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

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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
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In-situ Microbial Community Assessment
Classical Plate Count lt 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-physio
logical status, evidence for metabolic activity
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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
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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

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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

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Signature Lipid Biomarker Analysis

• NEW Expanded Lipid Analysis
• Utilizes HPLC not GC Greatly expanded Molecular
  Sizes
• Semi-automated, Flash Extraction 1 hr with
  fractionation gt 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
• ltlt Chemical Noise

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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
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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
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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
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Feasibility of Flash Extraction
ASE vs BD solvent extraction Bacteria BD,
no distortion Fungal Spores 2 x BD Bacterial
Spores 3 x BD Eukaryotic 3 x polyenoic
FA 2 cycles 80oC, 1200 psi, 20 min vs BD
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 from pure culture
and environmental samples using pressurized
accelerated hot solvent extraction. J.
Microbial Methods 31 19-27(1997)
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Microbial Insights, Inc.
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Respiratory Ubiquinone (UQ)
Gram-negative Bacteria with Oxygen as terminal
acceptor LOQ 225 femtomole/uL, LOD 75
femtomole/uL 100 E. coli
Isocratic 95.5/4.5 methanol/aqueous 1 mM
ammonium acetate
Q7
Q10
Q6
197 m/z
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Pyridinium Derivative of 1, 2 Dipalmitin
M92-109
M mass of original Diglyceride LOD 100
attomoles/ uL
M92
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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
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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
150, 160, 161, 170, 171, 181, 191 (see Fig
5) (C) EIC for ions diagnostic of PE from the
soil used in B.
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HPLC/ESI/MS

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

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ESI (cone voltage)
Q-1
CAD
Q-3
ESI/MS/MS
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Parent product ion MS/MS of synthetic PG
Q-1 1ppm PG scan m/z 110-990
(M H) -
Sn1 160, Sn2 182
Q-3 product ion scan of m/z 747 scanned m/z
110-990 Note 50X gt sensitivity
SIM additional 5x gt sensitivity 250X
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Archaebacterial Tetraether Lipid
FW 1640.4
In sim LOQ 50 ppb
ES
M-2HNaK
MH
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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

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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 140
• Pseudomonad's 3OH 100 3OH 120
• (Should Dog Drink from Toilet
  Bowl?)

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ESI Spectrum of 2, 6-Dimethyl Dipicolinate
LOD 103 spores 0.5 femtomoles/ul
MH
ES
Mobile phase MeOH 1mM ammonium acetate Cone
40V
MNa
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Signal Optimization for 2,6 Dimethyl Dipicolinate
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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
i150/a150 (lt 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.

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Signature Lipid Biomarker Analysis
Microniche Properties from Lipids 5.
Microniches with carbon terminal electron
acceptors with limiting N or Trace growth factors
high ( gt 0.2) poly ß-hydroxyalkonate
(PHA)/PLFA ratios 6. Microniches with
suboptimal growth conditions (low water activity,
nutrients or trace components) high ( gt 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

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ANN Analysis of CR impacted Soil Microbial
Communities

1. Cannelton Tannery Superfund Site, 75 Acres on the
   Saint Marie River near Sault St. Marie, Upper
   Peninsula, MI
2. Contaminated with Cr3 and other heavy metals
   between1900-1958 by the Northwestern Leather Co.
3. Cr3 background 10-50 mg/Kg to 200,000 mg/Kg.
4. Contained between 107-109/g dry wt. viable
   biomass by PLFA no correlation with Cr
   (Pgt0.05)
5. PLFA biomass correlated (Plt001) with TOM TOC but
   not with viable counts (P0.5)

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Sensitivity analysis ranks the inputs by
importance in predicting Cr3 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
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ANN Analysis of CR impacted Soil Microbial
Communities
SENSITIVITY (from ANN) 20 of the variables
accounted for 50 of the predictive of Cr3
concentration Of these 20 181w9c (6.6)
Eukaryote (Fungal) correlated with 182?6
(Plt0.02) 10Me 160 (2.5) correlated with i170
(4.8), 161 ?11c (2.9), i150 (3.1) (Plt0.001).
Thus all are most likely indicative of SRBs or
MRBs. 181?7c (4.6) Gram negative
bacteria 10Me 180 (4.3) (Actinomycetes)
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NABIR
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ANN Analysis of CR impacted Soil Microbial
Communities
CONCLUSIONS 1. Non-Linear ANN gtgt predictor than
Linear PCA (principal Components Analysis) 2. No
Direct Correlation (Pgt0.05) Cr3 with Biomass
(PLFA), Positive correlation between biomass
(PLFA) and TOC,TOM 3. ANN Sensitivity to Cr3
Correlates with Microeukaryotes (Fungi)181?9c,
and SRB/Metal reducers (i150, i 170, 161w11,
and 10Me 160) 4. SRB Metal reducers peaked
10,000 mg/Kg Cr3 5. PLFA of stress gt trans/cis
monoenoic, gt aliphatic saturated with gt Cr3
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NABIR
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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

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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
161 253 269 same as
12C 170 160 255 271 Unusual
12C 170 (269) 2 13C ? cy170 267
284 12C 180 (283) 13C 181 281
299 12C 206 , 12C 190 with 2 13C
? 191 295 314 12C 215 (315),
12C 216 (313)
?
13C bacteria added
?
No 13C bacteria added
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Cannelton Tannery Superfund Site
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Cr3 Concentrations Site map
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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
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Good Predictive Accuracy at gt 100 mg Cr3 /Kg
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Tandem Mass Spectrometers
Ion trap MSn (Tandem in Time) Smaller, Least
Expensive, gtSensitive (full scan)
Quadrupole/TOF gt Mass Range, gt 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. gt Sensitivity for SIM 6.
gt Dynamic Range
JPL
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