ASTM D6469-20

Standard Guide for Microbial Contamination in Fuels and Fuel Systems

SIGNIFICANCE AND USE
5.1 This guide provides information addressing the conditions that lead to fuel microbial contamination and biodegradation and the general characteristics of and strategies for controlling microbial contamination. It compliments and amplifies information provided in Practice D4418 on handling gas-turbine fuels. More detailed information may be found in Guidelines for the Investigation of Microbial Content of Liquid Fuels and for the Implementation of Avoidance and Remedial Strategies, 3rd Ed.,10 ASTM Manual 47, and Passman, 2019.11  
5.2 This guide focuses on microbial contamination in refined petroleum products and product handling systems. Uncontrolled microbial contamination in fuels and fuel systems remains a largely unrecognized but costly problem at all stages of the petroleum industry from crude oil production through fleet operations and consumer use. This guide introduces the fundamental concepts of fuel microbiology and biodeterioration control.  
5.3 This guide provides personnel who are responsible for fuel and fuel system stewardship with the background necessary to make informed decisions regarding the possible economic or safety, or both, impact of microbial contamination in their products or systems.
SCOPE
1.1 This guide provides personnel who have a limited microbiological background with an understanding of the symptoms, occurrence, and consequences of chronic microbial contamination. The guide also suggests means for detection and control of microbial contamination in fuels and fuel systems. This guide applies primarily to gasoline, aviation, boiler, industrial gas turbine, diesel, marine, furnace fuels and blend stocks (see Specifications D396, D910, D975, D1655, D2069, D2880, D3699, D4814, D6227, and D6751), and fuel systems. However, the principles discussed herein also apply generally to crude oil and all liquid petroleum fuels. ASTM Manual 472 provides a more detailed treatment of the concepts introduced in this guide; it also provides a compilation of all of the standards referenced herein that are not found in the Annual Book of ASTM Standards, Section Five on Petroleum Products and Lubricants.  
1.2 This guide is not a compilation of all of the concepts and terminology used by microbiologists, but it does provide a general understanding of microbial fuel contamination.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Aug-2020
• ICS: 27.060.10 - Liquid and solid fuel burners
• ICS: 43.060.40 - Fuel systems
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels

Relations

• Refers: ASTM D3326-07(2024) - Standard Practice for Preparation of Samples for Identification of Waterborne Oils
• Effective Date: 01-Apr-2024
• Refers: ASTM D445-24 - Standard Test Method for Kinematic Viscosity of Transparent and Opaque Liquids (and Calculation of Dynamic Viscosity)
• Effective Date: 01-Apr-2024
• Refers: ASTM D4412-19(2024) - Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• Effective Date: 01-Apr-2024
• Refers: ASTM D1655-24 - Standard Specification for Aviation Turbine Fuels
• Effective Date: 15-Mar-2024
• Refers: ASTM D6227-24 - Standard Specification for Unleaded Aviation Gasoline Containing a Non-hydrocarbon Component
• Effective Date: 01-Mar-2024
• Refers: ASTM D910-24 - Standard Specification for Leaded Aviation Gasolines
• Effective Date: 01-Mar-2024
• Refers: ASTM D3241-24 - Standard Test Method for Thermal Oxidation Stability of Aviation Turbine Fuels
• Effective Date: 01-Mar-2024
• Refers: ASTM D4814-24 - Standard Specification for Automotive Spark-Ignition Engine Fuel
• Effective Date: 01-Jan-2024
• Refers: ASTM D975-23a - Standard Specification for Diesel Fuel
• Effective Date: 15-Dec-2023
• Refers: ASTM D4814-23a - Standard Specification for Automotive Spark-Ignition Engine Fuel
• Effective Date: 01-Dec-2023
• Refers: ASTM D445-23 - Standard Test Method for Kinematic Viscosity of Transparent and Opaque Liquids (and Calculation of Dynamic Viscosity)
• Effective Date: 01-Nov-2023
• Refers: ASTM D3242-23 - Standard Test Method for Acidity in Aviation Turbine Fuel
• Effective Date: 01-Nov-2023
• Refers: ASTM D4012-23a - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
• Effective Date: 01-Nov-2023
• Refers: ASTM D1655-23a - Standard Specification for Aviation Turbine Fuels
• Effective Date: 01-Oct-2023
• Refers: ASTM D3241-23ae1 - Standard Test Method for Thermal Oxidation Stability of Aviation Turbine Fuels
• Effective Date: 01-Oct-2023

Overview

ASTM D6469-20: Standard Guide for Microbial Contamination in Fuels and Fuel Systems provides essential guidance for understanding, detecting, and controlling microbial contamination in a wide range of fuels and fuel systems. Developed by ASTM International, this standard is designed for personnel with limited backgrounds in microbiology, offering foundational knowledge on fuel microbiology, causes of fuel biodegradation, and contamination control strategies.

Microbial contamination is an underappreciated but significant issue affecting all stages of the petroleum industry, from crude oil production through storage, transportation, and end-use. Microbes such as bacteria and fungi can proliferate in the presence of water, leading to a variety of challenges including fuel deterioration, filter plugging, and system corrosion. The economic and safety implications make proper management and prevention vital for fuel quality and system reliability.

Key Topics

• Sources and Occurrence of Contamination

  • Microbes can enter fuels via condensation, atmospheric exposure, or contaminated water, especially at storage and handling interfaces.
  • Biofilms form where water and fuel interface, creating environments for further microbial proliferation and system damage.

• Symptoms and Consequences

  • Common indicators include filter and pipeline blockage, fuel phase turbidity, the presence of slimes or mats, fuel degradation, and unusual odors such as hydrogen sulfide.
  • Microbial contamination can affect physical properties like filterability, viscosity, and stability, potentially leading to engine failure or fuel system leaks.

• Detection and Testing

  • The standard discusses methods for sampling, gross observation, and laboratory testing as means to detect microbial presence.
  • Representative sampling, particularly at the fuel-water interface, is crucial.
  • Adopting a systematic approach to analysis, including both field and laboratory procedures, enables accurate diagnosis and response.

• Control and Prevention Strategies

  • Effective fuel system design to minimize water entry, regular water removal, and scheduled cleaning and inspection form the baseline for prevention.
  • Remediation techniques may include fuel filtration, treatment with biocides, removal and cleaning of tanks, and disposal of contaminated material.
  • Routine monitoring, risk assessment, and cost-effective implementation of control measures are strongly recommended.

Applications

ASTM D6469-20 applies to:

• Gasoline, diesel, furnace, aviation, marine, boiler, industrial gas turbine fuels, and their blend stocks
• Fuel storage and handling systems across the petroleum supply chain
• Crude oil and all liquid petroleum fuels, with relevance for storage terminals, distribution systems, fleet operations, and end-user fuel tanks

Practical users of this guide include:

• Fuel system operators
• Maintenance personnel in transportation, aviation, or marine industries
• Fuel quality assurance and laboratory staff
• Anyone responsible for fuel storage, handling, or stewardship

Employing this standard helps industries:

• Maintain fuel reliability and performance
• Reduce maintenance and corrosion-related costs
• Improve operational safety by minimizing risks of leakage or hazardous conditions
• Ensure compliance with quality and environmental regulations

Related Standards

Key referenced and associated standards include:

• ASTM D4418 - Practice for Receipt, Storage, and Handling of Fuels for Gas Turbines
• ASTM D396, D975, D1655, D2880, D3699, D4814, D6227, D6751 – Specifications for various fuel types
• ASTM D4057, D7464 – Practices for fuel sampling and sample handling
• ASTM D5452, D6217, D2068 – Test methods for particulate and microbial contamination
• ASTM Manual 47 – In-depth guidelines on fuel microbiology and control measures
• Energy Institute IP 385, IP 472 – International protocols for microbial content determination in fuels

For comprehensive fuel contamination management, refer to these documents alongside ASTM D6469-20 to ensure an integrated approach to fuel quality and microbial risk mitigation.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D6469 − 20
Standard Guide for
Microbial Contamination in Fuels and Fuel Systems
This standard is issued under the fixed designation D6469; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* 2. Referenced Documents
1.1 This guide provides personnel who have a limited 2.1 ASTM Standards:
microbiological background with an understanding of the D130Test Method for Corrosiveness to Copper from Petro-
leum Products by Copper Strip Test
symptoms, occurrence, and consequences of chronic microbial
contamination. The guide also suggests means for detection D396Specification for Fuel Oils
D445Test Method for Kinematic Viscosity of Transparent
and control of microbial contamination in fuels and fuel
systems. This guide applies primarily to gasoline, aviation, andOpaqueLiquids(andCalculationofDynamicViscos-
ity)
boiler, industrial gas turbine, diesel, marine, furnace fuels and
blend stocks (see Specifications D396, D910, D975, D1655, D515Test Method for Phosphorus In Water (Withdrawn
1997)
D2069, D2880, D3699, D4814, D6227, and D6751), and fuel
systems. However, the principles discussed herein also apply D664Test Method for Acid Number of Petroleum Products
by Potentiometric Titration
generally to crude oil and all liquid petroleum fuels. ASTM
Manual47 providesamoredetailedtreatmentoftheconcepts D888Test Methods for Dissolved Oxygen in Water
D910Specification for Leaded Aviation Gasolines
introducedinthisguide;italsoprovidesacompilationofallof
thestandardsreferencedhereinthatarenotfoundinthe Annual D974Test Method for Acid and Base Number by Color-
Book of ASTM Standards, Section Five on Petroleum Products Indicator Titration
and Lubricants. D975Specification for Diesel Fuel
D1067Test Methods for Acidity or Alkalinity of Water
1.2 Thisguideisnotacompilationofalloftheconceptsand
D1126Test Method for Hardness in Water
terminology used by microbiologists, but it does provide a
D1293Test Methods for pH of Water
general understanding of microbial fuel contamination.
D1298Test Method for Density, Relative Density, or API
1.3 The values stated in SI units are to be regarded as
Gravity of Crude Petroleum and Liquid Petroleum Prod-
standard. No other units of measurement are included in this
ucts by Hydrometer Method
standard.
D1331Test Methods for Surface and Interfacial Tension of
Solutions of Paints, Solvents, Solutions of Surface-Active
1.4 This standard does not purport to address all of the
Agents, and Related Materials
safety concerns, if any, associated with its use. It is the
D1426Test Methods for Ammonia Nitrogen In Water
responsibility of the user of this standard to establish appro-
D1655Specification for Aviation Turbine Fuels
priate safety, health, and environmental practices and deter-
D1744Test Method for Determination of Water in Liquid
mine the applicability of regulatory limitations prior to use.
Petroleum Products by Karl Fischer Reagent (Withdrawn
1.5 This international standard was developed in accor-
2016)
dance with internationally recognized principles on standard-
D1976Test Method for Elements in Water by Inductively-
ization established in the Decision on Principles for the
Coupled Plasma Atomic Emission Spectroscopy
Development of International Standards, Guides and Recom-
D2068Test Method for Determining Filter Blocking Ten-
mendations issued by the World Trade Organization Technical
dency
Barriers to Trade (TBT) Committee.
D2069Specification for Marine Fuels (Withdrawn 2003)
D2274TestMethodforOxidationStabilityofDistillateFuel
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom-
mittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Sept. 1, 2020. Published October 2020. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1999. Last previous edition approved in 2017 as D6469–17. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/D6469-20. the ASTM website.
2 4
MNL 47, Fuel and Fuel System Microbiology: Fundamentals, Diagnosis, and The last approved version of this historical standard is referenced on
Contamination Control, Passman, F. J., ed., ASTM International, 2003. www.astm.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6469 − 20
Oil (Accelerated Method) D7463TestMethodforAdenosineTriphosphate(ATP)Con-
D2276Test Method for Particulate Contaminant inAviation tent of Microorganisms in Fuel, Fuel/Water Mixtures, and
Fuel Associated Water
Fuel by Line Sampling
D2880Specification for Gas Turbine Fuel Oils D7464Practice for Manual Sampling of Liquid Fuels, As-
sociated Materials and Fuel System Components for
D3240Test Method for Undissolved Water In Aviation
Microbiological Testing
Turbine Fuels
D7687Test Method for Measurement of CellularAdenosine
D3241Test Method for Thermal Oxidation Stability of
Triphosphate in Fuel and Fuel-associated Water With
Aviation Turbine Fuels
Sample Concentration by Filtration
D3242Test Method for Acidity in Aviation Turbine Fuel
D7978TestMethodforDeterminationoftheViableAerobic
D3325Practice for Preservation of Waterborne Oil Samples
Microbial Content of Fuels and Associated Water—
D3326PracticeforPreparationofSamplesforIdentification
Thixotropic Gel Culture Method
of Waterborne Oils
D8070Test Method for Screening of Fuels and Fuel Asso-
D3328Test Methods for Comparison of Waterborne Petro-
ciated Aqueous Specimens for Microbial Contamination
leum Oils by Gas Chromatography
by Lateral Flow Immunoassay
D3414Test Method for Comparison of Waterborne Petro-
E177Practice for Use of the Terms Precision and Bias in
leum Oils by Infrared Spectroscopy (Withdrawn 2018)
ASTM Test Methods
D3699Specification for Kerosine
E1259Practice for Evaluation of Antimicrobials in Liquid
D3867Test Methods for Nitrite-Nitrate in Water
Fuels Boiling Below 390°C
D3870PracticeforEstablishingPerformanceCharacteristics
E1326Guide for Evaluating Non-culture Microbiological
for Colony Counting Methods in Microbiology (With-
4 Tests
drawn 2000)
2.2 Energy Institute Standards:
D4012TestMethodforAdenosineTriphosphate(ATP)Con-
IP385Determinationoftheviableaerobicmicrobialcontent
tent of Microorganisms in Water
of fuels and fuel components boiling below 390°C—
D4057Practice for Manual Sampling of Petroleum and
Filtration and culture method
Petroleum Products
IP472Determination of fungal fragment content of fuels
D4176TestMethodforFreeWaterandParticulateContami-
boiling below 390°C
nation in Distillate Fuels (Visual Inspection Procedures)
2.3 Government Standards:
D4412TestMethodsforSulfate-ReducingBacteriainWater
40 CFR 152Pesticide Registration and Classification Proce-
and Water-Formed Deposits
dures
D4418Practice for Receipt, Storage, and Handling of Fuels
2.4 Other Standards:
for Gas Turbines
Test Method 2540D Total Suspended Solids Dried at
D4454Test Method for Simultaneous Enumeration of Total
103–105°C
andRespiringBacteriainAquaticSystemsbyMicroscopy
98/8/ECBiocidal Products Directive
(Withdrawn 2015)
TPC Publication No. 3The role of bacteria in the corrosion
D4814Specification for Automotive Spark-Ignition Engine
of oil field equipment
Fuel
D4840Guide for Sample Chain-of-Custody Procedures 3. Terminology
D4860TestMethodforFreeWaterandParticulateContami-
3.1 Definitions:
nation in Middle Distillate Fuels (Clear and Bright Nu-
3.1.1 aerobe, n—an organism that requires oxygen to re-
merical Rating)
main metabolically active.
D4870Test Method for Determination of Total Sediment in
3.1.1.1 Discussion—Aerobes use oxygen as their terminal
Residual Fuels
electron acceptor in their primary energy-generating metabolic
D4952Test Method for Qualitative Analysis for Active
pathways. Aerobes require oxygen for survival, using aerobic
Sulfur Species in Fuels and Solvents (Doctor Test)
metabolic processes to generate energy for growth and sur-
D5304Test Method for Assessing Middle Distillate Fuel
vival.
Storage Stability by Oxygen Overpressure
3.1.2 aggressiveness index (A.I.), n—the value computed
D5452Test Method for Particulate Contamination in Avia-
fromthesumofthepH+logalkalinity+loghardnessofwater
tion Fuels by Laboratory Filtration
sample where both alkalinity and hardness are reported as
D6217TestMethodforParticulateContaminationinMiddle
milligram CaCO L.
Distillate Fuels by Laboratory Filtration
D6227Specification for Unleaded Aviation Gasoline Con-
Available from Energy Institute, 61 New Cavendish St., London, W1G 7AR,
taining a Non-hydrocarbon Component
U.K., http://www.energyinst.org.
D6426Test Method for Determining Filterability of Middle
Available from U.S. Government Printing Office, Superintendent of
Distillate Fuel Oils
Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.
Available from American Public Health Association, 800 I Street, NW
D6751Specification for Biodiesel Fuel Blend Stock (B100)
Washington, DC 20001.
for Middle Distillate Fuels
Official Journal of the European Communities, 24.4.98, L123/1–63(1998).
D6974Practice for Enumeration of Viable Bacteria and
Available from NACE International (NACE), 1440 South Creek Dr., Houston,
Fungi in Liquid Fuels—Filtration and Culture Procedures TX 77084-4906, http://www.nace.org.
D6469 − 20
3.1.2.1 Discussion—AsA.I.decreases,waterbecomesmore /cm )sample);whenusedforproductsderivedfromorganisms
corrosive. At A.I. ≥ 12, water is noncorrosive. At 10 ≤ A.I. < biomass is typically expressed in terms of mass (kg, MT, etc.)
12, water is moderately corrosive. At A.I. < 10, water is or volume (L, m , bbl, etc.).
strongly corrosive. 3.1.11.2 Discussion—Products of living organisms include
those materials produced directly by living organisms as
3.1.3 anaerobe, n—an organism that cannot grow or prolif-
metabolites (for example, ethanol, various carbohydrates and
erate in the presence of oxygen.
fatty acids), materials manufactured by processing living
3.1.3.1 Discussion—Anaerobes use molecules other than
organisms (for example, pellets manufactured by shredding
oxygen in their primary energy-generating metabolic
and pelletizing plant material) and materials produced by
pathways, such as sulfate, nitrate, ketones, and other high-
processing living organisms, their components or metabolites
energy organic molecules.Although anaerobes may survive in
(for example, transesterified oil; also called biodiesel).
thepresenceofoxygen,anaerobicgrowthtypicallyoccursonly
in an oxygen depleted environment.
3.1.12 biosurfactant, n—a biologically produced molecule
that acts as a soap or detergent.
3.1.4 anoxic, adj—oxygen free.
3.1.13 consortium (pl. consortia), n—microbial community
3.1.5 antimicrobial, n—see biocide.
comprised of more than one, species that exhibits properties
3.1.6 bacterium (pl. bacteria), n—a single cell microorgan-
not shown by individual community members.
ism characterized by the absence of defined intracellular
3.1.13.1 Discussion—Consortia often mediate biodeteriora-
membranes that define all higher life forms.
tion processes that individual taxa cannot.
3.1.6.1 Discussion—All bacteria are members of the bio-
3.1.14 depacifying, adj—the process of removing hydrogen
logical diverse kingdoms Prokaryota and Archaebacteriota.
ions (protons) from the cathodic surface of an electrolytic cell,
Individual taxa within these kingdoms are able to thrive in
thereby promoting continued electrolytic corrosion.
environments ranging from sub-zero temperatures, such as in
frozen foods and polar ice, to superheated waters in deep-sea
3.1.15 deplasticize, v—the process of breaking down poly-
thermal vents, and over the pH range <2.0 to >13.0. Potential
mers in plastics and similar materials, resulting in loss of the
food sources range from single carbon molecules (carbon
material’s structural integrity.
dioxideandmethane)tocomplexpolymers,includingplastics.
3.1.16 facultative anaerobe, n—amicroorganismcapableof
Oxygen requirements range from obligate anaerobes, which
growing in both oxic and anoxic environments.
die on contact with oxygen, to obligate aerobes, which die if
3.1.16.1 Discussion—Facultative anaerobes use oxygen
oxygen pressure falls below a species specific threshold.
when it is present, and use either organic or inorganic energy
3.1.7 bioburden, n—the level of microbial contamination
sources (nitrate, sulfate, and so forth) when oxygen is depleted
(biomass) in a system.
or absent.
3.1.7.1 Discussion—Typically,bioburdenisdefinedinterms
3.1.17 fungus (pl. fungi), n—single cell (yeasts) or filamen-
of either biomass or numbers of cells per unit volume or mass
tous (molds) microorganisms that share the property of having
or surface area material tested (g biomass/mL; g biomass/g;
the true intracellular membranes (organelles) that characterize
cells/mLsample, and so forth). The specific parameter used to
all higher life forms (Eukaryotes).
define bioburden depends on critical properties of the system
3.1.18 metabolite, n—a chemical substance produced by
evaluated and the investigator’s preferences.
any of the many complex chemical and physical processes
3.1.8 biocide, n—a poisonous substance that can kill living
involved in the maintenance of life.
organisms.
3.1.19 microbial activity test, n—any analytical procedure
3.1.8.1 Discussion—Biocides are further classified as bac-
designed to measure the rate or results of one or more
tericides (kill bacteria), fungicides (kill fungi), and microbio-
microorganism processes.
cides (kill both bacterial and fungi). They are also referred to
3.1.19.1 Discussion—Examples of microbial activity tests
as antimicrobials.
include loss or appearance of specific molecules or measuring
3.1.9 biodeterioration, n—the loss of commercial value or
the rate of change of parameters, such as acid number,
performance characteristics, or both, of a product (fuel) or
molecular weight distribution (carbon number distribution),
material (fuel system) through biological processes.
and specific gravity.
3.1.10 biofilm, n—a film or layer of microorganisms,
3.1.20 microbially influenced corrosion (MIC),
biopolymers,water,andentrainedorganicandinorganicdebris
n—corrosion that is enhanced by the action of microorganisms
that forms as a result of microbial growth and proliferation at
in the local environment.
phase interfaces (liquid-liquid, liquid-solid, liquid-gas, and
soforth) (synonym: skinnogen layer). 3.1.21 mold, n—form of fungal growth, characterized by
long strands of filaments (hyphae) and, under appropriate
3.1.11 biomass, n—biological material including any mate-
growth conditions, aerial, spore-bearing structures.
rial other than fossil fuels which is or was a living organism or
3.1.21.1 Discussion—In fluids, mold colonies typically ap-
component or product of a living organism.
pear as soft spheres; termed fisheyes.
3.1.11.1 Discussion—Inbiologyandenvironmentalscience,
biomassistypicallyexpressedasdensityofbiologicalmaterial 3.1.22 obligate aerobe, n—microorganism with an absolute
per unit sample volume, area, or mass (g biomass/g (or /mLor requirement for atmospheric oxygen in order to function.
D6469 − 20
3.1.22.1 Discussion—Obligate aerobes may survive periods for growth. Although bacteria and fungi can be present in the
inanoxicenvironmentsbutwillremaindormantuntilsufficient fuel phase, their growth and activity is restricted to the water
oxygen is present to support their activity. phase of fuel systems. The water phase includes volumes
ranging from trace (several µL) to bulk (>1 m ) accumulations
3.1.23 obligate anaerobe, n—microorganism that cannot
and water entrained within deposits that accumulate on system
function when atmospheric oxygen is present.
surfaces. Typically, fuel and system deterioration is caused by
3.1.23.1 Discussion—Obligate anaerobes may survive peri-
the net activity of complex microbial communities living
ods in oxic environments but remain dormant until conditions
within slimy layers called biofilms. Biofilms may be found on
become anoxic.
tankroofs,shells,atthefuel/waterinterface,andwithinbottom
3.1.24 oxic, adj—an environment with a sufficient partial
sludge/sediment. Section 7 provides greater detail.
pressure of oxygen to support aerobic growth.
4.3 Obtaining representative samples may be challenging.
3.1.25 shock treatment, n—the addition of an antimicrobial
Forbestresults,samplesshouldbecollectedfromtheinterface
agentsufficienttocauserapidandsubstantial(severalordersof
zones, especially the fuel/water interface, described in 4.2.
magnitude) reductions in number of living microbes in a fluid
Refer to Section 8 for more details.
or system receiving that concentration.
4.4 Sampleanalysisincludesgrossobservationsaswellasa
3.1.26 skinnogen, n—synonymous with biofilm.
battery of physical, chemical, and microbiological tests. Be-
3.1.26.1 Discussion—Generally applied to a biofilm formed
cause biodeterioration shares symptoms with other fuel and
at the fuel-water interface.
fuel-system degradation processes, it is critical to subject
3.1.27 sour, v—to increase the concentration of hydrogen
samples to a sufficient range of appropriate tests to permit
sulfide.
accurate root-cause diagnosis. Section 9 provides more infor-
3.1.28 sulfate reducing bacteria (SRB), pl., n—any bacteria
mation on examining and testing samples.
with the capability of reducing sulfate to sulfide.
4.5 Microbial contamination control requires a well de-
3.1.28.1 Discussion—The term SRB applies to representa-
signed strategy that considers system design, sampling and
tives from a variety of bacterial taxa that share the common
analysis, and preventive and remedial treatment. See Section
= =
feature of sulfate reduction (SO to S ). SRB are major
11 for details.
contributors to MIC.
4.5.1 Goodsystemdesignminimizescontaminantentryand
3.1.29 taxa, pl., n—the units of classification of organisms,
provides for adequate sampling, water removal, and periodic
based on their relative similarities.
cleaning and inspection.
3.1.29.1 Discussion—Each taxonomic unit (group of organ-
4.5.2 Effective monitoring programs cost-effectively bal-
isms with greatest number of similarities) is assigned, begin-
ance biodeterioration risks with sampling and analytical costs.
ningwiththemostinclusivetokingdom,division,class,order,
4.5.3 Remedial efforts may include fuel filtration,
family, genus, and species. Bacteria and fungi are often further
reconditioning, disposal, biocide treatment, or tank/system
classified by strain and biovariation.
cleaning, or combination thereof. Health, safety, and environ-
3.1.30 viable titer, n—thenumberoflivingmicrobespresent mental considerations are critical to proper tank remediation.
per unit volume, mass, or area.
5. Significance and Use
3.1.30.1 Discussion—Viable titer is reported in terms of
either colony forming units (CFU) or most probable number
5.1 This guide provides information addressing the condi-
(MPN) per millilitre, milligram, or centimetre squared.
tions that lead to fuel microbial contamination and biodegra-
dation and the general characteristics of and strategies for
4. Summary controlling microbial contamination. It compliments and am-
plifies information provided in Practice D4418 on handling
4.1 Microbes may be introduced into fuels as products cool
gas-turbine fuels. More detailed information may be found in
inrefinerytanks.Bacteriaandfungiarecarriedalongwithdust
GuidelinesfortheInvestigationofMicrobialContentofLiquid
particles and water droplets through tank vents. In seawater
Fuels and for the Implementation of Avoidance and Remedial
ballasted tanks, microbes are transported with the ballast.
rd 10 11
Strategies, 3 Ed., ASTM Manual 47, and Passman, 2019.
Vessel compartments ballasted with fresh, brackish, or
seawater, all of which may contain substantial numbers of 5.2 This guide focuses on microbial contamination in re-
microbes, may easily become contaminated with the microbes
fined petroleum products and product handling systems. Un-
transported with the ballast water. See Section 6 for more a controlled microbial contamination in fuels and fuel systems
detailed discussion.
remainsalargelyunrecognizedbutcostlyproblematallstages
of the petroleum industry from crude oil production through
4.2 Afterarrivinginfueltanks,microbesmayeitherstickto
overhead surfaces or settle through the product. Some mi-
crobes will adhere to tank walls, whereas others will settle to 10
Available from Energy Institute, 61 New Cavendish St., London, W1G 7AR,
the fuel/water interface. Most growth and activity takes place
U.K., http://www.energyinst.org.
Passman, F. J. “Chapter 35 – Biodeterioration.” In: G. Totten, R. Shah and D.
where fuel and water meet. The tank bottom fuel/water
Forester, Eds. ASTM Manual 37 – Fuels and Lubricants Handbook: Technology,
interface is the most obvious fuel/water boundary. However,
Properties, Performance, and Testing, 2nd Edition, ASTM International, West
there is also a considerable area of fuel/water interface on the
Conshohocken, www.astm.org, pp: 1231-1273 (2018). DOI: 10.1520/
interior surface of tank-shells. Microorganisms require water MNL3720150021.
D6469 − 20
fleet operations and consumer use. This guide introduces the 7. Occurrence and Impact
fundamental concepts of fuel microbiology and biodeteriora-
7.1 Microbes require water as well as nutrients.
tion control.
Consequently, they concentrate at sites within fuel systems
where water accumulates (see Fig. 1).
5.3 This guide provides personnel who are responsible for
7.1.1 Water is essential for microbial growth and prolifera-
fuel and fuel system stewardship with the background neces-
tion. Even negligible traces of water are sufficient to support
sary to make informed decisions regarding the possible eco-
microbial populations.
nomic or safety, or both, impact of microbial contamination in
7.1.2 Nutrients are divided into macro-nutrients and micro-
their products or systems.
nutrients. Carbon, hydrogen, oxygen, nitrogen, sulfur, and
phosphorus (CHONSP) comprise the macro-nutrients, and
6. Origins of Microbial Contamination
most of these are readily available in fuels. Only phosphorous
6.1 The high temperature characteristic of distillation and
is likely to be growth limiting in most fuel systems.Avariety
other refinery processes sterilize refinery stocks used in fuel
of elements, including calcium, sodium, potassium, iron,
blending. However, conditions in refinery tankage, transport
magnesium, manganese, copper, cobalt, nickel, and other
systems,terminaltankage,andusers’systemtankagemaylead
metals,arerequiredintracequantities.Noneoftheseelements
to microbial contamination and possible biodeterioration.
is limiting in fuel systems. Fuel systems that provide both the
requisitewaterandnutrientswillsupportmicrobialgrowthand
6.2 In refinery tankage, water can condense and coalesce as
proliferation.
product cools. Tank vents draw moisture from the outside
7.1.3 Therateofmicrobialgrowthincreaseswithincreasing
atmosphere and may allow precipitation to enter the tank.
temperature within the physiological range (temperature range
Moreover, product withdrawal creates a partial vacuum that
within which growth occurs) of a given microorganism.
pulls pollen, dust, and other microbe-carrying particulates
Microbes are generally classified into three groups, based on
through tank vents. Consequently, refinery products tanks are
their temperature preferences/requirements. Some microbes
the first stage of petroleum handling where significant micro-
require low temperatures (<20°C). Others thrive in super-
bial contamination can occur.
heated environments (>100°C). However, the physiological
6.3 In transport by means of tanker or pipeline, additional range of the microbes most commonly recovered from fuel
water may be introduced by condensation. In contrast to
tanksis0°Cto35°C,withgrowthoptimalbetween25°Cand
pipelines, condensate is not the major source of additional 35°C.
water. Rather, inadequate cargo compartment stripping, use of
NOTE 1—The risk of uncontrolled microbial contamination is generally
water as false bottoms to facilitate complete cargo discharge,
greatest in tropical regions. However, in the absence of adequate house-
and other incidental, intentional water use provide substantial
keeping practices, microbial contamination problems can also occur in
fuel systems located in cold climates.
water to fuel tanks. Biofilms can form on tanker or pipeline
surfaces where they entrain water, inorganic particles, and
nutrientstosupportgrowth.Suchgrowthcansloughoffandbe
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
turnoverratesincrease,thelikelihoodofdrawingbiomasswith
fuel also increases, due to reduced settling times. Population
densities of less than two million 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
(<3L)onpowerappliances(chain-saws,mowers,andsoforth)
to large (>4000L) day tanks feeding major power generation
andpropulsionengines.Location(aboveorbelowground)and
(a) = refinery distillation towers
proximity to the point of combustion will also vary. End-use
(b) = refinery product tanks
tanks accumulate water and bioburden that can lead to engine
(c) = fueltransportationpipeline(lowpointsinpipelinetrap
failure through fuel starvation resulting from filter or feed line
water)
plugging, or both. Moreover, MIC may compromise fuel tank
(d) = distribution terminal tanks
integrity,leadingtoleakage.Substantialwatervolumesmaybe
(e) = commercial dispensing rack and tank truck
introducedintofueltanksintentionally.Insomeships,wateris
(f) = retail/fleet underground storage tank
used as ballast and may occupy greater than 80% of the total
(g) = retail/fleet dispensing system; arrows indicate sites
tank volume. At some tank farms, a layer of water is used to
where water and biologicals tend to accumulate
reduce the risk of ground water, contamination due to fuel
leakage. FIG. 1 Fuel Distribution System
D6469 − 20
7.1.4 Water pH is generally not a controlling factor in fuel 7.2.4.8 First, the heterogeneity of biofilm accumulation
systems.MostcontaminantmicrobescantoleratepH’sranging creates electropotential gradients between zones of covered
from 5.5 to 8.0. As with temperature, there are microbes that and uncovered surfaces.
preferacidicenvironments(somegrowintheequivalentof2N 7.2.4.9 SRB and other anaerobes use the hydrogen ions,
sulfuricacid)andothersthatgrowinalkalinesystemswithpH thereby depacifying the electrolytic cell and accelerating the
>11. Fuel tank bottom-water pH is usually between 6 and 9. corrosion reactions (TPC Publication No. 3). The hydrogen
sulfidegeneratedbybiologicalsulfatereductionsoursthefuel,
7.2 As water activity tends to be greatest at interface zones,
causing copper corrosion test (see Test Method D130) failure.
this is where microbes are most likely to establish
Moreover,toxichydrogensulfidetrappedwithinbottomsludge
communities, or biofilms. Numbers of microbes within bio-
can be a safety hazard to personnel entering gas-freed tanks.
films are typically orders or magnitude greater than elsewhere
7.2.4.10 Microbes growing anaerobically produce low mo-
in fuel systems. Biofilms can form on tank overheads, at the
lecular weight organic acids (formate, acetate, lactate,
bulk-fuel, bottom-water interface, and on all system surfaces.
pyruvate, and others). These acids accelerate the corrosion
7.2.1 Using fuel hydrocarbon vapors as their carbon source,
processbychemicallyetchingthemetalsurface.Therearedata
microbes can colonize tank overheads, where condensation
demonstrating that biofilm communities can deplasticize the
provides the necessary water activity. Biofilms on overheads
polymers used in fiberglass synthesis. Such activity can result
generally look like slimy stalactites.
incatastrophictankfailureandismostlikelytooccuralongthe
7.2.2 The biofilm that develops at the fuel-water interface
longitudinal centerline (the same place of the greatest fre-
(sometimes called the skinnogen layer because of its tough
quency of MIC pinholes).
membranous characteristics) represents a unique micro-
7.3 Biodeterioration shares many symptoms with nonbio-
environment relative to either the overlying fuel or underlying
logical fuel deterioration processes. Without an adequate bat-
water. Nutrients from both the overlying fuel and underlying
teryoftests,therootcauseofagivenfueldegradationproblem
water are concentrated in this third-phase.
maybemisdiagnosed.Thefollowingparagraphsdiscusssymp-
7.2.3 Whereas a 1mm thick biofilm on a tank wall may
toms caused by microorganisms. However, many of these
seemnegligible,itis100timesthethicknessofmostfungi,and
symptoms may also be caused by nonbiological factors.
500to1000timesthelongestdimensionofmostbacteria.This
7.3.1 Biosurfactants facilitate water transport into the fuel
seemingly thin film provides a large reservoir for microbial
phase and some fuel additive partitioning into the water phase.
activity.Within the biofilm micro-environment, conditions can
Other metabolites may accelerate fuel polymerization. Pro-
be dramatically different from those in the bulk product.
duced at concentrations that are difficult to detect against the
7.2.4 The microbial ecology of biofilms is complex. Micro-
complex chemistry of fuel components, these metabolites can
bial consortia (communities) give the biofilm community
have a significant deleterious effect on fuel stability.Although
characteristics that cannot be predicted from analysis of its
most of the change occurs within a few centimeters of the
individual members.
biofilm-fuel interface, product mixing can distribute metabo-
7.2.4.1 Biofilms are formed when early colonizers, or
lites throughout the fuel system.
pioneers, secrete mucous-like biopolymers that protect cells
7.3.2 The most commonly recognized symptom of micro-
from otherwise harsh environmental conditions.
bial contamination is filter plugging. Two distinct mechanisms
7.2.4.2 These biopolymers trap nonpolymer producing
can cause this problem.When flocs of biomass are transported
microbes,thatthenbecomepartofthebiofilmcommunity,and
through the fuel system and are trapped in the filter medium,
cations that act as ligands that strengthen biofilm structural
they can restrict flow. Direct observation of filters plugged by
integrity.
this mechanism reveals masses of slime on the filter element’s
7.2.4.3 Aerobes and facultative anaerobes (bacteria that
external surfaces. Alternatively, microbial contaminants may
grow aerobically under oxic conditions and anaerobically
colonizefiltermedia.Thebiopolymerstheyproducewithinthe
underanoxicconditions)scavengeoxygen,creatingconditions
filter medium’s matrix eventually plug the filter.
necessary for obligate anaerobes to grow and proliferate.
7.2.4.4 Some bacterial and fungal species produce biosur-
8. Sampling
factants that create invert emulsions, which in-turn make
nonpolar fuel components available for use as food. 8.1 Bottom samples, as described in Practices D4057 and
7.2.4.5 Microbes able to attack hydrocarbons directly ex- D7464, provide the best material for evaluating microbial
crete waste products that other consortium members use as contamination. Practice D7464 provides guidance specific to
food. The net effect is a change in pH, oxidation-reduction (o
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D6469 − 17 D6469 − 20
Standard Guide for
Microbial Contamination in Fuels and Fuel Systems
This standard is issued under the fixed designation D6469; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope*
1.1 This guide provides personnel who have a limited microbiological background with an understanding of the symptoms,
occurrence, and consequences of chronic microbial contamination. The guide also suggests means for detection and control of
microbial contamination in fuels and fuel systems. This guide applies primarily to gasoline, aviation, boiler, industrial gas turbine,
diesel, marine, furnace fuels and blend stocks (see Specifications D396, D910, D975, D1655, D2069, D2880, D3699, D4814,
D6227, and D6751), and fuel systems. However, the principles discussed herein also apply generally to crude oil and all liquid
petroleum fuels. ASTM Manual 47 provides a more detailed treatment of the concepts introduced in this guide; it also provides
a compilation of all of the standards referenced herein that are not found in the Annual Book of ASTM Standards, Section Five on
Petroleum Products and Lubricants.
1.2 This guide is not a compilation of all of the concepts and terminology used by microbiologists, but it does provide a general
understanding of microbial fuel contamination.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D130 Test Method for Corrosiveness to Copper from Petroleum Products by Copper Strip Test
D396 Specification for Fuel Oils
D445 Test Method for Kinematic Viscosity of Transparent and Opaque Liquids (and Calculation of Dynamic Viscosity)
D515 Test Method for Phosphorus In Water (Withdrawn 1997)
D664 Test Method for Acid Number of Petroleum Products by Potentiometric Titration
D888 Test Methods for Dissolved Oxygen in Water
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcommittee
D02.14 on on Stability, Cleanliness and Compatibility of Liquid Fuels.
Current edition approved June 1, 2017Sept. 1, 2020. Published July 2017October 2020. Originally approved in 1999. Last previous edition approved in 20142017 as
D6469 – 14.D6469 – 17. DOI: 10.1520/D6469-17.10.1520/D6469-20.
MNL 47, Fuel and Fuel System Microbiology: Fundamentals, Diagnosis, and Contamination Control, Passman, F. J., ed., ASTM International, 2003.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6469 − 20
D910 Specification for Leaded Aviation Gasolines
D974 Test Method for Acid and Base Number by Color-Indicator Titration
D975 Specification for Diesel Fuel
D1067 Test Methods for Acidity or Alkalinity of Water
D1126 Test Method for Hardness in Water
D1293 Test Methods for pH of Water
D1298 Test Method for Density, Relative Density, or API Gravity of Crude Petroleum and Liquid Petroleum Products by
Hydrometer Method
D1331 Test Methods for Surface and Interfacial Tension of Solutions of Paints, Solvents, Solutions of Surface-Active Agents,
and Related Materials
D1426 Test Methods for Ammonia Nitrogen In Water
D1655 Specification for Aviation Turbine Fuels
D1744 Test Method for Determination of Water in Liquid Petroleum Products by Karl Fischer Reagent (Withdrawn 2016)
D1976 Test Method for Elements in Water by Inductively-Coupled Plasma Atomic Emission Spectroscopy
D2068 Test Method for Determining Filter Blocking Tendency
D2069 Specification for Marine Fuels (Withdrawn 2003)
D2274 Test Method for Oxidation Stability of Distillate Fuel Oil (Accelerated Method)
D2276 Test Method for Particulate Contaminant in Aviation Fuel by Line Sampling
D2880 Specification for Gas Turbine Fuel Oils
D3240 Test Method for Undissolved Water In Aviation Turbine Fuels
D3241 Test Method for Thermal Oxidation Stability of Aviation Turbine Fuels
D3242 Test Method for Acidity in Aviation Turbine Fuel
D3325 Practice for Preservation of Waterborne Oil Samples
D3326 Practice for Preparation of Samples for Identification of Waterborne Oils
D3328 Test Methods for Comparison of Waterborne Petroleum Oils by Gas Chromatography
D3414 Test Method for Comparison of Waterborne Petroleum Oils by Infrared Spectroscopy (Withdrawn 2018)
D3699 Specification for Kerosine
D3867 Test Methods for Nitrite-Nitrate in Water
D3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)
D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
D4057 Practice for Manual Sampling of Petroleum and Petroleum Products
D4176 Test Method for Free Water and Particulate Contamination in Distillate Fuels (Visual Inspection Procedures)
D4412 Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
D4418 Practice for Receipt, Storage, and Handling of Fuels for Gas Turbines
D4454 Test Method for Simultaneous Enumeration of Total and Respiring Bacteria in Aquatic Systems by Microscopy
(Withdrawn 2015)
D4814 Specification for Automotive Spark-Ignition Engine Fuel
D4840 Guide for Sample Chain-of-Custody Procedures
D4860 Test Method for Free Water and Particulate Contamination in Middle Distillate Fuels (Clear and Bright Numerical
Rating)
D4870 Test Method for Determination of Total Sediment in Residual Fuels
D4952 Test Method for Qualitative Analysis for Active Sulfur Species in Fuels and Solvents (Doctor Test)
D5304 Test Method for Assessing Middle Distillate Fuel Storage Stability by Oxygen Overpressure
D5452 Test Method for Particulate Contamination in Aviation Fuels by Laboratory Filtration
D6217 Test Method for Particulate Contamination in Middle Distillate Fuels by Laboratory Filtration
D6227 Specification for Unleaded Aviation Gasoline Containing a Non-hydrocarbon Component
D6426 Test Method for Determining Filterability of Middle Distillate Fuel Oils
D6751 Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels
D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
D7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel
Associated Water
D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological
Testing
D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With Sample
Concentration by Filtration
D7978 Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated Water—Thixotropic Gel
Culture Method
D6469 − 20
D8070 Test Method for Screening of Fuels and Fuel Associated Aqueous Specimens for Microbial Contamination by Lateral
Flow Immunoassay
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E1259 Practice for Evaluation of Antimicrobials in Liquid Fuels Boiling Below 390 °C
E1326 Guide for Evaluating Non-culture Microbiological Tests
2.2 Energy Institute Standards:
IP 385 Determination of the viable aerobic microbial content of fuels and fuel components boiling below 390 °C—Filtration and
culture method
IP 472 Determination of fungal fragment content of fuels boiling below 390 °C
2.3 Government Standards:
40 CFR 152 Pesticide Registration and Classification Procedures
2.4 Other Standards:
Test Method 2540D Total Suspended Solids Dried at 103–105 °C
98/8/EC Biocidal Products Directive
TPC Publication No. 3 The role of bacteria in the corrosion of oil field equipment
3. Terminology
3.1 Definitions:
3.1.1 aerobe, n—an organism that requires oxygen to remain metabolically active.
3.1.1.1 Discussion—
Aerobes use oxygen as their terminal electron acceptor in their primary energy-generating metabolic pathways. Aerobes require
oxygen for survival, using aerobic metabolic processes to generate energy for growth and survival.
3.1.2 aggressiveness index (A.I.), n—the value computed from the sum of the pH + log alkalinity + log hardness of water sample
where both alkalinity and hardness are reported as milligram CaCO L.
3.1.2.1 Discussion—
As A.I. decreases, water becomes more corrosive. At A.I. ≥ 12, water is noncorrosive. At 10 ≤ A.I. < 12, water is moderately
corrosive. At A.I. < 10, water is strongly corrosive.
3.1.3 anaerobe, n—an organism that cannot grow or proliferate in the presence of oxygen.
3.1.3.1 Discussion—
Anaerobes use molecules other than oxygen in their primary energy-generating metabolic pathways, such as sulfate, nitrate,
ketones, and other high-energy organic molecules. Although anaerobes may survive in the presence of oxygen, anaerobic growth
typically occurs only in an oxygen depleted environment.
3.1.4 anoxic, adj—oxygen free.
3.1.5 antimicrobial, n—see biocide.
3.1.6 bacterium (pl. bacteria), n—a single cell microorganism characterized by the absence of defined intracellular membranes
that define all higher life forms.
3.1.6.1 Discussion—
All bacteria are members of the biological diverse kingdoms Prokaryota and Archaebacteriota. Individual taxa within these
kingdoms are able to thrive in environments ranging from sub-zero temperatures, such as in frozen foods and polar ice, to
superheated waters in deep-sea thermal vents, and over the pH range <2.0 to >13.0. Potential food sources range from single carbon
molecules (carbon dioxide and methane) to complex polymers, including plastics. Oxygen requirements range from obligate
anaerobes, which die on contact with oxygen, to obligate aerobes, which die if oxygen pressure falls below a species specific
threshold.
3.1.7 bioburden, n—the level of microbial contamination (biomass) in a system.
Available from Energy Institute, 61 New Cavendish St., London, W1G 7AR, U.K., http://www.energyinst.org.
Available from U.S. Government Printing Office, Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.
Available from American Public Health Association, 800 I Street, NW Washington, DC 20001.
Official Journal of the European Communities, 24.4.98, L123/1–63(1998).
Available from NACE International (NACE), 1440 South Creek Dr., Houston, TX 77084-4906, http://www.nace.org.
D6469 − 20
3.1.7.1 Discussion—
Typically, bioburden is defined in terms of either biomass or numbers of cells per unit volume or mass or surface area material
tested (g biomass/mL; g biomass/g; cells/mL sample, and so forth). The specific parameter used to define bioburden depends on
critical properties of the system evaluated and the investigator’s preferences.
3.1.8 biocide, n—a poisonous substance that can kill living organisms.
3.1.8.1 Discussion—
Biocides are further classified as bactericides (kill bacteria), fungicides (kill fungi), and microbiocides (kill both bacterial and
fungi). They are also referred to as antimicrobials.
3.1.9 biodeterioration, n—the loss of commercial value or performance characteristics, or both, of a product (fuel) or material (fuel
system) through biological processes.
3.1.10 biofilm, n—a film or layer of microorganisms, biopolymers, water, and entrained organic and inorganic debris that forms
as a result of microbial growth and proliferation at phase interfaces (liquid-liquid, liquid-solid, liquid-gas, and soforth) (synonym:
skinnogen layer).
3.1.11 biomass, n—biological material including any material other than fossil fuels which is or was a living organism or
component or product of a living organism.
3.1.11.1 Discussion—
In biology and environmental science, biomass is typically expressed as density of biological material per unit sample volume,
area, or mass (g biomass/g (or /mL or /cm ) sample); when used for products derived from organisms biomass is typically
expressed in terms of mass (kg, MT, etc.) or volume (L, m , bbl, etc.).
3.1.11.2 Discussion—
Products of living organisms include those materials produced directly by living organisms as metabolites (for example, ethanol,
various carbohydrates and fatty acids), materials manufactured by processing living organisms (for example, pellets manufactured
by shredding and pelletizing plant material) and materials produced by processing living organisms, their components or
metabolites (for example, transesterified oil; also called biodiesel).
3.1.12 biosurfactant, n—a biologically produced molecule that acts as a soap or detergent.
3.1.13 consortium (pl. consortia), n—microbial community comprised of more than one, species that exhibits properties not shown
by individual community members.
3.1.13.1 Discussion—
Consortia often mediate biodeterioration processes that individual taxa cannot.
3.1.14 depacifying, adj—the process of removing hydrogen ions (protons) from the cathodic surface of an electrolytic cell, thereby
promoting continued electrolytic corrosion.
3.1.15 deplasticize, v—the process of breaking down polymers in plastics and similar materials, resulting in loss of the material’s
structural integrity.
3.1.16 facultative anaerobe, n—a microorganism capable of growing in both oxic and anoxic environments.
3.1.16.1 Discussion—
Facultative anaerobes use oxygen when it is present, and use either organic or inorganic energy sources (nitrate, sulfate, and so
forth) when oxygen is depleted or absent.
3.1.17 fungus (pl. fungi), n—single cell (yeasts) or filamentous (molds) microorganisms that share the property of having the true
intracellular membranes (organelles) that characterize all higher life forms (Eukaryotes).
3.1.18 metabolite, n—a chemical substance produced by any of the many complex chemical and physical processes involved in
the maintenance of life.
3.1.19 microbial activity test, n—any analytical procedure designed to measure the rate or results of one or more microorganism
processes.
D6469 − 20
3.1.19.1 Discussion—
Examples of microbial activity tests include loss or appearance of specific molecules or measuring the rate of change of parameters,
such as acid number, molecular weight distribution (carbon number distribution), and specific gravity.
3.1.20 microbially influenced corrosion (MIC), n—corrosion that is enhanced by the action of microorganisms in the local
environment.
3.1.21 mold, n—form of fungal growth, characterized by long strands of filaments (hyphae) and, under appropriate growth
conditions, aerial, spore-bearing structures.
3.1.21.1 Discussion—
In fluids, mold colonies typically appear as soft spheres; termed fisheyes.
3.1.22 obligate aerobe, n—microorganism with an absolute requirement for atmospheric oxygen in order to function.
3.1.22.1 Discussion—
Obligate aerobes may survive periods in anoxic environments but will remain dormant until sufficient oxygen is present to support
their activity.
3.1.23 obligate anaerobe, n—microorganism that cannot function when atmospheric oxygen is present.
3.1.23.1 Discussion—
Obligate anaerobes may survive periods in oxic environments but remain dormant until conditions become anoxic.
3.1.24 oxic, adj—an environment with a sufficient partial pressure of oxygen to support aerobic growth.
3.1.25 shock treatment, n—the addition of an antimicrobial agent sufficient to cause rapid and substantial (several orders of
magnitude) reductions in number of living microbes in a fluid or system receiving that concentration.
3.1.26 skinnogen, n—synonymous with biofilm.
3.1.26.1 Discussion—
Generally applied to a biofilm formed at the fuel-water interface.
3.1.27 sour, v—to increase the concentration of hydrogen sulfide.
3.1.28 sulfate reducing bacteria (SRB), pl., n—any bacteria with the capability of reducing sulfate to sulfide.
3.1.28.1 Discussion—
=
The term SRB applies to representatives from a variety of bacterial taxa that share the common feature of sulfate reduction (SO
=
to S ). SRB are major contributors to MIC.
3.1.29 taxa, pl., n—the units of classification of organisms, based on their relative similarities.
3.1.29.1 Discussion—
Each taxonomic unit (group of organisms with greatest number of similarities) is assigned, beginning with the most inclusive to
kingdom, division, class, order, family, genus, and species. Bacteria and fungi are often further classified by strain and biovariation.
3.1.30 viable titer, n—the number of living microbes present per unit volume, mass, or area.
3.1.30.1 Discussion—
Viable titer is reported in terms of either colony forming units (CFU) or most probable number (MPN) per millilitre, milligram,
or centimetre squared.
4. Summary
4.1 Microbes may be introduced into fuels as products cool in refinery tanks. Bacteria and fungi are carried along with dust
particles and water droplets through tank vents. In seawater ballasted tanks, microbes are transported with the ballast. Vessel
compartments ballasted with fresh, brackish, or seawater, all of which may contain substantial numbers of microbes, may easily
become contaminated with the microbes transported with the ballast water. See Section 6 for more a detailed discussion.
4.2 After arriving in fuel tanks, microbes may either stick to overhead surfaces or settle through the product. Some microbes will
D6469 − 20
adhere to tank walls, whereas others will settle to the fuel/water interface. Most growth and activity takes place where fuel and
water meet. The tank bottom fuel/water interface is the most obvious fuel/water boundary. However, there is also a considerable
area of fuel/water interface on the interior surface of tank-shells. Microorganisms require water for growth. Although bacteria and
fungi can be present in the fuel phase, their growth and activity is restricted to the water phase of fuel systems. The water phase
includes volumes ranging from trace (several μL) to bulk (>1 m ) accumulations and water entrained within deposits that
accumulate on system surfaces. Typically, fuel and system deterioration is caused by the net activity of complex microbial
communities living within slimy layers called biofilms. Biofilms may be found on tank roofs, shells, at the fuel/water interface,
and within bottom sludge/sediment. Section 7 provides greater detail.
4.3 Obtaining representative samples may be challenging. For best results, samples should be collected from the interface zones,
especially the fuel/water interface, described in 4.2. Refer to Section 8 for more details.
4.4 Sample analysis includes gross observations as well as a battery of physical, chemical, and microbiological tests. Because
biodeterioration shares symptoms with other fuel and fuel-system degradation processes, it is critical to subject samples to a
sufficient range of appropriate tests to permit accurate root-cause diagnosis. Section 9 provides more information on examining
and testing samples.
4.5 Microbial contamination control requires a well designed strategy that considers system design, sampling and analysis, and
preventive and remedial treatment. See Section 11 for details.
4.5.1 Good system design minimizes contaminant entry and provides for adequate sampling, water removal, and periodic cleaning
and inspection.
4.5.2 Effective monitoring programs cost-effectively balance biodeterioration risks with sampling and analytical costs.
4.5.3 Remedial efforts may include fuel filtration, reconditioning, disposal, biocide treatment, or tank/system cleaning, or
combination thereof. Health, safety, and environmental considerations are critical to proper tank remediation.
5. Significance and Use
5.1 This guide provides information addressing the conditions that lead to fuel microbial contamination and biodegradation and
the general characteristics of and strategies for controlling microbial contamination. It compliments and amplifies information
provided in Practice D4418 on handling gas-turbine fuels. More detailed information may be found in the IP Guidelines and in
Guidelines for the Investigation of Microbial Content of Liquid Fuels and for the Implementation of Avoidance and Remedial
rd 10 11
Strategies, 3 Ed., ASTM Manual 47.47, and Passman, 2019.
5.2 This guide focuses on microbial contamination in refined petroleum products and product handling systems. Uncontrolled
microbial contamination in fuels and fuel systems remains a largely unrecognized but costly problem at all stages of the petroleum
industry from crude oil production through fleet operations and consumer use. This guide introduces the fundamental concepts of
fuel microbiology and biodeterioration control.
5.3 This guide provides personnel who are responsible for fuel and fuel system stewardship with the background necessary to
make informed decisions regarding the possible economic or safety, or both, impact of microbial contamination in their products
or 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
Available from Energy Institute, 61 New Cavendish St., London, W1G 7AR, U.K., http://www.energyinst.org.
Passman, F. J. “Chapter 35 – Biodeterioration.” In: G. Totten, R. Shah and D. Forester, Eds. ASTM Manual 37 – Fuels and Lubricants Handbook: Technology, Properties,
Performance, and Testing, 2nd Edition, ASTM International, West Conshohocken, www.astm.org, pp: 1231-1273 (2018). DOI: 10.1520/MNL3720150021.
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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 million 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
(a) = refinery distillation towers
(b) = refinery product tanks
(c) = fuel transportation pipeline (low points in pipeline trap water)
(d) = distribution terminal tanks
(e) = commercial dispensing rack and tank truck
(f) = retail/fleet underground storage tank
(g) = retail/fleet dispensing system; arrows indicate sites where water and biologicals tend to accumulate
FIG. 1 Fuel Distribution System
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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 housekeeping
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 2 N 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-mm1 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.
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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 (TPC Publication No. 3). 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
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