BE/IDEA: Development of a Instrument for in situ Measurement of Microbial Enzyme Activities in Aquatic Ecosystems

微生物细胞表面酶(胞外酶)是水环境中聚合物水解的重要介质，也是微生物碳、氮或磷营养状况的指示剂。然而，与水环境中大多数其他微生物代谢率的测量一样，胞外酶活性的测量通常仅限于对离散的水样进行手动分析。最近开发了一种使用高灵敏度荧光底物连续测量微生物酶活性的仪器。该仪器已在船上用于绘制密西西比河羽流表层水中胞外酶活性、碱性磷酸酶和亮氨酸氨基肽酶的图谱。有了这台仪器，这些酶的活性就可以像在航中的考察船绘制温度、盐度、浮游植物荧光和其他参数一样绘制出来。然而，该系统仅限于从地面连续抽出的水样的水平测绘，需要频繁的操作员干预。这个用于环境活动的生物复杂性仪器开发(IDEA)项目将迈出更大的一步。研究和教育团队包括一名微生物生态学家(J.Ammerman博士)和一名地球化学家(G.Klink Hammer博士)，他们都对传感器开发有兴趣和专业知识，一名沿海物理海洋学家和模型师(R.Chant博士)，以及一名非教职教育专家(E.Simms先生)。该项目的目标是开发一种用于测量微生物酶活性的远程操作仪器，并将其部署在罗格斯大学长期生态系统天文台15米深处，称为LEO-15，持续数周至数月。这种仪器被称为多酶分析仪(MEA)，应该能够同时测量多达四种不同的酶活性。一个带有生物、化学和物理传感器的简单系泊装置将部署在酶分析仪附近，以帮助提供微生物速率测量的背景(以及来自LEO-15的其他数据)。将使用时间序列方法、光谱分析技术、统计建模和其他技术对酶和相关数据进行分析，以深入了解沿海地区的微生物过程。MEA的第二次部署将在新的玛莎葡萄园岛海岸天文台(伍兹霍尔海洋研究所)进行，同时还将配备用于浮游生物细胞计数和分类的自动流式细胞仪和图像分析仪。这个仪器是由R.Olson博士和他的同事在BioComplex Idea的支持下开发的。这种酶分析仪将在目前无法获得的时间尺度上提供连续的实时微生物代谢率测量。这种测量将为海洋观测站可以进行的广泛的化学和物理测量增加一个重要的生物组成部分。此外，水生微生物学方面的教育工作将包括为中小学教师举办培训讲习班，随后进行课程开发和实施，以及大学本科生和研究生的研究和教育。

Microbial cell-surface enzymes (ectoenzymes) are important agents of polymer hydrolysis in aquatic environments and indicators of the state of microbial carbon, nitrogen, or phosphorus nutrition. However, like most other microbial metabolic rate measurements in aquatic environments, ectoenzyme activity measurements have usually been limited to manual assays with discrete water samples. An instrument for continuous underway measurements of microbial enzyme activities using high-sensitivity fluorescent substrates has recently been developed. This instrument has been used aboard ship to map the ectoenzyme activities alkaline phosphatase and leucine aminopeptidase in the surface waters of the Mississippi River plume. With this instrument these enzyme activities can be mapped in much the same way that temperature, salinity, phytoplankton fluorescence, and other parameters are mapped from a research ship while underway. However, this system is limited to horizontal mapping of water samples continuously pumped from the surface and requires frequent operator intervention. This Biocomplexity Instrumentation Development for Environmental Activities (IDEA) project will take the next much larger step. The research and education team includes a microbial ecologist (Dr. J. Ammerman) and a geochemist (Dr. G. Klinkhammer), both of whom have interests and expertise in sensor development, a coastal physical oceanographer and modeler (Dr. R. Chant), and a non-faculty education specialist (Mr. E. Simms). The goal of this project is to develop a remotely operated instrument for measurement of microbial enzyme activities and to deploy it at the Rutgers University Long-term Ecosystem Observatory at 15 meters depth, called LEO-15, for periods of weeks to months. This instrument is called the Multiple Enzyme Analyzer (MEA), and should be capable of the simultaneous measurement of up to four different enzyme activities. A simple mooring with biological, chemical, and physical sensors will be deployed near the enzyme analyzer to help provide a context for the microbial rate measurements (along with additional data from LEO-15). The enzyme and related data will be analyzed with time-series methods, spectral analysis techniques, statistical modeling, and other techniques to gain insight into microbial processes in the coastal zone. A second deployment of the MEA will take place at the new Martha's Vineyard Coastal Observatory (Woods Hole Oceanographic Institution), along with an automated flow cytometer and image analyzer used for plankton cell counting and sorting. This instrument is under development by Dr. R. Olson and colleagues with Biocomplexity IDEA support. This enzyme analyzer will provide continuous real time microbial metabolic rate measurements on time scales that are currently unavailable. Such measurements will add an important biological component to the extensive chemical and physical measurements that can be made at ocean observatories. In addition, education efforts in aquatic microbiology will include training workshops for primary and secondary school teachers followed by lesson development and implementation, as well as university undergraduate and graduate student research and education