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

在过去的二十年里，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的作用的了解。这包括发现新的生物能力，并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--包括氧气最少区、深海温泉和其他化学成分相似的生境--的研究揭示了它们对海洋健康的关键影响(无论是好是坏)。然而，当从这样的环境中采样时，研究人员面临着可能由于生物变化或分析前现有样品收集和运输手段导致的化学降解而产生的偏差。对于在样本收集和保存之间可能迅速降解的遗传物质来说，这是一个特别的挑战。这里提出的技术开发正是针对这一挑战，次要目标是向更广泛的社区提供使用这项技术的机会。采样器的开发与各种水样平台兼容--价格非常有竞争力--使海洋科学大众化，为地方、州和联邦沿海资源管理者、非营利性组织、以教学为重点的机构以及任何设备预算不高的调查人员带来了采样工具。此外，这项研究将为高中生参与尖端研究和工程提供机会。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与其地球化学环境之间的关系。这项研究的数据将为哈佛大学剑桥林奇分校和多元化公立高中拉丁学校的研究人员现有的合作伙伴关系中的学生项目做出贡献。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？在海面控制实验中的原型。海洋在化学和生态上是异质的。例如，在热液喷口和碳氢化合物渗漏处发现的化学减少的环境是海洋的显著特征。这些地区拥有广阔的微生物群落，它们在从浮游生物健康到地球温度的过程中发挥着关键作用。海洋的氧气最低限度区域(OMZ)也是如此。人们越来越意识到，上层海洋和其他氧气充足的栖息地以及海洋中耗尽或缺乏氧气的区域之间存在着生物地球化学联系。为了加深对这些关系的理解，研究人员需要继续研究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)相应的地球化学成分。要做到这一点，对微生物基因表达产物(如RNA、蛋白质)和代谢物的分析是必不可少的。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在采样过程中由于基因表达的变化或化学降解而产生的偏差。这对于信使RNA(信使RNA)这样的分子来说尤其具有挑战性，因为它们可以在样本采集和保存之间迅速降解。这项研究旨在设计、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玫瑰花环“即插即用”。一般说来，我们提出了一种原位取样器，它可以代替单个尼斯金瓶安装在CTD玫瑰花环上。最重要的是，它将通过绳索驱动，就像标准的尼斯金瓶一样，从而消除了对电气接口的需要。一旦被部署到适当的深度，并释放绳索，该系统将开始其预先编程的程序，收集水样并进行适当的防腐步骤。该设备还将能够延迟采样事件，这将允许用户将CTD玫瑰花环重新定位到另一个深度，并收集另一个样本。部署和操作测试将沿着深度梯度在东热带北太平洋主要缺氧OMZ的一个现成地点进行。采样器将安装在标准的玫瑰花环上，并在三个深度进行成功点火、注入防腐剂和样品混合的测试，三个深度跨越缺氧亚硝酸盐最大深度(~100-130米)、缺氧OMZ核心(~300-700米)和亚OMZ(~1500米)。众所周知，这三个区域支持着生态和分类上截然不同的微生物群落。除了系统验证外，对这一梯度上的微生物和化学变化的有效分析将提供重要的信息，微生物如何对溶解氧的变化做出反应。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，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.