Copper Nanoparticle Interactions with Nitrogen-Cycling Bacteria

这项由纳米技术环境健康与安全项目授予的国家科学基金会奖支持Shaily Mahendra教授的工作，该研究旨在研究铜纳米颗粒（NPs）对自然和工程环境系统中参与氮循环的细菌的多样性和功能的影响。背景与意义。我们预计，在未来的几十年里，含有纳米材料的工程产品进入废物流的数量将会增加，并且将需要更多的工厂来进行废水的三级处理。环境中氮的转化是由微生物驱动的；硝化、反硝化和固氮是由特定的酶催化的，这些酶对环境干扰和污染很敏感。此外，氮管理在农业和废水处理中是一个重大挑战。纳米材料在商业产品中的生产和使用的快速增长引起了人们对其对环境的潜在不利影响的关注。我们目前对微生物的摄取和毒性机制的理解是有限的。此外，大多数毒理学研究评估？制造材料?而不是经过环境转变后所获得的形式。我们将研究纯培养物和群体中相关酶的调控，从而解决NSF的重要任务，即建立一种预测方法，以理解和减轻NPs与环境相互作用的潜在有害影响。知识的优点。本研究将验证以下假设：铜NPs会选择性地影响参与氮循环的细菌群落的数量、多样性和活性，这是基于NP-细胞相互作用的特定性质（离子铜摄取与NP摄取，外部与内部溶解，然后是活性氧（ROS）的产生和膜/DNA/ATP的损伤）；等等)。虽然最近有一些关于NPs对实验室纯培养物的短期毒性作用的报道，但该建议的新颖方面包括评估NPs？对一类本地微生物的影响，以及它们在较长时间尺度上所扮演的环境角色。此外，这将是第一个探索高通量筛选（HTS）评估剂量反应特征以及NP毒性机制的研究。具体目标包括：(a)通过标准生理和HTS实验确定铜NPs对微生物的影响，(b)研究铜NPs对几种纯硝化、反硝化、厌氧氨氧化和固氮细菌功能基因表达的影响，以确定氮循环中最敏感的微生物过程，(c)量化和模拟氮循环细菌群落数量和多样性的变化。包括离子和颗粒铜、营养物质和电子受体的可用性对离子和颗粒铜的传输阻力的影响。纯培养和微观环境下，NPs对相关基因表达的影响将通过酶特异性测定和RT-qPCR来确定。最终，本文采用的方法将作为解决NP危害识别和风险评估、纳米技术的安全设计和实施以及全球碳和氮循环管理的模板。更广泛的影响。拟议研究的更广泛影响超出了对NP与环境相关细菌相互作用的更好理解。除了在科学会议上发表论文和报告外，这些发现还将被翻译成一系列讲座，在新的环境生物技术研究生课程和重新设计的环境纳米技术本科课程中发表（两者都由PI教授）。此外，PI将与洛杉矶联合学区的一所中学合作，为一门新的环境科学课程开发课程，并在Go Green园艺俱乐部开展实践活动，描述细菌在基本元素的环境循环中的作用，并评估有毒化学品的影响。最后，PI将担任加州大学洛杉矶分校女工程师协会（SWE）分会的指导教师，并与受资助的博士生一起参加会议和推广活动。

1134355MahendraThis NSF award by the Environmental Health and Safety of Nanotechnology program supports work by Professor Shaily Mahendra to examine the effects of copper nanoparticles (NPs) on the diversity and function of bacteria involved in nitrogen cycling in natural and engineered environmental systems.Background and Significance. We anticipate that in the next few decades, the inputs of engineered nanomaterial-containing products to waste streams will increase, and more plants will be required to carry out tertiary treatment of wastewater. Transformations of nitrogen in the environment are microbially driven; nitrification, denitrification, and nitrogen fixation are catalyzed by specific enzymes, which are sensitive to environmental disturbances and pollution. Additionally, nitrogen management is a significant challenge in agriculture and in wastewater treatment. The rapid growth in production and use of nanomaterials in commercial products has raised concerns about their potential adverse effects on the environment. Our current understanding of the mechanisms of uptake and toxicity towards microorganisms is limited. Furthermore, most toxicological studies evaluate ?manufactured materials? rather than the forms attained after undergoing environmental transformations. We will investigate the regulation of relevant enzymes in pure cultures as well as consortia, thus, addressing issues important to NSF missions of generating a predictive methodology of understanding and mitigating the potential harmful effects of the interaction of NPs with the environment. Intellectual Merits. This study will test the hypothesis that copper NPs will selectively affect the population, diversity, and activity of bacterial communities involved in nitrogen cycling based on specific nature of NP-cell interactions (ionic copper uptake versus NP uptake; external versus internal dissolution followed by reactive oxygen species (ROS) generation and membrane/DNA/ATP damage; etc.). While there are a few recent reports of short-term toxic effects of NPs on laboratory pure cultures, the novel aspects of this proposal include evaluating NPs? effects on a class of indigenous microorganisms, and the environmental roles they play, over longer time scales. In addition, this will be the first study to explore high-throughput screening (HTS) for evaluating dose-response characteristics as well as mechanisms of NP toxicity. Specific objectives include: (a) to determine the microbial impacts of copper NPs via standard physiological and HTS assays, (b) to investigate the effect of copper NPs on functional gene expression in several pure cultures of nitrifying, denitrifying, anammox, and nitrogen-fixing bacteria in order to identify most susceptible microbial process in the N cycle, and (c) to quantify and model changes in population and diversity of N-cycling bacterial communities, including the effect of intrafloc transport resistance on availability of ionic and particulate copper, nutrients and electron acceptors. pure cultures and microcosms, effect of NPs on the expression of relevant genes will be determined using enzyme-specific assays as well as RT-qPCR. Ultimately, the methodology employed herein will serve as a template to address NP hazard identification and risk assessment, safe design and implementation of nanotechnology, as well as management of global carbon and nitrogen cycles.Broader Impacts. The broader impacts of the proposed research extend beyond a better understanding of NP interactions with environmentally relevant bacteria. In addition to publications and presentations at scientific meetings, the findings will be translated into a series of lectures to be presented in a new graduate course on Environmental Biotechnology and a redesigned undergraduate course on Environmental Nanotechnology (both taught by the PI). Further, the PI will collaborate with a middle school in Los Angeles Unified School District to develop curriculum for a new environmental science class as well as hands-on activities in the Go Green horticultural club to describe the role of bacteria in environmental cycling of essential elements and assessing the impacts of toxic chemicals. Finally, the PI will serve as the faculty adviser for UCLA chapter of the Society of Women Engineers (SWE), and participate, with the funded Ph.D. students, in meetings and outreach activities.