Skip to main content

Biogenic iron-silver nanoparticles inhibit bacterial biofilm formation due to Ag+ release as determined by a novel phycoerythrin-based assay


Silver nanoparticles (Ag-NPs) can be considered as a cost-effective alternative to antibiotics. In the presence of Fe(III)-citrate and Ag+, Klebsiella oxytoca DSM 29614 produces biogenic Ag-NPs embedded in its peculiar exopolysaccharide (EPS). K. oxytoca DSM 29614 was cultivated in a defined growth medium–containing citrate (as sole carbon source) and supplemented with Ag+ and either low or high Fe(III) concentration. As inferred from elemental analysis, transmission and scanning electron microscopy, Fourier transform infrared spectrometry and dynamic light scattering, Ag-EPS NPs were produced in both conditions and contained also Fe. The production yield of high-Fe/Ag-EPS NPs was 12 times higher than the production yield of low-Fe/Ag-EPS NPs, confirming the stimulatory effect of iron. However, relative Ag content and Ag+ ion release were higher in low-Fe/Ag-EPS NPs than in high-Fe/Ag-EPS NPs, as revealed by emission-excitation spectra by luminescent spectrometry using a novel ad hoc established phycoerythrin fluorescence–based assay. Interestingly, high and low-Fe/Ag-EPS NPs showed different and growth medium–dependent minimal inhibitory concentrations against Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 15442. In addition, low-Fe/Ag-EPS NPs exert inhibition of staphylococcal and pseudomonal biofilm formation, while high-Fe/Ag-EPS NPs inhibits staphylococcal biofilm formation only. Altogether, these results, highlighting the different capability of Ag+ release, support the idea that Fe/Ag-EPS NPs produced by K. oxytoca DSM 29614 can be considered as promising candidates in the development of specific antibacterial and anti-biofilm agents.

Key points

Klebsiella oxytoca DSM 29614 produces bimetal nanoparticles containing Fe and Ag.

Fe concentration in growth medium affects nanoparticle yield and composition.

Phycoerythrin fluorescence–based assay was developed to determine Ag+ release.

Antimicrobial efficacy of bimetal nanoparticle parallels Ag+ ions release.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Data availability

All the data are available in the manuscript and in Supplementary Material.


Download references


TEM experimental data were provided by ATeN Center of University of Palermo. The FE-SEM microanalysis was performed by Davide Cristofori, Scientific Campus DSMN Cà Foscari University.

Code availability

Not applicable


This work was supported by the University of Palermo and Italian MIUR to GG and by Ca’ Foscari University of Venice to FB.

Author information

Authors and Affiliations



MGC, FA, GN, MG and AS performed the experiments. DCM, DS, FB and GG supervised the experiments and analysed the data. FB and GG conceived and designed research. All the authors contributed to writing the manuscript.

Corresponding author

Correspondence to Giuseppe Gallo.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

Not applicable

Consent to participate

Not applicable

Consent for publication

All the authors red and approved the manuscript for publication.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cusimano, M.G., Ardizzone, F., Nasillo, G. et al. Biogenic iron-silver nanoparticles inhibit bacterial biofilm formation due to Ag+ release as determined by a novel phycoerythrin-based assay. Appl Microbiol Biotechnol 104, 6325–6336 (2020).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Biogenic bimetal nanoparticles
  • Bacterial exopolysaccharide
  • Nanoparticle production yield
  • Silver ion release
  • Phycoerythrin fluorescence–based assay
  • Biofilm formation inhibition