Industry guidance on monitoring

and control of microbial

contamination in the aviation fuel
supply chain
DLA Energy Worldwide Energy Conference
Gaylord Convention Center
April 11th 2017

Leon O’Malley, Ph.D

Laboratory Manager
ECHA Microbiology Ltd.
United Kingdom

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Presentation overview

— Microbiological
Contamination
◦ Overview of problem
◦ Options for control
— Monitoring
◦ Field based monitoring
◦ Ideal properties of
testing methods
◦ Available test method
overview

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Microbiological Contamination

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The Microbial Growth Triangle

FOOD;
• Fuel & Oil
MICROBES • Additives
• Other
Contaminants
MICROBIAL
GROWTH
SPOILAGE &
CORROSION

WATER
Control !!

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 Types of Microorganisms
Bacteria Yeasts Molds

10 mm bar
Fungi

All types may be

involved in a single
incident, simultaneously
or in succession.

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 Manifestation of the Problem

“Leopard Skin” Spotting Biofilm on Tank Surfaces

Of Fuel Coalescer Elements

Slime in Jet A-1 Storage Tank Pitting Corrosion by SRB in Fuel Pipe
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Monitoring

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 Risk Based Routine Monitoring
◦ IATA recommends limit values for microbiological contamination
in aircraft fuel tanks.
◦ IATA limit values as such are not appropriate in the fuel supply
chain;
e.g. A heavy contamination from a sump sample in a supply
tank is not necessarily a confirmation that fuel supply is unfit
for service.

JIG Bulletin 83 / Technical Information Document recommends;

1. Evaluate “normal” background levels;
• e.g. Initial testing program over 1 - 2 years.
2. Monitor for change;
• Routine testing at defined sample points and frequencies
according to risk assessed from;
• Initial testing.
• Operational experience.
• Operating conditions (temperature, humidity, risk of water
ingress etc.).

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Risk Based Routine Monitoring

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 Risk Based Routine Monitoring

Item Sampling location Sampling Frequency

High risk facilities Moderate risk Low Risk facilities

facilities
Fixed Storage Storage Tank sump Monthly 3 - 6 monthly Annual monitoring after
Tanks drain line or dead advisable. initial (at least) quarterly
bottom sample screening for 12 months
to determine background
contamination level

Product Recovery Storage Tank sump Monthly 3 monthly. Quarterly where visual
Tanks drain line or dead inspection is not possible
bottom sample

Defuelling Vehicle Vehicle Tank sump drain Monthly 3 monthly 6 monthly for vehicles
line routinely used for
defuelling

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Risk Based Routine Monitoring

— When interpreting results always consider;

◦ Trends,
◦ Operational data / experience

Monthly test of tank bottom fuel by ASTM D7978

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Visual Assessment is the Primary Check
“Cling film” like
lacy material
(bacterial
polysaccharide)
at fuel - water
interface.

Fungal Further
Growth at the
fuel-water Contamination
interface for
Jet A-1 fuel
Spore
(hydrophobic)

• Best Samples are Tank Bottoms, Filter Sump, Low Point etc.
• However, significant microbial contamination may not always be seen!
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 Ideal Properties of a (Field)
Microbiological Test Method
— Reliable
— Reproducible
◦ ASTM Precision Statement
— Sensitive
◦ Detection Limits
— Wide Range of
Detection Training required?
◦ Bacteria, Yeasts, Moulds
(SRB)
— Cost effective
— Rapid
◦ Relatively Speaking

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IATA Recommended Test Kits for Aircraft
Operators
Fuel and Water Phase
— ASTM D7978 (MicrobMonitor2)
◦ Growth Method in Nutrient Gel
— ASTM D7463 (Hy-Lite Jet A-1)
◦ ATP detection by Bioluminescence
— ASTM D8070 (Fuelstat Resinae PLUS)
◦ Immunoassay type method

Water Phase only

— Dip –slides
◦ San-AI Biochecker FC (Bacteria and Fungi)
◦ Easicult TTC (Bacteria)
◦ Easicult M (Fungi)
◦ Only approved for WATER samples!

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ASTM D7978 (ECHA Microbiology
MicrobMonitor2 ®)
— Technology
◦ Growth of Microorganisms in Nutrient Gel
and Counting of Resultant Colonies
— Detects
◦ Bacteria, Yeasts, Mold Growth, Spores
— Equipment
◦ Basic Incubator
— Speed
◦ 1 to 4 days
— Units
◦ CFU/L
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ASTM D7463 (Merck Hy-Lite® Jet A-1)
— Technology
◦ ATP Detection in Water Phase or Water
Drops by Extraction Method
— Detects
◦ Bacteria, Yeasts, Mold Growth (not Spores)
— Equipment
◦ Meter
— Speed
◦ < 10 minutes
— Units
◦ Relative Light Units

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Laboratory Reference Methods
— Filtration methods IP 385 and ASTM D6974 (technically similar)
are considered by IATA to be the reference methods.
— Microscope methods can be used for investigation
◦ eg Light Microscopy or fluorescent microscopy using stains/probes (FISH).
— Molecular based profiling methods to assess microbiological
diversity

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Molecular Methods
Culture Sequencing
Standardized method Many elements may
between labs differ e.g. DNA
extraction, PCR primers
etc.
Bacteria and Fungi Bacteria (and Fungi
possible, but needs the
correct target gene)
Identification requires Less time required (Once
many manual isolation methodology optimised)
steps
Live Microorganisms Live and Dead
Microorganisms
Limited Spectrum of Wider Spectrum of
Microorganisms Microorganisms
Percentage of so-called “unculturables”?

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 Comparison of Culture and Sequencing
Techniques

White et al (2011) AEM Vol 77 (13) p. 4527

Cultivation DGGE Pyrosequencing
a-proteobacteria 13.8 % 3.9 % 6.6 %
b-proteobacteria 21.1 % 19.2 % 45.0 %
g
-proteobacteria 42.8* % 53.9 % 32.1 % * High Pseudomonas
Bacilli 11.8 % 15.4 % 5.1 %
Total 89.5 % 92.4 % 88.8 %

Denaro et al (2005) Wright-Patterson Air Force Base/Uni of Dayton

Direct PCR Cultivation Both
61.9% 4.8% 33.3%

— Culturing can detect a significant proportion of microbial

contaminants
— Power of molecular methods for investigative work
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References
Aviation Fuel Industry Guidance
Aircraft fuel tanks
— IATA Guidance Material on Microbiological
Contamination in Aircraft Fuel Tanks
◦ 5th Edition December 2015.
Aviation Fuel Supply Chain - US
— API RP 1595 Design, Construction, Operation,
Maintenance, and Inspection of Aviation Pre-Airfield
Storage Terminals.
◦ 6 month check of storage tank bottoms by microbial test.
— A4A 103 Standards for Jet Fuel Quality Control at Airports.
◦ Microbial testing recommended if there are indicators of
microbial growth (e.g. during tank inspection).

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References
Aviation Fuel Industry Guidance
Aviation Fuel Supply Chain – RoW (EI/JIG)
— EI / JIG 1530 Quality Assurance Requirements for the
Manufacture, Storage and Distribution of Aviation Fuels to
Airports.
— JIG Guidelines for Aviation Fuel Quality Control & Operating
Procedures for Into-plane Fuelling Services (JIG 1), Airport
Depots (JIG 2) and Smaller Airports (JIG 4).
— JIG Bulletin 83 and Technical Information Document issued
October 2015

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Thank you for your attention!!

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