Bioremediation of Heavy Metals and Toxic Chemicals …
69
2 Methods
2.1 Sample Collection
Water sample was collected from Muttukadu lake, Chennai, for the estimation of
heavy metals and its concentrations and also to detect the presence of toxic functional groups. The water sample was collected in a large sterile screw cap bottle.
Estimation of heavy metals in untreated sample was carried out by using Atomic
absorption spectroscopy and the chemical groups were determined by FTIR analysis
respectively.
2.2 Isolation of Indigenous Bacteria from Metal/Iron
Contaminated Water Sample
For the isolation of indigenous microbe, the water sample was collected from metal
contaminated effluent at Kandigai, Chennai. Nutrient broth was prepared in sterile
conical flask and sterilized by autoclaving at 121
◦ C for 20 min. Meanwhile the
water sample were serially diluted by tenfold dilution. Sterile petriplates were taken
and marked as 10
–2 , 10
–3 , 10
–4 , 10
–5 . 1 ml of the diluted samples were added to the
sterile petriplates respectively and molten nutrient agar was poured then allowed for
solidification. Then the plates were incubated at 37
◦ C for 24–48 h. Morphologically
differing various colonies were observed and further pure cultured.
2.3 Identification of Isolated Bacteria
Based on Bergey’s manual of systematic Bacteriology the unknown candidate was
identified. The unknown bacteria was identified by morphological characteristics,
Gram’s staining followed by biochemical characterization.
2.4 Production of Biosurfactant by Selected Strain
The selected strain was used to produce biosurfactant by submerged fermentation in
a stirred tank reactor and purified by acid precipitation method. 100 ml of isolation
medium for Pseudomonas spp., (cetrimide broth) was prepared, additionally 2%
glycerol was supplied as the carbon source and sterilized by autoclaving at 121
◦ C
for 20 min, 15 lbs. Loop full of selected culture was inoculated and incubated for
3–4 days at 37
◦ C in a shaking incubator and 130 rpm was maintained.
69
2 Methods
2.1 Sample Collection
Water sample was collected from Muttukadu lake, Chennai, for the estimation of
heavy metals and its concentrations and also to detect the presence of toxic functional groups. The water sample was collected in a large sterile screw cap bottle.
Estimation of heavy metals in untreated sample was carried out by using Atomic
absorption spectroscopy and the chemical groups were determined by FTIR analysis
respectively.
2.2 Isolation of Indigenous Bacteria from Metal/Iron
Contaminated Water Sample
For the isolation of indigenous microbe, the water sample was collected from metal
contaminated effluent at Kandigai, Chennai. Nutrient broth was prepared in sterile
conical flask and sterilized by autoclaving at 121
◦ C for 20 min. Meanwhile the
water sample were serially diluted by tenfold dilution. Sterile petriplates were taken
and marked as 10
–2 , 10
–3 , 10
–4 , 10
–5 . 1 ml of the diluted samples were added to the
sterile petriplates respectively and molten nutrient agar was poured then allowed for
solidification. Then the plates were incubated at 37
◦ C for 24–48 h. Morphologically
differing various colonies were observed and further pure cultured.
2.3 Identification of Isolated Bacteria
Based on Bergey’s manual of systematic Bacteriology the unknown candidate was
identified. The unknown bacteria was identified by morphological characteristics,
Gram’s staining followed by biochemical characterization.
2.4 Production of Biosurfactant by Selected Strain
The selected strain was used to produce biosurfactant by submerged fermentation in
a stirred tank reactor and purified by acid precipitation method. 100 ml of isolation
medium for Pseudomonas spp., (cetrimide broth) was prepared, additionally 2%
glycerol was supplied as the carbon source and sterilized by autoclaving at 121
◦ C
for 20 min, 15 lbs. Loop full of selected culture was inoculated and incubated for
3–4 days at 37
◦ C in a shaking incubator and 130 rpm was maintained.