Supporting Materials

Remediation of antimony-rich mine waters: assessment of antimony removal and shifts in

the microbial community of an onsite field-scale bioreactor

Weimin Sun a,b,c, Enzong Xiao a,d, Margarete Kaline, Valdis Kruminsf, Yiran Dongg, Zengping

Ninga, Tong Liu c, Min Sun a,d, Yanlong Zhaoh, Shiliang Wuh, Jianzhong Maoi, Tangfu Xiao a,*

a. State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese

Academy of Sciences, Guiyang 550081, China

b. Guangdong institute of Eco-environment and Soil Sciences, Guangzhou, 510650, China

c. Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New

Jersey, 08901, USA

d. University of Chinese Academy of Sciences, Beijing, 100049, China

e. Boojum Research Ltd., Toronto, Ontario, M4T 1L9, Canada

f. Department of Environmental Sciences, Rutgers University, New Brunswick, New Jersey,

08901, USA

g. Department of Geology, University of Illinois-Urbana Champaign, Urbana, Illinois, 61801,

USA

h. School of Environment and Sustainability, University of Saskatchewan, Saskatoon, S7H

4Z9, Canada
h. Water Resources Protection Bureau of Pearl River Water Resources Commission, Guangzhou,

510611 , China

i. Yunnan Provincial Bureau of Hydrology and Water Resources, Kunming 650106, China

*Corresponding author.

99 Lincheng Road West, Guiyang 550081, Guizhou Province, China. Phone: +86-851-85895318. Fax:

+86-851-85891334. Email: xiaotangfu@vip.gyig.ac.cn

Materials and methods

Total genomic DNA was extracted from the sediment samples using the FastDNA® spin kit (MP

bio, Santa Ana, CA, USA) following the manufacturer’s protocol. All DNA extracts were stored

at -80°C until further analysis. DNA concentration and purity was monitored on 1% agarose gels.

The V4 region of the SSU rRNA gene was amplified using the 515f/806r primer set (Caporaso,

Lauber et al. 2011). Amplification conditions were as follows: Initial incubation at 95 ºC for 3

minutes; 30 cycles of 95 ºC for 45 s, 57 ºC for 45s, and 72 ºC for 1 minute. A final extension at

72 ºC for 5 minutes. SSU rRNA tag-encoded high-throughput sequencing was carried out on the

Illumina MiSeq platform at Novogene (Beijing, China). Sequences were analyzed with the

Quantitative Insights Into Microbial Ecology (QIIME) software and UPARSE pipeline

(Caporaso, Kuczynski et al. 2010). Default settings for Illumina processing in QIIME were used

(r = 3 p = 0.75 total read length; q = 3; n = 0).The UPARSE pipeline was used to cluster

operational taxonomic units (OTUs) at 97% similarity and taxonomy was assigned using the

Ribosome database project (RDP) classifier (Wang, Garrity et al. 2007). The reads were

deposited into the NCBI short reads archive database under accession number of SRP064908.

The similarity of microbial communities among different sediment samples was determined

using both weighted and unweighted UniFrac. UPGMA (Unweighted Pair Group Method with

Arithmetic mean) clustering was conducted on weighted UniFrac (Kuczynski, Stombaugh et al.

2012). We also used weighted UniFrac distance for Principal Coordinate Analysis (PCoA),

which helps to get principal coordinates and visualize them from complex multidimensional data

(Lozupone, Hamady et al. 2007; Lozupone, Lladser et al. 2011). Canonical correspondence

analysis (CCA) performed by CANOCO 4.5 (Microcomputer Power, Ithacha, NY) was used to

measure the major physicochemical parameters that had the most substantial influence on
microbial community structure. CCA was done on abundant bacteria genera (i.e., relative

abundance >1% in at least one sequencing library) and selective physicochemical parameters. A

symbol's position in relation to a vector head indicates the correlation between the community

and the environmental factors. The length of a vector reflects the relative importance of those

environmental factors in discriminating the overall microbial community within one library

(Zhang, Wan et al. 2008). Manual forward selection with Monte Carlo permutation tests was

then performed to determine the significance of the environmental variables with 999

permutations (Lepš and Šmilauer 2003). CCA was performed focusing on interspecies

difference. The CCA bipolar were generated by CanoDraw 4.0 (Microcomputer Power, Ithaca,

NY).
Table S1. Concentrations of metal(loid)s in the water samples. Acronyms: BPX-I: the influent of unit BPX (X could be 2 to 6); BPX-
E: the effluent of unit BPX (X could be 2 to 6).

Sampling Al Ca K Mg Mn Na Si Sr
Sample name
time mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L
BP2-I 9.42 73.9 4.87 26.9 0.69 3.59 0.63 0.15
BP3-E 15.2 105 4.51 41.8 0.96 2.34 11.6 0.44
September-
BP4-E 15.4 105 5.43 41.6 0.96 2.12 14.8 0.34
2013
BP5-E 9.75 121 8.11 43.2 1.06 2.67 16.7 0.41
BP6-E 0.89 139 12 45.5 1.17 3.73 12.7 0.49
BP2-I 25.4 115 6.04 49.4 1.28 4.59 1.33 0.39
BP3-E 10.7 130 6.5 45.1 1.06 3.14 13.1 0.41
Ocotober-
BP4-E 9.18 130 7.34 45.2 1.09 3.12 10.7 0.44
2013
BP5-E 1.49 133 9.19 43.9 1.04 3.19 13.3 0.42
BP6-E 0.18 138 10.2 43.7 1.13 2.99 11.1 0.42
BP2-I 22.1 114 8.1 46.7 1.13 3.07 1.75 0.38
BP3-E 11.9 116 7.27 42.4 0.96 3.25 2.45 0.37
November-
BP4-E 3.49 117 9.7 39.1 0.8 5.02 12.7 0.45
2013
BP5-E 0.68 131 10.4 42.2 0.97 3.23 11 0.44
BP6-E 0.03 126 11.2 40.4 0.79 2.94 10.4 0.41
BP2-I 6.33 52.6 2.24 20.6 0.3 3.19 0.88 0.11
BP3-E 6.26 95.2 7.95 33.2 0.73 3.05 12.8 0.47
December-
BP4-E 13.7 130 8.96 45.5 1.09 4.52 37.2 0.44
2013
BP5-E 1.08 138 15.2 42.4 0.76 8.6 7.46 0.57
BP6-E 0.54 144 17.6 43.6 0.27 6.48 8.02 0.58
BP2-I 19.3 102 7.07 43.3 1.09 5.77 0.61 0.37
BP3-E 7.99 122 10.8 41.1 0.92 5.69 14.4 0.47
BP4-E 3.81 134 13 43.3 0.92 5.65 12.5 0.55
Januaruy-
BP5-E 2014 0.72 139 15.7 42 0.74 7.21 8.48 0.59
BP6-E 0.25 145 19.2 42.4 0.29 6.27 6.45 0.59
Table S2. Concentrations of anions in the water samples. Acronyms: BPX-I: the influent of unit
BPX (X could be 2 to 6); BPX-E: the effluent of unit BPX (X could be 2 to 6).

F- Cl- NO3- SO42-

Sample name Sampling time
mg/L mg/L mg/L mg/L
BP2-I 1.7 2.6 1.9 989
BP3-E 1.7 4.3 2.0 992
BP4-E July-2013 1.9 4.1 1.1 953
BP5-E 1.5 3.6 0.5 950
BP6-E 1.2 3.7 0.9 944
BP2-I 1.3 3.4 2.3 859
BP3-E 1.9 4.5 3.9 1052
BP4-E August-2013 1.7 4.7 2.2 1006
BP5-E 0.5 4.7 1.6 876
BP6-E 0.9 5.1 2.1 865
BP2-I 0.5 2.7 5.7 513
BP3-E 0.9 2.7 4.8 629
September-
BP4-E 1.2 3.0 3.9 743
2013
BP5-E 1.3 3.7 4.4 717
BP6-E 0.8 3.9 6.5 738
BP2-I 1.3 3.2 4.9 1054
BP3-E 1.4 3.1 4.3 827
BP4-E Ocotober-2013 1.3 3.0 3.3 839
BP5-E 1.0 2.5 4.3 770
BP6-E 0.5 2.4 3.6 760
BP2-I 1.3 2.3 4.8 991
BP3-E 1.2 2.9 3.6 816
Novomber-
BP4-E 1.4 3.2 3.4 727
2013
BP5-E 0.7 3.7 3.4 774
BP6-E 0.6 2.5 3.6 740
BP2-I 1.2 2.4 4.0 727
BP3-E 1.2 3.0 4.0 704
BP4-E December-2013 1.0 2.9 2.9 703
BP5-E 0.6 3.1 3.3 632
BP6-E 0.6 3.6 4.2 680
BP2-I 1.1 2.6 4.0 850
BP3-E 0.9 7.2 4.3 691
BP4-E Januaruy-2014 0.8 3.3 3.8 704
BP5-E 0.6 4.2 3.9 694
BP6-E 0.6 3.2 4.8 682
Table S3. The relative abundances of various taxonomic groups at phylum level.
Taxonomy BP1 BP2 BP3 BP4 BP5 BP6
Proteobacteria 65.60 60.33 53.69 59.76 46.47 61.53
Cyanobacteria 0.55 5.17 13.12 6.43 20.00 23.21
Bacteroidetes 15.70 3.56 3.05 5.00 5.04 2.21
Firmicutes 8.03 5.12 9.27 6.25 3.47 3.01
WPS-2 0.58 1.09 0.07 0.70 5.73 0.72
Actinobacteria 2.04 3.04 5.53 5.29 2.99 2.05
Acidobacteria 1.13 4.71 2.29 3.79 2.97 1.25
Planctomycetes 0.81 1.41 0.26 1.47 3.81 1.39
Nitrospirae 0.23 2.61 0.07 0.61 0.24 0.26
Chlorobi 0.16 1.23 0.10 1.47 0.51 0.21
Verrucomicrobia 1.41 0.46 0.17 0.63 1.34 0.17
Gemmatimonadete
s 1.19 0.72 0.00 1.05 0.61 0.51
TM6 0.30 0.27 0.00 0.75 1.03 0.83
Chloroflexi 0.35 0.38 0.26 0.99 0.72 0.20
Armatimonadetes 0.37 0.95 0.17 0.68 0.93 0.66
Elusimicrobia 0.07 0.74 0.03 0.49 0.16 0.08
Spirochaetes 0.11 0.33 0.00 0.58 0.44 0.12
OD1 0.02 0.17 0.00 0.15 0.51 0.41
Lentisphaerae 0.04 0.49 0.00 0.10 0.04 0.03
Thermi 0.43 0.20 0.00 0.31 0.21 0.20
TM7 0.02 0.02 0.03 0.02 0.33 0.01
Chlamydiae 0.05 0.20 0.00 0.25 0.15 0.13
Crenarchaeota 0.02 0.03 0.00 0.20 0.01 0.00
OP9 0.06 0.20 0.00 0.10 0.02 0.02
Euryarchaeota 0.00 0.06 0.00 0.15 0.00 0.00
Fusobacteria 0.01 0.00 0.10 0.00 0.00 0.00
WS3 0.01 0.00 0.00 0.09 0.01 0.00
AD3 0.00 0.00 0.00 0.02 0.00 0.00
BRC1 0.01 0.01 0.00 0.01 0.01 0.00
Fibrobacteres 0.00 0.00 0.00 0.00 0.01 0.00
OP11 0.00 0.00 0.00 0.00 0.01 0.00
OP3 0.00 0.00 0.00 0.01 0.00 0.00
Hyd24-12 0.00 0.00 0.00 0.00 0.00 0.01
WS2 0.00 0.00 0.00 0.00 0.00 0.00
Others 0.69 6.48 11.79 2.66 2.25 0.76
 120
Sulfate reduction

Acidity reduction
100
Removal rates (%)

80

60

40

20

0
CL CL+RS RS

Treatments

Figure S1. Removal rate of sulfate and acidity in treatment of CL (chicken litter only), CL+RS

(chicken litter + rice straw), and RS (rice straw only) to treat coal-mine polluted water. Three

treatments were set up: chicken litter alone, chicken litter and rice straw, and rice straw alone. The

most sulfate reduction was observed in treatments inoculated with chicken litter and rice straw (>99%)

while the rice straw only treatment exhibited ~20% sulfate reduction. This observation indicated the

effectiveness of chicken litter in facilitating SRB and some other anaerobic bacteria.
Figure S2. Total soluble Fe concentrations (mg/L) in each monitoring point of the onsite field-scale

bioreactor. Acronyms: BPX-I: the influent of unit BPX (X could be 2 to 6); BPX-E: the effluent of

unit BPX (X could be 2 to 6).

 100%

90%

80%
Other
TM6
70% Armatimonadetes
Thermi
60% Chloroflexi
WPS-2
Cyanobacteria
50%
Planctomycetes
Acidobacteria
40%
Verrucomicrobia
Gemmatimonadetes
30% Actinobacteria
Firmicutes
20% Bacteroidetes
Proteobacteria

10%

0%
BP1 BP2 BP3 BP4 BP5 BP6

Figure S3. Taxonomic classification of bacterial reads retrieved from sediment samples within

each treatment unit. Classification was done at the phylum level using RDP classifier. Other: the

phyla with relative abundance less than 0.5%.

 PC2-Percent variation explained
29.88%

PC1-percent variation explained

42.62%

Figure S4. PCoA plot demonstrated the differences of microbial communities within each unit

and mine portal. The percentages are the percentage of variation explained by the components.
 Ferrovum
Thiomonas
Gallionella
Leptospirillum
Rhodobacter
Acidithiobacillus
Peptococcaceae (f)
Halomonas
Acidocella
Acidovorax
Hydrogenophaga
Methylosinus
Legionella
Rhodanobacter
Shewanella
Geobacter
Clostridium
Granulicella
Novosphingobium
Giesbergeria
Prevotella
Gemmata
Cellulomonas
Bdellovibrio
Desulfomonile
Escherichia
Acidiphilium
Fusibacter
Flavobacterium
Gemmiger
BP3

BP2

BP4

BP1

BP5

BP6

0 1 2 3 4
Square root of relative abundances
Figure S5. Heatmap of the distribution of abundant phylotypes with relative abundances >1% in

at least one sample. Double hierarchical dendrogram showed the microbial distribution of the six

samples. The relative percentage values for the microbial genera are indicated by hue. Please

note the relative abundances were square-root transformed.

 B
P2

B
P3

B
P4
 B
P5

BP6

Figure S6. Representative SEM images (left panel) and the corresponding EDX spectra (right

panel) of the sediment samples. Sampling sites were labeled on the bottom of each figure.
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