Microalga Broths Synthesize Antibacterial and Non-Cytotoxic Silver Nanoparticles Showing Synergy with Antibiotics and Bacterial ROS Induction and Can Be Reused for Successive AgNP Batches

Abstract

The era of increasing bacterial antibiotic resistance requires new approaches to fight infections. With this purpose, silver-based nanomaterials are a reality in some fields and promise new developments. We report the green synthesis of silver nanoparticles (AgNPs) using culture broths from a microalga. Broths from two media, with different compositions and pHs and sampled at two growth phases, produced eight AgNP types. Nanoparticles harvested after several synthesis periods showed differences in antibacterial activity and stability. Moreover, an evaluation of the broths for several consecutive syntheses did not find relevant kinetics or activity differences until the third round. Physicochemical characteristics of the AgNPs (core and hydrodynamic sizes, Z-potential, crystallinity, and corona composition) were determined, observing differences depending on the broths used. AgNPs showed good antibacterial activity at concentrations producing no or low cytotoxicity on cultured eukaryotic cells. All the AgNPs had high levels of synergy against Escherichia coli and Staphylococcus aureus with the classic antibiotics streptomycin and kanamycin, but with ampicillin only against S. aureus and tetracycline against E. coli. Differences in the synergy levels were also dependent on the types of AgNPs. We also found that, for some AgNPs, the killing of bacteria started before the massive accumulation of ROS.

Keywords: AgNPs from recycled broths; ROS production; Tinto River; acid-tolerant; antibacterial activity; antibacterial synergistic activity; cytotoxicity; green synthesis; microalgae; silver nanoparticles.

Figure 1

Microalga growth curves in two media and at two pHs.

Figure 2

Kinetics of AgNP synthesis followed by UV–Vis spectroscopy. Graphs inserted in the upper right corner of each panel show the kinetics of synthesis from the increase in absorbance at the corresponding λ_max.

Figure 3

UV–Vis spectra of AgNPs harvested at different times of synthesis before and after washing.

Figure 4

Analysis of AgNPs obtained by successive syntheses reusing broth. Upper panels: synthesis kinetics; lower panels: UV–Vis spectra of the AgNPs from the consecutive batches.

Figure 5

Representative TXRF analyses of the elemental composition of AgNPs. AU: arbitrary units.

Figure 6

Representative XRD patterns of the AgNPs. Crystals’ planes are indicated for Ag⁰ in blue and AgCl in red.

Figure 7

TEM representative images of the AgNPs. Lower-right corner insets in each panel show an amplified image of a nanoparticle with internal parallel patterns.

Figure 8

Distribution of AgNPs’ core diameters.

Figure 9

Characterization of the corona components of the AgNPs and the broths by FTIR. AgNPs’ spectra are shown in blue, the broths before the synthesis in green, and the broths after the synthesis in red.

Figure 10

Effect of the presence of the antibiotics on the UV–Vis spectra of the AgNPs.

Figure 11

The hydrodynamic diameters of AgNPs in the presence of antibiotics. C(−) negative control, without antibiotic. (*) indicates a significant difference between the AgNPs’ sizes in the mixes with antibiotic and the C(−). p-value < 0.05.

Figure 12

ROS production and viable cells after treatment with AgNPs. Viable cells % are shown in blue and fluorescence is in red. Statistically significant differences of each value with the untreated control (*) and with the immediately lower AgNPs’ concentration value (#) are indicated.