1316

Effects of remediation on the bacterial community

of an acid mine drainage impacted stream
Suchismita Ghosh, Moumita Moitra, Christopher J. Woolverton, and Laura G. Leff

Abstract: Acid mine drainage (AMD) represents a global threat to water resources, and as such, remediation of AMD-
impacted streams is a common practice. During this study, we examined bacterial community structure and environmental
conditions in a low-order AMD-impacted stream before, during, and after remediation. Bacterial community structure was
examined via polymerase chain reaction amplification of 16S rRNA genes followed by denaturing gradient gel electrophoresis.
Also, bacterial abundance and physicochemical data (including metal concentrations) were collected and relationships to bacterial
community structure were determined using BIO-ENV analysis. Remediation of the study stream altered environmental
conditions, including pH and concentrations of some metals, and consequently, the bacterial community changed. However,
remediation did not necessarily restore the stream to conditions found in the unimpacted reference stream; for example, bacterial
abundances and concentrations of some elements, such as sulfur, magnesium, and manganese, were different in the remediated
stream than in the reference stream. BIO-ENV analysis revealed that changes in pH and iron concentration, associated with
remediation, primarily explained temporal alterations in bacterial community structure. Although the sites sampled in the
remediated stream were in relatively close proximity to each other, spatial variation in community composition suggests that
differences in local environmental conditions may have large impacts on the microbial assemblage.
Key words: acid mine drainage, remediation, denaturing gradient gel electrophoresis, bacterial community structure.
Résumé : Le drainage minier acide (DMA) représente une menace globale aux ressources en eau et de ce fait, la remédiation
des ruisseaux affectés par le DMA est devenue pratique courante. Lors de cette étude, nous avons examiné la structure de la
communauté bactérienne et les conditions environnementales des ruisseaux affectés par un DMA d’ordre faible, avant, durant et
après la remédiation. La structure de la communauté bactérienne a été examinée par une amplification par PCR des gènes
d’ARNr 16S suivie d’une électrophorèse sur gel en gradient dénaturant. Des données sur l’abondance des bactéries et les
conditions physicochimiques (incluant les concentrations de métaux) ont aussi été recueillies et leurs relations avec la structure de
la communauté bactérienne ont été déterminées par une analyse par BIO-ENV. La remédiation du ruisseau modifiait les
conditions environnementales dont le pH et les concentrations que quelques métaux, et conséquemment, la communauté
bactérienne changeait. Cependant, la remédiation ne rétablissait pas nécessairement le ruisseau aux conditions trouvées dans un
ruisseau de référence non affecté ; par exemple, l’abondance des bactéries et les concentrations de certains éléments comme le
soufre, le magnésium et le manganèse du ruisseau remédié différaient de celles du ruisseau de référence. L’analyse par BIO-
ENV a révélé que les changements de pH et de la concentration de fer associés à la remédiation expliquaient essentiellement les
modifications temporelles de la structure de la communauté bactérienne. Même si les sites échantillonnés du ruisseau remédié
étaient relativement près les uns des autres, la variation spatiale de la composition de la communauté suggère que des différences
de conditions environnementales locales peuvent avoir des impacts importants sur l’assemblage microbien.
Mots-clés : drainage minier acide, remédiation, électrophorèse sur gel en gradient dénaturant, structure de la communauté
bactérienne.
[Traduit par la Rédaction]

Introduction AMD occurs upon exposure of pyrite (FeS2) to O2, which

results in sulfuric acid and Fe2⫹ production (Nordstrom and
Acid mine drainage (AMD) occurs widely around the world Alpers 1999; Akcil and Koldas 2006). Formation and mainte-
(Sasowsky et al. 2000; Kimball et al. 2002; Neculita et al. nance of AMD is a geomicrobial phenomenon, wherein sulfur
2007) and is a major threat to water resources (USDA Forest and iron serve as sources of electrons to chemolithotrophic
Service 1993). Low pH and leaching of heavy metals at bacteria (Goltsman et al. 2009). Oxidation of iron via oxygen
AMD-affected sites deteriorates water quality and has adverse exposure is the dominant pathway for aqueous dissolution of
effects on aquatic life (Jennings et al. 2008). pyrite but is limited by low pH (Baker and Banfield 2003).
Received 13 May 2012. Revision received 28 August 2012. Accepted 15 September 2012. Published at www.nrcresearchpress.com/cjm
on 7 November 2012.
S. Ghosh, M. Moitra, and L.G. Leff. Department of Biological Sciences, Kent State University, Kent, OH 44242, USA.
C.J. Woolverton. Department of Environmental Health Sciences, Kent State University, Kent, OH 44242, USA.
Corresponding author: Suchismata Ghosh (e-mail: sghosh3@kent.edu).

Can. J. Microbiol. 58: 1316 –1326 (2012) doi:10.1139/w2012-110 Published by NRC Research Press
Ghosh et al.
Decreased abiotic rates of iron oxidation occur under acidic
conditions (Bond et al. 2000b), but iron-oxidizing bacteria
accelerate this process at low pH (Nordstrom and Southam
1997; Baker and Banfield 2003). Thus, microorganisms that
inhabit AMD environments contribute to maintenance of acid-
ity (Baker and Banfield 2003).
Because of the negative impacts of AMD on water resources
(Jennings et al. 2008), various remediation approaches have
been adopted (reviewed by Johnson and Hallberg 2005 and
Skousen et al. 1998). Abatement of AMD requires alteration
of physicochemical conditions via constructed systems
(Costello 2003). Such engineered systems used in AMD mit-
igation are generally effective in improving water quality, and
the mechanisms responsible may be biotic or abiotic (summa-
rized by O’Sullivan 2005). Abiotic remediation approaches
include active systems, like aeration and lime addition, and
passive systems, like anoxic limestone drains in which mine
water is passed through a bed of limestone gravel (Johnson
and Hallberg 2005). The latter is among the most common meth-
ods used to mitigate AMD, since it is low maintenance and relatively
low cost (Costello 2003; Johnson and Hallberg 2005).
Despite the adoption of several AMD abatement strategies,
improvement in environmental conditions is not always achieved
(Chadwick et al. 1986; Watanabe et al. 2000; McClurg et al.
2007), because remediation success depends on the method
employed and adequate characterization of the site (Evanko
and Dzombak 1997; Skousen et al. 2000; Kuipers et al. 2006).
Overall, knowledge of biological, hydrological, and geochem-
ical characteristics is important in monitoring and restoring
AMD-impacted systems (Edwards et al. 2000).
Given the importance of bacterial communities to the func-
tion of AMD-impacted streams (Baker and Banfield 2003;
Druschel et al. 2004) as well as unimpacted streams (Peterson
et al. 2001; Crump and Hobbie 2005; Hullar et al. 2006),
responses of bacterial communities may influence the success
of AMD remediation. In general, bacterial community struc-
ture is influenced by an array of physical and chemical con-
ditions, like pH; temperature; and concentrations of heavy
metals, oxygen, nutrients, and electron acceptors (Compton
et al. 2004; Fey and Conrad 2000; Köpke et al. 2005; Zhou
et al. 2002). Previous studies have also revealed that AMD-
associated metals, like iron, copper, lead, and zinc, impact
microbial communities (Foster et al. 2008; Wang et al. 2009).
However, alteration of bacterial community structure by re-
mediation of AMD-impacted streams is unstudied.
Therefore, in this study, we evaluated bacterial community
structure of a low-order AMD-impacted stream before, during,
and after remediation (by passive, limestone treatment). To
examine bacterial community structure, 16S rRNA genes were
amplified by polymerase chain reaction (PCR) and subjected
to denaturing gradient gel electrophoresis (DGGE; Muyzer
et al. 1993). This technique generates a genetic profile or
“fingerprint” of the bacterial community, which was used to
track changes in community composition as a result of reme-
diation. In addition, bacterial abundance and physicochemical
data (including metal concentrations) were examined and re-
lationships to bacterial community structure were determined
using BIO-ENV analysis. BIO-ENV is a multivariate tech-
nique for relating community structure to environmental data
(Clarke and Ainsworth 1993) and has been used for this
purpose in a variety of microbial ecology studies (Van der
1317
Gucht et al. 2005; Polymenakou et al. 2005; Bremner et al.
2006; Gilbert et al. 2009).
Materials and methods
Study site
Huff Run is a tributary (15.9 km long) of Conotton Creek (a
tributary of the Tuscarawas River) near Mineral City in north-
eastern Ohio. Discharge from abandoned coal mines and unre-
claimed coal refuse piles are responsible for acid loading to Huff
Run (Huff Run Watershed Restoration Partnership 2000).
Samples were collected from an unnamed, AMD-impacted,
first-order tributary of Huff Run (40°36.290=N, 81°18.407=W),
with an approximate gradient of 400 feet/mile (1 foot ⫽
0.3048 m; 1 mile ⫽ 1.609344 km) (http://huntersdb.com/OH/
County/Tuscarawas/topo), that drains an abandoned coal mine.
Discharge from this stream was responsible for substantial acid
loading to Huff Run, and thus, the stream was subject to reme-
diation (Maureen Wise, Huff Run Watershed Restoration Part-
nership Coordinator, personal communication, March 2008) via
steel slag leach beds and open limestone channels (a widely used
remediation approach (Costello 2003)). Specifically, water from
seeps was passed through alkalinity-producing vertical-flow
wetlands, and mine spoils were also reclaimed. Remediation
commenced in summer 2008 and ended in fall 2008.
In addition, 2 reference streams in the Huff Run watershed were
examined. One was an AMD-impacted, nonremediated stream and
the other was not impacted by AMD and had near neutral pH.
Sample collection and processing
Sediment and water samples were collected in triplicate
during November 2007 (preremediation), April 2008 (pre-
remediation), June 2008 (during remediation), and June 2009
(postremediation) from 3 sites (upstream, midstream, down-
stream) along the length of the study stream. Sites within the
stream were approximately 10 m apart. Sediment and water
samples were also collected from the reference (AMD impacted and
non-AMD impacted) streams in July 2009. Water temperature, pH,
conductivity, dissolved oxygen (DO) (VWR Symphony meter,
model 11388-314), and turbidity (Hach turbidometer, model 2100P)
were measured in the field (in triplicate) on each date.
For inductively coupled plasma optical emission spectrom-
etry (ICP-OES) analysis, water samples were filtered through
0.45 ␮m polycarbonate filters (Whatman, Piscataway, New
Jersey) and stored in 2% nitric acid. Sediment samples were
divided into 3 parts: 1 for organic matter determination, another
was frozen at – 80 °C for DNA extraction, and the remainder was
preserved with 8% paraformaldehyde in phosphate-buffered sa-
line (pH 7.2) for bacterial enumeration.
Sample analysis
Concentrations of calcium, magnesium, manganese, sulfur,
iron, and aluminum in water samples were determined using
ICP-OES (Perkin Elmer Optima 3300 DV ICP-OES). The
instrument failed to detect barium, mercury, arsenic, cobalt,
chromium, nickel, lead, zinc, cadmium, and selenium because
concentrations of these elements were lower than our stan-
dards. The lowest concentrations used were 2.5 ppm (mg/L)
for each barium, arsenic, cobalt, chromium, nickel, lead, and
zinc; 0.125 ppm (mg/L) for mercury; and 0.625 ppm (mg/L)
for cadmium and selenium. Only dissolved concentrations
were determined. Benthic organic matter content of
Published by NRC Research Press
1318
sediments was determined by loss on ignition analysis
(Eaton et al. 2012).
To enumerate bacteria, paraformaldehyde-preserved sam-
ples were treated with 0.1% tetrasodium pyrophosphate fol-
lowed by sonication at 40 kHz for 5 min (Ultrasonic cleaner,
model 2210; Branson Ultrasonics Co., Danbury, Connecticut)
to detach bacterial cells from sediment particles (McNamara
et al. 2002). Then samples were filtered through 0.2 ␮m pore
size black polycarbonate filters (Poretics, Livermore, Califor-
nia) and stained with DAPI (4,6-diamidino-2-phenylindole)
(Porter and Feig 1980). Bacteria in 10 fields per filter were
enumerated via epifluorescence microscopy.
DNA was extracted with the Power-Soil DNA extraction kit
(MoBio Laboratories, Carlsbad, California) as per the manu-
facturer’s instructions with minor modifications as in Feinstein
et al. (2009). 16S rRNA genes were amplified using 968f⫹GC
clamp (5=-GCCCCGCCGCGCGCGGCGGGCGGGGCGGGGGA
CGGGGGGAACGCGAAGAACCTTAC-3=; Heuer and Smalla
1997) and 1392 R (5=-ACGGGCGGTGTGTRC-3=; Brosius et al.
1981); primers were synthesized by Integrated DNA Technol-
ogies (Coralville, Iowa). PCR (Heuer et al. 1997 as modified
by van Dillewijn et al. 2002) was performed in a programma-
ble thermal controller (Bio-Rad Laboratories, Hercules, Cali-
fornia) with Go Taq Flexi DNA Polymerase (Promega
Corporation, Madison, Wisconsin). PCR master mixes were
prepared and each 25 ␮L of reaction mixture contained
15.125 ␮L of water, 5 ␮L of 5⫻ Green Go Taq Flexi buffer,
2 ␮L of 25 mmol/L MgCl2, 0.5 ␮L of 10 mmol/L dNTP mix,
0.125 ␮L of each primer, 2 ␮L of template DNA extract, and
0.125 ␮L of Go Taq DNA Polymerase. Negative controls
(sterile deionized water) and positive controls (Pseudomonas
aeruginosa genomic DNA) were run with each set of PCR
reactions. Success of PCR amplification was verified by gel
electrophoresis.
DGGE (Muyzer et al. 1993) of PCR products was per-
formed with a D-Code apparatus (Bio-Rad). DGGE was per-
formed with 6% polyacrylamide gels with urea–formamide
denaturing gradients of 30% to 60%. Electrophoresis was
carried out at 150 V for 6 h in 1⫻ Tris–acetate–EDTA buffer
(pH 8.0) at 60 °C. SYBR gold (1:10 000 dilution, Molecular
Probes, Eugene, Oregon) was used to stain the gels for 40 min.
Digital images of the stained gels were captured with a Gel
Doc imaging system (Bio-Rad).
Statistical analysis
Two-way ANOVA (analysis of variance) was used to ex-
amine differences among dates and sites for bacterial abun-
dance and pH, turbidity, conductivity, DO, organic matter, and
metal ion concentrations before, during, and after remediation
using JMP 8 software (SAS Institute Inc. 2010). Tukey’s test
was used for post hoc analysis, and p values ⱕ0.05 were
considered significant.
To compare bacterial community structure among sites in
the study stream, dates, and the reference streams, DGGE profiles
were analyzed using cluster analysis with the unweighted pair-
group method using arithmetic averages (UPGMA), to derive
linkage dendrograms. Similarity between DGGE profiles was
determined by calculating Sørensen’s pairwise similarity coeffi-
cient, Cs, based on the mean number of bands in common
between samples (Gillan et al. 1998). The pairwise similarity
coefficient was calculated as follows: Cs ⫽ 2D/(A ⫹ B) ⫻
Can. J. Microbiol. Vol. 58, 2012
100, where A was the total number of bands in sample 1, B was
the total number of bands in sample 2, and D was the number
of DGGE bands in common. Based on this calculation, 2
identical DGGE profiles would have a Cs ⫽ 100%, and 2
completely different profiles would have a Cs ⫽ 0%.
To determine the relationship between the environmental
parameters and bacterial community structure, BIO-ENV
(Clarke and Ainsworth 1993), Primer V5 (version 5.2.9,
Plymouth, UK), analysis was used. BIO-ENV was used to determine
the degree of association between the similarity matrix ob-
tained from DGGE profile and the Euclidean distance matrix
created after normalization of the obtained environmental data
(Bowen et al. 2009). Abiotic variables (pH, temperature, tur-
bidity, conductivity, DO, and metal ion concentrations) were
log transformed, and similarity matrices were created using
Euclidean distances. A given DGGE gel contained all samples
from 1 site from each date; therefore, each site was analyzed
separately to eliminate gel to gel variation in migration dis-
tances. To evaluate the degree of association between similar-
ity matrices, rank correlation (Spearman’s correlation
coefficient, ␳) was used; values range from –1 (maximum
incongruity) to ⫹1 (maximum congruity) (Bowen et al. 2009).
Results
Over the course of this study, pH changed significantly
(p ⬍ 0.05) over time, ranging from below 4.0 before and during
remediation to near neutral after remediation (Fig. 1). Before
remediation there were significant differences in pH among
sites in the study stream (p ⬍ 0.05); at the most downstream
site, pH was roughly double that of the most upstream site.
However, pH became more uniform among sites during and
after remediation (as reflected in the significant site-by-date
interaction). Postremediation, pH became uniform along the length
of the stream; pH was similar to the unimpacted reference stream and
higher than the AMD-impacted reference stream.
Other physicochemical data are summarized in Table 1.
Temperature varied significantly (p ⬍ 0.05) among dates and
displayed a typical seasonal pattern; lowest temperatures were
in November and higher temperatures were in April and June.
Unlike pH, conductivity did not vary significantly among sites
on a given date but decreased significantly over time; there
was a 3-fold decline postremediation. Postremediation, con-
ductivity of the study stream was significantly (p ⬍ 0.05)
lower than that of the AMD-impacted reference stream and
similar to that of the unimpacted reference stream (Table 2).
Like conductivity, turbidity did not vary among sites but
varied among dates (Table 1). Turbidity increased signifi-
cantly, more than 20-fold during remediation (p ⬍ 0.05), as
the water passed through limestone beds. Turbidity of the
study stream was similar to the AMD-impacted reference
stream before remediation but became more similar to the
unimpacted reference stream after remediation (Table 2).
DO differed significantly among sites and dates (p ⬍ 0.05,
Table 1). Prior to remediation, DO was lowest at the upstream
site and about 1.5-fold higher at the downstream site. During
and after remediation the DO increased significantly (p ⬍
0.05), and the differences among sites diminished.
There were no significant (p ⬎ 0.05) differences in elemen-
tal ion concentrations among sites on any given date, but they
differed significantly (p ⬍ 0.05) among dates (Fig. 2). Con-
centrations of magnesium, calcium, and sulfur were highest on
Published by NRC Research Press
Ghosh et al. 1319

Fig. 1. The pH of 3 sampling sites (U, upstream; M, midstream; D, downstream) in the study stream on 4 dates (Nov. 2007, Apr. 2008,
June 2008, and June 2009) compared with the pH of 2 sites in each reference stream sampled on 1 date (July 2009). ⫹, acid mine drainage
impacted reference stream; –, unimpacted reference stream. Values are means ⫾ standard errors.

Table 1. Physicochemical variables measured at each site on 4 dates.

Conductivity Turbidity Dissolved

Date Site Temp. (°C) (␮S/cm) (NTU) oxygen (mg/L)
Nov. 2007 U 7.30⫾0.06 902.00⫾3.06 0.31⫾0.01 4.70⫾0.32
M 7.40⫾0.23 877.33⫾6.36 0.47⫾0.03 4.63⫾0.35
D 7.30⫾0.00 899.33⫾2.33 0.71⫾0.06 6.01⫾0.53
Apr. 2008 U 15.20⫾0.21 752.67⫾29.34 0.59⫾0.02 4.97⫾0.12
M 15.50⫾0.15 691.00⫾2.65 0.44⫾0.03 5.27⫾0.19
D 15.20⫾0.11 696.00⫾4.04 0.87⫾0.03 6.43⫾0.24
June 2008 U 13.10⫾0.12 498.33⫾8.95 28.20⫾1.13 7.53⫾0.19
M 13.00⫾0.20 503.67⫾5.36 18.81⫾2.86 7.27⫾0.20
D 13.00⫾0.17 497.33⫾9.39 24.50⫾2.80 7.23⫾0.09
June 2009 U 20.10⫾0.32 246.67⫾29.17 5.86⫾0.51 8.31⫾0.15
M 19.50⫾0.09 239.67⫾9.53 14.07⫾1.09 7.06⫾0.09
Note: Values are means (n ⫽ 3) ⫾ standard errors. U, upstream; M, midstream; D, downstream.
November 2007 and April 2008 are preremediation sampling dates, June 2008 during remediation, and
June 2009 postremediation.

Table 2. Physicochemical variables measured at 2 sites in each reference stream in July 2009.

Conductivity Turbidity Dissolved

Stream Site Temp. (°C) (␮S/cm) (NTU) oxygen (mg/L)
AMD impacted 1 7.37⫾0.24 2280⫾40.41 1.32⫾0.26 5.13⫾0.18
(⫹ control) 2 6.97⫾0.28 2283⫾12.02 0.57⫾0.20 5.37⫾0.24
Not AMD impacted 1 8.81⫾0.01 337⫾40.37 7.21⫾0.79 8.70⫾0.15
(– control) 2 8.20⫾0.51 361⫾5.90 11.73⫾0.34 8.47⫾0.15
Note: Values are means (n ⫽ 3) ⫾ standard errors.

Published by NRC Research Press

1320 Can. J. Microbiol. Vol. 58, 2012

Fig. 2. Ion-coupled plasma analysis of concentrations of (A) magnesium, (B) sulfur, (C) calcium, (D) iron, (E) manganese, and
(F) aluminum preremediation, during remediation, and postremediation. Each bar in a graph represents the mean concentration from 3 sites
on that date. ⫹, acid mine drainage impacted reference stream; –, unimpacted reference stream. Values are means ⫾ standard errors.

Published by NRC Research Press

Ghosh et al.
Fig. 3. Bacterial numbers at each site in the study stream (U, upstream; M, midstream; D, downstream) on 4 dates (Nov. 2007, Apr. 2008,
June 2008, June 2009) compared with bacterial numbers at 2 sites in each reference stream sampled on July 2009. ⫹, acid mine drainage
impacted reference stream; –, unimpacted reference stream. Values are means ⫾ standard errors.
the first sampling date, and both magnesium and calcium were
similar to that of the AMD-impacted reference stream. Con-
centrations declined on the second sampling date prior to
remediation; however, this decline was statistically significant
only for sulfur and not significant for magnesium and calcium.
There was a significant decrease in concentrations of these
elements during remediation (p ⬍ 0.05). Postremediation there
was a significant increase in calcium concentrations, while
magnesium and sulfur concentrations remained low.
Iron concentrations were significantly (p ⬍ 0.05) lower in
the study stream than the AMD-impacted reference stream and
showed a sharp decline (28-fold) on the second sampling date
before remediation (Fig. 2). During remediation iron concen-
trations dropped, and postremediation, iron concentrations
were similar to the unimpacted reference stream.
On the first sampling date, manganese concentration was
not significantly different from the AMD-impacted reference
stream (Fig. 2). Subsequently, concentrations dropped 2-fold
on the second sampling date, and concentrations postremediation
were significantly (p ⬍ 0.05) lower than prior to remediation.
Aluminum concentrations were similar to the AMD-impacted
reference stream before remediation. Postremediation aluminum
declined dramatically and was below detection limit, similar to
the unimpacted reference stream.
Benthic organic matter content in the study stream ranged
from 8.3% to 10.3% before, during, and after remediation, and
there were no significant differences among dates and sites.
1321
Values were similar to the AMD-impacted reference stream
(9.8%–10.2%). However, the unimpacted reference stream
had a significantly higher percentage of organic matter
(24.0%–25.5%) when compared with the study stream post-
remediation.
Bacterial abundance changed significantly (p ⬍ 0.05) over
the course of the study (Fig. 3). There were no differences in
bacterial abundance among sites, but bacterial counts post-
remediation (June 2009) were significantly higher (10-fold)
than before and during remediation. Bacterial abundance be-
fore and during remediation were similar to the AMD-
impacted reference stream. Although bacterial numbers
increased by an order of magnitude after remediation, numbers
were still 104-fold higher in the unimpacted reference stream.
DGGE fingerprints obtained were used to compare the
structure of microbial community at the 3 sites over the course
of the remediation. Cluster analysis revealed that there was a
distinct community after remediation compared with before
remediation (Fig. 4). Also, bacterial community structure was
different among sites before and during remediation. Spatial
differences in community structure were observed both before
and during remediation: the upstream and midstream sites
were similar and differed from the downstream site. These
differences in community structure before and during reme-
diation decreased postremediation as the communities across
the sites became more uniform. Community structure before
and during remediation clustered with the AMD-impacted
Published by NRC Research Press
1322
Fig. 4. UPGMA (unweighted pair-group method using arithmetic
averages) dendrogram of bacterial DGGE (denaturing gradient gel
electrophoresis) bands showing patterns of similarity in bacterial
community structure on different dates and with the acid mine
drainage (AMD)-impacted and unimpacted reference streams.
Sampling dates (preremediation: Nov. 2007 and Apr. 2008; during
remediation: June 2008; postremediation: June 2009), sites (U,
upstream; M, midstream; D, downstream), AMD impacted
reference sites 1 and 2, and unimpacted reference sites 1 and 2 are
given at the end of branches.
reference stream, whereas postremediation community struc-
ture clustered with the unimpacted reference stream.
Similarity indices (Cs) were used to compare pairs of DGGE
fingerprints. DGGE profiles preremediation, when compared
with the AMD-impacted reference stream, had 35%– 44% simi-
larity. Bacterial communities postremediation compared with that
of the unimpacted reference stream had the highest similarity
(Cs ⫽ 80%). In contrast, when the bacterial community of the
unimpacted reference stream was compared with the community
of the AMD-impacted reference stream before and during reme-
diation, Cs ranged from 43% to 52%.
The number of DGGE bands varied significantly among
dates. Prior to remediation, 5–10 DGGE bands were obtained,
which increased to 14 –17 bands postremediation (Fig. 5). The
number of bands obtained from the study stream prior to reme-
diation was not significantly different when compared with the
AMD-impacted reference stream (8 –9 bands). The unimpacted
Can. J. Microbiol. Vol. 58, 2012
Fig. 5. Number of denaturing gradient gel electrophoresis (DGGE)
bands obtained on 4 dates (Nov. 2007, Apr. 2008, June 2008, June
2009). Each bar represents a mean of 3 sites on that date. ⫹, acid
mine drainage impacted reference stream; –, unimpacted reference
stream. Values are means ⫾ standard errors.
reference stream had the highest number of DGGE bands, 22–23,
which is significantly different from the number of bands ob-
tained from the study stream.
To examine the relationships of the DGGE results to the
environmental data, BIO-ENV analysis was performed and
revealed that a combination of different variables explained
the observed temporal alteration in bacterial community struc-
ture associated with remediation. At the upstream site, changes
in community structure were explained primarily by pH along
with changes in manganese and iron concentration (Spear-
man’s correlation coefficient ␳ ⫽ 0.943). Alteration in com-
munity structure at the midstream and downstream site was
related to pH and temperature along with changes in concen-
tration of magnesium and iron (␳ ⫽ 0.94).
Discussion
The immediate goal of AMD remediation is to increase the
pH of the stream and alter the biogeochemical cycle that
promotes acidification. In this study, remediation of the AMD-
impacted stream altered water chemistry and the bacterial
community. As observed in this and other studies (Runkel
et al. 2012; Cravotta and Trahan 1999; Hallberg and Johnson
2005), the pH of the study steam was altered as a result of the
remediation.
Beyond the effects of remediation on pH, concentrations of
metal ions are also altered because of the impacts of pH on
metal mobilization (Salomons 1995; El Khalil et al. 2008).
Consistent with this finding, in the present study, aluminum
and manganese concentrations declined dramatically as pH
changed during remediation. In contrast, concentrations of
iron declined greatly before remediation; rain and snow melt
lead to increased discharge of the stream prior to remediation
concurrently with drops in iron concentrations. A combination of
dilution resulting in oxidation and increased precipitation
Published by NRC Research Press
Ghosh et al.
(Stumm and Lee 1961; Watzlaf and Casson 1990; Earle and
Callaghan 1998) is likely responsible for this pattern. Calcium
and magnesium concentrations declined during remediation
but then rebounded, likely because of continuous dissolution
of leftover limestone (CaCO3) and dolomite (CaMg(CO3)2)
from channels constructed during remediation (Cravotta and
Trahan 1999; Kim et al. 2003). A similar pattern was observed
with sulfur concentration, which increased postremediation.
Perhaps neutralization by the addition of carbonates resulted in
precipitation of amorphous iron oxyhydroxide and goethite,
which, in turn, released significant quantities of soluble SO4
due to desorption (Rose and Elliott 2000).
Bacterial communities changed along with the environ-
mental conditions after remediation. Specifically, bacterial
abundance increased, and community structure became
more similar to the unimpacted reference stream. Low bacte-
rial abundance prior to remediation is likely related to the
restricted number of microbial species that AMD habitats
harbor (Baker and Banfield 2003; Yin et al. 2008; Druschel et
al. 2004). Postremediation abundances did not achieve the
values observed in the unimpacted reference, perhaps because
benthic organic matter was much higher in the reference
stream.
Bacterial abundance is not reflective of the composition of
the microbial community, and during remediation bacterial com-
munity structure also changed. In AMD-impacted streams, mi-
crobial community structure is shaped by environmental factors
like pH, temperature, iron, sulfur, lead, zinc, copper, and calcium
concentrations (Baker and Banfield 2003; Bond et al. 2000a;
Zhang et al. 2007a). Based on BIO-ENV analysis, pH along
with changes in temperature and elemental ion concentrations
(iron, manganese, and sulfur) played a significant role in
driving changes in bacterial community structure. However,
surprisingly, there were differences among sites in the rela-
tionship of bacterial community structure to environmental
conditions. Specifically, community composition of the down-
stream site, which had a higher pH before remediation than the
2 upstream sites, was independent of pH perhaps as the result
of groundwater entering the stream. Spatial variation in bac-
terial community structure postrestoration may result from the
local environment controlling microbial composition (Martiny
et al. 2006).
The maximum number of DGGE bands was obtained from
postremediation samples (15.3), and lowest numbers (8.5)
were found prior to remediation. In other freshwater environ-
ments that are not impacted by AMD, prior studies have found
DGGE band numbers ranging from 18 –21 in a study of
bacterial communities in lakes (Van der Gucht et al. 2001) to
28 –31 in a study of river biofilms (Lyautey et al. 2005), which
is similar to what we found in the unimpacted reference
stream. In prior studies of AMD-impacted environments, re-
gardless of the approach used (i.e., culture based or culture
independent), AMD bacterial communities have lowered di-
versity (Baker and Banfield 2003; Bond and Banfield 2001;
Druschel et al. 2004; Goebel and Stackebrandt 1994; Johnson
et al. 2001), likely reflective of a lower number of bacterial
niches and strict physiological requirements for success under
these conditions (Doelman et al. 1994; Zhang et al. 2007b).
Each DGGE band was assumed a phylotype or operational
taxonomic unit. Although this method allows us to estimate
the number of dominant phylotypes, collection of 16S rRNA
1323
gene sequence data in future studies can facilitate determina-
tion of shifts in dominant taxonomic groups of bacteria during
remediation.
Conclusion
Remediation of the study stream altered environmental con-
ditions, including pH and concentrations of some metals, and
consequently, the bacterial community changed. However,
remediation did not necessarily restore the stream to condi-
tions found in the unimpacted reference stream, but bacterial
community structure postremediation became more similar to
that of the unimpacted reference stream. Although the sites
sampled in the remediated stream were in relatively close
proximity to each other, spatial variation in community com-
position suggests that differences in local environmental con-
ditions have large impacts on the microbial assemblage.
Acknowledgements
This research was funded by Kent State University via a
Graduate Student Senate research grant and Art and Margaret
Herrick grant. Thanks to Maureen Wise, Elizabeth Griffith,
Xiaozhen Mou, Curtis Clevinger, Larry Feinstein, Alyssa Bax-
ter, Heather Kirkpatrick, and Oscar Valverde for their assis-
tance. The Author(s) declare(s) that there is no conflict of
interest.
References
Akcil, A., and Koldas, S. 2006. Acid mine drainage (AMD): causes,
treatment and case studies. J. Clean. Prod. 14(12–13): 1139 –1145.
doi:10.1016/j.jclepro.2004.09.006.
Baker, B.J., and Banfield, J.F. 2003. Microbial communities in acid
mine drainage. FEMS Microbiol. Ecol. 44(2): 139 –152. doi:
10.1016/S0168-6496(03)00028-X. PMID:19719632.
Bond, P.L., and Banfield, J.F. 2001. Design and performance of
rRNA targeted oligonucleotide probes for in situ detection and phy-
logenetic identification of microorganisms inhabiting acid mine drain-
age environments. Microb. Ecol. 41(2): 149 –161. doi:10.1007/
s002480000063. PMID:12032620.
Bond, P.L., Druschel, G.K., and Banfield, J.F. 2000a. Comparison of
acid mine drainage microbial communities in physically and
geochemically distinct ecosystems. Appl. Environ. Microbiol.
66(11): 4962– 4971. doi:10.1128/AEM.66.11.4962-4971.2000.
PMID:11055950.
Bond, P.L., Smriga, S.P., and Banfield, J.F. 2000b. Phylogeny of
microorganisms populating a thick, subaerial, predominantly litho-
trophic biofilm at an extreme acid mine drainage site. Appl.
Environ. Microbiol. 66(9): 3842–3849. doi:10.1128/AEM.66.9.
3842-3849.2000. PMID:10966399.
Bowen, J.L., Crump, B.C., Deegan, L.A., and Hobbie, J.E. 2009. Salt
marsh sediment bacteria: their distribution and response to external
nutrient inputs. ISME J. 3(8): 924 –934. doi:10.1038/ismej.2009.
44. PMID:19421233.
Bremner, J., Rogers, S.I., and Frid, C.L.J. 2006. Matching biological
traits to environmental conditions in marine benthic ecosystems.
J. Mar. Syst. 60(3–4): 302–316. doi:10.1016/j.jmarsys.2006.02.004.
Brosius, J., Dull, T.J., Sleeter, D.D., and Noller, H.F. 1981. Gene
organization and primary structure of a ribosomal RNA operon
from Escherichia coli. J. Mol. Biol. 148(2): 107–127. doi:10.1016/
0022-2836(81)90508-8. PMID:7028991.
Chadwick, J.W., Canton, S.P., and Dent, R.L. 1986. Recovery of
Published by NRC Research Press
1324
benthic invertebrate communities in Silver Bow Creek, Montana,
following improved metal mine wastewater treatment. Water Air
Soil Pollut. 28(3– 4): 427– 438. doi:10.1007/BF00583505.
Clarke, K.R., and Ainsworth, M. 1993. A method of linking multi-
variate community structure to environmental variables. Mar.
Ecol. Prog. Ser. 92: 205–219. doi:10.3354/meps092205.
Compton, J.E., Watrud, L.S., Porteous, L.A., and DeGrood, S. 2004.
Response of soil microbial biomass and community composition
to chronic nitrogen additions at Harvard forest. For. Ecol. Manage.
196(1): 143–158. doi:10.1016/j.foreco.2004.03.017.
Costello, C. 2003. Acid mine drainage: innovative treatment technol-
ogies. U.S. Environmental Protection Agency Report. U.S. EPA,
Technology Innovation Office, Washington, D.C., USA.
Cravotta, C.A., III, and Trahan, M.K. 1999. Limestone drains to
increase pH and remove dissolved metals from acidic mine drain-
age. Appl. Geochem. 14(5): 581– 606. doi:10.1016/S0883-2927(98)
00066-3.
Crump, B.C., and Hobbie, J.E. 2005. Synchrony and seasonality in
bacterioplankton communities of two temperate rivers. Limnol.
Oceanogr. 50(6): 1718 –1729. doi:10.4319/lo.2005.50.6.1718.
Doelman, P., Jansen, E., Michels, M., and van Til, M. 1994. Effects
of heavy metals in soil on microbial diversity and activity as
shown by the sensitivity-resistance index, an ecologically relevant
parameter. Biol. Fertil. Soils, 17(3): 177–184. doi:10.1007/
BF00336319.
Druschel, G.K., Baker, B.J., Gihring, T.M., and Banfield, J.F. 2004.
Acid mine drainage biogeochemistry at Iron Mountain, California.
Geochem. Trans. 5(2): 13–32. doi:10.1186/1467-4866-5-13.
Earle, J., and Callaghan, T. 1998. Effects of mine drainage on aquatic
life, water uses, and man-made structures. Chap. 4. In Coal mine
drainage prediction and pollution prevention in pennsylvania.
Pennsylvania Department of Environmental Protection, Penn.,
USA. pp. 4-1– 4-10.
Eaton, A.D., Rice, E.W., and Baird, R.B. (Editors). 2012. Standard
methods for the examination of water and wastewater. American
Public Health Association, American Water Works Association,
and Water Environment Federation. 22nd ed. Washington, D.C.,
USA.
Edwards, K.J., Bond, P.L., Druschel, G.K., McGuire, M.M., Hamers,
R.J., and Banfield, J.F. 2000. Geochemical and biological aspects
of sulfide mineral dissolution: lessons from Iron Mountain, Cali-
fornia. Chem. Geol. 169(3– 4): 383–397. doi:10.1016/S0009-
2541(00)00216-3.
El Khalil, H., El Hamiani, O., Bitton, G., Ouazzani, N., and Boularbah, A.
2008. Heavy metal contamination from mining sites in South
Morocco: monitoring metal content and toxicity of soil runoff and
groundwater. Environ. Monit. Assess. 136(1–3): 147–160. doi:
10.1007/s10661-007-9671-9. PMID:17375271.
Evanko, C.R., and Dzombak, D.A. 1997. Remediation of metals-
contaminated soils and groundwater. The Ground-Water Remedia-
tion Technologies Analysis Center (GWRTAC) Technology
Evaluation Report. E Series, TE-97-01.
Feinstein, L.M., Sul, W.J., and Blackwood, C.B. 2009. Assessment of
bias associated with incomplete extraction of microbial DNA from
soil. Appl. Environ. Microbiol. 75(16): 5428 –5433. doi:10.1128/
AEM.00120-09. PMID:19561189.
Fey, A., and Conrad, R. 2000. Effect of temperature on carbon and
electron flow and on the archaeal community in methanogenic rice
field soil. Appl. Environ. Microbiol. 66(11): 4790 – 4797. doi:
10.1128/AEM.66.11.4790-4797.2000. PMID:11055925.
Foster, A.L., Munk, L., Koski, R.A., Shanks, W.C., III, and Stillings,
Can. J. Microbiol. Vol. 58, 2012
L.L. 2008. Relationships between microbial communities and en-
vironmental parameters at sites impacted by mining of volcano-
genic massive sulfide deposits, Prince William Sound, Alaska.
Appl. Geochem. 23(2): 279 –307. doi:10.1016/j.apgeochem.2007.
10.012.
Gilbert, J.A., Field, D., Swift, P., Newbold, L., Oliver, A., Smyth, T.,
et al. 2009. The seasonal structure of microbial communities in the
Western English Channel. Environ. Microbiol. 11(12): 3132–
3139. doi:10.1111/j.1462-2920.2009.02017.x. PMID:19659500.
Gillan, D.C., Speksnijder, A.G.C.L., Zwart, G., and Deridder, C.
1998. Genetic diversity of the biofilm covering Montacuta ferrugi-
nosa (Mollusca, Bivalvia) as evaluated by denaturing gradient gel
electrophoresis analysis and cloning of PCR-amplified gene frag-
ments coding for 16S rRNA. Appl. Environ. Microbiol. 64(9):
3464 –3472. PMID:9726898.
Goebel, B.M., and Stackebrandt, E. 1994. Cultural and phylogenetic
analysis of mixed microbial populations found in natural and
commercial bioleaching environments. Appl. Environ. Microbiol.
60(5): 1614 –1621. PMID:7517131.
Goltsman, D.S., Denef, V.J., Singer, S.W., VerBerkmoes, N.C., Lefsrud,
M., Mueller, R.S., et al. 2009. Community genomic and proteomic
analyses of chemoautotrophic iron-oxidizing “Leptospirillum
rubarum” (group II) and “Leptospirillum ferrodiazotrophum”
(group III) bacteria in acid mine drainage biofilms. Appl. Environ.
Microbiol. 75(13): 4599 – 4615. doi:10.1128/AEM.02943-08.
PMID:19429552.
Hallberg, K.B., and Johnson, D.B. 2005. Biological manganese re-
moval from acid mine drainage in constructed wetlands and pro-
totype bioreactors. Sci. Total Environ. 338(1–2): 115–124. doi:
10.1016/j.scitotenv.2004.09.011. PMID:15680632.
Heuer, H., and Smalla, K. 1997. Application of denaturing gradient
gel electrophoresis and temperature gradient gel electrophoresis
for studying soil microbial communities. In Modern soil microbi-
ology. Edited by J.D. van Elsas, J.T. Trevors, and E.M.H.
Wellingon. Marcel Dekker, New York, USA. pp. 353–373.
Heuer, H., Krsek, M., Baker, P., Smalla, K., and Wellington, E.M.
1997. Analysis of actinomycete communities by specific amplification of
genes encoding 16S rRNA and gel-electrophoretic separation in dena-
turing gradients. Appl. Environ. Microbiol. 63(8): 3233–3241. PMID:
9251210.
Huff Run Watershed Restoration Partnership. 2000. Acid mine drain-
age abatement and treatment plan for the Huff Run Watershed.
Huff Run, Mineral City, Ohio, USA.
Hullar, M.A., Kaplan, L.A., and Stahl, D.A. 2006. Recurring seasonal
dynamics of microbial communities in stream habitats. Appl.
Environ. Microbiol. 72(1): 713–722. doi:10.1128/AEM.72.1.713-
722.2006. PMID:16391111.
Jennings, S.R., Neuman, D.R., and Blicker, P.S. 2008. Acid mine
drainage and effects on fish health and ecology: a review. Recla-
mation Research Group Publication, Bozeman, Mont., USA.
Johnson, D.B., and Hallberg, K.B. 2005. Acid mine drainage reme-
diation options: a review. Sci. Total Environ. 338(1–2): 3–14.
doi:10.1016/j.scitotenv.2004.09.002. PMID:15680622.
Johnson, D.B., Rolfe, S., Hallberg, K.B., and Iversen, E. 2001.
Isolation and phylogenetic characterization of acidophilic micro-
organisms indigenous to acidic drainage waters at an abandoned
Norwegian copper mine. Environ. Microbiol. 3(10): 630 – 637.
doi:10.1046/j.1462-2920.2001.00234.x. PMID:11722543.
Kim, J.J., Kim, S.J., and Choo, C.O. 2003. Seasonal change of
mineral precipitates from the coal mine drainage in the Taebaek
Published by NRC Research Press
Ghosh et al.
coal field, South Korea. Geochem. J. 37(1): 109 –121. doi:10.2343/
geochemj.37.109.
Kimball, B.A., Runkel, R.L., Walton-Day, K., and Bencala, K.E.
2002. Assessment of metal loads in watersheds affected by acid
mine drainage by using tracer injection and synoptic sampling:
Cement Creek, Colorado, USA. Appl. Geochem. 17(9): 1183–
1207. doi:10.1016/S0883-2927(02)00017-3.
Köpke, B., Wilms, R., Engelen, B., Cypionka, H., and Sass, H. 2005.
Microbial diversity in coastal subsurface sediments: a cultivation
approach using various electron acceptors and substrate gradients.
Appl. Environ. Microbiol. 71(12): 7819 –7830. doi:10.1128/AEM.
71.12.7819-7830.2005. PMID:16332756.
Kuipers, J.R., Maest, A.S., MacHardy, K.A., and Lawson, G. 2006.
Comparison of predicted and actual water quality at Hardrock
Mines: the reliability of predictions in environmental impact state-
ments. Kuipers & Associates, Butte, Mont., USA. pp. 12–14.
Lyautey, E., Lacoste, B., Ten-Hage, L., Rols, J.-L., and Garabetian, F.
2005. Analysis of bacterial diversity in river biofilms using 16S
rDNA PCR-DGGE: methodological settings and fingerprints inter-
pretation. Water Res. 39(2–3): 380 –388. doi:10.1016/j.watres.2004.
09.025. PMID:15644246.
Martiny, J.B., Bohannan, B.J., Brown, J.H., Colwell, R.K., Fuhrman,
J.A., Green, J.L., et al. 2006. Microbial biogeography: putting
microorganisms on the map. Nat. Rev. Microbiol. 4(2): 102–112.
doi:10.1038/nrmicro1341. PMID:16415926.
McClurg, S.E., Petty, T.J., Mazik, P.M., and Clayton, J.L. 2007.
Stream ecosystem response to limestone treatment in acid im-
pacted watersheds of the Allegheny Plateau. Ecol. Appl. 17(4):
1087–1104. doi:10.1890/06-0392. PMID:17555220.
McNamara, C.J., Lemke, M.J., and Leff, L.G. 2002. Culturable and
non-culturable fractions of bacterial populations in sediments of a
South Carolina stream. Hydrobiologia, 482(1–3): 151–159. doi:
10.1023/A:1021268516231.
Muyzer, G., de Waal, E.C., and Uitterlinden, A.G. 1993. Profiling of
complex microbial populations by denaturing gradient gel electro-
phoresis analysis of polymerase chain reaction-amplified genes
coding for 16S rRNA. Appl. Environ. Microbiol. 59(3): 695–700.
PMID:7683183.
Neculita, C.M., Zagury, G.J., and Bussiere, B. 2007. Passive treat-
ment of acid mine drainage in bioreactors using sulfate-reducing
bacteria: critical review and research needs. J. Environ. Qual.
36(1): 1–16. doi:10.2134/jeq2006.0066. PMID:17215207.
Nordstrom, D.K., and Alpers, C.N. 1999. Negative pH, efflorescent
mineralogy, and consequences for environmental restoration at the
Iron Mountain Superfund site, California. Proc. Natl. Acad. Sci.
U.S.A. 96(7): 3455–3462. doi:10.1073/pnas.96.7.3455. PMID:
10097057.
Nordstrom, D.K., and Southam, G. 1997. Geomicrobiology of sulfide
mineral oxidation. Vol. 35. In Geomicrobiology: interactions be-
tween microbes and minerals. Edited by J.F. Banfield and K.H.
Nealson. Mineralogical Society of America, Washington, D.C.,
USA. pp. 361–390.
O’Sullivan, A. 2005. Passive treatment technologies for managing
metal mine wastes: lessons learnt from global applications. In
Metal contaminants in New Zealand, sources, treatments, and
effects on ecology and human health. Edited by T.A. Moore, A.
Black, J.A. Centeno, J.S. Harding, D.A. Trumm, T.A. Moore, A.
Black, J.A. Centeno, J.S. Harding, D.A. Trumm. Resolutionz
Press, Christchurch, New Zealand. pp. 279 –300.
Peterson, B.J., Wollheim, W.M., Mulholland, P.J., Webster, J.R.,
Meyer, J.L., Tank, J.L., et al. 2001. Control of nitrogen export
1325
from watersheds by headwater streams. Science, 292(5514): 86 –
90. doi:10.1126/science.1056874. PMID:11292868.
Polymenakou, P.N., Bertilsson, S., Tselepides, A., and Stephanou,
E.G. 2005. Links between geographic location, environmental
factors, and microbial community composition in sediments of the
Eastern Mediterranean Sea. Microb. Ecol. 49(3): 367–378. doi:
10.1007/s00248-004-0274-5. PMID:16003476.
Porter, K.G., and Feig, Y.S. 1980. The use of DAPI for identifying
and counting aquatic microflora. Limnol. Oceanogr. 25(5): 943–
948. doi:10.4319/lo.1980.25.5.0943.
Rose, S., and Elliott, W.C. 2000. The effects of pH regulation upon the
release of sulfate from ferric precipitates formed in acid mine drainage.
Appl. Geochem. 15(1): 27–34. doi:10.1016/S0883-2927(99)00015-3.
Runkel, R.L., Kimball, B.A., Walton-Day, K., Verplanck, P.L., and
Broshears, R.E. 2012. Evaluating remedial alternatives for an acid
mine drainage stream: a model post audit. Environ. Sci. Technol.
46(1): 340 –347. doi:10.1021/es2038504. PMID:22074087.
Salomons, W. 1995. Environmental impact of metals derived from
mining activities: processes, predictions, prevention. J. Geochem.
Explor. 52(1–2): 5–23. doi:10.1016/0375-6742(94)00039-E.
SAS Institute Inc. 2010. JMP® 8 introductory guide. Release 8
[computer program]. SAS Institute Inc., Cary, N.C., USA.
Sasowsky, I.D., Foos, A., and Miller, C.M. 2000. Lithic controls on
the removal of iron and remediation of acidic mine drainage. Water
Res. 34(10): 2742–2746. doi:10.1016/S0043-1354(00)00019-1.
Skousen, J., Rose, A., Geidel, G., Foreman, J., Evans, R., and Hellier,
W., and Members of the Avoidance and Remediation Working
Group. 1998. Introduction. In A handbook of technologies for
avoidance and remediation of acid mine drainage. National Mine
Land Reclamation Center, West Virginia University, Morgantown,
W.Va., USA. pp. 4 –5.
Skousen, J.G., Sexstone, A., and Ziemkiewicz, P.F. 2000. Overview
of passive systems for treating acid mine drainage. In Acid mine
drainage control and treatment. Chap. 6. In Reclamation of dras-
tically disturbed lands. Edited by S. Frank. The American Society
for Agronomy and the American Society for Surface Mining and
Reclamation, Madison, Wis., USA. pp. 131–168.
Stumm, W., and Lee, G.F. 1961. Oxygenation of ferrous iron. Ind.
Eng. Chem. 53(2): 143–146. doi:10.1021/ie50614a030.
USDA Forest Service. 1993. Acid mine drainage from impact of
hardrock mining on the national forests: a management challenge.
Program Aid 1505.
Van der Gucht, K., Sabbe, K., De Meester, L., Vloemans, N., Zwart, G.,
Gillis, M., and Vyverman, W. 2001. Contrasting bacterioplankton
community composition and seasonal dynamics in two neighbour-
ing hypertrophic freshwater lakes. Environ. Microbiol. 3(11): 680–
690. doi:10.1046/j.1462-2920.2001.00242.x. PMID:11846758.
Van der Gucht, K., Vandekerckhove, T., Vloemans, N., Cousin, S.,
Muylaert, K., Sabbe, K., et al. 2005. Characterization of bacterial
communities in four freshwater lakes differing in nutrient load and
food web structure. FEMS Microbiol. Ecol. 53(2): 205–220. doi:
10.1016/j.femsec.2004.12.006. PMID:16329941.
van Dillewijn, P., Villadas, P.J., and Toro, N. 2002. Effect of a
Sinorhizobium meliloti strain with a modified putA gene on the
rhizosphere microbial community of alfalfa. Appl. Environ. Micro-
biol. 68(9): 4201– 4208. doi:10.1128/AEM.68.9.4201-4208.2002.
PMID:12200266.
Wang, Q., Zhou, D., Cang, L., Li, L., and Zhu, H. 2009. Indication of
soil heavy metal pollution with earthworms and soil microbial
biomass carbon in the vicinity of an abandoned copper mine in
Published by NRC Research Press
1326
Eastern Nanjing, China. Eur. J. Soil Biol. 45(3): 229 –234. doi:
10.1016/j.ejsobi.2008.12.002.
Watanabe, N.C., Harada, S., and Komai, Y. 2000. Long-term recov-
ery from mine drainage disturbance of a macroinvertebrate com-
munity in the Ichi-kawa River, Japan. Hydrobiologia, 429(1–3):
171–180. doi:10.1023/A:1004027201667.
Watzlaf, G., and Casson, L. 1990. Chemical stability of manganese
and iron in mine drainage treatment sludge: effects of neutraliza-
tion chemical, iron concentration, and sludge age. In Proceedings,
1990 Mining and Reclamation Conference. West Virginia Univer-
sity, Morgantown, W.Va., USA. pp. 3–9.
Yin, H., Qiu, G., Wu, L., Xie, M., Zhou, J., Dai, Z., et al. 2008.
Microbial community diversity and changes associated with a
mine drainage gradient at the Dexing copper mine, China. Aquat.
Microb. Ecol. 51(1): 67–76. doi:10.3354/ame01172.
Can. J. Microbiol. Vol. 58, 2012
Zhang, D., Xu, G., Zhang, W., and Golding, S.D. 2007a. High
salinity fluid inclusions in the Yinshan polymetallic deposit from
the Le-De metallogenic belt in Jiangxi Province, China: their
origin and implications for ore genesis. Ore Geol. Rev. 31(1– 4):
247–260. doi:10.1016/j.oregeorev.2004.11.002.
Zhang, H.B., Yang, M.X., Shi, W., Zheng, Y., Sha, T., and Zhao,
Z.W. 2007b. Bacterial diversity in mine tailings compared by
cultivation and cultivation-independent methods and their resis-
tance to lead and cadmium. Microb. Ecol. 54(4): 705–712. doi:
10.1007/s00248-007-9229-y. PMID:17333426.
Zhou, J., Xia, B., Treves, D.S., Wu, L.-Y., Marsh, T.L., O’Neill,
R.V., et al. 2002. Spatial and resource factors influencing high
microbial diversity in soil. Appl. Environ. Microbiol. 68(1): 326 –
334. doi:10.1128/AEM.68.1.326-334.2002. PMID:11772642.
Published by NRC Research Press
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