Adenosine Triphosphate (ATP) is an excellent biomarker present in all living cells. During the past several years, several ATP test methods have been developed to overcome interferences that have historically made ATP testing of fuels impractical. This paper compares two of the new ATP test methods, presenting sensitivity and precision data for each method. Additionally, for one of the methods it examines the effect of fuel type on ATP test results. This method has a lower detection limit of 20 pg ATP/mL (approximately 100 bacterial cells/mL). Conventional gasoline, ethanol-gasoline blends, ultra-low-sulfur diesel and biodiesel blends carrying a range of bioburdens are examined.
At the 2007 TAE Fuels Colloquium  Passman and Eachus reviewed the importance of effective, real-time microbial contamination condition monitoring for fuels, and described a new adenosine triphosphate (ATP) test protocol that provided reliable ATP data from fuel and fuel-associated bottom-water samples. Subsequent field work using that method revealed that a key component of the test kit was incompatible with ethanol-blended fuels. An alternate method - currently in ballot as a new ASTM standard test method  circumvents the material compatibility issue with conventional fuels by extracting the fuel-phase biomass and other hydrophilic particles into an aqueous capture solution. However the protocol depends on extraction of fuel-phase biomass into an aqueous capture solution. This works well in conventional fuels, but adequate phase separation does not occur in high water-retaining fuels such as ethanol-blended gasoline. This paper describes an ATP test method that addresses the limitations of the two previous protocols.
Four 3,600 mL microcosms were set up in 3.8 L glass jars. The fuels used were: conventional 87 octane gasoline (87UNL), 87 octane gasoline with 10% (v/v) ETOH (87E10), ultra low sulfur diesel (ULSD) and ULSD with 20% (v/v) soy-derived B-100 biodiesel base stock (B-20). The fuel blends were prepared in-microcosm so that each microcosm contained 3,400 mL of the required blend. After preparing the fuel blends, 200 mL of contaminated-water was added to each microcosm. Microcosms were maintained at room temperature (19±2 °C). All tests were run within two-days after the microcosms were set up.
The contaminated-water inoculum (CWI) was prepared by inoculating commercial spring water with an uncharacterized commercial preparation of freeze-dried microbes (Rid-X, Reckitt Benckiser, Inc., Parsippany, NJ, USA) used to stimulate septic tank activity. To start a CWI preparation, 1 g of freeze dried material was dispersed into 1L of spring water. This preparation incubated at room temperature in the dark for two-weeks before being used to inoculate the microcosms.
To test ATP concentration, either a bottom-water (1.0 mL) or a fuel sample (25 mL) was drawn into a disposable syringe (5 mL for water samples; 60 mL for fuel samples). The sample was then filtered through an in-line 0.7 µm filter. The filter is removed from the syringe, the plunger is removed from the syringe's barrel, the filter is replaced and 5.0 mL of a proprietary cleaning agent (all reagents are from LuminUltra Technologies, Ltd., Fredericton, NB, Canada) is dispensed into the syringe barrel. The plunger is then reinserted into the syringe barrel and the cleaning agent is pressed through the filter.
A clean 60 mL syringe is then used to air dry the filter. The barrel is removed from the syringe, the in-line filter affixed to the syringe, the barrel is replaced and air is passed through the filter. This step is repeated two or three times, until the filter is dry. The air-dried filter is reaffixed to the original syringe, from which the barrel has again been removed. Then 1.0 mL of a proprietary lysing agent is dispensed into the syringe, the barrel is replaced and the fluid is pressure-filtered into a 17 x 100 mm culture tube. Next, 9.0 mL of dilution buffer is added to the culture tube. The tube is capped and shaken to mix its contents. A 100 µL portion of the diluted sample is transferred to a 12 x 55 mm culture tube to which 100 µL luciferase enzyme reagent has been added previously. The culture tube is swirled gently for 10 sec and placed into a luminometer. Data are in relative light units (RLU) which are converted to pg ATP/mL by comparison against data from an ATP standard.
For ULSD and B-100 bottom-waters, dilution series were run by two different analysts; each in triplicate. Sampled bottom water was diluted in bottled spring water: undiluted, 1:5, 1:10, 1:50 and 1:100.
LuminUltra 1.0 ng ATP/mL standard was used to calibrate the luminometer and compute Log10 pg ATP/mL from raw RLU data:
(1) Log10 pg ATP/mL = Log10 [(RLUsmpl ÷ RLUctrl) . (10,000 ÷ mL sample)]
where RLUsmpl was the RLU from the test sample and RLUctrl was the average of triplicate 1.0 ng ATP/mL control samples. The 10,000 value was derived from the 10-fold dilution of the extracted ATP times the conversion of ng to pg (1,000 pg/ng). To run the control test, 100 µL of 1.0 ng ATP/mL standard was dispensed into a reaction tube containing 100 µL luciferase enzyme reagent.
Dr. Frederick Passman, PhD is a Certified Metalworking Fluids Specialist with over 35 years experience in Environmental & Industrial Microbiology. His company, Biodeterioration Control Associates, Inc. (BCA) provides clients with unparalleled expertise in Microbial Contamination Control.
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