**3. Determination of U(VI)**

U(VI) reductase activity was determined by measuring the decrease in U(VI) in the solution using UV/vis spectrophotometer (WPA, Light Wave II, and Labotech, South Africa). Arsenazo III (Sigma-Aldrich, St. Louis, Missouri, USA) (1, 8-dihydroxynaphthalene-3, 6 disulphonic acid-2, 7-bis [(azo-2)-phenylarsonic acid]), a non-specific chromogenic reagent, was selected as the complexing agent for facilitating U(VI) detection. The accuracy and the precision of the method on the UV/vis spectrophotometer was determined by measuring the concentration of standard U(VI) solution in the range of (0-80 mg/L). A linearized U(VI) standard curve was generated by plotting the absorbance at 651 nm versus the known U(VI) standard concentra‐ tion. Standard curve for U(VI) measurement demonstrated high degree of accuracy with R2 = 99.7% and was used to estimate unknown U(VI) concentration.

bacterial strains were isolated by serial dilution of the cultivated culture. U(VI) reduction activity was evaluated for the isolates starting with evaluation for abiotic processes to make sure that physical-chemical processes are taken into consideration when analysing the

Bioremediation of Radiotoxic Elements under Natural Environmental Conditions

Heat-killed cultures and sodium azide exposed cultures were used to determine the extent of abiotic U(VI) reduction in batch experiments. For U(VI) reduction experiments cultures were grown over night in a sterile nutrient or Luria-Bettani (LB) broth under anaerobic conditions. Overnight grown cells were heat killed by autoclaving at 121°C for 20 minutes and another

harvested by centrifuging at 6000 rpm (2820 *g*) for 10 minutes. The supernatant was then decanted and the remaining pellet was washed three times with sterile 0.85% NaCl solution. The washed pellet was then re-suspended in 100 mL serum bottle containing U(VI) stock solution and sterile basal mineral medium (BMM) amended with D-glucose as sole carbon

residual oxygen before sealing the bottle with rubber stoppers and aluminium seal. The experiments were all conducted at 30±2°C with continuous shaking on lateral shaker (Labotec, Gauteng, South Africa). The experimental conditions in the abiotic controls and the live cells experiments were kept the same (100 mL serum bottles containing BMM amended with Dglucose, 100 mg/L U(VI) solution, and incubated at 30±2°C under anaerobic conditions). A sample was withdrawn at regular time interval using a disposable syringe for U(VI) reduction

The results showed insignificant difference U(VI) reduction between live and heat-killed cells (Figure 3). The instantaneous U(VI) reduction in heat-killed cells may be due to the presence of the cells that escaped destruction by heat. The reduction of U(VI) observed during the first 2 hours in all treatment containing biomass presented an anomaly. It was clear from these results that another mechanism rather than the direct metabolic process was involved in the U(VI) removal from solution. On the other hand abiotic control (without bacteria) showed that

Reverting back to the biosorption studies, it is suggested that functional groups on the cell wall

the cell surface. U(VI) reduction may serve as a step towards this complexation step. To evaluate these effects we conducted experiments where the pH was varied and the oxidation reduction potential (ORP) was measured with time. Results presented in Figure 4 show that that the rate of U(VI) reduction was pH dependent (Figure 4a). Electronegative conditions under anaerobic conditions created a strongly reducing environment as expected, after which the ORP increased to electropositive values (Figure 4b). As a result insignificant change over time in ORP indicated poor oxidation-reduction, while significant change in ORP over time

indicated that the oxidation-reduction process approaches completeness.

, and –COOH) may serve as ligands for U(VI)-U(IV) complexation with

). Cells were then

http://dx.doi.org/10.5772/56909

191

) for about 5-10 minutes to expel

set of overnight grown cells were incubated with 0.1% of sodium azide (NaN3

source. The serum bottles were then purged with 99.9% (N2

U(VI) reduction process is biologically mediated.

biological process.

**5. Abiotic U(VI) reduction**

analysis as described above.

surfaces (-OH, -NH2

Measurement of U(VI) was carried out by withdrawing 2 mL of homogenous solution from a 100 mL serum bottle using a disposable syringe. The sample was then centrifuged for about 10 minutes at 6000 rpm (2820 *g*) using a Minispin® Microcentrifuge (Eppendorf, Hamburg, Germany). The sample (0.5-1 mL) was then diluted with 0.4 mL of 2.5% diethylene-triamine‐ penta acetic acid (DTPA) and then diluted up to mark with basal mineral medium (BMM) (1:10) dilution. The homogenous solution was then mixed with 2 mL of complexing reagent (Arsenazo III ) and then allowed to stand for full development of the pink-violet color prior analyses for U(VI) at a wavelength of 651 nm (10 mm light path) against a reagent blank. Total uranium in the unfiltered sample was measured using inductively-coupled plasma mass spectrometry (ICP-MS).
