嗜热脂肪地芽孢杆菌DM-2产生物乳化剂的合成机制及其乳化原油机制研究

Crude oil was a non-renewable energy resource. The crude oil exploitations have been arriving at the high-water content stage after long term water flooding in most oil fields in China. It is essential to develop new technologies that were non-pollution and sustainable development for enhancing oil recovery, which have important significance to the stabilization of national resources and also put forward the new challenge to microbial enhanced oil recovery (MEOR). Although it has been proved validity in field trial, MEOR technology also had some limitations, such as low efficiency and unstable during enhancing oil recovery, because of lacking of guidance of enough theories. Recent research showed that crude oil emulsifying is the dominate way in MEOR, which indicated a new direction to this technology. In this item, metabolism mechanism of synthesizing a new type bioemulsifier by Bacillus Stearothermophilus was investigated through complete genome analysis; crude oil bioemulsified model was also constructed, in order to elucidate the mechanism at gene and interphase level respectively, and reveal the biological and non-biological factors that affect crude oil emulsifying process. All of these researches above could enrich mechanism of MEOR, lay the foundation of resolving the unstable efficiencies during MEOR, and provide scientific guidance to industrial application and significantly increase of flooding efficiencies of MEOR.

石油是一种不可再生能源，我国许多油田均已进入注水开发后期，如何研究和开发环境友好、可持续发展的采油技术，对国家资源的稳定具有及其重要的意义，也为微生物采油技术的研究和应用提出了现实要求。微生物采油的有效性已经在矿场得到证实，但是现阶段缺乏大量的基础理论研究，微生物提高采收率的幅度有限，技术水平不高。研究发现，原油乳化是微生物采油的主要方式之一，这为微生物采油技术提供了一条新的研究方向。本项目以嗜热脂肪地芽孢杆菌为主要研究对象，通过全基因组破译及分析，揭示大分子生物乳化剂产物的合成机制；同时构建原油生物乳化模型，从基因水平和分子结构水平阐明原油生物乳化的机制，揭示油藏条件下影响原油生物乳化的分子基础，为进一步充实微生物采油机理，解决微生物驱油效率不稳定的问题奠定基础，也为微生物采油技术早日实现工业化应用、大幅度提高驱油效率提供科学的指导。

微生物提高原油采收率（microbial enhanced oil recovery, MEOR），是利用微生物在油藏中生长繁殖及其代谢过程中产生的生物表面活性剂、有机酸及气体等来提高油藏中的残余原油采收率。其中生物乳化剂可以改变油藏的润湿性和乳化原油，因此可以显著提高原油的流动性。油藏是一个高温贫瘠的生态系统，缺乏合适的营养物质和足够的氧气，并不适合微生物生长代谢产生乳化剂，而能有效利用油藏中的有机酸作为碳源来生产生物乳化剂的兼性厌氧菌具有极高的研究和应用价值。本项目的研究对象Geobacillus stearothermophilus DM-2是一株嗜热、兼性厌氧、高产新型生物乳化剂的菌株，其基因组中含有一个3.5 Mb的染色体，包含了3773个蛋白编码基因，占总体比例的86.21%，27个rRNA合成操纵子和88个tRNA合成基因。构建了含有接合转移起始位点、耐热的卡那抗性基因及upp反向筛选标签的基因敲除载体pK18-UK，实现了G. stearothermophilus DM-2的基因无痕敲除的目的，进一步通过对代谢途径中功能基因的敲除以及转录组的分析，成功破译了菌株DM-2合成生物乳化剂的途径。通过对乳化剂的纯化和鉴定，确定其是一种新型的糖蛋白类大分子物质，可以牢固地包裹在油滴的周围，防止其聚并，达到增加乳状液稳定性的目的。该乳化剂具有较广泛的乳化活性，可以乳化多种碳氢化合物（正己烷、十六烷、二甲苯、柴油、橄榄油和原油），且乳化稳定性能较好，在较广范围的温度、pH、盐离子浓度，以及长时间放置条件下依旧可以保持其良好的乳化活性和乳状液稳定性。填砂模型物理模拟驱替试验证明菌株DM-2可提高原油采收率6.8％。