Pedosphere 18(6): 731740, 2008

ISSN 1002-0160/CN 32-1315/P

c 2008 Soil Science Society of China

Published by Elsevier Limited and Science Press

Changes of Bacterial Community Structure in Copper Mine

Tailings After Colonization of Reed (Phragmites communis)1
CHEN Yu-Qing1 , REN Guan-Ju2 , AN Shu-Qing2,2 , SUN Qing-Ye3 , LIU Chang-Hong2 and
SHUANG Jing-Lei2
1 College

of Life Science, Nanjing Normal University, Nanjing 210046 (China). E-mail: chenyuqing@njnu.edu.cn
State Key Laboratory of Pollution Control and Resource Reuses, School of Life Science, Nanjing University, Nanjing
210093 (China)
3 School of Life Science, Anhui University, Hefei 230039 (China)
2 The

(Received February 16, 2008; revised June 10, 2008)

ABSTRACT
Soil samples were collected from both bare and vegetated mine tailings to study the changes in bacterial communities
and soil chemical properties of copper mine tailings due to reed (Phragmites communis) colonization. The structures of
bacterial communities were investigated using culture-independent 16S rRNA gene sequencing method. The bacterial
diversity in the bare mine tailing was lower than that of the vegetated mine tailing. The former was dominated by sulfur
metabolizing bacteria, whereas the latter was by nitrogen xing bacteria. The bare mine tailing was acidic (pH = 3.78),
whereas the vegetated mine tailing was near neutral (pH = 7.28). The contents of organic matter, total nitrogen, and
ammonium acetate-extractable potassium in vegetated mine tailings were signicantly higher than those in the bare mine
tailings (P < 0.01), whereas available phosphorus and electrical conductivity were signicantly lower than those in the bare
mine tailings (P < 0.01). The results demonstrated that 16S rRNA gene sequencing could be successfully used to study
the bacterial diversity in mine tailings. The colonization of the mine tailings by reed signicantly changed the bacterial
community and the chemical properties of tailings. The complex interactions between bacteria and plants deserve further
investigation.
Key Words:

16S rRNA gene, bacterial diversity, bacterial community, functional group, mine tailings

Citation: Chen, Y. Q., Ren, G. J., An, S. Q., Sun, Q. Y., Liu, C. H. and Shuang, J. L. 2008. Changes of bacterial
community structure in copper mine tailings after colonization of reed (Phragmites communis). Pedosphere. 18(6):
731740.

INTRODUCTION
With the rapid development of copper mining, the impacts of copper mine tailings have been expanding worldwide (Simpson et al., 1998; Lee and Correa, 2005). The mine tailings have been a major
source of heavy metal contamination and thus the restoration of the tailings is an unavoidable task for
both mining companies and governments (Manz and Castro, 1997; Bradshaw and Huttl, 2001; Sharma
and Al-Busaidi, 2001). Meanwhile, mine tailings as an accessible extreme environment have become
an ideal place for microbiologists and ecologists to explore the physiological and genetic diversities of
acidophilic bacteria (Okibe et al., 2003; Dopson et al. 2004).
A great deal of study on the mine tailings has been done to prevent the contamination and to restore
the wasteland. Restoring the vegetation has been the main approach to prevent the heavy metals and
other pollutants from releasing into the neighboring healthy ecosystems (Elberling et al., 2000; Tordo
et al., 2000; Bradshaw and Huttl, 2001). However, there still lies a great diculty for ecologists to
complete the task, because of the harsh physical properties, lack of nutrients, and the presence of toxic
1 Project

supported by the National Natural Science Foundation of China (Nos. 39830310 and 30070134) and the National
Key Basic Research Support Foundation (NKBRSF) of China (No. 2002CB111504).
2 Corresponding author. E-mail: anshq@nju.edu.cn.

732

Y. Q. CHEN et al.

substances (Tordo et al., 2000; Ye et al., 2002). The main challenge has been how to prevent the
acidication of mine tailings and how to deal with the acidied tailings and mine drainages (Romano
et al., 2003; Margarete, 2004). There have been very few successful examples in practical use so far.
Currently, biometallurgy is widely used (Krebs et al., 1997; Patra and Natarajan, 2003). Some
understanding of the biochemical mechanisms and the microbial function in the acidication process of
the tailings has been reported (Brett et al., 2004). Microbes, especially the sulfur-metabolizing bacteria,
play a key role in the acidication of the mine drainages and lead to environmental hazards (Benner
et al., 2000; Elberling et al., 2000). The microbes also play an important role in the mobilization and
immobilization of the heavy metals as well as the detoxication of metals and metalloids (Unz and
Shuttleworth, 1996; Rosen, 2002). Therefore, further research on microbial diversity in mine tailings is
important for the successful restoration of mine tailings. As the interactions exist between microbes,
it is necessary to study the microbial community as a whole (Liu et al., 2006). However, less research
has been done on the microbial communities in mine tailings so far. There are only limited reports on
the Uranium mines and the sulfur metabolizing bacteria (Vrionis et al., 2005; Skidmore et al., 2005).
Studies are needed to establish relationships among the vegetation, microbes, and chemical properties
of mine tailings.
In these experiments, the total bacterial DNA was extracted from both bare acidic copper mine
tailings and the same mine tailings with 20 years of reed (Phragmites communis) colonization. The
objectives of the present research were to document the changes of bacterial communities after vegetation
establishment, and to explore the eects of bacterial changes on the chemical properties of mine tailings.
MATERIALS AND METHODS
The research site is located in the Tongling Copper Mine area (30 54 N, 117 53 E), Anhui
Province, East China. The mine is one of the biggest ve copper mines in China. In the mining area,
the average annual rainfall is 1 346 mm, and the rainy season is from May to September. The average
annual temperature is 16.2 C. January is the coldest month while July is the hottest. The frost-free
period is 237258 days (Sun et al., 2004). A heap of mine tailings covers an area of 20 ha, and has
been abandoned for more than 3035 years. Natural reed population with 2025 years of history can
be found in part of the mine tailings. The reed population has an average height of 1.5 meters, and the
coverage of the reed population is approximately 30%.
Two plots were chosen for the studies. Plot 1 for the control sampling was completely bare without
any higher plants, and Plot 2 for the vegetated sampling was covered by the reed population. The two
plots were 20 m apart. Each plot was divided into four quadrats, and two sets of samples were collected
from each quadrat using a sterile soil core that was driven to a depth of 10 cm. A total of 16 cores were
collected. The samples were stored at 0 C immediately after being collected. Then these samples were
brought into lab within 24 h. One set of samples was stored in a deep freezer at 80 C for microbial
analysis, and the other set of samples was air-dried for chemical analysis.
The pH was measured by a pH meter equipped with a combination electrode (sediment:deionized
water = 1 g:5 mL). The electrical conductivity (EC) was determined by a conductance meter equipped
with a platinum electrode at 25 C (tailings:deionized water = 1 g:5 mL). Organic matter (OM) was
evaluated using K2 Cr2 O7 -H2 SO4 oxidation technique. Total nitrogen (TN) was determined by the Kjeldahl method (ISSCAS, 1978). Available phosphorus (AP, extracted with 0.5 mol L1 NaHCO3 ) was
determined using the molybdenum blue method (Institution of Soil Science, 1978). Ammonium acetateextractable potassium (EK, extracted with 1 mol L1 NH4 OAc) was measured using ame spectrophotometry (ISSCAS, 1978).
Four samples collected from the same plot were completely homogenized for DNA extraction. A
mixed sample of 0.5 g (wet weight), without roots, was mixed with 2 mL DNA extraction buer (100
mmol L1 pH 8.0 Tris-HCl, 100 mmol L1 pH 8.0 EDTA-Na2 , 100 mmol L1 pH 8.0 Na3 PO4 , 1.5 mol
L1 NaCl, 1% CTAB) in a 10 mL centrifuge tube, and then incubated in a shaker at 30 C with 200 r

BACTERIAL CHANGES AFTER REED COLONIZATION

733

min1 for 30 min. Subsequently, lysozyme was added to a nal concentration of 1 mg mL1 , and the
tubes were incubated at 37 C for 1 h. After that, SDS solution was added to a nal concentration of
1.5%, after being incubated at 65 C for 2 h. Then the tubes were centrifuged at 6 000 g for 10 min
to collect the supernatant in clean tubes, and the pellets were re-extracted. After that, a total of 9 mL
of the supernatant was obtained for DNA purication using a series of organic solutions: Tris buer
saturated phenol (pH 8.0), phenol-chloroform-isoamyl alcohol (25:24:1), and chloroform-isoamyl alcohol
(24:1), respectively. The crude DNA was precipitated with ethanol (4 C, 30 min). All pellets were
washed with ethanol and dissolved in 150 L TE buer (10 mmol L1 Tris and 1 mmol L1 Na2 EDTA,
pH 8.0). The crude DNA was puried using DNA Gel Purication Kit (Tianwei Inc., China) following
the protocol of the manufacturer. DNA was then dissolved with 20 L sterile H2 O and quantied under
UV light after agarose gel electrophoresis using a calibrated set of standard DNA of known concentrations.
Two bacteria-specic 16S rDNA primers, 8f (5 -AGAGTTTGATCMTGGC-3 ) and 1542r (5 -AAAGGAGGTGATCCA-3 ) were used to amplify the almost full-length 16S rDNA from the sampling sites
by polymerase chain reaction (PCR) (Skidmore et al., 2005). All reactions were carried out in 50 L
volumes, containing 0.4 mol L1 of each primer, 200 mmol L1 of each deoxyribonucleoside triphosphate, 5 mL of 10 PCR buer (100 mmol L1 Tris-HCl, 15 mmol L1 MgCl2 , 500 mmol L1 KCl;
pH 8.3), 2 U of Taq DNA polymerase (Tianwei Inc., China), and 1 L of the extracted DNA. PCR was
performed in a GeneAmp PCR System 2 400 (Perkin Elmer, German), with the following thermocycling
program: 5 min denaturizing at 94 C, followed by 30 cycles of 1 min denaturizing at 94 C, 1 min
annealing at 56 C, 1 min extension at 72 C, and a nal extension step of 10 min at 72 C. PCR
products were puried with a PCR Purication Kit (Tianwei Inc., China). A puried PCR product of
3 L was visualized by 1% (w/v) agarose gel electrophoresis to conrm their purity.
To avoid contamination, all solutions were prepared with sterile water, autoclaved twice and all
steps were performed in a sterilized incubator. The preparation of the master mix, the addition of
template, and the gel electrophoresis of PCR products were carried out in two separate incubators. For
each master mix, two negative controls were carried out through the whole procedure, in which water
instead of sample material was used, to exclude the possibility of false-positive PCR results through
cross contamination.
Ten microlitres of PCR product, about 1.5 kilobase, was cloned in the pGMT easy T-A Vector System (Tianwei Inc., China), following the protocol of the manufacturer. Five microlitres of the ligation
products was subsequently transformed into E. coli JM109, which allowed blue-white screening on the
LB plate containing antibiotic ampicillin 100 g mL1 , X-Gal 20 mg mL1 , and 40 mmol L1 IPTG,
to screen for positive clones.
The clones were amplied by PCR, with the vector-specic primers M13 according to the manufacturers instruction (Tianwei Inc., China). The PCR products were puried with a PCR Purication Kit
(Tianwei Inc., China), and then sequenced with an ABI 3100-Avant Genetic Analyzer (Applied Biosystems, USA). Sequencing reactions were carried out by cycle sequencing with the BigDye Terminato
(Applied Biosystems, USA) and 8f primer. These sequences, and their closely related 16S rDNA sequences, retrieved from the DNA databases using the BLAST program, were aligned with the ClustalW
program (ver. 1.60) and then realigned manually. Nucleotide positions, where there were ambiguous
alignments, were omitted from subsequent phylogenetic analysis. The phylogenetic tree of each sample
was constructed using the MEGA program (ver. 2.0), and the distance matrix was calculated by parsimony. The candidate taxonomy was further checked by a Naive Bayesian rRNA Classier (Ver. 1.0),
based on Bergeys Manual of Systematic Bacteriology.
The statistic analysis was carried out using the SPSS package (ver. 13.0). The calculation of Shannon
index was carried out using the following formula:


H =

s

i=1

pi ln pi

734

Y. Q. CHEN et al.

where H  is the value of the Shannon-Wiener diversity index; pi is the proportion of the ith species;
and s is the number of species in the community.
The selected sequences determined in this study have been deposited in GenBank under accession
Nos. DQ336025DQ336053.
RESULTS
Chemical properties of the copper mine tailings
The pH value of the bare mine tailings was 3.78, whereas that of the vegetated mine tailings was
7.21, and the dierence was statistically signicant (P < 0.01). The electrical conductivity (EC) of
bare mine tailings was 1.27 dS m1 , and the EC of vegetated mine tailings was as low as 0.13 dS m1
(Table I). The results indicated that bare mine tailings were acidic, whereas vegetated mine tailings
were near neutral. The contents of OM, TN and EK were 0.19 g kg1 , 0.26 g kg1 and 4.9 mg kg1 ,
respectively, in the bare mine tailings, whereas the values were 0.57 g kg1 , 0.93 g kg1 and 77.8 mg
kg1 , respectively, in the vegetated mine tailings. The results showed that the colonization of reed
brought more nutrients to the mine tailings. The concentration of AP of the bare mine tailings was 2.05
mg kg1 , whereas the value of vegetated mine tailings was 1.25 mg kg1 . The contents of OM, TN,
and EK of vegetated mine tailings were 3.0, 3.5, and 15.8 times higher than those of bare mine tailings,
respectively (P < 0.01). The content of AP was decreased by 40% from bare mine tailings to vegetated
mine tailings (P < 0.05).
TABLE I
The chemical properties of bare and vegetated copper mine tailings
Tailings

pH

Bare tailings
Vegetated tailings

3.781.39a)
7.210.51**

Electrical
conductivity

Organic
matter

Total N

dS m1
1.270.53**
0.130.08

g kg1
0.190.05
0.260.15
0.570.08** 0.930.24**

Ammonium
acetate-extractable K

Available P

mg kg1
4.91.0
77.824.1**

2.050.45*
1.250.44

*, **Signicant at P < 0.05 and P < 0.01, respectively.

a) Averagestandard deviation.

Dierent gene sequences between two mine tailings

After the PCR products of ligation were incubated, 86 clones and 115 clones were obtained from the
bare mine tailings and vegetated mine tailings, respectively. After blue-white screening, the numbers of
sequenced clones for the bare mine tailings and vegetated mine tailings were 44 and 51, respectively.
Among the clones 600 bases were determined on an average.
16S rRNA genes sequenced from bare mine tailings
All strains obtained from bare mine tailings were divided into 13 groups, among which Acidithiobacillus, Comamonas, sulfate-reducing bacterium, Alcaligenes, and Brevundimona dominated. The ve groups
together made up 78% of all the obtained strains (Table II). Meanwhile, there were 73% of sequences
obtained from the bare mine tailings that were closely related to the known sequences (the similarity
= 98%). If the similarity limit was expanded to 95%, there were 86% of the obtained sequences from
the bare mine tailings that were closely related to known sequences. The Shannon diversity index of the
bacteria species was 2.13.
16S rRNA genes sequenced from vegetated mine tailings
All the obtained strains from vegetated mine tailings were divided into 18 groups, among which Nitrosospira, acid streamer bacterium PK51, and Dechloromonas took the advantage in quantity, together
occupying 47% of all the obtained strains in the vegetated tailings (Table III). However, only 27% of

BACTERIAL CHANGES AFTER REED COLONIZATION

735

TABLE II
Identication and classication of 16S rRNA gene sequences obtained from bare tailings
Strains from bare tailings

Best matched strain in NCBI database (accession No.)

Similarity

T26,
T22,
T45,
T19,
T32
T10,
T11,
T13
T25
T29
T33
T4
T40
T5

Alcaligenes sp. (AJ002802)

Acidithiobacillus ferrooxidans (Y11595)

98%99%
98%99%

Comamonas testosteroni (AB007996)

Delftia tsuruhatensis (AY738262)
Sulfate-reducing bacterium Na82 (AB077817)
Brevundimonas diminuta (X87274)
Gram-positive iron-oxidizing acidophile Y0010 (AY140235)
Sulfobacillus sp. PK1 (AY534603)
Uncultured Nitrospira sp. clone 4-1 (AF351225)
Uncultured Rubrobacteridae clone glen99 25 (AY150872)
Bacillus fusiformis (AY472114)
Uncultured Chloroexi bacterium clone AKYG644 (AY922047)
Arthrobacter sp. GOL01(AY940423)

99%100%
100%
93%95%
93%99%
95%
89%
94%
93%
99%
90%
98%

T27, T3, T14, T39

T12, T20, T34, T9, T35, T44,
T7, T17, T1, T24, T30, T23
T41, T18, T8, T15, T31, T37
T43, T6, T42, T16, T38
T2, T21, T28

TABLE III
Identication and classication of 16S rRNA gene sequences obtained from vegetated tailings
Strains from vegetated tailings

Best matched strain in NCBI database (accession No.)

Similarity

P13, P46, P22, P40, P45, P48

Acid streamer bacterium PK51 (AY765997)
95%100%
P41, P9, P1, P32
Acidithiobacillus sp. NO-37 (AF376020)
98%99%
P16, P17, P18, P26
Dechloromonas sp. JJ (AY032611)
91%
P47, P23, P27
Gallionella (L07897)
95%
P5, P11, P29, P35, P52
Nitrosospira sp. FJI423 (AY631270)
92%93%
P4, P43, P44
Azoarcus sp. (AJ007007)
91%93%
P14, P25, P33
Janthinobacterium sp. IC161 (AB196254)
91%93%
P30, P34, P51, P8, P42, P39, P6, P49, P31 Nitrosospira sp. Ka3 (AY123806)
91%93%
P38, P21, P7
Thiomonas sp. B3 (AJ549220)
98%
P20, P24
Alcaligenes sp. (AJ002802)
98%99%
P3, P37
Uncultured Acidobacterium UA1 (AF200696)
97%
P12
Uncultured bacterium clone d021 (AF422632)
98%
P15
Rhodoblastus acidophila (M34128)
97%
P19
Desulfobulbus elongates (X95180)
95%
P2
Uncultured Chloroexi bacterium clone AKYG659 (AY921673) 92%
P28
Sulfuricurvum kujiense (AB080645)
97%
P36
Geothrix fermentans (U41563)
95%
P50
Enterobacter aerogenes (AB099402)
99%

sequences obtained from the vegetated mine tailings were closely related to the known sequences (similarity 98%), and when the similarity limit was expanded to 95%, only 47% were closely related to the
known sequences. The Shannon diversity index of the bacteria species was 2.56, being higher than that
in the bare mine tailings. These results suggested that the species diversity of bacteria in the vegetated
mine tailings was higher than that in the bare mine tailings.
Major subphyla of the bacteria communities
Proteobacteria, constituted by -, -, -, -, -Proteobacteria subdivisions, was the largest group,
but in dierent proportions in both clone libraries. There were 70% sequences in the bare mine tailings
belonging to the Proteobacteria phylum, whereas 78% of sequences from the vegetated mine tailings
could be ascribed to the same phylum. Although the proportion of Proteobacteria from the two plots
was almost the same, the distributions of these sequences in subdivisions were completely dierent. In
the bare mine tailings, four strains of -Proteobacteria, 13 strains of -Proteobacteria, and 14 strains of

736

Y. Q. CHEN et al.

-Proteobacteria were obtained, whereas in the vegetated mine tailings, one strain of -Proteobacteria,
32 strains of -Proteobacteria, 5 strains of -Proteobacteria, and 2 strains of /-Proteobacteria were
detected. The - and -Proteobacteria held major proportions in the Phylum. There were 47% of total
sequences in both libraries belonging to -Proteobacteria, and 20% belonging to -Proteobacteria. The
phylogenetic tree of selected sequences and known strains belonging to -Proteobacteria is shown in
Fig. 1a, whereas the phylogenetic tree of selected sequences and known strains belonging to -Proteoba-

Fig. 1 The phylogenetic trees of selected partial 16S rDNA sequences belonging to -Proteobacteria (a) and -Proteobacteria (b). The sequences marked with T were obtained from bare tailings, those marked with P were from vegetated tailings,
whereas those marked with taxon names were obtained from the NCBI (National Centre for Biotechnology Information)
gene bank.

BACTERIAL CHANGES AFTER REED COLONIZATION

737

cteria is shown in Fig. 1b. Through the two gures, no clear pattern between the two plots (Fig. 1) and
no obvious clusters were found among the sequences derived from the bare mine tailings or among those
derived from the vegetated mine tailings.
The functional groups of bacteria in the mine tailings
There were 14 out of 44 sequences from the bare mine tailings closely related to Acidithiobacillus
ferrooxidanss 16S rRNA gene and one sequence closely related to Sulfobacillus. All the 15 strains could
2
2
oxidize various sulfur compounds (S2 , S0 , S2 O2
4 , and SO3 ) into sulfate (SO4 ). Although there were
six sequences closely related to sulfate-reducing bacterium Na82s. Therefore, the 15 strains of sulfur
bacteria and six strains of sulfate reducing bacteria (SRB) constituted the most important functional
group of the local sulfur-cycling. Another functional group, having the function of nitrogen-xation, was
composed by the strains whose sequences were closely related to Nitrospira, Arthrobacter and Delftia
tsuruhatensis. However, the amount of the strains was much lower, and there was only one strain for
each genus.
Unlike the groups in the bare mine tailings, the dominant group changed from the sulfur metabolizing
group to the nitrogen xing group in the vegetated mine tailings. Twenty strains out of 51 sequences
were related to Nitrospira, Azoarcus, Enterobacter, and Alcaligenes, respectively, which constituted the
nitrogen xing group. Both the number of species and quantity of the functional group in the vegetated
mine tailings were higher compared to those in the bare mine tailings. Although, in vegetated mine
tailings there were only eight strains whose sequences were related to Acidithiobacillus, Thiomonas,
Desulfobulbus, and Sulfuricurvum, and belonged to the sulfur metabolism group, Moreover, there were
two more functional groups in the vegetated mine tailings: the photoautotrophic bacteria group and
the iron-oxidization group, which could not be found in the bare mine tailings. The photoautotrophic
bacteria group was composed of ve strains, whose sequences were closely related to Janthinobacterium,
Rhodoblastus, and Chloroexi, respectively, and they could use solar energy to transfer CO2 to organic
carbon. However, the iron oxidization group was composed of four strains whose sequences were closely
related to Gallionella and Geothrix, and it could utilize the energy from oxidizing Fe2+ to Fe3+ when
they transferred the CO2 to organic carbon.
DISCUSSION
The application of 16S rRNA gene in detecting microbes in tailings
In the application of the 16S rRNA gene to soil science, the extraction of puried, integral DNA is
always one of the hardest nuts to crack. The complex physical structure of soil makes it rather dicult
to accomplish high recovery rate and integrity at the same time (Paget et al., 1992). Moreover, the
humus and polysaccharide are always dicult to eliminate and the contamination of humus often causes
the failure of the following molecular experiments (Malik et al., 1994; Holben, 1997; Ogram, 1998). In
this study, the authors have demonstrated that, when applying the 16S rRNA gene in studying mine
tailings, no such problems, as mentioned earlier, have been encountered. The homogeneous texture
and extremely low content of humus make the extraction of qualied DNA very easy, and the following
molecular experiments have been carried out successfully. In addition to the easiness of DNA extraction,
the culture-independent 16S rRNA gene sequencing method has many advantages compared with any
culture-dependent approach. The sulfur metabolizing bacteria, as the most arresting organisms in mine
tailings, have been reported to have a bad growth condition in cold weather (Juszczak et al., 1995).
This characterization might lead to biased results when using the culture-dependent methods. In this
study, the samples have been collected in winter, when the soil temperature is about zero, although the
results display that a large amount of acidophiles do exist in the mine tailings, alive or not. Therefore,
the ability to cover and analyze 16S rRNA gene directly from environmental DNA could provide an
appropriate and convenient way for investigating microbial populations in tailing or sterile environment

738

Y. Q. CHEN et al.

without need for culture, eliminating the absolute dependence on the isolation of pure cultures.
Eects of plant colonization on the bacterial communities and chemical properties of mine tailings
The results showed that the bacterial communities of the bare mine tailings were quite dierent
from those in the vegetated mine tailings, although the two plots were close to each other and probably
had the same original substrate. However, without the changes of biota in the tailings, the process of
the physical-chemical properties of tailings would not change (Tordo et al., 2000; Stoltz and Greger,
2003). As the substrata of the two plots were discharged by the same factory, and the substrate was
treated by the same procedure, the bacterial community in the two plots, when discharged, could be
regarded as the same. The reed population grew up in one plot, and they changed the destination
of their habitat. For the historical reason it cannot be gured out as to why one plot was colonized
by reed and the other was not. Species-specic properties of plant and the properties of the root
exudates might have played very important roles in the selection of microbial communities in the metalcontaminated soils (Kunito et al., 2001). For example, the microbial community inhabiting the boreal
coniferous forest was inuenced by changes in heavy metal concentrations (Pennanen, 2001). There were
also some reports conrming that the colonization of plant populations could lead to the decrease of
acid-volatile suldes (Donna and Otte, 2004), and the rhizosphere of reed could reduce the toxicity of
Cu to bacteria (Kunito et al., 2001). Thus it can be inferred that the change of bacterial community
was initiated and stimulated by the colonization of reed population. As the results here showed, the
chemical properties of the two plots were also totally dierent. The changes in bacterial community
played an important role in the chemical properties of mine tailings. As widely reported, bacteria played
an important role in the process of oxidizing pyrite (Fortin and Beveridge, 1997; Elberling et al., 2000),
and Acidithiobacillus ferrooxidans, which dominated the bare mine tailings, could oxidize the sulfur
2
2
compounds (S2 , S0 , S2 O2
4 , SO3 ) into sulfate (SO4 ) (Cornelius et al., 2001), and oxidize the ferrous
to ferric (Gabriel and Vargas, 2003). During the process, the pH of mine tailings decreased, and the
mine tailings became acidic (Kupka and Kupsakova, 1999). The dominant bacteria in the bare mine
tailings were sulfur-metabolism bacteria, which might accelerate the process of acidication, and lead to
acidic mine tailings. Therefore, it was possible that with the change of bacterial community structure,
the existence of other bacteria prohibited the sulfur-metabolism bacterias eect on mine tailings, and
the process of acidication of mine tailings was stopped or delayed for at least 30 years. Moreover, the
nitrogen-xing and photoautotrophic bacteria, together with the reed population, made the mine tailing
nutrients richer than ever before.
As an accessible extreme environment, research on microbes in mine tailings opens up a new window
to look for valuable microbes for human use. In the present study, sequences that are closely related
to Comamonas and Brevundimonas were obtained, the two guilds have the ability to degrade benzoic
acid derivatives and organic phosphate, respectively (Bellinaso et al., 2003; Sondossi et al., 2004). As
only found in bare tailings with heavily acidic condition and extremely high content of heavy metals,
the two strains have probably developed the ability to adapt the acidic and heavy metal toxicity. Thus,
they may play a role in the restoration of contaminated soil of the mine tailings.
CONCLUSIONS
The bacteria showed an unimaginable viability and a higher diversity than expected. The plant
colonization changed the natural successional process of mine tailings thoroughly. After 20 plus years of
reed growth, the bacterial community changed from sulfur-metabolism bacteria dominant community to
nitrogen-xing and photoautotrophic bacteria dominant community. The change of bacterial community
could probably be attributed to the completely dierent chemical properties between bare mine tailings
and vegetated mine tailings. However, the interactions between plant colonization and changes of
bacterial community are still not clear. Further research is needed.

BACTERIAL CHANGES AFTER REED COLONIZATION

739

ACKNOWLEDGMENTS
Great thanks to Drs. Li Yuan and Fan Ning-Jiang in the Nanjing University, China for their assistance in the eld work; and to Dr. Q. X. Lin in the Louisiana State University, USA and Dr. W. X.
Cheng in the University of California at Santa Cruz, USA for their English editing.
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