主持/主持完成国家自然科学基金项目6项,发表SCI论文60余篇,合作出版专著2部。研究方向:矿区微生物群落及其介导的元素地球化学循环;洞穴及湿地系统微生物组的时空分布及其对气候变化的响应;微生物与矿物的相互作用及其对储层物性的影响
Hongmei Wang, Fax: +86-27-67883452; E-mail:
本文从胜利油田沾3区块的高温油藏的原油采出液中分离得到一株嗜热菌,通过其与膨润土的相互作用,尝试探讨油藏微生物作为油藏储层中水敏性矿物(如蒙皂石)改性剂的可能性。
研究结果将在降低水敏矿物的膨胀性能,为解决水驱采油中遇到的水敏效应的瓶颈问题提供微生物的新途径。
所得菌株为革兰氏阳性菌,呈杆状,具芽孢,兼性厌氧,鉴定为
To decrease the swelling of water-sensitive minerals via microorganisms in oil reservoir, one thermophilic bacterial strain was isolated from a high-temperature reservoir in the Zhan 3 block of the Shengli Oilfield. Further experiments were conducted to evaluate the interaction between this isolate and bentonite with smectite as the main mineral.
The isolated strain was thermophilic, facultative anaerobic, Gram-positive, and rod-shaped. It could form spores and was identified as
These results confirm that pristine bacteria in oil reservoir can play an important role in mineral transformation, subsequently decreasing the swelling properties of clay minerals. Therefore, our results offer a promising way to deal with the water-sensitive issues in oil recovery process.
水驱采油是我国石油开采过程中的重要手段之一[
自20世纪30年代以来,储层防膨研究已经得到重视,采用的主要措施是向油藏中添加正电荷化合物,常见储层防膨剂可分为无机盐类、阳离子表面活性剂和有机阳离子聚合物等三类[
事实上,微生物与黏土矿物相互作用的研究已经是地质微生物领域的热点研究方向之一。前人的工作表明,一些厌氧菌可以将水敏性黏土矿物蒙皂石中的结构Fe(Ⅲ)还原为Fe(Ⅱ),从而导致蒙皂石溶解破坏,并且在一定水化学条件下可催化形成一些非膨胀性或弱膨胀性次生矿物(如伊利石、长石、滑石和非晶SiO2等)[
针对以上问题,本文以我国胜利油田沾3区块的高温油藏为研究对象,尝试分离厌氧铁还原菌,评估菌株对膨润土(以蒙皂石为主)的缩膨效能。这一研究对高含水、高温油田的开采具有重要的理论和实际指导意义。
胜利油田位于山东东营市黄河三角洲,是我国重要的石油工业基地和第二大高产油田。自1961年发现并勘探以来,已在胜利油田发现81个油气田,累积生产原油12亿t,为推动我国经济发展提供了有力支撑[
用于嗜热菌分离的油井采出液于2016年12月采集于胜利油田沾3区块,油藏温度为60 ℃,属于高温油藏。用无菌桶收集采出液20 L,用于后续嗜热菌的室内分离。待样品运抵实验室后,将采出液静置分离成油层与水层。其中,将油层样品小心转移至500 mL离心瓶中,再以异辛烷为萃取剂纯化油样。最后,对混合液进行离心处理(5000 r/min,6 min,Allegra X-30R,美国Beckman公司),重复数次并收集沉淀,用于后续油相微生物的分离和培养。
配制液体培养基用于分离采出液油相样品中的嗜热菌,其成分为(每升含):NaNO3 (2 g),K2HPO4 (0.5 g),KH2PO4 (1 g),MgSO4 (0.05 g),CaCl2 (0.01 g),NaCl (10 g),FeSO4·7H2O (0.01 g),酵母提取物(2.5 g),C36正构烷烃(0.01 g),乳化剂吐温80 (15 mL)。调节培养液pH至7.0。待高温灭菌并冷却后,按5% (
好氧生长:待高温灭菌的培养基冷却后,按照5% (
厌氧生长:将配置好的培养基分装至150 mL血清瓶中,通入高纯氮除氧并密封。高温灭菌冷却后,将血清瓶转移至厌氧手套箱内(COY Lab,美国)。将有氧条件下生长至对数中后期的菌液转移至厌氧手套箱(含98% N2及2% H2),离心(5000 r/min,6 min)收集菌体,用0.8%生理盐水洗涤3次转接到血清瓶中,设置无菌对照组和含菌实验组各3组平行,于60 ℃静止培养11 d,间隔48 h取样,用分光光度计(UV-8000A,上海元析仪器有限公司)于600 nm波段读取吸光度值,绘制嗜热菌的厌氧生长曲线。
利用扫描电镜(SEM)和透射电镜(TEM)观察菌体形貌。其中,SEM样品经固定、脱水、临界点干燥、镀金等步骤处理再放置于SEM(TESCAN VEGA3 LMU/XMU)下观察。SEM的工作条件为:加速电压为10 kV,激发电子束为2 mA。TEM样品经固定、脱水、包埋和切片处理,再用TEM (Hitachi H-700FA,加速电压100 kV)观察切片形貌。
采用FastDNA®试剂盒(QIAGEN)提取纯菌株总DNA,并利用细菌通用引物(27F/1492R)[
为了验证获得的纯菌株是否具备在厌氧条件下还原黏土矿物结构铁的能力,将该菌株接种至预先灭菌和除氧处理的矿物悬浊液中。该矿物悬浊液的组成如下(每升含):NaNO3 (2 g),K2HPO4 (0.5 g),KH2PO4 (1 g),MgSO4 (0.05 g),CaCl2 (0.01 g),NaCl (10 g),酵母提取物(2.5 g)以及膨润土(5 g),其中膨润土样品由中石化胜利油田分公司采油工艺研究院提供,该样品来源于胜利油田沾3区块储层。
矿物悬浊液的处理过程如下所述:小心研磨膨润土,按照5 g/L浓度称取样品至上述培养液中,磁力搅拌1 d以上,确保矿物均匀悬浮在培养液中,并调节pH=7。将上述矿物悬浊液分装至150 mL厌氧瓶中,通入高纯氮除氧并密封。为了避免高温灭菌对矿物结构的破坏,本实验采用巴斯德消毒法对矿物悬浊液灭菌处理,即在70 ℃水浴锅进行低温灭菌(重复3 d,每天处理0.5 h)[
用于检测微生物在添加了膨润土的实验体系中的生长情况。首先建立蛋白质浓度标准曲线,具体步骤如下:准备不同浓度梯度的牛血清溶液(用纯水配制),且各浓度条件下设置2个平行样,利用分光光度计在波长为595 nm检测其吸光度值,获得的标准曲线中吸光度值与已知的蛋白浓度呈线性关系。本实验建立的标准方程为
样品蛋白质浓度的检测:取0.1 mL 0.2 mol/L NaOH溶液加入0.9 mL菌-矿悬浊液中,100 ℃下加热10 min裂解细胞。待充分冷却后,离心悬浊液,取0.8 mL上清组分与0.2 mL Bradford反应液(Sigma公司)混合,利用分光光度计于595 nm波段读取吸光度值,依据标准曲线计算样品中蛋白质的浓度。
首先建立Fe2+标准曲线,即配制不同浓度梯度的柠檬酸亚铁溶液,且各浓度条件下设置2个平行样,利用分光光度计在510 nm的条件下读取器吸光度值,获得的标准曲线中吸光度值与已知的Fe2+浓度呈线性关系。本实验建立的标准方程为
样品Fe2+浓度的测定:在厌氧手套箱中,用1 mL一次性无菌注射器移取0.2 mL菌-矿悬浊液,注入0.48 mL 1.8 mol/L H2SO4溶液中,然后加入0.04 mL 48% HF溶液及0.08 mL邻菲罗啉显色剂(1, 10-phenanthroline),混匀后在100 ℃恒温条件下加热30 min。冷却后,向待测样品加入0.4 mL H3BO3溶液中和多余HF。摇匀样品,移取0.1 mL加入1 mL 1%柠檬酸钠溶液中,待显色充分后用紫外分光光度计检测Fe2+浓度,检测波长为510 nm。
实验结束后,取矿物悬液,进行SEM前处理。采用不同浓度梯度的乙醇对微生物-矿物复合体进行脱水,再用临界点干燥仪(Quorum K850,英国)去除乙醇。干燥完毕后的样品经上桩粘接和喷金处理,用于矿物及微生物细胞的形貌扫描电镜观察(SEM,TESCAN VEGA3 LMU/XMU)。SEM工作条件为:加速电压10 kV,激发电子束为2 mA。将微生物作用前后的膨润土悬液用乙二醇蒸汽饱和法进行前处理[
精确称取0.5 g微生物作用前后的膨润土干重粉末分别装入10 mL量筒内,加入4.5 mL的蒸馏水,并设置3个平行样,待96 h后观察样品在量筒中的固液界面变化。读取各量筒界面处刻度,即各样品的自由膨胀体积。将反应后产物膨胀终点时的固液界面刻度记为V1,原始膨润土在水中的自由膨胀时固液界面高度记为V0,利用公式(1)计算反应后产物的缩膨率[
生长曲线结果显示,SL-1在厌氧与好氧条件下均能生长,为兼性厌氧菌,且好氧和厌氧条件下SL-1生长曲线趋势相同,均在前3 d显著上升,第3天达到最高值,之后则缓慢下降(
好氧和厌氧条件下SL-1的生长曲线
Growth curves of SL-1 under aerobic and anaerobic conditions
基于16S rRNA系统发育树可知,本实验分离的菌株隶属于
rRNA构建的单菌株SL-1邻接法系统发育树
Neighbor-Jointing phylogenetic tree of SL-1 based on 16S rRNA
扫描电镜(A)和透射电镜(B)下菌株SL-1的形态
SEM (A) and TEM (B) images of SL-1
实验所用膨润土的主要矿物成分为蒙皂石,含量高达70.4% (
蛋白质浓度(A)和Fe2+浓度(B)随培养时间的变化
Variation of protein concentration (A) and Fe2+ concentration (B) with time
实验前后对照组和实验组矿物相及其百分含量的变化(n.d., 未检出)
Changes in mineral phases and their percentages before and after experiments (n.d.: not detected)
Sample | Cells | Smectite/% | Illite/% | Kaolinite/% | Mixed-layer illite-smectite/% |
Pristine bentonite | 70.4 | 19.8 | 9.7 | n.d | |
After treatments | without cells | 70.4 | 19.8 | 9.7 | n.d |
with cells | 47.7 | 29.1 | 17 | 6.2 |
SEM结果显示,对照组中蒙皂石样品为不规则薄片状,断口处花瓣卷曲磷片状、棉絮状,轮廓不清楚(
对照组扫描电镜形貌图(A)及能谱图(B)和实验组扫描电镜形貌图(C)及能谱图(D)
SEM images (A, C) and energy spectrums (B, D) of samples after experiments without microbes (A, B) and with microbe (C, D)
对照组和实验组样品中主要元素的EDS分析
EDS analysis of major elements in abiotic and biotic samples
Sample | Al/Si | Fe/% | K/% |
Without cells | 0.5 | 1.3 | 0.5 |
With cells | 0.4 | 0.6 | 2.2 |
实验中所用的膨润土的主要成分有蒙皂石、伊利石和高岭石(
实验前和实验后样品XRD图谱
XRD spectrums of samples before and after experiments
实验结束后,对照组0.5 g固体样品在4.5 mL去离子水溶液中静置96 h后,液面上升至8.64 mL;而同等条件下实验组0.5 g固体样品对应的液面高度则为6.40 mL (
对照组和实验组矿物膨胀性能
Mineral swelling properties of samples in biotic and abiotic groups after experiments
Sample | Triplicate 1 | Triplicate 2 | Triplicate 3 | Average | Swelling inhibition/% |
Without cells (V1) | 6.34 mL | 6.40 mL | 6.46 mL | 6.40 mL | 25.9 |
With cells (V0) | 8.72 mL | 8.60 mL | 8.60 mL | 8.64 mL |
微生物对矿物结构铁的还原作用早在1998年就被发现,目前为止,已有多种微生物被证实可以通过胞外呼吸或发酵等方式进行铁还原,这些微生物包括典型异化铁还原菌(如
研究发现,伴随着铁还原菌对含铁矿物晶格Fe3+的还原,可溶性Fe2+离子浓度在前期快速升高。随后,Fe2+离子浓度进入平稳阶段,之后逐渐降低[
由于蒙皂石与伊利石矿物结构的差异,导致它们的膨胀性能不同,蒙皂石遇水膨胀,而伊利石遇水不膨胀。因此微生物作用导致的蒙皂石向伊利石转化,理论上能够降低样品的膨胀性,有利于解决油田储层的水敏性问题。本研究膨胀性能检测结果也证实了这一点,相对于原始样品,微生物作用后矿物缩膨率达到25.9%。与从中温油藏分离得到的四种不同微生物的作用相比,本研究中微生物作用后矿物缩膨率较低,前者作用后缩膨率可达48.5%[
对微生物异化铁还原机制的认识目前主要包括直接接触机制、螯合促溶机制、电子穿梭机制和纳米导线辅助机制[
本研究以酵母提取物作为电子供体,蒙皂石结构中的Fe(Ⅲ)为电子受体,SL-1不具有菌毛、性毛等结构,且体系中并不存在NTA、EDTA和多磷酸盐等螯合剂,加上在缺氧条件下该属菌株主要依靠发酵作用获得能量维持细胞生长等信息[
蒙皂石向伊利石转化机理示意图
Conversion mechanism of smectite to illite
从山东省胜利油田沾3区块高温油藏采出液油相样本中分离得到一株嗜热的异化铁还原菌
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