ROLE AND MECHANISMS OF BACTERIAL BIOFILMS IN SILVER NANOPARTICLE RESISTANCE

Abstract

The use silver nanoparticles (AgNPs) may release AgNPs into wastewater and natural water systems where they can be harmful to biofilms. Therefore, it is important to understand the mechanisms of biofilm resistance to AgNPs. This research aims to investigate the mechanisms of AgNP resistance in biofilms by focusing on the roles of biofilm maturity, biofilm physical characteristics, and AgNP properties. A representative for environmental bacteria used in this study was Pseudomonas putida KT2440. Biofilms at three stages of maturity were identified and selected based on adenosine triphosphate (ATP) activity, extracellular polymeric substance (EPS) amount, and expression of EPS-associated genes (csgA and alg8). Exposing to synthesized AgNPs, more mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity whereas the same treatment reduced more than 90% of ATP activity in the less mature biofilms (stage 1). The biofilms stripped of their EPS were more susceptible to AgNPs than controls with intact EPS, showing the critical role of EPS in AgNP resistance. Not only was the AgNP resistance of biofilms related to the stage but also physical characteristics. The mature biofilms forming in different carbon sources (glucose, glutamic acid, and citrate), glucose concentrations (5 and 50 mM of glucose), and temperature (25° and 30°C) had different physical characteristics. Biofilms with more thickness, more biomass volume, less surface/volume ratio, and less roughness are more resistant to AgNPs. The AgNP resistance of biofilms forming in glucose was similar to glutamic acid (1-log reduction in cell number), which was higher than citrate (2-log reduction in cell number). At 25°C, biofilms forming at higher glucose concentrations exhibited higher AgNP resistance (3-log reduction for 5 mM biofilms and less than 1-log reduction for 50 mM biofilms). Similarly, when the temperature was increased, the 5 mM glucose biofilms had higher AgNP resistance (3 log reduction at 25°C and 1 log reduction at 30°C). The toxicity of AgNPs in this study was mostly from the nanosized AgNPs combining with some toxicity from the released Ag+. The released Ag+ showed less toxicity (<1-log reduction) than AgNPs (3 to 4-log reduction) to biofilms in 5 mM of glucose at 25°C. The effect of AgNPs on biofilms increased when AgNPs had more Ag+ release and highly negative or positive charges. Three AgNPs were synthesized to have different Ag+ release, which are listed as AgNPs#1, AgNPs#2, and AgNPs#3, respectively. The sizes of the AgNPs were similar while the Ag+ releases were different, which are 5.3% for AgNPs#1, 7.7% for AgNPs#2, and 9.1% for AgNPs#3. On average, all AgNPs exhibited nearly neutral charge (-1.4 mV for AgNPs#1, -5.5 mV for AgNPs#2, and -2.4 mV for AgNPs#3). However, AgNPs#3 had mainly highly negative (-60 to -95 mV) and positive charges (+140 mV) (but the average charge is close to neutral). After exposing biofilms to AgNPs, the Ag+ release increased to 7% for AgNPs#1, 13% for AgNPs#2, and 12% for AgNPs#3. The AgNPs#3 exhibited highest toxicity to biofilms. The effect of AgNPs#1 on biofilms was less (1 to 2-log reduction) than that of AgNPs#2 and AgNPs#3 (3-log reduction). Overall, this study demonstrates that biofilm stage, physical characteristics, and AgNP properties must be considered altogether when determining the impact of AgNPs on environmental biofilms.