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PHYTOREMEDIATION: USING GREEN PLANTS TO CLEAN UPCONTAMINATED SOIL, GROUNDWATER, AND WASTEWATER

Ray R. HinchmanEnergy Systems DivisionArgonne National Laboratory

9700 S. Cass AvenueArgonne, Illinois 60439

M. Cristina NegriEnergy Systems DivisionArgonne National Laboratory

9700 S. Cass AvenueArgonne, Illinois 60439

Edward G. GatliffApplied Natural Sciences, Inc.7225 Dixie Hwy - Suite C

Fairfield, Ohio 45014

ABSTRACT

Phytoremediation, an emerging cleanuptechnology for contaminated soils, groundwater, andwastewater that is both low-tech and low-cost, is definedas the engineered use of green plants (including grasses,forbs, and woody species) to remove, contain, or renderharmless such environmental contaminants as heavymetals, trace elements, organic compounds, andradioactive compounds in soil or water. A greenhouse

experiment on zinc uptake in hybrid poplar (Populus sp.)was initiated in 1995. These experiments are beingconducted to confirm and extend field data from AppliedNatural Sciences, Inc. (our CRADA partner), indicatinghigh levels of zinc (4,200 g/g [ppm]) in leaves of hybridpoplar growing as a cleanup system at a site with zinccontamination in the root zone of some of the trees.Analyses of soil water from lysimeter pots that hadreceived several doses of zinc indicated that the zinc wastotally sequestered by the plants in about 4 hours during asingle pass through the root system. The data alsoshowed concentrations of sequestered metal of >38,000g/g (ppm) Zn in the dry root tissue. Above-groundorgans contained less metal. A similar experiment

evaluating zinc uptake in Eastern gamagrass (Tripsacumdactyloides), a large, robust native grass was conducted in1996. This study found similar patterns of partitioningand sequestration as the poplar experiments, but growthand transpiration were more suppressed at the highestlevels of accumulation in gamagrass. These levels ofsequestered zinc observed in both hybrid poplar andEastern gamagrass exceed the levels found in either rootsor tops of many of the known "hyperaccumulator"species. Because the roots sequester most of thecontaminant taken up in most plants, a major objective ofthis program is to determine the feasibility of rootharvesting as a method to maximize the removal ofcontaminants from soils. Currently ongoing studies

include heavy metal uptake and fate from soil by willowtrees as influenced by natural and chemical chelatingagents, rooting patterns in hybrid poplar, and the uptakeand fate of halogenated organics in hybrid poplar. Otherresearch includes the development and successful fielddemonstration of a plant bioreactor for processing thesalty wastewater from petroleum wells; the demonstrationis currently under way at a natural gas well site inOklahoma, in cooperation with Devon EnergyCorporation.

INTRODUCTION AND BACKGROUND

At many hazardous waste sites requiring cleanup, thecontaminated soil, groundwater, and/or wastewater containa mixture of contaminant types, often at widely varyingconcentrations. These may include salts, organics, heavymetals, trace elements, and radioactive compounds. Thesimultaneous cleanup of multiple, mixed contaminantsusing conventional chemical and thermal methods is bothtechnically difficult and expensive; these methods also

destroy the biotic component of soils.

Phytoremediation, an emerging cleanup technologyfor contaminated soils, groundwater, and wastewater, isboth low-tech and low-cost. We define phytoremediationas the engineered use of green plants, including grasses,forbs, and woody species, to remove, contain, or renderharmless such environmental contaminants as heavymetals, trace elements, organic compounds, andradioactive compounds in soil or water. This definitionincludes all plant-influenced biological, chemical, andphysical processes that aid in the uptake, sequestration,degradation, and metabolism of contaminants, either byplants or by the free-living organisms that constitute the

plant's rhizosphere. Phytoremediation takes advantage ofthe unique and selective uptake capabilities of plant rootsystems, together with the translocation,bioaccumulation, and contaminant storage/degradationabilities of the entire plant body. Plant-based soilremediation systems can be viewed as biological, solar-driven, pump-and-treat systems with an extensive, self-extending uptake network (the root system) that enhancesthe below-ground ecosystem for subsequent productiveuse.

Examples of simpler phytoremediation systems thathave been used for years are constructed or engineeredwetlands, often using cattails to treat acid mine drainage or

municipal sewage. Our work extends to more complicatedremediation cases: the phytoremediation of a sitecontaminated with heavy metals and/or radionuclidesinvolves "farming" the soil with selected plants to"biomine" the inorganic contaminants, which areconcentrated in the plant biomass (Ross 1994, Salt et al.1995). For soils contaminated with toxic organics, theapproach is similar, but the plant may take up or assist inthe degradation of the organic (Schnoor et al. 1995).Several sequential crops of hyperaccumulating plantscould possibly reduce soil concentrations of toxicinorganics or organics to the extent that residual

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Phytoremediation - Hinchman, Negri, and Gatliff 2Argonne National Laboratory and Applied Natural Sciences, Inc.

concentrations would be environmentally acceptable andno longer considered hazardous. The potential also existsfor degrading the hazardous organic component of mixedcontamination, thus reducing the waste (which may besequestered in plant biomass) to a more manageableradioactive one.

For treating contaminated wastewater, thephytoremediation plants are grown in a bed of inertgranular substrate, such as sand or pea gravel, usinghydroponic or aeroponic techniques. The wastewater,supplemented with nutrients if necessary, trickles throughthis bed, which is ramified with plant roots that functionas a biological filter and a contaminant uptake system.An added advantage of phytoremediation of wastewater isthe considerable volume reduction attained throughevapotranspiration (Hinchman and Negri, 1994a).

Though it is not a panacea, phytoremediation is wellsuited for applications in low-permeability soils, wheremost currently used technologies have a low degree of

feasibility or success, as well as in combination withmore conventional cleanup technologies (electromigration,foam migration, etc.). In appropriate situations,phytoremediation can be an alternative to the muchharsher remediation technologies of incineration, thermalvaporization, solvent washing, or other soil washingtechniques, which essentially destroy the biologicalcomponent of the soil and can drastically alter its chemicaland physical characteristics as well, creating a relativelynonviable solid waste. Phytoremediation actually benefitsthe soil, leaving an improved, functional, soil ecosystemat costs estimated at approximately one-tenth of thosecurrently adopted technologies.

RESEARCH APPROACH

The current project objectives are:

To identify the plant species best adapted for theuptake, sequestration, or degradation of specificcontaminants and evaluate these species incontrolled greenhouse and field experiments.

To elucidate the physical, chemical,physiological and metabolic mechanisms ofcontaminant uptake, translocation,sequestration/detoxification, partitioning andbioaccumulation in phytoremediation plants.This research is focusing on heavy metal,radionuclide, and organic contaminants that areproblems at DOE and other sites.

To demonstrate that plant-based cleanup systemsfor heavy metals, radionuclides and organics arelow-cost, low tech, environmentally friendly, andwill operate economically at actual contaminatedsites.

A Cooperative Research and Development Agreement(CRADA) has been established between Argonne NationalLaboratory (ANL) and Applied Natural Sciences, Inc.(ANS), which uses trees as phytoremediation plants to

explore deeper (down to 12m [40 feet]) soil horizons in atrademarked process called Treemediation

(Nyer and

Gatliff, 1996).

The phytoremediation concept is based on thewell-known ability of plants and their associatedrhizospheres to concentrate and/or degrade highly dilute

contaminants. Critical components of the rhizosphere, inaddition to a variety of free-living microorganisms,include root exudates. These complex root secretions,which "feed" the microorganisms by providingcarbohydrates, also contain natural chelating agents (citric,acetic, and other organic acids) that make the ions of bothnutrients and contaminants more mobile in the soil. Rootexudates may also include enzymes, such as nitroreductasedehalogenases, and laccases. These enzymes haveimportant natural functions, but they may also degradeorganic contaminants that contain nitro groups (e.g.,TNT, other explosives) or halogenated compounds(e.g., chlorinated hydrocarbons, many pesticides).

Plant roots and rhizosphere microorganisms "sense"the immediate soil environment in which they aregrowing and have complex feedback mechanisms thatpermit them to adapt to changing conditions as they grow.In some plants growing in phosphorus-deficient soil, theroot exudates contain large amounts of citric acid, in anattempt to mobilize and make available for uptake anyphosphorus compounds present. Some rhizospheremicroorganisms secrete plant hormones that increase rootgrowth, and thereby the secretion of root exudates thatcontain metabolites they use as an energy source.

Large green plants have the capability to move largeamounts of soil solution into the plant body through the

roots and evaporate this water out of the leaves as purewater vapor in a process called transpiration. Plantstranspire water to move nutrients from the soil solution toleaves and stems, where photosynthesis occurs, and tocool the plant. During this process, contaminants presentin the soil water are also taken up and sequestered,metabolized, or vaporized out of the leaves along with thetranspired water. Low water use is a trait considereddesirable in most economically important plants.However, some plants are notoriously poor at waterconservation, usually because they normally grow inmoist environments (e.g., hybrid poplars, willows,bulrush, marsh grasses). Such species are good candidatesfor phytoremediation plants because they take up and

"process" large volumes of soil water. For example, datashow that a single willow tree can, on a hot summer day,

transpire more than 19 m3

of water (19,000 liters or5,000 gal), and one hectare of a herbaceous plant like

saltwater cordgrass evapotranspires up to 80 m3

of water(80,000 liters or 21,000 gal) daily.

When we grow selected, adapted plants incontaminated substrates, the root system functions as ahighly dispersed, fibrous uptake system. Contaminantsover a large range of concentrations are taken up alongwith the water and degraded, metabolized, and/or

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sequestered in the plant body, while evapotranspirationfrom aerial parts maximizes the movement of soilsolution or wastewater through the plant. Through theprocess of bioaccumulation, contaminants can beconcentrated thousands of times higher in the plant than inthe soil or wastewater. The contaminated plant biomasscan be digested or ashed to reduce its volume (95%), and

the resulting small volume of material can be processed asan "ore" to recover the contaminant (e.g., valuable heavymetals, radionuclides). If recycling the metal is noteconomically feasible, the relatively small amount of ash(compared to the original biomass or the extremely largevolume of contaminated soil) can be disposed of in anappropriate manner.

Our studies are screening both hyperaccumulator andnonaccumulator plant species to identify candidate plantsfor phytoremediation of heavy metals, radionuclides, andorganics. It is well known that hyperaccumulator plantscan accumulate metals in large amounts. Demonstratingthat large plants that are not currently classified as

hyperaccumulators can compensate for somewhat loweraccumulation factors in above-ground organs with greaterbiomass production and high transpiration rates is criticalin proving the viability of field expolitability ofphytoremediation. This research is also increasing thenumber of potential phytoremediation plants by expandingthe type, range, and adaptability of species considered tobe hyperaccumulators.

Understanding contaminant uptake mechanismsincludes the study of root physiology and morphology,uptake kinetics, translocation in roots, stems, and leaves,and bioaccumulation in specific organs, as well as the roleof mycorrhizae and the rhizosphere. Many known

hyperaccumulator species are small and have specialgrowth requirements, such as species in the familyBrassicaceae (mustard family). Selected nonaccumulatorplants, though their specific toxic metal accumulationratios may be lower than those of hyperaccumulators, mayactually sequester more total metal because of their greaterbiomass and water uptake rates. Total contaminantremoval and binding capability are determined by manyplant attributes and factors being investigated by thisproject. These include tolerance to the contaminant;selectivity; accumulation capability; transpiration rate;plant size, biomass, and growth form; aerial surface area;root type, fibrosity, rooting depth, and harvestability;duration (annual, biennial, or perennial); dormancy; and

resistance to pests and disease.

The evaluation of plant potential for metalaccumulation by several selected plant species is beinginvestigated by growing plants in an inert substrate (quartzsand) in lysimeter pots. The inert substrate is used toeliminate any possible interferences with soil components(e.g., clay micelles, organic matter) that might affect thecontaminant availability to plants. Replicated treatmentsin a random scheme are used. Plants are irrigated withnutrient solution, and contaminants are supplied either atlow but constant doses in the nutrient, or at higher doses,

at specific application times. Leachate is drained from thepots each time nutrient is added. This is done at regularintervals, frequent enough so that measurable leachatevolumes are always present. Volume, pH, andconductivity are measured each time leachate is drained,and a sample is taken for metal analysis. Measuringleachate volume permits us to determine

evapotranspiration rates throughout the experiments.Plant leaves and branches are collected at regular intervalsfor metal analysis, and at the end of the experiments(about 4 months each), the pots are disassembled, andsamples are collected of the sand substrate, leachate, roots,and above-ground biomass.

For many contaminants, passive uptake viamicropores in the root cell walls (the apoplastic pathway)may be a major route into the root, where sequestration ordegradation can take place. The apoplast is a hydrated freespace continuum between the external soil solution andthe cell membranes of the root cortex and vascular tissue.The cell wall micropores exist within a network of

cellulose, hemicellulose, pectins, and glucoproteincontaining many negative charges (generated by carboxylicgroups) that act as cation binding sites and exchangers andas anion repellers. Di- and polyvalent cations (the form ofmany heavy metal and radionuclide contaminants) arepreferentially attracted to, and bound on, these cationexchange sites within the root cortex cell walls. Formetal ions to be metabolized or translocated to theaboveground parts of the plant, they must pass throughthe plasma membrane of a living cell, and this can onlyoccur by active transport processes. The inner limit of theroot cell wall free space, and hence of the passiveapoplastic radial transport of ions, is the endodermis,which forms the outer limit of the root vascular system,

or stele.

A major objective of this program is to determine thefeasibility of root harvesting as a method to maximize theremoval of contaminants from soils, since in most plantsthe roots sequester most of the contaminant that is takenup. Available techniques and equipment for harvestingplant roots, including young tree roots, are beingevaluated and modified as necessary for use withphytoremediation plants. Sequestration of heavy metalsin roots, as opposed to translocation to aboveground plantparts, reduces the likelihood of environmental dispersionof the contaminants via food chains by wildlife, domesticanimals, or birds.

We are also evaluating the use of large, robust plantswith high transpiration rates for the treatment ofwastewater, using a variety of hydroponic and aeroponicgrowth systems. The mobility of heavy metals and othercontaminants in soils by means of chelating agents, rootexudates, and other compounds is also being studied.

RESEARCH STATUS AND PLANS

ANL has been conducting basic and applied researchin phytoremediation since 1990. Initial greenhouse

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studies evaluated salt-tolerant wetland plants to clean upand reduce the volume of salty "produced water," thewastewater brought to the surface from wells producingnatural gas and crude oil (Hinchman and Negri, 1994b). Afield demonstration of a plant bioreactor for processingproduced water is under way at a natural gas well site inOklahoma. This system uses salt-tolerant marsh species

that simultaneously reduce the wastewater volume throughhigh rates of evapotranspiration, and remove contaminantsthrough uptake, degradation, metabolism and/orcomplexation. In this small-scale, two-compartmentbioreactor, each compartment processes wastewater ofincreasing salinity and contains a different plant species ora combination of species. The effluent water isconsiderably reduced in both volume and contaminants,while its salt concentration is increased. The usualdisposal method for produced water is deep well injection,the cost of which depends on the volume of wastewaterinjected. The bioreactor is based on a model that usesconservative greenhouse data and predicts a produced-watervolume reduction of 75% in less than eight days (Negri

and Hinchman, 1996). This represents a major potentialcost saving for the petroleum industry.

Currently, we are extending these studies and thephytoremediation concept to a wider variety of hazardouscontaminants and other waste site problems, throughgreenhouse studies of plant uptake of heavy metals inboth woody and herbaceous species, and throughpartitioning of the metal in specific plant organs. Ourcurrent research is expanding the plant hyperaccumulatorconcept to include large, robust species with highevapotranspiration rates. The plants investigated to datein our greenhouse experiments process more soil water (inwhich contaminants are dissolved) and sequester in their

tissues as much, or more, heavy metal than the"traditional" hyperaccumulators, many of which are small,shallow-rooted plants in the Brassicaceae (mustard) family.

In addition, we are developing a database ofphytoremediation literature. We have also identifiedcommercial sources for seed and transplants of many ofthe promising phytoremediation species.

Zinc Uptake in Hybrid Poplar

Recent greenhouse experiments on heavy metaluptake and sequestration by green plants include anexperiment studying zinc uptake in hybrid poplar

(Populus sp.) cuttings growing in inert quartz sand inlysimeter pots (Negri, Hinchman, and Gatliff, 1996).These experiments (Fig. 1), initiated in late March 1995,seek to confirm and extend field data from ANS indicatinghigh levels of zinc in leaf tissue of hybrid poplar growingat a cleanup site that had zinc contaminationapproximately 4.6m (15 ft) below the surface. Detaileddata were collected on contaminant uptake, translocation,and partitioning in plant organs, as well as onevapotranspiration rates, nutrient use, and biomassincrease of the rapidly growing poplar shoots. To obtainthese data, leachate volume, conductivity, and pH were

measured each day. To replace water lost viaevapotranspiration, fresh nutrient or water was added to theleachate each day to bring the total volume to either 1 or1.5 liters. The transpiration rate of potentialphytoremediation plants is considered to be a criticalfactor, because the transpiration rate determines the rate atwhich contaminated soil solution is drawn into the plant

to be processed.

In June 1995, when the poplars were growing welland had developed a normal root system, a series oftreatments was started in which three groups of plantswere given increasing doses of zinc ion in nutrientsolution over a period of about two months. These doseswere given in five zinc additions, with concentrationsranging from 50 to 2,000 g/g (ppm) Zn. Each groupconsisted of three replicates. On each zinc addition date,two groups received increasing doses up to 1,500 and2,000 g/g (ppm) Zn, respectively, while a control group(0 g/g [ppm]) received nutrient only.

The zinc was added to three replicate pots, in 500 mLof nutrient solution spiked with ZnSO4. These levels of

zinc, totally dissolved in nutrient solution, are equivalentto many times the available levels in "normal" soils withthe same amount of zinc, as revealed by available zincanalyses (DTPA extraction). In our experiments, there areessentially no binding sites in the sand growth substrate,as internal controls showed, in which the zincconcentration of nutrient solution remained unchangedwhen passed through lysimeter pots with quartz sandsubstrate but no plants. Prior to each zinc addition, sandsubstrate samples and plant tissues (roots, leaves, andbranches) were collected to determine how much zincremained in the substrate, and to track zinc translocation

and partitioning of bioaccumulation in poplaraboveground tissues.

Leachate analyses for zinc by atomic absorptionspectrophotometry indicate that in all cases, up to800 g/g (ppm) Zn added in nutrient solution, the zincwas totally and selectively absorbed and sequestered by theplants in about 4 hours during a single pass through theroot system contained in the lysimeter pot (Fig. 2). Atlevels of zinc above 1,000 g/g (ppm) in nutrient added tothe pots, leachate levels were always below 100 g/g(ppm) in samples making one pass through the lysimeterpot and taken the same day as the zinc addition; theselevels increased the following day, to concentrations up to548 g/g (ppm) for the 2,000 g/g (ppm) zinc addition,and then decreased sharply the second day after the zincaddition, to concentrations less than 100 g/g (ppm).Thereafter, the zinc concentration steadily decreased as theplants apparently reabsorbed the zinc as the nutrient wascycled through the pots on subsequent days. During theexperiment, this pattern of zinc concentration changes inleachate following a zinc addition was observed twice(Fig. 2).

The hybrid poplar plants in the lysimeter pots wereharvested in early September 1995. All of the

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aboveground tissue was harvested and divided into fourcategories (mature leaves, expanding leaves, old wood, andnew wood). The sand substrate was removed from eachpot as a "cylinder," with the undisturbed roots remainingwhere they had grown. After the sand cylinder wasphotographed with the roots in place, sand samples werecollected, and the sand was washed from the root system

with deionized water. Four types of roots (A, B, C, andD) were identified, on the basis of appearance,morphology, and color, and collected as separate samples.These types were clumps or wefts of white orlight-colored, new roots (A) originating at the perimeter orthe ends of large, woody roots (B). Very fine, hairlike,dark-colored roots (C) densely covered the surfaces of thewoody roots. Large-diameter, non-woody roots and fineroots that were grayish to brownish in color and occurredprimarily in the bottom of the lysimeter pots (which weresaturated most of the time) were grouped into a singlecategory (D). All tissue samples were dried at 80C andanalyzed for zinc by digestion, extraction, and atomicabsorption spectrophotometry.

Several leaf harvests were made during the course ofthe experiment. Initial leaf analyses showed 528 g/g(ppm) Zn in mature (large) leaves, 300 g/g Zn inmedium-sized leaves, and 140 g/g Zn in small leaves ofplants that received a single dose of 50 g/g (ppm) Zn innutrient solution. In aboveground plant parts (leaves andbranches) harvested at the end of the experiment, zincconcentrations did not exceed 2,250 g/g (ppm) Zn in thedry leaf tissue, or 900 g/g (ppm) Zn in the woodybranches, on a dry-weight basis.

Even at the highest zinc uptake levels, there wereonly subtle visual toxicity symptoms (slight leaf

chlorosis and some leaf "drooping") in the abovegroundparts of the experimental plants when compared tocontrols. However, in plants that received more than 800g/g (ppm) Zn, the evapotranspiration rate decreasedsignificantly as zinc concentration in the roots increased(Fig. 3); this physiological effect could have complexcauses.

The root tissues harvested at the end of theexperiment showed much higher concentrations ofaccumulated and sequestered metal than did theaboveground parts. In the pots receiving the highest doseof zinc (2,000 g/g [ppm]), the D roots contained a meanconcentration of 38,055 g/g (ppm) Zn in the dry root

tissue, while the C roots had 15,470, the A roots had12,225, and the B roots had 2,814 g/g (ppm) Zn,respectively. The pots receiving the lower zinc doses(maximum of 1,500 g/g [ppm]) had a similar pattern ofdistribution of zinc among root types; in this case, the Droots contained a mean concentration of 17,053 g/g(ppm) Zn. The control pots also showed the same zincuptake pattern among root types, the mean for the D rootsbeing 232 g/g (ppm) Zn. The small amount of zinc inthe controls is from the zinc present in the nutrientsolution as an essential nutrient. Figure 4 summarizesbiomass zinc content.

The hybrid poplar greenhouse experimentscomplement field studies and data collection on zincuptake by hybrid poplar trees growing under fieldconditions in installed remediation systems(Treemediation

), conducted by Applied Natural Sciences,

Inc. The implications for engineered soil and wastewatercleanup systems that use hybrid poplar growing either

directly in the soil or in special hydroponic systems arevery encouraging. Results from several Treemediation

systems that have been in place five years indicate thehydraulic control of a downgradient plume in a tight soilmatrix. Currently, approximately 18 systems are in placein the field, with additional sites being established during1997. Soil and groundwater as deep as 9m (30 ft) arebeing treated, and plants are under investigation for deeperconditions. With a properly engineered system, such asTreemediation

rooting activity in the contaminated area

has been realized in one to two years.

Zinc Uptake in Eastern Gamagrass

In a recent experiment, Eastern gamagrass transplantswere grown in inert quartz sand in lysimeter pots (Fig. 5).In April 1996, when the gamagrass was growing well andhad developed a normal root system, a series of treatmentswas started in which three groups of plants, each groupconsisting of five replicates, were continuously given zincion in nutrient solution at concentrations of 160 g/g Zn,600 g/g Zn, and 0 g/g Zn (control), respectively, over aperiod of about two months. Detailed data were collectedon zinc uptake, translocation, and partitioning in thegamagrass roots and shoots, as well as onevapotranspiration rates, nutrient use, and biomassincrease of the rapidly growing plants. Methods wereessentially identical to those described for the hybrid

poplar experiment.

Leachate analyses for zinc by atomic absorptionspectrophotometry indicate that initially plants subjectedto both levels of zinc were removing up to 70% of thezinc from the leachate. After two months, the plantsreceiving 160 g/g Zn had grown considerably and werealmost the same size as the controls (no zinc), but someof the mature leaf blades were rolled; the mean zincremoval rate for these plants was 50% of the zinc in theleachate. The plants receiving 600 g/g Zn were smallerthan the controls after two months, their color was adarker green, most of the mature leaf blades were rolled,and the mean zinc removal rate was about 30% of the zinc

in the soil solution (leachate).

In mid-June 1996 three replicates of each Easterngamagrass treatment were harvested. The sand substratewas removed from each pot as a "cylinder," with theundisturbed roots remaining where they had grown. Afterthe sand cylinder was photographed with the roots inplace, sand samples were collected, and the sand waswashed from the root system with deionized water. Theplants were divided into tops (leaves and crowns) androots. The root systems of all the plants had the typicalstructure of large primary roots and smaller, lateral,

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Figure 1. Hybrid Poplar (Populus sp.) Cuttings Growing in Lysimeter Pots

with Connecting Tubes and Leachate Collection Bottles Below

Figure 2

Zinc Concentration in Added Solution and Leachate

from Hybrid Poplar Lysimeter Pots

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Figure 3

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Hybrid Poplar Evapotranspiration, Mean Volume per Week

Figure 4

leaves new roots woody

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secondary roots. In the controls, most of the roots werelight colored, with the newest roots being white.However, in the plants receiving the 160 g/g Zntreatment, although the root systems were almost as largeas the controls, some of the secondary roots were dark orblack, while in the 600 g/g Zn treatment the rootsystems were small with many black roots. All tissue

samples were dried at 80C and analyzed for zinc bydigestion, extraction, and atomic absorptionspectrophotometry.

As was observed in the hybrid poplar zinc uptakeexperiment, the Eastern gamagrass root tissues harvestedin June 1996 showed much higher concentrations ofaccumulated and sequestered metal than did theaboveground parts (Fig. 6). In the pots receiving thehighest level of zinc in the nutrient solution (600 g/gZn), the roots contained a mean concentration of about10,000 g/g Zn in the dry root tissue, while the topscontained about 1000 g/g Zn. In the plants receiving160 g/g Zn, the roots contained about 4000 g/g Zn,

and the tops about 400 g/g Zn. The roots of the controlplants contained about 75 g/g Zn, and the tops about 50g/g Zn. The small amount of zinc in the controls isfrom the zinc present in the nutrient solution as anessential nutrient.

The two remaining replicates in each treatment wereallowed to continue growing under the same treatmentregime after the tops were pruned back to a length of 10cm above the substrate surface, to observe regrowthcharacteristics and any changes in zinc uptake.

The levels of sequestered zinc observed in both thegamagrass and hybrid poplar roots exceed the levels found

in either roots or tops of many of the knownhyperaccumulator species. We hypothesize that thegreater portion of the zinc is sequestered in the cell walltissue of the root cortex through internal complexationand detoxification, with subsequent translocation of arelatively small amount of the metal to the leaves andbranches. In hybrid poplar the evapotranspiration ratedecreased significantly as zinc concentration in the rootsincreased; while in gamagrass both top growth andevapotranspiration rate decreased at the higher zinc level(600 g/g Zn) as a constant dose.

In any case, a reduction of zinc from the soil solutionof between 90-50% (Eastern gamagrass), and 99+%

(hybrid poplar) when the concentration in the soil solutionis several hundred ppm has major implications for thedevelopment of effective plant-based cleanup systems forcontaminated soils, groundwater, and wastewater that isboth low-tech and low-cost. These data demonstrate avery effective uptake, bioaccumulation, and sequestrationsystem for zinc in both hybrid poplar and Easterngamagrass, as well as high evapotranspiration rates and awide range of adaptability for both of these versatileplants.

Eastern gamagrass is ideally suited to plant-based,hydroponic wastewater treatment systems consisting ofabove-ground open top tanks in which the plants aredensely grown in a bed of pea gravel, through which thewastewater is trickled for purification. Gamagrass canattain a height of over 2 meters, and develops anextensive, fibrous root system when grown

hydroponically. This very large root biomass providesnumerous sites for contaminant sequestration and/ordegradation. Because both tops and roots of gamagrass areuniform in development, the harvest of either of theseorgans will be relatively easy.

Bioremediation of Contaminated Soils b yEnhanced Plant Accumulation: Chelation-Assisted Metal Accumulation in Plants

Key factors that control the rate at which thecontaminants are removed from the soil by plant-basedcleanup systems are the ability of the plant to accumulate,without suffering toxic effects, large concentrations of

contaminant, and the rate and extent at which thecontaminants are made available for plant uptake.Phytoremediation is the result of matching the growth ofselected, adapted plants with the gradual but completerelease of the contaminants from the soils binding sitesinto the soil solution in plant-available forms.

Heavy metals are bound to soil components invarying degrees, depending on soil conditions such as pH,clay content, organic matter, redox potential. Plant-available metals include species that are readily soluble orexchangeable, while metals that are more stronglyadsorbed/bound to organic matter or co-precipitated withoxides are generally not available for plant uptake. One

way to increase metals availability is to selectivelyleach them into the soil solution using chelating agents.

Chelating agents increase metals diffusion in the soilsolution and keep them in plant available forms byforming large, less reactive ions, by increasing theconcentration of these larger chelated ions in solution, andby decreasing the ability of the free ions to react with thesoil. Natural chelating agents (organic acids such as citricand acetic) released by plant roots make the ions of bothnutrients and contaminants more mobile in the soil.Plants can usually break the chelation bond, take up themetal, and release the chelant back in the soil solution.

Current greenhouse experiments aim atinvestigating the feasibility of utilizing various chelatingagents to induce the release of heavy metals from soilbinding sites and increase the rate and extent of metalaccumulation in plant tissues. These experiments areinvestigating the changes in concentration factors ofseveral heavy metals (Zn, Cd, Pb, Cu) and radionuclides(U and Th) from soil/sediment from the Miami-ErieCanal, near Miamisburg, Ohio. This soil, althoughconsidered "clean, has concentrations of thesecontaminants sufficient to provide information on the

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Figure 5. Zinc Uptake in Eastern gamagrass (Tripsacum dactyloides)

Growing in Lysimeter Pots, Experimental Setup

Figure 6. Zinc Concentrations in Eastern gamagrass (Tripsacum dactyloides)

Tissues, Mean Values

0 g/gZn

160 g/gZn

600 g/gZn

0

2000

4000

6000

8000

10000

g/gZn

dry wt

0 g/gZn

160 g/gZn

600 g/gZn

Zn concentration in nutrient solution

RO O T S

TO PS/LEA V ES

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relative changes in plant accumulation induced by theaddition of chelating agents. Willow plants growing inthis soil in lysimeter pots are periodically irrigated withconstant doses of dilute chelating agents in aqueoussolution. At predefined time intervals, samples of soiland plant tissues are taken and analyzed for metals andradionuclides. The soil samples are analyzed via

sequential extraction techniques, which provide theempirical information on the probable species in whichthe contaminants are present, and the relative percentage ofthe total content. Plant concentration is considered as anadditional sequential extraction step, which represents thesum of metal species that are effectively taken up by theplant.

The Uptake of Lead and Arsenic byPhreatophytic Trees

In this recent greenhouse experiment we evaluated theuptake, translocation, partitioning, and sequestration oflead and arsenic in the organs of hybrid willow (Salix sp.)

and hybrid poplar (Populus sp.) trees grown incontaminated soil from a field site. Another group ofboth trees was grown in quartz sand and watered withsolutions containing soluble compounds of lead andarsenic. The contaminated soil contained about 1000 g/glead and up to 200 g/g arsenic on a total basis, but theplant-available concentrations in the soil water were below2 g/mL for both metals. The goal of this project,conducted in collaboration with our CRADA partner,Applied Natural Sciences, Inc., was to determine the roleof deep rooting phreatophytic trees in thephytoremediation of sites contaminated with heavymetals, and to develop optimal tree-based cleanupmethodologies to be tested in the field.

Rooted cuttings of hybrid willow and hybrid poplarwere grown in replicated sets of lysimeter pots containingeither contaminated soil or quartz sand for a period of fourweeks under greenhouse conditions. The pots containingcontaminated soil were watered with deionized water only,while separate sets of sand pots were watered withsolutions of lead nitrate and sodium arsenite, respectively.The concentrations of the metals in the watering solutionsused on the sand pots were 10 and 100 g/mL for bothlead and arsenic. At these concentrations, arsenic wasmore toxic than lead to both species. No toxicitysymptoms were observed in either tree species for bothconcentrations of lead.

Both willow and poplar showed uptake andpartitioning of both lead and arsenic in the sand media andin the contaminated soil. In the one-month test period,the willows were able to remove approximately 9.5percent of the available lead and about one percent of thetotal arsenic from the contaminated soil. In the same timeperiod, the less mature poplars removed about one percentof the available lead and 0.1 percent of the total arsenicfrom the same soil. in the sand experiment, the willows

took up about 40 percent of the administered lead and 30to 40 percent of the administered arsenic in the one-monthtest period. Considerably more of both lead and arsenicwas sequestered in the roots of both plants than in theabove ground parts ( branches and leaves).

Evaluation of Rooting Patterns and Bi om as s

Production of Hybrid Poplar as a Function o fPlanting Method of Cuttings.

This experiment is evaluating the rate and success ofroot generation and aboveground biomass production inhybrid poplar cuttings planted in quartz sand in bothvertical ("normal") and horizontal patterns at differentdepths. This experiment is aimed at developing plantingtechniques that may enhance or facilitate root recoveryduring whole-plant harvesting.

Data generated so far confirm that hybrid poplar canroot extensively and produce multiple vertical shoots fromhorizontally planted stem cuttings. The roots produced by

horizontal cuttings are formed all along the length of theburied cutting and explore the shallow soil horizons morecompletely than roots of vertically-planted cuttings.Thus, hybrid poplar rooting patterns can be relativelyeasily molded to match the existing contamination patternand the needs of root harvesting machines.

Evaluation of TCE and PCE Uptake and Fatein Plants.

For many organic contaminants such astrichloroethylene (TCE) and tetrachloroethylene (PCE),,there is evidence that plants can degrade a portion of theorganohalide that is taken up to form less volatile

compounds such as trichloroacetic acid (TCAA) which aresequestered in the plant tissue, while passing theremainder out of the leaf tissue with the transpirationstream.

In hybrid poplar and several other species there isevidence for an enzyme that breaks down TCE and PCE toTCAA in the plant. We are developing new gaschromatographic analytical methods for the detection ofTCAA and TCEh in woody plant tissue that is rapid andeasy. A number of samples of plant tissue have beenanalyzed using this method, and studies are ongoing tocorrelate the presence and concentration of TCAA inplants and its contamination "history" in the underlying

groundwater or soil.

These methods have the potential for tracking theplant-based cleanup of groundwater and soil contaminatedwith organohalides, as well as providing a very quick andeasy method of monitoring a site for organohalidecontamination using plant tissue samples from the siterather than obtaining soil and groundwater samples bycoring or drilling operations.

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Future Plans

Future plans include the following research andapplication studies on plant-based cleanup systems:

Field demonstration of heavy metal removal byselected tree species, including root harvesting, atour CRADA partner's sites.

Conduct high resolution microanalyses ofhyperaccumulator species (scanning ortransmission electron microscopy with x-rayenergy dispersive spectroscopy) to determine thediscrete sites of metal sequestration andbioaccumulation in specific plant organs, tissues,cells, and organelles.

Evaluate procedures for the disposal, processing,and volume reduction of metal-contaminatedplant biomass.

Conduct studies of root and other plant biomassdecomposition in soils to understand kinetics andcycling of contaminants.

Evaluate the removal and sequestration ofradionuclides from soils using plant-basedcleanup systems.

Continue the evaluation of demonstration unitsof an ANL-developed plant bioreactor forwastewater (produced water) treatment at naturalgas well sites in Oklahoma.

Extend investigations of the most promisinglines of current research on plant-based cleanupsystems:

- phytoremediation using trees and large,robust, herbaceous plants.

- degradation of halogenated organics.

- bioreactors for wastewater cleanup andvolume reduction.

ACKNOWLEDGMENTS

Work supported by the U.S. Department of Energy,Assistant Secretary for Energy Efficiency and RenewableEnergy, under Contract W-31-109-Eng-38.

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PHYTOREMEDIATION BIBLIOGRAPHY

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1998. Phytoremediation of Selenium Contaminated Soils.Soil and Groundwater Cleanup pp. 6-10, Feb./March1998.

Elsenheimer, D. 1998. Alfalfa Used to RemediateContaminated Railway Site. Soil and GroundwaterCleanup pp. 11-14, Feb./March 1998.

Goldsmith, W. 1998. Lead Contaminated SedimentsProve Susceptible to Phytoremediation. Soil andGroundwater Cleanup pp. 15-18, Feb./March 1998.

Medina, V.F., R. Rivera, S.L. Larson, and S.C.McCutcheon. 1998. Phytoreactors Show promise in

Treating Munitions Contaminants. Soil and GroundwaterCleanup pp. 19-24, Feb./March 1998.

Environmental Protection Agency. 1997. RecentDevelopments for In Situ Treatment of Metal-Contaminated Soils. Document EPA-S 42-R-97-004.

Hinchman, Ray R. and M. Cristina Negri. 1997.Providing the Baseline Science and Data For Real-LifePhytoremediation Applications - Partnering for Success,.Chapter 1.5. In, Proceedings of the 2nd Intl. Conferenceon Phytoremediation, Seattle WA, June 18-19, 1997.

Negri, M. Cristina, Ray R. Hinchman, and Jerry

Mollock. 1997. Biotreatment of Produced Waters forVolume Reduction and Contaminant Removal. In,Proceedings of the 4th Intl. Petroleum Conference,Sept. 9-12, 1997.

Newman, L.A., et al.. 1997. Uptake andBiotransformation of Trichloroethylene by HybridPoplars. Environmental Science and Technology31(4):1062-1067.

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Sytsma, L., J. Mulder, J. Schneider, C. Negri, R.Hinchman, and E. Gatliff. 1997. Uptake and Fate ofOrganohalogens from Contaminated Groundwater inWoody Plants. In, Book of Abstracts, 213th American

Chemical Society National Meeting, San Francisco, April13-17, 1997.

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Wiley, J.P. 1997. Wastewater Problem? Just Plant a

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Anon. 1997. Phytoremediation Becoming Quite "Poplar".The Hazardous Waste Consultant 15(3):1.16-1.20 (May-June 1997).

Adler, T. 1996. Botanical Cleanup Crews - Using plantsto tackle polluted water and soil. Science News 150:42-43(July 20, 1996)

Carey, J. 1996. Can Flowers Cleanse the Earth?BusinessWeek, Feb. 19, 1996, p.54.

Cunningham, S.D. and D.W. Ow. 1996. Promises and

Prospects of Phytoremediation - Update onBiotechnology. Plant Physiology 110:715-719.

Cunningham, S.D., T.A. Anderson, A.P. Schwab, andF.C. Hsu. 1996. Phytoremediation of Soils Contaminatedwith Organic Pollutants. Advances in Agronomy 56:55-114.

Dutton, G. 1996. Stemming the Toxic Tide. CompressedAir101(4):38-42 (June 1996).

McCleary, H. 1996. CRADA Partners Root for Field ofTrees. The Bio-Cleanup Report5(3):6 (February 1, 1996).

Negri, M.C. and R.R. Hinchman. 1996. Plants ThatRemove Contaminants From the Environment.Laboratory Medicine 27(1): 36-40.

Anon. 1996. Phytoremediation Gets to the Root of SoilContamination. The Hazardous Waste Consultant pp.1.22-1.24 (May-June 1996).Black, H. 1995. Absorbing Possibilities:Phytoremediation. Environmental Health Perspectives103(12):1106-1108 (December 1995).

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Salt, D.E., M. Blaylock, N.P.B.A. Kumar, V.Dushenkov, B.D. Ensley, I. Chet, and I. Raskin. 1995.Phytoremediation - A Novel strategy for the Removal ofToxic Metals from the Environment Using Plants.Biotechnology 13(5): 468-474.

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Johnson. 1994. Biotreatment of Produced Water UsingGreen Plants and Hydroponics. p.171In AAPG 1994Annual Convention, Denver, CO (June 12-15, 1994).

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CONTACT INFORMATION:

Argonne National Laboratory :

Ray R. HinchmanPhone: (630) 252-3391FAX: (630) 252-6407E-mail: [email protected]

M. Cristina NegriPhone: (630) 252-9662FAX : (630) 252-9281E-mail: [email protected]

Applied Natural Sciences. Inc.:

Edward G. Gatliff:Phone: (513) 942-6061

FAX: (513) 942-6071E-mail: [email protected]

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