1.   
Material
and Methods

 

2.1 Microorganism and
culture condition

            A
previously reported thermotolerant bacterial strain Bacillus sp. ISTVK1 (gene bank accession number- KJ544559)
isolated from Vasant Kunj Sewage Treatment Plant, New Delhi, was used for the
study of PHA production by using municipal
wastewater. Bacterial strain was augmented at 30 °C, 150 rpm and pH 7.6
in Minimal salt medium (MSM) containing (g L?1): Na2HPO4·2H2O,
7.8; KH2PO4, 6.8; MgSO4, 0.2; NaNO3,
0.085; ZnSO4.7H2O, 0.05; ZnCl2, 0.02; Ca (NO3)2.4H2O,
0.05 and 20 g/L Glycerol as the sole carbon source. Pre-LB cultured
biomass of the strain was centrifuged (7000 rpm, 10 min) and the cell pellet transferred
to mineral medium (MM) (pH  7.6)1 containing (g L?1 ):
KH2PO4, 1.5; Na2HPO4, 6.78; NaCl,
0.5; NH4 (CH3COO)3Fe, 0.06; 1M MgSO4,
2 mL L?1; 1M CaCl2, 0.1 mL L?1; 1mL L?1  trace metal solution consisting of (g L?1):
ZnSO4, 0.1; H3BO3, 0.3; CuSO4, 0.006;  NiCl2.6H2O,
0.020; Na2MoO4.2H2O, 0.030;  MnCl2.2H2O, 0.25,
various concentrations ranging from 1,3,5,7,10% Pure Glycerol was used as
carbon source and flask was put on shaking at 150 RPM and at 30oC
temperature for several days.

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2.2 Selection of strain for PHA production

 

2.2.1 Microscopic
visualization

            Bacterial
cells grown in Minimal Media was collected after 72 h incubation, transferred on a clean glass slide,
heat-fixed, stained was done with crystal
violet for a duration of 10 sec crystal
violet was washed with running water (10 sec) and then visualized under
microscope2.

 

2.2.2 Fluorometric
visualization

            PHA
accumulation was observed fluorometrically with Nile red according to 3 with slight
modification. Briefly, 1 mL of the bacterial
strain cultured in Minimal Media and 5 % Glycerol was used as carbon source and
cell pellet was harvested at 72 h by centrifugation. Pellet was washed 3-4
times using Phosphate buffer saline, pH 7.4 to remove the media, fixed in (1:3)
Ethanol: Acetic acid solution for 10 min, again washed with deionized water and
lastly stained with Nile red (10 µg mL-1) for 5 min. Cells were mixed with 1 % low melting agarose in 1:1
ratio and mounted on glass slides for visualization under Zeiss HBO 100
fluorescence microscope to detect the intracellular PHA as indicated by the intensity
of Nile-red orange fluorescence as measured at 560 nm and 590 nm excitation and
emission wavelengths respectively.

 

2.2.3.
Spectrofluorometric analysis of PHA accumulation

            The spectrofluorometric study was carried out
by using Nile red following slight modification
of the protocol described by4.
Briefly, Bacillus sp.ISTVK1 was
cultured in Minimal Media with 5% Glycerol as a carbon
source and incubated at 30 °C and 150 rpm for seven days. Fluorescence of bacterial culture measured after staining with 10 µg mL-1Nile red at 488 nm
excitation wavelength and 575 nm and 590 nm emission wavelengths for short and
medium chain length PHA respectively, every 24 h with 0 h taken as control. The
intensity of Nile-red orange fluorescence as measured using Shimadzu RF-5301 PC
spectrofluorophotometer, indicated the presence of PHA within the cells. Absorbance was recorded at 600 nm on Cary 100
BIO UV-Visible Spectrophotometer simultaneously.

 

2.4 Confirmatory analysis of PHA production

 

2.4.1 FTIR
analysis

            Bacillus sp. ISTVK1 cultured
in Mineral Media with 5 % Glycerol as carbon source harvested after 72 h washed
adequately with deionized water. PHA accumulated in the bacteria, extracted via
the method as stated by 5
 from the powdered cell biomass with
chloroform for 72 h at 70 °C at 150 rpm, and then precipitated with 10 volumes
of hexane, air dried and weighed. Fourier-transform infrared spectroscopy spectrum recorded of
the dried PHA sample using Varian 7000 FTIR spectrometer (Perkin-Elmer Inc.,
Wellesley, MA, USA). 0.01 g of the dried sample
mixed with KBr, and the spectrum was read in the range 400 to 4000 cm-1,
applied for 64 scans per sample at 4 cm-1 resolution

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