Previously established unpublished data show that biofilm cells are more resistant to silver nanoparticles (AgNPs) than planktonic cells. The goal of this research is to determine the fate and transport of AgNPs in a single species biofilm system and to examine the role of AgNPs in the proliferation of antibacterial resistance in attached growth systems. The specific objectives and associated tasks include (1) characterizing AgNPs and examining microbially induced particle aggregation/dissolution and determining the spatial/temporal distribution of AgNPs in biofilms, (2) examining the spatial and temporal variation of microbial activity within the biofilm exposed to AgNPs, and (3) evaluating the impact of AgNPs on gene expression of the stress related genes and antimicrobial resistance genes in biofilms. The research will use microrespirometry, two-photon laser scanning fluorescence microscopy (2P-LSM) and laser capture microdissection microscopy to determine bacterial activity, AgNPs and cell activity within the biofilm, and the levels of antimicrobial resistance gene expression at different biofilm depth, respectively. The research will further examine particle dissolution/aggregation/transformation by microorganisms using advanced electron microscopy (TEM/SEM/ESEM) coupled with energy dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS). Two bacterial species Escherichia coli and Pseudomonas aeruginosa will be used for single species biofilm studies because P. aeruginosa is a well-studied opportunistic pathogen that is commonly found in the environment to form biofilms while E. coli is still used as the principle indicator for drinking water pollution monitoring. Significant broader impacts involve student support.
Microbial cells in aquatic ecosystems often live in densely clustered and surface-attached communities known as biofilms to protect themselves from environmental changes. Extracellular polymeric substances produced by the cells contribute to biofilm formation. Biofilms are resistant to toxic chemicals such as heavy metals and antimicrobial agents. Biofilms can be up to 1,000 times more resistant to toxicants than the planktonic cells. Although biofilms are ubiquitous in the environment and important in water/wastewater treatment systems, the fate of nanoparticles in biofilms and their toxicity to biofilm cells are unclear and less studied. Silver nanoparticles (AgNPs or nanosilver) are zero-valent silver with size less than 100 nm in at least one of its dimensions. Due to its small size and high specific surface area, nanosilver has unique physicochemical characteristics compared to its bulk counterpart, such as excellent catalytic activity, high nonlinear optical performance, and specific electronic properties. Nanosilver has very strong antimicrobial properties, which are mainly attributed to the release of silver ions (Ag+) from the dissolution of silver nanoparticles. Because regular water treatment process only partially removes these nanoparticles, there is a concern that the wide use of nanosilver products may affect wastewater treatment and select bacteria with resistance to different kinds of antimicrobial agents including nanosilver. The main objective of this research is to determine the fate, transport and toxicity of AgNPs in a bacterial biofilm. Results of this NSF project have shown that biofilm cells are more resistant to AgNPs than planktonic (free-swimming) cells. At the same bacterial concentrations (3×108 CFU/mL), biofilms were about four times more resistant to nanosilver inhibition than planktonic cells. The minimum bactericidal concentrations (MBCs) of nanosilver (size from 15 to 21 nm), defined as the lowest concentration that kills at least 99.9% of a planktonic or biofilm bacterial population, were 38 and 10 mg/L Ag, respectively. Nanosilver was aggregated in the presence of planktonic or biofilm-forming cells resulting in an increase of average particle size by a factor of 15 and 40, respectively. The nanosilver particles were able to penetrate to approximately 40 mm in a thick biofilm after 1-hour exposure. These findings suggested that biofilm resistance to nanosilver could be at least partially due to nanoparticle aggregation and retarded silver ion/particle diffusion. The project also determined the long-term effects of AgNPs on membrane bioreactor (MBR) wastewater treatment performance. At the influent AgNP concentration of 0.10 mg Ag/L, there was no significant difference in effluent water quality or bacterial activities before and after AgNP exposure. Nitrifying bacterial community structure was relatively stable before and after the long-term AgNP loading. Abundance of silver resistance gene silE in the MBR, however, increased by 50-fold after the AgNP exposure. The results suggest that AgNPs at the influent concentrations of 0.10 mg/L and below have almost no impact on wastewater treatment performance, as microbial cells in MBRs can effectively reduce nanosilver toxicity by adsorbing or precipitating AgNPs and Ag+released from the dissolution of AgNPs. This research helps better understand the nano-microbial interactions in biofilms and membrane bioreactor wastewater treatment systems. The results of this research covering nano-microbial interactions provide new information and guidance for real-word applications. The project’s research has been integrated into education by training two graduate students and providing hands-on learning experience for three undergraduates. Results of this research are also used for renovation of two environmental engineering courses. The research findings have been disseminated via student-led presentations in national/international conferences and published in peer-reviewed, high-impact journals.
