The influence of in situ chemical oxidation on microbial community composition in groundwater contaminated with chlorinated solvents
Bram Sercu, Antony D.G. Jones, Cindy H. Wu, Mauricio H. Escobar, Carol L. Serlin, Timothy A. Knapp, Gary L. Andersen,Patricia A. Holden
Microbial Ecology
Electronic Supplemental Information
I. Supplemental Tables and Figures
Table S1 Concentrations tetrachloroethylene (PCE), trichloroethylene (TCE) and cis-1,2-dichloroethylene (cDCE) in groundwater samples (µg/L). Vinyl chloride and ethylene concentrations were always below the limit of detection (0.4-50 µg/L for vinyl chloride; 2-800 µg/L for ethylene). Time is expressed as days after start of NaMnO4 injection (February 22, 2007). All samples are taken within two days of the time indicated. NA: not analyzed.
Time / MW-52 / MW-54 / MW-55 / MW-55B / MW-57 / MW-57B / MW-6PCE / -21 / NA / 4.1 / 3.9 / 2 / NA / NA / 8.3
-14 / 14 / NA / NA / NA / <10 / <10 / NA
30 / 2.9 / <5 / <1 / <1 / 3 / <10 / 8.4
56 / 12 / 4.2 / <10 / <10 / <50 / <10 / 8
89 / <100 / <5 / <10 / <10 / 2.6 / <10 / 8.6
117 / 9.1 / 2.1 / 5.9 / <10 / 2.6 / <10 / 9.3
154 / <100 / 2.2 / 7.1 / <10 / <100 / <10 / 23
191 / 3.2 / <0.5 / 4.4 / <0.5 / 2.6 / <0.5 / 9.2
223 / <50 / <0.5 / 8.8 / <0.5 / <50 / <0.5 / 12
251 / <50 / <0.5 / 6 / <0.5 / <50 / <0.5 / 9.6
300 / <0.5 / 0.82 / 0.93 / <0.5 / <50 / <0.5 / 12
435 / 11 / 3.4 / 4 / NA / NA / <0.5 / NA
TCE / -21 / NA / 440 / 760 / 2000 / NA / NA / 180
-14 / 12000 / NA / NA / NA / 13000 / 12000 / NA
30 / 7400 / 390 / 1 / 1 / 3100 / 10 / 190
56 / 15000 / 370 / 10 / 10 / 5300 / 10 / 200
89 / 18000 / 280 / 10 / 10 / 5100 / 10 / 210
117 / 17000 / 260 / 140 / 10 / 5500 / 10 / 190
154 / 17000 / 140 / 1 / 10 / 6200 / 10 / 270
191 / 5900 / 51 / 370 / 1 / 5000 / 1 / 200
223 / 6100 / 1.3 / 570 / 1 / 6600 / 1 / 260
251 / 5000 / 4.7 / 380 / 1 / 5150 / 1 / 230
300 / 190 / 110 / 1 / NA / 13000 / 1 / 180
435 / 27000 / 320 / NA / NA / NA / 0.61 / NA
cDCE / -21 / NA / 49 / 46 / 41 / NA / NA / 6
-14 / 220 / NA / NA / NA / 580 / 330 / NA
30 / 540 / 57 / <1 / <1 / 110 / <10 / 6
56 / 670 / 41 / <10 / <10 / 210 / <10 / 6.1
89 / 690 / 41 / <10 / <10 / 240 / <10 / 5.6
117 / 590 / 36 / 14 / <10 / 240 / <10 / 6.6
154 / 480 / 16 / <1 / <10 / 280 / <10 / 6.4
191 / 280 / 3.8 / 24 / <0.5 / 270 / <0.5 / 5.5
223 / 210 / <0.5 / 37 / <0.5 / 400 / <0.5 / 6.2
251 / 200 / <0.5 / 27 / <0.5 / 300 / <0.5 / <2.5
300 / 6.7 / 15 / <0.5 / <0.5 / 620 / <0.5 / 4.7
435 / 640 / 43 / 8.3 / NA / NA / <0.5 / NA
Table S2Oxidation-reduction potential (ORP) (mV), chloride (mg/L),nitrate(mg/L), dissolved iron (mg/L), dissolved manganese (mg/L), sulfate (mg/L), sulfide (mg/L) and dissolved methane(µg/L) concentrations in groundwater samples. Time is expressed as days after start of NaMnO4 injection. All samples are taken within two days of the time indicated. NA: not analyzed.
Time / MW-52 / MW-54 / MW-55 / MW-55B / MW-57 / MW-57B / MW-6Chloride / -21 / NA / 240 / 150 / 190 / NA / NA / 190
-14 / NA / NA / NA / NA / 230 / 200 / NA
28 / 78 / 240 / 180 / 200 / 230 / 230 / 200
56 / 87 / 220 / 150 / 210 / 220 / 220 / 190
89 / 110 / 230 / 130 / 190 / 215 / 200 / 190
117 / 130 / 230 / 54 / 200 / 220 / 210 / 210
162 / 170 / NA / NA / NA / NA / NA / NA
223 / 200 / 210 / 86 / 190 / 215 / 190 / 200
300 / 2.2 / 260 / 160 / 210 / 220 / 210 / 200
ORP / -21 / NA / 42 / 140 / 90 / NA / NA / 9
-14 / 60 / NA / NA / NA / 176 / 189 / NA
28 / 276 / 95 / 525 / 547 / 490 / 655 / 305
56 / 122 / 150 / 625 / 691 / 370 / 642 / 62
89 / 89 / 205 / 565 / 588 / 420 / 688 / 86
117 / 127 / 159 / 320 / 576 / 263 / 604 / 122
223 / 436 / 605 / 226 / 595 / 390 / 639 / 338
300 / 118 / 233 / 567 / 596 / 436 / 677 / 404
435 / 308 / 193 / 222 / NA / NA / 615 / NA
Nitrate / -21 / NA / 20 / 17 / 18 / NA / NA / 16
-14 / 20 / NA / NA / NA / 19 / 17 / NA
28 / 14 / 21 / 18 / 18 / 23 / 14 / 18
56 / 15 / 20 / 20 / 24 / 22 / 16 / 15
89 / 16 / 15 / 16 / <11 / 16 / <11 / 16
117 / 15 / 21 / 23 / 18 / 22 / 17 / 19
162 / 17 / NA / NA / NA / NA / NA / NA
223 / 24 / 22 / 21 / 21 / 23 / 20 / 19
300 / 0.73 / 26 / 21 / 110 / 21 / 22 / 21
Manganese / -21 / NA / 0.029 / <0.02 / 0.039 / NA / NA / 0.23
-14 / <0.02 / <0.02 / NA / NA / <0.02 / 0.21 / NA
28 / 0.03 / <0.02 / 3.1 / 7.2 / <0.02 / 230 / 0.035
56 / <0.02 / <0.02 / 4.5 / 340 / 0.029 / 680 / <0.02
89 / <0.02 / <0.02 / 2.7 / 440 / <0.02 / 550 / <0.02
117 / <0.02 / <0.02 / <0.02 / 20 / <0.02 / 200 / <0.02
154 / <0.02 / 0.15 / 74 / 260 / <0.02 / 12 / <0.02
162 / <0.02 / NA / NA / NA / NA / NA / NA
190 / <0.02 / 0.39 / <0.02 / 68 / <0.02 / 2.3 / <0.02
223 / <0.02 / 0.34 / <0.02 / 58 / 0.026 / 49 / <0.02
300 / <0.02 / <0.02 / 0.98 / 220 / 0.33 / 16 / 0.058
Iron / -21 / NA / <0.04 / <0.04 / <0.04 / NA / NA / 9.9
-14 / <0.04 / NA / NA / NA / <0.04 / <0.04 / NA
28 / <0.04 / <0.04 / <0.04 / <0.04 / 0.36 / <4.0 / <0.04
56 / <0.04 / <0.04 / <0.04 / <4.0 / <0.04 / <4.0 / <0.04
89 / <0.04 / <0.04 / <0.04 / <2.0 / 0.07 / <4.0 / <0.04
117 / <0.04 / <0.04 / <0.04 / <0.40 / <0.04 / <4.0 / <0.04
154 / <0.04 / <0.04 / <0.40 / <1.6 / <0.04 / <0.08 / <0.04
162 / <0.04 / NA / NA / NA / NA / NA / NA
190 / <0.10 / <0.10 / <0.10 / <0.5 / <0.10 / <0.50 / <0.10
223 / <0.10 / 0.12 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
300 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / 0.11
Sulfate / -21 / NA / 110 / 81 / 89 / NA / NA / 82
-14 / 190 / NA / NA / NA / 82 / 84 / NA
28 / 170 / 120 / 79 / 83 / 89 / 80 / 110
56 / 170 / 110 / 86 / 130 / 87 / 84 / 99
89 / 160 / 100 / 76 / 75 / 82 / 81 / 95
117 / 160 / 120 / 120 / 100 / 105 / 110 / 120
162 / 110 / NA / NA / NA / NA / NA / NA
223 / 100 / 94 / 85 / 96 / 87 / 110 / 97
300 / 32 / 120 / 93 / 110 / 88 / 99 / 96
Sulfide / -21 / NA / <0.1 / <0.1 / <0.1 / NA / NA / <0.1
-14 / <0.1 / NA / NA / NA / <0.1 / <0.1 / NA
28 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / 0.12 / <0.1
56 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1
89 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1
117 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1 / <0.1
162 / NA / NA / NA / NA / NA / NA / NA
223 / <0.05 / <0.05 / <0.05 / <0.5 / <0.05 / <0.5 / <0.05
300 / <0.05 / <0.05 / <0.05 / <0.5 / <0.05 / <0.05 / <0.05
Methane / 28 / <1 / 1.6 / NA / <1 / 54 / 540 / <1
56 / <1 / 1.2 / 2.5 / 10 / <1 / 320 / <1
89 / <1 / NA / NA / NA / NA / NA / <1
117 / <1 / <1 / <1 / 24 / 4.3 / 310 / <1
223 / <2 / <2 / <2 / 19 / 300 / 550 / <2
300 / <2 / 2.4 / <2 / 230 / 3400 / 1100 / <2
Table S3 DNA yields in groundwater (prefix “G”, ng/L) and RVC (prefix “R”, ng/80 cm3) samples. Yields in samples with NaMnO4 > 1 mg/L at the time of sampling are boldfaced.
Sample Time / 0 / 1 / 2 / 3G6 / 10 / 5 / 66 / 2641
G52 / 2896 / 929 / 9210 / 4501
G54 / 395 / 162 / 53 / 2584
G55 / 1597 / 55 / 493 / 420
G55B / 7267 / 32 / 40 / 91
G57 / 1976 / 3331 / 8424 / 2398
G57B / 2363 / 226 / 64 / 992
R6 / 13 / 122 / 1274 / 5245
R52 / 1286 / 1029 / 862 / 4903
R54 / 466 / 548 / 43 / 4323
R55 / 395 / 80 / 274 / 88
R55B / 708 / 27 / 68 / 66
R57 / 743 / 774 / 3374 / 4325
R57B / 1056 / 1311 / 0 / 79
Figure S1 TCE (grey bars) and NaMnO4 (black bars) concentration time series in all monitoring wells. NaMnO4 concentrations were measured by grab sampling, and were not available during months 9-11 (indicated by *). All x-axes indicate time, in months following permanganate injection.
Figure S2 Distribution of OTUs detected uniquely in RVC (RVC not GW) and in RVC and groundwater (RVC and GW) at the family level. Families are indicated in different colors, y-axis shows percent of all OTUs detected.
FigureS3 MDS plot of TRFLP-based microbial communities of groundwater (G) and RVC (R) samples for MW-54, MW-55 and MW-57. Samples before NaMnO4 exposure are indicated by black triangles (▲), samples after NaMnO4 exposure are indicated by open circles (○). Arrows link the sample before and the first sample after NaMnO4 exposure in each well.
FigureS4 Relative fluorescence intensities (%) of Sulfurospirillum spp. (groundwater and RVC) andDesulfuromonasspp. (groundwater), at PF > 0.9.
Time 0 / Time 1 / Time 2 / Time 3G52 / / / /
G54 / /
G55 / /
G57 / /
G6 / /
R52 / / / /
R54 / /
R55 / /
R57 / /
R6 / /
FigureS5 Relative fluorescence intensities of OTUs associated with cometabolic TCE oxidation in groundwater and RVC samples, at PF > 0.9. The number of bars on the x-axes indicates OTU richness within each genus. Y-axes indicate relative fluorescence intensities, and are all on the same scale.
II. Optimization of DNA extraction methods for RVC samples
Experimental
Three different protocols were tested for DNA extraction from the RVC material. In a first experiment, 10 cm3 of RVC sampler was spiked with identical amount Pseudomonas putida mt-2 (ATCC 33015) and extracted using the 3 DNA extraction protocols, in duplicate. P. putida mt-2 was grown overnight in 20 ml LB medium (37 °C) after inoculation from a stock culture on LB agar. The overnight culture was centrifuged for 5 min at 5000 x g, and the pellet was suspended in a final volume of 2 ml of sterile 0.9% NaCl. Two hundred μl of this bacterial suspension was added per replicate. In a second experiment, groundwater and RVC samples (20 cm3) were obtained from different monitoring wells the study site and extracted using DNA extraction protocols 1 and 3 (see below).
DNA extraction protocol 1 was based on the protocol of Griffiths et al.[1]. After crushing the RVC using a sterile glass stick in a 14 ml tube, 2 g of sterile 0.1 mm zirconia-silica beads (BioSpec Products, Bartlesville, OK), 2 ml of CTAB buffer (120 mM potassium phosphate buffer [pH 8], 0.35 M NaCl, 5% cetyltrimethyl ammonium bromide), 500 μg of polyadenylic acid and 2 ml phenol/chloroform/isoamylalcohol (25:24:1) were addedto each tube. Subsequently, the tubes were vortexed at maximum speed for 10 min, using a vortex adapter (MoBio, Carlsbad, CA). After centrifugation for 15 min at 4000 x g, the supernatant was collected. The remaining RVC pellet was extracted again by adding another 2 ml of CTAB buffer, vortexing at maximum speed for 5 min, and centrifuging for 10 min at 4000 x g. The two supernatants were pooled, mixed with 1 volume of phenol/chloroform/isoamylalcohol (25:24:1) and centrifuged for 5 min at 4000 x g. The supernatant was mixed with 1 volume of chloroform/isoamylalcohol and centrifuged again for 5 min at 4000 x g. The final supernatant was collected and stored at -20 °C until DNA was quantified.
DNA extraction protocol 2 was based on the method of Zhou et al.[2]. After crushing the RVC using a sterile glass stick in a 50 ml tube, 10 ml extraction buffer (100 mMTris-HCl [pH 8], 100 mM sodium EDTA [pH 8], 100 mM NaH2PO4 [pH 8], 1.5 M NaCl, 1% cetyltrimethyl ammonium bromide), 750 μg proteinase K, and 1 mg polyadenylic acid were added to each tube. After incubation for 30 min at 37 °C (225 rpm), 1.1 ml of 20% sodium dodecyl sulfate was added to each tube, and second incubation was performed for 2 hours at 65 °C, while tubes were inverted every 15 to 20 min. The solution was centrifuged for 10 min at 6000 x g, and the supernatant was collected. To the remaining pellet another 6 ml of extraction buffer and 0.7 ml of 20% sodium dodecyl sulfate was added. After vortexing for 10s, the solution was incubated for 10 min at 65 °C and centrifuged for 10 min at 6000 x g. The two supernatants were pooled, and further processed as described in the first protocol.
DNA extraction protocol 3 used the UltraClean Water DNA Kit (MoBio, Carlsbad, CA), with some modifications. After crushing the RVC material in a 14 ml tube, beads and reagents were added, including 500 μg of polyadenylic acid. The final DNA solution was stored at -20 °C until DNA was quantified.
Results
The DNA yields obtained from the spiked RVC samples decreased in the order protocol 1protocol 3protocol 2 (Table S4). Protocols 1 and 3 were selected for further optimization using field samples. Final optimization of the DNA extraction protocols was performed by adjusting reagent volumes, centrifugation speeds/times, and optimizing the ethanol precipitation step (results not shown). For groundwater and RVC samples, protocol 1 consistently showed higher DNA yields compared to protocol 3 (Table S5).
Table S4 DNA yields from RVC – proof of principle and comparison methods (duplicate)
Sample / Method / DNA yield (ng)RVC + P. putida / Protocol 1 / 85000 – 86000
Protocol 2 / 1000 – 2000
Protocol 3 / 17000 – 33000
Table S5 DNA yields from groundwater/RVC – method optimization
Sample / Method / DNA yieldGroundwatera
MW52 / Protocol 3 / 141
MW57 / Protocol 3 / 327
MW52 / Protocol 1 / 2374
MW57 / Protocol 1 / 4493
RVCb,c
MW8 / Protocol 3 / 4162 - 6183
MW57 / Protocol 3 / 5255 - 9252
MW8 / Protocol 1 / 7790 - 8402
MW57 / Protocol 1 / 20701 - 21072
aDNA yield in ng/L
bDNA yield in ng/RVC sampler
cextracted in duplicate
References
1. Griffiths RI, Whiteley AS, O'Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol66:5488-5491
2. Zhou JZ, Bruns MA, Tiedje JM (1996)DNA recovery from soils of diverse composition.Appl Environ Microbiol 62:316-322
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