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Enhanced degradation of TCE on a Superfund site using endophyte-assisted poplar tree phytoremediation Sharon Lafferty Doty, John Leonard Freeman, Christopher M. Cohu, Joel Gerard Burken, Andrea Firrincieli, Andrew Simon, Zareen Khan, Jud G. Isebrands, Joseph Lukas, and Michael J. Blaylock Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01504 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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Environmental Science & Technology

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Enhanced degradation of TCE on a Superfund site using endophyte-assisted

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poplar tree phytoremediation

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Sharon L. Doty1*#, John L. Freeman2#, Christopher M. Cohu3, Joel G. Burken4, Andrea

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Firrincieli5, Andrew Simon6, Zareen Khan1, J. G. Isebrands7, Joseph Lukas8 and Michael J.

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Blaylock6#.

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#Authors contributed equally to this work

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1

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Intrinsyx Technologies Corporation, NASA Ames Research Park, Moffett Field, CA 940351000

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3

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Edenspace Systems Corporation, Purcellville, Virginia 20134

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Environmental Forestry Consultants LLC, New London, WI 54961 USA

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Earth Resources Technology, Inc., Moffett Field, CA. 94035

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Environment, University of Washington, Seattle, WA 98195-2100. (206) 616-6255.

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[email protected]

University of Washington, Seattle, WA 98195-2100 USA

Phytoremediation and Phytomining Consultants United, Grand Junction, Colorado 81507

Department of Civil, Architectural, and Environmental Engineering, University of Missouri, Rolla, MO 65409 Dept. for Innovation in Biological, Agro-Food, and Forest Systems, University of Tuscia, Viterbo, Italy

* Corresponding author: School of Environmental and Forest Sciences, College of the

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Abstract:

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Trichloroethylene (TCE) is a widespread environmental pollutant common in groundwater

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plumes associated with industrial manufacturing areas.

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characterized a natural bacterial endophyte, Enterobacter sp. strain PDN3, of poplar trees, that

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rapidly metabolizes TCE, releasing chloride ion. We now report findings from a successful

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three-year field trial of endophyte-assisted phytoremediation on the Middlefield-Ellis-Whisman

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Superfund Study Area TCE plume in the Silicon Valley of California. The inoculated poplar

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trees exhibited increased growth and reduced TCE phytotoxic effects with a 32% increase in

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trunk diameter compared to mock-inoculated control poplar trees. The inoculated trees excreted

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50% more chloride ion into the rhizosphere, indicative of increased TCE metabolism in planta.

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Data from tree core analysis of the tree tissues provided further supporting evidence of the

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enhanced rate of degradation of the chlorinated solvents in the inoculated trees. Test well

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groundwater analyses demonstrated a marked decrease in concentration of TCE and its

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derivatives from the tree-associated groundwater plume. The concentration of TCE decreased

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from 300 µg/L upstream of the planted area to less than 5 µg/L downstream of the planted

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area. TCE derivatives were similarly removed with cis-1,2-dichloroethene decreasing from 160

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µg/L to less than 5 µg/L and trans-1,2-dichloroethene decreasing from 3.1 µg/L to less than 0.5

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µg/L downstream of the planted trees. 1,1-dichloroethene and vinyl chloride both decreased from

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6.8 and 0.77 µg/L, respectively, to below the reporting limit of 0.5 µg/L providing strong

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evidence of the ability of the endophytic inoculated trees to effectively remove TCE from

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affected groundwater. The combination of native pollutant-degrading endophytic bacteria and

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fast-growing poplar tree systems offers a readily deployable, cost-effective approach for the

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degradation of TCE, and may help mitigate potential transfer up the food chain, volatilization to

We had previously isolated and

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the atmosphere, as well as direct phyto-toxic impacts to plants used in this type of

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phytoremediation.

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Table of Contents/Abstract Art

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Photo courtesy of Edenspace Systems Corporation (M. Blaylock)

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Introduction:

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The United States Environmental Protection Agency (EPA) prioritizes sites for federal

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remediation assistance through the Superfund program based on criteria related to environmental

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risk and need for remediation (1). One of the most common organic pollutants at Superfund sites

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is trichloroethylene (TCE)(2), a known human carcinogen (3). Used extensively as a solvent and

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degreaser (2), its unconfined use, spills, landfill leachate, and improper disposal resulted in

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contamination of the soil and groundwater. The U.S. EPA has estimated that TCE is a soil or

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water contaminant on more than 1000 National Priorities List sites (1). The EPA has set the

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drinking water maximum contaminant level at 5 µg/L (4). The prevalence of TCE contamination

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coupled with the low action levels creates a substantial demand for effective remediation

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approaches.

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Current remediation methods of contaminated groundwater rely on enhancing volatilization of

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TCE through air stripping or sparging, sorption using activated carbon or zero valent iron

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barriers, or reductive dechlorination and degradation through bioremediation (5). While

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remediation costs vary widely depending on site conditions and method, at an estimated capital

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cost of installing conventional systems (i.e., pump and treat) of between USD $700,000 and USD

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$3,000,000 per site plus annual operating costs averaging approximately USD $200,000 (6), the

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addressable market for TCE remediation on the currently identified NPL alone exceeds USD

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$2.5 billion. Phytoremediation is an alternative strategy that utilizes a plant’s natural ability to

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take up chemicals from water and soil with its expansive root system, to degrade organic

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pollutants or sometimes to sequester inorganic pollutants (7). Most phytoremediation projects

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anticipate a cost savings of approximately 50 to 75% compared to traditional engineering



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methods (8). Poplar (Populus sp.) is an attractive plant for phytoremediation of TCE and other

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organic contaminants due to its compatibility with bioaugmentation systems, high growth rate,

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extensive root system, and high rates of water uptake from the soil (9). Poplar’s high water use

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can be used to contain contaminated ground water plumes and also to take up and degrade TCE

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to less toxic forms (10). However, in some cases, the pollutant concentration is phytotoxic,

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limiting the ability of the plant to effectively remove the chemical.

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Phytoremediation can be potentially improved by using genetic engineering and by bio-

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augmenting with pollutant-degrading bacteria (11-18). Transgenic trees (19-23) and other

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suitable phytoremediation plant species (24,25) engineered to overexpress key enzymes can

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potentially have substantially improved pollutant detoxification and removal. Alternatively,

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partnering plants with effective pollutant-degrading microorganisms has also shown promise,

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and research in this area has increased dramatically in the last decade (26) demonstrating near-

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term application potential without the need for the high-cost and lengthy deployment process

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associated with transgenic plants.

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In natural systems, plants can use symbiosis with internal microorganisms, termed endophytes

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(27), to adapt to environmental challenges, including pollutants (28-30). Plants in contaminated

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areas have a high number of endophytes which are capable of degrading the pollutant (31-34).

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This effect can be plant genotype-specific, suggesting that some plant species or ecotypes are

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better able to recruit or stably interact with the beneficial microorganisms (35-38). With the first

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demonstration that inoculation of plants with a specific pollutant-degrading endophyte can lead

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to improved removal and detoxification of the pollutant (39), lab-scale tests have demonstrated


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that specific endophyte strains improve host plant removal and/or detoxification of PAHs (40-

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43), toluene (44-47), diesel and petroleum (36,48), TCE (49,50), explosives (51,52), textile

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effluent (53), and metals (54-59).

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phytoremediation success with indirect benefits (60,61) including increased nutrient acquisition,

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more extensive root systems, and increased environmental stress tolerance (62-65).

The use of endophytes potentially also increases

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Despite these numerous lab or greenhouse-based studies, there are few reports of field trials of

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endophyte-assisted phytoremediation (26). The in situ addition of a poplar root endophyte strain

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engineered to degrade TCE resulted in reduced evapotranspiration of TCE (66). However, no

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direct evidence was provided on increased removal of groundwater TCE or degradation of TCE

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in planta.

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resulted in increased removal of TNT under laboratory conditions but had no impact under field

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conditions (67). Bioaugmentation in general is thought to have less success at field scale due to

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the possibility of native soil microorganisms harming or out-competing the artificially added

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strains (5). The complexity of field trials where the pollutants are often heterogeneous can also

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make precise assessments of phytoremediation success challenging.

Axenic plants inoculated with a TNT-degrading strain of Pseudomonas putida

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We previously reported on the isolation and characterization of a TCE-degrading endophyte

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strain (68). This Enterobacter sp. strain PDN3, isolated from hybrid poplar collected from a

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TCE-contaminated site, grows well on high levels of TCE.

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dechlorinated TCE without the addition of co-metabolite inducers. We report herein on the first

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successful phytoremediation field trial of bio-augmentation with this natural TCE-degrading


Strain PDN3 aerobically


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endophyte strain, demonstrating a low-cost, simple approach to improving tree growth and

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pollutant degradation.

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Materials and Methods

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Bacterial strain. Endophyte strain Enterobacter sp. PDN3 was originally isolated from hybrid

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poplar (Populus deltoides x nigra) in a screen for endophytes tolerant to 5.5 mM TCE (68).

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Cryogenically stored microbial stocks had been prepared previously by growing the strain in

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MG/L broth (69) containing approximately 300 µg/ml TCE, freezing in 33% sterile glycerol, and

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storing at -70ΕC.

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Verification of colonization ability. Prior to the field testing, the ability of strain PDN3 to

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colonize hybrid poplar clone OP367 (P. deltoides x nigra) was tested using fluorescent

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microscopy. The pBHR:gfp plasmid (70) was introduced into strain PDN3 using triparental

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mating (71) and transconjugants were selected on MG/L agar containing carbenicillin to select

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for PDN3 and kanamycin to select for the plasmid. gfp-PDN3 was grown in MG/L broth for

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24hrs and washed twice in deionized water by centrifugation. The pellet was resuspended in

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0.5X Hoagland’s solution (72), adjusted to an optical density (OD) at 600 nm of 0.1 using Unico

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spectrophotometer model 1000 (Fisher), and exposed to poplar roots for one week at room

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temperature. Colonization was visualized after 48 hours and one week using a Zeiss Imager M2

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equipped with an AxioCam MRM, and recorded with Zeiss AxioVision software (Carl Zeiss,

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LLC, USA). The microscopy experiments were done twice with two biological replicates.

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Field trial experimental design and tree plot establishment. A field site at Moffett

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Field/NASA-Ames Research Park was selected for a demonstration site, and permitting was

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completed with assistance from NASA environmental division and facilities groups. The

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Middlefield-Ellis-Whisman (MEW) Superfund Study Area was contaminated due to industrial,

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manufacturing, and military operations, and has been the subject of remediation studies since the

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1980's (73). As shown in Figure 1 and Supplementary Figure S1, planting sites were chosen in

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three areas near the center of the plume where the TCE concentration was in the 100-1000 µg/L

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range.

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In 2013, the planting areas were prepared by mowing and removal of existing vegetation. Three

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commercial varieties of poplar were selected for the study based on the geographical area and

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site conditions. Nine-inch dormant cuttings of OP-367, 15-029, and 311-93 were obtained for

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planting from Segal Nurseries (Grandview, WA). Half the number of cuttings of each variety

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were soaked in quarter-strength Hoagland’s solution (Control Poplar, CP) and the other half were

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inoculated (Endophyte-inoculated Poplar, EP) with the endophyte strain by placing the cuttings

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upright in an 8 L container and soaking in a 0.1 OD600 suspension of the PDN3 culture in

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quarter-strength Hoagland’s solution at a depth of approximately 10 cm for 24 hours prior to

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planting. Details of the planting and maintenance are in the Supplementary Materials.

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Tree Growth Assay. Tree growth was measured periodically throughout the study using tree

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base and breast height trunk diameter measurements.

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Assessing chloride level in the rhizosphere.

As a means of assessing phytoremediation

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performance with respect to TCE degradation, chloride levels were measured as a breakdown

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product from the TCE in samples collected from the rhizosphere of Control Poplar (CP) and

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Endophyte-Inoculated Poplar (EP) trees sixteen months from the beginning of the field trial.

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The samples were collected by moving aside the surface of the soil localized around the tree

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stems, collecting the rhizospheric soil from around the visible roots, and placing the samples in

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sterile 50-ml conical tubes. Approximately 2.0 grams of soil were ground with a mortar and

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pestle to pass through a 10 mesh sieve (< 2 mm) and mixed in order to homogenize the sample.

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Two-hundred mg of homogenized soil were weighed using an analytical balance (resolution ±

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0.0001 milligrams) and suspended in 25 mL of 4% acetic acid solution in 125 mL Erlenmeyer

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flasks. The flasks were sealed and incubated at room temperature under shaking conditions at

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110 rpm for 30 minutes. Soil particles were then removed by filtering the suspension through a

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Whatman® No. 1 filter paper (Sigma-Aldrich). Filtered samples were collected into glass tubes,

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sealed, and stored at 4 ° C for further analysis. Chloride (Cl) levels were assessed using a

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chloride-selective electrode (Thermo-Fisher Scientific). A calibration curve for low-level

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measurements was prepared as described by the manufacturer using a NaCl solution (chloride

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standard) prepared in deionized water (DQ3, Millipore) supplemented with 1 mL of Ionic

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Strength Adjustor (ISA) solution (1.0 M NaNO3). After each increment, millivolt potential was

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registered and plotted against Cl concentration. Finally, chloride concentration of each sample

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was calculated interpolating millivolt values to the corresponding calibration curve.

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Tree Core Measurements of TCE and breakdown products. Tree cores were taken using

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standard coring methods from both EP and CP trees by using a 5-mm diameter increment borer


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inserted to a minimum depth of 1½ (4 cm) inches. Within seconds of collection, the cores were

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placed into 20-mL PTFE-sealed head space vials. Samples were shipped the next day to the

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Missouri University of Science and Technology Environmental Engineering. Analysis of TCE

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and other chlorinated solvents in the transpiration stream of the trees was carried out using solid-

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phase micro extraction (HS-SPME) coupled with gas chromatography and electron capture

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detection (GC-µECD). Quantification was accomplished using a 5 point standard curve with

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external standards. An R2 > 0.98 was deemed acceptable. This equilibrium passive sampling

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method was developed speciﬁcally for tree core cVOC sampling using a 100 µm

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polydimethylsiloxane (PDMS) ﬁber (74). Method detection limits were below 8 ng/l for TCE

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and 0.5 ng/l for PCE in the aqueous solution in plant tissues (75).

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Groundwater test wells. Well borings were drilled to a depth of 17-feet below ground surface

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(bgs) using 8-inch hollow stem augers. The well screens, casings and completion materials were

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installed as per the attached well completion logs. The water samples were collected via “Hydro-

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sleeves”, which are plastic-like baggies that are collapsed when deployed to the bottom of the

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well, and then raised quickly for approximately 1-foot which causes the sleeve to open at the top

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allowing the well water to completely fill the sleeve within the screened aquifer zone (zone of

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interest). The wells were installed on 9/20/16. Test well 1 is approximately 20-feet south of the

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plot (the up-gradient/incoming well), while test well 2 is to the north of the plot (the

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downgradient/outgoing well). Groundwater samples were collected on 9/28/16 and again on

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11/21/2016 immediately prior to leaves turning yellow and winter senescence. The analytical

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method for the groundwater samples was EPA Method 624 normally utilized for Volatile

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Organic Compounds (VOCs) analysis.



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Verification of PDN3 colonization of the field site trees. To verify that the EP plants at the

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field test site had been colonized with PDN3, branch samples collected from the trees in spring

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2016 were shipped from the field site to the University of Washington, surface sterilized with

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0.6% sodium hypochlorite for 10 minutes and rinsed four times with sterile water. We chose to

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test branch samples because PDN3 can effectively colonize poplar vascular tissue, migrating

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from root to shoot or shoot to root depending on inoculation site (unpublished data). One node

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per tree sample was weighed and extracted with 0.5 ml 0.1X Hoagland’s solution per gram of

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tissue in separate sterile mortars. Resulting extracts were stored at -70ΕC. Samples of the

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extracts from 6 CP trees and 7 EP trees were randomly chosen, with strain PDN3 included as a

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reference, and inoculated into M9 medium containing 0.4% peptone (4) and 0.5% mannitol.

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After 3 days at 30ΕC, 1 ml samples were pelleted in a microcentrifuge, the cells resuspended in 1

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ml M9 with peptone, and the OD600 was measured. Cultures were adjusted to an OD600 of 0.1 in

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3 ml M9 medium with 0.4% peptone and 730 µg/ml TCE in 40-ml amber VOA vials. After 15

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days exposure to the high level of TCE, samples were plated on M9 with peptone, and restreaked

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for colony purification.

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subjected to colony PCR using 16S rRNA gene primers as described previously (76). PCR

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products were treated with ExoSAP-IT (Affymetrix) after verification by electrophoresis to be

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single 1.5 Kb products with no PCR product from a water blank control.

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sequenced by GeneWiz (Seattle) and identified using NCBI BLAST (77). For samples identified

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as Enterobacter species, PCR was repeated and the products cloned into pGEM-T-Easy

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(Promega), and sequenced twice. The 16S rRNA sequences were compared to that of PDN3

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(GenBank #JN634853) using the alignment program of NCBI BLAST.

Colonies of the dominant morphology and similar to PDN3 were


Samples were

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Comparative genomic analysis of Enterobacter sp. Strain PDN3. The PDN3 genome was

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sequenced and assembled at the Department of Energy Joint Genome Institute (JGI) as part of

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the research topic “Defining the poplar root microbiome”. Genome annotation, performed at the

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JGI, was carried out using the DOE-JGI Microbial Annotation Pipeline (DOE-JGI MAP) (78).

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Enzyme Commission (EC) numbers were used as annotation terms to define a functional profile

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to investigate the genetic potential of PDN3 to metabolize or co-metabolize TCE. The

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chloroalkane and chloroalkene degradation pathway and the metabolism of xenobiotics by

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cytochrome P450 from the KEGG database were used as reference pathways. Once the

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functional profile was defined, the “missing” enzymes were further analyzed using the ‘Find

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Candidate Genes for Missing Function’ tool from the IMG/ER platform. This step was

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performed using two different approaches; for search by homology, a percent identity cutoff of

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30 % and an E-value cutoff of 1e-2 were used, while a search by functional orthologs was

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performed with a percent identity cutoff of 10 %. Both steps were executed by querying the IMG

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database. Similarly, the genetic potential of PDN3 to regulate heavy metal homeostasis was

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investigated.

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Statistical analysis. Data analysis was carried out in R v3.2.3, performing a Tukey’s HSD

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(Honest Significant Difference) as post-hoc test in conjunction with a One Way ANOVA. For

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TCE levels in core samples the non-parametric test Kruskal-Wallis rank sum test was used.

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RESULTS AND DISCUSSION

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Verification of the colonization ability of endophyte strain PDN3. To verify that PDN3 was

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capable of colonizing the hybrid poplar, the fluorescently tagged PDN3 (pBHR:gfp) strain was

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used. After 48h of co-cultivation with poplar roots, the strain was observed to begin colonization

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through the formation of a biofilm at the junctions between the primary and secondary roots

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(Figure 2a). Crack entry at lateral root junctions is a common mode of colonization by

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endophytes (60,79). After one week of co-inoculation, gfp-PDN3 was verified to be within the

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host plant (Figure 2b). Since strain PDN3 was originally isolated from another hybrid P.

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deltoides x nigra poplar clone, it was expected that it would be able to effectively colonize the

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hybrid poplar used in the study.

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Observable growth differences in EP and CP trees. The preparation and plantings on the

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MEW plume resulted in successful establishment of the three poplar test plots directly over the

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MEW plume in an area of TCE concentrations ranging from 100-1000 µg TCE / L (Figure 1).

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In 2014 after one year of growth, it was apparent that the PDN3 inoculated poplar (EP) trees

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were more robust than the un-inoculated control (CP) trees (Figure 3). This may have been due

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to several factors. The endophyte may have reduced the TCE load within the trees by rapidly

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metabolizing the pollutant taken up by the plant, thereby directly reducing phytotoxic effects.

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Although the TCE concentration in the plume itself may not have been high enough to be

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phytotoxic, the condition of the CP trees indicated that TCE did impact plant growth. TCE may

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have been present in the upper rhizosphere possibly due to the nature of the volatile organic

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contaminant, a persistent dry soil condition and an unconfirmed volatilization up through

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cracked dry soil. In addition to benefitting the plant through detoxification of TCE, the

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endophyte may have enhanced root growth and overall health due to its ability to produce


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phytohormones. PDN3 has the biosynthetic genes for butane 2,3-diol and acetoin (80), volatile

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organic compounds shown to be important for increasing plant stress tolerance (81-83). The

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strain also produces indole acetic acid (unpublished data), an auxin that induces root formation

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and plant growth (64). The combination of both the endophyte strain and the right tree variety

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was necessary for success. The OP367 variety was the hybrid of choice for this site due to its

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larger, more robust growth when compared to the other two varieties.

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In order to assay the difference between CP and EP trees after two and three years of tree

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growth, the tree trunk diameter at breast height was recorded for all blocks on site for the OP-367

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variety. This data showed a statistically significant 32% increase in trunk diameter growth of the

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PDN3 inoculated trees after two and three years of growth when compared directly to the control

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un-inoculated trees (Figure 4. p

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