Antibacterial effect of copper nanoparticles produced in a Shewanella-supported non-external circuit bioelectrical system on bacterial plant pathogens

Abstract

The use of copper nanoparticles for the inhibition of plant pathogens Ralstonia solanacearum, which causes wilt disease, and Xanthomonas axonopodis, which causes citrus canker, was investigated in this study. To avoid the inhibiting effect of Cu²⁺ ions on the bacterial cells, the copper nanoparticles were synthesized in the cathode chamber of a non-external circuit bioelectrochemical system (nec_BES) inoculated with Shewanella sp. HN-41 at the anode. The electrons produced by the oxidation of lactate by Shewanella sp. HN-41 were directly transferred to the anolyte in the cathode via a graphite electrode connecting the anode and cathode chambers. SEM images of the produced particles revealed that the copper nanoparticles were aggregated into spherical shapes with an average size of 2.9 μm from smaller particles with a size range from 30 nm to approximately 190 nm. X-ray diffraction demonstrated that the copper nanoparticles were mainly in the form of a single-phase crystal mixture of atacamite (Cu₂Cl(OH)₃) and paracatamite (Cu₂Cl(OH)₃). Finally, for the application of synthesized nanoparticles, an agar diffusion test was applied to assess the antibacterial activity of the formed copper nanoparticles in propylene glycol solvent against R. solanacearum and X. axonopodis. The results showed that the nanoparticles damaged the cells of R. solanacearum, with a half maximum inhibition (IC₅₀) value of 42 ppm, but did not damage X. axonopodis cells.

Fig. 1. The configuration of the non-external circuit bioelectrochemical system.

Fig. 2. Changes in sodium lactate concentration (A) and pH value (B) in anolyte (DC: control sample (without bacteria added), nec_BES: experimental sample).

Fig. 3. Changes in Cu²⁺ ion concentration in catholyte (DC: control sample (without bacteria added), nec_BES: experimental sample).

Fig. 4. SEM image of Cu nanoparticle produced from nec_BES system supported by Shewanella sp. HN-41 (scale bar: A – 10 μm. B – 500 nm); nanoparticle diameter distribution (C); EDX picture of Cu nanoparticles (D).

Fig. 5. XRD image of Cu nanoparticles produced from nec_BES system supported by Shewanella sp. HN-41.

Fig. 6. Effect of different Cu nanoparticle concentrations (10, 25, 50, 100, 200 and 500 ppm) on bacteria (2012.001: Xanthomonas axonopodis; 2012.002: Ralstonia solanacearum; PG: polyethylene glycol).

Fig. 7. Effect of different Cu nanoparticle concentrations (ppm) on the growth of Ralstonia solanacearum (DC_PG + number: cells incubated with different propylene glycol concentrations; VK_NANO + number: cells incubated with different nanoparticle concentrations).

Fig. 8. SEM and cross-section TEM images: Xanthomonas axonopodis (A) and Ralstonia solanacearum (B) cells incubated with 100 ppm Cu nanoparticles in the liquid. Dead (C) and live (D) Ralstonia solanacearum cells (scale bar: 500 nm). CuNs: copper nanoparticles, CM: cell membrane.