University of Pretoria Reduction of Uranium(VI) under Micro-aerobic Conditions using an Indigenous Mine Consortium University of Pretoria Energy Postgraduate Conference 2013

Aim & Objectives To utilise indigenous cultures of bacteria from the local environment to biologically reduce U(VI) to U(IV) Isolation and purification of microorganisms for use in further experiments Characterization of microorganisms in order to identify and classify the microorganisms involved in the reduction of uranium (VI) Investigation of the reduction potential of microorganisms that reduce uranium (VI) to uranium (IV) using the consortium in a batch system to establish kinetic parameters for use in reactor scale-up

Introduction Among all elements currently in use in the energy industry worldwide, uranium is the most abundant Uranium-containing wastes are produced at various steps of the nuclear fuel cycle, and vary considerably from low level radioactive effluents produced during uranium mining to intensely radioactive levels in nuclear power plant, spent fuel, and liquid wastes Discharge of radio-nuclides such as uranium from contaminated sites and their subsequent mobility in the environment is a subject of paramount concern The primary radiation health effect of concern is an increased probability of the exposed individual developing cancer during their lifetime

Introduction Physical/chemical processes Treatment Options Pump and treat processes of U(VI)-contaminated water Involves extraction of contaminated water, followed by a separation process on the surface Separation processes include: Ion exchange Chemical precipitation Reverse osmosis Limitation: Expensive to apply

Introduction Biological processes Offer the potential for removing metal/radionuclide pollutants from dilute solutions, where physical chemical methods may not be feasible. 4 mechanisms by which bacteria immobilize metals or Radio-nuclides namely; Bio-sorption, Bioaccumulation, Precipitation by reaction with inorganic ligands Microbial reduction

Introduction Bioreduction Involves the reduction of an element from a higher to a lower oxidation state or to an elemental form affects its solubility, resulting in its precipitation using bacteria Advantages: It is not limited by saturation Many radio-nuclides are less soluble when reduced

Materials & Methods Elemental Analysis of Soil Uranium contaminated soil was collected from a closed uranium mine was analyzed by ICP-OES Isolation of Indigenous Bacteria Mixed culture was obtained by inoculating basal mineral medium (BMM) amended with glucose with mine soil Bacterial cultures were purified and then incubated Batch studies Pure culture batch studies were conducted in basal mineral medium (BMM) supplemented with glucose as a carbon source with different concentrations of U(VI) 75, 100, 200, 400, 600 and 800 mg/L

Materials & Methods Sampling A 0.5 mL sample of the homogenous solution was collected using a syringe and then centrifuged The sample was then diluted with 4.5 mL of BMM (1:10 dilution), mixed with 2 mL of complexing reagent and analyzed in duplicate for U6+ immediately on the UV spec Total uranium level in each sample (U(IV) and U(VI)) was determined by oxidizing an unfiltered sample with nitric acid prior to uranium measurement

Results Elemental analysis of soil Soil was found to contain 168 mg/kg Uranium Preliminary culture characterization Gram staining – Gram- & Gram+ Culture characterization Purified and sequenced rRNA genes from bacteria 4 species were found: Pantoea agglomerans Enterobacter cloacae Pseudomonas stutzeri

Removal of Uranium at Low Concentrations Figure 2. Uranium(VI) reduction for the three pure cultures of bacteria Pseudomonas stutzeri, Pantoea agglomerans and Enterobacter cloacae under an initial concentration of 75 mg/L

Removal of Uranium at High Concentrations Figure 3. Uranium(VI) reduction for the three pure cultures of bacteria Pseudomonas stutzeri, Pantoea agglomerans and Enterobacter cloacae under varying concentrations; A: 100 mg/L B: 200 mg/L C: 400 mg/L and D: 600 and 800 mg/L

Conclusion Microorganisms play important roles in the environmental fate of toxic - array of physical-chemical and biological mechanisms effecting transformations between soluble and insoluble phases Further work - Identify and characterize of cytosolic and outer membrane proteins involved in U(VI) reduction at a cellular level Kinetic modelling of uranium reduction and cumulative removal studies should help us to better predict and model how uranium will behave in situ

Contribution to Scientific Community Achieving U(VI) reduction reaction under near zero oxidation reduction potential (ORP) under facultative conditions Developing a simplified and more reliable method for measuring U(VI) using the Arsenazo III method Acknowledgements Prof EMN Chirwa (University of Pretoria) National Research Foundation (NRF) South African Nuclear Human Asset & Research Programme (SANHARP)