Chen Et Al. - 2008 - Changes of Bacterial Community Structure in Copper Mine Tailings After Colonization of Reed (Phragmites Communis)

8/18/2019 Chen Et Al. - 2008 - Changes of Bacterial Community Structure in Copper Mine Tailings After Colonization of Reed…

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Pedosphere 18(6): 731–740, 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 MineTailings 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

1College of Life Science, Nanjing Normal University, Nanjing 210046 (China). E-mail: [email protected] 2The State Key Laboratory of Pollution Control and Resource Reuses, School of Life Science, Nanjing University, Nanjing

210093 (China)3School of Life Science, Anhui University, Hefei 230039 (China)

(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 fixing 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 significantly higher than those in the bare mine

tailings (P


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732 Y. Q. CHEN et al .

substances (Tordoff et al., 2000; Ye et al., 2002). The main challenge has been how to prevent the

acidification of mine tailings and how to deal with the acidified 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 acidification process of

the tailings has been reported (Brett et al., 2004). Microbes, especially the sulfur-metabolizing bacteria,

play a key role in the acidification 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 detoxification 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 effects 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 five 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 237–258 days (Sun et al., 2004). A heap of mine tailings covers an area of 20 ha, and hasbeen abandoned for more than 30–35 years. Natural reed population with 20–25 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:deionizedwater = 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 K2Cr2O7-H2SO4 oxidation technique. Total nitrogen (TN) was determined by the Kjel-

dahl method (ISSCAS, 1978). Available phosphorus (AP, extracted with 0.5 mol L−1 NaHCO3) was

determined using the molybdenum blue method (Institution of Soil Science, 1978). Ammonium acetate-

extractable potassium (EK, extracted with 1 mol L−1 NH4OAc) was measured using flame spectropho-

tometry (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 buffer (100

mmol L−1 pH 8.0 Tris-HCl, 100 mmol L−1 pH 8.0 EDTA-Na2, 100 mmol L−1 pH 8.0 Na3PO4, 1.5 mol

L−1 NaCl, 1% CTAB) in a 10 mL centrifuge tube, and then incubated in a shaker at 30 ◦C with 200 r


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BACTERIAL CHANGES AFTER REED COLONIZATION 733

min−1 for 30 min. Subsequently, lysozyme was added to a final concentration of 1 mg mL−1, and the

tubes were incubated at 37 ◦C for 1 h. After that, SDS solution was added to a final 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 purification using a series of organic solutions: Tris buffer

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 µL TE buffer (10 mmol L−1 Tris and 1 mmol L−1 Na2EDTA,

pH 8.0). The crude DNA was purified using DNA Gel Purification Kit (Tianwei Inc., China) following

the protocol of the manufacturer. DNA was then dissolved with 20 µL sterile H2O and quantified under

UV light after agarose gel electrophoresis using a calibrated set of standard DNA of known concentra-

tions.

Two bacteria-specific 16S rDNA primers, 8f (5-AGAGTTTGATCMTGGC-3) and 1542r (5-AAA-

GGAGGTGATCCA-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 µL

volumes, containing 0.4 µmol L−1

of each primer, 200 mmol L−1

of each deoxyribonucleoside triphos-phate, 5 mL of 10 × PCR buffer (100 mmol L−1 Tris-HCl, 15 mmol L−1 MgCl2, 500 mmol L

−1 KCl;

pH 8.3), 2 U of Taq DNA polymerase (Tianwei Inc., China), and 1 µL 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 final extension step of 10 min at 72 ◦C. PCR

products were purified with a PCR Purification Kit (Tianwei Inc., China). A purified PCR product of

3 µL was visualized by 1% (w/v) agarose gel electrophoresis to confirm 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 Sys-

tem (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 µg mL−1, X-Gal 20 mg mL−1, and 40 mmol L−1 IPTG,

to screen for positive clones.

The clones were amplified by PCR, with the vector-specific primers M13 according to the manufac-

turer’s instruction (Tianwei Inc., China). The PCR products were purified with a PCR Purification Kit

(Tianwei Inc., China), and then sequenced with an ABI 3100-Avant Genetic Analyzer (Applied Biosys-

tems, 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 se-quences, 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 par-

simony. The candidate taxonomy was further checked by a Naive Bayesian rRNA Classifier (Ver. 1.0),

based on Bergey’s 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


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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. DQ336025–DQ336053.

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 difference was statistically significant (P < 0.01). The electrical conductivity (EC) of

bare mine tailings was 1.27 dS m−1, and the EC of vegetated mine tailings was as low as 0.13 dS m−1

(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 kg−1, 0.26 g kg−1 and 4.9 mg kg−1,

respectively, in the bare mine tailings, whereas the values were 0.57 g kg−1, 0.93 g kg−1 and 77.8 mg

kg−1, 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 2.05mg kg−1, whereas the value of vegetated mine tailings was 1.25 mg kg−1. 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


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TABLE II

Identification and classification of 16S rRNA gene sequences obtained from bare tailings

Strains from bare tailings Best matched strain in NCBI database (accession No.) SimilarityT26, T27, T3, T14, T39 Alcaligenes sp. (AJ002802) 98%–99%

T22, T12, T20, T34, T9, T35, T44, Acidithiobacillus ferrooxidans (Y11595) 98%–99%

T45, T7, T17, T1, T24, T30, T23

T19, T41, T18, T8, T15, T31, T37 Comamonas testosteroni (AB007996) 99%–100%

T32 Delftia tsuruhatensis (AY738262) 100%

T10, T43, T6, T42, T16, T38 Sulfate-reducing bacterium Na82 (AB077817) 93%–95%

T11, T2, T21, T28 Brevundimonas diminuta (X87274) 93%–99%

T13 Gram-positive iron-oxidizing acidophile Y0010 (AY140235) 95%

T25 Sulfobacillus sp. PK1 (AY534603) 89%

T29 Uncultured Nitrospira sp. clone 4-1 (AF351225) 94%

T33 Uncultured Rubrobacteridae clone glen99 25 (AY150872) 93%

T4 Bacillus fusiformis (AY472114) 99%

T40 Uncultured Chloroflexi bacterium clone AKYG644 (AY922047) 90%

T5 Arthrobacter sp. GOL01(AY940423) 98%

TABLE III

Identification and classification 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 Chloroflexi 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 (simi-

larity ≥ 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 thatin 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 α-, β -, γ -, δ -, ε-Proteobacteria subdivisions, was the largest group,

but in different 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 different. In

the bare mine tailings, four strains of α-Proteobacteria , 13 strains of β -Proteobacteria , and 14 strains of


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γ -Proteobacteria were obtained, whereas in the vegetated mine tailings, one strain of α-Proteobacteria ,

32 strains of β -Proteobacteria , 5 strains of γ -Proteobacteria , and 2 strains of δ/ε-Proteobacteria were

detected. The β - and γ -Proteobacteria held major proportions in the Phylum. There were 47% of total

sequences in both libraries belonging to β -Proteobacteria , and 20% belonging to γ -Proteobacteria . The

phylogenetic tree of selected sequences and known strains belonging to β -Proteobacteria is shown in

Fig. 1a, whereas the phylogenetic tree of selected sequences and known strains belonging to γ -Proteoba-

Fig. 1 The phylogenetic trees of selected partial 16S rDNA sequences belonging to β -Proteobacteria (a) and γ -Proteobacte-ria (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.


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cteria is shown in Fig. 1b. Through the two figures, 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

ferrooxidans ’s 16S rRNA gene and one sequence closely related to Sulfobacillus . All the 15 strains could

oxidize various sulfur compounds (S2−, S0, S2O2−4 , and SO

2−3 ) into sulfate (SO

2−4 ). Although there were

six sequences closely related to sulfate-reducing bacterium Na82’s. 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-fixation, 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 fixing 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 fixing 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 five strains, whose sequences were closely related to Janthinobacterium ,

Rhodoblastus , and Chloroflexi , 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+

whenthey 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 purified, integral DNA is

always one of the hardest nuts to crack. The complex physical structure of soil makes it rather difficult

to accomplish high recovery rate and integrity at the same time (Paget et al., 1992). Moreover, the

humus and polysaccharide are always difficult 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 minetailings, no such problems, as mentioned earlier, have been encountered. The homogeneous texture

and extremely low content of humus make the extraction of qualified 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


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without need for culture, eliminating the absolute dependence on the isolation of pure cultures.

Effects 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 differentfrom 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 (Tordoff 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 figured out as to why one plot was colonized

by reed and the other was not. Species-specific properties of plant and the properties of the root

exudates might have played very important roles in the selection of microbial communities in the metal-

contaminated soils (Kunito et al., 2001). For example, the microbial community inhabiting the boreal

coniferous forest was influenced by changes in heavy metal concentrations (Pennanen, 2001). There were

also some reports confirming that the colonization of plant populations could lead to the decrease of acid-volatile sulfides (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 different. 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

compounds (S2−, S0, S2O2−4 , SO

2−3 ) into sulfate (SO

2−4 ) (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 Kupsáková, 1999). The dominant bacteria in the bare mine

tailings were sulfur-metabolism bacteria, which might accelerate the process of acidification, and lead toacidic mine tailings. Therefore, it was possible that with the change of bacterial community structure,

the existence of other bacteria prohibited the sulfur-metabolism bacteria’s effect on mine tailings, and

the process of acidification of mine tailings was stopped or delayed for at least 30 years. Moreover, the

nitrogen-fixing 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-fixing and photoautotrophic bacteria dominant community. The change of bacterial community

could probably be attributed to the completely different 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.


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ACKNOWLEDGMENTS

Great thanks to Drs. Li Yuan and Fan Ning-Jiang in the Nanjing University, China for their assis-

tance in the field 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.

REFERENCES

Bellinaso, M. D. L., Greer, C. W., Carmo Peralba, M., Henriques, J. A. P. and Gaylarde, C. C. 2003. Biodegradation of

the herbicide trifluralin by bacteria isolated from soil. FEMS Microbiol. Ecol . 43: 191–194.

Benner, S. G., Gould, W. D. and Blowes, D. W. 2000. Microbial populations associated with the generation and treatment

of acid mine drainage. Chem. Geol. 169: 435–448.

Bradshaw, A. 1997. Restoration of mined lands—Using natural processes. Ecol. Eng. 8: 255–269.

Bradshaw, A. D. and Huttl, R. F. 2001. Future minesite restoration involves a broader approach. Ecol. Eng . 17: 87–90.

Brett, J. B., Lutz, M. A., Dawson, S. C., Bond, P. L. and Banfield, J. F. 2004. Metabolically active eukaryotic communities

in extremely acidic mine drainage. Appl. Environ. Microbiol . 70: 6264–6271.

Cornelius, G. F., Rother, D., Bardischewsky, F., Quentmeier, A. and Fischer, J. 2001. Oxidation of reduced inorganicsulfur compounds by bacteria: Emergence of a common mechanism? Appl. Environ. Microbiol . 67: 2873–2882.

Donna, L. J. and Otte, M. L. 2004. Long-term effects of submergence and wetland vegetation on metals in a 90-year old

abandoned Pb-Zn mine tailings pond. Environ. Pollut . 130: 337–345.

Dopson, M., Baker-Austin, C., Hind, A., Bowman, J. P. and Bond, P. L. 2004. Characterization of Ferroplasma isolates

and Ferroplasma acidarmanus sp. Nov., extreme acidophiles from acid mine drainage and industrial bioleaching

environments. Appl. Environ. Microbiol . 70: 2079–2088.

Elberling, B, Schippers, A. and Sand, W. 2000. Bacterial and chemical oxidation of pyritic mine tailings at low tempera-

tures. J. Contam. Hydrol. 41: 225–238.

Fortin, D. and Beveridge, T. J. 1997. Role of the bacterium Thiobacillus in the formation of silicates in acidic mine

tailings. Chem. Geol . 141: 235–250.

Gabriel, M. and Vargas, T. 2003. Bacterial oxidation of ferrous iron by Acidithiobacillus ferrooxidans in the pH range

2.5–7.0. Hydrometallurgy . 71: 149–158.

Holben, W. E. 1997. Isolation and purification of bacterial community DNA from environmental samples. In Hurst,

C. J., Knudsen, G. R., McInerney, M. J., Stetzenbach, L. D. and Walter M. V. (eds.) Manual of EnvironmentalMicrobiology. American Society for Microbiology, Washington, D.C. pp. 431–436.

Institute of Soil Science, Chinese Academy of Sciences (ISSCAS). 1978. Physical and Chemical Analysis Methods of Soils

(in Chinese). Shanghai Science and Technology Press, Shanghai.

Juszczak, A., Domka, F., Kozlowski, M. and Wachowska, H. 1995. Microbial desulfurization of coal with Thiobacillus

ferrooxidans bacteria. Fuel . 74: 725–728.

Krebs, W, Brombacher, C., Bosshard, P., Bachofen, R. and Brandl, H. 1997. Microbial recovery of metals from solids.

FEMS Microbiol. Rev . 20: 605–617.

Kunito, T., Saeki, K., Nagaoka, K., Oyaizu, H. and Matsumoto, S. 2001. Characterization of copper-resistant bacterial

community in rhizosphere of highly copper-contaminated soil. Eur. J. Soil Biol . 37: 95–102.

Kupka, D. and Kupśaková, I. 1999. Iron(II) oxidation kinetics in Thiobacillus ferrooxidans in the presence of heavy

metals. In Amils, R. and Ballester, A. (eds.) Biohydrometallurgy and the Environment Toward the Mining of the

21st Century, Part A. Elsevier Press, Amsterdam. pp. 387–396.

Lee, M. R. and Correa, J. A. 2005. Effects of copper mine tailings disposal on littoral meiofaunal assemblages in the

Atacama region of northern Chile. Mar. Environ. Res. 59: 1–18.

Liu, B. R., Jia, G. M., Chen, J. and Wang, G. 2006. A review of methods for studying microbial diversity in soils.

Pedosphere. 16(1): 18–24.

Malik, M., Kain, J., Pettigrew, C. and Ogram, A. 1994. Purification and molecular analysis of microbial DNA from

compost. J. Microbiol. Method . 20: 183–196.

Manz, M. and Castro, L. J. 1997. The environmental hazard caused by smelter slags from the Sta. Maria de la Paz mining

district in Mexico. Environ. Pollut . 98: 7–13.

Margarete, K. 2004. Passive mine water treatment: The correct approach? Ecol. Eng . 22: 299–304.

Ogram, A. 1998. Isolation of nucleic acids from environmental samples. In Burlage, R. S., Atlas, R., Stahl, D., Geesey,

G. and Sayler, G. (eds.) Techniques in Microbial Ecology. Oxford University Press, New York. pp. 273–288.

Okibe, N., Gericke, M., Hallberg, K. B. and Johnson, D. B. 2003. Enumeration and characterization of acidophilic

microorganisms isolated from a pilot plant stirred-tank bioleaching operation. Appl. Environ. Microbiol . 69: 1936–

1 943.

Paget, E., Monrozier, L. J. and Simonet, P. 1992. Adsorption of DNA on clay minerals: protection against DNase I and

influence on gene transfer. FEMS Microbiol. Lett . 97: 31–40.


10/10


Patra, P. and Natarajan, K. A. 2003. Microbially-induced flocculation and flotation for pyrite separation from oxide

gangue minerals. Miner. Eng . 16: 965–973.

Pennanen, T. 2001. Microbial communities in boreal coniferous forest humus exposed to heavy metals and changes in soil

pH—A summary of the use of phospholipid fatty acids, Biolog(R) and 3H-thymidine incorporation methods in field

studies. Geoderma . 100: 91–126.

Romano, C. G.,Ulrich Mayer, K., Jones, D. R., Ellerbroek, D. A. and Blowes, D. W. 2003. Effectiveness of various cover

scenarios on the rate of sulfide oxidation of mine tailings. J. Hydrol. 271: 171–187.

Rosen, B. P. 2002. Transport and detoxification systems for transition metals, heavy metals and metalloids in eukaryotic

and prokaryotic microbes. Comp. Biochem. Physiol. 133(Part A): 689–693.

Sharma, R. S. and Al-Busaidi, T. S. 2001. Groundwater pollution due to a tailings dam. Eng. Geol . 60: 235–244.

Simpson, S. L., Apte, S. C., Hortle, K. G. and Richards, D. G. 1998. An evaluation of copper remobilization from mine

tailings in sulfidic environments. J. Geochem. Explor . 63: 203–215.

Skidmore, M., Anderson, S. P., Sharp, M., Foght, J. and Lanoil, B. D. 2005. Comparison of microbial community

compositions of two subglacial environments reveals a possible role for microbes in chemical weathering processes.

Appl. Environ. Microbiol. 71: 6986–6997.

Sondossi, M., Barriault, D. and Sylvestre, M. 2004. Metabolism of 2,2- and 3,3-dihydroxybiphenyl by the biphenyl

catabolic pathway of Comamonas testosteroni B-356. Appl. Environ. Microbiol . 70(1): 174–181.

Stoltz, E. and Greger, M. 2002. Accumulation properties of As, Cd, Cu, Pb and Zn by four wetland plant species growing

on submerged mine tailings. Environ. Exp. Bot . 47: 271–280.

Sun, Q. Y., An, S. Q., Yang, L. Z. and Wang, Z. S. 2004. Chemical properties of the upper tailings beneath biotic crusts.Ecol. Eng . 23: 47–53.

Tordoff, G. M., Baker, A. J. and Willis, A. J. 2000. Current approaches to the revegetation and reclamation of metalliferous

mine wastes. Chemosphere . 41: 219–228.

Unz, R. F. and Shuttleworth, K. L. 1996. Microbial mobilization and immobilization of heavy metals. Curr. Opin.

Biotech . 7: 307–310.

Vrionis, H. A., Anderson, R. T., Ortiz-Bernad, I., O’Neill, K. R., Resch, C. T., Peacock, A. D., Dayvault, R., White,

D. C., Long, P. E. and Lovley, D. R. 2005. Microbiological and geochemical heterogeneity in an in situ uranium

bioremediation field site. Appl. Environ. Microbiol . 71: 6308–6318.

Ye, Z. H., Shu, W. S., Zhang, Z. Q., Lan, C. Y. and Wong, M. H. 2002. Evaluation of major constraints to revegetation

of lead/zinc mine tailings using bioassay techniques. Chemosphere . 47: 1 103–1 111.

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