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P. Nag et al.
at specific time intervals and the degradation was monitored by weight loss of the
sample using weighing balance (model no. BT 224 S, Max 220 gm, d = 0.1 mg,
made by Sartorius) as described by Sil and Chakrabarti 2010.
2.2.3 Collection of Soil Sample
Soil sample was collected from a depth of 4–5 cm from Captain Bhery Eco & Aquatic
Hub located at Tapuriaghata, E. M. Bypass Road, Kolkata-700105 for microbial
isolation that was used for biodegradation of plastics. The site was used for dumping
municipal solid waste along with huge amount of plastic materials either openly or
partially buried in the soil. Sample was collected using a sterile spatula and aseptically
taken to the laboratory for further analysis.
2.2.4 Isolation and Identification of PVC-Degrading Microbes
from Soil
A stock solution of the collected soil sample was prepared by mixing 1gm soil with
10 ml of normal saline water (0.9%). Single colonies were obtained after 24 h incubation at 37 °C temp. Single colonies were further subcultured on freshly prepared
nutrient agar slant to obtain pure cultures. Mineral Salt Media (MSM) was used for
screening bacteria from the isolated pure culture those were able to degrade PVC.
PVC was used in the media as a major carbon source because MSM contain all
nutrients except carbon necessary for bacterial growth for 4–8 weeks at 37 °C for
isolating PVC-degrading bacteria. A control set was maintained (bacterial inoculation on media without polymer) simultaneously. The culture was identified by MTCC
(CSIR), Chandigarh for 16S rRNA gene sequencing (1434 bases) and Blast search.
2.2.5 Biodegradation of the Film Using Isolated Microorganisms
Accurately weighed and sterilized plastic composite films were aseptically transferred to the 250 ml conical flasks having 50 ml of sterile MSM broth with 0.1%
yeast extract in the laboratory. Except control each conical flask was inoculated
with 5% of isolated PVC-degrading bacteria at approximately the same time. All
the flasks were then incubated in a temperature-controlled shaker at 160 rpm at
37 °C for 25 days. After every 5 days, the films were recovered from the media and
adhered bacterial cells, if any, were washed off from the surface of the film with 2%
(v/v) SDS (sodium dodecyl sulphate) and further washed with distilled water ( Gilan
et al. 2004). The washed films were then dried overnight at 60 °C and finally the
degradation was monitored by loss of weight.
P. Nag et al.
at specific time intervals and the degradation was monitored by weight loss of the
sample using weighing balance (model no. BT 224 S, Max 220 gm, d = 0.1 mg,
made by Sartorius) as described by Sil and Chakrabarti 2010.
2.2.3 Collection of Soil Sample
Soil sample was collected from a depth of 4–5 cm from Captain Bhery Eco & Aquatic
Hub located at Tapuriaghata, E. M. Bypass Road, Kolkata-700105 for microbial
isolation that was used for biodegradation of plastics. The site was used for dumping
municipal solid waste along with huge amount of plastic materials either openly or
partially buried in the soil. Sample was collected using a sterile spatula and aseptically
taken to the laboratory for further analysis.
2.2.4 Isolation and Identification of PVC-Degrading Microbes
from Soil
A stock solution of the collected soil sample was prepared by mixing 1gm soil with
10 ml of normal saline water (0.9%). Single colonies were obtained after 24 h incubation at 37 °C temp. Single colonies were further subcultured on freshly prepared
nutrient agar slant to obtain pure cultures. Mineral Salt Media (MSM) was used for
screening bacteria from the isolated pure culture those were able to degrade PVC.
PVC was used in the media as a major carbon source because MSM contain all
nutrients except carbon necessary for bacterial growth for 4–8 weeks at 37 °C for
isolating PVC-degrading bacteria. A control set was maintained (bacterial inoculation on media without polymer) simultaneously. The culture was identified by MTCC
(CSIR), Chandigarh for 16S rRNA gene sequencing (1434 bases) and Blast search.
2.2.5 Biodegradation of the Film Using Isolated Microorganisms
Accurately weighed and sterilized plastic composite films were aseptically transferred to the 250 ml conical flasks having 50 ml of sterile MSM broth with 0.1%
yeast extract in the laboratory. Except control each conical flask was inoculated
with 5% of isolated PVC-degrading bacteria at approximately the same time. All
the flasks were then incubated in a temperature-controlled shaker at 160 rpm at
37 °C for 25 days. After every 5 days, the films were recovered from the media and
adhered bacterial cells, if any, were washed off from the surface of the film with 2%
(v/v) SDS (sodium dodecyl sulphate) and further washed with distilled water ( Gilan
et al. 2004). The washed films were then dried overnight at 60 °C and finally the
degradation was monitored by loss of weight.