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（ecto酶）是水生环境中聚合物水解的重要因子，也是微生物碳、氮、磷营养状态的指示物。然而，与水生环境中大多数其他微生物代谢率测量一样，外酶活性测量通常仅限于用离散水样进行人工分析。最近开发了一种利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地表水中的外酶活性、碱性磷酸酶和亮氨酸氨基肽酶。有了这个仪器，这些酶的活性就可以像在航行中从研究船上绘制温度、盐度、浮游植物荧光和其他参数一样被绘制出来。然而，该系统仅限于从地面连续抽取水样的水平制图，并且需要频繁的操作人员干预。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. Klinkhammer博士），他们都对传感器开发感兴趣并具有专业知识，一名海岸物理海洋学家和建模师（R. Chant博士），以及一名非教师教育专家（E. Simms先生）。该项目的目标是开发一种远程操作的仪器，用于测量微生物酶的活性，并将其部署在罗格斯大学15米深的长期生态系统观测站，称为LEO-15，为期数周至数月。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。酶分析仪附近将部署一个带有生物、化学和物理传感器的简单系泊装置，以帮助提供微生物率测量的背景（以及来自LEO-15的额外数据）。酶和相关数据将通过时间序列方法、光谱分析技术、统计建模和其他技术进行分析，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观测站（伍兹霍尔海洋研究所）进行，以及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。该仪器由Dr. R. Olson及其同事在Biocomplexity IDEA的支持下开发。这种酶分析仪将提供持续的实时微生物代谢率测量的时间尺度，目前是不可用的。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重要的生物成分。此外，水生微生物学方面的教育工作将包括为小学和中学教师举办培训讲习班，然后是课程编制和实施，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