1. RESEARCH POSTER PRESENTATION DESIGN © 2011
www.PosterPresentations.com
ABSTRACT METHODS
Bright field light microscopy showed that PW9 was
more numerous in 10% NaCl media than 5%. Bright
white undissolved salt crystals were found (Figure 2).
RESULTS RESULTS
FUTURE DIRECTIONS
ACKNOWLEDGEMENTS
REFERENCES
Results
CONCLUSIONS
BACKGROUND
Figure 4.6: Phylogenetic tree of aligned sequencing results for the SWPA isolateFigure 4.6: Phylogenetic tree of aligned sequencing results for the SWPA isolate
Figure 4.6: Phylogenetic tree of aligned sequencing results for the SWPA isolate
•W.
Isolation and Characterization of an Antibiotic Resistant Bacterium
from Produced Water
Jenna Kuhn1 and John F Stolz1,2
1 Department of Biological Sciences
2 Center for Environmental Research and Education
Duquesne University, Pittsburgh PA 15282
Development of natural gas through unconventional
drilling and hydraulic fracking well fluid generates
production fluids as well as releases formation water.
These fluids are not sterile and microbial activity can
result in potentially harmful compounds as well as well
deterioration and decreased gas production. Produced
water from hydraulically fracked wells contains a
variety of microbes able to withstand a high total
dissolved salt concentration (TDS) environment. This
study focused on isolating a single strain by growing
cultures in high TDS liquid media and streak plating,
analyzing microbial morphology using light microscopy
and gram staining techniques, testing for antibiotic
resistance, and optimal growth conditions. Antibiotic
resistance testing found the strain to be sensitive to
erythromycin, but resistant to oxacillin and nalidixic
acid. The microbe grew optimally at 10.0 % NaCl, pH of
6, and 24oC.
Unconventional drilling methods are used to
extract oil and natural gas from source pools
underground, often within organic-rich rock1. A highly
pressurized solution containing high total dissolved
salts (TDS) known as frac fluid is pumped into less
permeable rock to release the gas within pores1. The
fluid contains salts, chlorides, metals, biocides,
propping agents such as silica sand, and various
chemical additives that could be potentially harmful for
the environment in the case of a pipe leak and
groundwater contamination2.
Some of the fluid is retrieved and stored in
impoundments, where it can be treated to remove
contaminants. However, the water is not completely
sterilized and contains a high level of microbial activity.
Biocides are often used to limit the growth of various
microorganisms within frack fluid to prevent
degradation of pipes2.
In previous studies (Joshi, 2013; Dugas, 2014)
two strains of microbes were isolated from samples of
impoundment water and flowback from gas well
operations in southwest Pennsylvania, LP-1. This study
focuses on the isolation and characterization of an
antibiotic resistant bacteria isolated from produced
water, identified as PW9.
Nikon Eclipse light microscope bright field images
revealed that the isolated PW9 microbe was a small,
approximately two µm in diameter, bean-shaped, thick
walled halophilic bacteria. The morphology of the
bacteria seemed to change depending on the salinity
concentration of the media (Figure 1). The high salt
environment could cause the bacteria to become
hypotonic to its environment and absorb more water.
The bacteria tend to group together and were more
numerous in the 10% NaCl media inoculated at 24° C.
Formulate media,
inoculated with
previously isolated
culture PW 9
• Verify growth using light microscopy
• Streak plate until culture is pure
• Gram stain procedure for characterization
• Control for gram stain was K-12 E. coli
Inoculate solid plate
media with PW 9
and complete
antibiotic testing
• Apply 3 disks to each plate: TE30, P10, S10,
AM10, OX1, E15, NA30, C30.
• Incubate at 37°C for at least 48 hrs.
• Measure and record zone of inhibition in mm.
• Use UV imager to take pictures of plates
Test growth of PW9
bacteria under
certain conditions
• Temperature of incubation at 4, 15, 24, 37,
and 65°C.
• Liquid media pH of 4, 5, 6, 8, and 10.
• NaCl (%) concentration of 0, 0.5, 1, 5, and
10%.
Measure cell growth
using
Spectrophotometer
at 600 nm.
• Test each condition at time zero
• Record cell growth vs. absorbance and plot in
Microsoft Excel
• Compare bar graphs to find most optimal
growth conditions
• Analyze liquid cultures under light microscopy
An aerobic halophilic bacterium was enriched on high
salt medium (“frac attack”) from a produced water
sample generated from an unconventional gas well. It
was isolated using streak plating. Designated PW9, it
was found to grow optimally in a high salt media
containing 10% NaCl, 20 mM lactate, at 6 pH and
incubated at 24 °C.
The bacteria is halophilic, bean shaped,
approximately 2 µm in diameter, with a thick outer cell
wall, and was unable to absorb a gram stain.
PW9 bacteria showed resistance to nalidixic acid
and oxacillin and sensitivity to erythromycin antibiotic
disks.
1) What is Unconventional Oil and Gas? Alberta Energy
Regulator (2015). https://www.aer.ca/about-aer/spotlight-
on/unconventional-regulatory-framework/what-is-
unconventional-oil-and-gas
2) Hydraulic Fracturing Fluids - Composition and Additives
Geology.com (2015). http://geology.com/energy/hydraulic-
fracturing-fluids/
I would like to thank Dr. Stolz, a professor of the Center
for Environmental Research and Education (CERE) for
mentoring my progress, and the Bayer School of Natural
and Environmental Sciences for funding this research.
Figure 1: Three images taken at 60 X of PW9 microbes in A) 0%
NaCl, B)5% NaCl and C) 10% NaCl. The morphology of the
bacteria changed with an increase in number and size of bacteria
with a greater salinity environment.http://fexoildrilling.com/
Antibiotic resistance was found when nalidixic acid
(NA30) and oxacillin (OX1) disks were applied. PW9
bacteria were highly sensitive to erythromycin (E15),
which caused the largest diameter of zone of inhibition
(Figure 3).
Figure 3: 8 solid “frac attack” media plates inoculated with PW9.
Resistance was found in plates A and B, which contain nalidixic acid
and oxacillin antibiotic disks respectively. In image C, PW 9 bacteria
were most sensitive to erythromycin. The following antibiotics were
also tested with a clear zone of inhibition: D) penicillin, E)
streptomycin, F) tetracycline, G) chloramphenicol, and H) ampicillin.
Liquid media inoculated with PW9 culture had the highest cell
count when incubated at 24°C, measured with
spectrophotometer readings (Table 1, Figure 3). Two trials of
the experiment were completed with similar, definitive results.
The pH range test did not lead to any significant results and
was inconclusive due to a large white formation of precipitate
and little growth. A blue-green color change for mid-pH and
the pH of test tubes increased over time of incubation.
Table 1: Test tubes containing 10 mL of liquid FA media were
incubated at varying temperatures. Cell count was measured using a
spectrophotometer. The lowest absorbance readings were found at
4°C, and highest at 24°C.
Temp (˚C) 4 15 24 37 65
1 0.048 0.039 0.368 0.095 0.117
2 0.045 0.063 0.16 0.086 0.104
3 0.044 0.056 0.597 0.157 0.036
4 0.047 0.056 0.416 0.196 0.044
5 0.047 0.06 0.235 0.106 0.068
6 0.046 0.061 0.16 0.17 0.042
Time 0 0.095 0.099 0.099 0.099 0.099
Median 0.0465 0.058 0.3015 0.1315 0.056
St. Dev. 0.001472 0.00870441 0.171249 0.045325 0.034559
Absorbance
Figure 3: Bar graph showing the results of incubation temperature
(°C) vs. Absorbance. For each trial, the incubation temperature of
24°C, resulted in the highest cell count.
The pH range experiment should be repeated to assess
for optimal growth conditions for PW9 bacteria in FA
media.
Biocide testing will be completed to test how
certain concentrations affect microbial growth. This
could help to reduce microbial degradation of pipes used
in the process of hydraulic fracking, possibly preventing
groundwater contamination.
Cell harvesting of the produced water PW9 strain
microbe on solid “frac attack” media will be done to
complete DNA extraction.
10 µm
10 µm
Figure 2: Both bright field light microscopy images taken at 60 X
comparing the microbial activity within A) 10 % NaCl and B) 5% NaCl
media.