1. Claflin University
NMR based metabolomics study of chromium(VI)
treated Pseudomonas fluorescens (Pf-5)
Yugaananthy Thanaiah
Thesis Defense
Research Mentors: Dr. Randall Harris
Dr. Arezue Boroujerdi

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Outline
• Introduction
– Bioremediation
– Chromium
– Pseudomonas fluorescens – Pf5
– Metabolomics study
• Methodology
– Sample Collection and Chemical Preparation
– Data collection using NMR
– Data analysis using PCA
• Results and Discussion
• Future Work
• Acknowledgements

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Bioremediation
• “Bio-remediate" means to use biological organisms to solve an
environmental problem such as cleaning of contaminated soil or
groundwater.
• Bioaugmentation - bacterial cultures are externally added to the
waste site to speed up the rate of degradation of a contaminant.
• Bioremediation is an environmental friendly and less expensive way
to clean the contaminated sites.
• EPA had listed nearly 1700 sites as
NPL sites under Super Fund.
http://www.epa.gov/superfund/sites/npl/

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Chromium
• Chromium contamination was found in 1127 sites out of the 1699 NPL
sites.
• Chromium is a heavy metal. Naturally occurring Cr forms are Cr(III),
Cr(VI). Most prevalent is Cr(III).
• Chromium(III): Less toxic, trace amount is essential nutrient in human
body.
Chromium(VI): highly toxic, imposes many health hazards, has been
discovered as a strong carcinogen.
• Chromium is mainly released to the environment as industrial waste.
Main contributors are chemical, leather and textile manufacturing.

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Chromium Contamination
• Chromium(VI) levels found in some NPL sites:
– Ground water sample near Aircraft plant in NY: 1400 µg/L Cr(IV) = 1400 ppb
– The sample from NOVACO Industries:
• Due to Chromic acid spill, the concentration of Chromium in wells and ground
water was increased up to 9.40 x 108 ppb (940 000 mg/L)
• The standard concentration of Chromium(VI) after remediation is
expected to be < 50 ppb
• Drinking water standard for Chromium according to WHO Standard:
– 50 ppb total chromium
– 0.2ppb Cr(VI)
• Pseudomonas fluorescens (LB300) has been reported to reduce highly
toxic hexavalent chromium (Cr6+)to a less toxic trivalent chromium(Cr3+).
http://www.epa.gov/superfund/sites/npl/

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 Hypothesis
We hypothesized that the metabolism pattern observed in Pseudomonas
fluorescens (Pf5) will be different because of the effect of chromium(VI).
 Objective
To identify the metabolic pathway differences between the Pf-5 control
sample and stress sample(Chromium (VI)) at different time intervals with
NMR based metabolomics.
“Lawrence H. Bopp, and Henry L. Ehrlich. "Chromate Resistance and Reduction in Pseudomonas Fluorescens Strain LB300-
Springer." Chromate Resistance and Reduction in Pseudomonas Fluorescens Strain LB300 - Springer. Archives of Microbiology, 01 Sept. 1988. Web. 06
June 2014.”

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Pseudomonas fluorescens – Pf5
• Obligate gram-negative bacteria
• Optimum growth temperature : 25°C-30 °C
• Has been found to have role in destroying certain toxins and
pollutants: styrene, polycyclic aromatic hydrocarbons, and TNT.
• Exhibits bio-control properties and produces antibiotic
Mupirocin.

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Metabolomics study
• Study of collection of all metabolites in a
biological cell, tissue, organ or organism.
• Metabolites are the end products of cellular
processes which act as specific fingerprints.
• Environmental metabolomics focuses on
the study of interactions between
organisms and their environment.
• This approach has many advantages for
studying organism–environment
interactions and for assessing the effect of
environmental stress at the molecular level.

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• Sample collection and chemical preparation
• Data collection using NMR
• Data analysis using PCA
• Significant metabolite identification
• Probable pathway Analysis
• Biomarker Identification and Biological Interpretation
Methodology

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Sample collection and chemical preparation
5 mL LB
Broth
Control:
20 mL LB Broth
Chromium Stress:
19.5 μL of K₂Cr₂O₇
20 mL LB Broth
Polar Layer
Non-Polar Layer

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 NMR Spectra
 1D and 2D spectra
 1D spectra: 1H or 13C information
 2D spectra: 1H and 13C information
 1D: Shows metabolic profile or metabolic
fingerprint at given state
Each sample = one spectra
Data Collection using NMR

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Data analysis using PCA
Principal Component Analysis (PCA)
• Statistical technique that takes a large number of data sets and
identifies the pattern in the data.
• Extract the most important information from the data table.
• Compress the size of the data set by keeping only the important
information.

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• Address the chemical shift variability across spectra
• Spectra are divided into small regions called “Buckets”
• Ideally contains one peak per bucket
• Compare the changes within the bucket from each NMR spectra
96,000 buckets to analyze
0.0-10.0ppm NMR spectrum
0.005ppm/variable bucket widths
2000 buckets
x 48 samples
Principal Component Analysis (PCA)

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Scores Plot: each NMR sample = NMR spectrum = one point
Loadings plot: each bucket = one point = ~ one metabolite
Directions in the Scores plot correspond to the directions in the Loadings plot
Principal Component Analysis (PCA)
Identification of significant MetabolitesIdentification of metabolic Profile Separation
A
B
C
D

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Growth Curve and Survival Rate of Pf-5
upon exposure to different Chromium(VI) levels
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 4 8 12 16 20 24
Absorbance
Time (hrs)
Growth Curve of Pf 50 ppm
25 ppm
50 ppm
100 ppm
200 ppm
1 ppm = 1000 ppb
0.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
0 25 50 100 200
BacterialCulture/mL
Chromium(VI) Concentration (ppm)

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Study of Biological Variability among the group:
Constant OD Vs Variable OD
The biological
variability among
the group was
decreased by
matching the ODs
during sample
collection.

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Growth Curve of Pf-5
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0.00 3.00 6.00 9.00 12.00 15.00 18.00 21.00 24.00
Absorbance
Time (hrs)
Control
Chromium stressed
Comparative to the control sample, lower growth rate and survival rate was
observed in the stress sample(Chromium(VI)).
0.00E+00
5.00E+08
1.00E+09
1.50E+09
2.00E+09
0 6 12 24
0 ppm
50 ppm

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7.50 5.00 2.50 ppm
24h Cont.
6h Cr
24h Cr
6h Cont.
Data Collection : 1D Spectra

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PCA : Control Vs Stress sample(Chromium(VI))
at different time intervals
Distinct metabolic profile separation is observed between each
sample groups.

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Distinct metabolic profile
separation is observed
between control sample and
stress sample at 6hrs.
Distinct metabolic profile
separation is observed
between control sample
and stress sample at 24hrs.
DM = 6.1137
DM = 8.075
Effect of Chromium(VI) Stress: PCA Scores Plot of
Control sample Vs Stress samples at 6hrs and 24 hrs

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Distinct metabolic profile
separation is observed
between stress samples at
6hrs and 24hrs.
Distinct metabolic profile
separation is observed between
control samples at 6hrs and 24hrs.
DM = 5.6314
DM = 5.203
Time Effect: PCA Scores Plot of Control sample and
Stress samples at 6hrs Vs 24 hrs

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Mahalanobis Distance and F-Critical values
Statistical Parameters 24HCr6+_24HControl 6HCr6+_6HControl 24HCr6+_6HCr6+ 24HControl_6HControl
DM 6.1137 8.075 5.6314 5.203
T2 112.1317 195.6154 95.1381 81.2131
F-True 50.4593 88.0269 42.8122 36.5459
F-Critical (95%) 4.96 4.96 4.96 4.96
Significant Status Yes Yes Yes Yes
 The F-true value greater than the F-critical value revealed that the
separation between the groups are statistically significant.

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Potential Significant Metabolites
Metabolites FC Avg 6H FC Avg 24H
Glutamate 1.7483 0.3915
acetate 1.6237 1.0000
lactate 1.5412 1.0000
succinate 1.2832 1.0000
betaine 0.7597 0.8685
ornithine 0.4610 1.0000
glycerol 1.5329 1.7735
alanine 1.0000 2.0295
3-hydroxyisovalerate 2.2283 2.4960
dimethylsulfone 1.4476 2.5784
isobutyrate 2.0363 2.6977
formate 1.7860 2.8607
valine 1.8719 2.9586
adenine 1.0000 3.5554
leucine 1.9470 3.5792

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Fold Change Analysis of the Metabolites
 Fold change is equal to the concentration ratio of the metabolite expressed in
the Stress sample to the Control sample at a given time point.
 The fold change value greater than one means the concentration of a specific
metabolite is higher in the stress sample than the control sample.

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Probable Significant Metabolic Pathways

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Discussion
• The metabolism pattern observed in Pseudomonas fluorescens (Pf5)
was hypothesized to be different because of the effect of chromium.
• PCA analysis confirmed that there is distinct metabolic profile
separation between the control and stress samples.
• The metabolic profile separation suggests that the chromium(VI) stress
could have induced a change in the metabolic pathway.
• Potential significant metabolites have been identified by assigning the
corresponding NMR peaks using Chenomx NMR suite.
• Probable metabolic pathways which could have been changed due to
chromium(VI) stress were identified using MetaboAnalyst.

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• Reconfirmation of the significant metabolites
• Identification of the critical metabolic pathway responsible for
Chromium(VI) resistance in Pseudomonas fluorescens Pf -5.
• Biomarker Identification and Biological Interpretation.
Future Work

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Acknowledgement
• My research mentors Dr. Randall Harris and Dr. Arezue Boroujerdi.
• My committee members Dr. Chowdhury and Dr. Raja.
• Claflin University, Department of Biology and Department of
Chemistry.
• Department of Energy-Environmental Management (DE-
EM0000479) .
• My research partners Jessica A. Fuller and Kareem Altidor.

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Thank You