Investigation of the electrochemical activity of chromium tolerant mutants of Geobacter metallireducens
Pastorella, Gabriele (2014) Investigation of the electrochemical activity of chromium tolerant mutants of Geobacter metallireducens. PhD thesis, Dublin City University.
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Dissimilatory metal reducing bacteria (DMRB) can reduce insoluble electron acceptors, such as metals and electrodes, through extracellular electron transfer. The DMRB Geobacter metallireducens can reduce Cr(VI) to Cr(III) via direct electron transfer through transmembrane cytochrome complexes. Extracellular electron transfer is relevant to both energy production and Cr(VI) detoxification in subsurface environments and groundwater. However, the toxicity of Cr(VI) to G. metallireducens limits its application to field bioremediation, where Cr(VI) concentration may exceed the toxicity threshold (few mg L-1).
In this study, we improved the G. metallireducens electroactivity and its tolerance to Cr(VI) through genome shuffling. The parent strain was exposed to nitrosoguanidine (NTG) for 60 minutes. Then, protoplasts obtained by lysozyme recombined their genomes.
Following protoplast recombination, the mutants obtained are screened by biological, molecular, and electrochemical assays, to select those with higher Cr(VI) tolerance and electroactivity. After one round of genome shuffling, the best performing mutants could grow at 5 mM of Cr(VI), that is, two fold the Cr(VI) concentration tolerated by the wild type G. metallireducens.
Most of the Cr(VI)-tolerant mutants obtained by genome shuffling showed a two-fold decrease of electroactivity when grown in potentiostat-controlled electrochemical cells. This suggests that resistance to Cr(VI) and electroactivity may be partially overlapping pathways, therefore a screening method based on resistance to Cr(VI) is not adequate to select only electroactive strains. Out of ten Cr(VI)-tolerant mutants, we select the M23 mutant that shows two-fold increase in electroactivity with respect to the wild type strain.
In this study we show for the first time that genome shuffling can be applied to strict anaerobes, metal-reducing bacteria, to improve metal resistance and electroactivity. The mutants obtained could be applied to Cr(VI) bioremediation in highly contaminated soil and groundwater.
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