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Molecules 2020, 25, 2023 3 of 14 Molecules 2020, 25, x FOR PEER REVIEW 3 of 15 microscopy (ESEM), and confocal Raman microscopy (CRM). Raman spectroscopy provides us with molecular fingerprint information and permits us to study subtle changes in cellular structure, as a their Raman band profiles. This technique has been used to provide an image of the antimicrobial result of modification of proteins, lipids and nucleic acids that undergo changes in their Raman band effect and was recently used to study molecular changes within bacterial cells after antibiotic profiles. This technique has been used to provide an image of the antimicrobial effect and was recently treatment [19,20]. used to study molecular changes within bacterial cells after antibiotic treatment [19,20]. The aim of this work was to produce silver nanoparticles using the extracellular cell free extracts The aim of this work was to produce silver nanoparticles using the extracellular cell free extracts of Phanerochaete chrysosporium and evaluate their antimicrobial and antibiofilm properties. To achieve of Phanerochaete chrysosporium and evaluate their antimicrobial and antibiofilm properties. To achieve this, our study focused on the interactions between the biogenic silver nanoparticles (PchNPs) and E. this, our study focused on the interactions between the biogenic silver nanoparticles (PchNPs) and coli cells using TEM, ESEM and CRM, in order to ascertain the antimicrobial mechanisms of these E. coli cells using TEM, ESEM and CRM, in order to ascertain the antimicrobial mechanisms of these nanoparticles; while the effect of these antimicrobial nanoparticles on the disruption and eradication nanoparticles; while the effect of these antimicrobial nanoparticles on the disruption and eradication of of E. coli and C. albicans biofilms was also evaluated. E. coli and C. albicans biofilms was also evaluated. 2. Results and Discussion 2. Results and Discussion 2.1. Synthesis of Biogenic Silver Nanoparticles (PchNPs) 2.1. Synthesis of Biogenic Silver Nanoparticles (PchNPs) The biomass from cultures of the fungus Phanerochaete chrysosporium was harvested, washed and The biomass from cultures of the fungus Phanerochaete chrysosporium was harvested, washed and suspended in sterilized distilled water. After incubation, the cell-free filtrate was added to a silver suspended in sterilized distilled water. After incubation, the cell-free filtrate was added to a silver nitrate nitrate solution and the mixture was incubated in dark. The absorbance spectrum was measured in solution and the mixture was incubated in dark. The absorbance spectrum was measured in the range of the range of 250–800 nm and the maximum peak was determined. Color change in the reaction 250–800 nm and the maximum peak was determined. Color change in the reaction mixture (Figure 1a,b) mixture (Figure 1a,b) as well as the appearance of an absorption band between 400 and 450 nm as well as the appearance of an absorption band between 400 and 450 nm corresponding to the surface corresponding to the surface plasmon resonance (SPR) (Figure 1c) were indicative of the formation plasmon resonance (SPR) (Figure 1c) were indicative of the formation of silver nanoparticles (Figure 1b). of silver nanoparticles (Figure 1b). Figure 1. Color change obtained for silver nanoparticles synthesized (a) control, (b) PchNPs and Figure 1. Color change obtained for silver nanoparticles synthesized (a) control, (b) PchNPs and (c) (c) UV-Vis absorption spectra, after 24 h of reaction. UV-Vis absorption spectra, after 24 h of reaction. After a 24 h period an 87% yield was obtained from the synthesis. The reaction mixture was After a 24 h period an 87% yield was obtained from the synthesis. The reaction mixture was centrifuged and the pellet containing the silver nanoparticles was washed with and resuspended in centrifuged and the pellet containing the silver nanoparticles was washed with and resuspended in water. The UV-vis spectra after centrifugation showed an absorption band at 442 nm, corresponding to water. The UV-vis spectra after centrifugation showed an absorption band at 442 nm, corresponding the SPR band of silver nanoparticles (Figure 2). to the SPR band of silver nanoparticles (Figure 2). 2.2. Evaluation of the Incidence of Reaction Conditions in the Biosynthesis of PchNPs The evolution of silver nanoparticle synthesis over time was monitored by measuring the formation of SPR band via UV-vis spectroscopy absorbance at 440 nm. The incidence of AgNO3 concentration, reaction temperature and incubation time of the mycelium with water are shown in Figure 3. 5 mM AgNO3 was the concentration that reached the highest yield for the nanoparticle synthesis (Figure 3a). In addition, the conditions for the greatest nanoparticle production in the shortest time found at a reaction temperature of 37 ◦C (Figure 3b) and 41 h of incubation of the mycelium with water (Figure 3c). All subsequent biosyntheses were carried out using these conditions to obtain the highest production of stable PchNPs in the shortest time.PDF Image | Biofilm Eradication Using Biogenic Silver Nanoparticles
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