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Aberdeen Section30th October 2012
Michael Horne
Senior Microbiologist
Intertek Commercial Microbiology
The Pro’s & Con’s of Current Oilfield Bacterial Monitoring Techniques
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Our Heritage
Caleb Brett founds a marine surveying business
1885
Thomas Edison establishes what is later renamed as the Electrical Testing Laboratories (ETL)
1896
Virginius Daniel Moody establishes Moody Engineering for construction and electrical engineering projects
1911
Intertek and Moody International join forces
2011
Intertek Today: Valued Quality. Delivered.
Today
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An Extensive Global Network
• FTSE 100 company in the Support Services sector
• Market capitalization at £4.5 billion
• Revenue generation of over £1.7bn in 2011
100More than
countries
1,000More than
laboratoriesand offices
30,000people
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Intertek E&P Services
Operating Centres
• USA (Houston)
• UK (*Aberdeen and Manchester)
• Norway (Stavanger)
• Middle East (Sharjah, Fujairah, Qatar and Abu Dhabi)
• Libya (Tripoli)
• SE Asia (Singapore, KL, Jakarta, Perth)
• Azerbaijan (Baku)
E&PServices
Analytical Support Services
Reservoir ServicesProduction & Integrity
AssuranceMetering & Measurement
Allocation
Westlab
Aberdeen*
Westport
Geotech
UKCAPCIS* & CML*
Middle EastDubai & Fujairah
SE AsiaKuala Lumpur
LibyaTripoli
EMIS
IMM
SREL*
M&MMiddle East
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Production & Integrity Assurance
UMIST Corrosion & Protection Centre Industrial Services unit founded – later to become CAPCIS Ltd
Commercial Microbiology Ltd started trading
2008200719851973 2010 2012
Fujairah Lab opened in UAE
CAPCIS acquired by Intertek
Commercial Microbiology Ltd acquired by Intertek
New Lab opens in Malaysia –Kuala Lumpur
Corrosion Lab opened in Houston Texas
Libya Joint Venture formed
Production & Integrity
Assurance Business Stream
Our Heritage
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Presentation Outline
• Introduction to Microbial Issues
• Sample Collection
• Traditional Culturable Enumeration of Viable Bacteria
• Enumeration by Direct Counting
• Activity Determination
• Molecular Techniques
• Summary
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Introduction
• Microbiologically Influenced Corrosion (MIC) is the degradation of a material through the presence and activity of microorganisms
• Industries affected by MIC include;
• Chemical, Food, Pulp & Paper, Sewage
• Conventional & Nuclear Power Generation
• Exploration, Production, Transportation & Storage
• Marine & Aviation Industries
• Economic Impact untold, potentially 10-50% of all failures include microbiology
• 1998 it is estimated that 'all corrosion’ cost the US $276 billion dollars
• UK, Japan, Germany estimate cost between 1-5% gross national product
• Traditionally in petroleum production the major threat from MIC comes from sulphate reducing bacteria (SRB)
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Samples
• Sampling & Sample Points
• Sample Type
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Sampling & Sample Points
1. Sample containers should be sterile or at the very least new
2. Sample points should be working & well suited
3. Sample point should be flowed for several minutes prior to sampling
4. Sample bottle should be fully filled to remove air
5. Sample should be analysed without delay preferably on site
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Sample Type
Planktonic Samples
‘Bulk’ fluid sampling to detect the ‘free-swimming’
bacteria that can attach to the surface of a system.
Sessile Samples
‘Solid’ sampling to detect the bacteria attached to the surface of a system
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Culturable Techniques
• Traditional Culturable Enumeration of Viable Bacteria
• Plate Counts
• Extinction dilution (TMO194-04)
• Most Probable Number (MPN)
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Plate Counts
Positives
� Cheap
� Quick Turn Around
� Ideal for hydrotest water quality
Negatives
� Low recovery rate (sometimes < 0.1 %)
� Limited selectivity (Legionella, Yeast, Mould)
� Space & disposal requirements
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Sample
1 A,B,C2 A,B,C
3 A,B,C 4 A,B,C
Syringe 2
Syringe 1
Syringe 4Syringe 3
Most Probable Number (MPN)
MPN +/- 0.7 Log
Single Series +/- 1.5 Log
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MPN Reading
SRB (Sulphate Reducing Bacteria)
Black insoluble FeS within bottle
GHB (General Heterotrophic Bacteria)
Red & Orange Turbidity
APGHB (Acid producing GHB)
Yellow Turbidity Only
NRB (Nitrate reducing Bacteria)
Turbidity (cloudy appearance) & Strip Test
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Culturable Techniques
Positive
� Long term use = lots of historical data
� Greater ease of result interpretation
� Cheap & user friendly
Negative
� Low recovery rate (sometimes < 0.1 %)
� SRB Incubation periods (28 days) = long turn around time
� Space & disposal requirements
� Knowledge of system (salinity, temperature)
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Rapid Methods
• Direct Counts
• ATP
• Antibodies
• Activity determination
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Positive
� Quick turn around
� Rapid method of cell quantification
Negative
� Requires high cell concentrations
(e.g. 107 / ml)
� Potentially counting dead and living cells with equal probability
� Time consuming
� Microscope required
� No differentiation
Phase contrast Epi-fluorescence
Direct Count
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ATP Analysis
Positive
� Quick method analysis ~10 min per sample
� Easy to use
� Can highlight problem areas rapidly
Negative
� Initial outlay for equipment
� Swab storage issues
� No species differentiation (bacteria, yeast mould)
� Accuracy still in question, difficulties with quantification (dormant cells?)
www.CDEworld.com
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Rapid Methods
Antibodies specific for certain species of SRB can be used in rapid method kits, i.e. RapidChek® II and immuno-magnetic methods
Positive
� Cheap
� Quick
� User friendly
Negative
� Minimum detection levels > 103
� Storage issues, require refrigeration
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Activity Determination
• Nutrients removed from the system
• Products accumulate in the system
• Sulphide
• Nutrients
• Analyse for decrease in sugar concentration
• Analyse for oxygen consumption
• Products
• Acids – Follow decrease in pH
• CO2 – Follow increase in partial pressure
• Polymers – Increase in protein
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• RNA based methods• Fluorescence in situ Hybridization (FISH)
• DNA based Methods:• Quantitative Polymerase Chain Reaction (qPCR)
• Denaturing Gradient Gel Electrophoresis (DGGE)
• Pyrosequencing
Molecular Methods
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Enumeration is done by pixel analyses on the images obtained from fluorescent markers fixed to cell RNA% area covered with cells converted back to cells per ml or cm2
FISH Analysis
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qPCR
• The aim of PCR technology is to specifically amplify a target (gene) from an undetectable amount of starting material
• Quantitative, or real time, PCR allows you to quantify whilst this proceeds
• Like photocopying a page from a book, the process allows you to determine how many pages ‘genes’ where generated by the photocopier meter. The meter is a fluorescent marker increasing in intensity with each generation.
• At the end of the run the light intensity is directly related to an initial amount of target in the sample, expressed as gene copy per (ml/g/cm2)
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DGGE
Sample Extract DNA FingerprintPCR DGGE
• DGGE creates a fingerprint of the population
• Target is typically DNA, but RNA is possible
• Monitoring population changes
• Sequencing of bands possible
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Pyrosequencing
• Relatively new molecular technique (1996)
• Allows for amplification and sequencing of DNA from an undetectable amount of starting material.
• Whilst the amplification is being performed the DNA sequence is also determined and matched against the DNA bank
• Microorganisms not in the database will not be identified
• Allows identification of a population profile
• Although a new technique cost and turn around times are continually getting better
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Pyrosequencing
Planktonic
Other, 0.1
Eukaryota, 0.5
Archaea , 0.5
Actinobacteria , 2.3
Firm icutes , 0.6
Bacteroidetes, 16.8
Proteobacteria, 79.2
Other
Eukaryota
Archaea
Proteobacteria
Firmicutes
Actinobacteria
Bacteroidetes
Chlorof lexi
26.27
1.31
72.30
0.09 0.00
0.00
20.00
40.00
60.00
80.00percent (%)
α β γ δ ε
15.37
82.63
0.00
0.00
20.00
40.00
60.00
80.00
100.00
percent (%)
Methanobacteria Methanomicrobia Thermoplasmata
Proteobacteria
Archaea
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Positives
� Avoids culturing step
� Detection of un-culturable organisms
� Quicker, matter of days turnaround
� RNA more likely to detect active cells (FISH)
� More sensitive
� No need for knowledge of system under test
� Better understanding of all microorganism involved in oilfield
Why Molecular Methods?
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Negatives
� You have to know what you are looking for
� Bias choice of probes (FISH)
� DNA analysis may also detect dead and dormant cells (PCR based methods)
� Costs (Labour Intensive)
� Special requirement of sample preservation & handling
� Understanding data & comparing to historical data
� Only known microorganisms can be detected with certainty
� Unknown microorganisms require very laborious sequencing to identify
Why Molecular Methods?
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Issues with DNA Analysis
Does the DNA die with the cell?
Current estimates suggest in ideal preservation environments DNA may have an upper stability limit as high as 1,000,000 years
(Source: Natural History Museum)
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• Expect variable data
• Establish a baseline
• Always consider additional information
• Present results
• Long term – Sessile monitoring
• Short term – Planktonic monitoring
• Long term trending,
• Set KPIs & respond proactively
Data and Reporting
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Summary
• Is the right monitoring regime in place for your system?
• How reliable are the samples taken from your system?
• Is the data planktonic or sessile, can I generate sessile data?
• Consider which analysis suits your needs best, bear in mind no one monitoring technique is the ‘holy grail’ you need to consider all analysis as your tool box and get the right tools for the right job.
• Monitor the data generated, is there value?
• Set KPIs and put in place remedial actions
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Intertek Microbiology & Chemistry Laboratory Tour
Includes;
Guided Tour of Facility
Demonstration of Analysis
Food & Drink Reception with Q&A
Evening of Tuesday 26/03/2013
*Pre-Registration Required
ICorr – Industrial Visit
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Valued Quality. Delivered.
