Edward F. Askew PhD
easkew@askewscientific.org

Baseline Industries provided the support for
this study including the use of their
Greenlight® 930 to measure oxygen uptake
rate.



Biocides are used to control bacteria growth
in drilling water reuse.
The amount of Biocide used depends on the
amount of control desired by the driller.
Traditional American Petroleum Institute (API
RP 38) Bacteriostatic Time to Kill and NACE
TM0194 tests rely on traditional
microbiological methods to measure efficacy.



Toxicity: The degree to which something is
poisonous.
Inhibition: An inhibitor is a substance that
delays or slows the bacteria metabolism.
Therefore: A chemical may slow down the
biological treatment of a waste (inhibit) or
stop it all together (toxic).

Aerobic and Facultative Anaerobic bacteria
metabolize carbon, nitrogen, and sulfur
organic compounds and also perform redox
on inorganic compounds utilizing oxygen.




Follows “Standard Methods for the
Examination of Water and Wastewater, 22nd
Edition, Method 2710”
Utilizes bio-organisms from the drilling
process or those specified in API RP 38.
Measures rates of metabolism of affected
organisms.
Can be performed with instruments that are
thermostated and measure dissolved
oxygen.

GreenLight Vial with an internal oxygen
sensor and thermostatic cell holder(s)



Determine if the OUR can operate in a
fracking water matrix. (Synthetic Fracking
Water Matrix AOAC posters, 2013-2014)
Determine if the biocide (Glutaraldehyde)
efficacy on bacteria activity can be measured.
Develop a secondary calibration check on
biocide of interest (Glutaraldehyde).
Glutaraldehyde Calibration
545 nm
2.5
y = 0.0001x2 + 0.0049x
R² = 0.9987
Absorbance
2
1.5
1
0.5
0
0
20
40
60
80
Glutaraldehyde Concentration (mg/L)
100
120
Recommended Practice for
Biological Analysis of Subsurface
Injection Waters
Prepare Growth Media
Inoculate and Grow
Bacteria
(3 Cycles-TSB)
Inoculate Time to Kill
Samples
< 12 Hours
Count Bacteria by OUR

Develop the bacteria matrix as per API RP 38.
◦ Growth media
 Tryptic Soy Broth for 3 Cycle Growth, API RP 38 for TTK
Test.
◦ Bacteria.
 Pseudomonas (aeruginosa, not fluorescens).
 Bacillus cereus.

Run a calibration curve of time to threshold
vs. bacterial count by traditional methods.
◦ IDEXX Pseudalert®
◦ R-biompharm X-BC®
◦ GreenLight 930


Inoculate 100 mL samples of API RP 38 media
with set amounts of live bacteria.
Samples:
◦ Control
No Biocide
◦ Time to Kill Samples
Know amount of
Glutaraldehyde

Measure time to threshold.

Calculate % Bacterial Killed

Percent Killed = 1 
 Active Bacteria in TTK Sample  

  × 100
 Active Bacteria in ControlSample  
100%
100%
100%
100%
% Bacteria Killed
100%
100%
99%
90%
80%
70%
66%
60%
50%
40%
40%
30%
25%
20%
10%
0%
100
50
25
20
10
5
Glutaraldehyde (mg/L)
1
0.75
0.5
Field Monitoring of Bacterial
Growth in Oil and Gas Systems

Develop the bacteria matrix as per NACE
TM1094.
◦ Growth media
 Tryptic Soy Broth.
◦ Bacteria (Facultative anaerobes and aerobes).





E Coli
Pseudomonas aeruginosa
HPC
Bacillus cereus.
Run a calibration curve of time to threshold vs.
bacterial count by traditional methods.
◦ IDEXX Colilert® and Pseudalert®
◦ R-biompharm X-BC®
◦ GreenLight 930
GreenLight® Model 930 NACE TM1094 Calibration
12.00
10.00
y = -1.20x + 13.07
Average Time (Decimal Hrs.)
R² = 0.98
8.00
6.00
4.00
2.00
0.00
0
2
4
6
8
Log 10 of the CFU per 100 mL
10
12
% Bacteria Killed
45%
40%
38%
33%
35%
30%
25%
20%
15%
11%
10%
5%
0%
5 mg/L
1 mg/L
Glutaraldehyde
0.1 mg/L

OUR can provide a Time To Kill result that
mirrors the API RP 38 or NACE TM 0194 tests
with the following improvements:
◦ Results in 12 hours or less.
◦ Quantitative results obtained under GALP or ISO
17025 protocols.
◦ Test can be run by laboratory personnel or field
operational staff with minimal training.
Edward F. Askew PhD
easkew@askewscientific.org
Thanks Again for
Baseline Industries Support