ECHA Microbiology LTD

THE IDENTITY and IDENTIFICATION of
MICROBES in FUEL
WHERE ARE WE NOW and WHERE ARE
WE GOING?
Ted Hill & Graham Hill
ECHA Microbiology Ltd., Cardiff CF24 5EJ
Judith White
Cardiff School of Biosciences, CF10 3TL
ISMOS, 17-18 September 2007

© ECHA Microbiology Ltd, 2006
solving microbiological problems in industry

THE OPERATIONAL PROBLEMS
GROWTH ONLY OCCURS WHERE THERE IS FREE

WATER - This may be condensate, rain or sea water
• GROWTH Occurs in Free Water, on Surfaces and at
Interfaces - Growth Disperses into the Fuel - CAUSING
• FOULING – Filters, Pipelines and Injectors
• MALFUNCTIONS – Restricted Fuel Flow (Engine may
Shut Down or Disintegrate), Erratic Fuel Measurements,
Pump Failures, Difficulty Separating Water from Fuel
• CORROSION
DIFFERENT PHYSICAL NICHES HAVE DIFFERENT
MICROBIAL ECOLOGIES
DIFFERENT FUELS/FUEL BATCHES HAVE DIFFERENT
CHEMISTRIES WHICH SUPPORT DIFFERENT MICROBES

REFINERY
Filter
Pipeline
TO AIRPORT
Filters
TERMINAL
Filter Filter Filter
MICROBES CAN GROW

IN MOST NICHES IN FUEL Pipeline
SUPPLY CHAIN
FILTER/WATER SEPARATORS
FOR AVIATION FUEL

Used with permission Exxon Mobil Aviation
AIRPORT FUEL DISTRIBUTION
Via FUEL Via
BOWSER HYDRANT
FWS Storage Tank
Storage Tank
FWS
FWS
FWS = Filter Water Separator
MICROBES CAN GROW IN MOST NICHES IN AIRPORT

FUEL DISTRIBUTION – AND IN THE AIRCRAFT

Used with permission Air BP
Each Niche Will Have a Different
Physical and Chemical Environment
and a Different Microbial Flora
– Bacteria 10 μm
– Yeasts 10 μm
– Moulds –
including
spores
Bacteria may be: 10 μm
aerobic, facultative
or anaerobic
Only some of flora
will be hydrocarbon
degraders © ECHA Microbiology Ltd, 2006

In a Settled Storage Tank Physical Conditions
Differ from Top to Bottom
– and So Do the Microbes
Sampling at Different Levels Gives Different Results


GROWTH IN JET A-1
OXYGENATED AIRCRAFT FUEL OXYGEN DEFICIENT

CARGO TANK BOTTOM

WITHIN AN AIRCRAFT THERE WILL ALSO
BE DIFFERENT ECOLOGICAL NICHES
• Fuel tanks turned-over rapidly will be most aerated;

some fuel used mainly for trimming will be less aerated
• Outboard tanks will be colder in flight than fuselage
tanks, and will be substantially below freezing on long
haul flights
• Tanks will be warm/hot on supersonic flights
• Length and altitude of flight will control fuel temperature
• Condensate will form at different rates in different tanks
– and will be drained off with differing frequency/efficacy
• Fuel uplifts will vary in their chemistry and microbial
content

MICROBIOLOGICAL GROWTH IN
AIRCRAFT WING FUEL TANK
• Microbial growth
“Biofilm”
• Coating is attacked
and fails
• Microbially
Influenced Corrosion
(MIC) is local, rapid &
intense due to
– Microbially
generated acids
– Oxygen gradients
cause electrons to
flow – anodic
pitting

FIRST CONCLUSION
MICROBIAL ECOLOGY IS COMPLEX

AND VARIES WITH
• NICHE LOCATION
• TIME
• FUEL COMPOSITION
IDENTIFICATION OF MICROBES IS
COSTLY, SLOW AND NOT NORMALLY
JUSTIFIED

SAMPLING ISSUES – FUEL IN USE
MAJOR DEDUCTIONS WILL HAVE TO BE MADE FROM

THE RESULT OF TESTS ON SMALL SAMPLES
FOR EXAMPLE, ON AN AIRCRAFT, A TEST ON A TANK

DRAIN SAMPLE IS THE ONLY ONE AVAILABLE
BUT WE WANT TO KNOW HOW MUCH SUSPENDED
GROWTH, INTERFACIAL GROWTH AND BIOFILM
THERE IS IN THE TANK

DISTRIBUTION OF MICROBES IN
AIRCRAFT FUEL TANK
FUEL
Biofilm Interfacial Suspended
Material Material
ONLY A DRAIN SAMPLE

DRAIN WATER CAN BE TESTED © ECHA Microbiology Ltd, 2006

SAMPLING ISSUES – FUEL IN
SUPPLY CHAIN
MAJOR DEDUCTIONS WILL HAVE TO BE MADE FROM

THE RESULT OF TESTS ON SMALL SAMPLES FROM
LARGE SYSTEMS TAKEN AT A SPECIFIC TIME
FOR EXAMPLE, TESTS ON SMALL SAMPLES OF FUEL

DELIVERED FROM A LARGE STORAGE TANK WHICH
IS BEING CONTINUOUSLY EMPTIED AND REFILLED

Distribution Of Microbes In Fuel Storage Tanks
- Sampling at Different Times Gives Different Results
In Presence of water microbial growth occurs at fuel : water
interface
Fuel receipts disturb the Interface
SUCTION
LEVEL
SAMPLE
Heavily
Not
Contaminated
Contaminated

Distribution Of Microbes In Fuel Storage Tanks
Sampling at Different Times Gives Different Results
With time Microbes settle to bottom of tank
•Individual microbes at 0.2 cm per hour
•Aggregates (>25 µm) at 30 cm per hour
ONLY AFTER
VERY LONG
SETTLING
TIME
SUCTION LEVEL
SAMPLE
Not
Contaminated

CURRENT INDUSTRY MICROBIOLOGICAL
TESTING DOES NOT RECOGNISE THAT
DIFFERENT NICHES WILL HAVE
DIFFERENT FLORA
•Total AEROBIC viable counts - Colonies counted
and rarely differentiated into bacteria, yeasts, moulds
– ONE COLONY EQUALS ONE UNIT
•Fixed culture media, temperature, pH, oxygen
•No routine testing for hydrocarbon degrading
capability – but all microbes contribute to fouling etc.
•Testing for anaerobes rare
•An assumption that what grows in one niche will
grow in other niches downstream © ECHA Microbiology Ltd, 2006

Laboratory test IP 385 / 99
(ASTM test is similar)
1. Fuel sample filtered
through 0.45 µm
membrane filters
2. Membranes are
washed and rinsed
3. Membranes are
transferred to nutritive
agar plates for
• Bacteria
• Fungi (yeasts &
moulds)
4. Incubated 25°C 3 – 5
days
5. Membranes are
examined and colonies
of growth (CFU)
counted © ECHA Microbiology Ltd, 2006

ON-SITE MICROBIOLOGICAL TESTS
• Ideal properties are;
– Detect appropriate microbial contaminants
• Moulds (hyphae & spores)
• Yeasts
• Bacteria
– Sufficiently sensitive to detect small numbers
– Quantitative
– Reliable and reproducible
– Can be used for Fuel & Water Phase samples
– Distinguish live microbes from dead microbes
– Easy to use and interpret
– Quick
– Safe in unskilled hands
Preferably – No expensive Instrumentation

FUEL QUALITY ISSUES
• Although there are no petroleum industry Microbial

Specifications for fuel supplied
• Military and airlines are most pro-active in setting in-
house standards for fuel in use
• Since 2002 airlines have tested aircraft fuel with on-
site tests, have set limit values and specified
treatment procedures (IATA Guidance Handbook)
• Few other end-users (except the military) monitor fuel
in their tanks and systems
• Several Fuel Suppliers have now instigated
programmes of ROUTINE microbiological monitoring;
some are under pressure from end-user to validate
quality of fuel supplied

Industry Guidance for Aircraft Operators
from I.A.T.A
IATA Technical Fuels Group
• IATA Guidance Material on
Microbiological Contamination
in Aircraft Fuel Tanks,
December 2002; 2nd Edition
Effective 01 February 2004
• Guide primarily covers
microbiological issues in
Aircraft Tanks
– Describes problem
– Risk factors
– On-site detection of
microbes
– Limit values for actions
– Remedial measures

MICROBIOLOGICAL TESTING
• Three on-site tests recommended in IATA Guidelines
• Objective is to detect a DEVELOPING contamination
BEFORE it causes problems
• If contamination is treated at an early stage of
development;
– No operational problems
– Biocide is more effective (no thick biofilm)
– Less aircraft downtime (no tank cleaning)
– Less disruption to operation
– More cost effective

MICROBMONITOR2 - ON-SITE TEST FOR
FUELS and WATER
1. Measuring sample
into MM2
1 2
2. Shaking MM2
containing sample
3. Tapping MM2 bottle
to produce flat layer
of gel – then allow to
set and incubate
4. Counting ‘colonies’
of microbes (cfu) after
incubation
3 4

Fuelstat RESINAE Test – Detects
Hormoconis resinae antigens
Lines appear in long ‘window’ and

are interpreted as negligible,
moderate or heavy for aircraft tanks

FuelstatTM RESINAE
• The FuelstatTM RESINAE provides real time
assessment of contamination by Hormoconis resinae
• Speed is advantageous where a rapid assessment is
required (e.g. quick check of fuel uplift or de-fuel
quality).
• Limitations
– The test only detects active Hormoconis resinae (not
spores)
– in recent years, operational problems have been caused
by a variety of different microbes with or without
presence of Hormoconis resinae
– Will give positive results for days after biocide treatment;
therefore, when confirming efficacy, of treatment all
biocide treated fuel should be burnt before testing.
– Fixed and arbitrary limit values
– Extraction into water phase needed and efficacy may
vary

ATP TESTS
• Adenosine Tri Phosphate (ATP) is a component
present in all living active cells
• ATP can be assessed by mixing aqueous samples
with a reagent (luciferase) which reacts with ATP to
emit light
• The amount of light emitted (measured using a
portable instrument) correlates with the amount of
ATP present
• A number of companies have developed ATP tests
for fuels
• An extraction or filtration stage is used to concentrate
microbes from fuel into an aqueous phase – efficacy
not substantiated
• Variable limit values and broader detection spectrum
for ATP - but other limitations similar to FuelstatTM
RESINAE test

ACCURACY
• Thorough tank inspection provides the best assessment of
the extent of contamination present but this is costly and
time consuming
• The aim of test kits is to provide a reasonable assessment
of contamination status without the need for tank inspection
• The limitations of the test method employed are usually not
clearly understood by the user
• NONE of the Microbiological Tests can be considered to be
highly accurate
• Rapid methods and cfu methods assess different
parameters and hence the methods are not directly
comparable
• Test user does not usually appreciate important influence of
sampling location and sampling time
• IATA recommends that a positive result should always be
confirmed by a RETEST – but this is rarely done
CAN MOLECULAR METHODS IMPROVE ON CURRENT
METHODS???

IDENTIFICATION IS AN AID TO
UNDERSTANDING ECOLOGY OF NICHES
AND TRACING SOURCES OF
CONTAMINATION - NICHE TO NICHE
• Carried out primarily to trace sources of contamination
and allocate blame
• Currently identification is mostly by API biochemical test
packages – yielding a numerical profile and (hopefully)
an identity
• API tests give little information on
spoilage/fouling/corrosion potential
• ECHA is sponsoring research on molecular methods at
Cardiff University

Aims of project
Aim 1. Molecular systematics of hydrocarbon
degrading bacteria
-Diversity analysis using 16S rRNA PCR
Aim 2. Application of genetic fingerprinting and strain

typing to trace the source of bacterial contamination
of hydrocarbons.
- Random Amplified Polymorphic DNA (RAPD)
- Multi Locus Sequence Typing (MLST)
Aim 3. Cultivation-independent analysis of microbial
species found in various fuel/oil environments
-16S rRNA PCR
- DGGE (Denaturing Gradient Gel Electrophoresis)
Aim 4. Molecular characterisation of bacterial

factors required to survive in the presence of
hydrocarbons.

General scheme of work
SAMPLES FROM ECHA
Culture Dependent Culture Independent
Create collection of Single Strains

recovered from Fuel/Water bottom DNA Extraction directly from sample
Plate, grow, Assessment of DNA quality and quantity

pick single colonies
PCR amplification of 16S rDNA
DNA Extraction followed by RAPD
DGGE Clone Library

Culture of single strain types
Analysis of bacterial diversity
Frozen Stocks PCR Targeting 16S rDNA
Sequencing and Bioinformatics © ECHA Microbiology Ltd, 2006

Conclusions
Bacterial Systematics and Genetic Fingerprinting
• >60 fuel samples from a range of sources investigated
• >200 individual bacterial isolates
• 16S rRNA gene targeted for identification at genus/species
level
• Dominant genera include Pseudomonas sp., Alcaligenes sp.,
Burkholderia., Brevundimonas., Staphylococcus sp., Bacillus sp.
• Genetic fingerprinting (using RAPD) used to generate strain-
specific profile of all isolates
• May be used to trace source and spread of contamination –
industrial epidemiology
Culture Independent Assessment of Diversity
• Total community DNA extracted directly from samples
• DGGE profile produced for >10 samples
• Culture-dependent results compared to culture-independent
• Too few samples for general conclusions to be made

THE END

ECHA Microbiology LTD

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THE IDENTITY and IDENTIFICATION of

ISMOS, 17-18 September 2007

solving microbiological problems in industry

GROWTH ONLY OCCURS WHERE THERE IS FREE

solving microbiological problems in industry

Filter Filter Filter

MICROBES CAN GROW

solving microbiological problems in industry

FWS = Filter Water Separator

MICROBES CAN GROW IN MOST NICHES IN AIRPORT

solving microbiological problems in industry

Bacteria may be: 10 μm

solving microbiological problems in industry

Sampling at Different Levels Gives Different Results

solving microbiological problems in industry

OXYGENATED AIRCRAFT FUEL OXYGEN DEFICIENT

solving microbiological problems in industry

• Fuel tanks turned-over rapidly will be most aerated;

solving microbiological problems in industry

solving microbiological problems in industry

MICROBIAL ECOLOGY IS COMPLEX

solving microbiological problems in industry

MAJOR DEDUCTIONS WILL HAVE TO BE MADE FROM

FOR EXAMPLE, ON AN AIRCRAFT, A TEST ON A TANK