Microbiological contamination of jet fuel Examples and tips for an effective presentation What s the problem? 2 Fuel provides a rich carbon source for microbial growth Microbes may degrade fuel (in storage) Microbes form biofilms that foul sensors, block filters, corrode metals (MIC) Microbes degrade light oils in reservoirs 3 Biological activity in the deep subsurface and the origin of heavy oil Head, Jones & Larter (2003) Nature 426, 344-352 1
Microbes degrade fuel in aircraft tanks 4 Aero magazine: Boeing Microbes degrade spilt fuels - bioremediation Aircraft fuel tank 5 Lots of different surfaces and crevices Multiple material types Airbus ASTM Standards 6 ASTM standard D 6469 D 6974 D 7463 D 7464 D 7687 Title Standard Guide for Microbial Contamination in Fuels and Fuel Systems Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels - Filtration and Culture Procedures Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures and Fuel Associated Water Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing Standard Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel, Fuel/Water Mixtures, and Fuel-Associated Water with Sample Concentration by Filtration 2
Water is a problem but inevitable 7 Drain water but often frozen after long-haul Test regularly Recommended once per year More frequently if contamination is found Treat quickly if a problem found What do we find? 8 Bacteria, fungi, yeast Hormoconis resinae Pseudomonas sp. Candida tropicalis Rhodococcus sp. We can t sample/measure everything 9 What is present in the tank Sample Cultured If what is measured is indicative of the total contamination, incomplete sampling is not a problem. 3
Culture-based methods 10 Colony forming units (CFUs) must be alive must be able to grow in culture Aggregates grow as 1 cfu but may contain 1000s of organisms Only a small proportion can be cultured Culture-independent methods 11 Measure biomarkers ATP, catalase, endotoxin production Antibody based detection of organisms Enzyme Linked Immuno Sorbant Assay (ELISA) Nucleic acid based detection of organisms 16S rrna genes Metabolites ATP based assay 12 Active cell ATP Luciferase Extract +ATP Inactive cell ATP Light 4
Antibody based assay 13 Antibodies are very specific Active cell Extract Inactive cell Fuelstat 14 FuelStat resinae Antibodies against H. resinae Manufacturers say this is indicative of most contamination FuelStat Kerosene Antibodies against H. resinae, bacteria and fungi Undefined FuelStat resinae PLUS Antibodies against something a metabolite? Could potentially sample activity in the whole tank Undefined so we don t know what it is Will it work in the future? DNA-based assay 15 Active cell Polymerase chain reaction Extract Inactive cell Amplification Detect specific organisms Detect all organisms (sequence) 5
Polymerase Chain Reaction (PCR) 16 Uses short DNA sequences (primers) to exponentially amplify genes 16S rrna millions of copies Specificity from selecting primers or Amplify everything and then sequence Cultured vs non-cultured? 17 Amplified sequences are known as Operational Taxonomic Units (OTUs) Denaro et al (2005) Identified 36 OTUs in JP-8 28 never previously described Only 10 were cultured Other studies show fuel type is important (JetA1 vs JP-8) Local soil is a major source of microbes Work at Sheffield: Direct sequencing of samples 18 5 fungi and 21 bacterial OTUs dominant in water samples provided from aircraft and ground tanks These are the dominant organisms (>1%) Many more present at lower proportions 6
The future of DNA-based diagnostics? 19 PCR technology not well suited for the field Technologies being driven by medical diagnostics Can potentially identify all organisms Could potentially identify function Correlations 20 log ATP log CFU Log Catalase Log ATP 1 Log CFU 0.633 1 Log Catalase 0.630 0.919 1 Correlations between different methods are variable. Passman et al (2003) Therefore use multiple methods. Change in fuels results in change in microbes 21 Tetra-ethyl lead is inhibitory to microbes Replacement of EGME with DiEGME caused biodeterioration problems (USAF) Novel fuels will likely alter the microbial population (bacteria will change more quickly than fungi/yeasts) Additives are potential nutrients. 7
Fuel H. resinae from stock centres does not grow in GTL 22 Hormoconis resinae A McFarlane Pseudomonas putida Pseudomonas graminis Hormoconis resinae A McFarlane Candida tropicalis Biofilm formation is influenced by fuel type 24 Water Interface BH 4 weeks GTL JetA1 K Bloxham 8
Fuel Fuel Biofilm formation is influenced by additives 25 Water GTL no additives GTL + MDA 4 weeks GTL + Stadis GTL + LIA K Bloxham Biofilm formation is influenced by additives 26 MDA Stadis Z = N, P or S P is likely to be limiting in most systems Summary 27 A better understanding of what is out there are how they function can improve our confidence in controlling microbial contamination Any change in the system is likely to change the community for better or worse but certainly different New technologies provide unparalleled insight into microbial communities 9
References 28 Microbial contamination and its control in fuels and fuel systems since 1980 - a review Passman FJ (2013) International Biodeterioration and Biodegradation 81 88-104 10