GTEC UST Management &Compliance Assistance Seminar August 22, 2019

1. GTEC UST Management &Compliance Assistance SeminarAugust 22, 2019 Fuel System Biological Test Kits How They Work Pro’s & Con’s Presented by Howard Chesneau Fuel Quality Services, Inc.

2. No one has a Biological problem in their fuel What they have is a water PROBLEM in their Fuel System NO WATER, NO LIFE !!! What are the Types of Microorganisms Associated with Fuel System Corrosion and Fuel Degradation?

3. Typical Fuel System Microorganisms Fungi Bacteria

4. Spores vs. Active Microbes Active microbes thrive and propagate in the water phase Spores are dormant and move with the fuel from up stream to down stream.

5. Microorganisms ALGAE Doesn’t Count Algae does not grow in fuel systems. Requires Sunlight “Photosynthesis”

6. Energy Source Fuel Interface (Bio surfactant) Biofilm & water Water Phase Factors That Promote Microbial Growth In Fuel Systems Can you SAY WATER!!!! Primarily Dormant Spores Active Microbes Remember this phrase- NO Water, NO Life !!!

7. Microorganisms In Water Droplets Active Microbes Emulsified Jet Fuel Created By Microbes Water droplets ~40μm diameter

8. Vertical Distribution of Microorganisms Greatest Population at Dead Bottom/Tank Floor

9. Take Aways • Bacteria and fungus contaminate fuel systems • Bacteria and fungus require water to survive and multiply. • Bacteria and fungus live in the water phase in or near the fuel water interface while feeding on the fuel for energy. • Microbes are in the water at the bottom of the fuel storage tank. They do not grow in the fuel but can move within it. • Algae does not grow in fuel system. Algae requires sunlight for photosynthesis to grow • NO WATER, NO LIFE !!!!!

10. Where do Microorganisms Come From and How do They Get into the Fuel?

11. 4. After refining fuel cools along the distribution system condensing water and microorganisms to form active pools/biofilms distributing microorganisms and their corrosive metabolites downstream to ASTs and USTs 5. Poor ballast stripping sends water and microbes with offloaded fuel. 6. Pipelines and storage tanks are contaminated from upstream product sources, Source and Distribution 3. Moisture and microbial spores enter refinery storage tank via ventilation, insufficient stay time to remove water and microbes. 2. Refining sterilizes the products 1. Microbes enter the crude from the field Fuel Storage Retail

12. Rising/falling ground water H20 contaminants into tank Some External Sources of Contaminationat the end user level Water and Microbial Contamination Steel Tank Institute: “Keeping Water Out of Your Storage System,” March 2004

13. Microbial Contamination of Fuel Storage and Distribution Systems • Microbial Contamination Pathways • Present in the environment soil • Vents – Air, Water, Dust • Tanks with Floating Roofs that Leak • Ships Ballast / Seepage Water • Transfer Piping • Cross Contamination Between Fuel System Tankage

14. Take Aways • Microbes are present in the environment around us. • They can enter a fuel system through a variety of pathways. • Poor site management can lead to microbial issues • NO WATER, NO LIFE!!!!

15. What is the Proper Sampling and Various Detection Techniques for Fuel Related Microorganisms

16. Detection of Microbial Activity • Microbial population in the water phase is significantly greater than in the fuel phase • Microbes live in the water layer • Not typically found active in fuel layer • Types of microbes • Bacteria & Fungus (mold & yeast) • Not Algae (requires sunlight for photosynthesis) • Various Detection Techniques • Results are only as good as the sample and technique

17. Typical Techniques for The detection of microbiologial’s in a fuel system

18. Laboratory Technique • 1 liter of fuel is filtered on a sterile disc and place on growth media. • Incubate in the range of 1-5 days for bacteria and fungi. • Test complies with IP 385 and ASTM D6974

19. Pros & Cons Pros • Done in a Sterile Environment by a professional • May provide a numerical number Cons • Samples Require special handling and shipping • Susceptible to cross contamination by dirty sample containers • Susceptible to samples changing during shipment time.

20. Dip Slides Nutrient Growth Media • Incubate in a range of 1-4 days for bacteria and fungi. • Semi-Quantitative results

21. Pros & Cons Pros Cons • Incubate in a range of 1-4 days for bacteria and fungi. • Semi-Quantitative results • May be done on site with minimally trained people • Dip slide only effective with water samples • Most dip slides do not work well with fuel samples very low probability of worthwhile results. • Fuel contamination of dip slide can ruin a dip slide test. • test kits have Approximately a six month shelf life

22. Liquid TestNutrient Growth Media • Inject the fuel / water sample into bottle with growth media and color indicator sensitive to pH changes. • Incubate sample 30-72 hrs. growth is reported as a power of 10 • Semi-Quantitative

23. Pros & Cons Pros Cons • Incubate in a range of 1-3 days for bacteria and fungi. • Semi-Quantitative results • May be done on site with minimally trained people • Requires the use of a syringe • Fuel additives can potentially produce false positive results.

24. Dehydrated Nutrient Growth Media • Can test both fuel or water samples. • Incubate sample 1- 3 days to express microbes colonies (cfu/Fuel or cfu/mlH20) • Semi-Quantitative Bacteria Yeast & Mold

25. Pros & Cons Pros Cons • Incubate in a range of 1-5 days for bacteria and fungi. • Semi-Quantitative results • May be done on site with minimally trained people • Requires pre-wetting with supplied sterile water when testing fuel only • Works better with water. Fuel has a tendency to skew the results unless carefully applied to pre-wetted pad • Fuel additives can potentially produce false positive results.

26. ATP Mg2+ AMP Firefly Luciferase CO2+ PPi O2 Luciferin Oxy-luciferin Light (λ) Rapid ATP Test Bioluminescence (all fuels)(ASTM D 7463) • Can test fuel, fuel/water mixtures and water only. • Test time 10 minutes. • One (1) microbial ATP molecule produces one (1) photon of light. • Number of Photons αre proportional to microbial ATP, Which is proportional to the number microbes in sample. • Complies with ASTM D7463, D7687

27. Pros & Cons Pros Cons • Easy to use • Test is fast -time approx. 10 minutes. • Quantitative results • One May be done on site in the field or in the office with minimally trained people. • Requires an instrument to read the results • The two vetted ASTM test methods are different and may require a different number of steps and training. One is field friendly the other is not. • Test does not differentiate between bacteria and fungus

28. New Test Kit for the Diesel Industry Previously only Marketed to the aviation Industry and approved for use by IATA (International Aviation Transport Association) Aviation version has an ASTM D-7978

31. Speed GelNutrient Growth Media • Can test both fuel or water samples. • Incubate sample 1-4 days to express microbes colonies (cfu/Lfuel or cfu/mlH20) • Semi-Quantitative and Quantitative. • Fuel additives can potentially produce false positive results. • Originally designated for the Aviation industry. Now a version available for the Diesel industry with a clear, action or danger designation.

32. Pros & Cons Pros Cons • Easy to use • Does not require special equipment to read the results • Work with fuel, water or fuel water mix • Easy to incubate in a range of 1-5 days for bacteria and fungi. • Semi-Quantitative results • May be done on site with minimally trained people • More reliable results than similar growth medias • Certain additives may influence results to give false positives. • More expensive than some others tests

33. Take Away • Microbes live in the water phase of the fuel storage tank not the fuel layer. • Fuel samples obtained for testing, the further from the water layer the less reliable the sample. • Usefulness of test information is dependent on sample quality and the limitations of the test kit. • These tests should be considered assays of either a problem or a potential problem. They are an indication of the presence of water!!!! Once again - No water, NO life!!!!

34. Questions? water biological testing