MO: Collaborative Research: Transitions in the Surface Layer and the Role of Vertically Stratified Microbial Communities in the Carbon Cycle- An Oceanic Microbial Observatory

本提案的重点是浮游细菌微生物群落分层在海洋碳循环中的作用。复杂的生物、化学和物理过程控制着碳从光带海洋向深海转移的效率，而深海则有可能进行封存。大多数从透光带输出的有机碳从未离开地表500米，大约90%输出的有机碳在中层带（140 - 1000米）被再矿化。微生物群落在海洋中是垂直分层的，特别是在表层（0-300米），它跨越了深层混合事件的区域，并从光带过渡到中上层-碳再矿化活动最高的区域。这一建议的前提是分层的浮游细菌枝参与专门的生物地球化学活动，可以通过综合海洋学和微生物学方法来识别。具体来说，本提案的目的是评估中上层微生物群落是否依赖于成岩作用改变的有机物和亚细胞碎片，这些有机物和亚细胞碎片是由泛光区微生物过程产生的，并通过下沉或混合传递到中上层。在过去的努力中，这个微生物观察站在培养嗜光带微生物群落成员方面取得了更大的成功，并揭示了SAR11分支的α变形菌中对还原硫化合物的意外生长需求。基因组信息显示，对基质的激烈竞争迫使浮游细菌进行权衡——在N维营养空间中，有一些区域是专家获胜的。我们假设特定的生长需求可以解释在中上层浮游细菌群落中观察到的一些规则的空间和时间模式，以及培养其中一些生物的困难。该项目的具体目标是：1)从光合浮游生物培养物中产生13C和15N标记的亚细胞（如可溶性、细胞壁和膜）和DOM组分，并使用稳定同位素探测来识别表面和上层中上层微生物群落中吸收不同组成和不稳定性组分的特定分支。2)利用荧光原位杂交方法监测SIP和HTC实验中鉴定的特定微生物种群的时空变异性。为了提高分辨率，我们将使用CARD-FISH协议。3)测定浮游细菌群落的蛋白质组学，鉴定表层和中上层的高翻译基因，以及群落对季节性营养限制的响应。4)通过高通量培养（HTC）来培养这些生物，假设它们需要特定的营养因子和/或成岩改变的有机基质。将从关键生物体中寻找完整的基因组序列，并将其用作研究与生物地球化学重要功能相关的基因和种群自然变异模式的查询。这个项目将为科学界提供新的浮游细菌培养和基因组序列。这项研究的发现可以直接用于食物网和海洋碳循环模型。这项研究的教育部分通过暑期课程和直接参与研究的研究生和本科生的专门培训，为来自多个学科的学生提供微生物海洋学培训。

The focus of this proposal is the role of bacterioplankton microbial community stratification in the ocean carbon cycle. Complex biological, chemical and physical processes control the efficiency of carbon transfer from the euphotic zone ocean to the deep sea, where sequestration is a possibility. Most organic carbon exported from the euphotic zone never leaves the surface 500 m, with approximately ninety percent of the exported organic matter being remineralized in the mesopelagic zone (140 - 1000 m). Microbial communities are vertically stratified in the oceans, particularly in the surface layer (0-300 m), which spans the region of deep mixing events and transition from the euphotic zone to the upper mesopelagic - the region of highest carbon remineralization activity. The premise of this proposal is that stratified bacterioplankton clades engage in specialized biogeochemical activities that can be identified by integrated oceanographic and microbiological approaches. Specifically, the objective of this proposal is to assess if the mesopelagic microbial community rely on diagenetically altered organic matter and subcellular fragments that are produced by microbial processes in the euphotic zone and delivered into the upper mesopelagic by sinking or mixing. In past efforts this microbial observatory had greater success cultivating members of the euphotic zone microbial community, and revealed an unanticipated growth requirement for reduced sulfur compounds in alphaproteobacteria of the SAR11 clade. Genomic information showed that intense competition for substrates imposes trade-offs on bacterioplankton - there are regions of N dimensional nutrient space where specialists win. We postulate that specific growth requirements may explain some the regular spatial and temporal patterns that have been observed in upper mesopelagic bacterioplankton communities, and the difficulties of culturing some of these organisms.The specific objectives of this project are: 1) to produce 13C and 15N labeled subcellular (e.g., soluble, cell wall, and membrane) and DOM fractions from photosynthetic plankton cultures and use stable isotope probing to identify specific clades in the surface and upper mesopelagic microbial community that assimilate fractions of varying composition and lability. 2) to use fluorescence in situ hybridization approaches to monitor temporal and spatial variability of specific microbial populations identified from the SIP and HTC experiments. To increase resolution we will use CARD-FISH protocols. 3) to measure the proteomes of bacterioplankton communities to identify highly translated genes in the surface layer and upper mesopelagic, and community responses to seasonal nutrient limitation. 4) and, to cultivate these organisms via high throughput culturing (HTC) by pursuing the hypothesis that they require specific nutrient factors and/or diagenetically altered organic substrates. Complete genome sequences from key organisms will be sought and used as queries to study patterns of natural variation in genes and populations that have been associated with biogeochemically important functions.This project will make cultures of novel bacterioplankton and genome sequences available to the scientific community. Findings from this research may be used directly in foodweb and ocean carbon cycle models. The educational component of this research brings microbial oceanography training to students from many disciplines, through a summer course, and specialized training to graduate and undergraduate students involved directly in research.