Development of a simple, low-cost device for sample collection and on-site preservation using a common oceanographic deployment platform

在过去二十年中，先进的遗传分析技术加速了社会对海洋微生物及其在海洋化学中所起作用的了解。这包括发现新的生物能力和认识到它们在以前未知的化学过程中的作用。特别是，对低氧海洋环境的研究--包括氧气最低区、深海温泉和其他化学性质相似的栖息地--揭示了它们对海洋健康的关键影响（无论是好是坏）。然而，当从这样的环境中取样时，研究人员面临着可能由于分析前目前的样品收集和运输方式造成的生物变化或化学降解而产生的偏差。这对于在样品收集和保存之间可能迅速降解的遗传材料来说是一个特别的挑战。这里提出的技术开发正是针对这一挑战，第二个目标是为更广泛的社区提供获得这一技术的机会。一个采样器，是兼容的各种各样的水采样平台的发展-在一个非常有竞争力的价格点-民主化的海洋科学，使采样工具，以地方，州和联邦沿海资源管理人员，非营利组织，教学为重点的机构，和任何调查人员与适度的设备预算。此外，这项研究将提供机会，让高中学生从事尖端研究和工程。虽然学生经常在课堂上接触微生物学和化学，但他们很少有机会研究生物体及其地球化学环境之间的关系。这项研究的数据将有助于哈佛大学剑桥里奇和拉丁学校（一所多元化的公立高中）的研究人员之间现有的合作伙伴关系中的学生项目。最后，研究人员将与哈佛自然历史博物馆和CRLS合作，举办一场青少年“XPRIZE”风格的比赛。参与的学生将建造一个水采样器来收集和保存海洋中的微生物，他们的设备将与研究人员一起进行测试。海洋在化学和生态上是不均匀的。例如，在热液喷口和碳氢化合物渗漏处发现的化学还原环境是海洋的突出特征。这些地点拥有广泛的微生物群落，在从浮游生物健康到行星温度的过程中发挥着关键作用。这也适用于海洋的氧气最小区（OMZ）。人们越来越意识到上层海洋和其他含氧良好的栖息地以及氧气耗尽或缺乏氧气的海洋区域之间存在的生物地球化学联系。要进一步理解这些关系，研究人员需要继续调查A）这些化学还原环境中的微生物多样性、分布和代谢活动;以及B）相应的地球化学组成。为了实现这一点，分析微生物基因表达产物（例如，RNA，蛋白质）和代谢物是必不可少的。然而，在从这样的环境中采样生物分子时，研究人员面临的挑战是避免由于样品收集期间基因表达或化学降解的变化而可能产生的偏差。这对于信使RNA（mRNA）等分子尤其具有挑战性，因为它们可以在样品收集和保存之间快速降解。本研究旨在设计、开发和验证一种低成本的mRNA/蛋白质/地球化学保存系统，该系统将与大多数海洋研究船上发现的尼斯金轮虫“即插即用”。一般来说，我们提出了一个原位采样器，可以安装在代替一个单一的尼斯金瓶的CTD玫瑰花。最重要的是，它将通过挂绳驱动，就像一个标准的尼斯金瓶，从而消除了对电气接口的需要。一旦部署到适当的深度并且其系索被释放，该系统将开始其预先编程的例行程序，即收集水样并进行适当的防腐步骤。该设备还将能够进行延时采样事件，这将允许用户将CTD玫瑰花重新定位到另一个深度并收集另一个样本。部署和操作测试将在热带北太平洋东部主要缺氧OMZ的一个大陆架外地点沿沿着深度梯度进行。取样器将安装在标准玫瑰花上，并在缺氧亚硝酸盐最大值（~100-130 m）、缺氧OMZ岩心（~300-700 m）和亚OMZ（~1500 m）的三个深度进行成功点火、防腐剂注入和样品混合测试。已知这三个区域支持生态学和分类学上不同的微生物群落。除了系统验证外，对该梯度内微生物和化学变化的有效分析将提供微生物如何响应溶解氧变化的重要信息。此外，这将有助于准确划定OMZ和其他氧化还原分层栖息地的过程边界。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Over the past two decades, advanced genetic analysis techniques have accelerated society's understanding of marine microorganisms and the roles they play in ocean chemistry. This includes the discovery of new biological capacities and the recognition of their role in previously unknown chemical processes. Particularly, research on low-oxygen marine environments - including oxygen minimum zones, deep-sea hot springs, and other chemically-similar habitats - has revealed the critical influence they have on ocean health (for better and for worse). However, when sampling from such environments, researchers face biases that may arise due to biological changes or chemical degradation resulting from current means of sample collection and transport prior to analysis. This is a particular challenge for genetic materials that can degrade rapidly between sample collection and preservation. The technology development proposed here is aimed squarely at this challenge, with a secondary objective of providing the broader community with access to this technology. The development of a sampler that is compatible with a wide variety of water sampling platforms - at a very competitive price point - democratizes ocean science, bringing sampling tools to local, state, and federal coastal resource managers, non-profit organizations, teaching-focused institutions, and any investigator with a modest equipment budget. Further, this research will provide opportunities for engaging high school students in cutting edge research and engineering. While students are often exposed to microbiology and chemistry in the classroom, they are rarely afforded the opportunity to study the relationships among living organisms and their geochemical environment. The data from this research will contribute to student projects in an existing partnership between the researchers at Harvard University at the Cambridge Rindge and Latin School, a diverse public high school. Finally, in collaboration with the Harvard Museum of Natural History and CRLS, the researchers will conduct a Junior "XPRIZE" style competition. The participating students will build a water sampler to collect and preserve microbes at sea, and their devices will be tested alongside the researchers? prototype during the sea surface control experiments.The ocean is chemically and ecologically heterogeneous. For example, chemically reduced environments like those found at hydrothermal vents and hydrocarbon seeps are prominent features in the ocean. These locations host expansive microbial communities that play critical roles in processes from plankton health to planetary temperature. This is also true for the ocean's oxygen minimum zones (OMZs). There is a growing awareness of the biogeochemical connections that exist among the upper ocean and other well-oxygenated habitats, and those regions of the ocean that are depleted in or devoid of oxygen. Advancing the understanding of these relationships requires that researchers continue investigating A) the microbial diversity, distribution, and metabolic activity within these chemically-reducing environments; and B) the corresponding geochemical composition. To accomplish this, analysis of microbial gene expression products (e.g., RNA, proteins) and metabolites is essential. However, in sampling biomolecules from such environments, researchers are challenged to avoid biases that may arise due to changes in gene expression or chemical degradation during sample collection. This is especially challenging for molecules such as messenger RNA (mRNA) that can degrade rapidly between sample collection and preservation. This research aims to design, develop, and validate a low-cost, mRNA/protein/geochemical preservation system that will be "plug-n-play" with the Niskin rosettes that are found on most oceanographic research vessels. Broadly speaking, we propose an in situ sampler that can be mounted in lieu of a single Niskin bottle on a CTD rosette. Most importantly, it will be actuated via lanyard, like a standard Niskin bottle, thus eliminating the need for electrical interfacing. Once deployed to the appropriate depth and its lanyard is released, the system will begin its pre-programmed routine of collecting water samples and conducting the appropriate preservative steps. The device will also be capable of time-delayed sampling events, which will allow the user to re-position the CTD rosette to another depth and collect another sample. Deployment and operations testing will be conducted along a depth gradient at an off-shelf site in the major anoxic OMZ of the Eastern Tropical North Pacific. The sampler will be mounted onto a standard rosette and tested for successful firing, injection of preservative, and sample mixing at three depths spanning the anoxic nitrite maximum (~100-130 m), anoxic OMZ core (~300-700 m), and sub-OMZ (~1500 m). These three zones are known to support ecologically and taxonomically distinct microbial communities. In addition to system validation, effective analysis of microbial and chemical changes across this gradient will provide important information how microbes respond to changes in dissolved oxygen. Further, this will help to accurately delimit the boundaries of processes in OMZs and other redox-stratified habitats.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.