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A. Das et al.
2.3 Preparation of Consortium and Plasmid Curing
A consortium is defined as two or more microbial groups living symbiotically. They
could be endosymbiotic or ectosymbiotic. The concept of consortium was first introduced by Johannes Reinke in 1872. In this study, the consortium was prepared using
the bacterial isolates having high metal (arsenic) tolerance.
Plasmids are defined as extrachromosomal structure that replicates independently.
Most of the essential genes required for the normal survival of bacteria are found in
chromosomes. Many bacteria also carry different mobile genetic elements (MGE),
that contribute to specialized functions as antibiotic resistance, metal resistance, etc.
Thus, plasmid curing is a method to substantiate the relationship between the genetic
trait and carrying that specific trait in the plasmid. Numerous agents Viz. chemical
and physical are used, namely, acridine orange, acriflavin, SDS, etc. In this study
ethidium bromide is used as the curing agent. The nutrient broth was prepared and
filter sterilized ethidium bromide was added at concentrations ranging from 1.0–
100 µg. Then the culture was added and incubated at 37 °C. The highest turbidity
showing tube was used for plating and testing for survivability check.
2.4 Removal of Arsenic from Simulated Solution
The metal biosorption study of selected isolates was performed by measuring the As
removal by the bacterial cells using methods proposed by Pazirendeh et al. (1998) and
Bhakta et al. (2012). Fresh bacterial culture was harvested in 2mL centrifuge tube,
tubes were centrifuged (13000 g/min) and the cells were washed three times with
sterilized MiliQ water. Pellets of the cell (40 mg wet weight) of each bacterial isolates
were resuspended in 5 ml sterilized As (1 mg/L) solution. Samples were incubated
for 4 h at 37 °C. After incubation, the samples were centrifuged 13000 g/min) and
the supernatants were filtered through a 0.25 µm filter paper. The As content was
measured using spectrophotometer using safranin O as a reagent at 532 nm. The
metal removal percentage was calculated using equations described by Bhakta et al.
(2012).
3 Results and Discussion
3.1 Isolation of Metal Resistant Bacteria
Seven arsenite resistant strains were isolated from sample 1 and sample 2 after
cultured in minimal salt media and TSB media. The isolates are AsR1 AsR2, AsR3,
AsR4, AsR(i), Asr(i), AsR(iii) (Fig. 1). The minimum inhibitory concentration of all
the isolates has been presented in Table 1. The higher MIC values indicated that the
A. Das et al.
2.3 Preparation of Consortium and Plasmid Curing
A consortium is defined as two or more microbial groups living symbiotically. They
could be endosymbiotic or ectosymbiotic. The concept of consortium was first introduced by Johannes Reinke in 1872. In this study, the consortium was prepared using
the bacterial isolates having high metal (arsenic) tolerance.
Plasmids are defined as extrachromosomal structure that replicates independently.
Most of the essential genes required for the normal survival of bacteria are found in
chromosomes. Many bacteria also carry different mobile genetic elements (MGE),
that contribute to specialized functions as antibiotic resistance, metal resistance, etc.
Thus, plasmid curing is a method to substantiate the relationship between the genetic
trait and carrying that specific trait in the plasmid. Numerous agents Viz. chemical
and physical are used, namely, acridine orange, acriflavin, SDS, etc. In this study
ethidium bromide is used as the curing agent. The nutrient broth was prepared and
filter sterilized ethidium bromide was added at concentrations ranging from 1.0–
100 µg. Then the culture was added and incubated at 37 °C. The highest turbidity
showing tube was used for plating and testing for survivability check.
2.4 Removal of Arsenic from Simulated Solution
The metal biosorption study of selected isolates was performed by measuring the As
removal by the bacterial cells using methods proposed by Pazirendeh et al. (1998) and
Bhakta et al. (2012). Fresh bacterial culture was harvested in 2mL centrifuge tube,
tubes were centrifuged (13000 g/min) and the cells were washed three times with
sterilized MiliQ water. Pellets of the cell (40 mg wet weight) of each bacterial isolates
were resuspended in 5 ml sterilized As (1 mg/L) solution. Samples were incubated
for 4 h at 37 °C. After incubation, the samples were centrifuged 13000 g/min) and
the supernatants were filtered through a 0.25 µm filter paper. The As content was
measured using spectrophotometer using safranin O as a reagent at 532 nm. The
metal removal percentage was calculated using equations described by Bhakta et al.
(2012).
3 Results and Discussion
3.1 Isolation of Metal Resistant Bacteria
Seven arsenite resistant strains were isolated from sample 1 and sample 2 after
cultured in minimal salt media and TSB media. The isolates are AsR1 AsR2, AsR3,
AsR4, AsR(i), Asr(i), AsR(iii) (Fig. 1). The minimum inhibitory concentration of all
the isolates has been presented in Table 1. The higher MIC values indicated that the