1. NMR BASED METABOLOMICS STUDY OF CHROMIUM (IV) TREATED
PSEUDOMONAS FLUORESCENS PF-5
BY
YUGAANANTHY THANAIAH
A Thesis Submitted in Partial Fulfillment of the Requirements for the
Degree of Master of Science in Biotechnology in the
Department of Biology, School of Natural Sciences and Mathematics,
Claflin University,
Orangeburg, South Carolina.
April 2015
Dr. Randall H. Harris Dr. Arezue Boroujerdi
Thesis Advisor Thesis Co-Advisor
Dr. Muthukrishna Raja Dr. Kamal Chowdhury
Committee Member Committee Member
Received by: Date:
Dr. Ewen McLean, Chairperson, Department of Biology
Received by: Date:
Dr. Verlie Tisdale, Dean, School of Natural Sciences and Mathematics
Received by: Date:
Dr. Angela Peters, Vice Provost for Academic Programs

2. ii
ACKNOWLEDGEMENTS
I would like to thank God, for all the guidance and invaluable opportunities he had given
me throughout my life in Claflin. I would like to express my sincere gratitude to Dr. Randall H.
Harris for accepting me as his mentee and for the invaluable opportunities and memories
obtained during this study. Also I would like to thank Dr. Arezue Boroujerdi for her continuous
encouragement and guidance throughout the implementation of this research project. Further, I
would like to thank my parents for always supporting me and encouraging me to follow my
dreams. In addition I would like to thank my better half for being so understanding and
supportive throughout the days. Furthermore, I would like to thank my family and friends for
their understanding, encouragement and support. I wish all my classmates a successful and
happy future and would like to thank them all for all their support, guidance and the sweet
memories. Finally I would like to thank my research partners Jessica A. Fuller and Kareem
Altidor for their great assistance in pursuing this research.

3. iii
THESIS STATEMENT
Pseudomonas fluorescens (LB300) has been reported to reduce highly toxic hexavalent
chromium to a less toxic trivalent chromium (Bopp and Ehrlich 1988, DeLeo and Ehrlich 1994).
We hypothesized that the metabolism pattern observed in P. fluorescens (Pf-5) will be different
because of the effect of hexavalent chromium. The objective of the research is to identify the
metabolic pathways that are altered due to the hexavalent chromium stress in P. fluorescens Pf-5
using NMR based metabolomics.

4. iv
ABSTRACT
DEPARTMENT OF BIOLOGY
YUGAANANTHY THANAIAH M.S. CLAFLIN UNIVERSITY, 2015
Advisors: Dr. Randall H. Harris, Dr. Arezue Boroujerdi.
Thesis Dated: April, 2015
Soil and groundwater contamination by heavy metals from nuclear and industrial wastes
is one of the major problems found at sites within the United States. Out of the 1699 sites on the
National Priorities List from the Superfund Program administered by the Environmental
Protection Agency, 1127 sites were reported to be highly contaminated with the heavy metal
chromium. Even though chromium (VI) has been discovered as a strong carcinogen, chromium
(III) is reported to be less toxic. Bioremediation uses microorganisms to transform hazardous
contaminants into forms that are less toxic than the parent materials and is considered to be a
cheap and environmental friendly method. Pseudomonas fluorescens species were reported to
reduce highly toxic chromium (VI) to a less toxic chromium (III).
In this study, we use NMR based metabolomics to study the changes in metabolic
pathways due to chromium stress on P. fluorescens Pf-5. P. fluorescens Pf-5 overnight cultures
containing 50 ppm K2Cr2O7 were incubated at 25 o
C with shaking (200 rpm) for 6h and 24h. At
each time point, samples were collected and processed to obtain bacterial pellets. The polar
metabolites were extracted from the pellets through a methanol/chloroform/water two phase
solvent extraction process. The polar phase was dried and dissolved in NMR buffer. The NMR
samples were analyzed using Bruker 700 MHz NMR. The results were statistically analyzed
using Principal Component Analysis (PCA). Distinct metabolic profile separation was observed
between each sample group (6h control versus 6h chromium stressed, 24h control versus 24h
chromium stressed). Among all combinations, the metabolic profile separation observed between
control samples at 24 h and chromium stressed samples at 24 h was most prominent. The
metabolic profile separation observed in PCA suggests that the chromium stress could have
induced a change in the metabolic pathways of P. fluorescens Pf-5. Currently, further research is
being conducted to analyze and identify potential critical metabolic pathways responsible for
chromium resistance in P. fluorescens Pf-5 that can serve as possible biomarkers of chromium
resistance.

5. v
KEYWORDS AND ABBREVIATIONS
Keywords: chromium (VI), Pseudomonas fluorescens, bioremediation, growth analysis, NMR,
PCA analysis, metabolomics, pathway analysis.
Abbreviations
EPA Environmental Protection Agency
PCA Principal Component Analysis
NMR Nuclear Magnetic Resonance Spectroscopy
LB Luria Bertani
OD Optical Density
VOD Variable Optical Density
COD Constant Optical Density

6. vi
LIST OF TABLES
Table 1. Mahalanobis Distance and F-Critical values..............................................................24
Table 2. Significant Metabolites and Fold change....................................................................26
Table 3. Probable Metabolic Pathways......................................................................................29

7. vii
LIST OF FIGURES
Figure 1. Growth Curve of Pf-5..................................................................................................14
Figure 2. The survival rate of Pf-5 treated with different concentrations of K2Cr2O7.........15
Figure 3. Growth Curve of the Control and the Chromium stressed sample........................16
Figure 4. The survival rate of Pf-5 treated with 50 ppm K2Cr2O7..........................................17
Figure 5. PCA Score Plot of Control Sample at 6hrs and 24 hrs with variable OD
readings (VOD) and Constant OD readings (COD).................................................................19
Figure 6. PCA Score Plot of Control and Chromium stressed sample at 6hrs and 24 hrs...20
Figure 7. PCA Score Plot of Control Vs Chromium stressed sample at 6hrs and 24hrs......21
Figure 8. PCA Score Plot of Control and Chromium stressed samples at 6hrs Vs 24 hrs....22
Figure 9. Fold Change of the Metabolites..................................................................................27
Figure 10. Pathway Analysis.......................................................................................................30

8. viii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS..........................................................................................................ii
THESIS STATEMENT................................................................................................................iii
ABSTRACT...................................................................................................................................iv
KEYWORDS AND ABBREVIATIONS.....................................................................................v
LIST OF TABLES........................................................................................................................vi
LIST OF FIGURES.....................................................................................................................vii
TABLE OF CONTENTS...........................................................................................................viii
INTRODUCTION.........................................................................................................................1
BACKGROUND AND LITERATURE REVIEW.....................................................................5
METHODOLOGY........................................................................................................................8
RESULTS.....................................................................................................................................13
DISCUSSION...............................................................................................................................28
REFERENCES.............................................................................................................................33

9. 1
INTRODUCTION
Chromium is one of the main heavy metals that is widely used by industries such as
electroplating, leather tanning, corrosion inhibition, wood treatment and textile dying (Joutey et
al., 2015; DeLeo and Ehrlich, 1994). Chromium is predominantly available in the environment
in the forms of chromium (III) and chromium (VI). The trivalent chromium combines with the
organic matter available in the environment and forms insoluble amorphous hydroxide
complexes (Cheung and Gu, 2007). Hexavalent chromium is highly soluble and mobile in
ground water systems. Thus it gets into the biological ecosystem easily. Hexavalent chromium is
essential for human beings, animals and plants in trace amounts, but at high concentrations it is
highly toxic, carcinogenic and mutagenic (Joutey et al., 2015; Deepali, 2011). The
Environmental Protection Agency (EPA) has enforced that the total concentration of chromium
in drinking water should be less than 0.1 mg/L or 100 ppb. The World Health Organization
(WHO) designated guideline value for total chromium is 0.05 mg/L or 50 ppb.
Large quantities of solid and liquid waste containing hexavalent chromium are being
dumped into the environment from industries. In a ground water sample collected near an aircraft
plant in New York, the hexavalent chromium level was found to be 1400 ppb. After a chromic-
acid spill happened at NOVACO chemical industries in Michigan, the chromium concentration
observed in the nearby wells and groundwater had increased up to 9.40 x 108
ppb. Different
kinds of technologies are being used to remove heavy metal contaminations such as soil removal
and land filling, precipitation followed by sedimentation, addition of chemical reductant,
physico-chemical extraction, soil washing, flushing and phytoremediation (Jeyasingh and Philip,
2005). These conventional methods fails to meet the complete satisfaction of waste management

10. 2
associations because they only offer temporary solutions, or the capital and operational costs
become high, or the cost of disposal of metal sludge becomes costly.
Bioremediation is considered to be one of the most promising and economical ways to
clean up chromium contaminated waste sites (Jeyasingh and Philip, 2005; Kamaludeen et al.,
2003). The detoxification of chromium through bioremediation mainly focuses on converting the
toxic hexavalent chromium into less toxic trivalent chromium through the usage of
microorganisms. The reduced trivalent chromium is immobilized by forming amorphous
hydroxide complexes with the available organics and thus eliminate the toxicity (Jeyasingh and
Philip, 2005). In bioremediation, microorganisms such as bacteria or fungi are used to degrade or
transform the hazardous contaminants present in the soil, sediments, ground water and sludge
into less harmful forms.
Bioremediation could be achieved through in situ or ex situ treatment of the contaminated
sites. In an ex situ process, the contaminant is removed from the contamination site and treated in
a laboratory or a waste management site. In situ processes involve the treatment of contaminants
at the original place of contamination. Even though, the in situ process is less expensive, it is
difficult to control the environmental conditions during the cleaning process. During the in situ
process the efficiency of the bacterial activity could be enhanced through two major ways called
biostimulation and bioaugmentation (Joutey et al., 2015). In biostimulation, nutrients, air,
organic substances and other essential substances are externally added to the contaminated site to
promote/enhance bacterial growth and bioremediation activity. In bioaugmentation, active
bacterial cultures are externally added to the waste site where the bacteria necessary to degrade
the contaminant are not naturally available.

11. 3
Development of a feasible bioremediation strategy to treat chromium contaminated site
involves many processes such as isolation of efficient chromate-reducing bacterial strains,
evaluation of their ability to survive, multiply, and reduce hexavalent chromium, and the
development of efficient economical treatment methods using these bacterial strains (Ganguli
and Tripathi, 2002). The identification of P. dechromaticans as active chromium resistant
bacteria from sewage sludge in 1970 opened up the possibility of the usage of bioremediation to
treat chromium waste (Joutey et al., 2015; Bopp and Ehrlich, 1998). Many different kinds of
chromium-resistant microorganisms such as Bacillus cereus, B. subtilis, Ps. aureginosa, Ps.
ambigua, P. fluorescens, Escherichia coli, Mocrococcus roseus, Enterobacter cloacae,
Desulfovibrio desulfuricans and D. vulgaris have been successfully isolated from waste sites
(Cheung and Gu, 2007). Different strains of P. fluorescens have been reported to have chromium
resistance and to reduce chromium aerobically and anaerobically (DeLeo and Ehrlich, 1994;
Cheung and Gu, 2007; Deepali, 2011; Bopp and Ehrlich, 1998).
Currently, NMR based metabolomics has become a leading analytical technique to study
the alterations of metabolic pathways due to an external stress (Kim et al., 2007). NMR is an
efficient non-destructive method to analyze small quantities of polar or non polar
compounds/metabolites. The identification of different metabolites present in a sample can
reveal the cellular/metabolic pathway adjustments that have been undergone due to an external
stress (Viant et al., 2008). Each NMR spectrum contains large amounts of information about
different kinds of metabolites available in the sample. The unbiased statistical approach based on
pattern recognition technique called principal components analysis (PCA) is used to extract the
necessary information from the given NMR data. This extracted information could be used to
identify the metabolites whose concentrations were changed significantly due to the applied

12. 4
stress (Nicholson et al., 2008). These significant metabolites are used in the identification of the
probable metabolic pathways that could be used as biomarkers for a particular condition.
P. fluorescens strain LB300 was reported to reduce chromate both aerobically and
anaerobically (Bopp and Ehrlich, 1998; DeLeo and Ehrlich, 1994). In our study, we analyzed
the chromium resistance and reduction capability of P. fluorescens strain Pf-5, and to proposed
metabolic pathways responsible for chromium reduction.

13. 5
BACKGROUND AND LITERATURE REVIEW
In the study carried out by Deepali the bioremediation capability of P. putida was
examined. The bacterial growth was examined under solutions having 10, 20, 30, 40 and 50
mg/L chromium concentrations at 30 o
C. Nine different strains were screened for chromium
tolerance. Bacillus sp., P. aeruginosa and P. putida expressed high tolerance even at 50.0 mg/L.
Chromium metal uptake by different bacterial strains was monitored under 10, 20, 30, 40 and 50
mg/L chromium concentrations up to 96 hours. For Bacillus sp. chromium removal of nearly
75% was observed at 10 mg/L. Pseudomonas aeruginosa removed nearly 70% chromium at 40
mg/L. The chromium removal efficiency was initially higher and got saturated with time, due to
the binding of chromium at the active sites (Deepali 2011).
In the study by Ganguli and Tripathy (2002) the effect of chromium reduction in P.
aeruginosa was examined by introducing the bacterial culture in three different types. The
bacterial culture was introduced to 10, 25, 50, and 100 mg/L chromium as batch culture, in a
dialysis bioreactor and as a biofilm on a rotating biocontractor (Ganguli and Tripathy 2002). At
high chromium concentrations the chromate reduction rate was lower due to the adverse effects
caused by the chromium on the bacterial metabolism. Bacterial cells inside the dialysis sac were
better protected from the chromium toxicity.
In the growth study performed by DeLeo and Ehrlich (1994), P. fluorescens LB300 strain
under different chromium concentrations revealed that the bacteria culture is capable of reducing
high chromium concentrations. In the shaken mode the reduction rate was drastically decreased.
This observation reveals that in the aerobic chromium reduction, Cr (VI) and O2 are competing
with each other to be efficient terminal electron acceptors (DeLeo and Ehrlich 1994).

14. 6
Chromium detoxification by microorganisms could be achieved through different
biochemical mechanisms. Due to the structural resemblance between chromate and sulfate,
hexavalent chromium is taken up by the cells through the sulfate transport channels available in
the membrane. This hexavalent chromium will react with the intracellular reductants such as
ascorbate or glutathione aerobically and will form short lived intermediates, free radicals and
trivalent chromium (Cheung and Gu 2007). The chromium resistance in P. aeruginosa was due
to the enhancement in the efflux of the hexavalent chromium or due to the reduction of the
uptake. In the absence of oxygen, the hexavalent chromium will act as the terminal electron
acceptor in the respiratory chain of sulfur reducing bacteria (SRB). The energy generated during
the chromium detoxification process in the SRB will be used up for the growth of the bacteria.
In the study carried out by Alvarez et al. (1999) they have identified that the chromate
efflux systems plays a main function in chromium resistance. When the ChrA protein was
expressed in P. aeruginosa chromate accumulation was increased by four-fold. It was also stated
that the chromium uptake by the vesicles was dependant on NADH oxidation and the uptake was
affected by energy inhibitors and sulfate (Joutey et al. 2015). Several enzymes with different
metabolic functions have been reported to be able to catalyze chromium (VI) reduction in the
bacteria; such enzymes include Cr (VI) reductase, aldehyde oxidase, cytochrome P450,
nitroreductase, iron reductase, quinone reductases, flavin reductases and NADH/NADPH
dependant reductases.
Recent studies have revealed that the chromium reduction efficiency could be affected by
several environmental factors such as competing electron acceptors, pH, temperature, redox
potential, presence of other metals, complexing agents and type of electron donor. The
complexing agents will chelate the trivalent chromium and other intermediates and thus will

15. 7
increase the chromium reduction efficiency. Low molecular weight carbohydrates, amino acids
and fatty acids were identified as optimal electron donors. The presence of biogenic sulfide and
Fe (II) also helped to promote the metabolic pathways which have an indirect positive impact on
the chromium reduction (Lloyd 2003).

16. 8
MATERIALS AND METHODS
Materials
P. fluorescens Pf-5 was purchased from American Type Culture Collection (Manassas,
VA). Luria Bertani (LB) and potassium dichromate (K2Cr2O7) were purchased from Fisher
Scientific (Pittsburg, PA).
Growth Curve Analysis
P. fluorescens Pf-5 was streaked for isolation onto LB Agar and the LB plate was
incubated at 25 o
C for two days. A single colony was picked from the LB plate and was
inoculated into 3 mL of LB broth to prepare the overnight culture. The prepared overnight
culture sample was incubated at 25 o
C with 200 rpm shaking for 24 hrs. The experiment was
planned to measure the OD readings of the bacterial sample exposed to different chromium
concentrations (0, 25, 50, 100, and 200 ppm) at different time points (0, 2, 4, 6, 8, 10, 12, 16, and
24 hrs). Aliquots of 15 mL of the LB broth were added to all the tubes and corresponding
volumes of 51200 ppm K2Cr2O7 (0 µL = 0 ppm, 7.32 µL = 25 ppm, 14.65 µL = 50 ppm, 29.30
µL = 100 ppm, 58.60 µL = 200 ppm) were added to the broth and mixed well. Aliquots of 150
µL of the bacterial overnight culture were added to all the tubes. The time was recorded and the
samples were incubated at 25 o
C with 200 rpm shaking. At each time point, aliquots of 1 mL
were transferred into cuvettes and the absorbance was recorded at 600 nm using fresh LB broth
as the blank.

17. 9
Drop plate Analysis
Aliquots of 225 µL of 0.9% NaCl solution were added into the wells (8 wells for one
sample and were numbered 1 through 8) in a 96 well plate and 25 µL of the bacterial sample was
added to well #1. Well #1 was mixed well and 25 µL was transferred into well #2. This serial
dilution was performed up to well #8. The quadrants were marked on the LB agar plate and were
numbered from 1 through 8 (Plate1: 1 through 4, Plate2: 5 through 8). In each quadrant three
spots of 10 µL of the sample from the appropriate well was placed and allowed to dry. The plates
were incubated at 25 o
C for two days and the colonies were calculated.
Sample Preparation for Metabolomics Study
P. fluorescens Pf-5 was streaked for isolation onto LB agar and the LB plate was
incubated at 25 o
C for two days. A single colony was picked from the LB plate and was
inoculated into 5 mL of LB Broth to prepare the overnight culture. The prepared overnight
culture sample was incubated at 25 o
C with 200 rpm shaking for 24 hrs. The experiment was
planned to have sampling at 6 hrs and 24 hrs for control and chromium stressed samples. The 50
mL conical tubes (8 tubes for each time point) were obtained and 20 mL of LB broth was added
into each of them. For the chromium stressed samples (6 hrs, 24 hrs containing 8 tubes each)
19.5 µL of 51200 ppm K2Cr2O7 was added and mixed well. Then, 200 µL of the bacterial
overnight culture was added into all the samples and the time was noted. The samples were
mixed well and were incubated at 25 o
C with 200 rpm shaking. At each time point 1 mL of the
sample was transferred into a cuvette and the absorption was measured at 600 nm using a
spectrophotometer. When the OD readings of the samples were almost similar, 15 mL of each of
the samples were transferred into fresh 50 mL conical tubes. Aliquots of ~ 3-4 mL of liquid

18. 10
nitrogen were poured into the sample directly to quickly quench metabolism. The tubes were
kept on ice to keep metabolism quenched. The samples were centrifuged at 5000 rpm at 4 o
C for
10 min. A 1 mL aliquot of supernatant was removed from each tube and transferred into a 1.5
mL microcentrifuge tube and was stored at - 80 o
C. The rest of the supernatant was discarded and
the pellet was washed with ~ 3 mL of 0.9% NaCl solution and was centrifuged at 5000 rpm at 4
o
C for 10 min. The supernatant was discarded and the cells were redissolved in 1 mL of 0.9%
NaCl. The solution was mixed well and the cells were transferred into the pre-weighed
microcentrifuge tube. These were centrifuged at 16000 rpm at 4 o
C for 5 min and the supernatant
was discarded and the pellet was dipped in liquid nitrogen and the tubes were kept on ice. The
wet weights of the tubes containing the sample were recorded and the tubes were frozen at -80 º
C
for 2-3 hours. The tubes were freeze-dried overnight using a lyophilizer to permanently quench
the metabolism. The dry weight of the tubes were recorded and the tubes were stored at -80 º
C.
Metabolite Extraction
Methanol-chloroform two phase extraction was performed as described by Kim et al.
(2010). Approximately 20 mg of dried and homogenized bacterial cells were used for the
extraction and the solvent volumes were calculated according to Bligh and Dyer method (Bligh
et al. 1959; Wu et al. 2008). The dry sample was rehydrated with the appropriate ice-cold
methanol and water solution. The mixture was mixed well and transferred into the ice-cold
chloroform and water solution in glass tubes. The tubes were incubated on ice for 10 min and
were centrifuged at 10000 rpm at 4 º
C for 10 min. The top layer consisting the polar metabolites
was transferred into the new microcentrifuge tube. The bottom layer consisting the non-polar

19. 11
metabolites was transferred into the glass tubes. The polar metabolites were dried using
Centrivap overnight and stored at -20 º
C until further preparation for NMR analysis.
NMR Sample preparation and NMR Data Collection
The dried polar metabolites were resuspended in 620 µL of NMR buffer (100 mM
sodium phosphate buffer - pH 7.3, 1 mM TMSP and 0.1% sodium azide in D2O). The mixture
was vortexed and centrifuged at 1000 rpm 4 º
C for 5 min. Aliquot of 600 µL of the solution was
transferred into the NMR tubes without disturbing the bottom. The 1D 1
H NOESY NMR spectra
were recorded with Bruker 700 MHz NMR Spectrometer. All NMR data were collected with 2.9
s acquisition time and spectral width of 16.0 ppm. The NOESY spectra were collected with 120
scans, 4 dummy scans, 3 s relaxation delay. On-resonance pre-saturation was used for solvent
suppression during the relaxation delay. The 90º pulse widths were measured using TopSpin3.2.
NMR Data Analysis
The NMR spectra were phased using Bruker TopSpin 3.2 Software and the buckets for
the PCA analysis were created using AMIX 3.9.7 software. The spectra were normalized to total
intensity and were binned into 0.01 ppm wide buckets over the region of 0.5 ppm - 9.0 ppm
using advanced bucketing in AMIX. The water region (4.7505 - 4.8558 ppm) was excluded in all
the bucket creations and further analysis. Analysis was done with and without Betaine region
(3.2480 - 3.3070 ppm, 3.8988 - 3.9171 ppm). Betaine was removed because it was an extremely
large peak compared to all other metabolites and therefore suppressed the significance of less
intense metabolic peaks. PCA was performed on the bucket tables created using MetaboAnalyst
3.0 (http://www.metaboanalyst.ca/MetaboAnalyst/) (Xia et al. 2015).

20. 12
Spectral analysis and Metabolite Identification
To identify the significant metabolites between control and Cr (VI) stressed samples, fold
change and p- values were determined for each of the buckets using MetaboAnalyst and AMIX.
Buckets with corresponding p-values less than 0.5 were considered statistically significant and
metabolites corresponding to these peaks were identified using Chenomx NMR Suite. These
significant metabolites were used to establish the significant metabolic pathway using the
Metabolic Pathway Analysis component of MetaboAnalyst 3.0 (Xia et al. 2015).

21. 13
RESULTS
Growth Curve Analysis and Drop plate Analysis
The results of the growth curve analysis of P. fluorescens Pf-5 under different
concentrations of K2Cr2O7 is shown in Figure 1. The growth pattern under different
concentration of K2Cr2O7 was studied to analyze the effect of K2Cr2O7 on the growth of Pf-5.
According to Figure 1, when the K2Cr2O7 concentration was increased, the growth rate of Pf-5
was decreased. The growth rate was higher in the control and the 25 ppm conditions. But a
decrease in the growth rate was observed at 50 ppm and 100 ppm. At 200 ppm the growth curve
was almost flat.
To further confirm these results, drop plate analysis was performed for the samples after
24 hrs incubation at 25 º
C. The survival data of Pf-5 (Figure 2A, 2B) with different
concentrations of K2Cr2O7 clearly depicts the decrease of the survival rate with increase in the
K2Cr2O7 concentration. With the growth curve data and drop plate analysis, 50 ppm was selected
as the sub-lethal concentration to pursue the experiments, because at 50 ppm the growth rate of
the bacteria was decreased but still the bacteria were able to survive.
To see the effect of the selected concentration on the growth rate, the growth curve
analysis (Figure 3) and drop plate analysis (Figure 4A, 4B) were performed for the control (0
ppm of K2Cr2O7 ) and the chromium stressed sample (50 ppm of K2Cr2O7). According to both
results, it was concluded that 50 ppm chromium stress has a negative effect on the growth of P.
fluorescens Pf-5.

22. 14
Figure 1. Growth Curve of Pf-5
Figure 1. The absorbance readings of the Pf-5 incubated at 25 º
C with different concentrations of
the K2Cr2O7 were measured at 600 nm at corresponding time periods.

23. 15
Figure 2. The survival rate of Pf-5 treated with different concentrations of K2Cr2O7
Figure 2. The drop plate analysis of Pf-5 under different chromium concentrations after 24 hr of
incubation at 25 º
C.

24. 16
Figure 3. Growth Curve of the Control and the Chromium stressed sample
Figure 3. The absorbance reading of the Pf-5 incubated at 25 º
C with 0 or 50 ppm K2Cr2O7 at
600 nm at 0, 6, 12, and 24 hr time periods.
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
-1.00 4.00 9.00 14.00 19.00 24.00
Absorbance
Time (hrs)
Control
Chromium stressed

25. 17
Figure 4. The survival rate of Pf-5 treated with 50 ppm K2Cr2O7
Figure 4. The drop plate analysis of the Pf-5 incubated with 0 and 50 ppm K2Cr2O7 at 25 º
C was
done at 0, 6, 12, and 24 hr time periods.

26. 18
PCA Analysis
PCA analysis was done to create scores plots to identify the metabolic profile separation
among the different sample groups. Initially the PCA was created for the control samples after 6
hrs and 24 hrs using two different sampling methods. In method 1, the sampling was done
exactly after 6 hrs and 24 hrs (VOD). In method 2, the sampling was done when the OD reading
of the samples were almost equal (COD). According to the PCA results (Figure 5), the biological
variability among the group was higher in the VOD samples comparative to COD. Therefore the
future experiments were planned to continue with the sampling method 2.
Figure 6 shows the PCA scores plot of all the four groups (the control sample at 6 hrs,
control sample 24 hrs, chromium stressed sample at 6hrs and chromium stressed sample at 24
hrs). Significant profile separation was observed in between all the four groups. To further
analyze the separation, pair wise PCA analysis was performed.
Initially, the pair wise profile separation between the control sample and chromium
stressed sample at 24 hrs was analyzed (Figure 7A). Significant profile separation was observed
with 6.1137 mahalanobis distance. The pair wise profile separation between the control sample
and chromium stressed sample at 6 hrs is shown in Figure 7B. Significant profile separation was
observed with 8.075 mahalanobis distance. The separation observed at 6 hrs was higher than the
separation at 24 hrs.
To analyze the time effect on the samples, initially the pair wise profile separation of the
chromium stressed sample at 6 hrs and 24 hrs was analyzed (Figure 8A). Significant profile
separation was observed with 5.6314 mahalanobis distance. Figure 8B shows pair wise profile
separation of the control sample at 6hrs and 24 hrs. Significant profile separation was observed
with 5.203 mahalanobis distance. Higher separation was found in the chromium stressed sample.

27. 19
Figure 5. PCA Score Plot of Control Sample at 6hrs and 24 hrs with variable OD readings
(VOD) and Constant OD readings (COD)
Figure 5. The profile separation between the control sample after 6hrs and 24hrs analyzed using
PCA Scores plot. For the VOD samples the mechanism was quenched and samples were
collected exactly after 6hrs and 24hrs. For the COD samples the sampling was done after 6hrs
and 24hrs, when the OD readings of the samples were almost equal.

28. 20
Figure 6. PCA Scores Plot of Control and Chromium stressed sample at 6hrs and 24 hrs
Figure 6. The profile separation between all the four groups (the control sample at 6hrs, control
sample 24hrs, chromium stressed sample at 6hrs and chromium stressed sample at 24 hrs) were
analyzed in the above PCA score plot. The chromium stressed was prepared by introducing the
Pf-5 to 50 ppm K2Cr2O7.

29. 21
Figure 7. PCA Scores Plot of Control Vs Chromium stressed sample at 6hrs and 24hrs
Figure 7. In Figure 7A - The pair wise profile separation between the control sample and
chromium stressed sample at 24 hrs was analyzed. In Figure 7B - The pair wise profile
separation between the control sample and chromium stressed sample at 6 hrs was analyzed.
The chromium stressed was prepared by introducing the Pf-5 to 50 ppm K2Cr2O7.

30. 22
Figure 8. PCA Scores Plot of Control and Chromium stressed samples at 6hrs Vs 24 hrs
Figure 8. In Figure 8A - The pair wise profile separation of the chromium stressed sample at
6hrs and 24 hrs was analyzed. The chromium stressed was prepared by introducing the Pf-5 to 50
ppm K2Cr2O7. In Figure 8B - The pair wise profile separation of the control sample at 6hrs and
24 hrs was analyzed.

31. 23
Mahalanobis Distance and F-value calculation
The T2
value and F-value were calculated using MatLab (Table 1). The F-Critical
value was calculated as 4.96 using the standard F value calculation methodology. F-true value of
50.4593 was observed between the control and chromium stressed sample at 24 hrs. F-true value
of 88.0269 was observed between the control and chromium stressed sample at 6 hrs. F-true
value of 42.8122 was observed between chromium stressed sample at 6 hrs and 24 hrs. F-true
value of 36.5459 was observed between control sample at 6 hrs and 24 hrs. The F-true value
greater than the F-critical value revealed that the separation between the groups are statistically
significant.

32. 24
Table 1. Mahalanobis Distance and F-Critical values
Statistical
Parameters
24HCr6+ Vs
24HControl
6HCr6+ Vs
6HControl
24HCr6+ Vs
6HCr6+
24HControl Vs
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 4.96 4.96 4.96 4.96
Significant
Status
Yes Yes Yes Yes
Mahalanobis Distance (DM) and F values were calculated by MatLab

33. 25
Significant Metabolite Identification and Fold Change Analysis
To identify the significant metabolites the loading plot and volcano plots were created.
By assigning the significant buckets to the corresponding metabolites using Chenomx the
significant metabolites were identified (Table 2). Fold change is equal to the concentration ratio
of the metabolite expressed in the chromium stressed sample and control sample at a given time
point. According to the fold change analysis of metabolites at 6 hrs and 24 hrs the effect of
metabolite on the chromium stress could be confirmed (Figure 9). The fold change value greater
than one means the concentration of a specific metabolite is higher in the chromium stresses
sample than the control sample. At 24 hrs glutamate and betaine were expressed in high
concentration in the control sample. But at 24 hrs 3-hydroxyisovalerate, dimethylsulfone,
formate, glycerol, Iiobutyrate, leucine, valine, adenine and alanine were expressed in high
concentrations in the chromium stressed sample. The concentration of 3-hydroxyvalerate,
acetate, dimethylsulfone, formate, glutamate, glycerol, isobutyrate, lactate, leucine, succinate and
valine were higher in the chromium stressed sample at 6hrs, but the concentration of betaine and
ornithine were lower than the control sample. At 6 hrs the concentration of glutamate was higher
in the chromium stress sample but with time the concentration was decreased. At 24 hrs the
concentration of Glutamate observed in the control sample was higher than the chromium stress
sample.

34. 26
Table2: Significant Metabolites and Fold change
Significant Metabolites FC 6 hrs FC 24 hrs
3-hydroxyisovalerate 2.2283 2.496006
Acetate 1.6237 1
Adenine 1 3.555429
Alanine 1 2.029509
Betaine 0.75972 0.868496
Dimethylsulfone 1.4476 2.578449
Formate 1.786 2.860739
Glutamate 1.748273 0.391482
Glycerol 1.5329 1.773528
Isobutyrate 2.03625 2.697745
Lactate 1.54115 1
Leucine 1.947 3.579194
Ornithine 0.461013 1
Succinate 1.2832 1
Valine 1.8719 2.95858
Fold change of the significant metabolites was analyzed using MetaboAnalyst 3.0. Fold change
is equal to the concentration ratio of the metabolite expressed in the chromium stressed sample
and control sample.

35. 27
Figure 9. Fold Change of the Metabolites
Figure 9. Fold change of the significant metabolites was analyzed using MetaboAnalyst 2.5.
Fold change is equal to the concentration ratio of the metabolite expressed in the chromium
stressed sample to control sample. Fold change of the significant metabolites at 6 hrs is depicted
in blue. Fold change of the significant metabolites at 24 hrs is depicted in red.

36. 28
Pathway Analysis
These significant metabolites were used to identify the probable metabolic pathways
which could have been changed due to chromium stress. The twenty-four probable metabolic
pathways resulted from the initial investigation are listed in Table 3. The interconnection
between these metabolic pathways were created using common metabolites and shown in Figure
10. The metabolic pathways are shown in oval and metabolites in rectangular shape. The
significant metabolites are shown in blue color.
From the literature it had been reported that chromium resistance could be commonly
achieved in microorganisms via different kind of mechanisms such as efflux mechanism,
reduction of chromate and chromium (VI) uptake. Chromium reduction could be achieved
through direct reduction where chromium (VI) acts as the terminal electron acceptor of the
electron transport chain. The iron and sulfur reducing bacteria reduces the chromium through
indirect reduction mechanisms where chromium is reduced by biogenic Fe( II) and sulfides. In
some gram-negative bacteria chromium reduction is achieved extracellularly. NADH serves as
the electron donor in these extracellular reductions and they are mediated by soluble reductase
enzymes. In the aerobic chromium reduction NADH, NADPH and electrons from endogenous
reserve acts electron donors (Joutey et al. 2015).

37. 29
Table 3. Probable Metabolic Pathways
Probable Metabolic Pathways Total Metabolites Hits
Pyruvate metabolism 21 2
Glyoxylate and dicarboxylate metabolism 22 2
Valine, leucine and isoleucine biosynthesis 26 2
Phosphonate and phosphinate metabolism 7 1
D-Glutamine and D-glutamate metabolism 7 1
Valine, leucine and isoleucine degradation 37 2
Methane metabolism 11 1
Glycerolipid metabolism 12 1
Sulfur metabolism 13 1
Cyanoamino acid metabolism 14 1
Benzoate degradation via CoA ligation 15 1
Glutathione metabolism 17 1
Selenoamino acid metabolism 17 1
Alanine, aspartate and glutamate metabolism 18 1
Citrate cycle (TCA cycle) 19 1
Pantothenate and CoA biosynthesis 20 1
Butanoate metabolism 21 1
Aminoacyl-tRNA biosynthesis 66 2
Purine metabolism 66 2
Propanoate metabolism 22 1
Tyrosine metabolism 23 1
Glycolysis or Gluconeogenesis 23 1
Glycine, serine and threonine metabolism 34 1
Arginine and proline metabolism 56 1
The metabolic pathways were identified using Pathway Analysis technique available in
MetaboAnalyst 3.0.

38. 30
Figure 10. Pathway Analysis
Figure 10. The interconnection between each metabolic pathways were created using the
common metabolites. The metabolic pathways are shown in oval and metabolites in rectangular
shape. The significant metabolites are shown in blue color.

39. 31
DISCUSSION
The increase in the chromium concentration resulted in a decrease in the growth rate of
Pf-5. Higher growth rate was observed in the control and the 25 ppm conditions. But a
significant decrease in the growth rate was observed at 50 ppm and 100 ppm. At 200 ppm the
growth curve was almost equal to zero. According to the data from the drop plate analysis, the
survival rate was decreased with increasing chromium concentration. With the growth curve data
and drop plate analysis, 50 ppm was selected as the sub-lethal concentration to pursue the future
experiments, because at 50ppm the growth rate of the bacteria was decreased but still the bacteria
was able to survive.
The effect of chromium on the growth pattern of P. fluorescens Pf-5 was studied through
PCA analysis. PCA analysis was carried out with and without Betaine, because the concentration
of betaine was so high that it suppressed the expression of the other metabolites. Significant
profile separation was observed among all four sample groups (the control sample at 6 hrs,
control sample 24 hrs, chromium stressed sample at 6 hrs and chromium stressed sample at 24
hrs). The pair wise PCA analysis between control sample and chromium stressed sample at 24
hrs and control sample and chromium stressed sample at 6 hrs revealed that chromium stress has
an effect on the growth pattern of P. fluorescens Pf-5. Profile separation with the mahalanobis
distance of 6.1137 was observed at 24 hrs and a profile separation with the mahalanobis distance
of 8.075 was observed at 6 hrs. The F-value calculations revealed that these separations are
statistically significant.
Fold change analysis revealed that the expression of some metabolites was increased at
24 hrs whereas the expression of some other metabolites was suppressed. The concentration of

40. 32
betaine in the chromium stressed sample was lower than the control at both 6 hrs and 24 hrs time
point. The expression of glutamate was higher at 6 hrs but with time it was decreased and was
suppressed in the chromium sample at 24 hrs. These significant metabolites were used in the
identification of the probable metabolic pathways. These significant metabolites and other
common metabolites were used to create a connection in-between these probable metabolic
pathways. In Joutey et al. (2015) it had been reported that the microorganisms show chromium
resistance through chromate reduction, efflux mechanisms and Cr (VI) uptake. The NADPH is
widely been used as the electron donor in the chromium reduction mechanisms.

41. 33
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