SUPPLEMENTAL MATERIAL
Fig. S1 Photograph of a high flow pipe; water is pumped from the goaf at Mine A (samples
GOAF-A7 and GOAF-A8).
Fig. S2 a) Photograph of a mine slime (taken using a spark-free camera) where water is flowing
through the coal wall in Mine A (sample CWB-A3). Note the iron staining of the rock dust,
which is applied to the coal mine wall.
Fig. S3 Rarefaction analysis of 16S rRNA gene amplicon libraries from Bowen Basin coal mine
samples.
Table S1 Number of sequences and diversity index values for 16S rRNA amplicon gene libraries
from Bowen Basin mine samples
Sample ID
FW-B5
CWB-A3
CWB-A6
CWB-B3
MMW-B4
GOAF-A7
GOAF-A8
Total unique reads
2035
5494
2095
7704
4947
2381
3158
Da
0.3533
0.0124
0.0046
0.0138
0.0065
0.1151
0.0153
1/D
2.8
80.5
217.0
72.5
153.3
8.7
65.3
Hˊa
1.5
5.6
6.1
5.6
6.3
3.4
5.3
D is the Simpson’s diversity value. Hˊ is the Shannon index value. All diversity indices were
calculated after subsampling libraries to 2035 OTU, which allowed for a more appropriate
comparison of groups (Gihring et al., 2012).
a
Fig. S4. A principle components analysis of the coal mine libraries. Red numbers indicate OTU.
The source (r2 = 0.5440 P=0.05) where samples are divided into waters collected from piped
environments compared to the coal wall samples. P values are based on 999 permutations.
Fig. S5 A principle components analysis of the coal mine libraries with geochemical data. Red
numbers indicate OTU. Microbial community variance and geochemistry is associated for pH (r2
= 0.9099 P=0.03), Mg (r2 = 0.857 P=0.05), Ca (r2 = 0.8783 P=0.03) and K (r2 = 0.8762 P=0.04)
but not for the other geochemical parameters listed in Table 2. P values are based on 999
permutations.
Growth of microbial cultures
All biological samples for culturing were collected at the mine site in sterile Falcon tubes
without a gas headspace and stored at 4°C for transport. Microbial cultures were grown on a
simple salt mixture (based on the mine geochemistry) and processed coal from the Mine B coal
pile. The minimal salts solution was based on the geochemistry of coal mine formation water
(FW-B2; Table 2) and contained (per L): 2.5293 g NaCl, 0.4216 g NaHCO3, 0.1980 g
MgSO4•7H2O, 0.0477 g CaCl2•2H2O, 0.0666 g NH4Cl, 0.0076 g KCl, and 0.0031 g NaF.
Anaerobic cultures were grown in 10 ml crimp top vials containing 0.25 g of coal (dried and
sieved between 350 and 150 µm) and 4.5 ml of minimal salts solution. The media was reduced
by bubbling with 100% N2(g) and was dispensed in an anaerobic chamber with an atmosphere of
97% N2 and 3% H2 (COY Laboratory Products Inc., Ann Arbor, MI). Vials were inoculated with
0.5 ml of field sample, including coal fines, and grown for at least 4 weeks in the dark at room
temperature, without shaking. The presence of aerobic methanotrophs was tested in CWB-B3 by
growing cultures in crimp top vial containing 4.5 ml of minimal salts solution, atmospheric
headspace (21% O2) and a 0.5%(vol./vol.) methane supplement. The presence of methanogenesis
was indicated by a methane concentration in the vial being at least 50% greater than baseline
methane desorbed from the coal. The presence of methanotrophy was confirmed by a decrease in
methane to below the detection limit. Methane was measured using a Shimadzu 2014 gas
chromatography (Shimadzu, Japan) with a Shincarbon micropack column (no. 19808; Restek)
and flame ionisation detector (GC-FID).
References
Gihring TM, Green SJ, Schadt CW (2012) Massively parallel rRNA gene sequencing
exacerbates the potential for biased community diversity comparisons due to variable
library sizes. Environmental Microbiology, 14, 285-290.