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Assessment of Subsurface in-situ Microbial Communities by Biomarkers for Remediation Potential, Monitoring Effectiveness, and as Rational End-Points.

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  1. Assessment of Subsurface in-situ Microbial Communities by Biomarkers for Remediation Potential, Monitoring Effectiveness, and as Rational End-Points • David C. White, Cory Lytle, Sarah J. Macnaughton, John R. Stephen, Aaron Peacock, Carol A. Smith, Ying Dong Gan,Yun-Juan Chang, Yevette M. Piceno • Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, Microbial Insights, Inc., Rockford, TN, Microbial Insights, Inc. -CEB

  2. In-situ Microbial Community Assessment Classical Plate Count < 1.0 to 0.1% of community, takes days, lose community interactions & Physiology Two Biomarker Methods: DNA: Recover from surface, Amplify with PCR using rDNA primers , Separate with denaturing gradient gel electrophoresis (DGGE), sequence for identification and phylogenetic relationship. Great specificity Lipids: Extract, concentrate, structural analysis Quantitative, Insight into: viable biomass, community composition, Nutritional-physiological status, evidence for metabolic activity

  3. LIPID Biomarker Analysis 1. Intact Membranes essential for Earth-based life 2. Membranes contain Phospholipids 3. Phospholipids have a rapid turnover from endogenous phospholipases . 4. Sufficiently complex to provide biomarkers for viable biomass, community composition, nutritional/physiological status 5. Analysis with extraction provides concentration & purification 6. Structure identifiable by Electrospray Ionization Mass Spectrometry at attomoles/uL (near single bacterial cell) 7. Surface localization, high concentration ideal for organic SIMS mapping localization

  4. Signature Lipid Biomarker Analysis Cathedral from a Brick Predict impact of Cr contamination (from 50-200,000 ppm) on soil microbial community by artificial neural network (ANN) analysis PLFA (phospholipid fatty acid) excellent ~x 102-103 ppm Cr with (PLFA). DNA is “non compressible” ~ perfect code not so influenced By microniche conditions as cell membranes PLFA is compressible as contains physiological status input Contains “holistic’ information & responds to perturbations Predict it is a Cathedral or a Prison : DNA a perfect brick PLFA a non-linear mixture of bricks and a window

  5. Signature Lipid Biomarker Analysis • Phospholipid Fatty Acid [PLFA] Biomarker Analysis = Single most quantitative, comprehensive insight into in-situ microbial community • Why not Universally utilized? • Requires 8 hr extraction with ultrapure solvents [emulsions]. • Ultra clean glassware [incinerated 450oC]. • Fractionation of Polar Lipids • Derivatization [transesterification] • 5. GC/MS analysis ~ picomole detection ~ 104 cells LOD • 6. Arcane Interpretation [Scattered Literature] • 7. 3-4 Days and ~ $250

  6. Signature Lipid Biomarker Analysis • NEW Expanded Lipid Analysis • Utilizes HPLC not GC [Greatly expanded Molecular Sizes] • Semi-automated, ~ “Flash” Extraction ~ 1 hr with fractionation & > recovery from spores • 3. Direct analysis of intact lipids [no derivatization] • 4. Sensitivity ~ Electrospray Ionization [sub femtomolar near single cell as 100% of analyte ionize not 1%] • 5. Specificity ~ Tandem Mass Spectrometry • Neutral loss or gain • Select parent ions • Analysis of specific product ions • Structural analysis of components in MS/MS • [<< Chemical Noise]

  7. Lyophilized Soil Fractions, Pipe Biofilm 1. Neutral Lipids SFECO2 UQ isoprenologues ESE Chloroform.methanol Derivatize –N-methyl pyridyl Diglycerides Sterols Ergostrerol Cholesterol 2. Polar Lipids Transesterify PLFA Intact Lipids Phospholipids PG, PE, PC, Cl, & sn1 sn2 FA Amino Acid PG Ornithine lipid Archea ether lipids Plamalogens 3. In-situ Derivatize in SFECO2 CG/MS PHA Thansesterify & Derivatize N-methyl pyridyl 2,6 DPA (Spores) LPS-Amide OH FA HPLC/ESI/MS/MS

  8. Sequential Extraction & HPLC/ESI/MS analysis ~ 1-2 hrs Extraction SFE/ESE Concentration/ Recovery Fractionation Separation HPLC/in-line Detection HPLC/ESI/MS(CAD)MS or HPLC/ESI/IT(MS)n CEB Microbial Insights, Inc.

  9. Lipid Biomarker Analysis Sequential High Pressure/Temperature Extraction (~ 1 Hour) Supercritical CO2 + Methanol enhancer Neutral Lipids, (Sterols, Diglycerides, Ubiquinones) Lyses Cells Facilitates DNA Recovery (for off-line analysis 2. Polar solvent Extraction Phospholipids CID detect negative ions Plasmalogens Archeal Ethers 3). In-situ Derivatize & Extract Supercritical CO2 + Methanol enhancer 2,6 Dipicolinic acid Bacterial Spores Amide-Linked Hydroxy Fatty acids [Gram-negative LPS] Three Fractions for HPLC/ESI/MS/MS Analysis

  10. Feasibility of “Flash” Extraction ASE vs B&D solvent extraction* Bacteria = B&D, no distortion Fungal Spores = 2 x B&D Bacterial Spores = 3 x B&D Eukaryotic = 3 x polyenoic FA [2 cycles 80oC, 1200 psi, 20 min] vs B&D = 8 -14 Hours *Macnaughton, S. J., T. L. Jenkins, M. H. Wimpee, M. R. Cormier, and D. C. White. 1997. Rapid extraction of lipid biomarkers frompure culture and environmental samples using pressurized accelerated hot solvent extraction. J. Microbial Methods 31: 19-27(1997) CEB Microbial Insights, Inc.

  11. Respiratory Ubiquinone (UQ) Gram-negative Bacteria with Oxygen as terminal acceptor LOQ = 225 femtomole/uL, LOD = 75 femtomole/uL ~ 100E. coli Isocratic 95.5/4.5 % methanol/aqueous 1 mM ammonium acetate Q7 Q10 Q6 197 m/z

  12. Pyridinium Derivative of 1, 2 Dipalmitin [M+92-109]+ M = mass of original Diglyceride LOD ~100 attomoles/ uL [M+92]+

  13. Membrane Liability (turnover) VIABLE NON-VIABLE O O || || H2COC H2COC O O phospholipase | | || || cell death C O CH C O CH | O | || H2 C O H H2 C O P O CH2CN+ H3 | Neutral lipid, ~DGFA O- Polar lipid, ~ PLFA

  14. PE PE PG A PC PG B Separation on HAISIL reverse phase HL C-18 column, 30 mm x 1mm x 3 μ, 95/5 methanol + 0.002% piperidine/water 50 μL/min, post-column modifier 0.02% piperidine in methanol, 10 μL/min. PE C (A) Chromatogram of purified brain and egg yolk derived authentic PG, PE, and PC; (B) Extracted ion chromatogram (EIC) of PG from soil containing 15:0, 16:0, 16:1, 17:0, 17:1, 18:1, 19:1 (see Fig 5); (C) EIC for ions diagnostic of PE from the soil used in B.

  15. HPLC/ESI/MS • Enhanced Sensitivity • Less Sample Preparation • Increased Structural Information • Fragmentation highly specific i.e. no proton donor/acceptor fragmentation processes occurring CEB

  16. ESI (cone voltage) Q-1 CAD Q-3 ESI/MS/MS

  17. Parent product ion MS/MS of synthetic PG Q-1 1ppm PG scan m/z 110-990 (M –H) - Sn1 16:0, Sn2 18:2 Q-3 product ion scan of m/z 747scanned m/z 110-990 Note 50X > sensitivity SIM additional 5x > sensitivity ~ 250X

  18. Archaebacterial Tetraether Lipid FW 1640.4 In sim LOQ ~ 50 ppb ES+ [M-2H+Na+K]+ [M+H]+

  19. Lipid Biomarker Analysis Expanded Lipid Analysis Greatly Increase Specificity ~ Electrospray Ionization ( Cone voltage between skimmer and inlet ) In-Source Collision-induced dissociation (CID) Tandem Mass Spectrometry Scan Q-1 CID* Q-3 Difference Daughter ion Fix Vary Vary Parent ion Vary Fix Vary Neutral loss Vary Vary Fix Neutral gain Vary Vary Fix Select-ion monitoring Fix Fix Fix *Collision-induced dissociation (CID) is a reaction region between quadrupoles

  20. Problem: Rapid Detection of Bacterial Spores & LPS Amide-Linked OH Fatty Acids in Complex Matrices • From the lipid-extracted residue - - - - derivatize (acid methanolysis) & Supercritical Carbon Dioxide + methanol Extract • Detect 2,6 dipicolinate with HPLC/ESI/MS/MS 1 hour and 100% not 3 days and ~ 20% viable • 2. Detect 3-OH Fatty Acids Amide-linked to KDO in LPS of Gram-negative Bacteria with HPLC/ESI/MS/MS • Enterics & Pathogens 3OH 14:0 • Pseudomonad's 3OH 10:0 & 3OH 12:0 • (Should Dog Drink from Toilet Bowl?)

  21. ESI Spectrum of 2, 6-Dimethyl Dipicolinate LOD ~ 103 spores ~ 0.5 femtomoles/ul [M+H]+ ES+ Mobile phase: MeOH + 1mM ammonium acetate Cone: 40V [M+Na]+

  22. Signal Optimization for 2,6 Dimethyl Dipicolinate

  23. Signature Lipid Biomarker Analysis Microniche Properties from Lipids 1. Aerobic microniche/high redox potential.~ high respiratory benzoquinone/PLFA ratio, high proportions of Actinomycetes, and low levels of i15:0/a15:0 (< 0.1) characteristic of Gram-positive Micrococci type bacteria, Sphinganine from Sphingomonas 2. Anaerobic microniches ~high plasmalogen/PLFA ratios (plasmalogens are characteristic Clostridia), the isoprenoid ether lipids of the methanogenic Archae. 3. Microeukaryote predation ~ high proportions of phospholipid polyenoic fatty acids in phosphatidylcholine (PC) and cardiolipin (CL). Decrease Viable biomass (total PLFA) 4. Cell lysis ~ high diglyceride/PLFA ratio.

  24. Signature Lipid Biomarker Analysis Microniche Properties from Lipids 5. Microniches with carbon & terminal electron acceptors with limiting N or Trace growth factors ~ high ( > 0.2) poly β-hydroxyalkonate (PHA)/PLFA ratios 6. Microniches with suboptimal growth conditions (low water activity, nutrients or trace components) ~ high ( > 1) cyclopropane to monoenoic fatty acid ratios in the PG and PE, as well as greater ratios of cardiolipin (CL) to PG ratios. 7. Inadequate bioavailable phosphate ~ high lipid ornithine levels 8. Low pH ~ high lysyl esters of phosphatidyl glycerol (PG) in Gram-positive Micrococci. 9. Toxic exposure ~ high Trans/Cis monoenoic PLFA

  25. ANN Analysis of CR impacted Soil Microbial Communities • Cannelton Tannery Superfund Site, 75 Acres on the Saint Marie River near Sault St. Marie, Upper Peninsula, MI • Contaminated with Cr+3and other heavy metals between1900-1958 by the Northwestern Leather Co. • Cr+3 background ~10-50 mg/Kg to 200,000 mg/Kg. • Contained between ~107-109/g dry wt.viable biomass by PLFA; no correlation with [Cr] (P>0.05) • PLFA biomass correlated (P<001) with TOM &TOC but not with viable counts (P=0.5) -CEB

  26. Sensitivity analysis ranks the inputs by importance in predicting [Cr+3] PLFA have a significant larger predictive value than environment parameters (marked with arrows). PLFA profiles are a can be used as a general purpose biosensor

  27. ANN Analysis of CR impacted Soil Microbial Communities • SENSITIVITY (from ANN) • 20% of the variables accounted for 50% of the predictive of Cr+3 concentration • Of these 20 %: • 18:1w9c (6.6%) Eukaryote (Fungal) correlated with 18:26 (P<0.02) • 10Me 16:0 (2.5%) correlated with i17:0 (4.8), 16:1 11c (2.9), i15:0 (3.1) (P<0.001). Thus all are most likely indicative of SRBs or MRBs. • 18:17c (4.6%) = Gram negative bacteria • 10Me 18:0 (4.3%) (Actinomycetes) -CEB NABIR

  28. ANN Analysis of CR impacted Soil Microbial Communities • CONCLUSIONS: • 1. Non-Linear ANN >> predictor than LinearPCA(principal Components Analysis) • 2. No Direct Correlation (P>0.05) Cr+3 with Biomass (PLFA), Positive correlation between biomass (PLFA) and TOC,TOM • 3. ANN: Sensitivity to Cr+3 Correlates with Microeukaryotes(Fungi)18:19c, and SRB/Metal reducers (i15:0, i 17:0, 16:1w11, and 10Me 16:0) • 4. SRB & Metal reducers peaked 10,000 mg/Kg Cr+3 • 5. PLFA of stress > trans/cis monoenoic, > aliphatic saturated with > Cr+3 -CEB NABIR

  29. Signature Lipid Biomarker Analysis Expand the Lipid Biomarker Analysis 1. Increase speed and recovery of extraction “Flash” 2. Include new lipids responsive to physiological status HPLC (not need derivatization) Respiratory quinone ~ redox & terminal electron acceptor Diglyceride ~ cell lysis Archea ~ methanogens Lipid ornithine ~ bioavailable phosphate Lysyl-phosphatidyl glycerol ~ low pH Poly beta-hydroxy alkanoate ~ unbalanced growth 3. Increased Sensitivity and Specificity ESI/MS/MS

  30. Detection of specific per 13C-labeled bacteria added to soils Extract lipids, HPLC/ESI/MS/MS analysis of phospholipids detect specific PLFA as negative ions PLFA 12C Per 13C 16:1 253 269 same as 12C 17:0 16:0 255 271 Unusual 12C 17:0 (269) + 2 13C  cy17:0 267 284 12C 18:0 (283) + 13C 18:1 281 29912C 20:6 , 12C 19:0 with 2 13C  19:1 295 314 12C 21:5 (315), 12C 21:6 (313)  13C bacteria added  No 13C bacteria added

  31. Wooded Wetland Grassy Wetland Swampy/Cattails Running Water Woodland Grass Pond Beach Removed A Cannelton Tannery Superfund Site

  32. ND 1-50 51-100 101-500 501-1,000 1,001-2,000 2,001-3,000 3,001-5,000 5,001-7,000 7,001-10,000 10,001-25,000 25,001-50,000 50,001-75,000 75,001-100,000 100,001-300,000 Cr+3 ConcentrationsSite map

  33. ANN are universal predictors Schematic architecture of a three layer feedforward network used to associate microbial community typing profiles (MCT) with classification vectors. Symbols correspond to neuronal nodes Capable of learning from examples Generalization is assured by cross-validation

  34. 1E+006 slope = 1.09 100000 R2 = 0.98 10000 1000 Predicted Cr3+ concentration (mg Kg-1) 100 training set validation set regression 10 identity 1 1 10 100 1000 10000 100000 1E+006 Observed Cr3+ concentration (mg Kg-1) Good Predictive Accuracy at > 100 mgCr+3 /Kg

  35. Tandem Mass Spectrometers Ion trap MSn (Tandem in Time) Smaller, Least Expensive, >Sensitive (full scan) Quadrupole/TOF > Mass Range, > Resolution MS/CAD/MS (Tandem in Space) 1. True Parent Ion Scan to Derivative Ion Scan 2. True Neutral Loss Scan 3. Generate Neutral Gain Scan 4. More Quantitative 5. > Sensitivity for SIM 6. > Dynamic Range JPL CEB

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