基于环境模拟策略的未培养微生物的可培养方法与微系统构筑

The need to close the gap between microbial diversity and large number of uncultivated microorganisms is an urgent practical issue, as environmental and pharmaceutical industries appear to have exhausted what the limited numbers of cultivable species have to offer. Towards micro-scale exploring in environmental science and environment simulation techniques, we here introduce a new concept of “artificial microenvironment” and a useful microfluidic platform. To simulating natural environment in micro scale from a new point of view, a series of microfluidic devices, including anti-adhesion material and structures, micro mixer, concentration / temperature / pH gradient regulator, inertial separator, capillary electrophoretic chip and microsensors will be fabricated. Furthermore, the micro devices will be highly integrated into a system, and then it will be applied into the research on environmental pollution control microbiology to give solution to the bottle necks of microbial cultivation, for example, different condition from nature, quantitative control of the concentration/temperature/pH of nutrient solution, and in-situ detection and analysis.. Our method will make a breakthrough in the separation and concentration of microorganisms with high fidelity, high throughput, and fast analysis. Moreover, we aim to analyze the effects of the condition changing in the microenvironment, also including the anti-adhesion treatment to the microbe growth, and optimize the artificial microenvironment system. We also aim to evolve the development of our own intellectual properties. The simple operation steps make the method easy to use, the samples can be pipetted in and play cultivation, and the cultivated samples are real-time detected, and in-situ analyzed automatically, then finally ready for downstream processing. . The concept and the system can be adapted to the research on physiological features of microorganisms, and also to a wide range of applications, such as in the fields of bioanalysis, ecosystem and related applications.

随着医药、环境、食品以及工农业等领域对微生物资源需求的日益增大，未培养（不可培养或难培养）微生物种群的可培养性研究已经不仅是微生物学需要研究的首要问题。针对目前尚缺乏对未培养微生物生长环境及其规律性的了解，以及实验室培养面临的自然环境条件还原失真问题，本课题拟构筑一个基于微流控技术的自然条件模拟微环境平台。首先通过明晰微生物界面黏附机理，探索微环境静态与动态下抗黏附新方法，解决由于黏附导致的微通道堵塞以及传热传质性能下降问题；其次建立复杂微生物种群的分离筛选原则与方法，揭示微生物尺寸、带电电荷以及趋化性随生长繁殖的变化规律，从而解决培养前的筛选问题；最后通过微环境的多参数模拟与精确调控，实现培养条件的快速优化。本课题拟完成3株以上未培养菌株的培养条件的快速优化，并实现该系统在微生物连续培养中的应用，为未培养微生物生理生态习性研究提供参考依据并奠定相关基础。

针对医疗、制药以及环境污水处理面临的微生物资源短缺需求，项目通过开展微流控芯片内细菌黏附抗黏附机理研究、细菌筛选检测与培养条件优化研究，建立了一系列微流控器件、技术方法与集成微系统，为解决未培养与难培养微生物面临的微环境调控与细菌生长特性原位检测等关键问题提供了重要工具和研究手段。. 项目研发了用于细菌连续流培养研究的内置微柱阵列的膛线型微混合结构器件、浓度梯度形成器件、基于叶脉仿生的且具有城垛结构的百纳升级培养芯片、具有凸缘结构亚微升级低扰动连续流培养腔体阵列等器件与微流控芯片，以及具有高集成的非接触电导检测器件与高感度自发光检测螺旋芯片。. 建立了基于不同尺度微培养腔体的连续流培养与原位检测一体化系统，以及连续流培养方法及细菌检测计数方法，包括相应于亚微升级培养腔体的低光程高精度OD检测系统、对应百纳升级培养腔体的光学检测与图像处理方法，针对细菌筛选分离用的亚微升级液滴生成及液滴计数检测系统，针对微升甚至亚微升级的低样品量和低密度细菌样品的电容耦合非接触电导检测方法。. 构建了微生物生物膜初期形成与原位观察的连续流微反应器系统，揭示了微流体与抗生素胁迫下生物膜的生长特性。研发了一种高深宽比黑硅材料，发现了区别于现有一维纳米材料抗菌的细菌细胞膜损伤结果，从而提出了新的黑硅抗菌机制。. 在应用方面，基于所研发芯片器件与系统，针对医疗制药领域开展了抗生素的抗菌药物敏感性分析应用研究；与环境学科结合，开展了活性污泥中的细菌趋化性分离，以及活性污泥中混合细菌的生物膜形成研究；研发了一系列微流控芯片及原理样机，解决了作为急性毒性分析生物指示的发光细菌的稳定性差等问题；从发光细菌的细胞水平生物毒性分析方法出发，拓展到克氏原螯虾靶器官的肝胰腺、肠道组织水平、肠道菌群以及分子转录水平，开展了重金属类、天然毒素类以及抗炎药物类污染物的毒理学研究，有效将微流控芯片技术与毒理学结合起来。