GEOBACTER METALLIREDUCENS MUTANTS FOR CHROMIUM BIOREMEDIATION Ilaria J. Chicca, Gabriele Pastorella and Enrico Marsili School of Biotechnology, DCU GEOBACTER METALLIREDUCENS MUTANTS FOR CHROMIUM BIOREMEDIATION Ilaria J. Chicca, Gabriele Pastorella and Enrico Marsili School of Biotechnology, DCU Abstract Geobacter metallireducens is known to use oxidized metals such as Fe(III) and Mn(IV) as terminal extracellular electron acceptors. G. metallireducens can reduce the highly soluble and toxic Cr(VI) to its insoluble form Cr(III), thus enabling chromium removal from contaminated water and soil. This process is limited by the low toxicity resistance of G. metallireducens to Cr(VI). Genome shuffling can generate mutants with improved phenotype. In our work we are carrying out G. metallireducens genome shuffling, in order to increase its resistance to Cr(VI). In Geobacter species, metal toxicity resistance is correlated to the respiration rate, hence to the reduction rate of the metallic electron acceptor, when soluble electron acceptors are depleted. Our results show that mutants obtained are more resistant than wild type strain to Cr(VI). We characterized the metal reduction process of G. metallireducens wild type and mutants in potentiostat-controlled electrochemical cells through microbial biofilm voltammetry. Introduction Material and methods Aim Geobacter metallireducens strain GS15 (DSMZ 7210) was sub-cultured using strict anaerobic protocols in NBFC medium added by 50 µg/ml NTG and 1 mM Cr(VI). The mutagenized colonies were collected and transferred to NBFC. The cell wall was removed by lysozyme in presence of an isosmotic protoplast buffer. The protoplast fusion was promoted by PEG6000 and EDTA. The recombined cell were transferred to a Geobacter Protoplast Buffer added by NAG to re-build the cell wall. A population of the best performing mutants was selected according to the Cr(VI) resistance. This population was used to start the next round of genome shuffling. The mutant most resistant to Cr(VI) that show the highest growth (respiration) rate on solid medium was grown as a thin biofilm on electrode poised at oxidative potential. +50µg/ml NTG NTG +Lysozime +PEG mM CrO 3 Cr(VI) Result and discussion Chromium has been widely used in various industries. Hexavalent chromium (Cr +6 ) is a toxic, mutagenic and carcinogenic chemical released into water and sediments. Furthermore, its high solubility make it difficult to remove. The reduction to trivalent chromium (Cr +3 ), less bioavailable and completely insoluble, enables chromium removal from groundwater and its long-term immobilization in soils. Geobacter metallireducens is a Cr(VI)- reducing microorganism. As a dissimilatory metal reducing bacterium (DMRB), Geobacter reduce metals during anaerobic respiration through a cell membrane-associated electron transport system (ETS) and Cr(III) precipitates in amorphous or nanocrystalline form on the bacterial membrane during this microbial reduction process. The main obstacle to the large-scale application of this method for bioremediation is the low toxicity resistance of G. metallireducens to Cr(VI). In fact, even at low concentrations, hexavalent chromium is inhibitory to growth and activity of Geobacter. Improve Cr(VI) tolerance in Geobacter metallireducens using genome shuffling technology Figure 3. Diagram of a genome shuffling cycle:  Mutagenesis: G. metallireducens was grown in presence of 50mg/l NTG and 1,2mM Cr(VI);  Shuffling: the cell wall is removed by lysozyme, and the cell membrane fusion is promoted by detergent;  Selection: after the cell wall restoration, the recombined mutants are selected according to the improved maximal Cr(VI) tolerance phenotype Figure 1. Example of bioremediation by G. metallireducens [1]. As other electroactive microorganisms, G. metallireducens can be grown as biofilms at graphite electrodes in potentiostat-controlled electrochemical cells. In absence of soluble electron acceptors, the microorganisms conserve energy through extracellular electron transfer (EET) to the electrode, which is maintained at oxidative potential. Chronoamperometry (CA) measures the respiration rate of G. metallireducens biofilms; cyclic voltammetry (CV) provides qualitative information about the respiration mechanism in the bacteria. The combination of CA, CV, and other electrochemical methods will be used to characterize the G. metallireducens mutants after each round of genome shuffling. Figure 4 and 5 show typical CA and CV for wild type G. metallireducens. Figure 4. Chronoamperometry of a G. metallireducens wild type biofilm growing on a graphite electrode. Following inoculation, the residual iron determines the high oxidation current. After 2 changes of growth medium, the current start increasing as the biofilm accumulate on the electrode. The maximum current is independent from acetate concentration. Figure 5. Cyclic Voltammetry of a G. metallireducens wild type biofilm growing on a graphite electrode. After removal of residual iron, a sigmoidal catalytic curve develops and grows with time, indicating the formation of a stable G. metallireducens electroactive biofilm at the electrode. The midpoint potential of EET is about 0.15 V vs. SHE. Figure 2. Generic scheme of genome shuffling technology [2]. References [1] R. Mahadevan, B. Palsson‡, D.R. Lovley. Nat. Rev. Microbiol. 9 (2011) 39. [2] J.Gong, H. Zheng, Z. Wu, T. Chen, X. Zhao. BIotechnol. Adv. 27 (2009) 996.