Transcript PowerPoint slide presentation

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
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
DGGE3 White et al
Increasing sample depth
Bridging
capillary
13.5-14.3’
Saturated
15-16’
Migration Standards
Vadose/
Capillary
fringe
12.2-12.8’
Saturated
17-18’
Migration Standards
Background samples,
single bore-hole,
triplicate sub-samples
Whey-barrier samples,
single bore-hole, triplicate
sub-samples.
(45 days after amendment)
12.4-13.1’
Vadose/
Capillary
fringe
13.7-14.6’
Bridging
capillary
16-17’
Saturated
Key: Prominent bands
derived from:
Background
(No treatment)
Whey-Barrier
Burkholderia and relatives
Bacteroides-FlexibacterCytophaga phylum
Vadose
Saturated
No box = capillary
Arthrobacter and relatives
Neighbor-joining analysis of sequences recovered
from DGGE gels and reference sequences
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
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
Signature Lipid Biomarker Analysis
Phospholipid Fatty Acid [PLFA] Biomarker Analysis =
Single most quantitative, comprehensive insight into insitu microbial community
Why not Universally utilized?
1. Requires 8 hr extraction with ultrapure solvents [emulsions].
2. Ultra clean glassware [incinerated 450oC].
3. Fractionation of Polar Lipids
4. Derivatization [transesterification]
5. GC/MS analysis ~ picomole detection ~ 104 cells LOD
6. Arcane Interpretation [Scattered Literature]
7. 3-4 Days and ~ $250
Signature Lipid Biomarker Analysis
NEW Expanded Lipid Analysis
1. Utilizes HPLC not GC [Greatly expanded Molecular Sizes]
2. 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]
Lyophilized Soil Fractions, Pipe Biofilm
1. Neutral Lipids
SFECO2
UQ isoprenologues
ESE Chloroform.methanol
2. Polar Lipids
Transesterify
Intact Lipids
Derivatize –N-methyl pyridyl
Diglycerides
Sterols
Ergostrerol
Cholesterol
PLFA
CG/MS
Phospholipids
PG, PE, PC, Cl,
& sn1 sn2 FA
Amino Acid PG
Ornithine lipid
Archea ether lipids
Plamalogens
3. In-situ Derivatize in SFECO2
PHA
Thansesterify &
Derivatize
N-methyl pyridyl
2,6 DPA (Spores)
LPS-Amide OH FA
HPLC/ESI/MS/MS
Sequential Extraction & HPLC/ESI/MS analysis ~ 1-2 hrs
Concentration/
Recovery
Extraction Fractionation
SFE/ESE
Separation
Detection
HPLC/in-line
HPLC/ESI/MS(CAD)MS
or
HPLC/ESI/IT(MS)n
CEB
Microbial
Insights, Inc.
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
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 from pure culture and
environmental samples using pressurized accelerated hot solvent
extraction.
J. Microbial Methods 31: 19-27(1997)
Microbial
Insights, Inc.
CEB
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
Q6
Q10
O
H3OC
CH3
H3OC
O
197 m/z
H
]n
Pyridinium Derivative of 1, 2 Dipalmitin
SO3
O
CH 2O C
CH 2(CH 2)13CH 3
O
C
CH 2(CH 2)13CH 3
CHO
+
F
N
CH 3
CH 3
[M+92-109]+
CH 2OH
O
CH 2O C
CH 2(CH 2)13CH 3
O
C
CH 2(CH 2)13CH 3
CHO
C6H7NO
Exact Mass: 109.05
Mol. Wt.: 109.13
neutral loss
C41H73NO5+
Exact Mass: 659.55
Mol. Wt.: 660.02
OCH
N
CH 3
O
N
CH 3
M = mass of original
Diglyceride
O
CH 2O C
CH 2(CH 2)13CH 3
O
C
CH 2(CH 2)13CH 3
CHO
CH 2
C35H67O4+
Exact Mass: 551.50
Mol. Wt.: 551.90
LOD ~100 attomoles/ uL
[M+92]+
Membrane Liability (turnover)
VIABLE
NON-VIABLE
O
O
||
||
H2COC
O H2COC
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
OPolar lipid, ~ PLFA
PG
PG
C
PE
PE
PC
A
B
PE
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.
(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.
HPLC/ESI/MS
• Enhanced Sensitivity
• Less Sample
Preparation
• Increased Structural
Information
• Fragmentation highly
specific i.e. no proton
donor/acceptor
fragmentation
processes occurring
O
X O
P
O
CH2
O
O
HC O C R1
O
R2
C O CH2
CEB
ESI
(cone voltage)
Q-1
ESI/MS/MS
CAD
Q-3
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 747 scanned
m/z 110-990
Note 50X > sensitivity
SIM additional 5x > sensitivity ~ 250X
Archaebacterial Tetraether Lipid
HO
HO
CH2OH
O
O
OH
CH2
CH
O
O
O
CH2 O
CH2
CH2
H2C
OH
CH2
O
CH2
O-
CH2 O P
O
FW 1640.4
In sim LOQ ~ 50 ppb
5 ppm
ES+
1704
100
[M-2H+Na+K]+
1701
1698
[M+H]+
%
1707
16381641
1643
0
1600
1620
1640
1706
1664
1660
1680
1680
1695
1713
1700
m/z
1720
1740
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
Daughter ion
Fix
Parent ion
Vary
Neutral loss
Vary
Neutral gain
Vary
Select-ion monitoring Fix
CID*
Q-3
Vary
Fix
Vary
Vary
Fix
Difference
Vary
Vary
Fix
Fix
Fix
*Collision-induced dissociation (CID) is a reaction region
between quadrupoles
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
1. 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?)
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
H3OC
OCH 3
N
O
O
C9H9NO4
Exact Mass: 195.05
[M+Na]+
Signal Optimization for 2,6 Dimethyl Dipicolinate
1.2E+08
196 m/z
1.0E+08
Response
218 m/z
8.0E+07
168 m/z
6.0E+07
4.0E+07
2.0E+07
0.0E+00
0
20
40
60
Cone Voltage, V
80
100
120
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.
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
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 Cr+3 and other heavy metals
between1900-1958 by the Northwestern Leather Co.
3. Cr+3 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] (P>0.05)
5. PLFA biomass correlated (P<001) with TOM &TOC but
not with viable counts (P=0.5)
-CEB
7.0%
6.0%
90%
5.0%
80%
70%
4.0%
60%
3.0%
50%
40%
2.0%
30%
1.0%
20%
Cummulative sensitivity
0.0%
C181W9C
CI170
C181W7C
C10ME180
CA170
CI151W11
CI151A
C161W5C
CI150
C201W9C
C161W11C
C10ME160
CBR181
CA150
CI160
%TOM
C160
CCY170B
C170
C150
C203W6
CA
C210
PH
C12ME160
C161W7T
C183W3
%TOC
CI171W8
CBR150B
CBR170B
C181W7T
C182A
CBR170A
BIOMASS
C151
P
CI151B
WETLAND
CBR150A
CCY190
MG
CI140
C180
C161W7C
C230
CBR160
K
C11ME160
C205W3
C12ME180
C200
CCY170A
CI151C
C182W6
C140
C220
CI161
C162
C240
CBR150C
C204W6
VIABLE_C
C181W5C
Relative sensitivity
Sensitivity analysis ranks the inputs by importance in predicting [Cr+3]
PLFA have a significant larger predictive value than environment
parameters (marked with arrows).
110%
100%
10%
0%
PLFA profiles are a can be used as a general purpose biosensor
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)
NABIR
-CEB
ANN Analysis of CR impacted Soil Microbial
Communities
CONCLUSIONS:
1. Non-Linear ANN >> predictor than Linear PCA (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
NABIR
-CEB
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
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
cy17:0 267
18:1 281
19:1 295
271 Unusual 12C 17:0 (269) + 2 13C
284 12C 18:0 (283) + 13C
299
314
13C
12C
20:6 , 12C 19:0 with 2 13C
12C 21:5 (315), 12C 21:6 (313)
bacteria added

No 13C bacteria added



U26
T27
Wooded Wetland
Grassy Wetland
Q24 Q26
P23 P25
Swampy/Cattails
O22 O24
N21 N23
Running Water
Woodland
M20
L21
Grass
Pond
K20 K22
J19
J21 J23
I20 I22
Beach
Removed
H15 H17
H19 H21
G18
G14
D9
C8
C4
B5
B7
D11
C10
E16 E18
D17
C16
K28
N
0
400ft
B9
TANNERY
Cannelton Tannery Superfund Site
A
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 Concentrations Site map
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
na
by cross-validation
int
erp
o
lat
Generalization is assured
Predictive error
Capable of
learning from examples
Stop !
int
sig erpo
na
l + latin
no g
ise
sig
h id d e n la y e r
ing
In p u t
p ro file
l
c la ssific a tio n
v e c to r
testing
cross-validated error
training
regression
Good Predictive Accuracy at > 100 mg Cr+3 /Kg
Predicted Cr3+ concentration (mg Kg-1)
1E+006
slope = 1.09
100000
R2 = 0.98
10000
1000
100
training set
validation set
regression
identity
10
1
1
10
100
1000
10000
Observed Cr3+ concentration (mg Kg-1)
100000
1E+006
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