ASTM D6469 standard guide for microbial contamination in fuels and fuel systems
6. Origins of Microbial Contamination
6.1 The high temperature characteristic of distillation and other refinery processes sterilize refinery stocks used in fuel blending. However, conditions in refinery tankage, transport systems, terminal tankage, and users' system tankage may lead to microbial contamination and possible biodeterioration.

6.2 In refinery tankage, water can condense and coalesce as product cools. Tank vents draw moisture from the outside atmosphere and may allow precipitation to enter the tank. Moreover, product withdrawal creates a partial vacuum that pulls pollen, dust, and other microbe-carrying particulates through tank vents. Consequently, refinery products tanks are the first stage of petroleum handling where significant microbial contamination can occur.

6.3 In transport by means of tanker or pipeline, additional water may be introduced by condensation. In contrast to pipelines, condensate is not the major source of additional water. Rather, inadequate cargo compartment stripping, use of water as false bottoms to facilitate complete cargo discharge, and other incidental, intentional water use provide substantial water to fuel tanks. Biofilms can form on tanker or pipeline surfaces where they entrain water, inorganic particles, and nutrients to support growth. Such growth can slough off and be carried to terminal and end user tankage (see 6.4). In terminal tanks, turnover rates may be a week or longer, allowing particulates (including biofilm flocs) to settle into the sludge and sediment zone before product is drawn from the tank. As turnover rates increase, the likelihood of drawing biomass with fuel also increases, due to reduced settling times. Population densities of less than two millions cells/mL will have no effect on fuel clarity. Consequently, contaminated fuel is rarely detected visually at the terminal rack.

6.4 End-user tank materials and configurations are varied, reflecting use applications that range from small reservoirs (< 3 L) on power appliances (chain-saws, mowers, and so forth) to large (> 4000 L) day tanks feeding major power generation and propulsion engines. Location (above or below ground) and proximity to the point of combustion will also vary. End-use tanks accumulate water and bioburden that can lead to engine failure through fuel starvation resulting from filter or feed line plugging, or both. Moreover, MIC may compromise fuel tank integrity, leading to leakage. Substantial water volumes may be introduced into fuel tanks intentionally. In some ships, water is used as ballast and may occupy greater than 80 % of the total tank volume. At some tank farms, a layer of water is used to reduce the risk of ground-water, contamination due to fuel leakage.

7. Occurrence and Impact
7.1 Microbes require water as well as nutrients. Consequently, they concentrate at sites within fuel systems where water accumulates (see Fig. 1).

7.1.1 Water is essential for microbial growth and proliferation. Even negligible traces of water are sufficient to support microbial populations.

7.1.2 Nutrients are divided into macro-nutrients and micro-nutrients. Carbon, hydrogen, oxygen, nitrogen, sulfur, and phosphorus (CHONSP) comprise the macro-nutrients, and most of these are readily available in fuels. Only phosphorous is likely to be growth limiting in most fuel systems. A variety of elements, including calcium, sodium, potassium, iron, magnesium, manganese, copper, cobalt, nickel, and other metals, are required in trace quantities. None of these elements is limiting in fuel systems. Fuel systems that provide both the requisite water and nutrients will support microbial growth and proliferation.

7.1.3 The rate of microbial growth increases with increasing temperature within the physiological range (temperature range within which growth occurs) of a given microorganism. Microbes are generally classified into three groups, based on their temperature preferences/requirements. Some microbes require low temperatures (<20°C). Others thrive in superheated environments (>100°C). However, the physiological range of the microbes most commonly recovered from fuel tanks is 0°C to 35°C, with growth optimal between 25°C and 35°C.

NOTE 1 - The risk of uncontrolled microbial contamination is generally greatest in tropical regions. However, in the absence of adequate house-keeping practices, microbial contamination problems can also occur in fuel systems located in cold climates.

7.1.4 Water pH is generally not a controlling factor in fuel systems. Most contaminant microbes can tolerate pH's ranging from 5.5 to 8.0. As with temperature, there are microbes that prefer acidic environments (some grow in the equivalent of 2N sulfuric acid) and others that grow in alkaline systems with pH > 11. Fuel tank bottom-water pH is usually between 6 and 9.

7.2 As water activity tends to be greatest at interface zones, this is where microbes are most likely to establish communities, or biofilms. Numbers of microbes within biofilms are typically orders or magnitude greater than elsewhere in fuel systems. Biofilms can form on tank overheads, at the bulk-fuel, bottom-water interface, and on all system surfaces.

7.2.1 Using fuel hydrocarbon vapors as their carbon source, microbes can colonize tank overheads, where condensation provides the necessary water activity. Biofilms on overheads generally look like slimy stalactites.

7.2.2 The biofilm that develops at the fuel-water interface (sometimes called the skinnogen layer because of its tough membranous characteristics) represents a unique micro-environment relative to either the overlying fuel or underlying water. Nutrients from both the overlying fuel and underlying water are concentrated in this third-phase.

7.2.3 Whereas a 1-mm thick biofilm on a tank wall may seem negligible, it is 100 times the thickness of most fungi, and 500 to 1000 times the longest dimension of most bacteria. This seemingly thin film provides a large reservoir for microbial activity. Within the biofilm micro-environment, conditions can be dramatically different from those in the bulk product.

7.2.4 The microbial ecology of biofilms is complex. Microbial consortia (communities) give the biofilm community characteristics that cannot be predicted from analysis of its individual members.

7.2.4.1 Biofilms are formed when early colonizers, or pioneers, secrete mucous-like biopolymers that protect cells from otherwise harsh environmental conditions.

7.2.4.2 These biopolymers trap nonpolymer producing microbes, that then become part of the biofilm community, and cations that act as ligands that strengthen biofilm structural integrity.

7.2.4.3 Aerobes and facultative anaerobes (bacteria that grow aerobically under oxic conditions and anaerobically under anoxic conditions) scavenge oxygen, creating conditions necessary for obligate anaerobes to grow and proliferate.

7.2.4.4 Some bacterial and fungal species produce biosurfactants that create invert emulsions, which in-turn make nonpolar fuel components available for use as food.

7.2.4.5 Microbes able to attack hydrocarbons directly excrete waste products that other consortium members use as food. The net effect is a change in pH, oxidation-reduction (or redox) potential, water activity, and nutrient composition that has little resemblance to the environment outside the biofilm.

7.2.4.6 The biofilm consortium acts like a complex bioreactor, causing several types of significant changes to the fuel and fuel system.

7.2.4.7 Biofilm communities are directly involved in MIC that can result in pinhole leaks in tanks and pipelines. The problem of MIC is a consequence of several microbial processes.

7.2.4.8 First, the heterogeneity of biofilm accumulation creates electropotential gradients between zones of covered and uncovered surfaces.

7.2.4.9 SRB and other anaerobes use the hydrogen ions, thereby depacifying the electrolytic cell and accelerating the corrosion reactions. The hydrogen sulfide generated by biological sulfate reduction sours the fuel, causing copper corrosion test (see Test Method D130) failure. Moreover, toxic hydrogen sulfide trapped within bottom sludge can be a safety hazard to personnel entering gas-freed tanks.

7.2.4.10 Microbes growing anaerobically produce low molecular weight organic acids (formate, acetate, lactate, pyruvate, and others). These acids accelerate the corrosion process by chemically etching the metal surface. There are data demonstrating that biofilm communities can deplasticize the polymers used in fiberglass synthesis. Such activity can result in catastrophic tank failure and is most likely to occur along the longitudinal centerline (the same place of the greatest frequency of MIC pinholes).

7.3 Biodeterioration shares many symptoms with nonbiological fuel deterioration processes. Without an adequate battery of tests, the root cause of a given fuel degradation problem may be misdiagnosed. The following paragraphs discuss symptoms caused by microorganisms. However, many of these symptoms may also be caused by nonbiological factors.

7.3.1 Biosurfactants facilitate water transport into the fuel phase and some fuel additive partitioning into the water phase. Other metabolites may accelerate fuel polymerization. Produced at concentrations that are difficult to detect against the complex chemistry of fuel components, these metabolites can have a significant deleterious effect on fuel stability. Although most of the change occurs within a few centimeters of the biofilm-fuel interface, product mixing can distribute metabolites throughout the fuel system.

7.3.2 The most commonly recognized symptom of microbial contamination is filter plugging. Two distinct mechanisms can cause this problem. When flocs of biomass are transported through the fuel system and are trapped in the filter medium, they can restrict flow. Direct observation of filters plugged by this mechanism reveals masses of slime on the filter element's external surfaces. Alternatively, microbial contaminants may colonize filter media. The biopolymers they produce within the filter medium's matrix eventually plug the filter.