RESEARCH ARTICLE
Biodiversity, abundance, and activity of nitrogen-fixing bacteria
during primary succession on a copper mine tailings
Li-Nan Huang, Feng-Zao Tang, Yong-Sheng Song, Cai-Yun Wan, Sheng-Long Wang, Wei-Qiu Liu &
Wen-Sheng Shu
School of Life Sciences and State Key Laboratory of Biocontrol, Sun Yat-Sen University, Guangzhou, China
Correspondence: Wen-Sheng Shu, School
of Life Sciences, Sun Yat-Sen University,
Xingang West Road 135, Guangzhou
510275, China. Tel.: +86 20 39332933;
fax: +86 20 39332944;
e-mail: [email protected]
Received 23 July 2010; revised 3 June 2011;
accepted 21 July 2011.
Final version published online 25 August
2011.
DOI: 10.1111/j.1574-6941.2011.01178.x
MICROBIOLOGY ECOLOGY
Editor: Alfons Stams
Keywords
mine tailings; primary succession; nitrogenfixing bacteria; clone library; real-time PCR;
nifH gene.
Abstract
Microorganisms are important in soil development, inputs and biogeochemical
cycling of nutrients and organic matter during early stages of ecosystem development, but little is known about their diversity, distribution, and function in
relation to the chemical and physical changes associated with the progress of
succession. In this study, we characterized the community structure and activity
of nitrogen-fixing microbes during primary succession on a copper tailings. Terminal fragment length polymorphism (T-RFLP) and clone sequencing of nifH
genes indicated that different N2-fixing communities developed under primary
succession. Phylogenetic analysis revealed a diversity of nifH sequences that were
mostly novel, and many of these could be assigned to the taxonomic divisions
Proteobacteria, Cyanobacteria, and Firmicutes. Members of the Cyanobacteria,
mostly affiliated with Nostocales or not closely related to any known organisms,
were detected exclusively in the biological soil crusts and represented a substantial fraction of the respective diazotrophic communities. Quantitative PCR (and
statistical analyses) revealed that, overall, copy number of nifH sequences
increased with progressing succession and correlated with changes in physiochemical properties (including elementary elements such as carbon and nitrogen) and the recorded nitrogenase activities of the tailings. Our study provides
an initial insight into the biodiversity and community structure evolution of
N2-fixing microorganisms in ecological succession of mine tailings.
Introduction
Waste tailings dumps from either inactive or abandoned
mine sites are prevalent in various parts of the world.
The global impact of such tailings dumps is enormous, as
unreclaimed mining sites generally remain unvegetated
for tens to hundreds of years (Mendez & Maier, 2008).
Natural recolonization of plants on mine tailings is
difficult, as these degraded materials typically have no
aggregate structure or organic matter, and they are deficient in nutrients (N and P), but rich in toxic heavy metals and metalloids (Pb, Zn, Cu, Cd, Mn, Ni, and As). The
successively colonized mine tailings present an ideal
opportunity for the study of primary succession, i.e. ecosystem development in situations where no previously
developed soil exists (Dobson et al., 1997) and ecological
reconstruction practice (Marrs & Bradshaw, 1993; Shu
et al., 2005).
FEMS Microbiol Ecol 78 (2011) 439–450
The accumulation of nitrogen (N) in the initial
degraded materials is frequently the limiting factor controlling ecosystem development (Dobson et al., 1997).
Consequently, N2-fixing microorganisms often serve as
early and abundant colonizers in these N-deficient terrestrial habitats that then give way to other species over time
(Walker & del Moral, 2003). It has been demonstrated that
the presence of N2-fixing organisms is tightly linked to the
accumulation of soil N (Bormann & Sidle, 1990; Matthews,
1992), and increases in soil N availability may facilitate colonization by other later successional species (Chapin et al.,
1994; Walker & del Moral, 2003). The below-ground
microbial communities are expected to be important in
the initial stages of progressive succession on mine tailings,
because they are the primary driving force for the improvement of substrate nutritional status and other physiochemical properties. Importantly, early investigations have
indicated that nitrogen-fixing bacteria may have a role in
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L.-N. Huang et al.
440
soil formation in the mining spoils, and the functioning of
the nitrogen cycle within the disturbed ecosystem that
mine environments constitute is necessary for revegetation
and long-term stability (Ledin & Pedersen, 1996).
The soil microbial communities in primary succession
have been characterized with phospholipid fatty acid
(PLFA) technique and traditional activity measurements
(Ohtonen et al., 1999; Schipper et al., 2001; Tscherko
et al., 2004). Recent applications of nucleic acid-based
molecular approaches have provided more detailed
insights into the succession of specific phylogenetic
groups, e.g. bacteria (Edwards et al., 2006; Lazzaro et al.,
2009), fungi (Jumpponen, 2003), and archaea (Nicol
et al., 2005; Merilä et al., 2006), in various successional
systems. Due to their significance in the biogeochemical
processes associated with primary succession, the composition and activity of N-cycling groups have been investigated in the past years by analyzing the relative encoding
genes (Deiglmayr et al., 2006; Kandeler et al., 2006; Duc
et al., 2009). Ecological studies reveal that metalliferous
mine waste, such as tailings, can also be colonized by
plants as a consequence of primary succession (Marrs &
Bradshaw, 1993). As this process is desirable for stabilizing the tailings surface, it has been a topic of intensive
research for many years (Shu et al., 2005). However, most
of the conducted studies on ecological restoration of tailings wastelands have been concentrated in the establishment and succession of natural vegetation. In contrast,
research with specific focus on the below-ground microbial communities and their correlation with soil processes
and plant establishment remains limited in the literature
(Héry et al., 2005). Importantly, to date, there have been
no investigations on community structure and evolution
of N-cycling bacteria associated with ecosystem development on mine tailings, despite the fact that these functional groups may play a role in the early stages of
primary succession. We anticipated that mine tailings
may habour specific assemblages of N2-fixing bacteria due
to their unique physical and geochemical properties, and
that these functional groups evolve with progressing
primary succession in response to the changing environmental conditions. To test this, we studied the biodiversity, activity, and relative abundance of N2-fixing bacteria
associated with different successional series of a copper
(Cu) mine tailings area, and explored the key environmental determinants for the observed patterns in the
distribution of diazotrophic communities. Insights from
this and other comparable studies will further our understanding of the underlying mechanisms of ecosystem
development in tailings primary succession systems. Such
knowledge is crucial for the development of effective
strategies for the ecological restoration of these degraded
environments.
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Published by Blackwell Publishing Ltd. All rights reserved
Materials and methods
Site description, sample collection, and
physio-chemical analyses
The Yangshanchong tailings dump is located in the Tongling massive Cu mine area (30°54′N, 117°53′E), Anhui
Province, which represents the most important base of
Cu resource in China (Fig. 1). The climate is humid with
an annual precipitation around 1300 mm and an annual
mean temperature of 16 °C. The country rock of the Cu
ore body in this area is mainly comprised of carbonatite
(carbonate rock). The tailings dump was in operation for
about 30 years and has been left abandoned since 1990,
occupying an area of about 20 ha (Sun et al., 2004).
Gradually, natural biological colonization (of microbes,
biotic crusts, and pioneer plants) of substrates takes place,
displaying a sparse distribution in the early stage of colonization. Our continuous investigations and monitoring
over the past 7 years indicate that biotic community succession at this site is in fact determined by a combination
of factors including microhabitat variation, inoculation
and colonization by propagules, and microbial, biological
soil crust, and vascular plant colonization. This results
from the physiochemical heterogeneity of the tailings
materials, wind and water erosion, and man-made interferences. Although not strictly a chronosequence, typical
series ranging from bare land, biological crusts (algal,
algal-moss, and moss), and a vegetation stage exist on
this single tailings dump that collectively represent a
primary successional sequence (Fig. 1). As such, we consider the tailings impoundment as a suitable model for a
primary succession study.
Bare tailings, the three crust types, and a vascular plant
community series were sampled (0–10 cm layer) using
corers (3 cm diameter, 20 cm long) in May 2007. One
site outside the tailings dump typical of local vegetation
type served as reference. Three field replicates, each composited from five soil cores, were taken for each tailings/
soil type. All samples were kept in cooler boxes for transportation to the laboratory, where they were homogenized and passed through a 2-mm sieve. Subsamples for
molecular microbial analysis were stored at 40 °C prior
to DNA extraction.
The tailings samples were air-dried and analyzed
using standard methods for pH, electrical conductivity
(EC), 2 M KCl-extractable NO
3 -N and NH4 -N, total
organic carbon (TOC) (TOC-VCPH; Shimadzu, Columbia, Maryland), N (Kjeltec TM 2300; Foss, Hilleroed,
Denmark), and total and DTPA-extractable Zn and Cu
concentrations (Page et al., 1982). All heavy metals were
analyzed using ICP-OES (OPTIMA 2100; Perkin-Elmer,
Wellesley, MA, USA).
FEMS Microbiol Ecol 78 (2011) 439–450
Ecological succession of diazotrophs on mine tailings
441
Fig. 1. Map of the Yangshanchong tailings dump at the Tongling copper mine and examples of the distinctive successional series. BT, bare
tailings; AC, algal crusts; AMC, mixed moss and algal crusts; MC, moss crusts; VEG, vegetation stage.
Microbial biomass analysis
Total lipids were extracted from the soil samples (5 g)
using a modified Bligh and Dyer solution of methanol,
chloroform, and phosphate buffer (Petersen & Klug,
1994). The lipid extract was fractionated into glyco-, neutral, and polar lipids (Ibekwe & Kennedy, 1998). The
polar lipid fraction was transesterified with mild alkali to
recover the PLFAs as methyl esters in hexane. The PLFAs
were separated, quantified, and identified using gas chromatography-flame ionization detection (Ibekwe & Kennedy, 1998; MacNaughton et al., 1999). Soil microbial
biomass was calculated by summing up PLFAs specifically
attributed to bacteria and fungi, respectively (Frostegård
& Bååth, 1996).
sten, 1977; Patriquin & Denike, 1978). Organic glass
cylinders (20 cm diameter and 25 cm height) were buried
15 cm depth in the soil at randomly chosen positions.
After balancing overnight, 10% of the above-ground cylinder volume was replaced with acetylene by a tailormade injector through a rubber stopper equipped with
the cylinder. Subsamples of the headspace were then
withdrawn after 24 h into vaccutainers and analyzed back
in the laboratory for acetylene and ethylene content on
a gas chromatograph (Agilent HP-6890, Agilent Technologies, Wilmington, DE, USA) equipped with a hydrogen
flame ionization detector, using nitrogen as a carrier gas.
The mean value of replicate measurements for each series
was converted into inputs to biological N2 fixation.
Nitrogenase activity measurement
DNA extraction and terminal fragment length
polymorphism (T-RFLP) analysis
Nitrogenase activity in the tailings series was determined
in situ using the acetylene reduction assay (Hardy & Hol-
Total community genomic DNA was extracted from
c. 0.5 g of soil from each sample using a FastDNA SPIN
FEMS Microbiol Ecol 78 (2011) 439–450
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L.-N. Huang et al.
442
Table 1. Soil physicochemical properties, PLFA biomass, and nitrogenase activities in the six sampling series
Parameters
Bare tailings
Sand (%)
Silt (%)
Clay (%)
pH
EC (dS cm1)
C/N
TOC (%)
TN (mg kg1)
TP (mg kg1)
TK (mg kg1)
TCa (g kg1)
1
NHþ
4 N (mg kg )
NO3 N (mg kg1)
TCu (mg kg1)
TZn (mg kg1)
DTPACu (mg kg1)
DTPAZn (mg kg1)
Bacterial PLFA
(nmol g1)
Fungal PLFA (nmol g1)
Nitrogen fixation
(kg N ha1 year1)
95
2.1
3.7
8.0
2.4
28
0.09
34
384
433
137
1.5
2.3
2493
273
36
15
3.74
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.5 a
0.1 c
0.5 b
0.1 ab
0.4 bc
2.6 a
0.01 b
3.0 b
11 b
45 bc
0.8 c
0.8 d
0.6 d
303 a
49 b
3.3 b
2.1 b
1.02 a
0.13 ± 0.06 a
1.9 ± 0.2 c
Algal crusts
88
5.0
4.3
8.4
2.7
18
0.13
78
471
580
136
8.7
4.2
937
260
28
17
7.99
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.5 b
0.9 bc
0.3 b
0.03 a
0.2 ab
3.4 bc
0.00 b
14 b
8.5 b
15 b
1.8 c
0.6 a
0.2 c
107 b
36 b
6.8 b
4.1 b
1.41 a
1.76 ± 0.51 bc
3.8 ± 0.7 ab
Mixed moss and
algal crusts
93
4.5
3.7
8.0
1.6
20
0.10
50
414
373
143
4.9
2.4
683
253
45
11
7.35
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.4 ab
0.6 bc
1.0 b
0.01 ab
0.1 cd
0.6 b
0.01 b
5.1 b
13 b
28 c
5.0 bc
0.4 c
0.1 d
157 b
8.8 b
5.1 ab
0.9 bc
1.63 a
0.77 ± 0.22 ab
5.1 ± 0.7 a
Moss crusts
93
2.7
2.6
7.8
3.4
19
0.12
63
405
487
149
1.3
4.1
1047
307
60
17
6.26
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.1 ab
0.6 c
0.3 b
0.1 b
0.3 a
1.2 bc
0.01 b
6.8 b
18 b
34 bc
1.9 b
0.1 d
0.05 c
269 b
41 ab
11 a
1.7 b
0.61 a
0.73 ± 0.26 ab
2.5 ± 0.4 bc
Vegetation
series
89
7.3
4.1
8.1
1.0
18
0.30
167
567
590
163
1.1
6.1
73
400
31
29
22.88
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.3 ab
1.2 b
0.4 b
0.02 ab
0.3 d
1.1 bc
0.05 b
18 b
18 ab
87 b
2.6 a
0.1 d
0.3 b
29 c
36 a
3.7 b
1.0 a
3.61 b
2.81 ± 0.64 c
nd
Reference
site
± 3.7 c
± 2.1 a
± 2.9 a
± 0.3 c
± 0.3 d
± 1.0 c
± 0.21 a
± 245 a
± 196 a
± 60 a
± 0.5 d
± 0.4 b
± 0.5 a
nd
90 ± 5.8 c
8.3 ± 0.4 c
4.3 ± 1.7 c
31.47 ± 5.08 c
54
24
22
6.2
1.2
13
1.7
1298
769
2560
9.6
7.0
21
4.66 ± 0.83 d
nd
EC, electrical conductivity; TOC, total organic carbon; TN, total nitrogen; C/N, carbon and nitrogen ratio; TP, total phosphorus; TK, total
potassium; NHþ
4 -N, ammonium; NO3 -N, nitrate; TCu and TZn, total Cu and Zn; DTPACu and DTPAZn, DTPA-extractable-Cu and Zn; nd, not
determined.
Different letters in the same row indicate significant difference between successional series (P < 0.05). Values given are means ± standard error
(n = 3).
kit for soil (Qbiogene, Carlsbad, CA, USA) as specified
by the manufacturer. A region of the nifH gene (365 bp
in length) was amplified using primers PolF and PolR
(Poly et al., 2001). The forward primer PolF was labeled
5′ terminally with FAM (6-carboxyfluorescein). The
50-lL PCR reaction mixture contained 19 PCR buffer,
200 lM of each dNTPs, 0.5 mM of each primer, 2.5 U
of Taq DNA polymerase (Takara Biotechnology, Dalian,
China), and 1 lL of DNA templates (10–100 ng). The
thermal profile for amplification was: 4 min at 94 °C; 35
cycles of 60 s at 94 °C, 60 s at 55 °C, and 120 s at 72 °C;
and final 10 min at 72 °C. Triplicate PCRs per sample
were performed, and the labeled PCR products were
pooled, purified with a Qiaex II gel extraction kit
(Qiagen, Hilden, Germany), and digested at 37 °C for
6 h using the tetrameric enzyme HaeIII (Takara Biotechnology, Dalian, China). Digested PCR products were
resolved by electrophoresis using an ABI 3730xl sequencer (Applied Biosystems, Foster City, CA, USA). GS-500
ROX was loaded as internal size standard in each lane.
GENESCAN software (version 3.7, Applied Biosystems, Foster City, CA, USA) was used to analyze fragment sizes
and peak fluorescence intensities. The relative abundance
of individual terminal restriction fragments (T-RFs) was
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Published by Blackwell Publishing Ltd. All rights reserved
calculated as the percentage of total peak height in a
given T-RFLP profile. Only those T-RFs with the relative
abundance > 1% were considered in the analysis.
nifH cloning and phylogenetic analysis
Clone libraries were constructed for DNA extracts from
the bare tailings, algal-moss and moss crust series, and
the reference site. Triplicate DNA extracts from each sampling series were pooled, and fragments of nifH were
amplified using the same conditions as described above
and non-fluorescence labeling primers. Triplicate PCR
products were pooled (to minimize bias), purified with a
QIAquick PCR purification kit (Qiagen, Hilden, Germany), and cloned using a TOPO TA cloning kit
(Invitrogen, Carlsbad, CA, USA). nifH inserts from
recombinant clones were reamplified with vector primers,
and the PCR products were subjected to separate enzymatic digestions with HhaI and HaeШ endonucleases
(Takara Biotechnology, Dalian, China). Digested DNA
fragments were resolved using electrophoresing on 2%
agarose gels, and the resulting restriction fragment length
polymorphism (RFLP) profiles were compared manually.
Clones representing each unique RFLP pattern were
FEMS Microbiol Ecol 78 (2011) 439–450
443
Ecological succession of diazotrophs on mine tailings
Table 2. Diversity indices for diazotrophic communities based on T-RFLP analysis
Sample
Shannon’s index (H)*
Bare tailings
Algal crusts
Mixed moss and algal crusts
Moss crusts
Vegetation series
Reference site
1.95
2.26
2.06
2.37
1.90
2.47
±
±
±
±
±
±
0.05
0.16
0.09
0.07
0.29
0.01
bc
abc
abc
ab
c
a
Evenness
0.82
0.86
0.82
0.84
0.81
0.94
±
±
±
±
±
±
0.02
0.03
0.01
0.02
0.08
0.02
Simpson’s index
b
ab
b
ab
b
a
5.23
7.65
5.83
7.16
5.81
10.22
±
±
±
±
±
±
0.48 b
1.41 ab
0 .46 b
0.85 ab
1.87 b
0.64 a
Richness†
11.00
13.67
12.33
16.67
10.33
14.00
±
±
±
±
±
±
0.00
1.33
0.88
0.67
1.45
0.58
cd
bc
bcd
a
d
ab
Different letters within the same column indicate significant difference between successional series (P < 0.05). Values given are means ± standard
error (n = 3).
*H = ΣPi (ln Pi) and Simpson’s index = 1/ΣPi2, where Pi = the proportion of each T-RF in a sample. Evenness = H/ln (richness).
†
Richness: average number of T-RFs in each sample.
Fig. 2. Principal component analysis of diazotrophic communities in
the six sampling series based on nifH T-RFLP patterns. Sampling
series: BT, bare tailings; AC, algal crusts; AMC, mixed moss and algal
crusts; MC, moss crusts; VEG, vegetation stage; CK, reference site.
sequenced using ABI 3730xl sequencer. Operational taxonomic units (OTUs) were defined as sequences with at
least 95% of nucleotide sequence similarity using DOTUR
software (Schloss & Handelsman, 2005). nifH sequences
were compared with those available in GenBank using the
BLAST network service to determine their closest relatives.
Sequences were translated into amino acid sequences
using MEGA3.1 and these were used for the construction of
neighbor-joining phylogenetic trees using Poisson correction distances and pair-wise deletion of gaps and missing
data. Bootstrap confidence values were obtained based on
500 replicates.
Real-time quantitative PCR assays
nifH abundance was quantified using real-time PCR with
the same primers described above. Amplification was carried out in a total volume of 10 lL containing 1 lL of
diluted template DNA, 0.2 lL of each primer (concentraFEMS Microbiol Ecol 78 (2011) 439–450
tion of 200 nM), 3.6 lL of PCR-grade water, and 5 lL of
SYBR® Premix Ex Taq (29) (Takara Biotechnology,
Dalian, China). All sample and standard reactions were
performed in triplicate using a LightCycler480 instrument
(Roche, Penzberg, Germany). Thermal cycling for the
assays consisted of 95 °C for 30 s, followed by 35 cycles
of denaturation at 95 °C for 5 s, annealing at 55 °C for
15 s, and elongation at 72 °C for 10 s. After each qPCR
assay, the specificity of amplification was verified via generation of melting curves and agarose gel electrophoresis.
Standard curves were constructed based on triplicate 10fold dilutions of a DNA standard (plasmids containing
cloned nifH PCR amplicons previously sequenced) containing a known nifH copy number. Negative controls
without template DNA were included in all experiments
to exclude contamination.
Statistical analysis
Principal component analysis (PCA) and hierarchical cluster analysis were performed using the SPSS statistical package (version 15.0, SPSS Inc, Chicago, IL, USA) to cluster or
separate samples on the basis of the abundance and size of
T-RFs in the T-RFLP profiles. Library coverage (Good,
1953) was calculated using C = 1 n/N, where n is the
number of sequence types that occur only once in the
library and N is the total number of clones examined.
LIBSHUFF software (Singleton et al., 2001) was used to determine the significance of differences between the nifH clone
libraries. One-way ANOVA was used with different successional series as the categorical variable to test for differences in soil diazotrophic diversity and abundance along
the notional chronosequence. Correlation analyses were
applied to evaluate the relationship between nifH densities
and soil physicochemical properties. Canonical correspondence analysis (CCA) was performed using CANOCO
(version 4.0, Microcomputer Power, Ithaca, NY, USA) to
show how diazotrophs respond to the biogeochemical variations. Significance levels were within confidence limits of
0.05 or less.
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Published by Blackwell Publishing Ltd. All rights reserved
L.-N. Huang et al.
444
Nucleotide sequence accession numbers
The nifH sequences determined in the present study have
been deposited in the EMBL/GenBank/DDBJ databases
under accession numbers FN985167-FN985220.
Results
Mine tailings physicochemical characteristics,
PLFA biomass, and nitrogenase activities
All tailings samples were slightly alkaline with the pH values fluctuating around 8.0 and showing little variation
between series (P < 0.05) (Table 1). In contrast, the reference soils had a weakly acidic pH of 6.1. The analysis of
selected soil physicochemical characteristics revealed that
nutrient contents, including organic carbon, total and
extractable N (nitrate and ammonium) concentrations
were significantly higher in the reference site than in the
tailings series. The only exception was that ammonium
concentration was higher in the algal crust series than the
reference soils. In contrast, total and DTPA-extractable
Cu and Zn concentrations were generally lower in the reference soils than in the tailings, and total Cu concentration decreased as the primary succession progressed from
the bare tailings to biological soil crusts and finally to the
vegetation stage (P < 0.05). Soil total PLFA represent an
estimate of microbial biomass. The total bacterial PLFA
content was consistently higher than the fungal PLFA
content in each tailings series (Table 1). Total PLFA of
both bacteria and fungi showed an increase with the progress of succession from bare tailings to biological soil
crusts and then to the vegetation series. Nitrogen fixation
evaluated by acetylene reduction activity averaged
3.32 kg N ha1 year1, with the lowest values recorded
in the bare tailings and the highest values in the algalmoss crust samples (Table 1).
T-RFLP analysis
A total of 45 distinct T-RFs were identified in all T-RFLP
profiles obtained for this experiment. The average num-
ber of T-RFs per sample was about 13, but varied from
sample to sample with a minimum number of 10 (vegetation series) and a maximum of 17 (moss crust series)
(Table 2). Only three T-RFs were detected in all samples
analyzed, and nine unique T-RFs were observed exclusively in the biological soil crust series. In general, nifH
diversity (as indicated by the three diversity indices
calculated) showed no significant increase or decrease in
the tailings series. However, nifH diversity at the reference
site was significantly higher than the bare tailings and
vegetation series that showed the lowest levels of diazotrophic diversity (Table 2). PCA analysis showed that all
of the sites combined could explain 22.2% of the T-RF
variability by PC1 and 15.8% by PC2 (Fig. 2). Along axis
1, samples in this study grouped relatively closely together
except for the reference site that was significantly separated from the other sites. Along axis 2, however, the
samples could be generally separated into four groups:
algal crusts, mixed algal and moss crusts, and the vegetation series clustered together, and the reference soils, bare
tailings, and the moss crusts deviated from each other.
This grouping pattern was well supported by the hierarchical cluster analysis (data not shown).
Clone library analysis
nifH libraries were constructed for four selected soil samples. Randomly selected clones were grouped based on
their RFLP patterns and representatives subjected to
sequencing. A few non-specific PCR products were identified, and the corresponding clones were therefore
excluded from further analyses. Finally, comparative
sequence analysis of 204 nifH clones from the four
libraries revealed 54 unique phylotypes at the 95%
sequence similarity level (Table 3). The coverage of
libraries ranged from 73% to 98%, indicating that the
N2-fixing communities were sufficiently covered by the
clone library analysis. Although many nifH sequences
were affiliated with cultivated N2-fixing organisms distributed among the Proteobacteria, Cyanobacteria, and Firmicutes (Fig. 3), a significant portion of the retrieved nifH
sequences (44% of the OTUs) (not shown in the phyloge-
Table 3. Diversity and predicted richness of nifH sequences from the four clone libraries*
Sample
No. of
clones
analyzed
No.
of
OTUs
%
Coverage
Chao1
value
ACE
value
Shannon
index
Reciprocal of
Simpson’s
index
Bare tailings
Mixed moss and algal crusts
Moss crusts
Reference site
58
55
26
65
9
19
12
26
98.3
80.0
73.1
83.1
9.44
32.94
23
34.83
9.32
44.45
23
34.93
1.85
2.40
2.21
3.04
4.85
7.05
7.19
17.68
*OTUs were defined by a 5% difference in the nucleic acid sequence alignment for the nifH gene.
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Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Ecol 78 (2011) 439–450
445
Ecological succession of diazotrophs on mine tailings
netic tree) were remotely related to known bacterial
species, and represent novel phylotypes (i.e. displaying
< 95% sequence identity to already existing sequences in
public databases) never described in previous studies. The
bare tailings sample had the lowest estimated richness,
and the clone library was dominated (approximately
60%) by two abundant sequences that display low levels
of similarity (< 90%) to any known nifH sequences. In
contrast, samples from the reference site had overall the
highest species richness, and clones were more evenly distributed among the detected OTUs in the library. These
results were generally in agreement with the T-RFLP analysis. Overall, the bare tailings, biological soil crusts
(mixed algal and moss crusts and moss crusts), and the
reference samples were distinct from each other, as there
was not a single OTU detected in all clone libraries, and
there were only four OTUs shared by the biological soil
crust and the reference soil libraries. Consistent with this,
Fig. 3. Phylogenetic tree of the translated nifH sequences with Methanothermococcus thermolithotropicus as the outgroup. Clones from the
present study are shown in bold and marked with bare tailings (BT), mixed moss and algae crusts (AMC), moss crusts (MC), or reference site
(CK) to indicate their origins. Additional symbols show the relative frequency (%) of a sequence in their respective clone libraries ( , CK; , MC;
▲, AMC; , BT). Bootstrap values of >50% are indicated at branch points. To save space, clones that display very low levels of similarity to nifH
sequences of any cultivated diazotrophs are not included in the tree.
○
FEMS Microbiol Ecol 78 (2011) 439–450
■
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L.-N. Huang et al.
446
Fig. 4. nifH abundance in the Cu mine tailings series as measured
using real-time PCR. Bars (standard errors; n = 3) labeled with
different letters indicate statistical difference (P < 0.05) between
successional stages. Tailings series: BT, bare tailings; AC, algal crusts;
AMC, mixed moss and algal crusts; MC, moss crusts; VEG, vegetation
stage.
Fig. 5. CCA biplot of correlation between the nifH parameters and
geochemical variations of the tailings. nifH copy: copy numbers of
nifH as revealed by quantitative PCR (see Fig. 4); T-RF Richness and H′
(nifH): numbers of TRFs and Shannon’s index based on the nifH
T-RFLP analysis of diazotrophic communities (see Table 2).
comparative sequence analysis revealed that some of the
retrieved nifH sequences formed sample-specific clusters
in the phylogenetic tree (data not shown). Statistical analysis using LIBSHUFF confirmed that the observed differences in diazotroph community composition indeed
represented statistically significant community differences.
Of note, Cyanobacteria-affiliated nifH sequences were
detected exclusively in the biological soil crust samples
(Fig. 3), and they constituted a substantial proportion
(23–39%) of the corresponding clone libraries.
Quantification of nifH sequences
Quantitative real-time PCR assays revealed that the abundance of nifH sequences in the tailings series ranged from
5.06 9 105 (bare tailings) to 3.41 9 107 (vegetation series) copies per gram of dry soil (Fig. 4). There was an
obvious increase of nifH copy number in the biological
soil crusts over the bare tailings series, and diazotroph
numbers were significantly higher in the vegetation area
than in the biological soil crusts and bare soils. These
results suggest that the presence of crusts or plants promotes the growth of diazotrophs in the tailings. In addition, the abundance of nifH detected in the reference site
was significantly higher than the tailings series, by one to
approximately three orders of magnitude. Correlation
between nifH copy numbers and physicochemical parameters was established using the two-tailed Pearson’s correlation coefficient (P < 0.05 and 0.01) (data not shown).
Soil C and N concentrations directly or indirectly indicate
the nutrient status of the study sites. It was observed that
TOC, TN, and NO
3 -N showed a significant positive correlation with the nifH copy number (P < 0.01), which
was not affected by NHþ
4 -N. However, C/N presented a
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Fig. 6. Relation between abundance of soil diazotrophs and nitrogen
fixation in the Cu mine tailings series. Sampling series: BT, bare
tailings; AC, algal crusts; AMC, mixed moss and algal crusts; MC,
moss crusts.
negative correlation with the nifH abundance (P < 0.05).
In addition, soil pH (P < 0.01) and both total and
extractable heavy metals (Cu and Zn, P < 0.05 or 0.01)
showed negative effects on the abundance of diazotrophs.
These results were in good agreement with the CCA analysis (Fig. 5). Additional statistical analysis revealed that
the increase in the abundance of nifH sequences associated with progressing succession was positively related to
the recorded nitrogenase activities of the mine tailings
(Fig. 6).
Discussion
Successional colonization on mine tailings is often
accompanied by a gradual change in major physiochemical characteristics of the tailings substrate. The analysis of
selected biogeochemical parameters in our study indicated
FEMS Microbiol Ecol 78 (2011) 439–450
Ecological succession of diazotrophs on mine tailings
a heterogeneous nature of the Cu tailings series probably
affected by soil age and the presence of pioneer plants.
Variations in the prevailing environmental conditions
may have significant influence on the diversity and abundance of indigenous soil microorganisms, including those
involved in the N-cycle. This in turn will greatly affect
soil processes and the progress of primary succession of
this nutrient-limiting ecosystem.
Overall, our nifH T-RFLP analysis revealed that diazotrophic diversity was low in the bare tailings, and there
was an increase in the biological soil crusts followed by a
decrease in the vegetation series. Likewise, in the study of
diazotrophs in the forefield of a receding alpine glacier,
Duc et al. (2009) found that the nifH diversity was higher
in 70-year bulk soil compared with that either in 8-year
bulk soil or rhizosphere soil. It was believed that the
intermediate disturbance hypothesis (Connell, 1978), i.e.
environments with intermediate rates of disturbance display highest diversity, would be a likely explanation for
the observed pattern. Similar findings were reported for a
nutrient-rich agricultural soil by Marilley & Aragno
(1999) who showed that low rates of disturbance associated with rhizosphere soils (e.g. higher substrate availability) lead to bacterial communities dominated by a few
species. In our study, biological soil crusts may correspond to the intermediate stage for the reason that they
underwent less abiotic disturbance than bare soil, but
more abiotic disturbance than vegetation soil according
to different environmental stresses such as available nutrients and heavy metal levels. Relatively higher nifH diversity was found in the reference soils than in the tailings
series. This observation is in agreement with previous
studies which demonstrated that microbial diversity was
significantly lower in mine tailings than the undisturbed
vegetated area adjacent to the tailings pile (Moynahan
et al., 2002; Mendez et al., 2008). These results indicate
that the harsh ecological factors prevailing in the tailings
impose strong selective pressure on the indigenous
microbial communities.
In a pioneer study investigating the diversity and activity of N-cycling groups in nickel mine spoils, almost all
of the environmental nifH sequences retrieved clustered
with known organisms, with the dominant phylotypes
closely related to the Bradyrhizobium and Beijerinckia spp.
(Héry et al., 2005). In marked contrast, the majority of
the sequenced clones in the current study were only
remotely related to nifH sequences of cultivated bacteria
and thus may represent novel diazotrophs. Cultivation
and characterization of these as yet uncultured organisms
will lead to a better understanding of their roles in the
biogeochemical cycle of N and primary succession at this
site. Uncultured microorganisms have been shown to
dominate the diazotrophic communities and may conFEMS Microbiol Ecol 78 (2011) 439–450
447
tribute significantly to the global N input in soils of
diverse physical environments (Widmer et al., 1999; Poly
et al., 2001; Buckley et al., 2007), indicating that the diazotrophic diversity in the environments remains largely
unexplored. In addition, not a single OTU was found
common to all analyzed samples, and sample-specific
clusters were identified in the nifH phylogenetic tree (data
not shown). These results suggest that the succession progress may have opened up new environmental niches in
the tailings, and the differentiated soil physicochemical
conditions would have determined the diversity and distribution of diazotrophic populations.
Both clone library and quantitative PCR analysis of
nifH demonstrated the presence of diazotrophs in the
bare tailings series, although with a low phylogenetic
diversity. A handful of previously unrecognized bacteria
probably dominated the early-stage diazotrophic community. These pioneer N2 fixers may play a role in relieving
the N deficiency stresses associated with the unaltered
tailings materials, thus facilitating the subsequent colonization of other organisms and their functioning in soil
stabilization. Biological soil crusts represent important
stages of ecologic succession on degraded land. They play
critical roles in nutrient cycling, particularly fixed-N
input, in these nutrient-poor ecosystems. Our results
indicated that cyanobacteria were significant members of
the diazotrophic communities associated with the biological crust stages of the successional tailings series.
Many of the cyanobacterial OTUs detected are affiliated
with Nostocales that have been determined as dominant
N2 fixers in mature, nitrogen-producing crusts from the
Colorado plateau and Chihuahuan Desert (Redfield et al.,
2002; Yeager et al., 2004). Nitrogen-fixing genera Nostoc
and Leptolyngbya have also been identified as the abundant members of the cyanobacterial community at the
young sites of the foreland of Midre Love’n glacier on
Svalbard, indicating that N2-fixation is important during
early stages of primary succession (Turicchia et al., 2005).
Additional nifH sequences affiliated with the Oscillatoriales, Stigonematales, and other uncultured cyanobacteria
were also recovered from the biological crust tailings,
indicating the availability of diverse microhabitats and
ecological niches for the colonization of other cyanobacterial groups. Plant establishment often lead to an elevation of soil stability and nutrient concentration by root
exudation and vegetation cover (Tscherko et al., 2004).
However, no cyanobacterial nifH clones were detected in
the reference site library. Similarly, comparative 16S
rRNA sequence analysis of bacterial communities at an
abandoned semi-arid lead/zinc mine tailings site also
revealed no occurrence of cyanobacteria in the vegetated
off-site control sample (Mendez et al., 2008). In fact,
cyanobacteria were not detected even in the N2-fixing
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L.-N. Huang et al.
448
bacterial communities from plateau areas least disturbed
by human activities (Zhang et al., 2006).
Quantitative nifH PCR indicated that diazotrophs were
abundant in the Cu mine tailings and showed an increase
with the progress of succession from bare tailings to biological soil crusts and then to the vegetation series correlating with the nitrogenase activities recorded in situ.
Total bacterial and fungal biomass as measured by the
PLFA analysis showed a similar trend during ecosystem
development on the mine tailings. Asymbiotic N2 fixation
has been found to be increased significantly during the
earliest stages (4–5 years) of ecosystem succession in
recently deglaciated soils at the Puca Glacier (Schmidt
et al., 2008) and in the presence of pioneer plants in the
older soil age class in the forefield of a receding alpine
glacier (Duc et al., 2009). Our observations are also in
agreement with previous investigations of evolution of
total microbial community and other N-cycling groups
during primary succession of glacier forelands, where
increases with progressing ecosystem development in the
microbial biomass and respiration (Ohtonen et al., 1999)
and in the abundance of eubacteria and denitrifiers
(Kandeler et al., 2006) have been demonstrated. It should
be noted, however, that nitrogenase activity could be controlled differentially in the tailings ecosystem due to variations in localized environmental conditions. Indeed, a
lack of agreement between the density and activity of the
nitrate reducer community has been reported in the studies of microbial succession across the glacier foreland of
the Rotmoosferner in the Ötz valley (Austria) (Deiglmayr
et al., 2006; Kandeler et al., 2006). However, the observed
correlation between the nifH copy number and nitrogenase activity in the current study implies that the enzymatic activity per nifH copy may not differ significantly
among distinct stages of ecological succession at this site.
Ecosystem development involves successional biological
colonization and the dynamic interactions between the
established biological communities and the abiotic environment. As pioneer colonizers, microorganisms are critical in the ensuing development of soil, biogeochemical
cycling, and facilitating colonization by plants (Schütte
et al., 2009). It has been demonstrated that inputs of
nutrients and organic matter during early ecosystem succession are dominated by microbial carbon and nitrogen
fixation (Schmidt et al., 2008). On the other hand, the
continuous improvement in the overall substrate nutrient
status and other physiochemical properties would significantly influence the growth and metabolic activity of a
broad range of microorganisms. This may explain the
parallel increase in the diazotroph numbers and bacterial/
fungal biomass during progressing succession of the Cu
tailings and the correlation between major physiochemical
parameters and nifH abundance. Nevertheless, statistical
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
analysis did not reveal significant correlation between
nifH copy number and bacterial or fungal biomass (data
not shown), indicating that these microbial groups may
respond differently to the environmental changes associated with progressive tailings succession. The capability of
fixing atmospheric nitrogen would render diazotrophs less
affected by the availability of fixed-N in the nutrientlimited tailings ecosystem. Identification of the major
environmental determinants for the distribution and
relative abundance of N-cycling populations is an important step for revealing how the changing geochemical
conditions associated with progressing succession structure these potentially important functional communities.
This represents the first report using cultivationindependent molecular approaches to elucidate the
phylogenetic composition and abundance of N2-fixing
microorganisms associated with primary succession on
mine tailings. Our data have revealed novel and shifting
diazotrophic communities in the Cu mine tailings, with
cyanobacteria being exclusively detected in and constituting a substantial fraction of the biological crust N2-fixing
communities. Results have demonstrated an increase in the
abundance and activity of the nitrogenase encoding nifH
gene with progressive succession corresponding to changes
in environmental conditions. Future investigations are
needed to explore temporal variations in the dominant
N2-fixing populations and how they interact with the fluctuating geochemical and physical conditions in different
successional stages, as these may provide important insights
into the mechanisms and functional consequences of
diazotroph succession in the mine tailings ecosystem.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 30970548 and 30770398). We
thank the two anonymous reviewers for providing
thoughtful and constructive comments on the manuscript.
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