1. Edward F. Askew PhD easkew@askewscientific.org

2.  Baseline Industries provided the support for this study including the use of their Greenlight® 930 to measure oxygen uptake rate.

3.  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.

5.  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).

6.  Aerobic and Facultative Anaerobic bacteria metabolize carbon, nitrogen, and sulfur organic compounds and also perform redox on inorganic compounds utilizing oxygen.

7.  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.

8.  GreenLight Vial with an internal oxygen sensor and thermostatic cell holder(s)

9.  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).

14. Recommended Practice for Biological Analysis of Subsurface Injection Waters

16.  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

18.  Inoculate 100 mL samples of API RP 38 media with set amounts of live bacteria.  Samples: ◦ ControlNo Biocide ◦ Time to Kill SamplesKnow amount of Glutaraldehyde  Measure time to threshold.  Calculate % Bacterial Killed

23. Field Monitoring of Bacterial Growth in Oil and Gas Systems

24.  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

27.  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.

28. Edward F. Askew PhD easkew@askewscientific.org Thanks Again for Baseline Industries Support