1249

Mine tailings are moderately to severely impacted sites that lack normal plant cover, soil structure and development, and the associated microbial community. In arid and semiarid environments, tailings and their associated contaminants are prone to eolian dispersion and water erosion, thus becoming sources of metal contamination. One approach to minimize or eliminate these processes is to establish a permanent vegetation cover on tailings piles. Here we report a revegetation trial conducted at a moderately impacted mine tailings site in southern Arizona. A salt and drought-tolerant plant, four-wing saltbush [Atriplex canescens (Pursh) Nutt.], was chosen for the trial. A series of 3 by 3 m plots were established in quadruplicate on the test site to evaluate growth of four-wing saltbush transplants alone or with compost addition. Results show that >80% of the transplanted saltbush survived after 1.5 yr in both treatments. Enumeration of heterotrophs and community structure analysis were conducted to monitor bacterial community changes during plant establishment as an indicator of plant and soil health. Th e bacterial community was evaluated using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA PCR gene products from tailings samples taken beneath transplant canopies. Signifi cant diff erences in heterotrophic counts and community composition were observed between the two treatments and unplanted controls throughout the trial, but treatment eff ects were not observed. Th e results suggest that compost is not necessary for plant establishment at this site and that plants, rather than added compost, is the primary factor enhancing bacterial heterotrophic counts and aff ecting community composition.

Bacterial Community Changes during Plant Establishment at the San Pedro River Mine

Tailings Site

Karyna Rosario, Sadie L. Iverson, David A. Henderson, and Shawna Chartrand University of Arizona

Casey McKeon and Edward P. Glenn Environmental Research Laboratory

Raina M. Maier* University of Arizona

There is emerging concern regarding the environmental

consequences of mine tailings sites, especially those in

sensitive arid and semiarid riparian or wildlife refuge areas. Tailings

are produced during ore processing and are characterized by

little or no vegetation, poor soil structure, low nutrient content,

surface crusting, and high levels of contaminants including heavy

metals (Ledin and Pedersen, 1996). Th e impact of tailings goes

beyond the immediate site because erosion and leaching processes

release and spread tailings contaminants to other areas. Removal

of tailings materials is costly largely because they are extensive in

nature and considered to be hazardous waste. Th us, remediation

eff orts have focused on stabilization of the tailings.

One approach to stabilization is phytoremediation or establish-

ment of a plant cover that acts to reduce all spreading mechanisms:

eolian dispersion, water erosion, and leaching. Concomitantly, a

plant cover acts to stabilize soil contaminants by altering the water

fl ux through the soil, by incorporating free contaminants into the

roots and rhizosphere, and by improving ecosystem structure and

function within the tailings (Cunningham et al., 1995). Th e chal-

lenge with phytostabilization in arid and semiarid environments is

that plant establishment is often limited by high levels of metals and

extreme pH in addition to the other challenges that arid environ-

ments pose to plants (i.e., low water availability, low organic matter,

salinity, and high temperatures). In general, the development of a

persistent vegetative cover depends on selecting plant species suitable

to site conditions and amending tailings with organic matter that

improves soil properties that favor plant growth.

Th e improvements made to mine tailings sites through phytosta-

bilization rely, in part, on activities of soil microorganisms that are

essential to healthy ecosystems (Crowley et al., 1997). Bacteria play

an important role in the success of phytostabilization by promoting

Abbreviations: ANOVA, analysis of variance; BLM, Bureau of Land Management; C, plots

containing transplants with compost; CECL, University of Arizona Chorover Enviromental

Chemistry Laboratory; CFU, colony-forming unit; DGGE, denaturing gradient gel

electrophoresis; HIC, University of Arizona Superfund Basic Research Program Hazard

Identifi cation Core; ICP-MS, inductively coupled plasma–mass spectrometry; KNMDS,

Kruskal’s Non-Metric Multidimensional Scaling; NC, plots containing transplants with

no compost, OSC, off -site control plots; SPRNCA, San Pedro River National Conservation

Area; U, unplanted control plots; WQCL, University of Arizona Water Quality Center Lab.

K. Rosario, S.L. Iverson, S. Chartrand, and R.M. Maier, Dep. of Soil, Water, and

Environmental Science, Univ. of Arizona, Tucson, AZ 85721-0038; D.A. Henderson, Dep.

of Animal Sciences, Univ. of Arizona, Tucson, AZ 85721-0038; and C. McKeon and E.P.

Glenn, Environmental Research Lab, 2601 E. Airport Drive, Tucson, AZ 85706.

Copyright © 2007 by the American Society of Agronomy, Crop Science

Society of America, and Soil Science Society of America. All rights

reserved. No part of this periodical may be reproduced or transmitted

in any form or by any means, electronic or mechanical, including pho-

tocopying, recording, or any information storage and retrieval system,

without permission in writing from the publisher.

Published in J. Environ. Qual. 36:1249–1259 (2007).

doi:10.2134/jeq2006.0315

Received 7 Aug. 2006.

*Corresponding author (rmaier@ag.arizona.edu).

© ASA, CSSA, SSSA

677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS: BIOREMEDIATION & BIODEGRADATION

Published online July 17, 2007

1250 Journal of Environmental Quality • Volume 36 • September-October 2007

plant growth through the mineralization of inorganic nutrients (Xu

and Johnson, 1995), production of growth regulatory substances

like phytohormones (Pishchik et al., 2002), facilitation of nutri-

ent uptake from the soil (Burd et al., 2000), improvement of soil

structure by soil aggregation (Maier et al., 2000), and reduction

of metal toxicity (Salt et al., 1999; Sandrin et al., 2000). In return,

established plants provide a nutrient-rich environment that can

stimulate bacterial activity (Glick, 2003; Kuiper et al., 2004). Th us,

monitoring changes in the microbial community can aid in assess-

ing the success of reclamation strategies.

Th e goal of this study was to use the local native shrub four-

wing saltbush to revegetate barren areas in a former silver-gold mill

site located adjacent to the San Pedro River in southern Arizona

and determine if plant establishment had a measurable infl uence

on the bacterial community. Th e site is in the San Pedro riparian

area, which was designated as a National Conservation Area in

1988 to help protect the fast disappearing desert riparian ecosystem

in the Southwest. A revegetation trial was conducted using four-

wing saltbush transplants to investigate if this species was suitable

for phytostabilization purposes. Th e specifi c objectives of the re-

vegetation trial were (i) to determine if four-wing saltbush could be

successfully established in the tailings, (ii) to evaluate if the addition

of compost amendment in the tailings was necessary to achieve

this goal, and (iii) to follow changes in the bacterial community

during the revegetation process. Bacterial community responses

beneath transplant canopies were studied by monitoring changes

in the abundance of heterotrophic bacteria (culturable counts) and

in the bacterial community structure based on DGGE community

fi ngerprints using universal bacterial primers.

Materials and Methods

Site Background and Study Area DescriptionTh e Boston mill mine tailings site (Fig. 1) is adjacent to the

San Pedro River in the San Pedro River National Conservation

Area (SPRNCA), approximately 3.2 km (2 miles) south of Fair-

bank, AZ, and 12.6 km (8 miles) southwest of Tombstone, AZ,

at an elevation of 1192 m (31°40′38.6″N, 10°10′53.4″W). It

is one of fi ve mill sites along the upper

San Pedro River, within the SPRNCA,

that were active from 1879 to 1887 for

processing silver and gold ore that was

brought from mines in Tombstone, AZ.

Th e site covers approximately 2.8 ha (7

acres) and can be divided into three areas;

an upper tier near the mill foundation,

a lower tier at a slightly lower elevation

south of the upper tier, and tailings across

the railroad tracks in the fl oodplain. Pre-

liminary data provided by the Bureau of

Land Management (BLM), which cur-

rently manages the site indicated that the

upper tier has the highest concentrations

of heavy metals. Th is area has a shallow

layer of tailings (10–20 cm) and includes

completely barren areas to the west of

the mill foundation and some areas with

patchy vegetation on the eastern side composed primarily of big

sacaton grass (Sporobolus wrighti Munro ex Scribn) and mesquite

(Prosopis spp.). Th is partial vegetation suggests that natural at-

tenuation has slowly occurred on the tailings during the past

century. For this reason the Boston mill site is considered to be

a moderately impacted mine tailings site. Nevertheless, barren

areas still pose a source of concern for eolian dispersion and water

erosion of tailings into the San Pedro River and the surrounding

region, which is a wildlife refuge and hiking area.

Experimental Design and Sampling SummaryA 24 by 30 m study area in the upper tier of the Boston Mill

site was selected for the revegetation trial (Fig. 1). One of the objec-

tives of this work was to determine whether compost amendment

was necessary for establishment of four-wing saltbush on the site.

Th erefore, two treatments were evaluated: four-wing saltbush

transplants with compost amendment (C), and unamended trans-

plants (NC). A 6 by 12 m off -site control (OSC) area (no mine

tailings) was chosen southeast of the study site. For each treat-

ment (compost, C; no compost, NC; and off -site control, OSC)

quadruplicate plots (n = 4) were established on the relevant study

area following a complete randomized design. Each plot received

fi ve transplants and plant growth was evaluated by height and

crown measurements made on each plant at time 0, 11, and 18

mo (n = 20). A soil sample was taken from each plot for chemical,

physical, and microbiological analysis before planting (n = 4). Fol-

lowing planting, root zone soil samples were taken from one plant

in each plot at 3, 4, 5, 7, 8.5, 11, 13, 16, and 18 mo for microbial

analysis (n = 4). Shoot tissue samples were collected at 18 mo to

determine plant shoot metal content. Tissue from each of the fi ve

plants in a plot was harvested and combined into a single com-

posite sample for each NC and C treatment plot (n = 4). It should

be noted that transplants did not survive in the OSC area, and so

for shoot metal content comparison four composite samples were

taken from nearby indigenous four-wing saltbush shrubs to obtain

background shoot tissue metal levels (n = 4).

For the microbial analysis a third treatment, unplanted con-

Fig. 1. Photos of the study area (A) showing an aerial view of the site and the relative locations of the transplant plot area, the control site, and their proximity to the San Pedro River. The line bisecting the site from the lower left of the photo (northwest) to the mid-right (southeast) is a railroad track. Photo (B) shows a closeup of two of the 3 by 3 m plots just after having received the transplants. The inset photo is an example of a transplant.

Rosario et al.: Bacterial Community Changes during Plant Establishment 1251

trol (U), was added to account for natural variations in the bac-

terial community. Th e U samples were initially taken from two

areas (n = 2). Th e fi rst was an area 1.7 m outside the west side

of the 24 by 30 m trial area and the second was just inside the

24 by 30 m trial area on the west side. Th irteen months follow-

ing planting, two more U sites were randomly selected on the

east side of the plot and monitored from 13 to 18 mo (n = 4).

TransplantsTh e plant chosen for this study was four-wing saltbush, which

is a perennial halophytic subshrub native to Arizona, California,

Nevada, and Utah (USDA-NRCS 2005). Th is plant is considered

drought tolerant and has been observed encroaching into other

historical mine tailings sites (USDA-SCS, 1977; Booth et al.,

1999; Arunachalam et al., 2004; Jeff erson, 2004) as well as the

Boston Mill site. Four-wing saltbush transplants were germinated

and grown in the greenhouse for 2 mo in a 2:1 sand/compost mix

in 0.375-L pots. Th e compost was a commercial composted mulch

(Sunshine mix no. 1, Sun Gro Horticulture). Th is material was also

used as the compost amendment in the fi eld trial (Tables 1 and 2).

Field StudyPlanting took place on 31 Oct. 2003. Th e study area was di-

vided into eight 7.5 by 12 m plots. A point within each plot was

selected using random X and Y coordinates and marked with a pin

fl ag to serve as a reference to later establish the 3 by 3 m treatment

areas in each plot (Fig. 1). Quadruplicate plots per treatment were

established on the study area following a complete randomized

design. To establish the treatments on the fi eld, a 3 by 3 m quadrat

was centered on a random point that was previously marked with

a pin fl ag in each of the eight 7.5 by 12 m plots. Th e four corners

of the quadrat were fl agged and one transplant was placed near the

inside of each of the quadrat corners and one in the middle. Th e

transplants were planted by digging a hole deep enough to cover

the roots, centering the transplant in the hole, and fi lling it back

with the original soil (Treatment NC) or with approximately 3.8 L

(1 gallon) of compost (Treatment C). For Treatment C, half a gal-

lon of compost was added fi rst, followed by a thin layer of soil, the

other half of the compost, and fi nally a thin layer of soil at the top.

Th e 6 by 12 m OSC area similarly received transplants in four 3

by 3 m plots that were evenly spaced in the area. After planting, all

transplants were covered with chicken wire cages for approximately

6 mo to protect against foraging animals. Over the next 8 mo the

transplants were irrigated with a total of 44 L of water per plant

using the following schedule: Day 0 and 3, 1.5 L; Day 7, 4 L; Day

14, 3 L; Day 24, 4 L; Days 40, 45, 52, 3 L; Days 83, 100, 4 L;

Days 114, 131, 181, 3 L; and on Day 218, 4 L.

Th e height (h) and crown diameters (d1 and d

2) of the

transplants were measured immediately after planting and at

11 and 18 mo to assess growth. Th e height measurement was

made from the ground to the highest point on the shrub. Th e

two crown diameter measurements were made perpendicular

to each other at the widest part of the shrub canopy. Shrub

volume was estimated by assuming an elliptical shrub shape

(V = 4/3π(h/2 × d1/2 × d

2/2) (Th omson et al., 1998).

Soil and Plant SamplingSoil samples were taken from each of the C and NC treat-

ment plots (n = 4) before planting and from the U plots

(n = 4) at the end of the trial to determine selected physical

and chemical characteristics. A 500-g surface sample (5–10

cm) was taken from each plot using a closed bucket auger and

stored in a plastic bag for analysis.

For microbiological analysis, 5-g soil samples were taken

Table 1. Selected characteristics for treatment plot tailings samples, the off -site control soil, and the compost used in this study.

Treatment plot†

pH‡ CEC§ Sand¶ Silt¶ Clay¶ TOC# TN††

cmolc kg−1 ––––––––––%–––––––––– ––g kg−1––

Off -site 8.7 8.0 62.5 26.9 10.6 4.8 0.2

NC-1 8.7 25.1 37.8 43.4 18.8 11.6 0.4

NC-2 8.4 28.7 45.3 40.9 13.8 11.0 0.7

NC-3 8.6 25.5 52.9 32.5 14.7 10.5 0.3

NC-4 9.0 24.5 74.6 18.9 6.5 10.1 0.3

C-1 8.8 22.2 47.4 40.5 12.1 8.1 0.3

C-2 8.5 30.4 26.4 47.2 26.4 21.4 0.8

C-3 9.0 32.5 61.4 30.1 8.5 8.6 0.3

C-4 8.3 28.5 45.3 38.3 16.3 11.2 0.8

U1 9.0 27.2 52.1 33.9 13.9 9.4 0.3

U2 9.0 29.1 40.8 41.6 17.7 9.7 0.3

U3 8.9 21.8 57.4 30.4 12.3 9.6 0.2

U4 9.8 25.5 41.6 39.8 18.5 17.4 0.5

Compost 8.3 ND‡‡ ND ND ND 169 3.7

† Treatment plots include no compost (NC), compost (C), and unplanted

controls (U).

‡ pH- soil to deionized water 1:2 ratio after 1 h mixing.

§ Cation exchange capacity (CEC) determined by the magnesium nitrate

method (University of Arizona Water Quality Center Lab, WQC).

¶ Texture determined by the hydrometer method (WQC).

# Total organic carbon (TOC) determined by the diff erence in total carbon

and inorganic carbon (Chorover Environmental Chemistry Lab, CECL).

†† Total nitrogen (TN) determined by high temperature combustion (CECL).

‡‡ Not determined.

Table 2. Total metal concentrations (mg kg−1) for the treatment plot tailings, the off -site control soil, and the compost used in this study.

Treatment Pb As Hg Zn Cu Fe Mn Cd Al

Off -site control† 127 (3) 7 (1) 3 (0) 989 (139) 97 (8) 10 500 (1450) 730 (69) 1 (0) 14 300 (3500)

NC‡ 11 100 ± 6100 576 ± 302 503 ± 191 5410 ± 3070 2440 ± 1370 15 600 ± 5810 44 500 ± 22 000 93 ± 50 11 500 ± 3590

C‡ 15 800 ± 958 845 ± 94 560 ± 83 8100 ± 1370 3620 ± 404 19 500 ± 4007 67 300 ± 24 000 138 ± 30 12 900 ± 4490

U‡ 14 100 ± 463 474 ± 35 178 ± 77 6570 ± 506 2290 ± 200 16 000 ± 1520 50 400 ± 7 780 86 ± 12 8000 ± 885

Compost† 21 (1) 3 (0) 2 (1) 200 (4) 28 (5) 2040 (1400) 0 0 1750 (110)

† Samples (n = 1) were analyzed in duplicates. The number in parentheses represents the deviation from the mean of the duplicates.

‡ Values are the average ± standard deviation (n = 4).

1252 Journal of Environmental Quality • Volume 36 • September-October 2007

(5–10 cm) using a sterile spatula (washed with 95% ethanol and

fl amed between samples), placed into sterile vials, and stored at

4°C within 4 h of collection for microbial analysis. Th ese samples

were collected at 0, 3, 4, 5, 7, 8.5, 11, 13, 16, and 18 mo to fol-

low bacterial community changes during plant establishment.

Th e time zero samples were taken before planting as described

above. Following planting, one plant was chosen from each treat-

ment plot (NC and C treatments, n = 4) for the microbial analy-

sis. Selected plants looked healthy, and with the exception of plot

C3, were at least 0.5 m away from any encroaching vegetation.

Soil samples were taken beneath the transplant canopy of the

chosen plant within 2 cm of the stem and approximately 5 cm

deep. For the unplanted controls, two U plots were sampled

from 3 to 11 mo (n = 2) and two more U plots were added from

13 to 18 mo (n = 4). U plots samples were taken within 5 cm of

the pin fl ag marking a random point.

Shoot tissue samples were collected 18 mo after planting

from each NC and C treatment plot to determine plant shoot

metal content. Since transplants did not survive in the off -site

control area, four composite samples were taken from nearby

indigenous four-wing saltbush shrubs to obtain background

shoot tissue metal levels.

Soil and Plant AnalysisSoil physical and chemical properties were determined by

the Water Quality Center Lab (WQCL) and by the Chorover

Enviromental Chemistry Laboratory (CECL) of the Univer-

sity of Arizona using standard soil analysis methods (Tables 1

and 2). Total metal concentrations were determined follow-

ing microwave-assisted digestion of dry soil samples (USEPA

Method 3051) and analysis by inductively coupled plasma–

mass spectrometry (ICP-MS).

For plant tissue total metal content, samples were carefully

rinsed with nanopure water to remove soil particles, dried at 65°C

for 2 d, and digested following USEPA Method 3051. Samples

were analyzed using ICP-MS by the University of Arizona Super-

fund Basic Research Program Hazard Identifi cation Core (HIC).

Heterotrophic Bacterial CountsHeterotrophic counts were performed using R2A agar

medium (Becton, Dickinson and Co., Sparks, MD) with

200 mg L−1 of cyclohexamide to prevent fungal growth. Each

sample was plated in triplicate. Briefl y, inocula were prepared

by mixing 1 g (wet wt.) of soil with 9.5 mL sterile Zwittergent

(CalBiochem, La Jolla, CA) extractant (per L H2O: 2 g NaCl,

0.0002% Zwittergent). Th e sample was vortexed for 2 min

and then serially diluted in sterile water, plated on R2A, incu-

bated at 23°C in the dark, and enumerated after 4 d.

Soil Bacterial Community DNA Extraction and Purifi cationDNA was extracted from soil bacterial communities by direct

lysis using the Fast DNA Spin Kit for Soil (Qbiogene, Carlsbad,

CA). Th e manufacturer’s protocol was followed with some modi-

fi cations. Before the extraction all sterile tubes, caps, and solu-

tions used in the protocol were exposed to UV light for 5 min in

a UVC-508 Ultraviolet Crosslinker (Ultra-Lum, Claremont, CA)

to remove any potential DNA contamination. Th e bead beating

procedure was performed by attaching the lysing matrix tubes

to a Vortex Genie II vortexer (VWR, West Chester, PA) using a

Mo Bio adaptor (Mo Bio Laboratories, Solana Beach, CA) and

vortexing the tubes at maximum speed for 10 min. Cell debris,

soil particles, and the lysing matrix beads were separated from

the DNA-containing supernatant by centrifugation at 14 000 × g

for 15 min. An alternative manufacturer’s protocol was followed

to enhance removal of soil humic materials that could inhibit

PCR amplifi cation. Samples with supernatants retaining a brown

color were assumed to contain humic material. For these samples

the Binding Matrix-DNA complex was rinsed repeatedly with

saturated 5.5 M guanidine thiocyanate (GTC, Sigma, St. Louis,

MO) until the supernatant lost its brown tint.

16S rDNA PCR for Denaturing Gradient Gel ElectrophoresisA 391 bp (including the GC clamp) portion of the 16S

rRNA gene (rDNA) encompassing the hypervariable V9 region

of the domain Bacteria was amplifi ed to target the soil bacterial

community in DNA extracts using primers 1070f and 1406r-

GC (Ferris et al., 1996). Th e PCR for DGGE was accomplished

using a modifi ed protocol from Colores et al. (2000). Briefl y,

50 μL reactions contained 1X Expand Hi Fidelity PCR Buff er

with 1.5 mM MgCl2 (Roche, Indianapolis, IN), 0.5 μM of each

primer, 0.4 g L−1 unacetylated bovine serum albumin (Sigma, St.

Louis, MO) to relieve PCR inhibition (Kreader, 1996), 200 μM

of each deoxynucleoside triphosphate, 5% dimethyl sulfoxide,

1.4 U Expand High Fidelity PCR System Enzyme (Roche, Indi-

anapolis, IN), and 0.2 to 0.28 mg L−1 soil bacterial DNA extract.

Th e reactions were exposed to 30 cycles at 95°C for 45 s, 55°C

for 45 s, and 72°C for 30 s in a GeneAmp 2400 PCR System

thermal cycler (PE Applied Biosystems, Foster City, CA).

Th e PCR products (5 μL per lane) were visualized and

evaluated using electrophoresis through a 2% GenePure LE

agarose gel (Intermountain Scientifi c Corp., Kaysville, UT)

followed by ethidium bromide staining. Products in lanes that

showed two bands, the desired product and another of slight-

ly larger size, were rejected because DGGE analysis indicated

that the extra band was associated with template overloading.

Rejected extracts were reamplifi ed using a reduced template

concentration. Most of the soil DNA extracts produced single

bands using an approximate concentration of 0.28 mg L−1,

but those requiring reamplifi cation were diluted incrementally

down to 0.2 mg L−1 until only a single band was observed.

Denaturing Gradient Gel ElectrophoresisTh e DGGE analysis was performed using a Dcode Universal

Mutation Detection System (Bio-Rad Laboratories, Hercules, CA).

Acrylamide gels (6%) were prepared with a 50 to 60% urea–for-

mamide denaturing gradient according to the manufacturer’s

protocol. Gel lanes were loaded with 25 to 35 μL of PCR product

depending on its intensity on the agarose gel. Th e gels were run at

a constant voltage of 50 V for 19 h at 60°C, stained with 3X SYBR

Green I (Molecular Probes, Eugene, OR) for 40 min, and then

imaged. To compensate for inconsistencies in electrophoresis, the

Rosario et al.: Bacterial Community Changes during Plant Establishment 1253

gels were aligned by loading an external reference ladder at regular

intervals. Th e ladder was composed of pooled PCR product from

isolated heterotrophic bacteria from tailings samples. Th e DGGE

profi les (banding patterns) were manually scored according to the

method of Konopka et al. (1999) and evaluated using Quantity

One 4.5.2 software (Bio-Rad Laboratories, Hercules, CA).

Statistical AnalysisAll statistics were performed using the statistical software

package R (R Foundation for Statistical Computing, Vienna,

Austria). Data were all found to be normally distributed with

constant variance. Results of all F tests were considered signifi -

cant at 95% confi dence level. For the revegetation trial, data

were subjected to a two-way analysis of variance (ANOVA) for a

completely randomized design, with sample date (11 or 18 mo)

and soil treatment (compost or no compost) as independent

variables and plant volume as the dependent variable. Dead

plants were excluded from the data analysis. Th e ANOVA had

1 df due to sample date, 1 df due to soil treatment, and 64 df

due to error. Systat 11.0 software (Systat Software, San Jose, CA)

was used to conduct the analysis of variance. Plant volumes were

log transformed to equalize variances among treatments. Th e

hypotheses of unequal variance between soil treatments or sample

dates were not signifi cant (p > 0.05) for transformed data. At the

end of the trial, diff erences in shoot metal concentrations were

analyzed using one way ANOVA of a completely randomized

design. Pearson’s correlation coeffi cient (r) was used to screen

linear relationships between plant and soil metal concentrations.

For the bacterial analysis, treatment eff ects on heterotrophic cul-

turable counts as a function of time were analyzed using a nested

ANOVA model with a maximum likelihood estimator on log

transformed data. Treatment was set as a fi xed factor, while plant

and time eff ects were set as random factors. Treatment diff erences

were evaluated using pairwise t tests on weighted means calcu-

lated for each treatment level (i.e., least squares means, α = 0.05).

Th e profi les obtained from DGGE gels were evaluated in

three ways. First, ANOVA analysis of the number of bands in

each profi le was used to evaluate whether there were treatment

eff ects on species richness (the number of populations present).

Second, Dice coeffi cient analysis was used to evaluate the similar-

ities between individual profi les within each treatment plot (C1,

C2, C3, C4, NC1, NC2, NC3, NC4, U1, and U2) as a func-

tion of time. Th is analysis examined the extent of the community

structure change that occurred in each plot between consecutive

sampling times during the fi eld trial. Th e Dice coeffi cient is ex-

pressed as S = 2a/(b + c) where a represents the number of bands

present in both profi les, b refers to the number of bands present

in Profi le 1, and c represents the number of bands present in

Profi le 2. Finally, Kruskal’s Non-Metric Multidimensional Scal-

ing (KNMDS; Venables and Ripley, 2002) was used to evaluate

similarities among profi les from all treatments at each time point.

Th is nonparametric ordination method was used to visualize

and interpret changes in the bacterial community during the

revegetation trial based on binary distance where similar bacterial

communities (i.e., profi les) cluster more closely than those with

low similarity in multidimensional space. Diff erences between

bacterial communities were evaluated with a permutation test (α

= 0.05). A stress factor (sf) was also calculated to refl ect good-

ness-of-fi t of the model (sf < 0.1 was considered a good fi t). Th e

confi gurations in multidimensional space were evaluated using

three or four dimensions (D) to minimize stress. Comparisons

of the bacterial community could be performed only for samples

within the same gel. Th erefore, treatment and time eff ects were

analyzed individually due to a limited number of wells per gel.

Results

Site CharacterizationSelected physical and chemical characteristics were determined

for samples from each treatment plot: NC, C, U, and OSC (Table

1). Th e alkaline pH and the presence of organic carbon are key pa-

rameters that indicate this as a moderately stressed site. In severely

stressed sites, the pH is usually acidic (<3 to 4) and there is essen-

tially no organic matter. In this site the pH is moderately alkaline,

ranging from 8.3 to as high as 9.8 in one sample and total organic

carbon values ranged from 8.6 to 21.4 mg kg−1 with an average

of 11.6 mg kg−1. Th ese values can be compared very favorably to

the OSC, which has a pH of 8.7 and a total organic C content of

4.8 mg kg−1. Th e big diff erence between the tailings and the OSC

is the total metal concentrations, which were greater in the mine

tailings samples than the OSC sample for all metals measured ex-

cept Al (Table 2). In particular, Pb, Hg, and Cd were elevated up

to 100-fold over the OSC.

Revegetation TrialOne goal of this 18-mo revegetation fi eld trial was to evaluate

whether four-wing saltbush transplants would survive and grow

in a moderately impacted mine tailings site and if so, whether

compost addition was necessary. Results from this study show

that after 1.5 yr, more than 80% of the transplants survived in

the tailings area regardless of whether or not compost was added

during planting. A signifi cant increase in growth (p < 0.05) was

observed during the fi rst 11 mo of the study with average plant

volumes increasing from 0.00047 ± 0.000067 m3 to 0.015 ±

0.012 m3. Th ere was no signifi cant diff erence in growth between

the transplants in the NC and C treatments (p > 0.05) (Fig. 2).

From 11 to 18 mo, plants did not grow further and even lost

volume to an average of 0.0080 ± 0.0067 m3, although this

volume loss was not signifi cant (p > 0.05) (Fig. 2). It should be

noted that the plants were irrigated only from 0 to 8 mo and did

not receive water for the latter part of the study. Further, during

this fi eld trial, the SPRNCA was experiencing a drought. Infor-

mation from the closest weather station, 8 miles to the northeast

in Tombstone, AZ, indicates that the area received a total of

362 mm of precipitation during the 18 mo of this study with ap-

proximately 44% of the precipitation occurring in the fi rst 8 mo.

None of the four-wing saltbush transplants survived in the

OSC plots, which was unexpected since they are indigenous

to the site. Failure to establish transplants in the OSC plots

could have been due to a fairly dense growth of indigenous

grass. Grasses, like western wheatgrass, have been found to

negatively aff ect four-wing saltbush establishment (Sabey et

1254 Journal of Environmental Quality • Volume 36 • September-October 2007

al., 1990). Th us, it is possible that naturally established grass

in this area either out-competed the transplants for nutrients

or had an inhibitory eff ect on their growth.

At the end of the trial, shoot metal contents of transplants were

elevated from 2 to 80-fold over levels found in saltbush naturally

established in nearby areas (Table 3). Th e highest levels of metal

accumulation were for Mn (?880 mg kg−1), Pb (?310 mg kg−1),

Fe (?265 mg kg−1), Al (?230 mg kg−1), and Zn (?200 mg kg−1),

refl ecting the high total concentration of these metals in the tail-

ings (Tables 2 and 3). Th ere was a positive correlation between the

uptake of Fe and the uptake of other metals including Pb, Cu, As,

Mn, Al, and Hg (r = 0.89–0.98, Table 4). Th ere were also correla-

tions between uptake of some metals pairs including Pb and As

(r = 0.92), Pb and Hg (r = 0.93), As and Hg (r = 0.91), and As

and Mn (r = 0.99). However, there was no correlation between soil

metal concentrations and plant metal uptake for individual metals

(r = −0.22 to 0.45, data not shown)

Microbial AnalysisSoil samples were taken from beneath transplant canopies

throughout the revegetation trial to monitor changes in the bacte-

rial community. Before the trial began, heterotrophic bacterial

numbers in bulk tailings averaged 2.1 ± 0.9 × 105 CFU g−1 dry soil

(CFU, colony-forming unit). Th is is similar to other studies that

have reported heterotrophic bacteria in untreated alkaline (pH > 8)

tailings (106 CFU g−1) (Shetty et al., 1994; Noyd et al., 1995)

and in acidic (pH < 4.5) tailings (105 CFU g−1) (Moynahan et al.,

2002). Within 3 mo, a 1 to 1.5 log increase was observed in bacte-

rial numbers under transplant canopies in both the C and NC

treatments in comparison to unplanted controls (U). Th is diff er-

ence was signifi cant (p < 0.001) and was observed throughout the

trial (Fig. 3). It should be noted that the U plots were not irrigated.

Despite this diff erence in irrigation, the enhancement in heterotro-

phic counts continued when irrigation ceased after 8 mo. Th us, the

diff erence in numbers between planted and unplanted plots was

not associated with irrigation eff ects alone.

Changes in the dominant bacterial populations during the

revegetation trial were monitored by DGGE. Th e PCR prod-

ucts amplifi ed from samples obtained before the trial started

were resolved best with a 50 to 60% denaturing gradient.

Th erefore, all other time points were evaluated using this gradi-

ent. Th e ANOVA analysis showed no signifi cant diff erences in

the total number of bands between the planted treatments and

the unplanted controls (p < 0.05) throughout the trial except

for 5 mo. At 5 mo, planted community profi les (NC and C)

had an average of 30 bands, which was signifi cantly more than

for unplanted (U) profi les, which averaged 24 bands (p = 0.02)

(Fig. 4). As shown in Fig. 4, the negative control lanes generally

had a small number of faint bands. In each case, these bands

were subtracted from the profi les during analysis.

Examination of the banding patterns in each of the DGGE

profi les shows that the bacterial community changed with time

for each treatment including the unplanted controls. Th is is not

surprising since samples were taken during a 13 mo period that

had large moisture and temperature variations (for precipitation a

total of 288 mm was measured in nearby Tombstone, AZ, from

31 Oct. 2003 to 31 Nov. 2003, ranging from 0 to 60 mm mo−1;

for temperature the high was 38.7°C, the low was −6.8°C, with an

average of 20.5°C; these data are from www.wunderground.com;

verifi ed 2 May 2007). Dice similarity coeffi cients were determined

for each treatment plot using binary sets of profi les as a function of

Fig. 2. Average plant volumes for no compost and compost treatments at times 0, 11, and 18 mo where n = 4. Error bars represent one standard deviation.

Table 3. Total metal content (mg kg−1) in four-wing saltbush and transplant shoots 18 mo following planting.†

Treatment Fe Cu Zn As Cd Hg Pb Al Mn

Off -site‡ 102 ± 8 a 5.7 ± 2.5 a 67.5 ± 35.4 a 0.1 ± 0.1 a 0.6 ± 0.5 a 0.04 ± 0.02 a 4.1 ± 5.6 a 57.5 ± 29.5 a 200 ± 46 a

NC§ 268 ± 71 b 33.4 ± 6.9 b 203 ± 48 b 5.3 ± 2.7 b 6.4 ± 2.4 b 1.0 ± 0.6 b 300 ± 139 b 253 ± 103 b 844 ± 210 b

C§ 264 ± 98 b 33.4 ± 14.6 b 186 ± 46 b 6.1 ± 4.5 b 6.6 ± 1.6 b 0.9 ± 0.5 b 327 ± 153 b 214 ± 84 b 923 ± 418 b

† In all cases n = 4. Means followed by the same letter in each column are not signifi cantly diff erent at 5% level of signifi cance as determined by

analysis of variance (ANOVA).

‡ Shoot materials were collected from indigenous four-wing saltbush in an area adjacent to the tailings pile to represent background metal concentrations.

§ NC, no compost treatment; C, compost treatment.

Table 4. Pearson’s correlation coeffi cient (r) for metals in four-wing saltbush transplant shoots in tailings treatment plots. Samples were collected at 18 mo.

Metalr

Fe Cu Zn As Cd Hg Pb Al Mn

Fe 1.00

Cu 0.94 1.00

Zn 0.01 0.31 1.00

As 0.98 0.93 −0.01 1.00

Cd 0.75 0.69 −0.05 0.69 1.00

Hg 0.92 0.86 −0.09 0.91 0.80 1.00

Pb 0.90 0.88 −0.02 0.92 0.85 0.93 1.00

Al 0.89 0.73 −0.22 0.81 0.83 0.87 0.85 1.00

Mn 0.95 0.90 −0.04 0.99 0.57 0.85 0.87 0.76 1.00

Rosario et al.: Bacterial Community Changes during Plant Establishment 1255

time. Th is analysis indicated that the largest changes in community

profi les occurred in the fi rst 5 mo of the study for most of the plots

(i.e., the biggest diff erence in % similarities occurred between 0

and 3 mo or between 0 and 5 mo, data not shown).

Th e DGGE profi les were compared using KNMDS to

determine treatment and time eff ects on the bacterial commu-

nities in NC, C, and U plots (Fig. 5). Each KNMDS graph

illustrates the fi rst two out of three or four dimensions. In

these graphs, the relative distance between samples indicates

the amount of similarity or diff erence among data points.

Th e KNMDS analysis of DGGE profi les suggests that there

were shifts in the dominant bacterial populations associated with

the diff erent treatments (NC, C, U) during the trial. Th e bacte-

rial communities that developed under plant canopies (NC or

C) became more similar to each other and more diff erent from

unplanted controls with time (Fig. 5). Before planting, the bacterial

community in bulk soil in the tailings treatment plots was divided

into three groups or clusters that were quite random (p = 0.06)

(Fig. 5, Time–0 Months). Th is plot indicates that the bacterial

communities in treatment plots that clustered together are more

similar to each other than to the communities that were not in the

cluster. Th ree months after initiation of the revegetation trial, the

original relationships (clusters) had disappeared and no signifi cant

clusters were observed (Fig. 5). However, by 5 mo, the profi les

from samples taken under transplant canopies became more simi-

lar to each other, regardless of treatment (NC or C), than to the

unplanted controls (p = 0.02). Seven months following planting

the same trend was observed, although the diff erences were not

statistically signifi cant (p = 0.18), and thus clusters are shown with

dashed trend lines. At 11 mo, three signifi cantly diff erent clusters

were found (p = 0.01). Although planted treatments were divided

into two diff erent clusters, is not possible to distinguish between

treatments since there are samples from compost and no compost

treatments in both clusters (Fig. 5, Time–11 Months). In addition,

it can be seen that the relative distance between the two clusters

containing communities in planted treatments is smaller than the

distances between each cluster and the unplanted controls. Th ere-

fore, the communities under transplant canopies were still more

similar to each other than to the unplanted controls continuing the

trend fi rst observed at 5 mo. Th e fi nal sampling for DGGE analy-

sis took place at 13 mo. Two additional unplanted control samples

were collected at this time, U3 and U4. Th e KNMDS analysis of

this time point again shows the trend (not signifi cant, p = 0.41)

that the communities in the four unplanted controls are more

similar to each other than to any of the transplanted samples.

To determine whether the community structure under trans-

plant canopies was infl uenced by the compost that the transplants

were originally grown in, the compost DGGE profi le was com-

pared to a time series (0, 3, 5, and 11 mo) of DGGE profi les from

two treatment plots, NC-1 and C-1 (Fig. 6). Th e KNMDS analy-

sis shows that the compost community is signifi cantly diff erent

from all profi les except the NC1 before planting (0 mo) (p = 0.03).

Discussion

RevegetationTh is study demonstrates the feasibility of establishing four-

wing saltbush transplants in moderately impacted mine tailings

sites with minimal inputs of water (a total of 44 L plant−1) and

no fertilizer or organic C amendments. Four-wing saltbush trans-

plants showed a 30-fold increase in plant volume in the fi rst 11

mo of this study and then stopped growing. It is not clear why

growth stopped. Water stress may have been a factor since irriga-

tion was terminated after the monsoon rains began in summer

2004 (after 8 mo) and the monsoon delivered only 110 mm of

Fig. 3. Heterotrophic plate counts beneath transplant canopies for no compost (NC) (circle) and compost (C) (triangle) treatments and for unplanted controls (U) (square) throughout the revegetation trial. Error bars represent one standard deviation (n = 4 for treatments NC and C, n = 2 for the unplanted controls except for *n = 1, **n = 4).

Fig. 4. Denaturing gradient gel electrophoresis (DGGE) profi les of PCR-amplifi ed 16S rDNA fragments from tailings treatment plots 5 mo after transplants were established. Labels on top of the gel indicate the sample identity: C, compost plot; NC, no compost plot; U, unplanted control; M, marker ladder composed of heterotrophic mine tailings isolates; and N, negative control.

1256 Journal of Environmental Quality • Volume 36 • September-October 2007

precipitation (measured in Tombstone, AZ) during July, August,

and September 2004. Metal toxicity is also a possible factor in

the observed growth inhibition (Hagemeyer, 1999), given that

there were elevated levels of shoot tissue metal concentrations in

the four-wing saltbush transplants, which will be discussed fur-

ther below. Th ese results suggest that long-term monitoring (e.g.,

>1.5 yr) is necessary to assess if saltbush transplants can establish

an enduring and successful vegetative cover.

In regard to phytotoxicity, Table 2 shows that several metals

in the Boston Mill site exceed reported soil plant toxicity levels

including Pb (500 mg kg−1), As

(15 mg kg−1), Cd (3 mg kg−1), Hg

(3 mg kg−1), Cu (200 mg kg−1),

Zn (400 mg kg−1), and Mn

(3000 mg kg−1) (Kataba-Pendias

and Pendias, 2001; Munshower,

1993; Mulvey and Elliott, 2000).

However, no toxicity studies have

been performed specifi cally with

four-wing saltbush so it is not clear

what levels of soil metal concentra-

tions cause toxicity in this plant.

While it was beyond the scope of

this study to explore the mecha-

nism of metal uptake into shoot

tissue, we did make the following

observations. Metal uptake was

not attributed to total soil metal

concentrations because there was

no correlation between soil metal

concentration and plant metal up-

take (r = −0.22 to 0.45). However,

there was a positive correlation

between Fe uptake and uptake of

other metals including Pb, Cu,

As, Mn, Al, and Hg (r = 0.89 to

0.98, Table 4). Th e exception to

this observation was Zn uptake,

which was not correlated with the

uptake of Fe or any other metal. It

is possible that root exudates from

plants were responsible for solubi-

lizing the metals from the soil. At

the alkaline pH of the tailings used

in this study, Fe is not soluble and

therefore is unavailable for plant

uptake. Plants adapted to alkaline

soils have been shown to solubilize

Fe by releasing H+ from their roots

(Brown, 1976). Th is eff ectively

lowers the pH in the root zone,

which enhances solubilization of

Fe as well as other metals.

Of all the metals present, only

Pb was translocated into shoot

material in levels that exceed the

guidelines for metal toxicity limits

for domestic animals, specifi cally cattle. A recent National Re-

search Council report (National Research Council, 2005) states

the limit for Pb is 100 mg kg−1. In this study, measured Pb levels

in four-wing saltbush transplant shoot tissues exceed this guide-

line by up to threefold (Table 3). A comparison of available data

indicates that Pb uptake may be saltbush species dependent. For

example, while four-wing saltbush has been reported to take up

Pb from acidic mine tailings in greenhouse experiments (Sabey

et al., 1990), other studies indicate that two other Atriplex spe-

Fig. 5. Kruskal’s Non-Metric Multidimensional Scaling (KNMDS) analysis of denaturing gradient gel electrophoresis (DGGE) profi les from tailings treatment plots as a function of time. Symbols include no compost (NC) (circle), compost (C) (triangle), and unplanted control (U) (square). For each plot, solid lines delineate clusters that are signifi cantly diff erent from each other and dashed lines delineate clusters that show a trend but are not signifi cantly diff erent from each other. The 0 mo plot represents samples taken before transplants were established (stress factor = 0.013, D = 3) (D, dimension) and shows three clusters that are signifi cantly diff erent (p = 0.06). The 3 mo plot (stress factor = 0.026, D = 3) shows no signifi cant clusters. The 5 mo plot (stress factor = 0.069, D = 3) shows two clusters that are signifi cantly diff erent from each other (p = 0.02). The 7 mo plot (stress factor = 0.071, D = 3) shows two clusters that are not signifi cantly diff erent (p = 0.18). The 11 mo plot (stress factor = 0.026, D = 3) shows three signifi cantly diff erent clusters (p = 0.01) and the 13 mo plot (stress factor = 0.023, D = 4) shows three clusters (p = 0.41).

Rosario et al.: Bacterial Community Changes during Plant Establishment 1257

cies, A. numularia, and A. lentiformis, did not shoot accumulate

Pb from alkaline and acidic tailings, respectively (Williams et al.,

1994; Jordan et al., 2002; Mendez et al., 2007). Th e observed

shoot accumulation of Pb in this study is undesirable as wildlife

may be negatively impacted. Th us, other native species that

naturally establish in the tailings area, such as big sacaton grass or

other Atriplex species, should be investigated.

Microbial Community AnalysisOne way in which the introduction of plants into mine

tailings sites improves ecosystem function is by enhancing mi-

crobial activity, which in turn enhances soil productivity and

supports plant establishment and succession. Th us, we hypoth-

esized that following planting the presence of root exudates

and compost C inputs would lead to an increase in culturable

heterotrophic bacterial numbers in planted treatments. Th is

hypothesis was confi rmed with a 1 to 1.5 log increase in het-

erotrophic numbers (CFUs) in planted treatment plots. Th is

increase occurred whether or not compost was added, sug-

gesting that the plants, rather than added compost, enhanced

the heterotrophic bacterial community beneath transplant.

canopies. Th is is further supported by the data showing that the

compost community DGGE profi le was quite diff erent from

the NC and C community profi les (Fig. 6). Note that both of

these communities had exposure to the compost. For the NC

treatments the plants were grown in compost before transplant-

ing and for the C treatments the plants were both grown in the

compost and were amended with compost during transplanting

We also hypothesized that plant establishment would lead

to an increase in species richness in the Boston Mill site. Th is

hypothesis was not confi rmed. An ANOVA analysis compar-

ing the number of bands among DGGE profi les suggests

that there was no signifi cant increase (p > 0.05) in richness

between planted areas and the unplanted controls (except at

5 mo, p = 0.02). While DGGE profi les do not provide total

richness indices, as one band may represent more than one

species (Jackson et al., 2000), these profi les can serve as a

screening tool to observe gross diff erences in species richness

between multiple samples. Interestingly, some studies have

suggested that root exudates can serve as selective compounds

decreasing richness indices rather than increasing them

(Marilley et al., 1998; Kozdrój and van Elsas, 2000).

As just discussed, an increase in heterotrophic bacterial numbers

is not equivalent to an increase in species richness, nor is it equiva-

lent to an increase in community functional diversity (e.g., nutrient

cycling, response to environmental stress). In fact it has been sug-

gested that heterotrophic bacterial numbers recover more readily

than functional diversity during reclamation (Moynahan et al.,

2002). Th is may be due to a slower response in the bacterial ge-

netic pool. Although species richness is not equivalent to functional

diversity, there is some indication that there may be a critical level

of species richness below which function is impaired (Griffi ths et

al., 1997). Th is is an important issue because low functional diver-

sity might compromise the ability of the bacterial community to

normally degrade organic matter, and thus, aff ect nutrient cycling.

One impact would be an accumulation of organic matter in the

site. Th is question can only be resolved by long-term monitoring

of the site to investigate if there is organic matter accumulation.

Spatial heterogeneity and seasonal changes often makes it dif-

fi cult to fi nd signifi cant diff erences among treatments in fi eld stud-

ies. In this study, although KNMDS results were not signifi cant

for every time point, a consistent trend was observed regarding

bacterial community structure from 5 mo on. In general, DGGE

profi les from planted treatments were more similar to each other

than to the unplanted controls. In contrast, there was no consistent

diff erence in profi les taken beneath transplant canopies in compost

and no compost treatments (similar to the plant growth results).

Th us, KNMDS analysis suggests that the establishment of plants,

in either the absence or presence of compost amendment, caused

a similar change in the bacterial community structure beneath

transplant canopies. Several other groups studying bacterial com-

munity structure have also observed community changes during

plant growth (e.g., Marschner et al., 2002; Kang and Mills, 2004;

Lerner et al., 2006). It would be intriguing to determine whether

the community structure changes observed in this study could be

used to develop biomarkers to predict the stage and success of phy-

tostabilization in arid and semiarid mine tailings sites.

Temporal analysis of unplanted control DGGE profi les in-

dicates that shifts occurred in the bulk soil bacterial community

(e.g., the Dice similarity coeffi cient for time 0 and time 13 mo

was 62%). Th e magnitude of this shift is actually quite similar to

that found for the NC (58.5%) and the C (52.6%) treatments

suggesting that there are natural seasonal changes in the bacte-

rial community structure in both the control and treated plots.

Such temporal shifts in soil communities have also been observed

previously in biological soil crusts (Nagy et al., 2005) and in an

upland grassland soil in the UK (Griffi ths et al., 2003). In the

latter study, profi les obtained from 5 to 10 cm and from 10 to 15

cm changed more than profi les obtained from 0 to 5 cm. In the

present study, samples were obtained from approximately 5 cm.

Th e results of this study along with similar fi ndings from

other groups show that natural temporal variations in the bac-

Fig. 6. Kruskal’s Non-Metric Multidimensional Scaling (KNMDS) analysis of the denaturing gradient gel electrophoresis (DGGE) profi le from the compost used to germinate and grow the transplants (compost only) and the profi les from one of the no compost plots (NC1) and one of the compost plots (C1) at times 0, 3, 5, and 11 mo. The plot (stress factor = 0.021, D = 4) (D, dimension) shows two signifi cantly diff erent clusters (p = 0.025).

1258 Journal of Environmental Quality • Volume 36 • September-October 2007

terial community can aff ect the interpretation of results. For

example, there was a single time point, 5 mo, where we observed

signifi cant diff erences in the number of bands among the DGGE

profi les of unplanted and planted areas. If this was the only time

point analyzed, this fi nding would have suggested that there were

increases in bacterial diversity associated with planted areas but

not unplanted controls. Th erefore, to assess reclamation success

through microbial community changes it is important to moni-

tor changes at various times throughout the year.

ConclusionsPrevious work has shown that reclamation strategies such as

liming, seeding, and compost addition can increase heterotro-

phic numbers and change community structure in both acidic

and alkaline mine tailings (Noyd et al., 1995; Mummey et al.,

2002; Moynahan et al., 2002; Kelly et al., 2003). Here we have

shown that the establishment of four-wing saltbush transplants

in the Boston Mill mine tailings site caused similar changes in

the bacterial community as detected by heterotrophic counts

and DGGE profi le analysis. In this moderately impacted mine

tailings site, compost amendment was not essential to either

establishment of plants or to the development of the bacterial

community. Study results also suggest that increased heterotro-

phic bacterial abundance and shifts in the dominant bacterial

populations did not translate into an increased bacterial species

richness. Long-term monitoring needs to be performed to inves-

tigate whether the observed changes are sustained over time and

to determine whether the observed shifts in community structure

refl ect potential increases in functional diversity. In summary,

the practical implication of this work is that reclamation eff orts

in alkaline mine tailings should take into consideration that the

addition of compost may not provide any benefi t for plant es-

tablishment. Th is should be a cost-reducing advantage for imple-

menting phytostabilization in moderately impacted mining sites.

AcknowledgmentsTh is research was supported by Grant 2 P42 ES04940-11

from the National Institute of Environmental Health Sciences

Superfund Basic Research Program, NIH. Our thanks to Karl Ford

and William L. Auby of the Bureau of Land Management for their

support and cooperation in giving us access to the Boston Mill

Site. We also wish to thank Michael Kopplin of the University of

Arizona Superfund Basic Research Program Hazard Identifi cation

Core for performing all ICP-MS total metal analyses.

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