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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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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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
5
Firrincieli5, Andrew Simon6, Zareen Khan1, J. G. Isebrands7, Joseph Lukas8 and Michael J.
6
Blaylock6#.
7
#Authors contributed equally to this work
8
1
9 10
2
Intrinsyx Technologies Corporation, NASA Ames Research Park, Moffett Field, CA 940351000
11
3
12 13
4
14 15
5
16 17 18 19 20 21 22 23
6
Edenspace Systems Corporation, Purcellville, Virginia 20134
7
Environmental Forestry Consultants LLC, New London, WI 54961 USA
8
Earth Resources Technology, Inc., Moffett Field, CA. 94035
24
Environment, University of Washington, Seattle, WA 98195-2100. (206) 616-6255.
25
[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.
34
characterized a natural bacterial endophyte, Enterobacter sp. strain PDN3, of poplar trees, that
35
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
46
area. TCE derivatives were similarly removed with cis-1,2-dichloroethene decreasing from 160
47
µg/L to less than 5 µg/L and trans-1,2-dichloroethene decreasing from 3.1 µg/L to less than 0.5
48
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.
72 73
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.
99 100
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
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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.
139 140
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
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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
253
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
255
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
258
investigated.
259 260
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.
263 264
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
272
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.
275 276
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
292
and plant growth (64). The combination of both the endophyte strain and the right tree variety
293
was necessary for success. The OP367 variety was the hybrid of choice for this site due to its
294
larger, more robust growth when compared to the other two varieties.
295 296
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
298
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
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Environmental Science & Technology
1
Enhanced degradation of TCE on a Superfund site using endophyte-assisted
2
poplar tree phytoremediation
3 4
Sharon L. Doty1*#, John L. Freeman2#, Christopher M. Cohu3, Joel G. Burken4, Andrea
5
Firrincieli5, Andrew Simon6, Zareen Khan1, J. G. Isebrands7, Joseph Lukas8 and Michael J.
6
Blaylock6#.
7
#Authors contributed equally to this work
8
1
9 10
2
Intrinsyx Technologies Corporation, NASA Ames Research Park, Moffett Field, CA 940351000
11
3
12 13
4
14 15
5
16 17 18 19 20 21 22 23
6
Edenspace Systems Corporation, Purcellville, Virginia 20134
7
Environmental Forestry Consultants LLC, New London, WI 54961 USA
8
Earth Resources Technology, Inc., Moffett Field, CA. 94035
24
Environment, University of Washington, Seattle, WA 98195-2100. (206) 616-6255.
25
[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
33
plumes associated with industrial manufacturing areas.
34
characterized a natural bacterial endophyte, Enterobacter sp. strain PDN3, of poplar trees, that
35
rapidly metabolizes TCE, releasing chloride ion. We now report findings from a successful
36
three-year field trial of endophyte-assisted phytoremediation on the Middlefield-Ellis-Whisman
37
Superfund Study Area TCE plume in the Silicon Valley of California. The inoculated poplar
38
trees exhibited increased growth and reduced TCE phytotoxic effects with a 32% increase in
39
trunk diameter compared to mock-inoculated control poplar trees. The inoculated trees excreted
40
50% more chloride ion into the rhizosphere, indicative of increased TCE metabolism in planta.
41
Data from tree core analysis of the tree tissues provided further supporting evidence of the
42
enhanced rate of degradation of the chlorinated solvents in the inoculated trees. Test well
43
groundwater analyses demonstrated a marked decrease in concentration of TCE and its
44
derivatives from the tree-associated groundwater plume. The concentration of TCE decreased
45
from 300 µg/L upstream of the planted area to less than 5 µg/L downstream of the planted
46
area. TCE derivatives were similarly removed with cis-1,2-dichloroethene decreasing from 160
47
µg/L to less than 5 µg/L and trans-1,2-dichloroethene decreasing from 3.1 µg/L to less than 0.5
48
µg/L downstream of the planted trees. 1,1-dichloroethene and vinyl chloride both decreased from
49
6.8 and 0.77 µg/L, respectively, to below the reporting limit of 0.5 µg/L providing strong
50
evidence of the ability of the endophytic inoculated trees to effectively remove TCE from
51
affected groundwater. The combination of native pollutant-degrading endophytic bacteria and
52
fast-growing poplar tree systems offers a readily deployable, cost-effective approach for the
53
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
55
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:
62
The United States Environmental Protection Agency (EPA) prioritizes sites for federal
63
remediation assistance through the Superfund program based on criteria related to environmental
64
risk and need for remediation (1). One of the most common organic pollutants at Superfund sites
65
is trichloroethylene (TCE)(2), a known human carcinogen (3). Used extensively as a solvent and
66
degreaser (2), its unconfined use, spills, landfill leachate, and improper disposal resulted in
67
contamination of the soil and groundwater. The U.S. EPA has estimated that TCE is a soil or
68
water contaminant on more than 1000 National Priorities List sites (1). The EPA has set the
69
drinking water maximum contaminant level at 5 µg/L (4). The prevalence of TCE contamination
70
coupled with the low action levels creates a substantial demand for effective remediation
71
approaches.
72 73
Current remediation methods of contaminated groundwater rely on enhancing volatilization of
74
TCE through air stripping or sparging, sorption using activated carbon or zero valent iron
75
barriers, or reductive dechlorination and degradation through bioremediation (5). While
76
remediation costs vary widely depending on site conditions and method, at an estimated capital
77
cost of installing conventional systems (i.e., pump and treat) of between USD $700,000 and USD
78
$3,000,000 per site plus annual operating costs averaging approximately USD $200,000 (6), the
79
addressable market for TCE remediation on the currently identified NPL alone exceeds USD
80
$2.5 billion. Phytoremediation is an alternative strategy that utilizes a plant’s natural ability to
81
take up chemicals from water and soil with its expansive root system, to degrade organic
82
pollutants or sometimes to sequester inorganic pollutants (7). Most phytoremediation projects
83
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
85
organic contaminants due to its compatibility with bioaugmentation systems, high growth rate,
86
extensive root system, and high rates of water uptake from the soil (9). Poplar’s high water use
87
can be used to contain contaminated ground water plumes and also to take up and degrade TCE
88
to less toxic forms (10). However, in some cases, the pollutant concentration is phytotoxic,
89
limiting the ability of the plant to effectively remove the chemical.
90 91
Phytoremediation can be potentially improved by using genetic engineering and by bio-
92
augmenting with pollutant-degrading bacteria (11-18). Transgenic trees (19-23) and other
93
suitable phytoremediation plant species (24,25) engineered to overexpress key enzymes can
94
potentially have substantially improved pollutant detoxification and removal. Alternatively,
95
partnering plants with effective pollutant-degrading microorganisms has also shown promise,
96
and research in this area has increased dramatically in the last decade (26) demonstrating near-
97
term application potential without the need for the high-cost and lengthy deployment process
98
associated with transgenic plants.
99 100
In natural systems, plants can use symbiosis with internal microorganisms, termed endophytes
101
(27), to adapt to environmental challenges, including pollutants (28-30). Plants in contaminated
102
areas have a high number of endophytes which are capable of degrading the pollutant (31-34).
103
This effect can be plant genotype-specific, suggesting that some plant species or ecotypes are
104
better able to recruit or stably interact with the beneficial microorganisms (35-38). With the first
105
demonstration that inoculation of plants with a specific pollutant-degrading endophyte can lead
106
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
109
effluent (53), and metals (54-59).
110
phytoremediation success with indirect benefits (60,61) including increased nutrient acquisition,
111
more extensive root systems, and increased environmental stress tolerance (62-65).
The use of endophytes potentially also increases
112 113
Despite these numerous lab or greenhouse-based studies, there are few reports of field trials of
114
endophyte-assisted phytoremediation (26). The in situ addition of a poplar root endophyte strain
115
engineered to degrade TCE resulted in reduced evapotranspiration of TCE (66). However, no
116
direct evidence was provided on increased removal of groundwater TCE or degradation of TCE
117
in planta.
118
resulted in increased removal of TNT under laboratory conditions but had no impact under field
119
conditions (67). Bioaugmentation in general is thought to have less success at field scale due to
120
the possibility of native soil microorganisms harming or out-competing the artificially added
121
strains (5). The complexity of field trials where the pollutants are often heterogeneous can also
122
make precise assessments of phytoremediation success challenging.
Axenic plants inoculated with a TNT-degrading strain of Pseudomonas putida
123 124
We previously reported on the isolation and characterization of a TCE-degrading endophyte
125
strain (68). This Enterobacter sp. strain PDN3, isolated from hybrid poplar collected from a
126
TCE-contaminated site, grows well on high levels of TCE.
127
dechlorinated TCE without the addition of co-metabolite inducers. We report herein on the first
128
successful phytoremediation field trial of bio-augmentation with this natural TCE-degrading
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Strain PDN3 aerobically
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endophyte strain, demonstrating a low-cost, simple approach to improving tree growth and
130
pollutant degradation.
131 132
Materials and Methods
133 134
Bacterial strain. Endophyte strain Enterobacter sp. PDN3 was originally isolated from hybrid
135
poplar (Populus deltoides x nigra) in a screen for endophytes tolerant to 5.5 mM TCE (68).
136
Cryogenically stored microbial stocks had been prepared previously by growing the strain in
137
MG/L broth (69) containing approximately 300 µg/ml TCE, freezing in 33% sterile glycerol, and
138
storing at -70ΕC.
139 140
Verification of colonization ability. Prior to the field testing, the ability of strain PDN3 to
141
colonize hybrid poplar clone OP367 (P. deltoides x nigra) was tested using fluorescent
142
microscopy. The pBHR:gfp plasmid (70) was introduced into strain PDN3 using triparental
143
mating (71) and transconjugants were selected on MG/L agar containing carbenicillin to select
144
for PDN3 and kanamycin to select for the plasmid. gfp-PDN3 was grown in MG/L broth for
145
24hrs and washed twice in deionized water by centrifugation. The pellet was resuspended in
146
0.5X Hoagland’s solution (72), adjusted to an optical density (OD) at 600 nm of 0.1 using Unico
147
spectrophotometer model 1000 (Fisher), and exposed to poplar roots for one week at room
148
temperature. Colonization was visualized after 48 hours and one week using a Zeiss Imager M2
149
equipped with an AxioCam MRM, and recorded with Zeiss AxioVision software (Carl Zeiss,
150
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
154
completed with assistance from NASA environmental division and facilities groups. The
155
Middlefield-Ellis-Whisman (MEW) Superfund Study Area was contaminated due to industrial,
156
manufacturing, and military operations, and has been the subject of remediation studies since the
157
1980's (73). As shown in Figure 1 and Supplementary Figure S1, planting sites were chosen in
158
three areas near the center of the plume where the TCE concentration was in the 100-1000 µg/L
159
range.
160 161
In 2013, the planting areas were prepared by mowing and removal of existing vegetation. Three
162
commercial varieties of poplar were selected for the study based on the geographical area and
163
site conditions. Nine-inch dormant cuttings of OP-367, 15-029, and 311-93 were obtained for
164
planting from Segal Nurseries (Grandview, WA). Half the number of cuttings of each variety
165
were soaked in quarter-strength Hoagland’s solution (Control Poplar, CP) and the other half were
166
inoculated (Endophyte-inoculated Poplar, EP) with the endophyte strain by placing the cuttings
167
upright in an 8 L container and soaking in a 0.1 OD600 suspension of the PDN3 culture in
168
quarter-strength Hoagland’s solution at a depth of approximately 10 cm for 24 hours prior to
169
planting. Details of the planting and maintenance are in the Supplementary Materials.
170 171
Tree Growth Assay. Tree growth was measured periodically throughout the study using tree
172
base and breast height trunk diameter measurements.
173
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Assessing chloride level in the rhizosphere.
As a means of assessing phytoremediation
175
performance with respect to TCE degradation, chloride levels were measured as a breakdown
176
product from the TCE in samples collected from the rhizosphere of Control Poplar (CP) and
177
Endophyte-Inoculated Poplar (EP) trees sixteen months from the beginning of the field trial.
178
The samples were collected by moving aside the surface of the soil localized around the tree
179
stems, collecting the rhizospheric soil from around the visible roots, and placing the samples in
180
sterile 50-ml conical tubes. Approximately 2.0 grams of soil were ground with a mortar and
181
pestle to pass through a 10 mesh sieve (< 2 mm) and mixed in order to homogenize the sample.
182
Two-hundred mg of homogenized soil were weighed using an analytical balance (resolution ±
183
0.0001 milligrams) and suspended in 25 mL of 4% acetic acid solution in 125 mL Erlenmeyer
184
flasks. The flasks were sealed and incubated at room temperature under shaking conditions at
185
110 rpm for 30 minutes. Soil particles were then removed by filtering the suspension through a
186
Whatman® No. 1 filter paper (Sigma-Aldrich). Filtered samples were collected into glass tubes,
187
sealed, and stored at 4 ° C for further analysis. Chloride (Cl) levels were assessed using a
188
chloride-selective electrode (Thermo-Fisher Scientific). A calibration curve for low-level
189
measurements was prepared as described by the manufacturer using a NaCl solution (chloride
190
standard) prepared in deionized water (DQ3, Millipore) supplemented with 1 mL of Ionic
191
Strength Adjustor (ISA) solution (1.0 M NaNO3). After each increment, millivolt potential was
192
registered and plotted against Cl concentration. Finally, chloride concentration of each sample
193
was calculated interpolating millivolt values to the corresponding calibration curve.
194 195
Tree Core Measurements of TCE and breakdown products. Tree cores were taken using
196
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
198
placed into 20-mL PTFE-sealed head space vials. Samples were shipped the next day to the
199
Missouri University of Science and Technology Environmental Engineering. Analysis of TCE
200
and other chlorinated solvents in the transpiration stream of the trees was carried out using solid-
201
phase micro extraction (HS-SPME) coupled with gas chromatography and electron capture
202
detection (GC-µECD). Quantification was accomplished using a 5 point standard curve with
203
external standards. An R2 > 0.98 was deemed acceptable. This equilibrium passive sampling
204
method was developed specifically for tree core cVOC sampling using a 100 µm
205
polydimethylsiloxane (PDMS) fiber (74). Method detection limits were below 8 ng/l for TCE
206
and 0.5 ng/l for PCE in the aqueous solution in plant tissues (75).
207 208
Groundwater test wells. Well borings were drilled to a depth of 17-feet below ground surface
209
(bgs) using 8-inch hollow stem augers. The well screens, casings and completion materials were
210
installed as per the attached well completion logs. The water samples were collected via “Hydro-
211
sleeves”, which are plastic-like baggies that are collapsed when deployed to the bottom of the
212
well, and then raised quickly for approximately 1-foot which causes the sleeve to open at the top
213
allowing the well water to completely fill the sleeve within the screened aquifer zone (zone of
214
interest). The wells were installed on 9/20/16. Test well 1 is approximately 20-feet south of the
215
plot (the up-gradient/incoming well), while test well 2 is to the north of the plot (the
216
downgradient/outgoing well). Groundwater samples were collected on 9/28/16 and again on
217
11/21/2016 immediately prior to leaves turning yellow and winter senescence. The analytical
218
method for the groundwater samples was EPA Method 624 normally utilized for Volatile
219
Organic Compounds (VOCs) analysis.
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220 221
Verification of PDN3 colonization of the field site trees. To verify that the EP plants at the
222
field test site had been colonized with PDN3, branch samples collected from the trees in spring
223
2016 were shipped from the field site to the University of Washington, surface sterilized with
224
0.6% sodium hypochlorite for 10 minutes and rinsed four times with sterile water. We chose to
225
test branch samples because PDN3 can effectively colonize poplar vascular tissue, migrating
226
from root to shoot or shoot to root depending on inoculation site (unpublished data). One node
227
per tree sample was weighed and extracted with 0.5 ml 0.1X Hoagland’s solution per gram of
228
tissue in separate sterile mortars. Resulting extracts were stored at -70ΕC. Samples of the
229
extracts from 6 CP trees and 7 EP trees were randomly chosen, with strain PDN3 included as a
230
reference, and inoculated into M9 medium containing 0.4% peptone (4) and 0.5% mannitol.
231
After 3 days at 30ΕC, 1 ml samples were pelleted in a microcentrifuge, the cells resuspended in 1
232
ml M9 with peptone, and the OD600 was measured. Cultures were adjusted to an OD600 of 0.1 in
233
3 ml M9 medium with 0.4% peptone and 730 µg/ml TCE in 40-ml amber VOA vials. After 15
234
days exposure to the high level of TCE, samples were plated on M9 with peptone, and restreaked
235
for colony purification.
236
subjected to colony PCR using 16S rRNA gene primers as described previously (76). PCR
237
products were treated with ExoSAP-IT (Affymetrix) after verification by electrophoresis to be
238
single 1.5 Kb products with no PCR product from a water blank control.
239
sequenced by GeneWiz (Seattle) and identified using NCBI BLAST (77). For samples identified
240
as Enterobacter species, PCR was repeated and the products cloned into pGEM-T-Easy
241
(Promega), and sequenced twice. The 16S rRNA sequences were compared to that of PDN3
242
(GenBank #JN634853) using the alignment program of NCBI BLAST.
Colonies of the dominant morphology and similar to PDN3 were
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Comparative genomic analysis of Enterobacter sp. Strain PDN3. The PDN3 genome was
245
sequenced and assembled at the Department of Energy Joint Genome Institute (JGI) as part of
246
the research topic “Defining the poplar root microbiome”. Genome annotation, performed at the
247
JGI, was carried out using the DOE-JGI Microbial Annotation Pipeline (DOE-JGI MAP) (78).
248
Enzyme Commission (EC) numbers were used as annotation terms to define a functional profile
249
to investigate the genetic potential of PDN3 to metabolize or co-metabolize TCE. The
250
chloroalkane and chloroalkene degradation pathway and the metabolism of xenobiotics by
251
cytochrome P450 from the KEGG database were used as reference pathways. Once the
252
functional profile was defined, the “missing” enzymes were further analyzed using the ‘Find
253
Candidate Genes for Missing Function’ tool from the IMG/ER platform. This step was
254
performed using two different approaches; for search by homology, a percent identity cutoff of
255
30 % and an E-value cutoff of 1e-2 were used, while a search by functional orthologs was
256
performed with a percent identity cutoff of 10 %. Both steps were executed by querying the IMG
257
database. Similarly, the genetic potential of PDN3 to regulate heavy metal homeostasis was
258
investigated.
259 260
Statistical analysis. Data analysis was carried out in R v3.2.3, performing a Tukey’s HSD
261
(Honest Significant Difference) as post-hoc test in conjunction with a One Way ANOVA. For
262
TCE levels in core samples the non-parametric test Kruskal-Wallis rank sum test was used.
263 264
RESULTS AND DISCUSSION
265
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Verification of the colonization ability of endophyte strain PDN3. To verify that PDN3 was
267
capable of colonizing the hybrid poplar, the fluorescently tagged PDN3 (pBHR:gfp) strain was
268
used. After 48h of co-cultivation with poplar roots, the strain was observed to begin colonization
269
through the formation of a biofilm at the junctions between the primary and secondary roots
270
(Figure 2a). Crack entry at lateral root junctions is a common mode of colonization by
271
endophytes (60,79). After one week of co-inoculation, gfp-PDN3 was verified to be within the
272
host plant (Figure 2b). Since strain PDN3 was originally isolated from another hybrid P.
273
deltoides x nigra poplar clone, it was expected that it would be able to effectively colonize the
274
hybrid poplar used in the study.
275 276
Observable growth differences in EP and CP trees. The preparation and plantings on the
277
MEW plume resulted in successful establishment of the three poplar test plots directly over the
278
MEW plume in an area of TCE concentrations ranging from 100-1000 µg TCE / L (Figure 1).
279
In 2014 after one year of growth, it was apparent that the PDN3 inoculated poplar (EP) trees
280
were more robust than the un-inoculated control (CP) trees (Figure 3). This may have been due
281
to several factors. The endophyte may have reduced the TCE load within the trees by rapidly
282
metabolizing the pollutant taken up by the plant, thereby directly reducing phytotoxic effects.
283
Although the TCE concentration in the plume itself may not have been high enough to be
284
phytotoxic, the condition of the CP trees indicated that TCE did impact plant growth. TCE may
285
have been present in the upper rhizosphere possibly due to the nature of the volatile organic
286
contaminant, a persistent dry soil condition and an unconfirmed volatilization up through
287
cracked dry soil. In addition to benefitting the plant through detoxification of TCE, the
288
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
290
organic compounds shown to be important for increasing plant stress tolerance (81-83). The
291
strain also produces indole acetic acid (unpublished data), an auxin that induces root formation
292
and plant growth (64). The combination of both the endophyte strain and the right tree variety
293
was necessary for success. The OP367 variety was the hybrid of choice for this site due to its
294
larger, more robust growth when compared to the other two varieties.
295 296
In order to assay the difference between CP and EP trees after two and three years of tree
297
growth, the tree trunk diameter at breast height was recorded for all blocks on site for the OP-367
298
variety. This data showed a statistically significant 32% increase in trunk diameter growth of the
299
PDN3 inoculated trees after two and three years of growth when compared directly to the control
300
un-inoculated trees (Figure 4. p
