中高温油藏典型采油微生物Geobacillus spp.的驱油功能基因及调控机制

Microbial enhanced oil recovery technology (MEOR) is a kind of more respected EOR technology in the world. It mainly has a wide range of applications, environmental compatibility, good biodegradability, low-cost, simple operation and other advantages, which may become the most potential technology for oil recovery. The major obstacle that has prevented the implementation of MEOR has been the difficulty in finding, isolating or engineering microorganisms which can survive the harsh variety of environmental conditions present in oil reservoirs. For the microorganisms to be suitable and useful in MEOR in situ, they must be able to grow under the severe environmental conditions encountered in oil reservoirs, such as high temperature, pressure, salinity and low oxygen. How to stimulate the organisms growing and producing biosurfactants in these conditions is the major work for MEOR. In our proposal, the thermophilic hydrocarbon- degrading bacterium Geobacillus sp.SL-1, isolated from high temperature reservoir of Shengli Oil Field, will be used as our research object. Genome-wide transcriptome analyses of strain SL-1 with regard to bioemulsan metabolism and hydrocarbon- degrading will be employed to detect genes that are differentially transcribed during bioemulsan biosynthesis in different cultural conditions. Based on the genome-wide transcriptome analyses, we will find the biosynthesis pathway of bioemulsan in strain SL-1 and inspect its correlation with hydrocarbon- degrading function. Bioemulsan production by strain SL-1 will be assessed by metabolic flux distribution analysis. Bioemulsan production will be tested under various nutrition and oxygen supply conditions to obtain the optimal conditions. Based on the metabolic flux analysis, we will develop the strategy of stimulating thermophilic hydrocarbon- degrading bacterium Geobacillus sp. SL-1 in situ for MEOR in high temperature reservoir. We are sure it is the first time to find the biosynthesis pathway of bioemulsan in thermophilic hydrocarbon- degrading Geobacillus.

微生物提高原油采收率技术(MEOR)具有工艺简单、成本低和无污染等优点；但中高温油藏(55-80oC)中微生物的生长代谢受到限制，能完全发挥驱油功能的微生物较少，导致MEOR的技术应用存在局限；深入研究中高温油藏内的土著嗜热微生物及其驱油机制成为拓展MEOR技术的一个重要途径。具有嗜热解烃功能的地芽孢杆菌属菌株Geobacillus spp.是中高温油藏的典型驱油功能菌株。本项目以分离自胜利油田的高温驱油菌株Geobacillus sp.SL-1为研究对象，重点研究乳化剂的代谢途径和调控机制，探索乳化功能与噬烃功能的互作关系；通过基因组及转录组研究，确定SL-1菌株乳化剂合成及烃代谢的功能基因（簇），分析不同模拟激活条件下乳化剂合成及烃代谢基因簇的转录特征，解析乳化剂合成途径的关键步骤，考察激活剂及环境对乳化剂合成及烃代谢途径的激活响应，通过物理模拟驱油建立SL-1高效驱油的激活策略。

本项目对从胜利油田分离出的嗜热MEOR菌株 SL-1进行了基因组测序及功能分析，对该菌株的烃代谢及产生物乳化剂等MEOR功能特性进行了深入研究，解析了生物乳化剂主要功能组分，基于仿生原理构建了新型廉价的生物乳化剂。.1.采油功能菌株SL-1的全基因组测序和功能分析。SL-1菌株含有一条4156266 bp的染色体，G+C含量为39.52%，共预测到的4681个编码基因，占基因组比例为84.43%。菌株SL-1基因组中含有烷烃及芳烃等降解基因，同时包含2条可能的生物乳化剂多糖组分合成途径。.2.菌株SL-1的烃代谢特征。菌株SL-1的细胞表面具有良好的疏水性，能够在60oC条件下以原油为唯一碳源生长，并能够优先降解原油中的短链烷烃和芳烃组分。在以多环芳烃为唯一碳源时，在5天内降解80%以上的萘(1000 mg/L)或菲(200 mg/L)，50%以上的芘(50 mg/L)。.3.菌株SL-1合成生物乳化剂SL-bioemlsifier的表征及功能解析。 SL-bioemulsifier是糖蛋白乳化剂，主要成分为多糖（65.6%）与蛋白质（13.2%），其中蛋白质组分为乳化功能主要活性剂成分。针对SL-bioemulsifier主要蛋白质组分的质谱解析获得了乳化功能蛋白质SDR家族氧化还原酶（26kDa），该乳化剂具有广泛的底物谱，能有效乳化短链烷烃以及芳香烃类以及良好的乳化稳定性，可以在温度（70ºC）、pH（3-9）和高盐浓度(300 mM )下保持乳化活性。该乳化剂能够增加多环芳烃在水中的溶解度和生物利用度。.4.通过乳化功能蛋白质和多糖组分的适当组合来构建新型生物乳化剂。来源于枯草芽孢杆菌CICC 20034中的酯酶AXE蛋白具有高效乳化活性，AXE蛋白在pH 3-9，高温（<85ºC）以及高盐度（0-1000 mM）条件下乳化活性非常稳定，能够有效乳化短链脂肪族和芳香族底物。AXE蛋白可以增加多环芳烃在水中的溶解度，添加40 mg/L的AXE蛋白能够促进菌株SL-1对菲和原油的降解，菲的降解率从29%提高到38%，原油降解率从30.19%提高到56.03%。AXE蛋白六聚体结构活性中心的疏水空腔是其具有乳化活性的基础。通过将AXE蛋白与多种多糖复配，仿生构建开发新型生物乳化剂，进一步降低了AXE蛋白的使用浓度，为低成本环保型生物乳化剂的应用开辟了广阔的新领域。

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