ASTM D6469-99
(Guide)Standard Guide for Microbial Contamination in Fuels and Fuel Systems
Standard Guide for Microbial Contamination in Fuels and Fuel 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 and control of microbial contamination in fuels and fuel systems. This guide applies primarily to gasoline, aviation, boiler, industrial gas turbine, diesel, marine, and furnace fuels (see Specifications D 396, D 910, D 975, D 1655, D 2069, D 2880, D 3699, D 4814, and D 6227) and fuel systems. However, the principals discussed herein also apply generally to crude oil and all liquid petroleum fuels.
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 in SI units are to be regarded as the 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 and health practices and determine the applicability of regulatory limitations prior to use.
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An American National Standard
Designation: D 6469 – 99
Standard Guide for
Microbial Contamination in Fuels and Fuel Systems
This standard is issued under the fixed designation D 6469; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope D 1067 Test Methods for Acidity or Alkalinity of Water
D 1126 Test Method for Hardness in Water
1.1 This guide provides personnel who have a limited
D 1293 Test Methods of pH of Water
microbiological background with an understanding of the
D 1298 Test Method for Density, Relative Density (Specific
symptoms, occurrence, and consequences of chronic microbial
Gravity), or API Gravity of Crude Petroleum and Liquid
contamination. The guide also suggests means for detection
Petroleum Products by Hydrometer Method
and control of microbial contamination in fuels and fuel
D 1331 Test Methods for Surface and Interfacial Tension of
systems. This guide applies primarily to gasoline, aviation,
Solutions of Surface-Active Agents
boiler, industrial gas turbine, diesel, marine, and furnace fuels
D 1426 Test Methods for Ammonia Nitrogen in Water
(see Specifications D 396, D 910, D 975, D 1655, D 2069,
D 1655 Specification for Aviation Turbine Fuels
D 2880, D 3699, D 4814, and D 6227) and fuel systems.
D 1744 Test Method for Water in Liquid Petroleum Prod-
However, the principals discussed herein also apply generally
ucts by Karl Fischer Reagent
to crude oil and all liquid petroleum fuels.
D 1976 Test Method for Elements in Water by Inductively-
1.2 This guide is not a compilation of all of the concepts and
Coupled Argon Plasma Atomic Emission Spectroscopy
terminology used by microbiologists, but it does provide a
D 2068 Test Method for Filter Blocking Tendency of Dis-
general understanding of microbial fuel contamination.
tillate Fuel Oils
1.3 The values in SI units are to be regarded as the standard.
D 2069 Specification for Marine Fuels
1.4 This standard does not purport to address all of the
D 2274 Test Method for Oxidation Stability of Distillate
safety concerns, if any, associated with its use. It is the
Fuel Oil (Accelerated Method)
responsibility of the user of this standard to establish appro-
D 2276 Test Method for Particulate Contaminant in Avia-
priate safety and health practices and determine the applica-
tion Fuel by Line Sampling
bility of regulatory limitations prior to use.
D 2880 Specification for Gas Turbine Fuel Oils
2. Referenced Documents
D 3240 Test Method for Undissolved Water in Aviation
Turbine Fuels
2.1 ASTM Standards:
D 3241 Test Method for Thermal Oxidation Stability of
D 130 Test Method for Detection of Copper Corrosion from
Aviation Turbine Fuels (JFTOT Procedure)
Petroleum Products by the Copper Strip Tarnish Test
D 3242 Test Method for Acidity in Aviation Turbine Fuel
D 396 Specification for Fuel Oils
D 3325 Practice for Preservation of Waterborne Oil
D 445 Test Method for Kinematic Viscosity of Transparent
Samples
of Opaque Liquids (the Calculation of Dynamic Viscos-
D 3326 Practice for Preparation of Samples for Identifica-
ity)
tion of Waterborne Oils
D 515 Test Methods for Phosphorus in Water
D 3328 Test Methods for Comparison of Waterborne Petro-
D 664 Test Method for Acid Number of Petroleum Products
leum Oils by Gas Chromatography
by Potentiometric Titration
D 3414 Test Method for Comparison of Waterborne Petro-
D 888 Test Methods for Dissolved Oxygen in Water
leum Oils by Infrared Spectroscopy
D 910 Specification for Aviation Gasolines
D 3699 Specification for Kerosine
D 974 Test Method for Acid and Base Number by Color-
D 3867 Test Methods for Nitrite-Nitrate in Water
Indicator Titration
D 3870 Practice for Establishing Performance Characteris-
D 975 Specification for Diesel Fuel Oils
tics for Colony Counting Methods in Microbiology
D 4012 Test Method for Adenosine Triphosphate (ATP)
This test method is under the jurisdiction of ASTM Committee D02 on
Petroleum Products and Lubricantsand is the direct responsibility of Subcommittee
D02.14on Stability and Cleanliness of Liquid Fuels.
Current edition approved Nov. 10, 1999. Published January 2000. Annual Book of ASTM Standards, Vol15.04.
2 5
Annual Book of ASTM Standards, Vol 05.01. Annual Book of ASTM Standards, Vol 05.02.
3 6
Annual Book of ASTM Standards, Vol 11.01. Annual Book of ASTM Standards, Vol 11.02.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6469–99
Content of Microorganisms in Water 40 CFR 79 Fuels and Fuel Additives Registration Regula-
D 4057 Practice for Manual Sampling of Petroleum and tions
Petroleum Products 40 CFR 152 Pesticide Registration and Classification Pro-
D 4176 Test Method for Free Water and Particulate Con- cedures
tamination in Distillate Fuels (Visual Inspection Proce- 2.4 Other Standards:
dures) Test Method 2540 D. Total Suspended Solids Dried at
D 4412 Test Methods for Sulfate-Reducing Bacteria in 103–105°C
Water and Water-Formed Deposits
D 4418 Practice for Receipt, Storage, and Handling of Fuels 3. Terminology
for Gas Turbines
3.1 Definitions:
D 4454 Test Method for Simultaneous Enumeration of Total
3.1.1 aerobe, n—an organism that requires oxygen to re-
Respiring Bacteria in Aquatic Systems by Microscopy
main metabolically active.
D 4478 Test Methods for Oxygen Uptake
3.1.1.1 Discussion—Aerobes use oxygen as their terminal
D 4814 Specification for Automotive Spark-Ignition Engine
electron acceptor in their primary energy-generating metabolic
Fuel
pathways. Aerobes require oxygen for survival, using aerobic
D 4840 Guide for Sampling Chain of Custody Procedures
metabolic processes to generate energy for growth and sur-
D 4860 Test Method for Free Water and Particulate Con-
vival.
tamination in Mid-Distillate Fuels (Clear and Bright Nu-
3.1.2 aggressiveness index (A.I.), n—the value computed
merical Rating)
from the sum of the pH + log alkalinity + log hardness of water
D 4870 Test Method for Determination of Total Sediment in
sample where both alkalinity and hardness are reported as
Residual Fuels
milligram CaCO L.
D 4952 Test Method for Qualitative Analysis for Active
3.1.2.1 Discussion—As A.I. decreases, water becomes more
Sulfur Species in Fuels and Solvents (Doctor Test)
corrosive. At A.I. $ 12, water is noncorrosive. At 10 # A.I. <
D 5304 Test Method for Assessing Distillate Fuel Storage
12, water is moderately corrosive. At A.I. < 10, water is
Stability by Oxygen Overpressure
strongly corrosive.
D 5452 Test Method for Particulate Contamination in Avia-
3.1.3 anaerobe, n—an organism that cannot grow or prolif-
tion Fuels by Laboratory Filtration
erate in the presence of oxygen.
D 6217 Test Method for Particulate Contamination in
3.1.3.1 Discussion—Anaerobes use molecules other than
Middle Distillate Fuels by Laboratory Filtration
oxygen in their primary energy-generating metabolic path-
D 6227 Specification for Grade 82 Unleaded Aviation
ways, such as sulfate, nitrate, ketones, and other high-energy
Gasoline
organic molecules. Although anaerobes may survive in the
D 6426 Test Method for Determining Filterability of Distil-
presence of oxygen, anaerobic growth typically occurs only in
late Fuel Oils
an oxygen depleted environment.
E 177 Practice for the Use of the Terms Precision and Bias
3.1.4 anoxic, adj—oxygen free.
in ASTM Test Methods
3.1.5 antimicrobial, n—see biocide.
E 1259 Test Method for Evaluation of Antimicrobials in
3.1.6 bacterium (pl. bacteria), n—a single cell microorgan-
Distillate Fuels (Based on Preliminary Screening and
ism characterized by the absence of defined intracellular
Compatibility)
membranes that define all higher life forms.
E 1326 Guide for Evaluating Nonconventional Microbio-
3.1.6.1 Discussion—All bacteria are members of the bio-
logical Tests Used for Enumerating Bacteria
logical diverse kingdoms Prokaryota and Archaebacteriota.
2.2 Institute of Petroleum Standards:
Individual taxa within these kingdoms are able to thrive in
IP 385 Determination of the Viable Microbial Content of
environments ranging from sub-zero temperatures, such as in
Fuels and Fuel Components Boiling Below 390°C—
frozen foods and polar ice, to superheated waters in deep-sea
Filtration and Culture Method
thermal vents, and over the pH range < 2.0 to > 13.0. Potential
IP Guidelines for the Investigation of the Microbial Content
food sources range from single carbon molecules (carbon
of Fuel Boiling Below 390°C and Associated Water
dioxide and methane) to complex polymers, including plastics.
IP Proposed Method BY Determination of Fungal Fragment
Oxygen requirements range from obligate anaerobes, which
Content of Fuels Boiling Below 390°C
die on contact with oxygen, to obligate aerobes, which die if
2.3 Government Standards:
oxygen pressure falls below a species specific threshold.
3.1.7 bioburden, n—the level of microbial contamination
(biomass) in a system.
3.1.7.1 Discussion—Typically, bioburden is defined in
Discontinued: see 1994 Annual Book of ASTM Standards, Vol 11.02.
terms of either biomass or numbers of cells per unit volume or
Annual Book of ASTM Standards, Vol 05.03.
Annual Book of ASTM Standards, Vol 05.04.
mass or surface area material tested (g biomass / mL; g
Annual Book of ASTM Standards, Vol 14.02.
biomass / g; cells / mL sample, and so forth). The specific
Annual Book of ASTM Standards, Vol 11.05.
Available from Institute of Petroleum, 61 New Cavendish St., London, W.I.,
England.
Available from Superintendent of Documents, U.S. Government Printing
Office, Washington, D.C. 20402. Available from American Public Health Association, Washington, D.C.
D6469–99
parameter used to define bioburden depends on critical prop- 3.1.21 mold, n—form of fungal growth, characterized by
erties of the system evaluated and the investigator’s prefer- long strands of filaments (hyphae) and, under appropriate
ences. growth conditions, aerial, spore-bearing structures.
3.1.21.1 Discussion—In fluids, mold colonies typically ap-
3.1.8 biocide, n—a poisonous substance that can kill living
pear as soft spheres; termed fisheyes.
organisms.
3.1.22 obligate aerobe, n—microorganism with an absolute
3.1.8.1 Discussion—Biocides are further classified as bac-
requirement for atmospheric oxygen in order to function.
tericides (kill bacteria), fungicides (kill fungi), and microbio-
3.1.22.1 Discussion—Obligate aerobes may survive periods
cides (kill both bacterial and fungi). They are also referred to
in anoxic environments but will remain dormant until sufficient
as antimicrobials.
oxygen is present to support their activity.
3.1.9 biodeterioration, n—the loss of commercial value or
3.1.23 obligate anaerobe, n—microrganism that cannot
performance characteristics, or both, of a product (fuel) or
function when atmospheric oxygen is present.
material (fuel system) through biological processes.
3.1.23.1 Discussion—Obligate anaerobes may survive peri-
3.1.10 biofilm, n—a film or layer of microorganisms,
ods in oxic environments but remain dormant until conditions
biopolymers, water, and entrained organic and inorganic debris
become anoxic.
that forms as a result of microbial growth and proliferation at
3.1.24 oxic, adj—an environment with a sufficient partial
phase interfaces (liquid-liquid, liquid-solid, liquid-gas, and so
pressure of oxygen to support aerobic growth.
forth) (synonym: skinnogen layer).
3.1.25 shock treatment, n—the addition of an antimicrobial
3.1.11 biomass, n—density of biological material per unit
agent sufficient to cause rapid and substantial (several orders of
sample volume, area, or mass (g biomass/g(or/mLor/cm )
magnitude) reductions in number of living microbes in a fluid
sample).
or system receiving that concentration.
3.1.12 biosurfactant, n—a biologically produced molecule
3.1.26 skinnogen, n—synonymous with biofilm.
that acts as a soap or detergent.
3.1.26.1 Discussion—Generally applied to a biofilm formed
3.1.13 consortium (pl. consortia), n—microbial community
at the fuel-water interface.
comprised of more than one, species that exhibits properties
3.1.27 sour, v—to increase the concentration of hydrogen
not shown by individual community members.
sulfide.
3.1.13.1 Discussion—Consortia often mediate biodeteriora-
3.1.28 sulfate reducing bacterial (SRB), pl., n—any bacteria
tion processes that individual taxa cannot.
with the capability of reducing sulfate to sulfide.
3.1.14 depacifying, adj—the process of removing hydrogen
3.1.28.1 Discussion—The term SRB applies to representa-
ions (protons) from the cathodic surface of an electrolytic cell,
tives from a variety of bacterial taxa that share the common
= =
thereby promoting continued electrolytic corrosion.
feature of sulfate reduction (SO to S ). SRB are major
contributors to MIC.
3.1.15 deplasticize, v—the process of breaking down poly-
mers in plastics and similar materials, resulting in loss of the 3.1.29 taxa, pl., n—the units of classification of organisms,
based on their relative similarities.
material’s structural integrity.
3.1.29.1 Discussion—Each taxonomic unit (group of organ-
3.1.16 facultative anaerobe, n—a microorganism capable of
isms with greatest number of similarities) is assigned, begin-
growing in both oxic and anoxic environments.
ning with the most inclusive to kingdom, division, class, order,
3.1.16.1 Discussion—Facultative anaerobes use oxygen
family, genus, and species. Bacteria and fungi are often further
when it is present, and use either organic or inorganic energy
classified by strain and biovariation.
sources (nitrate, sulfate, and so forth) when oxygen is depleted
3.1.30 viable titer, n—the number of living microbes
or absent.
present per unit volume, mass, or area.
3.1.17 fungus (pl. fungi), n—single cell (yeasts) or filamen-
3.1.30.1 Discussion—Viable titer is reported in terms of
tous (molds) microorganisms that share the property of having
either colony forming units (CFU) or most probable number
the true intracellular membranes (organelles) that characterize
(MPN) per millilitre, milligram, or centimetre squared.
all higher life forms (Eukaryotes).
3.1.18 metabolite, n—a chemical substance produced by
4. Summary
any of the many complex chemical and physical processes
4.1 Microbes may be introduced into fuels as products cool
involved in the maintenance of life.
in refinery tanks. Bacteria and fungi are carried along with dust
3.1.19 microbial activity te
...
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