Genomics 101: How genomics can assist in MIC management

Transcription

1 Genomics 101: How genomics can assist in MIC management Lisa Gieg Biological Sciences 1

2 Microbiological Monitoring ATP Assay Bug bottles Molecular Microbiological Methods (MMM) a.k.a. Genomics, NGS 2

3 Microbiological Monitoring Bug bottles e.g., BART TM, serial dilutions (MPN) Based on growth in specific medium SRB APB HAB 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 3

4 Microbiological Monitoring Bug bottles e.g., BART TM, serial dilutions (MPN) Based on growth in specific medium SRB APB HAB Major Limitation: Growth based, targets <1% of microbes in a sample -e.g., captures only some SRB but not S 0 or thiosulfate-reducers Most microorganisms will not be captured in growth-based tests 4

5 Microbiological Monitoring Molecular microbiological methods (MMM) a.k.a. Genomics, NGS - Newer approach, but now being widely used to identify microorganisms in O&G systems - Not growth based Based on DNA life s blueprint! 5

6 Microbiological Monitoring Genomics Based on DNA life s blueprint! Uses genetic information contained in microbial cells to determine what types of microorganisms and/or their potential metabolism (e.g., sulfide production) 6

7 Genomics as a Monitoring Tool 3 ways! (1) 16S rrna genes - Fingerprint molecule that identifies all microorganisms in a sample - Who is there? (2) Functional genes - Genes that encode for specific kind of microorganism or metabolism in a sample - e.g., gene for sulfide production Can quantify to get total numbers Sample DNA Can quantify to get total numbers (3) Metagenomic sequencing - Total DNA sequenced in a sample - Complete genetic potential 7

8 (1) 16S rrna or (2) functional genes Polymerase chain reaction (PCR) amplification Makes millions of copies of a piece of DNA so that we can detect it DNA primer Short piece of DNA designed to target a specific gene 8

9 (1) 16S rrna gene surveys commonly-used method - Biological molecule present in all microbial life (involved in protein synthesis) conserved and variable regions 16S rrna - EXTRACT DNA from a sample - AMPLIFY using primers in a PCR reaction - SEQUENCE on an instrument - LIST of microbial names who is there? 9

10 (1) 16S rrna gene surveys Relative abundance (% of total sequencing reads) From An et al.,

11 (1) 16S rrna gene surveys List of microorganisms infer metabolism! Pipe cutout from an offshore oil production facility From Vigneron et al.,

12 (1) 16S rrna genes surveys - qpcr - can quantify using quantitative PCR (qpcr) to get a total number of microorganisms qpcr From Skovhus et al

13 (2) Functional gene analysis commonly-used method - Targets genes specific to a certain kind of microorganism or metabolism - Can quantify these to obtain numbers of specific microbes - for sulfate-reducers, methanogens, acidproducers, iron-reducers, etc. - Total Bacteria, Archaea Sulfatereducers SO 4 2- dsra gene Oil organics HS - + H + FeS Fe Fe e- CO 2 steel 13

14 (2) Functional gene analysis qpcr qpcr From Skovhus et al

15 (3) Metagenomics less commonly used but gives complete information about a microbial community - All DNA sequenced in a sample - All the genetic potential of all microorganisms 15

16 (3) Metagenomics All sulfate-reducers All sulfate-reducing genes From Hu et al

17 Genomics as a Monitoring Tool Field Samples Extract DNA -16S rrna gene -Identifies all microbes - Functional genes -Identifies specific microbes/functions Can get quantitative information Metagenomes - All DNA sequenced - Complete genetic potential 17

18 Genomics as a Monitoring Tool Sampling & sample preservation very important! Surface solids samples where the key microbes are Very important to measure physical & chemical parameters Know operating conditions! 18

19 *Genomics data must be interpreted in context of a given operation and associated chemistry! Microbiologically-influenced corrosion = microbiology + corrosion *holds true for data from all microbial tests 19

20 Genomics in MIC Management Benefits of applying Genomics/MMM 20

21 A Genomics Case Study 21

22 Effect of Bisulfite on Biocorrosion Who is there? 16S rrna gene sequencing WATER TREATMENT FACILITY A Degassing & Filtration Sodium Bisulfite Addition B C D Utility Water Cooling Water Source Well Water Samples Undetectable sulfate Low sulfide concentrations Courtesy of Dr. Jaspreet Mand Pipeline Sample Elemental sulfur deposits Iron sulfide deposits 22

23 Microbial Community Composition Genus Desulfocapsa A Genus Acidovorax Upstream B Family Rhodocyclaceae Genus Desulfurivibrio Genus Geobacter Genus Desulfovibrio Downstream C D Desulfocapsa Genus Desulfomicrobium Genus Comamonadaceae Phylum Firmicutes Family Methanobacteriaceae Other 23 Courtesy of Dr. Jaspreet Mand

24 Sulfite and MIC It was assumed that sulfur, found downstream of bisulfite addition, was due to oxygen ingress However, sulfur may be result of bisulfite metabolism (disproportionation) by Desulfocapsa: 3HSO 3- S SO H 2 O + H + Resulting byproducts may be used by other microorganisms in the system 24

25 Microbial Enrichment on Sulfite Sulfite (mm) Sulfur (mg/l) Abiotic control Live Time (Days) Live Abiotic control Time (Days) Sulfite degradation is seen in incubations with the field sample relative to the abiotic control Elemental sulfur is produced Genomics helped to pinpoint the problem Altered KPI for bisulfite treatment 25 Courtesy of Dr. Jaspreet Mand

26 Genomics in MIC Management - Genomics is a powerful microbiological monitoring tool that captures broadest diversity in a field sample - contributes a piece of the corrosion puzzle to help guide corrosion management and mitigation - monitoring programs (KPI) - effectiveness of treatments - failure analysis - risk management Thank you! Questions? R. Eckert, Materials Performance 26