Diversity of sediment microbial community in response to acid mine drainage pollution in Hengshi River (Southeast China)
Song Tang1, Xiaohui Zhang2, Mao Wang3, Yuwei Xie2, Weimin Sun4, John P. Giesy2,5,6, Hongling Liu2, Markus Hecker1,5
School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada
2 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu, China
3 School of Public Health, Sun Yat-Sen University, Guangzhou, Guangdong, China
4 Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA
5 Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada
6 Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
1
Background
Conclusions
Results
 Acid mine drainage (AMD) is one of the most environmentally threatening
byproducts of the mining industry because of its low pH and elevated
concentrations of sulfate and metals, which severely impair water and soil
quality (1).
A.
C.
 Despite its extreme toxicity and acidity, many environments receiving
AMD harbor numerous acidophilic and metal-tolerant microorganisms (2,
3); however, our understanding of the roles that geochemical factors play
in shaping microbial community structure and the potential of
microorganisms in natural attenuation of AMD is limited.
 Located in Southeast China (Guangdong Province), the Hengshi River is
continually contaminated by AMD produced by the Dabaoshan mine,
which produces a high amount of acidic runoff (pH < 3) with extremely
high concentrations of Cu (7000 ppb), Cd (400 ppb), Pb (600 ppb), and etc.
(4). This river provides an opportunity to explore the effects of AMDimpacted environments on microorganism communities.
Figure 1. Detailed map of the Dabaoshan mine and the Hengshi river showing twentyseven sampling sites. Samples of 23-27 were collected from the reference river site.
 Results showed the most abundant prokaryotic organisms in all samples
belonged to the Firmicutes and Proteobacteria phyla. Other phyla, such as
Actinobacteria, Acidobacteria, Bacteroidetes, and Nitrospirae that have
previously been reported to be characteristic for AMD contaminated
environments were also detected in the libraries retrieved from Hengshi
samples.
B.
Figure 4. Bacteria (A) richness (Chao1) and (B) evenness
(Shannon) of sediment samples. (C) Multidimensional
scaling plot of the compositional dissimilarities
(weighted-UniFrac) between communities of all sediment
samples of the Hengshi river and the reference river. The
groups indicate the sampling distance to the main
pollution site of the DBS mine (Zone 1 < 3.95 km < Zone 2
< 10 km < Zone 3 < 19 km < Zone 4 < 26 km).
 Therefore, this study characterized the spatial distribution of microbial
communities along the Hengshi river using integrated geochemical and
molecular biological analyses.
Objectives
II.
III.
Characterize the microbial community structure and composition
along the polluted Hengshi river;
Explore the relationships between microbial structure and different
geochemical variables; and
Identify the active organisms and their metabolic potentials for natural
mitigation of the AMD contamination.
Methods
 The physicochemical parameters of water and sediments differed among
samples collected along the Hengshi river. The pH of water and sediments
collected from upstream sites was very low (< 3) and increased further
downstream (5 to 6). Similarly, concentrations of Cu, Zn, Cd and Pb in
water and sediment conductivity were much higher at upstream sites than
further downstream. However, concentrations of Cu and Zn in
downstream sediment samples were higher than upstream samples.
 16S rRNA-gene amplicon sequencing revealed that bacterial richness and
evenness (alpha diversity) gradually increased along the Hengshi river.
These trends were also supported by beta diversity analysis.
 A better understanding of the indigenous microbial diversity and their
potential roles in natural attenuation of AMD will enable the development
of molecular monitoring tools to evaluate and enhance bioremediation
processes of AMD treatment facilities and other AMD-contaminated
environments.
I.
Email song.tang@usask.ca
 At the genus level, Acidocella, Leptospirillum, Prevotella, Spirochaeta,
Thiomonas, Hydrogenophaga, Arcobacter, Acidiphilium and Meiothermus
were significantly dominant in samples from the Hengshi river compared
to the reference sites.
 MRT analysis that interpreted the relationship between the relative
abundance of dominant lineages and environmental conditions by
providing a tree with three terminal nodes indicated aquatic pH and Cu
appeared to be a strong predictor of relative lineage abundance among
samples.
 The whole microbiome approach featured in this study offered a direct
and reliable means to characterize the diversity of microbial communities
in the presence of extremely high metal concentrations. The spatial
distribution of microbial communities in AMD polluted Hengshi River
facilitates the understanding of the metabolic capacities of indigenous
microorganisms.
 It has been reported that bioreactor systems using acidophilic and
sulfidogenic bacteria that are indigenous to mine-impacted environments
could remove and precipitate metals from mine waters. Therefore, the
phylogenetically divergent lineages coexisting in the Hengshi river may
have the potential for in situ natural attenuation of AMD pollution.
Figure 2. The environmental parameters including pH, conductivity, concentration of total
organic carbon (TOC) and metals in water (Aq.) and/or sediment (Sd.) samples along the
Hengshi river.
Sample collection and analytical analysis
Surface sediment (0-10 cm) samples were collected in October, 2014.
Samples were dried by a vacuum freeze dryer until a constant dry weight was
obtained. Samples were homogenized with a ceramic mortar and pestle and
sieved through 2 mm (for physico-chemical parameters) and 63 μm (for
metals) mesh screen. Conductivity and pH were determined based on
electrical measurement by using 1:2 ratio sediment to double-deionized
water. For metal analysis, samples were digested using aqua regia (HCl:HNO3
= 1:3) and analyzed using Inductively Coupled Plasma Mass Spectrometry
(ICP-MS).
References
1.
Figure 5. Significant changes in abundance of bacteria between the Hengshi river and the reference
river. Bacteria were identified as significantly differentially abundant (adjusted P ≤ 0.005) via DESeq2.
Genus-level assignments are presented where available.
2.
3.
4.
DNA extraction, PCR amplification, and sequencing analysis
Genomic DNA was extracted using the FastDNA® spin kit (MP biomedicals,
Santa Ana, USA). The V3 region of the 16S rRNA gene was amplified. The
below flowchart illustrates the work-flow employed by this study (1, 5, 6).
5.
6.
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Acknowledgements
Extract DNA
Sediments
Barcode PCR
Ion proton
Sequence data analysis
Packages: phyloseq, ggplot2, DESeq2,
vegan, reshape2, mvpart, etc.
Created by Peter Downing – Educational Media Access and Production © 2011
OTU table
Figure 3. Circular representation of microbial communities in sediment samples at
phylum level. The thickness of each ribbon represents the abundance of each taxon. The
absolute tick above the inner segment and relative tick above the outer segment stand for
the reads abundances and relative abundance of each taxon, respectively. Others refer to
those phyla with abundance lower than 1%.
Figure 6. Multivariate regression tree analysis of the
relation between relative abundance of dominant lineages
and environmental parameters in microbial communities
of Hengshi river. The bar plots show the mean relative
abundance of specific lineages at each terminal nodes and
the distribution patterns of relative abundance represent
the dynamics of community composition among each
split. The numbers under the bar plots indicate the
number (n) of samples within each group.