Supporting information
Characterization of groundwater microbial communities, dechlorinating bacteria and in situ biodegradation of chloroethenes along a vertical gradient
Gwenaël Imfelda*, Hanna Piepera, Noam Shanic, Pierre Rossic. Marcell Nikolauszb, Ivonne Nijenhuisa, Heidrun Paschkec, Holger Weissc and Hans H. Richnowa
Departments of aIsotope Biogeochemistry, bBioremediation, cGroundwater Remediation
Helmholtz Centre for Environmental Research - UFZ. Leipzig D-04318, Germany
cLaboratory for Environmental Biotechnology. EPFL-ENAC-ISTE-LBE, Lausanne CH-1015, Switzerland
*Corresponding author:
Gwenaël Imfeld
Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS)
University of Strasbourg/ENGEES, CNRS
1 rue Blessig
F-67 084 Strasbourg
Tel: + 333 6885 0407
Fax: + 333 8824 8284
E-mail:
Manuscript for Water, Air, & Soil Pollution
Table of content
1. Materials and Methods 3.
1.1. Isotopic analysis using the purge and trap procedure 3.
1.2. Multiple Factor Analysis (MFA) 3.
1.3. T-RFLP analysis 4.
.
2. Results 5.
Table S1 5.
Table S2 6.
Table S3 7.
Figure S1 8.
Figure S2 9.
1. Materials and Methods
1.1. Isotopic analysis using the purge and trap procedure
Purge and trap technique was used to measure the isotope composition of trans– and cis–DCE,TCE and PCE for low concentrated samples employing a Tekmar LSC 3100 purge and trap concentrator (Varian, Palo Alto, CA, USA) connected to a gas chromatography-combustion-isotope ratio monitoring mass spectrometry system (GC-C-IRM-MS). The purge and trap procedure followed a modified EPA method 524.2 (EPA 1992): 100ml water sample were purged for 11 min with He gas flow rate of 40 ml min-1. The remaining water sample was discarded. The analytes were trapped at room temperature (25°C air conditioned laboratory temperature) with a VOCARB 3000 trap (Sigma-Aldrich, Saint-Louis, MI, USA) provided by the manufacturer. The desorption step lasted for 6 min at 250°C; the desorbed gases were transferred in a heated transfer line (200°C) to a cooled GC cryofocusing injector of the GC (6890 series; Agilent Technology). The injector was maintained at a temperature of -100°C by liquid nitrogen serving as cryofocusing unit. GC temperature programme started with heating the injector inlet to 250°C (at 650°C min-1). Separation was accomplished with a 100% dimethylpolysiloxane Zebron ZB 1 column (60m x 0.32 mm, 1 µm film; Phenomenex. Torrance, CA, USA). The temperature program was set as follows: 40°C, (5 min.) 3°C min-1 to 120°C; 20°C min-1 to 250°C (10 min.) and the helium gas flow rate was 1.5 ml min-1.
The analytical error was < 1 δ unit for the purge and the trap methods and < 0.5 δ unit for the manual injection method and incorporated both the accuracy of the measurement and the reproducibility on triplicate measurements of the sample. Although the analytical error of both methods is different, the same GC-C-IR-MS protocol was used for the measurement of isotope composition and the analytical errors of each method allow comparing the two data sets.
1.2. Multiple Factor Analysis (MFA)
MFA. Multiple factor analysis (MFA) is a PCA-based technique allowing the simultaneous ordination of a composite table obtained by the juxtaposition of the two species and the environmental data sets, after weighting the different matrices (Escofier & Pages, 1994). The matrices thus have the same importance, regardless of their size, prior to their conjugation in a new composite table. The newly constructed matrix is then used in the final PCA. The final ordination plot shows the relative positions of the objects in the reduced space at the centroïd of their positions according to PCA based on each of the weighted matrices. As for the previous PCA, the species data set was subjected to the Hellinger transformation.
1.3. T-RFLP analysis (modified from Rossi et al., 2009).
PCR products were generated with a combination of FAM-labelled Eub8f (Lane 1991) and non-labelled Univ518r (Edwards et al. 1989) primers according to the following conditions: 50 μl PCR reactions were composed of 5 μl 10x PCR buffer (Promega, Madison, WI. USA). 1.2 μl of both primer at 10 μM primer, 4 μl of 10mM dNTPs, 2.5 U of GoTaq DNA polymerase (Promega, Madison, WI, USA) and 0.1 ng/μl template DNA (final concentration), completed with sterilized and UV-treated MilliQ water (Millipore). PCR amplifications were conducted in a PTC200 Peltier Thermal Cycler (MJ Research, MA, USA) as follow: initial denaturing step at 94°C (4.5 min), followed by 25 cycles of 0.5 min denaturation at 94°C, 1 min annealing at 56°C. 1 min elongation at 72°C and a final elongation step of 10 min at 72°C. PCR products were purified with the EZNA Cycle-pure purification kit (PeqLab, Erlangen, Germany) according to the manufacturer instructions. Aliquots of 200 ng of purified PCR products were digested with 1 unit of HaeIII (Promega, Madison, WI, USA) (37°C, 4 h) in a 10 μl reaction volume. One μl of digested sample was mixed with 8.5 μl of HiDi formamid (ABI) and 0.5 μl of GS600-LIZ standard (ABI). Samples were denatured by heating to 95°C for 2 min followed by cooling on ice for 5 min. The denatured samples were loaded onto an ABI 3130xl DNA capillary sequencer equipped with 50 cm long capillaries and POP 7 electrophoresis matrix. Negative extraction and amplification control samples were composed of sterilized and UV treated water. The obtained datasets were analyzed with GeneMapper v4.0 (ABI). Electropherograms showing a background noise over 10 RFU (visual inspection) were discarded as well as peaks with height smaller than 50 Relative Fluorescence Units (RFU). The resulting profiles (percentage of contribution of all peak areas) were aligned semi-manually using Treeflap (http://www.sci.monash.edu.au/wsc/staff/walsh/treeflap.xls). T-RFs shorter than 50 bp and larger than 500 bp were discarded due to inconsistent results and lack of precision in their sizing respectively.
Table S1. Results of geochemical and chloroethenes concentration analyses of groundwater derived from wells a to e (refer to Figure 1 in the body text).
Well / Depth / Electricalconductivity / Temp. / pH / Redox
potential / Dissolved
oxygen / Sulfate / Nitrate / Iron(II) / Ethene / VC / trans-1.2-Dichlorethene / cis-1.2-Dichlorethene / Trichlorethene / Tetrachlorethene
[mbS] / [μS cm-1] / [°C] / [mV] / [mg l-1] / [mg l-1] / [mg l-1] / [mg l-1] / [μg l-1] / [μg l-1] / [μg l-1] / [μg l-1] / [μg l-1] / [μg l-1]
a / 53.0 / 999 / 13.6 / 4.8 / -123 / 2.4 / 34 / 6.3 / 13.5 / 526 / 4324 / 3666 / 15964 / 11595 / 21643
b / 39.0 / 4824 / 13.2 / 8.0 / -476 / 0 / 283 / 70 / 0.1 / 170 / 300 / 2700 / 7900 / 37000 / 2000
c / 45.7 / 3620 / 12.7 / 6.4 / -60 / 0.1 / 384 / 8.43 / 0.26 / 34.9 / 165 / 63.2 / 334 / 77.3 / 5.48
d / 47.2 / 1920 / 13.9 / 6.6 / 34 / 0.5 / 452 / 0.5 / 0.2 / 456 / 8287 / 4496 / 29145 / 14 / 0
e / 45.3 / 4185 / 12.7 / 6.3 / -205 / 0.0 / 83.6 / 9.67 / 0.50 / 1351 / 3994 / 10464 / 33311 / 33636 / 611
Table S2. Results of the carbon isotope signatures [‰] for the chloroethene species and carbon isotope signature [‰] of total chloroethenes (quantified as isotope balance) for groundwater samples derived from the 10 depths of the multilevel monitoring well and the five surrounding wells. Depths are numbered starting with 1 from the top towards the bottom 10 of the well for identification.
Well / Filter / Depth / ETH / VC / trans-1.2-DCE / cis-1.2-DCE / TCE / PCE / Isotope balanceCethene / δ 13Cethene / CVC / δ13CVC / CtDCE / δ13C-tDCE / C-cisDCE / δ13Ccis-DCE / CTCE / δ13CTCE / CPCE / δ13CPCE / ΣCchlorinated Ethene
[mbS] / [µM] / [‰] / [µM] / [‰] / [µM] / [‰] / [µM] / [‰] / [µM] / [‰] / [µM] / [‰] / [µM] / [‰]
Vertical gradient / 1a / 3.5 / 0.03 / n.d. / 0.08 / n.d. / 0.05 / -8.0 ± 1.0 / 0.53 / -13.8 ± 1.0 / < 0.01 / -20.8 ± 1.0 / < 0.01 / n.d. / 0.66 / -11.3 ± 0.8
2a / 11.5 / 0.03 / n.d. / 0.08 / n.d. / 0.05 / -8.6 ± 1.0 / 0.54 / -14.1 ± 1.0 / 0.01 / -20.8 ± 1.0 / < 0.01 / n.d. / 0.71 / -11.6 ± 0.8
3a / 15.0 / 0.03 / n.d. / 0.08 / n.d. / 0.05 / -9.2 ± 1.0 / 0.54 / -13.9 ± 1.0 / < 0.01 / -20.6 ± 1.0 / < 0.01 / n.d. / 0.70 / -11.4 ± 0.8
4a / 19.5 / 0.03 / n.d. / 0.12 / n.d. / 0.05 / -8.8 ± 1.0 / 0.52 / -14.0 ± 1.0 / 0.01 / -22.2 ± 1.0 / < 0.01 / n.d. / 0.72 / -10.8 ± 0.7
5b / 27.5 / 0.96 / -29.6 ± 1.0 / 0.68 / -25.3 ± 1.0 / 0.13 / -10.9 ± 1.0 / 0.58 / -15.2 ± 1.0 / 0.13 / -21.2 ± 1.0 / 0.01 / -33.6 ± 1.0 / 2.49 / -12.4 ± 0.5
6b / 32.5 / 1.96 / -29.9 ± 1.0 / 0.60 / -25.3 ± 1.0 / 0.21 / -11.5 ± 1.0 / 0.80 / -10.0 ± 1.0 / 0.17 / -18.4 ± 1.0 / 0.01 / -33.3 ± 1.0 / 3.75 / -7.8 ± 0.6
7b / 36.5 / 1.54 / -31.8 ± 1.0 / 0.60 / -12.4 ± 1.0 / 0.19 / -8.9 ± 1.0 / 0.68 / -2.1 ± 1.0 / 0.17 / -16.3 ± 1.0 / 0.02 / -32.8 ± 1.0 / 3.20 / -4.4 ± 0.6
8b / 40.5 / 2.04 / -35.0 ± 1.0 / 1.18 / -10.2 ± 1.0 / 0.26 / -11.0 ± 1.0 / 1.05 / -2.4 ± 1.0 / 0.22 / -15.0 ± 1.0 / 0.01 / -33.2 ± 1.0 / 4.77 / -4.4 ± 0.6
9b / 44.5 / 2.04 / -29.5 ± 1.0 / 0.95 / -24.2 ± 1.0 / 0.90 / -17.2 ± 1.0 / 4.20 / -18.3 ± 1.0 / 0.47 / -16.7 ± 1.0 / 0.01 / -32.6 ± 1.0 / 8.56 / -14.4 ± 0.6
10b / 48.5 / 1.49 / -27.1 ± 1.0 / 0.23 / -17.3 ± 1.0 / 0.36 / -15.2 ± 1.0 / 1.65 / -17.4 ± 1.0 / 0.28 / -17.3 ± 1.0 / 0.02 / -33.2 ± 1.0 / 4.03 / -11.1 ± 0.6
Surrounding wells / ab / 53.0 / 7.13 / -39.9 ± 1.0 / 4.48 / -35.2 ± 1.0 / 5.05 / -27.0 ± 1.0 / 15.5 / -24.6 ± 1.0 / 262.42 / -27.5 ± 1.0 / 96.50 / -39.1 ± 1.0 / 391.12 / -30.6 ± 1.0
bb / 39.0 / 6.06 / -39.0 ± 1.0 / 4.80 / -36.6 ± 1.0 / 27.90 / -24.1 ± 1.0 / 81.5 / -25.4 ± 1.0 / 27.34 / -25.0 ± 1.0 / 12.06 / -32.7 ± 1.0 / 410.34 / -26.0 ± 1.0
cb / 45.7 / 1.24 / -29.1 ± 1.0 / 2.64 / -24.1 ± 1.0 / 0.65 / -11.8 ± 1.0 / 3.45 / -26.0 ± 1.0 / 0.58 / -23.8 ± 1.0 / 0.03 / n.d. / 8.59 / -20.4 ± 1.0
db / 47.2 / 16.30 / -71.8 ± 1.0 / 132.60 / -32.8 ± 1.0 / 46.40 / -1.7 ± 1.0 / 300.00 / -20.8 ± 1.0 / 0.10 / n.d. / n.d. / n.d. / 495.16 / -23.9 ± 1.0
eb / 45.3 / 48.16 / -36.0 ± 1.0 / 63.90 / -16.7 ± 1.0 / 107.93 / -21.2 ± 1.0 / 343.59 / -24.8 ± 1.0 / 252.14 / -20.6 ± 1.0 / 3.69 / -37.5 ± 1.0 / 819.42 / -21.1 ± 1.0
n.d.: not detected
aMeasurement by purge and trap procedure (adapted from EPA. 1995)
bMeasurement by manual injection of head space samples
Table S3. Concentrations of BTEX, chlorinated ethanes and benzenes [µg L-1] for groundwater samples derived from the 10 depths of the multilevel monitoring well and the five surrounding wells. Depths are numbered starting with 1 from the top towards the bottom 10 of the well for identification and are indicated in brackets [mbS].
Compound / Code used in theMulti Factor Analysis (MFA)a / Depth of the vertical gradient [mbS]
1 [3.5] / 2 [11.5] / 3 [15.0] / 4 [19.5] / 5 [27.5] / 6 [32.5] / 7 [36.5] / 8 [40.5] / 9 [44.5] / 10 [45.3]
Trichlormethaneb / TCM / <0.10 / <0.10 / <0.10 / <0.10 / 0.45 / 0.28 / 0.50 / 0.97 / 169.60 / 76.38
1.1-dichloretheneb / 1.1-DCE / 0.22 / 0.20 / 0.18 / 0.21 / <0.10 / 0.11 / 0.10 / 0.15 / 1.19 / 0.55
Dichlormethaneb / DCM / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / 1.20 / 198.22 / 82.42
1.1-dichlorethaneb / 1.1.DCA / 0.17 / 0.24 / 0.18 / 0.21 / 0.15 / 0.21 / 0.20 / 0.19 / 0.39 / 0.28
1.2-dichlorethaneb / 1.2.DCA / 1.44 / 1.52 / 1.37 / 1.39 / 1.70 / 2.17 / 2.16 / 2.85 / 14.89 / 4.81
1.1.2-trichlorethaneb / 1.1.2.TCA / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / 0.20 / 12.08 / 5.67
1.1.1-trichlorethane / 1.1.1.TCA / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
Tetrachlormethane / PCM / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
1.1.2.2-tetrachlorethaneb / 1.1.2.2.TCA / <0.10 / 0.84 / <0.10 / <0.10 / 0.60 / 0.49 / 1.14 / 0.72 / 16.12 / 6.15
Chlorbenzeneb / CB / <0.10 / 0.10 / <0.10 / <0.10 / 1.08 / 1.70 / 3.88 / 1.59 / 0.87 / 0.88
1.2-dichlorbenzeneb / 1.2-DCB / <0.10 / <0.10 / <0.10 / <0.10 / 0.19 / 0.21 / 0.33 / 0.14 / <0.10 / 0.13
1.3-dichlorbenzene / 1.3-DCB / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
1.4-dichlorbenzeneb / 1.4-DCB / <0.10 / <0.10 / <0.10 / <0.10 / 0.23 / 0.31 / 0.44 / 0.13 / <0.10 / 0.15
Benzeneb / Benzene / 0.24 / 0.29 / 0.23 / 0.26 / 5.14 / 3.53 / 2.52 / 4.44 / 5.39 / 5.38
Ethylbenzene / Ethylbenzene / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
Tolueneb / Toluene / 0.13 / 0.12 / <0.10 / <0.10 / 0.13 / 0.19 / 0.30 / 0.20 / 0.17 / 0.20
p-xylene / p-xylene / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
o-xylene / o-xylene / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10 / <0.10
aThe MFA is displayed in Figure 4 of the manuscript
bContaminant with limit of quantification > 0.10 µg L-1 that was included in the Multi Factor Analysis (MFA, see Figure 4)