2012-10-30-Michael-Horne-of-Intertek_Pros-and-Cons-of-Various-Monitoring-Techniques.pdf
- 1. www.intertek.com 1 Aberdeen Section 30th October 2012 Michael Horne Senior Microbiologist Intertek Commercial Microbiology The Pro’s & Con’s of Current Oilfield Bacterial Monitoring Techniques
- 2. www.intertek.com 2 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
- 4. www.intertek.com 4 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
- 5. www.intertek.com 5 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
- 6. www.intertek.com 6 Presentation Outline • Introduction to Microbial Issues • Sample Collection • Traditional Culturable Enumeration of Viable Bacteria • Enumeration by Direct Counting • Activity Determination • Molecular Techniques • Summary
- 7. www.intertek.com 7 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)
- 8. www.intertek.com 8 Samples • Sampling & Sample Points • Sample Type
- 9. www.intertek.com 9 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
- 10. www.intertek.com 10 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
- 11. www.intertek.com 11 Culturable Techniques • Traditional Culturable Enumeration of Viable Bacteria • Plate Counts • Extinction dilution (TMO194-04) • Most Probable Number (MPN)
- 12. www.intertek.com 12 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
- 13. www.intertek.com 13 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
- 14. www.intertek.com 14 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
- 15. www.intertek.com 15 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)
- 16. www.intertek.com 16 Rapid Methods • Direct Counts • ATP • Antibodies • Activity determination
- 17. www.intertek.com 17 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
- 18. www.intertek.com 18 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
- 19. www.intertek.com 19 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
- 20. www.intertek.com 20 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
- 21. www.intertek.com 21 • RNA based methods • Fluorescence in situ Hybridization (FISH) • DNA based Methods: • Quantitative Polymerase Chain Reaction (qPCR) • Denaturing Gradient Gel Electrophoresis (DGGE) • Pyrosequencing Molecular Methods
- 22. www.intertek.com 22 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
- 23. www.intertek.com 23 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)
- 24. www.intertek.com 24 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
- 25. www.intertek.com 25 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
- 26. www.intertek.com 26 Pyrosequencing Planktonic Other, 0.1 Eukaryota, 0.5 Archaea , 0.5 Actinobacteria , 2.3 Firmicutes , 0.6 Bacteroidetes, 16.8 Proteobacteria, 79.2 Other Eukaryota Archaea Proteobacteria Firmicutes Actinobacteria Bacteroidetes Chloroflexi 26.27 1.31 72.30 0.09 0.00 0.00 20.00 40.00 60.00 80.00 percent (%) 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
- 27. www.intertek.com 27 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?
- 28. www.intertek.com 28 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?
- 29. www.intertek.com 29 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)
- 30. www.intertek.com 30 • 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
- 31. www.intertek.com 31 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
- 32. www.intertek.com 32 Intertek Microbiology Chemistry Laboratory Tour Includes; Guided Tour of Facility Demonstration of Analysis Food Drink Reception with QA Evening of Tuesday 26/03/2013 *Pre-Registration Required ICorr – Industrial Visit