Supporting Information
Effects of bioaugmentation on enhanced reductive dechlorination of 1,1,1trichloroethane in groundwater – A comparison of three sites
Charlotte Scheutz*, Neal D. Durant, and Mette M. Broholm*
*
Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Building 115, DK-2800 Kgs. Lyngby, Denmark

Geosyntec Consultants, 10220 Old Columbia Rd., Suite A, Columbia, Maryland 21046, USA

Corresponding Author, email:
+1.410.381.4499
ndurant@geosyntec.com, phone:
+1.410.910.7642, fax:
List of contents.
1. Procedures for groundwater and sediment sampling
2. Procedures for sampling and analysis of microcosm
3. Data handling
4. Procedures for extraction and analysis of chloroethenes and chloroethenes
5. Figures
6. Tables
1. Procedures for groundwater and sediment sampling
Groundwater was collected from wells screened in the clayey till and the underlying sand aquifers
in autoclaved blue-cap glass-bottles under nitrogen flush, capped and stored at 4C until
microcosms were set-up. Samples were collected using a peristaltic pump with dedicated tubing.
Two well and gravel pack volumes were purged from each well and purge stabilization parameters
(pH, temperature, oxidation reduction potential [ORP], dissolved oxygen [DO]) were monitored
via an in-line flow-through cell (WTW-electrodes and instruments, Fagerberg, Brøndby,
Denmark) prior to collection. Sediment cores from the clayey till and underlying sand aquifers
were collected immediately adjacent to these wells using direct push technology (i.e., GeoProbe®)
coring. The sediment cores were sampled in plastic core-barrels, capped under nitrogen flush, and
transferred to nitrogen-filled, diffusion-tight aluminum bags, sealed and stored at 4C until
microcosms were constructed.
2. Procedures for sampling and analysis of microcosm
All microcosms were incubated upside down at 10°C for 600 days, and sampled periodically to
analyze for donor consumption, (non-volatile organic carbon [NVOC], lactate, acetate, propionate,
formate), redox conditions (NO3-, SO42-, dissolved Fe, methane) and degradation of chlorinated
ethanes and ethenes (TCE, cDCE, tDCE, 1,1-DCE, VC, ethene, ethane, TCA, 1,1-DCA, CA).
Prior to each sampling event, the bottles were shaken, weighed (to account for water and
headspace volume changes) and left for sediment to settle. Samples were collected with sterile
syringe and needle through the rubber stopper after volume compensation with sterile filtered
nitrogen to prevent vacuum build up in batch flasks.
Samples for the determination of chloroethenes, chloroethenes (1,1,1-TCA, 1,1-DCA and
CA), ethene and ethane were analyzed with an Agilent 6890N gas chromatograph (GC) equipped
with an Agilent 5973 mass spectrometer (MS). Aqueous samples (1 mL) were injected into sealed
vials, acidified (with 0.5 mL 4% H2SO4), heated to 80oC, and the headspace was sampled and
injected via autosampler into the GC. Separation was performed on a 25.0 m  320 µm  1.00 µm
(nominal) capillary column (J&W GSQ) with helium (class 2) as carrier gas. Chloroform was used
as internal standard (0.5 mL of a 10 ppmv aqueous solution). Detection limits for the chlorinated
ethenes/ethanes and ethene were below 1 µg L-1 whereas for ethane it was 2.6 µg L-1.
Samples for analysis of short-chained organic acids (lactate, acetate, formate, and
propionate) were filtered through 0.45 µm nylon filter membrane acidified with 50 µL 17% H3PO4
per mL of sample, and frozen until analysis by suppressed ion chromatography (IC) using a
Dionex IonPac ICE AS1 (9  250 mm) ion exclusion column, Dionex AMMS ICE II suppressor,
and WATERS 432 conductivity detector (eluent: 4 mM heptafluorobutyric acid in 2% vol
isopropanol; chemical suppression: 5 mM tetrabutyl ammonium hydroxide). Detection limits for
the short-chained organic acids were below 0.01 mM.
Anions were analyzed by IC (Dionex DX-120) using a two column set-up (Dionex Ion Pac
AS14 (4  250 mm) column and an AG 14 (4  250 mm) column) with 3.5 mM Na2CO3 / 1 mM
NaHCO3 as eluent. Detection limits for anions were below 0.2 mg L-1.
Samples for analysis of cations (iron) were filtered through 0.45 µm nylon filters, acidified
with 1M HNO3 (4 drops per 20 mL sample), refrigerated at 4 oC until analysis, and analyzed by
atomic absorption (Perkin Elmer Instruments Analyst 200 AAS 5000) with flame detection at
wavelengths of 248.33 nm for iron. Detection limit was below 0.01 mM.
Headspace samples (200 µL) for analysis of methane were injected manually as direct oncolumn injections into a Shimadzu 14A GC equipped with a packed column (3% SP1500
Carbopack B) and a flame ionization detector. Methane was analyzed with an isothermal column
temperature of 100 °C. The detection limit was 0.003 mM. The headspace methane concentrations
were converted to aqueous concentrations by using Henry’s law (Syracuse Research Corporation
Environmental Science PhysProp Database: http://www.syrres.com/esc/physdemo.htm. (Accessed
April 2012).
3. Data handling
The aqueous concentrations determined on aqueous samples from the microcosms were corrected
based on Henry’s law to include the mass of the compounds in the headspace of the microcosms in
order to account for changes in the aqueous to gas volume ratio caused by sampling. Aqueous
concentrations were not corrected to account for sorption to or desorption from the sediment. As
such, a portion of the concentration changes in the microcosms, particularly in the sterile controls,
was attributed to sorption/desorption processes (e.g., increases in the aqueous concentration of
compounds not added but initially present were attributed to the presence of a sorbed phase). As
dechlorination daughter products are less hydrophobic than the parent compounds this can lead to
increasing total molar concentrations in the aqueous phase over time as the parent compounds
degrade.
4. Procedures for extraction and analysis of chloroethenes and chloroethenes
After completion of the incubation experiments by Day 601, the total mass (sorbed, dissolved in
water and in gas phase) of chloroethanes (TCA, 1,1-DCA) and chloroethenes (PCE, TCE, cDCE
and 1,1-DCE) was quantified in selected batches by solvent extraction (in pentane) and analysis
(GC-ECD) of the extract. CA, VC, ethene and ethane could not be quantified by this method, due
to their lower boiling point preventing chromatographic separation. Sorption of these constituents
was considered negligible, however, as they have high vapor pressure and are gases at the
experiment temperature. Pentane (50 mL) added an internal standard (bromotrichloromethane 10
mg/L) was injected through the septum to each individual batch and shaken for 24 hours in a
rotary box. The pentane phase was extracted through the septum and transferred to glass tubes
with Teflon coated screw caps. If needed samples were diluted with pentane. The extracts were
analyzed with a Shimadzo GC 2010 gas chromatograph equipped with an electron capture detector
(Nuclide 63Ni Quantity 370 MBq 10Ci). Extract samples (1 µL) were injected via autosampler
into the GC. Separation was performed on a 30.0 m  0.53 mm  3.00 µm capillary column
(VocolTM, 11389-03B) with nitrogen as carrier gas. The temperature program was as followed:
initial temperature: 45.0 ºC (1 min), temperature rate: 30.0 ºC/min (3.5 min), finale temperature
150 ºC (5.5 min). Detection limits for the chlorinated ethenes/ethanes were 0.15 µg L-1.
5. Figures
Fig. SI 1 - Trends in aqueous phase chloroethanes, redox parameters, and electron donor in Høje Tåstrupvej
source zone microcosms. First column; intrinsic control; second column; lactate-amended; and third
column; lactate and bioaugmented. Batch abbreviations refer to the microcosm set-up presented in Table 2
in the paper.
Fig. SI 2 - Trends in aqueous phase chloroethanes and chloroethenes in Høje Tåstrupvej source area
microcosms. First column; intrinsic control; second column; lactate-amended; and third column; lactate and
bioaugmented. Batch abbreviations refer to the microcosm set-up presented in Table 2 in the paper.
Fig. SI 3 - Total mass of chloroethanes and 1,1-DCE (extracted in pentane) compared to total mass
quantified in the aqueous and gas phase based on water and headspace analysis in source area Høje
Tåstrupvej microcosms. The difference between the bars represents the mass sorbed to the sediment. Batch
abbreviations refer to the microcosm set-up presented in Table 2 in the paper. Note that the mass of CA is
not included as CA cannot be extracted into pentane due to the lower boiling point of CA in comparison to
pentane. Also please note that during autoclaving of the sterile controls 1,1,1-TCA sorbed to the
contaminated sediment were abiotically transformed to 1,1-DCE.
Fig. SI 4 - Trends in aqueous phase chloroethanes, chloroethenes, redox parameters, and electron donor in
Vadsbyvej source area microcosms. First column; intrinsic control; second column; lactate-amended; and
third column; lactate and bioaugmented. Batch abbreviations refer to the microcosm set-up presented in
Table 2 in the paper.
Fig. SI 5 - Total mass of chloroethanes and c-DCE (extracted in pentane) compared to total mass quantified
in the aqueous and gas phase based on water and headspace analysis in Vadsbyvej source area microcosms.
The difference between the bars represents the mass sorbed to the sediment. Batch abbreviations refer to the
microcosm set-up presented in Table 2 in the paper. Note that the mass of CA is not included as CA cannot
be extracted into pentane due to the lower boiling point of CA in comparison to pentane.
6. Tables
Table SI 1. Microcosm results summary.
Experimental set-up
Redox conditions
Location
Microcosms
Compound
added
Baldersbæk
Source area
Sterile control
Chloroethane and chloroethene degradation
Days to complete
sulfate reduction
Days to
methane
production
Days to complete
conversion of
1,1,1-TCA
Days to
complete
conversion of
1,1-DCA
Days to
complete
conversion of
c-DCE
Days to mass
balance ethene
production
DOD,
TCA
DOD,
PCE
TCA
>600
>600
>600
>600
-
-
27
-
Intrinsic
TCA
>600
>600
339
>600
-
-
33
-
Stimulated
TCA
42
283
231
601
-
-
67
-
Stimulated
TCA+PCE
42
283
231
402
231
295
67
100
Bioaugmented
TCA
42
283
231
402
-
-
67
-
Bioaugmented
TCA+PCE
42
283
231
402
181
181
67
100
Intrinsic
TCA
>600
>600
>600
>600
-
-
25
-
Stimulated
TCA+PCE
42
470
339
>600
>600
>600
38
50
Bioaugmented
TCA+PCE
42
283
295
601
132
181
67
100
Location
Microcosms
Compound
added
Days to complete
sulfate reduction
Days to
methane
production
Days to complete
conversion of
1,1,1-TCA
Days to
complete
conversion of
1,1-DCA
Days to
complete
conversion of
1,1-DCE
Days to mass
balance ethene
production
DOD,
TCA
DOD,
DCE
Høje
Taastrup
Source area
Sterile control
TCA
>600
>600
>600
>600
-
-
18
-
Intrinsic
TCA
>600
>600
>600
>600
-
-
25
-
Stimulated
TCA
100
>600
>600
>600
-
-
25
-
Bioaugmented
TCA
42
333
402
470
-
-
67
-
Sterile control
1,1-DCE
>600
>600
>600
>600
>600
>600
30
50
Intrinsic
1,1-DCE
>600
>600
>600
>600
>600
>600
25
50
Stimulated
1,1-DCE
100
>600
>600
>600
>600
>600
30
50
Bioaugmented
1,1-DCE
42
331
181
339
100
132
67
100
Sterile control
CA
>600
>600
-
>600
-
-
50
-
Intrinsic
CA
>600
>600
-
>600
-
-
50
-
Stimulated
CA
178
331
-
>600
-
-
55
-
Bioaugmented
CA
42
282
-
402
-
-
67
-
Baldersbæk
Plume area
- : Compound not present
Experimental set-up
Redox conditions
Location
Microcosms
Compound
added
Vadsbyvej
Source area
Sterile control
Chloroethane and chloroethene degradation
Days to complete
sulfate reduction
Days to
methane
production
Days to complete
conversion of
1,1,1-TCA
Days to
complete
conversion of
1,1-DCA
Days to
complete
conversion of
c-DCE
Days to mass
balance ethene
production
DOD,
TCA
DOD,
TCE
TCA
>600
>600
>600
>600
-
-
20
-
Intrinsic
TCA
50
541
>600
>600
-
-
30
-
Stimulated
TCA
23
442
>600
>600
-
-
30
-
Stimulated
TCA+TCE
23
442
>600
>600
204
259
30
100
Stimulated
TCE
23
248
42
447
157
157
67
100
Bioaugmented
TCA
23
442
447
447
372
447
36
100
Bioaugmented
TCA+TCE
23
442
447
447
372
447
67
100
Bioaugmented
TCA+TCE
23
370
447
372
310
372
67
100
Bioaugmented
TCA+TCE
23
370
>600
>600
310
372
32
100
Bioaugmented
TCE
23
248
42
372
157
157
67
100
Vadsbyvej
Intrinsic
TCA
>600
>600
>600
>600
-
-
20
50
Plume area
Stimulated
TCA+TCE
105
541
>600
>600
590
590
25
100
Bioaugmented
TCA+TCE
50
370
447
>600
310
310
56
100
Sterile control
CA
>600
>600
-
>600
-
-
67
-
Intrinsic
CA
>600
>600
-
>600
-
-
67
-
Stimulated
CA
178
178
-
204
-
-
67
-
Bioaugmented
CA
255
255
-
310
-
-
67
-
- : Compound not present