一株地芽孢杆菌（Geobacillus sp.）在模拟油藏环境下的生长与运移实验研究
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摘要
微生物在油藏中有效地生长与运移是微生物提高原油采收率技术成功的关键。目前的微生物采油技术研究更多的集中在利用室内摇瓶培养实验研究微生物代谢产物对油、水理化性质的影响方面,而对于细菌在油藏环境中的生长、运移和代谢作用研究较少。
本论文以一株典型的好氧采油微生物G2菌为例,确定在适宜的营养和配气条件下,好氧微生物在模拟典型实验区块实际油藏条件下的生长状况,确定影响微生物在岩石毛细孔隙中运移的主要因素,并计算运移效率。主要研究结果与结论如下:
1.从胜利油田微生物研究中心菌种库中优选出一株地芽孢杆菌(Geobacillus sp. G2),通过室内评价实验确定了该菌株适宜作为典型的好氧采油微生物用于微生物驱油基础理论研究。
通过对菌种库的细菌进行筛选,从中挑选出一株G2菌,室内摇瓶实验结果表明,该菌具有耐高温、以不同类型原油为唯一碳源能够生长、降粘、产表面活性物质、并对原油和液蜡有较好的乳化和分散作用等利于提高原油采收率的功能。通过物理模拟驱油实验和盲管驱油实验考察了该菌株的驱油性能:物理模拟驱油实验表明,采用空气辅助驱(液气比1:10),在一次水驱的基础上,经过5周期50d培养驱替,该菌能够提高原油采收率12.9-15.9%;盲管驱油实验也表明该菌能够驱出盲管中的残余油。以上实验结果表明,优选的G2菌可以作为典型的好氧采油微生物用于微生物驱油基础理论研究。
2.采用单次单因子法确定了G2菌适宜的生长条件。采用单次单因子法,通过摇瓶培养实验确定了G2菌适宜的培养条件:蔗糖3%,NaCl 0.5%,NaNO3 0.2%,KH2PO4 0.14%,Na2HPO4·12H2O 0.37%,FeSO4·7H2O 0.001%,MgSO4·7H2O 0.02%,CaCl2 0.001%,酵母粉0.05%,pH 7.0,温度50-55℃。确定了该菌株生长的pH、温度和盐度范围:pH 5.5-9.5,温度35-70℃, NaCl浓度0-10%;同时确定了该菌株适宜生长的pH、温度和盐度的范围:pH 6.0-9.0,温度40-60℃,NaCl浓度0.5-8%。采用高压岩心管,利用高压空气加压,通过培养实验研究了高压对该菌株生长的影响,结果表明,该菌株能在10MPa高压下生长,但生长延滞期变长。以上实验结果表明,该菌株的pH、温度和盐度适应范围均较广且耐高压,说明该菌可能的油藏适应范围较广,进一步说明该菌株适宜用于好氧微生物驱油基础理论研究。
3.以G2菌为例,通过调整模拟油藏环境的营养和配气,研究其在模拟典型实验区块实际油藏环境下的生长状况。
1)通过对非生物因素耗氧实验研究和细菌生长耗氧量的估算,确定了好氧微生物在室内模拟胜利油田孤岛中一区Ng3区块油藏环境下生长实验的配气量(空气):在不饱和原油的情况下,配气量维持在常压下液气比1:10;在饱和原油的情况下,配气量维持在常压下液气比1:30就足以维持好氧微生物在模拟油藏环境下良好生长了。通过对蔗糖和NO3-等营养物在岩心中的运移实验,确定了在注入岩心3-4.5PV培养液时,岩心出口营养物的浓度基本达到注入浓度。
2)通过模拟目标油藏的温度、压力、孔隙度、渗透率、流体特征和开发程度等因素,进行了G2菌在模拟油藏环境下的生长实验,结果表明,在注入适宜的营养和配气条件下,G2菌能够在模拟Ng3区块油藏环境下良好生长,细菌密度由106个/mL增加至108个/mL,该菌株适宜作为该区块实施微生物采油技术的候选菌种。残余油的存在对G2菌的生长基本没有产生影响,但在模拟油藏环境下,该菌生长延滞期较摇瓶培养实验长,说明为特定油藏进行的各种微生物采油技术研究必须在模拟油藏环境下进行。
4.以G2菌为例,系统考察了驱替速度、实验温度、岩心渗透率、微生物菌体大小和聚集度以及油相存在与否等因素对微生物在岩石毛细孔隙中运移的影响,并探讨了微生物在岩心中运移过程中滞留的主要机制。
1)先用处于生长稳定期的菌液进行连续的岩心驱替,接着用无菌水驱替,在出口取样分析菌体密度,记录整个驱替过程中的压差变化。驱替实验结果表明:驱替速度、实验温度、岩心渗透率、微生物菌体大小和聚集度以及油相存在与否等因素对微生物在岩心中运移有重要影响。当驱替速度、实验温度和岩心渗透率升高时,微生物运移效率增加;当微生物菌体大小和聚集度增加时,微生物运移效率降低;脉冲驱替能够增加微生物在岩心中的运移效率;油相存在能增加细菌的滞留量,降低细菌的运移效率。微生物在岩心中运移过程中的滞留是筛分、架桥堵塞、界面吸附、粘附和聚集堵塞等作用机制共同作用的结果。当注入微生物浓度较高时,架桥和聚集堵塞是微生物在岩心中滞留的主要作用机制,微生物更易滞留在岩心入口段,形成外部或内部滤饼,并对该段岩心渗透率伤害较大。
2)微生物在模拟油藏环境下的生长和运移实验表明,即使在静止培养的条件下,扩散对微生物及其代谢产物的运移影响也很小,在连续驱替过程中,微生物运移的动力主要来自于水动力。
本论文的研究成果为建立空气辅助微生物驱油技术室内评价方法(尤其是物理模拟驱油体系方法)和数模软件提供基础。另外,本论文针对胜利油田孤岛中一区Ng3区块进行研究,研究成果为该区块微生物采油矿场试验设计提供一定的理论基础和技术支撑,对最终提高现场应用效果具有促进作用。
The effective transport and growth of bacteria in reservoir is crucial to the success of microbial enhanced oil recovery (MEOR) technology. In recent years considerable efforts have been put into the effect of microbial metabolic products on physical and chemical properties of oil and water using shake-flask culture experiments, while there’s little information about the growth, transport and metabolism of bacteria in reservoir environment.
The objectives of this study are to identify whether aerobic bacteria can grow in simulated reservoir conditions or not, and to determine the important factors that control the transport of bacteria through rock porous media. Furthermore, the mechanisms of bacteria retention and permeability reduction are investigated. The main results are summarized as follows:
1 One strain of bacterium, identified as Geobacillus sp., was screened and confirmed as a candidate aerobic microorganism for MEOR by laboratory study.
The bacterium Geobacillus sp., named G2, was screened from library of strains of Shengli Oilfield. The results of shake-flask experiments showed it exhibited good properties for MEOR: suffering high temperature, growing when different types of crude oil was added as the sole carbon source, reducing the viscosity of crude oil, producing biosurfactants, emulsifying and dispersing crude oil or liquid wax, and so on. Besides, Core flood experiments showed the oil recovery had been increased by 12.9-15.9% of original oil in place (OOIP) after 5 treatments of 50 days by adopting air-assistant technique (liquid/air 1:10 v:v). Blind tube-flood experiments also showed that the oil in the dead area could be effectively drived out by the strain G2. These results indicated that the strain G2 was suited for the theory study of MEOR.
2 The optimal medium components and suitable growth conditions for the strain G2 were investigated by using“one-variable-at-a-time”method.
The optimal culture conditions of this bacterium were studied by the shake-flask culture experiments. The results showed that the optimal culture conditions were as follows: 3% sucrose, 0.5% NaCl, 0.2% NaNO3, 0.14% KH2PO4, 0.37% Na2HPO4·12H2O, 0.001% FeSO4·7H2O, 0.02% MgSO4·7H2O, 0.001% CaCl2, 0.05% yeast extract, pH 7.0 and at 50-55℃. The ranges of the main factors that affected the growth of the bacterium were also investigated. The results were as follows: the temperature, salinity and pH range for growth of the strain G2 was 35-70℃, 0-10% NaCl and 5.5-9.5 respectively; suitable growth temperature, salinity and pH occurred at 40-60℃, 0.5-8% NaCl 6.0-9.0 respectively. Besides, the influence of high pressure on the growth of the strain G2 was studied by high-pressure culture experiments. The results showed that the strain G2 could grow at 10MPa, but the lag phase of growth became longer. These results further indicated that G2 was the suited experimental object for the theory study of MEOR.
3 The growth of the strain G2 was studied in the simulated reservoir conditions when the nutrients and air conditions were adjusted for the better growth of the bacterium.
1) Based on the experimental study on abiotic factor oxygen consumption and the estimation of biotic oxygen consumption, the amount of air injection for the growth of G2 in the simulated reservoir conditions of Block Ng3 of Shengli Oilfield was investigated. The results indicated that the amount of oxygen was sufficient for maintaining the growth of microorganisms in the simulated reservoir conditions when the ratio of Vwater to Vair was 1:10 and 1:30 at atmospheric pressure in the case of unsaturated oil and saturated oil respectively. In addition, the amount of nutrients injection was studied by the transport experiments of sucrose and nitrate. The results showed that the concentration of nutrients in the sandpack exports approached the injection concentration nearly when 3-4.5 pore volume (PV) of nutrition was injected. So the amount of nutrients was sufficient for maintaining the growth of the bacterium in the simulated reservoir conditions when 3PV of nutrition was injected.
2) The growth of the strain G2 was studied in the reservoir by simulating the temperature, pressure, porosity, permeability, fluid characteristics and development status of the aimed reservoir. The results showed that the cell density of the bacterium increased from 106 to 108 cells/mL in the simulated reservoir conditions of Block Ng3 when appropriate nutrients and gas were injected. This suggested that the strain G2 was suitable as a candidate aerobic microorganism for implementation of the MEOR technology in this block. Basically, the presence of residual oil had no impact on the growth of this bacteria. But the lag phase of growth was longer than in the shake-flask culture, which suggested that all MEOR studies pertaining to a specific reservoir should be evaluated under the in situ conditions of the reservoir.
4 The factors which affect the transport of the strain G2 were investigated systemically in sandpacks and cores. These factors included the linear velocity of injection, temperature, permeability, size and degree of the bacteria aggregation, presence of a residual oil phase and pulse injection. Furthermore, the major mechanisms of bacteria retention were discussed.
1) Firstly, the cellular suspension in stationary phase was continuously injected into the core. Subsequently, the cellular suspension was continuously displaced by sterile water. Effluent samples were collected at the export for analyzing the cell density, and the pressure across the core was monitored during the displacement process. The results showed that increasing the fluid flow velocity, temperature, permeability and pulse displacement increased the efficiency of microbial transport, while increasing the size and aggregation of the bacteria decreased the efficiency of transport. Besides, the presence of a residual oil phase increased the bacteria retention and decreased the efficiency of transport. Furthermore, the mechanisms of the bacteria retention during the transport process were studied by the former experiments. The mechanisms of the bacteria retention during the transport of the bacterial suspension included surface adhesion, size exclusion, pore bridging and multi-particle hydrodynamic exclusion. It was likely that all these mechanisms were involved to varying degrees in the transport of the bacterial suspension. When high concentrations (>108 cells/mL) of microorganisms were injected, multiparticle hydrodynamic exclusion and size exclusion of cell aggregates were the most likely operative mechanisms for bacteria retention. Under this condition, the injected bacteria were prone to retain in the entrance of the core, and the rapidly decrease of permeability occurred due to the forming of the external or internal filter cakes.
2) The growth and transport experiments showed that diffusive flux had little effect on the transport of microorganisms and their metabolites. This indicated that the main driving force of microbial transport came from hydrodynamic convection during the displacement process.
In summary, the results of this study will hopefully provide the technologic support for establishing evaluation methods (especially physical simulation experiment method) for MEOR which adopt air-assistant technique, and will also hopefully provide some data for mathematical model of MEOR. In addition, it will provide the theoretical basis and technologic support for the design of MEOR in the Block Ng3 of Shengli Oilfield.
本论文以一株典型的好氧采油微生物G2菌为例,确定在适宜的营养和配气条件下,好氧微生物在模拟典型实验区块实际油藏条件下的生长状况,确定影响微生物在岩石毛细孔隙中运移的主要因素,并计算运移效率。主要研究结果与结论如下:
1.从胜利油田微生物研究中心菌种库中优选出一株地芽孢杆菌(Geobacillus sp. G2),通过室内评价实验确定了该菌株适宜作为典型的好氧采油微生物用于微生物驱油基础理论研究。
通过对菌种库的细菌进行筛选,从中挑选出一株G2菌,室内摇瓶实验结果表明,该菌具有耐高温、以不同类型原油为唯一碳源能够生长、降粘、产表面活性物质、并对原油和液蜡有较好的乳化和分散作用等利于提高原油采收率的功能。通过物理模拟驱油实验和盲管驱油实验考察了该菌株的驱油性能:物理模拟驱油实验表明,采用空气辅助驱(液气比1:10),在一次水驱的基础上,经过5周期50d培养驱替,该菌能够提高原油采收率12.9-15.9%;盲管驱油实验也表明该菌能够驱出盲管中的残余油。以上实验结果表明,优选的G2菌可以作为典型的好氧采油微生物用于微生物驱油基础理论研究。
2.采用单次单因子法确定了G2菌适宜的生长条件。采用单次单因子法,通过摇瓶培养实验确定了G2菌适宜的培养条件:蔗糖3%,NaCl 0.5%,NaNO3 0.2%,KH2PO4 0.14%,Na2HPO4·12H2O 0.37%,FeSO4·7H2O 0.001%,MgSO4·7H2O 0.02%,CaCl2 0.001%,酵母粉0.05%,pH 7.0,温度50-55℃。确定了该菌株生长的pH、温度和盐度范围:pH 5.5-9.5,温度35-70℃, NaCl浓度0-10%;同时确定了该菌株适宜生长的pH、温度和盐度的范围:pH 6.0-9.0,温度40-60℃,NaCl浓度0.5-8%。采用高压岩心管,利用高压空气加压,通过培养实验研究了高压对该菌株生长的影响,结果表明,该菌株能在10MPa高压下生长,但生长延滞期变长。以上实验结果表明,该菌株的pH、温度和盐度适应范围均较广且耐高压,说明该菌可能的油藏适应范围较广,进一步说明该菌株适宜用于好氧微生物驱油基础理论研究。
3.以G2菌为例,通过调整模拟油藏环境的营养和配气,研究其在模拟典型实验区块实际油藏环境下的生长状况。
1)通过对非生物因素耗氧实验研究和细菌生长耗氧量的估算,确定了好氧微生物在室内模拟胜利油田孤岛中一区Ng3区块油藏环境下生长实验的配气量(空气):在不饱和原油的情况下,配气量维持在常压下液气比1:10;在饱和原油的情况下,配气量维持在常压下液气比1:30就足以维持好氧微生物在模拟油藏环境下良好生长了。通过对蔗糖和NO3-等营养物在岩心中的运移实验,确定了在注入岩心3-4.5PV培养液时,岩心出口营养物的浓度基本达到注入浓度。
2)通过模拟目标油藏的温度、压力、孔隙度、渗透率、流体特征和开发程度等因素,进行了G2菌在模拟油藏环境下的生长实验,结果表明,在注入适宜的营养和配气条件下,G2菌能够在模拟Ng3区块油藏环境下良好生长,细菌密度由106个/mL增加至108个/mL,该菌株适宜作为该区块实施微生物采油技术的候选菌种。残余油的存在对G2菌的生长基本没有产生影响,但在模拟油藏环境下,该菌生长延滞期较摇瓶培养实验长,说明为特定油藏进行的各种微生物采油技术研究必须在模拟油藏环境下进行。
4.以G2菌为例,系统考察了驱替速度、实验温度、岩心渗透率、微生物菌体大小和聚集度以及油相存在与否等因素对微生物在岩石毛细孔隙中运移的影响,并探讨了微生物在岩心中运移过程中滞留的主要机制。
1)先用处于生长稳定期的菌液进行连续的岩心驱替,接着用无菌水驱替,在出口取样分析菌体密度,记录整个驱替过程中的压差变化。驱替实验结果表明:驱替速度、实验温度、岩心渗透率、微生物菌体大小和聚集度以及油相存在与否等因素对微生物在岩心中运移有重要影响。当驱替速度、实验温度和岩心渗透率升高时,微生物运移效率增加;当微生物菌体大小和聚集度增加时,微生物运移效率降低;脉冲驱替能够增加微生物在岩心中的运移效率;油相存在能增加细菌的滞留量,降低细菌的运移效率。微生物在岩心中运移过程中的滞留是筛分、架桥堵塞、界面吸附、粘附和聚集堵塞等作用机制共同作用的结果。当注入微生物浓度较高时,架桥和聚集堵塞是微生物在岩心中滞留的主要作用机制,微生物更易滞留在岩心入口段,形成外部或内部滤饼,并对该段岩心渗透率伤害较大。
2)微生物在模拟油藏环境下的生长和运移实验表明,即使在静止培养的条件下,扩散对微生物及其代谢产物的运移影响也很小,在连续驱替过程中,微生物运移的动力主要来自于水动力。
本论文的研究成果为建立空气辅助微生物驱油技术室内评价方法(尤其是物理模拟驱油体系方法)和数模软件提供基础。另外,本论文针对胜利油田孤岛中一区Ng3区块进行研究,研究成果为该区块微生物采油矿场试验设计提供一定的理论基础和技术支撑,对最终提高现场应用效果具有促进作用。
The effective transport and growth of bacteria in reservoir is crucial to the success of microbial enhanced oil recovery (MEOR) technology. In recent years considerable efforts have been put into the effect of microbial metabolic products on physical and chemical properties of oil and water using shake-flask culture experiments, while there’s little information about the growth, transport and metabolism of bacteria in reservoir environment.
The objectives of this study are to identify whether aerobic bacteria can grow in simulated reservoir conditions or not, and to determine the important factors that control the transport of bacteria through rock porous media. Furthermore, the mechanisms of bacteria retention and permeability reduction are investigated. The main results are summarized as follows:
1 One strain of bacterium, identified as Geobacillus sp., was screened and confirmed as a candidate aerobic microorganism for MEOR by laboratory study.
The bacterium Geobacillus sp., named G2, was screened from library of strains of Shengli Oilfield. The results of shake-flask experiments showed it exhibited good properties for MEOR: suffering high temperature, growing when different types of crude oil was added as the sole carbon source, reducing the viscosity of crude oil, producing biosurfactants, emulsifying and dispersing crude oil or liquid wax, and so on. Besides, Core flood experiments showed the oil recovery had been increased by 12.9-15.9% of original oil in place (OOIP) after 5 treatments of 50 days by adopting air-assistant technique (liquid/air 1:10 v:v). Blind tube-flood experiments also showed that the oil in the dead area could be effectively drived out by the strain G2. These results indicated that the strain G2 was suited for the theory study of MEOR.
2 The optimal medium components and suitable growth conditions for the strain G2 were investigated by using“one-variable-at-a-time”method.
The optimal culture conditions of this bacterium were studied by the shake-flask culture experiments. The results showed that the optimal culture conditions were as follows: 3% sucrose, 0.5% NaCl, 0.2% NaNO3, 0.14% KH2PO4, 0.37% Na2HPO4·12H2O, 0.001% FeSO4·7H2O, 0.02% MgSO4·7H2O, 0.001% CaCl2, 0.05% yeast extract, pH 7.0 and at 50-55℃. The ranges of the main factors that affected the growth of the bacterium were also investigated. The results were as follows: the temperature, salinity and pH range for growth of the strain G2 was 35-70℃, 0-10% NaCl and 5.5-9.5 respectively; suitable growth temperature, salinity and pH occurred at 40-60℃, 0.5-8% NaCl 6.0-9.0 respectively. Besides, the influence of high pressure on the growth of the strain G2 was studied by high-pressure culture experiments. The results showed that the strain G2 could grow at 10MPa, but the lag phase of growth became longer. These results further indicated that G2 was the suited experimental object for the theory study of MEOR.
3 The growth of the strain G2 was studied in the simulated reservoir conditions when the nutrients and air conditions were adjusted for the better growth of the bacterium.
1) Based on the experimental study on abiotic factor oxygen consumption and the estimation of biotic oxygen consumption, the amount of air injection for the growth of G2 in the simulated reservoir conditions of Block Ng3 of Shengli Oilfield was investigated. The results indicated that the amount of oxygen was sufficient for maintaining the growth of microorganisms in the simulated reservoir conditions when the ratio of Vwater to Vair was 1:10 and 1:30 at atmospheric pressure in the case of unsaturated oil and saturated oil respectively. In addition, the amount of nutrients injection was studied by the transport experiments of sucrose and nitrate. The results showed that the concentration of nutrients in the sandpack exports approached the injection concentration nearly when 3-4.5 pore volume (PV) of nutrition was injected. So the amount of nutrients was sufficient for maintaining the growth of the bacterium in the simulated reservoir conditions when 3PV of nutrition was injected.
2) The growth of the strain G2 was studied in the reservoir by simulating the temperature, pressure, porosity, permeability, fluid characteristics and development status of the aimed reservoir. The results showed that the cell density of the bacterium increased from 106 to 108 cells/mL in the simulated reservoir conditions of Block Ng3 when appropriate nutrients and gas were injected. This suggested that the strain G2 was suitable as a candidate aerobic microorganism for implementation of the MEOR technology in this block. Basically, the presence of residual oil had no impact on the growth of this bacteria. But the lag phase of growth was longer than in the shake-flask culture, which suggested that all MEOR studies pertaining to a specific reservoir should be evaluated under the in situ conditions of the reservoir.
4 The factors which affect the transport of the strain G2 were investigated systemically in sandpacks and cores. These factors included the linear velocity of injection, temperature, permeability, size and degree of the bacteria aggregation, presence of a residual oil phase and pulse injection. Furthermore, the major mechanisms of bacteria retention were discussed.
1) Firstly, the cellular suspension in stationary phase was continuously injected into the core. Subsequently, the cellular suspension was continuously displaced by sterile water. Effluent samples were collected at the export for analyzing the cell density, and the pressure across the core was monitored during the displacement process. The results showed that increasing the fluid flow velocity, temperature, permeability and pulse displacement increased the efficiency of microbial transport, while increasing the size and aggregation of the bacteria decreased the efficiency of transport. Besides, the presence of a residual oil phase increased the bacteria retention and decreased the efficiency of transport. Furthermore, the mechanisms of the bacteria retention during the transport process were studied by the former experiments. The mechanisms of the bacteria retention during the transport of the bacterial suspension included surface adhesion, size exclusion, pore bridging and multi-particle hydrodynamic exclusion. It was likely that all these mechanisms were involved to varying degrees in the transport of the bacterial suspension. When high concentrations (>108 cells/mL) of microorganisms were injected, multiparticle hydrodynamic exclusion and size exclusion of cell aggregates were the most likely operative mechanisms for bacteria retention. Under this condition, the injected bacteria were prone to retain in the entrance of the core, and the rapidly decrease of permeability occurred due to the forming of the external or internal filter cakes.
2) The growth and transport experiments showed that diffusive flux had little effect on the transport of microorganisms and their metabolites. This indicated that the main driving force of microbial transport came from hydrodynamic convection during the displacement process.
In summary, the results of this study will hopefully provide the technologic support for establishing evaluation methods (especially physical simulation experiment method) for MEOR which adopt air-assistant technique, and will also hopefully provide some data for mathematical model of MEOR. In addition, it will provide the theoretical basis and technologic support for the design of MEOR in the Block Ng3 of Shengli Oilfield.
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