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Chapter III: Materials and Methods
CHAPTER III
MATERIALS AND METHODS
3.1 Isolation of chromium tolerant organisms
Chromium-tolerant microorganisms were isolated from liquid effluent and solid
waste and were collected separately from an electroplating industries located in Ambattur
Industrial estate, Chennai, India in sterile glass containers. 1 gm of the sludge and 1 ml of
the effluent was then added to 100ml of sterile M1 growth media. This media consists of
peptone (10gm), beef extract (2 gm), yeast extract (1 gm) and sodium chloride (5 gm) in 1
L of distilled water. The medium was mixed with potassium dichromate (1 ppm Cr (VI))
and incubated at 30 °C, 150 rpm. After significant growth, 1 ml of the culture was
transferred to 100 ml of fresh sterile media with increased concentration of chromium.
This process of enrichment was continued till no growth in the flask was observed. The
enriched culture was serially diluted and plated on 2% agar plates containing peptone (10
gm), yeast extract (1 gm), beef extract (2 gm) and sodium chloride (5 gm) in 1 L distilled
water. Chromium-tolerant strains representing different colony morphologies were
screened and maintained on same agar medium slants and stored at -20 °C. All the
chemicals were purchased from Hi Media and Merck.
3.2 Chromium toxicity effect on growth pattern of isolated organisms
Isolated chromium tolerant microorganisms were tested for the effect of chromium
toxicity on the growth of microorganisms. Increasing concentration of Cr(VI) (as
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Chapter III: Materials and Methods
potassium dichromate) from 1 ppm was added during the log phase of organism. The flask
was kept at shaker incubator at 150 rpm at 30°C. The growth characteristics were further
monitored at regular time intervals and the toxic effect of chromium on the growth pattern
was observed. The growth characteristics were evaluated by measuring the absorbance
value at 600nm at regular time intervals using a spectrometer (Shimadzu Biotech- Biospec
Mini). The absorbance and the concentration of the cell (g/L) was found to be linearly
related (1 absorbance value = 0.5 g/L).
3.3 16S rDNA Analysis
The 16S rDNA of the two most chromium tolerant microorganism was carried out.
The process consisted of isolation of the genomic DNA from pure culture pellet. The ~1.5
kb 16S rDNA fragment was amplified using high-fidelity PCR Polymerase. This is
followed by cloning of the PCR product and then bidirectional sequencing of the plasmid
DNA using forward, reverse and an internal primer. The sequenced data was aligned and
analyzed to find the closest homologs for the microorganisms.
3.4 Biochemical characterization:
3.4.1
Gram staining: The organisms were Gram stained and observed under an
optical microscope.
3.4.2
Starch hydrolysis test:
a) The organism was streaked (single) over starch agar plates and was
incubated for 24 hours at 37°C.
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Chapter III: Materials and Methods
b) After incubation, iodine solution was dispensed over the plate and
observed.
c) Clear zone around the growth indicated a positive result.
3.4.3
Catalase Test:
a) A loop full of culture from agar plates was dipped into a test tube
containing 3-4 ml of H2O2.
b) Bubbles evolving from the tube indicated a positive result.
3.4.4
Nutrient gelatin hydrolysis test:
a) Nutrient gelatin tubes was prepared, inoculated and incubated at 370C.
b) Following incubation, the tubes were placed in refrigerator (40C) for 30
min along with the control.
c) If the test culture fails to solidify, the organism has hydrolyzed the gelatin
(positive result).
d) If the test culture solidify then gelatin has not been hydrolyzed (negative
result)
3.4.5
Triple sugar iron (TSI) agar test:
a) TSI agar slant test tubes were prepared. The butt was stubbed down to the
bottom with culture and the slant surface was streaked.
b) The tubes were incubated at 370C for 18 to 24 hours.
c) If yellow butt and red slant is observed after incubation, only glucose has
been fermented.
d) If yellow butt and yellow slant is observed after incubation, lactose
and/or sucrose has been fermented.
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Chapter III: Materials and Methods
e) If red butt and red slant is observed after incubation, neither glucose,
lactose or sucrose has been fermented.
3.4.6
Citrate utilization test:
a) Simmons’ citrate medium (a modification of Koser’s medium with agar
and an indicator) was prepared and dispensed in test tubes. The tubes
were sterilized at 121οC for 15 minutes and the agar was allowed to set
as slants.
b) The organisms were inoculated onto agar slants.
c) The tubes were incubated for 96 hours at 37οC.
d) Blue color on the streak of growth indicated a positive result.
e) Retention of original green color and no growth on the line of streak
indicated negative result.
3.5 Total chromium removal experiments
Two of the most chromium tolerant microorganisms were screened and analyzed
for chromium uptake. The isolates were inoculated in M1 medium broth supplemented
with increasing range of Cr (VI) as K2Cr2O7 incubated at 30°C under continuous shaking
(120 rpm). Control experiments without the organism were also performed with the same
M1 media containing the same concentration of Cr (VI) as K2Cr2O7 to ensure that the
removal of the metal was due to the microorganism and not due to abiotic factors or
precipitation. At regular intervals, the culture was pelletized in a centrifuge at 6440×g for
15 min. The supernatant was collected and the decrease in Cr concentration was evaluated
using ICP- MS (Inductively coupled plasma- mass spectroscopy) (Agilent Technologies
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Chapter III: Materials and Methods
7500-Series ICP-MS) at the same intervals for total chromium removal. Standard
calibration curve was plotted using ICP multi-element standard solution including
chromium. The appropriate dilutions were made within a range of 0-20 mg/L of chromium
and standard curve was plotted as shown below in Figure 3.1.
Figure 3.1 Standard calibration curve for chromium analysis.
3.6 Intracellular compartmentalization of accumulated metal in the microorganism
Metal accumulated cells were fractionated to release the periplasmic, cytoplasmic
and membrane fractions in accordance to the method described by Choudhary and Sar
(2009). Metal-loaded cells were suspended in 40 ml each of Tris–HCl buffer (pH 7.1, 0.2
M) and sucrose (40%, w/v). Following equilibration (30 min), the cells were harvested
and resuspended immediately in 80 ml MgCl2 (0.5 mM) in an ice bath to release the
periplasmic fraction. Cell suspension was centrifuged at 15,000g (using Hitachi himac CR
22G) for 30 min and the periplasmic fraction was collected. Post-shock cells were
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Chapter III: Materials and Methods
disrupted by sonication (30 s pulse, 10 min) and the homogenate was centrifuged
(20,000g, 3 h) to separate total membrane components and the cytosol. The amounts of
metal in respective fractions were determined by ICP-MS as described above.
3.7 Analytical methods:
3.7.1. Fourier transform infrared (FTIR) spectroscopy
The biomass obtained from centrifugation of 48 hours grown cultures (40
ppm concentration chromium exposed and non-exposed) at 6440×g for 15 min was
lyophilized using a freeze dryer (Christ Alpha 1-2). The dry sample was then
mixed with 2% potassium bromide. The mixture was ground into fine powder and
compressed into translucent sample disks using a manual hydraulic press at a
pressure of 100 kg/cm2. The disk was then loaded in a Fourier Transform Infrared
Spectrometer (Perkin-Elmer spectrum FT/IR-4200 Type A) at a resolution of
4 cm-1 FTIR spectrum of the biomass (Cr exposed and non-exposed) was obtained
at a single scan. The shifts in the FTIR peaks were determined with reference to
reported standard values (Silverstein et al., 1991).
3.7.2 X-ray diffraction (XRD) analysis
The biomass obtained from centrifugation of 48 hours grown cultures (40
ppm concentration chromium exposed and non-exposed) was lyophilized as
described above. The dried samples were then placed in the sample holder for XRay diffraction (XRD) analysis using X-Ray diffractometer (XRD-6000,
Shimadzu). The scans were collected in a range of 2θ from 10° to 80° at a rate of
0.02°/min. The diffraction pattern were compared with that obtained from JCPDS
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Chapter III: Materials and Methods
(Joint committee for powder diffraction studies) - International center for
diffraction data for peak identification. The peaks were analyzed using PDF
(Powder diffraction file) - version 4.
3.7.3 X-ray photoelectron spectroscopy (XPS)
The biomass obtained from centrifugation of 48 hours grown cultures (40
ppm concentration chromium exposed and non-exposed) was lyophilized as
described above. The dried cells were examined using an XPS system (Kratos
Axis Hsi165) using monochromatized Al Kα radiation (1486.6 eV), the source
being operated at 15 kV and 10 mA (Yang and Chen., 2008). Survey scans were
collected from 0 to 1200 eV with pass energy of 80 eV. High-resolution scans for
the element Cr was performed over the 570–582 eV ranges, with the pass energy
adjusted to 0.1 eV. The spectral deconvolution was performed using the curvefitting program with the subtraction of Shirley background; the line-width (full
width at half-maximum) of the Gaussian peaks was maintained constant for all
components in a particular spectrum. Surface elemental compositions and their
quantification were determined from XPS peak area ratios using the software
Vision 2.1.3.
3.8 Characterization of exo-polysaccharides (EPS)
The effect of toxicity of chromium on cell membrane was analyzed. The sample
preparation was done in accordance with Fang et. al.(2002). The culture exposed to 40
ppm chromium and unexposed culture was centrifuged at 4×103 ×g for 20 min at 4°C. The
concentrated biomass was then re-suspended in a 10ml of aqueous solution containing
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Chapter III: Materials and Methods
0.85% NaCl and 0.22% formaldehyde at 80°C for 30 min for EPS extraction (Fang and
Jia, 1996; Jia et al., 1996). The EPS dissolved in the formaldehyde solution was recovered
by further centrifugation at 2×104 ×g for 30 min at 4°C. The carbohydrate content of EPS
in the extracted solution was measured using the phenol/sulfuric-acid method (Gaudy,
1962) and the protein content using the Lowry method (Lowry et. al., 1951).
3.8.1 Phenol/sulfuric-acid method (anthrone reagent test) for carbohydrate
estimation
Anthrone reagent was prepared by mixing 0.1% antrone solution in
73%H2SO4 by heating in a gently boiling steam bath. It was then allowed to cool
in an ice bath and stored at 4 °C in the dark. (The solution remains stable in this
condition for 2 months.) For standard calibration curve, different concentrations
ranging from 0 mg/L to 100 mg/L of glucose, fructose and sucrose was prepared.
Six ml of the color reagent was placed in the Falcon tube and chilled in ice water
for 5 min. 1 ml of the standard solution was added without mixing and allowed to
chill again before the tube is mixed by rapid swirling and returned to the ice water.
The tubes were closed and heated in a well-stirred water bath maintained at 80°±
0.5°C. After 30-60 mins, when a color change is observed, the tubes were returned
to the ice water to cool completely. The absorbance was recorded using a
spectrophotometer at 620 nm. The standard curve is then plotted as shown below
in Figure 3.2-a, b, and c. Samples were then prepared in the same manner as above
and the respective carbohydrate concentration was determined by correlating the
absorbance value against the standard calibration curve. All experiments were
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Chapter III: Materials and Methods
done in triplicates (including the calibration curve) and the average value is
reported.
0.5
0.45
0.4
OD (620 nm)
0.35
0.3
y = 0.0044x + 0.0271
R² = 0.9999
0.25
0.2
0.15
0.1
0.05
0
0
20
40
60
80
100
Concentration (mg/L)
Figure 3.2 (a) Standard calibration curve for glucose analysis
0.9
0.8
0.7
OD (620 nm)
0.6
0.5
y = 0.008x + 0.025
R² = 0.9999
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
70
80
90
100
Concentration (mg/L)
Figure 3.2 (b) Standard calibration curve for fructose analysis
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Chapter III: Materials and Methods
0.7
0.6
OD (620nm)
0.5
y = 0.006x + 0.0306
R² = 0.9994
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
70
80
90
100
Concentration (mg/L)
Figure 3.2 (c) Standard calibration curve for sucrose analysis
3.8.2 Lowry method for protein estimation
The standard calibration curve was plotted using different range (0 mg/L to
100 mg/L) of Bovine Serum Albumine (BSA). Lowry solution was freshly
prepared by mixing Sol A (2.8598 g NaOH and 14.3084 g Na2CO3 in 500 ml of
water), Sol B (1.4232 g CuSO4. 5H2O in 100 ml of water) and Sol C (2.85299 g
Na2C4H4O6.2H2O in 100 ml of water) in proportion of 100:1:1. 0.5 ml. Different
dilution of BSA solution was taken in 2 ml eppendorf tubes. 0.7 ml of lowry
solution (freshly prepared) was added, vortex briefly and incubated for 20 min at
room temperature in dark. In last five remaining minutes, Folin reagent in dark (5
ml of 2N folin and ciocalteu’s phenol reagent + 6 ml of DI water) was prepared.
After 20 minutes of incubation, 0.1 ml of diluted freshly prepared folin reagent
was added to each eppendorfs. The tubes were vortex immediately and incubated
at room temperature in dark for 30 minutes. After the incubation, an appropriate
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Chapter III: Materials and Methods
amount in a cuvette was transferred and measured in spectrophotometer at 750nm.
The calibration curve (Figure 3.5) was then plotted from the absorbance reading.
Test samples were then processed in the same manner as above and respective
protein content was evaluated by correlating the absorbance value against the
standard calibration curve. All experiments were done in triplicates (including the
calibration curve) and average value is plotted.
0.8
0.7
OD (750 nm)
0.6
0.5
0.4
y = 0.0063x + 0.0481
R² = 0.9905
0.3
0.2
0.1
0
0
20
40
60
80
100
120
Concentration (mg/L)
Figure 3.2 (d) Standard Calibration curve for protein analysis
3.9 Microscopic Analysis
3.9.1 Atomic force microscopy (AFM)
The biomass was obtained by centrifugation of 48 hours grown cultures (40
ppm chromium exposed and non-exposed) at 3500 rpm for 15 min. The cells were
then fixed in 4% paraformaldehyde for 16 to 18 h at ambient temperature (Chourey
et. al., 2006). After fixation, cells were pelleted by centrifugation at 3,000 rpm for
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Chapter III: Materials and Methods
3 min and then resuspended in distilled water. A drop of the resuspended cells was
placed on a small microscope glass slide and left undisturbed for 15 min. Slides
were washed briefly with distilled water, dried, and mounted for imaging. A
Nanoscope Controller IIIa AFM (Veeco multimode SPM) in contact mode was
used to image the fixed cells. Nanoprobe silicon nitride (Si3N4) cantilevers with a
spring constant of k=0.06 N/m were obtained from Digital Instruments. Force
measurements were carried out by engaging AFM with the scan rate of 0.7016 Hz.
The force measurements were evaluated and images were captured at different
scan sizes in order to compare Cr treated cells with control. The roughness and
height were measured for the control and chromium treated cells using the
software (V5.30r2 Nanoscope).
3.9.2 Scanning electron microscopy (SEM) and Energy dispersive X-ray
(EDX) spectroscopy
The biomass was obtained by centrifugation of 48 hours grown cultures (40
ppm chromium exposed and non-exposed) at 3500 rpm for 15 min. The cells were
fixed with 3% glutaraldehyde in a phosphate buffer solution (PBS, pH 7.3-7.4) for
more than four hours, and then washed with PBS for two changes (5 minutes
each), rinsed with distilled water for another two changes (again, 5 minutes each),
and dehydrated with using an ethanol gradient (at 50%, 75%, 95% and for 10
minutes) before being finally stored in a desiccator. A scanning electron
microscope (JEOL JSM-5600LV) with the beam voltage at 15 kV was used to
compare the morphological changes that have taken place in the Cr treated isolate
with the control.
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Chapter III: Materials and Methods
Simultaneously, EDX analysis was run for the same sample at a
magnification of 3000X and the concentration of chromium in the control
organism and the treated sample was compared. In EDX spectrum processing,
peaks possibly omitted were 1.609, 9.428 keV. Five iterations were taken to cover
larger scanning area.
3.9.3 Transmission electron microscopic (TEM) analysis
The biomass was obtained by centrifugation of 48 hours grown cultures (40
ppm chromium exposed and non-exposed) at 3500 rpm for 15 min. The cells were
fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (PBS, pH 7.2) for 4 hours.
The sample was centrifuged again at 3500 rpm for 15 min and was washed twice
with same buffer before being fixed in 1% OsO4 in phosphate buffer at 4 °C for 60
min and finally dehydrated with ethanol and embedded in epoxy resin. The
samples were thin sectioned (100 nm) with an ultramicrotome using a diamond
knife, and the sections were collected on copper grids. However, instead of
staining ultrathin sections with uranyl acetate and lead citrate, electron scattering
provided by the adsorbed metal ions acted as the contrasting agent. Micrographs
from the sectioned sample in copper grid were then recorded on TEM (JEOL JEM2100F). TEM data was analyzed from multiple samples and comparison between
chromium exposed and unexposed cells was drawn.
50
[...]... curve) and average value is plotted 0.8 0.7 OD (750 nm) 0.6 0.5 0.4 y = 0.0063x + 0.0481 R² = 0.9905 0 .3 0.2 0.1 0 0 20 40 60 80 100 120 Concentration (mg/L) Figure 3. 2 (d) Standard Calibration curve for protein analysis 3. 9 Microscopic Analysis 3. 9.1 Atomic force microscopy (AFM) The biomass was obtained by centrifugation of 48 hours grown cultures (40 ppm chromium exposed and non-exposed) at 35 00 rpm... using the software (V5 .30 r2 Nanoscope) 3. 9.2 Scanning electron microscopy (SEM) and Energy dispersive X-ray (EDX) spectroscopy The biomass was obtained by centrifugation of 48 hours grown cultures (40 ppm chromium exposed and non-exposed) at 35 00 rpm for 15 min The cells were fixed with 3% glutaraldehyde in a phosphate buffer solution (PBS, pH 7 .3- 7.4) for more than four hours, and then washed with PBS... 30 00X and the concentration of chromium in the control organism and the treated sample was compared In EDX spectrum processing, peaks possibly omitted were 1.609, 9.428 keV Five iterations were taken to cover larger scanning area 3. 9 .3 Transmission electron microscopic (TEM) analysis The biomass was obtained by centrifugation of 48 hours grown cultures (40 ppm chromium exposed and non-exposed) at 35 00... (Chourey et al., 2006) After fixation, cells were pelleted by centrifugation at 3, 000 rpm for 48 Chapter III: Materials and Methods 3 min and then resuspended in distilled water A drop of the resuspended cells was placed on a small microscope glass slide and left undisturbed for 15 min Slides were washed briefly with distilled water, dried, and mounted for imaging A Nanoscope Controller IIIa AFM (Veeco...Chapter III: Materials and Methods amount in a cuvette was transferred and measured in spectrophotometer at 750nm The calibration curve (Figure 3. 5) was then plotted from the absorbance reading Test samples were then processed in the same manner as above and respective protein content was evaluated by correlating the absorbance value against the standard calibration curve All experiments... silicon nitride (Si3N4) cantilevers with a spring constant of k=0.06 N/m were obtained from Digital Instruments Force measurements were carried out by engaging AFM with the scan rate of 0.7016 Hz The force measurements were evaluated and images were captured at different scan sizes in order to compare Cr treated cells with control The roughness and height were measured for the control and chromium treated... at 35 00 rpm for 15 min and was washed twice with same buffer before being fixed in 1% OsO4 in phosphate buffer at 4 °C for 60 min and finally dehydrated with ethanol and embedded in epoxy resin The samples were thin sectioned (100 nm) with an ultramicrotome using a diamond knife, and the sections were collected on copper grids However, instead of staining ultrathin sections with uranyl acetate and. .. ultrathin sections with uranyl acetate and lead citrate, electron scattering provided by the adsorbed metal ions acted as the contrasting agent Micrographs from the sectioned sample in copper grid were then recorded on TEM (JEOL JEM2100F) TEM data was analyzed from multiple samples and comparison between chromium exposed and unexposed cells was drawn 50 ... changes (again, 5 minutes each), and dehydrated with using an ethanol gradient (at 50%, 75%, 95% and for 10 minutes) before being finally stored in a desiccator A scanning electron microscope (JEOL JSM-5600LV) with the beam voltage at 15 kV was used to compare the morphological changes that have taken place in the Cr treated isolate with the control 49 Chapter III: Materials and Methods Simultaneously, ... within a range of 0-20 mg/L of chromium and standard curve was plotted as shown below in Figure 3. 1 Figure 3. 1 Standard calibration curve for chromium analysis 3. 6 Intracellular compartmentalization... Hitachi himac CR 22G) for 30 and the periplasmic fraction was collected Post-shock cells were 42 Chapter III: Materials and Methods disrupted by sonication (30 s pulse, 10 min) and the homogenate was... (620nm) 0.5 y = 0.006x + 0. 030 6 R² = 0.9994 0.4 0 .3 0.2 0.1 0 10 20 30 40 50 60 70 80 90 100 Concentration (mg/L) Figure 3. 2 (c) Standard calibration curve for sucrose analysis 3. 8.2 Lowry method for