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Three alternative, non-conventional test methods are evaluated for their ability to detect and quantify bioburdens in fuel and bottom-water samples. Two of the parameters, catalase activity and adenosine triphosphate (ATP) concentration have been used previously. This is the first report of the use of fluorescence polarization (FP) technology for fuel and fuel-associated water testing.

In this investigation, each parameter is assessed for repeatability and reproducibility. Covariance amongst the three non-conventional test method data is reported. Covariance between each of the non-conventional parameters and each of a variety of conventional parameters (viable bacteria and fungi enumeration data, fuel and bottom water chemistry) is also reported. Although each test method has limitations, the new methods reported in this paper may contribute significantly to fuel system microbial contamination condition monitoring and biodeterioration root cause analysis efforts.


Petroleum product biodeterioration was first reported in 18951. Most recently, Passman has reviewed the fundamentals of fuel and fuel system microbiology2. Uncontrolled microbial activity in fuel systems can cause product degradation and system damage. Passman et al. demonstrated substantial loss of oxygenate and n-alkanes from gasoline stored over microbially contaminated bottom-water3. Microbially influenced corrosion (MIC) of steel tanks, first described in 19454 and has been well documented subsequently5. More recently, Gu6 has described the biodeterioration of fiber reinforced polymers (FRP).

Detecting microbial contamination in fuel systems remains problematic. Samples collected routinely for fuel quality monitoring are generally ill-suited for microbiological testing7.

Moreover the most commonly used test method - viable recovery - suffers from several considerable limitations. Viable recovery methods depend on the ability of microbes collected from fuel or bottom-water samples to proliferate in or on a specific growth medium into or onto which they are suspended, spread or adsorbed. By definition, all growth media are selective. This means that only a fraction of viable microbes present in the original sample will proliferate in any given growth medium8. Although nominal time limitations are set for enumerating microbial growth in or on nutrient media (typically 48 to 72 hours for most bacteria and fungi; 1-week for sulfate reducing bacteria), substantial portions of the sampled system's indigenous population may require two or three times longer to become detectable (in broth media detection is based on fluid turbidity, color change or combination of both; on solid media detection is based on the formation of visible colonies). This time lag represents two problems. Under best conditions, there is a considerable delay between test initiation and data availability. Second, there is a substantial risk of population density underestimation. Failure to disperse individual cells from aggregates may also lead to significant population density underestimates. These limitations make a compelling case for alternative enumeration methods that reflect the total contaminant biomass, provide data speedily or accomplish both.

An ASTM document reviews the critical considerations necessarily considered when evaluating a new test method9. Optimally, any new method will generate data that covary with a previously accepted method. Additionally, the new microbiological method might covary with non-microbiological symptoms of biodeterioration. Finally, the new method should be reliable. It should have an adequate lower detection limit (LDL) and should not generate false-positive data.

With these considerations in mind, we evaluated three non-conventional test methods. The first method has a fifty-year history. With minor methodological improvements, adenosine triphosphate (ATP) has been used to quantify microbial biomass since the mid-1950's10. By the late 1960's, ATP had become an important tool for estimating microbial biomass in marine and other aquatic systems11, 12; 13. However, in complex fluids such as metalworking fluids, oilfield production water and fuel bottom-water, hydrocarbons and other non-ATP organic molecules interfered with the Luciferin-Luciferase bioluminescence reaction on which ATP quantification depended. Some molecules quenched (obscured) ATP-driven luminescence reaction. Other molecules auto-fluoresced, thereby causing positive interferences. These limitations continue to limit the ATP test's utility for fuel system biomass determination.

In 1997, Miller and Loomis were awarded a U.S. patent for a novel approach to eliminating certain ATP test interferences14; 15. The paper presents the results of a systematic evaluation of the applicability of the method taught my Miller and Loomis in these two patents.

Another cell constituent used to estimate bioburdens in environmental samples is lipopolysaccharide (LPS), also referred to as endotoxin. The LPS molecule is a characteristic component of Gram negative bacterial cell walls. In 1976, Levine and Bang16 reported that LPS caused the lysate of horseshoe crab (Limulus polyphemus) hemolymph (analogous to blood) to clot. Their test was called the Limulus lysate test after the horseshoe crab species from which the hemolymph was harvested. Shortly thereafter, Passman et al. used the Limulus lysate test to quantify microbial communities of the North Atlantic outer Continental Shelf17. During the past 30 years, the test has been improved and automated for use in a variety of applications18,18;20.

Recently, Sloyer et al.21 reported the use of an automated fluorescent method for estimating the numbers of viable bacteria in metalworking fluids. Based on the promising metalworking fluid data, the investigators evaluated the fluorescent method with fuel tank bottom-water samples. This paper presents the results of these tests.

Materials and Methods

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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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