Biodegradation of Lead from Accumulated Municipal Solid Waste …
105
Each solid waste sample was approximately 500 g of mixed waste and three samples
were collected from different sample points. Each solid waste sample was labeled
according to its location. Samples were gently placed into hygienic and sterilized
plastic zipper bags and were taken to the laboratory. 50 g of thoroughly mixed waste
were taken for microbial analysis and the rest of the samples were allowed to dry at
room temperature for ten days. After drying, solid waste was shredded and ground,
and were further analyzed for their heavy metals content.
2.3 Isolation and Identification of Bacteria
Solid waste samples were taken to the laboratory under aseptic conditions. Samples
were serially diluted and spread using sterile L-rod on pre-sterilized nutrient agar
plates in triplicates (Bergey et al. 1974; Yamina et al. 2012). After incubation of
inoculated plates for 24 h at 37 °C, the plates were observed for the appearance
of bacterial colonies. Morphologically different 42 colonies were selected and the
isolates were subcultured and maintained on nutrient agar slant and stored at 4 °C.
Among 42 isolates, the heavy metals resistant bacteria were selected and were identified based on morphological characteristics, gram staining, and biochemical tests
such as Indole, MR-VP, motility, and Covac’s oxidase tests were performed using
Bergey’s manual of determinative bacteriology (Bergey et al. 1974).
2.4 Heavy Metals Analysis
Exactly 2 g solid waste was weighed using a monopan digital balance and transferred
to a 500 ml round bottom flask. To this, a little amount of distilled water was added and
swirled gently to make a slurry. Then to this, a mixture of each 20 ml nitric acid, 4 ml
perchloric acid, and 2 ml sulphuric acid was added respectively. The sample was
then digested (Guna-Soxhlet extraction mantle) for 3 h at 90
◦ C. The appearance
of brown fumes during digestion was followed by the addition of 5 ml of 30%
Hydrogen peroxide. Bumping was controlled by putting glass beads in the sample
getting digested. Ensured digestion was followed by the addition of a little amount
of distilled water to the unit. The digested samples were then cooled and filtered into
a 50 ml standard flask. The final volume of 50 ml was made with distilled water.
The samples were then transferred to pre-treated, laboratory cleaned, plastic vials,
and stored at 4
◦ C till further analysis. The digested samples were aspirated in the
Atomic Absorption Spectrophotometer (iCE 3300 AA Spectrometer) and absorbance
was noted from which estimations were made for each metal concentration in mg/kg
(U.S. EPA. 1996). Working standards were prepared from 1000 ppm stock solution
of lead (Pb) to 2, 4, and 6 ppm.
105
Each solid waste sample was approximately 500 g of mixed waste and three samples
were collected from different sample points. Each solid waste sample was labeled
according to its location. Samples were gently placed into hygienic and sterilized
plastic zipper bags and were taken to the laboratory. 50 g of thoroughly mixed waste
were taken for microbial analysis and the rest of the samples were allowed to dry at
room temperature for ten days. After drying, solid waste was shredded and ground,
and were further analyzed for their heavy metals content.
2.3 Isolation and Identification of Bacteria
Solid waste samples were taken to the laboratory under aseptic conditions. Samples
were serially diluted and spread using sterile L-rod on pre-sterilized nutrient agar
plates in triplicates (Bergey et al. 1974; Yamina et al. 2012). After incubation of
inoculated plates for 24 h at 37 °C, the plates were observed for the appearance
of bacterial colonies. Morphologically different 42 colonies were selected and the
isolates were subcultured and maintained on nutrient agar slant and stored at 4 °C.
Among 42 isolates, the heavy metals resistant bacteria were selected and were identified based on morphological characteristics, gram staining, and biochemical tests
such as Indole, MR-VP, motility, and Covac’s oxidase tests were performed using
Bergey’s manual of determinative bacteriology (Bergey et al. 1974).
2.4 Heavy Metals Analysis
Exactly 2 g solid waste was weighed using a monopan digital balance and transferred
to a 500 ml round bottom flask. To this, a little amount of distilled water was added and
swirled gently to make a slurry. Then to this, a mixture of each 20 ml nitric acid, 4 ml
perchloric acid, and 2 ml sulphuric acid was added respectively. The sample was
then digested (Guna-Soxhlet extraction mantle) for 3 h at 90
◦ C. The appearance
of brown fumes during digestion was followed by the addition of 5 ml of 30%
Hydrogen peroxide. Bumping was controlled by putting glass beads in the sample
getting digested. Ensured digestion was followed by the addition of a little amount
of distilled water to the unit. The digested samples were then cooled and filtered into
a 50 ml standard flask. The final volume of 50 ml was made with distilled water.
The samples were then transferred to pre-treated, laboratory cleaned, plastic vials,
and stored at 4
◦ C till further analysis. The digested samples were aspirated in the
Atomic Absorption Spectrophotometer (iCE 3300 AA Spectrometer) and absorbance
was noted from which estimations were made for each metal concentration in mg/kg
(U.S. EPA. 1996). Working standards were prepared from 1000 ppm stock solution
of lead (Pb) to 2, 4, and 6 ppm.