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Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
414414
ISOLATION AND CHARACTERIZATION OF FLUORIDE RESISTANT
BACTERIAL STRAINS FROM FLUORIDE ENDEMIC AREAS OF
WEST BENGAL, INDIA: ASSESSMENT OF THEIR FLUORIDE
ABSORPTION EFFICIENCY
Goutam Banerjee,a,* Archya Sengupta,a Tathagato Roy,a Prajna Paramita Banerjee,a
Ansuman Chattopadhyay,a Arun Kumar Raya
West Bengal, India
ABSTRACT: In the present investigation, two highly fluoride ion (F) tolerant bacterial
strains, Bacillus cereus FT1 (GenBank Acc. KP729612) and Bacillus marisflavi FT2
(GenBank Acc. KP729613) were isolated from soil samples collected from F endemic
areas of Birbhum district (Rampurhat block II) of West Bengal, India. The F tolerance
limit and absorption efficiency exhibited by these two bacterial isolates were
monitored for 72 hr at 24 hr intervals. At lower F concentrations (10–100 ppm), the F
absorption efficiency of the bacterial strains was not affected. However, at a higher F
concentration (730 ppm) the absorption efficiency was significantly increased
compared to the control strain (B. licheniformis ONF2). The concentration of F in the
culture medium of B. cereus FT1 and B. marisflavi FT2 was reduced from 730 ppm to
570 ppm and 730 ppm to 580 ppm at 72 hr, respectively. To monitor F toxicity, growth
curves were prepared at two different concentrations, 1500 ppm and 3000 ppm of
NaF. In both cases, the lag phases in the growth curves were extended. However, the
bacterial growth was not completely inhibited. The F tolerance efficiency exhibited by
these two bacterial isolates was again confirmed by cell morphology study using a
scanning electron microscope. To the best of our knowledge, this is the first report
on F absorption by the bacterial strains, B. cereus and B. marisflavi.
Keywords: Absorption efficiency; Bacterial isolates; Fluoride tolerance; Scanning electron
microscopy.
INTRODUCTION
Fluorine is one of the most abundant elements on earth and acts as a major
environmental toxicant originating from both natural and industrial sources.1 The
level of the fluoride ion (F) in surface water is gradually increasing due to rapid
industrialization and contamination with pesticides like cryolite and sulfuryl
fluoride.2 The long term consumption of F containing water exerts various adverse
effects on health including skeletal and dental fluorosis in both man3-6 and
domestic animals.7-11 The fluoride ion acts as a protoplasmic poison and a very
small amount of this anion can alter several biochemical processes inside living
cells.12 From recent research, F is found to induce oxidative damage in cells by
producing reactive oxygen species (ROS) and modulating intracellular redox
homeostasis.13,14 F can also induce genotoxicity and bind with DNA leading to
DNA damage which could be the initial event of chemical carcinogenesis.15-17 In
addition, F can modulate expression of many genes related to the phase I and
phase II detoxification systems at the transcriptional level.18,19 Thus, the
increasing concentration of F in groundwater resources is now becoming an
important toxicological and geo-environmental concern.20
aDepartment of Zoology, Visva-Bharati University, West Bengal-731235, India; *For
correspondence: Dr Goutam Banerjee, Department of Zoology, Visva-Bharati University, West
Bengal-731235, India; E-mail: banerjee.goutam2@gmail.com
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
415415
People in a few villages (Noapara, Atla, Junidpur, etc.) in West Bengal (India)
consume water with a high F content and fluorosis remains as a major health
hazard in these villages.21 In recent years, a few microorganisms have been
reported to be an important bioremediation tool against xenobiotics. Microbes are
known to have a variety of mechanisms to tolerate and utilize toxic elements like
lead, cadmium, nickel, etc.22,23 They can adapt to toxic environments through
different ways like mineralization, metal sorption, enzymatic oxidation or
reduction, and the efflux of xenobiotics from the cells.24 However, reports
regarding the bio-absorption of F are scanty. Only a single literature is available
reporting F resistant B. flexus.25 The detoxifying mechanism exhibited by these
microorganisms might be a future tool for developing engineered bacteria for the
reduction of F levels. Therefore, the present investigation was undertaken with the
objectives of (i) screening, isolation, and characterization of F tolerant bacterial
strains; (ii) evaluating the absorption efficiency of F by these selected bacterial
strains; and (iii) studying the effect of F on bacterial growth kinetics and cellular
morphology using scanning electron microscopy.
MATERIALS AND METHODS
Sampling and isolation of bacteria: Soil and drinking water (from hand-pumps)
samples were collected in triplicate from six different sites in three villages,
namely Atla, Nowapara, and Junidpur, located in Rampurhat block II (24º10’34”N
and 87º52’56”E), Birbhum district, West Bengal, India. In order to isolate the F
tolerant bacteria, 1 g of soil was dissolved in 10 mL of sterile distilled water and
serial dilutions were made according to the standard method.26 The isolation of the
F tolerant bacteria was done in tryptone soya agar (TSA) plates containing 300
ppm of sodium fluoride (NaF). Six morphologically different bacterial colonies
were obtained and pure cultures made by the repeated streaking method. On the
basis of the F tolerance level, two bacterial strains named FT1 and FT2 were
selected for further studies. The minimum inhibitory concentration (MIC) for
these two selected bacterial strains was found with TSA plates prepared with
different concentrations of NaF (1500 ppm–5000 ppm).
Fluoride estimation in water samples: The collected water samples were
subjected to F concentration measurement using a Thermo Scientific ORION
STAR A214 ISE meter following the manufacturer’s protocol. In brief, 1 mL of
TISAB III reagent was added to 10 mL of water sample, mixed carefully, and
measured using a F selective electrode. The ion analyzer electrode was calibrated
using standard F solutions (1, 10, and 100 ppm) prepared from a 100 ppm F
standard solution (Orion 940907).
Characterization of the selected bacterial isolates: In order to determine the
colony morphology, these two selected bacterial strains, FT1 and FT2, were
separately cultured on TSA plates and incubated for 24 hr at 37ºC. The colour,
margin, elevation, and surface morphology were checked under a scanning
electron microscope. Gram staining was carried out according to the standard
method. The biochemical characterization of these selected bacterial strains was
done using biochemical characterization kits (HiCarbo kit, HiMedia).
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
416416
16S rRNA sequence analysis: The proper identification of the selected bacterial
strains was done by 16S rRNA sequence analysis26 with minor modifications. In
brief, genomic DNA was extracted following the SDS-lysozyme method. Both the
quantity and quality of the extracted DNA were checked in a nano-
spectrophotometer (Eppendorf Biospectrometer). The amplification of the 16S
rRNA gene was carried out in standard conditions in a thermal cycler (GeneAmp
9700, ABI). The PCR products were bi-directionally sequenced using forward
AGAGTTTGATCMTGGCTCAG (B27 F) and reverse
GGTTACCTTGTTACGACTT (1492 R) primers, respectively. The sequenced data
obtained were edited, aligned, and submitted to the NCBI GenBank for accession
numbers. The phylogenetic tree of the selected bacterial strains was prepared using
Mega 6 software. The guanine and cytosine content (G+C) was calculated and
plotted using an online calculator program (http://www.endmemo.com/bio/
gc.php).
F absorption by the selected bacterial strains: In order to check the F absorption,
the selected bacterial strains FT1 and FT2 were cultured in tryptone soya broth
(TSB) medium containing 1500 ppm NaF and incubated for 3 days at 37ºC. The F
concentration was estimated at 24, 48, and 72 hr using the Thermo Scientific
ORION STAR A214 ISE meter following the same protocol as described above.
The initial F concentration of the culture medium was estimated before inoculating
the bacterial strains. B. licheniformis ONF2 (Acc. No. JX912557) was used as a
control.
Effect of NaF on bacterial growth kinetics: The bacterial strains FT1 and FT2
were cultured separately in 100 mL of tryptone soya broth (TSB) medium for 16
hr. 100 µL of seed culture from each container was taken and mixed in freshly
prepared 100 mL of TSB medium containing 1500 and 3000 ppm of NaF. This
was followed by incubation at 37ºC under a continuous shaking mode (100 rpm).
In order to check the growth kinetics, 1 mL of culture from each container was
taken in a sterilized condition and the optical density (OD) was measured at 600
nm using a spectrophotometer for 12 hr at 1 hr intervals. A culture broth without
NaF was used as a positive control. The growth kinetics of B. licheniformis ONF2
(Acc. No. JX912557) at 1500 ppm of NaF were used as negative control.
Effect of NaF on bacterial cell morphology: The bacterial strains FT1 and FT2
were cultured in TSB medium for 10 hr in the presence of 0 (control) and 1500
ppm of NaF. 10 µL of bacterial culture was taken on a cover slip and a thin layer
made, followed by air drying and heat fixation. Dehydration of the sample was
done with increasing concentrations of alcohol (30, 50, 70, 90, and 100%; 30 min
each). The cover slip was then mounted in a stub, coated with gold particles, and
observed under a scanning electron microscope (Model Hitachi S530).
Statistical analysis: The calculation of the standard error, one way ANOVA, and
a DMRT analysis for significance testing were performed using Microsoft Excel
2010.
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
417417
RESULTS
Very high levels of F were found in the drinking water samples collected from
the three villages (Atla, Junidpur, and Nowapara) including a level of 16 ppm at
two sampling sites in Nowapara (Table 1).
Six bacterial strains were isolated from the F contaminated soil to check the F
tolerance ability. Two strains namely FT1 and FT2 showed the highest F tolerance
(1500 ppm), and thus were selected for further studies (Table 2).
The minimum inhibitory concentrations (MIC) of these two bacterial isolates,
FT1 and FT2, were measured to be 6000 and 3400 ppm NaF, respectively.
Primarily, these two bacterial strains were identified by their morphological and
biochemical properties as listed in Table 3. The colony morphology of FT1 was
round, convex, semitransparent, off-white in colour, smooth surfaced, and with
Table 1. Fluoride level in drinking water collected from three villages of Birbhum,
West Bengal, India
Concentration of fluoride (ppm) Sampling
sites
Sample
number 1
Sample
number 2
Sample
number 3
Sample
number 4
Sample
number 5
Sample
number 6
Atla 8 7 10 8 9 7
Nowapara 12 16 14 13 16 14
Junidpur 12 10 14 9 8 11
Table 2. Screening of F tolerant bacterial strains (+ = growth, – = no growth)
Sodium fluoride (NaF) concentrations (ppm) Bac teri al strains
300 600 900 1200 1500
FT1
FT2
FT3
FT4
FT5
FT6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
–
+
+
+
+
–
–
–
+
+
+
–
–
–
–
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
418418
irregular margins, whereas, FT2 was irregular, convex, transparent, and yellow in
colour. Both the isolates were Gram positive rods. It was observed that both FT1
and FT2 were able to efficiently utilize inulin, glycerol, rhamose, and esculin. FT2
could also utilize maltose, fructose, dextrose, galactose, trehalose, sucrose,
arabitol, and melezitose, whereas, it was negative for α-methyl-d-mannoside,
lactose, xylose, malonate, raffinose, melibiose, manose, l-arabinose, sodium
gluconate, inositol, α-methyl-d-glucoside, sorbitol, mannitol, adonitol, dulcitol,
erythritol, cellobiose, xylitol, and sorbose.
The guanine and cytosine (G+C) contents and their respective graphs are
presented in Table 4 and Figure 1, respectively.
Table 3. Morphological and biochemical characteristics of the selected strains
(+ = positive reaction, – = negative reaction)
Bacterial str ains Cha rac teristi cs
FT1 FT2
Gram staining property + +
surface smooth smooth
margi n ir regular irregul ar
elevation convex convex
col our o ff-whit e yel low
shape round irregular
Colony morphology
opacity semi-trans parent transparent
inulin, glycerol, rhammose, esculin
hydrolysis + +
citrate, sa licin + –
maltose, fructose, dextrose, galactose,
trehalose, sucrose, arabitol, melezitose – +
Biochemi cal
characterization
α-met hyl-d-mannoside, lactose, xylose,
malonate, raffi nose, melibiose, manose,
l – arabinose, sodi um gluconate,
inos itol, α-methyl-d-glucoside, sorbitol,
mannitol, adonitol, dulcitol, erythritol,
cellobiose, xylitol, sorbose
– –
Table 4. Guanine (G) + cytosine (C) content of the sel ected bacteri al strains
Bacterial strains G+C content (%) A+T content (%)
Bacillus cereus FT1
Bacillus marisflavi FT 2
53.29
54.53
46.71
45.47
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
419419
Finally, these bacterial strains were identified up to species level by 16S rDNA
sequence analysis (Figure 2).
a b
G + C content (%) G + C content (%)
Figure 1. Guanine (G) + cytosine (C) content graphs of two bacterial strains. a: B. cereus FT1
and b: B marisflavi FT2.
67.5
53.3
37.5
62.5
53.1
0
a b
0 640 1 Kb 0 640 1 Kb
Number of base pairs Number of base pairs
(
Kb = kilobase-
p
air
)
(
Kb = kilobase-
p
air
)
KP729612
KP729613
Figure 2. The phylogenetic relationship of the two bacterial isolates with their close homologs
taken from the NCBI database. The phylogenetic tree of these two bacterial strains was
constructed using Mega 6.0.
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
420420
The bacterial isolates FT1 and FT2 were identified as B. cereus (GenBank Acc.
KP729612) and B. marisflavi (GenBank Acc. KP729613), respectively. the F
absorption exhibited by these two bacterial, strains B. cereus and B. marisflavi, is
shown in Table 5. The F concentration in the medium decreased gradually at 24,
48, and 72 hr. The concentration of F in the culture medium of B. cereus and B.
marisflavi reduced from 730 ppm to 570 ppm and 730 ppm to 580 ppm at 72 hr,
respectively, while no such change was detected with the control strain.
The effect of F on bacterial growth at 1500 ppm and 3000 ppm of NaF is
represented in Figure 3. B. cereus showed an extended lag phase in the presence of
NaF compared to the control. A higher concentration of NaF (3000 ppm) was
found to be highly toxic in the case of B. marisflavi.
Table 5. F absorption efficiency exhibited by bacterial isolates. Data are presented as
mean±SEM, n=3.
Bacterial strains Concentrations of fluoride (ppm)
Initial Day 1 Day 2 Day 3
Bacillus cereus FT1
Bacillus marisflavi FT2
Bacillus licheniformis ONF2
taken as control
730±9.6a
730±9.6a
730±9.6a
680±9.25a
690±8.94a
728±9.4a
610±7.26b
590±7.12b
720±9.7a
570±7.79b
580±7.53b
722± 8.9a
a,bDifferent lower case letters indicate the presence of a significant difference, p<0.05,
whereas the same lower case letters denote no significant difference.
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
421421
gg
w
th curve in presence of 1500 ppm and 3000 ppm of NaF. a an
d
v
i, respectively. Bacterial culture without NaF was taken as pos
ence of 1500 ppm of NaF was taken as negative control.
1.2
1.0
0.8
0.6
0.4
0.2
0
Positive control
1500 ppm NaF
3000 ppm NaF
Negative control
1 2 3 4 5 6 7 8 9 10 11 12 13
Incubation period (hr)
c
urve in presence of 1500 ppm and 3000 ppm of NaF. a and b r
e
spectively Bacterial culture without NaF was taken as positive
Positive control
1500 ppm NaF
3000 ppm NaF
Negative control
1.2
1.0
0.8
0.6
0.4
0.2
0
1 2 3 4 5 6 7 8 9 10 11 12 13
Incubation period (hr)
b
Figures 3a and 3b. The bacterial growth curve in the presence of 1500 and 3000 ppm of NaF.
a: B. cereus, b: B. marisflavi. A bacterial culture without NaF was used as a positive control. The
growth kinetics of B. licheniformis in the presence of 1500 ppm of NaF was used as a negative
control.
a
OD at 600 nm
OD at 600 nm
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
422422
In order to examine the toxic effect of F, scanning electron microscopy was
performed. Interestingly, 1500 ppm of NaF failed to impose any adverse effect on
bacterial morphology which might be related their tolerance efficiency (Figure 4).
DISCUSSION
In recent years, water pollution has become a major threat for both animals and
human beings in many parts of the world. Due to overwhelming globalization and
competition, most industries directly deposit their waste without prior treatment
which increases the concentration of toxic metals such as Pb, Cd, Ni, and Co, etc.
in water bodies.27 Despite F toxicity being quite different to the toxicity of these
toxic metals and F being very difficult to remove from water bodies, several
relatively inexpensive mechanical and chemical procedures have been introduced
for F removal, such as electro-chemical methods, adsorption processes, and ion
exchange processes.28 Researchers have reported that there are some advantages
with methods of F removal involving the accumulation or absorption of F using
different material matrices like activated carbon, alumina, and zeolites.29-31 In the
present study, we have characterized two potent bacterial strains, B. cereus FT1
(a) Control (a) 1500 ppm NaF
(b) Control (b) 1500 ppm NaF
Figures 4a and 4b. Scanning electron micrographs of the two bacterial strains in the
presence of 1500 ppm NaF. a: the morphology of B. cereus FT1 (3000 ×) and b: the
morphology of B. marisflavi FT2 (3000 ×).
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
423423
and B. marisflavi FT2 isolated from F contaminated soil, where the average F
concentration was 1.5 mg/kg of soil.25 Both of these bacterial strains can tolerate a
high level of F in the form of NaF and can actively absorb F in a liquid medium. B.
cereus is a Gram positive, soil dwelling, spore forming, and facultative anaerobic
bacterium whereas B. marisflavi is an endospore forming, Gram positive, aerobic,
and non-pathogenic bacterium. In general, bacteria possess different types of
mechanisms to deal with toxic components. In the case of F, bacteria possess a
special type of channel protein named putative F-transporters that help them to
reduce the toxic effect of F on the bacterial cell.32 In fact, these genes are
riboswitches which are special type of metabolite binding RNA structure and are
activated by high F concentrations. It has also been evaluated that the channel
transporter has a high affinity with the fluoride ion but rejects other negative ions
like chloride.32 In the present study, it was observed that both of these isolated
bacterial strains could tolerate very high concentrations of F. Investigators have
also reported the F tolerance by Acinetobacter sp. isolated from soil samples.33
The F tolerance of these bacterial strains is due to the expression of riboswitch
genes or some other mechanisms. Further studies are necessary to understand the
tolerance mechanism in these two bacteria isolated by our group. The F absorption
efficiency exhibited by these two bacterial isolates B. cereus FT1 and B. marisflavi
FT2 was very high. However, no significant absorption was detected with low
concentrations of F. This might be due to the limitation of the detection limit in the
estimation method. In a similar study, researchers reported the absorption of F by
the bacterial strain Bacillus flexus MN25 (Acc. No. HQ875778).25 There are
several reports regarding the bioremediation of toxic metals by bacterial isolates,
but only a few studies have been conducted on F absorption.34 A F resistant
mutant Streptococcus mutans GS-5 isolated at low pH was also reported
previously.35 In a similar study, researchers reported the bioabsorption of F by five
bacterial strains namely, Micrococcus luteus, Aeromonas hydrophila, Micrococcus
varians, Pseudomonas aerugenosa, and Escherichia coli.24 Bacteria can absorb F
up to a certain degree and the absorbed F is not toxic for its cell division and
survival. The present investigation demonstrated the bacterial growth curve at two
different concentrations of NaF, 1500 and 3000 ppm. It was observed that in both
treatments, the lag phase was extended compared to the control, but after a certain
time period they started dividing actively. The effect of F on the bacterial cell
morphology was also observed using scanning electron microscopy. Both the
bacterial strains were able to maintain their normal cellular morphology, although
slight bulging characteristics were detected in a few bacterial cells.
CONCLUSION
In conclusion, the F tolerance efficiency of these two bacterial isolates was high
and this property might be useful for investigating the F related expression of
membrane channel protein, which will be important for developing F removing
engineered bacteria in the future. To the best of our knowledge, it is the first report
on F absorption by the bacterial strains, B. cereus and B. marisflavi.
Research report
Fluoride 49(4 Pt 1):414-425
October-December 2016
Isolation and characterization of fluoride resistant bacterial strains
from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
424424
ACKNOWLEDGEMENTS
The authors are thankful to the University Grants Commission, New Delhi,
India, and the Head, Department of Zoology, Visva-Bharati University, India, for
financial assistance and providing the necessary laboratory facilities, respectively.
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Fluoride 49(4 Pt 1):414-425
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from fluoride endemic areas
Banerjee, Sengupta, Roy, Banerjee, Chattopadhyay, Ray
425425
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