基于功能活性可视化追踪的芳烃降解微生物高通量筛选方法

Isolation and screening of microbial resources for aromatic hydrocarbon pollutants biodegradation is an important issue to environmental organic pollution control. The conventional methods for the isolation and screening of functional microbe are low efficiency and accuracy because they cannot rapidly and intuitively track the functional activity of microorganism. In the previous work, we labeled the target aromatic hydrocarbon pollutants with fluorescence probes, and realized the visual tracking of microbe during the substrate conversion process. This study intends to establish a set of high throughput screening method for efficiently, intuitively, and specifically tracking of microorganism with aromatic-degrading activity, in combination with the microfluidic droplets and flow sorting as well as our previous work. In this study, we will solve the non-specific tracking problems of microbe due to the light-emitting products diffusion by embedding a single cell and substrate probe using the microfluidic droplet technology. Then, in combination with flow sorting the micro-emulsion established by artificial consortia of bacteria with known function using Flow Cytometry, we optimize the screening parameters and determine the universality and sensitivity of our high-throughput screening tactics. Moreover, we analyze the microorganism from environmental samples using the conventional plate cultivation and amplicon sequencing technology, as well as our new high-throughput tactics in order to adjust and modify the operation conditions and obtain the new functional strains. This study provides new technique and thought for digging deeply the microorganism resources for aromatic hydrocarbon pollutants biodegradation.

芳烃降解功能微生物资源的分离筛选是环境有机污染治理的迫切需求。然而已有的微生物分离筛选方法因无法快速直观地追踪微生物的功能活性而存在效率低、准确性差等问题。前期的研究中我们将目标芳烃污染物进行荧光标记，实现了底物转化过程中微生物可视化追踪。本项目拟利用前期相关工作基础，结合微流控液滴和流式分选，建立一套高效直观、特异性追踪微生物芳烃降解功能活性的高通量筛选方法。项目采用微流控液滴技术包裹微生物单细胞和底物探针，防止发光产物的扩散，解决微生物非特异性追踪问题；结合人工复配菌群（已知功能）的微乳包埋和流式高通量分选，研究并明确功能微生物流式筛选的条件参数以及相关方法的灵敏性、普适性；进一步采用环境驯化菌群样品，结合扩增子测序和平板分离培养功能验证，调整和优化流式分选条件，获取芳烃降解新菌株；本项目的开展将为深入挖掘芳烃降解功能微生物资源提供新的技术手段和研究思路。

荧光激活细胞筛选技术因为稳定性差，背景干扰等原因难以实现对微生物细胞进行筛选。本项目以偶氮染料为目标化合物，以偶氮染料脱色菌株为目标菌株，将纳米二氧化硅荧光探针技术应用于功能微生物选育中，荧光探针初始是无荧光活性的，当偶氮染料降解后发出荧光。利用纳米颗粒在细菌细胞内偶氮染料被降解的显色过程结合流式细胞仪进行特异筛选功能活性细菌。研究结果表明荧光探针具有高灵敏度和较宽的检测范围，可以检测多种偶氮染料降解菌。通过激光共聚焦显微镜检测功能菌Shewanella decolorationis S12的检测范围为8.0 cfu/mL - 8.7 × 108 cfu/mL。利用荧光计数器检测功能菌检测范围为1.1 × 107 - 9.36 ×108 cfu/ mL。此外，其他功能菌Bosea thiooxidans DSM 9653 和 Lysinibacillus pakistanensis NCCP-54 也可以被检测到。人工构建复配菌（S12、C1、C2、大肠杆菌），其中只有S12对探针有脱色显荧光功能，其他的菌均不会对其进行还原显荧光，经16S rDNA测序所得基因组数据确认，分选所得的菌只有复配菌液中的S12，从而实现功能菌的筛选。基于高通量测序技术对芳烃污染物富集的微生物菌群进行遗传背景分析，为实现可视化筛选功能微生物提供参考。该研究的开展将建立一套全新的基于荧光标记技术的微生物选育研究模式，更高效直观地特异性对单个细胞进行快速、高灵敏度分析分选，能为功能微生物的高效选育提供新的工具，也能为功能微生物单细胞研究和深入挖掘未培养微生物提供技术支撑，对于毒性芳烃污染物防治有重大的实践意义和应用潜力。