Experimentation and Evaluation of Biodegradative Kinetic …
165
reported. Hence, this study was designed to remove NO 3
− and heterotrophic bacteria
from drinking water through biological and chemical treatment processes.
2 Material and Methods
2.1 Isolation of Nitrate Reducing Bacteria
The water and sediment samples were collected under aseptic conditions in sterile
bottles from Kodaikanal and Yercaud lakesin Tamil Nadu, India. Bacterial populations were estimated by pour plate technique using nutrient agar (per litre containing
3 g of beef extract, 3 g of yeast extract, 5 g of peptone, 5 g of NaCl,20 g of agar,
pH 7.0 ± 0.2). Well-defined colonies were isolated on the basis of morphological
characteristics, transferred to nutrient agar and were identified up to the generic level
after purification (Buchanan and Gibbons 1974). All the strains were tested for nitrate
reduction using potassium nitrate broth (per litre containing 5 g of peptone, 3 g of
beef extract, 5 g of NaCl, 5 g of KNO 3 , pH7.0 ± 0.2). The ability of the isolates to
reduce NO 3
− to nitrite (NO 2
− ) and ammonium (NH 4
+ ) was determined by the addition of Nessler’s reagent to the cultures. The appearance of yellow orange showed
that NO 3
− and/orNO 2
− has been reduced to NH 4
+ . Based on the intensity of the color,
the isolates were categorized (
+ low,
++ moderate and
+++ high NO 3
− reduction). The
bacterial species, Pseudomonas sp. (KW1) and Bacillus sp. (YW4) were found to
be most efficient for nitrate reduction and used as consortium for further studies of
nitrate reduction. After screening, the selected isolates were purified by repeated
streaking on nutrient agar and stored at 4 °C.
2.2 Inoculum Preparation
A loopful of cultures (KW1 and YW4)were inoculated in presterilized 100 ml nutrient
broth separately. The flasks were kept in a shaker at 120 rpm for 12 h at 30 °C. The
culture broths were centrifuged at 10,000 rpm for 20 min. Cell suspensions were
prepared using sterile distilled water and adjusted to 1OD (10
4 CFU/mL) using UV–
Visible Spectrophotometer (model: Hitachi U 3210) at 600 nm. One ml containing
10
4 CFU/mL of the cell suspension was used as inoculum.
165
reported. Hence, this study was designed to remove NO 3
− and heterotrophic bacteria
from drinking water through biological and chemical treatment processes.
2 Material and Methods
2.1 Isolation of Nitrate Reducing Bacteria
The water and sediment samples were collected under aseptic conditions in sterile
bottles from Kodaikanal and Yercaud lakesin Tamil Nadu, India. Bacterial populations were estimated by pour plate technique using nutrient agar (per litre containing
3 g of beef extract, 3 g of yeast extract, 5 g of peptone, 5 g of NaCl,20 g of agar,
pH 7.0 ± 0.2). Well-defined colonies were isolated on the basis of morphological
characteristics, transferred to nutrient agar and were identified up to the generic level
after purification (Buchanan and Gibbons 1974). All the strains were tested for nitrate
reduction using potassium nitrate broth (per litre containing 5 g of peptone, 3 g of
beef extract, 5 g of NaCl, 5 g of KNO 3 , pH7.0 ± 0.2). The ability of the isolates to
reduce NO 3
− to nitrite (NO 2
− ) and ammonium (NH 4
+ ) was determined by the addition of Nessler’s reagent to the cultures. The appearance of yellow orange showed
that NO 3
− and/orNO 2
− has been reduced to NH 4
+ . Based on the intensity of the color,
the isolates were categorized (
+ low,
++ moderate and
+++ high NO 3
− reduction). The
bacterial species, Pseudomonas sp. (KW1) and Bacillus sp. (YW4) were found to
be most efficient for nitrate reduction and used as consortium for further studies of
nitrate reduction. After screening, the selected isolates were purified by repeated
streaking on nutrient agar and stored at 4 °C.
2.2 Inoculum Preparation
A loopful of cultures (KW1 and YW4)were inoculated in presterilized 100 ml nutrient
broth separately. The flasks were kept in a shaker at 120 rpm for 12 h at 30 °C. The
culture broths were centrifuged at 10,000 rpm for 20 min. Cell suspensions were
prepared using sterile distilled water and adjusted to 1OD (10
4 CFU/mL) using UV–
Visible Spectrophotometer (model: Hitachi U 3210) at 600 nm. One ml containing
10
4 CFU/mL of the cell suspension was used as inoculum.