SUPPLEMENTARY INFORMATION
Dechlorinating Microorganisms in a Sedimentary Rock
Matrix Contaminated with a Mixture of VOCs
Gláucia Lima, Beth Parker and Jessica Meyer
School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada.
[email protected], [email protected], [email protected]
CORRESPONDING AUTHOR FOOTNOTE. Gláucia Lima, 50 Stone Road East, Guelph, ON, N1G
2W1, Canada. Current Address: 35 St George St. Toronto, ON, M5S 1A4, [email protected],
+1 416 978 4396.
(1) EP-PCR and PCR-DGGE Methods.
Table S1. PCR primers targeting the 16S rDNA gene and their published protocols. All protocols
adopted in the PCR analysis conducted with the samples can be found in the literature, as listed.
Primer pair (forward/reverse)
Target
1
8f/1541r
Universal Bacteria1
2
Dhc730f/Dhc1350r
Dehalococcoides sp.2
3
Sulf114f/Sulf421r
Sulfurospirillum sp.3
4
Dhb477f/Dhb647r
Dehalobacter sp.4
5
Geo73f/Geo485r
Geobacter sp.3
6
341f-GC/534r
DGGE Universal Bacteria3,5
7
1Af/1100Ar
Universal Archaea6
(2) VOC relative abundances.
Table S2. Relative abundance (%) of contaminants in the source (DNAPL) and rock core (MP-19D).
Other VOCs include methanol, mineral spirits, and naphtha. Chlorobenzenes were less than 0.05% each.
Comparisons between the abundances of different groups of contaminants were done by calculating
percent mass based on relative concentrations of quantifiable (above LOQ, Limit of Quantitation)
contaminants. Results are provided in Table S2.
Contaminants
MP-19D
Average DNAPL
Haloetha(e)nes
70
66
Halomethanes
16
5
Aromatics
3
21
Other VOCs
0
8
Ketones
11
0
(3) Physical properties. A total of 64 rock samples were collected from other research coreholes within
2 kilometers of the MP-19D corehole and analyzed for physical properties. These samples were
collected by the same methods as all other rock core samples, but were not split, as were the ones for
VOC and DNA samples. Wet bulk density and imbibition porosity were determined gravimetrically.7
Permeability was determined with a nitrogen gas permeameter according to the ASTM Standard
Method D 4525-04.8 Total and interparticle porosities and pore radius were determined by mercury
intrusion porosimetry9 on 20 samples. Images with back-scattered scanning electron microscopy
(BSEM) were obtained from the same 20 samples.9 Fraction of organic carbon (foc) was determined on
20 samples.10 Results are shown in Table S3.
Table S3. Physical properties of stratigraphic units/lithologies present in MP-19D.
K (cm/s)
Total
1.9 - 14.6
1.9-7.1
0.1-2.2
1.2-6.7
0.3-4.7
8×10-10 - 5×10-6
2.67±0.04
5.2 - 10.2
6.4-8.5
1.4-6.0
0.5-7.1
0.1-2.4
1×10-8 - 3×10-6
N.D.b
2.50±0.13
11.4 - 19.7
10.1
0.10
9.9
0.79
8×10-10 - 8×10-5
Sandstone
0.010
2.17
26.5
20.2
3.2
17.0
3.15
5×10-9
Sandstone
N.D.
2.50±0.08
12.4 - 20.8
N.A.d
N.A.
N.A.
N.A.
2×10-7 - 5×10-7
Sandstone
N.D.
2.41±0.10
10.1 – 21e
11.5-20.3
0.2-6.0
11-14
1.0-9.5
8×10-10 - 5×10-6
Mazomanie
Sandstone
0.012
2.35±0.11
9.5 - 24.7
7.7
6.9
0.8
18.9
1×10-6 - 6×10-5
Wonewoc
Sandstone
0.010
2.44±0.06
8.2 – 16.8
5.1-9.4
2.0-9.2
0.22-3.1
9.5-18.9
1×10-6 - 4×10-4
Eau Claire
Sandstone
Silstone
0.015
2.56±0.14
5.7 - 17.5
3.9-13
0.15-12.5
0.5-3.8
0.03-18.9
8×10-10 - 6×10-5
Dominant
Lithology
foc
(%)
Wet Bulk Imbibition
Densitya
Porosity (%)
Prairie du
Chien
Dolostone
N.D.
2.63±0.10
St. Lawrence
Dolostone
Siltstone
N.D.
Jordan
Sandstone
Lone Rock
(Laminated)
Lone Rock
(glauconitic
conglomerate)
Lone Rock
(bioturbated)
a
Porosity determined by
Mercury Intrusion Porosimetry (%)
Pore
Radius
Interparticle Intraparticle (µm)
Stratigraphic
Unit11
Result ± standard deviation. b Not detected - below detection limit. d Not available. e One of the samples resulted in 39.7% and was not included in the table.
Figure S1. Overall schematic for rock core sampling for DNA and VOC extractions, considering
lithological changes, presence of fractures, and bedding plane partings. The detail shows the trimmed
piece of core with sub-samples A, B, and C at increasing distances from natural fractures for DNA
extractions.
Chein
Prairie du
St.
Lone Rock Lawrence Jordan
Mazomanie
Wonewoc
Eau
Claire
(a)
(b)
(c)
(d)
(e)
Figure S2. Depth (m bgs): (a) Main stratigraphic11/lithological units: Prairie du Chien Grp. and St.
Lawrence Frm. – dolostones or siltstones, all other stratigraphic units are dominantly sandstones; (b)
VOC concentrations in µg per g of wet rock from 205 samples (sampling intervals 41± 28 cm). Solid
symbols > limit of quantification (LOQ), empty symbol < LOQ, light colored symbol - field or lab
contamination, small crosses < detection limit (MDL), TVOC (total VOC concentration). This profile
demonstrates that most of the detectable contaminant mass is within the Lone Rock Formation; (c)
Cementation12 for the clastic units (sandstones): 0 – none, 4 – highly cemented; (d) Number of fractures
per 1.5 m run; (e) Positive PCR: EUB - Universal Eubacteria, DHB - Dehalobacter, DHC Dehalococcoides, SUL - Sulfurospirillum, and ARC - Universal Archaea, horizontal arrows - negative
PCR. DNA extracted from 66 samples: 39 from the Lone Rock Formation (sampling intervals 0.24 ±
0.25 m) and 27 from above and below the Lone Rock Formation (sampling intervals 2.8 ± 1.8 m).
Nested PCR negative controls had no amplification product in all runs.
(j)
Figure S3. BSEM images from: (a) Lone Rock Formation
bioturbated glauconitic sandstone; (b) Lone Rock Formation
laminated sandstone; (c) Mazomanie Formation sandstone; (d)
Wonewoc Formation sandstone; (e) Prairie du Chien Group
dolostone; (f) Jordan Formation sandstone; and (g) St.
Lawrence Formation dolostone. Scale bars represent 100 µm.
White arrows show examples of pore throats.
Figure S4. (a) and (b) DGGE gels on Lone Rock Formation samples showing bands successfully
sequenced and with more than 95% match to known microorganisms. Lanes are named according to
sample depth and color coded according to their distance to a fracture (red label < 1 cm, green 1-3 cm,
purple > 3 cm). Sequence abbreviations: SHE-Shewanella, CLO-Clostridium, PEL-Pelomonas. For all
others, refer to Figure 1 in the paper.
Gels were scored by Gene Tools software (Syngene). The number of bands was counted by the software
by fixing the lowest intensity as 2% of the highest intensity band on each gel. Band alignments were
done assuming 1% tolerance. Distinction between Aquaspirillum and Methylophilaceae in (a) is not
clear.
Similarity (%)
10
19
28
37
46
55
64
73
82
91
100
39 – 43.73
43 – 44.88
41 – 44.61
36A – 42.87
36B – 42.86
37C – 43.04
26 – 39.58
38D – 43.47
45C – 45.24
30 – 41.33
34 – 42.09
38A – 43.43
29 – 40.60
45B – 45.22
37B – 43.03
27 – 39.77
35 – 42.64
42 – 44.75
47 – 45.92
37A – 43.02
38C – 43.45
45A – 45.21
25B – 38.92
40 – 44.28
32 – 41.86
38B – 43.44
24B – 38.80
25A – 38.89
10
19
28
37
46
55
64
73
82
91
100
Figure S5. Cluster analysis DGGE gels on Lone Rock Formation samples. The denaturing gradient was
30 to 60% and gels were run at 50V, for 16 hours, at 60°C. Sample IDs are on the right hand side with
respective depths in m bgs (numbers with two decimal places), i.e. 39 – 43.73 = sample 39 from 43.73
mbgs. Colors orange and green indicate samples that clustered together. The clustering does not show a
clear trend in sample depth (numbers with two decimal places beside the sample IDs), with samples
very close together in space being in different clusters. This indicates the high heterogeneity of the
communities within the pore matrix of the Lone Rock Formation.
The 100% similarity between samples from 43.73 and 44.88 m bgs is surprising because these two
samples are over 1 m apart. The next level of similarity is 73% between samples 36A and B (depths
42.87 and 42.86 m bgs). Clustering of the rock matrix samples does not show a particular trend when
comparing shallower and deeper portions of the core (samples in the figure are from 38.80 to 45.92 m
bgs), and neighboring samples did not have similarities higher than 73%. This indicates that the
microbial communities are heterogeneous throughout the core, even considering samples as closely
spaced as 1 cm apart.
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