2.3. Testing the ability of bacteria to utilize aminocyclopropane carboxylic acid (ACC) as a sole nitrogen source
Because ACC is an intermediate precursor in ethylene synthesis, the ability of cadmium-resistant PGPR to use ACC as a sole nitrogen source was tested. Briefly, DF salts minimal medium (Dworkin and Foster, 1958) containing 3.0mM ACC (Sigma, USA) instead of ammonium sulfate ((NH4)2SO4), as a nitrogen source (Penrose and Glick, 2003) was inoculated with the bacteria. DF salts minimal medium containing (NH4)2SO4 was used as a control (Rajkumar and Freitas, 2008). The cultures were incubated at 28 °C with shak- ing at 150 rpm for 24 h. The bacterial growth was monitored by measuring the optical density at a wavelength of 600 nm (OD600).
2.4. Determination of the minimum inhibitory concentration (MIC) of cadmium
The MIC of cadmium against the growth of Micrococcus sp. and Klebsiella sp. in culture broth was studied. The culture of each cad- mium-resistant PGPR was added to LB broth modified with differ- ent concentrations of cadmium in the form of Cd(NO3)2 ranging from 0 to 1200 mM. The cell cultures were incubated at 28 °C with shaking at 150rpm for 24h. The growth was monitored as a change in OD600 using a spectrophotometer. The lowest concentra- tion of cadmium in the broth that inhibited growth was recorded as the MIC.
2.5. Biosorption of the cadmium ion from an aqueous solution by cadmium-resistant PGPR
The biosorption of the cadmium ion by Micrococcus sp. and Klebsiella sp. was examined. Briefly, bacterial cells were added to a 25 mg L