2012-10-30-Michael-Horne-of-Intertek_Pros-and-Cons-of-Various-Monitoring-Techniques.pdf

SRB Technique

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Aberdeen Section 30
th
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

3. www.intertek.com 3
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
100
More than
countries
1,000 More than
laboratories
and offices
30,000
people

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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&P
Services
Analytical Support
Services
Reservoir Services
Production & Integrity
Assurance
Metering & Measurement
Allocation
Westlab
Aberdeen*
Westport Geotech
UK
CAPCIS* & CML* Middle East Dubai & Fujairah
SE Asia
Kuala Lumpur
Libya
Tripoli
EMIS IMM
SREL*
M&M
Middle 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
2008
2007
1985
1973
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 Bacter ia
•
Enumeration by Direct Counting
•
Activity Determination
•
Molecular Techniques
•
Summary

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Introduction
• Microbiologically Influenced Corrosion (MIC) is th e 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 th reat 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 ai r
5. Sample should be analysed without delay preferabl y 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 Bacter ia
•
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,C
2 A,B,C
3 A,B,C
4 A,B,C
Syringe 2
Syringe 1
Syringe 4
Syringe 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. 10
7
/ 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 > 10
3

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
• CO
2
– Follow increase in partial pressure
• Polymers – Increase in protein

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• RNA based methods
• Fluorescence in situHybridization (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 p er ml or cm
2
FISH Analysis

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qPCR
• The aim of PCR technology is to specifically ampli fy a target (gene) from an
undetectable amount of starting material
• Quantitative, or real time, PCR allows you to quan tify 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 intensi ty with each generation.
• At the end of the run the light intensity is direc tly related to an initial amount of
target in the sample, expressed as gene copy per (m l/g/cm
2
)

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DGGE
Sample
Extract DNA
Fingerprint
PCR
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 iden tified
•
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 A rchaea Proteobacteria Firmicutes A ctinobacteria Bacteroidetes Chlorof lexi
26.27
1.31
72.30
0.09
0.00
0.00
20.00
40.00
60.00
80.00
pe rce nt (%)
α β γ δ ε
15.37
82.63
0.00
0.00
20.00
40.00
60.00
80.00
100.00
pe rce nt (%)
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 & handli ng

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 sy stem?
•
How reliable are the samples taken from your system ?
•
Is the data planktonic or sessile, can I generate s essile 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 ri ght 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.