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Characterizing In Situ
Methane-Enhanced Biostimulation
Potential for 1,4-Dioxane Biodegradation
in Groundwater
Venus Sadeghi
Rebecca Mora
Priya Jacob
This study characterizes the 1,4-dioxane biodegradation potential for an in situ methane-enhanced
biostimulation field pilot study conducted at Air Force Plant 44, located south of the Tucson Inter-
Sheau-Yun (Dora) Chiang national Airport in Arizona. In this study, the use of methane as the primary substrate in aerobic
cometabolic biodegradation of 1,4-dioxane is evaluated using environmental molecular diagnostic
tools. The findings are compared to an adjacent pilot study, wherein methane was generated via
enhanced reductive dechlorination and where methane monooxygenase and methane-oxidizing
bacteria were also found to be abundant. This article also presents the use of 13 C and 2 H isotopic ra-
tio enrichment, a more recent tool, to support the understanding of 1,4-dioxane biodegradation in
situ. This study is the first of its kind, although alkane gas-enhanced biodegradation of 1,4-dioxane
has been evaluated extensively in microcosm studies and propane-enhanced biodegradation of
1,4-dioxane has been previously studied in the field. c⃝ 2016 Wiley Periodicals, Inc.
INTRODUCTION
extract, 2-propanol, and propane (Vainberg et al., 2006). These microcosm studies
verified 1,4-dioxane can be cometabolically biodegraded under aerobic conditions.
Shen et al. (2008) reported anaerobic biodegradation of 1,4-dioxane by sludge
enriched with iron-reducing microorganisms. The 1,4-dioxane biodegradation by
iron-reducing bacteria was enhanced by iron(III) oxide and humic acid (HA) or
iron(III)-ethylenediaminetetraacetic acid. According to this study, HA works as a catalyst
to increase the biological activities of iron-reducing bacteria for 1,4-dioxane degradation.
This assumes that the degradation of 1,4-dioxane first biologically transfers electrons to
The confirmation of HA via iron-reducing bacteria and that once HA is reduced, it abiotically transfers
biodegradation mech- electrons to iron(III), regenerating HA to the oxidized form. To the best of the authors’
anisms at groundwater knowledge, this is the only study that has documented the anaerobic biodegradation
contamination sites often potential of 1,4-dioxane.
relies on monitoring CB1190, an actinomycete grown on 1,4-dioxane as the sole carbon and energy
of contaminant con- source, was isolated from a 1,4-dioxane-contaminated sludge sample (Parales et al.,
centrations, daughter 1994). This CB1190 strain degrades 1,4-dioxane and mineralizes 59.5 percent of it to
compound generation,
carbon dioxide (CO2 ) following the same degradation pathways proposed for cometabolic
and biogeochemical pa-
biodegradation (Mahendra et al., 2007). Mahendra and Alvarez-Cohen (2005)
rameters. Environmental
molecular diagnostic
characterized CB1190, based on morphological, physiological, chemotaxonomic, and
(EMD) tools have been phylogenetic evidence, and proposed that it be classified as a novel species, Pseudonocardia
developed specifically for dioxanivorans sp. nov. CB1190 uses 1,4-dioxane as its source of carbon and energy for
evaluating 1,4-dioxane growth. In contrast, microorganisms that facilitate 1,4-dioxane biodegradation via
biodegradation. cometabolism require a primary substrate (e.g., methane or propane) as a carbon or
energy source. Other bacterial strains for metabolic biodegradation of 1,4-dioxane have
also been isolated (Kim et al., 2009; Huang et al., 2014).
Field demonstrations of 1,4-dioxane biodegradation potential are limited. Shrinkage
of a large 1,4-dioxane plume was first documented in 2008 with support of fate and
transport modeling; however, the mechanisms that governed 1,4-dioxane plume shrinkage
were not identified (Chiang et al., 2008). A field study of natural 1,4-dioxane
biodegradation potential under intrinsic aerobic conditions was conducted by using stable
isotope probing (SIP; Chiang et al., 2012). The study confirmed the incorporation of the
heavier stable carbon isotope (carbon 13 or 13 C) labeled 1,4-dioxane into biomass. The
13
C incorporation into biomass can be due to the bacterial utilization of bioavailable
intermediates during the 1,4-dioxane biodegradation process. In situ propane-enhanced
biostimulation and bioaugmentation for treating 1,4-dioxane was pilot tested at
Vandenberg Air Force Base (Lippincott et al., 2015). The 1,4-dioxane concentration
reductions observed during the pilot study shed light on the consideration of in situ
bioremediation (ISB) for 1,4-dioxane. However, the injection of methane for field-scale in
situ biostimulation and biodegradation of 1,4-dioxane has not been previously
documented, although microcosm studies using methane as a primary substrate were
previously documented (Mahendra & Alvarez-Cohen, 2006).
The confirmation of biodegradation mechanisms at groundwater contamination sites
often relies on monitoring of contaminant concentrations, daughter compound
generation, and biogeochemical parameters. Environmental molecular diagnostic (EMD)
tools have been developed specifically for evaluating 1,4-dioxane biodegradation. Primary
EMDs include microbial population analysis to identify the presence and abundance of
important microorganisms potentially involved in 1,4-dioxane biodegradation; SIP
analysis, which can unambiguously verify 1,4-dioxane biodegradation potential in the
METHODS
Site Description
The methane biostimulation pilot study was conducted at Air Force Plant 44 (AFP44, the
Site), located south of Tucson International Airport in Tucson, Arizona. AFP44 started Former waste manage-
operating as an industrial plant in 1951. Former waste management practices at AFP44 ment practices at AFP44
have impacted the regional groundwater in the vicinity and have contributed to a 7 mile have impacted the re-
long, ½-mile wide groundwater plume contaminated primarily with chlorinated volatile gional groundwater in
organic compounds (CVOCs) and 1,4-dioxane. the vicinity and have con-
Two aquifer zones have been defined within the basin fill sediments underlying the tributed to a 7 mile long,
½-mile wide groundwater
Site, including the upper zone of the regional aquifer (UZ) and the lower zone of the
plume contaminated
regional aquifer (LZ). The UZ is further subdivided into two distinct aquifer subunits, the
primarily with chlorinated
upper zone upper unit (UZUU) and the upper zone lower unit (UZLU). The UZUU is the volatile organic com-
most impacted subunit and is the subject of this study. It comprises the upper portion of pounds (CVOCs) and
the UZ, from the groundwater surface to a depth of approximately 140 to 160 ft. The base 1,4-dioxane.
of the unit generally coincides with the base of the deepest coarse-grained layer in the
UZUU. The UZUU has been further subdivided into two zones that are relevant to the
remediation program, the UZUU aquitard and the UZUU aquifer. Where the UZUU
aquitard is present beneath former AFP44 source areas, this fine-grained unit contains a
significant portion of the remaining mass of contaminants of concern (COCs) at AFP44. In
these areas, the UZUU aquitard acts as an ongoing contaminant source to the underlying
UZUU aquifer, slowly diffusing COCs into these more permeable sediments. The low
permeability of the UZUU aquitard makes source area remediation using a traditional
pump and treat approach difficult. Therefore, use of environmental hydraulic fracturing
with in situ remediation approaches was selected in this zone to effectively reduce mass
discharge from the UZUU aquitard into the underlying groundwater system or the UZUU
aquifer, which comprises the permeable channel sands and gravels that extend from the
base of the UZUU aquitard to the top of the fine-grained UZLU aquitard at a depth of
approximately 140 to 160 ft. Exhibit 1 shows the extents of the trichloroethene (TCE)
and 1,4-dioxane plumes in the UZUU in 2013 prior to the pilot studies.
Exhibit 1. TCE (above) and 1,4-dioxane (below) plume extents in UZUU aquifer – 2013. Concen-
trations are in micrograms per liter
The pilot study areas presented here pertain to the portions of the plume with the
highest 1,4-dioxane and TCE impacts at the known source area, Site DP-03, where
AECOM implemented ISB to reduce contaminant mass in fine-grained sediment. ERD, in
the form of biostimulation and bioaugmentation, was implemented where CVOC impacts
were dominant. An aerobic cometabolic pilot study of methane-enhanced biostimulation
was implemented in the vicinity of well M-69, where historic 1,4-dioxane concentrations
have been in the several parts per million range (Exhibit 2).
Eighty-eight injection borings were installed at Site DP-03. In the ERD area, ISB
injection points (numbered 16–50 and 61–98) were installed from the water table, at
approximately 100 ft bgs (ft bgs), to 145 ft bgs. ERD activities were completed in late
February 2015. Blank casings were installed in the boreholes, and hydrofracturing
technology was implemented using high-pressure water to create fissures, in
approximately 7-ft intervals, into the casing and the formation while injecting a slurry
containing sand/guar gum, vegetable oil (EDS-ERTM ; Tersus Environmental, Wake Forest,
NC) with a neutral pH to provide slow released carbon sources and buffering agent,
metabolic nutrients (Nurimens R⃝ ; Tersus Environmental), and a consortium of anaerobic
bacteria (KB-1 R⃝ ; SiREM, Guelph, Ontario, Canada) into the fissures. EDS-ERTM is an
extended release, soy-based, water-mixable electron donor and self-emulsifying oil with a
neutral pH that has a lower viscosity than EVO.
In the methane biostimulation area, injection points 1 to 15 were installed near the
vicinity of well M-69 that historically had the highest concentrations of TCE and
1,4-dioxane. Hydrofracturing consisted of injecting a sand/guar gum slurry and
amendments consisting of dissolved methane gas, sodium molybdate dihydrate (Mo), and
diammonium phosphate (DAP) at high pressure to create fissures in the borehole casing
and the formation (as described earlier). Based on pressure measurements, the radius of
fracture was found to be 25 ft or higher. Approximately 0.033 pounds of methane, 16
pounds of Mo, and 34 pounds of DAP, respectively, were injected into each borehole
during fracturing. The selection of amendments was based on the results of a bench-scale
treatability study performed prior to field implementation. The purpose of the amended
fracturing slurry was to “precondition” the area for air and methane-enhanced
M-69 Located within the methane Well contains the highest 1,4-dioxane
biostimulation pilot study area concentration
M-93 Located upgradient from the Well contains trace levels of
methane biostimulation pilot 1,4-dioxane and is not within the
study area target methane biostimulation pilot
study area, but the data suggest
this well could have been
influenced by the amendments
during fracturing activities
M-98 Located within the ERD pilot study Well is impacted by high
area concentrations of CVOCs and
1,4-dioxane
M-101 Located within the ERD pilot study Well is impacted by CVOCs but low
area level of 1,4-dioxane
M-104 Located outside of ERD and is Well has no CVOC data, contains very
considered as background well low level of 1,4-dioxane
A total of five wells were selected for this study (Exhibit 3). Well M-69 was selected to
represent conditions within the methane biostimulation area. Well M-93 represents
conditions upgradient of the methane biostimulation area. Wells M-98 and M-101 were
selected to evaluate conditions within the ERD area. Well M-104 was selected to evaluate
background conditions where no remediation is currently being performed. Exhibit 4
shows the study well locations.
Groundwater samples were collected for analysis of enzymes and 1,4-dioxane
biomarkers, DXMO and ALDH. DXMO mediates the first step of 1,4-dioxane
to biodegradation. The differences in isotopic ratios are typically expressed in “per mil”
(parts per thousand, or ‰) values, relative to a reference standard. A “delta” (𝛿) notation
is used to represent the per mil change in isotopic ratio when compared to the standard.
Groundwater samples were collected between July 11 and 21, 2016, using
HydraSleevesTM (GeoInsight; Las Cruces, NM). HydraSleevesTM are sampling devices that
Biotrap R⃝ samplers are allow groundwater to be collected from the screened interval without purging the well.
passive sampling tools Static groundwater levels and total well depths were recorded prior to the sampling
designed to collect rep- activities. Static water levels at the wells ranged from 99.88 ft bgs at well M-93 to
resentative samples of 111.62 ft bgs at well M-104. Groundwater temperature, pH, electrical conductivity,
the microbial community dissolved oxygen (DO) concentration, oxidation–reduction potential (ORP), total
over time. Most microbes dissolved solids concentration, and turbidity measurements were collected using a YSI
prefer to be attached to (Omni Controls, Tampa, FL) 556 multiparameter measuring tool with a downhole probe.
a surface rather than free- Following the collection of field water quality parameters, six to eight HydraSleevesTM
floating; therefore, the
tied in series were deployed in each well. Each HydraSleeveTM had a 4-inch diameter and
biotraps are constructed
2.5-liter capacity. The HydraSleevesTM were deployed along the screen interval, at depths
as small plastic canisters
containing Bio-Sep R⃝
ranging from 110 ft bgs to 142 ft bgs. After 48 hours, the HydraSleevesTM were removed
beads that provide a from the wells and a sample of collected groundwater was used for the field analysis of
large surface area for the ferrous iron (Fe[II]), using a HACH (Loveland, CO) color disc test kit.
microbes to colonize and Groundwater samples were transferred to laboratory sample containers, labeled and
form biofilms. logged on chain-of-custody forms, and shipped to Pace for the analysis of geochemical
parameters, dissolved gases, and 13 C and 2 H isotopic ratios for 1,4-dioxane. Groundwater
samples for the analysis of microbial gene targets were submitted following chain of
custody procedures to Microbial Insights (Knoxville, TN). CVOC and 1,4-dioxane
concentration data for the evaluation were based on the routine site monitoring results,
with the last samples having been collected in March 2016.
Following the collection of groundwater using HydraSleevesTM and submission of
samples to the aforementioned laboratories, standard Biotrap R⃝ samplers were lowered
into wells M-69 and M-93 on July 21 2016. Biotrap R⃝ samplers are passive sampling tools
designed to collect representative samples of the microbial community over time. Most
microbes prefer to be attached to a surface rather than free-floating; therefore, the
biotraps are constructed as small plastic canisters containing Bio-Sep R⃝ beads that provide a
large surface area for the microbes to colonize and form biofilms. The Biotrap R⃝ samplers
were deployed in the wells in the middle of the screen interval. After 30 days, the
samplers were removed from the wells on August 22, 2016, and submitted following chain
of custody procedures to Microbial Insights for the analyses of bacteria and gene targets.
Analytical Methods
For the field samples submitted to Microbial Insights, total DNA was extracted from
groundwater samples collected from AFP44 using a PowerSoil DNA Extraction Kit (MO
BIO, Carlsbad, CA) following the manufacturer’s instructions. DNA extracts were subjected
to CENSUS qPCR analysis (Microbial Insights) of microbial populations and enzymes
(DXMO, ALDH, sMMO, MOB, EtnC, EtnE, PPO, PHE, RMO). These qPCR targets
represent organisms or enzymes capable of degrading 1,4-dioxane, except Etn enzymes,
which have not been documented to be responsible for 1,4-dioxane biodegradation.
Each qPCR mixture contained 1× cloned Pfu buffer (Stratagene, LaJolla, CA), 0.2 mM of
each of the four deoxynucleoside triphosphates, SYBR green (diluted 1:30,000; Molecular
Probes, Eugene, OR), and 1 unit (U) of Pfu Turbo HotStart DNA polymerase (Stratagene).
Annealing temperatures, primer concentrations, and magnesium chloride concentrations
varied depending on the primer set used. All qPCR reactions were performed
on an ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA).
Groundwater samples were collected from the wells and sent to Pace for isotopic analysis
of 12 C and 13 C as well as 2 H and 1 H. 1,4-Dioxane in each sample was first preconcentrated
using techniques as recommended in USEPA Method 522 and then analyzed by direct
injection using gas chromatography isotope ratio mass spectrometry.
RESULTS
Geochemical Conditions
Water quality parameters were measured in each well prior to the deployment of
HydraSleevesTM in July 2016. As shown in Exhibit 6, the groundwater temperature was
approximately 25 degrees Celsius in all wells and pH was near neutral for both pilot study
areas.
Exhibit 5. Continued
In the methane biostimulation area, DO concentrations were 6.33 mg/L and 8.53
mg/L in wells M-69 and M-93, respectively, indicating the aquifer in the area is generally
aerobic. The ORP values were 36.6 millivolts (mV) and 78.9 mV at wells M-69 and
M-93, respectively. Methane was not detected in well M-69, even though this well is
located within the methane biostimulation pilot study area. This may have been due to the
consumption of methane at faster rates than methane replenishment rates by the iSOC R⃝
units.
Three monitoring wells, M-98 (inside ERD treatment area), M-101 (inside ERD
treatment area) and M-104 (background and cross-gradient, outside ERD treatment area)
were selected to evaluate ERD study area conditions. Well M-104 had DO and ORP of
6.21 mg/L and 54.6 mV, respectively, confirming the ambient aerobic conditions for the
Site. DO concentrations at M-98 and M-101 were 1.19 mg/L and 0.45 mg/L,
respectively, significantly lower than background levels, indicating the field conditions have
been altered due to the ERD treatment. ORP values were 41.9 mV and 28 mV in M-98
and M-101, respectively. ORP values less than 50 mV are considered to be representative
of mildly reducing conditions. (Although the focus of this study is on 1,4-dioxane
biodegradation, it is noteworthy that the weak reducing conditions may be due to mixing
in the wells, as the screen intervals in the performance wells only partially overlap with
the amendment injection intervals in the aquitard above the well screens.) Methane,
ethene, and ethane were detected at wells M-98 and M-101, but not in M-104
(background well). The detection of dissolved gases confirmed the formation of an anoxic
to anaerobic environment due to the ERD treatment.
The presence and abundance of bacteria and functional genes in groundwater samples and
in biotraps are summarized in Exhibit 7. For groundwater samples, abundances of ALDH,
DXMO, MOB, sMMO, EtnC, EtnE, PPO, PHE, and RMO were analyzed. Biotrap R⃝
samplers were only deployed in wells M-69 and M-93 (methane biostimulation pilot study
area) and not in the ERD study area. Ethene and ethane were not detected in the methane
Abbreviation: DO, dissolved oxygen; ND, not detected; TKN, total kjeldahl nitrogen.
biostimulation pilot study area, thus functional genes EtnC and EtnE were not analyzed for
in biotrap samples.
Biomarkers ALDH and DXMO were developed based on aerobic metabolic
biodegradation of 1,4-dioxane in CB1190 culture (Gedalanga et al., 2014). DXMO was
not detected or was detected at an estimated value (2.1E+00 J cells per milliliter
[cells/mL] at M-69). ALDH was also only detected at trace levels slightly above the
detection limit. These two 1,4-dioxane-specific biomarkers were not detected in biotrap
samplers deployed in wells M-69 and M-93.
MOB and sMMO were detected in all groundwater samples. MOB abundance was
very similar in wells M-69, M-93, M-98, and M-101, ranging from 2.05E+04 cells/mL in
M-93 to 3.31E+04 cells/mL in M-98. The MOB result for M-104 was two orders of
magnitude lower at 5.20E+02 cells/mL. Similar to MOB, sMMO abundance was within
an order of magnitude in the same four out of the five wells (ranging from 1.80E+02
Exhibit 7. Presence and abundance of bacterial and functional genes potentially responsible for 1,4-dioxane biodegradation
qPCR-DNA for
Groundwater
Samples Unit Sampling Date M-69 M-93 M-98 M-101 M-104
DXMO cell/mL Jul 2016 2.10E+00 J < 5.00E–01 6.00E–01 J 3.00E–01 J < 5.00E+00
ALDH cell/mL Jul 2016 8.00E–01 J 3.00E–01 J 1.00E–01 J 2.00E–01 J < 5.00E+00
sMMO cell/mL Jul 2016 1.80E+02 5.81E+02 3.17E+02 1.61E+03 3.24E+01
MOB cell/mL Jul 2016 2.48E+04 2.05E+04 3.31E+04 2.24E+04 5.20E+02
EtnC cell/mL Jul 2016 3.87E+01 2.05E+01 4.39E+02 2.29E+02 8.00E–01 J
EtnE cell/mL Jul 2016 5.57E+01 1.04E+02 1.16E+04 5.93E+03 4.15E+01
PPO cell/mL Jul 2016 5.22E+01 3.30E+00 J 1.54E+02 2.18E+02 2.70E+00 J
PHE cell/mL Jul 2016 2.86E+02 1.16E+03 7.61E+02 1.67E+03 6.87E+01
RMO cell/mL Jul 2016 1.00E–01 J 1.90E+00 J 1.5E+00 J 1.7E+00 J < 5.00E+00
DXMO cells/bead Aug 2016 <2.50E+02 <2.50E+02
ALDH cells/bead Aug 2016 <2.50E+02 <2.50E+02
sMMO cells/bead Aug 2016 1.59E+03 1.62E+03
MOB cells/bead Aug 2016 2.46E+03 5.32E+04 No Biotrap Data
PPO cells/bead Aug 2016 8.08E+01 J 1.38E+01 J
PHE cells/bead Aug 2016 3.03E+03 2.21E+04
RMO cells/bead Aug 2016 <2.50E+02 <2.50E+02
DO Concentration (mg/L)
1.00E+04 7
DO
6
(cells/mL)
1.00E+03
5
4
1.00E+02
3
1.00E+01 2
1.00E+00 0
M-69 M-93 M-98 M-101 M-104
Monitoring Well
Exhibit 8. MOB and sMMO abundance in aerobic methane-biostimulation (M-69 and M-93) and
anaerobic ERD (M-98, M-101, and M-104) pilot study areas
Additional qPCR targets PPO, PHE, and RMO were measured in samples collected
from all the wells in July 2016. These targets are ubiquitous in the environment, similar to
MOB and sMMO, and have no direct correlation with redox conditions or 1,4-dioxane
biodegradation potential. Based on these data, it is hypothesized that low DO in the anoxic
environment can sustain the presence and abundance of a wide range of monooxygenases.
The presence of ethene monooxygenases, EtnC and EtnE, has not been documented
in the literature to be relevant to 1,4-dioxane biodegradation. Owing to the ethene
generation from ERD treatment, EtnC and EtnE were detected in wells M-98 and M-101,
suggesting the ethene monooxidizing bacteria were present and utilized ethene as a
primary substrate while using low levels of oxygen in the ERD pilot study area as an
electron acceptor.
In summary, aerobic monooxygenase abundance is similar in both ERD and methane
biostimulation areas, suggesting these selected targets are ubiquitous and can be abundant
under both high and low DO concentrations. However, 1,4-dioxane was reduced (by
71 percent) in the aerobic methane biostimulation pilot study area only, and not in the
ERD study area, suggesting the presence and abundance of MOB, sMMO, PPO, PHE, and
RMO alone cannot be used as direct evidence of 1,4-dioxane biodegradation potential.
This may also suggest that DO concentrations are vital for supporting 1,4-dioxane
biodegradation potential. In other words, abundance of monooxygenase enzymes in the
presence of adequate DO concentrations could lead to conditions conducive to
1,4-dioxane biodegradation.
CSIA for 1,4-dioxane is a newly developed tool to verify 1,4-dioxane biodegradation. The
groundwater samples collected in July 2016 from all the wells were analyzed for both 13 C
and 2 H isotopic ratios for 1,4-dioxane. The results are provided in Exhibit 9. CSIA results
for 1,4-dioxane show that 𝛿 13 C values ranged from 30.02 ‰ to –31.63 ‰, suggesting
either a possible lack of 13 C isotope enrichment, or that 13 C isotopic shifts for 1,4-dioxane
are small, regardless of the degradation mechanism. However, 1,4-dioxane 𝛿 2 H values
ranged from –76.34 ‰ in the methane biostimulation treatment area well M-69 to
–103.68 ‰ in the ERD treatment area well M-101. The enrichment of the heavier 2 H
isotope (relative to other wells) in conjunction with the decreasing 1,4-dioxane
concentration trend observed in well M-69, points to the degradation of 1,4-dioxane.
DISCUSSION
The Site is currently undergoing two pilot studies, side by side. Both pilot study areas are
impacted by chlorinated solvents (primarily TCE and 1,1-DCE) and 1,4-dioxane
originating from the same contamination source.
The ERD pilot study area is primarily impacted by CVOCs; 1,4-dioxane
concentrations were detected at lower levels, thus, the ERD technology was selected to
treat CVOCs. EVO, nutrients, and KB-1 culture were injected into the ERD pilot study
area to accelerate anaerobic dechlorination of CVOCs. In response to the ERD pilot study,
a mild reducing environment has been created. DO and ORP have decreased and
methane, ethene, and ethane have been produced, as expected. Adjacent to the ERD pilot
study area, a methane biostimulation pilot study has been conducted. 1,4-Dioxane
concentrations are significantly higher, and CVOCs are also present at high concentrations.
The high 1,4-dioxane concentrations appear to be localized near well M-69. Methane gas
and air are cyclically injected into the subsurface to biostimulate 1,4-dioxane degradation.
It is known that 1,4-dioxane is primarily biodegraded under aerobic conditions.
Chemical, geochemical, and biological parameters have been used to verify aerobic
biodegradation of 1,4-dioxane. The understanding of the usefulness of these parameters is
still evolving, and the number of case studies that document biodegradation potential of
1,4-dioxane at the field scale is very limited. This study not only documents the first field
demonstration of methane biostimulation, but, more importantly, evaluates the role of
methane and EMDs in evaluating 1,4-dioxane biodegradation potential. Furthermore, the
same parameters were also applied to groundwater samples collected from the adjacent
anaerobic ERD pilot study area, which was not designed for 1,4-dioxane treatment. This
study was designed to understand the role of methane in both pilot study areas and to
determine whether the selected 1,4-dioxane–degrading monooxygenases were present
and abundant in both pilot study areas and whether their presence would impact
1,4-dioxane biodegradation.
The study included the collection of groundwater samples from five monitoring wells.
Three wells, including one background well, were in the ERD pilot study area, and two
wells were from the methane biostimulation area. The results reveal that 1,4-dioxane was
only reduced in well M-69, located in the methane biostimulation pilot study area.
1,4-Dioxane concentrations were stable in all wells in the ERD pilot study area and
showed no decreasing trends. MOB and sMMO abundance were elevated in both the
methane biostimulation and ERD pilot study areas when compared to the abundance in
the background well M-104. These findings suggest that methane (via methane injection
or generation from ERD treatment) has increased the abundance of MOB and sMMO in
both pilot study areas, even though the DO (electron acceptor for MOB and sMMO
growth) is significantly lower in the ERD area. Although MOB and sMMO were abundant
in both pilot study areas, 1,4-dioxane only biodegraded in well M-69. Therefore, DO
levels may play a vital role in evaluating 1,4-dioxane biodegradation potential, while MOB
and sMMO are ubiquitous under a wide spectrum of redox conditions. MOB and sMMO
are not likely strong indicators of 1,4-dioxane biodegradation potential. DXMO and
ALDH are two biomarkers based on metabolic biodegradation of 1,4-dioxane. DXMO
and ALDH in well M-69 were detected at trace levels, only slightly above detection limits,
indicating that the observed reductions in 1,4-dioxane concentrations were likely not due
metabolic biodegradation.
This is the first case study that reports the 2 H enrichment of 1,4-dioxane during a
field demonstration of ISB of 1,4-dioxane. The enrichment in the 2 H isotopic ratio appears
to be the strongest direct evidence of 1,4-dioxane biodegradation in this study. This
implies cleavage of the carbon–hydrogen bond during the 1,4-dioxane biodegradation
process, but this theory needs to be verified with additional data. More CSIA data (perhaps
including evaluation of enrichment in the heavier oxygen isotope [18 O] when that analysis
is available) should be collected to confirm the observations in this study.
ACKNOWLEDGMENTS
This study is funded by the AECOM Innovation Fund. We like to acknowledge Mr. John
Kim who is the AECOM project manager for AFP44 and provided data, background, and
support for this study.
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Venus Sadeghi, PhD, is a principal chemical engineer and leads the In Situ Bioremediation Technical Practice
Group at AECOM. She has a PhD in chemical engineering from University of California at Davis and has over 25
years of environmental consulting experience, with emphasis on innovative remediation practices and remedi-
ation of emerging contaminants such as 1,4-dioxane. Venus.Sadeghi@aecom.com 2020 L Street, Sacramento,
CA 95811.
Rebecca Mora is a senior technical leader at AECOM. She holds a BS in environmental engineering from the
University of Notre Dame and has over 18 years of consulting experience. She primarily focuses on innovative
remediation technologies for groundwater contaminated with chlorinated solvents and emerging contaminants.
Rebecca.Mora@aecom.com 999 W. Town and Country, Orange, CA 92868.
Priya Jacob is an environmental engineer (EIT) at AECOM. She holds an MS in environmental engineering
from Clemson University and has three years of consulting experience. She supports projects that focus on
remediation of groundwater and soils contaminated with petroleum hydrocarbons, chlorinated solvents, and
emerging contaminants. priya.jacob@aecom.com, 1000 Abernathy Road NE, Suite 900, Atlanta, GA 30328.
Sheau-Yun (Dora) Chiang, PhD, P.E., is director of emerging contaminants at AECOM. She holds a PhD
from Georgia Institute of Technology and over 15 years of consulting experience in the areas of contaminated
site investigation and remediation. She works collaboratively with senior management to assess and determine
the firm’s position in global contaminated site cleanup and closure. dora.chiang@aecom.com, 1360 Peachtree
Street NE, Atlanta, GA 30309.