Optimization of Process Parameters for Biodegradation …
361
2 Materials and Methods
2.1 Isolation of Mixed Bacterial Culture from Soil
10 g of the soil sample was collected from the outer limit of effluent discharge point
of tannery. The soil, which was rich in microbial diversity, was suspended in 0.8%
saline water and kept in a shaker overnight at 35 °C. The soil suspension was then
centrifuged and the supernatant consisting of mixed bacterial culture was added
to nutrient media. The culture was grown in the nutrient broth for 24 h at 35 °C.
100 ml of MS (Mineral salt) media was prepared and was inoculated with 10% of the
culture from nutrient broth. Composition of the MS media (mg/L) was (KH 2 PO 4 :
680, K 2 HPO 4 : 1730, FeSO 4 : 30, (NH 4 )NO 2 : 100, MgSO 4 : 100, CaCl 2 : 20, MnSO 4 :
30). The mixed bacterial culture was allowed to grow in the MS media for 24 h with
glucose as the sole carbon source.
2.2 Acclimatization of Mixed Bacterial Culture in Resorcinol
100 ml of MS media containing 1% glucose and 10 ppm of resorcinol was inoculated
with 10 ml of the culture from the above bacterial media. The resorcinol concentration
was gradually increased from 10 to 500 ppm and glucose concentration was decreased
till resorcinol became the sole source of carbon for the bacterial consortium. The
mixed bacterial consortium was allowed to grow in shaker at 100 rpm at 35 °C
till stationary phase of the cells was reached or concentration of residual resorcinol
dropped to below detection limit. Samples were collected at regular interval and
cell biomass and residual resorcinol concentration were checked. The cell mass was
obtained by spectrophotometer (Labman visible spectrophotometer LMSP V320)
by measuring the absorbance at 600 nm. From the standard curve of A 600 against
bacterial dry cell biomass (mg/ml), the cell O.D. was converted to dry biomass
of bacteria. Residual resorcinol concentration was analyzed from the supernatant
obtained by centrifugation of the above culture at 6000 rpm for 5 min. The resorcinol
in the supernatant was checked by antipyrine assay. The absorbance of the above
assay was checked at 490 nm.
2.3 Batch Kinetic Study of Removal of Resorcinol
by the Mixed Bacterial Culture in Presence of Different
Toxic Metals
Removal of resorcinol by mixed bacterial culture was studied in presence of different
toxic metals such as Mercury (Hg), Lead (Pb), and Chromium [Cr (VI)]. MS Media
361
2 Materials and Methods
2.1 Isolation of Mixed Bacterial Culture from Soil
10 g of the soil sample was collected from the outer limit of effluent discharge point
of tannery. The soil, which was rich in microbial diversity, was suspended in 0.8%
saline water and kept in a shaker overnight at 35 °C. The soil suspension was then
centrifuged and the supernatant consisting of mixed bacterial culture was added
to nutrient media. The culture was grown in the nutrient broth for 24 h at 35 °C.
100 ml of MS (Mineral salt) media was prepared and was inoculated with 10% of the
culture from nutrient broth. Composition of the MS media (mg/L) was (KH 2 PO 4 :
680, K 2 HPO 4 : 1730, FeSO 4 : 30, (NH 4 )NO 2 : 100, MgSO 4 : 100, CaCl 2 : 20, MnSO 4 :
30). The mixed bacterial culture was allowed to grow in the MS media for 24 h with
glucose as the sole carbon source.
2.2 Acclimatization of Mixed Bacterial Culture in Resorcinol
100 ml of MS media containing 1% glucose and 10 ppm of resorcinol was inoculated
with 10 ml of the culture from the above bacterial media. The resorcinol concentration
was gradually increased from 10 to 500 ppm and glucose concentration was decreased
till resorcinol became the sole source of carbon for the bacterial consortium. The
mixed bacterial consortium was allowed to grow in shaker at 100 rpm at 35 °C
till stationary phase of the cells was reached or concentration of residual resorcinol
dropped to below detection limit. Samples were collected at regular interval and
cell biomass and residual resorcinol concentration were checked. The cell mass was
obtained by spectrophotometer (Labman visible spectrophotometer LMSP V320)
by measuring the absorbance at 600 nm. From the standard curve of A 600 against
bacterial dry cell biomass (mg/ml), the cell O.D. was converted to dry biomass
of bacteria. Residual resorcinol concentration was analyzed from the supernatant
obtained by centrifugation of the above culture at 6000 rpm for 5 min. The resorcinol
in the supernatant was checked by antipyrine assay. The absorbance of the above
assay was checked at 490 nm.
2.3 Batch Kinetic Study of Removal of Resorcinol
by the Mixed Bacterial Culture in Presence of Different
Toxic Metals
Removal of resorcinol by mixed bacterial culture was studied in presence of different
toxic metals such as Mercury (Hg), Lead (Pb), and Chromium [Cr (VI)]. MS Media