The soil samples were collected from the sewage of Bangladesh Small and Cottage Industries Corporation (BSCIC) industrial zone, Sopura, Rajshahi, Bangladesh. The samples were collected in a sterile plastic container and transported to laboratory for bacteriological analysis. Then the sewage water was filtered by filter paper in a beaker and mixed bacterial cultures. Cadmium was collected as CdCl2.H2O from the department of Chemistry, University of Rajshahi, Bangladesh. The cadmium was collected in a glassware container and stored until use.
The mixed bacterial cultures were grown in MS medium at different concentrations of cadmium. Then the bacterial isolates were screened on nutrient agar (NA) plates supplemented withpeptone1gm/100 ml, yeast extract 0.5 gm/100 ml and agar 2 gm/100 ml. The growth and morphological characteristics of both strains were observed in LB (Lauria Bertani) and MS (Minimal Salt) media by standard plating and streaking techniques were incubated at 37°C for 24h. Then the bacterial strains will be isolated and suspended in LB liquid medium for storage and further use.
Colonial morphology such as shape, color, opacity and consistency appearance on the LB media will be recorded. Gram staining, motility test and viable cell counting will be conducted. Methyl red, catalase, macConkey, citrate utilization and SIM-medium (Sulphide-Indole-Motility medium) tests will be performed to characterize the isolates.
Effects of pH variations, temperatures and carbon sources on growth were performed to study the stability of the isolates for the biodegradation of cadmium and these experiments were conducted in a Erlenmeyer flask containing 10 µg/ml of the two isolated bacterial cultures were inoculated in 100 ml Minimal salt broth. After sterilization by autoclaving the flasks were cooled and inoculated with the bacterial cultures and maintained at different temperatures ranging from 10°C to 50°C for the test of temperature effects; maintained at different pH ranging from 0 to 10for checking the pH effects; added 1g of various carbon sources (glucose, sucrose, peptone and glycerol) for examining the effects of carbon sources of the bacterial isolate. After inoculation the cultural flasks were placed on orbital shaker at 150 rpm for 96 hours to observe their growth efficiency. The bacterial growth was measured by observing the optical density (OD) at 660 nm through UV-spectrophotometer (ANALYTIK JENA AG, SPOKOL 1500/1, GERMANY) after different time intervals.
The antibiotic sensitivity testing of the isolates were performed against seven antibiotics following the disk diffusion method (Bauer et al., 1966). The test material used is onion bulbs from a population of commercial varieties of Allium cepa. This species has somatically 16 large chromosomes (2n=16) with clear and characteristic morphologic features, suitable for rapid mitotic studies. The cytological study was carried out with the collected root tips using both control and treatment (Cd) medium roots were collected by a pair of fine forceps and fixed in aceto-alcohol (1:3) solution. After 24 hours of fixation, root tips were transferred from fixative to 70% ethanol and stored in the refrigerator until they were used in the laboratory.
A compound microscope was used for microscopic analysis. All the cells were observed in 40X magnification in the microscope (LABOMED Cxl). Cell counts were carried out considering visual fields scanning the whole slide. During the cell count, the cells were divided into two categories: regular (do not present damages in the chromosomes) and irregular (present damages in the chromosome, such as: chromosomal breakages, simple or multiple an aphasic bridges, laggard or lost chromosome, sticky metaphase etc.) and irregular (present damages in the chromosome, such as: chromosomal breakages, simple or multiple an aphasic bridges, laggard or lost chromosome, sticky metaphase etc.).
Percentage of mitotic abnormalities = Error!× 100
In case of two isolated bacterial strains (isolate A and isolate B), the MIC value was determined by broth tube dilution method against heavy metals (Cd). Based on the evaluation, minimum inhibitory concentration was determined at 37 ºC after 24 hours of incubation period. The lowest concentration that prevented bacterial growth was considered the MIC.
Cadmium degradation efficiency of both isolated bacterial strains observed in MS media. Isolates were grown in different 100ml shake flasks containing 40 ml of cadmium supplemented medium (0.01 g, 0.02 g, 0.03 g, 0.04 g, 0.05 g and 0.06 g Cd) inoculated with 0.5 ml of cells (1% inoculum). Flasks were incubated at 30°C and agitated at 200 rpm in a shaker bath. Optical density at 660 nm (OD660) and pH were monitored. Selected samples were transferred to Eppendorf tubes and centrifuged for 5 min at 8000 x g. The supernatant was drawn and stored at 20°C for cadmium analysis. The cadmium concentration in the supernatant was determined with an Analytik Jena, spekol 1500 UV-visible spectrometer at 228.8 nm with a cadmium lamp.