Inhibition of Nitrosomonas europaea by Ag+ and Ag-NP : Determining the influence of aqueous chemistry, capping agents, growth stage and gene expression on inhibition

Oregon State University, Corvallis OR

Investigators

Abstract

The use of silver nanoparticles (Ag-NP) as a broad spectrum biocide in a wide range of consumer goods has grown exponentially in recent years, which will result in an increased release of Ag-NP into wastewater streams and ultimately the receiving bodies. Ammonia oxidizing bacteria (AOB) play a critical role in the global nitrogen cycle and in the removal of nitrogen during wastewater treatment, thus preventing eutrophication of receiving waters. AOB are also widely considered to be the most sensitive fauna in wastewater treatment plants (WWTP) and are readily inhibited by many industrial contaminants including Ag-NP. This interdisciplinary proposal integrates the application of genomic and physiological assays with physical and chemical characterization techniques to elucidate the fate of Ag-NP in various water matrices, to identify the inhibition mechanism(s) of Ag-NP and to characterize the defense and recovery mechanisms employed by Nitrosomonas europaea, the model AOB, upon exposure to Ag-NP. The hypotheses that will be tested with the proposed experiments are: i) The fate of Ag-NP in natural and engineered systems, including dissolution, aggregation and partitioning to cells will depend on the capping agent used to stabilize the Ag-NP. ii) Ag-NP will inhibit N. europaea activity differently than silver ions (Ag+) and the inhibition is highly influenced by the capping agent, the size of the Ag-NP, the aqueous chemistry of the test media and the growth phase of the cells. iii) Transcriptional responses will differ between N. europaea cells exposed to Ag-NP and Ag+. iv) Sentinel genes can be identified and used to detect Ag+ and Ag-NP at sub-inhibitory concentrations in sequencing batch reactor and biofilm studies mimicking WWTP conditions. v) Biofilms will be more resistant to Ag+ and Ag-NP inhibition. Intellectual Merit This project will examine aggregation, partitioning to cells, and dissolution behavior of a suite of Ag-NPs with different capping agents in relevant water matrices. The inhibition of N. europaea by Ag+ and Ag-NP will then be evaluated in batch systems with tests focusing on the combinations of Ag-NP (with varying capping agents) and aqueous chemistries, including the presence of divalent They will then conduct microarray experiments to determine if Ag+ and Ag-NP cause a differential gene expression and will identify new sentinel genes for Ag+ and Ag-NP. Sequencing batch reactors (SBR) and biofilm experiments will be performed to determine the effects of both long- and short-term Ag+ and Ag-NP exposure on N. europaea. They will determine how well sentinel genes are correlated with Ag-NP concentrations. They will also investigate vertical profiles of nitrification within natural and artificial biofilms using microsensor techniques and will evaluate the spatial distribution of Ag-NP using a Laser Capture Microdissection Microscope. Spatial samples of the biofilm will be used to determine gene expression using qRT-PCR and TEM imaging coupled with elemental analysis will be used to determine the Ag-NP concentrations profiles. Broader Impacts The work includes: Sentinel genes will be identified that can be used to detect the presence of Ag-NP and Ag+, at sub-inhibitory levels. These genes might be used in the development of biosensors for use in wastewater treatment and the environmental sensing of Ag-NP. Physiological consequences of inhibitory effects of Ag-NP as well as damage to cellular functions will also be determined for N. europaea, a model AOB. This research will be among the first to characterize how different capping agents affect the partitioning of Ag-NP and will add to the limited knowledge of the inhibition observed in biofilms. The researchers are members of the Safer Nanomaterials and Nanomanufacturing Initiative (http://www.greennano.org/). Thus there is synergy with the activities being conducted as part of that Initiative, including interactions with companies manufacturing Ag-NP. A post-doctoral, graduate and undergraduate student will be involved in the research. As in their past NSF-funded work, mentoring opportunities for the post-doc and graduate student include the independent supervision of undergraduate research, participation in grant writing, presenting at conferences, and publishing manuscripts as the lead and communicating author. They also plan to continue their involvement of under-represented undergraduate and high school students in this research through their continued participation in the Science and Engineering Summer Experience for Youths (http://cbee.oregonstate.edu/ sesey/), Saturday Academy (http://academy.engr.oregonstate.edu/) and the OSU Engineering Women and Minorities Tektronix Scholars program (http://engr.oregonstate.edu/wme/).