did not exhibit a significant effect compared to plants harvested without the foliar amendment. The only significant effect found in this experiment was that external application of 20 mmol/kg citric acid to the soil surface in pots having control tobacco, simulating the procedure normally performed in commercial phytoextraction, induced significantly greater uptake of lead (up to 700 mg/kg) than the control tobacco without this application and much more uptake than that induced by the transgenic tobacco lines.
Additional Plant Characterization and Testing. Given the citrate production and lead uptake results, additional plant characterization and testing was conducted in the laboratory and growth chamber in lieu of a field demonstration. A literature search indicated that plants naturally exude citrate in response to either a phytotoxicity threat caused by soluble aluminum (Al3+) in acid soils or when searching for phosphorus (P) in P-deficient soils. Both such triggers were absent in the Las Cruces firing range soil (pH = 8.0, soil fertilized with triple super phosphate prior to transplanting), possibly leading to the similar uptake observed for the transgenic and control plants. To test the possibility of triggering greater uptake of lead using P-deficiency or Al toxicity, control and transgenic tobacco plants were grown in Fort Meade soil and fertilized twice a week with a P-deficient fertilizer (15-0-15). Each set of four replicates in each line was treated once with a different level of added AlCl3 (0, 0.5, 1.0, and 2.5 mmol/kg). For the most part, results showed no significant difference in the uptake of lead between the control and transgenic tobacco lines, suggesting that P and Al levels play little part in the citrate production and exudation rates of the transgenic plants. Another approach attempted was to stimulate citrate production using a foliar application of methyl jasmonate on tobacco plants containing the jasmonate-inducible ubi7 promoter. Results from this experiment also showed citrate levels identical to those of the control tobacco.
Despite a substantially greater expression of the Pseudomonas aeruginosa CS protein in the transformed tobacco, these results indicate that the transgenic plants evidenced neither increased internal citrate concentrations nor increased citrate efflux from roots. Published research by other researchers indicates highly variable results of CS insertion in other plants, with some researchers reporting high citrate exudation and others reporting results similar to those above. The difficulty in obtaining uniform results in different laboratories suggests that the activity of the P. aeruginosa CS in transgenic tobacco is either sensitive to environmental conditions or that additional pathways and genes, such as those involved in citrate transport within the plant, are insufficiently engaged.
Characterization of Transgenic B. juncea Lines. A good hyperaccumulator of heavy metals, Indian mustard (B. juncea) was transformed in Phase II with the CS gene. The transgenic plants were found to overexpress the CS gene, representing the first instance when a CS gene has functioned in a phytoremediation plant species. Following the end of the grant period, it is planned to grow T2 homozygous plants for analysis of citric acid production and lead uptake. The transgenic tobacco and B. juncea plants also will provide the basis for future transformation experiments with genes that complement CS, such as a citrate transporter.
Disposal and/or Recycling Options. Because little lead-contaminated biomass
was produced during the course of the project, the project’s investigation
of contaminant recovery used arsenic-contaminated biomass to compare the efficiency
of several methods to minimize landfill disposal costs and increase recycling
possibilities. Composting, blending, autoclaving, and pressing using a commercial
screw press were tested for extraction of arsenic from the biomass. Screw pressing
was observed to continually extract the arsenic from the biomass in the press
liquor, lowering the arsenic concentration in the residual press cake to less
than 100 mg kg-1 and thereby converting the classification of the biomass from
a hazardous waste requiring disposal in a hazardous waste landfill to a nonhazardous
waste that can be disposed in a municipal landfill, saving significant disposal
costs. More recent research has shown that near-total extraction of metals
from harvested biomass can be obtained when biomass is pulverized prior to
processing.
Conclusions:
Edenspace investigated a transgenic approach for synthesis and exudation of citric acid by inserting the CS gene in tobacco, which if successful, would reduce the required time and expense of external application of this soil amendment. Although the CS gene successfully was inserted and expressed in the transformed tobacco, exudation of citrate from the roots was not found to be significantly greater than in the control tobacco. Possible explanations include a negative feedback mechanism at the level of the protein, coupled with a paucity of transport proteins for citrate exudation. Identification of the mechanisms, including transporter genes, which are necessary to increase citrate exudation from plant roots would be a worthwhile follow-on study.
Because no measurable increase of citrate was found from the roots of the
transgenic plants, the technical feasibility of using citrate-exuding plants
for phytoremediation remains to be assessed. Commercialization of this technology
awaits additional knowledge about the mechanisms underlying the production,
transport, and exudation of citrate by transgenic plants. Results of these
investigations were presented at the 2004 Plant Biology Annual Meetings.
Supplemental Keywords:
citrate-producing plants, lead, phytoremediation, citric acid, soil, uranium, citrate synthase, heavy metals, chelate, tobacco, EPA, small business, SBIR,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Waste, TREATMENT/CONTROL, Water, Treatment Technologies, Contaminated Sediments, Microbiology, Analytical Chemistry, Environmental Microbiology, Hazardous Waste, Molecular Biology/Genetics, Bioremediation, Hazardous, degradation, bioavailability, biodegradation, transgenic plants, contaminated sediment, Brassica juncea, citric acid, lead, contaminated soil, contaminants in soil, bioremediation of soils, natural recovery, biochemistry, chlorinated organics, phytoremediationSBIR Phase I:
Transgenic Citrate-Producing Plants for Lead Phytoremediation | Final ReportThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.