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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.


Bernhardt and Diekmann (1991) conducted an early assessment of 1,4-dioxane

biodegradation and indicated that the isolated strains from forest soil or activated sludge
grown on 1,4-dioxane, tetrahydrofuran (THF), and other cyclic ethers could not be
enriched or isolated when 1,4-dioxane was used as the sole carbon substrate. Fam et al.
(2005) cultured a propanotroph from project sites in Salt Lake City, Utah, that can utilize
propane as a sole carbon source and aerobically degrade at least 10 milligrams per liter
(mg/L) of 1,4-dioxane within days. Mahendra and Alvarez-Cohen (2006) also verified the
cometabolic transformation of 1,4-dioxane for monooxygenase-expressing strains that
were induced with methane, propane, THF, or toluene. The kinetics of 1,4-dioxane
degradation and identification of the intermediates for cometabolic degradation of
1,4-dioxane by monooxygenase-containing bacteria were evaluated, and the results
indicated that the intermediates would not cause an accumulation of toxic degradation
products in the environment (Mahendra et al., 2007; Mahendra & Alvarez-Cohen, 2006).
In addition, Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on
THF and was found to degrade 1,4-dioxane after growth on sucrose, lactate, yeast

c ⃝ 2016 Wiley Periodicals, Inc.

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/rem.21500 115
In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

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
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

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environment; and, more recently, compound specific isotope analysis (CSIA).

Biodegradation biomarkers (i.e., dioxane monooxygenase [DXMO] and aldehyde
dehydrogenase [ALDH]) were recently developed to identify the presence and abundance
of 1,4-dioxane–degrading enzymes (Gedalanga et al., 2014).
This article documents an EMD-facilitated evaluation of a field pilot study in which
methane is being injected below the water table, approximately 100 feet below ground
surface (ft bgs), to stimulate cometabolic biodegradation of 1,4-dioxane. The
biostimulation results are compared to an adjacent pilot study in which methane is
generated biologically and anaerobically through injection of emulsified vegetable oil
(EVO) for enhanced reductive dechlorination (ERD) of chlorinated solvents in a plume
area where 1,4-dioxane is also present. The site setting (two side-by-side pilot studies)
provides a unique opportunity to look into the role of methane for biostimulating
1,4-dioxane biodegradation. Various EMDs were used to evaluate the biodegradation
characteristics of both pilot studies.


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.

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

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).

118 Remediation DOI: 10.1002/rem c ⃝ 2016 Wiley Periodicals, Inc.


Exhibit 2. ERD and methane biostimulation pilot study areas

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

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

Exhibit 3. Rationale of monitoring well selection for the study

Well ID Description of Well Locations Rationale of Well Selection

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
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

biostimulation, which was achieved by installation of in situ Submerged Oxygen Curtain

(iSOC R⃝ ; Tersus Environmental) units used to infuse adequate amounts of methane, air, and
nitrogen into groundwater. The iSOC R⃝ system has been operating continuously since
March 2015. Methane, air, and nitrogen are supplied by cylinders stored in cages
aboveground. Methane is injected for five days during the week, after which the lines are
purged with nitrogen, and air is injected over the weekend. The lines are again purged
with nitrogen before switching back to methane, and so on.
Monthly performance monitoring was conducted at performance monitoring wells
from March 2015 to June 2015 for CVOCs, 1,4-dioxane, and hexavalent chromium, then
the monitoring frequency was reduced to semiannual sampling after June 2015.
Exhibit 2 shows the locations of the performance monitoring wells and all injection
points installed at DP-03. It should be noted that 7 of 88 injection points were not
installed successfully due to the casing and grout failure during hydrofracturing.

Selection of Wells and EMDs for the Study

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

120 Remediation DOI: 10.1002/rem c ⃝ 2016 Wiley Periodicals, Inc.


Exhibit 4. Study well locations

metabolism, and ALDH catalyzes the metabolism of 1,4-dioxane breakdown products

(Gedalanga et al., 2014). Several groups of aerobic organisms can utilize primary
substrates such as methane, propane, or toluene and produce enzymes capable of
cometabolizing 1,4-dioxane (Mahendra & Alvarez-Cohen, 2006). These enzymes, for
which quantitative polymerase chain reaction (qPCR) primer sets have been developed,
include soluble methane monooxygenase (sMMO), propane monooxygenase (PPO), ring
hydroxylating toluene monooxygenase (RMO), and phenol hydroxylase (PHE).
Methane-oxidizing bacteria (MOB) can also be a useful qPCR target to evaluate the
potential for 1,4-dioxane biodegradation by methane oxidizers. Ethene monooxygenases
(EtnC and EtnE) have not been documented to biodegrade 1,4-dioxane, but their
presence and abundance were also monitored in this study.
CSIA for 1,4-dioxane is a recently commercialized EMD that can be utilized to
evaluate 1,4-dioxane degradation in environmental samples. CSIA is an analytical method
that measures the ratio of naturally occurring stable isotopes in environmental samples, as
changes in isotopic ratios occur due to breakage of bonds between atoms but not as a result
of physical processes, such as dilution and diffusion. As 1,4-dioxane degradation is thought
to involve cleavage of carbon–carbon bonds during metabolism or cometabolism, there is
expected to be enrichment of the heavier 13 C isotope and this can be measured by CSIA to
evaluate if 1,4-dioxane degradation has occurred. The current published 1,4-dioxane
degradation does not specifically document carbon–hydrogen bond cleavage; however, the
possibility cannot be ruled out completely. The method to measure the ratio of the heavier
hydrogen isotope (deuterium or 2 H) to hydrogen (1 H) for 1,4-dioxane was developed by
Pace Analytical laboratory in Pittsburgh, Pennsylvania (Pace), and the results are included
in this study to document potential 2 H/1 H (as well as 13 C/12 C) isotopic ratio changes due

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

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.

Field Sampling Methods

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

DNA Extraction and CENSUS qPCR Analysis

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

122 Remediation DOI: 10.1002/rem c ⃝ 2016 Wiley Periodicals, Inc.


(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).

Compound-Specific Isotope Analysis

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.


1,4-Dioxane Within approximately 12

months of starting the
Of the five wells sampled, well M-69 is located in the methane biostimulation pilot study methane biostimulation
area and wells M-98 and M-101 are located in the ERD pilot study area. These three wells pilot study (March 2015 to
have been routinely sampled as part of the basewide groundwater monitoring program March 2016), 1,4-dioxane
concentrations in well
(Exhibit 5). 1,4-Dioxane concentrations in well M-69 fluctuated and ranged from
M-69 decreased from
7,000 𝜇g/L to 2,220 𝜇g/L before the methane biostimulation pilot study, and
3,020 𝜇g/L in August 2014
1,4-dioxane concentrations were higher than CVOC concentrations. Within (baseline) to 873 𝜇g/L in
approximately 12 months of starting the methane biostimulation pilot study (March 2015 March 2016, or by about
to March 2016), 1,4-dioxane concentrations in well M-69 decreased from 3,020 𝜇g/L in 71 percent, representing
August 2014 (baseline) to 873 𝜇g/L in March 2016, or by about 71 percent, representing a degradation half-life of
a degradation half-life of approximately 314 days. approximately 314 days.
Historical 1,4-dioxane concentrations ranged from 52.3 𝜇g/L to 95.4 𝜇g/L in well
M-98 and ranged from 3 𝜇g/L to 16.2 𝜇g/L in well M-101 before the ERD pilot study.
CVOC concentrations were higher than 1,4-dioxane concentrations in these two wells.
For the ERD pilot study area, 1,4-dioxane concentrations have remained relatively stable
(95.4 𝜇g/L in August 2014 to 103 𝜇g/L in March 2016) at well M-98 and changed from
16.2 𝜇g/L in August 2014 to 3.1 𝜇g/L at well M-101. When comparing the post-ERD
injection data with historical data from March 2014 (with a concentration of 3 𝜇g/L),
1,4-dioxane concentrations are considered stable in well M-101.

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

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

Exhibit 5. Chemical data summary

Sample ID Analyte TCE 1,1-DCE 1,4-Dioxane

Study wells in the Methane Biostimulation Pilot Study Area

M-69 2/5/2010 890 160 NA
2/5/2010 910 180 NA
2/24/2011 270 240 NA
2/24/2011 1,200 260 4,500
8/4/2011 1,000 250 4,200
8/4/2011 510 270 NA
12/2/2011 1,300 290 7,000
12/2/2011 650 250 NA
7/12/2012 400 130 NA
7/12/2012 780 120 4,600
3/6/2013 771 136 2,340
8/26/2013 616 106 2,220
8/4/2014 1,520 272 3,020
Operation of Pilot Study in February 2015
3/9/2015 784 128 1,570
4/9/2015 964 166 1,340
5/11/2015 639 102 1,030
6/5/2015 851 138 1,100
8/18/2015 604 98.8 961
3/3/2016 503 69.3 873
M-93 2/18/2009 NA NA 5.40
6/4/2009 NA NA 8.60
Study wells in the ERD Pilot Study Area
M-98 12/2/2011 500 46 71
12/2/2011 370 48 NA
3/11/2013 404 29.5 52.3
8/26/2013 453 41.9 57.7
3/20/2014 460 35.7 80.6
8/4/2014 442 36.7 95.4
Operation of pilot study in February 2015
3/9/2015 317 29.2 86
4/9/2015 201 31.8 53.8
5/13/2015 338 42.4 94.5
6/5/2015 258 43.7 61.8
8/21/2015 333 38.7 121
3/8/2016 284 27.3 103
M-101 3/7/2013 277 10.6 4.8
3/20/2014 360 15.8 3
8/4/2014 331 12.4 16.2

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Exhibit 5. Continued

Sample ID Analyte TCE 1,1-DCE 1,4-Dioxane

Operation of pilot study in February 2015

3/18/2015 3.7 14 2.1
8/21/2015 234 15.6 3.7
3/2/2016 139 11.7 3.1
M-104 12/2/2011 NA NA ND
8/26/2013 NA NA 1.2
3/20/2014 NA NA 0.14

All concentrations are in micrograms per liter (𝜇g/L).

Abbreviations: 1,1-DCE, 1,1-dichloroethene; NA, not available or not analyzed; ND, not detected; TCE, trichloroethene.

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
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.

Presence and Abundance of Bacteria and Functional Genes

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

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

Exhibit 6. Geochemical data results: July 2016

Field Parameters Unit M-69 M-93 M-98 M-101 M-104

Specific conductivity mS/cm 1.533 0.713 1.079 1.038 0.823

DO mg/L 6.33 8.53 1.19 0.45 6.21
ORP mV 36.6 78.9 41.9 28 54.6
pH SU 7.35 7.4 7.03 6.61 7.24
Temperature ◦C 24.54 25.05 24.48 25.29 25.72
Turbidity NTU 0.9 1.4 2.9 11 5.4
Alkalinity mg/L 130 138 192 228 170
Total iron 𝜇g/L ND 481 1,890 808 622
Ferrous iron 𝜇g/L ND 0.59 ND 0.66 ND
Total manganese 𝜇g/L 33.9 63.6 461 2,310 124
Nitrate-nitrogen mg/L 8.9 14 10 1.5 5.5
Sodium (Na) 𝜇g/L 83,900 55,100 71,100 56,700 54,800
Sulfate (SO4 ) mg/L 460 130 260 160 150
Total organic carbon mg/L 3.1 ND ND ND ND
Phosphorous mg/L ND ND
Molybdenum 𝜇g/L ND ND ND ND ND
Acetylene 𝜇g/L ND ND ND ND ND
Methane ng/L ND ND 350 7,600 ND
Ethane 𝜇g/L ND ND 0.14 0.36 ND
Ethene 𝜇g/L ND ND 1.7 9.4 ND
Propane 𝜇g/L ND ND ND ND ND
Propene 𝜇g/L ND ND ND ND ND
n-Butane 𝜇g/L ND ND ND ND ND
iso-Butane 𝜇g/L ND ND ND ND ND

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

126 Remediation DOI: 10.1002/rem c ⃝ 2016 Wiley Periodicals, Inc.


Exhibit 7. Presence and abundance of bacterial and functional genes potentially responsible for 1,4-dioxane biodegradation

qPCR-DNA for
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

Note: The shaded table entries pertain to biotrap sampler results.

cells/mL in M-69 to 1.61E+03 cells/mL in M-101). Well M-104, considered the

background well, had the lowest sMMO abundance (3.24E+01 cells/mL). Biotrap R⃝
samplers in the methane biostimulation area confirmed MOB and sMMO abundance in
both wells M-69 and M-93 (Exhibit 7).
In the methane biostimulation area, methane was delivered into the subsurface
cyclically, as described earlier. Methane was not detected in well M-69, suggesting
methane may be consumed by the intrinsic methane-oxidizing bacteria faster than the
methane replenishment rate through injection. For the ERD pilot study area, methane was
detected in wells M-98 and M-101. Based on February 2009 methane concentration data
(data not shown), methane was not detected in well M-101 prior to ERD, indicating the
methane was generated due to ERD treatment in the study area. The ERD pilot study area
exhibited reducing conditions and low DO concentrations compared to wells M-69 or
M-93 (Exhibit 6). However MOB and sMMO have similar abundance in wells M-98 and
M-101 compared to wells M-69 and M-93. The MOB and sMMO data suggest these two
DNA targets are ubiquitous in the environment and can be abundant under both aerobic
and anoxic to anaerobic environments (Exhibit 8). This also suggests that commercially
available DNA targets MOB and sMMO are likely not good indicators for interpreting
1,4-dioxane biodegradation potential. However, they can provide basic information about
the presence of methane-oxidizing bacteria and related functional genes in the

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

Bacteria and Functional Gene Abundance

1.00E+05 9

DO Concentration (mg/L)
1.00E+04 7



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

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

128 Remediation DOI: 10.1002/rem c ⃝ 2016 Wiley Periodicals, Inc.


Exhibit 9. CSIA results for 1,4-dioxane

Well ID Sampling Date Pilot Study Area del13C del2H

M-69 7/13/2016 Methane biostimulation −30.34 −76.34

M-93 7/13/2016 Upgradient of methane −30.02 −83.66
M-98 7/19/2016 ERD −30.39 −99.24
M-101 7/13/2016 ERD −31.63 −103.68
M-104 7/13/2016 Sidegradient Not analyzed, 1,4-dioxane
(background) of ERD conc too low

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.


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

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

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.


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

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In Situ Methane-Enhanced Biostimulation Potential for 1,4-Dioxane Biodegradation

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.

132 Remediation DOI: 10.1002/rem c ⃝ 2016 Wiley Periodicals, Inc.