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

近年来，银纳米粒子(Ag-NP)作为一种广谱杀生剂在各种消费品中的使用呈指数级增长，这将导致Ag-NP释放到废水中，并最终释放到接收体中。氨氧化细菌(AOB)在全球氮循环和废水处理过程中的脱氮过程中发挥着关键作用，从而防止了受纳水体的富营养化。AOB也被广泛认为是污水处理厂(WWTP)中最敏感的动物，很容易受到包括Ag-NP在内的许多工业污染物的抑制。这一跨学科的方案综合了基因组和生理分析以及物理和化学表征技术的应用，以阐明Ag-NP在各种水基质中的去向，确定Ag-NP的抑制机制(S)，并表征模型AOB欧洲亚硝酸单胞菌在暴露于Ag-NP时所采用的防御和恢复机制。将用拟议的实验检验的假设是：i)银-NP在自然和工程系统中的命运，包括溶解、聚集和分配到细胞，将取决于用于稳定银-NP的封闭剂。2)银离子(Ag+)与银离子(Ag+)不同，Ag-NP对菜豆根结线虫的抑制作用与银离子(Ag+)不同，其抑制作用受封端剂、Ag-NP的大小、实验介质的水化学和细胞生长时相的影响较大。(Iii)银离子和银离子对神经节杆菌细胞的转录反应不同。Iv)在模拟污水处理厂条件的序批式反应器和生物膜研究中，可以识别前哨基因，并将其用于检测亚抑制浓度的Ag+和Ag-NP。V)生物膜对Ag+和Ag-NP的抑制能力更强。这个项目将研究一组不同封顶剂的银纳米颗粒在相关水基质中的聚集、向细胞的分配和溶解行为。然后，将在批处理系统中评估Ag+和Ag-NP对N.uropaea的抑制作用，测试重点是Ag-NP(具有不同封闭剂)和水化学的组合，包括二价的存在。然后，他们将进行微阵列实验，以确定Ag+和Ag-NP是否会导致基因表达差异，并将确定Ag+和Ag-NP的新前哨基因。将进行序批式反应器(SBR)和生物膜实验，以确定长期和短期的Ag+和Ag-NP暴露对欧洲夜蛾的影响。他们将确定前哨基因与Ag-NP浓度的相关性有多好。他们还将使用微传感器技术调查天然和人造生物膜内硝化作用的垂直分布，并将使用激光捕获显微解剖显微镜评估Ag-NP的空间分布。生物膜的空间样本将被用来通过qRT-PCR来确定基因表达，而电子显微镜成像和元素分析将被用来确定Ag-NP浓度分布。这项工作的更广泛影响包括：将识别可用于检测亚抑制水平上的Ag-NP和Ag+存在的前哨基因。这些基因可用于废水处理生物传感器的开发和银纳米粒子的环境传感。还将确定Ag-NP抑制效应的生理后果以及对细胞功能的损害，这是一种AOB模型。这项研究将是首批表征不同封顶剂如何影响Ag-NP分配的研究之一，并将增加对生物膜中观察到的抑制作用的有限知识。研究人员是更安全的纳米材料和纳米制造倡议(http://www.greennano.org/).)的成员因此，作为该倡议的一部分而开展的活动，包括与生产银-NP的公司的互动，产生了协同作用。一名博士后、研究生和本科生将参与这项研究。与过去NSF资助的工作一样，博士后和研究生的指导机会包括独立监督本科生的研究，参与助学金的撰写，在会议上发表报告，以及作为主要和交流作者出版手稿。他们还计划通过继续参加青年科学和工程暑期体验(http://cbee.oregonstate.edu/SESY/)、星期六学院(http://academy.engr.oregonstate.edu/))和俄亥俄州立大学工程妇女和少数族裔泰克学者计划(http://engr.oregonstate.edu/wme/).)，继续让未被充分代表的本科生和高中生参与这项研究

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/).