Sampling & Methods for MIC Determination · Sampling SOPs 3 •Important to have sampling SOPs in...
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Sampling & Methods for MIC Determination
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By Tom Jack & Lisa Gieg
Biological Sciences
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Sampling SOPs (Standard Operating Procedures)
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• Microbial/genomics and corrosion data are only as good as samples taken!
• NACE (e.g., TM0194-2014; TM0212-2018) and other standards (DNV GL RPs, etc.)
consistent, high quality sampling
Garbage
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Sampling SOPs
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• Important to have sampling SOPs in place for:
• Liquids (e.g., produced water)
• Solids (e.g., pig solids, corrosion coupons, pipeline scrapings)
• Ensure as high quality samples as possible
• Recommended materials and preservation methods
• Sterile vessels and sampling tools
• Filled to brim to minimize air
• Proper preservation and timely shipment
to lab for further analysis are critical
X
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Sampling SOPs
• Samples for conducting lab experiments/microbial corrosion experiments
• keep cold (on ice, do not freeze)
• can feasibly add samples to anoxically-prepared serum bottles already containing carbon steel beads or coupons (good for long transport times)
• Samples for genomic analysis
• can stay on ice for short time (24-48 h)
• add chemical preservative • DNAzolTM, RNAlaterTM, ethanol, isopropanol, etc.
• filter in-field and add preservative or keep cold
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Microbial community composition can change dramatically during transport if samples not
chemically-preserved after sampling
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10
20
30
40
50
60
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90
100
1 2 3
Unassigned + misc
Kosmotoga
Marinobacterium
Halothiobacillus
Desulfacinum
Desulfomicrobium
Thauera
Thermoanaerobacter
Caminicella
Methermicoccus
% R
ela
tive
Ab
un
dan
ce
8 days in transit, all samples kept cold
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Offshore Separator
Water, 55°C
Chemically-preserved & cold
Cold only Gieg et al, unpublished
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Where to Sample?
• Corrosion occurs at the metal surface therefore surface-attached/sessile microorganisms are the best samples to get
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Metal Surface
FLUID FLOW
slime Biofilm
Photo: Torben L. Skovhus
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Where to Sample?
• Sessile samples can be challenging to obtain but provide the most relevant information • Sampling corroded & uncorroded areas helps to assess
importance of MIC
• Liquid samples more common and sometimes are the only possible samples
To help understand MIC – need to obtain samples from lines/operations with history of MIC, and compare
with those with no history
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Where to Sample?
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Location 1: Leak point
Location 2: Adjacent to Leak point
Location 3: Uncorroded area
Location 4: Sediments away from leak
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Measuring & Diagnosing MIC
• Diagnosing the root cause of corrosion can be very challenging
• No single measurement can identify MIC
• Three types of evidence needed:
• Microbiological
• Chemical
• Metallurgical
All should be consistent with MIC
microbes
products
materials
MITIGATION
MITIGATION
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Microbiological Monitoring
ATP Assay
Molecular Microbiological Methods (MMM)
Genomics, qPCR
Bug bottles
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Microbiological Monitoring
ATP Assay Bug bottles
Advantages: • Most widely used, especially for baseline monitoring/KPI’s • Easy to use/interpret (if done right)
Metabolic Activity
microbial counts
Disadvantages: ATP assay: no information on types of microorganisms present Bug bottles: only counts those organisms that can be cultured (targets <1% of all microorganisms)
Microbial counts (SRB, APB, etc.)
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Microbiological Monitoring
Molecular Microbiological Methods (MMM)
Genomics, qPCR
Advantages: • No cultivation – based on genetic material • Total microbial community! • Some methods give microbial counts (qPCR) – common KPI
Disadvantages: • Data interpretation more complex
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(1) 16S rRNA genes - ’Fingerprint’ molecule that
identifies all microorganisms in a sample - ‘Who’ is there?
Genomics (MMM) as a Monitoring Tool
(2) ‘Functional’ genes - Genes that encode for specific kind of microorganism (or metabolism) in a sample - e.g., gene for sulfide production
Sample DNA
(3) Metagenomic sequencing - Total DNA sequenced in a sample - Complete genetic potential
Can quantify to get total numbers
Can quantify to get total numbers
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14 Modified from Skovhus et al. 2017, J. Biotechnol. 256: 31-45.
Genomic Analysis (qPCR) vs. Bug Bottles (MPN)
Lower numbers!
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% Relative Abundance
0 20 40 60 80 100 120
original
SRB MPN
SRB BART
Desulfovibrio (4.7%)
Enriched Only one
known sulfate-reducer
detected!
Tistrella
Arcobacter
Azospirillum
Parvibaculum
Unenriched
Shewanella
(genus level, >2% abundance)
106 cfu/mL
103 cfu/mL
(0.2% SRB)
Bug Bottles vs. Genomics Analysis
Gieg, unpublished
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Microbiological Monitoring
KPI – ‘number of bacteria’ for system monitoring
‘High’ numbers don’t necessarily mean MIC will be a problem
‘Low’ numbers don’t necessarily mean MIC won’t be a problem
You need to get the whole story multiple lines of evidence
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MIC
Microbiological Data
Samples
Unpreserved samples lab tests – what are they doing?
Preserved samples:
MMM - who is there?
Choice of primers for 16S MMM?
A: 926F, 1392R (V6-V8)
B: 515F, 926R (V4-V5)*
C: 341F, 785R (V3-V4)
Context
Chemical and operating
parameters
Water chemistry: organic acids, cations, anions, pH, total soluble iron etc.
Solid deposits:
X-ray diffraction (XRD) etc.
Flow rates, temperature, operational changes etc.
Corrosion damage
Field: pitting profiles, sampling
locations etc.
Lab Tests: corrosion damage
under various conditions
Ho
listi
c ap
pro
ach
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Summary
• Data are only as good as your samples!
• Important to have SOPs in place for sampling and preservation for field samples
• Take samples from appropriate locations (corroded vs. non-corroded area)
• Diagnosing MIC requires multiple lines of evidence
• Chemical
• Metallurgical
• Microbiological • Genomics – cultivation-free!
• ATP/bug bottles
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Thank you!
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