Molecular Characterization of Metal Tolerant Bacteria Isolated from Contaminated Soil

Abstract Soil pollution with toxic metals is an alarming threat to the agricultural sector as these are non-biodegradable. The problem is getting worse in developing countries like Pakistan. Several methods remove the toxic metals from the environment including physical, chemical, and biological processes. Physical and chemical methods are not only expensive but also produce secondary by-products that may damage the ecosystem. Bioremediation is a cost-effective method that reduced environmental pollution by using biodegradation processes. Therefore, this study was carried out for the isolation and characterization of metal tolerant bacteria from wastewater irrigated soil in Multan. Contaminated soil samples were collected from wastewater irrigated soil, and were inoculated on Nutrient agar poured with hexavalent chromium. Positive cultures were freshed on Nutrient agar on basis of colony morphology, and pure colonies were subjected to varying concentrations of metal to determine the Minimum Inhibitory Concentration (MIC). The broth dilution method was used for the determination of minimum inhibitory concentration. Out of four soil specimens, a total of six bacteria belonging to the two different species were isolated and identified on basis of gram staining and biochemical characterization. Differentiation of gram-positive rods on basis of colony morphology and biochemical tests were identified as Bacillus subtilis (n = 4) and Bacillus cereus (n = 2). Gene amplification through specific primers for 16S rDNA was performed according to the protocol. For further evaluation and sequencing, isolates have been sent to Macrogen (Korea).