2.6. Bioremediation of heavy metal
Five milliliters of the overnight pre-culture were inoculated into 45 mL of YA broth contained in 250 mL conical flasks. The YA broth contained 5 mM urea and 25 mM CaCl2, which were filter sterilized using a 0.22 m filter, and was supplemented with 2 mM of heavymetal mixture. The flasks were incubated in a shaking incubator(200 rpm) at 30◦C for 48 h. Control sets without any added bacterial cells were also included in all experiments. After incubation, the cultures were centrifuged at 8000 × g for 15 min. The heavy metal bioremediation by single cultures and bacterial mixtures (A3) werecalculated as the percentage difference between the initial and final concentrations of heavy metals present in the supernatants. Concentrations of heavy metals were measured using a Perkin-ElmerOPTIMA-7300DV inductively coupled plasma optical spectrometer(ICP-OES, PerkinElmer, Inc., USA).