海洋未培养功能微生物驱动碳素循环的单细胞分析

There exist a massive amount of unknown microorganisms in ocean environment participating in the Earth’s element cycling. It remains unclear which strains or species of microorganisms play the key role in the cycling of Carbon, Nitrogen, and Sulfur in ocean. It is also unknown what are the metabolism pathways of these ocean organisms, and how they response to the environmental changes and what are their cellular and molecular responses. On the other hand, almost 99.9% of microbial cells collected from ocean, especially from deep-sea or ocean hot vents, cannot be cultured under the standard laboratory conditions, which substantially constrains the understanding of these cells. These uncultured microbes most likely entered a metabolic dormant state, but remained alive. Only when exposed to the favor environmental triggers, they may return to the growing state and resume the active metabolism. In this project, we propose to apply the single-cell laser tweezers and Raman spectroscopy (LTRS) technique for (1) analyzing and identifying the functional microorganisms that are involved in carbon-cycling from a variety of ocean environments. In particular, we will focus on two types of microbes: one can synthesize PHB biopolymer and the other can produce carotene molecules. These special microbes can be identified based on their characteristic single-cell Raman spectra and then isolated by optical tweezers manipulation; (2) investigating the dormancy and the activation of these uncultured microbial cells by high temperature (70-90 degree) or high pressure (30-300 MPa) in the simulant ocean environment. We will measure in real-time the changes in cellular and molecular components in individual cells in response to these activations, so that we can understand the mechanism on how the uncultured ocean organisms response to the changes in environmental factors; (3) studying the kinetic processes of production or consumption of PHB or carotene biomolecules during the growing of two important isolated ocean microorganisms. We will investigate their pathways of matter metabolism in order to understand their contributions to the carbon-cycle in specific ocean environments.

海洋环境中蕴含大量未知微生物参与地球元素循环，人们不清楚哪些微生物类(群)起关键的驱动作用，也不了解其物质代谢途径及其对环境变化的适应与响应机制。从海洋(特别是深海或热液口等极端环境)中采集的微生物绝大部分无法在实验室条件下培养。这些未培养微生物可能处于休眠状态，只有获得合适的环境因子刺激，它们才可能从休眠进入代谢或生长状态。本项目运用单细胞光镊拉曼光谱技术，分析不同海域样品中参与碳素循环的单细胞功能微生物 (特别是两类分别合成PHB聚合物和类胡萝卜素的微生物)，通过特征拉曼光谱识别与发现它们，并利用光镊操纵技术进行分选；模拟海洋环境，采用高温或高压等方法，研究海洋重要微生物的休眠与激活，实时观察细胞的生命状态和分子响应，从单细胞层次认识未培养微生物对环境变化的响应机制；分析两类分选的功能微生物单细胞合成和降解PHB及类胡萝卜素的过程，研究它们的物质代谢途径和在特定环境碳素循环中的贡献。

海洋环境中蕴含大量未知微生物参与地球元素循环,从海洋中采集的微生物绝大部分无法在实验室条件下培养。本项目采用单细胞光镊拉曼光谱技术，分析不同海域海水样品中参与碳素循环的未培养功能微生物 (特别是两类分别合成PHB聚合物和类胡萝卜素的微生物)，通过特征拉曼光谱识别与发现它们，并利用光镊操纵技术进行分选；模拟海洋环境，采用高温或高压等方法，研究海洋重要微生物的休眠与激活，从单细胞层次认识未培养微生物对环境变化的响应机制；分析两类功能微生物单细胞合成和降解PHB及类胡萝卜素的过程。通过本项目的研究，我们采集了2000-6000米深层海水样品,发现了大量产PHB和类胡萝卜素的菌体, 并筛选出两株富含大量类胡萝卜素的深海芽孢菌(#2966、#2430); 经过拉曼光谱与相差显微成像结合分析，证明在4500-6000米深海高压环境中海洋细菌不以休眠态的芽孢方式出现, 而是以萌发态或生长态方式参与碳素循环;经高温或高压刺激后，深海细菌培养率能得到较大提高，且海洋菌对高压刺激有记忆效应。我们还利用荧光显微成像技术实现对深海菌单细胞生长过程的实时观测,发现菌体内类胡萝卜素的分布与合成。