1. Figure 2. Left: Microbial cell counts from day -1 (pre CH4 injection) to day 253. The stable
or increasing cell numbers at 2 m and 4 m depth was indicative of an active
methanotrophic population. Combined effects of CH4 concentration (Figure 3) and
anoxic conditions possibly contributed to the sharp decline in cell numbers at 6 m. No
visible change was observed at 8 m depth.
Middle: Green enhanced fluorescent image from 2 m depth of various cell morphologies at
day -1.
Right: Day 253 sample image showing dominance of large coccoid cells. The morphology
and cell diameter is consistent with Methylococcaceae and support their relative
abundance in Figure 1 as a direct result of CH4 release.Figure 3. Contour plot by depth at 113 days and
245 days showing CH4 migration and
persistence in the aquifer. Dashed line; CH4
injection horizon, Blue arrow; groundwater flow
direction, Diamond in green box; monitoring
well for this study.
Figure1. Left: Domain level taxonomic distribution. The relative sequence abundance of
methane oxidizers (red), other Bacteria (blue) and Archaea (green) are displayed per depth.
Right: Relative sequence abundance of bacterial taxa in the monitoring well. CH4 was
injected from day 0 to day 72 (dashed line). Methylococcaceae are shown in red,
Methylophilaceae in orange, sulfate reducers in yellow. Other taxa possibly involved in the
cycling of sulfur and iron are shown in blue and brown, respectively.
Methane injection strongly and persistently disturbed indigenous microbial communities with
no sign of recovery after 254 days.
Microbial community response to
fugitive methane in groundwater
Olukayode Kuloyo1
* (olukayode.kuloyo@ucalgary.ca)
Collaborators: Emil Ruff1
, Bernhard Mayer1
, Aaron Cahill2
, Beth Parker2
, Colby Steelman2
, John Cherry2
, Ulrich
Mayer3
, Olenka Forde3
, Cathryn Ryan1
, Bethany Ladd1
, Sara Cho1
, and Marc Strous1
1
Department of Geoscience, University of Calgary, Canada
2
G360 Centre for Applied Groundwater Research, University of Guelph, Ontario, Canada
3
Department of Earth, Ocean and Atmospheric Science, University of British Colombia, British Colombia, Canada
Abstract
The expansion of unconventional natural gas extraction (e.g. from shale) has raised concerns over potential impacts of fugitive methane migration
on shallow groundwater. To provide direct insight into the effect of methane (CH4) and the microbial processes involved in CH4 removal from
contaminated aquifers, we performed metagenomic and microbiological analyses of groundwater from multi-level depths during a controlled
methane-release experiment at a research aquifer in Borden, Ontario.
 Methane (51,350 liters) was injected at 4.5 m
and 9 m depth into the aquifer over a 72-day
period
 Groundwater from multi-level monitoring wells
was sampled for geochemical and molecular
analyses over 254 days
 Samples were serial-filtered through 0.22 mμ
and 0.1 m membrane filters for DNAμ
extraction
 The V3-V4 region of 16S rRNA gene was
amplified and sequenced using Illumina Miseq
platform
 Microbial cells were visualized and enumerated
using DAPI stain under a fluorescent
microscope
Materials and Methods
Microbial community profile of groundwater monitoring well site
2 m
4 m
6 m
8 m
Depth
(m)
113 Days 245 Days
Horizontal Distance (m)
CH4
(mg/l)
 CH4 release caused microbial community shift
and strongly stimulated the growth of
methanotrophs (up to 64% relative abundance)
 Methane oxidation decreased with depth due
to oxygen-limitation. Anaerobic methanotrophs
were undetected.
 Low microbial cell numbers and persistent CH4
after 8 months suggested that methane
oxidation was relatively ineffective in the
aquifer.
 Metagenomic analyses are an important and
complementary tool to geochemical and
isotopic techniques for assessing the fate of
CH4 in contaminated shallow groundwater.
Key Findings
Microbial cell numbers
Methane Plume Persistence
Day -1 Day 253