Stable Isotope Probing with Resonance Raman Cell Sorting to profile influence of ocean acidification on microbial carbon fixation

当我们想到光合作用时，我们通常会想到树木和绿色植物将二氧化碳转化为氧气。然而，还有一系列其他生物也同样有效地执行这一功能，每一种生物都适应了自己的环境。超过70%的地球表面被海洋覆盖，其中光合微生物是主要的初级生产者，并在二氧化碳固定中发挥关键作用。确定环境变化对这些处于食物链基础的生物的影响的技术将是环境科学工具包中的一项广泛资产。然而，识别环境因素影响的一个主要障碍是，自然环境中的绝大多数微生物无法培养。自然界中光合微生物的巨大多样性仍然是未知的。为了克服培养海洋微生物的需要，我们将联合收割机结合新兴的单细胞分选和宏基因组技术，开发一种使能技术，通过将单个微生物的生理活动与群落多样性和功能联系起来，研究环境因素对微生物群落的影响。作为概念验证，在该项目中，我们将重点关注海洋酸化（OA）对海洋光合微生物的影响，因为人们担心OA的全球影响。迄今为止的研究尚未报告OA对不同分类群有不同反应的光合微生物的一致影响。由于几乎所有的光合微生物都含有类胡萝卜素，类胡萝卜素可以作为光合微生物的内部生物标志物。通过向微生物提供稳定同位素标记的13 CO2作为碳源，使用拉曼光谱，我们可以识别活跃固定CO2的单个细胞。在这里，我们将结合联合收割机这种技术与拉曼激活单细胞分选筛选和分离社区成员的基础上，对单个细胞的CO2固定活性的定量测量。对这些功能性分选细胞的宏基因组分析将进一步确定每类细胞中的物种。OA对光合微生物的CO2固定活性有什么影响？2.哪些微生物及其CO2固定活性受OA影响？3.问题1和问题2会给社区带来什么样的变化？4.哪些酶与光合微生物的最佳CO2固定活性有关？虽然这项研究将具体回答有关有机农业影响的问题，这些问题是粮食安全和水产养殖等重大问题的基础，但这种方法可以作为一种通用技术，广泛应用于环境科学，以确定任何环境变化因素的影响。

When we think of photosynthesis we normally think of trees and green plants converting carbon dioxide into life giving oxygen. However, there is a panoply of other organisms that also perform this function equally efficiently, each having adapted to their own individual environment. More than 70% of Earth's surface is covered by ocean where photosynthetic micro-organisms are the major primary producers and play a critical role in CO2-fixation. A technique to determine the influence of environmental change on these organisms at the base of the food chain will be a wide-ranging asset in the environmental science toolkit. However, a major hurdle in identifying the influence of environmental factors is that the vast majority of microbes in natural environments cannot be cultivated. The enormous diversity of photosynthetic microbes in nature remains unknown. In a timely approach that overcomes the need to cultivate the marine micro-organisms, we will combine emerging single-cell sorting and metagenomic techniques to develop an enabling technology to investigate the influence of environmental factors on microbial communities by linking physiological activities of individual micro-organisms with community diversity and function. As a proof of concept, within this project, we will focus on the effect of ocean acidification (OA) on marine photosynthetic micro-organisms because of concerns over the global impact of OA. Studies thus far do not report a consistent OA effect on photosynthetic micro-organisms with different responses from different taxonomic groups. Since nearly all photosynthetic micro-organisms contain carotenoids, carotenoids could be used to as an internal biomarker to identify photosynthetic micro-organisms. By providing micro-organisms with stable isotope labelled 13CO2 as the carbon source, using Raman spectroscopy, we can identify individual cells that are actively fixing CO2. Here we will combine this technique with Raman activated single cell sorting to screen and isolate community members based on a quantitative measure of CO2 fixation activity of individual cells. Metagenomic analyses of these functionally sorted cells will further identify the species in each category.By developing this technology, we will address the following specific questions:1. To what extend will OA affect the CO2 fixation activities of photosynthetic microorganisms? 2. Which microorganisms and their CO2 fixation activities are affected by OA? 3. What changes to the community will come about as a consequence of question 1&2?4. Which enzymes are implicated in optimal CO2 fixation activities of photosynthetic microorganisms?While this research will specifically answers questions on the influence of OA that underpin major issues such as food security and aquaculture, the approach can serve as a generic technique to be applied widely in environmental sciences to determine the influence of any agent of environmental change.